Design, synthesis, and inhibitory activity of potent, photoswitchable mast cell activation inhibitors

Article abstract of DOI:10.1021/jm400115k

Allergic reactions affect millions of people worldwide. The need for new and effective antiallergic agents is evident, and insight into the underlying mechanisms that lead to allergic events is necessary. Herein, we report the design, synthesis, and activity of photoswitchable mast cell activation inhibitors. In mast cell degranulation assays, these inhibitors possess significantly greater potency than an original, chromone-based antiallergic agent. Furthermore, one of the photoswitchable inhibitors shows a significant difference in inhibitory activity between its two photoisomeric forms. Further optimization could ultimately lead to a photoswitchable compound suitable for studying mechanisms involved in allergic reactions in a novel manner, with activity addressable by light and with precise spatiotemporal control over events at the molecular level.

Full text of DOI:10.1021/jm400115k

Article
pubs.acs.org/jmc
Design, Synthesis, and Inhibitory Activity of Potent, Photoswitchable
Mast Cell Activation Inhibitors
Willem A. Velema,† Marco van der Toorn,‡ Wiktor Szymanski,*,† and Ben L. Feringa*,†
†Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The
Netherlands
‡Laboratory of Allergology and Pulmonary Diseases, Department of Pathology and Medical Biology, University Medical Centre
Groningen, 9713 GZ Groningen, The Netherlands
S
* Supporting Information
ABSTRACT: Allergic reactions affect millions of people worldwide. The need for new and effective antiallergic agents is evident,
and insight into the underlying mechanisms that lead to allergic events is necessary. Herein, we report the design, synthesis, and
activity of photoswitchable mast cell activation inhibitors. In mast cell degranulation assays, these inhibitors possess significantly
greater potency than an original, chromone-based antiallergic agent. Furthermore, one of the photoswitchable inhibitors shows a
significant difference in inhibitory activity between its two photoisomeric forms. Further optimization could ultimately lead to a
photoswitchable compound suitable for studying mechanisms involved in allergic reactions in a novel manner, with activity
addressable by light and with precise spatiotemporal control over events at the molecular level.
process.15 While the exact mechanism of their action is yet to
be elucidated, and other targets have been proposed, it is
INTRODUCTION
■
The prevalence of allergic diseases has increased over the last
hypothesized that chromones interfere with the calcium influx,
decades,1,2 and the search for suitable pharmacotherapeutical
which is necessary for degranulation.16,17
treatment is ongoing.3,4 Several options to treat allergic patients
Our aim was to design and synthesize a new class of
are available,5 including antihistamines, leukotriene inhibitors,
chromone derivatives which have higher potency than the
corticoids, and chromones. This last class of drugs has lost
available, chromone-based drugs such as disodium cromogly-
popularity, mainly due to their less pronounced effect and short
cate18 (DSCG, Figure 1A). DSCG inhibits mast cell activation,
duration of action.6 However, chromones show few side
and its structure−activity relationships are well studied.19 The
drug is a dimeric ligand and consists of two chromone moieties
linked by a spacer (Figure 1A). Altering the length of the spacer
changes the inhibitory activity of the drug.19,20 However, we
expected that the molecular structure of the spacer, rather than
length only, might also influence the drugs’ activity as was
shown before, for example, for opioid receptor antagonists.20
To investigate this, three different DSCG derivatives were
designed with an azobenzene photoswitch as a spacer (Figure
1A). Azobenzene molecules can be photoisomerized, changing
the molecule’s length, dipole moment, and geometry in a
effects,6 as opposed to the commonly used corticoids,7,8 which
renders them continuously interesting therapeutic agents.
Furthermore, they seem to have an interesting, yet not fully
understood, mechanism of action.9
Chromones stabilize mast cells,10 which are effector cells of
immediate hypersensitivity reactions and therefore play a key
role in allergic diseases.11 These cells can be found throughout
the human body, and they secrete granules upon activation.12,13
The granules contain inflammatory mediators, which induce
the symptoms of an allergic reaction, like edema, warmth, and
itchiness.12 Activation of mast cells occurs as a result of cross-
linking of high affinity IgE receptors on the cell surface by
allergens.14 Chromones bind to the cromolyn binding protein
(CBP) on the surface of mast cells and inhibit this activation
Received: January 24, 2013
Published: April 25, 2013
© 2013 American Chemical Society
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Figure 1. Principle and molecular structure of photoswitchable mast cell activation inhibitors. (A) Molecular structure of the mast cell activation
inhibitor disodium cromoglycate (DSCG) and the three designed photoswitchable inhibitors DAC, MAC and PAC. (B) Envisioned mode of action
of MAC and PAC. When two chromone groups (illustrated as red spheres) of the inhibitor in one of the photoisomeric forms are bound to the
cromolyn binding protein (CBP), mast cell activation is inhibited. When the molecule photoisomerizes to the other form, only one chromone group
can bind and mast cell activation is not inhibited.
a
Scheme 1
a
Reagents and conditions: (i) diethyl oxalate, NaOEt/EtOH, HCl (75%); (ii) H2SO4, HNO3, rt, 1 h (81%); (iii) H2, Pd/C, benzene, rt, 16 h (75%);
(iv) oxone, DCM/water, rt, 2 h (70%); (v) compound 3, glacial AcOH, rt, 2 d (43%); (vi) EtOH, NaOH, reflux, 2 h (91%).
reversible manner.21 In addition, their structure is essentially
different from that of the spacer of DSCG. This allows studying
the influence of both the structure and the length of the spacer
on inhibitory activity of the drug. Moreover, incorporating a
photoswitch into the DSCG molecule might allow changing its
inhibitory potency upon light irradiation. This eventually could
lead to optical control over mast cell activation with high
spatiotemporal resolution, as was shown with other bioactive
compounds.22,23 The information gained from these experi-
ments could provide more insight into the mechanisms by
which DSCG inhibits mast cell activation and the way it
interacts with the CBP.
