MediaChrom: Discovering a Class of Pyrimidoindolone-Based Polarity-Sensitive Dyes

Article abstract of DOI:10.1021/acs.joc.5b02066

A small library of six polarity-sensitive fluorescent dyes, nicknamed MediaChrom, was prepared. This class of dyes is characterized by a pyrimidoindolone core fitted out with a conjugated push-pull system and a carboxy linker for a conceivable coupling wi

Full text of DOI:10.1021/acs.joc.5b02066

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Article
MediaChrom: discovering a class of
pyrimidoindolone based polarity-sensitive dyes
Monica Dell'Acqua, Luca Ronda, Riccardo Piano, Sara Pellegrino, Francesca
Clerici, Elisabetta Rossi, Andrea Mozzarelli, Maria Luisa Gelmi, and Giorgio Abbiati
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b02066 • Publication Date (Web): 16 Oct 2015
Downloaded from http://pubs.acs.org on October 17, 2015
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MediaChrom: discovering a class of pyrimidoindolone based polarity-sensitive dyes
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Monica Dell’Acqua,a, § Luca Ronda,b, § Riccardo Piano, Sara Pellegrino, Francesca Clerici,
b
a
a
a
c, d
a
a,
Elisabetta Rossi, Andrea Mozzarelli, Maria Luisa Gelmi and Giorgio Abbiati *
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a
Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Generale e Organica “A. Marchesini”, Università degli Studi
di Milano, Via Venezian, 21, 20133 Milano, Italy
b
Dipartimento di Neuroscienze, Università degli Studi di Parma, Parco Area delle Scienze, 23/A, 43124 Parma, Italy
c
Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze, 23/A, 43124 Parma, Italy
d
Istituto di Biofisica, CNR, 56124 Pisa, Italy
§
These authors contributed equally to the work.
TOC graphic
ABSTRACT: A small library of six polarity-sensitive fluorescent dyes, nicknamed MediaChrom, was
prepared. This class of dyes is characterized by a pyrimidoindolone core fitted out with a conjugated
push-pull system, and a carboxy-linker for a conceivable coupling with biomolecules. The optimized
eight-step synthetic strategy involves a highly chemo- and regio-selective gold catalyzed
cycloisomerization reaction. The photophysical properties of MediaChrom dyes have been in-depth
evaluated. In particular, the MediaChrom bearing a diethylamino as electron-donating group and a
trifluoromethyl as electron-withdrawing group displays the most interesting and advantageous
spectroscopic features (e.g., absorption and emission in the visible range and a good quantum yield).
Promising results in terms of sensitivity have been obtained in vitro on this dye as membrane/lipophilic
probe and as peptide fluorescent label.
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INTRODUCTION
The modern biological research needs a continuous development of highly specific and sensitive
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fluorescent dyes for monitoring a wide range of molecular processes and events. A particular class of
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dyes, called environment-sensitive dyes, is able to change their spectroscopical properties in response to
the change of chemical-physical features of their surroundings. Among them, the polarity-sensitive dyes2
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(also called solvatochromic-dyes) have the unique features of displaying a different emission maximum
as a function of the polarity of the medium (i.e., solvent). This peculiarity makes polarity-sensitive dyes
the ideal probes to monitor the local properties of particular cell districts as well as biomolecular
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,5
interactions (e.g., peptide-nucleic acid, protein-protein, and peptide-lipid interactions).
Two main classes of polarity-sensitive dyes are available, single-band and two-band solvatochromic
dyes.6,7 The former are the most used, because the latter suffer from poor photostability. Single-band
solvatochromic dyes are usually characterized by a rigid aromatic backbone bearing conjugated electron
acceptor and electron donor groups at the opposite sides. In these molecules, the dipole moment increases
by electronic excitation due to an intramolecular charge transfer from the electron donating (ED) to the
electron withdrawing (EW) group. If their excited states are stabilized by dipole-dipole or H-bonding
interactions with the surrounding medium, these dyes exhibit a bathochromic effect of their emission
spectrum in response to an increase in solvent polarity (positive solvatochromism).
8
Several polarity-sensitive dyes have been developed, but most of them are far to meet simultaneously all
the optimal spectroscopic requirements for biological applications, i.e., a strong solvatochromism,
absorption close to the visible range, large Stokes shift, high extinction coefficient, high quantum yield
and good photostability. Among the most common and commercially available polarity-sensitive dyes,
9
Dansyl and its derivatives absorb in the UV region (around 340 nm) and display low extinction
coefficients as well as DMAP10 and its derivatives.11 Dapoxyl12 and Prodan,13 two of the best
solvatochromic dyes available, display only a slightly red-shifted absorption (373 nm and 360 nm,
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respectively). Furthermore, Dapoxyl and its derivatives are characterized by low extinction coefficients.
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Anthradan, a benzo-homologue of Prodan, solved the problem of the absorption in the UV-range
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around 450 nm) but its brightness is compromised by a low extinction coefficient. The performances of
Prodan-type solvatochromic dyes were recently strongly improved by the substitution of the polycyclic
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aromatic skeleton with a fluorene core. NBD displays an interesting red-shifted absorption (around
465 nm), but its solvatochromism is limited. Fluoroprobe,17 probably the molecule with highest
solvatochromic effect to date, suffers from a marked blue-shifted absorption (around 310 nm), a low
extinction coefficient and a strong quenching of fluorescence in polar media. These examples highlight
that the ideal polarity-sensitive dye is not yet been discovered. Beside the above mentioned well-
consolidated polarity-sensitive dyes, a number of new interesting solvatochromic D--A molecules have
been recently published, testifying that the interest of the scientific community in this field is
experiencing a dramatic growth.18
Since many years, we have been interested in the development of new strategies for the synthesis and the
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functionalization of indoles and polycyclic indole-based heterocycles. The barely investigated  but
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interesting  fluorescence properties of the pyrimidoindolone nucleus prompt us to design a new class
of polarity-sensitive dyes of general structure I, characterized by the presence of selected ED and EW
groups in a proper conjugate position (Figure 1).
Figure 1: General formula of the planned pyrimidoindolone based polarity-sensitive dyes.
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Our ambitious goal was to obtain a small library of original pyrimidoindolone-based solvatochromic
dyes with enhanced photophysical properties. The ED and EW groups were selected based on literature
findings. The most used ED groups in push-pull solvatochromic dye are secondary amines. When a
weaker donating group, such as the alkoxy group, has been used the spectroscopical performance was in
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general worse. Therefore, in our study we privileged the use of diethylammino and morpholine groups.
In particular, we preferred these amino groups with larger alkyl residues than the standard dimethylamino
group because recent studies demonstrated that this modification is able to improve brightness,
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photostability and QY of the dye. Different EW groups are present in most effective polarity-sensitive
dyes, ranging from carbonyl, to sulfonic, nitro and cyano groups. We planned to investigate the effect of
the latter three groups characterized by a strong mesomeric EW effect, and the trifluoromethyl, a strong
inductive EWG seldom encountered in this context.23 Moreover, our synthetic strategy allows the
functionalization of the lactam nitrogen with a suitable linker for a handy conjugation to biomolecules
(Figure 1). After the synthesis, the photophysical properties of the new solvatochromic dyes were
evaluated, and, as proof of concept, their applications as a membrane/lipophilic probe and as peptide
fluorescent label were briefly investigated. In this paper we describe our results.
