CHAPTER: 4. The Mass Spectrometric Investigation of Proton Induced Cyclisations of 2-nitro-N-phenyl anilines In Gas-Phase

Transcription

1 CAPTER: 4 The Mass Spectrometric Investigation of Proton Induced Cyclisations of 2-nitro--phenyl anilines In Gas-Phase

2 4.1. Scope In presence of concentrated sulfuric acid, 4 -chloro-2-nitro--phenyl aniline is reported to cyclise in condensed phase [1] to yield 2-chloro phenazine at C and 2-chloro phenazine-10-oxide at low temperatures Scheme This reaction is a method for the synthesis of substituted phenazine--oxide, which on subsequent reduction yielded phenazines. leum 2 S C C 2-chloro phenazine-10-oxide 2-chloro phenazine Scheme This type of acid-catalyzed reactions can be well investigated in the gas-phase using mass spectrometric experiments but the mechanisms may be different due to the absence of a solvent. Such mechanisms may be established using molecular orbital calculations. Ionization methods such as fast atom bombardment, chemical and electrospray ionizations can generate protonated molecules, [M + ] + ions, as in acid- catalyzed reactions, and the course of their rearrangements can be investigated by tandem mass spectrometric experiments. It is reported [2, 3] that the EI mass spectrum of 2-nitro--phenyl aniline exhibit peaks corresponding to eliminations of radical and 2 due to proximity effects. The EI mass spectra of 1,2-disubstituted aromatic compounds also exhibit peaks corresponding to unusual fragment ions due to proximity effects [3-6]. Many of these fragment ions have heterocyclic structures due to intramolecular cyclisations taking place within the molecular 143

3 ions in the gas phase [7, 8]. Investigations on such cyclisations have often led to the discovery of new synthetic routes [8, 9] for heterocyclic compounds. owever, proximity effects leading to cyclisation, by fast atom bombardment (FAB), electrospray ionization (ESI) or Chemical ionisation (CI) that produce [M+] + ions of aromatic compounds containing ortho substituents are not common. This is probably due to the lack of rearrangements in even electron molecular ions (closed-shell) generated by FAB, ESI or CI in comparison with the odd-electron ions produced by electron impact. Proton-induced cyclisations in mass spectrometry of even-electron ions have been reported in the case of 1,2-diacetoxy biphenyl [10] and recently for ortho nitro aromatic compounds [11]. Protonated 1,2-diacetoxy biphenyl eliminates a molecule of acetic acid leading to a cyclic product ion. A similar elimination of acetic acid from the [M+] + ion of acetylated hyperacine, a marine natural product, has lead to the establishment of its structure [12]. We have recently reported a novel mass spectrometry-induced cyclisation of protonated - [(2-benzoyloxy) phenyl)]- benzamide to yield protonated 2-phenyl benzoxazole by a process that is analogous to the acid catalyzed cyclisation [13]. Moreover, recent reports by Eberlin and coworkers [14] and by Cooks and coworkers [15] demonstrate that gas-phase synthetic chemistry is of current interest. The literature survey revealed that there are no reports on cyclisation of 2-nitro--phenyl aniline or their substituted derivatives on protonation under FAB, CI or ESI ionisations. In the previous chapter, we have established that protonated 2-nitrophenyl phenyl ether cyclises to a heterocyclic intermediate, which dissociates by competitive elimination of radical and 2 molecule. So the investigation of the mass spectral reactions of protonated 2-nitro--phenyl aniline (a compound in which ether oxygen is replaced by group) and their substituted derivatives was reasoned because this study would provide a useful mechanistic benchmark for 144

4 acid-catalyzed cyclisations in the gas phase. Moreover, we have identified this system as one that may be useful in extending our knowledge of gas-phase electrophilic cyclisation of nitro aromatic compounds upon protonation. ence the investigation of possible cyclisation reactions of 2-nitro--phenyl aniline and their substituted derivatives upon protonation by FAB, CI and ESI ionisations were under taken. Tandem mass spectrometric experiments and ab initio molecular orbital calculations were used to propose the structures for fragment ions and mechanisms for gas-phase synthetic reactions. 145

