MOLECULAR ORBITAL CALCULATIONS ON
CATECHOLAMINES; THEORY AND EXPERIMENT
Andrulis Research Corporation, 7315 Wisconsin Ave., Bethesda, MD 20014, U.S.A.
STEPHEN R. HELLER
Division of Computer Research and Technology,
National Institutes of Health,
Bethesda, MD 20014, U.S.A.
A. E. JACOBSON, A. ROTMAN, and C. R. CREVELING
Laboratory of Chemistry, NIAMDD,
National Institutes of Health,
Bethesda, MD 20014, U.S.A.
It is now well established that the primary site for the uptake of circulating 3H-norepinephrine is the noradrenergic nerve terminal in the peripheral autonomic nervous system . The properties of this uptake mechanism have been extensively explored in cardiac tissue. The process appears to result from a membrane carrier system which requires metabolic energy, is temperature dependent, stereospecific, saturable and has a high affinity for norepinephrine with an apparent Km of 0.2 to 1 micro molar. Many phenolic derivatives of both phenethylamine and B-phenethanolamine serve as substrates for this uptake system and are transported to the interior of the neuron. In addition, many phenethylamines which are not transported into the axoplasm of the neuron, nevertheless have a high affinity for the uptake process and are potent competitive inhibitors of uptake. Thus it appears that such compounds are able to bind to the uptake sites but lack the structural requirements necessary for complete transport which should follow the initial binding.
In a recent study of the structural requirements of compounds related to 2, 4, 5-trihydroxyphenethylamines (6-hydroxy dopamine) necessary to induce selective degeneration of noradrenergic nerve terminals, it was clearly shown that a significant affinity for the uptake process was an obligatory requirement for neurodegeneration . The inability of the 2, 4, 6- and 2, 3, 6-trihydroxyphenethylamines (see Figure 1) to inhibit uptake of norepinephrine suggested that the presence of two hydroxyl groups flanking the ethyl amine side chain greatly reduces the affinity of the amine for the uptake process. The presence of a single hydroxyl group ortho to the ethyl amine side chain did not markedly reduce the ability of the amine to inhibit uptake. However, when the investigation was extended to include the phenolic derivatives of the phenethanolamines, in particular 6-hydroxy norepinephrine, it was discovered that phenethanolamines with an ortho hydroxyl group were very poor or totally ineffective as inhibitors of uptake. Previous studies with 6-hydroxy norepinephrine [3, 4] had indicated the inability of this derivative to induce neurodegeneration of adrenergic nerve terminals. Extensive attempts in our laboratory to demonstrate inhibition of uptake, release of norepinephrine or to effect any long term alteration of the uptake system with large doses of 6-hydroxy norepinephrine, have been negative. However, the rapidity with which 6-hydroxy norepinephrine undergoes autoxidation in solution always raises a question concerning the authenticity of the preparation. The present preparation was deemed authentic on the basis of NMR, EI-MS and chromatographic behavior.
In order to circumvent the question of inactivation of 6-hydroxy norepinephrine the more stable 2-hydroxy and 2,5-dihydroxyphenethylamines were prepared. As illustrated in Figure 2, the dose response of 2,5-dihydroxyphenethylamine is markedly shifted to the right approximately 50-fold on introduction of a B-hydroxy group. The results of similar dose-response comparisons are shown in Table I. It should be noted that 3-, 4-hydroxy and 3,4-dihydroxyphenethylamines show no shift when compared to the respect ive phenethanolamine derivatives as indicated by ratios of the ED50 values of 13.8, 2.5 and 0.7 micro moles/kg respectively. However, the analogous phenethanolamines are markedly less effective. Indeed, no ED50 value could be obtained for 6-hydroxy norepinephrine although some inhibition (80% of control values) could be demonstrated with doses as high as 300 micro moles/kg. None of the phenethylamines possessing hydroxyl groups in both the 2-and 6-positions, showed any inhibition of norepinephrine uptake.
