Phenylethynyl-substituted heterocycles inhibit cyclin D1 and induce the expression of cyclin-dependent kinase inhibitor p21^Wif1/Cip1 in colorectal cancer cells

Abstract

Fluorinated phenylethynyl-substituted heterocycles that possessed either an N-methylamino or N,N-dimethylamino group attached to heterocycles including pyridines, indoles, 1H-indazoles, quinolines, and isoquinolines inhibited the proliferation of LS174T colon cancer cells in which the inhibition of cyclin D1 and induction of the cyclin-dependent kinase inhibitor-1 (i.e., p21^Wif1/Cip1) served as readouts for antineoplastic activity at a cellular level. At a molecular level, these agents, particularly 4-((2,6-difluorophenyl)ethynyl)-N-methylisoquinolin-1-amine and 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylisoquinolin-1-amine, bound and inhibited the catalytic subunit of methionine S-adenosyltransferase-2 (MAT2A).

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Introduction

In the course of studies focused on the mechanisms of colorectal cancer^1–3 development, we employed chemical biology^4–7 to probe cell signaling events^8,9 that regulate gene expression. Selective targeting of key steps in this process offered the potential for developing therapeutic agents to treat these cancers.⁹ We reported, for example, that 2′,6′-dihalostyrylanilines 1 (Fig. 1) inhibited the expression of Wnt target genes, such as c-myc,¹⁰ and repressed colon cancer LS174T cell growth in vitro and in vivo.^11–13 We established that this agent exclusively targeted the catalytic subunit of the oligomeric enzyme, methionine S-adenosyltransferase-2 (MAT2), which furnishes S-adenosylmethionine (SAM) to regulatory methyltransferases. We further established that heterocyclic variants¹³ of these 2′,6′-dihalostyrylanilines 1 possessed minimal gross toxicity, no human ether-à-go-go-related protein (hERG)^14,15 activation, and reasonable bioavailability and pharmacokinetics. As desired, these stilbene-based agents did not affect methionine S-adenosyltransferase-1 (MAT1)^16–19 that served as the principal source of SAM necessary for other cellular needs. Unlike a recent report²⁰ on other MAT2A inhibitors, we also demonstrated in vivo potency in a xenograft model using colorectal cancer cells.¹²

Fig. 1 Fluorinated stilbene 1 and diphenylacetylene 2. Synthesis of heterocyclic-substituted diphenylacetylenes 4 and phenylethynyl-substituted heterocycles 5. Legend: a, aryl iodide, iPr₂EtN, Pd(PPh₃)₄, CuI, H₂O, 75 °C; b, heteroaryl iodide, iPr₂EtN, Pd(PPh₃)₄, CuI, H₂O, 75 °C; c, chloro-substituted heteroaryl iodide, iPr₂EtN, Pd(PPh₃)₄, CuI, H₂O, 75 °C followed by R₂NH, THF, 130 °C, 2–3 h, pressure tube.

Concerns regarding thermal or photochemical E/Z-isomerizations^21,22 in stilbenes 1 that could potentially complicate future pharmacodynamic studies led to synthesis and evaluation of the analogous diarylacetylenes²³2 (Fig. 1). In accord with the SAR study of the stilbenes 1, we found that the diarylacetylenes 2 bearing an N,N-dimethylamino group on one phenyl ring and either one or preferably two ortho-oriented fluoro or chloro groups on the other phenyl ring retained the in vitro potencies in a colorectal cancer LS174T cell proliferation assay. The diarylacetylenes 2 also possessed the desired property of having minimal effects on the cardiac potassium hERG channel.^14,15 Molecular docking studies suggested that diphenylacetylenes, like their stilbene counterparts,¹² targeted the catalytic subunit (MAT2A) of MAT2.²³

Results and discussion

We performed additional structure–activity studies of the diarylacetylene pharmacophore with a view to obtaining improved potency, solubility and bioavailability relative to the sparingly water-soluble 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylaniline (2). Initial efforts focused on replacing the N,N-dimethylamino group in 2 with a heterocyclic ring as in the heterocyclic-substituted diphenylacetylenes 4 (Fig. 1). The Sonogashira coupling^23–25 of 2,6-difluorophenylacetylene (3) with 4-iodophenyl-substituted heterocycles provided access to the oxopiperidinyl, morpholino, N-methylpiperazinyl, N-pyrrolyl, and 2,5-dimethyl-N-pyrrolyl analogs 4a–4e (Table 1). The N-pyrrolyl analog 4d showed activity in a cell proliferation assay comparable to 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylaniline (2) at 1 μM concentration but considerably diminished activity at 100 nM.

Table 1 Inhibition of proliferation (%) of LS174T cells by heterocyclic-substituted diphenylacetylenes 4 at various concentrations

Compound	C-4′ substituent	10 μM	1 μM	100 nM
a Ref. 23.
2	N,N-Dimethylamino		99.5 ± 0.1	85.2 ± 0.5
4a	1-Piperidinyl-2-one	68.1 ± 6.9	6.4 ± 11.0
4b	N-Morpholino	32.1 ± 9.8	13.0 ± 2.5
4c	N′-Methyl-N-piperazinyl	99.6 ± 0.2	19.9 ± 18.2
4d	N-Pyrrolyl	96.7 ± 1.9	77.5 ± 9.2	11.0 ± 18.1
4e	2,5-Dimethyl-N-pyrrolyl	78.8 ± 4.8	20.9 ± 10.0

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We also examined other halogenation patterns (e.g., 2-fluoro, 2-chloro, 2-trimethylfluoro, 2-chloro-6-fluoro and 2,6-dichloro) in other heterocyclic-substituted diphenylacetylenes 4, but these studies failed to produce a compound with an in vitro potency that exceeded that of 2 (data not shown). In summary, we were unable to identify a heterocyclic variant of 4 with potency equal to that of the parent compound, 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylaniline²³ (2).

Because heterocyclic variants in the stilbene series¹³ in which the N,N-dimethylaniline ring in 1 was replaced by either an N,N-dimethylaminopyridine or an N,N-dimethylaminopyrimidine ring exhibited improved solubility and minimal hERG activation, we next focused on replacing the N,N-dimethylaniline ring in the diarylacetylene series with a heterocyclic ring as in the phenylethynyl-substituted heterocycles 5 (Fig. 1). The Sonogashira coupling^24,25 of 2,6-difluorophenylacetylene with iodo-substituted heterocycles provided access to most of these analogs (Table 2). In some cases, such as the synthesis of 5mm–5oo, it was preferable to couple 2,6-difluorophenylacetylene to 1-chloro-4-iodoisoquinoline and subsequently utilize a nucleophilic aromatic substitution reaction to replace the chloro group with a primary or a secondary amine. Yet another approach involved the coupling of 2,6-difluorophenylacetylene to 1-amino-4-iodoisoquinoline to afford 4-((2,6-difluorophenyl)ethynyl)isoquinolin-1-amine (5ll) and the subsequent N,N-dimethylation of the amino group using 1,2-bis[(dimethylamino)methylene]hydrazine via an intermediate 1,2,4-triazole,²⁶ but this latter process proceeded in a lower overall yield than the yield in the nucleophilic aromatic substitution reaction. As summarized in Table 2, using these approaches, we synthesized 2,6-difluorophenylethynyl-substituted heterocycles including pyridines 5a–5c, pyrazine 5d, indoles 5e and 5f, 1H-indazoles 5g–5k, 1H-pyrrolo[2,3-b]pyridine 5l, quinolines 5m–5ee, isoquinolines 5ff–5oo, 1,6-naphthyridine 5pp, and quinazoline 5qq. The corresponding monofluorinated and monochlorinated analogs possessed comparable or diminished potency in cell proliferation assays at low concentrations relative to their difluorinated counterparts (data not shown).

Table 2 Inhibition of proliferation (%) of colon cancer LS174T cells by 2,6-difluorophenylethynyl-substituted heterocycles 5 at various concentrations

Compound	Heterocycle or aryl ring	Position^a	10 μM	1 μM	100 nM	30 nM
a Position of attachment of the 2,6-difluorophenylethynyl group. b Ref. 23. c Tested as the hydrochloride salt.
2	N,N-Dimethylaniline	C-4		99.5 ± 0.1	85.2 ± 0.5	57 ± 1.2
5a	N,N-Dimethylpyridin-3-amine	C-6	85.2 ± 3.8	68.9 ± 1.3	15.1 ± 7.4
5b	N,N-Dimethylpyridin-2-amine	C-5	83.6 ± 7.8	77.9 ± 7.0	0 ± 23.7
5c	2-(1H-Pyrrol-1-yl)pyridine	C-5	95.3 ± 0.8	80.5 ± 6.6	0 ± 27.7
5d	N,N-Dimethylpyrazin-2-amine	C-5	91.6 ± 3.0	22.9 ± 9.8
5e	1H-Indole	C-5	91.5 ± 3.5	52.9 ± 5.9
5f	N-Methylindole	C-5	95.2 ± 1.0	77.7 ± 13.6	3.0 ± 5.6
5g	1H-Indazole	C-4	96.2 ± 2.0	19.8 ± 13.6
5h	1H-Indazole	C-5	82.9 ± 9.8	17.6 ± 7.9
5i	1H-Indazole	C-6	97.0 ± 1.1	81.5 ± 2.9
5j	N-Methylindazole	C-5	94.6 ± 3.5	58.4 ± 9.6	0 ± 24.5
5k	N-Methylindazole	C-6	98.1 ± 0.4	85.9 ± 5.4	0 ± 22.7
5l	1H-Pyrrolo[2,3-b]pyridine	C-5	77.9 ± 8.9	17.3 ± 12.0
5m	Quinoline	C-2	93.8 ± 1.7	67.3 ± 13.6	6.1 ± 27.0
5n	Quinoline	C-3	90.4 ± 1.0	41.7 ± 6.5	0 ± 5.1
5o	Quinoline	C-4	59.0 ± 7.5	15.4 ± 9.1
5p	2-Chloroquinoline	C-3	85.1 ± 6.2	28.4 ± 10.5
5q	4-Chloroquinoline	C-3	80.4 ± 13.7	51.5 ± 20.4	0 ± 26.3
5r	Quinolin-6-amine	C-3	9.2 ± 15.7
5s	7-Fluoroquinoline	C-3	95.1 ± 1.6	69.4 ± 4.5	14.8 ± 5.5
5t	8-Fluoroquinoline	C-3	89.1 ± 8.2	63.6 ± 8.4	8.8 ± 5.1
5u	7-Chloroquinoline	C-4	74.3 ± 0.2	36.0 ± 0.4
5v	Quinoline	C-5	94.7 ± 2.9	43.1 ± 11.2
5w	Quinoline	C-6	80.1 ± 1.0	11 ± 30.4
5x	Quinoline	C-7	99.4 ± 0.3	90.7 ± 0.6	2.9 ± 14.0
5y	Quinoline	C-8	92.7 ± 2.9	47.6 ± 3.5
5z	2-Chloroquinoline	C-6	63.3 ± 2.3	21.2 ± 11.4
5aa	4-Chloroquinoline	C-6	89.7 ± 4.3	43.8 ± 10.8
5bb	4-Chloroquinoline	C-7	70.8 ± 4.8	23.1 ± 8.3
5cc	Quinolin-4-amine	C-6	93.9 ± 0.4	66.2 ± 3.1	0 ± 5.4
5dd	Quinolin-4-amine	C-7	97.7 ± 2.2	23.6 ± 2.5
5ee	N,N-Dimethylquinolin-2-amine	C-6	38.7 ± 4.3	2.0 ± 11.9
5ff	Isoquinoline	C-1	96.7 ± 0.2	35.4 ± 15.0
5gg	Isoquinoline	C-4	87.9 ± 1.0	16.5 ± 2.1
5hh	Isoquinoline	C-5	93.6 ± 0.5	64.2 ± 6.1
5ii	Isoquinoline	C-6	82.4 ± 10.2	11.6 ± 21.3
5jj	2-Chloroisoquinoline	C-4	94.0 ± 0.5	48.1 ± 18.1	0.8 ± 36.1
5kk	7-Fluoroisoquinoline	C-1	96.2 ± 0.1	71.1 ± 3.5	0 ± 5.2
5ll	Isoquinolin-2-amine	C-4	93.1 ± 1.6	86.2 ± 1.6	35.5 ± 1.7
5mm	N,N-Dimethylisoquinolin-2-amine	C-4	97.6 ± 1.5	99.5 ± 0.1	81.7 ± 2.1	86.0 ± 2.0
5nn	N-Methylisoquinolin-2-amine	C-4	98.9 ± 1.0	99.8 ± 0.2	99.5 ± 0.5	94.5 ± 1.5
5oo	1-(4-Methylpiperazin-1-yl)isoquinoline	C-4	100 ± 0^c	23.2 ± 9.4^c
5pp	N,N-Dimethyl-1,6-naphthyridin-5-amine	C-8	85.2 ± 1.0	87.9 ± 0.8	59.5 ± 3.5
5qq	Quinazoline	C-7	96.3 ± 0.8	33.8 ± 4.8

