Structure-activity relationship of anti-malarial spongean peroxides having a 3-methoxy-1,2-dioxane structure

Article abstract of DOI:10.1016/j.bmc.2004.04.051

In order to study the structure-activity relationship of anti-malarial spongean peroxides, several analogues concerning with the 6-methoxyacetyl moiety and the 3-pentyl residue in methyl 2-(3-methoxy-3-pentyl-1,2-dioxan-6-yl) acetate were synthesized and evaluated for anti-malarial activity. In order to study the structure-activity relationship of anti-malarial spongean peroxides, several analogues concerning with the 6-methoxyacetyl moiety and the 3-pentyl residue in methyl 2-(3-methoxy-3-pentyl-1,2-dioxan-6-yl)acetate were synthesized and evaluated for anti-malarial activity. The tert-butyl ester analogue 14 showed stability in mouse serum and a high selectivity index against the malaria parasite, Plasmodium falciparum, and the citronellyl analogue 31 exhibited the strongest in vitro anti-malarial activity among them, and the imidazole analogue 25 showed desirable in vivo anti-malarial activity against P. berghei infected mice.

Full text of DOI:10.1016/j.bmc.2004.04.051

Bioorganic & Medicinal Chemistry 12 (2004) 5297–5307
Structure–activity relationship of anti-malarial spongean
peroxides having a 3-methoxy-1,2-dioxane structure
Motoyuki Kawanishi,a Naoyuki Kotoku,a Sawako Itagaki,b Toshihiro Horiib and
Motomasa Kobayashia,*
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
bResearch Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Received 5 April 2004; revised 29 April 2004; accepted 29 April 2004
Available online 11 September 2004
Abstract—In order to study the structure–activity relationship of anti-malarial spongean peroxides, several analogues concerning
with the 6-methoxyacetyl moiety and the 3-pentyl residue in methyl 2-(3-methoxy-3-pentyl-1,2-dioxan-6-yl)acetate were synthesized
and evaluated for anti-malarial activity. The tert-butyl ester analogue 14 showed stability in mouse serum and a high selectivity
index against the malaria parasite, Plasmodium falciparum, and the citronellyl analogue 31 exhibited the strongest in vitro anti-
malarial activity among them, and the imidazole analogue 25 showed desirable in vivo anti-malarial activity against P. berghei
infected mice.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Malaria is one of the most deadly diseases for humans
worldwide and more than 2.5 million people die from
it each year.1 Due to the emergence and ongoing spread
of the chloroquine-resistant strains of Plasmodium falci-
parum,2 including multi-drug resistant strains to conven-
tional anti-malarial drugs, cyclic peroxides like
artemisinin (1) and its derivatives (2–3) are regarded as
new anti-malarial principals (Chart 1).3 In this context,
we studied the structure–activity relationship(SAR) of
the cyclic peroxide, methyl 2-(3-methoxy-3-pentyl-1,2-
dioxan-6-yl)acetate (7), as a scaffold.
Chart 1.
In our previous report, we disclosed that the spongean
peroxides, methyl esters (5, 6) of peroxyplakoric acid
A3 and B3 , showed potent in vitro anti-malarial activity
4
selectivity index against P. falciparum than those of 5
and 6.6 However, compound 7 did not exhibit in vivo
anti-malarial activity, since the methyl ester moiety in
7 was hydrolyzed in mouse serum to afford the corre-
sponding carboxylic acid 8, which showed significantly
reduced anti-malarial activity. The ester moiety in 7
was converted to an amide moiety, and the ethyl and
propyl amide analogues (9, 10) were found to exhibit
in vivo anti-malarial potency.7 This paper deals with a
more detailed SAR study of the ester function and the
3-alkyl side chain in 7.
and established a facile synthetic method for construc-
tion of their core skeleton, a 3-methoxy-1,2-dioxane
moiety, by Sc(OTf)3-mediated peroxyhemiacetalization
and intramolecular Michael addition.5 Furthermore,
the synthetic peroxide 7 was found to exhibit higher
Keywords: Anti-malarial; Cyclic peroxide; Structure–activity relation-
ship; Marine sponge.
*
Corresponding author. Tel.: +81 6 6879 8215; fax: +81 6 6879
8219; e-mail: kobayasi@phs.osaka-u.ac.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.051

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M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
2. Chemistry
drance circumjacent to the ester portion in 7 and the two
ketone analogues (16, 17) were designed.
The amide analogues (9, 10) showed in vivo anti-malar-
ial activity in a four-day suppressive test using Plasmo-
dium berghei infected mice; however, a tendency to
reduced in vitro activity (9: IC50 0.54lM, 10: 0.31lM)
in comparison with that of 7 (7: IC50 0.12lM) was seen.
In order to study SAR of the 6-methoxyacetyl portion
in the synthetic peroxide 7, we further designed and
synthesized several analogues (13–26) as depicted in
Chart 2.
Preparation of the ester analogues (13–15) and the
methyl ketone analogue 16 was conducted as illustrated
in Scheme 1. Treatment of the ketoaldehyde,6 which was
derived from ketoalcohol 32 by Swern oxidation, with
methyl 2-(triphenyl-k5-phosphanylidene)propionate8 or
commercially available tert-butyl (triphenyl-k5-phosph-
anylidene)acetate furnished keto-a,b-unsaturated esters
(33: 88%, 34: 92% for two steps), respectively. Conver-
sion from the keto-a,b-unsaturated esters (33, 34) to
3-methoxy-1,2-dioxanes (13, 14) was carried out via per-
oxyhemiacetal. Namely, the peroxyhemiacetalization of
33 and 34 in the presence of Sc(OTf)3 and subsequent
In consideration of metabolism in the serum,7 three ester
analogues: a-methyl analogue 13, tert-butyl ester ana-
logue 14, and phenyl ester analogue 15, with steric hin-
Chart 2. Design for structure–activity relationship study of spongean peroxides.
Scheme 1. Reagents and conditions: (a) DMSO, (COCl)2, Et3N, CH2Cl2; (b) Ph3P@C(R2)COR1, CH2Cl2, two steps 88% for 33, two steps 92% for
34, two steps 89% for 35; (c) H2O2ÆH2NCONH2, Sc(OTf)3, MeOH, 8.3% for 16; (d) Et2NH, CF3CH2OH, two steps 38% for 13, two steps 20% for 14;
(e) PhOH, EDCIÆHCl, pyridine, 98% for 15; (f) NaHMDS, THF, ꢀ78ꢁC; (g) MsCl, Et3N, CH2Cl2, two steps 73%; (h) H2O2ÆH2NCONH2, Sc(OTf)3,
MeOH, 17%.

M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
5299
intramolecular Michael addition of the peroxyhemiac-
etal provided two corresponding ester analogues (13:
38%, 14: 20% for two steps), respectively. Treatment
of the ketoaldehyde with commercially available 1-(tri-
phenyl-k5-phosphanylidene)propan-2-one furnished a
keto-a,b-unsaturated ketone 35 in 89% yield. Then, 35
was further converted to a methyl ketone 16 by peroxy-
hemiacetalization in the presence of Sc(OTf)3. On the
other hand, the carboxylic acid 8 was coupled with phe-
nol in the presence of N-ethyl-N0-3-dimethylaminoprop-
ylcarbodiimide hydrochloride (EDCIÆHCl) in pyridine
to give a phenyl ester analogue 15 in 98% yield. Next,
the analogue 17 was synthesized from the keto-a,b-
unsaturated ketone 38, which was prepared from ace-
tophenone (36) and ketoaldehyde 37 by aldol condensa-
tion, in the same fashion as for the preparation of 16.
