2-Amino-2,3-dihydro-1H-indene-5-carboxamide-Based Discoidin Domain Receptor 1 (DDR1) Inhibitors: Design, Synthesis, and in Vivo Antipancreatic Cancer Efficacy.

A series of 2-amino-2,3-dihydro-1H-indene-5-carboxamides were designed and synthesized as new selective discoidin domain receptor 1 (DDR1) inhibitors. One of the representative compounds, 7f, bound with DDR1 with a Kd value of 5.9 nM and suppressed the kinase activity with an half-maximal (50%) inhibitory concentration value of 14.9 nM. 7f potently inhibited collagen-induced DDR1 signaling and epithelial-mesenchymal transition, dose-dependently suppressed colony formation of pancreatic cancer cells, and exhibited promising in vivo therapeutic efficacy in orthotopic mouse models of pancreatic cancer.

Introduction

Pancreatic cancer, also known as the “king of cancer” with a 5 year survival rate less than 7%, is lacking effective therapies.[1] Intrinsic and/or acquired chemoresistance to the first-line drug gemcitabine is a major reason for the poor prognosis. Collective studies suggested that desmoplasia and tumor microenvironment (TME) are the key contributors to chemoresistance of pancreatic cancer patients. Desmoplasia (or desmoplastic reaction) is a prominent pathological characteristic of pancreatic cancer, resulting from the rapid expansion of cancer-associated fibroblasts and increased deposition of extracellular matrix (ECM) components. Numerous studies have shown that ECM is heavily involved in pancreatic cancer development, immune evasion, and therapy resistance. Therefore, stroma-targeting therapy becomes an attractive strategy to improve therapeutic response.[2] Collagens are the most abundant components in ECM.[3] Biological functions of collagens are mainly mediated by two types of receptors, integrins and discoidin domain receptors (DDRs).[4,5] DDRs belong to the receptor tyrosine kinase (RTK) family. Two members of DDRs (DDR1 and DDR2) have been identified to date.[6] DDR1 is predominantly expressed in epithelial cells, whereas DDR2 is typically found in cells of connective tissues. Dysregulation of DDR1 is frequently detected in a variety of human cancers and involved in several key cellular processes, such as cell differentiation, proliferation, adhesion, migration, and invasion.[7,8] Studies have shown that DDR1 overexpression mediates prosurvival signals and metastasis in breast cancer and gastric cancer and is also involved in the recurrence of certain types of cancers.[9−11] DDR1 has also been demonstrated to contribute to epithelial–mesenchymal transition (EMT) in pancreatic cancer.[12,13] Pharmacological inhibition of DDR1 by a selective DDR1 inhibitor 7rh (compound 1, Figure ) successfully slowed tumor progression and enhanced chemosensitivity to standard-of-care pancreatic cancer regimens.[14] Therefore, DDR1 is being considered as a novel molecular target for drug discovery against pancreatic cancer.

Figure 1

Reported selective DDRs inhibitors.

A number of DDR inhibitors have been reported (Figure ),[15−21] among which compound 1 (7rh)[16] represents one of the first disclosed selective DDR1 inhibitors. This compound demonstrated promising therapeutic potential in a variety of human cancer models including pancreatic cancer,[14] nonsmall cell lung cancer,[22] and gastric carcinoma.[10] Most recently, tetrahydroisoquinoline-7-carboxamide derivatives were also designed and synthesized as highly selective DDR1 inhibitors exhibiting in vivo efficacy in mouse models of inflammation-mediated pulmonary fibrosis and acute lung injury.[21,23] Compound 6 represents one of the most selective DDR1 inhibitors to date, which exhibited an half-maximal (50%) inhibitory concentration (IC50) value of 15.4 nM against DDR1 kinase, whereas it is significantly less potent for the majority of a panel of 403 other kinases. The major “off-targets” include tropomyosin receptor kinases B and C (TrkB and TrkC, respectively) with corresponding binding affinities (Kd values) of 22 and 18 nM, respectively. Further kinase assay determination confirmed that this molecule inhibited TrkA, B, and C, with IC50 values of 68.4, 36.8, and 30.2 nM, respectively. Trks are a family of receptor tyrosine kinases (RTKs), which are activated by neurotrophin hormones and regulate synaptic plasticity and strength in the mammalian nervous system.[24] Lack of Trks significantly affects the development of central and peripheral nervous systems.[25,26] For instance, the population of corneal sensory neurons is markedly depleted in TrkA (−/−) mice, animals lacking TrkB in parvalbumin-positive cells displayed sexually dimorphic behavioral phenotypes, whereas TrkC knockout mice exhibited profound deficiencies in CNS glial cells.[27] Thus, “off-targeting” Trks may raise some concerns about the potential neurotoxicity issues of the previously reported DDR1 inhibitors. With the aim to improve the target specificity, a series of 2-amino-2,3-dihydro-1H-indene-5-carboxamide derivatives were designed and synthesized as new Trks-sparing DDR1 inhibitors. Moreover, the potential therapeutic effects on pancreatic cancer were also investigated by utilizing in vitro and in vivo models (Figure ).

Figure 2

Design of 2-amino-2,3-dihydro-1H-indene-5-carboxamide derivatives 7 as new Trks-sparing selective DDR1 inhibitors.

Syntheses of the designed compounds 7a, 7b, and 7c–n are described in Schemes S1 and , respectively. Briefly, protection of 8 and 9 with (Boc)2O gave 10 and 11, respectively. Further alkylation of 10 generated 12–14. 10–14 were converted to 15–19 by using a carbonyl-extrusion reaction, which were deprotected by hydrochloric acid to produce intermediates 20–24. Coupling of 20–24 with 5-bromopyrimidine through a Buchwald–Hartwig amination reaction provided 25–29. The ester 27 was hydrolyzed to 30. 25–30 were further reacted with 31 to produce the final compounds 7c–n.

Scheme 1

Syntheses of Compounds 7c–n

Reagents and conditions: (a) di-tert-butyl dicarbonate (Boc2O), triethylamine (Et3N), 90–95%; (b) sodium hydride (NaH), N,N-dimethylformamide (DMF), iodomethane, 12; iodoethane, 13; 1-iodopropane, 14; 50–80%; (c) palladium(II) acetate (Pd(OAc)2), Et3N, carbon monoxide (CO), 100 °C, 50–70%; (d) 4 M HCl, 50 °C; (e) bis(dibenzylideneacetone)palladium (Pd(dba)2), 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (Ruphos), cesium carbonate (Cs2CO3), 5-bromopyrimidine, 80 °C, 30–40%; (f) 1 M sodium hydroxide (NaOH), dioxane, 98%; (g) potassium tert-butoxide (t-BuOK), −20 °C to room temperature (rt), 30–70%; (h) 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine (DIPEA), 20–55%.

Results and Discussion

Compound 6 is a selective DDR1 inhibitor designed and synthesized in our laboratory. However, this compound displayed similar off-target potency against TrkA, TrkB, and TrkC with IC50 values of 68.4, 36.8, and 30.2 nM, respectively (Table ). Off-target inhibition against Trks may raise some concern about the potential neurotoxicity issues of the molecule. Aiming to improve the target specificity, we first docked compound 6 into the adenosine triphosphate (ATP)-binding site of TrkC [Protein Data Bank (PDB) ID: 3V5Q].[28] It was shown that the compound could bind well to TrkC with a Asp-Phe-Gly (DFG)-out conformation, which was similar to the binding mode with DDR1 (Figure A,B).[21] The only small difference between binding modes of 6 with DDR1 and TrkC is that the methyl group on tetrahydroisoquinoline occupied a small hydrophobic pocket formed by Val552, Ala570, Lys572, and Phe617 to achieve some hydrophobic interactions in TrkC. Thus, it was predicted that diminishing this interaction might decrease the binding with Trks and improve DDR1 target selectivity of the molecule. Based on this hypothesis, an isoindoline scaffold was first utilized to replace the 1-methyl tetrahydroisoquinoline in 6 to generate compounds 7a and 7b. Disappointingly, both 7a and 7b lost all of the DDR1 inhibitory activity, although the modification indeed abolished the off-target inhibition against Trks (Table ). Further computational investigation demonstrated that 7a failed to fit nicely into the DDR1 ATP binding pocket (PDB ID: 5FDP, data not shown),[21] whereas compound 7b could form weak hydrogen bonds (HBs) with the hinge residue Met704, but there was no other interaction between the isoindoline scaffold and DDR1 (Figure S1). The computational study also indicated that the methylene moiety of 7b is accessible to the gatekeeper Thr701 residue, and it could be replaced by a hydrogen bond donor moiety to form a hydrogen bond interaction with Thr701 to potentially improve the DDR1 inhibitory activity. To validate this hypothesis, 2-amino-2,3-dihydro-1H-indene-5-carboxamide enantiomers 7c and 7d were designed and synthesized by moving the nitrogen atom out of the original isoindoline ring. It was also predicted that the R-isomer 7c might achieve a more favorable binding with DDR1 than that of the S-isomer 7d to form a hydrogen bond with the gatekeeper residue Thr701 (Figure S2A,B). Indeed, kinase inhibition determination showed that compound 7c potently inhibited DDR1 with an IC50 value of 5.6 nM, whereas the corresponding value for 7d was 42.5 nM. Compound 7c exhibited a similar suppressive activity against DDR2 with an IC50 value of 21.5 nM. It was also noteworthy that 7c almost totally abolished the inhibitory potencies against Trks with IC50 values greater than 1000 nM. However, introduction of a methyl group at the R3-position (7e) caused an approximately 129-fold potency loss, which might be due to the steric hindrance to interfere with potential hydrogen bond formation.

