Discovery of fluorescent 3-heteroarylcoumarin derivatives as novel inhibitors of anaplastic lymphoma kinase

Article abstract of DOI:10.1039/c8ob02874e

Altered expression or hyperactivation of anaplastic lymphoma kinase (ALK), as a consequence of translocations or point mutations, is one of the main oncogenic drivers in non-small cell lung cancer. Using structure-based design and in vitro enzyme assays, we identified 3-heteroarylcoumarin as a new template for the development of novel fluorescent ALK inhibitors. Molecular simulation provided structural insights for the design of 3-heteroarylcoumarin derivatives, which were easily prepared through efficient synthetic approaches including direct C-H cross coupling. Importantly, these coumarin-based ALK inhibitors can be tracked using microscopy techniques: we illustrated the use of the most potent compound in this series, 5a, (ALK/IC₅₀ = 0.51 μM, λ_emi = 500 nm, φ_F = 0.29) to monitor its subcellular distribution pattern by confocal fluorescence microscopy.

Full text of DOI:10.1039/c8ob02874e

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Takeharu Haino et al.
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DOI: 10.1039/C8OB02874E
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Discovery of Fluorescent 3-Heteroarylcoumarin Derivatives as
Novel Inhibitors of Anaplastic Lymphoma Kinase
Shinmee Mah,a,b Jaebong Jang,b Daesun Song,a Yongje Shin,a,b Muhammad Latif,b Yongwon Jung,*a
Received 00th January 20xx,
Accepted 00th January 20xx
and Sungwoo Hong*b,a
DOI: 10.1039/x0xx00000x
Altered expression or hyperactivation of anaplastic lymphoma kinase (ALK), as a consequence of translocations or point
mutations, is one of the main oncogenic drivers in non small cell lung cancer. Using structure-based design and in
vitro enzyme assays, we identified 3-heteroarylcoumarin as a new template for the development of novel fluorescent ALK
inhibitors. Molecular simulation provided structural insights for the design of 3-heteroarylcoumarin derivatives, which were
easily prepared through efficient synthetic approaches including direct C-H cross coupling. Importantly, these coumarin-
based ALK inhibitors can be tracked using microscopy techniques: we illustrated the use of the most potent compound in
this series, 5a, (ALK/IC50 = 0.51 μM, λemi = 500 nm, ϕF = 0.29) to monitor its subcellular distribution pattern by confocal
fluorescence microscopy.
www.rsc.org/
mutant through structure-based design strategy (
1).16
1 and 2, Fig
Introduction
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase
that belongs to the insulin receptor superfamily, and normally
plays a critical role in the development and maintenance of the
nervous system.1 Constitutive activation of ALK mainly caused
by translocations or point mutations has been highly associated
with various human cancers such as anaplastic large cell
lymphoma
(ALCL),2 non-small
cell
lung
cancer
(NSCLC),3 squamous cell carcinoma,4 and diffuse large B-cell
lymphoma.5 In particular, an oncogenic EML4-ALK protein,
generated from the fusion of the N-terminal part of echinoderm
microtubule-associated protein-like-4 (EML4) and the kinase
domain of ALK, has been intensively studied as a therapeutic
target in NSCLC. Accordingly, numerous ALK inhibitors have
been reported as recently reviewed in a comprehensive
fashion,6, 7 and five ALK inhibitors including crizotinib, ceritinib,
alectinib, brigatinib and Lorlatinib were approved by the US FDA
(Fig 1).8, 9 Patients have shown excellent response rates to these
ALK inhibitors; however, they inevitably relapse due to the
emergence of resistance.10-14 Therefore, identification of
structurally diverse ALK inhibitors is important for providing
novel therapeutic options for patients harboring oncogenic
ALK.15 Recently, our group reported new 4-phenoxyquinoline-
based inhibitors targeting ALK WT and the L1196M
Fig 1. Chemical structures of representative ALK inhibitors.
An understanding of intracellular drug behavior such as cell
membrane penetration, distribution in cells, and target
engagement in biological systems is of enormous value. A
tagged drug conjugated with a fluorescent dye can be broadly
used to monitor the time course of drug distribution and its fate,
and study biological processes in cells, animals, and humans
with a noninvasive and nonradioactive procedure.17-20 However,
tethering an additional fluorescent dye in the drug molecule
a.Department of Chemistry, Korea Advanced Institute of Science and Technology
(KAIST), Daejeon 34141, Republic of Korea; E-mail: hongorg@kaist.ac.kr (S.Hong)
ywjung@kaist.ac.kr (Y. Jung). Fax: 82 42 350 2810; Tel: 82 42 350 2811.
b.Center for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science
(IBS), Daejeon 34141, Republic of Korea.
