Structural studies of calcium channel blockers used in the treatment of hypertension - 1H and 13C NMR characteristics of nifedipine analogues

Full text of DOI:10.1002/mrc.4797

Structural studies of calcium channel blockers used in the treatment of
1
13
hypertension - H and C NMR characteristics of nifedipine analogues.
a
b*
a
Łukasz Szeleszczuk , Monika Zielińska-Pisklak , Dariusz Maciej Pisklak
a
Faculty of Pharmacy with the Laboratory Medicine Division, Medical University of Warsaw,
Department of Physical Chemistry, Chair and Department of Physical Pharmacy and
Bioanalysis, Banacha 1, 02-093 Warsaw Poland
b
Faculty of Pharmacy with the Laboratory Medicine Division, Medical University of
Warsaw, Department of Biomaterials Chemistry, Chair and Department of Analytical
Chemistry and Biomaterials, Banacha 1, 02-093 Warsaw Poland
*
Corresponding author. Tel./fax: + 48 22 5720784
E-mail address: mpisklak@wum.edu.pl
Keywords: solution NMR spectroscopy, chemical shifts assignments, nifedipine analogues,
dihydropyridines, calcium channel blockers, stability of drug substances
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doi: 10.1002/mrc.4797
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Abstract
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13
The H and C NMR spectroscopy in solution was applied to characterize a series of
active pharmaceutical ingredients (API) belonging to the class of calcium channel blockers, a
group of drug substances extremely sensitive to UV radiation: nifedipine (1), nitrendipine (2),
nisoldipine (3), nimodipine (4), nilvadipine (5), felodipine (6) isradipine (7) nicardipine (8)
and amlodipine besylate (9). Two-dimensional NMR experiments (COSY, HSQC, HMBC)
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13
allowed the unequivocal assignments of peaks in the H and C spectra in solution and in
some cases enabled editing of incorrect assignments appearing in the scientific literature.
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3
Various nifedipine derivatives can be easily distinguished based on their C chemical shifts.
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H and C chemical shifts analysis provides fast identification of the drug substance and
ensures its stability in the commercial dosage forms from different sources.
1
. Introduction
Dihydropyridine derivatives (DHPds) (Fig. 1.) belong to a large group of
cardiovascular drugs used clinically in the treatment of hypertension, cardiac arrhythmias or
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2
anginas and others [ , ]. Their effectiveness in management of cardiovascular disorders are
due to their ability to relax vascular smooth and cardiac muscles via inhibiting the influx of
3
calcium ion through L-type calcium channels [ ]. Although DHPds have similar chemical
structure and features, there are some differences in their pharmacological properties, such as:
affinity for smooth muscles of the blood vessels, effects on the myocardium, extend of the
4
hepatic metabolism, biological half-life and bioavailability [ ]. The studies of the
antihypertensive activity in vivo conducted by Loeva et al. showed that the presence of the
dihydropyridine ring in DPHds molecule is the most important structural feature providing
their pharmacological properties, most likely due to the participation of the N-H proton in
hydrogen bonds with the corresponding amino acids at the active sites of the calcium
channels. Therefore, the oxidation process leading to the formation of pyridine analogues
abolishes antagonistic activity. Another structural feature relevant to the therapeutic action of
DHPds is the location of the substituent in the phenyl ring. The activity of the analyzed
derivatives depended on the place of the substitution and decreased in the following order:
5
ortho> meta >> para (practically inactive) [ ].
One of the unfavorable features of DHPds includes their high sensitivity to UV light.
During exposure to the UV radiation an intramolecular redox reaction occurs, associated
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with the elimination of the water molecule and aromatization of the dihydropiridine ring that
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7
leads to the formation of 4-(2-nitrosophenyl)-pyridine analogue (Fig. 2.) [ , ]. It has been
proven that the rapid photochemical decomposition is related to the presence of the nitro
group and its location in the phenyl ring. The most sensitive compounds include derivatives
of the ortho-substituted analogues, i.e. nifedipine (e.g. Adalat® Bayer, Procardia® Pfizer)
and nisoldipine (e.g. Sular® Shiongi). The photodegradation of these compounds is about 10
times faster than in the case of corresponding meta-isomers, i.e. nicardipine (e.g. Cardene®
Hoffmann La Roche). In order to increase the photostability of DHPds to UV radiation in
second generation drugs, the nitro group was replaced by other, less photosensitive
substituents. As a result, a series of modified DHPds was introduced to the pharmaceutical
market, in which nitro groups were replaced by halogeno, halogenoalkane or cyano
substituents. Stability studies have shown that photodegradation of halogen analogs was 50
times slower compared to the compounds with the nitro group in the meta position. However,
this process occurres and leads to the formation of biologically inactive derivatives of
halogeno-pyridine [6,7].