Table 1

In Vitro Kinase Inhibitory Activities of Compounds 7a–n against DDR1, DDR2, TrkA, TrkB, and TrkCa,b

DDR1 and DDR2 inhibition assays were performed using the LANCE ULTRA kinase assay.

TrkA/B/C activity was performed using the fluorescence resonance energy transfer (FRET)-based Z′-Lyte assay. The data are mean values from at least three independent experiments.

Figure 3

(A) Molecular docking of 6 into DDR1 (PDB: 5FDP). (B) Molecular docking of 6 into TrkC (PDB: 3V5Q). (C) Cocrystal structures of DDR1 and 7c (PDB: 6HP9). (D) Molecular docking of 7c into TrkC. Compounds 6 and 7c are shown in gray, purple, and blue stick structure. The key residues of DDR1 and TrkC kinase are shown in blue and gray stick and ribbon representation. Hydrogen bonds are indicated by yellow dashed lines to key amino acids.

A 2.0 Å resolution X-ray crystallographic structure of 7c and the DDR1 complex was determined to elucidate details of the interaction (Figure C and Table S2). It was shown that 7c fitted nicely into the ATP binding pocket of DDR1 with a typical type II binding mode. The nitrogen atom of pyrimidine was observed to form a hydrogen bond with hinge residue Met704, whereas the amino group at 2-position of the indene formed a hydrogen bond with gatekeeper residue Thr701. Two additional hydrogen bonds were also observed, one between the amide nitrogen of 7c and the carboxylates of Glu672 and another between the amide carbonyl of 7c and the backbone amide nitrogen of Asp784. Additionally, the trifluoromethyl group was observed to fit snugly into the DFG-out pocket. A computational study was further performed to rationalize the significant potency loss for Trks (PDB ID: 3V5Q).[18] It was shown that the pyrimidinyl moiety of 7c would still be able to form a hydrogen bond (∼3.1 Å) with the key residue Met620 in TrkC (Figure D), but the gatekeeper Phe617 of TrkC altered the three-dimensional (3D) structure of the ATP pocket relative to DDR1, which would both preclude a key hydrogen bond and additionally introduce a steric clash with 7c.

Aiming to improve DDR1 target specificity, further structural optimization was conducted. Superposition of DDR1 (PDB: 6HP9, gray) and a homology model of DDR2 (orange)[29] indicated that DDR1 possessed a small hydrophobic groove formed by Ile685, Thr701, and Asp703, whereas no such pocket existed in DDR2 because of its outward shifting of Glu93 and Ile76 (Figure A). Based on this observation, it was hypothesized that a small hydrophobic substituent might be introduced at the nitrogen atom of 7c to improve the DDR1 selectivity. Thus, compounds 7f–h, in which a methyl, ethyl, or n-propyl group was introduced at the corresponding position, respectively, were designed and synthesized. It was shown that the methylated compound 7i indeed significantly decreased the DDR2 inhibitory potency with an IC50 of 933 nM, although it also caused a 2.6-fold potency loss for DDR1 with an IC50 value of 14.9 nM (Table ). However, both 7g and 7h resulted in 26.8- and approximately 200-fold DDR1 potency decreases, respectively. Computational investigation supported the idea that a methyl group (7f) could fit into a small hydrophobic pocket formed by Ile685, Thr701, and Asp702, whereas loss of a hydrogen bond with Thr701 would contribute to the DDR1 potency decrease (Figure B).

Figure 4

(A) Superposition of DDR1 (PDB: 6HP9) and DDR2. (B) Molecular docking of 7f into DDR1 (PDB: 6HP9). The key residues of DDR1 kinase are shown in gray stick representation and DDR2 kinase are shown in orange. Compounds 7c and 7f are shown in gray and green stick structures, respectively. Hydrogen bonds are indicated by yellow dashed lines to key amino acids.

Further structure–activity relationship investigation also revealed that the trifluoromethyl of 7f could be replaced by the ethyl (7j), isopropyl (7k), or tertiary butyl (7l) group without obviously affecting the DDR1 inhibitory potency. For instance, compounds 7j–l exhibited IC50 values of 14.7, 15.0, and 14.4 nM, respectively. However, their potencies against DDR2 and Trks were improved, which made the compound less selective. A replacement of the trifluoromethyl with a methyl group (7i) caused an approximately 43-fold decrease in DDR1 potency. When the trifluoromethyl was replaced with a cyclohexyl group, the resulting compound 7m was also 5-fold less potent than the lead molecule 7f. Significantly, the amantadine derivative (7n) totally abolished its activity against DDR1 with an IC50 value greater than 2.0 μM. Collectively, 7f represents one of the most potent and selective DDR1 inhibitors in the series for further biological investigation.

The binding affinity determination showed that 7f tightly bound to the ATP-binding sites of DDR1 with a Kd value of 5.9 nM (DiscoverX, San Diego, CA).[30] Further target specificity of 7f was investigated by KINOMEscan profiling with a panel of 468 kinases at 1.0 μM (Figure A). The results indicated that 7f displayed an extraordinary target selectivity with S (10) and S (1) scores of 0.015 and 0.007, respectively (Table S3). The major off-targets (inhibition > 90%, ctrl% < 10) included the DDR2, ABL1, KIT, PDGFRB, and TrkC. The inhibitory activities (IC50) of 7f against above-mentioned off-targets were further determined with our in-house kinase assays (Figure B). It was shown that 7f displayed significantly less potencies to all of the off-target kinases.

Figure 5

Target selectivity of 7f. (A) KINOMEscan profiles for 7f. (B) Inhibitory activities (IC50) of compound 7f against the potential off-targets.

To investigate the inhibitory effects of 7f on the biological functions of DDR1, we first examined the DDR1 signaling pathway with the drug treatment in a mouse pancreatic cancer cell line Pan02 (Figure ). It was found that 7f dose-dependently inhibited collagen-induced activation of DDR1 and its downstream signaling proteins, for example, proline-rich tyrosine kinase 2 (PYK2) and pseudopodium-enriched atypical kinase 1 (PEAK1).

Figure 6

7f inhibits DDR1 activation induced by collagen in vitro. Pan02 were treated with collagen I (50 μg/mL) and dimethyl sulfoxide (DMSO) or different concentrations of 7f for 8 h. Cell lysates were harvested and subjected to western blot, probing for p-DDR1, DDR1, p-PYK2, PYK2, p-PEAK1, PEAK1, and TUBULIN.

The activation of DDR1–PYK2–PEAK1 signaling has been shown to mediate the tumor-promoting functions of collagen, including an important process of EMT called cadherin switching,[31] in which cancer cells specifically upregulate the mesenchymal cell adhesion protein N-cadherin, whereas the epithelial cell adhesion protein E-cadherin may or may not be affected. As a result of N-cadherin upregulation, cancer cells become substantially more motile and aggressive.[32] Therefore, we performed immunofluorescence staining for E-cadherin and N-cadherin in Pan02 and another mouse pancreatic cancer cell line BMF-A3 derived from KPfC (Kras; Trp53; Ptf1a) mice, a genetically engineered mouse model of pancreatic cancer, to explore the effects of 7f on inhibiting DDR1-mediated cadherin switching (Figure A). Consistently, 7f markedly inhibited cadherin switching in each cell line regardless of the different genetic background. Moreover, we also found that 7f significantly inhibited the migratory capability of the cells in a wound-healing assay (Figure B).

