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Latest perspectives of orally bioavailable 2,4-diarylaminopyrimidine analogues (DAAPalogues) as anaplastic lymphoma kinase inhibitors: discovery and clinical developments

The course of anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) therapy has improved impressively. The Food and Drug Administration (FDA) has approved crizotinib (Xalkori, Pfizer) as a first-in-class tyrosine kinase inhibitor (TKI) that demonstrated a substantial objective response rate (ORR) and remarkable progression-free survival (PFS). However, acquired resistance to crizotinib is still a major concern especially as the central nervous system (CNS) remains the most common sites of relapse. To combat disease resistance, limited PFS and poor CNS exposure exhibited by crizotinib (Xalkori, Pfizer) led to the discovery of numerous next generation ALK-TKIs and surprisingly most of them are 2,4-Diarylaminopyrimidine Analogues (DAAPalogues). To date, DAAPalogues have been investigated extensively to display their superior potency against numerous kinase targets especially ALK/ROS1. This review describes hit-to-drug evolution strategies, activity spectra, milestones related to medicinal chemistry discovery efforts and scalable synthetic pathways of clinically emerging DAAPalouges which are either progressing as investigational or preclinical candidates. In addition, the significance of DAAPalogues to treat the patients with ALK+-NSCLC in clinical settings has been detailed. This review is beneficial for medicinal chemists and researchers contributing to discovering ALK-TKIs to overcome existing issues related to DAAPalouges in the drug discovery process.


Introduction
Lung cancer is a fatal malignancy that has a higher mortality rate than colorectal, breast, and prostate malignancies. [1][2][3][4][5] Lung cancer is associated with cancer related deaths globally with a truncated survival rate of about 15%. Smoking addiction is reported to be the predominant risk factor. [6][7][8][9][10] Historically, various classications of lung cancer have been reported but clinically, lung cancers are categorized into small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Reports have revealed that SCLC is responsible for 10-15% while NSCLC is accountable for 85-90% of lung malignancies. NSCLC, typically includes squamous cell carcinoma (10-15%), adenocarcinoma (25-30%) and large cell carcinoma (10-15%) as its three key subtypes. Among these the adenocarcinoma accounts for about 40% of NSCLC cases whereas squamous cell carcinoma is reported to be responsible for about 25% of cases. [11][12][13][14] Despite the devoted investigations to improve the diagnosis of lung carcinoma and treatment, the ve year survival rate for patients detected with NSCLC remains very low (10-15%). In spite of technological advancements in diagnostics and its availability, more than 50% patients are diagnosed at an advanced stage. It is pertinent to note that the therapy demonstrates a palliative aspect instead of a curative intent. [15][16][17] The lung cancer therapy, especially NSCLC has been revolutionized remarkably due to the emergence of molecular targeted therapy (MTT). 18 The emergence of activated gene rearrangements in ALK as an oncogenic driver of NSCLC patients (3-7%) have made the ALK a potential therapeutic target in medicinal chemistry discovery campaigns around the globe. The purpose of current article is to review hit-to-drug evolution strategies, activity spectrum, milestones related to medicinal chemistry discovery efforts and scalable (pilot plant) synthetic pathways of clinically emerging DAAPalouges. In addition, the signicance of DAAPalogues to treat the patients with ALK + -NSCLC in clinical settings has been detailed.

ALK in non-small cell lung cancer
ALK is a receptor tyrosine kinase (RTK) and a key member of the insulin receptor protein-tyrosine kinase superfamily. It was rst discovered in 1994 in the anaplastic large-cell lymphoma (ALCL) as a non-Hodgkin lymphoma subtype. [19][20][21] ALK is a novel orphan RTK which is considered to play an essential role in usual growth and in function of nervous system. The reports have revealed that ALK is usually expressed during the growth of nervous tissues and also down-modulates in the adult. [22][23][24] ALK is closely correlated to leukocyte tyrosine kinase (LTK) [25][26][27][28][29] and has been found to implicate in numerous solid and hematologic cancers. Considering oncogenic manifestations, the ALK rearrangements were rst identied as a fusion to a portion of the nucleophosmin (NPM) gene in 60% of ALCL, a small subset of non-Hodgkin lymphomas. 20,30 It has been revealed that in lymphoma cells containing t(2;5), the NPM gene plays a critical role as an active promoter to initiate or facilitate the expression of the ALK catalytic domain. It has also been envisioned that the truncated ALK is deregulated and phosphorylates the intracellular molecules (substrate) to prompt further malignant transformations. [31][32][33] Thus new NPM-ALK fusion protein was discovered while screening the patients associated with ALCL malignancy where the N-terminal portion of NPM was found to fuse with the kinase domain of ALK as a consequence of t(2;5)(p23;q35) translocation. 34,35 Since the identication of novel potential mutation driver observed in 5-10% NSCLC cases several other ALK fusion genes have also been identied recently. The fusion of the echinoderm microtubule-associated protein-like 4 gene (EML4) with the ALK gene resulted in the generation of a fusion protein (EML4-ALK) and in NSCLC it is considered to be the most signicant discovery being an oncogenic factor in solid tumors. 36 EML4-ALK fused protein was identied in 2007 in the Japanese patients with NSCLC. [37][38][39][40] Surprisingly, since the preliminary reports regarding the discovery of the NPM-ALK fusion protein in clinical settings pertaining to the patients investigated with ALCL, a plethora of ALK fusion proteins have been described in NSCLC, 37,41 inammatory myobroblastic tumors (IMTs), 42,43 squamous cell carcinoma (SCC) of the oesophagus, 44,45 renal cell carcinoma, 46 and diffuse large B-cell lymphomas (DLBCLs) 47 (Fig. 1). Furthermore, oncogenic progression via overexpression and point mutations has been investigated in various cancers. [48][49][50][51][52][53][54] Physiologically, the function of ALK in mammals is still under investigation and unclear. However, the signicance and key role of full-length ALK-RTK in the development or onset of numerous diseases is well investigated and consequently Sultan Ayoub Meo is a professor and consultant in clinical physiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia. He is a medical graduate and obtained M.Phil and PhD in human physiology. He also obtained masters in medical education from University of Dundee, Scotland. He is credited with 10 books and 140 articles in peer reviewed national and international bio-medical journals. Prof. Meo has been invited as a speaker and key note speaker to deliver talks at more than 100 national and international conferences in different countries including Pakistan, Saudi Arabia, Bahrain, UAE, China, Turkey, Indonesia, UK and the USA. known to be expressed in several types of tumors including NSCLC (Fig. 2). Numerous signal transduction pathways have been detected which are activated by the involvement of ALK. Initially, fusion of ALK ligand to extracellular domain occurs during the signal transduction process followed by dimerization and trans-phosphorylation which in turn prompts cellular process involved in oncogenesis. ALK signaling via several downstream pathways have been reported. Ras/Raf/MEK/ERK1/ 2 pathways, PI3K/Akt pathway, JAK/STAT pathway, and PLC-g pathway have been reported to be the principle pathways activated by ALK signal transduction process. Cell survival is mediated by the JAK/STAT and PI3K/Akt pathways while PLC-g and Ras/ERK1/2 pathways play a critical role in cell proliferation. 55 To date, ample number of ALK inhibitors with promising oncolytic activity against ALK + -NSCLC tumors have been investigated and developed in the recent decade.

