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Article

Synthesis of Degraded Limonoid Analogs as New Antibacterial Scaffolds against Staphylococcus aureus

by
Marta Ferrera-Suanzes
1,†,
Victoria Prieto
2,†,
Antonio J. Medina-Olivera
1,
José Manuel Botubol-Ares
1,
Fátima Galán-Sánchez
2,3,
Manuel A. Rodríguez-Iglesias
2,3,
Rosario Hernández-Galán
1,3 and
María Jesús Durán-Peña
1,*
1
Department of Organic Chemistry, Faculty of Sciences, Campus Universitario Río San Pedro s/n, Torre Sur, 4a; planta, University of Cádiz, 11510 Puerto Real, 11009 Cádiz, Spain
2
Department of Biomedicine, Biotechnology and Public Health, Hospital Puerta del Mar, University of Cádiz, 11009 Cádiz, Spain
3
Instituto de investigación e Innovación Biomédica de Cádiz (INIBICA), 11009 Cádiz, Spain
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Antibiotics 2020, 9(8), 488; https://doi.org/10.3390/antibiotics9080488
Submission received: 30 June 2020 / Revised: 26 July 2020 / Accepted: 4 August 2020 / Published: 6 August 2020
(This article belongs to the Special Issue Natural Products and Their Derivatives with Antibacterial, Antioxidant and Anticancer Activities)
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Versions Notes

Abstract

:
Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) have become serious infections in humans and ruminants. S. aureus strains are showing rapid changes to develop resistance in traditional antibiotic-containing systems. In the continuous fierce fight against the emergent multi-drug resistant bacterial strains, straightforward and scalable synthetic procedures to produce new active molecules are in demand. Analysis of molecular properties points to degraded limonoids as promising candidates. In this article, we report a simple synthetic approach to obtain degraded limonoid analogs as scaffolds for new antibacterial molecules. The minimum inhibitory concentrations against S. aureus were evaluated for the stereoisomer mixtures by the broth microdilution method. Analysis of results showed that the acetylated derivatives were the most active of them all.

Graphical Abstract

1. Introduction

Multidrug resistance (MDR) is one of the emergent problems in healthcare in recent times due to the loss of effective activity of some drugs against multiresistant bacteria [1]. Infections caused by multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), linezolid-resistant Staphylococcus spp. or vancomycin-resistant Enterococcus faecium represent a growing worldwide issue [2,3]. Developing new antimicrobial agents is too slow versus the urgent need to combat bacterial pathogens [4]. To achieve a meaningful control of these multiresistant strains, that become serious infections in humans and ruminants [5,6], new approaches to obtain active molecules are in demand.
Due to the diverse biological activities showed by natural products (NPs), they comprise a rich resource for studies about structural complexity and functional group diversity [7,8]. For this reason, NPs are still a potent source of inspiration in drug discovery [9,10,11,12]. Among NPs, secondary metabolites from plants with terpene structures have proven numerous clinical trials and drugs [13]. Limonoids are tetranortriterpenoids isolated from Meliaceae and Rutaceae plants [14]. In literature, two limonoids are described to exhibit multiple-drug resistance activity against eight MDR bacterial strains including S. aureus strains (Figure 1, swietenolide and 2-hydroxy-3-O-tigloylswietenolide) [15].
Unfortunately, the availability of limonoids from natural sources is limited and their total syntheses are a challenge because they usually require a considerable number of reaction steps [16,17].
When designing new antibacterial drugs some aspects should be kept in mind such as avoiding too long multi-step total synthesis or semi-synthesis starting from NPs averting biodisponibility problems to contribute to the scalability of synthetically-accessible drug-like molecules, as well as foreseeing good drug-likeness molecular properties. Currently, some sectors of the pharmaceutical industry continue producing compounds that have suboptimal physicochemical profiles [18], decreasing the likelihood of success in terms of the development of these molecules as drugs [18,19]. Recent studies have been described to predict the bioavailability of small molecules about physicochemical properties involve in the drug-likeness characteristics [20,21]. The well-known Lipinski’s rules—also named as the “rule of five”—has gained acceptance as an approach to design, discover, and develop new bioactive molecules [19]. Nevertheless, these properties and others have been analyzed or matched up in other approaches, which provide required parameters for drugability. The application of all these guidelines linked to the concept of drug-likeness could contribute to drawing up a preliminary analysis of the molecules to be considered as drug-like compounds based on these predictions.

2. Results

2.1. Design of Model Molecules Based on Degraded Limonoid Skeleton and Molecular Properties Prediction

Phytochemical studies have revealed a great variety of pharmacological activities [22,23,24] as well as health-promoting and disease-preventing properties exhibited by limonoids [25,26].
For designing new antibacterial compounds based on limonoid structures, we first evaluated the molecular properties of swietenolide and 2-hydroxy-3-O-tigloylswietenolide. The freely available ADMETlab database website was employed to predict the administration, distribution, metabolism, excretion, and toxicity properties (ADMET) from these chemical structures [27,28]. We analyzed a set of physicochemical descriptors for swietenolide and 2-hydroxy-3-O-tigloylswietenolide according to Lipinski’s rules [29,30], Ghose’s rules [31], Veber’s rules [32], Varma’s rules [33], and Oprea’s rules [34] that attempt to make predictions of drug-likeness (Table S1). With this aim, the range of drug-like parameters according to 11 molecular properties were studied (Table 1).
As opposed to these complex biologically-active limonoids, their simpler related degraded limonoids have also shown promising biological effects such as anticancer [35], antifungal [36], neuroprotective [37], anti-trypanosomal [38], and antibacterial activities [39].
A common feature in the different limonoid families and the degraded limonoids is the main motif δ-lactone with a 3-furyl substituent (Figure 2).
Based on the purpose of designing new active and non-complex molecules—avoiding unnecessary ornaments in the structures—in this study we proposed to use a degraded limonoid skeleton as a scaffold for the synthesis of new antibacterial agents.
Theoretical molecular properties were calculated to predict if some representative degraded limonoids (Table S2) could match the required parameters for drugability (Table 2). Some of these metabolites show relevant pharmacological or agrochemical effects (Figure 3) [40,41,42,43].
On the other hand, some natural phragmalin-type limonoids possess the D-ring δ-lactone demolished such as swiemahogins A and B [44], or chukvelutides [45] (Figure 4).
With all these above considerations, the degraded limonoid analogs 1–3 were proposed to be synthetized as model molecules (Scheme 1). The previous phragmalin-type limonoids share a common pattern with 1−hydroxyl group on the carbon connected to furanyl group−or 2−acetyl group on the carbon connected to furanyl group−, respectively. We proposed the synthesis of compounds 1–3 to evaluate if these modifications in the D ring could induce different results.
Physicochemical descriptors for the model compounds 1–3 were also calculated (Table S2) and they were compared with those belonging to the MDR limonoids swietenolide and its derivative 2-hydroxy-3-O-tigloylswietenolide.

