Metabolites of febrifugine and its synthetic analogue by mouse liver S9 and their antimalarial activity against Plasmodium malaria parasite

Article abstract of DOI:10.1021/jm0302086

Quinazolinone type alkaloids, febrifugine (1) and isofebrifugine (2), isolated from Dichroa febrifuga roots, show powerful antimalarial activity against Plasmodium falciparum. Unfortunately, their emetic effect and other undesirable side effects have precluded their clinical use for malaria. Because of their antimalarial potency, analogues were searched for, with the goal of preserving the strong antimalarial activity, while dramatically reducing side effects. We expected that compounds useful in drug development would exist in metabolites derived from 1 and Df-1 (3), the condensation product of 1 with acetone, by mouse liver S9. Feb-A and -B (4 and 5) were isolated as the major metabolites of 1. In addition to 4 and 5, feb-C and -D (6 and 7) were also purified from the metabolic mixture of 3. Compounds 4 and 5 were compounds oxidized at C-6 and C-2 of the quinazolinone ring of 1, respectively. Compounds 6 and 7, derived from 3, also bear febrifugine type structures in which the 4″- and 6″-positions of the piperidine ring of 1 were oxidized. In vitro antimalarial and cytotoxic tests using synthetically obtained racemic 4-6 and enantiomerically pure 7 demonstrated that 4 and 6 had antimalarial activity against P. falciparum, of similar potency to that of 1, with high selectivity. The antimalarial activity of 5 and 7, however, was dramatically decreased in the test. The in vitro antimalarial activity of analogues 22 and 43, which are stereoisomers of 4 and 6, was also evaluated, showing that 22 is active. The results suggest that basicity of both the 1- and the 1″-nitrogen atoms of 1 is crucial in conferring powerful antimalarial activity. Racemic 4 and 6 exhibited powerful in vivo antimalarial activity against mouse malaria P. berghei, and especially, no serious side effects were observed with 4. Thus, the metabolite 4 appears to be a promising lead compound for the development of new types of antimalarial drugs.

Full text of DOI:10.1021/jm0302086

J . Med. Chem. 2003, 46, 4351-4359
4351
Meta bolites of F ebr ifu gin e a n d Its Syn th etic An a logu e by Mou se Liver S9 a n d
Th eir An tim a la r ia l Activity a ga in st P la sm od iu m Ma la r ia P a r a site
Shingo Hirai,† Haruhisa Kikuchi,† Hye-Sook Kim,‡ Khurshida Begum,‡ Yusuke Wataya,‡ Hidehisa Tasaka,†
Yuriko Miyazawa,† Keisuke Yamamoto,† and Yoshiteru Oshima*,†
Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai 980-8578, J apan, and
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, J apan
Received April 29, 2003
Quinazolinone type alkaloids, febrifugine (1) and isofebrifugine (2), isolated from Dichroa
febrifuga roots, show powerful antimalarial activity against Plasmodium falciparum. Unfor-
tunately, their emetic effect and other undesirable side effects have precluded their clinical
use for malaria. Because of their antimalarial potency, analogues were searched for, with the
goal of preserving the strong antimalarial activity, while dramatically reducing side effects.
We expected that compounds useful in drug development would exist in metabolites derived
from 1 and Df-1 (3), the condensation product of 1 with acetone, by mouse liver S9. Feb-A and
-B (4 and 5) were isolated as the major metabolites of 1. In addition to 4 and 5, feb-C and -D
(6 and 7) were also purified from the metabolic mixture of 3. Compounds 4 and 5 were
compounds oxidized at C-6 and C-2 of the quinazolinone ring of 1, respectively. Compounds 6
and 7, derived from 3, also bear febrifugine type structures in which the 4′′- and 6′′-positions
of the piperidine ring of 1 were oxidized. In vitro antimalarial and cytotoxic tests using
synthetically obtained racemic 4-6 and enantiomerically pure 7 demonstrated that 4 and 6
had antimalarial activity against P. falciparum, of similar potency to that of 1, with high
selectivity. The antimalarial activity of 5 and 7, however, was dramatically decreased in the
test. The in vitro antimalarial activity of analogues 22 and 43, which are stereoisomers of 4
and 6, was also evaluated, showing that 22 is active. The results suggest that basicity of both
the 1- and the 1′′-nitrogen atoms of 1 is crucial in conferring powerful antimalarial activity.
Racemic 4 and 6 exhibited powerful in vivo antimalarial activity against mouse malaria P.
berghei, and especially, no serious side effects were observed with 4. Thus, the metabolite 4
appears to be a promising lead compound for the development of new types of antimalarial
drugs.
Ch a r t 1. Structures of 1-3
In tr od u ction
Malaria is caused by the protozoan parasite of the
genus Plasmodium and leads to mortality if patients
infected with Plasmodium falciparum are left un-
treated. Malaria affects approximately 300 million
patients worldwide, leading to more than 2 million
deaths a year. The treatment of P. falciparum malaria
has attracted global attention because of the rapid
growing resistance of the parasites toward all known
antimalarial drugs, such as chloroquine. The chemo-
therapy of malaria thus continues to be a matter of
utmost concern for medicinal chemists.1
Roots of Dichroa febrifuga, the plant belonging to the
family Saxifragaceae, have been used as a traditional
antimalarial in China. The quinazolinone type alkaloids
febrifugine (1) and its stereoisomer, isofebrifugine (2),
have been identified as the active components of these
roots (Chart 1).2 Although alkaloid 1 demonstrated
outstanding antimalarial activity, both in vitro and in
vivo with no resistant parasite to 1 reported, its use as
an antimalarial drug has been precluded due to its
strong emetic properties and other side effects.3 How-
ever, the potent antimalarial activity of 1 stimulated
medicinal chemists to pursue compounds derived from
1, which may be valuable leads for novel drugs. We also
made an effort to account for the role of the structural
components of 1 in its activity and proposed active
analogues such as the reductive product at C-2′, oxida-
tive product at C-3′′, and Df-1 (3), the condensation
product of 1 with acetone.4,5
Compounds 1 and 2 showed powerful in vitro anti-
malarial activity, with similar potencies against chlo-
roquine sensitive P. falciparum FCR-3 (EC50 of 1 and
2, 7.0 × 10-10 and 3.4 × 10-9 M, respectively). They also
exhibited promising activity against chloroquine resis-
tant P. falciparum K1 (EC50 of 1 and 2, 1.2 × 10-9 and
* To whom correspondence should be addressed. Tel: +81-22-217-
6822. Fax: +81-22-217-6821. E-mail: oshima@mail.pharm.tohoku.ac.jp.
† Tohoku University.
‡ Okayama University.
10.1021/jm0302086 CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/29/2003

4352 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Hirai et al.
Ch a r t 2. Structures of Metabolic Products, feb-A-D
(4-7), of 1 and 3
F igu r e 1. Correlations of the HMBC spectrum of feb-A (4).
F igu r e 2. Relative configuration of piperidine ring in feb-C
(6).
