Isolation and characterization of novel degradation products in cilnidipine by LC-QTOF-MS/MS, LCMSn, 2D-NMR and FTIR

Article abstract of DOI:10.1039/c7nj02781h

Cilnidipine (CIL) is a novel calcium antagonist accompanied with L-type and N-type calcium channel blocking functions. The drug was subjected to stress studies as per the international conference on harmonization guidelines (ICH) Q1A (R2) to understand the degradation profile of the drug. In the present study, seven degradation products were detected by simple mass compatible high performance liquid chromatography (HPLC) and tentatively characterized using high resolution mass spectrometry. Of these, three oxidative degradation (CD1, CD2 & CD3) products were isolated from the reaction mass using preparative HPLC and their structures were elucidated comprehensively using high resolution and multistage mass fragmentation studies, multidimensional nuclear magnetic resonance and in spectroscopic techniques. Formation of racemic mixtures for each CD1 and CD2 were verified by chiral HPLC. Stereo centers for CD1 and CD2 were fixed using ROESY data as 1S, 2S, 3S, 4R, 5R (and its enantiomer) and 1S, 2S, 3R, 4R, 5R (and its enantiomer). To the best of our knowledge, CD1 and CD2 were novel structures which have not been discussed in any form of publication as yet. The proposed structures of CD1, CD2 and CD3 have been rationalized by appropriate mechanisms.

Full text of DOI:10.1039/c7nj02781h

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Isolation and characterization of novel degradation products in Cilnidipine by
LC-QTOF-MS/MS, LCMSn, 2D-NMR and FTIR
Ch Krishnam Raju1,2, J V Shanmukha Kumar*1, Sanath Kumar Goud2
1Department of chemistry, K L University, Guntur, Andhra Pradeshꢀ522502, India.
2Analytical Research and Development, United States Pharmacopeial Convention – India (P)
Ltd., Plot No. D6 & D8, IKP, Genome Valley, Shameerpet, Hyderabad, Telanganaꢀ500078,
India.
*1Corresponding author: Dr. J V Shanmukha Kumar, Head of the department, Department of
chemistry, K L University, Guntur, Andhra Pradeshꢀ522502, India
Tel: +91 9000586007
Eꢀmail: shanmukh_fed@kluniversity.in
1

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Abstract
Cilnidipine (CIL) is a novel calcium antagonist accompanied with Lꢀtype and Nꢀtype calcium
channel blocking function. The drug was subjected to stress studies as per International
Conference on Harmonization guidelines (ICH) Q1A (R2) to understand degradation profile of
the drug. In the present study, seven degradation products were detected by a simple mass
compatible high performance liquid chromatography (HPLC) and tentatively characterized High
resolution mass spectrometry. Out of these, three oxidative degradation (CD1, CD2 & CD3)
products were isolated from the reaction mass using preparative HPLC and their structures were
elucidated comprehensively using high resolution and multistage mass fragmentation studies,
multidimensional nuclear magnetic resonance and in spectroscopic techniques. Formation of
racemic mixtures for each CD1 and CD2 were verified by chiral HPLC. Stereo centers for CD1
and CD2 were fixed using ROESY data as 1S,2S,3S,4R,5R (and its enantiomer) and
1S,2S,3R,4R,5R (and its enantiomer). To best of our knowledge, these CD1 and CD2 were novel
structures which are not discussed at any form of publication yet. The proposed structures of
CD1, CD2 and CD3 have been rationalized by appropriate mechanisms.
Keywords: Cilnidipine, Novel degradation products, Isolation and characterization, Mass, NMR,
FTIR.
2

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Introduction
Cilnidipine (CIL), 3ꢀOꢀ(2ꢀmethoxyethyl) 5ꢀOꢀ[(E)ꢀ3ꢀphenylpropꢀ2ꢀenyl] 2,6ꢀdimethylꢀ4ꢀ(3
nitroꢀ phenyl)ꢀ1,4ꢀdihydropyridineꢀ3, 5ꢀdicarboxylate, CIL is the novel calcium antagonist
accompanied with Lꢀtype and Nꢀtype calcium channel blocking function. CIL is a
dihydropyridine calciumꢀchannel blocker. It inhibits cellular influx of calcium, thus causing
vasodilatation. It has greater selectivity for vascular smooth muscle1ꢀ2. Stress studies using acid,
base, oxidative, photolytic, thermal and humid stress of the APIs, is a common way of simulating
the impurities, which may appear or increase during transportation or at shelf time beyond
unacceptable level. A thorough literature search revealed that, Ishijima et al3 discussed about
hydrolytic and photolytic stress studies for CIL. Photo degradation of cilnidipine was recently
studied by Hongxia et al4. Various assay procedures are available for detection of CIL5ꢀ7. To the
best of our information no attempts were made for the identification of oxidative stress
degradants. This prompted us to carry out comprehensive degradation study of the drug.
Recently, there has been many work published for characterization of potential drug
impurities in API and formulation using ICH Q3A(R2) based on modern and sophisticated
analytical protocols8ꢀ14. In the present study, a comprehensive stress degradation study was
carried out under hydrolytic (acidic & alkaline), oxidative, thermal, humid and photolytic
conditions to evaluate the intrinsic stability of CIL drug substance. CIL was stable for humid and
thermal conditions; it is susceptible to hydrolytic, photolytic and oxidative conditions.
A total of seven degradation products (DPs) were identified and tentatively characterized by
mass spectrometric studies with accurate mass measurements. The thorough literature search
revealed that hydrolytic and photolytic degradation products, KD1, KD2, KD3 and KD4 were
published by Ishijima et al3. While potential oxidative DPs, CD1, CD2 and CD3 were not
3

