Studies on Antimicrobial Sesquiterpenes in the Leaves of Rugosa

Title
Author(s)
Studies on Antimicrobial Sesquiterpenes in the Leaves of
Rugosa Rose (Rosa rugosa Thunb.)
Hashidoko, Yasuyuki
Citation
Issue Date
1990-03-24
DOI
Doc URL
http://hdl.handle.net/2115/36087
Right
Type
theses (doctoral)
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Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
Studies on Antlmicrobial Sesquiterpenes in the Leaves of Rugosa
Rose (Rosa rugosa Thunb.)
ハマナス (Rosa rugosa Thunb.) 葉中の抗菌性セスキテルペンに関する研究
Volume II
YASUYUKI HASHIDOKO
Doctor's Course, Division of
Agricultural Chemistry
March 1990
農芸化学専攻
橋床
泰之
博士課程
3-6 Rugosic Acid A Metab'olites in t"zosa rt'igosct- Lea-v-es
.t.
3-6-1 Introduction
As described in t,he previous section, rugosal A (1) is
probably synthesized from carota-1,4-dienaldehyde (3) and further
oxidized to rugosic acid A (2) in the leaf tissues of Rosa rugosa.
Those carotane peroxides 'tend to be 'accumulated in 'the tissues.
However, the author noticed that old and slightly senescent leaves
of rosa rugosa (for example, sample VIII) contained quite small
amount of those compounds. Albeit the dramatic decrease of 1 and
2, the leaves were still rich in oily substance. The author
therefore speculated that those peroxides were metabolized in the
senescent leaf tissues. Accordingly, the presuinable metabolites
were surveyed in t,he constituents of senescent Rosa rugosa leaves,
especially in the acidic extractives. In this section, the auther
describes isolat,ion and identification of those metabolites of
rugosic acid A (2), and discusses on the biogenesis of carotanolds
originated in Rosa rugosa, focussing on rugosal A (1) as a key
compound.
3-6-2 Rugosic Acid A Methyl Ester
Rugosic acid A methyl ester (5), a neutral metabolite of
rugosic acid A (2) was found during a survey of peroxides in Rosa
rugosa leaf-constituents, using a peroxide test with N,N=dimethylp-phenylenediamine sulfite reagent. Firstly, 9.6 kg of leaves
(sample V) were mechanically damaged and then soaked in 76 liters
of tap water for 24 hr, at whieh point the wat･er layer was
collected and the exudates were partitionated with an equal volume
of EtOAc. After being concentrated to ca 5 liters, t･he EtOAc
400
extracts were once washed with 10 liters of 5 % NaHC03 to ca 17 g
tt
of neutral
substances in the organic layer. The neutral
constituents coated on 70 ml of silica gel were chromatographed on
silica gel column settled in benzene (gel voleme 1500 inl) to obtain
9 fractions as shown in Table 3-90 and Fig. 3-235. Since an
unknown spot clearly positive to the peroxide reagent was detecetd
in Fr-V-1, t,he compound denoted as RL-PERO-3 was isolated by PTLC
successively in n-hexane-EtOAc 3:1, ,successive benzene-EtOAc 5:1,
n-hexane-EtOAc 4:1 and CHC13-MeOH 50:2; Rf O.66, O.58, O.36 and
O.81, respeetively) to give colorless needles (6.5 mg, from ca 1/2
of Fr-3) (Fig. 3-236).
Table 3-90
No
Fr-V-1
Fr-V-2
Fr-V-3
Fr-V-4
Fr-V-5
Fr-V-6
Fy-V-7
Fr-V-8
Fr-V-9
Fractionation of the extraetives from Samp1e V by
silica gel column chromatography
Volume
Solvent'
20
20
50
50
50
50
100
100
100
%
%
%
%
%
%
%
%
%
500
500
500
500
500
500
500
500
500
EA/B
EA/B
EA/B
EA/B
EA/BEA/B
EtOAc
EtOAc
EtOAc
401
ml
ml
ml
ml
ml
ml
ml
ml
ml
H-EA 9:1
o
RL-,,.,tbyekag
O quenching under
UV 254 nm
ZZlp peroxide test: +
8
6
1
2
3
4
5
6
7
8
9
Fig. 3-235 TL Chromatograpm of Column Eluants of Neutral
Constituents in the Exduates from Damaged Rosa rttgosa Leaves
The isolate showing the molecular ion at m/z 296 (O.8 %) was
agreeable with that of RSA-CN or RDA-ME lk in the EI-MS (Fig. 3237). In the IH-NMR spectrum, RL-PERO-3 was also indistinguishable
fvom them (Fig. 3-238). RL-PERO-3 was thus identified as 5. The
fifth naturally occurring carotanoid from Rosa rugosa named rugosic
acid A methyl ester was thus eonfirmed. As the optical rQtation of
'
the isolate [ct]D + 150 O indicates an absolute
eonfiguration
identical with that of rugosie acid A (2), 5 is presumed to be
derived from 2 in the plant. The possibility that 5 is an artifact
was reasonably rejected, because the leaf material was extracted
not with MeOH but with water.
Rugosic acid A methyl ester (5) was also isolated from sample
VI (ca 4.5 mg/kg). The content of 5 in the overmatured leaves
(eollected in late Augst) was about 3.5 times higher than that of
leaves during the flowering season ･(June-July). Therefore, this
methyl ester was regarded as an ordinary met･abolite of 'rugosic acid
A (2).
402
B-EA 5:1
H-EA 3;1
-
C-M 50:2
under
a quenching
UV 254 nm
do mo
ma peroxide test: +
a
RL-PERO---3 of do
o em
wh
O
O
" ny"--------
Fr-V-3 std.lk RL-PERO-3 lk
-'O---.
RL-PERO-3 lk
'
Fig. 3-236 TL Chromatograrns of Rugosic Acid A Methyl Ester (RLPERO-3)
IZZ
6
8Z
a7s
6Z
41
55
296
4Z
2Z
7Z
199
.83 g7
*2Z,Z
IZ9
91
12Z
175
se lzz lsz
2e3
2ZZ
221
264
235
25e
3ZZ
35Z
'
Fig. 3-237 EI-lyfass Spectrum of Rugosic Acid A Methyl Ester (RL-
PERO-3)
403
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404
山
Table 3-gl Physicochemieal properties of meth:vl rugosate A (5==
PDA-ME)
-VUiiJ"/
""PNtSNt
/
COOCH3
NN
:N:
OH
5= lk
Colorless needles, mp i44-147 "C
Rf: O.66 (H-EA 3:1), O.56 (B-EA 5:1), O.34 (H-EA 4:l), O.81
(C-M 50:2)
Vanillin-H2S04 eolor: pinkish grey
N,IVL-dimethyl-p-phenylenediamine sulfate test: positive (pink)
[a]D : + 150 O (c O.02 in MeOH)
FI-MS m/z (%): 296 (M+, 100)
EI-MS m/z (%): 296 (M', O.8), 278 (M+-H20, 2.0), 264 (4.0), 246
(2.9), 235 (3.8), 221 (9.6), 203 (5.7), 175 (6.7), 139
(31), 120 (17), 109 (19), 97 (27), 83 (29), 81 (20), 70
(29), 69 (100), 57 (20), 55 (54), 43 (39), 41 (59).
iH-NMR 6g"Di8i3(soo MHz): 4.3g7 (iH, dd,, J= n.s and 6.s Hz, c-2-H),
2.687 (IH, d, J= 11.5 Hz, C-2-OH), 7.064 (IH, dd, J= 6.4
and 1.2 Hz, C-3-H), 5.234 (IH, ddd, J= 5.1, 2.4 and 1,2 Hz,
C-5-H), 2.220 (IH, dd, .J= 14.2 and 5.1 Hz, C-6-Ha), 1.884
(IH, dd, .I= 14.2 and 2.4 Hz, C-6-Hb), 1.813 (IH, ddd, J=
12.8, 12.8 and 7.1 Hz, C-8-Ha), 1.706 (IH, dd, J= 12.1 and
6.5 Hz, C-8-Hb), 1.603 (IH, ddd, .1= C-9-Ha), 1.434 (IH,
dddd, J= 13.0, 13.0, 10.8 and 6.6 Hz, C-9-Hb), 1.873 (IH,
ddd, J= 10.3, 8.5 and 2.2 Hz, C-10-H), 2.611 (IH, double
sept., J= 6.8 and 2.2 Hz, C-11-H), O.966 (3H, d, J= 6.8 Hz,
C-12-H3), O.922 (3H, d, J= 6.8 Hz, C-13-H3), O.899 (3H, s,
C-15-H3), 3.776 (3H, s, C-7'-H3). These signals showed a
good accordance to those of RSA-CN (lk)･
405
3-6-2
Rugosic Acid B
As the metabolic fate of carotanoids, it is expectable that
rugosic acid A (2) is converted into some polar compounds through
some rearrangement at the endoperoxide bridge, since 2 is the
largesP pool of carotanoids in Rosa rugosa tissues. The leaf
materials (sample VH and VllI) were botl'} extracted with MeOH, and
the concentrated extract, was dissolved in EtOAc. The organic layer
of V (ca 1 liter) was once washed with 700 ml of 5 % NaHC03･ The'
washing was acidified to pH 3.0 with 5 N HCI, and then extracted
with 500 ml of EtOAc. The acidic substances (ca 8 g) were
successively chromatographed on a silica gel column. As shown in
Table 3-92, the acid eonstituents coated on 90 ml silica gel was
put onto 250 ml of Wako gel C-200 settled in n-hexane, and then
eluted with a EtOAc/hexane mixture containing O.33 % of formic
acid. The eluates were iinmediately concentrated to dryness to
remove the formie aeid, and then the residue was dissolved in small
volume of EtOAc. The TLC pattern of the each fraetion was shown in
Fig. 3-239.
Tab1e 3-92 Si1ica gel column chroma.tography of acidic constituents
from ,sample VU
No
Fr-VIIA-1
Fr-VIIA-2
Fr-VIIA-3
Fr-VIIA-4
Fr-VIIA-5
Fr-VIIA-6
Fr-VIIA-7
Fr-VIIA-8
Fr-VIIA-9
Fr-VIIA-1O
Solvent
CH2C12-HCOOH 300:1
CH2C12-HCOOH 300:1
CH2C12-MeOH-HcooH 400:10:1.5
CH2C12-MeOH-HcooH 400:10:1.5
CH2Cl2-MeOH-HCOOH 400:20:1.5
CH2C12-rvleOH-HcooH 400:20:1.5
CH2C12-MeOH-HcooH 400:60;i.5
CH2C12-MeOH-HcooH 400:60:1.5
CH2C12-MeOH-HcooH 400:60:1.5
acetone
406
Vo1ume
100
200
200
200
200
200
200
200
200
300
ml
ml
)
ml
ml
)
ml
ml
ml
ml
ml
ml
Weight (g)
o
o
o
5
.
9
.
9
.
4
In Fr-VUA-8 and -9, some spots showing lower Rf than that of
2 were detected on TLC, and one of them indicated a yellow
colo.ration by the vanillin-H2S04 test. As RDA-KOH (2e) was also
colorlized yellow with vanillin-H2S04 reagent, the acidic fraction
was again developed in H-EA-F 25:25:i with authentic 2e. As the
result, the focused compound and 2e were agreeable in their Rf
values (O.27) and responses to vanillin-H2S04 reagent･
Accordingly, the focused compound was isolated by PTLC in H-EA-F
30:30:1 and C-M-F 30:2:1 (Rf O.21, in Fig. 3-240), to give 10.4 mg
of a colorless syrup (ca 14 mg/kg of sample VH). EI-MS and IH-NMR
spectra of the isolate were indistinguishable £rom those of 2e
(Fig. 3-241 and 242, cL 13C-NMR spectrum in Fig. 3-243), and
accordingly the isoiate named rugosic acid B (6= 2e) wqs revealed
to be identical to RDA-KOH and present, in the fresh leaves of Rosa'
rugosa.
Although rugosic aeid B (6) is convertible from rugosic acid A
(2) by an alkali-reaction, the isolate (6) from the exudates seems
not to be an artifaet but a nat.urally oceurring earotane acid,
since the MeOH extract was ordinarily aeidic (> pH 5.5).
