Efficient one-step synthesis of 4-amino substituted phthalimides and evaluation of their potential as fluorescent probes

Article abstract of DOI:10.1039/c4ob00342j

The phthalimide scaffold is recognized from bioactive compounds and marketed drugs, but can also be used as fluorescent probes by introducing a 4-amino substituent. Unfortunately, a general and convenient method to synthesize various 4-amino substituted phthalimides has been lacking. To overcome this, an atom efficient one-step synthesis of 4-amino substituted phthalimides in good to excellent yields that tolerate a wide range of substituents has been developed. Several of the generated compounds display interesting solvatochromic properties with high quantum yield of fluorescence in non-polar solvents that are significantly reduced in polar protic solvents. Many of these compounds displayed non-toxic properties and non-detectable unspecific binding and can thus potentially be linked to a substrate and used as fluorescent probes. Furthermore, bioactive and fluorescent 4-amino substituted phthalimides with IC₅₀-values in the low micromolar range in cell-based assays have been identified and could be used to study uptake and distribution. The developed convenient synthetic method is thus valuable not only to construct fluorescent probes and fluorescent bioactive compounds to gain information about target binding, but also from a structure activity point of view in the various areas where the phthalimides have displayed activity. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00342j

Organic &
Biomolecular Chemistry
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Efficient one-step synthesis of 4-amino
substituted phthalimides and evaluation of their
potential as fluorescent probes†
Cite this: Org. Biomol. Chem., 2014,
12, 4461
Tomas Kindahl‡ and Erik Chorell*‡
The phthalimide scaffold is recognized from bioactive compounds and marketed drugs, but can also be
used as fluorescent probes by introducing a 4-amino substituent. Unfortunately, a general and convenient
method to synthesize various 4-amino substituted phthalimides has been lacking. To overcome this, an
atom efficient one-step synthesis of 4-amino substituted phthalimides in good to excellent yields that tole-
rate a wide range of substituents has been developed. Several of the generated compounds display
interesting solvatochromic properties with high quantum yield of fluorescence in non-polar solvents that
are significantly reduced in polar protic solvents. Many of these compounds displayed non-toxic pro-
perties and non-detectable unspecific binding and can thus potentially be linked to a substrate and used
as fluorescent probes. Furthermore, bioactive and fluorescent 4-amino substituted phthalimides with
IC50-values in the low micromolar range in cell-based assays have been identified and could be used to
study uptake and distribution. The developed convenient synthetic method is thus valuable not only to
construct fluorescent probes and fluorescent bioactive compounds to gain information about target
binding, but also from a structure activity point of view in the various areas where the phthalimides have
displayed activity.
Received 14th February 2014,
Accepted 29th April 2014
DOI: 10.1039/c4ob00342j
www.rsc.org/obc
investigated. This might be an effect of the lack of synthetic
methods to introduce amino substituents into the 4-position
Introduction
The phthalimide core structure can be recognized from mar- on the phthalimide central fragment. Up to this point, the
keted drugs and a broad spectrum of lead compounds with introduction of different 4-amino substituents has been based
different biological targets.1–3 In addition to this, the phthal- on a primary 4-amino substituent, often generated from a
imide scaffold has also been successfully used as a key com- 4-nitro substituent that has been transformed into dimethyl- or
ponent of materials and polymers,4–7 catalysts,8–10 and diethylamine substituents (Fig. 1).17,18 A few reports nicely
fluorescent probes, the latter by introducing
a 4-amino
substituent.11–16 In light of these various reported applications
of the phthalimide core structure, the possibility of introdu-
cing fluorescence by a 4-amino substituent is highly valuable,
e.g. as it could generate fluorescent bioactive compounds.
Such fluorescent bioactive compounds can enable uptake and
distribution studies, give information about compound
binding, allow for competition based assays, and can be deve-
loped into important chemical research tools for broader use.
However, the effect of different 4-amino substituents on the
fluorescence properties of the phthalimides has not been fully
Department of Chemistry, Umeå University, SE-90187 Umeå, Sweden.
E-mail: erik.chorell@chem.umu.se; Fax: +46 90 7867655; Tel: +46 90 7869329
†Electronic supplementary information (ESI) available: Photophysical data, cell
toxicity data for all compounds, cell imaging studies with compounds 19 and 20,
and 1H and 13C NMR spectra of all synthesized compounds. See DOI:
10.1039/c4ob00342j
‡These authors contributed equally to this work.
Fig. 1 Methods to synthesize 4-amino substituted phthalimides.
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describe methods to introduce anilines into 4-halogen substi-
tuted phthalimides by using transition metal based coupling
reactions,19–23 but to the best of our knowledge, there are no
papers describing methods to introduce amines into such sub-
strates. Introduction of various secondary amines into position
4 on the phthalimide core structure is important not only from
a structure activity perspective in the various biological set-
tings where the phthalimide core structure has displayed
activity but also from a fluorescence perspective, e.g. because
of their electron donating properties. For these reasons it
would be valuable to develop convenient and general synthetic
procedures to generate 4-amino substituted phthalimides. A
straightforward protocol with equimolar relationships between
phthalic anhydride and amine/s and/or aniline without any
additives would both be atom economical and simplify purifi-
cation of the target compounds. Herein, we describe the develop-
ment of such synthetic procedures (Fig. 1). Additionally, the
photophysical properties of the generated compounds and
how these can be used to identify and study bioactive com-
pounds are also presented.
Table 1 Screen to improve the reaction conditions
Entry
Solvent
Temp. (°C)
Time (min)
X
Yielda (%)
1
2
3
4
5
6
7
8
AcOH
DMF
DMSO
MeCN
NMP
Pyridine
NMP
NMP
NMP
NMP
NMP
NMP
NMP
NMP
NMP
NMP
200
200
200
200
200
200
150
180
200
220
220
200
200
200
200
200
60
60
60
60
60
60
75
75
75
75
30
30
120
75
75
75
F
F
F
F
F
F
F
F
F
F
F
F
Trace
34
47
56
84
77
45
88
95
96
66
57
94
36
31
24
9
10
11
12
13
14
15
16
F
Br
Cl
NO2
Results and discussion
a Isolated yield.
