Accepted Manuscript Cyclin Dependent Kinase (CDK) inhibitors as anticancer drugs Concepción Sánchez-Martínez, Lawrence M. Gelbert, María José Lallena, Alfonso de Dios PII: DOI: Reference:

S0960-894X(15)00584-3 http://dx.doi.org/10.1016/j.bmcl.2015.05.100 BMCL 22791

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

27 February 2015 21 May 2015 30 May 2015

Please cite this article as: Sánchez-Martínez, C., Gelbert, L.M., Lallena, M.J., Dios, A.d., Cyclin Dependent Kinase (CDK) inhibitors as anticancer drugs, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/ 10.1016/j.bmcl.2015.05.100

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Cyclin Dependent Kinase (CDK) inhibitors as anticancer drugs Concepción Sánchez-Martíneza, Lawrence M. Gelbertb, María José Lallenaa and Alfonso de c Dios a

Discovery Chemistry Research and Technologies, Eli Lilly and Company, Alcobendas (Madrid), 28108, Spain; bHerman B Wells Center for Pediatric Research, Section of Pediatric Hematology, Oncology, Indiana University, Indianapolis, IN 46202; cDiscovery Chemistry Research and Technologies, Eli Lilly and Company, Indianapolis, IN 46285. Key words: CDK inhibitors, cell cycle.

Corresponding Author: Concepción Sánchez-Martínez, Discovery Chemistry Research and Technologies, Eli Lilly and Company, Alcobendas (Madrid), Spain; Tel: (34) 91-6633425; Fax: (34) 91-6633411; Email: [email protected]

Notes about the manuscript: Abstract has 89 words; Discussion has 6076 words; 16 figures; 2 tables; 78 references

ABSTRACT Sustained proliferative capacity is a hallmark of cancer. In mammalian cells proliferation is controlled by the cell cycle, where cyclin-dependent kinases (CDKs) regulate critical checkpoints. CDK4 and CDK6 are considered highly validated anticancer drug targets due to their essential role regulating cell cycle progression at the G1 restriction point. This review provides an overview of recent advances on cyclin dependent kinase inhibitors in general with special emphasis on CDK4 and CDK6 inhibitors and compounds under clinical evaluation. Chemical structures, structure activity relationships, and relevant preclinical properties will be described. ------------------------------------------------------------------------------------------------------------------------------------------How living cells grow and divide has been a focus of biomedical research since the theory of spontaneous generation was disproved in the late seventeenth century. Interest only increased as it became clear that sustained cellular proliferation was central to the initiation and progression of cancer, and today sustained proliferative capacity is considered a hallmark of cancer.1 The cell cycle has been extensively studied and has been at the forefront of biomedical research (Figure 1). The underlying regulatory pathways controlling the mammalian cell cycle were initially identified through genetic and biochemical studies in model organisms.2-4. The work of Hartwell, Nurse and Hunt defined how cells regulate proliferation and the central role of cyclin dependent kinases (CDKs), for which they were awarded in the 2001 Nobel Prize in physiology and medicine.5 The cell cycle has four functional phases: S phase where DNA replication occurs; M phase (mitosis) where DNA and cellular components are divided to form two daughter cells; G2 phase, between S and M, where cells prepare for mitosis; G1 phase after mitosis and before S phase, where cells commit and prepare for another round of DNA and cellular replication (Figure 2). The human genome encodes 21 CDKs, although only seven (CDK1-4, 6, 10, 11) have been shown to have a direct role in the cell cycle progression. Other CDKs play an indirect role via activation of other CDKs (CDK3), regulation of transcription (CDK7-9) or neuronal function (CDK5). Different families of cyclins, the regulatory subunits required for CDK activity, have been identified and their expression fluctuates significantly throughout the phases of the cell cycle. Cyclin D/CDK complexes are functionally active in G1 and phosphorylate the retinoblastoma protein (pRb) allowing progression into S phase. Cyclins E and A accumulate at G1/S phase boundary where they activate CDK2 and CDK1 successively, promoting progression through to the G2 phase. At this point, B-type cyclins, especially cyclinB1 and CDK1 drives cells to mitosis.6 In mammalian cells the G1 restriction point (R) denotes where proliferation becomes independent of mitogens and growth factors.7 The normal function of the restriction point is essential for maintaining control of cellular proliferation,8 and is controlled by the retinoblastoma pathway (CDK4 and CDK6-cyclin D1-Rb-p16/ink4a). Retinoblastoma (Rb) is a tumor suppressor that inhibits proliferation through binding to the E2F family of transcription factors, thereby suppressing their activity.9 In early G1, when conditions are favorable for proliferation, D-type cyclin levels increase through transcriptional and posttranscriptional mechanisms.10 Increased cyclin D drives the formation of active kinase heterodimers with CDK4 and CDK6 catalytic subunits. Active CDK4 and CDK6 then phosphorylates Rb, partially relieving suppression of E2F to allow expression of genes required for passage through the restriction point.11 This includes expression of cyclin E, which activates another kinase (CDK2) leading to hyperphosphorylation of Rb, fully releasing the suppression of E2F, allowing cells to exit the G1 phase and initiate DNA replication. Additional restriction point control occurs through the action of the endogenous CDK inhibitors p16/ink4a and p21cip1. P16/ink4a blocks the binding of D-type cyclins to CDK4 and CDK6 while p21/cip1 both