5 4.2. Results and Discussion The 2-nitro--phenyl aniline and their substituted derivatives 2 to 4, Scheme were synthesized as per reported procedures. Compounds 1,5 and phenazine used for this study were purchased from Aldrich Chemical Co. and used without further purification. Their mass spectra were recorded to investigate the possible rearrangements leading to cyclisation reactions of the even-electron molecular ions generated by FAB, Electrospray and Chemical ionizations. R R 1 = ; R 2 = 2. R 1 = ; R 2 = C 3 3. R 1 = ; R 2 = 4. R 1 = ; R 2 = C 3 R 2 2 Scheme The FAB mass spectrum of 2-nitro--phenyl aniline (1) shows, Fig.4.2.1, peaks corresponding to ions of m/z 215, 214, 198, 197, 181, 180 and Relative Abundance + Fig The FAB mass spectrum of 2-nitro--phenyl aniline (The ion of m/z 154 is due to the matrix). m/z 146

6 The ion of m/z 215 corresponds to the protonated molecule, [M+] +, while the ion of m/z 214 is due to the radical cation of compound 1. The ion of m/z 167 can be attributed to the loss of elements of 2 2. The MI mass spectrum of the protonated molecule of m/z 215, Fig.4.2.2, shows fragment ions of m/z 198, 197, 181, 180, 169 and 167 indicating that these ions are produced by the fragmentation process of the molecular ion of m/z 215. The ion of m/z 169 is assigned to the loss of C from the ion of m/z 197. n collision activation, the abundance of ions of m/z 181 and 180 increases significantly at the expense of that of the ions of m/z 198 and 197, Fig This indicates that ions of m/z 181 and 180 are probably, generated by stepwise processes from the molecular ion whereas the ions of m/z 198 and 197 are formed from the molecular ion directly, Scheme Relative Abundance Fig.4.2.2, FAB-MI, mass spectrum of [M+] + ion of m/z 215. m/z The formation of ions of m/z 198 and 197 involves the eliminations of radical and 2 molecule respectively from the molecular ion [M+] +. The subsequent elimination of an 147

7 radical from the ion of m/z 197 produces ion of m/z 180 while the expulsion of C afford the ion of m/z 169 as indicated by its FAB-CAD mass spectra, Fig.4.2.4a. Relative Abundance m/z Fig FAB-CAD mass spectrum of [M+] +, m/z 215 Relative Abundance Fig.4.2.4a, FAB-CAD mass spectrum of the ion of m/z 197 z 148

8 The ions of m/z 180 and 181 are also generated by the elimination of 2 and radicals respectively, from the fragment ion of m/z 198 as indicated by its CI- CAD mass spectra, Fig Relative Abundance Fig.4.2.5, CI- CAD mass spectrum of the ion of m/z 198 The different pathways for the generation of the fragment ion of m/z 180 from the [M+] + ion are illustrated in Scheme [M+] + m/z m/z m/z m/z 169 -C m/z m/z 180 Scheme 4.2.2, Schematic representation of the fragmentation pathways for the [M+] + ion from compound

9 In addition, the major fragmentation pathway for the ion of m/z 181 is the elimination of hydrogen radical to afford the fragment ion of m/z 180 as indicated by its FAB-CAD mass spectrum, Fig Relative Abundance Fig.4.2.6, FAB- CAD mass spectrum of the ion of m/z 181 wing to the fact that the ion of m/z 214, M +., is more abundant than the ion of m/z 215, [M+] +, in the FAB mass spectrum, Fig.4.2.1, of Compound 1, there may be some contribution of the 13 C isotopomer of M +. in the MS/MS spectrum, Fig.4.2.3, of the [M+] + ion. (The abundance of m/z 215 due to [ 13 CC ] +. is 12.1 %). Better protonation efficiency could be achieved by performing the electrospray ionization of compound 1, Fig.4.2.7a. The ESI experiment was conducted by using a quadrupole time-offlight instrument (ESI-Q-Tof). The ESI-CAD mass spectrum of the [M+] + ion, Fig.4.2.7b, shows fragment ions of m/z 198, 197, and 169 similar to the mass spectral behaviour under FAB ionization. 150

10 (a) (b) % m/z 215 % m/z Fig The ESI (a) mass spectrum and (b) CAD Mass spectrum of 2-nitro--phenyl aniline The ESI-CAD mass spectrum was recorded at a resolution of and hence the exact masses of the fragment ions could be determined using the ion of m/z 215 as the internal standard. The high-resolution mass spectral data thus obtained are in good agreement with the calculated masses for the elemental compositions of the proposed fragment ions, Table To explore the structure of the [M+- 2 ] + ion, the CAD mass spectrum of the collisionally produced ion of m/z 197 was studied by performing MS 3 experiment on the ESI generated [M+] + ion of Compound

