Third-generation CDK inhibitors: A review on the synthesis and binding modes of Palbociclib, Ribociclib and Abemaciclib

European Journal of Medicinal Chemistry 172 (2019) 143e153

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Mini-review

Third-generation CDK inhibitors: A review on the synthesis and binding modes of Palbociclib, Ribociclib and Abemaciclib Melania Poratti, Giovanni Marzaro* Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via Marzolo 5, I-35131, Padova, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 February 2019 Received in revised form 18 March 2019 Accepted 31 March 2019 Available online 4 April 2019

The role of cyclin-dependent kinases (CDKs) in regulating the transition of cell cycle steps makes this class of enzymes a suitable target for cancer therapy. Three different generations of CDKs inhibitors have been developed so far. Third-generation compounds (i.e. selective CDK4/6 inhibitors) are the most promising ones, due to their limited toxicity and high in vivo activity. To date, three compounds have entered the therapy, namely Palbociclib, Ribociclib and Abemaciclib. Herein we review the medicinal chemistry aspects of these drugs, with some references to very similar analogues that have been published. © 2019 Published by Elsevier Masson SAS.

Keywords: Palbociclib Ribociclib Abemaciclib CDKs Third-generation inhibitors Synthesis

1. Introduction Cancer is still a major cause of death worldwide. While the first anticancer strategy was based on the rather unspecific induction of death in replicating cells (mainly targeting the DNA [1e3] and the duplicational machinery [4e7]), the more modern strategy (the socalled target therapy) aims at identifying specific biomarkers (i.e. mutated, deregulated or overexpressed proteins) fundamental for tumour cells [8]. In this respect a key role is played by protein

Abbreviations: AML, acute myeloid leukaemia; BINAP, (2,20 -bis(diphenylphosphino)-1,10 -binaphthyl); Boc, tert-butyloxycarbonyl; Bu4NI, tetrabutylammonium iodide; CDK, cyclin-dependent kinase; dba, dibenzylideneacetone; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; dppf, 1,1’-(diphenylphosphino)ferrocene; EGFR, epidermal growth factor receptor; ER, estrogen receptor; Et3N, triethylamine; FGFR1, fibroblast growth factor receptor 1; FLT3-ITD, Fms-like tyrosine kinase 3 - internal tandem duplication; HBTU, N,N,N0 ,N0 -tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HER2, human epidermal growth factor 2; iPrMgCl, iso-propylmagnesium chloride; LiHMDS, litium hexamethyldisilazane; MCL, mantle cell lymphoma; MeCN, acetonitrile; MeOH, methanol; MW, microwave; NBS, N-bromosuccinimide; NMO, Nmethylmorpholine oxide; NMP, N-methylpyrrolidone; OAc, acetate; PDB, protein data bank; PDGFRb, platelet-derived growth factor receptor b; PgP, P-glycoprotein; Ph, phenyl; SAR, structure activity relationship; TBAF, tetrabutylammonium fluoride; THF, tetrahydrofuran; TPAP, tetrapropylammonium perruthenate. * Corresponding author. E-mail address: [email protected] (G. Marzaro). https://doi.org/10.1016/j.ejmech.2019.03.064 0223-5234/© 2019 Published by Elsevier Masson SAS.

kinases [9], a family of enzymes that regulate almost all cell events [10]. Great attention has been paid to tyrosine kinases, due to their central role in regulating cell differentiation, tissue development and proliferative signal transduction [11]. Besides, the tyrosine kinase activity is deregulated in most cancer. The development of Imatinib (the first ATP-competitive kinase inhibitor to enter the therapy) has initiated the era of the target therapy [12]. The elucidation of cell cycle regulation has also highlighted the role of a subfamily of serine-threonine kinases, the cyclin-dependent kinases (CDKs) [13,14]. The activity of CDKs is mediated by the interaction with regulator proteins called cyclins. 21 different CDKs are present in the human kinome, that interact with 29 cyclins [15,16]. CDKs regulate either the transition of the cell cycle steps (CDK1,2 and 4), the gene transcription (CDK7,8,9 and 11) or both (CDK6) [15e17]. Inhibition of cell cycle CDKs has been proven an efficient anticancer strategy, in particular in combination with antihormone compounds [18]. Nevertheless, first-generation CDK inhibitors (pan-CDK inhibitors, e.g. Flavopiridol [19] and Roscovitine [20,21]; Fig. 1A) were characterized by a too high toxicity that impaired their full development. Second-generation CDK inhibitors (e.g. Dinaciclib [22]; Fig. 1B) were designed to be more specific for CDK1 and 2 or to be generally more potent inhibitors. Again, the development of second-generation inhibitors was discontinued for toxicity reasons [23]. The third-generation inhibitors (selective CDK4/6 inhibitors; e.g. Palbociclib, Ribociclib and Abemaciclib;

