Effect of reactants' concentration on the ratio and yield of E,Z isomers of isatin-3-(4-phenyl)semicarbazone and N-methylisatin-3-(4-phenyl)semicarbazone

Article abstract of DOI:10.2478/s11696-012-0248-x

In this work, the effect of inter- and intramolecular interactions of reactants and products, reactants concentration as well as the solvent effect on the ratio of E and Z isomers of isatinphenylsemicarbazones in the reaction mixture is examined. Theoreti

Full text of DOI:10.2478/s11696-012-0248-x

Chemical Papers 67 (1) 117–126 (2013)
DOI: 10.2478/s11696-012-0248-x
ORIGINAL PAPER
Effect of reactants’ concentration on the ratio and yield
of , isomers of isatin-3-(4-phenyl)semicarbazone
and -methylisatin-3-(4-phenyl)semicarbazone
b
a
a
aKlaudia Jakusová*, Martin Gáplovský, Jana Donovalová, Marek Cigáň,
aHenrieta Stankovičová, Robert Sokolík, Jan Gašpar, Anton Gáplovský
a
a
a
aInstitute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-2, 842 15 Bratislava, Slovakia
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University,
Kalinčiakova 8, 832 32 Bratislava, Slovakia
Received 17 April 2012; Revised 18 July 2012; Accepted 19 July 2012
Dedicated to Professor Štefan Toma on the occasion of his 75th birthday
In this work, the effect of inter- and intramolecular interactions of reactants and products,
reactants concentration as well as the solvent effect on the ratio of E and Z isomers of isatin-
phenylsemicarbazones in the reaction mixture is examined. Theoretical calculations proved that Z
isomers are more stable than E isomers. Experimental results confirmed the noncovalent intermolec-
ular donor-acceptor interactions of the reactants in the reaction mixture at concentrations above
0.1 mol L−1. The E/Z isomer ratio of isatin-3-(4-phenyl)semicarbazone (I ) and N-methylisatin-
3-(4-phenyl)semicarbazone (II ) depends on the initial concentrations of 3-amino-1-phenylurea
(phenylsemicarbazide; V ) and 1H-indole-2,3-dione (isatin; III ), or 3-methylindol-2,3(1H )-dion (3-
methylisatin; IV ), respectively. Both isomers exhibit high thermal stability. Thermal E–Z isomer-
ization takes place at temperatures above 70◦C in N,N-dimethylformamide.
c
ꢀ 2012 Institute of Chemistry, Slovak Academy of Sciences
Keywords: isatinphenylsemicarbazones, synthesis of E,Z isomers, concentration and solvent de-
pendence, intermolecular interactions
Introduction
Senthilkumar et al., 2005). Formation of these hydro-
gen bonds often leads to hydrogen transfer from a hy-
droxy or amino group to the acceptor, especially in
biological and photochemical processes (Levy, 1980;
Li & Fang, 2003; Otsubo et al., 2002). The trans-
fer of hydrogen (or proton) from the base or excited
state of a molecule occurs in both the solution and
the solid phase (Li & Fang, 2003; Alarcón et al., 1995;
Falkovskaia et al., 2002; Mehata et al., 2002; Chai et
al., 2005).
Considerable attention has recently been paid to
the preparation of compounds which can be subjected
to photoisomerization for their practical application in
electronics, e.g. switches, storage media, and sensors.
Inter- and intramolecular interactions, especially hy-
drogen bonds as relatively weak interactions, play an
important role in biology; they affect, amongst other
things, the course of chemical reactions and also the
stability and structure of compounds. The information
on their existence and their characteristics in the stud-
ied system often helps to understand the molecule’s
physical and chemical properties (Cubero et al., 1999;
Inter- or intramolecular hydrogen bonds represent
a very important group of interactions which affect
the physico–chemical properties of compounds. The
change of physico–chemical properties of chemical
*Corresponding author, e-mail: jakusova@fns.uniba.sk

118
K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
compounds influences also their reactivity (Epshtein,
1979; Brancato et al., 2002). Chemical reactivity of
molecules can be modified due to the change of charge
density on the reaction centre after hydrogen bond sta-
bilization/destabilization. A hydrogen bond can also
lock one of the isomers or conformers (locking ef-
fect) (Kolehmainen et al., 2000) influencing thus the
transition state of the chemical reaction. This specific
type of noncovalent interactions consequently influ-
ences the reaction rate and the ratio of the products
in the reaction mixture. Hydrogen bonds formed in
compounds containing a fragment of phenylcarbazide
affect the arrangement of molecules and the molecu-
lar geometry in crystals (Chai et al., 2005; Hadjoudis
et al., 1999). Since photochromic properties depend
on the molecule’s geometry, hydrogen bonds indirectly
affect also their properties (Wu et al., 2007). Addition-
ally, bioactivity depends so strongly on the molecular
conformation (molecular geometry) that the existence
of intra- and intermolecular hydrogen bonds in such
systems can significantly affect these processes.
Hydrazones and their derivatives, such as semicar-
bazones, represent a very attractive group of organic
compounds. Their characteristics predispose them for
practical use. An exhaustive literature review revealed
that these compounds have interesting biological prop-
erties including: anti-inflammatory, analgesic, anti-
convulsant, antituberculous, antitumour, anti-HIV,
and antimicrobial activities (Afrasiabi et al., 2005;
Sathisha et al., 2008; Verma et al., 2009; Cerchiaro
& da Costa Ferreira, 2006). Synthetic versatility of
hydrazone and also that of semicarbazone led to ex-
tensive use of these compounds in organic synthesis.