2.2. Synthesis of the Degraded Limonoid Analogs 1–3 and Chemical Characterization

Following the strategy used for the synthesis of some limonoids described in the literature [46,47], treatment of cyclohex-2-en-1-one with the sterically hindered base lithium diisopropylamide (LDA) in THF at −50 °C and subsequent condensation of its lithium enolate with the commercially available 3-furaldehyde afforded the mixture of stereoisomers (±)-1a and (±)-1b in 69% yield. Then, these β-hydroxy ketones (±)-1a and (±)-1b were acetylated under standard conditions—acetic anhydride and a catalytic amount of pyridine—to give quantitatively the compounds (±)-2a and (±)-2b. At this point, the mixture of (±)-2a and (±)-2b was subjected to an intramolecular aldol reaction employing LDA in THF at −50 °C to afford the δ-valerolactone (±)-3a in 7% yield as an isolated product. It is remarkable that in this cyclization reaction one diastereoisomer out of four possible could be only purified by chromatography column using mixtures of hexane:ethyl acetate in different proportions possibly due to the reaction crude was difficult to solubilize (Scheme 2).
Compounds 1 and 2 have two stereocenters. Therefore, these mixtures are made up of four isomers, which are two diastereoisomers and two enantiomer pairs. The structures of the synthesized compounds 1–3 were characterized by mass spectrometry, 1H NMR, 13C NMR, and Fourier transform IR (FT-IR) spectroscopy.
Furthermore, in the NMR spectra of compound (±)-3a there were nuclear Overhauser effect (n.O.e.) correlations between H-8a and H-1 that supported the relative configuration of the ring as 1S*,8aS* (Figure 5).

2.3. Antimicrobial Activity of the Degraded Limonoid Analogs 1–3

The minimal inhibitory concentration (MIC) of degraded limonoid analogs 1–3 was determined on 96 well culture plates for reference strain S. aureus ATCC 25923 and a set of methicillin-resistant S. aureus clinical isolates by using microdilution assay and a microorganism suspension (Table 3).
Then, we evaluated the behavior of stereoisomer mixture 2 ((±)-2a and (±)-2b) against other Gram-positive bacteria (Table 4).

2.4. Time–Kill Curves

Using time course assays, time-kill curve assays were performed and we examined the bactericidal activity of the stereoisomers 2 ((±)-2a and (±)-2-b) against S. aureus ATCC 25923 and MRSA 18032913 (Figure 6).
Stereoisomers 2 ((±)-2a and (±)-2b) presented bactericidal effects against S. aureus at 64 and 32 mg/L after 4 h. These effects are decreasing whenever the time is increasing. These reductions did not persist noteworthy at 24 h.
In the same way, 2 presented bactericidal effects against MRSA 18032913 showing a significant decrease in the first 8–10 h at both concentrations tested. After 24 h, the difference in bactericidal concentration became clearer than in previous measurements at 128 mg/L whereas at 64 mg/L the inhibition is slightly lower than the control measurement.

2.5. Synthesis and Antimicrobial Activity of More Lipophilic Degraded-Limonoid Analogs 4–6 and Chemical Characterization

The procedures carried out to synthetize the more lipophilic derivatives 4–6 by Steglich esterification and O-acylation reactions are shown on Scheme 3.
Further purification by high-performance liquid chromatography (HPLC) or chromatography column afforded compounds 4–6 as single diastereomers and the two series were labeled as “a” for the less polar enantiomer pair and ‘b’ for more polar enantiomer pair according to the TLC plates using 25% hexane:ethyl acetate as eluent. The structures of the diastereoisomers of furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl butyrate ((±)-4a and (±)-4b), furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl hexanoate ((±)-5a, (±)-5b), and furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl decanoate ((±)-6a and (±)-6b) were separately characterized by mass spectrometry, 1H NMR, 13C NMR and FT-IR spectroscopy.
Diastereoisomers (±)-4a-(±)-6a and (±)-4b-(±)-6b were tested against S. aureus ATCC 25923. The MIC50 values are presented in Table 5. These data showed that these compounds are all less active than mixture (±)-2a and (±)-2b (MIC50 = 16 mg/L).

3. Discussion

Oral bioavailability is correlated to Lipinski’s rules in terms of the number of hydrogen bond donors (≤5), the number of hydrogen bond acceptors (≤10), molecular weight (≤500 g/mol), and log P (≤5). According to these data, swietenolide falls within the typical Lipinski’s rules values giving 100% matches (Table S1), whereas its derivative 2-hydroxy-3-O-tigloylswietenolide only matches in 75% due to the molecular weight of this natural product is higher than 500 g/mol (Table S1). Unexpectedly, these naturally-occurring limonoids do not perfectly conform to all the remaining rules except in Veber’s rules for swietenolide. Swietenolide has four rotatable bonds (less than six) reducing it matches coincidence with Oprea´s rules (Table S1). 2-Hydroxy-3-O-tigloylswietenolide does not fit in Ghose´s rules due to the molar refractivity, the total number of atoms and molecular weight are all higher than the typical values for drugability (Table S1). The total polar surface area (TPSA)—a parameter included in both Veber and Varma´s rules—is used as a tool to estimate the transport pathway of the particular type of drug [48]. The TPSA value for 2-hydroxy-3-O-tigloylswietenolide is 149.57 Å, slightly higher than 140 Å—upper in Veber´s rules—and much higher than Varma´s predictions (TPSA ≤120 Å) (Table S1). According to Varma´s rules, this molecule is in concordance with just 40% with these rules (Table S1).
Despite the promising bacterial inhibition showed by swietenolide and its derivative, it would be desirable to improve the molecular properties as a key aspect to design new antibacterial compounds. Getting better molecular properties might increase the likelihood of success when producing a drug with good pharmacokinetic and pharmacodynamics parameters. Calculations of the physicochemical descriptors included in the mentioned rules for naturally degraded limonoids—dictamdiol, calodendrolide, fraxinellone, 8,14-epoxyfraxinellone, and melazolide A—resulted in excellent properties to be considered as interesting biologically-active structures (100% matches in Lipinski, Ghose, Veber, and Varma’s rules, although lower percentage matches are predicted for Oprea´s rules) (Table S2).
Based on the favorable properties calculated for degraded limonoids and to synthesize model molecules characterized by great structural simplicity, we chose degraded limonoids for the design of new antibiotics with simple structures, easy to synthesize and that would preserve those properties. Therefore, after a careful analysis of their drug-like properties, the synthesis of the degraded limonoid analogs 1–3 was proposed (Table S2). It was expected to anticipate good biological properties for 1–3 because most of the properties included in the rules for prediction of drug-like characteristics overlap for these model compounds (100% in 4 out of the 5 sets of rules, the same matches in the drugability rules exhibited by naturally degraded limonoids). Thus, in theory, they could be biologically relevant compounds. Furthermore, the physicochemical descriptors for compounds 1–3 seem to predict better ADMET characteristics in comparison to those expected for swietenolide and its derivative 2-hydroxy-3-O-tigloylswietenolide, which may be remarkable for preparing new drugs.
These model molecules 1-3 were synthetized and tested against S. aureus ATCC 25923 and MRSA strains. Assays did not show the antibacterial activity of (±)-3a against S. aureus and MRSA strains. The free hydroxyl-containing stereoisomers (±)-1a and (±)-1b displayed low activity against all the tested bacteria (MIC50 = 512 mg/L) while acetate derivatives (±)-2a and (±)-2b were more active showing MIC50 = 16 mg/L against S. aureus and values below 100 mg/L against MRSA 15019301 and MRSA 18032913. Stereoisomer mixture 2 was the most active of them. Because of this reason, it was also tested against other Gram-positive bacteria (Table 4). Once again, this mixture exhibit antibacterial effect against linezolid-resistant Staphylococcus epidermidis clinical isolates (MIC50 = 64 mg/L) and lower activity against Listeria monocytogenes (MIC50 = 128 mg/L), Enterococcus faecalis ATCC 29212 (MIC50 = 256 mg/L), and Enterococcus spp. clinical isolates (daptomycine-resistant E. faecalis and vancomycin-resistant E. faecium, showing both MIC50 = 256 mg/L).
These results encouraged us to design derivatives that could be more active, and lipophilicity was considered as a key descriptor. Some authors suggest that lipophilicity is one of the most important molecular properties to be considered on decision-making in medicinal chemistry [19], and certain studies pointed out that high hydrophilicity decreases the biological activity of certain natural products and, as a consequence, higher lipophilicity, a higher activity [12]. Lipophilicity is represented by the descriptors partition coefficient (log P)—which is often used in the analysis of structure-activity relationships (SAR and QSAR) [49,50,51,52]—and distribution coefficient (log D) that is considered as the most impactful parameter by some authors, rather than log P [52].
In the preliminary antibacterial assays, stereroisomer mixture of 2 exhibited much lower MIC50 value (16 mg/L) than the hydroxylated compounds 1 (512 mg/L) against S. aureus ATCC 25923. The acetylation of the free hydroxyl group of 1 to produce 2 involves a higher lipophilicity for 2.
Considering the previous, we proposed to introduce longer side-chains in the ester group to assess if the antibacterial activity could be positively affected. Starting from the mixture (±)-1a and (±)-1b, a set of esters containing butanoate ((±)-4a and (±)-4b), hexanoate ((±)-5a and (±)-5b)), and decanoate chains ((±)-6a and (±)-6b)) (Scheme 3) were proposed to be synthetized for obtaining a gradual increase of lipophilicity (Table 6) at the same time they conform in the druggability rules previously mentioned (Table S2).
Curiously—contrary to our hypothesis—the higher lipophilic character of molecules 4–6 respect to the previous molecules assayed (1-2), decreased the antibacterial activity against S. aureus ATCC 25923. For compounds 4–6 evidenced that the less polar enantiomer pairs a ((±)-4a-(±)-6a) were notably less active than enantiomeric pairs b ((±)-4b-(±)-6b) (Table 5). Stereochemical factors could play a key role in the inhibition process.
The results demonstrated that amongst the analyzed compounds 1–6, acetylated mixture 2 was the most active of them all against S. aureus ATCC 25923 and the acetate derivatives 2 could be further explored to develop new antimicrobial drugs to combat S. aureus bacterial infections.

