and Anaplastic Lymphoma Kinase (ALK) - ACS Publications

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Discovery of Clinical Candidate CEP-37440, a Selective Inhibitor of Focal Adhesion Kinase (FAK) and Anaplastic Lymphoma Kinase (ALK) Gregory R. Ott,*,† Mangeng Cheng,† Keith S. Learn,† Jason Wagner,† Diane E. Gingrich,† Joseph G. Lisko,† Matthew Curry,† Eugen F. Mesaros,† Arup K. Ghose,† Matthew R. Quail,† Weihua Wan,† Lihui Lu,† Pawel Dobrzanski,† Mark S. Albom,† Thelma S. Angeles,† Kevin Wells-Knecht,† Zeqi Huang,† Lisa D. Aimone,† Elizabeth Bruckheimer,‡ Nathan Anderson,‡ Jay Friedman,‡ Sandra V. Fernandez,§ Mark A. Ator,† Bruce A. Ruggeri,† and Bruce D. Dorsey† †

Teva Branded Pharmaceutical Products R&D, 145 Brandywine Parkway, West Chester, Pennsylvania 19380, United States Champions Oncology, Inc., One University Plaza, Suite 307, Hackensack, New Jersey 07601, United States § Thomas Jefferson University, 233 South 10th Street, 1002 BLSB, Philadelphia, Pennsylvania 19107, United States ‡

S Supporting Information *

ABSTRACT: Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.

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INTRODUCTION The identification of tumor-specific genetic aberrations, their validation as druggable targets, and therapeutic interdiction with small molecule drugs and/or biologics has led to a distinct clinical benefit for specific, genetically defined patient populations and has further advanced the field of personalized medicine.1 In particular, non-small-cell lung cancer (NSCLC) has seen the development of targeted small-molecule therapies to oncogenic driver mutations in the epidermal growth factor (EGFR) gene (gefitinib, erlotinib, afatinib),2,3 the oncogenic fusion protein echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase gene (EML4-ALK) (crizotinib, certinib, alectinib),4 as well as other oncogenic proteins.5 The emergence of tumor resistance through drug-induced activating mutations, gene amplification, and/or induced-resistance/ alternative signaling pathways has led to extensive effort in developing second and third generation therapies.6−9 Despite the ability to genotype and stratify patients to tailored therapy, significant medical need exists in NSCLC to address the abovementioned resistance to targeted therapy, metastasis, as well as © XXXX American Chemical Society

other genetic drivers/variants of this disease that have not been clinically validated with small-molecule/biologic therapeutics (e.g., KRas).10 The biological rationale, therapeutic intervention, and clinical proof of concept data with small−molecule ALK inhibitors have been well documented.11−13 Current approaches toward ALK+ cancers have focused on improving potency toward drug-induced activating mutations as well as incorporation of ancillary activity against other oncogenic signaling mechanisms14,15 to combat resistance and the recurrence of local disease and distal metastases. Our initial efforts in the ALK field led to the discovery of 1 (Figure 1), a selective ALK inhibitor which advanced to preclinical development.16,17 Several distinct challenges for our next generation of inhibitors were readily apparent which included not only addressing the limitations of the lead molecule itself (e.g., metabolic stability, high plasma protein binding) but the broader, changing landscape of the Received: April 1, 2016

A

DOI: 10.1021/acs.jmedchem.6b00487 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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specifically NSCL, breast, ovarian, prostate, and HNSCC carcinomas.21,22 Interest in small molecule FAK inhibitors has grown and is currently being evaluated at the clinical level.23 Furthermore, a very recent report identified FAK as a key mediator of the immune response in certain cancers and provides strong evidence that adjunctive administration of small-molecule FAK inhibitors would be beneficial with inhibitors of T-cell immune checkpoint antibodies (i.e., antiPD-1, CTLA-4).24 Herein, we describe the SAR, in vitro and in vivo properties, and the discovery of a clinical level compound, 27b (CEP37440). Compound 27b improved upon the pharmaceutic properties of 1 and also demonstrated an enhanced pharmacological and kinase selectivity profile with activity in ALK-positive NSCL tumor xenografts, favorable brain penetration, and both in vitro and in vivo biochemical and pharmacodynamic activity/antitumor efficacy against focal adhesion kinase (FAK) and FAK-dependent tumors. Furthermore, this activity is especially relevant to treating subsets of inflammatory breast cancer (IBC), an aggressive and highly metastatic subset of breast cancer that has recently been shown to have amplification FAK in the preponderance of tumors evaluated.25 This selective, dual-action inhibitor provides a novel therapeutic opportunity to treat ALK- and FAK-driven malignancies particularly in NSCLC, IBC, and potentially as an adjunctive to immunotherapy.

Figure 1. Teva small molecule ALK inhibitors.

small-molecule ALK inhibitor field, which has seen the approval of three distinct molecular entities to treat EML4-ALK defined NSCLC as first or second line therapy, as well as specific druginduced resistance. Toward this end, a multipronged strategy was developed which included new chemical architecture by constraining the small-molecule ligands into the putative active conformation,18−20 as well as fine-tuning the current lead series. This strategy made ample use of our broad knowledge base of structure−activity relationships (SARs) which engendered cross-fertilization among the architecturally unique core scaffolds (2−4). Ultimately, we addressed the pharmaceutical liabilities, demonstrated in vitro activity against clinically relevant ALK mutations, and also capitalized upon SAR surrounding ancillary activity against focal adhesion kinase (FAK). In tumors, FAK activation mediates anchorageindependent cell survival, one of the hallmarks of cancer cells, as well as migration, invasion, and angiogenesis, all critical components in the process of metastatic progression. FAK overexpression and activation are indicative of multiple solid tumors, particularly those with a propensity for bone metastasis,

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RESULTS AND DISCUSSION Our lead inhibitor 1, as well as other known26−28 ALK inhibitors, contains a diaminopyrimidine core motif. The synthetic strategy employed a highly convergent approach with fragment disconnections centered around the 5-chloro2,6-diaminopyrimidine designated as A−B−C shown in Figure 2. The first disconnection at the 2-position affords amines A and advanced 6-amino-2,5-dichloro fragments B−C. Further disconnection yields starting 2,5,6-tricholoropyrimidine B and the amines C. The synthesis of the key amines A and the elaboration to the final diaminopyrimidines are shown in Schemes 1−4. The intermediate aniline 8 (Scheme 1) was derived from the known ketone 529 through reductive amination with morpho-

Figure 2. Retrosynthetic disconnections for 4-chloro-2,6-diaminopyrimidines. B



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Scheme 1a

a

Conditions: (a) morpholine, NaBH(OAc)3, AcOH, THF (99%); (b) KNO3, TFAA, MeCN (19%); (c) H2, Pd/C, EtOH (99%).

Scheme 2a

a

Conditions: (a) KNO3, TFAA, MeCN (36%); (b) amine, NaBH(OAc)3, AcOH, THF (24−85%); (c) H2, Pd/C, EtOH (61−99%).

Scheme 3a

a

Conditions: (a) methanesulfonic acid, 1-methoxy-2-propanol, Δ or microwave.

line to provide 6 in near quantitative yield. Nitration provided a mixture of 2-nitro (7) and 4-nitro isomers (approximately 1:1) which were separable by silica gel chromatography. Reduction of the nitro group provided racemic 8. The 6-amino derivatives (Scheme 2) were synthesized from known ketone 9.30 A diverse set of substituents were desired; thus nitration provided 10 and 4-nitro, which were separable on silica gel. Reductive amination provided amines 11b−d; for 11a, the process was reversed. Reduction of the nitro group gave 12a−d.

The intermediate 13 (Scheme 3) was independently reacted with 8 and 12a−d to provide final compounds 14−18 via methanesulfonic acid promoted addition. Intermediates 21a−i31 (Scheme 4) were available from anilines 19a−i via base-promoted addition to 2,4,5-trichloropyrimidine. The final compounds 22−30 were then synthesized in an analogous fashion to that shown in Scheme 3. Separation of racemic 12d (Scheme 5) into individual enantiomers was accomplished using SFC (Chiralcel OJ-H). The individual enantiomers were then converted using C



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Scheme 4a

a

Conditions: (a) K2CO3, DMF; (b) 12a or 12d or (R)-12d or (S)-12d, methanesulfonic acid, 1-methoxy-2-propanol, Δ or microwave.

Scheme 5a

a

Conditions: (a) Chiralcel OJ-H (3 cm × 15 cm), 15% MeOH, 0.2% diethylamine, CO2 (SFC); (b) p-Br-benzoyl chloride.

microsome incubations provided clarity and direction for this approach. As shown in Figure 3, the two major locations for oxidative metabolism proved to be the morpholine ring, which was oxidized and degraded, and the olefin of the bicyclo[2.2.1] system which was epoxidized. The degree and location of metabolism in rat and human liver microsomes were quite similar and suggested that in vitro and in vivo screening in rats would be reasonably predictive. The in vitro potency and metabolic stability results for the initial series of analogues are shown in Table 1. A clear deleterious effect on potency was observed when the 6-position morpholine was moved to the α-position (relative to the morpholine of 1) on the benzocycloheptane ring (14). In contrast, when the morpholine was moved to the α′-position, providing the separable diastereomers 15a and 15b, potency was within 2- to 4-fold relative to 1. Selectivity for the insulin receptor (INSR) was maintained, though unfortunately so was the short t1/2 in liver microsomes. We decided to further pursue C6-substituted analogs based upon the maintenance of potency/selectivity relative to 1. A significant improvement in rat liver microsome stability was gained when the morpholine was switched to the N-methylpiperazine (t1/2 = 5 min for 15a,b,

procedures outlined in Scheme 4 to 27a,b. The absolute stereochemistry was determined using X-ray (anomalous dispersion) of a single crystal of the bis-p-bromobenzoyl derivative 31, which was available via reaction of the first eluting isomer with p-bromobenzoyl chloride.32 Thus, the first eluting enantiomer used in the preparation of 27a was determined to be the (R)-configuration; by analogy 27b was assigned (S)configuration. Concurrent with our strategy to advance backup compounds to the lead candidate by developing novel chemical architecture, we also focused on fine-tuning 1 by making minor structural modifications to address both pharmaceutical limitations and broader, pharmacological aspects. Despite moderate in vivo clearance values for 1 [CD-1 mouse, Cl = 29 mL min−1 kg−1; rat, Cl = 17 mL min−1 kg−1; cynomolgus monkey, Cl = 28 mL min−1 kg−1], the in vitro liver microsome stability values [liver microsome t1/2 (min): CD-1 mouse, 13; rat, 7; human, 10] suggested room for improvement. Enhanced metabolic stability would potentially lead to lower in vivo clearance and thus increase overall exposure and bioavailability, since permeability and solubility were not limiting factors. Furthermore, identification of metabolic hotspots from liver D



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Figure 3. Major sites of metabolism from in vitro liver microsome incubation (10 μM concentration for 120 min in the presence of NADPH and analysis by LC/MS/MS.).

