2-Pyridin-2-yl-1H-indole derivatives: Synthesis, estrogen receptor binding affinity, and photophysical properties

Article abstract of DOI:10.1016/j.bioorg.2005.10.005

A series of novel 2-pyridin-2-yl-1H-indole derivatives (4a-f) was prepared by intramolecular cyclodehydration of α-anilinyl (or 3-anisidyl)-2- pyridin-2-yl-ethanones (2a-f) and their optical spectroscopy and estrogen receptor (ER) binding properties were

Full text of DOI:10.1016/j.bioorg.2005.10.005

Bioorganic Chemistry 34 (2006) 1–14
www.elsevier.com/locate/bioorg
2-Pyridin-2-yl-1H-indole derivatives:
Synthesis, estrogen receptor binding affinity,
and photophysical properties
Konstantinos M. Kasiotis, Serkos A. Haroutounian *
Chemistry Laboratory, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece
Received 20 July 2005
Available online 1 December 2005
Abstract
A series of novel 2-pyridin-2-yl-1H-indole derivatives (4a–f) was prepared by intramolecular cyc-
lodehydration of a-anilinyl (or 3-anisidyl)-2-pyridin-2-yl-ethanones (2a–f) and their optical spectros-
copy and estrogen receptor (ER) binding properties were studied. These compounds showed long
wavelength fluorescent emission, which is sensitive to solvent polarity and pH, while indol-6-ols
4b, e, and f displayed reasonably good binding affinities to ER.
ꢀ 2005 Elsevier Inc. All rights reserved.
Keywords: Indoles; Cyclization; Fluorescence; Estrogen receptor
1. Introduction
The development and use of fluorescent probes as tools for the assay and characteriza-
tion of cellular binding sites of steroidal hormone receptors is a subject of considerable
research activity [1,2]. In this regard, particularly interesting is the development of a fluo-
rescence-based assay of the estrogen receptor (ER) [3]. Such a method would permit a cell-
by-cell assay of the quantity and distribution of ER in breast cancer cells using flow
cytometry or fluorescence microscopy, providing useful information for the prediction
*
Corresponding author. Fax: +30 210 5294265.
E-mail address: sehar@aua.gr (S.A. Haroutounian).
0045-2068/$ - see front matter ꢀ 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2005.10.005

2
K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
of responsiveness to hormonal therapy [4–6]. A suitable fluorescent probe should exhibit:
(i) a relatively high binding affinity (RBA) for the ER, (ii) fluorescence at wavelengths
greater than 430 nm (in order to be distinguishable from the background of cell autoflu-
orescence), and (iii) environmental sensitivity, which results in substantial changes in emis-
sion intensity and/or wavelength in media of different polarity and/or pH.
To date three major classes of fluorescent estrogenic probes have been developed: estro-
gen–fluorophore conjugates, inherently fluorescent estrogens, and photofluorogenic estro-
gens [7–9]. However, most of the agents described do not possess the desired optimal
photophysical properties or binding affinity. Thus, the search for lead structures and novel
compounds that will prove suitable for use as fluorescent probes for the assay of the ER
represents an attractive research goal.
Part of our earlier work in this field concerned the synthesis of novel indole derivatives,
since compounds of this structural framework have been found to possess pronounced
estrogenic and tumor inhibition properties [10–13]. In this context, we have synthesized
various novel 2,3-bis-(4-hydroxyphenyl)-indole derivatives that exhibit good binding affin-
ities and interesting photophysical properties. Among the compound synthesized, 2,3-bis-
(4-hydroxyphenyl)-1H-indol-6-ol (DPI) was the most efficient since has been found to pos-
ses the best photophysical properties and receptor binding affinity [14]. However, the
design of such fluorescent probes must include a donor–acceptor system. Since the pres-
ence of phenol ring (donor) is a prerequisite for good binding affinity to the ER, our atten-
tion was focused on the study of the acceptor moiety. Thus, we have considered
substituting one of the phenyl rings with a pyridine ring. The latter is expected to act as
a potent acceptor moiety because of the additional involvement of a (n, p, and *) state that
significantly enhances its photophysical properties. In this report, we describe the synthesis
of derivatives of 2-pyridin-2-yl-1H-indoles, their ER binding affinity measurements and
photophysical property studies.
2. Materials and methods
2.1. General
Air- and/or moisture-sensitive reactions were carried out under an argon atmosphere in
flame-dried glassware. All starting materials were purchased from Aldrich (analytical
reagent grades) and used without further purification. Solvents were distilled from the
appropriate drying agents prior to use. All reactions were monitored by thin-layer chro-
matography using TLC sheets coated with silica gel 60 F254 (Merck); spots were visualized
with UV light or by treatment with an ethanolic solution of anisaldehyde. Products were
purified by flash chromatography on Merck silica gel 60 (230–400 mesh ASTM). Prior the
performance of photophysical experiments and RBA assays, all compounds were purified
by semi-preparative HPLC (Column: Kromasil 10-5 C18 (25 cm · 10 mm); mobile phase:
CH3CN; detector: UV k 300 nm; flow: 1.5 mL/min; load: 3 mg/100 lL solution in mobile
phase). Melting points (uncorrected): Buchi melting point apparatus. FT-IR: Nicolet
¨
1
Magna 750, series II. Samples were recorded as KBr pellets, unless otherwise stated. H
NMR: Bruker DRX-400 (400 MHz) spectrometer, in the indicated solvents. Chemical
shifts are referenced to internal TMS. HPLC: Hewlett–Packard 1100 series instrument
with a variable wavelength UV detector and coupled to HP Chem. Station utilizing the
manufacturerÕs 5.01 software package.

