Structural elucidation of thermolysis products of methyl N-methyl-N-nitrosoanthranilate

Article abstract of DOI:10.1039/c5ra07612a

Although it is common knowledge that N-nitroso compounds are thermally (and otherwise chemically) labile, little or nothing is known about the specific reactions that occur during thermal treatment of a compound possessing this functionality. Methyl N-met

Full text of DOI:10.1039/c5ra07612a

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Methyl N-methyl-N-nitrosoanthranilate thermolysis in the vapor and condensed phases gave
different coupling products, dimethyl 2,2'-(1,2-dimethylhydrazine-1,2-diyl)dibenzoate and
methyl 5-methyl-6-oxo-(5H)-phenanthridine-4-carboxylate, respectively.

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Structural elucidation of thermolysis products of methyl N-methyl-N-nitrosoanthranilate
Ana B. Miltojevića,b and Niko S. Radulovića,*
aDepartment of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska
33, 18000 Niš, Serbia
bFaculty of Occupational Safety, University of Niš, Čarnojevićeva 10a, 18000 Niš, Serbia
Abstract
Although it is common knowledge that N-nitroso compounds are thermally (and otherwise
chemically) labile, little or nothing is known of the specific reactions that occur during a thermal
treatment of a compound possessing this functionality. Methyl N-methyl-N-nitrosoanthranilate
was found to undergo a complete thermal degradation under gas chromatographic (GC-MS)
conditions yielding a major unidentified coupling product and methyl N-methylanthranilate. In
an attempt to elucidate the structure of the formed degradation product, a preparative scale
thermolysis in an evacuated vessel at 220 °C of the nitroso compound was carried out. A
chromatographic separation of the thermolysate, followed by GC-MS and NMR (and other
spectral techniques) analyses enabled the identification of in total 46 different products. Among
them a novel coupling product, methyl 5-methyl-6-oxo-(5H)-phenanthridine-4-carboxylate, was
identified and fully spectrally characterized. Interestingly, the initially detected coupling product
that formed under GC conditions, tentatively identified as dimethyl 2,2'-(1,2-dimethylhydrazine-
1,2-diyl)dibenzoate, was not detected in the thermolysate. A careful consideration of the
structures of the identified thermolysate constituents led us to a proposition of major thermolysis
pathways of methyl N-methyl-N-nitrosoanthranilate both in condensed and vapor phases.
Generally, the identified products could be classified as those arising from fission of the N-NO
bond or rather unexpectedly, the Ar-NNO bond, i.e. products related to anthranilic and benzoic
acids, respectively. The latter represents a novel chemical transformation up to now unreported
* Corresponding author. Tel.: +381 18533015; fax: +381 18533014.
E-mail address: nikoradulovic@yahoo.com (N.S. Radulović).

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and unutilized. The structural diversity of the identified products and the noted marked
differences between vapor and condensed phases point to possible synthetic utility of thermolysis
of carefully designed N-nitroso compounds.
Keywords: methyl N-methyl-N-nitrosoanthranilate, thermal degradation products, gas
chromatography-mass spectrometry, 6-(5H)-phenanthridinone derivatives, hydrazine derivatives

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Introduction
Although the first report on N-nitroso compounds dates back to 1863,1 there was little interest in
the chemistry of this class of compounds for almost a century. As is frequently the case, their
biological properties (especially the knowledge that they are carcinogenic) led also to an
increased interest in other aspects of these compounds, including their synthesis, mechanisms of
formation, chemical and physical properties.2-7 Only a few N-nitroso compounds occur
naturally,7 while most of them can be readily synthesized by nitrosation of secondary amines.8
They can be formed in the environment, food, tobacco smoke or in vivo (e.g. in the stomach)
through simultaneous ingestion of nitrosation agents and nitrosable substances.6, 7, 9
N-nitroso compounds of low molecular mass (containing three carbon atoms or less) are
soluble in water, while most of them are soluble in common organic solvents. Moreover, in the
past, they were occasionally used as solvents.7 Generally, they have favorable partition
coefficients into most organic solvents versus water, and there are attempts to link their
“liposolubility“ to their carcinogenic activity in certain organs.10 Even though the N-nitroso
group is not without reactivity, there are few reactions it undergoes. N-nitroso compounds are
well-known alkylating agents; they can undergo cleavage to produce the parent amines and NO,
reduction to give di-substituted hydrazines, and oxidation to yield nitramines.7, 11, 12 Exposure of
nitrosamines’ solution to visible light, converts them to amine and nitrite. When it comes to their
thermal stability, they are deemed stable at room temperature, while they are being decomposed
at temperatures above 200 °C with liberation of NO.7
Two secondary amines, isopropyl N-methylanthranilate (ternanthranin) and methyl N-
methylanthranilate (1), were subjects of our previous studies in which they showed a number of
interesting pharmacological activities.13-18 Methyl N-methylanthranilate is a part of our everyday
diet as it naturally occurs in various fruits and plants and is used as a food flavor.19 Estimated
daily oral intake of 1 in Europe is 60 µg/day (1 µg/kg bodyweight/day).20 Within foodstuff,
compound 1 can react with nitrites, which are widely used as food preservatives, to form methyl
N-methyl-N-nitrosoanthranilate (2). Besides this, compound 2 can be formed in vivo, in the
stomach of humans, in the reaction of nitrosation of 1 by nitrites present in the saliva, as well as
by the ingestion of nitrite-rich foods.21 The nitrosation of methyl N-methylanthranilate with

