Phosphatidylinositol 3-Kinase (PI3K) and Phosphatidylinositol 3

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Phosphatidylinositol 3‑Kinase (PI3K) and Phosphatidylinositol 3‑Kinase-Related Kinase (PIKK) Inhibitors: Importance of the Morpholine Ring Martin Andrs,†,‡ Jan Korabecny,† Daniel Jun,†,‡ Zdenek Hodny,§ Jiri Bartek,§,∥ and Kamil Kuca*,† †

Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 81, 500 05 Hradec Kralove, Czech Republic Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic § Department of Genome Integrity, Institute of Molecular Genetics of the ASCR, v.v.i., Videnska 1083, 142 20 Prague, Czech Republic ∥ Danish Cancer Society Research Center, Strandboulevarden 49, DK-2100 Copenhagen, Denmark ‡

ABSTRACT: Phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 3-kinase-related protein kinases (PIKKs) are two related families of kinases that play key roles in regulation of cell proliferation, metabolism, migration, survival, and responses to diverse stresses including DNA damage. To design novel efficient strategies for treatment of cancer and other diseases, these kinases have been extensively studied. Despite their different nature, these two kinase families have related origin and share very similar kinase domains. Therefore, chemical inhibitors of these kinases usually carry analogous structural motifs. The most common feature of these inhibitors is a critical hydrogen bond to morpholine oxygen, initially present in the early nonspecific PI3K and PIKK inhibitor 3 (LY294002), which served as a valuable chemical tool for development of many additional PI3K and PIKK inhibitors. While several PI3K pathway inhibitors have recently shown promising clinical responses, inhibitors of the DNA damage-related PIKKs remain thus far largely in preclinical development.

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adaptor p101 and the subsequently discovered p84 and p87.9−13 Classes II and III have different structures and functions. Class II PI3Ks consist of only a p110-like catalytic subunit and have three isoforms. Class III PI3Ks consist of a single unit, Vps34 (vacuolar protein sorting 34). Their function and regulation are still under study.5,14−16 In addition to classes I−III, there is a group of related enzymes that are referred to as PI3K-related protein kinases (PIKK) or class IV PI3Ks. This is a family of serine/ threonine protein kinases, different from other protein kinases, and functionally distinct from PI3K, as none of them have been shown to phosphorylate lipids.17 The structure and function of PIKK members will be described later. The primary result of PI3K activation is the generation of PIP3 in the membrane. PIP3 serves as a second messenger and through phosphorylation activates, among other targets, Akt which is also known as protein kinase B.18 Akt targets many downstream proteins (Figure 1) resulting in cancer-relevant consequences, which involve cell survival, cell cycle progression, proliferation, and cell growth.19,20 The PIKK member mTOR is one of several Akt targets and represents a central regulator of cell growth, proliferation, and metabolism.21,22 mTOR was originally

PHOSPHATIDYLINOSITOL 3-KINASES The phosphatidylinositol (PI) kinases are a unique family of lipid kinases that catalyze phosphorylation of phosphatidylinositol and its metabolites. Phosphatidylinositol is a second messenger functionally implicated in processes related to cell proliferation, survival, membrane transport, and cytoskeletal remodeling.1 The major focus of studies concerned with various diseases has been the family of phosphatidylinositol 3-kinases (PI3Ks) which phosphorylate the hydroxyl group at position 3 in the inositol ring to produce phosphatidylinositol 3-phosphate (PIP), phosphatidylinositol (3,4)-bisphosphate (PIP2), and phosphatidylinositol (3,4,5)-trisphosphate (PIP3).2−4 PI3Ks are divided into three classes I, II, and III based on their structure, substrate preference, distribution, and function. Class I forms heterodimeric complexes consisting of a 110−120 kDa large catalytic subunit and a 50−100 kDa associated regulatory (adaptor) subunit. This class of PI3Ks is further subdivided into two subclasses, based on the form of the adaptor subunits.5,6 Class IA PI3K is linked to the growth factor tyrosine kinase receptors and consists of catalytic subunits p110α, p110β, and p110δ which bind adaptor subunits p85α and p85β. The p85α gene has several splice variants that encode smaller proteins p50α, p55α, and p55γ.7,8 Class IB PI3Ks, expressed in white blood cells, are activated by heterodimeric G-protein subunits and contain the catalytic subunit p110γ that associates tightly to © 2014 American Chemical Society

Special Issue: New Frontiers in Kinases Received: July 8, 2014 Published: November 11, 2014 41

dx.doi.org/10.1021/jm501026z | J. Med. Chem. 2015, 58, 41−71

Journal of Medicinal Chemistry

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Figure 1. Schematic representation of the PI3K/Akt/mTOR signaling pathway. The class IA PI3Ks receives signals from growth factors (GF) through stimulation of receptor tyrosine kinase (RTK). The PI3K complex is located at the plasma membrane where the p110 subunit converts PIP2 to PIP3. This activity is antagonized by PTEN. Subsequently, PIP3 and mTORC2 activate Akt through phosphorylation of Thr308 and Ser473, respectively. Phosphorylated Akt activates or inhibits a broad range of downstream targets including mTORC1, nuclear factor of κB (NF-κB), BCL2-associated agonist of cell death (BAD), forkhead box O1 (FKHR, also known as FOXO1), glycogen synthase kinase 3β (GSK3β), and mouse double minute 2 homolog (MDM2) affecting cell survival, proliferation, and metabolism. mTORC1, activated through phosphorylation and inhibition of mTORC1’s negative regulators tuberous sclerosis complex 1 and 2 (TSC1 and TSC2), further regulates S6kinase 1 (S6K) and eIF4E-binding protein 1 (4EBP1), thereby promoting initiation of protein translation and cell growth.

phosphatase (SHIP1 and SHIP2).29,30 Indeed, PTEN is one of the most frequently mutated tumor suppressor genes in human cancer, and its loss leaves PI3K activity without suppression.31 The frequency of monoallelic mutations of this gene has been estimated at 50%−80% in endometrial carcinoma, glioblastoma, and prostate cancer and 30%−50% in breast, colon, and lung cancer. Complete loss of PTEN is associated with advanced cancers and metastasis.32−35 Besides PTEN, there are many other gene alterations at the DNA level that cause hyperactivity of this pathway. One of the most frequent causes of PI3K alteration is a mutation of the PIK3CA gene that encodes the p110α catalytic subunit. This mutation leads to elevated activity of PI3K and Akt phosphorylation, promoting tumor cell proliferation and invasion.36 This mutation was first demonstrated in colorectal carcinoma by Samuels et al.37 This discovery led to the examination of PIK3CA mutations in other cancer types and was found most frequently in carcinomas of the endometrium, breast, and colon and in benign skin tumors (reviewed in refs 38−40). Another related oncogene PIK3R1, the gene for the p85α regulatory subunit, was discovered in human ovarian and colon tumors.41 Furthermore, also described have been mutations and overexpression of Akt,42,43 SHIP,44 and other components of the PI3K/Akt/mTOR pathway, leading to tumorigenesis and increased resistance to therapy.45,46 Hence, blocking this signaling pathway by inhibitors provides a promising approach for treating many types of cancer, an area of fruitful research highlighted in several recent review articles.47−52

identified as a target of the immunosuppressant rapamycin, a macrolide produced by a soil bacterium found on the island Rapa Nui.23 It is a large serine−threonine kinase, functionally distinct from the other members of the PIKK protein kinase family. The main role of mTOR is the integration of inputs from growth factor signaling pathways, energy, nutrients, and stress, channeled into regulation of metabolism, cell growth, and division. mTOR is a component of two functionally distinct multiprotein complexes: mTORC1 and mTORC2. mTORC1 activates several downstream effectors, resulting in potentiated protein translation and cell growth, while mTORC2 regulates the signaling pathway by phosphorylation of Akt. Overall, mTOR activity promotes cell survival and accelerated growth, both physiologically and in diverse pathologies, including cancer, metabolic disorders, neurodegeneration, and aging. Such intimate relationship with a spectrum of severe human disorders makes mTOR a prominent therapeutic target.24−26 Accumulating evidence supports the notion that the PI3K/ Akt/mTOR pathway is deregulated in many diseases. The best known examples relate to cancer, but dysregulation of PI3K activity contributes also to a variety of other pathologies including thrombosis, diabetes, and inflammatory and autoimmune diseases.27,28 PIP3 levels are tightly regulated by phosphatases, which hydrolyze PIP3 and generate PIP2. Among the most common reasons for dysregulation of PI3K control is deletion of the gene on chromosome 10, encoding the tumor suppressor phosphatase and tensin homolog (PTEN), and aberrations of SH2-domain-containing inositol polyphosphate 542



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single strand breaks (SSBs).59,60 DSBs do not occur as frequently as SSBs, but they are probably the most dangerous DNA lesions that may lead to a loss of genetic information and cell death. They are caused by exogenous agents such as ionizing radiation (IR) and certain chemotherapeutic drugs (e.g., topoisomerase inhibitors) and reactive oxygen species (ROS) and by mechanical stress. The repair of DSBs is performed by two distinct mechanisms: homologous recombination (HR), in which ATM is the primary kinase, and nonhomologous endjoining (NHEJ), regulated mainly by DNA-PK. On the other hand, the overall scenario is complex, and ATM, ATR, and DNAPK are often linked and cooperate to regulate both HR and NHEJ through various mechanisms.61−64 PIKKs are large and structurally very similar.54 The DNA-PK is a holoenzyme that comprises the DNA-PK catalytic subunit (DNA-PKcs) and two tightly associated subunits Ku70 and Ku80. Ku heterodimers form a ring to encircle the DNA with high-affinity binding and play a pivotal role in recognition of DSBs and recruitment of DNA-PKcs. 65 Once the Ku heterodimers are bound to the broken DNA, two DNA-PKcs are recruited to join the broken DNA ends, thereby stimulating the protein kinase activity and the ensuing autophosphorylation and regulation of multiple processing factors for DNA repair.66 The crystal structure of DNA-PKcs has recently been elucidated.67 Although this crystal structure has insufficient resolution for accurate molecular modeling (6.6 Å), it showed that DNA-PKcs forms a large open-ring cradle for embracing and binding the DNA with a forehead and kinase domain on top. As aforementioned, mTOR is a catalytic subunit of two distinct complexes mTORC1 and mTORC2. These two complexes contain, apart from mTOR, unique accessory proteins, regulatory-associated protein of mTOR (RAPTOR) and rapamycin-insensitive companion of mTOR (RICTOR), which define mTORC1 and mTORC2, as well as several unique components mostly different in the two complexes.68,25 Rapamycin binds to the small protein FKBP12 (FK506-binding protein),23 and in turn, rapamycin-FKBP12 binds to the FKBP12-rapamycin-binding protein (FRB)68 and blocks mTOR-RAPTOR interaction but not RICTOR.69 Thus, rapamycin inhibits the function of mTORC1 but not mTORC2. The name ATM kinase is derived from ataxia telangiectasia (AT), an autosomal recessive disorder caused by a mutation in the ATM gene. It is characterized by extreme radiosensitivity, chromosomal instability, cancer predisposition, cell cycle anomalies, immunodeficiency, and neuropathology.70,71 Studying this disorder has provided many insights into how human cells respond to IR. More than 35 years ago it was proposed that radiosensitivity of patients’ cells is due to unrepaired DSBs.72 Subsequent studies revealed that cells with a disabled ATM gene featured cell-cycle abnormalities and hypersensitivity to IR and to some cancer chemotherapeutics (e.g., topoisomerase inhibitors).73−76 Application of these findings provides an opportunity for designing innovative approaches to cancer therapy. The majority of current standard-of-care anticancer therapies (i.e., IR, chemotherapeutic agents) are based on their ability to cause DNA lesions, leading to cancer cell death. However, such DNA damage-inducing therapies are often associated with resistance, due to defective cell death pathways and DNA repair capacity of cancer cells. Thus, pharmacological inhibition of key components of the DNA repair system is an attractive way to sensitize tumors toward IR or chemotherapy. The PIKK family (ATM, ATR, and DNA-PK) and some other DNA damage response

PHOSPHATIDYLINOSITOL 3-KINASE-RELATED KINASES PIKKs are a family of six atypical serine/threonine protein kinases, which are structurally different from classical protein kinases. However, they are related to lipid PI3K because all PIKKs contain a kinase domain with motifs that are typical for the PI3K family. Members of this family are ataxia telangiectasia mutated (ATM), ataxia telangiectasia- and RAD3-related (ATR), DNA-dependent protein kinase (DNA-PK), human suppressor of morphogenesis in genitalia 1 (hSMG-1), transformation/ transcription associated protein (TRRAP), which is the only member without kinase activity, and mammalian target of rapamycin (mTOR).53−55 Four members of this group (ATM, ATR, DNA-PK, and hSMG-1) play key roles in cellular responses to genotoxic insults. The DNA damage response machinery is a complex network organized hierarchically and initiated through detection of the DNA damage lesions by sensor proteins, followed by activation of the apical signaling kinases ATM, ATR, and DNA-PK. These PIKKs then phosphorylate a large number of downstream proteins and thereby trigger a series of events that include activation of many signal transducers and effectors, resulting in activation of cell cycle checkpoints, DNA repair, and chromatin remodeling, and in the case of too severe or irreparable damage may evoke senescence or cell death (Figure 2).56−58 DNA-PK and ATM are activated mainly in response to DNA double strand breaks (DSBs), while ATR is activated by

