The conformation of the naproxen anion studied by 1H NMR and theoretical methods

Journal of Molecular Structure 559 (2001) 369–377 www.elsevier.nl/locate/molstruc

The conformation of the naproxen anion studied by 1H NMR and theoretical methods E. Bednarek a,b, W. Bocian a, J.Cz. Dobrowolski a,b, L. Kozerski a, N. Sadlej-Sosnowska a,*, J. Sitkowski a b

a Drug Institute, 30/34 Chełmska Street, 00-725 Warsaw, Poland Industrial Chemistry Research Institute, 8 Rydygiera Street, 01-793 Warsaw, Poland

Received 26 June 2000; accepted 18 August 2000

Abstract The conformation of naproxen and the importance of intramolecular hydrogen-bond-like (HBL) C–H···O–C interactions was studied by means of the ab initio HF/6-31G ⴱ, HF/3-21G ⴱ and semiempirical AM1 methods. Molecular structure optimisation including rotations around single bonds of naphthalene substituents, allowed us to find four stable conformers for which HBL interaction plays an important role. For all the four conformers the methoxy group is coplanar with the naphthalene ring. Conformational equilibrium related to the internal rotations was also studied by 1H NMR spectroscopy. The experimental NOE values as well as coupling constants 3J(C,H) were compared with the calculated values. 䉷 2001 Elsevier Science B.V. All rights reserved. Keywords: Ab initio; AM1; Anion; Conformation; 1H NMR; NOE; Naproxen; Semiempirical

1. Introduction Naproxen, (⫹)-6-methoxy-a-methyl-2-naphthalene acetic acid and its sodium salt, are non-steroidal antiinflammatory drugs. The two substituents at the naproxen naphthalene ring, i.e. the methoxy group at position C6 and the amethyl acetic group (CH(CH3)COO–) at position C2, are flexible. Two-dimensional 1H NMR spectra of naproxen have revealed that the interactions between the protons of a substituent and the nearest protons of the naphthalene ring are non-equivalent [1]. This holds true especially for the methoxy group. There* Corresponding author. Tel.: ⫹48-22-8-41-2940; fax: ⫹48-22-841-0652. E-mail address: [email protected] (N. Sadlej-Sosnowska).

fore, the NMR results prompted us to scan the naproxen potential energy surface generated by rotations of the substituents. Naproxen is a weak acid …pKa ˆ 4:2 [2]); at the neutral pH, it exists mainly in the anionic form and for that reason we performed our calculations for the anionic form of the molecule. It is known that in the absence of overriding steric effects from substituents on adjacent ring positions, a methoxy group attached to an aromatic ring adopts a conformation with its heavy atoms in the ring plane [3–9]. The stability of this coplanar conformation is accepted to be mainly defined by the conjugation between the oxygen in-plane lone pairs and the aromatic p system [9]. If the system is not symmetrical with respect to the methoxy Cipso –Cpara axis, both coplanar conformers are generally not equally populated [7,10,11]. In this

0022-2860/01/$ - see front matter 䉷 2001 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(00)00714-6

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was explained by the through-space interaction of the methyl group with the p system [12]. The aim of the present study is to find the reasons for non-equivalence of the interactions of the substituent protons with the nearest protons of the naphthalene ring as well as the conformational composition in naproxen molecules by means of 1H NMR, semiempirical, and ab initio studies. Scheme 1 — definition of internal coordinates for rotation around C2–C2 0 , C2 0 –C1 0 , and C6–O; bonds: dihedral angle C1–C2–C2 0 –C1 0 ˆ w; C2–C2 0 –C1 0 –O ˆ c; C5–C6–O–C4 0 ˆ x:†

2. Methods 2.1. Calculations

case, the s-cis orientation of the methoxy group to the aromatic C–C bond with the larger p bond order is preferred [7,11]. Interactions that determine the conformation of the methoxy group (with respect to the aryl ring in the family of anisole derivatives) were studied by 13C NMR spectroscopy and semiempirical MO calculations [7]. The conformation was found to be a result of the electrostatic interaction between the side-chain dipole moment induced on the p system of the aromatic Cipso –Cortho bond oriented s-cis to the methyl moiety of the methoxy group. In non-aromatic methoxycarbenium anions, the preferred orientation of the methoxy group relative to the double-bond