1.8 × 10-9 M, respectively). In vivo activity against
mouse malaria, Plasmodium berghei, is significantly
greater in 1 than in 2 (ED50 of 1 and 2, 0.3 and 79 mg/
kg, respectively).5 The different in vivo activities be-
tween the stereoisomers 1 and 2 prompted us to further
study metabolites of 1 generated by mouse liver S9. In
addition, a comparative metabolic study of the active
analogue 3 was carried out to search for suitable leads.
In vitro antimalarial activities of synthetic 1, 2, and
their antipodes were evaluated by Kobayashi et al.6 The
EC50 value of synthetic 1 against P. falciparum was
1/2632 of that of its enantiomer, and the EC50 of
synthetic 2 was 1/552 of that of its enantiomer. In
addition to the great difference in antimalarial activity
between synthetic 1, 2, and their antipodes, high
selectivities against P. falciparum were only reported
for synthetic 1 and 2. These results permitted us to use
racemic compounds in the antimalarial tests.
hydroxy group at C-6 or C-7. The two proposed struc-
tures were unambiguously distinguished by the HMBC
spectrum of 4, showing the H-C correlation peaks of
H-2-C-8a and H-7-C-8a, confirming the presence of the
C-6 hydroxy group in 4 (Figure 1).
The 1H NMR signals, due to the piperidine ring
protons of 5, closely resembled those of 1. No singlet
1
signal for H-2 was observed in the H NMR spectrum
of 5, and the aromatic proton signals of H-6 and H-8
resonated upfield (δ 7.25 and 7.20) relative to those of
1 (δ 7.58 and 7.72 (see Supporting Information, Table
S1)). A structure bearing a newly formed carbonyl
function at C-2 was thus proposed for 5.
The 1H NMR spectrum of 6 pointed out a methine
proton signal at δ 4.03 (1H, br.d, J ) 2.4 Hz), instead
of signals around δ 1.60-2.00 for methylene hydrogens
at C-4′′ of the piperidine ring of 1. The value of the
coupling constant (J 3′′,4′′ ) 2.4 Hz) along with nuclear
Overhauser effects (NOEs) of H-4′′ with H-3′′, H-5′′R,
and H-5′′â proved the axial configuration of the C-4′′
hydroxy group (Figure 2).
We report here the isolation, structure elucidation,
and synthesis of metabolic products, feb-A-D (4-7), of
1 and 3 (Chart 2). The antimalarial activity of the
metabolites and their analogues is also described in this
paper.
The molecular formula C16H17N3O4 for 7 was deter-
mined from its HREIMS, showing a molecular ion at
m/z 315.1244, which differed from 1 by 14 mass units.
Resu lts a n d Discu ssion
Isola tion a n d Str u ctu r e Elu cid a tion of Meta bo-
lites of 1 a n d 3 Gen er a ted by Mou se Liver S9.
Compound 1 was incubated with mouse liver S9, in the
presence of cofactor for the NADPH-regenerating sys-
tem, in phosphate-buffered solution at 37 °C. After 1 h,
the reaction was stopped with ethyl acetate and ex-
tracted with n-butanol. High-performance liquid chro-
matography (HPLC) separation of the extract by re-
versed phase chromatography using an ODS column
identified feb-A and -B (4 and 5) as the major metabolic
products of 1. Treatment of 3, prepared from 1 with
acetone by a condensation reaction,5 with mouse liver
S9 for 40 min, yielded feb-C and -D (6 and 7) as well as
4 and 5.
1
In the H NMR spectrum, no signal assignable to H-6′′
of the piperidine ring was detected, and the chemical
shifts of H-5′′ (δ 2.28-2.50 (2H, m)) shifted downfield
from those of 1 (δ 1.95-2.01 (1H, m) and 1.71-1.83 (1H,
m) (see Supporting Information, Table S1)), demon-
strating that 7 was an oxidized product of 1 at C-6′′.
Syn th esis of F eb-A-D, Meta bolites of 1 a n d 3.
The low yield of febrifugine and Df-1 metabolites 4-7
obtained from the metabolic experiments required their
synthesis for further biological evaluation. Racemic 4
was synthesized employing the synthetic strategy of 1
and 2 developed by Takeuchi et al.,7 which relies on an
unusual Claisen rearrangement of allyl enol ether 11
and the stereoselective reduction of 12 (Scheme 1).
3-Hydroxypyridine (8) was refluxed with benzyl chloride
in toluene to afford pyridinium chloride 9. Compound
10 was obtained by O-allylation followed by reduction
of 9. The benzyl group of 10 was replaced by a benzy-
loxycarbonyl group by treatment with benzyl chlorofor-
mate to generate 11. The unusual Claisen rearrange-
ment of 11 occurred upon treatment with boron
trifluoride-diethyl ether complex, resulting in the
formation of 12. Reduction of 12 by sodium borohydride
gave 13 as a sole product. The intramolecular bromo-
etherification of 13 using N-bromosuccinimide afforded
14 as a mixture of diastereomeric isomers. Then, a
The molecular formulas of the three metabolites 4-6
were established to be C16H19N3O4 based on analyses
of their high-resolution electron ionization mass spec-
trometry (HREIMS) and the H and 13C NMR spectral
1
data. Their molecular formulas differed from the parent
compound 1 by an oxygen atom, implying that 1 was
oxidized to form the metabolites 4-6. A comparative
1
study of the H NMR spectra of 4 and 1 indicated the
presence of a 1,2,4-trisubstituted benzene ring in 4 (δ
7.54 (1H, d, J ) 8.8 Hz), 7.42 (1H, d, J ) 2.9 Hz), and
7.30 (1H, dd, J ) 8.8, 2.9 Hz)) instead of a 1,2-
disubstituted benzene ring in 1. Compound 4 was thus
suggested to be an oxidized analogue of 1, bearing a

Metabolites of Febrifugine and Its Synthetic Analogue
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4353
Sch em e 1. Synthesis of the Piperidine Moiety of feb-A
(4) and feb-B (5)a
group to yield 18. A coupling reaction of 18 with
hemiacetal 16 provided compound 19. Hydrogenolysis
of 19 catalyzed by palladium hydroxide and boiling of
the residue in methanol gave 20 as well as its stereo-
isomer 21. After separation of 20 and 21, each com-
pound was deprotected in acidic condition resulting in
racemic feb-A (4) and isofeb-A (22) (Scheme 2).
Synthesis of the aromatic part in 5 was carried out
using isatoic anhydride (23) as a starting material.
Compound 23 was converted to 24 by treating with tert-
butylamine. Compound 24 was then reacted with meth-
yl chloroformate to yield methyl carbamate 25, which
was refluxed with potassium hydroxide to generate
3-tert-butyl benzoyleneurea 26. Reaction of 26 with
p-methoxybenzyl chloride, followed by deprotection of
the tert-butyl group, yielded 1-p-methoxybenzyl ben-
zoyleneurea 27. Similar to the synthesis of racemic 4, a
coupling reaction of 27 with hemiacetal 16 gave 28.
Hydrogenolysis of 28 followed by heating in methanol
produced 29 and its isomer 30. After separation of 29
and 30, the p-methoxybenzyl group of each compound
was deprotected with ceric ammonium nitrate resulting
in racemic feb-B (5) and isofeb-B (31). However, 31 was
too unstable to be purified with silica gel column
chromatography (Scheme 3).
a
Reagents and conditions: (a) Benzyl chloride, toluene, reflux.