K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
3
2.2. 2-Bromo-1-(4-methoxy-phenyl)-2-pyridin-2-yl-ethanone (1)
To
a
stirred solution of 1-(4-methoxy-phenyl)-2-pyridin-2-yl-ethanone (0.3 g,
1.32 mmol) and catalytic amount of anhydrous aluminum trichloride (2 mg), in anhy-
drous THF (2 mL), was added bromine (0.07 mL, 1.32 mmol). The reaction was run
at room temperature for 1 h, then the mixture was partitioned between EtOAc
(20 mL) and H2O (15 mL). The organic layer was separated, washed with brine, and
dried over MgSO4. Concentration under reduced pressure produced a yellow solid, which
was purified by flash chromatography (hexane/EtOAc 7:3) to afford 0.32 mg of bromide
1 (yield 80%) as a pale yellow solid. Mp 78–79 ꢁC (diethylether/hexane). Rf = 0.5
(EtOAc/hexane 2:3). IR: m ¼ 1698 cm (C@O). 1H NMR (CDCl3): d = 3.83 (s, 3H,
ꢀ1
~
–OCH3), 6.45 (s, 1H, H-2); pyridinyl: 7.21 (m, 2H, H-3, H-5), 7.70 (m, 1H, H-4), 8.51
(d, J = 4.8 Hz, 1H, H-6); phenyl: 6.90 (d, J = 9.1 Hz, 2H, Ar-H), 8.02 (d, J = 9.1 Hz,
2H, Ar-H). C14H12BrNO2 (305.9): calcd: C, 54.92; H, 3.95; N. 4.58. Found: C, 54.76;
H, 4.09; N, 4.71.
2.3. General procedure for the synthesis of amino 2-pyridin-2-yl-ethanones (2a and b)
To a stirred solution of 2-bromo-1-(4-methoxy-phenyl)-2-pyridin-2-yl-ethanone 1
(7.46 mmol) and triethylamine (9 mmol) in anhydrous CH2Cl2 (10 mL) was added aniline
or m-anisidine (9 mmol). The reaction was run at room temperature for 1 h, then the sol-
vent was evaporated under reduced pressure and the remaining slurry was partitioned
between EtOAc (50 mL) and H2O (30 mL). The organic layer was separated, washed with
brine, and dried over MgSO4. Concentration under reduced pressure and flash chromato-
graphic purification furnished the desired products.
2.4. 1-(4-Methoxy-phenyl)-2-phenylamino-2-pyidin-2-yl-ethanone (2a)
This compound was obtained as a yellow solid (0.22 g, 53%). Mp 114–115 ꢁC (dieth-
ylether/hexane). Rf = 0.65 (EtOAc/hexane 2:3). IR: ~m ¼ 3384 cmꢀ1 (N–H), 1673 (C@O).
1H NMR (CDCl3): d = 3.82 (s, 3H, –OCH3), 6.14 (s, 1H,–NH), 6.90 (s, 1H, H-2); pyrid-
inyl: 7.08 (m, 1H, H-5), 7.38 (d, J = 7.7 Hz, 1H, H-3), 7.54 (ddd, J = 1.8, 7.7, 7.7 Hz,
1H, H-4), 8.56 (d, J = 4.8 Hz, 1H, H-6); phenyl: 6.73 (m, 3H, Ar-H), 6.88 (d,
J = 8.8 Hz, 2H, Ar-H), 7.10 (m, 2H, Ar-H), 8.23 (d, J = 8.8 Hz, 2H, Ar-H).
C20H18N2O2 (318.4): calcd: C, 75.45; H, 5. 70; N, 8.80. Found: C, 75.61; H, 5.56; N,
8.67.
2.5. 1-(4-Methoxy-phenyl)-2-(3-methoxy-phenylamino)-2-pyidin-2-yl-ethanone (2b)
This compound was obtained as an amorphous yellow solid (0.25 g, 55%). Rf = 0.54
(N–H), 1679 (C@O). 1H NMR (CDCl3):
ꢀ1
~
(EtOAc/hexane 2:3). IR: m ¼ 3356 cm
d = 3.81 (s, 3H, –OCH3), 3.83 (s, 3H, –OCH3), 6.16 (s, 1H, –NH), 6.92 (s, 1H, H-2); pyrid-
ixnyl: 7.09 (d, J = 7.7 Hz, 1H, H-3), 7.36 (dd, J = 4.5, 7.7 Hz, 1H, H-5), 7.86 (ddd, J = 1.8,
7.7, 7.7 Hz, 1H, H-4), 8.59 (d, J = 4.5 Hz, 1H, H-6); phenyl: 6.25 (d, J = 8.8 Hz, 1H, Ar-
H), 6.57 (m, 1H, Ar-H), 6.85 (d, J = 8.8 Hz, 1H, Ar-H), 6.95 (d, J = 9.1 Hz, 2H, Ar-H),
7.11 (m, 1H, Ar-H), 7.78 (d, J = 9.1 Hz, 2H, Ar-H). C21H20N2O3 (348.4): calcd: C,
72.40; H, 5.79; N, 8.04. Found: C, 72.54; H, 5.65; N, 8.17.

