Reactivity of N-Methylidenemalonates of 3-Arylaminoindoles and p-Dimethylamino-N-Phenylaniline in the Course of Their Analysis by Electrospray Ionization Mass Spectrometry

The behavior of N-methylidenemalonates of 3-arylaminoindoles and p-dimetylamino-N-phenylanyline (M = ANa) was studied during their analysis with ESI mass spectrometer operated in negative (NI) and positive (PI) ion modes. Anions [A] and both [M + H] and [M + Na] were recorded under conditions of the NI-ESI and PI-ESI, respectively. The fragmentation processes of [A] and [M + H] were found that probably occurred as “insource collusion induced dissociation”. The main paths for [A] proved to be elimination of CO2 and breakage of the N-methylidenemalonate bond. A route [A] − CO2 − ROH (R = Me or Et) was less expressed and occurred for the indolyl-containing compounds with the NH bond only. Experiments employing heavy water demonstrated the isotope exchange to occur involving the hydrogen atom of this bond. This and other facts evidenced that the last fragmentation included abstraction of just this atom. Quantum-chemical calculations allowed picking out a structure for the product ion from the possible ones. The calculations also indicated that the protonation of M occurred at the anionic oxygen atom of the malonate moiety. The fragmentation of [M + H] ions included elimination of two water molecules that was supported by their MS spectra. A common feature of the NIand PI-ESI mass spectra was the presence of oligomeric ions, up to tetramers and trimers for the NIand PI-ESI ones, respectively. The oligomers were formed by interaction of the corresponding ions with neutral molecules. When ions contained extra hydrogen atoms, they were introduced by hydrolysis. *Died on 22 June 2012. How to cite this paper: Nekrasov, Y.S., Ikonnikov, N.S., Borisov, Y.A., Kiselev, S.S., Kornienko, A.G., Velezheva, V.S. and Lyakhovetsky, Y.I. (2017) Reactivity of NMethylidenemalonates of 3-Arylaminoindoles and p-Dimethylamino-N-Phenylaniline in the Course of Their Analysis by Electrospray Ionization Mass Spectrometry. International Journal of Analytical Mass Spectrometry and Chromatography, 5, 116. https://doi.org/10.4236/ijamsc.2017.51001 Received: November 26, 2016 Accepted: February 10, 2017 Published: February 13, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access Y. S. Nekrasov et al.


Introduction
3-Aminoindoles along with their derivatives and heterocycles with the embedded 3-aminoindole scaffold were found in a number of natural products and represented a group of pharmacologically promising compounds that show diverse biological activities, such as antimalarial, antimuscarinic, antibacterial, antiviral, antihypoglicemic, antiplasmodium, and PARP-inhibiting activities [1] [2].Earlier, some of us reported a new reaction for the synthesis of 3-aminoindoles and their aniline analogs tethered to methylidenemalonate fragments.They were prepared from easily available indole-3-carbaldehyde/p-dimetylaminobenzaldehyde aryl nitrones and sodium malonates [3].Mass spectrometry (MS) seems to be a good tool for their analysis and identification.Since they are sodium salts, a MS method with the desorption type ion source should be employed.Such instruments include, for example, one with the ion sources of field desorption (FD) [4], secondary ion mass spectrometry (SIMS) [5], those with desorption chemical ionization (DCI) [6], laser desorption/ionization (LDI) [7], fast atom bombardment (FAB) [8], and some other types [9] [10].For the analysis of low-and non-volatile organic, bioorganic, and organoelement compound, however, the most applied mass spectrometers are now the matrix assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) instruments [11] [12] [13] [14] [15].These ionization methods belong to the group of so-called "mild methods", for which the fragmentation of the precursor ions (molecular radical anions and cations, deprotonated, protonated, and cationated molecules) either does not occur or occurs to a small extent.In those cases when the fragmentation is of interest, the tandem technique (MS 2 ) or generally, MS n is employed.For the analysis of the aforementioned sodium malonates, the ESI method appears to be more appropriate since negative ions are already present in analyte solutions.Moreover, the construction peculiarities of the ESI instruments involve ionization at the atmospheric pressure (with the formation of positive ions in the case of the malonates), while ion filtration and their scanning over the ratio of mass to charge (m/z) occur in high vacuum.As a result, the ion sources have the region of a medium pressure where a background gas (often, N 2 ) is present.The electrodes designed for focusing ions toward the skimmer, for example, tube lens, are built in this zone.An additional potential between 0 and ± 200 V (for cations and anions, respectively) can be applied to the lens to focus and accelerate ions.Collisions of the accelerated ions with the background gas favor desolvation of the ions, thus increasing the sensitivity of the instrument.However, if the module of the potential is sufficiently high, the internal energy of ions may increase to such a level that the fragmentation of the ions occurs.Thus, in some cases, the fragmentation called "in-source collision-in-duced dissociation (in-source CID or source-CID)" may come into being under conditions of ESI [16].
Fragmentation patterns and the structures of fragment ions formed can help chemists to establish the analyte structure.Moreover, they provide valuable information explaining the behavior of the species studied in chemical or biochemical processes.
In the present work, we have studied the behavior of a number of sodium methylidenemalonates of 3-arylaminoindoles or p-dimethylamino-N-phenylaniline, obtained by the above mentioned reaction, under conditions of ESI in the positive and negative ion modes when in-source CID occurred.As it was told before, the results of the investigation could provide important information on the reactivity and the reaction mechanisms of these compounds in drug synthesis processes.