Here we present the design and synthesis of three analogues
of DSCG, with an azobenzene photoswitch incorporated in
their structure. The inhibitory capacity of the molecules, named
diazochromone (DAC), meta-azochromone (MAC), and para-
azochromone (PAC) (Figure 1A), in both photoisomeric forms
was tested on human mast cell cultures. Remarkably, the
molecules showed to have a considerably higher inhibitory
effect on an evoked degranulation in mast cell cultures, as
compared to the common drug DSCG. Furthermore, PAC
showed a significant difference in inhibitory capacity in two
isomeric forms. It is anticipated that external control of mast
cell activation can be achieved by switching between the two
forms with light
RESULTS
■
Design of Photoswitchable Inhibitors. The structure of
photoswitchable inhibitors DAC, MAC, and PAC was inspired
by the known mast cell activation inhibitor DSCG. This drug
consists of two chromone groups linked by a spacer (Figure
1A). The length of the spacer influences the inhibitory activity
of the drug.19 We chose an azobenzene unit as the photoswitch
to connect the chromone units because the difference in
structure and length between the two forms (the trans and cis
isomer) of this photoswitch is well-defined and relatively
large.21,24 Azobenzenes are privileged photoswitches for usage
in bioactive compounds for their small size and stability in
aqueous environments.21
We hypothesized that both chromone groups need to bind to
the CBP for the drug to have an optimal inhibitory effect
(Figure 1B), as was shown for other bivalent compounds.25,26
With this in mind, we designed three photoswitchable
inhibitors called DAC, MAC, and PAC (Figure 1A). The first
photoswitchable inhibitor, DAC, was designed in such a way
that the azobenzene photoswitch intergrates two chromone
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a
Scheme 2
a
Reagents and conditions: (i) dimethyl oxalate, NaOMe/MeOH, HCl (33%); (ii) oxone, DCM/water, rt, 1 h (8a, 54%; 8b, 25%); (iii) m/p-
toluidine, glacial AcOH, rt, 16 h (9a, 55%; 9b, 86%); (iv) NBS, AIBN, CCl4, reflux, 16 h (10a, 35%; 10b, 42%); (v) Cs2CO3, MeCN, reflux, 5 h
(11a, 70%; 11b, 35%); (vi) EtOH, NaOH, reflux, 2 h (12a, 86%; 12b, 91%).
Figure 2. Photochemical properties of compounds DAC, MAC and PAC. (A−C) UV−vis absorption spectra of DAC (125 μM), MAC (128 μM),
and PAC (100 μM) in water. The solid lines represent the spectra of the nonirradiated forms and the dashed lines of the 365 nm irradiated forms.
(D−F) Reversible photochromism of photoswitchable inhibitors. (G−I) Thermal cis−trans isomerization at 37 °C.
groups in its structure. This reduced the length of the spacer as
compared to DSCG and rendered the molecule more rigid,
which could result in a larger difference in inhibitory activity
between the two photoisomeric forms. Compounds MAC and
PAC (Figure 1A) consist of an azobenzene moiety with two
chromone groups linked to it in the meta and para positions,
respectively. A methylene group was placed in between the
azobenzene and chromone groups to introduce more flexibility
in the molecule. This flexibility might be favorable when two
chromone groups need to interact with the CBP at the same
time (Figure 1B).
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Synthesis. The photoswitchable inhibitor DAC was
synthesized starting from chromone ethyl ester (1) (Scheme
1), which was obtained from hydroxyacetophenone and diethyl
oxalate.25 Compound 1 was converted into nitroso-chromone 4
via sequential nitration,28 reduction,28 and oxidation reactions.
Azobenzene formation from precursors 3 and 4 and a
subsequent saponification step afforded the photoswitchable
inhibitor DAC (6).
therefore its activity is a reliable measure for the degree of mast
cell degranulation.33 LAD2 cells were incubated with different
concentrations of DSCG and nonirradiated DAC, MAC, and
PAC for 30 min, after which degranulation was evoked by the
secretagogue compound 48/80.34 From Figure 3A, it is
apparent that DAC, MAC, and PAC show significantly greater
inhibitory activity than the original drug DSCG. MAC and PAC
seem to have an almost 100 times greater inhibitory potency
than DSCG. Furthermore, MAC and DAC show greater
efficacy than PAC.
At the concentration of compound 48/80 (1.0 μM) that was
used in this assay, DSCG did not show any inhibitory activity.
At lower concentrations of compound 48/80 (0.1 μM), DSCG
was able to inhibit mast cell degranulation (Figure 3B), albeit
with lower activity than MAC (∼100 times).