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Fig. 1. Examples of chemical structure and CDKs inhibitory activity of first (A), second (B) and third (C) generation compounds.

Fig. 1C) is the most interesting and promising class of CDK inhibitors identified up to now [23]. The role of CDK4 and 6 in cancer has been extensively reviewed [18,23e26] as well as the pharmacological properties of Palbociclib, Ribociclib and Abemaciclib [27e32]. Hence, herein we will give only a brief overview of the pharmacology of the third-generation CDK inhibitors, while we will more specifically focus our attention on the medicinal chemistry aspects of the compounds. 2. Palbociclib Palbociclib (Ibrance, PD0332991) was the first orally bioavailable, highly selective CDK4/6 inhibitor to enter the therapy [31,33]. The pharmacokinetic properties of Palbociclib were determined in human subjects, both healthy and affected by solid tumours (e.g. advanced breast cancer) [34e36]. After oral administration of a 125 mg dose the absolute bioavailability of Palbociclib is about 46%, the Cmax is reached in 6e12h and the drug appears to penetrate extensively into peripheral tissues as demonstrated by the distribution volume. Binding of Palbociclib to human plasma proteins is 85% and, when administered daily, the steady state is reached after 8 days. Palbociclib is a substrate of CYP3A4 (of which it is also a moderate inhibitor) and SULT2A1 [37]. Palbociclib is mainly catabolized by oxidation and sulfonation [33]. Extensive UPLC-Q-

TOF/MS/MS analysis of in vivo and in vitro metabolism identified 14 different catabolites derived by oxidation, hydroxylation, sulfonation and piperazine ring opening [38]. The drug was approved in 2015 and underwent several clinical trials [18], both as single drug and in combination therapy. Among the trial, the PALOMA1 (enrolling postmenopausal woman with ERþ/HER2-advanced breast cancer) is worth of mention since after this study the FDA granted the accelerated approval to Palbociclib [39]. When tested on a panel of about 40 kinases, Palbociclib resulted active only against CDK4/D1, CDK4/D3 and CDK6/D6. G1 cell cycle block, inhibition of DNA synthesis and of pRb phosphorylation in cell models further strengthened the mechanism of action of Palbociclib. Besides, as expected for a selective CDK4/6 inhibitor, the effect of Palbociclib depends on the cell expression of Rb. Consequently, Palbociclib resulted active against a variety of Rb þ human tumour xenograft in mice [40]. As demonstrated by preclinical studies, Palbociclib is particularly active in haematological malignancies, both alone and in combination with other drugs [41,42]. Recently, a campaign of drug screening conducted on FLT3-ITD mutant AML cells revealed a surprising effect of Palbociclib [43]. In FLT3-ITD AML cells, short hairpin RNAemediated suppression of CDK4 was ineffective, whereas suppression of CDK6 led to effects comparable to that obtained with the treatment with Palbociclib. On these bases, the authors concluded that the effect of the drug on AML