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sodium nitrite under conditions similar to those in the human stomach was previously examined
by Sakai et al.22
Considering a possible formation of methyl N-methyl-N-nitrosoanthranilate in food and
the fact that food frequently undergoes thermal treatment, where thermolysis plays an important
role (e.g. baking, frying, grilling, and caramelizing), we decided to investigate the products of
methyl N-methyl-N-nitrosoanthranilate thermolysis. During thermolysis, organic matter
undergoes irreversible processes of simultaneous change of chemical composition and physical
state, where organic compounds, heated above certain temperature, are being decomposed.23-26
Depending on the structure of the initial molecule and thermolysis conditions, thermolysate may
contain compounds that are the results of decomposition of the initial material and/or compounds
formed by the decomposition or recombination of fragments formed from the initial compounds.
Having in mind the possible environmental and/or toxicological issues that compounds generated
during the process of thermolysis may pose,23 we decided to carry out a preparative scale
thermolysis of compound 2 and to isolate and structurally elucidate (by spectral means and
through synthesis) the thermolysate products.
Experimental
Material and methods
Methyl N-methylanthranilate (1)13 and methyl N-benzoylanthranilate27 were synthesized
according to previously published procedures. All other reagents (methyl anthranilate, benzoyl
chloride, sodium nitrite, sodium hydrogen carbonate, hydrochloric acid, anhydrous magnesium
sulfate) and solvents (diethyl ether, hexane, chloroform, methanol) were obtained from
commercial sources (Sigma-Aldrich, St. Louis, MO, USA; Merck, Darmstadt, Germany; Carl
Roth, Karlsruhe, Germany) and used as received, except the solvents were purified by
distillation.
Nuclear magnetic resonance (NMR) spectra were recorded at 25 °C on a Bruker Avance
13
III 400 MHz NMR spectrometer (1H at 400 MHz, C at 100 MHz) using CDCl3 as the solvent.
Chemical shifts were expressed as δ (ppm) using tetramethylsilane as an internal standard. 2D
experiments (1H-1H COSY, NOESY, TOCSY, HSQC and HMBC), as well as DEPT-90 and

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DEPT-135, were run on the same instrument with usual pulse sequences. UV spectra (in
acetonitrile) were measured using a UV-1800 Shimadzu spectrophotometer (Tokyo, Japan).
Infrared (IR) measurements (attenuated total reflectance) were carried out using a Thermo
Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). “Dry flash”
chromatography was performed using silica gel 20-45 μm (Carl Roth GmbH + Co.KG,
Karlsruhe, Germany). For thin layer chromatography (TLC) silica gel 60 on Al plates (Kieselgel
60 F254, layer thickness 0.2 mm; Merck, Darmstadt, Germany) was used. Spots on TLC were
visualized by UV light (254 nm) and by spraying with 10% (w/v) ethanolic solution of
phosphomolybdic acid, followed by heating. Microanalysis of carbon, hydrogen and nitrogen
were carried out on a Carlo Erba 1106 microanalyzer; their results agreed favorably with the
calculated values.
GC and GC-MS analyses
Gas chromatography/mass spectrometry (GC-MS) analyses were repeated three times for each
sample using an HP 6890N gas chromatograph coupled with an HP 5975B mass-selective
detector (Hewlett-Packard, Palo Alto, CA, USA). The gas chromatograph was equipped with a
DB-5MS fused silica capillary column (5% phenylmethylsiloxane, 30 m × 0.25 mm, film
thickness 0.25 µm; Agilent Technologies, Palo Alto, CA, USA). The oven temperature was
raised linearly from 70 to 315 °C at a heating rate of 5 °C min−1 and then held isothermally for
10 min. Helium at a flow rate of 1 mL min−1 was used as carrier gas. The injector and interface
were maintained at 250 and 320 °C respectively. The samples, 1 μL of the solutions of reaction
mixtures or pure compounds in diethyl ether (ca. 1 mg in 1 mL of Et2O), were injected in a
pulsed split mode (the flow rate was 1.5 mL min−1 for the first 0.5 min and then set to 1 mL
min−1 for the remainder of the analysis; split ratio 40:1). The mass-selective detector was
operated at an ionization energy of 70 eV in the m/z 35–500 range with a scanning speed of 0.34
s/scan. Analyses by GC with flame ionization detection (GC-FID) were carried out under the
same experimental conditions using the same column as described for GC-MS. The percentage
composition was computed from the GC-FID peak areas without the use of correction factors.
Qualitative analyses of the reaction mixtures and purified synthesized compounds were based on
the comparison of the compounds’ linear retention indices relative to the retention times of C11–

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C36 n-alkanes on the DB-5MS column28 with those reported in the literature13, 29, 30 comparison of
their mass spectra with those of authentic standards as well as those from Wiley 6, and NIST11;
the analysis of fragmentation patterns from mass spectra; the correlation of the calculated boiling
points of the tentatively assigned structures with the experimental RI values; and finally,
wherever possible, identification was achieved by GC co-injection with an authentic sample.
Synthesis of methyl N-methyl-N-nitrosoanthranilate
Synthesis of methyl N-methyl-N-nitrosoanthranilate (2) was accomplished following a slightly
modified literature method.22 A solution of 2.77 g (40 mmol) of sodium nitrite in 10 ml of water
was added slowly dropwise into a mixture of 3.31 g (20 mmol) of methyl N-methylanthranilate
(1) and 3.32 ml of 37% hydrochloric acid (40 mmol) in 10 ml of water, with stirring at ambient
temperature. Afterwards, the mixture was stirred for additional 30 min. The end of the reaction
was determined by TLC. The reaction mixture was extracted with diethyl ether; the organic layer
was washed with HCl (1%, w/w), water, solution of sodium hydrogen carbonate (2%, w/w) and
again with water. The organic layer was then separated, dried over anhydrous magnesium sulfate
and the solvent evaporated. The crude product (2.67 g) was purified by an isocratic “dry flash”
chromatography (hexane : diethyl ether = 2:1, v/v) to yield a pure sample of methyl N-methyl-N-
nitrosoanthranilate (2.0 g, 52%).
Thermolysis of methyl N-methyl-N-nitrosoanthranilate
Methyl N-methyl-N-nitrosoanthranilate (2) (0.12 g, 0.62 mmol) was sealed in an evacuated glass
tube and heated in an oil bath at 220 °C for 15 minutes. The contents of the vessel were taken up
with chloroform. The thermolysate (0.10 g), obtained after the removal of the solvent, was
subjected to “dry flash” chromatography on silica gel (20-45 μm) using n-hexane-Et2O (v/v)
mixtures of increasing polarity and finally with methanol to furnish 7 fractions (initially pooled
based on TLC). The fractions were analyzed by GC-MS and NMR.