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K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
2.6. General procedure for the reductive alkylation of amines
To a stirred solution of amine (14.8 mmol) and the corresponding aldehyde (29.6 mmol)
in acetonitrile (10 mL) were added sodium cyanoborohydride (29.6 mmol) and acetic acid
(0.12 mL). The reaction was run for 2–4 h (monitored by TLC) and the solvent was evap-
orated under reduced pressure. The residue was dissolved in diethylether (15 mL) and
extracted with a saturated solution of NaHCO3 (15 mL). The organic layer was separated,
dried over MgSO4, concentrated in vacuo, and purified by flash chromatography.
2.7. 1-(4-Methoxy-phenyl)-2-(methyl-phenylamino)-2-pyridin-2-yl-ethanone (2c)
This compound was obtained as a yellow solid (0.22 g, 50%). Mp 121–122 ꢁC (diethy-
1
ꢀ1
~
lether/hexane). Rf = 0.66 (EtOAc/hexane 1:4). IR: m ¼ 3327 cm (N–H), 1668 (C@O). H
NMR (CDCl3): d = 2.99 (s, 3H, –NCH3) 3.82 (s, 3H, –OCH3), 6.72 (s, 1H, H-2); pyridinyl:
6.99 (dd, J = 4.8, 7.6 Hz, 1H, H-5), 7.19 (d, J = 7.6 Hz, 1H, H-3), 7.54 (ddd, J = 4.8, 7.6,
7.6 Hz, 1H, H-4), 8.35 (d, J = 4.8 Hz, 1H, H-6); phenyl: 6.73 (m, 2H, Ar-H), 6.80 (m, 4H,
Ar-H), 7.21 (m, 1H, Ar-H), 7.60 (d, J = 9 Hz, 2H, Ar-H). C21H20N2O2 (332.4): calcd: C,
75.45; H, 5.70; N, 8.80. Found: C, 75.29; H, 5.54; N, 8.97.
2.8. 2-(Ethyl-phenylamino)-1-(4-methoxy-phenyl)-2-pyridin-2-yl-ethanone (2d)
This compound was obtained as an orange solid (0.44 g, 40%). Mp 85–86 ꢁC (diethyle-
ꢀ1
~
ther/hexane). Rf = 0.62 (EtOAc/hexane 1:4). IR (neat): m ¼ 3319 cm
(N–H), 1667
(C@O). 1H NMR (CDCl3): d = 0.94 (t, J = 7.2 Hz, 3H, –NCH2CH3) 3.25 (qd, J = 7.2 Hz,
1H, –NCH2CH3), 3.37 (qd, J = 7.2 Hz,1H, -NCH2CH3), 3.82 (s, 3H, –OCH3), 6.71 (s,
1H, H-2); pyridinyl: 6.94 (dd, J = 4.8, 7.7 Hz, 1H, H-5), 7.21 (d,J = 7.7 Hz, 1H, H-3), 7.52
(ddd, J = 4.1, 7.7, 7.7 Hz, 1H, H-4), 8.33 (d, J = 4.1 Hz, 1H, H-6); phenyl: 6.76 (m, 2H,
Ar-H), 6.80 (m, 4H, Ar-H), 7.19 (m, 1H, Ar-H), 7.55 (d, 2H, J = 9 Hz, Ar-H).
C22H22N2O2 (346.4): calcd: C, 76.28; H, 6.40; N, 8.09. Found: C, 76.47; H, 6.22; N, 8.28.
2.9. 1-(4-Methoxy-phenyl)-2-[(3-methoxy-phenyl)-methylamino]-2-pyridin-2-yl-ethanone
(2e)
This compound was obtained as a yellowish solid (0.16 g, 40%). Mp 120ꢀ–1121 ꢁC (diethy-
~
lether/hexane). Rf = 0.38 (EtOAc/hexane 1:4). IR (neat): m ¼ 3340 cm (N–H), 1671
1
(C@O). H NMR (CDCl3): d = 2.99 (s, 3H, –NCH3) 3.81 (s, 3H, –OCH3), 3.82 (s, 3H, –
OCH3), 6.39 (s, 1H, H-2); pyridinyl: 6.99 (d, J = 7.7 Hz, 1H, H-3), 7.10 (m, 1H, H-5), 7.51
(ddd, J = 4.1, 7.7, 7.7 Hz, 1H, H-4), 8.35 (d, J = 4.8 Hz, 1H, H-6); phenyl: 6.33 (m, 2H,
Ar-H), 6.80 (m, 2H, Ar-H), 6.82 (d, J = 9.1 Hz, 2H, Ar-H), 7.60 (d, J = 9.1 Hz, 2H, Ar-
H). C22H22N2O3 (362.4): calcd: C, 72.91; H, 6.12; N, 7.73. Found: C, 72.84; H, 6.22; N, 7.59.
2.10. 2-[Ethyl-(3-methoxy-phenyl)-amino]-1-(4-methoxy-phenyl)-2-pyridin-2-yl-ethanone
(2f)
This compound was obtained as a yellow amorphous solid (0.17 g, 40%). Rf = 0.43
1
(EtOAc/hexane 1:4). IR (neat): ~m ¼ 3340 cmꢀ1 (N–H), 1671 (C@O). H NMR (CDCl3):
d = 0.94 (t, J = 7.2 Hz, 3H, –NCH2CH3), 3.25 (qd, J = 7.2 Hz, 1H, –NCH2CH3), 3.37