We recently published an optimized method to synthesize
phthalimides by simply heating phthalic acids together with
an amine or aniline in acetonitrile, pyridine or acetic acid
without additives and in equimolar quantities.24 Based on
this, and the fact that SNAr reactions can be conducted under
similar conditions, we set out to develop a one step method to
synthesize various 4-amino substituted phthalimides. Test
reactions using 4-fluorophthalic anhydride, morpholine, and
p-toluidine in equimolar amounts without any additives gave
promising results. Encouraged by this, various solvents, temp-
eratures, times, and leaving groups were screened to set the
conditions for the reaction (Table 1).
fluorescent properties of the target compounds. The R1 substi-
tuent was first examined together with p-toluidine and 4-fluoro-
phthalic anhydride (Table 2, entries 1–8). Good to excellent
yields were obtained when diethylamine, morpholine, and
piperidine were introduced by using the corresponding
amines (Table 2, entries 2–4). The dimethylamine substituent
was introduced by using sarcosine that decarboxylates during
the reaction to give the desired product (2) in 81% yield
(Table 2, entry 1). This proved to be a more effective procedure
to introduce the dimethylamino substituent as compared to
the use of dimethylamine HCl salt in combination with a base,
which resulted in lower yields and more complicated purifi-
cations. An N-benzylmethylamine, a substituted piperidine,
and a Cbz protected piperazine were also well tolerated and
gave phthalimides 6, 7 and 8 in 92%, 87% and 72% yields
respectively (Table 2, entries 6–8). A heterocycle could also be
introduced using this method as exemplified by imidazole that
gave the desired 4-imidazolo substituted phthalimide 5 in 81%
yield (Table 2, entry 5). Interestingly, the reaction of N-methyl-
aniline with p-toluidine and 4-fluorophthalic anhydride gave no
conversion, probably due to the low nucleophilic properties of
the aniline. The herein presented method is thus a nice comp-
lement to previously developed transition metal coupling reac-
tions with anilines in this position of the phthalimide.19,21–23
The effect of the R2 substituent on the reaction outcome was
examined by using a variety of electron donating substituents
(Table 2, entries 9 and 10) and withdrawing substituents
(Table 2, entries 12 and 13) as well as a heterocycle (Table 2,
The screen showed that the choice of the solvent is highly
important for the reaction outcome with NMP and pyridine as
the most promising solvents of the tested set giving 4-morpho-
lino substituted phthalimide 1 in 84% yield and 77% yield,
respectively (Table 1, entries 5 and 6). All of the tested leaving
groups resulted in the formation of the desired product, but
fluorine proved to be by far the best leaving group giving 94%
yield of 1 compared to 24–36% for Br, Cl, and NO2 (Table 1,
entry 9 vs. entries 14–16). Furthermore, it is clear that the reac-
tion needs high temperatures and times around 60 minutes
to proceed in high yields, which also show that the target
4-morpholino substituted phthalimide 1 is very thermally stable.
Hence, by using NMP as a solvent, fluorine as a leaving
group, microwave heating at 200 °C for 75 min, substrates in
equimolar amounts, and no additives, the scope of the reac-
tion was next evaluated with various amines and anilines
(Table 2).
The R1 and R2 substituents were selected to probe both the
scope and limitations of the developed synthetic method but
also to investigate how the substituents affect the potential
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Table 2 Evaluating the scope of the reaction
Table 2 (Contd.)
R1
R2
Yielda (%)
81b
ID
R1
R2
Yielda (%)
90
ID
20
2
3
4
1
5
6
89
83
95
81
92
21
93
a Isolated yield. b Using sarcosine as dimethylamine source.
entry 14), sterically hindered substituents (Table 2, entries
19–21) and alkyl substituents (Table 2, entries 15–18). All of
these substituents gave the target compounds in very good
yields, thus indicating that the reaction tolerates a very wide
span of substituents in this position. Overall, the reaction
proved to be very general and smoothly gave the desired com-
pounds in good to excellent yields after straightforward
purifications.
7
87
8
72
94
74
96
79
67
89
Evaluation of the photophysical properties of the new
phthalimide derivatives in chloroform revealed that many of
them have very high quantum yields (up to 81%) and un-
usually long lifetimes (up to 19.1 ns) (Table 3). All the 4-amino
substituents generated compounds with high quantum yields
except for the 4-imidazole substituted phthalimide 5 that dis-
played a quantum yield of 1%, thus emphasizing the need for
non-aromatic electron donating substituents in this position.
The substituent on the phthalimide nitrogen proved to be
important for the fluorescent properties and, surprisingly,
electron donating aryl substituents as in compounds 9 and 10
gave quantum yields close to zero. Compounds 1, 2, 6, 8, and
13 gave quantum yields of 21%, 49%, 37%, 13%, and 17%
respectively whereas the rest of the compounds display
quantum yields between 60 and 81% in chloroform (Table 3).
Interestingly, when the compounds’ photophysical properties
were evaluated in other solvents (methanol, acetonitrile, and
cyclohexane) the compounds displayed solvatochromic pro-
perties, i.e. low fluorescence quantum yields and lifetimes in
polar protic solvents compared to those in nonpolar solvents.
In methanol, all compounds’ quantum yields as well as life-
times decrease to 1–3% and below 1 ns, respectively, except for
the N-benzylmethylamine substituted phthalimides 6 and 20
that retain slightly more of their fluorescent properties
(quantum yields of 5% and 6% respectively). The compounds
also shift in the wavelength of maximum emission from
around 500 nm in chloroform to 550–570 in methanol, which
is typical of solvatochromic fluorophores as they are sensitive
9
10
11
12
13
14
15
95
16
17
18
19
92
68
54
94
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Table 3 The compounds photophysical properties
Quantum yielda (%)
Emission maximum (nm)
Lifetime (ns) [%]
ID CHCl3 CH MeCN MeOH CHCl3 CH MeCN MeOH CHCl3
CH
MeCN
MeOH
1
2
21
49
4
9
38
37
2
3
500
497
472 517
462 510
562
565
6.9 [96]
15.9
2.9 [86] 14.1 [14]
7.2
9.2
9.7
0.5 [99]
0.9 [87] 5.3 [7]
0.5 [6]
3
4
5
6
7
8
9
60
62
1
37
65
13
1
21
20
<1
10
16
3
14
10
<1
46
44
34
<1
2
1
<1
6
3
2
501
510
496
496
509
498
501
468 512
473 529
562
574
—
18.9
12
9.1
—
3.9
3.8
—
0.6 [98]
18.2 [99]
17.5 [87] 0.8 [8]
12.1
16.2
4.4
0.4 [85] 6.6 [13]
b
b
b
b
b
b
—
—
—
461 505
477 519
480 513
454 518
555
576
562
6.9 [87] 15.7 [13] 12.7
8.5
2.5 [96]
13.3
1.8
10.1
7.9
0.9 [50] 0.7 [46]
0.7 [71] 0.4 [29]
b
1
<1
—
17.6
8.5 [63] 5.1 [35] 0.4 [51] 7.7 [24]
2.4 [11]
0.1 [77] 9.4 [19] 0.2 [98]
8.1
4.9
b
b
10 <1
11 68
12 68
<1
33
57
<1
33
21
<1
1
1
507
503
505
—
520
—
17.0 [88] 0.3 [9] 13.2 [84] 0.3 [9]
19.1
17.9
460 516
457 521
560
562
13.6
17.0 [91] 7.3 [8]
0.4 [95]
0.3 [68] 6.6 [21]
2.3 [11]
b
13 17
14 63
31
48
<1
19
<1
1
501
500
459 521
454 523
—
5.5
12.8 [79]
6.4 [20]
16
6.1 [55] 0.1 [25] 0.2 [82] 0.8 [15]
2.5 [14]
5.1
551
18.2
0.3 [61] 7.9 [28]
2.0 [10]
15 75
16 71
17 75
8
46
52
26
37
34
1
2
3
496
494
509
489 520
448 513
454 517
555
565
569
16.7
18
18
12.2
13.8
8.8
9.7
1.3 [97]
0.6 [73] 6.4 [27]
16.2 [62] 11.1 [37]
8.4 [81] 14.8[19] 0.6 [54] 6.7 [37]
3.6 [9]
18 72
19 78
20 77
21 81
54
53
54
53
17
30
54
11
2
1
5
1
512
495
487
491
458 528
448 514
446 499
448 506
570
569
556
546
17.3
16.5
16.3
17.1
17.2 [70] 9.6 [30]
4.9
7.2
12.3
2.7
0.4 [96]
0.4 [99]
1.3
13.7
13.8
14.1
0.3 [97]
a See ESI for details. b Not possible to determine.
