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analogues, 113, 114 & 117, are the most active inhibitors of Mdm2. The best

substituent on the meta N10phenyl for activity is the chloro group (as shown by

113), possibly due to the increased hydrophobic nature of the compounds [Table 9]. There is a link between activity as an Mdm2 inhibitor and partition

coefficient (a measure of hydrophilicity, as illustrated in [Table 9]). 10-(3,4-

Dichlorophenyl)-9-trifluoromethyl-5-deazaflavin, 119, has chloro groups at the

meta and para positions of the N10 phenyl, is active but not as active as the

singly substituted meta- or para-chloro N10 phenyl 9-trifluoromethyl-5-

deazaflavin analogues 10-(3-chlorophenyl)-9-trifluoromethyl-5-deazaflavin,

Table 9

Compound Inhibition of Ub of p53 ( M) CLog P

114 13 4.2 117 4.5 4.5 113 2.5 4.6 HN N N O O F CF₃ HN N N O O CF₃ HN N N O O Cl CF₃ 113 117 114

Table 9. The link between compound activity as an inhibitor of Mdm2 E3 ubiquitin ligase activity to Log P.CLog P calculated from Chem Draw Pro 10.0. With IC50data for inhibiting

ubiquitinylation of p53. Ub stands for ubiquitinylation.

A noticeable exception to the SAR rule that the 9-trifluoromethyl group is a

prerequisite for activity, is for the ortho-chloro and ortho-methyl 9-

trifluoromethyl-5-deazaflavin analogues, 112 & 116, possibly due to the size of

the substituent at the ortho position forcing the 5-deazaflavins into a

conformation than cannot act in the site of action of inhibiting Mdm2. 10-(2-

Fluorophenyl)-9-trifluoromethyl-5-deazaflavin, 47 act as an inhibitor of Mdm2

(even with the ortho position substituted) possibly due to the smaller size of the

fluorine atom, leading to the compound having the required conformation to

act as an inhibitor.

These biological results also show clear hit optimization, with the current

hit compound, 10-(3-chlorophenyl)-9-trifluoromethyl-5-deazaflavin, 113,

being 30 times more active than the previously identified hit compound, 10-

(4-chlorophenyl)-7-nitro-5-deazaflavin, 25.

3-Substituted-5-Deazaflavin Analogues Synthesis and Biological Results

To investigate the role of the third position (N3) [Figure 9] of the 5-deazaflavin

synthesized, containing a methyl or ethyl group at the N3 position with either

the exposed quinoline ring unsubstituted, 120 & 121, or 9-trifluoromethyl and

para-chloro substitution of the N10phenyl, 122 & 123.

N N N O O R₅ R R'₃ (a) HN N N O O R₅ R'₃ 64; R₅& R'₃= H. 51; R₅= CF₃& R'₃= Cl.

120; R = Me, R₅& R'₃= H - 33% Yield.

121; R = Et, R₅& R'₃= H - 22% Yield.

122; R = Me, R₅= CF₃& R'₃= Cl - 18% Yield.

123; R = Et, R₅= CF₃& R'₃= Cl - 43% Yield. Figure 27. Synthesis of 3-substituted-5-deazaflavin analogues.(a) ROH, NaOR, RIand . Where R = Me or Et.

The N3methyl and ethyl 5-deazaflavin analogues, 120 & 121, were inactive at

250 µM concentration on the qualitative in vitro biological assay as inhibitors

of Mdm2. Due to the fact of these analogues, 120 & 121, not having the

required trifluoromethyl group at the nine position. The N3methyl or ethyl-10-

(4-chlorophenyl)-9-trifluoromethyl-5-deazaflavin analogues, 122 & 123, have

not, at the time of writing, been tested on the qualitative in vitro biological

assay.

These results will be interesting as they will establish if the N3 position is

required for inhibition of Mdm2 E3 ubiquitin ligase ability. If the N3position is

not required for inhibitor activity, this would allow the addition of acidic and

basic solubilising groups to be added to the N3 position to improve the poor

9-Substituted-5-Deazaflavin Analogues Synthesis and Biological Results

Rationale

At present having an electron-withdrawing and hydrophobic substituent at the

nine position of 5-deazaflavin is a prerequisite for inhibitor activity, with the

most active substituent being the trifluoromethyl group. The majority of 5-

deazaflavin analogues synthesised and tested contain an electron-withdrawing

and hydrophobic substituent at the nine position. Apart from the 9-methyl-5-

deazaflavin analogues, 52-63, which have electron-donating and hydrophobic

substituent and are inactive as inhibitors of Mdm2 E3 ubiquitin ligase activity.

Other substituents with different electronic and hydrophobic character at the

nine position of 5-deazaflavin require further investigation.

To this end, the fluoro and bromo substituents at the nine position of 5-

deazaflavin with the N10 phenyl either unsubstituted, 124 & 125 [Figure 28],

the least active substituent pattern, or bearing para-chloro, 126 & 127 [Figure

28], at the time the most active substituent pattern, were synthesised. These

compounds, 124-127 [Figure 28], will compare the three different halogen

atoms as substituents at the nine position of 5-deazaflavin on inhibitor activity

towards Mdm2. Using Craig plots [284] the hydroxy and cyano substituents

were selected at the nine position of 5-deazaflavin as these substituents have

different electronic and hydrophilic character to the previous substituents at the

nine position. A Craig plot compares the Hammet substituent constant ( ), a

measure of electronic effects of a substituent and the substituent

hydrophobicity constant ( ) [285]. The hydroxy substituent is electron-

donating and hydrophilic while the cyano group is electron-withdrawing and

will have the N10phenyl either unsubstituted, 128 & 129 [Figure 28], the least

active substituent pattern, or para-chloro, 130 & 131 [Figure 28], at the time of

synthesis the most active substituent pattern.

HN N N O O R₅ R'₃ 124; R₅= F & R'₃= H. 125; R₅= Br & R'₃= H. 126; R₅= F & R'₃= Cl. 127; R₅= Br & R'₃= Cl. 128; R₅= OH & R'₃= H. 129; R₅= CN & R'₃= H. 130; R₅= OH & R'₃= Cl. 131; R₅= CN & R'₃= Cl.

Figure 28. The 9-Substituted-5-Deazaflavin Analogues, 124-131. Synthesis