Structural investigation, molecular structure and molecular docking of solifenacin succinate, flavoxate hydrochloride and tolterodine tartrate anti-cholinergic drugs: Correlation using thermal analysis and mass spectral fragmentation

Article abstract of DOI:10.1007/s10973-017-6592-x

In this work, solifenacin succinate (SOLS), flavoxate HCl (FLXHC) and tolterodine tartrate (TOLT) drugs were investigated using thermal analysis (TA) measurements in comparison with electron impact mass spectral fragmentation at 70?eV. Also chemical purity, melting point (using differential scanning calorimetry), activation energy and enthalpy in the process of characterizing medicines were important requirements evaluated in quality control of the pharmaceutical industry. The thermal decomposition of these drugs revealed a moderate stability up to 161, 215 and 195?°C for SOLS, FLXHC and TOLT drugs, respectively, before a complete decomposition in the temperature ranges of 161–800, 215–650 and 195–650?°C. The initial decomposition can be accounted for the loss of C₇H₁₂NO molecule, followed by loss of C₂₀H₂₀NO₅ molecule for SOLS, loss of HCl, followed by loss of C₂₄H₂₅NO₄ molecule for FLXHC, and loss of C₂₃H₃₀NO₇ molecule followed by loss of C₃H₇ for TOLT drug. On the other hand, the molecular ion can easily fragmented by succinate, HCl and tartrate loss followed by loss of C₂H₅, C₄H₈ and C₂H₄ for SOLS, FLXHC and TOLT drugs, respectively. This is the best-selected pathway comparable with decomposition using TA. In addition, computational method including molecular docking was carried out to investigate the E. coli bacterial RNA (4p20) binding to the drug compounds under study. Molecular docking calculation indicated the existence of hydrogen bond and π-interaction between active sites of E. coli bacterial RNA (4p20) and O and or N and aromatic ring in all drug compounds which lead to their stabilization.

Full text of DOI:10.1007/s10973-017-6592-x

J Therm Anal Calorim
DOI 10.1007/s10973-017-6592-x
Structural investigation, molecular structure and molecular
docking of solifenacin succinate, flavoxate hydrochloride
and tolterodine tartrate anti-cholinergic drugs
Correlation using thermal analysis and mass spectral fragmentation
1
2
1
Ali K. Attia
•
Gehad G. Mohamed
•
Heba E. Ahmed
Received: 26 February 2017 / Accepted: 15 July 2017
Ó Akad e´ miai Kiad o´ , Budapest, Hungary 2017
Abstract In this work, solifenacin succinate (SOLS),
flavoxate HCl (FLXHC) and tolterodine tartrate (TOLT)
drugs were investigated using thermal analysis (TA) mea-
surements in comparison with electron impact mass spec-
tral fragmentation at 70 eV. Also chemical purity, melting
point (using differential scanning calorimetry), activation
energy and enthalpy in the process of characterizing
medicines were important requirements evaluated in
quality control of the pharmaceutical industry. The thermal
decomposition of these drugs revealed a moderate stability
up to 161, 215 and 195 °C for SOLS, FLXHC and TOLT
drugs, respectively, before a complete decomposition in the
temperature ranges of 161–800, 215–650 and 195–650 °C.
The initial decomposition can be accounted for the loss of
C H NO molecule, followed by loss of C H NO
drug compounds under study. Molecular docking calcula-
tion indicated the existence of hydrogen bond and p-in-
teraction between active sites of E. coli bacterial RNA
(4p20) and O and or N and aromatic ring in all drug
compounds which lead to their stabilization.
Keywords Solifenacin succinate Á Flavoxate HCl Á
Tolterodine tartrate Á Thermal analysis Á Mass spectral
fragmentation Á Molecular docking
Introduction
Solifenacin succinate (SOLS) is a urinary antispasmodic
(anti-muscarinic class) drug. It acts as a direct antagonist at
muscarinic acetylcholine receptors in cholinergically
innervated organs. Its anti-cholinergic parasympatholytic
action decreases the tonus of smooth muscle in the bladder,
effectively reducing the number of required voids, urge
incontinence episodes and urge severity and improving
retention, facilitating increased volume per void [1].
Flavoxate HCl (FLXHC) is an antispasmodic drug that
works by counteracting smooth muscle spasms of the uri-
nary tract through directly acting on the muscle. It prevents
spasms in the urinary tract and relieves painful or difficult
urination, urinary urgency, excessive nighttime urination,
pubic area pain, frequent urination and the inability to hold
urine caused by urinary tract infections [2].
7
12
20 20
5
molecule for SOLS, loss of HCl, followed by loss of
C H NO molecule for FLXHC, and loss of C H NO
7
2
4
25
4
23 30
molecule followed by loss of C H for TOLT drug. On the
3
7
other hand, the molecular ion can easily fragmented by
succinate, HCl and tartrate loss followed by loss of C H ,
2
5
C H and C H for SOLS, FLXHC and TOLT drugs,
2 4
4
8
respectively. This is the best-selected pathway comparable
with decomposition using TA. In addition, computational
method including molecular docking was carried out to
investigate the E. coli bacterial RNA (4p20) binding to the
Tolterodine tartrate (TOLT) is an anti-muscarinic drug
used in the treatment of urinary urge incontinence and
other symptoms of an overactive bladder (Fig. 1) [3].
Literature survey revealed that few chromatographic
methods for SOLS [4–15], FLXHC [16–19] and TOLT
[20–27], spectrophotometric methods for SOLS [28, 29],
FLXHC [30, 31] and TOLT [32–34], potentiometric
&
Gehad G. Mohamed
ggenidymohamed@sci.cu.edu.eg
1
2
National Organization for Drug Control and Research,
P.O. Box 29, Cairo, Egypt
Chemistry Department, Faculty of Science, Cairo University,
Giza 12613, Egypt
123