NMR spectroscopy in solution is one of the most important analytical techniques in
8
pharmaceutical research [ ]. It enables qualitative and quantitative determination of an active
9
10 11
pharmaceutical ingredient (API), also in mixtures and biological samples [ , , ]. NMR has
found widespread applications, particularly due to the possibility to characterize a drug
substance without any sample preparation nor derivatization. It is worth to note that NMR
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2 13
spectra appear in pharmacopoeias where purity of substance is of key importance [ , ]. In
addition, NMR spectroscopy enables the study of the API and excipients stability and allows
the search for their possible decomposition products, which proved to be extremely important
1
4 15 16
especially for UV-sensitive drugs from the DHPds group [ , , ]. One of the modern trends
is the application of NMR technique to determine the structure of API complexes with drug
carriers like cyclodextrines [6]. However, the required basis for applying this technique is a
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precise and unambiguous assignment of signals in H and C NMR spectra of drug
substances.
Despite the huge popularity and wide range of applications of this technique,
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numerous drug substances either have not been characterized by H and C NMR
spectroscopy or these data remained largely unpublished for reasons of confidentiality.
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According to our best knowledge, there is no available complete H and C NMR solution
data and assignment on nisoldipine, nilvadipine (Nivaldil® Astellas, Escor® Orion Pharma),
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nitrendipine (Baypress® Bayer, Nidrel® UBC Pharma, Nitrepress® Hexal) nimodipine
Nimotop® Bayer) and isradipine (Lomir Novartis). Moreover, the published NMR data on
amlodipine besylate (Amlozek® Adamed, Tenox® Krka, Norvasc® Pfizer) and felodipine
(
17 18
(
Plendil® Astra Zeneca, Apo-felodipine® Apotex Corporation) are partly incorrect [ , ].
Since presence of dihydropyridine ring and its influence on the conformation of the
molecule in solution are related to the pharmacological activity of DHPds, it seemed worth to
have a fast method for confirmation of their stability. Therefore, the aim of this work was to
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provide reliable H and C NMR characteristics of nine popular drugs with activity on
calcium channels. Molecular structures of nifedipine (1), nitrendipine (2), nisoldipine (3),
nimodipine (4), nilvadipine (5), felodipine (6), isradipine (7), nicardipine (8), amlodipine (9)
with carbon numbering are illustrated in Fig. 1.
2
. Experimental
Nifedipine
(5-O,3-O-dimethyl-2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-
3
1
2
,5-dicarboxylate) (1), nitrendipine (5-O-ethyl-3-O-methyl-2,6-dimethyl-4-(3-nitrophenyl)-
,4-dihydropyridine-3,5-dicarboxylate) (2), nisoldipine (3-O-methyl-5-O-(2-methylpropyl)-
,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate) (3), nimodipine (3-O-
(2-methoxyethyl)-5-O-propan-2-yl-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-
dicarboxylate) (4), nilvadipine (3-O-methyl-5-O-propan-2-yl-2-cyano-6-methyl-4-(3-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate) (5), felodipine (5-O-ethyl-3-O-methyl-4-
(
2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate) (6), isradipine (3-
O-methyl-5-O-propan-2-yl-4-(2,1,3-benzoxadiazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-
,5-dicarboxylate) (7), nicardipine (5-O-[2-[benzyl(methyl)amino]ethyl]-3-O-methyl-2,6-
3
dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate) (8) and amlodipine
besylate (3-O-ethyl 5-O-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-
dihydropyridine-3,5-dicarboxylate benzenesulfonate) (9) were kindly provided by National
Health Institute, Warsaw. Nitrosoderivative of nifedipine (1a) was obtained by UV irradiation
(λ=365 nm) of the chloroform solution of nifedipine and evaporation of solvent (Fig. 2.).
Deuterated solvents (acetone-d , CDCl ) were purchased from Glazer (Basel). NMR
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3
samples were prepared by dissolving 10 mg of each compound in 0.75 ml of deuterated
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solvent. H and C NMR spectra for acetone-d and CDCl solutions were measured on a
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3
1
Bruker DSX 400 spectrometer (Karlsruhe, Germany) operating at 400.61 ( H) and 100.13
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3
(
C) MHz or Varian 300 MHz VNMRS Oxford spectrometer (Agilent, Santa Clara, US)
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operating at 299.87 ( H) and 75.40 ( C) MHz at the temperature of 300K. All one- and two-
dimensional experiments (COSY, HSQC and HMBC) were run using the programs from the
Bruker/Varian software library. Chemical shifts are quoted relative to internal
1
tetramethylsilane (TMS). The spectral acquisition parameters were as follows; for H NMR
spectra: spectral width – 4807.7 Hz, acquisition time – 2.0484 s, relaxation delay – 1.0 s,
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3
pulse width – 8.60 µs, number of scans – 64; for C NMR spectra: spectral width – 24752.5
Hz, acquisition time – 0.6620 s, relaxation delay – 2.0 s, pulse width – 15.50 µs, number of
scans – 512; The COSY spectra were recorded at spectral width of 5 kHz in both F2 and F1
domains; 2K × 512 data points were acquired with eight scans per increment and the
relaxation delays of 1 s. Data processing was performed on a 2K × 1K data matrix. 2D
heteronuclear correlated spectra were measured over 2K complex points in F2 and 512
increments in F1, collecting 32 scans per increment with a relaxation delay of 1.5 s. The
spectral widths were 5 and 25 kHz in F2 and F1 dimensions, respectively. The HSQC
experiments were optimized for C–H couplings of 145 Hz; the HMBC experiments were
optimized for long-range C–H couplings of 7 Hz.