Figure 7

7f inhibits collagen-induced cadherin switching and migration in pancreatic cancer. (A) Pan02 and primary mouse pancreatic cancer cell line BMF-A3 were treated with collagen I (50 μg/mL) and DMSO or 1 μM 7f for 24 h. Cells were fixed with methanol and stained with E-cadherin (green), N-cadherin (red), and 4′,6-diamidino-2-phenylindole (DAPI, blue). Fluorescent images were captured at 20× magnification. (B) Wound-healing assay was performed with Pan02 cells. Cells were treated with collagen I (50 μg/mL) and DMSO or 1 μM 7f for 24 h, and relative rates of migration were measured after a 24 h period of time. **** P < 0.0001.

Cell heterogeneity is a major cause of therapy resistance in most types of cancers.[33] To understand whether 7f had similar effects on inhibiting cadherin switching in different cancer cell populations derived from the same tumor, we performed a 3D culture experiment with two different cancer cell clones derived from a KPfC tumor (BMF-A3 and CT1A-C11). In the presence of ECM that mimics the in vivo tumor microenvironment.[34] We found that BMF-A3 represented a more epithelial phenotype, which remained as clusters under 3D culture, whereas CT1A-C11 was more mesenchymal and aggressive, with an elongated and fibroblastic morphology. However, we found that 7f strongly inhibited such a mesenchymal phenotype (Figure A). We also examined the effect of 7f on DDR1-induced cadherin switching in the two cell lines by probing for E-cadherin and N-cadherin expressions in cell lysates. We found that although the two cell lines had different phenotypes in 3D culture, 7f inhibited the upregulation of N-cadherin similarly in a dose-dependent manner (Figure B). This suggests that 7f may have effects on many cancer cell populations despite cellular heterogeneity.

Figure 8

7f inhibits collagen-induced cadherin switching in different cancer cell clones from KPfC mice. (A) BMF-A3 and CT1A-C11 were embedded in ECM consisting of 5 mg/mL matrigel and 2.1 mg/mL collagen I. Cultures were overlaid with Dulbecco’s modified Eagle’s medium (DMEM) + 10% fetal bovine serum (FBS) containing 2% matrigel. After 48 h, cells were fixed with methanol and stained with phalloidin (red) and DAPI (blue). Fluorescent images were captured at 20× magnification. (B) Primary mouse pancreatic cancer cell lines BMF-A3 and CT1A-C11 were treated with collagen I (50 μg/mL) and DMSO or different concentrations of 7f for 18 h. Cell lysates were harvested and subjected to western blot, probing for N-cadherin, E-cadherin, and ACTIN.

In addition, we examined the effects of 7f on the tumorigenicity of pancreatic cancer cells using an in vitro colony formation assay. As shown in Figure A,B, 7f dose-dependently inhibited colony formation significantly in BMF-A3 and Pan02 cells. However, the direct effect against proliferation of 7f seemed to be moderate, measured by cell proliferation in two-dimensional with BMF-A3 and Pan02 cells showing IC50s values of 4.26 and 11.92 μM, respectively (Figure S4).

Figure 9

7f inhibits colony formation in BMF-A3 (A) and Pan02 (B) pancreatic cancer cells. Colony formation for cells was grown in DMEM with 10% FBS ± 7f at the indicated doses for 10 days. Mean ± standard deviation (S.D.) colonies are shown. ** P < 0.01, **** P < 0.001, ***** P < 0.0001 by Student’s t-test vs DMSO.

Given the effects of 7f on pancreatic cancer cells in vitro, we further studied its effects in vivo. First, we profiled the in vivo pharmacokinetics (PK) of 7f in Sprague-Dawley (SD) rats (Table ). 7f displayed an ideal PK profile with an oral area under curve (AUC) value of 80 535 μg/(L h), a T1/2 value of 1.7 h, and an oral bioavailability of 89.9%, which provides the foundation for an in vivo dosing regimen.

Table 2

Pharmacokinetic Profile of Compound 7f in Ratsa

	po (25 mg/kg)	iv (5 mg/kg)
AUC(0–∞) μg/(L h)	80 535.0	16 855.5
T1/2 (h)	1.7	0.9
Tmax (h)	4.0	0.083
Cmax (μg/L)	15 352.5	14 746.0
BA (%)	89.9

SD rats (male, three animals per group) weighted 180–220 g were used for the study.

We then established syngeneic models of pancreatic cancer by orthotopically implanting the BMF-A3 and Pan02 cells in C57BL/6 mice and evaluated the efficacy of 7f on pancreatic cancer progression. The animals were orally (po) administered vehicle or 7f [twice per day (bid), 25 and 50 mg(kg day)] for 3 weeks. In both tumor models, 7f inhibited the progression of the tumors significantly without obviously caused animal body weight loss (Table S4), highlighting the in vivo efficacy of the molecule in pancreatic cancer (Figure ).

Figure 10

7f inhibits pancreatic cancer progression in vivo. Mouse pancreatic cancer cell lines BMF-A3 and Pan02 were implanted orthotopically in C57BL/6 mice. 10 days after implantation, mice (n = 4–5 per group) were orally administered with vehicle or 7f (25 and 50 mg/kg) twice per day for 3 weeks. Data were analyzed by analysis of variance (ANOVA) and presented as the mean ± S.D. * P < 0.05.

Conclusions

In summary, a series of 2-amino-2,3-dihydro-1H-indene-5-carboxamide derivatives were designed and synthesized as novel highly selective DDR1 inhibitors with a structure-based drug design method. Compound 7f strongly suppressed DDR1 with an IC50 value of 14.9 nM, but it was significantly less potent against a panel of other 403 nonmutated kinases at 1.0 μM. 7f also potently inhibited the collagen-induced cadherin switching event induced by DDR1 and dose-dependently suppressed colony formation of pancreatic cancer cells. Moreover, 7f demonstrated good pharmacokinetic properties and promising therapeutic effect by oral administration in orthotopic syngenic models of pancreatic cancer. Notably, several somatic mutations of DDR1 had been clinically identified, but most of them are located out of the kinase domain.[35] Thus, 7f may achieve similar inhibition against these disease-related mutants. Extensive biological investigation is undergoing to validate 7f as a lead molecule for further development.

Experimental Section

General Chemistry

Reagents and solvents were purchased from commercial sources and used directedly. Flash chromatography was performed using 300-mesh silica gel. Reactions were monitored by thin-layer chromatography using silica gel plates with fluorescence F254 and UV light visualization. Low-resolution electrospray ionization mass spectrometry (ESI-MS) was performed on an Agilent 1200 high-performance liquid chromatography (HPLC)-mass selective detector mass spectrometer and high-resolution ESI-MS on an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. 1H NMR spectra were performed on a Bruker AV-400 spectrometer at 400 MHz or a Bruker AV-500 spectrometer at 500 MHz. 13C NMR spectra were performed on a Bruker AV-500 spectrometer at 125 MHz. Coupling constants (J) were expressed in hertz (Hz). Chemical shifts (δ) of NMR were reported in parts per million units relative to an internal standard (tetramethylsilane). Purity of the compounds was determined by reverse-phase high-performance liquid chromatography (HPLC) analysis to be >95%. HPLC instrument: Dionex Summit HPLC (column: Diamonsil C18, 5.0 μm, 4.6 × 250 mm2 (Dikma Technologies); detector: PDA-100 photodiode array; injector: ASI-100 autoinjector; pump: p-680A). A flow rate of 1.0 mL/min was used with mobile phase of MeOH in H2O with a 0.1% modifier (ammonia, v/v).

N-(3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-2-(pyrimidin-5-yl)isoindoline-5-carboxamide (7a)

To a solution of methyl 2-(pyrimidin-5-yl)isoindoline-5-carboxylate 38 (32 mg, 0.12 mmol) and 3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline (30 mg, 0.12 mmol) in tetrahydrofuran (THF) (1 mL) was added t-BuOK (42 mg, 0.36 mmol) at −20 °C. The mixture was stirred at rt overnight. Then, the reaction mixture was poured into ice-water and extracted with ethyl acetate three times. The combined organic layer was washed with brine, dried over Na2SO4, concentrated in vacuo, and purified by flash chromatography on silica gel to give 7a (18 mg, 32% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 8.57 (s, 1H), 8.30 (s, 3H), 8.21 (s, 1H), 8.17 (s, 1H), 8.05 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.74 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.49 (s, 1H), 4.79 (s, 4H), 2.19 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 165.69, 146.85, 141.30, 141.27, 140.51, 139.74, 138.89, 137.91, 137.53, 134.93, 133.42, 130.84 (q, J = 32.1 Hz), 129.60, 127.24, 123.61 (q, J = 271.3 Hz), 122.88, 122.09, 114.93, 114.19, 111.63, 52.73, 52.59, 13.50. High-resolution mass spectrometry (HRMS) (ESI) [M + H]+, calcd: 465.1645, found: 465.1642. HPLC analysis: MeOH/H2O (70:30), 4.32 min, 96.7% purity.