H---EA-F 30:10:1
o
Q
o
8
8
o
D
(z)
a
O
Fig. 3-239 TL Chromatogram of Column Eluants of Acidic
Constituents in the Methanol Extracts of Rosa rugosa Leaves
(Sample V)
407
H--EA-F 25:25:1
C-M-F 30:2:1
H-･EA-F 30:30;i
O
guenching under
UV 254 nm
c; i:[I/:ti
iiiiii
O
@
vaniliin"-H2SO4
test: posltlve
1i,T 11･
pt..------e.-
Fr-VIIA-8
Std., 2e
Fig. 3-240
Isolated 2e
Isolated 2e
TL Chromatograms of Rugosic acid B and Authentic
Compound 2e
IZZ
1
9
se
69
6e
4Z
a64
41
97
181
55
282
*3Z.Z
81
91
2Z
5Z
IZ9
IZZ
Fig. 3-24i
121
a53
2Z5
15Z
2ZZ
EI-lyfass Speet･rum o£
408
22Z
246
asz
3ZZ
Rugosic Acid B
S5Z
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8
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8
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410
In this extractives (Sample VIII), rugosic acid B (6)
only as an ambiguous spot detected by Vanillin-H2S04 test,
rugosic acid C (7) was undetected. On the contrary, 8 was
detectable in the acidiq extractives of the younger leaves
VII). These facts allowed a speculation that rugosic acid
further transformed to various minor metabolites including
acids B･-D (6, 7 and 8), and that only 8 among them can be
accumulated in the old leaves. Probably, 8 is one of the
stable and physiologically inactive carotane acids in Rosa
tissues,
411
appeared
whi1e
also
(samp1e
A (2) is
rugos1c
most
rugosa
Table 3-93 Physicochemical properties of rugosic acid B (6= RDA-
KOH)
OH
NN
"N
"x lc1i COOH
"s""NO
N'-"
ssl
OH
6= 2e
Colorless columns, mp
Vanillin-H2S04 color: clear yellow
EI-MS m/z (%): 282 (M', O.1), 264 (M"-H20, 1.2), 246 (5.6), 231
' (3.7), 220 (13), 205 (9.5), 204 (8.6), 203 (9.0), 181 (44),
140 (37), 139 (100), 121 (22), 109 (17), 97 (58), 95 (19),
91 (20), 83 (17), 81 (30), 79 (16), 69 (73), 55 (40), 44
(49), 43 (40), 41 (63)
1H-NMR 6[i{Mlil8s2 (soo MHz): 6.7g1 (IH, d, .T= 4.g Hz, c-3-H), 4.o1g (.J=
4.9 Hz, C-2-H), 2,414 (IH, d sept., J= 6.9 and 3.9 Hz, C11-H), 2.172 (IH, d, J= 11.9 Hz, C-6-Ha), ca 2.09 (IH,
overlapped, C-10-H), l.966 (IH, d, .T= 11.9 Hz, C-6-Hb),
1.966 (IH, m, C-9-Ha), 1.753 (IH, m, C-8-Ha), 1.662 (IH,
ddd, J= 15,8 and 7.2 Hz, C-8-Hb), 1.638 (IH, ddd, J= 12.5,
11.4 and 7.0 Hz, C-9-Hb), 1.112 (3H, s, C-15-H3), 1･O02
(3H, d, J= 6.9 Hz, C-12-H3), O.911 (3H, d, J= 6.9 Hz, C-12H3)'
13c-NMR 6Elit!i18s2'(125 lvfHz): 167.2 (C-14), 138･9 (C--4), 129･2 (C-3),
102.0 (C-5), 97.0 (C-1), 65.1 (C-2), 56.0 (C-10), 53.4 (C6), 48.9 (C-7), 44.8 (C-8), 25.7 (C-12), 25.6 (C-11), 24.7
(C-15), 24.0 (C-9), 20.7 (C-13).
412
3-6-4 Rugosic Acid C
From the column fractior} (Fr-VIIA-8 and -9) eontainir}g rugosie
acid B, a substance with.Rf O.23 in n-hexane-EtOAe-HCOOH 25:25:1
.t .
identical to that of authentic RSA-TU (2a) was also found. After
PTLC in the solvent, the substance was further purified by PTLC in
CHCI3-MeOH'HCOOH 30:2;1 (Rf O.30) and finally isolated by PTLC in
the first solvent syst,em to give 1.9 mg of a colorless syrup (Fig.
3-244), The isolate was elucidated as 7, by direct comparison with
2a in EI-mass and IH-NMR spectra, and was named rugosic acid C
(Ftg. 3'245 and 246), Rugosic acid C (7) was involved in a
comparatively small amount in the leaf tissues (ca 5 mg/kg). This
compound was considered as one of the metabolites of rugosic acid A
(2),' as with rugosic acid B (6). Since these acidic metabolites
were hardly found in senescent leaves colleeted in late September
(Sample VIII), it was expected that these compound were further
metabolized or modified in the tissues while the leaves were
funetioning.
H-EA-F 30:30;1 C-M-F 30:2:1
s
H-EA-F 25:25:1
o
quenching under
UV 254 nm
:"i3tl.1:.:
va egsU"sggi 92.e
`liiii>
Qtk..
s.
Fr-VIIA-8
Fig. 3-244
.v･･
@-@
@
Std.
s.'::.':'lt
2a Isolated 2a
------ k-----
Isolated 2a
TL Chromatogram of Rugosic Acid C and Authentic
CompQund 2a
413
lze
9
14e
BZ
6Z
41
4Z
55
189
69
81
2Z
*IZ.Z
162
izsii7 i3i
77
177
5Z
266
-L-"--'-･---r-'"---"--v---
91
IZZ
Fig. 3-245
2Z5
2ZZ
15Z
a17
232
248
25Z
3ez
EI-Mass Spectrum of Rugosic Acid C
414
35Z
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d
鵠
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8
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（Qり
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二
外
臼
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8・8
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呂
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o
ぜ
9
8
K
90
Φ
の
髪
8
器
ご
ぜ
8葦
ま
rく【L
ぜ
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8
κ
φ
8め 〒
寓
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6
N
I
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b幻
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山
415
Tabie 3-94l
Physicochemdca1 properties of rugosic acid C (7=
RDA-TU)
.OH
t
.,s`'
""NOH lc11 COOH
s
=-OH
7= 2a
A eolorless syrup
Vanillin-H2S04 color: grayish yellow -> graish purple
EI--MS m/z (%): 266 (M+-H20, O.4), 248 (M"-2H20, 6.4), 232 (4.7),
230 (4.1), 217 (9,7), 205 (13), 190 (15), 189 (36), 162
(20), 145 (25), 140 (83), 131 (17), 117 (17), 97 (100), 96
(17), 9i (27), 81 (23), 69 (35), 55 (33), 44 (28), 43 (28),
41 (so),
IH-NMR 6EiSlsC13(500 MHz): 7,O05 (IH, br., C-3-H), 4.900 (IH, br･, C-
5-H), 4.436 (IH, br., C-2-H), '2.461 (IH, br., C-11-H), ca
2,21 (IH, ill, C-6-Ha), 2.188 (IH, m, C-10-H), 2.016 (IH, dd,
J= 15.4 and 7.1 Hz, C-6-Hb), 1.959 (IH, m, C-9-Ha), 1.769
(IH, m, C-9-Hb), 1,551 (IH, m, C-8-Ha), 1.438 (IH, dd, .r=
ll.6 and 7.0 Hz, C-8-Hb), O.947 (3H, d, .I= 6.8 Hz, C-12H3), O.901 (3H, d, .1= 6.8 Hz, C-l3-H3), O.878 (3}I, s, C-15-
H3)'
416
3-6-5 Rugosic Acid D
Rugosic acid D (8), a naturally occurring carotanoic acid
equal to RDA-TSA (2c) was isolated from the exdudates of old Rosa
rugosa leaves. Sample VII[ (550 g) collected in late Sept,ember was
once extracted for 3 weeks with ca 4 liters of MeOH. The '
extraetives were defatted twice by shaking with a 1/3 volume of nhexane, and the resulting lower layer was concentrated to ca 500 ml
of a water suspension. The water layer was saturated with NaCl and
extraeted with 600 ml of EtOAc (x 2). The combined EtOAc extracts
were repeatedly washed with 700 ml of 5 % NaHC03 solution. The
washings were acidified with 5 N HCI' to pH 3.5 and re-extracted
with 500 ml of EtOAc. The EtOAe layer contain･ing the acidic
constituents was then dried over Na2S04, and concentrated in vacuo
to give ca 3 g of acidic extracts.
' 'acidic extractives were chromatographed (pTLc) in nThe
hexane-EtOAc-HCOOH 25:25:1 (Fig. 3-247), As a constituent
corresponding to RSD-TSA (2c) was detected as the major one, the
compound was focused to isolate. After the PTLC in the H-EA-F
system, the focused compound (Rf O.51) was further purified by PTLC
in CHC13-MeOH-HCOOH 50:2:1 (Rf O.34). By re-PTLC in the first
solvent system, the compound was eventually isolated as 11.2 mg of
a colorless syrup (calculated ca 41 ing/kg). In EI-mass and IH-NMR
speetra, the isolate and RDA-TSA was completely identical (Fig. 3248 and 249). The fourth naturally occurring carotanoic acid named
rugosic acid D was thus formulated as 8 (= 2c).
In this extractives (Sample VII[), rugosic acid B (6) appeared
only as an ambiguous spot detected by Vanillin-H2S04 test,, while
rugosic acid C (7) was undetected. On the contrary, 8 was also
deteetable in the acidic extraetives of the younger leaves (sample
V). These facts allowed a speeulation that rugosic acid A (2) is
further transformed to various minor metabolites including rugosic
417
acids B-D (6, 7 and 8), and that only 8 among them
can be
rnACH+
accumulated in t,he old lea-v-es. Pi'obab]y, 8 is orie of the "iUOL
st,able and physiologically inactive carotane acids in Rosa rugosa
tissues.
C-M-F 50:2;i
H-EA-F 25:25:1
O
quenching under
UV 254 nm
cl
$n･,'iis';
vanillin-H2S04
test: posltxve
iiii (ES) {g:) @)
,.;,,i･ll'
SampleVI[I Std.2c Isolated 2c
-
Fig. 3-247 TL Chromatographic Detection of Rugosic Acid D in
Acidic Extractions (Sample
IZZ
VIll)
7
BZ
6Z
41
S5
4Z
156
87
B3
2Z
l
lsilI
9
IZ9
1
'
141
122 i35
1l]
lee
Fig. 3-248
'191
175
alB
a33 a49 264
,
2ZZ 25Z
15Z
3ZZ
EI-Mass Speetrum of Rugosic Acid D
418
3SZ
L：
． 一
一 ｝ 一
8
噛
8
C
丙
（⑩
8
雨
Q⑩
o
q
｝イ
い
N
8
q
ooゆ
）
P
〔⊃マ
。己
ロ；【L
∩
℃
・▼→
o
00欄m
＜
o
8
め
・P1
の
o
bO
鰍
。
8
臼
べ
ρ
o
①
8
q
の
面
1
「
@
@
@
8
£
氏
匹
φ
目
①
寸
N
@ 一
5
Qり
8
噌⊂；
一
6
419
■
bO
。H
国
Tab1e 3-95
Physicochemical properties of rugosic acid P (8= RDATSA)
"N
"N
NN""sdN
>NN"i""
o
/
COOH
s
NS
NOH
2c
A colorless syrup
Rf; O.51 (H-EA--F 25:25:1), O,34 (C-N 100:5:2)
Vanillin-H2S04 color: grayish brown
EI-)!S n?/z (%): 264 (M'-H20, 2.1 Hz), 249 (M'-CH3-H20, 2,8), 233
(2.8), 218 (6.3), 203 (3.0), 191 (10), 175 (7.2), 165
(7.9), 156 (33), 141 (15), 138 (10), 135 (10), 122 (12),
109 (15), 91 (13), 87 (32), 83 (26), 70 (100), 69 (62), 55
(54), 53 (16), 43 (31), 41 (61).
