The Effect of Oxidizing Agents in the Preparation of 2,3,5-triaryl-2H-tetrazolium Salts from 1,3,5-triarylformazans

Article abstract of DOI:10.1002/jhet.2441

Tetrazolium salts are a group of versatile molecules with a relatively limited application due to synthesis difficulty and high cost of production. Here, we have comparatively examined several approaches for the preparation of 2-(4-nitrophenyl)-3,5-diphenyl-2H-tetrazolium chloride (NTC), as a model tetrazolium salt, through the oxidation of 1-(4-nitrophenyl)-3,5-diphenylformazan (NF), by means of interphase oxidation, dehydrogenation, and direct chlorination. Our findings indicate that chlorination of NF with 1-chlorobenztriazole to be the best choice to prepare NTC from small-scale and scaled-up considerations.

Full text of DOI:10.1002/jhet.2441

Month 2015
The Effect of Oxidizing Agents in the Preparation of 2,3,5-triaryl-2H-
tetrazolium Salts from 1,3,5-triarylformazans
Kalin I. Peneva and Kibret Mequaninta,b
*
aDepartment of Chemical and Biochemical Engineering, The University of Western Ontario, London,
Ontario N6A5B9, Canada
bBiomedical Engineering Graduate Program, The University of Western Ontario, London, Ontario N6A5B9, Canada
*
E-mail: kmequani@uwo.ca
Additional Supporting Information may be found on the online version of this article.
Received August 14, 2014
DOI 10.1002/jhet.2441
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
Tetrazolium salts are a group of versatile molecules with a relatively limited application due to synthesis
difficulty and high cost of production. Here, we have comparatively examined several approaches for the
preparation of 2-(4-nitrophenyl)-3,5-diphenyl-2H-tetrazolium chloride (NTC), as a model tetrazolium salt,
through the oxidation of 1-(4-nitrophenyl)-3,5-diphenylformazan (NF), by means of interphase oxidation,
dehydrogenation, and direct chlorination. Our findings indicate that chlorination of NF with
1-chlorobenztriazole to be the best choice to prepare NTC from small-scale and scaled-up considerations.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
respective chloride salts. Moreover, the oxidation of
formazans to tetrazolium chlorides has been reported using
interfacial oxidation with permanganate (KMnO4) [16,17]
and sodium nitrite (NaNO2) [18], dehydrogenation by
thionyl chloride (SOCl2) [19,20], and direct chlorination
with chlorine [21], tert-butyl hypochlorite [22], and
1-chlorobenzotriazole (CBT) [23]. However, the most
convenient and efficient oxidation process remains to be
determined. In view of this, and given the variety of
methods reported with limited success for preparing
scalable TTz, we conducted comparative studies with the
aim of preparing 2-(4-nitrophenyl)-3,5-diphenyl-2H-tetra-
zolium chloride (NTC) from 1-(4-nitrophenyl)-3,5-
diphenylformazan (NF). NTC was chosen as a model
compound rather than the commonly studied 2,3,5-
triphenyltetrazolium chloride [16–23], because NTC is of
higher practical interest in recent TTz studies [7] and the
addition of an electron-withdrawing substituent at N(2) would
hinder the oxidation reaction [24], therefore representing a
wider group of tetrazolium compounds. For our studies, the
direct chlorination of NF with either chlorine or tert-butyl
hypochlorite was deemed impractical, because of the high
reactivity and hazard properties of these reagents. Instead,
we tested formazan oxidation using (1) KMnO4 in 5% HCl
(aq)–chloroform, (2) NaNO2 in 5% HCl(aq)–chloroform, (3)
thionyl chloride, and (4) 1-chlorobenzotriazole. In the last
two cases, the typically used benzene was replaced with the
less hazardous toluene and ethyl acetate, respectively.
