Synthesis of bisboron compounds and their strong inhibitory activity on store-operated calcium entry

Article abstract of DOI:10.1016/j.bmcl.2009.12.108

Store-operated calcium entry (SOCE) is an important mechanism for replenishing intracellular calcium stores and for sustaining calcium signaling. We developed a method for synthesis of bisboron compounds that have two borinic acids or their esters in one molecule. These compounds are analogues of 2-APB, which is widely used as a membrane-permeable SOCE inhibitor. Further, we examined the effect of the newly synthesized bisboron compounds on SOCE in Jurkat T cells. All the bisboron compounds showed strong inhibitory activity on SOCE, with IC₅₀ values of less than 1 μM, which were 20-45 times lower than observed with 2-APB.

Full text of DOI:10.1016/j.bmcl.2009.12.108

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1395–1398
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of bisboron compounds and their strong inhibitory activity
on store-operated calcium entry
Akinobu Z. Suzuki a, Shoichiro Ozaki a, Jun-Ichi Goto a, Katsuhiko Mikoshiba a,b,
*
a Laboratory for Developmental Neurobiology, Brain Development Research Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and Technology, Japan Science and Technology Agency,
Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Store-operated calcium entry (SOCE) is an important mechanism for replenishing intracellular calcium
stores and for sustaining calcium signaling. We developed a method for synthesis of bisboron compounds
that have two borinic acids or their esters in one molecule. These compounds are analogues of 2-APB,
which is widely used as a membrane-permeable SOCE inhibitor. Further, we examined the effect of
the newly synthesized bisboron compounds on SOCE in Jurkat T cells. All the bisboron compounds
Received 30 November 2009
Revised 28 December 2009
Accepted 29 December 2009
Available online 11 January 2010
showed strong inhibitory activity on SOCE, with IC50 values of less than 1
lower than observed with 2-APB.
lM, which were 20–45 times
Keywords:
Calcium
SOCE
Ó 2010 Elsevier Ltd. All rights reserved.
2-APB
Inhibitor
Boron
Intracellular calcium signaling plays a key role in various phys-
iological phenomena, including contraction of smooth muscle,
secretion of granules, transcription, cell differentiation, and cell
death.1 Store-operated calcium entry (SOCE; also termed capacita-
tive calcium entry, CCE), which is activated by depletion of intra-
cellular calcium stores caused by repetitive calcium release,
provides the means to replenish calcium from the extracellular
milieu.2,3 Recent studies have identified the molecular components
of the calcium release-activated current (ICRAC) channel, which is
the major Ca2+ influx pathway for SOCE in T lymphocytes and mast
cells. The endoplasmic reticulum-resident protein STIM and the
channel pore-forming subunit Orai (also termed CRACM) consti-
tute functional ICRAC channels,4–8 and the ablation of either mark-
edly alters immune system function, including T cell activation
and anaphylactic responses in mast cells.6,9,10 Thus, the production
of inhibitors against SOCE may provide a potential therapeutic
strategy including use as immunosuppressants and allergy treat-
ment. Two-aminoethyl diphenylborinate (2-APB) is a commonly-
used SOCE inhibitor that was initially identified as a membrane-
permeable inhibitor of inositol 1,4,5-trisphosphate (IP3)-induced
calcium release (IICR),11 then later identified as an SOCE inhibi-
tor.12,13 Other lines of studies also identified inhibitors of
SOCE.14–17 Recently, in an attempt to improve the activity and
the specificity of 2-APB, we analyzed the effects of more than
600 boron-containing 2-APB analogues on IICR and SOCE, and re-
ported that some inhibitors exhibited 100-fold more potent SOCE
inhibitory activity than 2-APB, while having less effect on IICR.18,19
We also reported that two structural isomers of 2-APB analogues,
DPB162-AE and DPB163-AE, showed different effects on SOCE in
DT40, CHO, HeLa, and other cell types, and also on ICRAC, which
was reconstituted by STIM and CRACM subtypes in HEK293 cells,
suggesting that these inhibitors may be useful tools to elucidate
the mechanisms regulating CRAC channel activation.19 In the pres-
ent study, we developed a novel method for synthesis of 2-APB
analogues that contain two boron atoms in the form of borinic
acids and/or their esters, termed ‘bisboron compounds’, and exam-
ined their effects on SOCE in Jurkat T cells.
