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[D6]DMSO): d=8.39 (t, J=13.0 Hz,
1H), 7.63 (d, J=7.0 Hz, 2H), 7.48–
7.40 (m, 4H), 7.29 (td, J=8.0,
1.5 Hz, 2H), 6.53 (d, J=13.5 Hz,
2H), 4.34 (t, J=5.5 Hz, 4H), 3.26 (s,
6H), 1.70 ppm (s, 12H); 13C NMR
(125 MHz, [D6]DMSO): d=174.5,
149.9, 142.2, 140.3, 128.4, 125.0,
122.3, 111.8, 103.0, 68.8, 58.6, 48.8,
44.1, 27.4 ppm; UV/Vis (EtOH):
lmax =550.0 nm; MS (EI, 70 eV): m/z
445 [M]+; HRMS: m/z [M]+ calcd
for C29H37N2O2: 445.2850, found:
445.2855.
Figure 5. Neuropathological staining of Bowman’s glands of the olfactory epithelium containing tau aggregates
extracted from a deceased AD patient (male, 78 years old, Braak: V, CERAD-Score: 3). Probes 5a (A and E), 5b (B
and F), 5c (C and G) and 5 f (F and H) are clearly stained tau deposits. Scale bar=25 mm (except C and G, where
scale bar=50 mm). Filter set 09 (A–D) and filter set 15 (E–H) were used for fluorescence microscopy. Staining pro-
tocol: All sections were immersed in 1 mm solution of compound in DMSO for 10 min and then washed with
methanol. The sections were differentiated in 1% CH3CO2H for 20 min. After washing with running water for
5 min, the sections were treated with Roti-Mount FluorCare (Sigma–Aldrich) and covered with a coverslip.
1-(2-(2-(2-Methoxyethoxy)ethox-
y)ethyl)-2-((1E,3E)-3-(1-(2-(2-(2-
methoxyethoxy)ethoxy)ethyl)-
3,3-dimethylindolin-2-ylidene)-
prop-1-enyl)-3,3-dimethyl-3H-in-
dolium chloride (5e):[28] Colorless
1
waxy solid (125mg, 38%): H NMR
(500 MHz, [D6]DMSO): d=8.38 (t,
J=13.0 Hz, 1H), 7.63 (d, J=7.0 Hz, 2H), 7.46 (d, J=8.0 Hz, 2H),
7.42 (td, J=8.0, 1.0 Hz, 2H), 7.29 (td, J=8.0, 1.0 Hz, 2H), 6.55 (d,
J=13.5 Hz, 2H), 4.34 (t, J=5.0 Hz, 4H), 3.82 (t, J=5.0 Hz, 4H),
3.55–3.50 (m, 4H), 3.44–3.40 (m, 4H), 3.39–3.36 (m, 4H), 3.31–3.27
(m, 4H), 3.16 (s, 6H), 1.70 ppm (s, 12H); 13C NMR (125 MHz,
[D6]DMSO): d=174.5, 149.7, 142.1, 140.3, 128.3, 125.0, 122.2, 111.9,
103.1, 71.1, 70.2, 69.7, 69.5, 67.1, 57.9, 48.8, 44.1, 27.4 ppm; UV/Vis
(EtOH): lmax =551.5 nm; MS (EI, 70 eV): m/z 622 [M+1]+.
reflux for 16 h under argon. The reaction mixture was cooled to
room temperature, pyridine was removed in vacuo, and the resi-
due was purified by column chromatography (SiO2; CH2Cl2/MeOH,
10:1).
1,3,3-Trimethyl-2-((1E,3E)-3-(1,3,3-trimethylindolin-2-ylidene)-
prop-1-enyl)-3H-indolium iodide (5a):[26] Purple solid (201 mg,
83%): 1H NMR (500 MHz, [D6]DMSO): d=8.34 (t, J=13.5 Hz, 1H),
7.62 (d, J=7.0 Hz, 2H), 7.46–7.42 (m, 4H), 7.32–7.27 (m, 2H), 6.44
(d, J=13.5 Hz, 2H), 3.64 (s, 6H), 1.68 ppm (s, 12H); 13C NMR
(125 MHz, [D6]DMSO): d=174.4, 149.6, 142.7, 140.6, 128.7, 125.3,
5-Bromo-2-((1E,3E)-3-(5-bromo-1,3,3-trimethylindolin-2-ylidene)-
prop-1-enyl)-1,3,3-trimethyl-3H-indolium iodide (5 f):[26] Purple
1
solid (186 mg, 58%): H NMR (500 MHz, [D6]DMSO): d=8.30 (t, J=
122.5, 111.5, 102.7, 48.9, 31.4, 27.4 ppm; UV/Vis (EtOH): lmax
=
547.0 nm; MS (EI, 70 eV): m/z 356 [MÀ1]+; HRMS: m/z [M]+ calcd
13.5 Hz, 1H), 7.93 (d, J=2.0 Hz, 2H), 7.64 (dd, J=8.5, 2.0 Hz, 2H),
7.42 (d, J=8.5 Hz, 2H), 6.45 (d, J=13.5 Hz, 2H), 3.63 (s, 6H),
1.68 ppm (s, 12H); 13C NMR (125 MHz, [D6]DMSO): d=174.1, 149.6,
142.7, 141.9, 131.2, 125.5, 117.4, 113.2, 103.1, 48.9, 31.5, 26.9,
26.2 ppm; UV/Vis (EtOH): lmax =556.0 nm; MS (EI, 70 eV): m/z 514
[MÀ1]+
for C25H29N2: 357.2325, found: 357.2331.
