A base promoted multigram synthesis of aminoisoxazoles: valuable building blocks for drug discovery and peptidomimetics

Abstract

A practical multigram metal free synthesis of isoxazole-containing building blocks from commercially available amino acids was elaborated. The key reaction was a regioselective [3 + 2]-cycloaddition of in situ generated nitrile oxides with alkynes/enamines. The obtained building blocks were used in the preparation of bioactive compounds and peptidomimetics.

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Introduction

The isoxazole core is a structural element in many commercial pharmaceuticals (Fig. 1). The range of biological activities of the isoxazole-containing drugs is broad: analgesic,¹ anti-inflammatory,^1a,c,2 anthelmintic,³ anti-depressant,⁴ anti-bacterial,⁵ anticancer,⁶ insecticidal,⁷ nootropic,⁸ anxiolytic,^8a neuroprotective,⁹ and HIV-inhibitory agents.¹⁰ In this context, the development of practical methods towards isoxazole-based building blocks from common cheap starting materials is truly desirable.

Fig. 1 Commercialized biologically active derivatives of isoxazole.

Nitrile oxides are useful intermediates towards the synthesis of a wide plethora of isoxazole-based architectures.^11a–h However; most of these methods either utilize toxic metals or hypervalent iodine compounds^11h which in turn, are not environmentally friendly. At the same time, there are just a few known reports dealing with metal-free conditions and most common are based on the use of triethylamine^12a–b and sodium perchlorate/triethylamine.^12c Therefore, the development of metal-free protocols towards the synthesis of compounds bearing isoxazole core is of particular interest.^12d,e

The use of amino acids^13a–h in construction of isoxazole-containing compounds has been known before. However, these reports, dealt with the specific amino acids, and the studies have not been performed systematically on large variety of substrates. Taking into account the need of exploration of metal-free protocols as well as search for easily available substrates, herein we would like to elaborate on a general practical multigram metal-free synthesis of isoxazole-core building blocks starting from commonly available and diverse α-, β- and γ-amino acids.

Results and discussion

Synthesis

1. Synthesis of chloroximes from amino acids. The initial synthesis step was reduction of the amino acid carboxyl-groups in a number of N-Boc protected amino acids. Resulting N-Boc amino alcohols were subsequently oxidized into the corresponding aldehydes (Table 1). These aldehydes formed oximes upon treatment with hydroxylamine under mildly basic conditions (sodium hydrogen carbonate). The oximes were then treated with N-chlorosuccinimide under acidic conditions to afford the target N-Boc protected chloroximes 1–7 in good yields. The products were white powders that could be stored at room temperature for at least several months as exemplified in Table 1.

Table 1 Chloroximes synthesized starting from N-Boc amino acids

Boc-amino acid		Chloroxime		Yield^a	Storage^b
a Overall yield over four steps.b The products were stored in closed flasks at the room temperature.c The products were stored at 0 °C under argon. After six months ca. 10% decomposition was found according to ^1H NMR.
Boc-Gly		1		49%	>2 years
Boc-(S)-Ala		2		68%	≤0.5 years^c
Boc-(R)-Ala		3		67%	≤0.5 years^c
Boc-(S)-Pro		4		70%	>2 years
Boc-(R)-Pro		5		71%	>2 years
Boc-Aze		6		65%	>2 years
Boc-Pip		7		73%	>2 years

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2. Synthesis of N-Boc aminoisoxazoles via [3 + 2]-cycloaddition. It is worth mentioning that our methodology for the synthesis of variety isoxazoles is known and here we would just mention some selected examples where the similar conditions like base or reaction temperatures were applied. Johnson et al. reported on the regioselective synthesis of 3,4-disubstituted isoxazoles between enolsilanes and nitrile oxides.¹⁴ In the same vein, the Zhu group and later Wang and co-workers reported on the synthesis of 3,4-disubstituted isoxazoles in high yields.^15a,b The key point of the reported protocols was the reaction of in situ generated enamines from corresponding ketones or β-keto esters and nitrile oxides. Caddick and co-workers reported and interesting strategy towards the synthesis of 3,5-disubstituted isoxazoles bearing sulfonamide group at the 5-position.¹⁶ Later the Shibata group developed the strategy for the synthesis of isoxazole triflones.¹⁷ Hamme et al. reported on the synthesis of 3,5-disubstituted isoxazoles with electron withdrawing group at the 5-position by the reaction of 1,1-disubstituted bromoalkenes with nitrile oxides.^18a,b Last but not least Schmidt and co-workers reported on the regioselective synthesis of 4-trifluorosubstituted isoxazoles in good yields.¹⁹ All the aforementioned methods avoided the usage of metal-mediated reactions which prompted us to follow the similar conditions in order to synthesize a large panel of aminoisoxazoles in large scale.

In our case, synthesis of isoxazoles from the corresponding in situ generated nitrile oxides and alkynes/enamines was analogous to the mentioned above methods. Namely, treatment of chloroximes 1–7 with a mild base (sodium hydrogen carbonate or triethylamine) at either the room temperature or at 0 °C led to generation of corresponding nitrile oxides (Table 2). Subsequent addition of enamines 8–10 (1.2 eq.) to these in situ generated nitrile oxides lead to [3 + 2]-cycloaddition intermediates that readily eliminated dimethylamine to give the 3,4-disubstituted isoxazoles in a regioselective fashion as previously reported with push–pull enamines (Table 2, A).²⁰ In contrast, terminal alkynes 11, 12 afforded a mixture of 3,4-/3,5-isomers ∼30/70 when reaction was performed at the room temperature.²¹ Nevertheless, the same reaction at 0 °C exhibited an improved regioselectivity of ∼10/90, and the major 3,5-disubstituted isomers were easily isolated by column chromatography.²²

Table 2 The synthesis of N-Boc aminoisoxazoles via [3 + 2]-cycloaddition

a Et₃N was used instead of NaHCO₃ to generate the nitrile oxide, 5 eq. of 12 were used.

