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

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.


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 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.

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-Otigloylswietenolide 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). 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.

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].
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].  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.
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).  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. 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 2hydroxy-3-O-tigloylswietenolide.

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, 1 H NMR, 13 C 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).  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.

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). 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 2hydroxy-3-O-tigloylswietenolide.

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).

Scheme 2. Synthesis of model molecules 1-3.
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, 1 H NMR, 13 C 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). 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, 1 H NMR, 13 C 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). 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 2hydroxy-3-O-tigloylswietenolide.

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).

Scheme 2. Synthesis of model molecules 1-3.
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, 1 H NMR, 13 C 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).

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).   (Table 4).

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).  (Table 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). 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.
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). 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.

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. 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.

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 (MIC 50  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 MIC 50 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.
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.

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 ). 1 H and 13 C NMR measurements were recorded on Agilent 500 MHz NMR spectrometer with SiMe 4 as the internal reference. Chemical shifts were referenced to CDCl 3 (δ H 7.25, δ C 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. 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). 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. 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 NH 4 CI (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).