Microwave Mediated Synthesis and Analytical Method Development for the Estimation of Novel 1,4-Dihydropyridines in Bulk by RP-HPLC

Article abstract of DOI:10.1055/s-0043-120663

The present work describes a rapid and green microwave mediated method for the synthesis and a simple and precise isocratic reverse phase HPLC method for the estimation of the biologically significant dihydropyridines. The conventional synthesis of these

Full text of DOI:10.1055/s-0043-120663

Thieme
Original Paper
Microwave Mediated Synthesis and Analytical Method
Development for the Estimation of Novel 1,4-Dihydropyridines
in Bulk by RP-HPLC
Authors
Anupreet Kaur1, Jaspreet Kaur1, Ranju Bansal2
Supporting Information for this article is available online at
Affiliations
http://www.thieme-connect.de/products
1
University Institute of Engineering & Technology, Panjab
University, Chandigarh, India
ABStRAct
2
University Institute of Pharmaceutical Sciences, Panjab
University, Chandigarh, India
The present work describes a rapid and green microwave medi-
ated method for the synthesis and a simple and precise iso-
cratic reverse phase HPLC method for the estimation of the
biologically significant dihydropyridines. The conventional
synthesis of these dihydropyridines has been previously re-
ported from our lab. The analysis of a standard solution (1mg/
ml) was accomplished on a symmetry (4.6mm I.D x 250 mm)
C-18 column using mobile phase acetonitrile:water:triethylam
ine (TEA) (70:30:0.1 v/v/v) at a flow rate of 0.7ml/min. Detec-
tion was monitored at 354 nm. The retention time for all the
compounds was accomplished as less than 10 min. The com-
pounds showed the linear response over the concentration
range 10–100 µg/ml. The study is aimed to develop a rapid
method for the quantification of these potent molecules. Var-
ious parameters like linearity (10–100 µg/ml), USP tailing and
plate count were found to be satisfactory. The investigated
parameters were studied with the freshly prepared solutions.
Key words
Calcium channel blockers, 1,4-dihydropyridines, microwave
synthesis, vasodilation, RP-HPLC
Bibliography
DOI https://doi.org/10.1055/s-0043-120663
Published online: 2017 | Drug Res
© Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Correspondence
Ranju Bansal
University Institute of Pharmaceutical Sciences
Panjab University
Sector 14
Chandigarh-160014
Tel.: + 91/172/2541 142, Fax: + 91/172/2543 101
ranju29in@yahoo.co.in
Introduction
tion using various catalysts such as copper (II) triflate [11], molecu-
lar iodine [12], organocatalyst [13], trifluoroacetic acid [14]. Further
to overcome inherited drawbacks of longer reaction times and poor
yields, microwave assisted organic synthesis (MAOS) [15, 16] has
been adopted. Syntheses of DHP and pyridines have been demon-
strated using microwave in batch [17] and flow reactor in the pres-
ence of a catalyst to replace the traditional two-step synthesis [18].
Synthesis of some new analogues of 4-aryl-1,4-dihydropyridines
(1,4-DHPs) possessing significant calcium channel blocking activ-
ity along with good vasodilatory profile has been reported [14]
from our lab. With chemical synthesis, achieving high yields in rea-
sonable times is a great challenge, therefore there is a need to de-
velop an efficient method to synthesize these compounds with min-
imum hazards and less time. We have tried to develop an efficient
and greener method for the preparation of novel 1,4-DHPs exhib-
iting excellent vasodilation activity as an environment friendly ap-
proach. As the regulatory authorities have made the complete as-
Antihypertensive drugs are prescribed worldwide to cure the com-
mon cardiovascular diseases and related complications [1]. Calcium
channel blockers (CCBs) have emerged as the most favourable
choice for treating hypertension as they have been proven to con-
trol the various coronary and stroke related risks [2,3]. CCBs of the
type 4-aryl-1,4-dihydropyridine are of interest to medicinal chem-
ists for the treatment of cardiovascular disorders because of their
high potency, selectivity of action and heterogeneity [4, 5]. CCBs
control the calcium influx through voltage-dependant calcium chan-
nels in the smooth muscles and dilate coronary and peripheral ar-
teries and reduce heart after-load [6–8]. Dihydropyridines (DHPs)
have been reported to be the most frequently prescribed CCBs for
the treatment of hypertension in many regions of eastern Asian
countries including China [9,10]. The fascinating biological spectra
of the calcium channel antagonists have led to the development of
newer technologies to improve conventional methods of prepara-
Kaur A et al. Studies on 1,4-dihydropyridines… Drug Res

