Letter pubs.acs.org/OrgLett
Synthesis of Sugar−Boronic Acid Derivatives: A Class of Potential Agents for Boron Neutron Capture Therapy Daniela Imperio, Erika Del Grosso, Silvia Fallarini, Grazia Lombardi, and Luigi Panza* Department of Pharmaceutical Sciences, Università del Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy S Supporting Information *
ABSTRACT: To date, sugar analogues that contain boronic acids as substitutes for hydroxyl groups are a class of compounds nearly unknown in the literature. The challenging synthesis of two sugar−boronic acid analogues is described, and data are retrieved on their solution behavior, stability, and toxicity. As these compounds were expected to mimic the behavior of carbohydrates, they were tested in regards to their future development as potential boron neutron capture therapy agents.
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the whole molecule, the reported general information about their stability gives a broad overview.6 A detailed review by Matteson systematically depicts the compatibility of boronic acids/esters in the presence of other functional groups.7 From this review, it appears that β-alkoxyboronates are reasonably stable, with some surprising exceptions, as described in a previous paper.8 The β-hydroxyboronates appear to easily give elimination reactions;7 however, other reports describe the synthesis and isolation of β-hydroxyboronates9 or even β-hydroxyboronic acids,10 mainly isolated after their conversion to trifluoroborates. Keeping in mind the previous considerations, we decided to explore the synthesis of sugar−boronic acids, initially focusing on the replacement of the 6-OH with a boronic acid moiety, and herein, we describe the synthesis of two new sugar derivatives whose structures are presented in Figure 1.
oron neutron capture therapy (BNCT) is a binary approach for cancer treatment based on the nuclear reaction between two nontoxic species: low-energy (thermal or epithermal) neutrons and a compound containing 10B atom(s). The neutron capture reaction produces an α-particle and a lithium ion, along with 2.4 MeV of kinetic energy and a 480 keV photon. These high-linear-energy transfer ions dissipate their kinetic energy before traveling one cell diameter (5−9 μm) in biological tissues, which gives them the potential for precise cell killing. Highly selective delivery, accumulation of boron in tumor tissues, and proper subcellular distribution are among the most important requirements for successful neutron capture therapy.1 Sodium mercaptoundecahydrododecaborate (Na2B12H11SH, BSH) and L-boronophenylalanine (BPA), the two compounds currently used for clinical trials, have been demonstrated to be safe in humans. However, clinical results from the two compounds are not universally attractive mainly because of their rapid clearance from blood.2 In fact, both are only moderately selective for cancer cells and exhibit short retention times in tumors. Therefore, research efforts are underway to identify new candidates for BNCT with better tumor selectivity and more cell retention. Boronated carbohydrate derivatives have received consideration in BNCT due to the sugars’ preferential uptake by tumor cells.3 Most cancer cells produce energy by high rates of glycolysis, increasing the glucose utilization with respect to normal cells. Galactose and fructose also allow cell tumor proliferation in the absence of glucose. Boronic acids have gained interest in the past few years for the development of enzyme inhibitors, drug delivery polymers, and saccharide sensors and as a boron source for BNCT agents, such as amino acid derivatives or DNA binders.4 Within a more general research program, we planned the synthesis of boronic acid derivatives of monosaccharides, where the boronic acid moiety substitutes one of the sugar’s hydroxyl groups. A careful literature survey was performed, revealing that such compounds are not described and very few examples of related structures are reported.5 As the stability of boronic acids appears to be strongly dependent on the structural features of © XXXX American Chemical Society
Figure 1. Structures of the newly synthesized sugar−boronic acids.
