Structural diversity and similar bioactivity in synthetic bicyclononanes

Article abstract of DOI:10.1080/00397911.2016.1141429

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

Full text of DOI:10.1080/00397911.2016.1141429

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Structural Diversity and Similar Bioactivity in
Synthetic Bicyclo Nonanes
Liliana E. Luna, Pamela S. Forastieri, Patricia Marchiaro, Adriana Limansky &
Raquel M. Cravero
To cite this article: Liliana E. Luna, Pamela S. Forastieri, Patricia Marchiaro, Adriana Limansky
& Raquel M. Cravero (2016): Structural Diversity and Similar Bioactivity in Synthetic Bicyclo
Nonanes, Synthetic Communications, DOI: 10.1080/00397911.2016.1141429
To link to this article: http://dx.doi.org/10.1080/00397911.2016.1141429
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Date: 22 January 2016, At: 11:36

Structural Diversity and Similar Bioactivity in Synthetic Bicyclo Nonanes
Liliana E. Luna1, Pamela S. Forastieri1, Patricia Marchiaro2, Adriana Limansky2, Raquel
M. Cravero1
1IQUIR (Instituto de Química Rosario), Facultad de Ciencias Bioquímicas y
Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina 2IBR (Instituto de
Biología Molecular y Celular de Rosario). Facultad de Ciencias Bioquímicas y
Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
Corresponding author Raquel M. Cravero E-mail: cravero@iquir-conicet.gov.ar
Supplemental data for this article can be accessed on the publisher’s website.
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 IR, NMR (1H and 13C) and ESI-HRMS. 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.
KEYWORDS: bicyclo nonanes, diketones, Michael-aldol-alkylation, antimicrobial
activity
1

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 derivated 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] Due to
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-
10]
Exploring synthetic strategies to build oxygenated polinuclear 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 antifeedant compound which was compared with related natural and
semi-synthetic products.[11] (Scheme 1).
2

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,[11-15] 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.
RESULTS AND DISCUSSION
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] as well as 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 CH2Cl2 at room temperature, also afforded adducts 3 (66%) and 4 (33%).
3

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 1H-NMR spectrum
shows a double doublet at 4.02 ppm corresponding to the CH axial coupled with the near
CH2 (3J = 2.6 (Hax-Hec) and 3J = 12.0 (Hax-Hax)), and signals at 3.22 (CH3O), 2.20
(CH3C=O), 1.35 (CH3) and at 1.90-1.30 appears a multiplet for other methylene groups
of the ring.
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 3 dissolved in acetone with K2CO3 at 50ºC, 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 r.t., the known allyl Wieland-
4

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
K2CO3, dissolved in MeOH at -18°C at r.t.[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 1H and 13C NMR. The
structure elucidation of compounds 5 and 6 was performed by extensive spectroscopic
analysis; including 1D (BB and DEPT), 2D NMR (HSQC and HMBC) and their relative
stereochemistries were readily determined by NOE measurements.
In 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
diatereostereotopic character of this methylene group. Besides, the irradiation of methine
C-1’ gave a nuclear Overhauser enhancement of de methylic protons on C-7’ and the
methylene protons of C-2’.
From the HSQC spectrum of 6, we observed the correlation between the methylene
carbon at 39.4 ppm with proton resonances at 2.57 and 2.31, the methylene carbon at 38.7
5

ppm with proton resonances at 2.59 and 2.25 and the methylene carbon at 18.9 ppm also
correlates with proton resonances at 2.09 and 1.70. Thus, we have assigned the chemical
shifts for all de protons of 6 and correlated them with the resonance from 13C spectrum
with the proposal to define the diastereotopic protons for each of the methylene groups.
In addition, the irradiation of methyl at peak = 1.35 ppm (CH3C-OH) gave a nuclear
Overhauser enhancement of de methine H-5 ( = 2.65 ppm) and axial proton of
methylene H-8 ( = 1.70 ppm) (Fig. 3.)
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-1,3-
cyclopentanedione 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 alkylations reactions 13 and 14 were used as representative simple models of
functional groups for the in vitro evaluation of biological activity.
Antimicrobial Activity. Fig. 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), Staphylococcus aureus ATCC 25923 (S. au), and Gram-negative
6

