A Study on Hydrolytic Stability of Isatin N-Mannich Bases

Article abstract of DOI:10.1134/S1070363218010085

Stability parameters of biologically active compounds with N-Mannich base motif in their structure, which is susceptible to hydrolysis, have been studied. A procedure for the synthesis of small molecule compounds reactivating the function of the p53 tumor suppressor protein was developed, and the dependence of the Mannich base degradation rate on the structure of the compounds was established. For the hydrolysis reaction we determined the rate constants and calculated ρ, ΔG#, ΔH, and ΔS. Modifications of active compounds that retain the activity at the maximum possible stability were proposed.

Full text of DOI:10.1134/S1070363218010085

ISSN 1070-3632, Russian Journal of General Chemistry, 2018, Vol. 88, No. 1, pp. 48–56. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © D.D. Orlova, D.S. Novikova, A.V. Garabadzhiu, V.G. Tribulovich, 2018, published in Zhurnal Obshchei Khimii, 2018, Vol. 88,
No. 1, pp. 52–60.
A Study on Hydrolytic Stability of Isatin N-Mannich Bases
D. D. Orlova*, D. S. Novikova, A. V. Garabadzhiu, and V. G. Tribulovich
St. Petersburg State Institute of Technology (Technical University), Moskovskii pr. 26, St. Petersburg, 190013 Russia
*e-mail: orlova.daria.d@gmail.com
Received November 21, 2017
Abstract—Stability parameters of biologically active compounds with N-Mannich base motif in their
structure, which is susceptible to hydrolysis, have been studied. A procedure for the synthesis of small
molecule compounds reactivating the function of the p53 tumor suppressor protein was developed, and the
dependence of the Mannich base degradation rate on the structure of the compounds was established. For the
hydrolysis reaction we determined the rate constants and calculated ρ, ΔG#, ΔH, and ΔS. Modifications of
active compounds that retain the activity at the maximum possible stability were proposed.
Keywords: p53, MDM2, hydrolysis of N-Mannich bases, isatin
DOI: 10.1134/S1070363218010085
The development of drugs that inhibit tumor
formation by inhibiting the p53-MDM2 protein-protein
interaction is one of the priority areas of medicinal
chemistry. Screening of the library of chemical
compounds allowed the identification of hit
compounds, and the evaluation of their activity in cell
models made it possible to determine a number of the
most promising compounds based on indoline-2,3-
dione [1]. It should be noted that indolinone or
isoindolinone core is a popular scaffold for compounds
with a similar spectrum of biological activity [2–4].
The structure of the studied compounds contains a
Schiff base motif at position 3 of indole-2,3-dione and
a Mannich base motif at position 1, obtained by
N-aminomethylation of the indole ring with substituted
piperazines. It is known that both Schiff base and Mannich
base fragments are present in the structures of various
compounds, which possess biological activity [5, 6].
One of the characteristic features of such a struc-
tural fragment as N-Mannich base is the possibility of
its use as a transport group within a prodrug [5]. Our
studies showed that the activity of lead compounds is
significantly dependent on the presence of a
substituent at position 1 of indole-2,3-dione.
The activity of the compounds chosen for the study
was higher than that of Nutlin-3a, the known MDM2-
p53 protein-protein interaction inhibitor, which was
used as a positive control.
To obtain a focused library of compounds of the
studied type, a scheme for the synthesis was proposed
(Scheme 1), where the Mannich base was synthesized
in the first stage, and the Schiff base was synthesized
in the second stage. After the first stage, the yield was
about 80%, but in the second stage poorly separable
mixture was obtained with a low yield (~5%).
O
R2
N
H
N
R1
According to the data of NMR spectroscopy, the
product without piperazine fragment predominated in
the mixture, indicating the hydrolysis of the Mannich
base during the reaction.
O
N
N
N
The most common method for the synthesis of
N-Mannich bases is the interaction of equimolar
amount of an amine, carbonyl compound and aqueous
formaldehyde in ethanol under reflux for several hours
R3
48

49
A STUDY ON HYDROLYTIC STABILITY OF ISATIN N-MANNICH BASES
Scheme 1.
O
R
N
H
O
O
N
H+
CH2O
O
O
O
i-PrOH,
reflux, 6 h
BuOH,
reflux, 12 h
N
N
N
H
N
N
N
N
[7–13]. At the same time, the structure of some
considered compounds also contained a Schiff base
motif [11, 12]. However, the authors did not face the
problem of hydrolysis of the reaction products, and the
yields of the target products were often quantitative.
Analysis of the obtained compounds 3a–3f by TLC
and HPLC showed the decomposition of the reaction
products both on silica gel and under conditions of
reversed-phase chromatography. In this regard, the
study of the obtained compounds was carried out by
NMR spectroscopy.
In the aqueous medium N-Mannich bases are
quantitatively decomposed into initial components
with the rate that depends on pH of the medium and
the structural features of the substrate, such as the
acidity of the amide, the basicity of the amine, and
steric factors [14–18]. To achieve the optimum
stability–activity relationship of the studied com-
pounds, it is necessary to select the structure with high
binding energy to the MDM2 protein and, at the same
time, a half-life sufficient for the manifestation of their
inhibitory properties.
