Synthesis and properties of sulfo-containing tetrazolium betaines and their formazan precursors

Article abstract of DOI:10.1134/S1070363215090066

Chromogenic redox indicators - 3,5-diphenyl-2-(4-sulfophenyl)-2H-tetrazolium betaine and 2-(4-nitrophenyl)-5-phenyl-3-(4-sulfophenyl)-2H-tetrazolium betaine - were synthesized by the oxidative cyclization of 1,3-diphenyl-5-(4-sulfophenyl)formazan and 5-(4

Full text of DOI:10.1134/S1070363215090066

ISSN 1070-3632, Russian Journal of General Chemistry, 2015, Vol. 85, No. 9, pp. 2048–2057. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © V.M. Ostrovskaya, L.K. Shpigun, Ya.V. Shushenachev, A.K. Buryak, A.S. Peregudov, 2015, published in Zhurnal Obshchei Khimii,
2015, Vol. 85, No. 9, pp. 1465–1475.
To the 85th Anniversary of birthday of late Yu.G. Gololobov
Synthesis and Properties of Sulfo-Containing Tetrazolium
Betaines and Their Formazan Precursors
V. M. Ostrovskayaa, L. K. Shpiguna, Ya. V. Shushenacheva,
A. K. Buryakb, and A. S. Peregudovc
a Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences,
Leninskii pr. 31, Moscow, 119991 Russia
e-mail: ostr@igic.ras.ru
b Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia
c Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia
Received July 23, 2015
Abstract—Chromogenic redox indicators―3,5-diphenyl-2-(4-sulfophenyl)-2Н-tetrazolium betaine and 2-(4-
nitrophenyl)-5-phenyl-3-(4-sulfophenyl)-2Н-tetrazolium betaine―were synthesized by the oxidative cyclization
of 1,3-diphenyl-5-(4-sulfophenyl)formazan and 5-(4-nitrophenyl)-3-phenyl-1-(4-sulfophenyl)formazan under
the action of N-bromosuccinimide. The synthesis of sulfoformazans is accompanied by formation of azo
coupling products with substitution of the sulfo group in the hydrazone molecule by the arylazo group. Spectral
and voltage–current characteristics of the synthesized compounds were studied.
Keywords: tetrazolium betaines, formazan, redox indicators
DOI: 10.1134/S1070363215090066
At present tetrazolium salts as electron acceptors
have found diverse applications in biochemical,
chemical analytical, and other research as chromogenic
markers and redox indicators whose principle of action
is based on the conversion of colorless redox indicators
into brightly colored formazans. In particular, this
property forms the basis for the use of tetrazolium salts
for screening of anticancer drugs and assessment of the
metabolic activity of live cells, and, therewith, special
focus is on nitrotetrazolium salts which are highly
sensitive to reduction under the action of dehydro-
genases and apophorases [1–6].
one case, a sulfo-substituted phenyl ring and, in the
other, nitro- and sulfo-substituted phenyl rings and
compare the spectral and electrochemical character-
istics of the synthesized compounds.
Tetrazolium salts are generally synthesized in two
stages: the fierst involves synthesis of the cor-
responding formazans and the second, oxidation of the
latter in various systems [11]. From the viewpoint of
practical applications, of the greatest interest are water-
soluble tetrazolium salts containing sulfo substituents.
Fichter and Schiess described the synthesis of an inner
tetrazolium salt, involving the synthesis of the sodium
salt of 1,3-diphenyl-5-(4-sulfophenyl)formazan by the
reaction of benzaldehyde (4-sulfophenyl)hydrazone
with diazobenzene in aqueous sodium carbonate and
subsequent oxidation of the synthesized salt with
nitrous acid [12]. However, the authors of the cited
work did not provide the procedure of the synthesis
and any characteristics of the products (except for
analyses for nitrogen and sulfur) to confirm their
individuality and structure. The syntheses of a water-
It was found that tetrazolium salts are highly
sensitive indicators of superoxide radicals [7, 8].
Useful applications of the chemical systems on the
basis of tetrazolium salts in inorganic analysis [9] and
preparative heterocyclic chemistry [10] have been
described.
In the present work we set ourselves the goal to
synthesize aryltetrazolium betaines by oxidative intra-
molecular cyclization of arylformazans containing, in
2048

SYNTHESIS AND PROPERTIES OF SULFO-CONTAINING TETRAZOLIUM BETAINES
2049
Scheme 1.
23
14
13
11
24
25
22
21
NH2
12
15
HN
Ph
20
_
10
BF4
8 N
9
O
10N
N8
N7
9
+
Ph
N
N
7
HN
11N
2
5
17
14
pH = 3
Py, NaOH
1
1
16
15
3
H
12
2
6
5
4
13
6
SO3H
3
SO3H
4
SO3H
1
2
15
14
13
16
17
12
3
Br_
2 N
N4
NBS
23
11
+N1 N5
10
9
22
21
6
7
18
19
SO3H
8
20
3
Scheme 2.
Ph
Ph
Br_
Ph
N
+ N
N
N
N
N
NBS
N
N
Ph
H
N N
N N
Ph
Ph
3a
2a
soluble 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-
[(phenylamino)carbonyl]-2H-tetrazolium hydroxide
[13] and a series of water-soluble tetrazolium salts
containing SO3, NO2, COOH, OCH3 substituents in the
phenyl rings [14–16] were reported.
diazobenzene to obtain 1,3-diphenyl-5-(4-sulfophenyl)
formazan (2), and oxidation of the latter with N-
bromosuccinimide (NBS) to betaine 3 (Scheme 1).
