Synthesis, protolytic equilibria, and antimicrobial action of nifuroxazide analogs

Article abstract of DOI:10.1016/j.molliq.2021.116911

The present paper reports on the synthesis of four hydrazones derived from 5-nitro-2-furfural, 5-nitro-2-thiophenal, isoniazid, 2,4- and 3,4-dihydroxy-N′-methylenebenzohydrazide. The acid-base dissociation constants of these compounds were determined in an aqueous solution. The protolytic equilibria-related ability of hydrazones to “sense” anions in dimethyl sulfoxide-containing water of different concentrations is studied using spectrophotometry, NMR spectroscopy, and quantum chemistry methods. The antimicrobial action of the hydrazones was tested and compared with that of the known drug nifuroxazide.

Full text of DOI:10.1016/j.molliq.2021.116911

Journal of Molecular Liquids xxx (xxxx) xxx
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Synthesis, protolytic equilibria, and antimicrobial action of nifuroxazide
analogs
b
a
a
G.A. Gamov a, , A.N. Kiselev , A.E. Murekhina , M.N. Zavalishin , V.V. Aleksandriiskii a,b, D.Yu. Kosterin c
⇑
a Research Institute of Thermodynamics and Kinetics of Chemical Processes, Ivanovo State University of Chemistry and Technology, 153000 Ivanovo, Sheremetevskii pr. 7, Russia
b G.A. Krestov Institute of Solution Chemistry, Russian Academy of Sciences, 153045 Ivanovo, Akademicheskaya str. 1, Russia
c D.K. Belyaev Ivanovo State Academy of Agriculture, 153012 Ivanovo, Sovetskaya str. 45, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
The present paper reports on the synthesis of four hydrazones derived from 5-nitro-2-furfural, 5-nitro-2-
thiophenal, isoniazid, 2,4- and 3,4-dihydroxy-N0-methylenebenzohydrazide. The acid-base dissociation
constants of these compounds were determined in an aqueous solution. The protolytic equilibria-
related ability of hydrazones to ‘‘sense” anions in dimethyl sulfoxide-containing water of different con-
centrations is studied using spectrophotometry, NMR spectroscopy, and quantum chemistry methods.
The antimicrobial action of the hydrazones was tested and compared with that of the known drug
nifuroxazide.
Received 12 April 2021
Revised 29 June 2021
Accepted 1 July 2021
Available online xxxx
Keywords:
Hydrazone
Nifuraxozide
Ó 2021 Elsevier B.V. All rights reserved.
5-nitro-2-furfural
Acid-base equilibria
1. Introduction
ing way of increasing the solubility is the introduction of more
hydroxyl groups into the molecule.
Nifuroxazide
(5-nitrofurfurylidene
hydrazide
of
4-
In this paper, we aim to synthesize nifuroxazide analogs that
contain more hydroxylic groups in the benzene ring or are replaced
by a 4-pyridinyl moiety. Thiophene-containing compounds were
also obtained (Fig. 1). We observe difficulties in the synthesis of
the di- or tri-hydroxybezoyl derivatives and propose mechanism
of the side product accumulation, which might be helpful to the
researchers making efforts to improve nifuroxazide. We also focus
on the protolytic equilibria of these ligands, as the protonation
constants are associated with pharmacokinetic characteristics [9],
and determination of pKa is among the most important require-
ments in drug discovery [9]. Moreover, we demonstrate that the
anion ‘‘sensing” ability of hydrazone is an indication of the change
in pH of a solution. In other words, the anion discovery with the
help of hydrazones is also related to their acid-base properties. A
primary antimicrobial activity test was performed to assess the
feasibility of the selected chemical modification of nifuroxazide.
It should be noted that compound 3 [10–13] had been previ-
ously synthesized. A derivative of 5-nitro-2-formylthiophene sim-
ilar to compound 4 was also obtained [14]. Moreover, their
antimicrobial action was tested against mycobacteria [11] and
other bacteria and fungi [12–14]. However, compounds 1, 2, and
4 are synthesized for the first time.
hydroxybenzoic acid) is an oral antibiotic [1] used for intestinal
infection curation [2]. It is effective against both gram-positive (Sta-
phylococcus spp., Streptococcus spp.) and gram-negative (Salmonella
spp., Klebsiella spp., Enterobacter spp., Escherichia coli, Shigella spp.,
Proteus spp., Hemophilus influenzae) microorganisms. However, it
should be noted that nifuroxazide along with other nitrofuran
derivatives increases the capacity of Pseudomonas aeruginosa and
Burkholderia cenocepacia pathogenic bacteria to form biofilms at
concentrations not inhibiting bacterial growth [3]. In addition to
antibiotic action, nifuroxazide can be useful for the treatment of
myeloma, as it inhibits the survival of multiple myeloma cells [4].
Moreover, recently, the potent antitumor and antimetastatic activ-
ity of nifuroxazide in melanoma was described [5]. Nifuroxazide is
low-toxic, safe for oral administration [6], and highly bioavailable
(>90%) [7]. However, its poor solubility in water makes chemists
search for soluble crystal forms of nifuroxazide [8], while perme-
ability enhancement requires chemical modification of the mole-
cule [7]. A significant increase in permeability was achieved using
a thiophene moiety instead of the furan cycle [7]. Another promis-
⇑
Corresponding author.
E-mail address: ggamov@isuct.ru (G.A. Gamov).
https://doi.org/10.1016/j.molliq.2021.116911
0167-7322/Ó 2021 Elsevier B.V. All rights reserved.
Please cite this article as: G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al., Synthesis, protolytic equilibria, and antimicrobial action of nifuroxazide analogs,
Journal of Molecular Liquids, https://doi.org/10.1016/j.molliq.2021.116911

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
Fig. 1. Structures of compounds synthesized and studied in the present work.
separated using column chromatography on silica gel. The
mixture of THF:hexane (1:4) was used as an eluent. The first
bright yellow fraction containing 4-nitro-2-formylthiophene
2. Experimental
2.1. Chemicals
yielded
a light brown solid after solvent evaporation. Yield
0.537 g (38%).
