In search of novel agents for therapy of tropical diseases and human immunodeficiency virus

Article abstract of DOI:10.1021/jm070763y

Malaria, sleeping sickness, Chagas' disease, Aleppo boil, and AIDS are among the tropical diseases causing millions of infections and cases of deaths per year because only inefficient chemotherapy is available. Since the targeting of the enzymes of the polyamine pathway may provide novel therapy options, we aimed to inhibit the deoxyhypusine hydroxylase, which is an important step in the biosynthesis of the eukaryotic initiation factor 5A. In order to identify new lead compounds, piperidines were produced and biologically evaluated. The 3,5-diethyl piperidone-3,5-dicarboxylates 11 and 13 substituted with 4-nitrophenyl rings in the 2 and 6 positions were found to be active against Trypanosoma brucei brucei and Plasmodium falciparum combined with low cytotoxicity against macrophages. The corresponding monocarboxylates are only highly active against the T. brucei brucei. The piperidine oximether 53 demonstrated the highest plasmodicidal activity. Moreover, compounds 11 and 53 were also able to inhibit replication of HIV-1.

Full text of DOI:10.1021/jm070763y

238
J. Med. Chem. 2008, 51, 238–250
In Search of Novel Agents for Therapy of Tropical Diseases and Human Immunodeficiency
Virus
Tim Goebel,† Daniela Ulmer,† Holger Projahn,† Jessica Kloeckner,† Eberhard Heller,† Melanie Glaser,‡ Alicia Ponte-Sucre,‡
Sabine Specht,§ Salem Ramadan Sarite,§ Achim Hoerauf,§ Annette Kaiser,# Ilona Hauber, Joachim Hauber, and
Ulrike Holzgrabe*,†
Institute of Pharmacy and Food Chemistry, UniVersity of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany, Laboratory of Molecular
Physiology, UniVersidad Central de Venezuela, Caracas, Venezuela, Institute of Medical Microbiology, Immunology and Parasitology,
UniVersity of Bonn, Sigmund-Freud-Strasse 29, 53105 Bonn, Germany, FH-Bonn-Rhein-Sieg, Von-Liebig-Strasse 20, 53359 Rheinbach,
Germany, and Heinrich-Pette-Institute for Experimental Virology and Immunology, Martinistrasse 52, 20251 Hamburg, Germany
ReceiVed June 27, 2007
Malaria, sleeping sickness, Chagas’ disease, Aleppo boil, and AIDS are among the tropical diseases causing
millions of infections and cases of deaths per year because only inefficient chemotherapy is available. Since
the targeting of the enzymes of the polyamine pathway may provide novel therapy options, we aimed to
inhibit the deoxyhypusine hydroxylase, which is an important step in the biosynthesis of the eukaryotic
initiation factor 5A. In order to identify new lead compounds, piperidines were produced and biologically
evaluated. The 3,5-diethyl piperidone-3,5-dicarboxylates 11 and 13 substituted with 4-nitrophenyl rings in
the 2 and 6 positions were found to be active against Trypanosoma brucei brucei and Plasmodium falciparum
combined with low cytotoxicity against macrophages. The corresponding monocarboxylates are only highly
active against the T. brucei brucei. The piperidine oximether 53 demonstrated the highest plasmodicidal
activity. Moreover, compounds 11 and 53 were also able to inhibit replication of HIV-1.
Scheme 1. Polyamine Pathway and Available Inhibitors of the
Introduction
Enzymesa
The malaria parasite Plasmodium is a major cause of global
human mortality with Plasmodium falciparum being the deadli-
est protozoan to humans, causing one to two million deaths per
year, mostly in subsaharian Africa.1 Trypanosomatids such as
Trypanosoma brucei gambiense and T. b. rhodesiense are the
causative agents of African sleeping sickness, T. cruzi of South
American Chagas’ disease, and Leishmania donoVani, L. major,
and L. tropica of different forms of leishmaniasis, e.g., Aleppo
boil. Worldwide, millions of people are infected by these
parasites and tens of thousands of patients die every year.2 The
chemotherapy against all these diseases is very limited and
unsatisfactory because of the emerging levels of resistance
against plasmodicidal drugs and the lack of safe drugs against
all forms of trypanosomiasis.3 Even with the increasing number
of infections with resistant and multiresistant pathogens, the
major pharmaceutical companies are mostly neglecting this
urgent medical problem and are frequently leaving the field of
antiinfective drug discovery, especially in the case of tropical
diseases.4,5
Thus, the purpose of this study was the search for novel agents
active against plasmodia, trypanosomatids, and human immu-
nodeficiency virus type 1 (HIV-1) by targeting the enzymes of
* To whom correspondence should be addressed. Phone: 0931-888-5460.
Fax: 0931-888-5494. E-mail: holzgrab@pharmazie.uni-wuerzburg.de.
†
University of Wuerzburg.
Universidad Central de Venezuela. Current address: Institute for
‡
a DHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase.
Molecular Infection Biology, University of Wuerzburg, Roentgenring 11,
97070 Wuerzburg, Germany.
§
University of Bonn.
FH-Bonn-Rhein-Sieg.
the polyamine pathway (see Scheme 1). In fact, targeting of
enzymes of this pathway like the ornithine decarboxylase
(ODCa) [EC 4.1.1.17], adenosylmethionine decarboxylase
(AdoMetDC) [4.1.1.50], and the spermidine synthase (SPDS)
[EC 2.5.1.16] turned out to be valuable for both antiparasitic
chemotherapy and prevention;6–8 e.g., difluoromethylornithine,
#
Heinrich-Pette-Institute for Experimental Virology and Immunology.
Abbreviations: eIF5A, eukaryotic initiation factor 5A; DOHH, deoxy-
a
hypusine hydroxylase; DHS, deoxyhypusine synthase; ODC, ornithine
decarboxylase; AdoMetDC, adenosylmethionine decarboxylase; CQS, chlo-
roquine sensitive.
10.1021/jm070763y CCC: $40.75
2008 American Chemical Society
Published on Web 12/27/2007

NoVel Agents for Therapy
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 239
Complexing the essential catalytic metal ion (iron) was
hypothesized to be the mechanism of inhibition by the DOHH
by Park et al.,18 because the hydroxamic acid moiety and the
hydroxyl keton moiety of ciclopiroxolamine and mimosine,
respectively, are putatively iron chelating subunits.19 Following
the iron complexing hypothesis, 4-oxopiperidine 3-carboxylates
(Figure 1a), being chelators of the metal either via the enolizable
ꢀ-ketoester moiety (cf. acetylacetonate complexes of iron20) or
via the three nitrogens, in position 1 and in the pyridines18,21
were recently tested for the inhibitory properties in P. falciparum
and found to be more active than ciclopiroxolamine and
mimosine in the same test.16 However, this finding is an indirect
proof-of-principle of the hypothesis only because the piperidones
were not tested at the isolated enzyme. Some of the piperidones
lacked sufficient water solubility.
Moreover, the eIF-5A is an essential cofactor of the viral
regulatory protein Rev, which is important for HIV-1 replica-
tion.22 Consequently, the direct inhibition of AdoMetDC, DHS,
and DOHH with small molecules has been already shown to
block the Rev activity and thus the virus replication,23–26
indicating that inhibition of hypusine (especially DHS and
DOHH) is a preferred strategy for the development of anti-
HIV drugs.
Figure 1. (a) Structural formulas of the DOHH inhibitors ciclopirox-
olamine (1), L-mimosine (2), and 4-oxopiperidone 3-carboxylate (3).
(b) Points of variation of the piperidine skeleton.
These findings suggested the hypusine biosynthesis pathway
to be a target for antimicrobial therapy27 and therefore prompted
us to produce a target-oriented library of piperidine compounds
being able to complex the catalytic metal ion of the DOHH
and being adequately water-soluble. The library (see Figure 1b)
is composed of a variety of 4-oxopiperidine 3,5-dicarboxylates
and 3-monocarboxylates carrying different substituents in the
2 and 4 positions and on the nitrogen, of spiropiperidine
compounds, and of piperidine oximes and related ethers. The
library was tested for their in vitro and in vivo activity against
Plasmodium falciparum (NF-54), Trypanosoma brucei brucei,
Leishmania major, and bacteria, such as Staphylococcus aureus
and Staphylococcus epidermidis, Escherichia coli, and Pseudomo-
nas aeruginosa. Some representative compounds with high
activity in plasmodia or trypanosomatids were also subjected
to the inhibition assay of HIV-1 replication. Finally, most active
compounds were tested for their capacity to inhibit the growth
of the macrophage cell line J774.1. By application of this
strategy, we aimed to find lead structures for the development
of small compounds of selective activity against one of the
aforementioned microorganisms and low cytotoxicity.
which is a specific inhibitor of ODC, blocks the erythrocytic
schizogony of P. falciparum in culture, and recent results have
shown that spermidine synthase from the malaria parasite can
be inhibited by trans-4-methylcyclohexylamine with an inhibi-
tory effect on cell proliferation.9,10 In contrast to the enzyme
of the parasite, the drug inhibits the mammalian enzyme without
any antiproliferative effect.
The unusual amino acid hypusine (see Scheme 1) is a
posttranslational modification of the eukaryotic initiation factor
5A (eIF-5A) and necessary for eIF-5A activity.11 Hypusine is
formed in two steps under catalysis of deoxyhypusine synthase
(DHS) [EC 1.1.1.249] and by deoxyhypusine hydroxylase
(DOHH) [EC 1.14.99.29].12 DHS transfers an aminobutyl
moiety from the triamine spermidine to a specific lysine residue
in the eIF-5A precursor protein to give deoxyhypusine,13 and
subsequently DOHH hydroxylates this molecule, completing the
hypusine biosynthesis.
Previous studies have shown that mature eIF-5A formation
in plasmodia can be blocked by inhibition of DHS by means of
1,7-diaminoheptane in vitro.14 Additionally, the eIF-5A forma-
tion can be prevented by inhibition of DOHH. Findings in rat
testis have demonstrated that the DOHH can be inhibited by
the antifungal drug ciclopiroxolamine (Figure 1). Ciclopirox-
olamine showed antiangiogenetic effects in human vascular
endothelial cells (HUVEC) and antiproliferative effects in the
chick aortic arch sprouting assay.15 Moreover, ciclopiroxolamine
inhibits the in vitro proliferation of the chloroquine sensitive
(CQS) NF54 P. falciparum strain with an IC50 of 8.2 µM.
However, ciclopiroxolamine was ineffective in vivo in a rodent
malaria model.16
The plant amino acid L-mimosine (Figure 1) can reversibly
block mammalian cells at the late G1 phase and leads to a
notable reduction in the steady-state level of mature eIF-5A by
means of DOHH inhibition.17 These findings prompted the
determination of the antiplasmodial effect, resulting in an IC50
of 32 µM for the chloroquine susceptible (CQS) strain and 39
µM for the chloroquine resistant (CQR) strain. However,
mimosine was toxic in a rodent malaria model.16
Chemistry
The 4-oxopiperidine 3,5-dicarboxylates 1–18 (see Figure 2)
were achieved via a double Mannich condensation of 1 mol of
a corresponding primary amine, 2 mol of pyridine-2-carbalde-
hyde and 3- or 4-nitrophenylcarbaldehyde, and dimethyl oxo-
glutarate in analogy to Merz and Haller,28 Ashauer-Holzgrabe
and Haller,29 and Kuhl et al.30 Here, we tried to enhance the
water solubility of the dicarboxylates by introduction of polar
substituents attached to the nitrogens, e.g., carboxylate and
hydroxyl residues, and by attachment of nitro groups to the
aromatic ring in the 2 and 6 positions (Table 1a). In particular,
the latter variation led to compounds of higher water solubility.
Moreover, the diesters are pretty stable; they can be hydrolyzed
and subsequently decarboxylated only under strongly acidic
conditions (cf. compound 31 below).
The analogous monocarboxylates 19–24 (Table 1b) can be
obtained by conversion of 2,4,6-trioxotetrahydropyrane, formed
by solvolytic cleavage with methanol, with freshly destilled
pyridine-2-carbaldehyde and the correspondingly substituted

