3-[N,N-Bis(sulfonyl)amino]isoxazolines with Spiro-Annulated or 1,2-Annulated Cyclooctane Rings Inhibit Reproduction of Tick-Borne Encephalitis, Yellow Fever, and West Nile Viruses

Spirocyclic compounds containing heterocyclic moieties represent promising 3D scaffolds for modern drug design. In the search for novel anti-flaviviral agents, we have obtained a series of 3-[N,N-bis(sulfonyl)amino]isoxazolines containing spiro-annulated cyclooctane rings and assessed their antiviral activity against tick-borne encephalitis (TBEV), yellow fever (YFV), and West Nile (WNV) viruses. The structural analogs of spirocyclic compounds with a single sulfonyl group or 1,2-annulated cyclooctane ring were also investigated. Almost all the studied 3-[N,N-bis(sulfonyl)amino]isoxazolines revealed antiviral activity against TBEV and WNV. The most active against TBEV was spiro-isoxazoline derivative containing p-nitrophenyl groups in the sulfonyl part (EC50 2.0 ± 0.5 μM), while the highest potency against WNV was found for the compounds with lipophilic substituents in sulfonyl moiety, naphtyl being the most favorable one (EC50 1.3 ± 0.5 μM). In summary, two novel scaffolds of anti-flaviviral agents based on N,N-bis(sulfonyl)amino]isoxazoline were proposed, and the compounds of this type demonstrated activity against TBEV and WNV.


Introduction
In the past two decades, spirocyclic compounds appeared at the focus of research in the field of drug design and discovery as promising 3D scaffolds, enlarging the chemical space, allowing the construction of new types of lead compounds, and improving their potency, selectivity, and pharmacokinetic properties [1]. Spiroheterocyclic motifs are present in a number of approved drugs and natural compounds [1][2][3][4] (Figure 1). Such an interest in spirocyclic structures echoed in the field of synthetic organic chemistry, giving rise to a number of elegant preparative approaches to compounds of this type [5][6][7][8][9][10][11].
On the other hand, medium-sized rings (8-11 atoms) are rarely used for the construction of spirocyclic structures, reflecting the general tendency concerning medium rings. Although they possess appealing properties, such as a unique balance between rigidity and flexibility and diverse geometry, improve binding affinity to target enzymes, and are present in both bioactive and natural compounds ( Figure 1) [12][13][14], application of medium Isoxazoles and isoxazolines reveal a broad range of biological activities, including antitumor, antibacterial, antimalarial, antisclerotic, antidiabetic, and others (Figure 1), and a number of examples exist of how the insertion of an isoxazoline moiety increases the potency of the bioactive compounds [18][19][20][21][22][23]. The most general approach to isoxazoline derivatives is 1,3-dipolar cycloaddition, though other methods, taking advantage of metal-mediated and radical processes, are constantly gaining more significance [24,25]. Recently we have elaborated the preparative approach to spiro-and 1,2-annulated 3-aminoisoxazoles 1,2, containing an 8-membered ring, and demonstrated their potent antiviral activity against influenza A virus ( Figure 2) [26]. A series of 3-(N-sulfonylamino)isoxazolines 3 was also obtained and found to possess low-to-no cytotoxicity, making them attractive for the search of other applications in lead discovery [26]. Flaviviruses are arthropod-borne viruses that are mainly transmitted between birds and wild animals by the bite of infected blood-sucking arthropods, such as mosquitoes or ticks. Unfortunately, humans could be bitten and infected too, resulting in fevers and encephalitides of different severity, from mild to even lethal, therefore representing a constant epidemic threat [27]. Currently, there are no specific medications for the treatment of flavivirus-borne diseases, so patient care is usually symptomatic, and the main way of preventing infection is vaccination [28]. Nevertheless, vaccines do not provide complete protectivity [29], and there is still no human vaccine against West Nile virus [30]. Thus, the development of specific anti-flaviviral drugs is still very important. Pre- Isoxazoles and isoxazolines reveal a broad range of biological activities, including antitumor, antibacterial, antimalarial, antisclerotic, antidiabetic, and others (Figure 1), and a number of examples exist of how the insertion of an isoxazoline moiety increases the potency of the bioactive compounds [18][19][20][21][22][23]. The most general approach to isoxazoline derivatives is 1,3-dipolar cycloaddition, though other methods, taking advantage of metalmediated and radical processes, are constantly gaining more significance [24,25]. Recently we have elaborated the preparative approach