Expedient Organocatalytic Syntheses of 4-Substituted Pyrazolidines and Isoxazolidines

Abstract

The efficient organocatalytic synthesis of heterocyclic systems of biological relevance is a subject of growing interest. We have found that the pyrrolidine/benzoic acid-catalyzed reaction of α-substituted propenals such as methacrolein, 2-benzylpropenal and 2-(n-hexyl)propenal with activated hydrazines takes place in very good yields (83%–99.6%) under very mild conditions to afford 4-substituted pyrazolidin-3-ols (as diastereomer mixtures); subsequent oxidation with PCC affords the corresponding-4-substituted-3-pyrazolidinones in essentially quantitative yields. In a similar way, 4-substituted isoxazolidinones are obtained with N-Cbz-hydroxylamine as a reagent. The use of chiral diarylprolinol trimethylsilyl ethers as catalysts allows the synthesis of several of these compounds in optically active form, in some cases with excellent enantioselectivity (up to 96:4 er). A preliminary evaluation of the biological activity shows that some of these compounds exhibit interesting antibacterial and antifungal activities.

1. Introduction

The pyrazolidinone (pyrazolidin-3-one) moiety is an important structural motif that can be found both in natural products and in synthetic compounds with interesting pharmacological activity (analgesic, antibiotic, anticonvulsant, and with inhibitory cyclooxygenase, lipoxygenase, and γ-aminobutyrate transferase activity, among others) [1,2,3,4,5,6,7,8,9,10]. On the other hand, the selective oxidation of pyrazolidinones affords the corresponding pyrazolones (pyrazol-5-ones), a class of compounds that also exhibits very relevant pharmacological properties [11,12,13,14,15,16,17,18]. Figure 1 shows structures of some biologically active pyrazolidinones and pyrazolones.

It is therefore not surprising that efficient and selective syntheses of pyrazolidinone derivatives have been actively pursued [19]. Recently, organocatalytic approaches to these compounds, based either on the amine-catalyzed [20,21] or on the carbene-catalyzed [22] reaction of α,β-unsaturated aldehydes with hydrazine derivatives and subsequent oxidation of the intermediate pyrazolidinols, have been described, enabling their preparation in optically active form [23,24,25]. Although these approaches are highly interesting, all of them suffer from the common limitation that they have been applied only to β-substituted enals, so that they can exclusively lead to 5-substituted-3-pyrazolidinones.

In the course of our studies on the organocatalytic synthesis of α,β-disubstituted-β-amino acids we had found that the pyrrolidine-promoted aza-Michael addition/cyclization of N-protected hydroxylamines to acyclic α,β-unsaturated, α,β-disubstituted enals took place in several instances with excellent yields and good diastereoselectivities [26], so that we set out to investigate if the secondary amine-catalyzed reaction of these branched enals with substituted hydrazines, followed by oxidation of the resulting pyrazolidin-3-ols, would provide a general route to 4,5-disubstituted-3-pyrazolidinones (Scheme 1).

2. Results and Discussion

2.1. Organocatalytic Synthesis of 4-Substituted Pyrazolidinones and Isoxazolidinones

We began our research by examining the pyrrolidine-catalyzed addition/cyclization of cyclopentene-1-carboxaldehyde (1) with 1-Boc-2-(4-nitrobenzenesulfonyl)hydrazine (2a) [20] using benzoic acid as a co-catalyst. After some experiments, we found that by using 40 mol % of both pyrrolidine and benzoic acid the expected bicyclic pyrazolidin-3-ol 3 was obtained in excellent yield (87% isolated yield after chromatographic purification) and as a single isomer (dr > 30:1), after three days in toluene at room temperature (r.t.) (Scheme 2).

Next, we tried the same reaction conditions with acyclic α,β-substituted enals. While the reaction with tiglic aldeyhyde ((E)-2-methylbutenal, 4) led to the formation of the pyrazolidinol 5 (76% yield, 6:1 dr), we found that (E)-2-methyl-3-phenylbutenal 6 was completely unreactive, no product being detected after 7 days of stirring at r.t. (Scheme 3).

Contrary to what we had observed for the corresponding isoxazolidinols [26], attempted oxidation of both 3 and 5, either with pyridinium chlorochromate (PCC) or with pyridinium dichromate (PDC), failed to cleanly give the desired pyrazolidinones, so we decided to reduce the steric hindrance of the intermediate pyrazolidinols by suppressing the β-substituent in the starting enal.

We were pleased to find that the pyrrolidine/benzoic acid-catalyzed reaction of the α-substituted enals methacrolein (7a), 2-benzylpropenal (7b), or 2-(n-hexyl)propenal (7c) with the activated hydrazines 2a and 1,2-bis(p-toluenesulfonyl)hydrazine (2b) took place in very good yields (83%–99.6%), affording the 4-substituted pyrazolidin-3-ols 8aa–8bc as diastereomer mixtures; without further purification, oxidation with PCC was performed to afford the corresponding-4-substituted-3-pyrazolidinones 9aa–9bc in essentially quantitative yields (Scheme 4 and

Table 1. Pyrrolidine-catalyzed synthesis of pyrazolidin-3-ols 8aa–8bc from α-substituted enals 7a–7c and hydrazines 2a and 2b, and oxidation to the corresponding pyrazolidin-3-ones 9aa–9bc.

Entry	Reactants	R	X	Y	Pyrazolidinol	Yield 1	Reaction Time (Day)	dr 2	Pyrazolidinone	Yield 1
1	7a, 2a	Me	Ns	Boc	8aa	93%	4	4:1	9aa	94%
2	7b, 2a	PhCH2	Ns	Boc	8ab	83%	2	>20:1	9ab	98%
3	7c, 2a	n-Hexyl	Ns	Boc	8ac	99.6%	2	5:1	9ac	99%
4	7a, 2b	Me	Ts	Ts	8ba	88%	2	8:1	9ba	98%
5	7b, 2b	PhCH2	Ts	Ts	8bb	85%	2	9:1	9bb	97%
6	7c, 2b	n-Hexyl	Ts	Ts	8bc	98%	2	10:1	9bc	98%

¹ Yield of isolated product after chromatographic purification; ² By ¹H-NMR.

).

As it can be seen, the aza-Michael addition/cyclization step took place in excellent yield and with high diastereoselectivity, irrespective of the nature of the hydrazine (2a or 2b) and of the acrolein substituent (methyl, benzyl, or n-hexyl). It should be noted however that when the more hindered α-isopropyl acrylaldehyde (3-methyl-2-methylenebutyraldehyde (7d)) was used under the same conditions, no reaction with hydrazine 2a was observed after 7 days at rt. In our hands, N,N’-bis(tert-butoxycarbonyl)hydrazine (2c), reported to give good yields of pyrazolidin-3-ols upon pyrrolidine-catalyzed reaction with cinnamaldehyde derivatives [21], failed to react with the α-substituted enals 1, 4, and 7a–7d.