240 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Goebel et al.
Figure 2. Synthesis pathway to 4-oxopiperidine 3,5-dicarboxylates 1–18 (reagents and conditions, (a) ArCHO, R2NH2, –5 °C; (b) Ce(SO4)2, 0–5
°C), monocarboxylates 19–24, and dihydropyridine and tetrahydropyridine compounds 25–28 (reagents and conditions, (a) absolute MeOH, 25 °C;
(b) 2 equiv of 2-pyridinealdehyde, RNH2, –5 °C; (c) Ce(SO4)2, 0–5 °C).
primary amines (cf. ref 31). The monocarboxylates were always
isolated in the enol form. The corresponding dihydropyridine
and tetrahydropyridine compounds 25-28 (see Figure 2) can
be achieved by means of oxidation using cerium(IV) sulfate.28,29,31
In order to obtain the spiropiperidines 33–41, 44, 45, and 47
(see Figure 3), the corresponding 4-oxopiperidine 3,5-dicar-
boxylates 30a-d have to be synthesized via a double Mannich
condensation of ammonium bromide, 2 mol of the corresponding
freshly destilled aliphatic aldehyde, and dimethyl oxoglutarate.
The dicarboxylates were isolated as a mixture of keto–enol
tautomers mostly occurring as trans isomers with regard to the
alkyl groups. Ester hydrolysis and decarboxylation were per-
formed with concentrated hydrochloric acid to give the 2,6-
alkyl substituted and nonsubstituted 4-piperidones 31, which
are mainly isolated as cis isomers indicating a configurational
change of the alkyl groups during the course of the reaction,
which is in accordance with findings of Siener et al.32 N-
Benzylation can be achieved by means of benzyl bromide in
acetonitrile in the presence of an excess of potassium carbonate,
resulting in high yields of the piperidone 42. Via a Strecker
synthesis33 the nitrile compounds 32a-f and 43a,b were
synthesized in glacial acid using trimethylsilyl cyanide and using
aniline and benzylamine as amine components. In order to avoid
the hydrolysis of the amine, the reaction was stopped by addition
of a concentrated ammonia solution. Only the cis isomer of all
compounds was isolated because in the case of the trans isomer,
the carbonyl group is difficult to attack because of one axial
alkyl group. Conversion of the compounds 32a-f and 43a,b
with chlorosulfonyl isocyanates and subsequent refluxing in 1
M hydrochloric acid lead directly to the spiro derivatives 33–36
and 44. The nitrogen N3 in the imidazolidione skeleton can be
selectively alkylated to give 37–41 and 45. The spiro compound
47 was obtained via the amide 46 being formed by conversion
with concentrated sulfuric acid and working-up with concen-
trated ammonia solution. Ring closure was achieved with
formamide at 200 °C in analogy to Röver et al.34 In contrast to
Röver et al., the reduced compound 47 was forthwith isolated.
Thus, the reduction step with NaBH4 was dispensable.
Since oximes are known to complex iron ions, e.g., [2,2′]-
furildioxime,35 corresponding piperidone oximes were synthe-
sized (see Figure 4). The N-alkylated oxime of the 4-piperidone
48 was synthesized starting from the piperidone, which can
be alkylated with phenylpropyl bromide. Subsequently, the
ketone can be converted to the oxime by using hydroxylamine
hydrochloride. 48 was alkylated with dichlorobenzyl bromide
to give the oxime ether 49.
The synthesis of the corresponding 4-piperidinecarbaldehyde
oximes started from pyridine-4-carbaldehyde whose aldehyde
function was first protected by means of ethylene glycol. The
resulting acetal was N-alkylated with phenylpropyl bromide in
the microwave within 2 h to give compound 50, which can be
hydrogenated with PtO2/H2 to 51. Acetal cleavage and subse-
quent oxime formation with hydroxylamine hydrochloride gave
the piperidone oxime 52, which was alkylated again with
dichlorobenzyl chloride to yield the oxime ether 53.
Results and Discussion
All piperidine compounds were subjected to the aforemen-
tioned microbiological assays. None of the compounds showed
considerable activity against the bacteria S. aureus, S. epider-
midis, P. aeruginosa, and the fungi C. albicans (data not shown).
Additionally, none of the compounds inhibited the formation
of biofilm produced by S. aureus (data not shown). The
inhibition activity against P. falciparum, T. brucei brucei, and
L. major is summarized in Table 1 as well as the toxicity against
macrophages after 48 h. Structure–activity relationships (SAR)
will be qualitatively discussed by chemical group.
Whereas all piperidone dicarboxylates were not active against
L. major, many of them show activity against T. brucei brucei
and P. falciparum. For both parasites the activity (IC50 and ED50
values) is felt in the higher micromolar range of concentration if
the piperidones are armed with pyridines rings in the 2 and 6
positions (1–7) and regardless of the substitution on the nitrogen.
There are two exceptions, which are the compounds having a
benzylic moiety (i.e., benzyl for 9 and pyridinylmethyl for 8); both

NoVel Agents for Therapy
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 241
Table 1. Antimicrobial Activity of All Piperidone Compounds against P. falciparum (NF-54), T. brucei brucei, and L. major and Cytotoxicity Measured
in Macrophages (J774.1)a
(a) 4-Oxopiperidine 3,5-Dicarboxylates
ED50 (µM)
compd
Ar
R1
R2
IC50 (µM)P. falciparum
T. brucei brucei
L. major
macrophage after 48 h
1
2
3
4
5
6
7
8
9
2-pyridine
2-pyridine
2-pyridine
2-pyridine
2-pyridine
2-pyridine
2-pyridine
2-pyridine
2-pyridine
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
na
28.09 ( 1.52
26.48 ( 7.64
22.84 ( 5.57
18.29 ( 2.59
24.14 ( 4.27
19.29 ( 2.28
23.98 ( 4.48
4.62 ( 1.39
11.13 ( 1.91
>100
>100
allyl
na
>100
>100
2-hydroxyethyl
3-hydroxypropyl
2-(2-hydroxy-ethoxy)ethyl
3-carboxypropyl
7-carboxypentyl
2-pyridinylmethyl
benzyl
na
>100
31.15 ( 0.14
32.02 ( 0.63
38.48 ( 6.17
46.00 ( 5.92
33.83 ( 2.81
29.87 ( 2.32
33.99 ( 1.56
na
>100
na
65.04 ( 3.60
>100
na
na
50.34 ( 8.26
>100
na
10.88 (72 h)
55.99 ( 6.45
10.20 (24 h)
10
11
12
13
14
15
16
17
18
4-NO2phenyl
4-NO2phenyl
4-NO2phenyl
4-NO2phenyl
3-NO2phenyl
3-NO2phenyl
3-NO2phenyl
3-NO2phenyl
phenyl
CH3
C2H5
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
allyl
na
2.41 ( 0.39
3.06 ( 0.55
3.01 ( 0.27
2.72 ( 0.51
10.57 ( 2.83
3.93 ( 1.10
2.66 ( 0.49
2.49 ( 0.79
4.86 ( 0.63
>100
nd
50.29 ( 11.70
>100
allyl
11.03 (72 h) + (m)
benzyl
11.91 (72 h) + (m)
>100
18.39 ( 2.93
>100
benzyl
10.06 (72 h) + (m)
nd
4-chlorbenzyl
4-methoxybenzyl
4-methylbenzyl
benzyl
na
na
na
na
na
>100
>100
>100
>100
nd
46.64 ( 9.86
>100
49.78 ( 16.80
>100
allyl
>100
(b) 4-Oxopiperidine 3-Monocarboxylates
ED50 (µM)
compd
R
IC50 (µM)P. falciparum
T. brucei brucei
L. major
macrophage after 48 h
19
20
21
benzyl
4-methylbenzyl
4-chlorbenzyl
na
nd
1.37 ( 0.02
0.47 ( 0.17
0.32 ( 0.09
25.97 ( 6.76
83.83 ( 10.38
87.93 ( 32.99
33.17 ( 0.69
34.87 ( 0.45
32.84 ( 0.49
12.03 (72 h)
1.7 (48 h)
nd
22
23
24
4-methoxybenzyl
3-methoxybenzyl
allyl
0.43 ( 0.17
>100
1.08 ( 0.48
30
>100
50
29.86 ( 0.33
>100
(35)
nd
>40 1.4 (24 h)
4-Oxo-1,4,5,6-tetrahydropyridine 3-Monocarboxylates
25
26
benzyl
4-chlorbenzyl
4.7 (24 h)
18 (24 h)
>100
nd
nd
nd
nd
nd
4-Oxo-1,4-dihydropyridine 3-Monocarboxylates
27
28
4-chlorbenzyl
4-methylbenzyl
9.4 (36 h)
40 (48 h)
9.1 (24 h)
>100
nd
>100
nd
>100
nd
(c) Spiropiperidines
ED50 (µM)
L. major
>100
>100
>100
>100
>100
nd
>100
41.10 ( 3.57
>100
>100
>100
nd
compd
R
R1
R2
IC50 (µM)P. falciparum
T. brucei brucei
macrophage after 48 h
33
34
35
36
37
38
39
40
41
44
45
47
CH3
phenyl
benzyl
phenyl
phenyl
benzyl
phenyl
benzyl
phenyl
phenyl
na
na
na
na
na
na
>100
>100
>100
36.02 ( 3.97
nd
>100
nd
C2H5
C3H7
C4H9
CH3
C2H5
CH3
>100
>100
24.36 ( 14.10
>100
2-hydroxyethyl
benzyl
4-nitrobenzyl
2-phenylethyl
3-phenylpropyl
20.28 ( 3.55
3.74 ( 0.77
18.05 ( 5.34
13.44 ( 6.69
30.97 ( 2.18
25.37 ( 1.61
44.65 ( 15.50
14.57 (72 h)
42.44 ( 14.49
32.30 ( 2.52
>100
C2H5
C2H5
na
na
na
na
na
>100
>100
nd
(d) Oximes and Corresponding Ethers
ED50 (µM)
L. major
nd
>100
>100
30.72 ( 3.15
IC50 (µM)P. Falciparum
T. brucei brucei
macrophage after 48 h
48
49
52
53
26.0
31.0
nd
8.29 (72 h)
11.83 (24 h)
nd
>100
>100
nd
>100
>100
3.86 ( 1.95
28.39 ( 5.92
a For comparison, pentamidine diisethionate ED50 (T. brucei brucei) ) 0.0027 µM; suramine ED50 (T. brucei brucei) ) 0.3 µM; eflornitine ED50 (T.
brucei brucei) ) 22.9 µM; mimosine IC50 (P. falciparum chloroquine-sensitive) ) 32 µM, IC50 (P. falciparum chloroquine-resistent) ) 39 µM, ED50
(Trypanosoma brucei brucei) > 100 µM; cyclopiroxolamine IC50 (P. falciparum chloroquine-sensitive) ) 8.2 µM), ED50 (T. brucei brucei) ) 0.62 ( 0.22
µM, ED50 (macrophages, 48 h) ) 4.62 ( 0.97 µM; amphotericin B IC50 (L. major) ) 5.07 ( 0.05 µM, IC50 (J774.1 macrophages) ) 68.6 ( 16.48 µM. na
) no activity. + (m) ) activity in mice. nd ) not determined.