Scheme 3. Reagents and conditions: (a) MeNHOMeÆHCl, Me2AlCl,
CH2Cl2; (b) BrMg-R, THF; (c) DMSO, (COCl)2, Et3N, CH2Cl2; (d)
Ph3P@CHCOOCH3, CH2Cl2, four steps 45% for 41, four steps 63%
for 42, four steps 59% for 43, four steps 77% for 44, four steps 69% for
45; (e) H2O2ÆH2NCONH2, Sc(OTf)3, MeOH; (f) Et2NH, CF3CH2OH,
two steps 1.1% for 27, two steps 42% for 28, two steps 28% for 29, two
steps 58% for 30, two steps 51% for 31.
Artemisinin (1), a typical cyclic peroxide with potent
anti-malarial activity, is proved to be degradated by
one-electron reduction with Fe(II) of heme in the food
vacuole,9 which is the characteristic acidic organelle
(pH5.0–5.4) in the malaria parasite.10,11 The resulting
radical species were presumed to form covalent adducts
with proteins in the food vacuole to inhibit the prolifer-
ation of the malaria parasites. OꢀNeill and co-workers12
noted this character of the heme-rich parasite food vac-
uole, and they synthesized an artemisinin derivative 4
with the N atom (Chart 1) and assessed the ꢁion-trap-
pingꢀ effect in expecting the accumulation of 4 in the
food vacuole. Watanabe and co-workers reported that
the imidazole moiety facilitated oxidation of heme
iron.13 We designed amide analogues (18–26) having
the N atom as depicted in Chart 2. Preparation of the
eight amide analogues (18–25) having the N atom and
the dimeric analogue 26 was carried out by treatment
of the pentafluorophenyl ester 397 with commercially
available amines having the N atom, in good yields
(Scheme 2).
The syntheses of the analogues (27–31) having a differ-
ent alkyl moiety at the C-3 position of the 3-methoxy-
1,2-dioxane were conducted as illustrated in Scheme 3.
Treatment of the Weinreb amide, which was prepared
from c-butyrolactone 40, with each Grignard reagent
provided the corresponding ketoalcohols. The Swern
oxidation of the ketoalcohols and subsequent Wittig
reaction with methyl (triphenyl-k5-phosphanylid-
ene)acetate furnished the corresponding keto-a,b-unsat-
urated esters (41: 45%, 42: 63%, 43: 59%, 44: 77%, 45:
69% for four steps). The peroxyhemiacetalization of
the keto-a,b-unsaturated esters (41–45) in the presence
of Sc(OTf)3 and subsequent intramolecular Michael
addition provided the corresponding analogues possess-
ing a different alkyl moiety at the C-3 position of the 3-
methoxy-1,2-dioxane (27: 1.1%, 28: 42%, 29: 28%, 30:
58%, 31: 51% for two steps). Generally, the reaction of
peroxyhemiacetalization takes two days, while the reac-
tion for 41 proceeded very slowly to afford 27 in poor
yield because of the steric hindrance.
When the 3-pentyl analogue 7 was treated with FeSO4 as
a model experiment in the food vacuole, 11 and 12 were
afforded as major products (Chart 2).14 This result sug-
gested that the alkyl radical i, which was a plausible ac-
tive principal against the malaria parasite, was generated
in this reaction. Previously, we reported that the anti-
malarial activity of the analogue with a methyl residue
at C-3 in the 3-methoxy-1,2-dioxane moiety was weaker
than those of the analogues with a pentyl or nonyl resi-
due,6 since the methyl radical is less stable than pentyl or
nonyl radicals. For the purpose of production of a more
stable radical, we designed 3-cyclohexyl analogue 27, 3-
phenyl analogue 28, 3-benzyl analogue 29, and two ana-
logues (30, 31) with an olefin portion (Chart 2).
3. Biological properties and discussion
The in vitro anti-malarial activity against P. falciparum
and cytotoxicity against human epidermoid carcinoma
cells (KB 3-1) of the synthesized analogues (13–31) were
evaluated.15 The in vitro anti-malarial activity of the
three ester analogues (13–15) and the related analogues
(16, 17) was depicted in Table 1. Compounds 13 and 14
showed similar activity to that of the methyl ester ana-
logue 7, while the phenyl ester analogue 15 and two re-
lated analogues (16, 17) showed only weak activity.
Moreover, the selectivity index of 14 against P. falcipa-
rum was higher than that of 7. These results indicated
that the ester function is related with anti-malarial activ-
ity. As a result of the assessment for stability in the
mouse serum, tert-butyl ester analogue 14 was shown
to be free from metabolism in comparison with the
methyl ester analogue 7 and the phenyl ester analogue
15 (Fig. 1). Hence, 14 is expected to exhibit in vivo
anti-malarial activity.
Scheme 2. Reagents and conditions: (a) R-NH2, THF, 91% for 18;
quant. for 19; quant. for 20; 85% for 21; quant. for 22; 95% for 23; 88%
for 24; quant. for 25; quant. for 26.

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M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
Table 3. In vivo anti-malarial activity of analogues
Table 1. In vitro anti-malarial activity of analogues (13–17)
Compd
R1
R2
IC50 (lM)
Selectivity
index
Compd
ED50 (mg/kg) P. berghei
T/Ca
P. falciparum KB 3-1
14
22
32
>10 (27%)
125
117
125
122
136
81
13
14
15
16
17
7
CH3O Me
0.26
0.12
26
70
100
583
23
24
>10 (20%)
>10 (15%)
18
t-BuO
PhO
Me
H
H
H
H
H
>3.1
N. T.
N. T.
N. T.
43
25
>4.1
>3.3
7
10
>30
9.3
5
Ph
CH3O
138
145
0.12
360
140
230
Artemisinin
5
0.15
0.12
21
28
a Dose of 14, 22–25, 7, and 10: 10mg/kg, artemisinin: 5mg/kg T/C is
the quotient of the survival days of the treated animals (T) and those
of the control animals (C). T/C values of >120 are considered to be
active.
6
N.T.: not tested.
little in vivo activity at 30mg/kg by intraperitoneal
administration, the tert-butyl ester analogue 14 showed
in vivo potency (T/C 125). Among the amide analogues
(22–25) having the N atom, the imidazole analogue 25
showed the highest in vivo potency (ED50 = 18mg/kg,
T/C 136). In the case of the propyl amide analogue 10,
which showed potent in vivo anti-malarial activity, the
mortality of mice was observed at the 100mg/kg dose,
while, no obvious sign of acute toxicity such as decrease
of body weight and diarrhea was observed at the same
dose of 25. This result suggests that the toxicity of
the 3-methoxy-1,2-dioxane for mice was decreased
appreciably.
On the other hand, the analogues possessing a different
3-alkyl side chain (27–31) were evaluated for in vitro
anti-malarial activity and cytotoxicity (Table 4). The
anti-malarial activities of 27, 28, and 29 were reduced
in comparison with that of 7, while the pentenyl ana-
logue 30 showed similar activity to that of 7 and the
citronellyl analogue 31 showed stronger activity. More-
over, the methyl ester analogue 7 showed no in vitro
anti-malarial activity at the dose less than 0.01lg/mL,
while the two analogues (30, 31) having an olefin func-
tion showed about 40% growth inhibitory activity
against malaria parasites at the same dose (Fig. 2). On
the other hand, this tendency was not observed against
KB 3-1 cells (Fig. 3).
Figure 1. Stability of esters (7, 14, and 15).
Similarly, the analogues (18–26) having the N atom were
evaluated for in vitro anti-malarial activity. Each ana-
logue (18–26) showed similar activity, which was some-
what weaker than that of 7 (Table 2).
