Abemaciclib, a Recent Novel FDA-Approved Small Molecule Inhibiting Cyclin-Dependant Kinase 4/6 for the Treatment of Metastatic Breast Cancer: A Mini-Review

: Abemaciclib (Verzerio ® ) is a cell cycle inhibitor of both CDK4 and CDK6. In 2017, abemaciclib was approved by the Food and Drug Administration (FDA) and, in 2018 by the European Medicines Agency (EMA) for the treatment of postmenopausal women with hormone receptor positive (HR + ), human epidermal growth factor receptor 2 negative (HER2 -) advanced breast cancer. In this mini-review, we provide a series of information for respectively their targets and its selectivity, results on preclinical trial, clinical phase I, II and III trials, an some perspectives. We also describe the batch and flow steps used for the synthesis of this cancer drug.


Introduction
Imatinib (1) (Figure 1; Gleevec ® from Novartis, Basel, Switzerland) was the first approved protein kinase inhibitor [1][2] in May 2001 by the Food and Drug Administration for the treatment of myeloid leukemia [3]. To date, the number of protein kinase inhibitors (PKIs) approved worldwide continue to grow steadily between 2001 to January 2018 period: 39 drugs have been approved [4] by Food and Drug Administration, Chinese and European regulatory authorities for their respective markets.   (1) and, the CDK4/6 inhibitor drugs palbociclib (2) and ribociclib (3) In 2017, Food and Drug Administration approved the palbociclib (2) [5], a first-in-class inhibitor of CDK4/6 [6] developed by Pfizer for the treatment of postmenopausal women with hormone-receptor positive, HER-2 negative advanced breast cancer after initial endocrinebased therapy. During the Phase 2 PALOMA-1/TRIO 18 study, treatment with palbociclib (2) was associated to letrozole [7]. During the same period, ribociclib (3) owned by Novartis, the US Food and Drug Administration and the European Medicines Agency, also approved a direct competitor of palbociclib (2), in March 2017 for the treatment of metastatic breast cancer [8][9]. Now the purpose of this mini-review is to provide information's on the third CDK4/6 inhibitor approved by Food and Drug Administration in 2017 named as abemaciclib (4) (LY2835219), for the treatment of certain breast cancer [10][11].

Uses
Abemaciclib (4) was designated as a breakthrough therapy for metastatic breast cancer after successful Phase I [12], and Phase II [13] trials realized respectively in May and December 2014. Food and Drug Administration approved it for use in USA in September 2017 [14] and the European Medicines Agency regulatory authorities in September 2018.

Targets and Selectivity
For regulating the cell cycle, the G1 restriction point is critical and is controlled by the retinoblastoma (Rb) pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). The retinoblastoma protein is a tumor suppressor, which inhibits proliferation through binding to and suppressing the activity of the E2F family of transcription factors. The central role of the Rb pathway for controlling cellular proliferation has been demonstrated by its dysregulation in human cancer. Transition through the restriction point requires phosphorylation of Rb by CDK4/6, which validated cancer drug targets [15].
Eli Lilly and Company identified the 2-anilino-2,4-pyrimidine-[5-benzimidazole] scaffold as potent inhibitors of CDK4/cyclin D1 and CDK6/cyclin D1 by compound screening. Optimization of the scaffold has been realized by structure-activity relationship (SAR) studies associated to computing structure-based design and biochemical screening against a small panel of kinases to improve potency and selectivity, and with a colo-205-cell high content imaging monitoring inhibition of its phosphorylation. Compounds with good physicochemical and pharmacokinetic properties were then evaluated for in vivo tumor activity against xenograft tumors in immunodeficient mice. In this context, LY2835219 emerged as a good candidate for its potential biological activities and optimal pharmacological properties. In biochemical assays (Table 1), abemaciclib (4) (or LY2835219) inhibited respectively CDK4/cyclin D1 and CDK6/cyclin D1 with IC 50 2 nM and 9.9 nM. K i ATP constants were also determined through kinetic studies; for CDK4/cyclin D1, it showed K i ATP = 0.6 nmol/l and 2.4 nmol/l for CDK6/cyclin D1. This means that abemaciclib (4) is a competitive ATP inhibitor [16].

Preclinical and Clinical Trials
In preclinical development [17], pharmacokinetic and pharmacodynamic properties of methane sulfonate salt of abemaciclib (4)  In Phase I trial [18], effect of abemaciclib (4) has been explored in five different types (n = 132) with a cohort of 47 patients (median age: 55 years) with metastatic breast cancer who received a median of 7 prior-therapeutics. In the cohort of 47 patients, 74% had visceral metastases and 76% (36 patients) were hormone receptor-positive (HER + ). Among this HER + patient's group, 25% (9 patients) had confirmed partial responses and 56% (20 patients) had stable disease (including 2 patients with unconfirmed responses). The clinical benefit rate was 61% and disease central rate was 81%. The maximum tolerated dose was 200 mg every 12 hours (fatigue was the dose-limiting toxicity).
The MONARCH 2 clinical Phase III study (Clinical Trials.gov: NTC 02107703) of women with HER + and human epidermal growth factor-receptor 2-negative ABC was realized between August 2014 and December 2015 with 669 patients [19]. This Phase III, randomized, double-blind, placebo-controlled study of fulvestrant with abemaciclib (4) (n = 446) or without (n = 223) was conducted in 142 centers and in 19 countries. Treatment of patients randomly assigned 2:1 to receive abemaciclib (4)  In MONARCH 3 Final PFS (Progression Free Survival) study [21], a total of 125 women patients (38.1%) in the abemaciclib arm and 35 patients (21.2%) in the placebo arm remained in treatment. Following 24 cycles of treatments, the mean decrease in tumor size was 76.1% in the abemaciclib arm and 50% in the placebo arm. For patients responded to abemaciclib (4), the response typically occurred within the first 8 months of treatment (Figure 4a) Results of this clinical Phase III trial come in light for CD4/6 cyclin D in the regulation of cancer immune surveillance [22][23].