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While the above described [3 + 2] cycloaddition reactions were performed in ethyl acetate the synthesis also worked well in tetrahydrofuran, dichloromethane, dimethoxymethane or chloroform, though with compromised regioselectivity. At the same time synthesis of isoxazoles in water as environmentally benign solvent is of high interest.²³ It is also worth mentioning, that these reactions were performed under obligatory high dilution – substrate/solvent = 1/10 (wt/v) – otherwise formation of side furoxans was observed.²⁴

As the result we obtained a variety of N-Boc aminoisoxazoles initially starting from amino acids, and the synthetic approach was scalable such that up to 50 g of a product (Boc-6-H) was obtained in a single synthesis run.

3. Synthesis of isoxazole-containing amines and amino acids. We also made several forays toward the cleavage of TMS-group utilizing K₂CO₃ or trifluoroacetic acid (TFA) as TMS-scavengers,²⁵ but unfortunately in our case the described procedure was not that efficient, furnishing the desired products in moderate yields. Moreover, attempts to cleave TMS-group with 4 M HCl in dioxane were also not optimal giving low yields. Though, these forays were not successful, it allowed us to improve our tactics. As a result, we found that cleavage of the TMS-group from the isoxazole core was easily achieved by treatment with catalytic amounts of KHF₂ in methanol–water mixture. Resulting 3-substituted isoxazoles were obtained in excellent yields (Table 3).

Table 3 Cleavage of TMS-group from the isoxazole ring

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As next, cleavage of N-Boc group from all compounds was performed under acidic conditions to afford the target building blocks – aminoisoxazoles (Table 4).

Table 4 Cleavage of the N-Boc protection group from the amino moiety

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Carboxymethyl-substituted isoxazoles were saponificated with sodium hydroxide in methanol to afford corresponding free amino acids in good yields (Table 5).

Table 5 Synthesis of isoxazole-containing amino acids

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The structures of two final compounds were confirmed by X-ray analysis (Fig. 2). Interestingly, the structure of Boc-4A-COMe indicates a repulsion occurring between the carbamate carbonyl oxygen and the isoxazole nitrogen, similar to what has been recently reported for analogous structures with 1,3-oxa/thiazine in place of the isoxasole.²⁶

Fig. 2 X-ray crystal structures of Boc-4A-COMe and Boc-6A-CO₂H.

Practical applications

After having established a practical general synthetic protocol towards isoxazole-containing building blocks, we also aimed to demonstrate applicability of the synthesized structures in other chemical areas.

1. Synthesis of a drug candidate. ABT-418 is a known nootropic agent.^27a Recently, chemists at Abbott Laboratories synthesized its bioisostere 16 in four steps and 7.3% total yield starting from 4 and 2-bromopropene.^27b Herein, we have developed an alternative approach to afford compound 16 (Scheme 1). First, [3 + 2]-cycloaddition between allylbromide and nitrile oxide generated from chloroxine 4 gave isoxazoline 13 in 95% yield. Next, elimination of hydrogen bromide under basic conditions gave isoxazole 14. Cleavage of the N-Boc group produced free amine 15, which was then subjected to reductive amination with aqueous formaldehyde to afford the target product 16 in gram quantities. The overall yield of the synthesis was 55% over 5 steps.

Scheme 1 Synthesis of isoxazole 16 – bioisostere of the known nootropic agent ABT-418.

2. Synthesis of peptidomimetics. We also envisioned that chiral aminoisoxazoles could be used to prepare conformationally restricted peptidomimetics.²⁸ In this case the heterocyclic fragment will act as a rigid peptide bond substitute²⁹ (Scheme 2).

Scheme 2 (a) α-Peptide, and its conformationally-restricted analogue with isoxazole backbone; (b) construction of a post-translationally modified peptide with oxa/thiasole backbone.

In addition, isomeric oxazoles and thiazoles are common in the backbones³⁰ of various ribosomally synthesized and post-translationally modified peptides.³¹ Construction of an azole ring is generally afforded in nature by cyclization of serine or threonine side chain on the downstream amide bond.^32–34 Total synthesis of respective peptidomimetics has been developed in order to provide naturally occurring antimicrobial agents or toxins as well as their chemically modified analogues.^35,36 Though, oxa/thiazole to isoxazole mutations have not been explored. Therefore, next we attempted the synthesis of isoxazole-containing peptidomimetics by solid phase peptide synthesis (SPPS) in order to highlight this opportunity.

Initially we synthesized the Fmoc-protected amino acid Fmoc-AlaIso-OH (17) (Scheme 3). Peptide chains were grown on Rink-amide resin, and TBTU/HOBt/DIPEA system was used for coupling. Only in the case of Ac-Leu-AlaIso-Val-NH₂ (18) we observed minor degree of epimerization (≤15%), whereas in Ac-Leu-Val-AlaIso-NH₂ (19) no racemization was observed.³⁷ In addition, no degradation was found after cleavage from the resin upon treatment with TFA/TIS. Generally the amino acid Fmoc-AlaIso-OH turned out to be compatible with the standard SPPS, allowing synthesis and isolation of both target peptides by conventional methods.³⁸

Scheme 3 Synthesis of isoxazole-containing peptidomimetics 18 and 19.

Conclusions

We have developed a general metal-free practical protocol towards isoxazole-containing building blocks starting from commonly available α-, β- and γ-amino acids. The target products were obtained in up to 50 g scale. The high potential of the developed method was demonstrated by synthesis of compound 16 – bioisostere of the known nootropic agent ABT-418, – and preparation of isoxazole-containing peptidomimetics 18, 19 by solid phase peptide synthesis.