Two main aspects had to be considered when planning their synthesis. First, the introduction of the boronic acid/boronate ester would have to be performed using reaction conditions compatible with the sugar structure. In fact, the common methods used for the synthesis of alkylboronic acid derivatives,6,11 when applied to carbohydrates, could cause degradation or give multiple regio- and stereoisomers. Moreover, a proper set of protecting groups would have to be installed on the sugar. Accordingly, we planned to exploit a cross-coupling reaction between a halosugar and the diboron compound B2pin2, as it would introduce the boronate moiety while respecting the Received: February 16, 2017
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DOI: 10.1021/acs.orglett.7b00382 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
This unexpected result, although disappointing, demonstrated that the choice of the protecting groups was crucial. Moreover, the formation of the dimer could be explained by the generation of a radical at C-6 from 1, followed by a migration of a hydrogen atom from the benzylic position of the protecting benzyl group at O-4 to C-6. Finally, homocoupling of the newly generated, more stable benzyl radical gave rise to dimer 8. Although not fully demonstrated, this result strongly supports the involvement of a radical mechanism in the Cu-catalyzed borylation, as was already purposed (but essentially ruled out) in ref 12b. Therefore, various substrate structures and protecting groups were investigated, such as benzyl ethers, esters, silyl ethers, and acetals (Table 1). Again, when tried on other bromosugars, the Pd-catalyzed reaction returned only the starting compound. Therefore, the couplings were performed using CuI as a catalyst. From the obtained results, it appears that the reaction is very sensitive to both steric (see entry 5) and electronic effects (see entries 2 and 4). The best results were obtained using isopropylidene protecting groups (entries 6 and 7). With compounds 9 and 10 in hand, we then moved toward the final, apparently simple, deprotection steps (Scheme 2).
sugar structure. Deprotection of the boronic ester and sugar would give the desired compounds. From previous considerations, we devised the following general synthetic approach shown in Scheme 1. We initially Scheme 1. General Synthetic Approach
tested two recently developed cross-coupling reactions12 on 6bromo-6-deoxy-1,2,3,4-tetra-O-benzyl-β-D-glucopyranoside 1, obtained from the treatment of the easily available 1,2,3,4tetra-O-benzyl-β-D-glucopyranoside13 with PPh3/CBr4.14 The Pd-catalyzed reaction12a did not give any products; the use of copper as a catalyst12b afforded a complex mixture from which the main TLC-observed product was carefully purified and characterized and determined to be the dimer 8 (Table 1, entry 1).
Scheme 2. Synthesis of the Galactose−Boronic Acid Derivative
Table 1. Sugar−B2Pin2 Coupling Reactionsa
We decided to remove the pinacol ester first, as Bpin deprotection can sometimes be cumbersome. An initial attempt with diethanolamine18 on compound 9 gave no reaction, while a more traditional oxidative cleavage of the pinacol ester at neutral pH, using a slightly modified19 literature procedure,20 afforded us the free boronic acid 11 in good yield. Final deprotection of the isopropylidene groups was attempted using a variety of conditions (HCl aq/dioxane, dioxane/H2O/Amberlite IR120/MeOH, and TFA). The best results in terms of limiting byproduct formation were observed in a mixture of trifluoroacetic acid, water, and dichloromethane. Under these conditions, the pure galactose−boronic acid derivative 12 was obtained after precipitation by the addition of acetonitrile. Other attempts to purify compound 12 were unsuccessful, as the crude product was contaminated by an impurity whose amount increased with crude manipulations. A similar decomposition of compound 12, but with much slower kinetics, was observed when it was left in aqueous solution at rt, which resulted in the formation of the same impurity. The 1 H NMR spectrum of compound 12 after 24 h in solution showed signals of a terminal vinyl group, which can be ascribed to the formation of the elimination product 17. These data were also supported by MS on the water solution of 12 left at rt for 1 week: the minor presence of ions at m/z = 147 [M + H]+, m/z = 164 [M + NH4]+, and m/z = 169 [M + Na]+ strongly suggest the presence of the elimination product 17 (see Figure 2 and Supporting Information). An analogous pathway was chosen for the synthesis of the glucose−boronic acid derivative. Boronic ester 10, obtained by
a Typical conditions: CuI (0.1 equiv), PPh3 (0.13 equiv), B2pin2 (2 equiv), LiOMe (2 equiv), DMF, and 25 °C. bIsolated yield. cNo reaction.
