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Decomposition mechanism of annelated triazinones bearing the

para-nitrophenyl group

Marta Worzakowska (Conceptualization) (Methodology)

(Investigation) (Writing - original draft) (Writing - review and editing),
Małgorzata Sztanke (Conceptualization) (Methodology)
(Resources) (Writing - original draft) (Writing - review and editing),
Krzysztof Sztanke (Conceptualization) (Methodology) (Resources)
(Writing - original draft) (Writing - review and editing)

PII: S0165-2370(20)30021-8
DOI: https://doi.org/10.1016/j.jaap.2020.104856
Reference: JAAP 104856

To appear in: Journal of Analytical and Applied Pyrolysis

Received Date: 14 January 2020

Revised Date: 12 May 2020
Accepted Date: 16 May 2020

Please cite this article as: { doi: https://doi.org/

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© 2020 Published by Elsevier.

Decomposition mechanism of annelated triazinones bearing the para-nitrophenyl group

Marta Worzakowskaa, Małgorzata Sztankeb, Krzysztof Sztankec

a
Department of Polymer Chemistry, Institute of Chemical Sciences, Faculty of Chemistry,

Maria Curie-Sklodowska University in Lublin, Gliniana 33 Street, 20-614 Lublin, Poland

b
Chair and Department of Medical Chemistry, Medical University, 4A Chodźki Street, 20-

093 Lublin, Poland

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c
Laboratory of Bioorganic Synthesis and Analysis, Chair and Department of Medical

Chemistry, Medical University, 4A Chodźki Street, 20-093 Lublin, Poland

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Highlights
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 Decomposition mechanism of fused azaisocytosine-containing congeners was studied.
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 One-stage degradation of compounds took place in inert conditions.
 Two-stage degradation of compounds took place in oxidative conditions.

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Simultaneous, radical degradation processes run during their pyrolysis and

combustion.
 Volatiles formed under their pyrolysis and combution by the FTIR/QMS were
analyzed.
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ABSTRACT
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A novel class of fused azaisocytosine-containing congeners, i.e. 3-(4-nitrophenyl)-8-(R-

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phenyl)-7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones, acting as anticancer

antimetabolites, were subjected to the heating in order to evaluate their decomposition

mechanism. The pyrolysis and oxidative decomposition course was studied with a use of

simultaneous thermal analysis: thermogravimetry/differential scanning calorimetry

(TG/DTG/DSC) coupled online with Fourier transform infrared spectroscopy (FTIR) and

1
Quadrupole mass spectrometry (QMS). The performed analyses allowed to conclude that, the

pyrolysis and oxidation processes of the tested compounds run through simultaneous, radical

processes in one, main stage (inert conditions) or in two stages (oxidative conditions)

resulting in the emission of NH3, RNCO, HCN, CO, CO2, NO2, aromatics and alkenes (inert

atmosphere) and NH3, RNCO, HCN, CO, CO2, NO2, NO, N2O, H2O, aromatics and alkenes

(oxidative atmosphere). The results confirmed the simultaneous breaking of C-C, C-N, N-H,

C-H and C-Cl bonds in the starting compounds and the created intermediates, and then

additional oxidation processes of some, formed products to the corresponding nitrogen oxides

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and water. The connection between the thermal behaviour of the tested compounds and their

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possible use as candidates for pharmaceutical applications was emphasized.

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Keywords: fused azaisocytosine-containing congeners, thermal studies, decomposition course
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1. Introduction
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3-(4-Nitrophenyl)-8-(R-phenyl)-7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones

(1-9) (Fig. 1) are a recently identified novel class of fused azaisocytosine-containing

congeners, acting as anticancer antimetabolites, whose molecular structures in solution are

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completely defined. These small molecular-mass compounds possess the medical utility as

their remarkable cytotoxic activities are of benefit in the treatment of human solid tumours of
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lung, cervix, breast and ovary. All the title molecules reveal stronger in vitro inhibitory
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effects on the growth of lung, cervical and breast tumour cells than that of pemetrexed (a

clinically useful anticancer antimetabolite). In addition, the significant QSAR equations

describing the important pharmacokinetic properties of the title molecules in terms of

lipophilicity values are disclosed. In this class of anticancer agents, some derivatives

containing the 4-methyl, 2,3-dimethyl, 2-methoxy, 2-chloro and 3,4-dichloro group as R at

2
the phenyl moiety (i.e. compounds 3, 4, 5, 6 and 9, respectively) (Fig. 1) are the most

promising drug candidates for pharmaceutical use because of the confirmed less toxic effects

on normal cells [1].

Despite the current state of knowledge about the structure, anticancer activities

(including selectivity data), physico-chemical properties (such as molecular masses, retention

times and logarithmic retention factors determined on some reversed phase columns imitating

biosystems) and molecular descriptors related to important pharmacokinetic properties of the

title compounds (1-9) [1], their thermal decomposition mechanism as well as thermal

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behaviour in inert and oxidative conditions still remains unknown.

