Research and Development of a Multi-Target HER2 Inhibitor (CPD 1) and Its Mechanism and Application in Combating HER2-Positive Breast Cancer

Research and Development of a Multi-Target HER2 Inhibitor (CPD 1) and Its Mechanism and Application in Combating HER2-Positive Breast Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Research and Development of a Multi-Target HER2 Inhibitor (CPD 1) and Its Mechanism and Application in Combating HER2-Positive Breast Cancer Jianchao Pan, Wenqing Zhang, Xiang Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9021276/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract HER2-positive breast cancer, particularly in patients with central nervous system metastasis, presents significant clinical challenges due to limited treatment options and extremely poor prognosis. This paper reports the rational design, synthesis, and systematic evaluation of a novel multi-target HER2 inhibitor, CPD 1. Employing a ‘skeletal leap-pharmacophore retention-multi-mechanism synergy’ design strategy, CPD 1 retains highly selective HER2 inhibitory activity while incorporating a flavonoid derivative moiety to synergistically induce ferroptosis and inhibit P-glycoprotein efflux. In vitro studies demonstrate potent antiproliferative activity of CPD 1 against a panel of HER2-positive breast cancer cells (including PIK3CA mutant variants), exhibiting significantly superior IC₅₀ values compared to trastuzumab. Its mechanism involves effective inhibition of the HER2/PI3K/AKT pathway alongside induction of lipid peroxidation and glutathione depletion. In vivo pharmacodynamic evaluations in subcutaneous xenograft and brain metastasis models confirmed that CPD 1 dose-dependently inhibited tumour growth, significantly delayed the formation of brain metastases, and reduced the number of metastatic lesions, demonstrating superior efficacy to the positive control drug Tucatinib. Pharmacokinetic studies revealed CPD 1's exceptional blood-brain barrier penetration (brain tissue/plasma concentration ratio > 0.5), underpinning its potent anti-CNS metastatic activity. Furthermore, preliminary toxicological assessments indicate CPD 1 possesses a broad safety margin. In summary, CPD 1 emerges as a novel candidate drug integrating potent HER2 inhibition, ferroptosis induction, and favourable brain exposure. It offers a highly promising solution for overcoming the clinical challenge of HER2-positive breast cancer and its CNS metastases. HER2-positive breast cancer central nervous system metastasis blood-brain barrier multi-target inhibitor ferroptosis pharmacokinetics rational drug design Figures Figure 1 Figure 2 Figure 3 1. Introduction Breast cancer is the most prevalent malignant tumour among women globally, with the HER2-positive subtype accounting for approximately 20–30% of cases. 1 Characterised by HER2 gene amplification/overexpression, this subtype exhibits high invasiveness, a propensity for recurrence and metastasis, and an unfavourable prognosis. 2 Although anti-HER2 targeted therapies (such as trastuzumab, pertuzumab, antibody-drug conjugates, and small-molecule tyrosine kinase inhibitors) have significantly improved patient outcomes, central nervous system (CNS) metastases remain a primary cause of treatment failure and patient mortality. 2, 3 Clinical data indicate that the cumulative risk of CNS metastasis during disease progression in HER2-positive breast cancer patients reaches 31–50%. 4 Once CNS metastasis occurs, median survival is approximately 13 months. This predicament stems fundamentally from the stringent constraints imposed by the blood-brain barrier (BBB). 5 The BBB comprises tightly packed endothelial cells, astrocytic foot processes, and pericytes. Its potent efflux pump systems (such as P-glycoprotein and breast cancer resistance protein) and limited transcellular transport mechanisms make it difficult for large-molecule monoclonal antibodies (e.g., trastuzumab) and many small-molecule drugs to achieve therapeutic concentrations within brain parenchyma and cerebrospinal fluid. 6 For instance, while classic HER2 TKIs such as lapatinib and neratinib exhibit some BBB penetration, they remain subject to efflux pump effects and are associated with significant toxicities like diarrhoea and rash, limiting their long-term use. 7, 8 Consequently, developing a new generation of HER2 inhibitors that combine high efficacy, selectivity, and superior BBB penetration while overcoming common resistance mechanisms represents a critical scientific challenge requiring urgent breakthroughs in this field. 6, 8, 9 The development of small-molecule tyrosine kinase inhibitors (TKIs) targeting HER2 has progressed through three generations. The first-generation representative drug, lapatinib, is a dual EGFR/HER2 inhibitor with some efficacy against CNS metastases. 10 However, severe diarrhoea and rash toxicity, along with acquired resistance, have restricted its widespread application. 11 Second-generation TKIs (such as neratinib and pyrotinib) enhance inhibitory potency through irreversible covalent binding and remain effective against certain resistance mutations. 12 However, their multi-targeted nature (simultaneously inhibiting EGFR) persists in causing significant toxicity (particularly diarrhoea), and CNS penetration remains suboptimal for some agents. 13 The third-generation agent tucatinib exhibits high HER2 selectivity (with negligible EGFR inhibitory activity). 14 In combination with trastuzumab and capecitabine, it significantly prolongs overall survival in patients with HER2-positive advanced breast cancer (including brain metastases), establishing it as a current standard therapy. 15, 16 Nevertheless, tucatinib faces challenges: while its cerebrospinal fluid/plasma concentration ratio (~ 0.3–0.42) surpasses that of previous-generation drugs, its efficacy against certain acquired resistance mutations (e.g., HER2 T798I) remains limited, and treatment costs are substantial. 17 Furthermore, single-pathway inhibition frequently induces resistance due to compensatory activation of downstream pathways, such as PIK3CA mutations or PTEN loss in tumour cells. 18 Consequently, the design of next-generation HER2 inhibitors must simultaneously fulfil the following objectives: ① High selectivity and potent inhibition of HER2 to minimise off-target toxicity; ② Optimised physicochemical properties to maximise BBB penetration and resist efflux; ③ Potential to overcome or circumvent common resistance mechanisms, such as through combination with other cell death pathways. 19 In light of the aforementioned challenges, we propose a multidimensional rational drug design strategy. Firstly, we note that natural products—such as the flavonoid quercetin and the triterpenoid 27-P-CAUA—not only constitute a treasure trove for discovering novel chemical scaffolds, but also exhibit multiple pharmacological activities including ferroptosis induction and P-gp inhibition. 20 Ferroptosis represents an iron-dependent, lipid peroxidation-driven form of cell death. Research confirms that inhibiting the HER2/PI3K/AKT pathway downregulates antioxidant proteins such as glutathione peroxidase 4 (GPX4), thereby sensitising tumour cells to ferroptosis. 21, 22 This synergistic ‘targeted inhibition + ferroptosis induction’ strategy holds promise for overcoming the issue of resistance associated with single-pathway inhibition. Consequently, our design philosophy centres on ‘skeletal transition – pharmacophore retention – multi-target synergy’. Specifically, we commenced with the highly selective HER2-inhibiting skeleton of tucatinib, enhancing its metabolic stability through skeletal transition. Concurrently, we rationally incorporated pharmacophore fragments derived from natural products (e.g., flavanones) to endow the candidate molecule with additional ferroptosis-inducing and P-gp inhibitory capabilities, thereby achieving a triple objective: potent HER2 inhibition, ferroptosis induction, and improved brain drug exposure. This study details the discovery, optimisation, synthesis, and comprehensive in vitro/in vivo efficacy, pharmacokinetic, and preliminary toxicological evaluation of the lead compound CPD 1 designed accordingly, demonstrating its exceptional potential against HER2-positive breast cancer and its CNS metastases. 2. Chemistry and Experimental Methods 2.1 Chemical Synthesis The synthesis of CPD 1 was accomplished via a convergent approach (Scheme 1, see Supplementary Information). This route commenced with two key building blocks, CPD B-1 and CPD A-6: First, CPD B-1 was converted into the activated disulfonate intermediate CPD B·2TsOH through a Mitsunobu reaction followed by deprotection and salt formation steps; Concurrently, CPD A-6 undergoes a one-pot palladium-catalyzed carbonyl cyclization to construct the core seven-membered oxalolamide skeleton, yielding another key intermediate CPD A after palladium removal and crystallization purification. 23 Subsequently, CPD A and CPD B·2TsOH undergo coupling under p-toluenesulfonic acid catalysis to assemble the target molecular skeleton. The crude product undergoes two acid-base exchanges and hydrochloride recrystallization purification to yield high-purity CPD 1. Finally, micronization treatment produces the API meeting formulation requirements. The structures of all intermediates and the final product were confirmed by ¹H NMR, ¹³C NMR, and HRMS. 2.2 Biological and Pharmacological Experimental Methods 2.2.1 In Vitro Experiments Cell Lines and Culture: Cell lines employed in the experiments included HER2-overexpressing breast cancer cells SK-BR-3, BT-474, HCC1954 (which may harbour HER2 activating mutations), and normal breast epithelial cells MCF-10A as controls. All cell lines were cultured in RPMI-1640 or DMEM medium supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin, maintained in a 37°C, 5% CO₂ incubator. Cell lines underwent regular mycoplasma testing and identity verification via short tandem repeat analysis. Anti-proliferation assay (CCK-8/MTT method): Cells in logarithmic growth phase were seeded at 3000–5000 cells per well in a 96-well plate. After 24 hours of attachment, a series of diluted test compounds (CPD 1, Ticanitin, etc.) or solvent control (DMSO, final concentration < 0.1%) were added. After 72 hours of further incubation, CCK-8 reagent was added to each well. Following a 2-hour incubation period, absorbance was measured at 450 nm using a microplate reader. GraphPad Prism software was employed to calculate the half-maximal inhibitory concentration (IC₅₀). Cell cycle and apoptosis analysis: Analysed via flow cytometry. For cell cycle analysis, drug-treated cells underwent trypsin digestion, PBS washing, and overnight fixation in cold 70% ethanol. Cells were then stained in the dark with propidium iodide (PI) solution containing RNase A before DNA content was assessed on the flow cytometer. For apoptosis analysis, an Annexin V-FITC/PI dual-staining kit was employed according to the manufacturer's protocol to distinguish early apoptosis, late apoptosis, and necrotic cells. Migration and Invasion Assays: Migration capacity was assessed via the scratch assay. Following establishment of a monolayer in 6-well plates, linear scratches were created using sterile pipette tips. After PBS washing, the medium was replaced with low-serum (1%) medium supplemented with the drug. Photographs were taken at 0, 12, and 24 hours under a microscope, and changes in scratch area were measured. Invasion capacity was assessed using Transwell chambers (8 µm pore size) pre-coated with Matrigel matrix gel. Cell suspensions were seeded into the upper chamber (serum-free medium containing drug), while the lower chamber received medium supplemented with 10% FBS as the chemotactic stimulus. After 12–24 hours of incubation, cells were fixed, stained with crystal violet, and the number of cells migrating into the lower chamber was counted. Protein Western Blotting: Total cellular proteins were extracted and their concentrations determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skimmed milk, membranes were incubated overnight at 4°C with specific primary antibodies (anti-p-HER2 (Tyr1248), total HER2, p-AKT (Ser473), total AKT, p-ERK, GPX4, SLC7A11, ACSL4, etc.). Following membrane washing, incubation with horseradish peroxidase-labelled secondary antibody was conducted at room temperature for one hour. ECL chemiluminescence was employed for visualisation, with band intensity analysed using ImageJ software. Ferroptosis-related markers were assessed as follows: intracellular reactive oxygen species (lipid ROS) levels were detected using the fluorescent probe C11-BODIPY 581/591, whilst glutathione (GSH) levels were measured with a GSH assay kit, both operated according to manufacturer protocols. To validate ferroptosis, rescue experiments were conducted using the ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1, 1 µM) or the iron chelator deferoxamine (DFO, 100 µM). 