Spiro-Lactams as Novel Antimicrobial Agents

: Introduction : Structural modulation of previously identified lead spiro-β-lactams with antimicrobial activity was carried out. Objective : The main objective of this work was to synthesize and evaluate the biological activity of novel spiro-lactams based on previously identified lead compounds with antimicrobial activity. Methods : The target chiral spiro-γ-lactams were synthesized through 1,3-dipolar cycloaddition reaction of a diazo-γ-lactam with electron-deficient dipolarophiles. In vitro activity against HIV and Plasmodium of a wide range of spiro-β-lactams and spiro-γ-lactams was evaluated. Among these compounds, one derivative with good anti-HIV activity and two with promising antiplasmodial activity (IC 50 < 3.5 µM) were identified. Results : A novel synthetic route to chiral spiro-γ-lactams has been established. The studied β-and γ-lactams were not cytotoxic, and three compounds with promising antimicrobial activity were identified, whose structural modulation may lead to new and more potent drugs. Conclusion : The designed structural modulation of biologically active spiro-β-lactams involved the replacement of the four-membered β -lactam ring by a five-membered γ-lactam ring. Although conformational and superimposition computational studies revealed no significant differences between β-and γ-lactam pharmacophoric features, the studied structural modulation did not lead to compounds with a similar biological profile. The observed results suggest that the β-lactamic core is a requirement for the activity against both HIV and Plasmodium .

In this work, previous studies have been extended to new spiro-lactams, in order to further explore structure-activity relationships.Life-threatening hypersensitivity reactions are a major problem in the use of β-lactams, therefore the manufacture of these drugs is subjected to ever-increasing de-manding requirements to avoid cross-contamination of other drugs [26][27].In order to overcome the stability-and toxicity-related problems of β-lactams, we sought to produce spirocyclic-γ-lactams 2, corresponding to the replacement of the four-membered β-lactam ring by the five-membered γlactam ring (Fig. 1).A synthetic strategy to produce these novel γ-lactams derivatives, as well as their evaluation as antimicrobial agents is described (Fig. 1).

Chemistry
Thin-layer Chromatography (TLC) analyses were performed using precoated silica gel plates.Flash column chromatography was performed with silica gel 60 as the stationary phase. 1 H Nuclear Magnetic Resonance (NMR) spectra (400 MHz) and 13 C NMR spectra (100 MHz) were recorded in CDCl 3 , CD 3 OD or hexadeuterated dimethylsulfoxide (DMSO) as solvents.Chemical shifts are expressed in parts per million (ppm) relatively to internal tetramethylsilane (TMS) and coupling constants (J) are expressed in hertz.Infrared spectra (IR) were recorded in a Fourier Transform spectrometer coupled with a diamond Attenuated Total Reflectance (ATR) sampling accessory.Elemental analyses were carried out with an Elemental Vario Micro Cube analyser.High-Resolution Mass Spectra (HRMS) were obtained on a TOF VG Autospect M spectrometer with electrospray ionization (ESI).Melting points (m.p.) were determined in open glass capillaries.Optical rotations were measured on an Optical Activity AA-5 electrical polarimeter.Benzyl (2S)-2-(tert-butoxycarbonylamino)-4-oxobutanoate 5 [28] was prepared as previously described [29].

(R)-Benzhydryl 2-amino-3-mercapto-3-methylbutanoate Hydrochloride 7
To a stirred solution of D-penicillamine (1.0 g, 9.2 mmol) in dry methanol (9.2 mL), a solution of diphenyldiazomethane (1.7 g, 9.2 mmol) in dry dichloromethane (28 mL) was added dropwise.After 24 h at room temperature, an additional portion of diphenyldiazomethane (0.7 g, 4.6 mmol) was added and the reaction mixture was stirred for further 96 h.The solvent was removed under reduced pressure and diethyl ether was added (30 mL).The solution was cooled in an ice bath and HCl (1 M) was added dropwise until pH 1 was reached.The product precipitated as a white solid which was then filtered.Yield: 55% (1.78 g).m.p.