Molecular orbital calculations, using the complete neglect of differential overlap (CNDO) method, were previously performed in our laboratory on protonated dopamine and (R)-norepinephrine  2,4,5-, 2,3,5-, 2,3,4-, 2,3,6-, and 2,4,6-trihydroxyphenethylamines, as well as 2,5-dihydroxy and 2,3,4,5-tetrahydroxyphenethylamines  (See Figure 1). These calculations led to the following conclusions:
(1) Whenever R2 was a hydroxyl group, the minimum energy conformations were gauche forms. The proton on the nitrogen atom at the end of the side chain, rotated 120o around the 14, 15-bond, was situated 1.29 A apart from the oxygen atom at the 2-position. The energy barrier between the gauche form and the extended trans (anti) conformation, with the 14,15-bond perpendicular on the benzene ring, was found to be approximately 16 kcal/mol. This barrier seems sufficiently high to preclude free passage from the gauche to the trans conformation.
(2) Whenever R2 and R6 were hydroxyls, we found two equally probable gauche minimum energy conformations, free passage between them was likely to be precluded (see Figures 3 and 4). Both these minimum energy conformations can be reached from the trans extended form, which was found to be the crystal structure for 2,4,5-trihydroxyphenethylamine  (6-hydroxy dopamine).
(3) Since neither of the compounds possessing two hydroxyl groups at the 2- and the 6- positions showed any measurable affinity for the uptake site into the adrenergic nerve terminal, it seemed that the deep energy minima at gauche conformations with a hydrophilic group on the R6 side of these molecules were responsible for their lack of affinity.
In order to check the above presumption, the protonated (R)-2,5-dihydroxyphenethanolamine was selected as a representative of the 2-, 2,5- and 2,4,5-substituted hydroxyphenethanolamines for molecular orbital calculations. As shown in Table I, these compounds possess very low affinity, or no affinity at all, for the uptake site. CNDO calculations should therefore provide two distinct deep minimum energy conformations, with one possessing a hydrophilic group on the side of R6.
The results obtained from the calculations are summarized in Figure 5. Both under-the-plane and over-the-plane rotations around the 14,15 bond were calculated, with energy minimization around the 1,14 and 15,16 bonds. The minimum energy gauge conformation obtained from the CNDO calculation, at the 240o under-the-plane rotation around the 14,15-bond, has all of the polar groups together on the side of the 2-hydroxyl group. There is no hydrophilic group on the side of the 6-position. However, when we follow the over-the-plane rotation around the 14,15 bond, the deep energy well occurred at the 120o gauche position with the 14-hydroxyl group is on the 6-position side (See Figure 6). It is apparent that again an approximately 13.0 kcal/mole barrier separates the two minimum energy gauche conformations, most probably precluding free passage between them. If the crystal structure of 2,5-dihydroxyphenethanolamine is the extended trans conformation, the passage to the gauche forms would be straightforward. This X-ray crystal structure has not been determined yet.
A direct flip-over of one gauche conformation into the other is unlikely, because of a 20.0 kcal/mole barrier for this conversion.
Although the 14-hydroxyl group and the 2-oxygen atom can form a 6-membered ring with an OH...O type hydrogen bond, this bond is not the one that in our CNDO calculations provided the minimum energy conformation. CNDO calculates the N+H...O hydrogen bond to be approximately 9.0 kcal/mole stronger than the OH...O hydrogen bond. The possibility that CNDO overestimates N+H...O, bonding beyond approximately 30% could be real. In this case, the deep energy well for the gauche conformation could perhaps be eliminated, or the OH...O bond would have a greater influence. Also, there exists the possibility that CNDO itself is perhaps inappropriate for obtaining minimum energy conformations of these particular molecules.
If, however, CNDO is more or less correct in postulating the above conformations, and if these conformations do, in fact, exist at the uptake sites, it is possible that some yet-unknown factors, not calculable by molecular orbital theory, disturb the affinity of these compounds toward the uptake sites.
Accepting the validity of the CNDO method and of the results obtained, it appears that the presence of a hydrophilic group in an undesirable area, precluded from free rotation by a high energy barrier, could be the reason for lack of affinity of the above biogenic amines for the uptake mechanism.
It seems to us that the above hypothesis of a hydrophobic site as a necessity for affinity for uptake will further the present state of drug design and will help us in our continuing efforts to elucidate the mechanism of uptake into the noradrenergic nerve terminals.