Tetrazolium salts are a class of generally colorless and
water soluble heterocyclic compounds that contain the
four-nitrogen, five-member tetrazolium moiety. Upon re-
duction, the tetrazolium ring opens to produce an intensely
colored formazan. Although different mono-, di-, and tri-
substituted derivatives are known, the most studied groups
are the 2,3,5-triaryl-2H-tetrazolium salts (TTz) and their
corresponding 1,3,5-triarylformazans [1–3] mainly for use
as histochemical dyes and markers of cellular metabolism
[2–6]. Recently, there has been a renewed interest in
TTz for applications such as ionizing radiation dosimetry
[7–11] and new drugs development [12]. TTz are obtained
in a three-step process from an aryl-aldehyde, an aryl-
hydrazine, and an arylamine (Fig. 1) [1,3]. The first step
involves condensation of an aryl-aldehyde with an
aryl-hydrazine to a hydrazone (1). A diazonated arylamine
is then reacted with 1 to produce the formazan (2). At the
final step, the formazan is oxidized to the tetrazolium salt (3).
A variety of different oxidizing agents such as lead (IV)
acetate, mercury (II) oxide, nitrous fumes, oxygen, nitrites,
hypochlorites, and halogenoimides has been reported for
the conversion of (2) to (3) [1]. Although formazan oxida-
tion with N-bromosuccinimide has emerged as the pre-
ferred method for TTz synthesis [4,5,13–15], the product
purification is arduous and inefficient, and the resulting
tetrazolium bromides are less water soluble than the
© 2015 HeteroCorporation

K. I. Penev and K. Mequanint
Vol 000
Figure 1. Synthesis of 2,3,5-triaryl-2H-tetrazolium salts (3) via arylaldehyde arylhydrazones (1) and 1,3,5-triarylformazans (2). R1, R2, and R3 – various
aromatic groups, X – usually halogen.
RESULTS AND DISCUSSION
calculations, it was assumed that the molar ratio between
NTC and HCl was 1:1 in the case of oxidation with SOCl2,
and that no bound HCl was present in the other cases.
Structural characterization.
The structures of all
synthesized compounds were confirmed by 1H-NMR
(Table 1 and Fig. 2). Where the peak assignments were
ambiguous, two-dimensional methods were used to
Experimental yields and general discussion.
Table 2
summarizes the tested methods. The yields are the best
achieved from two or three trials with the same method.
A discussion of each method follows.
determine the proper assignment, as needed: 1HÀ H
1
gradient correlation spectroscopy and/or HÀ13C gradient
1
heteronuclear single quantum correlation spectroscopy (not
shown). Regardless of the method of preparation, all
batches of NTC had practically identical NMR spectra and
mass fragmentation patterns (Table 1). However, the mass
spectrum of the product from the oxidation with SOCl2
showed an additional fragment peak, corresponding to the
hydrochloride salt of NTC (NTC.HCl) [19]. For yield
(i) Oxidation with potassium permanganate.
When
dissolved in dilute hydrochloric acid, KMnO4 reacted
immediately forming gaseous chlorine and a precipitate of
manganese (IV) oxide (MnO2). This issue could be
alleviated by working at lower temperature; however, in an
ice bath, the formazan oxidation was significantly retarded.
In any case, by the end of the reaction, large amounts of
Table 1
1H-NMR and mass spectra.
Compound
Spectrum
1. 1H-NMR (400 MHz), δ (ppm)
1-(4-nitrophenyl)-3,5-diphenylformazan, 2a
Acetone d6: 13.75 (br. s, 1H), 8.28 (d, J = 9.0 Hz, 2H), 8.11
(dd, J = 6.6, 2.7 Hz, 2H), 8.06 (d, J = 7.4 Hz, 2H), 7.74
(d, J = 9.0 Hz, 2H), 7.65 (t, J = 3.5 Hz, 3H), 7.50 (t, J = 7.4 Hz, 2H),
7.45 (t, J = 7.4 Hz, 1H)
2-(4-nitrophenyl)-3,5-diphenyl-2H-tetrazolium chloride, 3a
1-chlorobenzotriazole, 4
Dimethyl sulfoxide d6: 8.52–8.57 (m, 2H), 8.34 (dd, J = 7.0, 1.6 Hz, 2H),
8.20 (dd, J = 9.4, 7.0 Hz, 2H), 7.90–8.00 (m, 2H), 7.69–7.87 (m, 6H)
Chloroform d: 8.06 (dq, J = 7.8, 0.8 Hz, 1H), 7.54–7.63 (m, 2H), 7.43
(ddd, J = 8.3, 5.6, 2.5 Hz, 1H)
2. Electrospray ionization mass spectrometry of the final products, m/z
2-(4-nitrophenyl)-3,5-dipheny-2H-ltetrazolium chloride
[M]+ calculated for C19H14N5O2: 344.11, found: 344.1; [MCl-H]À cal.:
378.08, found: 378.1; [MCl + H]+ cal.: 380.09, found: 380.1; [MCl.HCl-H]À
cal.: 414.05, absent.