to changes in solvent polarity and relaxation rates as well as
their orientation to electric fields and the rigidity of the local
environment. These effects are highly valuable and can be
used to probe and monitor protein binding.13–16 In this
context, compounds 15 and 18 together with phthalimide 8,
after Cbz deprotection, are particularly attractive because they
are equipped with a chemical handle and can thus be linked
to various substrates and used as fluorescent probes to study
biological interactions.
The compounds were next evaluated for their toxicity in
whole-cell assays by using both the HeLa cervical cancer cell
line and the PC-3 prostate cancer cell line. This was performed
to investigate the potential use of the compounds as fluo-
rescent probes, for which purposes compounds that do not
affect cell viability generally are preferred. Compounds were
tested at 100, 25, 6, 1.5, and 0.4 μM and the results in HeLa
Fig. 2 The effect of diisopropylphenyl substituted compounds 19–21
on the HeLa cervical cancer cell line and the PC-3 prostate cancer cell
line after 48 h of incubation as measured by crystal violet staining. Com-
pounds 1–18 and 21 did not affect the HeLa cell line below 25 μM
whereas some toxicity can be observed also at 25 μM in the PC-3 cell
line (e.g. compound 17) (see ESI Fig. S1†).
cells show that compounds 1–18 are non-toxic even at 100 μM clear dose response with an EC50 value of around 7 μM in
except for the nitro-substituted compound 13 that displayed HeLa cells and 4 μM in PC-3 cells. The 4-amino substituent
toxic effects at 100 μM but was non-toxic at 25 μM. The same seems to be of high importance as the toxic effects are reduced
general non-toxic trend was observed in the PC-3 cell line even in compounds 20 and 21 (Fig. 2).
though more compounds displayed slight toxicity also at
To further evaluate the compounds as fluorescent probes
25 μM (e.g. compounds 13 and 17) (see ESI Fig. S1†). These and bioactive fluorescent compounds, live-cell imaging experi-
data in two different cell lines indicate that the generated ments were performed using the HeLa cervical cancer cell
4-amino substituted phthalimides 1–18 can be used as solvato- line. Cells were treated with 10 μM of compounds 1–21 and
chromic probes without affecting cell viability. Interestingly, uptake and distribution were monitored by fluorescence every
the diisopropylphenyl substituted 19–21 affects cell viability in 15 min. The solvatochromic properties of the compounds that
both cell lines. Compound 19 was most active and displayed a were revealed in the photophysical evaluation clearly showed
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Conclusion
The herein presented work has resulted in the following. Firstly,
it has presented a general, convenient, and atom efficient one-
step synthetic method that gives good to excellent yields of
4-amino substituted phthalimides. By using this method, the
reactions could be performed without any additives and in equi-
molar ratios and the pure compounds could thus in many cases
be isolated after simple work-up procedures without the need for
column chromatography. Secondly, this has opened up a possi-
bility of introducing novel 4-amino substituents on the phthali-
mide central fragment in combination with varying substituents
on the phthalimide nitrogen. Thirdly, this has generated highly
fluorescent and solvatochromic compounds, many of which
displayed non-toxic properties and non-detectable unspecific
binding. These compounds can thus potentially be linked to a
substrate and used as fluorescent probes (after controlling that
this does not alter the effect of the substrate). Lastly, the pre-
sented synthetic method proved to be useful to develop bioactive
fluorescent compounds to generate information about com-
pound uptake, distribution, and binding, which can be useful in
any of the numerous biological settings where phthalimides
have displayed activity. In light of this, and especially in the
context of the generated central fragment’s many applications
(e.g. drugs, a vast amount of bioactive compounds, materials,
polymers, catalysts, and fluorescent probes), the herein deve-
loped one-step synthesis of 4-amino substituted phthalimides
is expected to have broad utility to efficiently construct novel
compounds for future biological and chemical applications.
Fig. 3 (a) Live-cell images performed in the HeLa human cancer cell-
line at 15 min, 75 min, and 3 h post addition of compound 19. The DNA
stain Hoechst 33342 in blue (bottom), compound 19 in green (middle),
and overlay of both Hoechst 33342 and compound 19 (top) (see ESI
Fig. S2† for additional time points). (b) Confocal microscopy showing a
HeLa cell stained with compound 19 in green and the DNA stain
Hoechst 33342 in blue.
Experimental section
General considerations
Reagents were used as received unless otherwise noted. 1-
Methyl-2-pyrrolidone (NMP) was dried over 4 Å molecular
sieves prior to use. All reactions were carried out under an
inert atmosphere. Microwave reactions were carried out in an
Initiator+ microwave instrument from Biotage, using sealed
2–5 mL process vials. Reaction times refer to irradiation time
at the target temperature and not the total irradiation time.
that the compounds’ fluorescence is diminished in protic,
hydrophilic environments and as expected, the compounds are
not visible directly after addition to the cells in growth
medium. However, the bioactive compounds 19 and 20 start to
become visible inside the cells already after 15–30 min, indicat-
ing that they are cell permeable and accumulate in a hydro-
phobic niche. Compounds 1–18 are not detected even after
4 hours of incubation, indicating a low unspecific binding of
hydrophobic structures. Interestingly, compounds 19 and 20
seem to specifically stain the cell nucleus when compared to
the DNA stain Hoechst, thus suggesting that the compounds’
effect is cell nucleus related (Fig. 3a and ESI Fig. S2†). The dis-
tribution of the most active compound 19 was further studied
by confocal microscopy. This revealed that the compound does
in fact not overlap with the cell DNA stain Hoechst but seems
to accumulate around the DNA (Fig. 3b). Additional studies to
identify the mode of action of compound 19 as well as further
structure activity studies based on this compound are currently
being pursued within our laboratory.