Naproxen structure was first optimised by using the semiempirical AM1 method. The semiempirical calculations were executed by the use of the program Spartan 4.0.3 [13]. During this optimisation the most stable conformer was found with respect to the rotation of substituents of the naphthalene ring around three single-bonds (Scheme 1). The first two bonds were simultaneously rotated every 30⬚, which was equivalent to scanning two dihedral angles, C1–C2– C2 0 –C1 0 , w , and C2–C2 0 –C1 0 –O, c . Next, for the most stable of the 144 conformers thus obtained, the C6–O bond was rotated every 10⬚ (equivalent to scanning of dihedral angle C5–C6–O–C4 0 , x ). This structure optimised with respect to the rotation around three marked bonds, was used as a starting point to scan further geometry optimisations relative to the rotation around the C2–C2 0 bond (new scan of w ). All the other molecular parameters were relaxed. These new scans were performed at the AM1 as well as at the ab initio level by using STO-3G and 3-21G ⴱ basis sets. The scan step was equal to 2⬚ in the vicinity of energy extrema and equal to 10⬚ between them. For the conformations corresponding to the energy minima (Fig. 1) the structure and energy refinements were carried out at the HF level by using 6-31G ⴱ and 6-31⫹G ⴱ basis sets. Calculations were performed by using Gaussian 98 software package [14] run on a Silicon Graphics workstation. 2.2. 1H NMR

Fig. 1. Dependence of potential energy of naproxen anion on dihedral angle x (the angle of the out-of-plane twist of the methoxy group) obtained from the semiempirical AM1 calculation.

2.2.1. Steady-state NOE measurements Steady-state NOEs for naproxen were measured in D2O at 300 K on a Varian INOVA 500 spectrometer by using a routine program for multiplet irradiation.

E. Bednarek et al. / Journal of Molecular Structure 559 (2001) 369–377 Table 1 Longitudinal relaxation time 1H T1 for naproxen anion Observed proton

1

H T1

H1

H3

H4

H5

H7

H8

OCH3

H2 0

CH3

2.0

2.3

2.0

1.8

3.4

2.0

1.6

2.4

0.8

The longitudinal relaxation times 1H T1 (Table 1) determined for the sample were used for setting up total irradiation time necessary to produce steadystate NOEs. The experimental conditions were as follows: 15 s total irradiation time, 3.2 s acquisition by using a 5000-Hz spectral window and 64K data points; 400 transients in blocks of eight were acquired without interleaving the blocks or irradiated multiplets. Line broadening of 1 Hz was used for processing FIDs and the same phase parameters were used for reference and irradiated runs. The NOE enhancements were calibrated by using the reference signal unaffected by the irradiation. The irradiation power was kept at minimum to avoid the direct saturation effects on closely lying multiplets in the crowded region of aromatic resonances. The experimental NOEs were calculated by the program NOE [15].

Fig. 2. Dependence of potential energy of naproxen anion on dihedral angle f (Scheme 1) obtained from semiempirical AM1 calculation (⫹⫹⫹) and ab initio RHF calculation using 3-21G ⴱ basis set (SSS).

371

2.2.2. Double-pulse-field gradient spin echo NOE The double-pulse-field gradient spin echo (DPFGSE) NOE experiments were run on a Varian INOVA 500 spectrometer by using the pulse sequence published by Stott et al. [16]. In the present case, 35– 75 ms r-SNOB [17] pulse was used for selectively refocusing appropriate proton signals. The mixing times were 250, 500, 1000 and 2000 ms.