They are often used in the synthesis of heterocyclic
compounds (Verma et al., 2004; Farghaly et al., 2009;
Sridhar et al., 2001; Pandeya et al., 2000, 2002, 2006;
Pal et al., 2011; Dimmock et al., 2000; Sonawane et al.,
2009; Jafri et al., 2012). Wide practical application of
hydrazones and their derivatives also encouraged the
study of azo–hydrazone tautomeric equilibrium and
E–Z isomerization of these compounds. Influence of
the molecular structure, temperature, and solvent on
these equilibriums was thus investigated.
tra were recorded at 300 MHz on a Varian Gem-
ini 200 spectrometer (Varian, USA) using CDCl3 or
DMSO-d6 as solvents and trimethylsilane as an inter-
nal standard. Chemical shifts are given in δ related
to the internal standard. IR spectra were recorded on
a spectrometer Nicolet FT-IR 6700 (Nicolet, USA).
UV spectra were measured on an Agilent 8543 (Agi-
lent Technologies, USA). Elemental analyses were per-
formed on a Carlo Erba Strumentacione 1106 appara-
tus (Carlo Erba Strumentacione, Italy).
HPLC chromatography was carried out using a
chromatographic system (Agilent Technologies, USA)
consisting of a quaternary pump, thermostated col-
umn compartment, a diode array detector (VWDG
1314A), manual injector (Rheodyne model 7725i) with
20 µL sample loop, and a degasser (g1379A) all of the
1100 series. For all experiments, Column ZORBAX-
SB-Phenyl (150 mm × 4.6 mm i.d.) was used. For
analyses of I, mobile phase A was a methanol/water
mixture (ϕr = 1 : 99) and phase B was acetonitrile. In
the analysis of isomers, the isocratic gradient A/B (ϕr
= 1 : 1) at the flow rate of 0.6 mL min−1 at 22◦C and
detection at 236 nm was used. The injection volume
was 20 µL. For analyses of II, mobile phase A was
a methanol/water mixture (ϕr = 1 : 99) and phase
B was methanol. In the analysis of isomers, the iso-
cratic gradient A/B (ϕr = 37 : 13) at the flow rate of
0.6 mL min−1 at 22◦C and detection at 236 nm was
used. The injection volume was 20 µL.
Chemicals and solvents were purchased from the
major chemical suppliers. EtOH, MeOH, CHCl3,
and DMF from Merck (Germany), 3-methylisatin
(IV ) from Acros Organics (Belgium), isatin (III ),
phenylsemicarbazide (V ), and KClO4 from Sigma–
Aldrich (USA) as the highest purity grade. Deionized
water for HPLC analysis was purified by a Pro-PS
water purification system (Labconco, USA) and sub-
sequently purified by Simplicity (Millipore, France).
Unless otherwise noted, all materials were used as re-
ceived without further purification. All solvents were
dried by standard methods and distilled prior to use.
All reactions were performed using oven-dried glass-
ware. Reactions were monitored using Merck analyti-
cal thin layer chromatography (TLC) plates (silica gel
In this study, the synthesis of E and Z iso-
mers of I and II, and the ability of reactants’ con-
centration and solvent to affect the ratio of these
isomers in the reaction mixture during their syn-
thesis from 1H-indole-2,3-dione (isatin; III ) (or 3-
methylindol-2,3(1H )-dione (3-methylisatin (IV ), re-
spectively), and 3-amino-1-phenylurea (phenylsemi-
carbazide; V ) were investigated.
60 F
254; Merck, Germany) with eluent (hexane/ethyl
acetate ϕr = 1 : 1) and analyzed with 254 nm UV light.
For column chromatography, Silica gel 60 (Merck, 230-
400 mesh ASTM, Merck, Germany) with indicated sol-
vent was used.
Characterization of E and Z isomers of I and
II
Experimental
Materials and methods
A solution of V (6 mmol) in hot absolute ethanol
(20 mL) was added to III or IV (6 mmol) in hot ab-
solute ethanol (130 mL). The reaction mixture was
refluxed for 2 h and the formed precipitate (Ia; E iso-
mer) was filtered off from the hot solution, washed
Melting points (uncorrected) were measured on a
Kofler hot stage (Nagema, Germany). H NMR spec-
1

K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
Table 1. Characterization data of prepared compounds Ia, Ib, IIa, and IIb
119
wi(calc.)/%
wi(found)/%
Yield
M.p.
