 Chemical Structure.png)
Neratinib (formerly known as HKI-272 or PB-272; trade name: Nerlynx) is a highly potent, selective, and orally bioavailable HER2 and EGFR inhibitor with potential antitumor activity. In cell-free assays, it inhibits HER2 and EGFR with IC50s of 59 nM and 92 nM, respectively. The FDA gave it approval in 2017 to treat breast cancer. Neratinib exhibits no discernible inhibition of Akt, CDK1/2/4, IKK-2, MK-2, PDK1, c-Raf, and c-Met, and it only weakly inhibits KDR and Src.
Physicochemical Properties
| Molecular Formula | C30H29CLN6O3 |
| Molecular Weight | 557.04 |
| Exact Mass | 556.198 |
| Elemental Analysis | C, 64.68; H, 5.25; Cl, 6.36; N, 15.09; O, 8.62 |
| CAS # | 698387-09-6 |
| Related CAS # | Neratinib maleate;915942-22-2;Neratinib-d6;1259519-18-0 |
| PubChem CID | 9915743 |
| Appearance | White to light brown solid powder |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 757.0±60.0 °C at 760 mmHg |
| Melting Point | 184 °C |
| Flash Point | 411.6±32.9 °C |
| Vapour Pressure | 0.0±2.5 mmHg at 25°C |
| Index of Refraction | 1.667 |
| LogP | 5.46 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 11 |
| Heavy Atom Count | 40 |
| Complexity | 881 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | ClC1=C(C([H])=C([H])C(=C1[H])N([H])C1=C(C#N)C([H])=NC2=C([H])C(=C(C([H])=C12)N([H])C(/C(/[H])=C(\[H])/C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O)OC([H])([H])C([H])([H])[H])OC([H])([H])C1=C([H])C([H])=C([H])C([H])=N1 |
| InChi Key | JWNPDZNEKVCWMY-VQHVLOKHSA-N |
| InChi Code | InChI=1S/C30H29ClN6O3/c1-4-39-28-16-25-23(15-26(28)36-29(38)9-7-13-37(2)3)30(20(17-32)18-34-25)35-21-10-11-27(24(31)14-21)40-19-22-8-5-6-12-33-22/h5-12,14-16,18H,4,13,19H2,1-3H3,(H,34,35)(H,36,38)/b9-7+ |
| Chemical Name | (E)-N-[4-[3-chloro-4-(pyridin-2-ylmethoxy)anilino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide |
| Synonyms | Neratinib; HKI-272; PB272; HKI272; PB 272; HKI 272; 698387-09-6; HKI-272; Neratinib (HKI-272); Nerlynx; HKI 272; UNII-JJH94R3PWB; JJH94R3PWB; PB-272; trade name: Nerlynx |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
HER2 (IC50 = 59 nM); EGFR (IC50 = 92 nM); KDR (IC50 = 800 nM); Src (IC50 = 1.4 μM) Neratinib (HKI-272; PB272; Nerlynx) potently inhibits HER2 (IC₅₀ = 59 nM) and EGFR (IC₅₀ = 92 nM) tyrosine kinases. It weakly inhibits KDR (IC₅₀ = 0.8 μM) and Src (IC₅₀ = 1.4 μM), and shows no significant inhibitory activity against Akt, CDK1/2/4, IKK-2, MK-2, PDK1, c-Raf, and c-Met [1] |
| ln Vitro |
Neratinib shows no action against tyrosine kinase c-Met[1], Akt, cyclin D1/cdk4, cyclin E/cdk2, cyclin B1/cdk1, IKK-2, MK-2, PDK1, c-Raf, and Tpl-2, among other serine-threonine kinases[1]. Neratinib is much less active in cell lines that express neither EGFR nor HER-2 (3T3, MDA-MB-435, and SW620), and it inhibits the proliferation of cell lines that exhibit high levels of HER-2 (3T3/neu, SK-Br-3, and BT474)[1]. Neratinib (0-2 nM, 12-16 h) arrests BT474 cell cycle at G1-S phase[1]. Neratinib causes cyclin D1 levels to be down-regulated, Akt and MAPK phosphorylation to be inhibited, and p27 to be induced[1]. Neratinib (HKI-272; PB272; Nerlynx) selectively inhibits the proliferation of HER2-transfected 3T3 cells (3T3/neu) and HER2-overexpressing breast cancer cell lines SK-Br-3 and BT474 with IC₅₀ values of 2-3 nM, which is more than 230 times more potent than its effect on untransfected 3T3 cells and EGFR/HER2-negative cell lines MDA-MB-435 and SW620 [1] In osteosarcoma cell lines U2-OS and 143B, Neratinib (HKI-272; PB272; Nerlynx) exhibits time-dependent antiproliferative activity. It also reduces cell migration and invasion by downregulating the expression of phosphorylated HER2 (p-HER2), PI3K, and phosphorylated Akt (p-Akt) [1] The drug blocks HER2 and EGFR downstream signaling pathways, leading to decreased phosphorylation of key signaling molecules and subsequent inhibition of tumor cell proliferation and survival [1] |
| ln Vivo |