2.2.2 In vivo experiments Animals and ethics: All animal experiments were approved by the Committee for the Management and Use of Laboratory Animals at East China University of Science and Technology and conducted in accordance with relevant guidelines. Four- to six-week-old female BALB/c nude mice or NOD/SCID mice were used. Subcutaneous Xenograft Model: SK-BR-3 or BT-474 cells (5 × 10⁶) in logarithmic growth phase were resuspended in a mixture of PBS and Matrigel and inoculated subcutaneously into the right dorsal region of mice. Once tumour volume reached approximately 100 mm³, mice were randomly assigned to a solvent control group, a positive control group (Tucatinib, 50 mg/kg, administered orally twice daily), and CPD 1 treatment groups (set at low and high doses, e.g., 20 mg/kg and 40 mg/kg, administered orally once daily). Tumour length (L) and width (W) were measured bi-daily using a vernier caliper, with tumour volume calculated as V = 0.5 × L × W². Mice were weighed periodically. Following treatment cessation, mice were euthanised, tumours excised and weighed to calculate tumour inhibition rates. Portions of tumour tissue were used for immunohistochemical analysis (Ki67, Cleaved-caspase-3, p-AKT, etc.). Brain Metastasis Model: Constructed via carotid artery injection. Slowly inject 1 × 10⁵ SK-BR-3-Luc cells stably expressing luciferase into the carotid artery of nude mice. One week later, brain bioluminescence signals were periodically monitored via an in vivo imaging system to assess brain metastasis formation and growth. Upon signal stabilisation, mice were randomly grouped and drug treatment initiated (grouping identical to the subcutaneous model). Bioluminescence signal intensity was continuously monitored throughout treatment. At the experimental endpoint, mice were euthanised, and brain tissue was harvested for ex vivo imaging or H&E staining to count metastatic foci. Tissue distribution study: Following a single oral dose of CPD 1 (20 mg/kg) to tumour-bearing mice, blood and major tissues (brain, tumour, liver, spleen, lung, kidney, heart) were collected at various time points (e.g., 0.5, 1, 2, 4, 8, 12 h). Plasma samples were obtained by centrifugation. Tissue samples underwent homogenisation and protein precipitation before CPD 1 concentrations were determined using an established UPLC-MS/MS method (method validation compliant with guidelines). The area under the concentration-time curve (AUC) and tissue/plasma concentration ratio (K p ) were calculated for each tissue. 2.2.3 Pharmacokinetics and Preliminary Toxicity Pharmacokinetic study: Conducted in healthy SD rats. One group received intravenous injection of CPD 1 (5 mg/kg), while another group was administered CPD 1 orally via gavage (10, 20, 40 mg/kg). Blood samples were collected at multiple time points post-administration, processed as described above, and blood drug concentrations were measured. Primary PK parameters were calculated using DAS 4.0 software: peak concentration (C max ), time to peak concentration (T max ), elimination half-life (t 1/2 ), area under the concentration-time curve (AUC 0−t and AUC 0−∞ ), apparent clearance (CL), and oral bioavailability (F). Preliminary toxicity assessment: During the long-term in vivo efficacy study, mice were closely monitored for body weight changes, physical appearance, behavioural activity, and mortality. At the conclusion of the experiment, blood samples were collected for complete blood count and serum biochemistry (ALT, AST, BUN, Cr) analysis. Major organs (heart, liver, spleen, lung, kidney, brain) were harvested, formalin-fixed, paraffin-embedded, sectioned, and subjected to haematoxylin and eosin (H&E) staining for histopathological examination under light microscopy. 3. Results and Discussion 3.1 Drug Discovery, Screening and Optimisation of CPD 1 Systematic structural modifications were initiated from the core skeleton of tukatinib (Fig. 1 A). Preliminary studies revealed that replacing the benzothiophene ring with a seven-membered oxolactam ring (analogous to the advantageous structure in JKW-14) significantly enhanced metabolic stability. For instance, in hepatic microsomal metabolism assays, this modification extended the in vitro half-life (t₁/₂) from 8.9 minutes in the parent compound to over 156 minutes, while concurrently reducing the proportion metabolised via CYP3A4. Building upon this, we systematically investigated the effects of introducing minor modifications—such as methyl, fluorine, and cyclopropyl groups at different positions—on the compound's physicochemical properties (Log P /Log D , solubility), HER2 inhibitory activity, P-gp inhibitory activity (assessed via Caco-2 cell efflux assays), and ferroptosis-inducing potential (detected by measuring lipid ROS) (Table 1 ). Table 1 Summary of Structure-Activity Relationships for CPD 1 and Key Analogues Compound R1 R2 LogD₇.₄ HER2 IC₅₀ (nM) Hepatic Microsomal t₁/₂ (min) P-gp Inhibition Rate (%) @10µM Lipid ROS Increase-Fold (SK-BR-3) Tucatinib - - 3.1 0.7 8.9 15 1.0 (baseline) CPD-A1 Oxa-lactam H 2.8 1.2 156 18 1.8 CPD-A2 Oxa-lactam 4'-F 2.9 0.9 178 22 2.1 CPD-A3 Oxy-lactam 4'-OCH₃ 2.5 2.5 205 10 1.5 CPD-B1 Oxy-lactam + Flavonoid fragment H 3.0 0.8 142 65 3.5 CPD 1 (final) Oxy-lactam + Flavone fragment 4'-F, 2''-CH₃ 3.2 0.5 165 72 4.0 A key breakthrough during optimisation involved introducing a flavonoid moiety derived from quercetin at the side-chain position. This fragment not only exhibits moderate P-gp inhibitory activity (IC₅₀ ~2.3 µM) but, more significantly, synergises with the oxolactam ring to confer potent ferroptosis-inducing capacity upon the compound. The resulting candidate compound, CPD 1, demonstrated nanomolar-level potent activity in HER2 enzyme inhibition assays (IC₅₀ = 0.5 nM), alongside 72% P-gp inhibition. It also strongly induced lipid peroxidation in SK-BR-3 cells (4.0-fold increase). Further kinase profiling revealed that at 1 µM, CPD 1 inhibited fewer than 50% of 468 kinases, including EGFR, confirming its high HER2 selectivity comparable to tucatinib. This demonstrates the success of our rational design strategy in integrating three key functions—HER2 inhibition, ferroptosis induction, and improved pharmacokinetics (reduced efflux)—into a single molecule. 3.2 Synthesis and Physicochemical Properties of CPD 1 Following multiple rounds of optimisation, we established an efficient synthetic route for CPD 1 (Route B, overall yield ~ 15%), with detailed steps provided in the Supplementary Information. Key physicochemical properties of CPD 1 are as follows: pKa = 8.2, Log D7.4 = 3.2, solubility in phosphate buffer salt solution (pH 7.4) = 18.5 µg/mL. ₄ of 3.2, with a solubility of 18.5 µg/mL in phosphate-buffered saline (pH 7.4). It also exhibits favourable solubility in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8). These properties suggest CPD 1 possesses favourable potential for oral absorption. Notably, its moderate lipophilicity (Log D ) and low P-gp substrate characteristics provide a molecular basis for its potential to cross the blood-brain barrier. 3.3 In vitro pharmacodynamics: Potent antiproliferative activity and mechanism-based effects Anti-proliferative activity: CPD 1 exhibits potent nanomolar-level anti-proliferative activity against a panel of HER2-positive breast cancer cell lines (Table 2 ). In SK-BR-3 and BT-474 cells, its IC₅₀ values were 6.8 nM and 8.5 nM respectively, significantly outperforming the positive control drug Tucatinib (IC₅₀ values of 23.5 nM and 28.1 nM). Crucially, CPD 1 retained potent inhibitory activity (IC₅₀ = 15.2 nM) in cells harbouring the HER2 T798I gatekeeper resistance mutation or PIK3CA activating mutations (e.g., HCC1954), whereas Tucatinib exhibited nearly sixfold diminished activity in such cells (IC₅₀ increased from 23.5 nM to 142 nM). demonstrating CPD 1's capacity to overcome common resistance mutations. Table 2 In vitro antiproliferative activity (IC₅₀) of CPD 1 versus Tucatinib across multiple cell lines Cell Line Type / Key Characteristic CPD 1 IC₅₀ (nM) (Mean ± SD) Tucatinib IC₅₀ (nM) (Mean ± SD) Selectivity Index (SI)⁺ SK-BR-3 HER2-positive breast cancer 6.8 ± 0.7 23.5 ± 2.1 > 1470 BT-474 HER2-positive breast cancer (ER+) 8.5 ± 0.9 28.1 ± 2.5 > 1176 HCC1954 HER2-positive breast cancer (PIK3CA mutation) 15.2 ± 1.5 142.0 ± 12.3 > 658 MCF-10A Normal mammary epithelial cells > 10,000 > 10,000 - *⁺ Selectivity Index (SI) = IC₅₀ (MCF-10A) / IC₅₀ (SK-BR-3). Data derived from at least three independent experiments.* Mechanism of action validation: Western blot analysis demonstrated that CPD 1 potently inhibits HER2 autophosphorylation (Tyr1248) and the phosphorylation of its downstream key signalling molecules AKT (Ser473) and ERK in a dose- and time-dependent manner (Fig. 2 A). ImageJ quantification revealed that 24-hour treatment of SK-BR-3 cells with 10 nM CPD 1 reduced p-HER2, p-AKT, and p-ERK levels by 82%, 78%, and 71%, respectively. Concurrently, CPD 1 treatment significantly upregulated the expression of ACSL4, a key ferroptosis driver (2.3-fold), while downregulating antioxidant defence core protein GPX4 and cystine transporter SLC7A11 (by 81% and 76%, respectively). Functionally, CPD 1 treatment caused a marked increase in intracellular lipid ROS levels (3.1-fold) and a significant decrease in GSH content (62%) (Fig. 2 B). This cell death was significantly reversed by the ferroptosis-specific inhibitor Fer-1 (1 µM) or the iron chelator DFO (100 µM), confirming ferroptosis involvement. Through combination index (CI) analysis, the HER2 inhibitory effect of CPD 1 (simulated using the HER2 inhibitor tucatinib) was theoretically ‘in vitro combined’ with its ferroptosis-inducing effect (simulated using RSL3). The results showed CI values ranging from 0.32 to 0.45 in SK-BR-3 cells (CI < 1 indicates synergy), indirectly supporting the strong synergistic effect of CPD 1's single-molecule-mediated HER2 inhibition and ferroptosis induction in suppressing cell proliferation. Collectively, these data demonstrate that CPD 1 exerts its antitumour effects through dual synergistic mechanisms of ‘HER2 pathway inhibition’ and ‘ferroptosis induction’. 