N-(3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-2-(pyrimidin-5-ylmethyl)isoindoline-5-carboxamide (7b)

Compound 7b was prepared by following a similar procedure to that for 7a. Yield, 39%. 1H NMR (500 MHz, CDCl3) δ 9.15 (s, 1H), 8.88 (s, 1H), 8.77 (s, 2H), 8.18 (s, 1H), 7.79 (s, 1H), 7.78–7.76 (m, 3H), 7.33 (s, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.06 (s, 1H), 3.99 (d, J = 6.5 Hz, 4H), 3.95 (s, 2H), 2.25 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 166.26, 158.14, 157.26, 144.70, 140.89, 140.68, 140.29, 138.64, 134.66, 133.27, 133.09 (q, J = 32.9 Hz), 132.20, 126.47, 123.34 (q, J = 271.5 Hz), 122.89, 121.71, 115.52, 115.14, 114.65, 113.06, 58.89, 58.75, 55.01, 13.69. HRMS (ESI) [M + H]+, calcd: 479.1802, found: 479.1802. HPLC analysis: MeOH/H2O (80:20), 7.72 min, 97.3% purity.

(R)-N-(3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-2-(pyrimidin-5-ylamino)-2,3-dihydro-1H-indene-5-carboxamide (7c)

To a solution of methyl (R)-2-(pyrimidin-5-ylamino)-2,3-dihydro-1H-indene-5-carboxylate 25 (50 mg, 0.19 mmol) and 3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline (45 mg, 0.19 mmol) in dry THF (1 mL) was added t-BuOK (64 mg, 0.56 mmol) at −20 °C. The mixture was stirred overnight at rt. Ice-water was added to the mixture, and then the solution was extracted with ethyl acetate three times. The combined organic layer was washed with brine, dried over Na2SO4, concentrated in vacuo, and purified by flash chromatography on silica gel to give the title compound 7c (31 mg, 34%). [α]D20 −23.1 (c 0.156, MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.42 (s, 1H), 8.30 (s, 1H), 8.19 (s, 3H), 8.17 (s, 1H), 7.89 (s, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.72 (s, 1H), 7.48 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 6.46 (d, J = 6.8 Hz, 1H), 4.41–4.34 (m, 1H), 3.46–3.40 (m, 2H), 2.93–2.86 (m, 2H), 2.18 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 166.43, 148.74, 146.03, 141.97, 141.34, 141.06, 140.69, 140.27, 138.64, 134.62, 133.20, 133.08 (q, J = 30.4 Hz), 126.43, 125.52, 124.20, 123.33 (q, J = 271.2 Hz), 115.48, 115.08, 114.63, 113.06, 53.63, 40.12, 39.91, 13.69. HRMS (ESI) [M + H]+, calcd: 479.1802, found: 479.1798. HPLC analysis: MeOH/H2O (70:30), 4.06 min, 100.0% purity.

(S)-N-(3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-2-(pyrimidin-5-ylamino)-2,3-dihydro-1H-indene-5-carboxamide (7d)

Compound 7d was prepared by following a similar procedure as that of 7c. Yield, 60%. [α]D20 +21.1 (c 0.152, MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.42 (s, 1H), 8.30 (s, 1H), 8.20–8.19 (m, 3H), 8.17 (s, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.72 (s, 1H), 7.48 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 6.46 (d, J = 6.8 Hz, 1H), 4.41–4.35 (m, 1H), 3.46–3.40 (m, 2H), 2.93–2.86 (m, 2H), 2.18 (s, 3H), 1.91 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 166.58, 148.56, 146.00, 141.91, 141.26, 141.14, 140.80, 140.13, 138.49, 134.61, 133.17, 133.01 (q, J = 32.7 Hz), 126.50, 125.43, 124.24, 123.34 (q, J = 271.3 Hz), 115.50, 115.23, 114.64, 112.95, 53.57, 40.07, 39.85, 13.63. HRMS (ESI) [M + H]+, calcd: 479.1802, found: 479.1798. HPLC analysis: MeOH/H2O (80:20), 5.49 min, 97.0% purity.

2-Methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-2-(pyrimidin-5-ylamino)-2,3-dihydro-1H-indene-5-carboxamide (7e)

Compound 7e was prepared by following a similar procedure as that of 7c. Yield, 35%. 1H NMR (500 MHz, CDCl3) δ 9.02 (s, 1H), 8.56 (s, 1H), 8.18 (s, 1H), 8.04 (s, 2H), 7.77 (s, 2H), 7.71 (d, J = 9.2 Hz, 1H), 7.33 (s, 1H), 7.28 (d, J = 7.9 Hz, 1H), 7.04 (s, 1H), 4.34 (s, 1H), 3.41–3.32 (m, 2H), 3.08–3.01 (m, 2H), 2.25 (s, 3H), 1.57 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 166.72, 148.18, 145.92, 142.33, 141.72, 140.84, 140.25, 140.12, 138.47, 134.58, 132.95 (q, J = 33.0 Hz), 126.57, 125.41, 124.20, 123.32 (q, J = 271.0 Hz), 115.51, 115.22, 114.62, 112.91, 61.49, 45.49, 45.35, 27.56, 13.60. HRMS (ESI) [M + H]+, calcd: 479.1802, found: 479.1798. HPLC analysis: MeOH/H2O (80:20), 8.17 min, 100.0% purity.

(R)-2-(Methyl(pyrimidin-5-yl)amino)-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-2,3-dihydro-1H-indene-5-carboxamide (7f)

Compound 7f was prepared by following a similar procedure as that of 7c. Yield, 71%. [α]D25 +21.64 (c 0.102, MeOH). 1H NMR (400 MHz, CDCl3) δ 9.08 (s, 1H), 8.59 (s, 1H), 8.29 (s, 2H), 8.23 (s, 1H), 7.81 (s, 1H), 7.79–7.76 (m, 3H), 7.35–7.33 (m, 2H), 7.07 (s, 1H), 4.81–4.74 (m, 1H), 3.34–3.23 (m, 2H), 3.14–3.06 (m, 2H), 2.78 (s, 3H), 2.25 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 166.37, 148.15, 146.11, 143.32, 142.05, 141.78, 140.75, 140.28, 138.62, 134.65, 133.19, 133.05 (q, J = 33.1 Hz), 126.38, 125.05, 123.81, 123.34 (q, J = 271.3 Hz), 115.48, 115.09, 114.63, 112.94, 58.39, 36.29, 36.04, 32.02, 13.69. HRMS (ESI) [M + H]+, calcd: 493.1958, found: 493.1954. HPLC analysis: MeOH/H2O (70:30), 4.75 min, 99.7% purity.

(R)-2-(Ethyl(pyrimidin-5-yl)amino)-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-2,3-dihydro-1H-indene-5-carboxamide (7g)

Compound 7g was prepared by following a similar procedure as that of 7c. Yield, 35%. [α]D25 +7.3 (c 0.11, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 8.40 (s, 1H), 8.26 (s, 2H), 8.23 (s, 1H), 7.83 (s, 1H), 7.79 (s, 1H), 7.75–7.73 (m, 2H), 7.38–7.36 (m, 2H), 7.09 (s, 1H), 4.75–4.67 (s, 1H), 3.40–3.31 (m, 4H), 3.17–3.11 (m, 2H), 2.29 (s, 3H), 1.17 (t, J = 7.0 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 166.47, 147.68, 145.90, 141.80, 141.61, 141.57, 140.81, 140.25, 138.58, 134.63, 133.22, 133.02 (q, J = 33.2 Hz), 126.48, 125.12, 123.93, 123.34 (q, J = 271.2 Hz), 115.48, 115.10, 114.62, 112.89, 58.61, 41.03, 36.68, 36.40, 13.69. HRMS (ESI) [M + H]+, calcd: 507.2115, found: 507.2110. HPLC analysis: MeOH/H2O (70:30), 5.43 min, 99.0% purity.