Furfural, 2-formylthiophene, 2,3- or 2,4-dihydroxybenzoic
acids, and isoniazid (Sigma-Aldrich, USA) were used without addi-
tional purification. The purity of reagents claimed by the manufac-
turer was >99% wt. Dimethyl sulfoxide (EKOS-1, Russia, wt. >99.9%)
was also used as purchased. Na3PO4ꢀ12H2O (Spektr-Khim, Russia)
and preliminarily standardized HCl were used for the fine-tuning
of pH solutions. The buffer acidity was controlled potentiometri-
cally. Small volumes (25 mL typically) of aqueous solutions of Na
or K salts containing different anions were added to the DMSO
solution of ligands 2 and 3 (25 mL typically). No precipitation of
salts was observed.
5-nitro-2-formylfuran (h). 32.2 mL of acetic anhydride and
10.9 mL of ice acetic acid were placed into a three-necked round-
bottom flask equipped with a drip funnel, thermometer, and stir-
rer. The mixture was cooled to 0 °C, and the nitrating mixture
(3.2 mL of fuming nitric acid and 0.12 mL of concentrated sulfuric
acid) was slowly added under intensive stirring keeping the tem-
perature below 10 °C. Then, the reaction mixture was cooled to
0 °C, and 5.5 mL (66.5 mmol) of freshly distilled furfural was grad-
ually added while keeping the temperature within the range of 0–
5 °C. The reaction mixture was stirred at 0 °C for 30 min, and then
41 mL of cool (7 °C) water was added. During 1 h, 16 mL of 25%
NaOH was added while maintaining the temperature at 35–
40 °C. After alkali addition, the reaction mixture was heated to
50 °C, allowed to stay at this temperature for 1 h, and cooled.
The precipitated 5-nitrofurfural diacetate was filtered and washed
with water. Yield 9.7 g (60%).
2.2. Synthesis of hydrazones 1–4
The precursors, as well as the hydrazones, were synthesized
according to Scheme 1. The procedures in detail are described
below:
Hydrazones 1, 2. First, 1.4 g (5.75 mmol) of 5-nitrofurfural diac-
etate was gradually added to the boiling mixture of 3 mL of water
and 0.550 mL of concentrated H2SO4. The mixture was refluxed for
10 min. Then, 13.5 mL of water was added, and the mixture was
refluxed again until complete 5-nitrofurfural dissolution. The
resulting solution was treated with activated carbon and filtered.
The filtrate was neutralized by the saturated NaHCO3 solution.
The yellow solution became reddish-brown after that. Then, the
solution was divided into two equal parts, and boiling solutions
of 0.7 g (4.16 mmol) of d were added. Reddish-brown crystals were
formed. After cooling, the product heavily contaminated with 1,2-
bis((5-nitrofuran-2-yl)methylene)hydrazine (see the Discussion)
was filtered and recrystallized multiple times from ethanol. Yield
120–250 mg (10–20%).
Hydrazone 3. First, 1.4 g (5.75 mmol) of 5-nitrofurfural diac-
etate was gradually added to the boiling mixture of 3 mL of water
and 0.550 mL of concentrated H2SO4. The mixture was refluxed for
10 min. Then, 13.5 mL of water was added, and the mixture was
refluxed again until complete 5-nitrofurfural dissolution. The
resulting solution was treated with activated carbon and filtered.
The filtrated solution was heated until boiling and added to a boil-
ing aqueous solution (30 mL) of 0.788 g (5.75 mmol) of isoniazid.
The readily precipitated product was filtered and recrystallized
from ethanol. Yield 897 mg (60%).
2,4- and 3,4-dihydroxybenzoic chloro anhydrides (b). Ten
grams (64.8 mmol) of dihydroxybenzoic acid was placed into a
single-necked round-bottom flask. Three drops of DMF and
15 mL of freshly distilled thionyl chloride were then added, and
the reaction mixture was stirred at room temperature for 2 h.
Finally, the excess SOCl2 was driven away using the rotor evapora-
tor at reduced pressure. Yields were not determined at this stage.
Ethyl ethers of 2,4- and 3,4-dihydroxybenzoic acids (c). The
flask containing precursor b synthesized in the previous step was
cooled in an ice bath. Then, 15 mL of ethanol was added gradually
while maintaining the temperature at 0 °C. The reaction mixture
was stirred for 30 min, and then the excess ethanol was evapo-
rated. The precipitated residue in the flask was washed with a
small quantity of cool water and dried. The crude product was then
recrystallized from ethanol. Yield 95%.
Hydrazides of 2,4- and 3,4-dihydroxybenzoic acids (d). Six
grams (32.9 mmol) of ethyl ether of dihydroxybenzoic acid was
placed into a single-necked round-bottom flask. Then, 20 mL of
ethanol and 4.3 mL of 80% hydrazine hydrate (115 mmol) were
added, and the reaction mixture was refluxed for 9 h. The excess
ethanol and N2H4ꢀH2O were driven away using the rotor evapora-
tor at reduced pressure. The oily crude product was again dissolved
in hot ethanol and cooled; the precipitated light brown crystals
were filtered. Yield 4.03 g (73%).
Hydrazone 4. Five hundred milligrams (3.18 mmol) of f was
placed into a glass and dissolved in 20 mL of ethanol under heating.
Into the second glass, 436 mg (3.18 mmol) of isoniazid and 20 mL
of water were placed. Then, the solutions were boiled and united. A
bright yellow hydrazone precipitated after cooling was filtered,
washed with water, and recrystallized from ethanol. Yield
571 mg (65%).