11 Table 4.2.1, Measured accurate masses of the fragment ions from the ESI-CAD Mass spectrum of the [M+] +, ion of 2-nitro--phenyl aniline. Fragment ion ominal mass Molecular Formula bserved mass Calculated mass [M+-] C [M+- 2 ] C [M+-2] C [M+- 2 -] C [(M+- 2 )-C] C The CAD mass spectrum of collisionally generated ion of m/z 197, Fig b shows intense peaks corresponding to the ions of m/z 180 and 169 indicating that [M+- 2 ] + ion dissociate via the expulsions of and C respectively. Therefore the ion of m/z 197, having only one oxygen atom (C ), must be a mixture of two isomeric structures. ne of the structures eliminates a molecule of while the other looses C Relative Abundance m/z Fig b, CAD MS of the collisionally produced ion of m/z 197 by MS 3 Experiments on the ESI generated [M+] + ion of compound 1 152

12 Based on the results of the above experiments, various fragmentation pathways, for the [M+] + ion are proposed, Scheme The proposed cyclisation of the [M+] + ion to form the intermediate M1a is analogous to that of protonated 2-nitrophenyl phenyl ether. + [Μ+Η] + ; m/z M1a M1; m/z M4; m/z 197 -C m/z 169 [(Μ+Η Η 2 Ο)] + +. m/z 198 [Μ+Η ΟΗ] M2; m/z181 [Μ+Η 2ΟΗ] M3; m/z180 [(Μ+Η Η 2 Ο) ΟΗ] + +. Scheme Competitive eliminations of radical and 2, from the [M+] + ion yield the fragment ions of m/z 198 and 197 respectively. Further expulsion of 2 from the ion of m/z 198 as well as the elimination of radical from the ion of m/z 197 afford the cyclic fragment ion of m/z 180 as shown in Scheme The ion of m/z 180 can as well be generated by the loss of hydrogen radical from the ion of m/z 181. It is envisaged that the fragment ion of m/z 197 is an isomeric mixture of ions of structure M1 and M4 while ions of m/z 181 and 180 possess structures of protonated phenazine M2 and phenazine radical cation M3 respectively. 153

13 A comparison of the CAD mass spectrum of the radical cation of phenazine obtained by EI with that of the ion of m/z 180 obtained as a fragment ion from compound 1 by FAB ionization, show that the two spectra are closely similar, Fig a and b. m/z Fig (a) The FAB-CAD mass spectrum of ion of m/z 180 from Compound 1, +.. m/z Fig (b) The CAD mass spectrum of ion of m/z 180 from Phenazine radical cation This indicates that the ion of m/z 180 has indeed posses the phenazine radical cation structure and hence supports the proposed cyclisation of compound 1 upon protonation, Scheme 4.2.3, followed by competitive eliminations of radicals and

14 To have more insight into the mechanism of the gas-phase cyclisation, the chemical ionization mass spectrum of 2-nitro--phenyl aniline (1) was recorded Fig by using methane as the reagent gas. +. Fig.4.2.9, The CI mass spectrum of 2-nitro--phenyl aniline The CI mass spectrum of Compound 1, Fig.4.2.9, also exhibited peaks corresponding to the ions of m/z 215, 214, 198, 197, 181, 180 and 169 very similar to FAB mass spectral behaviour. To get more evidence for the structure of the ion of m/z 180, the CAD mass spectra of the ion of m/z 180 generated from compound 1 by chemical ionisation, Fig was also compared with that of the radical cation of phenazine obtained by EI, Fig (b). It is observed that the two spectra are closely similar, indicating that the ion of m/z 180 from compound 1 has indeed posses the structure of phenazine radical cation M3 and hence supports the proposed cyclisation accompanied by competitive eliminations of radical and 2 to yield heterocyclic fragment ion. 155

15 +. m/z Fig (b) The CAD mass spectrum of the ion of m/z 180 from Phenazine radical cation m/z Fig , The CAD mass spectrum of the ion of m/z 180 from Compound 1 by CI To establish the structure M2 proposed for the ion of m/z 181, the CI-CAD mass spectrum of the fragment ion of m/z 181 from Compound 1, Fig (b), and that of the [M+] + ions of phenazine, Fig (a), (appropriate reference) obtained by protonation in methane CI was compared. The two spectra are closely similar indicating that the fragment ion of m/z 181 from compound 1 has the same structure as protonated phenazine as proposed in Scheme This observation further supports the proposed cyclisation. 156