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Molecular docking and molecular dynamics (MD) studies furnished a binding model for the phenylethynyl-substituted heterocycles 5 with the dimeric catalytic structure of human methionine S-adenosyltransferase-2 (pdb code: 2P02). A pocket formed at the interface between two α2 subunits of the MAT2A complex contained the SAM-binding site. The MD-simulated MAT2A–5mm or MAT2A–5nn complexes displayed a stable binding mode for these ligands in this pocket (Fig. 2 in which residues within 4 Å of the ligand are highlighted). A serine residue (Ser269) that is also part of this pocket within the 4 Å radius lies in front of the ligands in Fig. 2 and was removed for the sake of clarity. The ligands 5mm and 5nn exhibit hydrophobic van der Waals interactions with portions of the backbone and in particular with the side chains of phenylalanine-272 (F272) and isoleucine-274 (Ile274). Computational modeling studies provided guidance with respect to synthetic design but required experimental confirmation. Prior modeling work and pull-down assays confirmed MAT2A as the sole target of stilbene analogs,¹² but it was important to confirm experimentally that the acetylene analogs, such as phenylethynyl-substituted heterocycles 5, shared the same target as these stilbene analogs. In support of the binding of phenylethynyl-substituted heterocycles 5 to MAT2A, we synthesized and utilized a biologically active D-(+)-biotin derivative 6 (Fig. 3A) of isoquinoline 5mm in a pull-down assay. The synthesis of biotin derivative 6 followed a similar pattern to that reported previously¹² in which the biotin moiety was attached to one of the N-methyl groups in 5mm through a polyethylene glycol (PEG) spacer (Fig. 3A). We selected isoquinoline 5mm for biotinylation based on its potency in inhibiting LS174T cell proliferation (vide infra).

Fig. 2 Binding model of phenylethynyl-substituted heterocycles 5mm and 5nn. Compounds appear as orange stick models. Residues surrounding these ligands are colored in green. Green balls indicate the chloride ion in the MAT2A binding site. The contact surface between ligands and MAT2A appears as a semitransparent white surface. (Panel A) Binding model of isoquinoline 5mm with the receptor. (Panel B) Binding model of isoquinoline 5nn with the receptor.

Fig. 3 Identification of MAT2A as the binding target of phenylethynyl-substituted heterocycle 5mm. (Panel A) D-(+)-Biotin derivative 6 used for affinity purification. (Panel B) Analysis of MAT2A binding by western blotting.

The utilization of biotin-labeled molecules for the isolation and identification of protein targets is well established.^27,28 Incubation of recombinant MAT2A with or without 6 and the use of streptavidin beads facilitated the pull-down of MAT2A bound to the biotinylated 6 attached to the beads. Elution of the MAT2A bound to these beads with 2.5 mM D-(+)-biotin, separation on a 10% SDS-PAGE gel, and analysis of the western blot using an anti-MAT2A Ab, as described previously,¹² identified the MAT2A protein. In summary, the biotin derivative 6 functioned as a biologically competent analog of isoquinoline 5mm and bound MAT2A (Fig. 3B).

At 10 μM concentration, most of the 2,6-difluorophenylethynyl-substituted heterocycles 5 in Table 2 displayed potent effects on LS174T colon cancer cell proliferation assays, and at 1 μM concentration, several pyridines 5b and 5c, one indole 5f, two 1H-indazoles 5i and 5k, one quinoline 5x, three isoquinolines 5ll–5nn, and one naphthyridine 5pp showed greater than 75% inhibition. Two candidates that stood out with respect to potency among the 2,6-difluorophenylethynyl-substituted heterocycles 5 were 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylisoquinolin-1-amine (5mm) and 4-((2,6-difluorophenyl)ethynyl)-N-methylisoquinolin-1-amine (5nn). We tested the isoquinolines 5mm and 5nn against other colon cancer cell lines (i.e., HT-29 and Caco2) (Table 3) and against normal cell lines (i.e., lung epithelial BEAS-2B and fibroblast HEL 299 cells) (Table 4). Isoquinolines 5mm and 5nn showed better selectivity than 5-fluorouracil (5-FU) and cisplatin against colon cancer cells and in particular against colon LS174T cells where MAT2A levels are upregulated (Tables 3 and 4). In addition, several of the difluorinated phenylethynyl-substituted heterocycles 5, particularly 5oo and 5pp, met another of our developmental goals and furnished completely water-soluble hydrochloride salts. In summary, we identified phenylethynyl-substituted heterocycles 5 with potency and/or water solubility surpassing that of the parent compound, 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylaniline²³ (2).

Table 3 Percentage inhibition of the proliferation of colon cancer cells by isoquinolines 5mm and 5nn and by the chemotherapeutic drugs 5-FU and cisplatin

Cell line	5mm	5nn	5 -FU		Cisplatin
	1 μM	1 μM	10 μM	1 μM	10 μM	1 μM
LS174T	99.5 ± 0.1	99.8 ± 0.2	82.3 ± 2.5	6.5 ± 8.2	82.1 ± 1.4	24.2 ± 5.6
HT-29	35.9 ± 3.8	76.8 ± 0.8	82.9 ± 1.8	11.6 ± 17.7	72.7 ± 3.5	4.4 ± 5.9
Caco-2	54.5 ± 2.7	58.3 ± 1.7	60.2 ± 11.5	37.7 ± 25.9	69.8 ± 0.8	49 ± 9.6

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Table 4 Percentage inhibition of the proliferation of normal cells by isoquinolines 5mm and 5nn and by the chemotherapeutic drugs 5-FU and cisplatin

Cell line	5mm		5nn		5 -FU		Cisplatin
	10 μM	1 μM	10 μM	1 μM	10 μM	1 μM	10 μM	1 μM
HEL 299	11.7 ± 3.1	13.9 ± 0.1	9.5 ± 6.3	6.3 ± 8.5	64.6 ± 3.2	12.1 ± 84.1	57.8 ± 0.3	37.5 ± 4.1
BEAS-2B	16.2 ± 7.2	1.5 ± 22.7	19.2 ± 12.4	5.8 ± 17.3	57.5 ± 9	4.5 ± 16	82.4 ± 0.2	39.2 ± 14.9

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Variability in a MAT2A inhibition assay using a molybdate colorimetric assay for phosphate¹² made the measurement of cell-cycle inhibition a preferred analytical tool for assessing the potency of diarylacetylenes. We tested the effect of these heterocyclic-substituted diphenylacetylenes 4 (Table 1) and fluorinated phenylethynyl-substituted heterocycles 5 (Table 2) on the proliferation of LS174T colon cancer cells. As shown in the western blot (Fig. 4), the most active compounds (i.e., 2, 5mm and 5nn) inhibited cyclin D1 at 300 nM concentration and, as expected for a cell-cycle inhibitor, induced cyclin-dependent kinase inhibitor p21^Wif1/Cip1 at the same time. Consistent with results in prior studies,^13,23 the phenylethynyl-substituted heterocycles 5 lacking fluorine substituents or possessing only one fluorine substituent at the ortho-position relative to the acetylenic linkage had lower potency than the 2,6-difluorinated counterparts (data not shown). Unlike the diarylacetylene series in which the N-methylanilino and N,N-dimethylanilino analogs of 2 showed comparable activity in inhibiting LS174T colon cancer cell proliferation (i.e., 2, IC₅₀ = 25.1 ± 1.3 nM), the N-methylation pattern in the phenylethynyl-substituted heterocycles indicated that the N-methylaminoisoquinoline analog 5nn (IC₅₀ = 4.2 ± 0.2 nM) was more potent than the N,N-dimethylaminoisoquinoline analog 5mm (IC₅₀ = 11.8 ± 1.5 nM) (Fig. 4).

Fig. 4 Effects of phenylethynyl-substituted heterocycles 5mm and 5nn on cyclin D1 and p21^Wif1/Cip1 and dose response study. Effects of 2, 5mm, and 5nn on cyclin D1 and p21^Wif1/Cip1 in LS174T cells at 300 nM concentration. Dose responses of 2, 5mm, and 5nn in a colon cancer LS174T cell proliferation assay. IC₅₀ values for 2, 5mm, and 5nn were 25.1 ± 1.3 nM, 11.8 ± 1.5 nM and 4.2 ± 0.2 nM, respectively.

Conclusions

Phenylethynyl-substituted heterocycles 5 inhibited the proliferation of LS174T colon cancer cells in which the inhibition of cyclin D1 and induction of p21^Wif1/Cip1 served as readouts for antineoplastic activity. At a molecular level, these agents act directly on the catalytic subunit of methionine S-adenosyltransferase-2 (MAT2A), consistent with prior findings using halogenated stilbenes.^11–13 The absence of isomerization in 5 relative to stilbenes and the improved potency, and physical properties suggest that phenylethynyl-substituted heterocycles repress colon cancer proliferation through an epigenetic mechanism involving an alteration in histone methylation patterns. Future efforts will focus on the identification of the genes regulated by MAT2A inhibitors and the detailed molecular mechanisms that regulate the selectivity of these MAT2A inhibitors against different cell lines.