ALK inhibitors in NSCLC
ALK is one of the highly competitive drug targets in oncologic drug discovery and development campaigns. ALK-fused genes such as NPM-ALK, EML4-ALK, and KIF5-ALK are putative oncogenes which are accountable for several cancers such as anaplastic large-cell lymphomas (ALCL), inammatory myobroblastic tumors (IMT), diffuse large B cell lymphoma (DLBCL) and a variety of solid tumor types. 39,[56][57][58] Genetic abnormalities in ALK have been investigated as key drivers of oncogene addicted lung adenocarcinoma and have boosted the researchers to develop novel and targeted therapies. Nevertheless, growing interest to develop synthetic ALK inhibitors has led to a bigger number of research reports related to new drug candidates for cancer therapy. Some of the exciting entities for ALK inhibition approved by FDA with substantial impact in the eld of medical oncology are listed in Fig. 3.
Crizotinib (PF2341066, Xalkori), the Pzer's rst-generation dual c-Met/ALK inhibitor 59 was given approval by FDA in 2011 as rst line treatment for ALK + -NSCLC. 60 About 1% of ROS1 rearrangements have been reported in NSCLC patients. FDA in 2016, approved the crizotinib for the management of ROS1positive metastatic NSCLC. 61 Although crizotinib demonstrated substantial objective response rate (ORR ¼ 74%, N ¼ 343, phase III) and remarkable progression-free survival (PFS ¼ 10.9 months). Unfortunately, resistance to crizotinib has been reported as one of the critical issues to date. 62- 66 The clinical investigations have revealed that the most common identied secondary mutations are L1196M (gatekeeper mutation), G1202R, L1152R, G1269A, 1151T-ins, C1156Y, S1206Y, and F1174L. [67][68][69][70][71][72] To overcome the limitations exhibited by crizotinib Constitutively active ALK receptor signalling due to EML4 fusion. The fusion arises due to paracentric inversion between EML4 and ALK genes located in the short arm of chromosome 2. EML4-ALK translocation is thought to lead to the constitutive activation of oncogenic signaling through multiple pathways such as PI3K/Akt, JAK/STAT, and RAS/RAF/MEK/ERK. Aberrant activation of PI3K-Akt initiate mTOR, FOXO and GSK3b signalling which lead to survival and proliferation of tumour cells. PI3K/Akt activation also lead to MEKK2/3/MEK5/ERK5 pathway which subsequently promote the expression of the oncogene MYCN. The STAT3 signaling pathway has shown a key role in NPM-ALK mediated alteration however the significance of STAT3 activation in EML4-ALK + -NSCLC is unclear. ALK has also been shown to phosphorylate PLCg to mediate its mitogenicity. (mTOR; mechanistic target of rapamycin, S6K; ribosomal S6 kinase, TD; trimerisation domain, TAPE; tandem atypical propeller domain, MAM; meprin, A5 protein, and protein tyrosine phosphatase Mu domain).
(Xalkori, Pzer), extensive drug discovery campaigns have led to the innovation of numerous next generation ALK inhibitors to combat disease resistance including alectinib, ceritinib and lorlatinib. It is noteworthy, once crizotinib therapy is failed and then lorlatinib is reported to overcome entire secondary point mutations. Reportedly, point mutations such as L1196M, G1269A, S1206Y and I1171T can be overcome by ceritinib whereas L1196M, G1269A, F1174L and C1156Y can be overcome by alectinib. Although crizotinib (Xalkori, Pzer), alectinib (CH5424802, AF-001JP, Alecensa) 73 and lorlatinib (PF-06463922, 3 rd -generation ALK/ROS1 macrocyclic inhibitor, Pzer) 74 are not   Table 1 Key features of DAAPalogues as ALK inhibitor advancing to clinical study Topological polar surface area (TPSA), hydrogen bond donors (HBD), breakthrough therapy designation (BTD), cellular system ¼ (EML4-ALK), discontinued (Disc), the letter S symbolizes mutations which are "sensitive" (clinical and/or preclinical data) to a given drug entity, and "R" symbolizes resistance, data not available (NA), not determined (ND). a In 2011, FDA granted accelerated approval to crizotinib to treat patients with ALK-positive cancer but traditional approval was given in 2013. However on March 11, 2016 2,4-diarylaminopyrimidines (DAAPalogues) yet they show outstanding potential in clinical settings. Crizotinib (acyclic) and lorlatinib (macrocyclic) are among the FDA approved ALK inhibitors (Fig. 3) which comprised of similar central core such as aminopyridine. Crizotinib (approved by FDA in 2016) is an ALK/ROS1/c-MET tyrosine kinase inhibitor that has revealed a high activity in ALK + -NSCLC. Lorlatinib is a 3 rd -generation chemotype that was given breakthrough therapy designation (BTD) by FDA in 2017 and has been used for the treatment of patients with ALK-positive metastatic NSCLC which were treated earlier with one or more ALK inhibitors. [75][76][77] We recently documented the strategic development of lorlatinib from crizotinib. 78 It is noteworthy that both crizotinib and lorlatinib have aminopyridine core and are non-DAAPalogues. Notably, ceritinib (Zykadia®) is the rst FDA approved DAAPalogue for ALK/ROS1 inhibition 79-81 which has been described comprehensively elsewhere. [82][83][84][85][86][87] Interestingly, ceritinib being a member of DAAPalogues showed potency to overcome the gatekeeper mutation of L1196M which is still considered a resistance to crizotinib (non-DAAPalogue agent). In crizotinib, the existence of the polar-NH 2 (aromatic amine) substituent at C2-position of pyridine core is considered not to provide favorable interactions with a large lipophilic residue at the gatekeeper position, such as methionine. On the contrary, in ceritinib the chloro (Cl) substituent at C5-position of pyrimidine core could interact more favorably with a methionine gatekeeper and has revealed a remarkable interaction to gatekeeper mutation. On the other hand, alectinib is another FDA approved, highly selective, orally active, potent, and CNS penetrant ALK inhibitor which has shown a promising therapeutic window for the patients treated with crizotinib harboring ALK secondary mutations. [88][89][90][91][92][93] Alectinib is non-DAAPalogue drug which comprised of benzo[b]carbazolone structural motif 94-97 that is not ubiquitous template in second or even third generation TKIs. In medicinal chemistry campaign limited structure-activity relationship (SAR) investigation has been reported for benzo[b]carbazolone core possibly due to difficulty in chemical synthesis. [97][98][99][100][101] However, recently reported JH-VIII-157-02 has been potent against the G1202R mutant and comprised of benzo[b]carbazolone core. 102 In the designing and development of next-generation ALK-TKIs, the 2,4-diarylaminopyrimidine (DAAP) structural moiety has been incorporated extensively. The literature survey has revealed that majority of the next-generation TKIs being evaluated clinically or currently at preclinical phases have been derived from DAAP scaffold and referred as DAAPalogues. Such ALK-TKIs were developed to potently inhibit all acquired ALK mutants reported previously for rst and second generation ALK-TKIs. In recent decade, rapid progress of pharmacological strategies targeting ALK has shown marvelous achievements in clinical settings. Continued research efforts of researchers have made it possible to advance several DAAPalogues to clinical trials. To date, numerous ALK inhibitors have been discovered and diversied chemical classes have been evaluated at pre-clinical or clinical phases and most of them are DAAPalogues. 60,83,103-107 Some novel, potent and highly selective DAAPalogues for ALK inhibition are shown in Fig. 4.
