Febrifugine analogue compounds: Synthesis and antimalarial evaluation

Article abstract of DOI:10.1016/j.bmc.2011.11.053

Febrifugine is an alkaloid isolated from Dichroa febrifuga Lour as the active component against Plasmodium falciparum, but exhibits toxic side effects. In this study novel febrifugine analogues were designed and efficiently synthesized. New compounds unde

Full text of DOI:10.1016/j.bmc.2011.11.053

Bioorganic & Medicinal Chemistry 20 (2012) 927–932
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Bioorganic & Medicinal Chemistry
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Febrifugine analogue compounds: Synthesis and antimalarial evaluation
Shuren Zhu a, , Gudise Chandrashekar a, Li Meng a, Katie Robinson a, Dipsanker Chatterji b
⇑
a Radix Pharmaceuticals, Inc., 20271 Goldenrod Lane, Suite 2035, Germantown, MD 20876, USA
b Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Febrifugine is an alkaloid isolated from Dichroa febrifuga Lour as the active component against Plasmo-
dium falciparum, but exhibits toxic side effects. In this study novel febrifugine analogues were designed
and efficiently synthesized. New compounds underwent efficacy and toxicity evaluation. Some com-
pounds are much less toxic than the natural product febrifugine and existing antimalarial drugs and
are expected to possess wide therapeutic windows. In Aotus monkeys infected with the chloroquine
resistant FVO strain of P. falciparum, one interesting compound possesses a 50% curative dose of 2 mg/
kg/day and a 100% curative dose of 8 mg/kg/day. These compounds, as well as the underlying design
rationale, may find usefulness in the discovery and development of new antimalarial drugs.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 13 September 2011
Revised 16 November 2011
Accepted 23 November 2011
Available online 2 December 2011
Keywords:
Antimalarial
Febrifugine
Natural products
Preclinical evaluation
1. Introduction
withdrawing substitution group on the aromatic ring reduces tox-
icity while retaining desired biological activity; (II) the original
Malaria is the best known protozoal disease. It is one of the
most common infectious diseases in many tropical and subtropical
countries in Africa, Southeast Asia, and South America. Worldwide,
malaria infects 225 million people and kills about 781,000 in a
year.1 The increasing prevalence of multiple drug resistant strains
in most malaria endemic areas has significantly reduced the
efficacy of current anti-malarial drugs for prophylaxis and treat-
ment of this disease.2 Medicinal agents based on novel mode of
action are required to overcome the emergence of resistance and
to control an ever-increasing number of epidemics caused by the
malaria parasite.
Febrifugine and isofebrifugine (Fig. 1) were isolated as the active
components against malaria in the Chinese herb Chang Shan
(Dichroa febrifuga Lour),3,4 which has been employed by the local
people as medicine against fevers caused by malaria parasites for
a long time. Febrifugine acts by impairing haemazoin formation re-
quired for maturation of the parasite at the trophozoite stage. The
use of febrifugine as anti-malarial agent is initially appealing not
only because of its rapid effect and no drug resistance, but also be-
cause of its availability. Subsequent pre-clinical researches have
found that febrifugine possesses adverse side effects. Strong liver
toxicity has precluded febrifugine as a clinical drug.5–7
piperidine ring can be replaced by a pyrrolidine ring; (III) the pres-
ence of the 30-methylene group is not necessary for desired
antimalarial activity.
Incorporating the above findings, we wish to report herein the
synthesis and preclinical evaluation of a library of febrifugine ana-
logues (compounds 1–12, shown in Figure 2). Overall, these com-
pounds closely resemble febrifugine itself by possessing a planar
aromatic ring, a 100-amino group and C-20, C-300 O-functionality
and are therefore expected to possess same or similar mode of ac-
tion. All new compounds possess a pyrrolidine ring instead of the
original piperidine ring and are devoid of the 30-methylene group.
These newly designed compounds are much easier to synthesize so
they have the potential to become an affordable drug should
further clinical trials succeed. For compounds 2–12, an extra nitro-
gen atom or a substitution group was introduced on the aromatic
ring to block the C-5 or C-6 position of the quinazolinone ring or
to increase the oxidation potential of the molecule. Therefore, low-
er toxicity will be achieved by reducing or eliminating the ten-
dency to form chemically reactive and toxic intermediates.
In our previous studies, we have elucidated the essential role
played by the 4-quinazolinone ring and 100-amino group in the
appearance of activity. In separate reports,8–10 we have demon-
strated (I) introducing an extra nitrogen atom or an electron
⇑
Corresponding author. Tel.: +1 301 675 3714; fax: +1 301 528 4022.
E-mail address: shuren.zhu@gmail.com (S. Zhu).