Experimental

Chemicals were purchased from Sigma Aldrich or Fisher Scientific or were synthesized according to literature procedures. Solvents were acquired from commercial vendors and used without further purification unless otherwise noted. Nuclear magnetic resonance spectra were determined on Varian (¹H, 400 MHz; ¹³C, 101 MHz) and Bruker (¹H, 700 MHz; ¹³C, 176 MHz) instruments. High-resolution electrospray ionization (ESI) mass spectra were recorded on an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The FT resolution was set at 100 000 (at 400 m/z). Samples were introduced through direct infusion using a syringe pump with a flow rate of 5 μL min⁻¹. MALDI mass spectra were obtained on a Bruker ultrafleXtreme time-of-flight mass spectrometer (Billerica, MA), using a DHB (2,5-dihydroxybenzoic acid) matrix. The purity of compounds was established by combustion analyses by Atlantic Microlabs, Inc., Norcross, GA. The compounds were chromatographed on preparative layer Merck silica gel F254 unless otherwise indicated.

General procedure for the Sonogashira coupling of 2,6-difluorophenylacetylene with aryl iodides or heteroaryl iodides

To a mixture of 2.1 mmol of an aryl iodide or heteroaryl iodide, 3 mmol of diisopropylethylamine, 0.02 mmol of Pd(PPh₃)₄, and 0.02 mmol of CuI in 7 mL of water was added 2 mmol of 2,6-difluorophenylacetylene. The mixture was stirred for 1–2 h at 75 °C. The mixture was cooled, and the product was collected by filtration or extraction using dichloromethane. The products 4 or 5 were purified by recrystallization and/or chromatography as noted below.

1-(4-((2,6-Difluorophenyl)ethynyl)phenyl)piperidin-2-one (4a). Yield 75%; preparative layer chromatography using 1 : 10 methanol–dichloromethane (R_f = 0.52); mp 148–150 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.58 (d, J = 8.5 Hz, 2H), 7.56–7.47 (m, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.26 (t, J = 8 Hz, 2H), 3.64 (t, J = 5.6 Hz, 2H), 2.42 (t, J = 6.3 Hz, 2H), 1.94–1.77 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.01, 162.01 (dd, J = 5.2, 251.5 Hz, 2C), 144.53, 131.78 (2C), 131.39 (t, J = 10 Hz), 126.19 (2C), 118.35, 111.94 (dd, J = 5.3, 18.2 Hz, 2C), 100.77 (t, J = 19.8 Hz), 98.63 (t, J = 3.1 Hz), 75.77, 50.29, 32.70, 22.91, 20.79. HRMS (ESI) calcd for C₁₉H₁₆F₂NO [MH⁺]: 312.1194. Found: 312.1195. Anal. calcd for C₁₉H₁₅F₂NO: C, 73.30; H, 4.86; N, 4.50. Found: C, 73.04; H, 4.85; N, 4.52.

4-(4-((2,6-Difluorophenyl)ethynyl)phenyl)morpholine (4b). Yield 67%; column chromatography using 1 : 5 ethyl acetate–hexane and recrystallized from hexane; mp 144–145 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.53–7.44 (m, 1H), 7.42 (d, J = 8.9 Hz, 2H), 7.22 (t, J = 7.9 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 3.73 (t, J = 4.8 Hz, 4H), 3.2 (t, J = 4.9 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.85 (dd, J = 5.3, 250.6 Hz, 2C), 151.36, 132.57 (2C), 130.51 (t, J = 10.1 Hz), 114.35 (2C), 111.89 (dd, J = 5.4, 250.6 Hz, 2C), 110.38, 101.55 (t, J = 19.9 Hz), 100.09 (t, J = 3 Hz), 74.28, 65.91 (2C), 47.21 (2C). HRMS (ESI) calcd for C₁₈H₁₆F₂NO [MH⁺]: 300.1194. Found: 300.1195. Anal. calcd for C₁₈H₁₅F₂NO: C, 72.23; H, 5.05; N, 4.68. Found: C, 71.98; H, 5.09; N, 4.72.

1-(4-((2,6-Difluorophenyl)ethynyl)phenyl)-4-methylpiperazine (4c). Yield 60%, preparative layer chromatography using 1 : 10 methanol–dichloromethane (R_f = 0.43); mp 126–128 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.53–7.42 (m, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.22 (t, J = 7.9 Hz, 2H), 6.96 (d, J = 8.9 Hz, 2H), 3.24 (t, J = 4.9 Hz, 4H), 2.43 (t, J = 5.1 Hz, 4H), 2.21 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.82 (dd, J = 5.4, 250.6 Hz, 2C), 151.21, 132.56 (2C), 130.43 (t, J = 10.1 Hz), 114.49 (2C), 111.8 (dd, J = 5.3, 18.2 Hz, 2C), 109.85, 101.59 (t, J = 19.9 Hz), 100.22 (t, J = 3 Hz), 74.17, 54.35 (2C), 46.87 (2C), 45.73. HRMS (ESI) calcd for C₁₉H₁₉F₂N₂ [MH⁺]: 313.1511. Found: 313.1489. Anal. calcd for C₁₉H₁₈F₂N₂: C, 73.06; H, 5.81; N, 8.97. Found: C, 72.84; H, 5.75; N, 8.89.

1-(4-((2,6-Difluorophenyl)ethynyl)phenyl)-1H-pyrrole (4d). Yield 79%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.68); mp 122–124 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.76–7.61 (m, 4H), 7.60–7.49 (m, 1H), 7.48 (t, J = 2.2 Hz, 2H), 7.32–7.17 (m, 2H), 6.31 (t, J = 2.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.01 (dd, J = 5, 251.5 Hz, two C), 140.3, 132.98 (2C), 131.36 (t, J = 10.1 Hz), 119.15 (2C), 118.92 (2C), 117.61, 111.93 (dd, J = 5.4, 18.3 Hz, 2C), 111.19 (2C), 100.91 (t, J = 19.7 Hz), 98.45 (t, J = 3 Hz), 76.09. HRMS (ESI) calcd for C₁₈H₁₂F₂N [MH⁺]: 280.0932. Found: 280.0924. Anal. calcd for C₁₈H₁₁F₂N: C, 77.41; H, 3.97; N, 5.02. Found: C, 77.20; H, 4.21; N, 4.89.

1-(4-((2,6-Difluorophenyl)ethynyl)phenyl)-2,5-dimethyl-1H-pyrrole (4e). Yield 84%; column chromatography using 1 : 10 ethyl acetate–hexane; mp 124–126 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.71 (d, J = 8.4 Hz, 2H), 7.62–7.51 (m, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.30–7.25 (m, 2H), 5.83 (s, 2H), 2.00 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.07 (dd, J = 5.2, 251.8 Hz, 2C), 139.2, 132.43 (2C), 131.66 (t, J = 10.2 Hz), 128.49 (2C), 127.55 (2C), 120.42, 111.96 (dd, J = 5.3, 19 Hz, 2C), 106.48 (2C), 100.69 (t, J = 19.7 Hz), 98.05 (t, J = 3 Hz), 76.71, 12.86 (2C). HRMS (ESI) calcd for C₂₀H₁₆F₂N [MH⁺]: 308.1245. Found: 308.1229. Anal. calcd for C₂₀H₁₅F₂N: C, 78.16; H, 4.92; N, 4.56. Found: C, 77.91; H, 5.03; N, 4.42.

6-((2,6-Difluorophenyl)ethynyl)-N,N-dimethylpyridin-3-amine (5a). Yield 68%; preparative layer chromatography using 1 : 2 ethyl acetate–hexane (R_f = 0.53); mp 120–122 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (d, J = 3 Hz, 1H), 7.55–7.46 (m, 1H), 7.44 (d, J = 8.7 Hz, 1H), 7.29–7.2 (m, 2H), 7.06 (dd, J = 3.1, 8.8 Hz, 1H), 3 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.01 (dd, J = 5.3, 251.2 Hz, 2C), 145.52, 134.97, 130.95 (t, J = 10.1 Hz), 127.79, 127.47, 117.6, 111.96 (dd, J = 5.3, 18.2 Hz, 2C), 101.11 (t, J = 19.8 Hz), 99.79 (t, J = 3.1 Hz), 72.88, 39.33 (2C). HRMS (ESI) calcd for C₁₅H₁₃F₂N₂ [MH⁺]: 259.1041. Found: 259.1023. Anal. calcd for C₁₅H₁₂F₂N₂: C, 69.76; H, 4.68; N, 10.85. Found: C, 69.61; H, 4.85; N, 10.83.

5-((2,6-Difluorophenyl)ethynyl)-N,N-dimethylpyridin-2-amine (5b). Yield 79%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.41); mp 110–112 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.29 (d, J = 2.1 Hz, 1H), 7.64 (dd, J = 2.4, 8.9 Hz, 1H), 7.53–7.43 (m, 1H), 7.22 (t, J = 7.9 Hz, 2H), 6.68 (d, J = 8.9 Hz, 1H), 3.08 (s, 6H). ¹³C NMR (176 MHz, DMSO-d₆) δ 161.74 (dd, J = 5.2, 250.7 Hz, 2C), 158.12, 150.93, 139.51, 130.56 (t, J = 10.1 Hz), 111.81 (dd, J = 3.9, 20.2 Hz, 2C), 105.54, 104.57, 101.48 (t, J = 19.8 Hz), 98.02 (t, J = 3 Hz), 76.11, 37.52 (2C). HRMS (ESI) calcd for C₁₅H₁₃F₂N₂ [MH⁺]: 259.1041. Found: 259.1023. Anal. calcd for C₁₅H₁₂F₂N₂: C, 69.76; H, 4.68; N, 10.85. Found: C, 69.49; H, 4.81; N, 10.76.

5-((2,6-Difluorophenyl)ethynyl)-2-(1H-pyrrol-1-yl)pyridine (5c). Yield 66%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.47) and recrystallization from abs. ethanol; mp 121–123 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.63 (d, J = 2.2 Hz, 1H), 8.12 (dd, J = 2.3, 8.6 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.73 (t, J = 2.3 Hz, 2H), 7.62–7.51 (m, 1H), 7.28 (t, J = 8 Hz, 2H), 6.34 (t, J = 2.3 Hz, 2H). ¹³C NMR (176 MHz, DMSO-d₆) δ 162.02 (dd, J = 5, 252 Hz, 2C), 151.04, 150.19, 141.74, 131.8 (t, J = 10.1 Hz), 118.35 (2C), 114.63, 112.07 (m, 4C), 111.27, 100.54 (t, J = 19.7 Hz), 95.5 (t, J = 2.9 Hz), 78.6. HRMS (ESI) calcd for C₁₇H₁₁F₂N₂ [MH⁺]: 280.0885. Found: 280.0886. Anal. calcd for C₁₇H₁₀F₂N₂: C, 72.85; H, 3.60; N, 10.00. Found: C, 72.74; H, 3.69; N, 9.84.