K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
5
(qd, J = 7.2 Hz, 1H, –NCH2CH3), 3.77 (s, 3H, –OCH3), 3.79 (s, 3H, –OCH3), 6.55 (s, 1H,
H-2); pyridinyl: 6.92 (d, J = 8.2 Hz, 1H, H-3), 6.99 (dd, J = 5.1, 8.2 Hz, 1H, H-5), 7.52 (m,
1H, H-4), 8.33 (d, J = 5.1 Hz, 1H, H-6); phenyl: 6.31 (m, 2H, Ar-H), 6.43 (dd, J = 8.5,
2.1 Hz, 1H, Ar-H), 6.79 (d, J = 8.9 Hz, 2H, Ar-H), 7.13 (t, J = 8.8 Hz, 1H, Ar-H), 7.57
(d, J = 8.9 Hz, 2H, Ar-H). C23H24N2O3 (376.4): calcd: C, 73.38; H, 6.43; N, 7.44. Found:
C, 73.58; H, 6.57; N, 7.58.
2.11. General procedure for the cyclization of ketones
A mixture of a-anilinyl (or 3-anisidyl)-desoxyanisoin 2a–f (0.5 g) and PPA (10 g) was
mechanically stirred at 70 ꢁC for 5–10 h (monitored by TLC). The reaction was quenched
with an ice-cold solution of Na2CO3 and extracted repetitively with EtOAc (3· 25 mL).
The combined organic layers were dried over MgSO4, concentrated in vacuo, and purified
by flash chromatography.
2.12. 3-(4-Methoxy-phenyl)-2-pyridin-2-yl-1H-indole (3a)
This compound was obtained as pale white needles (0.3 g, 63%). Mp 101–102 ꢁC (dieth-
ylether/hexane). Rf = 0.56 (EtOAc/hexane 1:4). 1H NMR (CDCl3): d = 3.79 (s, 3H,
–OCH3), 7.07 (d, J = 8.7 Hz, 2H, Ar-H), 7.12 (m, 2H, H-6, H-4), 7.46 (d, J = 8.7 Hz,
2H, Ar-H), 7.48 (m, 1H, H-5), 7.52 (d, J = 7.7 Hz, 1H, H-4), 9.89 (s, 1H, NH); pyridinyl:
7.27 (ddd, J = 4.1, 7.9, 7.9 Hz, 1H, H-4), 7.42 (d, J = 7.9 Hz, 1H, H-3), 7.48 (dd, J = 5.1,
7.9 Hz, 1H, H-5), 8.62 (d, J = 5.1 Hz, 1H, H-6). C22H16N2O (300.3): calcd: C, 79.98; H,
5.37; N, 9.33. Found: C, 79.81; H, 5.19; N, 9.47.
2.13. 3-(4-Methoxy-phenyl)-1-methyl-2-pyridin-2-yl-1H-indole (3c)
This compound was obtained as an amorphous solid (0.26 g, 55%). Rf = 0.6 (EtOAc/
1
hexane 1:4). H NMR (CDCl3): d = 3.80 (s, 3H, –NCH3), 3.86 (s, 3H, –OCH3), 6.85 (d,
J = 8.2 Hz, 2H, ArH), 7.19 (m, 3H, H-5, H-6, H-7), 7.41 (d, J = 8.2 Hz, 2H, ArH), 7.69
(d, J = 7.7 Hz, 1H, H-4); pyridinyl: 7.21 (d, J = 7.1 Hz, 1H, H-3), 7.30 (dd, J = 4.1,
7.1 Hz, 1H, H-5), 7.51 (ddd, J = 4.1, 7.1, 7.1 Hz, 1H, H-4), 8.75 (d, J = 4.1 Hz, 1H, H-
6). C21H18N2O (314.4): calcd: C, 65.02; H, 6.21; N, 2.61. Found: C, 65.15; H, 6.32; N, 2.45.
2.14. 1-Ethyl-3-(4-methoxy-phenyl)-2-pyridin-2-yl-1H-indole (3d)
This compound was obtained as a white solid (0.23 g, 62%). Mp 98–99 ꢁC (diethylether/
1
hexane). Rf = 0.47 (EtOAc/hexane 1:4). H NMR (CDCl3) d = 1.25 (t, J = 7.1 Hz, 3H,
–NCH2CH3), 3.79 (s, 3H, –OCH3), 4.42 (q, J = 7.1 Hz, 2H, –NCH2CH3), 6.85 (d,
J = 8.8 Hz, 2H, ArH), 7.12 (m, 1H, H-5), 7.21 (d, J = 8.8 Hz, 2H, ArH), 7.29 (t,
J = 7.4 Hz, 1H, H-6), 7.44 (d, J = 8.1 Hz, 1H, H-7), 7.69 (d, J = 8.1 Hz, 1H, H-4); pyridinyl:
7.14 (m, 2H, H-3, H-5), 7.51 (ddd, J = 4.2, 7.7, 7.7 Hz, 1H, H-4), 8.74 (d, J = 4.2 Hz, 1H, H-
6). C22H20N2O (328.4): Calcd. C, 80.46; H, 6.14; N, 8.53. Found: C, 80.71; H, 6.29; N, 8.39.
2.15. 6-Methoxy-3-(4-methoxy-phenyl)-1-methyl-2-pyridin-2-yl-1H-indole (3e)
This compound was obtained as a yellowish solid (0.28 g, 60%). Mp 74–76 ꢁC
(diethylether/hexane). Rf = 0.21 (EtOAc/hexane 1:4). 1H NMR (CDCl3): d = 3.79 (s,