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.
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, this is, the prenylated-
analog 11, maintained its Gram-positive activity on E. fa (104 dilution) and also
diminished bacterial growth as seen on E. co.
These data show an antibacterial effect of the compounds 5, 6, 9 against Gram-positive
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 more broad spectrum of activity.
CONCLUSIONS
By means of simple reactions and few steps in respect to the known synthesis, we
synthesized new bicycle[3.3.1]nonanes of interest as core of prenylated acyl
phloroglucinols to further our understanding of structure-activity relationships in
7

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, have 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 above mentioned structural features are needed for antibacterial activity. Further
synthetic manipulations will be improve the biological activity of these compounds.
EXPERIMENTAL
General. All solvents were dried and distilled before use. All reactions were carried out
under anhydrous conditions under N2 atmosphere. All the organic extracts were dried
over anh. Na2SO4. Reactions were monitored by thin layer chromatography (TLC) on
aluminum-foil plates coated with Merck Kieselgel 60 F254, and spot visualization
performed under UV light. Column chromatography (CC): Analtech silica gel for flash
chromatography, under low N2 pressure. Elution was carried out with hexane/AcOEt
mixtures. IR Spectra (in cm-1) were recorded on a Shimadzu Prestige-21 FTIR
spectrophotometer. 1H- and 13C-NMR spectra (300 MHz and 75 MHz) were recorded on
a Bruker AC-300 spectrometer. Samples were dissolved in CDCl3 as solvent and Me4Si
8

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; uncorrected.
Preparation Of 2-(5,7-Dimethyl-6,8-Dioxabicyclo[3.2.1]Oct-7-Yl)-3-Hydroxy-
Cyclohex-2-Enone (5).
Michael reaction in solution. MVK (125 l, 1.5 mmoles) was added slowly to a mixture
of 1 (112 mg, 1 mmol), in anh. MeOH (1mL) and NaOH (1.2 mg, 0.03 mmol) at r.t., and
the mixture was left at r.t. for 2 h. MeOH was removed under vacuum and the resulting
solid was suspended in CH2Cl2, washed with a sat. aq. soln. of NaCl, dried (anh.
Na2SO4), 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.
Compound 5
White crystals (30%), Hex/EtAcO 50:50, Rf = 0.90, mp: 93.5-94.0 C. IR (film): 3109
(OH vinylogous acid), 2941, 1685 (C=O ketone), 1645, 1615, 1360, 1182, 1113, 1044,
969, 837. 1H-NMR (CDCl3): 10.98 (s, 1H, OH); 4.66 (bs, 1H, H-C(1′)); 2.48 (t, 2H, J =
6.2, H-C(4)); 2.32 (dt, 2H, J = 6.6, 2.0, H-C(6)); 2.01-1.87 (m, 2H, H-C(5)); 1.74-1.35
(m, 6H, H-C(2′), H-C(3′), H-C(4′)); 1.49 (s, 3H, H3C-C(5′)), 1.41 (s, 3H, H3C-C(7′)). 13C-
9