5-((2,6-Difluorophenyl)ethynyl)-N,N-dimethylpyrazin-2-amine (5d). Yield 73%; column chromatography using 1 : 10 ethyl acetate–hexane; mp 137–138 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.3 (d, J = 1.4 Hz, 1H), 8.19 (d, J = 1.4 Hz, 1H), 7.52 (m, 1H), 7.25 (t, J = 8 Hz, 2H), 3.13 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.93 (dd, J = 251.5, 5.2 Hz, 2C), 152.96, 145.35, 131.25 (t, J = 10.1 Hz), 130.64, 123.25, 111.93 (dd, J = 5.2, 18.6 Hz, 2C), 100.85 (t, J = 19.8 Hz), 97.31 (t, J = 3 Hz), 75.74, 37.2 (2C). HRMS (ESI) calcd for C₁₄H₁₂F₂N₃ [MH⁺]: 260.0994. Found: 260.0994. Anal. calcd for C₁₄H₁₁F₂N₃: C, 64.86; H, 4.28; N, 16.21. Found: C, 64.63; H, 4.25; N, 16.39.

5-((2,6-Difluorophenyl)ethynyl)-1H-indole (5e). Yield 63%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.44); mp 131–133 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.4 (s, 1H), 7.82 (s, 1H), 7.52–7.44 (m, 3H), 7.27 (dd, J = 1.6, 8.5 Hz, 1H), 7.23 (t, J = 8 Hz, 2H), 6.51–6.49 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.94 (dd, J = 5.4, 250.5 Hz, 2C), 136.08, 130.44 (t, J = 10.1 Hz), 127.63, 126.95, 124.24, 124.18, 112.06, 111.81 (dd, J = 5.4, 18.3 Hz, 2C), 111.35, 101.69 (t, J = 19.7 Hz), 101.59, 101.49 (t, J = 3 Hz), 73.26. HRMS (ESI) calcd for C₁₆H₁₀F₂N [MH⁺]: 254.0776. Found: 254.0760. Anal. calcd for C₁₆H₉F₂N: C, 75.88; H, 3.58; N, 5.53. Found: C, 76.07; H, 3.63; N, 5.41.

5-((2,6-Difluorophenyl)ethynyl)-1-methyl-1H-indole (5f). Yield 76%; column chromatography using 1 : 2 ethyl acetate–hexane; mp 100–102 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.82 (d, J = 1.6 Hz, 1H), 7.55–7.45 (m, 2H), 7.43 (d, J = 3 Hz, 1H), 7.33 (dd, J = 1.6, 8.5 Hz, 1H), 7.24 (t, J = 7.9 Hz, 2H), 6.5 (d, J = 2.6 Hz, 1H), 3.82 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.94 (dd, J = 5.4, 250.6 Hz, 2C), 136.45, 131.28, 130.51 (t, J = 10.1 Hz), 127.96, 124.41, 124.24, 111.83 (dd, J = 5.3, 19.6 Hz, 2C), 111.45, 110.42, 101.63 (t, J = 20.0 Hz), 101.28 (t, J = 3 Hz), 100.92, 73.52, 32.63. HRMS (ESI) calcd for C₁₇H₁₂F₂N [MH⁺]: 268.0932. Found: 268.0917. Anal. calcd for C₁₇H₁₁F₂N: C, 76.39; H, 4.15; N, 5.24. Found: C, 76.17; H, 4.31; N, 5.04.

4-((2,6-Difluorophenyl)ethynyl)-1H-indazole (5g). Yield 71%; column chromatography using 1 : 2 ethyl acetate–hexane; mp 178–179 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.45 (s, 1H), 8.11 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.63–7.52 (m, 1H), 7.47–7.37 (m, 2H), 7.3 (t, J = 8 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.05 (dd, J = 5.2, 251.8 Hz, 2C), 139.64, 132.2, 131.67 (t, J = 10.2 Hz), 126.16, 124.5, 123.35, 112.98, 112.22, 112.05 (dd, J = 5.3, 18.2 Hz, 2C), 100.9 (t, J = 19.7 Hz), 96.74 (t, J = 3 Hz), 79.12. HRMS (ESI) calcd for C₁₅H₉F₂N₂ [MH⁺]: 255.0728. Found: 255.0712. Anal. calcd for C₁₅H₈F₂N₂: C, 70.86; H, 3.17; N, 11.02. Found: C, 70.60; H, 3.25; N, 10.92.

5-((2,6-Difluorophenyl)ethynyl)-1H-indazole (5h). Yield 25%; preparative layer chromatography using 1 : 2 ethyl acetate–hexane (R_f = 0.63); mp 193–195 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 13.34 (s, 1H), 8.15 (s, 1H), 8.08 (s, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.54–7.45 (m, 2H), 7.25 (t, J = 7.9 Hz, 2H). ¹³C NMR (176 MHz, DMSO-d₆) δ 162.01 (dd, J = 5.2, 251 Hz, 2C), 139.52, 134.15, 130.94 (t, J = 10.1 Hz), 128.85, 124.98, 122.82, 112.96, 111.88 (dd, J = 3.8, 20.2 Hz, 2C), 110.94, 101.24 (t, J = 19.8 Hz), 100.03 (t, J = 3 Hz), 74.15. HRMS (ESI) calcd for C₁₅H₉F₂N₂ [MH⁺]: 255.0728. Found: 255.0727. Anal. calcd for C₁₅H₈F₂N₂: C, 70.86; H, 3.17; N, 11.02. Found: C, 70.64; H, 3.22; N, 11.09.

6-((2,6-Difluorophenyl)ethynyl)-1H-indazole (5i). Yield 87%; column chromatography using 1 : 1 ethyl acetate–hexane; mp 192–194 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.31 (s, 1H), 8.15 (s, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.77 (s, J = 1.2 Hz, 1H), 7.6–7.48 (m, 1H), 7.33–7.21 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.09 (dd, J = 5.2, 251.5 Hz, 2C), 139.31, 133.87, 131.44 (t, J = 10.2 Hz), 123.20, 123.04, 121.37, 118.43, 113.63, 111.99 (dd, J = 5.3, 19 Hz, 2C), 100.94 (t, J = 19.7 Hz), 99.67 (t, J = 3.1 Hz), 75.66. HRMS (ESI) calcd for C₁₅H₉F₂N₂ [MH⁺]: 255.0728. Found: 255.0712. Anal. calcd for C₁₅H₈F₂N₂: C, 70.86; H, 3.17; N, 11.02. Found: C, 70.61; H, 3.06; N, 10.88.

5-((2,6-Difluorophenyl)ethynyl)-1-methyl-1H-indazole (5j). Yield 47%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.31); mp 107–109 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.12 (d, J = 0.9 Hz, 1H), 8.06 (s, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.55 (dd, J = 1.5, 8.7 Hz, 1H), 7.53–7.48 (m, 1H), 7.25 (t, J = 7.9 Hz, 2H), 4.08 (s, 3H). ¹³C NMR (176 MHz, DMSO-d₆) δ 162.01 (dd, J = 5.2, 251.0 Hz, 2C), 139.19, 133.06, 130.98 (t, J = 10 Hz), 128.78, 125.17, 123.39, 113.04, 111.89 (dd, J = 3.8, 20.2 Hz, 2C), 110.54, 101.2 (t, J = 19.8 Hz), 99.87 (t, J = 3 Hz), 74.38, 35.53. HRMS (ESI) calcd for C₁₆H₁₁F₂N₂ [MH⁺]: 269.0885. Found: 269.0885. Anal. calcd for C₁₆H₁₀F₂N₂: C, 71.64; H, 3.76; N, 10.44. Found: C, 71.41; H, 3.75; N, 10.53.

6-((2,6-Difluorophenyl)ethynyl)-1-methyl-1H-indazole (5k). Yield 75%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.35); mp 117–119 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.12 (s, 1H), 7.99 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.63–7.47 (m, 1H), 7.32–7.2 (m, 3H), 4.09 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.09 (dd, J = 5.2, 251.6 Hz, 2C), 139.09, 132.68, 131.44 (t, J = 10.2 Hz), 123.56, 123.20, 121.49, 118.42, 113.46, 111.94 (dd, J = 5.2, 18.6 Hz, 2C), 100.88 (t, J = 19.7 Hz), 99.65 (t, J = 3 Hz), 75.81, 35.58. HRMS (ESI) calcd for C₁₆H₁₁F₂N₂ [MH⁺]: 269.0885. Found: 269.0886. Anal. calcd for C₁₆H₁₀F₂N₂: C, 71.64; H, 3.76; N, 10.44. Found: C, 71.48; H, 3.83; N, 10.39.

5-((2,6-Difluorophenyl)ethynyl)-1H-pyrrolo[2,3-b]pyridine (5l). Yield 45%; column chromatography using 1 : 2 ethyl acetate–hexane; mp 192–194 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.99 (s, 1H), 8.41 (d, J = 2 Hz, 1H), 8.22 (d, J = 2 Hz, 1H), 7.59 (t, J = 3 Hz, 1H), 7.57–7.47 (m, 1H), 7.26 (t, J = 8 Hz, 2H), 6.52 (dd, J = 1.8, 3.5 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.95 (dd, J = 5.2, 251.2 Hz, 2C), 147.80, 145.04, 131.21, 131.02 (t, J = 10.3 Hz), 127.83, 119.20, 111.76 (dd, J = 5, 19 Hz, 2C), 109.53, 101.29 (t, J = 19.9 Hz), 100.42, 98.22 (t, J = 3 Hz), 75.88. HRMS (ESI) calcd for C₁₅H₉F₂N₂ [MH⁺]: 255.0728. Found: 255.0710. Anal. calcd for C₁₅H₈F₂N₂: C, 70.86; H, 3.17; N, 11.02. Found: C, 70.57; H, 3.17; N, 10.88.

2-((2,6-Difluorophenyl)ethynyl)quinoline (5m). Yield 83%; column chromatography using 1 : 5 ethyl acetate–hexane; mp 127–129 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.48 (d, J = 8.5 Hz, 1H), 8.09–8.02 (m, 2H), 7.89–7.82 (m, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.72–7.66 (m, 1H), 7.66–7.58 (m, 1H), 7.32 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.44 (dd, J = 5, 252.8 Hz, 2C), 147.66, 141.73, 137.07, 132.57 (t, J = 10.2 Hz), 130.62, 128.75, 128.05, 127.82, 127.15, 124.25, 112.16 (dd, J = 4.6, 19 Hz, 2C), 99.97 (t, J = 19.7 Hz), 98.51 (t, J = 3 Hz), 75.62. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0756. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 76.73; H, 3.38; N, 5.17.