IH-NrviR 6CiMDs6 (soo MHz); 7.o76 (IH, d, J= 4.1 Hz, c-3-H), 5.025
(IH, br. d, .T= 7.7 Hz, C-5-H), 3.940 (IH, d, J= 4.1 Hz, C2-H), 3.647 (IH, ddd, J= 8.5, 6.5 and 3.0 Hz, C-10-H),
1.824 (IH, dd, Jr= 12.2 and 7.7 Hz, C-6-Ha), 1.560 (IH, d
sept., .T= 6.8 and 6.5 Hz, C-ll-H), 1.394 (IH, in, C-8-Ha),
1.350 (IH, br. d, J= 12.2 Hz, C-6-Hb), ca 1.26 (IH, m, C-8Hb), ca 1.21 (IH, m, J= C-9-Ha), 1.070 (IH, ill, C-9-Hb),
O.873 (3H, d, .T= 6.8 Hz, C-12-H3), O.775 (3H, d, J= 6.8 Hz,
C-13-H3), O.698 (3H, s, C-15-H3).
420
3-6-6 Conclusion
Rugosic acid A (2) as a large pool of carotanoids in Rosa
rugosa was found to be further metabolized into more polar and
stable compounds. As the result of these surveys, at least four
metabolic pathways for 2 were presumed (Scheme 3-32). Those
metabolic reaction, involving methylation at the carboxyl group and
peroxide rearrangements, were presumed as only a part of the
complicated carotanoid metabolism in Rosa rugosa, since more and
more constituents were detectable on TLC. However, those
metabolites are considered to oecupy an initial part in the
metabolism of 2. Significance of those metabolit･es are not known.
As Rosa rugosa can control the pathogenic fungus, those
sesquiterpenes related to 2 may be immune from pathogens.
""ON d::
1 CHO
6, ×
NS
SON :
ol
.,
SN
NO. .S
",
cooH
o/I
cooMe
6'"'
,
OH
OH
×
s
s
,OH
"N
"N
NSS
>s,"i
N /44
,,"sO
o
N"
CHO
.OH
N"
tSH
"""
ON
/
COOH
OH
t:
OH
Scheme 3'46 Metabolic pathways from rugosic acid A (2)
421
/
COOH
3-7 Minor Carotanoids in Rosa rugosa
'
3-7-1 Introduction
According to the survey of terpenoids in Rosa rugosa leaves,
it was revealed that the pathway from carota-1,4-dienaldehyde (3)
to rugosic acids A-D and rugosic acid A methyl ester (2 and 5-8) is
the main flow in earotanoid metabolism. However, Rosa rugosa
leaves also contain various kinds of minor carotanoids, which are
considered to be originated in some isomers of 3 found in the RL
fraction. Here, minor carotanoids isolated and identified by
spectroscopic and/or chemical methods are described.
3L7:2 Daucenaldehyde and Its Related Compounds
1) Daueenaldehyde
As described in Section 5 of t,his chapter, some less polar
constituents idetected in GC-MS analysis of RL fraction indieated a
sesquiterpene nature isomeric to carota-1,4-dienaldehyde (3).
Following by the HPLC for isolation of carota-1,4-dienaidehyde (3),
these minor constit,uents were also purified. A compound appearing
as the second peak in HPLC (Unisil Q 100-5, 2.5 % EtOAc/n-hexane)
was isolated as a colorless oii (Fig. 3-250) identical with RL-A in
MS (the former EI-MS and the latter GC-MS, Fig. 3-25' 1). RL-A was
considered as an isomer of carota-1,4-dienaldehyde (3) because of'
the same molecular weight (M+ 218, ClsH220) as that of 3 and
similar MS fragmentation to that of 3. Furthermore, RL-A showed
xMmeaOxH 229 nm, which was indicative of the presence of an a,&
unsaturated aldehyde group in the molecule.
In IH-NMR spectrum, an isopropyl group (6H O.897 and O.869
each 3H, d, J= 6.9 Hz, and 2.515, IH, sept., J= 6.9 Hz ), an
422
t
;
i
i
i
;･
l･
11
i'
l
f
t
tt-- '---- - ' '- -- --- L '------H- r------L±･
:
->
o
t'
RL-A
/
･I,
'
1
;-
l
l
iiY
･!
1
l
.11
!
t
;
--
:
1.
Ii'
:
･;
:.
l 8l
l･
1-H
l.
-11
i
K
:･
Fig. 3-250 HPL-Chromatogram of RL Fraction (at UV 230 nm); The
second peak isolated by HPLC revealed the compound was
identical to RL-A depicted in Fig. 3-162.
423
aldehyde group (6H 9.263, IH, s) and bridgehead methyl group (6H
O.811, 3H, s) all characteristic of a carotane aldehyde were
obse.rved (Fig. 3-252 and Table 3-95). As a clear difference of
RL-A from 3 showing two olefinic protons in the IH-NMR spectrum,
only one olefinic proton was detected at 6H 6.101 (IH, ddd, .T= 8.2,
5.2 and 2.0 Hz). In addition, the methine proton of the ･isopropyl
group was observed as a clear septet signal (6H 2.515, tJ= 6･9 Hz),
which suggested that the isopropyl group was attached to a nonhydrogen-bearing carbon, Therefore, a carotane structure for RL-A
which possesses a C,C-double bond on C(10)-C(1) was tentatively
proposed. To confirm the estimation, decoupling experiments were
carried out.
As the results, a part of proton sequence was newly deduced as
shown in Fig. 3-253. The olefinic proton was vicinally coupled
with a pair of methylene prot,ons at 6H 2.053 (IH, br. dd, .J= 14.3
and 5.2 i{z) and 1.935 (IH, dd, J= 14.3 and 8.2 Hz), and further
coupled with one of allylic methylene protons at 6H 1.763 (IH, br･
dd, J= 14.9 and 14.5 Hz, geminally eoupled with 6H 3.049) by J= 2.0
Hz of an allyl coupling. This methylene protons being relayed to
another pair of methylene protons with further vicinal couplings,
the partial structure -CH2-CH=C-CH2-CH2- was event,ually revealed.
Because of the deshielding effects on the olefinic proton and the
allylic methylene protons (6H 3.049 and 1.763), the ct,B-unsaturated
aldehyde group was allocated to the disubstituted non-hydrogen
bearing olefinic carbon.
Furthermore, another proton sequence was also revealed.
Analysis of the proton coupling patterns revealed part strueture
--CH2'CH2-N, and equivalent methylene protons at 6H 2.125 showed a
homoallyl coupling with a proton at 6H 1.616. This result was
suggestive that t,he tetrasubstitut,ed olefinie bonds locate between
two earbons bearing these protons (See Fig. 3-253).
on the other hand, 13c-NMR spectra (coM, DEPT and CH-COSY)
were taken to assign all the proton bearing earbons (Fig. 3-254,
424
IZZ
11
162
8Zz
175
6ee
93
4ee
41
136
IZ7
79
147
2Zz
55
67
19Z2Z3
218
25Z3ZZ
'
se
2ZZ
15Z
IZZ
IZZ
1
1
8Z
41
6Z
16a
93
175
55
4Z
79
59
2Z
IZ5
135
67
147
185 2Z3
5Z
Fig. 3-251
lez
ISZ
2ZZ
218
25Z
GC-Mass Spectrum of RL-A (top) and EI-Mass Spectrum
of the Isolated Compound (bottom)
425
1｝．
o
00
一
om
0
め
N
ロ
00
N
ー
。
ω
冒Σ
員cL
一
NΩ一
00
め
。
「＼
。
い
（り
‘
一
囚
呂Σ
“【L
マ＿4⊂」
00
自
⑩
o
噂
・甲→
oo
O
o
一
め
Σ
江
【L
“
Σ
〔L
⊂）a－
o
m
00
N
〇
一
め
N
o
o
ゆ
）
＜
〇
一
N
Oo
1
日
ω
偏
。
00
へ
祠
。
Φ
q
の
oΣ＝
oo
一．a一
o⊃
Σ
z
l
（ρaF
一
00
㎝
Nゆ
N
l
σり
■
bD
・7→
隔
426
Tab1e 3-96
Proton chemical shift values of RL-A
(500 MHz, in C6D6, TMS as an int. std.)
6H
9
6
3
2
2
2
263
s
,
101
ddd J,= 8. 2, 5
.
049
ddd J= 14
.
512
sept J= 6. 9 Hz
.
330
ddd J= 14 .o,
.
125
053
br ddd J= 7
br dd J= 14
.
1
1
1
1
o
.9,
2
5
.
4
2. 9 Hz
'
.7,
.3,
5. 2
Hz
935
dd J= 14 .3,
8. 2
Hz
763
br.dd J= 14 .9, ca
br.dd J= ca 14. 5,
14.
616
'
550
.
471
.
869
dd J= 12. 4,
dd J= 12. 4,
d J= 6.9 Hz
d J= 6.9 Hz
.
811
s
897
C-14-H
C-5-H
C-3-Ha
C-11-H
2.0 Hz
'
,3
7. 4 ,2
.
o
o
Assignment
.
2
1
Coupling
5. 4
.
o
Hz
.
9
Hz
5
14. o
7
.4
Hz
7
.7
Hz
･427
'
'
3
2
.
o, 2
.
9
Hz
.O Hz
C-2-Ha
C-9-H2
C-6-Ha
C-6-Hb
C-3-Hb
C-2-Hb
C-8-Ha
C-8-Hb
C-12-H3
C-13-H3
C-15-H3
触＿
／コ
oり
頃
。
N
，
＼
め
◎り
O一工
§竃（ r詳一ノ
エ
①
●
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Φ
ρ
エーO
／皇
℃
o
F→ρ
ω
国
N
O一〇
＼／野
oり
⑩
工
ト
H
■
o
◎り
翰
o
N
。〆k
ゆ
Φ
、
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寸
o
O一工
●
ぐ一
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＼
σり
へ・
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、『
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Φ→ρ℃口
の⊆1日目
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¢・H 麟
・r→臼oo
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’
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、、
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＼胤ノ
臼
oΩ4qo
臼⇒
’
、
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qΦ のζ＝
＋⊇⇒
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・H ρ
HΦ申）o
側Ωi
ゴ図①bO
ρ・州
oO国H¢
bO・HH
q自のΩl
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＋）「→oo
ε
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bO
戯
＋） 償Φ
。H一4
傷。
』 F→ρ
つて1
ω
o目5
o
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監
1 コ
3
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め
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H
N
l
o
bO
・H
隔
目
428
卜
融
霞Q
Qり
狽p
o
N頃o
9
自
需
尋
oc6N
（
臼
山
富国
∩
で
8¢
：Σ1．
⊂）
一
N
Nの帽
顧7
Nm4〕N
一
（⊃
『一
帆
呂8
8
・H
8£‘
匙国
一
呈o
o
ゆ
爲
）
一ぐ
1
需冨
転
o
一
審
一
ρ
累〇
一 Φ
の
婁餌
琵
宝る
蜀
雪
欝
8㌣
一cつ
の
8bむ
N
・r→
山
429
Tab1e 3-97 13C-Al?!IR chemica1 shift values of RL-A
(COM and DEPT, 125 MHz , in C6D6, TMS as an int . std.)
6c
Properties
8
-CHO
=CH-
7
=C
o
=C
2
=C
C-14
C-5
C-1 (or C-4, C-10)
C-10 (or C-1, C-4)
C-4 (or C-1, C-10)
8
-C-
C-7
193 o
.
152
.
146
.
141
.
140
'
49
41
39
27
26
2 ･,i
24
22
21
21
.
Assignment
7
CH2
2
CH2
4
CH2
7
CH
2
CH3
C-8 (or C-6)
C-6 (or C-8)
C-9 (or C-2, C-3)
C-11
C-15
.
o
CH2
C-2 (or C-3, C-9)
.
4
CH2
C-3 (or C-2, C-9)
.
7
CH3
C-12 (or C-13) -
2
CH3
.
.
.
.
.
.
C-13 (or C-12)
430
255 and Table 3-97, 98). Three (one quarternary carbon at 6c 49･8
and two of disubstituted olefinic carbons) non-hydrogen bearing
earbons out of four were contributed to the connection of these
partial structures. Accordingly, the partial structures including
the isopropyl group and the bridgehead methyl group were combined
with those non-hydrogen bearing carbons. Since the bridgehead
methyl group must be allocates to the quarternary carbon and the
homoallyl coupling must be present over t,wo partial structures,
only two possible forms were depicted - one bas a carotane skeleton
(A) and the other a 4,8-bicyclic skeleton (B) (Fig. 3-256)･
To elucidate the correct structure, a COLOC expriment on RL-A
was carried out. The COLOC spectrum of RL-A, indicative of a H-C-C
and H-C-C-C sequenee together with H-C, showed some long range
cros' s peaks that made the non-hydrogen-bearing carbons assignable
as shown in Fig. 3-257, Accordingly, it was revealed that the
strueture A only fulfilled a eross peak between 6H 3.049 proton and
6c 141.0 (Table 3-99 and Fig. 3-258). Furthermore, a cross peak
between 6H 2.330 and 6c 49.8 was also explicable only with the
strueture A. Thus, the planar structure of RL-A was established as
9. Since RL-A was a novel carotane aldehyde having C-14-oxodaucene strueture, to t,his compound was given a trivial name,
daucenaldehyde. All the carbon signals were finally assinable as
shown in Table 3-97.
