Anti-Plasmodium activity of imidazole-dioxolane compounds

Article abstract of DOI:10.1016/j.bmcl.2006.01.122

A series of imidazole-dioxolane compounds, which we hypothesize should bind to heme and thus interfere with heme catabolism in the parasite, were assayed for inhibitory activity in Plasmodium falciparum cultures and the results were compared to those obta

Full text of DOI:10.1016/j.bmcl.2006.01.122

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2396–2406
Anti-Plasmodium activity of imidazole–dioxolane compounds
Jason Z. Vlahakis,a Robert T. Kinobe,b Kanji Nakatsu,b Walter A. Szareka,*
and Ian E. Crandallc,*
aDepartment of Chemistry, Queen’s University, Kingston, Ont., Canada K7L 3N6
bDepartment of Pharmacology and Toxicology, Queen’s University, Kingston, Ont., Canada K7L 3N6
cDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., Canada M5S 1A8
Received 3 November 2005; revised 26 January 2006; accepted 27 January 2006
Available online 21 February 2006
Abstract—A series of imidazole–dioxolane compounds, which we hypothesize should bind to heme and thus interfere with heme
catabolism in the parasite, were assayed for inhibitory activity in Plasmodium falciparum cultures and the results were compared
to those obtained with Chinese hamster ovary (CHO) cells. The majority of the compounds displayed a similar ratio of inhibitory
activity in the two culture systems; however, a number of the compounds tested showed promising anti-Plasmodium activity. The
mechanism of action of these compounds remains unclear, however their inability to act synergistically with chloroquine suggests
that, if they are inhibiting heme detoxification, they do so in a manner that does not complement the action of chloroquine.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Malaria accounts for an estimated 300–500 million cases
and up to 2.7 million deaths annually,1 particularly
among young children.2–4 Despite optimistic predictions
of the eventual control and eradication of malaria made
in the past, the spread of drug-resistant parasites, partic-
ularly those resistant to chloroquine, has led to a resur-
gence of the disease.5–9 Plasmodium falciparum, the
species responsible for the majority of malaria-related
deaths in humans, digests the hemoglobin present in
an erythrocyte, and while it is able to safely export a
majority of the liberated amino acids10 this process also
creates a source of heme that must be detoxified to pre-
vent the destabilization of membranes and inhibition of
enzymes that will lead to parasite death.11 The degrada-
tion of hemoglobin liberates free heme (or hemin)12
which can be oxidized to hematin (ferriprotoporphyrin
IX). Free hematin is toxic to the parasite and can inter-
fere with cellular metabolism by enzyme inhibition,
membrane peroxidation, and also through the genera-
tion of oxidative free radicals.13 Despite the lack of a
parasite-encoded heme oxygenase enzyme (HO, EC
1.14.99.3),14,15 other methods of heme catabolism in
P. falciparum are utilized in this parasite. Through the
formation of a complex with malarial histidine-rich pro-
tein (HRP), some (30–50%) of the free hematin in the
food vacuoles of plasmodial species is sequestered into
the non-toxic crystal hemozoin.5 In this process, several
units of hematin are linked via carboxylate–Fe(III) and
carboxylic acid–carboxylic acid interactions forming an
insoluble aggregate. The remaining free heme can also
be efficiently decomposed by complexation with reduced
glutathione (GSH) once it passes through the membrane
of food vacuoles and into the parasite cytosol.16,17 The
aggregation into hemozoin is a primary method of
detoxification and has been used as a target for drug
therapy.18–20 Many antimalarial compounds such as
quinine, chloroquine, and other 4-aminoquinolines
work by stabilizing (associating with) hematin or a
hematin derivative (a l-oxo-dimer), thus suppressing
the hematin aggregation process which forms
hemozoin.21
Antifungal agents such as econazole, ketoconazole,
miconazole, and clotrimazole have been reported22 to
inhibit the growth of P. falciparum through an interac-
tion with hematin. Each of these compounds is a
1-substituted imidazole; ketoconazole also incorporates
a 1,3-dioxolane ring. In detailed studies,23 it was deter-
mined that the axial coordination of the imidazole moi-
eties of two clotrimazole molecules to the heme iron
formed a stable heme–(clotrimazole)2 complex which
Keywords: Antimalarial; Plasmodium falciparum; Imidazole–dioxolane
compounds.