242 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Goebel et al.
Figure 3. Synthesis pathway to the spiro compounds 33–41, 44, 45, and 47. Reagents and conditions: (a) MeOH, EtOH, MeOH/H2O or THF,
0-25 °C; (b) 37% HCl, 70 °C; (c) RBr, K2CO3, acetonitrile, 60 °C; (d) glacial acetic acid, aniline or benzylamine, TMSCN, 25 °C; (e) (1)
chlorosulfonyl isocyanate, absolute CHCl3, 25 °C; (2) 1 M HCl, reflux, 90 °C; (f) R2Br, K2CO3, acetonitrile, reflux, 70 °C; (g) (1) concentrated
H2SO4, 25 °C; (2) concentrated NH3, 0 °C; (h) formamide, 200 °C.
are active against T. brucei brucei, and 9 is additionally active
against P. falciparum. The in vitro effect of 9 on P. falciparum
chloroquine sensitive NF-54 strains results in the percentage of
parasitemia to be close to 0 after day 1 posttreatment with 40 µM
and after 2 day posttreatment with 20 µM (see Figure 5a).
Additionally, the percentage of survival of C57BI/6 mice treated
iv with 9 could be substantially prolonged (data not shown).
However, the effective doses for parasites and for the toxicity
against macrophages are close together, which makes 8 and 9 out
of the question as new lead compounds.

NoVel Agents for Therapy
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 243
Figure 4. Synthesis pathway to oximes 48 and 52 and their corresponding ethers 49 and 53. Reagents and conditions for 48 and 49: (a) Ph(CH2)3Br,
K2CO3, CH3CN, 60 °C; (b) K2CO3, NH2OH. HCl, absolute EtOH; (c) 2,6-dichlorobenzylchloride, KOtBu, 55 °C. Reagents and conditions for 52
and 53: (a) ethylene glycol, pTosOH, toluene; (b) Ph(CH2)3Br, 2 h, 90 °C, microwave; (c) PtO2, H2, 60 °C, 20 bar, microwave; (d) H2SO4, microwave;
(e) NH2OH-HCl; (f) KOtBu; (g) 2,6-dichlorobenzyl chloride; (h) MeOH/HCl.
The replacement of the pyridine rings with 4-nitrophenyl and
with some restriction with 3-nitrophenyl rings enhances the
activity against T. brucei brucei and P. falciparum considerably
in connection with low cytotoxicity against macrophages. In
addition, these compounds are much more soluble in water and
buffer than the pyridine substituted analogues. In particular, the
diethyl dicarboxylates 11 and 13 rather than the corresponding
dimethyl dicarboxylates 10 and 12 show activity against both
microorganisms in the lower micromolar concentration range
with concomitant very low cytotoxicity and can thus be regarded
as lead compounds for further improvement. This is supported
by the facts that the percentage of P. falciparum parasitemia of
C57BL/6 mice 4 days after treatment was close to 0 in the case
of 11 and the number of days of survival was increased (see
parts b and c of Figure 5). The 3-nitrophenyl substituted
compounds are only active against T. brucei brucei but exhibit
a higher toxicity.
Since in a preliminary study the corresponding 3-monocar-
boxylates 4-piperidones 21 and 24 have shown high activity
against the plasmodia after 48 h, compounds were synthesized
having varying substituents attached to the nitrogen. The
aforementioned compounds showed a weaker activity after 72 h
and did not completely kill the plasmodia in vivo. All other
monocarboxylates did not show any activity against plasmodia
and demonstrated a low activity against leishmania. However,
the monocarboxylates are among the compounds with the
highest activity against T. brucei brucei. With exception of the
N-3-methoxybenzyl substituted compound 23, the trypanocidal
activity was in the lower and submicromolar range of concen-
tration, more or less independent from substitution of the benzyl
substituent. Since the cytotoxicity in macrophages was found
to be 100 times higher than the trypanocidal activity, these
compounds can be regarded as leads for the development of
trypanocidal drugs. Unfortunately, using the classical synthesis
pathway, we were not yet able to produce the 2-and 6-(p-
nitrophenyl) substituted analogues whose corresponding dicar-
boxylates showed higher activity and water solubility in
comparison to the pyridine substituted ones. Thus, these
compounds are being currently produced via an independent
synthesis pathway.
Since mimosine and ciclopiroxolamine can be regarded as
dihydropyridine derivatives, structurally corresponding com-
pounds were considered in our library. The antiplasmodial
activity of the dihydro- and tetrahydropyridine monocarboxylates
25–28 was previously reported16 and was now found to be low
for 28 after 48 h. The trypanocidal activity of all compounds
25–28 is rather low. Since these compounds are difficult to
isolate from oxidation of the corresponding piperidone, we did
not embark on this strategy. The lack of activity may be due to
the fact that the ꢀ-ketocarboxylate is not enolizable because of
double bonds next to this moiety. Thus, complexation of the
DOHH via the metal ion is difficult.
A similar reason can be given for the missing antiplasmodial
activity of all spiro compounds. There is only one exception,
compound 39 having a p-nitrobenzyl substituent attached to the
imidazolidione ring. The antiplasmodial activity is in the same
range of concentration as that found for the most promising
compound 11. However, the therapeutic distance to cytotoxicity
of the compound is small. In contrast, some of the spiro
compounds show an interestingly high trypanocidal activity
being in the same concentration range as eflornitine. In
particular, a phenylalkyl substitution in position N3 of the
imidazolidione ring seems to be advantageous. With increasing
length of the alkyl spacer, the inhibitory activity increases (cf.

244 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Goebel et al.
Figure 5. (a) In vitro parasitemia of 9 on Pf/NF-54 strains. (b) In vitro parasitemia of C57BL/6 mice treated iv with 11. (c) Survival of C57BL/6
mice treated orally with 11.
38, 40, and 41). The activity can be enhanced by a nitrobenzyl
substitution resulting in 39, a compound with the highest activity
within this series and a therapeutic index of 10, which has to
be improved. Further SARs are difficult to derive. Because of
the multistep synthesis of these compounds and the fact that
activity is often connected with cytotoxicity, the spiro com-
pounds were discarded from the list of potential lead compounds
even though some active compounds were found.
Because of the hypothesis that compounds complexing metal
ions are able to inhibit the DOHH and to show antiplasmodial
activity, oximes with piperidone moiety (48 and 52) were
synthesized in addition to the corresponding dichlorobenzyl
ethers (49 and 53). Interestingly, neither the oximes 48 and 52
nor the oxime ether 49 is active against trypanosomes and
plasmodia, respectively. In contrast, the oxime ether of the
piperidine aldehyde 53 is active against plasmodia, trypano-
somes, and leishmania; however, the activity against leishmania
is low. In addition, 53, showing an IC50 of 8.3 µM after 72 h,
is able to kill the plasmodia completely (see Figure 6). Thus,
Figure 6. Parasitemia of C57BL/6 mice treated iv with 53.