Electronic Supplementary Information (ESI) available: Experimental procedures,
characterization data, and 1H and 13C NMR spectra for all compounds. See
DOI: 10.1039/x0xx00000x
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often causes negative effects on binding affinity, cell benzothiazole substituted analogs, showed that installation of
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DOI: 10.1039/C8OB02874E
permeability, toxicity, and in vivo activity. For this reason, these groups induces a red-shift in the emission wavelength and
inherently autofluorescent drugs are more beneficial than a significant increase in quantum yield (3f: ϕF = 0.32, 3g: ϕF =
fluorophore-conjugated compounds and can be considered as 0.72, 3h: ϕF = 0.72). Furthermore, the addition of a 7-alkoxy or
an ideal species for tracking its translocation and disposition in 7-amino substituents influences the pathway of radiative decay,
cells.21-28 Coumarin constitute a major class of naturally resulting in an increased value of quantum yield.41 It is
occurring compounds, and various synthetic derivatives exhibit remarkable to note that the installation of 7-amino substituent
a broad range of biological activities as well as photophysical improved the inhibitory activities with respect to ALK.
properties.29-34 Therefore, significant efforts have been directed Because 3h displayed promising potency against ALK and good
toward constructing and designing the coumarin structural photophysical
properties
among
tested
coumarin
motif having valuable optical properties and biological analogs, 3h was selected as a promising starting point for the
activities.35-40 In this study, we aim to identify novel fluorescent development of new fluorescent ALK inhibitors.
ALK inhibitors in which the coumarin fluorophore was modified
to be a pharmacophore capable of inhibiting ALK. Herein, we Table 1. Structures and absorption/excitation wavelength of coumarin
report studies on the identification of new coumarin-based derivatives.
fluorescent ALK inhibitors and demonstrate visualization of
subcellular localization in living cells by fluorescence
microscopy.
Compd
Structure
λabsa (nm) λemib (nm)
3a
324
435
Results and discussion
Identification of 3-heteroarylcoumarin derivatives as
fluorescent ALK inhibitors. With the goal of developing novel
fluorescent ALK inhibitors, we initiated our study by testing
fluorophore derivatives in our in-house coumarin-based library
which was constructed by the direct cross-coupling of a
coumarin core and various heteroarenes.30 To identify the
suitable structures having both inhibitory activity and
fluorescence, percent enzyme activity at 100 μM against ALK
and photophysical properties were examined as a primary
screen, summarized in Fig 2 and Table 1. Among these coumarin
derivatives that exhibited a broad emission range over 400-600
3b
3c
3d
295
256
326
N.D. c
N.D. c
403
3e
3f
305
383
460
538
nm, 4-methylcoumarin
derivative 3a showed
desirable
fluorescent efficiency but weak ALK inhibitory activity (ϕF = 0.46,
enzyme inhibition = 15%). Compared to the 4,7,8-substituents
(
3a), substitution of an amide or phenyl group at the C3-position
of coumarin led to weakening optical properties (3b 3d), albeit
3-phenylcoumarin derivative 3d displayed enhanced ALK
inhibition. A comparison of 3d with 3e 3h, the C3 thiazole or
-
-
3g
394
469
% enzyme inhibition
Quantum Yield (FF)
100
80
60
40
20
0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
3h
446
494
F
a Only the longest absorption maxima are shown. b Excited at the maximum
excitation wavelength. c N.D. = not detected.
As a strategy to increase potency, we initially explored the C3
substituent space on coumarin and designed several 3-
heteroarylcoumarin derivatives. To obtain the structural insight into
the inhibitory activities, the interaction patterns of coumarin in the
ATP-binding sites of ALK were investigated in the comparative
fashion. Fig 3 shows the lowest energy conformation of 3h in the ATP
binding pocket of ALK (PDB ID: 2XP2)42 as calculated with the
3a 3b 3c 3d 3e 3f 3g 3h
Fig 2. Percent enzyme activity at 100 μM and fluorescence quantum yield of
coumarin derivatives.
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Discovery Studio 4.5 software. Molecular simulation of 3h-ALK Arg1120 is located at the upper lip of the ATP biVniedwinAgrticpleoOcnklienet
DOI: 10.1039/C8OB02874E
complex revealed that the carbonyl oxygen of coumarin core and stretches out into the solvent exposed exterior.
establishes a hydrogen bond with the backbone amide group of a Interestingly, the side chain of Arg1120 shows conformational
residue (Met1199) in the hinge region of ALK. As a check for the variation in several X-ray crystal structures of the ALK kinase
presence of a peripheral binding pocket nearby a catalytic lysine, the domain: the downstretched mode of Arg1120 (Fig 4)43,
calculated binding modes in this site were analyzed in detail. Because 44 could cause a putative steric clash with the rotatable
Lys1150 is located adjacent to the 6-position of the benzothiazole diethylamine group. Based on our structural analysis, the
subunit, maintaining a distance within 3.9 Å, we hypothesized that diethylamine group was replaced with cyclic functionalities to
the introduction of a suitable hydrogen bond acceptor at the C6- evaluate the substituent space of the C7 position of coumarin.
position of benzothiazole might improve the overall ALK inhibitory
activity. To assess this hypothesis, we installed a methoxy group into Table 2. Structure-activity relationship information of 3-
this position (4d), which led to a slight enhancement of inhibitory heteroarylcoumarin derivatives.
