Borylated methyl cinnamates: Expedited synthesis, characterization, crystallographic analysis and biological activities in glycosidase inhibition and in cancer cells lines

Three cinnamate derivatives bearing a boronate pinacol ester group para, meta and ortho to the ,-unsaturated ester group have been synthesized via a solventless, expedited Wittig protocol in the best stereoisomeric ratios yet reported, purified by recrystallization, characterized and analyzed by X-ray crystallography. These are valuable building blocks to biologically active derivatives and are themselves biologically active drug leads, displaying excellent selectivities as glycosidase modulators and for future testing as boron neutron capture therapy (BNCT) 2 agents. In a panel of 15 glycosidases, IC50 values of 351 M and 374 M were shown for para 2 against almond -glucosidase and bovine liver -galactosidase, respectively. For meta 2 the selectivity profile is improved with only inhibition of bovine liver -galactosidase with an IC50 of 780 M. These borylated derivatives also possess the capability to be used as BNCT agents. This occurs via irradiation with slow neutrons, thus granting them a switch-on/switch-off toxicity. This is an important new capability imbued into anticancer drugs, too many of which are too toxic in their therapeutic window. BNCT drugs bearing the organic boron pharmacophore have the potential to fine-tune the timing of toxicity delivery.


Introduction
The Wittig Reaction, with its variations (e.g. the Horner-Wadsworth-Emmons (HWE) reaction), provides a particularly reliable synthetic tool for the stereocontrolled formation of ethylenic bonds. It allows preparation of an alkene by reaction of an aldehyde or ketone with the ylide generated from a phosphonium salt. 1 The geometry of the resulting alkene depends on the reactivity of the ylide. If the ylide is stabilized by an electron-withdrawing group then predominantly (E)-alkenes are formed. Non-stabilized ylides lead to (Z)-alkenes. The flexibility of the reaction allows to obtain simple as well as substituted molecular scaffolds. 2,3 In the HWE reaction, the carbonyl compound of aldehydes or ketones reacts with stabilized phosphorus ylides (phosphonate carbanions) leading to olefins with excellent E-selectivity. The nucleophilic carbon needs to bear an anion stabilizing group (e.g. CO2Me, COMe, COH, phenyl).