4-nitro-2-formylthiophene (f). Twelve milliliters of concen-
trated H2SO4 was placed into a two-necked round-bottom flask
with a thermometer and stirrer. Then, 1 g (9 mmol) of e was grad-
ually added while maintaining the temperature at 0 °C. Then, 1.2 g
(11.8 mmol) of KNO3 was gradually added at intense stirring and a
temperature of 0 °C. The resulting orange solution was stirred for
30 min at 0 °C and then poured out onto ground ice. The precipitate
formed was filtered and washed with water until the neutral reac-
tion of the medium. The crude product contains a mixture of 4- and
5-nitro regioisomers. The required 4-nitro-2-formylthiophene was
1H, 13C NMR, 2D 1H, 13C HSQC analysis performed at pD ca. 13
confirmed the success of synthesis (see data below) and the
absence of impurities.
2

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
(4J = 1.8 Hz, 1H, Phenyl3). 13C NMR (DMSO d6), d, ppm: 165.8
(C@O), 163.5, 162.3 (Phenyl2, 4), 152.4 (Furan5), 152.2 (Furan2),
135.9 (ACH@), 130.7 (Phenyl6), 115.8 (Furan4), 115.1 (Furan3),
108.2 (Phenyl5), 106.9 (Phenyl1), 103.3 (Phenyl3). IR, cmꢁ1
:
3380vs, 2920s, 2851 m, 1644 m, 1597vs, 1535 m, 1458 m,
1377 m, 1257s, 1186 m, 1151 m, 810 m, 622w.
5-Nitro-2-furoyl-3,4-dihydroxybenzocarbohydrazone (ligand
2): m/z = 290.49 [L-Hꢁ], 1H NMR (DMSO d6), d, ppm: 11.98s (1H,
NH), 9.74s, 9.33s (2H, OH), 8.38s (1H, ACH@), 7.78 d (3J = 3.9 Hz,
1H, Furan4), 7.37d (4J = 2.0 Hz, 1H, Phenyl2), 7.31dd (3J = 8.2 Hz,
4J = 2.0 Hz, 1H, Phenyl6), 7.22d (3J = 8.2 Hz, 1H, Phenyl5), 6.85d
(3J = 3.9 Hz, 1H, Furan3). 13C NMR (DMSO d6), d, ppm: 163.7
(C@O), 152.6 (Furan5), 152.3 (Furan2), 150.0 (Phenyl4), 145.6
(Phenyl3), 134.8 (ACH@), 124.1 (Phenyl1), 120.4 (Phenyl6), 116.0
(Phenyl2), 115.2 (Phenyl5), 115.6 (Furan3), 115.1 (Furan4). IR,
cmꢁ1: 3474vs, 3421vs, 2920vs, 2851s, 1665 m, 1603 m, 1552 m,
1510 m, 1472s, 1359 m, 1292vs, 1250s, 1141 m, 1025w, 812w,
643w.
5-Nitro-2-furoyl-isonicotinoylhydrazone (ligand 3): m/
z = 259.43 [L-Hꢁ], 1H NMR (DMSO d6), d, ppm: 8.81d (3J = 4.8 Hz,
2H, Pyridine2,6), 7.78s (1H, ACH@), 7.83d (3J = 4.8 Hz, 2H,
Pyridine3,5), 7.80d (3J = 3.9 Hz, 1H, Furan4), 7.32d (3J = 3.9 Hz, 1H,
Furan3). 13C NMR (DMSO d6), d, ppm: 162.5 (C@O), 152.5 (Furan5),
151.8 (Furan2), 151.4 (Pyridine2,6), 140.3 (Pyridine4), 137.7
(ACH@), 122.0 (Pyridine3,5), 116.5 (Furan3), 115.0 (Furan4). IR,
cmꢁ1
: 3422s, 2920vs, 2851s, 1668s, 1623 m, 1555s, 1519s,
1480s, 1385 m, 1353s, 1321 m, 1281s, 1145 m, 814w, 687w.
4-Nitrothiophenyl-2-methylene isonicotinoylhydrazone (li-
gand 4): m/z = 275.49 [L-Hꢁ], 1H NMR (DMSO d6), d, ppm: 12.35s
(1H, ANHA), 8.87s (1H, ACH@), 8.80 d (3J = 5.9 Hz, 2H, Pyridine2,6),
8.67d (3J = 1.1 Hz, 1H, Thiophene5), 8.17d (3J = 1.1 Hz, 1H, Thio-
phene3), 7.82d (3J = 5.9 Hz, 2H, Pyridine3,5). 13C NMR (DMSO d6),
d, ppm: 162.2 (C@O), 150.9 (Pyridine2,6), 148.2 (Thiophene4),
142.6 (Thiophene5), 140.8 (Thiophene2), 140.5 (Pyridine4), 132.2
(ACH@), 125.2 (Thiophene3), 122.0 (Pyridine3,5). IR, cmꢁ1: 3421s,
2920s, 2850s, 1670vs, 1605w, 1538s, 1390vs, 1344s, 1301s,
1182w, 843w, 586 m.
2.3. Spectroscopic studies
The UV–Vis spectra of dimethyl sulfoxide solutions of ligands 2
and 3 (10ꢁ4 mol Lꢁ1) and anions (PO34ꢁ, HPO42ꢁ, H2PO4ꢁ, Clꢁ, HFꢁ2 ,
SO42ꢁ, SO23ꢁ, OH–, CH3COO–, CN–, NO–3 of 10ꢁ4 mol Lꢁ1), as well as
aqueous solutions of ligands 1–4 at different pH values, were reg-
istered on a double-beamed Shimadzu UV1800 spectrophotometer
in the wavelength range of 220–700 nm and absorbance range of
0–2. The pure solvent was used as a blank solution. The error of
wavelength determination did not exceed 0.5 nm, and the maximal
inaccuracy of absorbance measurements was 0.006 units. The
temperature maintained at 298.2
thermostat.