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stabilizes CDK4 and CDK6 cyclin D complexes and is sequestered thus preventing the inhibition of CDK2/cyclin E.8 Phosphorylation of Rb by CDK4 and CDK6 also leads to transcription of genes involved in cell cycle-independent activities including signal transduction, DNA repair transcriptional control, and mRNA processing.12 The role of the Rb pathway in tumor cell initiation and progression is well established. In 1971 Knudson first proposed the two-hit hypothesis for recessive tumor suppressors through the analysis of familial and sporadic retinoblastoma tumors.13 The two-hit hypothesis was confirmed by loss of heterozygosity (LOH) studies by Cavenee and White,14 subsequently leading to the cloning of the Rb gene.. Eventually other components of the Rb pathway were shown to be associated with numerous cancers including cyclinD1,15 CDK4, and CDK6. It has been estimated that the Rb pathway is deregulated in greater than 80% of human tumors, and has been recently reviewed by Malumbres and Barbacid.8, 16 CDK inhibitors have therapeutic potential for several diseases including cancer, diabetes, renal, neurodegenerative and infectious diseases. However, the focus has been on their development as anticancer drugs, with emphasis on the cell cycle and transcriptional CDKs. Academic and industry drug discovery programs have generated potent smallmolecule CDK inhibitors since the early 1990s, and CDK4 and CDK6 in particular are considered highly validated anticancer drug targets. CDK4 and CDK6 and the associated Rb pathway are deregulated in a majority of human tumors which provides multiple therapeutic opportunities. Knowledge of the genetic alterations in the Rb pathway allows for a tailored therapeutic approach, such as the use of biomarkers for assessment of pharmacodynamic response in the clinic and identification of patients most likely to respond.17 Compound screening, X-ray analysis of apo-CDK2-ligand complexes and virtual screening technology were the initial tools used for the identification of firstgeneration CDK inhibitors. These CDK inhibitors lacked selectivity within the CDK family, some inhibited CDK4 and CDK6, but also inhibited numerous other kinases. These off-target kinase interactions and the non-selective inhibition of CDKs have detrimental effects on normal cells and likely explain the appearance of numerous side effects seen in clinical trials.18, 19 In the last ten years the use of multiplexed biomarkers and phenotypic assays (e.g. cell cycle arrest concomitant to target engagement) has led to the identification of more specific CDK inhibitors, particularly for CDK4, CDK6 and CDK7/9.20 However, reported CDK specificities are dependent on assay conditions and can be difficult to compare from research group to research group. Some groups have claimed selectivity based on enzymatic ratios while others have used cell-based assays with multiplexed biochemical and phenotypic readouts to achieve cell-based functional selectivity in the CDK family. A variety of chemical classes, typically planar hetero-aromatic structures, which include flavonoid, purine, indenopyrazole, arylcarbazole, indolinone, oxindole, pyrimidine, thiazole, indirubin, hymenialdisine or paullone derivatives, among others, have been described as small-molecule ATP-competitive (type I) CDK inhibitors. Most showed activity as CDK1/2 inhibitors presumably because the structure-based design was developed predominantly through studies on monomeric CDK2. The first inhibitors resulted promiscuous across the CDK family and other kinases. Research has also focused on inhibitors that do not compete with ATP. This mechanisms, represented primarily by pharmacologically active peptides, has been summarized by Orzaez, Cirillo and Abate,21, 22 Also, few small molecules have been described.23 This manuscript reviews the latest advances on CDK inhibitors design since 2011 (ATP and ATP noncompetitive), with an overview of the chemical structures, relevant structure-activity relationships (SAR) and development status with emphasis on CDK4 and CDK6 inhibitors and other CDK inhibitors under clinical evaluation. Over the last few years, most of the reported molecules are being described as CDK4 and CDK6 or CDK7/9 inhibitors. For additional information the reader is directed to review articles for comprehensive coverage of the CDK arena and references therein.15, 24-32 CDKs are unique in the protein kinase family because their activity depends on the association with their partner cyclins, and this could be a potential advantage for inhibitor design. The recognition of CDK-cyclin complexes by different interacting proteins (tumor suppressors, transcription factors) occurs at least in part through protein-protein interaction (PPI). Blocking this recruitment site prevents recognition and subsequent phosphorylation of CDK substrates. This recruitment binding-site has been identified in A, D, and E cyclins (partners of CDK1, 2, 4 and 6). Cyclin-CDK binding is relatively tight and, to date, no small-molecule has been reported as an effective inhibitor to disrupt this PPI interface yet. Peptidomimetic molecules have been designed to mimic endogenous CDK inhibitors (p16, p21, p27) or endogenous substrates (EF2, p53, pRb, p130, p107) to interfere with the interface between the CDK and cyclin partner or to interrupt conformational changes required for activation of the CDK-cyclin complexes.23 Despite the high specificity observed, the use of these peptides has been limited by the poor pharmacokinetic properties of these large, often charged molecules. Achieving effective doses of therapeutic peptides at the tumor site is often prevented by their rapid degradation and poor tumor cell penetration properties. However, Wang et al recently described the antitumor effects in vitro and in vivo for new key peptide motifs derived from cyclin D1, cyclin D3 and CDK4 conjugated to a membrane-permeable peptide such as PTD4 (protein transduction domain 4 derived from HIVTat) (Table 1).33 These chimeric peptides were designed to specifically inhibit the activity of cyclin D/CDK4 complexes by targeting the protein-protein interface. In in vitro assays they induced cell cycle arrest and apoptosis of cancer cells. In in vivo experiments they showed antitumor effect with fewer side effects. Also, Metamax has recently