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However, the stability during heating in inert or oxidative conditions, thermal

properties and decomposition products have already been investigated in three

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pharmacologically important classes of small molecules bearing the privileged scaffold of

7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-one [2-4]. In addition, the most likely

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mechanisms of thermal decomposition, based on the experimental results, have been
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previously suggested in two classes of these possible pharmaceutics [2,4]. None of these

specific molecules contained the nitrophenyl moiety in their structures.

These results prompted us to extend our investigations on the thermal stability and
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decomposition mechanism to other novel class of 3-(4-nitrophenyl)-8-(R-phenyl)-7,8-

dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones (1-9) with anticancer activity. These

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investigations are necessary for the thermal characterization of these conjugated small
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molecules bearing the para-nitrophenyl moiety, and the determined thermostability data will

be an additional important parameter taken into account when assessing the usefulness of

these potential drugs.

The literature survey revealed that there are research papers describing factors

affecting the chemical and physical stability, decomposition products, thermal degradation

3
kinetics and mechanism of thermal decomposition of nitrophenyl-containing drugs [5-15] –

that are commonly used in human and veterinary medicine as: i) antiparkinsonian agents (e.g.

entacapone [5,6]), ii) angiotensin-converting enzyme inhibitors (e.g. delapril [7]), iii) calcium

channel blockers (e.g. manidipine [7], nisoldipine, nitrendipine, nicardipine, felodipine,

amlodipine [8], nifedipine [8,9,10], nimodipine [8,11]), iv) antihelminthic agents (e.g.

niclosamide [12,13]), v) skeletal muscle relaxants (e.g. dantrolene [14]) or vi) anticoccidial

agents (e.g. nicarbazin [15]) – as well as biologically active molecules [16,17], such as

antibacterially effective 2-(3- or 4-nitrophenyl)-5-(thiophen-2-yl)-1,3,4-oxadiazoles or an

of
antimicrobially active 3,6-bis(4-nitrophenyl)bis([1,2,4]triazolo)[3,4-a:4’,3’-c]phthalazine.

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These reports emphasize the importance of thermal analysis techniques in thermal

characteristics, purity assessment, identification of polymorphism in the molecules of these

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drugs and drug-like compounds, and show that they undergo various degradation pathways

due to their significantly diverse molecular structures. Furthermore, there are experimental
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and in silico studies devoted to thermal stability and decomposition of various organic
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compounds (including energetic materials) bearing the nitrophenyl group or groups [18-23].

The present investigation is aimed at assessing, for the first time, the range of thermal

stability of all novel compounds (1-9) under pyrolysis in inert or oxidative conditions, using
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the reliable TG/DSC/FTIR/QMS method. In addition, the study is aimed at explaining not

only the thermal degradation mechanism of pharmaceutically important molecules but also
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identifying the decomposition reactions observed during all stages of chemical degradation
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and volatile decomposition products under pyrolysis in helium or synthetic air atmosphere. It

should be noted that performing the experimental thermal analysis of this class of compounds

was a big challenge since all small molecules investigated may be also regarded as

nitrobenzene derivatives substituted in the para position by heterobicyclic scaffold. It has

4
been reported earlier that a number of nitrobenzene derivatives with high decomposition

enthalpy, as energetic materials, can cause difficulties when studied thermally [18,19,22].

2. Experimental

2.1. Short characteristic of the studied small molecules (1-9)

All anticancer active annelated triazinones bearing the para-nitrophenyl group (1-9)

have been resynthesized for the purposes of thermal studies following a general annelation

approach described in an earlier paper [1]. This synthetic procedure and medical application

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of molecules has been previously patented too. The structures of all crystalline compounds

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intended for thermal studies (1-9) have been characterized by consistent proton nuclear

magnetic resonance (1H NMR), carbon-13 nuclear magnetic resonance (13C NMR) spectra
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and elemental analyses (being within ±0.4 percent of the theoretical values). Moreover, all

compounds subjected to thermal investigations have been previously characterized in

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reversed-phase high performance liquid chromatography (RP-HPLC) measurements by their
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retention times and peak asymmetry values in acceptable ranges. All the above-mentioned

experimental data have been published in an earlier paper directed on the design, synthesis,

structural, pharmacological, physico-chemical and pharmacokinetic characterization of the

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targeted molecules [1]. All the investigated compounds (1-9) have been shown to possess

enhanced growth inhibitory effects in human solid tumour cells of lung (A549), cervix
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(HeLa), breast (T47D) and ovary (TOV112D). In addition, their less toxic effects on African
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Green Monkey kidney cells (i.e. non-tumoural epithelial cells) have been already reported in

a previous paper [1].