The synthetic methods for some bisboron compounds have been
described previously.20–22 The first method involves the preparation
of diborinicacidsin threesteps20,21: (1) preparation of diboronicacid
[(HO)2B–X–B(OH)2; X represents aromatic derivatives such as phe-
nyl or biphenyl] by reacting di-aryl magnesium bromide with trim-
ethoxyborane, and subsequent hydrolysis of boronate dimethyl
ester; (2) esterification of diboronic acid by 2,2-dimethyl-propane-
1,3-diol; and (3) treatment of the ester with aryl magnesium bro-
mide to produce diborinic acid [RB(OH)–X–B(OH)R]. As this overall
method requires purification of intermediates at each step, the total
yield is low and a much longer reaction time is required. A second
method uses purified diisopropoxyphenylborane (Scheme 1, 3) to
further react with diaryllithium (Scheme 1, 5).22 Although
diisopropoxyphenylborane is purified by distillation at reduced
* Corresponding author. Tel.: +81 3 5449 5767; fax: +81 3 5446 5768.
E-mail address: mikosiba@brain.riken.jp (K. Mikoshiba).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.108

1396
A. Z. Suzuki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1395–1398
Scheme 1. Reagents and conditions: (a) sec-BuLi, ether, ꢀ98 °C; (b) B(OiPr)3, ether, ꢀ78 °C; (c) compound 5, ether, gradual warming from ꢀ78 °C to rt; (d) 1 M HCl; (e) 2-
aminoethanol, ethanol, rt; (f) sec-BuLi, ether, ꢀ78 °C.
Figure 1. Chemical structures of 2-APB and synthesized borinate esters.
pressure,23 aryldiisopropoxyborane cannot be distilled if the molec-
ular weight of the aryl group is high. As such, this method is unsuit-
able for the synthesis of a wide variety of bisboron compounds. To
overcome these problems, we developed a convenient method for
the production of diborinic acids from commercially available or
easily prepared materials, without purification of the intermediates.
This method is summarized in Scheme 1. Lithiation of bromoben-
zene 1 with sec-butyllithium (sec-BuLi) at ꢀ98 °C in diethylether
and subsequent reaction with triisopropoxyborane at ꢀ78 °C
afforded diisopropoxyphenylborane3,24 which was used in a further
reaction without purification to avoid loss of yield. Treatment of
diisopropoxyphenylborane 3 with the bis-phenyllithium 5, which
was prepared by lithiation of 3,30-dibromodibenzyl ether 4 under
the same conditions, produced diborinic acid 6 by gradual warming
of the reaction mixture to room temperature and following
additionof 1 M HCl. Esterificationof theborinicacid 6 with 2-amino-
ethanol produced the bisboron compound 7.25 The different reactiv-
ities of triisopropoxyborane, aryldiisopropoxyborane, and
Table 1
Total yield of synthesized borinate esters and their inhibitory activities on SOCE in
Jurkat T cells
Compound
Yield (%)
IC50 (lM)
2-APB
7
8
—
15
31.9
24.1
52.3
41.1
47.1
41.1
60.2
0.32
0.43
0.45
0.41
0.31
0.64
1.9
9
10
11
12
13

A. Z. Suzuki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1395–1398
1397
Figure 2. Effects of 2-APB and bisboron compound 7 on TG-induced SOCE in Jurkat T cells.
diarylisopropoxyborane allowed step-by-step addition of arylli-
thium to triisopropoxyborane,23 resulting in selective production
of bisboron compound 6. The advantage of this method is that dibo-
rinic acid is prepared in a one-pot reaction from readily available
starting materials, and therefore in high yield. For example, we im-
proved the yield of borinate esters 9 to 52% (Fig. 1) from the previ-
ously described 13%.20,21 Further, we produced DPB162-AE and
DPB163-AE (7, 8), as well as newly synthesized bisboron compounds
9–12, in high yield using the same method (Table 1).26 Borinate ester
13, in which one boron was substituted with oxygen, was synthe-
sized using 3-bromobenzyl-30-phenoxybenzyl ether instead of bis-
arylbromide.26
od of bisboron compound synthesis will allow us to prepare a wide
variety of bisboron compounds for further screening to identify
more potent and selective SOCE inhibitors, which may be more
suitable as therapeutics for autoimmune diseases and allergic
disorders.