At the second stage we obtained, along with
formazan 2, two by-products formed by azo coupling
of diazobenzene with the sulfo group and substitution
of the sulfo group by phenylazo group: benzaldehyde
[4-(4-phenylazo)-phenyl]hydrazone (1а) and 1,3-di-
phenyl-5-[4-(phenylazo)phenyl]formazan (2а), which
were separated from the target product by extraction
with chloroform. Formazan 2а was oxidized with NBS
to 3,5-diphenyl-2-(4-(phenylazo)phenyl)-2Н-tetrazol-3-
ium bromide (3а) (Scheme 2).
We synthesized two sulfo-containing tetrazolium
betaines and their formazan precursors and studied
their structure and properties by means of cyclic
voltammetry, electronic absorption (EA), IR, and
NMR spectroscopy, and mass spectrometry.
The synthesis of 3,5-diphenyl-2-(4-sulfophenyl)-
2Н-tetrazolium betaine (3) involved three stages: con-
densation of (4-hydrazinobenzene)sulfonic acid with
benzaldehyde to obtain benzaldehyde (4-sulfo-phenyl)
hydrazone (1), azo coupling of hydrazone 1 with
The synthesis of 2-(4-nitrophenyl)-5-phenyl-3-(4-
sulfophenyl)-2Н-tetrazolium betaine 6 involved two
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

2050
OSTROVSKAYA et al.
Scheme 3.
2
6
3
8
N
H7
18
16
1
19
15
4
N
NO2
+
9
17
+
N
N
SO3H
5
14
Br4_
COOH
4
23
_O3S
21
22
22
21
24
25
20
23
5
20
19 18
H
N4
N
9
16
14
17
13
2
5
10N
N
N
N
NBS
1
3
3
4
7
8
11N
12
17
14
Py, NaOH
N
1
16
15
6
12
6
7
NO2
13
11
HO3S
10
9
O2N
5
Scheme 4.
Ph
Ph
HO
N
N
HOOC
N
N
+
NBF4_
O
HO3SC6H4N
N
N
N
Py, NaOH
N
O2N
O2N
N
N
SO3H
SO3H
4b
4a
stages: azo coupling of phenylglyoxalic acid (4-
nitrophenyl)hydrazone (4) with 4-diazobenzene-
sulfonic acid to obtain 5-(4-nitrophenyl)-3-phenyl-1-
(4-sulfophenyl)formazan (5) and oxidation of the latter
to betaine 6 (Scheme 3).
Apparently, these reactions are associated with the
deprotonation of the hydrazono NH group under the
action of the electron-acceptor nitro group, as well as
with the presence of still unsubstituted carboxylic group
which prevents the tetrazene–formazan rearrangement.
The yield of fomazan 5 at the first stage involving
substitution of the carboxylic group in hydrazone 4 by
the arylazo group is low because of the occurrence of
two side azo coupling reactions: substitution of the
imino proton in hydrazone 4 under the action of 4-
diazobenzenesulfonic acid by the 2-[2-(4-nitrophenyl)-
4-(4-sulfophenyl)tetraazylidene]-2-phenylacetic acid
(4а) and subsequent substitution of the sulfo group in
tetrazene 4a by the 4-sulfophenylazo group to form 2-
[2-(4-nitrophenyl)-4-(4-sulfophenylazo)phenyltetraazyl-
idene]-2-phenylacetic acid (4b) (Scheme 4).
Compound 4b is insoluble in acetone, which
allowed us to separate it from formazan 5 by extraction
of the latter with acetone. Formazan 5 was obtained in
a low yield, and, therefore, it was synthesized with a
higher yield by azo coupling of benzaldehyde (4-nitro-
phenyl)hydrazone with 4-diazobenzenesulfonic acid.
We studied the spectral characteristics of com-
pounds 1–6.
Tetrazolium betaines 3 and 6 are insoluble in
absolute ethanol and soluble in aqueous ethanol; the
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

SYNTHESIS AND PROPERTIES OF SULFO-CONTAINING TETRAZOLIUM BETAINES
2051
Table 1. Parameters of the EA spectra of tetrazolium betaines 3 and 4 and typical tetrazolium salts
λ, nm
εmax, L mol–1cm–1
εmax1 × 104 εmin × 104 εmax2 × 104
Compound
λmax
1
λmin
λmax
2
3
4
200
203
201
224
252
260
250
3.64
3.92
3.64
1.04
1.03
0.67
1.91
226
220
2.60
1.64
2,3,5-Triphenyl-2Н-tetrazolium chloride
2-(4-Nitrophenyl)-3,5-diphenyl-2Н-tetrazolium chloride
203
225
259
3.70
1.08
2.27
solution of betaine 3 is colorless, like the solution of
2,3,5-triphenyl-2Н-tetrazolium chloride, while the
solution of betaine 6 has a light yellow color, like that
of 2-(4-nitrophenyl)-3,5-diphenyl-2Н-tetrazolium chloride.
As seen from Table 1, the EA spectra of the mentioned
two couples of compounds are similar to each other,
but the bands in the spectra of nitrophenyl derivatives
are slightly shifted red and have higher extinction
coefficients ε.
were overlapping multiplets, the NMR spectra of this
compound could not be assigned completely. We can
only believe that we have strong grounds to assign the
multiplet at 8.35 ppm to the С13,17Н proton, which
shows a double-intensity signal at 130.70 ppm in the
HMQC spectrum. This conclusion is based on the
observation in the HMBC spectrum of a cross-peak
between this multiplet and the signal at 164.6 ppm,
associated without any doubt with С3. The latter, in its
turn, gives a correlation signal with the С15 signal (δС =
134.10 ppm) from among other surrounding СН-
signals. This signal can be attributed to para-carbon
atoms in two phenyl rings. Thus, the second downfield
signal at 134.71 ppm is assignable to С9.
