If i

Figure 7. The influence of potential hydrogen bond acceptors as estimated by the number of O and N atoms on susceptibility of compounds to Pgp efflux. A compound is determined to be a Pgp substrate in this study if it has directional transport greater than 2 in transport studies with L-MDR1 cells. The number of compounds showing directional transport greater than 2 over the total number of compounds in each category is presented above the bar.

toward false positive or false negative results. The majority of incorrect predictions had transport ratios between two and three. The second modeling approach determined the probability that a compound would be subject to Pgp efflux based on calculated physical chemical parameters. This approach yeilded similar predictive potential to the KNN approach. Although both of these models could be useful for virtual screening, the greatest value came from the potential to resolve out of the model structural features conferring Pgp susceptibility. In the case of the KNN model specific functional groups could be identified which conferred susceptibility to Pgp. The functional groups identified were consistent with those identified through manual comparison of structures, and tended to show groups with greater number of hydrogen bond acceptors as liabilities which increased Pgp efflux. QSAR analysis using molecular operating environment (MOE) resolved 7 physical chemical descriptors which were most influential in determining susceptibility to Pgp. These descriptors were heavily weighted toward polarity (cLogP; sLog P) and hydrogen bond acceptors (number of nitrogen atoms, surface area of hydrogen bond acceptors, etc). This trend toward increasing hydrogen bond acceptors in compounds which are Pgp substrates is consistent with reported predicitive models for identifying Pgp substrates. Seelig et. al. (Seelig, 1998; Seelig and Landwojtowicz, 2000) evaluated the chemical properties of 100 Pgp substrates, inhibitors, inducers and non-substrates revealing a pattern where two hydrogen bond acceptors separated by 4.6 A or three acceptors positioned 2.5 A from each other were consistent motifs in compounds interacting with Pgp. Other predicitive models have also implicated hydrogen bonding as a determinant for Pgp substrate recognition, and studies on a homologous series suggest a correlation between hydrogen bond acceptor potency and potential to interact with Pgp (Gombar, Polli, Humphreys, Wring, and Serabjit-Singh, 2004; Osterberg and Norinder, 2000; Xue et al., 2004). Collectively these results suggest that one approach to reducing Pgp efflux of the compounds is to lower the hydrogen bond acceptor potency.

Applying the strategy of reducing hydrogen bond acceptors combined with the empirical SAR proved successful eventually resulting in over 70% of the new discovery compounds having good pharmacological activity and low Pgp transport. However due to metabolism related liabilities in the core structure, a new structural template had to be pursued which presented new challenges.

In contrast to the initial series in which Pgp susceptibility could be controlled by altering the substituents at two positions, Pgp liability was intrinsic to the core structure of the new series. The effects of specific substituents on Pgp transport did not transfer from the first structural series to the second. Instead, modification which increased the log P of compounds resulted in increased Pgp efflux. As noted previously Pgp-substrate interactions appear to entail partitioning of the drug into the membrane followed by interactions of the substrate with the protein. Increasing Pgp efflux with increasing Log P would be consistent with membrane partitioning being the primary limitation to transport for this series suggesting that determinants that confer high potential for recognition and transport by Pgp are intrinsic to the core structural template. In this series an amide bond was added to the core structure in place of a secondary amine. Addition of an amide bond in the first chemical series had previously been shown to dramatically increase susceptibility of compounds to Pgp. Initial attempts to replaced the amide, to introduce direct modifications to the amide bond, or to introduce steric hindrance to the amide bond reduced Pgp transport, but at the expense of pharmacological potency (figure 8a and b). Consequently, the structural features which conferred susceptibility to Pgp could not be separated from features required for pharmacological activity. The solution to this problem came through modification of the hydrogen bond acceptor potential of the amide bond. As noted previously, SAR from the initial series indicated that minimizing hydrogen bond acceptor potential could reduce susceptibility to Pgp efflux. Placing an electron withdrawing substituent (CF3) immediately adjacent to the amide bond reduces its electron density, making it a weaker hydrogen bond acceptor. Incorporating this modification into this series resulted in compounds which were not Pgp substrates and maintained high affinity to the biochemical target. Using this approach as well as other approaches to decrease the electron density of the amide bond, several compounds were prepared which maintained nanomolar potency and were not Pgp substrates. Thus, by combining empirically derived SAR with our current functional understanding of Pgp-substrate interactions strategies were developed for making potent compounds with high potential for CNS penetration such that 80-90% of compounds being prepared in this series were not substrates for human Pgp.