2.2. Methods and physical measurements

5
 The thermal behaviour of compounds investigated was determined using a

simultaneous TG/DSC/FTIR/QMS method on a STA 449 Jupiter F1 instrument produced by

Netzsch (Germany). The temperature and sensitivity calibrations of apparatus were done with

the following metals In, Sn, Bi, Zn, Al, Au and checked with calcium oxalate monohydrate

according to the manufacturer proceedings. The samples with a mass ca. 10 mg were heated

in open ceramic crucibles between 40°C and 750°C (synthetic air atmosphere, a flow rate 100

mL/min) or 40°C and 850°C (helium atmosphere, a flow rate 40 mL/min) and with a heating

rate 10 K/min. All the samples were heated three times. In order to evaluate the

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decomposition mechanism of the studied compounds, simultaneously with TG/DSC analyses,

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the FTIR and QMS spectra were recorded. TG/DSC instrument was coupled on-line with a

FTIR TGA 585 analyzer (Bruker, Germany) and a QMS 403 C Aëolos (Netzsch, Germany)
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analyzer. The FTIR spectrometer was connected on-line to a STA instrument by Teflon

transfer line with a diameter of 2 mm. The temperatures in the gas cell and transfer line were
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maintained at 200°C to reduce the possibility of condensation of evolved gases along the IR
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cell and the transfer line. The FTIR spectra were collected within the range of 600-4000 cm-1

with 16 scans per spectrum and with a resolution 4 cm-1. The QMS analyzer was connected

on-line to a STA instrument by quartz capillary which was heated to 300°C. The QMS
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analyzer was operated under electron ionization 70eV. The QMS spectra were gathered in the

range 10-300 m/z.

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3. Results and discussion

3.1. Melting process of the studied small molecules (1-9)

The melting process of the tested compounds was studied by the use of a DSC method. The

appropriate DSC results, such as the melting point onset temperature (Tonset), melting peak

temperature (Tpeak) and the estimated values of melting enthalpy (ΔH) received in inert

6
conditions (helium atmosphere) were presented in Table 1. The DSC curves were shown as

Fig. S1 in Supplementary material. According to the DSC data, the first observed process

during heating described by the endothermic peak is the melting process. The melting process

of all the tested anticancer active compounds starts at Tonset1. In addition, their solid-liquid

(melting) phase transition is described by one, sharp DSC peak which testifies to their high

purity [3]. The melting temperatures are completely dependent on the test compounds

structure (the substituent at the benzene ring in congeners). As can be seen, the compound 1

(i.e. the parent structure) that does not contain a substituent attached to the phenyl moiety –

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exhibits the melting temperature designated as Tpeak1 284ºC. Replacing the hydrogen atom at

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the benzene ring by one or two methyl groups or by one methoxy substituent or by one

chlorine atom in the ortho and meta position causes a decrease in the melting temperature.
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The melting temperature changes as follows: 226ºC (the ortho-CH3 derivative), 254ºC (the

meta-Cl derivative), 260ºC (the ortho-Cl derivative), 265ºC (the dimethyl derivative
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substituted at ortho and meta positions), 274ºC (the para-CH3 derivative) and 279ºC (the
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ortho-OCH3 derivative). On the other hand, the incorporation of the chloro group in the para

position (compound 8) or two chlorine atoms in meta and para-Cl positions (compound 9)

results in an increase of the melting temperature up to 294ºC and 325ºC, respectively as

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compared to the melting temperature of the parent compound (1). However, when we

compare, the melting points of small molecules containing the same type of substituent
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(methyl derivatives 2-4 and chloro derivatives 6-8), we can find the following correlation: the
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substitution of hydrogen at the phenyl moiety in the para position with methyl or chloro

group leads to an increase in the melting point as compared to that of ortho and meta

substituted ones.

Table 1

DSC data for compounds 1-9 (helium atmosphere)

Sample Melting process

7
 Tonset1/°C Tpeak1/°C ΔH1/kJ/mol
1 282 284 110
2 223 226 63.2
3 271 274 114.3
4 262 265 78.0
5 275 279 127.1
6 257 260 92.8
7 252 254 94.0
8 294 294 113.5
9 322 325 104.7

3.2. Decomposition course of the studied small molecules (1-9) in helium

According to the presented TG-DTG data (Table 2, Fig. 1), the studied compounds

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decompose in one, main decomposition stage in helium atmosphere. This stage, composed of

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at least two steps, is visible from Tonset1 up to Tfinal1. In addition, above the Tfinal1 temperature,

a slow, mass loss without the marked DTG peak is observed. It is worth noting that all the
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tested compounds exhibit high thermal stability. Their decomposition starts at 319-360ºC
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(Tonset1), which seems very important property for their possible application as drugs. It has

been confirmed that in case of pharmaceutics with high thermal stability at temperature much
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higher than ambient conditions, changes in the storage temperature have a negligible impact

on their shelf life [24,25]. The thermal stability marked as onset temperature (Tonset1) of the
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tested compounds is falling from the value 352ºC (the parent compound 1) up to 319ºC (the

compound 5) and then it is growing again for compounds containing chlorine atoms in their
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structures (compounds 6-9). The lowest thermal stability is observed for the compound 5

containing the methoxy group (as a substituent attached to the phenyl moiety. On the other
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hand, all the compounds bearing one or two chlorine atoms at the phenyl moiety (compounds

6-9) are characterized by the highest thermal stability. These compounds are thermally stable

up to the temperature ca. 356-360ºC (Tonset1).