activity against ALK (3h, IC50 = 2.63 μM; 4d, IC50 = 1.60 μM), indicating
that this enhancement of potency most likely stemmed from the
strengthening of hydrogen bonds between the inhibitor and Lys1150.
a
b
IC50
λabs
λemi
c
Compd
R
Ar
ϕF
(μM) (nm) (nm)
3h
2.63 446 494 0.54
2.80 433 482 0.36
70.9 454 503 0.78
4a
4b
4c
39.2 420 481 0.57
4d
5a
5b
1.60 451 502 0.76
0.51 426 500 0.29
4.10 428 497 0.50
Fig 3. Calculated binding mode of 3h in the ATP binding pocket of ALK (PDB
ID: 2XP2). The blue broken line indicates a hydrogen bond. A red dotted circle
shows the proximity of the C6-position of benzothiazole to Lys1150.
Optimization of ALK inhibitory activity via structure-based
drug design. With respect to improving the inhibitory activity,
we investigated the effect of 3-heteroaryl derivatives and
substituents attached to the C7-position. For the structure-
activity relationship (SAR) analysis, a survey of C3-benzothiazole
analogs was conducted and IC50 values determined. Conversion
of benzothiazole (3h) into benzoxazole (4a) showed a subtle
effect on inhibitory activity but a slightly negative effect on
fluorescence. Substitution of the phenyl group at the C6-
position of benzothiazole (4b, IC50 = 70.9 μM) was detrimental
5c
8.80 436 500 0.84
6a
6b
1.75 407 489 0.46
22.1 379 465 0.55
6c
> 100 nd
nd
nd
to the potency compared with the parent molecule (3h, IC50
=
2.63 μM). A caffeine-based analog (4c), which also provides
intense photonic luminescence, exhibited weak ALK inhibitory
activity with an IC50 value of 39.2 μM. The negative results
of 4b and 4c could be due to steric hindrance between the
inhibitor and amino acid residues in the ALK active site. To
improve the ALK inhibitory activity of 4d, we further analyzed
the structural features of the neighboring amino acids in the
solvent front of the ATP binding site. The calculated binding
mode of 4d (Fig 4) revealed that the 7-diethylamino group of
coumarin extends toward the solvent-exposed region and is
surrounded by several amino acid residues. For example,
a Only the longest absorption maxima are shown. b Excited at the
maximum excitation wavelength. c Fluorescence quantum yield. d nd:
not determined because of low potency.
The installation of the morpholine or piperazine moieties was
intended to improve the aqueous solubility of these derivatives,
which is required as a probe for real-time monitoring the
cellular distribution. Of particular significance is the observation
that the substitution with morpholine group yielded more
potent inhibitor 5a with submicromolar activity (5a, IC50 = 0.51
μM). According to the molecular docking study as depicted in
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IC50 = 22.1 μM). Investigation of alternate moieties such as 3-
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methoxyphenyl-thiazole group revealed that the 6-
DOI: 10.1039/C8OB02874E
methoxybenzothiazole was optimal for this series, which is in
turn responsible for a weaker biochemical potency of 6c (IC50
>
100 μM). These results confirm the importance of forming a
stable hydrogen bond of 6-methoxybenzothiazole with Lys1150
to enhance the inhibitory activity (Fig 5).
Next, the representative compounds 5a and 6a45 were
further tested against the most common secondary ALK
mutants such as L1196M, G1269A and G1202R (Table 3).
Compound 5a showed potent inhibitory activities against three
ALK mutants (L1196M IC50 = 0.27 μM; G1269A IC50 = 0.30 μM;
G1202R IC50 = 0.59 μM). Compound 6a displayed an IC50 value
of 0.45 μM for L1196M and compatible enzymatic inhibitory
activity against G1269A and G1202R with that against WT ALK.
Fig 4. Predicted binding mode of compound 4d in the ATP binding pocket
of ALK (PDB ID: 2XP2). Two possible locations of the Arg1120 side chain
reported in PDB 3AOX43 was superimposed.
Table 3. IC50 values of compounds against ALK WT and mutants.
ALK mutant IC50a (μM)
Compd
WT
0.51
1.75
L1196M
0.27
G1269A
0.30
G1202R
0.59
5a
6a
0.45
2.30
1.82
a The IC50 measurements were performed at the Reaction Biology Corp
(Malvern, PA, USA).
Monitoring molecular behavioral variability in cells by
confocal microscopy. Among the fluorescent ALK inhibitors
with
compounds 5a and 6a were selected for further investigation of
the imaging ability of its molecular behavior.