The inhibitory activity of the most potent inhibitor, MAC,
was further investigated using a histamine release assay35
(Figure 4). LAD2 cells were incubated with different
concentrations of DSCG and MAC for 30 min, after which a
degranulation was evoked by compound 48/80. At the assayed
concentrations DSCG did not show any inhibitory activity,
whereas MAC completely inhibited histamine release at a
concentration of 100 μM.
Next, the difference in inhibitory activity between the two
photoisomers of DAC, MAC, and PAC was determined. The
inhibitory activity was measured for each compound at two
concentrations that were on the steep part of the concentration
response curve, because at these concentrations a possible
difference in inhibitory activity is most pronounced. LAD2 cells
were incubated with either nonirradiated solutions of DAC,
MAC, and PAC or with solutions that were irradiated with 365
nm light prior incubation. The samples were irradiated in
DMSO and diluted in water to obtain a maximum
concentration of 1% DMSO (v/v). After evoking a
degranulation with compound 48/80, β-hexosaminidase release
was determined (Figure 5). We observed no significant
difference in inhibitory activity between the irradiated and
nonirradiated forms of MAC and DAC. However, a significant
difference was observed between the irradiated and non-
irradiated form of PAC. At a concentration of 10 μM, the
relative difference in inhibitory activity between the two forms
of PAC is 25%. The nonirradiated form, which consists of 91%
trans-PAC in the photostationary state (PSS), is more active
than the irradiated form, which is 81% cis-PAC in the PSS.
Compounds MAC and PAC were prepared in six steps as
depicted in Scheme 2. The key features of these syntheses
include a formation of a diazo compound by a reaction between
m/p-toluidine and m/p-nitrosotoluene (8a and 8b) to afford
methylated azobenzenes (9a and 9b), which were subjected to
radical bromination to yield compounds 10a and 10b. Via a
double Williamson ether formation, 5-hydroxychromone
methyl ester 7, obtained from 1,2-dihydroxyacetophenone
and dimethyl oxalate,29 was introduced to both methylene
groups of the azobenzenes 10a and 10b, resulting in 11a and
11b. A saponification step afforded the sodium salts of the
photoswitchable inhibitors MAC (12a) and PAC (12b).
Photoswitching of Designed Mast Cell Activation
Inhibitors. The photoswitchable behavior of DAC, MAC, and
PAC was studied using UV−vis spectroscopy and RP-HPLC.
The trans-isomers of DAC, MAC, and PAC have a character-
istic absorbance maximum between 300 and 400 nm in water.
This absorbance maximum decreases when the molecules are
photoswitched to their cis-isomers and a new absorbance
maximum appears around 430 nm (Figure 2A−C).30 Isosbestic
points at 405 nm (DAC), 270 nm (MAC), and 270 nm (PAC)
clearly illustrate the selective isomerization and reversibility of
the photoswitching process. Using RP-HPLC, the ratio of
photoisomers was determined for both the nonirradiated and
365 nm irradiated forms of DAC, MAC, and PAC (Table 1).
Table 1. Ratio of trans and cis Isomers of the
Photoswitchable Inhibitors under Different Conditions
a
DAC (trans:cis) MAC (trans:cis) PAC (trans:cis)
nonirradiated
99:1
88:12
45:55
38:62
91:9
irradiated (water)
irradiated (DMSO)
59:41
48:52
56:44
19:81
a
Ratios are determined at the isosbestic point (405 nm for DAC, 270
nm for MAC, 270 nm for PAC) with RP-HPLC (C18, flow 1 mL/min,
MeCN and H3PO4 pH = 3.0).
DISCUSSION
Higher photostationary states were obtained when the
photoswitchable inhibitors were irradiated with 365 nm light
in DMSO as compared to water (Table 1). Therefore, for
inhibitory activity studies (vide infra), the compounds were
switched at a high concentration in DMSO and diluted in water
to obtain a maximum concentration of 1% DMSO (v/v). The
inhibitors could be photoisomerized by alternating irradiation
at 365 nm and 400−700 nm for at least five times, showing
little fatigue (Figure 2D−F). Thermal cis−trans isomerization
was relatively slow at 37 °C (t1/2 ≥ 3 h for all compounds)
(Figure 2G−I). Because in the inhibitory activity studies (vide
infra) the incubation times were only 30 min, assaying the
activity of the cis-form without significant cis−trans conversion
was possible.
■
The chromone-based drugs are an attractive and routinely used
class of antiallergic agents because of their mast cell stabilizing
capability and the limited side effects.6 The main disadvantage
of this class of drugs is its low effectiveness.36 The novel,
azobenzene-bridged, bis-chromone compounds MAC and PAC
show an almost 100 times higher potency than the chromone
drug DSCG based on β-hexosaminidase release assays (Figure
3). This large increase in potency makes MAC and PAC
interesting potential antiallergic agents, especially if the side
effect pattern remains unchanged. Further investigation is
ongoing to determine the toxicity and side effects of these
compounds.
In earlier research by Hunter et al., the effect of the length of
the spacer between the two chromone groups on inhibitory
activity was explored.19 Inhibitors containing longer spacers,
consisting of alkylene chains, showed a decrease in activity
when the spacer exceeded six atoms.19 Spacer length is known
Inhibitory Activity. The inhibitory activity of DAC, MAC,
and PAC was determined on the LAD231 mast cell line using a
β-hexosaminidase release assay.32 β-Hexosaminidase is an
enzyme that is secreted by mast cells upon activation, and
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Figure 3. Concentration response curves of mast cell activation inhibitors (n = 3). (A) Degranulation was evoked by 1.0 μg/mL of cmpound 48/80.