Acknowledgments
We thank Ms. E. Ebisui, N. Matsumoto, and M. Yamada for
excellent technical help in measurement of intracellular calcium
in Jurkat T cells, and Dr. A. Mizutani, Dr. T. Nakamura, and Profes-
sor T. Furuta (Toho University) for helpful discussion.
Next, we examined the effect of the borinate esters on SOCE in
Jurkat T cells. Ca2+ entry in fura-2-loaded cells was measured after
depletion of internal Ca2+ stores induced by thapsigargin (TG), a sar-
coplasmic and endoplasmic reticulum calcium ATPase inhibitor,
with or without borinate esters.27 Consistent with previous Let-
ters,18,19 store depletion and subsequent addition of external Ca2+
evoked a rapid and large elevation in the concentration of cytoplas-
mic Ca2+. Application of 2-APB or borinate esters before the restora-
tion of external Ca2+ inhibited Ca2+ entry in a drug dose-dependent
manner (Fig. 2). The IC50 values of the different drugs can be seen
in Table 1. All bisboron compounds showed a marked inhibitory
References and notes
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effect on SOCE, with IC50 values of less than 1 lM, regardless of the
linker length between borinate esters (8–11), substituents in the
phenyl ring (10, 12), and orientation in benzyl ether (7, 8); these
IC50 values were 20–45 times lower than for 2-APB. The monoboron
compound 13, in which one boron atom was substituted with oxy-
gen, was more potent than 2-APB. However, compound 13 was less
effective than a half amount of bisboron compounds, suggesting that
the two boron atoms might exert a synergistic effect on the target
molecule, although the bulkiness of diarylborinate group or phen-
oxyphenyl group would be also important for the interaction be-
tween boron compound and the target molecule. Our previous
Letter indicated that compounds 7 and 8 inhibit SOCE via STIM pro-
tein, an activator of SOCE.19 Therefore, further study of the effect of
bisboron compounds for STIM will provide insights into where and
how bisboron compounds interact with STIM and inhibit SOCE.
In conclusion, we established a facile method for preparing bis-
boron compounds, which have been demonstrated to be potent
inhibitors of SOCE. These compounds may help to further elucidate
the molecular mechanism of CRAC channel activation.19 Our meth-
18. Zhou, H.; Iwasaki, H.; Nakamura, T.; Nakamura, K.; Maruyama, T.; Hamano, S.;
Ozaki, S.; Mizutani, A.; Mikoshiba, K. Biochem. Biophys. Res. Commun. 2007, 352,
277.

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19. Goto, J.; Suzuki, A. Z.; Ozaki, S.; Matsumoto, N.; Nakamura, T.; Ebisui, E.; Fleig,
A.; Penner, R.; Mikoshiba, K. Cell Calcium, in press. doi: 10.1016/
j.ceca.2009.10.004.
20. Coutts, I. G. C.; Goldschmid, H. R.; Musgrave, O. C. J. Chem. Soc. 1970, 488.
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22. Mikoshiba, K.; Iwasaki, H.; Maruyama, T.; Hamano, S. PCT Int. Appl. 2003,
WO2003033002.
116.8, 62.4, 41.3 HRMS(FAB) (m/z): calcd for C28H31O3N2B2+ [M+H+] 465.2520,
found 465.2502. Compound 11: light yellow powder; 1H NMR (400 MHz,
DMSO-d6) d 7.38–7.27 (m, 8H), 7.14–6.97 (m, 10H), 6.02 (br, 4H, NH2), 4.35 (s,
2H), 3.75 (t, 2H, J = 6.4 Hz), 3.74 (t, 2H, J = 6.4 Hz), 3.52 (t, 2H, J = 7.2 Hz), 2.82 (t,
2H, J = 6.4 Hz) 2.78 (t, 2H, J = 6.4 Hz) 2.71 (t, 2H, J = 7.2 Hz) 13C NMR (100 MHz,