*
Corresponding authors. Tel.: +1 613 533 2643; fax: +1 613 533 6532
(W.A.S.); tel.: +1 416 978 0356; fax: +1 416 978 8765 (I.E.C.); e-mail
addresses:
utoronto.ca
walter.szarek@chem.queensu.ca;
ian.crandall@
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.122

J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
2397
prevents heme association into hemozoin by HRP, in
addition to inhibiting completely GSH-dependent heme
degradation. Under the assumption that other 1-substi-
tuted imidazole compounds should bind to heme and
thus also interfere with heme catabolism in the parasite,
we have studied a series of imidazole–dioxolane com-
pounds with respect to anti-Plasmodium activity in P.
falciparum cultures. A number of compounds tested
showed good anti-Plasmodium activity, however the
mechanism involved in their action remains to be
defined.
values obtained in P. falciparum cultures. A ratio that
was greater than unity indicates that a compound was
preferentially toxic to P. falciparum parasites (Table
1). Further, the relative toxicity of these compounds
was compared graphically by plotting the IC50 values
obtained in P. falciparum cultures versus the values ob-
tained in CHO cell cultures (Fig. 1). The majority of the
data points in Figure 1 appear to fall parallel to and
about 1 log-unit above a diagonal line that represents
unity, suggesting that there is a common toxicity to both
CHO and P. falciparum cultures. The data points for six
of the compounds fell significantly above the diagonal
(about 2 log-units), indicating that the compounds were
preferentially toxic to P. falciparum cultures. Examina-
tion of P. falciparum cultures exposed to inhibitory con-
centrations of compounds QC-5, -18, -34, -35, and -36
indicated that the parasites present in the cultures had
formed compacted structures that lacked hemozoin
and had not progressed beyond the ring stage of devel-
opment (the results from QC-5 are shown in Fig. 2). We
hypothesized that 1-substituted imidazole compounds
should bind to heme and therefore interfere with heme
catabolism in the parasite. This mechanism of action is
similar to that of chloroquine,21 hence the effects of
the four most selective QC compounds (QC-34, -35,
-36, and -5) in the presence of chloroquine were investi-
gated. The presence of varying amounts of the QC com-
pounds had an additive rather than synergistic effect on
the action of chloroquine (results for QC-34 are shown
in Fig. 3), suggesting that, if these compounds inhibited
heme detoxification, they did not do so by inhibiting a
biochemical process that complements the action of
chloroquine (see also Ref. 29).
The synthesis of compounds QC-1, -3, -4–7, -9–12, -14,
and -16–24 has been described previously;24 the synthe-
sis of compounds QC-34–37 is reported here (see
Scheme 1),25 and the synthesis of the remaining com-
pounds will be reported separately. The racemic alcohol
4-(4-chlorophenyl)-1-(1H-imidazol-1-yl)butan-2-ol (A)
was prepared from 4-chlorobenzyl chloride26 and the
tosylate (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imi-
dazol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,3-
dioxolane (B) was prepared as previously reported.24
Both QC-34 and QC-35 were prepared from tosylate
B; the nucleophilic displacement reaction with 4-mer-
captophenol afforded QC-34, whereas the complete
reduction of tosylate B using an excess of LiAlH4 gave
QC-35. QC-36 was prepared by the nucleophilic
ring opening of 1,2-epoxybutane with imidazole,
followed by acid-catalyzed transesterification of the
resulting alcohol with ethyl acetate; the ring opening
of 1,2-epoxybutane gave preponderately 1-(1H-imidazol-
1-yl)butan-2-ol. QC-37 was prepared simply by the
alkylation of the racemic alcohol A with 4-fluorobenzyl
chloride. All compounds were isolated/characterized as
hydrochloride salts, with the exception of compounds
QC-6, -16, -17, -21, -34, and -36.