6
K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
3H, –OCH3) 3.81 (s, 3H, –OCH3), 3.92 (s, 3H, –NCH3), 6.79 (d, J = 2.1 Hz, 1H, H-7),
6.82 (m, 3H, Ar-H, H-4), 7.22 (d, J = 8.5 Hz, 2H, Ar-H), 7.48 (dd, J = 7.7, 2.1 Hz, 1H,
H-5); pyridinyl: 7.17 (m, 2H, H-3, H-5), 7.54 (m, 1H, H-4), 8.75 (d, J = 4.1 Hz, 1H, H-
6). C22H20N2O2 (344.4): calcd: C, 65.02; H, 6.21; N, 2.61. Found: C, 64.87; H, 6.35; N,
2.47.
2.16. 1-Ethyl-6-methoxy-3-(4-methoxy-phenyl)-2-pyridin-2-yl-1H-indole (3f)
This compound was obtained as a yellow solid (65 mg, 68%). Mp 130–131 ꢁC (diethy-
lether/hexane). Rf = 0.51 (EtOAc/hexane 1:4). 1H NMR (CDCl3): d = 1.23 (t, J = 7.1 Hz,
3H, –NCH2CH3), 3.79 (s, 3H, –OCH3), 3.91 (s, 3H, –OCH3), 4.36 (q, J = 7.1 Hz, 2H,
–NCH2CH3), 6.84 (d, J = 8.7 Hz, 2H, Ar-H), 6.88 (d, J = 2.1 Hz, 1H, H-7), 7.01 (dd,
J = 2.1, 8.3 Hz, 1H, H-5), 7.19 (d, J = 8.7 Hz, 2H, Ar-H), 7.31 (d, J = 8.3 Hz, 1H, H-
4); pyridinyl: 7.11 (d, J = 7.9 Hz, 1H, H-3), 7.16 (m, 1H, H-5), 7.49 (ddd, J = 4.1, 7.9,
7.9 Hz, 1H, H-4), 8.73 (d, J = 4.1 Hz, 1H, H-6). C23H22N2O2 (358.4): calcd: C, 77.07;
H, 6.19; N, 7.82. Found: C, 76.88; H, 6.32; N, 7.69.
2.17. General procedure for the deprotection of phenols
To a stirred solution of indole 3a–f (0.35 mmol) in CH2Cl2 (5 mL), at ꢀ78 ꢁC was
added a solution of BBr3 as a 1 N solution in CH2Cl2 (1.5 mL, 1.5 mmol). The reaction
mixture was allowed to warm to room temperature and stirred for 16–24 h. The reaction
was quenched by addition of H2O (5 mL), the layers were separated, and the aqueous
layer was extracted with EtOAc (3· 10 mL). The combined organic layers were
dried over MgSO4, concentrated under reduced pressure, and purified by flash
chromatography.
2.18. 4-(2-Pyridin-2-yl-1H-indol-3-yl)-phenol (4a)
This compound was obtained as yellow needles (44 mg, 81%). Mp 126–128 ꢁC (diethy-
1
lether). Rf = 0.49 (EtOAc/hexane 2:3). IR (neat): m ¼ 3329 cmꢀ1 (OH). H NMR (acetone-
~
d6): d = 3.80 (s, 3H, –NCH3), 6.89 (d, J = 8.6 Hz, 2H, ArH), 7.23 (d, J = 8.3 Hz, 2H,
ArH), 7.34 (t, J = 8.6 Hz, 2H, ArH), 7.49 (d, J = 8.1 Hz, 2H, ArH), 9.41 (br s, 1H,
–NH); pyridinyl: 7.01 (d, J = 7.3 Hz, 1H, H-3), 7.22 (m, 2H, H-4, H-5), 8.44 (d,
J = 4.8 Hz, 1H, H-6). C19H14N2O (286.3): calcd: C, 79.98; H, 5.43; N, 4.44. Found: C,
80.18; H, 5.31; N, 4.63.
2.19. 3-(4-Hydroxyphenyl)-2-pyridin-2-yl-1H-indol-6-ol (4b)
This compound was obtained as an orange solid (18 mg, 80%). Mp 107–108 ꢁC (dieth-
1
ꢀ1
~
ylether). Rf = 0.40 (EtOAc/hexane 1:1). IR (neat): m ¼ 3341 cm (OH). H NMR (ace-
tone-d6): d = 6.63 (dd, J = 2.1, 8.8 Hz, 1H, H-5), 6.96 (d, J = 8.5 Hz, 2H, Ar-H), 7.01
(d, J = 2.1 Hz, 1H, H-7), 7.27 (d, J = 8.5 Hz, 2H, Ar-H), 7.32 (d, J = 8.8 Hz, 1H, H-4),
8.11 (s, 1 H, OH), 8.45 (s, 1H, OH), 10.57 (br s, 1H, NH); pyridinyl: 7.09 (m, 1H, H-5),
7.18 (d, J = 7.9 Hz, 1H, H-3), 7.49 (ddd, J = 4.9, 7.9, 7.9 Hz, 1H, H-4), 8.53 (d,
J = 4.9 Hz, 1H, H-6). C19H14N2O2 (302.3): calcd: C, 75.48; H, 9.67; N, 9.27. Found: C,
75.66; H, 9.92; N, 9.37.