NoVel Agents for Therapy
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 245
P. falciparum. Moreover, the compounds 11 and 53 appear to
be promising leads for anti-HIV-1 drug development. Besides
the good antiifective activity, the main advantage of these
compounds is their simple synthesis route. Most of the
compounds were satisfyingly water-soluble for biological evalu-
ation. In the next step, it is necessary not only to increase the
antimicrobial activity but also to reduce the cytotoxicity,
especially of compound 53, by variation of the substitution
pattern.
However, the small molecules were only tested against entire
organisms so far. Thus, the mechanism of action is not fully
understood. Whereas the piperidone 3,5-dicarboxylate 11 is in
principle able to complex the iron ion via the enolizable
ꢀ-ketoester moiety, which is also typical for many HIV integrase
inhibitors,36 or the terpyridine skeleton, the piperidine oximether
53 cannot chelate an iron. Since the pKA value of the piperi-
dine nitrogen was determined to be approximately 7,37 53 will
be partially protonated at physiological conditions and therefore
able to interact with the negatively charged amino acids in the
active site of the DOHH.38 Taken together, it has to be analyzed
whether the growth inhibition of the microorganisms and
inhibition of the replication of HIV-1 were caused by the
interference with the polyamine pathway, especially the block
of the DOHH. Respective work, e.g., the cloning of the DOHH
for assay development, is in progress.
Figure 7. Inhibition of HIV-1 BaL replication by piperidones. HIV-1
BaL-infected PM1 cells were cultured in the presence of the indicated
concentrations of 11, 53, or DMSO as solvent control. Culture medium
was changed at days 6 and 12 postinfection, and the cells were split
1:1. p24Gag antigen levels and cell viabilities were determined at days
6, 12, and 18. (A) The percentage of inhibition of virus replication in
the drug-treated cell culture, compared to the untreated controls, is
shown. (B) Cell viabilities from the same cultures were determined by
alamarBlue assay (Serotec).
Experimental Section
Material and Methods. Melting points were determined on a
model B 540 Büchi or a Sanyo Gallenkamp melting point apparatus
(Sanyo Gallenkamp, U.K.) and are uncorrected. 1H NMR and 13
C
NMR spectra were recorded with a Bruker AV 400 spectrometer
(1H, 400.132 MHz; 13C, 100.613 MHz). Chemical shifts (δ) are
expressed in ppm and coupling constants (J) in hertz. Abbreviations
for data quoted are as follows: s, singlet; d, doublet; t, triplet; m,
multiplet; br, broad. The centers of the peaks of CDCl3 and DMSO-
d6 were used as an internal reference. FT-IR spectra were recorded
on a Bio-Rad PharmalyzIR equipped with an ATR unit. TLC
analyses were performed on commercial silica gel 60 F254 aluminum
sheets; spots were further evidenced by spraying with a dilute
alkaline potassium permanganate solution. Microanalysis results (C,
H, N) of the new compounds agreed with the theoretical values
within (0.4%.
The dimethyl-4-oxopiperidine 3,5-dicarboxylates 1 and 2 were
prepared according to ref 30, and the methyl 4-oxopiperidine
3-monocarboxylates 20, 21, and 24 and the dihydro- and tetrahy-
dropyridine compounds 25-28 were synthesized according to
ref 16.
53 is the most active compound against plasmodia in the entire
library, but it is also cytotoxic, having a therapeutic index of
about 10. However, 53 will be a new lead compound for further
development. Since 53 is not able to form a complex with metal
ions, the mode of action has to be elucidated in the future.
Since the host cell eIF-5A, a cofactor of the HIV-1 protein
Rev, is involved in the virus replication, the inhibition of DOHH
and DHS will reduce the multiplication rate of the virus.
Consequently, the most active compounds 11–13 and 53 were
tested against the R5-tropic HIV-1 strain BaL and/or against
the X4-tropic HIV-1 strain NL4/3 in the human T cell line PM1.
In contrast to the compounds 12 and 13, where only 20%
inhibition of virus replication was observed, the compound 11
was able to completely inhibit replication of HIV-1 BaL at 5
µM and HIV-1 NL4/3 at 6 µM compared to the respective
control cultures (which were treated with the drug solvent
DMSO). When HIV-1 BaL infected PM1 cells were exposed
to a concentration of 10 µM 53, virus replication was inhibited
by 85% (Figure 7). Finally, the parallel analysis of cellular
metabolic activities revealed that these antiviral effects were
not caused by deleterious effects of the respective drugs on the
host cell.
Synthesis. General Procedure for the Synthesis of the 3,5-
Dialkyl-2,6-di-2-aryl-4-oxopiperidine 3,5-Dicarboxylates 1–18
and 30a-d, Modified after Reference 39. The corresponding
amine (0.02 mol), pyridine-2-carboxaldehyde (0.04 mol), and m-
and p-nitrobenzaldehyde were dissolved in methanol (20 mL) and
cooled to 0 °C. Over the course of about 1 h, dimethyl oxoglutarate
and diethyl oxoglutarate (0.02 mol) were added dropwise. The
solution was allowed to stand overnight at 5 °C. The product was
obtained by filtration of the precipitate formed. In the case where
no precipitate appeared, the solvent was removed in vacuo at 40-50
°C and the remaining oil dissolved in methanol/diethyl ether or
treated with diethyl ether. The obtained crystals could be washed
with a mixture of methanol/diethyl ether and recrystallized from
methanol. The piperidones could be isolated in yields ranging from
28% to 81%.
Conclusion
The evaluation of the library revealed several promising lead
compounds for further drug development, i.e., the 3,5-dieth-
ylpiperidone 3,5-dicarboxylates 11 and 13 for drugs against T.
brucei brucei and P. falciparum, and the corresponding mono-
carboxylates against the T. brucei brucei and the dichlorobenzyl
ether of the piperidine oxime 53 antiproliferative activity against
Dimethyl (2R,6S)-4-oxo-2,6-di(pyridin-2-yl)piperidine-3,5-di-
carboxylate (1): yield 33% (67%); mp 175 °C (170–171 °C).29
Dimethyl (2R,6S)-1-allyl-4-oxo-2,6-di(pyridin-2-yl)piperidine-
3,5-dicarboxylate (2): yield 87%; mp 133 °C (134 °C).29

246 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Goebel et al.
Dimethyl (2R,6S)-1-(2-hydroxyethyl)-4-oxo-2,6-di(pyridin-2-
yl)piperidine-3,5-dicarboxylate (3): yield 55%; mp 135 °C. Anal.
(C21H23N3O6) C, H, N.
Methyl (2′R,6′S)-4′-hydroxy-1′-(4-methoxybenzyl)-1′,2′,5′,6′-
tetrahydro-2,2′;6′,2′′-terpyridine-3′-carboxylate (22): yield 43%
(46%);34 mp 140 °C (140 °C).43
Dimethyl (2R,6S)-1-(2-hydroxypropyl)-4-oxo-2,6-di(pyridin-
2-yl)piperidine 3,5-dicarboxylate (4): yield 39%; keto-enol ratio,
1:3; mp 136 °C. Anal. (C22H25N3O6) C, H, N.
Methyl (2′R,6′S)-4′-hydroxy-1′-(3-methoxybenzyl)-1′,2′,5′,6′-
tetrahydro-2,2′;6′,2′′-terpyridine-3′-carboxylate (23): yield 56%
(63%);34 mp 143 °C (144 °C).43
Dimethyl (2R,6S)-1-[2-(2-hydroxyethoxy)ethyl]-4-oxo-2,6-
di(pyridin-2-yl)piperidine-3,5-dicarboxylate (5): yield 63%; mp
175 °C. Anal. (C23H27N3O7) C, H, N.
Methyl (2′R,6′S)-1′-allyl-4′-hydroxy-1′,2′,5′,6′-tetrahydro-
2,2′;6′,2′′-terpyridine-3′-carboxylate (24): yield 44%; mp 158
°C.43
4-[(2R,6S)-3,5-bis(methoxycarbonyl)-4-oxo-2,6-di(pyridin-2-
yl)piperidin-1-yl]butanoic acid (6): yield 70%; mp 169 °C. Anal.
(C23H25N3O7) C, H, N.
Dimethyl (2R,6S)-4,4-dihydroxy-2,6-dimethyl-piperidinium-
3,5-dicarboxylate hydrobromide (30a): yield 62%; mp 178–
182 °C.
8-[(2R,6S)-3,5-bis(methoxycarbonyl)-4-oxo-2,6-di(pyridin-2-
yl)piperidin-1-yl]octanoic acid (7): yield 43%; mp 130 °C. Anal.
(C25H29N3O7) C, H, N.
Dimethyl (2R,6S)-4-oxo-2,6-di(pyridine-2-yl)-1-(pyridine-2-
ylmethyl)-piperidine-3,5-dicarboxylate (8): yield 47%; mp 152–153
°C. Anal. (C25H24N4O5) C, H, N.
Dimethyl (2R,6S)-1-benzyl-4-oxo-2,6-di(pyridin-2-yl)piperi-
dine-3,5-dicarboxylate (9): yield 65%; mp 154 °C. Anal.
(C26H25N3O5) C, H, N.
Dimethyl (2R,6S)-1-allyl-4-hydroxy-2,6-bis(4-nitrophenyl)-
1,2,3,6-tetrahydropyridine-3,5-dicarboxylate (10): yield 39%; mp
154–155 °C. Anal. (C24H23N3O9) C, H, N.
Dimethyl (2RS,6RS)-4-hydroxy-2,6-diethylpiperidine-3,5-di-
carboxylate (30b): yield 97% (oil); keto–enol ratio, 1:2.
Dimethyl (2RS,6RS)-4-hydroxy-2,6-dipropylpiperidinium-3,5-
dicarboxylate hydrobromide (30c): yield 50%; mp 142–143 °C.
Dimethyl (2RS,6RS)-4-hydroxy-2,6-dibutyl-3,5-piperidinium-
dicarboxylate hydrobromide (30d): yield 24%; mp 144–146 °C.
(2R,6S)-2,6-Dimethyl-4-oxopiperidinium chloride (31a): yield
95%; mp 227–229 °C.
(2R,6S)/(2RS,6RS)-2,6-Diethyl-4-oxopiperidinium chloride
(31b): yield 93%; cis/trans ratio, 5:1; mp 181–183 °C.
(2R,6S)/(2RS,6RS)-2,6-Dipropyl-4-oxopiperidinium chloride
(31c): yield 92%; cis-/trans ratio, 4:1; mp 194 °C.
(2R,6S)/(2RS,6RS)-2,6-Dibutyl-4-oxopiperidinium chloride
(31d): yield 90%; cis/trans ratio, 2:1; mp 212 °C.
Diethyl (2R,6S)-1-allyl-4-hydroxy-2,6-bis(4-nitrophenyl)-1,2,3,6-
tetrahydropyridine-3,5-dicarboxylate (11): yield 28%; mp 150–151
°C. Anal. (C26H27N3O9) C, H, N.
General Procedure for the Synthesis of the Carbon-
itrile Compounds 32a-f and 43. To the corresponding piperidone
(25 mmol) dissolved in glacial acid (80 mL) the corresponding
amine (25 mmol) and trimethylsilyl cyanide (3.1 mL, 25 mmol)
were added. The solution was stirred for 12 h at 25 °C. After
completion of the reaction, the solution was alkalized with
concentrated ammonia (pH 11), keeping the temperature under -5
°C. The solution was extracted 3 times with chloroform (100 mL).
The organic layer was dried over sodium sulfate, and the solvent
was removed in vacuo. The obtained residue was crystallized with
diethyl ether or tert-butyl ethyl ether (32a,c-e), or a column
chromatography with ethyl acetate on basic aluminum oxide was
performed (Rf between 0.5 and 0.9, 32f). 32b was forwarded to
the next reaction as an oil.
Dimethyl (2R,6S)-1-benzyl-4-hydroxy-2,6-bis(4-nitrophenyl)-
1,2,3,6-tetrahydropyridine-3,5-dicarboxylate (12): yield 12%; mp
163–171 °C. Anal. (C28H25N3O9) C, H, N.
Diethyl (2R,6S)-1-benzyl-4-hydroxy-2,6-bis(4-nitrophenyl)-
1,2,3,6-tetrahydropyridine-3,5-dicarboxylate (13): yield 7%; mp
181–184 °C. Anal. (C30H29N3O9) C, H, N.
Dimethyl (2R,6S)-1-(4-chlorobenzyl)-2,6-bis(3-nitrophenyl)-
4-oxopiperidine-3,5-dicarboxylate (14): yield 41%; mp 144–146
°C. Anal. (C28H24N3O9Cl) C, H, N.
Dimethyl (2R,6S)-1-(4-methoxybenzyl)-2,6-bis(3-nitrophenyl)-
4-oxopiperidine-3,5-dicarboxylate (15): yield 29%; mp 159–161
°C. Anal. (C29H27N3O10) C, H, N.
Dimethyl (2R,6S)-1-(4-methylbenzyl)-2,6-bis(3-nitrophenyl)-
4-oxopiperidine 3,5-dicarboxylate (16): yield 41%; mp 170–172
°C. Anal. (C29H27N3O9) C, H, N.
Dimethyl (2R,6S)-1-benzyl-2,6-bis(3-nitrophenyl)-4-oxopip-
eridine-3,5-dicarboxylate (17): yield 15%; mp 144–146 °C. Anal.
(C28H25N3O9) C, H, N.
Dimethyl (2R,6S)-1-allyl-4-oxo-2,6-diphenylpiperidine-3,5-di-
carboxylate (18): yield 81%; keto–enol ratio, 4:3; mp 142 °C. Anal.
(C24H25NO5) C, H, N.
(2R,6S)-2,6-Dimethyl-4-phenylaminopiperidine-4-carbonitrile
(32a): yield 71%; mp 126–128 °C.
(2R,6S)-4-Benzylamino-2,6-dimethylpiperidine-4-carboni-
trile (32b): yield 68% (oil); ratio of rotamers, 4:1; mp 126–
128 °C.
(2R,6S)-2,6-Diethyl-4-phenylaminopiperidine-4-carbonitrile
(32c): yield 52%; mp 142–143 °C.
(2R,6S)-4-Benzylamino-2,6-diethylpiperidine-4-carbonitrile
(32d): yield 47%; ratio of rotamers, 5:1; mp 109–111 °C.
(2R,6S)-4-Phenylamino-2,6-dipropylpiperidine-4-carboni-
trile (32e): yield 27%; mp 94 °C.
(2R,6S)-2,6-Dibutyl-4-phenylaminopiperidine-4-carbonitrile
(32f): yield 21%; mp 79 °C.
General Procedure for the Synthesis of the 3-Methyl 2,6-
Di(pyridine-2-yl)-4-piperidone-3-monocarboxylates 19–24, Modi-
fied after References 40 and 41. 2,4,6-Trioxotetrahydropyrane (20
mmol) were stirred in methanol (30 mL) till dissolution and
afterward cooled to -20 °C. The corresponding amine (20 mmol)
and pyridine-2-carboxaldehyde (40 mmol), each dissolved in
methanol (20 mL), were added to the solution in parallel such that
the reaction temperature did not exceed -20 °C. After the mixture
was stirred for 2 h at -10 °C, the solvent was removed in vacuo
at room temperature. The obtained residue was covered with a small
amount of methanol, and the product crystallized at 4 °C. The
analytical and spectroscopic data of 19–24 are in accordance with
refs 40–43.
Methyl (2′R,6′S)-1′-benzyl-4′-hydroxy-1′,2′,5′,6′-tetrahydro-
2,2′;6′,2′′-terpyridine-3′-carboxylate (19): yield 33% (53%);41 mp
155 °C (163–164 °C).43
Methyl (2′R,6′S)-4′-hydroxy-1′-(4-methylbenzyl)-1′,2′,5′,6′-tet-
rahydro-2,2′;6′,2′′-terpyridine-3′-carboxylate (20): yield 72%
(41%);41 mp 145 °C (144 °C).43
General Procedure for the Synthesis of the Spiro
Compounds 33–36 and 44. The corresponding carbonitrile com-
pounds (2.5 mmol) were suspended in nonaqueous chloroform (20
mL). Chlorosulfonyl isocyanate (0.22 mL, 2.5 mmol) was added
to achieve 33–36, and chlorosulfonyl isocyanate (0.44 mL, 5 mmol)
was added to obtain 44. The solution was stirred for 2 h at room
temperature and afterward evaporated to dryness. To the residue
hydrochloric acid (50 mL, 1 M) was added, and the obtained
solution was refluxed for 2 h. After neutralization with NaOH (5
M) under intensive cooling, the product crystallized directly or the
pH had to be adjusted to pH 9–10 and the solution extracted 5
times with dichloromethane (50 mL). The combined organic phases
were dried over sodium sulfate. The solvent was reduced in vacuo,
and the obtained crystals were collected and washed with diethyl
ether or ethyl acetate. If necessary, column chromatography on silica
gel was performed with chloroform/methanol ) 25/1, Rf ) 0.6–0.9.
Methyl (2′R,6′S)-1′-(4-chlorobenzyl)-4′-hydroxy-1′,2′,5′,6′-tet-
rahydro-2,2′;6′,2′′-terpyridine-3′-carboxylate (21): yield 68%
(49%);34 mp 141 °C (142 °C).43