Results and Discussion
The Wittig and HWE reactions are a mainstay reaction in organic synthesis and provides a useful synthetic strategy to access cinnamic acid analogues. There are limited green solventless Wittig reaction protocols to the production of substituted cinnamate scaffolds.
These involve either 1) a milling protocol by ball-milling, 16 or grinding, [17][18][19][20] or 2) simply stirring. [21][22][23] Although non-green reaction solvents were eliminated from reaction protocols, however they tend to be used during purifications involving mostly column chromatography on silica, where copious amounts of solvents tend to be used (Table 1, Supporting Information) compared to the re/crystallization protocols used by us.
Ball-milling protocols include one-pot preparation of stabilized ylides followed by 4 solventless Wittig reaction in 8-20 hours under an atmosphere of helium and product (with E:Z ratio 3.5:1 to 1.6:1) isolation via column chromatography. 16 The grinding of reactants with stabilized ylids provides mainly (E)-products, where isolation is achieved via recrystallization or column chromatography. [17][18][19][20] (Z)-Intermediates are produced only when coumarin target molecules were produced from reagents impregnated with sodium methoxide and magnesium oxide. 18 Under simple stirring conditions, the reaction can be carried out at room temperature with liquid aldehydes (e.g. benzaldehyde) 23 or, if heated, in a melt when using solid aldehydes, 23 e.g. 9-anthraldehyde. 21,24,25 Microwave irradiation can also be applied to accelerate reaction rates provided phosphorane 23,26 or phosphazene 27 stability. In dry conditions, the Wittig reaction has also been shown to occur in the presence of alumina or potassium fluoride supported on alumina, which after stirring for 48 hours afforded the alkene products in a E:Z ratio 92:8 to 1: 1. 22 In many cases the easiest way of separating the excess phosphorane and triphenylphosphine byproduct is still column chromatography using non-green solvents. 25 In our laboratory, we have a strong interest in organic boron 6 and the development of synthetic protocols to new drug leads containing organic boron pharmacophores. 4,5,28 In the synthesis of methyl cinnamate analogues (Scheme 1), a solventless Wittig reaction, modified from two original procedures 17,21 was employed with optimized reaction times and temperatures to ensure the rapid and efficient synthesis of boronate esters ortho 2, meta 2 and para 2 in the set of highest stereoisomeric ratios to date in literature, followed by isolation by recrystallization or flash chromatography.
The rapid green olefination of ortho 2, meta 2 and para 2 was effected with stabilized Wittig reagent (methoxycarbonylmethylene)triphenylphosphorane (Scheme 1) under normal atmosphere in an open round-bottom flask in a melt at 150 o C for 2.5 hours to give the corresponding methyl cinnamate products in 85, 89 and 91% yields respectively after purification via recrystallization in the best set of E:Z ratios in the literature, with 92.5:7.5, 99:1 and 92:8 respectively. Flash chromatography was also scoped out as a purification method that resulted in no loss of borylated products on silica. Flash chromatography can make purifications challenging in many cases due to over-adsorption of boron-containing compounds on silica, resulting in a reduction in yields. 29 Scheme 1. Reagents and Conditions: (i) (methoxycarbonylmethylene)triphenylphosphorane, 150°C, 2.5 hrs.
One literature protocol to the synthesis of ortho 2 is known to proceed via stirring in toluene at 90 o C for 18 hours, followed by purification by flash chromatography to give the product in an 87% yield, and in a E:Z ratio 85:15 as a light yellow solid. A pure sample of the Eisomer was obtained by recrystallization from hexanes and characterised by HRMS, IR, 1 H-and 13 C-NMR. 30 Our ortho 2 product's IR and NMR data match the literature data, apart from the location of the two pinacol Cq, at 77.5 ppm, whereas others 30 report them at 84.1 ppm.
One literature protocol to the synthesis of para 2 (used as intermediate towards aaminoquinazolin-4(3H)-ones as plasmepsin inhibitors) involves coupling of methyl (E)-3-(4bromophenyl)acrylate (1.0 eq) with bis(pinacolato)diboron (1.2 eq) using Pd(dppf)Cl2CH2Cl2 (0.1 eq) and KOAc (3.0 eq) in DMSO at 80 o C under an argon atmosphere, which -after extraction, drying and purification by flash-chromatography -gave the product in 52% yield as a white 6 amorphous solid and was characterised by 1 H-and 13 C-NMR. 31 Our product's NMR data match the literature data.
In related work, Molander and Oliveira carried out the Wittig reaction of aromatic organotrifluoroborates with methyl(triphenylphosphoranylidene) acetate in water at 90 o C, with a 12 hour reaction time, to give the corresponding trifluoroborate methyl cinnamates in variable diastereoselectivities (E:Z 70:30 (para), 100:0 (meta), 80:20 (ortho)) and isolated after trituration in DCM and acetone as mainly mixtures of E and Z products. 32

X-Ray Crystallography Data
As each of the three borylated cinnamates produced crystalline products, they were studied by X-ray crystallography. For ortho 2 the alkene moiety is in a trans configuration and the dioxaborolane 5-membered ring is in a puckered conformation with atoms C2A, B1A, O3A and O4A sitting on the same plane in trigonal planar geometry with angle O3A-B1A-C2A measuring the ideal 120.07(13)°, whereas angles O4A-B1A-O3A and O4A-B1A-C2A departing by ~6° from the ideal trigonal planar geometry angle and measuring respectively 113.34(12)° and 126.59(13)°; the dihedral angle B1A-C2A-C1A-C7A measures 20° thus displacing the pinacol ester group from the plane on which the aromatic ring and the ,-unsaturated ester lie.
The arrangement of the four molecules in the unit cell (Supporting Information Figure S1) involves the two B molecules sitting on parallel planes, head-to-tail to one another, so that the aromatic and pinacol ester moieties of one molecule are facing one another. The two A molecules are also sitting on parallel planes in such a way that the aromatic moiety and the chain containing the ,-unsaturated esters are facing head-to-tail to one another.  There are two molecules in the asymmetric unit of the para 2 structure and one of these is shown in Figure 4. The boronate ester groups were disordered between two different puckered conformations (Supporting Information Figure S3). Of the three geometrical isomers, para 2 was the only one that suffered disorder. Similarly to meta 2, the atoms forming the alkyl framework of both molecules in the asymmetric unit (C10 to B1) are sitting on the same plane ( Figure 3). The alkene is in trans conformation in this case as well.

