Synthesis and pharmacological characterization of 2-aminoethyl diphenylborinate (2-apb) derivatives for inhibition of store-operated calcium entry (soce) in mda-mb-231 breast cancer cells

Article abstract of DOI:10.3390/ijms21165604

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca²⁺ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP₃) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca²⁺ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca²⁺ channels that facilitate a measurable Ca²⁺ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.

Full text of DOI:10.3390/ijms21165604

International Journal of
Molecular Sciences
Article
Synthesis and Pharmacological Characterization
of 2-Aminoethyl Diphenylborinate (2-APB)
Derivatives for Inhibition of Store-Operated Calcium
Entry (SOCE) in MDA-MB-231 Breast Cancer Cells
Achille Schild 1,† , Rajesh Bhardwaj 2,† , Nicolas Wenger 2, Dominic Tscherrig 1,
Palanivel Kandasamy 2 , Jan Dernicˇ , Roland Baur 1, Christine Peinelt 1
,
2
Matthias A. Hediger 2,* and Martin Lochner 1,
*
1
Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28,
3012 Bern, Switzerland; achille.schild@dcb.unibe.ch (A.S.); dominic.tscherrig@gmail.com (D.T.);
roland.baur@ibmm.unibe.ch (R.B.); christine.peinelt@ibmm.unibe.ch (C.P.)
Department of BioMedical Research, University of Bern, Murtenstrasse 35, 3008 Bern, Switzerland;
rajesh.bhardwaj@dbmr.unibe.ch (R.B.); nicolas.wenger@students.unibe.ch (N.W.);
palanivel.kandasamy@dbmr.unibe.ch (P.K.); jan.dernic@students.unibe.ch (J.D.)
Correspondence: matthias.hediger@ibmm.unibe.ch (M.A.H.); martin.lochner@ibmm.unibe.ch (M.L.);
Tel.: +41-31-631-3311 (M.L.)
2
*
†
These two authors have equally contributed to the study.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 24 June 2020; Accepted: 3 August 2020; Published: 5 August 2020
Abstract: Calcium ions regulate a wide array of physiological functions including cell differentiation,
proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum
(ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion
(e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known
as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing
stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai
proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly
selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive
refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and
Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with
several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune,
and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending
on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of
2-APB, introducing halogen and other small substituents systematically on each position of one of
the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging
assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at
different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB
derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.
Keywords: store-operated calcium entry; inhibitors; synthetic chemistry; 2-APB; structure–activity
relationship; breast cancer cells; calcium imaging assay; patch-clamp electrophysiology
1. Introduction
Store-operated calcium entry (SOCE) is a ubiquitous pathway by which cells regulate basal
calcium (Ca2+), refill intracellular Ca2+ stores, and maintain Ca2+ signaling to control diverse functions
Int. J. Mol. Sci. 2020, 21, 5604; doi:10.3390/ijms21165604
www.mdpi.com/journal/ijms

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such as gene expression, cell proliferation, migration, and differentiation [
1]. The stimulation of cell
surface receptors coupled to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) hydrolysis produces
inositol 1,4,5-trisphosphate (IP3), a second messenger which upon binding to IP3 receptors (IP3Rs) on
the endoplasmic reticulum (ER) membrane, releases Ca2+ from the ER. The depletion of the ER Ca2+
stores, triggers the activation of ER Ca2+-sensing stromal interaction molecules (STIM1 and STIM2)
that are embedded in the ER membrane which in turn activate Orai channels (Orai1, Orai2, and Orai3)
at the plasma membrane (PM). STIM1 and Orai1 were identified as the predominant isoforms of
STIMs and Orais that mediate SOCE in the majority of cell types [
rearrangements in STIM expose the cytosolic STIM Orai-activating region (SOAR). SOAR physically
opens the PM Orai channels, resulting in Ca2+ entry into the cytosol [
]. The STIM/Orai-mediated
Ca2+ entry, electrophysiologically characterized as Ca2+ release-activated Ca2+ current (ICRAC) [
],
]. Experimentally, sarcoplasmic/endoplasmic
] is widely used for passive depletion of
2–5]. Upon activation, structural
1
6
thus allows the acute refilling of the ER Ca2+ stores [
7
reticulum Ca2+ ATPase (SERCA) blocker thapsigargin (Tg) [
ER Ca2+ stores and subsequent activation of SOCE.
8
Gain-of-function or loss-of-function mutations in STIM1 or Orai1, or alterations in expression ratios
of STIM and Orai isoforms as well as aberrations in other regulatory mechanisms of SOCE pathway lead
to its dysfunction [
muscle, immune, and neurodegenerative disorders as well as cancer [12
has been linked to STIM1/Orai1-mediated Ca2+ influx [17] and several studies also highlighted the
upregulation of STIM1 and Orai1 in basal breast cancers [18 19]. Our understanding on the remodeling
9–
11]. The disruption of SOCE has various pathophysiological implications such as
–16]. Breast cancer metastasis
,
of SOCE in breast cancer cells has increased over the years and has recently been reviewed [20].
In addition, SOCE has been shown to control the proliferation, migration, invasion, and metastasis in
different cancer cell lines [21
and selective SOCE modulators is of high therapeutic interest.
Several chemical inhibitors [23 27] as well as potentiators of SOCE [28
However, 2-aminoethyl diphenylborinate (2-APB, Figure 1) remains the best characterized compound
used to modulate store-operated CRAC channels, despite of its effects on other known targets [31 44].
Although originally described as an inhibitor of IP3 receptors [45 46], it was later shown that inhibition
of SOCE and ICRAC by 2-APB is independent of its action on IP3 receptors [36 47 51]. 2-APB is known
to exert a very interesting dose-dependent bimodal effect on SOCE. 2-APB when applied in 10
concentration potentiates whereas 30 M concentration blocks SOCE and STIM1/Orai1-mediated
CRAC in various cell lines such as HEK293, human Jurkat T cells, chicken DT40 B cells, and rat basophilic
leukemia cells [42 47 49 50 52 56]. Recent studies have shed some light on the underlying mechanisms
of action of potentiation and inhibition of SOCE/ICRAC by 5 M and 50 M 2-APB, respectively [57 59].
,22]. Given the role of SOCE in tumor progression [16], developing potent
–
–30] have been reported.
–
,
,
–
≤
µM
≥
µ
I
,
,
,
,
–
µ
µ
–
2-APB not only acts directly on the Orai1 channel for the inhibition of SOCE, but also enhances the
intramolecular interactions within STIM1, which leads to the disruption of the functional coupling
between STIM1 and Orai1 [59]. On the other hand, 5 µM 2-APB potentiates CRAC channels by a direct
dilation of the open pore of Orai1 [57]. The other STIM/Orai isoforms as well as heteromeric channels
formed by Orai1/Orai3 have been shown to respond differently to 2-APB [53,55,60–65].
Several derivatives of 2-APB that modulate SOCE have been reported previously [28
Among these derivatives, DPB162-AE (Figure 1), a dimeric, more specific as well as more potent variant
of 2-APB, did not exert the typical dose-dependent potentiating effect on SOCE [54 58]. Interestingly, in
addition to SOCE inhibition, DPB162-AE (1–10
M) also released Ca2+ from the ER stores of pancreatic
,52,54,58,66–68].
,
µ
acinar cells as well as HEK293, HeLa and B-cell lymphoma SU-DHL-4 cells [69,70]. In HEK293 and HeLa
cells, IP3Rs were partly responsible for this DPB162-AE elicited ER Ca2+ release, with a likely additional
role of other ER Ca2+ release channels [69]. A similar IP3R-dependent release of ER Ca2+ stores was
reported in DT40 cells by 2-APB at concentrations ranging from 10 to 75 µ
M [56]. The release of ER Ca2+ by
2-APB is most likely cell type-dependent, as pretreatment of Chinese hamster ovary-K1 cells with 100
2-APB did not affect the thapsigargin-mediated store-depletion [71].
µM

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(a)
(b)
(c)
Figure 1. Pharmacological characterization of store-operated calcium entry (SOCE) in MDA-MB-231
cells by fluorometric imaging plate reader (FLIPR) assay. ( ) Structures of known SOCE inhibitors used
in the validation of the assay. ( ) Recorded intracellular calcium signal over the course of the assay.
Black bars above traces indicate duration of applications. MDA-MB-231 cells were incubated with
thapsigargin (Tg, 1 M, 10 min) in nominal calcium-free (NCF) buffer, followed by DMSO (negative
control) or drug application (in NCF) for 10 min and finally, addition of 2 mM extracellular CaCl2. Note
how low concentration (5 M) of 2-APB enhanced SOCE (blue trace) whereas at high concentration
(50 M) SOCE is blocked (red trace). ( ) Quantification of calcium influx after CaCl2 application (area
under the curve (AUC)), normalized to DMSO control (n 9 for all data points). Data shown are mean
a
b
µ
µ
µ
c
≥
± SD. Statistical significance compared to DMSO control is indicated as “***” for p ≤ 0.001.
In this study, we used the MDA-MB-231 breast cancer cell line to screen our newly developed
2-APB analogues for their action on SOCE, using a fluorometric imaging plate reader (FLIPR)-based
Ca2+ imaging assay. FLIPR assays with MDA-MB-231 cells have been used in the past for discovery
of SOCE modulators [26
MDA-MB-231 cells is conducted by STIM1 and Orai1 [18
2-APB at concentrations 30 M blocks SOCE in MDA-MB-231 cell line [73–
,
30
,
72]. Earlier reports using RNA silencing have shown that SOCE in
26 73]. In addition, it was shown that
75]. Herein, we report
,
,
≥
µ
new derivatives of 2-APB, that are more potent than 2-APB in blocking SOCE in MDA-MB-231 cells.
We also report the effect of our new 2-APB derivatives on store-depletion as well as proliferation of
MDA-MB-231 cells.
2. Results and Discussion
2.1. Synthesis of 2-APB Analogues
The mono-halogenated 2-APB analogues were synthesized by following our previously optimized
route [67]. More specifically, mono-halogenated aryl bromides or iodides were transformed into