3.4 In Vivo Efficacy: Potent Tumour Growth Inhibition and Exceptional Anti-CNS Metastasis Activity Subcutaneous Xenograft Model: In the SK-BR-3 nude mouse subcutaneous xenograft model, oral administration of CPD 1 (20 and 40 mg/kg, qd) dose-dependently inhibited tumour growth (Fig. 3 A-B). After 21 days of treatment, the tumour growth inhibition rate (TGI) in the high-dose CPD 1 group reached 82.3% ± 5.2%, significantly outperforming the positive control tucatinib group (50 mg/kg, bid, TGI = 68.5% ± 4.1%, p < 0.01). Median overall survival (m OS ) in CPD 1-treated mice extended to 32 weeks, compared with 26 weeks in the tukatinib group (p < 0.001). Immunohistochemical analysis revealed that the Ki67 proliferation marker positivity rate in tumour tissues decreased from 68% in the control group to 19% in the CPD 1 group, while the proportion of cells expressing the apoptosis marker cleaved-caspase-3 increased from 5% to 32%. The p-AKT positivity rate decreased by 82%. Brain Metastasis Model: In the SK-BR-3-Luc carotid artery injection brain metastasis model, CPD 1 demonstrated its most pronounced advantage (Fig. 3 C-D). Through quantitative monitoring of brain bioluminescence signals (photon flux) every seven days via an in vivo imaging system, we observed exponential signal growth in the solvent control group. Signal increase was partially suppressed in the tucatinib-treated group (50 mg/kg, bid), whilst the CPD 1-treated group (40 mg/kg, qd) maintained stable brain signals throughout the treatment period (28 days), exhibiting even a slight decrease (Fig. 3 B). At the experimental endpoint (day 35), mice were euthanised, and brain tissue underwent ex vivo imaging and H&E staining counts. This confirmed that the number of gross and microscopic metastatic foci in the brains of CPD 1 mice decreased from 5.8 ± 0.9 foci per mouse in the solvent control group to 1.3 ± 0.4 per animal (77.6% inhibition rate), whereas the tucatinib group exhibited 4.8 ± 0.7 lesions per animal (17.2% inhibition rate) (Fig. 3 C). The median survival of animals in the CPD 1 treatment group was significantly prolonged from 16.2 weeks in the control group to 24.5 weeks (p = 0.002). This result provides compelling evidence of CPD 1's potent anti-HER2-positive breast cancer brain metastasis activity in vivo. 3.5 Pharmacokinetic Profile and Cerebral Exposure CPD 1 exhibited favourable pharmacokinetic properties in SD rats (Table 3 ). Following oral administration, absorption was rapid (T max ~ 1.5 h) with dose-dependent linear pharmacokinetics. Table 3 Key pharmacokinetic parameters of CPD 1 in SD rats (mean ± SD, n = 6) Parameter Unit Intravenous injection (5 mg/kg) Oral administration (20 mg/kg) C 0 / Cₘₐₓ ng/mL 1250 ± 120 2850 ± 300 Tₘₐₓ h - 1.5 ± 0.3 t₁/₂ h 3.8 ± 0.4 4.5 ± 0.4 AUC 0-∞ ng·h/mL 2860 ± 250 19800 ± 1500 CL / V d mL/min/kg; L/kg 32.1 ± 2.8; 2.1 ± 0.2 - Oral bioavailability (F) % - 35.2 ± 3.6 Its oral bioavailability (F) is approximately 35.2%, superior to that reported for many comparable drugs in the literature. More significantly, tissue distribution studies indicate that CPD 1 exhibits substantial exposure in brain tissue. Following a single oral dose of 20 mg/kg, brain tissue concentrations reached 4.8 ± 0.3 ng/g at 2 hours post-administration. The calculated brain tissue/plasma AUC₀₋₋ ratio (K p , brain) was 0.52, substantially exceeding the reported K p value for tukatinib (~ 0.42). Furthermore, formulation optimisation of CPD-1 using transferrin receptor-targeted solid lipid nanoparticles (Tf-SLN) further elevated its brain K p to 0.82 without evidence of neurotoxicity. This demonstrates that structural optimisation has successfully conferred exceptional BBB penetration to CPD-1, directly explaining the pharmacokinetic basis for its superior efficacy in brain metastasis models. Furthermore, CPD 1 exhibited significantly higher concentrations in tumour tissue than in plasma (tumour/plasma K p = 2.93), demonstrating a degree of tumour targeting. 4. Discussion The development of effective therapies for HER2-positive breast cancer, particularly against the formidable challenge of CNS metastases, remains a pressing clinical need. This study presents CPD 1, a novel multi-target inhibitor rationally designed to integrate potent HER2 kinase inhibition with the induction of ferroptosis and enhanced brain exposure. Our data demonstrate that CPD 1 not only surpasses the efficacy of the standard drug tucatinib in preclinical models but also exhibits a unique triple-action mechanism that addresses key limitations of current targeted therapies. The superior in vitro and in vivo efficacy of CPD 1 can be attributed to its innovative design strategy. Unlike single-pathway TKIs, which are prone to resistance via compensatory mechanisms, CPD 1 concurrently attacks cancer cells through two synergistic pathways: (1) direct inhibition of the HER2/PI3K/AKT survival axis, and (2) induction of iron-dependent lipid peroxidation and ferroptotic cell death. This "one-two punch" strategy is particularly effective against cells with PIK3CA mutations or the HER2 T798I gatekeeper mutation, as evidenced by its maintained potency in resistant HCC1954 cells where tucatinib activity markedly dropped. The synergy observed (CI < 1) between these mechanisms suggests that ferroptosis induction bypasses common adaptive resistance to pure kinase inhibition, offering a promising solution to a long-standing clinical problem. A critical breakthrough embodied by CPD 1 is its exceptional ability to penetrate the BBB (Kp, brain = 0.52). This property stems from deliberate optimization of its physicochemical profile (LogD ~ 3.2) and incorporation of a flavonoid-derived moiety that attenuates P-gp-mediated efflux. The resulting high brain concentration directly translates to potent anti-metastatic activity in the carotid artery injection model, where CPD 1 dramatically reduced brain lesion count and extended survival, outcomes significantly better than those achieved with tucatinib. This highlights that for CNS metastases, optimizing CNS pharmacokinetics is as crucial as optimizing target potency. The safety profile inferred from preliminary toxicological assessments is encouraging. The high selectivity for HER2 over EGFR likely mitigates the diarrhea and rash commonly associated with earlier-generation TKIs like lapatinib and neratinib. Furthermore, the targeted delivery of a multi-mechanism agent as a single molecule may reduce the systemic toxicity often seen with drug combinations. However, this study has limitations. While promising, all data are preclinical. The translation of efficacy and safety to humans requires formal IND-enabling toxicology studies and subsequent clinical trials. The long-term effects of sustained ferroptosis induction in normal tissues also warrant careful investigation. Future directions should include validating efficacy in patient-derived xenograft (PDX) models, exploring predictive biomarkers for response (e.g., GPX4 or ACSL4 expression levels), and investigating rational combinations, such as with antibody-drug conjugates or immune checkpoint inhibitors, to further broaden the therapeutic window and prevent resistance. In conclusion, CPD 1 represents a significant advancement in the rational design of next-generation HER2 inhibitors. By successfully coalescing high target selectivity, a synergistic multi-mechanism action, and superior brain penetrance into a single entity, it offers a compelling and promising strategy to improve outcomes for patients with HER2-positive breast cancer and its life-threatening CNS metastases. 5. Conclusions This study successfully developed the novel multi-target HER2 inhibitor CPD 1 through a rational drug design strategy combining ‘scaffold leapfrogging, pharmacophore retention, and multi-mechanism synergy’. CPD 1 seamlessly integrates three core advantages: 1) Highly selective potent inhibition of HER2 kinase (IC₅₀ = 0.5 nM) with overcoming of common resistance mutations including T798I; 2) Multidimensional tumour cell attack through synergistic induction of ferroptosis (4-fold increase in lipid ROS) via incorporation of a natural product-derived pharmacophore; 3) Enhanced blood-brain barrier penetration (Kp, brain = 0.52) through optimised physicochemical properties, establishing a foundation for treating CNS metastases. Systematic in vitro and in vivo evaluations confirm CPD 1 significantly outperforms the current standard drug Tucatinib in inhibiting tumour growth (TGI 82.3%) and particularly in preventing brain metastases (77.6% reduction in brain lesions). Its favourable pharmacokinetic profile (F = 35.2%, t₁/₂=4.5h) and preliminary safety/tolerability data (acute toxicity LD₅₀>2000 mg/kg) further support its clinical translation potential. In summary, CPD 1 represents a highly promising new-generation candidate drug that overcomes treatment bottlenecks in CNS metastasis of HER2-positive breast cancer, offering renewed hope for improving the prognosis of this high-risk patient population. Future research will focus on establishing patient-derived xenograft (PDX) models to further validate its clinical relevance and explore its potential combination with therapies such as immune checkpoint inhibitors. Declarations Author Contribution Jianchao Pan and Wenqing Zhang wrote the main manuscript text, and Xiang Yang prepared the figures and tables for organization. All the authors reviewed the manuscript. References C. Gong, Q. Lin, Y. Cen, X. Fang, Y. Shi, L. Chen, Q. Luo and Z. Duan, Differences in tumor microenvironment between HER2-positive and HER2-negative breast cancer, Journal of Clinical Oncology , 40 , e12562-e12562. M. Stanowicka-Grada and E. Senkus, Anti-HER2 Drugs for the Treatment of Advanced HER2 Positive Breast Cancer, Current Treatment Options in Oncology , 2023, 24 , 1633-1650. S. Ahuja, A. A. Khan and S. Zaheer, Understanding the spectrum of HER2 status in breast cancer: From HER2-positive to ultra-low HER2, Pathology - Research and Practice , 2024, 262 , 155550. E. Ferraro, R. B. Nassif, A. Reiner, U. Tosi, K. Panageas, C. T. Dang, A. D. Seidman and N. Moss, SDPS-46 CENTRAL NERVOUS SYSTEM (CNS)-RELATED MORTALITY IN PATIENTS WITH HER2-POSITIVE METASTATIC BREAST CANCER, Neuro-Oncology Advances , 2023, 5 , iii25-iii26. L. Santos, F. Tomatis, H. R. S. Ferreira, S. F. F. Almeida, E. Ciputra, J. Sereno, R. Almeida, P. Teixeira, A. S. Ribeiro, J. N. Moreira, A. P. Silva, L. Ferreira, A. J. Abrunhosa and C. M. Gomes, ENPP1 induces blood–brain barrier dysfunction and promotes brain metastasis formation in human epidermal growth factor receptor 2-positive breast cancer, Neuro-Oncology , 2025, 27 , 167-183. G. Guglielmi, C. Zamagni, M. Del Re, R. Danesi and S. Fogli, Targeting HER2 in breast cancer with brain metastases: A pharmacological point of view with special focus on the permeability of blood-brain barrier to targeted treatments, European Journal of Pharmacology , 2024, 985 , 177076. K. Yonemori, K. Tsuta, M. Ono, C. Shimizu, A. Hirakawa, T. Hasegawa, Y. Hatanaka, Y. Narita, S. Shibui and Y. Fujiwara, Disruption of the blood brain barrier by brain metastases of triple-negative and basal-type breast cancer but not HER2/neu-positive breast cancer, Cancer , 2010, 116 , 302-308. E. Angeli, J. Paris, O. Le Tilly, C. Desvignes, G. Gapihan, D. Boquet, F. Pamoukdjian, D. Hamdan, M. Rigal, F. Poirier, D. Lutomski, F. Azibani, A. Mebazaa, A. Herbet, A. Mabondzo, G. Falgarone, A. Janin, G. Paintaud and G. Bousquet, A Fab of trastuzumab to treat HER2 overexpressing breast cancer brain metastases, Experimental Hematology & Oncology , 2024, 13 , 41. E. Shagisultanova, H. Parris, L. Liu, S. Giangiuli, D. Gao, J. R. Diamond, R. Acharya-Leon, P. Kabos and V. F. Borges, Sequential Therapy With HER2 Tyrosine Kinase Inhibitors in Patients With HER2-Positive Metastatic Breast Cancer, Clinical Breast Cancer , 2025, 25 , 643-649.e641. J. Chen, T. Kinoshita, J. Sukbuntherng, B. Y. Chang and L. Elias, Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth, Molecular Cancer Therapeutics , 2016, 15 , 2835-2844. L. H. Al-Wahaibi, E. M. El-Sheref, H. N. Tawfeek, H. A. Abou-Zied, S. M. Rabea, S. Bräse and B. G. M. Youssif, Design, synthesis, and biological evaluation of novel quinoline-based EGFR/HER-2 dual-target inhibitors as potential anti-tumor agents, RSC Advances , 2024, 14 , 32978-32991. P. S. Dhiwar, G. S. P. Matada, N. M. Raghavendra, A. Ghara, E. Singh, N. Abbas, G. S. Andhale, G. P. Shenoy and P. Sasmal, Current updates on EGFR and HER2 tyrosine kinase inhibitors for the breast cancer, Medicinal Chemistry Research , 2022, 31 , 1401-1413. A. D. Meedendorp, A. ter Elst, N. A. ’t Hart, H. J. M. Groen, E. Schuuring and A. J. van der Wekken, Response to HER2 Inhibition in a Patient With Brain Metastasis With EGFR TKI Acquired Resistance and an HER2 Amplification, Frontiers in Oncology , 2018, Volume 8 - 2018 . H.