0.1 K using an external
Typically, a titration of 2.7 mL of 9.5ꢀ10ꢁ5 to 1.80ꢀ10ꢁ4 mol Lꢁ1
of 1–4 solution in water containing 0.01 mol Lꢁ1 Na3PO4 by 20 to
25 additions of 1.1885 mol Lꢁ1 HCl (4 mL each using the Transfer-
pette (Brand, Germany) pipette) was performed, which corre-
sponds to pH variation within the range of 11.5–1.5 units.
Phosphate was chosen due to its buffer properties to ensure the
smooth variation of [H+] during the experiment. pH values were
measured directly in the spectrophotometric cell using an I-160
MI laboratory ion meter (Izmeritel’naya Tekhnika, Russia)
equipped with a 3 mm InLab Microelectrode (Mettler Toledo,
Switzerland). The potentiometric setup was preliminarily cali-
brated using six standard buffer solutions with pH values of 1.65,
3.56, 4.01, 6.86, 9.18, and 12.43. An ionic strength value of
Scheme 1. Synthesis of hydrazones 1–4.
5-Nitro-2-furoyl-2,4-dihydroxybenzocarbohydrazone (ligand
1): m/z = 290.49 [L-Hꢁ], 1H NMR (DMSO d6), d, ppm: 11.99s (2H,
OH), 10.33s (1H, NH), 8.39s (1H, ACH@), 7.79 d (3J = 3.8 Hz, 1H,
Furan4), 7.77d (3J = 8.7 Hz, 1H, Phenyl6), 7.24d (3J = 3.8 Hz, 1H,
Furan3), 6.39dd (3J = 8.7 Hz, 4J = 1.8 Hz, 1H, Phenyl5), 6.34d
3

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
1.0 mol Lꢁ1 was maintained using NaCl as an indifferent elec-
trolyte. Such a high amount of background electrolyte was neces-
sary to negate the variations in ionic strength related to the
neutralization of phosphate during the titration.
3. Results and discussion
3.1. Protolytic equilibria interfere with hydrazone synthesis
The solution of ligand 2 in DMSO (5ꢀ10ꢁ5 mol Lꢁ1) and aqueous
dimethyl sulfoxide (XDMSO = 0.6–0.9 mol. fractions) was titrated by
10 injections of 0.0528 mol Lꢁ1 aqueous solution of CH3COONa
(0.5–12.5 mL each, the volume of injection depended on the water
content; the higher XH2O is the larger the Vtitr) without indifferent
electrolyte. The corresponding mixed solvent was used as a blank.
The protonation constants were calculated from the spec-
trophotometric data using KEV software [15].
Protons play an important role in several reactions in organic
chemistry as either reagents or catalysts [27]. Protolytic equilibria
strongly (and negatively) influence the synthesis of hydrazones 1
and 2. As we mentioned in Section 2.2, the crude product formed
after uniting the aldehyde and hydrazide solutions is heavily con-
taminated with 1,2-bis((5-nitrofuran-2-yl)methylene)hydrazine,
especially in the case of ligand 2. What is the reason for its forma-
tion? The most likely explanation is the following.
To determine the dissociating functional groups of different
hydrazones, NMR experiments for mixtures of compounds 2 and
3 (solvent DMSO d6) of ca. 0.1 mol Lꢁ1 with CH3COONa of 0.014
to 0.126 mol Lꢁ1 was performed on a Bruker Avance III 500 NMR
spectrometer with 1H and 13C operating frequencies of 500.17
and 125.77 MHz, respectively. Temperature control was achieved
using a Bruker variable temperature unit (BVT-2000). Experiments
were run at 298 K without sample spinning. The inaccuracy of
chemical shift measurement with respect to the external standard
HMDSO (Sigma Aldrich) was evaluated as 0.01 ppm for 1H NMR
spectra and 0.1 ppm for 13C NMR.
The proton of the para-phenolic OH group is labile and can
easily join the solvating molecules. Another proton of the meta-
OH group forms an intramolecular hydrogen bond with deproto-
nated oxygen. Emerging excess electron density through meso-
meric effects shifts toward the carbonyl group of hydrazone, thus
increasing its nucleophilicity. As a result, a hydrazone becomes a
stronger nucleophile than the hydrazide of the benzoic acid and
quickly attacks formyl carbon in aldehyde. The residue of dihy-
droxybenzoic acid (2,4-dihydroxybenzoyl, in particular) is a good
leaving group. In water, 2,4- or 3,4-dihydroxybenzoyl transforms
into the corresponding acid via the addition of OH–. One OH group
does not interfere with hydrazone formation (as in the case of
nifuroxazide resulting from 4-hydroxybenzoic acid), while two or
more hydroxyl groups are substituted into the benzene ring of
the molecule, leading to the prevailing formation of 1,2-bis((5-nitro
furan-2-yl)methylene)hydrazine. Scheme 2 illustrates the ideas
above. The unwanted side process can probably be prevented from
occurring if the second hydroxyl group is alkylated or involved in a
complex with d-metal ions, thus negating the influence of the
substituent.
The standard pulse sequence [16] from TopSpin 3.6.1 software
was used for 1H NMR spectra registration. The width of the spectral
window was 7183.908 Hz with a dot number TD of 32768. The 13
C
NMR spectra were also registered using a standard pulse sequence
with power-gated decoupling (waltz16) within the spectral range
of 22321.42857 Hz, and dot number TD of 32768.
The 2D 1H–13C HSQC (1H–13C correlation via double inept trans-
fer) [17–19] spectrum was recorded in phase-sensitive mode using
field gradient pulses. The spectrum was recorded with 2048 points
in the F2 direction and 256 points in the F1 direction. The spectral
range, as well as other parameters of this experiment, were taken
from 1D 1H and 13C NMR runs settings.
It is worth noting that the other ways of increasing the main
product yield, such as pH variation in the range of 5 to 10 and sol-
vent change from water to aqueous ethanol and aqueous DMSO up
to XH2O = 0.5 m.f., were tested – to no avail, unfortunately. Using
gallic acid hydrazide instead of dihydroxyl derivatives also leads
to zero hydrazone yield; 1,2-bis((5-nitrofuran-2-yl)methylene)hy
drazine is the only product.