RESULTS and DISCUSSION
Synthesis of the pyrimidoindolones (MediaChrom) library
We designed a first retrosynthetic approach (Scheme 1) taking in mind our experience in the
transformation of indolin-2-ones (V) in the indole-2-triflates (IV), useful building blocks for the
2
4
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preparation of 2-alkynyl indoles (III). The early synthetic steps have sound bases in the literature, but
involve the synthesis of a number on unknown compounds. In particular the synthesis and the reactivity
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of 6-amino substituted indoles (IV) and indolinones (V), is seldom explored. The target compounds (I)
should be finally obtained from intermediates III by the aminocarbonylation of the indole nitrogen with
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phosgene and a suitable amine to give compound II, followed by a metal-catalyzed cycloisomerization
Scheme 1).
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Scheme 1: Retrosynthetic approach.
We started our study trying to synthesize a properly substituted key intermediate IV, i.e., the N-protected
6
-dialkylaminoindole-2-triflate (4), (Scheme 2). 6-Aminoindolin-2-one 1 was prepared by the one-pot
reduction/lactamization of 2,4-dinitrophenylacetic acid.25 The following reductive amination with
acetaldehyde gave the corresponding 6-diethylaminoindolin-2-one 2 in 80 % yield. Surprisingly, this
approach broke down in the last step because every attempt to transform the N-protected 6-diethylamino
indolin-2-one 3 in the corresponding indole-2-triflate 4 was unsuccessful (see SI for details).
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Scheme 2: Tentative approach to intermediate 4.
This drawback prompted us to plan an alternative synthetic strategy. As iodine is a good leaving group
as triflate, we identify the N-protected 6-amino-2-iodoindoles 8 and 10 as suitable new key intermediates.
They were obtained in four steps starting from cheap and commercially available 6-nitroindole (Scheme
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).
Scheme 3. Alternative strategy to 2-iodinated key intermediates 8 and 10.
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The nitrogen group of 6-nitroindole was protected with a benzenesulfonyl group to give the 1-
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benzenesulfonyl-6-nitroindole 5 in excellent yield. The catalytic reduction of 5 in ethyl acetate led to
the 1-benzenesulfonyl-6-aminoindole 6 in 93 % yield, along with a small amount of the corresponding
1
-benzenesulfonyl-6-aminoindoline derived from the partial reduction of the C2-C3 indole double bond.
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The N,N-bis-alkylation of 6 by reductive amination was ruled-out because it was reported that using
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NaBH
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CN the reduction of indole C2-C3 double bond can occur. Therefore, the N,N-bis-alkylation of
6
was performed under basic condition with ethyl bromide at 50 °C,30 giving rise to the 1-
benzenesulfonyl-6-(N,N-diethylamino)-indole derivative 7 in 65 % yields. As mentioned above, to
modulate the properties of the EDG in position 6 of the indole, an alternative morpholine substituent was
introduced by the reaction of 6 with bis-(2-bromoethyl)ether in the presence of N,N-diisopropyl-
ethylamine to give the 1-benzenesulfonyl-6-(morpholin-1-yl)-indole derivative 9 in 65 % yield. Finally,
the N-protected 6-amino-2-iodoindoles 8 and 1031 were obtained by well-customized iodination
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reactions of compounds 7 and 9, respectively.
Starting from key intermediates 8 and 10, the library of target compounds 15 – nicknamed MediaChrom
–
was obtained in four steps, starting from 2-iodoindoles 8 and 10 (Scheme 4).
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Scheme 4: Synthesis of MediaChrom dyes 15a-f.
N-Protected 2-alkynyl-6-aminoindoles 12 a-f were obtained from fair to excellent yields (Table 1) by a
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typical Sonogashira cross-coupling reaction with four different alkynes (11 a-d) in the presence of
Pd(PPh , CuI, TEA in DMF. Alkynes 11 b-d are commercially available, while 1a was synthesized
)
3 4
from the corresponding aryl halide by a Pd-catalyzed cross-coupling with TMS-acetylene followed by
desilylation (see SI for details). The subsequent deprotection reactions of the indole nitrogen of
compounds 12 a-f to give the corresponding 6-amino-2-alkynylindoles 13 a-f were performed under
alkaline conditions, properly customized to overcome specific solubility (i.e., 12b,c,f) or hydrolysis (i.e.,
1
2d) problems of the substrates (Table 1).
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Table 1: Synthesis of compounds 12 a-f and 13 a-f.
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R1
Alkyne
R2
a
Yieldb
(%)
12  13 Time
c
Yieldb
(%)
Sub.
8/10  12
Time (h)
Method
(h)
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Et
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N-
11a
11b
11c
11d
11a
11c
-SO
2
-Me
2
5
6
4
16
24
12a (90)
12b (52)
12c (55)
12d (79)
12e (90)
12f (78)
A
B
C
D
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B
13a (96)
13b (57)
13c (87)
13d (52)
13e (97)
13f (91)
(0.016 mmol),
Et
Et
Et
N-
N-
N-
-NO
-CF
-CN
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morpholine
morpholine
-SO
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-Me
-CF
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a
3 4
Reaction conditions: 8 or 10 (0.4 mmol) in DMF (1.6 mL), 11 (0.48 mmol), TEA (8 mmol), Pd(PPh )
CuI (0.008 mmol), rt, N . Pure isolated products. Method A: 12a or 12e (0.4 mmol) in MeOH (8 mL), NaOH aq. (2 M,
2
2.5 mL), reflux. Method B: 12b or 12f (0.4 mmol) in MeOH (24 mL), NaOH aq. (6 M, 2.5 mL), reflux. Method C: 12c
0.4 mmol) in MeOH (16 mL), NaOH aq. (6 M, 0.8 mL), reflux. Method D: 12d (0.4 mmol) in dioxane (3 mL), t-BuONa
0.8 mmol), 80 °C, N
b
c
(
(
2
.
The insertion of the amido function at the nitrogen atom required for the final cyclization step  and the
simultaneous introduction of the suitable linker for a potential connection with a biomolecule  involved
the preparation of a proper spacer. Our idea was conceived to allow the easy insertion of different sized
linkers. This molecular architecture calls for a versatile use of MediaChrom dyes as fluorescent probe
dye as well as fluorescent labels for biomacromolecules (e.g., peptides, proteins or amino modified
oligonucleotides). At this stage, the four-carbon spacer derived from -aminobutyric acid (GABA) was
chosen as compromise model, with the aim to introduce in the molecule an unhindered linking point for
an amine containing biomolecule that was not too distant from the core of the dye.34 Thus, the p-
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toluenesulfonate salt of the benzyl 4-aminobutanoate was prepared by reaction of GABA with benzyl
alcohol in the presence of p-TSA. This was reacted with substrates 13 a-f in the presence of phosgene
and triethylamine, in dichloromethane at 0° C to give the compounds 14 a-f in fair to good yields (Table
2
).36
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Finally, the cyclization to MediaChrom 15 a-f was accomplished by a gold catalyzed reaction. The gold
catalyzed intramolecular addition of an amide group on an alkyne can show chemo- (O vs N) and regio-
(
6-endo vs 5-exo) selectivity problems, due to the bidentate nature of both the nucleophile (amide) and
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the electrophile (alkyne). In particular, it has been recently reported that the cycloisomerization of some
scaffolds closely related to 14, afforded either N-cyclization or O-cyclization products depending from
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the nature of the counterion of the metal catalyst. A brief screening of gold and silver based catalytic
systems (see SI for details) resulted in the selection of 1,3-bis(diisopropylphenyl)imidazol-2-ylidene
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3
4
5
6
7
8
9
0
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2
3
4
5
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7
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9
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gold(I) hexafluoroantimonate (IPrAuSbF , 5 mol%), as the catalyst of choice, able to give the
6
pyrimidoindolones 15 a-f in a chemo- and regio-specific fashion from fair to excellent yields (Table 2).