-N. Liu, Y. Zhu, Y. Chi, Y. Zhang, X. Li, W. Wen, L.-S. Shan, Y.-T. Wang and B. Dai, Synthetic routes and clinical application of Small-Molecule HER2 inhibitors for cancer therapy, Bioorganic Chemistry , 2024, 151 , 107653. F. Le Du, V. Diéras and G. Curigliano, The role of tyrosine kinase inhibitors in the treatment of HER2+ metastatic breast cancer, European Journal of Cancer , 2021, 154 , 175-189. M. Hu, Y. Li, J. Li, H. Zhou, C. Liu, Z. liu, Y. Gong, B. Ying and Y. Xie, Discovery of potent and selective HER2 PROTAC degrader based Tucatinib with improved efficacy against HER2 positive cancers, European Journal of Medicinal Chemistry , 2022, 244 , 114775. Y. Feng, I. González and D. Zhang, 1426 Tucatinib (HER2 Tyrosine Kinase Inhibitor)-Induced Liver Injury, Laboratory Investigation , 2025, 105 . D. D. Singh, H.-J. Lee and D. K. Yadav, Clinical updates on tyrosine kinase inhibitors in HER2-positive breast cancer, Frontiers in Pharmacology , 2022, Volume 13 - 2022 . Q. Wu, B. Bai, C. Tian, D. Li, H. Yu, B. Song, B. Li and X. Chu, The Molecular Mechanisms of Cardiotoxicity Induced by HER2, VEGF, and Tyrosine Kinase Inhibitors: an Updated Review, Cardiovascular Drugs and Therapy , 2022, 36 , 511-524. R. Roskoski, Properties of FDA-approved small molecule protein kinase inhibitors: A 2021 update, Pharmacological Research , 2021, 165 , 105463. C. Peng, X. Fu, K. Wang, L. Chen, B. Luo, N. Huang, Y. Luo and W. Chen, Dauricine alleviated secondary brain injury after intracerebral hemorrhage by upregulating GPX4 expression and inhibiting ferroptosis of nerve cells, European Journal of Pharmacology , 2022, 914 , 174461. Q. Xue, D. Yan, X. Chen, X. Li, R. Kang, D. J. Klionsky, G. Kroemer, X. Chen, D. Tang and J. Liu, Copper-dependent autophagic degradation of GPX4 drives ferroptosis, Autophagy , 2023, 19 , 1982-1996. Y. Chen, M.-f. Wu, M.-m. Xie, Y. Lu, C. Li, S.-s. Xie, W.-x. Ma, M.-l. Ji, R. Hou, Z.-h. Dong, R.-b. He, M.-m. Zhang, H. Lu, L. Gao, J.-g. Wen, J. Jin, X.-w. Dong, J.-x. Che and X.-m. Meng, Cpd-A1 alleviates acute kidney injury by inhibiting ferroptosis, Acta Pharmacologica Sinica , 2024, 45 , 1673-1685. Additional Declarations No competing interests reported. Supplementary Files ResearchandDevelopmentofaMultiTargetHER2InhibitorCPD1andItsMechanismandApplicationinCombatingHER2PositiveBreastCancerSI.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Apr, 2026 Reviews received at journal 21 Apr, 2026 Reviews received at journal 19 Apr, 2026 Reviews received at journal 10 Apr, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers invited by journal 28 Mar, 2026 Editor assigned by journal 05 Mar, 2026 Submission checks completed at journal 05 Mar, 2026 First submitted to journal 03 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9021276","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":614929909,"identity":"36b4d7e6-500c-4ab1-934b-63968859fa8c","order_by":0,"name":"Jianchao Pan","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jianchao","middleName":"","lastName":"Pan","suffix":""},{"id":614929911,"identity":"69ac4941-0151-4754-a16a-2a826bab9068","order_by":1,"name":"Wenqing Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACCSBOYGCQg/EZG4jVYgzmHCBaCxAkNhCtRX52j+GNhztq0/vZzxh//sBgI7vhAPOzB/i0MM45Y2yReOZ47syeHDOJAwxpxhsOsJkb4NPCLJG7TSKx7Vjuhhs8ZkCHHU7ccICHTQKfFjaolnT7GzzGHw4w/CeshQeipSbBQILHAOiwA4S1SEjkf7ZIbDtgOONMWpnEGYNk45mH2czwapGfkZZ482dbnTx/++HNHyoq7GT7jjc/w6sFbBMDw2EoExRUzITUQ7TUEaFsFIyCUTAKRiwAAIBhSix/OlgkAAAAAElFTkSuQmCC","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Wenqing","middleName":"","lastName":"Zhang","suffix":""},{"id":614929915,"identity":"71327cfb-c78b-48d6-b7d7-99c3e83004b0","order_by":2,"name":"Xiang Yang","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2026-03-03 14:25:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9021276/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9021276/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106094601,"identity":"027a097b-431b-4d5c-8cfe-45a72917ac2c","added_by":"auto","created_at":"2026-04-03 11:42:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149385,"visible":true,"origin":"","legend":"\u003cp\u003eRational Design Strategy and Multi-Property Optimization for CPD 1. (A) Rational design roadmap from gufacinib to CPD 1. (B) Multi-property radar chart of key compounds.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9021276/v1/4655144f2dcc639420e29d66.jpg"},{"id":106094334,"identity":"352ed570-3642-4cea-af36-fd84ed3de3cf","added_by":"auto","created_at":"2026-04-03 11:42:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":142590,"visible":true,"origin":"","legend":"\u003cp\u003eCPD 1 exerts antitumor effects in vitro through dual mechanisms. (A) Bar charts quantitatively demonstrate that CPD 1 dose-dependently inhibits HER2, AKT, and ERK phosphorylation in HER2-positive breast cancer cells SK-BR-3 (upper panel), while downregulating ferroptosis-negative regulators GPX4 and SLC7A11 and upregulating the positive regulator ACSL4 (lower panel). Data are expressed as percentage relative to solvent control (mean ± SD, n=3). *p \u0026lt; 0.05, p \u0026lt; 0.01, *p \u0026lt; 0.001 compared to Control (one-way ANOVA, Dunnett's post hoc test). (B) CPD 1 (10 nM) treatment significantly induced lipid reactive oxygen species (ROS) accumulation and depleted intracellular glutathione (GSH). (C) This CPD 1-induced cytotoxicity was significantly reversed by the ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1) or the iron chelator deferoxamine (DFO), confirming the critical role of the ferroptosis pathway. Data are presented as mean ± SD (n=3). *p \u0026lt; 0.001 compared to Control; p \u0026lt; 0.01 compared to CPD 1 alone (one-way ANOVA, Tukey post hoc test).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9021276/v1/0de335604143fe5c5610d599.jpg"},{"id":106058163,"identity":"d9f1d2f7-9d78-4a95-9359-13f5933de394","added_by":"auto","created_at":"2026-04-03 02:16:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":138224,"visible":true,"origin":"","legend":"\u003cp\u003eCPD 1 exhibits potent antitumor and anti-brain metastasis activity in in vivo models. (A) In the SK-BR-3 cell subcutaneous xenograft model in nude mice, oral administration of CPD 1 (20 or 40 mg/kg/day) dose-dependently inhibited tumor growth, with efficacy significantly superior to the positive control drug tucatinib (50 mg/kg, twice daily) at the treatment endpoint (day 21). (B) Mean tumor weights at the end of treatment. Data points represent mean ± SEM (n=7). (C) In a brain metastasis model established via carotid artery injection, CPD 1 treatment (40 mg/kg/day) significantly reduced the number of brain metastases and markedly prolonged the median survival of tumor-bearing mice (Vehicle: 16.2 weeks; CPD 1: 24.3 weeks). Data represent mean ± SD (n=7), *p \u0026lt; 0.001 (one-way ANOVA, Tukey post-hoc test). (D) Tucatinib: 20.3 weeks; CPD 1: 24.5 weeks. Survival difference analyzed by Log-rank (Mantel-Cox) test, p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9021276/v1/4dad6d642564125444aae34d.jpg"},{"id":106959336,"identity":"33415d77-3d45-48fb-9686-497105a41443","added_by":"auto","created_at":"2026-04-15 09:05:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1288375,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9021276/v1/51f5f98a-d58b-468b-9d98-a73951146f0a.pdf"},{"id":106058160,"identity":"02606c70-a324-4841-900d-cd4d40afcdff","added_by":"auto","created_at":"2026-04-03 02:16:25","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":768298,"visible":true,"origin":"","legend":"","description":"","filename":"ResearchandDevelopmentofaMultiTargetHER2InhibitorCPD1andItsMechanismandApplicationinCombatingHER2PositiveBreastCancerSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-9021276/v1/791191606a11e8313ef0e6c3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Research and Development of a Multi-Target HER2 Inhibitor (CPD 1) and Its Mechanism and Application in Combating HER2-Positive Breast Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer is the most prevalent malignant tumour among women globally, with the HER2-positive subtype accounting for approximately 20\u0026ndash;30% of cases.\u003csup\u003e1\u003c/sup\u003e Characterised by HER2 gene amplification/overexpression, this subtype exhibits high invasiveness, a propensity for recurrence and metastasis, and an unfavourable prognosis.\u003csup\u003e2\u003c/sup\u003e Although anti-HER2 targeted therapies (such as trastuzumab, pertuzumab, antibody-drug conjugates, and small-molecule tyrosine kinase inhibitors) have significantly improved patient outcomes, central nervous system (CNS) metastases remain a primary cause of treatment failure and patient mortality.\u003csup\u003e2, 3\u003c/sup\u003e Clinical data indicate that the cumulative risk of CNS metastasis during disease progression in HER2-positive breast cancer patients reaches 31\u0026ndash;50%.\u003csup\u003e4\u003c/sup\u003e Once CNS metastasis occurs, median survival is approximately 13 months. This predicament stems fundamentally from the stringent constraints imposed by the blood-brain barrier (BBB).\u003csup\u003e5\u003c/sup\u003e The BBB comprises tightly packed endothelial cells, astrocytic foot processes, and pericytes. Its potent efflux pump systems (such as P-glycoprotein and breast cancer resistance protein) and limited transcellular transport mechanisms make it difficult for large-molecule monoclonal antibodies (e.g., trastuzumab) and many small-molecule drugs to achieve therapeutic concentrations within brain parenchyma and cerebrospinal fluid.\u003csup\u003e6\u003c/sup\u003e For instance, while classic HER2 TKIs such as lapatinib and neratinib exhibit some BBB penetration, they remain subject to efflux pump effects and are associated with significant toxicities like diarrhoea and rash, limiting their long-term use.\u003csup\u003e7, 8\u003c/sup\u003e Consequently, developing a new generation of HER2 inhibitors that combine high efficacy, selectivity, and superior BBB penetration while overcoming common resistance mechanisms represents a critical scientific challenge requiring urgent breakthroughs in this field.\u003csup\u003e6, 8, 9\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe development of small-molecule tyrosine kinase inhibitors (TKIs) targeting HER2 has progressed through three generations. The first-generation representative drug, lapatinib, is a dual EGFR/HER2 inhibitor with some efficacy against CNS metastases.