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The plotted electrostatic potentials show a dipole moment across the A-ring, the magnitude and direction of which was calculated from an AMI single point calculation on the A-ring fragments using the geometries found in the parent molecules. The magnitudes are given in table 4.6 and directions in fig 4.7, while the potential derived charges for H20 are included in table 4.6.

Table 4.6 Activitv vs. Pinole Moments.

A-Ring In plane of A-ring W hole m olecule H2 0 charge Phorbol 2.275 2.168 3.188 0.322 4‘ Deoxy 3.016 2.999 1.235 0.324 16' Hydroxy _i 2.583 0.322 5p Hydroxy 1.026 0.314 6a,7 a Epoxy 1.751 0.312 6p,7(5 Epoxy 2.162 0.337 3a Hydroxy 1.747 1.173 4.037 0.320 3P Hydroxy 1.162 1.084 4.169 0.320 20’ Aldehyde 3.101 -0.035 1,2a Saturated 1.878 1.795 3.618 0.324 1,2P Saturated 1.914 1.859 3.550 0.326 la,2 a Epoxy 2.883 1.811 4.913 0.311 1P,2P Epoxy 1.751 1.474 2.303 0.327 4p Methoxy 1.859 1.831 1.559 0.318 20' Acid - - 2.730 0.109

1 The entries marked with a hyphen do not differ from phorbol in the A ring so the

Charges for the oxygen atoms as obtained from Mulliken analysis do not vary significantly between derivatives, but the charges obtained from the wavefuncdon ESP for a small fragment of the molecules have been compared below. The ESP's for the fragments have been calculated using a minimal basis (STO-3G) as a basis with more primitive Gaussian functions requires more disc space than was available.

Table 4.7 Activitv vs. Oxvgen Charges

O3 O4 O9 O2 0 Phorbol -0.397 -0.523 -0.574 -0.543 4' Deoxy -0.402 -0.556 -0.545 16' Hydroxy -0.397 -0.523 -0.574 -0.543 5p Hydroxy -0.410 -0.486 -0.588 -0.523 6a,7 a Epoxy -0.390 -0.499 -0.571 -0.490 6p,7P Epoxy -0.396 -0.504 -0.576 -0.536 3a Hydroxy -0.567 -0.569 -0.570 -0.541 3p Hydroxy -0.591 -0.588 -0.574 -0.540 20' Aldehyde -0.394 -0.514 -0.573 -0.316 1,2a Saturated -0.390 -0.562 -0.576 -0.538 l,2p Saturated -0.383 -0.552 -0.560 -0.542 la,2 a Epoxy -0.386 -0.556 -0.605 -0.518 ip,2p Epoxy -0.391 -0.489 -0.560 -0.546 4P Methoxy -0.394 -0.293 -0.569 -0.524 20' Acid -0.409 -0.491 -0.557 -0.628

It is noticeable, from fig 4.7, that the dipole moments for both phorbol and 4' deoxy phorbol lie almost on the plane of the A ring, while for 3p hydroxy phorbol and the inactive 1,2' epoxides the dipole moment has a substantial component perpendicular

to the ring. The dipole moment for 3 a hydroxy phorbol points between and C2

rather than between Ci and Cio as is the case with phorbol. The A-ring dipole moments of the 1,2 saturated derivatives differ from that of phorbol only in being

slightly smaller, while the dipole moment of the 4' methoxy derivative is, not surprisingly, almost identical. These differences could account for some of the differences in activity as the direction of the dipole moment will have some influence on the orientation of the A-ring on its approach to the site, while the magnitude will contribute to the binding energy. By comparison, the dipole moments for the whole molecule are not useful as a guide to activity. Though many of the low activity and inactive derivatives possess dipole moments that differ from that of phorbol, the magnitude of the dipole moment is very sensitive to rotation of key groups, for example rotation of the 20' aldehyde changes the magnitude of the dipole moments from 3.101 Debye to 3.935 and a change on a similar scale can be seen for a rotation of the 16' hydroxyl group, without affecting the activity.

It is difficult also to draw any conclusions from the oxygen charges beyond the observation that deviation from the charges found for phorbol is associated with a reduction in activity. None of the derivatives shows a change in the O9 charge and its

inclusion as one of the three hydrogen bonding contacts is based solely on the comparison with DAG. The 20' hydrogen of the aldehyde carries a positive charge when the charges are calculated using Mulliken analysis on the semi-empirical wavefunction, but direct calculation of the ESP from the wavefunction gives it a small negative charge. It is noticeable that at twice the Van der Waals radius, the potential over the hydrogen atom is completely dominated by the oxygen charge even when the potential is calculated from the Mulliken charges, so this difference should not alter the conclusions drawn from the ESP's.

4.4.3 Parameter Dependency of Results.

The ESP's, charges and dipole moments discussed above are calculated using the AMI parametrisation for the reasons mentioned eaiiier. In this section, some of the results obtained using the three different parameter sets are compared with each other and, where possible, with experimental data.

The phorbol ESP's calculated using each of the three parameter sets are shown in fig 4.8 and are remarkably similar. The potentials are calculated at the minimum energy geometries for each method yet the conformations differ only in the rotation of the 9' hydroxyl (and some small rotations elsewhere), which is able to freely rotate over a limited range before coming into close contact with the neighbouring ring systems. The potentials themselves differ only in that the AMI potential is of a slightly higher magnitude over much of the molecule, but without significant changes of sign. As all three parameter sets use a well balanced minimal basis set, and have been tested and parametrised using a wide range of carbon, oxygen and hydrogen systems, the Mulliken analysis would be expected to give similar atomic charges for all three methods.

The only experimental data for the phorbols, apart from tumour promoting activities, is the crystal structure [116]. In table 4.8* below, the deviations between the crystal structure and the optimised geometries are compared. AMI and PM3 give equally good results for the bond lengths, but MNDO is rather less accurate. It is noticeable however that MNDO consistently overestimated bond lengths especially for bonds within the ring systems, as is shown by the signed mean being rather large compared to the unsigned mean. The calculation of the bond angles is rather different as all three methods give similar accuracy with MNDO being most accurate. The signed mean is small in all cases showing that there are no systematic errors for the angles considered, but as the molecules are both rigid and in places strained, there is little scope for large variation in the bond angles. As the hydrogen atoms are not placed correctly in the crystal structure, hydrogen containing bonds and angles were not considered in the analysis.

k!i" 'm a f AMI b/ PM3 mBAisMiwW Wwhb’'! J .'||.,'1!1, c/ MNDO