Synthesis and evaluation of hybrid drugs for a potential HIV/AIDS-malaria combination therapy

Article abstract of DOI:10.1016/j.bmc.2012.06.038

Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia

Full text of DOI:10.1016/j.bmc.2012.06.038

Bioorganic & Medicinal Chemistry 20 (2012) 5277–5289
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of hybrid drugs for a potential HIV/AIDS-malaria
combination therapy
Makoah N. Aminake a, Aman Mahajan b, Vipan Kumar c, Renate Hans d, Lubbe Wiesner e, Dale Taylor e,
Carmen de Kock e, Anne Grobler f, Peter J. Smith e, Marc Kirschner g, Axel Rethwilm g, Gabriele Pradel a,
Kelly Chibale b,h,
⇑
a University of Würzburg, Research Center for Infectious Diseases, Josef-Schneider-Str. 2/D15, 97080 Würzburg, Germany
b Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
c Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India
d Department of Chemistry and Biochemistry, University of Namibia, Windhoek, Namibia
e Division of Pharmacology, University of Cape Town, Observatory 7925, South Africa
f North West University, Potchefstroom Campus, Potchefstroom 2520, South Africa
g University of Würzburg, Institute for Virology and Immunbiology, Versbacher Str. 7, 97078 Würzburg, Germany
h Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Malaria and HIV are among the most important global health problems of our time and together are
responsible for approximately 3 million deaths annually. These two diseases overlap in many regions
of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk
of co-infection. In this study, we generated and characterized hybrid molecules to target Plasmodium fal-
ciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules
were synthesized by the covalent fusion of azidothymidine (AZT) with dihydroartemisinin (DHA), a tet-
raoxane or a 4-aminoquinoline derivative; and the small library was tested for antiviral and antimalarial
activity. Our data suggests that compound 7 is the most potent molecule in vitro, with antiplasmodial
Received 8 May 2012
Revised 13 June 2012
Accepted 21 June 2012
Available online 29 June 2012
Keywords:
Malaria
HIV/AIDS
Combination therapy
Hybrid drugs
activity comparable to that of DHA (IC50 = 26 nM, SI >3000),
a
moderate activity against HIV
M).
(IC50 = 2.9 M; SI >35) and not toxic to HeLa cells at concentrations used in the assay (CC50 >100
l
l
Pharmacokinetics studies further revealed that compound 7 is metabolically unstable and is cleaved
via O-dealkylation. These studies account for the lack of in vivo efficacy of compound 7 against the
CQ-sensitive Plasmodium berghei N strain in mice, when administered orally at 20 mg/kg.
Ó 2012 Elsevier Ltd. All rights reserved.
Medicinal chemistry
Drug metabolism
Pharmacokinetics
1. Introduction
On the other hand Acquired immunodeficiency syndrome
(AIDS) is a disease of the human immune system caused by the hu-
Malaria caused by the human malaria parasite Plasmodium
falciparum is responsible for close to one million deaths annually.
It represents a fundamental threat to human health worldwide,
and its chemotherapeutic treatment is increasingly complicated
by frequent drug resistance.1 In order to gain control of drug-resis-
tant malaria, guidelines were released by WHO, which emphasized
the use of an artemisinin-based combination therapy (ACT) involv-
ing drugs with independent targets.1 In addition, co-infection with
other infectious diseases further complicates the treatment in
many regions of the world where malaria is endemic. It is therefore
important to understand the effect of co-infections on patients and
the consequences on treatment to design an effective drug regimen
that could be used to control mixed infections.
man immunodeficiency virus (HIV). The disease is a major health
problem in many parts of the world. In 2009, WHO estimated 2.0
million annual deaths due to AIDS.2 Malaria and HIV/AIDS, taken
together are two of the most devastating diseases of our time,
especially in sub-Saharan Africa. Given the increasing number of
people becoming infected with HIV, and the omnipresence of
malaria in areas where the two diseases co-exist, the prevalence
of co-infections is on the rise.
Over the past 10 years, research interest in malaria-HIV/AIDS
co-infections has increased and data on the effect of these co-infec-
tions on patient health, implications for treatment and control
programs are accumulating. HIV infection, through immunosup-
pression, affects the acquisition and persistence of immune
response to malaria. The highest proportion of co-infection cases
in adults are expected in areas with a high prevalence of HIV infec-
tion and a low occurrence of malaria because natural antimalarial
⇑
Corresponding author. Tel.: +27 21 6502553; fax: +27 21 6505195.
E-mail address: Kelly.Chibale@uct.ac.za (K. Chibale).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.038

5278
M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
immunity is not acquired.3 It has been observed that HIV-infected
people, in areas of malaria transmission, have more frequent epi-
sodes of symptomatic parasitemia and higher parasitemia than
those without HIV.4,5 Additionally, HIV infected people have an in-
crease in viremia during episodes of malaria,6,7 leading to a poten-
tial increase in the risk of HIV transmission. Finally, it appears that
malaria and HIV co-infections may work synergistically, amplify-
ing the prevalence and intensifying both in regions where they
co-exist.
The concept of hybrid drugs, which typically involve covalent fu-
sion of two or more existing drugs or pharmacophores to create a
single molecule with multiple, but not necessarily simultaneous
pharmacological targets,8 is relatively new and has shown some
success in cancer therapy.9 In addition, promising hybrid antimala-
rials are currently under development.10 A related approach is now
being developed to generate anti-HIV hybrid drugs to target the
virus’ reverse transcriptase (RT) and integrase, the newest validated
target against AIDS and retroviral infections.11–14 The design of hy-
brid molecules to target HIV and P. falciparum simultaneously has
not been reported; however, the intrinsic anti-HIV activity of some
antimalarials and the antimalarial activity of HIV protease inhibitors
(PIs) already reported could be exploited within this concept.15–17
Herein we report the design and synthesis of three groups of
hybrid molecules (Fig. 1) either using the available antimalarials
dihydroartemisinin (DHA) and scaffolds derived from clinically
used drugs such as chloroquine (CQ) or antimalarial experimental
drugs such as the tetraoxanes, in combination with the anti-HIV
drug azidothymidine (AZT). The first set of hybrids was inspired
by the findings of Boelaert et al.18 who reported an additive effect
on HIV when CQ is administered in combination with azidothymi-
dine (AZT) or another nucleoside reverse transcriptase inhibitor
(NRTI). Therefore, a hybrid molecule was generated by covalent
linkage of CQ-based 4-aminoquinoline diamines and AZT. The sec-
ond set was based on the hypothesis that covalent association of
DHA with AZT may yield a single molecule with an improved
in vivo half-life, and hence a better pharmacokinetic (PK) profile
compared to DHA, which is known to have a less favorable PK pro-
file. Furthermore, and in an analogous manner to artemisinin
derivatives such as artemether and artesunate, the biologically
unstable glycosidic bond was used as a linker in artemisinin-based
hybrids and was hypothesized to release dihydroartemisinin
(DHA) and AZT upon cleavage in vivo. The third set was deduced
from the second set and DHA was replaced by a tetraoxane-based
peroxide molecule. We evaluated the antimalarial and anti-HIV
O
O
H
O
H3C
HO
N
H3C
N
H3C
NH
NH
N
N
O
N
O
N
O
O
O
O
O
O
H
N
Cl
N3
RO
N
H
NH
N
RO
N
H
N
H
NH
N
n
n
O
3=R= OTBDPS
4=R= H
5
1= R= OTBDPS
2= R= H
Cl
Cl
n= 1-4
n= 2-6
H3C
H3C
H
H
O
N
H
H
H3C
HO
N
O
H
O
H3C
O
O
O
N
O
H
H
N
N
O
O
O
O
N
H3C
N
O
O
Cl
HO
N3
N3
O
O
N
O
CH3
N
O
6
7
8
N
HN
O
O
H
CH3
O
N
H3C
HO
NH
O
O
O
O
O
O
O
HN
9
Figure 1. General structure of the target bifunctional hybrids of AZT.

M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
5279
activity of the hybrids and further studied the PK and metabolism
properties of the most active hybrid.
The synthesis of AZT based bifunctional hybrids (Fig. 1) was
initiated by an initial silylation of AZT 17 using tert-butyldiphenyl-
silylchloride and imidazole in dry methylene chloride followed by
azide reduction using the zinc-ammonium chloride protocol to
yield the corresponding O-silyl protected amine 19 in excellent
yields.21,22 The silylated amine 19 was then converted to carba-
mate 20 by reacting with phenyl chloroformate in the presence
of dry pyridine in dry dichloromethane. Similarly 21 was obtained
by treatment of 19 with ethyloxalyl chloride in the presence of dry
dichloromethane at 0 °C. The progress of the reaction at each time
was monitored by tlc and on completion, the reaction mixture was
concentrated and chromatographed using a ethyl acetate: hexane
solvent system to obtain silylated intermediates 20 and 21 in good
yields (Scheme 2). The structures of the compounds have been as-
signed with the aid of spectral data.
2. Synthesis
2.1. AZT–CQ hybrids
The 4,7-dichloroquinoline based intermediates required for the
desired synthesis (Scheme 1) were obtained as mentioned in the
reported procedures.19,20 The intermediate 12 was prepared by
an initial reaction of ethanolamine with 4,7-dichloroquinoline 10
to produce 11 which was heated at 165 °C in the presence of HBr
and H2SO4. Similarly, the intermediate 15 was obtained by reflux-
ing 4,7-dichloroquinoline 10 and 3-aminobenzyl alcohol 13 to
yield 14, which was further reacted with thionyl chloride at 0 °C
to obtain 15. Synthesis of a range of 4-aminoquinoline diamines
16 was realized by refluxing 4,7-dichloroquinoline and diamines.
The purified 4-aminoquinoline based intermediates were re-
acted with silylated azidothymidine intermediates 20 and 21 in
OH
Br
HN
N
HN
(i)
(ii)
Cl
Cl
N
12
11
OH
Cl
N
HN
HN
(iv)
NH2
13
OH
Cl
(iii)
Cl
Cl
N
14
Cl
N
10
15
HN
NH2
n
(v)
n =2-6
Cl
N
16
Scheme 1. Reagents and conditions: (i) ethanolamine, reflux, 90%; (ii) HBr/H2SO4, 165 °C, 74%; (iii) 13, ethanol reflux, 78%; (iv) SOCl2, dry DMF, 76%; (v) diamino alkanes,
reflux, quantitative.
O
N
O
N
O
N
O
N
H3C
H3C
H3C
H3C
NH
NH
NH
NH
O
O
O
(iii)
O
(ii)
(i)
O
O
O
O
O
TBDPSO
O
TBDPSO
TBDPSO
HO
NH
NH2
N3
N3
17
18
19
20
(iv)
O
N
H3C
NH
O
O
O
OEt
TBDPSO
NH
O
21
Scheme 2. Reagents and conditions: (i) tert-butyldiphenylsilylchloride, imidazole, dry dichloromethane, N2 atm, 88%; (ii) zinc–ammonium chloride, C2H5OH/H2O reflux, 81%;
(iii) PhOCOCl, pyridine, CHCl3 reflux, 82%; (iv) ethyloxalylchloride, DCM, 0 °C, 87%.

