Molecules 2019, 24, 3661
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2
-Chloro-N,N-dimethylacetamide (11): Dark brownish liquid, yield: 62%. H NMR (400 MHz, CDCl ):
3
13
δ = 3.86 (s, 1 H), 2.81 (s, 3 H), 2.67 (s, 3 H). C NMR (CDCl ): δ = 166.06, 40.84, 36.92, 35.31.
3
1
2
-Chloro-1-(7-nitro-1H-indol-3-yl)ethanone (12): Brownish solid, yield: 17%. H NMR (400 MHz,
DMSO-d6):
.24–8.21 (dd, J = 8.04 Hz, 0.90 Hz, 1 H), 7.47 (t, J = 7.96 Hz, 1 H), 5.02 (s, 2 H). C NMR (DMSO-d6):
δ = 186.91, 137.11, 133.39, 129.35, 129.08, 128.82, 122.20, 120.18, 114.26, 46.80.
δ = 12.73 (s, 1 H), 8.64–8.62 (dd, J = 7.84 Hz, 0.92 Hz, 1 H), 8.57 (d, J = 2.08 Hz, 1 H),
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General procedure for synthesis of mIPP and its derivatives (3–6). Chloroacetylindole (1 equiv.) was
added to a stirred mixture of 4,6-dimethyl-2-(piperazin-1-yl)pyrimidine (1 equiv.), potassium carbonate
(
3 equiv.) and sodium iodide (1 equiv.) in 60 mL of acetonitrile. The reaction mixture was stirred under
reflux for 4 h. Upon completion of the reaction determined by TLC analysis, the reaction mixture was
cooled to room temperature and acetonitrile was removed in vacuo to give a dry residue. The dry
residue was washed with water to remove the salts and the precipitate was dissolved in methanol and
dry-loaded onto silica gel. Crude dry-loaded on silica gel were purified by column chromatography
(EA: Hexane = 1:100–3:1) to give mIPP and its derivatives.
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-(4-(4,6-Dimethylpyrimidin-2-yl)piperazin-1-yl)-1-(1H-indol-3-yl)ethanone (3): White solid, yield:
◦
8%, mp = 165 C (decomp): H NMR (400 MHz, DMSO-d ) δ11.91 (s, 1H), 8.49 (s, 1H), 8.20–8.18 (m,
6
1
1
H), 7.48–7.46 (m, 1H), 7.23–7.20 (m, 1H), 7.19–7.16 (m, 1H), 6.40 (s, 1H), 3.76–3.74 (m, 4H), 3.66 (s, 2H),
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2
.59–2.57 (m, 4H), 2.22 (s, 6H). C NMR (DMSO-d ):
δ
= 192.37, 166.63, 161.17, 136.28, 134.19, 125.60,
22.74, 121.72, 121.32, 115.23, 112.07, 108.72, 64.99, 52.79, 43.31, 23.69. HPLC analysis of compound
purity by two solvent systems: ACN:H O = 70:30 (98%), MeOH:H O = 70:30 (99%).
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1
2
2
2
-(4-(4,6-Dimethylpyrimidin-2-yl)piperazin-1-yl)-1-(7-methoxy-1H-indol-3-yl)ethanone(4): Whitesolid,
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1
yield: 77%, mp = 162 C (decomp): H NMR (400 MHz, DMSO-d ) δ12.05 (s, 1H), 8.36 (d, J = 3.16 Hz,
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1
4
H), 7.76 (d, J = 7.96 Hz, 1H), 7.12–7.08 (m, 1H), 6.80–6.78 (m, 1H), 6.39 (s, 1H), 3.93 (s, 3H), 3.74–3.72 (m,
13
H), 3.64 (s, 2H), 2.58–2.55 (m, 4H), 2.21 (s, 6H). C NMR (DMSO-d ):
δ = 192.43, 166.64, 161.17, 146.20,
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133.40, 127.16, 126.28, 122.53, 115.74, 113.91, 108.73, 103.49, 65.01, 55.29, 52.75, 43.30, 23.68. HPLC analysis
of compound purity by two solvent systems: ACN:H O = 70:30 (95%), MeOH:H O = 70:30 (95%).
2
2
2
-(4-(4,6-Dimethylpyrimidin-2-yl)piperazin-1-yl)-1-(7-chloro-1H-indol-3-yl)ethanone (5): White solid,
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1
yield: 59%, mp = 163 C (decomp): H NMR (400 MHz, DMSO-d )
δ12.31 (s, 1H), 8.56 (d, J = 2.84 Hz,
6
1
3
1
H), 8.17–8.15 (m, 1H), 7.32-7.30 (dd, J = 7.60 Hz, 0.86 Hz, 1H), 7.20 (t, J = 7.84 Hz, 1H), 6.39 (s, 1H),
13
.75–3.72 (m, 4H), 3.69 (s, 2H), 2.59–2.56 (m, 4H), 2.22 (s, 6H). C NMR (DMSO-d ):
δ
= 192.62, 166.61,
61.13, 134.99, 133.17, 127.51, 122.88, 122.36, 120.27, 116.43, 115.97, 108.72, 64.97, 52.72, 43.28, 23.68.
HPLC analysis of compound purity by two solvent systems: ACN:H O = 70:30 (89%), MeOH:H O =
6
2
2
7
0:30 (89%). Purification by prep-HPLC was conducted to obtain just the desired product (98% purity).
2
-(4-(4,6-Dimethylpyrimidin-2-yl)piperazin-1-yl)-1-(7-nitro-1H-indol-3-yl)ethanone (6): White solid,
◦
1
yield: 69%, mp = 165 C (decomp): H NMR (400 MHz, DMSO-d )
δ12.56 (s, 1H), 8.68 (d, J = 7.36 Hz,
6
0
6
.92 Hz, 1H), 8.65 (d, J = 3.00 Hz, 1H), 8.21 (dd, J = 8.04 Hz, 0.90 Hz 1H), 7.47–7.43 (t, J = 7.96 Hz, 1H),
13
.40 (s, 1H), 3.78 (s, 2H), 3.76 (dd, J = 4.84 Hz, 0.42 Hz, 4H), 2.61–2.59 (m, 4H), 2.22 (s, 6H). C NMR
= 166.64, 161.12, 136.62, 133.21, 129.53, 129.33, 128.45, 121.78, 119.80, 115.61, 108.78,
5.06, 52.68, 43.20, 23.68. HPLC analysis of compound purity by two solvent systems: ACN:H O =
(DMSO-d6): δ
6
7
2
0:30 (95%), MeOH:H O = 70:30 (95%).
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.2. Biological Evaluation
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.2.1. TGFα Shedding Assays for hA2AAR and hA AR
3
Expression vectors (250 ng of AP-TGF
α
plasmid, 100 ng of hA2AAR/hA AR plasmid and 50 ng of
3
G
α
protein) were transfected into HEK293 cells using Lipofectamine 2000 (1.25 µL/well) in 12-well plates.
Transfection of siRNA (final concentration: 10 nM) was conducted with Lipofectamine RNAiMAX