3. Results 3.1. Calculations Scanning of the molecule energy at the AM1 level as a function of rotation around the C6–O bond (dihedral angle x ), Fig.1, showed that for x ⬇ 3⬚; the plot has one minimum about 1 kcal/mol deep. As can be seen in Fig. 1, the scan of x reveals some less stable minima, which have not been studied further by ab initio methods. In each of the four stable structures found at the ab initio level by scanning the torsion angle w , the angle x decreased to a value less than 1⬚. Thus, indeed, the methoxy group in the stable conformers is nearly coplanar with the naphthalene system. The scans of the torsion angle w at the AM1 and HF/3-21G ⴱ levels (Fig. 2) allow us to select four energy minima. The minima predicted by both methods coincide: according to the AM1 method, they occur at w equal to ⫺127.7, ⫺46.1, 52.6 and 131.5⬚, whereas the HF/3-21G ⴱ calculations yield values equal to ⫺131.2, ⫺33.5, 41.0, and 141.6⬚, respectively. On the other hand, the two methods differ significantly in the predicted barriers for internal rotation, which are much higher in the Hartree–Fock calculations. This is, however, a known failure of the AM1 method, which produces satisfactory trends, but underestimates the rotational barriers [18,19]. Interestingly, the scan at the HF/ STO-3G level leads only to three low-energy minima, but the fourth minimum (which is the most shallow one) is lacking. Total energies of the four stable conformers at different calculation levels are given in Table 2. Dihedral angles w of these conformers are shown in Table 3. The conformation of the naproxen molecule was previously studied, through the MM scanning of the

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Table 2 Total energies (hartree) and relative energies (kcal/mol) of the four stable conformers calculated by using different calculation levels AM1

3-21G ⴱ

3-21⫹G ⴱ

6-31G ⴱ

6-31⫹G ⴱ

I

⫺0.189345 0.0

⫺758.092398 0.0

⫺758.224717 0.0

⫺762.350747 0.0

⫺762.383250 0.0

II

⫺0.188394 0.597

⫺758.091401 0.626

⫺758.222539 0.405

⫺762.349543 0.756

⫺762.381953 0.814

III

⫺0.188553 0.497

⫺758.091010 0.871

⫺758.224071 1.367

⫺762.349476 0.798

⫺762.381505 1.095

IV

⫺0.187122 1.395

⫺758.088859 2.220

⫺762.346969 2.370

w torsional angle, by Velazquez et al. [20]. They have found only two minima that correspond to w ⬇ ⫺131⬚ and w ⬇ 46⬚: Thus, using only the molecular mechanics method, two deepest minima are lost. All the four rotamers stable at the HF/3-21G ⴱ level were next optimised at the HF level with the 6-31G ⴱ basis set and the following w angles were obtained: ⫺119.3, ⫺39.4, 62.3, and 130.5⬚. The values of three of the four minimum energy structure angles are nearer to the values obtained by the semiempirical AM1 than to those resulting from the ab initio 321G ⴱ calculation. At this level, the relative energies referred to the most stable conformer are 0.798, 0.756, 2.37, and 0.0 kcal/mol. To calculate Boltzmann populations at a temperature of 298⬚, thermodynamic functions were calculated at the 6-31G ⴱ level together with the harmonic frequencies, which showed that the four stationary points were true minima because all the frequencies were positive. The populations calculated according to the Boltzmann formula on the basis of free energies are 12, 21, 1, and 66%, respectively, and except for the rotamer with w ˆ 62:3⬚; all the structures can be observed at room temperature. Table 3 Dihedral angles w (⬚) of the four stable conformers calculated by using different calculation levels