A. K. Attia et al.
O
SOLS
TOLT
FLXHC
OH
H
O
O
O
H C
CH3
.
OH
H
3
COOH
CH3
O
CH
.
HC OH
N
3
O
O
N
H C
HO CH
COOH
3
CH3
N
C
O
O
.
HCl
N
Fig. 1 Chemical structures of SOLS, FLXHC and TOLT drugs
method for the determination of FLXHC [35, 36], spec-
trofluorimetric method for the determination of FLXHC
The knowledge about drugs interaction mechanisms of
drug with plasma proteins as a biomacromolecule was
essential requirement to understand pharmacodynamics
and pharmacokinetics of drugs [43]. As regards function of
plasma proteins as drug carriers, there were a large number
of researches about binding of drugs to them [44, 45]. As a
result, plasma proteins interact with metal complexes in
evaluating the worth drug candidate. Generally, proteins
are known as the major targets for most of the drugs in
organisms [46]. Their interaction with drug affected on the
drug absorption, distribution and elimination in the circu-
latory system [47] and can prevent their rapid elimination
from blood stream [48]. In this work, the behavior of
SOLS, FLXHC and TOLT dugs was investigated using
electron impact mass spectrometry at 70 eV and thermal
analysis techniques such as TG, DrTG, DTA and DSC.
These techniques were spirited in material characterization,
and many authors have agreed these techniques to char-
acterize the materials [49–54]. Therefore, the correct
pathway of degradation (TA and MS) knowing this struc-
tural session of bonds can be used to predict the active sites
of these drugs which responsible for their chemical reac-
tivity. Correlation between the three techniques was dis-
cussed in order to throw more light on the degradation
behavior of these drugs. Molecular docking of the three
drugs under investigation was carried out using molecular
docking studies to predict their binding modes toward
E. coli bacterial RNA (4p20).
[
37] and voltammetric methods for the determination of
FLXHC [38] and TOLT [39, 40] were reported for their
determination in bulk, dosage forms and biological fluids.
Thermal decomposition measurements are used to study
the physical and chemical changes that occur in the sample
under temperature-controlled program. Therefore, one can
explain a thermal analysis curve by relating the features of
the property against temperature with possible chemical or
physical events that have taken place in the system under
observation. In thermogravimetric analysis (TG), the mass
of sample was measured as a function of temperature at a
predetermined rate where the sample may either lose mass
to the atmosphere or gain mass by reaction with the
atmosphere. In dynamic thermogravimetry (TG), the sam-
ple was subjected to continuous temperature changes under
usually linear time. Meanwhile, in static thermogravimet-
ric, the sample was maintained at a constant temperature
for a period of time where any changes in mass are
recorded. Derivative thermogravimetry (DrTG) can be
obtained from TG, and it is used to know the decomposi-
tion steps of compounds.
Differential thermal analysis (DTA) is the measurement
of the difference in temperature between a sample and a
reference as heat applied to the system. It provides infor-
mation on the chemical reaction, phase transformation and
structure changes that occur in a sample during a heat-up or
a cool-down cycle. The DTA measures the differences in
energies released or absorbed and the changes in heat
capacity of materials as a function of temperature. All
materials behave in certain predictable ways when exposed
to certain temperature.
Experimental
Materials
Differential scanning calorimetry (DSC) is used to
measure the temperature and heat flows associated with
transition in materials as a function of time or temperature.
These measurements provide qualitative and quantitative
information about the physical and chemical changes that
involve endothermic and exothermic processes or changes
in heat capacity on DSC technique [41, 42].
SOLS, FLXHC and TOLT drugs were obtained from
Multipharma Pharmaceutical Company (Egypt), Uni-
pharma Pharmaceutical Company (Egypt) and Pfizer
Pharmaceutical Company (Egypt), respectively, their pur-
ity values were 100.3, 100.5 and 99.72% for SOLS,
FLXHC and TOLT drugs, respectively.
1
23

Structural investigation, molecular structure and molecular docking of solifenacin succinate…
Equipment and procedures
Results and discussion
Thermal analyses
Because of their importance as anti-cholinergic drugs, the
chemistry and reactivity of SOLS, FLXHC and TOLT
drugs have always been of great interest. Knowledge of
thermal decomposition mechanism of SOLS, FLXHC and
TOLT drugs was very important to understand the chem-
ical processes that exact major fragmentation pathway in
EI using conventional MS will be discussed.
Thermogravimetric analysis, derivative thermogravimetry
and differential thermal analysis measurements were taken
by using simultaneous DTA-TG thermal analyzer appara-
tus (Shimadzu DTG-60H). The mass of samples was
ranging from 4 to about 7 mg, using a platinum pan.
Measurements were taken from ambient to 800 °C in
dynamic nitrogen atmosphere with the flow rate of
Thermal analysis decomposition of drugs
-
1
-1
3
0 mL min and heating rate of 10 °C min
DSC curves were obtained using Shimadzu DSC-50
.
Thermogravimetry is a kind of technology to measure the
change in the mass of the samples with the temperature
[56–59]. Figure 2 shows the thermal analyses data (TG,
DrTG and DTA) for SOLS, FLXHC and TOLT drugs
under the goal of this article. The TG data showed two
main mass losses for SOLS and TOLT drugs and four main
mass losses for FLXHC drug. The first stage occurred at
161–280 °C, 215–254 °C and 195–295 °C as a result of an
estimated mass losses of 26.53, 8.51 and 91.78% which
may be accounted for the loss of C H NO, HCl and
cell. Approximately 3 mg of samples was weighed and
placed in a sealed aluminum pan. An empty aluminum pan
was used as a reference. The purity determination was
performed in temperature range from 10 to 400 °C in
-
1
nitrogen atmosphere with flow rate of 30 mL min using
-
1
heating rate of 10 °C min . DSC equipment was prelim-
inary calibrated with standard of indium.
7
12
Mass spectrometry
C H NO molecules (calcd. mass loss = 26.22, 8.53 and
2
3
30
7
9
0.96%) for SOLS, FLXHC and TOLT drugs, respectively.
The second stage occurred at 280–800 °C, 254–307 °C and
95–650 °C as a result of 72.88, 27.043 and 9.14% esti-
The mass spectra of SOLS, FLXHC and TOLT drugs were
recorded using Shimadzu GC–MS-Qp 1000 PX quadruple
mass spectrometer with electron multiplier detector
equipped with GC–MS data system. The sample was ion-
ized by electron beam emitted from the filament, the gen-
erated ions being effectively introduced into the analyzer
by the focusing and extractor lenses system. The MS was
continuously scanned and the spectra obtained were stored.
Electron ionization mass spectra were obtained at ionizing
energy value of 70 eV, ionization current of 60 mA and
2
mated mass losses which may be due to the loss of
C H NO , C H and C H molecules (calcd. mass
2
0
20
5
9
8
3 7
loss = 73.67, 27.11 and 9.041%) for SOLS, FLXHC and
TOLT, respectively. For FLCHC, the third stage occurred
at 307–365 °C due to loss amount to 54.47% may be
assigned to the loss of C H O molecule (calcd. mass
1
3 13 4
loss = 54.44%). The fourth stage occurred at 365–650 °C
as a result of 9.87% estimated mass losses which may be
due to the loss of C H N molecule (calcd. mass
-
6
vacuum of 10 Torr.
2
4
loss = 9.81%). The thermal degradation patterns of these
drugs are shown in Schemes 1, 2 and 3. These mass losses
appeared as endothermic and exothermic peaks as given by
DTA data. The first mass loss appeared at 152, 236 and
Molecular docking simulation
In order to find out the possible binding modes of the drugs
under study against E. coli bacterial RNA (4p20), molec-
ular docking studies were performed using Autodock
MOA2008.10 software and it is rigid molecular docking
software [55] and is an interactive molecular graphics
program for calculating and displaying feasible docking
modes of drugs and E. coli bacterial RNA (4p20) receptor.
It necessitates the drugs and the receptor as input in PDB
format. The structure of the drugs in PDB file format was
created by Gaussian09 software. The crystal structure of
the E. coli bacterial RNA (4p20) receptor was downloaded
from the protein data bank (https://www.rcsb.org/pdb/
home/home.do).
2
10 °C as endothermic peaks due to melting point of
SOLS, FLXHC and TOLT drugs, respectively. Thus, these
drugs melt with decomposition. The second mass loss
appeared in DTA as endothermic peaks at 255 and 338 °C
for SOLS and at 300 and 238 °C for FLXHC and TOLT,
respectively. There were also exothermic peaks at 364,
4
4
01, 444 and 510 °C for SOLS and at 281, 313, 366, 451,
88 and 535 °C for TOLT. The third and fourth mass
losses appeared in DTA as endothermic peak at 331 °C and
exothermic peak at 511 °C for FLXHC.
The thermal analysis results showed that FLXHC, SOLS
and TOLT drugs started to decompose at 215, 195 and
161 °C, respectively. Therefore, the order of drugs under
123