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cells was exclusively due to CDK6 inhibition, fostering the evidences that CDK6 does not just overlap the CDK4 functions. Indeed, it is now recognized that CDK6 is implicated both in cell cycle regulation and gene transcription [43,44]. 2.1. Chemistry Palbociclib is a pyridopyrimidine compound bearing a pyridopyrazine side chain. Usefulness of pyridopyrimidine scaffold for kinase inhibitors was already published [45e47], in particular targeting EGFR, FGFR1, Src and PDGFRb. The binding mode of pyridopyrimidines with the ATP binding pocket was elucidated by the resolution of the crystal structure of the complex PD173955-Abl kinase [48], highlighting the fundamental role of both an exocyclic NH at position 2 and of the nitrogen at position 3 for the interaction with the kinase hinge region (Fig. 2). A key aspect of pyridopyrimidine inhibitors was the nature of the substituent in position 6, since this moiety occupies the selectivity pocket (or hydrophobic back pocket) bordered on the gatekeeper residue. A major difference between tyrosine kinases and CDKs is the steric hindrance of gatekeeper residue, being it bulkier in the latter family than in the former (e.g. a phenylalanine in CDK2,4,6 and a threonine in EGFR and PDGFRb). Hence, compounds not bearing a phenyl at position 6 were effective inhibitors of both receptor tyrosine kinases (e.g. FGFR1) and CDKs (Fig. 3). Early discovery of Palbociclib started with the identification of the nonselective inhibitor 8 [49]. Substitution of the aniline side chain with 2-aminopyridine led to a preferential inhibition of CDK4 over CDK2 and to a substantial reduction of affinity for FGFR1, allowing for the discovery of the first selective CDK4 inhibitor 10 (Fig. 3). The functionalization of the amino sidechain with a piperazine and the insertion of a bromine in position 6 led to compound 12, active in Rbþ MDA-MB43 cells and able to reduce the growth of human tumour xenografts in mice [51]. Investigation of N8 substituents showed the requirement of a cycloalkyl function, whose dimensions were proportional to potencies up to the norbornyl ring. However, considering the selectivity profile (CDK4 vs CDK2 inhibition), the cyclopentyl proved to be the optimal substituent. To improve the physical properties of the lead compound 12, the bromine atom was replaced with an acetyl group, thus reducing the overall lipophilicity of the molecule. Anyway, at this stage it was observed that this substitution was optimal when a methyl function was also present at position 5, suggesting the requirement of a precise orientation for the acetyl function and leading to the final discovery of Palbociclib [51] (see also “Binding modes with CDK6 section”). The SAR for pyridopyrimidine arising from Palbociclib development is depicted in Fig. 4. The original synthesis of Palbociclib started from ethyl 4-chloro2-(methylthio)pyrimidine-5-carboxylate (13; Scheme 1), that was condensed with cyclopentylamine [51e53]. LiAlH4 reduction of carboxylate to aldehyde intermediate and Grignard reaction afforded the acetyl analogue 15, that was in turn condensed with ethyl 2-(diethoxyphosphoryl)acetate to the desired heterocycle 16. Bromination at position 6 and subsequent sulfur oxidation led to