1
In the H NMR spectrum of compound 1, the р-
C6H4SO3H protons appear as an АХ system. The
observation in the NOESY spectrum of cross-peaks
between the С7Н and С9Н protons suggests their
spatial
proximity
and,
consequently,
trans
1
configuration of the enimine fragment. Note that the
NOESY spectrum also shows correlations between the
NH and С2Н и С6Н ortho-proton signals, as well as
between the С9Н and С11Н и С15Н ortho-proton
signals, which provides evidence for correct
assignment of the 1H NMR spectrum of compound 1.
The H NMR spectrum of compound 4 shows an
АХ system from the р-C6H4NO2 protons at 7.48
(С2,6Н) and 8.19 ppm (С3,5Н). The NH proton signal is
observed at 11.83 ppm, implying a lack of the
potentially possible NH···O=C hydrogen bond.
1
The H NMR spectrum of compound 5 contains
1
The H NMR spectrum of compound 2 contains an
signals of two АХ systems characteristic of para-
substituted benzene rings. According to the COSY
spectrum, the doublet at 8.25 ppm is associated with
the doublet at 7.69 ppm, whereas the doublet at
8.10 ppm is associated with the doublet at 7.83 ppm,
and, therewith, the J constant for the first АХ system
(9.2 Hz) is much larger than for the second (6.8 Hz). In
АХ system from the р-C6H4SO3H protons, as well as
characteristic signals of two unsubstituted phenyl
rings. The NH proton signals is observed at 14.17 ppm,
which suggests strong H-bonding in the 6-membered
heteroring. The assignment of the δС signals of two
different phenyl rings was based on the HMBC
correlations between the С12 and С20 signals at 148.83
and 136.25 ppm and the triplet proton signals at 7.54
and 7.48 ppm, respectively, as well as the cross-peak
between the С9Н signal and the doublet signal of the
1
the H NMR spectrum, the unsubstituted phenyl group
appears as a doublet from ortho protons at 7.79 ppm
(J = 7.0 Hz) and two overlapping triplets of the meta
and para protons at 7.44−7.49 ppm. The NH proton
gives a downfield signal (12.69 ppm), implying that
com-pound 5 contains a 6-membered ring with an
intra-molecular hydrogen bond between this proton
and the N1 atom. The presence in the spectrum of a
correlation signal between the NH proton and the
signal at δС = 114.85 ppm, which, according to the
С
21,25Н proton at 7.99 ppm
Some signals in the NMR spectra of compound 3
were assigned using 2D HMQC and HMBC
correlation techniques, as well as the 13С NMR spectra
registered in the JMODECHO mode. In view of the
fact that the aromatic proton signals at 7.7−7.9 ppm
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

2052
OSTROVSKAYA et al.
1
ε × 10–3, L mol–1cm–1
In the Н NMR spectra of compounds 1–6, larger
downfield shifts are characteristic of all the nitro
derivatives, as well as in the tetrazolium cation
compared to its formazan precursor. Signals of the aryl
substituent at the azo group are observed exclusively in
formazans and tetrazolium betaines.
Solid formazans 2 and 5 exist in a chelate EZZ
conformation with intramolecular hydrogen bonds. At
the same time, the tautomeric equilibria of these
compounds in solutions differ from each other
Aqueous ethanolic solutions of formazans 2 and 5 are
colored red and orange-red, respectively. The EA
maximum of nitroformazan 5 which has a weaker
intramolecular hydrogen bond is shifter blue with
respect to the EA maximum of formazan 2 (Fig. 1),
even though the nitro group usually imparts a more
intense color.
λ, nm
As known, the NH proton in an EZZ chelate
arylformazan alternately forms a covalent bond with
N1 or N5 due to intrachelate proton transfer (the
lifetime of the tautomers is about 10–3 s). Based on the
2J(13СNH) values we showed the introduction of the
strongly acceptor NО2 group into one N-aryl ring in
2,3,5-triphenyl-2Н-tetrazolium chloride almost com-
pletely shifts the tautomeric equilibrium to 4-
NО2С6Н4NН [17, pp. 29−32]. Therewith, the δН(NH)
decreases by 2 ppm. As the formazan is present as two
conformers (EZZ and and EЕZ), this proton chemical
shift is an average value due to fast (on the NMR
scale) prototropic tautomeric interconversions in the
formazan chain.
Fig. 1. Electronic absorption spectra of (1, 2) formazan 2
and (3, 4) formazan 5 at (1, 3) рН 6 and (2, 4) 12.
HMQC spectrum, corresponds to the proton signal at
7.69 ppm, shows that the АХ spin system at 7.69 and
8.25 ppm belongs to the p-C6H4NO2 substituent.
1
The H NMR spectrum of compound 6 shows
signals of two АХ systems from two para-substituted
phenyl rings. The first system (8.16 ppm, С7,11Н; 8.58
ppm, С8,10Н, J 9.2 Hz) belongs to the р-C6H4NO2
system. One component of the second АХ system of
the р-C6H4SO3H substituent is observed at 7.90 ppm
(С20,22Н, J = 8.6 Hz), and the other component of this
system (С19,23Н) overlaps with the multiplet (С14,15,16Н)
from the unsubstituted phenyl ring at 7.77−7.85 ppm.