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Original Paper
OH
CHO
OH
O
O
O
O
a
+
O
O
O
2
N
H
Methyl acetoacetate
4-Hydroxy benzaldehyde
1
b
R
O
O
O
O
N
H
2, 3
Compound
R
(2)
OCH2CH2N
(3)
OCH2CH2N(CH3)2
▶Fig. 1 Synthetic route to the synthesis of the dihydropyridines. Reagents and reaction conditions: a Microwave 75 ºC, 25-30min, Liq. NH3
b Microwave 75 ºC, 50min, EMK, anhyd. K2CO3, respective amine.
sessment even more rigorous for new drug candidates and under-
standing that the detection limits are completely varied for a vast
range of applications, consistent increase in the method develop-
ment for commercial calcium channel blockers such as amlodipine
[19–21] felodipine [22] and nimodipine is obvious [23].
tage). Solvents were dried and purified by standard methods prior
to use. The progress of all reactions was monitored by TLC using
glass plates pre-coated with silica gel 60 F254 with a thickness of
0.5 mm. The Veego melting point apparatus (VMP-D) was used to
measure melting points of the synthesized compounds. The melt-
ing points reported are uncorrected.
Analytical methods based on HPLC [24, 25] have long been the
methods of choice for the quantification of dihydropyridines as the
technique is sensitive and rapid. High therapeutic index of dihydro-
pyridines has promoted the analytical method development for
their assessment in bulk, dosage and in biological fluids. Literature
study reveals that several analytical methods for the quantitative
determination of dihydropyridines such as spectrophotometry
[26, 27], spectrofluorimetry [28], electrochemical methods [29]
and HPLC-MS [30,31] have been reported. The inherent simplicity,
reproducibility and highly selective nature of analysis make the liq-
uid chromatography based estimations as the most suitable for
quality control laboratories. The first phase of the research work is
aimed to develop a greener and rapid synthetic method for 1,4-
DHP via microwave irradiation and the second phase involves de-
velopment of a simple and efficient RP-HPLC method for the quan-
titative estimation of these potent dihydropyridines.
Microwave mediated synthesis
Synthesis of the parent 1,4-dihydropyridine scaffold 1 was carried
out by reacting 4-hydroxybenzaldehyde, methyl acetoacetate and
liquor ammonia under stirring at 75oC in a microwave vial for 25min
(▶Fig. 1). The residue obtained was washed with cold ethanol
(25 %) and crystallised in acetone. Further alkylation of the parent
dihydropyridine with hydrochlorides of 1-(2-chloroethyl)pyrroli-
dine and 2-(diethylamino)ethyl chloride in ethyl methyl ketone
(EMK) using anhydrous potassium carbonate afforded compounds
2 and 3, respectively. Reaction progress was monitored with the
help of the TLC. The comparative data for the formation of dihydro-
pyridines using conventional and microwave methods has been
provided in ▶table 1.
Analytical method development
Chromatographic conditions
Materials and Methods
The chromatographic conditions for analysis of the molecules
throughout the experimental work were maintained as detailed in
▶table 2. All analyses were performed in an air-conditioned lab.
General Information
All reactions were carried out under an inert atmosphere, unless
otherwise stated using microwave synthesizer (Initiator Exp. Bio-
Kaur A et al. Studies on 1,4-dihydropyridines… Drug Res

