- Email: [email protected]
Spatial structure of felodipine dissolved in DMSO by 1D NOE and 2D NOESY NMR spectroscopy
Accepted Manuscript Spatial structure of felodipine dissolved in DMSO by 1D NOE and 2D NOESY NMR spectroscopy I.A. Khodov, M.Yu. Nikiforov, G.A. Alper, D.S. Blokhin, S.V. Efimov, V.V. Klochkov, N. Georgi PII: DOI: Reference:
S0022-2860(12)01081-2 http://dx.doi.org/10.1016/j.molstruc.2012.11.040 MOLSTR 19377
To appear in:
Journal of Molecular Structure
Received Date: Revised Date: Accepted Date:
23 October 2012 8 November 2012 19 November 2012
Please cite this article as: I.A. Khodov, M.Yu. Nikiforov, G.A. Alper, D.S. Blokhin, S.V. Efimov, V.V. Klochkov, N. Georgi, Spatial structure of felodipine dissolved in DMSO by 1D NOE and 2D NOESY NMR spectroscopy, Journal of Molecular Structure (2012), doi: http://dx.doi.org/10.1016/j.molstruc.2012.11.040
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Spatial structure of felodipine dissolved in DMSO by 1D NOE and 2D NOESY NMR spectroscopy. I. A. Khodov a*, M. Yu. Nikiforov a, G. A. Alper a, D. S. Blokhin b, S. V. Efimov b, V. V. Klochkov b, N. Georgi c a
G.A. Krestov Institute of Solution Chemistry of the Russian Academy of
Sciences, ul. Akademicheskaya 1, 153045 Ivanovo, Russia b
Kazan Federal University, ul. Kremlevskaya 18, 420008 Kazan, Russia
c
Max Planck Institute for Mathematics in the Sciences, Inselstraße 22, 04103
Leipzig, Germany *
Corresponding author: [email protected]
Abstract Small organic molecules in dissolved state exist as an ensemble of conformers. In this work conformation of felodipine in dimethyl sulphoxide was studied and dominant stable conformers were determined. Effective interatomic distances were obtained by means of NOE spectroscopy. Fractions of different conformers were estimated by comparing effective distances and those obtained from quantum-chemical calculation [Teberekidis, 2007]; averaging of distances was made following the N-site jump model.
Highlights Internuclear distances in felodipine were obtained by analysis of 1D and 2D NOE data. Obtained effective distances were compared with quantum-chemical calculations. Fractions of different conformers of felodipine dissolved in DMSO were estimated.
Keywords Conformational analysis; Nuclear magnetic resonance; NOE; Jump model. 1
ACCEPTED MANUSCRIPT Introduction Accurate determination of interproton distances in relatively small, flexible molecules by 1D and 2D NOE NMR spectroscopy is the subject of increasing interest in recent years [1–3]. Results of such investigations can be applied to establishing conformational details for biologically active molecules in solutions. It is well known that polymorphism of drug compounds affects the biological activity and plays an important role in the production of pharmaceuticals. In turn, the properties of polymorphs are due to molecular structure of compounds and their ability to exist in different conformational forms in the solvent from which recrystallization is performed. Therefore, search for new polymorphic forms of drugs is closely associated with the study of conformational states of biologically active molecules in solutions. Felodipine (ethylmethyl-4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl3,5-pyridine-dicarboxylate) is widely used as antihypertensive and antianginal drug [4]. The possible existence of different polymorphic forms connected with conformations of the phenyl ring was predicted in [5]. Commercial felodipine has the melting point of 144°C and is a racemic mixture of enantiomers [6]. Some researches point to existence of three or more crystal structures [5, 7]. Conformational flexibility of felodipine is determined by two dihedral angles: (C3-C4-C1’-C2’), which defines the rotation of 2,4-dichlorophenyl group around C1’-C4 bond, and (C3a-O-C3b-C3c), related with the rotation of ethyl group around O-3b bond (Fig. 1). Teberekidis and Sigalas in 2007 published the result of a theoretical study of felodipine using a hybrid density functional method B3LYP [8]. They revealed six conformers with very close energies (within 4,2 kJ/mol), starting from number 1 (the global minimum) up to number 6 (with the maximal energy). It is important for the purposes of further discussion, that in the conformers 1, 2, and 5 chlorine atoms point to the same direction as NH1 proton, whereas in the conformers 3, 4, and 6 the dichlorophenyl ring is turned by 180° and chlorine atoms are closer to the H4 proton (Figs. 2, 3). Obvious progress was made recently in determining interproton distances from NOE data. Butts et al. [9] gave a convincing example of the fundamental possibility for quantitative determination of conformations of flexible molecules by NOE. This success is due to the fact that only five short interproton distances (<3 Å) are present in the molecule of arugosin C, which can be determined with a 2
ACCEPTED MANUSCRIPT high accuracy. The question remains, however, how effective are conventional NOE experiments for analysis of an arbitrary small molecule in a given solvent. In this work, conventional methods of 1D and 2D NOESY were employed to study the conformational state of felodipine in DMSO. The choice of this solvent is associated, in particular, with high solubility of felodipine in DMSO.
Fig. 1. Structure and atom nomenclature of felodipine. Arrows indicate rotations responsible for the diversity of conformations.
Fig. 2. Conformations 1, 2, and 5 obtained by quantum-chemical calculations [8]. Molecular structures are depicted in the Chimera software [10].
3
ACCEPTED MANUSCRIPT
Fig. 3. Conformations 3, 4, and 6 from [8], differing from 1, 2, and 5 by orientation of the dichlorophenyl ring.