Method A
A solution of compound 7 (1.08 g, 3.41 mmol) and aldehyde 5 (1.05 g, 3.41 mmol) in pyridine (6 mL) was stirred under reflux over 18 h.The pyridine was removed under reduced pressure, the reaction mixture was diluted with ethyl acetate and washed with water.The organic phase was separated, and the aqueous phase was extracted with ethyl acetate.The organic extracts were dried over Na 2 SO 4 and concentrated under reduced pressure to give compound 8. Yield: 24% (405.5 mg).

Method B
To a solution of thiazolidine 9 (119 mg, 0.2 mmol) in toluene (20 mL) a catalytic amount of p-toluenesulfonic acid monohydrate was added.The reaction mixture was refluxed over 70 h under N 2 atmosphere.The solvent was removed under reduced pressure and crystallization with ethyl acetate/hexane gave compound 8 as a white solid.Yield: 69% (67 mg).m.p. 203.5-204.8ºC.

General Procedure for the Cycloaddition Reactions of Diazo-γ-lactam 3 and Dipolarophiles
To an ice-cold solution of freshly prepared diazo-γlactam 3 in dry dichloromethane (7 mL), the appropriate dipolarophile was added.The reaction mixture was stirred at room temperature or at 0 ºC and under N 2 atmosphere for the time indicated in each case.After removal of the solvent under reduced pressure (no heat), the products were isolated by flash chromatography.

General Procedure for the Conversion to Carboxylic Acids
To a solution of the appropriate spiro-γ-lactam in anhydrous dichloromethane (7 mL) at -5 °C, anisole (7 equiv) and Trifluoroacetic Acid (TFA) (25 equiv) were added.The reaction mixture was stirred for 4 h at -5 ºC under N 2 atmosphere.The mixture was diluted with cold diethyl ether, and the solvent was evaporated.The residue was dissolved in tetrahydrofuran, a saturated aqueous solution of NaHCO 3 was added and the mixture was stirred at 0 °C for 15 min.Ethyl acetate was added, the organic phase was separated and the aqueous phase was extracted twice with ethyl acetate.The aqueous layer was then acidified to pH 3 in an ice bath with HCl (1 M) and extracted with ethyl acetate.The organic extracts were dried over Na 2 SO 4 and concentrated under reduced pressure to give the desired acid.

Viruses and Titration
The HIV-1 SG3.1 subtype B X4, which uses the CXCR4 coreceptor, the lab-adapted strain was obtained by transfection of HEK293T cells with pSG3.1 plasmid using jetPrime transfection reagent (Polyplus-transfection SA, Illkirch, France) according to the instructions of the manufacturer.The 50% Tissue Culture Infectious Dose (TCID 50 ) of each virus was determined in a single-round viral infectivity assay using a luciferase reporter gene assay in TZM-bl cells [30,31] and calculated using the statistical method of Reed and Muench.

Cellular Viability Assays
The in vitro cytotoxicity of test compounds was evaluated in TZM-bl cells using alamarBlue cell viability reagent (Life Technologies, USA).Cells were cultured in the presence and absence of serial-fold dilutions of the test compounds.Each dilution of each compound was performed in triplicate wells.Medium controls (only growth medium), cell controls (cells without test compound) and cytotoxicity controls (a compound that kills cells) were employed in each assay.The cytotoxicity of each test compound was expressed by the 50% cytotoxic concentration (CC 50 ), corresponding to the concentration of compound causing a 50% decrease of cellular viability.

Antiviral Assays
The antiviral activity of test compounds was determined in a single-round viral infectivity assay using TZM-bl reporter cells, as previously described [30,32].Briefly, TZMbl cells were infected with 200 TCID 50 of HIV-1 in the presence of serial fold dilutions of the compounds in the growth medium, supplemented with diethylaminoethyl-dextran (DEAE-dextran).After 48 h of infection, luciferase expression was quantified with Pierce Firefly Luc One-Step Glow Assay Kit (ThermoFisher Scientific, Rockford, USA) according to the instructions of the manufacturer.For each virus and compound dilution, the assay was set up in triplicate wells.Virus controls, cell controls and inhibitors controls (drugs with a known action against each virus) were employed.