t1/2 = 39−40 min for 16a,b) without eroding the potency or selectivity. A similar result was observed by “opening” the morpholine to give methoxyethylamine derivative 17. However, in each of these cases, the human liver microsome stability was still unfavorable (t1/2 ≤ 15 min). The inhibitors 16a,b were put through an expanded in vitro and in vivo assay regimen to benchmark against 1. Table 2 shows the in vitro and in vivo profiling of the two individual distereomers. Along with ALK (enzyme, cell assays), kinase selectivity was assessed against an internal panel (INSR, JAKs 1, 2, 3, PYK2, TYK2, FAK, and cMet)33 as well as an external panel (1 μM concentration, DiscoveRx).34 In vitro ADME/ toxicology profiling was also completed (hERG, CYP inhibition, expanded liver microsome stability) as well as iv/ po pharmacokinetics in rats. Relative to compound 1 (ALK IC50 = 1.9 nM, cell IC50 = 25 nM), 16a,b were about 4-fold less potent in enzyme and 9- to 10-fold less potent in cells. Overall kinome selectivity (1 μM screening concentration) was improved relative to 1 with S(90) < 0.10 for both. Surprisingly, the modification of moving the heterocycle from the 7-position to the α-position resulted in introduction of near equivalent activity against focal adhesion kinase (FAK) compared to the 60-fold separation for ALK over FAK observed for compound 1. The structural basis of this activity will be discussed in the context of further optimized analogs (vide infra). Importantly, reasonable hERG selectivity and CYP inhibition profiles were maintained as well as an improvement in liver microsome stability in mouse and rat; however, monkey and human stability was less than desired. Pharmacokinetics in rat35 demonstrated high iv AUCs and low Cl (data not shown) and reasonable oral AUCs for both compounds and thus low overall bioavailability (F = 11%). Upon the basis of these promising results, we focused our strategy on further improving the potency of these inhibitors for ALK, maintaining the ancillary FAK potency and improving upon the human liver microsome metabolic stability. A subtle, yet noteworthy, SAR point was identified in the series by

changing the N-methyl substituent on the piperazine to a hydroxyethyl substituent. The resultant analogues 18a,b proved to be slightly more potent in the enzyme assay and 4- to 5-fold more potent in cells, and potency was now in a similar range as 1. Other properties remained similar including selectivity (INSR and kinome), hERG (IC50 > 10 μM), metabolic stability, and oral bioavailability (10%). As noted above, the other main metabolic hot spot in 1 proved to be the olefin of the bicyclo[2.2.1]heptene systems. Previous efforts28 and literature reports36 had demonstrated that aromatic sulfonamides/sulfones as well as N-alkylsulfonamides and other hydrogen bond acceptor motifs37 were acceptable substituents on the aromatic group in the 2-position. Toward defining the favored substituent on the piperazine, another direct comparator set was synthesized with the 2-(N-methylmethanesulfonamide)phenyl ring in place of the bicyco[2.2.1]heptene system (cf. 22 and 23). This set proved equipotent against enzyme (ALK and FAK) and in ALK cells and demonstrated similar selectivity against INSR and the kinome. However, hydroxyethyl derivative 23 demonstrated roughly 3fold higher oral exposure (22, AUC0−6h = 1300 ng·h/mL, vs 23, AUC0−6h = 3959 ng·h/mL). This higher exposure resulted in about 4-fold enhanced oral bioavailability for 23 relative to 22. In vitro liver microsome stability for 23 across multiple species, however, continued to show issues with metabolic liabilities. In rodent liver microsomes (mouse and rat) 23 was quite stable (t1/2 = 37 min each), but in higher species (cynomolgus monkey and human) half-lives were significantly lower (t1/2 = 8 and 13 min, respectively). Metabolite identification studies in liver microsomes 23 (data not shown) revealed that loss of the methyl group from the N-methylmethanesulfonamide was the major product in both human and monkey liver microsomes for 23; this metabolite was minor in rodent liver microsomes. Upon the basis of these results, we profiled the des-methyl derivative 24, which proved to be 9-fold less potent against ALK and outside our desired range (IC50 < 10 nM). Another tactic to potentially circumvent this metabolism involved tying E



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Table 1. Effects of Position and Nature of Substituent on Benzocycloheptane Ring System

a

IC50 values reported as the average (±SD if ≥3 determinations). bLM = liver microsome.

Table 2. Expanded Profiling of 16a and 16ba

compd

ALK/cell,b IC50 (nM)

1 16a 16b

1.9 ± 0.5/42 14/200 9 ± 3/200

INSR IC50 (nM)

FAK IC50 (nM)

1257 382 508 ± 209

25 ± 5 8.5 6.5

S(90)c (1 μM)

hERG IC50 (μM)

CYPf IC50 (μM)

LM t1/2g (min), M, R, Mo, H

F (%),h rat

oral AUCh (ng·h/mL)

>10 9.1 7.9

>10 >10 >10

8, 16, 17, 11 40, 40, 9, 15 40, 39, 7, 11

25 11 11

1203 ± 221 4405 ± 1704 5526 ± 682

d

0.136 0.093e 0.057e

IC50 values are reported as the average (±SD if ≥3 determinations). bBiochemical cell activity measuring inhibition of NPM-ALK phosphorylation. S(90) = no. kinases with >90% inhibition/total number kinases tested. dDiscoveRx 256 kinase panel. eDiscoveRx 402 kinase panel. fCYP isozymes: 1A2, 2C9, 2C19, 2D6, 3A4. gLM (liver microsome): M = mouse, R = rat, Mo = monkey, H = human. h1 mg iv dose, 5 mg/kg po dose. a c

for this analog was noted (IC50 = 2.7 μM), potentially as a result of the increased lipophilicity. The 2-N,N-dimethylsulfonamide derivative (26) and the N-methylamide (27) demonstrated similar ALK enzyme and cell potency (IC50 = 4 and 40 nM, respectively), and both showed decreased INSR

the methyl group back onto the sulfonamide to give a cyclic sultam (25). This proved to be acceptable for potency against ALK and FAK, as well as yielded acceptable oral bioavailability in rats (48%). However, metabolic stability was similar to the acylic variant (t1/2 = 16 min, hLM). Furthermore, hERG activity F



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Table 3. Optimized Analogs at C2 and C7

IC50 values are the average of at least 2 determinations (±SD if ≥3 determinations). bBiochemical cell activity measuring inhibition of NPM-ALK phosphorylation. c1 mg iv dose, 5 mg/kg po dose.

a

respectively), though hERG activity was an issue for 26 (IC50 = 0.24 μM) relative to 27 (IC50 ≥ 10 μM). Importantly, 27 demonstrated acceptable in vitro liver microsome stability across species (rat and human t1/2 > 40 min). The amide

selectivity in the enzymatic assay relative to the N-linked sulfonamides. Overall kinome selectivity was better for the Nmethylamide 27 (S90 = 0.08 vs 0.16). Analogues 26 and 27 displayed acceptable oral bioavailability (28% and 47%, G



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Table 4. Expanded Profiling of Individual Enantiomers 27a,ba

compd

ALK IC50 (nM)

1 (R)-27a (S)-27b

1.9 ± 0.5 5.7 3.1 ± 0.5

ALK cellb IC50 (nM) 42 50 22

INSR IC50 (nM) 662 81 65 ± 9

INSR cellb IC50 (nM) >10000 2000

FAK IC50 (nM) 30 1.0 2.0 ± 0.2

FAK cellb IC50 (nM)

S(90)c (at 1 μM) d

944

0.136 0.095e 0.084e

80

hERG IC50 (μM)

CYPsf IC50 (μM)

LMg (t1/2 min), M, R, Mo, H

F (%),h rat

oralh AUC0−∞ (ng·h/mL)

>10 >10 >10

>10 >5.6 >5.6

8, 16, 17, 11 40, 40, 24, 40 40, 40, 21, 40

25 18 42

1203 1196 8360

IC50 values are reported as the average (±SD if ≥3 determinations). bBiochemical cell activity measuring inhibition of phosphorylation. cS(90) = no. kinases with >90% inhibition/total number kinases tested. dDiscoveRx 256 linase panel. eDiscoveRx 442 kinase panel. fCYP isosymes: 1A2, 2C9, 2C19, 2D6, 3A4. gLM (liver microsome): M = mouse, R = rat, Mo = monkey, H = human. h1 mg iv dose and 5 mg/kg, po dose. a

potency within 10-fold of ALK (DiscoveRx Kd = 2.3 nM): FAK (2×), FLT3(D835Y) (7×), LTK (4×), PLK4 (7×), PYK2 (3×), RSK1 (4×), RSK2 (1×), STK33 (4×), TNK1 (4×). These data confirmed the activity against FAK and also demonstrated that 27b displays high selectivity for ALK and FAK with only RSK2 (kinase domain) having equipotency. Given the clinically derived mutations that have been reported for patients undergoing treatment with small-molecule ALK inhibitors, compound 27b was screened for activity using the DiscoveRx platform against known ALK activating mutations. Shown in Table 5 are the activities with DiscoveRx

isostere derivatives pyrazole (28) and imidazole (29) provided similar ALK and FAK potencies and improved INSR selectivity to that of 27 but suffered from hERG activity for 28 (IC50 = 3.7 μM) and poor metabolic stability for 29 (hLM t1/2 = 18 min). Both analogues, though orally bioavailable (F = 19% for each), were inferior to 27. The 2-methoxy derivative 30 was tested but proved to be significantly less active against ALK (IC50 = 44 nM) (Table 3). As noted above, analogues 22−30 were screened as racemic mixtures to expedite discovery. Upon the basis of the overall profile of 27, with the dual ALK and FAK activity, improved metabolic stability and promising oral PK profile, individual enantiomers 27a and 27b were synthesized from intermediates (R)- and (S)-12d that were separated using SFC (Chiralcel OJH). Expanded profiling of the two enantiomers is shown in Table 4. The profiles for 27a and 27b are quite similar across on-target potency, off-target selectivity, and in vitro ADME/ toxicity parameters. Given that the predicted binding mode of these inhibitors places the piperazinyl moiety in a solvent accessible region of the kinase, this similarity is not entirely surprising. However, what was unexpected were the oral PK profiles in which the (S)-enantiomer 27b displayed a 2- to 3fold improvement in oral biovailability and significantly higher oral AUC despite similar in vitro metabolic stability values; this result was also consistent with mouse PK/PD studies (data not shown). For both enantiomers, Caco-2 permeability was classified as high (Papp(A→B) > 1.0 × 10−6 cm/s) with similar efflux ratios (90% inhibition at the screening concentration of 1 μM is in Supporting Information. The following kinases in the panel demonstrated

Table 5. DiscoveRx Profiling of 27b against WT ALK and ALK Mutations

a

target

Kd (nM)a

ALK (WT) ALK(1151Tins) ALK(C1156Y) ALK(F1174L) ALK(L1196M)

3.6b 2.6 1.6 1.1 3.3

Average value (n = 2). bData from independent experiment.