2-(4-nitrophenyl)-3,5-dipheny-2H-ltetrazolium chloride hydrochloride [M]+ calculated for C19H14N5O2: 344.11, found: 344.1; [MCl-H]À cal.:
378.08, found: 378.1; [MCl + H]+ cal.: 380.09, found: 380.1; [MCl.HCl-H]À
cal.: 414.05, found: 414.0.
Figure 2. 1H-NMR spectral shifts of the synthesized compounds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Oxidizing Agents for Preparation of Tetrazolium Salts
Table 2
Methods for formazan oxidation to tetrazolium chlorides, using 2-(4-nitrophenyl)-3,5-diphenyl-2H-tetrazolium chloride (NTC) as a model compound.
Oxidizing reagent (Ox)
Molar ratio Ox: NF
Solvents
Temperature
Yield of NTC
Potassium permanganate
Sodium nitrite
Thionyl chloride
1:1
1:1
Chloroform – 4–5% HCl(aq)
Chloroform – 4-5% HCl(aq)
Toluene
5–25°C
5–25°C
75–80°C
reflux
40%
59%
79%
72%
Large excess
2:1
1-Chlorobnezotriazole
Ethyl acetate
MnO2 had been formed and co-precipitated with the
tetrazolium salt. It has been suggested that the entrapped
product should be extracted with dichloromethane [16],
and that the tetrazolium salt from the organic and aqueous
phases should then be precipitated and purified as an iodide
[16,17]. Arguably, such treatment is problematic, as it
requires additional labor and may result in diminished
practical applicability for the water-insoluble tetrazolium
iodide. In our case, the product was not extracted from the
liquid phases, as they were continuing to release gaseous
products. Following extraction from the precipitate with
dichloromethane, the final yield of NTC was only 40%.
refluxing ethyl acetate at 2:1M ratio. Most importantly, as
explained in the experimental section, the purification steps
were very simple, and a high purity product was obtained
in only two steps.
(v) Scaled-up oxidations with SOCl2 and CBT. Given the
potential utility of TTz compounds in low-dose radiation
dosimetry, where large amounts of material may be
required [7], it is beneficial to scale-up the synthesis. The
reaction scale-up was attempted in limited amounts of
solvent. It was hypothesized that any undissolved
formazan will be exchanged for tetrazolium salt in the
precipitate and would eventually oxidize completely.
However, it became apparent that key to the success of
the scale-up was the complete dissolution of the formazan
before addition of the oxidizing agent, with as much as
80mL of solvent required per gram of formazan in this
work. At the 4-g scale, both oxidation reactions proceeded
readily with near complete discoloration of the solutions,
but the follow-up product purification and recovery were
challenging compared with small-scale preparation. In the
case of thionyl chloride oxidation, no pure product was
recovered. Given the difficulty we encountered to purify
and recover the NTC after oxidation with thionyl chloride
in small scale (refer to iii earlier), this was not surprising.
The yield from the CBT oxidation was 55% and may be
further improved if column separation is used. It may be
concluded that for high yield, a complete dissolution of
the formazan precursor is a necessary condition and the
formazan oxidation with 1-chlorobenzotriazole was the
most successful among all tested methods.
(ii) The interphase oxidation with sodium nitrite.
This
process produces no solid by-product, and therefore the
purification of the product was easier, and the yield was
higher than that obtained with potassium permanganate
(59% vs. 40%). However, there was a significant formation
of gaseous by-products. Furthermore, when sodium
chloride was added at the end of the reaction in order to
desalt the tetrazolium salt from the aqueous layer into the
organic layer [18], the gas release became vigorous, which
necessitated careful treatment of the waste solutions.
(iii) Oxidation with thionyl chloride. Thionyl chloride is
moisture sensitive, so all reagents and solvents had to be
pre-dried otherwise the product yield would drop.