A. K. Attia et al.
investigation was FLXHC [ TOLT [ SOLS according to
their stability.
z = 392, R.I. = 53%) and C H to produce C H NO
4
9
8
15 17
(m/z = 275, R.I. = 42%) for FLHXC which agree with the
results of thermal analysis of these drugs. The important
ions are represented in Table 1.
Mass spectral fragmentation
Electron ionization (EI) mass spectra of SOLS, FLXHC
and TOLT drugs at 70 eV were recorded and investigated
Determination of purity of SOLS, FLXHC
and TOLT drugs
(
Fig. 3). Schemes 4, 5 and 6 show the mass fragmentation
patterns of the studied drugs, the loss of C H NO mole-
7 12
For the determination of purity of drugs, DSC technique
was utilized. It was based on the assumption that the
impurities will lower the melting point of a pure substance.
cule to produce C H NO (m/z = 355, R.I. = 32%) for
5
2
0
20
SOLS and the loss of HCl to produce C H NO (m/
2
4
25
4
Fig. 2 TG, DrTG and DTA
curves of SOLS, FLXHC and
TOLT drugs
DTG/mg min–1
.00
TG/%
DTA/µV
1
Mass Loss
1
00.00
4
44.01°C
5
DTA
TGA
DrTGA
–
26.533%
SOLS
09.98°C
3
64.07°C
Exo
20.00
400.55°C
8
0.00
160.73°C
0.00
Mass Loss
72.881%
3
71.83°C
–
6
0.00
0.00
280.25°C
56.58°C
1
0.00
0.00
2
–
1.00
2.00
4
3
37.68°C
–
Endo
2
0.00
–
255.42°C
152.00°C
–
0.00
–
340.79°C
0.00
200.00
600.00
400.00
800.00
T/°C
DTG/mg min–1
.00
TG/%
DTA/µV
1
DTA
TGA
60.00
1
00.00
FLXHC
11.18°C
Mass Loss
–8.508%
Mass Loss
DrTGA
5
–
27.043%
2
15.00°C
254.00°C
40.00
0
.00
3
85.42°C
Exo
5
13.96°C
2
32.71°C
2
0.00
2
98.97°C
06.50°C
–
1.00
2.00
50.00
3
3
31.11°C Mass Loss
0
.00
–
54.468%
–
2
99.90°C
31.87°C
3
Mass Loss
Endo
–
9.872%
–20.00
–0.00
2
36.42°C
–
3.00
–0.00
200.00
400.00
T/°C
600.00
800.00
1
23

Structural investigation, molecular structure and molecular docking of solifenacin succinate…
Fig. 2 continued
DTG/mg min–1
TG/%
DTA/µV
Mass Loss
91.781%
TOLT
DTA
TGA
DrTGA
100.00
451.44°C
487.83°C
–
2
80.73°C
12.62°C
66.27°C
195.00°C
3
535.10°C
0
.00
3
2
0.00
460.82°C
Exo
295.00°C
0
.00
–
1.00
2.00
50.00
2
38.34°C
–
20.00
Mass Loss
–9.142°C
–
Endo
2
48.84°C
–0.00
–
40.00
2
10.58°C
–0.00
200.00
400.00
600.00
T/°C
Scheme 1 Thermal
decomposition pattern of SOLS
drug
O
O
N
OH
HO
.
N
O
O
O
N
OH
HO
280–800 °C
Complete decomposition
O
.
–
O
The melting transition of pure substance (100% crystalline)
should be infinitely sharp, but impurities or defects in the
crystal structure will broaden the melting range and lower
the melting point [60]. In a system that contains impurities,
Van’t Hoff equation approximately held and allowed the
purity value to be calculated as follows: T = T -
f 0
2
[(R T 9 /DH )Á 1/F], where T is the melting temperature
0
f
f
of the sample, T is the melting point of a pure substance in
0
Kelvin (K), R is the gas constant, DH is the heat of fusion,
f
F is fraction of sample melted at T and x is mole fraction
f
123