The DAAP skeleton as a conventional kinase inhibitor core is well recognized and such DAAPalogues has demonstrated high potency against many kinase targets, particularly ALK, c-Met and ROS1. To this end, the drug designing strategy stemmed from our literature survey has demonstrated that ceritinib (LDK-378, 2 nd -generation ALK/ROS1 inhibitor Zykadia®, Novartis), brigatinib (Alunbrig™, AP26113), NVP-TAE684, Cephalon 2812, Cephalon 1, and CEP-37440 comprised of DAAP-motif are clinically novel ALK-TKIs. 87,[108][109][110][111][112][113] It is signicant to note that on DAAP template chloro (Cl) substituent have been installed as an optimal substituent at C-5 position of pyrimidine which in most of the DAAPlouges interacts with ALKL1196 gatekeeper residue. The designing strategies, biological characteristics, therapeutic exploration, and clinical data of DAA-Palogues as ALK inhibitors is well-detailed in literature. In the following sections, we have summarized hit-to-drug evolution strategies, activity spectrum, milestones related to medicinal chemistry discovery efforts, scalable synthetic pathway, and development of some potent ALK-TKIs comprised of DAAP template which are either progressing as investigational or preclinical candidates. Some key features of DAAPalogues as ALK inhibitor advancing to clinical study are shown in Table 1.

Brigatinib (Alunbrig™, AP26113)a second-generation DAAPalogue
Brigatinib (Alunbrig™, AP26113), developed by ARIAD Pharmaceuticals as antineoplastic ALK inhibitor is a 2 nd -generation ALK inhibitor with DAAP structural motif. It is a potent DAA-Palogue with imperious in vitro potential against ALK (IC 50 ¼ 0.6 nM L À1 ), ROS1 (IC 50 ¼ 1.9 nM L À1 ), FLT3 (IC 50 ¼ 2.1 nM L À1 ) and EGFR (IC 50 ¼ 1.9 nM L À1 ). Being a potent DAAPalogue, brigatinib received an accelerated FDA approval for therapy of patients suffering from ALK + -NSCLC who either progressed on or were intolerant to crizotinib therapy. The occurrence of resistance to crizotinib posed a therapeutic task and has led to the advancement of 2 nd -generation ALK-TKIs such as brigatinib. Brigatinib have exhibited activity against treatment-resistant ALK mutants (Table 1). Brigatinib showed favorable clinical potential and satisfactory safety prole in the patients treated with crizotinib and crizotinib-naïve ALK-rearranged NSCLC patients. 117, 124 The structure-activity relationship reported by Katayama et al., have revealed that brigatinib also demonstrated potency against the triple-mutant EGFR (IC 50 < 100 nM). Brigatinib suppresses EGFR phosphorylation and its downstream signaling pathway in cells which express several types of EGFR mutations such as EGFR C797S/del19 (IC 50 ¼ 28.4 nM), IGF1R (IC 50 ¼ 32 nM), and EGFR del19 (IC 50 ¼ 36.8 nM). 125,126 This feature has shown that brigatinib will be a promising candidate to overcome the acquired resistance of 3 rd -generation EGFR-TKIs if it is used as combination therapy with anti-EGFR antibody. In the following section, description related to hit-to-drug evolution design and the milestones achieved during the development of brigatinib are summarized. These advancements ultimately led the brigatinib to its rst global approval for the therapy of patients with ALK + -NSCLC who either progressed on or remained intolerant to crizotinib. 126 The synthetic strategy of brigatinib (Alunbrig™) from one of the potent ALK inhibitor (AP23464, IC 50 ¼ 0.45 nM) discovered in early discovery efforts is described rst.

Synthetic strategy of brigatinib (Alunbrig™, AP26113) from (1)
Although phosphorous is an abundant element in human body but rarely employed in drug discovery strategies. Poor cell penetration, biological instability or low oral bioavailability have narrowed down the clinical utilization of monophosphonic or bisphosphonic acid-based drugs, phosphonates, phosphinates, and phosphate-containing prodrugs in drug discovery.
Ridaforolimus (mTOR inhibitor), Src-kinase inhibitors (antiresorptive agents) have been reported in recent decade having phosphine oxide (P]O) functionality. The P]O bond has been investigated as a H-bond acceptor in designing of kinase inhibitors. To this end, analogous efforts related to the discovery of AP23464 (IC 50 ¼ 0.45 nM), a potent ATP-based oncogenic protein kinase inhibitor, 127-129 further stimulated the discovery of potent DAAPalogue (1) where dimethylphosphine oxide (DMPO) onto the 2-anilinopyrimidine template was surveyed. 111 Aforementioned investigations provoked to incorporate DMPO motif in drug designing which led to the discovery of several DAAPalouges (small-molecules) as ALK inhibitor from a known SRC inhibitor (AP23464) (Scheme 1). The DMPO installation at C-4 0 enhanced the ALK activity by approximately 7-fold compared to the unsubstituted derivatives in a small focused library closely related to (1). The H-bonding ability of the DMPO functional group with the Lys-NH (K1150) on the protein was explored near the hinge region by synthesizing double-phosphorylated analog (2) which did not exhibit substantial increase in ALK activity (ALK IC 50 ¼ 1.06 nM) but demonstrated remarkably improved selectivity over both IGF1R and InsR (IGF1R IC 50 ¼ 139 nM, InsR IC 50 ¼ 726 nM). 111 In subsequent investigations, the inuence on ALK activity and cellular potency with variety of basic amines containing solubilizing groups as C-ring was surveyed by keeping DMPO motif at ortho-position on A-ring. To this end, solubilizing group screening investigation efforts led to the identication of numerous equipotent amines similar to (3). Among them DAAP (5) was most promising analog with promising potency and selectivity prole (ALK IC 50 ¼ 0.37 nM, IGF1R IC 50 ¼ 24.9 nM, InsR IC 50 ¼ 196 nM, Karpas-299 (ALK + ) ¼ 29 nM, U937 (ALK À ) ¼ 3194 nM). Subsequent lead optimization explored at other positions on the inhibitor core by maintaining the C-ring resulted in (4) bearing a methyl (Me) group at C-5 0 position and it was equipotent to (5) in cellular assays but exhibited moderate enzymatic potency loss. 2 0 -Methoxy (ortho-MeO) at C-ring and 5chloro (Cl) on pyrimidine core was optimal in terms of potency and selectivity. Finally to conrm the critical role DMPO functionality at C-4 of pyrimidine core, a systematic modication of DMPO was carried out with numerous H-bond acceptors (nitrile, amides, t-amides, sulfonamides, methylsulfone, isopropylsulfone acetyl etc.). The DMPO at C-4 revealed as an crucial structural unit indispensable to attain the appropriate balance between potency and selectivity of DAAPalouge (5).