Among the analogues (13–17) with steric hindrance cir-
cumjacent to the ester portion and the analogues (18–26)
with the N atom on the amide portion, tert-butyl ester
analogue 14 and several amide analogues (22–25) were
evaluated for in vivo anti-malarial activity against P.
berghei infected mice. The results are summarized in
Table 3. Although the methyl ester analogue 7 showed
As shown in Chart 3, the radical species ii and v first de-
rived from the olefin analogues (30, 31) could be easily
converted to secondary stable radicals (iii or iv, vi or
vii). From these evidences, we presumed that the pentyl
Table 2. In vitro anti-malarial activity of analogues (18–26)
Compd
IC50 (lM)
P. falciparum
Selectivity index
KB 3-1
Table 4. In vitro anti-malarial activity of analogues (27–31)
18
19
20
21
22
23
24
25
26
10
0.48
0.42
0.63
0.85
0.38
0.36
0.36
0.26
0.46
0.31
12
12
16
17
18
6
26
28
26
20
47
16
79
47
10
39
Compd
IC50 (lM)
P. falciparum
Selectivity index
KB 3-1
27
28
29
30
31
7
1.2
1.2
N.T.
N.T.
N.T.
78
>3.6
0.28
0.033
0.12
279
158
360
29
12
5
5.2
43
12
N.T.: not tested.

M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
5301
4. Experimental
The following instruments were used to obtain physical
data: a Hitachi 330 spectrophotometer for UV spectra; a
JASCO FT/IR-5300 infrared spectrometer for IR spec-
tra; a JEOL JMS SX-102 mass spectrometer for FAB-
MS; a JEOL JNM AL-500 (500MHz) NMR spectro-
meter for 1H NMR (CDCl3 solution with tetramethysilane
(TMS) as an internal standard unless otherwise speci-
fied). HPLC was performed using a Hitachi L-6000
pump equipped with Hitachi L-4000H UV detector. Sil-
ica gel (Merck 60–230mesh) and pre-coated thin layer
chromatography (TLC) plates (Merck, Kiesel gel,
60F254) were used for column chromatography and
TLC. Spots on TLC plates were detected by spraying
acidic p-anisaldehyde solution (p-anisaldehyde: 25mL,
c-H2SO4: 25mL, AcOH: 5mL, EtOH: 425mL) with sub-
sequent heating.
Figure 2. In vitro anti-malarial activity of analogues (7, 30, and 31).
4.1. General procedure A: preparation of Weinreb amide
followed by Grignard reaction
Me2AlCl (1.0M in n-hexane, 1.9equiv) was treated with
the anhydrous CH2Cl2 solution of MeONHMeÆHCl
(1.5M, 1.9equiv) at 0ꢁC, and the whole was stirred for
1h at room temperature. After adding an anhydrous
CH2Cl2 solution of c-butyrolactone (40, 0.17M), the
whole was stirred for 1h at room temperature. After
quenching with phosphate buffer (pH8.0) at 0ꢁC, the
whole was stirred for 10min. The reaction mixture was
diluted with anhydrous CHCl3, and the resulting residue
was removed by Celite column. The filtrate was ex-
tracted with CHCl3, dried over MgSO4, and concen-
trated under reduced pressure to afford a Weinreb
amide.
Figure 3. In vitro cytotoxic activity of analogues (7, 30, and 31).
A THF solution of the Weinreb amide (0.18M) was
treated with Grignard reagent (8.0equiv), which was
prepared from commercially available alkylbromide, at
room temperature for 30min. The reaction mixture
was quenched with 25% aqueous H2SO4 at 0ꢁC, and
the whole was stirred for 10min at room temperature.
The reaction mixture was poured into saturated aqueous
NaHCO3, and the whole was extracted with EtOAc. The
EtOAc extract was washed with saturated aqueous
NaCl and dried over MgSO4. Removal of solvent from
the EtOAc extract under reduced pressure gave a crude
product, which was further purified by SiO2 column (n-
hexane–EtOAc) to furnish a ketoalcohol.
4.2. General procedure B: Swern oxidation
DMSO (6.0equiv) was added to the anhydrous CH2Cl2
solution of (COCl)2 (0.06M, 3.0equiv) at ꢀ78ꢁC, and
the whole was stirred for 20min. After adding an anhy-
drous CH2Cl2 solution of ketoalcohol (0.028M) to the
reaction mixture at ꢀ78ꢁC, the whole was stirred for
30min. Then, the reaction mixture was treated with
Et3N (8.0equiv) at ꢀ78ꢁC for 2h. After the reaction
mixture was diluted with anhydrous Et2O, the filtrate
given through Na2SO4 column was concentrated under
reduced pressure to afford a ketoaldehyde.
Chart 3. Plausible radical production from 3-methoxy-1,2-dioxane
moiety.
radical i produced from the 3-pentyl analogue 7 is unsta-
ble and short-lived, while the radical species produced
from the olefin analogues (30, 31) would be more
long-lived to form covalent adducts with a parasiteꢀs
protein.

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M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
4.3. General procedure C: Wittig reaction
Compound 13: colorless oil. IR mmax (KBr) cmꢀ1: 1744.
1H NMR (500MHz, CDCl3) d: 0.87 (3H, t, J = 7.3Hz,
CH3-a), 0.88 (3H, t, J = 7.3Hz, CH3-b), 1.22 (3H, d,
J = 7.3Hz, 10-CH3-a), 1.29 (3H, d, J = 6.7Hz, 10-CH3-
b), 1.13–1.44, 1.46–1.68, 1.74–1.89, and 2.15 (total
12H · 2, m), 2.50 (1H, dq, J = 12.2, 7.3Hz, 10-Ha),
3.06 (1H, dq, J = 9.8, 6.7Hz, 10-Hb), 3.24 (3H, s, 3-
OCH3-a), 3.27 (3H, s, 3-OCH3-b), 3.67 (3H, s,
CO2CH3-a), 3.68 (3H, s, CO2CH3-b), 4.00 (1H, m, 6-
Ha), 4.17 (1H, m, 6-Hb). FAB-MS m/z: 297
(M + Na)+. FAB-HRMS m/z: calcd for C14H26NaO5:
297.1678, found: 297.1694.
An anhydrous CH2Cl2 solution of ketoaldehyde
(0.016M) was treated with phosphonylidene reagent
(1.2–2.3equiv) at room temperature over night. Re-
moval of solvent under reduced pressure gave a crude
product, which was further purified by SiO2 column
(n-hexane–EtOAc) to furnish a keto-a,b-unsaturated
ester.
4.4. General procedure D: preparation of peroxy-
hemiacetal
4.8. tert-Butyl (E)-6-oxoundec-2-enoate (34)
An anhydrous MeOH solution of keto-a,b-unsaturated
ester (0.025M) was treated with Sc(OTf)3 (0.003M)
and H2O2ÆH2NCONH2 (7.5equiv) at room temperature
for 48h. The reaction mixture was diluted with CH2Cl2,
and the resulting residue was removed by column
packed with aluminum oxide 90 (neutral, 70–230mesh,
Merck Co. Ltd). Removal of solvent from the filtrate
under reduced pressure gave a crude product, which
was further purified by SiO2 column (n-hexane–EtOAc)
rapidly to furnish a peroxyhemiacetal.
As described in procedures B and C, 32 (200mg,
1.3mmol) was converted to 34 (303mg, two steps 92%).
Compound 34: colorless oil. IR mmax (KBr) cmꢀ1: 1717,
1655. 1H NMR (500MHz, CDCl3) d: 0.85 (3H, t,
J = 6.7Hz, 11-CH3), 1.17–1.31 (4H, m), 1.43 (9H, s,
tert-Bu), 1.54 (2H, m), 2.36 (2H, t, J = 7.3Hz, 7-H),
2.40 (2H, td, J = 7.3, 6.7Hz, 4-H), 2.52 (2H, t,
J = 7.3Hz, 5-H), 5.70 (1H, d, J = 15.8Hz, 2-H), 6.78
(1H, dt, J = 15.8, 6.7Hz, 3-H). FAB-MS m/z: 277
(M + Na)+. FAB-HRMS m/z: calcd for C15H26NaO3:
277.1779, found: 277.1779.