In the light of these results, we explored the possible use of the α-substituted acroleins 7 as starting materials for the synthesis of 4-substituted isoxazolidin-5-ones, potentially interesting compounds both from the biological point of view and as advanced intermediates towards α-substituted-β-amino acids [26], by the amine-catalyzed reaction with a suitable protected hydroxylamine. To our satisfaction, we found that in fact α-substituted acroleins 7, with the sole exception of the isopropyl derivative 7d, reacted smoothly with N-Cbz-hydroxylamine 10 (Cbz = benzyloxycarbonyl) to afford the isoxazolidinols 11 in good yields (78%–83% isolated yields) as diastereomer mixtures. Subsequent oxidation of these compounds with PCC took place uneventfully, providing the target 4-substituted isoxazolidin-5-ones 12 (Scheme 5 and

Table 2. Pyrrolidine-catalyzed synthesis of isoxazolidin-5-ols 11a–11c from α-substituted enals 7a–7c and N-Cbz-hydroxylamine 10, and oxidation to the corresponding isoxazolidin-5-ones 12a–12c.

Entry	Reactants	R	Isoxazolidinol	Yield 1	dr 2	Isoxazolidinone	Yield 1
1	7a, 10	Me	11a	83%	2:1	12a	98%
2	7b, 10	PhCH2	11b	79%	3:1	12b	99%
3	7c, 10	n-Hexyl	11c	78%	3:1	12c	87%

¹ Yield of isolated product after chromatographic purification. ² By ¹H-NMR.

).

Previous studies from our group [26] and those of Córdova [27,28] and Vicario [20] had shown that the use of chiral diarylprolinol-silyl ethers as catalysts led to high enantioselectivities in the aza-Michael/cyclization reactions of β-substituted [20,27,28] and of α,β-disubstituted [26] enals with hydroxylamines or hydrazines. However, α-branched vinyl carbonyls remain a very challenging substrate for this type of reaction and only one successful example of aminocatalytic asymmetric aza-Michael addition to α-substituted vinyl ketones has been reported so far [29,30]. Bearing these precedents in mind, we proceeded to examine the performance of the Jørgensen-Hayashi catalysts 13 and 14 (Figure 2) in the reaction between aldehydes 7a–7c and hydrazines 2a and 2b (

Table 3. Enantioselective synthesis of pyrazolidin-3-ones 9.

Entry	Reactants	Catalyst	Pyrazolidinol	Yield 1	dr 2	Pyrazolidinone	Yield 1	er 3
1	7a, 2a	13	8aa	82%	4:1	9aa	87%	50:50
2	7a, 2a	14	8aa	99%	3:1	9aa	94%	90:10
3	7b, 2a	13	8ab	77%	>20:1	9ab	98%	44:56
4	7b, 2a	14	8ab	98%	>20:1	9ab	98%	45:55
5	7b, 2b	13	8bb	85%	9:1	9bb	97%	- 4
6	7b, 2b	14	8bb	85%	9:1	9bb	97%	- 4
7	7a, 2b	13	8ba	84%	8:1	9ba	98%	96:4
8	7a, 2b	14	8ba	0% 5		9ba	-	-
9	7c, 2b	13	8bc	93%	10:1	9bc	98%	78:22
10	7c, 2b	14	8bc	77%	10:1	9bc	98%	50:50

¹ Yield of isolated product after chromatographic purification; ² By ¹H-NMR; ³ By chiral HPLC analysis (See the Supplementary Materials for details); ⁴ No satisfactory conditions for the HPLC analysis of this compound could be found; ⁵ No reaction was observed after 7 days.

), using a 20 mol % of the catalyst together with a 20 mol % of benzoic acid co-catalyst in toluene at r.t. (typical reaction time 3 days). The stereoisomeric mixture of pyrazolidinols 8 was then submitted to oxidation with PCC as before, and the enantiomeric purity of the corresponding pyrazolidinones 9 was determined by chiral HPLC analysis. After 7 days of stirring at rt, no product was observed in the attempted reactions of aldehyde 7c with hydrazine 2a, either when using 13 or 14 as catalysts. On the other hand, we could not find satisfactory HPLC conditions for the separation of the enantiomers of 9bb, so that the enantioselective catalysis for the reaction of aldehyde 7b with hydrazine 2b was not attempted.

As it can be seen in Table 3, the enantioselectivity of the reaction depends strongly both in the nature of the reactants and of the catalyst. The highest enantiomeric purities were obtained for the less hindered methyl-substituted enal 7a, either with hydrazine 2a (entry 2, 90:10 er, catalyst 14) or with hydrazine 2b (entry 7, 96:4 er, catalyst 13). In any case, these preliminary results demonstrate for the first time the feasibility of the asymmetric synthesis of 4-substituted-3-pyrazolidinones by the organocatalytic aza-Michael/cyclization of activated hydrazines to α-substituted acroleines.

A simplified mechanistic proposal for the formation of enantiomerically enriched 4-substituted pyrazolidinols 8aa–8ac is depicted in Scheme 6. While in the formation of the first carbon-nitrogen bond by the aza-Michael reaction of the chiral iminium intermediate A (formed from the α-substituted enal 7a–c and the chiral pyrrolidine catalyst 13 or 14) with the hydrazine 2a no new chiral center is formed, the protonation of the intermediate enamine B can take place on the two diastereotopic faces of the C–C double bond. When using the trifluoromethyl-substituted catalyst 14, we assume (based on our previous studies on the asymmetric organocatalytic synthesis of isoxazolidines) [26] that when R = Me protonation will take place under kinetic control from the face opposite to the bulky pyrrolidine substituent, leading to the second iminium intermediate C as the major diastereomer. Fast, irreversible hydrolysis and cyclization of this intermediate would predominantly give the (4R) enantiomer of 8aa. When the steric bulk of the R substituent is increased (7b or 7c), the formation of the diastereomeric iminium ion C’ will be relatively favoured (in this intermediate the steric interactions of the α-substituent R with the pyrrolidine substituent are minimized), resulting of the competitive formation of the (4S) enantiomer of the pyrazolidinol 8ab or 8ac.

When the asymmetric synthesis of isoxazolidinones 12a–12c was attempted by means of the use of the chiral catalysts 13 and 14 in the reactions between enals 7a–7c and N-Cbz-hydroxylamine 10, the results were much less satisfactory, since after oxidation of the intermediate isoxazolidinols 11a–11c to the corresponding isoxazolidinones 12, we were not able to find suitable HPLC conditions for the determination of the enantiomeric purity of 12a; on the other hand, 12b was obtained in essentially racemic form with both catalysts, and scalemic 12c could be prepared but only in low enantiomeric purity (57:43 er) when using 14 as the chiral catalyst (see details in the Experimental Section).