For the rapid analysis of the reaction course and
purification by flash chromatography, we developed a
procedure for the TLC plate preparation; the use of this
procedure minimized hydrolysis of the target com-
pounds. The TLC plate was heated at 120°C for
30 min, then the active groups of silica gel were
deactivated by eluting the plate with chloroform–
triethylamine (95 : 5), and the plate was dried at a
reduced pressure. The sample was eluted with
chloroform–triethylamine (95 : 5). To obtain a reliable
result, the analysis was carried out several times; the
lowest decomposition of the product was considered as
the most reliable result (see figure).
We were aimed at the development of a procedure
for the synthesis of Mannich bases, whose implementa-
tion would not result in hydrolysis of the reaction
product, and also at the investigation of the stability of
final compounds.
According to TLC data, the Schiff base is
practically not hydrolyzed, while the Mannich base
decomposes faster than the final compound. Thus, the
introduction of a Schiff base fragment into the
structure of the compound stabilizes the molecule.
A significant increase in the yield of the reaction
product was achieved when using the approach, which
involves the preparation of the Mannich base as the
last stage of the synthesis. The solvent used in the
reaction (isopropanol) was mixed with an excess of
37% formaldehyde and dehydrated over molecular
sieves 4 Å. The reaction was carried out in the
presence of molecular sieves and a small amount of
triethylamine (Scheme 2). The desired products 3a–3f
were isolated by flash chromatography. Depending on
the carefulness of the preliminary operations, the yield
of the reaction product ranged from 40 to 80%.
During TLC in the chloroform–triethylamine
system (95 : 5), the decomposition of both the Mannich
base and the final compound (plate 1) was observed.
When plate 1 was repeatedly eluted, the decomposition
of the final compound and the Mannich base was
observed again (plate 2). Under preliminary
deactivation of active hydroxyl groups on silica gel,
the decomposition of the compounds becomes less
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50
ORLOVA et al.
Scheme 2.
O
OH
HO
O
O
N
R1
R1
H+
O
+
O
BuOH,
reflux, 4 h
N
N
H
H
NH2
1a, 1b
O
R2
N
H
R2
N
NH2
R1
O
DMF,
0°С, 12 h
N
H
2a_2d
O
R2
N
H
N
R1
TEA
CH2O
O
i-PrOH, reflux, 6 h,
R3
ο
N
molecular sieves 4 Α
N
N
3a_3f
R1 = H (1а, 2а, 2c, 2d, 3a, 3c–3f), Cl (1b, 2b, 3b); R2 = H (2а, 2b, 3а, 3b, 3e), CH3(R) (2c, 3c), CH3(S) (2d, 3d, 3f); R3 = H
(3a–3d), F (3e, 3f).
pronounced (plate 3), and when using dried plates that
have been previously deactivated, the compounds are
practically not subjected to the decomposition, and the
more thoroughly the preparation of the plates, the less
the observed decomposition of the compounds, which
is absent in the ideal case.
4–13, which differ in the nature of the substituents in
the benzyl moiety of the piperazine ring, were chosen
for the study of the decomposition kinetics of
N-Mannich bases. Compounds 4–13 were obtained by
reacting isatin with formaldehyde and the cor-
responding secondary amine in ethanol in the presence
of molecular sieves 4 Å and a small amount of TEA at
room temperature for 3 h (Scheme 3).
During the preliminary study of the hydrolysis rate
of Mannich bases and Schiff bases, it was found that
compounds 3a–3f, which contain both the Mannich
base and the Schiff base, are more stable than
compounds with the Mannich base in position 1 and
without the Schiff base in position 3 of indole-2,3-
dione. Taking this fact into account, model compounds
The degree of hydrolysis was detected in NMR spectra
by a change in the signal intensity corresponding to two
protons of the methylene bridge NCH2N. The change
in the integral signal intensity was compared with the
total integral intensity of aromatic protons. The experi-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 1 2018

51
A STUDY ON HYDROLYTIC STABILITY OF ISATIN N-MANNICH BASES
(a) (b) (c)
(d)
front
start line
O
O
R2
N
N
H
N
H
H
N
N
O
O
N
O
O
O
O
N
N
N
N
H
H
N
N
N
N
2
4
5
1
3
Effect of TLC conditions on results of analysis: (a) the first elution of the sample on unprepared plate; (b) the second elution of the
sample on unprepared plate; (c) plate pre-eluted with chloroform–triethylamine 95 : 5; (d) plate preliminarily dried at 120°C and
eluted with chloroform–triethylamine 95 : 5. In all plates: the first track is Schiff’s base, the second one is the Mannich base, the
third one is the final product. The eluent is chloroform–triethylamine.
ment was carried out in the ethanol–water system (1 : 1).
The investigation of the stability of the compounds in
anhydrous ethanol showed complete absence of
decomposition products under 8-hour refluxing.
were determined based on the Gibbs activation energy
ΔG# (Table 2). The determination of these kinetic
parameters of the decomposition allows revealing the
structural elements that determine the hydrolytic
stability of the studied compounds.
Kinetic parameters such as the rate constant k of the
reaction, half-life τ1/2, Gibbs activation energy ΔG#
were calculated based on the experimental data for
each temperature (40, 60 and 80°C). In addition, based
on the Hammett σ constants, the values of the reaction
sensitivity to the change of the substituent ρ were
calculated for two series of compounds 4–7 and 8–11
(Table 1) [19, 20]. The entropy and enthalpy factors
The kinetic parameters of the hydrolysis reaction
were calculated from the first order equations, since
the solvent, which was also a reagent, was taken in a
large excess. In such cases, the order of the reaction is
considered to be pseudo-first because of a slight
change in the concentration of one of the reagents. To
obtain the reaction rate constant, the data were
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52
ORLOVA et al.