Compound 5a seems to possess more favorable
desirable
inhibitory
activities,
two
leading
physicochemical properties than 6a based on calculated AlogP
values (5a, ALogP = 3.70; 6a, ALogP = 3.84, predicted by
Discovery Studio 4.5 Client). In the present study, HeLa cells was
used to monitor the general behavior of our fluorescent
inhibitors such as cell penetration and cellular distribution. HeLa
Fig 5. Predicted binding mode of compound 5a in the ATP binding pocket of
ALK (PDB ID: 2XP2).
cells
were
incubated
with 5a or 6a at
10
μM
Fig 5, 5a appears to be stabilized by multiple hydrophobic
interactions with various residues including Leu1122, Val1130,
and Leu1256. The improved binding affinity can be rationalized
by additional hydrophobic interaction of cyclic fragment and
Ala1200, Arg1120 and Glu1132 that stabilizes ligand
concentration for 1 h. Unbound compounds were washed, and
the resulting cells were further incubated for 4 h and 12 h. After
treatment of 5a for 1 h, visible fluorescence was clearly
detected in both plasma membrane and cytoplasm of HeLa cells,
which maintained even after 12 h incubation (Fig 6). To our
delight, we also observed the fluorescence at membrane region
that might be evidence of membrane penetration that provided
the feasibility of monitoring a time-dependent morphology
change of membrane (Fig 6B and C). On the other
hand, 6a seems to be attached to cell surface plasma
membrane after 1 h treatment due to the high lipophilicity.
Therefore, the cell membrane penetration of 6a appear to be
inferior than that of 5a. When the fluorescence of 6a was
monitored after 4 h or 12 h treatment, the compound was
gradually distributed into the cytoplasm (Fig 7B and 7C). These
results provide a visible insight of molecular behavior such as
cell membrane penetration and intracellular distribution of
fluorescent compounds as well as a way to monitor the cell
morphology changes. The present 5a can be used to not only
functionally alter ALK activity but also monitor drug
binding.
Moreover,
compound 5a showed
promising
fluorescence emission (5a, λemi = 500 nm, ϕF = 0.29) and Stokes
shift (Δλ ≥ 70 nm), which can be beneficial as a biological
imaging study tool.21 The enzymatic inhibitory activity of the
derivatives was decreased when more sterically hindered
heterocycles
such
as N-methyl
or N-Boc
piperazine moiety were introduced (5b, IC50 = 4.1 μM; 5c, IC50
=
8.8 μM), indicating that the morpholine group is most suitable
for this position in benzothiazole core. Replacement of the
morpholine with a methoxy group resulted in reduction of
inhibitory activity against ALK (6a, IC50 = 1.75 μM). The binding
energy of 6a in docking simulation was much lower than that
of 5a (5a, -CDocker energy = 21.8214; 6a, -CDocker energy =
17.6135, calculated by Discovery Studio 4.5), revealing that loss
of van der Waals interaction between morpholine and small
pocket located in solvent front inevitably culminates in the
weakening of the binding affinity of 6a. Next, importance of the
hydrogen bond network in 6-methoxybenzothiazole was distributions, which are known to be important indicatives of
further demonstrated by removal of the methoxy group (6b
,
various cancerous cells.
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preparation of 3-heteroarylcoumarin derivatives.30 The focused
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DOI: 10.1039/C8OB02874E
compound library was established by this synthetic method allowing
for rapid exploration of the SAR profile of 3-heteroarylcoumarin
derivatives (Scheme 1). To install heteroaromatic rings, the C3
position of coumarins 7 was first brominated using a mixture of
oxone and hydrobromic acid in dichloromethane at room
temperature, and then treating the product with Et3N. Subsequently,
various heteroaryl groups were installed at the C3 position through
a Pd-catalyzed direct cross-coupling of 3-bromocoumarins with
heteroarenes to afford the desired products. For compounds bearing
morpholine or piperazine rings at the C7 position, the 7-fluoro-
coumarin intermediate 9 was further converted to target
compounds (5a-5c) using the SNAr reactions with appropriate amines.
Fig
6. Confocal imaging of the intracellular disposition of
compound 5a in HeLa cells. Cells were incubated with a 10 μM solution
of compound for 1 h. (A) Cells after 1 h upon treatment of 5a. (B) Cells
after 4 h upon treatment of 5a. (C) Cells after 12 h upon treatment
of 5a. Left: transmitted light image; middle: fluorescence microscopic
image; right: overlaid image. Scale bar, 10 µm
Scheme 1. Synthetic route for 3-heteroarylcoumarins. Reagents and
conditions: (a) Oxone, 2N HBr, CH2Cl2, rt, 2 h, then TEA, rt, 2h; (b)
Heteroarenes, Pd(PPh3)4, CuI, Cs2CO3, 1,4-Dioxane, 110 oC, 6 h; (c) DIPEA,
Acetonitrile, 90 oC, 12 h.
Conclusions
In summary, we discovered a family of coumarin-based ALK
inhibitors serving as a dual function in exhibiting fluorescence
and inhibiting ALK. Guided by structure-based design and
analysis of photophysical properties, we successfully identified
compound 5a with potent activity and significant fluorescence
properties (ALK IC50 = 0.51 μM, λemi = 500 nm, ϕF = 0.29). Cell
imaging after treatment of 5a and 6a showed the insight of
molecular behavior such membrane penetration and
intracellular distribution and provided a way to monitor cellular
morphology changes such as deconstruction of lipid bilayer. The
combined features of newly discovered fluorescent ALK
inhibitors would be useful in biological or pharmaceutical
research enabling visualization of subcellular localization in
living cells by fluorescence microscopy.
