Synthesis of plasmepsin II inhibitors - Potential antimalarial agents

Article abstract of DOI:10.3390/80700556

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 resista

Full text of DOI:10.3390/80700556

Molecules 2003, 8, 556–564
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis of Plasmepsin II Inhibitors – Potential Antimalarial
Agents
Reto Mueller 1, Marianne Huerzeler 1 and Christoph Boss 2,*
1 University of Applied Sciences Basel, Gründenstrasse 40, CH-4132 Muttenz, Switzerland;
E-mail: m.huerzeler@fhbb.ch
2 Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, CH-4123 Allschwil, Switzerland;
Tel. (+41)-61-487-4561, Fax (+41)-61-487-4500
* Author to whom correspondence should be addressed; E-mail: christoph.boss@actelion.com
Received: 16 June 2003; in revised form: 25 June 2003 / Accepted: 25 June 2003 / Published: 15 July
2003
Abstract: 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.
Keywords: Malaria, Plasmepsin II, Reductive Amination, Suzuki Coupling.
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

Molecules 2003, 8
557
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.
Endocytosis of host cell cytosol
H+ O2
OxyHemoglobin
-
H2O + 1/2O2
2O2
H2O2
Plasmepsin I, II
Food Vacuole
Ferroproto-
porphyrin
Heme
Large Peptides
Falcipain
Polymerization
Hemozoin
Small peptides
Amino peptidase
Amino acids
Scheme 1 presents a simplified overview of processes in the food vacuole and shows many
interesting targets for drug development [3-5]. 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 IC50 on PM II of 3.6 µM. By
variation of different parameters like ring size, functional groups of substituents or chain length of

Molecules 2003, 8
558
alkyl groups, the preferred key intermediate identified for further optimization cycles was 4-amino-
piperidine-tert-butylcarbamate (1) [7].
Scheme 2.
O
R'
NH2
R'''
N
N
R''
CF3
m(H2C)
(CH2)n
N
O
N
N
RIV
O
Initial Lead
1
for definitions of R', R'',
R''' and RIV see
IC50 (PM II): 3.6 µM
WO 02/24649
Part of the work presented here, was undertaken to elaborate the Structure-Activity-Relationships
(SAR) with respect to variations of RII and RIV (Scheme 2, RI=H; for RIII either the trans-4-pentyl-
cyclohexanoyl- or the 4-pentyl[2.2.2]bicyclooctanoyl-unit was introduced as a replacement of the 4-
pentyl-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 RIII 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 4-
alkoxy-substituted benzyl group were also prepared (see Tables 1 and 2).

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559
Scheme 3.
Suzuki couplings:
O
O
Br
3 mol% Pd(PPh3)4
H
H
+
N
K2CO3 (aq.)
Toluene, i-PrOH
.
HCl
B(OH)2
N
2
3
4
Scheme 4. Representative synthetic pathways for the preparation of 4-amino-piperidine
based PMII inhibitors
O
NH2
N
HN
C5H11
Cl
a) 4, MeOH
6
N
b) NaBH4
NEt3, CH2Cl2
N
O
O
O
O
5
1
O
O
N
N
C5H11
C5H11
4 M HCl / dioxane
N
N
N
H
N
.
HCl
O
O
7
8
O
N
O
H
OH
C5H11
9
N
N
NaBH(OAc)3
CH2Cl2
OH
10

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560
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-pentyl-
cyclohexanoyl unit as RIII. With respect to R1 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 R2 = 2-imidazolyl, where already the pyridyl substituted compound exhibits low
activity). With respect to R2, 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.
O
Table 1:
N
R1
N
R2
IC50 (PM II) in nM
R1
Row
1
N
O
R2
N
189
32
50
64
OH
2
3
117
651
151
189
OH
N
4
5
6
137
61
937
N
N
2255
2736
HN

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561
Table 2 gives the results for the PM II inhibitors bearing the 4-pentyl[2.2.2]bicyclooctanoyl moiety
in position RIII. The compounds possessing a 4-pyridyl group in position R1 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 R2 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 4-
pyridyl (row 3, Table 2) unit results in derivatives exhibiting comparable activities as analogous
compounds from Table 1. The influence on changes in activity by moving from trans-4-pentyl-
cyclohexanoyl as RIII to 4-pentyl[2.2.2]bicyclooctanoyl as RIII seems to be weak. Both substituents
lead to the same SAR within the respective series of PM II inhibitors.
O
Table 2:
N
R1
N
R2
IC50 (PM II) in nM
R1
Row
1
N
R2
313
37
92
OH
2
3
808
N
4799
107
108
N
4
5
6
697
1247
651
HN
S

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562
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.
Acknowledgements
The authors would like to thank Dr. T. Weller and Dr. F. Spindler for helpful discussions, Prof H.
Ramuz for his interest in the present work and S. Meyer for performing the plasmepsin II screening.
Experimental
General
1H-NMR spectra were recorded at 300 MHz using a Varian Oxford NMR 300 and chemical shifts
are reported in δ values (ppm) using DMSO-d6 as internal standard (unless otherwise specified).
Purities of all compounds were over 95% and were determined by either 1H-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.
Selected Procedures [11]
4-Pyridin-4-yl-benzaldehyde (4)
To a solution of 3-bromopyridine hydrochloride (3) (2.92 g, 15 mmol, 1 eq) in toluene (30 mL) was
added iso-propanol (30 mL), 4-formylphenylboronic acid (2) (2.48 g, 16.5 mmol, 1.1 eq) and 2 M
aqueous K2CO3 (40 mL, 80 mmol, 5.3 eq). The solution was degassed with N2 for 15 minutes. After
adding Pd(PPh3)4 (0.519 g, 0.45 mmol, 0.03 eq), the solution was refluxed for 7 h under argon. The
reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined
organic layers were washed twice with brine, dried over MgSO4 and the solvent was evaporated.

