NMR Studies on [ 2 + 3 ] Cycloaddition of Nitrile Oxides to Polyunsaturated Medium Size Rings

Site selectivity, regioselectivity and stereoselectivity of [2 + 3] cycloaddition of 4-trifluoromethylbenzonitrile oxide to polyunsaturated medium size rings including 1,5,9-cyclododecatriene, 11-membered sesquiterpenes, 1,3-cyclooctadiene and 5-vinyl-2norbornene were examined. Site selectivity was correlated with electron charges of alkenyl carbon atoms. Structure of the products has been established by an extensive application of 1D and 2D H and C nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry. Some of the obtained products showed moderate fungicidal activities.


Introduction
Isoxazolines are one of major classes of five-membered nitrogen containing heterocycles, found in a large number of natural products and biologically active compounds.A variety of synthetic methods has been elaborated for preparation of 2-isoxazolines, of which the most convenient and attractive route is the [2 + 3] dipolar cycloaddition of nitrile oxides to alkenes. 1 2-Isoxazolines can be easily reduced to several synthetically important compounds such as b-hydroxy ketones, b-hydroxy esters, a,b-unsaturated carbonyl compounds or iminoketones. 2he nitrile oxides can be formed either by Huisgen method from aldoximes by chlorination and base-induced dehydrochlorination 1 or by dehydration of primary nitro compounds by phenyl isocyanates (Mukayama method) 3 or ethyl chloroformate (Shimizu method). 4 key feature of the cycloaddition is the cis-stereospecificity -from E-alkenes 4,5-anti isomers are produced and from Z-alkenes 4,5-syn products are obtained. 5eactions of monosubstituted and 1,1-disubstituted alkenes are very regioselective favoring strongly 5-substituted 2-isoxazolines.On the other hand, 1,2-disubstituted olefins usually afford mixtures of regio-and stereoisomers.Two methods have been used to solve these problems.One approach was a substrate control, an application of appropriately functionalized cycloaddends.The second more effective approach relied on metal complexes acting as catalysts or on organocatalysts.Shortage of reports on metal assisted 1,3-dipolar cycloadditions of nitrile oxides was due to interference of catalyst with generation of these dipoles and formation of unreactive complexes. 6ite-selectivity of nitrile oxides cycloaddition to polyunsaturated alkenes was examined in several laboratories.In reactions of nitrile oxides with dimethyl 7-(diphenylmethylene)bicyclo[2.2.1]hept-2-ene-5,6dicarboxylateonly disubstituted norbornene double bond participated. 7Similarly, in recently examined cycloadditions of aryl nitrile oxides to norbornenes substituted with an acrylate-derived moiety, only adducts to norbornene system were formed with good site and exo selectivity. 8ite selectivity and regioselectivity of cycloaddition to cyclohexene derivatives was studied in our group. 9,6-Dichlorobenzonitrile oxide reacts with isothiazolones at the ethylenic double bond.Mesitonitrile oxide, on the other hand, adds preferentially to the carbonyl double bond. 10o [2 + 3] cycloaddition reactions of nitrile oxides to medium ring cycloalkenes was reported before, to the best of our knowledge, presumably because of the low reactivity of these dipolarophiles.However, other types of cycloadditions to these systems are known.Diels-Alder cycloaddition of benzotropolone with humulene (11-membered sesquiterpene) was described. 11Acetylacetonatoboron difluoride and oxalate undergo cycloaddition from their singlet excited state with cyclic olefins to give non-regiospecific products.A conjugated diene, 1,3-cyclooctadiene, reacted similarly but with slower rates. 12Singlet excited dibenzoyl(methanato)boron difluoride underwent also cycloaddition to 1,3-cyclooctadiene. 13Reaction of SO with 1,3-cyclooctadiene gave the corresponding [1 + 4] adduct in low 2% yield. 141,3-Cyclooctadiene was unreactive in titanium complex catalyzed [2 + 6] cycloaddition reaction with cycloheptatriene. 15Reaction of unconjugated triene 1,5,9-cyclododecatriene with chlorosulfonyl isocyanate (CSI) gave an unsaturated b-lactam product, where only one double bond had reacted. 16Acetylacetone underwent a photochemical cycloaddition to 1,5,9-cyclododecatriene (the de Mayo reaction). 17The sterically controlled cycloaddition of dichloroketene to unreactive olefin, 5-methylene-2-norbornene, has been accomplished. 18It resulted in a spirocyclobutanone derivative formed by addition across the exocyclic double bond.
The sesquiterpenes possess fifteen carbons, derived from three isoprenoid units.This is a vast group of naturally occurring substances, containing an immense range of structural diversity which includes acyclic, monocyclic, bicyclic, tricyclic and tetracyclic compounds.Many sesquiterpenes display biological activity including antimicrobial, antitumor and cytotoxic properties. 19Some bisnor-sesquiterpenes exhibit allelopathic activity. 20erein we present results of our research concerning site selectivity, regioselectivity and stereoselectivity of 1,3-dipolar cycloaddition of 4-trifluoromethylbenzonitrile oxide to polyunsaturated medium rings including 1 1 -m e m b e r e d s e s q u i t e r p e n e s 5 , 8 a n d 1 0 , 1,5,9-cyclododecatriene (13), 1,3-cyclooctadiene (18)  and 5-vinyl-2-norbornene (21) envisaging to obtain new biologically active compounds.Structure of the products has been established by an extensive application of 1D and 2D 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS).

