Synthesis of Plasmepsin II Inhibitors – Potential Antimalarial Agents

A new series of plasmepsin II (PM II) inhibitors has been prepared based on 4-aminopiperidine-tert-butyl-carbamate (1). These compounds might be useful as antimalarial drugs acting via a new mechanism, and therefore be less susceptible to parasite resistance now often observed with current antimalarial therapies. Some of the final compounds prepared exhibited encouraging inhibitory activity towards PM II.


Introduction
The World Health Organization (WHO) estimates that more than 280 million people are infected with malaria. This situation worsens because drug resistant strains of the malaria parasite are spreading quickly. Among four different strains, Plasmodium falciparum causes the largest number of lethal infections and often shows substantial resistance against Chloroquin, still the most important drug used to treat malaria infections.
Plasmodia are transmitted by Anopheles gambia mosquitoes, which are developing greater pesticide resistance. Climate changes, changing agricultural habits and increased mobility are assumed to be responsible for the spread of the mosquito and the disease into formerly unaffected regions. Political conflicts in regions affected by malaria have made disease control difficult. Thus, possible new drug targets are urgently needed which allow for the development of new medicines acting via different mechanisms in order to avoid fast development of resistance [1].
Among the various targets, the hemoglobin degradation pathway -involving several parasite specific enzymes like the plasmepsins [6] and the falcipains [2] -is of major interest. One possibility to strike against the parasite is to inhibit an enzyme within this pathway [2], interrupting the nutrition source and therefore eliminating the parasite by starvation. Scheme 1 presents a simplified overview of processes in the food vacuole and shows many interesting targets for drug development [3][4][5]. PM II has been chosen because its inhibition follows a completely new and promising pathway of fighting against the malaria parasite having a good potential of being devoid of fast emerging resistance. This target fits well within our core competencies in the field of aspartic protease inhibition as a therapeutic strategy [6], which has been proven especially valid in the field of HIV-protease inhibition to treat AIDS. The lead compounds depicted below (Scheme 2) emerged from a high throughput screening (HTS) of a commercial library containing 50,000 compounds for PM II inhibitory activity.
One of the preferred lead structures found in the HTS effort considered as being suitable for optimization as PM II inhibitors is depicted in Scheme 2 exhibiting an IC 50 on PM II of 3.6 µM. By variation of different parameters like ring size, functional groups of substituents or chain length of alkyl groups, the preferred key intermediate identified for further optimization cycles was 4-aminopiperidine-tert-butylcarbamate (1) [7]. Part of the work presented here, was undertaken to elaborate the Structure-Activity-Relationships (SAR) with respect to variations of R II and R IV (Scheme 2, R I =H; for R III either the trans-4-pentylcyclohexanoyl-or the 4-pentyl[2.2.2]bicyclooctanoyl-unit was introduced as a replacement of the 4pentyl-benzoyl-group for which results are published in [6]). Therefore the synthesis of biaryl aldehydes 4 by Suzuki coupling reactions [8] (Scheme 3) was investigated and optimized. Aldehydes 4 were then coupled in a reductive amination reaction onto N-Boc-4-aminopiperidine (1) [7,9]. The subsequent acylation of amine 5 with suitable carboxylic acids or carboxylic acid chlorides (e.g. 6) under standard reaction conditions resulted in the amides 7. Cleavage of the Boc-protecting group with 4 M HCl in dioxane gave the precursors 8. Final reductive amination with a diverse set of aldehydes (e.g. 9) following standard literature procedures in a parallel chemistry set-up resulted in the target inhibitors 10 which were tested for inhibition of plasmepsin II in a FRET-assay [10]. In order to assess the specificity and the selectivity of the PM II inhibitors (e.g. 10), the compounds were tested in FRET-assays for the inhibition of other aspartic proteases like plasmepsin IV, cathepsin D or cathepsin E. For the whole set of final compounds depicted in Tables 1 and 2, substantially lower or no inhibition of these aspartic proteases was detected.
The main goal of this investigation was to find out what types of biaryl units in combination with either the trans-4-pentylcyclohexanoyl-or the 4-pentyl[2.2.2]bicyclooctanoyl-unit in position R III led to the most active PM II inhibitors (e.g. 10; Scheme 4). In order to confirm the superior influence of a biaryl moiety on the inhibitory activity, a limited number of compounds bearing a 4-alkyl-or 4alkoxy-substituted benzyl group were also prepared (see Tables 1 and 2).