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Spectral characterization of the synthesized/detected compounds
Methyl N-methyl-N-nitrosoanthranilate (2) (2.0 g, 52%). Yellowish liquid; UV λmax
(CH3CN)/nm 283sh (log ɛ 4.59) and 218 (5.20); FTIR (neat) νmax/cm-1 2952.4, 1720.7, 1601.0,
1495.6, 1432.9, 1395.4, 1293.3, 1264.1, 1192.1, 1116.7, 1066.9, 1040.9, 952.6, 762.1, 711.5,
702.7; EIMS m/z 194 (M+, 1.4%), 164 (25.7, M−NO), 133 (14.6, M−OCH3−NO), 132 (100.0,
M−CH3OH−NO), 105 (19.5, M−COOCH3−NO), 104 (18.5, M−CH3OH−NO−CO), 77 (38.8,
M−C3H5N2O3); Found: C, 55.70; H, 5.22; N, 14.39; O, 24.69%. Calc. for C9H10N2O3: C, 55.67;
H, 5.19; N, 14.43; O, 24.72%; 1H and 13C NMR data, see Table 1.
Dimethyl 2,2'-(1,2-dimethylhydrazine-1,2-diyl)dibenzoate (4). EIMS m/z 328 (M+,
10.0%), 165 (63.9, M−C9H9NO2), 164 (18.2, M−C6H4(NCH3)COOCH3), 163 (22.4,
M−C9H11NO2), 148 (24.4, M−C9H11NO2−CH3), 133 (27.6, M−C9H9NO2−CH3OH), 132 (100.0,
M−C9H10NO2−CH3OH), 118 (10.3, M−C11H16NO3), 105 (69.0, M−C9H9NO2−CH3OH−CO), 104
(58.8, M−C9H9NO2−CH3−H2O−CO), 91 (12.5, M−C11H13N2O4), 77 (62.4, M−C12H15N2O4).
Methyl 5-methyl-6-oxo-(5H)-phenanthridine-4-carboxylate (5); Yellowish amorphous
solid; UV λmax (CH3CN)/nm 328 (log ɛ 4.58), 242 (5.19) and 224 (5.18); FTIR (neat) νmax/cm-1
2920.8, 2849.9, 1722.5, 1651.8, 1608.0, 1584.9, 1513.6, 1431.0, 1325.6, 1293.1, 1254.3, 1144.1,
1104.8, 1060.6, 753.3, 722.7, 691.4; Rt 36.941 min, RI (DB-5MS) 2570; EIMS m/z 267 (M+,
100.0%), 252 (25.1, M−CH3), 236 (78. 7, M−OCH3), 222 (94.0), 208 (40.6, M−COOCH3), 206
(44.4), 180 (20.7), 179 (31.2), 178 (48.0), 164 (10.2), 152 (37.8), 151 (31.2), 139 (11.8), 117
(10.0), 103 (12.6), 90 (10.6), 89 (11.6), 76 (16.3), 63 (5.9), 51 (2.3); Found: C, 71.86; H, 4.88; N,
5.27; O, 17.99%. Calc. for C16H13NO3: C, 71.90; H, 4.90; N, 5.24; O, 17.96%. 1H and 13C NMR
data, see Table 2. IR, 1H and 13C NMR spectroscopic data are in accordance with Han et al.31
Methyl N-benzoylanthranilate (6) (1.2 g, 90%). White amorphous solid; UV λmax
(CH3CN)/nm 314 (log ɛ 4.22), 267 (4.37), 234 (4.64) and 212 (4.72); FTIR (neat) νmax/cm-1
3263.9, 2949.4, 1692.5, 1666.9, 1608.4, 1589.1, 1531.3, 1494.1, 1453.7, 1432.9, 1323.3, 1295.8,
1268.0, 1251.3, 1189.7, 1178.4, 1079.9, 965.8, 896.1, 766.7, 696.4, 664.6; Rt 31.951 min, RI
(DB-5MS) 2249; EIMS m/z 255 (M+, 34.0%), 224 (2.2, M−OCH3), 196 (6.1, M−COOCH3), 105