2.2. Primary Antimicrobial Screening

Finally, and in the framework of the Community for Open Antimicrobial Drug Discovery (CO-ADD) program [31], the new heterocyclic systems synthesized by us (both in racemic and when possible in enantiomerically enriched form) were submitted to a primary antimicrobial screening study by whole cell growth inhibition assays. The inhibition of growth was measured against five bacteria: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus, and two fungi: Candida albicans and Cryptococcus neoformans. The results of the study are summarized in

Table 4. Active compounds ¹ in the primary antimicrobial screening (bacterial and fungal).

Entry	S. aureus	E. coli	K. pneumoniae	A. baumannii	P. aeruginosa	C. albicans	C. neoformans
1	-	-	-	-	-	12b	-
2	8aa (rac)	-	-	-	-	-	-
3	8aa (90:10 er)	-	-	-	-	-	-
4	-	-	-	-	-	-	12a
5	-	-	-	-		11c	-

¹ Compounds showing inhibition > 80% and Z-score > 2.5 in two replicate experiments, at a concentration of 32 μg/mL (water/DMSO).

, which shows the active compounds found for each microbial strain.

Among the samples tested, five showed antimicrobial activity and have been selected for hit confirmation. It is worth noting that antifungal activity was found only for isoxazolidinones (12a and 12b) and for isoxazolidinol 11c, and that the pyrazolidinol 8aa, both in racemic and in enantiomerically enriched form, was active against Staphylococcus aureus. The fact that neither the corresponding pyrazolidinone 9aa nor the 4-methyl-1,2-bis-(p-toluenesulfonyl)pyrazolidin-3-ol 8ba show any growth inhibition for the same bacterial strain strongly suggests that 4-methyl-1-(4-nitrobenzenesulfonyl)-4,5-dihydro-1H-pyrazole, resulting from acid-catalyzed deprotection/ dehydration of 8aa [20], could be the biologically active species.

3. Experimental Section

3.1. General Information

Reactions were generally performed at room temperature, either in round-bottomed flasks or in loosely stoppered glass vials, with magnetic stirring and open to the air. Commercially available reagents, catalysts, and solvents were used as received with the exception of dichloromethane, which was distilled from calcium hydride under nitrogen. Aldehydes 1 [32] and 7b–7d [33,34] were prepared according to literature procedures. Yields refer to products isolated after chromatographic purification. Reactions were monitored both by ¹H-NMR and by thin-layer chromatography, carried out on silica gel plates Merck 60 F254 (Sigma-Aldrich Química SL, Madrid, Spain), and compounds were visualized by irradiation with UV light and/or treatment with a solution of KMnO₄ as developing agent followed by heating. Flash column chromatography was performed using silica gel Merck 60 (particle size: 0.040–0.063 mm). ¹H (400 MHz) and ¹³C (100.6 MHz) NMR spectra were recorded with a Mercury 400 spectrometer (Varian Inc., Palo Alto, CA, USA). Chemical shifts (δ) are given in ppm relative to tetramethylsilane (TMS), and coupling constants (J) are given in Hz. The spectra were recorded in CDCl₃ as solvent at room temperature. TMS served as an internal standard (δ = 0.00 ppm) for ¹H-NMR spectra, and CDCl₃ (δ = 77.0 ppm) for ¹³C-NMR spectra. Data are reported as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad signal. Where appropriate, 2D techniques (COSY, NOESY) were also used to assist in structure elucidation. High-resolution mass spectra (HRMS) were recorded with a MicrOTOF spectrometer (Bruker, Billerica, MA, USA) by the Unitat d’Espectrometria de Masses, CCiT-UB. Specific rotations were determined at room temperature with a 241 MC polarimeter (Perkin-Elmer, Waltham, MA, USA). Chiral HPLC analyses were performed with a LC Series 20 apparatus (Shimadzu Corp., Kyoto, Japan) with an M20 diode array UV/Vis detector, using Chiralpak^® IC, IA and IB columns (Daicel Corporation, Tokio-Osaka, Japan). The homogeneity of the peaks corresponding to the two enantiomers of the product was thoroughly checked by comparison of the UV spectra.

3.2. General Procedures for the Preparation of Pyrazolidines

3.2.1. Racemic Pyrazolidinols from Enals and Hydrazines

An ordinary glass vial equipped with a magnetic stirring bar was charged with pyrrolidine (1.42 mL, 14 mg, 0.20 mmol, 40 mol %), PhCOOH (24 mg, 0.20 mmol, 40 mol %) and toluene (2 mL). The hydrazine 2a or 2b (0.50 mmol, 1 eq) and the α,β-unsaturated aldehyde (1.0 mmol, 2 eq) were added sequentially. Stirring was maintained at room temperature until the reaction was complete (TLC and or ¹H-NMR monitoring, 24 h–4 days) and the crude reaction mixture was diluted with 10 mL of ethyl acetate. The organic phase was washed first with 10% w/w aqueous sodium bicarbonate solution (10 mL), then with brine (10 mL), and dried over MgSO₄. Filtration and evaporation of solvents under reduced pressure afforded the crude reaction product that was purified by column chromatography (silica gel; hexane/ethyl acetate mixtures of increasing polarity) to afford the intermediate pyrazolidinol as a diastereomer mixture.

3.2.2. Asymmetric Synthesis of Pyrazolidinols from Enals and Hydrazines

An ordinary glass vial equipped with a magnetic stirring bar was charged with the chiral prolinol silyl ether 13 or 14 (0.05 mmol, 20 mol %), PhCOOH (6 mg, 0.05 mmol, 20 mol %) and toluene (1 mL). The hydrazine 2a or 2b (0.24 mmol, 1 eq) and the α,β-unsaturated aldehyde (0.48 mmol, 2 eq) were added sequentially. The stirring was maintained at room temperature until the reaction was complete (TLC and or ¹H-NMR monitoring, 3–6 days) and the crude reaction mixture was diluted with 10 mL of ethyl acetate. The organic phase was washed first with 10% w/w aqueous sodium bicarbonate solution (10 mL), then with brine (10 mL), and dried over MgSO₄. Filtration and evaporation of solvents under reduced pressure afforded the crude reaction product that was purified by column chromatography (silica gel; hexane/ethyl acetate mixtures of increasing polarity) to afford the intermediate pyrazolidinol as a diastereomer mixture.

3.2.3. Pyrazolidinones by Oxidation of Pyrazolidinols

The pyrazolidinol diastereomer mixture (0.15 mmol) and pyridinium chlorochromate PCC (158 mg, 0.73 mmol, 5 eq) were added sequentially to a stirred suspension of activated 4 Å molecular sieves (300 mg) in anhydrous dichloromethane (2 mL), and the reaction mixture was stirred at room temperature until completion (24–48 h). After the addition of diethyl ether (10 mL) to precipitate the chromium salts, the solution was decanted and filtered through a short pad of Celite^® eluting with a 1:10 mixture of ethyl acetate and diethyl ether). After removal of the solvents in vacuo, the reaction product was purified by column chromatography (silica gel, hexane/ethyl acetate mixtures) to give the desired pyrazolidinone.