Fig 7. Confocal imaging of the intracellular disposition of compound 6a in
HeLa cells. Cells were incubated with 10 μM solution of compound for 1 h.
(A) Cells after 1 h upon treatment of 6a. (B) Cells after 4 h upon treatment
of 6a. (C) Cells after 12 h upon treatment of 6a. Left: transmitted light image;
middle: fluorescence microscopic image; right: overlaid image. Scale bar, 10
µm.
Synthesis of coumarin derivatives. Our group reported an efficient
direct C–H cross-coupling approach to heteroaryl coumarins using a
Pd/Cu catalytic system, which enabled the straightforward
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MHz, Chloroform-d) δ 8.92 (d, J = 1.9 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H),
7.95 (d, J = 8.0 Hz, 1H), 7.49 (dd, J = 7.3, 4.1 Hz, 2H), 7.36 (t, J = 7.6
Experimental
General
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DOI: 10.1039/C8OB02874E
Hz, 1H), 6.68 (dt, J = 9.0, 2.2 Hz, 1H), 6.57 (d, J = 2.3 Hz, 1H), 3.47
(q, J = 7.4 Hz, 4H), 1.26 (t, J = 7.2 Hz, 6H). 13C NMR (150 MHz,
Chloroform-d) δ 161.8, 161.0, 157.0, 152.6, 152.1, 142.0, 136.3,
130.8, 126.0, 124.4, 122.1, 121.6, 112.5, 109.9, 108.7, 97.0, 45.1,
12.5. HRMS (ESI+) m/z calcd. for C20H18N2NaO2S+ [M + Na]+: 373.0981,
found: 373.0984.
Unless otherwise stated, reactions were performed in flame-dried
glassware and monitored by TLC. Analytical thin layer
chromatography (TLC) was performed on pre-coated silica gel 60
F254 plates and compounds were visualized by UV light (254 and 365
nm). Flash column chromatography was undertaken on silica gel
(400-630 mesh) and ethyl acetate, n-hexane were used as eluents for
product purification. 1H and 13C NMR were recorded in CDCl3 or
CD2Cl2 solution on 400 or 600MHz instrument. All chemical shifts
were quoted in parts per million (ppm) referenced to the appropriate
solvent peak or 0.0 ppm for tetramethylsilane. The following
abbreviations were used to describe peak splitting patterns when
appropriate: br = broad, s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, dd = doublet of doublet, td = doublet of triplet, ddd =
doublet of doublet of doublet. Coupling constant, J values were
reported in hertz unit (Hz). Mass spectral data were obtained from
KAIST Basic Science Institute by using ESI method. Commercial grade
reagents and solvents were used without further purification except
as indicated below.
3-(1,3-Benzoxazol-2-yl)-7-(diethylamino)chromen-2-one
(4a). Compound 4a was prepared (yellow solid, 8.4 mg, 2 steps- 24%
overall yields) according to GP I and GP II. 1H NMR (600 MHz,
Methylene Chloride-d2) δ 8.60 (s, 1H), 7.73 (dd, J = 5.9, 3.2 Hz, 1H),
7.59 (dd, J = 5.9, 3.3 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.34 (dd, J = 6.0,
3.2 Hz, 2H), 6.67 (dd, J = 8.9, 2.4 Hz, 1H), 6.53 (d, J = 2.3 Hz, 1H), 3.46
(q, J = 7.1 Hz, 4H), 1.23 (t, J = 7.2 Hz, 6H). 13C NMR (150 MHz,
Methylene Chloride-d2) δ 160.1, 157.8, 157.6, 152.6, 150.4, 145.4,
142.1, 130.6, 124.8, 124.4, 119.7, 110.3, 110.0, 109.7, 108.1, 96.7,
45.1, 12.2. HRMS (ESI+) m/z calcd. for C20H18N2NaO3+ [M + Na]+:
357.1210, found: 357.1219.
7-(Diethylamino)-3-(6-phenyl-1,3-benzothiazol-2-yl)chromen-2-
one (4b). Compound 4b was prepared (dark red solid, 8.1 mg, 2
steps- 18% overall yields) according to GP I and GP II. 1H NMR (600
MHz, Methylene Chloride-d2) δ 8.93 (s, 1H), 8.15 (d, J = 1.7 Hz, 1H),
8.02 (d, J = 8.4 Hz, 1H), 7.73 (dd, J = 8.6, 1.7 Hz, 1H), 7.71 – 7.65 (m,
2H), 7.52 (d, J = 8.9 Hz, 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.4 Hz,
1H), 6.71 (dd, J = 8.9, 2.3 Hz, 1H), 6.57 (d, J = 2.3 Hz, 1H), 3.47 (q, J =
7.2 Hz, 4H), 1.24 (t, J = 7.1 Hz, 6H). 13C NMR (150 MHz, Methylene
Chloride-d2) δ 162.0, 160.9, 157.1, 152.3, 151.9, 142.0, 140.7, 137.6,
136.9, 130.8, 128.8, 127.3, 127.2, 125.7, 122.0, 119.6, 112.1, 110.0,
108.6, 96.8, 45.1, 12.2. HRMS (ESI+) m/z calcd. for C26H23N2O2S+ [M +
H]+: 427.1475, found: 427.1488.