Scheme 3.
O
R1
O
H
N
R1
Et3N
CH2O
O
+
O
N
EtOH, 3 h,
ο
N
molecular sieves 4 A
N
H
N
R2
N
R2
4−13
R1 = H (4, 5, 6, 7, 12, 13), Cl (8, 9, 10, 11); R2 = CH2-4-piperonyl (4, 8), CH2C6H5 (5, 9), CH2-4-Cl-C6H4 (6, 10), CH2-3-Cl-
C6H4 (7, 11), Me (12), Ph (13).
processed by the method of least squares, after which
the half-life value was calculated. The most stable
compound 12 had the half-life of 16.4 h at 80°C (260 h
at 40°C); the half-life value for the least stable
compound 11 fluctuated in the range of 1.4–1.7 h.
According to the results of the study, compound 12
with N-methylpiperazine as the amine component of
the Mannich base was found to be the most stable. In
addition, N-phenylpiperazine derivative 13 turned out
to be more stable than N-benzylpiperazine derivative 5.
According to the value of ρ, the reaction of hydrolysis
of Mannich bases 4–11 based on N-benzylpiperazine
proved to be very sensitive to the electronic effect of
substituents in the benzyl fragment, in spite of its
remoteness from the reaction center. Thus, ρ for
compounds 4–7 was 2.91 at 40°C, and for compounds
8–11 with chlorine atom at position 5 of the indolinone
ring, the value of ρ was 3.38 at the same temperature,
indicating an increase in the sensitivity of the reaction
in the presence of halogen in the indolinone ring.
To study the effect of substituents on the decom-
position rate of the studied compounds, the correlation
of the rate constants of hydrolysis with various types
of σ constants of substituents was evaluated (Table 1).
The correlation of the Gammet σ0 constants (r 0.998)
turned out to be the best compared with the induction σI
constants (r 0.975), the resonance σR constants (r 0.789)
and the Brown σ+ constants (r 0.871).
The obtained value of ρ was positive, that is the rate-
determining stage of the reaction is accelerated in the
presence of electron-withdrawing substituents.
The calculated decomposition rate of the model
compounds allows us assuming that during the study
Table 1. Calculation of kinetic parameters for compounds 4–13
k, h–1
τ1/2, h
r
log (k/k0)
ρ
Comp.
no.
σ0
40°С 60°С 80°С 40°С 60°С 80°С 40°С 60°С 80°С 40°С 60°С 80°С 40°С 60°С 80°С
4
5
6
7
8
9
0.0027 0.0086 0.059 258 80.8 11.8 0.997 0.998 0.999 –0.468 –0.315 –0.179
–0.160
0
0.0079 0.0180 0.089 87.7 39.1 7.8 0.999 0.998 0.999 0
0
0
2.91 1.95
0.93
1.29
0.0370 0.0500 0.145 18.8 13.9 4.8 0.999 0.999 0.999 0.674 0.450 0.184
0.0940 0.0930 0.196 7.3 7.4 3.5 0.999 0.998 0.999 1.086 0.718 0.329
0.0062 0.0230 0.080 112.7 30.0 8.7 0.990 0.975 0.999 –0.541 –0.371 –0.247
0.230
0.370
–0.160
0
0.0210 0.0550 0.142 32.5 12.7 4.9 0.998 0.999 0.997 0
0
0
3.38 2.33
10 0.1280 0.1880 0.280 5.4 3.7 2.5 0.999 0.995 0.995 0.785 0.542 0.255
11 0.3800 0.4000 0.420 1.4 1.7 1.6 0.990 0.991 0.993 1.245 0.864 0.451
0.230
0.370
12
13
0.042
0.080
16.4
8.6
0.999
0.994
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A STUDY ON HYDROLYTIC STABILITY OF ISATIN N-MANNICH BASES
Table 2. Gibbs activation energy, entropy and enthalpy for compounds 4–11
ΔG, kJ/mol
60°С
100.1
98.1
ΔS,
ΔH,
kJ/mol
Comp. no.
kJ mol–1 deg–1
40°С
96.9
94.1
90.1
87.7
94.8
91.5
86.9
84.1
80°С
100.6
99.4
97.9
97.1
99.7
98.3
96.0
94.8
4
0.09
0.13
0.20
0.24
0.12
0.16
0.23
0.27
93.7
89.3
82.7
78.6
89.9
85.1
77.7
73.3
5
6
95.2
7
93.5
8
97.3
9
95.0
10
11
91.5
89.4
of biological activity in cell test systems there is no
significant decrease in the concentration of active
compounds due to hydrolysis of Mannich bases during
the experiment. However, it is rational to use com-
pounds based on benzyl piperazines having electron-
donating substituents in the benzyl moiety for further
optimization. This is due to the fact that having close
values of the calculated energy of the complex of the
active molecule with the target MDM2 protein, they
possess much higher stability. If it is necessary to
increase the lipophilicity of the compound without a
significant change in the distribution of the electron
density in the binding fragment, the replacement of
chlorine by fluorine both in the benzyl moiety of the
amine component of the Mannich base and at position
5 of the indolinone ring will be optimal.
and distilled. Triethylamine was dried over sodium
hydroxide and distilled. The eluent and silica gel were
prepared immediately prior to the separation. The
separation was started after the elution of the column.
