Design and synthesis of novel pinacolylboronate containing combretastatin ‘antimitotic agent’ analogues

Abstract

We developed a procedure to synthesize pinacolyl boronate containing stilbene derivatives and used this procedure to synthesize boron-containing combretastatin analogues. The key step involves the Wittig reaction of the ylide 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboratophenyl)-methyl triphenylphosphonium bromide 11 with 3,4,5-trimethoxy benzaldehyde in the presence of ^tBuONa in DMF, providing 88% yield. We are now in a position to evaluate the biological activity of these derivatives as modulators of TGF-beta signaling pathways.

For an ongoing chemical biology project involving the study of the TGF-beta (Transforming Growth Factor-beta) pathways using developing zebrafish embryos, we synthesized a small library of 2-substituted 2H-chromene derivatives, and after screening, we identified a lead molecule BT7 capable of modulating a specific relevant pathway, namely p-SAPK/JNK, that is known to be down-stream of TGF-beta and can mediate Smad-independent signaling.^1,2 To increase the activity and potency of our lead molecule, and for structure/activity relationship studies, we envisioned developing a boron-based stilbene-containing small molecule library (Fig. 1) based on a hypothesis that (a) introducing chromene isosteres like stilbene may increase potency and (b) introducing a boron atom into a biologically active framework, might allow interaction with a target protein not only through hydrogen bonds but also through covalent bonds, and this interaction would produce potent biological activity, and (c) from a literature search we also found that combretastatin A modulates TGF-beta signaling pathway. Therefore, we considered if different CA4 analogues could provide additional insight into TGF-beta signaling pathways to identify new gene targets or co-factors. In this context we undertook a project to develop synthetic methodology to synthesize boron-containing stilbene derivatives, and use those methods to synthesize CA4 analogues.

Figure 1.

Structure of BT7.

Stilbene (1,2-diphenylethene) represents a prototype of a 1,2-disubstituted olefin. Stilbene itself does not occur in nature, but substituted stilbenes (Fig. 2) such as pinosilvin 2, resveratrol 3,^3,4 diethylstilbestrol 4,⁵ 4,4-diaminostilbene-2,2-disulfonic acid,⁶ nitro- substituted stilbene boronate pinacol esters,⁷ arotinoids⁸, combretastatin 5, 6⁹ (Fig. 2) are among the many synthetic and natural stilbene derivatives that are biologically active compounds.

Figure 2.

Biologically important stilbene derivatives.

The use of boron atoms in pharmaceutical drug design possesses a high potential for discovery of new biological activity.^10–13 Among various boron compounds synthesized, much attention has been paid to boronic acid containing peptides such as Velcade and DPP-IV inhibitors.¹² In these boropeptides, a carboxylic acid has been replaced by a boronic acid group.

Combretastatin A-4 (CA-4) has been found to be a potent inhibitor of tubulin polymerization through binding to the colchicine binding site. It is also a cytotoxic agent toward a wide variety of human cancer cell lines and also modulates TGF-beta signaling pathways.¹⁴ From a structure/activity relationship point of view, CA-4 belongs to the class of natural compounds related to biphenyls and contains, as a key structural feature, the cis-stilbene motif.

Due to the strong biological significance of stilbene-containing compounds, a general applicable synthetic protocol to synthesize these highly demanding molecules with boronic acid groups would be useful. The protocol should tolerate a large number of functional groups in different positions on the aromatic ring. The most familiar and general strategy for synthesis of boron-containing stilbenes 8 is based on disconnection A (Fig. 3) and involves the Wittig reaction of the various substituted benzyl phosphonium ylide 9 with the pinacol ester of boronate aldehyde 10.¹⁵

Figure 3.

Retrosynthetic analysis.

Although Wittig and Horner–Wadsworth–Emmons reactions have been carried out on aldehyde derivatives of boronate esters^16,17 the problem with this approach (A) for the synthesis of a library of boron-containing stilbene derivatives is the need to utilize various substituted benzyl phosphonium ylides and boron-containing aldehydes. Boron-containing aldehydes are very prone to self-dimerization and oxidation and are unstable in air, so this method is limited in scope. To overcome this problem, we undertook the disconnection B approach (Fig. 3), envisaging the use of the pinacolylboronate phosphonium ylide 11, which has not previously been explored at all. However, organotrifluoroborato phosphonium ylides are used to synthesize alkene derivatives by Wittig reactions.¹⁸

To synthesize our model compound 1, we first synthesized compound 11. 4-(4,4,5,5-tetramethyl-1,3,2 dioxaboratophenyl)- methyl triphenylphosphonium bromide 11 was synthesized from 2-[4′-(bromomethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 13 in the presence of 1.01 equiv of triphenylphosphine in acetonitrile at reflux conditions. The minor excess PPh₃ was removed from the product by trituration with ether 2–3 times and the product is stable under normal atmospheric conditions (Scheme 1).