General procedure I (GP I) for 3-bromocoumarins.
Coumarin derivative (1 equiv.) and Oxone (1.2 equiv.) were
dissolved in CH2Cl2 (0.25 M). 2 N HBr solution (HBr 2.2 equiv.) was
added and the reaction mixture was stirred at room temperature.
After 2 h, Et3N (3 equiv.) was added and stirred for additional 2 h. The
reaction mixture was diluted with CH2Cl2 and water, and then the
organic compounds were extracted by CH2Cl2. The organic layer was
dried over Na2SO4 and evaporated. The residue was purified by flash
column chromatography on silica gel (hexanes : EtOAc = 6 : 1) to give
the desired product.
General procedure II (GP II) for 3-heteroarylcoumarins.
3-Bromocoumarin derivative (1 equiv.), heteroarene (2 equiv.),
Pd(PPh3)4 (2.5 mol%), CuI (3 equiv.), and Cs2CO3 (2 equiv.) were
combined in 1,4-Dioxane under air. The reaction mixture was heated
to 110 oC with stirring, and monitored by TLC using ethyl acetate and
n-hexane as the mobile phase. After disappearance of starting
material, the reaction mixture was diluted and filtered through Celite
with CH2Cl2. After removal of solvent, the residue was purified by
flash column chromatography on silica gel to provide the desired
product.
8-[7-(Diethylamino)-2-oxo-chromen-3-yl]-1,3,7-trimethyl-purine-
2,6-dione (4c). Compound 4c was prepared (yellow solid, 14.5 mg, 2
steps- 34% overall yields) according to GP I and GP II. 1H NMR (400
MHz, Methylene Chloride-d2) δ 8.02 (s, 1H), 7.39 (d, J = 8.9 Hz, 1H),
6.67 (dd, J = 8.9, 2.5 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 3.91 (s, 3H), 3.55
(s, 3H), 3.47 (q, J = 7.1 Hz, 4H), 3.36 (s, 3H), 1.24 (t, J = 7.1 Hz, 6H). 13
C
NMR (100 MHz, Methylene Chloride-d2) δ 159.7, 158.0, 155.7, 152.6,
152.0, 148.6, 148.4, 147.7, 130.5, 110.0, 109.0, 108.4, 97.2, 45.4,
34.0, 29.8, 28.0, 12.6. HRMS (ESI+) m/z calcd. for C21H23N5NaO4+ [M +
Na]+: 432.1642, found: 432.1667.
General procedure III (GP III) for 7-substituted 3-
heteroarylcoumarins.
7-(Diethylamino)-3-(6-methoxybenzo[d]thiazol-2-yl)-2H-chromen-
2-one (4d). Compound 4d was prepared (yellow solid, 9.1 mg, 2
steps- 17% overall yields) according to GP I and GP II. 1H NMR (400
MHz, Methylene Chloride-d2) δ 8.83 (s, 1H), 7.86 (d, J = 8.9 Hz, 1H),
7.50 (d, J = 8.9 Hz, 1H), 7.41 (d, J = 2.5 Hz, 1H), 7.08 (dd, J = 8.9 Hz,
2.5 Hz, 1H), 6.70 (dd, J = 8.9, 2.5 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 3.89
(s, 3H), 3.47 (q, J = 7.1 Hz, 4H), 1.25 (t, J = 7.1 Hz, 6H). 13C NMR (100
MHz, Methylene Chloride-d2) δ 161.4, 159.6, 157.8, 157.3, 152.5,
147.7, 141.6, 138.1, 131.0, 123.1, 116.1, 112.9, 110.3, 108.9, 104.0,
97.2, 56.2, 45.4, 12.6. HRMS (ESI+) m/z calcd. for C21H20N2NaO3S+ [M
+ Na]+: 403.1087, found: 403.1092.
7-Fluoro-3-(6-methoxy-1,3-benzothiazol-2-yl)chromen-2-one
(1
equiv.) was dissolved in Acetonitrile at room temperature under air.
To this solution, cyclic aliphatic amines (2 equiv.) and DIPEA (3 equiv.)
were added. The reaction mixture was stirred at 90 oC for 12 h. After
completion of reaction, the mixture was cooled to rt. Then, the
precipitate was collected by filtration with cold Acetonitrile and
water to give the desired product.
