2,2′-(Arylmethylene)bis(3-hydroxy-5,5-dimethylcyclohex-2-enone) crystals formation via atom economy reaction and their antioxidant activity

Article abstract of DOI:10.1007/s00706-021-02767-x

Tetraketones with their diversified biological potencies became a highly significant class of oxygen-containing organic compounds. These are prepared by applying a simple Knoevenagel-Michael cascade procedure with 1,3-dicarbonyl compound and aldehydes. In

Full text of DOI:10.1007/s00706-021-02767-x

Monatshefte für Chemie - Chemical Monthly (2021) 152:537–543
https://doi.org/10.1007/s00706-021-02767-x
ORIGINAL PAPER
2,2′‑(Arylmethylene)bis(3‑hydroxy‑5,5‑dimethylcyclohex‑2‑enone)
crystals formation via atom economy reaction and their antioxidant
activity
Deepa Thakur1 · Manvinder Kaur1 · Dharambeer Singh Malhi1 · Sonali Garg1 · Ajay Sharma1 ·
Harvinder Singh Sohal1
Received: 4 March 2021 / Accepted: 5 April 2021 / Published online: 27 April 2021
© Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
Tetraketones with their diversifed biological potencies became a highly signifcant class of oxygen-containing organic com-
pounds. These are prepared by applying a simple Knoevenagel-Michael cascade procedure with 1,3-dicarbonyl compound
and aldehydes. In the due course of time, numerous methods for the synthesis of these compounds, have been developed
which having their own advantages and disadvantages. So, the development of an efcient, simple, and ecologically benign
method for their preparation in the presence of the novel catalytic agent is still in great demand. In the present report, direct
crystals of 2,2′-(arylmethylene)bis(3-hydroxy-5,5-dimethyl-2-cyclohexene-1-one) (a tetraketone) were obtained via a simple
procedure using 2-aminopyrazine as a catalyst. The prepared compound shows signifcant antioxidant checked by diferent
procedures like DPPH, ABTS, and TAC.
Graphic abstract
R
CHO
O
O
H
3C
CH3
CH3
O
O
H
3C
N
N
NH2
12 potent examples all with
crystal forma on
Keywords Lewis base · Multi-component reaction · Dimedone · Anti-oxidant activity · Single crystal XRD · 2D NMR
Introduction
condensation of three or more than three components takes
place without the introduction of the poisonous intermedi-
ate in the atmosphere such that the product contains a nota-
ble portion of atoms got from the entirety of the beginning
material [8]. Strecker frstly reported primary MCR, i.e.,
synthesis of α-aminocyanides from carbonyl compounds,
ammonia, and hydrogen cyanide [9]. Nowadays, combina-
torial synthesis [10] and diversity-oriented synthesis [11]
are preferred via MCRs because they reduce the number
of reaction steps, waste creation, human labor, the expense
of building highly diverse and complex molecules. Due
to their experimental simplicity, they have the chance of
automatization [12] and allow experimental variations.
Multi-component reactions (MCRs), which are also known
as the Multi-component Assembly Process (MCAP) [1],
are progressively becoming one of the frontiers of organic
synthesis [2–4], as they address the complexity and diver-
sity in organic transformations [5, 6]. By varying reagents,
molecular complexity, diversity, as well as predefned func-
tionality, can be easily attained in MCRs [7]. In MCRs, the
* Harvinder Singh Sohal
drharvinder.cu@gmail.com
1
Department of Chemistry, Chandigarh University, Gharuan,
140413 Mohali, Punjab, India
Vol.:(0123456789)
1 3

538
D. Thakur et al.
Multi-component reactions have been broadly applied to the
preparation of bioactive [13] and complex molecules [14].
MCRs play a signifcant function in diferent examination
felds like biomedical, synthetic organic, industrial chemis-
try, and so forth.
Efect of catalysis and solvent
To standardize the reaction conditions, to get an appropri-
ate combination of catalyst, reaction time, percentage yield,
and solvent, various combinations were investigated multiple
times under varied conditions (Scheme 1). However, none
of the combinations replicated the same results, as only raw
solid was observed on working up with tedious methods and
still satisfactory results were not obtained even after recrys-
tallization (Table 1, entries 1–8). Further, the obtained yield
with various combinations of solvent and catalysts were low
in contrast to the combination of 2-aminopyrazole with ACN
(Table 1, entry 9).