(R)-N-(3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-2-(propyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indene-5-carboxamide (7h)

Compound 7h was prepared by following a similar procedure as that of 7c. Yield, 11%. [α]D25 +1.4 (c 0.146, MeOH). 1H NMR (500 MHz, CDCl3) δ 9.39 (s, 1H), 8.49 (s, 1H), 8.16 (s, 1H), 8.14 (s, 2H), 7.79 (s, 1H), 7.75 (s, 1H), 7.73–7.71 (m, 2H), 7.23 (d, J = 10.2 Hz, 1H), 7.00 (s, 1H), 4.63–4.57 (m, 1H), 3.26–3.20 (m, 2H), 3.18–3.14 (m, 2H), 3.08–3.00 (m, 2H), 2.19 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 166.59, 147.54, 145.75, 141.83, 141.63, 140.98, 140.11, 138.45, 134.63, 133.25, 132.95 (q, J = 33.1 Hz), 126.58, 125.04, 124.03, 123.36 (q, J = 271.1 Hz), 115.53, 115.27, 114.64, 112.79, 59.11, 48.78, 36.46, 36.19, 21.50, 13.60, 11.25. HRMS (ESI) [M + H]+, calcd: 521.2271, found: 521.2269. HPLC analysis: MeOH/H2O (70:30), 6.62 min, 98.0% purity.

(R)-2-(Methyl(pyrimidin-5-yl)amino)-N-(3-methyl-5-(4-methyl-1H-imidazol-1-yl)phenyl)-2,3-dihydro-1H-indene-5-carboxamide (7i)

To a solution of (R)-2-(methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indole-5-carboxylic acid 30 (100 mg, 0.37 mmol) in 2 mL of CH2Cl2 were added 3-(adamantan-1-yl)-5-(4-methyl-1H-imidazol-1-yl)phenylamine (82.2 mg, 0.44 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (211 mg, 0.56 mmol), and N,N-diisopropylethylamine (0.2 mL, 1.11 mmol). The mixture was stirred overnight at room temperature and diluted with CH2Cl2. The resulting solution was washed with water and brine, dried over Na2SO4, concentrated in vacuo, and purified by column chromatography to give compound 7i. (51 mg, 32% yield). [α]D25 +21.9 (c 0.082, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.33 (s, 3H), 8.10 (s, 1H), 7.95 (s, 1H), 7.81 (s, 1H), 7.79 (s, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.40 (s, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 6.97 (s, 1H), 4.84–4.78 (m, 1H), 3.36–3.30 (m, 2H), 3.16–3.10 (m, 2H), 2.80 (s, 3H), 2.42 (s, 3H), 2.32 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 166.14, 148.12, 145.61, 143.33, 141.92, 141.75, 140.84, 139.61, 137.97, 133.80, 126.24, 124.92, 123.70, 119.29, 117.46, 114.77, 110.22, 58.33, 36.29, 36.07, 31.96, 21.70, 13.68. HRMS (ESI) [M + H]+, calcd: 439.2241, Found: 439.2239. HPLC analysis: MeOH/H2O (70:30), 3.82 min, 99.6% purity.

(R)-N-(3-Ethyl-5-(4-methyl-1H-imidazol-1-yl)phenyl)-2-(methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indene-5-carboxamide (7j)

Compound 7j was prepared by following a similar procedure as that of 7i. Yield, 54%. [α]D25 +24.5 (c 0.1098, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.34 (s, 2H), 8.05 (s, 1H), 7.97 (s, 1H), 7.85 (s, 1H), 7.81 (s, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.38–7.36 (m, 1H), 7.09 (s, 1H), 6.99 (s, 1H), 4.86–4.79 (m, 1H), 3.37–3.31 (m, 2H), 3.17–3.10 (m, 2H), 2.81 (s, 3H), 2.72 (q, J = 7.6 Hz, 2H), 2.34 (s, 3H), 1.29 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 166.11, 148.15, 147.20, 145.63, 143.34, 141.96, 141.86, 139.56, 138.16, 134.71, 133.84, 126.21, 124.95, 123.68, 118.07, 116.42, 114.83, 110.50, 58.40, 36.30, 36.09, 31.97, 29.02, 18.57, 15.41, 13.76. HRMS (ESI) [M + H]+, calcd: 453.2397, found: 453.2398. HPLC analysis: MeOH/H2O (70:30), 4.34 min, 98.1% purity.

(R)-N-(3-Isopropyl-5-(4-methyl-1H-imidazol-1-yl)phenyl)-2-(methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indene-5-carboxamide (7k)

Compound 7k was prepared by following a similar procedure as that of 7i. Yield, 19%. [α]D25 +21.0 (c 0.114, MeOH). 1H NMR (500 MHz, CDCl3) δ 8.62 (s, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.31 (s, 2H), 7.80 (s, 2H), 7.77–7.75 (m, 2H), 7.39 (s, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.05 (s 1H), 6.98 (s, 1H), 4.82–4.76 (m, 1H), 3.33–3.28 (m, 2H), 3.13–3.08 (m, 2H), 2.97–2.92 (m, 1H), 2.78 (s, 3H), 2.27 (s, 3H), 1.28 (d, J = 6.9 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 166.10, 152.00, 148.16, 145.64, 143.36, 141.97, 141.87, 139.70, 138.07, 133.82, 126.23, 124.96, 123.69, 116.87, 115.11, 110.76, 58.35, 36.31, 36.10, 34.41, 31.96, 23.92, 13.64. HRMS (ESI) [M + H]+, calcd: 467.2554, found: 467.2558. HPLC analysis: MeOH/H2O (80:20), 8.54 min, 95.7% purity.

(R)-N-(3-(tert-Butyl)-5-(4-methyl-1H-imidazol-1-yl)phenyl)-2-(methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indene-5-carboxamide (7l)

Compound 7l was prepared by following a similar procedure as that of 7i. Yield, 32%. [α]D25 +20.5 (c 0.078, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.32 (s, 1H), 8.21 (s, 1H), 8.14 (s, 1H), 7.85 (s, 1H), 7.83–7.79 (m, 2H), 7.74 (s, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.15 (s 2H), 4.85–4.80 (m, 1H), 3.39–3.28 (m, 2H), 3.19–3.07 (m, 2H), 2.79 (s, 3H), 2.38 (s, 3H), 1.38 (s, 9H). 13C NMR (125 MHz, CDCl3) δ 166.18, 154.47, 148.02, 145.60, 143.38, 141.98, 141.71, 139.63, 133.70, 126.33, 124.93, 123.64, 116.38, 114.05, 110.63, 58.51, 58.28, 55.19, 43.23, 36.35, 36.08, 35.28, 31.89, 31.32, 29.83, 18.56, 13.41, 12.44. HRMS (ESI) [M + H]+, calcd: 481.2710, Found: 481.2712. HPLC analysis: MeOH/H2O (80:20), 10.38 min, 95.3% purity.

(R)-N-(3-Cyclohexyl-5-(4-methyl-1H-imidazol-1-yl)phenyl)-2-(methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indene-5-carboxamide (7m)

Compound 7m was prepared by following a similar procedure as that of 7i. Yield, 28%. [α]D25 +23.7 (c 0.076, MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 8.51 (s, 1H), 8.45 (s, 2H), 8.02 (s, 1H), 7.888–7.86 (m, 2H), 7.82 (d, J = 7.8 Hz, 2H), 7.62 (s, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.34 (s 1H), 7.18 (s, 1H), 5.03–4.96 (m, 1H), 3.27–3.25 (m, 2H), 3.12–3.07 (m, 2H), 2.77 (s, 3H), 2.58–2.56 (m, 1H), 2.17 (s, 3H), 1.87–1.71 (m, 5H), 1.51–1.23 (m, 5H). 13C NMR (125 MHz, DMSO-d6) δ 165.59, 149.84, 146.93, 145.53, 143.24, 141.61, 141.25, 140.36, 138.27, 137.06, 134.53, 133.09, 126.32, 124.29, 123.56, 116.56, 114.27, 113.96, 109.66, 57.26, 43.84, 35.18, 35.02, 33.70, 31.09, 26.22, 25.55, 13.52. HRMS (ESI) [M + H]+, calcd: 507.2867, Found: 507.2872. HPLC analysis: MeOH/H2O (80:20), 22.43 min, 96.3% purity.