Figure 1. Molecular structures of febrifugine and isofebrifugine.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.053

928
S. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 927–932
Figure 2. Molecular structures of novel febrifugine analogues.
from previously published procedures.13–15 For synthesis of com-
pounds 6–8, the 100-amine was protected with tert-butyl carbamate
(BOC) group. Selective cleavage of the BOC group was achieved by
the combination of HCl/EtOAc.16 Synthesis of compounds 9–12 will
need a diastereomer or the enantiomer of alkene 13. These starting
compounds can be readily synthesized via previously described
protocols.11
3. Preclinical evaluation and discussion
Synthesized new compounds were first tested against a panel of
strains of Plasmodium falciparum. The IC50 values (50% inhibitory
concentrations) were summarized in Table 1. 3D7 is a chloroquine
sensitive strain of P. falciparum. The rest of the strains (TM6, K1,
and V1S) are chloroquine resistant. The antimalarial activity of
compounds 1–5 was comparable to the parent natural product
febrifugine. So the antimalarial activity was preserved when the
original piperridine ring was replaced with a pyrrolidine ring and
the 30-methylene group was removed. Compounds 6–8 have exhib-
ited superior and equally potent antimalarial activity against chlo-
roquine sensitive and resistant malaria strains. These compounds
bear an ether functionality on C-300 position of the pyrrolidine ring.
Apparently, a compound’s antimalarial potency was improved
when lipophilicity was increased. Compounds 9 and 10 are diaste-
reomers of compounds 2 and 5, respectively. The tenfold decrease
in efficacy for compounds 9 and 10 indicates that the stereochem-
istry at C-300 position is crucial for desired biological activity.
Compounds 11 and 12 are enantiomers of compounds 2 and 5,
respectively. Interestingly, the enantiomers have shown almost
identical antimalarial potency towards all tested strains of P.
falciparum.
Scheme 1. Synthesis of A representative compound (1).
Compounds 6–8 each bear an alkoxyl group on C-300 position of the
pyrrolidine ring. These compounds possess increased lipophilicity.
Compounds 9–12 possess different stereochemistry at either the
C-200 or the C-300 position and it would be interesting to see if such
stereochemistry difference will cause changes in antimalarial effi-
cacy or toxicity.
2. Chemistry
Scheme 1 illustrated the synthesis of compound 1. Although ini-
tial oxidation of the known and readily available alkene 1311 by
mCPBA to give oxirane 14 was not successful, the transformation
was smooth and high-yielding via a three-step sequence: (i)
dihydroxylation; (ii) selective tosylation of the primary alcohol;
and (iii) epoxide ring formation facilitated by potassium carbonate
in methanol. 3H-Quinazolin-4-one then condensed with oxirane
14, to furnish corresponding alcohol 15, as a pair of diastereomers.
Compound 15 then underwent TPAP oxidation12 and hydrogenoly-
sis, to afford compound 1. The synthesis of compounds 2–12 fol-
lows similar route. The respective aromatic moieties are available
In vivo antimalarial efficacy data was obtained by evaluating se-
lected new compounds 1–8 in rodent infected with blood stage
malaria strain Plasmodium berghei NK65. The ED50’s (the dose
leading to 50% parasite growth inhibition compared to the blank
control) and MCDs (minimum clearance dose), commonly used
efficacy measurements, were calculated. The acute toxicity study

S. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 927–932
929
Table 1
In vitro antimalarial activities of new compounds versus a panel of P. falciparum isolates [IC50 (nM)]
Strain
PH3
1.6 0.3
1.4 0.2
1.1 0.2
1.0 0.3
2.5 0.4
3D7
1.9 0.4
DD2
2.1 0.4
4.5 0.8
2.3 0.3
2.2 0.5
8.1 1.4
TM6
3.1 0.6
3.1 0.5
2.1 0.4
3.4 0.7
3.9 0.6
K1
TM4
5.4 0.8
7.9 0.7
4.1 0.4
3.0 0.5
6.2 1.1
V1S
4.2 0.7
5.6 1.0
3.6 0.5
4.1 0.7
5.3 1.0
11.2 1.9
0.13 0.03
0.25 0.05
0.68 0.12
28.7 4.8
36.5 5.2
3.7 0.5
11.0 1.9
152.2 14.1
8.7 2.5
Febrifugine
2.0 0.3
3.9 0.4
3.1 0.3
2.8 0.4
5.9 0.9
4.7 0.9
0.24 0.04
0.24 0.05
0.61 0.10
19.8 5.0
24.2 3.6
3.3 0.3
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
Chloroquine
Amodiaquine
2.8 0.4
1.8 0.3
1.4 0.3
5.7 0.9
2.7 0.2
0.21 0.05
0.21 0.04
0.36 0.07
16.3 2.4
19.6 3.3
1.9 0.3
2.6 0.2
9.7 3.4
4.9 1.8
3.2 0.5
6.3 0.8
2.5 0.3
7.4 1.3
0.20 0.04
0.18 0.04
0.54 0.11
11.4 1.9
21.2 4.0
1.0 0.2
3.4 0.5
33.4 9.1
15.9 3.2
0.17 0.03
0.26 0.07
0.78 0.14
21.9 4.1
31.8 5.6
2.4 0.3
6.1 0.8
45.8 10.2
9.1 1.2
0.08 0.02
0.17 0.03
0.31 0.05
13.2 2.5
25.6 4.1
2.0 0.4
2.7 0.3
96.4 6.9
7.5 2.4
0.33 0.07
0.22 0.04
0.39 0.06
32.7 5.3
51.9 9.4
4.0 0.4
7.3 1.3
115.2 12.7
15.2 2.6
4.8 0.9
165.6 19.3
21.2 2.1
Table 2
Table 3
Efficacy of test compounds against blood stages of P. falciparum in Aotus monkeys
Efficacy and toxicity evaluation of compounds in rodent modelsa
MCDb
MTDb
Therapeutic indexc
Test compounds
Dosea (mg/kg/day)
Cure rateb (%)
b
Compound
ED50
Febrifugine
2.3
1.7
1.2
1.0
12
35
100
450
350
400
400
250
250
250
240
600
250
3
10
63
47
63
Compound 6
2
4
8
16
2
4
1/4 (25)
5/7 (71)
4/5 (80)
5/5 (100)
2/4 (50)
4/6 (67)
5/5 (100)
5/5 (100)
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Chloroquine
Mefloquine
Artesunatea
9.9
7.1
7.5
6.3
6.7
1.7
1.9
2.4
40
1.1
Compound 7
0.95
0.19
0.31
0.23
4.5
60
147
132
104
6
61
37
8
16
a
P. falcipanum-infected Aotus monkeys were administered drug orally once a day
for 3 days beginning the day after a parasitemia level of approximately 5,000
1.1
0.55
9.9
6.8
organisms per mm3 was reached.