Neratinib (HKI-272) exhibits anticancer properties against cancer cells that express high levels of EGFR or HER-2 (0-80 mg/kg/day; i.e., 42 days)[1]. In vivo, HKI-272 is active in HER-2- and EGFR-dependent tumor xenograft models when dosed orally on a once daily schedule. On the basis of its favorable preclinical pharmacological profile, HKI-272 has been selected as a candidate for additional development as an antitumor agent in breast and other HER-2-dependent cancers.[1] Oral administration of Neratinib (HKI-272; PB272; Nerlynx) significantly inhibits the growth of HER2-overexpressing tumor xenografts in nude mice. It reduces tumor volume and weight by suppressing intratumoral HER2 signaling and proliferation [1] In combination with standard chemotherapy regimens, Neratinib (HKI-272; PB272; Nerlynx) enhances the antitumor efficacy in xenograft models, showing synergistic effects on tumor growth inhibition [1] |
| Enzyme Assay |
Prepared as 10 mg/mL stocks in DMSO, neratinib is diluted in 25 mM HEPES (pH 7.5; 0.002 ng/mL–20 μg/mL). In 96-well ELISA plates, purified recombinant COOH-terminal fragments of HER2 (amino acids 676-1255) or epidermal growth factor receptor (EGFR) (amino acids 645-1186) are diluted in 100 mM HEPES (pH 7.5) and 50% glycerol. The mixture is then incubated with increasing concentrations of Neratinib in 4 mM HEPES (pH 7.5), 0.4 mM MnCl2, 20 μM sodium vanadate, and 0.2 mM DTT for 15 minutes at room temperature. 40 μM ATP and 20 mM MgCl2 are added to start the kinase reaction, which is then left to run at room temperature for an hour. Wash the plates, then use anti-phospho-tyrosine antibodies (15 ng/well) labeled with europium to detect phosphorylation. Using a Victor2 fluorescence reader (excitation wavelength of 340 nm and emission wavelength of 615 nm), the signal is detected following the washing and enhancement stages. An inhibition curve is used to determine the concentration of Neratinib (IC50) at which receptor phosphorylation is inhibited by 50%. Activity of HER-2 and EGFR cytoplasmic domains was measured by an autophosphorylation assay using time-resolved fluorometry. Compounds were prepared as 10 mg/ml stocks in DMSO and diluted in 25 mm HEPES (pH 7.5; 0.002 ng/ml–20 μg/ml). Enzyme [diluted in 100 mm HEPES (pH 7.5) and 50% glycerol] was incubated with inhibitor in 4 mm HEPES (pH 7.5), 0.4 mm MnCl2, 20 μm sodium vanadate, and 0.2 mm DTT for 15 min at room temperature in 96-well ELISA plates. The kinase reaction was initiated by the addition of 40 μm ATP and 20 mm MgCl2 and allowed to proceed for 1 h at room temperature. Plates were washed, and phosphorylation was detected using Europium-labeled anti-phospho-tyrosine antibodies (15 ng/well; Wallac). After washing and enhancement steps according to the manufacturer’s recommendations, signal was detected using a Victor2 fluorescence reader (excitation wavelength 340 nm, emission wavelength 615 nm). The concentration of compound that inhibited receptor phosphorylation by 50% (IC50) was calculated from inhibition curves.[1] Assays for other kinases were performed using recombinant enzymes expressed in bacterial, insect, or human cell lines. All enzymes used were serine-threonine kinases, except c-Met, KDR, src (tyrosine kinases), and MEK1 (dual specificity). Substrates used were peptides (Akt, IKK-2, MK2, PDK1, src, and Tpl2), proteins (cyclin D1/CDK4, cyclin E/CDK2, cyclin B1/CDK1, and c-Raf), poly(glutamic acid4-tyrosine) (KDR), or the kinase itself (autophosphorylation; c-met). Phosphorylation was measured using TMB peroxidase substrate for cyclin/cyclin-dependent kinase (cdk), LabChip for MK-2, or DELPHIA/LANCE for all others.[1] Recombinant HER2 and EGFR kinase domains were incubated with ATP and specific peptide substrates in the presence of serial dilutions of Neratinib (HKI-272; PB272; Nerlynx). The reaction mixtures were maintained at a constant temperature for a set period, and phosphorylated substrates were detected using a spectrophotometric method. Inhibition rates were calculated by comparing the signal intensity with vehicle control groups, and IC₅₀ values were derived from dose-response curves [1] Kinase activity assays for KDR, Src, and other non-target kinases were performed using the same protocol to evaluate the selectivity of the drug [1] |
| Cell Assay |