Virtually all of the compounds tested were found to be
selectively toxic to Plasmodium, however, only a few
were highly selective for P. falciparum. It is recognized
that five of these compounds do not contain the 1,3-
dioxolane ring. Interestingly, QC-9, -10, and -33 were
least potent against P. falciparum cultures, and QC-37
was only modestly selective. In contrast, QC-36 proved
to be highly selective for P. falciparum cultures. These
compounds, that lack the dioxolane moiety, provide
other avenues worthy of investigation and will be the
subject of a separate study. Most of the compounds
The antimalarial activity of the QC compounds was
determined in quadruplicate using P. falciparum cul-
tures,27 whereas the general toxicity of the QC com-
pounds was determined also in quadruplicate using
Chinese hamster ovary (CHO) cell cultures.28 The IC50
values of the compounds listed in Table 1 were deter-
mined, and a selectivity index was calculated by dividing
the IC50values obtained in CHO cell cultures by the IC50
Scheme 1. Reagents and conditions: (a) Cs2CO3, acetone, reflux, 8.5 h; (b) LiAlH4, THF, reflux, 9 h; (c) imidazole, NaH, DMF, 130 ꢁC, 1 h;
(d) EtOAc, HCl, heat; (e) NaH, THF, reflux, 1.5 h.

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
Table 1. Inhibitory activity of imidazole–dioxolane compounds in CHO cell and Plasmodium falciparum cultures
Structure
IC50 (lM)
IC50 CHO/IC50
P. falciparum
P. falciparum
CHO cells
S
NH2
N
O
O
O
O
2HCl
0.8 0.1
13
5
16.3
N
Cl
QC-1
S
N
1.2 0.2
9
1
7.5
HCl
O
N
Cl
H2N
QC-2
S
N
0.6 0.1
6
1
10
O
2HCl
N
Cl
QC-3
O
S
O
O
N
O
O
0.67 0.05
7
1
10.4
HCl
N
Cl
QC-4
S
NH2
N
O
O
HCl
1.3 0.1
374 49
287.7
N
Cl
O
S
QC-5
O
O
N
O
O
1.2 0.2
3.8 0.9
3.2
N
Cl
QC-6
S
N
NH2
2HCl
O
O
0.3 0.1
18
6
60
N
Cl
QC-7

J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
2399
Table 1 (continued)
Structure
IC50 (lM)
IC50 CHO/IC50
P. falciparum
P. falciparum
CHO cells
O
OCH3
N
HCl
O
O
0.8 0.1
15
5
18.8
N
Cl
QC-8
N
O
HCl
N
25
2
168 13
6.7
Cl
QC-9
N
OH
N
70 19
314 36
4.5
HCl
Cl
QC-10
H2N
S
N
2 HCl
3.9 0.5
16
2
4.1
O
O
N
Cl
QC-12
CH3
HCl
N
O
O
N
7
1
51
3
7.3
Cl
QC-13
S
N
NH2
O
O
2.0 0.7
10
4
5
N
HCl
Cl
QC-14
N
O
O
HCl
N
9
2
84
9
9.3
Cl
QC-15
(continued on next page)

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
Table 1 (continued)
Structure
IC50 (lM)
IC50 CHO/IC50
P. falciparum
P. falciparum
CHO cells
O
S
O
O
N
O
O
2.9 0.2
8
2
2.8
N
Cl
QC-16
O
S
O
O
N
O
O
2.8 0.2
13
3
4.6
N
Cl
QC-17
S
NH2
N
HCl
O
O
3.0 0.8
254 34
6.6 0.1
9.2 0.7
5.7 0.4
84.7
N
Cl
QC-18
H2N
S
N
3.3 0.6
2.5 0.3
1.9 0.3
2
HCl
O
O
N
Cl
QC-19
S
N
NH2
HCl
O
O
3.7
N
Cl
QC-20
O
S
O
O
N
O
O
3
N
Cl
QC-21

J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
2401
Table 1 (continued)
Structure
IC50 (lM)
IC50 CHO/IC50
P. falciparum
P. falciparum
CHO cells
H2N
S
N
2.3 0.3
11
1
4.8
2 HCl
O
O
N
Cl
QC-22
S
N
NH2
O
O
1.2 0.1
9
1
7.5
N
2 HCl
Cl
QC-23
S
NH2
N
O
O
2.7 0.6
6
2
2.2
N
2 HCl
Cl
QC-24
CH3
HCl
N
O
O
N
4.1 0.4
39
7
9.5
Cl
QC-25
CH3
HCl
N
O
O
N
18
2
82 16
4.6
Cl
QC-26
CH3
HCl
N
O
O
N
23
4
102 25
4.4
Cl
QC-27
S
N
O
O
3.3 0.3
7
4
2.1
N
HCl
Cl
QC-30
(continued on next page)

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
Table 1 (continued)
Structure
IC50 (lM)
IC50 CHO/IC50
P. falciparum
P. falciparum
CHO cells
N
S
N
2HCl
O
O
4.9 0.3
27 12
5.5
N
Cl
QC-32
N
OH
N
38
6
116 66
3.1
HCl
H3CO
QC-33
S
OH
N
O
O
0.06 0.03
0.34 0.02
8.3 0.8
10.9 2.5
181.6
N
Cl
QC-34
CH3
N
O
O
HCl
N
668 378
1848 437
5.3 0.5
2226.7
222.7
6.6
QC-35
N
N
O
O
QC-36
F
HCl
N
O
0.8 0.1
N
Cl
QC-37
OH
N
O
