Drugs in Clinical Pipeline: Neratinib | Dual Kinase Inhibitor | Breast Cancer Drug | EGFR Inhibitor | ERBB2 Inhibitor

Neratinib [(2E)-N-[4-[[3-Chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide] is an orally available small-molecule irreversible inhibitor of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2; also known as erbB2, CD340, ERBB2, HER2/neu) tyrosine kinases (TKs) [1, 2].

Neratinib : 2D and 3D Structure

It also blocks activity of HER4 (also known as erbB4). In vitro, it potently and selectively inhibits the erb-B receptor TKs at nanomolar concentrations (IC₅₀ ERBB2, EGFR = 59, 92 nM, respectively).

The activity and selectivity of Neratinib is attributed to a Michael acceptor at the C-6 position, which covalently bonded to the cysteine residue (Cys-773 in EGFR and Cys-805 in HER-2) in the ATP binding pocket of those enzymes. The dimethylamino group present at the end of the Michael acceptor group can serve as an intramolecular catalyst for the addition of Neratinib to the protein, which may accelerate the reaction between the bound drug and the protein. This entropic effect probably underlies the selectivity of Neratinib for its target enzymes.

HER-2 belongs to the ErbB family of receptor tyrosine kinases (RTKs), which has been implicated in a variety of cancers. Overexpression of HER-2 is seen in 25 – 30% of breast cancer patients and predicts a poor outcome in patients with primary disease. Blocking HER-2 function by a small molecule kinase inhibitor, therefore, represents an attractive alternate strategy to inhibit the growth of HER-2-positive tumors.

 Neratinib is a potent inhibitor of HER-2 and is highly active against HER-2-overexpressing human breast cancer cell lines in vitro. It also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. Neratinib reduces HER-2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation and functions as an irreversible binding inhibitor.

Neratinib has been selected as a candidate for development as an antitumor agent in breast and other HER-2-dependent cancers. It is under clinical trials [1, 2].

Dual Kinase (EGFR, ERBB2) Inhibition for Cancer Treatment:

The ErbB family of proteins contains four receptor tyrosine kinases (RTKs), and in humans, the family includes Her1 (EGFR, ErbB1), Her2 (Neu, ErbB2), Her3 (ErbB3), and Her4 (ErbB4). All members are structurally related to the epidermal growth factor receptor (EGFR), its first discovered member. The gene symbol, ErbB, is derived from the name of a viral oncogene to which these receptors are homologous: erythroblastic leukemia viral oncogene [3].

Excessive ErbB signaling is associated with the development of a wide variety of types of solid tumor whereas insufficient ErbB signaling in humans is associated with the development of neurodegenerative diseases, such as multiple sclerosis and Alzheimer's Disease (AD). In mice, loss of signaling by any member of the ErbB family results in embryonic lethality with defects in organs including the lungs, skin, heart, and brain.

Excessive ErbB signaling is associated with the development of a wide variety of types of solid tumor. ErbB-1 and ErbB-2 are found in many human cancers, and their excessive signaling may be critical factors in the development and malignancy of these tumors.

Several malignancies are associated with the mutation or increased expression of members of the ErbB family including lung, breast, stomach, colorectal, head and neck, and pancreatic carcinomas and glioblastoma. Gefitinib, erlotinib, and afatinib are orally effective protein-kinase targeted quinazoline derivatives that are used in the treatment of ERBB1-mutant lung cancer. Lapatinib is an orally effective quinazoline derivative used in the treatment of ErbB2-overexpressing breast cancer. Trastuzumab, pertuzumab, and ado-trastuzumab emtansine, which are given intravenously, are monoclonal antibodies that target the extracellular domain and are used for the treatment of ErbB2-positive breast cancer; ado-trastuzumab emtansine is an antibody-drug conjugate that delivers a cytotoxic drug to cells overexpressing ErbB2. Cetuximab and panitumumab are monoclonal antibodies that target ErbB1 and are used in the treatment of colorectal cancer. Cancers treated with these targeted drugs eventually become resistant to them [4].

Neratinib comes from the same laboratory that gave Pelitinib, an irreversible-binding inhibitor of EGFR. Pelitinib compound is predicted to covalently modify a cysteine residue (Cysteine-773) within the ATP binding site of the kinase.  Pelitinib currently in clinical trials for EGFR-dependent tumors but shows poorer efficacy in HER-2-dependent tumor models than in EGFR-dependent models. Therefore, it is felt that a compound such as Neratinib that is more potent than Pelitinib in HER-2-expressing tumors will complement the activity in the clinic.

Neratinib is under clinical trials for various cancers, including breast cancer and solid tumors.

Mechanism of Action in Neratinib:

Neratinib is a small-molecule irreversible inhibitor of the Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases (TKs) that is available in an oral formulation. In vitro, Neratinib potently and selectively inhibits the erb-B receptor (HER) TKs at nanomolar concentrations [1].

