Complexation of tetrandrine with calcium ion probed by various

spectroscopic methods and molecular modeling

Ioana Stanculescua, Cristina Mandravela, David Landyb, Patrice Woiselb,Gheorghe Surpateanub,*

aDepartment of Physical Chemistry, University of Bucharest, 4-12 Bd. Regina Elisabeta, 70346 Bucharest, RomaniabOrganic Synthesis Laboratory, M.R.E.I.D., 145 avenue Maurice Schumann, 59140 Dunkerque, France

Received 6 January 2003; revised 13 January 2003; accepted 20 March 2003

Abstract

The formation of the complex between tetrandrine and the calcium ion, in solution, was studied using FTIR, UV–Vis, 1H

NMR, 13C NMR and electrospray mass spectroscopy spectroscopic methods and molecular modeling. The calcium salts used

were: Ca(ClO4)2·4H2O and Ca(Picrate)2 in the solvents: acetonitrile (CH3CN), deuterated acetonitrile (CD3CN) and

tetrahydrofurane (THF). The determined complex stability constant was: 20277 ^ 67 dm3 mol21 and corresponding free

energy DG0 ¼ 25:820 ^ 0:002 kcal mol21. The molecular simulation of the complex formation with the MM3 Augmented

force field integrated in CAChe provided useful data about its energy. Combining the experimental results and molecular

modeling we propose a model for the structure of tetrandrine–Ca complex with an eight coordinated geometry.

q 2003 Elsevier Science B.V. All rights reserved.

Keywords: Calcium complexes; Electrospray mass spectroscopy; Molecular modeling; Molecular spectroscopy (FT-IR, NMR, UV–Vis);

Tetrandrine

1. Introduction

Tetrandrine (TET) (6,60,7,12-tetramethoxy-2,20-

dimethyl-berbaman), a bis-benzylisoquinoline alka-

loid, isolated from the root of Stephania tetrandrae

S. Moore, is used in China to treat angina and

hypertension [1].

This compound, according to data in recent

literature, exerts a variety of pharmacological actions:

anti-hypertensive, anti-arrhythmia, anti-ischemia, anti

inflammatory, anti-fibrosis of the lung, immunosup-

pressive and antiplasmodial [2–5].

The chemical structure of TET, obtained by X-ray,

shows a dimer of two benzylisoquinoline subunits

condensed in a head to head, tail to tail fashion,

forming a macrocycle, with 1S, 10S stereochemistry at

the chiral isoquinoline carbon atoms (Fig. 1) [6]. The

spectroscopic and X-ray studies of TET have demon-

strated that its structure is very similar both in solution

and in solid state [7].

The known biological properties of TET can be

explained by its calcium channel blocker activity, but

the corresponding mechanism it is not completely

elucidated [8,9].

0022-2860/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0022-2860(03)00216-3

Journal of Molecular Structure 655 (2003) 81–87

www.elsevier.com/locate/molstruc

* Corresponding author. Tel./fax: þ33-3286-58254.

E-mail address: surpatea@univ-littoral.fr (G. Surpateanu).

In this study we demonstrate that TET binds the

calcium ion and acts as a host molecule for this

metal cation in a molecular recognition process.

This behavior can be related to the three-dimen-

sional characteristics of such cyclophane type

structure [10].

Calcium ions are key intracellular messengers in

the cardiovascular system. Calcium homeostasis is

regulated by an extracellular cycle which controls the

entry and removal of calcium between the cytosol

(cell) and extracellular space, and an intracellular

cycle, which controls calcium fluxes between the

cytosol and intracellular stores in the sarcoplasmic

reticulum [11].

The probability of formation of the TET–calcium

complex lead us to the idea that TET may acts as a

mobile carrier and thus, it can interfere in the calcium

cycle [12].

Forces that contribute to molecular complex

assembly can be: hydrogen bonding, ion pairing,

van der Waals interactions, metal–ligand binding,

solvent reorganization, partial covalent bonds and

noncovalent cation-pi type interactions [13].

Thus, in this paper we evidence the calcium

complex formation of TET, in solution, using two

calcium salts and different solvents.