4. Materials and Methods

4.1. General

Unless otherwise noted, materials and reagents were obtained from commercial suppliers and were used without further purification. Infrared spectra were recorded on an FT-IR spectrophotometer and reported as the wave number (cm−1). 1H and 13C NMR measurements were recorded on Agilent 500 MHz NMR spectrometer with SiMe4 as the internal reference. Chemical shifts were referenced to CDCl3 (δH 7.25, δC 77.0). NMR assignments were made using a combination of 1D and 2D techniques. High-resolution mass spectroscopy (HRMS) was performed in a QTOF mass spectrometer in the positive ion ESI mode. Purification by analytical HPLC was performed with a Hitachi/Merck L-6270 apparatus equipped with a differential refractometer detector (RI-7490). A LiChrospher® Si gel 60 (10 µm) LiChroCart® (250 mm × 4 mm) were used in isolation experiments. Silica gel (Merck) was used for column chromatography. TLC was performed on Merck Kiesegel 60 F254, 0.25 mm thick.

4.2. Reaction Procedures

4.2.1. Preparation of 6-(furan-3-yl-(hydroxy)methyl)cyclohex-2-en-1-one (Mixture of Stereoisomers (±)-1a and (±)-1b)

A solution of n-BuLi (1.6 M in hexane, 11.4 mL, 18.2 mmol) was added dropwise at –50 °C to a solution of N,N-diisopropylamine (2.6 mL, 19.08 mmol) in dry THF (22 mL) under an argon atmosphere to prepare lithium diisopropylamide (LDA) in situ. Then cyclohex-2-en-1-one (1 g, 10.4 mmol) was added dropwise and, 3-furaldehyde (0.92 mL, 10.40 mmol) resulting in a pale yellow solution. The mixture was stirred for 3 h at –50 °C, and then was allowed to warm to room temperature. Then, water was added (10 mL), the layers were separated and the aqueous layer was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate. Filtration and evaporation of the solvent under reduced pressure yielded the crude material that was purified by silica gel chromatography to give (±)-1a and (±)-1b (1.37 g, 69% yield).
6-(furan-3-yl-(hydroxy)methyl)cyclohex-2-en-1-one (mixture of stereoisomers (±)-1a and (±)-1b). Amorphous solid. IR (KBr) νmáx (cm−1): 3439 (ν O-H), 2929 (ν C-H), 1667 (ν C=O (α,β-unsaturated ketone)),1160, 1024, 874, 602; 1H NMR δ (CDCl3, 500 MHz): 7.41–7.36 (4H, m, H-2′ and H-5′), 7.05–6.97 (2H, m, H-3), 6.42 (1H, dd, J = 1.9, 0.9 Hz, H-4′), 6.29 (1H, t, J = 1.4 Hz, H-4′), 6.08–6.02 (2H, m, H-2), 5.28 (1H, dd, J = 6.4, 3.2 Hz, CHOH), 4.89 (1H, dd, J = 8.5, 1.9 Hz, CHOH), 4.55 (d, J = 1.9 Hz, OH), 3.20 (d, J = 6.4 Hz, OH), 2.70–2.62 (1H, m, H-6), 2.55 (1H, ddd, J = 13.5, 8.5, 4.7 Hz, H-6), 2.49–2.34 (4H, m, H-4), 1.93–1.85 (2H, m, H-5), 1.84–1.75 (1H, m, H-5a), 1.63–1.51 (1H, m, H-5b); 13C NMR δ (CDCl3, 125 MHz): 203.3 (s, C-1), 201.3 (s, C-1), 151.6 (d, C-3), 151.3 (d, C-3), 143.4 (d, C-5′), 143.1 (d, C-5′), 140.2 (d, C-2′), 139.7 (d, C-2′), 130.0 (d, C-2), 129.6 (d, C-2), 126.1 (s, C-3′), 125.6 (s, C-3′), 108.8 (d, C-4′), 108.6 (d, C-4′), 67.7 (d, CHOH), 66.5 (d, CHOH), 52.0 (d, C-6), 51.9 (d, C-6), 25.9 (t, C-4), 25.8 (t, C-4), 25.3 (t, C-5), 22.8 (t, C-5); HRMS (ESI+): calcd for C11H13O3 [M+H]+ 193.0865, found 193.0846.