Formulation of the Problem NOE spectroscopy is a powerful tool of studying the spatial structure and conformations of molecules in solution. The method of estimating internuclear distances for different proton pairs in a molecule is based on the existence of a strong dependence of the cross-relaxation rate constant σij on the distance rij between the interacting nuclear spins. Normally, such a dependency is approximated by the simple formula σ ij ~ 1 rij6 , and then internuclear distances are obtained according to the expression
⎛σ rij = r0 ⎜ 0 ⎜ ⎝ σ ij
1
⎞6 ⎟ , ⎟ ⎠
(1)
where r0 and σ0 refer to the proton pair chosen for calibration; the distance r0 is supposed to be known from some independent source (e.g., quantum chemical calculations). However, for small, conformationally flexible molecules, of great importance is the problem of more accurate determination of internuclear distances in order to obtain information on relative population of various conformers. This goal may be obtained by both a more accurate interpretation of the 2D NOESY experiment and taking into account anisotropic molecular rotation. A more advanced approach to calculate distances rij was developed in [11– 13] based on relative integral intensities; cross-peak intensities aij are normalized by the intensity of corresponding diagonal peaks. The matrix of integral intensities A with elements aij from 2D NOE spectrum is related to the mixing time τm by the formula 4
ACCEPTED MANUSCRIPT A(τ m ) = exp(− Rτ m ) ,
(2)
where R is the cross-relaxation matrix of the form
⎛ ρ11 σ 12 ⎜ ⎜ σ 21 ρ 21 R=⎜ ⎜ ⎜ σ σ s2 s1 ⎝
σ 1s ⎞
⎟ ⎟ ⎟. ⎟ ρ ss ⎟⎠
(1)
Here matrix elements ρij and σij correspond to the longitudinal and crossrelaxation rates, respectively:
ρ ij =
2π 4 2 γ ∑ J ij0 (ω ) + J ij1 (ω ) + J ij2 (ω ) , 5 j ≠i
(2)
2π 4 2 γ ∑ + 6 J ij2 (ω ) − J ij0 (ω ) . 5 j ≠i
(3)
[
σ ij =
]
[
]
J(ω) is the spectral density function, which for the case of isotropic tumbling of the molecule has the form
Jijm (ω) =
⎤ 1 ⎡ τc ⎢ ⎥. 4π rij6 ⎣1+ m2ω2τ c2 ⎦
(4)
Here τc is correlation time, which is on the order of 10-10 s for small molecules; m stands for the order of relaxation transitions (0, 1, or 2). As was shown in [11–13], where series expansion into powers of exp(-Rτm) is used, dependency of the averaged integral intensity Ī on the mixing time τm I (τ m ) =
1 ⎛⎜ 1 aij (τ m ) 1 a ji (τ m ) ⎞⎟ + 2 ⎝⎜ n j aii (τ m ) ni a jj (τ m ) ⎟⎠
(5)
may with a high degree of accuracy be considered linear with the τm values much higher than those employed usually in the initial rate approximation. Having recorded several 2D NOE spectra with various τm values, one may plot the dependence Ī(τm) and then determine more accurately the values of σij and, applying Eq. (1), the distances rij.
5
ACCEPTED MANUSCRIPT Another factor that should be taken into account when accurately determining internuclear distances is the anisotropic molecular rotation. When considering the interactions of two groups of equivalent spins I and S (each containing nI and nS spins), the effective distance reffij, obtained from an experiment, should be understood as denoting the average over all possible i–j distances. For the isotropic rotation, in view of Eqs. (1) and (6), we have [14]
rijeff
⎡ 1 =⎢ ⎣⎢ nI nS
−
1
1⎤ 6 ∑ij r 6 ⎥ . ⎥ ij ⎦
(6)
Anisotropy of the molecular rotation makes direct use of the formula (6) incorrect; this expression is applicable to the case of isotropic rotation only. Tropp [15] obtained some general relations for the spectral density function J(ω) when internal molecular rotation is taken into account. In particular, for the fast rotation of the methyl group in the slope of the three-site jump model, the expression for J(ω) has the form
τc 1 Jijm (ω) = 5 1+ m2ω2τ c2
(
(
)
)
2
i i ,ϕmol 1 3 Y2k θmol . ∑ ∑ r3 k=−2 3 N=1 ij 2
(7)
Here Y2k are second rank spherical harmonics; θ and φ are polar coordinates of internuclear vectors connecting each nucleus pair IS subject to averaging. Therefore, when considering the interaction of one spin I and a group of equivalent spins S in the methyl group, we obtain the following relation for the effective distance
rijeff
(
)
⎡1 2 1 3 Y θ i ϕ i ⎤ = ⎢ ∑ ∑ 2 k mol3 mol ⎥ rij ⎢⎣ 5 k =−2 3 N =1 ⎦⎥
−
1 6
(8)
Experimental
Felodipine and deuterated DMSO were purchased from Aldrich and used without further purification. Samples were prepared with concentration of 0.077 g/L. Solution volume was 0.6 mL.
6
ACCEPTED MANUSCRIPT All NMR experiments were performed on a Bruker Avance II-500 NMR spectrometer equipped with a 5 mm probe using standard Bruker TOPSPIN Software. Temperature control was performed using a Bruker variable temperature unit (BVT-2000) in combination with a Bruker cooling unit (BCU05) to provide chilled air. Experiments were performed at 298 K without sample spinning. 1
H NMR (500 MHz) spectra were recorded using 90° pulses and relaxation
delay of 1 s; spectral width was 12.02 ppm; 128 scans were acquired.
13
C NMR
spectra were recorded using 45° pulse sequence with power-gated decoupling for suppression of the scalar couplings with protons. 13C NMR spectra were acquired with relaxation delay of 1 s; spectral width was 236 ppm; 1024 scans were acquired. Total correlation spectroscopy (TOCSY) [16], one dimensional gradient homonuclear selective total correlation spectroscopy (1D ge-TOCSY) [17], heteronuclear multiple-bond correlation (HMBC) [18], and gradient heteronuclear single-quantum coherence (ge-HSQC) [19] were used to assign signals in 1H and 13
C NMR spectra. Two-dimensional nuclear Overhauser effect spectroscopy (2D ge-NOESY)
[20] experiments were performed with pulsed filtered gradient techniques [21]. The spectra were recorded in a phase-sensitive mode with 2048 points in the F2 direction and 512 points in the F1 direction. Mixing time values were 0.30, 0.50, 0.70, and 0.90 s. The spectra were acquired with 24 scans and relaxation delay of 2 s. Selective one-dimensional nuclear Overhauser effect spectroscopy (ge-1D NOESY) experiments were carried out by using the double-pulse field gradient spin-echo NOE (1D-DPFGSENOE) [22] pulse sequence. The 1D NOESY spectra were acquired with 64k data points, 160 scans, 12-ppm spectral width, relaxation delay of 2 s, acquisition time of 5.45 s, and mixing times of 0.3, 0.5, 0.7, and 0.9 s. The pulse programs for all NMR experiments were taken from the Bruker software library.
