Exploration of a new type of antimalarial compounds based on febrifugine

Article abstract of DOI:10.1021/jm0601809

Febrifugine (1), a quinazoline alkaloid, isolated from Dichroa febrifuga roots, shows powerful antimalarial activity against Plasmodium falciparum. The use of 1 as an antimalarial drug has been precluded because of side effects, such as diarrhea, vomiting

Full text of DOI:10.1021/jm0601809

4698
J. Med. Chem. 2006, 49, 4698-4706
Exploration of a New Type of Antimalarial Compounds Based on Febrifugine
Haruhisa Kikuchi,† Keisuke Yamamoto,† Seiko Horoiwa,† Shingo Hirai,† Ryota Kasahara,† Norimitsu Hariguchi,‡
Makoto Matsumoto,‡ and Yoshiteru Oshima*,†
Graduate School of Pharmaceutical Sciences, Tohoku UniVersity, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan, and
Microbiological Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan
ReceiVed February 16, 2006
Febrifugine (1), a quinazoline alkaloid, isolated from Dichroa febrifuga roots, shows powerful antimalarial
activity against Plasmodium falciparum. The use of 1 as an antimalarial drug has been precluded because
of side effects, such as diarrhea, vomiting, and liver toxicity. However, the potent antimalarial activity of
1 has stimulated medicinal chemists to pursue compounds derived from 1, which may be valuable leads for
novel drugs. In this study, we synthesized a new series of febrifugine derivatives formed by structural
modifications at (i) the quinazoline ring, (ii) the linker, or (iii) the piperidine ring. Then, we evaluated their
antimalarial activities. Thienopyrimidine analogue 15 exhibited a potent antimalarial activity and a high
therapeutic selectivity both in vitro and in vivo, suggesting that 15 is a good antimalarial candidate.
Introduction
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 300-500 million patients
worldwide and leads to more than 2 million deaths each year.
Although malaria has been widely eradicated in many parts of
Figure 1. The three parts of febrifugine (1).
the world, the number of patients continues to rise mainly due
to 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
Chart 1. Structures of Febrifugine (1) and Isofebrifugine (2)
The roots of Dichroa febrifuga, a plant that belongs to the
Saxifragaceae family, have been used as a traditional antima-
larial drug in China. The quinazoline-type alkaloids, febrifugine
1 and its stereoisomer isofebrifugine 2, have been identified as
the active components of these roots (Chart 1).2 Although 1
and 2 show powerful in vitro antimalarial activity against both
chloroquine-sensitive P. falciparum FCR-3 and chloroquine-
resistant P. falciparum K1, the in vivo activity of 1 against
mouse malaria, P. berghei, is approximately 200 times more
potent than that of 2. Because of side effects, such as diarrhea,
vomiting,3 and liver toxicity,4 1 has been precluded as an
antimalarial drug. However, the potent antimalarial activity of
1 has stimulated medicinal chemists to pursue derivatives of 1,
which may provide valuable leads for novel drugs. We have
tried to create active febrifugine analogues.5-7 However, the
role of the structural components of 1 in its antimalarial activity
has yet to be elucidated.
8 are based on febrifugine metabolites feb-A and -B,5 respec-
tively, which prevent metabolic oxidation. In compounds 9 and
14-16, the quinazoline ring of 1 is replaced by purine,
benzotriazine, thienopyrimidine, and quinoline rings, respec-
tively. The antimalarial activity and cytotoxicity of the racemic
analogues of 1 are due to the natural enantiomers because it
was reported that both activities of the synthesized unnatural
enantiomer of 1 were approximately 1/2000 and 1/100 of those
of natural 1, respectively.8 Therefore, we synthesized 7-9 and
14-16 as racemic compounds.
Racemic 7-9 were synthesized by employing the synthetic
strategy for 1 developed by Takeuchi et al.9 The starting
material, 3-hydroxypyridine, was used to produce hemiacetal 4
via compound 3. A coupling reaction of 4 with the aromatic
component of 7 provided compound 5a, which was hydrogeno-
lized to give 6a. As in the isomerization of isofebrifugine (2)
into febrifugine (1), the boiling of the MeOH solution of 6a
isomerized 6a into 7.9b,10 Analogues 8 and 9 were synthesized
in a similar manner using the corresponding heterocyclic
component.
Therefore, we decided to synthesize febrifugine derivatives
formed by structural modifications at (i) the quinazoline ring,
(ii) the linker, or (iii) the piperidine ring (Figure 1). In this
article, we report the syntheses of these febrifugine derivatives
and evaluate their in vitro and in vivo antimalarial activities.
However, the syntheses of 14-16 were carried out in a
different manner from those of 7-9 because the coupling
reaction or catalytic hydrogenation was unsuccessful. Compound
3 was converted into Boc-derivative 10 in two steps. After the
MOM-etherification of 10, the epoxidation by 3-chloroperben-
zoic acid afforded 11. A coupling reaction with the aromatic
component of 14 and subsequent oxidation gave 12a. The acidic
deprotection of 12a and refluxing the residue in MeOH gave a
mixture of 13a and 14. The isomerization of 13a into 14 also
Results and Discussion
Syntheses of Febrifugine Analogues. (i) Quinazoline Ring.
We synthesized quinazoline ring-modified derivatives 7-9 and
14-16 (Scheme 1). 6-Fluoro analogue 7 and 2-methyl analogue
* To whom correspondence should be addressed. Phone: +81-22-795-
6822. Fax: +81-22-795-6821. E-mail: oshima@mail.pharm.tohoku.ac.jp.
† Tohoku University.
‡ Otsuka Pharmaceutical Co., Ltd.
10.1021/jm0601809 CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/06/2006

Antimalarial Compounds Based on Febrifugine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4699
Scheme 1. Synthesis of 7-9 and 14-16a
a Reagents and conditions: (a) corresponding aromatic compound, K2CO3, DMF, rt; (b) H2 (1 atm), Pd(OH)2/C, MeOH, rt (65% (6a), 80% (6b) and 78%
(6c) (2 steps)); (c) MeOH, reflux; (d) Boc2O, iPr2EtN, CH2Cl2, rt; (e) 10% HCl-MeOH, rt (48% (7), 42% (8) and 40% (9) (3 steps)); (f) 6 M HCl, reflux;
(g) Boc2O, 5 M NaOH, rt (61% (2 steps)); (h) MOMCl, iPr2EtN, CH2Cl2, rt; (i) mCPBA, CH2Cl2, NaHCO3, 0 °C (44% (2 steps)); (j) corresponding aromatic
compound, KH, DMF, rt; (k) Dess-Martin periodinane, CH2Cl2, rt (71% (12a), 31% (12b) and 39% (12c) (2 steps)); (l) 10% HCl-MeOH, rt (97% (13a),
98% (13b) and 95% (13c)); (m) MeOH, reflux; (n) Boc2O, iPr2EtN, CH2Cl2, rt; (o) 10% HCl-MeOH, rt (41% (10), 28% (11) and 30% (12) (3 steps)).