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intermediate 18 suitable for the SNAr with the amine side chain 21, that was in turn prepared starting from 5-bromo-2-nitropyridine 19. In the final step, a Stille coupling followed by acid hydrolysis led to both the insertion of the acetyl function and the removal of the Boc protection, affording Palbociclib. More efficient synthetic strategies were developed to accomplish industrial production requirements. Three major differences were reported: 1 The use of 5-bromo-2,4-dichloropyrimidine (23) as starting material [53,54], laying the basis for a more efficient SNAr and avoiding the oxidation step. This modification led to a complete revision of the entire synthetic process (Scheme 2). Indeed, a Heck reaction followed by intramolecular cyclization was used to synthesize the heterocycle 27. 2. The improvement of the SNAr step (Scheme 3), since the 2aminopyridine sidechain proved to be a poor nucleophile. A first attempt was based on the use of 1M LHMDS in THF to activate the amine function of compound 21 [53]. Reaction success was strictly related to the order of addition of reagents: indeed, it was necessary to first mix the amine 21 with LHMDS in equimolar amount, and then to add the pyridopyrimidine scaffold 27 (half molar ratio with respect to the amine), since both the starting chloro derivative 27 and the final compound 22 were unstable to LHMDS. Further studies showed that the use of a Grignard reagent as base [54] (in particular iPrMgCl or cyPrMgCl) resulted in the optimal condensation conditions. In these conditions, two equivalents of base were still necessary, but only a slight molar excess of amine was required. 3. The use of the Heck condensation in place of the Stille coupling followed by hydrolysis with isethionic acid, furnishing Palbociclib isethionate (Scheme 4). Later on, the use of isethionic acid to accomplish the final hydrolysis was abandoned due to production reasons. Indeed, the management of Palbociclib free base was found easier than those of Palbociclib salts, thus the use of the more readily accessible and economical hydrochloric acid was finally preferred [54]. Heck reaction was deeply investigated, finally finding that the best catalyst for the reaction was Pd(OAc)2/DPEPhos [54]. Several alternative synthetic routes were also tested, comprising different annulation strategies, the inversion of SNAr reaction and final Heck condensation [55e60]. None of these alternative routes led to a substantial improvement of the overall process and, in several cases, the desired product was not even obtained. Thus, these alternative ways were not investigated further. With the goal of enhancing pharmacokinetic properties, as previously reported for other kinase inhibitors [61], in 2014 Concert Pharmaceuticals patented deuterated analogues of Palbociclib where 2H atom replaced 1H atoms of either cyclopentyl or piperazine moieties. Although claiming a superior metabolic stability, however, the patent lacks experimental data. Compounds were synthesized according to Scheme 1. Palbociclib analogues modified at piperazine moiety (either acyl or alkyl derivatives) have also been reported [62]. Compounds were synthesized directly starting from Palbociclib, but no analogue proved to be superior than the parent compound. 3. Ribociclib

Fig. 2. A) Chemical structure and depiction of interaction with ATP-pocket of PD173955; B) numbering of pyridopyrimidine scaffold.

Ribociclib (Kisqali, LEE011) is another orally bioavailable selective CDK4/6 inhibitor able to suppress the Rb phosphorylation, thus causing G1 arrest. After oral administration, the Cmax is reached in

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Fig. 3. Early development of selective CDK4/6 inhibitors [49,50].

Fig. 4. SAR for Palbociclib in context with the main features of ATP binding site.

Scheme 1. Early synthetic strategy to Palbociclib. Reaction conditions: (a) cyclopentylamine, Et3N, THF, yield 76% [52]; (b) 1. LiAlH4, THF; 2. MnO2, CH2Cl2; 3. CH3MgBr, THF; 4. TPAP, NMO, yield 53% over four steps [52]; (c) (EtO)2P(O)CH2COOEt, NaH, THF, yield 66% [52]; (d) NBS, DMF, yield 66% [52]; (e) Davis oxaziridine, CH2Cl2, yield 94% [52]; (f) 1. piperazine, Bu4NI, K2CO3, DMSO; 2. Boc2O, NaHCO3, THF, H2O, yield 62% over two steps [51] OR Boc-piperazine, Et3N, DMSO, yield 79% [53]; (g) H2 (50 psi), Ni-Raney, MeOH, yield 83% [51] OR 20% Pd(OH)2/C, H2, iPrOH, yield 84% [53]; (h) toluene, yield 38% [51]; (i) 1. tributyl(1-ethoxyvinyl)tin, Pd(PPh3)4, toluene; 2. HCl(g), CH2Cl2, yield 92% [51].

1e4 h and, when administered daily, the steady state is reached after 8 days. Binding of Ribociclib to human plasma proteins is 70% and shows about half the distribution volume of Palbociclib. Ribociclib is catabolized by CPY3A4 (of which it is a weak inhibitor), the main catabolites being N-hydroxy and N-demethyl derivatives [63]. At clinically relevant concentration Ribociclib has a low effect on PgP. The drug was approved in 2017 and is indicated for the

combination treatment of pre/perimenopausal or postmenopausal women with HRþ/HER2-metastatic breast cancer. The ability of the compound in combination with Alpelisib (a PI3K inhibitor), Fluvestant and Letrozole to inhibit the growth of tumour xenografts in mice was assessed [64,65]. Ribociclib entered several clinical trials, both alone and in combination therapies [18,64]. Phase I studies also suggested a potential use for the treatment of lymphoma [66].