2.4. Quantum chemical computation details
The geometry optimization, calculation of harmonic frequen-
cies, and IR spectra of probable 2 and 3 molecular models were car-
ried out by the Gaussian 09W software package [20] employing the
hybrid DFT computational method B3LYP [21]. The O, N, C, and H
atomic electronic shells were described by the cc-pvtz [22] basis
set. The correspondence of the optimized structures to the global
minima on the potential energy surface was checked via vibra-
tional spectra calculations. All the Hessians for the models were
positive. To take into account the effect of the solution of dimethyl
sulfoxide, all calculations were performed using the polarizable
continuum model (PCM). The calculations of ligands 2 and 3 and
their anions were performed in the framework 0 or ꢁ1.00e charge
value and singlet electronic state. The visualization of molecular
models was made using ChemCraft software [23]. NMR shielding
constants were calculated using the GIAO method [24]. According
to recommendations [25,26], benzene and methanol were chosen
as the standards for calculating the chemical shifts of sp2- and
sp3-hybridized carbons, respectively. The reference compound
geometry and shielding parameters were calculated at the same
theoretical level as the studied hydrazones. The chemical shifts di
of the hydrazones were calculated according to the equation:
Compounds 1 and 2 can still be advantageous even considering
their costly synthesis and purification due to potentially better sol-
ubility and bioavailability (which requires additional experiments
to be proven).
However, our results suggest that, probably, other ways of nifu-
raxozide chemical modifications should be preferred.
3.2. Hydrazones protonation in water
UV–Vis spectra of different hydrazones 1–4 dissolved in alkali
medium show similar alterations under strong acid addition and
decreasing pH (Fig. 2):
For all studied hydrazones, deprotonated species are present at
the beginning of the titration in the alkali pH range. The absorption
bands 413; 324 nm for 1 (Fig. 2a), 388; 317 nm for 2 (Fig. 2b), 407;
273 nm for 3 (Fig. 2c), 340 nm for 4 (Fig. 2d) in the spectra are
assigned to the anions. The gradual addition of the strong acid
leads to a decrease in the intensity of these bands, while new peaks
blue-shifted by 15–40 nm appear, namely, 391; 286 nm for 1
(Fig. 2a), 373; 284 nm for 2 (Fig. 2b), 365; 241 nm for 3; 325;
278 nm for 4. The only consistent smooth transition is observed
in the spectra of 1 and 2 (Fig. 2a, b), corresponding to the only pro-
tonation process that occurs during the transition from alkali to
neutral medium. In acidic solution, the spectral changes are negli-
gible. However, the situation is different for pyridine-containing 3
(Fig. 2c) and 4 (Fig. 2d) hydrazones. In strongly acidic media,
di ¼ rref
ꢁ
ri þ dref
ð1Þ
where rref and ri are the shielding constants calculated for hydra-
zones and standards, and dref is an experimental chemical shift of
the reference compound (128.5 ppm for benzene 13C NMR).
4

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
Scheme 2. Probable pathway of side product formation interfering with 1,2 synthesis.
additional spectral changes could be observed, namely, a decrease
in absorbance at 273 (3) or 278, 325 (4) nm and an increase in
absorbance at 241 (3), 265 (4) nm, while the peak at 365 nm
remained intact for hydrazone 3. These spectral variations result
from the second protonation process, occurring in the pyridine
moiety at low pH values.
Fig. 2. Changes in UV–Vis spectra of aqueous solutions of hydrazones 1 with C0 = 1.02ꢀ10ꢁ4 mol Lꢁ1 (a), 2 with C0 = 1.74ꢀ10ꢁ4 mol Lꢁ1 (b), 3 with C0 = 9.63ꢀ10ꢁ5 mol Lꢁ1 (c),
and 4 with C0 = 1.13ꢀ10ꢁ4 mol Lꢁ1 (d) under decreasing pH at T = 298.2 K and I = 1.0 mol Lꢁ1 (NaCl).
5

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
Interestingly, the longwavelength maximum of compound 4
experiences a strong hypsochromic shift compared to hydrazone
3. This is mostly the influence of different placement of nitro
groups in the five-membered heterocycle (site 4 instead of site
5). Quantum chemical calculations (TD DFT, Fig. S3) confirm the
hypsochromic shift by ca. 40 nm while transferring from 5-
nitrothiophene to a 4-nitrothiophene-containing moiety. Geome-
try optimization shows that position 4 of the nitro group disturbs
the conjugation between aromatic cycles, making the hydrazone
molecule highly nonplanar.
The calculated values of protonation constants are given
(Table 1).
The errors in Table 1 are the half-widths of the confidence inter-
val at the confidence probability of 0.95 and the sample size of 3–5
experiments.
NMR spectra of 2 and 3 ligands dissolved in DMSO d6 in the pres-
ence of an increasing amount of acetate. The largest changes in
chemical shifts could be expected for nuclei neighboring the func-
tional groups involved in dissociation. For example, if hydroxyl
groups of the dihydroxybenzoic acid moiety lose a proton, the most
significant changes should be observed for carbons bound to those
groups. In contrast, if the ANHA group dissociates, the resonances
of ACH@ as well as other carbons from both six- and five-
membered aromatic cycles should shift.
Indeed, for different hydrazones 2 and 3, the different nuclei
resonances shift (Table 2).
The most significant changes in the 13C NMR spectrum of hydra-
zone 2 (DMSO d6) upon the addition of acetate are observed for the
C4 and C1 nuclei of the benzoic acid residue (and somewhat large
alteration is noted for C3). Other signals remain almost undisturbed.
The similarity of the spectral changes under strong acid addi-
tion to the alkali solution of hydrazones suggests approximately
the same dissociation mechanism for different hydrazones.
Namely, we believe the central ANHA group dissociates in an
aqueous solution for both dihydroxybenzoic acid and pyridine
derivatives. However, the situation is different in dimethyl sulfox-
ide solution (see the following Section for details).