Figure 2. Simplified schematic representation of the DNA damage response machinery. The ATM, ATR, and DNA-PK are at the top of a complex signaling network. They are rapidly activated in response to DNA DSBs and ssDNA structures, respectively. The list of subsequently activated substrates is large and contains dozens of proteins. Prominent among their targets are the effector kinases Chk2 and Chk1, activated by ATM and ATR, respectively. The ATM-Chk2 and ATR-Chk1 signaling modules along with DNA-PK also phosphorylate the central regulator and tumor suppressor p53, which regulates many genes instrumental for DNA repair, cell cycle arrest (eventually inhibiting the cyclin dependent kinases CDK2 and CDK1), and apoptosis. The primary goal of the DNA damage response machinery is to delay cell cycle and repair the damage, e.g., DSBs by either HR or NHEJ. The HR pathway is activated mainly by ATM and involves many proteins including breast and ovarian susceptibility protein (Brca2), Rad51, or X-ray repair cross complementing protein 2 (XRCC2). The NHEJ pathway is regulated primarily by DNA-PK and includes among other proteins DNA ligase IV and XRCC4.54,57,59,60 43



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of 1.2 μM), mTOR (IC50 value of 2.5 μM), and also several unrelated kinases and proteins, e.g., casein kinase 2 (CK2), glycogen synthase kinase 3 (GSK3), and bromodomains (BRD).101−104 In vitro and in vivo studies revealed that this compound possesses a number of potential effects: cell growth inhibition,105 G1 phase cell cycle arrest,106 antiangiogenic activity,107 and radiosensitivity induction.91,92 However, poor solubility, bioavailability and very fast metabolic degradation precluded further biological evaluation and prompted the search for more convenient inhibitors. One solution for avoiding these physicochemical disadvantages was the preparation of 4 (SF1126), a 3 prodrug, at Semafore Pharmaceuticals (now SignalRx Pharmaceuticals Inc.).108 This prodrug was designed to increase solubility and also to target tumors via binding to specific integrins. Compound 4 was prepared by conjugation of 3 with an arg-gly-asp-ser tetrapeptide chain, resulting in superior pharmacokinetic properties and also more favorable antitumor and antiangiogenic efficacy without severe toxicity in vivo.109 In summary, 4 had suitable properties for clinical trials and has successfully finished phase I clinical trials. The structure of 3 and the PI3K isoform p110γ complex revealed valuable insights into the structure−activity relationship for 3 and related inhibitors (Figure 4).110 Compound 3 is a

proteins, especially checkpoint kinases Chk1 and Chk2,77−79 poly ADP-ribose polymerase (PARP),80−82 and methylguanine methyltransferase (MGMT),83,84 are promising targets for breaking the resistance to standard-of-care treatments, with relatively modest, tolerable side effects.85−92 The most recently discovered PIKK member hSMG-1 could also be a potential target for therapy, but there is still lack of information about the modulation of the action of this kinase. Inhibition of some DNA damage response components can even be exploited as a monotherapy, in a personalized manner to target subsets of tumors whose specific DNA repair defects make them particularly vulnerable to inhibitors of the remaining operational repair pathway, based on the principle of synthetic lethality. This principle says that two genes are synthetic lethal if mutation of either alone is compatible with viability but mutation of both leads to death.93 As examples are synthetic lethal interactions between ATR and ATM/p53, or ATM and DNA-PK pathway.94−96

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LY294002: A WAY TO MORPHOLINE CONTAINING INHIBITORS The first discovered inhibitor of PI3K and PIKK was a fungal metabolite 1 (wortmannin, Figure 3).97 Kinetic analysis showed

Figure 4. Insight into the PI3K isoform p110γ active site and its interaction with 3 (blue sticks, PDB code 1E7V). Important amino acid residues are displayed as sticks in magenta. The morpholino ring of 3 is hydrogen-bonded to Val882 (1.5 Å). This moiety overlaps the volume occupied by the adenine in the ATP−enzyme complex. The chromone scaffold mimics the adenine ring of ATP: the carbonyl group provides hydrogen bonding with Lys833 (3.2 Å), and the 8-phenyl ring is located in a space corresponding to the ribose of ATP. It is stacked between Trp812 (π−π interaction, 3.6 Å) and Met804 (hydrophobic contact, 4.2 Å) on one side and Met953 (hydrophobic contact, 4.0 Å) on the other side. This figure was created with PyMol Viewer 1.30.114

Figure 3. Structures of first generation PI3K inhibitors.

that 1 is a noncompetitive irreversible inhibitor of PI3K with IC50 values in the low nanomolar range. Irreversible inhibition with a lack of selectivity (inhibition of PI3K, ATM, ATR, DNA-PK, mTOR, hSMG-1, and other unrelated proteins) and instability resulted in high toxicity, and hence, this agent was not further clinically studied.97,98 A great step forward was the discovery of the first synthetic specific inhibitor of PI3K and PIKK: 3 (LY294002, Figure 3) at Eli Lilly and Company.99 Its structure was derived from 2 (quercetin), a naturally occurring bioflavonoid, which was previously shown to inhibit PI3K with IC50 values at low micromolar levels.100 The advantage of 3 over 2 was its structurally simple molecule and more specific inhibition of the PI3K family with a 2.7-fold greater potency (class IA IC50 = 1.4 μM).99 Subsequent studies showed that 3 inhibits all PI3K class I subtypes (IC50 values of 0.55, 16, 12, and 1.6 μM for isoforms p110α, -β, -γ, and -δ, respectively) and also class II and class III, related protein kinases DNA-PK (IC50 value

competitive inhibitor for the ATP binding site of PI3K, which is very similar to that of the related protein kinases (e.g., DNA-PK, mTOR). The most crucial part of its structure is the morpholine ring which forms a key hydrogen bond with Val882. This kinase− ligand interaction plays a key role in the enzyme inhibition representing a cornerstone in drug development of novel PI3K inhibitors. Unlike the morpholine ring, the other parts of the 44



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structure are not crucial for affinity and their modification did not result in significant decrease in functional kinase inhibition.99,110 These findings led to the preparation of many inhibitors related to 3, some directly derived from 3 and others of different origin with several dissimilar structural features. The fundamental characteristic of these derivatives is the critical interaction between the morpholino oxygen and the NH of valine in the hinge region of the ATP-binding domain of both PI3K and PIKK family members. These inhibitors are bound to the hinge region by a single point interaction giving this H-bond the highest level of binding energy. This explains the fact that the morpholine moiety is essential for inhibition and substitution of oxygen by sulfur, nitrogen, hydroxymethyl, or methylene as well as other structural modifications (with few exceptions discussed in the section on mTOR inhibitors) resulting in significant loss of affinity. Replacement of morpholine for a 4-pyridyl moiety seemed to provide similar binding mode and potency (compound 48, Figure 12).111 However, ongoing studies were performed with the morpholine analogue (47, Figure 12),111 probably because of the better physicochemical properties. Moreover, these inhibitors generally retained very high selectivity over kinases out of the PI3K family with very low number of off targets.112,113 There are two major directions in the development of morpholine based inhibitors, one focused on DNA-PK inhibition and the other focused on the PI3K and mTOR inhibition. Despite several attempts to develop target-selective inhibitors, many of these compounds possess dual PI3K/DNA-PK or PI3K/mTOR activity. In turn, efforts centered in the development of potent selective or dual-targeting (specifically PI3K/ mTOR) inhibitors with good pharmacokinetics and tolerance. From all these inhibitors, almost none exhibited affinity against ATM or ATR kinases, despite their similar origin. To this date, only a few specific and potent inhibitors of ATM and ATR kinases have been reported, mostly having different nonmorpholine structures. In this review, these unique specific ATM and ATR inhibitors will be placed according to their structures with DNA-PK (ATM) and mTOR (ATR) inhibitors. Similarly, mTOR inhibitors have structures related to those of PI3K inhibitors and many of them possess dual activity, and thus, they will follow the PI3K inhibitors coverage in this review.

Figure 5. Structures of fused core DNA-PK inhibitors.

the core (Figure 6) was found to be well tolerated for DNA-PK inhibition and for which these derivatives served as new

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DNA-PK AND ATM KINASE INHIBITORS Most of the DNA-PK inhibitors are, unlike the PI3K inhibitors, very similar to the structure of 3. This may be due to the fact that to date there is no high-resolution DNA-PK crystal structure available, and for the purpose of molecular modeling, a homology model approach has been mostly used based on the X-ray crystal structure of PI3Kγ. Hence, DNA-PK inhibitors were designed mainly through structure−activity relationship studies (SARs) around the 3 structure. The main structural modification was the replacement of the chromone core with a different heterocycle containing carbonyl oxygen or substitution within the phenyl group. Morpholine ring modifications generally did not provide target affinity enhancement. The majority of these developed morpholine DNA-PK inhibitors are linked to research conducted at the Northern Institute for Cancer Research in Newcastle Upon Tyne and KuDOS Pharmaceuticals Ltd. (now part of AstraZeneca). The absence of crystal structure information for DNA-PK led to strategic ligand-based SAR mapping around the template structure. As a result, fusion of the chromone core with an additional benzene ring (Figure 5) or conversely minimization of

Figure 6. Structures of single core DNA-PK inhibitors.

templates for further inhibitor development. Besides these modifications, also tested were fused coumarin templates (11 and 12), but they exhibited inferior target potency compared to 3 (Figure 5).115−117 All fused-ring chromones showed promising potency to DNAPK inhibition and selectivity over PI3K isoforms, which correlates with previously reported SARs with fused-ring 3 derivatives.99 The 7,8- and 6,7-fused-ring chromones 5 (NU7026) and 8 exhibited submicromolar IC50 DNA-PK inhibitory activity (IC50 values of 0.23 and 0.39 μM, respectively) with more than 50-fold selectivity over PI3K (5, p110α IC50 = 13 μM), whereas the 5,6-fused chromone (9) proved to have approximately equipotent activity with 3.116 The promising activity of 5 led to the synthesis of the saturated 7,8-fused ring derivative (7) and isosteric pyrimido[2,1-a]isoquinolin-4-one (10), both of which showed inhibitory activity comparable with 45



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Cellular activity of 18 was confirmed by examining the phosphorylation of p53 on Ser15, which is a key substrate of activated ATM.124,125 A mapping approach, using small libraries to define SARs, was focused on the basic 2-morpholinochromen-4-one pharmacophore (Figure 7). The 6- and 7-alkoxychromenones (19) were

that of 5. Interestingly, the 4-thiochromone derivative (6) showed no significant decrease in potency, suggesting that carbonyl oxygen does not form a critical hydrogen bond as does the oxygen of the morpholine ring. The importance of the morpholine moiety was probed by replacing it with a diverse range of amine substituents in order to improve pharmacokinetics. Most modifications led to a large decrease in activity, with only the introduction of a 2-methyl substituent to the morpholine ring creating a slight increase in the DNA-PK inhibitory activity.116,117 The cellular activity profiles of 5 and its methylated derivative were assessed for DNA-PK inhibition and radiosensitizing properties using human tumor cell lines in vitro. Both inhibitors enhanced the cytotoxicity induction by ionizing radiation against tumor cells, whereas the inhibitors themselves had no effect on cell survival.116 Compound 5 was subjected to many further investigations as a chemosensitizer of various tumor cells to commonly used cancer therapeutics.118−121 Despite the promising in vitro results, preclinical in vivo pharmacokinetic studies showed that 5 is rapidly cleared from circulation. The main reasons for this rapid clearance are attributed to multiple oxidations and glucuronidation, predominantly at C-2 of the morpholine ring.122 The single core inhibitors (Figure 6) offered more opportunities for introducing structural diversity to the aromatic region in comparison to the fused-ring derivatives. As a starting point, 2-morpholinyl-6-arylpyran-4-ones 13 and thiopyran-4ones 14 were examined. The 6-aryl ring was expected to have a similar role as the A ring of 5 in the ATP-binding domain.115 All tested pyran-4-ones and thiopyran-4-ones with lipophilic aryl substituents showed better or equipotent inhibition compared with 3, while compounds 13A, 13B, 14A, and 14B were comparable to 5 (IC50 values of 0.18, 0.22, 0.53, and 0.28 μM, respectively). Interestingly, both pyran-4-ones and thiopyran-4ones proved to have very similar profiles, with the pyran-4-ones tending to be slightly more potent.115 Later, this subset was extended to various 6-substituted derivatives, followed by the single-core pyran-2-one (15) and 4-pyridone (16) derivatives and the substituted cyclohexenone (17). The pyran-2-one derivatives (15) proved to be almost equipotent with pyran-4ones, suggesting that the oxygen in the core does not participate in key interaction with amino acid residues in the ATP-binding pocket. The importance of the 4-carbonyl oxygen was corroborated by the reduced potency of 4-pyridones (16) and 4-alkoxy pyridines (16A). Similarly, the cyclohexenone derivative (17) showed only modest potency, supporting the importance of a planar heteroaromatic ring core for inhibition.123 Screening of the 6-substituted series confirmed the need for a lipophilic aromatic substituent. All these inhibitors were counterscreened against other selected members of the PIKK family (ATM, ATR, and mTOR) and PI3K (p110α), with no significant off-target activity being observed. Only the 1thianthrenyl derivative (18, KU-55933) displayed remarkably increased inhibition of the ATM kinase.123,124 This derivate, 18, exhibited excellent potency against ATM kinase (IC50 value of 13 nM) with 100-fold selectivity over DNA-PK and other PIKK family members. It should be noted that out of all the morpholine derivatives tested, no other inhibitor showed comparable activity against ATM. Even the corresponding thiopyran derivative (18A) was 30-fold less potent (IC50 in value of 0.35 μM) as an ATM inhibitor compared to 18, while the DNA-PK inhibition of these two compounds was comparable (IC50 values of 1.8 and 2.1 μM, respectively). This observation suggests that the ATPbinding domains of DNA-PK and ATM differ considerably.