As can be seen from the presented results (Table 2, Fig. 1), the first decomposition stage for

all the tested compounds is visible as a huge DTG signal (at ca. 339-380ºC) with a small tail

8
or tails at ca. 400-446ºC, except for compounds 3 and 5, where two not-well subdivided DTG

signals at 366ºC and 416ºC (compound 3) and at 339ºC and 435ºC (compound 5) are

observed. For all the studied compounds, the major mass loss (53-68.0%) occurs at this

temperature range. Interestingly, on DSC curves only exothermic signals in the temperature

range from Tonset1 to Tfinal1 are observed. This may testify to some chemical reactions between

the decomposition products leading to the formation of not only the pyrolysis products but

also the other gaseous products which are the result of the additional reactions. Further

heating of the compounds from Tfinal1 up to 800ºC causes very slow mass loss, without any

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DTG peak. This mass loss is due to the pyrolysis of remaining parts of the tested compounds.

The mass loss (Δm2) is from 4.2% to 11.5%. The recorded residual masses are from 26.8% to

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40.3% at 800ºC. It proves the formation of some char residues during the heating of the

compounds in inert conditions.

Table 2
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TG data of compounds 1-9 (helium atmosphere)
Sample Degradation process
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First decomposition stage Second decomposition

stage
Tonset1 Tmax1/Tmax1a/Tmax1a’/°C Tfinal1 Δm1 Tonset2 Δm2 rm
/°C /°C /% /°C /% /%
1 352 369/415/446 523 63.3 523 9.8 26.9
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2 348 369/400 580 68.0 580 5.2 26.8

3 346 366/416 640 29.5/25.0 640 5.2 40.3
4 341 362/401 577 60.6 577 4.2 35.2
5 319 339/435 500 33.1/19.9 500 9.1 37.9
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6 360 380/401 580 66.6 580 6.0 27.4

7 358 376/408 582 63.7 582 6.8 29.5
8 356 376/410/427 573 61.0 573 10.6 28.4
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9 360 379/408 575 58.7 575 11.5 29.8

Tmax1a – the maximum temperature of a tail/small peaks, rm – residual mass at 800°C

According to the gaseous FTIR spectra recorded together with TG/DTG/DSC curves at

Tmax1/Tmax1a, the emission of the gaseous products associated with degradation of the central

core system is well seen. For all the studied compounds the emission of NH3 (two

9
characteristic signals at 931 cm-1 and 966 cm-1 coming from the deformation vibrations of N-

H groups and at ca. 3350 cm-1 derived from the stretching vibrations of N-H groups)

[3,26,27], CO2 (peaks at 2300-2365 cm-1 (valence vibration) and 669 cm-1 (deformation

vibration)), CO (2050-2270 cm-1) and H2O (several, jagged, weak signals at the range 3450-

4000 cm-1 and 1300-1950 cm-1) is observed, Fig. 2. The emission of above-mentioned

gaseous molecules is also confirmed based on the QMS analysis. The most characteristic m/z

ions for NH3 (17,16,15,14), CO2 (44), CO (28) and H2O (18,17,16) are observed on the

presented curves, Fig. 3. One of the decomposition products is nitrobenzene which is

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confirmed based on the presence of m/z ions 123, 77, 51 on the QMS spectra, Fig. 3.

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According to the literature survey, the pyrolysis of nitrobenzene includes a lot of radical

processes, such as isomerization of the nitro group into nitrite group, pyrolysis of N-O in the
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nitrite group, pyrolysis of C-N bonds, etc. and the formation of the following products: NO,

NO2, benzene, aniline, nitrosobenzene, biphenyl or dibenzofuran [28]. Based on the

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performed FTIR and QMS analyses, we have found that the additional emission of benzene
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(m/z 78) at higher temperature than nitrobenzene and NO2 (m/z 30) proceeds as a result of the

cleavage of C-N bonds in nitrobenzene. It is also verified by the low intensity bands at 2850-

2930 cm-1 and 1570-1630 cm-1, Fig. 2, Scheme 1. It is worth noting that at Tmax1a, the creation
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of a new gaseous decomposition product HNCO and its methyl and ethyl derivatives (RNCO)

is confirmed based on the absorption bands with the maximum centred at 2270 cm-1 and 2290
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cm-1, Fig. 2, as well as the presence of the following m/z ions: 71, 57, 56, 43 on the QMS
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spectra, Fig. 3 [4,29,30]. In addition, at Tmax1/Tmax1a, the attendance of the band at 713 cm-1

may indicate the delivery of HCN molecule. However, this band is also characteristic for CO2

(699 cm-1) which emission at Tmax1 is high. Therefore, the additionally performed QMS

analysis allows us to verify the type of this gaseous decomposition product. Analysing the

QMS results, one can conclude the presence of the following m/z ions 27, 26 which are

10
characteristic for the defragmentation of HCN molecule. This emission for the compound 1

(the parent structure) is registered up to ca. 800ºC, meanwhile for other derivatives up to ca.