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(100.0, M−C6H4(NH)COOCH3), 90 (3.5), 77 (38.8, M−C9H8NO3); Found: C, 70.56; H, 5.12; N,
5.52; O, 18.80%. Calc. for C9H10N2O3: C, 70.58; H, 5.13; N, 5.49; O, 18.80%. 1H and 13C NMR
data, see Table 3. 1H and 13C NMR spectroscopic data are in accordance with Hinsberger et al.32
Methyl N-formylanthranilate (7). White amorphous solid; UV absorptions: λmax
(CH3CN)/nm 311 (log ɛ 4.34), 251 (4.77), 244 (4.78) and 221 (5.01) nm; FTIR (neat) νmax/cm-1
3320.1, 2950.0, 1685.4, 1586.1, 1516.2, 1435.0, 1292.0, 1259.5, 1193.3, 1127.3, 1082.4, 963.4,
755.5, 700.7; Rt 18.307 min, RI (DB-5MS) 1568; EIMS m/z 179 (M+, 25.9), 151 (58.9, M−CO),
148 (8.2, M−OCH3), 146 (10.8), 120 (21.2, M−COOCH3), 119 (100.0, M−OCH3−CHO), 92
(39.8, M−COOCH3−CO), 90 (10.4), 77 (4.0, M−C3H4NO3), 65 (16.2); Found: C, 60.37; H, 5.04;
1
N, 7.84; O, 26.75%. Calc. for C9H9NO3: C, 60.33; H, 5.06; N, 7.82; O, 26.79%. H and 13C
NMR data, see Table 3. 1H and 13C NMR spectroscopic data are in accordance with Chen et al.33
Although, we observed the presence of two rotamers in the NMR spectra, as it was previously
published,33 herein we give only the data for the major rotamer, as the fraction 3 was a complex
mixture of several compounds.
Results and discussion
Methyl N-methyl-N-nitrosoanthranilate (2) was prepared by direct nitrosation of methyl N-
methylanthranilate (1, Scheme 1). An NMR analysis of the reaction products verified that there
was no methyl N-methylanthranilate contamination. However, the 1H NMR contained two sets of
analogous signals of unequal intensity (ratio 1 : 12.9). Both sets of signals appeared to
correspond to compound 2 based on the chemical shifts and signal multiplicities. An inspection
of HSQC and HMBC spectra confirmed the expected C-H connectivity. The most pronounced
differences between the chemical shift values belonging to analogous pairs of signals were
observed for the NCH3 and H-3 protons. We believe that the two sets of signals belong to the
stereoisomers arising from a hindered rotation about the NN bond (Scheme 1). Table 1
summarizes the NMR data for the two diastereoisomers. We believe that the isomer with the
lower chemical shift of NCH3 protons could be assigned to the (E)-isomer based on the
antiperiplanar (trans) relationship between the lone pair on the nitrogen atom and of the
corresponding methyl group (further work in this direction is underway).34 Although this is a

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known compound,22, 35, 36 there was no mention of the existence of stereoisomers and no NMR
data provided in the previous work. We did not attempt to separate the two diastereoisomers, and
used the obtained mixture in all further experiments.
Scheme 1 Synthesis of methyl N-methyl-N-nitrosoanthranilate (2).
1
Table 1 H (400 MHz) and 13C (100 MHz) NMR spectroscopic data (CDCl3; Me4Si) for
compounds (E)-2 and (Z)-2
(E)-Methyl N-methyl-N-
nitrosoanthranilate ((E)-2)
(Z)-Methyl N-methyl-N-
nitrosoanthranilate ((Z)-2)
Position
δC, mult.
127.1 C
δH (J in Hz)
δC, mult.
δH (J in Hz)
1
-
127.0 C
138.9 C
-
2
141.6 C
-
-
3
126.0 CH
133.0 CH
128.9 CH
131.4 CH
166.1 C
7.40, dd (7.9, 1.1)
7.67, td (7.7, 1.6)
7.53, td (7.7, 1.1)
8.01, dd (7.8, 1.6)
–
127.1 CH
133.7 CH
129.6 CH
131.3 CH
164.9 C
7.05, dd (7.8, 1.1)
7.64, td (7.7, 1.6)
7.49, td (7.7, 1.2)
8.03, dd (8.1, 1.5)
–
4
5
6
-COOCH3
-COOCH3
-N(NO)CH3
52.6 CH3
35.3 CH3
3.82, s
52.5 CH3
41.0 CH3
3.82, s
3.41, s CH3
4.15, s CH3
A GC-MS analysis (relevant temperatures: injector 250 °C, column 70-315 °C, transfer
line 320 °C) of the product revealed an interesting conduct of the compound upon heating. There
was no peak corresponding to the expected compound, but instead, the TIC chromatogram (Fig.
1) displayed two peaks eluting at 14.75 min (RI 1419) and 18.66 min (RI 1583) interconnected
with an elevated baseline. The first peak (14.75 min), broadened and tailing, belonged to methyl
N-methylanthranilate (1).13 The mass spectrum of the second, more abundant, compound (18.66

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min) was also very similar to the mass spectrum of 1, but it possessed the highest m/z value at
328. An even m/z value of M+• pointed to a compound containing an even number of nitrogen
atoms. The number of carbon atoms per molecule (18 C) was inferred from the relative intensity
of the [M+1]+ ion to the M+ ion (the ratio of M : [M+1] was found to be 5.0). This and a more
precise value of m/z of M+ (328.20) indicated that its molecular formula could be C18H20N2O4.
Inferred from the fragmentation pattern (similar to 1) and the fact that the molecular formula
corresponded to 2 molecules of methyl N-methylanthranilate minus 2 H atoms, we could assume
that the unknown compound represents the product of an oxidative coupling of two molecules of
1. Such a reaction can be envisaged as a recombination reaction of two free radicals formed by
thermal degradation of compound 2 during the GC experiment where N-NO bond was
homolytically cleaved (Scheme 2). As the unpaired electron is expected to be delocalized into
the benzene ring, free radicals (3a-3d) could be recombined in, in total, six different manners
(Fig. 2). Except for 4, all other structures (4a-e) require additional proton transfer steps leading
to rearomatization. Sterically and energetically speaking, C-C and C-N coupling of the type in 4a
and 4d are the most favorable. We believe that the likely structure of this compound would be 4,
and not 4a-e. Although an N-N bond is significantly weaker than C-N or C-C bonds, the mode of
formation (as we have shown below) of this compound limits the complexity (number of steps)
of the coupling mechanism. Another possible fate of methyl N-methylanthranilate free radicals
was an abstraction of a hydrogen atom to give 1 (Rt = 14.754 min).
Scheme 2 Homolytic cleavage of N-NO bond and free radical delocalization.