431
r-ttrrr･T-rrrT-t-T-ttT-tTt-TTTT･nTT-Ttt-t-T･-･TT.-t
rrTrrTTTTt.t.tTrrrrTT.TrT
2.00
s.ao
o.o
1.00
PPM
-
1
thf
g
-
Si
8
o
eit -
-
-
-
tsNb'
as
.t.
3
:-
--..
2}:
.
or--
6-
.
ti1 --
-
.-
Fig. 3-255
o-
CH-COSY Spectrum, of RL-A (500 and 125 MHz , in C6D6)
432
Table 3-98
CoreZations between carhons and protons in CH-COSY of
RL-A
Carbon
193.0
Proton
9.263
152.8
6.10l
41.7
2.053, 1.935
39.2
1.550, 1.471
27.4
26.7
2.125
24.2
O.811
24.0
3.049, 1.763
22.4
21.7
2.330, 1.616
21.2
O.869
2.512
O,897
CHO
structure B
structure A
Fig･ 3-256
Possible Structure of RL-A: Strueture A affords
a earotadiene skeleton
433
ro
8--
l-iIwn'-
5
rmm'rrrmm
I-,llii
l-/-FMnMI
i'ii
o
1
T"'
ol'l
th.
o
;;
o
･I･i..,,,..i
rs
i,Iilii
o
1,.1.ilr.r
g r-
x8
g
Ii
'i/''u'1'''
li'
llii
}.tt
lu....-...t..t
11I.....L...J.L.-...-
-----=------------･---
1''
t.--........-..t+....".."-.t.4..
i
IIS
lr･Havni-"'-
i+
1/
E;
ii'
i/QI'mr
ll'Il･-･ll--t
ll･l,
lii･l,I
il
lli･
ll...
g
r-u'-wn"rm
11iIlimh-rm--
+-
iil･ii-+'i''"-"'i"-----T----
i･-ii-
-1･
o-
j
v
i'
8
6
P
o
fi)
o
y
,o
9,
fJ:
.p
fH
o
o
o
o
N
=
Fig. 3-257 COLOC Spectrum of RL-A (500 and 125 MHz , in C6D6):
The long range cross peaks (C-C-H, or C-C-CH) were marked with
a circle.
434
Table 3-99
Proton-carbon peaks in COLOC expl'iment of RL-A
Proton
Carbon
(6H)
(6c)
--- >
--- >
141.0
811 ---d >
o. 811 ---- >
49.8
3.
049
o.
811
o.
141.0
41.7
o.
39.2
9.
49.8
146.7
811 ---- >
2. 330 ---- >
263
--- >
CHO
Fig. 3-258 Correiation between Non-Hydrogen-Bearing Carbons and
Protons in RL-A Revealed by CH-COSY and COLOC Experiment:()-m.t>
indicates a long range correlation (Proton to carbon).
435
Tab1e 3-100 Physicochemical properties of daucenaldehyde
(RL-A, 9)
CHO
9
A colorless oil
Vanillin-H2S04 color: grayish purple
GC-MS m/lz (%): 218 (M', 11), 203 (M'-CH3, 5.2), 190 (6.0), 176
' (12), 175 (M'-C3H7, 58), 162 (84), 147 (19), 136 (32), 135
(28), 133 (17), 122 (11), 121 (100), 107 (31), 105 (23), 9fl
(29), 93 (44), 91 (33), 79 (25), 77 (23), 55 (15), 53 (14),
41 (32).
EI-MS m/z (%): 218 (M', 9.8), 203 (M'-CH3, 5.4), 190 (4.9), 185
(5.9), 176 (11), 175 (M'-C3H7, 55), 162 (58), 147 (23), 136
(27), 135 (27), 133 (18), 122 (11), 121 (leO), 107 (35),
105 (35), 94 (30), 93 (53), 91 (51), 79 (34), 77 (33), 55
(42), 53 (21), 43 (45), 41 (69).
IH- and 13c-NMR data are shown in Tables 3-97 and 3-98,
respectively.
436
2) Epoxydaucerialdehydes
While constituents of the rugosa rose leaves were surveyed, a
quenching spot was detected on TLC around Rf O.55 in H-EA 4:1 <RL109), and the eompound corresponding to the spot was accordingly
isolated from Fr-B-5 (Sample VII, See Section 3-5, pp. 324) and Fr'
' See Section 3-8, pp. 529). The isolated coinpound
I-5 (Sample I,
(ea 30 mg) afforded the parent ion at m/z 234 in FI-MS. RL-109 was
however found to be a mixture of two compounds in IH-NMR analysis.
For separation of two constituents from each other, some solvent
systems for TLC were examined to find n-hexane-dichloromethaneaeetone 25:25:1 (H-DCM-AT) as an effective slvent (Fig. 3-259).
Aeeordingly, the mixture was separated to RL-109A (8.2 mg) and RL109B (18.2 mg) by HPLC (Unisil Q 100-5, n-hexane-iso-PrOH 100:O.5
and H-DCM-AT 100:100:10) equipped with a UV detecter at 230 mn
(Fig. 3-260).
The' major const,ituent RL-109B, providing M+ 234 in FI- and EI-
MS (Fig. 3-261, 262) showed some signals characteristic of carotane
aldehydes (cL carota-1,4-dienaldehyde, 3) in IH-NMR and HH-COSY
spectra as shown in Fig. 3-263, 264 and Table 3-101. .Furthermore,
some methylene protons were quite similar to those of daucenaldehyde (9) in the signal pattern. The presence of clear septet
signai assignable to C-11-H (6H 1.468) and isolated -CH2-CH=C-CH2-
CH2- coupling sequence aHowed to presume that C-1 and C-10 were
replaced by non-hydrogen-bearing sp3 earbons. since two-nonhydrogen-bearing and oxygenated carbons were detected at 6c 76.8
and 7s.4 in place of the two sp2 carbons in the 13c-NMR spectruin
(Fig. 3-265 and Table 3-101), those were attributable to C-1 and C10. According to the 13C-NMR (INEPT), RL-109B was deduced to have
the inolecular formula ClsH2202 (CH x 3, CH2 x 5, CH3 x 3 and C x
4), which was only compatible with a structure having an epoxy
group on C(1)-C(10). Thus, structure 10 was proposed for RL-109B.
437
H-EA 4:1
RL-1O9
H-DCM-AT 25:25:1 x 3
Q
o
1' i
- ' N"'i
quenching under
UV 254 nm
R.L,1i,Z9,A,-.'..)'Ziii]t
O
Fr-VIIB-5 1
Fig.
RL-1O9
3-259 TL Chromatograms of RL-109A and RL-1O9B
RL-109A
N.. ARL-109B
'iWl'r'r''' , + ,
l ,･l
I il 1･ L,
l. Pl --1
ki F,
tii' I･/i
..-"-;･-,.
,l--+,l,-.-I,
ili'li--･･'i'
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li･liii
l'!lilil,i1
lilttl/i:tti/
-l--･;ii
sv[
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t
i''iii
iiii'i'Ll
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.Lll,
,'
tltt
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't
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l,-
111
l'i"'
ll-･･･/
it''
::
't'
1/llt/t/
li/l'
ltttt1i'i,ll-11･l･l,･･
F
Fig･ 3-260
HPL-Chromatogram of RL-1O9 Mixture (Unisil Q 100-5,
H-DCM-AT 25:25:i (-TV 230 nm)
'
438
M+
'l ?ee
fi,
3e.pt. ?.
>:'t
!fS, i}-
bu-
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z
.t
e
i-rrttrT-rrrrrrrrrrr;'rr-r-r-rrrrTT--TTr`r--n"r:-Tft--ii}lrtf'tit-rrti
-f r:mr･''''rrrrrrr"T'-Trrr"-T:-
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ui
tr-
H- i
?
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I
Bgg
35e
Fig. 3-261
IZZ
4se
4･ee
5n-eM/E
FI-Mass Spectrum of RL-109B
7
43
se
6e
lse
4e
91
55
79
az
IZ9
125 '137
163
21s 2B4
173
1912el
se
IZZ
15Z
2ez
Fig. 3-262 EI-Mass Spectrum of RL-1O9B
439
25e
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Fig. 3-264a
q.o3.s3.o2.s2.o1.st.o.s-･
PPM
HH-COSY Spectrum of RL--109B (500 MHz , in C6D6)
441
PM
.
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Continued (Magnified in High Magnetic Field)
442
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443
Tab1e 3-Z OI
Physicochemical properties of epoxydaucenaldehyde A
( -m-- R L -1 0 9B, 1 0 .>
N
CHO
tttt :"r"
o
10
A colorless syrup
EI-MS m/z (%): 234 (M', 17), 216 (15), 201 (6.4), 191 (6.4), 173
15), 163 (11), 152 (46), 149 (33), 148 (28), 137 (24), 125
(27), 119 (26), 109 (32), 107 (27), 105 (23), 91 (29), 81
'(22), 79 (22), 71 (100), 55 (28), 44 (43), 43 (95), 41
(64).
IH-NMR 6TCM6Ds6' (50o MHz): 1.407 (IH, ddd, J= 14.1, 13.3 and 2･2 Hz,
C-2-Ha), 1.173 (IH, br. ddd, J= 14.1, 5.5 and 2.2 Hz, C-2Hb), 3.149 (IH, m-dvid. dd, J= 14.8 and 5.4 Hz, C-3-Ha),
2.202 (IH, m-dvid. dd, J= 14.8 and 13.2 Hz, C-3-Hb), 6.033
(IH, br. m, C-5-), 2.516 (IH, br. dd, J= 14.4 and 4.7 Hz,
C-6-Ha), 1.781 (IH, dd, J= 14.4 and 8.8 Hz, C-6-Hb), 1.260
(IH, br. ddd, .r= 11.7, 10.5 and 8.0 Hz, C-8-Ha), O.918 (IH,
dd, J= 11.7 and 7.8 Hz, C-8-Hb), 1.617 (IH, dd, k 13.4 and
8.0 Hz, C-9-Ha), 1.357 (IH, ddd, J= 13.4, 10.5 and 7.8 Hz,
C-9-Hb), 1.469 (IH, sept., J= 6.9 Hz, C-11-H), O.991 (3H,
d, J= 6.9 Hz, C-12-H3), O.666 (3H, d, .J= 6.9 Hz, C-13-H3),
9.240 (IH, s, C-L14-H), O.511 (3H, s, C-15-H3).
13c-NMR 6TC M6Ds6 (12s MHz): 76.8 (C, C--1), 23.3 (CH2, C-2), 19.0
(CH2, C-3), 147.0 (C, C-4), 152.5 (CH, C--5), 36.2 (CH2, C6), 42.8 (C, C-7), 34.6 (CH2, C-8), 22.9 (CH2, C-9), 75.3
(C, C-10), 28.8 (CH, C-11), 18,8 (CH3, C--12), 18.6 (CH3, C13), 192.6 (CH, C-14), 21.0 (CH3, C-.15).
444
While RL-109B was elucidated as 10, the other isoiate, RL-109A
was also analyzed spectroscopically. RL-109A whose FI-MS showed
the .parent ion at m/z 234 (100 %) (Fig. 3-266) resembled to 10 in
the EI-MS £ragmentation (Fig. 3-267). Although the IH-NMR spectrum
of RL-109A (in C6D6) was quite different from that of RL-109B (Fig.
3-268), in CDCI3 the minor isolate showed a similar IH-NMR signal
pattern (Fig. 3-269), HH-COSY of RL-A was there£ore measured in
CDC13 (Fig. 3-270) to prove two 1-CH2.-CH2-- coupling systems on RL1O9A. 13C-NMR data of RL-109A were indicative of its
diastereoisomeric nature to 10 at the Cl/CIO-epoxy ring, showing a
good aceordanee of carbon chemical shift, values to those of 10
(Fig. 3-271). Accordingly, structure 11 was proposed for RL-109A.