Experimental section

General

All reagents were available from Enamine Ltd. Solvents were purified according to standard procedures. When organic solutions were concentrated under reduced pressure, 35–40 °C bath temperature was used. Column chromatography was performed with silica gel 60 (230–400 mesh) as the stationary phase. ¹H, ¹³C NMR spectra were recorded at the NMR spectrometers operating at 400 and 500 ¹H frequency (101 and 126 MHz for ¹³C experiments). NMR chemical shifts are reported in ppm, in the δ scale and are referenced using residual NMR solvent peaks at 7.26 and 77.16 ppm for ¹H and ¹³C in CDCl₃, 2.50 and 39.52 ppm for ¹H and ¹³C in DMSO-d₆, and 4.79 ppm for ¹H in D₂O. For peptides NMR analysis was performed in 5 mM peptide solutions in CD₃OD at 700 MHz (¹H frequency) spectrometer. ¹H NMR spectra were recorded 25 min after the peptides were dissolved (fresh) and therefore contained few of the non-exchanged amide resonances. The ¹H NMR spectra were in addition recorded after overnight measurements when the NH-to-ND exchange was already completed. ¹H HOHAHA (dipsi2 of 60 ms) and ROESY (spin-lock 300 ms) spectra were recorded to complete the assignment of the ¹H resonances. ¹³C {¹H} dept45 and ¹H {¹³C} HSQC experiments were performed in order to assign accompanied ¹³C resonances. The following abbreviations are used in reported NMR data: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), br (broad), brs (broad singlet). Coupling constants (J) are in Hz. Spectra are reported as follows: chemical shift (δ, ppm), multiplicity, integration, coupling constants (Hz). Measured melting points are uncorrected. Boiling points were measured at 0.8 mmHg unless otherwise specified herein. LC-MS data were acquired on Agilent 1200 HPLC system equipped with DAD/ELSD/LCMS-6120 diode-matrix and mass-selective detector, column: Poroshell 120 SB-C18, 4.6 mm × 30 mm. Eluent, A, acetonitrile–water with 0.1% of FA (99 : 1); B, water with 0.1% of FA. Optical rotations were measured on polarimeter in methanol using 1 dm cell; optical rotation values are given in 10⁻¹ deg cm² g⁻¹; concentrations (c) are given in mmol L⁻¹, wavelength 589 nm at 20 °C. The enantiomeric excess and retention time (t_R) was determined for major signal by HPLCs: Daicel CHIRALPACK IA, 5 μm, 4.6 × 250 mm, Daicel CHIRALPACK IB, 5 μm, 4.6 × 250 mm, Daicel CHIRALPACK OJ-H, 5 μm, 4.6 × 250 mm, Daicel CHIRALPACK AS-H, 5 μm, 4.6 × 250 mm chiral columns, injection volume 0.1 μL, eluent (hexanes: 2-propanol). Solid compounds were recrystallized from acetonitrile unless other is specified.

Synthesis of 3,4-disubstituted isoxazoles, representative procedure

Methyl 3-(((tert-butoxycarbonyl)amino)methyl)isoxazole-4-carboxylate (Boc-1A-CO₂Me). A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding chloroxime 1 (2 g, 9.6 mmol) and EtOAc (20 mL). To the resulting vigorously stirred homogeneous solution (E)-methyl 3-(dimethylamino) acrylate 8 (1.48 g, 11.5 mmol) was added followed by addition of NaHCO₃ (1.6 g, 19.2 mmol) at the ambient temperature. The resulting mixture was stirred overnight. The progress of the reaction was monitored by NMR spectroscopy. After the reaction was complete, the organic phase was washed with 10% NaHSO_4(aq) (3 × 5 mL), 10% K₂CO_3(aq) (2 × 5 mL) and brine (1 × 5 mL). Organic phase was dried over anhydrous Na₂SO₄. The resulting mixture was filtered through a silicagel pad and concentrated to yield Boc-1A-CO₂Me (95%, 2.3 g) as a beige powder, mp 65 °C.

¹H NMR (500 MHz, DMSO-d₆): δ 9.59 (s, 1H, isoxaz), 7.24 (brs, 1H, NH), 4.41 (d, 2H, CH₂), 3.80 (s, 3H, OCH₃), 1.39 (s, 9H, CH₃-Boc). ¹³C NMR (125 MHz, DMSO-d₆): δ 164.9, 161.1, 160.0, 155.4, 111.9, 78.2, 51.9, 35.9, 28.1. MS (APCI) m/z [M − Boc + 1H]⁺ calculated for C₁₀H₁₃N₂O₅: 255.1; found: 255.4. Anal. calcd for C₁₁H₁₆N₂O₅: C, 51.56; H, 6.29; N, 10.93. Found: C, 51.35; H, 6.07; N, 10.52.

Synthesis of 3,5-disubstituted isoxazoles, representative procedure

Methyl 3-(((tert-butoxycarbonyl)amino)methyl)isoxazole-5-carboxylate (Boc-1B-CO₂Me). A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding chloroxime 1 (2 g, 9.6 mmol) and EtOAc (20 mL). Then, the resulting vigorously stirred homogeneous solution was cooled to −5–0 °C and methyl propiolate (0.9 mL, 10.1 mmol) was added followed by addition of NaHCO₃ (1.6 g, 19.2 mmol) (in case of reaction 7 (2 g, 7.6 mmol) with ethynyltrimethylsilane 12 (5.4 mL, 38 mmol) dropwise addition of Et₃N (1.6 mL, 11.5 mmol) as a solution in EtOAc (100 mL) was required). The resulting mixture was stirred overnight. The progress of the reaction was monitored by NMR spectroscopy. Work-up was done by simple filtration through silica pad, then the resulting solution was concentrated and dried in vacuo to yield the corresponding product Boc-1B-CO₂Me (89%, 2.2 g) as a white powder, mp 84–86 °C. For reaction of compound 7 with 12 when the reaction was complete, the organic phase was washed with 10% K₂CO_3(aq) (2 × 20 mL) and brine (1 × 20 mL). Organic phase was dried over anhydrous Na₂SO₄. ¹H NMR (500 MHz, DMSO-d₆): δ 7.49 (s, 1H, NH), 7.05 (s, 1H, isoxaz), 4.24–4.23 (d, 2H, J = 5.5, CH₂), 3.89 (s, 3H, OCH₃), 1.39 (br, 9H, CH₃-Boc). ¹³C NMR (125 MHz, DMSO-d₆): δ 163.7, 159.5, 156.7, 155.7, 109.0, 78.5, 52.9, 35.6, 28.1. MS (APCI) m/z [M − Boc + 1H]⁺ calculated for C₁₀H₁₃N₂O₅: 255.1; found: 255.4. Anal. calcd for C₁₁H₁₆N₂O₅: C, 51.56; H, 6.29; N, 10.93. Found: C, 51.27; H, 6.54; N, 10.63.

