Stereochemical effects in the EI mass spectra of cis-and trans-fused octa/hexahydro-3,l-benzoxazines and -benzothiazines

The electron ionization mass spectra of cis-and trans-fused 2-oxo-( 1 , 2 ), and 2-thioxo-4-phenyloctahydro-2 H -3,l-benzoxazines ( 3,4 ), 2,4-diphenylhexahydro-3,1-benzoxazines ( 5,6 ) and 2-phenylimino-4-phenylhexahydro-3,l-benzoxazines ( 7 , 8 ) and -benzothiazines ( 9,10 ) were recorded, and the fragmentation pathways established and compared to their 4-unsubstituted counterparts. Effects of stereochemistry, substitution, and (in the case of 2-phenylimino derivatives) of an intramolecular cyclization of the [M-H] + ions were observed in the mass spectra. In general, mass spectral behavior was similar for the isomeric compounds although they could usually be differentiated from each other based on the relative abundances of their characteristic fragment ions.


Introduction
Saturated benzoxazines and -thiazines form a class of heterocyclic compounds which possess remarkable pharmaceutical activity. 1 They are also interesting from an organic synthesis and structural chemistry point of view.
It has been shown earlier 5 that in the case of 2-thioxoperhydro-3,l-benzoxazines, cis-and trans-fused isomers gave similar spectra, although the cis-type compounds fragmented more easily.The isomers could, however, be distinguished from each other.The gas phase enolisa-tion or thioenolisation was not found which probably also reflects the lack of ring-chain tautomerism.This was also true for octahydro-3,1-benzoxazines 6 with a heteroaryl moiety at position 2. It was shown previously 7 that an intramolecular cyclization could occur between the ring nitrogen atom and the ortho-carbon of the phenylimino moiety at position 2 which was always accompanied 8 with a hydrogen loss from position 1.
The aim of this study was to find out whether the phenyl substitution at position 4 has any significant effect on the mass-spectral fragmentations and whether any cyclization occurs between an ortho-carbon of the phenylimino group and the ring nitrogen.It was also interesting to see how much the higher polarizability of sulfur as compared with that of oxygen atom alters the fragmentations.The possibility to differentiate cis-and trans-fused isomers based on their low-resolution mass spectra was also a question of interest.

Experimental Section
Synthesis.The synthetic pathways to the aliphatic cyclocondensed 1,3-oxazines and -thiazines studied are shown in Schemes 2 and 3.The syntheses of all compounds were described earlier 9 except for thiazines 9 and 10.The reactions of A and B with ethyl chloroformate followed with sodium methoxide gave 2-oxo-octahydro-2H-3,1-benzoxazines 1 and 2 in good yields.The corresponding 2-thiones 3 and 4 were prepared by reacting A or B with carbon disulfide.The cyclizations of A and B with ethyl benzimidate resulted in 2,4-diphenyl-4a,5,6,7,8,8a-hexahydro-4H-3,1-benzoxazines 5 and 6 and the treatment of A and B with phenyl isothiocyanate provided the thiourea derivatives 11 and 12, which were treated with methyl iodide and alkali to give 2phenylimino-3,l-benzoxazines 7 and 8 with retention of configuration of the 4-phenyl substituent. 9hen thioureas 11 and 12 were refluxed in ethanol containing dry HC1, thiazines 9 and 10 were obtained in good yields.In the ring closure it is possible to have either retention or inversion of configuration.According to NMR-measurements, both 9 and 10 attain inverted configurations.

NH
NMR spectroscopy.The NMR spectra of compounds 9 and 10 were recorded on a Bruker Avance 500 NMR spectrometer (500.13MHz for 1 H and 125.76 MHz for 13 C) in CDCl 3 using TMS as an internal reference.The spectra were measured at 25 °C both with and without adding a drop of trifluoroacetic acid (TFA); for 10, 1 H spectrum without TFA was recorded also at 55 °C.The experiments consisted of standard 1 H and 13 C{ 1 H} NMR, 1D gs-NOESY or NOEdifference, dqf-COSY, 1 H{ 13 C}-HSQC and 1 H{ 13 C}-HMBC, using vendor-supplied pulse programs.

