Atropselective syntheses of (-) and (+) rugulotrosin A utilizing point-to-axial chirality transfer.

Chiral, dimeric natural products containing complex structures and interesting biological properties have inspired chemists and biologists for decades. A seven-step total synthesis of the axially chiral, dimeric tetrahydroxanthone natural product rugulotrosin A is described. The synthesis employs a one-pot Suzuki coupling/dimerization to generate the requisite 2,2'-biaryl linkage. Highly selective point-to-axial chirality transfer was achieved using palladium catalysis with achiral phosphine ligands. Single X-ray crystal diffraction data were obtained to confirm both the atropisomeric configuration and absolute stereochemistry of rugulotrosin A. Computational studies are described to rationalize the atropselectivity observed in the key dimerization step. Comparison of the crude fungal extract with synthetic rugulotrosin A and its atropisomer verified that nature generates a single atropisomer of the natural product.

Due to hindered rotational barriers of highly substituted biaryls and related compounds, a type of stereochemistry in the form of axial chirality is generated, often termed atropisomerism.[1] Atropisomerism widely exists in nature and can lead to biological stereoselectivity for molecular targets.[2,3,4] Moreover, in contrast to traditional sp3 carbon-centered chirality, control of axial chirality and atropisomer selectivity remains highly challenging especially in the context of complex natural product synthesis.[5,6,7,8]

Tetrahydroxanthones,[9] secondary fungal metabolites, are an emerging family of natural products. Dimeric tetrahydroxanthones[10] including the secalonic acids[11,12] (Figure 1, a) display intriguing anticancer and antibacterial properties. During the last decade, tetrahydroxanthones containing biaryl axial chirality including the 2,2’-linked natural product[13] rugulotrosin A (1) and the 2,4’-linked congener rugulotrosin B (2) (Figure 1, b) have been isolated and characterized. Related hybrid chromone lactone/tetrahydroxanthones including the 2,4'-linked heterodimer gonytolide E (3)[14] and the 4,4'-linked chromone lactone homodimer gonytolide A (4)[15] have also been reported. Recently, these interesting molecules have attracted significant interest in the synthetic community as evidenced by reports of syntheses of both monomeric[16,17,18,19,20,21,22] and dimeric natural products. [23] However, syntheses of tetrahydroxanthones bearing axial chirality including rugulotrosin A (1) and related compounds have not been reported.

Figure 1

Axially Chiral, Dimeric Tetrahydroxanthone Natural Products and Point-to-Axial Chirality Transfer Strategy. a) Structures of the dimeric tetrahydroxanthone natural products secalonic acids B and D; b) Structures of representative axially chiral tetrahydroxanthone natural products; c) Select literature examples of remote point-to-axial chirality transfer; d) Strategy for the synthesis of rugulotrosin A.

In order to establish axial chirality for rugulotrosin A, we envisioned that a point-to-axial chirality transfer strategy could be implemented. In the last few decades, point-to-axial chirality transfer has been widely used in atropselective synthesis. There are several different approaches for point-to-axial chirality transfer: (a) use of an ortho-auxilary[5,6] or chiral leaving group [24] for intermolecular, atropselective coupling; (b) utilization of low-energy conformers derived from existing chirality centers[25,26] or chiral auxiliaries[5,6] to execute intramolecular couplings; (c) transfer of chiral sp3 carbon to axial chirality;[27, 28] (d) and use of remote and existing chirality centers to influence atropselectiviy using intermolecular oxidative/metal-mediated couplings.[29,30,31,32,33] For example, Shaw and coworkers reported that the chiral monomer 5 underwent oxidative coupling to generate atropisomer 6 in 76:24 dr which could be improved to higher selectivity using a chiral catalyst system[29] (Figure 1, c). Likewise, Lipshutz and coworkers performed an atropselective cross coupling to access the korupensamine B precursor 9 using boronic acid 7 and iodotetrahydroisoquinoline partner 8[31] (Figure 1, c). In our studies, we considered a remote asymmetric induction strategy for point-to-axial chirality transfer for the rugulotrosin synthesis in the absence of chiral auxiliaries. In the case of dimerization of tetrahydroxanthone substrate 10, it was of interest to determine whether the chirality of the remote stereocenters could be transferred to the axial chirality of ortho, ortho dimer 11 (Figure 1, d), a product bearing a different coupling pattern than previous examples. In this case, we planned to employ transition metal catalysis to bring the two monomers in close proximity to enhance potential for chirality transfer.