4.2.2. Preparation of furan-3-yl-(2-oxocyclohex-3-en-1-yl)-methyl acetate (Mixture of Stereoisomers (±)-2a and (±)-2b)

Pyridine (2 drops) was added to a solution of the mixture of stereoisomers (±)-1a and (±)-1b (100 mg, 0.52 mmol) in acetic anhydride (0.5 mL) at room temperature for 18 h. Then, cyclohexane was added (2 mL) and the solvent was evaporated under reduced pressure. This procedure was repeated three times to give (±)-2a and (±)-2b in >99% yield.
Furan-3-yl-(2-oxocyclohex-3-en-1-yl)-methyl acetate (mixture of stereoisomers (±)-2a and (±)-2b). Yellow oil. IR (KBr) νmáx (cm−1): 2938 (ν C-H), 1745 (ν C=O, aliphatic ketone), 1675 (ν C=O (α,β-unsaturated ketone)), 1239, 1025, 875, 602; 1H NMR δ (CDCl3, 500 MHz): 7.40–7.32 (4H, m, H-2′ and H-5′), 6.96 (1H, dddd, J = 9.6, 5.2, 2.9, 0.9 Hz, H-4″), 6.90 (1H, dddd, J = 10.0, 4.6, 3.3, 0.9 Hz, H-4″), 6.46 (1H, d, J = 4.2 Hz, CHOCO), 6.44 (1H, d, J = 5.8 Hz, CHOCO), 6.33 (1H, dd, J = 1.8, 0.8 Hz, H-4′), 6.31 (1H, brs, H-4′), 6.03 (1H, ddd, J = 9.6, 2.7, 1.4 Hz, H-3″), 5.97 (1H, dt, J = 10.0, 1.9 Hz, H-3″), 2.86 (1H, ddd, J = 11.6, 5.8, 4.6 Hz, H-1″), 2.63 (1H, dt, J = 11.7, 4.2 Hz, H-1″), 2.51–2.29 (4H, m, H-5″), 2.13–2.06 (2H, m, H-6″a), 2.05 (3H, s, H-2), 2.01 (3H, s, H-2), 1.76–1.64 (2H, m, H-6″b); 13C NMR δ (CDCl3, 125 MHz): 197.8 (s, C-2″), 197.1 (s, C-2″), 169.7 (s, C-1), 166.4 (s, C-1), 150.1 (d, C-4″), 149.7 (d, C-4″), 143.2 (d, C-5′), 142.9 (d, C-5′), 140.6 (d, C-2′), 139.8 (d, C-2′), 129.8 (d, C-3″), 129.6 (d, C-3″), 123.7 (s, C-3′), 122.2 (s, C-3′), 109.3 (d, C-4′), 108.9 (d, C-4′), 67.5 (d, CHOCO), 66.4 (d, CHOCO), 51.0 (d, C-1″), 50.6 (d, C-1″), 25.3 (t, C-5″), 24.9 (t, C-5″), 23.5 (t, C-6″), 22.7 (t, C-6″), 21.1 (q, C-2), 20.9 (q, C-2); HRMS (ESI+): calcd for C13H14O4Na [M+Na]+ 257.0790, found 257.0790.

4.2.3. Preparation of (1S*,8aS*)-1-(furan-3-yl)-4a-hydroxy-1,4,4a,7,8,8a-hexahydro-3H-isocromen-3-one ((±)-3a)

A solution of the stereoisomer mixture (±)-2a and (±)-2b (50 mg, 0.22 mmol) in dry THF (0.6 mL) was added dropwise to the LDA solution (0.26 mmol) at –50 °C. The reaction mixture was stirred at –50 °C for 4 h, and then saturated aqueous NH4CI (5 mL) was added and the mixture was stirred and gradually warmed to room temperature. The layers were separated and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate. Filtration and evaporation of the solvent under reduced pressure yielded the crude material that was purified by silica gel chromatography to give (±)-3a (3.5 mg, 7% yield).
(1S*,8aS*)-1-(furan-3-yl)-4a-hydroxy-1,4,4a,7,8,8a-hexahydro-3H-isochromen-3-one ((±)-3a). Yellow oil. IR (KBr) νmáx (cm−1): 3419 (ν O-H), 2933 (ν C-H), 1732 (ν C=O, aliphatic ketone), 1022, 875, 755, 603; 1H NMR δ (CDCl3, 500 MHz): 7.50 (brs, H-2′, 1H), 7.45 (1H, d, J = 1.8 Hz, H-5′), 6.50 (1H, dd, J = 1.8, 0.9 Hz, H-4′), 5.99 (1H, dt, J = 9.9, 3.9 Hz, H-6), 5.68 (1H, dt, J = 9.9, 2.1 Hz, H-5), 4.94 (1H, d, J = 10.0 Hz, H-1), 2.77 (1H, d, J = 16.0 Hz, H-4α),† 2.72 (1H, d, J = 16.0 Hz, H-4β),† 2.26 (1H, ddd, J = 10,0, 7.9, 4.4 Hz, H-8a), 2.07–2.00 (2H, m, H-7), 1.87 (1H, ddt, J = 13.5, 6.3, 4.4 Hz, H-8α),‡ 1.44 (1H, ddt, J = 13.5, 7.9, 5.9 Hz, H-8β)‡.†,‡Interchangeable signals; 13C NMR δ (CDCl3, 125 MHz): 170.2 (s, C-3), 144.0 (d, C-5′), 140.8 (d, C-2′), 131.4 (d, C-6), 130.3 (d, C-5), 123.1 (s, C-3′), 108.7 (d, C-4′), 75.2 (d, C-1), 68.6 (s, C-4a), 45.9 (d, C-8a), 42.9 (t, C-4), 22.6 (t, C-8), 22.1 (t, C-7); HRMS (ESI+): calcd for C13H14O4Na [M+Na]+ 257.0790, found 257.0794.