(R)-N-(3-(Adamantan-1-yl)-5-(4-methyl-1H-imidazol-1-yl)phenyl)-2-(methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indene-5-carboxamide (7n)

Compound 7n was prepared by following a similar procedure as that of 7i. (90 mg, 25%). [α]D25 +5 (c 0.1, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.45 (s, 1H), 8.32 (s, 2H), 7.88 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.56 (s, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.10 (s 1H), 7.07 (s, 1H), 4.83–4.76 (m, 1H), 3.34–3.28 (m, 2H), 3.14–3.08 (m, 2H), 2.79 (s, 3H), 2.30 (s, 3H), 2.11 (s, 3H), 1.92 (s, 6H), 1.82–1.73 (m, 6H). 13C NMR (125 MHz, CDCl3) δ 166.17, 154.58, 148.14, 145.53, 143.36, 139.70, 137.73, 134.59, 133.81, 126.36, 124.87, 123.76, 116.05, 115.24, 113.85, 110.82, 58.37, 43.22, 36.76, 36.72, 36.27, 36.05, 31.96, 28.94. HRMS (ESI) [M + H]+, calcd: 559.3180, found: 559.3183. HPLC analysis: MeOH/H2O (80:20), 20.76 min, 100.0% purity.

tert-Butyl (R)-(5-Bromo-2,3-dihydro-1H-inden-2-yl)carbamate (10)

To a mixture of (R)-5-bromo-2,3-dihydro-1H-inden-2-amine 8 (40 mg, 0.19 mmol) and Et3N (39 μL, 0.28 mmol) in 1 mL of dichloromethane (DCM) was added (Boc)2O (131 μL, 0.57 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was then poured into water and extracted three times with dichloromethane. The combined organic phase was dried over Na2SO4 and purified by column chromatography to give 10 (53 mg, 90%). 1H NMR (400 MHz, DMSO-d6), 7.38 (s, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.17 (s, 1H), 7.14 (d, J = 8.0 Hz, 1H), 4.22–4.17 (m, 1H), 3.14–3.02 (m, 2H), 2.79–2.68 (m, 2H), 1.38 (s, 9H). MS (ESI) m/z 335.3 [M + Na]+.

tert-Butyl (R)-(5-Bromo-2,3-dihydro-1H-inden-2-yl)(methyl)carbamate (12)

tert-Butyl (R)-(5-bromo-2,3-dihydro-1H-inden-2-yl)carbamate 10 (600 mg, 1.92 mmol) was dissolved in 2 mL of DMF, and sodium hydride (89 mg, 2.22 mmol, in 60% mineral oil) was added. The reaction was stirred for 40 min, and then CH3I (0.13 mL, 2.12 mmol) was added dropwise in an ice bath. The reaction was stirred overnight at room temperature. The mixture was quenched by water and extracted with ethyl acetate three times. The combined organic layer was dried over magnesium sulfate, filtered, evaporated, and purified by silica gel column chromatography to give 12 (501 mg, 80%). 1H NMR (500 MHz, DMSO-d6), δ 7.41 (s, 1H), 7.32 (d, J = 1.6 Hz, 1H), 7.31 (d, J = 1.5 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 4.85 (s, 1H), 3.09–2.99 (m, 2H), 2.96–2.85 (m, 2H), 2.64 (s, 3H), 1.39 (s, 9H). MS (ESI) m/z 348.3 [M + Na]+.

Methyl (R)-2-((tert-Butoxycarbonyl)amino)-2,3-dihydro-1H-indene-5-carboxylate (15)

A mixture of tert-butyl (R)-(5-bromo-2,3-dihydro-1H-inden-2-yl)carbamate 10 (100 mg, 0.32 mmol), Pd(OAc)2 (3.6 mg, 0.016 mmol), MeOH, DMF, and triethylamine (89 μL, 0.64 mmol) was stirred overnight at 100 °C under carbon monoxide. The solids were filtered off, and the filtrate was concentrated under vacuo. The residue was purified by column chromatography to give 15 (55 mg, 59%). 1H NMR (500 MHz, DMSO-d6) δ 7.77 (s, 1H), 7.75 (s, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 6.5 Hz, 1H), 4.26–4.22 (m, 1H), 3.82 (s, 3H), 3.19–3.14 (m, 2H), 2.84–2.78 (m, 2H), 1.39 (s, 9H). MS (ESI) m/z 314.6 [M + Na]+.

Methyl (R)-2-((tert-Butoxycarbonyl)(methyl)amino)-2,3-dihydro-1H-indene-5-carboxylate (17)

A mixture of tert-butyl (R)-(5-bromo-2,3-dihydro-1H-inden-2-yl)(methyl)carbamate 12 (2.39 g, 7.35 mmol), PdCl2dppf (548 mg, 0.7 mmol), MeOH (6 mL), DMF (35 mL), and triethylamine (3.1 mL, 22 mmol) was stirred overnight at 100 °C under carbon monoxide. The solids were filtered off, and the filtrate was concentrated under vacuo. The residue was purified by column chromatography to give 17 (1.9 g, 85%). 1H NMR (500 MHz, DMSO-d6), δ 7.80 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 4.90 (s, 1H), 3.83 (s, 3H), 3.17–3.10 (m, 2H), 3.02–2.96 (m, 2H), 2.65 (s, 3H), 1.39 (s, 9H).

Methyl (R)-2-Amino-2,3-dihydro-1H-indene-5-carboxylate Hydrochloride (20)

A mixture of methyl (R)-2-((tert-butoxycarbonyl)amino)-2,3-dihydro-1H-indene-5-carboxylate 15 (126 mg, 0.43 mmol) and 4 M HCl (1 mL) in dioxane (1 mL) was stirred at 60 °C for 2 h. The reaction mixture was concentrated to give the amine, which was used directly for the next step without further purification. MS (ESI) m/z 192.2 [M + H]+.

Methyl (R)-2-(Methylamino)-2,3-dihydro-1H-indene-5-carboxylate (22)

A mixture of methyl (R)-2-((tert-butoxycarbonyl)(methyl)amino)-2,3-dihydro-1H-indene-5-carboxylate 17 (1.9 g, 6.22 mmol) and 4 M HCl (13 mL) in dioxane (13 mL) was stirred at 50 °C for 4 h. The solvent was evaporated in vacuum. The residue was diluted with H2O and extracted with dichloromethane three times. The combined organic phase was washed with saturated NaHCO3. The aqueous layer was extracted twice with dichloromethane. The combined organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated in vacuo to give the free amine 22 (1.0 g, 78%), which was used directly in the next step without further purification.

Methyl (R)-2-(Pyrimidin-5-ylamino)-2,3-dihydro-1H-indene-5-carboxylate (25)

A sealed tube containing methyl (R)-2-amino-2,3-dihydro-1H-indene-5-carboxylate hydrochloride 20 (630 mg, 2.77 mmol), 5-bromopyrimidine (656 mg, 4.1 mmol), Pd(dba)2 (10 mmol %), Ruphos (20 mmol %), and Cs2CO3 (2.7 g, 8.31 mmol) in toluene was flushed with argon and then heated overnight at 80 °C. The reaction mixture was cooled to room temperature, filtered, concentrated in vacuo, and purified by flash column to obtain 25 (450 mg, 60%). 1H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 8.17 (s, 2H), 7.83 (s, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 6.44 (d, J = 6.4 Hz, 1H), 4.36–4.30 (m, 1H), 3.83 (s, 3H), 3.42–3.37 (m, 2H), 2.88–2.83 (m, 2H). MS (ESI) m/z 270.3 [M + H]+.

Methyl (R)-2-(Methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indene-5-carboxylate (27)

A sealed tube containing methyl (R)-2-(methylamino)-2,3-dihydro-1H-indene-5-carboxylate 22 (500 mg, 2.4 mmol), 5-bromopyrimidine (578 mg, 3.6 mmol), Pd(dba)2 (10 mmol %), Ruphos (20 mmol %), and Cs2CO3 (1.6 g, 4.8 mmol) in toluene was flushed with argon and then heated overnight at 80 °C. The reaction mixture was cooled to room temperature, filtered, concentrated in vacuo, and purified by flash column to give 27 (382 mg, 56%). 1H NMR (400 MHz, DMSO-d6), δ 8.50 (s, 1H), 8.43 (s, 2H), 7.84 (s, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 7.9 Hz, 1H), 5.00–4.93 (m, 1H), 3.83 (s, 3H), 3.29–3.22 (m, 2H), 3.10–3.03 (m, 2H), 2.74 (s, 3H). MS (ESI) m/z 284.3 [M + H]+.

(R)-2-(Methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indene-5-carboxylic Acid (30)

To a solution of methyl (R)-2-(methyl(pyrimidin-5-yl)amino)-2,3-dihydro-1H-indole-5-carboxylate 27 (1 g, 3.5 mmol) in dioxane was added 1 M sodium hydroxide solution. The reaction mixture was stirred at 50 °C overnight. The solvent was evaporated under vacuo, and the resulting residue was dissolved in water. The solution was acidified to pH 4, and a white precipitate was formed, which was collected, washed with water, and dried to give 30 (772.9 mg, 82%). 1H NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H), 8.50 (s, 1H), 8.43 (s, 2H), 7.82 (s, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 5.09–4.89 (m, 1H), 3.25 (dd, J = 17.6, 8.0 Hz, 2H), 3.12–2.98 (m, 2H), 2.74 (s, 3H).