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their corresponding aryl lithium species by halogen-lithium exchange at low temperature and then
reacted with phenylboronic acid pinacol ester (Scheme 1). This gave the crude aryl phenyl borinic
acids after work up and quick flash chromatography purification, which were subsequently esterified
with 2-aminoethanol to yield the stable, mono-halogenated 2-APB analogues. This protocol allowed
the synthesis of almost all possible mono-halogenated 2-APB analogues (Scheme 1a) in isolated
yields ranging from 6 to 65%. For the synthesis of o-Br-2APB it was important to use a mixture of
toluene/THF 4:1 [76] in the lithiation and subsequent borination step, in order to significantly reduce
the formation of a biphenyl side product that is very difficult to separate from the desired product
(Appendix A). We were unable to isolate the ortho-iodo 2-APB derivative o-I-2APB. All our conditions
gave inseparable mixtures of ortho-iodophenyl borinic acid and significant amounts of the side product
(Appendix A).
Scheme 1. Synthesis of (
and conditions: (i) n-BuLi or t-BuLi, Et2O, or THF,
or THF,
78 ◦C to rt; then 1 M HCl, rt. For the synthesis of o-Br-2APB toluene/THF 4:1 was used as
a
) mono-halogenated and (
b) para-substituted 2-APB analogues. Reagents
−
78 ◦C; then phenylboronic acid pinacol ester, Et2O
−
solvent. (ii) 2-Aminoethanol, EtOH, MeCN, or THF, rt or reflux. Isolated yields over two steps: 6–66%.
p-NO2-2APB was synthesized via different route (see text and Materials and Methods).
We were also interested in accessing 2-APB congeners with small substituents other than halogens
in order to probe the influence of size and electron-withdrawing or electron-donating properties at
the para-position on SOCE activity. These compounds were generated using the same route as above,
except for p-NO2-2APB, and yielded 2-APB analogues shown in Scheme 1b in 8–66%. The para-nitro
2-APB analogue was obtained by reacting 4-nitrophenylborinic acid ester with phenyllithium, followed
by borinate formation of the isolated borinic acid addition product.
For comparison, we also synthesized the dimeric 2-APB compound DPB162-AE, reported by
the Mikoshiba group [54
heterocyclic SOCE blockers GSK-7975A and Synta66 were also synthesized (Schemes S2 and S3)
applying procedures in the corresponding patents [78 79]. Notably, we have obtained a crystal
,77], but slightly adapted their procedure (Scheme S1). Recently reported
,
structure for GSK-7975A which was not described before (Figure S1).
Others and we have shown previously that amino acid adducts of 2-APB, forming acyloxyboranes,
are less dynamic in solution than the corresponding amino alcohol esters [67,80]. Furthermore, our
studies indicated that amino acid 2-APB adducts with large side chain might block SOCE in HEK293

Int. J. Mol. Sci. 2020, 21, 5604
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cells efficiently. In order to study this more systematically, we expanded our 2-APB library in this
direction and synthesized several amino acid complexes shown in Scheme 2. Conveniently, such
derivatives can be synthesized in one step from diphenylborinic anhydride.
Scheme 2. Synthesis of 2-APB amino acid analogues. Reagents and conditions: (i) amino acid, EtOH,
reflux. Isolated yields: 16%-quantitative.
The cyclic nature of the synthesized 2-APB derivatives in solution is apparent from their 1H and
11B NMR spectra (in DMSO-d6). Donation of electron density of the amino alcohol or amino acid
nitrogen to the electron deficient boron atom leads to deshielding of the N-H protons and thus they
are low field shifted to 6.1–7.2 ppm. Likewise, the 11B chemical shift of the central boron atom is
recorded at 3.7–4.7 ppm for all isolated 2-APB derivatives, except for βAla-2APB which is at 2.05 ppm.
For comparison, the “uncoordinated” boron atom in diphenyl borinic acid (Ph2B(OH)) shows a 11
chemical shift of 45 ppm [67]. The cyclic form of the 2-APB derivatives is also visible in the crystal
structures of cBu-2APB p-SO2NMe2-2APB o-F-2APB, and o-Cl-2APB (Figures S2–S5). For the latter
B
,
,
two derivatives the phenyl and ortho-substituted phenyl groups are mutually disordered in the structure
(Figures S4 and S5).
2.2. Influence of 2-APB Analogues on Store-Operated Ca2+ Entry in MDA-MB-231 Breast Cancer Cells
For evaluating the activity of the synthetic 2-APB analogues on SOCE, we used a fluorescence-based
calcium influx assay into the triple negative breast cancer cells MDA-MB-231. In our assay protocol,
1 µM thapsigargin in nominal calcium-free (NCF) buffer was first applied to the cells for several
minutes, which irreversibly blocks SERCA, passively depletes the intracellular ER-stores and initiates
the SOCE cascade. This was followed by compound application in NCF buffer for several minutes
and a subsequent application of 2 mM extracellular CaCl2 to measure external calcium entry through
CRAC channels (Figure 1b). We validated our assay using known SOCE borinic acid inhibitors
2-APB, DPB162-AE and heterocyclic compounds GSK-7975A
concentrations between 5 and 20 M (Figure 1c). As reported previously in other cell lines, 2-APB
showed a similar bimodal effect on SOCE in MDA-MB-231 cells wherein it enhanced Ca2+ entry at low
concentrations (5 and 10 M) and blocked at higher concentrations (partial block at 20 and maximum
block at 50 M) [49 56]. The dimeric 2-APB derivative DPB162-AE exerted a comparable inhibition
, Synta66, and Ro2959 [81] (Figure 1a) at
µ
µ
µ
,

Int. J. Mol. Sci. 2020, 21, 5604
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of SOCE at lower concentration, as 2-APB at 50
µM, confirming its higher activity than 2-APB [77].
The inhibition of SOCE at 10 µM by DPB162-AE was even stronger than by heterocyclic Ro2959 and
Synta66. Even doubling the concentration of the latter two compounds did not increase the SOCE block.
In our assay, GSK-7975A was the most potent heterocyclic SOCE blocker. We were surprised at the
somewhat low activity of Ro2959 and Synta66. The inhibition activity of these heterocyclic compounds
has been previously characterized by measuring the influence on the calcium release-activated calcium
current (ICRAC) using electrophysiology.
The reported values for Synta66 (IC50 1.4
µM, ICRAC in RBL cells) [82
,83], GSK-7975A (IC50 4 µM,
I
CRAC in HEK293 cells) [84], and Ro2959 (IC50 0.4
µM, ICRAC in RBL-2H3 cells) [85] suggest that they
should fully block calcium entry at the concentrations applied in our SOCE assay in MDA-MB-231
cells. The electrophysiology studies have also revealed that all three heterocyclic compounds have a
slow onset of inhibition, requiring cells to be preincubated for long periods (e.g., >1 h for Synta66).
Indeed, when we preincubated 0.3–0.5 µM GSK-7975A or 1.25–10 µM Synta66 for 20 min before the
thapsigargin application (30 min total incubation time), SOCE in MDA-MB-231 cells was inhibited by
50% or 80%, respectively, which is in the range of the reported IC50 values (Figure S6).
Next, we investigated whether the SOCE observed in MDA-MB-231 cells that we could block by
inhibitors shown in Figure 1 is conducted by STIM1 and Orai1. Indeed, knock-out of either STIM1 or
Orai1 in MDA-MB-231 cells using CRISPR/Cas9 gene editing technique, led to significantly diminished
SOCE (Figure S7). This suggests that SOCE in these cells is mainly facilitated by Orai1/STIM1,
confirming the RNA interference studies by Motiani et al. [73].
After having validated the SOCE assay, we first tested all our synthetic 2-APB analogues
at a single concentration (50
efficiently blocked SOCE, some even exerted a more pronounced reduction of the Ca2+ entry
than the parent compound (e.g., m-Cl-2APB o-Cl-2APB o-Br-2APB, and m-I-2APB). Compared
µM, Figure 2). Our data show that all halogenated compounds
,
,
to the para-halogenated analogues, most of the other para-substituted congeners did not block SOCE
significantly. In fact, 2-APB derivatives with large, polarized substituents in the para-position rather
enhanced SOCE (e.g., p-CN-2APB, p-NO2-2APB, and p-SO2NMe2-2APB). The amino acid 2-APB
derivatives βAla-2APB His-2APB, and Tyr-2APB could not be tested because of very poor solubility in
,
the assay buffer. Only few derivatives with polar side chains (e.g., Gln-2APB, Lys-2APB, and Cys-2APB)
were able to inhibit SOCE in MDA-MB-231 cells (Figure 2).

Int. J. Mol. Sci. 2020, 21, 5604
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Figure 2. Screening of synthetic 2-APB analogues on SOCE in MDA-MB-231 cells using the FLIPR-based
SOCE assay. Quantification of calcium influx after CaCl2 application (AUC), normalized to DMSO
control (n
≥
9 for all data points). Data shown are mean
±
SD. All compounds were tested at 50 µM
concentration, unless otherwise noted. Statistical significance compared to DMSO control is indicated
as “***” for p ≤ 0.001, “*” for 0.01 < p ≤ 0.05 and “ns” not significant for p > 0.05.
Given the encouraging results, we turned our attention to the mono-halogenated 2-APB analogues.
In order to test if any of them have higher SOCE blocking potency than 2-APB, we applied them at
lower concentrations (10 and 20 µM, Figure 3). At 10 µM compound concentration all fluorinated
2-APB derivatives enhanced SOCE, similar to 2-APB at the same concentration. This effect was also
observed for some of the chloro-compounds, depending on the substitution position, whereas the
bromo- and iodo-analogues were overall rather inhibiting SOCE at this low concentration. In terms of
the halogen substitution position on one of the phenyl rings of 2-APB, the chloro, bromo, and iodo
2-APB derivatives affect SOCE with the following trend: ortho enhances SOCE, meta blocks efficiently,
and para shows only partial or no block of SOCE. This efficient block of SOCE by the meta-substituted
derivatives is also apparent at 20
(Figure 3).
µM compound concentration, at which 2-APB only partially blocks

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Figure 3. Select halogenated 2-APB analogues block SOCE more potently at lower concentration than
2-APB. Influence on SOCE in MDA-MB-231 cells after prior depletion of the intracellular Ca2+ store,
using the FLIPR-based SOCE assay. Data show quantification of Ca2+ entry (AUC, normalized to
DMSO control) after extracellular application of 2 mM CaCl2. Compound concentrations: 10
bars), 20 M (grey bars), 50 M (dark grey bars). All data points from n 9 independent experiments.
Data shown are mean SD. Statistical significance is compared to the respective DMSO control and
indicated as “***” for p ≤ 0.001 and “*” for 0.01 < p ≤ 0.05.
µM (white
µ
µ
≥
±
When comparing the SOCE blocking effect at 10 M compound concentration with the different
µ
halogen substituents at the same position, all ortho-halogenated derivatives did not block, but rather
potentiated SOCE. For the meta- and para-positions, inhibiting potency increased with increasing size
and decreasing electronegativity (m-F-2APB << m-Cl-2APB
<< p-Cl-2APB < p-Br-2APB < p-I-2APB).
< m-Br-2APB < m-I-2APB and p-F-2APB
2.3. Store Depletion in MDA-MB-231 Breast Cancer Cells by 2-APB Analogues
We also investigated if the synthesized 2-APB derivatives have an effect on intracellular Ca2+
stores, as it has been observed in some cell lines with DPB-162AE 69] and non-boron based SOCE
inhibitors [72]. To this end, we treated MDA-MB-231 cells with compounds at 50 M in NCF
[
µ
buffer first and then added thapsigargin to deplete the stores (Figure 4). The FLIPR recordings of
intracellular [Ca2+] show that 2-APB, for instance, does not lead to any detectable mobilization of
Ca2+ from intracellular stores of MDA-MB-231 cells. Conversely, p-Br-2APB produced a substantial
Ca2+ release signal during application and subsequent store depletion by thapsigargin was unable
to mobilize more calcium. Quantification of this effect revealed that dimeric 2-APB compound
DPB162-AE mobilizes stored calcium significantly at high concentration compared to 2-APB. From the
halogenated compounds, p-Br-2APB and p-I-2APB were as strong or even stronger Ca2+ mobilizers
than DPB162-AE.