Materials and Methods
The syntheses of species studied were carried out by the method described in reference [3].
The ESI mass spectra were taken on a Finnigan LCQ Advantage mass spectrometer with ion trap mass analyzer (USA) and equipped with a Surveyor MS pump, and a Schmidlin-Lab nitrogen generator (Germany).Nitrogen served as a sheath gas, while no auxiliary gas was used.A flow of acetonitrile (purchased from the Merk company) of 25 µL•min −1 was maintained.The temperature of the ion transfer capillary was 150˚C; the electric voltages between the needle and the counter electrode were ±4.5 kV and ±55 V for tube lens (for the positive and negative ion polarity modes, respectively).The samples with the concentration of 10 −4 mol•L −1 in acetonitrile:water 9:1 were introduced into the ion source through the Reodyne injector with a 5 µL loop.The data acquisition and treatment were fulfilled using the program X Calibur, version 1.3.The removal of the contributions of the 13 C-and other natural heavy-isotope-containing ions from the peak intensities of the deuterium labeled compounds was performed iteratively using the program SCIPE [17].
The quantum chemical calculations were made in the framework of the density functional theory (DFT) [18].The Becke-Lee-Yang-Parr hybrid method (B3LYP/6-31G*) was employed [19] [20].All calculations with total optimization of the geometry of molecules and computation of normal vibrational frequencies were carried out with the GAUSSIAN 09 program operated under LINUX [21].

Results and Discussion
The N-methylidenmalonates of tertiary 3-arylaminoindoles and p-dimethylamino-N-phenylaniline the ESI mass spectra of which have been studied are presented in Figure 1 (compounds 1 -5).
All Figure 1

ESI Mass Spectra in the Negative Ion Polarity Mode
The NI mass spectra of all compounds examined showed peaks of anions   To solve this problem and for the elucidation of other reactions peculiarities, experiments with compounds 1 and 4 were performed in the presence of heavy water.Four modes were employed; in the first variant, D 2 O was introduced in the ion source as a solvent for the analytes (method A).Protocols C and D involved additional introduction in the ion source of heavy water and water via a syringe pump, respectively.By application of method B, the analytes were introduced in the source in water solutions, while heavy water was admitted through the syringe pump.In the case of compound 1, ion [A] − and its fragment ions save one contained deuterium-labeled components thus indicating hydrogen-deuterium exchange to have occurred (Table 2).The comparison of the data for method A with those for method B demonstrates that the H,D-exchange proceeded inside the ion source to a noticeable extent.With that, however, the percentage of deuterium containing ions was greater in each deuterated entry of this ionic group when method A was employed.It can be explained by the fact that under the protocol A conditions, the exchange occurred in pure D 2 O, while a mixture of D 2 O and H 2 O was present in the ion source when method B was employed.This finds support in the points that the percentages of deuterated components in the ions increased or decreased when methods C or D were used, respectively.It reveals especially clear by the example of ions [A] − with m/z 335 and 336 (Table 2).However, the results obtained can't exclude the possibility that the exchange could partly proceed in the solution of the analyte in D 2 O before its introduction into the ion source.
Since ion [A − CO 2 − MeOH] − in the case of compound 1 and all ions listed in Table 2 for compound 4 did not contain deuterium atoms, these experiments strictly supported two points: 1) the hydrogen-deuterium exchange in compound 1 occurred with the hydrogen atom located at the nitrogen atom of the indole ring; 2) the fragmentation of [A] − to give the above mentioned first ion involved this ion exactly.The latter is apparently valid for ion [A 2 ] − of compound 2.
In the cases of compounds 1, 2, and 4 the dissociation patterns of [A] − , [A 2 ] − , and [A 4 ] − ions, respectively, occurred due to in-source CID turned out to be virtually the same as in MS 2 experiments differing in the intensities of the corresponding peaks only (cf. Figure 2   [ ] [ ]

[ ] [ ]
Trimeric and tetrameric ions appear to be formed by the analogous schemes involving addition the corresponding dimeric or trimeric ions to neutral molecules and hydrolysis processes.