16 (a) + (b) Fig (a) CI-CAD mass spectrum of the ion of m/z 181 from [phenazine + ] + (b) CI- CAD mass spectrum of the ion of m/z 181 from [M+] + ion of compound 1 Moreover, the CI-CAD mass spectra, Fig (a) and (b) are similar to the FAB- CAD mass spectrum Fig of the fragment ion of m/z 181 from Compound 1. owever, Fig (a) and (b) are plotted in such a manner that ion of m/z 77 appears to be the base peak while Fig is plotted in different mass range with ion of m/z 180 as the base peak and hence appears slightly different. In all the three mass spectra, the most abundant fragment ions, m/z 180 and 179, are due to the loss of radical and

17 For establishing the mechanism of the proposed cyclisation and consequent fragmentations yielding heterocyclic ions, molecular orbital calculations were performed by using Density Functional Theory, (DFT). This is part of Gaussian 98 package. In the calculations the heats of formations of the intermediates, transition states and products are stated relative to that for the [M + ] + ion. The protonated molecule [M+] + rearranges to a cyclic structure M1a through a C- bond formation via an electrophilic substitution reaction in which the nitro group upon protonation participates as an electrophile. M1a then isomerises to M1b by a reversible 1,3- migration. The isomerisation of M1b to M1d involves reversible -shifts as shown in Scheme The intermediate, M1d either eliminates a molecule of 2 to form the ion of m/z 197, M1 {[M+- 2 ] + } or rearranges to M1f via M1e by two consecutive migrations. The successive eliminations of two radicals take place from structure M1f to form the ion of m/z 198 {[M+-. ] +. } first and then the ion of m/z 181, M2, {[M+-2. ] + }. The highest energy transition state TS3, for the eliminations of 2 and. is 39.1 Kcal/mol above the energy of the molecular ion [M+] +. Towards the last part of the mechanism, M2 loses a hydrogen radical producing the ion of m/z 180, with a phenazine radical cation structure, M3. It can be seen from the mechanism that M1a transforms to another intermediate M1g, in which there are two groups on the nitrogen atom, through a reversible 1,3-hydrogen shift from carbon to oxygen. M1g can eliminate a molecule of 2 to form the ion of m/z 197, but this pathway is thermodynamically less favorable. It may be assumed that the [M+- 2 ] + ion has a structure M1 which can aromatize to protonated 1-hydroxy phenazine, Q, Scheme

18 + [Μ+Η] + ; m/z 215 : ΔΗf = 0.0Kcal/mol TS1: TS2: + ΔΗf = 30.2 Kcal/mol ΔΗf =37.4 Kcal/mol + - M1a: ΔΗf = 24.5 Kcal/mol + M1b: ΔΗf = -5.1 Kcal/mol TS3: ΔΗf = 39.1Kcal/mol + M1g: ΔΗf = 2.2 Kcal/mol TS8; 42.3 Kcal/mol + M1d; ΔΗf = Kcal/mol ΔΗf = -1.3 Kcal/mol TS5: - 2 TS4: ΔΗf =4.0 Kcal/mol TS6 : ΔΗf = 5.6 Kcal/mol + Μ1c; ΔΗf=-22.6 Kcal/mol + + m/z 197 Μ1:[Μ+Η Η 2 Ο] + ΔΗf = Kcal/mol M1e: ΔΗf = Kcal/mol TS7: ΔΗf = -0.4 Kcal/mol +. o TS. - + m/z 180 Μ3; [Μ+Η 35] m/z 198 [Μ+Η ΟΗ] +. : o TS ΔΗf = 9.9 Kcal/mol M1f: ΔΗf = Kcal/mol m/z 181 Μ2; [Μ+Η 2ΟΗ] +. ΔΗf = 30.1 Kcal/mol Scheme 4.2.4; Proposed mechanism for formation of ions in ESI and FAB ionisation. Moreover, the presence of the ion of m/z 169 in the FAB-CAD mass spectrum, Fig.4.2.4a of the [M+- 2 ] + ion of m/z 197 from compound 1, corresponding to the loss of 159