The mast cell activation inhibitors MAC and PAC show inhibitory activity in the same concentration range with IC50 values of 10 and 28 μM,
respectively. DAC shows to be a less potent inhibitor with an IC50 value of 272 μM but is still more potent than the original drug DSCG, which did
not show any inhibitory activity under these conditions (IC50 > 1 mM) (see Supporting Information Figure S1). MAC and DAC show higher
efficacy than PAC. (B) Inhibitory activity of MAC and DSCG. Degranulation was evoked by 0.1 μg/mL of compound 48/80. MAC shows complete
inhibition at a concentration of 10 μM, whereas DSCG shows complete inhibition at a concentration of 1000 μM.
to modulate selectivity in bivalent ligands.20 Our results suggest
that the length of the spacer plays a role in the inhibitory
activity because there is a change in activity between the two
isomeric forms of PAC, which have a distinct difference in
distance between the chromone units. However, our results also
suggest that the molecular structure of the spacer, rather than
spacer length only, might be of great importance for activity.
Similar observations have already been reported earlier for
opioid receptor antagonists.20 MAC and PAC, which include an
azobenzene group as a spacer and might be considered lengthy
compared to the original linker in DSCG, showed to be almost
100 times more potent than the model compound DSCG in a
degranulation assay based on the activity of β-hexosaminidase
released from granules.
To exclude the false positive result which could stem from
the inhibition of β-hexosaminidase activity by MAC, we also
assayed the amount of histamine released from the granules
(Figure 4). The results obtained in this experiment confirmed
the much higher activity of MAC in stabilizing mast cell
activation than DSCG.
Several modes of actions for chromones have been proposed,
apart from CBP binding, including the blockage of chloride
channels37 and G-protein-coupled-receptor 35 binding.38 It is
Figure 4. Histamine release assay of MAC and DSCG (n = 3).
Degranulation was evoked by 1.0 μg/mL of compound 48/80. MAC
completely inhibited histamine release at a concentration of 100 μM.
The fluorescence detected was comparable to the control measure-
ment without compound 48/80. DSCG did not show any inhibitory
activity at the studied concentrations.
Figure 5. Inhibitory activity of the nonirradiated and 365 nm light-irradiated forms of DAC, MAC and PAC. (A,B) No difference between the two
forms of DAC and MAC were observed. (C) A difference in activity was observed between the two forms of PAC. Nonirradiated PAC was 25%
more active than irradiated PAC at a concentration of 10 μM.
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supernatant was transferred to a 96-well plate. To this, 100 μL of a
2 mg/mL solution of p-nitrophenyl-N-acetyl-D-glucosaminidine in
citrate buffer pH 4.5 was added. The plate was incubated at 37 °C for
1 h. The reaction was stopped by adding 100 μL of 0.4 M glycine
solution, and the absorbance was measured on a fluorescent plate
reader at a wavelength of 405 nm. Subsequently, the percentage of
released β-hexosaminidase was determined by comparison with the
absorption of cell lysate of the same cells using the following formula:
possible that the newly designed inhibitors exhibit an effect via
binding to multiple receptor types, which makes it harder to
explain the effect of changes in molecular structure on activity.
For instance, the effect via binding to various receptor types
could provide an explanation for the observed lower efficacy of
PAC, as compared to DAC, MAC, and DSCG.
The lack of difference in inhibitory activity between the two
forms of MAC, which differ significantly in length, also
confirms our observation that the structure and geometry of
the spacer, rather than its length, is important for the
interaction between mast cell activation inhibitors and the CBP.
Because there is a difference in inhibitory activity between
the cis and trans isomer of PAC, this photoswitchable inhibitor
might be a useful tool to study the events involved in mast cell
degranulation. To completely control the state of mast cell
activation as an “on/off” event, it is necessary to enhance the
difference in activity between the two forms of a photo-
switchable inhibitor. To achieve this, first the PSS of the least
active isomer should be optimized. In case of PAC, the least
active isomer is the cis form. The PSS of irradiated PAC
consists of 81% cis and 19% trans. This means that when PAC is
photoisomerized, still 19% of the more potent isomer is
present. If this percentage can be reduced, a larger difference in
activity would be obtained. One possibility to achieve this is the
use of bidirectional azobenzenes.39−42 In this class of
azobenzenes, the UV−vis absorption maxima of the trans and
cis form do not overlap, which allows better control over
photoisomerization and results in a high PSS for both forms.
Second, the optimal distance between the two chromone
groups in the molecular structure of the inhibitor needs to be
investigated. Then a photoswitchable inhibitor could be
designed with maximum activity in one photoisomeric form
and minimum activity in the other form.
supernatant
× 100% = %released β‐hexosaminidase
supernatant + lysate
Histamine Assay. Histamine release was measured according to a
published method.35 In short, 80 μL of supernatant was transferred to
a 96-well plate. To this, 20 μL of a 1 M aqueous NaOH solution was
added. Next, 20 μL of an 0.25% o-phthalaldehyde in MeOH was added
and incubated for 4 min at room temperature. Then 10 μL of a 3 M
aqueous HCl solution was added and the fluorescence was measured
on a fluorescent plate reader (λex = 360 nm, λem = 450 nm).