Final DAAPalouge (5) (brigatinib) emerged as most the advanced clinical candidate loaded with phosphine oxide (P] O) warhead and to date it is being investigated in a global phase II registration trial in patients detected with advanced malignancies and has showed activity in crizotinib-resistant patients. Brigatinib revealed almost 10-fold higher potency and selectivity compared to crizotinib. 130 Brigatinib has advanced to phase I/II clinical trials which for this unique inhibitor were initiated 131 in September 2011.
Brigatinib, a pyrimidine-based molecule, is a potent and orally bioavailable 2 nd -generation DAAPalogues (an ALK inhibitor) which occupies the ATP-binding site of ALK preferably in U-shaped conformation. 111,132 Brigatinib incorporates many chemical characteristics that enhance its affinity towards ALK inhibition that were lacked in crizotinib. These features include ortho-methoxy (OMe) group on C-ring, an extended solubilization group, an optimal chloro (Cl) substituent at C-5 and a distinctive ortho-DMPO functionality on the C-4 aniline (Scheme 1). Brigatinib demonstrated potent inhibition with a kinase activity of ALK (IC 50 ¼ 0.62 nmol L À1 ), IGF-1R (IC 50 ¼ 38 nmol L À1 ), insulin receptor kinase (IC 50 ¼ 262 nmol L À1 ) and for other ve tested mutant variants (C1156Y IC 50 . Brigatinib also revealed a higher selectivity and only inhibited eleven additional mutants or native kinases (out of 289 screened) with IC 50 < 10 nmol L À1 . These included ROS1 (IC 50 ¼ 1.9 nmol L À1 ), FLT3 (IC 50 ¼ 2.1 nmol L À1 ), and mutant variants of FLT3 (D835Y) and EGFR (L858R; IC 50 , 1.5-2.1 nmol L À1 ). Brigatinib exhibited more modest activity against EGFR with a T790M resistance mutation (L858R/T790M), native EGFR, IGF1R, and INSR with (IC 50 , 29-160 nmol L À1 ) but did not inhibit c-MET (IC 50 > 1000 nmol L À1 ). This DAAPalouge has exhibited dual inhibition of ALK and EGFR T790M. Due to this inhibitory mechanism brigatinib could be a promising therapeutic option for patients progressing on crizotinib for the activation of the EGFR bypass track. In vitro and in cellular assays against select kinases, the brigatinib has also showed a consistent data for ALK (IC 50 ¼ 0.62 nm L À1 in kinase assay, IC 50 ¼ 14 nmol L À1 in EML4-ALK as a cellular system) and ROS1 (IC 50 ¼ 1.9 nm L À1 in kinase assay, IC 50 ¼ 18 nmol L À1 in CD74-ROS1 cellular system). 117

Medicinal chemistry discovery pathway of brigatinib
Pd-catalyzed phosphorous arylation (P-arylation) methodology was employed to synthesize phosphorus-containing aniline (6) directly from iodoaniline and dimethylphosphine oxide without competitive N-arylation though reactive anilino NH 2 substitutent was there and such kind of transformations were reported recently. 133,134 To this end, S N -Ar reaction of phosphoruscontaining aniline (6) with 2,4,5-trichloropyrimidine using base mediated conditions delivered a known intermediate (7) in the following step. In another pathway, S N -Ar displacement of uoro (F) group of 2-nitro-5-uoroanisole (8) with secondary amine (9) using potassium carbonate as basic media yielded nitro-intermediate (10), followed by common hydrogenation provided aniline (11) in high yield. Finally, subsequent substitution at C-2 of (7) was achieved via an acid-promoted S N -Ar reaction in good yield to provide brigatinib. A summarized drug discovery synthetic pathway of brigatinib recently reported by Huang et al., is depicted in Scheme 2.

Therapeutic trial of brigatinib (Alunbrig™)
4.3.1 Phase I/II trails. In a preliminary dose-nding study, the patients were given 30-300 mg daily of brigatinib (in the phase I) and in the phase II patient received 90, 180, or 90 mg per day for 7 days followed by 180 mg day À1 where 70 patients were crizotinib-resistant and 8 patients were crizotinib-naïve. The most common post-treatment-adverse events were nausea (50%), diarrhea (40%), headache (30%), and cough (30%). Interestingly, in 8% of patients early-onset pulmonary events occurred with dyspnea, pneumonia, and hypoxia, usually within seven days aer starting the treatment. This peculiar pulmonary toxicity was considered to drug related and not a class-related event, observed with a higher starting dose of the drug. 126,135 Scheme 2 Medicinal chemistry discovery pathway of brigatinib.
The promising clinical activity of brigatinib in the patients pre-treated with crizotinib and crizotinib-naïve ALK rearranged NSCLC has been reported. During the advancement in phase I/ II trial, single-arm study, an open label and multicenter study, the brigatinib has revealed signicant clinical activity against brain metastases (NCT01449461). In order to establish the recommended dose in phase II trials, dose escalation evaluation in phase I revealed total doses of 30-300 mg per day. Phase II evaluation demonstrated three once daily dosage regimens; 90 mg, 180 mg, and 180 mg with a 7 days lead in at 90 mg. A cohort of 137 patients (n ¼ 79, 58% were ALK-rearranged NSCLC) enrolled from academic hospitals or cancer centres in the USA and Spain in phase I/II during the period from September 2011 to July 2014 and all were treated. In phase II clinical study, among the total patients established with ALKrearranged NSCLC (n ¼ 79), 52 patients (66%) showed conrmed objective response. In the patients pre-treated with crizotinib (n ¼ 71), a population of patients (n ¼ 51) showed an objective response of 72% and objective response of 100% has been reported for crizotinib naïve patients (n ¼ 8) (NCT01449461).