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reported MM-D37K a new chimeric peptide that act as a surrogate of the endogenous p16/ink4a inhibitor (Table 1, 2).34 MM-D37K consists of p16/ink4a derived short sequence and cell penetrating peptide (CPP)-Antp (Penetratin). In vitro Rb phosphorylation was inhibited and cell cycle was blocked at G1 phase which suggests that MM-D37K mechanism of action is inhibition of cyclin D/CDK4 and CDK6 complex activation. MM-D37K induced apoptosis in tumor cell lines of different histologies. In vivo it showed antitumor effect in lung and colon xenograft models using three times a week dosing regimen (5 and 10 mg/kg). Additive effects of MM-D37K for 5-fluorouracil (5-FU) and etoposide and amplification for taxol cytotoxic activity were observed.35 This is the first chimeric peptide entering in clinical development. Interestingly in this PPI context, the REPLACE (Replacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been developed.36 This strategy was designed for the iterative conversion of peptidic blockers of PPI into pharmacologically relevant compounds and could accelerate the generation of new leads for the development of novel non-ATP competitive CDK inhibitors. McInnes et al. applied the REPLACE-mediated conversion of the known octapeptide cyclin groove inhibitor (HAKRRLIF) into N- and C-terminal di-capped or Nterminal mono-capped peptides to develop non-ATP competitive CDK2/cyclin A inhibitors.37, 38 Some recent examples are exemplified in Figure 3A. Most recently Schönbrunn reported a new approach to identify small-molecule ligands of CDK2 with a potential allosteric mode of action (type II and type III) using the fluorophore 8-anilino-1-naphthalene sulfonate (ANS)39 that binds to a large allosteric pocket adjacent to the ATP site.40 To distinguish pure allosteric from ATP-site-directed ligands, a follow-up assay was employed in which a potent type I inhibitor such as staurosporine, with weak ANSdisplacement potential, served to block the ATP site. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC50 values) ranging from 6 to 44 µM, but all of them were found to be ATP-site-directed ligands. Some new CDK2 type I inhibitor scaffolds were confirmed by X-ray crystallography (Figure 3B). Using the same methodology, Rastelli described in 2014 the first examples of truly Type III allosteric ligands of CDK2.41 Through virtual screening of commercially available compounds in the allosteric pocket of the CDK2-ANS binary complex, and using a combination of docking (AutoDock) and post docking, the authors identified new core inhibitors. Competition experiments performed in the presence of staurosporine confirmed the allosteric mode of action. The most potent compound inhibited CDK2 mediated retinoblastoma phosphorylation, confirming its mechanism of action is fully compatible with an inhibition of CDK2 in cells (Figure 3B). These compounds inhibit the growth of breast cancer cells in the micromolar range. In 2014, Gray´s group used a phenotypic screen to identify covalent inhibitors for any kinase that could suppress proliferation, regardless of the target, and identified THZ1, a CDK7 inhibitor (Figure 4, Table 1).42 THZ1, contains an acrylamide moiety, that binds covalently to a cysteine residue far outside the canonical kinase domain leading to the first covalent and irreversible CDK inhibitor, and suggesting a novel approach for designing small molecules to target the CDK family. THZ1 showed potent antiproliferative activity on T-ALL (T cell acute lymphoblastic leukemia) cell lines and other hematologic cancers, where oncogenic transcription factors play a prominent role in pathophysiology. THZ1 exhibited efficacy in a bioluminescent xenograft model using the human KOPTK1 cell-line when dosed twice daily at 10 mg/kg. Importantly, THZ1 was well tolerated with no observable body weight loss or behavioral changes, suggesting an acceptable toxicology profile for this molecule. Syros Pharmaceuticals Inc. licensed this inhibitor and incorporated it into its preclinical CDK7 inhibitor program. This body of work indicates that the identification of non-classical CDK inhibitors (ATP non-competitive, allosteric and covalent) has potential to achieve specificity among the members of the CDK family. Currently the first ATP-non-competitive inhibitor, MM-D37K, has entered clinical trials to evaluate safety, tolerability and pharmacokinetics. A number of ATP-competitive pan-CDK inhibitors are currently undergoing preclinical studies or have been advanced to clinical development for cancer treatment since the 1990’s. In February 2015, the US Food and Drug Administration granted approval accelerated to palbociclib (Ibrance®, formerly termed PD-0332991, Pfizer). Table 2 and Figures 5 and 6 contain the CDKs inhibitors advanced to clinical evaluation.43 Data was generated with results from Citeline (Trialtrove, 2014 Citeline), Cortellis (www.cortellis.thomsonreuterslifesciences.com) and Clinical trials (www.clinicaltrials.gov) and it is current as of November 2014. To our knowledge there are 11 inhibitors under clinical evaluation. Most CDK inhibitors in clinical development target several CDKs, with CDK9 being a frequent component of the target profile. Potent inhibition of transcription through CDK9 inhibition could potentially result in toxic effects in non-tumor cell, and that could significantly limit their therapeutical application.19. Despite the promising preclinical results, the first-generation inhibitors (flavopiridol,44 R-roscovitine,45 SNS-032,46 PHA79388747) and second generation inhibitors, more specifically active against the CDK family, with or without CDK4 AND CDK6 inhibitory activity but with reduced off-target activities (RGB-286638,48 R547,49 AZD5438,50 ZK304709,51 AG-024322,52 PD-0183812,53 P1446A-0554) were discontinued during phase I or phase II trials.43 These clinical discontinuations can be tentatively attributed to unfavorable pharmacological properties and presumably low specificity for a certain cell cycle phase-selective profile, resulting in generalized cytotoxicity with concomitant undesirable adverse effects. The withdrawal of P276-00 from clinical development in 2014 was attributed by the