The doublet of the ortho protons С13,17Н appears at
8.36 ppm.
In an alkaline medium (рН = 12), the absorption
maximum of formazan 2 remains remains almost
unchanged, because the strong intramolecular bond is
not broken. By contrast, an alkaline solution of
formazan 5 acquires a dark blue color, and its
absorption maximum shifts red by 163 nm, and its ε
becomes markedly higher (Fig. 1). This result is
explain by the fact that the electron-acceptor nitro
group causes breakage of the intramolecular hydrogen
bond, deprotonation of the NО2С6Н4NН group, and
rearrangement of the chromophoric system of the
molecule.
Table 2. Comparative characteristics of formazans 2 and 5
and crown formazans 7−10
Comp. no.
Conformer
δH(NH), ppm
λmax, nm
2
EZZ
14.17
482
5
7
EEZ
EZZ
EZZ
EEZ
EEZ
12.69
14.21
14.00
11.56
12.18
450
552
495
480
454
Comparison of the δH(NH) and λmax values for 2
and 5 with the respective values for crown formazans
7−10 which exist as a single conformer having the
formazan fragment is rigidly incorporated into the ring
[18, 19] (Table 2) provides further evidence for the
8
9
10
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

SYNTHESIS AND PROPERTIES OF SULFO-CONTAINING TETRAZOLIUM BETAINES
2053
Table 3. Parameters of the IR spectra (ν, cm–1) of compounds 1–6
Assignment
N–H
1
3320
3070
−
2
3
−
4
5
6
−
−
3280
−
O–Hsulfo
C=OCOOH
C=CAr
3060
3064
−
−
3080
3070
−
−
1680 s
−
1608 s
1596 s
1568 m
1644 w, br
1596 s
1448 m
1608
1600 s
1580 m
1560 m
1596 s
1444 m
1584 m
C=N
1524 s
1492 m
−
1512 s
1492 s
1456 s
1356 m
−
1528
1488
1456 s
1344 w
−
1536 s
1504
1516 s. ш
1516
1536 s
1488 m
1456 s
1348 s
1307 sh
1288 m
C–CAr
N=N
−
1456 s, sh
1344 s
νs(C–N)
νs(C–NO2)
N–N
1356 w
−
1332 s, br
1300 m
1240 s, br
−
1300 w
1280 s
1290 m
1228 s
1232 s
SO3H
1260 s
1192 vs
1036 s
1210 sh
1184 s
1040 s
1208 s
1180 s, sh
1036 s
1230 s, br
1188 s
1108 s
1232 s
1212 s
1108 m
C–Cdisubst
1124 vs
932 m
1124 vs
984 m
1116 s
996 m
1112 s
992 m
1124 s
1180 s
–С–Сhydrazone
1008 m
1008 m
γ(СНmonosubst
)
760 m
696 m
760 s
688 m
772 m
692 m
792 m
688 m
752 m
696 m
764 m
692 m
γ(СНAr)
636 m
640 m
640 s
−
620 m
644 m
EZZ configuration of formazan 2 and ЕЕZ configura-
tion of formazan 5.
Electrochemical characteristics of compounds 2,
3, 5, and 6. The electrochemistry of the tetrazolium
salt–formazan system is fairly complicated, because
the electrode process involves many stages and certain
intermediate products are unstable [20–25]. Figure 2
shows the cyclic voltammograms of the aqueous
alcoholic solutions of compounds 2, 3, 5, and 6,
measured on a glassy-carbon electrode. As seen, in the
range from –1.4 V to +1.0 V formazans 2 and 5
undergo quasiequilibrium oxidation (Fig. 2а), whereas
betaines 3 and 6 under go quasiequilibrium reduction
(Fig. 2b). Therewith, all curves contain two pairs of
redox peaks, implying two-stage electrode processes
which are likely to involve intermediate formation of
tetrazolyl radicals. In view of the data in [25], the
electrooxidation of formazans can be presented by the
Scheme 5.
Comparison of the IR spectra of hydrazones,
formazanes, and tetrazolium salts 1–6 (Table 3)
confirms the molecular structures and the presence in
them of azo, sulfo, or nitro groups. Characteristic ν
(NH) bands are observed only in the spectra of
hydrazones, which suggests that solid formazans 2 and
5 have a stable chelate structure.
R
R
N
N
N
N
N
N
N N
H
H
O
O
O
O
O
O
The cathodic and anodic peak potentials for the
synthesized formazans 2 and 4 are listed in Table 4. As
seen from the data in the table, the anodic peak
potentials which relate to the tendency of these
O
7, 8
9, 10
R = Ph (7), CN (8, 9), H (10).
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

2054
OSTROVSKAYA et al.
Scheme 5.
Ar1
Ar1
Ar1
Ar2
N
N
N
N
N
N
N
N
N
Ph
Ph
Ph
_
H
_
_e
:
:
_e, _H+
N
N
N
+
Ar2
Ar2
compounds for oxidation are almost the same, whereas
the cathodic peak potentials measured on the reverse
potential sweep much differ from each other.
Therewith, it is obvious that the introduction of the
nitro group into sulfo-substituted formazan 2 results in
an appreciable cathodic shift of the electroreduction
potential. The difference of the formal redox pontials
in this case is about 50 mV. These data correlate with
the characteristic electrochemical parameters of the
ferences in the reaction kinetcs (Fig. 3). Thus, com-
pound 3 was reduced within 25 min, while compound
6, within 2 min.