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(a)
(b)
Figure 4. Store-depletion by selected 2-APB analogues in MDA-MB-231 cells. (a) Examples of recorded
intracellular calcium responses for m-Br-2APB and p-Br-2APB in FLIPR assay. Black bars above traces
indicate duration of compound applications. MDA-MB-231 cells were incubated with compounds
(50
in NCF buffer. (
of the curves, by compounds (white bars) and by subsequent thapsigargin (dark bars) application.
µ
M, unless otherwise noted) in NCF for 10 min, followed by thapsigargin (Tg, 2.5
µM) application
b
) Quantifications of Ca2+ mobilization effect from intracellular stores, using the slopes
Normalized to post-thapsigargin response by DMSO, n
≥
9 for all data points. Data shown are mean
±
SD. Statistical significance is compared to the respective DMSO control and indicated as “***” for p
0.001, “**” for 0.001 < p ≤ 0.01 and “*” for 0.01 < p ≤ 0.05. ns, not significant for p > 0.05.
≤
The compound-induced (pre-thapsigargin) store depletion increases with increasing size and
decreasing electronegativity of the halogen substituent (F < Cl < Br < I), in particular at the para-position.
Concerning the halogen substitution position, the Ca2+ mobilization effect was strongest for the
para-compounds, less for the meta-, and not substantial for the ortho-analogues.

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In addition, we checked the ability of m-Br-2APB and p-Br-2APB to block ICRAC in HEK293 cells
overexpressing STIM1/Orai1 (Figure 5). Both compounds rapidly inhibited ICRAC in a similar fashion
as the parent compound 2-APB, which suggests that in addition to store-depleting activity (Figure 4)
SOCE was blocked as well.
(a)
(b)
(c)
Figure 5. Inhibition of ICRAC in HEK293 cells overexpressing human STIM1/Orai1 by 2-APB (black
traces) and analogues m-Br-2APB (blue) and p-Br-2APB (red). ( ) ICRAC was evoked with 20 mM
BAPTA and 50 M IP3 in the patch pipette, currents at 130 mV were normalized to cell capacity and
plotted as current density (CD) versus time. Upon ICRAC development, 2-APB (black), m-Br-2APB
(blue) or p-Br-2APB (red) were applied from 120–180 s (black bar above traces). ( ) I-V curves extracted
from recordings in ( ) when ICRAC had developed at t = 118 s. ( ) Inhibition of ICRAC was calculated
from CD at 180 s normalized to the point in time before compounds were applied (t = 118 s) and plotted
as inhibition in %. All compounds applied at 50 M, number of experiments (n) in brackets in panel ( ).
a
µ
−
b
a
c
µ
a
Data shown are mean ± SD. Statistical significance is indicated as ns for not significant (p > 0.05).
2.4. Synthetic 2-APB Analogues Effect MDA-MB-231 Cell Viability
We wondered if the propensity of the 2-APB analogues to mobilize calcium from internal stores
is affecting cell viability. MDA-MB-231 cells as well as MCF-10A, a non-tumorigenic mammary
epithelial cell line (control), were incubated with 2-APB or halogenated derivatives thereof at 50 µM
concentration for different time points ranging from 24 to 120 h. The XTT cell viability assay showed
that 2-APB did not have any substantial effect on proliferation of any of the cell lines (Figure 6a).
While the more specific SOCE blocker GSK-7975A had only minor cytotoxic effect in both cell lines,
50 µM DPB162-AE strongly affected the proliferation of MDA-MB-231 cells in comparison to MCF-10A
(Figure 6a). On the other hand, the ortho- and para-halogenated analogues did not show contrasting
effects on proliferation of MDA-MB-231 cells compared to MCF-10A cells (Figure 6b,d). Conversely,
m-Cl-2APB, m-Br-2APB, and m-I-2APB exhibited a stronger cytotoxic effect similar to that of 50 µM
DPB162-AE on MDA-MB-231 cells compared to MCF-10A cells (Figure 6c). These compounds also
induced changes in morphology of MDA-MB-231 cells (Figure 7). The difference between the ortho-
and para-halogenated regioisomers for example, p-Br-2APB and m-Br-2APB is particularly striking
considering that the bromine atom is moved by only one carbon position (Figures 6 and 7). In any
case, the data suggest that compounds which empty internal calcium stores (e.g., p-Br-2APB) do not
influence MDA-MB-231 cell viability much stronger than compounds which empty internal stores very
little (e.g., p-F-2APB). Furthermore, we do not see a direct relation between SOCE inhibitory activity
of 2-APB analogues or control compounds (e.g., GSK-7975A) and MDA-MB-231 cell proliferation.
We cannot rule out the possible off-target effects of our 2-APB analogues in the observed cytotoxicity.

Int. J. Mol. Sci. 2020, 21, 5604
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Figure 6. Dose-response curves of 20
µ
M
GSK-7975A, 5
) 50
p-Br-2APB, and p-I-2APB (d
µ
M and 50
µ
M
DPB162-AE, and 50
m-Cl-2APB m-Br-2APB
) in non-tumorigenic
µM
2-APB (
a), 50
µM
o-F-2APB
,
o-Cl-2APB, and o-Br-2APB
p-F-2APB p-Cl-2APB
(b
µM
m-F-2APB
,
,
,
and m-I-2APB
(c), and 50
µM
,
,
breast epithelial cell line MCF-10A and triple negative breast cancer cell line MDA-MB-231. Relative
cell survival in case of each compound treatment was calculated as a percentage of DMSO control.
Data shown are mean
±
SD (n = 12/compound for each time point) of two independent experiments.
Significance levels compared to the DMSO control are indicated as “*” for 0.01 < p
< p ≤ 0.01, or “***” for p ≤ 0.001 and not indicated if non-significant (p > 0.05).
≤
0.05, “**” for 0.001

Int. J. Mol. Sci. 2020, 21, 5604
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Figure 7. Photomicrographs show the morphological aspects of the cultures of MDA-MB-231 cells after
72 h treatment with control (DMSO) or the indicated compounds. The shown bright-field microscopy
images were captured on Nikon Eclipse TiU inverted microscope.

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3. Materials and Methods
3.1. Chemical Synthesis
Commercial solvents and reagents were used without further purification. Dry solvents were
obtained by filtration over columns of dried alumina under a positive pressure of argon. The glassware
was dried by heating while flushing with argon. In general, reactions were run under argon atmosphere.
Flash chromatography was performed on a TeledyneISCO CombiFlash Rf+ (Lincoln, NE, USA) using
RediSepRf (Lincoln, NE, USA) pre-packed silica gel columns and cyclohexane/EtOAc gradients to
1
elute compounds, unless otherwise noted. H, 11B, 13C, and 19F NMR spectra were measured on a
Bruker Avance 300 III HD or Avance II 400 spectrometer. Chemical shifts given in ppm, are referenced
to residual solvent peaks (1H and 13C) or internal standard (11B and 19F), and coupling constants J
in Hz. (high resolution) mass spectra were recorded on a Thermo Fisher Scientific (Waltham, MA,
USA) LTQ Orbitrap XL spectrometer consisting of a linear ion trap (LTQ) featuring a HCD collision
cell, coupled to the Orbitrap mass analyzer, equipped with a nanospray ion source (NSI). All X-ray
structure determination measurements were made on an Oxford Diffraction SuperNova area-detector
diffractometer using mirror optics monochromated Mo Kα radiation (λ = 0.71073 Å) and Al filtered.
Reference compound Ro2925 was purchased from AK Scientific and DPB162-AE
GSK-7975A were resynthesized (Supplementary Materials) by adapting literature [54] and patent
protocols [78 79]. The synthesis and spectroscopic characterization of 2-APB derivatives p-Me-2APB
, Synta66 and
,
and p-I-2APB was reported previously [67].
3.1.1. General Procedure A for the Synthesis of Mono-substituted Phenyl 2-APB Analogues
Dry Et2O or THF (60 mL) was cooled to
(10.2 mmol, 1.7 M in pentane) was added dropwise. The resulting mixture was stirred at
5 min. Then, a solution of iodide or bromide (4.96 mmol) in dry Et2O or THF (10 mL) was added to the
−
78 ◦C and, unless otherwise noted, t-BuLi solution
78 ◦C for
−
reaction mixture at a drop rate so that the internal temperature remained below
75 ◦C. The mixture
−
was stirred for another hour at −78 ◦C. A solution of phenylboronic acid pinacol ester (4.96 mmol) in
dry Et2O or THF (10 mL) was then added, again taking care that the internal temperature did not raise
above
by the addition of aq. 1 M HCl (100 mL). The two resulting phases were separated, and the aqueous
phase extracted with Et2O or EtOAc (2 20 mL). The combined organic layers were dried over Na2SO4,
−
75 ◦C. The reaction mixture was allowed to warm up to r.t. overnight and was then quenched
×
filtered and evaporated. The crude, oily borinic acid was purified by flash chromatography (silica gel,
cyclohexane/EtOAc gradient). The resulting purified borinic acid (oil or solid) was dissolved in MeCN
or abs. EtOH (5 mL) and 2-aminoethanol (3.48 mmol, 1 equiv. with respect to purified borinic acid)
was added to the solution. The resulting solution was stirred at r.t. For 30 min–1 h to produce the
corresponding borinic acid 2-aminoethyl ester, which either precipitated or was isolated after removal
of the solvent (for details see individual compounds below).
3.1.2. General Procedure B for the Synthesis of 2-APB Amino Acid Analogues
A mixture of amino acid (0.70 mmol) and diphenylborinic anhydride (0.58 mmol) in abs. EtOH
(10 mL) was heated at reflux for 30 min–3 h. The clear solution was allowed to cool to r.t. during
which the product often precipitated. For some derivatives, further cooling to 0 ◦C or 20 ◦C and
−
concentration of the solution was necessary to initiate precipitation or crystallization. The solid
products were washed (e.g., with cold EtOH, n-hexane or Et2O) or recrystallized if necessary (for
details see individual compounds below).
3.1.3. 2-(4-Fluorophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (p-F-2APB)
Following general procedure A, 4-fluoroiodobenzene (572
µL, 4.96 mmol) in dry Et2O was lithiated
with t-BuLi (6 mL, 1.7 M in pentane, 10.2 mmol) and reacted with phenylboronic acid pinacol ester