to spiro-and 1,2-annulated 3-aminoisoxazoles 1,2, containing an 8-membered ring, and demonstrated their potent antiviral activity against influenza A virus ( Figure 2) [26]. A series of 3-(N-sulfonylamino)isoxazolines 3 was also obtained and found to possess low-to-no cytotoxicity, making them attractive for the search of other applications in lead discovery [26]. gidity and flexibility and diverse geometry, improve binding affinity to target enzymes, and are present in both bioactive and natural compounds ( Figure 1) [12][13][14], application of medium rings in drug design is limited by the lack of synthetic approaches. Successful efforts to obtain the compounds containing medium rings, in most cases with heteroatoms, have been recently reported, illustrating the arising interest in such structures and the need for synthetic methods for them [15][16][17]. Isoxazoles and isoxazolines reveal a broad range of biological activities, including antitumor, antibacterial, antimalarial, antisclerotic, antidiabetic, and others (Figure 1), and a number of examples exist of how the insertion of an isoxazoline moiety increases the potency of the bioactive compounds [18][19][20][21][22][23]. The most general approach to isoxazoline derivatives is 1,3-dipolar cycloaddition, though other methods, taking advantage of metal-mediated and radical processes, are constantly gaining more significance [24,25]. Recently we have elaborated the preparative approach to spiro-and 1,2-annulated 3-aminoisoxazoles 1,2, containing an 8-membered ring, and demonstrated their potent antiviral activity against influenza A virus ( Figure 2) [26]. A series of 3-(N-sulfonylamino)isoxazolines 3 was also obtained and found to possess low-to-no cytotoxicity, making them attractive for the search of other applications in lead discovery [26]. Flaviviruses are arthropod-borne viruses that are mainly transmitted between birds and wild animals by the bite of infected blood-sucking arthropods, such as mosquitoes or ticks. Unfortunately, humans could be bitten and infected too, resulting in fevers and encephalitides of different severity, from mild to even lethal, therefore representing a constant epidemic threat [27]. Currently, there are no specific medications for the treatment of flavivirus-borne diseases, so patient care is usually symptomatic, and the main way of preventing infection is vaccination [28]. Nevertheless, vaccines do not provide complete protectivity [29], and there is still no human vaccine against West Nile virus [30]. Thus, the development of specific anti-flaviviral drugs is still very important. Pre- Flaviviruses are arthropod-borne viruses that are mainly transmitted between birds and wild animals by the bite of infected blood-sucking arthropods, such as mosquitoes or ticks. Unfortunately, humans could be bitten and infected too, resulting in fevers and encephalitides of different severity, from mild to even lethal, therefore representing a constant epidemic threat [27]. Currently, there are no specific medications for the treatment of flavivirus-borne diseases, so patient care is usually symptomatic, and the main way of preventing infection is vaccination [28]. Nevertheless, vaccines do not provide complete protectivity [29], and there is still no human vaccine against West Nile virus [30]. Thus, the development of specific anti-flaviviral drugs is still very important. Previously we have found several structural types of compounds showing antiviral activity against tick-borne encephalitis (TBEV), yellow fever (YFV), and West Nile (WNV) viruses [28,[31][32][33][34][35][36][37][38]. As can be seen in examples (Figure 3), they possess quite a diverse structure. viously we have found several structural types of compounds showing antiviral activity against tick-borne encephalitis (TBEV), yellow fever (YFV), and West Nile (WNV) viruses [28,[31][32][33][34][35][36][37][38]. As can be seen in examples (Figure 3), they possess quite a diverse structure. A heterocyclic core, spiro-annulated or 1,2-annulated to carbocycle and furnished with lipophilic substituents, may be distinguished as a peculiar pattern. For example, spiropyrazolopyridone derivatives were identified as dengue virus type 2 (DENV-2) inhibitors [40], while substituted 4,6-dihydrospiro[ [1,2,3]triazolo [4,5-b]pyridine-7,3′-indoline]-2′,5(3H)-dione analogs such as JMX0254 (Figure 3) effectively inhibit NS4B protein activity of dengue virus type 1 (DENV-1) [39]. That inspired us to synthesize a series of novel 3-[N,N-bis(sulfonyl)amino]isoxazolines 4,5, containing spiro-annulated or 1,2-annulated cyclooctane rings (Figure 4), and probe them as novel anti-flaviviral agents along with the previously obtained series of 3-(N-sulfonylamino)isoxazolines 3.