4.5. General procedure E: preparation of 3-methoxy-1,2-
dioxane
4.9. tert-Butyl 2-(3-methoxy-3-pentyl-1,2-dioxan-6-yl)-
acetate (14)
Et2NH (0.005M) was added to a CF3CH2OH solution
of peroxyhemiacetal (0.016M) at 0ꢁC, and the whole
mixture was stirred at room temperature for 8h. Re-
moval of solvent from the whole mixture under reduced
pressure gave a product, which was purified by SiO2 col-
umn (n-hexane–EtOAc) to furnish a 3-methoxy-1,2-di-
oxane.
As described in procedures D and E, 34 (100mg,
0.39mmol) was converted to 14 (24mg, two steps 20%).
The tert-butyl ester analogue 14 was obtained as a mix-
ture of syn and anti isomers. The physicochemical data
were analyzed as a mixture. In the H NMR spectra,
1
4.6. Methyl (E)-2-methyl-6-oxoundec-2-enoate (33)
only the signal due to 3-OCH3 was definitely separated
between the two isomers.
As described in procedures B and C, 32 (98mg,
0.63mmol) was converted to 33 (152mg, two steps 88%).
Compound 14: colorless oil; syn:anti = 5:1. IR mmax
(KBr) cmꢀ1: 1744. 1H NMR (500MHz, CDCl3) d:
0.87 (3H, t, J = 6.7Hz, CH3), 1.20–1.36 and 1.44–1.89
(total 12H, m), 1.43 (9H, s, tert-Bu), 2.24 (1H, dd,
J = 15.3, 6.1Hz, 10-Ha), 2.40 (1H, dd, J = 15.3, 7.3Hz,
10-Hb), 3.24 (3H, s, 3-OCH3, syn), 3.26 (3H, s, 3-
OCH3, anti), 4.42 (1H, m, 6-H). FAB-MS m/z: 325
(M + Na)+. FAB-HRMS m/z: calcd for C16H30NaO5:
325.1991, found: 325.1987.
Compound 33: colorless oil. IR mmax (KBr) cmꢀ1: 1715,
1646. 1H NMR (500MHz, CDCl3) d: 0.87 (3H, t,
J = 7.3Hz, H-11), 1.20–1.32 and 1.56 (total 6H, m),
1.83 (3H, d, J = 1.2Hz, 2-CH3), 2.38 (2H, t,
J = 7.3Hz, 7-H), 2.41 (2H, td, J = 7.3, 7.3Hz, 4-H),
2.52 (2H, t, J = 7.3Hz, 5-H), 3.70 (3H, s, CO2CH3),
6.65 (1H, td, J = 7.3, 1.2Hz, 3-H). FAB-MS m/z: 249
(M + Na)+. FAB-HRMS m/z: calcd for C13H22NaO3:
249.1467, found: 249.1475.
4.10. Phenyl 2-(3-methoxy-3-pentyl-1,2-dioxan-6-yl)ace-
tate (15)
4.7. Methyl 2-(3-methoxy-3-pentyl-1,2-dioxan-6-yl)pro-
panoate (13)
A pyridine (0.5mL) solution of 8 (5mg, 0.02mmol) was
treated with phenol (2.3mg, 0.024mmol) in presence of
EDCIÆHCl (7.7mg, 0.04mmol) at room temperature
for 4h. Removal of solvent under reduced pressure gave
a crude product, which was further purified by SiO2 col-
umn (n-hexane–EtOAc = 10:1) to furnish 15 (6.3mg,
98%). The phenyl ester analogue 15 was obtained as a
mixture of syn and anti isomers. The physicochemical
As described in procedures D and E, 33 (80mg,
0.35mmol) was converted to 13 (36mg, two steps
38%). The a-methyl analogue 13 was obtained as a dia-
stereo-mixture in a ratio of 1:1. The physicochemical
1
data were analyzed as a mixture. In the H NMR spec-
tra, only the signals due to 3-OCH3 and CO2CH3 were
definitely separated between the two isomers of 10-
CH3. The ratio of syn and anti isomers was difficult to
determine.
1
data were analyzed as a mixture. In the H NMR spec-
tra, only the signal due to 3-OCH3 was definitely sepa-
rated between the two isomers.

M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
5303
Compound 15: colorless oil; syn:anti = 4.8:1. IR mmax
(KBr) cmꢀ1: 1763. 1H NMR (500MHz, CDCl3) d:
0.83 (3H, t, J = 6.7Hz, CH3), 1.17–1.31, 1.54–1.69,
and 1.79–1.90 (total 12H, m), 2.56 (1H, dd, J = 15.9,
5.5Hz, 10-Ha), 2.68 (1H, dd, J = 15.9, 7.8Hz, 10-Hb),
3.22 (3H, s, 3-OCH3, syn), 3.25 (3H, s, 3-OCH3, anti),
4.54 (1H, m, 6-H), 7.03 (2H, d, J = 8.5Hz, Ph), 7.16
(1H, d, J = 7.3Hz, Ph), 7.30 (2H, dd, J = 8.5, 7.3Hz,
Ph). FAB-MS m/z: 345 (M + Na)+. FAB-HRMS m/z:
calcd for C18H26NaO5: 345.1678, found: 345.1662.
poured into saturated aqueous NaHCO3, extracted with
CH2Cl2, dried over MgSO4. Removal of solvent under
reduced pressure gave a crude product, which was fur-
ther purified by SiO2 column (n-hexane–EtOAc = 5:1)
to furnish 38 (358mg, two steps 73%).
Compound 38: colorless oil. IR mmax (KBr) cmꢀ1: 1715,
1
1671, 1622. H NMR (500MHz, CDCl3) d: 0.87 (3H, t,
J = 7.3Hz, 11-CH3), 1.20–1.34, and 1.46–1.61 (total 6H,
m), 2.41 (2H, t, J = 7.9Hz, 7-H), 2.55–2.64 (4H, m, 4-H
and 5-H), 6.88 (1H, d, J = 15.3Hz, 2-H), 6.98 (1H, dt,
J = 15.3, 6.1Hz, 6-H), 7.45 (2H, dd, J = 7.3, 6.7Hz,
Ph), 7.54 (1H, d, J = 6.7Hz, Ph), 7.89 (2H, d,
J = 7.3Hz, Ph). FAB-MS m/z: 259 (M + H)+. FAB-
HRMS m/z: calcd for C17H23O2: 259.1698, found:
259.1705.
4.11. (E)-Dodec-3-ene-2,7-dione (35)
As described in procedures B and C, 32 (142mg,
0.91mmol) was converted to 35 (158mg, two steps 89%).
Compound 35: colorless oil. IR mmax (KBr) cmꢀ1: 1715,
1
1676, 1628. H NMR (500MHz, CDCl3) d: 0.86 (3H, t,
4.14. 2-(3-Methoxy-3-pentyl-1,2-dioxan-6-yl)-1-phenyl-
ethan-1-one (17)
J = 7.3Hz, 12-CH3), 1.19–1.34 (total 4H, m), 1.56 (2H,
tt, J = 7.9, 7.3Hz, 9-H), 2.20 (3H, s, COCH3), 2.38
(2H, t, J = 7.3Hz, 8-H), 2.46 (2H, td, J = 7.3, 6.7Hz,
5-H), 2.56 (2H, t, J = 7.3Hz, 6-H), 6.04 (1H, d,
J = 15.9Hz, 3-H), 6.76 (1H, dt, J = 15.9, 6.7Hz, 4-H).
FAB-MS m/z: 197 (M + H)+. FAB-HRMS m/z: calcd
for C12H21O2: 197.1542, found: 197.1555.
As described in procedure D, 38 (18.3mg, 0.07mmol)
was converted to 17 (3.6mg, 17%).
The phenyl ketone analogue 17 was obtained as a mix-
ture of syn and anti isomers. The physicochemical data
were determined as a mixture. In the H NMR spectra,
1
4.12. 1-(3-Methoxy-3-pentyl-1,2-dioxan-6-yl)propan-2-
one (16)
only the signal due to 3-OCH3 was definitely separated
between the two isomers.