Representative procedure for cleavage of tert-butyloxycarbonyl group

1-(3-(Aminomethyl)isoxazol-4-yl)ethan-1-one hydrochloride (1A-COMe·HCl). A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding Boc-protected isoxazole Boc-1A-COMe (2.5 g, 9.8 mmol) and EtOAc (25 mL). The flask was submerged into an ice cooling bath; and to the vigorously stirred homogeneous solution equimolar ratio of MeOH (1.05 eq.) was added followed by dropwise addition of AcCl (1.05 eq.). As the result white precipitate was formed. After the reaction was complete (NMR control) resulting solid was filtered and washed with cold EtOAc and recrystallized from MeCN (if it was necessary) affording 1A-COMe·HCl as a white powder. In case of EWG = CO₂Me-group AcCl (1.05 eq.) in MeOH was used.

1A-COMe·HCl: 95%, white powder, mp 94–96 °C. ¹H NMR (500 MHz, D₂O-d₂): δ 9.53 (s, 1H, isoxaz), 4.54 (s, 2H, CH₂), 2.60 (s, 3H, CH₃-Ac). ¹³C NMR (125 MHz, DMSO-d₆): δ 192.0, 166.1, 155.9, 119.8, 34.4, 29.0. MS (APCI) m/z [M + 1H]⁺ calculated for C₆H₉N₂O₂: 141.1; found: 141.2. Anal. calcd for C₆H₉ClN₂O₂: C, 40.81; H, 5.14; N, 15.86. Found: C, 40.25; H, 5.33; N, 15.96.

Representative procedure for the hydrolysis of isoxazole-based esters

3-(((tert-Butoxycarbonyl)amino)methyl)isoxazole-4-carboxylic acid (Boc-1A-CO₂H). A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding isoxazole (Boc-1A-CO₂Me, 2 g, 7.8 mmol) and MeOH (18 mL). Next, the flask was submerged into an ice cooling bath and to the resulting vigorously stirred homogeneous solution 2 equiv. of 10% NaOH_(aq) was added dropwise. After the NMR control indicated the reaction completion (10–24 h), the solution was concentrated in vacuo. HCl_(aq), or NaHSO_4(aq), was added dropwise until pH = 3 was reached and solid NaCl was added until the resulting solution become saturated. The product was extracted by EtOAc (3 × 20 mL). Combined organic fractions were dried over anhydrous MgSO₄, concentrated and dried in vacuo yielding Boc-1A-CO₂H as a white powder, (95%, 1.8 g), mp 129–131 °C. ¹H NMR (500 MHz, DMSO-d₆): δ 13.25 (br, 1H, C–OOH), 9.45 (s, 1H, isoxaz), 7.17 (brs, 1H, NH-major rotamer), 6.80 (brs, NH-minor of rotamer), 4.40 (d, 2H, J = 4.5, CH₂), 1.38 (s, 9H, CH₃-Boc). ¹³C NMR (125 MHz, DMSO-d₆): δ 164.6, 162.4, 160.3, 155.5, 113.2, 78.2, 36.1, 28.2. MS (APCI) m/z [M − 1H]⁺ calculated for C₁₀H₁₃N₂O₅: 241.1; found: 241.2. Anal. calcd for C₁₀H₁₄N₂O₅: C, 49.58; H, 5.83; N, 11.56. Found: C, 49.38; H, 5.66; N, 11.43.

Representative procedure for the for the synthesis of 3-substituted isoxazoles

tert-Butyl (isoxazol-3-ylmethyl)carbamate (Boc-1-H). A round bottomed flask (100 mL) was charged with a magnetic stirrer, substrate Boc-1-H-TMS (5.5 g, 20.3 mmol) and MeOH (25 mL). KHF₂ 5 mol% (80 mg, 1 mmol) with H₂O_(cat) (100 μL) were added and the resulting homogeneous solution was vigorously stirred for 24 h at the ambient temperature while the progress was monitored by NMR spectroscopy. After the reaction was completed the solvent was evaporated and the remaining residue was dissolved in EtOAc (50 mL). Organic phase was filtered through anhydrous Na₂SO₄ pad and concentrated to yield Boc-1-H as a white powder (94%, 3.8 g). ¹H NMR (500 MHz, DMSO-d₆): δ 8.79 (m, 1H, CH–isoxaz), 7.43–7.35 (brs, 1H, NH), 6.42 (s, 1H, isoxaz), 4.20–4.19 (m, 2H, CH₂), 1.39 (s, 9H, CH₃-Boc). ¹³C NMR (125 MHz, DMSO-d₆): δ 161.5, 159.8, 155.7, 103.8, 78.3, 35.5, 28.2. Anal. calcd for C₉H₁₄N₂O₃: C, 54.53; H, 7.12; N, 14.13. Found: C, 54.36; H, 7.18; N, 14.01.

Synthesis of the ABT-418 bioisostere 16

tert-Butyl (2S)-2-(5-(bromomethyl)-4,5-dihydroisoxazol-3-yl)pyrrolidine-1-carboxylate (13). A 500 mL round bottomed flask was charged with a magnetic stirrer, corresponding chloroxime 4 (10 g, 40.2 mmol) and EtOAc (100 mL). To the resulting vigorously stirred homogeneous solution allyl bromide (5.6 g, 46.23 mmol) was added followed by addition of NaHCO₃ (6.7 g, 80.4 mmol). The resulting mixture was stirred overnight. The progress of the reaction was monitored by NMR spectroscopy. After the reaction was complete, organic phase was dried over anhydrous Na₂SO₄ and filtered through the silica gel pad. The resulting solution was concentrated to yield 13 (95%, 12.95 g) as a white powder, mp 60 °C.