It reduces EGFR and HER2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation. Neratinib inhibits the proliferation of cell lines that show high levels of HER-2 (3T3/neu, SK-Br-3, and BT474) and is much less active in cell lines that express neither HER-2 nor EGFR (3T3, MDA-MB-435, and SW620). Neratinib inhibits the growth of HER-2-dependent tumors in vivo. The minimum dose, which causes a statistically significant inhibition of tumor growth, is estimated to be 5-10 mg/kg/day. In these xenograft studies, Neratinib was well tolerated by the animals, and no weight loss or other compound-related toxicity was observed [1].

The irreversible binding nature of Neratinib plays an important part in its activity and selectivity. As an irreversible inhibitor is noncompetitive with ATP, it would be unaffected by high concentrations of ATP within the cell. Also, the biological effects of an irreversible inhibitor should persist even after the drug leaves the circulation, once the target enzyme has been deactivated by covalent bond formation. Finally, irreversible inhibitors should have better selectivity for their targets since they form a covalent interaction with an active-site cysteine residue.

Dosages and Approvals:

Neratinib (Tradename: -) an EGFR/ERBB2 kinase inhibitor is discovered and developed by Wyeth Pharmaceuticals. It follows Pelitinib, an EGFR inhibitor from Wyeth Pharmaceuticals. Neratinib is designed in a manner to improve the efficiency of Pelitinib.

It is under clinical trials for breast cancer and other solid tumors.

Reported activities for Neratinib:

In a cell-free autophosphorylation assay using the recombinant cytoplasmic domain of HER-2, Neartinib reduced kinase activity by 50% (IC₅₀) at a concentration of 59 nM. It also inhibited the kinase activity of the EGFR cytoplasmic domain under similar assay conditions at 92 nM.

Neratinib did not significantly inhibit several serine-threonine kinases tested (Akt, cyclin E/cdk2, cyclin B1/cdk1, IKK-2, PDK1, c-Raf, and Tpl-2 (IC₅₀ was more than 9 uM), cyclin D1/cdk4, MK-2 (IC₅₀ was more than 45 uM) or the tyrosine kinase, c-Met (IC₅₀ greater than 35 uM). It weakly inhibited two other tyrosine kinases tested, KDR and Src (IC₅₀ = 800 and 1400 nM, respectively), but was 14- and 24-fold less active against these kinases, compared with HER-2. Neratinib is, therefore, a highly selective inhibitor of HER-2 and EGFR [1].

Neratinib repressed the proliferation of a mouse fibroblast cell line (3T3) transfected with the HER-2 oncogene (3T3/neu) by IC₅₀ at 3 nM. This value was 230-fold lower than that obtained with the isogenic untransfected cells (IC₅₀ = 700 nM), demonstrating that Neratinib has a high degree of selectivity for this oncogenic pathway.

Neratinib also inhibited two other HER-2-overexpressing breast cancer cell lines, SK-Br-3 and BT474 (IC₅₀ = 2 nM), but was much less active on MDA-MB-435 and SW620 (a breast and a colon cancer cell line, respectively) that are EGFR- and HER-2-negative. Consistent with its activity against EGFR, Neratinib inhibited proliferation of the epidermal carcinoma cell line, A431, that overexpresses EGFR (IC₅₀ = 81 nM) [1].

IC₅₀ (Inhibition of HER-2 Kinase Activity) = 59 ± 13 nM

IC₅₀ (Inhibition of EGFR Kinase Activity) = 92 ± 17 nM

IC₅₀ (Inhibition of KDR Kinase Activity) = 800 nM

IC₅₀ (Inhibition of SRC Kinase Activity) = 1400 nM

Summary

Common name: HKI-272; HKI 272; HKI272; Neratinib

Trademarks: -

Molecular Formula: C30H29ClN6O3

CAS Registry Number: 698387-09-6

CAS Name: (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide

Molecular Weight: 557.04

SMILES:O=C(NC1=C(OCC)C=C2N=CC(C#N)=C(NC3=CC=C(OCC4=NC=CC=C4)C(Cl)=C3)C2=C1)/C=C/CN(C)C

InChI Key: JWNPDZNEKVCWMY-VQHVLOKHSA-N

InChI: 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+

Mechanism of Action: Kinase Inhibitor; Epidermal growth factor receptor (EGFR) Inhibitor; Human epidermal growth factor receptor 2 (HER2) Inhibitor

Activity: Antineoplastics; Breast Cancer Drug; Treatment for Solid Tumors

Status: Under Phase Trials

Chemical Class: Small molecules; Amide containing; Chloro containing; Cyano containing; Nitrile containing; Anilinoquinoline derivatives; Pyridine containing; Quinoline derivatives; Aniline derivatives

Originator: Wyeth Pharmaceuticals

Neratinib Synthesis

The literature reports few syntheses for Neratinib. The synthesis is divided into intermediates and final synthesis.

First Intermediate: from Ref. 8

Neratinib Intermediate

Second Intermediate: from Org Process Res Dev 2012, 16(12), 1970-1973.

Neratinib Intermediate

Final Synthesis from WO2006127207A1: The patent reports the first known synthetic procedure for Neratinib synthesis.

Neratinib Synthesis

J Med Chem 2005, 48(4), 1107-31: The article reports three synthetic routes were used to prepare Neratinib and its derivatives. The depicted route is preferred one for the synthesis. It also reports the biochemistry and design of the potent HER-2 inhibitors.