In phase I/II clinical ndings, brigatinib demonstrated an objective response rate (ORR ¼ 70%, n ¼ 70, the patient were on pretested with crizotinib), and median progression free survival (PFS ¼ 13.5, n ¼ 8, previously on crizotinib). The one-year overall survival rate (OS ¼ 80%, patients previously on crizotinib) and (OS ¼ 100%, in crizotinib-naïve). 136 In a phase II single arm trial (ALTA) study, patients (n ¼ 222) resistant to crizotinib received brigatinib, the ORR (45%) at 90 mg day À1 and ORR (55%) at 180 mg day À1 was observed in two cohorts. In rst dose-nding trial (in ALTA investigation), within the rst 7 days pulmonary toxicity occurred in the 6% of patients (3% were grade 3-4) and it was correlated to the higher dose of the drug (180 mg day À1 ). 137 In phase I/II trial and ALTA investigations in patients (n ¼ 32), the relationship between brigatinib efficacy and ALK mutational status has also been demonstrated. 138 In the samples obtained at baseline (before treatment with brigatinib), the secondary ALK mutations were detected in the 30% of patients, and the ORR was >60% both in patients with or without secondary ALK mutations which demonstrated the activity of brigatinib regardless of the presence of a secondary ALK mutations conditioning resistance to crizotinib. Although two specic mutations (F1174L and G1202R) have been detected aer long-term treatment with brigatinib (in two out of the ve post-brigatinib samples), however the responses were also observed when these mutations were present at baseline. 139 Brigatinib is currently under evaluation in both rst-line setting versus crizotinib in a phase III trial (NCT02737501) and in phase II study (NCT02706626) aer unfruitful outcome of other second-generation ALK inhibitors (ceritinib and alectinib). This last study aims to explore the possibility of brigatinib to overcome the resistance to ALK inhibitors mediated by secondary ALK mutations such as G1202R and I117N/S/T. 140, 141 The clinical activity of brigatinib may further inspire application of phosphorus oxide group as a unique functionality more broadly in the eld of medicinal chemistry. In patients with measurable CNS disease, 53% (8 out of 15) demonstrated an intracranial objective response with an intracranial disease control rate of 87%, and median duration of response was 18.9 months. Therapy emergent side effects were fatigue, nausea, diarrhea, cough and hypertension. ALTA, a phase II trial of brigatinib in patients with ALK + -NSCLC who progressed on crizotinib, has completed recruitment of patients (NCT02094573). In this trial preliminary data include 67% patient with CNS disease and 74% with prior treatment who were treated at 180 mg daily. The reported PFS was 12.9 months (95% CI: 11.1-NR), ORR 54% (97.5% CI:  and CNS response of 67% provides a favorable comparision with previously reported outcomes with ceritinib and alectinib. 141,142 4.3.2 Brigatinib safety prole. In ongoing phase I/II clinical study pertaining to brigatinib, adverse effects (AEs) such as diarrhea (40%), headache (33%), nausea (52%), cough (32%), and fatigue (42%) were most common. The most common grade $ 3 AEs were increased lipase (9%), dyspnea (7%), fatigue (4%) and increased amylase (4%). The most common serious AEs were dyspnea (7%) and pneumonia (6%). Seven patients out of 79 (9%) discontinued the study due to AEs. Overall, 9% of patients showed pulmonary events within seven days of therapy commencement. The rate was lower (4%) in patients who started on 90 mg daily (QD) than those who started immediately on the full 180 mg QD dose (14%). No reports of pulmonary events aer dose escalation in patients who received 90 mg QD for about 7 days followed by 180 mg QD were observed. 124,126,[141][142][143] 4.3.3 Ongoing clinical trials of brigatinib. Phase III ALTA-1L trial (NCT02737501) commenced in April 2016 which were designed to draw a comparision between brigatinib and crizotinib in the patients conrmed with advanced NSCLC. This phase III multicenter open-label study of brigatinib is expected to be completed primarily in April 2019. On the other side, phase II study funded by ARIAD started in March 2017 is underway and recruiting patients. This phase II trial is planned to evaluate drug safety and effectiveness in patients with advanced NSCLC who either had rst-line cancer treatment but got worse, even aer, or while taking 2 nd -generation ALK inhibitors. This trial is expected to be completed in June 2020 (NCT02706626).

Discovery and clinical development of NVP-TAE 684 DAAPalouge
This DAAPalouge was discovered by Novartis and had 2,4-diarylaminopyrimidine (DAAP) core decorated with chloro (Cl) at C-5 position and exhibits anticancer chemotherapeutic activity. It was characterized as a highly potent and specic inhibitor of NPM-ALK. The DAAPalouge (NVP-TAE684) is an inhibitor of ALK and exhibited Ba/F 3 -NPM-ALK IC 50 ¼ 3.0 nM, Ba/F 3 -Tel-InsR IC 50 ¼ 44 nM, Ba/F 3 -WT IC 50 ¼ 1336 nM and Karpas-299 IC 50 ¼ 2.2 nM. Leucine-rich repeat kinase 2 (LRRK2) is another highly attractive therapeutic target and its association with Parkinson's disease has been reported. NVP-TAE684 is also a highly potent inhibitor of LRRK2 and has demonstrated promising LRRK2 kinase activity (LRRK2 wt IC 50 ¼ 7.8 nM, G2019S mutant IC 50 ¼ 6.1 nM). NVP-TAE684 remarkably inhibited Ser935 and Ser910 phosphorylation of both LRRK2 wt and G2019S mutant at a concentration ranging from 0.1 to 0.3 mM in cells, and in mouse spleen and kidney, but not in brain, at an oral doses of 10 mg kg À1 . 144 In patients who demonstrated anti-VEGF refractory cancers, c-Fes plays central role in angiogenic cellular responses of endothelial cell and has been reported another potential target. Beside this bone marrowderived monocytic lineage regulates angiogenesis and c-Fes has been responsible to express in these cells. Investigations pertaining to c-Fes has made TAE-684 a potent inhibitor of c-Fes. 145-147 NVP-TAE684 induces apoptosis and inhibited growth of diffuse large B-cell lymphoma cells in animal models and induces tumor regression. NVP-TAE684 also induces cell cycle arrest and apoptosis in animal models of anaplastic large-cell lymphoma, preventing lymphomagenesis and inducing regression.
NVP-TAE684 was discovered through high throughput screening (HTS) of a kinase-directed small-molecule library aimed to explore potent entity selectively cytotoxic to BaF 3 NPM-ALK but should not be toxic to non-transformed parental BaF 3 cells. 56 An inhibition for proliferation of BaF 3 NPM-ALK cells (IC 50 ¼ 3 nM) and for parental BaF 3 cells (IC 50 > 1 mM) was observed which facilitated the initiation of discovery process of NVP-TAE684. Interestingly, NVP-TAE684 inhibited the human Karpas-299 ALCL cell line (IC 50 ¼ 3 nM) and showed an improvement on crizotinib. Additionally, NVP-TAE684 exhibited from 100-1000 times higher IC 50 values for non-ALK kinases. The assay was comprised of a panel of thirty ve (35) BaF 3 cells which were transformed with several tyrosine kinases constitutively activated by fusion to the TEL oncogene. This specicity was associated as a consequence of the bulky hinge region residue at the 1198 position of the non-ALK kinases (e.g. Tyr or Phe compared to Lys existed in ALK).
