2-Aminoethyl diphenylborinate (2-APB) analogues: part 2.regulators of Ca2+ release and consequent cellular processes

In order to obtain compounds with modified 2-APB activities, we synthesized number of bis-boron 2-APB analogues and analyzed their inhibitory activities for SOCE and IICR. Adducts of amino acids with bis-borinic acid showed the highest activity. The IC50 of 2-APB for SOCE inhibition was 3 μM, while the IC50 of 2051 bis(4,4’(phenyllysineboryl)benzyl) ether was 0.2 μM. By using these compounds, we may be able to regulate Ca2+ release and consequent cellular processes more efficiently than with 2-APB.

In 1997, we identified 2-aminoethyl diphenylborinate (2-APB) as being an IP 3 receptor inhibitor which regulates IP 3 -induced calcium release [21][22]. This discovery led to substantial interest as it led to more than 600 citations and more than 1000 studies on 2-APB have been published so far (examples are references 23-37). This was supported by increasing sales of 2-APB by Sigma-Aldrich as membrane-permeable modulator of intracellular IP 3 -induced cellular calcium release. In this study, we aimed to generate better modulator of calcium release than 2-APB.
Here we analyzed SOCE and IICR inhibitory activities of our bis-boron compounds collection. The analysis of poly-boron compounds will be reported in our upcoming publications.
We believe that by regulating Ca 2+ release and associated cellular processes by boron compounds, we could therapeutically intervene in many diseases, such as heart diseases and Alzheimer`s disease.

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← Click for updates doi: 10.7243/2055-0898-1-1 added to the mixture of diisopropoxyphenylborane, gradually warmed to room temperature, and then stirred overnight. The reaction was quenched with 1N 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 MgSO 4 and concentrated. The crude residue was purified by flash column chromatography on a silica gel (n-hexane/ EtOAc = 3:1) to give bis-(4,4'-(hydroxyphenylboryl) phenyl) ether 1012 (1122 mg, 3.15 mmol, 58.8%) as an oil.

Methods
We have assayed the inhibitory activity of the 2-APB analogues for SOCE and IICR using our improved assays described previously [45].

Results and discussion
We measured inhibitory activities of the 2-APB analogues for SOCE and IICR. The results are shown in (Supplement Table S1).
We can verify the efficiency of these sample by comparing with IC 50 of 2-APB for SOCE inhibition 3 µM, and IC 50 of 919 :best sample of our previous paper (45)

Comparison of 2APB, mono-boron compounds and bis-boron compounds
The IC 50 of 2-APB for SOCE inhibition is 3 µM. The IC 50 of best mono-boron compound (example 919) at previous paper (45) is 0.2 µM. The IC 50 of best bis-boron compound reporting at this paper (example 1024) is 0.2µM. That is, the bis-boron compound reporting at this paper and mono-boron compound reported at previous paper (45) showed almost same activity and about 10 times stronger activity than 2APB. Mono-boron compounds are easy to prepare. But bis-boron compounds are somewhat difficult to prepare.

Comparison of bis-phenyl ether and bis-benzyl ether
When we compare compounds mentioned at 3.1 and at 3.2 and when we compare 1022, 4020, 162AE, we can tell that there is not so much difference between bis-phenyl ether type compound (1022) and bis-benzyl ether type compounds (4020,162AE).

Comparison of amino acids and ethanol amine
As a reagent to add on to the dihydroxy boron compound, we used amino acid 3.1 3.2)and ethanol amine (3.3). Activities of both compounds were quite similar, but the stabilities of the compound obtained are different. Amino acid adducts are much more stable and easy to purify. We recommend amino acids derivatives over ethanol amine derivatives as regulators of Ca 2+ and cellular process. Also among amino acid, basic amino acid having extra amino group or amide group like lysine, ornithine, asparagine, glutamine gave compounds with strong activity.
We have synthesized many bis-boron compounds. These compounds showed as active as mono-boron compounds on molar basis. But when we consider on a weight basis, bis-boron compounds are half as active as mono-boron compounds, because the molecular weight of bis boron compound is about twice that of mono-boron compound.
These compounds can thus regulates the Ca 2+ release and consequent cellular response more efficiently than 2-APB at pharmacological concentrations. Some of these compounds were shown to inhibit the calcium dependent enzyme transglutaminase [44]. Transglutaminase inhibitors block the abnormal cross-link of protein [43,44] and therefore they might slow down or even stop the progression of diseases caused by misfolded proteins, such as Huntington`s disease.
The 2-APB analogues presented in this study could be proven to be excellent lead compounds for many human diseases including heart disorders [59], Alzheimer`s [60,61] and Huntington`s disease [62,63].
We have shown different kinds of active compounds with IC 50 ranging 0.1 to 5 µM. By choosing the compound we would be able to control the release of Ca 2+ and regulate many cellular processes such as secretion, cardiac contraction, fertilization, proliferation, synaptic plasticity, atrial arryhythmiss [31], inhibition of calcium entry channel [25], excitation-contraction coupling in the heart [32], arrhythmogenic action of endothelin-1 on ventricular cardiac myocytes [34], dysreguration of neural calcium signaling in Alzheimer disease [61], Huntington aggregation [62,63] and protein cross-link by transglutaminase [43].
We believe that many investigators will find these reagents regulating Ca 2+ release and related cellular processes very useful.

Competing interests
The author declares that he has no competing interests.