Results and Discussion

Our synthesis was initiated with chromone 12 which was prepared in greater than 50 gram batches and purified by recrystallization (Figure 2). Utilizing diisopropylsilyl ditriflate to activate chromone 12 to a siloxybenzopyrylium species,[22] vinylogous addition of 2-trimethylsiloxyfuran was achieved which was followed by hydrogenation to afford chromone lactone 13 on adecagram scale (89 % yield, dr > 10:1) after purification by trituration (Supplementary Information S4–S19). Further treatment of 13 with NaH in THF led to the production of epi-blennolide C 14 through Dieckmann cyclization. Due to the propensity of thevinylogous acid moiety of tetrahydroxanthone 14 to both epimerization and tetrahydroxanthone-to-chromone lactone rearrangement, we utilized the neutral methylating reagent trimethylsilyldiazomethane to obtain the desired O-methylated product 16 in 61 % yield along with the separable isomer 15 (20 % yield). After phenol-directed ortho-iodination of 16, the derived aryl iodide 17 was subjected to kinetic acylative resolution using Birman's catalyst [34] on a gram scale (s factor >200).[35] Chiral monomer (−)-17 was isolated in 46 % yield (99 % ee) and the O-acylated product (+)-18 was obtained in 46 % yield (99 % ee) after recrystallization.

Figure 2

Scalable Syntheses of Enantiopure Tetrahydroxanthone Monomers: Key steps involve siloxyfuran addition to a benzopyrylium species (12 to (±)-13) followed by kinetic acylative resolution of (±)-17 using the Birman catalyst.

After obtaining the two enantiopure tetrahydroxanthone monomers, our initial plan was to employ our stannane dimerization strategy which successfully led to syntheses of secalonic acids A and D.[23] However, after evaluation of various conditions, we observed only trace stannylation of iodides 17 or 18 (Table 1) which may be due to steric hindrance of the aryl iodide substrate. Subsequently, we considered that the less bulky borate may more readily undergo transmetallation. Gratifyingly, iodide (−)-17 was found to be stable under basic conditions and to produce an inseparable mixture of dimeric products (dr = 88: 12) under one-pot Suzuki coupling conditions using Pd(OAc)2/SPhos[36] and bis (pinacolato) diboron. We subsequently attempted to establish the stereochemistry of the major atropisomer by NMR analysis and by acid-mediated deprotection of both atropisomers 19 and 20 to 1 and 23 (Figure 4). However, both efforts failed as the axial chirality center is remote from existing stereocenters leading to almost identical 1H NMR spectra for both a tropisomers of rugulotrosin A. Fortunately, a single crystal of the major atropisomer (−)-19 was obtained by recrystallization (CH2Cl2/MeOH) from its mixture with 20. X-ray crystal structure analysis of (−)-19 (CCDC#1022610) was achieved which verified that the relative stereochemistry was identical to that found in natural rugulotrosin A.[13]

Table 1

One-pot Suzuki Dimerization of Chiral Tetrahydroxanthone Monomer (−)-17.

Entry	Ligand	Conversion (yield)	d.r.	Entry	Ligand	Conversion (yield)	d.r.
1		68 % (45 %)	88 : 12	7		44 % (24 %)	95.5 : 4.5
2		<10 % conv		8		40 % conv	90 : 10
3		50 % (27 %)	93 : 7	9a		72–82 % (61 %)	89 : 11
4		65 % (44 %)	87 : 13	10a		45 % (~30 %)	80 : 20
5		no reaction
6		36 % conv	81 : 19	11a,b		62 % (44 %)	95.5 : 4.5

Conditions for Pd precatalyst: 0.2 equiv Pd precatalyst, 0.7 BPin2, 3 equiv K3PO4, THF/water 4:1, 70 °C, 5 h.

0.6 equiv BPin2 was used.

Figure 4

Syntheses of (−) and (+)-rugulotrosin A and atrop-rugulotrosin A. a) Synthesis of ent-rugulotrosin A; b) Synthesis of rugulotrosin A and comparison rotation data with natural sample; c) Synthesis of atrop-rugulotrosin A; d) Synthesis of ent-atroprugulotrosin A; e) Comparison between the natural extract and synthetic rugulotrosins. HPLC-DAD (210 nm) (Zorbax C18 column, gradient elution H2O/MeCN plus 0.05% HCO2H)analysis of 21 dayPenicillium nov. sp. (MST-F8741) cultures extracted with (a) MeCN, or (b) MeOH, compared against (c) natural (+)-1, (d) synthetic (+)-1, (e) (−)-23, and (f) a mixture of (+)-1, (−)-1, (−)-23 and (+)-23).