Results and Discussion
The structures of compounds 1-8 have been verified earlier. 9Compound 9 displayed large vicinal diaxial couplings between protons H4 and H4a, and between H4a and H8a (10.7 and ca. 10 Hz, respectively), thus confirming both the trans-fusion and the axial position of H4 for this compound.Consistently with this, presaturation of the H4 resonance resulted in an NOE enhancement of protons H5ax and H8a. 1 H and 13 C signals were broadened at 25 °C to the point that some 13 C signals could not be observed, indicating the presence of dynamic processes (e.g.endo-exo-tautomerism of the double bond or E-Z-isomerism with respect to it).Addition of a drop of TFA resulted in sharper NMR lines with all carbon signals being visible.Compound 10 was only slightly soluble in CDCl 3 at 25 °C showing very broad 1 H resonances.At 55 °C, signals were sharper, though still broadened.Again, adding TFA increased the signal resolution, and dramatically improved as well the solubility, allowing measurements at 25 °C.In these conditions, 10 showed a large coupling (9.2 Hz) between H4 and H4a, indicating their mutual trans orientation, and thus also the predominance of N-out conformation (i.e. a conformation where N1 is equatorial w.r.t. the cyclohexane moiety).1D NOESY confirmed that in the predominant ring-invertomer H4 is facing the cyclohexane ring (e.g. an NOE in H8ax due to H4 was observed).At 55 °C without the presence of TFA, the coupling between H4 and H4a was resolved in the H4 signal; J(H4,H4a) = 6.6 Hz.Assuming this coupling has a value of 10.7 Hz in the limiting N-out conformer (axial-axial relationship, cf.9) and 2.5 Hz in the N-in conformer (equatorial-equatorial relationship as shown in Scheme 3), we conclude that 10 exists in these conditions as a roughly 50:50 mixture of these conformers.A further indication for the fact that indeed the Ph is axial in compounds 3, 5, and 7 is their C-8a chemical shift which is around 50 ppm whereas for 10 it is ca.58 ppm -so one can see the shielding effect of axial Ph in the former.To further prove this situation we remeasured the H4,H4a couplings for compounds 3 (5.0Hz) and 5 (5.3 Hz) which are what one can expect from an eq,ax-type coupling between these protons.
The 70-eV EI mass spectra of compounds 1-10 are listed in Table 1 which shows that all compounds except 2-oxo-derivatives 1 and 2 exhibit intense molecular ion peaks under the EI conditions.The M +• peaks are especially abundant for sulphur-containing derivatives 3, 4, 9 and 10 and give rise to the base peaks in thiazines 9 and 10.Note that [M-H] + ions were detected only in the spectra of 2-phenylimino-substituted compounds (7−10).An abundant loss of hydrogen occurred from their molecular ions ([M-H] + ions, 40-90% RA).This loss is most probably due to the intramolecular cyclization between the ortho-carbon of the iminophenyl group and the ring nitrogen (Scheme 4) -a phenomenon often observed in oxazines, thiazines and pyrimidinones with e.g. a benzyl or 2-phenylimino substituent in a position next to the ring nitrogen atom. 7However, elimination of one of the bridgehead or benzylic hydrogen atoms is also likely.The possible hydrogen migration between 1-NH and 2-NPh moieties can further stabilize the structure thus formed.Some similarities exist between the fragmentations of 2-phenylimino oxazines and corresponding thiazines.However, due to the greater ability of sulfur to maintain the charge some of the main fragmentation routes are different.The fragment ions appear to be less abundant and more numerous in the thiazine spectra.The differences between the LR spectra of cis-and trans-fused isomers are very small in thiazines but in case of oxazines the isomers can be differentiated based on their LR spectra.
The main difference between the fragmentation routes of 2-phenylimino-oxazines (7 and 8) and -thiazines (9 and 10) is that the M +• of oxazines do not release hydroxyl radicals although SH • is released from the M +• of thiazines.Also, the ion C 13 H 16 N + at m/z 186, which in oxazines is formed directly from M +• , can be obtained from the M +• of thiazines only after the loss of SH • .Also, the ions C 7 H 5 NS +• (m/z 135) observed in the CID spectrum of M +• of thiazines, although there is no counterpart C 7 H 5 NO +• ions in the CID spectra of oxazines.This reflects the role of the sulfur atom which localizes the charge in the phenyliso-thiocyanate ions.