We have successively used, for this purpose

Fourier Transform Infrared and UV– Vis spec-

troscopy, 1H NMR, 13C NMR and mass spectrometry

methods. We have applied molecular modeling on the

structure of complex in vacuo, obtaining the variation

of the total energy in molecular mechanics as function

of calcium ion distance from the plane of TET cavity.

Finally, we propose a model for the TET–calcium

complex structure. Surely, this approach neglects the

interactions which appear in solution, especially in

polar solvents.

2. Experimental

TET (purity 98%) was obtained from Fluka

(Germany). The calcium salt: Ca(ClO4)2·4H2O, was

purchased from Across (USA). The calcium picrate

(CaPic2) was prepared in our laboratory, after

methodic described below. The deuterated aceto-

nitrile solvent was obtained from SDS (France). Other

reagents were of the highest purity grade available.

2.1. Spectroscopic measurements

Infrared spectra were recorded on a FT-IR Perkin

Elmer 2000 spectrophotometer in potassium bromide

discs. UV–Vis spectra were recorded on a Perkin

Elmer Lambda 2S spectrophotometer. The 1H mag-

netic resonance spectra (250 MHz) and 13C magnetic

resonance spectra (62.7 MHz) were recorded on a

Bruker AM 250 spectrometer. Mass spectra were

performed with a Platform II Micromass spectrometer

using the electrospray (ES) technique.

2.2. Molecular modeling

The molecular modeling was performed on a PC

with a Genuine Intel Family 6 Model 7601 processor

with the CAChe (Computer Aided Chemistry)

program and the MM3 Augmented force field.

2.3. Synthesis of CaPic2

To 40 ml solution of 2 g (0.2182 M) picric acid in

water maintained at <78 8C we have added 0.437 g of

CaCO3 (0.1092 M) after the method described in Ref.

[14]. We have maintained the reaction mixture,

stirring continuously, at this temperature for 1 h. As

the solution is cooled gradually to 0 8C the CaPic2

crystallizes. Yield can be maximized by boiling under

vacuum until the solution reduces to half of its

original volume. Before utilization the CaPic2 was

recrystallized in water.

2.4. Syntheses of complexes

The calcium TET complexes were prepared by

mixing 4 mg (1.2846 £ 1022 M) of ligand and 4 mg

(2.5692 £ 1022 M) of calcium perchlorate in 0.5 ml

CD3CN. This solution was directly used for NMR

Fig. 1. Molecular structure of TET with the numbering scheme.

I. Stanculescu et al. / Journal of Molecular Structure 655 (2003) 81–8782

records. Then 0.1 ml of the solution was poured on

250 mg of dry KBr, 1 ml chloroform was added and

the solvents were removed by a dry nitrogen stream

[15].

The resulting solid was compressed and used as

KBr pellets for infrared spectrum. The remaining

0.4 ml solution of complex was used for electrospray

mass spectroscopy (ESMS) analysis.

In the case of the CaPic2 complex study we have

recorded the UV–Vis spectra (i) of CaPic2 in

tetrahydrofurane (THF) in the range 310–470 nm

and (ii) the spectra obtained by adding of increasing

amounts of TET [16].

3. Results and discussion

The TET/Ca2þ complexes were synthesized as

described above.

The complexes formed by neutral macrocyclic

ligands with metallic cations are themselves cationic

(frequently, they have the charge of the complexed

cation) and are extracted in organic solvents as ion

pairs with an anion having large molecular weight and

lypophilic character. The solvents with small dielec-

tric constants (THF) inhibit the dissociation of the ion

pair favoring the formation of the complex [17].

The calcium salt used firstly for complexation, the

calcium perchlorate, is tetrahydrated and this fact can

influence the acid–base equilibrium of the two

tertiary amine functional groups of TET, even in the

non-aqueous solvent used. Thus, we have utilized for

complexation the calcium picrate whose changes in

the molar absorptivities in the 310–470 nm region in

presence of increased concentrations of TET have

permitted us to calculate the formation constant of the

complex in THF solutions.

3.1. Infrared study

The IR spectra were recorded in the region 4000–

450 cm21 (0.5 cm21 resolution).