Scheme 2. Synthesis of the Piperidine and Linker Parts of 28-30a
a Reagents and conditions: (a) BnBr, K2CO3, NaOH, MeOH-H2O (1:1), reflux; (b) LiAlH4, THF, 0 °C (34% (18a) and 62% (18b) (2 steps)); (c)
TBDMSCl, imidazole, CH2Cl2, 0 °C (42% (19a) and 56% (19b)); (d) (COCl)2, DMSO, Et3N, CH2Cl2, -78 °C; (e) (1,3-dioxan-2-ylethyl)magnesium bromide,
THF, -78 °C (56% (20a) and 59% (20b) (2 steps)); (f) 2M HCl, acetone, rt; (g) H2 (1 atm), Pd(OH)2/C, MeOH, rt; (h) Boc2O, Et3N, MeOH, rt (66% (22a)
and 33% (22b) (3 steps)); (i) TBDMSCl, imidazole CH2Cl2, 0 °C (82% (23a) and 55% (23b)); (j) MOMCl, iPr2EtN, DME, 50 °C; (k) TBAF, THF, 0 °C
(83% (24a) and 93% (24b) (2 steps)); (l) Dess-Martin periodinane, CH2Cl2, 0 °C; (m) triethyl phosphonoacetate, NaH, toluene, 0 °C (76% (2 steps)); (n)
H2 (1 atm), Pd(OH)2/C, THF, rt; (o) DIBAL-H, toluene, -78 °C (84% (2 steps)).
occurred. Analogues 15 and 16 were synthesized in a similar
manner from 3.
the piperidine and linker parts of 29. In Scheme 2, tosylation
of 24b afforded 26b, which was coupled with 4-hydroxy-
quinazoline to produce 27b. Finally, compound 27b was
deprotected under acidic conditions, which resulted in a decar-
bonylated febrifugine 29 as a hydrochloride.
In the same manner, analogues 28 and 30, which contain
shorter and longer linkers, respectively, were synthesized.
D-Aspartic acid (17a) was used as the starting material to
generate 24a (Scheme 2). The oxidation of 24a followed by
the Horner-Emmons reaction with triethyl phosphonoacetate
yielded 25. After the catalytic hydrogenation of 25, the reduction
by diisobutylaluminum hydride gave 24c. Compounds 24a and
24c were converted into 28 and 30, respectively (Scheme 3).
We synthesized compound 33, which has an amide bond for
the linkage between the quinazoline ring and the linker (Scheme
4). The successive oxidation of 24b by pyridinium chlorochro-
mate and sodium hypochlorite gave carboxylic acid 31. A
(ii) Linker Part. To evaluate the role of the C-2′ carbonyl
group in antimalarial activity, we synthesized chiral compound
29 (Scheme 3), which is a decarbonylated derivative of
febrifugine 1. The piperidine and linker parts of 29 were
synthesized by using N,N-dibenzylamino aldehyde11 as a chiral
building block (Scheme 2). D-Glutamic acid (17b) was converted
into amino alcohol 18b by a reaction with benzyl bromide and
a reduction using lithium aluminum hydride. Compound 18b
was reacted with tert-butyldimethylsilyl chloride to yield 19b.12
The Swern oxidation of 19b gave N,N-dibenzylamino aldehyde,
which was then reacted with (1,3-dioxan-2-ylethyl)magnesium
bromide13,14 to diastereoselectively give 20b. The hydrolysis
of the acetal 20b gave hemiacetal 21b. Catalytic hydrogenation
followed by N-protection afforded 22b. Three successive
reactions of 22b gave mono-MOM ether 24b, which contained

4700 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Scheme 3. Synthesis of 28-30a
Kikuchi et al.
trifluoroacetate of 41. H and 13C NMR spectra showed that
1
compound 41 exists as a hemiacetal form.
The position of the linkage between the piperidine ring and
linker is changed in febrifugine analogues 46 and 50. To
synthesize 46 (Scheme 6), the hydroxy group of 3-hydroxypi-
peridine 42 was protected as TIPS ether by three sequential
reactions to give 43. The treatment of 43 with epibromohydrin
afforded 44, which was coupled with 4-hydroxyquinazoline to
yield 45. The oxidation of 45 and subsequent deprotection gave
racemic 46 as the hydrochloride.
To synthesize 50, the N-Boc derivative of 42 was treated with
2,2-dimethyl-1,3-dioxolan-4-ylmethyl tosylate to give 47. After
deprotection under acidic conditions and N-reprotection of 47,
the epoxidation by N-tosylimidazole afforded 48. Finally, three
successive reactions, a coupling with 4-hydroxyquinaoline,
Dess-Martin oxidation, and acidic deprotection, yielded racemic
50 as the hydrochloride (Scheme 7).
a Reagents and conditions: (a) pTsCl, pyridine, rt (69% (26a), 80% (26b)
and 78% (26c)); (b) 4-hydroxyquinazoline, K2CO3, DMF, 50 °C (30% (27a),
72% (27b) and 84% (27c)); (c) 10% HCl-MeOH, 50 °C (90% (28a), 88%
(28b) and 93%(28c)).
Scheme 4. Synthesis of 33a
Antimalarial Activity in Vitro of the Synthetic Analogues
of Febrifugine. The in vitro antimalarial activity of febrifugine
analogues 7-9, 14-16, 28-30, 33, 41, 46, and 50 against P.
falciparum and the cytotoxicity against mouse L929 cells were
evaluated (Table 1). Compounds 7-9, 14, and 15 displayed
antimalarial activity (EC50 <0.5 µg/mL). The antimalarial
activity of 6-fluoro analogue 7 and thienopyrimidine ring-
containing analogue 15 were equivalent to that of febrifugine
(1). These results suggest that a suitable method to create more
active derivatives is to modify the quinazoline ring of 1. On
the contrary, compound 16 did not display any activity,
indicating that the nitrogen atom at position 3 in the quinazoline
ring is crucial to activity. The antimalarial activity of decarbo-
nylated derivative 29 was 1/15 of that of 1. Therefore, the
carbonyl group at C-2" was effective but not critical to activity.
Compounds 28 and 30, which have two and four carbon linkers,
respectively, did not display activity, suggesting that a three
carbon linker is important for potent antimalarial activity. In
addition, compounds 33, 41, 46, and 50 did not exhibit activity.
The piperidine ring of febrifugine 1 should be maintained to
create useful derivatives.
Antimalarial Activity in Vivo of the Synthetic Analogues
of Febrifugine. The in vivo antimalarial activities against rodent
malaria P. berghei and the acute toxicity in mice was examined
for 7, 15, and 29, which were more potent in vitro than
chloroquine (Table 2). Because 7 was highly toxic in mice (LD50
<2.5 mg/kg), the in vivo antimalarial activity of 7 was not
evaluated. The antimalarial activity (ED50 2.95 mg/kg) of 15
was comparable to that of chloroquine (ED50 1.53 mg/kg), a
clinically used medicine. The ED50 value of 15 was also similar
to the reported value of artemisinin derivatives (ED50 3-10 mg/
kg).16 In addition, the toxicity of 15 was much weaker (LD50
a Reagents and conditions: (a) Dess-Martin periodinane, CH2Cl2, 0 °C;
(b) NaClO2, 2-methyl-2-butene, NaH2PO4, tBuOH, H2O, rt (83% (2 steps));
(c) 2-amino-4(3H)-quinazolinone, HATU, iPr2EtN, DMF, CH2Cl2, rt (38%);
(d) 10% HCl-MeOH, rt (45%).
coupling reaction of 31 with 2-amino-4(3H)-quinazolinone15
provided compound 32, which was deprotected under acidic
conditions to afford 33.
(iii) Piperidine Ring. Piperidine ring-opened analogue 41
was synthesized as shown in Scheme 5. The Swern oxidation
of amino alcohol 19a, which was prepared from D-aspartic acid
17a, and a subsequent reaction with ethylmagnesium bromide
diastereoselectively gave 34. Four successive reactions, O-
protection by MOM group, debenzylation, N-protection by a
Boc group, and deprotection of the TBDMS group, afforded
37. The Dess-Martin oxidation of 37 gave the aldehyde, which
was treated with trimethylsulfoxonium iodide and sodium
hydride to yield epoxide 38. After N-methylation of 38, a
coupling reaction with 4-hydroxyquinazoline and subsequent
oxidation gave 40. Deprotection of 40 under 10% hydrogen
chloride in MeOH, formed the unstable hydrochloride of 41.