Z
1eze
>
H
Ho
dz
i
Fz
F-t
se.ee
e
.ee
,
se
1eeo
1ee
1se
M+
n.!E
Z
se.ez
F>
s--t
oz
z
tu
le
HZ
.ee
BSSM!E
Fig. 3-266 FI-Mass Spectrum of RL-109A
445
3eB
7
4
BZ
6Z
158
4Z
2e9
55
79
2Z
91
125 137
163
99
5Z
Fig. 3-267
173
179 191ezl
6i
izz
ISZ
216
234
2ZZ
EI-Mass Spectrum of RL-1O9A
446
2SZ
3ez
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Fig・3－270a HH－COSY Spectrum・f Rレ109A（500 MHz・in CDCI3）
449
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Continued （Magnified’in High Magnetic Field）
450
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Tab1e 3-102
Physicochemical properties of epoxydaucenaldehyde B
(11)
k
N
"
"
N
CHO
o
11
A colorless syrup
EI-MS m/z (%): 234 (N', 12), 216 (7.5), 201 (3.6), 19! (5.3), 173
(8.4), 163 (13), 152 (41), 149 (29), 148 (25), 137 (25),
125 (26), 119 (24), 109 (34), 107 (26), 91 (27), 79 (23),
71 (100), 55 (31), 44 (46), 43 (99), 41 (65).
IH-NMR.6TC M6Ds6 (5oO MHz): 9.211 (IH, s, C-14-H), 6.028 (IH, m, C5-H), 3,068 (IH, m, C-3-Ha), 1.840 (IH, dd, J= 14.1 and 5.0
Hz, C-6-Ha), 1.522 (IH, sept., .J= 6.9 Hz, C-11-H), 1.028
(3H, d, J= 6.9 Hz, C-12-H3), O.828 (3H, s, C-15-H3), O.763
(3H, d, .J= 6.9 Hz, C-13-H3). Ot,her signals were mostly
overlapped in C6D6･
IH-NMR 6QrDMCsl3(500 MHz): 1.712 (IH, br. dd, .J= 13.4 and 12･9 Hz, C-
2-Ha), ca 1.58 (IH, overlapped with H20 peak, C-2-Hb),
3.000 (IH, m-dvid dd, .T= 14.8 and 6.3 Hz, C-3-Ha), 1.829
(IH, t-like m, C-3-Hb), 6.832 (IH, in, C-5-H), 2.388 (IH,
dd, J= 14.1 and 5.2 Hz, C-6-Ha), 2.250 (IH, dd, J= 14.1 and
8.8 Hz, C-6-Hb), 1,393 (IH, ddd, J= 12.5, 10.5 and 8.3 Hz,
C-8-Ha), 1.273 (IH, br. dd, J= 12.5 and 7.9 Hz, C-8-Hb),
1.851 (IH, br. dd, J= 13.8 and 8.3 Hz', C-9-Ha), 1.746 (IH,
m, C-9-Hb), 1.751 (IH, sept., J= 6.9 Hz, C-11-H), 1.097
(3H, d, J= 6.9 Hz, C-12-H3), 1.092 (3H, d, .J= 6.9 Hz, C-13H3), 9.409 (IH, s, C-14-H), O.994 (3H, s, C-15-H3).
i3c-NMR 6Prlii8i3(i2s MHz): 7s.7 (c, c-1), 22.s (cH2, c-2), 20･i
(CH2, C-3), 146.3 (C, C-4), 152.3 (CH, C-5), 37.1 (CH2, C6), 44.2 (C, C-7), 35.7 (CH2, C-8), 25.4 (CH2, C-9), 28.5
(CH, C-11), 19.5 (CH3, C-l2), 18.9 (CH3, C-13), 191.8 (CH,
C-14), i9.7 (CH3. C-15)･
452
The proposed structures 10 for RL-109B and 11 for RL-109A was
conCimiled by direct comparison with the epoxidation products of 9
with m-CPBA as shown in Scheme 3-33 (TLC in Fig. 3-272).
RL-A 3.2 mg)
dissolved in 1.5 ml of CHCI3
cooled to O oc
added 2.4 mg of m-CPBA
stirred 30 min.
concentrated and dissolved in ca O .2 ml of EtOAc
subjected to PTLC (n-hexane-EtOAc 20:1)
Product RL-A-CPBA, Rf O.06, 2.1 mg, 61 % yield)
Scheme 3-33
Process of RL-A epoxidation and isolation of the
product
The product showing m/z 234 as the parent ion in EI-MS was, as
expected, found to be a mixture of diastereomers by the IH-NMR
analysis with a ratio o£ 3:1 (Fig. 3-273). The product was
therefore separated by HPLC (H-DCM-AT 100:100;10, UV 230 nm, Fig.
3-274) to obtain RL-A-CPBA-1 (O.5 mg) and RL-A-CPBA-2 (1.5 mg).
The EI-MS fragmentation and proton signals of the product,s were
each coincident well wit,h those of RL-109A and RL-109B,
respectively, proving their proposed structures (11 and 10) (Fig.
3-275, 276, 277 and 278).
453
C-M 50:4
Vatneisiti :in+-H2So4
:･l･:･li:;･･
q peroxide test:
ua
+
tl.- l' i･:･}
Reaction Std. lc
.
MIX.
Fig.
TL Chromatograms of Reaction Product
3-272
s
.Aii[A,
,
.
!.Ea t.so'
!.ia t..sa !..'a
FFn
nyt
.
io 2.ta 2.!
o
tsa
--
'
.
.
'"ny-'-T"nyT.-'-"T-'･-･-･"T-･-.
3.2e 3.!O :･
'
-
r,mTr--T-v.,
""
E.la Eae
V-rv
9.sa1,-"-=-..'. 9.::.
#=H
"l
2.En r. t.;o ;
---"'-'
-
T'"'"-""""T-M"'･--'--r"-"'T""-T-----`'---
t.ae .le .so .,o
t
:slee
Fig. 3-273
?.'
1
3a
s.'
o:･ 7.ao
E 'ae ,i ･.le... ,e ,
a.bo 3.'n･:
:･･::: !
H-NMR Spectrum of RL-A-CPBA Mixture (500 MHz,
454
bs:
m
elo
C6D6)
Thus, it was suggested that the epoxy carotanoids RL-109B (10)
and .RL-109A (11) were biogenetically derived froin 9, and were named
epoxydaucenaldehyde A (RL-109B) and epoxydaucenaldehyde B (RL109A), respectively. .Carbon ehemical shift values of 10 and 11
were quite similar to those of a known epoxycarotane (46) found in
Ferula communis subsp. communis by rvliski et al. (Table 3-103) [46]･
iiiii.,11.[-,,,..l,tlilll"/l'..il/llli/tl'10Lu/p.X/., t!t
iltlii
ll
liE
IUI･ i i, 1 4i'.1li li i
ii
ir
11 il
i/
d
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1
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p
1//
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l"',IillT[,,,,e-
Fig. 3-274 HPL-Chromatogram of RL-A-CPBA Mixture (UV 230 nm)
455
IZZ
q
.
91
8Z
53
179
12B
1e5
77
4Z
145
119
6Z
216
71
65
azl
159
t83
21
165
2sa
se
Fig. 3-275
2ez 2se
ISZ
lee
3ZZ
EI-Mass Speetrum of RL-A-CPBA--2
teee
91
z
119
6Zz
77
4ee
1Z5
12e
14S
S3
173
216
71
15e
2ee
6
5Z
Fig. 3-276
ezl
165
IZZ
15Z
183
e6234
2zz 2se
EI-Mass Spectrum of RL-A-CPBA-1
456
31Z
’c三
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2
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457
ch
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21
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51
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q
l
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く1
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一
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458
Table 3-103
Carbon chemical shift of an epoxy carotanoid (46]
isolated from an Uhiberifellae plant
15
8
Xi4
7
CH20-Anisate
1
11 o2
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
46*
10**
76.0
24.7
22.2
138.1
127.2
33.3
41.9
23.4
28.0
76.8
33.9
18.2
17.2
69.3
20.3
76.8
23.3
19.0
147.0
152.5
36.2
42.8
34.6
22.9
75.3
28.8
18.8
18.6
192.6
21.0
11***
75.7
22.8
2e.1
146.3
152.3
37.1
44.2
35.7
25.4
75.0
28.5
19.5
18.9
l91.8
19.7
* 50 MHz in CDC13
**125 MHz in C6D6, assigned by CH-COSY
***68 MHz in C6D6
'
459
Tab1e 3-104 Physicochemical properties of RL-A-CPBA--2 (10)
t
N
CHO
Z,tt--
10
A colorless syrup
Rf: O.35 (H-EA 4:1), O.19 (H-DCM-AT 25:25:1)
Vanillin-H2S04 color: brown
EI--MS m/z (%): 234 (M+, 4.3), 216 (51), 201 (29), 183 (22), 173
(56), 159 (25), 145 (62), 143 (49), -1.31 (51), 129 (49), 128
(54), 119 (62), 117 (37), 115 (46), 105 (53), 91 (80), 79
(40), 77 (52), 71 (34), 53 (36), 43 (66), 41 (100).
IH-NMR 6CTff?9 (500 MHz): 1.403 (IH, ddd, J= 14.2, 13.8 and 1.8Hz,
C-2-Ha), 1.171 (IH, br. dd, J= 14.1, 5.5 and 2.3 Hz, C-2Hb), 3.155 (IH, m-dvid. dd, J= 14.8' and 5.5 Hz, C-3-Ha),
2.204 (IH, m-divid. dd, J= 14.8 and 13.8 Hz, C-3-Hb), 6.023
(IH, br. m, C-5-H), 2.511 (IH, dd, J= 14.4 and 5.1 Hz, C-6Ha), 1.775 (IH, dd, J= 14.4 and 8.7 Hz, C-6-Hb), ca 1.25
(IH, overlapped, C-8-Ha), O.914 (IH, dd, J= 11.7 and 8.2
Hz, C-8-Hb), 1.617 (IH, dd, J= 13.2 and 8.2 Hz, C-9-Ha),
1.355 (IH, ddd, J= 13.2, 10.7 and 7.8 Hz, C-9-Hb), 1.403
(IH, sept., J= 6.9 Hz, C-11-H), O.991 (3H, d, Jt 6.9 Hz, C12-H3), O.663 (3H, d, J= 6.9 Hz, C-13-H3), 9.238 (IH, s, C14-H), O.507 (3H, s, C-･15-H3).
460
Ta bJ e
3-105 Physicochemical properties of RL-A-CPBA-1 (10
CHO
11
A colorless syrup
Rf: O.35 (H-EA 4:1), O.21 (H-DCM-AT 25:25:i)
Vanillin-H2S04 color: brown
EI-MS m/z (%): 234 (M+, 4.1), 216 (35), 201 (20), 183 (14), 173
(39), 145 (48), 143 (40), 131 (37)'i 129 (39), 128 (44), 119
(65), l15 (36), 105 (44), 91 (81), 79 (40), 77 (48), 53
(38), 44 (48), 43 (57), 41 (100).
IH-NMR 6*llil2i6 (soo MHz): g.211 (IH, s, c-14-H), 6.o2o (IH, m, c-s"
H), 3.072 (IH, m, C-3-Ha), 1.837 (IH, br. dd, k 13.7 and
4.5 Hz, C-6-Ha), 1.507 (IH, sept., J= 6.9 Hz, C-11-H),
1.028 (3H, d, J= 6.9 Hz, C-12-H3), O.827 (3H, s, C--15-H3),
O.761 (3H, d, .7t 6.9 Hz, C-13-H3).
461
By the NOE experiments, the relat･ive configuratien were
revealed as *rel. [1(S*), 7(R*), 10(S*) in 10, and 1(R*), 7(R*),
10(RI) in 11, respectively (Fig. 5-ZYU). Tendency to be formed
more 10 in the epoxidation of 9 is explicable with a steric
hindrance on C-15 bridgehead ]nethyl group. Compared with highly
restricted stereo-seleetivi'ty during epoxidation of carota-1,4dienaldehyde (3), t,his stereo-selection is rather loose. This fact
is suggested that the stereo-selectivity in the oxygenations of 3,
including the epoxidation and peroxylation, was mainly due to the
steric hindance of the isopropyl group on C-10 (Fig. 3-280)･
H-CH3NH
N
CHO
.SN((lll,
't
･.