Table 2: Synthesis of compounds 14 a-f and 15 a-f.
a
b
c
c
Sub.
R1
R2
13  14
Yield (%)
14  15
Yield (%)
Time(h)
Time(h)
1
3a
13b
3c
3d
Et
2
2
2
2
N-
-SO
-NO
2
-Me
4
4
3
4
24
3
14a (70)
14b (70)
14c (53)
14d (46)
14e (20)
14f (76)
6
8
8
4.5
4
2
15a (53)
15b (45)
15c (78)
15d (78)
15e (90)
15f (99)
Et
Et
Et
N-
N-
N-
2
1
1
1
-CF
-CN
-Me
-CF
3
3e morpholine -SO
2
13f morpholine
3
a
Reaction conditions: 13 a-f (0.2 mmol) in CH
, 30 min, then p-toluenesulfonate salt of the benzyl 4-aminobutanoate (0.8 mmol) in CH
1 mL), TEA (0.4 mmol) was added, rt. Pure isolated products. Reaction conditions: 14 a-f (0.1
2
Cl
2
(2 mL), COCl
2
(0.4 mmol), TEA (0.8 mmol),
0
(
°C, N
2
2
Cl
2
b
c
6 2
mmol) in DCE (3.5 mL), IPrAuSbF (0.005 mmol), 80 °C, N .
MediaChrom dyes 15 were designed with the linker protected as benzyl ester, to be easily deprotected
so transforming the MediaChrom dyes 15 in the carboxy-free MediaChrom labels 15’. Since the
MediaChrom 15c displayed the most interesting photophysical proprieties (see below), it was chosen as
lead compound to be transformed in the carboxy-free derivative 15’c for biomolecular labelling. The
debenzylation of the carboxy-terminus was obtained by treatment of 15c under heterogeneous reductive
conditions (H
2
, Pd/C) in methanol at rt to give 15’c in 76 % yield (Scheme 5).
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Scheme 5: Deprotection of carboxy-terminus of MediaChrom 15c.
The structure of product 15’c was unequivocally confirmed by NOESY and HSQC experiments (see
supporting information for details) giving indirectly insights on the entire library of MediaChrom dyes
1
5.
Photophysical evaluation of MediaChrom dyes
The spectroscopic properties of MediaChrom dyes 15 were first characterized by collecting absorption
spectra (Table 3). The well-known polarity sensitive dye Prodan was chosen as basis for comparison.
Table 3: Onsager radius and absorption properties of MediaChrom dyes 15a-f compared to Prodan (20
g/mL, ethanol).
a
Onsager
radius (Å)
5.7162

absorption
(nm)
401

Dye
5a
-1
-1
(mM cm )
1
8.5
b
1
5b
15c
5d
5.5919
456
7.6
5.6943
393
13.8
1
5.6234
409
15.4
c
1
5e
5.6323
369
17.1
15f
Prodan
5.6100
4.3628
377
360
17.5
d
d
18.4d
a
b
as the wavelength of the most red-shifted peak. C = 0.5
c
d
mg/mL poor solubility. ref. 13.
All MediaChrom dyes showed intense absorption peaks, spanning from 369 nm to 456 nm. MediaChrom
1
5b, which is the dye with the most red-shifted absorption band, exhibits the lower absorption intensity.
In order to obtain acceptable quality spectra a 25-fold more concentrated solution was prepared. The
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presence of the stronger EWG (nitro group) is responsible of the marked red-shift, typically observed
when the conjugation of the chromophoric system is increased. The comparison of the spectra of
MediaChrom 15a and 15c, characterized by the presence of the same EDG (-NMe
2
) but different EWGs
(
-SO Me vs –CF ), indicates that the presence of a mesyl resulted in a 8 nm red-shifted absorption peak,
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with respect to trifluoromethyl group. A similar effect was observed by comparison of the absorption
peaks of MediaChrom 15e and 15f, where the same EDG (morpholine) was introduced. In these dyes,
the presence of the morpholine substituent causes a blue-shift of the absorption peaks. MediaChrom 15d
seemed to be the most promising fluorophore for imaging applications, since an interesting red-shifted
absorption peak is accompanied by a good absorption intensity (Table 3). In fact, the availability of a dye
that can be excited in the visible range with many cheap and accessible light excitation sources represent
a remarkable benefit.
Next, all dyes were characterized for their fluorescence solvatochromic properties by collecting
fluorescence spectra at 20 °C in solvents at different polarity, i.e., hexane, n-octanol, ethanol and DMF
(Table 4). All MediaChrom dyes exhibited fluorescence emissions in all solvent with a solvatochromic
shift spanning over 90 nm from hexane to DMF. The only exception is 15b, which showed significant
fluorescence only in hexane (Table 4). This high sensitivity is comparable to that observed for many
commercially available solvatochromic probes (e.g., Prodan, Laurdan, Dansyl).
Table 4: Solvatochromic properties of MediaChrom dyes 15a-f.

emission (nm)a
Solvent
Hexane
n-Octanol
Ethanol
15a
525
585
595
605
15b
15c
15d
525
585
595
615
15e
512
568
582
597
15f
482
514
528
553
594
490
525
540
565
-
-
-
DMF
a
excitation wavelength, see Table 3
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For each MediaChrom dye, the integration of the emission peak in different solvents was calculated and
normalized to the peak with the maximum intensity between them to evaluate the relative intensity of
fluorescence emission in solvents with different polarity (Figure 2).
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Figure 2: Normalized fluorescence emission peak of MediaChrom dyes at 1.6 g/mL concentration in
different solvent, at 20°C. 15a (black circles), 15b (red triangles-down), 15c (green squares), 15d
(
yellow diamonds), 15e (magenta hexagon) and 15f (blue triangles-up).
Figure 2 shows that only MediaChrom 15c and 15f preserve an almost constant fluorescence emission in
all tested solvents over a wide range of polarity, together with a simultaneous large change in the Stokes
shift. These properties make both these molecules good candidates for several purposes, ranging from
structural and dynamic studies on biological macromolecules to cell imaging applications Combining
absorption and emission properties, 15c is considered the best candidate, since it requires a red-shifted
excitation respect to 15f (see Table 3). In fact, in applications such as fluorescent labeling for user-
friendly detection kits or dye for cell imaging, the possibility to work at longer wavelength guarantees a
reduced biological tissues auto-florescence and scattering, and/or a decreased cell damage. For these
reasons, we focused on MediaChrom 15c, and its properties were compared to the properties of the
commercial solvatochromic probe Prodan. The polarity sensitive properties of 15c were investigated by
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collecting absorbance and fluorescence spectra in solvents at different polarities. Fluorescence quantum
13
yields were determined by taking Prodan in ethanol (quantum yield, QY = 71 %) as a reference. For
spectroscopic measurements, solutions of the dyes at different concentrations (from 2.5 µM to 5 µM)
were both excited at 380 nm, an average excitation wavelength between the max of absorbance of Prodan
and MediaChrom 15c. Results are reported in Table 5 and Figure 3.
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Table 5: Comparison of the absorbance and fluorescence properties of 15c and Prodan in four solvents
with different polarities.