Two organic molecules hanged with the help of single
aldehyde units is a widely explored research area in synthetic
organic chemistry. These molecules fnd various applica-
tions in the biological and pharmacological felds. These
compounds are found efective as tyrosinase inhibitors and
also have considerable applicability in laser technology
[15, 16]. Synthesis of numerous tetraketone which includes
condensation of dimedone and aldehydes under varied reac-
tions conditions and catalysis like sodium dodecyl sulfate
(SDS) [17], molecular iodine [18], silica-diphenic acid [19],
hexafouro-2-propanol [20], [HClO4-SiO2] [21], and ethyl-
enediammonium diacetate (EDDA) [22] have been reported
in the literature. The existing methods have advantages one
over other but still the urge for neat and clean procedures is
the demand of an hour. So, in search of new methodology
and in continuation on our previous work [23, 24], we are
reporting a convenient, simple, and economical procedure
for the production of 2,2′-(phenyl methylene)bis(3-hydroxy-
5,5-dimethylcyclohex-2-enone) molecules into highly puri-
fed form, i.e., crystals.
Efect of temperature
Thereafter, to produce the best results in context to perfect
temperature and time duo, it was observed that stirring at
80 °C for 2 h gave 96% yield (Table 2, entry 4). Afterward,
no increase in the yield was observed at 90 °C (Table 2,
entry 5). However, on further increase in the temperature,
Table 1 Various combinations of catalyst-solvents to optimize the
condition and get the optimal time and percentage yield
Entry
Catalyst
Solvent
Time/h
Yielda/%
1
2
3
4
5
6
7
8
9
–
Ethanol
Ethanol
Water
Water
Water
Ethanol
ACN
9
7
11
12
12
7
6
4
2
68
78
37
49
45
65
74
67
96
Result and discussion
β-Cyclodextrin
SDS
Al(DS)3
Chemistry
Sc(DS)3
Starting with the intent of synthesizing fused ring 1,4-dihy-
dropyridine via Hantzsch one-pot multi-component conden-
sation reaction of dimedone, 2-aminopyrazine, and substi-
tuted aromatic aldehyde. After running the reaction for few
hours and leaving it overnight, crystals were observed. Dur-
ing the early investigation of spectral data, it was observed
some signature peaks were missing. Further analysis con-
firmed that the ring cyclization was not completed and
the desired 1,4-DHP molecule was not formed. Rigorous
brainstorming led us to the idea that 2,2′-(phenylmethyl-
ene)bis(3-hydroxy-5,5-dimethylcyclohex-2-enone) were
obtained, justifying the low isolated yield too. The curiosity
to understand the reaction behaviors completely changed the
direction of our research.
Piperidine
β-Cyclodextrin
Piperidine
2-Aminopyrazine
ACN
ACN
aYield refers to the pure isolated product
bReaction was performed in a silicon oil bath to maintain reaction
temperature
Table 2 Reaction conditions optimization in terms of appropriate
temperature, yield, and time
Entry
Temperature/°C
Yielda /%
Time/h
1
2
3
4
5
6
7
50
60
70
80
90
100
110
76
81
85
96
96
94
90
9–10
7
6
2
3
3
3
Scheme 1
aYield refer to combined amounts of diferent crops
1 3

2,2′‑(Arylmethylene)bis(3‑hydroxy‑5,5‑dimethylcyclohex‑2‑enone) crystals formation…
539
the compound starts decomposing and crystallization was
also afected. At lower temperatures, the reaction was not
complete even after providing the extended hours (Table 2,
entries 1 and 2).
molecule of dimedone II to form intermediate III. In
resulted intermediate III deprotonation of active methylene
by lone pair of 2-aminopyrazine results in the formation of
Knoevenagel adduct IV with the removal of a water mol-
ecule. Then intermediate IV is further activated by another
molecule of 2-aminopyrazine followed by the insertion of
the second dimedone molecule II. After that, lone pair of
activated intermediate abstracts hydrogen from tautomeric
enol form of dimedone leading to electron shift to form car-
banion. Then resulted carbanion undergoes Michael addi-
tion gives the compound V. Afterward, fnal molecule VI is
obtained by tautomerization of compound V.