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Scheme 2. Improved synthesis of pyridopyrimidine scaffold of Palbociclib. Reaction conditions: (a) cyclopentylamine, EtOH, yield not reported [53]; (b) crotonic acid, PdCl2 [PhCN]2, o-Tolyl3P, DIPEA, THF, intermediate not isolated [53] OR crotonic acid, Pd(OAc)2, Et3N, NMP, intermediate not isolated [54]; (c) Ac2O, yield 68% over steps b-c [53] OR 81% over steps b-c; (d) Br2, KOAc, AcOH, yield 61% [53] OR NBS, (COOH)2, MeCN, yield 88% [54].

Scheme 3. SNAr step in the synthesis of Palbociclib. Reaction conditions: (a) LHMDS, THF, toluene, yield 92% [53] OR iPrMgCl, THF 84% [54].

3.1. Chemistry Ribociclib is a pirrolopyrimidine compound bearing the same pyridopyrazine side chain as Palbociclib. The usefulness of pyrrolopyrimidine for CDK4/6 inhibition was patented by Novartis in 2007 (see for example compound 30 in Fig. 5) [67]. Claimed compounds showed also JAK3 inhibitory potency and a low selectivity profile against CDK members. In 2010 the same company patented selective CDK4/6 inhibitors, comprising Ribociclib [68]. Although being less potent than the originally claimed compound 30, Ribociclib showed a higher selectivity toward CDK4 (Fig. 5) as also demonstrated by comparing the effects on cell cycle exerted by the two compounds [68]. As previously seen in the development of Palbociclib [51], the replacement of the aniline moiety with a 2-aminopyridine had a pivotal role to improve selectivity profile. Another Novartis’ patent in 2011 reinforced the claims on Ribociclib [69]. The two patents reported also two different synthetic strategies to obtain the key chloropyrimidine intermediate 35 (Scheme 5). The pyrrolopyrimidine system was obtained in both cases through Sonogashira coupling followed by TBAF mediated cyclization. The latter synthetic strategy proved to be more convenient due to the lower number of synthetic steps. In particular, the conversion of hydroxymethyl function to dimetylformamide function was accomplished in a single step, by treating compound 37 with dimethylamine and sodium cyanide, followed by MnO2 mediated

Fig. 5. Chemical structures and CDK inhibitory activities for compound 30 and Ribociclib.

oxidation. In any case, the key chlopyrimdine intermediate 35 was condensed by means of Buchwald reaction with the same amino sidechain as Palbociclib. A final deprotection step was required to obtain Ribociclib (Scheme 6). As for Palbociclib, deuterated analogues of Ribociclib were also patented [70]. Deuterium atoms were introduced in the moiety subjected to in vivo metabolism, i.e. the dimethylamide function. d6-Ribociclib was prepared according to the synthetic scheme reported in WO2010020675 [68] using dimethyl-d6-amino hydrochloride. Comparison of main pharmacokinetic properties are listed in Table 1. Although not directly referring to Ribociclib, the authors of the present review would like to briefly mention an additional patent filed by G-Zero therapeutics claiming tricyclic analogues of Ribociclib, selective for CDK4 over CDK2, whose general structure is reported in Fig. 6 [71]. Finally, merging the chemical structure of Ribociclib with that of Cabozantinib (VEGFR2 inhibitor) [72] or with that of Vorinostat (HDAC inhibitor) [73] led to multi-targeting compounds. Remarkably, in both cases, the developed compounds resulted active also against CDK9. Both papers focused the attention on compounds bearing an aniline side chain rather than a 2-aminopyridine one (Fig. 7).

Scheme 4. Synthetic strategies for acetyl function insertion. Reaction conditions: (a) tributyl (1-ethoxyvinyl)tin, Pd(PPh3)4, toluene, yield 78% [51]; (b) HCl(g), CH2Cl2, yield 92% [51]; (c) butyl vinyl ether, Pd (dppf)2Cl2, DIPEA, n-butanol, yield 83% [53]; (d) isethionic acid, MeOH, quantitative yield [53].