As described in procedure D, 35 (10mg, 0.05mmol) was
converted to 16 (1.0mg, 8.3%). The methyl ketone ana-
logue 16 was obtained as a mixture of syn and anti iso-
mers. The physicochemical data were analyzed as a
mixture. In the H NMR spectra, only the signal due
to 3-OCH3 was definitely separated between the two
isomers.
Compound 17: colorless oil; syn:anti = 3.9:1. IR mmax
(KBr) cmꢀ1: 1687. 1H NMR (500MHz, CDCl3) d:
0.83 (3H, t, J = 6.7Hz, CH3), 1.16–1.32 and 1.45–1.86
(total 12H, m), 2.87 (1H, dd, J = 16.5, 6.7Hz, 10-Ha),
3.17 (1H, dd, J = 16.5, 6.1Hz, 10-Hb), 3.21 (3H, s, 3-
OCH3, syn), 3.23 (3H, s, 3-OCH3, anti), 4.64 (1H, m,
6-H), 7.39 (2H, t, J = 7.3Hz, Ph), 7.50 (1H, t,
J = 7.3Hz, Ph), 7.87 (2H, d, J = 7.3Hz, Ph). FAB-MS
m/z: 329 (M + Na)+. FAB-HRMS m/z: calcd for
C18H26NaO4: 329.1728, found: 329.1732.
1
Compound 16: colorless oil; syn:anti = 12.5:1. IR mmax
(KBr) cmꢀ1: 1717. 1H NMR (500MHz, CDCl3) d:
0.82 (3H, t, J = 6.7Hz, CH3), 1.13–1.30 and 1.40–1.63
(total 10H, m), 1.68 (1H, ddd, J = 15.9, 11.4, 4.3Hz),
1.80 (1H, ddd, J = 12.2, 5.5, 1.8Hz), 2.12 (3H, s,
COCH3), 2.36 (1H, dd, J = 16.5, 5.5Hz, 10-Ha), 2.57
(1H, dd, J = 16.5, 6.7Hz, 10-Hb), 3.19 (3H, s, 3-OCH3,
syn), 3.24 (3H, s, 3-OCH3, anti), 4.43 (1H, m, 6-H).
FAB-MS m/z: 267 (M + Na)+. FAB-HRMS m/z: calcd
for C13H24NaO4: 267.1573, found: 267.1573.
4.15. General procedure F: preparation of amide analog-
ues having N atom
An amine having N atom (1.0equiv) was added to the
THF solution (0.2mL) of 39 (0.09M) at room tempera-
ture, and the whole was stirred for 10min. Removal of
solvent under reduced pressure gave a crude product,
which was further purified by SiO2 column (n-hexane–
EtOAc = 3:1, CHCl3–MeOH = 3:1) to furnish an amide
analogue having N atom.
4.13. (E)-1-Phenylundec-2-ene-1,6-dione (38)
NaHMDS (1.0M in THF, 1.54mL, 1.54mmol) was
added to the THF solution (3mL) of 1-phenylethan-1-
one (36, 221lL, 1.9mmol) at ꢀ78ꢁC, and the whole
was stirred for 30min. Ketoaldehyde (37, 297mg,
1.4mmol) in THF (100mL) was added to the reaction
mixture at ꢀ78ꢁC, and the whole was stirred for 5h.
The reaction mixture was poured into 5% aqueous
HCl solution, extracted with EtOAc, washed with brine,
and dried over MgSO4. Removal of solvent under re-
duced pressure gave a crude product. The crude product
was dissolved in CH2Cl2 (3mL) and treated with MsCl
(164lL, 2.1mmol) and Et3N (793lL, 5.7mmol) at room
temperature overnight. The reaction mixture was
The amide analogues (18–26) were obtained as a mix-
ture of syn and anti isomers. The physicochemical data
were analyzed as a mixture. In the H NMR spectra,
only the signal due to 3-OCH3 was definitely separated
between the two isomers. The ratio of syn and anti ziso-
mers of 21 was difficult to determine.
1
4.16. N-[2-(Ethylamino)ethyl]-2-(3-methoxy-3-pentyl-1,2-
dioxan-6-yl)acetamide (18)
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 18 (5.2mg, 91%).

5304
M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
Compound 18: colorless solid; syn:anti = 4.1:1. IR mmax
4.20. 2-(3-Methoxy-3-pentyl-1,2-dioxan-6-yl)-N-[2-(1,4-
oxazinan-4-yl)ethyl]acetamide (22)
1
(KBr) cmꢀ1: 1649, 1543. H NMR (500MHz, CDCl3)
d: 0.82 (3H, t, J = 7.3Hz), 1.09 (3H, t, J = 7.3Hz),
1.22–1.29, and 1.50–1.85 (total 12H, m), 2.26 (1H, dd,
J = 15.3, 4.3Hz, 10-Ha), 2.65 (1H, dd, J = 15.3, 7.9Hz,
10-Hb), 2.32 (2H, q, J = 7.3Hz, NCH2CH3), 2.75 (2H,
dt, J = 6.1, 3.1Hz), 3.19 (3H, s, 3-OCH3, syn), 3.21
(3H, s, 3-OCH3, anti), 3.33 (2H, dt, J = 5.5, 3.1Hz),
4.37 (1H, m, 6-H), 6.52 (1H, br s, NH). FAB-MS m/z:
317 (M + H)+. FAB-HRMS m/z: calcd for
C16H33N2O4: 317.2440, found: 317.2442.
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 22 (6.4mg, quant.).
Compound 22: colorless solid; syn:anti = 4.3:1. IR
mmax (KBr) cmꢀ1: 1649, 1541. 1H NMR (500MHz,
CDCl3) d: 0.83 (3H, t, J = 6.7Hz), 1.15–1.40 and
1.47–1.85 (total 12H, m), 2.24 (1H, dd, J = 15.3,
3.7Hz, 10-Ha), 2.32 (1H, dd, J = 15.3, 8.5Hz, 10-Hb),
2.46 (6H, br s, NCH2), 3.20 (3H, s, 3-OCH3, syn),
3.22 (3H, s, 3-OCH3, anti), 3.32 (2H, dt, J = 6.1,
5.5Hz, CONHCH2), 3.68 (4H, br s, CH2OCH2), 4.36
(1H, m, 6-H). FAB-MS m/z: 359 (M + H)+. FAB-
HRMS m/z: calcd for C18H35N2O5: 359.2545, found:
359.2552.
4.17. N-[2-(Diethylamino)ethyl]-2-(3-methoxy-3-pentyl-
1,2-dioxan-6-yl)acetamide (19)
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 19 (6.2mg, quant.).
Compound 19: colorless solid; syn:anti = 3.9:1. IR mmax
4.21. 2-(3-Methoxy-3-pentyl-1,2-dioxan-6-yl)-N-(2-phenyl-
ethyl)acetamide (23)
1
(KBr) cmꢀ1: 1672, 1541. H NMR (500MHz, CDCl3)
d: 0.82 (3H, t, J = 7.3Hz), 1.00–1.12, 1.15–1.29, and
1.49–1.85 (total 18H, m), 2.23 (1H, dd, J = 15.3,
4.9Hz, 10-Ha), 2.32 (1H, dd, J = 15.3, 8.5Hz, 10-Hb),
2.62 (6H, br s, NCH2), 3.19 (3H, s, 3-OCH3, syn), 3.21
(3H, s, 3-OCH3, anti), 3.33 (2H, br s, CONHCH2),
4.39 (1H, m, 6-H). FAB-MS m/z: 345 (M + H)+. FAB-
HRMS m/z: calcd for C18H37N2O4: 345.2753, found:
345.2753.
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 23 (6.0mg, 95%).