Results and Discussion
Tables 1 and 2 summarize the results for the two series of PM II inhibitors prepared according to the pathway depicted in Scheme 4. Table 1 shows compounds bearing the trans-4-pentylcyclohexanoyl unit as R III . With respect to R 1 the differences in activity between the compounds containing a 4-pyridyl moiety and the compounds containing a 3-pyridyl moiety are in the range of a factor of 2, with no clear preference for either pyridyl moiety. Replacement of the pyridyl units by an ethoxy group results in a 6-to 10-fold loss of activity (with the exception of the compounds given in row 6, Table 1, with R 2 = 2-imidazolyl, where already the pyridyl substituted compound exhibits low activity). With respect to R 2 , the small lipophilic 2-methyl-propyl unit leads to more active compounds than the introduction of larger, heteroatom containing (e.g. hydroxy-phenyls (rows 2, 3, Table 1) or pyridyls (rows 4, 5, Table 1) or highly hydrophilic units like 2-imidazolyl, leading to an almost complete loss of activity in this series.    Table 2 gives the results for the PM II inhibitors bearing the 4-pentyl[2.2.2]bicyclooctanoyl moiety in position R III . The compounds possessing a 4-pyridyl group in position R 1 are generally more active than the compounds having an isopropyl unit in this position. This reflects the same SAR tendencies already elaborated for the first series of inhibitors given in Table 1. In the second group of inhibitors ( Table 2) the introduction of a 2-imidazolyl unit in position R 2 is much less detrimental for the inhibitory activity (col. 1, row 4, Table 2). But again the small lipophilic 2-methyl-propyl group leads to the most active derivatives. The introduction of the 4-hydroxy-phenyl (row 2, Table 2) or the 4pyridyl (row 3, Table 2) unit results in derivatives exhibiting comparable activities as analogous compounds from   The most active compounds prepared within these optimization efforts exhibit potencies which are a 100-fold higher than the activity of the initial lead compound depicted in Scheme 2. It has been shown that 4-aminopiperidine might well serve as a template to develop non-peptidic inhibitors of PM II allowing for the use of a parallel chemistry setup for the fast preparation of small focused libraries. In future work the potency of the PM II inhibitors series described needs to be further improved. In addition the physicochemical properties of the inhibitors (e.g. clogP, polar surface area or molecular weight) have to be optimized towards more drug-like values. Investigations are ongoing and results will be published in due course. The rather poor physicochemical properties of the final compounds depicted in Tables 1 and 2 could raise questions about a non-specific interaction of the inhibitors with the enzyme. According to preliminary results obtained from X-ray analysis there is strong evidence for a specific interaction of the inhibitors with the active site of the enzyme. This fact confirms the results obtained in the selectivity screening discussed above. 1 H-NMR spectra were recorded at 300 MHz using a Varian Oxford NMR 300 and chemical shifts are reported in δ values (ppm) using DMSO-d 6 as internal standard (unless otherwise specified). Purities of all compounds were over 95% and were determined by either 1 H-NMR or LC-MS (Waters Micromass; ZMD-platform with ESI-probe with Alliance 2790 HT; Colum: 2x30mm, Gromsil ODS4, 3µm, 120A; Gradient: 0 -100% acetonitrile in water, 6 min, with 0.05% formic acid, flow: 0.45mL/min). All solvents were stored over molecular sieves, except wet solvents used in Suzuki reactions. All reagents were used as received from commercial sources without further purification.