Materials and physical measurements
Reagent-grade chemicals were used without further purification unless otherwise noted.Compounds 5, 8, 10, 13, 18, 21 were purchased from Aldrich.Hydroximinoyl acid chlorides were prepared from the corresponding aryl aldehyde oximes and N-chlorosuccinimide (NCS) in N,N-dimethylformamide (DMF). 21pectra were recorded as follows: Fourier transform (FT) infrared (IR) spectra on a JASCO FTIR-420 spectrometer; 1 H, 13 C NMR, 2D correlation spectroscopy (COSY), 2D heteronuclear single quantum coherence (HSQC), 2D heteronuclear multiple bond correlation (HMBC) and 2D rotating frame nuclear Overhauser effect spectroscopy (ROESY) analyses on a Bruker AVANCE III 500 MHz and a Varian VNMRS 600 spectrometers in deuterated chloroform.The 1 H NMR spectra were recorded using single-pulse sequence and spectral width (SW) of 10000 Hz, 30° pulse width (pw) of 9.7 ms, an acquisition time (at) of ca.3.3 s and 64 k complex points.The free induction decays (FIDs) were processed with zero filling.The 13 C spectra were obtained using a spectral range of ca.31250 Hz, 30° pulse width (3.3 ms), an acquisition time of ca.1.05 s, a relaxation delay of 1.5 s and collecting 64 k complex points.Chemical shifts are given in ppm (d) relative to tetramethylsilane (TMS) as an internal standard and coupling constants are reported in Hz.The 2D gradient selected (g)COSY and ROESY spectra were run using spectral width (ca.6000 Hz) in both dimensions, at = 0.18 s, 2-4 (COSY) and 4-8 (ROESY) transients per 512 increments, relaxation delay (d1) of 2.0 s.Prior to FT the data were processed using squared sinebell (COSY) and gaussian (ROESY) multiple function.In the case of the ROESY experiments spinlock time ca.200 ms was chosen.The echo-antiecho phase-sensitive 1 H/ 13 C gHSQC correlation were obtained with an at of 0.18 s, spectral window of 5500 Hz (F2) and 25500 Hz (F1), 512 increments in the 13 C dimension, d1 = 2.0 s and 2 transients per t 1 increment.Experiments were optimized for 1 J(C-H) = 145 Hz.The data were zero-filled to 2048 points and processed using cosine-squared window function in both dimensions prior to Fourier transformation.The proton and carbon 90° pulse lengths were 8 and 15 ms, respectively.The 1 H- 13 C HMBC experiments with pulse field gradient (PFG) coherence selection using two PFG pulses were recorded with the following parameters: an at of 0.18 s, spectral windows of 5500 Hz (F2) and 25500 Hz (F1), 512 increments in the 13 C dimension, 4 transients per increment and d1 of 2 s.This kind of experiment was optimized for n J(C-H) = 10 Hz.The proton and carbon 90° pulse lengths were 8 and 15 ms, respectively.The 1 H- 15 N HMBC experiments with PFG coherence selection using two PFG pulses were recorded with the following parameters: an at of 0.25 s, spectral windows of 6500 Hz (F2) and 12000 Hz (F1); 2 × 256 increments in the 15 N dimension, 16-32 transients per increment and d1 of 1.5 s, optimized for n J(N-H) = 5.0 Hz.The proton and nitrogen 90° pulse lengths were 7.2 and 30.0 ms, respectively.
Electron ionization (EI) MS were run on an AMD M-40 instrument, ESI-MS on an LCT (Micromass) apparatus.Flash chromatography was carried out using silica gel S 230-400 mesh (Merck) using hexane-ethyl acetate mixtures as an eluent.Calculations of electron charges on alkenyl carbon atoms, molecular modelling Vol. 27, No. 11, 2016   and substrate HOMO/LUMO energies were calculated with the Hyperchem 7.5 program using semiempirical AM1 method. 22cloaddition reaction of dipolarophiles 5, 8, 10, 13, 18 and 21 with 4-trifluoromethylbenzonitrile oxide (4).A general procedure for preparation of 6, 7, 9, 11, 12, 14-17, 19, 20, and 22-27 4-Trifluoromethylbenzonitrile oxide (4) was generated as follows: a solution of the corresponding chloroxime (0.25 g, 1.12 mmol) in dry dichloromethane was passed through an Amberlyst-21 column and added dropwise over 30 min to the solution of a dipolarophile in dry dichloromethane, and the solution was stirred overnight at room temperature.Water was added, organic layer was separated and the aqueous one extracted with dichloromethane.The combined organic layers were dried (MgSO 4 ) and the product was purified by flash column chromatography.