-1
-1
a
b
c
Solvent
max (abs), nm
 (mM cm )
max (fluo), nm
Q.Y.
Prodan
Prodan
346
15c
396
397
393
396
Prodan
36.8
15c
14.6
14.0
13.8
13.7
Prodan
412
15c
480
522
540
562
15c
51.5
52.4
39.9
36.9
Hexane
60.4
59.9
71.0
64.2
n-Octanol
Ethanol
362
33.6
473
368
19.9
487
DMF
355
39.3
455
a
absorption spectra acquired at four different concentrations (15c from 139 µM to 13.9 µM, Prodan from 100 µM to 10
µM); b excitation wavelength: 380 nm, [dye] = from 2.5 µM to 5 µM; c quantum yield values were corrected for the
solvent refractive index.
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Figure 3: Absorption (A) and normalized fluorescence (B) spectra of 15c in different solvents at 20°C.
15c absorbance spectra in hexane (black line), n-octanol (red line), DMF (green line) and methanol
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(
yellow line). B): 15c normalized fluorescence spectra (excitation wavelength = 380 nm) in hexane (black
line), triethylamine (red line), n-octanol (green line), chloroform (grey line), n-butanol (light blue line),
ethanol (yellow line), methanol (brown line), acetone (magenta line) and DMF (blue line).
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The absorption spectra of 15c in different solvents did not show significant differences (Figure 3A). In
all investigated solvents, the most red-shifted absorption peak lies around 395 ± 2 mn. This property
guarantees that 15c can be excited at the same excitation wavelength (for example, with a common 405
nm diode laser), irrespective to the polarity of the solvent. Conversely, Prodan displays a pronounced
difference in the maximum of absorbance depending on the nature of the solvent (from 346 nm to 368
nm), together with an unfavorable blue-shifted absorption maximum (ranging from -25 nm in ethanol to
-50 nm in hexane, compared to 15c). The absorption coefficients () of 15c in the four solvents tested are
-1
-1
from 13.7 to 14.6 mM cm . In all solvents (except for ethanol) they are lower than those observed for
Prodan. The fluorescence spectra of MediaChrom 15c in solvents with different polarities show that the
dye exhibits a Stokes shift from 84 nm in hexane to 166 nm in DMF (Table 5, Figure 3-B). This large
solvatochromic shift (82 nm) suggests that the chromophore is able to detect even small polarity changes.
Moreover, also the emission maxima are strongly red shifted with respect of those of Prodan. Despite
lower than those of Prodan, the quantum yields of MediaChrom 15c in all examined solvents are good.
To describe quantitatively the effects of the physical properties of the solvent on the fluorescent emission
4
0
spectra of the dye, Lippert-Mataga equation was used (see eq. (2) in Experimental Section) This
correlation is based on the assumption that the solvent is a continuum in which the fluorophore is
contained, and solvent-specific interactions are not considered. It can be approximated that the energy
difference between the ground and the excited states is a property of the refractive index (n) and the
dielectric constant () of the solvents. In Figure 4 the orientational polarizability (f) is plotted against
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-
1
Stokes shift (in cm ) for MediaChrom 15c and compared to Prodan under the same experimental
conditions.
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Figure 4: Lippert plot for MediaChrom 15c (triangles-down) and Prodan (circles) in aprotic (black)
and protic (red) solvents.
For both fluorophores, a clear dependence of the Stokes shift on orientational polarizability of the solvent
was observed. It is worth noting that the Stokes shifts for MediaChrom 15c in protic solvents are weaker
than for aprotic solvents of similar polarity (Figure 4, red triangles vs black triangles) while Prodan
displays exactly the opposite trend (Figure 4, red circles vs black circles). These phenomena are usually
retraced to specific interactions of the fluorophore with the solvent. For example, in Prodan this
phenomenon was been related to H-bonding of the protic solvents with the carbonyl group of the
14
fluorophore. The reverse effect observed in MediaChrom 15c could be related to a particular interaction
with the solvent, probably due to the presence of the uncommon trifluoromethyl in the D--A system.
Since Lippert-Mataga equation usually well describes fluorophores behavior in aprotic solvents, only
data for both dyes in aprotic solvents were used to fit and determine the change in dipole moment upon
*
excitation ( -).
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MediaChrom 15c and Prodan Stokes shifts showed a well-defined dependence to orientational
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polarizability, considering all solvents. In particular, data obtained in aprotic solvents showed a good
*
correlation, with a ( -) of 13.3  0.6 and 8.1  0.4 Debye, respectively. For all other MediaChrom
dyes, which showed similar solvatochromic shifts, the dipole moment changes fall in the 13.0-13.4
Debye range (see table S1 in SI). This finding demonstrates that, regardless the substituent pattern, the
charge transfer process is similar. Moreover, the differences in the Stokes shift suggests an effect of
substituents on the non-emitting energy loss in the excited state.
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To estimate the photostability of MediaChrom 15c, we made a photodegradation test. Ethanol solutions
of 15c and Prodan were prepared in two different quartz cuvette and illuminated at 380 nm with a Xenon
lamp for 100 min, while the fluorescence signal was recorded as a function of time. The power density
2
applied to the sample was 6.7 mW/cm . As depicted in Figure 5, MediaChrom 15c decays significantly
slower than Prodan showing a halved emission after 2800 seconds, while the latter after 1750 seconds.
Figure 5: Photodegradation test for MediaChrom 15c (red line) and Prodan (black line) in ethanol (c =
5
µM, excitation wavelength = 380 nm).
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Based on these results, the comparison between MediaChrom 15c and Prodan properties suggests that,
in spite of exhibiting a slightly reduced quantum yield, the former possesses photochemical properties
comparable with the commercial dye Prodan with the advantage of a strong, red-shifted absorption and
fluorescence emission and an increased photostability.
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Behavior of MediaChrom 15c in dipalmitoylphosphatidylcholine (DPPC) vesicles
Prodan dye is widely used for probing membrane state, including transition phases and oxidation
processes of phospholipid systems, due to its capability of penetrating in the lipophilic layer and sensing
41
polarity changes. In order to evaluate the potential of MediaChrom 15c as a membrane probe, we
collected fluorescence spectra of both Prodan and MediaChrom 15c after their addition to a DPPC
vesicles suspension at different temperatures. It has been already observed that an increase in temperature
cause a change in the fluorescence spectrum of Prodan. This is due to the transition of the phospholipid
system from the gel to the liquid phase that allows more water molecules to enter into the lipid phase.42
The fluorescence spectra of MediaChrom 15c embedded in DPPC vesicles showed an emission peak
centered at 528 nm (Figure 6), very similar to that observed when it is dissolved in n-octanol (525 nm)
at the same temperature (20 °C). This result suggests that the fluorophore lies in the lipid phase of DPPC
vesicles, probing this region. The comparison of the behavior of MediaChrom 15c and Prodan clearly
demonstrates that the former is able to sense transition phase of phospholipid systems similarly to Prodan,
with the advantage of an emission wavelength red-shifted by 100 nm, significantly reducing scattering
effects that are considerably high in micellar suspension as well as when cellular membranes are present.
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Figure 6: Fluorescence spectra of 1.6 g/mL MediaChrom 15c (A) and Prodan (B) in DPPC vesicles at
different temperatures: 20°C (black lines) and 37°C (red lines). Excitation wavelength = 380 nm.