ALK (WT) as a benchmark (Kd = 3.6 nM); this result is in good agreement with our internal isolated enzyme assay (IC50 = 3.1 nM) and that reported from previous independent experiments at DiscoveRx (Kd = 2.6 nM). Of particular note, 27b displayed similar potencies for the ALK mutants tested, ALK(1151Tins)38 Kd = 2.6 nM, ALK(C1156Y)39 Kd = 1.6 nM, ALK(F1174L)40 Kd = 1.1 nM, as well as the gatekeeper mutation ALK(L1196M)38 Kd = 3.3 nM. The majority of analogues in Table 4 displayed similar ALK and FAK potency in contrast to 1. Examination of the structural basis for the dual ALK/FAK potency via docking into published crystal structures found a potentially significant set of molecular interactions to explain this activity. Shown below (Figure 4) are both 1 and 27b docked (Glide XP) in crystal structures of ALK (PDB code 3LZT) and FAK (PDB code 3BZ3). By examination of the ALK structure, an acidic residue in the solvent exposed region is within reasonable distance to the basic heterocyclic amine of both 1 (yellow) and 27b (gray) to pick up an interaction from Glu1210. This type of interaction is H



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Figure 4. Glide docking of 27b (gray) and 1 (yellow): left panel, ALK (PDB code 3LZT); right panel, FAK (PDB code 3BZ3).

supported with other ALK ligand/protein structures.41 On the right side of Figure 4 is the docking in a crystal structure of FAK. Since the FAK activity was highly determinant on the location of the heterocycle attached to the benzocycloheptane and appeared to have little activity dependence on the amine attached to the chloropyrimidine core, we were not surprised to find an acidic residue, Glu506, in this region with which the amine of the piperazine of 27b (gray) could potentially form a favorable interaction. However, the morpholine of 1 (yellow) is displayed in an alternative trajectory and would not be able to form this same interaction. Furthermore, in FAK, the residue corresponding to Glu1210 in ALK is Val513 which offers a potential explanation as to the decreased activity of 1 in FAK. There are reports that suggest that the high energetic penalty due to desolvation could be overcome by free energy binding gains with such favorable electrostatic interactions.42 In the absence of other obvious ligand/protein structural features impacting the observed selectivity, the modeling offers a potential explanation. In vivo pharmacokinetic parameters for 27b in three species (CD-1 mouse, Sprague-Dawley rat, and cynomolgus monkey) are shown in Table 6. Following iv dosing, low clearance was noted in rodents and moderate to high clearance was seen in monkey which is consistent with the liver microsome results. Volume of distribution (Vd) was also significantly higher in

monkeys. It was unclear why there was such a large shift in Vd between rodents and non-human primates. Oral bioavailability was acceptable across all three species with CD-1 mouse roughly 2-fold that of rat and cynomolgus monkey. Overall plasma exposure was lower in cynomolgus monkey (both iv and oral) relative to the rodents, ostensibly due to the higher clearance and very high distribution, since oral bioavailability was similar to that of rat. Importantly, from tissue distribution studies in rats at 30 mg/kg oral dose, total brain exposure relative to plasma was roughly 1.5- to 2-fold [brain Cmax (3160 ng/g) vs plasma Cmax (2116 ng/g); brain AUC0−48h (61 584 ng· h/mL) vs plasma AUC0−48h (31 539 ng·h/mL)] which is significant, since one aspect of clinically derived resistance of NSCLC to crizotinib is manifested in the emergence of brain metastases.43,44 Compared to 1, a significant improvement in multiple pharmacokinetic parameters was noted across species.16 For example, 27b displays lower iv clearance values in CD-1 mouse (10 mL min−1 kg−1 vs 29 mL min−1 kg−1) and rat (3 mL min−1 kg−1 versus 17 mL min−1 kg−1) and similar values to cyno (30 mL min−1 kg−1 vs 28 mL min−1 kg−1). Furthermore, higher oral (dose normalized) AUC values45 across all three species were observed for 27b vs 1 in mouse (1643 ng·h/mL vs 299 ng·h/mL), rat (1672 ng·h/mL vs 246 ng·h/mL) and cyno (276 ng·h/mL vs 95 ng·h/mL). To support the advancement of 27b and position the clinical program appropriately, it was profiled in a variety of in vitro assays and in vivo pharmacological models pertaining to the ALK component (NPM-ALK+ ALCL models, EML4-ALK+ NSCLC cancer models). Furthermore, 27b was evaluated in FAK-activated in vivo tumor models (both xenograft and patient derived xenografts46) and demonstrated robust activity. 27b was screened against both NPM-ALK+ ALCL cell lines (Sup-M2 and Karpas-299) and NPM-ALK− hematologic cell lines (Hut-102 and K562) and demonstrated selective cytotoxicity in the oncogene-addicted cell lines (Figure 5). The calculated cellular IC50 values for cell growth inhibition by 27b were 84 nM (n = 2) in Sup-M2 cells and 131 nM (n = 2) in Karpas-299 cells. Furthermore, this concentration-dependent induction of proapoptotic caspases was also observed for the ALK+ cell lines but was absent in tumor cell lines lacking oncogenic ALK fusion proteins. These data suggest that this selectivity is driven through inhibition of ALK kinase activity. Furthermore, 27b inhibited EML4-ALK tyrosine phosphorylation in two different EML4-ALK+ NSCLC-derived cell lines, NCI-H2228 and NCI-H3122, with mean IC50 values of 175 nM (n = 2) and 85 nM (n = 2), respectively. Inhibition of EML4-

Table 6. Pharmacokinetic Parameters of 27b in CD-1 Mouse, Sprague-Dawley (SD) Rats, and Cynomolgus Monkeys

iv

po

PK parameter

CD-1 mousea

SD ratb

cynomolgus monkeyb

dosec (mg/kg) t1/2 (h) AUC0−∞ (ng·h/mL) Vd (L/kg) CL (mL min−1 kg−1) dosec (mg/kg) Cmax (ng/mL) tmax (h) AUC0−∞ (ng·h/mL) F (%)

1 3.0 1612 2.7 10 10 1533 2 16429 102

1 2 ± 0.4 4005 ± 237 0.8 ± 0.2 4 ± 0.2 5 1340 ± 107 3.3 ± 0.7 8360 ± 540 42 ± 3

1 5.4 ± 0.6 554 ± 11 13.2 ± 1.9 30 ± 0.5 10 239 ± 6 6 2757 ± 114 50 ± 3

a

Values are the mean from three animals from each time point. Values ± SEM are the mean from three animals. cFormulated as solutions: iv, 3% DMSO, 30% Solutol, 67% PBS (mouse, rat), dH2O (monkey); oi, dH2O (mouse, monkey), PEG400 (rat). b

I



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Figure 5. In vitro cytotoxicity and caspase 3/7 activation results for 27b.

Figure 6. PK/PD of 27b (single 30 mg/kg, po dose) in NPM-ALK+ ALCL tumor xenograft.

improve upon the activity of compound 1, which demonstrated target inhibition following a single, oral dose of 30 mg/kg out to 12 h and thus required b.i.d. dosing of 30 mg/kg to achieve robust responses. Upon the basis of the improvement in pharmacokinetic parameters in mice, we predicted that sustained and prolonged plasma levels would be achieved at this dose. Indeed, a single oral dose (30 mg/kg) with 27b in an NPM-ALK+ ALCL tumor xenograft (Sup-M2) (Figure 6) demonstrated significant (>85%) knockdown of NPM-ALK phosphorylation out to 24 h. Plasma and tumor levels remained high to this time point.

ALK phosphorylation in H2228 and NCI-H3122 cells by 27b resulted in concentration-related cytotoxicity and growth inhibition, with a mean IC50 values (n = 2) of 2900 nM in H2228 cells and 1779 nM in H3122 cells. The shift in IC50 values between biochemical inhibition and the cytotoxicity/ growth inhibition was more significant for the EML4-ALK+ NSCLC lines; however, tumor growth inhibition was still robust (see Figure 7). Prior PK/PD and efficacy studies in NPM-ALK ALCL models demonstrated that sustained and significant (>90%) target inhibition was required in vivo to elicit a robust antitumor response.28 Furthermore, we were hoping to J



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Figure 7. In vivo efficacy of 27b on ALK+ ALCL and NSCLC animal xenograft models.

Figure 8. PK/PD of 27b (single 55 mg/kg, po dose) in FAK+ CWR22 tumor xenograft.

clinically in NSCLC and inflammatory breast cancer, we decided to explore efficacy in those preclinical models with the reasonable assumption that 27b would display similar PK/PD effects as those reported above. Toward this end 27b was evaluated for biochemical inhibition of phospho-FAK in HCC827 cells that have high levels of constitutively activated FAK (ELM4-ALK negative) and displayed an IC50 = 357 nM which is reasonably consistent with the IC50 in our screening assay (IC50 = 82 nM). Thus, in vivo antitumor efficacy in a HCC-827 NSCLC tumor xenograft (Figure 9) was evaluated in SCID mice at 30 and 55 mg/kg (b.i.d) oral doses of 27b. Dose-related antitumor efficacy was observed with 60% incidence of partial tumor regressions at 55 mg/kg (b.i.d.) and significant tumor growth inhibitory activity at 30 mg/kg (b.i.d.). The dosing regimens were well-tolerated, with no overt toxicity or weight loss. 27b does not inhibit EGFR phosphorylation in HCC-827 cells (activated EGF-R due to E746-A750 deletion), which suggests the activity is related to FAK inhibition. The tumor PD response observed in the FAK+ CWR22 tumor xenograft and the marked antitumor activity achieved in the FAK activated HCC-827 NSCLC xenograft led us to further explore the FAK inhibitory component of 27b in patient-derived xenograft (PDX) models46 that had a defined pFAK expression profile. Shown in Figure 10 are the screening results at a single dose (55 mg/kg, b.i.d.) from two separate PDX models, CTG-0142 squamous cell carcinoma head and neck (SCCHN) and CTG-0785 sarcoma model. Relative to

Dose-dependent antitumor efficacy of 27b with oral dosing was confirmed in the NPM-ALK+ ALCL Sup-M2 model (Figure 7, left panel) with dosing at 3 mg/kg (b.i.d.), 10 mg/kg (b.i.d), 30 mg/kg (b.i.d and q.d.), and 55 mg/kg (q.d.). As expected, the once daily 30 mg/kg dose resulted in tumor regressions during the 12-day dosing paradigm. Furthermore, the higher 55 mg/kg once daily regimen effected complete tumor ablation. No significant effect was observed with the 3 and 10 mg/kg b.i.d. regimens. In an EML4-ALK+ NSCLC H2228 tumor xenograft (Figure 7, right panel), oral dosing at 30 and 55 mg/kg (b.i.d.) and 55 mg/kg (q.d.) demonstrated significant tumor responses at all doses. On day 12, all animals were switched to the 55 mg/kg, q.d. dosing regimen and continued to day 40. The animals were followed out to day 80 with no tumor reemergence. Upon the basis of the in vitro enzyme and cellular activity against FAK, 27b was evaluated for single dose PK/PD effects (Figure 8) in the CWR22 prostate CA xenograft which had very high expression levels of both total and activated (pFAK) FAK. Time-dependent inhibition of FAK phosphorylation at a 55 mg/kg oral dose out to 24 h was observed; this PD effect was comparable but of slightly diminished magnitude to the pALK inhibition profile in ALK-positive ALCL xenografts over a similar time course, consistent with the differences in cellular potency of 27b for ALK versus FAK. The CWR22 PK/PD study validated the in vivo biochemical FAK activity of 27b. Since 27b was going to be positioned K



Article

rational approach, liabilities associated with the advanced lead 1 were diminished, including specific metabolic liabilities of both amino moieties attached to the pyrimidine core ring system. Furthermore, we identified a small structural change on the benzocycloheptyl ring that provided consistent, potent FAK inhibition while maintaining high levels of kinome selectivity. The structural basis for this potency was identified through molecular docking experiments. Compound 27b displayed favorable in vitro ADME properties and acceptable oral bioavailability in three species. Furthermore, 27b imparts equipotent in vitro activity against clinically-derived resistance mutations and favorable brain exposure which may impact physiologically relevant escape pathways such as brain metastases. Dose-dependent antitumor efficacy was observed in multiple animal models of ALK+ and FAK-driven cancers. This dual FAK and ALK active compound is of unique interest in light of the recent findings in inflammatory breast cancer where subsets have ALK or FAK oncogenic components. Additionally, exploration of the clinical significance of FAK inhibition in conjunction with the recent findings in immunotherapy is warranted. 27b is currently undergoing human clinical trials.48

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Figure 9. Activity of 27b in HCC-827 human NSCLC xenograft (ALK−/FAK+).