Chemistry
Target 3-[N,N-bis(sulfonyl)amino]isoxazolines were obtained via sulfonylation of 3-aminoisoxazolines 1,2 or, in some cases, 3-(sulfonylamino)isoxazolines 3 upon treatment with corresponding sulfonyl chlorides (Table 1). Depending on the starting amine and sulfonyl chloride, the chosen method, reaction time, and reagent ratio were varied in order to improve the preparative yield (see Section 3.2).

Chemistry
Target 3-[N,N-bis(sulfonyl)amino]isoxazolines were obtained via sulfonylation of 3aminoisoxazolines 1,2 or, in some cases, 3-(sulfonylamino)isoxazolines 3 upon treatment with corresponding sulfonyl chlorides (Table 1). Depending on the starting amine and sulfonyl chloride, the chosen method, reaction time, and reagent ratio were varied in order to improve the preparative yield (see Section 3.2).
The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.   The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod-   The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod-   The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod- The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod- The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod- The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod- The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod- The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod- The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod- The interaction of amines 1 and 2 with mesyl chloride and benzyl sulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) (Method A) gave hardly separable mixtures of the products of one-and two-fold sulfonylation, even when an excess of sulfonyl chloride was used. That resulted in lowering the yields of compounds 4a,b, and 5b, and despite all the efforts, we were not able to obtain compound 5a in analytically pure form.
Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in mod- Spirocyclic isoxazoline 1 interacted with aryl sulfonyl chlorides in the presence of DIPEA (Method A), affording the products of two-fold sulfonylation, generally in moderate yields. Nevertheless, bis(sulfonyl)amines 4e,h could not be obtained in these conditions. Compound 4e was successfully obtained via sulfonylation of 3-(sulfonylamino)isoxazoline 3e using the mixture of triethylamine (TEA) and 4-dimethylaminopyridine (DMAP) as the bases (Method C); this method was also probed for the synthesis of bis(sulfonyl)amine 4f more the doubling the yield of the target compound. 2-Nitrobenzenesulfonyl chloride did not give products of two-fold sulfonylation in the presence of N-bases (DIPEA, TEA, DMAP). We managed to obtain compound 4h, containing o-nitrophenyl substituents, only using pyridine as a solvent (Method B).
The reactions of isoxazoline 2 with both EWG-and EDG-substituted aryl sulfonyl chlorides in the presence of DIPEA (Method A) in most cases proceeded smoothly, giving 3-[N,N-bis(sulfonyl)amino]isoxazolines 5c-l in the yields up to quantitative. The exceptions were sterically hindered o-substituted aryl sulfonyl and naphtyl sulfonyl chlorides-the reactions of compound 2 with the latter afforded target heterocycles 5e,h,j in moderate yields.
Such a difference in reactivity between spirocyclic and bicyclic amines 1,2 could be explained according to their geometry. The spiro-annulated carbocycle in compound 1 hinders both sides of the isoxazoline ring, while 1,2-annulated cyclooctane leaves one side of the ring unhindered, thus not preventing the amino group from serving as a nucleophile.

Antiviral Activity Assessment
For two series of 3-[N,N-bis(sulfonyl)amino]isoxazolines 4a-l and 5b-l, antiviral activity was investigated against TBEV on porcine embryo kidney (PEK) cells using plaque reduction assay based on the estimation of the plaque-forming efficiency of a virus in the presence of different concentrations of a compound. Acute and chronic cytotoxicity of these compounds was studied using resazurin cell viability assay-viable cells turn resazurin into fluorescent resorufin ( Table 2; see also Supplementary Materials). In order to compare the bioactivity of mono-and bissulfonylated derivatives, the same tests were performed for the series of previously obtained [26] 3-(N-sulfonylamino)isoxazolines 3a-h,j-l. Previously studied isoxazole derivative 6 ( Figure 4) that showed anti-TBEV activity [41] was used as a positive control. Despite it not being structurally similar to the compounds studied in this work, it allowed us to confirm the validity of the obtained EC 50 values.