EXPERIMENTAL
The electronic absorption (EA) spectra of
formazans and tetrazolium betaines (5 × 10–5 М) were
registered on a Jenway spectrophotomer (Bibby
Scientific, England), solvent ethanol–water (1 : 1). The
1Н and 13С NMR spectra were measured on a
BrukerAvanceTM600 spectrometer at 600.22 and
150.93М Hz, respectively; solvent DMSO-d6, internal
reference TMS. The mass spectra (MALDI) were
obtained on a Bruker Ultraflex instrument. The IR
spectra in the range 3800–400 cm–1 were recorded on a
Specord М in KBr. Elemental analysis was performed
on a Carlo Erba instrument. Cyclic voltammograms
were registered on an Ekotest-VA (Ekoniks-Ekspert,
Moscow) with a glassy carbon working electrode, a
synthesized tetrazolium betaines
3 and 6 and
unsubstituted 2,3,5-triphenyl-2Н-tetrazolium chloride
(Table 5). All other factors being equal, the peak
currents for nitrophenyl derivatives are much higher
than for the other compounds.
Reductive properties of compounds 3 and 6. The
photometric study of the chemical reduction of the
synthesized sulfo-containing tetrazolium betaines to
the corresponding formazans in solutions under the
action of sodium sufide revealed considerable dif-
I, μA
I, μA
(a)
(b)
40
20
20
0
0
–20
–40
–60
–20
–40
–60
–1500 –1000
–500
0
500
1000
–1500 –1000
–500
0
500
1000
Е, mV
Е, mV
Fig. 2. Cyclic voltammograms of formazans (а, 1) 2 and (а, 2) 5 and tetrazolium betaines (b, 1) 3 and (b, 2) 6.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

SYNTHESIS AND PROPERTIES OF SULFO-CONTAINING TETRAZOLIUM BETAINES
2055
Table 4. Voltage–current characteristics of formazans 2
and 5
Table 5. Characteristic electroreduction parameters of the
synthesized tetrazolium betaines and 2,3,5-triphenyl-2Н-
tetrazolium chloride
Oxidation peak Reduction peak Formal redox
Reductionpeak Formal redox po-
Comp.
no.
potential, V
potential, V
potential, V
potential, V
tential, V
Compound
Е1pa Е2pa
Е10' Е20'
Е2pc Е1pc
Е2pc Е1pc
Е10' Е20'
2
5
–0.348 0.008 –0.437 –0.826 –0.597 –0.259
–0.350 0.011 –0.602 –0.984 –0.667 –0.295
2,3,5-Triphenyl-2Н- –0.438 –0.873 –0.428 –0.868
tetrazolium chloride
3
6
–0.442 –0.835
–0.640 –1.017
silver–silver chloride electrode (Аg/AgCl, KClsat)
reference electrode, and platinum auxiliary electrode.
Cyclic chronovoltammograms were registered in 40%
aqueous ethanolic solutions (0.15 М NaCl) at the
polarization potential sweep rate 0.1 V/s. The optical
density of the solutions in the kinetic study of the
reduction of tetrazolium betaines (3 × 10–4 М) in
aqueous ethanol (1 : 1) with 1% sodium sulfide was
measured in quartz cells (10 mm) on an Ekspert-003
miniphotometer (Ekoniks-Ekspert, Moscow) at 524 nm.
sodium nitrite (15 mmol) was stirred at 0−2°С for 1 h
and then added in portions to a solution of 4.14 g
(15 mmol) of hydrazine 1 in 40 mL of 6% sodium
hydroxide. After 3-h stirring at 1−3°С the pH of the
reaction mixture was adjusted to 2 with HCl. The
precipitate that formed was treated with chloroform
and methylene chloride to remove compounds
containing no sulfo group. The reaction product was
extracted with a methanol–acetone mixture (1 : 1) and
reprecipitated with diethyl ether from methanol. Yield
0.8 g (14%), dark red powder, mp 195−197°С
Benzaldehyde (4-sulfophenyl)hydrazone (1). A
mixture of 18.8 g (0.1mol) of (4-hydrazinobenzene)
sulfonic acid, 40 mL of water, 10 mL of acetic acid,
and 11.7 g (0.1 mol) of benzaldehyde was stirred at
60°С for 3 h. The reaction product was precipitated
with saturated aqueous sodium chloride and twice
recrystallized from 70% ethanol. Yield 19 g (72%), mp
1
(decomp.). Н NMR spectrum, δ, ppm (J, Hz): 7.38 t
(2Н, С14,16Н, J 4.6), 7.40 t (2Н, С22,24Н, J 4.6), 7.48 t
(1Н, С23Н, J 5.0), 7.54 t (1Н, С15Н, J 5.1), 7.70 d (2Н,
С2,6Н, J 5.5), 7.77 d (2Н, С13,17Н, J 5.7), 7.91 d (2Н,
1
243−246°С (decomp.). Н NMR spectrum, δ, ppm (J,
Hz): 7.01 d (2Н, С2,6Н, J 8.6), 7.30 t (Н, С13Н, J 7.4
Hz), 7.39 t (2Н, С12,14Н, J 7.6 Hz), 7.50 d (2Н, С3,5Н, J
8.6), 7.65 d (2Н, С11,15Н, J 7.3), 7.88(1H, C9), 10.49 s
(1H, NH). 13С NMR spectrum, δС, ppm: 111.10 (С2,6),
126.18 (С11,15), 127.31 (С3,5), 128.53 (С13), 129.14
(С12,14), 136.13 (С10), 137.56 (С9), 139.11 (С4), 145.85
(С1). Mass spectrum, m/z (Irel, %): 196 (16) [М – SO3]+,
178 (100) [М – SO3 – NH3]+. Mcalc 31. Found, %: С
49.38; H 4.87; N 8.45; S 10.42. C13H12N2O3S·2H2O.