In contrast, for hydrazone 3, Dd > 0.5 ppm is observed for
different carbons located in both aromatic cycles and the central
–(C@O) ANHAN@CHA group. Therefore, it could be assumed that
when dissolved in DMSO, hydrazone 2 dissociates through the 4-
OH group in the dihydroxybenzoic acid moiety, while pyridine
derivative 3 loses protons from the ANHA group when acetate is
added.
These conclusions were confirmed by quantum chemical NMR
calculations (Table S1). Comparing the carbon chemical shifts of
neutral molecule 2 with those obtained for the structure resulting
from OH dissociation, one can see a strong downfield shift for C4
and C3 of the phenyl residue and an upfield shift of C1 and C2 car-
bon resonances. In contrast, NH group dissociation leads to the
shielding of C4 and deshielding of C1, which is not observed in
the experiment. Interestingly, GIAO calculations predict no change
in the ACH@ chemical shift after NH dissociation.
Since the NMR study was performed in dimethyl sulfoxide solu-
tion, where dissociation might be hindered in favor of the associa-
tion, we also calculated the chemical shifts for hydrazone 2
associated with acetate. The most stable associate involving the
phenyl moiety is the one where two hydrogen bonds with 3- and
4-OH groups are formed. Although the chemical shifts of C4 and
C1 behave in the same way as in the experiment, such an associa-
tion can be rejected, as it also predicts strong deshielding of C2 and
C5, which is not supported by the experimental data. Acetate asso-
ciation with NH groups leads to the relatively slight alteration of
furan C2 and C3 and phenyl C2 and C5 carbons as well as shielding
of ACH@. The changes in 13C NMR spectra observed for double
associate (one acetate binds to 4-OH group while another binds
to ANHA group) are also inconsistent with the experimental data
as the computation predicts the shielding of Phenyl6 and an upfield
shift for Furan3 carbon while both nuclei resonances undergo a
downfield shift.
3.3. Hydrazones protonation in DMSO and aqueous DMSO
We observed previously [28] that pyridoxal-derived hydrazones
dissolved in DMSO are sensitive to the presence of anions of weak
(in water) acids if no buffer solution is used. This made us assume
that the reason for the sensing ability of the compounds is pro-
tolytic equilibria between ligand and anion, i.e., ligand acts as an
acid-base indicator. In this regard, it is interesting to check
whether the same applies to nifuroxazide analogs and shed more
light on ligand-anion interactions using NMR techniques combined
with quantum chemical calculations. For that aim, two different
hydrazones, 2 and 3, were chosen.
First, we ensured that hydrazones indicate only weak acid
anions. Bright yellow solutions of hydrazones 2 and 3 become
brownish (2) and crimson (3) in the presence of anions such as
PO34ꢁ, OH–, CH3COO–, and CN– and remain of the same color if Clꢁ
or NO–3 is added. The corresponding changes in UV–Vis spectra
are shown (Fig. 3). Ligand 3 can sense even SO24ꢁ, as it is (in water)
a medium-strength base on its first protonation stage.
The addition of water to the mixture of hydrazones 2 and 3 and
acetate quickly decreased the intensity of the longwavelength
absorption band (Fig. 3c, d). This can be explained by the decrease
in the basicity of the anion when it gradually transfers from DMSO
to water. The base (anion) weakening due to the medium change is
less capable of inducing the dissociation of the hydrazone. Acetate
basicity decreases faster than that of phosphate ions (Fig. S1).
Despite the similar spectral change in the aqueous solutions of
different hydrazones (Fig. 2b, c), dimethyl sulfoxide makes UV–Vis
spectra of hydrazones 2 and 3 behave differently under the addi-
tion (or removal) of anions of different basicities. The probable rea-
son can be the dissociation of different functional groups under a
base addition. To investigate this further, we measured the 13C
Analogously, it is confirmed that hydrazone 3 dissociates and
forms no associate with acetate, as the GIAO NMR results predict
deshielding of Furan2 and Pyridine4 and shielding of Furan3 car-
bons for anion compared to the acid (which is consistent with
the observed trends), while associate shows the opposite trend of
these carbons (shielding of Furan2 and Pyridine4, and deshielding
of Furan3).
Table 1
Protonation constants of hydrazones 1–4 in aqueous solution at T = 298.2 K and I = 1.0 mol Lꢁ1 (NaCl).
Hydrazone
1
2
3
4
a
lg b1
11.22 0.11
–
11.75 0.14
–
11.21 0.01
12.95 0.23
11.37 0.04
13.33 0.24
b
lg b2
a
b1 = [HL]ꢀ[H]ꢁ1ꢀ[L]ꢁ1
.
b
b2 = [H2L]ꢀ[H]ꢁ2ꢀ[L]ꢁ1
.
6

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
Fig. 3. UV–Vis spectra of ligands 2 (a) and 3 (b) in the presence of different anions in dimethyl sulfoxide at T = 298.2 K and anion/hydrazone ratio = 2:1. Changes in UV–Vis
spectra of aqueous DMSO solutions of hydrazone 2 + acetate (c) or hydrazone 3 + acetate (d) with increasing water content from 0.01 to 0.5 mol fraction.