Statistical Analysis
Statistical analysis was performed using Prism version 5.01 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com)with a level of significance of 5%.
For infection assays, 1.0 × 10 4 Huh-7 cells (per well) were seeded in 96-well plates the day before drug treatment and infection.Serial dilutions of each compound were then prepared in the infection medium.On the day of infection, the culture medium was replaced by the appropriate compound concentration and incubated for 1 h.Next, 1.0 × 10 4 firefly luciferase-expressing P. berghei sporozoites, freshly obtained through disruption of salivary glands of infected female Anopheles stephensi mosquitoes, were added to each well.An amount of the DMSO solvent equivalent to that present in the highest compound concentration was diluted in the infection medium and used as control.Sporozoite addition was followed by centrifugation at 1700×g for 5 min and subsequent incubation for 48 h at 37 ºC with 5% CO 2 .The effect of the compounds on the viability of Huh-7 cells was assessed by the Alamar Blue assay (Invitrogen, U.K.) according to the protocol of the manufacturer, followed by the measurement of parasite infection load by a bioluminescence assay (Biotium, USA).Nonlinear regression analysis was employed to fit the normalized results of the dose-response curves, and half-maximal inhibitory concentration (IC 50 ) values were determined using Prism version 5.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com).

Computational Methodology
Quantum chemical calculations were carried out in order to explore the structure and the preferred conformations of molecules 18b, 19, BSS-930A and BSS-939.The structures were optimized at the Density Functional (DFT) level of theory, using the B3LYP hybrid functional [35][36][37] and the standard 6-31G(d) basis set.All calculations were performed using the GAMESS program package [38] and graphical representations were produced with GaussView.The optimized structures are depicted in Fig.