MediaChrom 15’ as peptide labels
As described above, MediaChrom dyes 15 were designed with a benzyl ester, to be easily deprotected
4
3
transforming the MediaChrom dyes 15 in the carboxy-free MediaChrom labels 15’. This transformation
makes these dyes suitable for an easy conjugation with proteins, peptides or an amino-modified
oligonucleotide. These labeled bio-conjugates could be thus used for the detection of interaction
involving a change of polarity of the system, such as the protein/DNA binding.
As a proof of concept, we selected the protein Cro, whose interaction with its consensus DNA sequence
4
4
(O
R
3) is known and widely investigated. In particular, Cro is a 66 amino acids dimeric protein, which
plays a key role in the switch from lysogenic to lytic cycle in bacteriophage . Its interaction with DNA
4
5
is mainly restricted to a small helix-turn-helix motif spanning from residue 15 to 38, with a Kd of
4
6
around 25 M. The wild-type DNA binding sequence (GQTKTAKDLGVYQSAINKAIHAG) was
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prepared by microwave-assisted solid phase synthesis and on resin labelled at the N-terminus with the
MediaChrom 15’c using standard protocols.48
The fluorescence spectra of 15’c labeled Cro:1 in the presence and in the absence of O 3 consensus DNA
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sequence were recorded (Figure 7).
Figure 7: Normalized fluorescence spectra of peptide in the presence (red line) and in the absence (black
line) of O 3 consensus sequence (15’c-Cro:1 50 M, O 3 50 M, PBS buffer, pH 7.4, 20°C). Excitation
wavelength = 380 nm.
R
R
In the absence of a cognate DNA sequence, the labeled peptide 15’c-Cro:1 displayed a maximum of
fluorescence at 545 nm (Figure 7, black line). Upon addition of the O 3 consensus sequence, a strong
R
hypsochromic shift (93 nm) was observed (Figure 7, red line) thus indicating that the MediaChrom 15’c
sensitively detected the change in the polarity of the environment when the peptide interacts with the
DNA sequence. The minor peak present in the 15’c-Cro:1 spectrum and the shoulder in the peak of the
1
5’c-Cro:1-O 3 complex suggests that there is in an equilibrium of two discrete peptide conformations,
R
clearly signaled by the dye. The binding of O
equilibrium from one state to the other.
R
3 consensus sequence shifts the peptide conformational
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The Journal of Organic Chemistry
1
2
CONCLUSIONS
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We synthesized a six-membered library of polarity-sensitive fluorescent dyes called MediaChrom,
characterized by a pyrimidoindolone skeleton endowed with a conjugated push-pull system. The modular
synthesis involves eight steps starting from simple and commercially available materials, with overall
yields up to 19%. An add value of MediaChrom dyes is the presence of a linker (whose length can be
modulated) with a protected carboxy terminus that allows the handy conjugation with biomolecules,
making possible the transformation of the dyes in useful fluorescent solvatochromic labels. All
MediaChrom dyes display interesting photophysical profile. Among them, MediaChrom 15c shows the
best features for biological applications: a high absorption coefficient in the visible range almost constant
in solvents with different polarities, a wide solvatochromic effect combined with an almost constant
fluorescence emission, a good QY and a noteworthy photostability.49 In general, MediaChrom 15c
displays some advantage compared to well-known solvatochromic fluorophore Prodan. The
photophysical features of 15c made it competitive also with the more recently developed small-size
0
1
2
3
4
5
6
7
8
9
0
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2
3
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9
0
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0
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9
0
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9
0
1
4
polarity-sensitive organic dyes, such as the structural analogues of Prodan based on anthracene and
fluorene15 cores. For these reasons, MediaChrom 15c was take as lead compound to test some
conceivable applications. MediaChrom 15c demonstrated to be a versatile solvatochromic dye that can
be used as a membrane/lipophilic probe as well as its parent carboxy-free MediaChrom 15’c
demonstrated to be a highly sensitive label for peptide tagging, useful for study the interaction between
peptides (or proteins) and other target biomolecules, such as DNA. Current efforts in our laboratories are
now devoted to investigate some other biological applications in vivo of MediaChrom dyes, e.g., as
prokaryote/eukaryote cell stains and probes.
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1
2
3
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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5
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5
5
6
EXPERIMENTAL SECTION
General. Anhydrous solvents are commercially available and stored in a protected atmosphere of
nitrogen. All the reactions that involve the use of reagents sensitive to oxygen or hydrolysis, were carried
out under nitrogen. The glassware was previously dried in an oven at 110 °C and set with cycles of
vacuum and nitrogen. The chromatographic column separations were performed by a flash technique,
using silica gel (pore size 60 Å, particle size 230−400 mesh). TLC Alu foils with fluorescent indicator
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(254 nm) were used for TLC analysis, and the detection was performed by irradiation with UV light (λ
1
= 254 nm and/or 366 nm). H NMR analyses were performed with 200 MHz or 300 MHz spectrometers
1
13
at rt. Spectra were referenced to residual chloroform (7.27 ppm, H, 77.0 ppm, C). The coupling
1
3
constants (J) are expressed in Hertz (Hz), the chemical shifts (δ) in ppm. C NMR analysis were
performed with the same instruments at 50.3 and 75.45 MHz. Attached Proton Test (APT) sequence was
used to distinguish the methine and methyl carbon signals from those arising from methylene and
1
3
1
quaternary carbon atoms. All C NMR spectra were recorded with complete proton decoupling. The H
NMR signals of MediaChrom 15’c described in the following have been attributed by correlation
spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) techniques. Infrared spectra
were recorded using discs of NaCl for liquid samples and KBr tablets for solid samples. The absorbance
-1
-1
is reported in wavenumbers (cm ) with values between 4000 and 400 cm . Low-resolution MS spectra
were recorded with electron impact source and electrospray/ion trap equipped instrument, using a syringe
pump device to directly inject sample solutions. The values are reported as mass-charge ratio and the
relative intensities of the most significant peaks are shown in brackets. The melting points of the solid
products are uncorrected. UV-visible and fluorescence spectra were collected at 20 °C. 6-Aminoindolin-
2-one (1) was prepared according to the literature.25
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Synthesis of 6-(diethylamino)indolin-2-one (2).
3
4
5
6
7
8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
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9
0
NaBH
3
CN (190 mg, 3.02 mmol) was added to a solution of 6-aminoindolin-2-one (1) (180 mg, 1.21
mmol) in glacial AcOH (2 mL) followed by acetaldehyde (0.48 mL, 373 mg, 8.47 mmol), and the mixture
was stirred at rt for 24 h. The suspension was concentrated in vacuo, poured into H O (25 mL) and
extracted with EtOAc (3 × 20 mL). The organic layers were collected, dried over Na SO , filtered and
the solvent was removed at reduced pressure. The crude material was purified by flash chromatography
over a silica gel column using CH Cl /MeOH (99:1) to afford the desired product 2 as yellow solid (198
mg, 80%). Mp 106–108 °C. R
2
2
4
2
2
f
= 0.13 (silica gel, EtOAc/AcOH 3%), 0.31 (silica gel, CH
2
Cl
2
/MeOH
-1
1
98:2). IR (KBr): νmax = 3183, 2966, 2934, 1701, 1632, 1514, 1353, 1123, 1111, 770 cm . H NMR
(DMSO-d
6
, 200 MHz): δ = 1.04 (t, 6H, CH
3
, J = 7.0 Hz), 3.20–3.30 (m, 6H, CH
2
), 6.12 (d, 1H, CH, J =
1
3
2
.3 Hz), 6.19 (dd, 1H, CH, J = 8.2, 2.3 Hz), 6.92 (d, 1H, CH, J = 8.2 Hz), 10.09 (s, 1H, NH). C NMR
(DMSO-d
6
, 50.3 MHz): δ = 177.8 (C = O), 148.1 (C
), 35.7 (CH ), 13.1 (CH
O: C, 70.56; H, 7.90; N, 13.71; found: C, 70.67; H, 7.98; N, 13.54.