Table 2
Chemical shifts of hydrazones 2 and 3 at different equivalents of AcOꢁ added. The most significant increments in the chemical shift are bolded.
equivalents of AcOꢁ added
0
0.25
0.5
0.75
1.0
Parameter
Hydrazone 2
Furan5
Furan2
Phenyl4
d0, ppm
D
d = d – d0, ppm
152.6
152.3
150
0.1
0.1
0.1
0.1
ꢁ0.1
ꢁ0.1
ꢁ0.1
ꢁ0.1
0.8
1.3
1.4
1.5
Phenyl3
145.6
134.8
0.3
ꢁ0.1
0.4
ꢁ0.1
0.4
ꢁ0.1
0.4
ꢁ0.1
ACH@
Phenyl1
Phenyl6
Phenyl2
Furan3
Phenyl5
Furan4
124.1
120.4
115.9
115.6
115.2
115.1
ꢁ0.7
0.1
ꢁ0.1
0.1
0.1
0
ꢁ1.1
0.2
ꢁ0.2
0.2
0.1
0
ꢁ1.2
0.2
ꢁ0.2
0.2
0.1
0
ꢁ1.4
0.2
ꢁ0.3
0.2
0.1
0
Hydrazone 3
162.5
0.6
1.1
–
–
C@O
Furan5
Furan2
Pyridine4
Pyridine3,5
152.6
151.8
140.4
122.1
137.2
ꢁ0.2
ꢁ0.3
–
–
–
–
–
–
–
–
–
–
0.6
0.9
0.7
0.1
ꢁ0.3
ꢁ0.1
ꢁ0.6
0.2
1.1
0.1
ꢁ0.5
ACH@
Pyridine2,6
Furan3
150.9
116.5
115
ꢁ0.2
–
–
–
–
–
–
ꢁ1
Furan4
0.3
7

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
O
2OꢁDMSO
D
GHLꢁ
DtrGHL
ꢁ
DtrGL ¼ ð
D
GHHL2OꢁDMSO
ꢁ
D
GHHL2 Þ ꢁ ð
ꢁ
Therefore, hydrazones 2 and 3 dissociate in dimethyl sulfoxide
medium under acetate addition, and different groups are involved:
4-OH for Schiff base 2 and ANHA for hydrazone 3.
2O
ꢁ
D
GHLꢁ
Þ
ð6Þ
ð7Þ
Acetic acid dissociation in aqueous dimethyl sulfoxide is well
studied [29–36] (Fig. 4). As shown in Fig. 4, the protonation con-
stant increases almost linearly when the DMSO concentration
increases.
The known dissociation constants for acetic acid (determined
using the linear regression equation pKa = a + bXDMSO) in aqueous
dimethyl sulfoxide make it possible to determine the protonation
constants of hydrazones using the following chemical model:
D
Gr ¼ ꢁRTlnK
where DtrGr is the change in Gibbs energy of the reaction transfer
from water (where it is described by DtrGHr 2O) to aqueous dimethyl
sulfoxide (DtrGHr 2O-DMSO), DtrGH+, HL, L- is a change in Gibbs energy of
proton (adopted from [37]), protonated ligand and anion transfer
from water to aqueous dimethyl sulfoxide. The results are given
(Fig. 5).
ꢁ
AcO + Hþ = AcOH
ð2Þ
As expected, the protonated ligand is much more efficiently sol-
vated in DMSO solutions than the anion. Proton transfer con-
tributes mainly to the observed Gibbs energy of the reaction
transfer except for XDMSO = 0.99, where the compensation of DtrGH+
Hydr + Hþ = HydrH
ð3Þ
ꢁ
Examples of UV–Vis titration of hydrazone 2 by acetate in aque-
ous DMSO at different XDMSO (mol. f.) are given in ESI (Fig. S2). The
protonation constants determined were collected (Table 3).
The errors in Table 3 are the half-widths of the confidence inter-
val at the confidence probability of 0.95 and the sample size of 3–5
experiments.
The protonation constant of hydrazone 2 depends on the binary
solvent composition nonlinearly. It decreases when the DMSO con-
tent increases from 0 to 0.6 mol. f. then increases in the range of
and DtrGHL
- DtrGL- is observed.
3.4. Antimicrobial action of hydrazones 1–4
The antimicrobial action of hydrazones 1–4 synthesized in the
present work and nifuroxazide as a standard antibiotic was tested
using a zone of inhibition method (diffusion of the test compound
into agar inhibiting the growth of test cultures). S. aureus, B. subtilis
(gram-positive bacteria), E. coli (gram-negative bacteria), and C.
albicans (fungi) were inoculated on meat peptone agar and placed
in Petri dishes. Solutions of hydrazones, either aqueous in phos-
phate buffer, pH 7.0 (diluted solutions) or dimethylformamide as
in the original patent [1] (concentrated solutions), were applied
to 5 mm pits in the medium. After one day of incubation in the
thermostat at 37 °C, the diameters of growth inhibition zones were
measured. Six concentrations of each hydrazone were tested: 10,
50, 250, 1000, 5000, and 10000 mg mLꢁ1 (except for 2, where only
10, 50, and 250 mg mLꢁ1 concentrations were used). The effect of
DMF itself was also controlled in the blank experiment. It slightly
inhibited only the growth of B. subtilis (d = 11 mm taking into
account the pit diameter, 5 mm). No synthesized hydrazone at
XDMSO = 0.6–0.8 mol. f., then reaches a minimum at XDMSO = 0.9 mol.
f., and returns to the same value as in water at XDMSO = 0.99.
We performed a thermodynamic analysis of this system consid-
ering the changes in Gibbs energy of transfer of the reaction, pro-
ton, and difference between protonated ligand and anion:
2OꢁDMSO
2O
DtrGr ¼
D
GrH
ꢁ
D
GrH
ð4Þ
ð5Þ
2OꢁDMSO
GHHþ
2O
GHHþ
þ
DtrGH
¼
D
ꢁ
D
Fig. 5. Changes in the Gibbs energy of the transfer of protons [37], protonated
ligands, anions, and protonation processes from water to aqueous dimethyl
sulfoxide.
Fig. 4. Literature data [29–36] on acetic acid protonation in aqueous dimethyl
sulfoxide at different ionic strength values.
Table 3
Protonation constants of hydrazone 2 in aqueous dimethyl sulfoxide at different XDMSO concentrations.
XDMSO, mol.f.
0.99
0.9
0.8
0.7
0.6
lg b1
11.76 0.13
10.08 0.12
10.71 0.10
10.69 0.10
10.46 0.13
8

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
Table 4
The diameters of the growth inhibition zones for hydrazones 1–3 and nifuroxazide (including pit diameter, 5 mm).