3.3. General Procedures for the Preparation of Isoxazolidines

3.3.1. Racemic Isoxazolidinols from Enals and Hydroxylamine 10

Pyrrolidine (from a 10 mg/mL solution in toluene; 1.48 mL, 0.21 mmol, 40 mol %) and N-Cbz-hydroxylamine 10 (87 mg, 0.52 mmol, 1 eq) were added sequentially to a magnetically stirred solution of the α,β-unsaturated aldehyde 7a, 7b, or 7c (1.04 mmol, 2 eq) in toluene (2 mL), and the resulting solution was stirred at room temperature. The progress of the reaction was monitored both by ¹H-NMR spectroscopy and by TLC. When the starting hydroxylamine 10 was not detected (2 days), toluene and pyrrolidine were removed in vacuo, and the crude residue was directly purified by column chromatography (silica gel; hexane/ethyl acetate mixtures) to give the corresponding isoxazolidinol as a diastereomer mixture.

3.3.2. Asymmetric Synthesis of Isoxazolidinols from Enals and Hydroxylamine 10

The chiral prolinol silyl ether 13 or 14 (0.104 mmol, 20 mol %) and N-Cbz-hydroxylamine 10 (87 mg, 0.52 mmol, 1 eq) were added sequentially to a magnetically stirred solution of the α,β-unsaturated aldehyde 7a, 7b, or 7c (1.04 mmol, 2 eq) in toluene (2 mL), and the resulting solution was stirred at room temperature. The progress of the reaction was monitored both by ¹H-NMR spectroscopy and by TLC. When the starting hydroxylamine 10 was not detected (3 days), toluene was removed in vacuo, and the crude residue was directly purified by column chromatography (silica gel; hexane/ethyl acetate mixtures) to give the corresponding isoxazolidinol as a diastereomer mixture.

3.3.3. Isoxazolidinones by Oxidation of Isoxazolidinols

The isoxazolidinol diastereomer mixture (0.126 mmol) and pyridinium chlorochromate PCC (82 mg, 0.38 mmol, 5 eq) were added sequentially to a stirred suspension of activated 4Å molecular sieves (150 mg) in anhydrous dichloromethane (2 mL), and the reaction mixture was stirred overnight at room temperature. After the addition of diethyl ether (5 mL) to precipitate the chromium salts, the solution was decanted and filtered through a short pad of Celite^® eluting with a 4:1 mixture of hexane and ethyl acetate. After removal of the solvents in vacuo, the reaction product was purified by column chromatography (silica gel, hexane/ethyl acetate mixtures) to give the desired isoxazolidinone.

3.4. Characterization of the Products

tert-Butyl (3RS,3aRS,6aSR)-3-hydroxy-1-((4-nitrophenyl)sulfonyl)hexahydrocyclopenta[c]pyrazole-2(1H)-carboxylate, 3. Obtained in 87% yield (450 mg, 1.09 mmol) according to Procedure 3.2.1. Colorless solid, m.p. 83–85 °C (from 1:9 chloroform/hexanes). ¹H-NMR (CDCl₃): δ = 1.24 (s, 9H), 1.47–1.54 (m, 3H), 1.67–1.75 (m, 1H), 1.77–1.83 (m, 1H), 1.86–1.95 (m, 1H), 2.72–2.77 (m, 1H), 3.38 (br d, 1H, OH), 4.73–4.78 (m, 1H), 5.42 (m, 1H), 8.11 (d, J = 8.0 Hz, 2H), 8.29 (d, J = 8.0 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 24.5, 27.8, 30.5, 33.7, 53.6, 62.2, 65.5, 83.0, 91.9, 123.7, 131.1, 141.8, 150.7 ppm. HRMS (ESI): Calculated for C₁₇H₂₇N₄O₇S [M + NH₄]⁺ = 431.1595; found, 431.1592.

tert-Butyl 3,4-trans-5-hydroxy-3,4-dimethyl-2-((4-nitrophenyl)sulfonyl)-pyrazolidine-1-carboxylate, 5. Obtained as a 6:1 diastereomer mixture in 76% yield (380 mg, 0.95 mmol) according to Procedure 3.2.1. Colorless solid, m.p. 71–73 °C (major isomer, 1:9 chloroform/hexanes). ¹H-NMR (CDCl₃): δ = 1.00 (d, J = 7.0 Hz, 3H), 1.24 (s, 9H), 1.36 (d, J = 7.0 Hz, 3H), 1.80–1.87 (m, 1H), 2.86 (br d, 1H, OH), 3.72–3.79 (m, 1H), 5.46–5.48 (m, 1H), 8.07 (d, J = 8.0 Hz, 2H), 8.28 (d, J = 8.0 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 11.1, 20.7, 27.81, 27.83, 47.4, 62.2, 83.2, 86.6, 123.6, 130.9, 141.8, 150.6, 154.7 ppm. HRMS (ESI): Calculated for C₁₆H₂₄N₃O₇S [M + H]⁺ = 402.1337; found, 402.1340.

tert-Butyl 5-hydroxy-4-methyl-2-((4-nitrophenyl)sulfonyl)-pyrazolidine-1-carboxylate, 8aa. The racemic compound was obtained as a 4:1 diastereomer mixture in 93% yield (180 mg, 0.47 mmol) according to Procedure 3.2.1. Colorless solid, m.p. 111–114 °C (major isomer, 1:9 chloroform/hexanes). ¹H-NMR (CDCl₃): δ = 1.09 (d, J = 7.0 Hz, 3H), 1.35 (s, 9H), 2.25–2.30 (m, 1H), 3.03–3.09 (m, 1H), 3.22 (br d, 1H, OH), 3.29–3.36 (m, 1H), 3.83–3.87 (m, 1H), 5.47 (m, 1H), 8.12 (d, J = 8.0 Hz, 2H), 8.34 (d, J = 8.0 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 15.0, 27.9, 43.0, 55.3, 83.2, 91.9, 123.9, 131.0, 141.8, 150.8, 154.5 ppm. HRMS (ESI): Calculated for C₁₅H₂₁N₃NaO₇S [M + Na]⁺ = 410.0992; found, 410.1000. When using 14 as a catalyst and following Procedure 3.2.2, scalemic 8aa was obtained in 98.6% yield (3:1 diastereomer mixture). Colorless solid, m.p. = 135–137 °C (major isomer, 1:9 chloroform/hexanes).