EXPERIMENTAL SECTION

Unless otherwise indicated, all reagents and solvents were obtained from commercial sources and used as received. 1H and 13C NMRs were obtained on a Bruker Avance at 400 and 101 MHz, respectively, in the solvent indicated with tetramethylsilane as an internal standard. Analytical HPLC was run using a Zorbax RX-C8, 5 mm × 150 mm column, eluting with a mixture of acetonitrile and water containing 0.1% trifluoroacetic acid with a gradient of 10−100% over 5 or 20 min, wavelength of 254 and 290 nm to assess purity of final targets. LC/MS results were obtained on either of two instruments. LC/MS analysis was performed on a Waters Aquity Ultra Performance LC with a 2.1 mm × 50 mm Waters Aquity UPLC BEH C18 1.7 μm column. The target column temperature was 45 °C, with a run time of 2 min, a flow rate of 0.600 mL/min, and a solvent mixture of 5% (0.1% formic acid/ water):95% (acetonitrile/0.1% formic acid). The mass spectrometry data were acquired on a Micromass LC-ZQ 2000 quadrupole mass spectrometer. Alternatively, LCMS analysis was performed on a Bruker Esquire 200 ion trap. Purity was determined for final compounds using a combination of the analytical HPLC methods described above. All final compounds were determined to have ≥95% purity as assessed by area percent (A%) as noted in the tabular data. Automated column chromatography (SiO2) was performed on a CombiFlash Companion (ISCO, Inc.). Reverse phase HPLC was carried out on Gilson GX-281 series HPLC with Phenomenex Gemini-NX, C18 reverse phase 150 mm × 30 mm, 5 μm column, acetonitrile/water gradient, 0.1% TFA

undosed control mice, significant tumor growth inhibitory activity (p < 0.05) was observed in both PDX models. In the left-hand panel, over 28 days of dosing at of 55 mg/kg (b.i.d), tumor stasis and a 75% TGI were observed. In the sarcoma PDX, a 70% TGI was realized with only 13 days of dosing. Finally, in regard to the pharmacology of 27b and FAK inhibition, a potentially attractive therapeutic avenue for the dual ALK and FAK activity is in inflammatory breast cancer (IBC), a particularly aggressive subtype of breast cancer refractory to conventional treatment regimens.25 Elevated levels of pFAK have also been recently identified in a subset of IBC (in the absence of ALK activity).47 In vitro testing of 27b against a panel of FAK-activated IBC cell lines demonstrated concentration dependent inhibition of cell proliferation as well as robust in vivo antitumor activity against mammary glandimplanted xenografts in SCID mice and importantly the absence of brain metastases relative to control.47

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CONCLUSIONS In summary, we have advanced 27b, a selective inhibitor of FAK and ALK, as a novel anticancer therapeutic. Following a

Figure 10. Activity of 27b in CTG-0977 human SCCHN PDX (ALK−/FAK+) and CTG-0785 sarcoma PDX (ALK−/FAK+). L



Article

Hz, 1H), 2.87−2.70 (m, 4H), 2.61−2.47 (m, 3H), 2.30 (br t, J = 10.5 Hz, 1H), 2.15−2.03 (m, 2H), 1.93−1.78 (m, 1H), 1.47−1.32 (m, 1H). (8) 4-Methoxy-6-morpholino-6,7,8,9-tetrahydro-5H-benzo[7]annulen-3-amine. Using the general procedure for hydrogenation, 4-(4-methoxy-3-nitro-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl)morpholine (7) (0.44 g, 1.42 mmol) provided 8 (0.39 g, 99% yield) as a light tan solid which was used without further purification. LC/MS (ESI+): 277.07 (M + H). 1H NMR (400 MHz, CDCl3) δ 6.67 (d, J = 7.8 Hz, 1H), 6.51 (d, J = 7.8 Hz, 1H), 3.80−3.66 (broad m, 9H), 3.44 (br d, J = 12.9 Hz, 1H), 2.76 (br s, 2H), 2.67−2.52 (m, 4H), 2.44−2.26 (m, 2H), 2.13−1.96 (m, 2H), 1.85−1.72 (m, 1H), 1.42− 1.29 (m, 1H). (10) 1-Methoxy-2-nitro-5,7,8,9-tetrahydrobenzo[7]annulen6-one. Using the general procedure for nitration, 1-methoxy-5,7,8,9tetrahydrobenzo[7]annulen-6-one (25 g, 0.131 mol) provided 10 (10.7 g, 34.6% yield). 1H NMR (400 MHz, CDCl3) 7.70 (d, J = 8.3 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 3.92 (s, 3H), 3.80 (s, 2H), 3.13− 3.09 (m, 2H), 2.60 (t, J = 7.0 Hz, 2H), 2.10−2.03 (m, 2H). (11a) 4-(1-Methoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)morpholine. Using the general procedure for reductive amination followed by the general procedure for nitration, 1-methoxy-5,7,8,9-tetrahydrobenzo[7]annulen-6-one (9) (0.51 g, 1.95 mmol) provided 11a as a yellow oil (0.18 g, 24%, 2 steps). 1H NMR (400 MHz, CDCl3) δ 7.09 (t, J = 7.8 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 8.1 Hz, 1H), 3.81 (d, J = 1.3 Hz, 3H), 3.75 (t, J = 4.2 Hz, 4H), 3.40 (dd, J = 14.0, 7.2 Hz, 1H), 2.94−2.83 (m, 2H), 2.74− 2.66 (m, 2H), 2.61−2.54 (m, 2H), 2.44−2.36 (m, 1H), 2.35−2.26 (m, 1H), 2.14−2.00 (m, 1H), 1.87−1.75 (m, 1H), 1.38−1.25 (m, 1H). (11b) 1-(1-Methoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)-4-methylpiperazine. Following the general procedure for reductive amination, 1-methoxy-2-nitro-5,7,8,9-tetrahydrobenzo[7]annulen-6-one (10) (3.09 g, 13.1 mmol) and 1-methylpiperazine (13.1 g, 131 mmol) provided 11b (4.19 g, 99%). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.3 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H), 3.31 (br dd, J = 14.7, 7.6 Hz, 1H), 3.03−2.88 (m, 2H), 2.81−2.68 (m, 2H), 2.60 (br dd, J = 10.4, 4.8 Hz, 2H), 2.54−2.37 (m, 6H), 2.32 (s, 3H), 2.19−2.07 (m, 2H), 1.93−1.80 (m, 1H), 1.45−1.25 (m, 1H). (11c) 1-Methoxy-N-(2-methoxyethyl)-2-nitro-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-amine. Following a general procedure for reductive amination, 1-methoxy-2-nitro-5,7,8,9-tetrahydrobenzo[7]annulen-6-one (10) (1.85 g, 7.87 mmol) and 2-methoxyethylamine (5.91 g, 78.7 mmol) were converted to 11c (1.86 g, 80% yield) as a brown oil. 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.3 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 3.93−3.85 (m, 3H), 3.31 (br dd, J = 14.7, 7.6 Hz, 1H), 3.02−2.88 (m, 2H), 2.78−2.70 (m, 2H), 2.60 (br dd, J = 10.4, 4.8 Hz, 2H), 2.54−2.38 (m, 4H), 2.32 (s, 4H), 2.17−2.05 (m, 2H), 1.95−1.79 (m, 1H), 1.44−1.26 (m, 1H). (11d) 2-[4-(1-Methoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)piperazin-1-yl]ethanol. Following the general procedure for reductive amination, 1-methoxy-2-nitro-5,7,8,9tetrahydrobenzo[7]annulen-6-one (10) (15.1 g, 64.2 mmol) and 2piperazin-1-ylethanol provided 11d (19 g, 85% yield). LC/MS (ESI+) m/z = 350 (M + H)+; 1H NMR (400 MHz, CDCl3) 7.56 (d, J = 8.2 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 3.82 (s, 3H), 3.63−3.06 (m, 2H), 3.29−3.24 (m, 1H), 3.00−2.86 (m, 3H), 2.72−2.67 (m, 2H), 2.60− 2.51 (m, 8H), 2.46−2.37 (m, 2H), 2.12−2.07 (m, 2H), 1.87−1.78 (m,1H), 1.37−1.29 (m, 1H). (12a) 1-Methoxy-6-morpholino-6,7,8,9-tetrahydro-5Hbenzo[7]annulen-2-amine. Following the general procedure for hydrogenation, 4-(1-4-(1-methoxy-2-nitro-6,7,8,9-tetrahydro-5Hbenzo[7]annulen-6-yl)morpholine (11a) (1.5 g, 4.9 mmol) gave 12a as a light brown solid (0.8 g, 61%). 1H NMR (400 MHz, CDCl3) δ 6.76 (d, J = 8.1 Hz, 1H), 6.54 (d, J = 8.1 Hz, 1H), 3.76−3.72 (m, 5H), 3.71 (d, J = 1.3 Hz, 3H), 3.23 (br dd, J = 14.5, 7.2 Hz, 1H), 2.92−2.64 (m, 4H), 2.63−2.53 (m, 2H), 2.44−2.30 (m, 2H), 2.16−2.03 (m, 2H), 1.84−1.71 (m, 1H), 1.42−1.29 (m, 1H). (12b) 1-Methoxy-6-(4-methylpiperazin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-amine. Following the general procedure for hydrogenation, 1-(1-methoxy-2-nitro-6,7,8,9-tetrahydro-5Hbenzo[7]annulen-6-yl)-4-methylpiperazine (11b) (1.18 g, 3.68 mmol)