3-(1,3-Benzothiazol-2-yl)-7-(diethylamino)chromen-2-one
(3h). Compound 3h was prepared (yellowish orange solid, 8.0 mg, 2
steps- 21% overall yields) according to GP I and GP II. 1H NMR (600
6 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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Journal Name
3-(6-Methoxy-1,3-benzothiazol-2-yl)-7-morpholino-chromen-2-
one (5a). Compound 5a was prepared (yellowish orange solid, 15.0 332.0352, found: 332.0371.
mg, 3 steps- 34% overall yields) according to GP I, GP II and GP III. 1H
ARTICLE
100.6, 56.1. HRMS (ESI+) m/z calcd for C17H11NNaO3S+ [M + Na]+:
View Article Online
DOI: 10.1039/C8OB02874E
NMR (400 MHz, Chloroform-d) δ 8.89 (s, 1H), 7.93 (d, J = 8.9 Hz, 1H), 7-Methoxy-3-[5-(2-methoxyphenyl)thiazol-2-yl]chromen-2-one
7.55 (d, J = 8.8 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.12 (dd, J = 8.9, 2.5 (6c). Compound 6c was prepared (yellow solid, 17.0 mg, 2 steps- 19%
Hz, 1H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H), 6.78 (d, J = 2.3 Hz, 1H), 3.91 (s, overall yields) according to GP I and GP II. 1H NMR (600 MHz,
3H), 3.90 – 3.84 (m, 4H), 3.41 – 3.33 (m, 4H). 13C NMR (150 MHz, Chloroform-d) δ 8.99 (s, 1H), 7.69 (s, 1H), 7.68 (d, 1H), 7.62 (t, 1H),
Chloroform-d) δ 160.7, 158.5, 157.6, 156.1, 154.4, 147.2, 140.7, 7.59 (d, 1H), 7.39 (t, 1H), 6.98 (dd, 1H), 6.95 (d, 1H). 6.93 (dd, 1H),
138.0, 130.2, 123.0, 116.2, 115.4, 111.8, 111.0, 103.6, 100.1, 66.4, 3.93 (s, 3H), 3.91 (s, 3H). 13C NMR δ (150 MHz, Chloroform-d) δ 193.2,
55.8, 47.3. HRMS (ESI+) m/z calcd. for C21H18N2NaO4S+ [M + Na]+: 163.9, 160.1, 159.3, 155.5, 154.9, 139.6, 135.7, 130.2, 129.7, 118.6,
417.0880, found: 417.0872.
116.8, 115.9, 113.6, 112.8, 112.0, 110.0, 100.5, 56.0, 55.3. HRMS
(ESI+) m/z calcd for C20H15NNaO4S+ [M + Na]+: 388.0614, found:
388.0626.
3-(6-Methoxy-1,3-benzothiazol-2-yl)-7-(4-methylpiperazin-1-
yl)chromen-2-one (5b). Compound 5b was prepared (yellowish
orange solid, 12.0 mg, 3 steps- 26% overall yields) according to GP I, Virtually screened fluorophore derivatives including 3a-3g were
GP II and GP III. 1H NMR (600 MHz, Chloroform-d) δ 8.84 (d, J = 1.3 purchased from commercial source (InterBioScreen Ltd.,
Hz, 1H), 7.91 (dd, J = 8.9, 1.3 Hz, 1H), 7.50 (dd, J = 8.9, 1.4 Hz, 1H), Chernogolovka, Russia).
7.38 (dd, J = 2.5, 1.4 Hz, 1H), 7.10 (ddd, J = 8.9, 2.6, 1.3 Hz, 1H), 6.86
(ddd, J = 8.9, 2.5, 1.4 Hz, 1H), 6.75 (t, J = 1.7 Hz, 1H), 3.90 (d, J = 1.4 Fluorescence quantum yield measurement
Hz, 3H), 3.51 – 3.21 (m, 4H), 2.67 – 2.42 (m, 4H), 2.36 (d, J = 1.4 Hz,
Fluorescence quantum yields (ϕF) were measured by a combination
3H). 13C NMR (150 MHz, Chloroform-d) δ 160.8, 158.7, 157.5, 156.2,
system
of
a
spectrophotometer
(UV-2600
UV/Vis
154.3, 147.2, 140.8, 137.9, 130.2, 122.9, 116.1, 114.8, 112.0, 110.5,
103.6, 100.0, 55.8, 54.5, 47.1, 46.1. HRMS (ESI+) m/z calcd. for
C22H22N3O3S+ [M + H]+: 408.1376, found: 408.1383.
Spectrophotometer, SHIMADZU Corp. and V-530 UV/Vis
Spectrophotometer, Jasco, Inc.) with a fluorimeter (RF-6000 Spectro-
fluorophotometer, SHIMADZU Corp. and RF-5301PC Spectro-
fluorophotometer, SHIMADZU Corp.). Samples were prepared as
solutions by dilution of the fluorescent compound in DMSO (Table 1,
Fig 2) or CH2Cl2 (Table 2). Absorption λmax was measured with a
spectrophotometer scanning the 700-250 range wavelength while
fluorescence emission λmax and integrated intensity were analyzed
with MATLAB R2014b and Origin2018b software. The Sample
absorbance was maintained < 0.1 to minimize internal absorption.