Fungicidal testing
The compounds were screened for fungicidal activity in vitro.The tests were carried out for Fusarium culmorum (F.c.), Phytophthora cactorum (P.c.), Rhizoctonia solani (Rh.s.), and Botrytis cinerea (B.c.), and 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 L -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.
Cycloaddition of the sesquiterpene C 15 trans,trans,trans-2,6,6,9-tetramethyl-1,4,8-cycloundecatriene (5) afforded two mono-adducts to different double bonds, 6 and 7, in 3:1 ratio (Scheme 1, Figure 1).Structure 6 was assigned to the major isomer based on analysis of 2D NMR HSQC and COSY spectra.Presence of three H-C correlations in an olefinic area in HSQC spectrum and presence of HC=HC group in the COSY spectrum indicated that an addition to one of two tri-substituted double bonds took place.Observation of a CH 1 -CH 2 11 -CH 2 10 and not of a CH 8 -CH 2 7 moiety in COSY spectrum showed that the addition occurred to C1-C2 double bond.Finally, regiochemistry of the cycloaddition was established as that shown in structure 6 by lack of H-C correlation in HSQC spectrum for carbon 2 connected to isoxazoline oxygen atom.This direction of addition was favored by the orbital factors.The reaction is LUMO-dipole controlled and oxygen atom of the dipole tends to attack the more substituted carbon atom of the dipolarophile.Structure 7 was assigned to the minor isomer based on analysis of 2D NMR HSQC, COSY and HMBC spectra.Presence of two H-C correlations in an olefinic area in HSQC spectrum proved the addition to the di-substituted C4-C5 double bond.Correlations in the COSY spectrum between H5 and H13/H14 protons from the methyl groups 3', 5' 125.8 (q, 3.8) 125.8 (q, 3.8) 125.8 (q, 3.8) 125.9 (q, 3.8)
A complete assignment of 1 H and 13 C NMR spectra of 9 was based on analysis of 1D and 2D 1 H and 13 C NMR spectra (COSY, HSQC and ROESY) and is presented in Experimental.Presence of HC=HC unit in E configuration (a large coupling constant of 15.5 Hz in 1 H NMR spectrum) confirmed by the 2D COSY spectrum and two olefinic H-C correlations in HSQC spectrum indicated that C4-C5 double bond was preserved.Lack of H-C correlation in HSQC spectrum for carbon 9 connected to the isoxazoline oxygen atom and presence of H-C correlation for C8 have shown the same regiochemistry of the reaction as in case of compound 6.Oxygen atom of the second molecule of the dipole attacked the mono-adduct exocyclic double bond at the di-substituted carbon atom as expected.The 2D HSQC spectrum showed the presence of two H14a,b-C14 (43.1 ppm) correlations.Facial selectivity was proved to be as shown in the structure 9 by observation in the 2D ROESY NMR spectrum of a proximity between H3 and H14a/H14b.
Cycloaddition to the bicyclic C15 sesquiterpene trans-(1R,9S)-8-methylene-4,11,11-trimethylbicyclo[7.2.0]undec-Vol.27, No. 11, 2016 Scheme 4. Cycloaddition of the dipole 4 to trans,trans,cis-1,5,9-cyclododecatriene (13).(i) CH 2 Cl 2 , r.t.Non-systematic numbering of atoms has been used in adducts 14-17 for ease of comparison of spectral data.Scheme 5. Cycloaddition of the dipole 4 to 1,3-cyclooctadiene (18).(i) CH 2 Cl 2 , r.t.Non-systematic numbering of atoms has been used in adducts 19 and 20 for ease of comparison of spectral data.4-ene (10) afforded a mixture of a mono-adduct 11 (45%) and a bis-adduct 12 (30%) (Scheme 3, Figure 1).Structure of the mono-adduct 11 was established based on analysis of 1D and 2D 1 H and 13 C NMR spectra (COSY, HSQC and ROESY); a complete assignment of 1 H and 13 C NMR spectra is presented in Experimental.Cycloaddition to the exocyclic double bond via attack of the oxygen atom of the dipole on the di-substituted carbon atom afforded a spiro system, where carbon atom C1 attached to oxygen did not show any H-C correlations in the 2D HSQC NMR spectrum.Facial selectivity was established by observation in the ROESY NMR spectrum of correlations between H13a,b at 3.32 and 3.19 ppm and H10a,b at 2.07 and 1.96 ppm, as well as between H13b and aryl H2'/H6' protons.The C4-C5 double bond was preserved as witnessed by observation in the 13 C NMR spectrum signals of the quaternary C5 at 133.8 ppm and C4 at 120.2 ppm coupled in the HSQC spectrum to H4 at 5.21 ppm.
Mass spectrometry of the other product showed molecular weight of 578 mu, indicating a bis-adduct structure 12.It showed a similar regiochemistry and geometry of the spiro isoxazoline ring as the mono-adduct 11.The 2D ROESY NMR spectrum showed a proximity of H13a (3.43) and H11 (1.92), H13b (3.20) and H10a (1.81), as well as H13a,b and aryl H2'/H6' protons (7.71 ppm).Regiochemistry of the second cycloaddition step resulted from attack of the dipole oxygen atom at the more substituted carbon atom C5.
Cycloaddition to trans,trans,cis-1,5,9-cyclododecatriene (13) afforded a mixture of two inseparable regioisomeric mono-adducts to cis double bond 14 and 15 (53:47) as indicated by mass spectrometry giving a molecular weight of 349 mu, and two inseparable regioisomeric monoadducts (60:40) to the trans double bond 16 and 17 in an overall 62:38 ratio (Scheme 4).A complete assignment of 1 H and 13 C NMR spectra of the products was based on analysis of 1D and 2D 1 H and 13 C NMR spectra (COSY, HSQC, HMBC, and ROESY NMR) and is presented in Tables 1 and 2.
Structures 14 and 15 were proposed to the major pair of adducts assuming a preferred addition to the cis-double Scheme 6. Cycloaddition of the dipole 4 to 5-vinyl-2-norbornene (21).(i) CH 2 Cl 2 , r.t.Non-systematic numbering of atoms has been used in adducts 22-27 for ease of comparison of spectral data.bond.Calculations carried out with the molecular modelling program showed lower energies for these isomers and a much smaller difference of energy between endo and exo forms than for the adducts to the trans bonds of the dipolarophile 13.Larger coupling constants (12.5 Hz) were observed for the ring junction protons cis H9/H10 in these regioisomers than for the corresponding trans H1/H2 in isomers 16 and 17 (10.5 Hz).Calculations carried out with HNMR Predictor software v.12 confirmed higher J values for these protons in cis adducts 14 and