(b) (i) Allyl bromide, NaH, MeOH, reflux; (ii) NaBH4, MeOH, 0
°C. (c) Benzyl chloroformate, THF, room temperature. (d) BF3‚OEt2,
acetonitrile, room temperature. (e) NaBH4, MeOH, 0 °C. (f) NBS,
acetonitrile, room temperature. (g) (i) tBuOK, THF, 0 °C; (ii) NBS,
MeOH, room temperature. (h) 10% HCl, acetonitrile, room tem-
perature.
diastereomeric mixture of methyl acetal 15 was obtained
by the following two reactions: dehydrobromination of
14 using potassium tert-butoxide and bromoetherifica-
tion using N-bromosuccinimide and methanol. Hemi-
acetal 16 was prepared by hydrolysis of 15 and used in
the next reaction without purification. The quinazoli-
none component of 4 was prepared from 5-hydroxy-
anthranilic acid (17). Compound 17 was condensed with
formamide and then protected with a triisopropylsilyl
The synthesis of 6 is described in Scheme 4. Com-
pound 13 was deprotected under acidic condition, and
the product was reacted with di-tert-butyl dicarbonate,
resulting in 32. Compound 32 was acetylated to produce
33. The allyl group of 33 was oxidized with osmium
tetroxide, and protection of the formed diol group as
acetonide by 2,2-dimethoxymethane gave 34. After
deacetylation of 34, the resulting product was mesylated
Sch em e 2. Synthesis of feb-A (4)a
a
Reagents and conditions: (a) (i) Formamide, 160 °C; (ii) Triisopropylsilyl chloride, imidazole, DMF, 50 °C. (b) Compound 16, K2CO3,
DMF, room temperature. (c) (i) H2 (1 atm), Pd(OH)2/C, EtOH, room temperature; (ii) MeOH, reflux. (d) 10% HCl-MeOH, 50 °C.
Sch em e 3. Synthesis of feb-B (5)a
a
Reagents and conditions: (a) tert-Butylamine, DMAP, DMF, room temperature. (b) Methyl chloroformate, 1 M NaOH-dioxane (1:1),
room temperature. (c) KOH, EtOH, reflux. (d) (i) p-Methoxybenzyl chloride, NaH, DMF, room temperature; (ii) 6 M HCl-dioxane (2:3),
reflux. (e) Compound 16, K2CO3, DMF, room temperature. (f) H2 (1 atm), Pd(OH)2/C, EtOH, room temperature. (g) CAN, acetonitrile-
H2O (2:1), room temperature.

4354 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Sch em e 4. Synthesis of feb-C (6)a
Hirai et al.
a
Reagents and conditions: (a) (i) 10% HCl-MeOH, 50 °C; (ii) Boc2O, triethylamine, room temperature. (b) Ac2O, pyridine, room
temperature. (c) (i) Catalytic OsO4, NMO, acetone-acetonitirle-H2O (1:1:1), room temperature; (ii) 2,2-dimethoxypropane, pTsOH, DMF,
room temperature. (d) (i) MeONa, MeOH, room temperature; (ii) MsCl, triethylamine, CH2Cl2, 0 °C. (e) DBU, THF, reflux. (f) (i) 10%
HCl-MeOH, reflux; (ii) Boc2O, triethylamine, room temperature. (g) N-Tosylimidazole, KH, DMF, room temperature. (h) (i) Compound
39, KH, DMF, 80 °C; (ii) Dess-Martin oxidation. (i) Catalytic OsO4, NMO, acetone-acetonitrile-H2O (1:1:1), room temperature. (j) TFA-
CH2Cl2 (1:1), room temperature, and then 10% HCl-MeOH, room temperature. (k) 10% HCl-MeOH, room temperature.
Sch em e 5. Synthesis of feb-D (7)a
a
Reagents and conditions: (a) (i) Benzyl bromide, K2CO3, NaOH, MeOH-H2O (1:1), reflux; (ii) LiAlH4, THF, room temperature. (b)
tert-Butyldimethylsilyl chloride, triethylamine, DMAP, CH2Cl2, 0 °C. (c) (i) Oxalyl chloride, DMSO, -78 °C, and then triethylamine; (ii)
vinylmagnesium bromide, THF, -78 °C. (d) MOMCl, KI, N,N-diisopropylethylamine, DME, reflux. (e) Catalytic OsO4, trimethylamine
N-oxide, THF-tBuOH-H2O (1:1:1), room temperature. (f) (i) NaIO4, Et2O-H2O (1:1), 0 °C; (ii) benzyl diethylphosphonoacetate, NaH,
toluene, 0 °C. (g) (i) H2 (1 atm), Pd(OH)2/C, EtOH, room temperature; (ii) EDCI‚HCl, THF, 0 °C. (h) p-Methoxybenzyl chloride, NaH,
DMF, 0 °C. (i) TBAF, THF, room temperature. (j) (i) Dess-Martin oxidation; (ii) trimethyl sulfoxonium iodide, NaH, DMSO, room
temperature. (k) (i) Compound 39, KH, DMF, 80 °C; (ii) Dess-Martin oxidation. (l) CAN, acetonitrile-H2O (2:1), room temperature. (m)
10% HCl-MeOH, 50 °C.
with methanesulfonyl chloride to form 35. Compound
36 was obtained by refluxing of 35 in the presence of
DBU. The acetonide and Boc groups of 36 were cleaved
in acidic conditions, and the product was reacted with
di-tert-butyl dicarbonate to reprotect the amino func-
tional group, generating diol 37. Treatment of 37 with
toluensulfonylimidazole afforded epoxide 38. Coupling
of 38 with 4-hydroxyquinazoline (39) and Dess-Martin
oxidation of the resulting compound yielded 40. Diols
41 and 42 were prepared from 40 using osmium
tetroxide, and each diol was deprotected in acidic
condition, generating racemic feb-C (6) and isofeb-C (43)
(Scheme 4).
was reacted with tert-butyldimethylsilyl chloride to yield
46.9 Swern oxidation of 46 followed by Grignard reaction
with vinylmagnesium bromide yielded 47 diastereose-
lectively. Compound 47 was protected with a meth-
oxymethoxy group to give 48, which was converted to
diol 49 by osmium tetroxide. Oxidative cleavage of 49
followed by the Horner-Emmons reaction yielded ben-
zyl ester 50. After 50 was subjected to hydrogenolysis,
cyclization in the presence of 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride generated pi-
peridone 51. Protection of the amide function of 51 with
p-methoxybenzyl chloride yielded 52, which was depro-
tected with tetrabutylammonium fluoride to produce 53.