modifier gradient elution with automatic fraction collection. Melting points were taken on a Mel-Temp apparatus and are uncorrected. General Procedure for Reductive Amination. Ketone (1 equiv) and amine (1.1 equiv) were treated with sodium triacetoxyborohydride (1.4 equiv) and acetic acid (1 equiv) in THF (10 mL/ mmol substrate) and stirred at room temperature until completion. The reaction mixture was then concentrated and partitioned between dichloromethane and saturated sodium bicarbonate solution. The organic phase was washed with water, dried on MgSO4, and concentrated. Automated flash chromatography purification (ISCO, silica cartridge; gradient elution, 0−10% MeOH/CH2Cl2 or 10−100% ethyl acetate−hexane) afforded amine products. General Procedure for Nitration.49 Aromatic substrate (1 equiv) was treated with trifluoroacetic anhydride (30 equiv) and potassium nitrate (1 equiv) in acetonitrile (3 mL/mmol) at 0 °C for 3 h. The reaction mixture was partitioned between sodium bicarbonate solution and dichloromethane and the aqueous phase extracted with additional dichloromethane. The organics were dried (Na2SO4) and concentrated. Automated flash chromatography purification (ISCO, silica cartridge; gradient elution, 0−10% MeOH/CH2Cl2 or 10−100% ethyl acetate−hexane) afforded separated nitro regioisomers. General Procedure for Hydrogenation. Nitro compound (1 equiv), 10% palladium on carbon (50% wet) (0.1 equiv), and ethanol (20 mL/mmol substrate) were combined and subjected to hydrogenation at 50 psi in a Parr apparatus until hydrogenation was complete. The reaction mixture was filtered through Celite. Evaporation of solvent under reduced pressure provided the amines which were generally used without further purification. General Procedure for SN Ar Displacement on 2,4,5Trichloropyrimidine. 2,4,5-Trichloropyrimidine (1 equiv), 2-pyrazol-1-ylphenylamine (1 equiv), N,N-dimethylformamide (10 mL/ mmol substrate), and potassium carbonate (1.3 equiv) were combined and stirred heated to 75 °C until completion. The mixture was poured onto ice and stirred until all the ice had melted. The mixture was filtered on a Buchner and air-dried and the solid used without further purification or purified by automated flash chromatography purification (ISCO, silica cartridge; gradient elution, 0−10% MeOH/CH2Cl2 or 10−100% ethyl acetate−hexane). General Procedure for SNAr Displacement on 2,5-DichloroN-arylpyrimidin-4-amines. Amine (0.468 mmol), 2,5-dichloro-Narylpyrimidin-4-amine (0.312 mmol), 1-methoxy-2-propanol (1 mL/ mmol), and methanesulfonic acid (0.111 mL, 1.71 mmol) were combined in a sealed tube and heated at 90 °C o/n. The reaction mixture was diluted with DCM and washed with saturated aq NaHCO3. The organic layer was dried, filtered, and concentrated. Purification via reverse phase HPLC (Gilson GX-281 series HPLC with Phenomenex Gemini-NX, C18 reverse phase 150 mm × 30 mm, 5 μm column, 5−50% acetonitrile/water gradient, 0.1% TFA modifier). The desired fractions were either lyophilized to give the desired product as a trifluoroacetic acid salt or free-based (partitioned between dichloromethane and saturated aq NaHCO3, dried (Na2SO4), filtered, and concentrated). (6) 4-(4-Methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6yl)morpholine. Using the general procedure for reductive amination, 4-methoxy-5,7,8,9-tetrahydrobenzocyclohepten-6-one (5) (2.25 g, 11.8 mmol) and morpholine (1.13 mL, 13.0 mmol) gave 6 as a white solid (3.09 g, 99% yield). LC/MS (ESI+) m/z 262.0 (M + H)+. 1 H NMR (400 MHz, CDCl3) δ 7.06 (t, J = 8.0 Hz, 1H), 6.72 (dd, J = 11.0, 8.0 Hz, 2H), 3.82 (s, 3H), 3.74 (dt, J = 5.7, 3.7 Hz, 4H), 3.61 (br d, J = 12.9 Hz, 1H), 2.81−2.66 (m, 4H), 2.56−2.48 (m, 2H), 2.37− 2.29 (m, 1H), 2.27−2.19 (m, 1H), 2.08−1.98 (m, 2H), 1.89−1.76 (m, 1H), 1.48−1.34 (m, 1H). (7) 4-(4-Methoxy-3-nitro-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)morpholine. Using the general procedure for nitration, 4-(4-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)morpholine (6) (2.00 g, 7.65 mmol) gave 7 (second eluting peak) isolated as a yellow oil (0.43 g, 19%). LC/MS (ESI+) m/z 307.05 (M + H)+. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 3.89 (s, 3H), 3.78−3.67 (m, 4H), 3.50 (br d, J = 13.4 M



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LC/MS (ESI+) m/z 539 (M + H)+; 1H NMR (CDCl3, 400 MHz) δ 8.21 (d, J = 8 Hz, 1H), 7.90 (s, 1H), 7.42 (s, 1H), 7.03 (d, J = 8 Hz, 1H), 6.93 (d, J = 8 Hz, 1H), 6.39 (m, 1H), 6.34 (m, 1H), 4.37 (t, J = 8 Hz, 1H), 3.73 (m, 7H), 3.23 (m, 1H), 3.08 (s, H), 2.96 (s, 1H), 2.88 (m, 2H), 2.69 (m, 2H), 2.56 (m, 2H), 2.50 (d. J = 8 Hz, 1H), 2.37 (m, 2H), 2.25 (d, J = 9 Hz, 1H), 2.12 (m, 2H), 1.81 (m, 1H), 1.64 (d, J = 9 Hz, 1H), 1.34 (m, 1H). HPLC purity: 97 A%. (16a and 16b) (1S,2S,3R,4R)-3-[[5-Chloro-2-[[(6S)-1-methoxy-6-(4-methylpiperazin-1-yl)-6,7,8,9-tetrahydro-5Hbenzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide and (1S,2S,3R,4R)-3-[[5Chloro-2-[[(6S)-1-methoxy-6-(4-methylpiperazin-1-yl)-6,7,8,9tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amine, 12b (3.82 g, 13.2 mmol) and 13 (3.95 g, 13.2 mmol) gave two separable diastereomers. The stereochemistry at the 6position was not assigned. First eluting diasteromer from RP-HPLC, 16a (0.850 g, 12% yield): LC/MS (ESI+) m/z 552.22 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 8.1 Hz, 1H), 7.90 (s, 1H), 7.40 (s, 1H), 6.97 (br d, J = 7.3 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.41−6.37 (m, 1H), 6.35−6.31 (m, 1H), 5.60 (br s, 1H), 5.35 (br d, J = 6.1 Hz, 1H), 5.31 (s, 3H), 4.37 (t, J = 7.3 Hz, 1H), 3.72 (s, 3H), 3.28−3.21 (m, 1H), 3.08 (br s, 1H), 2.95 (br s, 1H), 2.89−2.83 (m, 2H), 2.73 (br d, J = 5.1 Hz, 2H), 2.65 (br s, 2H), 2.55−2.39 (m, 5H), 2.32 (s, 3H), 2.25 (d, J = 9.3 Hz, 1H), 2.13 (br d, J = 12.1 Hz, 2H), 1.85−1.71 (m, 1H), 1.68−1.53 (m, 2H), 1.42−1.29 (m, 1H). HPLC purity 95 A%. Second eluting diastereomer from RP-HPLC, 16b (0.52 g, 7% yield): LC/MS (ESI+) m/z 552.24; 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 8.3 Hz, 1H), 7.90 (s, 1H), 7.41 (s, 1H), 6.99 (br d, J = 8.1 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.41−6.37 (m, 1H), 6.35− 6.32 (m, 1H), 5.68 (br s, 1H), 5.46 (br s, 1H), 4.40−4.34 (m, 1H), 3.73 (s, 3H), 3.25 (br dd, J = 13.8, 7.2 Hz, 1H), 3.09 (br s, 1H), 2.96 (br s, 1H), 2.90−2.85 (m, 2H), 2.73 (br d, J = 5.3 Hz, 2H), 2.68−2.64 (m, 2H), 2.63 (s, 2H), 2.56−2.37 (m, 5H), 2.32 (s, 3H), 2.26 (d, J = 9.3 Hz, 1H), 2.13 (br dd, J = 10.5, 4.7 Hz, 2H), 1.91−1.72 (m, 2H), 1.65 (br d, J = 9.1 Hz, 1H), 1.43−1.25 (m, 1H). HPLC purity: 96 A%. (17) (1S,2S,3R,4R)-3-[[5-Chloro-2-[[1-methoxy-6-(2-methoxyethylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amine, 12c (0.2 g, 0.8 mmol) and 13 (0.3 g, 0.8 mmol) gave 17 as a tan solid (30 mg, 8%). NMR and HPLC appeared coincident for diastereomers. Mp 98−99 °C; LC/MS (ESI+) m/z 527 (M + H)+; 1H NMR (CDCl3, 400 MHz) δ 8.20 (m, 1H), 7.92 (s, 1H), 7.43 (s, 1H), 7.00 (d, J = 8 Hz, 1H), 6.91 (d, J = 8 Hz, 1H), 6.40 (m, 1H), 6.35 (m, 1H), 5.70 (br s, 1H), 5.56 (br s, 1H), 4.37 (m, 1H), 3.73 (s, 3H), 3.52 (m, 2H), 3.37 (s, 3H), 3.10 (m, 2H), 2.96−2.82 (m, 5H), 2.62 (m, 2H), 2.51 (d, J = 8 Hz, 1H), 2.25 (d, J = 8 Hz, 1H), 2.10 (m, 1H), 1.96 (m, 1H), 1.77 (m, 2H), 1.64 (d, J = 8 Hz, 1H), 1.50 (m, 1H). HPLC purity: 95 A% (18a and 18b) (1S,2S,3R,4R)-3-[[5-Chloro-2-[[(6S)-6-[4-(2hydroxyethyl)piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide and (1S,2S,3R,4R)-3[[5-Chloro-2-[[(6R)-6-[4-(2-hydroxyethyl)piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide. Following the general procedure for SNAr displacement on 2,5dichloro-N-arylpyrimidin-4-amine, 12c (0.2 g, 0.8 mmol) and 13 (0.2 g, 0.8 mmol) gave a diastereomeric mixture which was not separable using C18 RP-HPLC but could be separated employing SFC (Chiralpak AD-H column and 40% ethanol, 0.1% DEA modifier). The stereochemistry at the 6-position was not assigned. The first eluting peak 18a (108 mg, 30% yield) was isolated as a tan foam. LC/ MS (ESI+) m/z 582.16 (M + H)+. 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 8.1 Hz, 1H), 7.91 (s, 1H), 7.42 (s, 1H), 7.00 (br d, J = 9.9 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.39 (br d, J = 3.3 Hz, 1H), 6.37− 6.32 (m, 1H), 5.62 (br s, 1H), 5.35 (br s, 1H), 4.42−4.35 (m, 1H), 3.74 (s, 3H), 3.65 (br s, 2H), 3.32−3.22 (m, 1H), 3.09 (br s, 1H), 2.96 (br s, 1H), 2.91−2.84 (m, 2H), 2.74 (br s, 3H), 2.59 (broad m, 8H),