1-Butyl-2-((1E,3E)-3-(1-butyl-3,3-dimethylindolin-2-ylidene)prop-
1-enyl)-3,3-dimethyl-3H-indolium iodide (5b):[27] Purple solid
(179 mg, 61%): 1H NMR (500 MHz, [D6]DMSO): d=8.36 (t, J=
13.5 Hz, 1H), 7.64 (d, J=7.0 Hz, 2H), 7.49–7.41 (m, 4H), 7.30 (td, J=
7.5, 1.5 Hz, 2H), 6.58 (d, J=13.5 Hz, 2H), 4.14 (t, J=7.5 Hz, 4H),
1.77–1.71 (m, 4H), 1.70 (s, 12H), 1.48–1.40 (m, 4H), 0.94 ppm (t, J=
7.5 Hz, 6H); 13C NMR (125 MHz, [D6]DMSO): d=173.7, 149.8, 141.8,
140.5, 128.5, 125.1, 122.4, 111.5, 102.5, 48.8, 43.6, 29.1, 17.4, 19.5,
13.7 ppm; UV/Vis (EtOH): lmax =550.5 nm; MS (EI, 70 eV): m/z 442
[M+1]+; HRMS: m/z [M]+ calcd for C31H41N2: 441.3264, found:
441.3270.
Biological evaluation
Immunohistochemical staining: Staining was carried out on 4 mm
thick sections using a BenchMark automated stainer (Ventana,
Tucson, AZ, USA). Antibodies anti-PHF-Tau clone AT8 (Thermo Sci-
entific Pierce Protein Research Products, Rockford, USA), TAU Ab-3
(Neomarkers, Freemont, USA), and amyloid A4 (BAM10, Sigma, St.
Louis, USA), and the Ultraview Universal DAB Detection Kit (Venta-
na) were applied in staining.
1-Decyl-2-((1E,3E)-3-(1-decyl-3,3-dimethylindolin-2-ylidene)prop-
1-enyl)-3,3-dimethyl-3H-indolium bromide (5c): Purple solid
(235 mg, 68%): 1H NMR (500 MHz, [D6]DMSO): d=8.35 (t, J=
13.5 Hz, 1H), 7.65 (d, J=7.5 Hz, 2H), 7.47 (d, J=7.5 Hz, 2H), 7.43
(td, J=8.0, 1.0 Hz, 2H), 7.29 (td, J=8.0, 1.0 Hz, 2H), 6.65 (d, J=
13.5 Hz, 2H), 4.15 (t, J=7.5 Hz, 4H), 1.76–1.68 (m, 4H), 1.69 (s,
12H), 1.44–1.35 (m, 4H), 1.35–1.26 (m, 4H), 1.26–1.14 (m, 20H),
0.81 ppm (t, J=7.0 Hz, 6H); 13C NMR (125 MHz, [D6]DMSO): d=
173.6, 149.7, 141.7, 140.5, 128.5, 125.1, 122.4, 111.5, 102.7, 48.7,
43.7, 31.1, 28.8, 28.7, 28.7, 28.5, 27.3, 26.9, 25.9, 21.9, 13.8 ppm; UV/
Vis (EtOH): lmax =551.0 nm; MS (EI, 70 eV): m/z 610 [M]+; HRMS: m/
z [M]+ calcd for C43H65N2: 609.5142, found: 609.5148.
In vitro neuropathological staining of AD brain sections: Serial sec-
tion (4 mm thickness) of paraffin-embedded brain sections from AD
patients were used in these studies. The hippocampus (71 year old
male) and olfactory epithelium (78 year old male) sections were de-
paraffinized and hydrated in distilled water. All sections were im-
mersed in 1 mm solution of test compound in DMSO for 10 min
and then washed with methanol. The sections were differentiated
in 1% CH3CO2H for 20 min. After washing with running water for
5 min, the sections were treated with Roti-Mount FluorCare
(Sigma–Aldrich) and covered with a coverslip. All sections were an-
alyzed by fluorescence microscopy using a Axioskop microscope
with a HBO100 fluorescence illuminator (Carl Zeiss, Oberkochem,
Germany) with the bandpass filter set 09 BP450-490, FT510, LP515,
1-(2-Methoxyethyl)-2-((1E,3E)-3-(1-(2-methoxyethyl)-3,3-dimethy-
lindolin-2-ylidene)prop-1-enyl)-3,3-dimethyl-3H-indolium chlo-
1
ride (5d):[28] Colorless waxy solid (70mg, 29%): H NMR (500 MHz,
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 891 – 897 895