13-S-Isomer: 95%, white powder, [α]²⁰_D = −80.00 (c = 35.42). 99.6% ee, t_R = 12.2 min;

13-R-Isomer: 97%, white powder, [α]²⁰_D = +81.84 (c = 35.42), 99.8% ee, t_R = 10.5 min.

¹H NMR (500 MHz, CDCl₃): δ 4.85–4.78 (m, 1H, isoxazoline–CH), 4.63 (brs, 1H, –pyrrolidine–CH), 3.48–3.32 (m, 4H, CH₂–pyrrolidine + CH₂–Br), 2.94–2.90 (m, 2H, CH₂–isoxazoline), 2.17–1.91 (m, 4H, CH₂–pyrrolidine), 1.46 (s, 9H, CH₃-Boc). ¹³C NMR (125 MHz, DMSO-d₆, mixture of both rotamers): δ 160.3, 159.7, 153.5, 153.1, 78.8, 78.3, 54.0, 46.3, 46.0, 38.7, 35.7, 35.2, 34.8, 30.4, 30.2, 29.6, 29.3, 28.0, 23.9, 23.0. MS (APCI) m/z [M − Boc]⁺ calculated for C₈H₁₃BrN₂O: 235.0; found: 235.1. Anal. calcd for C₁₃H₂₁BrN₂O₃: C, 46.86; H, 6.35; N, 8.41. Found: C, 46.77; H, 6.44; N, 8.57.

tert-Butyl (S)-2-(5-methylisoxazol-3-yl)pyrrolidine-1-carboxylate (14). A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding isoxazoline 13 (12.5 g, 37.5 mmol) and DMF (50 mL). K₂CO₃ (10.4 g, 75 mmol) was added and the resulting mixture was stirred overnight at 100 °C. The progress of the reaction was monitored by NMR spectroscopy. After the reaction was complete, the reaction mixture was concentrated the residue was poured into ice-bath and aqueous phase was extracted with EtOAc. The combined orhanic phases were washed with brine (4 × 20 mL). Organic phase was dried over anhydrous Na₂SO₄. The resulting mixture was filtered through the silicagel pad and the solution was concentrated. The residue is colorless oil which was solidified upon standing 77%.

14-S-Isomer: 77%, [α]²⁰_D = −78.8 (c = 3.96), 99.9% ee, t_R = 9.5 min;

14-R-Isomer: 75%, [α]²⁰_D = +79.63 (c = 3.96), 99.9% ee, t_R = 7.6 min.

¹H NMR (500 MHz, DMSO-d₆): δ 6.10 (brs, 1H, isoxaz), 4.83–4.77 (m, 1H, CH–pyrrolidine), 3.43–3.38 (m, 2H, CH₂–pyrrolidine), 2.36 (s, 3H, CH₃), 2.21 (br, 1H, CH–pyrrolidine), 1.85 (br, 3H, CH–pyrrolidine), 1.39, (s, 9H, CH₃-Boc, major rotamer), 1.24 (s, 9H, CH₃-Boc, minor rotamer). ¹³C NMR (125 MHz, DMSO-d₆): δ 169.02, 166.4, 166.0, 153.5, 153.2, 100.4, 99.8, 78.6, 78.5, 53.3, 53.1, 46.3, 46.0, 32.6, 31.3, 28.1, 27.9, 23.6, 22.9, 11.7.

MS (APCI) m/z [M + 3H]⁺ calculated for C₁₃H₂₃N₂O₃: 255.3; found: 255.2. Anal. calcd for C₁₃H₂₀N₂O₃: C, 61.88; H, 7.99; N, 11.10. Found: C, 61.95; H, 7.83; N, 11.14.

(S)-5-Methyl-3-(pyrrolidin-2-yl)isoxazole hydrochloride (15·HCl). The same experimental procedure as previously described for 3,4- and 3,5-analogues for the cleavage of Boc-group was used.

15-S-Isomer, white powder, 92%, [α]²⁰_D = −21.63 (c = 53.00);

15-R-Isomer, white powder, 93%, [α]²⁰_D = +22.95 (c = 53.00).

¹H NMR (500 MHz, DMSO-d₆): δ 10.63 (br, 1H, NH·HCl), 9.57 (br, 1H, NH·HCl), 6.55 (s, 1H, isoxaz), 4.66 (brs, 1H, CH), 3.27–3.22 (m, 2H, CH₂–pyrrolidine), 2.43 (s, 3H, CH₃), 2.35–2.33 (m, 1H, CH–pyrrolidine), 2.02 (br, 3H, CH₂–pyrrolidine). ¹³C NMR (125 MHz, DMSO-d₆): δ 170.6, 160.1, 101.4, 54.0, 44.4, 29.4, 22.8, 11.9. MS (APCI) m/z [M − Cl + H]⁺ calculated for C₈H₁₉N₂O: 153.1; found: 153.2. Anal. calcd for C₈H₁₃ClN₂O: C, 50.93; H, 6.95; N, 14.85. Found: C, 50.87; H, 7.08; N, 14.89.

(S)-5-Methyl-3-(1-methylpyrrolidin-2-yl)isoxazole (16). A 500 mL round bottomed flask was charged with magnetic stirrer and corresponding isoxazole 15 (2.1 g, 10.60 mmol) which was suspended in dichloroethane (250 mL). MeOH (50 mL) was added followed by addition of 30% CH₂O_(aq) (10 mL) and NaBH(OAC)₃ (7.0 g, 33.03 mmol). Then, the resulting mixture was stirred overnight at the ambient temperature. Next, the reaction mixture was concentrated, EtOAc (250 mL) was added and K₂CO₃ was added until the pH = 10 was reached (Caution: carbon dioxide evolution!). The reaction mixture was then stirred over Na₂SO₄ for ca. 10 min filtered and concentrated. The product was purified by distillation yielding 16 (1.52 g, bp 75–80 °C at 5.5 mmHg) as a colorless liquid (with pyrrolidine odour) which is extremely well soluble in water.