Figure 7. Structures of chromone, quinolone, and pyridopyrimidone DNA-PK inhibitors.

examined, as was their parent 7-hydroxychromone derivative. They exhibited promising inhibitory potency starting at an IC50 value of 0.45 μM. Despite the encouraging 7-hydroxy derivative, all introduced substituents led to loss of activity with only compounds bearing benzylic substituents displaying comparable activity.117 More potent compounds were obtained from 6-, 7-, and 8-substituted chromenone libraries (20). Although 6- and 7substituted subsets of 20 did not display significant inhibition, 8substituted derivatives led to identification of two compounds: the dibenzothiophenyl (21, NU7441) and dibenzofuranyl (21A) derivatives, which exhibited superior improvement in potency with IC50 values of 14 and 41 nM against DNA-PK, respectively, and selectivity comparable to that of 5.117,126 The more potent compound 21 was submitted to further research, which determined that cellular radio- and chemosensitization by this compound on various tumor cell lines was efficient in 10 times lower concentration than 5. Compared to ATM inhibitor 18, 21 exhibited a greater degree of radiosensitization in MCF7 breast carcinoma cells, and no significant additive effect was observed when these two compounds were combined.124 Data from further pharmacodynamic in vitro studies confirmed 21 to be a very potent candidate for chemo- and radiosensitization in clinical use.128,129 However, the limited aqueous solubility and low oral bioavailability restricted further biological evaluation of this compound.130 The remarkable activity of 21 led to the preparation of several compounds with similar structural features in order to find inhibitors with comparable or even better inhibition properties. Quinolin-4-one (23) and pyridopyrimidin-4-one (24) derivatives were synthesized to assess whether heterocyclic core 46



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DNA-PK inhibition with IC50 value of 14 nM. In addition to this derivative, a series of O-alkoxyphenylchromen-4-ones was synthesized. Although substitutions with small polar groups led to less potent and nonselective compounds, small nonpolar groups were seen to be beneficial for the enzyme affinity and led to the discovery of relatively selective cyclopropylmethoxy derivative (27), with DNA-PK and p110α IC50 values of 8 and 70 nM, respectively.134 Pyridopyrimidin-4-one (28) and quinazolin4-one (29) derivatives also exhibited equipotent potencies with 25. Inhibitors based on the pyridopyrimidin-4-one template were generally more potent than those with quinazolin-4-one core.135 Interestingly, the most suitable polar groups for DNA-PK inhibition potency on compounds 26, 27, 28, and 29 proved to be morpholin-4-yl or cis-2,6-dimethylmorpholin-4-yl, unlike the ethylpiperazine group found in 25.134 In silico docking studies and minimization of 25 at the ATP-binding domain in the homology DNA-PK model and PI3Kγ model indicate that the 1substituent is located within a region of structural similarity for both kinases, rationalizing the high potency observed against both DNA-PK and PI3K. Exhibiting a 500-fold higher solubility than 21, 25 emerged as a very potent cell-permeable dual inhibitor of DNA-PK and PI3K (class I), which inhibits cell growth and enhances the cytotoxicity of radiation therapy and topoisomerase II poisons in a cell-line-dependent manner.133 Moreover, preclinical studies showed 25 to have very good oral bioavailability and pharmacokinetic profiles.136 Similar solubility improvement was applied for ATM inhibitor 18. Inclusion of a 2,6-dimethylmorpholin-4-yl group to position 2 on thioxanthene led to improved, water-soluble derivative 30 (KU-60019). This derivative showed a 2-fold improvement in ATM inhibition (IC50 value of 6 nM) and about 10 times greater effectiveness in radiosensitization of human glioma cells than its predecessor 18. Unlike the previous water-soluble DNA-PK inhibitors, 30 retained its ATM kinase specificity. Moreover, 30 in monotherapy showed an effect on glioma cell migration and invasion, perhaps acting on the Akt and MEK/ERK signaling pathways.137,138 It remains to be determined what these advantageous effects would translate to in vivo. A recent preclinical study shows that 30 is an efficient and relatively safe radiosensitizer suitable to proceed to clinical trials.139 In the past 10 years of drug development, there has been an increased interest in enantioselective binding of chiral drugs with their biological targets, as enantioselectivity improves activity, selectivity, and off-target driven toxicity. This strategy was also applied in DNA-PK inhibition through addition of small alkyl side chains (i.e., allyl, n-propyl, and methyl, which proved to be most suitable) to 21 at the 3-position of the dibenzothiophene moiety and the 7-position of the chromen-4-one moiety (Figure 9, 31 and 32, respectively).140,141 These small alkyl groups generated stable pairs of atropisomers due to restricted rotation between the core and the dibenzothiophene ring. Biological evaluation of these atropisomers showed that biological activity

alteration, other than the carbonyl group, would be tolerated. The potencies of these inhibitors (IC50 values of 28 and 13 nM for 19 and 24, respectively)131 showed that isosteric changes in the core aimed at retaining similar atomic distances and angles do not significantly affect the biological activity, and likewise for the fused-core derivative 10. Another approach was to explore extended aromatic planar systems that overlap the dibenzothienyl moiety at the 8- position. A structural lead was pointed out as 8-(biphenyl-3-yl)chromen-4-one (22).117 Although it was less potent than 21 (22 IC50 values of 0.18 μM versus 14 nM of 21), this biphenyl motif led to the discovery of several derivatives with comparable potency as 21. Specifically, replacement of 3phenyl with an isosteric thiophen-2-yl (22A) or thiophen-3-yl (22B) improved DNA-PK inhibitory activity to both, exhibiting similar IC50 values (18 and 20 nM, respectively).132 The poor aqueous solubility and unsatisfactory pharmacokinetic profile of these previous compounds prevented their further biological evaluation. An attempt to increase solubility was carried out by adding polar substituents onto the lipophilic moieties (Figure 8). For this purpose, 21 was selected as the most

Figure 8. Structures of DNA-PK inhibitors with improved watersolubility profiles.

potent compound. On the basis of homology modeling of the ATP-binding domain derived from the crystal structure of PI3Kγ, which predicted tolerance to substitution at position 1 of dibenzothiophene, various substituents expected to increase water solubility were introduced via acetamido, acetoxy, or alkoxy linker. Surprisingly, these structural modifications significantly increased the inhibitory potential in enzymatic assays to DNA-PK and other members of PI3K family, resulting in the identification of a new generation of dual DNA-PK/PI3K inhibitors. The majority of these derivatives proved to have at least comparable enzymatic potency as parent 21. Compound 25 (KU-0060648) with an ethylpiperazine group bound via acetamido linker was chosen as a representative of this series. This derivative showed remarkably high potencies with IC50 values of 5 nM for DNA-PK and 4 nM for PI3K p110α.133 Similarly, addition of water-solubilizing groups to other potent derivatives (22, 23, and 24) was investigated. The 8-biphenyl derivative with morpholine ring (26) exhibited good potency for

Figure 9. Structures of (−)-atropisomeric derivatives of 21. 47



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substituted chromone core. The starting point for the modification was structure 35, which exhibited the same potency against DNA-PK as parent compound 3.143 Substitution at the 8position led to derivatives with low micromolar and submicromolar DNA-PK activities. Compounds with substitution at the 7-position showed expectedly lower activities; however, the introduction of a methyl group at the 8-position, resulting in derivatives 36 and 37, increased activity (IC50 values of 0.28 and 0.5 μM, respectively). It is important to note that compound 37 is selective for DNA-PK over PI3K (p110α IC50 value of ≥100 μM) in contrast to the nonselective 36 (p110α IC50 value of 0.13 μM).144,145 This may be caused by the difference between the flexible methylpiperazine group and planar pyridine group. The most active compound of this series, 38 (with DNA-PK IC50 value of 0.089 μM), differed from the other DNA-PK inhibitors by its tricyclic structure with substitution at position 7; moreover, this compound showed no platelet inhibitory effect.146 A different type of derivative was identified from the ICOS Corporation (now Eli Lilly and Company) small molecule library (Figure 11).89 These simple derivatives differ from the 3

resides exclusively in the (−)-atropisomer, whereas the antipodal (+)-atropisomer proved to be inactive. The introduction of a methyl substituent at the 3-position of the dibenzothiophene ring (31) was not very advantageous, possibly as a consequence of a steric clash with amino acid residues.140 In contrast, substitution at the 7-position (32) was well tolerated to substitution.141 The racemate of compound 32 showed a great increase in potency against the parent 21 (IC50 value of 5 nM against 14 nM, respectively). The (−)-enantiomer of 32 showed a 2-fold increase of potency with an IC50 value of 2 nM. More studies on these derivatives are needed to find out whether the free rotation restriction also has an effect on the selectivity of these inhibitors. Alternative heterocyclic core scaffolds were also investigated for identification of new DNA-PK inhibitors (Figure 10).

Figure 11. Structures of other morpholine DNA-PK inhibitors.

Figure 10. Structures of DNA-PK inhibitors with modified heterocyclic core.

structure and combine a morpholine moiety with a hydroxybenzaldehyde motif, similar to vanillin. Vanillin is a naturally occurring compound, which proved to be a modestly potent inhibitor of DNA-PK with IC50 values in the submillimolar range.147 The initial library hit was the moderately potent arylmorpholine 2-hydroxy-4-morpholin-4-ylbenzaldehyde (39, DNA-PK IC50 value of 0.4 μM). Optimization of this template structure led to DNA-PK inhibitors 40−42. These derivatives proved to be potent DNA-PK inhibitors with IC50 values of 120 nM for 40 (IC86621), 35 nM for 41 (IC87102), and 34 nM for 42 (IC87361).89 These derivatives also exhibited activity against PI3K. Compound 40 was found to be selective for PI3K isoform p110β over other isoforms (20-fold against p110α), with p110β potency almost equivalent to that of DNA-PK inhibition. The more advanced morpholinoflavonoid derivative 42 was 50-fold more selective against DNA-PK over p110β and 100-fold over other PI3K isoforms.89 Similar derivatives were further investigated at University of California, where a small set of eight arylmorpholines was prepared and tested for their selectivity. Among them, analog AMA37 (43) was the most potent and selective (IC50 values of 0.27 μM for DNA-PK inhibition and 3.7 μM for p110β), with no activity against other related kinases of PI3K and PIKK family. The structurally simple nitro-derivative 44 (AMA56) was only about 3-fold less active against DNA-PK than 43.148

Following the discovery that the ring oxygen of chromone- and pyranone-based DNA-PK inhibitors does not directly contribute to inhibitor binding,123 coumarin (33) and isocoumarin (34) scaffolds were examined. Despite the promising parent 6methoxycoumarin (33A), which had an approximately equipotent activity to the isomeric 8-methoxychromen-4-one (DNAPK IC50 values of 1.8 and 1.2 μM, respectively),116 substitution at 6- or 7-positions on 33 led to a loss of inhibitory activity. Only the 7-(thienyl-2-yl)coumarin derivative (33B) exhibited submicromolar potency comparable with that of the isomeric 8-(thienyl-2yl)chromen-4-one (IC50 value of 0.6 μM). The most dramatic drop in activity was observed for larger aryl substituents (e.g., dibenzothienyl, biphenyl), resulting in a significant deterioration of potency. While the isocoumarin series (34) proved to be more tolerant to substitution at the 5-position, this only led to moderate potency (34A IC50 value of 0.34 μM).142 1,3-Benzoxazin-4-ones, representing another core scaffold, were assessed in the course of studying the antiplatelet effects of these compounds and 3 at La Trobe University in Australia.143 This study led to the identification of new potent collageninduced platelet aggregation inhibitors as well as DNA-PK inhibitors with structures differing from the conventional 848