DMSO-d6) d 135.1, 134.8, 131.5, 131.5, 131.4, 127.2, 127.2, 126.6, 126.5, 126.3,
+
124.9, 124.8, 72.3, 70.8, 62.4, 41.3 HRMS(FAB) (m/z): calcd for C31H37O3N2B2
23. Brown, H. C.; Colet, T. E. Organometallics 1983, 2, 1316.
24. Mori, Y.; Kobayashi, J.; Manabe, K.; Kobayashi, S. Tetrahedron 2002, 58, 8263.
[M+H+] 507.2990, found 507.3000. Compound 12: white powder; 1H NMR
(270 MHz, CDCl3) d 7.40 (s, 2H), 7.33 (d, 4H, J = 7.0 Hz), 7.12 (d, 2H, J = 7.5 Hz),
7.12 (d, 2H, J = 7.5 Hz), 6.97 (d, 4H, J = 7.0 Hz), 3.97 (t, 4H, J = 5.9 Hz), 3.02 (t,
4H, J = 5.9 Hz), 2.32 (s, 6H) 13C NMR (100 MHz, DMSO-d6) d 155.1, 132.8, 132.4,
131.6, 131.2, 130.2, 129.8, 116.9, 62.4, 41.3, 19.3 HRMS(FAB) (m/z): calcd for
25. To a solution of bromobenzene (211
added 0.99 M sec-BuLi (2.15 mL, 2.13 mmol) at ꢀ98 °C, gradually warmed to
ꢀ78 °C to complete lithiation, and then added triisopropoxyborane (459 L,
lL, 2.0 mmol) in diethyl ether (8 mL), we
l
C30H31O3N2Cl2B2 [MꢀH+] 559.1898, found 559.1906. Compound 13: light
1
2.0 mmol). The reaction mixture was stirred for 80 min at ꢀ78 °C. At the same
time, 3,30-dibromodibenzyl ether (355 mg, 1.0 mmol), which was prepared by
conventional methods, was dissolved in diethyl ether (10 mL), then reacted
with 0.99 M sec-BuLi (2.15 mL, 2.13 mmol) and stirred for 1 h at ꢀ78 °C. The
reaction mixture added to the mixture of diisopropoxyphenylborane, gradually
warmed to room temperature, and then stirred overnight. The reaction was
quenched with 1 N HCl, the diethyl ether layer was collected, and the water
layer then extracted twice with diethyl ether. The combined diethyl ether
layers were dried over MgSO4 and concentrated. The crude residue was
yellow oil; 1H NMR (400 MHz, CDCl3) d 7.34–7.15 (m, 12H), 7.10–6.89 (m, 6H),
4.49 (s, 2H), 4.47 (s, 2H), 4.14 (br, 2H, NH2), 3.72 (t, 2H, J = 6.0 Hz), 2.73 (br, 2H)
13C NMR (100 MHz, DMSO-d6) d 140.3, 136.5, 131.6, 131.5, 131.4, 129.8, 129.8,
127.7, 127.6, 126.4, 126.3, 123.2, 122.7, 118.9, 118.2, 118.0, 73.4, 72.1, 63.0,
42.1 HRMS(FAB) (m/z): calcd for C28H29O3N1B1 [M+H+] 438.2241, found
+
438.2244.
27. Jurkat
T
cells (1.25 ꢁ 107 cells) grown in RPMI-1640 medium (Gibco)
containing 10% fetal calf serum were collected by centrifugation (420g),
washed with Ca2+(+) assay buffer (137 mM NaCl, 5.4 mM KCl, 4.2 mM NaHCO3,
1.0 mM CaCl2, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 5.6 mM glucose, 20 mM
purified by flash column chromatography on
a silica gel (n-hexane/
EtOAc = 3:1) to give 3,30-(hydroxyphenylboryl)dibenzyl ether (153 mg,
0.377 mmol, 37.7%) as an oil. The borinic acid was dissolved in ethanol
HEPES, pH 7.4), and then loaded with 5 lM Fura-2/acetoxymethyl ester (Fura-
(4 mL), and 2-aminoethanol (49.8
l
L, 0.831 mmol) then added. The reaction
2-AM) at room temperature in the dark for 60 min. The fura-2-loaded cells
were washed twice with Ca2+(ꢀ) assay buffer (137 mM NaCl, 5.4 mM KCl,
4.2 mM NaHCO3, 0.1 mM EGTA, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 5.6 mM
glucose, 20 mM HEPES, pH 7.4), resuspended at 1.25 ꢁ 106 cells/mL with
mixture was stirred for 15 min at room temperature, the ethanol removed in
vacuo, and the mixture then purified by reprecipitation in diisopropylether/
CHCl3 to give 3,30-[(2-aminoethoxy)phenylboryl]dibenzyl ether 7 (157 mg,
0.319 mmol, 84.6%, total 31.9%) as a light yellow powder.