16-S-Isomer, 82%, [α]²⁰_D = −8.00 (c = 60.16);

16-R-Isomer, 83%, [α]²⁰_D = +7.86 (c = 60.16).

¹H NMR (500 MHz, DMSO-d₆): δ 6.14 (brs, 1H, isoxaz), 3.24–3.19 (t, 1H, J = 10.0, CH–pyrrolidine), 3.07–3.03 (m, 1H, CH–pyrrolidine), 2.36 (s, 3H, CH₃–NH), 2.26–2.19 (m, 1H, CH–pyrrolidine), 2.11 (s, 4H, CH₃ + CH), 1.84–1.71 (m, 3H, CH–pyrrolidine). ¹³C NMR (125 MHz, DMSO-d₆): δ 169.1, 166.1, 99.7, 61.3, 56.0, 31.7, 22.4, 11.8. MS (APCI) m/z [M + H]⁺ calculated for C₉H₁₅N₂O: 167.1; found: 167.2. Anal. calcd for C₉H₁₄N₂O: C, 65.03; H, 8.49; N, 16.85. Found: C, 65.14; H, 8.33; N, 16.94.

Notes and references

1. (a) S. Kankala, R. K. Kankala, P. Gundepaka, N. Thota, S. Nerella, M. R. Gangula, H. Guguloth, M. Kagga, R. Vadde and C. S. Vasam, Bioorg. Med. Chem. Lett., 2013, 23, 1306–1309; (b) B. Frølund, A. T. Jørgensen, L. Tagmose, T. B. Stensbøl, H. T. Vestergaard, C. Engblom, U. Kristiansen, C. Sanchez, P. Krogsgaard-Larsen and T. Liljefors, J. Med. Chem., 2002, 45, 2454–2468; (c) S. Dadiboyena and A. Nefzi, Eur. J. Med. Chem., 2010, 45, 4697–4707.
2. J. J. Talley, D. L. Brown, J. S. Carter, M. J. Graneto, C. M. Koboldt, J. L. Masferrer, W. E. Perkins, R. S. Rogers, A. F. Shaffer, Y. Y. Zhang, B. S. Zweifel and K. Seibert, J. Med. Chem., 2000, 43, 775–777.
3. J. B. Carr, H. G. Durham and D. K. Hass, J. Med. Chem., 1977, 20, 934–939.
4. (a) J. I. Andrés, J. Alcázar, J. M. Alonso, R. M. Alvarez, M. H. Bakker, I. Biesmans, J. M. Cid, A. I. De Lucas, J. Fernández, L. M. Font, K. A. Hens, L. Iturrino, I. Lenaerts, S. Martínez, A. A. Megens, J. Pastor, P. C. M. Vermote and T. Steckler, J. Med. Chem., 2005, 48, 2054–2071; (b) H. S. Youn, E. J. Lee, J. E. Lee, W.-K. Park, D.-J. Baek, Y. S. Cho, H. Y. Koh, H. Choo and A. N. Pae, Bull. Korean Chem. Soc., 2009, 30, 1873–1876.
5. (a) M. Ma, Y. Cheng, Z. Xu, P. Xu, H. Qu, Y. Fang, T. Xu and L. Wen, Eur. J. Med. Chem., 2007, 42, 93–98; (b) Ö. Temiz-Arpacı, İ. Yıldız, S. Özkan, F. Kaynak, E. Akı-Şener and İ. Yalçın, Eur. J. Med. Chem., 2008, 43, 1423–1431.
6. (a) W.-T. Li, D.-R. Hwang, C.-P. Chen, C.-W. Shen, C.-L. Huang, T.-W. Chen, C.-H. Lin, Y.-L. Chang, Y.-Y. Chang, Y.-K. Lo, H.-Y. Tseng, C.-C. Lin, J.-S. Song, H.-C. Chen, S.-J. Chen, S.-H. Wu and C.-T. Chen, J. Med. Chem., 2003, 46, 1706–1715; (b) J. Yong, C. Lu and X. Wu, Med. Chem. Commun., 2014, 5, 968–972.
7. N. Jacobsen, L.-E. K. Pedersen and A. Wengel, Pestic. Sci., 1990, 29, 95–100.
8. (a) D. S. Garvey, J. T. Wasicak, M. W. Decker, J. D. Brioni, M. J. Buckley, J. P. Sullivan, G. M. Carrera, M. W. Holladay, S. P. Arneric and M. Williams, J. Med. Chem., 1994, 37, 1055–1059; (b) A. Carenzi, D. Chiarino, D. D. Bella, M. Napoletano, A. Sala, US Pat. 4985428, Jan 15, 1991.
9. M. Koufaki, A. Tsatsaroni, X. Alexi, H. Guerrand, S. Zerva and M. N. Alexis, Bioorg. Med. Chem., 2011, 19, 4841–4850.
10. (a) S. Srivastava, L. K. Bajpai, S. Batra, A. P. Bhaduri, J. P. Maikhuri, G. Gupta and J. D. Dhar, Bioorg. Med. Chem., 1999, 7, 2607–2613; (b) K. A. Kumar and P. Jayaroopa, Int. J. Pharm., Chem. Biol. Sci., 2013, 3(2), 294–304.
11. (a) F. Hu and M. Szostak, Adv. Synth. Catal., 2015, 357, 2583–2614; (b) M. S. Mohamed Ahmed, K. Kobayashi and A. Mori, Org. Lett., 2005, 7, 4487–4489; (c) T. V. Hansen, P. Wu and V. V. Fokin, J. Org. Chem., 2005, 70, 7761–7764; (d) S. Grecian and V. V. Fokin, Angew. Chem., Int. Ed., 2008, 47, 8285–8287; (e) A. V. Dubrovskiy, P. Jain, F. Shi, G. H. Lushington, C. Santini, P. Porubsky and R. C. Larock, ACS Comb. Sci., 2013, 15, 193–201; (f) R. Huisgen, Angew. Chem., Int. Ed. Engl., 1963, 2, 565–598; (g) T. M. Vishwanatha and V. V. Sureshbabu, J. Heterocycl. Chem., 2015, 52, 1823–1833; (h) A. Singhal, S. K. R. Parumala, A. Sharma and R. K. Peddinti, Tetrahedron Lett., 2016, 57, 719–722.