b
Number of animals cured/total number tested (cure is defined as clearance of
a
Route of administration for Artesunate is iv. All others were administered
orally.
parasitemia with no recrudescence).
b
ED50‘s, MCD’s, and MTD’s are reported in mg/kg.
Therapeutic indice were calculated as MTD divided by MCD.
c
excellent efficacy in a relevant primate model and possess a wider
therapeutic index than that of the commonly used antimalarial
drugs. Detailed study of pharmacokinetics and pharmacodynamics
will be presented in a separate report. These compounds, as well as
the underlying design rationale, may find usefulness in the discov-
ery and development of new antimalarial drugs.
of test compounds was conducted with Sprague–Dawley rats.
MTDs (maximum tolerated dose) and therapeutic indices were
obtained. The data are summarized in Table 2. The antimalarial
activities of compounds 1–5 are comparable to the natural product
febrifugine and some commonly used antimalarial drugs. The more
lipophilic derivative compounds 6–8 exhibited superior antimalar-
4. Experimental
4.1. Chemistry
0
ial activity. Their ED50 s and MCD’s are smaller then artemisinin
derivative. New compounds 1–8 are much less toxic than the
parent natural product febrifugine. Compounds 2–5, with either
an extra nitrogen atom or an electron-withdrawing substitution
group on the aromatic ring, are over 10 times less toxic than feb-
rifugine. The more lipophilic compounds 6–8, bearing an alkoxyl
group on C-300 position of the pyrrolidine ring, are slightly more
toxic as compared to compound 2–5. However, these compounds
(6, 7, and 8) still possess wide therapeutic window since they are
very efficacious (low ED50 and MCD value).
Compounds 6 and 7 (with highest therapeutic index value)
were chosen for further efficacy studies in Aotus monkey model.
The monkeys selected for the study had active infections of the
chloroquine-resistant FVO strain of P. falciparum. Once parasite-
Melting points were determined on a Mettler FP62 melting point
apparatus and are uncorrected. Unless otherwise noted, all non-
aqueous reactions were performed under an oxygen-free atmo-
sphere of nitrogen with rigid exclusion of moisture from reagents
and glassware. Analytical thin layer chromatography (TLC) was per-
formed using EM Reagents 0.25 mm silica gel 60-F plates. Visualiza-
tion of the developed chromatogram was performed by UV
absorbance, aqueous potassium permanganate, or ethanolic anisal-
dehyde. Liquid chromatography was performed using a force flow
(flash chromatography) of the indicated solvent system on EM Re-
agents Silica Gel 60 (70–230 mesh). Preparative TLC was performed
using Whatman Silica Gel C8 TLC plates (PLK5F). 1H NMR spectra
were recorded in deuteriochloroform, unless otherwise noted, on
a Bruker Avance 600 spectrometer at the frequency of 600.1 MHz.
Chemical shifts are reported in parts per million on the d scale from
an internal standard of tetramethylsilane. Data are reported as fol-
lows: chemical shifts, multiplicity (s = singlet, d = doublet, t = trip-
let, q = quartet, qn = quintet, m = multiplet, and br = broad),
coupling constant in Hertz, integration, and assignment. 13C NMR
spectra were recorded in deuteriochloroform, unless otherwise
mias reached 5,000/ll (or as deemed appropriate by the attending
veterinarian), they were treated orally with compound 6 or com-
pound 7 at 2, 4, 8, or 16 mg/kg/day for 3 days. For compound 6,
the dosage needed to cure 50% of animals was 4 mg/kg/day and
the dosage needed to cure 100% of animals was 16 mg/kg/day.
For compound 7, the 50% curative dose was 2 mg/kg/day and the
100% curative dose was 8 mg/kg/day (Table 3).