(−)-tert-Butyl 4-methyl-2-((4-nitrophenyl)sulfonyl)-pyrazolidin-5-one-1-carboxylate, 9aa. Oxidation of 8aa obtained by catalyst 14 gave (−)-9aa (90:10 er) in 94% yield (101 mg, 0.26 mmol) according to Procedure 3.2.3. Colorless solid, m.p. 131–134 °C (from 1:9 chloroform/hexanes). ¹H-NMR (CDCl₃): δ = 1.16 (d, J = 7.0 Hz, 3H), 1.47 (s, 9H), 2.54–2.64 (m, 1H), 3.38–3.45 (m, 1H), 4.40–4.45 (m, 1H), 8.18 (d, J = 8.0 Hz, 2H), 8.41 (d, J = 8.0 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 13.4, 27.7, 36.8, 53.7, 85.6, 124.4, 130.7, 141.3, 151.4, 173.4 ppm. HRMS (ESI): Calculated for C₁₅H₁₉N₃NaO₇S [M + Na]⁺ = 408.0836; found, 408.0847. [α]${}_{\mathrm{D}}^{25}$ = −56.0 (1.23, DCM). Conditions for the HPLC analysis: Chiralpak^® IC column, 90:10 hexane/2-propanol, 1 μL/min, 25 °C, λ = 254 nm. t_R (major enant.) = 58.1 min, t_R (minor enant.) = 84.4 min.

tert-Butyl 4-benzyl-5-hydroxy-2-((4-nitrophenyl)sulfonyl)-pyrazolidine-1-carboxylate, 8ab. The racemic compound was obtained in 83% yield (165 mg, 0.36 mmol, >20:1 dr) according to procedure 3.2.1. Colorless solid, m.p. 122–126 °C (major isomer, recrystallized from 1:2:7 dichloromethane/chloroform/hexanes). ¹H-NMR (CDCl₃): δ = 1.36 (s, 9H), 2.53–2.58 (m, 2H), 3.14–3.19 (m, 1H), 3.24 (br d, 1H, OH), 4.01–4.06 (m, 1H), 5.39–5.41 (m, 1H), 7.09 (d, J = 7.8 Hz, 2H), 7.20–7.33 (m, 3H), 8.13 (d, J = 8.0 Hz, 2H), 8.34 (d, J = 8.0 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 27.9, 36.7, 50.1, 53.7, 83.3, 89.9, 123.8, 126.9, 128.4, 128.8, 131.0, 137.8, 141.7, 150.8, 154.3 ppm. HRMS (ESI): Calculated for C₂₁H₂₅N₃NaO₇S [M + Na]⁺ = 486.1305; found, 486.1298. When using 13 as a catalyst and following Procedure 3.2.2, scalemic 8ab was obtained in 77% yield. Colorless solid, m.p. = 132–136 °C (major isomer, 1:2:7 dichloromethane/chloroform/hexanes).

(−)-tert-Butyl 4-benzyl-2-((4-nitrophenyl)sulfonyl)-pyrazolidin-5-one-1-carboxylate, 9ab. Oxidation of 8ab obtained by catalyst 13 gave (−)-9ab (44:56 er) in 98% yield (108 mg, 0.23 mmol) according to Procedure 3.2.3. Colorless solid, m.p. 115–118 °C (recrystallized from 1:2:7 dichloromethane/chloroform/hexanes). ¹H-NMR (CDCl₃): δ = 1.46 (s, 9H), 2.61–2.67 (m, 1H), 2.86–2.92 (m, 1H), 3.17–3.21 (m, 1H), 3.46–3.52 (m, 1H), 4.16–4.21 (m, 1H), 7.07 (d, J = 6.7 Hz, 2H), 7.25–7.32 (m, 3H), 8.13 (d, J = 7.8 Hz, 2H), 8.37 (d, J = 7.8 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 27.7, 34.8, 43.6, 51.7, 85.7, 124.3, 127.2, 128.5, 130.7, 136.7, 141.2, 147.5, 151.4, 172.1 ppm. HRMS (ESI): Calculated for C₄₂H₅₀N₇O₁₄S₂ [2M + NH4]⁺ = 940.2852; found, 940.2847. [α]${}_{\mathrm{D}}^{25}$ = −11.4 (1.19, DCM). Conditions for the HPLC analysis: Chiralpak^® IC column, 90:10 hexane/2-propanol, 1 μL/min, 25 °C, λ = 254 nm. t_R (minor enant.) = 62.4 min, t_R (major enant.) = 73.2 min.

tert-Butyl 4-hexyl-5-hydroxy-2-((4-nitrophenyl)sulfonyl)-pyrazolidine-1-carboxylate, 8ac. Obtained in 99.6% yield as a diastereoisomer mixture (227 mg, 1.00 mmol, 5:1 dr) according to Procedure 3.2.1. Colorless solid, m.p. 83–85 °C. ¹H-NMR (CDCl₃, major isomer): δ = 0.86 (t, J = 7Hz, 3H), 1.25–1.32 (m, 8H), 1.33 (s, 9H), 1.49–1.56 (m, 2H), 2.15–2.19 (m, 1H), 3.01–3.07 (m, 1H), 3.25 (br d, 1H, OH), 4.19–4.24 (m, 1H), 5.25–5.28 (m, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.36 (d, J = 8.0 Hz, 2H) ppm. HRMS (ESI): Calculated for C₄₀H₆₂N₆NaO₁₄S₂ [2M + Na]⁺ = 937.3658; found, 937.3654.

tert-Butyl 4-hexyl-2-((4-nitrophenyl)sulfonyl)-pyrazolidin-5-one-1-carboxylate, 9ac. Obtained in 99% yield (150 mg, 0.33 mmol) according to Procedure 3.2.3. Colorless solid, m.p. 123–126 °C. ¹H-NMR (CDCl₃): δ = 0.87 (t, J = 6.5 Hz, 3H), 1.24–1.30 (m, 9H), 1.47 (s, 9H), 1.78–1.83 (m, 1H), 2.40–2.47 (m, 1H), 3.42–3.49 (m, 1H), 4.37–4.42 (m, 1H), 8.19 (d, J = 7.8 Hz, 2H), 8.42 (d, J = 7.8 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 13.9, 22.5, 26.8, 27.7, 28.8, 29.1, 31.4, 41.8, 52.6, 124.4, 130.7, 141.3, 147.7, 151.3, 172.9 ppm. HRMS (ESI): Calculated for C₂₀H₂₉N₃NaO₇S [M + Na]⁺ = 478.1618; found, 478.1627.

4-Methyl-1,2-bis-(4-toluenesulfonyl)-pyrazolidin-3-ol, 8ba. The racemic compound was obtained as a 8:1 diastereomer mixture in 88% yield (75 mg, 0.18 mmol) according to Procedure 3.2.1. Yellow solid, m.p. 131–134 °C. ¹H-NMR (CDCl₃, major isomer): δ = 0.86 (d, J = 7.0 Hz, 3H), 2.17–2.25 (m, 2H), 2.45 (s, 3H), 2.46 (s, 3H), 3.33–3.40 (m, 1H), 4.04–4.11 (m, 1H), 5.21–5.22 (m, 1H), 7.29–7.33 (m, 4H), 7.70 (d, J = 8.0 Hz, 2 H), 7.82 (d, J = 8.0 Hz, 2 H) ppm. HRMS (ESI): Calculated for C₃₆H₄₄N₄NaO₁₀S₄ [2M + Na]⁺ = 843.1832; found, 843.1832. When using 13 as a catalyst and following Procedure 3.2.2, scalemic 8ba was obtained in 89.4% yield (8:1 diastereomer mixture).