5280
M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
the presence of dry methanol as solvent to obtain silylated target
compounds 1a–e and 3a–d following purification by column chro-
matography.23 These silylated hybrids were then desilylated using
K2CO3 in refluxing methanol,23 to yield the corresponding 2a, 2d
and 4d as shown in Scheme 3.
Quinoline-based hybrids 5 and 6 were obtained by substitution
at the N-3 position of AZT, which was conducted in the presence of
dry DMF using the synthetic sequence shown in Scheme 4.
reported by Li et al.24 was used. It involved the reaction of dihyd-
roartemisinin and propargyl alcohol in dry DCM at 0 °C and then
25 °C for 10 h in the presence of BF3 etherate. The AZT–ART hybrid
8 was prepared using the classical ‘click’ conditions, and involved
the reaction of the b-isomer of 23 with AZT in the presence of
5 mol % of CuSO4ꢀH2O and 10 mol % of sodium ascorbate in DCM/
H2O (1:1) at 25 °C.25 The synthesis of the AZT–tetraoxane hybrid
was performed using AZT amine 19. The amine 19 was converted
to the corresponding silylated tetraoxane–AZT hybrid 25 by reac-
tion with ethyl chloroformate in the presence of dry triethylamine
and tetraoxane carboxylic acid 24 in dry dichloromethane using
the protocols described by Opsenica et al. and O’Neill et al.26,27 This
was followed by desilylation using K2CO3/ethanol to yield 9
(Scheme 5).23
2.2. AZT-artemisinin
The targeted compound 7 was obtained by BF3 etherate-medi-
ate O-glycosylation of dihydroartemisinin (Scheme 5). For the syn-
thesis of the acetylenic artemisinin intermediate 23, a procedure
O
N
O
N
O
H3C
H3C
NH
H3C
NH
NH
O
O
O
N
(i)
(ii)
O
O
O
O
O
O
O
TBDPSO
TBDPSO
HO
N
N
H
NH
N
N
N
NH
N
HN
n
n
H
H
H
20
2a= n=2
2d= n=5
1a
= n=2
1b= n=3
1c= n=4
Cl
Cl
1d
= n=5
1e= n=6
O
O
O
N
H3C
H3C
NH
NH
H3C
NH
N
O
N
O
(ii)
O
(i)
O
O
O
O
H
N
O
H
N
TBDPSO
OC2H5
N
NH
N
HO
O
N
NH
N
n
TBDPSO
H
n
H
HN
O
O
4d= n=4
3a= n=1
3b
21
O
Cl
= n=2
3c= n=3
3d
Cl
= n=4
Scheme 3. Reagents and conditions: (i) compound 16, dry methanol reflux; (ii) 2a, K2CO3, C2H5OH/H2O, 81%; (ii) 2d, K2CO3, C2H5OH/H2O, 89%; (ii) 4d, K2CO3, C2H5OH/H2O,
68%.
O
H
O
N
H3C
N
N
H3C
HO
Br
NH
HN
N
N
(i)
N
O
O
+
O
O
Cl
Cl
HO
N3
N3
5
17
12
O
O
H3C
H
Cl
NH
N
H3C
HN
N
N
O
(i)
N
+
N
O
O
N
Cl
O
HO
Cl
N3
HO
N3
6
17
15
Scheme 4. Reagents and conditions: (i) compound 5, dry DMF, NaH, N2, 26%; (i) 6, dry DMF, NaH, N2, 80%.

M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
5281
8
O
H
O
H
O
(vi)
O
CH3
OH
O
O
N
H
O
CH3
H3C
23
NH
HO
O O
O O
O
N
H3C
O
H
H
NH
O
24
O
O
(i)
O
(ii), (iii), (iv)
7
O
O
CH3
+
O
TBDPSO
O
O
HN
O
H
25
(v)
CH3
O O
22
HO
N3
17
9
Scheme 5. Reagents and conditions: (i) BF3*O(CH2CH3)2, ꢂ10 °C, 63%; (ii) tert-butyldiphenylsilylchloride, imidazole, dry dichloromethane, N2 atm, 88%; (iii) zinc–ammonium
chloride, C2H5OH/H2O, reflux, 81%; (iv) ethyl chloroformate, 24, triethylamine, 0 °C, 57%; (v) K2CO3, C2H5OH/H2O, reflux, 62%; (vi) CuSO4ꢀ5H2O, sodium ascorbate, DCM/H2O
(1:1), 25 °C, 24 h, 45%.
Compounds
compounds, but 3d was discontinued due to higher cytotoxicity
(CC50 = 28.6 M) and weaker antiplasmodial activity compared to
7 and 3d appeared to be the most active
3. Biological evaluation
l
3.1. Compound screening for cytotoxicity, antiplasmodial and
anti-HIV activity
compound 7. Since DHA is reported to have an additional effect
on young and mature gametocytes,28 gametocytocidal activity of
compound
7 was also evaluated. It was apparent that the
The series of hybrid molecules generated was evaluated for
their inhibitory activity against pseudotyped HIV-1, P. falciparum
3D7 and P. falciparum Dd2. Target compounds and relevant
intermediates were pre-screened at three concentrations (50, 5
activity of compound 7 on developing gametocytes is moderate
and comparable to that of DHA, but weaker than
primaquine, a known gametocytocidal drug used as a control
(Fig. 2).
and 1
Compounds toxic to HeLa cells at 5
the study. Compounds with no activity against HIV and P. falci-
parum at 50 M (i.e., with IC50 >50 M for both) were not further
l
M) to establish their activity and cytotoxicity (Table 1).
Since HIV-1 (VSV-G) enters cells through the endocytic
pathway and can be inhibited by compounds that block
lM were removed from
endosomal acidification,29 the activity of compound
7 was
l
l
further evaluated on a full replicative wild type NL43 HIV-1.
Compound 7 was 72ꢁ and 600ꢁ less potent than AZT against
pseudotyped viruses and wild type NL43 HIV-1, respectively
(Table 3 and Fig. 3). Viral infection was measured in the pres-
ence of drugs tested at six different concentrations and com-
pared to AZT, non-infected TZM cells and non treated-infected
TZM cells were used as 100% activity and 0% activity controls,
respectively.
screened since the exact IC50 could not be determined. Com-
pounds with acceptable activities were further evaluated for
their cytotoxicity on HeLa cells. Compounds with IC50 >15
lM
for anti-HIV activity and IC50 >10 M for antiplasmodial activity
l
were classified as not effective in our study. The selectivity index
(SI; Table 2) was also used as a cut-off parameter to prioritize
the most potent and selective compounds. Therefore a com-
pound with a SI <100 in both assays was considered not effective
(NE; Table 1).
3.2. In vivo antimalarial efficacy studies
Seventeen (17) compounds exhibited strong to moderate inhibi-
tion against HIV-1 (VSV-G) and P. falciparum (Table 1). The integr-
ase inhibitor raltegravir and the reverse transcriptase inhibitor
AZT were used as positive controls in the HIV assay while CQ and
DHA were used as positive controls in Malstat assays. Eleven (11)
compounds had IC50 values in the nanomolar range for both strains
of P. falciparum. Among these, compounds 3d, 1a and 1d showed
significant reduction in the growth of HeLa cells. Nonetheless, 3d
Hybrid 7 was evaluated in vivo using the mouse model de-
scribed by Peters.30 Plasmodium berghei N strain infected mice
were treated for 4 days and the parasitemia was monitored on
days 2, 4 and 7 after the beginning of the treatment. The parasi-
temia (%) was calculated for each mouse in different groups and
the mean parasitemia and standard deviations calculated
(Table 4).
showed high activity against pseudotyped HIV-1 (IC50 = 0.9 lM;
Treatment with compound 7 (in pheroid formulation) did not
reduce parasitemia at the administered concentration 7 days
after the beginning of the treatment. These data suggest that
the compound 7 does not suppress parasite growth in vivo after
oral administration. Also, compound 7 appeared not to be toxic
to mice as mice in the compound 7 treated group became dis-
tressed at the same time as mice from the untreated control.
For a drug to be effective, enough of the active form must reach
the target to elicit the desired effect. We therefore sought to
understand why the molecule was not active in vivo after formu-
lation. Drug metabolism and pharmacokinetic studies were thus
carried out.
SI = 31.8) while 1a showed no effect and 1d exhibited moderate
activity on pseudotyped HIV (IC50 = 7.92; SI = 4.6). Both compounds
7 and 8 were not toxic to HeLa cells at 100 lM. Interestingly, hybrid
7 was fourfold more active than 8 against pseudotyped HIV-1
which, speculatively, may be indicative of the importance of the
azido group on AZT or the detrimental effect of the triazole ring sys-
tem on anti-HIV activity. Additionally, compound 7 displayed anti-
plasmodial activity comparable to reference antimalarial drugs CQ
and DHA and was considered our priority since moderate activity
against HIV was also observed (IC50 = 2.86 lM; SI >35). For further
characterization, we decided to focus only on the compound 7 with
the most optimal profile.