was hydrogenated to give 12b (1.1 g, 99% yield). 1H NMR (400 MHz, CDCl3) δ 6.76 (d, J = 7.8 Hz, 1H), 6.53 (d, J = 8.1 Hz, 1H), 3.67 (s, 3H), 3.23 (br dd, J = 14.1, 7.1 Hz, 1H), 2.76 (br d, J = 13.6 Hz, 4H), 2.67−2.59 (m, 2H), 2.57−2.46 (m, 3H), 2.45−2.34 (m, 3H), 2.32 (s, 3H), 2.16−2.03 (m, 2H), 1.85−1.72 (m, 2H), 1.42−1.30 (m, 2H). (12c) 1-Methoxy-N6-(2-methoxyethyl)-6,7,8,9-tetrahydro5H-benzo[7]annulene-2,6-diamine. Following the general procedure for hydrogenation, 1-methoxy-N-(2-methoxyethyl)-2-nitro6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-amine (11c) (1.86 g, 6.31 mmol) was hydrogenated to give 12c (1.61 g, 97% yield) as a tan solid. 1 H NMR (400 MHz, CDCl3) δ 6.83 (d, J = 8.1 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 3.80 (br t, J = 5.1 Hz, 2H), 3.68 (s, 3H), 3.47 (s, 3H), 3.35 (s, 3H), 3.22 (t, J = 5.2 Hz, 2H), 3.16 (br s, 4H), 2.51 (br t, J = 12.9 Hz, 1H), 2.42 (br d, J = 10.6 Hz, 1H), 2.05 (br d, J = 7.6 Hz, 2H), 1.50−1.36 (m, 1H). (12d) 2-[4-(2-Amino-1-methoxy-6,7,8,9-tetrahydro-5Hbenzo[7]annulen-6-yl)piperazin-1-yl]ethanol. Following the general procedure for hydrogenation, 2-[4-(1-methoxy-2-nitro-6,7,8,9tetrahydro-5H-benzo[7]annulen-6-yl)piperazin-1-yl]ethanol (11d) (19.0 g, 54.4 mmol) gave 12d as a foamy white solid (17.25 g, 99% yield). LC/MS (ESI+) m/z = 320 (M + H)+. 1H NMR (400 MHz, CDCl3) 6.76 (d, J = 7.9 Hz, 1H), 6.53 (d, J = 7.9 Hz, 1H), 3.72 (broad s, 3H), 3.71 (s, 3H),3.64 (t, J = 5.4 Hz, 2H), 3.26−3.20 (m, 1H), 2.84−2.72 (m, 5H), 2.62−2.56 (m, 8H), 2.42−2.35 (m, 2H), 2.40− 2.37 (m, 1H), 1.81−1.74 (m, 1H), 1.70 (broad s,1H), 1.41−1.33 (m, 1H). (R)-12d and (S)-12d. An amount of 34 g of racemic 12d was separated using SFC (supercritical fluid CO2) chromatography using a Chiralcel OJ-H (3 cm × 15 cm) 808041 column with 15% methanol (0.2% DEA)/CO2, 100 bar eluent at 80 mL/min flow rate, monitoring the wavelength of 220 nm with an injection volume of 0.5 mL, 20 mg/ mL ethanol. 16.9 g of the first-enantiomer and 17 g of the second eluting enantiomer were isolated with a chemical purity of >99% and an enantiomeric excess (ee) of >99% (measured using a Chiralcel OJH analytical column). NMR and mass spectrometry results were equivalent to those of the racemic material. (14) (1S,2S,3R,4R)-3-[[5-Chloro-2-[(4-methoxy-6-morpholino-6,7,8,9-tetrahydro-5H-benzo[7]annulen-3-yl)amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide. Following the general procedure for SNAr displacement on 2,5dichloro-N-arylpyrimidin-4-amine, 8 (150 mg, 0.54 mmol) and (1S,2S,3R,4R)-3-[(2,5-dichloropyrimidin-4-yl)amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide (13) (162 mg, 0.54 mmol) gave 14 as a light tan solid (155 mg, 53% yield) as an inseparable ∼1:1 mixture of diastereomers. Mp: 139−148 °C; LC/MS (ESI+) m/z 539.16 (M + H)+; 1H NMR (CDCl3) 8.17 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.37 (s, 1H), 6.97 (m, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.37 (m, 1H), 6.31 (m, 1H), 5.61 (br, 1H), 5.38 (br s, 1H), 4.36 (m, 1H), 3.76 (s, 3H), 3.74 (br s, 4H), 3.43 (d, J = 13.1 Hz, 1H), 3.07 (s, 1H), 2.94 (s, 1H), 2.72 (m, 4H), 2.58 (m, 2H), 2.49 (m, 2H), 2.33 (m, 1H), 2.23 (d, J = 9.2 Hz, 1H), 2.07 (m, 2H), 1.79 (m, 1H), 1.62 (m, 1H), 1.39 (m, 1H). HPLC purity: 98 A%. (15a and 15b) (1S,2S,3R,4R)-3-[[5-Chloro-2-[[(6S)-1-methoxy-6-morpholino-6,7,8,9-tetrahydro-5H-benzo[7]annulen2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2carboxamide and (1S,2S,3R,4R)-3-[[5-Chloro-2-[[(6R)-1-methoxy-6-morpholino-6,7,8,9-tetrahydro-5H-benzo[7]annulen2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2carboxamide. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amine, 112a (200 mg, 0.70 mmol) and 13 (190 mg, 0.65 mmol) gave 15a (48 mg, 14% yield) as a mauve solid as the first eluting diastereomer from the RP-HPLC. The stereochemistry at the 6-position was not assigned. Mp 146−148 °C. LC/MS (ESI+) m/z 539 (M + 1); 1H NMR (CDCl3, 400 MHz) δ 8.21 (d, J = 8 Hz, 1H), 7.91 (s, 1H), 7.42 (s, 1H), 7.01 (br s, 1H), 6.93 (d, J = 8 Hz, 1H), 6.40 (m, 1H), 6.34 (m, 1H), 4.37 (t, J = 8 Hz, 1H), 3.74 (m, 7H), 3.23 (m, 1H), 3.09 (s, H), 2.96 (s, 1H), 2.85 (m, 2H), 2.71 (m, 2H), 2.60 (m, 2H), 2.51 (d. J = 8 Hz, 1H), 2.42 (m, 2H), 2.26 (d, J = 9 Hz, 1H), 2.14 (m, 2H), 1.81 (m, 1H), 1.64 (d, J = 9 Hz, 1H), 1.40 (m, 1H). HPLC purity: 99 A%. The second eluting diastereomer 15b (65 mg, 18% yield) was isolated as a white solid. Mp 148−51 °C. N



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amino]pyrimidin-4-yl]amino]phenyl]methanesulfonamide. Following the general procedure for SNAr displacement on 2,5dichloro-N-arylpyrimidin-4-amines, 12d (190 mg, 0.59 mmol) and 21c36 (132 mg, 0.40 mmol) gave 24 (65 mg, 26% yield) as an off-white foam. LC/MS (ESI+) m/z 616.18 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 7.71 (s, 2H), 7.57−7.53 (m, 1H), 7.51 (s, 1H), 7.46 (s, 1H), 7.41−7.35 (m, 2H), 6.73 (d, J = 8.3 Hz, 1H), 3.70 (s, 3H), 3.65 (t, J = 5.4 Hz, 2H), 3.51 (s, 1H), 3.26−3.17 (m, 1H), 2.94 (s, 3H), 2.86−2.79 (m, 2H), 2.72 (br d, J = 5.1 Hz, 2H), 2.66−2.53 (m, 8H), 2.45−2.34 (m, 2H), 2.10 (br d, J = 10.9 Hz, 2H), 1.78 (q, J = 11.2 Hz, 1H), 1.40−1.26 (m, 1H). HPLC purity >95 A%. (25) 2-[4-[2-[[5-Chloro-4-[2-(1,1-dioxo-1,2-thiazolidin-2-yl)anilino]pyrimidin-2-yl]amino]-1-methoxy-6,7,8,9-tetrahydro5H-benzo[7]annulen-6-yl]piperazin-1-yl]ethanol. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amines, 12d (0.18 g, 0.57 mmol) and 21d (0.12 g, 0.33 mmol) gave 25 (73.6 mg, 34% yield) as a white foam. LC/MS (ESI+) m/z 642 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 8.41−8.35 (m, 2H), 8.11 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.46 (br s, 3H), 7.27−7.20 (m, 1H), 6.87 (d, J = 8.3 Hz, 1H), 3.76−3.69 (m, 5H), 3.65 (t, J = 5.3 Hz, 2H), 3.46 (t, J = 7.7 Hz, 2H), 3.25 (br dd, J = 14.3, 6.9 Hz, 1H), 2.93−2.80 (m, 2H), 2.74 (br d, J = 5.1 Hz, 2H), 2.67−2.55 (m, 10H), 2.48−2.37 (m, 2H), 2.18−2.07 (m, 2H), 1.87−1.73 (m, 2H), 1.36 (br d, J = 11.9 Hz, 1H). HPLC purity >95 A%. (26) 2-[[5-Chloro-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-1methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]-N,N-dimethylbenzenesulfonamide. Following the general procedure for SNAr displacement on 2,5-dichloroN-arylpyrimidin-4-amines, 12d (92 mg, 0.3 mmol) and 21e29 (0.1 g, 0.3 mmol) gave 26 (76 mg, 24% yield) as a tan solid. Mp 196−197 °C; LC/MS (ESI+) m/z 630.18 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 9.42 (s, 1H), 8.57 (d, J = 8.3 Hz, 1H), 8.16 (s, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.31−7.25 (m, 2H), 6.92−6.87 (m, 1H), 3.74 (s, 3H), 3.69 (br t, J = 5.2 Hz, 2H), 3.26 (br dd, J = 14.1, 6.8 Hz, 1H), 2.95−2.82 (br s, 4H), 2.76 (s, 6H), 2.75−2.61 (m, 8H), 2.54−2.37 (m, 2H), 2.20−2.10 (m, 2H), 1.82 (br s, 1H), 1.45−1.30 (m, 1H). HPLC purity 98 A%. (27) 2-[[5-Chloro-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-1methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]-N-methylbenzamide. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin4-amines, 12d (0.13 g, 0.42 mmol) and 21f (0.10 g, 0.30 mmol) gave 27 (39 mg, 20% yield) as a pale yellow foam. LC/MS (ESI+) m/z 580.17 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 11.02 (s, 1H), 8.69 (d, J = 8.9 Hz, 1H), 8.13 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H),7.59− 7.50(m, 2H), 7.41 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.21 (s, 1H), 3.74 (m, 3H), 3.66−3.63 (m, 2H), 3.29−3.23 (m, 1H), 3.06 (d, J = 4.3 Hz, 3H), 2.92−2.72 (m, 5H), 2.66−2.55 (m, 8H), 2.48−2.39 (m, 2H), 2.16−2.10 (m, 2H), 1.87−1.77 (m, 1H), 1.42−1.32 (m, 1H). HPLC purity 99 A%. (27a) 2-[[5-Chloro-2-[[(6R)-6-[4-(2-hydroxyethyl)piperazin-1yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]-N-methylbenzamide. Following the general procedure for SNAr displacement on 2,5-dichloro-Narylpyrimidin-4-amines, (R)-12d (91 mg, 0.28 mmol) and 21f (56 mg, 0.19 mmol gave 27a (56 mg, 50% yield). LC/MS (ESI+) m/z 580.1 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 11.02 (s, 1H), 8.69 (d, J = 8.9 Hz, 1H), 8.13 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H),7.59−7.50(m, 2H), 7.41 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.21 (s, 1H), 3.74 (m, 3H), 3.66−3.63 (m, 2H), 3.29−3.23 (m, 1H), 3.06 (d, J = 4.3 Hz, 3H), 2.92−2.72 (m, 5H), 2.66−2.55 (m, 8H), 2.48−2.39 (m, 2H), 2.16−2.10 (m, 2H), 1.87−1.77 (m, 1H), 1.42−1.32 (m, 1H). HPLC purity: 95 A%. (27b) 2-[[5-Chloro-2-[[(6S)-6-[4-(2-hydroxyethyl)piperazin-1yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]-N-methylbenzamide. To a sealed vessel (S)-12d (2.69 g, 8.41 mmol) and 21f (2.00 g, 6.73 mmol) were combined in 1-methoxy-2-propanol (120 mL) followed by the addition of methanesulfonic acid (2.44 mL, 37.7 mmol). The reaction was then heated at 90 °C for 18 h. The reaction mixture was added to a separatory funnel and diluted with sat. aq NaHCO3 until a cloudy