Compounds 33–37 were prepared according to the published methods.[36]

5-Bromoisoindoline-1,3-dione (33)

4-bromophthalic anhydride 32 (1 g, 4.4 mmol) and formamide were combined in a sealed tube. The reaction mixture was stirred overnight at 200 °C. After cooling to room temperature, 10 mL of water was added to the reaction mixture. The precipitate was filtered, washed with methanol, and dried in a vacuum oven to give 33 (788 mg, 79%). 1H NMR (500 MHz, DMSO-d6) δ 11.46 (s, 1H), 8.04–8.00 (m, 1H), 8.01 (d, J = 3.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H). MS (ESI) m/z 247.2 [M + Na]+.

5-Bromoisoindoline (34)

To a solution of 5-bromoisoindoline-1,3-dione 33 (788 mg, 3.5 mmol) in THF (20 mL) was added the brane–THF complex (1 M, 14 mL). The reaction was heated to reflux overnight. After cooling to room temperature, the solvent was concentrated in vacuo. Water was added to the residue. The aqueous phase was extracted with DCM three times. The combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo to give 34 (282 mg, 41%), which was used for the next step without further purification. MS (ESI) m/z 198.4 [M + H]+.

tert-Butyl 5-Bromoisoindoline-2-carboxylate (35)

To 5-bromoisoindoline 34 (282 mg, 1.42 mmol) in 6 mL of THF were added (Boc)2O (1 mL, 4.3 mmol) and NaHCO3 (360.8 mg, 4.29 mmol). The reaction mixture was stirred at room temperature overnight. The reaction was quenched by water and extracted three times with CH2Cl2. The combined organic phase was washed with brine, dried over Na2SO4, concentrated in vacuo, and purified by silica gel column chromatography to give 35 (238 mg, 56%). 1H NMR (400 MHz, DMSO-d6) δ 7.55 (d, J = 4.4 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.28 (dd, J = 8.0, 4.0 Hz, 1H), 4.55 (dd, J = 14.8, 9.2 Hz, 4H), 1.45 (s, 9H). MS (ESI) m/z 320.3 [M + Na]+.

2-(tert-Butyl) 5-Methyl Isoindoline-2,5-dicarboxylate (36)

A mixture of tert-butyl-5-bromoisoindoline-2-carboxylate 35 (238 mg, 0.8 mmol), Pd(dppf)Cl2–CH2Cl2 (66 mg, 0.08 mmol), MeOH (1 mL), DMF (4 mL), and triethylamine (0.3 mL, 2.4 mmol) was stirred overnight at 100 °C under carbon monoxide. The solids were filtered off, and the filtrate was concentrated under vacuo. The residue was purified by column chromatography to give 36 (159 mg, 72%). 1H NMR (400 MHz, DMSO-d6) δ 7.90 (dd, J = 12.4, 8.8 Hz, 2H), 7.47 (dd, J = 7.6, 4.8 Hz, 1H), 4.63 (d, J = 2.8 Hz, 4H), 3.85 (s, 3H), 1.46 (s, 9H).

Methyl Isoindoline-5-carboxylate Hydrochloride (37)

2-(tert-Butyl) 5-methylisoindoline-2,5-dicarboxylate 36 (154 mg, 0.2 mmol) was stirred in a mixture of 4 M HCl (1.5 mL) and dioxane (1.5 mL) at 50 °C for 4 h. The reaction mixture was concentrated in vacuo to give 37, which was used directly for the next step without further purification. MS (ESI) m/z 178.1 [M + H]+.

Methyl 2-(Pyrimidin-5-yl)isoindoline-5-carboxylate (38)

5-Bromopyrimidine (93.6 mg, 0.59 mmol), Pd(dba)2 (10 mmol %), Ruphos (20 mmol %), Cs2CO3 (703.8 mg, 2.16 mmol), methyl isoindoline-5-carboxylate hydrochloride 37 (115 mg, 0.54 mmol), and toluene (3 mL) were combined in a sealed tube. The vessel was evacuated and replaced with argon. The reaction was stirred overnight at 80 °C. After cooling to room temperature, the reaction mixture was filtered, and the filtrate was concentration in vacuo and purified by flash column to afford 38 (40 mg, 29%). 1H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.25 (s, 2H), 7.99 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 4.76 (d, J = 3.2 Hz, 4H), 3.87 (s, 3H). MS (ESI) m/z 256.2 [M + H]+.

Methyl 2-(Pyrimidin-5-ylmethyl)isoindoline-5-carboxylate (39)

To methyl isoindoline-5-carboxylate hydrochloride 37 (90 mg, 0.42 mmol) in DMF were added 5-chloromethylpyrimidine (65 mg, 0.41 mmol) and potassium carbonate (145 mg, 1.05 mmol). The reaction was stirred overnight at 50 °C. Then, water (10 mL) was added to the reaction mixture and extracted with ethyl acetate three times. The combined organic phase was dried over Na2SO4, concentrated in vacuo, and purified by column chromatography to give 39 (44 mg, 41%). 1H NMR (500 MHz, CDCl3) δ 9.17 (s, 1H), 8.79 (s, 2H), 7.95–7.90 (m, 1H), 7.87 (s, 1H), 7.25 (s, 1H), 4.00 (s, 4H), 3.95 (s, 2H), 3.90 (s, 3H). MS (ESI) m/z 270.3 [M + H]+.

In Vitro Kinase Assay

Inhibition activity of compounds against TrkA/TrkB/TrkC was determined using the FRET-based Z′-Lyte assay system according to the manufacturer’s instructions (Invitrogen). Tyr1 peptide was used as the substrate. The reactions were carried out in 384-well plates in a 10 μL reaction volume with appropriate amount of kinases in 50 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) (pH 7.5), 10 mM MgCl2, 1 mM ethylene glycol–bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 0.01% Brij-35. TrkA/TrkB/TrkC kinase reactions were incubated 1.5 h at room temperature in the presence of 2 μM of substrate with 400, 25, and 50 μM ATP, respectively, and in the presence of various concentrations of the compounds; then, 5 μL of the development reagent was added for further 1 h room-temperature incubation followed by the addition of 5 μL of the stop solution. The fluorescence-to-signal ratio of 400 nm (excitation)/445 nm and 520 nm (emission) was examined with an EnVision Multilabel Reader (PerkinElmer, Inc.). Data analysis and curve fitting were performed using GraphPad Prism5 (GraphPad Software, Inc).

The effects of compounds on the kinases DDR1 and DDR2 were assessed using a LanthaScreen Eu kinase activity assay technology (Invitrogen). Kinase reactions are performed in a 10 μL volume in low-volume 384-well plates. The kinases in the reaction buffer consist of 50 mM HEPES pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA; the concentration of the fluorescein–poly-GAT substrate (Invitrogen) in the assay is 100 nM. Kinase reactions were initiated with the addition of 100 nM ATP in the presence of serials of dilutions of compounds. The reactions were allowed to proceed for 1 h at room temperature before a 10 μL preparation of EDTA (20 mM) and Eu-labeled antibody (4 nM) in time-resolved (TR)-FRET dilution buffer are added. The final concentration of antibody in the assay well is 2 nM, and the final concentration of EDTA is 10 mM. The plate is allowed to incubate at room temperature for one more hour before the TR-FRET emission ratios of 665/340 nm were acquired on a PerkinElmer EnVision multilabel reader (Perkin-Elmer, Inc.). Data analysis and curve fitting were performed using GraphPad Prism 7 software.

KINOMEscan

Kinase-tagged T7 phage strains were prepared in an Escherichia coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with the T7 phage and incubated with shaking at 32 °C until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for quantitative polymerase chain reaction (qPCR) detection. Streptavidin-coated magnetic beads were treated with biotinylated small-molecule ligands for 30 min at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer [SeaBlock (Pierce), 1% bovine serum albumin (BSA), 0.05% Tween 20, 1 mM dithiothreitol (DTT)] to remove the unbound ligand and to reduce nonspecific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and compound 7i in 1× binding buffer [20% SeaBlock, 0.17× phosphate-buffered saline (PBS), 0.05% Tween 20, 6 mM DTT]. All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 mL. The assay plates were incubated at room temperature with shaking for 1 h, and the affinity beads were washed with wash buffer (1× PBS, 0.05% Tween 20). The beads were then resuspended in an elution buffer (1× PBS, 0.05% Tween 20, 0.5 μM nonbiotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by qPCR. For primary screening, compound 6j was screened at the concentration of 1 μM, and the results are reported as “% Ctrl”.