4.2.4. General Procedure for Steglich Esterification. Preparation of (±)-4a, (±)-4b, (±)-5a and (±)-5b

A mixture of 6-(furan-3-yl-(hydroxy)methyl)cyclohex-2-en-1-one (mixture of stereoisomers (±)-1a and (±)-1b) (100 mg, 0.43 mmol), the corresponding carboxylic acid (butanoic acid (0.06 mL; 0.64 mmol) or hexanoic acid (0.08 mL; 0.64 mmol)) and dimethylaminopyridine (DMAP) (10.50 mg, 0.09 mmol) in dry CH2Cl2 (6.5 mL) was stirred for 10 min. To the resulting mixture was added N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) (98 mg, 0.52 mmol). After stirring for 18 h, the reaction mixture was evaporated and then added Et2O (10 mL). The crude was sequentially washed with H2O (10 mL), twice with saturated sodium bicarbonate solution (10 mL), H2O (10 mL), dried over anhydrous sodium sulphate, filtered and evaporated under reduced pressure to afford the stereoisomer mixture (±)-4a-(±)-4b, and (±)-5a-(±)-5b. The crude material of 4 was purified by HPLC to give (±)-4a and (±)-4b as single diastereoisomers (27.3 mg, 20% yield). The crude material of 5 was purified by silica gel chromatography to yield (±)-5a and (±)-5b as single diastereoisomers (84.5 mg, 56% yield).
Furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl butyrate (diastereoisomer (±)-4a). (13.4 mg, 10.2% yield of isolated product). Colorless oil. tR = 12 min, petroleum ether: ethyl acetate (80: 20), flow = 1.0 mL.min−1; Rf: 0.55 (petroleum ether: AcOEt (3: 1)); IR (KBr) νmáx (cm−1): 2964 (ν C-H), 2933 (ν C-H), 1738 (ν C=O, aliphatic ketone), 1677 (ν C=O (α,β-unsaturated ketone)), 1168, 1025, 875, 601; 1H NMR δ (CDCl3, 500 MHz): 7.38 (1H, dt, J = 1.6, 0.8 Hz, H-2′), 7.34 (1H, t, J = 1.6 Hz, H-5′), 6.94–6.88 (1H, m, H-4″), 6.46 (1H, d, J = 5.7 Hz, CHOCO), 6.34 (1H, brs, H-4′), 5.98 (1H, ddd, J = 10.1, 2.4, 1.6 Hz, H-3″), 2.87 (1H, ddd, J = 11.6, 5.7, 4.6 Hz, H-1″), 2.40–2.34 (2H, m, H-5″), 2.29 (2H, t, J = 7.4 Hz, H-2), 2.13–2.05 (2H, m, H-6″), 1.65 (2H, sext, J = 7.4 Hz, H-3), 0.93 (3H, t, J = 7.4 Hz, H-4); 13C NMR δ (CDCl3, 125 MHz): 197.8 (s, C-2″), 172.4 (s, C-1), 149.6 (d, C-4″), 142.9 (d, C-5′), 140.6 (d, C-2′), 129.7 (d, C-3″), 122.3 (s, C-3′), 109.4 (d, C-4′), 67.3 (d, CHOCO), 50.7 (d, C-1″), 36.4 (t, C-2), 24.9 (t, C-5″), 23.5 (t, C-6″), 18.4 (t, C-3), 13.6 (q, C-4); M.S. (E.I.) m/z (relative intensity): 191.1 (28.0), 174.1 (28.5), 123.1 (32.3), 96.1 (64.8), 95.0 (100), 77.7 (45.7), 71.1 (48.3),68.1 (30.1), 67.2 (42.2), 43.2 (98.7), 41.2 (64.3); HRMS (ESI+): calcd for C15H18O4Na [M+Na]+ 285.1103, found 285.1101.
Furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl butyrate (diastereoisomer (±)-4b). (13.9 mg, 9.8% yield of isolated product). Colorless oil. tR = 14 min, petroleum ether: ethyl acetate (80: 20), flow = 1.0 mL.min−1; Rf: 0.48 (petroleum ether: AcOEt (3: 1)); IR (KBr) νmáx (cm−1): 2964 (ν C-H), 2933 (ν C-H), 1738 (ν C=O, aliphatic ketone), 1677 (ν C=O (α,β-unsaturated ketone)), 1168, 1025, 875, 601; 1H NMR δ (CDCl3, 500 MHz): 7.36 (1H, t, J = 1.8 Hz, H-5′), 7.33 (1H, dt, J = 1.8, 0.9 Hz, H-2′), 7.00–6.94 (1H, m, H-4″), 6.48 (1H, d, J = 4.2 Hz, CHOCO), 6.32 (1H, dd, J = 1.8, 0.9 Hz, H-4′), 6.03 (1H, ddd, J = 10.0, 2.6, 1.4 Hz, H-3″), 2.64 (1H, dt, J = 11.6, 4.2 Hz, H-1″), 2.52–2.43 (1H, m, H-5″a), 2.41–2.30 (1H, m, H-5″b), 2.26 (2H, t, J = 7.4 Hz, H-2), 2.15–2.05 (2H, m, H-6″), 1.62 (2H, sext, J = 7.4 Hz, H-3), 0.86 (3H, t, J = 7.4 Hz, H-4); 13C NMR δ (CDCl3, 125 MHz): 197.0 (s, C-2″), 172.2 (s, C-1), 150.0 (d, C-4″), 143.2 (d, C-5′), 139.8 (d, C-2′), 129.9 (d, C-3″), 123.9 (s, C-3′), 109.0 (d, C-4′), 66.2 (d, CHOCO), 51.1 (d, C-1″), 36.2 (t, C-2), 25.3 (t, C-5″), 22.8 (t, C-6″), 18.5 (t, C-3), 13.6 (q, C-4); M.S. (E.I.) m/z (relative intensity): 191.1 (28.5), 174.1 (30.9), 96.1 (58.8), 95.0 (86.5), 77.1 (29.3), 71.1 (47.0), 65.4 (33.6), 43.2 (98.8), 41.3 (100); HRMS (ESI+): calcd for C15H18O4Na [M+Na]+ 285.1103, found 285.1101.
Furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl hexanoate (diastereoisomer (±)-5a). (43.2 mg, 28.6% yield of isolated product). Colorless oil. Rf: 0.67 (petroleum ether: AcOEt (3: 1)); IR (KBr) νmáx (cm−1): 2956 (ν C-H), 2932 (ν C-H), 1738 (ν C=O, aliphatic ketone), 1676 (ν C=O (α,β-unsaturated ketone)), 1163, 1023, 874, 602; 1H NMR δ (CDCl3, 500 MHz): 7.38 (1H, dt, J = 1.6, 0.8 Hz, H-5′), 7.34 (1H, m, H-2′), 6.91 (1H, dddd, J = 10.0, 4.6, 3.2, 1.0 Hz, H-4″), 6.46 (1H, d, J = 5.7 Hz, CHOCO), 6.36–6.31 (1H, m, H-4′), 5.97 (1H, ddd, J = 10.0, 2.3, 1.4 Hz, H-3″), 2.87 (1H, ddd, J = 11.6, 5.7, 4.5 Hz, H-1″), 2.40–2.34 (2H, m, H-5″), 2.33–2.29 (2H, m, H-2), 2.13–2.05 (2H, m, H-6″), 1.65–1.56 (2H, m, H-3), 1.32–1.26 (4H, m, H-4 and H-5), 0.91 (3H, t, J = 7.4 Hz, H-6). 13C NMR δ (CDCl3, 125 MHz): 197.8 (s, C-2″), 172.6 (s, C-1), 149.6 (d, C-4″), 142.9 (d, C-5′), 140.6 (d, C-2′), 129.7 (d, C-3″), 122.3 (s, C-3′), 109,4 (d, C-4′), 67.3 (d, CHOCO), 50.7 (d, C-1″), 34.4 (t, C-2), 31.2 (t, C-4),¥, 24.9 (t, C-5″), 24.6 (t, C-3), 23.5 (t, C-6″), 22.3 (t, C-5),¥, 13.9 (q, C-6) ¥Interchangeable signals; M.S. (E.I.) m/z (relative intensity): 191.1 (54.0), 174.1 (56.0), 123.1 (35.5), 96.1 (71.8), 95.0 (100), 71.2 (43.3), 43.2 (63.4), 41.2 (27.3); HRMS (ESI+): calcd for C17H22O4Na [M+Na]+ 313.1416, found 313.1417.
Furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl hexanoate (diastereoisomer (±)-5b). (41.3 mg, 27.4% yield of isolated product). Colorless oil. Rf: 0.58 (petroleum ether: AcOEt (3: 1)); IR (KBr) νmáx (cm−1): 2956 (ν C-H), 2932 (ν C-H), 1738 (ν C=O, aliphatic ketone), 1676 (ν C=O (α,β-unsaturated ketone)), 1163, 1023, 874, 602; 1H NMR δ (CDCl3, 500 MHz): 7.36 (1H, d, J = 1.8 Hz, H-5′), 7.33 (1H, d, J = 0.8 Hz, H-2′), 7.00–6.94 (1H, m, H-4″), 6.48 (1H, d, J = 4.3 Hz, CHOCO), 6.32 (1H, dd, J = 1.8, 0.8 Hz, H-4′), 6.05–6.01 (1H, m, H-3″), 2.64 (1H, dt, J = 11.6, 4.3 Hz, H-1″), 2.53–2.43 (1H, m, H-5″), 2.40–2.30 (1H, m, H-5″), 2.27 (2H, t, J = 7.4 Hz, H-2), 2.15–2.00 (2H, m, H-6″), 1.60 (2H, quint, J = 7.4 Hz, H-3), 1.32–1.20 (4H, m, H-4 and H-5), 0.87 (3H, t, J = 7.4 Hz, H-6). 13C NMR δ (CDCl3, 125 MHz): 197.0 (s, C-2″), 172.4 (s, C-1), 150.0 (d, C-4″), 143.2 (d, C-5′), 139.8 (d, C-2′), 129.9 (d, C-3″), 123.9 (s, C-3′), 109.0 (d, C-4′), 66.2 (d, CHOCO), 51.1 (d, C-1″), 34.3 (t, C-2), 31.2 (t, C-4),¥ 25.3 (t, C-5″), 24.6 (t, C-3), 22.8 (t, C-6″), 22.3 (t, C-5),¥ 13.9 (q, C-6) ¥Interchangeable signals; M.S. (E.I.) m/z (relative intensity): 191.1 (43.9), 174.1 (49.5), 123.1 (32.9), 96.1 (58.1), 95.0 (100), 71.2 (37.1), 43.2 (56.3), 41.2 (26.0); HRMS (ESI+): calcd for C17H22O4Na [M+Na]+ 313.1416, found 313.1417.