In conclusion, new febrifugine analogues were designed and
synthesized. Some of these compounds (6 and 7) have exhibited

930
S. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 927–932
noted, on a Bruker Avance 600 spectrometer at the frequency of
150.9 MHz. Chemical shifts are reported in parts per million on
the d scale from an internal standard of tetramethylsilane. Combus-
tion analyses were performed by Atlantic Microlab, Inc. (Norcross,
Georgia). All purified compounds possess a purity of at least 95%.
When necessary, solvents and reagents were dried as follows:
ether, tetrahydrofuran, benzene, and toluene were stored and dis-
tilled from sodium benzophenone ketyl; dichloromethane, triethyl-
amine, pyridine, and hexane were distilled over calcium hydride.
Unless otherwise stated, the reagents were purchased from Fisher
Scientific, Aldrich Chemical Company, Lancaster, or Fluka, and used
as received.
1H), 3.91 (m, 2H), 3.58 (m, 2H), 3.21 (m, 1H), 1.77 (m, 2H). 13C
NMR: 168.1, 164.2, 157.1, 145.9, 140.3, 139.1, 134.6, 129.8,
128.8, 128.4, 127.9, 127.5, 127.3, 127.1, 126.9, 126.6, 124.2, 79.9,
78.1, 70.1, 66.7, 58.2, 52.3, 38.2, 27.1. HRESIMS m/z 522.2011
[M+Na]+ (calcd for C29H29N3NaO5+, 522.2005) (100%). Anal. Calcd
for C29H29N3O5.HCl: C, 64.98; H, 5.64; Cl, 6.61; N, 7.84. Found: C,
64.93; H, 5.66; Cl, 6.56; N, 7.88.
4.1.3. (2S, 3S)-3-[2-(3-Hydroxy-pyrrolidin-2-yl)-2-oxo-ethyl]-3
H-quinazolin-4-one (1)
A solution of alcohol 15 (3.6 g) in 10 mL of CH2Cl2 was added
into a stirred slurry of tetrapropylammonium perruthenate (TPAP,
90 mg), N-methylmorpholine N-oxide (NMO, 1.8 g), and grounded
molecular sieve (1.8 g) in 20 mL of CH2Cl2 at room temperature.
After 2 h, the reaction mixture was loaded directly into a short col-
umn of silica gel and eluted with 5% MeOH/EtOAc. Concentration of
the eluant under reduced pressure afforded the corresponding ke-
tone. This ketone (3.3 g) was dissolved in 20 mL of 95% EtOH/H2O.
300 mg of 10% Pd on carbon was added in. It was then treated with
hydrogen (60 psi) in a Parr apparatus for 12 h. Solid was filtered off
and the solution was evaporated under vacuum to dryness. Recrys-
tallization from ethanol–water (with addition of dilute aqueous
HCl solution, 4–5 equiv of HCl) furnishes compound 1 (HCl salt)
as off-white crystals. Yield: 1.87 g, 84% (two steps). Mp: 206–
4.1.1. 3-Benzyloxy-2-oxiranyl-pyrrolidine-1-carboxylic acid be-
nyl ester (14)
Alkene 13 (9.6 g), osmium tetraoxide (OsO4, 4% in water,
1.0 mL), and N-methyl morpholine N-oxide (NMO, 10.8 g) were
dissolved in 80 mL of water and 80 mL of tetrahydrofuran (THF).
The resulting heterogeneous solution is stirred vigorously for
3 days at room temperature. After partition between ethyl acetate
(140 mL) and water (100 mL), the separated organic layer was
washed with brine (60 mL), dried over anhydrous sodium sulfate,
and evaporated in a rotary evaporator under reduced pressure to
furnish the essentially pure diol. Tosyl chloride (TsCl, 5.1 g) was
added into a stirred solution of this diol (9.7 g), triethylamine
(Et3N, 6.0 mL) and 4-dimethylaminopyridine (DMAP, 200 mg) in
100 mL of methylene chloride (CH2Cl2) in an ice-water bath. The
reaction mixture was then warmed to room temperature and stir-
red for additional 2 h. Solvent was evaporated. The resulting slurry
was dissolved in ethyl acetate (100 mL), washed with water
