6, (Continued)

k W r n y m i* !""! ''I lÜiÜiilü-'iiÜlii m/ 20' Phorbol Acid rI i'ÿ. rlSlira n/ 20' Phorbol Aldehyde Fiq 4,6. (Continued)

The AMI point charges of the derivatives were used to generate electrostatic potentials at both the Van der Waals radius and at twice the radius, shown in fig 4.6. Similarity indices, given in table 4.5, for the A and B rings, compared with that of phorbol, were calculated from these point charges without optimisation by the simplex method [101]. Instead the different molecules were fitted using the procedure in CHEM-X, which gave very small mean deviations for equivalent groups. The similarity indices do not show a good correlation with promoting activity showing ESP is not equally important for binding over the whole two rings. This may be caused by some parts of the rings not being involved in the site, or because an unfavourable steric interaction prevents a molecule with a complementary ESP from entering the binding site. In this particular case it is more useful to consider the detail of the ESP rather than similarity indices over sizeable fragments of the molecule.

The discussion of the ESP's themselves is largely qualitative both because the activity data is also essentially qualitative and because most of the derivative potentials differ from that of phorbol only in the immediate vicinity of the structural change. The potentials shown in fig 4.6 are all drawn to show the 3', 9' and 20' groups with the convention that negative potentials are displayed in red and positive in blue, while the terms 'a' and '(3* are replaced by 'a' and 'b', as the colour printer is unable to produce the Greek characters. At twice the Van der Waals radius, the ESP of phorbol shows very little detail, being dominated by the strongly negative potentials of the 3' carbonyl and the 12', 13' hydroxyls. At the single radius,* rather more detail is shown, the polarisation of the 9' and 20' hydroxyls can be seen clearly, while Hi has a strong positive potential. Apart from these regions, most of the carbon skeleton possesses a neutral potential.

Both of the modifications considered to the A-ring double bond (l',2') lead to complete inactivation of the ester, while structurally both alter the geometry of the Arring, forcing the 19' methyl group out of the plane of the ring. Epoxidation includes a second strongly electronegative atom into the A-ring and thus produces a substantial effect on its potential. The potential on the same side of the ring becomes more negative, while that on the opposite side, especially Hi, becomes more positive, which

is also accompanied in the P form by an increase in the positive potential on the hydrogen of the 9' hydroxyl. By comparison, saturation of the double bond does not change the ESP of the A-ring to any extent, except to reduce the positive potential at position 1. Thus it appears that the change in A-ring geometry alone is sufficient to abolish activity, rather than being an electrostatic effect.

As the 3' carbonyl was one of the key contacts identified in section 4.4.1, a change in ESP, or modification would be expected to have an effect on activity. Conversion of a hydroxyl group would not abolish its ability to accept a hydrogen bond, but as the positions of the lone pairs are different with the lone pair-oxygen- carbon angle decreasing from 120“ to nearer 109“ typical of tetragonal geometries, the energy of such an interaction would be altered. The rotation of the hydroxyl group is also restricted by the presence of neighbouring ‘groups so that there is only one minimum energy position for the p form, while those of the a form lie close together and in neither case would the lone pairs be expected to lie in the plane of the A-ring. That both forms have comparable activities is in keeping with the absence of change to other parts of the ESP and suggests that the optimum positions of the lone pairs for hydrogen bonding lies roughly midway between the positions which, from the conformations shown in fig 4.6, is virtually in the plane of the ring.

Some earlier studies [120,123,124] suggested that the 4' hydroxyl was one of the key hydrogen bonding contacts with the site, but this view is not supported by the results of the fitting to DAG, section 4.4.2, and by the activity data [112] which shows that the deoxy derivative possesses full activity. The potentials of both the 4' deoxy and methoxy derivatives are virtually identical, and-are similar to that of phorbol, thus the difference in activity cannot be due to an electrostatic effect. Rather, the larger methoxy group projects further from the rest of the molecule than does either the hydr oxyl group or hydrogen atom, and thus the reason for the inactivity of the methoxy derivative lies in the fact that it is unable to fit within the site. This requires the presence of site groups close to the 4' position, though little can be said about the potential except that it clearly does not interact strongly with either the hydroxyl group or hydrogen atom, and in view of the proximity of the 3' hydrogen bonding contact,

this site group may well be part of the hydrogen bond donor. Rotation of the 4' substituent is severely restricted (barrier height >200 kcal mol'^) by the presence of the A and B rings and results in the oxygen substituents pointing almost directly away from the rest of the molecule, so the effect of the extra size of the methyl group is exaggerated, and it is simultaneously prevented from rotation to relieve the unfavourable steric interaction. The 5' hydroxyl group also projects from the molecule in a position close to that of the 4' substituent, but the derivative retains almost complete activity. Like the 4' derivatives, there is little change in the ESP except at the site of modification itself, but the 5' group is not constrained by the rest of the molecule and is free to rotate. As it is also further from the 3' contact, it would appear to lie further from the site groups and not form an unfavourable steric interaction.

Epoxidation of the B-ring double bond (6’,7') as for its A-ring counterpart alters both the geometry of the ring and its ESP. However, unlike the corresponding A-ring modification, it does not abolish activity, merely reducing it by a small extent. As with the A-ring, the epoxide group causes the ring to acquire a negative potential on the same side and a positive potential on the opposite side of the ring to the substitution, with the effect that the a form has a region of negative potential between the 9' and 20' hydrogen bonding contacts, while the equivalent region in the P form has a positive potential. As both forms possess a high activity, this region cannot be involved in binding to the site, except perhaps as part of a non-specific polar interaction. Though the geometry of the ring, including the position of the 20' hydroxyl relative to the other hydrogen bonding contacts, is also altered, the flexibility of the 20’ group is such that the energy cost on changing the conformation at 6' and 20' to optimise the hydrogen bond would be quite small, and apparent in the small reduction in activity.

Oxidation of the 20' hydroxyl to either the aldehyde or acid reduces the promoting activity considerably, but without changing the ESP except at position 20. The almost complete inactivity of the aldehyde is consistent with the 20' group being a hydrogen bond donor. This derivative is still able to form the other hydrogen bonds to the site and would not suffer from the unfavourable steric interactions in the manner of the 4b methoxy derivative and derivatives with a saturated T,2' bond, so should still be

able to bind to the site though with a much lower affinity. Its low level of activity can be compared with that of the 3' hydroxyl derivatives, where the hydrogen bond is weakened rather than being lost completely. The acid derivative should be able to both accept and donate hydrogen bonds unless it is deprotonated, which is unlikely at physiological pH, and though the orientation of the hydroxyl group is the opposite of that of phorbol, the difference between the two acid conformations is only 0.04 kcal mol E Thus the lack of promoting activity of the acid is more likely to stem from an unfavourable interaction with the binding site. The potential at the acid carbonyl oxygen is the most negative found for the phorbol derivatives, so will be repelled by the hydrogen bond acceptor within the site if it too possess a sizeable negative potential.

The only modification outside the A and B rings to be considered was that of hydroxylation at position 16. The calculated similarity indices show that the A and B rings are essentially identical to those of phorbol which is consistent with their identical activities. This is not sufficient proof that the interactions involve solely these two rings as this is the assumption underlying the choice of model.

H

H

HO

,Ç 4 --- C 3

b/ la Epoxy Phorbol

c/ Ib Epoxy Phorbol

d/ la Saturated Phorbol

e/ lb Saturated Phorbol

f/ 3a Hydroxy Phorbol

g/ 3b Hydroxy Phorbol