4.2.5. Preparation of (±)-6a and (±)-6b

Triethylamine (0.18 mL; 1.29 mmol) was added to a solution of a mixture of 6-(furan-3-yl-(hydroxy)methyl)cyclohex-2-en-1-one (mixture of stereoisomers (±)-1a and (±)-1b) (100 mg, 0.43 mmol) in dry CH2Cl2 (5 mL). The reaction mixture was stirred at room temperature for 15 min. To the resulting mixture was added decanoyl chloride (0.14 mL, 0.64 mmol). After stirring for 18 h, H2O (5 mL) was added to the reaction mixture. The crude was sequentially washed with H2O (10 mL), twice with brine (10 mL), H2O (10 mL), dried over anhydrous sodium sulphate, filtered and evaporated under reduced pressure to afford the stereoisomer mixture (±)-6a and (±)-6b. The crude material was purified by silica gel chromatography to yield (±)-6a and (±)-6b as single diastereoisomers (79.3 mg, 44% yield).
Furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl decanoate (diastereoisomer (±)-6a). (41.3 mg, 22.9% yield of isolated product). Colorless oil. Rf: 0.44 (petroleum ether: AcOEt (9: 1)); IR (KBr) νmáx (cm−1): 2953 (ν C-H), 2925 (ν C-H), 1743 (ν C=O, aliphatic ketone), 1681 (ν C=O (α,β-unsaturated ketone)), 1161, 1026, 875, 723, 602; 1H NMR δ (CDCl3, 500 MHz): 7.38 (1H, dt, J = 1.7, 0.8 Hz, H-2′), 7.33 (1H, t, J = 1.7 Hz, H-5′), 6.91 (1H, dddd, J = 10.1, 4.6, 3.3, 1.0 Hz, H-4″), 6.46 (1H, d, J = 5.7 Hz, CHOCO), 6.33 (1H, dd, J = 1.7, 0.8 Hz, H-4′), 5.98 (1H, ddd, J = 10.1, 2.4, 1.6 Hz, H-3″), 2.87 (1H, ddd, J = 11.7, 5.7, 4.5 Hz, H-1″), 2.41–2.33 (1H, m, H-5″a), 2.33–2.29 (2H, m, H-2), 2.15–2.04 (1H, m, H-6″a), 1.76–1.66 (3H, m, H-3 and H-6″b), 1.65–1.56 (1H, m, H-5″b), 1.31–1.21 (12H, m, H-4, H-5, H-6, H-7, H-8 and H-9), 0.87 (3H, t, J = 6.8 Hz, H-10); 13C NMR δ (CDCl3, 125 MHz): 197.7 (s, C-2″), 172.6 (s, C-1), 149.6 (d, C-4″), 142.9 (d, C-5′), 140.6 (d, C-2′), 129.7 (d, C-3″), 122.3 (s, C-3′), 109.4 (d, C-4′), 67.3 (d, CHOCO), 50.7 (d, C-1″), 34.5 (t, C-2), 31.8 (t, C-4),¥ 29.4 (t, C-5),¥ 29.2 (t, C-6 and C-7, 2C),¥ 29.1 (t, C-8),¥ 24.9 (t, C-3 and C-5″, 2C), 23.5 (t, C-6″), 22.7 (t, C-9),¥ 14.1 (q, C-10).¥Interchangeable signals; M.S. (E.I.) m/z (relative intensity): 191.1 (52.2), 174.1 (44.6), 123.1 (43.5), 96.1 (64.6), 95.1 (100), 57.2 (36.9), 55.1 (43.6), 43.2 (65.7), 41.2 (65.2); HRMS (ESI+): calcd for C21H30O4Na [M+Na]+ 369.2042, found 369.2053.
Furan-3-yl-(2-oxocyclohex-3-en-1-yl)methyl decanoate (diastereoisomer (±)-6b). (38.0 mg, 21.1% yield of isolated product). Colorless oil. Rf: 0.36 (petroleum ether: AcOEt (9: 1)); IR (KBr) νmáx (cm−1): 2953 (ν C-H), 2925 (ν C-H), 1743 (ν C=O, aliphatic ketone), 1681 (ν C=O (α,β-unsaturated ketone)), 1161, 1026, 875, 723, 602; 1H NMR δ (CDCl3, 500 MHz): 7.36 (1H, t, J = 1.6 Hz, H-5′), 7.33 (1H, dd, J = 1.6, 0.9 Hz, H-2′), 6.97 (1H, ddd, J = 10.0, 5.0, 2.7 Hz, H-4″), 6.47 (1H, d, J = 4.2 Hz, CHOCO), 6.32 (1H, brs, H-4′), 6.03 (1H, ddd, J = 10.0, 2.7, 1.4 Hz, H-3″), 2.64 (1H, dt, J = 11.5, 4.2 Hz, H-1″), 2.52–2.43 (1H, m, H-5″a), 2.40–2.30 (1H, m, H-5″b), 2.27 (2H, t, J = 7.8 Hz, H-2), 2.14–2.04 (2H, m, H-6″), 1.62–1.55 (2H, m, H-3), 1.31–1.21 (12H, m, H-4, H-5, H-6, H-7, H-8 and H-9), 0.87 (3H, t, J = 6.8 Hz, H-10). 13C NMR δ (CDCl3, 125 MHz): 196.8 (s, C-2″), 172.6 (s, C-1), 150.3 (d, C-4″), 143.3 (d, C-5′), 140.0 (d, C-2′), 129.8 (d, C-3″), 123.1 (s, C-3′), 108.8 (d, C-4′), 67.1 (d, CHOCO), 51.0 (d, C-1″), 34.8 (t, C-2), 31.9 (t, C-4),¥ 29.6 (t, C-5),¥ 29.2 (t, C-6 and C-7, 2C),¥ 29.1 (t, C-8),¥ 24.9 (t, C-3 and C-5″, 2C), 23.4 (t, C-6″), 22.6 (t, C-9),¥ 14.1 (q, C-10). ¥Interchangeable signals; M.S. (E.I.) m/z (relative intensity): 191.1 (46.1), 174.1 (44.6), 123.1 (40.4), 96.1 (53.7), 95.1 (100), 57.2 (36.9), 55.2 (43.8), 43.2 (64.1), 41.2 (64.4); HRMS (ESI+): calcd for C21H30O4Na [M+Na]+ 369.2042, found 369.2053.