630-640ºC. These results are also confirmed based on the FTIR analysis. The wide range of

HCN emission will suggest uneven breaking of C-N bonds in the tested and formed

intermediate compounds. In addition, it is well seen, that at Tmax1a the emission of CO2 stops.

Due to this, on the FTIR spectrum at Tmax1a, the absorption band characteristic for the HCN

(713 cm-1) is well seen.

Besides the above-mentioned gaseous decomposition products, the emission of some

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aromatic fragments could be seen. Among them the following main aromatic products

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created during pyrolysis of N-C bonds such as aniline (in case of compound 1), (Fig. S2a), o-

toluidine (in case of compound 2), (Fig. S2b), p-toluidine (in case of compound 3), (Fig.
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S2c), 2,3-xylidine (in case of compound 4), o-anisidine (in case of compound 5), (Fig. S2d)

and o-, m-, p-chloroanilines (in case of compounds 6-9) (Fig. S2e, S2f) are formed. Beyond
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these aromatic products, the emission of fused aromatic rings as a result of radical reactions
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between the intermediates and/or minor amounts of other aromatics with a lower molecular

mass is very likely [31, 32]. The emission of some aromatics from the studied compounds (1-

9) in the range Tonset1-Tmax1/Tmax1a is confirmed by the presence of the characteristic

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absorption bands at 3030-3097 cm-1 (the stretching vibrations of C-H), 1619-1638 cm-1 (the

deformation vibrations of N-H), 1500-1587 cm-1 (the stretching vibrations of CAr=CAr), 1254-
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1274 cm-1 (the stretching vibrations of C-N), and at 640-900 cm-1 (out-of-plane deformation
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vibrations of N-H and CAr-H) [33].

Moreover, from the temperature ca. 390ºC the creation of small amount of HCl molecule, due

to the pyrolysis and radical reactions of compound 9, is confirmed by the occurrence of the

absorption bands at 2640-3100 cm-1 [27] on the FTIR spectrum, Fig. 2 and the following m/z

ions 38, 36 from the QMS spectra, Fig. 3.

11
However, in the case of chloro-substituted derivatives (compounds 6-9), the most realistic

aromatic products are chloroanilines. When one looks at the FTIR spectrum, the absorption

bands at 1620-1645 cm-1 (the deformation vibrations of N-H), 1484-1580 cm-1 (the stretching

vibrations of CAr=CAr), 1255-1278cm-1 (the stretching vibrations of CN), 1000-1065 cm-1 (in-

plane deformation vibrations of aromatic =C-H), 885-770 cm-1 (the out-of-plane deformation

vibrations of CArH) and 669 cm-1 (the stretching vibrations of CCl) are clearly observed.

These results are confirmed by the presence of m/z ions 129, 127 and 65 on the QMS spectra,

Fig. 3. The proposed pyrolysis mechanism of compounds 1-9 is presented in Scheme 1.

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The radical pyrolysis process of the tested compounds is also supported by the presence of an

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exothermic DSC signal which directly indicates the chemical reactions between the

decomposition products.
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When we look at the gaseous FTIR spectrum for compounds 1-9 gathered at the temperatures

higher than Tfinal1, we can see the continuous emission of low molecular mass molecules inter
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alia NH3, HCN, CO, CO2. This testifies further pyrolysis processes connected with the
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decomposition of the formed residues at the first decomposition stage, Figs. 2 and 3.

R
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N N
N
N NO 2
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Fig. 1. Structures of the investigated compounds: 1. R = H, 2. R = 2-CH3, 3. R = 4-CH3, 4. R

= 2,3-(CH3)2, 5. R = 2-OCH3, 6. R = 2-Cl, 7. R = 3-Cl, 8. R = 4-Cl, 9. R = 3,4-Cl2
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12
 R

N N
N
N NO2

R
N N
+ N + NO2
R + N N
N N
N
O
O

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HNCO + HCN + NCO + CO + CO2 + R + NH2 + NO2 + HCl* + H2O

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NH2
+ NH3
+ NO2

* for compound 9
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+

Scheme 1. The pyrolysis path of the tested compounds.

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na
ur
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13
 compound 2
(5)
compound 1 (7)
(1) (1)
(7)
(1) 700˚C
(1)

Absorbance/a.u.
665˚C (5) (6)
Absorbance/a.u.

(2) (2)(4)
(4) (8)
(1) H2O (6)
(6) aromatics
(2) CO2 (7) HCN (8)
(3) CO (8) alkenes
(4) RNCO (9) NO2 Tmax1a
(5) NH3 (9)
Tmax1a
(6)
(6)

Tmax1
(6)(8)
(6)(8) (3)
Tmax1 (3)

3600 3100 2600 2100 1600 1100 600 3600 3100 2600 2100 1600 1100 600
-1 -1
Wavenumber/cm Wavenumber/cm

of
compound 4
(7)
compound 5

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(1) (1) (8)
Absorbance/a.u.