Results and discussion
The 1H NMR data were completely assigned by Yiu and Knaus [23], but H-2a and H-6a were not distinguished. In addition to [23], the 13C NMR data were 7
ACCEPTED MANUSCRIPT also presented in [24]. We performed a proper assignment of lines in the 1H and 13
C NMR spectra with the aid of experiments HMBC, HSQC, and TOCSY; only
insignificant differences in results compared to [24] were found. The list of atomic coordinates for the various conformations of felodipine from supplementary data [8] enabled us to calculate the effective distances required for comparison with experiment (calculated distances are presented in Tables 1–4). It was shown [14, 25] that the calculation of the effective distance between spin systems containing no methyl protons can be carried out using simple averaging formula (6), whereas the presence of methyl protons requires the application of Eq. (8) due to the fast internal rotation of the methyl group. Calculations reported in [8] refer to an isolated felodipine molecule rather than solution in DMSO. We suppose, however, that it is reasonable to assume that spatial structures of the conformers do not alter when solvent is changed, and that solvent influences only the conformational equilibrium (that is, choice of the solvent determines relative fractions of the conformational states of the dissolved compound). Effective distances shown in tables as calibration distances were chosen because of their equality for all conformations of felodipine. It should be noted that the distance between aromatic protons should not be taken for the purpose of calibration due to artifacts observed in the NOE intensities [2]. Results of 2D NOE experiments are given in Table 1. Evidently, calculated values of the distances for conformations 1, 2, and 5 match the experimental data quite well, while for the conformations 3, 4, and 6 agreement with experiment is very poor. The experimental value of rij for the pair of protons 4-6’ lies from 2.12 Å (conformations 1, 2, 5) to 3.73 Å (conformations 3, 4, 6), while the difference between 2.21 Å and 2.12 Å lies beyond the experimental error (~3%). In fact, 2D NOE experiments show that at the employed concentration conformations 1, 2, and 5 prevail, although some of the conformations 3, 4, and 6 are also present. Results of the 1D NOE experiment are in favor of the existence of a significant amount of conformations 3, 4, and 6: note the agreement between the experimental and calculated rij values for protons NH-6’ (Table 2).
Table 1. Distances based on 2D NOESY of felodipine in DMSO
8
ACCEPTED MANUSCRIPT Atomic
Experimental
group
distance Å
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
3b-3c
calibration
2.69
2.69
2.69
2.69
2.69
2.69
4-6’
2.21
2.12
2.12
3.73
3.73
2.12
3.73
Calculated distances Å
Table 2. Analysis of 1D NOE spectra with selective irradiation of NH proton
Calculated distances Å
Atomic
Experimental
group
distance Å
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
NH-2a
calibration
2.48
2.48
2.48
2.48
2.48
2.48
NH-6a
2.50 ± 0.08
2.48
2.48
2.48
2.48
2.48
2.48
NH-6’
3.44 ± 0.06
-
-
3.50
3.48
-
3.37
Table 3. Analysis of 1D NOE spectra with selective irradiation of proton 3c
Atomic
Experimental
group
distance A
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
3b-3c
calibration
2.69
2.69
2.69
2.69
2.69
2.69
4-3c
4.49±0.18
4.92
4.50
-
4.98
4.13
4.08
Calculated distances Å
Table 4. Analysis of 1D NOE spectra with selective irradiation of proton 4
Calculated distances Å
Atomic
Experimental
group
distance A
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
4-6’
calibration
2.70
2.70
2.70
2.70
2.70
2.70
4-3b
4.08±0.09
4.13
4.24
4.24
-
4.33
4.13
4-5b
4.20±0.03
4.20
3.92
4.19
4.19
4.15
4.17
4-3c
4.33±0.17
4.92
4.50
-
4.98
4.13
4.08
Here, results for proton groups only confirm the correctness of calibration. Corresponding distances for the conformations 1, 2, and 5 are too large for NOE (>6 Å) and not given in Table 2. The data presented in Tables 3 and 4 refer to distances greater than 4 Å. In our opinion, comparison of experimental and calculated data only shows that the conformation 5 cannot be dominant. Allowing for the very strong dependency of the cross relaxation rate on the distance (σ~1/r6), we have come to the conclusion 9
ACCEPTED MANUSCRIPT that the population level, which corresponds to the conformation 5, should be substantially less than the populations levels of 1 and 2 (Table 3), since the effective distance for conformation 5 (4,13 Å) is substantially less than the experimental value (4,49 Å). This result is in agreement with theoretical [8] calculations which show that the difference between the energies of formation of conformations 1 and 2, on the one hand, and 5, on the other hand, is quite large (~2 kJ/mol).
Conclusions
Thus, the results of 1D and 2D NOE studies of felodipine in DMSO solution (0.077 g/L) show the dominant presence of felodipine conformations in which chlorine atoms are closer to the NH1 proton. However, the existence of a certain amount of conformations in which chlorine atoms point to the opposite direction is clear. We were not able to make quantitative evaluation of populations of the felodipine conformations in DMSO because of insufficient accuracy in the range of relatively large distances (~4 Å). Unfortunately, in our case, for each conformation we have only one distance shorter than 4 Å (if not count r(4-5b) = 3.92 Å for conformation 2 in Table 4). It is well known that errors in measuring distances by NOE in this range (>4 Å) increase significantly, mainly due to spin diffusion. Therefore the problem of the suppression of spin diffusion in NOE measurements of interproton distances for small molecules is very important. Acknowledgements. This work was performed under the auspices of the Marie Curie International Research Staff Exchange Scheme PIRSES-GA-2009-247500. I.A.K. expresses his thanks for Prof. S. Berger for fruitful discussion. Financial support was from the Russian Foundation for Basic Research, project no. 12-03-31001_mol_a.