H
H3C
CH3
Fig. 3-279 Some NOEs Observed on RL-109B
462
Φ
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463
3-7-3 Isodaueenaldehyde and Its Related Compounds
1) Isodaucenaldehyde
Third peak of RL £raction (Fig. 3-281) isolated as colorless
oil (ca 3 mg) was dentical to RL-E in MS analysis (Fig･' 3-282)･
This ininor compound was revealed to possess an isopropenyl group
instead of an isopropyl group (6H 4.811 and 4.720, each IH,
coupling with J= 2.4 Hz, assignable to exoinethylene protons, and
1･690, 3H, br s, allylmethyl protons) in the IH-NMR spectruin. In
addition, a methyl group on a quarternary carbon (6H O.814, 3H, s)
and a formyl proton at 6H 9.348 both characteristic of Rosa rugosa
carotane skeleton were detected (Fig. 3-283 and Table 3-!06).
Furthemnore, its 13 C-NMR spectrum revealed the presence of two
methine carbons at 6c 56.2 and 5e.3, in spite of the disappearance
of the isopropyl methine carbon (Fig. 3-284 and Table 3-107).
i
-r----i'
---
;･
fi
----
i
T""'--'-'"--
i
pt'''"'-'H''''-
l･:b-
.-.--"..
l･
'･･-------l----･---･--.
---
/i･
1
o------
i
-･-- RL--E･----------.--."-za-
1',/JI
l-･-----･------l----.･-.".
'b"7'-'-
-''--'---b--"---.-,..
;
lttetZIZI://ts---t-------.-----------
r
rr･--'----
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ii
i;
IIIbo
H-----H---.
--･-
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:-'"t'--"'-'
--
l
----'-' 1l--------------
--･
1･
.
l--
'i
Fig. 3-281 HPL-Chromatogram of RL-E Detected at UV 230 nm
464
IZZ
6
8Z
6Z
4Z
79
41
93 lz7
53
21
lal
5Z
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136
149
175
162
189
21e
2ZZ
15Z
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2e3
2SZ
3ZZ
6
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6Z
4Z
4Z
4
79 g3
2Z
55
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121
175,
136 149
16a
5Z
Fig.
IZZ
21B
leg 2Z3
2ZZ
15Z
25Z
3ze
35e
3-282 GC-Mass (top) and EI-Mass (bottom) Spectra of RL-E
465
3 I
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ぐつ
（つ
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⊂つ
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（つ
m
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Ho
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と・．
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o
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（⊃
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国
Tab1e 3-106
1
H-NMR chemical shift values of RL-E
(500 MHz , in CDCI3, TMS as an int. std.)
6H
1
'
1
1
.
3
1
6
2
2
1
4
･
751
.
619
.
195
IH br. d J= 15.3 Hz
617
IH br. dd J= 11.7 and 6.7 Hz
IH ddd J= 12.4, 11.7 and 8.2 Hz
IH ddd J= 12.4, 9.2 and 8.2 Hz
IH dddd J= 12.4, 12.4, 6.7 and O.9 Hz
IH ddd J= 11.6, 9.2 and 9.1 Hz
IH dd J= 2.4 and 1.2 Hz
.
502
.
757
821
.
020
.
811
4
720
1
690
9
o
IH m
IH ddd J= 8.8, 3.3 and 3.0 Hz
666
1
3
IH ddd .T= 12.3' 11.6 and 2.0 Hz
182 IH dddd .T= 13.6, 12.8, 12 3 and 1.7 Hz
71 (approx.) IH m
073 IH m dvid. d .T= 16.5 Hz
921
'
1
i
Assignment
Coupling
.
348
.
814
IH dd J= 15.3 and 8.8 Hz
IH d J= 2.4 Hz
3H d J= 1,2 Hz
IH s
3H s
467
C-1-H
C-2-Ha
C-2-Hb
C-3-Ha
C-3-Hb
C-5-H
C-6-Ha
C-6-Hb
C-8-Ha
C-8-Hb'
C-9-Ha
C-9-Hb
C-10-H
C-12-Ha
C-12-Hb
c-13-H3
C-14-H
C-15-H3
Since the single oxygen was att,ributable to the aldehyde group
these methine carbons rather resonating in a lower magnetic field
were deduced to be the C-10 tertiary methine and the C-1 bridgehead
methine carbons, respectively. Spin-spin decoupling experiments
and CH-COSY speetrum of RL-E (Fig. 3-285) clearly revealed a
sequence of proton coupling to form a carotane skeleton as shown in
Fig. 3-286. The methine carbons at, 6c 56.3 and 6c 50.3 each showed
a cross peak with proton signals of C-1-H (6H !.921) and C'10-H (6H
3.020), respectively. Thus, RL-E was proved as an isomerie
carotane dienaldehyde (12) and was named isodaucenaldehyde.
Stereostructure of 12 was determined by NOE experiments. By
the irradiation on the C-15 methyl protons, an NOE's were observed
on C-6-Ha, C-8-Hb, C-12-Ha and C-13-H3, suggesting rel* [7(R*),
10(R*) for 12 (Fig. 3-287). On the ot,her hand, stereostructure at
C-1 was determined by its proton coupling eonstant with vicinal C10-H (J= 11.6 Hz). If C-i are R* (namely cis-fusion on C-1 and C7), the dihedral angle between CIO-Cl-CIH and Ci-CIO-CIOH provides
cH, Hi(Cl)/--l)i
7(IN,
(-･-･b
H
N
N
77
([{tH
CHO
H. C]>
<H
×
H3C
.:/ <-XN
vH
'
/AJ
H
!I]i>
H H,ltYX
(l>/
H
,kv>
Fig.
3-286 Proton-Proton Coupling Sequence Elucidated by
Decoupling Experiment on RL-E
468
一1
o
ぎ｛
予
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o
6
o
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垂
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ミ
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→
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uo
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重
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h
重
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垂
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哺
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o
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「「
一
の
噂
一
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一
．‘、
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一
じ
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一
z
o
l
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嗣
罰
酋
一
。
、
儒
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ρ
h．
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l
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469
・H
h
Table 3-107 13C-?VMR chemical shift va1ues
fOl-
RL-E
(78 MHz, COM and INEPT, in CDC13, TMS as an int. std.)
'
6c CHx Assignment*
195.0 - CH
C-14
'
154.7 CH C-5
147.2 C C-4
145 ,.9 C C-11
113.4 CH2 C-12
56.2 CH C-1
50.3 CH C-10
43.7
CH2 C-6
'
42.7 C C-7
42.0' CH2 C-8
28.2
24.9
23.0
22.9
19.7
CH2
CH2
CH3
CH2
CH3
C-9
C-3
C-13
C-2
C-15
* The assignment was confirmed by the CH-COSY experiment on RL-E
470
1
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cr
-
6.S 6.0 S.5 5.0 4.5 4.0 3.5 PPM 3.0 2.5 2.0 1.5 1.0 .5 O.O
Fig. 3-285 CH-COSY Spectrum Qf RL-E (500 MHz, in C6D6): The
methylene carbon at 6c 28,2 unexpectedly showed no cross peak
with proton signals ; however, the carbon was eventually
assigned to be C-9 whose protons were also not attributable by
the spectrum.
471
nearly 90 O in tl}e inolecular model, while a model possesing S*
configuration at C-1 (trans-fused 5,7-bieyclic) indicates a small
angl.e (Fig. 3-288). In the Iight of Jackman-Sternhell function,
the coupling constant J= 11.6 }Iz between C-1-H and C-10-H is
*.
obviously compatible with C-10S configuration.
CH3
N
CHO
=-
H
N, ,
H3C
H
3-287 NOE's Observed on RL-E: The mutual NOE between C-15-H3
and C-13-H3 indieated that those groups were on･the same side of
Fig.
the five-membered ring.
15
15
'
'
'
'
H
7
L
x
x
t
t
7
'
1.
tl
11
10
T". 1OOP
'
t
t
t
1
t
t
2
t
1
11
t
z
t
'
t
10' k?T= 2Hoo
2
tt
,
'
t
H
t
t
tls
.v.2t
s
H
3-288 Dihedral Angles between CIO-Cl-CIH and cl-cio-cloH in
the Cases of C-IR* and C-IS*
Fig.
472
TabZe 3-108 Physicocheinical properties of isodaucenaldehyde (12)
CHO
12
A colorless oil
Vanillin-H2S04 color: brown
GC-MS m/z (%): 218 (M', 12), 203 (6.3), 189 (4.6), 176 (6. 8), 175
(14), 162 (6.4), 149 (11), 147 (9. 7), 136 (14), 135 (11),
133 (14), 121 (17), 109 (15), 107 (26), 105 (16), 95 (13),
94 (15), 93 (28), 91 (22), 81 (14) , 79 (30), 77 (16) , 68
(100), 67 (35), 55 (17), 53 (21), 41 (28).
EI-}4S m/z (%): 218 (M', 13), 203 (4.9), 189 (3.8), l76 (6. 2), 175
(14), 162 (5.8), 149 (12), 147 (9. 0), 136 (14), 135 (12),
133 (12), 121 (17), 109 (17), 107 (25), 105 (16), 95 (12),
94 (15), 93 (27), 91 (26), 81 (16) , 79 (28), 77 (16) , 68
(100), 67 (31), 55 (22), 53 (20), 44 (37), 41 (34), 40 (48)
IH- and 13c-NMR data are shown in Table 3-106 and 3-107,
respectively.
473
2) Isodaucenoic Aeid i
As described in Section 5, the fraction con-t,ainlng carota-l,4ttt
dienoic acid (4) furthec involved an unknown substanee isomeric to
4 and indicative of some carotane nature: After 4 was autoxidized
to yield RL-115MA-OX (4b) and rugosic acid A (2), the residue
recovered by PTLC yielded fine crystallines during the concentration (Fig. 3-289). When the crystallines were carefully washed
with cool n-hexane, ca 2.0 mg of colorless plates were obtatned.
This acidic substance (RL-115M-B) indicating M+ 234 in EI-MS (Fig.
3-2go) was revealed by IH- and 13c-NMR analyses to be a carotane
acid (structure 13) corresponding to isodaucenaldehyde (12) (Fig.
3-291 and 292). Most of the proton signals were similar to those
of 12, except C-14 formyl proton. The acid was the sub-inajor
constituent of the RL-115M fraction, unlike the carotane diene
aldehyde' mixture (RL faction) in whieh 12 was only a minor
eonstituent.
From the mixture of RL-115-M met,hylation products (See pp.
382), the methyl ester of 13 (RL-115M-B-ME) was also isolated. Its
spectroscopic data are shown in Fig. 3-293, 294.
474
H-EA-F 30:10:1
O
quenching under
UV 254 nm
ua
peroxide test: +
RL-115M-B
th
op
""-" r"-"-----･--)-F--m------.-.-.-
Reaction
MIX.
Std. RL-115}I
Fig. 3--289 . TL Chromatograins of RL-115M Fraet'ion Contained RL-115M- B
12Z
6
8Z
6Z
4Z
41
2Z
79
55
121
93
IZ5
191
145
5Z
Fig, 3-290
lel
164
219 234
2ZZ
15Z
EI-Mass Spectrum of RL-115M-B
475
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3ZZ
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41
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9
248
163
189
178
2Z
233
'
217
S7
se
lez
Fig. 3-293
2ez
15Z
25Z3ZZ35Z
EI-Mass Spectrum of RL-115M--B--ME
478
1養1・
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t
り
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民
＼
⊇
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479
h
Tab1e 3-109 Physicochemdcal properties of isodaucenoic acid (RL-
115M-B, 13?
N
COOH
13
Colorless powder
Vanillin-H2S04 color: clear pink
EI-MS m/z (%): 234 OI', 4.0), 219 (4.2), 191 (17), 164 (6.0), 149
'
IH-NMR
(8.3), 145 (9.1), 136 (9.9), 123 (11), 121 (27), 109 (13),
107 (14), 105 (17), 94 (15), 93 (22), 91 (20), 81 (15), 79
(-23), 77 (i2), 69 (21), 68 (100), 67 (26), 55 (15), 53
(15), 53 (15), 44 (27), 41 (28).