NMR (CDCl3) 197.2 (C=O); 174.6 (C(3)); 115.1 (C(2)); 108.4 (C(5′)); 87.4 (C(7′)); 82.1
(C(1′)); 37.5 (C(6)); 34.1 (C(4′)); 29.7 (C(4)); 27.4 (CH3(7′)); 25.8 (CH3(5′)); 24.7
(C(3′)); 20.7 (C(5)); 17.2 (C(2′)). EIMS: 252 (M+), 237 (M+- CH3, 5), 177 (18), 163 (33),
139 (15), 98 (43), 82 (25), 79 (28), 77 (28), 69 (40), 67 (37), 65 (25), 55 (100). ESI-
HRMS: calcd for (M+H+) C14H21O4 253.14344; found 253.14261.
Preparation Of 6-Hydroxy-6-Methyl-1-(3-Oxobutyl)Bicyclo[3.3.1]Nonane-2,9-Dione
(6).
Compound 4 (30 mg, 0.12 mmol) in MeOH (1.5 mL) was added to a soln. of K2CO3 (30
mg, 0.12 mmol) in MeOH (0.3 mL), maintained at -18°C, and left in the freezer
overnight. The mixture was filtered through a funnel containing a pad of celite and
washed with EtAcO, dried and concentrated. Purification by flash chromatography of the
crude rendered compound 6 in quantitative yield. White solid, mp: 113–114 C. IR (film):
3446 (OH), 2946, 2931, 2878, 1701 (C=O), 1375, 1166. 1H-NMR (CDCl3): 2.65-2.51
(m, 3H); 2.41-2.27 (m, 2H); 2.10 (s, 3H, CH3CO); 2.04-1.94 (m, 5H); 1.72-1.64 (m, 3H);
1.35 (s, 3H, CH3C-OH). 13C-NMR (CDCl3) 211.6, 211.2 (C=O, C(2), C(9)); 208.2
(C(3′)); 78.9 (C-OH); 64.4 (C(1)); 57.0 (C(5)); 39.4 (C(3)); 38.7 (C(2′)); 36.3(C(4)); 32.0
(C(7)); 29.8 (CH3CO); 27.7 (CH3C-OH); 25.7 (C(1’)); 18.9 (C(8)). EIMS: 222 (M+, 8)
204 (M+ - H2O, 25), 166 (10), 155 (20), 95 (36), 67(50) 55 (55), 43 (CH3CO+, 100). ESI-
HRMS: calcd for (M+Na+) C14H20NaO4 275.12538; found 275.12475.
10

Preparation Of 1-Allyl-6-Hydroxy-6-Methylbicyclo[3.3.1]Nonane-2,9-Dione (9).
Compound 7 (180 mg, 0.81 mmoles) dissolved in xylene (18 mL) was heated under
reflux for 10 h. Solvent evaporation under vacuum and further flash chromatography of
crude furnished 9 (92%) as oil. Compound 8 could also be converted into 9 by refluxing
in xylene for 5 h. Yellow oil (87%). IR (film): 3420 (OH), 3080, 2933, 1703 (C=O
ketone), 1639 (C=C), 1000, 910. 1H-NMR (CDCl3) ): ABCDX spin system 5.83
(complex signal, 1H, HX); 5.08 (complex signal, 1H, HB); 5.03-4.98 (m, 1H, HA); 4.50
(bs, 1H, OH); 2.64 (t, 1H, J = 1.0, H-C(5)); 2.54-1.92 (m, 6H, H-C(4), H-C(7), H-C(8));
1.72-1.62 (m, 4H, H-C(3), H-C(1’), CD spin system); 1.35 (s, 3H, CH3). 13C-NMR
(CDCl3) 211.1 (C=O); 211.0 (C=O); 133.2 (C(2’)); 118.2 (C(3’)); 78.7 (C-OH)); 65.1
(C(1)); 56.8 (C(5)); 39.8 (C(3)); 35.8 (C(1’)); 35.3 (C(7)); 31.5 (C(8)); 27.5 (CH3);
18.7(C(4)). ESI-HRMS: calcd for (M+Na+) C13H18NaO3 245.11482; found 245.11108.
Preparation Of 6-Hydroxy-6-Methyl-9-(3-Methylbut-2-Enyloxy)-1-(3-Oxobutyl)
Bicyclo [3.3.1]Non -5(9)-En-2-One (11).
A soln. of 6 (50 mg, 0.2 mmol) in anh. THF/HMPA (7 : 1, 0.25 mL) was slowly added to
a soln. of LDA at –78ºC, (prepared by treating a soln. of diisopropylamine (0.05 mL) in
anh. THF (0.3 mL) with n-BuLi (9.9 mL), 1.6 M in hexane for 20 min). The resulting
11