Synthesis of various tetraketones 3a‑3l
Several aromatic aldehydes were selected to undergo the
Knoevenagel condensation with dimedone in the presence
of 2-aminopyrazine in ACN at 80 °C and results were given
in Table 3. This efcient methodology afords products
in excellent yield from 90 to 97%. However, the electron-
withdrawing substituents on the aldehyde ring accelerate the
reaction process in shorter times to produce a high yield of
the product; on the contrary, the electron-donating group
decelerates the reaction. Therefore, in Table 3 the substrates
3b, 3f with electron-withdrawing substitutions give a higher
yield up to 97%, while substrates 3c, 3d having electron
releasing group produced lower yield.
The structure of the compounds 3a-3l was examined by
IR, NMR (1H, 13C, COSY, NOSY, HSQC), mass, elemental
analysis, and single-crystal XRD. The data obtained from
the infrared spectrum are very helpful in structure elucida-
tions of these compounds. The characteristic data obtained
from the infrared spectrum exhibit bands for four main func-
tional groups at 3430–3650 (O–H), 3087–3055 (C=C–H),
2981–2867 (sp3 C–H), and 1608–1558 cm−1 (C=O). In its
1H NMR spectrum (500 MHz, CDCl3), a singlet due to OH
group appeared at 11.92–11.70 ppm.
Proposed mechanism
In the probable depicted mechanistic pathway (Scheme 2),
initially, the aldehyde carbonyl group is activated by the
hydrogen bond formation with the amino group of 2-ami-
nopyrazine. This activation leads to the insertion of one
In compound 3a, the protons of aromatic phenyl region
(H-2′’, H-6′’) gives doublet at 7.10–7.08 ppm (Jo =8.40 Hz),
while a clear triplets of proton H-3′’, H-5′’, and H-4′’
observed at 7.25–7.24, 7.17–7.15 ppm. In p-substituted
Table 3 Structures of various tetraketone derivatives 3a-3l from three-component aldehydes, dimedone, 2-aminopyrazine at ambient tempera-
ture in acetonitrile
Compound
R
Yielda/%
M.p./°C
Lit. m.p./°Cb
3a
3b
3c
3d
3e
3f
3g
3h
3i
C6H5
96
97
94
94
96
97
95
93
90
92
94
95
191
169
128
148
172
196–198
199
188–190
213
177
190–192 [25]
167–171 [25]
126–128 [25]
142–144 [25]
172–174 [25]
201–203 [26]
205–206 [27]
193–195 [26]
215–217 [27]
175–177 [28]
207–209 [25]
155–157 [29]
4-NO2–C6H4
4-Me–C6H4
4-OMe–C6H4
4-Br–C6H4
3-NO2–C6H4
2-OH–C6H4
4-OH–3-OMe–C6H3
C6H4–CH=CH
Pyridin-2-yl
2-Furyl
3j
3k
3l
210
159–161
Thiophen-2-yl
aYield refers to the pure isolated product
bProducts were characterized with spectral techniques and compared with authentic samples
1 3

540
D. Thakur et al.
Scheme 2
N
H
C
O
3
3
N
N
H
N
H
HN
H
C
O
R
O
O
II
O
H
O
HN
H
C
3
H
H
3C
H
O
R
H
O
H
H
C
R
3
H
O
3C
II
I
IV
H
C
H
3
H
O
C
3
III
HN
N
N
R
O
O
H
R
H
O
H
C
H3
O
C
3
H
C
3
H
3C
H
H
3C
O
O
O
H
H
C
3
H
H
3C
H
3C
O
V
VI
ruc ure on rme rom
t
S
t
C
f
i
d f
n e cr s a
t l XRD
y
an
2D NMR
d
i
S
l
g
compound, 3b protons H-3′’, 5′’, and H-2′’, 6′’were present
at 8.13 (d, Jo =6.9 Hz) and 7.26 (Jo =6.5 Hz) ppm, respec-
tively. While in compound, 3i protons present at carbon
(C-1′’’), (C-2′’’) appeared at 6.82 (Jtrans = 15.1 Hz) and δ
7.01 (Jtrans =15.4 Hz) ppm. The signal of benzylic methene
proton as a singlet appeared at 5.60–5.49 ppm. In dime-
done ring, a multiplet at 2.55–2.20 ppm observed due to four
methylene groups present at (C-4,4′–C-6,6′) respectively.