Dess-Martin oxidation of 53 produced the aldehyde,
which after treatment with trimethylsulfoxonium iodide
and sodium hydride gave epoxide 54. Coupling of 54
with 4-hydroxyquinazoline (39) and Dess-Martin oxi-
dation of the resulting compound yielded 55. The
The piperidine ring of 7 was synthesized using N,N-
dibenzylamino aldehyde8 as a chiral building block
(Scheme 5). D-Aspartic acid (44) was converted to amino
alcohol 45 by reaction with benzyl bromide and reduc-
tion using lithium aluminum hydride. Compound 45

Metabolites of Febrifugine and Its Synthetic Analogue
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4355
Ta ble 1. Antimalarial Activities of Febrifugine (1) and Df-1 (3)
Metabolites against P. falciparum In Vitro
Ta ble 2. Antimalarial Activities of Febrifugine (1) and Df-1 (3)
Metabolites against P. berghei In Vivoa
antimalarial
i.p. (mg/kg)
ED90
p.o. (mg/kg)
ED50 ED90
activitya
EC50 (M)
cytotoxicityb
EC50 (M)
compd
ED50
compd
selectivityc
1c
2c
3c
4
6
22
0.3
79
1.5
1d
2d
3d
4
5
6
7
22
43
7.0 × 10-10
3.4 × 10-9
1.6 × 10-9
2.2 × 10-9
6.6 × 10-6
2.2 × 10-8
>5.2 × 10-5
2.7 × 10-10
1.5 × 10-7
1.8 × 10-8
1.0 × 10-8
1.7 × 10-7
1.8 × 10-7
3.8 × 10-7
2.7 × 10-7
>8.5 × 10-5
3.6 × 10-5
>5.2 × 10-5
2.9 × 10-6
2.5 × 10-5
3.2 × 10-5
1.0 × 10-5
243
53
238
123
>13
1636
>100 (70%)b
2.5
0.6
2.4
6.0
5.4
2.0
6.6
5.2
8.3
37
13
3.3
15
8.0
27
23
chloroquine
artemisinin
32
3.0
89
8.0
10 741
167
1778
1000
a
Various concentrations of the test compounds were prepared
in distilled water or olive oil. The test compounds were adminis-
tered to groups of five mice once a day starting on day 0 and
thereafter on days 1-3. Parasitemia levels were determined on
the day following the last treatment (on day 4), and ED values of
the antimalarial activities indicated above were determined by a
chloroquine
artemisinin
a
b
Against P. falciparum FCR-3. Against FM3A mouse mam-
mary cells. c Selectivity ) cytotoxicity/antimalarial activity. Data
d
from ref 5.
b
previously reported protocol.10 The value in parentheses shows
the growth inhibition (%) of each dose. c Data from ref 5.
p-methoxybenzyl group was removed from 55 with ceric
ammonium nitrate to yield 56, which was hydrolyzed
to give enantiomerically pure feb-D (7).
of the five treated mice was actually cured with no
parasites. These results imply that 4 might be an
effective antimalarial candidate with low toxicity.
In conclusion, we demonstrated that 4, 6, and 22 are
strongly effective against P. falciparum in vitro, with
N-1 and N-1′′ atoms found to be crucial for significant
antimalarial activity. Compounds 4 and 6 also exhibited
strong antimalarial activity against P. berghei-infected
mice. Furthermore, 4 was less toxic than the parent
compound 1, suggesting that 4 could be a good candidate
as a new antimalarial drug.
An tim a la r ia l Activity of F ebr ifu gin e a n d Df-1
Meta bolites a n d Th eir Syn th etic An a logu es. In
vitro antimalarial activity of febrifugine metabolites
4-7 and their analogues 22 and 43 against P. falci-
parum (FCR-3 strain) and cytotoxicity against mouse
mammary FM3A cells were evaluated (Table 1). Com-
pounds 4 and 22, whose hydrogen at C-6 of the quinazoli-
none ring was substituted by a hydroxy group, exhibited
powerful antimalarial activities (EC50, 2.2 × 10-9 and
2.7 × 10-10 M). Compound 22 also showed extremely
high selectivity (selectivity, 10 741) as compared to that
of 1 (selectivity, 243). These data suggest that the
hydroxy group at C-6 in 1 has the advantage of low
toxicity for mammalian cells and still maintains anti-
malarial activity against P. falciparum. The antima-
larial activity of 5, which had an amido group at C-2,
decreased dramatically (EC50, 6.6 × 10-6 M), implying
that basicity of the N-1 atom on the quinazolinone ring
in 1 was important for antimalarial activity. Compound
6, a 4′′-hydroxy analogue of 1, exhibited significant
antimalarial activity with high selectivity (EC50, 2.2 ×
10-8 M; selectivity, 1636), while 43, a 4′′-hydroxy
analogue of 2, showed weaker activity (EC50, 1.5 × 10-7
M) than 6. However, 7, which was formed by oxidation
of C-6′′ of 1, did not demonstrate antimalarial activity
(EC50, >5.2 × 10-5 M).
Exp er im en ta l Section
Gen er a l Meth od s. Analytical thin-layer chromatography
was performed on silica gel 60 F254 (Merck) and Aluminum
oxide 150 F254 neutral (Type T, Merck). Column chromatog-
raphy was carried out on silica gel 60 (70-230 mesh, Merck)
and Aluminum oxide 150 basic (type T, Merck). NMR spectra
were recorded on J EOL J NM GX-500, AL-400, and Varian
Gemini 2000. Mass spectra were measured on J EOL J MS DX-
303, AX-500, and AX-700. HPLC was carried out on Cosmosil
5C18-AR 10 mm × 250 mm (Nacalai Tesquie, Inc., Kyoto,
J apan). ICR mice (male, 5 weeks old) were used for preparation
of s9.
Meta bolism of 1 a n d 3. Febrifugine hydrochloride (1, 115
mg) was incubated at 37 °C for 1 h with mice s9 mix (480 mL,
2.5 mg protein/mL), 1.3 mM NADP, 10 mM glucose 6-phos-
phate, 0.4 unit/mL glucose 6-phosphate dehydrogenase, and
5 mM magnesium chloride in phosphate-buffered saline (final
volume 800 mL). The reaction was quenched with ethyl acetate
(1.6 L) and 28% ammonia solution. The mixture was extracted
with ethyl acetate and n-butanol, respectively. Each organic
layer was concentrated to give ethyl acetate and n-butanol
solubles, respectively. In the same manner, Df-1 hydrochloride
(3, 112 mg) was metabolized to afford ethyl acetate and
n-butanol solubles.
Isola tion of F eb-A-D (4-7). The n-butanol solubles of
metabolites of 1 were chromatographed by HPLC under the
gradient condition as following: 5% acetonitrile for 0-15 min,
5-20% for 15-30 min, 20-30% for 30-40 min, 30-50% for
40-50 min, and 50-100% for 50-60 min), containing 0.01%
trifluoroacetic acid at a flow rate of 2.0 mL/min, to give feb-A
(4, 2.3 mg) and feb-B (5, 1.0 mg). In the same manner, feb-C
(6, 1.7 mg) and feb-D (7, 0.5 mg) were afforded from ethyl
acetate and n-butanol solubles of metabolites of 3, respectively.
6-(Tr iisop r op ylsilyloxy)qu in a zolin -4-on e (18). A mix-
ture of 5-hydroxyanthranilic acid (17, 5.16 g, 33.7 mmol) and
formamide (34 mL) was heated at 160 °C for 6 h. The mixture
was cooled to room temperature and poured into water and
filtered. The precipitate was washed with water and dried to
give quinazoline-4,6-diol (4.73 g, 87%) as a dark purple solid.