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reported by any publishers until recently. Hence, this prompted us to carry out comprehensive
characterization of oxidative DPs by using hyphenated techniques. CD1, CD2 and CD3 were
isolated from the reaction mass using preparative HPLC and then characterized using all modern
sophisticated spectral techniques. A simple mass compatible high performance liquid
chromatography (HPLC) method has been developed to monitor the degradation profile.
Alternately the HPLC method was transferred to UPLC for UPLCꢀQꢀTOFꢀMS analysis to
identify the degradants with accurate mass measurements.
Experimental
Drugs and reagents
CIL sample was obtained from USPꢀIndia, Hyderabad, India. HPLC grade solvents such
as acetonitrile (ACN), methanol (MeOH), nꢀhexane, isopropyl alcohol (IPA), ethanol (EtOH)
and other chemicals such as ammonium acetate (Amm ace), 30% v/v hydrogen peroxide (H2O2),
hydrochloric acid (HCl), sodium hydroxide (NaOH) and deuterated dimethyl sulfoxide (DMSOꢀ
d6) were purchased from Merck (Merck, Mumbai, India). HPLC grade purified water was
obtained using a MilliꢀQ water purification system (Millipore Corporation, Billerica, MA, USA).
Instrumentations
All stability samples were analyzed by using Waters Alliance analytical HPLC (Waters
Corporation, Milford, MA, USA) equipped with photodiode array (PDA) detector using a
Zorbax SB C18, 250 mm × 4.6 mm column packed with 5ꢁm particles. Chromatographic data
was acquired using Empower 3 software.
An Agilent HPLC (Agilent technologies, Santa Clara, CA 95051, United States)
equipped with VWD was used for the separation of enantiomers of each CD1, CD2 and CIL
compounds. Analytical Chiralpak IC (Cellulose trisꢀ(3,5ꢀ dichlorophenylcarbamate) immobilised
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on 5µm silicaꢀgel) 250 × 4.6 mm column packed with 5 ꢁm particles was used with the solvent
flow 1.0 mL/min and the detection was carried out at 240 nm using isocratic run with mobile
phase nꢀhexane: IPA: Ethanol (70: 15: 15, v/v/v). Furthermore, column oven temperature was
ambient, whilst the diluent was IPA.
A Waters preparative HPLC (Waters Corporation) equipped with UVꢀVis detector was
employed for the isolation of CD1 and CD2 using Phenomenex Luna PhenylꢀHexyl 100A, 250
mm × 21.20 mm packed column with 5ꢁm particles. The flow rate was 18 mL/min and the
detection was carried out at 240 nm. The mobile phases consisted part A and B where part A was
water while B was acetonitrile. A timed program of T/%B: 0/40, 17/60, 22/60, 22.1/95, 27/95,
27.1/40, 30/40 was followed for linear gradient. While isolation of CD3 was done using
Phenomenex Luna C18 100A, 250 mm × 21.20 mm, 5ꢁm particles with a timed program of
T/%B: 0/40, 3/40, 12/80, 18/80, 18.1/40, 23/40 was followed for linear gradient.
LCMSn analysis was carried out with Thermo Ion Trap Mass spectrometer (Thermo
scientific, Waltham, MA, USA) coupled to an UHPLC quaternary gradient. LCꢀMS analysis
performed to study degradation samples. LCꢀMSn experiments performed to study multi level
fragmentation patterns of CD1, CD2, CD3 and CIL which creates systematic linkage to draw the
fragment structures. MS analysis was performed by applying Heated Electrospray Ionization
(HESI) source in positive and negative ion modes. HESI source parameters setting involved
source voltage 4.0 kV, capillary temperature 350°C, source heater temperature 350°C, sheath gas
flow 45.0, auxiliary gas flow 20.0, SꢀLens RF level 60.0 %. The data was acquired using
Xcalibur software.
LC–QꢀTOFꢀMS analysis was carried out on SYNAPT G2 QꢀTOF Mass spectrometer
(Waters Corporation) coupled to an ACQUITY HꢀClass UPLC having quaternary gradient pump
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to identify accurate mass measurements with mass accuracy and chemical formula suggestion for
degradants. QꢀTOFꢀMS/MS analysis was carried out to study the fragmentation patterns for
CD1, CD2, CD3 and CIL. MS analysis was performed by applying electrospray ionization (ESI)
source in positive ion mode. ESI source parameters setting involved capillary voltage 3.2 kV,
source temperature 120 °C, desolvation temperature 250°C, sampling cone 40 V, extraction cone
3.0 V, and desolvation gas flow 800 L/hour. The data was acquired using Masslynx 4.1 software.
GCꢀMS analysis for acid degradation sample of CIL (KD2) was carried out on Agilent
GCꢀMS (Agilent technologies) in order to identify the mass of KD2. Chromatography was
carried out using a Restex stabilwax, 30m x 0.25 mm, 0.25µm. The flow rate was 1.0 mL/min,
injection volume was 1.0 µL and split ratio was 10:1. Inlet temperature was 200°C, run time was
39 min and column oven program was 60°C for 5 min, then 10°C/min to 200°C for 20 min. MS
analysis was performed by applying Electron Impact (EI) source. EI source parameters setting
involved M/S Quad: 150°C, MS source: 230°C and turbo speed: 100.The data was acquired
using MSD chemstation software.
NMR spectral studies were carried out on Bruker Ascend 500, Avance III HD
spectrometer (Bruker BioSpin AG, Industriestrasse 26, 8117 Fallanden, Switzerland) with
Topspin software, version 3.2. Spectra were recorded using 500 MHz for 1H NMR and 126 MHz
13
for C NMR. The studies were undertaken at 298 K in DMSOꢀd6 solvent for CD1, CD2, CD3
1
and CIL. The H NMR chemical shift values were referenced to the tetramethylsilane (TMS) (δ
= 0.00 ppm) present in DMSOꢀd6 solvent, whereas 13C NMR to DMSOꢀd6 (δ = 39.50 ppm).
Multiplicities of NMR signals are designated as s (singlet), d (doublet), t (triplet), q (quartet), br
(broad), m (multiplet, for unresolved lines) etc. The proton and carbon chemical shift
assignments were carried out with the help of twoꢀdimensional (2D) correlation spectroscopy
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(COSY)15 correlates chemical shift of two hydrogen nuclei located on two different atoms that
are separated by a single bond, total correlation spectroscopy (TOCSY)16 Provide correlations
between all protons in a given spin system, hetero nuclear single quantum correlation
spectroscopy (HSQC)17 correlates chemical shift of hetero nuclei with hydrogens through bond,
hetero nuclear multiple bond correlation spectroscopy (HMBC)18 correlates chemical shift of
hetero nuclei with hydrogens through a multiple bond, nuclear overhauser effect spectroscopy
(NOESY) and rotating frame overhauser effect spectroscopy (ROESY) correlates between the
nuclei which are spatially close. All the experiments were carried out in the phase sensitive
mode19. The spectra were acquired with 2×256 or 2×192 free induction decays (FID) containing
8–16 transients with relaxation delays of 1.0–1.5 sec. The 2D data were processed with Gaussian
apodization in both the dimensions.
The FTIR spectra were recorded in the range of 4000 – 400 cmꢀ1 using PerkinꢀElmer
Spectrum 100 spectrophotometer (Waltham, MA, USA). 1% sample pellet was prepared using
dried KBr powder.
The hydrolytic stress degradation studies were carried out using a high precision water
bath and magnetic stirrer & hot plate with temperature controller (Deepali United Mfg. Pvt. Ltd.
India). The photolytic stress study was carried out in a Atlas SUNTEST XLS+ photo stability
chamber (Atlas Material Testing, Illinois, USA). The humid stress study was carried out in a
Espec SHꢀ642 Temperature and Humidity Benchtop Chamber (Advanced test equipment corp,
CA, USA). The thermal stress study was carried out in a Memmert hot air oven, modell 100ꢀ800
(Memmert GmbH + Co. KG, Schwabach, Germany).
Stress degradation studies and its sample preparation
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Stress studies were carried out on 500 µg mLꢀ1 solution of CIL as per ICH recommended
conditions of hydrolysis, oxidation, thermal and photolysis. Concentration of stressor was
initiated with milder concentration followed stronger concentration so as to get sufficient
degradation. As the drug was sparingly soluble in water and freely soluble in acetonitrile (ACN),
all the stress samples were prepared in a mixture of ACN and the aqueous stressor (HCl, NaOH,
H2O2) at ratio of 1:1 %v/v to the final concentration of 500 µg mLꢀ1. Hydrolytic stress
degradation study was carried out in 0.1 mol Lꢀ1 NaOH and 1 mol Lꢀ1 HCl at 60°C for 4h and 2h
respectively. Both the acid and base degradation samples were neutralized with NaOH and HCl,
respectively. For oxidative stress degradation, CIL was subjected to 15 % (v/v) H2O2 at room
temperature for 48 h in dark. Thermal degradation study was also carried out in solid state by
exposing pure CIL in a petri plate with a very thin layer to dry heat at 80°C for 5 days. A humid
stress study was carried out with 85% RH humidity and 85°C for 3 days. A photolytic stress
study was carried out in solid state by exposing pure CIL in a petri plate with a very thin layer to
visible light for about 1.2 Million Luxꢀhours and UV light for about 200 Wattꢀhours / meter
square. Samples were prepared by filtering the solution through 0.22 µ filter paper prior to HPLC
and LCMS analysis.
Isolation and preparation of oxidative degradants, CD1, CD2 and CD3
About 1g of CIL drug substance was dissolved in 250 mL of 15 % hydrogen peroxide
solution at room temperature for 48 h in dark, later studied using HPLC. It is found to be having
CD1 with 0.67 RRT, CD2 with 0.70 RRT and CD3 with 1.06 RRT (Figure 1). Suitable
preparative HPLC methods (refer instrumentation section) were developed to separate and
isolate CD1, CD2 and CD3 from residual drug substance and other minor impurities The
fractions containing CD1, CD2 and CD3 were evaporated individually to remove organic solvent
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and then lyophilized the aqueous portion to get the solid material of each CD1, CD2 and CD3
respectively.
Figure 1: HPLC chromatograms Cilnidipine (a) and Oxidative degradation (b)
Results and discussion
Optimization of the chromatographic conditions
The aim of the method to separate all degradants from each other and CIL, the selection of
mobile phase should be mass compatible so that the developed method could be transferred to
mass spectrometry studies. The method was optimized by varying the different selectivity factors
such as pH of mobile phase, combination and ratios of organic solvents (Acetonitrile and
methanol), flow rate, column oven temperature and different columns. During scouting
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experiments, it was found that Hypersil ODS and Zorbax SB C18 columns were found as
suitable columns to separate all degradants from CIL. The peak shapes of oxidative degradants
were not good in acidic pH (0.1% HCOOH in water) then the peak shapes of oxidative
degradants were improved by using 10 mM ammonium acetate (pH about 6.8). Combination of
organic solvents, acetonitrile: methanol, (50:50, v/v) required get better separation of degradants.
Acceptable separation was achieved using a Zorbax SB C18 (250 × 4.6 mm, 5 µm) with mobile
phases having components A and B where A was 10 mM ammonium acetate in water while B
was acetonitrile: methanol, (50:50, v/v) with a flow rate of 1.0 mL/min in a linear gradient
elution mode. Column oven temperature and injection volume was 50°C and10 µL, respectively.
A linear flow gradient program was set as follows: (Tmin / % B): 0/40, 3/40, 10/60, 40/80, 41/40
and 45/40. All the stress samples were analyzed using a PDA detector in a scan mode covering a
range of 200ꢀ400 nm and final chromatogram was extracted at 240 nm to detect the peaks of all
the degradation products. The same method was transferred for LCMSn studies by optimizing all
the MS parameters as discussed in experimental section. Alternatively the HPLC method was
transferred to ultra performance liquid chromatography (UPLC) for UPLCꢀQꢀTOFꢀMS/MS
studies by using Zorbax SB C18 (100 × 2.1 mm, 1.8 µm) with same mobile phases (components
A and B as mentioned above) and column oven temperature with a flow rate of 0.25 mL/min in a
linear gradient elution mode. A linear flow gradient program was set as follows: (Tmin / % B):
0/40, 1/40, 4/60, 15/80, 16/40 and 19/40. Injection volume is 2ul. QꢀTOFꢀMS parameters as
discussed in experimental section.
Degradation behavior of CIL under various stress conditions
The stress degradation behavior of CIL was investigated by using HPLCꢀPDA method.
These studies shown that CIL was stable for humid and thermal conditions; it is susceptible to
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acidic, alkaline, photolytic and oxidative conditions (Table 1). These degradation samples were
analyzed using LCMS (Ion trap). In order to achieve accurate mass measurements, a mixed
sample solution was prepared by mixing the degraded samples and analyzed using UPLCꢀQꢀ
TOFꢀMS (Table 2). Acid degradant peak (KD2) was not ionized in LCMS; hence GCꢀMS
analysis was carried out to determine the m/z value of acid degradant.
Table 1: Stress study results for CIL by HPLC
Degradation
Name KD1 KD2 KD3 CD1 CD2 KD4 CD3 CIL
Conditions
RRT 0.24 0.31 0.59 0.67 0.71 1.03 1.06 1.0
Control
ND ND ND
ND 29.29 ND
ND
ND
ND 0.14 ND 99.80
ND 1.10 0.59 52.69
Stressed with 1 mol Lꢀ1 HCl
at60° C for 2 hrs
Stressed with 0.1 mol Lꢀ1
NaOH at 60° C for 4 hrs
3.61 7.39 11.09 ND
ND 0.12 ND 76.56
%
purity
by
Stressed with 15% H2O2 at
room temperature for 48 hrs
ND ND 0.11 7.31 6.09 0.09 7.61 77.39
Thermal at 80° C for 5 days
ND ND ND
0.02 0.02 ND
ND
ND
ND 0.22 ND 99.70
ND 18.97 0.31 80.01
TDA
Exposed to Visible light for
about 1.2 Million Luxꢀhours
and UV light for about 200
Wattꢀhours / meter square
Humidity 85% RH and 85°
C for 3 days
ND ND ND
ND
ND 0.13 ND 99.80
Note: ND: Not detected, TDA: Total detection area
Table 2: HRMS data of Cilnidipine (CIL) and its degradation products KD1 to KD4 and CD1 to
CD3
Name RRT Suggested Observed Calculated Error DBE
Structure
formula
m/z value m/z value (mDa)
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+
KD1 0.24 C18H21N2O7 377.1367 377.1349
[M+H]+
1.8
9.5
KD2 0.31
C9H10O+.
134 M+.
134
NA* 5.0
+
KD3 0.59 C24H23N2O6 435.1570 435.1556
[M+H]+
1.4
14.5
+
CD1 0.67 C27H31N2O10 543.1973 543.1979 ꢀ0.6 13.5
[M+H]+
Unknown
+
CD2 0.71 C27H31N2O10 543.1973 543.1979 ꢀ0.6 13.5
Unknown
[M+H]+
H
N
+
CIL 1.00 C27H29N2O7 493.1970 493.1975 ꢀ0.5 14.5
O
O
[M+H]+
O
O
O
NO2
+
KD4 1.03 C27H29N2O7 493.1970 493.1975 ꢀ0.5 14.5
[M+H]+
+
CD3 1.06 C27H27N2O7 491.1822 491.1818
[M+H]+
0.4
15.5
Unknown
*As data generated from GCMS with quadrupole analyser system
The drug was degraded under alkaline and acid hydrolytic stress conditions. Overall, the drug
forms three DPs (KD1 to KD3) and one DP (KD2) under alkaline and acidic stress conditions
respectively. Photolytic stress condition resulted with one DP (KD4) and three DPs (CD1, CD2
and CD3) were formed under oxidative stress condition. KD1 to KD4, CD1 to CD3 were studied
by HRMS and further the oxidative degradants (CD1 to CD3) were fully characterized by mass
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fragmentation studies and NMR studies. In addition the novel structures of CD1 and CD2 are
further verified by IR studies and the three dimensional structures of CD1 and CD2 were studied
by chiral HPLC and ROESY NMR data.
Mass spectrometric studies on the drug
HRMS spectrum of CIL showed accurate mass of m/z 493.1970, which had difference of ꢀ0.5
mDa with respect to the exact mass of 493.1975 Da (Table 2). High resolution MS2 studies and
multistage fragmentation studies (upto MS4 level) were further strengthens the structure of CIL
(Table 3). The molecular ion of m/z 493 fragmented into product ions of m/z 475, 417, 375 and
117 indicated the presence of substituents; 2ꢀmethoxy ethyl formate, cinnamyl formate and
nitrobenzene in CIL. The remaining fragment ions were further reinforced the structure of CIL.
Table 3: HRMS and MSn data of Cilnidipine (CIL) and its degradation products CD1, CD2
and CD3
HRMS data
MSn data
Accurate
mass
Accurate masses of
fragments
475.1837, 417.1492, 493
Precursor ion
Product ion(s)
[CIL + H]+ 493.1970
475, 417, 375, 117
301, 210
375.1214, 317.0787, 493→417
301.0831, 117.0698 493→417→301
493→375
255
317
493→375→317
271, 226
[CD1 + H]+ 543.1973
499.2076, 482.2050, 543
465.2050, 349.1441, 543→499
214.1287, 142.0491, 543→499→465
499, 393, 117
482, 465, 405
389, 214
117.0698
543→499→465→214 172, 155
543→499→405
543→393
271, 243
349, 142
543→393→349
525.1940, 481.1988, 543
273, 245
[CD2 + H]+ 543.1973
525, 499, 332, 117
495, 481, 375, 200
302
427.1336, 393.1334,
349.1359, 200.0932,
133.0681, 117.0698
543→525
543→525→495
543→525→495→302 244
543→525→200 142
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543→525→375
271
543→499
543→332
465, 405
133
543→332→133
447.1887, 433.1372, 491
105
[CD3 + H]+ 491.1822
447, 433, 389, 375,
389.1526, 375.1214,
117
317.0787, 271.0738, 491→375
317
117.0698
491→375→317
271, 226
491→375→317→226 208
NMR spectroscopic studies on the drug:
1H NMR chemical shifts (δ in ppm), coupling constants (J in Hz), 13C NMR chemical shifts (δ in
ppm), DEPT information, COSY, HSQC and HMBC connections for CIL were tabulated in
1
13
Table 4. H and C NMR data revealed, CIL contains 28 protons and 27 carbons respectively.
DEPT90, DEPT135 and 13C NMR experiments verified that CIL has 3–CH3 (primary), 3–CH2
(secondary), 12–CH (tertiary) and 9–C (quaternary) carbons. The experimentally obtained
number of carbons precisely matched with the calculated number of carbons for proposed
chemical structure of CIL. The presence of 3ꢀbond HMBC connection between H6→C29 & C30
and H3→C23 & C27 (Figure 2) further confirms the chemical structure. In addition HSQC,
HMBC, COSY and TOCSY spectral correlations are in full agreement with the CIL structure
(Table 4).
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Figure 2: HMBC spectra of CIL (a), CD1 (b), CD2 (c) and CD3 (d) with labeling of
significant correlations.
Table 4: 1H (δ in ppm/J in Hz), COSY, 13C, DEPT, HSQC and HMBC data of CIL
No of
Protons
ꢀ
Position
ppm/J
COSY
13C
DEPT HSQC HMBC
1
2
3
ꢀ
ꢀ
ꢀ
ꢀ
146.59
100.81
39.17
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1H
ꢀ
5.05 / s
CH(1) Cꢀ3 Cꢀ1, 2, 4, 5,
7, 16, 23, 27
4
5
6
ꢀ
ꢀ
1H
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
101.04 ꢀ
146.88 ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
9.09 / s
ꢀ
ꢀ
Cꢀ2, 4, 29,
30
ꢀ
7
ꢀ
ꢀ
ꢀ
166.16 ꢀ
ꢀ
9a, 9b 2H
4.63 /ddd, 13.4, 6.0, 1.3 Hꢀ10
4.69 /ddd, 13.4, 6.0, 1.3
63.67 CH2(1) Cꢀ9 Cꢀ7, 10, 11
10
11
1H
1H
6.29 /dt, 16.0, 6.0
Hꢀ9a, 9b, 11 124.10 CH(1) Cꢀ10 Cꢀ9, 12
6.51 /dt, 16.0, 1.3
Hꢀ10
132.43 CH(1) Cꢀ11 Cꢀ9, 12, 13
15