4.3. Bacterial Strains

For the antimicrobial evaluation, two strains from the American Type Culture Collection (S. aureus ATCC 25923 and E. faecalis ATCC 29212), as well as, 9 clinical isolates (MRSA 15012406, 15019301, 17045463 and 18032913, daptomycine-resistant E. faecalis 16028257, vancomycin-resistant E. faecium 16051635, linezolid-resistant S. epidermidis 17101107 and 18031123, and L. monocytogenes 17035151) were used in this study.

4.4. In Vitro Susceptibility Testing

The minimal inhibitory concentration (MIC) was determined on 96 well culture plates by a microdilution method according to Clinical and Laboratory Standards Institute (CLSI) procedures [53], using Mueller–Hinton Broth (Sigma, Spain). 50 µL of inoculum (5 × 105 CFU/mL) was added to each well. Proper blanks were assayed simultaneously. The MIC was determined as the lowest concentration, which showed no visible growth. Fosfomycin was used as a reference standard. The procedures were performed in independent triplicates to validate the results.

4.5. Time–Kill Kinetic Assays

Time–kill curves of S. aureus ATCC 25923 and MRSA isolate 18032913 were carried out following the procedure described by CLSI [53]. Concentration equal to MIC, twice the MIC and four times the MIC of the extracts were prepared. An inoculum size of 2 × 106 CFU/mL was conducted on Mueller Hinton broth (Sigma, Spain). Aliquots of 0.5 mL of the medium were taken at time intervals of 0, 4, 8 10, 12, and 24h. A graph of the log CFU/mL was plotted against time.

Supplementary Materials

The following are available online at https://www.mdpi.com/2079-6382/9/8/488/s1, Table S1. Calculated physicochemical properties of the naturally-occurring limonoids swietenolide and 2-hydroxy-3-O-tigloylswietenolide by ADMETlab, Table S2. Calculated physicochemical properties of naturally degraded limonoids and the degraded limonoid analogs 1–6 by ADMETlab, Figure S1. NMR and HRMS Spectra of diastereoisomers (±)-1a and (±)-1b, Figure S2. NMR and HRMS Spectra of diastereoisomers (±)-2a and (±)-2b, Figure S3. NMR and HRMS Spectra of (±)-3a, Figure S4. NMR Spectra of diastereoisomers (±)-4a and (±)-4b, Figure S5. NMR Spectra of diastereoisomers (±)-5a and (±)-5b, Figure S6. NMR Spectra of diastereoisomers (±)-6a and (±)-6b, Figure S7. HRMS Spectra of compounds 4, 5, and 6.

Author Contributions

Conceptualization M.J.D.-P., F.G.-S., M.A.R.-I., R.H.-G.; methodology: M.F.-S., V.P., A.J.M.-O., J.M.B.-A., F.G.-S., M.A.R.-I., R.H.-G. and M.J.D.-P.; formal analysis: M.F.-S., V.P., A.J.M.-O., J.M.B.-A., F.G.-S., M.A.R.-I., R.H.-G. and M.J.D.-P.; writing—original draft preparation M.J.D.P., F.G.-S., M.A.R.-I., R.H.-G., J.M.B.-A.; funding acquisition M.J.D.-P., F.G.-S., M.A.R.-I., R.H.-G. All authors have read and agreed to the published version of the manuscript.

Funding

We would like to thank the research programme ‘Programa de fomento e impulso de la investigación y la transferencia en la Universidad de Cádiz’ from University of Cádiz for the funding for this project PR2017-046.

Acknowledgments

Use of the NMR and MS facilities at the Servicio Centralizado de Ciencia y Tecnología of the University of Cádiz is acknowledged.

Conflicts of Interest

The authors declare no conflict of interest.