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Scheme 5. Synthetic strategies to chloropyrimidine intermediate 35 (highlighted with dashed box) reported in WO2010020675 [68] (left) and in WO2011101409 [69] (right). Reaction conditions: (a) cyclopentylamine, DIPEA, EtOH, yield 89% [68]; (b) 3,3-diethoxypropine, PdCl2(PPh3)2, CuI, Et3N, DMF, yield 43% [68]; (c) TBAF 1M THF, yield 82% [68]; (d) HCl, dioxane, yield 82% [68]; (e) oxone, DMF, yield 85% [68]; (f) Et2NH 2M EtOH, HBTU, DIPEA, DMF, yield 79% [68]; (g) propargyl alcohol, TBAF 1M THF, PdCl2(PPh3)2, yield not reported [69]; (h) TBAF 1M THF, yield not reported [69]; (i) 1. Et2NH 2M THF, NaCN, DMF; 2. MnO2, yield not reported [69].

Scheme 6. Synthesis of Ribociclib. Reaction conditions: (a) Pd2 (dba)3, BINAP, NaOtBu, dioxane, MW [68]; (b) HCl 4M dioxane, yield 36% over two steps [68].

4. Abemaciclib Abemaciclib (Verzenio, LY2835219) is the last approved orally bioavailable CDK4/6 inhibitor. As anticipated by its ability to inhibit CDKs, Abemaciclib suppresses Rb phosphorylation, thus causing G1 arrest. Among the compounds herein described, Abemaciclib is the least selective inhibitor, being able to inhibit also DYRK, PIM, HIPK and CAMK kinase family (Ki < 10 nM) [74]. Considering a slightly higher cut-off (Ki < 50 nM), Abemaciclib resulted active against 29 human protein kinases [74,75]. After oral administration, the Cmax is reached in 4e24 h and, when administered daily, the steady state is reached after 5 days. Binding of Abemaciclib to human plasma proteins is about 96%. Abemaciclib is catabolized by CPY3A4, the main catabolite being N-desethyl derivative. Additional catabolites are hydroxyabemaciclib and hydroxy-N-desethylabemaciclib [76]. At clinically relevant concentration Abemaciclib inhibits PgP, of which it is also a substrate. The drug was approved in 2017 and is indicated for the treatment of HRþ/HER2-advanced or metastatic breast cancer in combination with Fluvestrant [76]. In xenograft

Fig. 6. General structure of tricyclic analogues of Ribociclib claimed in WO2012061156 [71].

tumours, Abemaciclib demonstrated remarkable activity against a number of tumours, comprising MCL, colorectal, lung, glioblastoma and AML [75]. Abemaciclib showed additive antitumor activity with Gemcitabine [75]. More recently, a promising activity against esophageal adenocarcinoma was also reported [77]. A review collecting data from clinical trials involving Abemaciclib for the treatment of breast cancer has been reported elsewhere [28]. Abemaciclib is a phenylpyrimidine compound structurally related to Palbociclib and Ribociclib. As mentioned before, Abemaciclib shows a broader spectrum of inhibition. Indeed, the original patent from Eli Lilly claimed CDK4/6 inhibitors endowed with additional anti PIM-1 kinase potency [78]. 4.1. Chemistry The heterocycle scaffold 43 of Abemaciclib was synthesized by means of condensation of the suitable acetamide 40 to 4-bromo2,6-difluoroaniline followed by potassium tert-butoxide mediated intramolecular cyclization (Scheme 7); bromine atom of 43 was displaced by pinacol borane and the boronate ester 44 was

Table 1 Pharmacokinetic properties for Ribociclib and its deuterated analogue claimed in WO2011101417 [70]. IV, 1 mg/kg (rats)

PO, 5 mg/kg (rats)

AUC0-7h (nM*h)

CL (ml/min/Kg)

Vdss (L/Kg)

T1/2 (h)

AUC0-7h (nM*h)

CL (ml/min/Kg)

T max (h)

F (%)

1028

34

5.4

2.3

1618

348

1.5

31

883

37

8.6

3.2

5432

549

2

55

Ribociclib

d6-Ribociclib

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Fig. 7. General structures of multi-target compounds derived from Ribociclib and VEGFR2 (top) or HDAC (bottom) inhibitors.