Beside, a modest amount of the corresponding mono-ethylated derivative 2’ (6-(ethylamino)indolin-2-
one) was obtained: yellow/orange solid (21 mg, 10%). Mp 165–169 °C (with decomposition). R = 0.40
(silica gel, EtOAc/AcOH 3%), 0.19 (silica gel, CH Cl /MeOH 98:2). IR (KBr): νmax = 3371, 3193, 2965,
917, 1693, 1628, 1519, 1335, 1193, 1172, 1110, 801 cm . H NMR (DMSO-d
H, CH , J = 7.0 Hz), 2.95 (dq, 2H, CH , J = 7.0, 5.1 Hz), 3.24 (s, 2H, CH ), 5.46 (bt, 1H, NH, J = 5.1
q
), 145.5 (C
q
), 125.5 (CH), 112.3 (C ), 105.2 (CH),
q
+
9
4.4 (CH), 44.6 (CH
2
2
3
). ESI-MS m/z (%): 205 [M + 1] (100). Calcd for
C
12
H
16
N
2
f
2
2
-1
1
2
3
6
, 200 MHz): δ = 1.12 (t,
3
2
2
1
3
Hz), 6.06–6.10 (m, 2H, CH), 6.84 (d, 1H, CH, J = 8.1 Hz), 10.09 (s, 1H, NH). C NMR (DMSO-d
6
, 50.3
MHz): δ = 177.9 (C = O), 149.6 (C ), 145.1 (C ), 125.2 (CH), 112.6 (C ), 105.2 (CH), 94.8 (CH), 38.2
q
q
q
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
(
CH
2
), 35.8 (CH
2
), 15.0 (CH
3
). ESI-MS m/z (%): 177 [M + 1] (100). Calcd for C10
H
12
N O: C, 68.16;
2
H, 6.86; N, 15.90; found: C, 67.96; H, 6.72; N, 15.73.
Synthesis of ethyl 6-(diethylamino)-2-oxoindoline-1-carboxylate (3).
0
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2
3
4
5
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8
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9
0
To a solution of 6-(diethylamino)indolin-2-one (2) (250 mg, 1.22 mmol) and TEA (0.39 mL, 285 mg,
.81 mmol) in THF (6 mL) was added ethyl chlorocarbonate (0.26 mL, 292 mg, 2.69 mmol) dropwise.
The temperature was kept below 30 °C during the addition. After stirring for 4.0 h at rt, the solvent was
evaporated. Water (5 mL) was added to the residue and the mixture was extracted with CH Cl (3 × 10
mL). The organic layers were collected, dried over Na SO , filtered and the solvent was removed at
2
2
2
2
4
reduced pressure. The crude material was purified by flash chromatography over a silica gel column
using Hex/EtOAc (90:10) to afford the intermediate N,O-diacylated product 2’’ (ethyl 6-(diethylamino)-
2
-((ethoxycarbonyl)oxy)-1H-indole-1-carboxylate) as yellow wax (404 mg, 95%), which darkened
rapidly on exposure to air and light. Mp 50–51 °C. R = 0.49 (silica gel, EtOAc/AcOH 3%), 0.51 (silica
gel, Hex/EtOAc 80:20). IR (KBr): νmax = 2976, 2934, 1779, 1739, 1615, 1502, 1378, 1326, 1275, 1239,
f
-1 1
1
133, 1102, 1028 cm . H NMR (CDCl
3
, 200 MHz): δ = 1.18 (t, 6H, CH
3
, J = 7.0 Hz), 1.36–1.47 (m,
6H, CH ), 3.39 (q, 4H, CH , J = 7.0 Hz), 4.35 (q, 2H, CH
3
2
2
, J = 7.0 Hz), 4.44 (q, 2H, CH
2
, J = 7.0 Hz),
6
2
1
.16 (s, 1H, CH), 6.71 (dd, 1H, CH, J = 8.7, 2.2 Hz), 7.29 (d, 1H, CH, J = 8.7 Hz), 7.51 (d, 1H, CH, J =
1
3
.1 Hz). C NMR (CDCl
3
, 50.3 MHz): δ = 153.1 (C
q
), 150.8 (C
q
), 146.5 (C
q
), 139.4 (C
q
), 135.0 (C
), 14.4
: C, 62.05;
q
),
21.2 (CH), 116.7 (C
), 14.3 (CH
H, 6.94; N, 8.04; found: C, 62.35; H, 7.12; N, 7.82.
q
), 110.8 (CH), 99.8 (CH), 97.2 (CH), 65.7 (CH
2
), 63.2 (CH
2
), 45.4 (CH
2
+
(CH
3
3
), 12.7 (CH
3
). ESI-MS m/z (%): 349 [M + 1] (100). Calcd for C18
H
24
N
2
O
5
To a solution of N,O-diacylated product 2’’ (375 mg, 1.08 mmol) in DMF (3 mL) was added finely
powdered ammonium carbonate (103 mg, 1.08 mmol) at 0-5 °C. The mixture was stirred for 5.0 h at rt
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The Journal of Organic Chemistry
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then poured into ice-water (20 mL) and extracted with EtOAc (3 × 20 mL). Combined organic phases
were washed with water (100 mL), dried over anhydrous Na SO and concentrated under reduced
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4
pressure. The crude product was purified by flash chromatography over a silica gel column using
Hex/EtOAc (90:10) to afford the desired product 3 as yellow solid (212 mg, 71%), which darkened
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
rapidly on exposure to air and light. Mp 58–60 °C. R
max = 2972, 2905, 1760, 1732, 1624, 1513, 1369, 1305, 1266, 1055, 767 cm . H NMR (CDCl
MHz): δ = 1.17 (t, 6H, CH , J = 7.0 Hz), 1.44 (t, 3H, CH , J = 7.0 Hz), 3.37 (q, 4H, CH , J = 7.0 Hz),
.57 (s, 2H, CH ), 4.46 (q, 2H, CH , J = 7.0 Hz), 6.45 (dd, 1H, CH, J = 8.0, 2.2 Hz), 7.04 (d, 1H, CH, J
= 8.0 Hz), 7.32 (d, 1H, CH, J = 2.2 Hz). C NMR (CDCl
f
= 0.42 (silica gel, Hex/EtOAc 80:20). IR (KBr):
-1 1
ν
3
, 200
3
3
2
3
2
2
1
3
3
, 50.3 MHz): δ = 174.4 (C
q
), 151.4 (C
), 45.0 (CH ), 36.1 (CH
: C, 65.20; H, 7.30;
q
), 148.3
(
C
q
), 142.2 (C
q
), 124.8 (CH), 109.6 (C
q
), 108.1 (CH), 99.8 (CH), 63.3 (CH
2
2
2
),
+
1
4.5 (CH ), 12.7 (CH
3
3
). ESI-MS m/z (%): 277 [M + 1] (100). Calcd for C15
H
20
N
2
O
3
N, 10.14; found: C, 65.09; H, 7.24; N, 10.24.