5282
M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
Table 1
100
Estimation of IC50 (concentration resulting in 50% inhibition of cell growth or viral
replication) obtained after inhibition of P. falciparum sensitive (3D7) strain, resistant
(Dd2) strain, pseudotyped HIV-1 and HeLa cells with serial dilution of hybrid
molecules. IC50s and standard deviations were calculated using the software GrapPad
prism.
80
60
40
20
0
IC50 (lM)
Compounds P. falciparum
P. falciparum
Dd2
IC50 SEM
HIV-1 (VSV- HeLa cells
G)
3D7
IC50 SEM
IC50 SEM
IC50 SEM
1a, n = 2
1d, n = 5
2a, n = 2
2d, n = 5
3d, n = 4
4d, n = 4
5
6
7
8
9
11
0.37 0.15
0.58 0.17
3.53 0.56
5.18 0.42
0.38 0.22
1.94 0.49
0.46 0.01
0.16 0.06
0.03 0.01
0.27 0.06
0.22 0.17
2.20 0.14
1.42 0.28
0.03 0.02
0.13 0.10
11.12 2.77
0.27 0.09
NE
0.34 0.20
0.08 0.02
31.60 14.71
8.02 2.43
0.08 0.02
10.57 4.95
0.65 0.14
0.22 0.06
0.01 0.00
0.10 0.03
0.10 0.08
1.72 0.04
1.46 0.30
0.23 0.04
0.23 0.12
12.10 1.43
0.19 0.08
—
NE
23.78 1.70
7.92 2.34 36.13 1.43
NE
NE
>100
>100
0.90 0.11 28.65 5.09
NE
>100
7.15 2.57 >100
1.76 0.46 >100
2.86 0.39 >100
11.10 0.46 >100
Figure 2. Inhibition of gametocytes maturation. Compounds at IC50s or a 0.5%
volume of DMSO were added to stage II gametocyte cultures for 2 days. The
numbers of stage IV and V gametocytes were counted after 7 days and correlated to
the gametocytemia of the DMSO control (normalized to 100%). The graph
represents results of two independent experiments in triplicate for PQ, compound
NE
NE
NE
NE
NE
NE
23.38 4.97
>100
>100
63.44 6.28
56.64 17.88
63.97 11.07
>100
7, CQ (mean SEM) and
a single experiment for DHA (mean SD). Statistical
14
analysis was performed using one way ANOVA followed by a Tukey test and mean
gametocytemia of compounds tested once were compare with mean gametocyt-
emia in DMSO control using t-test (GraphPad Prism 5). Asterisks represent a
significant difference between tested compounds and DMSO control (annex), where
⁄⁄⁄correspond to P <0.001; ⁄⁄correspond to 0.001 < P < 0.01; ⁄correspond to
0.01 < P < 0.05 and for p >0.05 the difference in not considered significant and
there is no asterisk.
16, n = 2
16, n = 5
21
25
AZT
DHA
Tetraoxane
CQ
NE
0.04 0.02 >100
0.03 0.01
0.32 0.28
0.03 0.01
NE
0.01 0.01
0.12 0.03
0.71 1.19
—
NE
NE
>100
>100
12.48 2.45 >100
0.01 0.00 >100
Raltegravir
Table 3
IC50s values of compound 7 obtained from the two HIV in vitro assays
IC50 (nM)
Table 2
Selectivity index values (SI = *CC50/IC50) of active hybrid molecules
HIV-1 (VSV-G)
Wild type (NL43)
Compounds
Selectivity index (CC50/IC50
)
AZT
40 20
0.8 0.7
7
2860 390
485.5 16.5
P. falciparum 3D7 P. falciparum Dd2 Pseudo HIV-1
1a, n = 2
1d, n = 5
2a, n = 2
2d, n = 5
3d, n = 4
4d, n = 4
5
6
7
8
9
11
64.3
62.3
>28.3
>19.3
75.4
>51.5
>217.4
>625
>3333.3
>370.4
106.3
>45.5
>70.4
2114.7
>435.7
69.9
451.6
>3.2
>12.5
358.1
>9.5
ND
4.6
ND
ND
31.8
ND
>14
>56.8
>35
>9
ND
ND
ND
ND
ND
ND
100
80
60
40
20
0
AZT
>153.8
>454.5
>10,000
>1000
233.8
>58.1
>68.5
275.8
>246.3
5.3
Compound 7
14
16, n = 2
16, n = 5
21
5.75
25
AZT
>370.4
ND
>526.3
—
10,000
>833.3
140.8
—
ND
>2500
ND
ND
>8
4
6
8
10
Dihydroartemisinin >3333.3
Tetraoxane
CQ
log [pM]
>312.5
>3333.3
ND
-20
Raltegravir
>10,000
Figure 3. Inhibition of wild type HIV replication. NL43 virus co-culture was
maintained in the presence of different concentrations of AZT and compound 7 for
48 h and the supernatant was used to infect TZM cells to quantify the amount of
particles formed in the presence of inhibitors. The mean count of wells with ‘no
drug and no virus’ was normalized to 100% activity and the mean count of wells ‘no
drug and with virus’ was normalized to 0% activity. Percentage activities were
deducted for treated wells.
*
CC50 = IC50 on HeLa cells: cytotoxic concentration 50, which is concentration
resulting in 50% inhibition of HeLa cells growth. ND: Not determined.
3.3. Evaluation of the metabolic stability and PK properties of
compound 7 in vitro and in vivo
of mice treated with compound 7 dissolved in 10% DMSO (Fig. 4A),
but after being formulated into Pheroid™ vesicles, a maximal con-
centration of approximately 40 ng/ml was measured in mice plas-
ma (Fig. 4B). This suggests that absorption of the compound 7 is
enhanced by Pheroid™ formulation, but the peak-like shape indi-
cate a rapid clearance of the parent drug (Fig. 4B). The 2 drugs were
Compound 7 was administered to mice in two different formu-
lations: the first group of mice received compound 7 dissolved in
a mixture of DMSO and distilled water (1:9, v/v) and the second
group received compound 7 formulated into Pheroid™ vesicles.
We found no trace of the compound 7 in the plasma from the group

M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
5283
quantified in the same plasma samples collected from two groups
of mice; AZT was detected at a lower concentration when com-
pound 7 was diluted in 10% DMSO (Fig. 5A) and in a much higher
concentration when administered in pheroids (Fig. 5B). DHA was
not detected after administration of the compound 7 when dis-
solved in 10% DMSO or pheroids formulation (Fig. 5C,D). AZT was
present in the blood of both groups of mice but not DHA (Fig. 5).
Compound 7, like other artemisinin derivatives appeared to
have poor bioavailability properties. Despite the apparent im-
proved bioavailability observed when formulated in Pheroid™ ves-
icles, the concentration of 7 in mice plasma remained low to
suppress parasite growth in infected mice. The maximal concentra-
tion in the blood was estimated to be 40 ng/ml, which is the equiv-
alent of 75 nM; the IC50 in vitro was 40 nM, which theoretically is
enough to eliminate approximately 50% of the parasites. However,
compound 7 did not reduce parasitemia in vivo as mice in the
group treated with this compound showed the same parasitemia
levels as the untreated group. Data from in vitro metabolism stud-
ies clearly showed that compound 7 was a very unstable drug and
potentially cleaved by CYP 450 enzymes. In vitro and in vivo stud-
ies correlated well and showed that compound 7 releases its pre-
cursor drug AZT but not DHA. While AZT was detectable in the
mice plasma (in vivo) or in the buffer (in vitro), DHA was not de-
tected in the same plasma or buffer. In vitro studies showed that
the presence of AZT in buffer was detected after 15 min incubation
while more than 90% of compound 7 was already metabolized
(Fig. 6). This further supports the rapid clearance already observed
with in vivo studies.
Table 4
In vivo activity of the compound 7 against the erythrocytic forms of P. berghei N
Compounds
Dose (mg/kg per day)
Parasitemia day 2 (%)
Parasitemia day 4 (%)
Parasitemia day 7 (%)
7
20
10
0
4.24 0.6
<1%
3.2 1.3
11.43 2.6
<1%
9.9 1.8
35.2 3.6
<1%
33.1 3.4
Chloroquine
Untreated control
Figure 4. Plasma concentration–time profiles of the compound 7. (A) Plasma concentration of the compound 7 in mice treated by oral administration of 20 mg/kg dissolve in
10% DMSO; (B) plasma concentration in mice treated by oral administration of 20 mg/kg dissolve in pheroids formulation.
Figure 5. Plasma concentration–time profiles of AZT and DHA. (A) Plasma concentration of AZT in mice treated by oral administration of 20 mg/kg dissolve in 10% DMSO; (B)
plasma concentration of DHA in mice treated by oral administration of 20 mg/kg dissolve in 10% DMSO; (C) plasma concentration of AZT in mice treated by oral
administration of 20 mg/kg dissolve in Pheroids) and (D) plasma concentration of DHA in mice treated by oral administration of 20 mg/kg dissolve in Pheroids.