O
HCl
9
2
227 14
25.2
N
Cl
QC-38

J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
2403
Table 1 (continued)
Structure
IC50 (lM)
IC50 CHO/IC50
P. falciparum
P. falciparum
CHO cells
O
NH2
N
O
O
1.3 0.2
14
1
10.8
2HCl
N
Cl
QC-39
S
CH3
N
O
O
HCl
2.1 0.4
9.3 0.8
4.7
N
Cl
QC-40
S
Br
N
O
O
0.9 0.2
12
3
13.3
N
HCl
Cl
QC-41
N
S
S
N
HCl
2.3 0.6
25
6
10.9
Cl
QC-42
O
OH
N
O
O
1.1 0.1
0.9 0.3
0.8
N
HCl
Cl
QC-46
F
N
O
O
HCl
20
3
24
4
1.2
1.1
1.3
N
Cl
QC-47
S
OCH3
N
O
O
3.5 0.2
3.7 0.5
HCl
N
Cl
QC-48
S
Cl
N
O
O
2.5 0.3
3.3 0.4
HCl
N
Cl
QC-49
Values represent means of four independent determinations with the standard deviation indicated.

2404
J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
were active in both cultures in the low micromolar range
10000
1000
100
10
and therefore show low selectivity. Selectivity comes
about either because of low inhibitory activity in CHO
cell cultures, as for example in the case of QC-5, QC-
18, and QC-36, or alternatively because of increased
potency in P. falciparum cultures, as for example in
the case of QC-34 and QC-35. The overall examination
of the results in Table 1 clearly suggests that the combi-
nation of 1,3-dioxolane and imidazole moieties has the
potential to bestow significant anti-P. falciparum
activity. Indeed, QC-35 displayed both high potency in
P. falciparum cultures (<1 lM) and low CHO cell
inhibitory activity (>100 lM).
1
0.1
0.01
1
0.01
0.1
10
100
1000 10000
The mechanism by which these agents kill P. falciparum
cultures is not immediately clear. The importance of the
stereochemistry of the compounds, for instance, as in
QC-1 versus QC-18, and the morphology of the para-
sites present in treated cultures (Fig. 2) are consistent
with the hypothesis that some aspect of an enzymatic
heme detoxification process is being targeted. However,
if heme detoxicification is being inhibited or the heme-
dependent hemolysis is being enhanced23 it would be
expected that these agents might display either additive
or synergistic behavior with chloroquine. For example,
Srivastava et al.29 have suggested that inhibiting alterna-
tive pathways of heme detoxification15,16 or export30
might have a synergistic effect with chloroquine since
this would increase the amount of heme in the chloro-
quine-sensitive pathway. The linear relationship be-
tween the concentration of QC-34 and the IC90 for
chloroquine shown in the isobologram in Figure 3
demonstrates the apparent lack of synergy between
chloroquine and the QC compounds studied. This
observation suggests that the QC compounds cannot
be used to potentiate the effects of chloroquine by pro-
moting the effects of oxidative stress present in a parasit-
ized erythrocyte.30 An initial determination of the effect
P. falciparum IC50
(μM)
Figure 1. log–log plot of the IC50 values obtained for the compounds
in Plasmodium falciparum cultures (X axis) versus CHO cell cultures
(Y axis). Points above the diagonal represent compounds that
selectively inhibit Plasmodium falciparum cultures.
Figure 2. The inhibition of Plasmodium falciparum cultures with
chloroquine or QC-5. Plasmodium falciparum line ItG was cultured
and used for viability assays as described in the text. Untreated
parasite cultures (left panel) developed normally and produced visible
hemozoin crystals. Cultures were treated with either 250 nM chloro-
quine (center panel) or 15 lM QC-5 (right panel) and incubated for
48 h. Treated cultures failed to develop past the ring stage of their
intra-erythrocytic cycle.