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12
ꢀ
ꢀ
ꢀ
135.96 ꢀ
ꢀ
ꢀ
13,13’ 2H
14,14’ 2H
7.37/ m
7.33/ m
Hꢀ14, 14’
126.28 CH(2) Cꢀ13 Cꢀ11, 15
128.57 CH(2) Cꢀ14 Cꢀ12
Hꢀ15, 13,
13’
15
16
1H
ꢀ
7.26/ m
ꢀ
Hꢀ14, 14’
ꢀ
127.84 CH(1) Cꢀ15 Cꢀ13
166.38 ꢀ
ꢀ
ꢀ
18a,
18b
19a,
19b
21
22
23
2H
4.03 /ddd,12.1, 6.0, 3.2 Hꢀ19a, 19b 62.41 CH2(1) Cꢀ18 Cꢀ19, 16
4.10 /ddd, 12.1, 6.0, 3.2
3.43 /ddd,11.2, 6.0, 3.2 Hꢀ18a, 18b 69.86 CH2(1) Cꢀ19 Cꢀ21
3.48 /ddd, 11.2, 6.0, 3.2
2H
3H
ꢀ
1H
ꢀ
3.18/ m
ꢀ
8.01/ m
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
57.90 CH3(1) Cꢀ21 Cꢀ19
150.09 ꢀ
121.88 CH(1) Cꢀ23 Cꢀ25, 27, 24
147.47 ꢀ
ꢀ
ꢀ
24
ꢀ
25
26
27
1H
1H
1H
7.99/ m
7.53/ t, 7.7
7.66/ dt,7.7, 1.4
2.34/ s
2.30/ s
Hꢀ26
Hꢀ25, 27
Hꢀ26
ꢀ
121.10 CH(1) Cꢀ25 Cꢀ23, 27, 24
129.57 CH(1) Cꢀ26 Cꢀ22, 24
134.27 CH(1) Cꢀ27 Cꢀ23, 25
18.17, CH3(2) Cꢀ29 Cꢀ1, 2, 4, 5,
29, 30 6H
18.27
Cꢀ30 7, 16
CIL
Structure
IR studies on the drug
IR spectrum of the drug showed bands for N–H stretching at 3463 cmꢀ1, C–H stretching at 2932
cmꢀ1, C=O stretching at 1698 cmꢀ1, while scissoring, twisting, wagging and rocking vibrations
were observed below 1500 cmꢀ1.
Characterization of the degradation products KD1 to KD4 and CD1 to CD3:
KD1:
HRMS data indicated that the protonated ions (M+H)+ of KD1 and CIL at m/z 377.1367 Da and
493.1970 Da (Table 2). The significant loss of 116.0621 Da in KD1 against CIL is due to the
ester hydrolysis at cinnamyl side chain in CIL. Molecular formula of protonated KD1 worked
16