Inhibitory Activity Tests. LAD2 cells were suspended in HEPES
buffer at a concentration of 50000 cells/mL for the β-hexosaminidase
release assay and 1000000 cells/mL for the histamine release assay.
Then 80 μL of cell suspension was transferred to a microreaction cup,
to which 10 μL of inhibitor solution was added. This was incubated at
37 °C for 30 min, after which 10 μL of a compound 48/80 solution
was added. Next, the cell suspension was incubated for 30 min at 37
°C and the β-hexosaminidase or histamine release was assayed.
Photoswitching Experiments. Irradiation experiments were
performed with a spectroline ENB-280C/FE UV lamp (365 nm)
and Thor Laboratories OSL1-EC fiber illuminator (white light).
Synthesis. General. For synthesis, all chemicals were obtained
from commercial sources and used as received unless stated otherwise.
Solvents were reagent grade. Thin-layer chromatography (TLC) was
performed using commercial Kieselgel 60, F254 silica gel plates. Flash
chromatography was performed on silica gel (Silicycle Siliaflash P60,
40−63 m, 230−400 mesh). Drying of solutions was performed with
MgSO4, and solvents were removed with a rotary evaporator.
Chemical shifts for NMR measurements were determined relative to
the residual solvent peaks. The following abbreviations are used to
indicate signal multiplicity: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; brs, broad signal; appt, apparent triplet. HRMS (ESI)
spectra were obtained on a Thermo Scientific LTQ Orbitrap XL.
Melting points were recorded using a Buchi melting point B-545
apparatus. UV−vis absorption spectra were recorded on an Agilent
8453 UV−visible spectrophotometer using Uvasol grade solvents. All
compounds whose IC50 values were determined had purities ≥95%.
Purities were determined with analytical RP-HPLC performed on a
JASCO PU-980 chromatography system using a GRACE Alltima HP
C18 5 μ column. Compounds were detected with a JASCO UV-1575
UV−vis intelligent detector.
CONCLUSIONS
■
Three photoswitchable mast cell activation inhibitors have been
designed and synthesized. All three inhibitors are more potent
than the commercially available drug DSCG. Especially the
compounds MAC and PAC show a large increase in potency
when compared to DSCG, which renders them interesting
candidates for antiallergic agents. Our results suggest that the
molecular structure of the spacer connecting the two chromone
groups is of great importance for activity. Furthermore, a
difference in inhibitory activity was observed between the
nonirradiated and irradiated form of PAC. This result
comprises the first step on the way to our far reaching goal,
i.e., obtaining complete control over mast cell activation with
light. In addition, our results show how molecular photo-
switches can be used to study and interfere in a noninvasive
manner with biological processes and how they might be used
as a tool in chemical biology.
Ethyl 4-Oxo-4H-chromene-2-carboxylate (1). 2-Hydroxyacetophe-
none (2.04 g, 15 mmol) and diethyl oxalate (5.12 g, 35 mmol) were
dissolved in ethanol (10 mL) and added to a solution of sodium (1.49
g, 65 mmol) in ethanol (100 mL). The reaction mixture was heated at
reflux for 1 h, after which it was cooled down and acidified with
concentrated HCl until a white precipitate was formed. The precipitate
was filtered off, and the filtrate was concentrated and extracted with
ethyl acetate, washed with brine, and dried (MgSO4). After
evaporation, a solid was obtained, which was recrystallized from
methanol/di-iso-propylether (4:1) to yield 2.45 g (75%) of a white
solid; mp 66−68 °C. 1H NMR (400 MHz, CDCl3): δ 8.21 (dd, J = 7.9,
1.7 Hz, 1H), 7.75 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.62 (dd, J = 8.6, 1.0
Hz, 1H), 7.46 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 7.12 (s, 1H), 4.47 (q, J =
7.1 Hz, 4H), 1.41 (t, J = 7.2 Hz, 3H). 1H NMR spectrum in agreement
with published data.27
EXPERIMENTAL SECTION
■
LAD2 Cell Culture. The human mast cell line LAD2 was kindly
provided by Dr. A. Kirshenbaum (National Institute of Allergy and
Infectious Diseases, Bethesda, MD). LAD2 cells were cultured in
suspension at 37 °C, 95% relative humidity, and 5% CO2 in StemPro-
34 serum-free medium (Invitrogen) supplemented with 2 mM L-
glutamine (Sigma Aldrich), 100 IU/mL penicillin (Sigma Aldrich),
100 μg/mL streptomycin (Sigma Aldrich), and 100 ng/mL rhSCF
(Invitrogen). Cells were not allowed to grow beyond a density of 5.0 ×
105 cells/mL, and fresh medium was supplied once a week.
Ethyl 6-Nitro-4-oxo-4H-chromene-2-carboxylate (2). Compound
1 (200 mg, 0.92 mmol) was suspended in 65% nitric acid (0.12 mL),
and the mixture was cooled on ice. Concentrated sulfuric acid (1.5
mL) was added, and the mixture was stirred for 2 h at room
temperature and poured on ice-cooled water, and the formed
β-Hexosaminidase Assay. β-Hexosaminidase release was meas-
ured according to a published method.33 In short, 50 μL of
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precipitate was filtered off to yield 195 mg (81%) of a white solid; mp
178−179 °C. H NMR (400 MHz, CDCl3): δ 9.07 (d, J = 2.8 Hz,
1H), 8.57 (dd, J = 9.2, 2.8 Hz, 1H), 7.78 (d, J = 9.2 Hz, 1H), 7.19 (s,
1H), 4.50 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.1 Hz, 3H). 13C NMR (100
MHz, CDCl3): δ 176.8, 159.7, 158.7, 152.8, 145.2, 128.9, 124.4, 122.5,
120.6, 115.0, 63.5, 14.1. HR-MS (ESI, [M + H]+): calcd for
C12H10NO6, 264.0508; found, 264.0502.
mixture was stirred at room temperature for 30 min. The organic layer
was separated, and the aqueous layer was extracted twice with DCM.