condensed with 2,4-dichloro-5-fluoropyrimidine 45 by means of Suzuki coupling to get 46. The amino sidechain 50 was in turn synthesized by means of reductive amination of 2-bromo-5formylpiridine 48 and ethylpiperazine. Replacement of bromine atom of 49 with amine function to get 50 and then Buchwald condensation of 50 with 46 yield Abemaciclib. In 2015 [79], Eli-Lilly reported the same synthetic strategy as in US20100160340 [78], with the only exception of the bromopyridine 49 amination: in the latter patent the reaction was conducted with liquid ammonia and copper oxide as catalyst in place of LiHMDS. In the same year, the same company published a slightly different synthetic strategy in which the last synthetic step was the reductive amination of 51 with ethylpiperazine (Scheme 8) [80], with a special focus on the optimization of the Leuckart-Wallach reductive amination step. In particular, the authors found that addition of two equivalents of trimethyl orthoformate led to reaction completion, due to the ability of the reactant to wrap the water produced during the reaction. The synthesis of starting aldehyde 51 was not reported in the paper.

In 2017, Rathiopharm focused the attention on the preparation of suitable crystalline forms of Abemaciclib [81]. Indeed, the patent disclosed only the last synthetic step (derived from US20100160340 [78]) and the recrystallization of the desired compound from acetonitrile. As for the other third-generation inhibitors, the deuterated version of Abemaciclib was also patented [82]. Deuterium atoms were introduced in different positions of the drug, leading to compounds with enhanced metabolic stability (data are summarized in Table 2). Compounds deuteration led to 11e45% improvement of metabolic stability as demonstrated by the higher halftimes. 5. Binding modes with CDK6 The crystallographic complexes between CDK6 and the herein reviewed compounds were recently resolved by Chen et al. (Fig. 8) [74]. Based on the high sequence homology between the ATP site of CDK4 and CDK6 it is also reasonable that the binding modes described with CDK6 would reflect those with CDK4.

Scheme 7. Synthesis of Abemaciclib. Reaction conditions: (a) Ac2O, Et3N, CH2Cl2, yield 91% [78]; (b) POCl3, Et3N, toluene, quantitative yield [78]; (c) KOtBu, N-methylformamide, quantitative yield [78]; (d) bis(pinacolato)diboron, tricyclohexylphosphine, Pd(OAc)2 KOAc, DMSO, yield 73% [78]; (e) PdCl2(PPh3)2, Na2CO3 2M H2O, 1,2-dimethoxyethane, yield 65% [78]; (f) NaBH(OAc)3, CH2Cl2, yield 99% [78]; (g) LiHMDS, dicyclohexylphosphinobiphenyl, Pd2 (dba)3, THF, yield 56% [78] OR NH3(l), CuO, MeOH, yield 54% [79]; (h) Pd2 (dba)3, Xantophos, Cs2CO3, dioxane, yield 89% [78].

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Scheme 8. Alternative route to Abemaciclib. Reaction conditions [80]: (a) PdCl2, K2CO3, 2-methyl-2-butanol, yield 66%; (b) ethylpiperazine, trimethyl orthoformate, HCOOH, MeCN, yield 74%.

Table 2 Pharmacokinetic properties for Abemaciclib and its deuterated analogues claimed in WO201808120 [82]. Percent remaining (microsome treatment)

T1/2 (min)

CL (ml/min/Kg)