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+
out as C18H21N2O7 , with mass error, 1.8 mDa (exact mass 377.1349 Da). Double bond
equivalence (DBE) 9.5 and even number (2) nitrogens were considered to determine the
structure. It was characterized as 5ꢀ((2ꢀmethoxyethoxy)carbonyl)ꢀ2,6ꢀdimethylꢀ4ꢀ(3ꢀ
nitrophenyl)ꢀ1,4ꢀdihydropyridineꢀ3ꢀcarboxylic acid. This product previously reported as
hydrolysis product3 of CIL.
KD2:
GCMS data indicated that the molecular ion (M+.) of KD2 at m/z 134 Da (Table 2). The
significant loss of 358 Da in KD2 against CIL (492 Da) is due to the ester hydrolysis at cinnamyl
side chain in CIL. Its molecular formula worked out as C9H10O+.. It was characterized as (E)ꢀ3ꢀ
phenylpropꢀ2ꢀenꢀ1ꢀol. This product previously reported as hydrolysis product of CIL3.
KD3:
HRMS data indicated that the protonated ions (M+H)+ of KD3 and CIL at m/z 435.1570 Da and
493.1970 Da (Table 2). The significant loss of 58.0418 Da in KD3 against CIL is due to the ester
hydrolysis at 2ꢀmethoxy ethyl formate side chain in CIL. Molecular formula of protonated KD3
+
worked out as C24H23N2O6 , with mass error, 1.4 mDa (exact mass 435.1556 Da). Double bond
equivalence (DBE) 14.5 and even number (2) nitrogens were considered to determine the
structure. It was characterized as (E)ꢀ5ꢀ((cinnamyloxy)carbonyl)ꢀ2,6ꢀdimethylꢀ4ꢀ(3ꢀnitrophenyl)ꢀ
1,4ꢀdihydropyridineꢀ3ꢀcarboxylic acid. This product previously reported as hydrolysis product of
CIL3.
KD4:
HRMS data indicated that the protonated ions (M+H)+ of KD4 and CIL at m/z 493.1970 Da and
493.1970 Da (Table 2). The same m/z value indicated it is isomer of CIL. Molecular formula of
+
protonated KD4 worked out as C27H29N2O7 , with mass error, ꢀ0.5 mDa (exact mass 493.1975
17