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M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
in compound 7 not having a free hydroxyl group, which would con-
tribute to improved solubility.
Despite the apparent improved bioavailability observed when
formulated in pheroid vesicles, the concentration of compound 7
in mice plasma remained low to suppress parasite growth in in-
fected mice and rapid clearance was observed. This suggested poor
stability and a short half life in vivo. The O-dealkylation of the oxy-
gen linking AZT and DHA in compound 7 appeared to be the main
reason for the poor metabolic stability of compound 7. In vitro and
in vivo studies correlated well and showed that compound 7 re-
leases AZT, but not DHA.
DHA is reported to be metabolized extensively to hydroxylated
derivatives by rat liver microsomes.35 However, a study carried out
in vivo and in vitro using human microsome preparations revealed
that DHA is mainly glucuronidated.36 AZT is also known to be
Figure 6. Metabolism of compound 7 in mice liver microsomes.
metabolized by glucuronide conjugation to
a major inactive
4. Discussion and conclusion
metabolite, 3-azido-3-deoxy-5-O-beta- -glucopyranuronosylt-
D
hymidine.37 Compound 7 was synthesized by linking AZT and
DHA at their respective glucuronidation sites,37,38 on this basis,
Compound 7 cannot be conjugated via glucuronidation unless it
is first hydroxylated. The DHA moiety released as the result of a
cleavage product might have undergone some hydroxylations prior
to cleavage, and could not be detected in mouse plasma in vivo or
in the buffer in vitro, as the method was strictly designed to detect
DHA and not hydroxylated products.
The interaction between HIV and malaria infection is bidirec-
tional and synergistic,31 and currently there are no specific recom-
mended treatment regimens for malaria and HIV/AIDS co-
infection. In this study, we investigated hybrid molecules for their
ability to target both P. falciparum and HIV. We anticipated that
these molecules could be used to treat malaria infected individuals
while offering protection against HIV. This strategy could be partic-
ularly beneficial for intermittent preventive treatment in HIV in-
fected pregnant women. Hybrid molecules synthesized in this
study showed strong to moderate potency in vitro against both,
HIV and P. falciparum, but most were not investigated further be-
cause of their cytotoxicity. Compound 7, the most potent hybrid
in our in vitro assays has poor aqueous solubility and displayed
poor bioavailability in vivo. However, our preliminary data are
encouraging as the antiplasmodial activity of compound 7 was
comparable to that of DHA in vitro. DHA is practically insoluble
in water just like compound 7 and because of its poor aqueous sol-
ubility, DHA has only been formulated as oral and rectal tablet
preparations.32 A combination of DHA with piperaquine is cur-
rently an option for the treatment of uncomplicated malaria. These
data suggest that compound 7 could be developed further and re-
quires appropriate formulation for its delivery. If successfully
developed as a drug, compound 7 could have an advantage over
DHA, since it might confer some additional protection against HIV.
Compound 7 did not suppress parasite growth in vivo when
administered orally in pheroid vesicles. Taking into account that
compound 7 was administered in pheroid vesicles to improve
absorption, moderate antimalarial activity was at least expected.
Since unacceptable PK properties is one of the main reasons for
clinical failure of drug candidates,33 it was therefore necessary to
evaluate other factors contributing to the poor PK profile of com-
pound 7, and to better understand the poor in vivo activity.
Our attempt to study PK properties was to use the data as a guide
in determining whether or not compound 7 was likely to be im-
proved. The hybridization of AZT and DHA obviously leads to the
generation of a higher molecular weight (MW) compound which
might explain the predicted poor solubility of compound 7, as the
trend towards increased MW is likely to worsen both aqueous solu-
bility and intestinal permeability.34 The reduction in MW is a useful
approach in drug development for increasing solubility and might
also improve metabolic stability. In our current context we envis-
aged that the two moieties would remain linked and thus preclude
the need to reduce the MW. Compound 7 displayed poor solubility
and prompted us to dissolve in DMSO for in vitro assays and to pre-
pare a formulation for in vivo studies since the latter can be used to
increase the absorption of the molecule. However, the observed poor
solubility is not surprising, considering the point of attachment of
AZT to DHA through the primary hydroxyl group of AZT, resulting
We have demonstrated the use of hybrid molecules to inhibit
two non-related organisms and our data showed that appropri-
ately designed hybrid molecules could have applications in the
treatment of HIV/malaria co-infections. It could be advantageous
to exploit compounds targeting aspartic proteases as recent studies
have shown that clinically approved HIV Protease Inhibitors (PIs)
were able to inhibit malaria parasite growth and to augment the
antimalarial action of artemisinin in vitro.
5. Experimental data
Chemicals and reagents were purchased from either Sigma–Al-
drich or Merck, South Africa. Chromatography solvents were pur-
chased from Kimix Chemicals or Protea Chemicals, South Africa,
Chemically Pure (CP grade) solvents and distilled before use.
5.1. Procedures of hybrid molecules synthesis
5.1.1. Procedure for the synthesis of phenyl 2-((tert-
butyldiphenylsilyloxy) methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-ylcarbamate 20
To a well stirred solution of 19 (200 mg, 0.417 mmol, 1 equiv) in
dry DCM was added (36.2 mg, 0.458 mmol, 1.1 equiv) of dry pyri-
dine. The resulting solution was stirred for 5 min followed by the
addition of (72.0 mg, 0.458 mmol,1.1 equiv) of phenyl chlorofor-
mate. The resulting reaction mixture was then refluxed for 16 h
and the progress of the reaction was monitored by TLC. On comple-
tion, the reaction mixture was diluted with DCM (200 ml) and
washed with 1 M sodium bicarbonate solution, water and brine
in succession. The organic layer was dried over anhydrous magne-
sium sulphate and the solvent was removed under reduced pres-
sure. The concentrated reaction mixture was subjected to further
reaction without purification. dH (400 MHz, CDCl3) 9.01 (s, 1H,
NH, exchangeable with D2O), 8.59 (d, 1H, J 8.09, NH, exchangeable
with D2O), 7.46 (m, 11H, ArH), 7.14 (m, 5H, ArH), 6.57 (m, 1H), 4.93
(m, 1H), 4.01 (m, 3H), 2.37 (m, 2H), 1.50 (d, 3H, J 1.12), 1.08 (s, 9H);
dC (100.6 MHz) 163.4, 161.0, 156.6, 151.5, 135.8, 135.2, 132.8,
133.6, 133.5, 130.5, 130.3, 128.9, 128, 127.9, 125.3, 121.4, 112.8,
86.5, 84.6, 64.1, 50.1, 37.5, 27.1, 13.7, 12.1; Mass 599.34. Elemental
analysis for; C33H37N3O6Si Calculated: C, 66.09; H, 6.22; N, 7.01;
Found: C, 65.95; H, 6.19; N, 7.10.