References
[1]
C.P. Butts, C.R. Jones, E.C. Towers, J.L. Flynn, L. Appleby, and N.J. Barron, Org. Biomol. Chem., 9 (2011) 177.
[2]
C.R. Jones, C.P. Butts, and J.N. Harvey, Beilstein J. Org. Chem., 7 (2011) 145.
10
ACCEPTED MANUSCRIPT [3]
M.G. Chini, C.R. Jones, A. Zampella, M.V.D. Auria, B. Renga, S. Fiorucci, C.P. Butts, G. Bifulco, M. Clinica, U. Perugia, N. Facolta, V. Gerardo, and D.S. Andrea, J. Org. Chem., 77 (2012) 1489.
[4]
B. Edgar, P. Collste, K. Haglund, and C.G. Regardh, Clin. Invest. Med., 10 (1987) 388.
[5]
S. Srčič, J. Kerč, U. Urleb, I. Zupančič, G. Lahajnar, B. Kofler, and J. Šmid-Korbar, Int. J. Pharm., 87 (1992) 1.
[6]
J.M. Rollinger and A. Burger, J. Pharm. Sci., 90 (2011) 949.
[7]
A.O. Surov, K.A. Solanko, A.D. Bond, G.L. Perlovich, and A. BauerBrandl, Cryst. Growth & Design, 12 (2012) 4022.
[8]
V.I. Teberekidis and M.P. Sigalas, J. Mol. Struc.: THEOCHEM, 803 (2007) 29.
[9]
C.P. Butts, C.R. Jones, Z. Song, and T.J. Simpson, Chem. Commun., 90 (2012) 9023.
[10] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.N. Greenblatt, E.C. Meng, and T.E. Ferrin, J. Comput. Chem., 25 (2003) 1605. [11]
J. Fejzo, Z. Zolnai, S. Macura, and J.L. Markley, J. Magn. Reson., 82 (1989) 518.
[12]
T.A. Gadiev, B.I. Khairutdinov, I.S. Antipin, and V.V. Klochkov, Appl. Magn. Reson., 30 (2006) 165.
[13]
M.P. Williamson and D. Neuhaus, J. Magn. Reson., 72 (1987) 369.
[14]
C.M. Fletcher, D.N.M. Jones, R. Diamond, and D. Neuhaus, J. Biomol. NMR, 8 (1996) 292.
[15]
J. Tropp, J. Chem. Phys., 72 (1980) 6035.
[16]
A.D. Bax and D.G. Davis, J. Magn. Reson., 65 (1985) 355.
[17]
K. Stott, J. Stonehouse, J. Keeler, T.-L. Hwang, and A. J. Shaka, J. Am. Chem. Soc., 117 (1995) 4199.
[18]
A. Bax and M.F. Summers, J. Am. Chem. Soc., 108 (1986) 2093.
[19]
J. Schleucher, M. Schwendinger, M. Sattler, P. Schmidt, O. Schedletzky, S. J. Glaser, O. W. Sørensen, and C. Griesinger, J. Biomol. NMR, 4 (1994) 301.
[20]
M.J. Thrippleton and J. Keeler, Angew. Chem. Int. Ed., 42 (2003) 3938.
[21]
R. Wagner and S. Berger, J. Magn. Reson., 123 (1996) 119.
[22]
K. Stott, J. Keeler, Q.N. Van, and A. J. Shaka, J. Magn. Reson. 125 (1997) 302.
[23]
S. Yiu and E.E. Knaus, Org. Prepar. Proc. Int., 28 (1996) 91. 11
ACCEPTED MANUSCRIPT [24]
J. Jung, L. Kim, C.S. Lee, Y. Hwan, S. Ahn, and Y. Lim, Magn. Reson. Chem., 39 (2001) 406.
[25]
S. Macura and R.R. Ernst, Mol. Phys., 100 (2002) 135.
Table 1. Distances based on 2D NOESY of felodipine in DMSO
Calculated distances Å
Atomic
Experimental
group
distance Å
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
3b-3c
calibration
2.69
2.69
2.69
2.69
2.69
2.69
4-6’
2.21
2.12
2.12
3.73
3.73
2.12
3.73
Table 2. Analysis of 1D NOE spectra with selective irradiation of NH proton
Calculated distances Å
Atomic
Experimental
group
distance Å
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
NH-2a
calibration
2.48
2.48
2.48
2.48
2.48
2.48
NH-6a
2.50 ± 0.08
2.48
2.48
2.48
2.48
2.48
2.48
NH-6’
3.44 ± 0.06
-
-
3.50
3.48
-
3.37
Table 3. Analysis of 1D NOE spectra with selective irradiation of proton 3c
Calculated distances Å
Atomic
Experimental
group
distance A
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
3b-3c
calibration
2.69
2.69
2.69
2.69
2.69
2.69
4-3c
4.49±0.18
4.92
4.50
-
4.98
4.13
4.08
Table 4. Analysis of 1D NOE spectra with selective irradiation of proton 4
Calculated distances Å
Atomic
Experimental
group
distance A
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
4-6’
calibration
2.70
2.70
2.70
2.70
2.70
2.70
4-3b
4.08±0.09
4.13
4.24
4.24
-
4.33
4.13
4-5b
4.20±0.03
4.20
3.92
4.19
4.19
4.15
4.17
4-3c
4.33±0.17
4.92
4.50
-
4.98
4.13
4.08
12
ACCEPTED MANUSCRIPT Highlights
Internuclear distances in felodipine were obtained by analysis of 1D and 2D NOE data. Obtained effective distances were compared with quantum-chemical calculations. Fractions of different conformers of felodipine dissolved in DMSO were estimated.