Compound
Formula
Mr
C
H
N
%
54
45
59
40
◦C
Ia
Ib
C15H12N4O2
C15H12N4O2
C16H14N4O2
C16H14N4O2
280.28
280.28
294.31
294.31
64.28
64.15
64.28
64.26
65.30
65.28
65.30
65.32
4.32
4.35
4.32
4.19
4.79
4.47
4.79
4.52
19.99
19.78
19.99
19.82
19.04
18.81
19.04
18.95
193–196
210–213
170–172
198–201
IIa
IIb
Table 2. Spectral data of Ia, Ib, IIa, and IIb
Compound
Spectral data
IR (CHCl3) ν˜/cm−1: 1448 (C N), 1536 (C C), 1602 (C O), 1735 (C O), 3380 (N—H), 3442 (N—H)
— — —
— — —
—
—
Ia
1H NMR (DMSO-d6), δ: 6.91–6.93 (d, 1H, J = 7.8 Hz, Ar-H), 7.04–7.12 (m, 2H, Ar-H), 7.32–7.41 (m, 3H, Ar-H),
7.58 (dd, 2H, J = 8.7 Hz, J = 1.2 Hz, Ar-H), 8.09 (d, 1H, J = 7.5 Hz, Ar-H), 9.49 (s, 1H, NH), 10.39 (s, 1H, NH),
10.76 (s, 1H, NH)
IR (CHCl3) ν˜/cm−1: 1448 (C N), 1533 (C C), 1621 (C O), 1706 (C O), 3255 (N—H)
— — —
— — —
—
—
Ib
1H NMR (DMSO-d6), δ: 6.94 (d, 1H, J = 7.8 Hz, Ar-H), 7.04–7.13 (m, 2H, Ar-H), 7.31–7.37 (m, 3H, Ar-H), 7.61–7.64
(m, 2H, Ar-H), 7.67 (d, 1H, J = 6.9 Hz, Ar-H), 9.84 (s, 1H, NH), 11.99 (s, 1H, NH), 12.25 (s, 1H, NH)
IR (CHCl3) ν˜/cm−1: 1448 (C N), 1535 (C C), 1604 (C O), 1716 (C O), 3380 (N—H)
— — —
— — —
—
—
IIa
1H NMR (CDCl3), δ: 3.30 (s, 3H, CH3), 6.93 (d, 1H, J = 3.9 Hz, Ar-H), 7.10–7.15 (m, 2H, Ar-H), 7.34–7.37 (m,
2H, Ar-H), 7.44–7.47 (ddd, 1H, J = 3.9 Hz, J = 0.6 Hz, J = 0.3 Hz, Ar-H), 7.61 (dd, 2H, J = 4.5 Hz, J = 0.6 Hz,
Ar-H), 7.79 (d, 1H, J = 3.9 Hz, Ar-H), 8.79 (s, 1H, NH), 9.10 (s, 1H, NH)
IR (CHCl3) ν˜/cm−1: 1448 (C N), 1533 (C C), 1617 (C O), 1691 (C O), 3249 (N—H), 3394 (N—H)
— — —
— — —
—
—
IIb
1H NMR (CDCl3), δ: 3.30 (s, 3H, CH3), 6.90–6.93 (d, 1H, J = 7.8 Hz, ArH), 7.10–7.19 (m, 2H, Ar-H), 7.34–7.44
(m, 3H, Ar-H), 7.58–7.66 (m, 3H, Ar-H), 8.31 (s, 1H, NH), 12.09 (s, 1H, NH)
with ethanol and dried. Z isomer of I (Ib) was iso-
lated and purified by column chromatography using
silica gel as the stationary phase, hexol (a mixture of
methylpentanes and hexane; ϕr ≈ 2 : 1)/ethyl acetate
(ϕr = 1 : 1) as the mobile phase. Isomers IIa (E isomer)
and IIb (Z isomer) were isolated from the evaporated
reaction mixture and purified using the same method
as for Ib. Experimental yields and spectral data of Ia,
Ib, IIa, and IIb are summarized in Tables 1 and 2.
Although I is not a new compound (Borsche &
Meyer, 1921; Kang et al., 2011), its isolation or a more
detailed study of Ib and the noncovalent interactions
of both isomers have not been performed until now (to
the best of our knowledge).
(0.1 mol L−1, 0.01 mol L−1, or 0.001 mol L−1) in
the corresponding solvent (10 mL). The reaction mix-
ture was refluxed for 3 h in EtOH, EtOH + KClO4
(5 mass %), and CHCl3; in N,N-dimethylformamide
(DMF), the reaction mixture was tempered at 50◦C
(due to the back isomerization of Z isomers). Ex-
perimental yields are summarized in Tables 3 and
4.
Quantum-chemical calculations
The quantum-chemical calculations were perfor-
med on the B3LYP/6-31G+(d,p) level of theory for
isatinphenylsemicarbazone isomers and the M062X/6-
31+G(d,p) level of theory was applied for the reac-
tants configurations as it includes the dispersion term
for better treatment of noncovalent interactions (Zhao
& Truhlar, 2008). The calculations were performed us-
ing the Gaussian version 03 (Gaussian, USA) and the
Jaguar version 7.7 (Schro¨dinger, USA), respectively.
The local energy minima were confirmed by vibra-
tional analysis of all structures.
General procedure for the synthesis of I and II
(HPLC analysis)
HPLC analysis was used for the determination
of the ratio of E/Z isomers in the reaction mixture
in various solvents. A solution of V (0.1 mol L−1
,
0.01 mol L−1, or 0.001 mol L−1) in different solvents
(15 mL) was mixed with the solutions of III or IV

120
K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
Table 3. Overall and individual yields of the reaction of IV with V
N-methylisatin
EtOH
EtOH + KClOa4
CHCl3
DMFb
c/(mol L−1
)
YE+Z/% YE/% YZ/% YE+Z/% YE/% YZ/% YE+Z/% YE/% YZ/% YE+Z/% YE/% YZ/%
0.1
0.01
0.001
58.9
39.9
32.7
59.4
72.9
75.2
40.6
27.1
24.8
76.3
71.8
72.4
76.7
72.9
75.2
23.3
15.6
13.0
90.3
87.9
83.3
84.4
85.9
88.1
15.6
14.1
11.9
1.0
0.2
0.4
89.4
55.5
16.9
10.6
44.5
83.1
a) 5 mass % of KClO4; b) At 50◦C; c – initial concentration of reactants; YE+Z – overall yield of both (E + Z) isomers; YE – yield
of E isomer, YZ – yield of Z isomer.