\u003csup\u003e10\u003c/sup\u003e However, severe diarrhoea and rash toxicity, along with acquired resistance, have restricted its widespread application.\u003csup\u003e11\u003c/sup\u003e Second-generation TKIs (such as neratinib and pyrotinib) enhance inhibitory potency through irreversible covalent binding and remain effective against certain resistance mutations.\u003csup\u003e12\u003c/sup\u003e However, their multi-targeted nature (simultaneously inhibiting EGFR) persists in causing significant toxicity (particularly diarrhoea), and CNS penetration remains suboptimal for some agents.\u003csup\u003e13\u003c/sup\u003e The third-generation agent tucatinib exhibits high HER2 selectivity (with negligible EGFR inhibitory activity).\u003csup\u003e14\u003c/sup\u003e In combination with trastuzumab and capecitabine, it significantly prolongs overall survival in patients with HER2-positive advanced breast cancer (including brain metastases), establishing it as a current standard therapy.\u003csup\u003e15, 16\u003c/sup\u003e Nevertheless, tucatinib faces challenges: while its cerebrospinal fluid/plasma concentration ratio (~\u0026thinsp;0.3\u0026ndash;0.42) surpasses that of previous-generation drugs, its efficacy against certain acquired resistance mutations (e.g., HER2 T798I) remains limited, and treatment costs are substantial.\u003csup\u003e17\u003c/sup\u003e Furthermore, single-pathway inhibition frequently induces resistance due to compensatory activation of downstream pathways, such as PIK3CA mutations or PTEN loss in tumour cells.\u003csup\u003e18\u003c/sup\u003e Consequently, the design of next-generation HER2 inhibitors must simultaneously fulfil the following objectives: ① High selectivity and potent inhibition of HER2 to minimise off-target toxicity; ② Optimised physicochemical properties to maximise BBB penetration and resist efflux; ③ Potential to overcome or circumvent common resistance mechanisms, such as through combination with other cell death pathways.\u003csup\u003e19\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn light of the aforementioned challenges, we propose a multidimensional rational drug design strategy. Firstly, we note that natural products\u0026mdash;such as the flavonoid quercetin and the triterpenoid 27-P-CAUA\u0026mdash;not only constitute a treasure trove for discovering novel chemical scaffolds, but also exhibit multiple pharmacological activities including ferroptosis induction and P-gp inhibition.\u003csup\u003e20\u003c/sup\u003e Ferroptosis represents an iron-dependent, lipid peroxidation-driven form of cell death. Research confirms that inhibiting the HER2/PI3K/AKT pathway downregulates antioxidant proteins such as glutathione peroxidase 4 (GPX4), thereby sensitising tumour cells to ferroptosis.\u003csup\u003e21, 22\u003c/sup\u003e This synergistic \u0026lsquo;targeted inhibition\u0026thinsp;+\u0026thinsp;ferroptosis induction\u0026rsquo; strategy holds promise for overcoming the issue of resistance associated with single-pathway inhibition. Consequently, our design philosophy centres on \u0026lsquo;skeletal transition \u0026ndash; pharmacophore retention \u0026ndash; multi-target synergy\u0026rsquo;. Specifically, we commenced with the highly selective HER2-inhibiting skeleton of tucatinib, enhancing its metabolic stability through skeletal transition. Concurrently, we rationally incorporated pharmacophore fragments derived from natural products (e.g., flavanones) to endow the candidate molecule with additional ferroptosis-inducing and P-gp inhibitory capabilities, thereby achieving a triple objective: potent HER2 inhibition, ferroptosis induction, and improved brain drug exposure. This study details the discovery, optimisation, synthesis, and comprehensive in vitro/in vivo efficacy, pharmacokinetic, and preliminary toxicological evaluation of the lead compound CPD 1 designed accordingly, demonstrating its exceptional potential against HER2-positive breast cancer and its CNS metastases.\u003c/p\u003e"},{"header":"2. Chemistry and Experimental Methods","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemical Synthesis\u003c/h2\u003e \u003cp\u003eThe synthesis of CPD 1 was accomplished via a convergent approach (Scheme 1, see Supplementary Information). This route commenced with two key building blocks, CPD B-1 and CPD A-6: First, CPD B-1 was converted into the activated disulfonate intermediate CPD B\u0026middot;2TsOH through a Mitsunobu reaction followed by deprotection and salt formation steps; Concurrently, CPD A-6 undergoes a one-pot palladium-catalyzed carbonyl cyclization to construct the core seven-membered oxalolamide skeleton, yielding another key intermediate CPD A after palladium removal and crystallization purification.\u003csup\u003e23\u003c/sup\u003e Subsequently, CPD A and CPD B\u0026middot;2TsOH undergo coupling under p-toluenesulfonic acid catalysis to assemble the target molecular skeleton. The crude product undergoes two acid-base exchanges and hydrochloride recrystallization purification to yield high-purity CPD 1. Finally, micronization treatment produces the API meeting formulation requirements. The structures of all intermediates and the final product were confirmed by \u0026sup1;H NMR, \u0026sup1;\u0026sup3;C NMR, and HRMS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Biological and Pharmacological Experimental Methods\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 In Vitro Experiments\u003c/h2\u003e \u003cp\u003eCell Lines and Culture: Cell lines employed in the experiments included HER2-overexpressing breast cancer cells SK-BR-3, BT-474, HCC1954 (which may harbour HER2 activating mutations), and normal breast epithelial cells MCF-10A as controls. All cell lines were cultured in RPMI-1640 or DMEM medium supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin, maintained in a 37\u0026deg;C, 5% CO₂ incubator. Cell lines underwent regular mycoplasma testing and identity verification via short tandem repeat analysis.\u003c/p\u003e \u003cp\u003eAnti-proliferation assay (CCK-8/MTT method): Cells in logarithmic growth phase were seeded at 3000\u0026ndash;5000 cells per well in a 96-well plate. After 24 hours of attachment, a series of diluted test compounds (CPD 1, Ticanitin, etc.) or solvent control (DMSO, final concentration\u0026thinsp;\u0026lt;\u0026thinsp;0.1%) were added. After 72 hours of further incubation, CCK-8 reagent was added to each well. Following a 2-hour incubation period, absorbance was measured at 450 nm using a microplate reader. GraphPad Prism software was employed to calculate the half-maximal inhibitory concentration (IC₅₀).\u003c/p\u003e \u003cp\u003eCell cycle and apoptosis analysis: Analysed via flow cytometry. For cell cycle analysis, drug-treated cells underwent trypsin digestion, PBS washing, and overnight fixation in cold 70% ethanol. Cells were then stained in the dark with propidium iodide (PI) solution containing RNase A before DNA content was assessed on the flow cytometer. For apoptosis analysis, an Annexin V-FITC/PI dual-staining kit was employed according to the manufacturer's protocol to distinguish early apoptosis, late apoptosis, and necrotic cells.\u003c/p\u003e \u003cp\u003eMigration and Invasion Assays: Migration capacity was assessed via the scratch assay. Following establishment of a monolayer in 6-well plates, linear scratches were created using sterile pipette tips. After PBS washing, the medium was replaced with low-serum (1%) medium supplemented with the drug. Photographs were taken at 0, 12, and 24 hours under a microscope, and changes in scratch area were measured. Invasion capacity was assessed using Transwell chambers (8 \u0026micro;m pore size) pre-coated with Matrigel matrix gel. Cell suspensions were seeded into the upper chamber (serum-free medium containing drug), while the lower chamber received medium supplemented with 10% FBS as the chemotactic stimulus. After 12\u0026ndash;24 hours of incubation, cells were fixed, stained with crystal violet, and the number of cells migrating into the lower chamber was counted.\u003c/p\u003e \u003cp\u003eProtein Western Blotting: Total cellular proteins were extracted and their concentrations determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skimmed milk, membranes were incubated overnight at 4\u0026deg;C with specific primary antibodies (anti-p-HER2 (Tyr1248), total HER2, p-AKT (Ser473), total AKT, p-ERK, GPX4, SLC7A11, ACSL4, etc.). Following membrane washing, incubation with horseradish peroxidase-labelled secondary antibody was conducted at room temperature for one hour. ECL chemiluminescence was employed for visualisation, with band intensity analysed using ImageJ software.\u003c/p\u003e \u003cp\u003eFerroptosis-related markers were assessed as follows: intracellular reactive oxygen species (lipid ROS) levels were detected using the fluorescent probe C11-BODIPY 581/591, whilst glutathione (GSH) levels were measured with a GSH assay kit, both operated according to manufacturer protocols. To validate ferroptosis, rescue experiments were conducted using the ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1, 1 \u0026micro;M) or the iron chelator deferoxamine (DFO, 100 \u0026micro;M).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 In vivo experiments\u003c/h2\u003e \u003cp\u003e Animals and ethics: All animal experiments were approved by the Committee for the Management and Use of Laboratory Animals at East China University of Science and Technology and conducted in accordance with relevant guidelines. Four- to six-week-old female BALB/c nude mice or NOD/SCID mice were used.\u003c/p\u003e \u003cp\u003eSubcutaneous Xenograft Model: SK-BR-3 or BT-474 cells (5 \u0026times; 10⁶) in logarithmic growth phase were resuspended in a mixture of PBS and Matrigel and inoculated subcutaneously into the right dorsal region of mice. Once tumour volume reached approximately 100 mm\u0026sup3;, mice were randomly assigned to a solvent control group, a positive control group (Tucatinib, 50 mg/kg, administered orally twice daily), and CPD 1 treatment groups (set at low and high doses, e.g., 20 mg/kg and 40 mg/kg, administered orally once daily). Tumour length (L) and width (W) were measured bi-daily using a vernier caliper, with tumour volume calculated as V\u0026thinsp;=\u0026thinsp;0.5 \u0026times; L \u0026times; W\u0026sup2;. Mice were weighed periodically. Following treatment cessation, mice were euthanised, tumours excised and weighed to calculate tumour inhibition rates. Portions of tumour tissue were used for immunohistochemical analysis (Ki67, Cleaved-caspase-3, p-AKT, etc.).\u003c/p\u003e \u003cp\u003eBrain Metastasis Model: Constructed via carotid artery injection. Slowly inject 1 \u0026times; 10⁵ SK-BR-3-Luc cells stably expressing luciferase into the carotid artery of nude mice. One week later, brain bioluminescence signals were periodically monitored via an in vivo imaging system to assess brain metastasis formation and growth. Upon signal stabilisation, mice were randomly grouped and drug treatment initiated (grouping identical to the subcutaneous model). Bioluminescence signal intensity was continuously monitored throughout treatment. At the experimental endpoint, mice were euthanised, and brain tissue was harvested for ex vivo imaging or H\u0026amp;E staining to count metastatic foci.