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(1.01 g, 4.96 mmol). Purification of the crude borinic acid by flash chromatography afforded a brownish
oil (663 mg), which was dissolved in MeCN and treated with 2-aminoethanol (210 L, 3.48 mmol) at r.t.
µ
For 30 min. Removing the solvents under reduced pressure and drying the residue under high vacuum
1
afforded the title compound as a colorless powder (742 mg, 62%). H NMR (300 MHz, DMSO-d6)
δ
7.43–7.34 (m, 4H), 7.20–7.09 (m, 2H), 7.08–6.99 (m, 1H), 6.97–6.88 (m, 2H), 6.07 (s, 2H), 3.76 (t, J = 6.5,
2H), 2.82 (p, J = 6.3, 2H). 11B NMR (96 MHz, DMSO-d6) 4.27. 13C NMR (75 MHz, DMSO-d6)
133.20,
-118.69 (p,
δ
δ
133.11, 131.46, 126.66, 124.98, 113.23, 112.98, 62.43, 41.35. 19F NMR (282 MHz, DMSO-d6)
δ
J = 9.1). MS (ESI+) m/z 305.18 [M+C2H8NO]+, 266.11 [M+Na]+, 244.13 [M+H]+, 166.08 [M-C6H5]+,
148.09 [M-C6H4F]+. HRMS (ESI+) m/z calc. For C14H16ONBF [M+H]+: 244.1303; found 244.1303.
3.1.4. 2-(3-Fluorophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (m-F-2APB)
According to general procedure A, 1-bromo-3-fluorobenzene (392 µL, 5 mmol) in dry THF was
lithiated with t-BuLi (6 mL, 1.7 M in pentane, 10.2 mmol) and condensed with phenylboronic acid
pinacol ester (1.02 g, 5 mmol). After aqueous work up, the crude borinic acid was purified by flash
chromatography and gave a pale yellow oil (188 mg). This was dissolved in abs. EtOH and treated with
2-aminoethanol (70 µL, 1.16 mmol). After solvent removal and drying the residual oil in a Kugelrohr
apparatus under high vacuum at 80 ◦C, the title compound was obtained as off-white powder (223 mg,
1
17%). H NMR (300 MHz, DMSO-d6)
δ
7.43–7.35 (m, 2H), 7.23–6.99 (m, 6H), 6.86–6.75 (m, 1H), 6.13 (s,
4.01. 13C NMR (75 MHz,
163.59, 160.38, 131.41, 128.45, 128.36, 127.32, 127.30, 126.70, 125.08, 117.32, 117.10, 111.52,
2H), 3.76 (t, J = 6.5, 2H), 2.83 (p, J = 6.2, 2H). 11B NMR (96 MHz, DMSO-d6)
δ
DMSO-d6)
δ
111.25, 62.44, 41.35. 19F NMR (282 MHz, DMSO-d6) -115.64 (m). MS (ESI+) m/z 244.13 [M+H]+, 266.11
δ
[M+Na]+, 166.08 [M-C6H5]+. HRMS (ESI+) m/z calc. For C14H16ONBF [M+H]+: 244.1303; found
244.1300.
3.1.5. 2-(2-Fluorophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (o-F-2APB)
1-Bromo-2-fluorobenzene (547 µL, 5 mmol) was lithiated in dry THF using t-BuLi, following
general procedure A, and reacted with phenylboronic acid pinacol ester (1.02 g, 5 mmol). After work
up and purification of the crude borinic acid by flash chromatography an almost colorless oil was
isolated (841 mg) that was mixed with abs. EtOH and 2-aminoethanol (275 µL, 4.56 mmol) to generate
the borinate. Drying the crude product in the Kugelrohr under high vacuum at 80 ◦C gave a slightly
brown solid (997 mg), which was recrystallized from MeCN (7 mL), yielding off-white crystals (837 mg,
1
65%) suitable for X-ray structure determination (Figure S4). H NMR (300 MHz, DMSO-d6)
δ 7.47–7.33
(m, 3H), 7.17–6.84 (m, 6H), 6.33–6.06 (m, 2H), 3.87–3.75 (m, 1H), 3.73–3.61 (m, 1H), 2.95–2.73 (m, 2H).
11B NMR (96 MHz, DMSO-d6) 3.70. 13C NMR (75 MHz, DMSO-d6)
133.60, 133.43, 131.58, 131.55,
127.26, 127.15, 126.57, 125.13, 123.15, 123.12, 114.00, 113.66, 62.36, 41.69. 19F NMR (376 MHz, DMSO-d6)
δ
δ
δ
-105.62 (m). MS (ESI+) m/z 244.13 [M+H]+, 266.11 [M+Na]+, 305.18 [M+C2H8NO]+. HRMS (ESI+)
m/z calc. For C14H16ONBF [M+H]+: 244.1303; found 244.1305.
3.1.6. 2-(4-Chlorophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (p-Cl-2APB)
Following general procedure A, the title compound was synthesized from 1-chloro-4-iodobenzene
(1.1 g, 4.61 mmol) using dry Et2O as reaction solvent. Work up and purification by flash chromatography
yielded a colorless solid (620 mg) which was treated with 2-aminoethanol (200 µL, 3.32 mmol) in
MeCN. The mixture was heated to reflux for 1 h in order to dissolve all the solid. Removing the solvent
under reduced pressure afforded a foam, which was dried in vacuo. The solidified foam was crushed
and washed with n-hexane. Further drying under high vacuum gave the title compound as a colorless
1
powder (743 mg, 62%). H NMR (300 MHz, DMSO-d6)
δ
7.43–7.33 (m, 4H), 7.20–7.10 (m, 4H), 7.08–7.00
4.14.
(m, 1H), 6.11 (s, 2H), 3.75 (t, J = 6.5, 2H), 2.82 (p, J = 6.2, 2H). 11B NMR (96 MHz, DMSO-d6)
δ
13C NMR (75 MHz, DMSO-d6)
δ 133.38, 131.43, 129.85, 126.69, 126.44, 125.05, 62.45, 41.36. MS (ESI+)
m/z 260.10 [M+H]+, 282.08 [M+Na]+, 300.13 [M+C2H3N]+, 321.15 [M+C2H8NO]+, 182.05 [M-C6H5]+,
148.09 [M-C6H4Cl]+. HRMS (ESI+) m/z calc. For C14H16ONBCl [M+H]+: 260.1008; found 260.1011.

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3.1.7. 2-(3-Chlorophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (m-Cl-2APB)
1-Bromo-3-chlorobenzene (587
µL, 5 mmol) was lithiated in dry THF and reacted with
phenylboronic acid pinacol ester according to general procedure A. Work up and purification by flash
chromatography gave a pale yellow oil (741 mg) that was treated with 2-aminoethanol in abs. EtOH
for 1 h at r.t. After solvent removal and drying of the residual oil in a Kugelrohr under high vacuum
1
at 80 ◦C the title compounds was isolated as a colorless powder (746 mg, 58%). H NMR (300 MHz,
DMSO-d6)
δ
7.42–7.29 (m, 4H), 7.20–7.10 (m, 3H), 7.10–7.01 (m, 2H), 6.16 (s, 2H), 3.76 (t, J = 6.5, 2H),
132.07,
2.83 (p, J = 6.2, 2H). 11B NMR (96 MHz, DMSO-d6)
δ
4.17. 13C NMR (75 MHz, DMSO-d6)
δ
131.37, 130.99, 129.93, 128.62, 126.74, 125.12, 124.72, 62.45, 41.37. MS (ESI+) m/z 260.10 [M+H]+, 282.08
[M+Na]+, 321.15 [M+C2H8NO]+, 182.05 [M-C6H5]+, 148.09 [M-C6H4Cl]+. HRMS (ESI+) m/z calc.
For C14H16ONBCl [M+H]+: 260.1008; found 260.1006.
3.1.8. 2-(2-Chlorophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (o-Cl-2APB)
The title compound was synthesized from 1-bromo-2-chlorobenzene (584 µL, 5 mmol) following
general procedure A using THF as the reaction solvent. Work up and flash chromatography purification
of the borinic acid afforded a colorless oil (450 mg) that was subjected to 2-aminoethanol treatment in
abs. EtOH. After removing the solvent and drying of the oily product (Kugelrohr, 80 ◦C, high vacuum)
a colorless solid was isolated, which was suspended in Et2O (10 mL) using an ultrasonic bath. After the
precipitate settled, the supernatant was removed. Washing with Et2O was repeated two more times
and the remaining solid (490 mg) was recrystallized from MeCN (3 mL), yielding colorless crystals
(235 mg, 18%) suitable for X-ray diffraction analysis (Figure S5). 1H NMR (300 MHz, DMSO-d6)
7.70–7.59 (m, 1H), 7.37–7.27 (m, 2H), 7.24–6.93 (m, 6H), 6.39 (s, 1H), 6.14 (s, 1H), 3.84–3.68 (m, 1H),
3.68–3.57 (m, 1H), 2.88 (dd, J = 15.4, 8.2, 2H). 11B NMR (96 MHz, DMSO-d6) 4.38. 13C NMR (75 MHz,
δ
δ
DMSO-d6)
δ 137.62, 133.77, 131.94, 131.52, 128.20, 127.27, 126.34, 125.55, 125.00, 62.09, 42.25. MS (ESI+)
m/z 260.10 [M+H]+, 282.08 [M+Na]+, 336.13 [M+C6H5]+, 182.05 [M-C6H5]+. HRMS (ESI+) m/z calc.
For C14H16ONBCl [M+H]+: 260.1008; found 260.1005.
3.1.9. 2-(4-Bromophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (p-Br-2APB)
The starting material 1,4-dibromobenzene (1 g, 4.24 mmol) was purified by flash chromatography
(silica gel, cyclohexane/EtOAc gradient) prior to use in the reaction. General procedure A was followed
using dry Et2O as the reaction solvent and n-BuLi solution (2.5 M in hexanes) for the lithiation.
The thus obtained pale yellow, oily borinic acid (502 mg) after work up and flash chromatography
was transformed into its borinate by 2-aminoethanol treatment in abs. EtOH. The oily product was
dried (Kugelrohr, 80 ◦C, high vacuum), which yielded the title compound as colorless powder (472 mg,
1
37%). H NMR (300 MHz, DMSO-d6)
δ
7.39–7.27 (m, 6H), 7.17–7.09 (m, 2H), 7.08–7.00 (m, 1H), 6.11 (s,
4.11. 13C NMR (75 MHz,
2H), 3.75 (t, J = 6.5, 2H), 2.82 (t, J = 6.3, 2H). 11B NMR (96 MHz, DMSO-d6)
δ
DMSO-d6) δ
133.83, 131.41, 129.33, 126.69, 125.05, 118.56, 62.44, 41.36. MS (ESI+) m/z 304.05 [M+H]+,
326.03 [M+Na]+, 226.00 [M-C6H5]+, 148.09 [M-C6H4Br]+. HRMS (ESI+) m/z calc. For C14H16ONBBr
[M+H]+: 304.0503; found 304.0500.
3.1.10. 2-(3-Bromophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (m-Br-2APB)
Following general procedure A, the title compound was synthesized from 1,3-dibromobenzene (550
µL, 4.55 mmol) using dry Et2O as reaction solvent. Work up and purification by flash chromatography
gave the borinic acid as a yellow oil (1.06 g), which was dissolved in abs. EtOH and esterified with
2-aminoethanol. Solvent removal and drying the oily product in the Kugelrohr at 80 ◦C in high vacuum
yielded the final borinate as a colorless powder (887 mg, 64%). 1H NMR (400 MHz, DMSO-d6)
δ
7.54–7.49 (m, 1H), 7.39–7.35 (m, 3H), 7.24–7.19 (m, 1H), 7.17–7.02 (m, 4H), 6.17 (s, 2H), 3.76 (t, J = 6.5,
2H), 2.88–2.78 (m, 2H). 11B NMR (96 MHz, DMSO-d6) 3.84. 13C NMR (75 MHz, DMSO-d6)
133.91,
131.37, 130.30, 129.07, 127.61, 126.75, 125.14, 121.46, 62.45, 41.38. MS (ESI+) m/z 304.05 [M+H]+, 326.03
δ
δ