0.40
0.35
0.30
0.25
0.20
0.15
300
250
200
150
100
50
0
0
200 400 600 800 1000
[QC-34] (nM)
1
10
100
[chloroquine] (nM)
1000
10000
Figure 3. The interaction of chloroquine and QC-34 in Plasmodium falciparum cultures. The effect of increasing concentrations of compound QC-34
in the presence of varying amounts of chloroquine was determined. Varying concentrations of QC-34 (700 nM d, 600 nM s, 500 nM j, 400 nM h,
300 nM n, 200 nM m, 100 nM $, and 0 nM .) were added and the corresponding IC90 values for chloroquine were calculated. An isobologram
(inset) plotted for 90% inhibition supports a lack of synergy between the two compounds.

J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
2405
22. Chong, C. R.; Sullivan, D. J., Jr. Biochem. Pharmacol.
2003, 66, 2201.
23. Huy, N. T.; Kamei, K.; Yamamoto, T.; Kondo, Y.;
Kanaori, K.; Takano, R.; Tajima, K.; Hara, S. J. Biol.
Chem. 2002, 277, 4152.
of QC-34 and QC-35 on chloroquine, and mefloquine-
resistant P. falciparum cultures suggests that these
compounds have similar activities in these lines.
It should be noted that most of the compounds in Table 1
have been found24 to display inhibitory activity against
heme oxygenase, an enzyme that is not present in P. fal-
ciparum.14,15,31 Consequently, their use as antimalarials
might be viewed with caution since infection of the host
with Plasmodium leads to several conditions in which
an active heme oxygenase may be a potential benefit.32,33
Thus, the incorporation of selectivity is a target for the
future design of useful antimalarial compounds.
24. Vlahakis, J. Z.; Kinobe, R. T.; Bowers, R. J.; Brien, J. F.;
Nakatsu, K.; Szarek, W. A. Bioorg. Med. Chem. Lett.
2005, 15, 1457.
25. Synthetic procedures and characterization of the com-
pounds outlined in Scheme 1: (2R,4S)-2-[2-(4-Chlorophe-
nyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[{(4-hydroxyphe-
nyl)thio}methyl]-1,3-dioxolane (QC-34). To a solution of
tosylate B24 (100 mg, 0.21 mmol) in acetone (4 mL) were
added cesium carbonate (137 mg, 0.42 mmol) and 4-
mercaptophenol (90 mg, 0.71 mmol). The mixture was
heated at reflux temperature for 8.5 h and then filtered.
The filter cake was washed with acetone and ethyl acetate,
and the filtrate and washings were concentrated to a
yellow residue. Flash chromatography on silica gel
(EtOAc) followed by recrystallization from acetone gave
60 mg (0.14 mmol, 67%) of QC-34 as a white solid:
Acknowledgments
The authors wish to acknowledge funding provided by
the CIHR in the form of an Operating Grant MOP
64305 to K.N. and W.A.S., and Proof-of-Principle
Grant PPP-62029 to I.C.
22
Rf = 0.15 (EtOAc); mp 153–154 ꢁC; ½aꢀD ꢁ11.5 (c 0.70,
1
CD3OD); H NMR (400 MHz, CD3OD): d 1.84–1.89 (m,
2H), 2.60–2.76 (m, 2H), 2.83 (dd, J = 13.8, 7.0 Hz, 1H),
2.98 (dd, J = 13.6, 5.6 Hz, 1H), 3.54 (t, J = 7.8 Hz, 1H),
3.59–3.66 (m, 1H), 3.88 (dd, J = 7.8, 5.8 Hz, 1H), 4.12 (s,
2H), 6.74 (d, J = 8.8 Hz, 2H), 6.91 (s, 1H), 7.08 (s, 1H),
7.14 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.26 (d,
J = 8.8 Hz, 2H), 7.59 (s, 1H); 13C NMR (100 MHz,
CD3OD): d 29.8, 39.4, 39.6, 53.3, 71.2, 77.9, 110.6,
117.2, 122.5, 125.0, 128.4, 129.5, 131.0, 132.7, 135.4,
139.8, 141.8, 158.7; HRMS (ES) [M+H]+ Calcd for
C22H24ClN2O3S: 431.1196. Found: 431.1198; Anal. Calcd
for C22H23ClN2O3S: C, 61.31; H, 5.38; N, 6.50. Found: C,
61.10; H, 5.34; N, 6.66. 1-Acetoxy-2-(1H-imidazol-1-yl)bu-
tane (QC-36) and 1-(1H-imidazol-1-yl)butan-2-ol hydro-
chloride. To a suspension of NaH (1.33 g, 55.47 mmol) in
DMF (20 mL) was added imidazole (3.77 g, 55.47 mmol).