The two sharp singlets observed from 1.06 to 1.30 ppm
could be ascribed to two CH3 groups at C-5,5′, respectively.
The characteristics spectroscopic 13C NMR data of com-
pounds 3a-3l showed that the signals of enone moiety were
present at 190.98–190.40 ppm. The signal of C-3 appeared
at 189.5–189.2 ppm due to the direct attachment of the
electronegative oxygen atom of the hydroxyl group. Signals
resonating at 115.7–114.9 ppm are assigned to C-2 and the
signals at 47.4–46.1 and 46.5–46.4 ppm represent C-6, C-4,
respectively. Moreover, the carbon C-5 appeared upfeld
at 41.8–31.1 ppm owing to the presence of two electron-
donating methyl groups. In the aromatic region, the signal
of carbon atom C-4′’ was found as high as 146.54 ppm with
nitro substitutions and as low as 124.8 ppm with methoxy
group substitution. Similarly, the signal for other carbon
atoms appeared in the range of 128.2–146.14 ppm for
C-3′’,5′’, 127.6–138.1 ppm for C-1′’, and 123.5–135.3 ppm
for C-2′’,6′’ as per the various group’s substitution. While
the upfeld signals of benzylic methylene carbon and carbon
of two methyl groups (C-5,5′–2CH3) resonating at 32.2–33.5
and 27.30–29.80 ppm, respectively. Finally, the ESI–MS
spectrum of compounds 3a-3l proved concrete evidence in
support of the dimeric existence of these compounds with
the (M+1), (M+Na) peaks along with (2M+1), (2M+Na).
Antioxidant activity
Examination of the antioxidant potential of the tetrake-
tone derivatives 3a-3l was carried out using phosphomo-
lybdenum reducing assay, ABTS, and DPPH free radical
scavenging assays. The results of different antioxidant
assays were presented in Table 4. The antioxidant poten-
tial of DPPH, ABTS, and TAC assays was ranged between
17.96 3.23 and 93.77 0.51%, 0 and 61.68 1.68%, and
0 and 63.5 0.42 mg of AAE/1 g of the compound, respec-
tively. The highest DPPH radical scavenging potential was
shown by tetraketone derivative 3h (93.77 0.51), followed
by 3a (66.20 0.22), 3i (65.32 0.073), 3g (59.16 0.22),
3d (54.15 0.17), and 3k (48.24 0.59) respectively. DPPH
scavenging potential of 3h was higher than the standard
used, i.e., gallic acid (91.76 0.62). On the other hand,
3a and 3i exhibited the comparable DPPH scavenging
potential with that of the standard used, while derivative
3l showed the lowest DPPH scavenging potential. Alike
results were also reported in case of tetraketones deriva-
tives 4-(N,N-dimethylamino)phenyl-2,2′-methylenebis-(5,5-
dimethylcyclohexane-1,3-dione) (67%), 2-chlorophenyl-2,2′-
methylenebis-(5,5-dimethylcyclohexane-1,3-dione) (77%),
and 4-methoxyphenyl-2,2′-methylenebis-(5,5- dimethylcy-
clohexane-1,3-dione) (84%) at concentration of 1 mM by
1 3

2,2′‑(Arylmethylene)bis(3‑hydroxy‑5,5‑dimethylcyclohex‑2‑enone) crystals formation…
541
Table 4 The results of various antioxidants assay for diferent synthe-
Table 4 (continued)
sized compounds
The results of DPPH and ABTS assays were expressed as % inhibi-
tion and that of TAC as mg AAE/g compound
Compound
R
DPPH
ABTS
TAC
7.45 0.08
TAC total antioxidant capacity, ABTS 2,2′-azino-bis(3-ethylbenzothi-
azoline-6-sulfonic acid), DPPH 2,2-diphenyl-1-picrylhydrazyl radical
scavenging assay
3a
66.20 0.22
34.79 0.67
3b
3c
3d
3e
3f
45.38 1.24
46.77 0.15
54.15 0.17
45.08 0.366
35.04 0.29
61.68 1.68
30.92 1.17
40.16 1.34
39.49 0.34
57.31 1.00
–
[30]. Further, [16] also reported comparable results of the
antioxidant potential of various tetraketone derivatives.