(−)-4-Methyl-1,2-bis-(4-toluenesulfonyl)-pyrazolidin-3-one, 9ba. Oxidation of 8ba obtained by catalyst 13 gave (−)-9ba (96:4 er) in 98% yield (58 mg, 0.14 mmol) according to Procedure 3.2.3. Colorless solid, m.p. 156–160 °C (recrystallized from 1:2:7 dichloromethane/chloroform/hexanes). ¹H-NMR (CDCl₃): δ = 0.76 (d, J = 7.0 Hz, 3H), 2.12–2.23 (m, 1H), 2.46 (s, 6H), 2.83–2.90 (m, 1H), 4.28–4.33 (m, 1H), 7.34–7.36 (m, 4H), 7.89 (d, J = 8.1 Hz, 2H), 7.89 (d, J = 8.1 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 15.3, 21.8, 34.7, 65.8, 127.0, 128.3, 128.7, 128.8, 129.6, 129.8, 130.1, 146.0, 146.2, 176.5 ppm. HRMS (ESI): Calculated for C₃₆H₄₀N₄NaO₁₀S₂ [2M + Na]⁺ = 839.1519; found, 839.1516. [α]${}_{\mathrm{D}}^{25}$ = −64.7 (0.67, DCM). Conditions for the HPLC analysis: Chiralpak^® IA column, 90:10 hexane/2-propanol, 1 μL/min, 25 °C, λ = 254 nm. t_R (major enant.) = 33.6 min, t_R (minor enant.) = 51.5 min.

4-Benzyl-1,2-bis-(4-toluenesulfonyl)-pyrazolidin-3-ol, 8bb. The racemic compound was obtained as a diastereomer mixture (>9:1 dr) in 85% yield (85 mg, 0.18 mmol) according to Procedure 3.2.1. Yellow solid, m.p. 110–113 °C. ¹H-NMR (CDCl₃, major isomer): δ = 2.14–2.20 (m, 1H), 2.35–2.42 (m, 2H), 2.46 (s, 3H), 2.47 (s, 3H), 2.82–2.87 (m, 1H), 3.45–3.47 (br d, 1H, OH), 3.89–3.94 (m, 1H), 5.36–5.40 (m, 1H), 7.28–7.31 (m, 5H), 7.67 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H) ppm. HRMS (ESI): Calculated for C₄₈H₅₆N₅O₁₀S₄ [2M + NH₄]⁺ = 990.2805; found, 990.2805. When using 13 or 14 as catalysts and following Procedure 3.2.2, scalemic 8bb was obtained in the same yield and diastereomeric ratio.

4-Benzyl-1,2-bis-(4-toluenesulfonyl)-pyrazolidin-3-one, 9bb. Oxidation of 8bb according to Procedure 3.2.3 gave 9bb in 97% yield (58 mg, 0.12 mmol). Yellow solid, m.p. 156–160 °C. ¹H-NMR (CDCl₃): δ = 2.01–2.07 (m, 1H), 2.45–2.47 (m, 1H), 2.47 (s, 3H), 2.48 (s, 3H), 2.90–2.95 (m, 1H), 2.98–3.01 (m, 1H), 4.05–4.011 (m, 1H), 6.81 (m, 1H), 7.20 (m, 3H), 7.34 (d, J = 6 Hz, 4H), 7.73 (d, J = 7.6 Hz, 2H), 7.86 (d, J = 7.6 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 21.79, 21.82, 34.7, 42.7, 54.0, 127.0, 128.3, 128.7, 128.8, 129.6, 129.8, 130.1, 131.2, 134.67, 134.73, 146.0, 146.2, 176.5 ppm. HRMS (ESI): Calculated for C₂₄H₂₈N₃NaO₅S₂ [M + NH₄]⁺ = 502.1465; found, 502.1466. No satisfactory conditions for the chiral HPLC analysis of this compound could be found.

4-Hexyl-1,2-bis-(4-toluenesulfonyl)-pyrazolidin-3-ol, 8bc. The racemic compound was obtained as a diastereomer mixture (10:1 dr) in 98% yield (97 mg, 0.20 mmol) according to Procedure 3.2.1. Yellow solid, m.p. 78–80 °C. ¹H-NMR (CDCl₃, major isomer): δ = 0.87 (t, J = 7.0 Hz, 3H), 0.95–1.00 (m, 1H), 1.13–1.18 (m, 6H), 1.22–1.27 (m, 3H), 2.01–2.14 (m, 1H), 2.44 (s, 3H), 2.45 (s, 3H), 2.85–2.87 (m, 1H), 3.35–3.37 (br d, 1H, OH), 4.08–4.13 (m, 1H), 5.24–5.28 (m, 1H), 7.27–7.32 (m, 4H), 7.68 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H) ppm. HRMS (ESI): Calculated for C₂₃H₃₂N₂NaO₅S₂ [M + Na]⁺ = 503.1642; found, 503.1645. When using 13 as a catalyst and following Procedure 3.2.2, scalemic 8bc was obtained in 93% yield (104 mg, 0.23 mmol; 10:1 dr).

(−)-4-Hexyl-1,2-bis-(4-toluenesulfonyl)-pyrazolidin-3-one, 9bc. Oxidation of 8bc (obtained by catalyst 13) according to Procedure 3.2.3 gave (−)-9bc (78:22 er) in 98% yield (58 mg, 0.12 mmol). Colourless solid, m.p. 160–164 °C. ¹H-NMR (CDCl₃): δ = 0.83–0.86 (m, 5H), 1.05–1.12 (m, 4H), 1.19–1.25 (m, 3H), 1.40–1.52 (m, 1H), 2.08–2.11 (m, 1H), 2.45 (s, 3H), 2.46 (s, 3H), 2.93–3.00 (m, 1H), 4.24–4.29 (m, 1H), 7.33–7.36 (m, 4H), 7.78 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 8.0 Hz, 2H) ppm. ¹³C-NMR (CDCl₃): δ = 13.9, 21.7, 21.8, 22.4, 26.2, 28.6, 28.7, 31.3, 41.0, 54.1, 128.7, 129.6, 129.8, 130.1, 131.3, 134.8, 145.9, 146.2, 177.5 ppm. HRMS (ESI): Calculated for C₂₃H₃₀N₂NaO₅S₂ [M + Na]⁺ = 501.1488; found, 501.1491. [α]${}_{\mathrm{D}}^{25}$ = −8.0 (1.01, DCM). Conditions for the HPLC analysis: Chiralpak^® IB column, 93:7 hexane/2-propanol, 1 μL/min, 25 °C, λ = 220 nm. t_R (major enant.) = 21.5 min, t_R (minor enant.) = 27.6 min.