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Da). Double bond equivalence (DBE) 14.5 and even number (2) nitrogens were considered to
determine the structure. It was characterized as (Z)ꢀ3ꢀ(2ꢀmethoxyethyl) 5ꢀ(3ꢀphenylallyl) 2,6ꢀ
dimethylꢀ4ꢀ(3ꢀnitrophenyl)ꢀ1,4ꢀdihydropyridineꢀ3,5ꢀdicarboxylate. This product previously
reported as Zꢀisomer of CIL3ꢀ4.
CD1:
HRMS data indicated that the protonated ions (M+H)+ of CD1 and CIL at m/z 543.1973 Da and
493.1970 Da (Table 2). The significant addition of 50.0003 Da in CD1 against CIL is due to
addition of H2O3 [1H2O + 2 (O)] in CIL. Molecular formula of protonated CD1 worked out as
+
C27H31N2O10 , with mass error, ꢀ0.6 mDa (exact mass 543.1979 Da). Double bond equivalence
(DBE) 13.5 and even number (2) nitrogens were considered to determine the structure.
NMR studies conducted to determine the structure; 1H NMR chemical shifts (δ in ppm), coupling
constants (J in Hz), 13C NMR chemical shifts (δ in ppm), DEPT information, COSY, HSQC and
1
HMBC connections for CD1 were tabulated in Table 5. H and 13C NMR revealed that CD1
contained 30 protons and 27 carbons respectively. DEPT90, DEPT135 and 13C NMR
experiments verified that CD1 has 3–CH3 (primary), 3–CH2 (secondary), 13–CH (tertiary) and
8–C (quaternary) carbons. The experimentally obtained number of carbons precisely matched
13
with the calculated number of carbons for proposed chemical structure of CD1. The C NMR
values of both CIL and CD1 differ significantly at C1, C2, C4 and C5 atoms. In addition; C4 (q)
has been changed into CH and one OH group has been attached at C2 carbon in CD1 (Table 5).
These data indeed indicates that the dihydropyridine ring is involved during the formation of
degradation product while the substituents viz., 2ꢀmethoxy ethyl formate, cinnamyl formate and
nitrobenzene were left unchanged. The presence of 3ꢀbond HMBC connection between
H4→C25, H9→C3, H4→C19 (Figure 2, Table 5) and 2ꢀbond COSY connection between
18