Ca2+(ꢀ) assay buffer, and then transferred into 96-well plates (80
lL per well;
26. Spectroscopic data for 7: light yellow powder; 1H NMR (270 MHz, DMSO-d6) d
7.40–7.23 (m, 8H), 7.14–7.00 (m, 10H), 6.06 (br, 4H, NH2), 4.40 (s, 4H), 3.75 (t,
4H, J = 6.3 Hz), 2.81 (tt, 4H, J = 6.3 Hz, 5.9 Hz) 13C NMR (67.5 MHz, DMSO-d6) d
136.0, 131.3, 130.9, 130.6, 126.4, 126.3, 124.7, 124.4, 72.2, 62.4, 41.4
ꢂ105 cells). Ca2+ measurements were performed using the Functional Imaging
Cell-Sorting System (FDSS/IMACS; Hamamatsu Photonics).Fura-2 fluorescence
intensities were measured at 510 nm with excitation at 340 nm (F340) and
380 nm (F380), and emission ratios (F340/F380) were monitored in each well.
Experiments were performed according to the following time schedule; 1 min:
HRMS(FAB) (m/z): calcd for C30H35O3N2B2 [M+H+] 493.2834, found
+
493.2849. Compound 8: light yellow powder; 1H NMR (400 MHz, DMSO-d6)
d 7.39–7.35 (m, 8H), 7.14–7.00 (m, 10H), 6.04 (br, 4H, NH2), 4.37 (s, 4H), 3.75 (t,
4H, J = 6.0 Hz), 2.81 (t, 4H, J = 6.0 Hz) 13C NMR (100 MHz, DMSO-d6) d 134.9,
131.5, 131.4, 126.6, 126.2, 124.9, 71.6, 62.4, 41.3 HRMS(FAB) (m/z): calcd for
addition of 20
l
L of 5X concentration of borinate ester in Ca2+(ꢀ) assay buffer,
which was prepared as a 1000ꢁ stock solution in DMSO; 3 min: addition of
20
l
L of 6
l
M TG in Ca2+(ꢀ) assay buffer containing borinate ester of interest
concentration (total 1 lM of TG); 16 min: addition of 20 lL of 14 mM CaCl2 in
C30H35O3N2B2 [M+H+] 493.2834, found 493.2838. Compound 9: white
Ca2+(ꢀ) assay buffer containing borinate ester of interest concentration (total
2 mM of Ca2+); and 22 min: end of measurement. Control experiments were
performed in each plate, in which 0.1% DMSO was added instead of borinate
ester solutions. The maximal emission ratio in control wells were regarded as
the maximal Ca2+ entry of Jurkat T cells in same plate, and the inhibitory
activities of borinate esters were calculated from dividing the maximal
emission ratio in the presence of borinate esters by the maximal emission
ratio in control.
+
powder; 1H NMR (400 MHz, CD3OD) d 7.10–7.07 (m, 12H), 6.86–6.75 (m,
6H), 3.61 (t, 4H, J = 6.0 Hz), 2.69 (t, 4H, J = 6.0 Hz) 13C NMR (100 MHz, CD3OD) d
140.5, 133.4, 133.0, 128.1, 126.7, 126.5, 64.0, 42.6 HRMS(FAB) (m/z): calcd for
C28H31O2N2B2+ [M+H+] 449.2571, found 449.2570. Compound 10: light yellow
powder: 1H NMR (400 MHz, DMSO-d6) d 7.40–7.23 (m, 8H), 7.15–7.02 (m, 6H),
6.73 (d, 4H, J = 6.8 Hz), 6.03 (br, 4H, NH2), 3.75 (t, 4H, J = 6.0 Hz), 2.82 (t, 4H,
J = 6.0 Hz) 13C NMR (100 MHz, DMSO-d6) d 155.1, 132.8, 131.4, 126.6, 124.8,