Synthesis of 6-nitro-1-(phenylsulfonyl)-1H-indole (5). PhSO
2
Cl (4.7 mL, 6.53 g, 37.00 mmol) was
CO (6.39 g, 46.25
mmol) in acetone (66 mL). The reaction mixture was stirred for at rt for 24 h. The residue was poured
into H O (200 mL) and extracted with EtOAc (3 × 150 mL), washed with brine and dried over Na SO .
added dropwise at 0 °C to a mixture of 6-nitroindole (3.00 g, 18.50 mmol) and K
2
3
2
2
4
The solvent was removed at reduced pressure and then the crude product was purified by flash
chromatography over a silica gel column using Hex/EtOAc (from 90:10 to 1:1) to afford the desired
product 5 as pale yellow solid (5.37 g, 96%). Mp 199–201 °C (dec.). R
80:20). IR (KBr): νmax = 3117, 3107, 1593, 1508, 1371, 1342, 1144, 727 cm . H NMR (CDCl
MHz): δ = 6.77 (d, 1H, C indole-H, J = 3.7 Hz), 7.46–7.58 (m, 3H, CH), 7.63 (d, 1H, CH, J = 8.8 Hz),
indole-H, J = 3.7 Hz), 7.94 (d, 2H, CH, J = 7.0 Hz), 8.14 (dd, 1H, CH, J = 8.8, 1.9 Hz),
f
= 0.24 (silica gel, Hex/EtOAc
-1 1
3
, 200
3
7
8
.84 (d, 1H, C
2
13
.91 (d, 1H, CH, J = 1.8 Hz). C NMR (CDCl
3
, 50.3 MHz): δ = 145.4 (C
q
), 137.9 (C
q
), 135.7 (C ), 134.8
q
(CH), 133.8 (C
q
), 131.4 (CH), 129.9 (CH), 127.2 (CH), 121.8 (CH), 118.9 (CH), 110.1 (CH), 109.0 (CH).
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
ESI-MS m/z (%): 325 [M + Na] (100), 303 [M + 1] (15). Calcd for C14
H
10
N
2
O S: C, 55.62; H, 3.33;
4
N, 9.27; found: C, 55.39; H, 3.36; N, 8.95.
Synthesis of 6-amino-1-(phenylsulfonyl)-1H-indole (6).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To a solution of 6-nitro-1-(phenylsulfonyl)-1H-indole (5) (2.50 g, 8.27 mmol) in EtOAc (120 mL), Pd/C
0% (10% weight, 250 mg) was added. The mixture was charged with hydrogen and stirred at rt for 7.0
1
h. The crude was filtered over celite and washed with EtOAc. The solution was concentrated under
reduced pressure. The crude product was purified by flash chromatography over a silica gel column using
Hex/CH
Cl
2 2
/TEA (60:40:10) to afford the desired product (6) as pale orange wax (2.09 g, 93%)
(
containing inseparable traces of a by-product derived from the reduction of the C2-C3 indole double
bond). R
f
= 0.24 (silica gel, Hex/EtOAc 70:30). IR (NaCl): νmax = 3384, 2927, 1622, 1359, 1172, 1120,
-1
1
7
27 cm . H NMR (CDCl
3
, 200 MHz): δ = 3.68 (bs, 2H, NH
2
), 6.52 (d, 1H, C indole-H, J = 3.7 Hz),
3
1
3
6.62 (dd, 1H, CH, J = 8.4, 1.8 Hz), 7.24–7.56 (m, 6H, CH), 7.86 (d, 2H, CH, J = 7.0 Hz). C NMR
(CDCl
3
, 50.3 MHz): δ = 144.7 (C
q
), 138.7 (C
q
), 136.7 (C
q
), 133.8 (CH), 129.4 (CH), 126.9 (CH), 124.2
+
(CH), 123.3 (C
q
), 122.1 (CH), 113.2 (CH), 109.6 (CH), 99.6 (CH). ESI-MS m/z (%): 273 [M + 1] (100),
+
295 [M + Na] (50). Elem. Anal. was not performed because the product contains traces of the indoline
derivative.
Synthesis of 6-(N,N-diethylamino)-1-(phenylsulfonyl)-1H-indole (7). Under a nitrogen atmosphere,
to a solution of 6-amino-1-(phenylsulfonyl)-1H-indole (6) (2.00 g, 7.34 mmol) in dry DMSO (30 mL),
KOH 85% (969 mg, 14.68 mmol) was added. The solution was stirred under rt, then ethyl bromide (2.2
mL, 3.20 g, 29.36 mmol) was added and the reaction mixture was stirred at 50 °C. After 6.0 h other 2
equivalents of ethyl bromide were added and the reaction mixture was stirred overnight at 50 °C until no
more starting product was detectable by TLC analysis. Upon finished the reaction mixture was quenched
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2
by H
2
O (400 mL) and extracted with EtOAc (3 × 300 mL). Combined organic phases were dried over
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
anhydrous Na
2
SO and concentrated under reduced pressure. The crude product was purified by flash
4
chromatography over a silica gel column using Hex/EtOAc (95:5) to afford the desired product 7 as
yellow solid (1.57 g, 65%) beside traces of the corresponding monoethylated derivative (not isolated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
neither characterized). Mp 78–80 °C. R
f
= 0.46 (silica gel, Hex/EtOAc 80:20). IR (KBr): νmax = 3413,
-1 1
2971, 2930, 1622, 1501, 1359, 1173, 1119, 728 cm . H NMR (CDCl
3
, 200 MHz): δ = 1.18 (t, 6H, CH
3
,
J = 7.0 Hz), 3.40 (q, 4H, CH
2
, J = 7.0 Hz), 6.50 (d, 1H, C
3
indole-H, J = 3.7 Hz), 6.67 (dd, 1H, CH, J =
13
8
.8, 2.2 Hz), 7.27–7.52 (m, 6H, CH), 7.85 (d, 2H, CH, J = 7.0 Hz). C NMR (CDCl
3
, 50.3 MHz): δ =
146.5 (C ), 138.8 (C ), 137.4 (C ), 133.7 (CH), 129.3 (CH), 127.0 (CH), 123.6 (CH), 121.8 (CH), 121.0
q
q
q
+
(C
q
), 110.8 (CH), 109.4 (CH), 96.8 (CH), 45.3 (CH
2
), 12.7 (CH ). ESI-MS m/z (%): 329 [M + 1] (100),
3
+
3
8
51 [M + Na] (20). Calcd for C18
H
20
N
2
2
O S: C, 65.83; H, 6.14; N, 8.53; found: C, 65.55; H, 6.03; N,
.53.