Table 4. Overall and individual yields of the reaction of III with V
Isatin
EtOH
EtOH + KClOa4
CHCl3
DMFa
c/(mol L−1
)
YE+Z/% YE/% YZ/% YE+Z/% YE/% YZ/% YE+Z/% YE/% YZ/% YE+Z/% YE/% YZ/%
0.1
0.01
0.001
94.8
54.7
14.3
54.4
77.7
89.5
45.7
22.3
10.5
51.6
22.1
16.0
76.4
80.1
89.7
23.6
19.9
10.3
66.5
30.7
16.0
87.0
87.2
95.2
13.0
12.8
4.8
95.6
41.6
30.3
90.1
90.4
90.2
9.9
9.6
9.8
a) 5 mass % of KClO4; c – initial concentration of reactants; YE+Z – overall yield of both (E + Z) isomers; YE – yield of E isomer,
YZ – yield of Z isomer.
Fig. 1. Synthesis of I and II in different solvents (CHCl3, EtOH, EtOH + KClO4, or DMF).
Results and discussion
mechanism of E or Z isomers and their stabilities. This
is necessary because other isomers can also be formed,
and their properties may not be suitable for the re-
quired application.
I and II and their E and Z isomers were prepared
by the reaction of isatin (III ) or methylisatin (IV )
with phenylsemicarbazide (V ), as shown in Fig. 1.
The reactions were carried out in chloroform,
ethanol, ethanol + KClO4, and DMF. UV-VIS spectra
of E and Z isomers of I and II in methanol are shown
in Fig. 2.
UV-VIS absorption spectra of E isomers of both
compounds, I and II, exhibit long-wavelength absorp-
tion bands located at 410 nm and 326 nm, respec-
tively, and a shoulder containing short-wavelength
bands with the maximum at 240 nm. The shapes
Under conditions often used in the preparation of
isatinsemicarbazones derivatives via the condensation
of 1H-indole-2,3-dione (isatin) derivatives and car-
bazides (Pandeya et al., 2002), the less soluble prod-
uct, here the less soluble geometric isomer, is directly
isolated by filtration from the reaction mixture. De-
pending on the thermodynamic stability of the iso-
lated isomer of I (isatin-3-(4-phenyl)semicarbazone)
and II (N-methylisatin-3-(4-phenyl)semicarbazone),
respectively, a thermally or photochemically initiated
isomerization can occur after its re-dissolution. Iso-
merization is completed after reaching the equilib-
rium. Due to the practical applications of these com-
pounds, it is important to understand the formation

K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
121
the non-methylated isatine derivative I. The increas-
ing polarity of the solvent (chloroform–methanol ex-
change) leads to a small bathochromic shift of the ab-
sorption band of E isomers at 326 nm (≈ 4 nm), and
to a hypsochromic shift of the absorption band of Z
isomers located at approximately 342 nm (≈ 5 nm).
Methanol as a polar protic solvent disturbs the in-
tramolecular hydrogen bond in Z isomers, which leads
to a hypsochromic shift of the absorption maxima.
If the reaction is not affected by other factors (ex-
cept for the stability of isomers), the reaction mix-
ture should contain only the more stable isomer, or
at least a very high percentage of it. According to the
bathochromic shift in the UV-VIS spectra, higher in-
tensity of the absorption band at 342 nm and IR spec-
tra for these isomers indicates that Z isomers are more
stable than E isomers. The slightly increased stability
of Z isomers was confirmed by the quantum-chemical
calculations using the Gaussian 03 program. Geome-
try optimization and frequency calculation of the E
and Z isomers were done at the B3LYP/6-31+G(d,p)
level of theory. Z isomers were found to be more sta-
ble than E isomers by approximately 19.9 kJ mol−1
for IIb and 18.8 kJ mol−1 for Ib, respectively. The in-
tramolecular hydrogen bonds significantly contribute
to the higher stability of Z isomers compared to E
isomers (Fig. 3).
Fig. 2. UV-VIS spectra of compounds Ia, Ib, IIa, and IIb in
MeOH (concentration of 10−4 mol L−1).
and positions of the absorption maxima in the spec-
tra are identical. They differ only in the extinction
coefficients; IIa has higher extinction coefficient at
410 nm (3535 L mol−1 cm−1) compared to I (2533
L mol−1 cm−1). At shorter wavelengths (326 nm and
240 nm), the extinction coefficients of methyl deriva-
tive IIa are lower (13722 L mol−1 cm−1 and 15959
L mol−1 cm−1, respectively) compared to those of Ia
(14519 L mol−1 cm−1 and 18069 L mol−1 cm−1, re-
spectively).
Z isomers of I and II have absorption max-
ima at 342 nm (16617 L mol−1 cm−1 and 18154
L mol−1 cm−1, respectively), 271 nm (14475 L mol−1
cm−1 and 17665 L mol−1 cm−1, respectively), and
232 nm (16121 L mol−1 cm−1 and 16304 L mol−1
cm−1, respectively). The absorption maximum lo-
cated at 326 nm in the spectra of E isomers is more
bathochromically shifted for Z isomers (≈ 24 nm). The
methyl substituent does not affect the position of the
absorption maxima. Instead of one complex absorp-
tion band at 240 nm noted in the spectra of E iso-
mers, there are two well resolved absorption bands
with maxima at 271 nm and 232 nm in the Z isomers’
spectra. The absorption maximum for the Z isomer of
II at 271 nm is bathochromically shifted by approx-
imately 4 nm compared to that for the Z isomer of
Experimental results show that the stability of the
isomers of derivatives I and II is not the only factor
affecting their ratio in the reaction mixture.
