A Strategy toward Icetexane Natural Products

Abstract

Icetexane diterpenoids are richly complex polycyclic natural products that have been described with a variety of biological activities. We report here a general synthetic approach toward the 6–7-6 tricyclic core structure of these interesting synthetic targets based on a two-step enolate alkylation and ring-closing metathesis reaction sequence.

Introduction

Icetexane diterpenoids are complex natural products that have been isolated from a variety of plant life known to several systems of traditional medicine in Asia.^[1] Teas and other preparations derived from several genera of icetexane producing plants have been used for their beneficial antioxidant, anti-inflammatory, and immunomodulatory effects, as well as for the treatment of hepatic disorders, hypertonia, and other ailments.^[2] Icetexanes are generally isolated as 6–7-6 tricyclic system, with geminal methyl substitution at C4, and an isopropyl unit at C13, with an aromatic ring comprising the C8–C14 ring and various units of unsaturation and oxidation in the tricyclic system.^[3] These decorated tricyclic diterpenoids have been described with a variety of medicinally significant biological activities, however a particularly interesting member of this family is premnalatifolin A (1), a novel dimeric diterpene isolated from Premna latifolia Roxb in 2011.^[4] Premnalatifolin A (1) was reported to have cytotoxic activity against seven human cancer cell lines in vitro including colon (HT-29), skin (A-431), liver (Hep-G2), prostate (PC-3), lung (A-549), kidney (ACHN), and breast cancer (MCF-7). The most potent activity was exhibited against breast cancer (MCF-7) with an IC₅₀ of 1.11 ± 0.23 μg/mL (1.77 μM), which compared favorably with doxorubicin (Adriamycin),^[5] one of the current standards of care and the positive control during that study (IC₅₀ = 2.01 ± 0.03 μg/mL, 3.68 μM).

Results and Discussion

We are interested in the synthesis of monomeric and dimeric icetexane diterpenoid natural products (e.g. 2, 3, Figure 1), and have developed a simple strategy for the synthesis of the 6-7-6 monomeric units. We describe here a two-step sequence for the construction of the central seven-membered ring based on an enolate alkylation and an olefin metathesis reaction.

Figure 1.

Dimeric and monomeric icetexane diterpenoids.

Icetexane diterpenoids have been examined by other synthetic organic chemistry groups in the past owing to their interesting structures and important biological activities. Several distinct strategies have been described including cycloadditions and metal-mediated cycloisomerizations^[6] and Friedel–Crafts type annulations and/or other cationic chemistry.^[7] Some of these strategies are reliant upon enolate alkylations, carbonyl additions, or otherwise metalated carbonyl compounds,^[8] but require several steps of processing to generate the central seven-membered ring and adjust functional groups. Our use of enolates derived from silylenol ether and prefunctionalized aromatic precursors ideally positions appropriate functional groups with the correct oxidation levels to make for short, convergent syntheses of monomeric icetexane terpenoids.

We originally intended to employ the enolate–OQM coupling reaction developed in our laboratories^[9] to join a vinyl-bearing silylenol ether derived from 4,4-dimethyl-2-cyclohexen-1-one with a highly functionalized tert-butyldimethylsilyloxybenzyl chloride under the action of tetramethylammonium fluoride (Scheme 1). The central seven-membered ring of the monomeric icetexanes would then be completed by a ring-closing metathesis reaction;^[10] our proposed two-step strategy is distinctly different from prior efforts and would represent the most convergent synthesis of these natural products to date.

Scheme 1.

An enolate–OQM coupling reaction followed by hemiacetal formation.

As a means of assessing the feasibility of this reaction sequence, we prepared a simplified model system with a silylenol ether derived from 2-cyclohexen-1-one and a silyloxy benzyl chloride derived from 2-chloro-6-fluorobenzaldehyde (Scheme 2a). The silylenol ether is available in a one-pot operation; conjugate addition of vinylmagnesium bromide in the presence of copper(I) iodide to 2-cyclohexen-1-one in THF at −78 °C gives the corresponding enolate which is then trapped with chlorotrimethylsilane to give the silyl ether 4 in 96 % yield.^[11] The silyloxybenzyl chloride 5 was synthesized in five steps. A nucleophilic aromatic substitution with potassium hydroxide and 2-chloro-6-fluorobenzaldehyde (12) gave the corresponding chlorosalicylic acid in 91 % yield.^[12] The vinyl group was installed by a Suzuki–Miyaura coupling with potassium vinyltrifluoroborate under the action of palladium(II) acetate with (±)-BINAP as ligand in DMF/water to give the styrene aldehyde 14 in 93 % yield.^[13] Finally, silyl protection, reduction of the aldehyde and conversion of the resultant alcohol to the corresponding benzyl chloride (5) was achieved in three steps and 79 % overall yield (See Supporting Information for the detailed preparation of each component).

Scheme 2.