M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
5285
5.1.2. Procedure for the synthesis of ethyl-2-(2-((tert-
sis for; C41H49ClN6O5Si Calculated: C, 64.00; H, 6.69; N, 10.92.
Found: C, 62.34; H, 6.44; N, 11.11.
butyldiphenylsilyloxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-ylamino)-2-
oxoacetate 21
5.1.5. N1-(2-((tert-butyldiphenylsilyloxy)methyl)-5-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-
yl)-N2-(5-(7-chloroquinolin-4-ylamino)pentyl)oxalamide 3d
To a solution of 21 (200 mg, 0.345 mmol, 1 equiv) was added of
(94.9 mg, 0.380 mmol, 1.1 equiv) 4,7-dicholoquniloine-based
amines in anhydrous MeOH. The resulting reaction mixture was
then refluxed for 12 h. The progress of the reaction was monitored
by TLC. On the completion of the reaction, the solvent was re-
moved under reduced pressure to yield the corresponding keto-
amides in good to excellent yields. dH (400 MHz, DMSO-d6) 11.30
(br s, 1H, NH, exchangeable with D2O), 9.14 (d, 1H, J 8.67, NH,
exchangeable with D2O), 8.77 (t, 1H, J 6.23, NH, exchangeable with
D2O), 8.37 (d, 1H, J 5.34), 8.26 (dd, 1H, J 3.02, 9.09), 7.76 (d, 1H, J
2.22), 7.60–7.62 (m, 4H), 7.36–7.45 (m, 8H), 6.45 (d, 1H, J 5.50),
6.25 (t, 1H, J 6.72), 4.62 (m, 1H), 3.99 (m, 1H), 3.81 (m, 2H), 3.23
(m, 2H), 3.13 (m, 2H), 2.30 (m, 2H), 1.69 (m, 2H), 1.61 (m, 2H),
1.53 (s, 3H), 1.38 (m, 2H), 0.98 (s, 9H); dC (100.6 MHz, DMSO-d6)
164.5 156.7, 151.9, 151.2, 150.9, 148.8, 136.7, 135.6, 135.5, 133.5,
133.1, 130.6, 128.5, 127.1, 125.0, 124.6, 115.7, 110.5, 99.2, 84.2,
83.6, 63.8, 48.8, 42.8, 36.8, 28.9, 27.9, 27.1, 24.4, 21.0, 19.4, 12.3;
Mass calculated 797.41. Found: 797.41; Elemental analysis for;
To the well stirred solution of 19 (200 mg, 0.417 mmol, 1 equiv)
in dry DCM, was added (62.8 mg, 0.459 mmol, 1.1 equiv) of ethyl 2-
chloro-2-oxoacetate. The reaction mixture was stirred at room
temperature for 6 h. Upon completion, as evidenced by TLC, a sat-
urated solution of sodium bicarbonate was added and the solution
was stirred vigorously until it turned slightly alkaline in nature.
The reaction mixture was then extracted with DCM and the organic
layer was dried over anhydrous magnesium sulphate. The solvent
was removed under reduced pressure resulting in the isolation of
crystalline white solid. The structure was assigned on the basis
of spectral data and analytical evidences. dH (400 MHz, CDCl3)
8.98 (s, 1H, NH, exchangeable with D2O), 8.57 (d, 1H, J 8.09, NH,
exchangeable with D2O), 7.65 (m, 4H), 6.54 (m, 1H), 7.39 (m,
7H), 4.88 (m, 1H), 4.38 (q, 2H, J 7.15), 3.98 (m, 3H), 2.35 (m, 2H),
1.51 (d, 3H,
J 1.12 Hz), 1.39(t, 3H, J 7.14), 1.09(s, 9H); dC
(100.6 MHz) 163.2, 161.2, 156.6, 151.4, 135.6, 135.2, 133.4,
133.3, 132.3, 130.1, 130, 128, 127.9, 112.3, 86.2, 84.4, 64.5, 63.9,
50.3, 37.8, 27, 19.5, 13.9, 11.9. Mass 579.25. Elemental analysis
for C30H37N3O7Si Calculated: C, 62.15; H, 6.43; N, 7.25. Found: C,
62.05; H, 6.32; N, 7.19.
C42H49ClN6O6 Si Calculated: C, 63.26; H, 6.19; N, 10.54. Found: C,
60.17; H, 6.25; N, 9.98.
5.1.3. 1-(2-((tert-Butyldiphenylsilyloxy)methyl)-5-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-
yl)-3-(2-(7-chloroquinolin-4-ylamino)ethyl)urea 1a
5.1.6. 1-[2-(7-Chloro-quinolin-4-ylamino)-ethyl]-3-[2-
hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-
To a solution of 20 (200 mg, 0.333 mmol, 1 equiv) was added
(81.4 mg, 0.367 mmol, 1.1 equiv) of 4,7-dichloroquinoline-based
amines in anhydrous methanol (MeOH). The resulting reaction
mixture was refluxed for 12 h. The progress of the reaction was
monitored by TLC. The solvent was removed under reduced pres-
sure on the completion of the reaction and the residue was column
chromatographed using 10% MeOH/DCM mixture, resulting in the
isolation of the corresponding amides in good yields. The structure
of the amides was assigned on the basis of spectral data and ana-
lytical evidences. dH (300 MHz, DMSO-d6) 11.28 (br s, 1H, NH,
exchangeable with D2O), 8.40 (d, 1H, J 5.54), 8.19 (d, 1H, J 9.11),
7.78 (d, 1H, J 2.21), 7.62–7.67 (m, 5H), 7.36–7.44 (m, 7H), 6.62
(br s, 1H, NH, exchangeable with D2O), 6.59 (br s, 1H, NH,
exchangeable with D2O), 6.57 (d, 1H, J 5.62), 6.23 (t, 1H, J 6.65,
NH, exchangeable with D2O), 6.15 (m, 1H), 4.4 (m, 1H), 3.86 (m,
3H), 3.23 (m, 4H), 2.21 (m, 2H), 1.53(d, 3H, J 0.82), 1.0(s, 9H); dC
(75.4 MHz- DMSO-d6) 163.5, 161.2, 158.1, 151, 150.6, 150.2,
135.4, 135.0, 134.8, 132.5, 129.8, 127.7, 126.7, 124.2, 123.8,
109.5, 117.1, 98.5, 84.6, 83.2, 63.9, 49.5, 43.6, 38.0, 37.3, 26.6,
18.8, 11.7; Mass calculated: 727.32, Found: 727.41; Elemantal
analysis for; C38H43ClN6O5Si Calculated: C, 62.75; H, 5.96; N,
11.5. Found: C, 59.85; H, 6.19; N, 11.1.
2pyrimidin-1-yl)-tetrahydro-furan-3-yl]-urea 2a
To a mixture of 1a (318 mg, 0.415 mmol, 1 equiv) in ethanol
(2 ml) and water (0.5 ml) was added potassium carbonate
(1.38 g, 10 mmol, 24 equiv). The reaction mixture was refluxed at
90 °C for 3 days. The completion of the reaction mixture was
checked using TLC with anisaldehyde spray in 10% MeOH/DCM sol-
vent system. The reaction mixture was concentrated and purifica-
tion was done using column chromatography with 15% MeOH:
DCM solvent system. white solid; mp 178 °C; dH (400 MHz, CD3OD)
8.36 (1H, d, J 5.6), 8.0(1H, J 9.0, d), 7.86 (1H, q, J 1.2), 7.8 (1H, d, J
2.1), 7.41(dd, 1H, J 2.1, J0 9.0), 6.58 (d, 1H, J 5.6), 6.17 (dd, 1H, J
5.6, J0 12.2), 4.33 (dd, 1H, J 6.46, 7.5), 3.48 (m, 4H,), 3.77 (3H, m),
2.23 (1H, m, 2H), 1.89 (3H, d, J 1.13), dC (75.4 MHz; CD3OD)
159.8, 151, 136.7, 135, 132.9, 130.1, 126.2, 124.7, 123.4, 122.8,
117.3, 110, 98.1, 85.5, 84.4, 60.9, 49.5, 43.7, 38.43, 37.9, 11; Calcu-
lated Mass 488.15, LCMS single peak, 3.65 min, m/z 489.2(M+1).
Elemental analysis for; C22H25ClN6O5 Calculated: C, 54.04; H,
5.15; N, 17.19. Found: C, 54.15; H, 5.05; N, 17.13.
5.1.7. 1-[5-(7-Chloro-quinolin-4-ylamino)-pentyl-3-[2-
hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-
pyrimidin-1-yl)-tetrahydro-furan-3-yl]-urea 2d
To a mixture of 1d (318 mg, 0.415 mmol, 1 equiv) in ethanol
(2 ml) and water (0.5 ml) was added potassium carbonate
(1.15 g, 8.4 mmol, 20 equiv). The reaction mixture was refluxed
for 7 days. The completion of the reaction mixture was checked
using TLC with anisaldehyde spray in 10% MeOH/DCM solvent sys-
tem. The reaction mixture was concentrated and purification was
done using column chromatography with 15% MeOH/DCM solvent
system. Yellow solid; mp 152 °C; dH (400 MHz, CD3OD) 8.36 (1H J
6.3, d), 8.24 (J 9.0, d, 1H), 7.85 (1H, q, J 1.19), 7.8 (1H, d, J 2.0),
7.53 (1H, dd, J 2.0, J0 9.0), 6.67 (1H, d, J 6.42), 6.16 (dd, J 5.5, J0
12.12), 4.33 (d, J 6.6 H), 3.75 (m, 3H), 3.49 (t, J 7.13), 3.15 (t, J
6.44,), 2.27 (2H, m), 1.88 (d, 3H), 1.82 (quintet, 2H, J 6.9, CH2),
1.51 (4H, m, CH2CH2); dC (75.4 MHz, CD3OD) 165, 159.28, 153.6,
150.9, 146.9, 143.84, 137, 136.6, 125.8, 123.6, 122.8, 116.5, 110,
98.2, 85.61, 84.4, 61.0, 49.5, 42.9, 39.3, 38.0, 29.6, 27.5, 23.8, 11.
5.1.4. 1-(2-((tert-Butyldiphenylsilyloxy)methyl)-5-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-
yl)-3-(5-(7-chloroquinolin-4-ylamino)pentyl)urea 1d
dH (400 MHz, DMSO-d6) 11.25 (s, 1H, NH, exchangeable with
D2O), 8.33 (d, 1H, J 5.45), 8.21 (dd, 1H, J 5.23, J0 9.05), 7.74 (d, 1H,
J 2.14); 7.60 (m, 5H), 7.38 (m, 7H), 6.43 (t, 1H, J 5.21), 6.24 (d,
1H, J 7.51, NH, exchangeable with D2O), 6.15 (t, 1H, J 6.44, NH,
exchangeable with D2O), 5.82 (t, 1H, J 5.75, NH, exchangeable with
D2O), 4.32 (m, 1H), 3.82 (m, 3H), 3.29 (m, 2H), 3.22 (m, 2H), 2.18–
2.27 (m, 2H), 1.65 (m, 4H), 1.47 (s, 3H), 1.37 (m, 2H), 0.96 (s, 9H,);
dC (75.4 MHz) 163.5, 157.4, 151.7, 150.2, 150.0, 148.9, 135.4, 134.9,
134.8, 132.5, 129.7, 127.7, 127.3, 123.9, 123.8, 117.3, 109.5, 98.5,
84.6, 83.2, 63.9, 49.4, 42.3, 37.4, 29.7, 27.5, 26.5, 24.2, 23.9, 18.7,
11.7; Mass calculated: 769.40 Observed: 769.45; Elemantal analy-