Int. J. Mol. Sci. 2020, 21, 5604
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[M+Na]+, 365.10 [M+C2H8NO]+. HRMS (ESI+) m/z calc. For C14H16ONBBr [M+H]+: 304.0503; found
304.0504.
3.1.11. 2-(2-Bromophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (o-Br-2APB)
A mixture of THF (10 mL), toluene (40 mL), 1,2-dibromobenzene (602
µL, 5 mmol),
and phenylboronic acid pinacol ester (2.04 g, 10 mmol) was cooled down to
−
78 ◦C. n-BuLi solution (2
mL, 2.5 M in hexanes, 5 mmol) was added dropwise over a period of 3 h. The cloudy white mixture
was allowed to warm up to r.t. overnight. The mixture was quenched by the addition of aq. 1 M HCl
(50 mL) and EtOAc (100 mL) was added. The resulting two phases were separated and the organic
phase was washed with aq. 1 M HCl (3
×
50 mL). The organic phase was dried over Na2SO4 and the
solvents were removed under reduced pressure. The residue was dried in vacuo, yielding a viscous
sticky solid. The solid was purified by flash chromatography (silica gel, with cyclohexane/EtOAc
gradient) affording an oil (446 mg). The oil was dissolved in abs. EtOH (5 mL) and 2-aminoethanol (120
µL, 2.00 mmol) was added to the solution. The resulting mixture was stirred at r.t. For 1 h. The solvent
was removed under reduced pressure and the residue was dried in the Kugelrohr under high vacuum
at 80 ◦C. The resulting solid was recrystallized from MeCN yielding the title compound as colorless
1
crystals (189 mg, 12%). H NMR (300 MHz, DMSO-d6)
δ
7.68 (dd, J = 7.4, 1.9, 1H), 7.38–7.27 (m, 3H),
7.22 (td, J = 7.3, 1.2, 1H), 7.12–6.97 (m, 4H), 6.40 (s, 1H), 6.12 (s, 1H), 3.80–3.69 (m, 1H), 3.66–3.56 (m,
1H), 3.02–2.80 (m, 2H). 11B NMR (96 MHz, DMSO-d6) 4.53. 13C NMR (75 MHz, DMSO-d6)
δ δ 134.25,
132.14, 131.61, 128.57, 127.64, 126.28, 126.00, 124.96, 62.10, 42.51. MS (ESI+) m/z 304.05 [M+H]+. HRMS
(ESI+) m/z calc. For C14H16ONBBr [M+H]+: 304.0503; found 304.0496.
3.1.12. 2-(3-Iodophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (m-I-2APB)
1,3-Diiodobenzene (1.65 g, 5 mmol) was lithiated with n-BuLi (2 mL, 2.5 M in hexanes, 5 mmol) in
dry THF, following general procedure A, and reacted with phenylboronic acid pinacol ester to yield
the corresponding borinic acid (604 mg) after work up and flash chromatography purification as a
pale yellow oil. Treatment with 2-aminoethanol in abs. EtOH yielded, after solvent removal and
drying (Kugelrohr, 80 ◦C, high vacuum), a sticky solid. This was reprecipitated with MeCN, filtered
and dried again in the Kugelrohr (80 ◦C, high vacuum). The title compound was thus obtained as a
1
colorless powder (434 mg, 25%). H NMR (300 MHz, DMSO-d6)
δ 7.76–7.69 (m, 1H), 7.44–7.31 (m, 4H),
7.20–6.92 (m, 4H), 6.15 (s, 2H), 3.75 (t, J = 6.5, 2H), 2.82 (p, J = 6.2, 4H). 11B NMR (96 MHz, DMSO-d6)
δ
3.74. 13C NMR (75 MHz, DMSO-d6)
δ 139.99, 133.49, 131.36, 130.71, 129.30, 126.74, 125.12, 95.30,
62.44, 41.38. MS (ESI+) m/z 352.04 [M+H]+, 374.02 [M+Na]+, 413.09 [M+C2H8NO]+, 273.99 [M-C6H5]+,
148.09 [M-C6H4I]+. HRMS (ESI+) m/z calc. For C14H16ONBI [M+H]+: 352.0364; found 352.0355.
3.1.13. 4-(2-Phenyl-1,3,2λ4-oxazaborolidin-2-yl)benzonitrile (p-CN-2APB)
Following general procedure A, the title compound was synthesized from 4-bromobenzonitrile (900
mg, 4.95 mmol) using dry THF as reaction solvent. Work up and purification by flash chromatography
yielded the borinic acid (712 mg) as a beige powder. It was dissolved in MeCN and treated with
2-aminoethanol, which gave the borinate as a sticky solid after solvent removal. This was redissolved
in CH2Cl2, the solvent evaporated and the residual solid was dried under high vacuum. This afforded
1
the title compound as a colorless powder (816 mg, 66%). H NMR (300 MHz, DMSO-d6)
δ
7.66–7.51 (m,
4H), 7.41–7.33 (m, 2H), 7.19–7.08 (m, 2H), 7.09–7.00 (m, 1H), 6.26 (s, 2H), 3.88–3.68 (m, 2H), 2.94–2.76
(m, 2H). 11B NMR (96 MHz, DMSO-d6) δ 3.94. 13C NMR (75 MHz, DMSO-d6) δ 132.19, 131.34, 130.14,
126.81, 125.25, 119.89, 107.53, 62.48, 41.40, 39.52. MS (ESI+) m/z 251.13 [M+H]+, 273.12 [M+Na]+, 312.19
[M+C2H8NO]+, 173.09 [M-C6H5]+. HRMS (ESI+) m/z calc. For C15H16ON2B [M+H]+: 251.1350; found
251.1349.

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3.1.14. 2-(4-Nitrophenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (p-NO2-2APB)
A solution of 4-nitrophenylboronic acid pinacol ester (500 mg, 2.01 mmol) in dry THF (20 mL)
was cooled to
78 ◦C. Phenyllithium solution (1.1 mL, 1.8 M in Bu2O, 1.98 mmol) was added dropwise
to the mixture. During the addition the drop rate was adjusted so that the temperature of the mixture
−
◦
◦
◦
remained below
−
75 C. The mixture was stirred at
−
78 C for 4 h. While still stirring at 78 C,
−
the mixture was quenched by the addition of aq. 1 M HCl (10 mL) over a period of 2 min. More aq.
1 M HCl (40 mL) and CH2Cl2 (100 mL) were added to the mixture. The resulting two phases were
separated. The clear yellow organic phase was dried over Na2SO4 and the solvent was removed under
reduced pressure. The remaining brown oil was purified by flash chromatography (silica gel, gradient
with CH2Cl2 and MeOH/CH2Cl2 1:19, (v/v)) affording a black oil (146 mg). The oil was dissolved in
abs. EtOH (4 mL) and 2-aminoethanol (40 µL, 0.66 mmol) were added to the solution. The resulting
mixture was stirred at r.t. For 30 min. The solvent was removed under reduced pressure and the
residue was dried in vacuo affording a sticky yellow solid with a slight greenish tint. The solid was
suspended in Et2O (20 mL) using an ultrasonic bath. After the solid settled, the supernatant was
removed. This washing step was repeated once. The residue was dried in the Kugelrohr at 80 ◦C
1
under high vacuum affording the title compound as a beige powder (47 mg, 9%). H NMR (300 MHz,
DMSO-d6)
δ
8.06–7.96 (m, 2H), 7.71–7.62 (m, 2H), 7.45–7.34 (m, 2H), 7.20–7.11 (m, 2H), 7.11–7.01 (m,
4.25. 13C NMR
1H), 6.30 (s, 2H), 3.87–3.69 (m, 2H), 2.92–2.79 (m, 1H). 11B NMR (96 MHz, DMSO-d6)
δ
(75 MHz, DMSO-d6)
δ 145.69, 132.35, 131.38, 126.86, 125.34, 121.39, 117.03, 62.53, 41.47. MS (ESI+)
m/z 271.12 [M+H]+, 293.11 [M+Na]+, 311.16 [M+C2H3N]+, 332.18 [M+C2H8NO]+, 193.08 [M-C6H5]+.
HRMS (ESI+) m/z calc. For C14H16O3N2B [M+H]+: 271.1248; found 271.1249.
3.1.15. 2-Phenyl-2-(4-(trimethylsilyl)phenyl)-1,3,2λ4-oxazaborolidine (p-TMS-2APB)
1-Bromo-4-(trimethylsilyl)benzene (400 µL, 2.05 mmol) was reacted according to general procedure
A in dry Et2O as reaction solvent. After work up and flash chromatography purification the borinic
acid was obtained as pale yellow oil (170 mg), which was subjected to 2-aminoethanol treatment
in abs. EtOH. The solid, isolated after solvent removal, was suspended in Et2O (10 mL) using an
ultrasonic bath. After the solid settled, the supernatant was removed. This washing procedure was
repeated two times with Et2O (10 mL) and once with n-hexane (5 mL). After drying the solid residue
1
in high vacuum the title compound was isolated as colorless powder (49 mg, 8%). H NMR (300 MHz,
DMSO-d6)
δ
7.44–7.35 (m, 4H), 7.33–7.24 (m, 2H), 7.17–7.07 (m, 2H), 7.06–6.98 (m, 1H), 6.06 (s, 2H),
4.42. 13C NMR
3.75 (t, J = 6.5, 2H), 2.81 (p, J = 6.4, 2H), 0.18 (s, 9H). 11B NMR (96 MHz, DMSO-d6)
δ
(75 MHz, DMSO-d6) δ 134.96, 131.55, 131.44, 131.01, 126.58, 124.87, 62.41, 41.37, -0.92. MS (ESI+) m/z
298.18 [M+H]+, 320.16 [M+Na]+, 359.23 [M+C2H8NO]+, 220.13 [M-C6H5]+. HRMS (ESI+) m/z calc.
For C17H25ONBSi [M+H]+: 298.1793; found 298.1793.
3.1.16. 2-(4-Methoxyphenyl)-2-phenyl-1,3,2λ4-oxazaborolidine (p-OMe-2APB)
Starting from 4-bromoanisole (669 µL, 5.35 mmol) the title compound was synthesized according
to general procedure A. The borinic acid (763 mg), obtained as pale yellow oil after work up and flash
chromatography purification, was treated with 2-aminoethanol in MeCN at r.t. Removal of solvent
gave a residual sticky solid that was redissolved in CH2Cl2. The solvent was evaporated under reduced
1
pressure yielding the borinate as a colorless powder (825 mg, 60%). H NMR (300 MHz, DMSO-d6)
δ
7.41–7.33 (m, 2H), 7.31–7.24 (m, 2H), 7.17–7.08 (m, 2H), 7.06–6.98 (m, 1H), 6.74–6.68 (m, 2H), 5.96
(s, 2H), 3.75 (t, J = 6.3, 2H), 2.81 (p, J = 6.3, 2H). 11B NMR (96 MHz, DMSO-d6) 4.40. 13C NMR (75
δ
MHz, DMSO-d6) δ 157.34, 132.63, 131.56, 126.58, 124.83, 112.34, 62.42, 54.65, 41.33, 39.52. MS (ESI+)
m/z 256.15 [M+H]+, 278.13 [M+Na]+, 178.10 [M-C6H5]+, 148.09 [M-C7H5O]+. HRMS (ESI+) m/z calc.
For C15H19O2NB [M+H]+: 256.1503; found 256.1500.

