Structural diversity and similar bioactivity in synthetic bicyclononanes

ABSTRACT Simple syntheses of diverse bicyclo[3.3.1]nonanes and related compounds as the minimal substructure of bioactive natural products via Michael, aldol, and alkylation reactions from diketones are described herein. The structures of the synthesized compounds were determined by infrared spectroscopy, NMR (1H and 13C), and electrospray ionization–high-resolution mass spectrometry. We also show the in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria. The qualitative analysis has revealed that the new synthesized compounds 5, 6, 9, and 11 present antibacterial properties. GRAPHICAL ABSTRACT


Introduction
Substituted bicyclo [3.3.1] nonanes are characteristic carbon skeletons of natural products. They belong to a class of interesting compounds with a remarkable diversity of biological activities against conditions such as cancer, HIV, bacterial infections, and depression, among others. [1,2] Members of prenylated acyl phloroglucinols derived from prolifenone, nemorosone, clusianone, [3,4] and particularly hyperforin isolated from Hypericum species, possess potent antibacterial activity and moderate cytotoxicity (Fig. 1). [5,6] Recently new cytotoxic and anti-inflamatory prenylated benzoylphloroglucinols were isolated from Garcinia esculenta and examined against human cancer and hepatic cells. [7] Because of manifest bioactivities of these compounds, many and innovative strategies have been developed towards the [3.3.1] bridged bicycle class of phloroglucinol natural products. [8][9][10] Exploring synthetic strategies to build oxygenated polynuclear heterocycles of biological interest, first we found a methodology on solid support for the chromenone 2 via Michael reaction on 1,3-cyclohexanedione 1. Biological evaluation of the heterocycle 2 led to its consideration as an antifeedant compound which was compared with related natural and semisynthetic products [11] (Scheme 1).
In this work we report the synthesis of bicyclic analogs of [3.3.1] bridged phloroglucinol natural products from 1,4-addition, alkylation, and cyclization reactions on 1,3-cyclohexanediones 1 and 12, and their biological activities to disclose minimal functional requirements as potential antibacterial agents (Fig. 1).
In spite of the existence of related synthetic studies, [11][12][13][14][15] the originality of this work is based on the low number of synthetic pathways to create functional diversity in highly oxygenated structures with similar biological activity.

Chemistry
The Michael addition continues to be a valuable synthetic method, which, in combination with alkylation and condensation reactions, may be used to obtain a wide variety of complex molecules from relatively simple starting materials. Consequently, new structures containing acetal, hemiketal, and carbonyl functions linked to a common ring have been isolated and identified. These structures are related to prolifenone A and B, [16] hyperforin, clusianone, [17] and the advanced intermediary phomoidride B, among others. [18] Michael addition of 1, methyl vinyl ketone (MVK), and NaOH in MeOH at room temperature led to compounds 3, [15] 4, [16] and the ketal 5 with 25:35:30% yield, respectively (Scheme 1). The same reaction carried out on neutral alumina as solid support in CH 2 Cl 2 at room temperature also afforded adducts 3 (66%) and 4 (33%). Alternatively, 3 could be transformed into 4 with prior isolation of the mixture, under the same reaction conditions.
As shown in Scheme 2, the reaction pathways to give a new compound 5 involves a dimerization of the methyl vinyl ketone followed by an aldol reaction between the resulting cyclic hemiacetal A and cyclohexane-1,3-dione. The formation of hemiacetal A was evidenced by literature data [19] and its structure was confirmed by spectroscopic characterization of the methylketal derivative B, as we discussed. The 1 H NMR spectrum shows a double doublet at 4.02 ppm corresponding to the CH axial coupled with the near CH 2 [ 3 J ¼ 2.6 (H ax -H ec ) and 3  By virtue of some substructures of natural products that contain prenyl side chains, we explored the incorporation of allyl and prenyl groups. After several attempts, introduction of the allylic function was exemplified by the base-catalyzed aldol process of the triketone Scheme 2. Sequence of chemical reactions for the formation of 5.
3 dissolved in acetone with K 2 CO 3 at 50 °C, and then addition of allyl bromide under reflux. [20] This reaction afforded products of O-alkylation (7) and C-alkylation (8) in a ratio 1:4, in quantitative yield (Scheme 3). The mixture of 7 and 8 could be easily converted into 8 by a Claisen rearrangement in boiling xylene during 5 h. [21] The same reaction conditions also allowed to transform 7 and 8 to the bicyclic allylic compound 9 in good yield by an aldol reaction on the side chain carbonyl group. When the reaction of 8 was carried out in the presence of L-pro in MeCN at rt, the known allyl Wieland-Miescher ketone 10 is formed by means of an aldol condensation on the carbonyl group of the ring. [12] Another approach was explored using tetraketone 4 as starting material, which led to two more advanced bicyclic substructures of interest (Scheme 4). Thus, reaction of 4 with K 2 CO 3 , dissolved in MeOH at À 18 °C at rt [22] rendered triketone 6 via intramolecular aldol condensation in quantitative yield. Then, treatment of 6 with LDA in THF and subsequent addition of prenyl bromide furnished only the O-alkylated 11 in good yield. [9] All compounds required a detailed analysis of their structures by 1 H and 13 C NMR. The structure elucidation of compounds 5 and 6 was performed by extensive spectroscopic analysis, including 1D (BB and DEPT) and 2D NMR (HSQC and HMBC) and their relative stereochemistries were readily determined by nuclear Overhauser effect (NOE) measurements.
In the case of the compound 5, from HSQC spectrum shows that the methylene carbon at 24.7 ppm correlates with proton resonances at 1.63 and 1.35 and the methylene carbon at 17.2 ppm correlates with proton resonances at 1.55 and 1.45, showing the diastereotopic character of this methylene group. Besides, the irradiation of methine C-1′ gave a NOE of de methylic protons on C-7′ and the methylene protons of C-2′ (Fig. 2).    shifts for all de protons of 6 and correlated them with the resonance from 13 C spectrum with the proposal to define the diastereotopic protons for each of the methylene groups.
In particular, structure 11 was elucidated by comparison with related Michael products previously reported by Hajos and Parrish in the Michael reaction between 2-methyl-1,3cyclopentanedione and MVK in refluxing MeOH and catalytic amounts of KOH. [23] Treatment of 12 with MVK, DBU, and prenyl bromide at room temperature resulted in the formation of the O-alkylated product 14 (78%) along with the C-and O-alkylated 13  (20%) and the methyl vinyl ketone dimer (2%), in a one-pot reaction (Scheme 5). These products of alkylation reactions 13 and 14 were used as representative simple models of functional groups for the in vitro evaluation of biological activity. Figure 4 shows the active compounds resulting from in vitro assays of antibacterial activity against Gram-positive organisms such as Enterococcus faecalis ATCC 29212 (E. fa) and Staphylococcus aureus ATCC 25923 (S. au), and Gram-negative bacteria Escherichia coli ATCC 25922 (E. co) and Pseudomonas aeruginosa ATCC 27853 (P. ae). [25,26] Monocyclic compounds 13 and 14, substituted with a gem-methyl on the ring and prenyl chains, did not exhibit activity in the in vitro tests.