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Fig. 1 Gas chromatogram (TIC) of the nitrosation product of 1.
Fig. 2 Possible structures of 4 formed by thermal degradation of 2 during the GC experiment.
The peak shapes (tailing of the methyl N-methylanthranilate peak) indicated that one part
of methyl N-methyl-N-nitrosoanthranilate was thermally degraded in the injector (250 °C), while
another portion (remaining part) evaporated and was introduced onto the chromatographic
column where after (column and/or transfer line) it was subject to further thermal degradation
took place. The sharpness of the peak corresponding to compound 4 indicated that it had not
form gradually during the GC run but in a short time period, most probably, in the most heated
instrument zone-the transfer line (320 °C). Thermal degradation of 2 that happened on the
column gave only compound 1 that significantly contributed to the elevated baseline. One can

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assume that the likelihood of hydrogen abstraction from the stationary phase was greater than the
probability of radical recombination.
To corroborate the assumption that compound 4 formed in the GC transfer line, and not
in the injector or the column, we conducted a series of experiments in which temperature of the
GC system was varied (injector, column and transfer line) from 190 to 250 °C. The highest
temperature corresponded, naturally, to the transfer line which was always held at least 10 °C
above the highest column temperature. Below 220 °C, no degradation of the nitroso compound
occurred and a peak of methyl N-methyl-N-nitrosoanthranilate (2) appeared (inferred from the
change of the mass scan detected in the same time window). Thus, the coupling product 4
formed in the short period when the nitroso compound reached the transfer line and most
probably in the vapor phase (i.e. in the heated helium gas). Such an environment (diluted radicals
in an inert gas) allowed only a recombination of two radicals to occur and probably did not
provide sufficient time for additional proton transfer reactions (these could not be achieved
intramolecularly as sigmatropic rearrangements since they are all forbidden by geometrical
constraints/symmetry).
Another fact that is in favor of the formation of the hydrazine derivative, and not
biphenyl or diphenylamine derivatives, is that only a single product is observed (no other peaks
were detected), under such kinetic control, since almost all other isomers (4a-e) are
thermodynamically more stable than 4. Consequently, the likelihood of hydrogen abstraction was
much lower and, hence, the formation of 1 was greatly diminished as noted. When making the
final decision on the structural assignment of 4, we took under consideration the general
appearance of the observed mass spectrum (strong resemblance to that of the “monomer”), and
argued that one should not expect such fragmentation in biphenyl and diphenylamino derivatives
(fragmentation that does not involve the cession of bonds leading to monomers should be
dominant and give raise to fragments of higher m/z than that of the monomer). Thus, we
concluded that the structure of 4 was that of dimethyl 2,2'-(1,2-dimethylhydrazine-1,2-
diyl)dibenzoate.
To confirm the structure of 4, we decided to perform a large scale-preparative
thermolysis of 2 and to analyze the formed thermolytic products by NMR. Thermolysis of 2 was
performed in an evacuated sealed glass vessel heated in an oil bath to 220 °C for 15 minutes.
Substantial degradation occurred at that temperature as evidenced by the change in color

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(darkening). The duration of the thermolysis was determined according to a series of experiments
with varying time of exposure to heat. The time of 15 min was sufficient for the complete
disappearance of 2 (monitored by TLC). After “dry flash” chromatography of the thermolysate,
the pooled fractions were analyzed by GC-MS and NMR.
Fraction 4 was pure enough to allow a direct NMR analysis. GC-MS analysis suggested
the presence of a major volatile compound eluting at 36.94 min (RI(DB-5MS) 2570). The
molecular ion peak at m/z 267 was also the base peak. An odd m/z value of M+• indicated that the
compound in question contained an odd number of nitrogen atoms. This was corroborated by
high resolution mass spectrometry that showed that its molecular formula was C16H13NO3. High
index of hydrogen deficiency (11 unsaturations) suggested that two molecules of methyl N-
methyl-N-nitrosoanthranilate were combined and that 8 of 11 unsaturations corresponded to 2
aromatic rings. The molecular formula of the investigated compound indicated that it contains 2
C, 7 H, 3 N and 3 O atoms less than those in two molecules of compound 2.
13
The presence of 16 C atoms in the molecule was confirmed by 16 peaks observed in C
NMR among which 5 were peaks of very low intensity. DEPT spectra showed only the existence
of CH3 and CH groups. The 1H NMR spectra of the compound showed 7 signals in the chemical
shifts range of protons attached to an aromatic ring: tree doublets of doublets (dd 7.74, 8.39, 8.54
ppm), two doublets of doublets of doublets (ddd 7.62 and 7.79 ppm), one doublet (d 8.29 ppm)
and one triplet (t, 7.33 ppm). This pattern was indicative of 2 aromatic cores, 1,2-disubstitued
and 1,2,3-trisubstituted ones. Additionally, there were two signals (singlets at 3.63 and 3.98
ppm) indicating the presence of N-methyl and O-methyl groups. The structural elucidation and
assignation of all proton and carbon-13 NMR signals was made possible only by the use of 2D
NMR (1H-1H COSY and HMBC spectra) and correlations observed in these spectra gave rise to
the assignations given in Table 2. The HMBC spectrum showed correlation between the protons
at 3.98 ppm and a carbon at 169.3 ppm showing the presence of an ester group. This was also
strengthened by the fragmentation patterns visible in the MS spectra, the presence of an ion
corresponding to the loss of the OCH3 group and the loss of COOCH3, and an IR absorption at
1722 cm−1.