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sponsor company to a commercial decision and not to lack of clinical activity.55 SCH-72796556 advanced to phase III in 2012 but it was also discontinued after it was granted an orphan drug status for the treatment of chronic lymphocytic leukemia (CLL).57 From this group of agents, only flavopiridol, which was granted orphan drug status as well (CLL),58 and (R)-roscovitine are still undergoing active clinical evaluations. PHA-848125,59 BAY-1000394,60 AT7519,61 and TG02,62 a set of non-specific CDK inhibitors, are in phase I or phase II. Even with the high level of interest in CDK inhibitors since the discovery of flavopiridol (summarized by Xu),29 very few specific CDK4 and CDK6 inhibitors were reported from 1990-2009. The following section summarizes the progress on the identification of highly specific, potent and ATP-competitive CDK4 and CDK6 inhibitors, which have been successful in the clinic. A few examples on preclinical work are also described herein. The crystal structure of CDK4 was not solved until 2009,63 coinciding with the identification of selective CDK4 and CDK6 inhibitors. Additional factors such as advances in structure-based drug design technologies and, the development of multiplexed cell based assays allowing for simultaneous target engagement and cell cycle analysis have played an important role in the identification of these selective CDK4 and CDK6 inhibitors.20 Recently, PD-0332991(palbociclib),64 LY2835219 (abemaciclib),65 and LEE-011 (ribociclib)66 advanced to phase III trials in breast and lung cancer. The three of them have CDK4 and CDK6 as primary kinase targets and show specific Rb phosphorylation inhibition leading to G1 cell cycle arrest in many different tumor types and contexts. The optimization of the pyrido[2,3-d]pyrimidine scaffold by scientists at Parke-Davis at positions C-2, C-6, C-7 and N-8 is particularly interesting and is summarized in Figure 7. The identification of 1a as an inhibitor of CDK4 led to the initiation of a program to evaluate this core for inhibition of CDKs. Modification to include a 2-amino-pyridine side chain at C2-position and a methyl at C-5 provided inhibitors with very high in vitro selectivity for CDK4 versus other cell cycle kinases in particular and protein kinases in general (1b-d). This work led to PD-0332991 (palbociclib), a potent inhibitor of cyclin D1/CDK4 and cyclin D2/CDK6 with IC50 values of 11 nM and 15 nM respectively in biochemical assays. In vitro, palbociclib prevented Rb phosphorylation by CK4/6 inhibition and induced a G1 cell-cycle arrest in Rb-proficient cell lines. It showed antiproliferative effects in multiple tumor cell lines with amplified CDK4 such as liposarcoma as well as in mantle cell lymphoma (MCL), myeloma, breast, ovarian and colon cancers. Palbociclib is an orally bioavailable drug that showed good pharmacokinetic properties in rats.64 With a desirable selectivity profile and pharmacokinetic behavior, it was identified as a drug candidate in 2004 for the treatment of cancer and is under evaluation in different tumor types, including mantle cell lymphoma (MCL), melanoma and sarcoma. On February 2015 and on basis of positive phase II data, the FDA granted an accelerated approval in the USA for palbociclib in combination with letrozole, an aromatase inhibitor, as a frontline treatment for postmenopausal women with ER-positive/ HER2-negative metastatic breast cancer (mBC).67, 68 Palbociclib (Ibrance® Pfizer), is the first molecule in CDK4 and CDK6 inhibitor class to achieve regulatory approval and it has paved the way for the exploration of the biology of these G1 targets which are emerging as the most promising ones in the cell cycle arena. LY2835219 (abemaciclib, Eli Lilly and Company) (Figure 8) is another orally bioavailable drug that selectively inhibited CDK4 and CDK6 in the nanomolar range (Ki= 0.6 nM and 8.2 nM for cyclin D1/CDK4 and Cyclin D3/CDK6 respectively).65, 69 Using extensive compound screening and data mining of the internal chemical collection, scientists at Eli Lilly identified the pyrimidine-benzimidazole series of molecules as a promising scaffold. Based on potency, ligand efficiency and in silico properties, compound 2a was selected as bona fide hit to build a new CDK4 and CDK6 pharmacophore. Replacement of the aniline by a 2-aminopyridine side chain at C2-position (2b) and posterior elongation through a methylene linker (2c) retained potency while improved selectivity against mitotic CDK1. Fluorine substitution of benzimidazole and pyrimidine rings resulted in beneficial modulation of specificity and pharmacokinetic properties. These SAR studies, combined with other variations on the piperazine side chain let to the identification abemaciclib in 2008. Abemaciclib demonstrated potent inhibition of Rb phosphorylation resulting in a G1 arrest in vitro and in vivo. This inhibition is specific for Rb-proficient cells. In vivo target inhibition studies showed abemaciclib induced a complete cell cycle arrest and suppressed expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of abemaciclib inhibited tumor xenograft growth in models representing different tumor histologies. Abemaciclib was efficacious and well tolerated when administered up to 56 continuous days in immunodeficient mice without significant loss of body weight or tumor outgrowth. Abemaciclib has also been shown to effectively cross the blood–brain barrier (BBB) in animal models and significant antitumor efficacy was shown in an intracranially implanted orthotopic glioma xenograft model.70 Clinical phase III studies in patients with mBC (including patients with brain metastases) and non-small cell lung cancer (NSCLC) are currently ongoing. In 2010 Novartis, in collaboration with Astex, reported LEE011 (ribociclib) (Figure 9) as an orally available, selective inhibitor of CDK4 and CDK6 kinases. No description of SAR studies has been reported.66 Ribociclib induced dephosphorylation of Rb, G1 arrest and senescence in cancer cells including melanoma, with BRAF or NRAS mutation, breast cancer, liposarcoma and neuroblastoma. The cell cycle arrest and senescence were attributed to dosedependent decreases in pRb and FOXM1 (another known target of CDK4 and CDK6). In preclinical in vivo tumor models, ribociclib has been shown to be active in cancers harboring aberrations that increase CDK4 and CDK6