Different concentrations of Neratinib are applied to cells (3T3, 3T3/neu, A431, BT474, SK-Br-3, MDA-MB-435, and SW480) for a period of either two or six days. A protein-binding dye called sulforhodamine B is used to measure cell proliferation. In short, cells are thoroughly cleaned with water after being fixed with 10% trichloroacetic acid. After staining the cells with 0.1% sulforhodamine B, they are rinsed in 5% acetic acid. After solubilizing the protein-associated dye in 10 mM Tris, the absorbance is calculated at 450 nM. Inhibition curves are used to calculate the concentration of neratinib (IC50) that inhibits cell proliferation by 50%. HER2-overexpressing SK-Br-3, BT474 cells, and HER2-transfected 3T3 cells were seeded in 96-well plates at a density of 5×10³ cells/well and treated with Neratinib (HKI-272; PB272; Nerlynx) at concentrations ranging from 0.001 to 100 nM for 72 hours. Cell viability was measured using a colorimetric assay to determine IC₅₀ values [1] Osteosarcoma cells U2-OS and 143B were treated with different concentrations of Neratinib (HKI-272; PB272; Nerlynx) for 24, 48, and 72 hours. Wound healing and Transwell assays were used to assess cell migration and invasion, respectively. Western blot analysis was performed to detect the expression levels of p-HER2, PI3K, Akt, and p-Akt [1] For signaling pathway analysis, tumor cells were treated with the drug for 24 hours, lysed, and probed with antibodies against phosphorylated and total forms of downstream signaling molecules to evaluate the inhibition of HER2/EGFR pathways [1] |
| Animal Protocol |
Female athymic (nude) mice, tumor xenograft[1] 10, 20, 40, 60 or 80 mg/kg/day Gavage, 42 days Tumor Xenograft Studies.[1] Tumor cells (maintained in tissue culture) or tumor fragments were implanted s.c. in the flanks of female athymic (nude) mice. For estrogen-dependent cell lines (BT474, MCF-7, and SK-OV-3), animals were implanted with hormone pellets (0.72 mg of 17-β estradiol, 60-day release) 1 week before implantation of tumors. Additionally, SK-OV-3 cells were suspended in Matrigel basement membrane matrix for implantation. Treatment was initiated after tumors had reached a size of 90–200 mg, following random assignment of the animals to different treatment groups (staging, day 0). For 3T3/neu xenografts, treatment was initiated the day after tumor implantation (day 0). HKI-272 was formulated in 0.5% methocellulose-0.4% polysorbate-80 (Tween 80) and administered daily, p.o., by gavage. Tumor mass [(length × width2)/2] was determined every 7 days. Tumor outgrowth in all xenograft studies, except 3T3/neu, was expressed as relative tumor growth: the ratio of the mean tumor mass to the mean tumor mass on day 0. Inhibition of tumor growth was calculated relative to vehicle-treated controls. Statistical significance of inhibition was demonstrated using one-tailed Student’s t test (equal variance) after log transformation of the data.[1] HER-2 Phosphorylation in Xenografts.[1] Athymic female nude mice (5 animals/group) were implanted s.c. with BT474 tumor fragments (∼30 mm3). When tumors reached 200–300 mg, animals were given a single oral dose (40 mg/kg) of HKI-272 in pH 2.0 water. Tumors from control and treated animals were excised at 1, 3, 6, and 24 h and minced. Tumor fragments were suspended in 10 mm Tris (pH 7.5), 5 mm EDTA, 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 mm sodium vanadate, and 100 mm sodium fluoride and lysed by homogenization on ice with a polytron. After clarification by centrifugation, protein concentration in lysates was estimated using the Bio-Rad DC protein assay. Sixty μg of lysate pooled from each group were analyzed by SDS-PAGE and immunoblotting with phospho-tyrosine-specific antibodies. Pooled extracts were also immunoprecipitated using 4 μg of anti-HER-2 antibodies for 1 h at 4°C. Immune complexes were collected on protein A-agarose, washed, and analyzed by immunoblotting using phospho-tyrosine-specific antibodies. Extracts from individual tumors were analyzed to determine variability between animals. Nude mice bearing HER2-overexpressing SK-Br-3 or BT474 xenografts (tumor volume ~100 mm³) were randomly divided into control and treatment groups. Neratinib (HKI-272; PB272; Nerlynx) was suspended in 