Rationalization of the observed site selectivity and regioselectivity
Table 5 gives electron charges at the alkenyl carbon atoms of the dipolarophiles 5, 8, 10, 13, 18, 21, and the observed site-selectivity in the cycloaddition reaction.
It was found that the amount of negative charges of both carbon atoms of the double bond correlated with the reactivity of the reaction.Cycloaddition to a-humulene (5) occurred mainly to the C1-C2 double bond with the greatest amount of electric charges (-0.303), and in the minor isomer the dipole added to one of the other two double bonds C4-C5 with a larger sum of electric charges (-0.263).Similarly, in cycloaddition to trans caryophylene (10) in the major adduct 11 more reactive was an exocyclic double bond C8-C12 with total electric charges of -0.321, while the other double bond had a charge of -0.273.The same trend was observed in case of 5-vinyl-2-norbornene (21), where higher electron densities were found in the C2-C3 bond (-0.349/-0.342,exo/endo isomers), than in the C8-C9 bond(-0.264/-0.256,exo/endo isomers).It correlated with a higher reactivity of the C2-C3 bond affording 41% of mono-adducts 22 and 23 to this bond, and 38% of bisadducts 24-27 to C2-C3 and C8-C9 bonds.Some discrepancy from this regularity was noticed for b-humulene (8), where the bis-adduct 9 was formed in reaction of the most electron-rich C8-C12 bond and the least electron rich C1-C2 double bond.It could be explained by the steric factors and a more difficult approach of the dipole to the hindered internal double bond of the dipolarophile flanked with two geminal methyl groups.