NoVel Agents for Therapy
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 247
(2R,6S)-7,9-Dimethyl-1-phenyl-1,3,8-triazaspiro[4.5]decane-
2,4-dione (33): yield 52%; mp 256 °C. Anal. (C15H19N3O2)
C, H, N.
(2R,6S)-1-Benzyl-7,9-diethyl-1,3,8-triazaspiro[4.5]decane-2,4-
dione (34): yield 84%; ratio of rotamers, 5:1; mp 284–286 °C. Anal.
(C18H25N3O2) C, H, N.
(2R,6S)-1-Phenyl-7,9-dipropyl-1,3,8-triazaspiro[4.5]decane-
2,4-dione (35): yield 66%; mp 216–218 °C. Anal. (C19H27N3O2)
C, H, N.
2R,6S)-7,9-Dibutyl-1-phenyl-1,3,8-triazaspiro[4.5]decane-2,4-
dione (36): yield 42%; mp 305 °C. Anal. (C21H31N3O2) C, H, N.
sulfate. After evaporation of the solvent, 47 was crystallized with
acetone/methanol. Yield 68%; mp 234–237 °C. Anal. (C20H23N3O)
C, H, N.
1-(3-Phenylpropyl)-4-piperidine Oxime (48). 4-Oxopiperidine
monohydrate hydrochloride (13.60 g, 100.0 mmol), 1-bromo-3-
phenylpropane (19.90 g, 100.0 mmol), and potassium carbonate
(69.10 g, 500.0 mmol) were mixed in acetonitrile (400 mL) and
stirred at 60 °C for 18 h. The solid was filtered off and washed
with acetonitrile, and the solvent was evaporated. The residue was
diluted with dichloromethane (100 mL) and saturated sodium
hydrogen carbonate solution (50 mL), and the layers were separated.
The aqueous layer was extracted twice with dichloromethane (50
mL), and the combined organic layers were dried and evaporated.
The pale-yellow solid was used for the next step without further
purification.
( 2 R , 6 S ) - 8 - B e n z y l - 7 , 9 - d i e t h y l - 1 - p h e n y l - 1 , 3 , 8 -
triazaspiro[4.5]decane-2,4-dione (44): yield 59%; mp 294–295
°C. Anal. (C24H29N3O2) C, H, N.
General Procedure for the Alkylation in Position 3 of the
Spiro Compounds 37–41 and 45. Compounds 33–36 and 44 (1.75
mmol) were each dissolved in acetonitrile (20 mL), and potassium
carbonate (0.62 g, 4.5 mmol) and the corresponding alkyl or
aromatic bromide (2 mmol) were added. The solution was refluxed
for 16 h and then cooled to 25 °C and filtered. The solvent was
partially removed in vacuo and the precipitate collected and washed
with diethyl ether or ethyl acetate.
Potassium carbonate (10.84 g, 156.0 mmol) and hydroxylamine
hydrochloride (21.60 g, 156.0 mmol) were dissolved with absolute
ethanol (200 mL). A solution of 1-(3-phenylpropyl)-4-piperidinone
(16.95 g, 78.0 mmol) in absolute ethanol (100 mL) was added,
and the mixture was refluxed for 1 h. The mixture was allowed to
cool to 25 °C, filtered, and washed with ethanol. The solvent was
evaporated, and a yellow solid was achieved. Yield 13.84 (59%);
mp 182.5 °C. Anal. (C14H20N2O) C, H, N.
(2R,6S)-1-Benzyl-3-(2-hydroxyethyl)-7,9-dimethyl-1,3,8-
triazaspiro[4.5]decane-2,4-dione (37): yield 48%; mp 195–196
°C. Anal. (C18H25N3O3) C, H, N.
( 2 R , 6 S ) - 3 - B e n z y l - 7 , 9 - d i e t h y l - 1 - p h e n y l - 1 , 3 , 8 -
triazaspiro[4.5]decane-2,4-dione (38): yield 55%; ratio of rota-
mers, 6:1; mp 74–75 °C. Anal. (C24H29N3O2) C, H, N.
(2R,6S)-1-Benzyl-7,9-dimethyl-3-(4-nitrobenzyl)-1,3,8-
triazaspiro[4.5]decane-2,4-dione (39): yield 71%; mp 161–162
°C. Anal. (C23H26N4O4) C, H, N.
(2R,6S)-7,9-Diethyl-3-phenethyl-1-phenyl-1,3,8-
triazaspiro[4.5]decane-2,4-dione (40): yield 62%; mp 146–147
°C. Anal. (C25H31N3O2) C, H, N.
1-(2-Phenylpropyl)-4-oxopiperidine O-(2,6-Dichlorobenzy-
l)oxime (49). 2,6-Dichlorobenzyl chloride (1.22 g, 6.25 mmol), 1-(3-
phenylpropyl)piperidin-4-oxime (1.20 g, 5.0 mmol), and potassium
tert-butoxide (0.71 g, 6.25 mmol) were dissolved in dry THF (100
mL) and stirred for 24 h at 55 °C in the dark under Ar. The solvent
was evaporated, and the residue was diluted with dichloromethane
(100 mL) and NaOH (50 mL, 2.0 M). The layers were separated,
and the aqueous layer was extracted with dichloromethane (2 ×
80 mL). The combined organic layers were dried and evaporated
to achieve a colorless solid. Yield 0.68 g (28%); mp 147 °C. Anal.
(C21H24N2OCl2) C, H, N.
4-(1,3-Dioxolan-2-yl)-N-(3-phenylpropyl)pyridinium Bromide
(50). 4-Pyridinecarbaldehyde (1.93 g, 18.0 mmol), ethylene glycol
(2.26 g, 36.0 mmol), and toluene-4-sulfonic acid (3.80 g, 20.0
mmol) were dissolved in toluene (100 mL) and refluxed for 4 h to
remove the water from the reaction mixture. The solution was
allowed to cool to 25 °C, and the solvent was evaporated. The
residue was diluted with NaOH (50 mL, 2.0 M) and dichlo-
romethane (50 mL). The layers were separated, and the aqueous
layer was extracted with dichloromethane (2 × 50 mL). The
combined organic layers were dried over anhydrous sodium sulfate,
filtered, and evaporated to achieve 4-(1,3-dioxolane-2-yl)pyridine
as a colorless oil, which was used in the next step without further
purification. This intermediate (2.07 g, 13.6 mmol) and bromo-3-
phenylpropane (4.06 g, 20.4 mmol) were dissolved in toluene (20
mL). The solution was sealed in a quartz glass vessel equipped
with a Weflon plate (diameter 1.7 cm, thickness 0.3 cm) and was
heated in the microwave oven at 110 °C for 2 h (5 min for 25–110
°C). After that time the solvent was evaporated, the oily residue
was dissolved in acetone (20 mL), and diethyl ether was added
until the solution became turbid. The solution was cooled to -20
°C for 24 h. The crystals were filtered off, washed with diethyl
ether, and dried. Yield, 1.78 g (37%) of colorless crystals; mp
78.1 °C.
(2R,6S)-7,9-Diethyl-1-phenyl-3-(3-phenylpropyl)-1,3,8-
triazaspiro[4.5]decane-2,4-dione (41): yield 63%; mp 118–120
°C. Anal. (C26H33N3O2) C, H, N.
1-Benzyl-4-piperidone (42a). 42a is commercially available but
was synthesized from 4-oxopiperidone-HCl. 4-Oxopiperidine-HCl
(20 mmol) and K2CO3(6.92 g, 50 mmol) were dissolved in
acetonitrile (50 mL), and benzyl bromide (1.8 mL, 24 mmol) was
added. After being heated for 16 h, the solution was cooled to 25
°C and filtered. To isolate the product, the remaining solid was
suspended in acetonitrile and filtered again. The filtrates were
combined and the solvent was evaporated in vacuo to obtain the
oily product that can be used for the following synthetic steps
without further purification. Yield 86%.
(2R,6S)/(2RS,6RS)-1-Benzyl-2,6-diethyl-4-piperidone (42b): yield
91% (oil); cis/trans ratio, 2:1.
1-Benzyl-4-phenylaminopiperidine-4-carbonitrile (43a): yield
62%; mp 146–148 °C. For spectroscopic data, see ref 44.
(2R,6S)-1-Benzyl-2,6-diethyl-4-phenylaminopiperidine-4-car-
bonitrile (43b): yield 59%; mp 123–125 °C.
(2R,6S)-8-Benzyl-7,9-diethyl-3-(4-nitrobenzyl)-1-phenyl-1,3,8-
triazaspiro[4.5]decane-2,4-dione (45): yield 46%; mp 185–187
°C. Anal. (C31H34N4O4) C, H, N.
(1,3-Dioxolane-2-yl)-N-(3-phenylpropyl)piperidine Hydrobro-
mide (51). 4-(1,3-Dioxolane-2-yl)-1-(3-phenylpropyl)pyridinium
bromide 50 (1.84 g, 8.18 mmol) and PtO2 (40 mg) were dissolved
in methanol (100 mL) and hydrogenated in a microwave hydro-
genation reactor at 60 °C and 20 bar for 1.5 h. The catalyst was
filtered off, and the solvent was evaporated. The residue was
dissolved in acetone and crystallized with diethyl ether at 4 °C to
give a colorless solid. Yield 1.84 g (98%); mp 132 °C (dec).
1-Benzyl-4-phenylaminopiperidine-4-carboxylic Acid Am-
ide (46). The corresponding carbonitrile compound 32 (7.5 mmol)
was stirred in concentrated sulfuric acid (60 mL) for about 12 h at
room temperature. During neutralization of the solution with
concentrated ammonia, the temperature is not allowed to exceed 0
°C. The resulting precipitate was collected, efficiently washed with
water, and dried in vacuo: C19H23N3O (309.4 g/mol), yield 96%;
mp 188–189 °C. For spectroscopic data, see ref 45.
1-[1-(3-Phenylpropyl)piperidine]-4-carbaldehyde
Oxime
8-Benzyl-1-phenyl-1,3,8-triazaspiro[4.5]decane-4-one (47). 46 (10
mmol) was dissolved in formamide (20 mL) and refluxed for 2 h.
The solution was allowed to cool to room temperature and was
then poured into ice–water. The aqueous solution was extracted 3
times with chloroform, and the organic layer was dried over sodium
(52). Acetal 51 (10.0 mmol, 3.55 g) was dissolved in water (30
mL), and sulfuric acid (0.2 mL, 98%) was added. The mixture was
heated in a microwave within 4 min to 110 °C. The temperature
was held for 30 min, and the solution was allowed to cool to 25
°C. Na2HPO4 (30 mL, 1.0 M) and hydroxylamine hydrochloride