2.52 (br d, J = 9.1 Hz, 1H), 2.48−2.38 (m, 2H), 2.26 (br d, J = 9.9 Hz, 1H), 2.13 (br d, J = 10.6 Hz, 2H), 1.84−1.75 (m, 1H), 1.65 (br d, J = 9.6 Hz, 1H), 1.43−1.33 (m, 1H). HPLC purity: 99 A%. The second eluting peak 18b (102 mg, 28% yield) was isolated as of a tan foam. LC/MS (ESI+) m/z 582.15 (M + H)+. 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.3 Hz, 1H), 7.91 (s, 1H), 7.42 (s, 1H), 7.05−6.98 (m, 1H), 6.94 (d, J = 8.1 Hz, 1H), 6.43−6.38 (m, 1H), 6.35 (br d, J = 2.8 Hz, 1H), 5.61 (br s, 1H), 5.34 (br s, 1H), 4.38 (t, J = 7.5 Hz, 1H), 3.73 (s, 3H), 3.69−3.63 (m, 2H), 3.30−3.22 (m, 1H), 3.09 (br s, 1H), 2.96 (br s, 1H), 2.91−2.82 (m, 2H), 2.79−2.71 (m, 2H), 2.60 (br s, 8H), 2.52 (d, J = 7.8 Hz, 2H), 2.44 (br d, J = 11.6 Hz, 2H), 2.27 (d, J = 9.3 Hz, 1H), 2.11 (m, 2H), 1.87−1.74 (m, 1H), 1.65 (br d, J = 8.3 Hz, 1H), 1.44−1.34 (m, 1H). HPLC purity 95 A%. (21d) 2,5-Dichloro-N-[2-(1,1-dioxo-1,2-thiazolidin-2-yl)phenyl]pyrimidin-4-amine. Following the general procedure for SNAr displacement on 2,4,5-trichloropyrimidine, 2-(1,1-dioxo-1,2thiazolidin-2-yl)aniline 19d (1.0 g, 5.4 mmol) and 2,4,5-trichloropyrimidine 20 (1.2 g, 5.6 mmol) gave 21d (1.15 g, 60% yield) as a yellow solid. LC/MS (ESI+) m/z 358.9 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.44 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.54 (br d, J = 7.8 Hz, 1H), 7.47 (br t, J = 7.7 Hz, 1H), 7.37−7.28 (m, 1H), 3.71 (t, J = 6.3 Hz, 2H), 3.45 (br t, J = 7.3 Hz, 2H), 2.37 (quin, J = 6.9 Hz, 2H). (21g) 2,5-Dichloro-N-(2-pyrazol-1-ylphenyl)pyrimidin-4amine. Following the general procedure for SNAr displacement on 2,4,5-trichloropyrimidine, 2-pyrazol-1-ylphenylamine 19g (2.00 g, 12.6 mmol) and 2,4,5-trichloropyrimidine 20 (2.30 g, 12.6 mmol) gave 21g as a tan solid (1.82 g, 47% yield). LC/MS (ESI+) m/z 306.03. 1H NMR (CDCl3, 400 MHz): δ 10.88 (s, 1H), 8.57 (d, J = 8.3 Hz, 1H), 8.19 (s, 1H), 7.85 (dd, J = 1.0, 6.44 Hz, 2H); 7.46 (t, J = 8.3 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.28−7.235 (m, 1H), 6.54 (s, 1H). (21h) 2,5-Dichloro-N-[2-(1-methylimidazol-2-yl)phenyl]pyrimidin-4-amine. Following the general procedure for SNAr displacement on 2,4,5-trichloropyrimidine, 2-(1-methylimidazol-2yl)aniline, 19h (220 mg, 1.27 mmol) and 2,4,5-trichloropyrimidine 20 (233 mg, 1.27 mmol) gave 21h (140 mg, 33% yield). LC/MS (ESI+) m/z 320.06 (M + H)+; 1H NMR (400 MHz, DMSO-d6) δ 11.69−11.37 (broad s, 1H), 8.40 (s, 1H), 8.33 (dd, J = 8.3, 1.0 Hz, 1H), 7.70 (dd, J = 7.8, 1.3 Hz, 1H), 7.52 (ddd, J = 8.5, 7.3, 1.5 Hz, 1H), 7.35 (d, J = 1.3 Hz, 1H), 7.31 (td, J = 7.6, 1.1 Hz, 1H), 7.14 (d, J = 1.3 Hz, 1H), 3.78 (s, 3H). (22) N-[2-[[5-Chloro-2-[[1-methoxy-6-(4-methylpiperazin-1yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]phenyl]-N-methylmethanesulfonamide. Following the general procedure for SNAr displacement on 2,5dichloro-N-arylpyrimidin-4-amines, 12b (63 mg, 0.22 mmol) and 21b24 (75 mg, 0.22 mmol) gave 22 (72 mg, 46% yield) isolated as a tan lyophilate, TFA salt. LC/MS (ESI+) m/z 600.10 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 9.42 (s, 1H), 8.58 (d, J = 8.3 Hz, 1H), 8.17 (s, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.64 (br t, J = 8.8 Hz, 1H), 7.51 (s, 1H), 6.89 (d, J = 8.3 Hz, 1H), 4.14 (q, J = 7.2 Hz, 1H), 3.75 (s, 3H), 3.65 (br s, 2H), 3.31−3.22 (m, 1H), 2.93−2.83 (m, 2H), 2.77 (s, 6H), 2.59 (m, 5H), 2.50−2.36 (m, 2H), 2.19−2.08 (m, 2H), 1.86−1.75 (m, 1H), 1.57 (br s, 3H), 1.43−1.33 (m, 1H), 1.28 (t, J = 7.1 Hz, 1H). HPLC purity >95 A% (23) N-[2-[[5-Chloro-2-[[6-[4-(2-hydroxyethyl)piperazin-1yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]phenyl]-N-methylmethanesulfonamide. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amines, 12d (68 mg 0.22 mmol) and 21b (75 mg, 0.22 mmol) gave 23 (106 mg, 65% yield) as a tan lyophilate, TFA salt. LC/MS (ESI+) m/z 630.16 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 10.44−10.37 (m, 1H), 9.15 (s, 1H), 8.06−7.99 (m, 1H), 7.97 (s, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.39 (d, J = 1.3 Hz, 3H), 6.81 (d, J = 8.3 Hz, 1H), 4.08−4.02 (m, 3H), 3.83 (br s, 4H), 3.73 (s, 4H), 3.39 (br dd, J = 14.4, 7.1 Hz, 1H), 3.31 (s, 6H), 3.19 (br s, 3H), 3.11−3.02 (m, 1H), 2.99 (s, 3H), 2.45−2.36 (m, 2H), 2.34−2.24 (m, 1H), 1.97−1.82 (m, 1H), 1.51−1.34 (m, 1H). HPLC purity 99 A% (24) N-[2-[[5-Chloro-2-[[6-[4-(2-hydroxyethyl)piperazin-1yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]O



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washed with water and brine, filtered, and concentrated. The solid was recrystallized from methanol to afford 31 (404 mg, 80% yield). Several of the crystals were suitable for X-ray. LC/MS (ESI+) m/z 686 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 8.19 (br d, J = 8.1 Hz, 1H), 7.92 (br d, J = 7.3 Hz, 2H), 7.77 (br d, J = 7.3 Hz, 2H), 7.66 (br d, J = 7.8 Hz, 2H), 7.61 (br d, J = 8.1 Hz, 2H), 7.00 (br d, J = 7.8 Hz, 1H), 4.48 (br t, J = 5.3 Hz, 2H), 3.75 (s, 3H), 3.21 (br dd, J = 14.7, 6.1 Hz, 1H), 2.97−2.70 (m, 6H), 2.64 (br s, 6H), 2.51−2.37 (m, 2H), 2.12 (br d, J = 11.4 Hz, 2H), 1.88−1.75 (m, 1H), 1.37 (br d, J = 10.6 Hz, 1H). In Vitro and in Vivo Screening Assays and in Silico Modeling. Experimental details regarding enzymatic, cellular, ADME screening assays, and in silico modeling have been reported previously; see refs 16−20, 28, 33, 46 and references therein. All in vivo experiments were conducted in accordance with Teva IUCUC protocols. Representative Procedures for Measuring FAK Activity in Vitro and in Vivo. the human prostate carcinoma cell lines, CWR22 and human non-small-cell lung cancer cell line HCC-827 were obtained from American Tissue Culture Collection (ATCC, Manassas, VA) and were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS). The rabbit phospho-FAK(Tyr397) (catalog no. 3283) and FAK antibodies (catalog no. 3285) were purchased from Cell Signaling Technology (Beverly, MA). Immunoblot analyses of phospho-FAK and total FAK were carried out according to the protocols provided by the antibody suppliers (Cell Signaling Technology). In brief, 2 mL of cells (at a density of 0.75 × 106 cells/mL in RPMI medium containing 10% FBS (regular culture medium) or 75% murine or human plasma diluted in regular culture medium were seeded in each well of 6-well plates and an amount of 2 mL of test compound diluted in culture medium at 2× indicated final concentrations was added to each well. After incubation for 2−2.5 h, the cells were collected into 15 mL centrifuge tubes. The cells were spun down by centrifugation at 233g for 5 min, the medium was aspirated, the cells were resuspended in 1 mL of PBS, transferred to 1.5 mL microcentrifuge, and centrifuged at 10 621g for 2 min at 40 °C. The cells were then lysed in 150 μL of Frak lysis buffer [10 mM Tris, pH 7.5, 1% Triton X-100, 50 mM sodium chloride, 20 mM sodium fluoride, 2 mM sodium pyrophosphate, 0.1% BSA, plus freshly prepared 1 mM activated sodium vanadate, 1 mM DTT, and 1 mM PMSF and the protease inhibitor cocktail III (1:100 dilution)] on ice for 10 min. After brief sonication, lysates were cleared by centrifugation at 20 817g for 10 min at 4 °C. The supernatants (90 μL) were transferred to fresh 1.5 mL microcentrifuge tubes containing 30 μL of 4× LDS sample buffer. The samples were heat-inactivated at 90 °C for 5 min; 20 μL of each sample was resolved by NuPAGE 7% Tris-acetate gels at 150 V until the dye front was out of the gels. The gels were transferred to nitrocellulose membranes for 2 h at 30 V constant using a wet XCell II blot module. The membranes were blocked in Tris-buffered saline (TBS) containing 0.2% Tween-20 (TBST) and 3% Nestle Carnation nonfat milk (Nestle USA Inc., Solon, OH) at room temperature (rt) for 1 h. The membranes were incubated with antiphospho-FAK (Tyr397) antibody (diluted 1:1000 in TBST containing 3% bovine serum albumin) for 1.5 h at rt or overnight at 4 °C while rocking gently. After washing 3 times with TBST for 10 min each time, the membranes were incubated with goat-anti-rabbit antibody conjugated with horseradish peroxidase (HRP) diluted in TBST containing 3% nonfat milk for 1 h at rt while rocking gently. After washing 3 times with TBST for 10 min each time and one time with TBS for 5 min, the membranes were incubated with 5 mL of ECL-Western blotting detection reagents for 5 min and exposed to Kodak chemiluminescence BioMax films for visualization. The membranes were then stripped by incubating with stripping buffer (62.5 mM Tris HCl, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol) for 30 min at 56 °C and reblotted with anti-FAK antibody similarly as described above for phospho-FAK antibody. Generation of Subcutaneous Human Tumor Xenograf ts in SCID/Beige or Nu/Nu Mice Female. SCID/Beige (6−8 weeks, Taconic, Hudson, NY) or Nu/Nu mice (6−8 weeks, Charles River Laboratory, Wilmington, MA) were maintained 5/cage in microisolator units on