K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
7
2.20. 4-(1-Methyl-2-pyridin-2-yl-1H-indol-3-yl)-phenol (4c)
This compound was obtained as pale white crystals (70 mg, 75%). Mp 150–152 ꢁC
(OH). 1H
ꢀ1
~
(diethylether). Rf = 0.43 (EtOAc/hexane 2:3). IR (neat): m ¼ 3343 cm
NMR (acetone-d6): d = 6.98 (d, J = 8.6 Hz, 2H, Ar-H), 7.26 (d, J = 8.3 Hz, 2H, Ar-
H), 7.38 (d, J = 8.6 Hz, 2H, Ar-H), 7.57 (d, J = 7.9 Hz, 2H, Ar-H), 9.52 (s, 1H,
–NH); pyridinyl: 7.02 (d, J = 7.3 Hz, 1H, H-3), 7.19 (m, 2H, H-4, H-5), 8.59 (d,
J = 4.8 Hz, 1H, H-6). C20H16N2O (300.3): calcd: C, 79.98; H, 5.37; N, 9.33. Found: C,
80.13; H, 5.22; N, 9.73.
2.21. 4-(1-Ethyl-2-pyridin-2-yl-1H-indol-3-yl)-phenol (4d)
This compound was obtained as an amorphous yellow solid (110 mg, 80%). Rf = 0.57
(EtOAc/hexane 3:2). IR: ~m ¼ 3338 cmꢀ1 (OH). 1H NMR (acetone-d6): d = 1.25 (t,
J = 7.1 Hz, 3H, –NCH2CH3), 3.47 (q, J = 7.1 Hz, 1H, –NCH2CH3), 4.4 (q, J = 7.1 Hz,
1H, –NCH2CH3), 6.95 (d, J = 8.4 Hz, 2H, Ar-H), 7.13 (m, 1H, Ar-H), 7.30 (d,
J = 8.8 Hz, 2H, Ar-H), 7.36 (d, J = 8.4 Hz, 2H, Ar-H), 7.43 (m, 1H, ArH), 9.65 (br s,
1H, OH); pyridinyl: 6.76 (d, J = 8.3 Hz, 1H, H-3), 7.10 (m, 1H, H-5), 7.14 (m, 1H, H-
4), 8.57 (d, J = 4.8 Hz, 1H, H-6). C21H18N2O (314.4): calcd: C, 80.23; H, 5.77; N, 8.91.
Found: C, 80.36; H, 5.62; N, 8.77.
2.22. 3-(4-Hydroxyphenyl)-1-methyl-2-pyridin-2-yl-1H-indol-6-ol (4e)
This compound was obtained as an orange solid (36 mg, 80%). Mp 107–108 ꢁC (dieth-
1
ꢀ1
~
ylether). Rf = 0.40 (EtOAc/hexane 1:1). IR (neat): m ¼ 3341 cm (OH). H NMR (ace-
tone-d6): d = 3.69 (s, 3H, –NCH3), 6.58 (dd, J = 2.1, 8.8 Hz, 1H, H-5), 6.89 (d,
J = 8.8 Hz, 2H, Ar-H), 6.96 (d, J = 2.1 Hz, 1H, H-7), 7.23 (d, J = 8.8 Hz, 2H, Ar-H),
7.32 (d, J = 8.1 Hz, 1H, H-4), 8.09 (s, 1H, OH), 8.46 (s, 1H, OH); pyridinyl: 7.03 (m,
1H, H-5), 7.12 (d, J = 8.3 Hz, 1H, H-6), 7.44 (ddd, J = 4.9, 8.3, 8.3 Hz, 1H, H-4), 8.53
(d, J = 4.9 Hz, 1H, H-6). C20H16N2O2 (316.3): calcd: C, 75.93; H, 5.10; N, 8.86. Found:
C, 75.83; H, 5.02; N, 8.97.
2.23. 1-Ethyl-3-(4-hydroxyphenyl)-2-pyridin-2-yl-1H-indol-6-ol (4f)
This compound was obtained as pale orange crystals (20 mg, 39%). Mp 201–203 ꢁC
1
ꢀ1
~
(diethylether). Rf = 0.45 (EtOAc/hexane 1:1). IR (neat): m ¼ 3347 cm (OH); H NMR
(acetone-d6): d = 0.94 (t, J = 7.1 Hz, 3H, –NCH2CH3), 4.36 (q, J = 7.1 Hz, 2H, –
NCH2CH3), 6.67 (dd, J = 2.1, 8.8 Hz, 1H, H-5), 7.01 (d, J = 8.3 Hz, 2H, ArH), 7.05 (d,
J = 2.1 Hz, 1H, H-7), 7.21 (d, J = 8.8 Hz, 1H, H-4), 7.31 (d, J = 8.3 Hz, 2H, ArH), 8.12
(s, 1H, OH), 8.51 (s, 1H, OH); pyridinyl: 7.12 (m, 1H, H-5), 7.34 (d, J = 7.9 Hz, 1H,
H-3), 7.49 (ddd, J = 4.1, 7.9, 7.9 Hz, 1H, H-4), 8.55 (d, J = 4.1 Hz, 1H, H-6).
C21H18N2O2 (330.4): calcd: C, 76.34; H, 5.49; N, 8.48. Found: C, 76.36; H, 5.62; N, 8.39.
2.24. Determination of the estrogen receptor binding affinity
Relative binding measurements were performed as previously reported, using lamb
uterine cytosol, diluted to ꢁ1.5 nM receptor [21]. The protein solution was incubated with

8
K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
buffer or several concentrations of unlabeled competitor together with 10 nM [3H]estradiol
at 0 ꢁC for 18–24 h. The free ligand was removed by adsorption to dextran-coated char-
coal. Unlabeled competitors were prepared and serially diluted in 1:1 (v/v) dimethylform-
amide/TEA buffer (10 mM Tris, 1.5 mM EDTA, and 3 mM sodium azide, pH 7.4, at
25 ꢁC) to ensure solubility. All data are reported relative to estradiol = 100%.
2.25. UV–Vis and fluorescence spectra
Ultraviolet–visible (UV–Vis) spectra were recorded on a Jasco V-550 spectrophotome-
ter. Fluorescence spectra were acquired by photon counting on a Jobin-Yvon Fluorolog-3
spectrophotometer. All spectra were recorded at room temperature and are corrected for
phototube sensitivity and by subtraction of the solvent background. Excitation was at the
wavelength of maximum absorbance. Samples were prepared from a stock solution
(10ꢀ3 M) of the corresponding compound in EtOH, giving final concentration of
5 · 10ꢀ6 M. Acidic or basic solutions were prepared by addition of 6 N HCl or 6 N
KOH solution in water, to give a final concentration of 0.1 N.
3. Results and discussion
3.1. Chemistry
The synthesis of the amine substrates (2a–f) is illustrated in Scheme 1. More specifically
treatment of 1-(4-methoxy-phenyl)-2-pyridin-2-yl-ethanone [15] with bromine in the pres-
ence of AlCl3 furnished the monobromide 1 [16], which was subsequently reacted with ani-
line (or 3-anisidine) in the presence of triethylamine, to produce the desired a-anilinyl-(or
3-anisydyl)-2-pyridin-2-yl-ethanones 2a and b. The latter, by reductive alkylation, provid-
ed efficiently the substrates 2c–f.
The synthesis of indoles was accomplished by an acid catalyzed intramolecular cyclode-
hydration of the corresponding a-anilinyl (or 3-anisidyl)-2-pyridin-2-yl-ethanone deriva-
tives (2a–f), a modified version of the classical Bischler method [17].
Scheme 1. Reagents and conditions: (a) Br2, AlCl3, THF; (b) RC6H4NH2, Et3N, CH2Cl2; (c) HCHO or
CH3CHO, NaBH3CN, AcOH, CH3CN.