(75 mL), then aqueous sodium bicarbonate (75 mL), and brine
(40 mL), dried over anhydrous sodium sulfate, and evaporated in
a rotary evaporator under reduced pressure to furnish the essen-
tially pure mono-tosylate. Potassium carbonate (K2CO3, 1.0 g)
was added into a stirred solution of this mono-tosylate (12.2 g)
in 30 mL of methanol (MeOH) at room temperature. After 4 h, reac-
tion mixture was partitioned between ethyl acetate (75 mL) and
water (75 mL), the separated organic layer was then washed with
brine (40 mL), dried over anhydrous sodium sulfate, and evapo-
rated in a rotary evaporator under reduced pressure to furnish
the crude product. Silica gel flash chromatography (20% ethyl ace-
tate in hexanes) furnishes 14 as viscous oil. Yield: 7.3 g, 72% (3
steps). 1H NMR: 7.29–7.20 (m, 10H), 5.39 (m, 2H), 4.63 (m, 2H),
4.05 (m, 1H), 3.49 (m, 2H), 3.24 (m, 1H), 3.07 (m, 1H), 2.64 (m,
2H), 1.82–1.77 (m, 2H). 13C NMR: 159.9, 141.1, 138.4, 128.7,
128.3, 127.8, 127.5, 127.1, 126.9, 75.5, 72.2, 70.6, 58.4, 49.4, 44.2,
38.8, 29.2. HRESIMS m/z 376.1521 [M+Na]+ (calcd for
207 °C. ½a 2D5
ꢁ
+51.6 (c 0.50, EtOH). 1H NMR (CD3OD): 8.21(d,
J = 7.9 Hz, 1H), 7.85 (s, 1H), 7.71 (t, J = 7.9 Hz, 1H), 7.65 (d,
J = 7.9 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 5.08 (d, J = 16.7 Hz, 1H),
4.92 (d, J = 16.7 Hz, 1H), 4.21 (d, J = 6.5 Hz, 1H), 4.11 (m, 1H),
3.47–3.41 (m, 2H), 2.21 (m, 1H), 1.92 (m, 1H). 13C NMR (CD3OD):
205.2, 168.3, 164.8, 147.5, 133.5, 128.6, 127.9, 126.1, 122.5, 74.7,
66.9, 55.2, 38.1, 33.6. HRESIMS m/z 296.1007 [M+Na]+ (calcd for
C
14H15N3NaO3+, 296.1011) (100%). Anal. Calcd for C14H15N3O3.HCl:
C, 54.29; H, 5.21; Cl, 11.45; N, 13.57. Found: C, 54.22; H, 5.23; Cl,
11.51; N, 13.53.
4.1.4. (2S, 3S)-3-[2-(3-Hydroxy-pyrrolidin-2-yl)-2-oxo-ethyl]-3
H-pyrido[3,2-d]pyrimidin-4-one (2)
Pale-yellow crystal. Mp: 244–245 °C. ½a D25
ꢁ
+18.4 (c 0.50, EtOH).
1H NMR (CD3OD): 9.55 (d, J = 7.2 Hz, 1H), 9.14 (d, J = 7.2 Hz, 1H),
8.71 (s, 1H), 8.15 (t, J = 7.2 Hz, 1H), 5.12 (d, J = 17.1 Hz, 1H), 5.02
(d, J = 17.1 Hz, 1H), 4.44 (d, J = 6.2 Hz, 1H), 4.22 (m, 1H), 3.49–
3.43 (m, 2H), 2.15 (m, 1H), 1.89 (m, 1H). 13C NMR (CD3OD):
204.5, 164.8, 161.5, 151.9, 148.4, 145.8, 133.7, 131.7, 78.1, 65.9,
52.7, 43.2, 35.8. HRESIMS m/z 297.0972 [M+Na]+ (calcd for
C
13H14N4NaO3+, 297.0964) (100%). Anal. Calcd for C13H14N4O3.HCl:
C, 50.25; H, 4.87; Cl, 11.41; N, 18.03. Found: C, 50.31; H, 4.84; Cl,
11.38; N, 18.10.
C
21H23NNaO4+, 376.1525) (100%). Anal. Calcd for C21H23NO4: C,
71.37; H, 6.56; N, 3.96. Found: C, 71.41; H, 6.53; N, 3.99.
4.1.5. (2S, 3S)-3-[2-(3-Hydroxy-pyrrolidin-2-yl)-2-oxo-ethyl]-
3H-pyrido[4,3-d]pyrimidin-4-one (3)
4.1.2. 3-Benzyloxy-2-[1-hydroxy-2-(4-oxo-4H-quinazolin-3-yl)-
ethyl]-pyrrolidine-1-carboxylic acid benzyl ester (15)
Pale-yellow crystal. Mp: 235–236 °C. ½a D25
ꢂ23.7 (c 0.50, EtOH).
ꢁ
1H NMR (CD3OD): 9.46 (s, 1H), 9.15 (d, J = 7.4 Hz, 1H), 8.76 (s, 1H),
8.21 (d, J = 7.4 Hz, 1H), 5.19 (d, J = 17.2 Hz, 1H), 5.05 (d, J = 17.2 Hz,
1H), 4.39 (d, J = 6.3 Hz, 1H), 4.21 (m, 1H), 3.44–3.39 (m, 2H), 2.21
(m, 1H), 1.82 (m, 1H). 13C NMR (CD3OD): 204.5, 167.1, 163.9,
158.4, 151.2, 148.6, 136.8, 128.1, 79.5, 59.8, 51.3, 42.6, 32.8. HRE-
SIMS m/z 297.0961 [M+Na]+ (calcd for C13H14N4NaO3+, 297.0964)
(100%). Anal. Calcd for C13H14N4O3.HCl: C, 50.25; H, 4.87; Cl,
11.41; N, 18.03. Found: C, 50.29; H, 4.89; Cl, 11.44; N, 17.98.