Glycosidase Assay
In our laboratory, we have an interest in the inhibition of glycosidases to the management of many disease types, including metabolism, 33 viral 34 and lysosomal storage disorders. 35 Cinnamates have been found to possess glycosidase inhibition properties 13 as "neutral" (i.e. lacking a basic nitrogen found in iminosugars/azasugars) glycosidase inhibitors. 15 Here the boron pinacol ester was explored as a pharmacophoric group in the para-, meta-and orthopositions to the unsaturated ester on the aromatic ring. Organic boron continues to emerge as a pharmacophore capable of not only intermolecularly interacting with active sites, but also intramolecularly through the establishment of dative bonds from nucleophilic atoms of the enzyme to the electrophilic boron atom. Introduction of a boronic acid can shift the glycosidase inhibitory profile of an iminosugar. 36 Free boronic acids can also improve inhibition by forming hydrogen bonds to an enzyme. 37 The kaleidoscopic chemistry associated with the organic boron atom grants huge potential in terms of the chemical and physical tuning of the drug molecule properties, but at the same time represents a huge challenge to the organic/medicinal chemist developing high Fsp 3 index 38 boron-bearing drug leads. 4,5,39,40 In Table 1, the three boron-bearing leads para 2, meta 2 and ortho 2, and the reference compound methyl cinnamate were screened against a panel of 15 glycosidases to assess both potency and selectivity of inhibition. It is shown that methyl cinnamate does not significantly inhibit any of the glycosidases assayed, with % inhibition at 1000 M ranging from 0% to 34%.
Ortho 2 has a similar inhibition profile to methyl cinnamate with % inhibition at 1000 M ranging from 0% to 42%. However, meta 2 and para 2 provide weak but highly or exclusively selective

Cancer Assay
In our laboratory we are interested in the use of BNCT as a potentially broad-spectrum approach to cancer management. Providing the drugs can selectively accumulate in cancer cells vs healthy cells, 41 it should be possible to manage the disease via BNCT, a non-invasive and least destructive radiation therapy currently available. [42][43][44] In order to use a drug in BNCT, it is ideal if, when not irradiated, the drug is non-toxic. In this preliminary study of synthesis, purification and toxicity of organic boron containing drug leads for BNCT applications, none of 10 the three borylated derivatives were found to be toxic in cancer cells lines and a normal cell line (MCF10A) ( Table 2). The organic boron pharmacophore provides promising results in this area because the currently in use BNCT agent sodium borocaptate, BSH, containing an inorganic boron pharmacophore, raises several toxicity concerns, 45-46 whereas boronophenylalanine, BPA, which contains the organic boronic acid pharmacophore has long been known to show no discernible toxicity. 47 Organic boron is also an essential element for plants and is probably essential for human and animal health. 48 When comparing toxicological data for organic boron containing molecules with their non-borylated congeners, it is also found that in general the presence of organic boron lowers toxicity profiles. For example, benzene has a lethal dose (LD50) = 125 mg/kg (human, oral) 49  Ortho 2 showed zero growth inhibition towards U87, BE2-C and SMA560 cancer cell lines.
Dose response experiments (GI50 as concentration (µM) that inhibits cell growth by 50%) were not completed for these compounds.

Conclusions
Three borylated derivatives of methyl cinnamate were produced via a green chemistry protocol with the best set of stereoisomeric ratios to date. They were fully characterized, including by X-ray crystallography, principally to study the behaviour of the boronate group. The drugs proved their glycosidase modulation capability to produce selective inhibition against a -glucosidase and a -galactosidase. Furthermore these compounds were screened against a panel of cancer cells and a normal cell line to ascertain their percent cell growth inhibition capabilities in view to their use as BNCT agents with the potential to fine-tune the timing of toxicity delivery.

Conflicts of interest
There are no conflicts to declare.