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DOI: 10.1021/acs.orglett.7b00382 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
The observed elimination clearly demonstrates that, at least on compound 12, the free β-hydroxy boronic acid must be present in aqueous solution. We therefore decided to further analyze the solution structures of compounds 12 and 23, and we will discuss data on these aspects later. From our observations, a proper strategy to overcome the drawback of the elimination in the glucose derivative could be to lock its anomeric position as a glucoside. Therefore, the synthesis of a stable glucoside−boronic acid derivative was performed, as shown in Scheme 4. Starting from the known Scheme 4. Synthesis of the β-Methyl Glucoside−Boronic Acid Derivative
Figure 2. Equilibria involved in the elimination reaction for compound 12.
cross-coupling as previously described (Table 1, entry 7), underwent hydrolysis and oxidation to give the free boronic acid 13. Isopropylidene deprotection with trifluoroacetic acid mainly afforded the β-elimination product 1521 (Scheme 3) instead of the desired glucose derivative 16. Scheme 3. Attempted Synthesis of the Glucose−Boronic Acid Derivative
compound 18, we obtained methyl 6-bromo-6-deoxy-2,3-di-Obenzyl-β-D-galactopyranoside under the described conditions25 in 80% yield. Attempts to perform the coupling reaction on the unprotected bromosugar 19 were unsuccessful. The 4-OH was then protected with TMS ether to obtain compound 20. The introduction of pinacol boronic ester to give 21 was achieved as previously described, avoiding any radical side reaction. Moreover, a TMS group was proven to be stable, and it did not give too much steric hindrance. Hydrolysis of the boronic ester and removal of the benzyl groups finally gave, as expected, the stable methyl 6-deoxy-6-dioxaboryl-β-D-glucopyranoside 23. With compounds 12 and 23 in hand, as previously stated, we decided to investigate their structures in solution. As it is difficult to distinguish between boronic acids and their cyclic monoesters by NMR, we used mass spectrometry. The mass spectrum of compound 12 in aqueous solution showed the presence of the stable cyclic boronic esters 12a/12c (m/z = 208 [M + NH4]+) and of the open boronic acids 12b/12d (m/ z = 226 [M + NH4]+ and m/z = 231 [M + Na]+). However, the MS spectrum of compound 23, obtained under the same conditions, revealed that it is mainly present as the open boronic acid 23a (m/z = 240 [M + NH4]+ and [M + Na]+) together with a small peak (m/z = 222 [M + NH4]+, approximately 5% of m/z = 240) ascribed to the cyclic boronic monoester 23b. These results suggest that it is possible to obtain qualitative information on boronic acid−cyclic ester equilibria in solution by MS. These results also allow for the comprehension of a hypothesized elimination scheme (see Supporting Information for details). Finally, the obtained compounds 12 and 23 were tested for their toxicity, as drug candidates for BNCT must have low systemic cytotoxicity. Therefore, derivatives 12 and 23 were evaluated for cytotoxicity on human primary fibroblasts using an MTT test and using commercial BPA as a reference. The
From these results, some general conclusions can be drawn on the stability of oxyboronic acids. While sugar-derived βalkoxyboronic acids and esters appear to be reasonably stable, the analogous structures with the boronic acids bearing free hydroxyl groups on the adjacent carbons are highly prone to elimination reactions. The elimination reaction was very fast on putative intermediate 14 but quite slow on compound 12. To explain this difference in stability, we must consider the equilibria that can occur, as shown in Figure 2. A β-elimination reaction can occur only on the intermediate 12d in which both the 5-OH and the free boronic acid are available simultaneously. Therefore, while in compound 12, before the elimination could occur, both the hemiacetal ring and the cyclic boronic monoester must convert to their open forms, in compound 14, the