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California (compound 46, MCA55).148 Screening of these libraries resulted in the identification of many potent p110βspecific inhibitors with IC50 values of 50−100 nM (Figure 12 shows two representatives 45, 46). Potency against other isoforms p110α and -δ was significantly decreased, and against p110γ these analogues showed almost no activity. This inherited low affinity to p110γ correlates with the lower affinity of 3 toward this isoform. Because of the structural similarities between 45, 46 and DNA-PK inhibitors 21, 23, 24, it was expected that they would also have an effect against DNA-PK. The majority of the prepared derivatives actually inhibited DNA-PK with similar or even better potency than PI3K, suggesting that DNA-PK active site is more susceptible to inhibition. Further development revealed two highly potent and selective compounds 47 (TGX221) and 48 (TGX286).111 Although compound 48 does not have a morpholine and still binds to the hinge region by pyridyl nitrogen, it exhibited comparable potency and selectivity with 47 (p110β IC50 values of 2 and 5, respectively, p110α IC50 > 1 μM).111 Compound 47 was submitted to detailed studies, which determined that the R-enantiomer of 47 was 100-fold more potent than the S-enantiomer, identifying the key role of the aniline group and its orientation for the interaction with the binding site of the p110β.155 As a p110β isoform-specific inhibitor, 47 proved to be a very efficient antithrombotic agent, and this inhibitor in vivo showed elimination of occlusive thrombus formation without prolonging the bleeding time.156 To study the role of selective p110β inhibitors on PTENdeficient tumor cells, a more suitable subset of analogues was prepared for in vivo testing. A representative compound from this series, 49 (KIN193, AZD6482), proved good anticancer activity of p110β inhibitors in p110β driven or PTEN-deficient tumor models (breast HCC70 and prostate PC3), without activity in wild-type PTEN cancer line harboring a mutant p110α (HCC1954).157 Compound 49, under AstraZeneca’s designation AZD6482, was further evaluated as an antithrombotic agent and found to be selective (approximately 100-fold over p110α and -γ) with the exception of PI3K p110δ (8-fold) and has mild and generalized antiplatelet effect. Compound 49 was the first p110β inhibitor validated in human during phase I clinical trial, which showed that this compound is well tolerated and could be useful as a parenteral antiplatelet agent in situations where a low bleeding risk is desirable.158 In order to devise new nonchiral p110β isoform selective analogs, researchers at GlaxoSmithKline designed imidazopyrimidin-5-one and triazolopyrimidin-7-one series with a substituted benzyl group at N1 or N3 (Figure 13). The idea was to replace the aniline residue of 47 with different substituents to obtain a nonchiral compound. Optimization of the 1- and 2substituents (2- and 3- in triazolopyrimidine) resulted in identification of very potent and selective p110β inhibitors 50 and 51 (IC50 values of 1.3 and 0.3 nM, respectively, with up to 1000-fold selectivity versus p110α and -γ and 5- to 10-fold versus p110δ).155,159 Generally, the most potent benzyl substituent appeared to be 2-methyl-3-trifluoromethylbenzyl, and a methyl substituent at the 2-position of the heterocyclic core increased potency 3-fold. Addition of a 2-(R)-methyl substituent at the morpholine cycle proved to be beneficial as well (51). The cellular activity of these inhibitors was demonstrated on PTENdeficient breast cancer lines (MDA-MB-468) by inhibiting phosphorylation of AKT and inhibiting cell proliferation. However, in vivo testing showed that these compounds had very high rat clearance rates, and thus, they were not suitable for

PI3K INHIBITORS The development of PI3K inhibitors is a domain of the past few years and together with mTOR inhibitors greatly prevail over the DNA damage response inhibitors. PI3K inhibitors display greater structural diversity than DNA-PK inhibitors, generally differing from the 3 template. The only feature shared with 3, the morpholine ring, is present in significant part of developed PI3K inhibitors in clinical trials. Unlike the inhibitors of DNA-PK and other PIKKs, PI3K inhibitors are further divided into categories based on their specificity against individual catalytic PI3K subunit subtypes. There are nonselective pan-PI3K inhibitors and specific p110α, -β, -γ, or -δ inhibitors; inhibitors of other classes of PI3K (class II and class III) are not common. The big question from a cancer therapeutics perspective is whether to inhibit simultaneously all isoforms or target selectively only one isoform. PI3K pan-inhibition is based on the theory that each isoform is able to sustain cell proliferation and survival, and thus only blocking all PI3K isoforms will effectively prevent tumor cell survival and proliferation.149 However, this can lead to the potential unwanted toxicity. On the other hand, development of specific inhibitors has been proposed for targeted therapy of the specific signaling-pathway deregulation. Thus, for PIK3CA genealtered tumors, p110α inhibitors should be effective; for PTENdeficient tumors, p110β inhibitors should be effective; and for hematologic malignancies, p110γ and -δ inhibitors should be effective; because of their presence in white blood cells. This approach has yet to be confirmed, since only blocking one PI3K isoform may not be therapeutically enough to stop the cancer.5,150,151 Besides cancer treatment, specific PI3K inhibitors can be used for therapy of other diseases such as thrombosis (p110β)152 and inflammation and immunity disorders (p110γ and -δ).153 PI3K inhibition is much related to inhibition of mTOR, both structurally and therapeutically. Thus, there are many inhibitors which act as dual PI3K/mTOR inhibitors. Early PI3K inhibitors were prepared in a similar way to DNAPK inhibitors, by substitution of the 8-phenyl group and modification of the chromone core. Trombogenix disclosed a series of chromone, quinolone, and pyridopyrimidine analogs (TGX compounds 45, 47, 48), similar to 21, 23, and 24, that differ in the core and in the replacement of the aryl group at the 8position with more extended aromatic substituents (Figure 12).154 This work was extended by researchers at University of

Figure 12. Structures of quinoline, chromone, and pyridopyrimidine specific p110β inhibitors. 49



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7H-thieno[3,2-b]pyran-7-one possessed a slightly better inhibition profile.163 Moreover, such analogues were more watersoluble and cell permeable. Likewise, against the previously mentioned imidazo- and triazolopyrimidines, there was an observed increase in activity for p110β and -δ isoforms, while p110α and -γ inhibition was approximately equipotent to that of 3. The study of this series was mainly focused on preparation of selective p110α inhibitors; however, the researchers also obtained several selective p110δ inhibitors. The most potent compounds are shown in Figure 13: selective p110α inhibitor 55 and selective p110δ inhibitor 56, with IC50 values of 34 and 12 nM, respectively. Exploration of the kinase specificity of structure 55 resulted in the finding that three additional kinases are potently inhibited by this compound: mTOR, DNA-PK, and serine/threonine-protein kinase PIM-1.163 Development of potent and selective p110β inhibitors was also carried out at AstraZeneca and Sanofi. Initial virtual screening gave rise to a 2-diethylamino-4-morpholinopyrimidin-1H-6-one fragment,164 which was evolved with the aim of improving p110β potency and selectivity. Structure modifications were performed on all variable positions of the parent scaffold. First, the other pyrimidine core structures were explored resulting in confirmation of the pyrimidin-6-one core as the most suitable moiety for p110β inhibition in term of potency. It emerged that bulky substituents such as naphthyl (57) or 2,3-dichlorophenyl (not shown in Figure 14) in the 2-position of the pyrimidin-6-one Figure 13. Structures of imidazopyrimidinone, triazolopyrimidinone, benzimidazole, thiazolopyrimidinone, and thiazolopyrane PI3K inhibitors.

further development.159 The final step in the evolution of these series represents carboxybenzimidazole compound 52 (GSK2636771), which is a very potent (p110β IC50 value of 0.89 nM), selective (>900-fold over p110α and -γ and >10-fold over p110δ isoforms), and orally bioavailable inhibitor of PI3K p110β. Compound 52 currently undergoes phase I/IIa open label dose escalation study to assess safety, pharmacokinetics, and efficacy in patients with PTEN deficient advanced tumors.160,161 Through the combination of molecular modeling and medicinal chemistry SAR studies, a novel series of thiazolopyrimidines was disclosed (53, 54).162 Despite the obvious change in the structure from triazolopyrimidines (51), homology modeling of p110β showed that these compounds overlay the same space region in the binding site, suggesting that this series could also be very potent and selective for the p110β isoform. Besides modification of the benzyl group, position 2 also proved to be very suitable for substitution. While bulky substituents (e.g., phenyl) were not tolerated, the inclusion of smaller polar functional groups significantly increased potency as demonstrated with compound 54 exhibiting extremely high potency to p110β isoform and high potency to p110δ isoform, with IC50 values of 0.05 and 3 nM, respectively. These compounds were also very effective in PTEN-deficient cell lines (PC3) with EC50 values reaching low nanomolar levels. Pharmacokinetic and pharmacodynamic studies in mice using compound 53 (IC50 value of 0.6 nM) showed low-to-moderate clearance, good oral availability, and complete tumor growth inhibition after 21 days of oral administration.162 In line with previously reported inhibitors, a series of compounds bearing a thienopyran-7-one moiety were identified through replacement of the benzo ring with a thieno residue in the chromone core of 3. The initial hit, 3-phenyl-5-morpholino-

Figure 14. Structures of pyrimidone specific p110β inhibitors.

core were the best moieties tested to occupy the lipophilic pocket created by side chains of Met-804 and Trp-812 (p110β IC50 values of 93 and 84 nM, respectively). Introduction of polar substituents or heterocycles to this position was not well tolerated resulting in weak potency.165 Related structures were developed by Sanofi’s research. A high-throughput screening campaign identified potent and selective anilide derivative 58, with p110β IC50 value of 42 nM and 50-fold selectivity over p110α.166 Subsequent efforts were devoted to replace the potentially labile anilide moiety by bioisosteric benzimidazole, benzoxazole, or indoline amide. Low nanomolar p110β inhibitors 59 and 60 (SAR260301) were identified through further SAR exploration and p110β homology modeling.167,168 Compounds 59 and 60 exhibited IC50 values of 76 and 23 nM, respectively, high selectivity over other PI3K isoforms (60, almost 70-fold over p110α) and other protein kinases, and efficiency in PTEN deficient tumor xenografts (PC3, melanoma UACC-62).167 60 possessed favorable pharmacokinetic properties (oral bioavailability 70%, T1/2 = 6.9 h in female nude rats) and entered phase I/Ib clinical trials in patients with advanced tumors.168 50



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Figure 15. Structures of PI3K inhibitors derived from quinazoline and thienopyrimidine.

fold less potent than 63, provided acetate, 2-propylpentanoate, and acrylate analogs that still retained similar inhibitory activity to 64.175 Compound 65 was discovered by Piramed Ltd. (later acquired by Genetech) by substitution focused on positions 6 and 7 of the thienopyrimidine core and the phenyl ring of 62.172 The primary goal of these substitutions was to maintain potency while improving the metabolic stability and solubility profile. The main focus was to replace the metabolically susceptible phenolic group with a more stable isostere capable of forming a potential hydrogen bond with the back end of the ATP binding pocket. The 4-indazolyl group displayed the best activity, albeit a 12-fold decrease in potency compared to the parent phenolic derivative. Position 6 of the thienopyrimidine core proved to be tolerant to a wide range of substituents, whereas substitution at position 7 showed a slight decrease in target affinity. Introduction of a 4methylpiperazin-4-ylmethylene moiety to position 6 improved solubility, oral bioavailability, and metabolic stability. Further optimization led to the sulfonylpiperazine derivate (65), which showed remarkable improvement in biochemical and cellular potency. This compound displayed equipotent activity against isoforms p110α and p110δ while demonstrating modest selectivity against p110β and p110γ (IC50 values of 3, 3, 33, and 75 nM, respectively). Lesser activities were observed against other PI3K members, DNA-PK and mTOR (IC50 values of 1.23 and 0.58 μM, respectively).172 Because of 65’s potent in vivo antitumor efficacy and good pharmacokinetics, this compound is currently in phase II clinical trials for the treatment of various types of tumors (e.g., breast and solid cancer).176 Further development led to second generation advanced compounds such as 68 (GNE-477)177 and 69 (GDC-0980).178 Substitution of the indazolyl moiety of 65 with aminopyrimidine resulted in derivative 66.179 This substitution preserved the PI3K inhibition and significantly increased potency to mTOR (PI3K p110α, -β, -γ, -δ, and mTOR IC50 values of 3.4, 12, 16, 16, and 30 nM, respectively). It should be noted that derivative 67 with methylated aminopyridine at 4-position exhibited a dramatic drop in activity related to mTOR while maintaining the same potency to PI3K (mTOR IC50 value of 750 nM). Both 66 and 67 were found to have an equivalent efficacy in a PI3K p110α mutated xenograft model (MCF7.1) after oral administration and proved to be excellent tools for investigation of therapeutic effectiveness of selective PI3K and dual PI3K/ mTOR inhibitors.179 Compound 68, a dual PI3K/mTOR inhibitor (with p110α and mTOR IC50 values of 4 and 21 nM,