13
S0022-2860(12)01081-2 http://dx.doi.org/10.1016/j.molstruc.2012.11.040 MOLSTR 19377
To appear in:
Journal of Molecular Structure
Received Date: Revised Date: Accepted Date:
23 October 2012 8 November 2012 19 November 2012
Please cite this article as: I.A. Khodov, M.Yu. Nikiforov, G.A. Alper, D.S. Blokhin, S.V. Efimov, V.V. Klochkov, N. Georgi, Spatial structure of felodipine dissolved in DMSO by 1D NOE and 2D NOESY NMR spectroscopy, Journal of Molecular Structure (2012), doi: http://dx.doi.org/10.1016/j.molstruc.2012.11.040
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Spatial structure of felodipine dissolved in DMSO by 1D NOE and 2D NOESY NMR spectroscopy. I. A. Khodov a*, M. Yu. Nikiforov a, G. A. Alper a, D. S. Blokhin b, S. V. Efimov b, V. V. Klochkov b, N. Georgi c a
G.A. Krestov Institute of Solution Chemistry of the Russian Academy of
Sciences, ul. Akademicheskaya 1, 153045 Ivanovo, Russia b
Kazan Federal University, ul. Kremlevskaya 18, 420008 Kazan, Russia
c
Max Planck Institute for Mathematics in the Sciences, Inselstraße 22, 04103
Leipzig, Germany *
Corresponding author: [email protected]
Abstract Small organic molecules in dissolved state exist as an ensemble of conformers. In this work conformation of felodipine in dimethyl sulphoxide was studied and dominant stable conformers were determined. Effective interatomic distances were obtained by means of NOE spectroscopy. Fractions of different conformers were estimated by comparing effective distances and those obtained from quantum-chemical calculation [Teberekidis, 2007]; averaging of distances was made following the N-site jump model.
Highlights Internuclear distances in felodipine were obtained by analysis of 1D and 2D NOE data. Obtained effective distances were compared with quantum-chemical calculations. Fractions of different conformers of felodipine dissolved in DMSO were estimated.
Keywords Conformational analysis; Nuclear magnetic resonance; NOE; Jump model. 1
ACCEPTED MANUSCRIPT Introduction Accurate determination of interproton distances in relatively small, flexible molecules by 1D and 2D NOE NMR spectroscopy is the subject of increasing interest in recent years [1–3]. Results of such investigations can be applied to establishing conformational details for biologically active molecules in solutions. It is well known that polymorphism of drug compounds affects the biological activity and plays an important role in the production of pharmaceuticals. In turn, the properties of polymorphs are due to molecular structure of compounds and their ability to exist in different conformational forms in the solvent from which recrystallization is performed. Therefore, search for new polymorphic forms of drugs is closely associated with the study of conformational states of biologically active molecules in solutions. Felodipine (ethylmethyl-4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl3,5-pyridine-dicarboxylate) is widely used as antihypertensive and antianginal drug [4]. The possible existence of different polymorphic forms connected with conformations of the phenyl ring was predicted in [5]. Commercial felodipine has the melting point of 144°C and is a racemic mixture of enantiomers [6]. Some researches point to existence of three or more crystal structures [5, 7]. Conformational flexibility of felodipine is determined by two dihedral angles: (C3-C4-C1’-C2’), which defines the rotation of 2,4-dichlorophenyl group around C1’-C4 bond, and (C3a-O-C3b-C3c), related with the rotation of ethyl group around O-3b bond (Fig. 1). Teberekidis and Sigalas in 2007 published the result of a theoretical study of felodipine using a hybrid density functional method B3LYP [8]. They revealed six conformers with very close energies (within 4,2 kJ/mol), starting from number 1 (the global minimum) up to number 6 (with the maximal energy). It is important for the purposes of further discussion, that in the conformers 1, 2, and 5 chlorine atoms point to the same direction as NH1 proton, whereas in the conformers 3, 4, and 6 the dichlorophenyl ring is turned by 180° and chlorine atoms are closer to the H4 proton (Figs. 2, 3). Obvious progress was made recently in determining interproton distances from NOE data. Butts et al. [9] gave a convincing example of the fundamental possibility for quantitative determination of conformations of flexible molecules by NOE. This success is due to the fact that only five short interproton distances (<3 Å) are present in the molecule of arugosin C, which can be determined with a 2
ACCEPTED MANUSCRIPT high accuracy. The question remains, however, how effective are conventional NOE experiments for analysis of an arbitrary small molecule in a given solvent. In this work, conventional methods of 1D and 2D NOESY were employed to study the conformational state of felodipine in DMSO. The choice of this solvent is associated, in particular, with high solubility of felodipine in DMSO.
Fig. 1. Structure and atom nomenclature of felodipine. Arrows indicate rotations responsible for the diversity of conformations.
Fig. 2. Conformations 1, 2, and 5 obtained by quantum-chemical calculations [8]. Molecular structures are depicted in the Chimera software [10].
3
ACCEPTED MANUSCRIPT
Fig. 3. Conformations 3, 4, and 6 from [8], differing from 1, 2, and 5 by orientation of the dichlorophenyl ring.