1
The temperature was measured with an IR sensor. The H and
13C NMR spectra were recorded on a 400 MHz spectrometer at
298 K, and the residual solvent peaks of CDCl3 (7.26 ppm and
77.16 ppm), MeOD-d4 (3.31 ppm and 49.00 ppm) and DMSO-
d6 (2.50 ppm and 39.52 ppm) were used as internal standards
1
for calibrating the H and 13C spectra, respectively. 19F spectra
were calibrated against an external standard of CF3COOH
(−76.55 ppm) in CDCl3. IR spectra were recorded on a Perkin-
Elmer BX FT-IR spectrophotometer using a Specac Golden
Gate ATR accessory. HRMS was conducted using a Bruker
microTOF II mass spectrometer with electrospray ionization.
UV-vis spectroscopic characterization
UV-vis absorbance spectra were recorded at 10 μM on a
Schimadzu UV-3101PC. Fluorescence spectra and lifetimes were
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obtained with a HORIBA Jobin Yvon Fluorolog TCSPC at a con- amine/aniline (1.0 mmol, 1.0 eq.) and the secondary amine
centration where the absorbance in the fluorescence wave- (1.0 mmol, 1.0 eq.). The vessel was sealed with a PTFE cap and
length range was lower than 0.1. The steady-state fluorescence heated to 200 °C for 75 minutes in a microwave reactor.
spectra were obtained by excitation at 370 nm, while fluo- Workup performed as in method A unless otherwise noted,
rescence decays were measured using a LED at 375 nm. The and purified according to the compound description.
quantum yields were determined relative to doubly recrystal-
Compound descriptions
lized quinine sulfate in 0.5 M H2SO4 (Φf = 0.546). The fluo-
rescence decays were observed at 550, 500, 450 and 500 nm in
2-(4-Methylphenyl)-5-(morpholin-4-yl)isoindole-1,3-dione
MeOH, MeCN, CHCl3 and cyclohexane, respectively. Decay life- (1). Method C. The reaction mixture was poured into 125 mL
times were analyzed using the Tcspcfit software package.25
water and stirred for 5 min before the precipitation was col-
lected by filtration to give the pure title compound as a yellow
solid in 95% yield. IR (cm−1): 3450w, 2957w, 2880w, 1766w,
Quantum yield measurements
1
1705s, 1613s. H NMR (400 MHz, CDCl3) δ 7.73 (1H, d, J = 8.5
UV-vis absorbance spectra were recorded at 10 μM on a Schi-
madzu UV-3101PC. Fluorescence spectra and lifetimes were
obtained with a HORIBA Jobin Yvon Fluorolog TCSPC at a con-
centration where the absorbance in the fluorescence wave-
length range was lower than 0.1. Steady-state fluorescence
spectra were obtained by excitation at 370 nm for the organic
solvents. Dilute solutions of the compounds were prepared
from stock solutions in CHCl3, giving residual stock solvent
concentrations of 1–3(−5)%. Stock solutions of compound 15
were prepared in DMSO and acetone due to its low solubility
in CHCl3. Quantum yields of compounds (subscript x) were
determined relative to doubly recrystallized quinine sulfate in
0.5 M H2SO4 (subscript ref; Φf = 0.546, η = 1.34), according to:
Hz), 7.33–7.25 (4H, m), 7.06 (1H, dd, J = 8.5, 2.4 Hz), 3.85–3.80
(4H, m), 3.35–3.31 (4H, m), 2.38 (3H, s); 13C NMR (100 MHz,
CDCl3) δ 167.7, 167.3, 155.6, 137.7, 134.2, 129.6, 129.4, 126.4,
125.1, 120.4, 117.9, 108.4, 66.3, 47.6, 21.2. HRMS calcd for
C19H18N2NaO3 [M + Na]+: 345.1210, found: 345.1196.
5-(Dimethylamino)-2-(4-methylphenyl)isoindole-1,3-dione
(2). Method C. Sarcosine was used as the secondary amine.
The reaction mixture was poured into 125 mL water and
stirred for 5 min before the precipitation was collected by fil-
tration to give the pure title compound as a yellow solid in
81% yield. IR (ATR, cm−1): 3446w, 3040w, 2916w, 1754m,
1
1701s, 1613s. H NMR (400 MHz, CDCl3) δ 7.70 (1H, d, J = 8.5
Hz), 7.33–7.25 (4H, m), 7.11 (1H, d, J = 2.4 Hz), 6.82 (1H, dd,
J = 8.5, 2.4 Hz), 3.10 (6H, s), 2.39 (3H, s); 13C NMR (100 MHz,
CDCl3) δ 168.2, 167.8, 154.5, 137.5, 134.5, 129.64, 129.62,
126.5, 125.2, 117.2, 115.0, 105.8, 40.5, 21.2. HRMS calcd for
C17H16N2NaO2 [M + Na]+: 303.1104, found: 303.1107.
5-(Diethylamino)-2-(4-methylphenyl)isoindole-1,3-dione
(3). Method C. The reaction mixture was poured into 125 mL
water and stirred for 5 min before the precipitation was col-
lected by filtration to give the pure title compound as a yellow
solid in 89% yield. IR (ATR, cm−1): 3451w, 3073w, 3031w,
2978w, 2905w, 1754m, 1703s, 1609s. 1H NMR (400 MHz,
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ðemx ꢀ emsolvent;xÞ=Ax
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ðemref ꢀ emsolvent;ref Þ=Aref
ηref
where Φ denotes quantum yield, em denotes the integrated
emission, A denotes absorbance at the excitation wavelength,
and η denotes the refractive index. η values used for cyclo-
hexane, chloroform, acetonitrile, and methanol were 1.424,
1.446, 1.344, and 1.329, respectively.