In vivo antimalarial activity against P. berghei in-
fected in mice was then determined for 4, 6, and 22,
the compounds showing notable activities by the in vitro
assay. As indicated in Table 2, intraperitoneal (i.p.)
administration of 4 and 6 exhibited comparable or
stronger antimalarial activity (ED50, 0.6 and 2.4 mg/
kg/day) than the clinically used medicines, chloroquine
and artemisinin (ED50, 5.4 and 2.0 mg/kg/day), while
the activity observed with 22 was moderate (ED50, 6.0
mg/kg/day). Oral administration of 4 and 6 was also
effective against murine malaria with ED50 values of
15 mg/kg/day for 4 and 8 mg/kg/day for 6. While a single
administration of 1 at 3 mg/kg i.p. ordinarily depau-
perated mice, no serious toxicities, such as diarrhea or
weight loss, were noted, even after 4 consecutive days
of treatment with 4, 6, and 22 (20, 100, and 50 mg/kg/
day each). In addition, P. berghei-infected mice treated
with 30 mg/kg/day i.p. of 4 showed no toxicity, and one

4356 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Hirai et al.
To a solution of quinazoline-4,6-diol (2.00 g, 12.3 mmol) in
dimethylformamide (DMF, 25 mL) was added imidazole (2.14
g, 30.8 mmol) and triisopropylsilyl chloride (3.26 mL, 14.8
mmol). The mixture was stirred at 50 °C for 14 h. After it was
cooled, the mixture was poured into 1 M hydrochloric 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 n-hexane-diethyl ether (1:2) to give 18 (3.60 g,
92%).
3-[3-[(2R*,3S*)-3-Hyd r oxyp ip er id in -2-yl]-2-oxop r op yl]-
6-(t r iisop r op ylsilyloxy)-3H -q u in a zolin -4-on e (20) a n d
3-[(3a S*,7a S*)-2-Hyd r oxyocta h yd r ofu r o[3,2-b]p yr id in -2-
ylm et h yl]-6-(t r iisop r op ylsilyloxy)-3H -q u in a zolin -4-on e
(21). To a solution of 16 (186 mg, 0.501 mmol) in DMF (1 mL)
were added 18 (161 mg, 0.505 mmol) and potassium carbonate
(83.8 mg, 0.606 mmol). After the solution was stirred at room
temperature 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 chromatographed over silica
gel eluted by n-hexane-chloroform (1:19) to give crude 19 (283
mg).
Palladium hydroxide on carbon (20 wt % Pd, 188 mg) was
added to a methanol solution (5 mL) of this crude 19. Under
hydrogen atmosphere, this solution was stirred at room
temperature for 1 h. The mixture was filtered and concen-
trated. The residue was chromatographed over silica gel eluted
by chloroform-methanol (19:1) to give 21 (446 mg, 46% from
16).
A methanol solution (15 mL) of 21 (472 mg, 0.996 mmol)
was refluxed for 3 h. The reaction mixture was concentrated.
The residue was chromatographed over aluminum oxide to
afford 21 (239 mg, 51%) eluted by n-hexane-chloroform (1:1)
and 20 (207 mg, 44%) eluted by chloroform.
3-[3-[(2R*,3S*)-3-Hyd r oxyp ip er id in -2-yl]-2-oxop r op yl]-
6-h yd r oxy-3H-qu in a zolin -4-on e (F eb-A) (4). Compound 20
(101 mg, 0.213 mmol) was dissolved in 10% hydrogen chloride
containing methanol (5 mL) and heated at 50 °C for 24 h. The
reaction mixture was concentrated, and the residue was
chromatographed over aluminum oxide eluted by methanol to
give 4 (79.7 mg, 100%).
3-[(3a S*,7a S*)-2-Hyd r oxyocta h yd r ofu r o[3,2-b]p yr id in -
2-ylm eth yl]-6-h ydr oxy-3H-qu in azolin -4-on e (Isofeb-A) (22).
By the use of 25 (128 mg, 0.271 mmol) as a substrate, 22 (56.2
mg, 65%) was afforded in the same manner as the synthesis
of 4.
2-Am in o-N-ter t-bu tylben za m id e (24). To a solution of
isatoic anhydride (23, 3.00 g, 18.4 mmol) in DMF (18 mL) were
added DMAP (225 mg, 1.84 mmol) and tert-butylamine (2.16
mL, 20.3 mmol). After it was stirred at room temperature for
3.5 h, the mixture was poured into water and filtered. The
precipitate was washed with water and methanol and dried
to give 24 (1.61 g, 46%).
N-ter t-Bu tyl-2-m eth oxyca r bon yla m in oben za m id e (25).
To a solution of 24 (746 mg, 3.88 mmol) in dioxane (4 mL)
and 1 M NaOH (4 mL) was added methyl chloroformate (330
µL, 4.27 mmol). After it was stirred at room temperature for
30 min, the mixture was poured into 1 M hydrochloric acid
and filtered. The precipitate was washed with water and
methanol and dried to give 25 (922 mg, 95%).
washed with water and methanol and dried to give 3-tert-butyl-
1-(4-methoxybenzyl)-1H-quinazolin-2,4-dione (965 mg, 100%).
3-tert-Butyl-1-(4-methoxybenzyl)-1H-quinazolin-2,4-dione
(1.42 g, 4.21 mmol) was dissolved in dioxane (12.6 mL) and 6
M hydrochloric acid (8.4 mL). After it was refluxed for 6 h,
the mixture was cooled to room temperature, poured into
water, and filtered. The precipitate was washed with water
and methanol and recrystallized from chloroform-methanol
to give 27 (1.14 g, 96%).
1-(4-Meth oxyben zyl)-3-[(3aS*,7aS*)-N-ben zyloxyca r bo-
n yl-2-h yd r oxyocta h yd r ofu r o[3,2-b]p yr id in -2-ylm eth yl]-
3H-qu in a zolin -2,4-d ion e (28). To a mixture of 16 (243 mg,
0.659 mmol) and 27 (186 mg, 0.659 mmol) was added potas-
sium carbonate (109 mg, 0.790 mmol). After it was stirred at
room temperature for 1.5 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 chromatographed over silica
gel eluted by n-hexane-ethyl acetate (1:1) to give 28 (127 mg,
34%).
3-[3-[(2R*,3S*)-3-Hyd r oxyp ip er id in -2-yl]-2-oxop r op yl]-
3H-qu in a zolin -2,4-d ion e (F eb-B) (5) a n d 3-[(3a S*,7a S*)-
2-Hyd r oxyocta h yd r ofu r o[3,2-b]p yr id in -2-ylm eth yl]-3H-
qu in a zolin -2,4-d ion e (Isofeb-B) (31). Palladium hydroxide
on carbon (20 wt % Pd, 50.5 mg) was added to a solution of 28
(289 mg, 0.505 mmol) in ethanol-ethyl acetate (4:1) (6.5 mL).
Under hydrogen atmosphere, this solution was stirred at room
temperature for 2.5 h. The mixture was filtered and concen-
trated. The residue was chromatographed over silica gel eluted
by chloroform-methanol (4:1) to give a mixture of 29 and 30.