elimination can occur immediately after acetonide removal, prohibiting the rearrangement from the furanosidic to pyranosidic forms of the sugar ring. It should be noted that our interpretation assumes the presence of the boronic acid both as the free acid and as its cyclic monoester. In the literature, differing data can be found on this aspect. The presence of cyclic boronic monoesters has been demonstrated in crystal structures22 and in solution,23 where the authors claimed that it was not possible to open the cyclic form, implicitly excluding the presence of an equilibrium. On the other hand, in similar cases, equilibria between the cyclic and open forms have been suggested.24 C
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(13) Lu, W.; Navidpour, L.; Taylor, S. D. Carbohydr. Res. 2005, 340, 1213−1217. (14) Mobarak, H.; Engstrom, O.; Widmalm, G. RSC Adv. 2013, 3, 23090−23097. (15) Khan, K. M.; Perveen, S. R.; Al-Qawasmeh, A. S.; Shekhani, M. S.; Ali Shah, S. T.; Voelter, W. Lett. Org. Chem. 2009, 6, 191−196. (16) Streicher, B.; Wünsch, B. Carbohydr. Res. 2003, 338, 2375− 2385. (17) Hodosi, G.; Podanyi, B.; Kuszmann, J. Carbohydr. Res. 1992, 230, 327−342. (18) Sun, J.; Perfetti, M. T.; Santos, W. L. J. Org. Chem. 2011, 76, 3571−3575. (19) Cox, C. D.; Dudkin, V.; Kern, J.; Layton, M. E.; Raheem, I. T. Patent Appl. WO2013028590, 2013 (Merck Sharp & Dohme). (20) Coutts, S. J.; Adams, J.; Krolikowski, D.; Snow, R. J. Tetrahedron Lett. 1994, 35, 5109−5112. (21) Skaanderup, P. R.; Hyldtoft, L.; Madsen, L. Monatsh. Chem. 2002, 133, 467−472 and refs cited therein. As the characterization of compound 15 was not consistent among different publications, its structure was confirmed by independent synthesis; see the Supporting Information. (22) (a) Maynard, A.; Crosby, R. M.; Ellis, B.; Hamatake, R.; Hong, Z.; Johns, B. A.; Kahler, K. M.; Koble, C.; Leivers, A.; Leivers, M. R.; Mathis, A.; Peat, A. J.; Pouliot, J. J.; Roberts, C. D.; Samano, V.; Schmidt, R. M.; Smith, G. K.; Spaltenstein, A.; Stewart, E. L.; Thommes, P.; Turner, E. M.; Voitenleitner, C.; Walker, J. T.; Waitt, G.; Weatherhead, J.; Weaver, K.; Williams, S.; Wright, L.; Xiong, Z. Z.; Haigh, D.; Shotwell, J. B. J. Med. Chem. 2014, 57, 1902−1913. (b) Hecker, S. J.; Reddy, K. R.; Totrov, M.; Hirst, G. C.; Lomovskaya, O.; Griffith, D. C.; King, P.; Tsivkovski, R.; Sun, D.; Sabet, M.; Tarazi, Z.; Clifton, M. C.; Atkins, K.; Raymond, A.; Potts, K. T.; Abendroth, J.; Boyer, S. H.; Loutit, J. S.; Morgan, E. E.; Durso, S.; Dudley, M. N. J. Med. Chem. 2015, 58, 3682−3692. (23) Matteson, D. S.; Soundararajan, R.; Ho, O. C.; Gatzweiler, W. Organometallics 1996, 15, 152−163. (24) Ness, S.; Martin, R.; Kindler, A. M.; Paetzel, M.; Gold, M.; Jensen, S. E.; Jones, J. B.; Strynadka, N. C. J. Biochemistry 2000, 39, 5312−5321. (25) Szolcsányi, P.; Gracza, T.; Koman, M.; Prónayová, N.; Liptaj, T. Tetrahedron: Asymmetry 2000, 11, 2579−2597.
derivatives illustrated no relevant cytotoxicity at the tested conditions, as reported in Supporting Information. In conclusion, we have reported the synthesis, stability evaluation, and preliminary biological testing of novel sugar− boronic acid derivatives. The synthesized compounds showed no toxicity on human primary fibroblasts. These compounds containing boronic acids as the boron source can find application as a new type of carrier for BNCT if they are demonstrated to be taken up by sugar transporters. Future studies are warranted to obtain information on their uptake in tumor cells and to develop the synthesis of new similar compounds.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00382. Experimental details and characterization data for all new compounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Luigi Panza: 0000-0002-0785-0409 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully thank Compagnia di San Paolo (GLYCOBNCT project CUP: C61J12000280007) for financial support. REFERENCES
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DOI: 10.1021/acs.orglett.7b00382 Org. Lett. XXXX, XXX, XXX−XXX