6,CrMDC
s13(500 MHz); 1,847 (IH, overlapped, C-1-H), 1･261 (IH,
m, C-2-Ha), 1.716 (IH, in, C-2-Hb), 3.050 (IH, m dvid. dd,
LT= 15.4 and 5.4 Hz, C-3-Ha), ca 1.68 (IH, m, C-3-Hb), ca
7.22 (IH, m, C-5-H), 2.469 (IH, dd, J= 14.8 and 9.3 Hz, C6-Ha), 2.039 (IH, br. d, J= 14.8 Hz, C-6-Hb), 1.577 (IH,
br. dd, J= 11.8 and 6.9 Hz, C-8-Ha), 1.455 (IH, ddd, J=
12.2, 11.9 and 8.0 Hz, C-8-Hb), 1.792 (IH, m, C-9-Ha), ca
1.88 (IH, m, C-9-Hb), 2.989 (IH, ddd, J= 11.4, 9.1 and 9.1
Hz, C-10-H), 4.797 (IH, dd, .T=2.3 and 1.3 Hz, C-12-Ha),
4.716 (IH, d, J= 1.3 Hz, C-12-Hb), 1.694 (3H, br. s, C-13H3), O.814 (3H, s, C-15-H3),
13c--NplR 6,CrrvD/813(7s MHz): s6.9 (CH, C-1), 22.8 (CH2, C-2), 27･9
(CH2, C-3), 134.0 (C, C-4), 144'.6 (CH, C-5), 42.7 (C-H2, C-
6), 42.3 (C, C-6), 41.9 (CH2, C-7), 28.2 (CH2, C-9), 50.1
(CH, C-10), 147.4 (C, C-11), 113.1 (CH2, C--12), 23.0 (CH3,
C-13), 172.2 (C, C-14), 19.5 (CH3, C-15).
480
Tab1e 3-110 Physicoehemical properties of RL-115MB-ne (13a?
N
COOCH3
13a
A colorless syrup
Vanillin-H2S04 color: clear pink
EI-MS m/z (%): 248 (M+, 33), 233 (19), 217 (8.4), 205 (45), 189
(28), 178 (21), 173 (21), 163 (32), 145 (30), 137 (47), 136
(53), 133 (34), 121 (100), 119 (50), 107 (48), 105 (65), 94
(51), 93 (82), 91 (54), 81 (43), 79 (61), 77 (35), 69 (42),
68 (88), 67 (62), 55 (37), 53 (37), 53 (37), 41 (60).
IH-NMR 6?6iDs6 (soo MHz): 3.o69 (IH , dddd, J= 15.0, 5.5, 2.2 and 1.1
Hz, C-3-Ha), 7.053 (IH, m, C-5-H), 2.431 (IH, dd, .J= 14.6
and 9.2 Hz, C-6-Ha), ca 2. OO (IH, br. d, J= 14.6 Hz, C-6Hb), 2.982 (IH, ddd, .T= ll .4, 9.2 and 9.2 Hz, C-10-H),
4.792 (IH, dd, J= 2.2 and 1.1 Hz, C-12-Ha), 4.712 (iH, d,
LI= 2.2 Hz, C-12-Hb), 1.692 (3H, d, .T= 1.1 Hz, C-13-H3),
3.724 (IH, s, C-14'-H3), O .806 (3H, s, C-15-H3)}
481
3) Isodaucenaldehyde Hydroxyl Derivative
Hydroxycarotaldehyde was found as a spot with Rf O.26 and
positive to DNPH reagent (orange) on silica gel/ pL/ates charged FrV-8 (from Sample V, See pp. 589) and developed in H-EA 3:1 (Fig. 3295). By column chromatography and PTLC of the fraction Sample V,
ca 4 mg of the foeused substance was isolated as a colorless syrup.
The compound denoted as RL-117C was also expected to be a carotane
aldehyde, which showed M+ 236 (18 %) with base peak at m/z 59 in
FI-MS (236.173 in FI-HR-MS, ClsH2202, calcd. 236.178)
On the other hand, a dehydration peak at in/z 214 was
EI-MS (Fig. 3-297). The presence of a hydroxyl group
by its IR spectrum appearing as a broad absorption at
(Fig. 3-296)･
observed in
was suggested
3430 cm-1
(Fig. 3-298).
' In t,he IH-NMR and HH-COSY analyses (Fiig. 3-299, 300 and
Table 3-111), RL-117C showed a similar signal pattern to that of
isodaucehaldehyde (12) except protons attributable to C-11, 12 and
13. The signals for the side-chain, revealed the presence of -C(OH)-(CH3)2. The 13C-NMR of the compound indicated a non-
hydrogen-bearing oxygenated carbon at 6c 73.3 assignable to the C11 (Fig. 3-301 and Table 3-112). Furtherinore, two methine carbons
resonating in a downfield at 6c 56.5 and 53.1 resonating downfield
were characteristic of a tertiary or a bridgehead carbon. As C-1
and C-10 methine carbons of 12 were detected at 6c 56.2 and 50･3,
respectively, the former methine carbons of RL-117C were similarly
assigned to those positions. Consequently, structure of RL-117C
was elucidated as 14, and this novel carotanoid was named
hydroxycarotanaldehyde.
482
"-EA 3:1
O
O
quenching under
UV 254 nm
"1tw
DNPH reagent
positive
RL-117C･ob
---------be--------------
Fr- V- 8 std. 1
3-295 TL Chromatogram of RL-117C
Fig.
2
1eez
.ez
g
t2
F-t e
.ee
se
1SZ
1ee
1eee
2e.ee
>oz
hi
Hz
M++1
}-rl
He
M!E
Z
M+
.ez
-i
t
2SZ
2ee
Fig. 3-296
FI-Mass Speetrum of RL-li7C
483
M!E
IZZ
5
se,
6Z
191
2Z3 218
95
43
4Z
t-
235
55
az
67
*5.Z
81
IZ7
lez
5Z
Fig. 3-297
121
16Z
147
178
15e 2ZZ
25Z
EI-Mass Spectrum of RL-117C
:･ i- 1 ;5 6
7 6a 1) 1･1i
1+
/, 1, I Ii[ri11
l.1-;illLi;,.+li I!l i l･:. l, ;i･ l･ 'l li. Is i, l -1.Il..ll-..ili .i
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Hrin rirrIi-=-r･r'rlrsT,=-t-nr,li,
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. i,il ;･ ` :;' '''::4::1
:U
IR Spectrum of RL-117C
484
./4/r."r::
!/Li:1':.tn:z/.
V,i･--,i.i･L･"･i
,t i ;E., .,:2:- 1
Fig. 3-298
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485
Tab1e 3-111
1
H-IVMR chemical shift values of RL-117C
(5oo MHz , in C6D6, TMS as an int . std.)
6H
9
6
3
.
283
.
067
.
361
345
2
2
'
056
2
1
1
074
.
708
.
569
1
482
1
466
1
1
1
1
o
Assignment
IH s
IH ddd J= 8.8, 3.3, 3.2 Hz
IH
IH
IH
IH
IH
IH
dddd .T= 15.3, 5.1, 2.3, O.8 Hz
m dvid. d .T= 13.9 Hz
dd J= 15.1, 8.8 Hz
ddd .T= 12.2, 9.1, 9.0 Hz
br. d J= 15,1 Hz
ddd .T= 12.2, 11.1, 1.7 Hz
IH m
IH m
IH m
.
245
.
20 (approx.) IH overlapped d-like
.
148
,
088
.
951
935
o
o
Coupling
'
564
IH dddd J= 12.6, 12.6, 12.5, 2.2 Hz
IH ddd J= 11,O, 10.5, 8.1 Hz
3H s
3H s
3H s
C-14-H
C-5-H
C-3-Ha
C-2-Ha
C-6-Ha
C-10-H
C-6-Hb
C-1-H
C-9-Ha
C-3-Hb
C-8-Ha
C-9-Hb
C-2-Hb
C-8-Hb
C-12-H3
C-13-H3
c-l5-H3
486
9
8
6
ア
5
2
3
4
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o
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o
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Fig・ 3－300
COSY Spectru班 of RL－117C （270 MHz・ in C6D6）
487
霧毒
一
葬
ぎ｛
鼻
｝
2
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9
妻
，
一
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488
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Tab1e 3-112
13C-ALISfR chemical shift values of RL-11 7C
(78 MHz, COM and INEPT , in C6D6, TMS as an int . std.)
6c
CHx
l93
Assignment*
3
CH
C-14
152
.
8
CH
C-5
146
.
6
C
C-4
73
.
3
C
C-11
56
.
5
CH
C--1
53
.
1
CH
C-1O
43
.
5
CH2
C-6
42
,
3
C
C-7
8
CH2
C-8
3
CH3
C-12
7
CH3
C-13
1
CH2
C-9
6
CH2
C-3
1
cH2
C-2
3
CH3
C-15
41
32
.
27
,
27
.
25
.
23
19
･
489
Tab1e 3-113 Physicochemical properties of hydroxycarotanaldehyde
(14)
N
CHO
;
:
H
OH
14
A colorless syrup
Vanillin-H2S04 color: brown --) pale pink
FI-MS m/z (%): 237 (M++1, 30), 236 (M+, 18), 221 (25),
219 o!+-
'
H20+1, 34), 177 (46), 59 (100).
EI-MS rdz (%): 236 (M', O.2), 235 (O.4), 221 (1.4), 218
4.2), 203 (4.0), 191 (5.3), 178 (8.4), 175 (8.2),
149 (12), 147 (12), 145 (11), 136 (11), 135 (11),
107 (15), 105 (13), 95 (43), 93 (20), 81 (24), 79
(20), 59 (100), 55 (23), 43 (40), 41 (32).
IH- and 13c-NMR data are shown in Tables 3in111 and 112,
respectively.
490
(M+-H20 ,
160 (17)
'
121 (15)
'
(20), 67
3-7-4 Carota-trienaldehyde and Its Related Compounds
1) Dehydrodaucenaldehyde
RL-D showing M+ 216 in GC-MS was a further dehydrogenated form
of carota-1,4-dienaldehyde (3), daucenaldehyde (10) or
isodaucenaldehyde (13). During the HPLC of the RL fraction, RL-D
was detected as the third peak (Fig. 3-302) and was obtained as a
colorless oil (1.8 mg). The EI-MS of the isolate was agreeable
with GC-MS of RL-D (Fig. 3-303), and its IH- and 13c-NMR spectra
were indicative of the triene form (Fig. 3-304, 305 and Tables 3114, ll5). Decoupling experiments revealed two -CH2-CH2- units
relayed by a homoallyl coup}ing each other. In addition,
-C(CHO)=CH-CH2-, -C(CH3)=CH2 and -C-CH3 moieties were visible.
Thes' e results formulated RL-D as 15, and the proposed structure was
supported by detection of four non-hydrogen-bearing olefinic
carbons ' at 6c 146.7, 144.7, 141.9 and 137.0. Compound 15, a
dehydrogenation product of 9 or i2 was given a trivial name,
dehydrodaucenaldehyde.
,
s
i.
:
l
I
-:t -
;
l
--.--L-..
;
/1
i
l･ RL-D
'f-
'J .tb,.
:.
o
i'
l
T
I-
･--- 1
:･
l''
N)
;
l -"'-r'',
;
t'------'-'-l
-iM'----'----'-----'---
;i .O
;.
i
'i
Fig. 3-302
HPL-Chromatogram of RL-D Detected at UV 230 nm
491
ez
1
216
1
145
8Z
91
6e
173
16Z
41
.
117
77
4Z
1Z5.
55
187
2Zl
69
65
2Z
181
5Z
lez
IZZ
2ZZ
15Z
1
251
3ZZ
2S.Z
3ZZ
9
8Z
146
6Z
4e
91
41
IZ5
133
le7
77
2e
216
55
161
65
5Z
Fig. 3-303
IZZ
173
2Zl
15Z 2ZZ
GC-Mass (top) and EI-Mass (bottom) Spectra of RL-D
492
11
匡
。
‘二）
8
日
雨
8
貯
諺
面
面
累
曾
8
純
L＄
附
呂
面
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9
純
（
⑩
o⑩
o
8
コ
。【1
9
繋
純
“
。＿
⊂》4．
，巳、．
Lρ〔；一
霧
9
丙
8
oo
鴎
雨
。
∩
1
ω
面
．日
幅
臼
雨
）
8
8
。
8
面
日
べ
ρ
o
9
Φ
ぜ
3
q
の
潟
8
諸
1
ぜ
禺‘
¶脚→ ・
8
d
8
唱
マ．
o
Qり
l
呂
8
5
oり
の
8
ヨ
（⊃
ω
493
舞
●
bO
・Fl
山
Tab1e 3-114
1
H-ALIttR
chemical shift･ va1ues for RL-D
(5oo MHz , in CDC13, TMS as an int . std.)