In another investigation, screening of more than six hundred cancer cell lines was performed for sensitivity to obtain selective ALK inhibitor. As an outcome of the aforesaid ndings, NVP-NVP-TAE684 was reported to be superior ALK-TKI over Xalkori (crizotinib) in Karpas-299 lymphoma and in the SU-DHL-1 cell lines. 16 NVP-TAE684 has also emerged as selective for oncogenic ALK containing tumors. 148 The evolution of NVP-TAE684 from the HTS cellular screening is summarized in Scheme 3.
Although the DAAPlouge TAE-684 is a potent ALK inhibitor but later ndings revealed that NVP-TAE684 upon metabolic oxidation could produce large number of reactive adducts. Metabolic oxidation was reported to be a potential threat for signicant toxicological liabilities. Approximately 20% of NVP-TAE684 was found to be converted into reactive products upon incubation in liver microsomes as determined by semiquantitative LC-MS analysis. The reactive species were determined using glutathione (GSH). 149 No relationship was established between hepatotoxic drugs and the development of GSH adducts. 150 However, it was hypothesized that reactive metabolites could have a critical role in idiosyncratic 151 and/or in other toxicities. 152 To address the further developmental deciencies of NVP-TAE684 (as a rst generation ALK inhibitor), modeling information, designing strategy, and focused investigation to achieve enhanced kinase selectivity of NVP-TAE684 was further surveyed extensively. The installation of iso-propoxy (i-Pr) instead of methoxy (MeO) revealed as well tolerated moiety to address selectivity issues. Further optimization of NVP-TAE684 and potential idiosyncratic toxicity issues led to the eventual discovery of ceritinib which got accelerated FDA approval for the patients with ALK-positive metastatic NSCLC in April 2014. However, on May 26, 2017, FDA declared regular approval to ceritinib for the patients suffering from ALK-positive metastatic NSCLC. 153

Discovery and clinical development of Cephalon DAAPalouges
In the following sections, medicinal chemistry efforts, early discovery description and clinical developments of novel Cephalon DAAPalouges such Cephalon 1, CEP-28122 and CEP-37440 is discussed. Based on reported investigation regarding the involvement of ALK gene rearrangements as an oncogenic driver of ALK-positive cancers, the inhibition of ALK with highly selective small molecular entities has emerged as an attractive area of research and this has led the multiple pharmaceutical companies to discover novel ALK inhibitors. To this end, plethora of unique chemical scaffolds and interesting smallmolecules for ALK inhibition were explored. Surprisingly, at that time the DAAPlouges were not well investigated rather tetrahydropyrido [2,3-b]pyrazine, 154 indolocarbazole, 155 pyridone, 156 1H-pyrrolo [2,3-d]pyrimidine, 157 dianilinopyrimidine, 108 and aminopyridine 158 were among the well probed pharmacophores. [159][160][161] Cephalon 1 (15) (Scheme 5) is a DAAPalogue derivative developed by Cephalon. It is a potent, selective, and orally bioavailable ALK inhibitor but this analog exhibited potential hERG liability. The  Review subsequent efforts for functional motif exploitation to balance out potency with promising physicochemical properties. 162 This approach resulted in another potent diarylaminopyrimidine ALK inhibitor (DAAPalogue) known as CEP-28122. Interestingly, CEP-28122 was another DAAPlouge developed by Cephalon. The synthesis of CEP-28122 (17) was conducted at the department of chemistry at Cephalon, Inc., and its synthetic routes have been reported elsewhere. 163 CEP-28122 is a potent, selective, and orally bioavailable ALK inhibitor. CEP-28122 (17) demonstrated ALK kinase activity (IC 50 ¼ 1.9 nM) in an enzyme-based TRF assay. It has shown concentration-dependent growth inhibition/cytotoxicity of ALK + -ALCL, NSCLC and neuroblastoma cells. CEP-28122 (17) also exhibited dose-dependent inhibition of ALK tyrosine phosphorylation in tumor xeno-gras in mice. 164 The strategic development of Cephalon 1 (15) and CEP-28122 (17) is shown (Scheme 5). Medicinal chemistry route as an initial strategy for the synthesis of CEP-28122 (17) exploited the coupling of three key structural motifs such as a morpholine substituted on benzocycloheptane fragment as Bring, a diaminopyrimidine central core as A-ring, and a bicyclic amino amide portion as C-ring. 163 However, to meet preclinical and clinical supplies, a unique eight-step (longest linear), convergent, and scalable manufacturing route discovered later. The development of scalable new route to CEP-28122 (17) was reported in 2012 which employed several distinctive synthetic approaches such as a selective nitration using para-blocking group methodology, a one-pot amination-transfer hydrogenation to provide four reductions approximately in simultaneous fashion, resolution based on an enzymatic reaction, and the leveraging of an in situ generated mixed mesylate hydrochloride salt of CEP-28122. 165 The nal pilot plant synthetic pathway to CEP-28122 is shown in Scheme 4.

Synthetic strategy of emerging DAAPalouge CEP-37440 from 12
In a quest to discover potent ALK inhibitors, 7-amino-1,3,4,5tetrahydrobenzo-[b]azepin-2-one (12) (Scheme 5) was identied as a novel DAAPalouge (a small molecule) which exhibited potent activity (ALK IC 50 ¼ 15 nM, IR IC 50 ¼ 23 nM, Karpas-299 cell IC 50 ¼ 30 nM) but the margin against IR was merely 1.5fold. Benzamide functional group was a key potency determinant for ALK in (12). Due to the involvement of insulin receptor (IR) in the regulation of glucose transport, glycogen, and fat biosynthesis, selectivity towards IR were of particular interest. Based on promising SAR, potent enzymatic/cellular ALK inhibition, and modulation of IR activity, improvement in modest oral bioavailability and optimization of IR selectivity was surveyed on azepinone core (B-ring) (Scheme 5). Ethyl substitution on nitrogen atom of azepinone for improved pharmacokinetic properties (PK) parameters was envisioned as essential group. For reasonable selectivity, ortho-MeO group either at 6-position or 8-position of the benzazepinone was proposed to be superior. Furthermore, focused modication on A-and C-rings, a bicyclo[2.2.1]hept-5-ene ring system was identied as an alternative C-ring partner for improved IR selectivity. Further optimization of potency, off-target selectivity, and PK parameters led to the discovery of a novel and potent bridged bicyclic derivative (13) (Scheme 5). Gratifyingly, (13) demonstrated impressive activity in an ALK assay (enzymatic or cellular) measuring oncogenic NPM-ALK autophosphorylation (ALK IC 50 ¼ 14 nM, IR IC 50 ¼ 597 nM, Karpas-299 cell IC 50 ¼ 45 nM) with modestly improved IR selectivity (IR/ ALK ¼ 43). Moreover, oral administration of lead inhibitor (13) to mice induced with ALK positive ALCL SCID xenogras resulted in near-complete/complete regressions. 166,167 Although (13) presented a balanced prole of ALK potency and exhibited global kinase selectivity but further efforts were needed to improve IR selectivity (enzymatic IR/ALK IC 50 ratio of >100). Despite of the promising features, (13) still lacked the capability of dose escalation in preclinical toxicological models which precluded its further improvement.