The comparison between the spectra of the free

ligand and the complex shows changes in the

vibrations of the TET upon complexation indicating

the presence of conformational distortion (Table 1).

Complex formation is accompanied by modifications

of form and intensity of bands and to a less extent in

their position. Despite of these not obvious spectral

changes one may conclude that the complex is

formed, considering the case of 4, 13 diaza-18-

crown-6 calcium chloride and bromide complexes

when no change in the vibrations of the macrocyclic

ligand were observed upon complex formation [18].

The fine bands corresponding to n(C–O–C)

asymmetric stretch in aromatic ethers which appear

in the spectrum of TET in the regions 1010–1050 and

1210–1280 cm21 loss their fine character upon

complexation becoming larger, showing the partici-

pation of the ethers oxygens to the coordination of the

calcium ion [19].

The ‘isolated’ perchlorate anion has a Td sym-

metry and shows a n(Cl–O) ðn3Þ single band at

1100 cm21 [20]. When the perchlorate anion is bond

to a cation, a two or three-fold band appears, related to

the loss of tetrahedral symmetry. In the spectrum of

the studied perchlorate complex we observed the

same multiple pattern for the n3 band. Thus the

perchlorate anions take part in the complexation of

calcium ions.

The band from 840 cm21 in the spectrum of the

free ligand, which corresponds to a in phase out of

plane CH wag modes of substituted benzenes is

shifted to 800 cm21 in the spectrum of complex. The

stretching of ring CyC bonds of para substituted

Table 1

FT-IR data (maxima) for pure ligand and calcium perchlorate

complex

Compound Wavenumber (cm21) and

transmittance ðT%Þ values

TET 524(42.3), 564(45.5), 624(46.8), 808(44.2),

840(42.5), 976(44.6), 1004(43.8), 1024(41.0),

1068(41.3), 1112(38.7), 1124(40.2), 1164(43.9),

1212(43.9), 1232(38.3), 1256(40.0), 1272(37.7),

1340(44.1), 1408(43.0), 1440(42.4), 1508(35.8),

1580(44.2), 2800(41.8), 2832(41.4), 2876(41.1),

2928(38.9), 2956(40.2)

TET–Ca 520(8.5), 556(9.0), 628(4.1), 752(12.6),

800(4.0), 864(13.1), 1016(4.1), 1080(2.4),

1140(3.2), 1212(8.2), 1232(7.4), 1260(3.3),

1356(9.2), 1412(8.0), 1460(7.0), 1508(4.4),

1620(5.0), 2792(12.2), 2852(10.1), 2924(8.2),

2960(8.1)

The CO2 band from 2360 cm21 is present in both spectra and a

large water band with 3420 cm21 absorption maximum appears

only in the complex spectrum.

I. Stanculescu et al. / Journal of Molecular Structure 655 (2003) 81–87 83

benzene gives in the spectra of the free ligand a band

at 1580 cm21 while in the complex it appears at

1620 cm21. The 624 cm21 vibration of TET attribu-

table to the in plane bend of para substituted aromatic

rings dramatically enhances in intensity (the shift is of

only 4 cm21). The band from 524 cm21 of pure ligand

which corresponds to the out of plane ring bending in

para substituted benzene decreases in intensity and is

shifted to 520 cm21 probing the change of confor-

mation of the ligand induced by the complexation

with Ca2þ ion.

3.2. UV–Vis study

The CaPic2 absorbance (A) in the region 310–

470 nm at 3.23 £ 1025 M in THF changes after

adding increasing amounts of TET as follows:

6.26 £ 1025, 1.292 £ 1024, 2.584 £ 1024 and

6.46 £ 1024 M.

Considering the presence of the 1:1 complex in

solution (we have obtained the best fit of the

experimental points, using this stoichiometry), the

interaction between the ligand (L) and alkaline earth

metal ion (M) is defined by the equilibrium equation:

L þ M , LM ð1Þ

and by the stability constant K associated with this

complex type:

K ¼½LM�

½L�·½M�ð2Þ

Using the values of the derived absorbance curves

at l ¼ 370 nm, where TET does not absorbs (see

Fig. 2) we have employed the following data

treatment [21]:

½LM� ¼ 21

2

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1

Kþ ½L�T þ ½M�T

� �2

24½L�T·½M�T

s

þ1

2

1

Kþ ½L�T þ ½M�T

� �ð3Þ

where T stands for total.