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H4→H3 of CD1, further confirms the proposed chemical structure. The structure of CD1 was
further verified with HSQC, COSY and TOCSY spectra where all the correlations are in full
agreement with proposed structure. Therefore, above NMR data substantially confirm the
proposed chemical structure for CD1 (Table 5). It was characterized as 2ꢀcinnamyl 4ꢀ(2ꢀ
methoxyethyl) 2ꢀhydroxyꢀ1,5ꢀdimethylꢀ3ꢀ(3ꢀnitrophenyl)ꢀ6,7ꢀdioxaꢀ8ꢀazabicyclo[3.2.1]octaneꢀ
2,4ꢀdicarboxylate.
Table 5: 1H (δ in ppm/Jin Hz), COSY, 13C, DEPT, HSQC and HMBC data of CD1
No of
Protons
ꢀ
Position
ppm/J
COSY
13C
DEPT HSQC HMBC
1
2
3
ꢀ
ꢀ
ꢀ
ꢀ
99.64
80.20
47.32 CH(1) Cꢀ3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1H
ꢀ
ꢀ
4.04/ d 12.3
Cꢀ1, 2, 4, 5,
10, 19, 25,
26, 30
Cꢀ2, 5, 19,
25, 33
ꢀ
4
5
8
1H
ꢀ
3.88/ d 12.3
ꢀ
ꢀ
ꢀ
54.05 CH(1) Cꢀ4
ꢀ
96.26
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1H
5.52/ s
ꢀ
Cꢀ1, 2, 4, 5,
32, 33
Cꢀ1, 2, 3, 10
9
1H
ꢀ
5.94/ s
ꢀ
Hꢀ10
ꢀ
ꢀ
ꢀ
ꢀ
10
169.34 ꢀ
ꢀ
12a,
12b
13
14
15
2H
4.57/ ddd 13.4, 6.0, 1.4 Hꢀ13
4.66/ ddd 13.4, 6.0, 1.4
64.94 CH2(1) Cꢀ12 Cꢀ11, 15
1H
1H
ꢀ
6.17 /dt, 16.0, 6.0
Hꢀ14, 12a, 12b 122.91 CH(1) Cꢀ13 Cꢀ12
6.60 /dt, 16.0, 1.4
ꢀ
Hꢀ13
ꢀ
133.01 CH(1) Cꢀ14 Cꢀ13
135.79 ꢀ
ꢀ
ꢀ
16, 16’ 2H
17, 17’ 2H
7.35/ m
Hꢀ17, 17’
126.32 CH(2) Cꢀ16 Cꢀ14, 18
7.34/ m
7.28/ m
ꢀ
Hꢀ18, 16 , 16’ 128.62 CH(2) Cꢀ17 Cꢀ15
18
1H
Hꢀ17, 17’
127.99 CH(1) Cꢀ18 Cꢀ16
19
ꢀ
ꢀ
169.60 ꢀ
ꢀ
ꢀ
21a,
21b
22a,
22b
2H
3.96 /ddd,12.1, 5.9, 3.2 Hꢀ22a, 22b
4.03 /ddd, 12.1, 5.9,3.2
3.24 /ddd,11.2, 5.9, 3.2 Hꢀ21a, 21b
3.28 /ddd, 11.2, 5.9,3.2
19
63.11 CH2(1) Cꢀ21 Cꢀ19, 22
2H
69.52 CH2(1) Cꢀ22 Cꢀ24

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24
25
26
27
28
29
30
32
33
3H
ꢀ
1H
ꢀ
1H
1H
1H
3H
3H
3.06/ s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
57.71 CH3(1) Cꢀ24 Cꢀ22
138.81 C(1)
124.20 CH(1) Cꢀ26 Cꢀ27, 28, 30
147.02 ꢀ
122.91 CH(1) Cꢀ28 Cꢀ26, 27, 30
128.87 CH(1) Cꢀ29 Cꢀ25, 27
136.63 CH(1) Cꢀ30 Cꢀ26, 28
15.56 CH3(1) Cꢀ32 Cꢀ1, 2
18.49 CH3(1) Cꢀ33 Cꢀ4, 5
ꢀ
ꢀ
8.20/ t, 2.1
ꢀ
8.02/ ddd 8.2,2.3,1.0
7.52/ t, 8.0
7.77/ m
ꢀ
Hꢀ29
Hꢀ28, 30
Hꢀ29
ꢀ
ꢀ
1.41/ s
1.51/ s
CD1
Structure
High resolution MS2 studies and multistage fragmentation studies (upto MS5 level) were
further strengthens the proposed structure of CD1 (Table 3). The molecular ion of m/z 543
fragmented into product ions of m/z 499, 393 and 117 revealed that the substituents; 2ꢀmethoxy
ethyl formate, cinnamyl formate and nitrobenzene were left unchanged in CD1 against CIL. The
precursor ion of m/z 499 further fragmented into 482, 465 and 405 provided evidences for the
formation of hydroxy and peroxy bond at dihydropyridine ring in CD1 against CIL. The
remaining fragment ions were further reinforced the structure of CD1.
An IR spectrum of CD1 was compared with CIL to predict major differences in functional
groups. IR spectrum of CIL showed a peak at 1698 cmꢀ1 corresponding to conjugated ester
group which is absent in CD1, in addition CD1 showed a peak at 1733 cmꢀ1 which correspond to
aliphatic non conjugated ester group, Moreover CD1 showed a broad peak at 3500 cmꢀ1 region
indicated the presence of OH group, these evidences indicated the involvement of
20

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dihydropyridine group in reaction, resulting into CD1. This further verifies the confirmed
chemical structures of these degradants by Mass and NMR.
CD2:
HRMS data indicated that the protonated ions (M+H)+ of CD2 and CIL at m/z 543.1973 Da and
493.1970 Da (Table 2). The significant addition of 50.0003 Da in CD2 against CIL is due to
addition of H2O3 [1H2O + 2 (O)] in CIL. Molecular formula of protonated CD2 worked out as
+
C27H31N2O10 , with mass error, ꢀ0.6 mDa (exact mass 543.1979 Da). This indicates the CD2 is
an isomer of CD1. Double bond equivalence (DBE) 13.5 and even number (2) nitrogens were
considered to determine the structure.
1H NMR chemical shifts (δ in ppm), coupling constants (J in Hz), 13C NMR chemical shifts (δ in
ppm), DEPT information, COSY, HSQC and HMBC connections for CD2 were tabulated in
1
Table 6. H and 13C NMR data revealed that CD2 contained 30 protons and 27 carbons
respectively. DEPT90, DEPT135 and 13C NMR experiments verified that CD2 has 3–CH3
(primary), 3–CH2 (secondary), 13–CH (tertiary) and 8–C (quaternary) carbons. The
experimentally obtained number of carbons precisely matched with the calculated number of
carbons for proposed chemical structure of CD2. The 13C NMR values of both CIL and CD2
differ significantly at C1, C2, C4 and C5 atoms. In addition; C2 (q) has been changed into CH
and one OH group has been attached at C4 carbon in CD2 (Table 6). These data indeed indicates
that the dihydropyridine ring is involved during the formation of degradation product while the
substituents viz., 2ꢀmethoxy ethyl formate, cinnamyl formate and nitrobenzene were left
unchanged (Table 6). The presence of 3ꢀbond HMBC connection between H2→C25, H9→C3,
H2→C10 as shown in Figure 2 and 2ꢀbond COSY connection between H2→H3 of CD2, further
21