ESI Mass Spectra in the Positive Ion Polarity Mode
Peaks of the protonated and cationated molecules [M + H] + and [M + Na] + of compounds 1 -5 (M = ANa) were present in their PI-ESI mass spectra.Besides them, the mass spectra showed peaks of fragment ions of the former ions, ions [AH + H] + (with significant peak abundances of such ion and its fragments for 1 only), and also ions of dimers and trimers (Table 3, Figure 3(a)).
With that, the peak abundances of protonated ions [M + H] + significantly exceeded those of cationated ions [M + Na] + , while the relative abundances of ion peaks of dimers at m/z 695, 751, 723, 751 and 703 for compounds 1 -5, respectively, were great, being even 100% for 1 -3, and 5.This suggests [MAH + H] + structure of these ions and the reaction 4 for their formation.

2
M M H M H M H H O MAH H Two other theoretically possible schemes involving the participations of [AH + H] + or [M + Na] + ions, and AH neutral molecules seem less likely for compounds 2 -5.Actually, the peak abundances of [AH + H] + were extremely small in their spectra (see Table 3).This means that the amounts of the neutral AH molecules were also small.Further, as we underlined above, the peak abundances 1 These AH compounds could also be formed directly in solution via hydrolysis of salts M occurred directly in solution before introduction of the samples into the ion source.1.
of [M + Na] + ions were small as compared with those of [M + H] + ones that speaks in favor of the fact that the reaction mostly occurred via the latter ions.
As relates to compound 1, the peak of [AH + H] + possessed a pronounced abundance in the mass spectrum.Thus, the reactions 5 could contribute to the process of the [MAH + H] + cation formation.

M H O AH NaOH
The participation of [M + Na] + is unlikely in this case by the same reason as above.
The origin of fragment ions of [M + H] + with m/z 319, 347, 333, 347, and 323 in the spectra of 1 -5, respectively, is worth discussion.The ions can be described as [M + H -NaOH] + , and this fragmentation seems quite reasonable.However, the MS 2 spectra of the precursor ions showed these products ions either to be present with negligible abundances, or to be absent.An alternative fragmentation process is [AH + H] + − H 2 O (see reaction 5 for a possible way of formation of this precursor ion; it could also be generated via hydrolysis of the [M + H] + ion.)The above MS 2 argument that would indirectly speak in favor of the latter process has a hint of doubt for compounds 2 -4, since the abundances of the precursor-product pair were rather small in these cases, while ions [M + H] + were abundant.Moreover, ion [A 5 H + H] + was virtually absent on the spectrum of 5, whereas a small abundance ion with m/z 323 was present and again and [M + H − CO 2 ] + were absent in the spectra, the MS 2 spectrum of [M 4 + H] + ion of 4 showed the former ion rather than the latter.Also, analogous ion [M 5 + H − MeOH] + and not [M 5 + H − CO 2 ] + was present in the MS 2 spectrum of [M 5 + H] + in the case of compound 5. Based on all this, we believe that the 297 Da ions discussed were formed due to the sequential elimination of the alcohol and carbon dioxide from the protonated molecules of 2 and 4. We also believe that this path took place in the cases of compounds 1, 3, and 5 producing ions 283, 297, and 287 Da, respectively.
An alternative fragmentation that could also have provided the above m/z ions in the mass spectra of these compounds seemed to be [M + H] + − H 2 O − H 2 O − NaOH.However, the mass spectra of 2 and 4, and the MS 2 spectrum of ion [M 4 + H] + of 4 lacked the corresponding product ions (311 Da).It allowed us to exclude such fragmentation from the consideration.
When 1 was introduced in D 2 O solution, the treatment of the mass spectrum as mentioned in section 2 showed ions of analyte 1 formed owing to addition of a proton or deuteron to contain between zero and two deuterium atoms [359 Da (12%), 360 Da (43%) and 361 Da (45%)].This means that the deuterium atoms were introduced into the analyte molecule by both H,D-exchange and the addition of a deuteron.At the same time, the corresponding ions of compound 4 obtained and registered under the same conditions contained maximum 1 deuterium atom [387 Da (25%) and 388 Da (75%)].Contrary to 1, this compound has no hydrogen atom at the nitrogen one of the indole ring.Thus, all of this is consisted with the conclusion drawn before when negative ions were examined that the H,D-exchange in compound 1 and its ions involved just this hydrogen atom.It is essential to note, that protonation (or deuteron addition) could hardly proceed in the solutions of analytes before introduction in the ion source, since the analytes are salts of weak acids and a strong base and thus provide basic mediums.Hence, the addition of proton or deuteron most likely occurred immediately in the ion source.