Biological activity of the products
Generally, only a modest or weak activity of the compounds synthesized in this project was recorded (Table 6).The highest fungicidal inhibitory potency was found against Botrytis cinerea strain of mono cycloadducts 6 and 19-20 pair.It was comparable to the activity of the reference compound, the commercial fungicide chlorothalonil.
Analysis of the data from Table 6 allows concluding that generally exo regioisomers show a higher activity than the endo regioisomers.As an example isomer 6 was more active than isomer 7, and compounds 22 and 25 were more active than, respectively, cycloadducts 23 and 24.One can see very clearly the influence of the spatial structure on the fungicidal activity.

Conclusions
High regio-and site selectivity of [2 + 3] dipolar cycloaddition reaction of 4-trifluoromethylbenzonitrile oxide (4) to sesquiterpene 8 were observed.A correlation of double bond reactivity with electron charges was found in reactions of most dipolarophiles.The addition to the exocyclic double bond was favored.All diastereoisomeric products were fully characterized by 1 H and 13 C NMR 1D and 2D spectroscopy.Some cycloadducts showed good fungistatic activity.Further research is in progress to analyze the biological potency of the new products, and to improve regioselectivity of the cycloaddition.

Table 5 .
Electric charges at the alkenyl carbon atoms of the dipolarophiles 5

Table 6 .
Fungicidal inhibitory activities of compounds 6