precipitate formed. This was extracted 3× with dichloromethane. The organic layer was then washed with brine, dried over MgSO4, filtered, and concentrated. The residue was pumped dry and then purified by automated flash chromatography (ISCO, gradient elution 0−10% dichloromethane/10% NH4OH in MeOH). The desired product eluted around 9−10%, and the 10% gradient was held until product eluted completely. Mixed fractions were concentrated and were purified by RP-HPLC (gradient elution, 0−40% CH3CN/water). Chromatography was repeated using normal phase silica and reverse phase HPLC to effect further purification as desired. Following neutralization and concentration of all the material, the resulting solid was obtained by taking the foam up into EtOAc and concentrating to dryness several times to give 27b (1.1 g, 28%) as an off-white foamy solid. LC/MS (ESI+) m/z = 580.0 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 11.02 (s, 1H), 8.69 (d, J = 8.9 Hz, 1H), 8.13 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.59−7.50 (m, 2H), 7.41 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.21 (s, 1H), 3.74 (m, 3H), 3.66− 3.63 (m, 2H), 3.29−3.23 (m, 1H), 3.06 (d, J = 4.3 Hz, 3H), 2.92−2.72 (m, 5H), 2.66−2.55 (m, 8H), 2.48−2.39 (m, 2H), 2.16−2.10 (m, 2H), 1.87−1.77 (m, 1H), 1.42−1.32 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 168.9, 158.5, 155.0, 154.7, 149.0, 139.3, 137.3, 135.6, 131.4, 130.3, 127.9, 124.4, 121.8, 121.3, 120.5, 104.8, 63.1, 61.0, 60.4, 58.5, 53.8, 47.7, 38.1, 33.9, 26.4, 26.3, 25.7, 25.5. High resolution mass spectrum m/z 580.2807 [(M + H) + calcd for C30H39ClN7O3 580.2803]. HPLC purity: 99 A%. (28) 2-[4-[2-[[5-Chloro-4-(2-pyrazol-1-ylanilino)pyrimidin-2yl]amino]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen6-yl]piperazin-1-yl]ethanol. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amines, 12d (0.19 g, 0.59 mmol) and 21g (0.12 g, 0.40 mmol) gave 28 (81 mg, 35% yield) as a white foam. LC/MS (ESI+) m/z 589.1 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 10.19 (s, 1H), 8.53 (d, J = 8.1 Hz, 1H), 8.10−8.05 (m, 2H), 7.84 (dd, J = 17.2, 1.5 Hz, 2H), 7.47−7.38 (m, 3H), 7.27−7.21 (m, 1H), 6.90 (d, J = 8.1 Hz, 1H), 6.53 (s, 1H), 3.72 (s, 3H), 3.65 (t, J = 5.3 Hz, 2H), 3.25 (br dd, J = 14.1, 7.1 Hz, 1H), 2.95−2.82 (m, 2H), 2.75 (br d, J = 5.3 Hz, 2H), 2.69−2.54 (m, 8H), 2.49−2.37 (m, 2H), 2.12 (br d, J = 11.1 Hz, 2H), 1.87−1.75 (m, 1H), 1.44−1.32 (m, 1H). HPLC purity >95 A%. (29) 2-[4-[2-[[5-Chloro-4-[2-(1-methylimidazol-2-yl)anilino]pyrimidin-2-yl]amino]-1-methoxy-6,7,8,9-tetrahydro-5Hbenzo[7]annulen-6-yl]piperazin-1-yl]ethanol. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin4-amines, 12d (0.15 g, 0.49) and 21h (0.10 g, 0.31 mmol) gave 29 (52 mg, 26% yield) as white crystals. Mp 95−96 °C. LC/MS (ESI+) m/z 603 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 10.70 (s, 1H), 8.60 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 8.06 (s, 1H), 7.51−7.41 (m, 3H), 7.25 (s, 1H), 7.24−7.19 (m, 1H), 7.02 (s, 1H), 6.92 (d, J = 8.3 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.65 (t, J = 5.3 Hz, 2H), 3.50 (q, J = 7.0 Hz, 1H), 3.25 (br dd, J = 14.1, 6.8 Hz, 1H), 2.94−2.70 (m, 4H), 2.67−2.55 (m, 8H), 2.49−2.37 (m, 2H), 2.17−2.08 (m, 2H), 1.82− 1.79 (m, 1H), 1.37 (q, J = 11.8 Hz, 1H). HPLC purity >95 A%. (30) 2-[4-[2-[[5-Chloro-4-(2-methoxyanilino)pyrimidin-2-yl]amino]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6yl]piperazin-1-yl]ethanol. Following the general procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amines, 12d (0.14 g, 0.45 mmol) and 21i (0.08 g, 0.30 mmol) gave 30 (74 mg. 45% yield) as a yellow solid. LC/MS (ESI+) m/z 553.17 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 8.47 (br d, J = 8.1 Hz, 1H), 8.10−8.06 (m, 2H), 7.82 (s, 1H), 7.43 (s, 1H), 7.16−7.07 (m, 1H), 7.06−6.99 (m, 1H), 6.95 (br dd, J = 14.0, 8.2 Hz, 2H), 3.96 (s, 3H), 3.74 (s, 3H), 3.65 (br t, J = 4.7 Hz, 2H), 3.26 (br dd, J = 14.0, 6.4 Hz, 1H), 2.97− 2.80 (m, 3H), 2.75 (br d, J = 4.5 Hz, 2H), 2.68−2.54 (m, 8H), 2.49− 2.37 (m, 2H), 2.12 (br d, J = 11.1 Hz, 2H), 1.81 (q, J = 11.6 Hz, 1H), 1.38 (br d, J = 11.6 Hz, 1H). HPLC purity 90 A%. (31) 2-[4-[(6R)-2-[(4-Bromobenzoyl)amino]-1-methoxy6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl]piperazin-1-yl]ethyl 4-Bromobenzoate. 12d (0.38 g, 0.75 mmol) was treated with 4-bromobenzoyl chloride, (0.20 g, 0.90 mmol), triethylamine (0.16 mL, 1.13 mmol) in dichloromethane (40 mL) at 0 °C, warmed to room temperature, and stirred overnight. The mixture was partitioned between sat. aq NaHCO3 and dichloromethane. The organics were P



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a standard laboratory diet (Teklad Labchow, Harlan Teklad, Madison, WI). Animals were housed under humidity- and temperaturecontrolled conditions and the light/dark cycle was set at 12 h intervals. Mice were quarantined at least 1 week prior to experimental manipulation. Briefly, the cells were collected and resuspended in RPMI medium at a density of 5 × 107/mL, and an aliquot (100 μL) of the cell suspension (5 × 106 cells) was inoculated subcutaneously to the left flank of each mouse with a 23 G needle. The mice were then monitored daily. When the tumor xenograft volumes reached approximately 300−500 mm3, the mice received a single oral administration of either PEG-400 vehicle or test compound at100 μL/dose. At indicated time point(s) after dosing, the mice (3−4 mice at each time point) were sacrificed and the blood was collected in 1.5 mL microcentrifuge tubes containing 20 μL of heparin sodium (10 000 unit/mL in H2O) and left on ice briefly. The tubes were centrifuged at 20 817g for 8 min at 4 °C, and the plasma was collected and transferred to 1.5 mL microcentrifuge tubes, which were then stored at −80 °C. The tumors were excised and weighed, cut into small pieces with a scalpel, and placed into a round-bottom 14 mL tube on ice. Two volumes of completed FRAK lysis buffer without detergent [10 mM Tris, pH 7.5, 50 mM sodium chloride, 20 mM sodium fluoride, 2 mM sodium pyrophosphate, 0.1% BSA, plus freshly prepared 1 mM activated sodium vanadate, 4 mM DTT, 1 mM PMSF, and the protease inhibitor cocktail III (1:100 dilution)] were added to 1 volume of tumor (e.g., an amount of 500 μL of FRAK lysis buffer was added to 250 mg of tissue). The tissues were then disrupted with a hand-held tissue blender 2−3 times, 10−15 s each time with 1−2 min interval. The lysates were then sonicated twice, 4−5 strokes each time. The tissue lysates were transferred to 1.5 mL microcentrifuge tubes and centrifuged at 20 817g for 10 min at 4 °C. The supernatants (12 μL) were transferred to 1.5 mL microcentrifuge tubes containing 108 μL of FRAK lysis buffer and 40 μL of 4× LDS sample buffer with freshly added 100 nM dithiotreitol. The samples were subjected for phospho-FAK and total FAK detection with immunoblotting as described above. The remaining tumor lysates were stored at −80 °C. The compound levels in both plasma and tumor lysates were measured by LC−MS/MS. Antitumor Activity Study with Test Compound with Oral Administration. The tumor-bearing mice were randomized into different treatment groups (8−10 mice/group) and were administered orally with either vehicle (PEG-400) or test compound formulated in PEG400 at indicated doses (expressed as mg/kg equivalents of free base) and with indicated dosing frequency, with 100 μL per dosing volume. The length (L) and width (W) of each tumor was measured with a vernier caliper, and the mouse body weight was determined every 2−3 days. The tumor volumes were then calculated with the formula 0.5236LW(L + W)/2. Statistical analyses of tumor volumes and mouse body weight were carried out using the Mann−Whitney rank sum test. The TGI values were calculated at the end of study by comparing the tumor volumes (TV) of each treatment group with those of vehicletreated group with the following formula: [1 − (the last day TV of compound-treated group/the last day TV of vehicle treated group)] × 100.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mark Olsen and Emily Kordwitz (SFC separations), Damaris Rolon-Steele (PK), Kelly Ziegler (PK), Maxime Siegler (Johns Hopkins University, X-ray) for their assistance.

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ABBREVIATIONS USED ALCL, anaplastic large-cell lymphoma; ALK, anaplastic lymphoma kinase; c-MET, mesenchymal epithelial transition factor; CYP, cytochrome P450; EGFR, epidermal growth factor receptor; EML4, echinoderm microtubule-associated proteinlike 4; EML4-ALK, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase; FAK, focal adhesion kinase; hERG, human ether-a-go-go-related gene; IBC, inflammatory breast cancer; INSR, insulin receptor kinase; JAK, Janus kinase; KRas, Kirsten rat sarcoma virus; LM, liver microsome; NPM, nucleophosmin; NSCLC, non-small-cell lung cancer; PDX, patient-derived xenograft; PYK, pyruvate kinase; TYK, tyrosine kinase; SAR, structure−activity relationship

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00487. Experimental conditions for X-ray data collection and solution, PK for compound 1, DiscoveRx kinase selectivity data for 27b (PDF)

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