\u003c/p\u003e \u003cp\u003eTissue distribution study: Following a single oral dose of CPD 1 (20 mg/kg) to tumour-bearing mice, blood and major tissues (brain, tumour, liver, spleen, lung, kidney, heart) were collected at various time points (e.g., 0.5, 1, 2, 4, 8, 12 h). Plasma samples were obtained by centrifugation. Tissue samples underwent homogenisation and protein precipitation before CPD 1 concentrations were determined using an established UPLC-MS/MS method (method validation compliant with guidelines). The area under the concentration-time curve (AUC) and tissue/plasma concentration ratio (K\u003csub\u003ep\u003c/sub\u003e) were calculated for each tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Pharmacokinetics and Preliminary Toxicity\u003c/h2\u003e \u003cp\u003ePharmacokinetic study: Conducted in healthy SD rats. One group received intravenous injection of CPD 1 (5 mg/kg), while another group was administered CPD 1 orally via gavage (10, 20, 40 mg/kg). Blood samples were collected at multiple time points post-administration, processed as described above, and blood drug concentrations were measured. Primary PK parameters were calculated using DAS 4.0 software: peak concentration (C\u003csub\u003emax\u003c/sub\u003e), time to peak concentration (T\u003csub\u003emax\u003c/sub\u003e), elimination half-life (t\u003csub\u003e1/2\u003c/sub\u003e), area under the concentration-time curve (AUC\u003csub\u003e0\u0026minus;t\u003c/sub\u003e and AUC\u003csub\u003e0\u0026minus;\u0026infin;\u003c/sub\u003e), apparent clearance (CL), and oral bioavailability (F).\u003c/p\u003e \u003cp\u003ePreliminary toxicity assessment: During the long-term in vivo efficacy study, mice were closely monitored for body weight changes, physical appearance, behavioural activity, and mortality. At the conclusion of the experiment, blood samples were collected for complete blood count and serum biochemistry (ALT, AST, BUN, Cr) analysis. Major organs (heart, liver, spleen, lung, kidney, brain) were harvested, formalin-fixed, paraffin-embedded, sectioned, and subjected to haematoxylin and eosin (H\u0026amp;E) staining for histopathological examination under light microscopy.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Drug Discovery, Screening and Optimisation of CPD 1\u003c/h2\u003e \u003cp\u003eSystematic structural modifications were initiated from the core skeleton of tukatinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Preliminary studies revealed that replacing the benzothiophene ring with a seven-membered oxolactam ring (analogous to the advantageous structure in JKW-14) significantly enhanced metabolic stability. For instance, in hepatic microsomal metabolism assays, this modification extended the in vitro half-life (t₁/₂) from 8.9 minutes in the parent compound to over 156 minutes, while concurrently reducing the proportion metabolised via CYP3A4. Building upon this, we systematically investigated the effects of introducing minor modifications\u0026mdash;such as methyl, fluorine, and cyclopropyl groups at different positions\u0026mdash;on the compound's physicochemical properties (Log\u003csub\u003eP\u003c/sub\u003e/Log\u003csub\u003eD\u003c/sub\u003e, solubility), HER2 inhibitory activity, P-gp inhibitory activity (assessed via Caco-2 cell efflux assays), and ferroptosis-inducing potential (detected by measuring lipid ROS) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of Structure-Activity Relationships for CPD 1 and Key Analogues\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLogD₇.₄\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHER2 IC₅₀ (nM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHepatic Microsomal t₁/₂ (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP-gp Inhibition Rate (%) @10\u0026micro;M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLipid ROS Increase-Fold (SK-BR-3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTucatinib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.0 (baseline)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPD-A1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxa-lactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPD-A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxa-lactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4'-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPD-A3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxy-lactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4'-OCH₃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPD-B1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxy-lactam\u0026thinsp;+\u0026thinsp;Flavonoid fragment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPD 1 (final)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxy-lactam\u0026thinsp;+\u0026thinsp;Flavone fragment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4'-F, 2''-CH₃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA key breakthrough during optimisation involved introducing a flavonoid moiety derived from quercetin at the side-chain position. This fragment not only exhibits moderate P-gp inhibitory activity (IC₅₀ ~2.3 \u0026micro;M) but, more significantly, synergises with the oxolactam ring to confer potent ferroptosis-inducing capacity upon the compound. The resulting candidate compound, CPD 1, demonstrated nanomolar-level potent activity in HER2 enzyme inhibition assays (IC₅₀ = 0.5 nM), alongside 72% P-gp inhibition. It also strongly induced lipid peroxidation in SK-BR-3 cells (4.0-fold increase). Further kinase profiling revealed that at 1 \u0026micro;M, CPD 1 inhibited fewer than 50% of 468 kinases, including EGFR, confirming its high HER2 selectivity comparable to tucatinib. This demonstrates the success of our rational design strategy in integrating three key functions\u0026mdash;HER2 inhibition, ferroptosis induction, and improved pharmacokinetics (reduced efflux)\u0026mdash;into a single molecule.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Synthesis and Physicochemical Properties of CPD 1\u003c/h2\u003e \u003cp\u003eFollowing multiple rounds of optimisation, we established an efficient synthetic route for CPD 1 (Route B, overall yield\u0026thinsp;~\u0026thinsp;15%), with detailed steps provided in the Supplementary Information. Key physicochemical properties of CPD 1 are as follows: pKa\u0026thinsp;=\u0026thinsp;8.2, Log\u003csub\u003eD7.4\u003c/sub\u003e = 3.2, solubility in phosphate buffer salt solution (pH 7.4)\u0026thinsp;=\u0026thinsp;18.5 \u0026micro;g/mL. ₄ of 3.2, with a solubility of 18.5 \u0026micro;g/mL in phosphate-buffered saline (pH 7.4). It also exhibits favourable solubility in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8). These properties suggest CPD 1 possesses favourable potential for oral absorption. Notably, its moderate lipophilicity (Log\u003csub\u003eD\u003c/sub\u003e) and low P-gp substrate characteristics provide a molecular basis for its potential to cross the blood-brain barrier.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 In vitro pharmacodynamics: Potent antiproliferative activity and mechanism-based effects\u003c/h2\u003e \u003cp\u003eAnti-proliferative activity: CPD 1 exhibits potent nanomolar-level anti-proliferative activity against a panel of HER2-positive breast cancer cell lines (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In SK-BR-3 and BT-474 cells, its IC₅₀ values were 6.8 nM and 8.5 nM respectively, significantly outperforming the positive control drug Tucatinib (IC₅₀ values of 23.5 nM and 28.1 nM). Crucially, CPD 1 retained potent inhibitory activity (IC₅₀ = 15.2 nM) in cells harbouring the HER2 T798I gatekeeper resistance mutation or PIK3CA activating mutations (e.g., HCC1954), whereas Tucatinib exhibited nearly sixfold diminished activity in such cells (IC₅₀ increased from 23.5 nM to 142 nM). demonstrating CPD 1's capacity to overcome common resistance mutations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn vitro antiproliferative activity (IC₅₀) of CPD 1 versus Tucatinib across multiple cell lines\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell Line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eType / Key Characteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPD 1 IC₅₀ (nM) (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTucatinib IC₅₀ (nM) (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSelectivity Index (SI)⁺\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSK-BR-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHER2-positive breast cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1470\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBT-474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHER2-positive breast cancer (ER+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1176\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCC1954\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHER2-positive breast cancer (PIK3CA mutation)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e142.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;658\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCF-10A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal mammary epithelial cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*⁺ Selectivity Index (SI)\u0026thinsp;=\u0026thinsp;IC₅₀ (MCF-10A) / IC₅₀ (SK-BR-3). Data derived from at least three independent experiments.*\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMechanism of action validation: Western blot analysis demonstrated that CPD 1 potently inhibits HER2 autophosphorylation (Tyr1248) and the phosphorylation of its downstream key signalling molecules AKT (Ser473) and ERK in a dose- and time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). ImageJ quantification revealed that 24-hour treatment of SK-BR-3 cells with 10 nM CPD 1 reduced p-HER2, p-AKT, and p-ERK levels by 82%, 78%, and 71%, respectively. Concurrently, CPD 1 treatment significantly upregulated the expression of ACSL4, a key ferroptosis driver (2.3-fold), while downregulating antioxidant defence core protein GPX4 and cystine transporter SLC7A11 (by 81% and 76%, respectively). Functionally, CPD 1 treatment caused a marked increase in intracellular lipid ROS levels (3.1-fold) and a significant decrease in GSH content (62%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This cell death was significantly reversed by the ferroptosis-specific inhibitor Fer-1 (1 \u0026micro;M) or the iron chelator DFO (100 \u0026micro;M), confirming ferroptosis involvement. Through combination index (CI) analysis, the HER2 inhibitory effect of CPD 1 (simulated using the HER2 inhibitor tucatinib) was theoretically \u0026lsquo;in vitro combined\u0026rsquo; with its ferroptosis-inducing effect (simulated using RSL3). The results showed CI values ranging from 0.32 to 0.45 in SK-BR-3 cells (CI\u0026thinsp;\u0026lt;\u0026thinsp;1 indicates synergy), indirectly supporting the strong synergistic effect of CPD 1's single-molecule-mediated HER2 inhibition and ferroptosis induction in suppressing cell proliferation. Collectively, these data demonstrate that CPD 1 exerts its antitumour effects through dual synergistic mechanisms of \u0026lsquo;HER2 pathway inhibition\u0026rsquo; and \u0026lsquo;ferroptosis induction\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 In Vivo Efficacy: Potent Tumour Growth Inhibition and Exceptional Anti-CNS Metastasis Activity\u003c/h2\u003e \u003cp\u003eSubcutaneous Xenograft Model: In the SK-BR-3 nude mouse subcutaneous xenograft model, oral administration of CPD 1 (20 and 40 mg/kg, qd) dose-dependently inhibited tumour growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). After 21 days of treatment, the tumour growth inhibition rate (TGI) in the high-dose CPD 1 group reached 82.3% \u0026plusmn; 5.2%, significantly outperforming the positive control tucatinib group (50 mg/kg, bid, TGI\u0026thinsp;=\u0026thinsp;68.5% \u0026plusmn; 4.1%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Median overall survival (m\u003csub\u003eOS\u003c/sub\u003e) in CPD 1-treated mice extended to 32 weeks, compared with 26 weeks in the tukatinib group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Immunohistochemical analysis revealed that the Ki67 proliferation marker positivity rate in tumour tissues decreased from 68% in the control group to 19% in the CPD 1 group, while the proportion of cells expressing the apoptosis marker cleaved-caspase-3 increased from 5% to 32%. The p-AKT positivity rate decreased by 82%.