Although the literature suggests refluxing benzene as the
solvent of choice [19], we found that the more benign
toluene at 80°C works equally well. However, attempts
with chloroform under reflux did not proceed
appreciably. Thionyl chloride was used in large excess,
and a high yield (79%) was achieved, but the product
purification was fairly difficult. Owing to the fact that
elemental sulfur is one of the reaction by-products, upon
vacuum drying, the reaction solution became resinous
and could not be evaporated to dryness. Hydrogen
chloride was another by-product of the reaction, and it
formed a hydrochloride salt of the tetrazolium chloride.
Therefore, further purification may be required should the
Treatment of waste streams. Finally, with large scale
synthesis, large amounts of waste can be generated.
Fortunately, most of the waste is combustible materials,
which can be effectively incinerated. The presence of
unreacted oxidants and acidic by-products should not be
problematic, as these compounds can be easily removed
from the incineration off-gasses using scrubbers. The
aqueous waste from the formazan synthesis can be
neutralized and treated with activated carbon or other
sorbents before disposal.
product be desired as a simple salt.
(iv) Oxidation with 1-chlorobenzotriazole. The CBT is not
readily available commercially and needs to be prepared,
purified, and dried in advance; however, these steps are
relatively simple [25]. The oxidation proceeded readily in
benzene, toluene, and ethyl acetate at (1.5 to 2):1M ratio of
oxidant to formazan with high yields, reaching 72% in
CONCLUSIONS
Considering that tetrazolium salts are often synthesized
using inefficient methods developed more than 60years
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

K. I. Penev and K. Mequanint
Vol 000
ago, we have conducted a detailed comparative investiga-
tion of four more recent methods for the synthesis of
NTC as a model compound for other tetrazolium salts.
We believe that this work will help future research with tet-
razolium salts and ultimately expand their practical appli-
cations. Based on the combination of product yield and
reaction convenience, the oxidations with thionyl chloride
and 1-chlorobenztriazole seem to be the best methods for
obtaining tetrazolium salts, although the purification and
product recovery were challenging in scale-up oxidation
with thionyl chloride. Taken together, we recommend the
use of 1-chlorobenztriazole oxidation, as the reagent is less
hazardous than thionyl chloride, the reaction is less sensi-
tive to moisture and impurities, and easy to scale-up.
precipitation was completely dissolved with 200mL
dimethylformamide, and the product was dispersed in
1.5 L H2O. To avoid formation of a colloidal solution or
phase separation, the pH of the mixture was carefully
adjusted to 5–6 (against pH paper) with dilute (10%) HCl
while keeping the reaction cool. With proper pH
(aq)
adjustment, the product precipitates fast. If needed, it can
be left to settle overnight. In our case, the precipitate
formed immediately; it was collected and washed with
cold water (150mL) and hot water (300mL), vacuum
dried, and used without further purification. Yield: 24.6 g
(89%).
Oxidation with KMO4. Finely ground 1-(4-nitrophenyl)-
3,5-diphenylformazan (1.01g, 2.9 mmol) was dissolved in
50mL CHCl3 and cooled to 15°C, and 0.46g (2.9 mmol)
of KMnO4 were dissolved in 35mL of H2O to which
3mL of concentrated HCl were added drop-wise with
constant stirring. Precipitation started immediately in the
KMnO4 solution, which was quickly added to the formazan
with vigorous mixing. The reaction was complete in less
than 30min. The precipitate was separated and collected in
a 100mL round bottom flask, where it was mixed and
shaken with two portions of 30mL CH2Cl2. The
dichloromethane solutions were filtered, and the filtrate
was vacuum-evaporated to 10mL, and then added to
250mL of methyl tert-butyl ether (MTBE). The pinkish
precipitate was filtered, dissolved in minimal amount of
methanol, and re-precipitated from ether to yield an off-
white product, which was collected and vacuum dried.
Yield: 442mg (40%).
EXPERIMENTAL
All chemicals were of reagent or higher grade, supplied
from Sigma Aldrich (Canada) or TCI America (Portland,
OR, USA), and used without further purification. The pu-
rity of the tetrazolium salt was tested primarily by thin
layer chromatography using a 1:1 acetone–methanol sol-
vent. Any remaining formazan moved with the solvent
front, and the tetrazolium salt lagged behind as an elon-
gated colorless spot that developed a reddish color after ir-
1
radiation with UV light at 265nm. H-NMR spectra were
obtained on a Varian Mercury VX 400 spectrometer.