Chemistry
The retrosynthetic analysis of chiral spirocyclic-γlactams is outlined in Scheme 1.The pyrazole ring could be formed by 1,3-dipolar cycloaddition of 6-diazo-γ-lactam 3 with electron-deficient dipolarophiles.In order to obtain this chiral 6-diazo-γ-lactam, a synthetic route starting from a naturally occurring amino acid was considered.Thus, following the methodology developed by Baldwin et al. for related systems, amine 4 could be obtained through a multistep strategy from D-penicillamine.Diazo-γ-lactam 3 could be prepared by reacting amine 4 with sodium nitrite and perchloric acid, as described for the synthesis of 6diazopenicillanate from 6-aminopenicillanate [40,41].
L-Aspartic acid derived aldehyde, benzyl (2S)-2-(tertbutoxycarbonylamino)-4-oxobutanoate (5) [28] was synthesized by using a method described for the preparation of the R enantiomer which involves the initial reduction of compound 6 to the corresponding alcohol, followed by a Swern oxidation (Scheme 2) [29].The use of the alcohol without purification by flash chromatography afforded aldehyde 5 in 76% overall yield.
The initial goal of this study was the synthesis of bicyclic-γ-lactam 8 which was prepared by two distinct methodologies.Firstly, the reaction of D-penicillamine benzhydryl ester hydrochloride 7 was carried out with aldehyde 5 in refluxing pyridine, leading to compound 8 in moderate yield (24%).Then a two-step approach was explored, which comprised of the synthesis of thiazolidine 9 followed by a cyclization step.Thus, the reaction of D-penicillamine benzhydryl ester hydrochloride 7 with aldehyde 5, in the presence of a base, was carried out giving thiazolidine 9 diastereoselectively and in 64% yield.The stereochemistry of thiazolidine 9 was assigned based on its two-dimensional NO-ESY (Nuclear Overhauser Effect Spectroscopy) spectrum, in which cross-peaks between protons H-2 and H-4 were observed.Thiazolidine 9 was then converted into bicyclic-γlactam 8 (69% yield) by heating in refluxing toluene for 70 h in the presence of catalytic amounts of p-toluenesulfonic acid (PTSA) (Scheme 3).In order to carry out the amino group deprotection, bicyclic-γ-lactam 8 was treated with an excess of HCl (2 M solution in diethyl ether) in dichloro- methane at room temperature to afford bicyclic-γ-lactam 10 in 70% yield.However, together with the N-Boc deprotection, the cleavage of the benzhydryl group was also observed.At this stage, since the cleavage of this group was not desirable a different strategy was considered.
The alternative approach was the use of D-penicillamine and to carry out the ester protection with diphenyldiazomethane at a later stage (Scheme 4).Thus, Dpenicillamine reacted with aldehyde 5 to give a mixture of diastereoisomers 11 (64:36) in 92% yield.
The structure of thiazolidines 11 was confirmed by their conversion into the corresponding methyl esters 15a and 15b by treatment with diazomethane, in 62% overall yield (Scheme 5).Isomers (2R,4S)-15a and (2S,4S)-15b were separated by flash chromatography allowing full characterization.The assigned stereochemistry was supported by the two-dimensional NOESY spectrum of compound 15b, which showed cross-peaks between proton H-2 and proton H-4.By refluxing the mixture of diastereoisomers 11 in toluene for 24 h, the bicyclic-γ-lactam 12 was obtained diastereoselectively in 83% yield.The deprotection of amino moiety of compound 12 was carried out using the strategy previously mentioned in Scheme 3. Treatment of 12 with an excess of HCl (2 M solution in diethyl ether) in tert-butanol at room temperature for 6 days, afforded γ-lactam 10 in 93% yield.However, a faster N-Boc deprotection could be achieved using p-toluenesulfonic acid in acetonitrile, as described by Chauvett et al. for other systems [42].In fact, the reaction of compound 12 under those conditions led to compound 13 in quantitative yield.The reaction of γ-lactam 13 with diphenyldiazomethane afforded γ-lactam benzhydryl ester 14 in 90% yield.Subsequent treatment of 14 with an aqueous solution of NaHCO 3 gave the amino-γ-lactam 4 in quantitative yield.Finally, the treatment of amino-γ-lactam 4 in a biphasic solvent system, as described by Sheehan and Commons for β-lactamic systems [43], with sodium nitrite in the presence of HClO 4 allowed the synthesis of the Dpenicillamine-derived diazo-γ-lactam 3 in 97% yield.
The 1,3-dipolar cycloaddition of diazo-γ-lactam 3 with methyl propiolate was also studied, leading to the desired spiro-γ-lactam 16b in a regioselective fashion and in moderate yields, regardless of the reaction conditions studied (entries 5-7).The work was also extended to the cycloaddition of diazo-γ-lactam 3 with N-substituted maleimides (Scheme 6).The reaction with an excess of N-methylmaleimide at room temperature gave spiro-γ-lactam 17a in 12% yield.Carrying out the cycloaddition reaction with N-phenylmaleimide under the same reaction conditions afforded compound 17b in 21% yield.The cycloaddition reactions with N-substituted maleimides were also studied at 0 ºC but the results obtained were unsatisfactory.The conversion of the spiro-γ-lactams 16 bearing a benzhydryl ester moiety into the corresponding carboxylic acid derivatives 18 was also studied (Scheme 7).Treatment of spiro-γ-lactams 16a and 16b with anisole and TFA in dichloromethane at -5 ºC gave spiro-γ-lactams 18a and 18b in 97% and 86% yield, respectively.

Anti-HIV Activity
Initially, in vitro cytotoxicity assays in TZM-bl cells were performed for a wide range of γ-lactams, and some βlactams previously synthesized (Fig. 2) [23].From a library of 19 compounds, 18 of them showed no cytotoxicity and their antiviral activity was evaluated (Table 2).
The antiviral activity of the selected 18 compounds was evaluated in a single-round viral infectivity assay against an HIV-1 isolate and resulted in the identification of three compounds (4, BSS-975B and BSS-1028) with moderate to good antiviral activity.Compound 4 was the only γ-lactam which exhibited activity, with a maximum percentage of inhibition (MPI) at 10 μg/mL of 64%.BSS-1028 exhibited an MPI at 10 μg/mL of 79%.Although these compounds showed some degree of anti-HIV activity, they were not considered promising enough and their IC 50 values were not calculated.Nevertheless, these molecules can still constitute a promising starting point for structural modulation on the search for additional compounds with anti-HIV activity.