Formulation of the Problem NOE spectroscopy is a powerful tool of studying the spatial structure and conformations of molecules in solution. The method of estimating internuclear distances for different proton pairs in a molecule is based on the existence of a strong dependence of the cross-relaxation rate constant σij on the distance rij between the interacting nuclear spins. Normally, such a dependency is approximated by the simple formula σ ij ~ 1 rij6 , and then internuclear distances are obtained according to the expression
⎛σ rij = r0 ⎜ 0 ⎜ ⎝ σ ij
1
⎞6 ⎟ , ⎟ ⎠
(1)
where r0 and σ0 refer to the proton pair chosen for calibration; the distance r0 is supposed to be known from some independent source (e.g., quantum chemical calculations). However, for small, conformationally flexible molecules, of great importance is the problem of more accurate determination of internuclear distances in order to obtain information on relative population of various conformers. This goal may be obtained by both a more accurate interpretation of the 2D NOESY experiment and taking into account anisotropic molecular rotation. A more advanced approach to calculate distances rij was developed in [11– 13] based on relative integral intensities; cross-peak intensities aij are normalized by the intensity of corresponding diagonal peaks. The matrix of integral intensities A with elements aij from 2D NOE spectrum is related to the mixing time τm by the formula 4
ACCEPTED MANUSCRIPT A(τ m ) = exp(− Rτ m ) ,
(2)
where R is the cross-relaxation matrix of the form
⎛ ρ11 σ 12 ⎜ ⎜ σ 21 ρ 21 R=⎜ ⎜ ⎜ σ σ s2 s1 ⎝
σ 1s ⎞
⎟ ⎟ ⎟. ⎟ ρ ss ⎟⎠
(1)
Here matrix elements ρij and σij correspond to the longitudinal and crossrelaxation rates, respectively:
ρ ij =
2π 4 2 γ ∑ J ij0 (ω ) + J ij1 (ω ) + J ij2 (ω ) , 5 j ≠i
(2)
2π 4 2 γ ∑ + 6 J ij2 (ω ) − J ij0 (ω ) . 5 j ≠i
(3)
[
σ ij =
]
[
]
J(ω) is the spectral density function, which for the case of isotropic tumbling of the molecule has the form
Jijm (ω) =
⎤ 1 ⎡ τc ⎢ ⎥. 4π rij6 ⎣1+ m2ω2τ c2 ⎦
(4)
Here τc is correlation time, which is on the order of 10-10 s for small molecules; m stands for the order of relaxation transitions (0, 1, or 2). As was shown in [11–13], where series expansion into powers of exp(-Rτm) is used, dependency of the averaged integral intensity Ī on the mixing time τm I (τ m ) =
1 ⎛⎜ 1 aij (τ m ) 1 a ji (τ m ) ⎞⎟ + 2 ⎝⎜ n j aii (τ m ) ni a jj (τ m ) ⎟⎠
(5)
may with a high degree of accuracy be considered linear with the τm values much higher than those employed usually in the initial rate approximation. Having recorded several 2D NOE spectra with various τm values, one may plot the dependence Ī(τm) and then determine more accurately the values of σij and, applying Eq. (1), the distances rij.
5
ACCEPTED MANUSCRIPT Another factor that should be taken into account when accurately determining internuclear distances is the anisotropic molecular rotation. When considering the interactions of two groups of equivalent spins I and S (each containing nI and nS spins), the effective distance reffij, obtained from an experiment, should be understood as denoting the average over all possible i–j distances. For the isotropic rotation, in view of Eqs. (1) and (6), we have [14]
rijeff
⎡ 1 =⎢ ⎣⎢ nI nS
−
1
1⎤ 6 ∑ij r 6 ⎥ . ⎥ ij ⎦
(6)
Anisotropy of the molecular rotation makes direct use of the formula (6) incorrect; this expression is applicable to the case of isotropic rotation only. Tropp [15] obtained some general relations for the spectral density function J(ω) when internal molecular rotation is taken into account. In particular, for the fast rotation of the methyl group in the slope of the three-site jump model, the expression for J(ω) has the form
τc 1 Jijm (ω) = 5 1+ m2ω2τ c2
(
(
)
)
2
i i ,ϕmol 1 3 Y2k θmol . ∑ ∑ r3 k=−2 3 N=1 ij 2
(7)
Here Y2k are second rank spherical harmonics; θ and φ are polar coordinates of internuclear vectors connecting each nucleus pair IS subject to averaging. Therefore, when considering the interaction of one spin I and a group of equivalent spins S in the methyl group, we obtain the following relation for the effective distance
rijeff
(
)
⎡1 2 1 3 Y θ i ϕ i ⎤ = ⎢ ∑ ∑ 2 k mol3 mol ⎥ rij ⎢⎣ 5 k =−2 3 N =1 ⎦⎥
−
1 6
(8)
Experimental
Felodipine and deuterated DMSO were purchased from Aldrich and used without further purification. Samples were prepared with concentration of 0.077 g/L. Solution volume was 0.6 mL.
6
ACCEPTED MANUSCRIPT All NMR experiments were performed on a Bruker Avance II-500 NMR spectrometer equipped with a 5 mm probe using standard Bruker TOPSPIN Software. Temperature control was performed using a Bruker variable temperature unit (BVT-2000) in combination with a Bruker cooling unit (BCU05) to provide chilled air. Experiments were performed at 298 K without sample spinning. 1
H NMR (500 MHz) spectra were recorded using 90° pulses and relaxation
delay of 1 s; spectral width was 12.02 ppm; 128 scans were acquired.
13
C NMR
spectra were recorded using 45° pulse sequence with power-gated decoupling for suppression of the scalar couplings with protons. 13C NMR spectra were acquired with relaxation delay of 1 s; spectral width was 236 ppm; 1024 scans were acquired. Total correlation spectroscopy (TOCSY) [16], one dimensional gradient homonuclear selective total correlation spectroscopy (1D ge-TOCSY) [17], heteronuclear multiple-bond correlation (HMBC) [18], and gradient heteronuclear single-quantum coherence (ge-HSQC) [19] were used to assign signals in 1H and 13
C NMR spectra. Two-dimensional nuclear Overhauser effect spectroscopy (2D ge-NOESY)
[20] experiments were performed with pulsed filtered gradient techniques [21]. The spectra were recorded in a phase-sensitive mode with 2048 points in the F2 direction and 512 points in the F1 direction. Mixing time values were 0.30, 0.50, 0.70, and 0.90 s. The spectra were acquired with 24 scans and relaxation delay of 2 s. Selective one-dimensional nuclear Overhauser effect spectroscopy (ge-1D NOESY) experiments were carried out by using the double-pulse field gradient spin-echo NOE (1D-DPFGSENOE) [22] pulse sequence. The 1D NOESY spectra were acquired with 64k data points, 160 scans, 12-ppm spectral width, relaxation delay of 2 s, acquisition time of 5.45 s, and mixing times of 0.3, 0.5, 0.7, and 0.9 s. The pulse programs for all NMR experiments were taken from the Bruker software library.