Additional insight into the above conclusions could be provided by recalculating the different compounds using the PCILO method, known to predict accurate minimum energy conformations. It is interesting to mention that PCILO predicted the gauche conformation as the energetically favorable one for ortho-methoxyamphetamine derivatives , in perfect agreement with our results.
1. Iversen, L. L.: Brit. Med. Bull. 29, 130 (1973).
2. Fuxe, K., Olson, and Zotterman: Dynamics of Degeneration and Growth in Neurons, Pergamon Press Ltd., London, 1974.
3. Sachs, C.: Europe. J. Pharmacol. 20, 149 (1973).
4. Lundstrom, J., Ong, H., Daly, J., and Creveling, C. R.: Mol. Pharmacol. 9, 505 (1973).
5. Katz, R., Heller, S. R., and Jacobson, A. E.: Mol. Pharmacol. 9, 486 (1973).
6. Katz, R. and Jacobson, A. E.: Mol. Pharmacol. 9, 495 (1973).
7. Kolderup, M., Mostad, A., and Romming, C.: Acta Chem. Scand. 26, 483 (1972).
8. Pullman, B. And Courriere, P.: Compt. Rend. Acad. Sci., Ser. D. 276, 1907 (1973).
Christoffersen R.: Would not there be a strong perturbation in the results obtained in these studies to the presence of solvent molecules?
Katz: Strong perturbations, possibly resulting in different minimum energy conformers, might occur if solvent molecules were included in these calculations. It is possible that, if solvation were considered in the calculation, the trans conformer of these molecules would be comparable in minimum energy to the gauche form. Indeed, B. Pullman et al., have noted (J. Med. Chem. 17, 439 (1974) that the N+-H bond to water molecules gives about kcal/mol per hydrogen bond lowering of energy. These calculations were obtained using both PCILO and ab initio studies. This 20 kcal/mole figure is quite analogous to the value we found for an intramolecular N+-H...O bond, using CNDO calculations.
We might note that the techniques which are now available for the calculation of the minimum energy conformations of biological molecules appears to be getting somewhat ahead of our knowledge as to how these molecules exist in vivo. It is fair enough to guess at the way solvation might occur, however we must not assume that we are calculating anything beyond this imaginative moiety, and we must keep in mind the fact that this moiety may not, in fact, exist in vivo as we draw it on paper.
A. Pullman: It is known through a number of studies that CNDO generally gives correct relative values of H bond energies. Moreover when the binding energy is compute for the experimental value of the H bone distances, the CNDO value is approximately satisfactory too (slightly too large). However, it is also known that if the proton donor is allowed to approach the proton acceptor until equilibrium, the value of the binding energy at the minimum of the curve is too large and the distance of approach to small in bydrogen bonds of the visual type. I suspect that these effects would be exaggerated in ionic hydrogen bonds of the N+H...O type.
You mentioned that in your case, the H...O distance is 1.29 A; this is a very close approach and may very well correspond to the position of the CNDO minimum where the binding energy is exaggerated. This might lead to a too deep minimum in the corresponding position on your map.
Katz: As we have noted in our publications on the subject (Mol. Pharmacol. 9, 486 and 495) using CNDO calculations, as we increase the distance between the N+-H and the 2-OH on the catechol ring in 6-hydroxy dopamine, the energy of the gauche conformation increases rapidly. We do not know, nor are we aware of any work directly related to it, what an average N+-H...O intramolecular distance should be in reality. It may be that we have exaggerated the H-bond effect by using too small (1.29 A) a distance. Nevertheless, this distance gave us our minimum energy conformation. It should be noted, however, that there is literature reference to intermolecular NH2...O hydrogen bonds at a distance of ca. 1.4 A. If this is true, then an N+H...O intramolecular H-bond might well occur at the shorter distances we have calculated.
B. Pullman: We have computed recently the conformational energy maps of 2,4,5- and 3,4,5- trihydroxyphenethylamines using the PCILO method (B. Pullman, H. Berthod and A. Pullman, Anales de Quimica, special issue in honor of Prof. Lora-Tamayo, in press) and I regret to have to say that our results are substantially different from your CNDO/2 ones.