3-((2,6-Difluorophenyl)ethynyl)quinoline (5n). Yield 64%; preparative layer chromatography using 1 : 10 ethyl acetate–hexane (R_f = 0.42); mp 115–117 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9 (d, J = 2.1 Hz, 1H), 8.72 (d, J = 2.2 Hz, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.85 (t, J = 7.6 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.64–7.52 (m, 1H), 7.30 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.12 (dd, J = 5, 252.2 Hz, 2C), 151.13, 146.64, 139.13, 132.02 (t, J = 10.2 Hz), 131.04, 128.87, 128.29, 127.7, 126.73, 115.41, 112.06 (dd, J = 5, 18.7 Hz, 2C), 100.46 (t, J = 19.7 Hz), 96.21 (t, J = 3.1 Hz), 78.9. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0757. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 76.68; H, 3.50; N, 5.11.

4-((2,6-Difluorophenyl)ethynyl)quinoline (5o). Yield 68%; column chromatography using 1 : 5 ethyl acetate–hexane; mp 101–102 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.97 (d, J = 4.4 Hz, 1H), 8.29 (d, J = 8.1 Hz, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.95–7.84 (m, 1H), 7.83–7.75 (m, 2H), 7.70–7.58 (m, 1H), 7.34 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.22 (dd, J = 4.9, 252.9 Hz, 2C), 150.26, 147.50, 132.8 (t, J = 10.2 Hz), 130.44, 129.78, 128.15, 127.1, 126.35, 124.97, 123.86, 112.21 (dd, J = 5.3, 19 Hz, 2C), 100.06 (t, J = 19.8 Hz), 94.17 (t, J = 3 Hz), 84.92. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0757. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 77.21; H, 3.43; N, 5.24.

2-Chloro-3-((2,6-difluorophenyl)ethynyl)quinoline (5p). Yield 53%; column chromatography using 1 : 5 ethyl acetate–hexane; mp 137–138 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.1 (d, J = 8.2 Hz, 1H), 8 (d, J = 8.5 Hz, 1H), 7.93–7.84 (m, 1H), 7.77–7.69 (m, 1H), 7.68–7.56 (m, 1H), 7.31 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.2 (dd, J = 5, 252.8 Hz, 2C), 148.86, 146.09, 143, 132.47 (t, J = 10.1 Hz), 132.3, 128.28, 128.16, 127.84, 126.14, 115.72, 112.17 (dd, J = 4.5, 19 Hz, 2C), 100.22 (t, J = 19.7 Hz), 94.07 (t, J = 2.9 Hz), 82.23. HRMS (ESI) calcd for C₁₇H₉ClF₂N [MH⁺]: 300.0386. Found: 300.0368. Anal. calcd for C₁₇H₈ClF₂N: C, 68.13; H, 2.69; N, 4.67. Found: C, 68.16; H, 2.73; N, 4.38.

4-Chloro-3-((2,6-difluorophenyl)ethynyl)quinoline (5q). Yield 40%; recrystallized from methanol; mp 140–142 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.03 (s, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.94 (t, J = 7.7 Hz, 1H), 7.84 (t, J = 7.7 Hz, 1H), 7.7–7.56 (m, 1H), 7.32 (t, J = 8.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.11 (dd, J = 4.9, 253.1 Hz, 2C), 151.25, 147.27, 143, 132.62 (t, J = 10.2 Hz), 131.84, 129.65, 129.22, 124.83, 124.17, 115.77, 112.16 (dd, J = 4.6, 19 Hz, 2C), 100.15 (t, J = 19.7 Hz), 93.43 (t, J = 3 Hz), 84.18. HRMS (ESI) calcd for C₁₇H₉ClF₂N [MH⁺]: 300.0386. Found: 300.0366. Anal. calcd for C₁₇H₈ClF₂N: C, 68.13; H, 2.69; N, 4.67. Found: C, 67.84; H, 2.82; N, 4.53.

3-((2,6-Difluorophenyl)ethynyl)quinolin-6-amine (5r). Yield 67%; column chromatography using 2 : 1 ethyl acetate–hexane; mp 184–185 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.54 (d, J = 2.1 Hz, 1H), 8.26 (d, J = 2.1 Hz, 1H), 7.73 (d, J = 9 Hz, 1H), 7.63–7.51 (m, 1H), 7.29 (t, J = 8 Hz, 2H), 7.22 (dd, J = 2.4, 9 Hz, 1H), 6.83 (d, J = 2.5 Hz, 1H), 5.82 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.10 (dd, J = 5.1, 251.9 Hz, 2C), 148.05, 145.57, 140.98, 135.88, 131.76 (t, J = 10.2 Hz), 129.62, 128.76, 123, 115.13, 112.07 (dd, J = 5, 18.7 Hz, 2C), 104.41, 100.8 (t, J = 19.7 Hz), 97.1 (t, J = 3.1 Hz), 78.02. HRMS (ESI) calcd for C₁₇H₁₁F₂N₂ [MH⁺]: 281.0885. Found: 281.0864. Anal. calcd for C₁₇H₁₀F₂N₂: C, 72.85; H, 3.60; N, 10.00. Found: C, 72.57; H, 3.64; N, 9.84.

3-((2,6-Difluorophenyl)ethynyl)-7-fluoroquinoline (5s). Yield 87%; column chromatography using 1 : 5 ethyl acetate–hexane; mp 142–143 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.03 (d, J = 2 Hz, 1H), 8.77 (d, J = 2.1 Hz, 1H), 8.17 (dd, J = 6.3, 9.1 Hz, 1H), 7.83 (dd, J = 2.5, 10.3 Hz, 1H), 7.66 (td, J = 2.6, 8.9 Hz, 1H), 7.61–7.54 (m, 1H), 7.3 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 163.16 (d, J = 250.1 Hz), 162.12 (dd, J = 5.1, 252.3 Hz, 2C), 152.29, 147.61 (d, J = 13 Hz), 139.24 (d, J = 1.3 Hz), 132.07 (t, J = 10.2 Hz), 131.14 (d, J = 10.3 Hz), 124.09, 118.13 (d, J = 25.5 Hz), 114.95 (d, J = 2.7 Hz), 112.58 (d, J = 20.6 Hz), 112.10 (dd, J = 5.3, 18.9 Hz, 2C), 100.4 (t, J = 19.7 Hz), 95.94 (t, J = 2.9 Hz), 78.94. HRMS (ESI) calcd for C₁₇H₉F₃N [MH⁺]: 284.0682. Found: 284.0661. Anal. calcd for C₁₇H₈F₃N: C, 72.09; H, 2.85; N, 4.95. Found: C, 71.83; H, 2.94; N, 4.70.

3-((2,6-Difluorophenyl)ethynyl)-8-fluoroquinoline (5t). Yield 79%; recrystallized from methanol; mp 136–137 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (d, J = 2 Hz, 1H), 8.8 (s, 1H), 7.97–7.84 (m, 1H), 7.75–7.65 (m, 2H), 7.64–7.56 (m, 1H), 7.31 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.15 (dd, J = 5, 252.4 Hz, 2C), 157.08 (d, J = 255.6 Hz), 151.38 (d, J = 1.5 Hz), 139.04 (d, J = 2.9 Hz), 136.37 (d, J = 12 Hz), 132.25 (t, J = 10.2 Hz), 128.44 (d, J = 2 Hz), 127.91 (d, J = 8 Hz), 124.26 (d, J = 4.6 Hz), 116.55, 115.22 (d, J = 18.4 Hz), 112.11 (dd, J = 5, 18.6 Hz, 2C), 100.27 (t, J = 19.7 Hz), 95.71 (t, J = 3 Hz), 79.59. HRMS (ESI) calcd for C₁₇H₉F₃N [MH⁺]: 284.0682. Found: 284.0661. Anal. calcd for C₁₇H₈F₃N: C, 72.09; H, 2.85; N, 4.95. Found: C, 71.79; H, 2.92; N, 4.95.

7-Chloro-4-((2,6-difluorophenyl)ethynyl)quinoline (5u). Yield 80%; column chromatography in 1 : 150 methanol–dichloromethane; mp 130–132 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.99 (d, J = 4.4 Hz, 1H), 8.25 (d, J = 8.9 Hz, 1H), 8.17 (d, J = 2.1 Hz, 1H), 7.83 (dd, J = 2.1, 8.9 Hz, 1H), 7.80 (d, J = 4.5 Hz, 1H), 7.7–7.59 (m, 1H), 7.34 (t, J = 8.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.24 (dd, J = 4.9, 253.1 Hz, 2C), 151.62, 147.83, 135.06, 133.04 (t, J = 10.3 Hz), 128.78, 128.42, 127.32, 127.01, 125.05, 124.22, 112.24 (dd, J = 4.5, 19 Hz, 2C), 99.89 (t, J = 19.6 Hz), 93.66 (t, J = 3 Hz), 85.43. HRMS (ESI) calcd for C₁₇H₉ClF₂N [MH⁺]: 300.0386. Found: 300.0366. Anal. calcd for C₁₇H₈ClF₂N: C, 68.13; H, 2.69; N, 4.67. Found: C, 67.96; H, 2.68; N, 4.68.

5-((2,6-Difluorophenyl)ethynyl)quinoline (5v). Yield 68%; preparative layer chromatography using 1 : 10 ethyl acetate–hexane (R_f = 0.45); mp 98–100 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.04–9 (m, 1H), 8.64 (d, J = 8.2 Hz, 1H), 8.15 (dd, J = 2.5, 8.5 Hz, 1H), 7.94 (dd, J = 2.8, 7.2 Hz, 1H), 7.9–7.78 (m, 1H), 7.78–7.69 (m, 1H), 7.66–7.52 (m, 1H), 7.40–7.23 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.06 (dd, J = 5.1, 251.9 Hz, 2C), 151.47, 147.34, 133.17, 131.9 (t, J = 10.2 Hz), 131.13, 131.06, 129.35, 127.62, 122.86, 119.2, 112.06 (dd, J = 5.3, 18.3 Hz, 2C), 100.56 (t), 95.63 (t, J = 3.1 Hz), 81.48. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0758. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 76.80; H, 3.47; N, 5.15.

6-((2,6-Difluorophenyl)ethynyl)quinoline (5w). Yield 80%; column chromatography using 1 : 2 ethyl acetate–hexane; mp 91–93 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (dd, J = 1.8, 4.2 Hz, 1H), 8.44 (d, J = 8.1 Hz, 1H), 8.33 (d, J = 1.9 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.86 (dd, J = 1.9, 8.7 Hz, 1H), 7.61 (dd, J = 4.2, 8.3 Hz, 1H), 7.59–7.52 (m, 1H), 7.29 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.12 (dd, J = 5.1, 251.9 Hz, 2C), 151.86, 147.35, 136.13, 132.05, 131.74 (t, J = 10.2 Hz), 131.38, 129.7, 127.75, 122.44, 119.23, 112.01 (dd, J = 4.9, 18.7 Hz, 2C), 100.67 (t, J = 19.7 Hz), 98.47 (t, J = 3 Hz), 76.91. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0758. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 76.97; H, 3.33; N, 5.29.