Compound
Concentration, mg mLꢁ1
Diameter of zone of growth inhibition, mm (including pit diameter,
5 mm) 2 mm
S. aureus
B. subtilis
E. coli
nifuroxazide
10
50
250
1000
5000
10,000
no effect
no effect
no effect
23
21
26
no effect
no effect
no effect
18
21
17
no effect
no effect
no effect
20
no effect
14
1
3
10
50
250
1000
5000
10,000
no effect
no effect
no effect
no effect
19
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
–
14
14
20
10
50
250
1000
5000
10,000
no effect
no effect
no effect
29
30
45
no effect
no effect
no effect
18
18
19
no effect
no effect
no effect
20
30
22
any concentration had any negative effect on C. albicans growth,
which is consistent with the data of [12]. The diameters of the
growth inhibition zones are given (Table 4).
The bacterial and fungal growth inhibition capability of hydra-
zones was tested. At any concentration, the hydrazone-containing
thiophene moiety did not affect the growth of S. aureus, B. subtilis
(gram-positive), or E. coli (gram-negative). In contrast, the com-
pound derived from 5-nitrofurfural and isoniazid showed activity
comparable to that of the standard analogous drug nifuroxazide.
Derivatives of dihydroxybenzoic acids are less active and more dif-
ficult to synthesize, making them less promising antimicrobial
agents. None of the hydrazones, including nifuroxazide, are active
against C. albicans.
Therefore, based on the results of the present contributions, it
could be concluded that the introduction of additional hydroxyl
groups alone is not a promising way of improving nifuroxazide
action. The decreased antimicrobial efficiency can probably be out-
weighed by the potential increase in solubility. However, the chal-
lenges in the synthesis of hydrazones containing di- or
trihydroxybenzoic moieties observed by us should be overcome
before further efforts to explore their biological action. The possi-
ble ways to solve the synthetic problems may include alkylation
of the additional OH groups or their involvement in chelate com-
plex formation with d-metal ions, which can also hinder side reac-
tions. Further studies in this field are required, and we will address
this problem in future contributions.
Hydrazone 4 has no effect on any of the studied strains (not
shown in Table 4), which is consistent with the results obtained
for the 5-nitro derivative [14]. Ligand 2 was also inefficient. Hydra-
zone 1 demonstrates weakened action when compared with
nifuroxazide, while ligand 3 is as efficient as the approved antibi-
otic. It should be noted that compounds 1 and 2 are difficult to syn-
thesize, show low gain in solubility in water compared with
nifuroxazide, and are less biologically potent than the latter. In
contrast, ligand 3 is simple for obtaining, good soluble, and rela-
tively efficient against different bacteria.
4. Conclusions and perspective
Four hydrazones, including three novel hydrazones, namely, 5-
nitro-2-furoyl-2,4-dihydroxybenzocarbohydrazone, 5-nitro-2-fur
oyl-3,4-dihydroxybenzocarbohydrazone, and 4-nitrothiophenyl-
2-methylene isonicotinoylhydrazone, were synthesized and char-
acterized using 1H, 13C, IR, and mass spectrometry.
The protolytic equilibria of the hydrazones were studied in an
aqueous solution. Hydrazones derived from dihydroxybenzoic
acids have the only protonation step, while compounds containing
pyridine moieties can accept additional protons in acidic media. In a
dimethyl sulfoxide medium, the hydrazones are capable of indicat-
ing only the presence of anions of weak (in water) acids. This capa-
bility is also explained by protolytic equilibria, namely, proton
transfer from hydrazone toward anions. 13C NMR measurements
combined with GIAO quantum chemical calculations suggest that
hydrazones dissociate rather than form associates with anions.
Moreover, different groups are involved in protolytic equilibria in
DMSO: the 4-OH group dissociates in hydrazone 2, while the ANHA
group dissociates in hydrazone 3. The sensitivity of hydrazones
toward anions quickly decreases when water is added to the DMSO
solution due to the decrease in the basicity of the anions. The pro-
tonation constants of hydrazone 2 in aqueous dimethyl sulfoxide
with various water contents were determined. Thermodynamic
analysis showed that proton transfer contributes mainly to the
observed Gibbs energy of the reaction transfer from water to the
binary solvent.
CRediT authorship contribution statement
G.A. Gamov: Conceptualization, Methodology, Formal analysis,
Writing - original draft, Funding acquisition. A.N. Kiselev: Investi-
gation, Resources. A.E. Murekhina: Investigation, Validation. M.N.
Zavalishin: Investigation, Validation. V.V. Aleksandriiskii: Investi-
gation, Validation. D.Yu. Kosterin: Investigation, Validation.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
The spectrophotometric experiments were carried out with the
financial support of the Ministry of Science and Higher Education
9

G.A. Gamov, A.N. Kiselev, A.E. Murekhina et al.
Journal of Molecular Liquids xxx (xxxx) xxx
potentiometric data, Talanta 198 (2019) 200–205, https://doi.org/10.1016/
j.talanta.2019.01.107.
of the Russian Federation (project FZZW-2020-0009). The theoret-
ical part of the work was performed thanks to the grant of the
Council on grants of the President of the Russian Federation (pro-
ject 14.Z56.20.2026-MK). The study of anion sensing and antimi-
crobial activity was performed with the support of the
Scholarship of the President of the Russian Federation (project
1556.2021.4-SP). The equipment of the Center of Joint Usage of Iva-
novo State University of Chemistry and Technology (ISUCT) was
used to perform the NMR, IR, MS experiments.
We thank Prof. G.V. Girichev, Dr. V.V. Sliznev, and Prof. A.V.
Belyakov for their help in conducting the quantum chemical calcu-
lations on computing resources of the Department of Physics of
ISUCT and Saint Petersburg State Institute of Technology (Technical
University). We also thank the anonymous Reviewer for the fruitful
discussion.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molliq.2021.116911.
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