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confirms the proposed chemical structure. The structure of CD2 was further verified with HSQC,
COSY and TOCSY spectra where all the correlations are in full agreement with proposed
structure. Therefore, above NMR data substantially confirm the proposed chemical structure for
CD2 as shown in Figure 2. It was characterized as 4ꢀcinnamyl 2ꢀ(2ꢀmethoxyethyl) 2ꢀhydroxyꢀ
1,5ꢀdimethylꢀ3ꢀ(3ꢀnitrophenyl)ꢀ6,7ꢀdioxaꢀ8ꢀazabicyclo[3.2.1]octaneꢀ2,4ꢀdicarboxylate.
Table 6: 1H (δ in ppm/Jin Hz), COSY, 13C, DEPT, HSQC and HMBC data of CD2
No of
Position Protons ppm/J
COSY
ꢀ
13C
96.30 ꢀ
DEPT HSQC HMBC
1
2
3
ꢀ
ꢀ
ꢀ
ꢀ
1H
1H
3.93/ d 12.3
4.05/ d 12.3
ꢀ
ꢀ
54.06 CH(1) Cꢀ4
47.35 CH(1) Cꢀ3
Cꢀ1, 4, 10, 25
Cꢀ1, 2, 4, 5,
10, 19, 25, 26,
30
ꢀ
ꢀ
4
5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
80.17 ꢀ
99.17 ꢀ
ꢀ
ꢀ
8
1H
5.45/ s
ꢀ
ꢀ
ꢀ
ꢀ
Cꢀ1, 2, 4, 5,
32, 33
9
1H
ꢀ
5.87/ s
ꢀ
Hꢀ10
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Cꢀ3, 4, 5, 19
10
169.50 ꢀ
ꢀ
12
13
14
15
2H
1H
1H
ꢀ
4.66/ dd, 6.2, 1.2
6.01 /dt, 16.0, 6.2
6.47 /dt, 16.0, 1.2
ꢀ
Hꢀ13
Hꢀ14, 12
Hꢀ13
ꢀ
64.46 CH2(1) Cꢀ12 Cꢀ11, 15
122.93 CH(1) Cꢀ13 Cꢀ12
133.20 CH(1) Cꢀ14 Cꢀ13
135.68 ꢀ
ꢀ
ꢀ
16,
16’
17,
17’
18
2H
2H
1H
7.27/ m
7.31/ m
7.27/ m
Hꢀ17, 17’
126.23 CH(2) Cꢀ16 Cꢀ14, 18
Hꢀ18, 16 ,16’ 128.55 CH(2) Cꢀ17 Cꢀ15
Hꢀ17, 17’
127.97 CH(1) Cꢀ18 Cꢀ16
169.50 ꢀ
19
21
22
24
25
26
27
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2H
2H
3H
ꢀ
1H
ꢀ
4.01/ m
3.43/ m
3.21/ s
ꢀ
8.22/ t, 2.0
ꢀ
Hꢀ22
Hꢀ21
ꢀ
ꢀ
ꢀ
ꢀ
63.57 CH2(1) Cꢀ21 Cꢀ19, 22
69.34 CH2(1) Cꢀ22 Cꢀ24
57.91 CH3(1) Cꢀ24 Cꢀ22
138.89 C(1)
124.44 CH(1) Cꢀ26 Cꢀ27, 28, 30
147.01 ꢀ
ꢀ
ꢀ
ꢀ
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28
29
30
32
33
1H
1H
1H
3H
3H
7.99/ ddd 8.2,2.3,0.8 Hꢀ29
121.96 CH(1) Cꢀ28 Cꢀ26, 27, 30
7.51/ t, 8.0
7.81/ m
1.50/ s
Hꢀ28, 30
Hꢀ29
ꢀ
ꢀ
128.84 CH(1) Cꢀ29 Cꢀ25, 27
136.58 CH(1) Cꢀ30 Cꢀ26, 28
18.59 CH3(1) Cꢀ32 Cꢀ1, 2
15.38 CH3(1) Cꢀ33 Cꢀ4, 5
1.40/ s
CD2
Structure
High resolution MS2 studies and multistage fragmentation studies data (upto MS5 level)
were further strengthens the proposed structure of CD2 (Table 3). The molecular ion of m/z 543
fragmented into product ions of m/z 525, 499, 332 and 117 revealed that the substituents; 2ꢀ
methoxy ethyl formate, cinnamyl formate and nitrobenzene were left unchanged in CD2 against
CIL. The precursor ion of m/z 525 further fragmented into 495, 481, 375 and 200 provided
evidences for the formation of hydroxy and peroxy bond at dihydropyridine ring in CD1 against
CIL. Rest of the fragment ions were further supported the structure of CD2.
An IR spectrum of CD2 was compared with CIL to predict major differences in
functional groups. IR spectrum of CIL showed a peak at 1698 cmꢀ1 corresponding to conjugated
ester group which is absent in CD2, in addition CD2 showed a peak at 1730 cmꢀ1 which
correspond to aliphatic non conjugated ester group, Moreover CD2 showed a broad peak at 3500
cmꢀ1 region indicated the presence of OH group, these evidences indicated the involvement of
dihydropyridine group in reaction, resulting into CD2. This further verifies the confirmed
chemical structures of these degradants by Mass and NMR.
CD3:
23