I II III IV

AM1

3-21G ⴱ

3-21⫹G ⴱ

6-31G ⴱ

6-31⫹G ⴱ

131.5 ⫺46.1 ⫺127.7 52.6

141.6 ⫺33.5 ⫺131.2 41.0

128.7 ⫺43.5 ⫺117.2

130.5 ⫺39.3 ⫺119.3 62.3

125.8 ⫺42.6 ⫺116.0

In the most stable rotamer I (Fig. 3), Mulliken bond orders (at the HF/6-31G ⴱ level) were 1.27 for the C6– C7 and 1.58 for the C6–C5 bond. The respective bond ˚ , and the electrostatic lengths were 1.418 and 1.357 A charges at the C7, C6, and C5 atoms were ⫺0.26, 0.41, and ⫺0.30e, respectively. Thus, indeed, the strength of the methoxy group interaction does depend on the p bond order of the bonds to the two ortho positions [6,10–12]. The C6–C5 bond was also more polarised than the C6–C7 bond. Three rotamers, the most stable at the HF/6-31G ⴱ level, were finally optimised by using the 6-31⫹G ⴱ basis set, and yielded w angles equal to ⫺116.0, ⫺42.6, and 125.8⬚ and the relative energies equal to 1.095, 0.814, and 0.0 kcal/mol, respectively. Like the w angles, the other geometrical features of these rotamers were similar to those obtained at the HF/6-31G ⴱ level. The inclusion of the diffusion functions did not allow us, however, to optimise the highest energy conformer, which had escaped from the shallow potential energy well. The same was true at HF/321⫹G ⴱ level (Table 2). In order to find the reasons for the stability of the conformers, a quantitative inspection was undertaken to check the distances between two carboxylic oxygens and all the neighbouring hydrogens. Table 4 lists these distances. For clarity, only those with ˚ were considered O·· ·H distance lower than 2.7 A ˚ (2.7 A is the sum of their van der Waals radii [21]). It turned out that in the three lowest energy structures, the one of the O·· ·H distances (where H originates from one of the naphthalene C–H bond) is lower ˚ , and for the highest-energy stable rotamer, than 2.4 A ˚ . This close O·· ·H–C such a distance is equal to 2.46 A

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373

contact goes together with: (a) the same O atom contact with a methyl proton and with the second carboxylate O atom contact with the H2 0 atom (conformers I, w ˆ 130:5⬚; Fig. 3, and II, w ˆ ⫺39:3⬚ at 6-31G ⴱ); (b) the same O atom contact with H2 0 atom and, simultaneously, the other carboxylate O atom contact with a methyl proton (conformers III, w ˆ ⫺119:3⬚; and IV, w ˆ 62:3⬚†: For the three most stable rotamers, the O distance to the naphthalene proton (H1 or H3) is shorter than that to the sidechain protons H3 0 or H2 0 , whereas for the rotamer ˚ ). IV, both distances are similar (2.47 and 2.46 A

Fig. 3. Structure of the lowest energy conformer with respect to rotation around three bonds marked in Scheme 1. The shortest hydrogen bond is marked by the broken line.

Because the distance between the atoms contributing in the presumed hydrogen bond is less than ˚ , one can conclude that the stabilisation of the 2.7 A conformers is correlated with the occurrence of the hydrogen bond between oxygen atoms and two naphthalene hydrogen atoms (H1 and H3) and substituent hydrogen atoms H2 0 and H3 0 . For conformers I– IV, the distance between O and H2 0 or methyl hydrogens H3 0 also points to the occurrence of the second shortest O·· ·H contact (weak hydrogen bond), which ˚ . These three-centre (bifuris also shorter than 2.7 A cated) interactions were recently evidenced spectroscopically in 2,6-disubstituted phenol derivatives [22]. Although for the highest energy maxima (Fig. 2) ˚ , a close O·· ·H the O·· ·H distance is not less than 3 A ˚ ) can also contact (the O···H distance less than 2.7 A be found for the structures other than those of the four most stable rotamers. The potential energy is shaped here not only by hydrogen-bond-like (HBL) interactions, but also by repulsive interactions: more specifically by the H···H repulsions. For example, for the

Table 4 The nearest interatomic distances of the non-bonded O and H atoms in the four naproxen rotamers corresponding to the energy minima in Fig. 2. This oxygen atom, which is nearer to a naphthalenic proton, H1 or H3, is numbered O1; the second one, O2 O1···H1 I II III IV