Compounds 4b-l and 5b-l, containing spiro-annulated or 1,2-annulated cyclooctane moieties, did not show a clear relation between antiviral activity and the configuration of the two-ring system. Spiro-isoxazolines 4c,d,f,i,l were more potent than 1,2-annulated analogs, while for compounds 4b,g,h,j,k, the relation is opposite. Nevertheless, the most potent compounds of all the investigated species, namely 4c and 4i, belong to the spiro-isoxazoline series.
For all the compounds 3-5, we have also determined the antiviral activity against WNV on African green monkey kidney (Vero) cells using the plaque reduction assay. The acute and chronic cytotoxicity of these compounds was studied using the resazurin cell viability assay (Table 3). Favipiravir was used as a positive control in this screening [42].
Almost all the tested compounds, except 5l, inhibited WNV reproduction. In sulfonylamine series 3, the most active were benzyl-and thiophenyl-substituted derivatives 3b and 3k. All the bis(sulfonyl)amines 4 were active in micromolar concentrations. Bis(sulfonyl)amines 4c-l bearing aromatic or heteroaromatic substituents demonstrated higher potency than methyl or benzyl substituted compounds 4a,b. The highest potency was found for compounds 4c-e,j with lipophilic substituents in sulfonyl moiety, naphtyl being the most favorable one. Isoxazolines 5 containing the 1,2-annulated cyclooctane moiety generally demonstrated lesser anti-WNV activity than their spiro-annulated analogs 4, except benzyl derivative 5b, that was more active than its analog 4b, and almost equally active compounds 4e and 5e. It is also interesting that the addition of the second pyridyl group to the sulfonylamine 3l, resulting in the compound 4l, did not affect the EC 50 value, while a change in the position of cyclooctane moiety from spiro-annulated to 1,2-annulated one (compounds 4l and 5l, respectively) resulted in the complete loss of anti-WNV activity. presence of different concentrations of a compound. Acute and chronic cytotoxicity of these compounds was studied using resazurin cell viability assay-viable cells turn resazurin into fluorescent resorufin ( Table 2; see also Supplementary Materials). In order to compare the bioactivity of mono-and bissulfonylated derivatives, the same tests were performed for the series of previously obtained [26] 3-(N-sulfonylamino)isoxazolines 3a-h,j-l. Previously studied isoxazole derivative 6 ( Figure 4) that showed anti-TBEV activity [41] was used as a positive control. Despite it not being structurally similar to the compounds studied in this work, it allowed us to confirm the validity of the obtained EC50 values. reduction assay based on the estimation of the plaque-forming efficiency of a virus in the presence of different concentrations of a compound. Acute and chronic cytotoxicity of these compounds was studied using resazurin cell viability assay-viable cells turn resazurin into fluorescent resorufin ( Table 2; see also Supplementary Materials). In order to compare the bioactivity of mono-and bissulfonylated derivatives, the same tests were performed for the series of previously obtained [26] 3-(N-sulfonylamino)isoxazolines 3a-h,j-l. Previously studied isoxazole derivative 6 ( Figure 4) that showed anti-TBEV activity [41] was used as a positive control. Despite it not being structurally similar to the compounds studied in this work, it allowed us to confirm the validity of the obtained EC50 values. reduction assay based on the estimation of the plaque-forming efficiency of a virus in the presence of different concentrations of a compound. Acute and chronic cytotoxicity of these compounds was studied using resazurin cell viability assay-viable cells turn resazurin into fluorescent resorufin ( Table 2; see also Supplementary Materials). In order to compare the bioactivity of mono-and bissulfonylated derivatives, the same tests were performed for the series of previously obtained [26] 3-(N-sulfonylamino)isoxazolines 3a-h,j-l. Previously studied isoxazole derivative 6 ( Figure 4) that showed anti-TBEV activity [41] was used as a positive control. Despite it not being structurally similar to the compounds studied in this work, it allowed us to confirm the validity of the obtained EC50 values. Most of bis(sulfonyl)amines 4 and 5 and several sulfonylamines 3 revealed anti-TBEV activity in micromolar concentrations. The most favorable substituents in sulfonyl moiety were found to be trimethylphenyl (3-5e), 2-or 4-nitrophenyl (3-5h, 4-5i) and naphtyl (3-5j)-all the types of isoxazolines containing these groups demonstrated antiviral activity.