19 C indicate that the [M+- 2 ] + can dissociate via expulsion of C. The measured accurate mass for the ion of m/z 169, obtained from ESI-CAD mass spectrum, Fig (b) of the [M+] + ion of compound 1, corresponds to the elemental composition of C , Table 4.2.1, (measured mass , calculated mass ) which indicates that the ion of m/z 169 is [(M+- 2 )-C] + and not [M+- 2 ] +. The feasibility of elimination of C from M1 was explored and confirmed by molecular orbital calculations and the results are presented in Scheme 4.2.5, justifying structure M1, for the ion of m/z 197. TS ΔΗf = Kcal/mol + Μ1: [Μ+Η Η 2 Ο] + m/z 197 ΔΗf = Kcal/mol + TS + ΔΗf = Kcal/mol + ΔΗf = Kcal/mol ΔΗf = Kcal/mol TS ΔΗf = Kcal/mol - C + Q P: ΔΗf = Kcal/mol Scheme 4.2.5, Proposed mechanism for formation extrusion of C from M1 ([M+- 2 ] + ). The possible isomeric structures of [M+- 2 ] + that are likely to fragment via losses of and C are -protonated phenazine-5-oxide (M4), Scheme and -protonated 1- hydroxyphenazine, Q, Scheme respectively. It is reported that the EI mass spectrum of 1-hydroxyphenazine [18] contains abundant (60%) fragment ion [M-C] +. along with a series of less abundant (<15%) fragment ions. The ESI-CAD mass spectrum [19] of 5- methyl-1-hydroxyphenazine shows abundant fragment ions due to the losses of C 3 and C. 160

20 + M1f: ΔΗf = Kcal/mol M4; m/z 197 M3; m/z 180 Scheme 4.2.6: Proposed mechanism for the extrusion of from [M+- 2 ] + To explore more about the mechanism of gas-phase cyclisation, the CI mass spectral behaviour of the molecular ion [M+D] + of compound 1 was studied. + D Fig. 4.7 CI-MI mass spectrum of the [M+D] + of compound 1, m/z Fig , The MI mass spectrum [M+D] + of compound 1 Fig , The CI- MI mass spectrum of [M+D] + ion of 2-nitro--phenyl aniline The MI mass spectrum [M+D] + of compound 1, Fig , shows peaks corresponding to the ions of 199, 198, 197, 182, 181 and 180. The peaks of high relative abundance correspond to the ions of m/z 198 and 181, one amu higher than the respective highly abundant ions obtained in ESI and FAB ionizations. This establishes that the 161

21 deuterium remains with fragment ions of m/z 197 and 180 predominantly. The formation of ions of m/z 180, 181 and 182 is attributed to the proton deuterium exchange prior to the eliminations of (D+), [( 2 +) or (+D)] and (+) respectively. Both structures M1c and M1d in the proposed mechanism, Scheme 4.2.4, for cyclisation contain three active hydrogen atoms each and these hydrogen atoms may be responsible for the /D exchange. The relative abundance of the peaks corresponding to ions of m/z 197, 198 and 199 show that there is /D exchange and consequent eliminations of D, D/ 2 and respectively, supporting the proposed mechanism. More evidence for the ortho stereo requirement for the gas-phase cyclisation is provided by the observation that 4-nitro- -phenyl aniline, the para isomer, compound 5, does not give fragment ions of m/z 180, 181 and 197 in the MI, Fig and CAD, Fig mass spectra, of the [M+] + ion in FAB ionization method. + Fig , The FAB- MI mass spectrum of [M+] + ion of 4-nitro--phenyl aniline. 162

22 + Fig , The CAD mass spectrum of [M+] + ion of 4-nitro-- Phenyl aniline Moreover, the ESI mass spectrum, Fig of 4-nitro--phenyl aniline shows the protonated molecule (m/z 215) and its ESI CAD (high-resolution) mass spectrum, Fig , shows the only fragment being the ion of m/z 198 with an accurate mass of (Calculated mass ). This is due to the ion [M+-] + formed by the expulsion of an radical from the protonated molecule which is the expected mass spectral behaviour. 4-ITR DIPEYL AMIE 100 % m/z Fig , The ESI mass spectrum of 4-nitro- -phenyl aniline 4-ITR DIPEYL AMIE MS/MS 100 % m/z Fig , The ESI -MS/MS R MS of 4-nitro- -phenyl aniline To understand further the characteristics of cyclisation, the FAB and ESI mass spectral fragmentations of methyl and chloro derivatives of 2-nitro--phenyl aniline were 163