650˚C
Absorbance/a.u

(2) (5)
(5)
(6)

Tmax1a
(4)
(6)

700˚C
(1)
-p (1)

(6)
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(6)(8)(9) (4)
Tmax1a
(6) (8)
Tmax1

(2)
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Tmax1 (6)(8) (9)

(3) (3)

3600 3100 2600 2100 -1 1600 1100 600 3600 3100 2600 2100 1600 1100 600
Wavenumber/cm Wavenumber/cm-1
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(7)
compound 6 compound 9
(1)
(1)
(1) (1)
680˚C
700˚C
(2)
Absorbance/a.u.

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Absorbance/a.u.

(5) (2)
(6) (4)
(6)
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(8) (6)
Tmax1a (7)
(8)
Tmax1a (6)(8) (3) (5)
(4) (6)
HCl
from ca. 390˚C

Tmax1
Tmax1 (3)

3600 3100 2600 2100 1600 1100 600 3600 3100 2600 2100 1600 1100 600
-1 -1
Wavenumber/cm Wavenumber/cm

Fig. 2. The gaseous FTIR spectra for the selected compounds in helium.
14
 1,5E-11
NH3 26 HCN
6,00E-11 16 H2O (compound 2)
17 1,2E-11 27
(compound 2)
18

QMID/A
15 9E-12
QMID/A

4,00E-11
14
6E-12
2,00E-11
3E-12

0,00E+00 0
40 240 440 640 840 40 240 440 640 840
Temperature/˚C Temperature/˚C

4,8E-11 5E-10
44 CO2 CO
4E-11 28
(compound 2) (compound 2)
4,5E-10
3,2E-11

QMID/A
QMID/A

2,4E-11 4E-10

1,6E-11
3,5E-10
8E-12

of
3E-10
0
40 240 440 640 840
40 240 440 640 840
Temperature/˚C
Temperature/ C

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3E-12 RNCO
30 NOx (compound 2)
2,4E-12 (compound 2) 1,8E-12 57
56
-p
QMID/A

QMID/A

1,8E-12 43
1,2E-12
71
1,2E-12
6E-13
6E-13
re
0
0
40 240 440 640 840 40 240 440 640 840
Temperature/˚C
Temperature/ºC
lP

1,2E-12
3E-13
65 aniline benzene
(compound 1) (compound 2)
9E-13 66
78
92
2E-13
QMID/A

QMID/A

93
na

6E-13

3E-13 1E-13

0
ur

40 240 440 640 840 0

Temperature/˚C 40 240 440 640 840
Temperature/˚C

toluidine
Jo

4E-13
nitrobenzene 106 (compound 2)
123 (compound 2) 107
51
QMID/A

77
QMID/A

4E-13 2E-13

0 0
40 240 440 640 840 40 240 440 640 840
Temperature/ºC Temperature/ºC

15
 o-anisidine
3E-13 127 chloroaniline 4E-13 80 (compound 5)
129 (compound 6)
123
2E-13 65 108

QMID/A
QMID/A

2E-13
1E-13

0 0
40 240 440 640 840 40 240 440 640 840
Temperature/ºC Temperature/ºC

HCl
36 (compound 9)
3E-11
38
QMID/A

2E-11

1E-11

of
40 240 440 640 840
Temperature/ºC

Fig. 3. The selected QMS spectra of the decomposition products gathered in helium.

ro
3.3. Decomposition course of the studied small molecules (1-9) in air

The decomposition course of the tested compounds under oxidative conditions occurs in two
-p
re
main stages (Table 3, Fig. 4). As with pyrolysis process, the oxidative degradation runs in at

least two steps visible as Tmax1 and Tmax1a. Similarly to pyrolysis, the oxidative stability of the
lP

tested compounds is significant. The beginning of their decomposition is visible from the

temperatures exceeding 310ºC (Tonset1). Comparing these results with those obtained in inert
na

atmosphere, it can be seen that thermal stabilities of the tested compounds in both

atmospheres are similar. In addition, the thermal stability of the compounds decreases from
ur

344ºC (compound 1) to 312ºC (compound 5) and then it grows up to 348-354ºC for

compounds containing the chloro substituent. This behaviour is exactly the same as that for
Jo

the compounds in helium atmosphere. Moreover, the mass loss marked at the first

decomposition stage in air atmosphere is lower (from ca. 38.6% to ca. 59%) than the mass

loss determined in helium atmosphere (from ca. 53 % to ca. 68%). Looking down on the

gaseous FTIR spectra gathered at Tmax1/Tmax1a, one can notice the emission of the same

gaseous products to that seen previously in analyses done in helium atmosphere.