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activity, including those directly linked to the kinases as well as activating alterations in the upstream regulators. Continued treatment inhibits growth of established xenograft models, as well as primary human liposarcoma tumor xenografts without detrimental effects on mouse weight. Clinical phase III studies in patients with breast cancer are currently ongoing. Very recently, in 2014, two new selective CDK4 and CDK6 inhibitors, MM-D37K34 and G1T28-1,71 entered in clinical development. However, neither structures nor SAR studies have been disclosed yet. Potential for combination with existing endocrine therapy (breast cancer) or other targeted therapies (HER2, MAPK pathway inhibitors) in other tumor types is under exploration to the expand clinical utility of CDK4 and CDK6 inhibitors.72 These combinations could result in increased therapeutic benefit, and delayed drug resistance emergence. In 2012, Novartis and Astex reported the identification and structure guided optimization of a series of 4(pyrazolyl)pyrimidines affording compound 3 as a highly selective and orally active CDK4 and CDK6 inhibitor (Figure 10).73 The starting point for this work was fragment 3a (Figure S2, Supporting Information). The fragment bound in the ATP pocket of CDK6 protein and formed hydrogen bonds with the backbone NH and the carbonyl of Val101. Selectivity over CDK1 and 2 was achieved by increasing interaction with residues His 100 and Thr 107 (Phe and Lys in CDK1 and 2 respectively). The 4-(dimethylamino)piperidine group at the 5-position placed a bulky amine in the vicinity of Thr 107 resulting in a selectivity improvement together with increased water solubility. Replacement of the benzimidazole with the 7-azabenzimidazole core provided a new key interaction with His 100. Further optimization afforded compound 3 which showed in vitro and in vivo inhibition of pRb-phosphorylation in Jeko-1 MCL model. Fluorescence-activated cell sorting analysis (FACS) showed a dose-dependent increase in G1 cell population with increasing concentration of compound. Clean G1 block was maintained over a range of 0.63-2.5µM compound concentration. Multiday repeat dosing in Jeko xenograft model demonstrated dose-dependent inhibition of pRb-phosphorylation as well tumor delay BID oral dose. As part of their program to search for potent and orally bioavailable CDK4 inhibitors, Daiichi Sankyo has designed and evaluated novel thieno[2,3-d]pyrimidine hydrazone analogs (Figure 10, Figure S3, Supporting Information).74 The introduction of a thiazole group at the hydrazone part led to marked enhancement of chemical stability under acidic conditions relative to the initial hit. Focusing on the optimization at the C4’-position of the thiazole ring and the C6position of the thieno[2,3-d]pyrimidine moiety, compound 4 was identified. Compound 4 induced an increase in G0/G1 population by FACS analysis and showed tumor growth delay (TGD) in HCT-116 xenograft model by intraperitoneal or oral dose. Blocking more than one aberrant key pathway in cancer cells is the mainstay of current standard of care, usually with combinations of therapies that increasingly include one or more targeted agents such as kinase inhibitors. A recent approach was the multi-target inhibition of additional tumor pathways combining them with cell cycle regulation through specific CDK4 and CDK6 inhibition (single compound polypharmacology). All the reported molecules are under preclinical characterization. Examples of these combined activities have been described by Onconova, Amgen and Texas Biomedical Research Institute. Onconova reported a multikinase inhibitor with activity against CDK4 and CDK6 and ARK5 kinases among others. A PK/PD-driven drug development strategy (cytoxicity to glioma cells and brain penetration) with the known pyrido[2,3-d]pyrimidone core led to the identification of ON-123300 (Figure 11).75 ON-123300 exhibited cross reactivity with a small number of kinases (FGFR1, PDGFRβ and PI3Kδ) that play critical role in growth, survival and metastasis in tumor cell lines, but little inhibitory activity against other CDKs. ON-123300 induced growth arrest in tumor cell lines and preferably in MCL. This polypharmacology could be associated to the amino-pyridine replacement by aniline. ON-123300 decreased expression of phosphorylated AKT, CREB and JNK whereas p-ERK and p-p38γ increased in cells. Compound activity was suggested to be related to the inhibition of tyrosine kinase receptors. In vivo efficacy studies in MDA-MB-231 breast xenograft model at 100 or 200 mg/kg, i.p., induced tumor growth inhibition without signs of toxicity or body weight loss. ON-123300 possessed favorable PK properties including the ability to penetrate the brain. Texas Biomedical Research Institute reported gossypin, a pentahydroxy flavone, as a potent antimelanoma agent (Figure 11).76 Gossypin inhibited in vitro human melanoma cell proliferation both in cell lines that harbor BRAFV600E mutation as well as in cells with BRAF wild-type allele. For cells harboring the BRAFV600E, gossypin inhibited cell proliferation through abrogation of the MEK-ERK-cyclinD1 pathway while in cells with BRAF wildtype allele, activity was related to attenuation of the retinoblastoma-cyclinD1 pathway. Gossypin (10 and 100 mg/kg, p.o.) treatment for ten days in human melanoma cell xenograft models harboring V600E significantly reduced tumor volume through induction of apoptosis and increased survival rate in mice, and the effect was significantly superior to that of vemurafenib (selective RAF mutant inhibitor) or roscovitine (pan-CDK inhibitor). In 2014, Amgen reported the discovery of substituted pyrido[4’3’:4,5]pyrrolo[2,3-d]pyrimidine 5a as a novel CDK4 and CDK6 inhibitor (Figure 12). Compound 5a also inhibited FLT3 with promising selectivity against other kinases. Lead optimization was conducted to address three main issues (CYP inhibition, suboptimal selectivity and