248 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Goebel et al.
(6.65 g, 100.0 mmol) were added, and the sodium hydroxide
solution was added until pH 8.5 was achieved. The mixture was
stirred at 25 °C for 18 h, and the solution was alkalized with
potassium carbonate and extracted with dichloromethane (3 × 100
mL). The combined organic layers were dried, filtered, and
evaporated. The oily residue was dissolved in diethyl ether and
filtered, and the filtrate was evaporated. Recrystallization from
cyclohexane gave a colorless solid. Yield 1.90 g (55%); mp 111
°C. Anal. (C15H22N2O) C, H, N.
4-[(2,6-Dichlorobenzyloxyimino)methyl]-1-(3-phenyl-
propyl)piperidinium Hydrochloride (53). The oxime 52 (0.7 g,
2.84 mmol) was dissolved in absolute MeOH (40 mL), and
potassium tert-butoxide was added. The solution was stirred at 25
°C for 3 h, and the solvent was removed in vacuo.The residue was
suspended in dry acetonitrile (50 mL), and 2,6-dichlorobenzyl
chloride (0.56 g, 2.84 mmol) was added. The solution was refluxed
for 2 h, and the solvent was removed in vacuo. Column chroma-
tography (silica gel, eluent 1:1 EtAc/EtOH + 0.7% triethylamine)
gave a colorless oil. This oil was dissolved in diethyl ether (10
mL), and 1.0 M hydrochloric acid (1 mL) in methanol was added.
After 12 h at 4 °C, a colorless solid was isolated. Yield 0.51 g
(40%); mp 198 °C (dec). Anal. (C22H27Cl3N2O) C, H, N.
mice. The percentage of the parasitemia was calculated by
examining Giemsa stained smears under a microscope with an oil
immersion lens (1000×).
Treatment of Mice. The treatment of P. berghei infected
BALB/c mice was started with the 4-piperidone compounds ip when
parasitemia in the mice had reached 5%. Five mice were used in
each group during the experiments. DOHH inhibitors were dissolved
in a concentration of 300 mg/kg in PBS (200 µL) with DMSO
(10% v/v). As a negative control, mice were treated with PBS with
DMSO (10% v/v) only. As a positive control, four mice were treated
with 25 mg/kg chloroquine dissolved in sterile PBS for four
days ip.
Detection of Parasites. Blood samples were collected daily (from
the beginning of the P. berghei infection) from experimental mice
via the tail vein. Giemsa stained (Merck, Darmstadt, Germany) thin
blood smears were prepared, and P. berghei parasitemia was
quantified as the percentage of infected red blood cells (iRBCs)
per 500 RBCs per slide.
Trypanosome Assay According to References 49 and 50.
Parasite Culture. Trypomastigote forms of T. brucei brucei
laboratory strain TC 221 were cultured in Baltz medium according
to standard conditions.51
In Vitro Cytotoxicity Assays. The test compounds were
dissolved in DMSO or NaOH (0.1 M). A defined number of
parasites (104 trypanosomes per milliliter) were exposed in test
chambers of 96-well plates to various concentrations of the test
substances in a final volume of 200 µL. Positive (trypanosomes in
culture medium) and negative controls (test substance without
trypanosomes) were run with each plate.
The plates were then incubated at 37 °C in an atmosphere of
5% CO2 for a total time period of 72 h. A reading was done at
48 h. The effect of test substances was quantified as ED50 values
by linear interpolation18 of three different measurements. The
activity of the test substances was measured by light absorption in
an MR 700 microplate reader at a wavelength of 550 nm with a
reference wavelength of 630 nm, using Alamar Blue.
Plasmodium Assays. Culturing of P. falciparum Chloroquine
Sensitive NF54 and Chloroquine Resistant R Strains. P. falci-
parum isolate NF54 and R strain were maintained in small Petri
dishes (5 cm) in a gaseous phase of 90% N2, 5% CO2, and 5% O2,
according to the protocol from refs 46 and 47. Parasites were
cultured in human erythrocytes (blood group A+) in RPM1640
medium (Sigma) supplemented with HEPES (25 mM), sodium
hydrogen carbonate (20 mM), and 10% heat inactivated human A+
plasma at hematocrit (10% v/v). The parasitemia of infected
erythrocytes was determined by light microscopy and estimated
by Giemsa stained smears. Parasitemias detected in the cultures
were scored visually with a 100-fold oil immersion objective,
counting at least 1000 infected erythrocytes to determine the
parasitemia.
Macrophage Assay According to Reference 52. The macroph-
age cell line J774.1 was maintained in complete Click RPMI
medium. For the experimental procedures, cells were detached from
the flasks with a rubber police, washed twice with PBS, and
suspended at 2 × 106 cells mL-1 in complete Click RPMI medium.
J774.1 macrophages were plated in complete RPMI medium (200
µL) without phenol red in 96-well plates in the absence or presence
of various concentrations of the compounds and incubated for 24 h
at 37 °C, 5% CO2, 95% humidity. Following the addition of of
Alamar Blue (20 µL), the plates were further incubated at similar
conditions. The plates were then read 24 and 48 h later. Control
experiments to examine the effect of cell density, incubation time,
and DMSO concentration were performed. Absorbance in the
absence of compounds was set as 100% of growth control.
Leishmania Assay According to Reference 53. Parasites. The
cloned virulent L. major isolate MHOM/IL/81/FE/BNI was main-
tained by passage in BALB/c mice. Promastigotes were grown in
blood agar cultures at 26 °C, 5% CO2, 95% humidity. For the
experiments described here, promastigotes were washed twice with
phosphate buffered saline (PBS) and suspended at 1 × 108 cells
mL-1 in Click RPMI 1640 medium (Invitrogen, Karlsruhe,
Germany) supplemented with 10% fetal calf serum (FCS) (PAA
Laboratories, Linz, Austria), L-glutamine (2 mM) (Biochrom, Berlin,
Germany), HEPES buffer pH 7.2 (10 mM) (Invitrogen), penicillin
(100 µg mL-1), gentamicin (160 µg mL-1), 7.5% NaHCO3, and
2-mercaptoethanol (5 × 10-5M) (Sigma-Aldrich) (complete me-
dium).
Inhibitor Experiments by Monitoring Multiplication and
Growth of Plasmodia. Cultures were adjusted to a parasitemia of
1.5%. Aliquots were diluted 1:10-fold in RPMI medium, dispensed
into 12-well microculture trays, and incubated at 37 °C in a candle
jar. Thereafter, the growth medium was changed once a day for 4
days and the total inhibitor treatment lasted 96 h. Inhibitors were
used in concentrations of 1-10 µM and dissolved in either DSMO
or RPMI medium before they were added to the media. Parasitemias
and stage distribution were estimated as triplicates daily from
Giemsa stained smears by counting 1000 erythrocytes. Control
experiments were performed with chloroquine (10-8 M) and the
appropriate P. falciparum strain without drug treatment.
Evaluation of Inhibitor Experiments by Determination of
the IC50 Values. Data obtained from the inhibitor-dependent
concentration growth curve of 96 h were plotted as percent
inhibition vs inhibitor concentration (µM) and analyzed with linear
regression according to ref 48.
Infection with Plasmodium berghei. The P. berghei ANKA
strain was used in all experiments. P. berghei ANKA was
maintained by periodic passages through the mosquito vector
Anopheles stephensi. A. stephensi mosquitoes were bred at the
animal facility of the Institute for Medical Microbiology, Immu-
noloy and Parasitology, University of Bonn. Stock blood-stage
parasites stored in liquid nitrogen were used to infect three mice.
Parasites were passaged to three new BALB/c mice, and 2 days
later mosquitoes were allowed to a blood meal on these mice.
Twenty-one days after the mosquito infection, salivary glands of
the mosquitoes were dissected to isolate the sporozoites. A dose
of 500 sporozoites in of phosphate buffer saline (200 µL) (PBS)
was administered intravenously to two new naive mice, and
parasitized erythrocytes were taken from these mice at a parasitemia
of 20% to generate frozen aliquots. For infection with parasitized
erythrocytes, mice were infected with the identical frozen aliquots
of parasite. The parasitized blood was diluted in PBS and injected
ip in concentrations of 50 000 P. berghei infected erythrocytes per
Cells and Cell Lines. The macrophage cell line J774.1 was
maintained in complete medium. For the experimental procedures,
cells were detached from the flasks with a police scraper, washed
twice with PBS, and suspended at 2 × 106 cells mL-1 in complete
medium.
Analysis of in Vitro Antiproliferative Activity. Promastigotes
were seeded into 96-well plates in complete medium without phenol
red (200 µL) in the absence or presence of increasing concentrations