A. K. Attia et al.
Scheme 2 Thermal
decomposition pattern of
FLXHC drug
O
O
O
O
CH3
CH3
– HCl
2
15–254 °C
C
O
O
C
O
O
.
HCl
N
N
–C9H8 254–307 °C
O
3
65–650 °C
O
3
07–365 °C
Complete decomposition
C
O
–
C H N
–C H O4
13 13
2
4
N
O
N
Scheme 3 Thermal
decomposition pattern of TOLT
drug
H3C
CH3
OH
COOH
HC
CH
COOH
OH
H
CH3
N
.
H3C
HO
CH3
1
95–295 °C
–
C H NO
2
3
30
7
H C
CH3
3
2
95–650 °C
C3H7
Complete decomposition
–
of impurities in the original sample. Figure 4 shows the
DSC curves of SOLS, FLXHC and TOLT drugs. It was
obvious that very strong and sharp endothermic peaks
appeared at 147.5, 236.2 and 213.70 °C which may be
attributed to the melting of SOLS, FLXHC and TOLT
drugs, respectively. Table 2 shows melting point values
and degree of purity of SOLS, FLXHC and TOLT which
agree with those of the reported methods.
1
23

Structural investigation, molecular structure and molecular docking of solifenacin succinate…
Fig. 3 Mass spectra of SOLS, FLXHC and TOLT drugs
123

A. K. Attia et al.
O
O
O
OH
OH
HO
HO
OH
HO
.
.
O
O
O
O
.
O
OH
HO
N
N
–
C7H12NO
N
O
N
6 4
–C H
O
O
–
C H11NO
10
O
C H N O
27
32
2
6
C H O
4 6 4
m/z = 118
R.I. = 30.59
m/z = 480.55
R.I = 60
C14H17NO5
m/z = 279
R.I = 38.82
–
C H O
C H NO5
m/z = 355
20 20
4
5
R.I = 31.76
C8H15NO2
m/z = 157 –CH O
O
N
C H11N
7
O
–
C H12N
1
5
4
2
N
N
m/z = 109
N
O
OH R.I. = 43.53
R.I. = 51.18
C23H27N2O2
m/z = 363
R.I. = 32.35
–H3
–C H
–
C3H2
2
5
O
N
N
O
O
N
C23H24N O2
2
N
m/z = 360
O
H
N
O
H
R.I. = 35.29
–
C H
N
6
4
O
C21H22N O2
2
m/z = 322
O
N
C20H25N O2
–C5H7N
2
R.I. = 33.53
m/z = 325
N
O
C17H20N O2
2
R.I. = 47.65
m/z = 284
–
C H
2
6
R.I. = 35.29
HO
N
–C H NO
8 11 2
O
–
C H
6 4
O
N
–
CH O
2 2
H N
C16H15NO2
m/z = 253
R.I. = 32.35
2
HN
O
HO
N
O
C H N
C10H11NO2
m/z = 177
R.I. = 36.47A
9 9
C18H N O
19
2
2
m/z = 131
m/z = 295
R.I. = 33.53
–
H N
2
R.I1. = 35.9
O
N
O
C14H13
C18H17NO2
m/z = 279
R.I. = 38.82
m/z = 181
R.I. = 31.76
–
C H
2 2
C H
6 6
–CH
m/z = 77
R.I. = 32.35
HO
N
–CHO2
HN
–C8H7
O
C15H14N+
C7H7+
C16H15NO2
m/z = 253
R.I. = 32.35
m/z = 208
m/z = 91
R.I. = 44.71
R.I. = 48.24
Scheme 4 The mass fragmentation pathways of the principle fragment ions of SOLS
1
23

Structural investigation, molecular structure and molecular docking of solifenacin succinate…
O
O
O
CH3
CH3
O
O
OH
–
HCl
–
C9H8
C
O
O
.
C
O
O
C
O
O
HCl
N
N
N
–
C24H25NO4.HCl
m/z = 427
R.I. = 67.2
C24H25NO4
m/z = 392
R.I = 52.8
C15H17NO4
m/z = 275
R.I. = 41.6
–C6H5
–C5H10N
O
O
CH3
CH3
C18H21NO4
m/z = 315
R.I. = 78.4
O
–
CO
O
C
O
O
C
O
O
O
N
–O
+
H2
C
O
O
C20H18O4
m/z = 322
R.I. = 41.6
O
N
CH3
–
C3H7
C17H21NO3
m/z = 287
R.I. = 59.2
–
C9H7O
–
C7H11NO2
O
C18H23NO3
C
O
O
O
m/z = 301
R.I. = 46.4
CH3
H2C
O
N
N
O
C8H14NO2
m/z = 156
R.I. = 73.6
O
–
C7H11N
C
O
C17H11O4
m/z = 279
R.I. = 49.6
OH
O
C10H10O
m/z = 146
R.I. = 46.4
–C HO
2
2
CH3
–
CO2
+
H3
N
C11H12O3
m/z = 192
R.I. = 46.4
O
OH
H2C
OH
C6H13N
m/z = 99
CH3
–
CHO
OH
R.I. = 16.8
C6H12O
m/z = 100
CH3
O
R.I. = 8.8
OH
O
C
H
O
m/z = 163
R.I. = 45.6
C16H14O2
m/z = 238
R.I. = 41.6
–
CH3
CH2O
O
H
–C5
C9H9O
–CH2
OH
m/z = 133
R.I. = 3.2
–
C4
O
+
H
CH3
O
H
–
C4
C7H14O2
m/z = 130
R.I. = 45.6
-CO
+H
O
C11H14O2
m/z = 178
R.I. = 52
–C2H4
C8H10
m/z = 106
R.I. = 59.2
O
C5H10O
m/z = 86
R.I. = 45.6
–C5H8O2
C6H6
m/z = 77
R.I. = 48
Scheme 5 The mass fragmentation pathways of the principle fragment ions of FLXHC
123