The combined organic layers were washed with 1 M aq HCl, saturated
NaHCO3, and brine, and dried (MgSO4). The crude product was
purified by flash chromatography (Silicagel, 40−63 μm, pentane/
1
1
AcOEt, 4:1, v/v), yielding 430 mg (54%) of a light-green solid. H
NMR (400 MHz, CDCl3): δ 7.77 (d, J = 6.4 Hz, 1H), 7.63 (s, 1H),
1
Ethyl 6-Amino-4-oxo-4H-chromene-2-carboxylate (3). Com-
pound 2 (300 mg, 1.14 mmol) was dissolved in benzene (100 mL)
and stirred overnight with 10% palladium on charcoal (15 mg) under a
hydrogen atmosphere (balloon) at room temperature. The mixture
was filtered over Celite. The filtrate was evaporated, yielding 300 mg of
7.48−7.54 (m, 2H), 2.50 (s, 3H). H NMR spectrum in agreement
with published data.43
1-Methyl-4-nitrosobenzene (8b). 4-Methylaniline (5.00 g, 33.1
mmol) was dissolved in DCM (100 mL), a solution of oxone (40.7 g,
66.2 mmol) in water (400 mL) was added to this, and the resulting
biphasic mixture was stirred at room temperature for 30 min. The
organic layer was separated, and the aqueous layer was extracted twice
with DCM. The combined organic layers were washed with 1 M aq
HCl, saturated NaHCO3, and brine, and dried (MgSO4). Evaporation
1
orange needles; mp 177−178 °C. H NMR (400 MHz, CDCl3): δ
7.45 (d, J = 9.0 Hz, 1H), 7.35 (d, J = 2.9 Hz, 1H), 7.11−7.03 (m, 2H),
4.45 (q, J = 7.1 Hz, 2H), 3.92 (brs, 2H), 1.43 (t, J = 7.1 Hz, 3H). 13C
NMR (100 MHz, CDCl3): δ 178.4, 160.8, 151.7, 149.6, 144.6, 125.4,
123.2, 119.8, 113.6, 107.4, 62.8, 14.1. HR-MS (ESI, [M + H]+): calcd
for C12H12NO4, 234.0766; found, 234.0762.
1
of the solvent yielded 2.34 g (59%) of a light-green solid. H NMR
(400 MHz, CDCl3): δ 7.81 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.0 Hz,
1
Ethyl 6-Nitroso-4-oxo-4H-chromene-2-carboxylate (4). Com-
pound 3 (100 mg, 0.43 mmol) was dissolved in DCM (2 mL), a
solution of oxone (422 mg, 0.69 mmol) in H2O (8 mL) was added,
and the biphasic mixture was stirred at room temperature for 2 h. The
organic layer was separated, and the aqueous layer was extracted twice
with DCM. The combined organic layers were washed with 1 M aq
HCl, saturated NaHCO3, and brine, and dried (MgSO4). Evaporation
of the solvent yielded 75 mg (70%) of a green solid; mp 144−145 °C.
1H NMR (400 MHz, CDCl3): δ 9.31 (d, J = 2.1 Hz, 1H), 7.77−7.65
(m, 2H), 7.21 (s, 1H), 4.50 (q, J = 7.1 Hz, 2H), 1.46 (t, J = 7.1 Hz,
3H). 13C NMR (100 MHz, CDCl3): δ 177.9, 161.6, 159.9, 159.2,
152.6, 125.6, 124.9, 120.8, 120.2, 115.0, 63.4, 14.1. HR-MS (ESI, [M +
H]+): calcd for C12H10NO5, 248.0559; found, 248.0552.
2H), 2.44 (s, 3H). H NMR spectrum in agreement with published
data.44
3,3′-Dimethylazobenzene (9a). Compound 8a (500 mg, 4.13
mmol) and 3-methylaniline (369 mg, 3.44 mmol) were dissolved in
glacial acetic acid (33 mL), and the mixture was stirred overnight. The
solution was diluted with water and extracted with ethyl acetate. The
organic phase was washed four times with water and once with brine
and dried (MgSO4). The crude product was filtered through silica,
yielding 400 mg (55%) of an orange solid. 1H NMR (400 MHz,
CDCl3): δ 7.70−7.74 (m, 4H), 7.41 (appt, J = 8.0 Hz, 2H), 7.29 (d, J
1
= 7.6 Hz, 2H), 2.46 (s, 6H). H NMR spectrum in agreement with
published data.45
4,4′-Dimethylazobenzene (9b). Compound 8b (300 mg, 2.48
mmol) and 4-methylaniline (292 mg, 2.73 mmol) were dissolved in
glacial acetic acid (20 mL) and stirred overnight. The solution was
diluted with water and extracted with ethyl acetate. The organic phase
was washed four times with water and once with brine and dried
(MgSO4). The crude product was purified by flash chromatography
(Silicagel, 40−63 μm, pentane/Et2O, 9:1, v/v), yielding 450 mg (86%)
of an orange solid. 1H NMR (400 MHz, CDCl3): δ 7.81 (d, J = 8.4 Hz,
Diethyl 6,6′-(Diazene-1,2-diyl)bis(4-oxo-4H-chromene-2-carbox-
ylate) (5). Compounds 3 (70.8 mg, 0.304 mmol) and 4 (75.0 mg,
0.304 mmol) were dissolved in glacial acetic acid (2.5 mL) and stirred
for 2 d. The solution was diluted with water and extracted with DCM.