Data in Tables 3 and 4 indicate that the overall
yield of I and II depends on the initial concentration
of the reactants and on the type of the solvent. The
effect of these factors on the overall yield varies de-
pending on the reaction partner of V is III or IV. If
III is the reactant in this condensation reaction, the
highest overall yield is achieved at the highest con-
centration of the reactants in all solvents. The overall
yield decreases with the decreasing concentration of
the reactants and reaches approximately 16 %, except
for DMF at the concentration of 10−3 M. In the reac-
tion of IV with V, the same trend on the overall yield
of products as for III was observed. The concentration
Fig. 3. Intramolecular hydrogen bonding in E and Z isomers of compounds I and II.

122
K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
Fig. 4. Possible noncovalent intermolecular interactions between molecules of derivatives I and II.
of reactants also affects the overall yield of the isomers;
however, this yield is in case of IV less dependent on
the initial concentration of reactants in comparison
to III. The overall yields are very low (< 1 %) and
practically independent of the initial concentration of
reactants for the reaction of IV in DMF.
unable to prove the presence of Z isomers using UV-
VIS, IR, or NMR spectra. Similar behavior of E iso-
mers was observed in DMF at 50◦C. This change in
the spectrum can be attributed to the alteration in
the molecule’s geometry as a result of intermolecular
interactions. This statement is based on the fact that
the scale of this change and its rate are proportional
to the concentration of I and II. Some possible in-
termolecular interactions of derivatives I and II are
shown in Fig. 4.
An increase in the concentration shifts the equilib-
rium to the right, which results in the formation of
a supramolecule. From structures depicted in Fig. 4,
structure VI is more feasible because this interac-
tion results in a six-membered ring which can share
six electrons localized on the heteroatoms providing
conditions for the existence of a quasi-aromatic sys-
tem. The higher stability of this structure in DMF
is caused by the high dielectric constant of the sol-
vent. On the contrary, proton–donor solvents, such as
ethanol, reduce the strength of hydrogen bonds in the
Thermodynamic stability of both isomers of I and
II is one of the parameters that can substantially af-
fect the ratio of the isomers in the reaction mixture.
Depending on the thermodynamic stability and on the
reaction conditions, the change from one isomer to the
other one occurs in the subsequent reaction. There-
fore, this problem was addressed by studying the effect
of temperature and solvent polarity on the isomeriza-
tion of E and Z isomers of I and II, respectively. Both
E and Z isomers are stable at the concentration of
10−4 mol L−1 in ethanol and chloroform at 55◦C or
65◦C. UV-VIS spectra of E isomers in ethanol at 65◦C
showed only a very small change in the absorbance
value at 326 nm. This change in the spectrum is not
caused by the geometric E–Z isomerization. We were

K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
123
Table 5. Rate constants k1 and k2 of product II (DMF, T =
100◦C)
c · 10−3/(mol L−1
)
k1 · 10−3/s−1
k2 · 10−3/s−1
0.1
0.02
0.05
0.9564
1.0700
1.1300
0.44
0.054
0.026
c – Initial concentration of reactants (c = cIV = cV ; where
cIV is the concentration of IV and cV is the concentration of
V ); k1 – rate constants of the breakdown of the supramolecule
aggregate; k2 – rate constant of the formation of the Z isomer
of isatinsemicarbazone II (IIb).
Fig. 5. Possible noncovalent intermolecular interactions of
derivatives Ia and IIa with ClO−4 (A−).
supramolecule and its structure becomes less stable
compared to that achieved in DMF. This explana-
tion is consistent with the experimental results ver-
ifying the stability of I and II in DMF at 100◦C. In
the initial phase of the reaction (at all initial concen-
trations) under the studied conditions, a decrease in
the absorbance was observed at 326 nm in the UV-
VIS spectra of E isomers (Table 5). However, no Z
isomers in the solution were detected. After approxi-
mately half an hour, an increase in the absorbance at
342 nm characteristic for Z isomers was observed, dur-
ing this second phase of the reaction (Table 5). The
presence of Z isomers in the solution was confirmed
by both NMR and FTIR spectroscopy. The scale of
the changes in the UV-VIS spectrum (absorbance at
326 nm) and also the rates of the changes depend on
the initial concentrations of I and II. At low concentra-
tions of I and II, the abundance of “supramolecules”
is limited and therefore it is not possible to observe
the first phase of the reaction in the UV-VIS spectra.
Accordingly, the effect of KClO4 on the stability of
I and II and on the ratio of their E and Z isomers
in ethanol can be explained by the interaction of the
ClO−4 anion (A−) with the I and II molecules (Fig. 5).
This interaction increases the conjugation and
thereby also the stability of the isomers (Jia et al.,
2010); on the other hand, the fact that under hetero-
Fig. 6. 1H NMR spectrum of the mixture (mole ratio, 1 : 1) of
IV a V in CDCl3 at t = 0 s, T = 20◦C; cIV = cV
=
10−1 mol L−1 (a); cIV = cV = 10−2 mol L−1 (b); cIV
= cV = 10−3 mol L−1 (c).
geneous conditions molecules of I and II are fixed on
the surface of KClO4 plays a significant role. A similar
interaction with the ClO−4 anion can also be expected
for V, explaining thus the almost independent concen-
tration of E isomers in the reaction mixture of ethanol

124
K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
Table 6. Dependence of the shift in the 1H NMR spectrum for hydrogens attached to nitrogen on the concentration of reactants
Compound
c/(mol L−1
)
V
III
IV
Mixture of IV and V (mole ratio, 1 : 1)
δN(1)
δN(2)
δN(3)
δH
δCH3
δN(1)
δN(2)
δN(3)
δCH3
0.1
0.01
0.001
3.818
3.854
3.858
6.687
6.086
5.750
8.200
8.169
8.140
8.178
7.915
7.607
3.261
3.262
3.262
3.158
3.856
3.859
5.253
6.057
5.755
8.201
8.157
8.140
3.235
3.259
3.261
c – Initial concentration of the corresponding compound; δN(1) – chemical shift for hydrogens attached to nitrogen N-1 of V, δN(2)
– chemical shift for hydrogens attached to nitrogen N-2 of V, δN(3) – chemical shift for hydrogens attached to nitrogen N-3 of V,
δH – shift for hydrogens attached to nitrogen of III, δCH3 – shift for CH3 hydrogens attached to nitrogen of IV.