The treatment of 40 with trifluoroacetic acid afforded the stable
Scheme 5. Synthesis of 41a
a Reagents and conditions: (a) (COCl)2, DMSO, Et3N, CH2Cl2, -78 °C; (b) EtMgBr, THF, -78 °C (66% (2 steps)); (c) MOMCl, iPr2EtN, CH2Cl2, rt
(79%); (d) H2 (1 atm), Pd(OH)2/C, MeOH, rt; (e) Boc2O, Et3N, MeOH, 0 °C (98% (2 steps)); (f) TBAF, THF, rt (67%); (g) Dess-Martin periodinane,
CH2Cl2, rt; (h) trimethylsulfoxonium iodide, NaH, DMSO, rt (75% (2 steps)); (i) MeI, NaH, DMF, 0 °C (91%); (j) 4-hydroxyquinazoline, NaH, DMF, 80
°C; (k) Dess-Martin periodinane, CH2Cl2, rt (41% (2 steps)); (l) TFA, CH2Cl2, rt (44%).

Antimalarial Compounds Based on Febrifugine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4701
Scheme 6. Synthesis of 46a
Table 2. Antimalarial Activities against P. berghei in Vivo and Acute
Toxicities of Synthesized Febrifugine Derivatives 7, 15, and 29a
antimalarial
activityb
acute
toxicityc
therapeutic
indexd
compound
ED50 (mg/kg)
LD50 (mg/kg)
febrifugine (1)
7
15
0.41
N.T.e
2.95
7.1
<2.5
88
17
30
29
22.5
1.53
>320
>14
chloroquine
N.T.e
a All compounds were administrated by po. b Against P. berghei (rodent
malaria). c Toxicity in mice. d Therapeutic index ) LD50 in mice/ED50 for
P. berghei in mice. e N.T., not tested.
a Reagents and conditions: (a) CbzCl, Et3N, CH2Cl2, 0 °C (96%); (b)
TIPSCl, imidazole, DMF, 0 °C; (c) H2 (1 atm), Pd(OH)2/C, MeOH, rt (71%
(2 steps)); (d) epibromohydrin, K2CO3, DMF, rt (71%); (e) 4-hydroxy-
quinazoline, NaH, DMF, 80 °C (68%); (f) (COCl)2, DMSO, Et3N, CH2Cl2,
-78 °C (72%); (g) 10% HCl-MeOH, 70 °C (80%).
as a metabolic analysis and the elucidation of the action
mechanism, are necessary to develop a novel antimalarial drug.
Table 1. Antimalarial Activities of Synthesized Febrifugine Derivatives
against P. falciparum in Vitro
Experimental Section
General Methods. Starting materials were either commercially
available or prepared as reported previously in the literature.
Analytical TLC was performed on silica gel 60 F254 (Merck) and
Aluminumoxid 150 F254 neutral (Type T, Merck). Column chro-
matography was carried out on silica gel 60 (70-230 mesh, Merck),
and aluminum oxide 150 basic (type T, Merck). NMR spectra were
recorded on JEOL JNM ECA-600, ECP-500, and AL-400. Mass
spectra were measured on JEOL JMS AX-500 and AX-700.
Elemental analyses were performed by Yanaco CHN Corder MT-
6.
6-Fluoro-3-[(3aS*,7aS*)-2-hydroxyoctahydrofuro[3,2-b]pyri-
din-2-ylmethyl]-3H-quinazolin-4-one (6a). To a solution of 49
(579 mg, 1.57 mmol) in DMF (6 mL) were added 6-fluoro-3H-
quinazolin-4-one17 (262 mg, 1.59 mmol) and K2CO3 (220 mg, 1.59
mmol) at room temperature. After stirring for 3 h, the mixture was
poured into brine and extracted three times with EtOAc. The organic
layer was washed with brine, dried over anhydrous Na2SO4, and
evaporated. The residue was chromatographed over silica gel eluted
with CHCl3-MeOH (49:1) to give crude 5a. 20% Pd(OH)2 on
carbon (150 mg) was added to the MeOH solution (12 mL) of this
crude product. After stirring for 1 h under a hydrogen atmosphere,
the mixture was filtered and concentrated. The residue was
chromatographed over silica gel eluted with CHCl3-MeOH (9:1)
to give 6a (314 mg, 65% (2 steps)). Using a similar procedure,
compounds 6b (yield: 80%) and 6c (78%) were prepared from
2-methyl-3H-quinazolin-4-one and 7-methylhypoxanthine, respec-
tively.
antimalarial activity
cytotoxicityc
FCR-3a
K1b
compound
EC50 (µg/mL)
EC50 (µg/mL) selectivityd
febrifugine (1)
7
8
9
14
15
16
28
0.00142
0.000730
0.116
0.506
0.154
0.00140
0.000916
0.190
0.167
0.0790
118
108
134
>198
106
15.6
>100
16.4
0.563
>100
>100
4.45
0.943
N.T.e
0.00306
0.00319
184
>1
>1
0.0225
>1
>1
0.0236
29
198
30
33
41
46
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>100
>100
6.44
<6.44
>100
50
>100
chloroquine
artemisinin
0.0475
0.0179
0.375
0.0117
18.6
392
>5590
>100
a Against P. falciparum FCR-3 (chloroquine-sensitive strain). b Against
P. falciparum K1 (chloroquine-resistant strain). c Against mouse L929 cells.
d Selectivity ) EC50 for L929 cells/EC50 for P. falciparum FCR-3. e N.T.,
not tested.
88 mg/kg) than that of 1, and the therapeutic index of 15 was
higher than that of 1. These results suggest that 15 may be a
good antimalarial candidate. Although no mice died after
receiving 320 mg/kg of 29 in the acute toxicity test, its
antimalarial activity was moderate (ED50 22.5 mg/kg).
In conclusion, we synthesized and evaluated a new series of
febrifugine derivatives. Among them, thienopyrimidine analogue
15 exhibited potent antimalarial activity and a high therapeutic
selectivity both in vitro and in vivo. Further studies on 15, such
6-Fluoro-3-[3-[(2R*,3S*)-3-hydroxypiperidin-2-yl]-2-oxopro-
pyl]-3H-quinazolin-4-one (7). Compound 6a (386 mg, 1.21 mmol)
was dissolved in MeOH (20 mL). After refluxing for 3 h, the
reaction mixture was concentrated. To a solution of this residue in
CH2Cl2 (12 mL) were added triethylamine (190 µL, 1.32 mmol)
and di-tert-butyl dicarbonate (526 mg, 2.41 mmol) at room
temperature. After stirring for 1.5 h, the mixture was poured into
Scheme 7. Synthesis of 50a
a Reagents and conditions: (a) Boc2O, Et3N, MeOH, rt (95%); (b) 2,2-dimethyl-1,3-dioxolan-4-ylmethyl p-toluenesulfonate, NaH, DMF, 80 °C (57%);
(c) 10% HCl-MeOH, 70 °C; (d) Boc2O, Et3N, CH2Cl2, 0 °C (80% (2 steps)); (e) N-tosylimidazole, NaH, THF, rt (32%); (f) 4-hydroxyquinazoline, NaH,
DMF, 80 °C; (g) Dess-Martin periodinane, CH2Cl2, rt (67% (2 steps)); (h) 10% HCl-MeOH, 70 °C (89%).

4702 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Kikuchi et al.
0.2 M HCl and extracted three times with EtOAc. The organic layer
was washed with saturated NaHCO3 solution and brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with EtOAc to give the Boc-derivative
of 7.