Synthesis of the Premnalatifolin A core structure. Conditions: (a) CuI, 10, THF, –78 °C, then TMSCl, Et₃N, −78 → 23 °C. (b) KOH, H₂O/DMSO, 23 °C. (c) Pd(OAc)₂, (±)-BINAP, 13, Cs₂CO₃, H2O/THF (1:9), 135 °C. (d) TBSCl, Et₃N, CH₂Cl₂, 23 °C. (e) NaBH₄, CH₃OH, 0 → 23 °C. (f ) SOCl₂, Et₂O, 0 → 23 °C or PBr₃, CH₂Cl₂, 0 °C. (g) CH₃Li, DME, 0 °C, then 15, 0 → 23 °C. (h) 18 (3–5 mol-%), CH₂Cl₂, 40 °C. (i) TBAF (1.0 M in THF), THF, 23 °C. (j) H₂, 10 % Pd/C, EtOAc, 23 °C.

We found that the silyl ether 4 and the silyloxybenzyl chloride 5 joined smoothly in minutes upon exposure to TMAF in cold dichloromethane, and did so diastereoselectively to give the 1,2-trans alkylation product 6 exclusively (Scheme 1).^[14] Despite this success, the enolate–OQM reaction strategy was undermined by a post-alkylation addition of the phenol to the carbonyl function to give a robust diastereomeric (and separable) pair of cyclic hemiketals 7. We were able to characterize one epimer by X-ray crystal diffraction, however each epimer slowly equilibrates back to a statistical mixture upon prolonged standing in protic solvent. We observed similar complications with other ketophenol substrates during our development of the enolate–OQM coupling reaction but unlike those simpler systems, we were unable to develop conditions under which the open ketophenol 6 could be captured.^[9]

We attempted the ring-opening process under basic and acidic conditions, as well as conditions under which the phenol might be captured as the corresponding ether in order to prevent reclosure.^[9,11] We overcame this complication by retreating to a more traditional enolate alkylation approach employing the silyloxybenzyl bromide 15 and the lithium enolate obtained by treating the silyl ether 4 with methyllithium in DME at −78 °C.^[15] In contrast to the rapid enolate–OQM reaction, this traditional alkylation proceeds very slowly and takes several hours to reach completion presumably due to the lower reactivity of the alkyl halide relative to the corresponding OQM, and the significant steric congestion. This reaction keeps the silyl group intact to prevent the unproductive carbonyl addition, and also proceeds diastereoselectively to give the 1,2-trans alkylation product 16 in 35 % yield. The balance of the material was a regioisomeric alkylation product that presumably arises via enolate isomerization that can occur upon prolonged stirring of starting materials and products under these reaction conditions. The alkylation also functioned well with the enolate generated from 4,4-dimethyl-2-cyclohexen-1-one, giving the highly substituted cyclohexanone 17 in 75 % yield; the balance of the material in that reaction was also the regioisomeric alkylation product. Our efforts to suppress the formation of the regioisomeric alkylation product and enhance the rate of these alkylation reactions with additives such as 1,3-dimethyl-3,4,5,6- tetrahydro-2(1H)-pyrimidinone (DMPU), hexamethylphosphoramide (HMPA), and metal salts were unsuccessful.^[16]

Following the alkylation events, the vinyl groups were joined under the action of the Grubbs second generation catalyst^[9] in straightforward ring-closing metathesis reactions to give the corresponding seven-membered rings 19 and 20, featuring the core structure of monomeric icetexane natural products in 92 % and 85 % yield, respectively. Alternatively, rigorous separation of the alkylation products proved unnecessary; the mixture of alkylation products could be subjected to the ring closing metathesis reaction directly to give the cycloheptenes in 32 % and 48 % yield, respectively, over two steps.^[17] Moreover we were able to characterize the tricyclic gem-dimethyl-substituted product by single-crystal X-ray diffraction.

Following both olefin metathesis reactions, we further diversified our scaffold preparations by generating the fully saturated icetexane skeletons. The analogues 23 and 24 were prepared in 93 % and 95 % yield, respectively, by hydrogenation of the cyclohexene products under the action of palladium on carbon in ethyl acetate. Finally, we removed the tert- butyldimethylsilyl protecting groups by treatment with tetra-n- butylammonium fluoride in tetrahydrofuran at 23 °C to give ketophenols 21, 22, 25, and 26 in 53–99 % yield.

We are now exploring the potential of icetexane natural products and simplified analogues as anticancer agents. To determine the effect of our simplified analogues on cell viability, the MCF-7 human breast cancer cell line was treated with increasing concentrations of each compound. Cell viability was determined 72 hours following treatment with compound using a standard commercially available MTT cell viability assay. Issues arising from the poor solubility of compounds 21, 22, and 26 in the cell culture media precluded detailed study of these materials; only the effect of compound 25 on cell viability was determined. Cell survival of the MCF-7s decreased with increasing concentrations of compound 25, and we determined a LD₅₀ of 250 μM (Figure 2). In addition, SUM159 cells treated with increasing concentrations of compound 25 showed a similar decline in cell viability (data not shown). This activity is far from that reported for the premnalatifolins and other similar icetexanes, but is important information nonetheless, as we assess these compounds for future analogue synthesis.

Figure 2.

MCF-7 cell viability following treatment with 25.

Conclusion

We are presently generating the fully functionalized monomeric subunits of premnalatifolin A as well as a series of closely related analogues in order to gain a more detailed survey of the biological potential of these interesting natural products. These studies will be reported in due course.

CCDC 1538503 (for 7), 1538504 (for 20), and 1538505 (for 21) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallo-graphic Data Centre.