After the evolution of gas had subsided, 1,2-epoxybutane
(3.50 g, 48.5 mmol) was added dropwise, and the reaction
mixture was stirred at rt for one week and then at 130 ꢁC
for 1 h. The mixture was concentrated and the brown
residue was washed with Et2O (3·). The residue was then
dissolved in boiling acetone and the mixture filtered hot,
and the filtrate concentrated. The residue was treated with
boiling acidic (HCl) EtOAc for a few minutes, and the
mixture was then cooled in the freezer. The mixture was
filtered and the filtrate was concentrated to an impure
dark oil (2.25 g) which was fractionated by flash column
chromatography on silica gel (EtOAc) giving preponder-
ately 1-(1H-imidazol-1-yl)butan-2-ol (Rf = 0.16) and also
370 mg (2.03 mmol, 4%) of 1-acetoxy-2-(1H-imidazol-1-
yl)butane (QC-36) as a clear oil: Rf = 0.22 (EtOAc); 1H
NMR (400 MHz, D2O): d 0.87 (t, J = 7.4 Hz, 3H), 1.44–
1.63 (m, 2H), 1.99 (s, 3H), 4.08 (dd, J = 14.8, 8.0 Hz, 1H),
4.22 (dd, J = 14.8, 2.8 Hz, 1H), 4.95–5.02 (m, 1H), 6.94 (s,
1H), 7.10 (s, 1H), 7.61 (s, 1H); 13C NMR (100 MHz,
D2O + CD3OD): d 9.7, 21.2, 25.1, 50.3, 76.1, 121.8, 128.7,
139.3, 174.3; HRMS (ES) [M+H]+ Calcd for C9H15N2O2:
183.1133. Found: 183.1138. All of the 1-(1H-imidazol-1-
yl)butan-2-ol isolated above was dissolved in EtOH
(5 mL) and to this solution was added a solution of 37%
aqueous HCl dropwise until the pH of the solution was
acidic. The solution was concentrated, and the residue was
dried under high vacuum and recrystallized from acetone
to give 1.41 g (7.98 mmol, 16%) of 1-(1H-imidazol-1-
yl)butan-2-ol hydrochloride as a hygroscopic white solid:
References and notes
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1
Rf = 0.45 (EtOH); H NMR (400 MHz, D2O): d 0.98 (t,
J = 7.4 Hz, 3H), 1.42–1.54 (m, 1H), 1.55–1.66 (m, 1H),

2406
J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2396–2406
3.87–3.96 (m, 1H), 4.15 (dd, J = 14.2, 8.2 Hz, 1H), 4.38
27. In vitro P. falciparum activity assay: P. falciparum cultures
were grown in A+ blood obtained by venipuncture of
volunteers. Cultures of the laboratory line ItG were
maintained by the method of Trager and Jensen34 using
RPMI 1640 supplemented with 10% human serum and
50 lM hypoxanthine (RPMI-A). In vitro P. falciparum
susceptibility testing was performed using an LDH
enzyme assay specific to LDH found in Plasmodium
(pLDH).35–37 Briefly, compounds to be tested were
dissolved in DMSO at a final concentration of 10 mg/
mL and were then serially diluted in duplicate in a 96-well
plate to produce a compound gradient with twofold
dilutions. Fifty microliters of parasite culture (5% hemat-
ocrit, 2% parasitemia from non-synchronous cultures)
were added to each well and the plates were then
incubated at 37 ꢁC in an atmosphere of 95% N2, 3%
CO2, and 2% O2 for 72 h. The contents of the wells were
then resuspended in 100 lL of tissue culture media and a
15-lL sample was removed and added to 100 lL of pLDH
enzyme assay mixture.37 After 1 h, the absorbance of the
wells at 540 nm was determined using a microplate reader
(BioRad, Mississauga, ON). The IC50 values of individual
compounds were determined using a non-linear regression
analysis of the data38 using the computer program
SigmaPlot (Jandel Scientific). The IC50 values represent
means standard deviation of four independent assays.