Electrospray mass spectra (ESI MS) were obtained on a
Perkin Elmer Sciex API 365 triple quadrupole mass
spectrometer.
Oxidation with NaNO2. Finely ground 1-(4-nitrophenyl)-
3,5-diphenylformazan (1.05g, 3.0 mmol) was dissolved in
50mL CHCl3 and cooled to 15°C, and 0.31g (4.5 mmol)
of NaNO2 were dissolved in 35mL of H2O to which 3 mL
of concentrated HCl were added drop-wise with constant
stirring. The aqueous solution was added at once to the
formazan solution with vigorous mixing. The reaction was
complete in less than 60min, as the bottom (organic) layer
changed from dark red to orange-yellow and an
amorphous mass was formed between the two layers. In
the meantime, the top layer started releasing gaseous by-
products. The solid mass was filtered and washed with
chloroform, which was added to the two-phase mixture.
The organic layer was separated and vacuum evaporated
to 10mL. The reddish solution was precipitated in MTBE,
and the product was filtered and dissolved in minimal
amount of methanol. This solution was used to dissolve
the previously separated solid and was mixed with
700 mL ether to give the final product. Yield: 655mg
(59%).
1-chlorobenzotriazole. To a 100mL solution of 10.0g
(83.9mmol) benzotriazole in 50% (v/v) CH3COOH/H2O,
125mL (83.9 mmol) of 5% NaClO were added. The
white crystals that precipitated almost immediately were
collected, washed with 80 mL H2O, and vacuum dried.
Yield: 11.08 g (85.9%).
1-(4-nitrophenyl)-3,5-diphenylformazan.
To a 100 mL
solution of 10.8 g (100 mmol) of phenylhydrazine in 2%
CH3COOH in CH3OH, benzaldehyde (10.6 g, 100mmol)
in 40mL CH3OH was added with vigorous stirring. The
product, benzaldehyde phenydrazone, precipitated almost
immediately as yellow needle-shaped crystals, which
were collected, washed with 60 mL CH3OH and 200mL
H2O and vacuum dried. Yield: 17.05 g (86%). From that
product, 15.68 g (80 mmol) were dissolved in 200mL
1:1 v/v pyridine-dimethylformamide and cooled in a dry
ice–acetone bath. Then, 4-nitroaniline (11.05 g, 80mmol)
was dissolved in 100 mL hot HCl(aq) (35%), cooled to
À25°C, and diazotized with a solution of NaNO2 (5.66g,
82mmol) in 10 mL H2O. The 4-nitrobenzenediazonium
solution was added portion-wise to the benzaldehyde
phenylhydrazone solution at temperatures below 0°C. At
the end, 10mL of cold 10 M NaOH were added to the
reaction mixture, which was left stirring overnight. Any
Oxidation with SOCl2. Finely ground 1-(4-nitrophenyl)-
3,5-diphenylformazan (1.05g, 3.0 mmol) was dissolved in
100 mL of warm toluene (at 70°C) in a round bottom
flask, and 2.0 mL of thionyl chloride were added at once.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Oxidizing Agents for Preparation of Tetrazolium Salts
After that, the flask was connected to a reflux condenser
and maintained at 75–80°C. The reaction was complete
in less than 60 min as the solution changed color from
dark red to orange and a precipitate was formed. The
reaction was left to cool down to room temperature, and
the precipitate was filtered and washed with 20mL of
toluene. The toluene fractions were collected and
vacuum-evaporated to less than 10 mL of red resinous
material. This was dissolved in 10 mL of methanol and
precipitated from MTBE. The precipitate was dissolved
with methanol, and this solution was used to dissolve the
solid fraction from the reaction. Following a second
precipitation from MTBE, the product was separated and
vacuum dried. Yield: 960 mg (79%) as 2-(4-nitrophenyl)-
3,5-diphenyl-2H-tetrazolium chloride-hydrochloride.
methanol and re-precipitated in 1.8 L of ether. The solids
were left to settle for a few hours, filtered, and dried to a
yield of 2.50g (55.8%) product.
Following the success at the 4-g level, scale-up was
attempted with 10.47 g (30 mmol) of formazan, which
was partially dissolved in only 350mL of ethyl acetate.