K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
9
Scheme 2. Reagents and conditions: (a) PPA, 70 ꢁC; (b) BBr3, CH2Cl2, ꢀ78 ꢁC.
Best yields were obtained when the cyclodehydration reaction was performed using a
polyphosphoric acid (PPA) solution at 70 ꢁC, for 5–10 h. Under these experimental con-
ditions the desired indole derivatives (3a and 3c–f) were obtained (Scheme 2) in satisfac-
tory yields, while there is no indication for the formation of any resinous side product.
However, the molecule of anisidyl derivative 2b under these (or more intense) experimental
conditions failed to undergo the cyclodehydration reaction, furnishing a complex mixture
of products.
Finally, the desmethylation of the indole derivatives was accomplished in excellent
yields using a slight excess of BBr3 in methylene chloride. On the other hand, the use of
larger excess of BBr3 resulted in the simultaneous cleavage of the N-alkyl bond to produce
the 3-(4-hydroxyphenyl)-2-pyridin-2-yl-1H-indol-6-ol (4b).
3.2. Absorption and fluorescence properties
The ultraviolet/visible absorbance spectra of the synthesized indoles (4a–f) were mea-
sured in tetrahydrofuran, acetonitrile, ethanol, and water, under neutral, acidic (0.1 N
HCl), and basic (0.1 N KOH) conditions. Absorbance measurements of indole 4a in base,
as well as all emission measurements in water were excluded from studies due to solubility
problems. The results of the measurements are summarized in Table 1 and the spectra of
compound 4b in CH3CN under neutral, acidic, and basic conditions are shown in Fig. 1.
Comparison of the absorption bands of indoles in different solvents indicates that their
absorbance spectra showed only limited solvatochromicity under neutral and acidic con-
ditions. However, these compounds show environmental sensitivity, since their absorption
intensities and wavelength were dependent on the pH of the solution.
The suitability of fluorescent probes for biological systems depends strongly on the
extent to which their fluorescent properties are sensitive to their environment (solvent
and pH) [18]. Thus, we have measured the fluorescence emission of all indoles (4a–f)
in each of the aforementioned solvents and pH conditions using the major-longwave-
length absorbance maximum as the excitation wavelength; in each case, wavelengths
of maximum emission were measured. As expected, the fluorescence emission is relatively

10
K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
Table 1
Long wavelength absorbance maxima for 2-pyridin-2-yl-1H-indol-6-ols (4a–f)
Conditionsa
k
ðeÞ
max
abs
Compound
THF
CH3CN
EtOH
239 (4800)
349 (3700)
260 (5000)
H2O
243 (5500)
324 (3400)
253 (5400)
4a
Neutral
Acid
266 (4700)
244 (4800)
280 (4500)
259 (5200)
353 (5900)
223 (9500)
268 (4100)
b
b
b
b
b
b
Basic
4b
Neutral
326 (1600)
247 (6100)
320 (4500)
240 (19,400)
312 (9000)
361 (11,400)
244 (9800)
313 (5300)
243 (10,100)
319 (4500)
362 (5600)
242 (10,100)
244 (5100)
318 (3000)
243 (5400)
320 (2200)
Acid
326 (6600)
367 (6200)
275 (12,900)
333 (6800)
Basic
b
4c
4d
Neutral
Acid
323 (2100)
326 (6720)
247 (6100)
320 (4500)
312 (9310)
358 (11,400)
243 (10,100)
309 (6100)
238 (10,230)
317(4600)
362 (5600)
244 (5100)
318 (3000)
242 (5400)
322 (3110)
367 (6200)
242 (10,100)
b
b
Basic
Neutral
Acid
228 (10,700)
323 (5200)
233 (37,300)
244 (7600)
323 (5500)
247 (7400)
365 (4200)
229 (31,300)
278 (7600)
322 (6000)
226 (51,000)
243 (7400)
313 (4300)
249 (4600)
278 (7600)
b
b
Basic
244 (9500)
319 (5900)
245 (8500)
313 (4700)
4e
Neutral
Acid
329 (2430)
249 (6720)
322 (5000)
242 (22,500)
313 (9900)
362 (12,900)
246 (10,100)
315 (6110)
245 (11,100)
322 (5110)
366 (6100)
245 (11,500)
246 (5870)
321 (3300)
245 (5400)
321 (2450)
330 (7600)
372 (6900)
277 (13200)
333 (7100)
Basic
b
b
4f
Neutral
Acid
331 (2490)
250 (6900)
324 (6000)
243 (22,500)
314 (9900)
364 (13,600)
248 (10,300)
318 (6230)
247 (11,400)
323 (5180)
368 (7000)
247 (12,500)
245 (6000)
322 (3400)
247 (6400)
322 (2652)
331 (8200)
374 (7300)
b
b
Basic
280 (13,330)
335 (8000)
DPI14
Neutral
242 (10,100)
325 (14,700)
242 (11,600)
315 (5300)
258 (33,300)
296 (20,500)
330 (9600)
244 (25,400)
311 (11,200)
Acid
239 (8900)
327 (10,500)
240 (15,200)
315 (6200)
251 (35,000)
297 (21,400)
334 (9500)
245 (23,800)
314 (9700)
Basic
267 (69,100)
309 (37,700)
351 (24,300)
264 (26,600)
307 (16,200)
345 (15,100)
b
b
a
Acid = 0.1 N HCl, base = 0.1 N KOH.
Not soluble.
b