All PI3K inhibitors previously described shared a high degree of similarity to 3 and DNA-PK inhibitors. As shown in Figure 15, while the morpholine ring remained constant, the carbonyl oxygen found in 3 is no longer present. These inhibitors without carbonyl oxygen were initially presented by Hayakawa et al. at Astellas Pharma, which disclosed the discovery via highthroughput screening of 4-morpholino-2-phenylquinazolin-6-ol (61) as a p110α isoform inhibitor (IC50 value of 1.3 μM).169 Structural modifications on this hit revealed that a 6-hydroxy group is not essential for activity. On the other hand, the presence of a 3-hydroxy group on the phenyl ring increased activity dramatically by 15-fold. Replacement of the benzo moiety of the quinazoline ring with other heterocycles (e.g., thiophene) also improved target affinity. The most active thieno[3,2-d]pyrimidine derivative (62) exhibited high potency against p110α isoform with IC50 value of 2 nM, and moderate selectivity over other isoforms (8-fold versus p110β, 300-fold versus p110γ).169 Although 62 possessed inadequate pharmacokinetics (plasma half-life after intraperitoneal administration was less than 10 min likely due to microsomal liability of the phenolic group170), it served as a starting point for further development leading to discovery of 63 (PI-103) or 65 (GDC-0941).171,172 Compound 63 was prepared also by Piramed Ltd. through extension of the thienopyrimidine core to pyrido[3′,2′:4,5]furo[3,2-d]pyrimidine (63). This derivative proved to be a potent inhibitor of all PI3K class IA isoforms p110α, p110β, p110δ and a slightly less potent inhibitor of class IB, p110γ (IC50 values of 2, 3, 3, 15 nM, respectively). Compound 63 was also potent against PI3K class II C2β with IC50 value of 10 nM.171 Subsequent studies showed inhibition activity against both mTORC1 and mTORC2 complexes (IC50 values of 20 and 83 nM, respectively) and DNA-PK (IC50 value of 14 nM). Compound 63 induced proliferative arrest in vitro and growth inhibition of human tumor xenografts in vivo at low doses with no compound-related side effects after 18 days of intraperitoneal administration. These studies also showed the beneficial effect of dual PI3K/mTOR inhibition in malignant glioma against single mTOR or PI3K inhibition.173 Unfortunately, the pharmacokinetic profile of 63 suffered from very rapid metabolism due to glucuronidation of the phenolic group and oxidation of the morpholine ring.174 Despite the pharmacokinetic and solubility limitation, 63 served as a valuable chemical tool for further drug development. Efforts to improve pharmacokinetic properties could be realized through acylation of the phenolic group. In this approach, acetylation of isosteric pyridopyrrolotriazine (64), which is approximately 251



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4-(2-hydroxypropyl)piperidine moiety and the core heterocycle. The result of core modification, analogue 71, was a potent, watersoluble inhibitor of p110δ (IC50 value of 2 nM) with 84-fold selectivity over p110α isoform.183 Unfortunately, 71 was positive in genotoxic micronucleus test (MNT) and human chromosomal aberration test (HCA) assays. Genotoxicity was suppressed through disruption of structural planarity by the modifications of the 2-substituent on benzimidazole and the methylene region. A series of oxygen-linked analogues turned out to possess the most favorable pharmacokinetic profiles together with excellent potency and selectivity. Compound 72 (GNE-293) with Ki value of 0.47 nM and 256-fold selectivity over p110α was submitted to further studies, which showed a very good pharmacokinetic profile (bioavailability of 82−100%, T1/2 of 2.59−11.6 h, rat/dog) and no genotoxic effect.184 In order to probe the activity of different scaffolds of bicycle core, a series of 62- and 65-related imidazopyrazines was synthesized by the Spanish Experimental Therapeutic Program (Figure 17). Apart from the core modification, these derivatives

respectively), was prepared in order to improve pharmacokinetic properties by methylation of the thienopyrimidine core of 66 at the 7-position.179 The development of 69 sought maximization of pharmacokinetic properties together with aqueous solubility while retaining the potency (IC50 values for p110α, -β, -γ, -δ, and mTOR were 4.8, 27, 6.7, 14, and 17 nM, respectively). This was accomplished by replacement of the methylsulfonyl group on the piperazine ring of 66 with a lactyl group.178,180 Compound 69 is currently undergoing phase II clinical trials for the treatment of various types of tumors (i.e., breast, renal, prostate, endometrial). PI3K inhibitors 70−72, displayed in Figure 16, developed in ongoing research by Genetech are somewhat different.181

Figure 16. Structures of pyridopyrimidine and imidazopyrimidine based selective p110δ inhibitors.

Although inhibitors in the series are derived from nonspecific 65 and 69, target of 70−72 is a specific inhibition of p110δ isoform, and thus they may be considered as potential therapeutics for diseases related to immune cell function.151,152 A basic structural feature for p110δ selectivity over other isoforms was found in replacement of the indazolyl moiety of 65 with indolyl. The core scaffold did not show a pronounced effect on selectivity or potency; however, the selectivity of pyridopyrimidine and imidazopyrimidine analogues was reinforced. For improvement of selectivity, potency, and physicochemical properties the 2-(piperidine-4-yl)propan-2-ol moiety was introduced resulting in compound 70. Compound 70 was a potent inhibitor of isoform p110δ with IC50 value of 3.8 nM and 340-fold selectivity over p110α. Such peculiarity in a selectivity increase is very surprising because the affinity pocket, where indole and indazole are expected to bind, is highly conserved across all class I of PI3K isoforms.181 As a structural lead, 70 was subjected to further studies in order to improve potency, selectivity, and pharmacokinetic profile.182−184 The first crucial modifications included replacement of indole moiety, which was found to be associated with the strong CYP3A4 time-dependent inhibition. The affinity pocket in p110δ was, unlike the other isoforms, very tolerant to various substituents, mostly represented by heteroaromatic bicycles, which exhibited moderate (ranging from 10- to 100-fold) selectivities over p110α. Out of them, a benzimidazol-1-yl group with a small substituent in the 2-position, as displayed in representative 71, was the most favored, given to no CYP3A4 inhibition and with a unique position for additional interaction with the Lys779 residue.182 Further modifications were concentrated on position 2 of benzimidazole, taking into account also replacement of the

Figure 17. Structures of imidazopyrazine PI3K inhibitors.

were also modified at position 2 (corresponding to the 6-position of 65) by introduction of various amines. Changes in the core resulted in a slight decrease of inhibitory activity, which could be explained by the geometric clashes and electron factors within the imidazopyrazine scaffold. The most potent derivative (73) showed activity with p110α IC50 value of 35 nM, but cellular activity was disappointingly diminished, possiblybecause of the presence of the basic amine chain.185 Both enzyme and cellular potencies were improved in subsequent studies by exploration of different substituents in the 2- and 6-positions of the core. Compound 74 (ETP-46321), analogous to 66, was obtained by the replacement of the basic amino chain with a methylsulfonylpiperidine moiety. By comparison of these two derivatives, the nonselective profile of 66 is in contrast to the moderate selectivity in compound 74 for isoforms p110α and p110δ and over 2000-fold selectivity over mTOR (IC50 values for p110α, -β, -γ, -δ, and mTOR were 2.3 nM, 170 nM, 14.2 nM, 179 nM, and 4.8 μM, respectively).186 Such an effect on selectivity is very unusual, considering the fact that core modifications in general do not have much of an effect on selectivity between PI3K isoforms or on mTOR. Further modification of the amine chain and methylation at the 3-position of core led to development of the next derivative 75 (ETP-46992). Both 74 and 75 exhibited comparable potencies against PI3K and favorable pharmacokinetic profiles (74 oral bioavailability in mice 88.6%, low clearance) for further in vivo studies.187,188 52



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Purine analogues of 62 (Figure 18) were developed at Wyeth Research Co. (now part of Pfizer Co.). Molecular modeling of

Subsequent efforts focused on purine modification and elimination of the metabolically liable hydroxyl group and afforded triazolopyrimidine compound 77 (PKI-402).190 The phenylureido moiety, initially reported by Zask et al.,191 emerged as a suitable surrogate for the hydroxyl group. Both nitrogens of the urea moiety form hydrogen bonds with Asp810, and the carbonyl oxygen interacts via hydrogen bond with Lys802 (p110α homology model). These interactions significantly improve activity against PI3K p110α and mTOR as well as metabolic stability. The suitability of a small substituent (i.e., ethyl, isopropyl, trifluoroethyl) bound to N-3 (corresponding N9 of purine) of triazolopyrimidine was explored in order to obtain a low molecular weight inhibitor with good microsomal stability. 77 showed potency against all PI3K class I isoforms p110α, -β, -γ, -δ, and mTOR, with IC50 values of 2, 7, 16, 14, and 3 nM, respectively. Although this compound was not the most potent kinase inhibitor in these series, 77 exhibited the best activity in human tumor cell lines with IC50 values below 10 nM (MDA361). Moreover, 77 had good physicochemical properties, and its efficacy was established in various in vivo models.192 Following a similar approach, also prepared was a series of analogous pyrrolopyrimidines represented by compound 78 as the most potent inhibitor (p110α and mTOR IC50 value of 2.4 and 1.7 nM, respectively).193 Figure 19 shows a class of PI3K inhibitors structurally based on triazine and pyrimidine cores. These inhibitors were first revealed by Zeynaku Kogyo Co. in Japan, when probing triaminosubstituted triazine derivatives as aromatase inhibitors. They found that triazines carrying benzimidazole and morpholine moieties possess strong antitumor activity while concomitantly being very weak aromatase inhibitors.194 Further studies identified that the molecular target of the most active compound 79 (ZSTK474) is PI3K.195 This compound acts as an ATPcompetitive pan class I PI3K inhibitor with IC50 values of 16, 44, 5, and 49 nM for isoforms p110α, -β, -γ, and -δ, respectively, with very weak activity against mTOR and DNA-PK.196,197 The crystal structure of the 79−PI3Kδ complex198 showed that the oxygen of one morpholine ring forms a hydrogen bond with Val828 (corresponding to Val882 in p110γ), in a similar way to the morpholine oxygen of 3. The benzimidazole moiety extends to the affinity pocket where its nitrogen acts as a hydrogen bond acceptor for the Lys779 residue (corresponding to Lys833 in

Figure 18. Structures of imidazopyrimidine, triazolopyrimidine, and pyrrolopyrimidine derivatives.

this initial compound suggested that introduction of a polar substituent to position N-9 of the purine cycle could increase potency against PI3K and mTOR. Besides that, the phenolic group was replaced with more microsomally stable primary alcohol. This modification did not affect PI3K inhibition, but it significantly decreased activity against mTOR. The most potent structure 76 exhibited inhibition of isoform p110α with IC50 value of 16 nM but with no activity against mTOR.189

Figure 19. Structures of triazine and pyrimidine PI3K inhibitors. 53



Perspective

over other PI3K classes and PIKK, favorable pharmacokinetic properties for oral use, and good toleration after daily administration in phase I clinical studies.207,208 85 is currently entering phase III clinical trials for treatment of advanced breast cancer. Triazine-based PI3K inhibitors bearing two morpholine rings were also developed by Wyeth Research Co. (Pfizer, Figure 20).

p110γ), and the second morpholine oxygen wedges in the ATPbinding pocket. Compound 79 displayed potent antitumor and antiangiogenic activity against human cancer xenografts (e.g., prostate PC3, renal RXF-631L) in mice after oral administration, without evidence of severe side effects,195,199 and despite its poor solubility, it is currently undergoing clinical trials for treatment of patients with advanced solid malignancies. Further studies showed that positions 4 and 6 of the benzimidazole cycle of 79 are tolerant for substitution,200 while substitution at position 5 led to deterioration of activity.201 The most potent derived compound was the 4-methoxy-6-amino derivative 80, with p110α IC50 value of 0.22 nM. Potency enhancement can be explained by increased electron density on the benzimidazole 3-nitrogen, resulting in stronger binding to the lysine amino residue. However, despite the improved aqueous solubility and potency of 80, unfavorable pharmacokinetic profile (AUC after ip injection of 80 reached approximately a quarter value of 79) caused less potency than 79 in human cancer xenograft (glioma U87MG).200 Among trisubstituted pyrimidines, a series related to 63 with a simplified tricyclic core was prepared by disconnecting the bicyclic system at position 5 while conserving the morpholine and phenolic groups. The most potent compound of the series 81 showed potency against p110α with IC50 value of 62 nM, and similar activity was observed against other isoforms p110β and -γ. The labile phenolic group was then replaced by bioisosteric heterocycle cores (e.g., indazole, not shown in Figure 19).202 Compound 82 (WDJ008) from Shanghai Institute of Materia Medica Co. is a representative compound of the 5-cyano-6morpholinopyrimidine series, which was also derived from structures of 62 and 63. Compound 82 was a dual PI3K/mTOR inhibitor with antiproliferative activity in cancer cells superior to 1.203 2,4,6-Trisubstituted pyrimidines researched by Novartis Co. were identified from a solid phase combinatorial library, proposing several potent nonselective compounds bearing a phenolic and heteroaromatic group with hydrogen bond acceptors. A very pronounced inhibitory effect was found in compound 83, with IC50 values of 17, 240, and 6 nM for p110α, -β, and -δ.204 Further development was mainly focused on replacement of the phenolic moiety to achieve better pharmacokinetic properties. Within this work, the phenolic moiety was replaced by 2-aminopyrimidin-5-yl resulting in analogue 84, which was a very potent pan class I PI3K inhibitor with IC50 values of 1, 92, 9, and 20 nM for p110α, -β, -γ, and -δ, respectively. Moreover, compound 84 demonstrated a good pharmacokinetic profile (i.e., oral bioavailability of 89%, half-life of 77 min) with a positive antiproliferative effect in human tumor xenograft (ovarian A2780).205 It should be noted that the 2aminopyrimidine group did not increase potency against mTOR and this compound remained PI3K-selective, in contrast to previously observed dual inhibitors 68 and 69.179 Further optimization of these series for in vivo testing resulted in identification of a clinical candidate, compound 85 (NVPBKM120, buparlisib). Compound 85 was designed by incorporation of the second morpholine group into position 4 of the central pyrimidine, which increased aqueous solubility while retaining the selectivity and potency. Addition of a trifluoromethyl group to position 4′ of aminopyridine enforces a nonplanar conformation between the central pyrimidine and C-6 heterocycle, thus disrupting the heterocyclic π-electron distribution.206 Compound 85 possessed IC50 values of 52, 166, 262, and 116 nM against p110α, -β, -γ, and -δ, respectively, selectivity

Figure 20. Structures of triazine based PI3K inhibitors.