Benzyl 5-hydroxy-4-methyl-isoxazolidine-2-carboxylate, 11a. The racemic compound was obtained as a 2:1 diastereomer mixture in 83% yield (102 mg, 0.43 mmol) according to Procedure 3.3.1. Colorless oil. ¹H-NMR (CDCl₃, major isomer): δ = 1.09 (d, J = 7 Hz, 3H), 2.60–2.67 (m, 1H), 3.61–3.64 (m, 1H), 4.26–4.29 (m, 1H), 5.16 (br s, 2H), 5.34–5.35 (m, 1H), 7.32–7.35 (m, 5H) ppm. ¹H-NMR (CDCl₃, minor isomer): δ = 1.13 (d, J = 7.0 Hz, 3H), 2.55–2.59 (m, 1H), 3.78–3.82 (m, 1H), 4.14–4.17 (m, 1H), 5.22 (br s, 2H), 5.55–5.56 (m, 1H), 7.37–7.39 (m, 5H) ppm. ¹³C-NMR (CDCl₃): δ = 9.4, 14.7, 40.4, 44.1, 67.8, 68.1, 73.5, 74.9, 82.1, 88.0, 128.29, 128.34, 128.43, 128.46, 128.5, 128.6, 135.38, 135.44, 141.8, 155.3, 159.0 ppm. When using 13 or 14 as catalysts and following Procedure 3.3.2, scalemic 11a was obtained in similar yield and diastereomer ratio.

Benzyl 4-methyl-5-oxo-isoxazolidine-2-carboxylate, 12a. Oxidation of 11a gave 12a in 98% yield (50 mg, 0.22 mmol) according to Procedure 3.3.3. Colorless oil. ¹H-NMR (CDCl₃): δ = 1.29 (d, J = 6.8 Hz, 3H), 3.01–3.11 (m, 1H), 3.96–4.01 (m, 1H), 4.54–4.58 (m, 1H), 5.33 (br s, 2H), 7.33–7.45 (m, 5H) ppm.¹³C-NMR (CDCl₃): δ = 12.2, 39.4, 68.8, 73.9, 98.0, 128.54, 128.6, 134.5, 147.7, 169.9 ppm. HRMS (ESI): Calculated for C₁₂H₁₄NO₄ [M + H]⁺ = 236.0917; found, 236.0916. No satisfactory conditions could be found for the chiral HPLC analysis of this compound.

Benzyl 4-benzyl-5-hydroxy-isoxazolidine-2-carboxylate, 11b. The racemic compound was obtained as a 3:1 diastereomer mixture in 79% yield (128 mg, 0.41 mmol) according to Procedure 3.3.1. Colorless oil. ¹H-NMR (CDCl₃, major isomer): δ = 2.74–2.78 (m, 2H), 3.02–3.06 (m, 1H), 3.57–3.61 (m, 1H), 3.77–3.80 (m, 1H), 4.19–4.23 (m, 1H), 5.25 (br s, 2H), 5.47–5.48 (m, 1H), 7.31–7.41 (m, 10H) ppm. ¹H-NMR (CDCl₃, minor isomer): δ = 2.59–2.68 (m, 2H), 2.94–2.98 (m, 1H), 3.01–3.27 (m, 1H), 3.67–3.71 (m, 1H), 4.01–4.05 (m, 1H), 5.36 (br s, 2H), 5.56–5.57 (m, 1H), 7.13–7.30 (m, 10H) ppm. ¹³C-NMR (CDCl₃): δ = 23.5, 31.2, 35.8, 48.0, 48.8, 51.0, 68.1, 71.9, 72.9, 81.3, 86.1, 97.2, 101.4, 126.5, 126.7, 128.1, 128.2, 128.4, 128.5, 128.6, 128.7, 135.5, 135.7, 138.2, 138.9, 155.2, 158.8 ppm.

Benzyl 4-benzyl-5-oxo-isoxazolidine-2-carboxylate, 12b. Oxidation of 11b gave 12b in 99% yield (68 mg, 0.22 mmol) according to Procedure 3.3.3. Colorless oil. ¹H-NMR (CDCl₃): δ = 2.78–2.84 (m, 1H), 3.24–3.28 (m, 2H), 4.07–4.11 (m, 1H), 4.35–4.39 (m, 1H), 5.33 (br s, 2H), 7.15–7.43 (m, 10H) ppm.¹³C-NMR (CDCl₃): δ = 30.7, 43.0, 65.7, 68.8, 123.9, 125.3, 125.4, 125.5, 125.7, 131.3, 134.0, 144.4, 165.4 ppm. HRMS (ESI): Calculated for C₁₈H₂₁N₂O₄ [M + NH₄]⁺ = 329.1496; found, 329.1494.

Benzyl 4-hexyl-5-hydroxy-isoxazolidine-2-carboxylate, 11c. The racemic compound was obtained as a 3:1 diastereomer mixture in 78% yield (120 mg, 0.39 mmol) according to Procedure 3.3.1. Colorless oil. ¹H-NMR (CDCl₃, major isomer): δ = 0.88 (d, J = 7.0 Hz, 3H), 1.28–1.31 (m, 9H), 1.34–1.38 (m, 1H), 2.45–2.55 (m, 2H), 3.62–3.66 (m, 1H), 4.26–4.28 (m, 1H), 5.22 (br s, 2H), 5.39–5.40 (m, 1H), 7.37–7.40 (m, 5H) ppm. ¹H-NMR (CDCl₃, minor isomer): δ = 0.87 (d, J = 7.0 Hz, 3H), 1.34–1.38 (m, 9H), 3.15–3.19 (m, 2H), 3.81–3.86 (m, 1H), 4.02–4.25 (m, 1H), 5.33 (br s, 2H), 5.39–5.40 (m, 1H), 7.32–7.36 (m, 5H) ppm. When using 14 as a catalyst and following Procedure 3.3.2, scalemic 11c was obtained in 87% yield (201 mg, 0.65 mmol) and in a 3:1 diastereomer ratio.

(−)-Benzyl 4-hexyl-5-oxo-isoxazolidine-2-carboxylate, 12c. Oxidation of 11c obtained with catalyst 14 gave (−)-12c (57:43 er) in 87% yield (87 mg, 0.28 mmol) according to Procedure 3.3.3. Colorless oil. ¹H-NMR (CDCl₃): δ = 0.88 (d, J = 7.0 Hz, 3H), 1.28–1.40 (m, 8H), 1.50–1.58 (m, 1H), 1.88–1.93 (m, 1H), 2.92–2.99 (m, 1H), 4.04–4.08 (m, 1H), 4.52–4.56 (m, 1H), 5.33 (br s, 2H), 7.33–7.45 (m, 5H) ppm. ¹³C-NMR (CDCl₃): δ = 14.0, 22.5, 27.0, 28.1, 29.0, 31.5, 44.5, 68.8, 72.6, 128.5, 128.6, 128.7, 134.6, 147.7, 169.5 ppm. HRMS (ESI): Calculated for C₃₄H₄₆N₂NaO₈ [2M + Na]⁺ = 633.3146; found, 633.3148. [α]${}_{\mathrm{D}}^{25}$ = −4.1 (0.41, DCM). Conditions for the HPLC analysis: Chiralpak^® IB column, 90:10 hexane/2-propanol, 1 μL/min, 25 °C, λ = 254 nm. t_R (minor enant.) = 13.6 min, t_R (major enant.) = 16.3 min.