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M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
Calculated mass 530, LCMS single peak, 3.65 min. Calculated mass
531.2(M+1). LCMS single peak, 3.65 min, m/e, 531.3(M+1). Elemen-
tal analysis for; C25H31ClN6O5 Calculated: C, 56.55; H, 5.88; N,
15.83. Found: C, 56.43; H, 5.85; N, 15.89.
H 12), 4.95 (1H, d, J 3.3, H 10), 4.29 (2H, d, J 2.4), 2.65 (1H, m),
2.40–2.30 (2H, m), 2.05–1.17 (10H, m), 1.42 (3H, s), 0.93 (3H, d, J
6.0), 0.91 (3H, d, J 7.2); dC (75 MHz, CDCl3) 104.1, 100.6, 88.0,
81.0, 79.7, 73.9, 54.9, 52.5, 44.3, 37.4, 36.4, 34.6, 30.6, 26.1, 24.7,
24.4, 20.3, 12.7; HRMS(ESI) found m/z 323.1207 [M+H]+ for
5.1.8. N-[5-(7-chloro-quinolin-4-ylamino)-pentyl]-N-[2-
C18H26O5 requires 322.17802; Anal. Calcd for C18H26O5: C, 67.06;
hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-
H, 8.13. Found: C, 66.70; H, 8.10.
pyrimidin-1-yl)-tetrahydro-furan-3-yl]-oxalamide 4d
To a mixture of 3d (318 mg, 0.415 mmol, 1 equiv) in ethanol
(2 ml) and water (0.5 ml) was added potassium carbonate
(1.38 g, 10 mmol, 24 equiv). The reaction mixture was refluxed at
90 °C for 3 days. The completion of the reaction mixture was
checked using T.L.C with anisaldehyde spray in 10% MeOH/DCM
solvent system. The reaction mixture was concentrated and purifi-
cation was done using column chromatography with 13% MeOH:
DCM solvent system. white solid; mp 257 °C; dH (400 MHz, CD3OD)
11.2 (br s, NH), 8.69 (J 6.11, t, 1H, NH), 8.35 (d, J 5.42, 1H), 8.23 (d, J
9.0, He), 7.72(d, 1H, J 2.2), 7.71 (q, 1H, J 1.2),7.39 (1H,dd, J 2.2, J0
8.96), 7.21 (t, 1H, J 5.0, NH), 6.42 (d, 1H, J 5.47), 6.17 (t, 1H, J
6.22), 4.97 (t, 1H, J 5.0, OH), 4.39 (m, 1H), 3.58(1H, m), 3.51 (1H,
m), 3.22 (2H, m), 3.12 (2H, m), 1.75 (3H, d, J 1.0), 1.61 (2H, quintet,
J 7.5), 1.5 (2H, quintet, J 6.84), 2.22 (2H, m), 1.34 (2H, q, J 6.88),
3.85(1H, m); dC (75.4 MHz, CD3OD) 164.2, 160.6, 160.1, 152.3,
150.8, 150.5, 149.5, 136.8, 133.8, 127.9, 124.5, 124.4, 117.9,
109.8, 99.1, 84.4, 84, 61.5, 49.2, 42.8, 37.1, 28.9, 27.9, 12.6; Calcu-
lated mass 559.2(M+1), LCMS single peak, 4.23, min, m/e,
559.5(M+1). Elemental analysis for; C26H31ClN6O6 Calculated: C,
55.86; H, 5.59; N, 15.03. Found: C, 56.83; H, 5.65; N, 15.14.
5.1.11. Hybrid 8
Copper(II) sulphate pentahydrate (0.0232 mmol, 5 mol %) and
sodium ascorbate (0.0696 mmol, 15 mol %) was added to a mixture
of AZT (0.464 mmol, 1.0 equiv) and the acetylene 23 (0.510 mmol,
1.1 equiv) in 3 mL of DCM/H2O (1:1). The resulting mixture was
stirred for 24 h at 25 °C. Upon completion, the product mixture
was diluted with water and extracted with EtOAc. The combined
organic layer was washed with water and brine, dried over anhy-
drous Na2SO4 and concentrated under reduced pressure. The crude
product so obtained was subjected to column chromatography
(0.5–10% MeOH/DCM) to give the b-isomer as a light green foam
(199.9 mg, 45%); mp 122 °C; dH (400 MHz, CDCl3) 9.27 (1H, s),
7.68 (1H, s), 7.46 (1H, s), 6.24 (1H, br s), 5.46 (1H, s), 4.93 (1H, br
s), 4.91 (1H, d, J 12.8), 4.71 (1H, d, J 12.8), 4.43 (1H, br s), 4.03
(1H, br d, J 11.2), 3.81 (1H, br d, J 11.6), 3.57 (1H, br s, OH), 3.00
(2H, br s), 2.65 (1H, m), 2.40–1.16 (11H, m), 1.93 (3H, s), 1.44
(3H, s), 0.94 (3H, d, J 5.6), 0.90 (3H, d, J 7.2); dC (100 MHz, CDCl3)
163.8, 150.5, 145.6, 137.9, 122.8, 111.3, 104.2, 102.1, 88.8, 88.0,
85.4, 81.1, 61.8, 61.6, 59.3, 52.5, 44.4, 37.5, 37.4, 36.4, 34.6, 30.9,
26.1, 24.7, 24.5, 20.3, 12.3, 12.4; HRMS(ESI) found m/z 590.2811
[M+H]+ for C28H39N5O9 requires 589.27478; Anal. Calcd for
5.1.9. 1-(4-Azido-5-hydroxymethyl-tetrahydro-furan-2-yl)-3-[3-
(7-chloro-quinolin-4-ylamino)-benzyl]-5-methyl-1H-
C28H39N5O9: C, 57.03; H, 6.67; N, 11.88. Found: C, 56.78; H, 6.61;
N, 11.49.
pyrimidine-2,4-dione 6
Sodium hydride (7.99 mg, 0.33 mmol, 1 equiv) was added at
0 °C to AZT (89 mg, 0.33 mmol, 1 equiv) in dry DMF under N2
atmosphere. The solution was stirred 25 min at 0 °C followed by
stirring at room temperature for 20 min. The (15) quinoline deriv-
ative (100.66 mg, 0.33 mmol, 1 equiv) in dry DMF was added drop-
wise at room temperature to the above mentioned solution. The
solution was stirred for 12 h and monitored with TLC using anisal-
dehyde spray. Extraction was done using water and ethyl acetated
solvent mixture. The compound was purified with 5% MeOH/DCM
solvent system. Yellow solid; mp 238 °C; yield 80%; dH (400 MHz,
DMSO-d6) 9.0 (br s, 1H), 8.44 (d, 1H, J 5.28, Hb), 8.38 (d, 1H, J
9.0), 7.88 (d, 1H, J 2.1), 7.55 (dd, J 2.1, J0 9.0), 7.79(d, 1H, J 1.0),
7.36 (m, 1H), 7.24 (m, 2H), 7.0 (d, J 7.65), 6.91 (d, J 5.34), 6.16 (t,
J 6.31), 5.2 (OH, br s), 5.01 (2H, m), 4.39 (dt, J 5.44, J0 12.0), 3.84
(dd, J 3.7, J00 8.67), 3.64 (2H, m), 2.42(1H,ddd, J 6.91, J0 13.64, J00
20.43), 2.31 (1H,ddd, J 6.54, J0 13.73, J00 19.44), 1.85 (br s, 3H); dC
(75.45 MHz, DMSO-d6) 163.1, 152.3, 150.9, 150.1, 148.2, 140.7,
138.9, 135.5, 134.3, 129.7, 128.15, 125.4, 124.9, 123.8, 121.9,
121.5, 118.9, 109.1, 102.4, 84.9, 84.6, 61.1, 60.4, 44.1, 36.8, 13.34.
Calculated Mass 534.16 (M+1). LCMS single peak, 5.65 min, m/e,
534.4 (M+1).
5.1.12. 1-(4-Azido-5-hydroxymethyl-tetrahydro-furan-2-yl)-3-
[2-(7-chloro-quinolin-4-ylamino)-ethyl]-5-methyl-1H-
pyrimidine-2,4-dione 5
Sodium hydride (12 mg, 0.5 mmol, 1 equiv) was added at 0 °C to
AZT (133 mg, 0.5 mmol, 1 equiv) in dry DMF under N2 atmosphere.
The solution was stirred for 25 min at 0 °C followed by stirring at
room temperature for 20 min. The (12) quinoline derivative
(142 mg, 0.5 mmol, 0.5 equiv) in dry DMF was added drop wise
at room temperature to the above mentioned solution. The solu-
tion was stirred for 12 h and monitored by TLC using anisaldehyde
spray. Extraction was done using water and ethyl acetated solvent
mixture. The compound was purified with 5% MeOH/DCM solvent
system. White solid; mp 157–159 °C; Yield 26%; dH (400 MHz,
DMSO-d6) 8.41(1H, d, J 5.41, Hb), 8.11 (1H, J 9.1, d), 7.77(1H, d, J
2.2), 7.75 (1H, Q, J 1.19), 7.48(t, J 5.6, NH), 7.43 (dd, 1H, J 2.2, J0
8.8), 6.69(1H, d, J 5.46), 6.0 (1H, t, J 6.3), 5.21 (1H, br s, OH), 4.35
(1H, dt, J 5.56, J0 6.9), 4.0 (2H, m,), 3.82 (1H, m), 3.66 (1H, m),
3.60 (1H, m), 3.48 (2H, m), 2.25(1H, ddd, J 5.86, J0 6.59, J00 13.7),
2.23(1H, ddd,
J J 1.0); dC
5.8, J0 7, J00 13.5), 1.82 (3H, d,
(75.45 MHz, DMSO-d6) 163.4, 152.3, 151, 150.6, 149.6, 135.3,
133.8, 128, 124.6, 124.3, 117.9, 109, 85, 84.7, 61.1, 60.2, 36.9,
13.3. Calculated mass 472.15 (M+1), LCMS, single peak, 4.94 min,
m/e, 472.4(M+1).
5.1.10. Compound/intermediate 23
Boron trifluoride diethyl etherate complex (5 drops) was added
slowly to a solution of dihydroartemisinin (1.00 g, 3.54 mmol,
1 equiv) and propargyl alcohol (794 mg, 14.2 mmol, 4 equiv) in
10 mL of dry DCM at 0 °C under a nitrogen atmosphere. The result-
ing mixture was allowed to warm to 25 °C and stirred at this tem-
perature for 10 h. The product mixture, after dilution with DCM,
was washed successively with 5% aqueous NaHCO3, water and
brine. The organic layer was dried over anhydrous Na2SO4 and con-
centrated. Purification of the crude product using column chroma-
tography (0–20% EtOAc/Hex) afforded the b-isomer as colourless
needles. mp 116 °C; yield 80%; dH (400 MHz, CDCl3) 5.39 (1H, s,
5.1.13. N-[2-(tert-Butyl-diphenyl-silanyloxymethyl)-5-(5-
methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-tetrahydro-
furan-3-yl]-2-(1,2,4,5-tetraoxa-3-bicyclo[3.3.1]nonane-
spiro[5.5]undec-9-yl)-acetamide 25
A solution of tetraoxane (80 mg, 0.24 mmol), Et3N (33
ll,
0.24 mmol) and ClCO2Et (22.52 l, 0.24 mmol) in dry DCM
l
(10 ml) was stirred for 90 min at 0 °C. Then the amine (223 mg,
0.46 mmol) was added and after 30 min of stirring, the reaction
mixture was warmed to room temperature. After an additional
90 min it was diluted with water and the layers were separated,