Syntheses
As depicted in the retrosynthetic approach ( Figure 5), abemaciclib (4) [24] involved a carbon-carbon Suzuki coupling as key step between boronic ester (5) and 2,4-dichloro-5fluoro pyrimidine (6) as readily available commercial building blocks [25]. For this, the authors used PdCl 2 (PPh 3 ) 2 with Na 2 CO 3 in DME at 80°C and they obtained the biaryl compound (9) after 4 h in 66% isolated yield (Scheme 1). For the second step, they investigated a carbon-nitrogen coupling via Buchwarld-Hartwing amidation. For optimization of this N,C-coupling, they explored successively the choice of appropriate solvent (MeOH, EtOH, t-AmylOH) due to solubility limitations of the starting reagents (9) and (7), the ligand and reaction temperature. Using the more hindered t-AmylOH (no undesired t-Amyl ether by-product was observed), DPEPhos (as sufficient active catalyst), they obtained successful carbon-nitrogen coupling after 18 h at 100°C and the aldehyde intermediate (10)   The third and last step in the synthesis of abemaciclib (4) is a classical reductive amination involving the aldehyde (10) and N-ethylpiperazine (8). After a few set of explorating experiences with NaBH(OAc) 3 as reductant, they observed a 97:3 distribution of (4) and reduced aldehyde (10) which, increased difficulties of separation and purification through crystallization. In this context, they dropped out these classical reductive reaction conditions and these setbacks led to the exploration of Leuckart-Wallach conditions without catalyst. Starting from 4 equivalents of formic acid as source of reducing reagent associated to trimethyl orthoester for removal of water, reductive amination of aldehyde (10) in the presence of 2 equivalents of N-ethylpiperazine afforded quantitatively after 16 h abemaciclib (4) in 74% isolated yield.
The authors continued to develop efforts on Leuckart-Wallach reductive amination conditions for a robust batch process and a more convergent approach for the kilogram scale preparation of abemaciclib (4) [26]. Briefly, they opted for modifications on the 6aminopyridine-3-carbaldehyde (7) used in the carbon-nitrogen coupling of Buchwarld-Hartwing amidation. In fact, a robust process (Scheme 2) was fully realized to make directly the 2-amino pyridine (7') through two telescoped steps: i) the first was the Leuckart-Wallach reductive amination using pipe-in-series reactors with near instantaneous heat-up times to maximize conversion and to reduce bye-products, ii) and the second was an Ullmann coupling with aqueous ammonia. Fortunately, the use of 2-methylaminoethanol at 100°C provided high conversion (99%).
For the intermediate (9) issued from the carbon-carbon Suzuki coupling (Scheme 1), the Eli Lilly's chemists, finally, developed also a telescoped Miyaura borylation and Suzuki coupling (Scheme 3) to maintain its high quality [27].  The use of bis(pinacolato)diboron (BisPin) and the aid of a Quality by Design (QbD) approach was the key point of success to control the risk of pinacol precipitation in THF and to provide a maximally flexible process for manufacturing in large scale.

Perspectives
Abemaciclib pharmacological profile as novel CDK4/6 inhibitor represented an optimal first line treatment for hormone receptor (HR) positive, human epidermal growth-factor receptor 2 (HER2) non-amplified metastatic breast cancer (MBC) in combination with endocrine therapy. Its indication can be extended for treatments of patients (clinical phase II) with brain metastases secondary to ER + breast cancer, non-small lung cancer, or melanoma (Clinical Trials.gov: NTC 02308020). Given the observed benefits of CDK4/6 inhibitors in combination with endocrine therapy for patients with HR + , HER2-non amplified MBC, adjuvant studies are now underway to investigate the combination in HR + , HER2-non amplified early breast cancer [28].

Conclusion
For the treatment of advanced or metastatic HR + breast cancer, CDK4/6 inhibitors are an effective option. Their oral administration and their toxicity profile [29] are convenient and manageable. In this context, abemaciclib appears to possess unique pharmacological properties and seems to obtain the best results in heavily pretreated with visceral disease and worse prognosis. The approval of abemaciclib by Food and Drug Administration adds another option to the armamentarium of effective CDK4/6 inhibitors [30][31] currently available.

Author Contributions:
The three authors contributed equally to this manuscript.
Funding: This research received no external funding.