16
The emission of NH3 (931 cm-1 and 966 cm-1), CO2 (699 cm-1 and 2300-2365 cm-1), CO

(2050-2270 cm-1) and HCN (713 cm-1) is clearly confirmed based on the FTIR spectra

gathered at Tmax1 and Tmax1a. Interestingly, the intensity of the absorption bands characteristic

for NH3 and HCN is significantly lower in oxidative conditions as compared to their emission

in inert conditions. These results may indicate the partial oxidation of above-specified

decomposition products in the gaseous state. According to the literature survey, NH3 and

HCN can undergo oxidation processes to the corresponding nitrogen oxides such as NO,

N2O, NO2 under appropriate conditions [34-36]. This supposition is confirmed by the

of
presence of low intensity absorption bands for nitrogen oxides at 1620-1650 cm-1 (NO2),

1850-1920 cm-1 (NO) and 2287 cm-1 (N2O) on the gaseous FTIR spectra, Fig. 5 and the

ro
attendance of the m/z 30 ion characteristic for nitrogen oxides on the QMS spectra, Fig. 6. In
-p
addition, as a result of the partial oxidation processes of NH3 and HCN, the emission of water

vapour can be observed. Analysing the results from the FTIR and QMS measurements, one
re
can notice the presence of the water bands (3450-4000 cm-1 and 1300-1950 cm-1) on the
lP

FTIR, Fig. 5 and the m/z ions characteristic for H2O (18,17,16), Fig. 6. In addition to the

emission of listed gaseous decomposition products, the occurrence of the absorption bands

characteristic for some aromatic compounds can be seen at 640-900 cm-1 (the out-of-plane
na

deformation vibrations of N-H and CAr-H), 1307-1351 cm-1 (the stretching vibrations of C-N),

1511-1600 cm-1 (the stretching vibrations of CAr=CAr), 1615-1635 cm-1 (the deformation
ur

vibrations of N-H) and 3030-3085 cm-1 (the stretching vibrations of C-H). These results are
Jo

confirmed by the QMS analysis. The following, selected m/z ions: 92, 78, 66, 65, 63, 51, 50

characteristic for aromatics are observed [33]. Moreover, at Tmax1a, the FTIR bands for

HNCO and RNCO at 2270-2290 cm-1 and the QMS m/z ions: 71, 57, 56, 43 and 42 are

indicated. Comparing the intensity of the HNCO/RNCO bands in both inert and oxidative

atmospheres, one can see its lower intensity when the analysis is done in air. This can prove

17
the oxidation processes of these gaseous molecules to the corresponding nitrogen oxides in

dependence on the oxygen accessibility [37,38]. In case of the compound 9, between the

temperatures 390-610ºC, the additional emission of HCl is clearly visible. This is confirmed

based on the FTIR bands at the range 2640-3100 cm-1 and the presence of the following m/z

ions: 38, 37 and 36, 35 on the QMS spectra, Figs. 5 and 6.

Under the second decomposition stage, mainly the emission of H2O, CO2, CO, RNCO and

nitrogen oxides is observed. This suggests the subsequent, simultaneous oxidation reactions

of the formed residues. The proposed oxidation path of the tested compounds is presented in

of
Scheme 2.

ro
Table 3
TG data of compounds 1-9 (air atmosphere)
Sample Degradation process

Tonset1
First decomposition stage

Tmax1/ Tmax1a Tfinal1 Δm1

-p
Tonset2
Second decomposition stage

Tmax2 Tfinal2 Δm2

re
/°C /°C /°C /% /°C /°C /°C /%
1 344 364/395 480 56.2 480 579 700 43.8
2 341 358/385 510 56.9 510 595 680 43.1
lP

3 338 354/412 484 38.6 484 592 690 61.4

4 333 350/374 491 47.8 491 612 690 52.2
5 312 329/431 462 44.6 476 572 697 55.4
6 354 371/391 477 58.7 477 591 680 41.3
7 349 369/403 470 53.9 470 577 695 46.1
na

8 348 363/398 475 53.4 475 595 695 46.6

9 354 369/392 468 51.3 468 579 690 48.7
ur
Jo

18
 R

N N
N
N NO2

N N
+ N + R + NO2
R
N
N N
O N
N

+O2 O

of
NCO + NH3 + NO2 + N2O + NO + H2O + HCl* + CO + CO2 + + R
HNCO + HCN +

NO2 NO2

ro
* for compound 9

Scheme 2. The oxidative decomposition path of the tested compounds. -p

re
100

85
compound 1
70 compound 2
Mass/%

compound 3
55
compound 4
lP

40 compound 5
compound 6
25 compound 7

10 compound 8
compound 9
-5
40 140 240 340 440 540 640 740
Temperature/°C
na

-2 compound 1
compound 2
-4
compound 3
DTG/%/min

-6 compound 4
ur

compound 5
-8
compound 6
-10 compound 7
compound 8
-12
compound 9
-14
Jo

40 140 240 340 440 540 640 740

Temperature/°C

Fig. 4. TG-DTG curves for the tested compounds in air.