General procedure for synthesis of compounds
3a–3f. A mixture of 150 mL of isopropanol and 0.21 mL
(2.86 mol) of 37% formaldehyde water solution was
dehydrated with molecular sieves 4 Å, than 2.6 mmol
of Schiff base 2, 2.6 mmol of substituted piperazine,
0.1 mL of triethylamine, and a small amount of fresh
molecular sieves were added thereto. The reaction
mixture was refluxed with stirring for 6–14 h. After the
reaction was complete, the precipitate was filtered off,
the filtrate was concentrated, and the residue was
purified by column chromatography in the chloroform–
triethylamine system, 95 : 5.
EXPERIMENTAL
N-Benzyl-4-({2-oxo-1-[(4-phenylpiperazin-1-yl)-
methyl]indolin-3-ylidene}amino)benzamide (3a). Yield
0.88 g (64%), isomer ratio E : Z = 75 : 25, orange
NMR spectra were recorded on a Bruker AVANCE
400 spectrometer (400 MHz) in DMSO-d6. Mass
spectra were taken on a Shimadzu LCMS 2020 single
quadrupole chromatographic mass spectrometer (EC
150/3 Nucleodur PolarTec column, Macherey-Nagel
Silica gel 3 μm, ionization temperature 200°C,
ionizing electron energy 70 eV). Flash chromato-
graphy purification of the reaction products was
carried out on an Isolera-4 Biotage instrument (Merck
Silica gel 60, 0.040–0.063 mm). Preliminary prepara-
tion was carried out for the preparative purification of
the reaction product on a flash chromatograph: silica
gel was dehydrated at 300°C for 2 h, then evacuated,
cooled and packed into a column. The column was
eluted with a prepared chloroform–triethylamine system,
95 : 5. Chloroform was washed with water, dried over
calcium chloride, and then over phosphorus(V) oxide
1
solid. H NMR spectrum, δ, ppm: E-isomer, 2.76 br.s
(4Н, CH2NCH2), 3.14 br.s (4Н, CH2NCH2), 4.52 d
(2Н, CH2, J = 5.9 Hz), 4.58 s (2Н, NCH2N), 6.44 d
(1Н, ArН, J = 7.3 Hz), 6.77 d (1Н, ArН, J = 7.2 Hz),
6.80 t (1Н, ArН, J = 7.6 Hz), 6.94 d (2Н, ArН, J =
8.1 Hz), 7.13 d (2Н, ArН, J = 8.4 Hz), 7.20 d (2Н,
ArН), 7.31–7.37 m (6Н, ArН), 7.46 t (1Н, ArН,
7.6 Hz), 8.03 d (2Н, ArН, J = 8.4 Hz), 9.09 t (1Н, NН,
J = 6.0 Hz); Z-isomer, 2.68 br.s (4Н, CH2NCH2), 3.10
br.s (4Н, CH2NCH2), 4.45 s (2Н, NCH2N), 4.52 d (2Н,
CH2, J = 5.9 Hz), 6.75 d (1Н, ArН), 6.90 t (1Н, ArН),
7.08 d (2Н, ArН, J = 8.5 Hz), 7.16–7.22 m (4Н, ArН),
7.24–7.30 m (6Н, ArН), 7.58 t (1Н, ArН, J = 7.8 Hz),
7.70 d (1Н, ArН, J = 7.4 Hz), 7.88 d (2Н, ArН, J =
8.5 Hz), 9.00 t (1Н, NН, J = 6.0 Hz).
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54
ORLOVA et al.
N-Benzyl-4-({5-chloro-2-oxo-1-[(4-phenylpiperazin-
4.45 s (2Н, NCH2N), 5.18 m (1Н, СН), 6.76 m (2Н,
ArН), 6.93 d (2Н, ArН), 7.08 d (2Н, ArН, J = 8.5 Hz),
7.19–7.21 m (6Н, ArН), 7.40–7.48 m (2Н, ArН), 7.58
t (1Н, ArН, J7.6 Hz), 7.69 d (1Н, ArН, J = 6.7 Hz),
7.88 d (2Н, ArН, J = 8.5 Hz), 8.75 d (1Н, NН, J =
8.1 Hz).
1-yl)methyl]indolin-3-ylidene}amino)benzamide (3b).
Yield 1.19 g (81%), isomer ratio E : Z = 70 : 30,
1
orange solid. H NMR spectrum, δ, ppm: E-isomer,
2.75 br.s (4Н, CH2NCH2), 3.12 br.s (4Н, CH2NCH2),
4.52 d (2Н, CH2, J = 5.8 Hz), 4.58 s (2Н, NCH2N),
6.33 s (1Н, ArН), 6.77 t (1Н, ArН, J = 7.2 Hz), 6.93 d
(2Н, ArН, J = 8.0 Hz), 7.15 d (2Н, ArН, J = 8.6 Hz),
7.17–7.25 m (2Н, ArН), 7.34–7.38 m (6Н, ArН), 7.54
d (1Н, ArН, J = 8.6 Hz)), 8.05 d (2Н, ArН, J =
8.6 Hz), 9.13 t (1Н, NН, J = 6.0 Hz); Z-isomer, 2.68
br.s (4Н, CH2NCH2), 3.12 br.s (4Н, CH2NCH2), 4.44 s
(2Н, NCH2N), 4.51 d (2Н, CH2, J = 5.8 Hz), 6.76 t
(1Н, ArН, J = 7.2 Hz), 6.91 d (2Н, ArН, J = 8.0 Hz),
7.11 d (2Н, ArН, J = 8.5 Hz), 7.17–7.25 m (2Н, ArН),
7.25–7.28 m (6Н, ArН), 7.64 d (1Н, ArН, J = 8.5 Hz),
7.70 s (1Н, ArН), 7.89 d (2Н, ArН, J = 8.5 Hz), 9.01 t
(1Н, NН, J = 6.1 Hz).