Compound 23: colorless solid; syn:anti = 4.2:1. IR mmax
1
(KBr) cmꢀ1: 1647, 1543. H NMR (500MHz, CDCl3)
d: 0.83 (3H, t, J = 7.3Hz), 1.18–1.36 and 1.44–1.86 (total
12H, m), 2.22 (1H, dd, J = 15.9, 4.3Hz, 10-Ha), 2.26
(1H, dd, J = 15.9, 8.5Hz, 10-Hb), 2.75 (2H, J = 7.3Hz,
CH2-Ph), 3.17 (3H, s, 3-OCH3, syn), 3.22 (3H, s, 3-
OCH3, anti), 3.44 (2H, m, CONHCH2), 4.30 (1H, m,
6-H), 7.10–7.27 (5H, m, Ph). FAB-MS m/z: 350
(M + H)+. FAB-HRMS m/z: calcd for C20H31NNaO4:
372.2151, found: 372.2130.
4.18. 2-(3-Methoxy-3-pentyl-1,2-dioxan-6-yl)-N-[2-tetra-
hydropyridin-1(2H)-ylethyl]acetamide (20)
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 20 (6.4mg, quant.).
Compound 20: colorless solid; syn:anti = 4.0:1. IR mmax
4.22. 2-(3-Methoxy-3-pentyl-1,2-dioxan-6-yl)-N-(2-pyridin-
4-ylethyl)acetamide (24)
1
(KBr) cmꢀ1: 1649, 1541. H NMR (500MHz, CDCl3)
d: 0.82 (3H, t, J = 6.7Hz), 1.12–1.28 and 1.30–1.83 (total
18H, m), 2.24 (1H, dd, J = 15.3, 4.3Hz, 10-Ha), 2.32
(1H, dd, J = 15.3, 8.5Hz, 10-Hb), 2.61 (6H, br s,
NCH2), 3.19 (3H, s, 3-OCH3, syn), 3.20 (3H, s, 3-
OCH3, anti), 3.40 (2H, dt, J = 5.5, 5.5Hz, CONHCH2),
4.40 (1H, m, 6-H). FAB-MS m/z: 357 (M + H)+. FAB-
HRMS m/z: calcd for C19H37N2O4: 357.2753, found:
357.2759.
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 24 (5.5mg, 88%).
Compound 24: colorless solid; syn:anti = 3.7:1. IR mmax
1
(KBr) cmꢀ1: 1651, 1555. H NMR (500MHz, CDCl3)
d: 0.83 (3H, t, J = 7.3Hz), 1.16–1.34 and 1.43–1.86 (total
12H, m), 2.25 (2H, d, J = 6.1Hz, 10-H), 2.78 (1H, dd,
J = 13.4, 6.7Hz, CH2-Py), 2.83 (1H, dd, J = 13.4,
6.7Hz, CH2-Py), 3.18 (3H, s, 3-OCH3, syn), 3.22 (3H,
s, 3-OCH3, anti), 3.42 (1H, ddt, J = 13.4, 6.7, 6.7Hz,
CONHCH2), 3.53 (1H, ddt, J = 13.4, 7.3, 6.1Hz,
CONHCH2), 4.27 (1H, m, 6-H), 7.17 (2H, d,
J = 4.9Hz, Py), 8.46 (2H, d, J = 4.9Hz, Py). FAB-MS
m/z: 351 (M + H)+. FAB-HRMS m/z: calcd for
C19H31N2O4: 351.2284, found: 351.2284.
4.19. 2-(3-Methoxy-3-pentyl-1,2-dioxan-6-yl)-N-[2-tetra-
hydropyrazin-1(2H)-ylethyl]acetamide (21)
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 21 (5.4mg, 85%).
Compound 21: colorless solid. IR mmax (KBr) cmꢀ1
:
1
1651, 1543. H NMR (500MHz, CDCl3) d: 0.83 (3H,
t, J = 7.1Hz), 1.10–1.30, 1.46–1.65, and 1.72–1.86 (total
12H, m), 2.26 (1H, dd, J = 15.8, 3.5Hz, 10-Ha), 2.32
(1H, dd, J = 15.8, 8.7Hz, 10-Hb), 2.35–2.57 (2H, m,
NCH2), 2.68 (4H, br s, NCH2), 3.11 (4H, br s, NCH2),
3.20 (3H, s, 3-OCH3), 3.29 (2H, dt, J = 6.3, 5.4Hz,
CONHCH2), 4.32 (1H, m, 6-H). FAB-MS m/z: 358
(M + H)+. FAB-HRMS m/z: calcd for C18H36N3O4:
358.2706, found: 358.2713.
4.23. N-[2-(1H-Imidazol-4-yl)ethyl]-2-(3-methoxy-3-pentyl-
1,2-dioxan-6-yl)acetamide (25)
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 25 (6.1mg, quant.).
Compound 25: colorless solid; syn:anti = 3.8:1. IR mmax
1
(KBr) cmꢀ1: 1647, 1543. H NMR (500MHz, CDCl3)

M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
5305
d: 0.82 (3H, t, J = 6.7Hz), 1.14–1.40 and 1.48–1.86 (total
4.27. Methyl (E)-6-oxo-6-phenylhex-2-enoate (42)
12H, m), 2.24 (1H, dd, J = 15.3, 4.9Hz, 10-Ha), 2.29
(1H, dd, J = 15.3, 7.9Hz, 10-Hb), 2.73 (1H, ddd,
J = 15.3, 6.7, 6.1Hz, CH2-Imi), 2.79 (1H, ddd,
J = 15.3, 7.3, 6.7Hz, CH2-Imi), 3.19 (3H, s, 3-OCH3,
syn), 3.22 (3H, s, 3-OCH3, anti), 3.38 (1H, ddd,
J = 12.8, 7.3, 6.7Hz, CONHCH2), 3.48 (1H, ddd,
J = 12.8, 6.7, 6.1Hz, CONHCH2), 4.38 (1H, m, 6-H),
6.77 (1H, br s, Imi), 7.54 (1H, br s, Imi). FAB-MS m/z:
340 (M + H)+. FAB-HRMS m/z: calcd for
C17H30N3O4: 340.2236, found: 340.2236.
As described in procedures A, B, and C, 40 (150mg,
1.7mmol) was converted to 42 (234mg, four steps 63%).
Compound 42: colorless oil. IR mmax (KBr) cmꢀ1: 1730,
1576. 1H NMR (500MHz, CDCl3) d: 2.64 (2H, dt,
J = 7.3, 7.3Hz, 4-H), 3.13 (2H, t, J = 7.3Hz, 5-H),
3.70 (3H, s, CO2CH3), 5.88 (1H, d, J = 15.9Hz, 2-H),
7.02 (1H, dt, J = 15.9, 7.3Hz, 3-H), 7.45 (2H, t,
J = 7.3Hz, Ph), 7.55 (1H, t, J = 7.3Hz, Ph), 7.93 (2H,
d, J = 7.3Hz, Ph). FAB-MS m/z: 219 (M + H)+. FAB-
HRMS m/z: calcd for C13H15O3: 219.1021, found:
219.1035.
4.24. 2-(3-Methoxy-3-pentyl-1,2-dioxan-6-yl)-N-[4-[{2-
(3-methoxy-3-pentyl-1,2-dioxan-6-yl)acetylamino}propyl]-
tetrahydropyrazin-1-(2H)-yl]propylacetamide (26)
4.28. Methyl 2-(3-methoxy-3-phenyl-1,2-dioxan-6-yl)ace-
tate (28)
As described in procedure F, 39 (7.6mg, 0.018mmol)
was converted to 26 (12mg, quant.).
As described in procedures D and E, 42 (11mg,
0.051mmol) was converted to 28 (5.7mg, two steps
42%). The 3-phenyl analogue 28 was obtained as a mix-
ture of syn and anti isomers. The physicochemical data
were analyzed as a mixture. In the 1H NMR spec-
tra, only the signal due to 3-OCH3 was definitely sepa-
rated between the two isomers.