19
 (2) (2)
compound 3 compound 4

(1) H2O
(2) CO2

Absorbance/a.u.
(3) CO
(4) RNCO
Absorbance/a.u.

(5) NH3
(6) aromatics
(1)
(7) HCN
Tmax2 (8) alkenes (1)(6)
(9) nitrogen (1)
oxides (3) Tmax2 (1)
(3)

Tmax1a (5)
(4)
(7) (4)
Tmax1a

of
(5) (7)
(6)
Tmax1
(6)(8)(9) (8)
(6) (6)
Tmax1

ro
3600 3100 2600 2100 1600 1100 600 3600 3100 2600 2100 -1 1600 1100 600
-1
Wavenumber/cm Wavenumber/cm

(2) (2)

compound 5 -p compound 7

(7)
re
Absorbance/a.u.

Absorbance/a.u.
lP

(1)
(1)
Tmax2 (3)
(5) (1)
(4)
Tmax2 (3)
Tmax1a
(4)
na

(6) (6) (7)

Tmax1a (5)
Tmax1 (8) (6)
(6)(8) (9) (6) (8)
Tmax1

3600 3100 2600 2100 -1 1600 1100 600 3600 3100 2600 2100 1600 1100 600
ur

-1
Wavenumber/cm Wavenumber/cm

Fig. 5. The gaseous FTIR spectra for the selected compounds in air.
Jo

20
 3,3E-08
8E-11 CO
17 H2O
16 NH3
18 28
2,9E-08
QMID/A

QMID/A
15
4E-11 14

2,5E-08

0
40 140 240 340 440 540 640 2,1E-08
Temperature/ºC 40 240 440 640
Temperature/ C

HCN
9E-11 6E-11
26
27

QMID/A
44
QMID/A

6E-11
3E-11

of
3E-11

0
0
40 140 240 340 440 540 640
40 240 440 640

ro
Temperature/ºC
Temperature/ C
4,5E-13
3E-11 nitrogen 50 aromatics
30 oxides
51
3,6E-13
63
2E-11 -p
QMID/A

65
QMID/A

2,7E-13
66
1,8E-13 78
1E-11
92
re
9E-14
0
40 140 240 340 440 540 640 0
Temperature/ºC 40 240 440 640
Temperature/˚C
lP

RNCO
56 3,6E-11
HCl
1,2E-12 43
42 35
2,7E-11
36
QMID/A

71
QMID/A

8E-13
na

57 37
1,8E-11 38

4E-13
9E-12

0
ur

0
40 140 240 340 440 540 640
Temperature/ºC 40 240 440 640
Temperature/˚C
Jo

Fig. 6. The QMS spectra of the decomposition products gathered in air.

21
4. Conclusions
The pyrolysis and oxidative degradation mechanism of nine, novel fused azaisocytosine-

containing congeners bearing the para-nitrophenyl moiety and different substituents, such as

phenyl, 2-methylphenyl, 4-methylphenyl, 2,3-dimethylphenyl, 2-methoxyphenyl, 2-

chlorophenyl, 3-chlorophenyl, 4-chlorophenyl and 3,4-dichlorophenyl were determined based

on the results obtained from TG/DTG/DSC/FTIR/QMS analyses. It was found that the

pyrolysis process of these compounds started from the temperatures 319-360ºC and happened

in two stages. The first, main stage was visible between the temperatures 319-360ºC and 500-

of
640ºC, however the second one above the temperatures 500-640ºC. This was connected with

the emission of a mixture of different volatile products. The type of formed gases allowed to

ro
conclude that the main degradation processes include the homolytic cleavage of C-C, C-N,

-p
N-H, C-H and C-Cl, formation of intermediate products, their further fragmentation,

pyrolysis and partial decarboxylation. The oxidative degradation occurred in two main stages
re
visible between the temperatures 312-354ºC and 462-510ºC (the first stage) and 462-510ºC

and 680-700ºC (the second stage). The performed analyses showed that the tested compounds
lP

are degraded according to the same radical mechanism as in inert conditions. However, due

to the oxygen access, the partial oxidation processes of some gaseous products (HNCO,
na

RNCO, HCN, NH3, amines and its derivatives) to the following final products: NO2, NO,

N2O, H2O, nitrobenzene and its derivatives were observed. The high thermal stability that
ur

was confirmed in our studies, is an additional beneficial property allowing the future
Jo

pharmaceutical application of the investigated anticancer drug candidates.

Author statement
Marta Worzakowska: Conceptualization, Methodology, Investigation, Writing - Original
Draft, Writing- Reviewing and Editing

22
Krzysztof Sztanke: Conceptualization, Methodology, Resources, Writing - Original Draft,
Writing- Reviewing and Editing

Małgorzata Sztanke: Conceptualization, Methodology, Resources, Writing - Original Draft,

Writing- Reviewing and Editing

Conflict of Interest

All the authors have reviewed and approved the original manuscript. No conflicts of interest
exist.

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