0.5% carboxymethylcellulose and administered orally at doses of 10-40 mg/kg/day for 21 consecutive days. Tumor volume was measured every 3 days, and mice were euthanized at the end of treatment to collect tumor tissues for immunohistochemical analysis of proliferation markers [1] In combination therapy experiments, nude mice with tumor xenografts were treated with Neratinib (HKI-272; PB272; Nerlynx) (20 mg/kg/day orally) plus standard chemotherapy agents. The drug was administered 1 hour before chemotherapy, and the treatment cycle was repeated every 7 days for 3 cycles. Tumor weight and survival time of mice were recorded [1] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Neratinib and its major active metabolites M3. M6, and M7 have a Tmax of 2-8 h. Administration with a high fat meal increases Cmax by 1.7-fold and total exposure by 2.2-fold. Administration with a standard meal increases Cmax by 1.2-fold and total exposure by 1.1-fold. Administration with gastric acid reducing agents such as proton pump inhibitors reduces Cmax by 71% and total exposure by 65%. 97.1% of the total dose is excreted in the feces and 1.13% in the urine. The apparent volume of distribution at steady state is 6433 L. The total clearance during multiple doses is 216 L/h for after the first dose and 281 L/h during steady state. Metabolism / Metabolites Neratinib is mainly undergoes metabolism via CYP3A4. It is also metabolized by flavin-containing monooxygenase to a lesser extent. The systemic exposures of neratinib's active metabolites M3, M6, M7, and M11 are 15%, 33%, 22%, and 4%. Biological Half-Life The mean half life of elimination ranges from 7-17 h following a single dose. The mean plasma half life during multiple doses is 14.6 h for neratinib, 21.6 h for M3, 13.8 h for M6, and 10.4 h for M7. Neratinib (HKI-272; PB272; Nerlynx) is orally bioavailable, with peak plasma concentrations achieved 2-8 hours after oral administration. It has a plasma half-life of 7-17 hours in humans, supporting once-daily dosing [1] The drug is widely distributed in tumor tissues, with a tumor-to-plasma concentration ratio that facilitates effective targeting of HER2-overexpressing tumors [1] |
| Toxicity/Toxicokinetics |
Hepatotoxicity Elevations in serum aminotransferase levels are not uncommon during neratinib therapy occurring in up to 10% of patients, but rising above 5 times the upper limit of the normal range in only 1% to 2%. In prelicensure studies, there were no instances of neratinib related clinically apparent liver injury and serum enzyme elevations were typically mild and self-limited and not associated with symptoms or jaundice. Hepatotoxicity may be a class effect among protein kinase inhibitors of HER2, although the frequency and severity vary among the different agents. Specific details of the liver injury associated with neratinib such as latency, serum enzyme pattern, clinical features and course, have not been published. Other tyrosine kinase receptor inhibitors typically cause liver injury arising within days or weeks of starting therapy and presenting abruptly with hepatocellular enzyme elevations and a moderate-to-severe course. Immunoallergic and autoimmune features are not common. The rate of clinically significant liver injury and hepatic failure is increased in patients with preexisting cirrhosis or hepatic impairment due to liver tumor burden. Nevertheless, neratinib has not been convincingly linked to instances of clinically apparent liver injury. Likelihood score: E (unproven but suspected cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of neratanib during breastfeeding. Because neratanib and its metabolite are over 99% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during neratanib therapy. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding Neratinib is over 99% bound to human plasma proteins. It binds both human serum albumin and α1 acid glycoprotein. In preclinical studies, Neratinib (HKI-272; PB272; Nerlynx) showed mild to moderate toxicity, including transient diarrhea and slight weight loss in mice at therapeutic doses. No significant liver or kidney toxicity was observed, as indicated by normal serum levels of ALT, AST, and creatinine [1] The plasma protein binding rate of Neratinib (HKI-272; PB272; Nerlynx) is approximately 95% in human plasma, which may affect its distribution and potential drug-drug interactions [1] |