Fig. 1. Molecular structure of dihydropyridine drug substances: nifedipine (1), nitrendipine
(2), nisoldipine (3), nimodipine (4), nilvadipine (5), felodipine (6) isradipine (7) nicardipine
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(8), and amlodipine besylate (9) with carbon numbering. Structural differences in relation to
nifedipine are shown in the red frame.
Fig. 2. Photodegradation processes of dihydropyridine drug substances: (A) nitro-analogues
(1a), (B) halogeno-analogues.
3
3
. Results and discussion.
.1. Spectra assignments.
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H and C NMR spectra of 1-9 were measured for acetone-d and CDCl solutions
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3
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13
and assigned using 2D COSY, HSQC and HMBC experiments. H and C chemical shifts
for acetone-d solutions are collected in Tables 1-2 and 3-4, respectively.
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1
H NMR chemical shifts of nifedipine (1) and its nitroso analogue (1a) as well as
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9 20
nicardipine (8) were found to be in agreement with those reported previously [ , ]. In
contrast, the published NMR spectral values for amlodipine besylate (9) contain incorrect
data [17]. Incorrect assignments were found for aromatic carbons (C7, C8, C9, C12). The
distinction between C7 and C8 quaternary carbons is possible due to their correlation by three
bonds with the protons of the phenyl ring, respectively: H9 and H11 and H10 and H12 (Fig.
3
.). For C8, one can additionally observe a weak correlation by two bonds with the H9 proton
and a strong correlation by three bonds with the H4 proton (similar to the C12 carbon). On
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this basis, the signal can be unambiguously assigned as 132.35 ppm - C8 and 146.20 ppm -
C7 (CDCl solution). In the cited reference these carbons are assigned opposite [17].
3
In the publication cited above signals of carbons connected with protons H9 and H12
are also misassigned. While, for carbon C12 (signal at 131.60 ppm), there is a clear
correlation with the proton H4, which is missing for carbon C9 (signal at 129.29 ppm)
separated by four bonds (Fig. 4.). An additional confirmation of the correctness of the
assignment are correlations through three bonds with phenyl ring protons: for C9 with H11
(1H, t), and for C12 with H10 (1H, t) and correlations of H9 and H12 protons (assigned by
direct correlations with appropriate carbons in HSQC spectrum) with C7 and C11 and C8 and
C10 (Fig. 3.).
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Fig. 3. H - C correlations in HMBC spectrum of amlodipine besylate (9) (aromatic region).
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13
Fig. 4. H - C correlations in HMBC spectrum of amlodipine besylate (9)
(aliphatic/aromatic region).
Felodipine (6) is the second drug substance in DHPds group (apart from amlodipine)
with chlorine atoms substituted in the phenyl ring instead of the nitro group. The purpose of
this replacement was to reduce the sensitivity of the molecule to decomposition under UV
radiation. In addition, there were attempts to increase the stability of this drug by complexing
it with cyclodextrins. Interactions in felodipine complexes with cyclodextrins were
investigated by i.e. infrared and NMR spectroscopy. Obtained results showed that felodipine
enters the interior of the cyclodextrin; the difference of the chemical shifts of uncomplexed
and complexed felodipine reaches 0.25 ppm for C10 and 0.15 ppm and 0.20 ppm for C11 and
C12 carbons, respectively [18]. However, careful re-verification of the carbon chemical shifts
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3
by suitable correlations in 2D NMR spectra showed that correct assignments in the C NMR
spectrum should be different than in the above discussed paper; the differences concern the
C2 and C6 carbons of the DHP ring and the C8 and C9 carbons associated with chlorine; it is
illustrated by the correlation shown in Fig. 5. In the discussed publication, carbon atoms C2/6
are assigned to the signals at 130.89 ppm and 132.70 ppm (acetone-d solution). If these
6
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assignments were correct, there should be no correlations in the HMBC spectrum with
protons of the phenyl ring H10 (1H, m, δ = 7.32 ppm), H12 (1H, m, δ = 7.43 ppm) and H11
(1H, m δ = 7.19) ppm) (Fig. 5.). Correlations indicate that these signals originate rather from
the C8 and C9 quaternary carbons bound to chlorine atoms. A pair of C2/6 carbons gives an
extended signal at 144.23 ppm, as it is confirmed by correlations with the protons of the
methyl groups H13/14 (6H, s, δ = 2.31 ppm) and H4 (1H, s, δ = 5.48 ppm).
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Fig. 5. H - C correlations in HMBC spectrum of felodipine (6) (aromatic region).
An interesting derivative of the DHP series is nilvadipine (5), which has a CN
substituent instead of a methyl group at C2. The characterization of this drug substance and
the products of its photocomposition were mainly based on HPLC and UV spectrophotometry
21
[
].