M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
5287
the organic layer was with water dried with MgSO4 and evaporated
to dryness. The purification was done using 90% ethylacetate and
hexane and TLC was checked using anisaldehyde stain. mp 98–
100 °C; dH (400 MHz, CD3OD) 10.18 (br s, 1H, NH), 7.58 (1H, Q, J
1.2, H6), 7.40 (6H, m); 7.68 (m, 4H), 6.43 (dd, 1H, J 5.9, J0 8.93),
4.61 (1H, t), 4.07 (m, 3H), 2.12 (2H, d, J 6.5), 2.27 (2H, m), 1.95
(m, 5H), 1.85 (2H, br s), 1.64 (14H, m), 1.55 (3H, d, J1.1, C5-Me),
1.26 (2H, m, adamantyliden), 1.10 (s, 9H, (CH3)3; dC (75.45 MHz,
CD3OD) 172.6, 164.2, 150.9, 135.7, 135.3133.5, 132.6, 130.4,
130.0, 128.4, 127.7, 111.6, 110.5, 107.9, 87.4, 87.37, 85.4, 84.9,
68.3, 65.2, 51.5, 42.6, 37.1, 34.1, 33.8, 33.3, 33.2, 32.9, 27.2, 19.6,
12.5.
5.2.2. Production of pseudotyped lentiviral particles
Viral particles were generated by co-transfection of HEK293T
cells with the transfer vector pWPXL (Addgene, 12257), the pack-
aging plasmid psPAX2 (Addgene, 12260), and the vesicular stoma-
titis virus glycoprotein (VSV-G) expression plasmid, pMD2.G
(Addgene, 12259). These 3 plasmids were used to transfect HEK93T
using the PEI (Polyethyleneimine) method as described.39 At 48 h
and 72 h posttransfection, the viral supernatant was harvested by
filtration through a 0.45-lm cellulose acetate filter to remove cell
debris and viral particles were further concentrated by using Ami-
con ultrafiltration concentrators with a pore size of 100 kDa (Mil-
lipore). The concentrated viral supernatant was used for the
subsequent infection of target cells.
5.1.14. 1-(4-Azido-5-(3R,5aS,6R,8aS,9R,12S,12aR)-decahydro-
3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-
5.2.3. Compound screening
benzodioxepin-10-oxymethyl-tetrahydro-furan-2-yl)-5-
methyl-1H-pyrimidine-2,4-dione 7
The reporter viruses were first used to screen hybrid molecules
for anti-HIV properties. 100 ll of culture containing approximately
To a solution of dihydroartemisnin 0.284 g (1 mmol) and
0.267 g (1 mmol AZT) in 150 ml of dry DCM was added 5 drops
of BF3ꢀEt3O at ꢂ10 °C. The mixture was stirred at room temp until
reaction completed. The solution was worked up using 5% NaHCO3
solution, and purified by column chromatography which resulted
1.2 ꢁ 104 HeLa cells were seeded in a 96-well plate and incubated
overnight at 37 °C. The next day, compounds were diluted in DMSO,
then in medium. Virus stock was thawed, diluted with DMEM and
plated in 96 well plates. Compounds were added to virus suspen-
sion and were immediately transferred to HeLa cells by replacing
old medium with virus suspension and incubated for 48 h at
37 °C. The final DMSO concentration was 1%. After 48 h incubation,
the plate was removed from the incubator and the medium con-
in a diastereomeric mixture of
a and b isomer (1:15) with a com-
bined yield of 63%. White solid; mp 85–87 °C; dH (300 MHz, CDCl3)
NMR for b isomer 8.5 (1H, br, s, D2O exchangeable NH), 7.17 (1H, q,
J 1.1), 6.13 (1H, t, J 6.6), 5.4 (1H, s), 4.80 (1H, d, J 3.5), 4.09 (3H, m),
3.64 (1H, dd, J 2.5, J0 9.9), 2.71 (1H, m), 2.42 (2H, m), 1.92 (d, 3H, J
1.0), 1.43 (br s, 3H), 0.96 (3H, br s), 0.94 (3H, d, J 1.5); dC
(75.45 MHz, CDCl3)163.3, 149.9, 134.6, 111.3, 104.3, 102.1, 87.9,
84.8, 82.6, 80.7, 67.7, 60.8, 52.4, 44.1, 37.55, 37.51, 36.34, 34.4,
30.7, 26, 24.6, 22, 20.2, 13.1, 12.7; FAB MS 534(M+1)+; Elemantal
analysis for; C25H35N5O8 Calculated: C, 56.27; H, 6.61; N, 13.13.
Obtained: C, 56.22; H, 6.40; N, 12.96. Calculated Mass 533, LCMS,
single peak, 8.78 min, m/z, 556(M+23), 572 (M+39).
taining virus particles was discarded and replaced with 100
cold PBS to rinse the cells. The washing step was performed twice
and 110 l lysis buffer (1 mM DTT, 0.1% triton X-100 in 20 mM
ll ice
l
HEPES, pH 7.4) was added and incubated at ꢂ80 °C for 45–60 min
to break cells membrane. The plate was then thawed at room tem-
perature with gentle shaking. Plates were subsequently centrifuged
at 1500 rpm for 10 min at 4 °C to pellet cell debris and 100 ll of
supernatant was then transferred to a black plate for fluorescence
measurement. Fluorescence intensity was measured using a spec-
trofluorometer (Safire2,Tecan) with the following settings. Mea-
surement mode: from the top, excitation wavelenght/bandwidth:
488 nm/10 nm, emission wavelenght/bandwidth: 509 nm/10 nm,
gain (manual): 100, at room temperature.
5.1.15. N-[2-Hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-tetrahydro-furan-3-yl]-2-(1,2,4,5-
tetraoxa-3-bicyclo[3.3.1]nonane-spiro[5.5]undec-9-yl)-
acetamide 9
To
a
mixture of (25) silylated AZT-tetraoxane compound
5.2.4. Infectivity assay using infectious wild type HIV-1 virus
For the most active compound, the anti-HIV activity was evalu-
ated on the replication of a wild type infectious virus in a biosafety
level 3 laboratory. MT4 cells are immortalized T-cells used to rep-
licate HIV in vitro.40 A co-culture is initiated by mixing a viral
suspension with an uninfected MT4 cells culture and a fresh co-
culture is prepared every 48 h by diluting an existing co-culture
with uninfected MT4 cells in a 1:10 ratio (5 ml of co-culture,
5 ml uninfected MT4 culture, 40 ml RPMI medium). A freshly pre-
pared co-culture was assayed in a 48 well plate at a final volume of
(50 mg, 0.062 mmol, 1 equiv) in ethanol (10 ml) and water
(0.5 ml) was added potassium carbonate (171 mg, 1.24 mmol,
20 equiv). The reaction mixture was refluxed at 90 °C for 2 days.
The completion of the reaction mixture was checked using TLC
with anisaldehyde spray in 10% MeOH/DCM solvent system. The
reaction mixture was concentrated and purification was done
using column chromatography with 8% MeOH/DCM solvent sys-
tem. White solid; mp 178 °C; dH (400 MHz, CD3OD) 8.37 (1H, br
s, NH), 7.70 (1H, q, J 1.2, H6), 6.14 (t, 1H, J 5.3), 5.91 (d, 1H, J 7.0,
NH), 4.47 (dd, 1H, J 7.4, J0 14.9), 3.93 (dd, 1H, J 2.2, J0 12.5), 3.75
(2H, m, 2H), 2.34 (2H, m), 2.12 (2H, d, J 6.5), 1.91 (3H, d, J1.1),
1.85 (2H, br s, 2H), 1.62 (14H, m), 1.92 (m, 5H), 1.28 (2H, m), dC
(75.45 MHz; CD3OD) 173.15, 169.1, 163.8, 148.9, 139.4, 135.5,
107.4, 86.7, 84.3, 61.5, 47.6, 43.1, 38.4, 36.7, 33.9, 33.5, 27, 12.5.
500
ll of diluted co-culture per well. After 4 h incubation at 37 °C,
5
ll of inhibitors dissolved in DMSO were added to each well and
plates were incubated for 48 h. Fourty eight (48) hours after incu-
bation, 1 ꢁ 104 TZM cells were seeded in a 96 well plate and
incubated at 37 °C to allow the cells to attach at the bottom and
the 48-well plate containing the co-culture was centrifuged at
Calculated mass 562
562.5(M+1).
2 (M+1), LCMS single peak, 7.59 m/z,
800 g for 10 min. One hundred (100)
ll of MT4 cell-free superna-
tant from each concentration was used to infect TZM cells in trip-
licate, which enables to determine the amount of infectious
particles that have been produced in the presence of inhibitor.41,42
After 2 days post infection, cells were fixed with aceton/ethanol
(1:1), washed three times with PBS. The infected TZM cells were
5.2. Antiviral activity of hybrid molecules
5.2.1. Cells
MT4 and TZM cells were cultured in RPMI 1640 containing glu-
tamine (Invitrogen) supplemented with 10% fetal calf serum (FCS)
(Invitrogen) and penicillin/streptomycin. HeLa and HEK293T cells
were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with glutamine, 10% FCS and penicillin/streptomy-
cin. The FCS was heat-inactivated at 56 °C before use.
further incubated with X-Gal (5-bromo-4-chloro-3-indolyl-b-D-
galactopyranoside) staining solution for 2–4 h. After incubation,
the number of blue cells in each well was counted under bright
field illumination and the mean number of blue cells in each well
and for each concentration was calculated.