To a solution of the Boc-derivative was added 10% HCl-MeOH
(20 mL) at room temperature. After stirring for 2 h, the mixture
was evaporated. The residue was chromatographed over silica gel
eluted with CHCl3-MeOH (4:1) to give 7 (73.5 mg, 48% from
6a) as a dihydrochloride. Using a similar procedure, compounds 8
(yield: 42%) and 9 (40%) were prepared from 6b and 6c,
respectively.
tert-Butyl (2S*,3S*)-2-allyl-3-hydroxypiperidine-1-carboxylate
(10). Compound 3 (4.39 g, 16.0 mmol) was dissolved in 6 M HCl
(32 mL). After refluxing for 1.5 h. the solution was cooled to 0
°C. Then, 5 M NaOH (56 mL) and di-tert-butyl dicarbonate (6.96
g, 31.9 mmol) were added at room temperature. After stirring for
1.5 h, the mixture was poured into 0.1 M HCl, and extracted three
times with EtOAc. The organic layer was washed with saturated
NaHCO3 solution and brine, dried over anhydrous Na2SO4, and
evaporated. The residue was chromatographed over silica gel eluted
with hexanes-EtOAc (2:1) to give 10 (2.36 g, 61%).
a similar procedure, compounds 13b (yield: 98%) and 13c (95%)
were prepared from 12b and 12c, respectively.
3-[3-[(2R*,3S*)-3-Hydroxypiperidin-2-yl]-2-oxopropyl]-3H-
benzo[d][1,2,3]-triazin-4-one (14). Compound 6a (45.1 mg, 0.149
mmol) was dissolved in MeOH (2.0 mL). After refluxing for 3 h,
the reaction mixture was concentrated. To a solution of this residue
in CH2Cl2 (3.0 mL) were added triethylamine (30 µL, 0.215 mmol)
and di-tert-butyl dicarbonate (47.0 mg, 0.215 mmol) at room
temperature. After stirring for 1.5 h, the mixture was poured into
0.2 M HCl and extracted three times with EtOAc. The organic layer
was washed with saturated NaHCO3 solution and brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with EtOAc to give the Boc-derivative
of 14.
To a solution of the Boc-derivative was added 10% HCl-MeOH
(2.0 mL) at room temperature. After stirring for 2 h, the mixture
was evaporated. The residue was chromatographed over silica gel
eluted with CHCl3-MeOH (4:1) to give 14 (18.6 mg, 41% from
13a) as a dihydrochloride. Using a similar procedure, compounds
15 (yield: 28%) and 16 (30%) were prepared from 13b and 13c,
respectively.
(3S,4R)-6-(tert-Butyldimethylsilyloxy)-4-(dibenzylamino)-1-
([1,3]-dioxan-2-yl)-hexan-3-ol (20a). Compound 19a was prepared
from D-aspartic acid (17a) according to the method reported by
Gmeiner et al.12 To a solution of oxalyl chloride (2.56 mL, 29.5
mmol) in CH2Cl2 (100 mL) were added DMSO (4.18 mL, 58.9
mmol) and 19a (7.85 g, 19.6 mmol) dissolved in CH2Cl2 (20 mL)
at -78 °C. After stirring for 1 h, triethylamine (13.7 mL, 98.2
mmol) was added to this mixture and stirred at 0 °C. The mixture
was poured into 1 M HCl and extracted three times with Et2O.
The organic layer was washed with saturated NaHCO3 solution and
brine, dried over anhydrous Na2SO4, and evaporated to give the
crude aldehyde of (2R)-4-(tert-butyldimethylsilyloxy)-2-(dibenzyl-
amino)butanal. This crude aldehyde was dissolved in THF (35 mL).
Then, 0.5 M (1,3-dioxan-2-ylethyl)magnesium bromide in THF
(45.5 mL) was added to this solution at -50 °C. After stirring for
2 h, the mixture was allowed to warm up to room temperature and
poured into a saturated NH4Cl solution. This mixture was extracted
three times with Et2O. The organic layer was washed with saturated
NaHCO3 solution and brine, dried over anhydrous Na2SO4, and
evaporated. The residue was chromatographed over silica gel eluted
with hexanes-EtOAc (4:1) to give 20a (5.61 g, 56% (2 steps)).
Using a similar procedure, compound 20b (yield: 59%) was
prepared from 19b.
tert-Butyl (2R,3S)-3-hydroxy-2-(2-hydroxyethyl)piperidine-1-
carboxylate (22a). Compound 20a (5.61 g, 10.9 mmol) was
dissolved in 2 M HCl (25 mL) and acetone (25 mL) at room
temperature. After stirring for 4 h, the solution was poured into a
1 M NaOH solution and extracted three times with EtOAc. The
organic layer was washed with a saturated NaHCO3 solution and
brine, dried over anhydrous Na2SO4, and evaporated to give the
crude product of 21a.
This crude product and 20% Pd(OH)2 on carbon (760 mg) in
MeOH (50 mL) were stirred at room temperature for 1 h under
hydrogen atmosphere. After filtration, the filtrate was evaporated
to give an oily residue. The residue was dissolved in MeOH (30
mL). Triethylamine (2.96 mL, 21.3 mmol) and di-tert-butyl
dicarbonate (3.48 g, 16.0 mmol) were added to this solution at room
temperature. After stirring for 1 h, the mixture was poured into 0.2
M HCl and extracted three times with EtOAc. The organic layer
was washed with saturated NaHCO3 solution and brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with EtOAc to give 22a (1.03 g,
66% (3 steps)). Using a similar procedure, compound 22b (yield:
33%) was prepared from 20b.
tert-Butyl (2S*,3S*)-3-methoxymethoxy-2-oxiranylmethylpi-
peridine-1-carboxylate (11). To a solution of 10 (2.45 g, 10.1
mmol) in CH2Cl2 (40 mL) were added N,N-diisopropylethylamine
(7.06 mL, 40.6 mmol) and chloromethyl methyl ether (3.85 mL,
50.7 mmol) at room temperature. After stirring for 7 h, the mixture
was poured into 0.2 M HCl and extracted three times with EtOAc.
The organic layer was washed with saturated NaHCO3 solution and
brine, dried over anhydrous Na2SO4, and evaporated to give the
crude MOM ether.
To a solution of this crude product in CH2Cl2 (50 mL) and water
(50 mL) were added NaHCO3 (1.89 g, 22.5 mmol) and m-
chloroperbenzoic acid (75%) (3.45 g, 15.0 mmol) at 0 °C. After
stirring for 48 h, the mixture was poured into 10% sodium
thiosulfate solution and extracted three times with EtOAc. The
organic layer was washed with 0.5 M NaOH and brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with hexanes-EtOAc (4:1) to give
11 (1.14 g, 44%).
3-[3-[(2S*,3S*)-1-tert-Butyloxycarbonyl-3-methoxymethoxypi-
peridin-2-yl]-2-oxopropyl]-3H-benzo[d][1,2,3]triazin-4-one (12a).
To a solution of 3H-benzo[d][1,2,3]triazin-4-one (68.0 mg, 0.462
mmol) in DMF (4.0 mL) was added potassium hydride (30% oil
suspension) (62.0 mg, 0.462 mmol) at 0 °C. After 30 min,
compound 11 (116 mg, 0.385 mmol) dissolved in DMF (1.0 mL)
was added to the solution. After stirring for 24 h at 80 °C, the
mixture was poured into water and extracted three times with
EtOAc. The organic layer was washed with water and brine, dried
over anhydrous Na2SO4, and evaporated. The residue was chro-
matographed over silica gel eluted with CHCl3-MeOH (39:1) to
give a mixture of 3-[3-[(2S*,3S*)-1-tert-butyloxycarbonyl-3-meth-
oxymethoxypiperidin-2-yl]-2-hydroxypropyl]-3H-benzo[d][1,2,3]-
triazin-4-one and 3H-benzo[d][1,2,3]triazin-4-one.
The mixture was dissolved in CH2Cl2 (2.0 mL). Then, 15%
Dess-Martin periodinane solution in CH2Cl2 (1.50 mL, 0.535
mmol) was added to this solution at room temperature. After stirring
for 3 h, the mixture was poured into 1 M NaOH solution and
extracted three times with EtOAc. The organic layer was washed
with water and brine, dried over anhydrous Na2SO4, and evaporated.
The residue was chromatographed over silica gel eluted with
hexanes-EtOAc (2:1) to give 12a (122 mg, 71% (2 steps)). Using
a similar procedure, compounds 12b (yield: 31%) and 12c (39%)
were prepared from 3H-thieno[3,2-d]pyrimidin-4-one18 and 4-hy-
droxyquinoline, respectively.
3-[(3aS*,7aS*)-2-Hydroxyoctahydrofuro[3,2-b]pyridin-2-yl-
methyl]-3H-benzo-[d][1,2,3]triazin-4-one (13a). To a solution of
the 12a (92.1 mg, 0.206 mmol) was added 10% HCl-MeOH (3.0
mL) at room temperature. After stirring for 2 h, the mixture was
evaporated. The residue was chromatographed over aluminum oxide
eluted with CHCl3-MeOH (4:1) to give 13a (59.8 mg, 97%). Using
tert-Butyl (2R,3S)-2-(2-tert-butyldimethylsilyloxyethyl)-3-hy-
droxypiperidine-1-carboxylate (23a). To a solution of 22a (1.29
g, 5.23 mmol) in CH2Cl2 (15 mL) were added imidazole (712 mg,
10.5 mmol) and tert-butyldimethylsilyl chloride (800 mg, 5.31
mmol) at 0 °C. After stirring for 1 h, the mixture was poured into
water and extracted three times with EtOAc. The organic layer was

Antimalarial Compounds Based on Febrifugine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4703
washed with brine, dried over anhydrous Na2SO4, and evaporated.
The residue was chromatographed over silica gel eluted with
hexanes-EtOAc (4:1) to give 23a (1.54 g, 82%). Using a similar
procedure, compound 23b (yield: 55%) was prepared from 22b.
tert-Butyl (2R,3S)-2-(2-hydroxyethyl)-3-methoxymethoxypi-
peridine-1-carboxylate (24a). To a solution of 23a (1.54 g, 4.30
mmol) in 1,2-dimethoxyethane (15 mL) were added N,N-diisopro-
pylethylamine (1.85 mL, 10.8 mmol) and chloromethyl methyl ether
(470 µL, 6.45 mmol). After stirring for 15 h at 50 °C, the mixture
was poured into 0.2 M HCl and extracted three times with EtOAc.
The organic layer was washed with 0.5 M HCl and saturated
NaHCO3 solution and brine, dried over anhydrous Na2SO4, and
evaporated to give the crude MOM ether.
over anhydrous Na2SO4, and evaporated. The residue was chro-
matographed over silica gel eluted with hexanes-EtOAc (2:1) to
give 27a (51 mg, 30%). Using a similar procedure, compounds
27b (yield: 72%) and 27c (84%) were prepared from 26b and 26c,
respectively.
3-[2-[(2R,3S)-3-Hydroxypiperidin-2-yl]ethyl]-3H-quinazolin-
4-one (28). To a solution of 27a (42.1 mg, 0.100 mmol) was added
10% HCl-MeOH (1.5 mL) at room temperature. After stirring for
2 h, the mixture was evaporated. The residue was chromatographed
over silica gel eluted with CHCl3-MeOH (4:1) to give 28 (25.2
mg, 90%) as a dihydrochloride. Using a similar procedure,
compounds 29 (yield: 88%) and 30 (93%) were prepared from
27b and 27c, respectively.
This crude MOM ether was dissolved in THF (10 mL). Then,
1.0 M tetrabutylammonium fluoride solution in THF (6.40 mL)
was added to the solution at room temperature. After stirring for 1
h, the mixture was poured into water and extracted three times with
EtOAc. The organic layer was washed with brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with hexanes-EtOAc (1:4) to give
24a (1.03 g, 83%). Using a similar procedure, compound 24b
(yield: 94%) was prepared from 23b.
tert-Butyl (2R,3S)-2-(3-ethoxycarbonyl-2-propenyl)-3-meth-
oxymethoxy-piperidine-1-carboxylate (25). To a solution of 24a
(425 mg, 1.47 mmol) in CH2Cl2 (5.0 mL) were added 15% Dess-
Martin periodinane solution in CH2Cl2 (6.25 g, 2.21 mmol) and
water (100 µL) at 0 °C. After stirring for 30 min, the mixture was
poured into 1 M NaOH solution, and extracted three times with
Et2O. The organic layer was washed with brine, dried over
anhydrous Na2SO4, and evaporated to give the crude aldehyde.
To a solution of triethyl phosphonoacetate (300 µL, 1.51 mmol)
in toluene (4.0 mL) was added sodium hydride (60% oil suspension)
(73 mg, 1.81 mmol) at 0 °C. After 15 min, the crude aldehyde in
toluene (2.0 mL) was added to the mixture. After stirring for 1.5 h
at room temperature, the mixture was poured into saturated a NH4-
Cl solution and extracted three times with EtOAc. The organic layer
was washed with brine, dried, and evaporated. The residue was
chromatographed over silica gel eluted with hexanes-EtOAc (9:
1) to give 25 (399 mg, 76% (2 steps)).
tert-Butyl (2R,3S)-2-(4-hydroxybutyl)-3-methoxymethoxypi-
peridine-1-carboxylate (24c). Compound 25 (206 mg, 0.576 mmol)
and 20% Pd(OH)2 on carbon (40 mg) in THF (3.0 mL) were stirred
at room temperature for 1 h under a hydrogen atmosphere. After
filtration, the filtrate was evaporated to give an oily residue. To a
solution of this residue in toluene (3.5 mL) was added a 1.0 M
diisobutylaluminum hydride solution in toluene (1.8 mL) at 0 °C.
After stirring for 2 h, a saturated Rochelle salt solution was added.
After stirring for an additional 30 min, the mixture was poured
into water and extracted three times with Et2O. The organic layer
was washed with brine, dried, and evaporated. The residue was
chromatographed over silica gel eluted with hexanes-EtOAc (2:
1) to give 24c (144 mg, 84% (2 steps)).
tert-Butyl (2R,3S)-3-methoxymethoxy-2-[2-(p-toluenesulfonyl-
oxy)ethyl]-piperidine-1-carboxylate (26a). To a solution of 24a
(172 mg, 0.594 mg) in pyridine (1.5 mL) was added p-toluene-
sulfonyl chloride (453 mg, 2.38 mmol) at room temperature. After
stirring for 1.5 h, the mixture was poured into 0.2 M HCl and
extracted three times with EtOAc. The organic layer was washed
with saturated NaHCO3 solution and brine, dried over anhydrous
Na2SO4, and evaporated. The residue was chromatographed over
silica gel eluted with EtOAc to give 26a (183 mg, 69%). Using a
similar procedure, compounds 26b (yield: 80%) and 26c (78%)
were prepared from 24b and 24c, respectively.
2-[(2R,3S)-1-tert-Butyloxycarbonyl-3-methoxymethoxypiperi-
din-2-yl]acetic acid (31). To a solution of 24a (163 mg, 0.562
mmol) in CH2Cl2 (2.5 mL) were added 15% Dess-Martin
periodinane solution in CH2Cl2 (2.4 mL, 0.843 mmol) and water
(50 µL) at 0 °C. After stirring for 40 min, the mixture was poured
into a 1 M NaOH solution and extracted three times with EtOAc.
The organic layer was washed with water and brine, dried over
anhydrous Na2SO4, and evaporated to give the crude aldehyde.
To a solution of the crude aldehyde in tert-butyl alcohol (3.5
mL) and water (1.1 mL) were added 2-methyl-2-butene (260 µL,
2.46 mmol), NaH2PO4‚2H2O (88.3 mg, 0.562 mmol), and NaClO2
(80%) (203 mg, 2.25 mmol) at room temperature. After stirring
for 40 min, the mixture was poured into water and extracted three
times with EtOAc. The organic layer was dried over anhydrous
Na2SO4 and evaporated. The residue was chromatographed over
silica gel eluted with hexanes-EtOAc (2:1) to give 31 (142 mg,
83% (2 steps)).
2-[2-[(2R,3S)-1-tert-Butyloxycarbonyl-3-methoxymethoxypi-
peridin-2-yl]-acetylamino]-3H-quinazolin-4-one (32). To a solu-
tion of 31 (59 mg, 0.195 mmol) in CH2Cl2 (4.8 mL) and DMF
(1.2 mL) were added 2-amino-4(3H)-quinazolinone15 (35.0 mg,
0.215 mmol), O-(7-azabenzotriazol-1-yloxy)-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate (222 mg, 0.584 mmol), and N,N-
diisopropylethylamine (100 µL, 0.583 mmol) at room temperature.
After stirring for 12 h, the mixture was poured into 0.3 M HCl and
extracted three times with EtOAc. The organic layer was washed
with saturated NaHCO3 solution and brine, dried over anhydrous
Na2SO4, and evaporated. The residue was chromatographed over
silica gel eluted with CHCl3-MeOH (49:1) to give 32 (33.1 mg,
38%).
2-[2-[(2R,3S)-3-Hydroxypiperidin-2-yl]acetylamino]-3H-
quinazolin-4-one (33). To a solution of 32 (25.2 mg, 0.056 mmol)
was added 10% HCl-MeOH (2.0 mL) at room temperature. After
stirring for 1.5 h, the mixture was evaporated. The residue was
chromatographed over silica gel eluted with CHCl3-MeOH (3:1)
to give 33 (10.3 mg, 45%) as a dihydrochloride.
(3S,4R)-4-Dibenzylamino-6-(tert-butyldimethylsilyloxy)hexan-
3-ol (34). DMSO (2.10 mL, 29.6 mmol) and 19a (3.94 g, 9.86
mmol) dissolved in CH2Cl2 (10 mL) were added to a solution of
oxalyl chloride (1.30 mL, 14.8 mmol) in CH2Cl2 (40 mL) at -78
°C. After stirring for 1 h, triethylamine (6.90 mL, 49.3 mmol) was
added to this mixture and stirred at room temperature. The mixture
was poured into 0.5 M HCl and extracted three times with EtOAc.
The organic layer was washed with saturated NaHCO3 solution and
brine, dried over anhydrous Na2SO4, and evaporated to give the
crude aldehyde of (R)-2-(dibenzylamino)-4-(tert-butyldimethylsi-
lyloxy)butanal. The crude aldehyde was dissolved in THF (30 mL)
and cooled into -78 °C. Ethylmagnesium bromide dissolved in
THF (1.0 M, 14.8 mL) was added to this solution. After stirring
for 1 h, the mixture was allowed to warm up to room temperature
and poured into 0.5 M HCl. This mixture was extracted three times
with EtOAc. The organic layer was washed with saturated NaHCO3
solution and brine, dried over anhydrous Na2SO4, and evaporated.
The residue was chromatographed over silica gel eluted with
hexanes-EtOAc (19:1) to give 34 (2.76 g, 66% (2 steps)).
(1R,2S)-N,N-Dibenzyl-1-[2-(tert-butyldimethylsilyloxy)ethyl]-
2-methoxy-methoxybutylamine (35). To a solution of 34 (2.69
g, 6.29 mmol) in CH2Cl2 (30 mL) were added N,N-diisopropyl-
3-[2-[(2R,3S)-1-tert-Butyloxycarbonyl-3-methoxymethoxypi-
peridin-2-yl]ethyl]-3H-quinazolin-4-one (27a). To a solution of
4-hydroxyquinazoline (61 mg, 0.414 mmol) in DMF (2.0 mL) was
added K2CO3 (113 mg, 0.818 mmol) at 0 °C. After 15 min,
compound 26a (183 mg, 0.414 mmol) dissolved in DMF (1.0 mL)
was added into the solution. After stirring for 4 h at 80 °C, the
mixture was poured into water and extracted three times with
EtOAc. The organic layer was washed with water and brine, dried

4704 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Kikuchi et al.
ethylamine (2.55 mL, 15.0 mmol) and chloromethyl methyl ether
(1.50 mL, 20.0 mmol) at room temperature. After stirring for 5 h,
the mixture was poured into water and extracted three times with
EtOAc. The organic layer was washed with 0.5 M HCl, saturated
NaHCO3 solution and brine, dried over anhydrous Na2SO4, and
evaporated. The residue was chromatographed over silica gel eluted
with hexanes-EtOAc (39:1) to give 35 (2.34 g, 79%).
tert-Butyl (1R,2S)-1-[2-(tert-butyldimethylsilyloxy)ethyl]-2-
methoxymethoxy-butylcarbamate (36). Compound 35 (2.25 g,
4.78 mmol) and 20% Pd(OH)2 on carbon (1.15 g) in ethanol (24
mL) were stirred at room temperature for 4 h under a hydrogen
atmosphere. After filtration, the filtrate was evaporated to give an
oily residue. The residue was dissolved in CH2Cl2 (25 mL).
Triethylamine (900 µL, 6.46 mmol) and di-tert-butyl dicarbonate
(1.36 g, 6.23 mmol) were added to this solution at 0 °C. After
stirring for 1 h, the mixture was poured into 0.1 M HCl and
extracted three times with EtOAc. The organic layer was washed
with saturated NaHCO3 solution and brine, dried over anhydrous
Na2SO4, and evaporated. The residue was chromatographed over
silica gel eluted with hexanes-EtOAc (9:1) to give 36 (1.83 g,
98%).
the mixture was poured into 1 M NaOH solution and extracted
three times with EtOAc. The organic layer was washed with water
and brine, dried over anhydrous Na2SO4, and evaporated. The
residue was chromatographed over silica gel eluted with CHCl3-
MeOH (39:1) to give 40 (272 mg, 41% (2 steps)).
(4R,5S)-3-(5-Hydroxy-4-methylamino-2-oxoheptyl)-3H-quinazo-
lin-4-one (41). To a solution of 50 (39.0 mg, 0.097 mmol) in CH2-
Cl2 (1.5 mL) was added trifluoroacetic acid (1.5 mL) at 0 °C. After
stirring for 1 h, the solution was evaporated. The residue was
chromatographed over silica gel eluted with CHCl3-MeOH (9:1)
to give 41 (22.2 mg, 44%) as a ditrifluoroacetate.
3-(Triisopropylsilyloxy)piperidine (43). To a solution of 3-hy-
droxypiperidine (42, 1.01 g, 9.99 mmol) in CH2Cl2 (20 mL) were
added triethylamine (1.53 mL, 10.9 mmol) and benzyloxycarbonyl
chloride (1.56 mL, 10.9 mmol) at 0 °C. After stirring for 30 min,
the mixture was poured into 0.2 M HCl and extracted three times
with EtOAc. The organic layer was washed with saturated NaHCO3
solution and brine, dried over anhydrous Na2SO4, and evaporated.