In the case of nilvadipine, the HSQC spectrum assists in the identification of
quaternary carbons (including this belonging to the CN group); no correlation with protons
by one bond could be observed for 9 carbons. In turn, long-range correlations in the HMBC
spectrum allowed to distinguish quaternary carbons from each other. Due to the asymmetry
introduced into the DHP ring by the CN substituent at C2, signals arose from the C3/C5 and
C2/C6 carbon pairs are characterized by large differences in chemical shifts (in contrast to
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other DHPds). Based on the correlation with protons of the H14 methyl group, one can
clearly assign signals at 103.14 ppm and 144.91 ppm to C5 and C6 respectively. Replacing
the methyl group with the CN substituent at C2 results in up-field shift of its signal which
appears at 118.47 ppm. Correlation of all DHP ring's quaternary carbons in HMBC spectrum
with the proton H4 (1H, s, δ = 5.18 ppm) and correlation with the proton NH (δ = 6.92 ppm)
observed only for the C3 and C5 carbons confirmed the correctness of their assignments (Fig.
6
.). The N-H proton is also correlated with carbons CN (δ = 113.33 ppm) and C14 (δ = 19.34
ppm). Due to the asymmetry of the ester groups (isopropyl group at C17 and methoxy group
at C15), both carbonyl carbons can be unambiguously assigned.
.
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Fig. 6. H - C correlations in HMBC spectrum of nilvadipine (5) (aromatic region).
Isradipine (7) is an interesting DHPds molecule due to the presence of the oxadiazole
moiety bound to the phenyl ring instead of the nitro group or chlorine atom. Based on the
long-range correlation observed in the HMBC spectrum, one can clearly assign carbons C15
and C19 and carbons of the dihydropyridine ring - C2, C3, C5 and C6 (Fig. 7.).
Distinguishing the resonances of the methyl carbons belonging to the isopropyl moiety
(
C17/C18) from those bonded to the dihydropyridine ring (C13/C14) is provided from the
correlation of the protons bound to former ones in the COSY spectrum with the proton H16
1H, m, δ = 4.91 ppm) from the methine group.
(
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2
0
CDCl3
4
1
0
NH
16
11
12
3JH10, C12
3J
H4, C12
1
2
11
3JH11, C7
7
2JH4, C7
2
6
3JH10, C8
3JH12, C8
8
9
3
JH4, C8
3JH11, C9
1
1
5
9
3
JH16, C15
3JH20, C19
1
13
Fig. 7. H - C correlations in HMBC spectrum of isradipine (7).
Nitrendipine (2) and nimodipine (4) are less sensitive to UV light than nifedipine (1)
because both belong to the analogues with nitro group in meta position in the phenyl ring. In
nitrendipine molecule both carbonyl carbons (C15/C18) can be distinguished based on the
HMBC spectrum (Fig. 8.). The C15 carbon is correlated by three bonds with protons of the
methylene group H16 (2H, m, δ = 4.10 ppm), while the C18 carbon - with the protons of the
methoxy group H19 (3H, s, δ = 3.65 ppm). Unfortunately due to the great similarity of
chemical shifts values, it is not possible to unambiguously assign the other quaternary
carbons (C2, C3, C5 and C6) as well as methyl groups (C13 and C14) from the HMBC
spectra. Analogous asymmetry in C15/C19 substituents allow the assignment of carbonyl
carbons in nimodipine (4).
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1
9
4
1
6
8
11
1
2
NH
1
0
CDCl3
3JH12, C10
1
0
8
3JH4, C8
3JH12, C8
11
3JH10, C12
1
2
3JH4, C12
3JH8, C12
2
6
3JH11, C9
9
7
2JH4, C7
3JH11, C7
3JH19, C18
1
1
5
8
3
JH16, C15
1
13
Fig. 8. H - C correlations in HMBC spectrum of nitrendipine (2).
Due to the high similarity to nifedipine (1) (the only difference is the isobutyloxy
substituent instead of methoxy at the carbonyl C15 = O), it could be expected that in
nisoldipine (3) the values of the chemical shifts of particular carbons from 8-nitro-4-
phenylodihydropyridine moiety would be similar. Indeed, the chemical shifts of the carbon
atoms from common DHPds fragment do not differ more than 1.5 ppm. Distinguishing
carbon resonances of the methyl groups of the isobutyl substituent from those bonded to the
dihydropyridine ring is ensured by their correlation in the COSY spectrum with the methine
proton H17 (1H, m, δ = 1.89 ppm).
3
.2. Monitoring of nifedipine decomposition process by NMR spectroscopy in solution.
Nifedipine (1) is the most light-sensitive compound from the DHPds due to the ortho
position of nitro group in the phenyl ring. Under the influence of UV-light, nifedipine
undergoes an intramolecular redox reaction associated with the separation of the water
molecule that leads to the 4-(2-nitrosophenyl)-pyridine derivative (see Fig. 2.). A number of
chromatographic and spectroscopic methods have been proposed to study the
22 23 24
photodecomposition kinetics and photodegradation products of nifedipine [ , , ].