Quantum-Chemical Calculations
As we mentioned above, the abundant peaks of fragment ions [A − CO 2 ] − and [ArN(C 6 H 4 R 2 )] − are present in the NI-ESI mass spectra of compounds 1 -4 (Table 1).At the same time, peaks of ions [A − CO 2 − R 3 OH] − , though of low intensities, were detected reliably for compounds 1 and 2. Their formation from ions [A] − ([A] − and [A 2 ] − , respectively) was supported by the corresponding MS 2 spectra.Quantum-chemical calculation performed in the case of compound 1 provided three possible structures of [A − CO 2 − MeOH] − (Figure 4).However, the ion with structure 1 turned out to be the most stable and even more stable than that with a cumulene fragment (such cumulene motif was reported, e.g., as propadienone [22]).The formation of this ion seems to be kinetically rather than thermodynamically controlled.However, during the period when this ion was present in the ion source, isomerization could occur for it to acquire the most stable structure 1.Moreover, according to the extended Hammond-Leffler postulate, [23] the more the reaction is endothermic the more the transition state for generating the [A − CO 2 − MeOH] − ion should be shifted to the reaction products When a compound lacked the hydrogen atom at the indole nitrogen one and the collision energy was high enough, an ion with the structure like 3 (e.g., with N-Me bond instead N-H one) might be formed as a result of the abstraction of the hydrogen located at the α-carbon atom of the indole ring.It seems to be the case of compound 3, for which fragmentation [A 3 ] − − CO 2 − MeOH was observed under MS 2 conditions.As indicated above, molecules of 1 -5 added a proton or sodium cation under conditions of PI-ESI (reactions 6 an 7, respectively).The abundances of ions [M + H] + were always higher than those of the [M + Na] + ions (see Table 3).As above, in the framework of the extended Hammond-Leffler postulate, this is consistent with the calculated values of proton affinity (PA) and the affinity to cation Na + for compound 1, the former being 3.5 times greater than the later.
[ ] M H M H H 1102.5 kJ mol [ ] M Na M Na H 312.7 kJ mol

M Na
H O M H NaOH H 180.5 kJ mol It is worthy of noting that the calculated PA of molecule 1 is very high prevailing that for ammonia (852.6 kJ•mol −1 ) [24].
There is little likelihood that the reaction of hydrolysis of [M + Na] + ion (reac- comprise the interaction of the molecules with their cationated forms and the hydrolysis of the dimers obtained (reaction 9).

[ ] [
] ( ) M M Na M Na Н 187.4 kJ mol for M Na H O M H NaOH Н 165.9 kJ mol for However, the abundances of [M + Na] + ions in the spectra were considerably less than those of [M + H] + ions, the ΔН of the interaction between M and [M + H] + was less than that of M and [M + Na] + exemplified by the case of 1. Besides, the formation of [M 2 + H] + ions involving the latter interaction would be a two-step process, the second step being highly endothermic.All this supports that the [M 2 + H] + ions were formed predominantly according reaction 4.