The residue was chromatographed over silica gel eluted with
hexanes-EtOAc (2:1) to give N-benzyloxycarbonyl-3-hydroxypi-
peridine (2.25 g, 96%).
tert-Butyl (1R,2S)-1-(2-hydroxyethyl)-2-methoxymethoxybu-
tylcarbamate (37). To a solution of 36 (1.53 g, 5.51 mmol) in
THF (15 mL) was added 1.0 M tetrabutylammonium fluoride
solution in THF (6.60 mL) at room temperature. After stirring for
1 h, the mixture was poured into water and extracted three times
with EtOAc. The organic layer was washed with brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with hexanes-EtOAc (4:1) to give
37 (1.02 g, 67%).
tert-Butyl (1R,2S)-2-methoxymethoxy-1-oxiranylmethylbu-
tylcarbamate (38). To a solution of 37 (467 mg, 1.69 mmol) in
CH2Cl2 (5.0 mL) was added Dess-Martin periodinane (855 mg,
2.02 mmol) at 0 °C. After stirring for 1 h, the mixture was poured
into a 0.5 M NaOH solution, and extracted three times with EtOAc.
The organic layer was washed with brine, dried over anhydrous
Na2SO4, and evaporated to give the crude aldehyde.
To a solution of sodium hydride (60% oil suspension) (75.0 mg,
1.88 mmol) in DMSO (3.0 mL) was added trimethylsulfoxonium
iodide (410 mg, 1.86 mmol) at room temperature. After stirring
for 1 h at 70 °C, the crude aldehyde in DMSO (3.0 mL) was added
to the mixture at room temperature. After stirring for 1.5 h at 50
°C, the mixture was poured into water and extracted three times
with EtOAc. The organic layer was washed with brine, dried, and
evaporated. The residue was chromatographed over silica gel eluted
with hexanes-EtOAc (4:1) to give 38 (263 mg, 75% (2 steps)).
tert-Butyl[(1R,2S)-2-methoxymethoxy-1-oxiranylmethylbutyl]-
methylcarbamate (39). To a solution of 38 (394 mg, 1.36 mmol)
in DMF (7 mL) were added sodium hydride (60% oil suspension)
(65.0 mg, 1.63 mmol) and CH3I (160 µL, 2.57 mmol) at 0 °C.
After stirring for 3 h, the mixture was poured into water and
extracted three times with EtOAc. The organic layer was washed
with water and brine, dried over anhydrous Na2SO4, and evaporated.
The residue was chromatographed over silica gel eluted with
hexanes-EtOAc (4:1) to give 39 (374 mg, 91%).
(4R,5S)-3-[5-Methoxymethoxy-4-(N-tert-butyloxycarbonyl-N-
methylamino)-2-oxoheptyl]-3H-quinazolin-4-one (40). To a solu-
tion of 4-hydroxyquinazoline (201 mg, 1.38 mmol) in DMF (3 mL)
was added sodium hydride (60% oil suspension) (55.1 mg, 1.38
mmol) at 0 °C. After 30 min, compound 39 (348 mg, 1.15 mmol)
dissolved in DMF (1 mL) was added to the solution. After stirring
for 20 h at 80 °C, the mixture was poured into water and extracted
three times with EtOAc. The organic layer was washed with water
and brine, dried over anhydrous Na2SO4, and evaporated. The
residue was chromatographed over silica gel eluted with hexanes-
EtOAc (1:1) to give a mixutre of (4R,5S)-3-[2-hydroxy-5-(meth-
oxymethoxy)-4-(N-tert-butyloxycarbonyl-N-methylamino)heptyl]-
3H-quinazolin-4-one and 4-hydroxy-quinazoline.
N-Benzyloxycarbonyl-3-hydroxypiperidine (561 mg, 2.39 mmol)
was dissolved in DMF (6.0 mL). Imidazole (287 mg, 4.21 mmol)
and triisopropylsilyl chloride (900 µL, 4.21 mmol) were added to
this solution at room temperature. After stirring for 4 h, the mixture
was poured into 0.2 M HCl and extracted three times with EtOAc.
The organic layer was washed with saturated NaHCO3 solution and
brine, dried over anhydrous Na2SO4, and evaporated.
The residue was dissolved in MeOH (6 mL), and 20% Pd(OH)2
on carbon (119 mg) was added to the solution. After stirring for 4
h under a H2 atmosphere, the reaction mixture was filtered through
a Celite pad, and the filter cake was washed with MeOH. The filtrate
was evaporated, and the residue was chromatographed over silica
gel eluted with CHCl3-MeOH (4:1) to give 43 (433 mg, 71% (2
steps)).
1-Oxiranylmethyl-3-(triisopropylsilyloxy)piperidine (44). To
a solution of 43 (396 mg, 1.54 mmol) in DMF (4.0 mL) were added
epibromohydrin (160 µL, 1.87 mmol) and K2CO3 (255 mg, 1.85
mmol) at room temperature. After stirring for 5 h, the mixture was
poured into water and extracted three times with EtOAc. The
organic layer was washed with water and brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with hexanes-EtOAc (9:1) to give
44 (344 mg, 71%).
3-{2-Hydroxy-3-[3-(triisopropylsilyloxy)piperidin-1-yl]propyl}-
3H-quinazolin-4-one (45). To a solution of 4-hydroxyquinazoline
(157 mg, 1.07 mmol) in DMF (4 mL) was added sodium hydride
(60% oil suspension) (43.0 mg, 1.07 mmol) at room temperature.
After 15 min, compound 44 (280 mg, 0.892 mmol) dissolved in
DMF (0.5 mL) was added to the solution. After stirring for 18 h at
80 °C, the mixture was poured into water and extracted three times
with EtOAc. The organic layer was washed with water and brine,
dried over anhydrous Na2SO4, and evaporated. The residue was
chromatographed over silica gel eluted with hexanes-EtOAc (1:
1) to give 45 (277 mg, 68%).
3-[3-(3-Hydroxypiperidin-1-yl)-2-oxopropyl]-3H-quinazolin-
4-one (46). To a solution of oxalyl chloride (60 µL, 0.69 mmol) in
CH2Cl2 (1.5 mL) was added DMSO (90 µL, 1.3 mmol) at -78 °C.
After 20 min, compound 45 (67.2 mg, 0.15 mmol) in CH2Cl2 (0.5
mL) was added to the solution. After 40 min, triethylamine (300
µL, 2.15 mmol) was added to the solution. The solution was stirred,
warmed to room temperature, poured into water, and extracted three
times with EtOAc. The organic layer was washed with water and
brine, dried over anhydrous Na2SO4, and evaporated. The residue
was chromatographed over silica gel eluted with CHCl3-MeOH
(99:1) to give 3-{3-[3-(triisopropylsilyloxy)piperidin-1-yl]-2-oxo-
propyl}-3H-quinazolin-4-one (48 mg, 72%).
The mixture was dissolved in CH2Cl2 (2 mL). Then, 15% Dess-
Martin periodinane solution in CH2Cl2 (3.90 g, 1.38 mmol) was
added to this solution at room temperature. After stirring for 2 h,
This ketone (11.1 mg, 0.024 mmol) was dissolved in 5% HCl-
MeOH (1.0 mL). After stirring for 2 h at 50 °C, the solution was
evaporated. The residue was chromatographed over silica gel eluted

Antimalarial Compounds Based on Febrifugine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4705
with CHCl3-MeOH (49:1) to give 46 (5.8 mg, 80%) as a
dihydrochloride.
silica gel eluted with CHCl3-MeOH (4:1) to give 50 (15.6 mg,
89%) as dihydrochloride.
Antimalarial Assay in Vitro. Type A+ human plasma and
erythrocytes were obtained from healthy volunteers at our institute.
Plasmodium falciparum strains FCR-3 (chloroquine sensitive) and
K1 (chloroquine resistant) were cultured in the human erythrocytes
in an RPMI medium (RPMI-1640 with 25 mM HEPES buffer, 24
mM NaHCO3, 0.2% glucose, 0.05% L-glutamine, 50 µg/mL of
hypoxanthine, and 25 µg/mL of gentamisin) supplemented with
10% human plasma at 37 °C, under 93% N2, 4% CO2, and 3% O2.
