multigram synthesis of aminoisoxazoles : valuable building blocks for drug discovery and peptidomimetics †

Enamine Ltd., Chervonotkatska 78, 01103 net. E-mail: A.tverdohlebov@mail.enamin Pavel.Mykhailiuk@gmail.com; Pavel.Mykha Department of Chemistry, Kyiv Polytechnic I Ukraine Department of Chemistry, Taras Shev Volodymyrska Street 64, Kyiv 01601, Ukrain Institute of Bioorganic Chemistry & Petroc Kyiv 02660, Ukraine Institute of Chemistry, Technical University 10623, Germany † Electronic supplementary information ( and characterization of all new com 1442716]. For ESI and crystallographic d see DOI: 10.1039/c6ra02365g Cite this: RSC Adv., 2016, 6, 25713

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 isoxazolecontaining compounds has been known before.However, these reports, dealt with the specic 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 a-, band g-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 exemplied in Table 1.
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 3,4disubstituted isoxazoles between enolsilanes and nitrile oxides. 14In 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 b-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. 16Later the Shibata group developed the strategy for the synthesis of isoxazole tri-ones. 17Hamme et al. reported on the synthesis of 3,5disubstituted 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-triuorosubstituted isoxazoles in good yields. 19All the aforementioned methods avoided the usage of metalmediated 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). 20In contrast, terminal alkynes 11, 12 afforded a mixture of 3,4-/3,5-isomers $30/70 when reaction was performed at the room temperature. 21Nevertheless, 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. 22hile 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. 23It is also worth mentioning, that these reactions were performed under obligatory high dilutionsubstrate/solvent ¼ 1/10 (wt/v)otherwise formation of side furoxans was observed. 24s 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 K 2 CO 3 or triuoroacetic 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 KHF 2 in methanol-water mixture.Resulting 3-substituted isoxazoles were obtained in excellent yields (Table 3).
As next, cleavage of N-Boc group from all compounds was performed under acidic conditions to afford the target building blocksaminoisoxazoles (Table 4).
Carboxymethyl-substituted isoxazoles were saponicated with sodium hydroxide in methanol to afford corresponding free amino acids in good yields (Table 5).
The structures of two nal compounds were conrmed 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.

Practical applications
Aer 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.
2. Synthesis of peptidomimetics.We also envisioned that chiral aminoisoxazoles could be used to prepare conformationally restricted peptidomimetics. 28In this case the heterocyclic fragment will act as a rigid peptide bond substitute 29 (Scheme 2).
In addition, isomeric oxazoles and thiazoles are common in the backbones 30 of various ribosomally synthesized and posttranslationally modied peptides. 313][34] Total synthesis of respective peptidomimetics has been developed in order to provide naturally occurring antimicrobial agents or toxins as well as their chemically modied analogues. 35,36hough, 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.
Leu-Val-AlaIso-NH 2 (19) no racemization was observed. 37In addition, no degradation was found aer 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. 38ble 3 Cleavage of TMS-group from the isoxazole ring Table 4 Cleavage of the N-Boc protection group from the amino moiety

Conclusions
We have developed a general metal-free practical protocol towards isoxazole-containing building blocks starting from commonly available a-, band g-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 16bioisostere of the known nootropic agent ABT-418,and preparation of isoxazole-containing peptidomimetics 18, 19 by solid phase peptide synthesis.

General
All reagents were available from Enamine Ltd.Solvents were puried 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. 1 H, 13 C NMR spectra were recorded at the NMR spectrometers operating at 400 and 500 1 H frequency (101 and 126 MHz for 13 C experiments).NMR chemical shis are reported in ppm, in the d scale and are referenced using residual NMR solvent peaks at 7.26 and 77.16 ppm for 1 H and 13 C in CDCl 3 , 2.50 and 39.52 ppm for 1 H and 13 C in DMSO-d 6 , and 4.79 ppm for 1 H in D 2 O.For peptides NMR analysis was performed in 5 mM peptide solutions in CD 3 OD at 700 MHz ( 1 H frequency) spectrometer. 1H NMR spectra were recorded 25 min aer the peptides were dissolved (fresh) and therefore contained few of the non-exchanged amide resonances.The 1 H NMR spectra were in addition recorded aer 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 1 H resonances. 13 C { 1 H} dept45 and 1 H { 13 C} HSQC experiments were performed in order to assign accompanied 13 C 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 shi (d, ppm), multiplicity, integration, coupling constants (Hz).Measured melting points are uncorrected.Boiling points were measured at 0.8 mmHg unless otherwise specied herein.LC-MS data were acquired on Agilent 1200 HPLC system equipped with DAD/ELSD/LCMS-6120 diodematrix and mass-selective detector, column: Poroshell 120 SB-C18, 4.6 mm Â 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 À1 deg cm 2
As the result white precipitate was formed.Aer the reaction was complete (NMR control) resulting solid was ltered 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 ¼ CO 2 Me-group AcCl (1.05 eq.) in MeOH was used.
Aer 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 Â 20 mL).Organic phase was dried over anhydrous Na 2 SO 4 .The resulting mixture was ltered through the silicagel pad and the solution was concentrated.The residue is colorless oil which was solidied upon standing 77%.

a
Overall yield over four steps.bThe products were stored in closed asks at the room temperature.c The products were stored at 0 C under argon.Aer six months ca.10% decomposition was found according to 1 H NMR.

Table 1
Chloroximes synthesized starting from N-Boc amino acids