Potassium hydride (KH, 30% in mineral oil, 4.0 g) was sus-
pended in 50 mL of dimethylformamide (DMF). It was cooled in
an ice-water bath, and solid 3H-quinazolin-4-one (4.3 g) was
added in. After 30 min, a solution of oxirane 14 (3.5 g) in 10 mL
of DMF was added in. The reaction mixture was then heated at
80 °C for 8 h under nitrogen atmosphere. It was partitioned be-
tween ethyl acetate (75 mL) and water (75 mL), separated organic
layer was washed with water (3 ꢀ 50 mL), then brine (50 mL),
dried over anhydrous sodium sulfate, and evaporated in a rotary
evaporator under reduced pressure to furnish the crude product.
Silica gel flash chromatography (75% ethyl acetate in hexanes) fur-
nished 15 as off white solid. Yield: 79%. 1H NMR: 8.31 (d, J = 7.6 Hz,
1H), 8.21 (s, 1H), 7.75 (m, 2H), 7.45 (m, 1H), 7.30–7.25 (m, 10H),
5.48 (m, 2H), 5.22 (br, 1H), 4.77 (m, 2H), 4.25 (m, 1H), 4.18 (m,
4.1.6. (2S, 3S)-5-Fluoro-3-[2-(3-hydroxy-pyrrolidin-2-yl)-2-oxo-
ethyl]-3H-quinazolin-4-one (4)
Off-white crystals. Mp: 223–224 °C. ½a D25
ꢁ
+12.5 (c 0.50, EtOH).
1H NMR (CD3OD): 8.38 (s, 1H), 8.21 (d, J = 7.1 Hz, 1H), 7.78 (d,
J = 7.2 Hz, 1H), 7.61 (t, J = 7.1 Hz, 1H), 5.17 (d, J = 17.0 Hz, 1H),
5.09 (d, J = 17.0 Hz, 1H), 4.41 (d, J = 6.3 Hz, 1H), 4.16 (m, 1H),

S. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 927–932
931
3.48–3.41 (m, 2H), 2.32 (m, 1H), 1.89 (m, 1H). 13C NMR (CD3OD):
206.2, 166.2, 161.5, 159.3, 149.6, 140.4, 130.1, 127.2, 121.2, 78.2,
62.2, 50.3, 40.4, 34.2. HRESIMS m/z 314.0922 [M+Na]+ (calcd for
8.62 (s, 1H), 8.04 (t, J = 6.9 Hz, 1H), 5.19 (d, J = 17.0 Hz, 1H), 5.08
(d, J = 17.0 Hz, 1H), 4.61 (d, J = 6.6 Hz, 1H), 4.32 (m, 1H), 3.44–
3.39 (m, 2H), 2.31 (m, 1H), 1.72 (m, 1H). 13C NMR (CD3OD):
207.1, 162.5, 160.9, 154.6, 147.1, 144.4, 135.6, 135.2, 79.6, 64.1,
53.6, 42.4, 34.1. HRESIMS m/z 297.0969 [M+Na]+ (calcd for
C
14H14FN3NaO3+, 314.0917) (100%). Anal. Calcd for C14H14FN3O3.
HCl: C, 51.31; H, 4.61; Cl, 10.82; F, 5.80; N, 12.82. Found: C,
51.26; H, 4.64; Cl, 10.85; F, 5.77; N, 12.87.
C
13H14N4NaO3+, 297.0964) (100%). Anal. Calcd for C13H14N4O3.HCl:
C, 50.25; H, 4.87; Cl, 11.41; N, 18.03. Found: C, 50.29; H, 4.85; Cl,
11.45; N, 17.98.
4.1.7. (2S, 3S)-3-[2-(3-Hydroxy-pyrrolidin-2-yl)-2-oxo-ethyl]-5-
trifluoromethyl-3H-quinazolin-4-one (5)
Off-white crystals. Mp: 252–253 °C. ½a D25
ꢂ17.2 (c 0.50, EtOH).
ꢁ
4.1.12. (2S, 3R)-3-[2-(3-Hydroxy-pyrrolidin-2-yl)-2-oxo-ethyl]-
5-trifluoromethyl-3H-quinazolin-4-one (10)
1H NMR (CD3OD): 8.32 (s, 1H), 7.81 (d, J = 7.2 Hz, 1H), 7.54 (d,
J = 7.2 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 5.06 (d, J = 16.9 Hz, 1H),
4.88 (d, J = 16.9 Hz, 1H), 4.49 (d, J = 6.2 Hz, 1H), 4.22 (m, 1H),
3.50–3.44 (m, 2H), 2.19 (m, 1H), 1.76 (m, 1H). 13C NMR (CD3OD):
203.4, 164.6, 159.5, 148.7, 140.5, 133.6, 132.2, 128.5, 125.2,
117.2, 76.4, 62.5, 52.3, 39.8, 33.1. HRESIMS m/z 364.0891
[M+Na]+ (calcd for C15H14F3N3NaO3+, 364.0885) (100%). Anal. Calcd
for C15H14F3N3O3.HCl: C, 47.69; H, 4.00; Cl, 9.39; F, 15.09; N, 11.12.
Found: C, 47.62 H, 4.02; Cl, 9.42; F, 15.05; N, 11.16.