Conclusions
Thus, both anions [A] − and cations [M + H] + of compounds 1 -5 (M = ANa) were detected under NI-ESI and PI-ESI conditions, respectively.These ions underwent fragmentation processes that occurred probably as in-source CID.
In the case of NI-ESI, the main fragmentation paths proved to be the loss of In the case of PI-ESI, ions [M + Na] + were recorded along with the [M + H] + ones, peaks of the former being considerably less abundant than those of the latter.Ion [AH + H] + was also present, but pronounced only in the case of 1.
Based on the MS 2 spectra of [M + H] + of 2 and 4, we believe that this ion successively lost alcohol and CO 2 molecules, contrary to the analogous fragmentation of A − that seems to occur in the opposite consequence.
A fragmentation of ions [M + H] + with elimination of two water molecules appeared to be rather surprising, but was supported by their MS 2 spectra for compounds 1, 3, and 5.
By the example of compound 1, quantum-chemical calculation indicated that protonation of molecule M occurred at the oxygen atom adjacent to the sodium one in Figure 1.
As in the case of NI-ESI mass spectra, peaks of oligomeric cations were rather greatly abundant in the PI-ESI spectra.Again, they were formed because of the interaction of the monomeric cations [M +H] + and their oligomeric cations with neutral molecules M. Those ions, where extra hydrogen atoms were present, acquired these atoms owing to hydrolysis processes.As an example, quantumchemical calculation made for 1 along with point that the abundance of ion [M + Na] + was rather small proved to be consistent with the proposal that ions [M 2 + H] + were formed via interaction of [M + H] + with M rather than the alternative route involving the reaction of [M + Na] + with M followed by the hydrolysis of the dimer obtained.
Completing the paragraph, it is worth to note that the depicted results can be of interest to solution chemists who work with compounds 1 -5 and similar species, since fragmentation and oligomerization processes observed in the ion source in this study and processes occurring in flasks can be akin.Moreover, the possibility of formation of oligomeric ions during ESI-MS analysis of similar compounds should be taken into consideration in order not to make identification mistakes.
compounds formed both negative and positive ions (NI and PI, respectively) under condition of the NI-ESI and PI-ESI, respectively.

Figure 1 .
Figure 1.The species subjected to this study.
(a) and Figure 2(b)).At the same time, besides for the formation of ions [A 2 H] − could be anticipated.The first path involved hydrolysis of ions [A 2 Na] − (reaction 2).The second implied the hydrolysis of neutral ANa or protonation in the ion source of [A] − by H 2 O and even CH 3 CN (a solvent in many experiments).AH formed then added [A] − (reaction 3) 1 .

Figure 4 .
Figure 4. Three possible structures of the [A − CO 2 − MeOH] − ion of compound 1 and their parameters obtained from the quantum-chemical calculations.

tion 8 )Figure 5 .
Figure 5.The protonated molecules of 1 with two lowest ∆Н of protonation (the protons added are marked by the asterisks).

CO 2
and the elimination of methylidenmalonate moiety from [A] − (the former with the exception of compound 5).The less expressed process found for compounds 1 and 2 was the successive elimination of CO 2 and an alcohol molecule (MeOH or EtOH).The absence of the corresponding product ions in the case of compound 4 lacking a hydrogen atom at the nitrogen atom of the indolyl moiety and for compound 5 containing no indolyl group at all suggested that just this atom was abstracted during this fragmentation.Experiments with 1 and 4 with the use of heavy water demonstrated the isotope exchange to occur, at least partly, in the ion source that involved the above hydrogen atom of 1. Also, these experiments supported the fact of the abstraction of this atom during the formation of [A − CO 2 − MeOH] − .Quantum-chemical calculations showed that the ion formed had to be of a cyclic structure rather than a linear one.Apart from ions [A] − and their fragment ions, protonated and cationated oligomeric ions [A 2 H] − , [A 2 Na] − , [A 3 Н n Na m ] − (n + m = 2) and [A 4 Н n Na m ] − (n + m = 3) were detected.The schemes of their formation include interaction of ions with neutral molecules, e.g., of [A] − with ANa, and hydrolysis to get species containing extra hydrogen atoms.

Table 1 .
M/z values and peak abundances with respect to the maximal peaks (I rel , %) in NI-ESI mass spectra of compounds 1 -5 a .

Table 2 .
Number of the deuterium atoms (n d ) in the ionic structures and the relative abundances of the corresponding ions (%) in these structures in the NI-ESI mass spectra from the experiments with the use of heavy water a .coincidedions (305 and 221 Da), other fragment ions of [A 3 ] − were present in its MS 2 spectrum (331, 323, 317, 291, and 273 Da).This difference again seems to be caused by the fact that the collision energies were different in these CID modes.Ions [A 2 Na] − were formed most likely due to interaction of ions [A] − with neutral molecules of analytes (reaction 1).
aThe contributions from ions containing13C and the other natural heavy isotopes were removed as described afore (see Section 2). the

Table 3 .
Values m/z and relative peak abundances of PI (I rel , %) with respect to the maximal peaks in the PI-ESI mass spectra of compounds 1 -5 a .
a See footnote "a" to Table