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Antibiotics 09 00488 g001 550
Figure 1. Structure of natural limonoids with antibacterial activity against multidrug resistance (MDR) bacterial strains.
Figure 1. Structure of natural limonoids with antibacterial activity against multidrug resistance (MDR) bacterial strains.
Antibiotics 09 00488 g001
Antibiotics 09 00488 g002 550
Figure 2. Limonoid and degraded limonoid skeleta.
Figure 2. Limonoid and degraded limonoid skeleta.
Antibiotics 09 00488 g002
Antibiotics 09 00488 g003 550
Figure 3. Chemical structures of some naturally-occurring degraded limonoids.
Figure 3. Chemical structures of some naturally-occurring degraded limonoids.
Antibiotics 09 00488 g003
Antibiotics 09 00488 g004 550
Figure 4. Chemical structures of some phragmalin-type limonoids.
Figure 4. Chemical structures of some phragmalin-type limonoids.
Antibiotics 09 00488 g004
Antibiotics 09 00488 sch001 550
Scheme 1. Retrosynthetic route for the synthesis of simplified model molecules 1–3.
Scheme 1. Retrosynthetic route for the synthesis of simplified model molecules 1–3.
Antibiotics 09 00488 sch001
Antibiotics 09 00488 sch002 550
Scheme 2. Synthesis of model molecules 1–3.
Scheme 2. Synthesis of model molecules 1–3.
Antibiotics 09 00488 sch002
Antibiotics 09 00488 g005 550
Figure 5. Key n.O.e. correlations for (±)-3a.
Figure 5. Key n.O.e. correlations for (±)-3a.
Antibiotics 09 00488 g005
Antibiotics 09 00488 g006a 550Antibiotics 09 00488 g006b 550
Figure 6. (a) Time-kill curve of S. aureus ATCC 25923; (b) Time-kill curve of MRSA 18032913.
Figure 6. (a) Time-kill curve of S. aureus ATCC 25923; (b) Time-kill curve of MRSA 18032913.
Antibiotics 09 00488 g006aAntibiotics 09 00488 g006b
Antibiotics 09 00488 sch003 550
Scheme 3. Synthesis of model molecules 4–6.
Scheme 3. Synthesis of model molecules 4–6.
Antibiotics 09 00488 sch003
Table
Table 1. Calculated physicochemical properties of the naturally-occurring limonoids swietenolide and 2-hydroxy-3-O-tigloylswietenolide by ADMETlab [28].
Table 1. Calculated physicochemical properties of the naturally-occurring limonoids swietenolide and 2-hydroxy-3-O-tigloylswietenolide by ADMETlab [28].
Molecular PropertiesLimonoidsMolecular PropertiesLimonoids
Swietenolide2-hydroxy-3-O-tigloylswietenolideSwietenolide2-hydroxy-3-O-tigloylswietenolide
HBD a22Nrotb g47
HBA b810TPSA h123.27149.57
MW c486.56584.66Log D i1.6791.674
Log P d3.1273.759NR j55
MR e122.54147.30NRigB k3539
TNA f6982
Matches in Drugability Rules
RulesLipinskiGhoseVeberVarmaOprea
Swietenolide100%75%100%80%66.7%
2-hydroxy-3-O-tigloylswietenolide75%25%66.7%40%100%
a HBD = hydrogen bond donors; b HBA = hydrogen bond acceptors; c MW = molecular weight; d Log P = partition coefficient; e MR = molar refractivity; f TNA = total number of atoms; g Nrotb = number of rotatable bonds; h TPSA = total polar surface area; i Log D = distribution coefficient; j NR = number of rings; k) NRigB = number of rigid bonds.
Table
Table 2. Calculated physicochemical properties of the naturally-occurring degraded limonoids dictamdiol, calodendrolide, fraxinellone, 8,14-epoxyfraxinellone, and melazolide A by ADMETlab [28].
Table 2. Calculated physicochemical properties of the naturally-occurring degraded limonoids dictamdiol, calodendrolide, fraxinellone, 8,14-epoxyfraxinellone, and melazolide A by ADMETlab [28].
Molecular PropertiesNatural Degraded Limonoids
DictamdiolCalodendrolideFraxinellone8,14-epoxyfraxinelloneMelazolide A
HBD a20002
HBA b54444
MW c278.304260.289246.262248.278212.245
Log P d1.7162.7622.5632.5950.238
MR e69.59666.24362.58961.7252.944
TNA f3835323431
Nrotb g11111
TPSA h79.951.9756.5151.9766.76
Log D i0.9781.5131.3831.3740.189
NR j34342
NRigB k2121192015
RulesMatches
Lipinski’s rules100%100%100%100%100%
Ghose’s rules100%100%100%100%100%
Veber’s rules100%100%100%100%100%
Varma’s rules100%100%100%100%100%
Oprea’s rules66.7%66.7%66.7%66.7%0%
a HBD = hydrogen bond donors; b HBA = hydrogen bond acceptors; c MW = molecular weight; d Log P = partition coefficient; e MR = molar refractivity; f TNA = total number of atoms; g Nrotb = number of rotatable bonds; h TPSA = total polar surface area; i Log D = distribution coefficient; j NR = number of rings; k NRigB = number of rigid bonds.
Table
Table 3. Minimal inhibitory concentrations are effective for ≥50% for 1–3 against S. aureus ATCC 25923 and MRSA strains.
Table 3. Minimal inhibitory concentrations are effective for ≥50% for 1–3 against S. aureus ATCC 25923 and MRSA strains.
PathogenStereoisomersMIC50 (mg/L)
S. aureus ATCC 25923(±)-1a and (±)-1b512
(±)-2a and (±)-2b16
(±)-3aND 1
MRSA 15012406(±)-1a and (±)-1b512
(±)-2a and (±)-2b128
(±)-3aND 1
MRSA 15019301(±)-1a and (±)-1b512
(±)-2a and (±)-2b64
(±)-3aND 1
MRSA 17045463(±)-1a and (±)-1bNT 2
(±)-2a and (±)-2b128
(±)-3aNT 2
MRSA 18032913(±)-1a and (±)-1bNT 2
(±)-2a and (±)-2b64
(±)-3aNT 2
1 ND = not detectable; 2 NT = not tested.
Table
Table 4. Minimal inhibitory concentrations are effective for ≥50% for 2 against other Gram-positive bacteria.
Table 4. Minimal inhibitory concentrations are effective for ≥50% for 2 against other Gram-positive bacteria.
PathogenL. monocytogenes 17035151E. faecalis
ATCC 29212		E. faecalis
DapR 1
16028257		E. faecium
VaR 2 16051635		S. epidermidis LzdR 3 17101107S. epidermidis LzdR 3 18031123
MIC50
(mg/L)		1282562562566464
1 DapR = daptomycine resistant; 2 VaR = vancomycin resistant; 3 LzdR = linezolid resistant.
Table
Table 5. Minimal inhibitory concentrations are effective for ≥50% for 4–6 against S. aureus ATCC 25923.
Table 5. Minimal inhibitory concentrations are effective for ≥50% for 4–6 against S. aureus ATCC 25923.
PathogenStereoisomersMIC50 (mg/L)
S. aureus ATCC 25923(±)-4a256
(±)-4b128
(±)-5a256
(±)-5b128
(±)-6a1024
(±)-6b256
Table
Table 6. Calculated log P and log D of 1 and esters 2 and 4-6 by ADMETlab [28].
Table 6. Calculated log P and log D of 1 and esters 2 and 4-6 by ADMETlab [28].
Molecular PropertiesModel Compounds
12456
Log P1.8482.4193.1993.9805.540
Log D0.8631.0421.4211.6412.151

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Ferrera-Suanzes, M.; Prieto, V.; Medina-Olivera, A.J.; Botubol-Ares, J.M.; Galán-Sánchez, F.; Rodríguez-Iglesias, M.A.; Hernández-Galán, R.; Durán-Peña, M.J. Synthesis of Degraded Limonoid Analogs as New Antibacterial Scaffolds against Staphylococcus aureus. Antibiotics 2020, 9, 488. https://doi.org/10.3390/antibiotics9080488

AMA Style

Ferrera-Suanzes M, Prieto V, Medina-Olivera AJ, Botubol-Ares JM, Galán-Sánchez F, Rodríguez-Iglesias MA, Hernández-Galán R, Durán-Peña MJ. Synthesis of Degraded Limonoid Analogs as New Antibacterial Scaffolds against Staphylococcus aureus. Antibiotics. 2020; 9(8):488. https://doi.org/10.3390/antibiotics9080488

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Ferrera-Suanzes, Marta, Victoria Prieto, Antonio J. Medina-Olivera, José Manuel Botubol-Ares, Fátima Galán-Sánchez, Manuel A. Rodríguez-Iglesias, Rosario Hernández-Galán, and María Jesús Durán-Peña. 2020. "Synthesis of Degraded Limonoid Analogs as New Antibacterial Scaffolds against Staphylococcus aureus" Antibiotics 9, no. 8: 488. https://doi.org/10.3390/antibiotics9080488

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