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poor oral absorption) using specific FLT3 and CDK4 cell assays as drivers. Lead optimization work, by combining structure-based drug design and traditional medicinal chemistry approaches (modification of the sugar pocket, the heteroaryl linker on the pyrimidine ring and the substitution in ortho to the pyridine nitrogen in the fused ring) let to the discovery of AMG-925.77 Although some substitutions turned out to be useful to ameliorate CYP activity (5b), the unsubstituted pyridine was critical for physicochemical and pharmacokinetic properties. Modification of the cyclopentyl ring to a substituted cyclohexyl ring (5c) led to a significant reduction of CYP3A4. FLT3 activity and bioavailability were improved through modifications on the distal amine function. Extension of the basic nitrogen further into the solvent channel, by making it exocyclic, increased cellular and FLT3 potency but diminished selectivity for CDK4 over CDK1. Selectivity was restored when the primary amine was bis-methylated (5d). Finally, replacement of the basic amine with a non-basic polar group resulted in largely improved pharmacokinetic properties, particularly drug exposure in high dose mouse experiments at the expense of modest reductions in potency. In cell assays, inhibition of pSTAT5 and pRb indicated that the observed efficacy in vitro was consistent with FLT3 and CDK4 and CDK6 inhibition. AMG-925 displayed potent antitumor efficacy in xenograft models mediated by inhibition of FLT3 and Rb phosphorylation in vivo. The authors suggested that combining inhibition of these essential kinases could have potential to overcome FLT3 inhibitor resistance in patients with AML. Multikinase inhibitors such as sorafenib, approved for renal cell (RCC) and hepatocellular carcinomas (HCC), or sunitinib, approved for RCC and gastrointestinal stromal tumors (GIST), are effective antiangiogenic and antitumor agents and owe their efficacy to blocking multiple signaling pathways. In this regard, some multikinase inhibitors with pan-CDK activity plus additional kinase inhibitory activities has been reported and advanced to clinical studies. PHA848125,59 BAY-1000394,60 AT7519,61 and TG0262 from Nerviano, Bayer, Astex and Tragara Pharmaceutical are representative examples. High throughput screening of the former Schering AG compound collection led to the identification of 2,4diaminopyrimidine derivative 6a (Figure 13) as a potent CDK1/2 inhibitor (IC50 = 100 nM) with moderate antiproliferative activity in vitro against the MCF7 cell line (IC50 = 4 µM). This compound was selected as lead for further optimization. In 2006 Schering presented the first multi-target tumor growth inhibitor ZK-304709 that blocked tumor cell proliferation and induced apoptosis by inhibiting a combination of disease progression driving pathways.51 That mode of action, different from standard chemotherapy at that time, was accomplished by potent pan-CDK inhibition (CDK1/2/4/7/9) and inhibition of tumor angiogenesis due to VEGFR1-3 and PDFGβ. Despite the great preclinical activity observed in vitro and in vivo in a broad panel of xenograft and orthotopic models, the compound was discontinued from clinical evaluation due to dose-limited absorption and high inter-patient variability, before the maximum tolerated dose was determined. Subsequent data analysis and work by Bayer led to the identification of the aminopyrimidine BAY-1000394 (Figure 13).60 Limited absorption at high doses and poor aqueous solubility were addressed by a significant improvement in potency and modification of the D-alaninol side chain at the 4-position of the pyrimidine core (6b, 6c). Undesirable off-target activity responsible for a high accumulation in the cellular blood fraction was observed and attributed to the interaction with carbonic anhydrases (CAs). This issue was solved by rational design around the sulfonamide moiety. A simple methyl group on it was sufficient to block the off-target activity in human CAs preserving the potent CDK and VEGFR inhibitory properties. However, it was quickly demethylated in vivo. Introduction of substitution at the ortho position of the arene sulfonamide group was also unsuccessful. Finally substitution of the sulfonamide group by the unusual sulfoximine (6d) moiety retained inhibitory properties, increasing water solubility, improving PK profile and showing no-CA activity in vitro. Roniciclib is a balanced pan-CDK (1/2/4/7/9) inhibitor able to arrest the cell cycle at multiple checkpoints (G1, S, G2, M) with potent, broad-spectrum antiproliferative activity against human cancer cell lines. Kinase profile indicated that 16 additional kinases were inhibited with IC50s below 100 nM. The physicochemical and pharmacokinetic properties of BAY-1000394 facilitated rapid absorption and moderate oral bioavailability. The compound potently inhibited growth of various human tumor xenografts, including models of chemotherapy resistance upon oral dosing. BAY-1000394 is currently in phase II clinical evaluation for small-cell lung cancer (SCLC). Expansion of the benzodipyrazole series (7a) as CDK2 inhibitors (Figure 14) allowed Nerviano to identify the new pyrazolo[4,3-h]quinazoline-3-carboxamide series represented by 7b, showing good potency as CDK inhibitor but being poorly selective against a panel of serine-threonine and tyrosine kinases.59 During the lead optimization phase, the primary carboxamide proved to be the most active compound. Introduction of the 4-methylpiperazinylmoiety (7c) at the para position of phenyl ring led to an improvement in antiproliferative activity in A2780 cell line as well as solubility in neutral buffer but not on overall kinase selectivity. Incorporation of the dimethyl substitution at the 4position (7d) resulted in a significant improvement of kinase selectivity. Further optimization on the physico-chemical properties and PK profile was achieved with amide substitution. PHA-848125 (milciclib) was able to show reduction of S-phase population associated with an increase in G1 arrest and suppression of Rb phosphorylation. Preclinically, it showed tumor growth inhibition against an ovarian xenograft model when orally administered bid for ten consecutive days. Authors suggested that TRKA (transforming tyrosine kinase) inhibition could be useful due to the involvement of this kinase in the development of solid tumors such as prostate, pancreatic and breast cancer. Milciclib blocked cell proliferation, and DNA synthesis in representative glioma cell lines. This compound demonstrated ability to cross the BBB, and good antitumor efficacy was observed by oral administration on different glioma models with either