12. (a) M. A. Weidner-Wells, T. C. Henninger, S. A. Fraga-Spano, C. M. Boggs, M. Matheis, D. M. Ritchie, D. C. Argentieri, M. P. Wachter and D. J. Hlasta, Bioorg. Med. Chem. Lett., 2004, 14, 4307–4311; (b) S. Dadiboyena and A. Nefzi, Tetrahedron Lett., 2012, 53, 2096–2099; (c) D. E. Kizer, R. B. Miller and M. J. Kurth, Tetrahedron Lett., 1999, 40, 3535–3538; (d) S. Mohammed, R. A. Vishwakarma and S. B. Bharate, RSC Adv., 2015, 5, 3470–3473; (e) P. K. Mykhailiuk, Org. Biomol. Chem., 2015, 13, 3438–3445.
13. (a) R. C. F. Jones, S. J. Hollis and J. N. Iley, Tetrahedron: Asymmetry, 2000, 11, 3273–3276; (b) Y. K. Kang, K. J. Shin, K. H. Yoo, K. J. Seo, C. Y. Hong, C.-S. Lee, S. Y. Park, D. J. Kim and S. W. Park, Bioorg. Med. Chem. Lett., 2000, 10, 95–99; (c) R. C. F. Jones, L. E. Seager and M. R. J. Elsegood, Synlett, 2011, 211–214; (d) R. C. F. Jones and T. A. Pillainayagam, Synlett, 2004, 15, 2815–2817; (e) R. C. F. Jones, J. P. Bullous, C. C. M. Law and M. R. J. Elsegood, Chem. Commun., 2014, 50, 1588–1590; (f) M. Falorni, G. Giacomelli and E. Spanu, Tetrahedron Lett., 1998, 39, 9241–9244; (g) M. Falorni, G. Giacomelli and A. M. Spanedda, Tetrahedron: Asymmetry, 1998, 9, 3039–3046; (h) L. De Luca, G. Giacomelli and A. Riu, J. Org. Chem., 2001, 66, 6823–6825.
14. D. C. Schmitt, L. Lam and J. S. Johnson, Org. Lett., 2011, 13, 5136–5139.
15. (a) Q.-f. Jia, P. M. S. Benjamin, J. Huang, Z. Du, X. Zheng, K. Zhang, A. H. Conney and J. Wang, Synlett, 2013, 79–84; (b) S. Zhu, S. Shi and S. W. Gerritz, Tetrahedron Lett., 2011, 52, 4001–4004.
16. C. C. Lee, R. J. Fitzmaurice and S. Caddick, Org. Biomol. Chem., 2009, 7, 4349–4351.
17. H. Kawai, Y. Sugita, E. Tokunaga and N. Shibata, Eur. J. Org. Chem., 2012, 1295–1298.
18. (a) S. Dadiboyena, J. Xu and A. T. Hamme II, Tetrahedron Lett., 2007, 48, 1295–1298; (b) J. Xu and A. T. Hamme II, Synlett, 2008, 919–923.
19. M. A. Schmidt, K. Katipally, A. Ramirez, O. Soltani, X. Hou, H. Zhang, B.-C. Chen, X. Qian and R. P. Deshpande, Tetrahedron Lett., 2012, 53, 3994–3997.
20. (a) A. O. Abdelhamid, A. A. Fahmi and K. N. M. Halim, Synth. Commun., 2013, 43, 1101–1126; (b) M. R. Shaaban, T. M. A. Eldebss, A. F. Darweesh and A. M. Farag, J. Heterocycl. Chem., 2008, 45, 1739–1744.
21. (a) A. Padwa and W. H. Pearson, Synthetic Applications of 1,3-Dipolar Cycloaddition Chemistry toward Heterocycles and Natural Products, John Wiley & Sons, Ltd, 2002; (b) R. Huisgen, Angew. Chem., Int. Ed. Engl., 1963, 2, 633–645; (c) L. N. Sobenina, D. N. Tomilin, M. D. Gotsko, I. A. Ushakov, A. I. Mikhaleva and B. A. Trofimov, Tetrahedron, 2014, 70, 5168–5174.
22. (a) C. Bryant, I. D. Kerr, M. Debnath, K. K. H. Ang, J. Ratnam, R. S. Ferreira, P. Jaishankar, D. M. Zhao, M. R. Arkin, J. H. McKerrow, L. S. Brinen and A. R. Renslo, Bioorg. Med. Chem. Lett., 2009, 19(21), 6218–6221; (b) P. Jones, M. E. Difrancesco, A. Petrocchi, C. L. Carroll, J. Marszalek, B. Czako, R. Johnson, J. Theroff, US Pat. US2015252058 (A1), Feb 27, 2014.
23. L. Han, B. Zhang, M. Zhu and J. Yan, Tetrahedron Lett., 2014, 55, 2308–2311.
24. (a) T. Pasinszki, B. Hajgató, B. Havasi and N. P. C. Westwood, Phys. Chem. Chem. Phys., 2009, 11, 5263–5272; (b) G. Romeo and U. Chiacchio, in Modern Heterocyclic Chemistry, ed. J. Alvarez-Builla, J. J. J. Vaquero and J. Barluenga, Wiley-VCH Verlag & Co., Weinheim, Germany, 2011, pp. 1047–1252.
25. B. Guillaume, K. M. Fiona, B. Renaud, L. Clemens, Q. Laura, T. Stephan, US Pat. US2014045890 (A1), Feb 13, 2014.
26. D. N. Reddy, R. Thirupathi, S. Tumminakatti and E. N. Prabhakaran, Tetrahedron Lett., 2012, 53, 4413–4417.