N-Benzyl-4-({1-[(4-{2-fluorophenyl}piperazin-1-
yl)methyl]-2-oxoindolin-3-ylidene}amino)benzamide
(3e). Yield 1.05 g (74%), isomer ratio E : Z = 78 : 22,
1
yellow fine needle crystals. H NMR spectrum, δ,
ppm: Е-isomer, 2.78 br.s (4Н, CH2NCH2), 3.02 br.s
(4Н, CH2NCH2), 4.52 d (2Н, CH2, J = 6.0 Hz), 4.59 s
(2Н, NCH2N), 6.44 d (1Н, ArН, J = 7.0 Hz), 6.85 t
(1Н, ArН, J = 7.3 Hz), 7.03 t (1Н, ArН, J = 8,8 Hz),
7.09–7.13 m (5Н, ArН), 7.33–7.37 m (6Н, ArН), 7.48
t (1Н, ArН, J = 7.9 Hz), 8.03 d (2Н, ArН, J = 8.6 Hz),
9.09 t (1Н, NН, J = 6.0 Hz); Z-isomer, 2.71 br.s (4Н,
CH2NCH2), 2.98 br.s (4Н, CH2NCH2), 4.45 s (2Н,
NCH2N), 4.52 d (2Н, СН2, J = 6.0 Hz), 6.94–6.99 m
(6Н, ArН), 7.18 t (1Н, ArН, J = 7.3 Hz), 7.24–7.29 m
(6Н, ArН), 7.58 t (1Н, ArН, J = 7.9 Hz), 7.70 d (1Н,
ArН, J = 6.7 Hz), 7.88 d (2Н, ArН, J = 8.5 Hz), 9.00 t
(1Н, NН, J = 6.0 Hz).
(R)-4-({2-Oxo-1-[(4-phenylpiperazin-1-yl)methyl]-
indolin-3-ylidene}amino)-N-(1-phenylethyl)benzamide
(3c). Yield 0.76 g (54%), isomer ratio E : Z = 78 : 22,
1
orange solid. H NMR spectrum, δ, ppm: Е-isomer,
2.60 br.s (4Н, CH2NCH2), 2.99 d (3Н, CH3, J = 6.0 Hz),
3.14 br.s (4Н, CH2NCH2), 4.58 s (2Н, NCH2N), 5.20
m (1Н, СН), 6.45 d (1Н, ArН, J = 7.0 Hz), 6.77 m
(2Н, ArН, J = 7.2 Hz), 6.84 t (1Н, ArН, J = 7.6 Hz),
6.95 d (2Н, ArН, J = 8.0 Hz), 7.11 d (2Н, ArН, J =
8.5 Hz), 7.19–7.21 m (6Н, ArН), 7.33–7.35 m (2Н,
ArН), 8.03 d (2Н, ArН, J = 8.6 Hz), 8.85 d (1Н, NН,
J = 8.1 Hz); Z-isomer, 2.76 br.s (4Н, CH2NCH2), 2.99
d (3Н, CH3, J = 6.0 Hz), 3.13 br.s (4Н, CH2NCH2),
4.45 s (2Н, NCH2N), 5.20 m (1Н, СН), 6.76 m (2Н,
ArН, J = 7.2 Hz), 6.93 d (2Н, ArН, J = 8.0 Hz), 7.08 d
(2Н, ArН, J = 8.5 Hz), 7.19–7.21 m (6Н, ArН), 7.40–
7.48 m (2Н, ArН), 7.58 t (1Н, ArН, J = 7.2 Hz), 7.69 d
(1Н, ArН, J = 6.7 Hz), 7.88 d (2Н, ArН, J = 8.5 Hz),
8.75 d (1Н, NН, J = 8.1 Hz).
(S)-4-({1-[(4-{2-Fluorophenyl}piperazin-1-yl)-
methyl]-2-oxoindolin-3-ylidene}amino)-N-(1-phenyl-
ethyl)benzamide (3f). Yield 1.11 g (76%), isomer
1
ratio E : Z = 77 : 23, orange solid. H NMR spectrum,
δ, ppm: Е-isomer, 1.50 d (3Н, СН3, J = 7.0 Hz), 2.87
br.s (4Н, CH2NCH2), 3.01 br.s (4Н, CH2NCH2), 4.59 s
(2Н, NCH2N), 5.21 m (1Н, СН), 6.46 d (1Н, ArН, J =
7.7 Hz), 6.85 t (1Н, ArН, J = 7.6 Hz), 7.02 t (1Н, ArН,
J = 8.4 Hz), 7.09–7.13 m (2Н, ArН), 7.13 d (2Н, ArН,
J = 8.6 Hz), 7.31–7.37 m (4Н, ArН), 7.38–7.47 m (4Н,
ArН), 8.03 d (2Н, ArН, J = 8.5 Hz), 8.85 d (1Н, NН,
J = 8.1 Hz); Z-isomer, 1.48 d (3Н, СН3, J = 7.0 Hz),
2.67 br.s (4Н, CH2NCH2), 2.98 br.s (4Н, CH2NCH2),
4.45 s (2Н, NCH2N), 5.20 m (1Н, СН), 6.96–7.01 m
(6Н, ArН), 7.18 t (1Н, ArН, J = 7.6 Hz), 7.20–7.32 m
(6Н, ArН), 7.57 t (1Н, ArН, J = 7.2 Hz), 7.69 d (1Н,
ArН, J = 7.6 Hz), 7.87 d (2Н, ArН, J = 8.5 Hz), 8.75 d
(1Н, NН, J = 8.1 Hz).