23 studied. The FAB-mass spectrum of - (4-methylphenyl)-2-nitroaniline (2) having a methyl label at the 4 -position, Fig , has shown peaks corresponding to the ions of m/z 194, 195 and 211. The ion of m/z 228, M +. is more abundant than the ion of m/z 229, [M+] + in the FAB mass spectrum. C 3 + Fig ; The FAB mass spectrum of - (4-methylphenyl)-2- nitro aniline Better protonation efficiency could be achieved by the electrospray ionization of compound 2. The ESI mass spectrum of compound 2 is shown in Fig (a). The ion of m/z 229 in the mass spectrum corresponds to the [M+] + ion. (a) - (4-methylphenyl)-2- nitro aniline MS 100 % 0 + C m/z (b) - (4-methylphenyl)-2- nitro aniline MS /MS 100 % m/z Fig (a) The ESI and (b) ESI-CAD mass spectrum of the [M+] + ion of compound 2 164

24 The ESI-CAD mass spectrum of the [M+] + ion, Fig (b) shows the fragment ions of m/z 212 and 211 corresponding to the eliminations of radical and 2. The ion of m/z 195 is due to the step-wise loss of two radicals and the ion of m/z 194 is assigned to the step-wise loss of 2 and radical. The fragment ion m/z 183 corresponds to the loss of C from the ion of m/z 211, [M+- 2 ] +. The accurate masses of these fragment ions obtained from the ESI-CAD mass spectrum, Table 4.2.2, are in good agreement with the proposed eliminations, Scheme Table Measured accurate masses of the fragment ions from the ESI-CAD mass Fragment ion Spectrum of the [M+] +, ion of 2-nitro-- (4-methylphenyl) aniline. ominal mass Molecular Formula bserved mass Calculated mass [M+-] C [M+- 2 ] C [M+-2] C [M+- 2 -] C The ions of m/z 211,195,194 and 183 observed in the ESI mass spectra of compound 2 are methyl analogues of ions of m/z 197, 181, 180 and 169 observed in the ESI-CAD mass spectrum of compound 1. ence, a mechanism is proposed, Scheme 4.2.7, for the cyclisation of protonated - (4-methylphenyl) 2-nitroaniline leading to the formation of ions of m/z 212, 211, 195 and 194, analogous to that proposed for the cyclisation of compound 1. The ion of m/z 211 is proposed to be a mixture of two ions having the same formula and structures, M2a and M2b while M2c is the structure proposed for the ion of m/z

25 + [Μ+Η] + ; m/z m/z 212 [Μ+Η ΟΗ] + C 3 -. C C 3 + m/z195 [Μ+Η 2ΟΗ] + C M2c; m/z194 M2a; m/z C 3 [Μ+Η Η 2 Ο] + + C 3. M2b; m/z C 3 Scheme Further, the ESI mass spectrum, Fig (a) and ESI-CAD mass spectrum, Fig (b) of - (4-chlorophenyl) 2-nitro- aniline (3) were recorded. The CAD mass spectrum shows peaks corresponding to the ions of m/z 249{[M+] + }, 232, 231, 215, 214 and 203. The ions of m/z 232 and 231 are due to eliminations of radical and 2, the ion of m/z 215 is due to the loss of a second radical, [M+-2] + and the ion of m/z 214 is assigned to the step-wise loss of 2 and radical {[M+- 2 -] + }. The ion of m/z 203 corresponds to [M+- 2 -C] + ion % m/z 203 Fig (a) The ESI and (b) ESI-CAD mass spectra of the [M+] + ion of compound 3 166

26 The high-resolution ESI-CAD mass spectral data, Table 4.2.3, are in good agreement with the proposed eliminations, Scheme Table Measured accurate masses of the fragment ions from the ESI-CAD Mass spectrum of the [M+] +, ion of - (4-chlorophenyl) 2-nitro aniline. Fragment ion ominal mass Molecular Formula bserved mass Calculated mass [M+-] C [M+- 2 ] C [M+-2] C [M+- 2 -] C [Μ+Η] + ; m/z m/z 232 [Μ+Η ΟΗ] m/z 215 [Μ+Η 2ΟΗ] M3a; m/z M3c; m/z 214 [Μ+Η Η 2 Ο] + M3b; m/z Scheme The abundant ions of m/z 231 and 214 observed in the ESI mass spectra of compound 3 are chloro analogues of ions of m/z 197 and 180 observed in the ESI mass spectrum of compound 1. ence a mechanism is proposed for the cyclisation leading to the formation of 167