Calculated, %: С 49.99; H 5.16; N 8.97; S 10.27. At
80°С and 10 mmHg the compound loses 11.2% of
weight, which corresponds to the fraction of the water
of crystallization. Found, %: С 56.11; H 4.58; N 9.85;
S 11.13. C13H12N2O3S. Calculated, %: С 56.51; H
4.38; N 10.14; S 11.60. The IR spectral data are listed
in Table 3.
А, abs. units
Reaction time, min
1,3-Diphenyl-5-(4-sulfophenyl)formazan (2). A
mixture of 1.91 g (15 mmol) of aniline hydrochloride,
12 mL of water, 3 mL of conc. HCl, 1.7 g (15 mmol)
of sodium tetrafluoroborate, and 10.4 mL of 10%
Fig. 3. Plots of optical density against time for solutions of
tetrazolium salts (1) 6, (2) 3, and (3) 2,3,5-triphenyl-2Н-
tetrazolium chloride in the course of reduction with sodium
sulfide.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

2056
OSTROVSKAYA et al.
С21,25Н, J 5.2), 7.99 d (2Н, С3,5Н, J 5.2), 14.17 s (1H,
NH). 13С NMR spectrum, δС, ppm: 118.07 (С2,6),
120.14 (С13,17), 126.88 (С21,25), 127.30 (С3,5), 128.40,
128.81 (С15,23), 128.93 (С22,24); 129.97 (С14,16), 136.25
(С20), 142.51 (С9), 146.98 (С4), 147.31 (С1), 148.83
(С12). Mass spectrum, m/z (Irel, %): 380.36 (70) [М]+,
301 (100) [М – SO3]+, 457 (48) [М + Ph]+. Mcalc
380.43. Found, %: С 58.63; H 4.35; N 14.36; S 7.99.
C19H16N4SO3. Calculated, %: С.99; H 4.24; N 14.73; S
8.43. The IR spectral data are listed in Table 3.
(С2,6), 126,24 (С3,5), 127.95 (С15,19), 128.74 (С16,18),
129.13 (С17), 135.47 (С14), 137.27 (С9), 140.85 (С4),
150.32 (С1), 164.88 (С10). Mass spectrum, m/z (Irel, %):
285 (100) [М]+, 241 (70) [М – СО2]+, 178 (100) [М –
СО2 – NО2 – NH3]+. Mcalc 285.26. Found, %: С 55.12;
H 4.25; N 13.17. C14H11N3O4 H2O. Calculated, %: С
55.45; H 4.32; N 13.86. At 80°С and 10 mmHg the
compound loses 5.2% of weight, which corresponds to
the fraction of the water of crystallization. Found, %:
С 58.73; H 3.66; N 14.81. C14H11N3O4. Calculated, %:
С 58.95; H 3.87; N 14.73. The IR spectral data are
listed in Table 3.
The extract was evaporated to leave a dark brown
powder. Thin-layer chromatography of the latter gave
benzaldehyde 4-[(4-phenylazo)phenyl]hydrazone (1а)
(Rf 0.72, m/z 300.36 [М]+, Mcalc 300.81), which was
previously obtained by azo coupling of benzaldehyde
phenylhydrazone with diazobenzene at рН 3 (mp 167–
169°С [26]), and 1,3-diphenyl-5-(4-(phenylazo)phenyl)-
formazan (2а) (Rf 0.21, mp 185°С, m/z: 404.60 [М]+,
Mcalc 404.44), which was previously obtained by azo
coupling of benzaldehyde phenylhydrazone with 4-
diazobenzene (mp 182°С [27]). Formazan 2а was
treated with N-bromosuccinimide to obtain
5-(4-Nitrophenyl)-3-phenyl-1-(4-sulfophenyl)for-
mazan (5). A mixture of 0.87 g (5 mmol) of sulfanylic
acid, 9 mL of water, 1.4 mL of conc. HCl, 0.88 g
(5 mmol) of 4-toluenesulfonic acid, and 3.5 mL of
10% sodium nitrite (5 mmol) was stirred at –1°С for
1 h, and then added in portions to a stirred suspension
of 1.21 g (5 mmol) of benzaldehyde 4-nitrophenyl-
hydrazone in 75 mL of DMF, 15 mL of pyridine, and
2 g of sodium carbonate. After 2-h stirring at 1−3°С,
the reaction product was precipitated with conc. HCl,
washed with water and isopropanol, extracted with
acetone, and reprecipitated with diethyl ether from
methanol. Yield 0.32 g (15%), dark red powder, mp
220−222°С. 1Н NMR spectrum, δ, ppm (J, Hz):
7.44−7.46 m (3Н, С22,23,24), 7.69 d (2Н С2,6Н, J 9.2)
7.79 d (2Н, С21,25Н, J 7.0), 7.83 d (2Н, С13,17Н, J.8),
8.10 d (2Н, С3,5Н, J 6.8), 8.25 d (2Н, С14,16Н, J 9.2),
12.69 s (1H, NH). 13С NMR spectrum, δС, ppm:
114.85 (С2,6), 123.69 (С14,16), 126.22 (С3,5), 127.14
(С13,17), 128.69 (С21,25), 128.76 (С23), 129.33 (С22,24),
133.80 (С20), 141.30(С4), 147.71 (С9), 149.98 (С1),
152.32 (С12), 152.40 (С15). Mass spectrum, m/z (Irel,
%): 425 (100) [М]+, 317 (100) [М – SO3 – N2]+, 178
(100) [М – SO3 – N2 – Ph – NO2 – NH3]+. Mcalc 425.43.
