Causas del fracaso en las franquicias

Several N-capping groups scaffolds have been identified however non are optimal in terms of mimicking the majority of the interactions of N-terminal tetrapeptide of HAKRRLIF. Even 35DCPT, the most effective N-cap only practically interacts with this region and hence there is a need for improvement. A previously published study described benzoic acid capped peptides with a chlorophenylcyclohexylamine replacement

ID N-cap PSA clogP

NC-1 21.05 2.29 NC-2 21.05 2.29 NC-3 33.22 2.95 NC-4 40.08 1.99 NC-5 27.91 2.03 NC-6 32.35 2.41 NC-7 32.35 2.41 NC-8 7.68 2.41 NC-9 7.68 4.32 NC-10 16.61 3.33

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for the phenylalanine. These derivatives included basic substituents which when incorporated into cyclin groove inhibitors resulted into potent compounds when tested in CDK2/cyclin A ELISA using retinoblastoma protein as substrate for competitive binding75 (Figure 5.2). This suggested that the benzoic acid scaffold would be an effective one for the presentation of the required functionality to interact more completely with the cyclin groove by including contacts with the secondary pocket while also making ion-pairing interactions with the acidic residues. As described in chapter 3, previous studies demonstrated that FLIP molecules containing 3,4 diethoxyphenylacetamido group as N-cap (530) were good inhibitors of CDK2/cyclin A (6.5µM) and CDK4/cyclin D (25.5µM). Incorporation of basic substituents including piperazinyl and alkyl amines onto heterocyclic scaffolds (5-((4-methylpiperazin-1- yl)methyl)furan-2-carboxamide-RLIF, 5589, 5-((diethylamino)methyl)furan-2- carboxamide-RLIF - 5581) resulted in moderate activity in both CDK2/cyclin A and CDK4/cyclin D68. Supportive docking results suggest that ion-pairing interactions with the negatively charged residues contributed to binding. Based on this result and literature reviews, benzoic acid derivatives were explored as N-capping groups. It was hypothesized that through effective substitutions on the 3,4 positions of benzoic acid core structure, ion-pairing, hydrogen bonding and van der Waals interactions might be retained.

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In the first steps to exploiting the potential of the benzoic acid scaffold, 3-alkoxy derivatives were designed so as to build on the success of 3,5-diethoxyphenylacetic acid as an Ncapping group. Although docking studies were undertaken with 3- alkyloxy or aryl oxy substituted benzoic acids (Overlays of 3-substituted benzoic acid derivatives are shown in figure 5.3), none of these FLIPs had significant activity in the FP binding assay with the exception of 3-ethoxybenzamido substituted FLIP which had marginal affinity for CDK4/cyclin D. The observed inactivity of this FLIP series could be because of weaker van der Waals interactions resulting from too much flexibility of the groups without the ion-pairing interactions to stabilize binding. For sufficient activity, N-capped peptides require more extensive interactions that are made by the potent CGI peptides including ion-pairing and H-bond contacts. In order to improve the affinity of these Ncapped peptides, and due to the synthetic versatility of the benzoic acid scaffold, additional substituents were appended at the 4 position to include groups capable of ion- pairing with the cyclin groove.

Figure 5.3 Overlays of 3-susbtituted benzoic acid derivatives

Overlay of docked poses of 3-susbtituted benzoic acid shows van der Waals interaction at the secondary pocket and hydrogen bonding with Gln 254.

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In the first instance, single 4-substituted benzoic acids were generated for incorporation as capping groups. Inclusion of basic groups (guanidomethyl (541), piperazin1-yl (5850), piperidin-4yloxy (5851) and 4-methylpiperazin-1ylmethyl (5919) shown in table 6.1 resulted in significant improvement in the activity compared to the 3- alkyl analogs. The guanidinomethyl substitution which has been previously reported, was generated as a RLNpfF FLIP and was the highest potency cyclin groove inhibitor in this series (0.69µM). The effectiveness of this compound is due to its very strong electrostatic interaction of the positively charged guanidine with the acidic region (Glu220, Glu224). Both the piperidin-4yloxymethyl (5851) and piperazinyl (5850) are similarly potent in binding to cyclin A in complex with CDK2 however 5851 exhibits higher activity for CDK4/cyclin D. The better activity in cyclin D could due to the proximity of piperidinyl group with Glu70 and GLu66. RRLNpfF is a better CBG inhibitor compared to RRLIF by 2 fold increased potency. The p-fluoro substituent on the phenylalanine ring enables increased hydrophobic contacts in the primary pocket69. The increased binding of the RLNpfF peptide was confirmed by the comparison of 5566 and 5919 since both of these FLIPs have (4-piperazin-1ylmethyl)benzoic acid as N-cap. When ligated to RLNpfF (5566) has 2.5 fold better potency in cyclin A compared to the one ligated to RLIF (5919). Incorporation of a methoxy or a hydroxyl substituent at 3 position in 5896 and 5920 improves cyclin A activity compared to the mono substituted counterparts (5851, 5919) (figure 5.4). Alkoxy or hydroxyl substitution provides additional hydrophobic complementarity with secondary pocket in addition to H-bonding interactions of the OH with Trp217. The activity profiles of these two compounds confirms the hypothesis that having a basic group at 4 position and an alkyl group at 3 position improves the activity

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of benzamide FLIPs through ion-pairing and van der Waals interactions. Based on improvement in the activity profiles of 3,4-disubstituted benzoic acid N-caps, 5896 and 5920, next generation fragment alternatives included modifications on the piperazine moieties for additional hydrophobic interactions. These compounds were designed with alkoxy or hydroxyl substituents at 3-position and piperazine with additional alkyl groups at the 4 position of benzoic acid as shown in scheme 5.10.

Figure 5.4 Docked pose of 5920 in CDK2/cyclin A

Examination of the binding mode of 5920 reveals that N4 of the piperazine- 1ylmethylbenzoic acid Ncap forms ion-pairing interactions with Glu224, the carbonyl contributes to an H-bond with Gln 254 and 3-hyrdoxyl can H-bond to Trp217.

Scheme 5.10 Design and optimization of benzoic acid derivatives for interaction with