7-((2,6-Difluorophenyl)ethynyl)quinoline (5x). Yield 74%; preparative layer chromatography using 1 : 10 ethyl acetate–hexane (R_f = 0.49); mp 123–125 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.98 (dd, J = 1.7, 4.2 Hz, 1H), 8.43 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 1.1 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.73 (dd, J = 1.6, 8.4 Hz, 1H), 7.66–7.5 (m, 2H), 7.29 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.13 (dd, J = 5, 252 Hz, 2C), 151.81, 147.12, 136.01, 132.15, 131.86 (t, J = 10.2 Hz), 129.08, 128.46, 128.24, 122.56, 122.21, 112.7–111.37 (dd, J = 5, 18.6 Hz, 2C), 100.62 (t, J = 19.8 Hz), 98.35 (t, J = 3.1 Hz), 77.65. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0757. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 77.15; H, 3.28; N, 5.31.

8-((2,6-Difluorophenyl)ethynyl)quinoline (5y). Yield 62%; column chromatography using 1 : 5 ethyl acetate–hexane; mp 108–110 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.04 (dd, J = 1.8, 4.2 Hz, 1H), 8.47 (dd, J = 1.8, 8.3 Hz, 1H), 8.11 (d, J = 8.2 Hz, 1H), 8.08 (dd, J = 1.4, 7.2 Hz, 1H), 7.7–7.62 (m, 2H), 7.6–7.5 (m, 1H), 7.28 (t, J = 8 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.07 (dd, J = 5.2, 251.6 Hz, 2C), 151.47, 147.17, 136.71, 134.39, 131.37 (t, J = 10.2 Hz), 130, 128.02, 126.3, 122.36, 121.25, 111.94 (dd, J = 5.3, 19 Hz, 2C), 101.33 (t, J = 19.6 Hz), 97.3 (t, J = 3 Hz), 80.76. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0757. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 76.68; H, 3.30; N, 5.29.

2-Chloro-6-((2,6-difluorophenyl)ethynyl)quinoline (5z). Yield 67%; column chromatography using 1 : 5 ethyl acetate–hexane; mp 137–138 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (d, J = 8.7 Hz, 1H), 8.39 (s, 1H), 8 (d, J = 8.7 Hz, 1H), 7.92 (dd, J = 8.7, 1.6 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.63–7.53 (m, 1H), 7.29 (t, J = 8.1 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.14 (dd, J = 252, 5.1 Hz, 2C), 151.17, 146.89, 140.01, 132.81, 131.97, 131.93 (t, J = 10.2 Hz), 128.64, 126.69, 123.53, 119.89, 112.06 (dd, J = 5.3, 19 Hz, 2C), 100.54 (t, J = 19.8 Hz), 98.05 (t, J = 3 Hz), 77.38. HRMS (ESI) calcd for C₁₇H₉ClF₂N [MH⁺]: 300.0386. Found: 300.0366. Anal. calcd for C₁₇H₈ClF₂N: C, 68.13; H, 2.69; N, 4.67. Found: C, 67.93; H, 2.70; N, 4.49.

4-Chloro-6-((2,6-difluorophenyl)ethynyl)quinoline (5aa). Yield 64%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.33) and recrystallization from hexane; mp 120–122 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.89 (d, J = 4.7 Hz, 1H), 8.33 (d, J = 1.8 Hz, 1H), 8.14 (d, J = 8.6 Hz, 1H), 7.96 (dd, J = 1.8, 8.6 Hz, 1H), 7.84 (d, J = 4.7 Hz, 1H), 7.67–7.5 (m, 1H), 7.29 (t, J = 8 Hz, 2H). ¹³C NMR (176 MHz, DMSO-d₆) δ 162.14 (dd, J = 4.9, 252.3 Hz, 2C), 151.75, 148.13, 140.97, 132.55, 132 (t, J = 10.1 Hz), 130.46, 127.06, 125.49, 122.61, 120.88, 112.00 (dd, J = 3.8, 20.1 Hz, 2C), 100.41 (t, J = 19.7 Hz), 97.85 (t, J = 3 Hz), 77.94. HRMS (ESI) calcd for C₁₇H₉ClF₂N [MH⁺]: 300.0386. Found: 300.0389. Anal. calcd for C₁₇H₈ClF₂N: C, 68.13; H, 2.69; N, 4.67. Found: C, 67.85; H, 2.79; N, 4.53.

4-Chloro-7-((2,6-difluorophenyl)ethynyl)quinoline (5bb). Yield 63%; column chromatography using 1 : 5 ethyl acetate–hexane and recrystallized from acetonitrile; mp 148–150 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (d, J = 4.7 Hz, 1H), 8.28 (d, J = 1.7 Hz, 1H), 8.25 (d, J = 8.7 Hz, 1H), 7.88 (dd, J = 1.7, 8.6 Hz, 1H), 7.84 (d, J = 4.7 Hz, 1H), 7.64–7.52 (m, 1H), 7.30 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.16 (dd, J = 5, 252.3 Hz, 2C), 151.83, 148.07, 141.2, 132.55, 132.16 (t, J = 10.1 Hz), 130.06, 125.83, 124.76, 123.5, 122.66, 112.08 (dd, J = 5.3, 19 Hz, 2C), 100.4 (t, J = 19.7 Hz), 97.62 (t, J = 3 Hz), 78.63. HRMS (ESI) calcd for C₁₇H₉ClF₂N [MH⁺]: 300.0386. Found: 300.0365. Anal. calcd for C₁₇H₈ClF₂N: C, 68.13; H, 2.69; N, 4.67. Found: C, 68.41; H, 2.97; N, 4.42.

6-((2,6-Difluorophenyl)ethynyl)quinolin-4-amine (5cc). Yield 93%; column chromatography using 1 : 10 methanol–dichloromethane; mp 220–222 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.35 (br s, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.73 (dd, J = 1.7, 8.6 Hz, 1H), 7.61–7.51 (m, 1H), 7.29 (t, J = 8 Hz, 2H), 7.27 (s, 2H), 6.62 (d, J = 5.3 Hz, 1H). ¹³C NMR (176 MHz, DMSO-d₆) δ 162.07 (dd, J = 5, 251.6 Hz, 2C), 152.28, 150.46, 147.61, 131.44, 131.41 (t, J = 10.1 Hz), 128.67, 126.82, 118.06, 116.27, 111.98 (dd, J = 5, 18.6 Hz, 2C), 103.1, 100.91 (t, J = 19.8 Hz), 99.23 (t, J = 3 Hz), 76.01. HRMS (ESI) calcd for C₁₇H₁₁F₂N₂ [MH⁺]: 281.0885. Found: 281.0887. Anal. calcd for C₁₇H₁₀F₂N₂: C, 72.85; H, 3.60; N, 10.00. Found: C, 72.84; H, 3.52; N, 10.06.

7-((2,6-Difluorophenyl)ethynyl)quinolin-4-amine (5dd). Yield 61%; column chromatography using ethyl acetate; mp 219–221 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.37 (br s, 1H), 8.23 (d, J = 8.6 Hz, 1H), 7.94 (s, 1H), 7.6–7.54 (m, 1H), 7.51 (dd, J = 1.5, 8.6 Hz, 1H), 7.29 (t, J = 8 Hz, 2H), 6.93 (s, 2H), 6.6 (d, J = 5.1 Hz, 1H). ¹³C NMR (176 MHz, DMSO-d₆) δ 162.1 (dd, J = 5.0, 251.8 Hz, 2C), 151.45, 151.4, 148.33, 132.32, 131.63 (t, J = 10.1 Hz), 125.22, 123.49, 121.57, 118.88, 111.98 (dd, J = 3.7, 20.3 Hz, 2C), 103.21, 100.78 (t, J = 19.7 Hz), 98.75 (t, J = 2.9 Hz), 76.85. HRMS (ESI) calcd for C₁₇H₁₁F₂N₂ [MH⁺]: 281.0885. Found: 281.0885. Anal. calcd for C₁₇H₁₀F₂N₂: C, 72.85; H, 3.60; N, 10.00. Found: C, 72.57; H, 3.73; N, 9.96.

General procedure for the synthesis of N,N-dimethylamino-substituted quinolines by nucleophilic aromatic substitution

A mixture of 130 mg (0.43 mmol) of an appropriate chloro-substituted quinoline and 2.2 mL (4.3 mmol, 10 eq.) of 2 M methylamine or dimethylamine in THF was stirred at 130 °C for 2–3 h in a pressure tube. The mixture was poured into water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, concentrated and purified by chromatography as noted for individual compounds (5ee, 5mm, 5nn, 5oo, and 5pp) described below.

6-((2,6-Difluorophenyl)ethynyl)-N,N-dimethylquinolin-2-amine (5ee). Yield 67%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.53) and recrystallization from abs. ethanol; mp 142–144 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.05 (d, J = 9.1 Hz, 1H), 7.98 (d, J = 1.9 Hz, 1H), 7.6 (dd, J = 2.0, 8.6 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.53–7.48 (m, 1H), 7.29–7.23 (m, 2H), 7.14 (d, J = 9.2 Hz, 1H), 3.19 (s, 6H). ¹³C NMR (176 MHz, DMSO-d₆) δ 161.97 (dd, J = 5.2, 251.1 Hz, 2C), 157.82, 147.85, 136.93, 131.54, 131.42, 130.93 (t, J = 10 Hz), 126.25, 121.85, 113.31, 111.88 (dd, J = 3.9, 20.2 Hz, 2C), 110.48, 101.25 (t, J = 19.8 Hz), 99.78 (t, J = 2.9 Hz), 75.07, 37.61 (2C). HRMS (ESI) calcd for C₁₉H₁₅F₂N₂ [MH⁺]: 309.1198. Found: 309.1198. Anal. calcd for C₁₉H₁₄F₂N₂: C, 74.01; H, 4.58; N, 9.09. Found: C, 74.04; H, 4.59; N, 9.09.

1-((2,6-Difluorophenyl)ethynyl)isoquinoline (5ff). Yield 72%; column chromatography using 1 : 5 ethyl acetate–hexane; mp 120–122 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.6 (d, J = 5.6 Hz, 1H), 8.45–8.37 (m, 1H), 8.13–8.05 (m, 1H), 7.97 (d, J = 5.6 Hz, 1H), 7.92–7.81 (m, 2H), 7.74–7.57 (m, 1H), 7.35 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.44 (dd, J = 5, 252.7 Hz, 2C), 143.08, 142.14, 135.38, 132.71 (t, J = 10.2 Hz), 131.22, 129.13, 128.6, 127.47, 125.45, 121.74, 112.21 (dd, J = 5.4, 18.3 Hz, 2C), 100.04 (t, J = 19.7 Hz), 96.00 (t, J = 3.1 Hz), 80. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0758. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 76.92; H, 3.50; N, 5.20.