O1···H3

O1···H3 0

2.32

2.62 2.61

2.29 2.38 2.46

O1···H2 0

O2···H3 0

O2···H2 0 2.42 2.41

2.48 2.47

2.58 2.56

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Table 5 The nearest interatomic distances of hydrogen atoms. Those lower ˚ are in bold type; for clarity the distances larger than 4 A ˚ than 2.4 A (not detected in NOE experiments) are omitted

I

H1–H3 0

H1–H2 0

H3–H3 0

H3–H2 0

H5–H4 0

3.42

2.32

2.64

3.74

2.35 2.35 3.62

II

2.93 3.95

3.71

III

2.39 2.33 3.63

3.56

IV

2.58

3.11

2.39 2.31

2.35

2.35 2.35 3.63

2.58

2.35 2.35 3.62

3.59

2.35 2.36 3.63

two lower maxima seen in Fig. 2 (the scan with HF/3˚ and this 21G ⴱ), the H·· ·H distance is lower than 2.5 A effect influences the energy more than does the attractive O···H contact with the distance shorter than ˚. 2.3 A Interpretation of the 1H NMR NOE spectra requires the close H···H distances to be checked. The distances between naphthalene’s H1 and H3, and the substituent hydrogens H2 0 and H3 0 in the four rotamers are therefore given in Table 5. Let us remark that in structure I, the closest contact is between the H1 and H2 0 protons; in structure II, between the H3 and H2 0 protons; for structure III, between the H1 and H3 0 protons; and in structure IV, between the H3 and H3 0 protons. Because conformation I is more populated than II,

Table 7 Comparison of back-calculated NOE data with experimental values for individual conformers and their averages by means of RMSNOE. ˚ RMSNOE factors calculated using external relaxation equal to 2.1 A Conformer

I II III IV I ⫹ II b I ⫹ III b I ⫹ II ⫹ III b II ⫹ III ⫹ IV b I ⫹ II ⫹ III c

Conformer ratio

Steady-state NOE

1 1 1 1 0.55 ⫹ 0.45 0.65 ⫹ 0.35 0.57 ⫹ 0.21 ⫹ 0.22 0.42 ⫹ 0.22 ⫹ 0.36 0.66 ⫹ 0.21 ⫹ 0.12

RMS

DRMS a

0.456 0.522 0.642 0.659 0.311 0.294 0.275 0.442 0.292

0.569–0.382 0.651–0.438 0.801–0.538 0.822–0.552 0.389–0.260 0.368–0.246 0.346–0.229 0.555–0.368 0.366–0.244

a DRMS defines the limits at the confidence level 0.95 of RMS giving a statistically valid conformer identity. b Conformer ratio based on minimisation using the experimental NOEs. c Conformer ratio calculated from HF/6-31Gⴱ free energies at 298 K.

the interaction of the substituent’s proton H2 0 with the proton H1 should be more intense than that with the H3. Similarly, the larger population of the conformation III with respect to IV should result in the more intense interaction of the substituent’s proton H3 0 with H1 than with H3. Values of the experimental NOE effects in Table 4 are in agreement with the anticipated trends on the basis of distance data. Table 5 displays also distances between the methoxy protons H4 0 and the naphthalene protons H5; all the distances H4 0 –H7 were omitted as longer ˚ (in methoxy s-cis conformation to the than 4 A

Table 6 Experimental steady-state NOEs for naproxen anion. In parentheses NOEs calculated for the weighted sum of the lowest energy conformers from free energies at 298 K calculated using HF/6-31Gⴱ (66% of I, 21% of II, and 12% of III). Estimated precision of experimental NOEs is 2% Irradiated proton

Observed proton H1

H1 H3 CH CH3 H5 H7 OCH3

⫺0.46 (0.03) 11.76 (9.35) 8.34 (3.80) 0.26 (0.07) 0.38 (0.02)

H3 ⫺0.37 (0.03) 8.34 (4.43) 5.99 (5.57) ⫺0.29 (0.02) 0.82 (0.00)