5288
M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
5.3. Evaluation of the antimalarial activity of hybrid molecules
liters of the stock solutions were diluted in human plasma to
obtain standard (S) 1 at a concentration of 10 g/ml. S2 (5 g/
g/ml), STD 5 (0.625 g/ml), S 6
g/ml) and S 9
l
l
5.3.1. Malstat assay
ml), S3 (2.5
(0.313 g/ml), S 7 (0.156
(0.0391
l
g/ml), S4 (1.25
l
l
Compounds were screened for growth inhibition activity
against P. falciparum using the Malstat assay as described.43,44 Syn-
chronized ring stages of P. falciparum strains 3D7 and Dd2 were
l
l
g/ml), S 8 (0.0781 l
l
g/ml) were prepared by serial dilution. Standards 4–9
were used to obtain calibration curves (for compound 7, AZT and
DHA) between 39.1 and 1250 ng/ml. The calibration standards
were briefly vortexed and used immediately or stored at ꢂ20 °C.
The calibration standards were analyzed in duplicate in each study
sample batch. The accuracies (%Nom) of the calibration standards
of compound 7, AZT and DHA were between 88.2% and 111.1%,
and the precision (%CV) were between 1.4% and 13.9% during study
sample analysis.
plated in triplicate in 96-well plates (200 ll/well) at a parasitemia
of 1% in the presence of the compounds dissolved in dimethyl sulf-
oxide (DMSO) and the Malstat assay was performed as described
by Aminake et al.45
5.3.2. Gametocyte toxicity test
P. falciparum NF54 parasites were grown at high parasitemia to
favor gametocyte formation as described.46 Upon the appearance
of gametocyte stage II, 1 ml of culture was aliquoted in triplicate
in a 24-well plate in the presence of compounds at their respective
IC50s. The gametocytes were cultivated for 7 days, and the medium
was replaced daily. For the first 48 h of cultivation, the gameto-
cytes were treated with test compounds, and subsequently the
medium was compound free. After 7 days, Giemsa-stained blood
smears were prepared and the gametocytemia was evaluated by
counting the numbers of gametocytes of stage IV and V in a total
number of 1000 erythrocytes.
5.6.2. Compounds extraction
To extract drugs from plasma samples collected from mice,
20
l
l of standards and plasma samples were thawed on ice and
l of internal standard solution (100 ng/ml of deox-
mixed with 80
l
ythymidine in acetonitrile). The samples were vortexed for 1 min,
and sonicated for 5 min, then centrifuged at 1500g for 5 min at
room temperature. The supernatant (approximately 60 ll) was
transferred to a clean tube and evaporated under vacuum in a rotor
evaporation system at 30 °C for 45 min or until the samples were
completely dried. The residue was reconstituted in 100 ll of mo-
bile phase (10 mM ammonium acetate/methanol, 85:15, v/v).
5.4. Evaluation of compounds cytotoxicity
5.7. Chromatography and mass spectrometry
The cytotoxicity of compounds was evaluated using MTT assay.
This assay was first described by Mosmann.47 MTT (3-(4, 5-
Dimethylthiazol-2-yl)-2, 5 Diphenyltetrazoliumbromid) is uptaken
by living cells and converted by mitochondrial dehydrogenases to a
violet formazan that cannot diffuse through cell membranes and
crystallizes in vital cells. The method was carried out as described
by Aminake et al.45
The compound 7, DHA and AZT were quantified using an Agilent
1200 HPLC (Agilent technologies, USA) connected in tandem to an
AB Sciex API 3200 mass spectrometer (AB SCIEX, 110 Marsh Drive,
Foster City, California, USA) equipped with a turbo ion spray (ESI)
source. The software Analyst 1.5.1 was used to control both, the li-
quid chromatogram and the mass spectrometer. The Phenomenex
Luna PFP (50 ꢁ 2.0 mm, 5
lm) column (Phenomenex, USA) was
5.5. Compound administration and blood collection
used for compounds separation. The mobile phase consisted of a
gradient of methanol and 10 mM ammonium acetate. The system
operated at a flow rate of 0.5 ml/min and the volume of injection
A stock solution of Pheroid™ vesicles containing the compound
7 were prepared at the Unit for Drug Research and Development
(North-West University, South Africa) and were dissolved in
NO2–water for the assay. The compound 7 was also dissolved in
10% DMSO, and the two formulations were compared. The com-
pound 7 was administered to two groups of male mice (C57/BL6)
at 20 mg/kg by oral gavage and blood was collected from mice
by tail bleeding at different time points. The blood collected was
transferred to heparanized tubes and immediately centrifuged at
1500g for 10 min at 4 °C. The plasma was separated from red blood
cells and stored at ꢂ80 °C until analysis. The study and all proce-
dures were approved by the Ethics Committee of the University
of Cape Town (REC REF: 010/026). The compound 7 dissolved in
DMSO–water (1:9) formed a white precipitate and was resus-
pended prior to administration. The total volume per administra-
was 20 ll. The ESI interface was used in positive mode and the tur-
bo ion spray source was heated to 450 °C.
The AB Sciex API 3200 mass spectrometer was operated at unit
resolution in the multiple reaction monitoring (MRM) mode, mon-
itoring the transition of the protonated molecular ions at m/z 534.1
to the product ions at m/z 126.9 for the compound 7, the proton-
ated molecular ions at m/z 268.1 to the product ions at m/z 127.0
for AZT, the ammonium adduct at m/z 302.2 to the product ions
m/z 267.1 for DHA, and the the protonated molecular ions at m/z
227.1 to the product ions at m/z 127.0 for the internal standard.
5.8. In vivo efficacy studies of the compound 7
To start an infection, a cryotube containing frozen of P. berghei N
(CQ sensitive) parasites was quickly thawed at room temperature,
tion was 200 ll and 20–25 ll of blood were collected at 30, 120,
240, 480 min after drug administration (compound 7 dissolved in
DMSO). For mice treated with the compound 7 entrapped in phe-
roid vesicles, blood was collected from one mouse at a time at
10, 20, 30, 40, 60, 90, 120, 180, 240, 300, and 360, 420 min after
drug administration to maximize the number of samples collected
and to reduce the gap between times of collection.
then mixed with equal volume of PBS and 200 ll of the resulting
mixture was injected immediately intra-peritoneally into a naive
recipient C57/BL6 male mouse. After 3–4 days, parasites were
monitored in peripheral blood by Giemsa staining, and when par-
asitemia was higher than 10%, blood was obtained by cardiac punc-
ture and diluted in equal volume of PBS. Each animal was
inoculated intraperitoneally with 200 ll of this blood suspension.
5.6. Quantification of plasma level of the compound 7, AZT and
dihydroartemisin
The test drug the compound 7 entrapped in Pheroid vesicles
(20 mg/kg), the standard drug chloroquine (10 mg/kg) and PBS
control were administered to parasitized mice 30 min after admin-
istration of infected erythrocytes on day zero. The same dose was
repeated on days 1, 2 and 3 after infection. Blood was collected
by tail bleeding and smears were prepared and stained with Giem-
5.6.1. Calibration standard
Stock solutions of test compounds (7, AZT and DHA) were pre-
pared in acetonitrile at a concentration of 1 mg/ml. Ten (10) micro-

M. N. Aminake et al. / Bioorg. Med. Chem. 20 (2012) 5277–5289
5289
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the compound at the tested dose. A dose resulting in survival times
greater than that of infected non treated mice was considered ac-
tive. Death occurring before day 6 of infected and treated mice
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5.9. In vitro metabolism studies
In vitro metabolism studies were performed to confirm metab-
olism observations made in vivo. A final concentration of 1
the compound 7 was incubated at 37 °C with mice liver micro-
somes. The incubation mix consisted of compound 7 (1 M),
lM of
l
0.2 mg/ml mice microsomal protein, and 100 mM KPO4 in
200 mM phosphate buffer, pH 7.4. The reaction was initiated by
the addition of NADPH (1 mM). The mixture was rotated at 37 °C.
Aliquots were collected before initiating the reaction. At varying
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ll of incubation mixture were removed and added
to 100 l ice cold acetonitrile to stop the reaction. The rate of loss
l
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Acknowledgement
We thank Trevor Finch (Scientific officer at UCT Pharmacology)
for assistance with animal experiments. M.N.A. was supported by
the IRTG1522 of the DFG. We also thank Professor Paul M. O’Neill
of the University of Liverpool (UK) for a generous sample of the tet-
raoxane carboxylic acid intermediate 24. The University of Cape
Town, South African Medical Research Council and South African
Research Chairs initiative of the Department of Science and Tech-
nology administered through the South African National Research
Foundation is gratefully acknowledged for support (K.C.).
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