Cephalon researcher during the course of their endeavors synthesized and proled a diversity of analogs closely related to NVP-TAE226 (ref. 168) but bis-adduct (16) (Scheme 5) was discovered accidentally. 169 This bis-adduct (16) demonstrated ALK inhibition equivalent to NVP-TAE226 and tremendous escalation in IR selectivity but lower (2.4-fold) cellular inhibition. The serendipitous ndings were capitalized and a morpholine-phenyl moiety was incorporated as C-ring fragment which led to emergence of novel compound (14) with benzazepinone as B-ring. Compound (14) exhibited drop-off in both ALK enzymatic activity and cellular potency was observed but improvement in potency while maintaining this high degree of IR selectivity was sought. Rather than benzazepinone moiety, a structural motif such as 2,3,4,5-tetrahydro-1H-benzo[d]azepine was perceived to be a synthetically tractable functionality with potential handles to modulate both potency and physiochemical properties and this was incorporated into the pharmacophore. Extensive SAR investigation and substitutions at benzazepine nitrogen of (15) and subtle variations to the nature of groups impacted on both enzyme and cell activity prominently. Numerous analogs were designed wherein basicity of benzazepine nitrogen emerged to be critical contributing factor. Gratifyingly, novel amide (15) was identied as orally bioavailable, most potent and highly selective ALK inhibitor (ALK enzyme IC 50 ¼ 4 nM, ALK cell IC 50 ¼ 30 nM, IR enzyme IC 50 ¼ 1380 nM, IR/ALK IC 50 ratio of 345) and displayed promising in vivo efficacy in ALCL tumor models. 169 Candidate (15) demonstrated tumor regressions at 30 mg kg À1 and 55 mg kg À1 b.i.d., po doses, at which (15) was found to be tolerated fairly. Neither evident toxicity nor body weight loss were observed. Although compound (15) exhibited potent in vitro ALK activity but potential hERG liability was main concern (IC 50 ¼ 3.9 mM for hERG, patch clamp assay) and against the most important CYP isoforms (15) demonstrated minimal inhibition for CYP1A2 and 2D6. 170 In subsequent investigations, the basic nitrogen atom was shied outside the 7-membered ring in (17) to further control the potency and selectivity variances that were associated with the analogs of azepine (15). The new B-ring was aminobenzocycloheptene motif with (S)-conguration at the morpholino carbon juncture. To generate potent ALK inhibition, enhanced IR selectivity, and dose proportional escalation in preclinical toxicology models, a dedicated medicinal chemistry efforts were therefore initiated to evaluate the amendments on aminobenzocycloheptene ring system. With this strategy Cephalon researchers identied (17) wherein at C-2 position of azepine or azepinone fragments were replaced with aminobenzocycloheptene motif as B-ring. Furthermore, earlier established bicyclo[2.2.1]hept-5-ene ring moiety was incorporated at C-4 position of pyrimidine as C-ring to afford the fully assembled HPLC separable (S)-enantiomer DAAPalouge scaffold. Compound (17) revealed as exceedingly potent and highly selective DAAPalouge (an ALK inhibitor). The PK of compound (17) was investigated in numerous species as shown in Table 2.

Pilot plant synthesis of CEP-37440 DAAPalouge
To provide early-phase clinical supplies of CEP-37440, Allwein et al., reviewed the previously described medicinal chemistry route of CEP-28122 and presented a scalable route to TEV-CEP-37440 (ref. 171) to obtain 22 kilograms of (19) as trihydrochloride dihydrate. The pilot plant synthesis involve the use of several steps such as a unique ring expansion approach, parablocking group strategy followed by selective nitration, amination-hydrogenation in a single-pot, resolution of diastereomeric salt, a step to avoid a dangerous intermediates, and a large-scale production of trihydrochloride dihydrate salt were the key highlighted features. The optimized pilot plant synthesis provided TEV-37440 as trihydrochloride dihydrate in >99.5% purity and in 10.7% overall yield (Scheme 6). 172
A single dose of novel CEP-28122 (17) given orally at 30 mg kg À1 revealed more than 90% inhibition of NPM-ALK phosphorylation in the tested NPM-ALK positive ALCL Karpas-299   (17) was well tolerated and at both dosing regimens neither evident toxicity nor a signicant body weight loss was observed. 112 Dose-dependent antitumor activity potential was also assessed in NPM-ALK-positive ALCL and EML4-ALK-positive NSCLC tumor xenogras in mice wherein CEP-28122 (17) was administered orally. It showed complete or near to complete tumor regression by following a therapy at a dose of 30 mg kg À1 b.i.d. or higher. Furthermore, CEP-28122 (17) induced growth inhibition and cytotoxicity of neuroblastoma cell lines harboring ALK activating mutants (F1174L in NB-1643 cells and R1275Q in SH-SY5Y cells, respectively). However, CEP-28122 (17) did not induce growth inhibition and cytotoxicity in those which contained ALK wild type (WT) without gene amplication (SKNAS cells). This indicated that (17) was active against the two ALK activating mutants usually found in human neuroblastoma. From the combination of entire favorable biological and pharmaceutical properties, the CEP-28122 (17) was discovered as a preclinical candidate. 112 The PK characteristics of novel analogs (13)(14)(15)(16)(17)(18)(19) discovered during the strategic development of CEP-37440 from (12) are presented in Table 3.