The antimalarial activity of the test compounds have been achieved
by a dose-response curve using the parasite lactate dehydrogenase
(pLDH) assay.19 Asynchronous parasites (2.0% hematocrit and 0.5%
parasitaemia) (90 µL) were seeded in a 96-well microplate, and a
solution of the test compound dissolved in distilled water or 5%
DMSO (10 µL) was added. After incubation at 37 °C for 72 h, the
parasite suspension (20 µL) was transferred to another plate
containing Malstat reagent (100 µL). The plate was further
incubated for 15 min at room temperature, and a 1:1 mixture of
nitroblue tetrazolium and phenazine ethosulfate (2 mg and 0.1 mg/
mL, respectively) (20 µL) was added to each well. After incubation
for 2 h at room temperature in darkness, the blue formazan product
was measured at 650 nm. The EC50 value was estimated from a
dose-response curve.
Cytotoxicity Test. The cytotoxicity test of compounds was
measured by the colorimetric MTT assay.20 Mouse L929 cell
suspension in RPMI-1640 with 10% FBS (90 µL) was added in
96-well microplates at 1.8 × 104 cells/well. Then, a solution of the
test compound dissolved in distilled water or 10% DMSO (10 µL)
was added to each well. After incubation for 48 h at 37 °C under
5% CO2, 2.5 mg/mL of MTT solution (10 µL) was added to each
well. The plate was incubated for a further 4 h. Then, the incubation
medium was aspirated, and DMSO (100 µL) was added to solubilize
the MTT formazan product. After mixing, the absorbances at 570
and 655 nm were measured. The EC50 value was estimated from a
dose-response curve.
1-tert-Butyloxycarbonyl-3-(2,2-dimethyl[1,3]dioxolan-4-yl-
methoxy)piperidine (47). To a solution of 3-hydroxypiperidine (42,
1.01 g, 9.95 mmol) in CH2Cl2 (20 mL) were added triethylamine
(1.66 mL, 11.9 mmol) and di-tert-butyl dicarbonate (2.63 g, 12.1
mmol) at 0 °C. After stirring for 1 h, the mixture was poured into
0.1 M HCl and extracted three times with EtOAc. The organic layer
was washed with saturated NaHCO3 solution and brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with hexanes-EtOAc (4:1) to give
1-tert-butyloxycarbonyl-3-hydroxypiperidine (1.90 g, 95%).
To a solution of 1-tert-butyloxycarbonyl-3-hydroxypiperidine
(998 mg, 4.96 mmol) in DMF (16 mL) were added sodium hydride
(60% oil suspension) (760 mg, 19.0 mmol) and 2,2-dimethyl-1,3-
dioxolan-4-ylmethyl tosylate (3.13 g, 10.9 mmol). After stirring
for 16 h at 90 °C, the mixture was poured into 0.2 M HCl and
extracted three times with EtOAc. The organic layer was washed
with saturated NaHCO3 solution and brine, dried over anhydrous
Na2SO4, and evaporated. The residue was chromatographed over
silica gel eluted with CHCl3-MeOH (9:1) to give 47 (891 mg,
57%).
1-tert-Butyloxycarbonyl-3-oxiranylmethoxypiperidine (48).
Compound 47 (812 mg, 2.58 mmol) was dissolved in 10% HCl-
MeOH (15 mL). After refluxing for 3 h, the solution was
evaporated. The residue was dissolved in CH2Cl2 (12 mL) and
MeOH (3.0 mL). Triethylamine (1.82 mL, 13.1 mmol), and di-
tert-butyl dicarbonate (683 mg, 3.13 mmol) was added to this
solution at 0 °C. After stirring for 2 h, the mixture was poured into
0.5 M HCl and extracted three times with EtOAc. The organic layer
was washed with saturated NaHCO3 solution and brine, dried over
anhydrous Na2SO4, and evaporated. The residue was chromato-
graphed over silica gel eluted with CHCl3-MeOH (99:1) to give
1-tert-butyloxycarbonyl-3-(2,3-dihydroxypropoxy)piperidine (565
mg, 80%).
To a solution of 1-tert-butyloxycarbonyl-3-(2,3-dihydroxypro-
poxy)piperidine (534 mg, 1.94 mmol) in DMF (15 mL) were added
sodium hydride (60% oil suspension) (194 mg, 4.85 mmol) and
1-(p-toluenesulfonyl)imidazole (603 mg, 2.71 mmol) at 0 °C. After
stirring for 5 h at room temperature, the mixture was poured into
0.5 M HCl and extracted three times with EtOAc. The organic layer
was washed with water and brine, dried over anhydrous Na2SO4,
and evaporated. The residue was chromatographed over silica gel
eluted with hexanes-EtOAc (4:1) to give 48 (159 mg, 32%).
3-[3-(1-tert-Butyloxycarbonylpiperidin-3-yloxy)-2-oxopropyl]-
3H-quinazolin-4-one (49). To a solution of 4-hydroxyquinazoline
(89.3 mg, 0.611 mmol) in DMF (4.0 mL) was added sodium hydride
(60% oil suspension) (24.6 mg, 0.615 mmol) at room temperature.
After 25 min, compound 48 (132 mg, 0.513 mmol) dissolved in
DMF (2.0 mL) was added to the solution. After stirring for 10 h at
80 °C, the mixture was poured into water and extracted three times
with EtOAc. The organic layer was washed with water and brine,
dried over anhydrous Na2SO4, and evaporated. The residue was
chromatographed over silica gel eluted with hexanes-EtOAc (2:
1) to give a mixutre of 3-[3-(1-tert-butyloxycarbonylpiperidin-3-
yloxy)-2-hydroxypropyl]-3H-quinazolin-4-one and 4-hydroxyquinazo-
line.
The mixture was dissolved in CH2Cl2 (4.0 mL). Then, 15%
Dess-Martin periodinane solution in CH2Cl2 (5.80 g, 2.05 mmol)
was added to this solution at room temperature. After stirring for
3 h, the mixture was poured into 10% sodium thiosulfate solution
and extracted three times with EtOAc. The organic layer was
washed with 0.5 M NaOH, water, and brine, dried over anhydrous
Na2SO4, and evaporated. The residue was chromatographed over
silica gel eluted with hexanes-EtOAc (1:1) to give 49 (137 mg,
67% (2 steps)).
3-[2-Oxo-3-(piperidin-3-yloxy)propyl]-3H-quinazolin-4-one (50).
Compound 49 (18.8 mg, 0.047 mmol) was dissolved in 5% HCl-
MeOH (2.0 mL). After stirring for 1 h at room temperature, the
solution was evaporated. The residue was chromatographed over
Antimalarial Assay in Vivo. The in vivo antimalarial activity
was determined against rodent malaria-derived P.berghei NK65
according to the 4-day suppressive test.21 Male ICR mice at weight
18-20 g were intravenously inoculated with 106 parasitized red
blood cells. Test compounds were suspended in 5% gum arabic
and orally administered to the mice 2 h after infection (Day 0).
The test compounds were successively administered to the mice
once a day for 3 consecutive days (Days 1-3). The day after the
last treatment (Day 4), blood was obtained from the tail vein of
the infected mice. Parasitaemia was determined by thin blood films
made from the blood samples. The ED50 value was estimated from
a dose-response curve.
Acknowledgment. This work was supported in part by a
Grant-in-Aid for Scientific Research (No. 15019007, 16017210,
and 16390030) from the Ministry of Education, Science, Sports
and Culture of Japan.
Supporting Information Available: Analytical data of new
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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