The organic phase was washed with water (4×) and brine and dried
(MgSO4). Recrystallization from DCM yielded 60 mg (43%) of a
light-orange solid; mp 254−255 °C (dec). 1H NMR (400 MHz,
CDCl3): δ 8.79 (d, J = 2.4 Hz, 2H), 8.35 (dd, J = 9.0, 2.4 Hz, 2H),
7.77 (d, J = 9.0 Hz, 2H), 7.18 (s, 2H), 4.50 (q, J = 7.2 Hz, 4H), 1.46 (t,
J = 7.1 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 178.1, 160.3, 157.5,
152.4, 149.4, 127.2, 125.0, 122.8, 120.2, 114.9, 63.2, 14.1. HR-MS
(ESI, [M + H]+): calcd for C25H18N2O8, 463.1136; found, 463.1131.
Sodium (E)-6,6′-(Diazene-1,2-diyl)bis(4-oxo-4H-chromene-2-car-
boxylate) (6) (DAC). To a solution of compound 5 (50 mg, 0.11
mmol) in ethanol (2 mL) was added aq NaOH (2.5 M, 0.128 mL)
dropwise at 0 °C. The reaction mixture was heated at reflux for 2 h,
after which the insoluble product was filtered off and purified by
recrystallization from methanol, yielding 45 mg (91%) of a red solid.
1H NMR (400 MHz, methanol-d4): δ 8.71 (d, J = 2.4 Hz, 2H), 8.43
1
4H), 7.30 (d, J = 8.4 Hz, 4H), 2.43 (s, 6H). H NMR spectrum in
agreement with published data.46
3,3′-Bis(bromomethyl)azobenzene (10a). To a solution of
compound 9a (1.30 g, 6.18 mmol) in 60 mL of CCl4 was added
NBS (2.50 g, 14.2 mmol) and AIBN (80 mg, 0.48 mmol). The
resultant solution was stirred overnight at 70 °C, then filtered, and the
filtrate was washed with hot water and brine and dried (MgSO4). After
evaporation the product was recrystallized from acetonitrile, yielding
1
800 mg (35%) of an orange solid; mp 140−141 °C. H NMR (400
MHz, CDCl3): δ 7.95 (m, 2H), 7.87 (m, 2H), 7.52 (m, 4H), 4.59 (s,
45
1
4H). H NMR spectrum in agreement with published data.
4,4′-Bis(bromomethyl)azobenzene (10b). To a solution of
compound 9b (300 mg, 1.43 mmol) in 20 mL of CCl4 was added
NBS (584 mg, 3.30 mmol) and AIBN (18 mg, 0.10 mmol). The
resultant solution was stirred overnight at 70 °C, then filtered, and the
filtrate was washed with hot water and brine and dried (MgSO4). After
evaporation of the solvent, the product was recrystallized from
acetonitrile, yielding 220 mg (42%) of an orange solid; mp 183−185
°C. 1H NMR (400 MHz, CDCl3): δ 7.89 (d, J = 8.4 Hz, 4H), 7.54 (d,
(dd, J = 9.0, 2.5 Hz, 2H), 7.90 (d, J = 9.0 Hz, 2H), 7.04 (s, 2H). 13C
NMR (100 MHz, methanol-d4): δ 180.2, 164.2, 160.3, 157.9, 149.3,
126.7, 124.2, 121.5, 120.3, 111.1. HR-MS (ESI, [M + Na]+): calcd for
C20H9N2O8Na, 451.0154; found, 451.0149.
Methyl 5-Hydroxy-4-oxo-4H-chromene-2-carboxylate (7). 2,6-
Dihydroxyacetophenone (1.50 g, 9.90 mmol) and dimethyloxalate
(5.80 g, 49.3 mmol) were dissolved in 0.5 M MeONa/MeOH (100
mL) and heated at reflux overnight. The solvent was removed in
vacuo, and the slurry was dissolved in water (100 mL) and
subsequently acidified with concentrated HCl. The precipitate was
filtered off and dissolved in MeOH (50 mL) and concentrated HCl
(10 mL). This solution was heated at reflux for 2 h, after which the
crude product was purified by flash chromatography (Silicagel, 40−63
μm, pentane/AcOEt, 9:1, v/v), yielding 762 mg (33%) of a yellow
powder. 1H NMR (400 MHz, CDCl3): δ 7.61 (appt, J = 8.4 Hz, 1H),
1
J = 8.4 Hz, 4H), 4.56 (s, 4H). H NMR spectrum in agreement with
published data.46
(E)-Dimethyl5,5′-(((diazene-1,2-diylbis(3,1-phenylene))bis-
(methylene))bis(oxy))bis(4-oxo-4H-chromene-2-carboxylate) (11a).