Results and discussion
The 1H NMR data were completely assigned by Yiu and Knaus [23], but H-2a and H-6a were not distinguished. In addition to [23], the 13C NMR data were 7
ACCEPTED MANUSCRIPT also presented in [24]. We performed a proper assignment of lines in the 1H and 13
C NMR spectra with the aid of experiments HMBC, HSQC, and TOCSY; only
insignificant differences in results compared to [24] were found. The list of atomic coordinates for the various conformations of felodipine from supplementary data [8] enabled us to calculate the effective distances required for comparison with experiment (calculated distances are presented in Tables 1–4). It was shown [14, 25] that the calculation of the effective distance between spin systems containing no methyl protons can be carried out using simple averaging formula (6), whereas the presence of methyl protons requires the application of Eq. (8) due to the fast internal rotation of the methyl group. Calculations reported in [8] refer to an isolated felodipine molecule rather than solution in DMSO. We suppose, however, that it is reasonable to assume that spatial structures of the conformers do not alter when solvent is changed, and that solvent influences only the conformational equilibrium (that is, choice of the solvent determines relative fractions of the conformational states of the dissolved compound). Effective distances shown in tables as calibration distances were chosen because of their equality for all conformations of felodipine. It should be noted that the distance between aromatic protons should not be taken for the purpose of calibration due to artifacts observed in the NOE intensities [2]. Results of 2D NOE experiments are given in Table 1. Evidently, calculated values of the distances for conformations 1, 2, and 5 match the experimental data quite well, while for the conformations 3, 4, and 6 agreement with experiment is very poor. The experimental value of rij for the pair of protons 4-6’ lies from 2.12 Å (conformations 1, 2, 5) to 3.73 Å (conformations 3, 4, 6), while the difference between 2.21 Å and 2.12 Å lies beyond the experimental error (~3%). In fact, 2D NOE experiments show that at the employed concentration conformations 1, 2, and 5 prevail, although some of the conformations 3, 4, and 6 are also present. Results of the 1D NOE experiment are in favor of the existence of a significant amount of conformations 3, 4, and 6: note the agreement between the experimental and calculated rij values for protons NH-6’ (Table 2).
Table 1. Distances based on 2D NOESY of felodipine in DMSO
8
ACCEPTED MANUSCRIPT Atomic
Experimental
group
distance Å
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
3b-3c
calibration
2.69
2.69
2.69
2.69
2.69
2.69
4-6’
2.21
2.12
2.12
3.73
3.73
2.12
3.73
Calculated distances Å
Table 2. Analysis of 1D NOE spectra with selective irradiation of NH proton
Calculated distances Å
Atomic
Experimental
group
distance Å
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
NH-2a
calibration
2.48
2.48
2.48
2.48
2.48
2.48
NH-6a
2.50 ± 0.08
2.48
2.48
2.48
2.48
2.48
2.48
NH-6’
3.44 ± 0.06
-
-
3.50
3.48
-
3.37
Table 3. Analysis of 1D NOE spectra with selective irradiation of proton 3c
Atomic
Experimental
group
distance A
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
3b-3c
calibration
2.69
2.69
2.69
2.69
2.69
2.69
4-3c
4.49±0.18
4.92
4.50
-
4.98
4.13
4.08
Calculated distances Å
Table 4. Analysis of 1D NOE spectra with selective irradiation of proton 4
Calculated distances Å
Atomic
Experimental
group
distance A
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
4-6’
calibration
2.70
2.70
2.70
2.70
2.70
2.70
4-3b
4.08±0.09
4.13
4.24
4.24
-
4.33
4.13
4-5b
4.20±0.03
4.20
3.92
4.19
4.19
4.15
4.17
4-3c
4.33±0.17
4.92
4.50
-
4.98
4.13
4.08
Here, results for proton groups only confirm the correctness of calibration. Corresponding distances for the conformations 1, 2, and 5 are too large for NOE (>6 Å) and not given in Table 2. The data presented in Tables 3 and 4 refer to distances greater than 4 Å. In our opinion, comparison of experimental and calculated data only shows that the conformation 5 cannot be dominant. Allowing for the very strong dependency of the cross relaxation rate on the distance (σ~1/r6), we have come to the conclusion 9
ACCEPTED MANUSCRIPT that the population level, which corresponds to the conformation 5, should be substantially less than the populations levels of 1 and 2 (Table 3), since the effective distance for conformation 5 (4,13 Å) is substantially less than the experimental value (4,49 Å). This result is in agreement with theoretical [8] calculations which show that the difference between the energies of formation of conformations 1 and 2, on the one hand, and 5, on the other hand, is quite large (~2 kJ/mol).
Conclusions
Thus, the results of 1D and 2D NOE studies of felodipine in DMSO solution (0.077 g/L) show the dominant presence of felodipine conformations in which chlorine atoms are closer to the NH1 proton. However, the existence of a certain amount of conformations in which chlorine atoms point to the opposite direction is clear. We were not able to make quantitative evaluation of populations of the felodipine conformations in DMSO because of insufficient accuracy in the range of relatively large distances (~4 Å). Unfortunately, in our case, for each conformation we have only one distance shorter than 4 Å (if not count r(4-5b) = 3.92 Å for conformation 2 in Table 4). It is well known that errors in measuring distances by NOE in this range (>4 Å) increase significantly, mainly due to spin diffusion. Therefore the problem of the suppression of spin diffusion in NOE measurements of interproton distances for small molecules is very important. Acknowledgements. This work was performed under the auspices of the Marie Curie International Research Staff Exchange Scheme PIRSES-GA-2009-247500. I.A.K. expresses his thanks for Prof. S. Berger for fruitful discussion. Financial support was from the Russian Foundation for Basic Research, project no. 12-03-31001_mol_a.