(S)-4-({2-Oxo-1-[(4-phenylpiperazin-1-yl)methyl]-
indolin-3-ylidene}amino)-N-(1-phenylethyl)benzamide
(3d). Yield 0.83 g (59%), isomer ratio E : Z = 77 : 23,
1
orange solid. H NMR spectrum, δ, ppm: Е-isomer,
2.60 br.s (4Н, CH2NCH2), 2.87 d (3Н, CH3, J = 6.0 Hz),
3.14 br.s (4Н, CH2NCH2), 4.58 s (2Н, NCH2N), 5.18
m (1Н, СН), 6.45 d (1Н, ArН, J = 7.0 Hz), 6.77 m
(2Н, ArН, J = 7.0 Hz), 6.84 t (1Н, ArН, J = 7.6 Hz),
6.95 d (2Н, ArН, J = 7.8 Hz), 7.11 d (2Н, ArН, J =
8.5 Hz), 7.19–7.21 m (6Н, ArН), 7.33–7.35 m (2Н,
ArН), 8.03 d (2Н, ArН, J = 8.6 Hz), 8.85 d (1Н, NН,
J = 8.1 Hz); Z-isomer, 2.76 br.s (4Н, CH2NCH2), 2.87
d (3Н, CH3, J = 6.0 Hz), 3.13 br.s (4Н, CH2NCH2),
General procedure for synthesis of compounds
4–13. To a solution of isatin (28 mmol) in 100 mL of
anhydrous ethanol with 1 g of dehydrated molecular
sieves and 37% aqueous formaldehyde (3.8 mmol),
secondary amine (28 mmol) was gradually added
under stirring. The reaction was carried out at room
temperature for 3 h. The product was recrystallized
from isopropanol.
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55
A STUDY ON HYDROLYTIC STABILITY OF ISATIN N-MANNICH BASES
1-(4-{Piperonylpiperazine-1-yl}methyl)indoline-
2,3-dione (4). Yield 87%. 1H NMR spectrum, δ, ppm:
2.34 br.s (4Н, CH2NCH2), 2.59 br.s (4Н, CH2NCH2),
3.36 s (2Н, CCH2N), 4.41 c (2H, NCH2N), 5.96 c (2H,
OCH2O), 6.70 d (1Н, ArН, J = 8.0 Hz), 6.76–6.81 m
(2H, ArН), 7.14 t (1H, ArН, J = 7.5 Hz), 7.25 d (1H,
ArН, J = 8.0 Hz), 7.55 d (1H, ArН, J = 7.4 Hz), 7.65 t
(1H, ArН, J = 7.9 Hz). Mass spectrum, m/z (Irel, %):
379 (100) [М + H]+.
ArН), 7.56–7.78 m (3Н, ArН). Mass spectrum, m/z
(Irel, %): 369 (100) [М + H]+.
5-Chloro-1-({4-[4-chlorobenzyl]piperazin-1-yl}-
methyl)indoline-2,3-dione (10) was obtained
similarly. Yield 77%. 1H NMR spectrum, δ, ppm: 2.38
br.s (4Н, CH2NCH2), 2.55 br.s (4Н, CH2NCH2), 3.44 s
(2Н, CCH2N), 5.01 s (2Н, NCH2N), 7.13–7.25 m (3H,
ArН), 7.31 d (1H, ArН, J = 7.6 Hz), 7.59 d (1H, ArН,
J = 8.0 Hz), 7.72 d (1H, ArН, J = 7.5 Hz), 7.85 s (1H,
ArН). Mass spectrum, m/z (Irel, %): 404 (100) [М + H]+.
1-({4-Benzylpiperazin-1-yl}methyl)indoline-2,3-
dione (5) was obtained similarly. Yield 76%. 1H NMR
spectrum, δ, ppm: 2.35 br.s (4Н, CH2NCH2), 2.59 br.s
(4Н, CH2NCH2), 4.42 s (2Н, CCH2N), 5.95 s (2H,
NCH2N), 6.69–6.81 m (5Н, ArН), 7.14 t (1Н, ArН, J =
7.5 Hz), 7.25 d (1H, ArН, J = 7.6 Hz), 7.54 d (1H,
ArН, J = 7.4 Hz), 7.64 t (1H, ArН, J = 8.0 Hz). Mass
spectrum, m/z (Irel, %): 335 (100) [М + H]+.
5-Chloro-1-({4-[3-chlorobenzyl]piperazin-1-yl}-
methyl)indoline-2,3-dione (11) was obtained similarly.
1
Yield 41%. H NMR spectrum, δ, ppm: 2.39 br.s (4Н,
CH2NCH2), 2.56 br.s (4Н, CH2NCH2), 3.44 s (2Н,
CCH2N), 5.01 s (2Н, NCH2N), 7.19 d (2H, ArН, J =
7.6 Hz), 7.33 d (2H, ArН, J = 7.6 Hz), 7.49 d (1H,
ArН, J = 8.0 Hz), 7.62 d (1H, ArН, J = 7.5 Hz), 7.85 s
(1H, ArН). Mass spectrum, m/z (Irel, %): 404 (100)
[М + H]+.