Found,%: С 54.04; H 3.40; N 16.28; S. 7.34.
C19H15N5O5S. Calculated, %: С 53.65; H 3.53; N 16.45;
S 7.52. The IR spectral data are listed in Table 3.
3,5-diphenyl-2-(4-(phenylazo)phenyl)-2Н-tetrazol-
3-ium bromide (3а). Mass spectrum, m/z: 483.20 [М]+,
403.37 [М – Br]+. Mcalc 483.25.
3,5-Diphenyl-2-(4-sulfophenyl)-2Н-tetrazolium
betaine 3. A mixture of 0.38 g (1 mmol) of formazan 2
in 15 mL of ethanol and 0.32 g (2 mmol) of N-
bromosuccinimide until the reaction mixture changed
color to light yellow. The solution was filtered off, and
the product was precipitated with diethyl ether and
recrystallized from 90% ethanol, washed with boiling
absolute ethanol, and dried to obtain an off-white
powder which did not melt up to 265°С. Yield 0.18 g
1
(47%). Н NMR spectrum, δ, ppm (J, Hz): 7.73–7.88
m (12Н, С7,8,10,11,14–16,19–23Н), 8.35 d (2Н, С13,17Н, J
7.3). 13С NMR spectrum, δС, ppm: 123.36 (С6), 126.67
(2СН), 126.80 (2СН), 127.77 (2СН), 127.89 (2СН),
130.70(С13,17), 130.99 (2СН), 133.05 (С12), 133.42
(С18), 134.10 (С15), 134.71 (С9), 153.50 (С21), 164.51
(С3). The EA and IR spectral data are listed in Tables 1
and 3, respectively.
Formazan 5 was also prepared by azo coupling of
4-diazobenzenesulfonic acid with hydrazone 4.
However, the yield of the target product in this syn-
thesis was as little as 3%; the main reaction products
were 2-[2-(4-nitrophenyl)-4-(4-sulfophenyl)tetraazylidene]-
2-phenylacetic acid (4а) and 2-([-(4-nitrophenyl)-4-(4-
sulfophenylazo)phenyltetraazylidene]-2-phenylacetic
acid (4b) formed by substitution of the sulfo group in
tetrazene 4a by the 4-sulfophenylazo group. Mass
spectrum, m/z (Irel, %): 4a, 469.20, Mcalc 469.44; 4b,
574.30, Mcalc 573.55.
Phenylglyoxalic acid (4-nitrophenyl)hydrazone
(4) was synthesized by the procedure in [28]. Yield
1
62%, mp 162−163°С. Н NMR spectrum, δ, ppm (J,
Hz): 7.42 t (2Н, С16,18, J 4.7), 7.45 d (2Н, С15,19Н, J
6.9), 7.48 d (2Н, С2,6Н, J 9.2), 7.71 t (1Н, С17Н, J 8.4),
8.19 d (2Н, С3,5Н, J 9.3), 10.40 s (1H, NH), 11.85 s
(1Н, COOH). 13С NMR spectrum, δС, ppm: 113.79
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

SYNTHESIS AND PROPERTIES OF SULFO-CONTAINING TETRAZOLIUM BETAINES
2057
12. Fichter, F.R. and Schiess, E., Chem. Ber., 1900, vol. 33,
2-(4-Nitrophenyl)-5-phenyl-3-(4-sulfophenyl)-
2Н-tetrazolium betaine (6) was prepared similarly to
tetrazolium betaine 3 from 0.4 mmol of formazan 5
and 0.8 mmol of N-bromosuccinimide. Yield 0.12 g
(70%), light yellow powder, doe not met up to 265°С.
1Н NMR spectrum, δ, ppm (J, Hz): 7.77–7.85 m
(5Н14,15,16,19,23), 7.90 (2Н, С20,22Н, J 8.6), 8.16 d (2Н,
С7,11Н, J 9.2), 8.36 d (2Н, С13,17Н, J 8.6), 8.58 d (2Н,
С8,10Н, J 9.2). 13С NMR spectrum, δС, ppm: 123.04
(С12), 126.46 (С13,17), 126.68 (С19,23), 127.84 (С13,17),
128.16 (С20,22), 128.78(С7,11), 130.76 (С14,16), 132.75
(С18), 134.33 (С15), 137.22 (С6), 150.87 (С9), 153.73
(С21), 165.97 (С3). Mass spectrum, m/z (Irel, %): 423
(95) [М]+, 317 (100) [М – SO3], Mcalc 423.41.
Found, %: С 53.37; H 2.91; N 16.21; S 7.18.
C19H13N5O5S. Calculated, %: С 53.96; H 3.09; N
16.54; S 7.57. The EA and IR spectral data are listed in
Tables 1 and 3, respectively.
no. 1, p. 747. DOI: 10.1002/ cber.190003301131.