soln. was stirred for 30 min at the same temperature. Prenyl bromide (0.05 mL) in anh.
THF (0.07 mL) was subsequently added and the temperature was slowly raised up to
20ºC over a 12 h period. The reaction was quenched with sat. aq. NH4Cl (1 mL) and
diluted with Et2O (3 mL). Layers were separated and the org. phase was washed with
brine (40 mL) and dried (Na2SO4). The solvent was removed in vacuo and the crude
residue was purified by flash column chromatography (silica gel, EtAcO/Hexane 1 : 3) to
furnish the desired product 11. Colorless oil (78%). IR (film): 3390 (OH), 3082, 2940,
2843, 1715 (C=O side chain), 1705 (C=O), 1610 (C=C), 1220, 1130. 1H-NMR (CDCl3) ):
5.34 (t, 1H, J = 7.3, H-C=); 4.57 (d, 2H, J = 7.5, OCH2); 2.74 (s, 1H, OH); 2.55 (t, 2H,
J = 7.5, H-C(3)); 2.40 (t, 2H, J = 5.3, H-C(2´)); 2.11 (s, 3H, CH3C=O); 2.00-1.84 (m,
8H, H-C(1´), H-C(4), H-C(7), H-C(8)); 1.66 (s, 3H, CH3C=C), 1.62 (s, 3H, CH3C=C);
1.22 (s, 3H, CH3COH). 13C-NMR (CDCl3) 211.1 (C=O side chain); 209.3 (C=O); 165.2
(=C-O); 138.2 (=C(CH3)2); 117.5 (=C); 113.1 (C(5)); 79.2 (C(OH)); 68.1 (C(1)); 67.4
(C(allyl)); 39.7 (C(3)); 37.9 (C(2´)); 35.2 (C(7)); 30.8 (C(8)); 29.2 (CH3C=O); 27.5
(CH3COH); 25.7 (C(1´)); 25.6 ((CH3)2C=); 20.0 (C(4)); 17.9 ((CH3)2C=). ESI-HRMS:
calcd for (M+H+) C19H29O4 321.20659; found 321.20232.
ACKNOWLEDGEMENT
The authors are grateful to the National University of Rosario (UNR), and CONICET
(Consejo Nacional de Investigaciones Científicas y Técnicas) by received financial
support. P. S. F. thanks CONICET for a fellowship.
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12

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Scheme 1. Products derived from the Michael reaction of cyclohexane-1,3-dione in
solution or on solid support.
15

Scheme 2. Sequence of chemical reactions for the formation of 5.
16

Scheme 3. Introduction of the allylic function into compound 3
17

Scheme 4. Introduction of the prenyl function into compound 4. Preparation of compound
11.
18

Scheme 5. C- and O-alkylation of 5,5-dimethyl cyclohexane-1,3-dione with prenyl
bromide.
19

Figure 1. Prenylated acyl phloroglucinol derivatives and Synthetic bicyclic analogs of
[3.3.1] bridged phloroglucinol natural products from cyclohexane-1,3-diones.
20

Figure 2. NOE measurement of the compound 5.
21

Figure 3. NOE measurement of the compound 6.
22

Figure 4. A-E Microbiological assays on Mueller-Hinton agar. Bacterial inocula of E.
faecalis and E. coli cells were prepared by making direct saline suspensions of isolated
colonies selected from an 18- to 24-h nutritive agar plate. After that, 10-fold serial
dilutions for each microorganism were made. Next, 2 μL aliquots of E. faecalis (top line)
and E. coli cells (bottom line) corresponding to dilutions 108 to 104 CFU/mL were added
to the Mueller-Hinton agar without supplement (A) or supplemented with 1000 μg/mL of
compound 5 (B), 1000 μg/mL of compound 9 (C), 1000 μg/mL of compound 6 (D) and
1000 μg/mL of compound 11 (E). Developing colonies were observed after 24 h
incubation at 37ºC. In B-E, white circle indicates diminished growth of bacterial
suspensions.
23