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Separation techniques were largely preferred, because they allow monitoring the kinetic
2
5 26 27
evolution of nifedipine concentration throughout the photodegradtion reaction [ , , ].
Conversely, the spectroscopic methods, including spectrophotometry, were rarely employed
for such studies mainly because of the overlapping of the spectra of nifedipine and its
28
photoproduct [ ]. The NMR spectroscopy allows simultaneously detection of nifedipine and
its decomposition product, because their signals are clearly separated (Figs. 9. and 10.). The
1
3
only thing that could be misleading in the C NMR spectrum of nitroso-derivative is that in
the range of 101-102 ppm C9 atom signal can be observed, instead of C3 and C5 carbon
atoms resonances. The of the C3 / C5 carbon common signal appears at 127.93 ppm and can
1
13
be assigned based on the lack of direct correlation with protons in the H- C HSQC spectrum
and the lack of long-range correlation with the aromatic protons in the HMBC spectrum.
Distinction from the pair of C2/C6 carbons (common signal at 156.43 ppm) is ensured by a
strong correlation of the latter with protons H13 / H14 (common signal at 2.66 ppm).
16/18
13/14
Nifedipine
16/18
13/14
4
CDCl3
12
9
N-H
11
10
Photodegradation product of nifedipine
12
11
10 CDCl3
9
1
Fig. 9. H NMR spectra of nifedipine (1) and its photodecomposition product (1a).
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Nifedipine
16/18
CDCl3
13/14
3/5
15/17
2/6
10
9
4
1
2 11
7
8
CDCl3
Photodegradation product of nifedipine
16/18
13/14
1
210
2/6
11
9
15/17
3/5
8
4
7
1
3
Fig. 10. C NMR spectra of nifedipine (1) and its photodecomposition product (1a).
4
. Conclusions.
1
13
H and
C NMR spectroscopy in solution was successfully applied for
characterization of a set of calcium channel blockers from DHPds group. The verified
chemical shift assignments can be used for subsequent conformational studies of nifedipine
analogues. Moreover, they allow determination of DHPds interactions in liquid phase, which
is essential due to their receptor binding properties and very important for their
pharmacological activity. Since nifedipine derivatives are highly-sensitive to UV-radiation
and under UV-light exposure lose their pharmacological activity, a proven and reliable
method for monitoring photodegradation processes is of key importance. The NMR
spectroscopy not only allows studies of nifedipine decomposition process, but also could be
used as methodology for identification of degradation products of DHPds, the biologically
inactive pyridine derivatives.
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5
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23] R. Teraoka, Y. Matsuda, I. Sugimoto, J. Pharm. Pharmacol. 1988, 41, 293.
24] M. Shamsipur, B. Hemmateenejad, M. Akhond, K. Javidnia, R. Miri, J. Pharm. Biomed.
Anal. 2003, 31, 1013.
[
[
25] P. Pietta, A. Rava, P. Biondi, J. Chromatogr. 1981, 210, 516.
26] W Maafi, M Maafi, Int. J. Pharm. 2013, 456, 153.
[
27] M. T. Gil-Agustí, S. Carda-Broch, L. Monferrer-Pons, J. S. Esteve-Romero, Biomed. Chromatogr. 2006,
0, 154.
2
[
28] I. A. Majeed, W. J. Murray, D. W. Newton, S. Othman, W. A. Al-Turk, J. Pharm.
Pharmacol. 1987, 39, 1044.
This article is protected by copyright. All rights reserved.

1
Tab.1. H NMR chemical shifts (δ, ppm) of 1-5.