28. In vitro CHO cell activity assay: CHO cells were grown in
RPMI-1640 supplemented with 10% fetal bovine serum
(Sigma, St. Louis, MO), 25 mM HEPES, and gentamicin.
Cells were grown to 50% confluency in 96-well plates prior
to the addition of individual samples in DMSO. After
24 h, the viability of the cells was determined by adding
1 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT, Sigma, St. Louis, MO) in
100 lL of culture media. The plates were incubated for a
further 30 min and then the media were removed and
100 lL of DMSO were added and the absorbance at
540 nm was read.39 The IC50 values of individual com-
pounds were determined using a non-linear regression
analysis of the data as in Ref. 27.
29. Srivastava, R.; Pandey, V. C.; Bhaduri, A. P. Trop. Med.
Parasitol. 1995, 46, 83; Srivastava, P.; Pandey, V. C. Int.
J. Parasitol. 1995, 25, 1061.
30. Becker, K.; Tilley, L.; Vennerstrom, J. L.; Roberts, D.;
Rogerson, S.; Ginsburg, H. Int. J. Parasitol. 2004, 34, 163;
Egan, T. J.; Combrinck, J. M.; Egan, J.; Hearne, G. R.;
Marques, H. M.; Ntenteni, S.; Sewell, B. T.; Smith, P. J.;
Taylor, D.; van Schalkwyk, D. A.; Walden, J. C. Biochem.
J. 2002, 365, 343.
31. Ginsburg, H. Exp. Parasitol. 1991, 73, 227.
32. Schluesener, H. J.; Kremsner, P. G.; Meyermann, R. Acta
Neuropathol. (Berl.) 2001, 101, 65.
33. McLaughlin, B. E.; Hutchinson, J. M.; Graham, C. H.;
Smith, G. N.; Marks, G. S.; Nakatsu, K.; Brien, J. F.
Placenta 2000, 21, 870.
34. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
35. Eda, S.; Eda, K.; Prudhomme, J. G.; Sherman, I. W. Blood
1999, 94, 326.
36. Makler, M. T.; Ries, J. M.; Williams, J. A.; Bancroft, J. E.;
Piper, R. C.; Gibbins, B. L.; Hinrichs, D. J. Am. J. Trop.
Med. Hyg. 1993, 48, 739.
37. Prudhomme, J. G.; Sherman, I. W. J. Immunol. Methods
1999, 229, 169.
38. Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat,
P.; Riscoe, M. Antimicrob. Agents Chemother. 2004, 48,
1803.
(dd, J = 14.0, 2.8 Hz, 1H), 7.48 (s, 1H), 7.52 (s, 1H), 8.73
(s, 1H); 13C NMR (100 MHz, D2O): d 9.3, 26.7, 54.6, 71.4,
119.8, 122.8, 135.3; HRMS (ES) [M+H]+ Calcd for
C7H13N2O: 141.1027. Found: 141.1022; Anal. Calcd for
C7H13ClN2O: C, 47.60; H, 7.42; N, 15.86. Found: C,
47.54; H, 7.21; N, 16.02. (2R,4R)-2-(2-Phenylethyl)-2-
[(1H-imidazol-1-yl)methyl]-4-methyl-1,3-dioxolane hydro-
chloride (QC-35). To a solution of tosylate B24 (215 mg,
0.45 mmol) in THF (4 mL) was added powdered lithium
aluminum hydride (102 mg, 2.69 mmol). The mixture was
heated at reflux temperature for 9 h and then carefully
quenched with H2O. The mixture was extracted with
EtOAc (3·) and the combined organic extracts were
washed sequentially with a saturated aqueous solution of
Na2CO3, and H2O, and then dried (MgSO4). The solution
was concentrated to a yellow oil, which was purified by
flash chromatography on silica gel (EtOAc) to give the free