Then for a given value of K; [LM] is known and the

spectral characteristic (1LM; molar absorptivity) of the

complex can be calculated, based on the following

Fig. 2. Variation of derived absorption of CaPic2 with the concentration of TET at l ¼ 370 nm.

I. Stanculescu et al. / Journal of Molecular Structure 655 (2003) 81–8784

relation:

A ¼ lð1M·½M� þ 1LM·½LM�Þ ð4Þ

The estimation of the 1LM is achieved for each

concentration of [L]. The difference over the par-

ameter of concern has to be minimized relative to K:

All calculations were performed in Excel (minimiz-

ation algorithm: Newton–Raphson).

The value of K is equal to 20277 ^ 67 dm3 mol21.

The complexation was accompanied by a weak

bathochromic shift of the picrate ion absorbance

from 333 to 336.6 nm related to the change of a tight

ion pair to a looser ion pair [16].

The value of DG0 was calculated using the

equation:

DG0 ¼ 2RT ln K ð5Þ

and the obtained value is 25.820 ^ 0.002 kcal mol21

at T ¼ 298 K (R ¼ 1:9872 cal mol21 K21) in good

agreement with literature data [17].

3.3. NMR studies

The 1H and 13C spectra of the free ligand showed

resonance signals fairly resolved. The complete

assignment of all the signals was realized using

previously reported data and new investigation

techniques: 13C DEPT 135 and RMN-2D spec-

troscopy: COSY HH, HMQC, HMBC and NOESY

[4,22].

The chemical shifts before ðdTETÞ and after

complexation ðdCOMPLEXEÞ are given in Table 2.

For the free ligand, the aliphatic protons are in the

range 2.24–3.92 ppm and the aromatic protons in the

5.99–7.45 ppm range, while for the complex these

intervals are, respectively, 2.30–4.11 and 6.03–

7.47 ppm.

In the case of 1H spectrum the protons: H(50),

H(80), H(10), H(16), H(160), H(110), H(130), H(140),

H(13) and H(14) have the greatest variations of the

chemical shifts, probing that the coordination of the

calcium ion by the ligand introduces shifts effects in

the protons close to the cavity. The signals corre-

sponding to carbon atoms: C(40), C(17), C(170),

Table 21H and 13C NMR data for TET and its complex with calcium perchlorate

Atom dH (ppm) Atom dC (ppm) Atom dC (ppm)

TET COMPLEXE TET COMPLEXE TET COMPLEXE

H140(dd) 7.45 7.47 C4 22.08 21.98 C110 122.24 122.39

H130(dd) 7.09 7.11 C40 26.42 25.26 C130 122.57 122.55

H13(d) 6.93 6.94 C150 37.49 37.55 C14 123.79 122.68

H14(dd) 6.82 6.84 C15 42.52 42.16 C20 123.92 123.97

H110(dd) 6.69 6.72 C16 42.60 42.40 C17 129.42 127.59

H50(s) 6.65 6.70 C160 42.95 42.49 C170 129.88 128.72

H10(d) 6.47 –a C3 44.37 44.44 C200 130.29 128.85

H5(s) 6.43 6.64 C30 46.04 46.08 C140 131.58 131.66

H100(dd) 6.36 6.38 C18 56.47 56.46 C100 133.56 133.56

H80(s) 5.99 6.03 C180 56.58 56.53 C9 135.88 134.91

H10(dd) 3.92 4.11 C21 56.63 56.56 C90 137.28 135.78

H21(s) 3.88 3.88 C19 60.38 60.36 C7 137.86 138.86

H18(s) 3.73 3.74 C1 62.63 62.82 C70 144.67 144.81

H1(d) 3.58 –a C10 64.32 64.42 C12 148.17 148.35

H180(s) 3.34 3.36 C5 107.35 107.39 C8 149.12 149.02

H150(dd) 3.24 –a C13 112.90 112.88 C60 149.21 149.80

H19(s) 3.14 3.15 C50 114.10 113.94 C11 150.43 150.37

H160(s) 2.58 2.70 C10 116.69 116.61 C6 152.51 152.72

H16(s) 2.24 2.30 C80 120.85 120.87 C120 154.39 154.63

dd—doublet of doublet, d—doublet, s—singlet.a These chemical shifts could not be distinguished because of signals superposition.