6

subcutaneous or intracranial implantation. The combination with temozolamide resulted in a synergistic effect. Compound showed good aqueous solubility and desirable PK properties. Authors suggested that all preclinical data indicated that milciclib could become a useful agent for glioma patients and supported its evaluation in phase II trials for this indication. Milciclib is in phase II studies in patients with thymic carcinoma (TC). Astex has described the use of fragment-based X-ray crystallographic screening to identify low-affinity fragment hits for a range of targets, including CDKs. Using apo crystals of CDK2 several hits with low potency were identified (40 µM to 1mM) but with high efficiency given their low molecular weight (15uM CDK2 IC50 >15uM

4 Thieno[2,3-d]pyrimidine hydrazine core (Daiichi Sankyo) CDK4 IC50 = 22nM CDK2 IC50 = 880nM HCT116 IC50 = 315nM

Figure 11: Identification of ON-123300 (Onconova) and Gossypin (Texas Biomedical Research Institute) as potent multi target CDK4 and CDK6 inhibitors.

21

CDK4 IC50 = 3.9nM CDK6 IC50 = 9.8nM ARK5 IC50 = 5nM FLT3 IC50 = 12nM FYN IC50 = 11nM FMS IC50 = 10nM PDGFRβ IC50 = 26nM FGFR1 IC50 = 26nM ABL IC50 = 53nM PI3Kδ IC50 = 144nM ON-123300 (Onconova)

Gossypin (Texas Biomedical Research Institute) Dual BRAF-CDK4/6 pathway inhibitor

Figure 12: AMG-925 (Amgen). Identification and Optimization of Pyrido[4’3’:4,5]pyrrolo[2,3-d]pyrimidine core as multitarget CDK4 and CDK6 inhibitor.