A. K. Attia et al.
OH
CH
OH
CH
COOH
H
C
HOOC
COOH
H
C
HOOC
OH
C
OH
.
OH
.
H C
3
CH3
CH3
COOH
OH
–
C H N
H
C
8
17
–
C14H14O
HOOC
OH
H
N
H C
3
OH
CH3
.
H C
3
+
C H O
4 6 6
m/z = 149
R.I. = 80.61
C H NO
7
m/z = 475
R.I. = 93.88
+
26
37
C H O
m/z = 347
1
8
20
7
R.I. = 63.27
C H O
4 6 6
H C
3
CH3
CH3
OH
H C
3
CH3
OH
OH
H
N
H
H
N
–
C H
N
H
2
4
H C
3
CH3
H3C
–C H
3 6
H C
3
C H NO
m/z = 297
R.I. = 71.43
2
0 27
C H NO
m/z = 325
R.I. = 56.12
22
31
C17H21NO
m/z = 255
–
C H
6 6
R.I. = 58.16
–
C H N
2 6
–
C H O
6
3
H C
3
CH3
CH3
OH
H C
3
CH3
OH
N
H
N
H
H C
3
CH3
H C
3
C H NO
1
6 25
m/z = 247
R.I. = 55.10
+
C H O+
C H N
1
4
24
15 15
–
C H N
–C H N
4 11
6
14
m/z = 206
R.I. = 56.12
m/z = 211
R.I. = 53.06
–
C H
1
0
13
–
C H
7 4
–
C H O
OH
7 6
H C
3
CH3
OH2
H C
3
HN
+
C H O
1
0 11
C H N
H C
3
4
11
m/z = 147
R.I. = 71.43
CH3
m/z = 73
R.I. = 66.33
–
C H
10 13
+
C H O
8 11
m/z = 133
R.I. = 67.35
C8H9+
m/z = 105
R.I. = 72.45
m/z = 123
R.I. = 63.27
–
C2
Scheme 6 The mass fragmentation pathways of the principle fragment TOLT
1
23

Structural investigation, molecular structure and molecular docking of solifenacin succinate…
Table 1 Relative intensity (R.I) and possible fragments for important
ions produced from SOLS, FLXHC and TOLT drugs
Table 1 continued
Drug
m/z
R.I
Formula
Drug
m/z
R.I
Formula
2
1
1
1
1
1
7
06
49
47
33
23
05
3
56
81
71
67
63
72
66
C
C
C
C
C
C
C
14
H
24
N
SOLS
480
60
32
35
32
48
33
35
36
39
32
45
36
32
44
34
51
31
48
32
67
53
42
78
46
59
50
42
42
46
52
46
74
46
3
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
23
23
23
20
20
21
18
17
14
16
15
10
14
H
H
H
H
H
H
H
H
H
H
H
H
H
26
27
24
N
N
N
2
2
2
O
O
O
2
2
2
ÁC
4 6 4
H O
4
H
6
O
6
3
3
3
3
3
2
2
2
2
2
1
1
1
1
1
1
9
7
63
60
55
25
22
95
84
79
53
08
77
81
57
31
18
09
1
10
H
11
O
10
H
13
20NO
5
8
8
4
11
H O
25
N
22
N
19
N
20
N
O
2 2
O
2 2
O
2 2
O
2 2
H
H
9
11
N
17NO
15NO
5
2
Molecular docking studies
14
N
The method of molecule simulation is an efficient way to
predict the correct binding mode and the interaction region.
Targeting the minor groove of DNA through binding to a
small molecule has long been considered as an important
tool in molecular recognition of a specific DNA sequence.
It was confirmed that the major factor affecting on the
distribution, metabolism, the free concentration and the
elimination rate of drugs was the binding affinity of drugs
to E. coli bacterial RNA (4p20). Moreover, the half-life of
drugs depends on their E. coli bacterial RNA (4p20)
binding that affected on the dosage of drugs. Therefore, the
study of interaction between E. coli bacterial RNA (4p20)
and SOLS, FLXHC and TOLT drugs was essential to
investigate the pharmacokinetics and availability of drugs
in tissues. The study of interaction of SOLS, FLXHC and
TLOT molecules and E. coli bacterial RNA (4p20) can
express carcinogens’ carcinogenic properties, contributing
to further understanding of the molecular mechanisms of
drug, as well as the drug–DNA interactions in rational drug
design. The big and small grooves are the mainly binding
sites of DNA. The interaction of binding mode can be non-
covalent binding, covalent binding and long-range assem-
bly. The first step is to explore the interaction of the
binding model of the series of compounds and DNA
molecule. Then, the binding energy must be calculated.
Molecular docking studies for the investigated drugs have
been performed using MOE 2008.10. software [55] to predict
their binding modes toward E. coli bacterial RNA (4p20)
11NO
13
2
8
9
7
4
7
6
H15NO
2
H
H
H
H
H
9
N
11
N
6
O
4
7
7
6
FLXHC
427
24
24
20
18
18
17
17
15
16
11
11
10
H
25NO
4
ÁHCl
3
3
3
3
2
2
2
2
1
1
1
1
1
1
1
1
1
9
8
7
92
22
15
01
87
79
75
38
92
78
63
56
46
33
30
06
00
9
H
H
H
H
H
H
H
H
H
H
H
26NO
4
18 4
O
21NO
23NO
21NO
4
3
3
11 4
O
17NO
4
14
12
14
11
O
O
O
O
2
3
2
2
8
H14NO
2
10 10
H O
9
7
8
6
6
5
6
9
H O
46
59
9
14 2
H O
H
10
H
12
H
13
H
10
H
6
O
N
O
17
46
48
94
63
56
71
58
55
53
6
[
61–63]. Figure 5 shows the binding models of SOLS,
7
FLXHC and TLOT to the bacterial RNA. The 2D interaction
maps (Fig. 6) showed the different modes of interactions
between each drug and the bacterial RNA (Table 3).
TOLT
476
22
H
18
H
22
H
20
H
17
H
16
H
15
H
31NOÁC
4 6 7
H O
347
325
297
255
247
211
20 7
O
31NO
27NO
21NO
25NO
The O14 and six-membered ring of SOLS compound
were the active sites and bound with O2P(Adenosine31, B)
and N4(Cytosine32, B) active sites of E. coli bacterial RNA
(4p20) via hydrogen bond and p-interaction, and the bind-
-
1
15
O
ing energy was found to be -3.6 and -1.5 kcal mol ,
123