General syntheses
Method A. To 4-fluorophthalic anhydride (166 mg, CDCl3) δ 7.69 (1H, d, J = 8.6 Hz), 7.33–7.25 (4H, m), 7.10 (1H,
1.0 mmol, 1.0 eq.) in 2 ml of NMP was added the primary d, J = 2.4 Hz), 6.81 (1H, dd, J = 8.6, 2.4 Hz), 3.46 (4H, q, J = 7.1
aniline (1.0 mmol, 1.0 eq.). The vessel was sealed with a PTFE Hz), 2.38 (3H, s), 1.22 (6H, t, J = 7.1 Hz); 13C NMR (100 MHz,
cap and heated to 100 °C for 10 minutes in a microwave CDCl3) δ 168.3, 167.7, 152.4, 137.5, 134.8, 129.7, 129.6, 126.5,
reactor. The secondary amine (1.2 mmol, 1.2 eq.) was added, 125.5, 116.4, 114.5, 105.4, 45.0, 21.2, 12.3. HRMS calcd for
and the vessel was heated to 200 °C for 60 minutes. The vessel C19H20N2NaO2 [M + Na]+: 331.1417, found: 331.1421.
contents were poured into EtOAc and washed three times with
2-(4-Methylphenyl)-5-(piperidin-1-yl)isoindole-1,3-dione
brine (if NaCl precipitated during the workup, a little water (4). Method C. The reaction mixture was poured into 125 mL
was added). The organic phase was dried over Na2SO4, filtered water and stirred for 5 min before the precipitation was col-
and concentrated, and purified according to the compound lected by filtration to give the pure title compound as a yellow
description below.
solid in 83% yield. IR (ATR, cm−1): 3447w, 3077w, 3034w,
Method B. To 4-fluorophthalic anhydride (166 mg, 2924m, 2856m, 1759m, 1704s, 1613s. 1H NMR (400 MHz,
1.0 mmol, 1.0 eq.) in 2 ml of NMP was added the primary aniline CDCl3) δ 7.68 (1H, d, J = 8.5 Hz), 7.33–7.25 (4H, m), 7.02 (1H,
(1.0 mmol, 1.0 eq.) and the secondary amine (1.2 mmol, 1.2 dd, J = 8.5, 2.3 Hz), 3.43–3.36 (4H, m), 2.38 (3H, s), 1.69–1.61
eq.). The vessel was sealed with a PTFE cap and heated to (6H, m); 13C NMR (100 MHz, CDCl3) δ 168.0, 167.4, 155.4,
200 °C for 75 minutes in a microwave reactor. Workup per- 137.4, 134.4, 129.54, 129.51, 126.4, 125.1, 118.4, 117.6, 108.2,
formed as in method A and purified according to the com- 48.7, 25.1, 24.2, 21.1. HRMS calcd for C20H20N2NaO2 [M +
pound description.
Na]+: 343.1417, found: 343.1417.
Method C. To 4-fluorophthalic anhydride (166 mg,
5-(Imidazol-1-yl)-2-(4-methylphenyl)isoindole-1,3-dione
1.0 mmol, 1.0 eq.) in 2 ml of NMP was added the primary (5). Method A. The aqueous phase was made basic by the
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addition of NaHCO3 during workup. Purified by column 7.08 (1H, dd, J = 8.5, 2.3 Hz), 6.79 (2H, d, J = 9.0 Hz), 3.89–3.85
chromatography on silica gel with a gradient of heptane– (4H, m), 3.39–3.34 (4H, m), 2.98 (6H, s); 13C NMR (100 MHz,
EtOAc 50 : 50 to 0 : 100 to give the title compound as a cream CDCl3) δ 168.4, 168.0, 155.6, 150.2, 134.5, 127.6, 125.1, 120.9,
colored powder (81% yield). IR (ATR, cm−1): 3470w, 3114w, 120.6, 118.0, 112.6, 108.6, 66.5, 47.9, 40.7. HRMS calcd for
1770m, 1706s, 1621m. 1H NMR (400 MHz, DMSO-d6) δ 8.59 C20H21N3NaO3 [M + Na]+: 374.1475, found: 374.1466.
(1H, s), 8.32 (1H, d, J = 1.8 Hz), 8.19 (1H, dd, J = 8.1, 2.0 Hz),
2-(4-Methoxyphenyl)-5-(morpholin-4-yl)isoindole-1,3-dione
8.08–8.05 (2H, m), 7.33 (4H, s), 7.18 (1H, s), 2.38 (3H, s); 13C (10). Method A. Purified by column chromatography on silica
NMR (100 MHz, DMSO-d6) δ 166.4, 166.3, 141.7, 137.8, 136.1, gel with a gradient of heptane–CH2Cl2 25 : 75 to 0 : 100, then
133.9, 130.7, 129.4, 129.2, 128.9, 127.2, 125.2, 125.1, 118.1, CH2Cl2–EtOAc 100 : 0 to 50 : 50 to give the title compound as a
114.5, 20.8. HRMS calcd for C18H14N3O2 [M + H]+: 304.1081, yellow solid (74% yield). IR (ATR, cm−1): 3452w, 3080w, 2956w,
found: 304.1069.
2839w, 1764m, 1707s, 1612m. 1H NMR (400 MHz, CDCl3)
5-[Benzyl(methyl)amino]-2-(4-methylphenyl)isoindole-1,3- δ 7.72 (1H, d, J = 8.5 Hz), 7.32–7.28 (3H, m), 7.05 (1H, dd, J = 8.5,
dione (6). Method C. The reaction mixture was poured into 2.4 Hz), 7.00–6.95 (2H, m), 3.85–3.81 (4H, m), 3.80 (3H, s),
125 mL water and stirred for 5 min before the precipitation 3.36–3.31 (4H, m); 13C NMR (100 MHz, CDCl3) δ 167.9, 167.5,
was collected by filtration to give the pure title compound as a 159.0, 155.6, 134.2, 127.9, 125.1, 124.7, 120.4, 117.9, 114.3,
yellow solid in 92% yield. IR (ATR, cm−1): 3453w, 3028w, 108.4, 66.3, 55.5, 47.6. HRMS calcd for C19H18N2NaO4 [M +
2917w, 1759m, 1705s, 1610s. 1H NMR (600 MHz, CDCl3) δ 7.72 Na]+: 361.1159, found: 361.1148.