To the formazan suspension, a slurry of 6.91 g (45 mmol)
1-chlorobenzotriazole in 50mL of ethyl acetate was added.
After 48h at refluxing conditions, the oxidation reaction
appeared incomplete, as the solution was still dark read; the
reaction was nevertheless cooled to room temperature and
filtered. The dark precipitate washed with two portions of
50mL methanol, allowing the recovery of unoxidized
formazan (3.10g, 29.6%). The product in the methanol frac-
tions was twice re-precipitated in 1.5L of ether to a final
yield of only 1.53g (13.4%), indicating that the reaction re-
quires complete dissolution of the formazan precursor.
Oxidation with 1-chlorobenzotriazole.
Finely ground
1-(4-nitrophenyl)-3,5-diphenylformazan (1.04g, 3.0mmol)
was dissolved in 70mL of warm ethyl acetate (at 60°C)
in a round bottom flask, and 0.92g (6.0mmol) of
1-chlorobenzotriazole were dissolved in 30mL of warm
ethyl acetate. The two solutions were mixed at once, and
the flask was connected to a reflux condenser and brought
to boiling temperature. The reaction was complete in less
than 30min, as the solution became orange and a
precipitate had formed. The flask was left to reach room
temperature, and the precipitate was separated, washed
with ethyl acetate, and left aside. The liquid fractions were
combined and vacuum evaporated to ~20mL. The
purification followed the same steps as in the case of
oxidation with thionyl chloride. Yield: 800mg (72%) as
2-(4-nitrophenyl)-3,5-diphenyl-2H-tetrazolium chloride.
Acknowledgments. We would like to acknowledge the funding
provided by the Natural Sciences and Engineering Research
Council (NSERC) and Mitacs Elevate Fellowship.
REFERENCES AND NOTES
[1] Nineham, A. W. The chemistry of formazans and tetrazolium
salts. Chem Rev 1955, 55, 355.
[2] Altman, F. P. Tetrazolium salts and formazans. Prog
Histochem Cytochem 1976, 9, 1.
[3] Zhivich, A. B.; Koldobskii, G. I.; Ostrovskii, V. A. Tetrazo-
lium salts (review). Chem Heterocycl Comp 1990, 26, 1319.
[4] Seidler, E. New nitro-monotetrazolium salts and their use in
histochemistry. Histochem J 1980, 12, 619.
[5] Berridge, M. V; Herst, P. M.; Tan, A. S. Tetrazolium dyes as
tools in cell biology: new insights into their cellular reduction. In: Bio-
technology Annual Review; El-Gewely M. R., Ed.; Elsevier: Oxford,
UK, 2005; Vol 11, 127.
[6] Albarella, J. P.; Felman, S. W.; Landi, J. J.; Marfurt, K. M.
Water-soluble tetrazolium salts. U.S. Patent 7,767,822, 2006.
[7] Pikaev, A. K.; Kriminskaya, Z. K. Use of tetrazolium salts in
dosimetry of ionizing radiation. Radiation Physics and Chemistry. Pro-
ceedings of the 10th International Meeting on Radiation Processing.
1998, 52, 555.
[8] Moussa, A.; Baranyai, M.; Wojnárovits, L.; Kovács, A.;
McLaughlin, W. L. Dosimetry characteristics of the nitro blue
tetrazolium-polyvinylalcohol film for high dose applications. Rad Phys
Chem 2003, 68, 1011.
[9] Basfar, A. A.; Rabaeh, K. A.; Moussa, A. A.; Msalam, R. I.
Dosimetry characterization of nitro-blue tetrazolium polyvinyl butyral
films for radiation processing. Radiat Phys Chem 2011, 80, 763.
[10] Kozicki, M.; Sąsiadek, E. Polyamide woven fabrics with 2,3,5-
triphenyltetrazolium chloride or nitro blue tetrazolium chloride as 2D ion-
izing radiation dosimeters. Radiat Meas 2012, 47, 614.
[11] Rabaeh, K. A.; Moussa, A. A.; Basfar, A. A.; Msalam, R. I.
Novel radio-chromic solution dosimeter for radiotherapy treatment plan-
ning. Phys Med 2013, 29, 374.