+ KClO4, with regard to the initial reactant concen-
trations.
The existence of the preliminary tautomeric equi-
librium of reactants (Fig. 4), where the equilibrium
constants vary due to the intermolecular interactions
between the reactants themselves and between the re-
actants and the molecules of the solvent, can also af-
fect the ratio of E and Z isomers in the reaction of
III or IV with V. Intermolecular interactions induce
(even prior to the formation of a transition state) the
arrangement of reactants ensuring the preference to
the formation of one isomer in the transition state
and thus affecting the ratio of the E and Z isomers in
the reaction mixture.
We were unable to clearly demonstrate the precise
intermolecular interactions between V and III or IV
by UV-VIS and FTIR spectroscopy. The effect of the
reactants’ concentration on the position and intensity
of signals for hydrogens of the aromatic ring, those
attached to nitrogens and those of the methyl group
1
in the H NMR spectrum of the mixture of IV a V,
is shown in Fig. 6. Chemical shifts of the signals for
hydrogens of the methyl group of isatin and also for
the change of the signals for all aromatic hydrogens of
both reactants were clearly evident at the 10−1 M con-
centration of reactants. The dependence of the shift in
hydrogens attached to nitrogen on the concentration
of reactants is shown in Table 6.
Fig. 7. 1H-NMR spectrum of the mixture (mole ratio, 1 : 1) of
IV a V in CDCl3 at t = 0 s; T = 10◦C (a); T = 50◦C
(b).
1H NMR spectrum of IV alone does not change
with its concentration. The change of the chemical
shifts for hydrogens attached to nitrogens of III and
V with the concentration of III (Table 6) indicates
the existence of intermolecular hydrogen bonds be-
tween the molecules of III and V. The dependence
the solvent, temperature, concentration of reactants,
and it is probably a factor which significantly affects
the ratio of E and Z isomers of I and II in the reaction
of III or IV, respectively, with V.
These experimental findings support the results of
the performed calculations (Fig. 8, Table 7). Fig. 8
demonstrates the optimized geometry for the most sig-
nificant reactant configurations of IV and V found at
the M062X/6-31+G(d,p) level of theory.
1
of the intensity of H NMR signals on the tempera-
ture (Fig. 7) proves that the shift of the NMR signals
(Fig. 6) is caused by the existence of the chemical
equilibrium between noncovalently bound IV with V
and noninteracting molecules IV and V. Data from the
1H NMR spectra confirmed the existence of noncova-
lent intermolecular interactions of type III-III, IV-IV,
V-V, III-V, and IV-V in the reaction system. The con-
centration of noninteracting reactants in the reaction
system, the equilibrium state, depends on the type of
Results of the calculations (Fig. 8, Table 7) and
changes of the shifts in the 1H NMR spectrum for

K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
125
Fig. 8. Optimized geometry for the most significant reactant configurations A, B, and C of IV and V found at the M062X/6-
31+G(d,p) level of theory; carbon – grey , hydrogen – white, nitrogen – blue, oxygen – red.
action with isatin in all employed solvents provides
the highest yield at the highest initial concentration
of the reactants; this yield decreases with the decreas-
ing reactants’ concentration. Similarly, the ratio of E
and Z isomers in the reaction mixture depends on
the solvent and on the initial concentration of the
reactants. This dependence is related with the in-
termolecular interactions between the reactants (in-
ducing mutual arrangement of the reactants ensur-
ing the preference to the formation of one isomer
in the transition state) and intermolecular interac-
tions between the molecules of the product (affect-
ing the stability of both isomers). E and Z isomers of
isatin-3-(4-phenyl)semicarbazone and N-methylisatin-
3-(4-phenyl)semicarbazone are stable up to 70◦C. In-
creasing the temperature above 70◦C causes thermal
E–Z isomerization. Under these conditions, the equi-
librium related to noncovalent intermolecular interac-
tions overtakes the following E–Z isomerization. The
range of this preliminary equilibrium depends on the
Table 7. M062X/6-31+G(d,p) optimized geometries for the
three lowest energy reactant configurations A, B, and
C of IV and V
Reactant geometry
∆G/(kJ mol−1
)
A
B
C
0.0
4.8a
4.9a
a) Geometry contains one weak negative frequency; ∆G – Gibbs
energy for the three lowest energy reactant configurations A, B,
and C of IV and V found at the M062X/6-31+G(d,p) level of
theory.
hydrogens of the reactants show a preference for the
donor–acceptor interaction in configuration A. This
interaction takes place over a space where III or IV
acts as the acceptor and V as the donor. Such arrange-
ment of the reactants favors the formation of E isomers
rather than of Z isomers. This conclusion is based on
the assumption that it is necessary to stabilize the
transition state, and consequently also the product,
by inter- and intramolecular interactions to achieve
the formation of the less stable E isomers. The ratio
of E and Z isomers of I and II in ethanol in the pres-
ence of KClO4 confirmed this assumption. Based on
these findings, it is impossible to definitely state which
configuration of the reactants leads to the preferential
formation of Z isomers, or indeed, if any configuration
does. Access of V to the reaction centre of III is spa-
tially unlimited for noninteracting reactants, in terms
of noncovalent intermolecular interactions. Therefore,
formation of Z isomers from noninteracting reactants
is quite possible since Z isomers are more stable than
E isomers.