Scheme 4
Major differences can also be found between the CID-oaTOF spectra of M +• ions of cis-and trans-fused isomers of these compounds.While the trans-fused isomers 7 (X = O) and 9 (X = S) give abundant cyclic C 7 H 6 N 2 X +• ions (100% and 65% RA, respectively), the base peak in the CID spectrum of cis-fused 2-phenylimino-oxazine 8 corresponds to the protonated phenylurea, C 7 H 9 N 2 O + (m/z 137), which is formed through loss of C 13 H 13 • from M +• .The cis-fused 2phenylimino-thiazine 10 loses SH • radical but very little of the (supposedly cyclic) ion C 7 H 6 N 2 S +• .Furthermore, the loss of hydrogen from M +• results in almost identical [M−H] + ions for the cis-and trans-fused isomers in both pairs of compounds.This agrees well with the [M−H] + cyclization hypothesis, but can also be due to a hydrogen rearrangement.
The behaviour of the 2-phenyl compounds 5 and 6 resembles that of the 2-oxo and 2-thioxo compounds.The ring-fusion isomers are easily differentiated on the basis of their EI spectra since the ions at m/z 200 and m/z 186 for the trans form 5 are much more abundant than for the cis form 6 (Scheme 6).Additional stereospecific fragmentations were revealed by their CID-oaTOF-spectra.Thus, the main fragmentation route of the cis form leads to the ions C 13 H 14 • (m/z 170), but the trans form gives the ions C 13 H 14 NO + (m/z 200) instead.Although the oaTOF-CID spectra of M +• for the cis-and trans-fused isomers are different, the loss of hydrogen makes the CID spectra of their [M-H] + ions quite similar.Stereochemical effects of ring annelation on the relative abundance of [M-H] + ions have been described in the literature, 10 but in this case the fragments and abundances of the high intensity ions are practically the same for both the cis-and trans-fused isomers, and only the relative abundances of some low intensity ions are different.
Thus, the CID spectra of compounds 7-10 show stereospecific fragmentation patterns unless the stereoisomers produce a common structure (in the case of [M-H] + ions).
The EI spectra of the 2-oxo isomers 1 and 2 show that they fragmented primarily via the loss of CO 2 from the M +• ions (Scheme 5).They differed more clearly from each other than the corresponding thioxo-isomers 3 and 4. For instance, the M +• ions are less stable, and the [M-CO 2 ] +• ions more abundant in the spectrum of the trans-fused 1 than in that of 2.

Scheme 5
However, the EI spectra of cis and trans-fused isomers of 2-thioxo compounds 3 and 4 also provided a few peaks with sufficiently different abundancies which allowed their differentiation.Fragmentations of compounds 3 and 4 proceeded mainly by the losses of HS

Conclusions
It was demonstrated for the 2-phenylimino substituted compounds that under EI conditions a hydrogen loss from their molecular ions is followed by cyclization to the ring nitrogen atom.This observation is in a good agreement with earlier results.
In general, the mass spectral behavior is similar for the isomeric compounds studied.The EI spectra of the isomeric 2-phenylimino thiazines are almost identical but the daughter-ion spectra of their molecular ions are different.The corresponding oxazines differ mainly by the higher stability of the molecular ion of the trans isomer as compared with that of the cis isomer, probably because the latter forms a protonated phenylurea ion more easily.The isomeric 2phenyl oxazines produce similar EI mass spectra except that the loss of benzyl radical, C 7 H 7 • , from the molecular ion is the main pathway only for the cis isomer but not for the trans isomer.The spectra of the isomeric 2-thioxo compounds exhibit only fairly small differences but those of the 2-oxo-compounds are clearly different.
The stabilities of the thioxo-compounds under electron ionization do not practically differ from those of the 4-unsubstituted homologues, probably because the sulfur atom better stabilizes the ions due to charge localization.In contrast, the difference between the isomeric oxo- Scheme 2