The corresponding conformational energy maps are reproduced in Figures 1 and 2. It may be observed that: (1) the two maps are similar, (2) on both of them the global energy minimum corresponds to an extended conformation, in close agreement with X-ray crystallographic results, (3) both maps present gauche minima only 0.5-1 kcal/mole above the global ones and (4) on both maps the barrier between the trans and gauche forms is of the order of 3-4 kcal/mole.
Our computation have been carried out using as input the geometries of the molecules in the crystals, while you have used, if I am right, standard geometries. This could be responsible partly for the discrepancies between your and our results, but I believe, that most of it comes from the short comings of the CNDO method for conformational studies, which are now quite evident on a number of examples.
We have also studied from the same viewpoint mescaline and related methoxyamphetamines (see my remark after Dr. Horn's lecture).
G. C. K. Roberts: We have recently done some preliminary NMR experiments on 6-hydroxy dopamine which indicate that its conformational distribution is very similar to that of dopamine (i.e. about 40% trans). This throws some doubt on the importance of the nitrogen-orthro hydroxyl interaction and is in much better agreement with the PCILO than the CNDO calculations.
Katz: Response to the question of B. Pullman. PCILO calculations on the conformation of 6-hydroxydopamine evidently do not show that a significant energy difference occurs between gauche and trans forms. Perhaps PCILO is giving a more correct answer. It is also possible, that PCILO underestimates the energy lowering due to N+H...O intramolecular H-bonds. It is difficult for us to state which is correct at the moment; perhaps the answer lies someplace between both types of calculations.
It is somewhat doubtful whether the use of X-ray diffraction data would account for the differences noted between PCILO and CNDO calculations on 6-hydroxydopamine. As can be observed, in our publications on the subject, our use of this diffraction data resulted in little essential difference in the variation of relative energy vs bond rotation, and in energy barriers. We merely found that the use of the diffraction data, with its perhaps too short N-H, O-H and C-H bonds gave us higher overall energies for each of the conformers, than did the use of standard bond distances and angles for these bonds.
Finally, it is most pleasant to note that PCILO calculations on protonated 3,4,5-trihydroxy-phenethylamine agree nicely with our published calculations on this molecule, using CNDO.
Response to the question of G. C. K. Roberts. These NMR experiments may be applicable to the conformation of 6-hydroxydopamine salts in solution. We have not, of course, done calculations which could be compared with this solvation study; solvation is undoubtedly of great importance (see our response to Dr. Chrisrofferson's question). It may be noted that PCILO calculations on 6 hydroxydopamine have also not been done, insofar as we are aware, taking hydration into account. It would indeed be interesting to see if the preliminary correspondence observed between the PCILO calculation on the isolated protonated 6-hydroxy dopamine and NMR experimental observations would continue to hold when the calculation of hydrated protonated 6-hydroxy dopamine is completed.
Horn: Using the cis and trans isomers of tranylcypromine (1-amino-2-phenyl cyclopropylamine) and 1 and 2-aminoindane we have shown that the preferred conformation of amphetamine for the inhibition of noradrenaline and dopamine uptake is trans rather than gauche.
Katz: There may, or may not, be a relationship between the inhibition of uptake of norepinephrine and dopamine with the tranylcypromines (rigid analogs of amphetamines) and with catecholamines. If the mechanism of action of the amphetamines and the catecholamines is the same, as regards this inhibition of uptake, then the work of Horn and Snyder (J. Pharmacol. Exp. Therapeut. 180, 523 (1972) would seem to indicate that inhibition relates to the trans, not the gauche conformer in the catecholamines.
It should be realized, however, that in the amphetamines the d-isomer is more biologically effective than the corresponding l-isomers, quite unlike the catecholamines.
May we also note that we have discussed in these Proceedings the possibility that an important factor in biological activity could be the presence of a hydrophilic moiety in a particular area of the molecule when the conformer exists in a relatively low energy state, having barriers to change from that conformation which can be overcome only with difficulty. Whether gauche or trans conformers exist predominantly, this difference (in the presence of a hydrophilic group) might still pertain.