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Table 2 1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data (CDCl3; Me4Si) for the
major constituent of fraction 4
Position
δC, mult.
125.2 CH
121.9 CH
130.9 CH
122.4 C
δH (J in Hz)
1
8.39, dd (8.3, 1.5)
2
7.33, t (7.8)
3
7.74, dd (7.5, 1.5)
4
–
4a
137.6 C
–
6
162.8 C
–
6a
125.4 C
–
7
128.8 CH
128.6 CH
132.8 CH
121.8 CH
133.1 C
8.54, dd (8.0, 1.0)
8
9
7.62, ddd (8.1, 7.2, 1.0)
7.79, ddd (8.3, 7.2, 1.5)
10
8.29, d (8.2)
10a
–
–
10b
121.2 C
-COOCH3
-COOCH3
-NCH3
169.3 C
–
52.8 CH3
36.4 CH3
3.98, s
3.63, s
1
The multiplicities and the corresponding coupling constants of protons in H NMR and
1
correlations observed in H-1H COSY spectrum revealed that the protons at 7.33, 7.74 and 8.39
ppm belong to a trisubstituted aromatic core and the protons at 7.62, 7.79, 8.29 and 8.54 ppm to a
disubstituted one. These data generally agreed with two possible structures, presented in Fig. 3.
Alongside with the value of chemical shift of the carbonyl which corresponded more to an amide
and less to the conjugated ketone, the cross peak between the proton at 3.63 ppm and the carbon
at 162.8 ppm in HMBC spectrum confirmed that the N-methylamino and carbonyl groups are
part of an amide group consistent with structure 5 and are not as distant as in structure 5a.
Structure 5 was additionally confirmed by the existence of cross peaks in the HMBC spectrum
1
(see Fig. 4) and in H-1H COSY spectrum (H1-H2, H2-H3, H7-H8, H9-H10). Very recently,
during the preparation of this manuscript, the synthesis of 5, alongside with the spectral data

RSC Advances
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1
(though without H and 13C NMR assignment), has been published.31 The NMR and IR data
published herein agreed favorably with this study.31
Fig. 3 Possible structures of the major constituent of fraction 4.
Fig. 4 Selected HMBC interactions of compounds 5, 6 and 7.
A compound eluting at 31.95 min (RI (DB-5MS) 2249), showing the highest m/z value at
255 in its mass spectrum, was detected in low amount during the GC-MS analysis of fractions 1,
2 and 3. An odd m/z value of M+• (255) pointed to an odd number of nitrogen atoms in the
investigated compound and corresponded to the molecular formula C15H13NO3. The comparison
of its mass spectra with those from MS libraries suggested that it contained an anthranilate
moiety and that it could be an amide of benzoic acid. Having this in mind we assumed that the
compound in question was methyl N-benzoylanthranilate (6) (Fig. 5). To confirm this tentative
identification we synthesized the mentioned compound starting from benzoyl chloride and
methyl anthranilate in the presence of Et3N following the procedure of Schneider et al.27 and the
structure of the synthetized compound was confirmed by 1D and 2D NMR (Fig. 4 shows
important HMBC interactions used to assign the spectra, while Table 3 summarizes the assigned

Page 17 of 36
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DOI: 10.1039/C5RA07612A
data). NMR data were in accordance with the literature.32 GC co-injection confirmed that
compound 6 was indeed the constituent of fractions 1-3.
Fig. 5 Structure of compound 6.
1
Table 3 H (400 MHz) and 13C (100 MHz) NMR spectroscopic data (CDCl3; Me4Si) for
compound 6 and 7
Methyl N-benzoylanthranilate (6)
Methyl N-formylanthranilate (7)a
Position
δC, mult.
115.3 C
δH (J in Hz)
δC, mult.
115.2 C
140.8 C
121.2 CH
134.8 CH
123.2 CH
130.9 CH
-
δH (J in Hz)
1
-
-
-
2
141.9 C
-
3
131.0 CH
122.6 CH
134.8 CH
120.5 CH
134.9 C
8.09, dd (8.1, 1.6)
7.12, ddd (8.1, 7.2, 0.9)
7.61, ddd (8.5, 7.2, 1.6)
8.94, dd (8.5, 0.9)
–
8.72 dd (8.6, 1.0)
4
7.57, ddd (8.6, 7.2, 1.6)
5
6
7.11, ddd (8.4, 7.2, 1.0)
8.05, dd (8.4, 1.6)
1’
-
2’, 6’
3’, 5’
4’
127.4 CH
128.8 CH
132.0 CH
169.1 C
8.06, dd (8.1, 1.6)
7.55, ddd (8.1, 7.6, 0.6)
7.53, br d (7.6)
–
-
-
-
-
-
-
-COOCH3
-COOCH3
168.6 C
52.5 CH3
159.6 NHCO
–
52.5 CH3
3.13, s
3.94, s
-NHCO 165.7 NHCO
12.04, br s
8.52, s NHCHO
7.55, s NHCHO
a
The presence of two rotamers was observed in the NMR spectra, as previously reported.33
However, herein we give only the data for the major rotamer, as the fraction 3 was a complex
mixture of several compounds.