This work was built on previously reported imidazo- and triazolopyrimidine PI3K/mTOR inhibitors, i.e., 77, which despite good efficacy in the in vivo model, suffered from poor solubility at neutral pH needed for advanced biological studies.190 Triazine as the core scaffold was chosen in order to increase polarity and aqueous solubility. The second morpholine group was added in a view of the frequent metabolic oxidation of this ring. Compound 86 (PKI-587, PF-05212384), with a 4dimethylaminopiperidine group on the phenylureido appendage, was selected as a representative compound for further in vitro and in vivo studies. The IC50 values against p110α, -β, -γ, -δ isoforms and mTOR were 0.4, 6, 5.4, 6, and 1.6 nM, respectively, and inhibition potencies in tumor cell lines (MDA-361, PC3) were in the low nanomolar range.209 Preclinical studies proved that 86 had a significant antitumor efficacy in many human tumor xenografts (e.g., MDA-361, U87MG, colon HCT116) and a favorable pharmacokinetic profile after intravenous administration (plasma half-life of 25 mg/kg single dose was 14.4 h against 3.5 h for 75) for clinical studies.210 Compound 86 has reached phase II clinical trials for solid tumors and endometrial and colorectal cancer. Unfortunately, 86 has to be administrated intravenously because of low plasma levels after oral administration, a consequence of poor permeability, low log P values, and high molecular weight.209 Hence, efforts were made to overcome these negative factors and obtain an orally efficacious compound. In order to increase the log P value, one of the morpholine rings 54



Perspective

Figure 21. Structures of PI3K inhibitors with different cores.

combining indoline ring, quinoline core, and morpholine, exhibited good starting potency against PI3K isoforms p110β and -δ (IC50 values of 60 and 360 nM, respectively), but lesser cellular activity and poor physicochemical properties especially in microsomal stability and solubility. This initial hit 91 was systematically optimized to enhance the p110β and -δ affinity and improve physicochemical and pharmacokinetic properties, which would be needed for in vivo evaluation. The initial SAR studies revealed positive effects of aryl introduction to position 2 and fluor to position 7 of quinoline on p110δ potency and in the case of 2-pyridyl as aryl also on solubility. Further improvements in overall physicochemical properties (cytochrome inhibition, microsomal stability, solubility) and biochemical activity were achieved by replacement of the indoline scaffold with pyrrolopyridine (92). Compound 92 showed potent and selective inhibition of PI3K p110β and -δ (IC50 values of 44 and 11 nM, respectively, versus 3.25 μM against p110α) with suitable pharmacokinetic profile (bioavailability of 65%, half-life of 4 h). The efficacy of this compound was positively evaluated in an animal model of rheumatoid arthritis, where 17 days administration of 92 reduced symptoms of this inflammatory disease.215 UCB Celltech Co. reported a series of 5,5-dimethyl-2morpholin-4-yl-5,6-dihydro-1,3-benzothiazol-7(4H)-ones (93− 96, Figure 21) as specific inhibitors of PI3K isoforms p110γ and -δ.216−219 Surprisingly, modification of the initial pharmacophore (93) was performed on the morpholine ring, which is not usually tolerated. Substitution on the central dihydrobenzothiazole core was not beneficial in term of potency except for the replacement of the carbonyl group at position 6 with a lactam (95, 96) to improve physicochemical properties. The pharmacophore 93 was generated by directed medium-throughput screening based on PI3K p110γ, which showed possible hydrogen bonds with amide NH of Val882 and the NH of the side chain of Lys833. Molecular modeling further showed a possible way to increase potency through hydrophobic interactions with the residues on

of 86 was replaced by a bridged-morpholine analogue. Molecular weight was reduced to a level below 500 Da by replacing the benzamide moiety of the urea appendage with a smaller suitable heterocycle. The resulting compound 87 (PKI-179) proved to be slightly less potent against p110α than the parent compound 86, but it was found to be orally efficient (MDA-361 xenograft model). Furthermore, the primary metabolic route was identified as oxidation of the ethylene bridge in the bridged-morpholine ring. Isolation and determination of this metabolite showed that it is still active with no significant decrease in potency.211 Another analogue of this series 88 was identified by a substitution of one the morpholine rings with an oxygen-linked tetrahydrofuran-3-yl moiety. This compound showed superior in vitro activity; however, in vivo it was not quite as potent as 86.212 A dihydropyrrolopyrimidine series (89, CH5132799, displayed as representative of this series in Figure 21), presented by Chugai Pharmaceutical Co.,213 was developed through modeling and SAR of previously reported inhibitors 63 and 81.171,202 The phenolic and pyridine rings of 63 and 81 were substituted by 2aminopyrimidine and methylsulfonyl moieties in order to improve physicochemical and pharmacokinetic properties, affording 89, a PI3K class I inhibitor (p110α IC50 value of 14 nM) with good microsomal stability and excellent bioavailability (100% in mouse).213 Imidazopyrimidines represented by 90 (VS-5584) are reminiscent of 89.214 They were identified by S*BIO Pte Ltd. in Singapore. 90 is a potent pan PI3K/mTOR inhibitor with IC50 values of 16, 68, 25, 42, and 37 nM for isoforms p110α, -β, -γ, -δ and mTOR, respectively. Moreover, this inhibitor proved efficacy in a wide range of tumor xenografts (e.g., PC3, colorectal COLO-205) and it has very favorable pharmacokinetic profile after oral administration (bioavailability of 100%, half-life of 10 h).214 Both 89 and 90 are currently in phase I clinical trials for treatment of advanced tumors (89) and nonhematologic malignancies or lymphoma (90). The indoline derivative 91 is an initial compound of Amgen Inc.’s PI3K inhibitor research. This initial hit, designed by 55



Perspective

the edge of the ATP binding site near the morpholine ring.217 These interactions were probed by modifying the morpholine moiety by introducing various substituents to position 3 (not shown) or by benzo-fusion (94). This fusion into 3,4dihydrobenzoxazine led to a 2-fold increase of potency against PI3K p110γ and -δ (IC50 values of 0.61 and 0.83 μM, respectively).218 It should be noted that the 3,4-dihydrobenzoxazine as a morpholine replacement was previously probed in DNA-PK inhibitors but revealed no inhibition ability.113 Further modifications were placed at positions 6, 7, and 8 of dihydrobenzoxazine. The most promising, in this sense, proved to be heteroaryl incorporation into position 6, which increased both potency and selectivity for isoforms p110γ and -δ. Figure 21 shows the most effective representatives in the series 95 and 96. Compound 95 exhibited potency against isoforms p110γ and -δ with IC50 values of 32 and 78 nM, respectively, and almost 10fold selectivity over other class I isoforms. Compound 96 is the result of further optimization focused on cellular activity, selectivity, and solubility, having IC50 values of 4 and 20 nM for p110γ and -δ, respectively. Both of these compounds (95, 96) possess favorable pharmacokinetic profiles for oral administration, and they are promising agents for chronic inflammatory disease therapy.219 The tetrasubstituted thiophene 97, Pfizer Co., is the result of a structure-based drug design and optimization approach.220 This derivative showed enhanced and specific p110α inhibition (p110α and mTOR IC50 values of 0.35 nM and 2.5 μM, respectively) and improved pharmacokinetic properties for oral administration. Molecular modeling of 97-related compounds within the p110α active site showed a similar binding mode to other previously described related derivatives. Morpholine oxygen forms a hydrogen bond with Val851 (corresponding to Val882 in p110γ) and likewise the morpholine oxygen in other inhibitors, and the nitrogen of the triazolyl moiety interacts with Tyr836 and Asp810 (Tyr867 and Asp841 in p110γ), since this spatial orientation can be found for phenolic or indazolyl scaffolds of 63 and 65. It is worth noting that the phenyl group of 97 attached to position C-4 of the thiophene core proved to be the key the element for PI3K selectivity over mTOR (7000-fold more potent against PI3K p110α).220 The inhibitor 98 is structurally different from all previously described morpholine-containing inhibitors. It differs in the origin of the structure and by the function and position of the morpholine ring in the PI3K-inhibitor interaction.221 Structure 98 was derived from a very potent (p110α IC50 of 1.2 nM) benzothiazol-2-ylacetamide PI3K/mTOR inhibitor (not shown in Figure 21).222,223 Molecular modeling showed that the key interaction with NH of Val882 was formed by quinoline nitrogen, and substituents in position 4 (morpholine) stick into a ribose pocket and affect only the physicochemical properties. Compound 98 is a very potent pan PI3K/mTOR inhibitor (IC50 values for p110α, -β, -γ, -δ, and mTOR were 4.6, 13, 8.1, 4.3, and 3.9 nM, respectively), with a good pharmacokinetics profile (bioavailability of 92%) and in vivo efficacy in human xenograft models (U87MG). This compound also exhibited high potency against DNA-PK and class III VPS34 (IC50 values of 2.3 and 11 nM respectively).221

by ATP-competitive inhibition. These inhibitors are expected to provide complete and efficient anticancer ability, whereas the analogues of rapamycin have only a modest efficacy because of increased activation of Akt by mTORC2 as a negative feedback. 224−226 Morpholine-containing small-molecule mTOR inhibitors bind to the kinase domain, reminiscent of all PI3K and PIKK members. Their structures are very similar to those of PI3K inhibitors, and their development was also carried out side by side in the same research centers. AstraZeneca Co., with KuDOS Pharmaceuticals Ltd., presented potent and selective mTOR inhibitors derived from the diamino-substituted pyridopyrimidine 99 (mTOR IC50 value of 1 μM, Figure 22), which was identified by high-throughput

Figure 22. Structures of pyridopyrimidine mTOR inhibitors.

screening. Initially, investigation of substitutions at positions 2 and 4 of the pyridopyrimidine core proved the necessity for a morpholine ring at C-4, whereas replacement of the piperidine ring at C-2 was well tolerated for retaining activity, with a positive influence on overall lipophilicity.227 Incorporation of an additional modified morpholine ring to position 2 displayed activity enhancement. Moreover, modification of core position 7 yielded access to the additional binding pockets within the ATPbinding site. Such arrangement resulted in increased potency (100, KU-63794).227 Compound 100 is a highly specific and potent inhibitor of both mTOR complexes with IC50 value of 16 nM and over 1000-fold selectivity over PI3K isoforms and other protein kinases. Moreover, 100 was found to induce greater dephosphorylation of mTORC1 substrates than rapamycin, suggesting that ATP-competitive mTOR inhibitors could be more effective in cancer treatment than rapamycin analogues.228 Improvements on 100 were basically focused on increasing aqueous solubility and potency while maintaining the selectivity. Modest alterations of both structurally conserved C-4 and tolerant C-2 morpholine rings led to discovery of 101 (AZD8055).229 Derivative 101 differs from 100 in the substitution of the morpholine rings by 3S-methylmorpholines, which increased aqueous solubility as well as potency. 101 is a selective and highly potent clinical candidate with IC50 value of 0.1 nM and great cellular efficacy with complete inhibition of phosphorylation of mTOR substrates.230 However, continuous in vivo studies pointed to a high turnover in human hepatocytes, combined with inconsistent rodent pharmacokinetics. The potential metabolic liabilities of the benzylalcohol and aryl methyl ether moieties were identified as a plausible reason for the high liver clearance. In order to decrease hepatocyte metabolism,

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mTOR AND ATR INHIBITORS Unlike rapamycin and its analogues, which are already used for treatment of different types of cancer and autoinflammatory diseases, the novel generation of specific small-molecule inhibitors of mTOR primarily targets both mTOR complexes 56



Perspective

Figure 23. Structures of pyrazolopyrimidine mTOR inhibitors.