Since the discovery of CEP-28122 DAAPalouge (17), the landscape of small molecule inhibitor had changed to broader extent and four distinct molecular entities had been approved by FDA to treat EML4-ALK positive NSCLC. Although (17) demonstrated overall moderate in vivo clearance potential [CD-1 mouse, Cl ¼ 29 mL min À1 kg À1 ; rat, Cl ¼ 17 mL min À1 kg À1 ; monkey, Cl ¼ 28 mL min À1 kg À1 ], and the in vitro liver microsomal stability values [liver microsome t 1/2 (min): CD-1 mouse, 13; rat, 7; human, 10] which prompted the further possibility for improvement. Innovative chemical architecture which could constrain the small-molecule ligands into the putative active conformation was executed which resulted in generation of architecturally distinctive template to overcome associated pharmaceutical liabilities. Recently focal adhesion kinase (FAK) has been investigated as a cytoplasmic protein tyrosine kinase and its overexpression and activation has been characterized in several advanced-stage solid cancers. FAK activation and overexpression are suggestive of numerous solid tumors, mainly those have susceptibility for bone metastasis especially NSCLC, ovarian, breast, prostate, and HNSCC carcinomas. [173][174][175] Due to the growing interest in focal adhesion kinase (FAK) inhibitors and their clinical development, 176 extensive SAR was sought on (17) which resulted in superior kinase selectivity prole, Scheme 6 Pilot plant synthesis of CEP-37440 DAApalouge to provide clinical supplies. 171 favorable brain penetration, and promising activity in ALK + -NSCLC tumor xenogras. Compound (18) also exhibited imperious biochemical and pharmacodynamic activity both in vitro and in vivo, and enhanced antitumor effectiveness in FAK and FAK-dependent tumors. 112 In further exploration, nature of substituents (morpholine, N-methyl piperazine and methoxyethylamine) on benzocycloheptane ring system and position renement of morpholine itself was surveyed. Compared to (19) (ALK IC 50 ¼ 3.1 nM, cell IC 50 ¼ 22 nM), both diastereomers of (18) were almost 4-fold lesser potent in enzymatic assays, and 10-fold lesser potent in cells based assays but maintained plausible hERG selectivity (7.9-9.1 mM) and CYP inhibition (>10 mM). Furthermore, an enhancement in liver microsomal stability in mouse and rat was observed but human and monkey liver microsomal stability was lesser than anticipated. Pharmacokinetics of (18) in rat demonstrated high iv AUCs but lower Cl and bioavailability (F ¼ 11%). Encouraging results compelled to improve ALK potency by maintaining the additional FAK effectiveness (potency) and human liver microsomal metabolic stability as next envisioned agenda. Subsequent investigation led to the optimization of substituent at piperazine and C-2 position of pyrimidine. Previously reported bicyclo[2.2.1]heptene motif at C-4 of pyrimidine was main metabolic hotspot in (17). To this end, to expedite discovery efforts the racemic mixtures of aromatic sulfones, sulfonamides, N-alkylsulfonamides, imidazoles, pyrazoles, and cyclic sultams were surveyed as novel H-bond acceptors on C-4 position of pyrimidine which resulted in Nmethylbenzamide as optimal C-ring partner. On the other hand, variation of N-methyl substituents on the piperazine ring to hydroxyethyl functionality was identied as a noteworthy group on B-ring.
Racemic mixture of (19) revealed as a novel lead candidate with promising ALK enzymatic activity (IC 50 ¼ 4 nM), cellular potency (IC 50 ¼ 40 nM), and acceptable dual ALK and FAK kinome selectivity. Candidate (19) proved adequate in vitro liver microsomal stability (rat and human t 1/2 > 40 min) across the tested species, adequate oral bioavailability (47%), and issues of hERG activity (IC 50 $ 10 mM) were also circumvented relative other analogs. Furthermore, (R) and (S)-enantiomers of B-ring were installed and individual enantiomers of (19) were synthesized and proled. Findings revealed both of the enantiomers fairly comparable across off-target selectivity, on-target potency, and in vitro ADME/toxicity parameters but in spite of comparable in vitro metabolic stability of both enantiomers, (S)enantiomer (19) showed 2-3-fold enhancement in oral bioavailability and considerable higher area under the curve (AUC). The structural basis for this observed potency was recognized using molecular docking. For clinically-derived resistance mutations against small-molecule, the ALK inhibitors compound (19) was tested for ALK mutants and showed equipotent potencies (ALK (1151Tins) K d ¼ 2.6 nM, ALK (C1156Y) K d ¼ 1.6 nM, ALK (F1174L) K d ¼ 1.1 nM, and ALK (L1196M) K d ¼ 3.3 nM) to that of FDA approved rst and 2 nd -generation ALK inhibitors. 112 Compound (19) exhibited promising in vitro ADME properties and appropriate oral bioavailability in three investigated Table 3 Pharmacokinetic parameters of novel analogs (13)(14)(15)(16)(17)(18)(19) in CD-1 mouse, Sprague-Dawley (SD) rats, and cynomolgus monkeys PK parameters    species. CEP-37440 (19) showed promising brain exposure which may inuence physiologically related escape pathways, for example brain metastases. Moreover, dose-dependent antitumor efficacy has also been reported in several animal models of anaplastic lymphoma kinase-positive and FAK-driven cancers. CEP-37440 (19) (19) also efficiently reduces the growth of the primary tumor xenogras and inhibits the development of brain metastases in mice. 177 In preclinical proling, CEP-37440 (19) has demonstrated advantageous metabolic stability, pharmacokinetic and proven the CNS brain penetration. 121

Conclusion and future prospectives
The trend and rapidity pertaining to drug discovery of anticancer agents has substantially changed the understanding of pathogenesis and cancer therapy. The extensive inclusion of computer-aided approaches has proven important involvement and has eliminated the requirement of expensive and timeconsuming tasks frequently used in experimental drug discovery processes. [178][179][180] To this end, widespread use of the techniques such as HTS, virtual screening (VS), pharmacophore modeling, and three-dimensional quantitative structureactivity relationships (3D QSAR) have made the lead compound discovery process much faster. Although the HTS is costly technique yet it has shown remarkable utilization in drug discovery campaigns since 1980. However, VS approach applied in the private sector and academia is a cost-saving technology, and has emerged as a versatile tool in the hit to lead identication and eventually becoming an integral part of drug discovery war, whereas the pharmacophore modeling has also been effectively used in recent years to identify a common pharmacophore hypothesis. In addition, the 3D QSAR model unfolds the statistical correlation between structure of designed analog and predictive functions. The 3D QSAR models are also playing signicant role to accomplish greater success rate of experimentation, cost reduction of development, and short cycle of research in drug discovery. [181][182][183] Due to inclusion of above said technologies in drug discovery campaigns, the current decade (2007-2017) has dragged prodigious progress globally in NSCLC pertaining to drug development and in clinical settings. [183][184][185][186][187] Novel targeted antineoplastic agents loaded with anticancer warhead to demonstrate imperious efficacy have been synthesized and introduced for clinical use. Second generation DAAPlouges including alectinib are known to be effective in greater than 50% NSCLC patients who were refractory to crizotinib. Unfortunately, the emergence of resistance to 2 nd -generation ALK-TKIs is a major concern to date. Among the identied multiple resistance mechanisms, the G1202R mutation of ALK is emerging as resistance to all currently available ALK-TKIs including DAA-Palouges. Lorlatinib (non-DAAPalouge) is a rst macrocyclic 3 rdgeneration ALK-TKI which have demonstrated effectiveness against the G1202R mutation of ALK but again compound mutation such as C1156Y/L1198F in the ALK kinase domain has been revealed as a major concern. Brigatinib is a DAAPalouge which has shown the potential to overcome the resistance mechanism to rst and second generation ALK inhibitors including the G1202R mutation of ALK in xenogra models. 117 However, many other newly discovered molecular entities have demonstrated effectiveness against the G1202R mutation. [188][189][190] Furthermore, the current median overall survival of patients with ALK-rearranged lung cancer is above 4 years only. This over survival is achieved through the treatment protocols of using sequential ALK-TKIs in addition to other FDA approved chemotherapeutics. Finally, despite the discovery the development of several potent ALK TKIs including DAAPalouges have led to the substantial increase of drugs but still it is believed that currently available therapies are palliative rather curative. Thus further discoveries are indispensable to develop superior therapeutic strategies.

Conflicts of interest
The authors also declare no conicts of interest.