1
initial concentration of the reactants. H NMR spec-
tra confirmed the existence of noncovalent intermolec-
ular interactions between the reactants, and the ex-
perimental results are consistent with the quantum-
chemical calculations.
Acknowledgements. This work was supported by the Slovak
Grant Agency for Science (Grant No. 1/1126/11).
References
Afrasiabi, Z., Sinn, E., Lin, W., Ma, Y., Campana, C., &
Padhye, S. (2005). Nickel (II) complexes of naphthaquinone
thiosemicarbazone and semicarbazone: Synthesis, structure,
spectroscopy, and biological activity. Journal of Inorganic
Biochemystry, 99, 1526–1531. DOI: 10.1016/j.jinorgbio.2005.
04.012.
Alarcón, S. H., Olivieri, A. C., Labadie, G. R., Cravero, R.
M., & González-Sierra, M. (1995). Tautomerism of repre-
sentative aromatic α-hydroxy carbaldehyde anils as studied
by spectroscopic methods and AM1 calculations. Synthesis
of 10-hydroxyphenanthrene-9-carbaldehyde. Tetrahedron, 51,
4619–4626. DOI: 10.1016/0040-4020(95)00002-p.
Borsche, W., & Meyer, R. (1921). Uber Desoxy-indigo. Berichte
der Deutschen Chemischen Gesellschaft (A and B Series),
54, 2854–2856. DOI: 10.1002/cber.19210541037.
Conclusions
The overall yield of isatin-3-(4-phenyl)semicarba-
zone (I ) and N-methylisatin-3-(4-phenyl)semicarba-
zone (II ) formation process depends on the initial
concentration of the reactants and on the type of
the solvent. The effect of these factors on the over-
all yield varies depending on the reaction partner of
phenylcarbazide is isatin or N-methylisatin. The re-
¨
Brancato, G., Coutrot, F., Leigh, D. A., Murphy, A., Wong,
J. K. Y., & Zerbetto, F. (2002). From reactants to products

126
K. Jakusová et al./Chemical Papers 67 (1) 117–126 (2013)
via simple hydrogen-bonding networks: Information trans-
Otsubo, N., Okabe, C., Mori, H., Sakota, K., Amimoto,
K., Kawato, T., & Sekiya, H. (2002). Excited-state in-
tramolecular proton transfer in photochromic jet-cooled N-
salicylideneaniline. Journal of Photochemistry and Pho-
tobiology A: Chemistry, 154, 33–39. DOI: 10.1016/s1010-
6030(02)00306-4.
Pal, M., Sharma, N. K., Priynka, P., & Jha, K. K. (2011). Syn-
thetic and biological multiplicity of isatin: A review. Journal
of Advanced Scientific Research, 2, 35–44.
Pandeya, S. N., Yogeeswari, P., & Stables, J. P. (2000). Synthe-
sis and anticonvulant activity of 4-bromophenyl substituted
aryl semicarbazones. European Journal of Medicinal Chem-
istry, 35, 879–886. DOI: 10.1016/s0223-5234(00)01169-7.
Pandeya, S. N., Raja, A. S., & Stables, J. P. (2002). Synthesis of
isatin semicarbazones as novel anticonvulsants – role of hy-
drogen bonding. Journal of Pharmacy and Pharmaceutical
Sciences, 5, 266–271.
Pandeya, S. N., Raja, A. S., & Nath, G. (2006). Synthesis and
antimicrobial evaluation of some 4- or 6-chloroisatin deriva-
tives. Indian Journal of Chemistry, 45B, 494–499.
Sathisha, M. P., Revankar, V. K., & Pai, K. S. R. (2008).
Synthesis, structure, electrochemistry, and spectral charac-
terization of bis-isatin thiocarbohydrazone metal complexes
and their antitumor activity against ehrlich ascites carci-
noma in swiss albino mice. Metal-Based Drugs, 1, 1–11. DOI:
10.1155/2008/362105.
mission in chemical reactions. PNAS, 99, 4967–4971. DOI:
10.1073/pnas.072695799.
Cerchiaro, G., & da Costa Ferreira, A. M. (2006). Oxin-
doles and copper complexes with oxindole-derivatives as
potential pharmacological agents. Journal of the Brazil-
ian Chemical Society, 17, 1473–1485. DOI: 10.1590/s0103-
50532006000800003.
Chai, H., Liu, G., Liu, L., Jia, D., Guo, Z., & Lang, J. (2005).
Crystal structure and spectroscopic study on photochromism
of 1,3-diphenyl-4-(4’-fluoro)benzal-5-pyrazolone N(4)-phenyl
semicarbazone. Journal of Molecular Structure, 752, 124–
129. DOI: 10.1016/j.molstruc.2005.04.047.
Cubero, E., Orozco, M., Hobza, P., & Luque, J. F. (1999). Hy-
drogen bond versus anti-hydrogen bond: A comparative anal-
ysis based on the electron density topology. Journal of Phys-
ical Chemistry A, 103, 6394–6401. DOI: 10.1021/jp990258f.
Dimmock, J. R., Vashishtha, S. C., & Stables, J. P. (2000). Ure-
lylene anticonvulants and related compounds. Pharmazie, 55,
490–494.
Epshtein, L. M. (1979). Hydrogen bonds and the reactivity of
organic compounds in proton transfer and nucleophilic sub-
stitution reactions. Russian Chemical Reviews, 48, 854–867.
DOI: 10.1070/rc1979v048n09abeh002417.
Falkovskaia, E., Pivovarenko, V. G., & del Valle, J. C. (2002).