CH 8.66 (6.90) 6.20 (3.43) 13.15 (16.87) 0.52 (0.01) 0.15 (0.00)

CH3 0.80 (0.41) 0.62 (0.34) 2.27 (1.32) 0.05 (0.00) 0.05 (0.00) 0.16 (0.00)

H5

H7

0.19 (0.06) 0.76 (0.00) 0.40 (0.03) ⫺0.86 (0.28) 21.20 (21.40)

⫺0.56 (0.02) 0.78 (0.00) 0.54 (0.02) ⫺1.02 (0.47) 4.17 (1.14)

OCH3 0.16 (0.00) 0.29 (0.02) 0.02 (0.00) 5.27 (1.78) 0.55 (0.06)

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Table 8 DPFGSE NOE data for naproxen anion in water solution. The distances, r, shown in the table refer to HF/6-31G ⴱ calculations and some of them are included in Tables 4 and 5. The experimental s denotes the rate of NOE buildup proportional to cross-relaxation. The x denotes the mole fraction of transient NOE Conformers, dihedral w

I, w ˆ ⫺130.5 II, w ˆ ⫺39.4 III, w ˆ ⫺119.3 IV, w ˆ 62.3

˚ ), NOE buildup, s (x s ⫺1) Distance, r (A

NOE buildup, s exp (x s ⫺1)

H1 ! H2 0

s 2 0 {1}

s 2 0 {3}

0.038

0.023

s OMe{5}

s OMe{7}

0.0910

0.0090

H2 0 ← H3

r

s calc. a

r

s calc. b

2.32 3.71 3.56 2.58

0.08833 0.00543 0.00643 0.0474

3.74 2.35 2.58 3.59

0.00439 0.06870 0.04094 0.00517

OCH3 ← H7

H5 ! OCH3

I, II, III, IV, dihedral x ˆ 0 a b c d

The The The The

rates rates rates rates

r

s calc.

2.65

0.054

of NOE buildup, calculated of NOE buildup, calculated of NOE buildup, calculated of NOE buildup, calculated

c

(s calc), were computed (s calc), were computed (s calc), were computed (s calc), were computed

r

s calc.

4.27

0.003

by using reference by using reference by using reference by using reference

C5–C6 bond). Hence, the interactions between H4 0 and H5 atoms should be much more intense than between H4 0 and H7. In order to verify these conclusions we looked for the experimental information on the conformational equilibrium provided by the NMR results. We have used three approaches to acquire experimental information on the conformational equilibrium along bonds of interests: the steady-state NOE, transient DPFGSE NOE, and 3J(C,H) vicinal coupling constant measurements. 3.2. NOE measurements Our strategy of using steady-state NOEs for uncovering the conformational space of flexible molecules was outlined elsewhere [23,24]. Accordingly, we have measured experimental NOEs (Table 6) and the backcalculated NOE enhancements for the conformations found theoretically (Table 7) using our NOE and ancillary programs [15]. Transient DPFGSE NOE [16] was also performed as an independent experimental probe of conformations involved (Table 8). Table 7 lists the RMS values (measure of goodness of fit of experimental and back-calculated NOEs) for each conformation and their average. Inspection of

d

 and s ref r…H1 ! H8† ˆ 2:48 A  and s ref r…H3 ! H4† ˆ 2:45 A  and s ref r…H4 ! H5† ˆ 2:45 A  and s ref r…H7 ! H8† ˆ 2:46 A