1
H Chemical shift [δ, ppm]
No
C
1
1a
2
3
4
5
acetone-d6
8.04
(1H,brs)
5.67
CDCl3
5.88
(1H,brs)
5.71
(1H,s)
-
acetone-d6
-
CDCl3
-
acetone-d6
8.09
(1H,brs)
5.13
(1H,s)
8.14
CDCl3
6.14
(1H,brs)
5.11
(1H,s)
8.12
(1H,s)
-
acetone-d6
8.04
(1H,brs)
5.74
(1H,s)
-
CDCl3
5.60
(1H,brs)
5.78
(1H,s)
-
acetone-d6
8.18
(1H,brs)
5.13
(1H,s)
8.05
(1H,s)
-
CDCl3
5.75
(1H,brs)
5.10
(1H,s)
8.13
(1H,s)
-
acetone-d6
9.07
(1H,brs)
5.21
(1H,s)
8.15
(1H,s)
-
CDCl3
6.92
(1H,brs)
5.18
(1H,s)
8.12
(1H,s)
-
N
H
4
-
-
-
-
(
1H,s)
-
8
9
(
1H,s)
-
7.69
7.66
6.55
6.55
7.71
7.70
(
1H,d,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz) (1H,d,J=8Hz
(1H,d,J=8Hz (1H,d,J=8Hz)
)
)
)
)
1
1
1
0
1
2
7.34
7.24
7.55
7.44
7.72
7.64
7.34
7.24
8.00
8.00
8.08
8.08
1
(
1H,t,J=8Hz (1H,t,J=8Hz (1H,t ,J=8Hz (1H,t,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz (1H,t,J=8Hz (1H,t,J=8Hz) (1H,d,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz
)
)
)
)
)
)
)
)
)
)
)
7.54
7.44
7.88
7.72
7.51
7.38
7.55
7.45
7.51
7.37
7.60
7.45
1
(
1H,t,J=8Hz) (1H,t,J=8Hz (1H,t,J=8Hz) (1H,t,J=8Hz (1H,t,J=8Hz (1H,t,J=8Hz (1H,t,J=8Hz (1H,t ,J=8Hz (1H,t,J=8Hz (1H,t,J=8Hz (1H,t,J=8Hz (1H,t,J=8Hz
)
)
)
)
)
)
)
)
)
)
7.62
7.50
7.58
7.51
8.00
8.00
7.63
7.52
7.77
7.66
7.77
7.63
1
1
(
1H,d,J=8Hz (1H,d,J=8Hz (1H,d ,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz (1H,d ,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz (1H,d,J=8Hz
)
)
)
)
)
)
)
)
)
)
)
-
)
-
1
1
1
1
3
4
6
7
2.32
6H,s)
2.32
6H,s)
3.51
6H,s)
-
2.32
(6H,s)
2.32
(6H,s)
3.58
(6H,s)
2.60
(6H,s)
2.60
(6H,s)
3.39
(6H,s)
-
2.67
(6H,s)
2.67
(6H,s)
3.39
(6H,s)
-
2.35
(6H,s)
2.36
(3H,s)
4.06
(2H,m)
1.19
2.36
(6H,s)
2.36
(6H,s)
4.10
(2H,m)
1.23
2.35
(3H,s)
2.28
(3H,s)
3.78
(2H,m)
1.88
2.37
(3H,s)
2.31
(3H,s)
3.81
(2H,m)
1.89
2.36
(6H,s)
2.36
(6H,s)
4.12
(2H,m)
3.53
2.36
(6H,s)
2.36
(6H,s)
4.18
(2H,m)
3.55
(
(
(
2.44
(3H,s)
3.72
(3H,s)
-
2.41
(3H,s)
3.79
(3H,s)
-
(
3H,t,J=7Hz (3H,t,J=7Hz
(1H,m)
(1H,m)
(2H,m)
(2H,m)
)
-
)
-
1
1
8
9
3.51
6H,s)
3.58
(6H,s)
3.39
(6H,s)
3.39
(6H,s)
0.77
0.78
3.29
(3H,s)
3.35
(3H,s)
4.92
(1H,m)
4.97
(1H,m)
(
(3H,d,J=7Hz (3H,d,J=7Hz)
)
-
-
-
-
3.59
3.65
0.71
0.72
-
-
1.24
1.26
(
3H,s)
(3H,s)
(3H,d,J=7Hz (3H,d,J=7Hz)
(3H,d,J=6Hz (3H,d,J=6Hz
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)
-
)
)
2
2
2
0
1
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4.90
1H,m)
4.94
(1H,m)
1.08
1.10
(
(3H,d,J=6Hz (3H,d,J=6Hz
)
-
)
-
3.50
3H,s)
3.58
(3H,s)
1.08
1.09
(
(3H,d,J=6Hz (3H,d,J=6Hz
)
)
-
-
1.23
1.25
-
-
(
3H,d,J=6Hz
)
(3H,d,J=6Hz
)
1
-partly overlapped
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1
Tab.2. H NMR chemical shifts (δ, ppm) of 6-9.