(1H, d, J = 8.5 Hz), 7.37–7.33 (2H, m), 7.31–7.27 (5H, m), 7.23
5-(Morpholin-4-yl)-2-phenylisoindole-1,3-dione (11). Method
(1H, d, J = 2.4 Hz), 7.18 (2H, d, J = 7.3 Hz), 6.91 (1H, dd, J = 8.5, C. The reaction mixture was poured into 125 mL water and
2.4 Hz), 4.71 (2H, s), 3.22 (3H, s), 2.39 (3H, s); 13C NMR stirred for 5 min before the precipitation was collected by fil-
(150 MHz, CDCl3) δ 168.2, 167.8, 154.2, 137.8, 136.8, 134.8, tration to give the pure title compound as a yellow solid in
129.8, 129.7, 129.1, 127.7, 126.6, 126.5, 125.5, 118.1, 115.6, 96% yield. IR (ATR, cm−1): 3455w, 3053w, 2954w, 2855w,
106.3, 56.4, 39.5, 21.3. HRMS calcd for C23H20N2NaO2 [M + 1767m, 1698s, 1609s. 1H NMR (400 MHz, CDCl3) δ 7.78 (1H, d,
Na]+: 379.1417, found: 379.1403.
J = 8.5 Hz), 7.52–7.34 (6H, m), 7.10 (1H, dd, J = 8.5, 2.4 Hz),
2-(4-Methylphenyl)-5-(3-phenylpiperidin-1-yl)isoindole-1,3- 3.89–3.85 (4H, m), 3.41–3.36 (4H, m); 13C NMR (100 MHz,
dione (7). Method B. Purified by column chromatography on CDCl3) δ 167.8, 167.3, 155.8, 134.4, 132.2, 129.1, 127.9, 126.7,
silica gel with a gradient of heptane–CH2Cl2 65 : 35 to 0 : 100 to 125.4, 120.6, 118.2, 108.6, 66.5, 47.8. HRMS calcd for
give the title compound as a yellow powder (87% yield). IR C18H16N2NaO3 [M + Na]+: 331.1053, found: 331.1040.
(ATR, cm−1): 3463w, 2918s, 2846w, 1764m, 1707s, 1611s. 1H
5-(Morpholin-4-yl)-2-[4-(trifluoromethyl)phenyl]isoindole-
NMR (400 MHz, CDCl3) δ 7.75 (1H, d, J = 8.5 Hz), 7.41–7.27 1,3-dione (12). Method C. The reaction mixture was poured
(10H, m), 7.11 (1H, dd, J = 8.5, 2.4 Hz), 4.08–4.01 (2H, m), into 125 mL water and stirred for 5 min before the precipi-
3.09–3.00 (2H, m), 2.93–2.84 (1H, m), 2.42 (3H, s), 2.15–2.09 tation was collected by filtration to give the pure title com-
(1H, m), 1.96–1.90 (1H, m), 1.85–1.76 (2H, m); 13C NMR pound as a yellow solid in 79% yield. IR (ATR, cm−1): 3464w,
(100 MHz, CDCl3) δ 168.0, 167.5, 155.2, 143.1, 137.7, 134.6, 3073w, 2976w, 2853w, 1771m, 1703s, 1613s. 1H NMR
129.7, 129.6, 128.7, 127.1, 127.0, 126.5, 125.4, 119.0, 117.9, (400 MHz, CDCl3) δ 7.79 (1H, d, J = 8.5 Hz), 7.74 (2H, d, J = 8.5
108.5, 55.2, 48.4, 42.2, 31.6, 24.9, 21.2. HRMS calcd for Hz), 7.63 (2H, d, J = 8.5 Hz), 7.35 (1H, d, J = 2.3), 7.11 (1H, dd,
C26H24N2NaO2 [M + Na]+: 419.1730, found: 419.1735.
J = 8.5, 2.4 Hz), 3.90–3.85 (4H, m), 3.42–3.37 (4H, m); 13C NMR
Benzyl 4-[2-(4-methylphenyl)-1,3-dioxoisoindol-5-yl]pipera- (100 MHz, CDCl3) δ 167.3, 166.7, 155.9, 135.5, 134.1, 129.4 (q,
zine-1-carboxylate (8). Method B. Purified by column chrom- J = 33 Hz), 126.4, 126.2 (q, J = 4 Hz), 125.7, 124.0 (q, J = 272 Hz),
atography on silica gel with a gradient of CH2Cl2–EtOAc 100 : 0 120.1, 118.3, 108.5, 66.4, 47.7; 19F NMR (376 MHz, CDCl3)
to 80 : 20 to give the title compound as a yellow powder (72% δ −63.20. HRMS calcd for C19H15F3N2NaO3 [M + Na]+: 399.0927,
yield). IR (ATR, cm−1): 3448w, 3034w, 2856w, 1760m, 1690s, found: 399.0916.
1614s. 1H NMR (400 MHz, CDCl3) δ 7.73 (1H, d, J = 8.5 Hz),
5-(Morpholin-4-yl)-2-(4-nitrophenyl)isoindole-1,3-dione
7.41–7.27 (10H, m), 7.05 (1H, dd, J = 8.5 Hz, 2.3 Hz), 5.18 (2H, (13). Method C. The reaction mixture was poured into 125 mL
s), 3.70–3.64 (4H, m), 3.43–3.35 (4H, m), 2.38 (3H, s); 13C NMR water and stirred for 5 min before the precipitation was col-
(100 MHz, CDCl3) δ 167.6, 167.2, 155.1, 155.0, 137.7, 136.4, lected by filtration to give the pure title compound as a yellow
134.3, 129.6, 129.3, 128.5, 128.2, 128.0, 126.4, 125.2, 120.3, solid in 67% yield. IR (ATR, cm−1): 3473w, 3124w, 2965w,
118.3, 108.7, 67.4, 47.3, 43.1, 21.1. HRMS calcd for 2851w, 1775m, 1719s, 1621s. 1H NMR (400 MHz, CDCl3) δ 8.34
C27H25N3NaO4 [M + Na]+: 478.1737, found: 478.1739.
(2H, ddd, J = 8.9, 2.8, 2.2 Hz), 7.81 (1H, d, J = 8.4 Hz), 7.76 (2H,
2-[4-(Dimethylamino)phenyl]-5-(morpholin-4-yl)isoindole- ddd, J = 9.0, 2.8, 2.1 Hz), 7.36 (1H, d, J = 2.2 Hz), 7.14 (1H, dd,
1,3-dione (9). Method C. The reaction mixture was poured into J = 8.5, 2.3 Hz), 3.92–3.87 (4H, m), 3.46–3.40 (4H, m); 13C NMR
125 mL water and stirred for 5 min before the precipitation (100 MHz, CDCl3) δ 167.0, 166.4, 156.1, 146.2, 138.2, 134.1,
was collected by filtration to give the pure title compound as a 126.2, 125.9, 124.5, 119.8, 118.5, 108.5, 66.4, 47.6. HRMS calcd
yellow solid in 94% yield. IR (ATR, cm−1): 3442w, 2873w, for C18H15N3NaO5 [M + Na]+: 376.0904, found: 376.0891.