Anti-Plasmodium activity
In order to evaluate the antiplasmodial potential of some γ-lactams and β-lactams, in vitro assays against the hepatic stage of P. berghei infection were performed (Table 3, Figures S31-S34).Compounds were tested at 1 and 10 μM, and DMSO was used as a negative control.The results showed that none of the tested compounds displayed cytotoxicity against the Huh-7 host cells.Among the 17 compounds evaluated, 9 had an IC 50 against P. berghei under 10 μM.
Since BSS-930A and BSS-939 appeared as the most potent of the 9 molecules showing anti-Plasmodium activity below 10 µM, their IC 50 values were determined and found to be 3.32 ± 0.23 and 2.67 ± 0.22 µM, respectively (Table 4).
These two compounds share a high structural similarity.In fact, BSS-930A is a precursor of BSS-939, the latter being obtained from the N 2 extrusion and subsequent ring contraction of the former's pyrazole ring.The fact that all pharma-

Computational Studies
Quantum chemical calculations, at the DFT level of theory, were carried out in order to explore the structure and the preferred conformations of selected molecules.Conformational studies of compounds 18b and 19 showed that both γand β-lactam compounds presented similar minimum energy conformations, despite the difference on the lactam ring size (Fig. 3).Similar results were obtained for the superposition studies between γ-and β-lactam minimum energy conformations, which presented an almost perfect overlap between the pharmacophoric features of both molecules.These results indicate that the γ-lactam molecule should be capable of reproducing the active β -lactam analogue pharmacophoric three-dimensional disposition and, consequently, it should also be able to reproduce its interactions with molecular targets.The molecular superposition and the pharmacophoric features shared between the two molecules are represented in Fig. (4).
Notwithstanding such structural, conformational and pharmacophoric similarities, the γ -lactam molecule showed no relevant anti-HIV and anti-Plasmodium activity, contrary to its β -lactam counterpart.Such a result suggests that the presence of the four-membered β-lactam ring appears to play a crucial role in both anti-HIV and anti-Plasmodium activity of spiro-lactams.The structural similarity of compounds BSS-930A and BSS-939 suggests that the minimum energy conformations of the two molecules are also similar (Fig. 5).This was con-firmed by the conformational study where both molecules showed a very similar conformation at their minimum energies, except for the orientation of the two phenyl substituents at spiro-rings.Such positional difference is related to the different rings sharing one carbon atom with the penicillanic bicyclic system, a 4,5-dihydro-3H-pyrazole (BSS-930A) vs. a cyclopropane (BSS-939) ring and may explain the slight difference observed between BSS-930A and BSS-939 anti-Plasmodium activities.

CONCLUSION
In summary, a structural modulation of previous lead compounds with antimicrobial activity was carried out, leading to the synthesis of new chiral spiro-γ-lactams.Assessment of the in vitro activity of a wide range of spiro-βlactams and spiro-γ-lactams against HIV and Plasmodium led to the identification of one derivative with good anti-HIV activity, and two with promising anti-Plasmodium activity.The results suggest that the β-lactamic core is an important structural feature to ensure activity against both HIV and Plasmodium.This information will be a good starting point for other structural modulations aiming at the development of new and more potent drugs against HIV and Plasmodium.

ETHICS APPROVAL AND CONSENT TO PARTICI-PATE
applicable.

HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION
Not applicable.
(3) andFig.(5), and in Figs.(S35 to S38) of the Supporting Information.Energy values and Cartesian coordinates are given in Supporting Information.Molecule superimposition studies were performed with LigandScout 4.3 software [39], and are represented in Fig. (4), and in Figs.(S39 to S50) of the Supporting Information.