Compound 26: colorless solid; syn:anti = 3.9:1. IR mmax
1
(KBr) cmꢀ1: 1647, 1541. H NMR (500MHz, CDCl3)
d: 0.82 (3H · 2, t, J = 6.7Hz), 1.13–1.40 and 1.48–1.86
(total 28H, m), 2.21 (1H · 2, dd, J = 15.3, 4.3Hz, 10-
Ha), 2.26 (1H · 2, dd, J = 15.3, 7.9Hz, 10-Hb), 2.34–
2.76 (12H, br s, NCH2), 3.19 (3H · 2, s, 3-OCH3, syn),
3.21 (3H · 2, s, 3-OCH3, anti), 3.23–3.35 (2H · 2, m,
CONHCH2), 4.37 (1H · 2, m, 6-H). FAB-MS m/z: 657
(M + H)+. FAB-HRMS m/z: calcd for C34H65N4O8:
657.4803, found: 657.4796.
Compound 28: colorless oil; syn:anti = 3.8:1. IR mmax
(KBr) cmꢀ1: 1742. 1H NMR (500MHz, CDCl3) d:
1.69, 1.83, 1.95, and 2.09 (total 4H, m), 2.44 (1H, dd,
J = 15.9, 4.9Hz, 10-Ha), 2.68 (1H, dd, J = 15.9, 7.9Hz,
10-Hb), 3.15 (3H, s, 3-OCH3, anti), 3.17 (3H, s, 3-
OCH3, syn), 3.72 (3H, s, CO2CH3), 4.65 (1H, m, 6-H),
7.25–7.41 (5H, m, Ph). FAB-MS m/z: 289 (M + Na)+.
FAB-HRMS m/z: calcd for C14H18NaO5: 289.1052,
found: 289.1055.
4.25. Methyl (E)-6-cyclohexyl-6-oxohex-2-enoate (41)
As described in procedures A, B, and C, 40 (150mg,
1.7mmol) was converted to 41 (171mg, four steps 45%).
Compound 41: colorless oil. IR mmax (KBr) cmꢀ1: 1714,
1659. 1H NMR (500MHz, CDCl3) d: 1.11–1.23 and
1.60–1.83 (total 10H, m), 2.30 (1H, tt, J = 11.0, 3.7Hz,
7-H), 2.43 (2H, dt, J = 7.3, 6.7Hz, 4-H), 2.57 (2H, t,
J = 7.3Hz, 5-H), 3.69 (3H, s, CO2CH3), 5.80 (1H, d,
J = 15.9Hz, 2-H), 6.91 (1H, dt, J = 15.9, 6.7Hz, 3-H).
FAB-MS m/z: 255 (M + H)+. FAB-HRMS m/z: calcd
for C13H21O3: 225.1491, found: 225.1486.
4.29. Methyl (E)-6-oxo-7-phenylhept-2-enoate (43)
As described in procedures A, B, and C, 40 (150
mg, 1.7mmol) was converted to 43 (232mg, four steps
59%).
Compound 43: colorless oil. IR mmax (KBr) cmꢀ1: 1717,
1601. 1H NMR (500MHz, CDCl3) d: 2.47 (2H, dt,
J = 7.3, 6.7Hz, 4-H), 2.64 (2H, t, J = 7.3Hz, 5-H),
3.73 (2H, s, CH2Ph), 3.74 (3H, s, CO2CH3), 5.81 (1H,
d, J = 15.9Hz, 2-H), 6.91 (1H, dt, J = 15.9, 6.7Hz, 3-
H), 7.23 (2H, t, J = 7.3Hz, Ph), 7.31 (1H, t,
J = 7.3Hz, Ph), 7.37 (2H, d, J = 7.3Hz, Ph). FAB-MS
m/z: 233 (M + H)+. FAB-HRMS m/z: calcd for
C14H17O3: 233.1177, found: 233.1185.
4.26. Methyl 2-(3-cyclohexyl-3-methoxy-1,2-dioxan-6-yl)-
acetate (27)
As described in procedures D and E, 41 (100mg,
0.45mmol) was converted to 27 (1.3mg, two steps
1.1%). The 3-cyclohexyl analogue 27 was obtained as a
mixture of syn and anti isomers. The physicochemical
1
data were analyzed as a mixture. In the H NMR spec-
tra, only the signal due to 3-OCH3 was definitely sepa-
rated between the two isomers.
4.30. Methyl 2-(3-benzyl-3-methoxy-1,2-dioxan-6-yl)ace-
tate (29)
Compound 27: colorless oil; syn:anti = 15:1. IR mmax
(KBr) cmꢀ1: 1728. 1H NMR (500MHz, CDCl3) d:
0.93–1.38 and 1.42–1.86 (total 15H, m), 2.35 (1H, dd,
J = 15.9, 5.5Hz, 10-Ha), 2.48 (1H, dd, J = 15.9, 7.6Hz,
10-Hb), 3.24 (3H, s, 3-OCH3, syn), 3.26 (3H, s, 3-
OCH3, anti), 3.68 (3H, s, CO2CH3), 4.42 (1H, m, 6-H).
FAB-MS m/z: 295 (M + Na)+. FAB-HRMS m/z: calcd
for C14H24NaO5: 295.1521, found: 295.1538.
As described in procedures D and E, 43 (7.1mg,
0.031mmol) was converted to 29 (2.4mg, two steps
28%). The 3-benzyl analogue 29 was obtained as a mix-
ture of syn and anti isomers. The physicochemical data
were analyzed as a mixture. In the H NMR spectra,
only the signal due to 3-CH3 was definitely separated
between the two isomers.
1

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M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
Compound 29: colorless oil; syn:anti = 4:1. IR mmax
(KBr) cmꢀ1: 1742. 1H NMR (500MHz, CDCl3) d:
1.55–1.83 (4H, m), 2.39 (1H, dd, J = 15.9, 5.5Hz, 10-
Ha), 2.53 (1H, dd, J = 15.9, 7.3Hz, 10-Hb), 2.74 and
3.02 (1H · 2, d, J = 14.0Hz, CH2Ph), 3.50 (3H, s, 3-
OCH3, syn), 3.51 (3H, s, 3-OCH3, anti), 3.73 (3H, s,
CO2CH3), 4.48 (1H, m, 6-H), 7.21–7.36 (5H, m, Ph).
FAB-MS m/z: 303 (M + Na)+. FAB-HRMS m/z: calcd
for C15H20NaO5: 303.1209, found: 303.1190.
(2H, t, J = 7.3Hz, 5-H), 3.69 (3H, s, CO2CH3), 5.05
[1H, t, J = 7.3Hz, CH@C(CH3)2], 5.81 (1H, d,
J = 15.9Hz, 2-H), 6.91 (1H, dt, J = 15.9, 6.7Hz, 3-H).
FAB-MS m/z: 303 (M + Na)+. FAB-HRMS m/z: calcd
for C17H28NaO3: 303.1936, found: 303.1924.
4.34. Methyl 2-[3-(3R)-(3,7-dimethyloct-6-enyl)-3-meth-
oxy-1,2-dioxan-6-yl]acetate (31)
As described in procedures D and E, 45 (12mg,
0.043mmol) was converted to 31 (7.2mg, two steps
51%). The 3-citronellyl analogue 31 was obtained as a
mixture of syn and anti isomers. The physicochemical
data were analyzed as a mixture. In the H NMR spec-
tra, only the signal due to 3-CH3 was definitely sepa-
rated between the two isomers.
4.31. Methyl (2E)-6-oxoundeca-2,10-dienoate (44)
As described in procedures A, B, and C, 40 (150mg,
1.7mmol) was converted to 44 (275mg, four steps 77%).
1
Compound 44: colorless oil. IR mmax (KBr) cmꢀ1: 1720,
1653. 1H NMR (500MHz, CDCl3) d: 1.62 and 1.99
(total 4H, m), 2.35 (2H, t, J = 7.3Hz, 7-H), 2.40 (2H,
dt, J = 7.3, 6.7Hz, 4-H), 2.49 (2H, t, J = 7.3Hz, 5-H),
3.65 (3H, s, CO2CH3), 4.91 (1H, d-like, J = ca. 10Hz,
CH@CH2), 4.94 (1H, d-like, J = ca. 17Hz, CH@CH2),
5.69 (1H, ddt, J = 17.1, 10.4, 6.7Hz, CH@CH2),
5.76 (1H, d, J = 15.9Hz, 2-H), 6.86 (1H, dt,
J = 15.9, 6.7Hz, 3-H). FAB-MS m/z: 211 (M + H)+.
FAB-HRMS m/z: calcd for C12H19O3: 211.1334, found:
211.1343.
Compound 31: colorless oil; syn:anti = 6.8:1. IR mmax
(KBr) cmꢀ1: 1744. 1H NMR (500MHz, CDCl3) d:
0.85 (3H, d, J = 6.1Hz, CH3), 1.03–1.44 and 1.50–2.03
(total 13H, m), 1.58 and 1.66 [3H · 2, s, CH@C(CH3)2],
2.36 (1H, dd, J = 15.9, 5.5Hz, 10-Ha), 2.49 (1H, dd,
J = 15.9, 7.9Hz, 10-Hb), 3.23 (3H, s, 3-OCH3, syn),
3.26 (3H, s, 3-OCH3, anti), 3.68 (3H, s, CO2CH3), 4.46
(1H, m, 6-H), 5.06 [1H, t, J = 5.5Hz, CH@C(CH3)2].
FAB-MS m/z: 351 (M + Na)+. FAB-HRMS m/z: calcd
for C18H32NaO5: 351.2148, found: 351.2165.
4.32. Methyl 2-[3-methoxy-3-(pent-4-enyl)-1,2-dioxan-6-
yl]acetate (30)
4.35. In vitro testing
As described in procedures D and E, 44 (9.8mg,
0.047mmol) was converted to 30 (7.0mg, two steps
58%). The 3-pentenyl analogue 30 was obtained as a
mixture of syn and anti isomers. The physicochemical
data were analyzed as a mixture. In the H NMR spec-
tra, only the signal due to 3-CH3 was definitely sepa-
rated between the two isomers.
A strain of P. falciparum (FCR3, cycloguanil-resistant
from Gambia) was used in sensitivity test. After
synchronization by the sorbitol treatment, 50lL of
the parasite culture at the ring stage (0.55% parasitemia
and 2% hematocrit) was added to each well in 96-well
microculture plates. The test samples were dissolved
in DMSO and diluted to the appropriate concentration
using complete medium, then 50lL of each sample
solution was inoculated. After incubation at 37ꢁC for
48h, the proliferation of P. falciparum was assessed
by Giemsa-stained smear by observing 10,000 erythro-
cytes per one thin blood film in triplicate. Quinine
was used as a reference anti-malarial. In this anti-
malarial assay, quinine inhibited the proliferation of
P. falciparum in a concentration-dependent manner
with IC50 of 40ng/mL and IC90 of 90ng/mL. Cytotox-
icity was evaluated by the colorimetric MTT assay, in
which mitomycin C used as a positive control showed
the IC50 of 0.1lg/mL.
1
Compound 30: colorless oil; syn:anti = 4.8:1. IR mmax
(KBr) cmꢀ1: 1744. 1H NMR (500MHz, CDCl3) d:
1.07–1.47 and 1.55–1.90 (total 8H, m), 2.04 (2H, td,
J = 7.3, 6.7Hz, CH2CH@CH2), 2.35 (1H, dd, J = 15.8,
5.5Hz, 10-Ha), 2.48 (1H, dd, J = 15.9, 7.3Hz, 10-Hb),
3.23 (3H, s, 3-OCH3, syn), 3.26 (3H, s, 3-OCH3, anti),
3.68 (3H, s, CO2CH3), 4.47 (1H, m, 6-H), 4.95
(1H, dd, J = 9.8, 1.8Hz, CH@CH2), 5.00 (1H, dd,
J = 15.3, 1.8Hz, CH@CH2), 5.75 (1H, ddt, J = 15.3,
9.8, 6.7Hz, CH@CH2). FAB-MS m/z: 281 (M + Na)+.
FAB-HRMS m/z: calcd for C13H22NaO5: 281.1365,
found: 281.1359.
4.36. Analysis for the stability of esters in mouse serum
4.33. Methyl (2E,9R)-9,13-dimethyl-6-oxotetradeca-2,12-
dienoate (45)
Each sample (30lL of 0.1mg/mL solution) was treated
with the fresh mouse serum (300lL) and incubated at
37ꢁC for 0, 30, 60, 180, 300min, respectively. After
extraction of the reaction mixture with EtOAc, each ex-
tract was concentrated under reduced pressure. The res-
idue was dissolved with 120lL of n-hexane–EtOAc
(1:1), then an aliquot (10lL for 7 and 14, 5lL for 15)
of the solution was analyzed by SiO2-phase HPLC (col-
umn: YMC-Pack SIL-06 4.6mm i.d. · 150mm, mobile
phase: n-hexane–EtOH = 200:1, flow rate: 0.5mL/min,
detection: UV 220nm) to determine the remaining
As described in procedures A, B, and C, 40 (150mg,
1.7mmol) was converted to 45 (328mg, four steps 69%).
Compound 45: colorless oil. IR mmax (KBr) cmꢀ1: 1717,
1659. 1H NMR (500MHz, CDCl3) d: 0.84 (3H, d,
J = 6.1Hz, 9-CH3), 1.08–1.41 and 1.85–2.03 (total 7H,
m), 1.57 and 1.65 [3H · 2, s, CH@C(CH3)2], 2.36 (1H,
dt, J = 15.3, 6.1Hz, 7-Ha), 2.39 (1H, dt, J = 15.3,
7.5Hz, 7-Hb), 2.44 (2H, td, J = 7.3, 6.7Hz, 4-H), 2.55

M. Kawanishi et al. / Bioorg. Med. Chem. 12 (2004) 5297–5307
5307
amounts of the test samples by the absolute calibration
method in triplicate.
Acknowledgements
This study was financially supported by Grants-in-Aid
for Scientific Research from the Ministry of Education,
Science, Sports, and Culture of Japan.
4.37. In vivo testing
In vivo anti-malarial activities of the compounds were
determined by the 4-day suppressive test using mice in-
fected with P. berghei (NK 65 strain). Five-week-old
ddY female mice obtained in sterile containers from
Charles River Breeding Laboratories Inc. (Yokohama,
Japan) weighing 24–27g were used. They were housed
under a natural day–night (12h each) cycle at 25ꢁC.
The mice were randomly assigned to treated groups
and housed in cages each containing five individuals.
Parasites are collected by cardiac puncture from a donor
mouse harboring about 15% parasitemia. The blood is
diluted with one-seventh volume of 3.2% trisodium cit-
rate solution, then a final concentration of the infected
erythrocytes was adjusted to 5 · 106 by adding 0.9%
NaCl solution. Initially, each mouse was inoculated
intravenously in the tail vein with 1 · 106 parasitized
erythrocytes in 0.2mL of the infected suspension. Test
compounds were prepared at doses of 1, 3, 10, and
30mg/kg in dimethylsulfoxide and administrated by
0.1mL once a day from day 0 to day 3. The first admin-
istration of the test compound started intraperitoneally
2h after parasite inoculation. Parasitemia levels were
determined on day 4. To evaluate the anti-malarial
activity of the compounds, we prepared tail blood
smears and stained them with Giemsa (E. Merck, Ger-
many). Total 1 · 104 erythrocytes per one thin blood
film were examined under microscopy. On day 4, para-
sitemia of control mice were between 30% and 35%.
The suppression of parasitemia was calculated by the
formula: [(average % of parasitemia for control ꢀ aver-
age % of parasitemia for treated mice)/average % para-
sitemia for control] · 100. Five infected and
dimethylsulfoxide-dosed mice were used as a control.
The data are determined from the five individuals in
duplicate.
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