1-(4-{4-Chlorobenzylpiperazin-1-yl}methyl)indoline-
1
2,3-dione (6) was obtained similarly. Yield 63%. H
1-({4-Methylpiperazin-1-yl}methyl)indoline-2,3-
NMR spectrum, δ, ppm: 2.38 br.s (4Н, CH2NCH2),
2.55 br.s (4Н, CH2NCH2), 3.44 s (2Н, CCH2N), 5.01 s
(2Н, NCH2N), 6.92 d (1H, ArН, J = 7.8 Hz), 7.07 t
(1H, ArН, J = 7.5 Hz), 7.29–7.38 dd (4H, ArН, J =
24.4 Hz, 8.2 Hz), 7.51 d (1H, ArН, J = 7.4 Hz), 7.59 t
(1H, ArН, J = 7.6 Hz). Mass spectrum, m/z (Irel, %):
369 (100) [М + H]+.
1
dione (12) was obtained similarly. Yield 69%. H
NMR spectrum, δ, ppm: 2.15 s (3Н, NСН3), 2.30 br.s
(4Н, CH2NCH2), 2.57 br.s (4Н, CH2NCH2), 4.39 s
(2Н, NCH2N), 7.15 t (1Н, ArН, J = 7.2 Hz), 7.26 d
(1Н, ArН, J = 8.0 Hz), 7.56 d (1Н, ArН, J = 7.4 Hz),
7.67 t (1Н, ArН, J = 7.8 Hz). Mass spectrum, m/z (Irel,
%): 259 (100) [М + H]+.
1-(4-{3-Chlorobenzylpiperazin-1-yl}methyl)indoline-
1
1-({4-Phenylpiperazin-1-yl}methyl)indoline-2,3-
2,3-dione (7) was obtained similarly. Yield 59%. H
1
dione (13) was obtained similarly. Yield 81%. H
NMR spectrum, δ, ppm: 2.38 br.s (4Н, CH2NCH2),
2.55 br.s (4Н, CH2NCH2), 3.44 s (2Н, CCH2N), 5.01 s
(2Н, NCH2N), 6.92 d (1H, ArН, J = 7.8 Hz), 7.07 t
(1H, ArН, J = 7.5 Hz), 7.18–7.29 m (4H, ArН), 7.55 d
(1H, ArН, J = 7.0 Hz), 7.61 t (1H, ArН, J = 7.6 Hz).
Mass spectrum, m/z (Irel, %): 369 (100) [М + H]+.
NMR spectrum, δ, ppm: 2.74 m (4Н, CH2NCH2), 3.12
m (4Н, CH2NCH2), 4.48 s (2Н, NCH2N), 6.77 t (1Н,
ArН, J = 7.3 Hz), 6.93 d (2Н, ArН, J = 7.9 Hz), 7.16–
7.21 m (3Н, ArН), 7.33 d (1Н, ArН, J = 8.0 Hz), 7.57
d (1Н, ArН, J = 7.4 Hz), 7.68 t (1Н, ArН, J = 7.8 Hz).
Mass spectrum, m/z (Irel, %): 321 (100) [М + H]+.
1-(4-{Piperonylpiperazine-1-yl}methyl)-5-chloro-
Procedure for kinetic studies. The studied com-
pound in an amount of (0.05×n) g, where n is the
number of samples (9–12), was dissolved in (5×n) mL
of anhydrous ethanol, then (5×n) mL of water was
added. The solution was incubated at a certain
temperature (80°C, 60°C or 40°C) and after 1 min a
sample was taken to determine the initial concentra-
tions of the studied compounds. Samples were then
collected at regular intervals to approximately 30%
conversion of the substance. The samples dried at
indoline-2,3-dione (8) was obtained similarly. Yield
78%. H NMR spectrum, δ, ppm: 2.35 br.s (4Н,
1
CH2NCH2), 2.59 br.s (4Н, CH2NCH2), 3.35 s (2Н,
CCH2N), 4.42 s (2Н, NCH2N), 5.95 s (2H, OCH2O),
6.68 d (1H, ArН, J = 8.4 Hz), 6.55–6.60 m (2Н, ArН),
7.28 d (1H, ArН, J = 8.5 Hz), 7.55 s (1H, ArН), 7.64 d
(1H, ArН, J = 8.5 Hz). Mass spectrum, m/z (Irel, %):
413 (100) [М + H]+.
1-({4-Benzylpiperazin-1-yl}methyl)-5-chloro-
1
room temperature and 1 mmHg were analyzed by H
indoline-2,3-dione (9) was obtained similarly. Yield
1
NMR on a Bruker AVANCE 400 spectrometer
(400 MHz) in DMSO-d6. The loss in the initial product
was determined by comparing the sum of the aromatic
72%. H NMR spectrum, δ, ppm: 2.36 br.s (4Н,
CH2NCH2), 2.60 br.s (4Н, CH2NCH2), 3.45 s (2Н,
CCH2N), 4.51 s (2Н, NCH2N), 7.22–7.35 m (5Н,
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 1 2018

56
ORLOVA et al.
3. Grigoreva, T.A., Garabadzhiu, A.V., and Tribulovich, V.G.,
Russ. J. Gen. Chem., 2016, vol. 86., p. 2454. doi
10.1134/S1070363216110098
4. Grigoreva, T., Novikova, D., Petukhov, A., Gureev, M.,
Garabadzhiu, A., Melino, G., Barlev, N., and Tribulo-
vich, V., Bioorg. Med. Chem. Let., 2017, vol. 27,
p. 5197. doi 10.1016/j.bmcl.2017.10.049
proton signals with the signal corresponding to the
protons of the N–CH2–N group.