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HRMS data indicated that the protonated ions (M+H)+ of CD3 and CIL at m/z 491.1822 Da and
493.1970 Da (Table 2). The significant loss of 2.0148 Da in CD3 against CIL is due to loss of
+
two hydrogens [2(H)] in CIL. Molecular formula of protonated CD3 worked out as C27H27N2O7 ,
with mass error, 0.4 mDa (exact mass 491.1818 Da). Double bond equivalence (DBE) 15.5 and
even number (2) nitrogens were considered to determine the structure.
1H NMR chemical shifts (δ in ppm), coupling constants (J in Hz), 13C NMR chemical shifts (δ in
ppm), DEPT information, COSY, HSQC and HMBC connections for CD3 were tabulated in
1
Table 7. H and 13C NMR data revealed that CD3 contained 26 protons and 27 carbons
respectively. DEPT90, DEPT135 and 13C NMR experiments verified that CD3 has 3–CH3
13
(primary), 3–CH2 (secondary), 11–CH (tertiary) and 10–C (quaternary) carbons. The C NMR
values of both CIL and CD3 differ significantly at C3 atom. In addition the loss two hydrogens
indicate that the dihydropyridine ring is involved during the formation of CD3 while the
substituents viz., 2ꢀmethoxy ethyl formate, cinnamyl formate and nitrobenzene were left
unchanged (Table 7). The experimentally obtained number of carbons precisely matched with
the calculated number of carbons for proposed chemical structure of CD3. The presence of 3ꢀ
bond HMBC connection between H23→C3 and H27→C3 further confirms the proposed
chemical structure. The structure of CD3 was confirmed with HSQC, HMBC, COSY and
1
TOCSY spectra where all the correlations are in full agreement with structure. H NMR
13
chemical shifts (δ in ppm), coupling constants (J in Hz), C NMR chemical shifts (δ in ppm),
COSY, HSQC and HMBC connections for CIL were tabulated in Table 7. It was characterized
as 3ꢀcinnamyl 5ꢀ(2ꢀmethoxyethyl) 2,6ꢀdimethylꢀ4ꢀ(3ꢀnitrophenyl)pyridineꢀ3,5ꢀdicarboxylate.
The resulted structure of CD3 is previously reported as a photolytic degradation product of the
drug4 and metabolite of the drug20.
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Table 7: 1H (δ in ppm/Jin Hz), COSY, 13C, DEPT, HSQC and HMBC data of CD3
No of
Position Protons ppm/J
COSY
ꢀ
13C
DEPT HSQC HMBC
1,5
2,4
3
7
9
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
125.78, 125.94 ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2H
ꢀ
ꢀ
ꢀ
155.63, 155.62 ꢀ
143.29
166.24
65.79
ꢀ
ꢀ
4.64 /dd, 6.6, 1.0 Hꢀ10
CH2(1)Cꢀ9
Cꢀ7, 10, 11
10
11
12
1H
1H
ꢀ
5.90 /dt,15.8, 6.6 Hꢀ9, 11
121.74
134.39
135.51
126.35
CH(1) Cꢀ10 Cꢀ9, 12
6.47 /d,15.8
Hꢀ10
CH(1) Cꢀ11 Cꢀ9, 12, 13
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
13, 13’ 2H
14, 14’ 2H
15
16
7.31/ m
7.34/ m
7.30/ m
ꢀ
Hꢀ14, 14’
Hꢀ15, 13 , 13’ 128.58
Hꢀ14, 14’
ꢀ
CH(2) Cꢀ13 Cꢀ11, 15
CH(2) Cꢀ14 Cꢀ12
CH(1) Cꢀ15 Cꢀ13
1H
ꢀ
128.15
166.37
ꢀ
ꢀ
ꢀ
18
19
2H
2H
4.07 /m
Hꢀ19
64.18
69.13
CH2(1)Cꢀ18 Cꢀ19, 16
CH2(1)Cꢀ19 Cꢀ21
3.25/dd,5.8, 3.6 Hꢀ18
21
3H
3.07/ s
ꢀ
57.74
CH3(1)Cꢀ21 Cꢀ19
22
23
24
25
26
27
ꢀ
1H
ꢀ
1H
1H
1H
ꢀ
ꢀ
ꢀ
ꢀ
136.89
122.44
147.23
123.52
130.06
134.39
22.66, 22.61
ꢀ
ꢀ
ꢀ
7.99/ m
ꢀ
8.12/ dt,7.6, 1.9 Hꢀ26
7.67/ t, 7.7
7.65/ m
2.58/ s
CH(1) Cꢀ23 Cꢀ25, 27, 24
ꢀ
ꢀ
CH(1) Cꢀ25 Cꢀ23, 27, 24
CH(1) Cꢀ26 Cꢀ22, 24
CH(1) Cꢀ27 Cꢀ23, 25
CH3(2)Cꢀ29 Cꢀ1, 2, 4, 5, 7,
Cꢀ30 16
Hꢀ25, 27
Hꢀ26
ꢀ
29, 30 6H
2.56/ s
CD3
Structure
High resolution MS2 studies and multistage fragmentation studies data (upto MS5 level)
were further strengthens the proposed structure of CD3 (Table 3). The molecular ion of m/z 491
fragmented into product ions of m/z 447, 433, 389, 375 and 117 revealed that the substituents; 2ꢀ
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New Journal of Chemistry
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DOI: 10.1039/C7NJ02781H
methoxy ethyl formate, cinnamyl formate and nitrobenzene were left unchanged in CD3 against
CIL. The remaining fragment ions were further reinforced the structure of CD3.
Three dimensional structure evaluation for CD1 and CD2
Enantiomeric purity:
CIL drug substance is a racemic compound with one stereo center whilst the mass, NMR
and IR proposed chemical structure of CD1 and CD2 shows five stereo centers. Due to this, each
CD1 and CD2 can exists with sixteen pairs of racemic compounds. However due to the presence
of rigid stereo centers at C1 and C5, only eight pairs of racemic compounds were possible.
Hence it was considered to explore the practicability for the formation of racemic mixture using
chiral HPLC. A new chiral HPLC method was developed to separate both the enantiomers using
Chiralpak IC column. Chiral HPLC study revealed that, each CD1 and CD2 exists with one pair
of racemic compound. The presence of both enantiomers in the ratio of 51.42% & 48.58% for
CD1 and 50.29% & 49.71% for CD2 (Figure 3). This data confirm that, though each CD1 and
CD2 contains five stereo centers, they practically exist with one pair of enantiomers (Figure 4).
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Page 27 of 35
New Journal of Chemistry
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DOI: 10.1039/C7NJ02781H
Figure 3: Chiral HPLC chromatograms of CD1 (a), CD2 (b) and ROESY NMR spectra of CD1
(c) and CD2 (d) with labeling of significant correlations.
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New Journal of Chemistry
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DOI: 10.1039/C7NJ02781H
Figure 4: Enantiomeric structures of CD1 (a), CD2 (b) and 3D structures of CD1 & CD2 (c).
ROESY NMR studies for CD1
ROESY experiment was recorded in NMR. Three dimensional structure of CD1 has
1
established based on the coupling constant and ROE information from H and ROESY NMR
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New Journal of Chemistry
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DOI: 10.1039/C7NJ02781H
experiment (Figure 3). The presence of strong ROE between H32/H9 and absence of ROE
between H33/H9 reinforces the OH position. The presence of strong ROE between H3/H9 and
weak ROE between H8/H4 confirms that they are in same plane, however the weak ROE
between H8/H4 is due to the hydrogen atom of NH in piperidine ring is oriented in the equatorial
position. The absence of ROE between H3/H8 and H8/H9 (Figure 3) evidenced that they are in
different plane. These facts confirmed the chemical structure for CD1 as shown in Figure 4.
ROESY NMR studies for CD2
ROESY experiment was recorded in NMR. Three dimensional structure of CD2 has
1
established based on the coupling constant and ROE information from H and ROESY NMR
experiment (Figure 3). The presence of strong ROE between H33/H9 and absence of ROE
between H32/H9 reinforces the OH position. The presence of strong ROE between H3/H9 and
weak ROE between H8/H2confirms that they are in same plane, however the weak ROE
between H8/H2is due to the hydrogen atom of NH in piperidine ring is oriented in the equatorial
position. The absence of ROE between H3/H8 and H8/H9 (Figure 3) evidenced that they are in
different plane. These facts confirm the chemical structure for CD2 as shown in Figure 4.
Proposed reaction mechanism for CD1, CD2 and CD3
The proposed mechanism for CD1 involves the following steps as shown in Figure 5. The carbon
atoms C1 and C4 are electrophilic and undergo attack by nucleophile i.e. hydroperoxide anion
(HOO−) [21ꢀ23]. In the first step, epoxide is formed from first peroxide molecule at C1=C2
double bond. In the second step, nucleophilic attack of hydroperoxide anion at C5 carbon atom
followed by the proton transfer produces the intermediateꢀ2. In the stepꢀ3, intramolecular
29