Off-white crystals. Mp: 233–234 °C. ½a D25
ꢂ24.6 (c 0.50, EtOH).
ꢁ
1H NMR (CD3OD): 8.19 (s, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.50 (d,
J = 7.2 Hz, 1H), 7.41 (t, J = 7.2 Hz, 1H), 5.01 (d, J = 16.8 Hz, 1H),
4.82 (d, J = 16.8 Hz, 1H), 4.41 (d, J = 6.5 Hz, 1H), 4.19 (m, 1H),
3.46–3.40 (m, 2H), 2.21 (m, 1H), 1.82 (m, 1H). 13C NMR (CD3OD):
204.1, 163.9, 158.8, 148.1, 141.8, 132.3, 131.7, 127.7, 125.4,
119.5, 77.8, 63.2, 52.8, 40.2, 34.5. HRESIMS m/z 364.0881
[M+Na]+ (calcd for C15H14F3N3NaO3+, 364.0885) (100%). Anal. Calcd
for C15H14F3N3O3.HCl: C, 47.69; H, 4.00; Cl, 9.39; F, 15.09; N, 11.12.
Found: C, 47.64 H, 4.03; Cl, 9.34; F, 15.13; N, 11.07.
4.1.8. (2S, 3S)-3-[2-(3-Benzyloxy-pyrrolidin-2-yl)-2-oxo-ethyl]-
3H-pyrido[3,2-d]pyrimidin-4-one (6)
Pale-yellow crystal. Mp: 266–267 °C. ½a D25
ꢁ
-33.1 (c 0.50, EtOH).
4.1.13. (2R, 3R)-3-[2-(3-Hydroxy-pyrrolidin-2-yl)-2-oxo-ethyl]-
3H-pyrido[3,2-d]pyrimidin-4-one (11)
1H NMR (CD3OD): 9.44 (d, J = 7.1 Hz, 1H), 9.09 (d, J = 7.1 Hz, 1H),
8.62 (s, 1H), 8.19 (t, J = 7.1 Hz, 1H), 7.22–7.17 (m, 5H), 5.41 (d,
J = 15.8 Hz, 1H), 5.33 (d, J = 15.8 Hz, 1H), 5.15 (d, J = 17.0 Hz, 1H),
5.04 (d, J = 17.0 Hz, 1H), 4.38 (d, J = 5.8 Hz, 1H), 4.18 (m, 1H),
3.45–3.39 (m, 2H), 2.08 (m, 1H), 1.71 (m, 1H). 13C NMR (CD3OD):
204.1, 163.3, 160.7, 151.2, 147.7, 144.1, 138.9, 135.3, 131.9,
130.1, 128.5, 127.2, 81.5, 76.3, 65.4, 53.1, 41.4, 34.2. HRESIMS m/
z 387.1429 [M+Na]+ (calcd for C20H20N4NaO3+, 387.1433) (100%).
Anal. Calcd for C20H20N4O3.HCl: C, 59.92; H, 5.28; Cl, 8.84; N,
13.98. Found: C, 59.86; H, 5.31; Cl, 8.80; N, 14.03.
Pale-yellow crystal. Mp: 242–243 °C. ½a D25
ꢂ18.2 (c 0.50, EtOH).
ꢁ
1H NMR (CD3OD): 9.53 (d, J = 7.1 Hz, 1H), 9.12 (d, J = 7.1 Hz, 1H),
8.70 (s, 1H), 8.18 (t, J = 7.1 Hz, 1H), 5.19 (d, J = 17.2 Hz, 1H), 5.05
(d, J = 17.2 Hz, 1H), 4.41 (d, J = 6.3 Hz, 1H), 4.20 (m, 1H), 3.48–
3.42 (m, 2H), 2.21 (m, 1H), 1.85 (m, 1H). 13C NMR (CD3OD):
204.9, 164.1, 161.3, 151.3, 148.1, 145.3, 133.2, 131.4, 78.8, 65.4,
52.4, 43.8, 35.2. HRESIMS m/z 297.0969 [M+Na]+ (calcd for
C
13H14N4NaO3+, 297.0964) (100%). Anal. Calcd for C13H14N4O3.HCl:
C, 50.25; H, 4.87; Cl, 11.41; N, 18.03. Found: C, 50.20; H, 4.90; Cl,
11.37; N, 18.09.
4.1.9. (2S, 3S)-3-[2-(3-Ethoxy-pyrrolidin-2-yl)-2-oxo-ethyl]-5-
fluoro-3H-quinazolin-4-one (7)
4.1.14. (2R, 3R)-3-[2-(3-Hydroxy-pyrrolidin-2-yl)-2-oxo-ethyl]-
5-trifluoromethyl-3H-quinazolin-4-one (12)
Off-white crystals. Mp: 242–244 °C. ½a D25
ꢁ
+19.6 (c 0.50, EtOH).
Off-white crystals. Mp: 254–255 °C. ½a D25
ꢁ
+17.4 (c 0.50, EtOH).