4.76 45.61

10.0 39.48

172.13 62.91

16.02 66.88

6.21 51.27

11.11 47.07

156.43 52.77

43.32 83

16.39 65.86

6.48 51.29

11.24 47.12

154.70 52.71

47.9 85.96

21.31 69.86

9.86 57.58

13.33 57.35

130.40 43.31

0

5

15

30

45

human rat

100.00 100.00

76.07 94.14

38.9 81.03

12.99 61.65

human rat

100.00 100.00

77.73 93.79

42.42 83.44

human rat

100.00 100.00

79.44 92.72

human rat

100.00 100.00

80.33 92.19

Abemaciclib

d5-Abemaciclibr

d8-Abemaciclib

d13-Abemaciclib

The three molecules share common features: they bind the inactive kinase conformation, establish a H-bond between 3Npyridine and the His100NH, and a H-bond between the Val101CO and the exocyclic NH of the side chain. In addition, Palbociclib and Ribociclib establish a H-bond between their carbonyl function and the DFG motif. As anticipated by the SAR on Palbociclib [51], the steric hindrance of the methyl function in position 5 forces the acetyl function to the most adequate orientation to interact with the enzyme. The resolution of crystallographic complexes accounted for the selectivity of compounds within the CDK family. The aminopyridine side chains face the Thr107 residue in CDK6; in CDK9 the residue is replaced by a glycine, and the compounds retain a certain activity. Conversely, in CDK1,2,3 and 5 the residue is replaced by a lysine. The resulting electrostatic repulsion with the nitrogen-containing side chain was proposed by Chen et al. as a

plausible reason for the inability to inhibit the enzymes. It was also postulated that His100 would play a key role in selectivity, since only CDK4 and 6 bear this amino acid in the hinge region [83]. The steric features of the heterocycle core directed towards the hydrophobic back pocket constitutes a major difference between the three compounds: Ribociclib and Palbociclib are characterized by larger substituents (dimethylamide and acetyl functions) than Abemaciclib (fluorine atom). Consequently, Abemaciclib is more readily buried in the ATP pocket; besides, the steric features of Ribociclib and Palbociclib account for their higher selectivity. Superimposition of crystallographic structures showed that, despite the very high structural similarity, the amino side chains occupy rather different positions whereas the pyrimidine cores overlap well (Fig. 9). Interaction of Abemaciclib with His100 is mediated by a water

Fig. 8. Binding modes of Palbociclib (A; PDB ID: 5L2I), Ribociclib (B; PDB ID: 5L2T) and Abemaciclib (C; 5L2S) with CDK6. Ligands, gatekeeper residue, DFG domain and hinge residues are depicted as stick. Hydrogen bonds are depicted as black dashed lines.

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Conflicts of interest The authors declare no conflict of interest. Acknowledgment This work has been carried out with the financial support of the University of Padova “PRID C92F1600250000S” to G.M. The authors gratefully acknowledge Dr Barbara Bertin for English language editing. References

Fig. 9. Superimposition of the crystallographic poses of third-generation CDK4/6 inhibitors in context with the main features of ATP pocket.

molecule. While this interaction was not revealed in the electron density maps for Palbociclib and Ribociclib, the authors postulated that this could be due just to resolution limit [74]. Another difference of interest is the lipophilicity of compounds, that was demonstrated to correlate with potency rather than with selectivity [84,85]: while Abemaciclib shows a higher cLogP (5.5), those of Ribociclib and Palbociclib resulted quite similar (cLogP ¼ 2.3 and 2.7 respectively). 6. Conclusion The development of Palbociclib and related compounds has highlighted the usefulness of CDK inhibitors for the treatment of breast cancer. In particular, the association of third-generation CDK inhibitors with other antineoplastic drugs seems to be a very promising pharmacological approach. Elucidation of binding modes by means of crystallographic resolution lays the basis for the development of more potent compounds. Differences in binding modes between high selective inhibitors (Palbociclib and Ribociclib) and a less selective one (Abemaciclib) may be useful for the design of “selectively nonselective” inhibitors [86], that are expected to be less prone to drug resistance onset. For Palbociclib (the first developed compound) a clear SAR can be derived from literature data. On the contrary, to the best of our knowledge, for Ribociclib and Abemaciclib only patented data are available that describe structurally similar compounds. It seems reasonable that pharmacodynamic and pharmacokinetic properties of such compounds could be improved taking also advantage from X-Ray data. From a synthetic point of view, Palladium-catalyzed reactions (namely Suzuki, Stille, Sonogashira and Buchwald reactions) play a key role in the preparation of third-generation CDK inhibitors. A lot of work has been done to improve reaction yields, identifying the most suitable conditions in terms of ligands, solvents, bases and temperatures. Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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