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Recrystallization from ethyl acetate gave 2.13 g (77%) of 4-pyridin-4-yl-benzaldehyde (4) as yellow
1
needles. LC: tr 2.41’; MS: 184 [M+H]+; H-NMR: 10.10 (s, 1H), 8.71 (d, 2H), 8.06 (s, 4H), 7.77 (d,
2H).
4-(4-Pyridin-4-yl-benzylamino)-piperidine-1-tert-butyl carbamate (5)
A solution of 4 (1.07 g, 5.33 mmol, 1 eq) and 1 (976 mg, 5.33 mmol, 1 eq) in methanol (30 mL)
was refluxed for 4 h. After cooling to r.t., NaBH4 (222 mg, 5.87 mmol, 1.1 eq) was added carefully
over 30 minutes. After 2 h of additional refluxing, the solution was carefully diluted with water and
extracted three times with ethyl acetate. The combined organic layers were washed twice with brine,
dried over MgSO4 and the solvent was evaporated to give 1.90 g (96%) of compound 5 as a yellow
solid which could be used in further transformations without purification [7]. LC: tr 2.68’; MS: 368
[M+H]+; 1H-NMR: 8.63 (d, 2H), 7.82-7.41 (m, 6H), 3.89-3.74 (m, 3H), 3.79 (s, 1H), 3.08-1.73 (m, 5H)
1.40 (br s, 9H) 1.28-1.09 (m, 2H).
4-[(4-trans-Pentyl-cyclohexanecarbonyl)-(4-pyridin-4-yl-benzyl)-amino]-piperidine-1-tert-butyl
carbamate (7)
To a solution of trans-4-pentylcyclohexanecarboxylic acid (485 mg, 2.45 mmol, 1 eq) in CH2Cl2
(20 mL) at 0°C was added oxalic acid chloride (0.35 mL, 4.14 mmol, 1.7 eq) and 3 drops of DMF. The
mixture was stirred in an N2 atmosphere for 1 h and carefully evaporated (40°C, 200 mbar, argon at
inlet) [12]. The obtained yellowish viscous acid chloride 6 was dissolved in an Ar atmosphere in
CH2Cl2 (10 mL), followed by the addition of NEt3 (1.2 mL, 8.61 mmol, 3.5 eq) and 5 (900 mg, 2.45
mmol, 1 eq) dissolved in CH2Cl2 (10 mL). Stirring was continued at r.t. for 12 h. The mixture was
diluted with ethyl acetate, washed once with saturated Na2CO3 solution and with brine, dried over
MgSO4 and the solvent was evaporated. The crude material was purified by column chromatography
(silica gel 60, DCM/Hex/MeOH 10:4:1) to give 623 mg (46%) of the amide 7 as white filaments [7].
1
LC: tr 5.85’; MS: 548 [M+H]+; H-NMR in CDCl3 (shows both amide rotamers with a ratio of ~1:1):
8.63-8.52 (m, 2H), 7.65-7.14 (m, 6H), 4.73-4.60 (m, 1H), 4.31, 4.24 (s, 2H, benzylic methylene of both
rotamers), 4.19-3.92 (m, 2H), 2.77-0.56 (m, 36H).
4-trans-Pentyl-cyclohexanecarboxylic acid piperidin-4-yl-(4-pyridin-4-yl-benzyl)-amide hydrochloride
(8)
A solution of compound 7 (617 mg, 1.13 mmol, 1 eq) in 4 M HCl / dioxane (10 mL, 40 mmol, 35.4
eq) was stirred for 4 h at r.t. under argon. The solvent was evaporated to give 8 as a white solid in
quantitative yield [7]. LC: tr 3.67’ (purity of >95%), MS: 448 [M+H]+.

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4-trans-Pentyl-cyclohexanecarboxylic acid [1-(4-hydroxy-benzyl)-piperidin-4-yl]-(4-pyridin-4-yl-
benzyl)-amide (10)
To a solution of compound 8 (63 mg, 0.131 mmol, 1 eq) in CH2Cl2 (10 mL) was added 4-hydroxy-
benzaldehyde (64 mg, 0.524 mmol, 4 eq) and NaBH(OAc)3 (138 mg) and stirring was continued at r.t.
under argon for 12 h. The reaction mixture was washed once with saturated NaHCO3 solution, dried
over MgSO4 followed by chromatographic purification to give 42.3 mg (58%) of the final compound
1
10 as a white solid. LC: tr 4.33’; MS: 555 [M+H]+; H-NMR: 8.68-8.47 (m, 2H), 7.66-6.61 (m, 10H),
5.27 (s, 1H), 4.59, 4.56 (s, 2H, benzylic methylene of both rotamers) 3.86 (m, 1H), 3.19 (s, 2H), 3.07-
2.79 (m, 2H), 2.62-2.41 (m, 1H), 2.31-0.59 (m, 26H).
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