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3.1.17. N,N-Dimethyl-4-(2-phenyl-1,3,2λ4-oxazaborolidin-2-yl)benzenesulfonamide
(p-SO2NMe2-2APB)
4-Bromo-N,N-diemthylbenzenesulfonamide (1 g, 3.79 mmol), which was freshly purified by flash
chromatography (silica gel, cyclohexane/EtOAc gradient), was used as starting material. Dry THF was
used as the reaction solvent and n-BuLi solution (2.5 M in hexanes) for the lithiation to synthesize the
corresponding borinic acid according to general procedure A. The borinic acid was obtained as brown
oil (930 mg) after work up and flash chromatography purification (silica gel, gradient with CH2Cl2
and MeOH/CH2Cl2 1:19 (v/v)). The oil was dissolved in abs. EtOH and 2-aminoethanol added to the
solution. Removal of solvent yielded a sticky solid that was dried (Kugelrohr, 80 ◦C, high vacuum),
affording an off-white solid. Recrystallization from MeCN gave the title compound (630 mg, 52%) as
1
off-white crystals, suitable for X-ray diffraction analysis (Figure S3). H NMR (300 MHz, DMSO-d6)
δ
7.68–7.63 (m, 2H), 7.53–7.49 (m, 2H), 7.43–7.37 (m, 2H), 7.20–7.12 (m, 2H), 7.09–7.02 (m, 1H), 6.24 (s,
2H), 3.78 (t, J = 6.5, 2H), 2.84 (p, J = 6.1, 2H), 2.55 (s, 6H). 11B NMR (96 MHz, DMSO-d6) 4.17. 13C
δ
NMR (75 MHz, DMSO-d6)
δ 131.96, 131.36, 126.82, 125.76, 125.21, 117.00, 62.51, 41.44, 37.59. MS (ESI+)
m/z 333.14 [M+H]+, 355.13 [M+Na]+, 394.20 [M+C2H8NO]+, 255.10 [M-C6H5]+; HRMS (ESI+) m/z calc.
For C16H22O3N2BS [M+H]+: 333.1439; found 333.1433.
3.1.18. 6,6-Diphenyl-7-oxa-5-aza-6λ4-boraspiro[3.4]octan-8-one (cBu-2APB)
Following general procedure B (reflux for 55 min), 1-amino-1-cyclobutanecarboxylic acid (80 mg,
0.70 mmol) was used as starting material. The crude precipitate was washed with Et2O and n-hexane,
reprecipitated in a mixture of EtOH and n-hexane and washed several times with EtOH and n-hexane.
Drying the solid product in high vacuum afforded the title compound (32 mg, 16%), containing crystals
1
suitable for X-ray structure determination (Figure S2). H NMR (300 MHz, DMSO-d6) δ 7.43–7.29 (m,
6H), 7.24–7.14 (m, 4H), 7.14–7.06 (m, 2H), 2.20–2.05 (m, 4H), 1.99–1.75 (m, 2H). 11B NMR (96 MHz,
DMSO-d6)
δ δ 176.39, 130.69, 126.97, 125.79, 59.16, 30.71, 14.72.
4.27. 13C NMR (75 MHz, DMSO-d6)
MS (ESI+) m/z 280.15 [M+H]+, 302.13 [M+Na]+. HRMS (ESI+) m/z calc. For C17H19O2NB [M+H]+:
280.1503; found 280.1502.
3.1.19. 2,2-Diphenyl-1,3,2λ4-oxazaborinan-6-one (βAla-2APB)
β-Alanine (60 mg, 0.67 mmol) was reacted as described in general procedure B (reflux for 1.5 h).
The product crystallized from the reaction solution upon cooling. The colorless crystals were washed
with n-hexane and dried in high vacuum. This gave the title compound as colorless crystals (88 mg,
1
52%). H NMR (300 MHz, DMSO-d6)
δ
7.41 (dd, J = 8.0, 1.3, 4H), 7.25–7.16 (m, 4H), 7.14–7.05 (m, 2H),
6.76 (s, 2H), 2.90–2.80 (m, 2H), 2.54–2.41 (m, 2H). 11B NMR (96 MHz, DMSO-d6)
δ
2.05. 13C NMR (75
MHz, DMSO-d6)
δ
169.23, 130.78, 127.02, 125.61, 35.48, 30.01. MS (ESI+) m/z 254.13 [M+H]+, 276.12
[M+Na]+, 176.09 [M-C6H5]+. HRMS (ESI+) m/z calc. For C15H17O2NB [M+H]+: 254.1347; found
254.1345.
3.1.20. (S)-3-(5-Oxo-2,2-diphenyl-1,3,2λ4-oxazaborolidin-4-yl)propanamide (Gln-2APB)
L-Glutamine (95 mg, 0.65 mmol) was treated as described in general procedure B (reflux for 3.75
h) and the product precipitated from the reaction mixture upon cooling. The solid was washed with
EtOH, Et2O, and n-hexane and dried in high vacuum. The residue was recrystallized from EtOH
affording the title compound as colorless crystals (96 mg, 48%). 1H NMR (300 MHz, DMSO-d6)
δ
7.43–7.28 (m, 6H), 7.21 (td, J = 7.1, 1.6, 4H), 7.18–7.07 (m, 2H), 7.03–6.90 (m, 1H), 6.86 (s, 1H), 3.56 (p, J =
7.5, 1H), 2.30 (td, J = 7.2, 2.7, 2H), 1.89 (ddt, J = 39.3, 14.7, 7.4, 2H). 11B NMR (96 MHz, DMSO-d6)
δ 174.07, 173.86, 131.00, 130.93, 127.08, 127.03, 126.01, 125.91, 54.44,
30.88, 25.31. MS (ESI+) m/z 311.16 [M+H]+, 333.14 [M+Na]+. HRMS (ESI+) m/z calc. For C17H20O3N2B
[M+H]+: 311.1567; found 311.1561.
δ
4.39. 13C NMR (75 MHz, DMSO-d6)