Scale-up of oxidation with SOCl2. Finely ground 1-(4-
nitrophenyl)-3,5-diphenylformazan
(4.10 g,11.9mmol)
was dissolved in 350mL of warm toluene (70°C). To the
toluene solution, ~4mL of SOCl2 were added, and the
reaction flask was connected to a reflux condenser and
brought to 80°C. The solution discolored almost
completely overnight with
a dark resinous mass
separating at the bottom of the flask after cooling.
Unfortunately, the purification of the SOCl2-reaction
mixture was unsuccessful, as the resinous material, which
was soluble in methanol, did not yield a single product
after re-precipitation form ether.
Scale-up of oxidation with 1-chlorobenzotriazole. Finely
ground 1-(4-nitrophenyl)-3,5-diphenylformazan (4.10g,
11.9mmol) was dissolved in 350 mL of warm ethyl
acetate (60°C), and 1-chlorobenzotriazole (3.65g,
23.8mmol) was added as slurry in 50 mL ethyl acetate.
Following an overnight reaction under reflux conditions
and cooling to room temperature, a pale solid precipitate
had separated from the largely discolored solution. The
product was filtered, and the filtrate was evaporated to
half its volume to afford more products. Both precipitates
were separated and dissolved in a total of 40mL of
[12] Cui, X.; Vlahakis, J. Z.; Crandall, I. E.; Szarek, W. A. Anti-
plasmodium activity of tetrazolium salts. Bioorg Med Chem 2008, 16, 1927.
[13] Seidler, E. Zur Analytik histochemisch gebräuchlicher
Tetrazoliumsalze. Fresenius’ Zeitschrift für analytische Chemie 1968, 237, 60.
[14] Brigati, D. J.; Nagubandi, S.; Arvanaghi, M. Chromagens for
Histological Staining, US Patent 5,116,732, 1992.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

K. I. Penev and K. Mequanint
Vol 000
[15] Kuhn, R.; Münzing, W. N-Halogen-acylamide zur Darstellung
von Tetrazolium-, Triazolium- und 8-Aza-indazolium-Salzen. Chem Ber
1953, 86, 858.
[21] Ostrovskaya, V. M.; Lushina, O. T.; Dziomko, V. M.;
Davydovskaya, Y. A. Method of Obtaining 2H-tetrazolium Salts. USSR
Patent 636,230, 1978.
[16] Zhivich, A. B.; Koldobskij, S. I.; Martynova, V. P.; Ostrovskij,
V. A. Method of Obtaining Tetrazolium Salts. USSR Patent 1,361,147, 1986.
[17] Zhivich, A. B.; Koldobskii, G. I.; Ostrovskii, V. A. Production
of tetrazolium salts under conditions of phase-transfer catalysis. J Org
Chem USSR (English Translation) 1988, 24, 203.
[18] Belyakov, S. A. Production of 2,3,5-triaryltetrazolium salts.
J Org Chem USSR (English Translation) 1990, 26, 217.
[19] Belyakov, S. A. Production of 2,3,5-triaryltetrazolium chlo-
rides. J Org Chem USSR (English Translation) 1989, 25, 2034.
[20] Belyakov, S. A.; Markovskij, L. N.; Silina, T. F.; Litvinov,
V. K.; Merzlyak, N. I. Method of Obtaining Derivatives of 2,3,5-
triaryltetrazolium Chloride. USSR Patent 1,549,952, 1990.
[22] Benson, F. R.; Otten, E. A.; Schachat, R. E. Oxidation of
formazans to tetrazolium chlorides with t-butyl hypochlorite. J Am Chem
Soc 1954, 76, 1695.
[23] Katritzky, A. R.; Belyakov, S. A.; Lam, J. N.; Durst, H. D.;
Karpenko, D. V. 1-Chlorobenzotriazole-mediated ring closure of 1,3,5-
triarylformazans: improved syntheses of 2,3,5-triaryl-2H-tetrazolium
salts. Heterocycles 1994, 34, 73.
[24] Hegarty, A. F.; Coy, J. H.; Scott, F. L. The oxidative cycliza-
tion of formazans to tetrazolium salts. J Chem Soc, Perkin Trans 2,
1975, 204.
[25] Rees, C. W.; Storr, R. C. 1-Chlorobenzotriazole: a new
oxidant. Chem Commun 1968, 21, 1305.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet