Synthesis of febrifugine derivatives and development of an effective and safe tetrahydroquinazoline-type antimalarial

Article abstract of DOI:10.1016/j.ejmech.2014.01.036

Febrifugine, a quinazoline alkaloid isolated from Dichroa febrifuga roots, shows powerful antimalarial activity against Plasmodium falciparum. Although the use of ferifugine as an antimalarial drug has been precluded because of its severe side effects, its potent antimalarial activity has stimulated medicinal chemists to pursue its derivatives instead, which may provide valuable leads for novel antimalarial drugs. In the present study, we synthesized new derivatives of febrifugine and evaluated their in vitro and in vivo antimalarial activities to develop antimalarials that are more effective and safer. As a result, we proposed tetrahydroquinazoline-type derivative as a safe and effective antimalarial candidate.

Full text of DOI:10.1016/j.ejmech.2014.01.036

European Journal of Medicinal Chemistry 76 (2014) 10e19
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of febrifugine derivatives and development of an effective
and safe tetrahydroquinazoline-type antimalarial
,
a
a
b
Haruhisa Kikuchi a , Seiko Horoiwa , Ryota Kasahara , Norimitsu Hariguchi ,
*
Makoto Matsumoto b, Yoshiteru Oshima a
a Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan
b Microbiological Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Febrifugine, a quinazoline alkaloid isolated from Dichroa febrifuga roots, shows powerful antimalarial
activity against Plasmodium falciparum. Although the use of ferifugine as an antimalarial drug has been
precluded because of its severe side effects, its potent antimalarial activity has stimulated medicinal
chemists to pursue its derivatives instead, which may provide valuable leads for novel antimalarial drugs.
In the present study, we synthesized new derivatives of febrifugine and evaluated their in vitro and
in vivo antimalarial activities to develop antimalarials that are more effective and safer. As a result, we
proposed tetrahydroquinazoline-type derivative as a safe and effective antimalarial candidate.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Received 3 October 2013
Received in revised form
12 January 2014
Accepted 19 January 2014
Available online 25 January 2014
Keywords:
Antimalarials
Quinazoline alkaloids
Plasmodium falciparum
Structureeactivity relationship
1. Introduction
drug has been precluded. Halofuginone (3) (Fig. 1), a 7-bromo-6-
chloro derivative of 1 that shows less toxicity, has been used as a
Malaria, which is caused by a protozoan parasite of the genus
Plasmodium, is a major parasitic infection in many tropical and
subtropical regions. Malaria affects about 200 million patients
worldwide and leads to more than half a million deaths each year.
Although malaria has been widely eradicated in many parts of the
world, the number of its victims continues to rise mainly because of
the emergence of chloroquine-resistant and multiple-drug-
resistant strains of malaria parasites. Thus, the discovery of new
and effective antimalarial drugs is urgently needed [1,2].
Roots of Dichroa febrifuga, a plant that belongs to the Saxi-
fragaceae family, have been used as a traditional antimalarial drug
in China. The quinazoline-type alkaloids, febrifugine (1) and its
stereoisomer, isofebrifugine (2), have been identified as the active
components of these roots (Fig. 1) [3,4]. Although 1 and 2 show
powerful in vitro antimalarial activity against both chloroquine-
sensitive Plasmodium falciparum FCR-3 and chloroquine-resistant
P. falciparum K1, the in vivo activity of 1 against mouse malaria
parasite, Plasmodium berghei, is approximately 200 times more
potent than that of 2. Because of side effects such as diarrhea,
vomiting [5,6], and liver toxicity [7], the use of 1 as an antimalarial
veterinary coccidiostat [8]. Recently, compound 3 has been devel-
oped as a medicine for scleroderma, and has been used in clinical
trials as a therapeutic in cancer and fibrotic disease [9e13]. Hal-
ofuginone (3) binds glutamyl-prolyl-tRNA synthetase and inhibits
prolyl-tRNA synthetase activity, revealing its anti-inflammatory
effect, which quenched by addition of an excess amount of pro-
line [14,15]. Addition of excess proline also lowers the antimalarial
activity of 3, but its nanomolar level IC50 is maintained. Thus,
additional molecular targets of febrifugine (1), halofuginone (3)
and their derivatives for antimalarial activity are remaining.
The potent antimalarial activity of 1 has stimulated medicinal
chemists to pursue derivatives of 1, which may provide valuable
leads for novel antimalarial drugs [5,6,16e21]. In our previous work
[19], we synthesized febrifugine derivatives via structural modifi-
cations at either (i) the quinazoline ring, (ii) linker, or (iii) piperi-
dine ring (Fig. 2). We found that modification of the quinazoline
ring was the most suitable method for creating the most effective
derivatives. Additionally, we recognized that the piperidine ring is
necessary for the antimalarial activity of 1; however, the ring size
has not been evaluated. In this paper, we describe the synthesis of
new derivatives of 1 and evaluate their in vitro and in vivo anti-
malarial activities in order to develop more effective and safer
antimalarials.
* Corresponding author. Tel.: þ81 22 795 6824; fax: þ81 22 795 6821.
E-mail address: hal@mail.pharm.tohoku.ac.jp (H. Kikuchi).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.036

H. Kikuchi et al. / European Journal of Medicinal Chemistry 76 (2014) 10e19
11
pyrrolidine 27 by borane reduction. Oxidation of 27 produced the
aldehyde, which, after treatment with dimethylsulfonium meth-
ylide [29], produced epoxide 28. Coupling reaction between 28 and
4-hydroxyquinazoline, followed by oxidation and acidic depro-
tection of the resulting compound, yielded 20.
The azepane and linker parts of 21 were synthesized from the
common intermediate 23 (Scheme 3). Oxidative N-debenzylation
of 23 by treatment with ceric ammonium nitrate [30] yielded 29,
which was then converted into N-allyl compound 30. Intra-
molecular olefin metathesis of 30 and subsequent hydrogenation
yielded azepane 32. Finally, coupling reaction between 32 and 4-
hydroxyquinazoline via an epoxide intermediate produced 21.
In our previous work [19], 20-decarbonylated derivative (33)
(Fig. 3) of 1 showed very low toxicity and sufficiently effective
antimalarial activity both in vitro and in vivo (Tables 1 and 2). Thus,
we synthesized 20-decarbonylated compounds 34 and 35 bearing
pyrrolidine and azepane rings, respectively (Scheme 4). Through
Fig. 1. Structures of febrifugine (1), isofebrifugine (2) and halofuginone (3).
2. Results and discussion
2.1. Syntheses of quinazoline ring-modified derivatives
the use of D-glutamic acid as a starting material, the aliphatic ring
and linker parts of compounds 34 and 35 were afforded in a
manner similar to those for 20 and 21, respectively. Finally, the
respective coupling reaction of parts with 4-hydroxyquinazoline
via tosylates produced 34 and 35.
In our previous works [19], we synthesized febrifugine de-
rivatives bearing a pyrimidine, an isoquinoline, substituted quina-
zolines, pyrimidines fused with heterocycles instead of the
quinazoline ring. In this work, new types of quinazoline ring-
modified derivatives 4e10 were synthesized. Compounds 4e6, as
well as 7 and 8, have pyrimidines fused with aliphatic rings and 5,6-
dialkylpyrimidines, respectively. The size of their aromatic moieties
is similar to that of the quinazoline ring, but there is no benzene
ring in them. Compounds 9 and 10 have 6- and 5-phenyl pyrimi-
dines, respectively, which are the benzo-cracked rings of the qui-
nazoline ring. These compounds were synthesized by a coupling
reaction between the corresponding heterocycle and a hemiacetal
11, which was reported by Takeuchi et al. (Scheme 1) [22,23].
Syntheses of the corresponding heterocycles are depicted in
Scheme 2. Enamines 15aec, which were prepared from 2-
methoxycarbonylcycloalkanone 14aec [24], were condensed with
formamide to produce the aliphatic-ring-fused pyrimidines 16aec.
Reductive desulfurization of thiouracil derivatives 18a,b [25],
2.3. In vitro antimalarial activities and cytotoxicities of the
synthetic febrifugine derivatives
The in vitro antimalarial activity of febrifugine analogs 4e10, 20,
21, 34, and 35 against P. falciparum and their cytotoxicity against
mouse L929 cells were evaluated (Table 1). The antimalarial activ-
ities of the aliphatic-ring-fused pyrimidine derivatives 4e6 was 1/
30e1/50 of that of 1. However, their cytotoxicities were very low
(EC50 > 50 mg/mL); consequently, these compounds showed very
high selectivities (about 1000). Therefore, a suitable method to
create antimalarials that are more effective and safer is to modify
the quinazoline ring of 1 through the aliphatic-ring-fused pyrimi-
dine. 5,6-Dialkylpyrimidine derivative 7 (diethyl) showed moder-
ate antimalarial activity whereas derivative 8 (di-n-hexyl) showed
none, indicating that a cyclic moiety of substituents on the py-
rimidine ring of 1 may be important for activity. The phenyl py-
rimidine compounds 9 and 10 had antimalarial activities 1/30e1/
60 of that of 1 and sufficient selectivities. On the contrary, com-
pounds 20, 21, 34, and 35, which have pyrrolidine or azepane rings
instead of the piperidine ring of 1, displayed only weak antimalarial
activity or none at all. Thus, the ring size of piperidine in 1 is crucial
for activity.
which were synthesized by condensation between
b-ketoesters
17a,b and thiourea [26], afforded 5,6-dialkylpyrimidines 19a,b.
Phenyl pyrimidines corresponding to 9 and 10 were synthesized as
reported previously in the literature [27].
2.2. Syntheses of piperidine ring-modified derivatives
In order to evaluate the role of the ring size of the piperidine
moiety, we synthesized derivatives 20 and 21, which have pyrro-
lidine and azepane rings, respectively. The pyrrolidine and linker
parts of 20 were synthesized by using N,N-dibenzylamino aldehyde
[28] as a chiral building block (Scheme 3). Oxidation of 22, which
2.4. In vivo antimalarial activity of the synthetic febrifugine
derivatives
had been prepared from D-aspartic acid, gave N,N-dibenzylamino
In vivo activities of synthesized febrifugine derivatives against
P. berghei, the malarial parasite of rodents, and their acute toxicity
to mice were examined (Table 2). As described above, the aliphatic
aliphatic-ring-fused pyrimidine derivatives 4e6 showed safer
antimalarial activities. Among these compounds, we chose cyclo-
hexane ring-fused derivative 5 as a test compound, because its ring
size is the same as that of febrifugine (1), and it is easy to prepare
sufficient amount of for in vivo experiment. The phenyl pyrimidine
compounds 9 and 10 also showed selective antimalarial activities,
and we chose more potent compounds 10 as a test compound for
in vivo experiment. As a result, the in vivo antimalarial activities of 5
(ED50 12.8 mg/kg) and 10 (ED50 6.3 mg/kg) were 1/15e1/30 of that
of 1. However, the acute toxicities of 5 and 10 were much weaker
than that of 1 and their therapeutic index were higher than 1.
Especially, therapeutic index of 5 was almost 100. In addition,
diarrhea and pale appearance of liver were not observed in mice
aldehyde, and subsequently reacted with allylmagnesium bromide
and chlorodimethyl ether to diastereoselectively give MOM ether
23. Dihydroxylation of 23 by osmium tetroxide and subsequent
oxidative cleavage produced
g-amino acid derivative 25. After
debenzylation of 25 by hydrogenolysis, intramolecular cyclization
gave
g-lactam 26, which was then converted into Boc-protected
Fig. 2. The three parts of febrifugine (1).

12
H. Kikuchi et al. / European Journal of Medicinal Chemistry 76 (2014) 10e19
Scheme 1. Synthesis of compounds 4e10.a
administered 200 mg/kg of compound 5. Therefore, the tetrahy-
droquinazoline derivative 5 is an effective and safe antimalarial
candidate.
AX-700. Elemental analyses were performed on Yanaco CHN Corder
MT-6.
In conclusion, we synthesized and evaluated a new series of
febrifugine derivatives, and found tetrahydroquinazoline derivative
5 to exhibit potent antimalarial activity with a very high thera-
peutic selectivity both in vitro and in vivo. Therefore, further studies
on 5 with focus on areas such as metabolic analysis and elucidation
of the action mechanism are necessary for the development of a
novel antimalarial drug.
3.1.1. (2R*,3S*)-tert-Butyl 3-hydroxy-2-[2-oxo-3-(4-oxo-4,5,6,7-
tetrahydro-3H-cyclopenta-[d]pyrimidin-3-yl)propyl]piperidine-1-
carboxylate (13a)
To a solution of 16a (62 mg, 0.454 mmol) in DMF (1 mL) were
added 11 [22] (168 mg, 0.454 mmol) and potassium carbonate
(69 mg, 0.500 mmol) at room temperature. After stirring for 2 h, the
mixture was poured into brine and extracted with ethyl acetate
three times. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and evaporated. The residue was dis-
solved in methanol (2.5 mL). Then, after refluxing for 3 h, trie-
3. Experimental section
thylamine (28
mL, 0.196 mmol) and di-tert-butyl dicarbonate
3.1. General methods
(78 mg, 0.357 mmol) were added at room temperature. After stir-
ring for 1 h, the mixture was poured into 0.2 M hydrochloric acid
and extracted with ethyl acetate three times. The organic layer was
washed with saturated sodium bicarbonate solution and brine,
dried over anhydrous sodium sulfate, and evaporated. The residue
was chromatographed over silica gel eluted by ethyl acetatee
methanol (9:1) to give 13a (27 mg, 0.068 mmol, 15% (4 steps)).
In a similar procedure, compounds 13b (yield: 24%), 13c (12%),
13d (10%), 13e (12%), 13f (18%) and 13g (21%) were prepared from
16aec, 19a,b, 6-phenylpyrimidine [27] and 5-phenyl pyrimidine
[27], respectively.
Starting materials were either commercially available or pre-
pared as reported previously in the literature. Analytical thin layer
chromatography was performed on silica gel 60 F254 (Merck) and
Aluminum oxide 150 F254 Neutral (Type T, Merck). Column chro-
matography was carried out on Silica Gel 60 (70e230 mesh, Merck),
and Aluminum Oxide 150 Basic (type T, Merck). Nuclear magnetic
resonance spectra were recorded on JEOL JNM ECA-600, ECP-500,
and AL-400. Mass spectra were measured on JEOL JMS AX-500 and
3.1.2. 3-[3-((2R*,3S*)-3-Hydroxypiperidin-2-yl)-2-oxopropyl]-6,7-
dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one (4)
Compound 13a (4.9 mg, 0.013 mmol) was dissolved in 10%
hydrogen chloride in methanol (2.0 mL) at room temperature. After
stirring for 2 h, the mixture was evaporated. The residue was
chromatographed over silica gel eluted by chloroformemethanol
(4:1) to give 4 (4.2 mg, 0.012 mmol, 92%) as dihydrochloride.
Data for 4: colorless amorphous solid; 1H NMR (400 MHz,
CD3OD)
d
8.64 (1H, s), 5.02 (1H, d, J ¼ 17.5 Hz), 4.96 (1H, d,
J ¼ 17.5 Hz), 3.64 (1H, ddd, J ¼ 13.3, 9.5, 3.7 Hz), 3.44 (1H, ddd,
J ¼ 9.5, 7.0, 4.5 Hz), 3.36 (1H, dd, J ¼ 18.3, 4.5 Hz), 3.29e3.31 (1H, m),
3.05 (1H, dd, J ¼ 18.3, 7.0 Hz), 2.91e2.96 (1H, m), 2.87e2.89 (2H, m),
2.70e2.74 (2H, m), 2.04e2.12 (2H, m), 1.96e2.00 (1H, m), 1.90e1.94
(1H, m), 1.62e1.69 (1H, m), 1.46e1.51 (1H, m); 13C NMR (100 MHz,
CD3OD) d 201.1, 165.0, 159.5, 153.4, 127.1, 68.2, 58.0, 56.0, 44.8, 40.1,
Scheme 2. Synthesis of compounds 16aec and 19a,b.a
34.1, 31.5, 28.6, 22.4, 21.4; HRFABMS m/z 292.1656 [M þ H]þ

H. Kikuchi et al. / European Journal of Medicinal Chemistry 76 (2014) 10e19
13
(292.1661 calcd for C15H22N3O3); Anal. (C15H25N3O4Cl2 (dihydro-
chloride hydrate)) C, H, N.
In a similar procedure, compounds 5 (yield: 94%), 6 (92%), 7
(91%), 8 (89%), 9 (91%) and 10 (91%) were prepared from 13beg,
respectively.
3.1.2.1. Data for 3-[3-((2R*,3S*)-3-hydroxypiperidin-2-yl)-2-
oxopropyl]-5,6,7,8-tetrahydro-quinazolin-4(3H)-one
(5). Colorless
amorphous solid; 1H NMR (600 MHz, CD3OD)
d
9.29 (1H, s), 5.17 (2H,
s), 3.60e3.65 (1H, m), 3.40e3.45 (1H, m), 3.37 (1H, dd, J ¼ 18.1,
4.7 Hz), 3.27e3.29 (1H, m), 3.06 (1H, dd, J ¼ 18.1, 7.1 Hz), 2.96e3.00
(1H, m), 2.68e2.72 (2H, m), 2.44e2.47 (2H, m), 2.02e2.07 (1H, m),
1.94e1.97 (1H, m), 1.81e1.87 (2H, m), 1.75e1.80 (2H, m), 1.71e1.76
(1H, m), 1.50e1.57 (1H, m); 13C NMR (150 MHz, CD3OD)
d 200.4,
159.4, 152.3, 151.7, 125.0, 68.3, 58.0, 56.7, 44.9, 40.3, 31.6, 27.6, 23.0,
21.8, 21.7, 21.4; HRFABMS m/z 306.1808 [M þ H]þ (306.1818 calcd for
C16H24N3O3); Anal. (C16H27N3O4Cl2 (dihydrochloride hydrate)) C, H,
N.
3.1.2.2. Data for 3-[3-((2R*,3S*)-3-hydroxypiperidin-2-yl)-2-
oxopropyl]-6,7,8,9-tetrahydro-3H-cyclohepta[d]pyrimidin-4(5H)-one
(6). Colorless amorphous solid; 1H NMR (600 MHz, CD3OD)
d 9.02
(1H, s), 5.15 (1H, d, J ¼ 17.5 Hz), 5.11 (1H, d, J ¼ 17.5 Hz), 3.64 (1H,
ddd, J ¼ 12.8, 9.5, 4.0 Hz), 3.44e3.48 (1H, m), 3.40 (1H, dd, J ¼ 18.2,
4.6 Hz), 3.28e3.30 (1H, m), 3.07 (1H, dd, J ¼ 18.2, 7.4 Hz), 2.98e
3.02 (1H, m), 2.86e2.91 (2H, m), 2.76e2.81 (2H, m), 2.05e2.11 (1H,
m), 1.98e2.03 (1H, m), 1.88e1.93 (2H, m), 1.73e1.80 (3H, m), 1.55e
1.63 (3H, m); 13C NMR (150 MHz, CD3OD)
d 200.8, 160.4, 159.3,
151.7, 129.4, 68.2, 58.0, 56.9, 44.9, 40.2, 34.8, 32.6, 31.5, 26.4, 25.8,
25.4, 21.4; HRFABMS m/z 320.1960 [M þ H]þ (320.1974 calcd for
C17H26N3O3); Anal. (C17H29N3O4Cl2 (dihydrochloride hydrate)) C,
H, N.
3.1.2.3. Data for 5,6-diethyl-3-[3-((2R*,3S*)-3-hydroxypiperidin-2-
yl)-2-oxopropyl]-pyrimidin-4(3H)-one (7). Colorless amorphous
solid; 1H NMR (600 MHz, CD3OD)
d 9.02 (1H, s), 5.15 (1H, d,
J ¼ 17.5 Hz), 5.11 (1H, d, J ¼ 17.5 Hz), 3.64 (1H, ddd, J ¼ 12.8, 9.8,
4.0 Hz), 3.44e3.49 (1H, m), 3.40 (1H, dd, J ¼ 18.2, 4.5 Hz), 3.28e
3.32 (1H, m), 3.06 (1H, dd, J ¼ 18.2, 7.4 Hz), 2.96e3.02 (1H, m),
2.74 (2H, q, J ¼ 7.6 Hz), 2.58 (2H, q, J ¼ 7.5 Hz), 2.06e2.11 (1H, m),
1.97e2.03 (1H, m), 1.72e1.81 (1H, m), 1.54e1.62 (1H, m), 1.29 (3H,
t, J ¼ 7.6 Hz), 1.11 (3H, t, J ¼ 7.5 Hz); 13C NMR (150 MHz, CD3OD)
d
200.8, 160.6, 157.5, 152.3, 128.1, 68.2, 58.0, 56.6, 44.9, 40.1, 31.5,
25.8, 21.4, 19.9, 13.2, 13.1; HRFABMS m/z 308.1959 [M þ H]þ
(308.1974 calcd for C16H26N3O3); Anal. (C16H29N3O4Cl2 (dihydro-
chloride hydrate)) C, H, N.
3.1.2.4. Data for 5,6-dihexyl-3-[3-((2R*,3S*)-3-hydroxypiperidin-2-
yl)-2-oxopropyl]-pyrimidin-4(3H)-one (8). Colorless amorphous
solid; 1H NMR (400 MHz, CD3OD)
d 9.30 (1H, s), 5.20 (2H, s), 3.66
(1H, ddd, J ¼ 13.5, 9.5, 3.8. Hz), 3.42e3.48 (1H, m), 3.42 (1H, dd,
J ¼ 18.3, 4.6 Hz), 3.29e3.33 (1H, m), 3.10 (1H, dd, J ¼ 18.3, 7.1 Hz),
2.94e3.01 (1H, m), 2.73 (2H, t, J ¼ 7.5 Hz), 2.55 (2H, t, J ¼ 7.5 Hz),
2.07e2.12 (1H, m), 1.98e2.04 (1H, m), 1.74e1.82 (1H, m), 1.65e1.73
(2H, m), 1.53e1.61 (1H, m), 1.42e1.51 (2H, m), 1.27e1.39 (12H, m),
0.93 (3H, t, J ¼ 7.0 Hz), 0.91 (3H, t, J ¼ 7.0 Hz); 13C NMR (100 MHz,
CD3OD)
d 200.8, 160.6, 156.0, 152.3, 127.4, 68.2, 58.0, 56.7, 44.9,
40.1, 32.7, 32.6, 31.5, 30.3, 30.1, 29.6, 29.3, 26.7, 23.7, 23.6, 23.5,
21.4, 14.4, 14.3; HRFABMS m/z 420.3236 [M þ H]þ (420.3226 calcd
for C24H42N3O3); Anal. (C24H45N3O4Cl2 (dihydrochloride hydrate))
C, H, N.
3.1.2.5. Data for 3-[3-((2R*,3S*)-3-hydroxypiperidin-2-yl)-2-
Scheme 3. Synthesis of compounds 20 and 21.a
oxopropyl]-6-phenylpyrimidin-4(3H)-one (9). Colorless amorphous
solid; 1H NMR (600 MHz, D2O)
d
8.38 (1H, s), 7.91 (2H, dd, J ¼ 8.3,

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H. Kikuchi et al. / European Journal of Medicinal Chemistry 76 (2014) 10e19
Table 2
In vivo antimalarial activities against P. berghei and acute toxicities of synthesized
febrifugine derivatives 5 and 10.a
Compound
Antimalarial activityb Acute toxicityc Therapeutic indexd
ED50 (mg/kg)
LD50 (mg/kg)
Fig. 3. Structure of 20-decarbonylated derivative 33.
Febrifugine (1)
5
10
0.41
12.8
6.3
7.1
17
94
63
e
1200
400
N.T.e
1.5 Hz), 7.53e7.61 (3H, m), 6.98 (1H, s), 5.11 (1H, d, J ¼ 18.1 Hz), 5.07
(1H, d, J ¼ 18.1 Hz), 3.70e3.76 (1H, m), 3.50e3.55 (1H, m), 3.45 (1H,
dd, J ¼ 18.6, 4.6 Hz), 3.32e3.36 (1H, m), 3.11 (1H, dd, J ¼ 18.6,
7.3 Hz), 3.00e3.06 (1H, m), 2.09e2.15 (1H, m), 1.97e2.03 (1H, m),
1.71e1.79 (1H, m), 1.54e1.63 (1H, m); 13C NMR (150 MHz, D2O)
Chloroquine
0.98
a
All compounds were administrated by p.o.
Against P. berghei (rodent malaria).
Toxicity in mice.
Therapeutic index ¼ LD50 in mice/ED50 for P. berghei in mice.
b
c
d
e
Not tested.
d
203.0, 164.0, 163.8, 152.8, 135.8, 132.0, 129.7 (2C), 127.8 (2C), 110.2,
67.8, 63.1, 56.8, 44.5, 39.5, 30.5, 20.6; HRFABMS m/z 328.1684
[M þ H]þ (328.1661 calcd for C18H22N3O3); Anal. (C18H25N3O4Cl2
(dihydrochloride hydrate)) C, H, N.
evaporated to give a crude enamine 15a. This crude was dissolved
in formamide (6 mL) and stirred for 9 h at 180 ꢀC. The mixture was
poured into brine and extracted with chloroform five times. The
organic layer was dried over anhydrous sodium sulfate, and evap-
orated. The residue was chromatographed over silica gel eluted by
chloroformemethanol (9:1) to give 16a (337 mg, 2.47 mmol, 39% (2
steps)).
3.1.2.6. Data for 3-[3-((2R*,3S*)-3-hydroxypiperidin-2-yl)-2-
oxopropyl]-5-phenylpyrimidin-4(3H)-one (10). Colorless amor-
phous solid; 1H NMR (600 MHz, CD3OD)
d 9.14 (1H, s), 8.27 (1H, s),
7.68 (2H, dd, J ¼ 7.7, 1.9 Hz), 7.43e7.49 (3H, m), 5.26 (1H, d,
J ¼ 17.7 Hz), 5.22 (1H, d, J ¼ 17.7 Hz), 3.64e3.69 (1H, m), 3.45e3.50
(1H, m), 3.43 (1H, dd, J ¼ 17.3, 4.4 Hz), 3.29e3.34 (1H, m), 3.12 (1H,
dd, J ¼ 17.3, 6.4 Hz), 2.97e3.03 (1H, m), 2.06e2.11 (1H, m), 1.96e
2.03 (1H, m), 1.74e1.82 (1H, m), 1.54e1.62 (1H, m); 13C NMR
In a similar procedure, compounds 16b (yield: 50%) and 16c
(34%) were prepared from 14b and 14c, respectively.
(150 MHz, CD3OD)
d 200.8, 159.6, 153.7, 142.9, 132.3, 130.7, 130.6
(2C), 129.7 (2C), 129.3, 68.2, 58.0, 57.0, 44.9, 40.3, 31.6, 21.4;
HRFABMS m/z 328.1681 [M þ H]þ (328.1661 calcd for C18H22N3O3);
Anal. (C18H25N3O4Cl2 (dihydrochloride hydrate)) C, H, N.
3.1.4. 5,6-Diethylpyrimidin-4(3H)-one (19a)
To a solution of methyl 2-ethyl-3-oxopentanoate (17a) (1.28 g,
8.10 mmol) in ethanol (70 mL) were added thiourea (9.25 g,
122 mmol) and sodium ethoxide (16.5 g, 243 mmol). After refluxing
for 1.5 h, the mixture was acidified until reaching pH 2 by 3 M
hydrochloric acid, and then a precipitate was collected by filtration
to give a crude thiourea 18a. This crude was dissolved in methanol
(20 mL) and nickel (II) chloride hexahydrate (1.35 g, 5.67 mmol) and
sodium borohydride (643 mg, 17.0 mmol) were added to the solu-
tion. After stirring for 3 h at room temperature, the mixture was
poured into saturate ammonium chloride solution and extracted
with chloroform three times. The organic layer was dried over
anhydrous sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel eluted by chloroformemethanol (19:1)
to give 19a (209 mg, 1.38 mmol, 17% (2 steps)).
3.1.3. 6,7-Dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one (16a)
To a solution of ethyl 2-oxo-1-cyclopentanecarboxylate (14a)
(1.00 g, 6.40 mmol) in toluene (15 mL) were added 1,1,1-trimethyl-
N-(triphenylphosphoranylidene)silanamine (2.24 g, 6.40 mmol), 2-
propanol (0.49 mL, 6.40 mmol) and p-toluenesulfonic acid mono-
hydrate (12.2 mg, 0.064 mmol) [24]. After refluxing for 10 h, the
mixture was poured into saturated sodium bicarbonate solution
and extracted with ethyl acetate three times. The organic layer was
washed with brine, dried over anhydrous sodium sulfate, and
Table 1
In a similar procedure, 5,6-dihexylpyrimidin-4(3H)-one (19b)
(yield: 22%) was prepared from 17b.
In vitro antimalarial activities of synthesized febrifugine derivatives against
P. falciparum.
Compound
Antimalarial activity
FCR-3a K1b
EC50
Cytotoxicityc
Selectivityd
3.1.5. (3R,4S)-N,N-Dibenzyl-1-(tert-butyldimethylsilyloxy)-4-
(methoxymethoxy)hept-6-en-3-amine (23)
(m
g/mL)
EC50 (mg/mL)
To a solution of oxalyl chloride (1.15 mL, 13.3 mmol) in
dichloromethane (40 mL) was added DMSO (1.88 mL, 26.5 mmol)
and 22 [19] (3.53 g, 8.84 mmol) dissolved in dichloromethane
(5.0 mL) at ꢁ78 ꢀC. After stirring for 1 h, triethylamine (4.47 mL,
44.2 mmol) was slowly added to this mixture, and stirred at 0 ꢀC.
The mixture was poured into 1 M hydrochloric acid and extracted
with ethyl acetate three times. The organic layer was washed with
saturated sodium bicarbonate solution and brine, dried over
anhydrous sodium sulfate, and evaporated to give the crude of (2R)-
4-(tert-butyldimethylsilyloxy)-2-(dibenzylamino)butanal.
The crude aldehyde was dissolved in THF (20 mL). 2 M allyl-
magnesium bromide in THF (8.84 mL, 17.7 mmol) was added to this
solution at ꢁ78 ꢀC. After stirring for 2 h, the mixture was allowed to
warm to 0 ꢀC, and poured into saturated ammonium chloride so-
lution. This mixture was extracted with ethyl acetate three times.
The organic layer was washed with saturated sodium bicarbonate
solution and brine, dried over anhydrous sodium sulfate, and
evaporated. The residue was chromatographed over silica gel
Febrifugine (1)
0.00142
0.0225
0.0475
0.0179
0.0747
0.0790
0.0411
0.197
0.00140
0.0236
0.375
0.0117
0.0911
0.0857
0.0452
0.243
>1
0.138
0.0475
>1
0.187
>1
0.725
0.167
4.45
18.6
>100
>100
73.8
58.4
72.3
N.T.e
29.4
6.3
118
198
392
>5590
1338
934
1421
367
e
33[19]
Chloroquine
Artemisinin
4
5
6
7
8
9
10
20
21
34
35
>1
0.0937
0.0422
0.611
0.123
>1
0.545
314
149
e
N.T.e
N.T.e
N.T.e
N.T.e
e
e
e
a
Against P. falciparum FCR-3 (chloroquine sensitive strain).
Against P. falciparum K1 (chloroquine resistant strain).
Against mouse L929 cells.
b
c
d
e
Selectivity ¼ EC50 for L929 cells/EC50 for P. falciparum FCR-3.
Not tested.

H. Kikuchi et al. / European Journal of Medicinal Chemistry 76 (2014) 10e19
15
9.68 mmol) and potassium iodide (771 mg, 4.64 mmol) at room
temperature. After refluxing for 4 h, the mixture was allowed to
room temperature, poured into saturated ammonium chloride so-
lution, and extracted with ethyl acetate three times. The organic
layer was washed with saturated sodium bicarbonate solution and
brine, dried over anhydrous sodium sulfate, and evaporated. The
residue was chromatographed over silica gel eluted by hexanee
ethyl acetate (39:1) to give 23 (1.52 g, 3.14 mmol, 81%).
3.1.6. (3S,4R)-6-(tert-Butyldimethylsilyloxy)-4-(dibenzylamino)-3-
(methoxymethoxy)-hexanoic acid (25)
To a solution of 23 (1.52 g, 3.14 mmol) in wateretert-butanole
THF (1:1:2) (8.0 mL) were added 4-methylmorpholine N-oxide
(920 mg, 7.85 mmol) and osmium tetroxide (2% solution in water)
(1.15 mL, 0.094 mmol) at 0 ꢀC. After being stirred for 2 h at room
temperature, the reaction mixture was poured into 10% sodium
sulfite solution, and extracted with ethyl acetate three times. The
organic layer was washed with brine, dried over sodium sulfate,
and evaporated. The residue was chromatographed over silica gel
eluted by hexaneeethyl acetate (1:1) to give a diastereomeric
mixture of a corresponding diol (1.54 g, 2.98 mmol, 95%).
Sodium periodate (1.09 g, 5.07 mmol) was added to a solution of
the diol (1.54 g, 2.98 mmol) in diethyl etherewater (5:1) (30 mL).
After vigorously stirring for 3 h at room temperature, the mixture
was poured into water and extracted with ethyl acetate three times.
The organic layer was washed with brine, dried over sodium sul-
fate, and evaporated to give a crude aldehyde.
The crude aldehyde was dissolved in tert-butanolewater (5:1)
(8.0 mL). 2-Methylbut-2-ene (1.26 mL, 11.8 mmol), sodium dihy-
drogen phosphate dihydrate (688 mg, 4.41 mol) and sodium chlo-
rite (80%) (365 mg, 3.23 mmol) were added to this solution at 0 ꢀC.
After stirring for 30 min, the mixture was poured into 0.2 M hy-
drochloric acid and extracted with ethyl acetate three times. The
organic layer was washed with brine, dried over anhydrous sodium
sulfate, and evaporated. The residue was chromatographed over
silica gel eluted by hexaneeethyl acetate (2:1) to give 25 (1.17 g,
2.32 mmol, 74% (2 steps)).
3.1.7. (4S,5R)-5-[2-(tert-Butyldimethylsilyloxy)ethyl]-4-
(methoxymethoxy)pyrrolidin-2-one (26)
Compound 26 (1.57 g, 3.13 mmol) and 20% palladium hydroxide
on carbon (523 mg) in methanol (30 mL) was stirred at room
temperature for 1 h under hydrogen atmosphere. After filtration
through a Celite pad, the filtrate was evaporated to give oily residue.
To a solution of this residue in THFedichloromethane (1:1) (30 mL)
were added triethylamine (0.65 mL, 4.70 mmol), 4-(dimethyla-
mino)pyridine (38.2 mg, 0.313 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (900 mg,
4.70 mmol) at 0 ꢀC. After stirring for 4 h, the mixture was poured
into 0.2 M hydrochloric acid and extracted with ethyl acetate three
times. The organic layer was washed with saturated sodium bi-
carbonate solution and brine, dried over anhydrous sodium sulfate,
and evaporated. The residue was chromatographed over silica gel
eluted by chloroformemethanol (49:1) to give 26 (1.17 g,
2.32 mmol, 74% (2 steps)).
Scheme 4. Synthesis of compounds 34 and 35.a
3.1.8. (2R,3S)-tert-Butyl 2-(2-hydroxyethyl)-3-(methoxymethoxy)
pyrrolidine-1-carboxylate (27)
eluted by hexaneeethyl acetate (19:1) to give (4S,5R)-7-(tert-
butyldimethylsilyloxy)-5-(dibenzylamino)hept-1-en-4-ol (3.59 g,
8.16 mmol, 91% (2 steps)).
To a solution of 26 (769 mg, 2.54 mmol) in dichloromethane
(25 mL) were added triethylamine (1.76 mL, 12.7 mmol), 4-(dime-
thylamino)pyridine (1.55 g, 12.7 mmol) and di-tert-butyl dicar-
bonate (5.54 g, 25.4 mmol). After stirring for 2 h at room
temperature, the mixture was poured into 0.5 M hydrochloric acid
and extracted with ethyl acetate three times. The organic layer was
washed with saturated sodium bicarbonate solution and brine,
To
a
solution of (4S,5R)-7-(tert-butyldimethylsilyloxy)-5-
(dibenzylamino)hept-1-en-4-ol (1.70 g, 3.87 mmol) in 1,2-
dimethoxyethane (20 mL) were added N,N-diisopropylamine
(2.70 mL, 15.5 mmol), chloromethyl methyl ether (0.700 mL,

16
H. Kikuchi et al. / European Journal of Medicinal Chemistry 76 (2014) 10e19
dried over anhydrous sodium sulfate, and evaporated. The residue
was chromatographed over silica gel eluted by hexaneeethyl ace-
tate (3:1) to give (2R,3S)-tert-butyl 2-[2-(tert-butyldimethylsily-
loxy)ethyl]-3-(methoxymethoxy)-5-oxopyrrolidine-1-carboxylate
(665 mg, 1.65 mmol, 65%).
(1.0 mL) at room temperature. After stirring for 2 h, the solution was
evaporated. The residue was chromatographed over silica gel
eluted by chloroformemethanol (3:1) to give 20 (15 mg,
0.042 mmol, 97%) as dihydrochloride.
Data for 20: colorless amorphous solid; 1H NMR (400 MHz,
To a solution of (2R,3S)-tert-butyl 2-[2-(tert-butyldimethylsily-
loxy)ethyl]-3-(methoxymethoxy)-5-oxopyrrolidine-1-carboxylate
(582 mg, 1.44 mmol) in THF (7.0 mL) was added 2 M solution of
boraneedimethyl sulfide complex in THF (2.16 mL, 4.32 mmol) at
room temperature. After refluxing for 8 h, the mixture was poured
into saturated ammonium chloride solution and extracted with
ethyl acetate three times. The organic layer was washed with
saturated sodium bicarbonate solution and brine, dried over
anhydrous sodium sulfate, and evaporated to give a crude of
piperidine derivative.
This crude was dissolved in THF (2 mL), and 1 M solution of
tetra-n-butylammonium fluoride in THF (2.1 mL, 2.1 mmol) was
added at 0 ꢀC. After stirring for 2 h at room temperature, the
mixture was poured into water and extracted with ethyl acetate
three times. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel eluted by hexaneeethyl acetate (1:1) to
give 27 (283 mg, 1.03 mmol, 71% (2 steps)).
CD3OD)
d
9.20 (1H, s), 8.34 (1H, d, J ¼ 8.0 Hz), 8.04 (1H, t, J ¼ 8.0 Hz),
7.81 (1H, d, J ¼ 8.0 Hz), 7.78 (1H, d, J ¼ 8.0 Hz), 5.22e5.29 (2H, m),
4.25e4.31 (1H, m), 3.82e3.87 (1H, m), 3.41e3.48 (2H, m), 3.25e
3.30 (1H, m), 3.18 (1H, dd, J ¼ 19.2, 9.9 Hz), 2.20e2.30 (1H, m),1.97e
2.06 (1H, m); 13C NMR (100 MHz, CD3OD)
d 201.2, 160.0, 152.0,
141.6,137.7,129.7,128.7,123.2,121.6, 74.9, 62.7, 56.2, 44.6, 41.0, 32.8;
HRFABMS m/z 288.1369 [M þ H]þ (288.1348 calcd for C15H18N3O3);
Anal. (C15H21N3O4Cl2 (dihydrochloride hydrate)) C, H, N.
3.1.10. (3R,4S)-3-(Benzylamino)-4-(methoxymethoxy)hept-6-en-1-
ol (29)
To a solution of 23 (1.12 g, 2.31 mmol) in acetonitrilee
dichloromethane (5:1) (24 mL) was added ceric ammonium nitrate
(3.17 g, 5.78 mmol). After stirring for 30 min at room temperature,
the mixture was poured into saturated sodium bicarbonate solu-
tion and extracted with ethyl acetate three times. The organic layer
was washed with brine, dried over anhydrous sodium sulfate, and
evaporated. The residue was chromatographed over silica gel
eluted by chloroformemethanol (99:1) to give 29 (458 mg,
1.64 mmol, 71%).
3.1.9. 3-[3-((2R,3S)-3-Hydroxypyrrolidin-2-yl)-2-oxopropyl]
quinazolin-4(3H)-one (20)
To a solution of 27 (283 mg, 1.03 mmol) in CH2Cl2 (6.0 mL) were
added 15 wt% solution of DesseMartin periodinane in dichloro-
methane (4.37 mL, 1.55 mmol) and water (20
stirring for 1 h, the mixture was poured into 1 M sodium hydroxide
solution, and extracted with ethyl acetate three times. The organic
layer was washed with brine, dried over anhydrous sodium sulfate,
and evaporated to give the crude aldehyde.
3.1.11. (3R,4S)-3-[Allyl(benzyl)amino]-4-(methoxymethoxy)hept-6-
en-1-ol (30)
m
L) at 0 ꢀC. After
To a solution of 29 (1.70 g, 3.87 mmol) in dichloromethane
(4.0 mL) were added imidazole (266 mg, 3.91 mmol) and tert-
butyldimethylchlorosilane (589 mg, 3.91 mmol). After stirring for
2.5 h at room temperature, the mixture was poured into saturated
ammonium chloride solution and extracted with ethyl acetate
three times. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel eluted by hexaneeethyl acetate (4:1) to
give a TBS ether of 29 (1.23 g, 2.83 mmol, 73%).
To a solution of a TBS ether of 29 (377 mg, 0.870 mmol) in DMF
(8.0 mL) were added sodium hydride (60% mineral oil suspension)
(104 mg, 2.61 mmol) and ally bromide (0.23 mL, 2.61 mmol). After
stirring for 4 h at 100 ꢀC, the mixture was allowed to cool to room
temperature, poured into saturated ammonium chloride solution
and extracted with dichloromethane three times. The organic
layer was washed with brine, dried over anhydrous sodium sul-
fate, and evaporated. The residue was dissolved in THF (2.0 mL),
and 1 M solution of tetra-n-butylammonium fluoride in THF
(1.0 mL, 1.0 mmol) was added at 0 ꢀC. After stirring for 2.5 h at
room temperature, the mixture was poured into water and
extracted with ethyl acetate three times. The organic layer was
washed with brine, dried over anhydrous sodium sulfate, and
evaporated. The residue was chromatographed over silica gel
eluted by hexaneeethyl acetate (3:1) to give 30 (172 mg,
0.538 mmol, 59% (2 steps)).
To a solution of sodium hydride (60% mineral oil suspension)
(12.7 mg, 0.318 mmol) in DMSOeTHF (1:1) (4.0 mL) were added
trimethylsulfonium iodide (64.9 mg, 0.318 mmol) and the crude
aldehyde at ꢁ15 ꢀC. After stirring for 30 min at room temperature,
the mixture was poured into water and extracted with ethyl acetate
three times. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel eluted by hexaneeEtOAc (2:1) to give a
diastereomeric mixture of a corresponding epoxide 28 (108 mg,
0.38 mmol, 36% (2 steps)).
To a solution of 4-hydroxyquinazoline (66.2 mg, 0.453 mmol) in
DMSO (1.0 mL) was added potassium hydride (30% mineral oil
suspension) (60 mg, 0.453 mmol) at 0 ꢀC. After 30 min, compound
28 (59.2 mg, 0.206 mmol) dissolved in DMSO (1.5 mL) was added
into the solution. After stirring for 4 h at 80 ꢀC, the mixture was
poured into saturated ammonium chloride solution and extracted
with ethyl acetate three times. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, and evaporated. The
residue was dissolved in dichloromethane (3.0 mL). 15 wt% solution
of DesseMartin periodinane in dichloromethane (0.68 mL,
0.240 mmol) was added to this solution at 0 ꢀC. After stirring for
1.5 h, the mixture was poured into 1 M sodium hydroxide solution
and extracted with ethyl acetate three times. The organic layer was
washed with brine, dried over anhydrous sodium sulfate, and
evaporated. The residue was chromatographed over silica gel
eluted by chloroformemethanol (49:1) to give 3-[3-[(2R,3S)-N-
(tert-butyloxycarbonyl)-3-(methoxymethoxy)pyrrolidin-2-yl]-2-
oxopropyl]quinazolin-4(3H)-one (27 mg, 0.072 mmol, 35% (2
steps)).
3.1.12. 2-[(2R,3S)-1-Benzyl-3-(methoxymethoxy)-2,3,4,7-
tetrahydro-1H-azepin-2-yl]ethanol (31)
Under argon atmosphere, Grubbs catalyst 1st generation
(84 mg, 0.102 mmol) was added to a solution of 30 (164 mg,
0.512 mmol) in chloroform (10 mL). After stirring for 2.5 h at 60 ꢀC,
the mixture was filtered through a Celite pad, and the filtrate was
evaporated. The residue was chromatographed over silica gel
eluted by hexaneeethyl acetate (1:1) to give 31 (109 mg,
0.374 mmol, 73%).
3-[3-[(2R,3S)-N-(tert-Butyloxycarbonyl)-3-(methoxymethoxy)
pyrrolidin-2-yl]-2-oxopropyl]quinazolin-4(3H)-one
(19
mg,
0.043 mmol) was dissolved in 10% hydrogen chloride in methanol

H. Kikuchi et al. / European Journal of Medicinal Chemistry 76 (2014) 10e19
17
3.1.13. (2R,3S)-tert-Butyl 2-(2-hydroxyethyl)-3-(methoxymethoxy)
azepane-1-carboxylate (32)
give a diastereomeric mixture of 38 (371 mg, 0.821 mmol, 80% (2
steps)).
To a solution of 31 (98 mg, 0.337 mmol) in methanol (3.5 mL)
was added 20% palladium hydroxide on carbon (33 mg), This
mixture was stirred at room temperature for 1 h under hydrogen
atmosphere. After filtration through a Celite pad, the filtrate was
evaporated to give oily residue. To a solution of this residue in
methanol (2.0 mL) were added triethylamine (0.12 mL,
0.843 mmol) and di-tert-butyl dicarbonate (221 mg, 1.01 mmol).
After stirring for 4 h at room temperature, the mixture was
poured into saturated ammonium chloride solution and extrac-
ted with ethyl acetate three times. The organic layer was washed
with saturated sodium bicarbonate solution and brine, dried over
anhydrous sodium sulfate, and evaporated. The residue was
chromatographed over silica gel eluted by hexaneeethyl acetate
(1:1) to give 32 (62 mg, 0.206 mmol, 61% (2 steps)).
Sodium periodate (230 mg, 1.07 mmol) was added to a solution
of 38 (244 mg, 0.538 mmol) in diethyl etherewater (2:1) (6 mL).
After vigorously stirring for 1 h at room temperature, the mixture
was poured into water and extracted with ethyl acetate three times.
The organic layer was washed with brine, dried over sodium sul-
fate, and evaporated to give a crude aldehyde.
The crude aldehyde was dissolved in methanol (3.0 mL), and
sodium borohydride (292 mg, 7.72 mmol) was added to the solu-
tion at 0 ꢀC. After stirring for 3 h, the mixture was poured into
saturated ammonium chloride solution and extracted with ethyl
acetate three times. The organic layer was washed with brine, dried
over sodium sulfate, and evaporated. The residue was chromato-
graphed over silica gel eluted by ethyl acetate to give 39 (166 mg,
0.394 mmol, 73% (2 steps)).
3.1.14. 3-[((3aR,8aS)-2-Hydroxyoctahydro-2H-furo[3,2-b]azepin-2-
yl)methyl]quinazolin-4(3H)-one (21)
3.1.17. (2R,3S)-tert-Butyl 2-(3-hydroxypropyl)-3-
(methoxymethoxy)pyrrolidine-1-carboxylate (40)
In the same manner as the synthesis of 20, compound 21
(4.1 mg, 0.011 mmol, 12% (5 steps)) was synthesized as dihy-
drochloride from 32 (27 mg, 0.088 mmol) by five successive
reactions.
To a solution of 39 (177 mg, 0.420 mmol) in pyridine (2.0 mL)
was added p-toluenesulfonyl chloride (120 mg, 0.630 mmol). After
stirring for 2.5 h at room temperature, the mixture was poured into
0.3 M hydrochloric acid and extracted with ethyl acetate three
times. The organic layer was washed with saturated sodium bi-
carbonate solution and brine, dried over sodium sulfate, and
evaporated to give a crude of tosylate.
The crude of tosylate was dissolved in THF (1.0 mL), and 1 M
solution of tetra-n-butylammonium fluoride in THF (0.63 mL,
0.63 mmol) was added at 0 ꢀC. After stirring for 1 h at room tem-
perature, the mixture was poured into water and extracted with
ethyl acetate three times. The organic layer was washed with brine,
dried over anhydrous sodium sulfate, and evaporated.
Data for 21: colorless amorphous solid; 1H NMR (400 MHz,
CD3OD)
d
8.94 (1H, s), 8.34 (1H, d, J ¼ 8.0 Hz), 7.98 (1H, t, J ¼ 7.4 Hz),
7.67e7.83 (2H, m), 4.66 (1H, d, J ¼ 14.4 Hz), 4.34 (1H, d, J ¼ 14.4 Hz),
4.11e4.23 (1H, m), 3.84e3.95 (1H, m), 3.12e3.31 (2H, m), 2.18e2.31
(2H, m), 1.80e1.99 (3H, m), 1.54e1.70 (3H, m); 13C NMR (100 MHz,
CD3OD)
d 161.2, 151.8, 143.1, 137.2, 130.2, 128.7, 124.2, 122.1, 107.8,
81.8, 60.0, 41.1, 38.9, 34.4, 33.2, 30.8, 24.2; HRFABMS m/z 316.1671
[M þ H]þ (316.1661 calcd for C17H22N3O3). Anal. (C17H25N3O4Cl2
(dihydrochloride hydrate)) C, H, N.
The residue was dissolved in THF (2.0 mL), and sodium
hydride (60% mineral oil suspension) (25 mg, 0.631 mmol) was
added at 0 ꢀC. After stirring for 3 h, the mixture was poured
into saturated ammonium chloride solution and extracted with
ethyl acetate three times. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, and evaporated.
The residue was chromatographed over silica gel eluted by
hexaneeethyl acetate (1:1) to give 40 (66 mg, 0.227 mmol, 54%
(3 steps)).
3.1.15. (4R,5S)-N,N-Dibenzyl-1-(tert-butyldimethylsilyloxy)-5-
(methoxymethoxy)oct-7-en-4-amine (37)
In the same manner as the synthesis of 23, compound 37 (3.43 g,
6.89 mmol, 65% (3 steps)) was synthesized from 36 [19] (4.39 g,
10.6 mmol) by three successive reactions.
3.1.16. (3R,4S)-7-(tert-Butyldimethylsilyloxy)-4-(tert-
butyloxycarbonylamino)-3-(methoxy-methoxy)heptan-1-ol (39)
To a solution of 37 (722 mg, 1.45 mmol) in wateretert-butanole
THF (1:1:2) (6.0 mL) were added 4-methylmorpholine N-oxide
(323 mg, 2.90 mmol) and osmium tetroxide (2% solution in water)
(0.46 mL, 0.036 mmol) at 0 ꢀC. After being stirred for 4 h at room
temperature, the reaction mixture was poured into 10% sodium
sulfite solution, and extracted with ethyl acetate three times. The
organic layer was washed with brine, dried over sodium sulfate,
and evaporated. The residue was chromatographed over silica gel
eluted by hexaneeethyl acetate (1:1) to give a diastereomeric
mixture of a corresponding diol (676 mg, 2.98 mmol, 88%).
To a solution of the diol (545 mg, 1.03 mmol) in methanol
(5.0 mL) was added 20% palladium hydroxide on carbon (142 mg),
This mixture was stirred at room temperature for 1 h under
hydrogen atmosphere. After filtration through a Celite pad, the
filtrate was evaporated to give oily residue. To a solution of this
residue in methanol (3.0 mL) were added triethylamine (0.40 mL,
2.86 mmol) and di-tert-butyl dicarbonate (421 mg, 1.93 mmol).
After stirring for 2 h at room temperature, the mixture was poured
into saturated ammonium chloride solution and extracted with
ethyl acetate three times. The organic layer was washed with
saturated sodium bicarbonate solution and brine, dried over
anhydrous sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel eluted by hexaneeethyl acetate (2:1) to
3.1.18. 3-[3-((2R,3S)-3-Hydroxypyrrolidin-2-yl)propyl]quinazolin-
4(3H)-one (34)
To a solution of 40 (52 mg, 0.181 mmol) in pyridine (1.0 mL) was
added p-toluenesulfonyl chloride (138 mg, 0.723 mmol). After
stirring for 4 h at room temperature, the mixture was poured into
0.3 M hydrochloric acid and extracted with ethyl acetate three
times. The organic layer was washed with saturated sodium bi-
carbonate solution and brine, dried over sodium sulfate, and
evaporated. The residue was chromatographed over silica gel
eluted by hexaneeethyl acetate (4:1) to give a tosylate of 40 (50 mg,
0.113 mmol, 64%).
To a solution of 4-hydroxyquinazoline (14 mg, 0.096 mmol) in
DMF (1.5 mL) were added potassium carbonate (27 mg,
0.193 mmol) and a tosylate of 40 (41 mg, 0.096 mmol). After stirring
for 5 h at 50 ꢀC, the mixture was poured into 0.3 M hydrochloric
acid and extracted with ethyl acetate three times. The organic layer
was washed with saturated sodium bicarbonate solution and brine,
dried over sodium sulfate, and evaporated. The organic layer was
washed with saturated sodium bicarbonate solution and brine,
dried over sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel eluted by chloroformemethanol (49:1)
to
give
3-[3-[(2R,3S)-1-(tert-Butyloxycarbonyl)-3-(methoxy

18
H. Kikuchi et al. / European Journal of Medicinal Chemistry 76 (2014) 10e19
methoxy)pyrrolidin-2-yl]propyl] quinazolin-4(3H)-one (19 mg,
0.050 mmol, 48%).
0.05% L-glutamine, 50 mg/mL hypoxanthine, and 25 mg/mL gen-
tamisin) supplemented with 10% human plasma at 37 ꢀC, under
93% N2, 4% CO2 and 3% O2. Antimalarial activity of the test
compound has been achieved by dose response curve using the
parasite lactate dehydrogenase (pLDH) assay [31]. Asynchronous
3-[3-[(2R,3S)-1-(tert-Butyloxycarbonyl)-3-(methoxymethoxy)
pyrrolidin-2-yl]propyl] quinazolin-4(3H)-one (11 mg, 0.026 mmol)
was dissolved in 10% hydrogen chloride in methanol (1.0 mL) at
room temperature. After stirring for 3 h, the solution was evapo-
rated. The residue was chromatographed over silica gel eluted by
chloroformemethanol (3:1) to give 34 (8.9 mg, 0.025 mmol, 97%) as
dihydrochloride.
parasites (2.0% hematocrit and 0.5% parasitemia) (90
seeded in a 96-well microplate, and a solution of test compound
dissolved in distilled water or 5% DMSO (10 L) was added. After
incubation at 37 ꢀC for 72 h, the parasite suspension (20
L) was
transferred to another plate containing Malstat reagent (100 L).
mL) was
m
m
Data for 34: colorless amorphous solid; 1H NMR (400 MHz,
m
CD3OD)
d
9.53 (1H, br. s), 8.40 (1H, dd, J ¼ 8.2, 1.2 Hz), 8.07 (1H, td,
The plate was further incubated for 15 min at room temperature,
and a 1:1 mixture of nitroblue tetrazolium and phenazine
J ¼ 8.2, 1.4 Hz), 7.79e7.86 (2H, m), 4.30 (2H, t, J ¼ 7.1 Hz), 4.23e4.28
(1H, m), 3.40e3.48 (3H, m), 2.21e2.31 (1H, m), 1.97e2.12 (3H, m),
ethosulfate (2 mg and 0.1 mg/mL, respectively) (20
mL) was
1.78e1.92 (2H, m); 13C NMR (100 MHz, CD3OD)
d
159.7, 152.3, 138.9,
added to each well. After incubation for 2 h at room temperature
in the dark condition, the blue formazan product was measured
at 650 nm. The EC50 value was estimated from a dose response
curve.
137.7, 130.0, 128.8, 121.5, 121.0, 74.8, 67.3, 44.3, 32.9, 28.3, 26.9, 18.4;
HRFABMS m/z 274.1569 [M þ H]þ (274.1556 calcd for C15H20N3O2);
Anal. (C15H23N3O3Cl2 (dihydrochloride hydrate)) C, H, N.
3.1.19. (4R,5S)-4-(Benzylamino)-5-(methoxymethoxy)oct-7-en-1-
ol (41)
3.3. Cytotoxicity test
In the same manner as the synthesis of 29, compound 41
(594 mg, 2.03 mmol, 82%) was synthesized from 37 (1.23 g,
2.47 mmol).
The cytotoxicity test of compounds was measured by the
colorimetric MTT assay [32]. Mouse L929 cell suspension in RPMI-
1640 with 10% FBS (90 mL) was added in 96-well microplates at
1.8 ꢂ 104 cells/well. Then, a solution of test compound dissolved in
3.1.20. (4R,5S)-4-[Allyl(benzyl)amino]-5-(methoxymethoxy)oct-7-
en-1-ol (42)
In the same manner as the synthesis of 30, compound 42
(320 mg, 0.960 mmol, 57% (3 steps)) was synthesized from 41
(494 mg, 1.68 mmol) by three successive reactions.
distilled water or 10% DMSO (10 mL) was added to each well. After
incubation for 48 h at 37 ꢀC under 5% CO2, 2.5 mg/mL MTT solution
(10 L) was added to each well. The plate was incubated further 4 h.
Then, the incubation medium was aspirated, and DMSO (100 L)
m
m
was added to solubilize the MTT formazan product. After mixing,
absorbances at 570 and 655 nm were measured. The EC50 value was
estimated from a dose response curve.
3.1.21. 3-[(2R,3S)-1-Benzyl-3-(methoxymethoxy)-2,3,4,7-
tetrahydro-1H-azepin-2-yl]propan-1-ol (43)
In the same manner as the synthesis of 31, compound 43
(164 mg, 0.439 mmol, 51%) was synthesized from 42 (287 mg,
0.860 mmol).
3.4. In vivo antimalarial assay
In vivo antimalarial activity was determined against rodent
malaria-derived P. berghei NK65 according to the 4-days suppres-
sive test [33]. Male ICR mice at weight 18e20 g were inoculated
with 106 parasitized red blood cells intravenously. Test compounds
were suspended in 5% gum arabic, and orally administered to the
mice two hours after the infection (Day 0). Test compounds were
successively administered to the mice once a day for 3 consecutive
days (Days 1e3). The day after the last treatment (Day 4), the blood
was obtained from the tail vein of the infected mice. The para-
sitemia was determined by thin blood films made from the blood.
The ED50 value was estimated from a dose response curve.
3.1.22. (2R,3S)-tert-Butyl 2-(3-hydroxypropyl)-3-
(methoxymethoxy)azepane-1-carboxylate (44)
In the same manner as the synthesis of 32, compound 44
(32 mg, 0.101 mmol, 50% (2 steps)) was synthesized from 43
(62 mg, 0.202 mmol) by two successive reactions.
3.1.23. 3-[3-((2R,3S)-3-Hydroxyazepan-2-yl)propyl]quinazolin-
4(3H)-one (35)
In the same manner as the synthesis of 34, compound 35
(5.5 mg, 0.015 mmol, 45% (3 steps)) was synthesized as dihydro-
chloride from 44 (10 mg, 0.033 mmol) by three successive
reactions.
Acknowledgment
Data for 35: colorless amorphous solid; 1H NMR (400 MHz,
This work was supported in part by the SUNBOR GRANT from
the Suntory Institute for Bioorganic Research, and Kobayashi In-
ternational Scholarship Foundation.
CD3OD)
d
9.24 (1H, br. s), 8.35 (1H, dd, J ¼ 8.1, 1.1 Hz), 8.07 (1H,
ddd, J ¼ 8.3, 7.4, 1.5 Hz), 7.71e7.79 (2H, m), 4.23 (2H, t, J ¼ 7.2 Hz),
3.90e3.97 (1H, m), 3.19e3.38 (3H, m), 1.78e2.12 (9H, m), 1.59e
1.70 (1H, m); 13C NMR (100 MHz, CD3OD)
d 161.0, 151.1, 142.9,
137.1, 130.1, 128.4, 123.8, 122.1, 70.6, 64.4, 49.5, 46.2, 33.5, 28.2,
26.7, 26.3, 21.0; HRFABMS m/z 302.1869 [M þ H]þ (302.1869 calcd
for C17H24N3O2); Anal. (C17H17N3O3Cl2 (dihydrochloride hydrate))
C, H, N.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.036.
3.2. In vitro antimalarial assay
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