I. Stanculescu et al. / Journal of Molecular Structure 655 (2003) 81–87 85

C(200), C(90) and C(7), which are defining the

intramolecular cavity of TET, changes most in

position in the complex spectrum towards the

spectrum of the free ligand [23,24]. In the 1H spectra

the induced shifts are in the range (0; 0.21 ppm) and in

the 13C spectra in the range (21; 1.83 ppm).

3.4. ESMS study

To obtain the ESMS spectrum we have diluted the

remaining solution with acetonitrile. The species

identified, conform to algorithms in use are presented

in Table 3 [25,26].

Peak intensities are cited as percentages of the

most intense peak ðI%Þ of the studied m=z domain

(700–1000). The instrument resolution is 1 a.u.

From Table 2 results that the calcium–TET

complex with 1:1 stoichiometry is present in solution

and two perchlorate anions are bonded to the

calcium ion.

3.5. Molecular modeling

The initial geometry of TET was obtained from the

atomic coordinates of the X-ray structure, published

in the literature [6].

TET forms an interior cage (a macrocycle with 18

atoms) defined by the atoms H(10), O(8), C(70), C(80),

H(80) and the inner surface of the phenyl ring C(90)–

C(100)–C(110)–C(120)–C(130)–C(140).We have used

for calculation the MM3 Augmented force field. The

lowest energy structure was found by geometry

optimization with the Block-diagonal Newton

Raphson algorithm, with the RMS gradient of

0.001 kcal mol21 A [27].

The atoms O(8) and O(11) define one axe in the

plane of the cavity. We have obtained the variation

of the total energy, in molecular mechanics, when

the calcium ion moves along an axe of 10 A, with

a step of 1 A, perpendicular to TET cavity in its

center (Fig. 3).

The conformation of TET is changing when the

calcium ion is approaching the cavity, and we can

observe a reduction of the total energy with

approximate 4.21 kcal mol21.

Considering these data and the obtained exper-

imental results we propose a structure with an eight

coordinated calcium ion (with the four ether oxygen

atoms (O(8), O(11), O(12) and O(60)), the two

oxyanions of the perchlorate counter-ion and two

water molecules). This coordination number is the

most favorable for the calcium ion [15].

Table 3

Positive ion ESMS of Ca–TET complex in CH3CN at CV 20V

Major ions

observed

m=z (theoretical) m=z (experimental)

ðI%Þ

[TETCa(ClO4)(H2O)2]þ 797.24 799, 21.74

[TETCa(ClO4)(CH3CN)2]þ 843.27 843, 59.63

[TETCa(ClO4)2 þ H]þ 861.17 861, 90.40

[TETCa(ClO4)2

þ (HClO4) þ H]þ961.13 963, 100

Fig. 3. The variation of the total energy when Ca2þ is docking in the cavity of TET.

I. Stanculescu et al. / Journal of Molecular Structure 655 (2003) 81–8786

The obtained bond lengths in the complex are as

follows: dðO8· · ·Ca2þÞ ¼ 2:8 A, dðO11· · ·Ca2þÞ ¼

2:74 A, dðO12· · ·Ca2þÞ ¼ 2:79 A, dðO60· · ·Ca2þÞ ¼

2:65 A, dðClO24 · · ·Ca2þÞ ¼ 2:50–2:52 A and dðO2

H· · ·Ca2þÞ ¼ 2:65–2:73 A, respectively.

4. Conclusions

Tetrandrine a natural macrocycle with a cyclo-

phane type structure can be engaged in calcium cation

recognition.

Following this study we can establish the stoichi-

ometry of the calcium–TET complex and we can

predict an eight coordinated geometry of the complex

as deduced from IR, NMR, UV–Vis, ESMS spectro-

scopic measurements and molecular modeling.

Acknowledgements

Support of this research by the O.N.B.S.S.-

Romania is gratefully acknowledged.

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