K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
11
Fig. 1. UV absorbance spectra of 3-(4-hydroxy-phenyl)-2-pyridin-2-yl-1H-indol-6-ol (4b) in acetonitrile
(5 · 10ꢀ6 M) under neutral, acidic (0.1 N HCl) and basic (0.1 N NaOH) conditions.
Table 2
Long wavelength emission maxima for indoles (4a–f)
Conditiona
k
ðrelative intensityÞ
max
em
b
Compound
THF
CH3CN
EtOH
4a
Neutral
Acid
Basic
433 (73)
432 (47)
436 (1.4)
c
c
c
d
d
435 (24)
4b
Neutral
Acid
Basic
437 (84)
434 (55)
445 (2.6)
c
c
c
d
441 (26)
437 (23)
4c
Neutral
Acid
Basic
436 (78)
434 (49)
438 (2.1)
c
c
c
d
d
437 (27)
4d
Neutral
Acid
Basic
439 (115)
437 (61)
445 (36)
c
c
c
d
d
441 (74)
4e
Neutral
Acid
basic
437 (79)
431 (48)
440 (2.4)
c
c
c
d
d
435 (27)
4f
Neutral
Acid
Basic
439 (99)
438 (67)
445 (3.6)
c
c
c
d
d
442 (76)
DPI14
Neutral
Acid
435 (29)
460e (22)
410 (31)
430 (35)
407e (22)
445 (31)
408e (23)
440 (24)
427 (37)
438 (46)
464e (40)
460 (24)
d
d
Basic
460 (39)
a
Acid = 0.1 N HCl, base = 0.1 N KOH. Excitation was always at the major long wavelength band.
b
c
max
Numbers in parentheses represent the relative intensity of emission (·104 cps) at k
.
em
Quenched.
Not soluble.
Shoulder.
d
e

12
K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
weak, because the aryl and pyridine ring are twisted out of planarity from one another.
For comparison reasons, we have included in Tables 1 and 2 the corresponding data of
DPI (2,3-bis-(4-hydroxyphenyl)-1H-indol-6-ol), the most efficient 4-hydroxyphenyl-in-
dole [14].
Fluorescence emission spectra of compound 4b in THF are shown in Fig. 2 and com-
plete results on fluorescence measurements are given in Table 2 showing that in all sol-
vents their emission maxima are slightly over 430 nm. The fluorescence emission of these
compounds however, is environmentally (solvent and pH) dependent indicating that
there is a large dipole moment in the excited state [19]. More specifically, the emission
intensity was decreased sharply when the polarity of the solvent was increased. On the
Fig. 2. Fluorescence emission spectra of 3-(4-hydroxy-phenyl)-2-pyridin-2-yl-1H-indol-6-ol (4b) in THF
(5 · 10ꢀ6 M) under neutral, acidic (0.1 N HCl) and basic (0.1 N NaOH) conditions.
Table 3
Estrogen receptor binding affinity of indole derivatives (4a–f)
OH
NR1
R2
N
Compound
RBA
R1
R2
4a
4b
0.82
4.26
0.95
1.73
9.51
H
H
Me
Et
Me
Et
H
OH
H
H
OH
OH
4c
4d
4e
4f
DPI14
14.30
10.37

K.M. Kasiotis, S.A. Haroutounian / Bioorganic Chemistry 34 (2006) 1–14
13
other hand, although in the excited state the formation of the ionic form of the molecules
is facilitated, no additional red shift was observed upon going from neutral to acidic and
basic conditions [20]. In all cases when indoles were diluted in an acidic solution, the
emission was quenched, while in basic pH the emission intensity was significantly
decreased.
3.3. Estrogen receptor binding affinity
The estrogen RBAÕs of the novel indoles were determined by a competitive binding
assay and are shown in Table 3. The affinities were obtained by competition with the tracer
compound [3H]estradiol and have been expressed on a percentage scale, relative to estra-
diol, whose affinity was considered to be 100%.
In every case, an increase in RBA is observed as the alkyl steric bulk and the lipophil-
icity in the center of the molecule are increased. As expected, the absence of a hydroxyl
group from the position 6 of the indole ring led to compounds with low binding affinities
for the estrogen receptor (compounds 4a, c, and d). Thus, the three indol-6-ols (com-
pounds 4b, e, and f) displayed much better affinities to the ER than the other indoles.
Their RBA values are comparable or better to those of the corresponding 2,3-bis-(4-
hydroxyphenyl)-1H-indoles (e.g., DPI) and may be considered suitable for use in biolog-
ical assays.
In conclusion, we have used a convenient and simple synthetic pathway to prepare a
variety of novel 2-pyridin-2-yl-1H-indolol derivatives. The target compounds have been
found to possess moderate to good affinities to the estrogen receptor, while their absor-
bance and fluorescence spectra display strong dependence on solvent polarity and pH.
Since their synthesis does not require complicated stereospecific methods or isomer sep-
arations, these compounds constitute attractive candidates for further development in
order to be used as fluorescent probes. Our current studies are directed towards the exten-
sion of these studies by covering additional derivatives and relevant heterocyclic systems.
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