NMR Studies on 1 , 3-Dipolar Cycloaddition of Nitrile Oxides to Norbornenes

A reação de cicloadição 1,3-dipolar de óxidos de nitrila a norbornanos substituídos com um grupo derivado de acrilato foi examinada. Somente adutos para o sistema norbornano foram formados com boa exo seletividade e completa seletividade ao sítio. As estruturas dos produtos foram elucidadas através das técnicas de espectrometria de massas com ionização por electrospray (ESI-MS) e de ressonância magnética nuclear (RMN) 2D de H e C.


Introduction
The 1,3-dipolar cycloaddition of nitrile oxides to alkenes is the most convenient method for the preparation of 2-isoxazolines 1 which can be easily reduced to several synthetically important compounds such as b-hydroxy ketones, b-hydroxy esters, a,b-unsaturated carbonyl compounds or iminoketones. 2Reactions of monosubstituted and 1,1-disubstituted alkenes furnish regioselectively 5-substituted 2-isoxazolines while 1,2-disubstituted olefins usually afford mixtures of regio-and stereoisomers.
The nitrile oxides can be formed either by the Huisgen method from aldoximes by chlorination and base-induced dehydrochlorination 1 or by dehydration of primary nitro compounds by phenyl isocyanates 3  (Mukayama method)  or ethyl chloroformate (Shimizu method). 4he 1,3-dipolar cycloaddition of nitrile oxides to bicyclic alkenes was examined before.For example, norbornadienes afforded mixtures of exo-and endo-adducts in ca.4:1 ratio in reaction with benzonitrile oxide and alphaoxophenylacetonitrile oxide.Reaction of nitrile oxides with norbornadienes substituted with electron deficient groups in 2,3-positions afforded only adducts to the more electron-rich unsubstituted double bond. 5On the other hand, 1,3-dipolar cycloaddition to norbornenes follows the exo rule without exception for symmetrically as well as for unsymmetrically substituted bicyclic systems, although generally mixtures of regioisomers were observed. 6nother problem is site-selectivity of nitrile oxide cycloaddition to polyunsaturated alkenes.In cycloaddition of benzonitrile oxide to 2-alkoxy-1,3-butadienes, only the unsubstituted vinyl group participated in the reactions, while in case of alpha-oxophenylacetonitrile oxide, both double bonds reacted. 7This result indicated the dominance of steric effects over electronic ones in the first case, in which more sterically demanding dipole did not interact with the activated, more electron-rich disubstituted double bond.In reactions of nitrile oxides with dimethyl 7-(diphenylmethylene)bicyclo[2.2.1]hept-2-ene-5,6-dicarboxylate,only disubstitured norbornene double bond partcipated affording exclusively exo cycloadducts. 8ome norbornane derivatives show biological activity.3-Methylene-2-norbornanone was identified as a potent anti-proliferative agent. 9Racemic gluco-configured norbornanes are inhibitors of b-glycosidases. 10ur group has been interested in studies on activity of 2-isoxazolinecarboxamides as plant protecting agents. 11herefore, it was envisaged that fusion of the norbornene system with the isoxazoline carboxamide moiety would be a promising approach to new biologically active products.Herein, the results of our work on 1,3-dipolar cycloaddition of 4-(trifluoromethyl)benzonitrile oxide to norbornenes substituted with alkenoyl groups are presented.Analysis of the reaction products was quite a challenge.There were two reactive double bonds in the dipolarophiles, all three dipolarophiles were mixtures of epimers, and two regioisomers were expected for each of the epimers.The problems were even more complex for dipolarophile 4, in which a chiral menthyl ester function was introduced to study effects of a remote group on regioselectivity and site-selectivity of the reaction.These structural problems were solved using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy.

E-3-(bicyclo[2.2.1]-hept-5-ene)-prop-2-ene-1-carboxylic
acid (1.595 g, 9.85 mmol), L-menthol (1.568 g, 10.10 mmol) and 4-dimethyloaminopyridine (0.742 g, 6.10 mmol) in a mixture of dry dichloromethane/acetonitrile (5 mL, 1:1) under dry argon.Stirring was continued for 24 h.The reaction mixture was filtered and the filter paper was washed with dichloromethane.The solution was washed with water, dilute HCl, water, aqueous solution of sodium bicarbonate, and finally several times with water.The solution was dried (Na 2 SO 4 ) and the product obtained after evaporation of the solvent was purified by flash chromatography on silica gel using mixtures of hexane-ethyl acetate as the mobile phase.The first fractions gave the expected methyl ester 4 as a yellowish wax (60-65%); IR (KBr) ν/cm

Fungicidal testing
The compounds were screened for fungicidal activity in vitro.The test was carried out for Fusarium culmorum Sacc., Phytophthora cactorum Schroek, Alternaria alternata Keissl.(Fr.),Rhizoctonia solani Kuhn, and Botrytis cinerea Pers.Ex Fr, which involved determination of mycelial growth retardation in potato glucose agar (PGA).Stock solutions of test chemicals in acetone were added to agar medium to give a concentration of 200 mg mL -1 and dispersed into Petri dishes.Four discs containing the test fungus were placed at intervals on the surface of the solidified agar and the dishes were then inoculated for 4-8 days depending on the growth rate of the control samples, after which fungal growth was compared with that in untreated control samples.The fungicidal activity was expressed as the percentage of fungi linear growth inhibition compared to that of the control.