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3.1.21. (S)-4-((1H-Imidazol-5-yl)methyl)-2,2-diphenyl-1,3,2λ4-oxazaborolidin-5-one (His-2APB)
L-Histidine (100 mg, 0.65 mmol) was used as starting material and general procedure B followed
to synthesize the title compound (reflux for 1 h). The crude product precipitated from the reaction
mixture upon cooling. It was washed with Et2O and n-hexane and dried in high vacuum. This gave
1
the title compound as colorless solid (92 mg, 45%). H NMR (300 MHz, DMSO-d6)
δ 12.04 (s, 1H),
7.62–7.54 (m, 1H), 7.48–7.07 (m, 12H), 6.91 (s, 1H), 3.84 (t, J = 7.2, 1H), 2.95 (d, J = 6.0, 2H). 11B NMR (96
MHz, DMSO-d6)
δ δ 173.32, 135.19, 131.18, 131.07, 127.04, 127.01,
4.40. 13C NMR (75 MHz, DMSO-d6)
126.01, 55.18. MS (ESI+) m/z 320.16 [M+H]+, 342.14 [M+Na]+, 242.11 [M-C6H5]+. HRMS (ESI+) m/z
calc. For C18H19O2N3B [M+H]+: 320.1565; found 320.1562.
3.1.22. (S)-4-(2-(Methylthio)ethyl)-2,2-diphenyl-1,3,2λ4-oxazaborolidin-5-one (Met-2APB)
Following general procedure B, L-methionine (100 mg, 0.67 mmol) was used as the starting
material. After refluxing for 45 min the clear solution was cooled to r.t. and then stored at
20 ◦C
−
to initiate precipitation of the crude product. The obtained crystals were washed with Et2O and
n-hexane, and dried in high vacuum. This afforded the desired product as colorless crystals (212 mg,
1
quantitative). H NMR (300 MHz, DMSO-d6)
δ
7.45–7.09 (m, 12H), 6.78 (t, J = 10.4, 1H), 3.69–3.53 (m,
1H), 2.72–2.52 (m, 2H), 2.14–1.78 (m, 4H). 11B NMR (96 MHz, DMSO-d6)
δ
4.92. 13C NMR (75 MHz,
DMSO-d6) δ 174.04, 130.99, 130.92, 127.14, 127.08, 126.08, 125.98, 53.60, 29.51, 28.95, 14.15. MS (ESI+)
m/z 314.14 [M+H]+, 336.12 [M+Na]+, 236.09 [M-C6H5]+. HRMS (ESI+) m/z calc. For C17H21O2NBS
[M+H]+: 314.1381; found 314.1380.
3.1.23. (R)-4-(Mercaptomethyl)-2,2-diphenyl-1,3,2λ4-oxazaborolidin-5-one (Cys-2APB)
L-Cysteine (75 mg, 0.62 mmol) was reacted according to general procedure B (reflux for 1 h).
The crude product, which precipitated from the reaction mixture upon cooling, was washed with
n-hexane. This gave the title compound as colorless solid (125 mg, 71%) after drying in high vacuum.
1H NMR (300 MHz, DMSO-d6)
δ 7.58–7.36 (m, 5H), 7.29–7.09 (m, 6H), 6.63 (t, J = 10.8, 1H), 3.79–3.63
(m, 1H), 3.04–2.65 (m, 3H). 11B NMR (96 MHz, DMSO-d6)
δ δ
4.58. 13C NMR (75 MHz, DMSO-d6)
172.29, 131.14, 131.08, 127.08, 127.06, 126.12, 126.07, 57.81, 23.83. MS (ESI+) m/z 286.11 [M+H]+, 308.09
[M+Na]+. HRMS (ESI+) m/z calc. For C15H17O2NBS [M+H]+: 286.1068; found 286.1066.
3.1.24. (S)-4-Benzyl-2,2-diphenyl-1,3,2λ4-oxazaborolidin-5-one (Phe-2APB)
The title compound was synthesized from L-phenylalanine (100 mg, 0.60 mmol) according to
general procedure B (reflux for 30 min). The precipitated crude product was washed with n-hexane
and dried in high vacuum. This yielded the final compound as colorless solid (128 mg, 64%). 1H
NMR (300 MHz, DMSO-d6)
δ
7.47–7.01 (m, 16H), 6.76 (t, J = 10.5, 1H), 3.77–3.61 (m, 1H), 3.17 (dd,
4.61. 13C NMR (75
J = 14.5, 4.0, 1H), 2.92 (dd, J = 14.5, 10.3, 1H). 11B NMR (96 MHz, DMSO-d6)
δ
MHz, DMSO-d6) δ 173.48, 137.26, 131.15, 130.81, 129.27, 128.47, 127.02, 126.95, 126.65, 125.95, 125.86,
56.68, 35.04. MS (ESI+) m/z 330.17 [M+H]+, 352.15 [M+Na]+. HRMS (ESI+) m/z calc. For C21H21O2NB
[M+H]+: 330.1660; found 330.1658.
3.1.25. (S)-4-(4-Hydroxybenzyl)-2,2-diphenyl-1,3,2λ4-oxazaborolidin-5-one (Tyr-2APB)
Following general procedure B, L-tyrosine (120 mg, 0.68 mmol) was reacted (reflux for 40 min)
and the reaction mixture cooled to r.t. and then
20 ◦C. The formed precipitate was washed with Et2O
−
and n-hexane. The isolated solid was suspended in EtOH, layered with n-hexane, and the mixture
stored at 5 ◦C. The obtained precipitate was filtered, washed with n-hexane, and dried in high vacuum.
1
This gave the title compound as shimmery beige solid (39 mg, 17%). H NMR (300 MHz, DMSO-d6)
δ
9.26 (s, 1H), 7.39 (td, J = 7.9, 1.6, 4H), 7.30–6.99 (m, 9H), 6.74–6.55 (m, 3H), 3.64–3.53 (m, 1H), 3.04
(dd, J = 14.5, 4.0, 1H), 2.79 (dd, J = 14.5, 10.3, 1H). 11B NMR (96 MHz, DMSO-d6) 4.73. 13C NMR (75
MHz, DMSO-d6) 173.56, 156.18, 131.15, 130.81, 130.22, 127.10, 127.00, 126.94, 125.93, 125.85, 115.26,
δ
δ

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57.04, 34.18. MS (ESI+) m/z 346.16 [M+H]+, 368.14 [M+Na]+. HRMS (ESI+) m/z calc. For C21H21O3NB
[M+H]+: 346.1609; found 346.1605.
3.1.26. (S)-4-((5-Hydroxy-1H-indol-3-yl)methyl)-2,2-diphenyl-1,3,2λ4-oxazaborolidin-5-one
(5-HO-Trp-2APB)
5-Hydroxytryptophan (191 mg, 0.87 mmol) was used as the starting material to synthesize the title
compound according to general procedure B (reflux until all the solids were dissolved). Precipitation
◦
of the crude product was initiated by first cooling to r.t. and then to
−
20 C. The precipitate was
washed with Et2O and n-hexane and the obtained solid recrystallized from EtOH. This gave the
desired compound as a colorless solid (220 mg, 66%) after drying in high vacuum. H NMR (300 MHz,
1
DMSO-d6)
(m, 7H), 7.03 (d, J = 2.3, 1H), 6.75 (d, J = 2.2, 1H), 6.59 (dd, J = 8.6, 2.2, 1H), 6.52–6.35 (m, 1H), 3.73–3.58
(m, 1H), 3.14 (dd, J = 15.1, 3.5, 1H), 2.93 (dd, J = 15.2, 10.1, 1H). 11B NMR (96 MHz, DMSO-d6)
8.58.
δ 10.67–10.58 (m, 1H), 8.60 (s, 1H), 7.42–7.34 (m, 4H), 7.29 (dd, J = 12.0, 6.2, 1H), 7.23–7.04
δ
13C NMR (75 MHz, DMSO-d6)
δ 174.01, 150.52, 131.19, 130.93, 127.47, 127.12, 127.05, 126.05, 125.99,
124.97, 111.99, 111.58, 107.80, 101.86, 55.71, 25.58. MS (ESI+) m/z 385.1726 [M+H]+, 407.1543 [M+Na]+,
307.1254 [M-C6H5]+. HRMS (ESI+) m/z calc. For C23H22O3N2B [M+H]+: 385.1718; found 385.1726.
3.1.27. (S)-4-(4-Aminobutyl)-2,2-diphenyl-1,3,2λ4-oxazaborolidin-5-one (Lys-2APB)
Following general procedure B, L-lysine (90 mg, 0.62 mmol) was reacted (reflux for 50 min) and
the solvents evaporated under reduced pressure. The residue was layered with a mixture of Et2O
and n-hexane, and the precipitation allowed to settle. The supernatant was removed and the solid
washed with Et2O and n-hexane. After drying the solid in high vacuum, it was recrystallized from
1
a mixture of i-PrOH and EtOH, affording the title compound as colorless crystals (96 mg, 50%). H
NMR (300 MHz, DMSO-d6)
δ
7.43–7.36 (m, 5H), 7.25–7.17 (m, 5H), 7.16–7.09 (m, 2H), 3.41 (dd, J =
9.4, 4.6, 1H), 2.48–2.44 (m, 2H), 1.80–1.21 (m, 8H). 11B NMR (96 MHz, DMSO-d6)
δ
4.42. 13C NMR
(75 MHz, DMSO-d6)
δ 174.27, 131.00, 130.92, 127.05, 126.99, 125.95, 125.86, 55.17, 41.21, 32.74, 29.37,
22.97. MS (ESI+) m/z 311.19 [M+H]+, 333.17 [M+Na]+, 233.15 [M-C6H5]+. HRMS (ESI+) m/z calc.
For C18H24O2N2B [M+H]+: 311.1925; found 311.1925.
3.2. Cell Culture
Cell culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
MDA-MB-231 cells (ATCC) were cultured at 37 ◦C in RPMI cell culture medium supplemented with
10% FBS, 10 mM HEPES, and 1% penicillin/streptomycin. HEK 293 cells stably expressing STIM1
were grown at 37 ◦C and 5% CO2 in MEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA,
USA) supplemented with 10% FCS and 500 µg/mL G418 (Gibco). Cells were transfected according
to manufacturer’s guidelines in a Nucleofector 4D device with Nucleofector Kit SF (Lonza, Basel,
Switzerland) with 1 µg DNA per 106 cells.
3.3. Fluorescent Ca2+ Influx Assay Using FLIPR® Tetra
3.3.1. General
The calcium indicator, Calcium-5 was purchased from Molecular Devices LLC and all other
chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). For loading the cells and subsequent
administrations during the FLIPR experiments a nominal calcium free (NCF) modified Krebs buffer
was used, containing 117 mM NaCl, 4.8 mM KCl, 1 mM MgCl2, 5 mM D-glucose, and 10 mM HEPES.
The pH of the NCF buffer was adjusted to 7.4 using HCl and NaOH. Thapsigargin (Tg) solutions
were always prepared freshly a few minutes before starting the FLIPR experiment. Cells were excited
using a 470–495 nm LED module, and the emitted fluorescence signal was filtered with a 515–575 nm
emission filter (manufacturer’s guidelines). Fluorescence signals were analyzed using the ScreenWorks
3.1.1.8 software (Molecular Devices).