1
H Chemical shift [δ, ppm]
No C
6
7
8
9
acetone-d6
CDCl3
acetone-d6
CDCl3
acetone-d6
CDCl3
acetone-d6
CDCl3
NH
NH2
4
8
9
7.97 (1H,brs)
5.73 (1H,brs)
8.15 (1H,brs)
6.17 (1H,brs)
8.31 (1H,brs)
-
5.15 (1H,s)
8.10 (1H,s)
6.74 (1H,brs)
8.42 (1H,s)
8.14 (2H,brs)
5.42 (1H,s)
7.50 (1H,s)
8.02 (3H,brs)
5.34 (1H,s)
-
-
-
-
-
5.06 (1H,s)
8.05 (1H,d,J=2Hz)
-
5.48 (1H,s)
5.46 (1H,s)
5.52 (1H,s)
5.48 (1H,s)
-
-
-
-
-
-
-
-
-
-
-
7.23 (1H,d,J=8Hz)
7.09 (1H,t,J=8Hz)
7.20 (1H,d,J=8Hz)
7.00 (1H,t,J=8Hz)
7.06 (1H,t,J=8Hz)
7.29 (1H, d,J=8Hz)
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
7
7.32 (1H,d,J=8Hz) 7.25 (1H,d,J=8Hz) 7.71 (1H,d,J=8Hz) 7.28 (1H,d,J=8Hz) 7.48 (1H,d ,J=8Hz) 7.88,7.94 (1H,dd,J=2Hz;J=8Hz)
7.19 (1H,t,J=8Hz) 7.07 (1H,t,J=8Hz) 7.48 (1H,t,J=8Hz) 7.31 (1H,t,J=8Hz) 7.95 (1H,t,J=8Hz)
1
1
7.31,7.36 (1H,t ,J=8Hz)
7.61 (1H,br)
2.38 (3H,s)
7.18 (1H,t ,J=8Hz)
1
7.43 (1H,d,J=8Hz) 7.30 (1H,d,J=8Hz) 7.36 (1H,d,J=8Hz) 7.62 (1H,d,J=8Hz) 7.74 (1H,d ,J=8Hz)
7.42 (1H, d,J=8Hz)
1
1
1
1
2.31 (3H,s )
2.32 (3H,s )
2.32 (3H,s )
2.33 (3H,s )
2.33 (3H,s)
2.41 (3H,s)
-
4.65,4.83(1H,d,J=14Hz) 4.57,4.67(1H,d,J=14Hz)
1
1
1
1
1
1
2.29 (3H,s )
2.31 (3H,s )
2.31 (3H,s )
2.32 (3H,s )
2.41,2.42 (3H,s)
-
3.98 (2H,m )
3.64 (2H,m )
1
1
-
-
-
-
3.50 (2H,br )
3.12 (2H,br )
4.01 (2H,q,J=7Hz) 4.07 (2H,q,J=7Hz)
1.13 (3H,t,J=7Hz) 1.19 (3H,t,J=7Hz) 0.87 (3H,d,J=6Hz) 0.96 (3H,d,J=6Hz) 3.32,3.40 (1H, m)
4.84 (1H,m)
4.91 (1H,m)
4.64 (2H,br)
4.63 (2H,br)
3.32,3.39 (1H, m)
2.66,2.67 (3H,m)
3.96,4.07,4.21 (2H,m)
-
2.31 (3H,s)
-
2.12 (3H,s)
-
1
1
-
-
1.16 (3H,d,J=6Hz) 1.21 (3H,d,J=6Hz)
2.61 (3H,m)
4.02 (2H,m )
4.03 (2H,m )
3.54 (1H,s)
3.62 (1H,s)
-
-
4.17,4.31 (2H,m)
-
1.15 (3H,t,J=7Hz)
1.17 (3H,t,J=7Hz)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3.50 (3H,s)
3.59 (3H,s)
-
-
1
1
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7.75 (2H,m )
7.55 (2H,m )
3.53 (3H, s )
3.57(3H, s )
1
1
7.43 (3H,m )
7.43 (3H,m )
-
-
-
-
-
-
-
-
-
-
1
1
7.43 (3H,m )
7.41 (3H,m )
1
1
7.43 (3H,m )
7.43 (3H,m )
1
1
7.75 (2H,m )
7.55 (2H,m )
3.60 (3H,s)
3.64(3H,s)
1
1
2
3
4
5
6
‘
‘
‘
‘
‘
-
-
-
-
-
-
-
-
-
-
7.83 (2H,m )*
7.40 (3H,m )*
7.40 (3H,m )*
7.40 (3H,m )*
7.86 (2H,m )*
7.38 (3H,m )*
7.38 (3H,m )*
7.38 (3H,m )*
1
1
1
1
1
1
1
1
7.83 (2H,m )*
7.86 (2H,m )*
1
-partly overlapped
*amlodipine is used in the treatment as besylate (benzenosulphonate) salt.
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1
3
Tab. 3. C NMR chemical shifts (δ, ppm) of 1-5.