Synthesis of 6-(N,N-diethylamino)-2-iodo-1-(phenylsulfonyl)-1H-indole (8). The LDA solution was
freshly prepared as follows: under a nitrogen atmosphere, a solution of diisopropylamine (0.41 mL, 294
mg, 2.90 mmol) in dry THF (3.8 mL) was cooled to -78 °C and a solution of n-butyllithium (1.6 M in
hexanes, 1.6 mL, 2.56 mmol) was added. The solution was stirred at -78 °C for 10 min, warmed to 0 °C
and stirred for 10 min, then cooled back to -78 °C. Under a nitrogen atmosphere, to a solution of 6-(N,N-
diethylamino)-1-(phenylsulfonyl)-1H-indole (7) (700 mg, 2.13 mmol) and TMEDA (0.38 mL, 297 mg,
2
.56 mmol) in dry THF (11 mL), the LDA solution freshly prepared was added via syringe over 10 min
at -78 °C. After being stirred for 1.5 h at -78 °C, the yellow solution was treated dropwise over 30 min
with a solution of iodine (541 mg, 2.13 mmol) in dry THF (3.8 mL) and the mixture was allowed to warm
slowly to rt overnight. The reaction mixture was cooled to 0-5 °C, treated with 5% aqueous sodium
thiosulfate (80 mL) and extracted with EtOAc (3 × 70 mL). Combined organic phases were washed again
with sodium thiosulfate (200 mL), dried over anhydrous Na
2
SO and concentrated under reduced
4
pressure. The crude product was purified by flash chromatography over a silica gel column using
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2
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8
9
1
1
1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
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5
6
Hex/EtOAc (98:2) to afford the desired product (8) as pale brown wax (774 mg, 80%). R = 0.21 (silica
f
-1
1
gel, Hex/EtOAc 95:5). IR (NaCl): νmax = 2970, 2928, 1615, 1503, 1373, 1197, 1173, 730 cm . H NMR
CDCl , 200 MHz): δ = 1.21 (t, 6H, CH , J = 7.0 Hz), 3.43 (q, 4H, CH , J = 7.0 Hz), 6.64 (dd, 1H, CH,
J = 8.8, 2.2 Hz), 6.82 (s, 1H, C indole-H), 7.18 (d, 1H, CH, J = 8.8 Hz), 7.36–7.54 (m, 3H, CH), 7.58 (d,
H, J = 2.2 Hz), 7.86 (d, 2H, CH, J = 7.0 Hz). C NMR (CDCl ),
(
3
3
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
3
, 50.3 MHz): δ = 175.5 (C
q
), 146.7 (C
), 120.2 (CH), 110.9
S:
q
141.0 (C
q
), 138.8 (C
q
), 133.9 (CH), 129.2 (CH), 127.3 (CH), 125.0 (CH), 122.4 (C
q
+
(CH), 99.1 (CH), 45.3 (CH
2
), 12.8 (CH
3
). ESI-MS m/z (%): 455 [M + 1] (100). Calcd for C18
H
19IN
2
O
2
C, 47.59; H, 4.22; N, 6.17; found: C, 47.93; H, 4.41; N, 6.30.
Synthesis of 6-morpholino-1-(phenylsulfonyl)-1H-indole (9). Under a nitrogen atmosphere, to a
solution of 1-(phenylsulfonyl)-1H-indol-6-amine (6) (800 mg, 2.94 mmol) in dry DMF (12 mL), N,N-
diisopropyl-ethylamine (1.02 mL, 759 mg, 5.88 mmol) and bis-(2-bromoethyl)ether (0.55 mL, 1.02 g,
4
.41 mmol) were added. The reaction mixture was stirred overnight at 90 °C until no more starting
product was detectable by TLC analysis. The residue was poured into sat. aqueous NaHCO (200 mL)
and extracted with EtOAc (3 × 150 mL), washed with brine and dried over Na SO . The solvent was
3
2
4
removed at reduced pressure and then the crude product was purified by flash chromatography over a
silica gel column using Hex/EtOAc (80:20) to afford the desired product (9) as pale yellow solid (650
mg, 65%). Mp 161–163 °C. R
853, 1615, 1488, 1448, 1367, 1176, 1128, 724 cm . H NMR (CDCl
J = 4.8 Hz), 3.91 (t, 4H, CH , J = 4.8 Hz), 6.56 (dd, 1H, C indole-H, J = 3.7, 0.7 Hz), 6.92 (dd, 1H, CH,
J = 8.8, 2.2 Hz), 7.37–7.55 (m, 6H, CH), 7.82–7.88 (m, 2H, CH). C NMR (CDCl
f
= 0.42 (silica gel, Hex/EtOAc 60:40). IR (KBr): νmax = 3436, 3140, 2967,
-1
1
2
3
, 200 MHz): δ = 3.20 (t, 4H, CH ,
2
2
3
1
3
3
, 50.3 MHz): δ =
1
49.7 (C
q
), 138.6 (C
q
), 136.5 (C
q
), 133.9 (CH), 129.4 (CH), 126.9 (CH), 125.2 (CH), 124.5 (C
q
), 121.9
(CH), 114.3 (CH), 109.4 (CH), 100.8 (CH), 67.1 (CH
2
), 50.6 (CH
2
). ESI-MS m/z (%): 343 [M + 1]+
(100). Calcd for C18
H
18
N
2
3
O S: C, 63.14; H, 5.30; N, 8.18; found: C, 62.88; H, 5.19; N, 8.00.
Synthesis of 2-Iodo-6-morpholino-1-(phenylsulfonyl)-1H-indole (10). The iodination reaction at the
position 2 was performed under the previously optimized reaction conditions (see the synthesis of
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1
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compound 8). The reagent 9 and the product 10 are not separable through a standard chromatographic
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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2
2
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2
2
2
2
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5
5
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5
6
1
column (R
f
= 0.13, silica gel, Hex/EtOAc 80:20). The yield (58%) was calculated via H-NMR (t
1
= 10s)
on the mixture of the two compounds obtained after a brief purification by flash chromatography over a
silica gel column using Hex/EtOAc (80:20). Since the 2-iodinated indoles are quite unstable, the next
Sonogashira coupling step was performed starting from this mixture, therefore the iodurate compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(10) was not fully characterized.
Synthesis of 1-Ethynyl-4-(methylsulfonyl)benzene 11a. 50
Under a nitrogen atmosphere, to a solution of 1-bromo-4-(methylsulfonyl)benzene (500 mg, 2.13 mmol)
in TEA (8.5 mL), ethynyltrimethylsilane (0.35 mL, 251 mg, 2.56 mmol) and trans-
dichlorobis(triphenylphosphine)palladium(II) (30 mg, 0.04 mmol) were added. The reaction was stirred
at rt for 15 min, and then CuI (4 mg, 0.023 mmol) was added. The reaction mixture was stirred at 50 °C
for 3.0 h until no more starting product was detectable by TLC analysis. The solvent was then evaporated
under reduced pressure and the crude material was purified by flash chromatography over a silica gel
column
(methylsulfonyl)phenyl)ethynyl)silane as pale yellow solid (489 mg, 91%). Mp 103–105 °C. R
silica gel, Hex/EtOAc 80:20). IR (KBr): νmax = 2959, 2160, 1591, 1309, 1145, 866, 837, 534 cm . H
NMR (CDCl , 200 MHz): δ = 0.26 (s, 9H, CH ), 3.04 (s, 3H, CH ), 7.62 (d, 2H, CH, J = 8.4 Hz), 7.87
, 50.3 MHz): δ = 140.1 (C
), 0.0 (CH
using
Hex/EtOAc
(85:15)
to
afford
the
desired
trimethyl((4-
f
= 0.30
-1
1
(
3
3
3
13
(d, 2H, CH, J = 8.4 Hz). C NMR (CDCl
3
q
), 132.9 (CH), 129.2 (C ), 127.5
q
+
(CH), 103.1 (Csp), 99.4 (Csp), 44.7 (CH
3
3
). ESI-MS m/z (%): 275 [M + Na] (100), 253 [M +
+
1] (10). Calcd for C12
H
16
2
O SSi: C, 57.10; H, 6.39; found: C, 57.33; H, 6.42.
To a stirred solution of trimethyl((4-(methylsulfonyl)phenyl)ethynyl)silane (400 mg, 1.58 mmol) in
MeOH (11 mL), K
2
CO
3
(438 mg, 3.16 mmol) was added. The reaction was stirred at rt for 3.0 h. The
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