A. K. Attia et al.
DSC/mW
.00
3
2
1
0
Exo
SOLS
–
–
163.18mJ
84.11J g
–
1
.00
.00
.00
Endo
–
–
1.00
2.00
1
47.47°C
0.00
100.00
200.00
T°C
DSC/mW
–
325.30mJ
Exo
–
302.33mJ
–169.42J g–1
2
.00
FLXHC
–157.46J g–1
3
00.12°C
0
.00
Endo
–
2.00
2
36.21°C
300.00
0.00
100.00
200.00
T°C
DSC/mW
Exo
–203.34mJ
–95.02J g
TOLT
–188.95mJ
–1
–
1
–
88.29J g
5
.00
2
28.34°C
0
.00
Endo
2
13.70°C
0.00
100.00
T°C
200.00
Fig. 4 DSC curves of SOLS, FLXHC and TOLT drugs
respectively. The minimum binding energies of the
(Cytosine10, A) of E. coli bacterial RNA (4p20) and O27
(pyrone ring), N48 (piperidine ring) and six-membered ring
of FLXHC compound, respectively. The minimum energy
docked pose revealed that the TLOT compound fitted into
FLXHC compound with DNA were -3.1, -1.1 and
-
1
-
0.7 kcal mol . There are a H-bond and p-interaction
between N4 (Cytosine8, A), N4 (Cytosine32, B) and N4
1
23

Structural investigation, molecular structure and molecular docking of solifenacin succinate…
Table 2 Melting point and degree of purity of SOLS, FLXHC and TOLT drugs
Drug
Melting point/°C
Degree of purity/%
DTA method
Melting point apparatus
DSC Method
Literature [53, 54]
DSC
Reported
Ref
SOLS
152.00
236.42
210.58
153
230
205
147.47
236.21
213.70
145–153
230–236
205–210
99.98
99.99
99.99
100.30
100.50
99.72
[8]
FLXHC
TOLT
[21]
[25]
Fig. 5 Binding models of a SOLS, b FLXHC and c TOLT to the
bacterial RNA
the curve contour of the targeted DNA in the groove.
Moreover, –C=O group (O32, carbonyl group) and N34
(
quinuclidine) of the TLOT compound acted as strong
H-bond donors or acceptors and were engaged in hydro-
gen-bonding interactions with N4 (Cytosine30, B) and N6
Fig. 6 2D interaction maps of a SOLS, b FLXHC and c TOLT to the
bacterial RNA
123

A. K. Attia et al.
Table 3 Molecular docking of SOLS, FLXHC and TLOT drugs with E. coli bacterial RNA (4p20)
-
1
Compound
SOLS
Active site
Receptor
Interaction
Distance/A°
E/ kcal mol
O14 (hydroxyl group)
Six-membered ring
O27 (pyrone ring)
O2P(Adenosine31, B)
N4(Cytosine32, B)
N4(Cytosine8, A)
N4(Cytosine32, B)
N4(Cytosine10, A)
N4(Cytosine30, B)
N6(Adenosine29, B)
Hydrogen bond
p-interaction
2.89
3.93
2.86
3.24
3.81
3.32
3.52
-3.6
-1.5
-3.1
-1.1
-0.7
-1.3
-1.2
FLXHC
TOLT
Hydrogen bond
Hydrogen bond
p-interaction
N48 (piperidine ring)
six-membered ring
O32 (carbonyl group)
N34 (quinuclidine)
Hydrogen bond
Hydrogen bond
(
Adenosine29, B) in the DNA. The binding energy was
5. Annapurna MM, Sowjanya G, Naidu MS, Lohithasu D. A vali-
dated liquid chromatographic method for the determination of
solifenacin succinate (urinary antispasmodic) in tablets. Chem
Sci Trans. 2014;3:602–7.
-
found to be -1.3 and -1.2 kcal mol , respectively.
1
6
. Vijayasree V, Kumar DA, Rao JVLNS. Validated RP-HPLC
method for the estimation of solifenacin succinate in tablet
dosage forms. Pharmanest. 2013;4:206–12.
Conclusions
7
. Krishna SR, Rao BM, Rao NS. A validated rapid stability indi-
cating method for the determination of related substances in
solifenacin succinate by ultra fast liquid chromatography.
J Chromatogr Sci. 2010;48:807–10.
. Desai N, Syed SH, Vasanthraju SG, Karthik A, Udupa N.
Development and validation of stability indicating method for
determination of solifenacin in bulk formulations. Int J Pharm
Pharm Sci. 2011;3:70–4.
This study provided further insights into application of
experimental TA and MS techniques on SOLS, FLXHC
and TOLT drugs. From the application of these techniques,
it was concluded that there were some similarities in TA
and MS fragmentation pathways for SOLS and FLXHC
drugs but there was difference in TA and MS fragmenta-
tion pathway for TOLT drug. DSC technique can be used
to determine purity of drugs giving compatible results with
the results obtained using the reported methods. This work
revealed the importance of the thermal analysis and DSC
techniques for the quality control of SOLS, FLXHC and
TOLT drugs. The way of interaction between the active a
sites in the drugs with E. coli bacteria was studied and
confirmed using molecular docking simulation program.
8
9. Israel DS, Krishnachaitanya K, Gowrisankar D. RP-HPLC
method for the estimation of tamsulosin and solifenacin in bulk
and its dosage forms. Int J Pharm Sci Res. 2013;4:4343–50.
0. PaliwalN,JainP, DubeyN, SharmaS, KhuranaS, MishraR, Paliwal
SK. Liquid chromatography tandem mass spectrometry method for
quantification of solifenacin in human plasma and its application to
bioequivalence study. Int J Drug Dev Res. 2013;5:91–101.
1. Babu PS, Parveen SKR, Chandrasekar KB, Challa BR, Awen
BZSh. In vitro-in vivo correlation studies of modified release
solifenacin tablet dosage form. J Sci Res Rep. 2014;3:1905–15.
2. Puttagunta SB, Shaik RP, Bannoth CK, Challa BSR, Awen BZSh.
Bioanalytical method for quantification of solifenacin in rat
plasma by LC-MS/MS and its application to pharmacokinetic
study. J Anal Sci Tech. 2014;5:1–8.
1
1
1
Acknowledgements The authors would like to express their grati-
tude to the National Organization for Drug Control and Research
(
NODCAR, Egypt) and Faculty of Science, Cairo University, for
providing instruments and the means necessary to accomplish
this work.
13. Oki T, Sato S, Miyata K, Yamada S. Muscarinic receptor binding,
plasma concentration and inhibition of salivation after oral
administration of a novel antimuscarinic agent, solifenacin suc-
cinate in mice. Br J Pharmacol. 2005;145:219–27.
1
4. Desai D, Mehta G, Ruikar D, Jain RA, Rajput SJ. Development
and validation of stability indicating HPTLC method of solife-
nacin succinate. Asian J Pharm Biol Res. 2011;1:310–6.
References
1
. Smulders RA, Krauwinkel WJ, Swart PJ, Huang M. Pharma-
cokinetics and safety of solifenacin succinate in healthy young
men. J Clin Pharmacol. 2004;44:1023–33.
1
5. Wankhede SB, Somani K, Chitlange SS. Stability indicating
normal phase HPTLC method for estimation of alfuzosin and
solifenacin in pharmaceutical dosage form. Int J Chem Tech Res.
2
3
. Pedersen E. Studies on the effect and mode of action of flavoxate
in human urinary bladder and sphincter. Urol Int. 1977;32:202–8.
. Gillberg PG, Sundquist S, Nilvebrant L. Comparison of the
in vitro and in vivo profiles of tolterodine with those of subtype-
selective muscarinic receptor antagonists. Eur J Pharmacol.
2
011;3:2003–10.
1
1
1
6. Attimarad MV, Harsha NS, Setty RS. Simultaneous determina-
tion of ofloxacin and flavoxate hydrochloride in human plasma by
RP-HPLC. J Liq Chromatogr Rel Tech. 2012;35:768–77.
7. El-Gindy A, Abdel-Salam ARA, Sallam S. High-performance
liquid chromatographic determination of flavoxate hydrochloride
and its hydrolysis product. Drug Dev Ind Pharm. 2008;34:1311–22.
8. Mahesh A. Liquid chromatographic determination of flavoxate
1
998;349:285–92.
4
. Reddy BVR, Reddy BS, Raman NVVSS, Subhash RK, Rambabu C.
Development and validation of a specific stability indicating high
performance liquid chromatographic methods for related com-
pounds and assay of solifenacin succinate. J Chem. 2013;2013:1–10.
HCl in pharmaceutical formulation.
010;2:280–3.
J
Young Pharm.
2
1
23