▶table 1 Physical, chemical and comparative data for the formation of investigated compounds.
R
O
O
O
O
N
H
time/Yield
conventional Method
crystallizing
Solvent
comp.
R
m.p. (oc)
Microwave Method
25 min /51.1 %
1
2
OH
231-233
Acetone
3 h / 87.43 %
8 h /41.48 %
155-156
Ether/ethyl acetate
50 min /40.21 %
OCH2CH2N
—OCH2CH2N(CH3)2
3
169-171
Ether/ethyl acetate
8 h/45.78 %
50 min /41.32 %
▶table 2 Details of the various parameters used for RP-HPLC method.
Standard Solution
System: Waters HPLC Model 2996
Column: Symmetry (4.6 mm I.D x 250 mm) C-18
Detector: Variable wavelength programmable UV/VIS detector PDA
Mobile phase: Acetonitrile + Water + Triethylamine (TEA) (70:30:0.1)
Detection wavelength: 354nm
An accurately weighed pure sample (10mg) was dissolved in meth-
anol (10 ml) to make a stock solution. The solution was sonicated
for 5 min. Dilutions (10–100 µg/ml) were further made using this
stock solution. The solutions were filtered through 0.22µm PTFE
membrane filter (Millipore) and were used for analysis.
Mode: Isocratic
Syringe: Hamilton syringe. (7202, 25 µL)
Flow rate: 0.7 ml/min.
Results and Discussion
Injector: Rheodyne-7725i
Microwave mediated synthesis
Type of Injector: Manual
Temperature: Room temperature
As observed from ▶table 1, the current synthetic method has a
clear advantage of shorter reaction time and higher yields in the
absence of any catalyst and solvent for the synthesis of dihydro-
pyridine analogues.
HPLC method development
To achieve the best chromatographic conditions, the detection
wavelength (200–400nm) and the mobile phase composition were
adequately selected. Pre-conditioning of the RP-phase columns be-
fore runs was always performed by gradually changing the flow
rates and mobile phase composition in the gradient mode. The
main objective was to develop a reverse phase liquid chromato-
graphic method allowing the determination of dihydropyridine an-
alogues in the shortest time. At 354 nm the dihydropyridines
showed enough absorption and the calibration has a good linear-
ity and the best S/N ratio. Because of less polar nature of dihydro-
pyridines they are readily soluble in organic solvents such as etha-
nol, methanol and DMSO and hence strongly retained on RP-HPLC
columns. The column selection has been done based on certain pa-
rameters including back-pressure, peak shape, theoretical plates
and reproducibility of retention times on these columns.
▶Fig. 2 Optimised chromatogram of compound 1 (100µg/ml).
Different types of C-18 columns (4.6×250mm) (BEH, SUN, Sym-
metry and Spherisorb from Waters) with various mobile phase com-
binations were investigated. This included methanol+water; ace-
tonitrile + water; methanol + water + TEA and acetoni-
trile + water + TEA systems in gradient mode. Though all the
columns performed satisfactorily for one or the other compound
but the aim was to develop one column for all the compounds under
investigation. The best choice came as symmetry column, which
could resolve all the compounds in less than 10 min of run time
(▶Figs. 2–ꢀ4). Selected stationary phase has shown better repeat-
ability with low back pressure. Acetonitrile was preferred as it has
shown lesser UV cut-off response compared to methanol. This was
Kaur A et al. Studies on 1,4-dihydropyridines… Drug Res

Thieme
Original Paper
further improved for the individual compounds and the run time
was brought down to less than 6 min. Addition of TEA led to im-
proved baseline and peak quality.
injected into the chromatographic conditions. Parameters studied
to evaluate the suitability of system were retention time, area
under curve, USP tailing and number of theoretical plates, statisti-
cal parameters and LOD/LOQ values (▶table 3).
Validation of system suitability parameters
The straight line graph (with zero intercept value) has been ob-
tained after plotting the peak area values as a function of the con-
centration values (in triplicates). This clearly confirms the linearity
through this measured range as well as lack of bias. The statistical
parameters of the calibration have been described in ▶table 3.
The mobile phase acetonitrile: water: TEA (70:30:0.1) (v/v/v %)
was selected as it gave sharp peaks of the compounds with reten-
tion time of less than 6 min. Wavelength was selected by scanning
standard solutions of drugs over 200 nm to 400 nm. The com-
pounds showed good response at 354 nm. The low value of stand-
ard deviation indicates that system suitability parameters are sta-
ble over the given chromatographic conditions. The value of coef-
ficient of correlation reflects the method is linear over the
concentration range of 10-100 µg/ml. The low relative standard
deviation values indicate a high accuracy of the method.
According to USP, system suitability tests were carried out on
standard stock solution of compounds. 20 µL of the solution was
Conclusion
A rapid, green and efficient microwave irradiated synthesis meth-
odology has been developed for the preparation of novel biologi-
cally active dihydropyridines in this study. Furthermore, for the es-
timations of these dihydropyridines a simple, sensitive and precise
RP-HPLC method has been developed. Since the analysis is com-
pleted within 6min, it clearly indicates that the method is rapid and
thus it could be used for routine studies of the potent dihydropyr-
idine based drug candidates.
▶Fig. 3 Optimised chromatogram of compound 2 (100µg/ml).
Acknowledgement
Authors are thankful to UGC, India and TEQIP –II for financial assis-
tance.
Ethical approval: This article does not contain any studies with
human participants performed by any of the authors.
Conflict of Interest
Authors declare no conflict of interest.
▶Fig. 4 Optimised chromatogram of compound 3 (100µg/ml).
▶table 3 Various chromatographic parameters obtained with symmetry column.
Parameters
(1)
3.28 min
(2)
5.6 min
(3)
4.6 min
Retention Time
Peak Area (mean)
1192170
1109994
1134391
Theoretical Plates(mean)
Tailing Factor(mean)
LOD;LOQ
2083
13277
6647
0.94
1.02
0.77
0.628 µg/ml; 2.09 µg/ml
0.45 µg/ml; 1.53 µg/ml
0.58 µg/ml; 1.19 µg/ml
Statistical parameters of the calibration
1st order linear curve;
r2 = 0.9989
1st order linear curve;
r2 = 0.9984
1st order linear curve;
r2 = 0.9970
Kaur A et al. Studies on 1,4-dihydropyridines… Drug Res

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