Calculation procedure. The calculations were
performed according to the standard procedure [19].
Kinetic equation of a first order reaction (1).
V1 = k1c = – dc/dτ.
Reduction of the equation to a linear form gave
Eq. (2).
(1)
5. Roman, G., Eur. J. Med. Chem., 2014, vol. 89, p. 743.
doi 10.1016/j.ejmech.2014.10.076.
6. Davidovich, P., Novikova, D., Tribulovich, V.,
Smirnov, S., Gurzhiy, V., Melino, G., and Garabadzhiu, A.,
J. Mol. Struct., 2014, vol. 1075, p. 450. doi 10.1016/
j.molstruc.2014.07.008
7. Sriram, D., Banerjee, D., and Yogeeswari, P., J. Enzyme
Inhib. Med. Chem., 2009, vol. 24, no. 1, p. 1. doi
10.1080/14756360701404159
8. Pau, A., Murineddu, G., Asproni, B., Murruzzu, C.,
Grella, G., Pinna, G., Curzu, M., Marchesi, I., and
Bagella, L., Molecules, 2009, vol. 14, p. 3494. doi
10.3390/molecules14093494
ln c = ln c0 – k1τ.
(2)
The obtained data were processed by the method of
least squares, after which linear equation (3), reaction
rate constant [Eq. (4)], half-life [Eq. (5)] were obtained:
y = ax + b,
k1 = –tan α,
(3)
(4)
(5)
τ1/2 = ln 2/k1.
After processing the experimental points, plots of
the linear dependence were obtained and the correlation
coefficient was calculated by the Hammett equation (6).
9. Tramontini, M., Synthesis, 1973, vol. 1973, no. 12,
p. 703. doi 10.1055/s-1973-22294
10. Vélez, Y., Díaz-Oviedo, C., and Quevedo R., J. Mol.
Struct., 2017, vol. 1133, p. 430. doi 10.1016/
j.molstruc.2016.12.039
ρ = (1/σ)log k/k0.
(6)
Here ρ is the reaction constant, k is the reaction rate
constant of the compound with the substituent in the
benzene ring, k0 is the reaction rate constant of the
compound with hydrogen instead of the substituent, σ
is the Hammett constant [20].
11. Bekircan, O., and Bektas, H., Molecules, 2008, vol. 13,
p. 2126. doi 10.3390/molecules13092126
12. Jothi Rani, P., Sundara Pandiyan, M., Senthamil Selvi, A.,
Aruna, A., and Venkatesan, N., World J. Pharm. and
Pharmaceut. Sci., 2016, vol. 6, no. 1, p. 1259. doi
10.20959/wjpps20171-8412
13. Bogdanov, A.V., Vazykhova, A.M., Khasiyatullina, N.R.,
Krivolapov, D.B., Dobrynin, A.B., Voloshina, A.D., and
Mironov, V.F., Chem. Heterocycl. Compd., 2016,
vol. 52, no. 1, p. 25. doi 10.1007/s10593-016-1826-6
Gibbs activation energy was calculated by Eq. (7),
the entropy factor and enthalpy factor by Eq. (8).
ΔG# = RT(ln kb/h – ln k/T),
(7)
(8)
ΔG = ΔH – TΔS.
14. Bundgaard, H., and Johansen, M., J. Pharm. Sci., 1980,
ACKNOWLEDGMENTS
vol. 69, no. 1, p. 44. doi 10.1002/jps.2600690112
15. Bundgaard, H., and Johansen, M., Int. J. Pharmaceut.,
This work was financially supported by the Russian
Science Foundation (project no. 16-13-10358).
1981, vol. 8, p. 183, doi 10.1016/0378-5173(81)90096-X
16. Bundgaard, H., Johansen, M., Stella, V., and Cortese, M.,
Int. J. Pharmaceut., 1982, vol. 10, p. 181. doi
10.1016/0378-5173(82)90068-0
REFERENCES
17. Koshy, K., and Mitchner, H., J. Pharm. Sci., 1964,
1. Davidovich, P., Aksenova, V., Petrova, V., Tentler, D.,
Orlova, D., Smirnov, S., Gurzhiy, V., Okorokov, A.L.,
Garabadzhiu, A., Melino, G., Barlev, N., and
Tribulovich, V., ACS Med. Chem. Lett., 2015, vol. 6,
no. 8, p. 856. doi 10.1021/acsmedchemlett.5b00011
vol. 53, no. 11, p. 1381. doi 10.1002/jps.2600531123
18. Orlova, D.D., Garabadzhiu, A.V., and Tribulovich, V.G.,
Bull. SPbSTI (TU), 2016, no. 36. p.72. doi 10.15217/
issn1998984-9.2016.36.72
19. Benson, S.E, The Foundations of Chemical Kinetics,
New York: McGraw-Hill, 1960.
20. Hansch, C., and Taft, R., Chem. Rev., 199l, vol. 97,
2. Ding, K., Lu, Y., Nikolovska-Coleska, Z., Wang, G.,
Qiu, S., Shangary, S., Gao, W., Qin, D., Stuckey, D.,
Krajewski, K., Roller, P., and Wang, S., J. Med. Chem.,
2006, vol. 49, no. 12, p. 3432. doi 10.1021/jm051122a
p. 165. doi 10.1021/ cr00002a004
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