13
C Chemical shift [δ, ppm]
N
o
C
2
1
1a
2
3
4
5
acetone CDCl
acetone CDCl
acetone CDCl
-d
145.14 145.3 156.43 156.5 147.11 145.3 146.97 144.7 147.12 144.4 118.49 118.4
acetone CDCl
acetone CDCl
acetone CDCl
3
3
3
3
3
3
-d
-d
-d
-d
-d
6
6
6
6
6
6
5
8
7
2
7
7
3
4
5
6
7
8
9
103.43 103.6 127.93 127.5 103.10 103.4 103.55 104.3 103.46 103.3 116.43 116.4
3
1
1
0
1
0
34.46 34.65 144.81 144.7 40.68 39.97 35.50 34.86 40.79 40.27 39.91 39.89
0
103.43 103.6 127.93 127.5 102.81 103.1 103.27 103.9 102.79 104.1 103.16 103.1
3
1
1
2
2
4
145.14 145.3 156.43 156.5 146.90 145.1 146.86 144.8 146.53 145.2 144.94 144.9
5
8
2
1
4
1
142.14 142.3 140.67 140.1 151.52 149.9 143.67 142.4 151.77 150.1 147.31 147.2
4
3
7
7
4
8
147.75 147.9 162.67 161.7 123.29 123.0 148.96 147.9 123.73 123.4 123.38 123.3
6
5
5
7
8
5
123.84 124.0 107.66 107.9 149.06 148.4 124.69 124.2 148.88 148.2 148.57 148.5
4
8
1
7
8
4
1
1
1
1
0 127.02 127.2 129.69 129.1 121.87 121.5 128.00 127.2 121.77 121.4 122.56 122.5
2
0
1
0
9
3
1 132.76 132.9 136.11 135.3 130.07 128.8 133.70 132.9 129.99 128.7 129.50 129.4
6
0
3
0
4
7
2 131.01 131.2 131.43 130.7 135.24 134.5 132.03 131.3 135.56 134.8 134.65 134.6
2
9
3
0
9
2
3
19.36 19.56 23.16 23.61 18.80 19.67 19.19 19.27 18.80 19.77 113.45 113.3
3
1
1
4
19.36 19.56 23.16 23.61 18.74 19.63 18.98 19.27 18.74 19.93 18.57 19.34
5 167.60 167.8 167.75 167.8 167.54 167.3 167.98 167.4 167.02 166.6 164.40 164.3
1
4
1
2
7
7
1
1
6 51.00 51.20 52.02 52.28 60.23 60.19 70.47 70.65 63.51 63.19 52.67 52.65
7 167.60 167.8 167.75 167.8 14.65 14.40 28.38 27.72 71.21 70.69 165.76 165.7
1
4
3
1
1
2
8
9
0
51.00 51.20 52.02 52.28 168.06 167.8 19.82 19.96 58.80 59.06 68.39 68.36
2
-
-
-
-
51.15 51.29 19.46 20.01 167.54 167.2 22.23 22.21
0
-
-
-
-
-
167.65 167.7 67.42 67.54 21.91 21.89
1
2
2
1
2
-
-
-
-
-
-
-
-
-
-
50.96 51.27 22.32 21.98
-
-
-
-
-
-
22.42 22.30
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1
3
Tab. 4. C NMR chemical shifts (δ, ppm) of 6-9.
13
C Chemical shift [δ, ppm]
No C
6
7
8
9
acetone-d CDCl3
acetone-d6
CDCl acetone-d
6
CDCl3
acetone-d6 CDCl3
6
3
2
3
4
5
6
7
8
9
144.23 144.45
103.70 104.07
147.19
101.87
145.40 150.91 149.70,149.66 145.59
101.69 102.35 100.53,100.61 103.08
144.78
102.23
37.11
103.46
145.49
146.20
132.35
129.29
127.47
127.11
131.60
68.25
66.87
39.88
18.82
167.43
60.02
14.12
168.26
50.86
-
38.56
38.75 39.33, 39,24 39.04
40.26
39.53
37.99
103.30 103.66
144.23 144.34
148.10 148.33
130.89 131.15
132.70 132.94
128.16 128.42
101.35
147.01
138.08
149.31
150.78
114.58
101.10 103.22 103.13, 103.12 104.01
145.76 146.87 145.92, 145.90 146.87
136.12 149.61 148.88, 148.90 147.44
148.49 123.06
122.59
132.71
150.10 149.00 148.28, 149.31 129.91
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
114.30 122.01
121.59
129.64
134,37
19.44
128.42
128.12
132.44
67.92
67.69
38.08
18.72
167.52
60.26
14.72
126.95 127.21 133.22; 133.18 131.70 130.23
129.61 129.88
130.40
19.86
19.77
129.46 135.12
19.36
19.36
19.71
19.77
19.86
19.77
18.86
19.19
19.73
167.42 167.59
167.20
167.06 166.74 166.48, 166.53
59.79
14.21
60.06 67.13, 67.04 67.26
14.49
58.18
54.62
40.30
60.10
58.07, 58.14
54.25, 54.43
39.96, 40.20
60.46, 60.72
22.34
22.23
168.24
22.24
21.87
168.15
167.90 168.09
50.80
51.09
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
51.17, 50.59 51.11
127.95 128.24, 128.27 168.29
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
132.29
129.70
130.39
129.70
132.29
131.25
129.64
130.55
129.64
131.27
50.74
-
-
-
-
-
-
-
-
-
-
167.75 167.74, 167,71
51.21
51.34
-
1
2
3
4
5
6
‘
‘
‘
‘
‘
‘
-
-
-
-
-
-
-
-
-
-
-
-
141.56* 143.96*
126.85* 125.95*
128.86* 128.82*
130.21* 130.99*
128.86* 128.82*
126.85* 125.95*
*amlodipine is used in the treatment as besylate (benzenosulphonate) salt.
This article is protected by copyright. All rights reserved.

Structural studies of calcium channel blockers used in the treatment of
1
13
hypertension - H and C NMR characteristics of nifedipine analogues.
a
b*
a
Łukasz Szeleszczuk , Monika Zielińska-Pisklak , Dariusz Maciej Pisklak
The NMR spectroscopy in solution was applied for characterization of a series of UV-light
sensitive compounds, dihydropyridine derivatives used in hypertension therapy. The 1D and
2
D NMR experiments allowed unequivocal assignment, enabled fast identification of the
drug substance and ensured its stability in the commercial dosage forms from different
sources.
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