27 ions of m/z 232, 231, 215 and 214, Scheme The mechanism involves the electrophilic cyclisation of the protonated - (4- chlorophenyl) 2-nitroaniline, analogous to that proposed for compound 1 (Scheme 4.2.4). For further support for the proposed mechanism of cyclisation, the ESI mass spectrum of 4-chloro-- (4-methylphenyl)-2-nitroaniline, (4), Fig (a) and its CAD mass spectrum, Fig (b) were investigated. 100 C % m/z 228 % m/z Fig (a) The ESI and (b) ESI-CAD mass spectra of the [M+] + ion of compound 4 ESI and ESI-CAD mass spectra of compound 4 are shown in Fig a and b. The CAD mass spectrum show peaks corresponding to the ions of m/z 263 {[M+] + }, 246, 245, 229, 228 and 217. The ions of m/z 246 and 245 are respectively due to eliminations of radical and 2 from the [M+] + ion. The ion of m/z 229 is due to the step-wise loss of two radicals, [M+-2] + and the ion of m/z 228 is assigned to the step-wise loss of 2 and radical [M+- 2 -] +. The ion of m/z 217 corresponds to [M+- 2 -C] +. The abundant ions of m/z 245 and 228 observed in the ESI mass spectra of compound 4 are chloro and methyl analogues of ions of m/z 197 and 180 observed in the ESI mass spectra of compound 1. The accurate masses of these fragment ions obtained from the ESI-CAD mass spectrum, Table 4.2.4, are in good agreement with the proposed eliminations. 168

28 Table Measured accurate masses of the fragment ions from the ESI-CAD Mass spectrum of the [M+] +, ion of Compound 4. Fragment ion ominal Molecular bserved Calculated mass Formula mass mass [M+-] C [M+- 2 ] C [M+-2] C [M+- 2 -] C [M+- 2 -C] C A mechanism is proposed for the electrophilic cyclisation of Compound 4, Scheme analogous to that proposed for the cyclisation of Compound 1. + [Μ+Η] + ; m/z 263 C C 3-2 M4a; m/z [Μ+Η Η 2 Ο] + C C 3 M4b; m/z C 3 C 3 C 3 m/z 246 [Μ+Η ΟΗ] + m/z 229 [Μ+Η 2ΟΗ] + m/z 228 M4c; Scheme

29 4.3 Conclusion The FAB and ESI mass spectra of 2-nitro--phenyl aniline and its 4 -methyl, 4 - choro, 4-chloro-4 -methyl analogues were investigated. The mass spectrum of the 4-nitro isomer was also examined for comparison. A novel electrophilic cyclisation of 2-nitro-phenyl aniline and its chloro, methyl and methyl- chloro derivatives take place in a mass spectrometer upon protonation. Protonated nitro group participates as an electrophile in the cyclisation reaction to yield a heterocyclic intermediate. The competitive eliminations of 2 (major process) and an radical occur from the cyclic intermediate. The [M+- 2 ] + ion is a mixture of two structures: (a) protonated phenazine-5-oxide, which loses an radical to afford phenazine radical cation (b) protonated 1-hydroxyphenazine, which dissociates by expelling C. The [M+-] + ion either extrudes 2 to yield phenazine radical cation or expels radical to afford [phenazine+] + ion. The feasibility of the proposed cyclisation and eliminations were substantiated by molecular orbital calculation by using DFT theory. The observed cyclisation is similar to that of protonated 2-nitrophenyl phenyl ether and its substituted analogues. The proposed mechanism and the structures of the product ion were based on tandem mass spectrometric experiments, high-resolution mass measurements, D- labeling, chemical substitution and molecular orbital calculations. The molecular ions of its methyl, chloro and methyl-chloro analogues exhibited similar mass spectral fragmentations but the para nitro isomer did not. This unusual reactions leading to cyclisation of [M+] + ions revealed that nitro group can participate in electrophilic cyclisations in the gas-phase. The cyclisation is analogous to the acid catalysed cyclisation of 4 -chloro-2-nitro--phenyl aniline in the solution-phase. 170