1
1765w, 1705s, 1614m. H NMR (400 MHz, CDCl3) δ 7.76 (1H,
5-(Morpholin-4-yl)-2-( pyridin-3-yl)isoindole-1,3-dione
d, J = 8.5 Hz), 7.34 (1H, d, J = 2.2 Hz), 7.23 (2H, d, J = 9.0 Hz), (14). Method C. The reaction mixture was poured into 125 mL
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water and stirred for 5 min before the precipitation was col- 1688s, 1613s. 1H NMR (400 MHz, CDCl3) δ 7.64 (1H, d, J =
lected by filtration to give the pure title compound as a yellow 8.4 Hz), 7.23 (1H, d, J = 2.2 Hz), 7.00 (1H, dd, J = 8.5, 2.2 Hz),
solid in 89% yield. IR (ATR, cm−1): 3446w, 3042w, 2950w, 3.86–3.83 (4H, m), 3.83–3.80 (4H, m), 3.34–3.31 (4H, m), 2.69
2877w, 1759m, 1708s, 1611s. 1H NMR (400 MHz, CDCl3) δ 8.77 (1H, t, J = 5.4 Hz); 13C NMR (100 MHz, CDCl3) δ 169.3, 169.0,
(1H, d, J = 2.0 Hz), 8.60 (1H, dd, J = 4.7, 1.0 Hz), 7.83 (1H, ddd, 155.5, 134.5, 125.0, 120.7, 117.7, 108.5, 66.5, 61.3, 47.7, 40.9.
J = 8.2, 2.5, 1.6 Hz), 7.79 (1H, d, J = 8.5 Hz), 7.43 (1H, ddd, J = HRMS calcd for C14H16N2NaO4 [M + Na]+: 299.1002, found:
8.2, 4.8, 0.6 Hz), 7.36 (1H, d, J = 2.4 Hz), 7.12 (1H, dd, J = 299.1001.
8.5 Hz, 2.4 Hz), 3.90–3.85 (4H, m), 3.43–3.38 (4H, m); 13C NMR
2-(2,6-Diisopropylphenyl)-5-(morpholin-4-yl)isoindole-1,3-
(100 MHz, CDCl3) δ 167.3, 166.7, 155.9, 148.4, 147.3, 134.2, dione (19). Method C. The reaction mixture was diluted in
133.6, 129.3, 125.7, 123.7, 120.1, 118.3, 108.6, 66.4, 47.7. HRMS CH2Cl2 and washed with water. The organic phase was dried
calcd for C17H16N3O3 [M + H]+: 310.1186, found: 310.1177.
over Na2SO4, filtered, and concentrated before being filtered
4-{[5-(Morpholin-4-yl)-1,3-dioxoisoindol-2-yl]methyl}benzoic through a short plug of silica, eluting with CH2Cl2–MeOH
acid (15). Method C. The reaction mixture was poured into 98 : 2 to give the pure title compound as a yellow solid in 94%
125 mL water and stirred for 5 min before the precipitation yield. IR (ATR, cm−1): 3451w, 3074w, 2962m, 2860m, 1762m,
1
was collected by filtration to give the pure title compound as a 1703s, 1615s. H NMR (400 MHz, CDCl3) δ 7.82 (1H, d, J = 8.5
yellow solid in 95% yield. IR (ATR, cm−1): 3442w, 3152w, Hz), 7.48–7.41 (2H, m), 7.29 (2H, d, J = 7.8 Hz), 7.14 (1H, dd,
3034m, 2967m, 2647w, 1758m, 1698s, 1618s. 1H NMR J = 8.5, 2.4 Hz), 3.86–3.82 (4H, m), 3.41–3.37 (4H, m), 2.76 (2H,
(400 MHz, CDCl3–MeOD-d4) δ 7.94 (2H, dt, J = 8.4, 1.7 Hz), sep, J = 6.9 Hz), 1.173 (6H, d, J = 6.9 Hz), 1.168 (6H, d, J = 6.9
7.66 (1H, d, J = 8.5 Hz), 7.39 (2H, dt, J = 8.4, 1.7 Hz), 7.26 (1H, Hz); 13C NMR (100 MHz, CDCl3) δ 168.7, 168.2, 155.6, 147.4,
d, J = 2.3 Hz), 7.04 (1H, dd, J = 8.5, 2.3 Hz), 4.81 (2H, s), 134.4, 130.0, 127.3, 125.4, 123.9, 120.6, 118.0, 108.7, 66.4, 47.7,
3.86–3.80 (4H, m), 3.37–3.32 (4H, m); 13C NMR (100 MHz, 29.3, 24.01, 23.96. HRMS calcd for C24H28N2NaO3 [M + Na]+:
CDCl3–MeOD-d6) δ 168.9, 168.7, 168.6, 155.9, 141.8, 134.6, 415.1992, found: 415.1991.
130.4, 130.1, 128.2, 125.3, 120.6, 118.0, 108.8, 66.6, 47.8, 41.3.
HRMS calcd for C20H18N2NaO5 [M + Na]+: 389.1108, found: 1,3-dione (20). Method C. The reaction mixture was diluted in
389.1105. CH2Cl2 and washed with water. The organic phase was dried
5-[Benzyl(methyl)amino]-2-(2,6-diisopropylphenyl)isoindole-
2-Isopropyl-5-(morpholin-4-yl)isoindole-1,3-dione (16). Method over Na2SO4, filtered, and concentrated before being filtered
C. The reaction mixture was poured into 125 mL water and through a short plug of silica, eluting with CH2Cl2–MeOH
stirred for 5 min before the precipitation was collected by fil- 98 : 2 to give the pure title compound as a yellow solid in 90%
tration to give the pure title compound as a yellow solid in yield. IR (ATR, cm−1): 3450w, 3064w, 2962m, 2868w, 1764m,
1
92% yield. IR (ATR, cm−1): 3428w, 3368w, 3072w, 2964m, 1706s, 1612s. H NMR (400 MHz, CDCl3) δ 7.79 (1H, d, J = 8.5
2867m, 1752m, 1686s, 1614s. 1H NMR (600 MHz, CDCl3) Hz), 7.48 (1H, t, J = 7.8 Hz), 7.42–7.36 (2H, m), 7.35–7.29 (4H,
δ 7.61 (1H, d, J = 8.4 Hz), 7.20 (1H, d, J = 2.4 Hz), 7.00 (1H, dd, m), 7.26–7.22 (2H, m), 6.98 (1H, dd, J = 8.5, 2.5 Hz), 4.74 (2H,
J = 8.4, 2.4 Hz), 4.44 (1H, sep, J = 7.0 Hz), 3.85–3.82 (4H, m), s), 3.24 (3H, s), 2.83 (2H, sep, J = 6.9 Hz), 1.22 (12H, d, J = 6.9
3.33–3.29 (4H, m), 1.43 (6H, d, J = 7.0 Hz); 13C NMR (125 MHz, Hz); 13C NMR (100 MHz, CDCl3) δ 169.0, 168.5, 154.2, 147.5,
CDCl3) δ 168.8, 168.5, 155.4, 134.6, 124.5, 121.3, 117.7, 108.2, 136.7, 134.8, 129.9, 129.0, 127.55, 127.53, 126.3, 125.5, 123.8,
66.4, 47.9, 42.8, 20.2. HRMS calcd for C15H18N2NaO3 118.0, 115.4, 106.2, 56.2, 39.3, 29.3, 24.06, 24.02. HRMS calcd
[M + Na]+: 297.1210, found: 297.1180.
