Fragmentation studies of tetrahydropyridocarbazole derivatives by EI , ESI-MS / MS and FAB

Four tetrahydropyridocarbazole derivatives were analysed by different mass spectrometry techniques: electrospray ionization, fast atom bombardment and by low and high resolution 70 eV electron ionization. Retro Diels Alder is the main fragmentation pathway, whereas other pathways leading to [M −1], [M−CH3] and double charge ions also occur to considerable extents. Semi-empirical calculation provided some evidence on the nature of tropylium ions [M −1]. Calculation of ∆Hf indicated that [M−1] could be formed preferentially when a hydrogen atom is lost from the methyl substituent of the homoaromatic ring.


Introduction
Ellipiticine and olivacine are two important pyridocarbazoles alkaloids that exhibit antitumor activity and their synthetic routes [1] and biological activity [2,3] have been widely studied.Although the pyridocarbazole system is present in several important compounds, few mass spectrometry studies have been performed on their fragmentation pattern.
Continuing our work toward the synthesis of substances with antitumor activity we synthesized new angular derivatives of pyridocarbazoles starting from alkylquinolines.Here we describe the preparation of 1-4 (Fig. 1) and report our results on their fragmentation pattern by EI, ESI-MS/MS and FAB.

Experimental
The infrared spectra were recorded in a Perkin Elmer model 1420 instrument.The nuclear magnetic resonance spectra, 13 C and 1 H, were obtained from a Varian-Unity Plus instrument (300 MHz for 1 H and 75.0 MHz for 13 C).Elemental analysis were obtained using a Carlo Erba instrument model 1104.Low-resolution EI mass spectra were recorded on a Finnigan MAT 711A instrument.The ionization energy used was 70 eV with the source at 200 • C and with an accelerating voltage of 8 kV.Samples were introduced by the standard direct insertion probe.High resolution data were obtained with the same instrument using 10,000 resolution.FAB positive-ion mass spectra were recorded on a Finnigan MAT-TSQ 70 triple quadrupole mass spectrometer.For FAB analysis, m-nitrobenzyl alcohol (NBA) was used as the matrix.Xenon was used to generate the bombarding atoms and the FAB gun was operated at 2 mA emission, providing fast atoms with an energy of 10 kV.Ionspray mass spectra were acquired with a triple quadrupole instrument, Perkin Elmer (API III TAGA 6000E) equipped with a ionspray source (Sciex, Toronto, Canada).Samples were infused into the electronspray source, in methanol solution, using a syringe inlet.The semi-empirical calculation were performed using the AM1 [23] and PM3 [24] methods as implemented in the MOPAC 93 program [25].All geometries were fully optimized using the EigenVector following optimization routine [26] until the gradient norm dropped bellow 0.05.Geometry optimizations were followed by frequency calculations to certify that the geometries found are true minima.

Results and discussion
The usual preparation of these substances is based on the well known Fischer indole cyclization between an acyclic or cyclic ketone and appropriated hydrazine derivative of quinoline.Then an appropriated alkylaminoquinoline, 2,4,8-trimethyl-5-aminoquinoline, 2,4,5,8-tetramethyl-6-aminoquinoline or 2methyl-6-aminoquinoline is diazotized and reduced in situ to give the hydrazine which was not isolated.When the hydrazine was reacted with cyclohexanone or α-tetralone the only product obtained were the angular tetrahydropyridocarbazoles 1-4.During the preparation of compound 2 the methyl group at position 5 was eliminated.Although this is an unusual result, similar observation had been already reported in the literature.

Mass spectrometry studies
Compounds 1, 2 and 4 in EI showed high intensities for the molecular ion M +. and the retro Diels Alder (RDA) ion, and low intensities for the [M−1] + ion.Compound 3 produced intense molecular and [M−1] + ions and no measurable RDA ion (Table 1).
Table 4 reports the ESI-MS spectra of 1-4, in which the RDA fragment [M−28] + was also observed for 1, 2, and 4. RDA also occurs from MH + by collision-induced dissociation (CID).When the protonated molecule is subjected to ESI-MS/MS, those for 1, 2 and 4 form the RDA ion as the major fragment (spectra not shown).
The  The [M−1] + fragment is observed by EI (Table 1) and FAB (Table 3).Hydrogen loss is common for benzyl derivatives [16][17][18], methylquinolines and isoquinolines [19][20][21].However, when a methyl group is attached to the homoaromatic ring the rearrangement of the benzyl cation to the tropylium ion often occurs, and when the methyl group is attached to the pyridine ring, the analogous azatropylium ion is observed.These alternative dissociations are exemplified for the model compound 2,4,8-trimethylquinoline 5 in Fig. 3, yielding either the tropylium ion 5f or the azatropylium ion 5b or 5d.
Draper and Maclean [22] studied the azatropylium mechanism by deuterium labeling dimethylquinoline.They suggested hydrogen atom loss and ring expansion, followed by loss of CH 3 CN or HCN.However, our results showed that the loss of hydrogen atom occurred but without HCN loss [?].
To stablish the more favorable H atom loss from M +. (Fig. 3), we performed semi-empirical AM1 and PM3 calculations for the model ions 5a-f.
Both the AM1 and PM3 results (Table 5) indicate thatthe most stable [M−H] + ion is 5e, that formed when the hydrogen atom is lost from the methyl group at position 8. Loss of a hydrogen atom from the methyl group in positions 2 (5a) or 4 (5c) leads to ions that are at least 10 kcal/mol less stable than 5e.Similarly, the tropylium ion 5f formed from 5e is more stable by near 5 kcal/mol than that originated from the rearrangement of the cation of position 2 (5b) or 4 (5d).
This finding for the model compound 5 suggests that the m/z [M−1] + in the tetrahydropyridocarbazoles 1, 2 and 3 correspond to H loss from the methyl group attached to the homoaromatic ring.Compounds 1, 2 and 4 (Table 1) also show doubly charged M 2+ and [M−28] 2+ ions, the latter corresponding to RDA fragmentation of M 2+ .Compound

Conclusions
This study showed that the [M−28] + fragment ions of tetrahydropyridocarbazolesare formed by RDA fragmentation.Although the formation of azatropylium was observed, it was not possible establish it subsequent loss of HCN.Semi-empirical calculation provided some evidence on the nature of tropy-lium ions [M−1] + .Calculation of ∆Hf 0 indicated that [M + −1] could be formed preferentially when a hydrogen atom is lost from the methyl substituent of the homoaromatic ring.
RDA fragment of m/z 236 from both 1 and 2, also subjected to CID, forms m/z 221 by methyl loss, a fragment of m/z 206 by CH 2 O loss, and a fragment of m/z 194 by the combined loss of [H+CH 3 CN].

Fig. 3 .
Fig. 3. Possible pathways for H loss of the model compound 5.