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Fraction 3 was a mixture of around twenty compounds among which methyl N-
formylanthranilate (7) (Fig. 6) was the major one (around 50%) (fragmentation patterns visible in
the MS spectra (ions corresponding to the loss of the OCH3 group and the loss of COOCH3
confirmed the presence of an ester group and an ion corresponding to the loss of CO confirmed
the presence of an N-formyl group)). NMR analysis of fraction 3 (without further purification)
1
corroborated the identity of 7. H NMR contained signals, two dd (8.05 and 8.72 ppm) and two
ddd (7.11 and 7.57 ppm), characteristic for an ortho-disubstituted aromatic core. All proton and
carbon NMR signals were assigned based on 2D NMR (1H-1H COSY and HMBC spectra). For
example, the chemical shifts of C1 and C2 were inferred from the HMBC spectrum as these
13
peaks were not observed (low intensity) in C NMR. The exchangeable proton attached to the
nitrogen atom of -NHCHO was located by two HMBC cross-peaks originating from the coupling
of the proton in question with C1 and C3 carbons. Although the presence of two rotamers was
1
13
observed in our NMR spectra of 7, as previously reported,33 herein we report only H and C
NMR data for the major rotamer (Table 3) unambiguously identified in the complex mixture.
Important HMBC interactions are presented on Fig. 4.
Fig. 6 Structure of compound 7.
Analysis (GC and GC-MS) of the seven pooled fractions further enabled the
identification of 46 different components, representing 25.2-94.7% of the peak areas detect for
the individual fractions. The identified constituents, their relative share in the fractions, molar
masses, Rt and RI values and methods of identification are listed in Table 4. Three arguments
were taken into account when a final decision was made to ascribe a certain structure to a peak.
The molecular mass and the MS fragmentation patterns were used to propose a tentative
chemically sound structure that might be expected from the thermolytic process. The
meaningfulness of the proposed structures was further tested by a correlation analysis of the

Page 19 of 36
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predicted boiling points (Bp) for these structures (ChemBio3D Ultra 13.0) and their experimental
retention indices (RI)37 since literature indices for the majority of the detected compounds could
not be found in the literature. All compounds that showed a significant deviation (30 K or more)
of the estimated Bp by the correlation equation from the calculated Bp were omitted from the list
of correctly identified compounds, and this was redone several times until a good correlation was
attained. The final correlation equation of predicted Bp vs RI are given below:
Bp = 351.73604 + 0.14209RI, R = 0.95742.
where Bp represents the predicted boiling point of a compound, RI represents its
experimental retention index, while R is the correlation coefficient.

RSC Advances
Page 20 of 36
Table 4 The preparative-scale thermolysis products of compound 2
M
Rt
Fr1
[%]
Fr2
[%]
Fr3
[%]
Fr4
[%]
Fr5
[%]
Fr6
[%]
Fr7
[%]
No
1.
Compound structurea
RI
IdenMethb
[g/mol] [min]
(8)
(9)
136
122
6.989 1103 RI, MS, CoI
8.979 1181 RI, MS, CoI
0.3
-
tr
-
-
-
-
-
-
-
-
-
2.
3.
0.1
0.3
(10)
135
11.545 1285
RIa, MS
-
tr
0.1
-
-
-
-
4.
5.
6.
(11)
(1)
151
165
181
13.181 1353 RI, MS, CoI
14.754 1419 RI, MS, CoI
4.0
82.7
-
10.7
15.8
18.8
0.2
5.4
2.0
-
-
-
tr
0.9
-
4.0
1.3
-
1.7
0.3
-
(12)
15.697 1458
RI, MS

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RSC Advances
7.
(13)
181
16.385 1486
RI, MS
-
0.2
-
-
-
-
-
RIa, MS,
CoI
8.
9.
(14)
(7)
151
179
16.808 1503
18.307 1568
-
-
0.8
0.2
-
-
-
-
-
-
RIa, MS,
NMR
18.3
48.7
tr
3.3
10.
11.
(15)
(16)
223
207
18.988 1597
19.152 1605
RIa, MS
RIa, MSa
-
-
-
-
-
-
1.1
-
-
-
-
0.1
0.7
-
12.
13.
(17)
(18)
193
210
19.241 1609
19.650 1627
RIa, MS
RIa , MS
-
-
0.2
1.4
0.2
1.1
-
-
-
-
-
-
24.8
tr

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14.
15.
(19)
(20)
196
241
21.001 1688
RIa, MS
RIa, MSa
-
tr
tr
-
-
-
tr
-
tr
-
-
-
24.767 1869
25.842 1923
tr
0.1
16.
17.
18.
(21a)
(21b)
(21c)
270
270
270
RIa, MS
RIa, MS
RIa, MSa
-
2.7
3.4
1.8
0.3
0.1
0.1
-
-
-
tr
tr
tr
-
-
-
-
tr
-
29.001 2081
29.434 2102
0.6
0.5

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19.
20.
(22)
(23)
227
269
26.343 1949
RIa, MSa
tr
-
tr
-
-
-
-
-
-
-
-
29.374 2099
RIa, MSa
tr
0.5
0.8
RIa, MSa
RIa, MSa
RIa, MSa
RIa, MSa
RIa, MSa
RIa, MSa
RIa, MSa
RIa, MSa
RIa, MSa
21.
22.
23.
24.
25.
26.
27.
28.
29.
29.924 2130
32.862 2304
32.964 2311
33.641 2353
33.694 2357
34.195 2389
35.985 2505
30.577 2167
30.790 2180
-
1.1
tr
0.1
0.6
0.1
0.3
tr
tr
0.2
-
1.6
tr
-
tr
-
tr
tr
-
-
-
(24)
(25)
tr
-
-
and/or
285
-
0.7
-
1.3
-
-
tr
tr
tr
0.2
tr
-
-
2.1
-
tr
0.4
tr
-
0.2
-
0.1
tr
0.1
tr
-
-
-
-
0.2
3.8
tr
-
tr
-
299
0.4
0.2
0.4
0.4
(26)