During our evaluation of dimerization conditions, we found that water was essential and that the ratio of atropisomers was dependent on ligands for palladium rather than solvent, temperature, or reaction time. Accordingly, we focused our efforts on the influence of ligands on atropisomeric selectivity (Table 1). After extensive ligand screening, we found that monophosphines were crucial for the success of there action, and that bidentate ligands such as XantPhos or DPEPhos (not shown) failed to provide the desired dimeric products. Besides SPhos(L1), the related ligand RuPhos (L2) provided dimeric products in even higher atropselectivity (93:7). Interestingly, we found when the 2' position of the monophophine ligand did not contain an oxygen atom (e.g. carbon atom in XPhos L2 or a nitrogen atom (DavePhos, not shown)), dimeric products were obtained in trace amounts. After experiments with the achiral ligand L4, which provided similar results to SPhos, we anticipated that related chiral ligands may achieve efficient control of atropselectivity.[37] We began this investigation with (R) and (S)-BI-DIME ligands L5,[38,39] which recently have shown excellent atropselectivities in biaryl couplings. However, neither ligand was able to perform the one-pot dimerization of (−)-12. Gratifyingly, chiral versions of the SPhos ligands, S-Cy-MOP (L6) and R-Cy-MOP (L7),[40, 41, 42] both successfully produced dimeric products. The ligand R-Cy-MOP provided a satisfactory dr (95.5:4.5) and the mismatched ligand S-Cy-MOP afforded a lower dr (81:19), both favoring the same major atropisomer. Even with the increased bulk of ligand L8, the dr did not noticeably improve. In order to enhance product yield, we prepared the Buchwald 3rd generation precatalysts[43] with SPhos, R-Cy-MOP, and S-Cy-MOP. Indeed, use of the precatalyst increased yields for these cases and maintained at ropselectivity. Use of bis (neopentyl glycolato) diboron ortetrahydroxydiboron[44, 45] as boron transfer reagents led only to trace amounts of dimeric products.

The acylated, enantiopure monomer (+)-18 was also investigated in the Suzuki dimerization which generated a separable 3:1 mixture of atropisomersin 36 % yield (Figure 3). Fortunately, an X-ray crystal structure of the minor atropisomer (−)-22 (CCDC#1022611) was obtained to verify relative and absolute stereochemistry. Introduction of a bulk yester onto the secondary alcohol of the monomer (e.g. OCOPh) did not affect the ratio of atropisomers.

Figure 3

One-pot Suzuki Dimerization of Chiral Tetrahydroxanthone Monomers (+)-18: Optimal conditions shown using Pd(OAc)2 and SPhos as achiral ligand.

After obtaining and establishing stereochemistry for several protected dimers, we anticipated that we could access all atropisomers and enantiomers of rugulotrosin A. Dimer (−)-19 was treated with 3M HCl/MeOH for 10 min which afforded rugulotrosin A ((−)-1) in 89 % yield (Figure 4, a). In a similar manner, (+)-rugulotrosin A was obtained from acidic hydrolysis of (+)-21 (Figure 4, b). With both enantiomers of rugulotrosin A in hand, natural rugulotrosin A was unambiguously assigned as (+)-1 though comparison of rotation data. Similarly, atrop-rugulotrosin A (−)-23 and entatrop rugulotrosin A (+)-23 were synthesized using similar protocols from the protected precursors (−)-22 and (+)-22 (Figure 4, c and d). To verify the stability of the axially chiral biaryl moiety, we heated rugulotrosin A (+)-1 in toluene (1 mg/mL) at 100 °C for 12 h and 150 °C for 3 h. Under both conditions, we did not observe formation of atrop rugulotrosin. This is supported by a high barrier of rotation of rugulotrosin A, calculated as 47.7 kcal/mol using the B3LYP/6-31G(d) level of theory (see Supplementary Information S52–S58).