Figure 24. Structures of triazine, pyrimidine, and cyclic sulfonylpyrimidine mTOR inhibitors.

motifs to those in dual inhibitors. Although the initial hit, structure 103 (WAY-001), identified by high-throughput screening was a modestly potent inhibitor of mTOR and PI3K, its potency against mTOR was about 6-fold lower than against PI3K (p110α). Modifications of 103 were performed at the piperidinyl nitrogen and at position 6 of the pyrazolopyrimidine core where there was a metabolically labile phenolic group. The most convenient substitution appeared to be inclusion of a 3pyridylmethyl substituent on piperidine nitrogen and a 5-indolyl substituent on core position 6. The resulting compound exhibited pronounced inhibition of mTOR (compound 104 (WAY-600) has IC50 value of 9 nM) and over 1000-fold selectivity over PI3K (p110α).232 SARs included a search for novel phenol bioisosteres. This effort resulted in discovery of

such substituents were replaced by more stable and soluble functional moieties (e.g., N-methylbenzamide). The resulting compound 102 (AZD2014) was a less potent inhibitor of mTOR (IC50 values of 2.8 nM) than 101, but it showed significantly decreased hepatocyte turnover and excellent aqueous solubility.229 Both 101 and 102 were submitted for clinical trials. Although 101 is no longer in clinical development, 102 is currently entering phase II for treatment of various advanced tumors (i.e., renal, endometrial, ovarian, breast). The effort of researchers at Wyeth Co. has been devoted not only to the development of dual PI3K/mTOR inhibitors (76− 78) but also to specific mTOR inhibitors (Figure 23).191,231,232 These mTOR inhibitors are based on the 4-morpholin-4ylpyrazolo[3,4-d]pyrimidine scaffold, and they carry very similar 57



Perspective

derivatives bearing a urea moiety at position 4 of phenyl.191 An increase in the mTOR potency and selectivity of these urea containing derivatives is caused by formation of three additional putative hydrogen bonds with amino acid residues (Asp2195 and Lys2187). Such interactions cannot be observed in other PIKK and non-PIKK kinases, thus supporting the theory for high selectivity over other kinases. Consequently, a urea moiety was employed in the development of several dual PI3K/mTOR inhibitors (i.e., 77, 86, 87). The methylurea derivative 105 exhibited subnanomolar biochemical potency against mTOR with 100-fold selectivity over PI3K (p110α). However, 105 suffered from poor metabolic stability due to dealkylation of the piperidine ring. Replacement of the pyridylmethyl residue with a carbomethoxy residue resulted in a microsomally stable and equally potent compound 107 (105 and 107 showed against mTOR IC50 values of 0.4 and 0.5 nM, respectively). Although substitution of the methylurea moiety by methyl carbamate led to less potent derivatives 106 (WYE-687) and 108 (WYE-354) with IC50 values of 4.6 and 4.3 nM, respectively, these compounds possessed higher selectivity, excellent microsomal stability, and in vivo antitumor efficacy (108 in PC3-MM2 and U87MG xenograft).191,233 Subsequent attention was focused on 6-ureidophenyl appendages, which resulted in a series of very potent, water-soluble 6-arylureidophenyl derivatives. These derivatives reached low-subnanomolar biochemical potencies, but they were less selective against mTOR when compared to short-chained alkylureido compounds. Compound 109 exhibited IC50 values of 0.3 and 15 nM for mTOR and PI3K p110α, respectively, and also good microsomal stability.232 The pyrroloquinazoline compound 110 should be noted within this review. This inhibitor exhibited low nanomolar potency against mTOR (IC50 value of 2 nM) and hSMG-1 (IC50 of 43 nM) while having almost 50-fold selectivity over PI3K (p110α).234 Pyrazolopyrimidine mTOR inhibitors were developed also at Shanghai Institute of Materia Medica Co. Among them, compound 111 (X-387) mimics the structural features of previously described pyrazolopyrimidine inhibitors, but the core has a different arrangement of substituents. 110 proved to be a cellularly efficient inhibitor of mTOR with more than 10-fold selectivity over class I PI3K (mTOR IC50 value of 23 nM).235 Triazine- and pyrimidine-based mTOR inhibitors are displayed in Figure 24. The bis-morpholino triazine 112 was identified as an attractive starting point for lead optimization of several analogs.236 Compound 112 exhibited moderate potency against mTOR (IC50 value of 0.27 μM) and complete selectivity over other PIKK and PI3K. Compound 113, obtained by replacement of the hydrazine moiety with a more stable cyclized moiety (i.e., imidazolyl or furanyl) and alterations in the core, demonstrated profound inhibitory effect toward mTOR (IC50 value of 23 nM) with retained selectivity. However, it exerted low cellular potency (U87MG cells). Modifications or substitutions of the 4-hydroxy-3,5-dimethoxyphenyl moiety were not successful in enhancing the inhibitory effect.236 Derivative 114, developed at AstraZeneca Co., showed modest potency and selectivity (IC50 values of 1.4 and 17.3 μM for mTOR and PI3K p110α, respectively), but a favorable pharmacokinetic profile.237 Subsequent studies showed that replacement of the phenyl group in the phenylsulfonyl moiety of 114 delivered a slight decrease in potency but at the same time increased the selectivity. Further modifications were carried out at the 2-position of pyrimidine. The 5-indolyl variant provided the moderately potent and selective inhibitor 115 (mTOR and PI3K p110α IC50 values of 0.29 and 26.3 μM, respectively);

however, pharmacokinetic studies revealed poor bioavailability and AUC, reflecting its low aqueous solubility. These drawbacks were partially solved by methylation of the morpholine ring in compound 116, such derivative showing improved aqueous solubility with only moderate potency (IC50 values of 0.3 and 23 μM against mTOR and PI3K p110α, respectively). Compound 117 containing the phenylureido moiety revealed 10-fold enhanced potency against mTOR, but because of poor solubility, it was not further studied.237 A family bearing conformationally restricted cyclic sulfones (118 and 119) has been developed by Pfizer Global Research Co.238 The five-membered sulfone ring derivatives generally exhibited double the potency to mTOR but lower selectivity over PI3K than the six-membered ones. It is of interest to note that methylation at position 3 of the morpholine ring in compounds 118 and 119 greatly influenced the activities of these derivatives. Methylation of five-membered ring analogue (118) increased potency almost 2-fold, independent of the stereochemistry (IC50 values for S and R analog of 118 were 13 and 15 nM, respectively). In contrast, introduction of an S-methyl group into the morpholine of six-membered ring derivative 119 (PF05139962) increased activity 6-fold (IC50 value of 8 nM), while the potency of the R-methyl analog was unchanged. 119 showed great selectivity (PI3K 110α IC50 > 5 μM) and in vitro efficacy. However, during in vivo studies in rats compound 119 suffered from low bioavailability and short half-life.238 After developing PI3K selective (65) and dual PI3K/mTOR (69) inhibitors, Genetech researchers focused on production of mTOR specific inhibitors.239,240 The starting point for development, compound 120, comprised the morpholinothienopyrimidine core as in 65 and urea moiety, which increased mTOR affinity. 120 exhibited good activity against mTOR with IC50 value of 3 nM and also 20-fold selectivity over PI3K (p110α). The initial efforts were devoted to replacement of the thienopyrimidine scaffold with other heterocycles, which could improve physicochemical properties of the compound. 7-Azatetrahydroquinoline scaffold seemed to be the most favorable in term of potency, selectivity over PI3K isoforms, and chemical stability. Modifications of the urea moiety were not tolerated and resulted in loss of mTOR binding. On the other hand, introduction of 2-S-methylmorpholine resulted in superior potency and selectivity against mTOR. Compound 121 appeared as the most attractive among the series differing in the substituents at position 7. 121 combined good potency and selectivity against mTOR (Ki value of 1.5 nM, 500-fold more than against p110α), the highest oral bioavailability (70%) with the lowest clearance.239 Unfortunately, further studies revealed time-dependent inhibition of cytochrome 3A4 by 121, which led to formation of series devoid of cytochrome inhibition and discovery of a clinical candidate 122 (GDC-0349). 122 exhibited slightly lowered potency (mTOR Ki was 3.8 nM) and great selectivity over other kinases including all PI3K isoforms (790fold over p110α). In vivo studies with 122 showed efficacy in several xenograft models (breast Her-2, A549), and 122 is undergoing phase I clinical trials (advanced solid tumors or nonHodgkin lymphoma).240 The effect of morpholine modification or substitution on potency against DNA-PK and PI3K is well established. Except for methylation, all efforts to enhance a compound’s activity usually have no or minor effect. However, different observations were made by researchers at Wyeth Co. during probing of morpholine modifications in mTOR inhibitors. Specifically, 3,5-ethylene and 2,6-ethylene bridged morpholines (Figure 25) showed slightly 58



Perspective

AstraZeneca’s compound 129 (AZ20, Figure 26) is a representative compound of a series of ATR inhibitors based

Figure 26. Structures of pyrimidine ATR inhibitors.

on the morpholine ring interaction.246 The ATR inhibitor 128 stands between the mTOR inhibitor 115 and 129. This analogue (128) was prepared by replacement of the 5-indolyl moiety of 115 with 4-indolyl. The explanation for the mTOR/ATR selectivity switch remains unknown, but it was hypothesized that 128 binds deeper into the back of the pocket reflecting the difference between the enzymes. The 5-indolyl scaffold was found to be crucial for ATR enzyme interaction, and all modifications decreased the potency. On the other hand, modification of the methylene unit in the sulfone side chain and the 3(R)-methyl modification of the morpholine ring proved to be beneficial in terms of potency and pharmacokinetic properties, leading to the compound 129. 129 (IC50 value of 5 nM and 13 μM against ATR and PI3K p110α, respectively) is a selective and efficient inhibitor of ATR in human tumor xenografts (colorectal LoVo) but also exhibits time-dependent inhibition of cytochrome 3A4 and is poorly soluble.247 Recently, there has been presented the most advanced inhibitor of this morpholinopyrimidine series, AZD6738 (130, structure not disclosed). This inhibitor has a good overall balance of potency, selectivity, and pharmacokinetic properties and has the potential to be clinically tested for efficiency and safety of ATR inhibitors as monotherapy for ATM-deficient tumors or in combination with radio- or chemotherapy. 130 is currently entering the phase I clinical trials for assessing the safety and tolerability of this agent.248,249

Figure 25. Structures of bridged morpholine and dihydropyrane mTOR inhibitors.

increased potency against mTOR while greatly decreasing potency against all PI3K isoforms, leading to highly mTORselective compounds (compound 123, with IC50 values of 0.2 and 5333 nM, respectively, for mTOR and PI3K p110α, has 27000-fold selectivity for mTOR).241 Such selectivity is ascribed to a single amino acid alteration between mTOR and PI3K in the vicinity of the hinge region. As a result, the mTOR active site becomes deeper and tolerant toward larger morpholines. This finding spurred development of several compounds with the same structural motifs as dual PI3K/mTOR inhibitors but differing in the morpholine moiety. The most advanced inhibitors 123−126 are shown in Figure 25.242−244 These compounds exhibited subnanomolar potencies against mTOR and high selectivity for this kinase while maintaining good pharmacokinetic profiles and in vivo efficacy in human xenograft models. It is noteworthy that introduction of an aryl or heteroaryl group to the urea appendage significantly reduced the selectivity over PI3K isoform p110α in compounds 124, 125, and 126 to 237-, 410-, and 899-fold, respectively. Compound 127 was prepared by replacement of the morpholino substituent with a 3,6-dihydro-2H-pyran-4-yl.245 Inhibitors containing the dihydropyranyl unit retained their activity against mTOR while partially losing their activities against PI3K (IC50 values for 127 were 1 and 2300 nM for mTOR and PI3K p110α, respectively). The inclusion of the tetrahydropyranyl scaffold instead of dihydropyranyl was associated with a great decrease in both mTOR and PI3K potency. This decline in activity was explained by the differing minimum energy conformation of these two cycles. The dihydropyran ring in compound 127 as expected was found to be coplanar with the pyrazolopyrimidine core, as has been observed in morpholinopyrazolopyrimidines, whereas the tetrahydropyran ring is rotated about 90° out-of-plane with the core.245

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CONCLUSION AND PERSPECTIVE To date, hundreds of potent and selective inhibitors of PI3K, mTOR, DNA-PK, and ATR kinases have been disclosed. An immense number of these inhibitors are based on the interaction of morpholine oxygen with the hinge region of the ATP binding domain, common to both PI3K and PIKK (summarized in Table 1). In the past few years the balance of interest in PI3K/mTOR versus DNA-PK, ATM, and ATR inhibitors has been shifted toward the PI3K/mTOR pathway. Progress in elucidation of the PI3K/Akt/mTOR pathway led to such inhibitors prevailing over those of DNA-PK, ATM, and ATR. In general, the most clinically advanced inhibitors are the inhibitors of mTOR, represented by rapamycin derivatives such as temsirolismus or everolimus that are used in therapy for both cancer and autoimmune diseases. Because of incomplete mTOR inhibition, such compounds suffer from only modest efficacy in most tumor types. Hence, there is now great emphasis on the results of clinical trials with the second generation of ATP-competitive inhibitors (102, 122, and non-morpholine INK-128/MLN0128/chemically 1-isopropyl3-(2-methylbenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine). These are expected to have better and more complex antitumor activity. However, questions still remain to be 59



Perspective

Table 1. The Most Important and Clinically Developed Morpholine-Containing Inhibitors compd 3

name LY294002

profile

potency, IC50 (μM)

nonselective PI3K, DNA-PK, mTOR, CK2, GSK3, BRD

4 21 25

SF1126 NU7441 KU-0060648

prodrug of LY294002 DNA-PK selective dual DNA-PK/PI3K

18 30 129 130 47 49 52 60 63

KU-55933 KU-60019 AZ20 AZD6738 TGX-221 AZD6482 GSK2636771 SAR260301 PI-103

ATM selective ATM selective ATR selective ATR selective p110β selective, thrombosis p110β selective, thrombosis p110β selective p110 selective PI3K, mTOR, and DNA-PK