Structural investigation, molecular structure and molecular docking of solifenacin succinate…
1
2
9. El-Shaheny RN, El-Enany NM, Belal FF. A green HPLC method
for the analysis and stability study of flavoxate HCl using
micellar eluent. Anal Methods. 2014;6:1001–10.
0. Kumar SA, Debnath M. Rao JVLNS. Method development and
validation of tolterodine tartarate in bulk as well as in pharma-
ceutical formulation by using RP-HPLC. Int. J Pharm Pharm Sci.
38. Ghoneim MM, El-Attar MA, Razeq SA. Voltammetric quanti-
tation at the mercury electrode of the anticholinergic drug
flavoxate hydrochloride in bulk and in a pharmaceutical formu-
lation. Central Eur J Chem. 2007;5:496–507.
39. Kul D. Sensitive and selective determination of tolterodine tar-
trate and its electrochemical investigation on solid carbon based
electrodes. J Anal Chem. 2014;69:970–81.
2
013;5:665–71.
2
1. Parveen SKR, Babu PS, Chandrasekar KB, Challa BR. Analytical
method development and validation of tolterodine in pharma-
ceutical dosage forms by RP-HPLC. Der Pharmacia Lett.
40. Macikova P, Skopalova J, Cankar P, Papouskova B, Strakova R,
Jirovsky D, Maier V. Electrochemical oxidation of tolterodine.
Electroanalysis. 2013;25:205–12.
2
014;6:246–54.
41. Khandpur RS. Handbook analytical instrumentation. 2nd ed. New
Delhi: Tata McGraw-Hill, India; 2006.
2
2
2
2
2. Ramathilagam N, Meeradevi M, Solairaj P, Rajesh SC. Devel-
opment and validation of HPLC method for the estimation of
tolterodine tartrate in tablets. Int J Pharm Bio Sci. 2012;2:332–7.
3. Shetty SK, Shah A. Development and validation of tolterodine by
RP-HPLC method in bulk drug and pharmaceutical dosage forms.
Int J Pharm Tech Res. 2011;3:1083–7.
4. Krishna SR, Rao BM, Rao NS. A validated stability indicating
HPLC method for the determination of related substances and
assay of tolterodine tartarate. Rasayan J Chem. 2009;2:144–50.
5. Mhamunkar SM, Vyavaharkar RY, Bhoir S. RP-HPLC method
development and validation for the simultaneous estimation of
tamsulosin HCl and tolterodine tartrate in pharmaceutical dosage
form. Int J Pharm Pharm Sci. 2012;4:319–22.
6. Rao MVB, Reddy BC, Rao TS. J.R. Mohanty JR. Determination
of tamsulosin hydrochloride 0.2% and tolterodine tartrate 0.2%
combination pellets by RP-HPLC method. Res. J. Pharm. Bio.
Chem Sci. 2010;1:136–40.
7. Yanamandra R, Vadla CS, Puppala U, Patro B, Murthy YLN,
Ramaiah PA. A new rapid and sensitive stability indicating
UPLC assay method for tolterodine tartrate, application in phar-
maceuticals, human plasma and urine samples. Sci Pharm.
42. Cazes J. Ewing’s analytical instrumentation handbook. 3rd ed.
Florida: Florida Atlantic University Boca Raton; 2005.
43. Cui F, Qin L, Zhang G, Liu Q, Yao X, Lei B. Interaction of
anthracycline disaccharide with human serum albumin: investi-
gation by fluorescence spectroscopic technique and modeling
studies. J Pharm Biomed Anal. 2008;48:1029–36.
44. Cui F, Kong X, Qin L, Zhang G, Liu Q, Lei B, Yao X. Specific
0
interaction of 4 -O-(aL-Cladinosyl) daunorubicin with human
serum albumin: the binding site II on HAS molecular using spec-
troscopy and modeling. J Photochem Photobiol B. 2009;95:162–9.
45. Khan SN, Islam B, Yennamalli R, Sultan A, Subbarao N, Khan
AU. Interaction of mitoxantrone with human serum albumin:
spectroscopic and molecular modeling studies. Eur J Pharm Sci.
2008;35:371–82.
2
2
46. Lu Y, Cui F, Fan J, Yang Y, Yao X, Li J. Interaction of human
0
serum albumin with N-(4-ethoxyphenyl)-N -(4-antipyrinyl)
thiourea using spectroscopies and molecular modeling method.
J Lumin. 2009;129:734–40.
47. McCallum MM, Pawlak AJ, Shadrick WR, Simeonov A, Jadhav A,
Yasgar A, Maloney DJ, Arnold LA. A fluorescence-based high
throughput assay for the determination of small molecule–human serum
albumin protein binding. Anal Bioanal Chem. 2014;406:1867–75.
48. Li F, Feterl M, Warner JM, Day AI, Keene FR, Collins JG.
Protein binding by dinuclear polypyridyl ruthenium (II) com-
plexes and the effect of cucurbit [10] uril encapsulation. Dalton
Trans. 2013;42:8868–77.
49. Yogam F, VethaPotheher I, Jeyasekaran R, Vimalan M, Antony
Arockiaraj M, Sagayaraj P. Growth, thermal, and optical prop-
erties of L-asparagine monohydrate NLO single crystal. J Therm
Anal Calorim. 2013;114:1153–9.
50. Suresh P, Janarthanan S, Sugaraj Samuel R, JestinLenus A,
Thangaraj K. Studies on the growth, spectral, optical, and thermal
aspects of a NLO crystal L-tryptophan hydrogen selenite (LTHS).
J Therm Anal Calorim. 2014;115:339–43.
51. Kannan V, Rakhikrishna R, Philip J, Brahadeeswaran S. Studies
on thermophysical and mechanical properties of hydrazonium
Ltartrate An organic nonlinear optical material. J Therm Anal
Calorim. 2014;116:339–40.
2
012;80:101–14.
2
2
8. Teja GD, Dasu ChD, Babu PS, Ravisankar P. Quantitative
analysis of solifenacin succinate in pharmaceutical dosage form
using UV absorption spectroscopy.
013;6:195–8.
J Chem Pharm Sci.
2
9. Seetharaman R, Lakshmi KS. Development and validation of first
order derivative spectrophotometric method for estimation of
solifenacin succinate in pharmaceutical formulation. Int J Res
Pharm Biomed Sci. 2011;2:1052–7.
0. Attimarad M. Simultaneous determination of ofloxacin and
flavoxate hydrochloride by absorption ratio and second derivative
UV spectrophotometry. J Basic Clin Pharm. 2011;2:53–61.
1. Attimarad M. Simultaneous determination of ofloxacin and
flavoxate hydrochloride by first and ratio first derivative UV
spectrophotometry. J Iranian Chem Soc. 2012;9:551–7.
2. Shetty SK, Shah A. New spectrophotometric method for esti-
mation of tolterodine in bulk and pharmaceutical formulation. Int
J Pharm Sci Res. 2011;2:1456–8.
3
3
3
3
3. Vanilatha S, Theresa MM, Prasanna N, Kumari DS, Harika B,
Sirisha P, Xavier MA, Kumari KGB. New method development
and validation of tolterodine using visible spectrophotometer. Int
J Sci Inov Disc. 2011;1:288–93.
52. Anitha B, Rathakrishnan S, Malliga P, Pragasam AJA. Crystal
growth, structural, optical, thermal and electrical properties of
organic NLO L-arginine acetamide single crystal. J Therm Anal
Calorim. 2015;119:785–9.
3
3
3
3
4. Fraihat SM, Khatib HS. Indirect spectrophotometric determina-
tion of tolterodine tartrate in pure and pharmaceutical prepara-
tions. Asian J Chem. 2013;25:1887–90.
5. Rizk MS, Abdel-Haleem FM. Plastic membrane electrodes for
the determination of flavoxate hydrochloride and cyclopentolate
hydrochloride. Electrochim Acta. 2010;55:5592–7.
53. Kazemi A, Hayaty M, Mousaviazar A, Samani KA, Keshavarz
MH. The synthesis and characterization of polyvinyl nitrate as an
energetic polymer and study of its thermal behavior. J Therm
Anal Calorim. 2015;119:613–8.
54. Yang T, Chen L, Chen W, Zhou Y, Gao H, Zhong T. Thermal
stability of 2-ethylhexyl nitrate with acid. J Therm Anal Calorim.
2015;119:205–12.
55. Molecular Operating Environment (MOE 2008.10), Chemical
Computing Group Inc., Montreal, QC, Canada.
56. Santos LB, Ribeiro CA, Capela JMV, et al. Kinetic parameters
for thermal decomposition of hydrazine. J Therm Anal Calorim.
2013;113:1209–16.
6. Sakr MM, El Nashar RM. Potentiometric determination of tol-
terodine in batch and flow injection conditions. Talanta.
2
012;96:153–60.
7. Nassar MW, Attia KA, Abou-Seada HM, Allam AE. Spectroflouri-
metric determination of tolterodine tartarate in pure form and phar-
maceutical preparation. Int J Pharm Sci Res. 2013;4:3845–9.
123