Compounds 4b-l and 5b-l, containing spiro-annulated or 1,2-annulated cyclooctane moieties, did not show a clear relation between antiviral activity and the configuration of Finally, all compounds 3-5 have been tested for anti-YFV activity. Spiro-isoxazoline 3b, bearing benzylsulfonyl moiety, was found to be active with EC 50 24 ± 11 µM, while other isoxazolines demonstrated no activity in concentrations up to 50 µM. Compound 6 was used as a positive control.
It should be noted that all the tested compounds demonstrated no acute cytotoxicity against PEK cells (see Supplementary Materials), and only compound 5i was slightly toxic during 7 days of incubation. In the case of Vero cells, compounds 3j, 4i, and 5k showed both acute and chronic cytotoxicity, while 4h was only acute toxic (Table 3; see also Supplementary Materials). Thus, the compounds studied here represent a substantially novel class of non-toxic antivirals, deserving further detailed studies of the mechanism of action and antiviral efficiency in vivo.

General Remarks
1 H and 13 C NMR spectra were recorded on a 400 MHz spectrometer Agilent 400-MR (400.0 and 100.6 MHz for 1 H and 13 C, respectively; Agilent Technologies, Santa Clara, CA, USA) at r.t. in CDCl 3 ; chemical shifts δ were measured with reference to the solvent (CDCl 3 , δ H = 7.26 ppm, δ C = 77.16 ppm). When necessary, assignments of signals in NMR spectra were made using 2D techniques. Accurate mass measurements (HRMS) were obtained on Bruker micrOTOF II (Bruker Daltonik GmbH, Bremen, Gemany) with electrospray ionization (ESI). Analytical thin-layer chromatography was carried out with silica gel plates supported on aluminum (ALUGRAM ® Xtra SIL G/UV 254 , Macherey-Nagel, Duren, Germany); the detection was performed by a UV lamp (254 nm). Column chromatography was performed on silica gel (Silica 60, 0.015-0.04 mm, Macherey-Nagel, Duren, Germany). 3-Aminoisoxazolines 1,2 and compounds 3a-l were obtained via described methods [26]. All other starting materials were commercially available. All reagents except commercial products of satisfactory quality were purified according to literature procedures prior to use.
Stock solutions of the compounds with a concentration of 5 mM were prepared in DMSO (Amresco, Cleveland, OH, USA).

Synthesis of Compounds 4,5 (General Methods)
Method A. To the solution of 3-aminoisoxazoline 1 or 2 (0.18 mmol) in dry DCM (2 mL), DIPEA (4 equiv, 0.72 mmol, 93 mg, 125 µL) was added under argon. The reaction mixture was cooled down to 0 • C, and sulfonyl chloride (0.9-5.5 equiv, 0.16-1.0 mmol) was added. Then the mixture was allowed to warm up to r.t., stirred for 3 h-8 days, and quenched with water (6 mL). The organic layer was separated, and the water layer was extracted with DCM (3 × 6 mL). Combined organic layers were washed with saturated aqueous NaHCO 3 (9 mL) and brine (9 mL) and dried over MgSO 4 . The solvent was evaporated under reduced pressure. The product was isolated via preparative column chromatography (SiO 2 ).
Method B. To the solution of 3-aminoisoxazoline 1 (0.28 mmol, 50 mg) in dry pyridine (1 mL), DIPEA (7.9 equiv, 2.2 mmol, 284 mg, 0.38 mL) was added under argon. The reaction mixture was cooled down to 0 • C, and 2-nitrobenzenesulfonyl chloride (5.9 equiv, 1.65 mmol, 365 mg) in dry pyridine (0.5 mL) was added. Then the mixture was allowed to warm up to r.t., stirred for 3 days, and quenched with 1M HCl (6 mL). The organic layer was separated, and the water layer was extracted with DCM (3 × 6 mL). Combined organic layers were washed with saturated aqueous NaHCO 3 (9 mL) and brine (9 mL) and dried over MgSO 4 . The solvent was evaporated under reduced pressure. The product was isolated via preparative column chromatography (SiO 2 ).