27. (a) S. P. Arneric, J. P. Sullivan, C. A. Briggs, D. Donnelly-Roberts, D. J. Anderson, J. L. Raszkiewicz, M. Hughes, E. D. Cadman, P. Adams, D. S. Garvey, J. Wasicak and M. Williams, J. Pharmacol. Exp. Ther., 1994, 270, 310–318; (b) D. S. Garvey, J. T. Wasicak, R. L. Elliott, S. Lebold, A.-M . Hettinger, G. M. Carrera, N.-H. Lin, Y. He, M. W. Holladay, D. J. Anderson, E. D. Cadman, J. L. Raszkiewicz, J. P. Sullivan and S. P. Arneric, J. Med. Chem., 1994, 37, 4455–4463.
28. (a) A. Plant, F. Stieber, J. Scherkenbeck, P. Lösel and H. Dyker, Org. Lett., 2001, 3, 3427–3430; (b) I. V. Komarov, A. O. Grigorenko, A. V. Turov and V. P. Khilya, Russ. Chem. Rev., 2004, 73, 785–810; (c) S. Pellegrino, A. Contini, M. L. Gelmi, L. Lo Presti, R. Soave and E. Erba, J. Org. Chem., 2014, 79, 3094–3102.
29. In particular, it was been recently demonstrated that terminal 2-oxasole-group is prone to induce β-conformation of the residue: (a) D. Siodłak, M. Staś, M. A. Broda, M. Bujak and T. Lis, J. Phys. Chem. B, 2014, 118, 2340–2350. 4,5-Thiasole-based building blocks with (R)-chirality have been used to promote β-turn structures in gramicidin (S)-analogues to construct peptidomimetics with reduced hemolytic activity: (b) B. Legrand, L. Mathieu, A. Lebrun, S. Andriamanarivo, V. Lisowski, N. Masurier, S. Zirah, Y. K. Kang, J. Martinez and L. T. Maillard, Chem.–Eur. J., 2014, 20, 6713–6720.
30. R. A. Hughes and C. J. Moody, Angew. Chem., Int. Ed., 2007, 46, 7930–7954.
31. P. G. Arnison, M. J. Bibb, G. Bierbaum, A. A. Bowers, T. S. Bugni, G. Bulaj, J. A. Camarero, D. J. Campopiano, G. L. Challis, J. Clardy, P. D. Cotter, D. J. Craik, M. Dawson, E. Dittmann, S. Donadio, P. C. Dorrestein, K.-D. Entian, M. A. Fischbach, J. S. Garavelli, U. Göransson, C. W. Gruber, D. H. Haft, T. K. Hemscheidt, C. Hertweck, C. Hill, A. R. Horswill, M. Jaspars, W. L. Kelly, J. P. Klinman, O. P. Kuipers, A. J. Link, W. Liu, M. A. Marahiel, D. A. Mitchell, G. N. Moll, B. S. Moore, R. Müller, S. K. Nair, I. F. Nes, G. E. Norris, B. M. Olivera, H. Onaka, M. L. Patchett, J. Piel, M. J. T. Reaney, S. Rebuffat, R. P. Ross, H.-G. Sahl, E. W. Schmidt, M. E. Selsted, K. Severinov, B. Shen, K. Sivonen, L. Smith, T. Stein, R. D. Süssmuth, J. R. Tagg, G.-L. Tang, A. W. Truman, J. C. Vederas, C. T. Walsh, J. D. Walton, S. C. Wenzel, J. M. Willey and W. A. van der Donk, Nat. Prod. Rep., 2013, 30, 108–160.
32. K. L. Dunbar and D. A. Mitchell, J. Am. Chem. Soc., 2013, 135, 8692–8701.
33. R. S. Roy, A. M. Gehring, J. C. Milne, P. J. Belshaw and C. T. Walsh, Nat. Prod. Rep., 1999, 16, 249–263.
34. E. M. Nolan and C. T. Walsh, ChemBioChem, 2009, 10, 34–53.
35. V. S. C. Yeh, Tetrahedron, 2004, 60, 11995–12042.
36. For instance see total synthesis of plantazolicin A: (a) S. Banala, P. Ensle and R. D. Süssmuth, Angew. Chem., Int. Ed., 2013, 52, 9518–9523 ; microcin B17:; (b) R. E. Thompson, F. Collin, A. Maxwell, K. A. Joliffe and R. J. Payne, Org. Biomol. Chem., 2014, 12, 1570–1578 ; sansavamide A:; (c) M. R. Davis, E. K. Singh, H. Wahyudi, L. D. Alexander, J. B. Kunicki, L. A. Nazarova, K. A. Fairweather, A. M. Giltrap, K. A. Jolliffe and S. R. McAlpine, Tetrahedron, 2012, 68, 1029–1051 ; (R)-telomestatin:; (d) T. Doi, M. Yoshida, K. Shin-ya and T. Takahashi, Org. Lett., 2006, 8, 4165–4167 ; or dendroamine A:; (e) Z. Xia and C. D. Smith, J. Org. Chem., 2001, 66, 3459–3466.
37. Curiously, epimerization of the AlaIso residue in Ac-Leu-AlaIso-Val-NH₂ (but not in Ac-Leu-Val-AlaIso-NH₂) would potentially lead to a β-turn structure rather than semi-extended β. This may be the driving force for the partial epimerization observed experimentally.
38. G. Chennakrishnareddy, B. Vasantha, N. Narendra and V. V. Sureshbabu, Int. J. Pept. Res. Ther., 2011, 17, 185–191.

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedure and characterization of all new compounds (PDF). CCDC [1442715 and 1442716]. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra02365g

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