Observation of a single proton transfer fluorescence in a biax-
ially symmetric dihydroxy diflavonol. Chemical Physics Let-
ters, 352, 415–420. DOI: 10.1016/s0009-2614(01)01490-7.
Farghaly, M., Abbel-Wahab, B. F., & Ahmed, E. M. (2009).
Synthesis, antiviral and antimicrobial screening of some new
2-oxoindoline derivatives. Chemistry of Heterocyclic Com-
pounds, 45, 539–544. DOI: 10.1155/2008/362105.
Senthilkumar, L., Ghanty, T. K., & Ghosh, S. K. (2005). Elec-
tron density and energy decomposition analysis in hydrogen-
bonded complexes of azabenzenes with water, acetamide, and
thioacetamide. Journal of Physical Chemistry A, 109, 7575–
7582. DOI: 10.1021/jp052304j.
Sonawane, A. E., Pawar, Y. A., Nagle, P. S., Mahulikar, P. P.,
& More, D. H. (2009). Synthesis of 1,4-benzothiazine com-
pounds containing isatin hydrazone moiety as antimicrobial
agent. Chinese Journal of Chemistry, 27, 2049–2054. DOI:
10.1002/cjoc.200990344.
Hadjoudis, E., Dziembowska, T., & Rozwadowski, Z. (1999).
Photoactivation of the thermochromic solid di-anil of 2-
hydroxy-5-methyl-isophthalaldehyde
in
β-cyclodextrin.
Journal of Photochemistry and Photobiology A: Chemistry,
128, 97–99. DOI: 10.1016/s1010-6030(99)00126-4.
Sridhar, S. K., Saravanan, M., & Ramesh, A. (2001). Syn-
thesis and antibacterial screening of hydrazones, Schiff and
Mannich bases of isatin derivatives. European Journal of
Medicinal Chemistry, 36, 615–625. DOI: 10.1016/s0223-
5234(01)01255-7.
Verma, M., Pandeya, S. N., Singh, K. N., & Stables, J. P. (2004).
Anticonvulsant activity of Schiff bases of isatin derivatives.
Acta Pharmaceutica, 54, 49–56.
Verma, K., Pandeya, S. N., Singh, U. K., Gupta, S., Prashant,
P., & Anurag Gautam, B. (2009). Synthesis and pharmaco-
logical activity of some substituted menthone semicarbazone
and thiosemicarbazone derivatives. International Journal of
Pharmaceutical Sciences and Nanotechnology, 1, 357–362.
Wu, D. L., Liu, L., Liu, G. F., & Jia, D. Z. (2007). Theo-
retical studies on geometrical properties and photochromic
mechanism of two photochromic compounds. Journal of
Molecular Structure: THEOCHEM, 806, 197–203. DOI:
10.1016/j.theochem.2006.11.027.
Zhao, Y., & Truhlar, D. G. (2008). The M06 suite of density
functionals for main group thermochemistry, thermochemical
kinetics, noncovalent interactions, excited states, and tran-
sition elements: two new functionals and systematic test-
ing of four M06-class functionals and 12 other functionals.
Theoretical Chemistry Accounts: Theory, Computation, and
Modeling (Theoretica Chimica Acta), 120, 215–241. DOI:
10.1007/s00214-007-0310-x.
Jafri, L., Ansari, F. L., Jamil, M., Kalsoom, S., Qureishi, S., &
Mirza, B. (2012). Microwave-assisted synthesis and bioeval-
uation of some semicarbazones. Chemical Biology and Drug
Design, 79, 1–10. DOI: 10.1111/j.1747-0285.2012.01360.x.
Jia, C., Wu, B., Liang, J., Huang, X., & Yang, X. J. (2010).
A colorimetric and ratiometric fluorescent chemosensor for
fluoride based on proton transfer. Journal of Fluorescence,
20, 291–297. DOI: 10.1007/s10895-009-0553-0.
Kang, I. J., Wang, L. W., Hsu, T. A., Yueh, A., Lee, C. C.,
Lee, Y. C., Chao, Y. S., Shih, S. R., Chern, J. H., & Lee,
C. Y. (2011). Isatin-β-thiosemicarbazones as potent herpes
simplex virus inhibitors. Bioorganic & Medicinal Chemistry
Letters, 21, 1948–1952. DOI: 10.1016/j.bmcl.2011.02.037.
Kolehmainen, E., O´smialꢀowski, B., Nissinen, M., Kauppinen,
R., & Gawinecki, R. (2000). Substituent and temperature
controlled tautomerism of 2-phenacylpyridine: the hydro-
gen bond as a configurational lock of (Z)-2-(2-hydroxy-2-
phenylvinyl)pyridine. Journal of Chemical Society, Perkin
Transactions 2, 2000, 2185–2191. DOI: 10.1039/b006879i.
Levy, D. H. (1980). Laser spectroscopy of cold gas-phase
molecules. Annual Review of Physical Chemistry, 31, 197–
225. DOI: 10.1146/annurev.pc.31.100180.001213.
Li, Q. S., & Fang, W. H. (2003). Theoretical studies on struc-
tures and reactivity of 8-hydroxyquinoline and its one-water
complex in the ground and excited states. Chemical Physics
Letters, 367, 637–644. DOI: 10.1016/s0009-2614(02)01791-8.
Mehata, M. S., Joshi, H. C.,
& Tripathi, H. B. (2002).
Excited-state intermolecular proton transfer reaction of 6-
hydroxyquinoline in protic polar medium. Chemical Physics
Letters, 359, 314–320. DOI: 10.1016/s0009-2614(02)00716-9.