In order to rationalize the axial chirality selectivity observed in the dimerization, we modeled the geometry of the expected intermediate diaryl palladium (II) complexes[46] using SPhos (L1) as ligand (Figure 5). Conformational analysis was carried out by systematic six-fold rotation about the C-Pd bonds, followed by optimization of candidate structures at the B3LYP/LanL2DZ level of theory.[47] The five lowest energy structures are represented in Figure 5. From this analysis, the three lowest energy structures 24a – 24c all have dihedral angles (C1, C2, C2’, C1’) for the monomer fragments which would afford atropisomer (−)-19 (precursor to rugulotrosin) after stereospecific reductive elimination (Figure 5,a). The next two higher energy structures 25a and 25b display a reversed dihedral angle which should lead to atropisomer 20 (Table 1). Relative energies correlate with the steric fit of components. Space filling models of structures 24a-c and 25a-b (Supplementary Information S34–S52) show a more crowded arrangement of the latter, in part because of steric interactions between the methyl ester and the cyclohexyl group. The distances between the hydrogen of the methyl ester to the hydrogen on the cyclohexyl of the SPhos ligand are 2.4 and 2.5 Å for conformers 25a and 25b, respectively. Based on literature precedent,[47] we assume that these catalyst-reactant complexes are under thermodynamic equilibration; additional computational studies will be required to predict barriers to interconversion. It thus appears that product stereochemistry is determined by reactant and catalyst assembly in the Pd (II) complex; this provides a basis for prediction of stereochemistry in future atropselective couplings.

Figure 5

Computational Studies for Atropselective Suzuki Dimerization. Conformational analysis was optimized at the B3LYP/LanL2DZ level of theory. Dihedral angles were measured by C1, C2, C2’, C1’and are shown in green. a) Conformers leading to (−)-19; b) Conformers leading to 20.

After assigning the absolute configuration of rugulotrosin A, we employed our library of synthetic rugulotrosins to determine whether Penicillium nov. sp. (MST-F8741) was capable of producing atrop-rugulotrosin A. To this end, separate 21 day fermentations of MST-F8741 were extracted with either MeCN (Figure 4,e(a)) or MeOH (Figure 4, e(b)) and were subsequently analyzed using an HPLC-DAD method optimized for the resolution of rugulotrosin A (Figure 4, e(c,d)) and atrop-rugulotrosin A (Figure 4, e(e)). To further enhance sensitivity, these analyses were also carried out using HPLC-DAD-ESIM Sprotocols augmented by single ion extraction (SIE) (Supplementary Information, S22). As these analytical studies failed to detect any trace of atrop-rugulotrosin in MST-F8741 extracts, we conclude that the rugulotrosin biosynthetic pathway present in Penicillium nov. sp. (MST-F8741) operates with high atropisomer fidelity.

As natural rugulotrosin A had previously been reported to exhibit antibacterial activity,[13] we also tested the synthetic rugulotrosins ((+)-1, (−)-1, (−)-23, and (+)-23) against several strains of Gram-positive and Gram-negative bacteria. Consistent with earlier reports,(+)-1 exhibited antibacterial activity against Bacillus subtilis (ATCC 6633) (IC50 = 2.1 µM) and Staphylococcus aureus (ATCC 25923) (IC50 = 6 µM). Of note, the enantiomer (−)-1 was also active against B. subtilis (ATCC 6633) (IC50 = 0.7 µM) and S. aureus (ATCC 25923) (IC50 = 18 µM), whereas the antibacterial activity of the atropisomer (−)-23 was weak and limited to B. subtilis (ATCC 6633) (IC50 = 10 µM) and (+)-23 lacked any appreciable antibacterial activity. None of the rugulotrosins (+)-1, (−)-1, (−)-23, or (+)-23 exhibited activity against the Gram-negative bacteria Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853), nor did they exhibit cytotoxicity (IC50>30 µM) against human colon (SW620) and lung (NCI-H460) cancer cells.

Conclusion

In conclusion, we have developed a concise, atropselective approach to rugulotrosin A and stereoisomers through point-to-axial chirality transfer which has facilitated determination of the absolute configuration of rugulotrosin A. Computational studies modelling the geometry of intermediate diaryl palladium (II) complexes have provided a rationale for the atropselectivity observed in the key Suzuki dimerization. These studies highlight the utility of SPhos and related bulky monophosphine ligands[48] in palladium couplings both to bring monomers in proximity and enhance remote steric effects leading to atropselectivity. Through HPLC analysis of fungal extracts and synthetic samples, we have determined that Penicillium nov. sp. (MST-F8741) generates rugulotrosin A in an atropselective manner. Moreover, the atropisomers and enantiomers of rugulotrosin A were found to have different activities against Gram-positive bacteria illustrating the importance of stereochemistry on target selectivity. Further studies regarding atropselective syntheses of dimeric natural products are currently under investigation and will be reported in due course.