For Kd determination, an 11-point 3-fold serial dilution of compound 7f was prepared in 100% DMSO at 100× final test concentration and subsequently diluted to 1× in the assay (final DMSO concentration = 1%). Binding constants (Kd values) were calculated with a standard dose–response curve using the Hill equation.

Western Blot Analysis

Pan02, BMF-A3, and CT1A-C11 cells were cultured in DMEM (Corning) containing 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified incubator with 5% CO2 and 95% air. For the collagen and 7f treatment, cells were cultured in 10 mL of complete media with 50 μg/mL collagen and indicated concentration of 7f for 8 or 18 h. Cells were lysed, supernatants were recovered by centrifugation at 13 000 rpm, protein concentration was measured, and equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to poly(vinylidene difluoride) membranes (Bio-Rad, Hercules, CA) followed by blockade for 1 h in 5% bovine serum albumin in TBS-T. Membranes were incubated overnight at 4 °C with primary antibody phospho-DDR1 (Tyr792, Cell Signaling #11994), DDR1 (Santa Cruz SC-532), phospho-PYK2 (Tyr402, Cell Signaling #3291), PYK2 (Cell Signaling #3292), PEAK1 (Millipore 09-274), phospho-PEAK1 (Tyr665, Millipore #ABT52), E-cadherin (24E10, Cell Signaling #3195), N-cadherin (13A9, Cell Signaling #3195), and TUBULIN α (Biorad, MCA77D800). Membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Pierce Biotechnologies, Rockford, IL) for 1 h. Specific bands were detected using the enhanced chemiluminescence reagent (ECL, PerkinElmer Life Sciences, Boston, MA) on an autoradiographic film.

Epithelial–Mesenchymal Transition Assay

For immunofluorescent staining, cells were treated with 50 μg/mL collagen and indicated concentration of 7f for 24 h. Cells were fixed with methanol and stained with E-cadherin (24E10, Cell Signaling #3195), N-cadherin (13A9, Cell Signaling #3195), and DAPI. Fluorescent images were captured with a Photometric CoolSNAP HQ camera using NIS Elements AR 2.3 Software (Nikon).

For 3D culture, cells were embedded in ECM consisting of 5 mg/mL matrigel (BD Biosciences) and 2.1 mg/mL collagen I (BD Biosciences). Cultures were overlaid with DMEM + 10% FBS containing 2% matrigel. For analysis of spheroid morphology, 48 h after plating onto ECM, cells were fixed with methanol and stained with phalloidin and DAPI. Fluorescent images were captured with a Photometric CoolSNAP HQ camera using NIS Elements AR 2.3 Software (Nikon).

Colony Formation Assay

Cells were cultured in six-well tissue culture plates at low density (1000 cells per well) in 2 mL of media with 10% FBS ± 7f at the indicated doses for 10 days. A DMSO control was added to the respective wells to demonstrate the vehicle-independent effect. Cells were then stained with crystal violet.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide Assay

Cells were plated at 2000 cells/well in tissue-culture-treated 96-well plates. The following day, different concentration of 7f was added to each plate in a dilution series across the plate. On day 5, 20 μL of thiazolyl blue tetrazolium blue was added, followed by a 3 h incubation at 37 °C, and then the medium was removed, and 100 μL of DMSO was added to each well. The absorbance was read at 540 nm on a plate reader.

Wound-Healing Assay

Cells were cultured in six-well tissue culture plates at high density (∼90% confluence) in 2 mL of media with 5% FBS. Uniform scratches were made down the center of each well with a p20 pipette tip, cells were gently washed with PBS to remove the loose cell debris, and drug was added in media containing 5% FBS. Each condition was in triplicates. Images from the center of each well were taken at 0 and 24 h. The wound width (μm) was measured using NIS Elements AR 2.30 software. The initial wound width was used to verify consistency in scratches.

Determination of Pharmacokinetic Parameters in Rats

All animal studies were performed according to the protocols and guidelines of the institutional care and use committee. The 4–6 week old male Sprague-Dawley rats were purchased from the Shanghai Laboratory Animal Research Center (Shanghai, China). All of the procedures related to animal handling, care, and treatment in this article were performed in compliance with Agreement of the Ethics Committee on Laboratory Animal Care and the Guidelines for the Care and Use of Laboratory Animals in Shanghai, China. Animals were maintained on standard animal chow and water ad libitum, in a climate-controlled room (23 ± 1 °C, 30–70% relative humidity, a minimum of 10 exchanges of room air/h and a 12 h light/dark cycle) for one week prior to experiments. The compound was dissolved in the solution containing 2% DMSO, 4% ethanol, 4% castor oil, and 90% ddH2O. Pharmacokinetic properties of SD rats (male) were determined following intravenous (iv) and oral administration. Animals were randomly distributed into two experimental groups (n = 3). The oral groups were given 25 mg/kg by gastric gavage. The other group was dosed by injection into the tail vein (5 mg/kg). After a single administration, whole blood samples (100–200 μL) were obtained from the orbital venous plexus at the following time points after dosing: 5, 10, and 30 min and 1, 2, 3, 4, 6, 8, 11, and 24 h (po); 2, 10, and 30 min and 1, 2, 3, 4, 6, 8, 11, and 24 h (iv). Whole blood samples were collected in heparinized tubes. The plasma fraction was immediately separated by centrifugation (8000 rpm, 6 min, 4 °C) and stored at −20 °C until liquid chromatography–mass spectrometry analysis. The rats were humanely euthanized by carbon dioxide 24 h after experiment without pain. The pharmacokinetics parameters were calculated by analyzing the compound concentration in plasma samples using the pharmacokinetic software DAS.2.0.

In Vivo Anticancer Activity Assay

All animals were housed in a pathogen-free facility with access to food and water ad libitum. C57BL/6 mice were purchased from the UT Southwestern Mouse Breeding Core. Pan02 and BMF-A3 cells (5 × 105) were injected orthotopically in 6–8 week-old C57BL/6 mice. Fourteen days after tumor cell injection, mice were randomized to receive treatment. Experiments were approved and performed in accordance with the Institutional Animal Care and Use Committee at UT Southwestern. Experiments were stopped after the designated time post tumor cell implantation. Tumors were harvested, and tumor weight was measured. Data sets were analyzed by ANOVA.

Molecular Modeling

All of the procedures were performed in Maestro 11.2 (version 11.2, Schrödinger, LLC, New York, NY, 2017). The DDR1 (PDB code: 5FDP and 6HP9) and TrkC (PDB code: 3V5Q) proteins were processed using the “Protein Preparation Wizard” workflow in Maestro 9.4 (version 11.2, Schrödinger, LLC, New York, NY, 2017) to add bond orders and hydrogens. All hetatm residues and crystal water molecules beyond 5 Å from the het group were removed. Compounds were built by the LigPrep module using the OPLS-2005 force field. The glide module was used as the docking program. The grid-enclosing box was placed on the centroid of the 0LI, which was extracted from the crystal structures of DDR1 and TrkC separately. The standard precision approach of Glide was adopted to dock compounds 6 and 7a–n to DDR1 and compound 7c to TrkC with the default parameters.

Crystallization and Structure Determination

The kinase domain of human DDR1 (Uniprot Q08345, residues 601–913) was expressed as an N-terminal 6× His fusion in Sf9 cells and purified by nickel affinity chromatography followed by tag cleavage with tobacco etch virus protease and then size exclusion chromatography on an S200 column (GE Healthcare). Protein at 12 mg/mL in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine, 2% DMSO was incubated with 1 mM compound 7c for 3 h on ice and then filtered to 0.22 μm. Then, 150 nL of sitting drops was set up with diffracting crystals being obtained from a 1:2 ratio of protein to mother liquor (10% ethylene glycol, 0.2 M sodium sulfate, 25% poly(ethylene glycol) 1500, 0.1 M bis–tris–propane pH 7.1). Crystals were cryoprotected in the mother liquor supplemented with 25% ethylene glycol and flash-frozen in liquid nitrogen. Diffraction was carried out at Diamond Light Source beamline I04 at 100 K. Data were indexed and integrated using XDS[37] and scaled using AIMLESS.[38] Initial phases were identified using molecular replacement in PHASER.[39] The structure was refined using iterative rounds of manual building using COOT[40] and refinement using PHENIX.REFINE.[41] The refined structure was validated with MolProbity,[42] and the atomic coordinate files were deposited in the Protein Data Bank with Autodep.[43]