base (90 mg, 0.33 mmol) as an oil (Rf = 0.22, EtOAc). To a
solution of this oil in hot 2-propanol (1 mL) was added a
solution of 37% aqueous HCl (40 mg, 0.41 mmol) in 2-
propanol (2 mL). The mixture was concentrated and dried
under high vacuum. The residue was recrystallized from 2-
propanol–Et2O to give 90 mg (0.29 mmol,64%) of QC-35
23
as a white solid: Rf = 0.02 (EtOAc); mp 163–164 ꢁC; ½aꢀD
ꢁ17.0ꢁ (c 0.83, CD3OD); 1H NMR (400 MHz, CD3OD): d
1.25 (d, J = 6.0 Hz, 3H), 1.91–2.00 (m, 2H), 2.70–2.82 (m,
2H), 3.48 (t, J = 8.4 Hz, 1H), 3.62–3.70 (m, 1H), 4.04 (dd,
J = 8.0, 5.6 Hz, 1H), 4.41 (s, 2H), 7.15–7.29 (m, 5H), 7.52
(s, 1H), 7.59 (s, 1H), 8.83 (s, 1H); 13C NMR (100 MHz,
CD3OD): d 18.1, 30.5, 39.7, 55.0, 73.0, 75.3, 109.3, 120.5,
125.1, 127.1, 129.3, 129.6, 137.7, 142.7; HRMS (ES)
[M+H]+ Calcd for C16H21N2O2: 273.1603. Found:
273.1609; Anal. Calcd for C16H21ClN2O2: N, 9.07. Found:
N, 8.81. 4-(4-Chlorophenyl)-2-(4-fluorobenzyloxy)-1-(1H-
imidazol-1-yl)butane hydrochloride (QC-37). To a solution
of alcohol A26 (70 mg, 0.28 mmol) in THF (2 mL) was
added a suspension of NaH (12 mg, 0.50 mmol) in THF
(1 mL). The mixture was stirred at rt for 1 h and then a
solution of 4-fluorobenzyl chloride (44 mg, 0.30 mmol) in
THF (1 mL) was added. The mixture was stirred at rt for
24 h, heated at reflux temperature for 1.5 h, and then
concentrated. After dilution with H2O, the mixture was
extracted with EtOAc (3·) and the combined organic
extracts were washed once with H2O, dried (MgSO4), and
concentrated. The resulting residue was purified by flash
column chromatography on silica gel (EtOAc) to give the
free base (50 mg, 0.14 mmol) as an oil (Rf ꢂ 0.2, EtOAc).
To a solution of this oil in hot 2-propanol (1 mL) was
added a solution of 37% aqueous HCl (16 mg, 0.16 mmol)
in 2-propanol (1 mL). The mixture was concentrated and
dried under high vacuum. The residue was recrystallized
from 2-propanol–Et2O to give 40 mg (0.10 mmol, 36%) of
QC-37 as a white solid: Rf = 0.18 (EtOAc); mp 125–
127 ꢁC; 1H NMR (400 MHz, CD3OD): d 1.80–1.94 (m,
2H), 2.74 (t, J = 7.8 Hz, 2H), 3.76–3.82 (m, 1H), 4.29 (dd,
J = 14.4, 7.6 Hz, 1H), 4.38 (d, J = 11.6 Hz, 1H), 4.48–4.53
(m, 1H), 4.54 (d, J = 11.6 Hz, 1H), 7.00–7.06 (m, 2H),
7.17–7.23 (m, 4H), 7.28 (d, J = 8.4 Hz, 2H), 7.51 (s, 1H),
7.56 (s, 1H), 8.85 (s, 1H); 13C NMR (100 MHz, CD3OD):
d 31.3, 34.2, 53.2, 71.8, 77.4, 116.2 (d, JCF = 21.6 Hz),
120.9, 124.1, 129.6, 131.0, 131.2 (d, JCF = 8.2 Hz),
132.9,135.1 (d, JCF = 3.0 Hz), 137.1, 141.5, 163.9 (d,
JCF = 245.2 Hz); HRMS (ES) [M+H]+ Calcd for
C20H21ClFN2O: 359.1326. Found: 359.1330; Anal. Calcd
for C20H21Cl2FN2O: N, 7.09. Found: N, 6.93.
26. Walker, K. A. M.; Braemer, A. C.; Hitt, S.; Jones, R. E.;
Matthews, T. R. J. Med. Chem. 1978, 21, 840.
39. Campling, B. G.; Pym, J.; Galbraith, P. R.; Cole, S. P. C.
Leukemia Res. 1988, 12, 823.