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and/or
RIa, MSa
30.
31.
32.
33.
31.792 2240
32.268 2269
34.085 2382
34.591 2414
-
tr
-
tr
-
-
-
-
-
-
-
RIa, MSa
RIa, MSa
RIa, MSa
0.3
2.1
1.2
0.7
1.9
2.3
-
(27)
0.1
tr
1.7
1.3
tr
tr
tr
tr
tr
tr
34.
(28)
209
31.016 2194
RIa, MSa
-
-
0.2
-
-
-
-
RIa, MSa,
35.
36.
(6)
255
298
31.951 2249
33.090 2319
0.8
0.5
0.4
0.5
tr
-
-
-
-
-
CoI, NMR
RIa, MSa
(29)
0.5
tr
tr
tr

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RSC Advances
RIa, MSa,
37.
38.
(5)
267
342
36.941 2570
37.020 2576
-
-
-
4.8
80.8
51.7
3.5
1.5
tr
-
NMR
(30)
RIa, MSa
tr
-
tr
-
39.
40.
(31)
(32)
(33)
296
37.060 2578
RIa, MSa
-
-
-
-
-
-
-
-
-
0.7
253
401
37.291 2594
RIa, MSa
tr
5.5
6.5
-
RIa, MSa
RIa, MSa
41.
42.
46.742 3320
46.866 3331
-
-
-
-
-
-
-
-
-
-
2.7
1.7
1.1
0.4

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RIa, MSa
43.
44.
45.
46.
48.008 3430
49.288 3544
-
-
-
-
tr
-
-
-
-
-
-
-
-
-
-
2.9
2.4
2.6
1.5
1.7
0.6
0.6
1.3
RIa, MSa
RIa, MSa
RIa, MSa
(34)
50.333
51.392
-
-
-
-
-
-
-
Total identified
94.7
85.4
66.2
92.6
65.8
25.2
34.2
a Compounds listed in order of elution on DB-5MS column (Rt: retention time (in min) and RI: experimentally determined retention
indices on the mentioned column by co-injection of a homologous series of n-alkanes C11–C36).
b RI, constituent identified by retention index matching; RIa, identification corroborated by the correlation of the calculated boiling
point of the tentatively assigned structure with the experimental RI value; MS, constituent identified by mass spectra comparison;
MSa, identification based on mass spectral fragmentation pattern; CoI, constituent identity confirmed by GC co-injection of authentic
sample; NMR, structure of an isolated pure constituent confirmed by 1H and 13C NMR spectral analysis.
tr, trace amount (less than 0.05%); -, compound not detected in this particular chromatographic fraction.

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Although compound 4 was not detected among the products of methyl N-methyl-N-
nitrosoanthranilate thermolysis in condensed phase, listed in Table 4, we correlated its predicted
boiling point (Bp) and experimental retention index (RI). Based on the above equation, the RI
value of 4 should be around 2500 which is almost 1000 RI units higher than the one we found
(RI 1583) in the early experiments. This discrepancy supported the fact that a significant portion
of 2 passed through the GC column unaltered and was completely decomposed in the much
warmer transfer line where the recombination of the methyl N-methylanthranilate radicals took
place (i.e. the part of the nitroso compound that did not decompose in the injector and column,
completely transformed to the coupling product 4 in the transfer line and this explains the fast
“elution” of 4 from the GC column since it formed only at the very end of the same).
A careful consideration of the structures of the identified thermolysate constituents led us
to a proposition of the major thermolysis pathways of methyl N-methyl-N-nitrosoanthranilate.
Since none of the identified products contained a nitroso group, compound 2 initially must have
undergone a cleavage of either N-NO or CAr-NNO bonds. N-NO bond appears to be a good
candidate for homolytic cleavage as one would expect from the stability of NO and a delocalized
methyl N-methylanthranilate radical (Scheme 2). A high number of products appears to have
formed in the reaction of the radical (3a-d) that was obtained as the result of the mentioned
homolytic cleavage of N-NO bond (1, 11, 14, 31, and in part 5, 6, 20, 22, 23, 24, 25, 26, 27, 28,
32, 33, 34).
Furthermore, we identified quite a few simple derivatives of benzoic acid, i.e. products
lacking the ortho-NHCH3 group (8, 9, 21a-c, in part 20, 22, 24, 25, 26, 27, 33, 34). These
products could have formed either from a hypothetical methyl benzoate radical (35) or in the
hypothetical reaction of methyl N-methylanthranilate radical (3) with methyl benzoate (8). The
origin of 8 and 35 is not clear to us, although there is a possibility that during the thermolysis,
besides the N-NO, CAr-NNO homolytic cleavage could have occurred, as well (Scheme 3). Other
more elaborate mechanisms of methyl benzoate-related species formation could be envisaged
(Scheme 4). However, at this moment, we cannot dismiss any of the possibilities or even rule out
their concurrent existence. Both N-NO and CAr-NNO cleavages seem to be likely as methyl N-
methylantranilate (1) and methyl benzoate (8) would arise from hydrogen abstraction by these
radicals (3a-d and 35, respectively). Beside hydrogen abstraction, these radicals could recombine
in several ways or could react with compounds 1 and 8 to give numerous products that could

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undergo further modifications (e.g. O- or N-demethylation and decarboxylation) (Scheme 3 and
5).

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Scheme 3 Possible pathways of thermolysate products formation.