4-((2,6-Difluorophenyl)ethynyl)isoquinoline (5gg). Yield 73%; column chromatography using 1 : 5 ethyl acetate–hexane; mp 102–104 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.41 (s, 1H), 8.79 (s, 1H), 8.25 (t, J = 9.1 Hz, 2H), 7.99 (t, J = 7.6 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.68–7.53 (m, 1H), 7.32 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.04 (dd, J = 5, 252.1 Hz, 2C), 153.34, 146.2, 134.33, 132.32, 132.06 (t, J = 10.2 Hz), 128.63, 128.57, 127.34, 123.75, 113.84, 112.09 (dd, J = 4.9, 18.6 Hz, 2C), 100.58 (t, J = 19.6 Hz), 94.01 (t, J = 3 Hz), 83.24. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0758. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 76.68; H, 3.49; N, 5.10.

5-((2,6-Difluorophenyl)ethynyl)isoquinoline (5hh). Yield 85%; column chromatography using 1 : 1 ethyl acetate–hexane; mp 115–117 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.44 (s, 1H), 8.7 (d, J = 5.8 Hz, 1H), 8.27 (d, J = 8.3 Hz, 1H), 8.11 (dd, J = 1.1, 7.2 Hz, 1H), 8.08 (d, J = 5.9 Hz, 1H), 7.81–7.72 (m, 1H), 7.66–7.55 (m, 1H), 7.33 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.06 (dd, J = 5.1, 252 Hz, 2C), 153.14, 144.54, 134.88, 134.71, 131.97 (t, J = 10.1 Hz), 129.62, 127.91, 127.36, 117.86, 117.53, 112.1 (dd, J = 5.3, 19 Hz, 2C), 100.63 (t, J = 19.6 Hz), 95.41 (t, J = 3 Hz), 81.86. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0756. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 76.75; H, 3.47; N, 5.23.

6-((2,6-Difluorophenyl)ethynyl)isoquinoline (5ii). Yield 75%; column chromatography using 1 : 1 ethyl acetate–hexane; mp 97–99 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.38 (s, 1H), 8.58 (d, J = 6.1 Hz, 1H), 8.3 (s, 1H), 8.2 (d, J = 8.5 Hz, 1H), 7.9 (d, J = 5.7 Hz, 1H), 7.79 (dd, J = 1.6, 8.5 Hz, 1H), 7.68–7.5 (m, 1H), 7.3 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.18 (dd, J = 5, 252.1 Hz, 2C), 152.35, 143.91, 134.84, 132.1 (t, J = 10.2 Hz), 130.25, 129.29, 128.44, 123.12, 120.19, 112.11 (dd, J = 5.0, 19 Hz, 2C), 100.37 (t, J = 19.7 Hz), 98.29 (t, J = 3.2 Hz), 78.06. HRMS (ESI) calcd for C₁₇H₁₀F₂N [MH⁺]: 266.0776. Found: 266.0757. Anal. calcd for C₁₇H₉F₂N: C, 76.98; H, 3.42; N, 5.28. Found: C, 77.16; H, 3.39; N, 5.21.

1-Chloro-4-((2,6-difluorophenyl)ethynyl)isoquinoline (5jj). Yield 48%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.62) and recrystallization from abs. ethanol; mp 145–146 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.63 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 8.3 (d, J = 8.3 Hz, 1H), 8.1 (ddd, J = 1.2, 6.9, 8.2 Hz, 1H), 7.95 (ddd, J = 1.1, 6.9, 8.3 Hz, 1H), 7.66–7.58 (m, 1H), 7.33 (t, J = 8.1 Hz, 2H). ¹³C NMR (176 MHz, DMSO-d₆) δ 162.06 (dd, J = 4.9, 252.5 Hz, 2C), 151.11, 144.73, 136.29, 133.31, 132.36 (t, J = 10.1 Hz), 130.35, 126.5, 125.37, 124.82, 114.66, 112.13 (dd, J = 3.7, 20.1 Hz, 2C), 100.31 (t, J = 19.7 Hz), 93 (t, J = 3.0 Hz), 84.26. HRMS (ESI) calcd for C₁₇H₉ClF₂N [MH⁺]: 300.0386. Found: 300.0388. Anal. calcd for C₁₇H₈ClF₂N: C, 68.13; H, 2.69; N, 4.67. Found: C, 68.06; H, 2.78; N, 4.61.

1-((2,6-Difluorophenyl)ethynyl)-7-fluoroisoquinoline (5kk). Yield 56%; column chromatography using 1 : 200 methanol–dichloromethane; mp 177–178 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (d, J = 5.5 Hz, 1H), 8.23 (dd, J = 5.5, 9.1 Hz, 1H), 8.07–7.96 (m, 2H), 7.83 (td, J = 2.6, 8.9 Hz, 1H), 7.71–7.57 (m, 1H), 7.35 (t, J = 8.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.43 (dd, J = 5, 252.8 Hz, 2C), 161.17 (d, J = 249.3 Hz), 142.77 (d, J = 2.4 Hz), 141.58 (d, J = 6 Hz), 132.93, 132.79 (d, J = 8.6 Hz), 131.15 (d, J = 8.9 Hz), 129.49 (d, J = 8.8 Hz), 121.79 (d, J = 25.6 Hz), 121.56 (d, J = 1.6 Hz), 112.23 (dd, J = 5, 18.7 Hz, 2C), 108.72 (d, J = 22.2 Hz), 99.91 (t, J = 19.7 Hz), 95.49 (t, J = 2.7 Hz), 80.54. HRMS (ESI) calcd for C₁₇H₉F₃N [MH⁺]: 284.0682. Found: 284.0662. Anal. calcd for C₁₇H₈F₃N: C, 72.09; H, 2.85; N, 4.95. Found: C, 71.98; H, 2.69; N, 4.91.

4-((2,6-Difluorophenyl)ethynyl)isoquinolin-1-amine (5ll). Yield 71%; column chromatography using 1 : 10 methanol–dichloromethane and recrystallization from methanol; mp 200–201 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.31 (d, J = 8.3 Hz, 1H), 8.17 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.55–7.43 (m, 3H), 7.26 (t, J = 7.9 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.04 (dd, J = 5.4, 250.5 Hz, 2C), 158.49, 147.85, 136.25, 131.71, 130.77 (t, J = 10 Hz), 126.86, 125, 124.33, 116.64, 111.85 (dd, J = 5.4, 18.3 Hz, 2C), 102.27, 102.23 (t, J = 19.9 Hz), 97.26 (t, J = 2.9 Hz), 80.27. HRMS (ESI) calcd for C₁₇H₁₁F₂N₂ [MH⁺]: 281.0885. Found: 281.0866. Anal. calcd for C₁₇H₁₀F₂N₂: C, 72.85; H, 3.60; N, 10.00. Found: C, 73.13; H, 3.69; N, 9.93.

4-((2,6-Difluorophenyl)ethynyl)-N,N-dimethylisoquinolin-1-amine (5mm). Yield 75%; preparative layer chromatography using 1 : 5 ethyl acetate–hexane (R_f = 0.47); mp 122–124 °C. ¹H NMR (700 MHz, DMSO-d₆) δ 8.31 (s, 1H), 8.2 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 8.1 Hz, 1H), 7.84 (ddd, J = 1.2, 6.9, 8.2 Hz, 1H), 7.62 (ddd, J = 1.3, 6.8, 8.3 Hz, 1H), 7.56–7.48 (m, 1H), 7.28 (t, J = 7.9 Hz, 2H), 3.19 (s, 6H). ¹³C NMR (176 MHz, DMSO-d₆) δ 161.72 (dd, J = 5.3, 250.9 Hz, 2C), 160.63, 145.03, 137.01, 130.96, 130.82 (t, J = 10.1 Hz), 126.8, 126.2, 124.21, 118.3, 111.91 (dd, J = 3.8, 20.1 Hz, 2C), 105.07, 101.45 (t, J = 19.8 Hz), 95.96 (t, J = 2.9 Hz), 81, 42.45 (2C). HRMS (ESI) calcd for C₁₉H₁₅F₂N₂ [MH⁺]: 309.1198. Found: 309.1200. Anal. calcd for C₁₉H₁₄F₂N₂: C, 74.01; H, 4.58; N, 9.09. Found: C, 73.75; H, 4.60; N, 9.00.

4-((2,6-Difluorophenyl)ethynyl)-N-methylisoquinolin-1-amine (5nn). Yield 60%, R_f = 0.48 (1 : 2 ethyl acetate–hexane), mp 118–120 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.28 (d, J = 8.6 Hz, 1H), 8.25 (s, 1H), 8.08 (t, J = 4.5 Hz, 1H), 8.05 (d, J = 9.1 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.55–7.44 (m, 1H), 7.26 (t, J = 7.9 Hz, 2H), 3.03 (d, J = 4.5 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.61 (dd, J = 5.4, 250.4 Hz, 2C), 156.29, 147.12, 135.26, 130.99, 130.33 (t, J = 10.1 Hz), 126.6, 124.05, 123.44, 117.01, 112.01 (dd, J = 3.8, 20.1 Hz, 2C), 101.78 (t, J = 19.9 Hz), 101.45, 96.88 (t, J = 2.9 Hz), 79.90, 28.29. HRMS (ESI) calcd for C₁₈H₁₃F₂N₂ [MH⁺]: 295.1041. Found: 295.1035. Anal. calcd for C₁₈H₁₂F₂N₂: C, 73.46; H, 4.11; N, 9.52. Found: C, 73.49; H, 4.23; N, 9.61.

4-((2,6-Difluorophenyl)ethynyl)-1-(4-methylpiperazin-1-yl)isoquinoline (5oo). Yield 79%, R_f = 0.42 (1 : 10 methanol–dichloromethane), mp 70–72 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 1H), 8.16 (d, J = 8.1 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.88 (ddd, J = 1.2, 6.9, 8.2 Hz, 1H), 7.68 (ddd, J = 1.3, 6.9, 8.3 Hz, 1H), 7.61–7.5 (m, 1H), 7.29 (t, J = 8 Hz, 2H), 3.47 (t, J = 5 Hz, 4H), 2.57 (t, J = 4.7 Hz, 4H), 2.27 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.82 (dd, J = 5.3, 251.1 Hz, 2C), 160.77, 144.84, 136.70, 131.31, 131.15 (t, J = 9.2 Hz), 127.19, 126.14, 124.54, 119.27, 112.12 (dd, J = 3.7, 20.1 Hz, 2C), 107.32, 101.20 (t, J = 19.7 Hz), 95.34 (t, J = 3 Hz), 81.52, 54.56 (2C), 50.65 (2C), 45.78. HRMS (ESI) calcd for C₂₂H₂₀F₂N₃ [MH⁺]: 364.1620. Found: 364.1614. Anal. calcd for C₂₂H₁₉F₂N₃: C, 72.71; H, 5.27; N, 11.56. Found: C, 72.91; H, 5.12; N, 11.67.