In ABTS scavenging assay, compound 3b (61.68 1.68)
exhibited the highest scavenging potential followed by 3f
(57.31 1.00), 3h (46.55 1.00), 3l (44.20 2.35), and
3g (43.19 3.36), respectively, while no ABTS scaveng-
ing potential was observed in the case of 3j. The order of
present results of ABTS scavenging potential for various
synthesized compounds was diferent from that of DPPH
scavenging potential this may be attributed to a diferent
mechanism of action and reaction conditions. Alike results
were also given by [31], where various substituted benzilic
acid derivatives have a diferent order of antioxidant poten-
tial in the case of DPPH and ABTS assay.
2.84 0.12
4.10 0.37
15.50 0.06
3.59 0.40
In the case of TAC, 3k (63.5 0.42) showed the highest
TAC values, whereas 3b showed the lowest TAC values. In
comparison with 3k, the TAC values of other derivatives are
very low and lie in the range from 0 to 17.36 0.12. These
values of TAC are much lower than the standard used. The
results of TAC values in the case of 3k were comparable to
the results of TAC values observed in the case of several
methanol extracts acquired from various parts of Nepeta leu-
cophylla, while the results of the rest of the derivatives were
comparable to the TAC values of hexane and chloroform
extracts obtained from various parts of Nepeta leucophylla
[32, 33].
3g
3h
59.16 0.22
93.77 0.51
43.19 3.36
46.55 1.00
13.47 0.08
8.12 0.10
The results of various antioxidant assays reveal that these
synthesized tetraketones derivatives have the tremendous
potential to act as an antioxidant, however, the necessary
investigation (in vivo evaluation and toxicological studies)
must be performed before proceeding further.
3i
3j
65.32 0.073
36.24 0.78
37.98 4.20
17.36 0.12
12.93 0.08
Conclusion
-
In conclusion, we have reported a novel route for the prepa-
ration of various tetraketones by condensation of dimedone
and aldehydes using 2-aminopyrazine via the Knoevenagel-
Michael cascade procedure. In this report, 12 examples of
tetraketones were collected in the form of pure crystals. The
yields of products varied from 90 to 97% depending upon
the substitutions on the aldehyde group. It was also observed
that the yield obtained through the electron-withdrawing
group is higher in comparison with electron releasing
groups. Moreover, the characteristic data obtained through
XRD, and ESI–MS shows that in the solvent medium the
3k
48.24 0.59
17.96 3.23
41.68 5.55
44.20 2.35
63.5 0.42
6.94 0.14
3l
Gallic acid
91.76 0.62
89.52 0.89
96.53 0.15
97.65 0.15
83.17 0.08
–
Ascorbic
acid
1 3

542
D. Thakur et al.
fnal structure exists in dimeric form. On the other hand, in
crystal form monomer molecule exists with intramolecular
hydrogen bonding. The evaluated data of antioxidant assay
show that these synthesized tetraketone derivatives have the
tremendous potential to acts as an antioxidant.
ranges from 1.359(4) Å to 1.536(3) Å and C–O bond lengths
range from 1.262(2) Å to 1.318(2) Å. The detailed bond
lengths and bond angles are attached to the supplementary
fle. The compound 3a CCDC no. is 1992004. Compound
3a crystal data details are given in Table 5.
Experimental
Antioxidant potential
The chemicals were obtained from Sigma Aldrich and they
were utilized as such. Solvents used in reaction and washing
of products were of analytical grade. For the preparation of
all the aqueous solutions, double distilled water was utilized.
Melting points were taken in an open capillary using digital
melting point apparatus. IR spectra were taken on Perkin
Elmer (spectrum II) using ATR mode. NMR (1H, 13C-NMR,
COSY, NOSY, HSQC) were recorded on a Bruker Advanced
NEO 500 MHz NMR spectrometer in CDCl3 and using TMS
as the internal standard. Coupling constants are expressed
in unit Hertz. The MS analysis was performed on LC–MS
Spectrometer Model Q-ToFMicromass, Waters. Single-
crystal XRD data were examined at 298 K on a Rigaku
SuperNova HyPix3000 difractometer with monochromatic
Mo-Kα radiation (λ=0.71073 Å).