Structural analysis of the cycloadducts
The cycloaddition of 4-trifluoromethylbenzonitrile oxide to the olefin 3 afforded two pairs of diastereoisomers which were separated by column chromatography.In the first pair (7a, 8a), the side chain was in endo configuration, and in the second pair (7b, 8b), the side chain was in exo configuration.Cycloaddition to the menthyl ester 4 yielded separable diastereoisomers 9a, 9b and 10a, 10b, and reaction with the urea derivative 5 gave mainly anti adducts 11a, 11b (vide supra) as well as very small amounts of syn isomers 12a,b (not shown).
Only adducts to the norbornene system were formed with a complete site-selectivity and exo selectivity which was proved by coupling pattern of H6 and H7 in the 1 H NMR spectra.Both protons were doublets coupled only to each other (J 8.0-8.4Hz).Coupling constants with the adjacent endo bridgehead protons H5 and H8 are very small Vol. 24, No. 5, 2013 Scheme 1. Cycloaddition products of the dipole 6 to the dipolarophiles 3, 4, 5.
(0-1 Hz) which corresponds to the value of the respective dihedral angles close to 90 o . 5egiochemistry of the cycloadducts was established by

Rationalization of the observed site-selectivity and regioselectivity
Calculations of electron charges on alkenyl atoms C2, C3 as well as C6 and C7 of the dipolarophiles 3-5 with the Hyperchem 7.5 program using the semiempirical AM1 method showed higher total electron densities at C6-C7 double bond, than at the more polarized conjugated C2-C3 bond (Table 7).This difference is probably responsible for the selective cycloaddition to the norbornenyl moiety.
The regioselectivity of the cycloaddition to this fragment is more difficult to explain.The difference of the negative charges between C5 and C6 is small and variable; only for the dipolarophile 4 the norbornene double bond polarization is consistent with the observed regioselectivity.The major syn isomer is formed via Although generally regarded as weak, 16 orbital control determines the observed regiochemistry of cycloaddition of nitrile oxides to acrylates and methacrylates overruling the steric and stereoelectronic factors. 2 Cycloaddition to norbornenes, an electron-rich system, is controlled by LUMO dipol -HOMO alkene interaction since LUMO alkene -HOMO dipol energy gap is higher. 17egioselectivity of the reaction is determined by the values of the atomic orbital coefficients of the olefinic carbon atoms.However, differences of FMO (frontier molecular orbital) coefficient values for sp 2 carbon atoms published for similar systems, 2-substituted norbornadienes, are too small to be a decisive factor. 18These results suggest that secondary orbital interactions are a plausible source of the regioselectivity.A similar conclusion was reached by Kurita and Takayama, 19 who examined electrophilic additions to norbornene.Our group did not find any positive remote group effect of the menthyl ester on regioselectivity of the cycloaddition rection compared to the results obtained for the methyl ester (Table 7), in which the observed level of regioselectivity decreased from 80:20 to 66:34.Such remote substituent effects were, on the other hand, recently reported in the Pauson-Khand [2 + 2 + 1] cycloaddition of 2-substututed-5-norbornenes. 6ological activity of the cycloadducts The biological activity of the compounds 7a-12b against several fungal strains was examined.Preliminary assays showed high fungistatic potency of cycloadducts 7a, 8a pair against Botrytis cinerea and Rhizoctonia solani (100% growth retardation at 200 mg L -1 concentration) which was most active of all the tested compounds (Table 8).The reference compound (chlorothalonil) showed 80 and 88% activities against these strains, respectively.

1 H
NMR and 2D NMR NOESY spectroscopy (Figure1).In compounds 7a,b, 9a,b and 11a,b with anti relationship of the phenyl ring and the methylene bridge, H6 showed cross peak with H5, and H7 showed cross peak with H8.On the other hand, in the regioisomeric cycloadducts 8a,b and 10a,b with syn relationship of the phenyl ring and the methylene bridge, the corresponding H7 exhibited cross peak with H8, and H6 exhibited cross peak with H5 [cf 6].In all, the anti adducts H5 in the vicinity of two electron-withdrawing bonds (C-O and C=C) showed larger chemical shifts than H8 as well as H5 in the syn regioisomer.The relevant 2D COSY, HMBC and NOESY correlations supporting structure elucidation for the diastereoisomers 7a, 10a,b and 11a are shown in Figure1.

Table 4 .
13C NMR signals d of the anti adducts 7a,b and 9a,b (in acetone-d 6 , J in Hz), and 11a,b (in CDCl 3 ; J in Hz)

Table 3 .
1 H NMR signals d of the anti adducts 7a,b and 9a,b (in acetone-d 6 , J in Hz), and 11a,b (in CDCl 3 ; J in Hz)

Table 8 .
Fungicidal inhibitory activities of compounds 3a

Table 7 .
Electron charges at the alkenyl carbon atoms of the dipolarophiles 3-5 (endo isomers) and anti/syn regioselectivity in the cycloaddition reaction atom of the dipole attacking the less negatively charged C5 atom of the dipolarophile.Other factors, such as orbital control and steric interactions, have to be considered, apart from stereoelectronic effects. oxygen