To a solution of compounds 7 (638 mg, 2.73 mmol) and 10a (400 mg,
1.09 mmol) in 80 mL of acetonitrile was added K2CO3 (452 mg, 3.27
mmol), and the resulting mixture was stirred overnight at 65 °C. The
solution was concentrated in vacuo, and the crude product was
purified using flash chromatography (Silicagel, 40−63 μm, DCM/
methanol, 95:5, v/v), yielding 520 mg (70%) of a light-orange solid.
1H NMR (400 MHz, CDCl3): δ 8.06 (s, 2H), 7.90−7.85 (m, 4H),
1
7.03−7.06 (m, 2H), 6.85 (d, J = 8.3 Hz, 1H), 4.02 (s, 3H). H NMR
spectrum in agreement with published data.29
1-Methyl-3-nitrosobenzene (8a). 3-Methylaniline (708 mg, 6.61
mmol) was dissolved in DCM (20 mL), a solution of oxone (8.10g,
13.2 mmol) in water (80 mL) was added, and the resulting biphasic
7.56−7.61 (m, 4H), 7.18 (d, J = 8.5 Hz, 2H), 7.02 (s, 2H), 6.91 (d, J =
8.3 Hz, 2H), 5.38 (s, 4H), 4.00 (s, 6H). 13C NMR (100 MHz,
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Journal of Medicinal Chemistry
Article
CDCl3): δ 177.6, 161.1, 158.4, 158.0, 152.7, 150.1, 137.5, 134.7, 129.7,
129.4, 122.6, 120.7, 116.7, 113.8, 111.1, 108.9, 70.6, 53.4. HR-MS
(ESI, [M + H]+): calcd for C36H27N2O10, 647.1660; found, 647.1603.
(E)-Dimethyl5,5′-(((diazene-1,2-diylbis(4,1-phenylene))bis-
(methylene))bis(oxy))bis(4-oxo-4H-chromene-2-carboxylate) (11b).
To a solution of compounds 7 (100 mg, 0.45 mmol) and 10b (75.8
mg, 0.21 mmol) in acetonitrile (25 mL) was added Cs2CO3 (201 mg,
0.62 mmol), and the resulting mixture was stirred at 65 °C for 3 h. The
solution was concentrated in vacuo, and the residue was dissolved in
DCM, washed with 1 M aq HCl, saturated NaHCO3, and brine, and
dried (MgSO4). After evaporation, the product was recrystallized from
DCM yielding 27 mg of a light-orange solid; mp 235−237 °C (dec).
1H NMR (400 MHz, CDCl3): δ 7.96 (d, J = 8.0 Hz, 4H), 7.76 (d, J =
8.1 Hz, 4H), 7.58 (t, J = 8.0, 2H), 7.18 (d, J = 8.2 Hz, 2H), 7.02 (s,
2H), 6.90 (d, J = 8.3 Hz, 2H), 5.36 (s, 4H), 4.00 (s, 6H). 13C NMR
(100 MHz, CDCl3): δ 177.7, 161.1, 158.4, 158.0, 152.2, 150.1, 139.3,
134.6, 127.2, 123.2, 116.6, 113.8, 111.1, 108.7, 70.5, 53.4. HR-MS
(ESI, [M + H]+): calcd for C36H27N2O10, 669.1485; found, 669.1480.
Sodium (E)-5,5′-(((Diazene-1,2-diylbis(3,1-phenylene))bis-
(methylene))bis(oxy))bis(4-oxo-4H-chromene-2-carboxylate) (12a)
(MAC). To a solution of compound 11a (100 mg, 0.15 mmol) in
ethanol (5 mL) was added aq NaOH (2.5 M, 186 μL) dropwise at 0
°C. The reaction mixture was heated at reflux for 2 h, after which the
insoluble product was filtered off, yielding 85 mg (86%) of an orange
solid. 1H NMR (400 MHz, methanol-d4): δ 8.12 (s, 2H), 7.86 (d, J =
8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.63 (m, 4H), 7.26 (d, J = 8.4
Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.88 (s, 2H), 5.42 (s, 4H). 13C
NMR (100 MHz, methanol-d4): δ 180.4, 164.7, 158.3, 158.1, 157.9,
152.7, 138.3, 134.5, 129.1, 121.2, 113.8, 112.5, 110.9, 108.5, 69.9. HR-
MS (ESI, [M + Na]+): calcd for C34H22N2O10Na, 641.1167; found,
641.1140.
ABBREVIATIONS USED
■
CBP, cromolyn binding protein; DSCG, disodium cromogly-
cate; DAC, di-azochromone; MAC, meta-azochromone; PAC,
para-azochromone; PSS, photostationary state; rhSCF, re-
combinant human stem cell factor
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S
* Supporting Information
β-Hexosaminidase release assay of DSCG. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone:+31 50 363 4235. Fax:+31 50 363 4296. E-mail: b.l.
feringa@rug.nl (B.L.F.); w.c.szymanski@rug.nl (W.S.).
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Dr. A. Kirshenbaum and Yun Bai
(National Institute of Allergy and infectious Diseases, Bethesda,
MD) for kindly providing us with the LAD2 cell line and for
their help with the β-hexosaminidase release assay. This work
was financially supported by the European Research Council
(ERC) advanced grant 227897 to B.L.F. and by the Ministry of
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