Determination of DPPH free radical scavenging potential
The free radical scavenging activity DPPH was done accord-
ing to the method given by [32]. According to the reported
procedure, 4 mg of DPPH was mixed in 100 cm3 of metha-
nol and the prepared solution was kept in dark for further
use. The standard solution and sample were prepared by dis-
solving 1 mg of sample or standard in 1 cm3 of DMSO. 200
mm3 of sample or standard or blank (DMSO) was mixed
with 3 cm3 of DPPH methanolic solution. Then, for 30 min
all the samples were incubated in dark at room temperature.
After incubation, sample absorbance was recorded at 517 nm
using a UV–Visible spectrophotometer. The calculation of
Table 5 Details of crystal Structure and compound Refnement of 3a
General experimental procedure
for synthesis of 2,2′‑(arylmethylene)‑
bis(3‑hydroxy‑5,5‑dimethylcyclohex‑2‑enone)
Empirical formula
C23H28O4
Formula weight
368.45
Temperature/K
Crystal system
Space group
a/Å
b/Å
c/Å
α/°
β/°
293(2)
Tetragonal
I41/a
20.9226(4)
20.9226(4)
36.9382(9)
90
In a one-pot synthesis of tetraketones, an aldehyde (5 mmol),
dimedone (10 mmol), and 2-aminopyrazine (3 mmol) were
mixed in an unproportionate ratio in ACN and stirred at
80 °C for 2–3 h (TLC: ethyl acetate: n-hexane 7:3). Thereaf-
ter the obtained mixture, after the reaction completion, was
allowed to cool at room temperature and allow standing for
overnight. Next day crystals of tetraketones were collected
by washing with ethanol. Colorless crystals were collected
for structure elucidation.
90
90
γ/°
Volume/Å3
16,169.9(7)
32
1.211
Z
ρcalc/g cm−3
XRD structure
μ/mm−1
0.082
Single crystals of 3a are suitable for the X-ray structure
examination. Single-crystal XRD data were obtained at
298 K on a Rigaku SuperNova HyPix3000 difractometer
with monochromatic Mo-Kα radiation (λ = 0.71073 Å).
The CIF has been deposited with the Cambridge Crystallo-
graphic Data Centre. From the Cambridge Crystallographic
Data Centre www.ccdc.cam.ac.uk/data_request/cif, the data
can be obtained without any charge. Single-crystal XRD
analysis revealed that compound 3a crystallized in the
tetragonal system with the I41/a space group. The asym-
metric unit contained two molecules with lattice parameters
a=20.9226(4) Å, b=20.9226(4) Å, c=36.9382(9) Å, and
16,169.9(7) Å3 is unit cell volume. The C–C bond distance
F(000)
Radiation
2Θ range for data collection/°
Index ranges
6336.0
MoKα (λ=0.71073)
6.542–54.776
− 26≤h≤23, − 26≤k≤26,
− 46≤l≤42
Refections collected
76,130
Independent refections
Data/restraints/parameters
Goodness-of-ft on F2
8701 [Rint =0.0984, Rsigma =0.0750]
8701/0/499
0.998
R1=0.0622, wR2=0.1420 Final
R indexes [all data] R1=0.1564,
wR2=0.1823
Final R indexes [I> =2σ (I)]
Largest dif. peak/hole/e Å−3
0.18/− 0.18
1 3

2,2′‑(Arylmethylene)bis(3‑hydroxy‑5,5‑dimethylcyclohex‑2‑enone) crystals formation…
543
Acknowledgements The authors are highly thankful to Chandigarh
University, Gharuan, Mohali, Punjab for providing all facilities to carry
out this research.
(I %) inhibition of DPPH free radical for diferent samples
was done according to the given equation:
[(
)
]
I% [DPPH free radical] = AC−AS ∕AC × 100
where AS and AC stand for absorbance of standards/samples
and control, respectively. Results for I% of standards/sam-
ples were represented as the mean standard deviation of
three values.
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The free radical scavenging potential ABTS was evaluated
according to the method given by [34]. To prepare ABTS
stock solution, equal volume (1 cm3 each) of 2 mM PPS
(potassium persulfate) solution and 7 mM ABTS solution
was mixed. The fnal solution was incubated for 12 h at
room temperature in dark. Afterward, the preparation of
the fnal working solution is done by mixing 1 cm3 of the
incubated stock solution and 22 cm3 of distilled water. After
this, standard (1 mg/cm3) or 400 mm3 of samples (1 mg/
cm3) or blank (DMSO) were allowed to react with an equal
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Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s00706-021-02767-x.
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