3.5. Methods for the Biological Activity Studies

3.5.1. Sample Preparation

Samples were provided as dry material, and were made to 10 mg/mL in DMSO solution and stored at −20 °C. An aliquot of each sample was diluted to 320 μg/mL in water, and plated in 384-well polypropylene plates. 5 μL was plated in duplicate into a 384-well non-binding surface plate for each strain or cell type assayed against. Once cells were added this gave a final compound concentration range of 32 μg/mL. The final DMSO concentration was 0.3%.

3.5.2. Antimicrobial Assay

Procedure

All bacteria were cultured in Cation-adjusted Müller Hinton broth (CAMBH) at 37 °C overnight. A sample of each culture was then diluted 40-fold in fresh broth and incubated at 37 °C for 1.5–3 h. The resulting mid-log phase cultures were diluted (CFU/mL measured by OD₆₀₀), and then 45 μL was added to each well of the compound containing plates, giving a cell density of 5 × 10⁵ CFU/mL and the nominated final compound concentration. All the plates were covered and incubated at 37 °C for 18 h without shaking.

Analysis

Inhibition of bacterial growth was determined measuring absorbance at 600 nm (OD₆₀₀), using a M1000 Pro monochromator plate reader (Tecan, Männedorf, Switzerland). The percentage of growth inhibition was calculated for each well, using negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references. The significance of the inhibition values was determined by Z-scores, calculated using the average and standard deviation of the sample wells (no controls) on the same plate. Samples with inhibition value above 80% and Z-Score above 2.5 (n = 2 on different plates) for either replicate were classed as actives.

3.5.3. Antifungal Assay

Procedure

Fungi strains were cultured for 3 days on Yeast Extract-Peptone Dextrose (YPD) agar at 30 °C. A yeast suspension of 1 × 10⁶ to 5 × 10⁶ cells/mL (as determined by OD₃₅₀) was prepared from five colonies. These stock suspensions were diluted with Yeast Nitrogen Base (YNB) broth to a final concentration of 2.5 × 10³ CFU/mL. Then, 45 μL of the fungi suspension was added to each well of the compound-containing plates, giving a final concentration of 32 μg/mL for the tested samples. Plates were covered and incubated at 35 °C for 24 h without shaking.

Analysis

Growth inhibition of C. albicans was determined measuring absorbance at 530 nm (OD₅₃₀), while the growth inhibition of C. neoformans was determined measuring the difference in absorbance between 600 and 570 nm (OD_600–570), after the addition of resazurin (0.001% final concentration) and incubation at 35 °C for additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate reader. The percentage of growth inhibition was calculated for each well, using negative control (media only) and positive control (fungi without inhibitors) on the same plate as references. The significance of the inhibition values was determined by Z-scores, calculated using the average and standard deviation of the sample wells (no controls) on the same plate. Samples with inhibition value above 80% and Z-Score above 2.5 (n = 2 on different plates) for either replicate were classed as actives.

3.5.4. Antibiotic Standards Preparation and Quality Control

Colistin and vancomycin were used as positive bacterial inhibitor standards for Gram-negative and Gram-positive bacteria, respectively. Fluconazole was used as a positive fungal inhibitor standard both for C. albicans and C. neoformans.

The antibiotics were provided in four concentrations, with two above and two below its MIC value, and plated into the first 8 wells of column 23 of the 384-well NBS plates. The quality control (QC) of the assays was determined by the antimicrobial controls and the Z’-factor (using positive and negative controls). Each plate was deemed to fulfill the quality criteria (pass QC), if the Z’-factor was above 0.4, and the antimicrobial standards showed full range of activity, with full growth inhibition at their highest concentration, and no growth inhibition at their lowest concentration.

3.5.5. Materials

Both the compound preparation plate and the assay plates were purchased from Corning (Corning, NY, USA). CAMHB from Bacto Laboratories (Mount Pritchard, Australia) was used as growth media for bacteria; culture agar and growth media for fungi were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Resazurin was provided by Sigma Aldrich (Sydney, Australia).

3.5.6. Microbial Strains

Escherichia coli	ATCC 25922 (FDA control strain)
Klebsiella pneumoniae	ATCC 700603 (MDR)
Acinetobacter baumannii	ATCC 19606 (type strain)
Pseudomonas aeruginosa	ATCC 27853 (Quality control strain)
Staphylococcus aureus	ATCC 43300 (MRSA)
Candida albicans	ATCC 90028 (CLSI reference)
Cryptococcus neoformans	ATCC 208821 (H99—Type strain)

4. Conclusions

The pyrrolidine-catalyzed reaction of the α-substituted enals methacrolein (7a), 2-benzylpropenal (7b), and 2-(n-hexyl)propenal (7c) with the activated hydrazines 1-Boc-2-(4-nitrobenzenesulfonyl)hydrazine (2a) and 1,2-bis(p-toluenesulfonyl)hydrazine (2b) takes place in very good yields (83%–99.6%) in toluene at room temperature and in the presence of benzoic acid as a co-catalyst, to afford the 4-substituted pyrazolidin-3-ols 8aa–8bc (generally as diastereomer mixtures); oxidation of these compounds with PCC leads to the corresponding-4-substituted-3-pyrazolidinones 9aa–9bc in essentially quantitative yields. In the same conditions α-substituted acroleins 7a–7c react smoothly with N-Cbz-hydroxylamine 10 to afford the isoxazolidinols 11 in good yields (78%–83% isolated yields) as diastereomer mixtures. Subsequent oxidation with PCC gives rise to the 4-substituted isoxazolidin-5-ones 12a–12c. The use of chiral diarylprolinol trimethylsilyl ethers 13 and 14 as catalysts allows the synthesis of several of these compounds in optically active form, in some cases with excellent enantioselectivity (up to 96:4 er). The 4-substituted pyrazolidinols, pyrazolidinones, isoxazolidinols and isoxazolidinones (both in racemic and when possible in enantiomerically enriched form) have been submitted to a primary antimicrobial screening study (antibacterial and antifungal) by whole cell growth inhibition assays. Antifungal activity has been found for the isoxazolidinones 12a and 12b, and for isoxazolidinol 11c, and the 4-methyl-pyrazolidinol 8aa, both in racemic and in enantiomerically enriched form, is highly active against Staphylococcus aureus.