ˆ s8{1} ˆ 0:059x s ⫺1 : ˆ s4{3} ˆ 0:063x s ⫺1 : ˆ s4{5} ˆ 0:057x s ⫺1 : ˆ s8{7} ˆ 0:054x s ⫺1 :

data in Table 7 allows us to suggest that the monoconformational model does not fit the experimental results because the RMS values of each single model I–IV are too high. On the contrary, the average of the conformations shown in Table 7 gives a plausibly low value of RMS. The averages shown cannot be distinguished, however, on the basis of our experiments, since the differences in RMS are not statistically significant. Table 7 gives in column 4 the values of the statistical confidence limits for the RMS. To be statistically meaningful, the RMS of the average must fall outside the indicated limits counted for a given average. Nevertheless, these results show that the biconformational or triconformational model fits the experimental data equally well and that conformation type I is an obligatory partner in any of the conformational averages that may be involved. Owing to the symmetry of the interatomic distances in conformers I–IV between H1–H2 0 and H2 0 –H3 (conform Table 5), the mixture II⫹III⫹IV was also checked as a possible average. It gives, however, a much higher RMS than the average of I⫹II⫹III, either calculated on the basis of experimental NOEs or theoretically derived populations. This finding strengthens the conclusion that the NOE-derived conformation

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E. Bednarek et al. / Journal of Molecular Structure 559 (2001) 369–377

Scheme 2.

populations for three conformation sets are in agreement with the calculated values. This is in harmony with the theoretical calculations showing a much higher energy for conformer IV. The conformational average proposed for the torsion C2–C2 0 is clearly contrasted with the single conformation preference on the torsion O–C6. Here, the respective values for the NOE values seen on protons H5 and H7 on methoxyl irradiation, are 21.4 and 1.1%, respectively, leaving no doubt as regards the preferred methoxyl orientation (see Section 1). The DPFGSE NOE gives complementary confirmation to the above conclusions on the two torsions. Table 8 collects the relevant information derived from the kinetics of NOE build-up in the two domains of the molecule by means of transient experiment. The rates of NOE rise observed on proton H2 0 on irradiation of H1 or H3 (expressed in mole fraction x per second (x s ⫺1) are different, in agreement with the steady-state NOEs, and fall in between the extreme values found in theoretical discrete conformations I– IV. No single conformational preference can be deduced. On the contrary, the rate of build-up of NOE on OCH3 is ca. 10 times faster on irradiation of the H5 with respect to the H7 irradiation, giving the same result as steady-state NOE. Two vicinal coupling constants 3J(C,H) shown in Scheme 2: 3J(C1,H2 0 ) and 3J(C3,H2 0 ), were also measured. They are equal to 5.2 and 4.9 Hz, respectively. We anticipated discriminating between the averages cited for NOEs by interpretation of the vicinal coupling constants shown in Scheme 2. The vicinal coupling constants are good measures of conformational constraint [25,26], providing there is appropriate theoretical modelling of a given coupling by means of the Karplus equation. In the absence of a good theoretical description of ⴱCar –Car –Csp3 – ⴱH torsion, we have tried to discuss the observed 3 J(C,H) coupling as an allylic type of coupling 1H– Csp 3 –Csp 2y 13Csp 2. A recent contribution on this type of

coupling [27] reports, however, on the breakdown of the Karplus relationship for this case, giving experimental evidences from terpenes. It is pointed out that two different mechanisms of coupling may be at work; one through the s-framework having maxima of 5 Hz for allylic torsion close to 0 and 180⬚, and other through the p-framework giving a maximum of 5 Hz within a range of 80–120⬚ torsion angles. In effect, if this relationship applies to our type of coupling, there is no dihedral dependence of the 3 J(C, H) coupling constants.

4. Conclusions The present results suggest that the energy profile with four minima for internal rotation around the C2– C2 0 bond calculated by the semiempirical AM1 method, exhibits the same behaviour as that obtained from the ab initio 3-21G ⴱ basis set calculations, even if in the former approach the energy differences between minima and maxima are significantly lower. Inspection of interatomic distances for various conformations showed that hydrogen-bond-like C– H·· ·O–C attractive interactions and repulsion of hydrogen atoms define the shape of the energy profile. On the other hand, the energy profile for internal rotation around the C6–OCH3 bond supports the occurrence of one planar conformation of the methoxy group, oriented towards the highest electronic density of the aromatic p bond. Experimental and calculated NOE effects converged with the values calculated for the conformational composition resulting from the relative conformer energies and the Boltzmann law.

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