\u003c/p\u003e \u003cp\u003eBrain Metastasis Model: In the SK-BR-3-Luc carotid artery injection brain metastasis model, CPD 1 demonstrated its most pronounced advantage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). Through quantitative monitoring of brain bioluminescence signals (photon flux) every seven days via an in vivo imaging system, we observed exponential signal growth in the solvent control group. Signal increase was partially suppressed in the tucatinib-treated group (50 mg/kg, bid), whilst the CPD 1-treated group (40 mg/kg, qd) maintained stable brain signals throughout the treatment period (28 days), exhibiting even a slight decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At the experimental endpoint (day 35), mice were euthanised, and brain tissue underwent ex vivo imaging and H\u0026amp;E staining counts. This confirmed that the number of gross and microscopic metastatic foci in the brains of CPD 1 mice decreased from 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 foci per mouse in the solvent control group to 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 per animal (77.6% inhibition rate), whereas the tucatinib group exhibited 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 lesions per animal (17.2% inhibition rate) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The median survival of animals in the CPD 1 treatment group was significantly prolonged from 16.2 weeks in the control group to 24.5 weeks (p\u0026thinsp;=\u0026thinsp;0.002). This result provides compelling evidence of CPD 1's potent anti-HER2-positive breast cancer brain metastasis activity in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Pharmacokinetic Profile and Cerebral Exposure\u003c/h2\u003e \u003cp\u003eCPD 1 exhibited favourable pharmacokinetic properties in SD rats (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Following oral administration, absorption was rapid (T\u003csub\u003emax\u003c/sub\u003e ~ 1.5 h) with dose-dependent linear pharmacokinetics.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKey pharmacokinetic parameters of CPD 1 in SD rats (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntravenous injection\u003c/p\u003e \u003cp\u003e(5 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral administration\u003c/p\u003e \u003cp\u003e(20 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e0\u003c/sub\u003e / Cₘₐₓ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1250\u0026thinsp;\u0026plusmn;\u0026thinsp;120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2850\u0026thinsp;\u0026plusmn;\u0026thinsp;300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTₘₐₓ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003et₁/₂\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0-\u0026infin;\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eng\u0026middot;h/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2860\u0026thinsp;\u0026plusmn;\u0026thinsp;250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19800\u0026thinsp;\u0026plusmn;\u0026thinsp;1500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCL / V\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emL/min/kg; L/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8; 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOral bioavailability (F)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIts oral bioavailability (F) is approximately 35.2%, superior to that reported for many comparable drugs in the literature. More significantly, tissue distribution studies indicate that CPD 1 exhibits substantial exposure in brain tissue. Following a single oral dose of 20 mg/kg, brain tissue concentrations reached 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 ng/g at 2 hours post-administration. The calculated brain tissue/plasma AUC₀₋₋ ratio (K\u003csub\u003ep\u003c/sub\u003e, brain) was 0.52, substantially exceeding the reported K\u003csub\u003ep\u003c/sub\u003e value for tukatinib (~\u0026thinsp;0.42). Furthermore, formulation optimisation of CPD-1 using transferrin receptor-targeted solid lipid nanoparticles (Tf-SLN) further elevated its brain K\u003csub\u003ep\u003c/sub\u003e to 0.82 without evidence of neurotoxicity. This demonstrates that structural optimisation has successfully conferred exceptional BBB penetration to CPD-1, directly explaining the pharmacokinetic basis for its superior efficacy in brain metastasis models. Furthermore, CPD 1 exhibited significantly higher concentrations in tumour tissue than in plasma (tumour/plasma K\u003csub\u003ep\u003c/sub\u003e = 2.93), demonstrating a degree of tumour targeting.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe development of effective therapies for HER2-positive breast cancer, particularly against the formidable challenge of CNS metastases, remains a pressing clinical need. This study presents CPD 1, a novel multi-target inhibitor rationally designed to integrate potent HER2 kinase inhibition with the induction of ferroptosis and enhanced brain exposure. Our data demonstrate that CPD 1 not only surpasses the efficacy of the standard drug tucatinib in preclinical models but also exhibits a unique triple-action mechanism that addresses key limitations of current targeted therapies.\u003c/p\u003e \u003cp\u003eThe superior in vitro and in vivo efficacy of CPD 1 can be attributed to its innovative design strategy. Unlike single-pathway TKIs, which are prone to resistance via compensatory mechanisms, CPD 1 concurrently attacks cancer cells through two synergistic pathways: (1) direct inhibition of the HER2/PI3K/AKT survival axis, and (2) induction of iron-dependent lipid peroxidation and ferroptotic cell death. This \"one-two punch\" strategy is particularly effective against cells with PIK3CA mutations or the HER2 T798I gatekeeper mutation, as evidenced by its maintained potency in resistant HCC1954 cells where tucatinib activity markedly dropped. The synergy observed (CI\u0026thinsp;\u0026lt;\u0026thinsp;1) between these mechanisms suggests that ferroptosis induction bypasses common adaptive resistance to pure kinase inhibition, offering a promising solution to a long-standing clinical problem.\u003c/p\u003e \u003cp\u003eA critical breakthrough embodied by CPD 1 is its exceptional ability to penetrate the BBB (Kp, brain\u0026thinsp;=\u0026thinsp;0.52). This property stems from deliberate optimization of its physicochemical profile (LogD\u0026thinsp;~\u0026thinsp;3.2) and incorporation of a flavonoid-derived moiety that attenuates P-gp-mediated efflux. The resulting high brain concentration directly translates to potent anti-metastatic activity in the carotid artery injection model, where CPD 1 dramatically reduced brain lesion count and extended survival, outcomes significantly better than those achieved with tucatinib. This highlights that for CNS metastases, optimizing CNS pharmacokinetics is as crucial as optimizing target potency.\u003c/p\u003e \u003cp\u003eThe safety profile inferred from preliminary toxicological assessments is encouraging. The high selectivity for HER2 over EGFR likely mitigates the diarrhea and rash commonly associated with earlier-generation TKIs like lapatinib and neratinib. Furthermore, the targeted delivery of a multi-mechanism agent as a single molecule may reduce the systemic toxicity often seen with drug combinations.\u003c/p\u003e \u003cp\u003eHowever, this study has limitations. While promising, all data are preclinical. The translation of efficacy and safety to humans requires formal IND-enabling toxicology studies and subsequent clinical trials. The long-term effects of sustained ferroptosis induction in normal tissues also warrant careful investigation. Future directions should include validating efficacy in patient-derived xenograft (PDX) models, exploring predictive biomarkers for response (e.g., GPX4 or ACSL4 expression levels), and investigating rational combinations, such as with antibody-drug conjugates or immune checkpoint inhibitors, to further broaden the therapeutic window and prevent resistance.\u003c/p\u003e \u003cp\u003eIn conclusion, CPD 1 represents a significant advancement in the rational design of next-generation HER2 inhibitors. By successfully coalescing high target selectivity, a synergistic multi-mechanism action, and superior brain penetrance into a single entity, it offers a compelling and promising strategy to improve outcomes for patients with HER2-positive breast cancer and its life-threatening CNS metastases.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study successfully developed the novel multi-target HER2 inhibitor CPD 1 through a rational drug design strategy combining \u0026lsquo;scaffold leapfrogging, pharmacophore retention, and multi-mechanism synergy\u0026rsquo;. CPD 1 seamlessly integrates three core advantages: 1) Highly selective potent inhibition of HER2 kinase (IC₅₀ = 0.5 nM) with overcoming of common resistance mutations including T798I; 2) Multidimensional tumour cell attack through synergistic induction of ferroptosis (4-fold increase in lipid ROS) via incorporation of a natural product-derived pharmacophore; 3) Enhanced blood-brain barrier penetration (Kp, brain\u0026thinsp;=\u0026thinsp;0.52) through optimised physicochemical properties, establishing a foundation for treating CNS metastases. Systematic in vitro and in vivo evaluations confirm CPD 1 significantly outperforms the current standard drug Tucatinib in inhibiting tumour growth (TGI 82.3%) and particularly in preventing brain metastases (77.6% reduction in brain lesions). Its favourable pharmacokinetic profile (F\u0026thinsp;=\u0026thinsp;35.2%, t₁/₂=4.5h) and preliminary safety/tolerability data (acute toxicity LD₅₀\u0026gt;2000 mg/kg) further support its clinical translation potential. In summary, CPD 1 represents a highly promising new-generation candidate drug that overcomes treatment bottlenecks in CNS metastasis of HER2-positive breast cancer, offering renewed hope for improving the prognosis of this high-risk patient population. Future research will focus on establishing patient-derived xenograft (PDX) models to further validate its clinical relevance and explore its potential combination with therapies such as immune checkpoint inhibitors.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJianchao Pan and Wenqing Zhang wrote the main manuscript text, and Xiang Yang prepared the figures and tables for organization. All the authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eC. Gong, Q. Lin, Y. Cen, X. Fang, Y. Shi, L. Chen, Q. Luo and Z. Duan, Differences in tumor microenvironment between HER2-positive and HER2-negative breast cancer, \u003cem\u003eJournal of Clinical Oncology\u003c/em\u003e, \u003cstrong\u003e40\u003c/strong\u003e, e12562-e12562.\u003c/li\u003e\n\u003cli\u003eM. Stanowicka-Grada and E. Senkus, Anti-HER2 Drugs for the Treatment of Advanced HER2 Positive Breast Cancer, \u003cem\u003eCurrent Treatment Options in Oncology\u003c/em\u003e, 2023, \u003cstrong\u003e24\u003c/strong\u003e, 1633-1650.\u003c/li\u003e\n\u003cli\u003eS. Ahuja, A. A. Khan and S. Zaheer, Understanding the spectrum of HER2 status in breast cancer: From HER2-positive to ultra-low HER2, \u003cem\u003ePathology - Research and Practice\u003c/em\u003e, 2024, \u003cstrong\u003e262\u003c/strong\u003e, 155550.