While in vitro studies indicated low susceptibility to human Pgp efflux and good passive permeability for these compounds, several of the compounds


MDR1 transport ratio (BtoA/AtoB)

Papp LLC PK1 (cm/s E-6)

















C h3




HsC C H,












Figure 8. : Influence of amide modifications on human Pgp mediated directional transport of new structural series. a) Pgp transport versus passive permeability for compounds containing an unmodified amide bond adjacent to R1 (♦) and compounds with modified amide bonds (■). b) Influence of amide associated substituents and modifications on Pgp transport, passive permeability, and pharmacological potency of compounds in the new structural series. Modifications which reduced Pgp efflux included replacement of the amide, methylation of the amide N, steric hindrance to the amide, and incorporation of electron withdrawing groups adjacent to the amide. Only incorporation of electron withdrawing groups retained potency while reducing Pgp efflux.

maintained significant transport by rodent Pgp and showed low brain penetration in rats. Therefore the decision to pursue clinical studies in man requires considerable confidence that the in vitro human Pgp results will extrapolate to brain penetration in man. Some confidence that the low Pgp transport would extrapolate to better brain penetration in man was obtained from analysis of brain tissue and plasma drug levels in monkeys used for receptor occupancy studies. Monkey MDR1 shares 96% homology with human Pgp in which most of the amino acid substitutions are in positions which are not anticipated to significantly alter substrate interaction. With few exceptions compounds which show Pgp B to A/ A to B transport ratios lower than 3 have brain tissue drug levels between 40% and 140% of the plasma drug levels. In contrast drug levels of compounds with transport ratios greater than 3 are all below 20% of the plasma drug concentrations. Although species differences between human and monkey CNS penetration may exist for some compounds, the general trend observed suggests that in spite of the species difference in rodent, the low Pgp efflux observed for human Pgp transport in vitro will translate to high CNS penetration in vivo.


The case study presented in this chapter illustrates the potential value of using in vitro transport models to establish structure based approaches to overcome Pgp efflux. The successful application of this approach requires that the in vitro model used has direct relevance to the barrier to CNS transport, and that development of SAR take into account mechanistic aspects of Pgp transport. In addition to this case study, we have found that establishment of structure transport relationships for Pgp have proven valuable in several CNS programs in which Pgp efflux has been an issue. Although specific SAR from one chemical series may not directly translate to other programs, general principles and strategies can be applied across different programs. This has allowed us to identify sites conferring Pgp susceptibility more rapidly and has yielded an assortment of approaches to overcome the liabilities in new structural series. With increased knowledge of Pgp-drug interactions and the establishment of better global SAR for Pgp transport, a broader array of strategies for limiting Pgp efflux should become available. However, it should be recognized that even with extensive SAR, identifying structural modifications to limit Pgp efflux which are compatible with maintenance of pharmacological potency, target specificity, and good drug metabolism and pharmacokinetic properties will remain a major challenge. The blood-brain barrier is an added complication to the already difficult task of transforming a pharmacologically active agent into a drug. Overcoming the numerous hurdles to discovery and development of a safe effective drug requires the coordinated efforts of medicinal chemists, biologist, pharmacologists, and drug metabolism scientist. In this regard effective communication is the most effective tool in the strategic development of CNS drugs.


The authors would like to thank Sergey Krymgold, Xiadong Shen Scott Fauty,

Todd Killino and William Neway for technical assistance on portions of this work.

The authors would also like to thank Chris Culberson, Rebecca Ann Perlow-

Poehnelt, Georgia B. McGaughey, , Chris Tong, Ken Korzekwa, Neville Anthony,

Raymond Evers and Tomoyuke Ohe for valuable discussions and suggestions.


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