Antimicrobial activity
The results observed in the microbiological assays indicate antibacterial activity of compounds 5, 6, and 9 on E. fa but not on E. co, and equivalent antimicrobial susceptibility testing results were obtained with S. au and P. ae, respectively.
The more advanced substructure of prenylated natural products, that is, the prenylated analog 11, maintained its Gram-positive activity on E. fa (10 4 dilution) and also diminished bacterial growth as seen on E. co.
These data show an antibacterial effect of the compounds 5, 6, and 9 against Grampositive bacteria, although no effect against Gram-negative bacteria was observed under the conditions tested, and the comparison between the compound 9 (allyl) and 11 (prenyl chain) shown the latter has a broader spectrum of activity.

Conclusions
By means of simple reactions and a few steps with respect to the known synthesis, we synthesized new bicycle[3.3.1]nonanes, which are of interest as core of prenylated acyl phloroglucinols, to further our understanding of structure-activity relationships in bioactivity profiles. In vitro analysis of antimicrobial activity allowed us to elucidate minimal functional requirements of the structural core to display bioactivity. The results demonstrate that the oxidation degree, this is β-hydroxyenone, β-hydroxyketone, β-diketone, and ketal moieties of the compounds 5, 6, 9 and 11, has a significant role similar to that of alkenyl chains in 9 and 11, which are present in the original structures. In addition, the negative tests for antibacterial activity of the monocyclic compounds 13 and 14, although with functional groups similar to those in 5, 6, 9, and 11, suggest that their antibacterial activity is also dependent on the number of rings and the three-dimensional arrangement (conformation) of these kind of molecules. Therefore molecular arrangements such as two separate rings (5) or fused ring systems (6, 9, and 11), along with the previously mentioned structural features, are needed for antibacterial activity. Further synthetic manipulations will improve the biological activity of these compounds.

Experimental
All solvents were dried and distilled before use. All reactions were carried out under anhydrous conditions under N 2 atmosphere. All the organic extracts were dried over anhydrous Na 2 SO 4 . Reactions were monitored by thin-layer chromatography (TLC) on aluminum-foil plates coated with Merck Kieselgel 60 F254, and spot visualization performed under ultraviolet (UV) light. Column chromatography (CC): Analtech silica gel for flash chromatography, under low N 2 pressure. Elution was carried out with hexane/AcOEt mixtures. Infrared (IR) spectra (in cm À 1 ) were recorded on a Shimadzu Prestige-21 FTIR spectrophotometer. 1 H and 13 C NMR spectra (300 MHz and 75 MHz) were recorded on a Bruker AC-300 spectrometer. Samples were dissolved in CDCl 3 as solvent and Me 4 Si as internal standard (δ in ppm, J in Hz). High-resolution mass spectrometry (HR-MS) was performed using a Bruker MicrOTOF-Q II 10223 instrument. Melting points were measured on a Ernst Leitz hot-stage microscope and are uncorrected. 2-(5,7-dimethyl-6,8-dioxabicyclo[3.2.1]oct-7-yl)-3-hydroxycyclohex-2-enone (5) Michael reaction in solution MVK (125 µl, 1.5 mmol) was added slowly to a mixture of 1 (112 mg, 1 mmol), in anhydrous MeOH (1 mL) and NaOH (1.2 mg, 0.03 mmol) at rt, and the mixture was left at rt for 2 h. MeOH was removed under vacuum and the resulting solid was suspended in CH 2 Cl 2 , washed with a saturated aqueous solution of NaCl, dried (anhydrous Na 2 SO 4 ), filtered, and concentrated. Purification of the residue obtained by flash chromatography rendered 3 (25%) as oil, 4 (35%) as a white solid, and compound 5 (30%) as white crystals.