To a solution of this mixture in acetonitrile (3 mL) and water
(1.5 mL) was added cerium ammonium nitrate (1.06 g, 1.93
mmol). The mixture was stirred at room temperature for 2 h
and extracted with n-butanol three times. The organic layer
was concentrated. The residue was chromatographed over
aluminum oxide to give 31 (5.7 mg, 3%) eluted by chloroform-
methanol (19:1) and 5 (75.6 mg, 40%) eluted by chloroform-
methanol (9:1).
(2S*,3S*)-N-ter t-Bu t yloxyca r b on yl-2-a llyl-3-h yd r oxy-
p ip er id in e (32). A solution of 13 (20.3 g, 73.7 mmol) in
methanol (10 mL) and 6 M hydrochloric acid (50 mL) was
refluxed for 5 h. The mixture was alkalinized with saturated
sodium bicarbonate solution and extracted with n-butanol
three times. The organic layer was evaporated. The residue
was dissolved in dichloromethane (70 mL) and cooled at 0 °C.
To the solution were added triethylamine (12.3 mL, 88.5 mmol)
and di-tert-butyl dicarbonate (19.3 g, 88.5 mmol). After it was
stirred for 1.5 h at room temperature, the mixture was
acidified with 0.1 M hydrochloric 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 n-hex-
ane-ethyl acetate (4:1) to give 32 (8.08 g, 45%).
(2S*,3S*)-N-ter t-Bu t yloxyca r b on yl-2-a llyl-3-a cet oxy-
p ip er id in e (33). To an ice-cooled solution of 32 (8.03 g, 33.3
mmol) in pyridine (20 mL) were added acetic anhydride (3.8
mL, 39.9 mmol) and DMAP (407 mg, 3.33 mmol). After it was
stirred for 4.5 h at room temperature, the mixture was
acidified with 0.1 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. The residue was
chromatographed over silica gel eluted by n-hexane-ethyl
acetate (49:1) to give 33 (8.04 g, 86%).
(2S*,3S*)-N-ter t-Bu tyloxyca r bon yl-3-a cetoxy-2-(2,2-d i-
m eth yl-1,3-d ioxola n -4-ylm eth yl)p ip er id in e (34). To an ice-
cooled solution of 33 (8.04 g, 28.3 mmol) in acetone-aceto-
nitrile-water (1:1:1) (30 mL) were added 4 wt % osmium
tetroxide solution in water (0.9 mL, 0.142 mmol) and 4-
methylmorpholine N-oxide (3.98 g, 34.0 mmol). After it was
stirred for 2.5 h at room temperature, the mixture was
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
3-ter t-Bu tyl-1H-qu in a zolin -2,4-d ion e (26). A mixture of
25 (870 mg, 3.48 mmol) and potassium hydroxide (1.84 g, 27.8
mmol) in ethanol (17 mL) was refluxed for 9 h. The mixture
was acidified with 1 M hydrochloric acid and filtered. The
precipitate was washed with water and methanol and dried
to give 26 (365 mg, 84%).
1-(4-Meth oxyben zyl)-1H-qu in a zolin -2,4-d ion e (27). To
an ice-cooled solution of 26 (606 mg, 2.78 mmol) in DMF (5
mL) were added sodium hydride (122 mg, 3.06 mmol) and
p-methoxybenzyl chloride (430 mL, 3.06 mmol). After it was
stirred at room temperature for 3 h, the mixture was poured
into 1 M hydrochloric acid and filtered. The precipitate was

Metabolites of Febrifugine and Its Synthetic Analogue
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4357
gel eluted by n-hexane-ethyl acetate (1:1) to give (2S*,3S*)-
N-tert-butyloxycarbonyl-3-acetoxy-2-(2,3-dihydroxypropyl)pi-
peridine (8.14 g, 90%).
the mixture was cooled to room temperature, 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 a solution of (2S*,3S*)-N-tert-butyloxycarbonyl-3-acetoxy-
2-(2,3-dihydroxypropyl)piperidine (8.14 g, 25.6 mmol) in DMF
(30 mL) were added 2,2-dimethoxypropane (31.5 mL, 256
mmol) and p-toluenesulfonic acid (1.46 g, 7.68 mmol). After it
was stirred for 3.5 h at room temperature, the mixture was
acidified with 0.1 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. The residue was
chromatographed over silica gel eluted by n-hexane-ethyl
acetate (9:1) to give 34 (8.78 g, 95%).
(2S*,3S*)-N-ter t-Bu tyloxyca r bon yl-2-(2,2-d im eth yl-1,3-
d ioxola n -4-ylm et h yl)-3-m et h a n esu lfon yloxyp ip er id in e
(35). To a solution of 34 (8.73 g, 24.2 mmol) in methanol (25
mL) was added sodium methoxide (1.44 g, 26.7 mmol). After
it was stirred for 3.5 h at room temperature, the mixture was
poured into water and extracted with ethyl acetate three times.
The organic layer was washed with saturated sodium bicar-
bonate solution and brine, dried over anhydrous sodium
sulfate, and evaporated. To an ice-cooled solution of the residue
in dichloromethane (25 mL) were added triethylamine (4.1 mL,
29.1 mmol) and methanesulfonyl chloride (2.25 mL, 29.1
mmol). After it was stirred for 2 h at room temperature, the
mixture was acidified with 0.1 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.
The residue was chromatographed over silica gel eluted by
n-hexane-ethyl acetate (9:1) to give 35 (6.69 g, 70%).
(2S*)-N-ter t-Bu t yloxyca r b on yl-2-(2,2-d im et h yl-1,3-d i-
oxola n -4-ylm eth yl)-1,2,5,6-tetr a h yd r op yr id in e (36). To an
ice-cooled solution of 35 (9.11 g, 23.2 mmol) in THF (55 mL)
was added DBU (34.4 mL, 232 mmol). After it was refluxed
for 12 h, the mixture was cooled to room temperature, 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 chromatographed over silica gel eluted by n-hexane-ethyl
acetate (19:1) to give 36 (4.05 g, 59%).
(2S*)-N-ter t-Bu tyloxycar bon yl-2-(2,3-dih ydr oxypr opyl)-
1,2,5,6-tetr a h yd r op yr id in e (37). A solution of 36 (1.93 g,
6.49 mmol) in 10% hydrogen chloride containing methanol (6.5
mL) was refluxed for 13 h. The mixture was cooled to room
temperature and concentrated. To an ice-cooled solution of the
residue in water (6.5 mL) were added triethylamine (2.7 mL,
7.79 mmol) and di-tert-butyl dicarbonate (1.71 g, 7.79 mmol).
After it was stirred at 0 °C for 5 h, the mixture was poured
into 0.1 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. The residue was chromato-
graphed over silica gel eluted by n-hexane-ethyl acetate
(4:1) to give 37 (1.30 g, 78%).
The residue was dissolved in dichloromethane (3 mL) and
reacted with Dess-Martin periodinane (777 mg, 1.83 mmol).
After it was stirred at 0 °C for 1.5 h, the mixture was quenched
with sodium thiosulfate solution, and the mixture was ex-
tracted 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 activated aluminum oxide
eluted by n-hexane-chloroform (9:1) to give 40 (134 mg, 57%
from 38).