NoVel Agents for Therapy
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 249
of the polyamine synthesis enzyme. Mol. Biochem. Parasitol. 2005,
142, 224–236.
(8) Casero, R. A., Jr.; Marton, L. J. Targeting polyamine metabolism and
function in cancer and other hyperproliferative diseases. Nature ReV.
Drug DiscoVery 2007, 7, 373–390.
(9) Das Gupta, R.; Krause-Ihle, T.; Bergmann, B.; Muller, I. B.;
Khomutov, A. R.; Müller, S.; Walter, R. D; Luersen, K. 3-Aminooxy-
1-aminopropane and derivatives have an antiproliferative effect on
cultured Plasmodium falciparum by decreasing intracellular polyamine
concentrations. Antimicrob. Agents Chemother. 2005, 49, 2857–2864.
(10) Kaiser, A.; Gottwald, A.; Wiersch, C.; Lindenthal, B.; Maier, W.; Seitz,
H. M. Effect of drugs inhibiting spermidine biosynthesis and metabo-
lism on the in vitro development of Plasmodium falciparum. Parasitol.
Res. 2001, 87, 963–972.
(11) Park, M. H.; Wolff, E. C.; Folk, J. E. Hypusine: its post-translational
formation in eukaryotic initiation factor 5A and its potential in cellular
regulation. BioFactors 1993, 4, 95–104.
(12) Abbruzzese, A.; Park, M. H.; Folk, J. E. Deoxyhypusine hydroxylase
from rat testis. Partial purification and characterization. J. Biol. Chem.
1988, 261, 3085–3089.
(13) Park, M. H.; Wolff, E. C. Cell-free synthesis of deoxyhypusine.
Separation of protein substrate and enzyme and identification of 1,3-
diaminopropane as a product of spermidine cleavage. J. Biol. Chem.
1988, 263, 15264–15269.
(14) Njuguna, J. T.; Nassar, M.; Hoerauf, A.; Kaiser, A. E. Cloning,
expression and functional activity of deoxyhypusine synthase from
Plasmodium ViVax. BMC Microbiol. 2006, 6, 91.
(15) Clement, P. M.; Hanauske-Abel, H. M.; Wolff, E. C.; Kleinmann,
H. K.; Park, M. H. The antifungal drug ciclopirox inhibits deoxyhy-
pusine and proline hydroxylation, endothelial cell growth and angio-
gensis in vitro. Int. J. Cancer 2002, 100, 491–498.
(16) Saeftel, M.; Sarite, R. S.; Njuguna, T.; Holzgrabe, U.; Ulmer, D.;
Hoerauf, A.; Kaiser, A. Piperidones with activity against Plasmodium
falciparum. Parasitol. Res. 2006, 99, 281–286.
(17) Dong, Z.; Arnold, R.; Yang, Y.; Park, M. H.; Hrncirova, P.; Mechref,
Y.; Novotny, M. V.; Zhang, J. T. Modulation of differentiation-related
gene-1 expression by cell cycle blocker mimosine, revealed by
proteomic analysis. Mol. Cell. Proteomics 2005, 4, 993–1001.
(18) Park, M. H. The post-translational synthesis of a polyamine-derived
amino acid, hypusine, in the eukaryotic translation initiation factor
5A (eIF-5A). J. Biochem. 2006, 139, 1–9.
(19) Du Moulinet d′Hardemare, A.; Torelli, S.; Serratrice, G.; Pierre, L.-
L. Design of iron chelators: synthesis and iron(III) complexing abilities
of tripodal tris-bidentate ligands. BioMetals 2006, 19, 349–366.
(20) Cotton, F. A.; Wilkinson, G. AdVanced Inorganic Chemistry, 5th ed.;
Wiley: New York, 1988, p 710.
of the compounds. They were then incubated for 24 h at 26 °C,
5% CO2, 95% humidity. Following the addition of Alamar Blue
(20 µL) (Trinova Biochem, Giessen, Germany), the plates were
incubated again and the optical densities (OD) measured 24 and
48 h later with a MultiAscent ELISA reader (Thermo Electon
Corporation, Dreieich, Germany) using a test wavelength of 540
nm and a reference wavelength of 630 nm. Absorbance in the
absence of compounds was set as 100% of growth. J774.1
macrophages, peritoneal macrophages, and BMDC were cultured
in complete medium without phenol red, and fibroblasts in DMEM
(200 µL) were cultured in the absence or presence of increasing
concentrations of the compounds for 24 h at 37 °C, 5% CO2, 95%
humidity. Following the addition of Alamar Blue (20 µL), the plates
were incubated again, and the OD was measured 24, 48, and 72 h
later as described. Amphotericin B (Sigma-Aldrich) was used as a
reference compound and positive control.
Statistical Analysis. Data on antiproliferative activity (from at
least two experiments) were analyzed with Ascent Software and
Microsoft Excel. OD values at 48 h were used to calculate, via
linear interpolation, the drug concentrations that inhibit 50% cell
growth or cell survival (IC50).50
HIV-1 Infection Experiments. HIV-1 infection experiments
using the T-cell tropic (X4) strain NL4/3 and the macrophage tropic
(R5) strain BaL were routinely performed using PM1 cells (virus
laboratory strains and cells were obtained from the NIH AIDS
Research and Reference Reagent Program). Cells were cultured in
RPMI medium containing 10% fetal calf serum (Pansystems
GmbH) and antibiotics (penicillin and streptomycin). For HIV-1
infection, 5 × 107 cells were resuspended in culture medium (500
µL) without drugs and incubated at 37 °C for 3 h with HIV-1 viral
stocks (100 ng). After infection, cells were washed twice with PBS
without Ca2+ and Mg2+ to avoid false positive p24 antigen
determination. Cells were resuspended, and identical aliquots (5 ×
105/mL) of infected cells were further cultured in 24-well plates
(triplicates) in the presence of the substances 11–13 and 53
(dissolved in DMSO) at various concentrations or in medium with
DMSO as a control for the calculation of the inhibition of virus
replication. Culture medium was changed, and cells were split twice
a week postinfection. Viability of the cells (as measured by Alamar
Blue) and p24 antigen levels (as measured by ELISA, Innogenetics
N.V.) were determined at different time points.
(21) Haller, R. Metallchelate pyridyl-(2)-substituierter piperidone und
piperidinole (Metal chelates of substituted piperidones and piperidi-
noles). Arch. Pharm. 1968, 301, 741–749.
Acknowledgment. This work was financially supported by
grants from the Deutsche Forschungsgemeinschaft to U.H.,
A.H., and J.H. and by the SFB 630 with respect to the testing
of the compounds against T. brucei brucei, L. major and
cytotoxicity.
(22) Bevec, D.; Jaksche, H.; Oft, M.; Wohl, T.; Himmelspach, M.; Pacher,
A.; Schebesta, M.; Koettnitz, K.; Dobrovnik, M.; Csonga, R.;
Lottspeich, F.; Hauber, J. Inhibition of HIV-1 replication in lympho-
cytes by mutants of the Rev cofactor eIF-5A. Science 1996, 271, 1858–
1860.
(23) Schäfer, B.; Hauber, I.; Bunk, A.; Heukeshoven, J.; Düsedau, A.;
Bevec, D.; Hauber, J. Inhibition of multi-resistant HIV-1 by interfer-
ence with cellular S-adenosylmethionine decarboxylase activity. J. In-
fect. Dis. 2006, 194, 740–750.
(24) Hauber, I.; Bevec, D.; Heukeshoven, J.; Krätzer, F.; Horn, F.; Choidas,
A.; Harrer, T.; Hauber, J. Identification of cellular deoxyhypusine
synthase as a novel target for antiretroviral therapy. J. Clin. InVest.
2005, 115, 76–85.
(25) Hart, R. A.; Billaud, J. N.; Choi, S. J.; Phillips, T. R. Effects of 1,8-
diaminooctane on the FIV Rev regulatory system. Virology 2002, 304,
97–104.
Supporting Information Available: Microanalysis and spec-
troscopic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Greenwood, B. M.; Bojang, K.; Whitty, C. J. M.; Targett, G. A. T.
Malaria. Lancet 2005, 365, 1487–1498.
(26) Andrus, L.; Szabo, P.; Grady, R. W.; Hanauske, A.-R.; Huima-Byron,
T.; Slowinska, B.; Zagulska, S.; Hanauske-Abel, H. M. Antiretroviral
effects of deoxyhypusyl hydroxylase inhibitors. Biochem. Pharmacol.
1998, 55, 1807–1818.
(27) Kaiser, A.; Ulmer, D.; Goebel, T.; Holzgrabe, U.; Saeftel, M.; Hoerauf,
A. Inhibition of hypusine biosynthesis in plasmodium: a possible, new
startegy in prevention and therapy of malaria. Mini-ReV. Med. Chem.
2006, 6, 1231–1241.
(28) Merz, K. W.; Haller, R. Synthesen mit Pyridin- und Chinolinaldehyden
(Synthesis with pyridine- and quinolinealdehyde). Pharm. Acta HelV.
1963, 38, 442–456.
(29) Ashauer-Holzgrabe, U.; Haller, R. Zur Umsetzung von N-benzylsub-
stituierten Piperidoncarbonsäuren mit Cer(IV)sulfat (Conversion N-
benzyl substituted piperidoncarboxylic acid with cerium(IV) sulfate).
Arch. Pharm. (Weinheim, Ger.) 1986, 319, 1079–1083.
(30) Kuhl,U.;Englberger,W.;Haurand,M.;Holzgrabe,U.Diazabicyclo[3.3.1]-
nonanone-type ligands for the opioid receptors. Arch. Pharm. 2000,
333, 226–230.
(2) See the following Web sites: http://www.who.int/tdr/diseases/tryp/,
http://www.who.int/tdr/diseases/chagas/diseaseinfo.htm, and http://
www.who.int/leishmaniasis/en/.
(3) D’Silva, C. Human African trypanosomiasis: future propects for
chemotherapy. Drugs Future 2007, 32, 149–160.
(4) Projan, S. J. Why is big Pharma getting out of antibacterial drug
discovery? Curr. Op. Microbiol. 2003, 6, 427–430.
(5) Nwaka, S.; Hudson, A. Innovative lead discovery strategies for tropical
diseases. Nat. Drug DiscoVery 2006, 5, 941–955.
(6) Müller, S.; Da’dara, A.; Lürsen, K.; Wrenger, C.; Das Gupta, R.;
Madhubala, R.; Walter, R. D. In the human malaria parasite Plasmo-
dium falciparum, polyamines are synthesized by a bifunctional
ornithine decarboxylase, S-adenosylmethionine decarboxylase. J. Biol.
Chem. 2000, 275, 8097–8102.
(7) Haider, N.; Eschbach, M. L.; Dias Sde, S.; Gilberger, T. W.; Walter,
R. D.; Luersen, K. The spermidine synthase of the malaria parasite
Plasmodium falciparum: molecular and biomchemical characterization