1H NMR (CD3OD): 8.51 (s, 1H), 8.29 (d, J = 7.0 Hz, 1H), 7.69 (d,
J = 7.0 Hz, 1H), 7.52 (t, J = 7.0 Hz, 1H), 5.10 (d, J = 17.1 Hz, 1H),
5.02 (d, J = 17.1 Hz, 1H), 4.53 (d, J = 6.4 Hz, 1H), 4.21 (m, 1H),
3.51 (q, J = 6.9 Hz, 2H), 3.44–3.38 (m, 2H), 2.24 (m, 1H), 1.75 (m,
1H), 1.21 (t, J = 6.9 Hz, 3H). 13C NMR (CD3OD): 205.1, 165.4,
161.9, 157.6, 147.1, 140.8, 131.4, 127.8, 120.1, 78.6, 69.1, 61.4,
1H NMR (CD3OD): 8.34 (s, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.52 (d,
J = 7.2 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 5.09 (d, J = 17.0 Hz, 1H),
4.86 (d, J = 17.0 Hz, 1H), 4.48 (d, J = 6.1 Hz, 1H), 4.19 (m, 1H),
3.51–3.45 (m, 2H), 2.16 (m, 1H), 1.77 (m, 1H). 13C NMR (CD3OD):
203.7, 164.4, 159.4, 148.7, 140.6, 133.4, 132.1, 128.6, 125.2,
117.3, 76.4, 62.6, 52.1, 39.8, 33.2. HRESIMS m/z 364.0881
[M+Na]+ (calcd for C15H14F3N3NaO3+, 364.0885) (100%). Anal. Calcd
for C15H14F3N3O3.HCl: C, 47.69; H, 4.00; Cl, 9.39; F, 15.09; N, 11.12.
Found: C, 47.71 H, 4.03; Cl, 9.34; F, 15.14; N, 11.09.
51.5, 41.6, 32.3, 16.2. HRESIMS m/z 342.1227 [M+Na]+ (calcd for
+
C16H18FN3NaO3
,
342.1230)
(100%).
Anal.
Calcd
for
C16H18FN3O3.HCl: C, 54.01; H, 5.38; Cl, 9.96; F, 5.34; N, 11.81.
Found: C, 53.94; H, 5.41; Cl, 9.91; F, 5.39; N, 11.83.
4.1.10. (2S, 3S)-3-[2-(3-Isopropoxy-pyrrolidin-2-yl)-2-oxo-ethyl]
-5-trifluoromethyl-3H-quinazolin-4-one (8)
4.2. Biological studies
Off-white crystals. Mp: 269–270 °C. ½a D25
ꢁ
-39.6 (c 0.50, EtOH). 1H
(i) In vitro drug susceptibility studies were conducted by using a
modification of the semiautomated microdilution techniques of
Desjardins and Chulay.17–19 (ii) Antimalarial efficacy and toxicity
evaluation in rodent models were carried out in procedures previ-
ously described.20,21 (iii) Compounds 6 and 7 underwent further effi-
cacy studies in Aotus monkey model. Detailed procedures of malaria
infection and drug tests were described in the previous reports.21–25
All animal experiments were conducted in compliance with the Ani-
mal Welfare Act and other federal statutes and regulations relating
to animals and experiments involving animals and adhere to princi-
ples stated in the Guide for the Care and Use of Laboratory Animals.26
NMR (CD3OD): 8.41 (s, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.50 (d,
J = 7.2 Hz, 1H), 7.39 (t, J = 7.2 Hz, 1H), 5.16 (d, J = 16.8 Hz, 1H),
4.92 (d, J = 16.8 Hz, 1H), 4.45 (d, J = 6.2 Hz, 1H), 4.27 (m, 1H),
3.49–3.42 (m, 2H), 3.28 (m, 1H), 2.25 (m, 1H), 1.79 (m, 1H), 1.19
(d, J = 5.9 Hz, 6H). 13C NMR (CD3OD): 204.3, 163.7, 160.1, 149.2,
140.8, 134.3, 131.8, 127.6, 124.7, 119.4, 77.1, 69.5, 66.5, 53.2,
39.2, 31.3, 24.2. HRESIMS m/z 406.1361 [M+Na]+ (calcd for
+
C18H20F3N3NaO3
,
406.1354)
(100%).
Anal.
Calcd
for
C18H20F3N3O3.HCl: C, 51.50; H, 5.04; Cl, 8.44; F, 13.58; N, 10.01.
Found: C, 51.47; H, 5.06; Cl, 8.39; F, 13.62; N, 10.04.
4.1.11. (2S, 3R)-3-[2-(3-Hydroxy-pyrrolidin-2-yl)-2-oxo-ethyl]-
3H-pyrido[3,2-d]pyrimidin-4-one (9)
Acknowledgments
Pale-yellow crystal. Mp: 221–222 °C. ½a D25
ꢁ
+27.1 (c 0.50, EtOH).
This research was partially supported by a grant from the Na-
tional Institutes of Health (R44 AI77109 to S.Z.). The content is
1H NMR (CD3OD): 9.71 (d, J = 6.9 Hz, 1H), 9.25 (d, J = 6.9 Hz, 1H),

932
S. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 927–932
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solely the responsibility of the authors and does not necessarily
represent the official views of the funding agency.
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