A. K. Attia et al.
5
7. Tabacof A, de Araujo Maria, Calado V. Thermogravimetric
analysis and differential scanning calorimetry for investigating
the stability of yellow smoke powders. J Therm Anal Calorim.
60. Hatakeyama T, Liu Z. Hand book of thermal analysis. London:
Wiley; 1998.
61. Anthony CM, Osselton MD, Widdop B. Clark’s analysis of drugs
and poisons. 3rd ed. London: Pharmaceutical Press; 2004.
62. Neil MJO. The merck index, an encyclopedia of chemicals, drugs
and biological. 14th ed. New Jersey: Merck Research Laborato-
ries, Whitehouse Station; 2006.
63. Kondo J, Fran c¸ ois B, Russell RJ, Murray JB, Westhof E. Crystal
structure of the bacterial ribosomal decoding site complexed with
amikacin containing the c-amino-a-hydroxybutyryl (haba) group.
Biochimie. 2006;88:1027–31.
2
017;128:387–98.
5
5
8. Zhang Y, Du Z, Han Z, Yao Q, Hu Z. Synthesis and thermal
analysis of 2-methyl-4,5-dicyano-2H-1,2,3triazole. J Therm Anal
Calorim. 2016;124:529–37.
9. Sangeetha MK, Mariappan M, Madhurambal G, Mojumdar SC.
TG–DTA, XRD, SEM, EDX, UV, and FT-IR spectroscopic
studies of L-valine thiourea mixed crystal. J Therm Anal Calorim.
2
015;119:907–13.
1
23