30 4.4 Experimental Details 2-itrodiphenylamine (1), 4-itro diphenylamine (5) and phenazine used for this study were purchased from Aldrich chemical Co. and used without further purification. 1. Preparation of - (4-methylphenyl)-2- nitro aniline (2) [1] 1-chloro-2-nitrobenzene g p -Toluidine g. Formamide - 8 cc A mixture of 3.2 g of 1-chloro-2-nitrobenzene (0.02mol), 3.0g of p -toluidine (0.023mol) and 8 ml of formamide were taken in a 100 ml R.B flask. About 2.0 g of Sodium Carbonate was added. The mixture is heated at about C for two hours and then cooled. The product obtained was washed small quantities of n-hexane repeatedly to remove the unreacted p-toluidine and then washed with water, filtered and dried. The product was purified by column chromatography on silica gel using a 1: 9 mixture of chloroform and hexane as eluent. Melting point was recorded. Yield was 50% Melting point bserved Reported [16] 69 C C IR (KBr) cm - 1: 3336 ( stretch), 3037 (aromatic C stretch), 2923, 2870 (C 3 - asymmetric and symmetric stretch), 1609, 1571 (aromatic C=C stretch), 1445 (C- stretch of amine), 1508, 1350 (asymmetric and symmetric = stretch of Ar- 2 ), 889 (C- stretch of Ar- 2 ). 1 MR (in CD 3 ): δ 8.2 (d, 1 ), (m, 7), 2.5 (s, 3 ), 1.6 (s, 1). 171

31 2. Preparation of - (4-chloro phenyl) -2-nitroaniline (Compound 3) [1] 1-chloro-2-nitrobenzene g 4-chloroaniline g. Formamide - 8 cc A mixture of 3.2 g of 1-chloro-2-nitrobenzene (0.02mol), 3.0g of 4-chloroaniline (0.23 mol) and 8 ml of formamide were taken in a 100 ml R.B flask. About 2.0 g of Sodium Carbonate was added. The mixture is heated at about C for two hours and then cooled. The product obtained was washed small quantities of n-hexane repeatedly to remove the unreacted p-toluidine and then washed with water, filtered and dried. The product was purified by column chromatography on silica gel using a 1: 9 mixture of chloroform and hexane as eluent. Melting point was recorded. Yield was 50% Melting point bserved Reported [1] 59 C 61 C IR (KBr) cm - 1: 3332 ( stretch), 3086 (aromatic C stretch), 2923, 2870 (C 3 - asymmetric and symmetric stretch), 1616, 1592 (aromatic C=C stretch), 1441 (C- stretch of amine), 1508, 1352 (asymmetric and symmetric = stretch of Ar- 2 ), 848 (C- stretch of Ar- 2 ). 776(Ar- stretch) 1 MR (in CD 3 ): δ 8.2 (d, 1 ), (m, 7), 2.5 (s, 3 ), 1.6 (s, 1). 172

32 3. Preparation of 4-chloro-- (4-methylphenyl)-2-nitroaniline. (Compound 4) [1] 1.4-dichloro-2-nitrobenzene g p -Toluidine g. Formamide - 8 cc A mixture of 3.8 g of 1.4-dichloro-2-nitrobenzene (0.02mol), 2.2 g of p -Toluidine (0.2 mol) and 8 ml of formamide were taken in a 100 ml R.B flask. About 2.0 g of Sodium Carbonate was added. The mixture is heated at about C for two hours and then cooled. The product obtained was washed small quantities of n-hexane repeatedly to remove the unreacted p-toluidine and then washed with water, filtered and dried. The product was purified by column chromatography on silica gel using a 1: 9 mixture of chloroform and hexane as eluent. Melting point was recorded. Yield was 50% Melting point bserved Reported [1] 121 C 122 C IR (KBr) cm - 1: 3351 ( stretch), 3091 (aromatic C stretch), 2913, 2870 (C 3 - asymmetric and symmetric stretch), 1616, 1605 (aromatic C=C stretch), 1488 (C- stretch of amine), 1508, 1346 (asymmetric and symmetric = stretch of Ar- 2 ), 846 (C- stretch of Ar- 2 ). 761(Ar- stretch). 1 MR (in CD 3 ): δ 8.5 (s, 1 ), (m, 6), 2.5 (s, 3 ), 1.6 (s, 1). 173

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