References
[1]
C.P. Butts, C.R. Jones, E.C. Towers, J.L. Flynn, L. Appleby, and N.J. Barron, Org. Biomol. Chem., 9 (2011) 177.
[2]
C.R. Jones, C.P. Butts, and J.N. Harvey, Beilstein J. Org. Chem., 7 (2011) 145.
10
ACCEPTED MANUSCRIPT [3]
M.G. Chini, C.R. Jones, A. Zampella, M.V.D. Auria, B. Renga, S. Fiorucci, C.P. Butts, G. Bifulco, M. Clinica, U. Perugia, N. Facolta, V. Gerardo, and D.S. Andrea, J. Org. Chem., 77 (2012) 1489.
[4]
B. Edgar, P. Collste, K. Haglund, and C.G. Regardh, Clin. Invest. Med., 10 (1987) 388.
[5]
S. Srčič, J. Kerč, U. Urleb, I. Zupančič, G. Lahajnar, B. Kofler, and J. Šmid-Korbar, Int. J. Pharm., 87 (1992) 1.
[6]
J.M. Rollinger and A. Burger, J. Pharm. Sci., 90 (2011) 949.
[7]
A.O. Surov, K.A. Solanko, A.D. Bond, G.L. Perlovich, and A. BauerBrandl, Cryst. Growth & Design, 12 (2012) 4022.
[8]
V.I. Teberekidis and M.P. Sigalas, J. Mol. Struc.: THEOCHEM, 803 (2007) 29.
[9]
C.P. Butts, C.R. Jones, Z. Song, and T.J. Simpson, Chem. Commun., 90 (2012) 9023.
[10] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.N. Greenblatt, E.C. Meng, and T.E. Ferrin, J. Comput. Chem., 25 (2003) 1605. [11]
J. Fejzo, Z. Zolnai, S. Macura, and J.L. Markley, J. Magn. Reson., 82 (1989) 518.
[12]
T.A. Gadiev, B.I. Khairutdinov, I.S. Antipin, and V.V. Klochkov, Appl. Magn. Reson., 30 (2006) 165.
[13]
M.P. Williamson and D. Neuhaus, J. Magn. Reson., 72 (1987) 369.
[14]
C.M. Fletcher, D.N.M. Jones, R. Diamond, and D. Neuhaus, J. Biomol. NMR, 8 (1996) 292.
[15]
J. Tropp, J. Chem. Phys., 72 (1980) 6035.
[16]
A.D. Bax and D.G. Davis, J. Magn. Reson., 65 (1985) 355.
[17]
K. Stott, J. Stonehouse, J. Keeler, T.-L. Hwang, and A. J. Shaka, J. Am. Chem. Soc., 117 (1995) 4199.
[18]
A. Bax and M.F. Summers, J. Am. Chem. Soc., 108 (1986) 2093.
[19]
J. Schleucher, M. Schwendinger, M. Sattler, P. Schmidt, O. Schedletzky, S. J. Glaser, O. W. Sørensen, and C. Griesinger, J. Biomol. NMR, 4 (1994) 301.
[20]
M.J. Thrippleton and J. Keeler, Angew. Chem. Int. Ed., 42 (2003) 3938.
[21]
R. Wagner and S. Berger, J. Magn. Reson., 123 (1996) 119.
[22]
K. Stott, J. Keeler, Q.N. Van, and A. J. Shaka, J. Magn. Reson. 125 (1997) 302.
[23]
S. Yiu and E.E. Knaus, Org. Prepar. Proc. Int., 28 (1996) 91. 11
ACCEPTED MANUSCRIPT [24]
J. Jung, L. Kim, C.S. Lee, Y. Hwan, S. Ahn, and Y. Lim, Magn. Reson. Chem., 39 (2001) 406.
[25]
S. Macura and R.R. Ernst, Mol. Phys., 100 (2002) 135.
Table 1. Distances based on 2D NOESY of felodipine in DMSO
Calculated distances Å
Atomic
Experimental
group
distance Å
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
3b-3c
calibration
2.69
2.69
2.69
2.69
2.69
2.69
4-6’
2.21
2.12
2.12
3.73
3.73
2.12
3.73
Table 2. Analysis of 1D NOE spectra with selective irradiation of NH proton
Calculated distances Å
Atomic
Experimental
group
distance Å
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
NH-2a
calibration
2.48
2.48
2.48
2.48
2.48
2.48
NH-6a
2.50 ± 0.08
2.48
2.48
2.48
2.48
2.48
2.48
NH-6’
3.44 ± 0.06
-
-
3.50
3.48
-
3.37
Table 3. Analysis of 1D NOE spectra with selective irradiation of proton 3c
Calculated distances Å
Atomic
Experimental
group
distance A
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
3b-3c
calibration
2.69
2.69
2.69
2.69
2.69
2.69
4-3c
4.49±0.18
4.92
4.50
-
4.98
4.13
4.08
Table 4. Analysis of 1D NOE spectra with selective irradiation of proton 4
Calculated distances Å
Atomic
Experimental
group
distance A
Conf 1
Conf 2
Conf 3
Conf 4
Conf 5
Conf 6
4-6’
calibration
2.70
2.70
2.70
2.70
2.70
2.70
4-3b
4.08±0.09
4.13
4.24
4.24
-
4.33
4.13
4-5b
4.20±0.03
4.20
3.92
4.19
4.19
4.15
4.17
4-3c
4.33±0.17
4.92
4.50
-
4.98
4.13
4.08
12
ACCEPTED MANUSCRIPT Highlights
Internuclear distances in felodipine were obtained by analysis of 1D and 2D NOE data. Obtained effective distances were compared with quantum-chemical calculations. Fractions of different conformers of felodipine dissolved in DMSO were estimated.
13