22

HN

N

N

N

N

N

N

N N

HN

N

HN

F

N

N

N

N

N H

N H

N H

5a, Lead compound CDK4 IC50 = 2nM CDK1 IC50 = 3uM FLT3 IC50 = 14nM Colo205 IC50 (Rb +) = 25nM MOLM13 IC50 (FLT3ITD ) = 5nM CYP3A4 IC50 = 550nM F% (rat) 9.4

5c CDK4 IC50 = 7nM CDK1 IC50 = 2.7uM FLT3 IC50 = 2nM Colo205 IC50 (Rb +) = 202nM MOLM13 IC50 (FLT3ITD ) = 13nM

5b CDK4 IC50 = 3nM CDK1 IC50 = 7.5uM FLT3 IC50 = 11nM Colo205 IC50 (Rb +) = 52nM MOLM13 IC50 (FLT3ITD ) = 21nM CYP3A4 IC50 >10uM

N

N N

N HN

N

N

N

N

N

N

N

HN

N

N

N

N

N OH O N

AGM-925 CDK4 IC50 = 3nM CDK1 IC50 = 2.2uM FLT3 IC50 = 1nM Colo205 IC50 (Rb +) = 55nM MOLM13 IC50 (FLT3ITD ) = 19nM F% = 75

5d CDK4 IC50 = 2nM CDK1 IC50 = 3.2uM FLT3 IC50 = 2nM Colo205 IC50 (Rb +) = 10nM MOLM13 IC50 (FLT3ITD ) = 10nM F% = 44

Figure 13: BAY-1000294 (Roniciclib, Bayer). Optimization of 2-Amino-pyrimidin-4-phenyl-sulfonamide core as multitarget CDK-based inhibitor.

23

Br

Br

Br

H N

H N

N

OH

OH N

N

N

HN

N

N

HN

HN

SONH2

SONH2

6a (HIT) CDK2 IC50 = 100nM CDK1 IC50 = 100nM MFC7 IC50 = 4uM

6b CDK2 IC50 = 10nM CDK1 IC50 = 10nM VEGF-R2 IC50 = 59nM CA-II IC50 = 403nM MFC7 IC50 = 30nM

ZK-304709 CDK2 IC50 = 4nM CDK1 IC50 = 500nM VEGF-R2 IC50 = 34nM CA-II IC50 = 514nM MFC7 IC50 = 266nM

Br O OH N

N

BAY-1000394 (Roniciclib)

N

HN

6c CDK2 IC50 = 9nM CDK1 IC50 = 4nM VEGF-R2 IC50 = 122nM MFC7 IC50 = 48nM CF3

SO2NH2

O OH N

6b

Br N OH N

N N

6d CDK2 IC50 = 2nM CDK1 IC50 = 3nM VEGF-R2 IC50 = 32nM CA-II IC50 >10uM MFC7 IC50 = 44nM

N N

S N O

BAY-1000394 (Roniciclib) CDK2 IC50 = 9nM CDK1 IC50 = 7nM VEGF-R2 IC50 = 163nM CA-II IC50 > 10uM MFC7 IC50 = 15nM

S N O

Figure 14: PHA-848125 (Milciclib, Nerviano). Optimization of Pyrazolo[4,3-h]quinazoline core as multitarget CDK-based inhibitor.

24

H N N

4

N

R2

R3

8 HN

N

N

O

O

N

N

N N

R1

N

HN 3

NH2

N N

1

N

7a, (HIT Series) CDK2 inhibitors N

7c CDK2 IC50 =2nM AurA IC50 =53nM A2780 IC50 =30nM sol pH7 156uM

7b CDK2 IC50 = 2nM AurA IC50 =50nM A2780 IC50 = 500nM sol pH7 156uM

N

N

O

O HN

HN

N N N

NH 2

N N N

N

N

N

N

N H

PHA-848125 = Milciclib CDK2 IC50 = 45nM TRKA IC50 = 53nM AurA IC50 = 1uM A2780 IC50 = 200nM sol pH7 191uM

7d CDK2 IC50 = 17nM AurA IC50 = 580nM A2780 IC50 = 50nM sol pH7 98uM

Figure 15: AT7519 (Astex). Optimization of 4-Acyl-pyrazole-3-carboxamide core as multitarget CDK-based inhibitor.

25

NH2

F

O

O

NH

N H

N

N

N

O

N H

N H

10a CDK2 IC50 = 185uM LE 0.57

H N

10b CDK2 IC50 = 3uM LE 0.42

N H

10c CDK2 IC50 = 0.85uM LE 0.44

F

H N

H N

F

O

F

N H

H N

Cl

O N H

H N

N F

O

N H

10d CDK2 IC50 = 0.03uM LE 0.45 HCT116 1.4uM

O

N H N

N F

O H N

Cl

N H

10e CDK2 IC50 = 0.14uM CDK1 IC50 = 0.98uM HCT116 IC50 = 0.3uM m-Plasma clearance 40ml/min/kg

O

N H

AT7519 CDK2 IC50 = 0.047uM CDK5 IC50 = 0.013uM CDK9 IC50