3-[3-[(2R*,3R*,4S*)-N-ter t-Bu tyloxyca r bon yl-3,4-d ih y-
d r oxyp ip er id in -2-yl]-2-oxop r op yl]-3H-qu in a zolin -4-on e
(41) a n d 3-[(3a S*,7S*,7a S*)-N-ter t-bu tyloxyca r bon yl-2,7-
d ih yd r oxyocta h yd r ofu r o[3,2-b]p yr id in -2-ylm eth yl]-3H-
qu in a zolin -4-on e (42). To an ice-cooled solution of 40 (37.0
mg, 0.09 mmol) in acetone-acetonitrile-water (1:1:1) (0.5 mL)
were added 1 wt % osmium tetroxide solution in water (110
µL, 0.004 mmol) and 4-methylmorpholine N-oxide (20.8 mg,
0.18 mmol). After it was stirred for 3 h, the mixture was
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 to give 41 (21.3 mg, 58%) eluted by chloroform-methanol
(19:1) and 42 (5.6 mg, 15%) eluted by chloroform-methanol
(199:1).
3-[3-[(2R*,3R*,4S*)-3,4-Dih yd r oxyp ip er id in -2-yl]-2-oxo-
p r op yl]-3H-qu in a zolin -4-on e (F eb-C) (6). Compound 41
(21.8 mg, 0.05 mmol) was dissolved in trifluoroacetic acid (0.2
mL) and dichloromethane (0.8 mL). After it was stirred at room
temperature for 1.5 h, the mixture was concentrated. The
residue was dissolved in 10% hydrogen chloride containing
methanol (1.5 mL) and then concentrated to give 6 (17.4 mg,
85%) as hydrochloride salt. When 41 was directly treated with
10% hydrogen chloride containing methanol at room temper-
ature, an unidentified product was formed together with 6.
3-[(3a S*,7S*,7a S*)-2,7-Dih yd r oxyocta h yd r ofu r o[3,2-b]-
p yr id in -2-ylm eth yl]-3H-qu in a zolin -4-on e (Isofeb-C) (43).
Compound 42 (13.3 mg, 0.03 mmol) was dissolved in 10%
hydrogen chloride containing methanol (1.5 mL) and stirred
at room temperature for 1.5 h. The reaction mixture was
concentrated, and the residue was chromatographed over
aluminum oxide eluted by chloroform-methanol (19:1) to give
43 (6.1 mg, 60%) as hydrochloride salt.
(3S,4R)-4-(N,N-Diben zyla m in o)-6-(ter t-bu tyld im eth yl-
silyloxy)-1-h exen -3-ol (47). (R)-2-(N,N-Dibenzylamino)-4-
(tert-butyldimethylsilyloxy)butan-1-ol (46) was prepared from
D-aspartic acid (44) according to the method reported by
Gmeiner et al.9 Dimethyl sulfoxide (DMSO, 3.4 mL, 48.3 mmol)
and 46 (6.43 g, 16.1 mmol) dissolved in dichloromethane (10
mL) were added to a solution of oxalyl chloride (2.1 mL, 24.1
mmol) in dichloromethane (80 mL) at -78 °C. After it was
stirred for 1 h, triethylamine (11.2 mL, 80.4 mmol) was added
to this mixture and stirred at room temperature. 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 crude (R)-
2-(N,N-dibenzylamino)-4-(tert-butyldimethylsilyloxy)buta-
nal. The crude aldehyde was dissolved in THF (30 mL) and
cooled to -78 °C. Vinylmagnesium bromide dissolved in THF
(1.0 M, 16.5 mL) was added to this solution. After it was stirred
for 1 h, the mixture was allowed to warm to room temperature
and poured into 1 M hydrochloric acid. 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 eluted by
n-hexane-ethyl acetate (19:1) to give 47 (4.87 g, 71% from 46).
(2S *)-N -t er t -Bu t yloxyca r b on yl-2-(oxir a n ylm e t h yl)-
1,2,5,6-tetr a h yd r op yr id in e (38). To an ice-cooled solution
of 37 (322 mg, 1.25 mmol) in THF (3 mL) were added sodium
hydride (150 mg, 3.76 mmol) and 1-(p-toluenesulfonyl)imida-
zole (251 mg, 1.13 mmol). After it was stirred for 15 h, the
mixture was extracted with ethyl acetate three times. The
organic layer was washed with brine, dried over anhydrous
sodium sulfate, and evaporated. The residue was chromato-
graphed over silica gel eluted by n-hexane-ethyl acetate (19:
1) to give 38 (146 mg, 49%).
3-[3-[(2S*)-N-ter t-Bu tyloxyca r bon yl-1,2,5,6-tetr a h yd r o-
p yr id in -2-yl]-2-oxop r op yl]-3H-qu in a zolin -4-on e (40). To
an ice-cooled suspension of potassium hydride (105 mg, 0.92
mmol) in DMF (2 mL) was added 4-hydroxyquinazoline (39)
(107 mg, 0.73 mmol), and the mixture was stirred at 0 °C for
1 h. A solution of 38 (146 mg, 0.61 mmol) in DMF (4 mL) was
added to the mixture. After it was stirred at 70 °C for 14 h,

4358 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Hirai et al.
(3S,4R)-4-(N,N-Diben zyla m in o)-6-(ter t-bu tyld im eth yl-
silyloxy)-3-m eth oxym eth oxy-1-h exen e (48). To a solution
of 47 (3.56 g, 8.36 mmol) in 1,2-dimethoxyethane (25 mL) were
added N,N-diisopropylethylamine (3.64 mL, 20.9 mmol), chlo-
romethyl methyl ether (1.27 mL, 16.7 mmol), and potassium
iodide (1.53 g, 9.19 mmol). After it was refluxed for 3 h, the
mixture was cooled to room temperature and poured into water
and extracted with ethyl acetate three times. The organic layer
was washed with 0.5 M hydrochloric acid, saturated sodium
bicarbonate solution, and brine, dried over anhydrous sodium
sulfate, and evaporated. The residue was chromatographed
over silica gel eluted by n-hexane-ethyl acetate (39:1) to give
48 (3.25 g, 83%).
chromatographed over silica gel eluted by n-hexane-ethyl
acetate (19:1) to give 52 (69.5 mg, 50%).
(5S,6R)-N-(p-Meth oxyben zyl)-6-(2-h ydr oxyethyl)-5-m eth -
oxym eth oxyp ip er id in -2-on e (53). To a solution of 52 (69.5
mg, 0.16 mmol) in THF (1.5 mL) was added 1.0 M tetrabuty-
lammonium fluoride solution in THF (0.56 mL). After it was
stirred at room temperature for 1 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
chloroform-methanol (99:1) to give 53 (44 mg, 86%).
(5S,6R)-N-(p-Meth oxyben zyl)-5-m eth oxym eth oxy-6-ox-
ir a n ylm eth ylp ip er id in -2-on e (54). To a solution of 53 (44
mg, 0.14 mmol) in dichloromethane (1.3 mL) was added Dess-
Martin periodinane (173 mg, 0.41 mmol). After it was stirred
at 0 °C for 1 h, the mixture was quenched with 10% sodium
thiosulfate 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.
To a solution of sodium hydride (5.4 mg, 0.14 mmol) in
DMSO (0.4 mL) were added a solution of the crude aldehyde
in DMSO (0.9 mL) and trimethylsulfoxonium iodide (30 mg,
0.14 mmol) at 0 °C. After it was stirred at room temperature
for 2 h, the mixture was poured into water and extracted with
ethyl acetate three times. The organic layer was washed with
brine, dried, and evaporated. The residue was chromato-
graphed over silica gel eluted by n-hexane-ethyl acetate (1:
3) to give 54 (18.6 mg, 41% from 53).
3-[3-[(2R,3S)-N-(p-Meth oxyben zyl)-3-m eth oxym eth oxy-
6-oxop ip er id in -2-yl]-2-oxop r op yl]-3H -q u in a zolin -4-on e
(55). To an ice-cooled suspension of potassium hydride (12.3
mg, 0.11 mmol) in DMF (0.3 mL) was added 4-hydrox-
yquinazoline (39) (15.7 mg, 0.11 mmol). After the mixture was
stirred at 0 °C for 30 min, a solution of 54 (17.3 mg, 0.05 mmol)
in DMF (0.7 mL) was added to this mixture. The mixture was
heated at 80 °C for 3.5 h. The mixture was cooled to room
temperature, quenched with a 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 in dichlo-
romethane (1.2 mL) was reacted with Dess-Martin periodi-
nane (65.7 mg, 0.15 mmol) and stirred at 0 °C for 2 h. The
mixture was quenched with 10% sodium thiosulfate solution,
and the mixture was extracted with ethyl acetate three times.
The organic layer was washed with brine, dried over anhy-
drous sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel eluted by chloroform-methanol
(49:1) to give 55 (19.8 mg, 80%).
3-[3-[(2R,3S)-3-Meth oxym eth oxy-6-oxop ip er id in -2-yl]-
2-oxop r op yl]-3H-qu in a zolin -4-on e (56). To a solution of 55
(18.9 mg, 0.023 mmol) in acetonitrile-water (2:1) (1.0 mL) was
added cerium ammonium nitrate (57.7 mg, 0.12 mmol). After
it was stirred at room temperature for 1.5 h, the mixture was
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 chloroform-methanol (97:3) to give 56 (8.5 mg,
60%).
(3S,4R)-4-(N,N-Diben zyla m in o)-6-(ter t-bu tyld im eth yl-
silyloxy)-3-m eth oxym eth oxy-h exa n e-1,2-d iol (49). Com-
pound 48 (3.21 g, 6.84 mmol) was dissolved in water (8 mL),
THF (8 mL), and tert-butyl alcohol (8 mL). Trimethylamine
N-oxide dihydrate (1.94 g, 17.1 mmol) and 4% osmium tetrox-
ide solution in water (870 µL, 0.137 mmol) were added to this
solution. After it was stirred at room temperature for 5 h, the
reaction was quenched with 10% sodium thiosulfate solution.
This mixture was extracted with ethyl acetate three times.
The organic layer was washed with 0.5 M hydrochloric acid,
saturated sodium bicarbonate solution, and brine, dried over
anhydrous sodium sulfate, and evaporated. The residue was
chromatographed over silica gel eluted by n-hexane-ethyl
acetate (7:3) to give 49 (2.80 g, 81%).
Ben zyl (2E,4S,5R)-5-(N,N-Diben zyla m in o)-7-(ter t-bu -
t yld im et h ylsilyloxy)-4-m et h oxym et h oxy-2-h ep t en oa t e
(50). Sodium periodate (1.10 g, 5.15 mmol) and water (7 mL)
were added to a solution of 49 (1.99 g, 3.96 mmol) in diethyl
ether (14 mL). After it was stirred vigorously for 3 h, the
mixture was extracted with diethyl ether three times, and the
organic layer was washed with brine, dried, and concentrated
to give crude (2S,3R)-3-(N,N-dibenzylamino)-5-(tert-butyldim-
ethylsilyloxy)-2-methoxymethoxypentanal. This crude alde-
hyde was added to a mixture of sodium hydride (174 mg, 4.35
mmol) and benzyl diethylphosphonoacetate11 (1.25 g, 4.35
mmol) in toluene (16 mL) at 0 °C. After it was stirred for 2 h,
the mixture was poured into water (20 mL) and extracted with
ethyl acetate three times. The organic layer was washed with
0.5 M hydrochloric acid, saturated sodium bicarbonate solu-
tion, and brine, dried over anhydrous sodium sulfate, and
evaporated. The residue was chromatographed over silica gel
eluted by n-hexane-ethyl acetate (19:1) to give 50 (1.96 g,
82%).
(5S,6R)-6-(2-ter t-Bu tyld im eth ylsilyloxyeth yl)-5-m eth -
oxym eth oxyp ip er id in -2-on e (51). Under hydrogen atmo-
sphere, 50 (1.40 g, 2.32 mmol) and 20% palladium hydroxide
on carbon (500 mg) in ethanol (24 mL) were stirred at room
temperature for 5 h. After the mixture was filtered, the filtrate
was evaporated to give an oily residue, which was crude
(4S,5R)-5-amino-7-tert-butyldimethylsilyloxy-4-methoxymethox-
yheptanoic acid (657 mg, 1.96 mmol). This oil was dissolved
in THF (10 mL), and this solution was cooled to 0 °C. 1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.50
g, 7.83 mmol) was added to the solution. After it was stirred
for 4 h, the mixture was poured into 0.5 M hydrochloric acid.
This mixture was extracted with ethyl acetate three times.
The organic layer was washed with saturated sodium bicar-
bonate solution and brine, dried over anhydrous sodium
sulfate, and evaporated. The residue was chromatographed
over silica gel eluted by chloroform-methanol (99:1) to give
51 (448 mg, 61% from 50).
3-[3-[(2R,3S)-3-Hyd r oxy-6-oxop ip er id in -2-yl]-2-oxop r o-
p yl]-3H-qu in a zolin -4-on e (F eb-D) (7). Compound 56 was
dissolved in trifluoroacetic acid (0.75 mL) and dichloromethane
(0.25 mL). After it was stirred at room temperature for 2.5 h,
the mixture was concentrated. The residue was dissolved in
10% hydrogen chloride containing methanol (1.5 mL) and then
concentrated to give 7 (6.8 mg, 100%) as hydrochloride salt.
An tim a la r ia l Assa ys In Vitr o a n d In Vivo. The antima-
larial activities in vitro and in vivo against P. falciparum and
P. berghei, respectively, were investigated as described in ref
10.
(5S,6R)-N-(p-Meth oxyben zyl)-6-(2-ter t-bu tyld im eth yl-
silyloxyet h yl)-5-m et h oxym et h oxyp ip er id in -2-on e (52).
To a solution of 51 (101 mg, 0.32 mmol) in DMF (1.5 mL) were
added p-methoxybenzyl chloride (50 µL, 0.35 mmol) and
sodium hydride (14.0 mg, 0.35 mmol). After it was stirred at
room temperature for 5.5 h, the mixture was quenched with
methanol and 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
Ack n ow led gm en t. This work was supported in part
by a Grant-in-Aid for Scientific Research (Nos. 15019007,

Metabolites of Febrifugine and Its Synthetic Analogue
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