250 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Goebel et al.
(31) Holzgrabe, U.; Piening, B.; Kohlmorgen, R.; Stoll, E. Synthese
verschieden substituierter 5-Oxo-2,6-methano-2-benzazocines (Syn-
thesis of differently substituted 5-Oxo-2,6-methano-2-benzazocines).
Arch. Pharm. (Weinheim, Ger.) 1988, 321, 917–920.
ierter 5-Oxo-2,6-methano-2-benzazocine (Cerium(IV) oxidation of
ꢀ-Aminoketones. 5. Synthesis of differently substituted 5-Oxo-2,6-
methano-2-benzazocines). Arch. Pharm. (Weinheim, Ger.) 1988, 321,
917–920.
(32) Siener, T.; Holzgrabe, U.; Drosihn, S.; Brandt, W. Conformational
and configurational behaviour of κ-agonistic 3,7-diazabicyclo-
[3.3.1]nonan-9-onesssynthesis, nuclear magnetic resonance studies
and semiempirical PM3 calculations. J. Chem. Soc., Perkin Trans.
1999, 2, 1827–1834.
(44) van Parys, M.; Vandewalle, M. The synthesis of 1,8-
diazaspiro[4,4]decanes. Bull. Soc. Chim. Belg. 1981, 90, 749–756.
(45) Taber, D. F.; Rahimizadeh, M. Amide to ester conversion: a practical
route to the carfentanil class of analgetics. J. Org. Chem. 1992, 57,
4037–4038.
(46) Trager, W.; Williams, J. Extracellular (axenic) development in vitro
of the erythrocytic cycle of Plasmodium falciparum. Proc. Nat. Acad.
Sci. U.S.A. 1992, 89, 5351–5355.
(47) Moloney, M. B.; Pawluk, A. R.; Ackland, N. R. Plasmodium
falciparum growth in deep culture. Trans. R. Soc. Trop. Med. Hyg.
1990, 84, 516–518.
(48) Singh, S.; Puri, S. K.; Singh, S. K.; Srivastava, R.; Das Gupta, R. C.;
Pandey, V. C. Characterization of simian malarial parasite (Plasmo-
dium knowlesi)-induced putrescine transport in rhesus monkey eryth-
rocytes. A novel putrescine conjugate arrests in vitro growth of simian
malarial parasite (Plasmodium knowlesi) and cures multidrug resistant
murine malaria (Plasmodium yoelii) infection in vivo. J. Biol. Chem.
1997, 272, 13506–13511.
(49) Räz, B.; Iten, M.; Grether-Bühler, Y.; Kaminsky, R.; Brun, R. The
Alamar Blue assay to determine drug sensitivity of African trypano-
somes (T. brucei rhodesiense and T. brucei gambiense) in vitro. Acta
Trop. 1997, 68, 139–147.
(50) Huber, W.; Koella, J. C. A comparison of three methods of estimating
EC50 in studies of drug resistance of malaria parasites. Acta Trop.
1993, 55, 257–261.
(51) Baltz, T.; Baltz, D.; Giroud, C.; Crocket, J. Cultivation in a semi-
defined medium of animal infective forms of Trypanosoma brucei, T.
equiperdum, T. eVansi, T. rhodesiense and T. gambiense. EMBO J.
1985, 4, 1273–1277.
(52) Ahmed, S. A.; Gogal, R. M.; Walsh, J. E. A new rapid and simple
non-radioactive assay to monitor and determine the proliferation of
lymphocytes: an alternative to [3H]thymidine incorporation assay.
J. Immunol. Methods 1994, 170, 211–224.
(33) Smith, M. B.; March, J. March′s AdVanced Organic Chemistry, 5th
ed.; Wiley-Interscience: New York, 2001; p 1240.
(34) Röver, S.; Adam, G.; Cesura, A. M.; Galley, G.; Jenck, F.; Monsma,
F. J.; Wichmann, J.; Dautzenberg, F. M. High-affinity, non-peptide
agonists for the ORL1 (orphanin FQ/nociceptin) receptor. J. Med.
Chem. 2000, 43, 1329–1338.
(35) Bisset, D. L.; McBride, J. F. Synergistic topical photoprotection by a
combination of the iron chelator 2-furildioxime and sunscreen. J. Am.
Acad. Dermatol. 1996, 35, 546–549.
(36) Pais, G. C. G.; Burke, T. R. Novel aryl diketo-containing inhibitors
of HIV-1 integrase. Drugs Future 2002, 27, 1101–1111.
(37) Holzgrabe, U.; Hoerr, V. Unpublished results.
(38) Kang, K. R.; Kim, Y. S.; Wolff, E. C.; Park, M. H. Specificity of the
deoxyhypusine hydroxylase-eukaryotic translation initiation factor
interaction. J. Biol. Chem. 2007, 282, 8300–8308.
(39) Holzgrabe, U.; Erciyas, E. Synthese und Stereochemie potentiell stark
analgetischer 2,4-m-diarylsubstituierter 3,7-diazabicyclo[3.3.1]nonan-
9-on-1,5-diester (Synthesis and stereochemistry of the potentially
strong analgesic 2,4-m-diaryl-substituted 3,7-diazabicyclo[3.3.1]nonan-
9-on-1,5-diester). Arch. Pharm. (Weinheim, Ger.) 1992, 325, 657–
663.
(40) Holzgrabe, U.; Piening, B.; Hesse, K.-F.; Höltje, H.-D.; Worch, M.
Stereochemistry of 2,6-dipyridine substituted N-benzyl-4-piperidone
mono- and dicarboxylates and the corresponding reduction products.
Z. Naturforsch. 1988, 44b, 565–574.
(41) Haller, R.; Kohlmorgen, R. Synthese und Struktur substituierter
N-Benzyl-piperidoncarbonsäureester (Synthesis and structure of sub-
stituted N-benzyl-piperidoncarboxylic acid ester). Arch. Pharm. (Wein-
heim, Ger.) 1976, 309, 206–214.
(42) Haller, R. Zur Synthese 2,6-disubstituierter Piperidonmonocarbon-
säureester (On the synthesis of 2,6-substituted piperidone monocar-
boxylic acid esters). Arch. Pharm. 1965, 298, 787–794.
(53) Ponte-Sucre, A.; Vicik, R.; Schultheis, M.; Schirmeister, T.; Moll, H.
Aziridine-2,3-dicarboxylates: peptidomimetic cysteine protease inhibi-
tors with antileishmanial activity. Antimicrob. Agents Chemother. 2006,
50, 2439–2447.
(43) Holzgrabe, U.; Piening, B.; Kohlmorgen, R.; Stoll, E. Cer(IV)oxida-
tionen von ꢀ-Aminoketonen. 5. Mitt. Synthese verschieden substitu-
JM070763Y