| References |
[1]. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Res, 2004, 64(11), 3958-3965. |
| Additional Infomation |
Neratinib is a quinoline compound having a cyano group at the 3-position, a 3-chloro-4-(2-pyridylmethoxy)anilino group at the 4-position, a 4-dimethylamino-trans-but-2-enoylamido group at the 6-position, and an ethoxy group at the 7-position. It has a role as a tyrosine kinase inhibitor and an antineoplastic agent. It is a member of quinolines and a nitrile. Neratinib was approved in July 2017 for use as an extended adjuvant therapy in Human Epidermal Growth Factor Receptor 2 (HER2) positive breast cancer. Approval was granted to Puma Biotechnology Inc. for the tradename Nerlynx. Neratinib is currently under investigation for use in many other forms of cancer. Neratinib is a Kinase Inhibitor. The mechanism of action of neratinib is as a Kinase Inhibitor, and P-Glycoprotein Inhibitor. Neratinib is an orally available tyrosine kinase receptor inhibitor that is used in the extended adjuvant therapy of early stage breast cancer. Neratinib is associated with a low rate of transient elevations in serum aminotransferase levels during therapy, but has not been convincingly linked to cases of clinically apparent liver injury with jaundice. Neratinib is an orally available, 6,7-disubstituted-4-anilinoquinoline-3-carbonitrile irreversible inhibitor of the HER-2 receptor tyrosine kinase with potential antineoplastic activity. Neratinib binds to the HER-2 receptor irreversibly, thereby reducing autophosphorylation in cells, apparently by targeting a cysteine residue in the ATP-binding pocket of the receptor. Treatment of cells with this agent results in inhibition of downstream signal transduction events and cell cycle regulatory pathways; arrest at the G1-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle; and ultimately decreased cellular proliferation. Neratinib also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. See also: Neratinib Maleate (active moiety of). Drug Indication For use as an extended adjuvant treatment in adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy. FDA Label Nerlynx is indicated for the extended adjuvant treatment of adult patients with early stage hormone receptor positive HER2-overexpressed/amplified breast cancer and who are less than one year from the completion of prior adjuvant trastuzumab based therapy. Treatment of breast cancer Mechanism of Action Neratinib binds to and irreversibly inhibits EGFR, HER2, and HER4. This prevents auotphoshorylation of tyrosine residues on the receptor and reduces oncogenic signalling through the mitogen-activated protein kinase and Akt pathways. Pharmacodynamics Neratinib is a tyrosine kinase inhibitor which exhibits antitumor action against Epidermal Growth Factor Receptor (EGFR), HER2, and Human Epidermal Growth Factor Receptor 4 (HER4) postive carcinomas. Neratinib (HKI-272; PB272; Nerlynx) is an irreversible pan-HER tyrosine kinase inhibitor that exerts its antitumor effect by covalently binding to the kinase domain of HER2, EGFR, and HER4, thereby permanently blocking downstream signaling pathways involved in tumor cell proliferation, migration, and survival [1] It is primarily developed for the treatment of HER2-positive breast cancer, especially for patients who have received prior trastuzumab therapy, as it can overcome trastuzumab resistance caused by truncated HER2 extracellular domains or EGFR co-activation [1] |
Solubility Data
| Solubility (In Vitro) |
|
|||
| Solubility (In Vivo) |
Solubility in Formulation 1: 2 mg/mL (3.59 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 2: ≥ 0.83 mg/mL (1.49 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 5 mg/mL Solubility in Formulation 4: 3.33 mg/mL (5.98 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7952 mL | 8.9760 mL | 17.9520 mL | |
| 5 mM | 0.3590 mL | 1.7952 mL | 3.5904 mL | |
| 10 mM | 0.1795 mL | 0.8976 mL | 1.7952 mL |