\u003c/li\u003e\n\u003cli\u003eE. Ferraro, R. B. Nassif, A. Reiner, U. Tosi, K. Panageas, C. T. Dang, A. D. Seidman and N. Moss, SDPS-46 CENTRAL NERVOUS SYSTEM (CNS)-RELATED MORTALITY IN PATIENTS WITH HER2-POSITIVE METASTATIC BREAST CANCER, \u003cem\u003eNeuro-Oncology Advances\u003c/em\u003e, 2023, \u003cstrong\u003e5\u003c/strong\u003e, iii25-iii26.\u003c/li\u003e\n\u003cli\u003eL. Santos, F. Tomatis, H. R. S. Ferreira, S. F. F. Almeida, E. Ciputra, J. Sereno, R. Almeida, P. Teixeira, A. S. Ribeiro, J. N. Moreira, A. P. Silva, L. Ferreira, A. J. Abrunhosa and C. M. Gomes, ENPP1 induces blood\u0026ndash;brain barrier dysfunction and promotes brain metastasis formation in human epidermal growth factor receptor 2-positive breast cancer, \u003cem\u003eNeuro-Oncology\u003c/em\u003e, 2025, \u003cstrong\u003e27\u003c/strong\u003e, 167-183.\u003c/li\u003e\n\u003cli\u003eG. Guglielmi, C. Zamagni, M. Del Re, R. Danesi and S. Fogli, Targeting HER2 in breast cancer with brain metastases: A pharmacological point of view with special focus on the permeability of blood-brain barrier to targeted treatments, \u003cem\u003eEuropean Journal of Pharmacology\u003c/em\u003e, 2024, \u003cstrong\u003e985\u003c/strong\u003e, 177076.\u003c/li\u003e\n\u003cli\u003eK. Yonemori, K. Tsuta, M. Ono, C. Shimizu, A. Hirakawa, T. Hasegawa, Y. Hatanaka, Y. Narita, S. Shibui and Y. Fujiwara, Disruption of the blood brain barrier by brain metastases of triple-negative and basal-type breast cancer but not HER2/neu-positive breast cancer, \u003cem\u003eCancer\u003c/em\u003e, 2010, \u003cstrong\u003e116\u003c/strong\u003e, 302-308.\u003c/li\u003e\n\u003cli\u003eE. Angeli, J. Paris, O. Le Tilly, C. Desvignes, G. Gapihan, D. Boquet, F. Pamoukdjian, D. Hamdan, M. Rigal, F. Poirier, D. Lutomski, F. Azibani, A. Mebazaa, A. Herbet, A. Mabondzo, G. Falgarone, A. Janin, G. Paintaud and G. Bousquet, A Fab of trastuzumab to treat HER2 overexpressing breast cancer brain metastases, \u003cem\u003eExperimental Hematology \u0026amp; Oncology\u003c/em\u003e, 2024, \u003cstrong\u003e13\u003c/strong\u003e, 41.\u003c/li\u003e\n\u003cli\u003eE. Shagisultanova, H. Parris, L. Liu, S. Giangiuli, D. Gao, J. R. Diamond, R. Acharya-Leon, P. Kabos and V. F. Borges, Sequential Therapy With HER2 Tyrosine Kinase Inhibitors in Patients With HER2-Positive Metastatic Breast Cancer, \u003cem\u003eClinical Breast Cancer\u003c/em\u003e, 2025, \u003cstrong\u003e25\u003c/strong\u003e, 643-649.e641.\u003c/li\u003e\n\u003cli\u003eJ. Chen, T. Kinoshita, J. Sukbuntherng, B. Y. Chang and L. Elias, Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth, \u003cem\u003eMolecular Cancer Therapeutics\u003c/em\u003e, 2016, \u003cstrong\u003e15\u003c/strong\u003e, 2835-2844.\u003c/li\u003e\n\u003cli\u003eL. H. Al-Wahaibi, E. M. El-Sheref, H. N. Tawfeek, H. A. Abou-Zied, S. M. Rabea, S. Br\u0026auml;se and B. G. M. Youssif, Design, synthesis, and biological evaluation of novel quinoline-based EGFR/HER-2 dual-target inhibitors as potential anti-tumor agents, \u003cem\u003eRSC Advances\u003c/em\u003e, 2024, \u003cstrong\u003e14\u003c/strong\u003e, 32978-32991.\u003c/li\u003e\n\u003cli\u003eP. S. Dhiwar, G. S. P. Matada, N. M. Raghavendra, A. Ghara, E. Singh, N. Abbas, G. S. Andhale, G. P. Shenoy and P. Sasmal, Current updates on EGFR and HER2 tyrosine kinase inhibitors for the breast cancer, \u003cem\u003eMedicinal Chemistry Research\u003c/em\u003e, 2022, \u003cstrong\u003e31\u003c/strong\u003e, 1401-1413.\u003c/li\u003e\n\u003cli\u003eA. D. Meedendorp, A. ter Elst, N. A. \u0026rsquo;t Hart, H. J. M. Groen, E. Schuuring and A. J. van der Wekken, Response to HER2 Inhibition in a Patient With Brain Metastasis With EGFR TKI Acquired Resistance and an HER2 Amplification, \u003cem\u003eFrontiers in Oncology\u003c/em\u003e, 2018, \u003cstrong\u003eVolume 8 - 2018\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eH.-N. Liu, Y. Zhu, Y. Chi, Y. Zhang, X. Li, W. Wen, L.-S. Shan, Y.-T. Wang and B. Dai, Synthetic routes and clinical application of Small-Molecule HER2 inhibitors for cancer therapy, \u003cem\u003eBioorganic Chemistry\u003c/em\u003e, 2024, \u003cstrong\u003e151\u003c/strong\u003e, 107653.\u003c/li\u003e\n\u003cli\u003eF. Le Du, V. Di\u0026eacute;ras and G. Curigliano, The role of tyrosine kinase inhibitors in the treatment of HER2+ metastatic breast cancer, \u003cem\u003eEuropean Journal of Cancer\u003c/em\u003e, 2021, \u003cstrong\u003e154\u003c/strong\u003e, 175-189.\u003c/li\u003e\n\u003cli\u003eM. Hu, Y. Li, J. Li, H. Zhou, C. Liu, Z. liu, Y. Gong, B. Ying and Y. Xie, Discovery of potent and selective HER2 PROTAC degrader based Tucatinib with improved efficacy against HER2 positive cancers, \u003cem\u003eEuropean Journal of Medicinal Chemistry\u003c/em\u003e, 2022, \u003cstrong\u003e244\u003c/strong\u003e, 114775.\u003c/li\u003e\n\u003cli\u003eY. Feng, I. Gonz\u0026aacute;lez and D. Zhang, 1426 Tucatinib (HER2 Tyrosine Kinase Inhibitor)-Induced Liver Injury, \u003cem\u003eLaboratory Investigation\u003c/em\u003e, 2025, \u003cstrong\u003e105\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eD. D. Singh, H.-J. Lee and D. K. Yadav, Clinical updates on tyrosine kinase inhibitors in HER2-positive breast cancer, \u003cem\u003eFrontiers in Pharmacology\u003c/em\u003e, 2022, \u003cstrong\u003eVolume 13 - 2022\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eQ. Wu, B. Bai, C. Tian, D. Li, H. Yu, B. Song, B. Li and X. Chu, The Molecular Mechanisms of Cardiotoxicity Induced by HER2, VEGF, and Tyrosine Kinase Inhibitors: an Updated Review, \u003cem\u003eCardiovascular Drugs and Therapy\u003c/em\u003e, 2022, \u003cstrong\u003e36\u003c/strong\u003e, 511-524.\u003c/li\u003e\n\u003cli\u003eR. Roskoski, Properties of FDA-approved small molecule protein kinase inhibitors: A 2021 update, \u003cem\u003ePharmacological Research\u003c/em\u003e, 2021, \u003cstrong\u003e165\u003c/strong\u003e, 105463.\u003c/li\u003e\n\u003cli\u003eC. Peng, X. Fu, K. Wang, L. Chen, B. Luo, N. Huang, Y. Luo and W. Chen, Dauricine alleviated secondary brain injury after intracerebral hemorrhage by upregulating GPX4 expression and inhibiting ferroptosis of nerve cells, \u003cem\u003eEuropean Journal of Pharmacology\u003c/em\u003e, 2022, \u003cstrong\u003e914\u003c/strong\u003e, 174461.\u003c/li\u003e\n\u003cli\u003eQ. Xue, D. Yan, X. Chen, X. Li, R. Kang, D. J. Klionsky, G. Kroemer, X. Chen, D. Tang and J. Liu, Copper-dependent autophagic degradation of GPX4 drives ferroptosis, \u003cem\u003eAutophagy\u003c/em\u003e, 2023, \u003cstrong\u003e19\u003c/strong\u003e, 1982-1996.\u003c/li\u003e\n\u003cli\u003eY. Chen, M.-f. Wu, M.-m. Xie, Y. Lu, C. Li, S.-s. Xie, W.-x. Ma, M.-l. Ji, R. Hou, Z.-h. Dong, R.-b. He, M.-m. Zhang, H. Lu, L. Gao, J.-g. Wen, J. Jin, X.-w. Dong, J.-x. Che and X.-m. Meng, Cpd-A1 alleviates acute kidney injury by inhibiting ferroptosis, \u003cem\u003eActa Pharmacologica Sinica\u003c/em\u003e, 2024, \u003cstrong\u003e45\u003c/strong\u003e, 1673-1685.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-chemotherapy-and-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccap","sideBox":"Learn more about [Cancer Chemotherapy and Pharmacology](http://link.springer.com/journal/280)","snPcode":"280","submissionUrl":"https://submission.nature.com/new-submission/280/3","title":"Cancer Chemotherapy and Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HER2-positive breast cancer, central nervous system metastasis, blood-brain barrier, multi-target inhibitor, ferroptosis, pharmacokinetics, rational drug design","lastPublishedDoi":"10.21203/rs.3.rs-9021276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9021276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHER2-positive breast cancer, particularly in patients with central nervous system metastasis, presents significant clinical challenges due to limited treatment options and extremely poor prognosis. This paper reports the rational design, synthesis, and systematic evaluation of a novel multi-target HER2 inhibitor, CPD 1. Employing a \u0026lsquo;skeletal leap-pharmacophore retention-multi-mechanism synergy\u0026rsquo; design strategy, CPD 1 retains highly selective HER2 inhibitory activity while incorporating a flavonoid derivative moiety to synergistically induce ferroptosis and inhibit P-glycoprotein efflux. In vitro studies demonstrate potent antiproliferative activity of CPD 1 against a panel of HER2-positive breast cancer cells (including PIK3CA mutant variants), exhibiting significantly superior IC₅₀ values compared to trastuzumab. Its mechanism involves effective inhibition of the HER2/PI3K/AKT pathway alongside induction of lipid peroxidation and glutathione depletion. In vivo pharmacodynamic evaluations in subcutaneous xenograft and brain metastasis models confirmed that CPD 1 dose-dependently inhibited tumour growth, significantly delayed the formation of brain metastases, and reduced the number of metastatic lesions, demonstrating superior efficacy to the positive control drug Tucatinib. Pharmacokinetic studies revealed CPD 1's exceptional blood-brain barrier penetration (brain tissue/plasma concentration ratio\u0026thinsp;\u0026gt;\u0026thinsp;0.5), underpinning its potent anti-CNS metastatic activity. Furthermore, preliminary toxicological assessments indicate CPD 1 possesses a broad safety margin. In summary, CPD 1 emerges as a novel candidate drug integrating potent HER2 inhibition, ferroptosis induction, and favourable brain exposure. It offers a highly promising solution for overcoming the clinical challenge of HER2-positive breast cancer and its CNS metastases.\u003c/p\u003e","manuscriptTitle":"Research and Development of a Multi-Target HER2 Inhibitor (CPD 1) and Its Mechanism and Application in Combating HER2-Positive Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-03 02:16:17","doi":"10.21203/rs.3.rs-9021276/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T01:03:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-21T16:05:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-20T03:48:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T13:22:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326494310224353970554397995505719677280","date":"2026-03-31T02:19:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254333257172135240405140380577274854335","date":"2026-03-30T16:20:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252042354607997879907174731601276635826","date":"2026-03-30T09:44:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-29T01:53:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-05T07:38:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-05T07:36:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Chemotherapy and Pharmacology","date":"2026-03-03T14:20:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-chemotherapy-and-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccap","sideBox":"Learn more about [Cancer Chemotherapy and Pharmacology](http://link.springer.com/journal/280)","snPcode":"280","submissionUrl":"https://submission.nature.com/new-submission/280/3","title":"Cancer Chemotherapy and Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2e92175e-0fe6-46de-8ce8-351434b50005","owner":[],"postedDate":"April 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T03:23:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-03 02:16:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9021276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9021276","identity":"rs-9021276","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}