The absolute quantum yield of a known fluorescent dye – fluorescein
and norharmane – was obtained to determine those of the sample
precisely (fluorescein : ϕF = 0.925 ± 0.015 in 0.1 N NaOH aqueous
solution, norharmane : ϕF = 0.58 in 0.1 N H2SO4 aqueous solution).
tert-Butyl 4-[3-(6-methoxy-1,3-benzothiazol-2-yl)-2-oxo-chromen-
7-yl]piperazine-1-carboxylate (5c). Compound 5c was prepared
(yellowish orange solid, 13.0 mg, 3 steps- 24% overall yields)
according to GP I, GP II and GP III. 1H NMR (400 MHz, Chloroform-d)
δ 8.87 (s, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.39
(d, J = 2.6 Hz, 1H), 7.11 (dd, J = 9.0, 2.6 Hz, 1H), 6.87 (dd, J = 9.0, 2.3
Hz, 1H), 6.76 (d, J = 2.4 Hz, 1H), 3.91 (s, 3H), 3.70 – 3.55 (m, 4H), 3.48
– 3.39 (m, 4H), 1.50 (s, 9H). 13C NMR (150 MHz, Methylene Chloride-
d2) δ 160.6, 158.5, 157.6, 156.2, 154.4, 154.2, 147.2, 140.8, 137.8,
130.3, 122.9, 120.1, 115.9, 114.8, 112.0, 110.5, 103.5, 99.8, 79.8,
55.7, 47.0, 28.1. HRMS (ESI+) m/z calcd. for C26H27N3NaO5S+ [M + Na]+:
516.1564, found: 516.1552.
Intracellular monitoring study
Human cervical cancer (HeLa) cells from ATCC with fewer than 15
passages were cultured as monolayers in complete growth media,
DMEM (Dulbecco’s Modified Eagle medium, Gibco, USA)
supplemented with 10% FBS (Fetal Bovine Serum, Gibco, USA) and 1 %
penicillin–streptomycin (Gibco, USA), at 37 °C under 5% CO2. Cells
were treated with 10 μM fluorescent compounds for 1 h and washed
3 times with DPBS. Treated cells were further incubated for 4 h and
12 h in complete growth media and fixed with 4% paraformaldehyde
for confocal imaging. Fluorescence images were obtained with a LSM
800 laser scanning confocal microscope (Carl Zeiss, LSM 800) using
100× oil objective lens. Optimized laser lines for 5a (λex 426 nm /
λem 500 nm) and 6a (λex 407 nm / λem 489 nm) were used.
7-Methoxy-3-(6-methoxy-1,3-benzothiazol-2-yl)chromen-2-one
(6a). Compound 6a was prepared (yellow solid, 11.0 mg, 2 steps- 33%
overall yields) according to GP I and GP II. 1H NMR (600 MHz,
Chloroform-d) δ 8.94 (s, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.61 (d, J = 8.6
Hz, 1H), 7.40 (d, J = 2.5 Hz, 1H), 7.13 (dd, J = 8.9, 2.5 Hz, 1H), 6.95
(dd, J = 8.6, 2.4 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 3.93 (s, 3H), 3.91 (s,
3H). 13C NMR (150 MHz, Chloroform-d) δ 164.0, 160.2, 157.9, 157.7,
155.8, 147.2, 140.7, 138.1, 130.3, 123.2, 117.1, 116.3, 113.8, 112.8,
103.5, 100.6, 56.0, 55.8. HRMS (ESI+) m/z calcd. for
C18H13NNaO4S+ [M + Na]+: 362.0458, found: 362.0464.
3-(Benzo[d]thiazol-2-yl)-7-methoxy-2H-chromen-2-one
(6b). Compound 6b was prepared (yellow solid, 15.5 mg, 2 steps- 53%
overall yields) according to GP I and GP II. 1H NMR (400 MHz,
Chloroform-d) δ 9.03 (s, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 8.2
Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.52–7.48 (m, 1H), 7.41–7.37 (m, 1H),
Conflicts of interest
There are no conflicts to declare.
6.93 (dd, J = 8.8, 2.4 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 3.90 (s, 3H). 13
C
NMR δ (100 MHz, Chloroform-d) δ 164.7, 160.9, 160.1, 156.2, 151.2,
142.7, 135.9, 130.9, 126.7, 125.4, 122.2, 121.8, 115.9, 114.1, 112.7,
Acknowledgements
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Organic & Biomolecular Chemistry
Page 8 of 9
ARTICLE
Journal Name
28. V. M. Yenugonda, T. B. Deb, S. C. Grindrod, S. Dakshanamurthy, Y.
This research was supported financially by the Institute for Basic
Science (IBS-R010-G1).
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Yang, M. Paige and M. L. Brown, BioorgD. OMI:e1d0..1C0h3e9m/C.8, O20B10128, 7149E,
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8 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02874E
N
S
OMe
N
H
R
[Pd]
S
+
N
O
O
Br
O
O
R
5a
O
IC50 = 0.51 M (ALK WT)
0.27 M (L1196M)
0.30 M (G1269A)
0.59 M (G1202R)
Coumarin-based ALK inhibitors were identified as a new template for the development of novel fluorescent ALK inhibitors,
which can be tracked using microscopy techniques.