Neratinib Synthesis

Res Chem Intermed 2013, 39(7), 3105-3110: The article reports an excellent synthesis route to Neratinib involving Wittig-Horner reaction.  Wittig-Horner reaction is a classic method to get alkenes by reaction phosphonates with carbonyl compounds. In the reported route, ethyl diethoxyphosphinylacetate and dimethylaminoacetaldehyde diethylacetal, instead of (E)-4- (dimethylamino)but-2-enoyl acid hydrochloride and oxalyl chloride, were used to synthesize the 6-position side chain of Neratinib.

The article also summarizes few synthetic route from literature, and brings out their drawbacks.

Neratinib Synthesis

Identifications:

1H NMR (Estimated) for Neratinib

Experimental: ¹H-NMR(DMSO-d₆, 500 MHz) δ: 9.60 (s, 1H, NH), 9.48 (s, 1H, NH), 8.96 (s, 1H, ArH), 8.60 (d, 1H, J = 4.8 Hz, ArH), 8.47 (s, 1H, ArH), 7.87 (t, 1H, J = 7.0 Hz, ArH), 7.58 (d, 1H, J = 7.8 Hz, ArH), 7.39–7.35 (m, 3H, ArH), 7.27–7.17 (m, 2H, ArH), 6.82–6.73 (m, 1H, –CH=), 6.59 (d, 1H, J = 15.5 Hz, =CH–), 5.28 (s, 2H, –OCH₂Py), 4.31 (q, 2H, J = 7.0 Hz, –OCH₂CH₃), 3.09 (d, 2H, J = 5.7 Hz, –CH₂CH=), 2.18 (s, 6H (CH₃)₂N–), 1.47 (t, 3H, J = 7.0 Hz, –OCH₂CH₃).

13C-NMR (Estimated) for Neratinib

Experimental: ¹³C-NMR (500 MHz, DMSO-d₆) δ: 163.3, 156.2, 152.4, 152.2, 151.0, 150.5, 149.1, 147.4, 137.0, 134.2, 127.7, 125.7, 123.9, 123.0, 121.5, 121.4, 117.1, 114.3, 113.3, 108.6, 88.8, 71.2, 64.6, 61.8, 35.0, 14.2.

Sideeffects: The most common Neratinib-related adverse events (AEs) reported in various clinical trials are diarrhea (89%), nausea (29%), vomiting (23%), fatigue (16%) and anorexia (15%). Of all the patients, 19% had greater than Grade 3 diarrhea. Dose reductions, mostly due to diarrhea, were necessary in 27% participants.

Diarrhea is likely a physiologic consequence of EGFR inhibition, and is common to other EGFR TKIs, such as Erlotinib and Lapatinib.

Others: 

1. Discovery of Neratinib: Neratinib is designed as a structural homologue of Pelitinib (also known as EKB-569) a 4-anilinoquinoline-3-carbonitrile compound. Pelitinib shows good activity and selectivity for EGFR kinase (IC₅₀ = 38.5 nM) with poor activity for HER-2 (IC₅₀ = 1.255 uM). The improved HER2 activity of Neratinib compared to Pelitinib may be due to the placement of a lipophilic 2-pyridinylmethyl moiety at the para-position of the aniline and a lipophilic chlorine atom at the meta-position.

Neratinib and Pelitinib

Neratinib also has a Michael acceptor functional group at the 6-position. Such groups are known to react with sulfhydryl groups, which likely explains the covalent interaction with the conserved cysteine (Cys) residues of the tyrosine kinases (TKs). The dimethylamino group present at the end of the Michael acceptor group can serve as an intramolecular catalyst for the addition of Neratinib to the protein, which may accelerate the reaction between the bound drug and the protein. This entropic effect probably underlies the selectivity of Neratinib for its target enzymes.

References:

1. Rabindran, S. K.; et. al. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Res 2004, 64(11), 3958 - 3965. (free article)

2. Wong, K. K.; et. al. A phase I study with neratinib (HKI-272), an irreversible pan ErbB receptor tyrosine kinase inhibitor, in patients with solid tumors. Clin Cancer Res 2009, 15(7), 2552 - 2558. (free article)

3. Sharma, P. S.; et. al. Receptor tyrosine kinase inhibitors as potent weapons in war against cancers. Curr Pharm Des 2009, 15(7), 758 - 776. (FMO)

4. Roskoski, R. Jr. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res 2014, 79, 34 - 74. (FMO)

5. Mao, Y.; et. al. A New and Improved Process for N-(4-Chloro-3-cyano-7-ethoxyquinolin-6-yl)acetamide. Org Process Res Dev 2012, 16(12), 1970 -1973. (FMO)

6. Tsou, H. R.; et. al. Optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity. J Med Chem 2005, 48(4), 1107 - 1131. (FMO)

7. Ji, M.; et. al. The Wittig–Horner reaction for the synthesis of neratinib. Res Chem Intermed 2013, 39(7), 3105 - 3110. (FMO)

8. Chew, W.; et. al. Methods of synthesizing substituted 3-cyanoquinolines and intermediates thereof. WO2006127207A1

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