65

GDC-0941

PI3K

69

GDC-0980

dual PI3K/mTOR

72 74

GNE-293 ETP-46321

p110δ selective p110α and p110δ selective

79

ZSTK474

PI3K

85

NVP-BKM120/buparlisib

PI3K

86

PKI587/PF-05212384

dual PI3K/mTOR only parenteral

89 90

CH5132799 VS-5584

PI3K PI3K/mTOR

100 101 102

KU-63794 AZD8055 AZD2014

mTOR selective mTOR selective mTOR selective

p110α 0.55 p110β 11 p110γ 12 p110δ 1.6 DNA-PK 1.2 mTOR 2.5 NA DNA-PK 0.014 DNA-PK 0.005, p110α 0.004 ATM 0.013 ATM 0.006 ATR 0.006 NA p110β 0.005 p110β 0.010 p110β 0.089 p110β 0.023 p110α 0.002 p110β 0.003 p110γ 0.015 p110δ 0.003 C2β 0.01 mTOR 0.02 DNA-PK 0.014 p110α 0.003 p110β 0.033 p110γ 0.075 p110δ 0.003 p110α 0.005 p110β 0.027 p110γ 0.007 p110δ 0.014 mTOR 0.017 p110δ 0.0004 (Ki) p110α 0.002 p110δ 0.008 p110α 0.016 p110β 0.044 p110γ 0.005 p110δ 0.049 p110α 0.052 p110β 0.166 p110γ 0.262 p110δ 0.116 p110α 0.0004 p110β 0.006 p110γ 0.005 p110δ 0.006 mTOR 0.002 p110α 0.014 p110α 0.016 p110β 0.068 p110γ 0.025 p110δ 0.042 mTOR 0.037 mTOR 0.016 mTOR 0.0001 mTOR 0.003

60

clinical trial (owner)

ref

preclinical (Lilly)

99, 101−106

phase I (Semafore) preclinical (KuDOS) preclinical (KuDOS)

107, 108 126−130 133, 136

preclinical (KuDOS) preclinical (KuDOS) preclinical (AstraZeneca) phase I (AstraZeneca) preclinical (Trombogenix) phase I (AstraZeneca) phase I/Ia (GlaxoSmithKline) phase I/Ib (Sanofi) preclinical (Piramed)

124, 125 137−139 246, 247 247, 249 111, 156 158 160, 161 168 171, 173, 174

phase I/II (Genetech)

172, 175, 176

phase I/II (Genetech)

178, 180

preclinical (Genetech) preclinical (Experimental therapeutic program)

184 186, 188

phase I/II (Zeynaku)

194−197, 199

phase II/III (Novartis)

206−208

phase II (Pfizer)

209, 210

phase I (Chugai Pharma) phase I (Verastem)

213 214

preclinical (KuDOS) phase I (AstraZeneca) phase I/II (AstraZeneca)

227, 228 229, 230 229



Perspective

Table 1. continued compd 108 122

name WYE-354 GDC-0349

profile

potency, IC50 (μM)

mTOR selective mTOR selective

mTOR 0.004 mTOR 0.0038 (Ki)

clinical trial (owner) preclinical (Wyeth, Pfizer) phase I (Genetech)

ref 191, 233 240

compounds developed. Currently, the most promising are ATM and ATR inhibitors 30 and 129, respectively, which were found to be suitable candidates for clinical trials. 129 is currently entering phase I for evaluation of safety and tolerability.136,249 It is of interest that a majority of DNA-PK inhibitors have essentially very similar structures based on 3 or vanillin, while the ATM and ATR inhibitors have mostly very different structures with a small number of morpholine derivatives. This suggests that the ATP-binding domains of PIKK kinases differ considerably. This is evidenced by the fact that almost no inhibitor has cross potency between these three kinases. As a consequence, ATM and ATR inhibitors are essentially very selective over other PIKK members. DNA-PK inhibitors are also very selective over other PIKKs, but the DNA-PK binding domain seems to be more similar to that of PI3K so that inhibitors of DNA-PK usually exhibit activity against PI3K isoforms. This is apparent in the most advanced morpholine DNA-PK inhibitor 25, which possesses equal activities against both DNA-PK and PI3K. This dual DNA-PK/PI3K activity could lead to a superior anticancer effect, but further studies for in vivo evaluation are needed. The limitations of developed inhibitors often result from poor pharmacokinetic profiles or low selectivities among the PIKK or PI3K kinases. The majority of inhibitors suffer from low aqueous solubility and metabolic liabilities. Particularly selective inhibitors of DNA-PK are burdened by poor solubility resulting from large aromatic moieties (e.g., dibenzothienyl, biphenyl in 21, 22), which appear to be necessary for the higher affinity to DNA-PK. The selectivity is also an important issue. In DNA damage response inhibitors are desirable to specifically target one kinase to avoid sensitization of healthy cells. In the case of PI3K/Akt/ mTOR pathway inhibitors, it is still needed to evaluate the benefits and risks of isoform selective, pan-class, or dual inhibitors. It is worth mentioning that both isoform selective and pan-PI3K inhibitors continue in clinical trials without evidence of severe toxicity, which partly undermines the primary concerns that pan-PI3K inhibition will lead to severe side effects. As to the overall kinase selectivity, developed PI3K and PIKK inhibitors were found to be, given their specific kinase domains, highly specific and generally exhibit no or very low activities against kinases out of the PI3K and PIKK family. Although inhibitors of ATM, ATR, and DNA-PK are still falling behind PI3K inhibitors, considerable progress has been made in understanding these complexes. First compound has already entered the clinical phase, and preliminary findings from other developed inhibitors are very promising. It is expected that further research will lead to inhibitors for clinical evaluation and then for specific oncology therapy. However, as in the case of PI3K/Akt/mTOR pathway inhibitors, new clinical biomarkers are needed for accurate identification of patients that are most likely to respond to these targeted therapies.

answered about efficacy, safety (through only partial kinase selectivity, in contrast to the extremely selective rapamycin analogues), and immunosuppressive effects. The PI3K inhibitors have become subjects of a very active research effort during the past few years, which have brought a quantum of promising compounds to clinical trials for treatment of cancer. The first PI3K inhibitor, p110δ selective idelalisib (CAL-101/GS-1101/chemically 5-fluoro-3-phenyl-2-[(1S)-1(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone), has been already approved for treatment of various hematological malignancies.153,250 The number is expected to grow, as two other inhibitors are already in phase III: isoform nonselective 85 and p110γ/δ selective duvelisib (IPI-145/chemically (S)-3-(1((9H-purin-6-yl)amino)ethyl)-8-chloro-2-phenylisoquinolin1(2H)-one).153,251 Similar remarks can be concluded for dual PI3K/mTOR and specific PI3K or mTOR inhibitors. Structurally, mTOR and PI3K binding domains share a high degree of homology, as is evidenced by the large number of developed dual PI3K/mTOR inhibitors; moreover, almost every PI3K inhibitor possesses slight mTOR activity and conversely. These dual inhibitors stand equally in clinical trials with selective PI3K inhibitors. Theoretically, simultaneous inhibition should be more effective than separate PI3K or mTOR inhibition, due to the fact that mTOR complexes can be activated through other signaling pathways. In most cases, preliminary efficacy data support dual inhibition.252,253 Nevertheless, without data from completed clinical trials the direction of development still remains to be thoroughly assessed. It is not only a matter of the prospects as anticancer or anti-inflammatory agents but also whether after prolonged administration other effects will be manifested resulting from a multilaterally acting PI3K/Akt/mTOR pathway (in particular immunosuppression, deterioration in glucose homeostasis, weight loss). In summary, inhibitors of PI3K/mTOR pathway offer a great hope for future treatment of cancer, inflammation, and autoimmune and other diseases. A great potential is expected from isoform-specific PI3K inhibitors, which are at present in a small number in clinical trials and which could enable safer and targeted therapy and not only for cancer. The decisive evidence for the advantage of either the specific or pan inhibitors will only be known after completion of the ongoing clinical trials. The DNA-damage response is also a very encouraging target for improvement of conventional cytotoxic cancer therapy and potentially as a monotherapy. The signaling network and DNA repair mechanisms are very complex processes and present many potential targets for inhibition. Several compounds affecting checkpoint kinases and DNA repair components MGMT and PARP are already in clinical trials in combination with various cytotoxic agents. However, MGMT inhibitors showed no efficacy due to potentiation of myelosuppression,254 and the therapeutic potential of Chk inhibitors still remains to be determined.88,255 Currently, enormous interest is being put into the apical kinases ATM, ATR, and DNA-PK, which play a central role in directing the whole DNA damage response cascade. Compared to the PI3K/mTOR inhibitors, inhibitors of these kinases did not achieve such advancement given the number of

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AUTHOR INFORMATION

Corresponding Author

*Phone: +420-495-832-923. Fax: +420-495-832-100. E-mail: [email protected]. Address: University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic. 61



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Notes

treatment of nerve agent poisoning, development of decontamination and disinfection methods, and drug design. He was/is principal investigator on numerous national and international projects (EU, NATO, etc.). He has published more than 300 research papers. He is working with several companies as a scientific consultant, focused on military chemistry and toxicology. He has a lot of cooperators throughout the world (e.g., Korea, Croatia, United Arab Emirates, U.S., France, Turkey, Singapore, Sweden, and Brazil).

The authors declare no competing financial interest. Biographies Martin Andrs graduated from the Faculty of Pharmacy (Charles University, Prague, Czech Republic). He is currently a postgraduate student at the Department of Toxicology Faculty of Military Health Sciences (University of Defence, Hradec Kralove, Czech Republic). His area of research is pharmaceutical/medicinal chemistry, biochemistry, and toxicology. He is interested in development of novel inhibitors of PIKK family members as potential anticancer agents.

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ACKNOWLEDGMENTS We thank Ian McColl for the help during the preparation of this manuscript. This work was supported by Ministry of Health, University Hospital Hradec Kralove (Grant 00179906), Faculty of Military Health Sciences, University of Defence (Grant SV/ FVZ201402), Institutional Grant (Project RVO 68378050), and European Social Fund and the State Budget (Grant CZ.1.07/ 2.3.00/30.0044).

Jan Korabecny graduated from the Faculty of Pharmacy (Charles University, Prague, Czech Republic) in 2008. He finished his Ph.D. studies at the Department of Pharmaceutical Chemistry and Drug Control at the same university in 2012. Recently, he was employed at the Department of Toxicology Faculty of Military Health Sciences (University of Defence, Hradec Kralove, Czech Republic). He is currently the head of the Laboratory of Chemistry at the Department of Toxicology and postdoctoral student at the Center of Biomedical Research (University Hospital, Hradec Kralove, Czech Republic). His area of research is pharmaceutical/medicinal chemistry, biochemistry, and toxicology. His interests are neuroscience, especially the development of novel antidotes for treatment of Alzheimer disease and organophosphorus poisoning, and anticancer agents.

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ABBREVIATIONS USED 4EBP1, eIF4E-binding protein 1; A-T, ataxia telangiectasia; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasiaand RAD3-related; BAD, BCL2-associated agonist of cell death; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNAdependent protein kinase catalytic subunit; DSB, double strand break; FKBP12, FK506-binding protein; FKHR, forkhead box O1; HR, homologous recombination; FRB, FKBP12-rapamycinbinding protein; HCA, human chromosome aberration test; hSMG-1, human suppressor of morphogenesis in genitalia; GSK3β, glycogen synthase kinase 3β; IR, ionizing radiation; MDM2, mouse double minute 2 homolog; MGMT, methylguanine methyltransferase; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor of κB; NHEJ, nonhomologous end joining; PARP, poly ADP-ribose polymerase; PI3K, phosphatidylinositol 3-kinase; PIKK, phosphatidylinositol 3kinase-related protein kinase; PIP, phosphatidylinositol phosphate; PTEN, phosphatase and tensin homolog; RAPTOR, regulatory-associated protein of mammalian target of rapamycin; RICTOR, rapamycin-insensitive companion of mammalian target of rapamycin; RTK, receptor tyrosine kinase; S6K, S6kinase 1; SHIP, SH2-domain-containing inositol polyphosphate 5-phosphatase; SSB, single strand break; TSC, tuberous sclerosis complex; TRRAP, transformation/transcription associated protein; Vps34, vacuolar protein sorting 34; XRRC, X-ray repair cross complementing protein

Daniel Jun is an Associate Professor at the Faculty of Military Health Sciences (FMHS, University of Defence, Czech Republic) and at the Center for Biomedical Research (University Hospital, Czech Republic). Since September 2014, he has moved to the Department of Toxicology and Military Pharmacy, FMHS, where he is Deputy Head and Head of Chemical Group. His area of research is analytical chemistry, biochemistry, and toxicology. He is focused on development of antidotes for the treatment of nerve agent poisoning, new disinfectants, and development of novel therapeutics for treatment of Alzheimer disease. He has published more than 130 research papers and was/is investigator on several national and international projects. Zdenek Hodny is the Deputy Head of the Laboratory of Genome Integrity at the Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic in Prague. He obtained his M.D. degree from the Masaryk University in Brno and Ph.D. in Physiology from the Academy of Sciences of the Czech Republic in Prague. His research interests focus on the role of DNA damage response in cellular senescence, tumorigenesis, tumor microenvironment, inflammation, and aging including search for novel therapeutic targets and biomarkers in aging and cancer treatment. He has published over 40 peer reviewed articles and was/is principal investigator on numerous national projects.

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Jiri Bartek is the Head of the Genome Integrity Unit at the Danish Cancer Society Research Center in Copenhagen, Denmark, and the Head of Laboratories of Genome Integrity at the Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic in Prague and the Institute of Molecular and Translational Medicine at the Palacky University in Olomouc, both in Czech Republic. His research interests focus on molecular mechanisms of the DDR including biology of the PIKKs, basic mechanisms of tumorigenesis, and search for novel therapeutic targets and biomarkers in cancer treatment. He has published over 300 research articles, and according to official surveys, he is the globally most cited Czech scientist across all disciplines and among the top 30 most cited European.

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Phosphatidylinositol 3-Kinase (PI3K) and Phosphatidylinositol 3

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