8-((2,6-Difluorophenyl)ethynyl)-N,N-dimethyl-1,6-naphthyridin-5-amine (5pp). Yield 76%, R_f = 0.29 (1 : 2 ethyl acetate–hexane), mp 149–150 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.04 (dd, J = 1.7, 4.2 Hz, 1H), 8.59 (dd, J = 1.7, 8.5 Hz, 1H), 8.45 (s, 1H), 7.57 (dd, J = 4.2, 8.5 Hz, 1H), 7.53–7.46 (m, 1H), 7.25 (t, J = 8 Hz, 2H), 3.25 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.9 (dd, J = 5.3, 250.9 Hz, 2C), 160.35, 153.88, 152.18, 148.92, 135.3, 130.67 (t, J = 10.1 Hz), 120.82, 113.48, 111.84 (dd, J = 3.8, 20.1 Hz, 2C), 106.58, 101.86 (t, J = 19.8 Hz), 96.37 (t, J = 3.0 Hz), 79.73, 42.31 (2C). HRMS (ESI) calcd for C₁₈H₁₄F₂N₃ [MH⁺]: 310.1150. Found: 310.1147. Anal. calcd for C₁₈H₁₃F₂N₃: C, 69.89; H, 4.24; N, 13.58. Found: C, 70.01; H, 4.44; N, 13.53.

7-((2,6-Difluorophenyl)ethynyl)quinazoline (5qq). Yield 68%; recrystallized from acetonitrile; mp 166–168 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.68 (s, 1H), 9.37 (s, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.2 (s, 1H), 7.89 (dd, J = 1.6, 8.4 Hz, 1H), 7.69–7.53 (m, 1H), 7.31 (t, J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.19 (dd, J = 4.9, 252.6 Hz, 2C), 160.85, 156.02, 148.92, 132.46 (t, J = 10.2 Hz), 130.75, 130.11, 128.79, 127.1, 124.50, 112.14 (dd, J = 5, 18.6 Hz, 2C), 100.21 (t, J = 19.7 Hz), 97.51 (t, J = 3 Hz), 79.73. HRMS (ESI) calcd for C₁₆H₉F₂N₂ [MH⁺]: 267.0728. Found: 267.0710. Anal. calcd for C₁₆H₈F₂N₂: C, 72.18; H, 3.03; N, 10.52. Found: C, 71.97; H, 3.22; N, 10.29.

N-(2-(4-((2,6-Difluorophenyl)ethynyl)isoquinolin-1-yl)-4-oxo-8,11-dioxa-2,5-diazatridecan-13-yl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (6). A mixture of 200 mg (0.66 mmol) of 1-chloro-4-((2,6-difluorophenyl)ethynyl)isoquinoline (5jj), 242 mg (1.33 mmol, 2 eq.) of sarcosine tert-butyl ester hydrochloride, and 0.35 mL (2 mmol, 3 eq.) of N,N-diisopropylethylamine was stirred under reflux for 3 h. An additional 2 equivalents of sarcosine tert-butyl ester hydrochloride and 3 equivalents of N,N-diisopropylethylamine were added. The mixture was stirred under reflux for an additional 3 h. After cooling, the mixture was poured into water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solution was filtered, concentrated, and chromatographed with 1 : 5 ethyl acetate–hexane (R_f = 0.4) to provide 220 mg (81%) of tert-butyl N-(4-((2,6-difluorophenyl)ethynyl)isoquinolin-1-yl)-N-methylglycinate as an oil with a pale greenish hue: ¹H NMR (400 MHz, DMSO-d₆) δ 8.25 (s, 1H), 8.22 (d, J = 8.5 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 7.86 (t, J = 7.3 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.58–7.47 (m, 1H), 7.28 (t, J = 8 Hz, 2H), 4.2 (s, 2H), 3.37 (s, 3H), 1.41 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.07, 161.74 (dd, J = 5.3, 251 Hz, 2C), 159.24, 144.56, 137.13, 131.11, 130.93 (t, J = 10.2 Hz), 126.49, 126.31, 124.28, 117.77, 111.92 (dd, J = 3.8, 20.1 Hz, 2C), 105.36, 101.39 (t, J = 19.7 Hz), 95.76 (t, J = 3 Hz), 81.12, 80.53, 55.11, 42.44, 27.75 (3C). HRMS (ESI) calcd for C₂₄H₂₃F₂N₂O₂ [MH⁺]: 409.1722. Found: 409.1719. A mixture of 100 mg (0.24 mmol) of tert-butyl N-(4-((2,6-difluorophenyl)ethynyl)isoquinolin-1-yl)-N-methylglycinate and 92 mg (0.24 mmol) of (+)-biotinyl-3,6-dioxaoctanediamine^29,30 in 1 mL of 1,4-dioxane was refluxed for 6 h. After cooling, the mixture was poured into water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solution was filtered, concentrated, and chromatographed with 1 : 10 methanol–dichloromethane (R_f = 0.31) to provide 19 mg (11%) of 6 as a viscous oil: ¹H NMR (400 MHz, CDCl₃) δ 8.36 (s, 1H), 8.31 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.84 (t, J = 5.4 Hz, 1H), 7.75 (ddd, J = 1.2, 6.9, 8.2 Hz, 1H), 7.56 (ddd, J = 1.3, 6.9, 8.3 Hz, 1H), 7.35–7.27 (m, 1H), 6.98 (dd, J = 7, 8.4 Hz, 2H), 6.51 (t, J = 5.6 Hz, 1H), 6.36 (s, 1H), 5.42 (s, 1H), 4.45–4.39 (m, 1H), 4.23–4.2 (m, 1H), 4.19 (s, 2H), 3.64–3.52 (m, 8H), 3.5 (t, J = 5.1 Hz, 2H), 3.4–3.33 (m, 2H), 3.27 (s, 3H), 3.11–3.01 (m, 1H), 2.84 (dd, J = 4.9, 12.8 Hz, 1H), 2.68 (d, J = 12.8 Hz, 1H), 2.15 (t, J = 7.5 Hz, 2H), 1.72–1.52 (m, 4H), 1.42–1.31 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 173.37, 170.67, 163.97, 162.88 (dd, J = 5.3, 253.5 Hz, 2C), 160.12, 144.64, 138.09, 131.02, 129.78 (t, J = 9.9 Hz), 126.79, 126.01, 125.85, 119.58, 111.45 (dd, J = 6, 18.5 Hz, 2C), 109.20, 102.69 (t, J = 19.7 Hz), 95.41 (t, J = 3.1 Hz), 82.16, 70.31, 70.19, 70.04, 70, 61.86, 60.28, 58.49, 55.64, 41.93, 40.6, 39.24, 39.17, 36.04, 28.28, 28.18, 25.68. HRMS (ESI) calcd for C₃₆H₄₃F₂N₆O₅S [MH⁺]: 709.2978. Found: 709.2983.

Cell proliferation assay

Colon cancer cells LS174T, HT-29 and Caco2 were obtained from Dr. B. Mark Evers, and BEAS-2B and HEL 299 cells were obtained from Dr. Jon Thorson at the University of Kentucky. These cells were grown in RPMI or DMEM medium (Mediatech) supplemented with 5% or 10% fetal bovine serum and 1% penicillin/streptomycin. For cell proliferation assays, 3 × 10⁴ cells per well grown in 12-well plates were treated with DMSO or inhibitors. The cell numbers and viability were analyzed using a Vi-Cell cell viability analyzer after 4 days. The IC₅₀ values were calculated with GraphPad Prism 5 (GraphPad Software).

Western blotting and streptavidin pulldown

Western blotting was performed as described previously.²³ The following antibodies were used: MAT2A (GTX112535), anti-cyclin D1 (Cell Signaling, 2922), anti-p21^Wif1/Cip1 (Cell Signaling, 2947), and anti-β-tubulin (Developmental Studies Hybridoma Bank, E7). Streptavidin pulldown was performed using recombinant MAT2A protein as described previously.¹²

Molecular modeling

The ligands binding to MAT2A were modeled by using a previously described open-state structure¹² of the MAT2A homodimer. Each ligand was docked into the active site of the enzyme using the AutoDock 4.2 program,³¹ as described previously for other enzyme–ligand binding structures.^32–34 During the docking process, the Solis and Wets local search method³⁵ was used for the conformational search, and the Lamarckian genetic algorithm (LGA)³⁶ was employed to deal with the enzyme–ligand interactions. The grid size was set to be 120 × 120 × 120. The final enzyme–ligand binding structures possessed the lowest binding free energies.

Author contributions

V. M. S. contributed to the design, synthesis, NMR characterization, and mass spectral characterization of some of the fluorinated phenylethynyl-substituted heterocycles of formula 5 (Table 2), design of the biotinylated analog 6 in Fig. 3, and writing the paper;

L. M. K. contributed to the synthesis of all of the phenylethynyl-substituted heterocycles of formula 4 (Table 1) and some of the heterocycles of formula 5 (Table 2);

W. Z. contributed to in vitro experimental biological studies (i.e., cell proliferation studies in Tables 1 and 2; pull-down experiment in Fig. 3);

Y. X. contributed to the synthesis of several phenylethynyl-substituted heterocycles and performed in vitro experimental biological studies (i.e., some of the cell proliferation studies in Tables 1 and 2);

P. W. contributed to the synthesis of the biotinylated analog 6 in Fig. 3;

L. P. contributed to in vitro testing of phenylethynyl-substituted heterocycles 5mm and 5nn and other antineoplastic agents in normal and in various cancer cell lines (Tables 3 and 4);

X. L. contributed to the design of biological studies;

Y. Y. performed the computational studies (Fig. 2);

C.-G. Z. designed and wrote the programs for the computational studies and contributed to writing the paper;

D. S. W. contributed to the design of fluorinated phenylethynyl-substituted heterocycles, the development of synthetic routes to these compounds, and writing the paper;

C. L. contributed to the design of the biological studies of biotinylated phenylethynyl-substituted heterocycles and writing the paper.

Conflicts of interest

The authors declare no competing interest.

Acknowledgements

CL and DSW were supported by CA172379 from the NIH. CGZ was supported by NSF grant CHE-1111761. CL and DSW have partial ownership of a new-start company, Epionc, Inc., that seeks to develop these compounds as commercial agents. CL and DSW disclosed this information and complied with requirements to mitigate any potential conflicts of interest in accord with University of Kentucky policy. DSW was also supported by the Office of the Dean of the College of Medicine, by the Center for Pharmaceutical Research and Innovation in the College of Pharmacy, and by NIH grant number P30 GM110787 from the National Institute of General Medical Sciences to L. Hersh, PI. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or the NIGMS. DSW was also supported by the Office of the Assistant Secretary of Defense for Health Affairs, through the Prostate Cancer Research Program under Award No. W81XWH-16-1-0635. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.

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Footnote

† These authors contributed equally to this work.

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