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3.3.2. SOCE Modulation Assay
The attached MDA-MB-231 cells from T75 flasks were trypsinized and plated onto Corning®
96-well black polystyrene clear bottom microplates (CLS3603 Sigma–Aldrich, St. Louis, MO, USA)
at a density 60,000 cells/well in a final volume of 100
supplemented with 10% FBS and 10 mM HEPES was used for seeding the cells. After culturing the
cells for 24 h, the medium of the confluent cells was removed and the cells were loaded with 50
µL per well. Phenol-red free RPMI medium
µL
NCF Krebs buffer containing calcium-5 dye (50 µL per mL). The cells then were incubated with the dye
in the dark for 1 h at 37 ◦C. Right after the dye-incubation period, the FLIPR experiment was started.
After establishment of stable baselines for 50 s, first ER Ca2+ stores were depleted by adding and
incubating 50
Next, 100 L of 2
or other SOCE inhibitor) in NCF Krebs buffer was applied and incubated for 10 min. Finally, 50
of NCF Krebs buffer containing 10 mM CaCl2 (2 mM final concentration) was administered while
maintaining the 1 concentration of the drug of interest. The subsequent response was monitored for
µL of 2
µM thapsigargin (Tg) in NCF Krebs buffer (1 µM final concentration) for 10 min.
µ
×
of the intended final concentration of the drug of interest (synthetic 2-APB analogue
µL
×
5 min after the addition of CaCl2. The modulation of the SOCE signal was quantified by calculating
the area under the curve (AUC) of the relative change of the fluorescence intensity over time (trace)
from the last 5 min of the experiment. The values were then normalized to the DMSO positive control
(DMSO response = 100%).
3.3.3. ER Ca2+ Mobilization Assay (Compound Followed by Thapsigargin)
The medium of the confluent cells was replaced by 50
calcium-5 dye (50
L per mL). The cells were incubated in the dark for 1 h at 37 ◦C. After the dye was
incubated, the cells were washed twice: first with 250 L of 500 M EGTA in NCF Krebs buffer and
afterwards with 250 L NCF Krebs buffer. Finally, the cells were loaded with 50 L NCF Krebs buffer
and subjected to the FLIPR experiment. Total of 50 L of 2 of the intended concentration of drug of
interest in NCF Krebs buffer was applied and incubated for 10 min while recording the Ca2+ response.
Afterwards 25 L of 12.5 M Tg (2.5 M final concentration) was applied and the subsequent response
µL buffer containing 2 mM CaCl2 as well as
µ
µ
µ
µ
µ
µ
×
µ
µ
µ
was monitored for 10 min. The response after each addition step was quantified by calculating the
slope of the curve and normalizing it to the slope of the response after Tg administration.
3.4. Electrophysiology
Currents from whole cell patch clamp recordings were acquired and filtered at 2.9 kHz with a
HEKA EPC-10 amplifier (HEKA Elektronik, Lambrecht (Pfalz), Germany). Currents were corrected
for a liquid junction potential of 10 mV, recorded and analyzed with HEKA Patchmaster (v2 53)
×
software and Igor Pro 6.37 (Wavemetrics, Lake Oswego, OR, USA). 50 ms voltage ramps spanning,
150 mV to 150 mV were delivered every 2 s from a holding potential of 0. Before each voltage ramp,
capacitive currents were corrected. Currents at 130 mV and 130 mV were extracted, normalized to
−
cell capacitance, and plotted versus time. Bath solution contained (in mM): 120 NaCl, 20 CaCl2, 10
TEA-Cl, 10 HEPES, 2 MgCl2. Pipette solution contained (in mM): 120 Cs-glutamate, 20 BAPTA, 10
HEPES, 3 MgCl2, and 0.050 IP3. Osmolarity was adjusted with glucose to 310 mOsm and pH was
adjusted to 7.2 with CsOH.
3.5. CRISPR/Cas9-Mediated Generation and Validation of STIM1 and Orai1 Knockout MDA-MB-231 Cells
Earlier described methods were used to generate STIM1 knockout [86] and Orai1 knockout
MDA-MB-231 cells [87]. Single-cell derived clones D11 and G10 were functionally confirmed to have
undergone knockout of STIM1 and Orai1, respectively by using SOCE functional assay on FLIPR
Tetra® (Molecular Devices). The knockouts were further confirmed by Western blotting. STIM1
was detected using the polyclonal hSTIM1 antibody raised in guinea pig [88] in a 1:1000 dilution.
Peroxidase-conjugated affinipure goat anti-guinea pig secondary antibody was used in a 1:10,000

Int. J. Mol. Sci. 2020, 21, 5604
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dilution (106-035-003, Jackson Immuno Research, West Grove, PA, USA). Orai1 was detected using
rabbit polyclonal hOrai1 antibody (O8264, Sigma-Aldrich) in a 1:1000 dilution. Horseradish peroxidase
(HRP)-conjugated secondary goat anti-rabbit IgG (W4011, Promega, Madison, WI, USA) was used in a
1:20,000 dilution. Tubulin (loading control) was detected using mouse monoclonal Tubulin antibody
(T9028, Sigma-Aldrich) in a 1:2000 dilution. HRP-conjugated goat anti-mouse secondary antibody was
used in a 1:3000 dilution (172-1011, Bio-Rad, Hercules, CA, USA).
3.6. Cell Viability Assay
The XTT assay was used to evaluate the effect of 2-APB analogues on cell proliferation. Cells
◦
were plated at a density of 5
×
103 cells/well in 96-well plates and were incubated at 37 C and
5% CO2 for 24 h. On the following day, the medium was carefully aspirated and replaced with
100 L medium supplemented with DMSO control or different 2-APB compounds and incubated
µ
over a range of different time points from 24 to 120 h. At each time point, 25
µL of sodium
30-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate
(XTT 1mg/mL prepared in Hank’s Balanced Salt Solution) containing 1.25% phenazine methosulphonate
◦
(PMS) was added to each well and incubated at 37 C for 2 h. Subsequently, the absorbance was
read at 650 nm and subtracted from the absorbance of 450 nm by spectrophotometry (Vmax Kinetic
Microplate Reader, Molecular Devices LLC). The resulting subtracted absorbance for each compound
was expressed as a percentage of viable cells relative to the DMSO control. XTT sodium salt and PMS
were purchased from Sigma-Aldrich. The MCF-10A cells were kindly provided by Prof. Dr. Olivier
Pertz, Institute of Cell Biology, University of Bern and were cultured in growth medium composed by
DMEM/F12 supplemented with 5% horse serum, 20 ng/mL recombinant human EGF (Sigma-Aldrich),
10 µg/mL insulin (Sigma-Aldrich), and 0.5 mg/mL hydrocortisone (Sigma-Aldrich).
3.7. Data Analysis
Statistical significance between the two groups was estimated using student’s T-test where both
groups were normally distributed and Mann Whitney test in case of data that were not distributed
normally. GraphPad Prism 8.4.3 was used to perform the statistical analysis as well as to generate the
bar graphs with scattered individual data points. Data are usually shown as mean ± SD.
4. Conclusions
In an effort to develop new potent inhibitors of store-operated calcium entry we have synthesized
several mono-substituted and amino acid analogues of 2-APB. We tested these 2-APB derivatives using
a FLIPR assay format in MDA-MB-231 cells. This breast cancer cell line already has been established
by others [26
mono-halogenated 2-APB derivatives, in particular, showed promising inhibitory activity compared to
2-APB. Some members of this series, e.g., m-Cl-2APB m-Br-2APB m-I-2APB, and p-I-2APB, exhibited
,72] as a viable tool for screening new SOCE inhibitors. The compound series of
,
,
efficient SOCE block at a five times lower concentration than 2-APB. It is worth noting that at this low
concentration 2-APB generally enhances SOCE. Throughout the course of our investigation we have also
demonstrated that some of our novel analogues (m-I-2APB, p-Cl-2APB, p-Br-2APB, p-I-2APB), but
also known 2-APB-type SOCE inhibitors (DPB162-AE), are able to mobilize calcium from intracellular
stores of MDA-MB-231 cells at high concentration. In this regard, m-Cl-2APB is a promising tool
compound from our synthetic series as it efficiently blocked SOCE at a low concentration (10
µM)
but did not completely deplete internal stores at higher concentrations (50 M). While structurally
µ
very close isomers like m-Br-2APB and p-Br-2APB did not show different behaviour in blocking ICRAC
they had a strikingly different effect on MDA-MB-231 cells viability.
,
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/16/5604/s1.
Supplementary data (adapted synthesis of DPB162-AE (Scheme S1), GSK-7975A (Scheme S2), and Synta66
(Scheme S3), crystal structures of GSK-7975A (Figure S1), cBu-2APB (Figure S2), p-SO2NMe2-2APB (Figure S3),
o-F-2APB (Figure S4), and o-Cl-2APB (Figure S5), SOCE blockade by GSK-7975A and Synta66 with pre-incubation

Int. J. Mol. Sci. 2020, 21, 5604
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(Figure S6), SOCE in STIM1 KO and Orai1 KO cells (Figure S7) and copies of 1H, 11B, 13C, and 19F NMR spectra of
all final compounds).
Author Contributions: Conceptualization, M.L.; methodology, A.S. and R.B. (Rajesh Bhardwaj); validation, A.S.,
R.B. (Rajesh Bhardwaj) and R.B. (Roland Baur); formal analysis, A.S., R.B. (Rajesh Bhardwaj), and R.B. (Roland
Baur); investigation, A.S., R.B. (Rajesh Bhardwaj), N.W., D.T., P.K., J.D., and R.B. (Roland Baur); data curation,
A.S. and R.B. (Rajesh Bhardwaj); writing—original draft preparation, A.S., R.B. (Rajesh Bhardwaj), C.P., and M.L.;
writing—review and editing, A.S., R.B. (Rajesh Bhardwaj), C.P., and M.L.; visualization, A.S. and R.B. (Rajesh
Bhardwaj); supervision, C.P., M.A.H., and M.L.; project administration, M.A.H. and M.L.; funding acquisition,
M.A.H. and M.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Swiss National Science Foundation (Sinergia grants CRSII3_160782
and CRSII5_180326 to M.A.H. and M.L.).
Acknowledgments: We thank the analytical services from the Department of Chemistry and Biochemistry,
University of Bern, Switzerland, for measuring the spectra of synthetic intermediates and final compounds: the
group of Chemical Crystallography (Piero Macchi) for the X-ray structure solutions and the Swiss National Science
Foundation (R’equip grant 206021_128724) for co-funding the single crystal X-ray diffractometer, the group of
Julien Furrer for measuring NMR spectra, and the group of Stefan Schürch for MS and HRMS spectra.
Conflicts of Interest: The authors declare no conflict of interest. The funder had no role in the design of the study,
in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish
the results.
Abbreviations
2-APB
AUC
CD
CRAC
ER
2-Aminoethyl diphenylborinate
Area under the curve
Current density
Ca2+ release-activated Ca2+ current
Endoplasmic reticulum
FLIPR
IP3
NCF
PI(4,5)P2
PM
Fluorometric imaging plate reader
Inositol 1,4,5-triphosphate
Nominal calcium-free (buffer)
Phosphatidylinositol 4,5-bisphosphate
Plasma membrane
SERCA
SOCE
STIM
Tg
Sarco/endoplasmic reticulum Ca2+-ATPase
Store-operated calcium entry
Stromal interaction molecule
Thapsigargin
Appendix A
When we used the general lithiation/borination protocol with 1,2-dibromobenzene (Scheme A1) we observed
significant attack of the aryl lithium species at the halogenated carbon of the borinic intermediate. This led to the
shown biphenyl side product which we were unable to separate from the desired borinic acid by chromatography
or crystallization. Changing the solvent to toluene/THF 4:1 to carry out the halogen lithium exchange and
subsequent reaction with phenylboronic acid pinacol ester suppressed the formation of the biphenyl side product
and enabled us to isolate pure (2-bromophenyl)(phenyl) borinic acid. The solvent change strategy did not work
with 1,2-diiodobenzene, however, and gave inseparable mixtures of desired (2-iodophenyl)(phenyl)borinic acid
and biphenyl side product.

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Scheme A1. Lithiation of 1,2-dibromo- or 1,2-diiodobenzene, and subsequent borination of the
organolithium species led to significant formation of biphenyl side products that were inseparable from
the desired (aryl)(phenyl)borinic acids.
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