13. Paull, K.D., Shoemaker, R.H., Boyd, M.R., Parsons, J.L.,
Risbood, P.A., Barbera, W.A., Sharma, M.N., Baker, D.C.,
Hand, E., Scudiero, D.A., Monks, A., Alley, M.C., and
Grote, M., J. Heterocycl. Chem., 1988, vol. 25, no. 3,
p. 911. DOI: 10.1002/ jhet.5570250340.
14. Ishiyama, M., Miyazono, Y., Shiga, M., Sasamoto, K.,
Ohkura, Y., and Ueno, K., Anal. Sci., 1996, vol. 12,
no. 3, p. 515. DOI: 10.2116/analsci.12.515.
15. Ishiyama, M., Sasamoto, K., Shiaga, M., Ohkura, Y.,
and Ueno, K., Analyst, 1995, vol. 120, no. 1, p. 113.
DOI: 10.1039/ AN9952000113.
16. Ishiyama, M., Miyazono,Y., Sasamoto, K., Ohkura, Y.,
and Ueno, K., Talanta, 1997, vol. 44, no. 7, p. 1299.
DOI: 10.1016/ S0039-9140(97)00017-9.
17. Buzykin, B.I., Lipunova, G.N., Pervova, I.G.,
Ostrovskaya, V.M., Lipunov, I.N., Maslakova, T.I.,
Stozhko, N.Yu., Barachevskii, V.A., and Sigeikin, G.I.,
Progress
v
khimii formazanov. Sintez–svoistva–
REFERENCES
primenenie (Progress in Formazan Chemistry. Syn-
thesis–Properties–Application), Moscow: Nauchnyi
Mir, 2009.
1. Butcher, R.G., Histochem. J., 1978, vol. 10, no. 6,
p. 739. DOI: 10.1007/BF01003123.
2. Plumb, J.A., Milray, R., and Kaye, S.B., Cancer Res.,
18. Dziomko, V.M., Ostrovskaya, V.M., Zhukova, I.A., and
Ryabokobylko, Yu.S., Zh. Obshch. Khim., 1981,
vol. 51, no. 10, p. 2324.
1989, vol. 49, no. 16, p. 4435.
3. Wan, H., Williams, R., Doherty, P., and Williams, D.F.,
J. Mater. Sci. Mater. Med., 1994, vol. 5, nos. 6–7,
p. 154. DOI: 10.1007/ BF00058980.
19. Belyakov, S.A., Ostrovskaya, V.M., Romanova, G.G.,
Bushtets, S.P., and Shmelev, L.V., Zh. Org. Khim.,
1992, vol. 28, no. 1, p. 164.
4. Mazhul’, M.M. and Danilov, V.S., Biotekhnologiya,
20. Umemoto, K., Bull. Chem. Soc. Jpn., 1989, vol. 62,
2002, no. 4, p. 91.
5. Aziz, D.M., Anim. Reprod. Sci., 2006, vol. 92, nos. 1–2,
p. 1. DOI: 10.1016/ j.anireprosci.2005.05.029.
6. Prokofieva, D.S. and Goncharov, N.G., Cell Tissue
Biol., 2014, vol. 8, no. 5, p. 389. DOI: 10.1134/
S1990519X1405006X.
7. Bielski, B.H.J., Shiue, G.G., and Bajuk, S., J. Phys.
Chem., 1980, vol. 84, no. 8, p. 830. DOI: 10.1021/
j100445a006.
8. Oritani, T., Fukuhara, N., Okajima, T., Kitamura, F.,
and Ohsaka, T., Inorg. Chim. Acta, 2004, vol. 357,
no. 2, p. 436. DOI: 10.1016/j.ica.2003.05.007.
9. Gavazov, K.B., Dimitrov, A.N., and Lekova, V.D.,
Russ. Chem. Rev., 2007, vol. 76, no. 2, p. 169. DOI:
10.1070/RC2007v076n02ABEH003655.
no. 12, p. 3783. DOI: 10.1246/ bcsj.62.3783.
21. Viseu, M.I., Simoes Goncalves, M.L.S., Costa, S.M.B.,
and Ferreira, M.I.C., J. Electroanal. Chem., 1990,
vol. 282, nos. 1–2, p. 201. DOI: 10.1016/0022-0728(91)
85098-A.
22. Gupta, P.N. and Raina, A., J. Indian Chem. Soc., 1990,
vol. 67, no. 3, p. 247.
23. Abou Elenien, G.M., J. Electroanal. Chem., 1994, vol. 375,
nos. 1–2, p. 301. DOI: 10.1016/0022-0728(94)03419-2.
24. Rapta, P., Brezova, V., Ceppan, M., Melnik, M., Bustin, D.,
and Stasko, A., Free Rad. Res., 1994, vol. 20, no. 2,
p. 71.
25. Tezcan, H., Ekmekci, G., and Aksu, L., Turk. J. Chem.,
2013, vol. 37, no. 1, p. 57.
10. Voitekhovich, S.V., Gaponik, P.N., Ivashkevich, O.A.,
Russ. Chem. Rev., 2002, vol. 71, no. 9, p. 721. DOI:
10.1070/RC2002v071n09ABEH000739.
26. Hauptmann, H. and Cid de Mello Persisse, A., Chem.
Ber., 1956, vol. 89, no. 5, p. 1081. DOI: 10.1002/
cber.19560890502.
11. Pechmann, H.V. and Runge, P., Chem. Ber., 1894,
27. Libman, D.D., Naineham, A.V., and Slack, R., J. Chem.
Soc., 1954, p. 1565. DOI: 10.1039/JR9540001565.
vol. 27, no. 1, p. 323. DOI: 10.1002/cber. 18940270165.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015