Scheme 1.

Synthesis of boronate ylide 11.

Compound 13, was synthesized by refluxing 14, NBS (N-bromosuccinimide) and AIBN (azobisisobutyronitrile) in carbon tetrachloride for six hours to provide the intermediate 13 (85% yield).¹⁹

In this study, we found that 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboratophenyl)- methyl triphenylphosphonium bromide 11, when treated with 2-hydroxy-3,5-dichloro benzaldehyde as a model substrate in the presence of 3 equiv of sodium tert-butoxide in DMF as solvent at room temperature produced the 2,4-dichloro-6-{2-[4- (4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-vinyl}-phenol 1 in 86% yield with a mixture of E and Z isomers (E:Z = 42:58) (Scheme 2).

Scheme 2.

Synthesis of model compound 1.

After successfully synthesizing our target compound 1, next we examined the scope of the Wittig reaction for the synthesis of pinacolylboronate- substituted stilbenes using various aryl aldehydes. ²⁰ The results are summarized in Table 1. The reaction proved tolerant of both electron-withdrawing (NO₂; Table 1, entry 4) and electron-donating groups (B(OH)₂, di-t-butyl; Table 1, entries 2 and 3) on the phenyl ring of the aldehyde. For the Wittig reaction of aryl aldehydes containing a heteroatom such as nitrogen (Table 1, entry 5) gave the product 1f with good yield with high E selectivity.

Table 1.

Wittig reaction of boronate ylide (11) with various aldehydes^a

Entry	Aldehyde	Product	Yieldb (%)
1		1b E:Z= 74:26	86
2		1c E:Z= 64:36	72
3		1dE:Z= 75:25	71
4		1e E:Z= 55:45	76
5		1f E:Z= 100:0	78

^aAll reactions were performed using 1 equiv of aldehyde and 3 equiv of ^tBuONa in DMF for 2–12 h.

^bIsolated yield (mixture of E and Z) refers to aldehyde and the E/Z ratios were determined by ¹H NMR.

Next we focussed on target compound CA4 analogues 7a and 7b, bearing a boronic acid system that replaces the OMe group of the natural CA-4 in ring B (Fig. 2). To synthesize 7a and 7b, the ylide 11 was reacted with aldehydes 12a and 12b. Wittig reaction between the aldehyde 12a and ylide 11 in the presence of ^tBuONa, afforded the corresponding products 7a and 7b with 88% and 82% yields, respectively. The products were a mixture of E and Z isomers (Scheme 3). As R_f value is very close to each other, it was very difficult to separate them, but from TLC we found R_f 0.69 is considerably more fluorogenic than R_f 0.76 (1:1 ethyl acetate/hexane; solvent system).

Scheme 3.

Synthesis of boronate containing combretastatin analogues 7a–b.

Moreover, we have also developed a one-pot Wittig reaction for synthesizing the compound 7a. In this case, 4-(4,4,5,5-tetramethyl- 1,3,2-dioxaboratophenyl)methyltriphenylphosphonium bromide 11, the intermediate generated by reaction of 13 with triphenylphosphine, was reacted directly with 3,4,5-trimethoxy benzaldehyde 12a in the presence of ^tBuONa as a base, and the desired boron-containing combretastatin analogue 7a was isolated in 74% overall yield as a one-pot Wittig reaction (Scheme 4).

Scheme 4.

One-pot synthesis of 7a.

To compare our approach B with the approach A in (Fig. 3), we synthesized the 7a starting from 16 using aldehyde 17 via the Wittig ylide 15 (Scheme 5), and found our approach gives higher yields with high stereoselectivity.

Scheme 5.

Synthesis of 7a using approach A.

In summary, we developed a procedure to synthesize pinacolylboronate containing stilbene derivatives and used this procedure to synthesize boron-containing combretastatin analogues. Experiments are currently underway to separate the E and Z isomers and test the biological activity of these derivatives and to determine their utility as modulators of TGF-beta signaling pathways.