for C28H30N2NaO2 [M + Na]+: 449.2199, found: 449.2213.
2-Cyclohexyl-5-(piperidin-1-yl)isoindole-1,3-dione (17). Method
5-(Diethylamino)-2-(2,6-diisopropylphenyl)isoindole-1,3-dione
A. Purified by column chromatography on silica gel with a gra- (21). Method C. The reaction mixture was diluted in CH2Cl2
dient of heptane–CH2Cl2 20 : 80 to 0 : 100, then in heptane– and washed with water. The organic phase was dried over
CH2Cl2 35 : 65 to 0 : 100 to give the title compound as a yellow Na2SO4, filtered, and concentrated before being filtered
powder, ca. 95% pure (68% yield). IR (ATR, cm−1): 3436w, through a short plug of silica, eluting with CH2Cl2–MeOH
3373w, 3076w, 2930m, 2918m, 2848m, 1759m, 1689s, 1615s. 98 : 2 to give the pure title compound as a yellow solid in 93%
1H NMR (400 MHz, CDCl3) δ 7.54 (1H, d, J = 8.5 Hz), 7.16 (1H, yield. IR (ATR, cm−1): 3450w, 3072w, 2964m, 2930w, 2869w,
d, J = 2.3 Hz), 6.93 (1H, dd, J = 8.5, 2.3 Hz), 4.00 (1H, tt, J = 1766m, 1703s, 1610s. 1H NMR (400 MHz, CDCl3) δ 7.76 (1H, d,
12.3, 3.9 Hz), 3.37–3.32 (4H, m), 2.21–2.08 (2H, m), 1.83–1.76 J = 8.6 Hz), 7.44 (1H, d, J = 7.8 Hz), 7.28 (2H, d, J = 7.8 Hz),
(2H, m), 1.69–1.58 (9H, m), 1.37–1.14 (3H, m); 13C NMR 7.17 (1H, d, J = 2.4 Hz), 6.89 (1H, dd, J = 8.6, 2.4 Hz), 3.50 (4H,
(100 MHz, CDCl3) δ 169.1, 168.6, 155.3, 134.6, 124.5, 119.2, q, J = 7.1 Hz), 2.79 (2H, sep, J = 6.8 Hz), 1.26 (6H, t, J = 7.1 Hz),
117.4, 108.1, 50.5, 49.0, 29.9, 26.1, 25.2, 25.2, 24.2. HRMS 1.180 (6H, d, J = 6.9), 1.178 (6H, d, J = 6.9); 13C NMR (100 MHz,
calcd for C19H24N2NaO2 [M + Na]+: 335.1730, found: 335.1721.
CDCl3) δ 169.2, 168.7, 152.4, 147.6, 135.1, 130.0, 127.7, 125.8,
2-(2-Hydroxyethyl)-5-(morpholin-4-yl)isoindole-1,3-dione 123.9, 116.8, 114.7, 105.8, 45.2, 29.3, 24.11, 24.06, 12.5. HRMS
(18). Method B. Purified by column chromatography on silica calcd for C24H30N2NaO2 [M + Na]+: 401.2199, found: 401.2198.
gel with a gradient of CH2Cl2–MeOH 95 : 5 to 90 : 10, then
Cell lines and culture conditions
CH2Cl2–MeOH 100 : 0 to 95 : 5 to give 150 mg of the title com-
pound as an orange powder, ca. 95% purity (54% yield). IR The HeLa cervical cancer cell line was cultured in RPMI
(ATR, cm−1): 3446m, 3110w, 3061w, 2940w, 2881w, 1747m, medium 1640 (1×) [+] L-glutamine (Gibco® by Life Techno-
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logies™) supplemented with 9% heat-inactivated fetal bovine for 30 min. Finally, cells were washed with PBS and mounted
serum (FBS; Sigma Aldrich, F6178), PEST (diluted from 100× using Dako Fluorescent Mounting Medium S3023 and then
stock, Sigma Aldrich P4333, with 10 000 units penicillin and examined by fluorescence using a confocal Nikon A1 coupled
10 mg streptomycin mL−1). The PC-3 prostate cancer cell line to an inverted Nikon eclipse Ti microscope.
was cultured in RPMI medium 1640 (1×) [+] L-glutamine
(Gibco® by Life Technologies™) supplemented with 9% heat-
inactivated fetal bovine serum (FBS; Sigma Aldrich, F6178),
PEST (diluted from 100× stock, Sigma Aldrich P4333, with
Acknowledgements
10 000 units penicillin and 10 mg streptomycin mL−1), 10 mM We gratefully thank IF:s stiftelse för farmacevtisk forskning,
HEPES, and 1 mM sodium pyruvate. Cells were grown at 37 °C Swedish Academy of Pharmaceutical Sciences, Stiftelsen Olle
under a 5% CO2 atmosphere and routinely split 1 : 5 at ∼80% Engkvist Byggmästare, the Carl Trygger foundation, and JC
confluence using 0.05% Trypsin-EDTA (1×) (Gibco® by Life Kempe Foundation (SJCKMS) for financial support.
Technologies™).
Cell viability assay
References
Cells (1000 cells per well) were seeded in 96-well plates and
allowed to grow for 24 h before treating with 100, 25, 6, 1.5,
and 0.4 μM of each compound (diluted from 20 mM stock
solutions in DMSO) or DMSO alone in triplicates. The cells
were next incubated for 48 h before the growth medium was
removed and the cells were fixed for 20 min at rt by using 1%
glutaraldehyde. The fixed cells were treated for 30 minutes at
rt with 100 μl of crystal violet (0.1% in water). Excess dye was
subsequently removed by gentle rinsing of the plates using
deionized water before the plates were air-dried. 200 μl of 1%
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