6H
Assignment
Coupling
247
IH
s
6
066
IH
4
935
IH
ddd .J= 8.1, 5.3, 2.0 Hz
dd .T= 2.9, L5 Hz
788
IH
O08
IH
.
693
IH
.
327
IH
.
248
IH
039
g22
IH
.
854
IH
.
726
3H
br. s
.
689
IH
.
539
IH
.
468
IH
br. dd .T= ca 16, ca 14 Hz
ddd J= 12.4, 8.4, 4.6 Hz
ddd .f= 12.4, 8.7, 7.3 Hz
.
800
3H
s
9
4
.
.
3
2
2
2
2
1
l
1
1
1
1
o
.
.
IH
d J= 1.5 Hz
ddd J= 15.1, 5.5, 31 Hz
ddd J= 16.2, 5.5, 3.3 Hz
dddd J= 15.8, 8.7, 8.4, 2
dddd J= 15.8, 7.3, 4.6, 1
dd J= 14.4, 53 Hz
dd J= 14.4, 8.1 Hz
br. dd J= 15 -p
1 ca 14 Hz
494
C-14-H
C-5-H
C-12-Ha
C-12-Hb
.
8
Hz
o
Hz
C-3-Ha
C-2-Ha
C-9-Ha
C-9-Hb
C-6-Ha
C-6-Hb
C-3-Hb
C-13-H3
C-2-Hb
C-8-Ha
C-8-Hb
C-15-H3
．・一
9り
国国
Q o
B，
σq
o
c｝
日
鶏
穿
鼠
躬習
℃
呂葱．
ド
o
ρo
ぎ
風
⑩
一9∩
⑩
o
・F→
需q
〔．，
．一
熟
N
ｮつマ＿
一・
錐
〔・Σ！
一ヨゆ
@（N
，・
州
）
1
臼
も
旧邸
一 』
臼
．尉
9
一 ζ為
の
呂因
j
ρ〇
一 力
ｰl
r’
8剛
8
一 ゆ
一塗あ
8響
国
e刈
495
Table 3-115
13C-IVMR chemical shift vaZues of RL-D
(125 MHz, COM and INEPT, in C6D6, TMS as an int. std.)
6c
192.8
152.1
146.7
144.7
141.9
137.0
113.9
50.2
41.4
39.0
32.6
24.1
24.0
23.3
22.6
CHx
Assignment
CH2
C-14
C-5
C-1
C-4
C-11
C-1O
C-12
C-7
C-6
CH2
C'-8
CH2
C-9
C-13
C-3
C-2
C-15
CH
CH
C
C
C
c
CH2
C
CH3
CH2
CH2
CH3
496
Table 3-116
Physicochemical properties of dehydrodaucenaldehyde
(15)
CHO
15
A colorless oil
Vani11in-H2S04 color: brown
GC-MS Jn/z (%): 218 (M", 12), 203 (6.3), 189 (4.6), 176 (6.8), 175
(14), 162 (6.4), 149 (11), 147 (9.7), 136 (14), 135 (11),
133 (14), 121 (17), 109 (15), 107 (26), 105 (16), 95 (13),
94 (15), 93 (28), 91 (22), 81 (14), 79 (30), 77 (16), 68
(100), 67 (35), 55 (17), 53 (21), 41 (28).
EI-MS m/z (%): 216 (M+, 27), 201 (M"-CH3, 17), 199 (6.2), 188
(11), 187 (M"-CHO, 38), 173 (16), 161 (20), 159 (16), 146
(64), 134 (26), 133 (41), 131 (28), 119 (100), 105 (39), 93
(26), 91 (57), 79 (22), 77 (27), 55 (20), 41 (43).
IH- and 13c'NMR data are shown in Table 3-114 and 3-115,
respectively.
497
2) Dehydrodaucenoic Acid
In the Fr-VIIA-6, a quenching spot was detected (Fig. 3-306).
From t.he fraction of 1/10 volume, the focused compound was
successfully isolated by PTLC (H-EA-F 30:20:1) to give a eolorless
syrup (ca 3 mg). The isolate showing M+ 232 in EI-MS (Fig. 3-307)
was tentatively deduced as 16, since the eompound exhibited a quit･e
similar IH-NMR spect,rum to that of dehydrodaucenaldehyde (15) (Fig･
3-308'
). As well as carota-1,4-dienoic acid (4) or isodaucenoic acid
(l3), t,his sesquiterpene acid whose corresponding aldehyde was also
contained was also present in Rosa rugosa leaves as a minor
constituents, and was named dehydrodaueenoic acid.
H-EA-F 30:10:1
O quenching under
UV 254 nm
Dehydrodaucenoic Acid
a
9
o
--------g,,F------.
Fr-VIIA-6
Fig. 3-306
TL Chromatogra]n of Fr-VIIA-6 Containing a Carotane
Trienoie Acid
lez
1
9
8Z
177
6Z
91
41
4Z
IZ5
77
55
ae
187
133
145
6e
232
15g 171
199
5Z
Fig. 3-307
IZZ
213
2zz 2se 3zz
i5Z
EI-Mass Spectrum of Dehydrodaucenoic Acid
498
蓄
」
旨
li
L’
｝
戸、
〉
一
薫
。
一
一扇 o
o
Q
藁
，
圏
一
一
～
驚
“童
《
の州
。
・H
ハN
『
め
対
こ＼」
しり o
の o
！r竃
噂
ゆ
二
ゆ
N
・r→
り
く
円
q．
麻ﾖ
己¢
L （レ
『 州
一
Lρ
GつΣ二
騨
・α＿
▼一cL
鴨
の o
oり
N
ロ
丙
達
麦
1．
o （⊃
の
鉢→
○
『
1ω
しニつ
ρ
雨
N
N
『
o
「＼ ω
の
Σ
oz
しり
隊
1
ミ
L
茎
ト
σ）
「i
ユ
の
一・一
一＝＝
てゴ
r
、
B
ln OO
レ．・
1
l
α） ○
σり
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『σつ
σヤ
b三
い
「許
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し
499
Tab1e 3-117 .Physicochemical properties of dehydrodaucenoic acid
(1 6?
COOH
16
Colorless syrup
Vanillin-H2S04 color: dark pink
EI--MS m/z (%): 232 (lv:[', 15), 217 (6.3), 187 (44), 177 (72), 145
(22), 134 (29), 133 (39), 131 (31), ,119 (100), 105 (36), 93
'(23), 91 (52), 77 (28), 55 (26), 44 (41), 41 (50).
iH-NMR 6gR8i3(soo MHz): 2.763 (iH, ddd, J= i4.i, 6.o and 2.g Hz,
C-2-Ha), 1.909 (IH, br. dd, J= 14.1 and 13.5 Hz, C-2-Hb),
2.852 (IH, ddd, J= 15.0, 6.0 and 2.9 Hz, C-3-Ha), 2.107
(IH, br. dd, .J=15.0 and 13.8 Hz, C-3-Hb), 7.233 (IH, ddd,
7.2, 7.2 and 1.8 Hz, C-5-H), 2.335 (2H, d, J= 7.2 Hz, C-6H2), 1･738 (IH, m, C-8-Ha), 1.681 (IH, m, C-8-Hb), 2.411
(IH, dddd, J= 16.1, 8.3, 8.2 and 2.9 Hz, C-9-Ha), ca 2.30
(IH, overlapped, C-9-Hb), 4.941 (IH, dd, J= 2.3 and 1.4 Hz,
C-12-Ha), 4.719 (IH, d, J=2.3 Hz, C-12-Hb), 1.842 (3H, br.
s, C-13-H3), 1.005 (3H, s, C-15-H3).
500
･ 3) Dehydrodaucer}aldehyde Hydroxyl Derivative
RL-119A was mainly isolated from Fr-VH-9 (See pp. 586) as a
minor constituents. By PTLC, the compound was isolated as a
colorless syrup (Fig. 3-309). In EI-MS, the parent ion (m/z '234,
11 %) and successive dehydration peak (m/z 216, 23 %) were
observed, suggesting a hydroxylated compound for RL-119A (Fig･ 3310). In the IH-NMR spectrum, some signals characteristic of a
tt
earotane aldehyde were visible [e.g. an a,B-unsaturated aldehyde
group at 6H 9.345, a bridgehead inet,hyl group at 6H O.822 and an
isopropenyl group at 6H 5.104, 4.988 (exomethylene) and 1.787
(allyl methyl)] (Fig. 3-311 and Table 3-118). As the signal
perttern was similar to that of isodaucenaldehyde (13, See Fig. 3275, pp. 486), the compound was expected to be a hydrate of 13.
By the 13C-NMR analyses of RL-119A (COM and INEPT), a
oxygenat'ed sp3 carbon was detected at 6c 86.7 (Fig, 3-312 and Table
3-ll9). On the other hand, only one methine carbon was observed at
c 66.5, unlike 13. The presence of these two carbons suggested
that either C-1 or C-10 was hydroxylated and gave a deshielding
effect on the adjacent methine carbon. The position of
hydroxylation was considered to be C-10 by comparison of t,he IH-NMR
spectrum with that of 13. Namely, C-10 ]nethine proton which may be
deshielded characteristically around 3 ppm by the C12/C13 olefinic
bond (e･g･ 6H 2.989 in 13) was not, detectable in RL-119A. The
methine proton of RL-119A resonated ln an upfield (6H 1.946, dd, J=
11.8 and 1.5 Hz) was rather favorably assigned tQ C-1-H (cf. C-1-H
of 13; 6H 1.921, ddd, .J= 12.3, 11.6 and 2.0 Hz). Thus, the
compound was proposed to be 17 which is a relative of dehydrodaucenaldehyde (15), and was named hydroxyisodaucenaldehyde.
501
H-EA 3:1
C-M 50:2
O
quenching under
UV 254 nm
N6
RL-119A
RL-119A -.
･v
.---･d---'-"'t'-"
Fr-VH-9
Isolated
Fig. 3-309
ze
RL-119A
TL Chrofiiatogram o£
6
41
84
se
6e
le9
91
119
55
4Z
79
97
133
146
2Z
151
216
lg7
l61173
'
2Zl
234
Z6
'
se
Fig. 3-310
lze
!5Z
2ZZ
EI-Mass Spectrufn ef RL-119A
502
2sz3ee
¶
まり
＼
＼
5
、
＼、
ノ
ほ
一
摯
＼．
層k
幽、＼
＼r
一
7｝
`・
㌔・，
’」
ヘロ
」
》
3
需闇廟胴r＝一．
一
～
～
、
ξ
蓬
一
、＼気
ノ
一』 黶ｻ．
≦
～
⑩
自
⑩
o
「
ぐ
」
・r一盛
｛
儀
『
ヘノ
矯、
L～
〉
う
。
卜
Q
｛
1
）
≦ノ…
＜
①
白
州
【
り
臼
㍉
ダ
輌
㍗』一
。
霞
、
≧
～
臼
鯉
～
。
Φ
の
ユ
1＝ま
“
H
、
ζ「
州
し7魑
鳥
503
l
。
州
Qり
1
0り
■
山
・セ→
Table 3-118 IH-IVMR cheinica2 shift values of RL-119B
'
'
-(500 MHz, in CDC13, T)IS as an int. std.)
6H Coupling
'
Assignment
9.345 IH s
6.713 IH ddd J= 8.8, 3.3 and 3.3 Hz
5.104 IH br. s
4.988 IH dd J= L5 and O.7 Hz
3.083 IH ddd J= 13.6 '4.8 and 2.9 Hz
2.643 IH dd .T= 15.4 and 8.8 Hz
2.354 IH br d J= 15.4 Hz
2.300 IH ddd .T= 13.6, 11.7 and 6.6 Hz
1.946 IH dd J= 11.8 and 1.5 Hz
1.912 IH ddd J= 12.5, 12.1 and 7.7 Ilz
1.787 3H d .T= O.7 Hz
1.77 (approx.) IH m
1.690 IH ddd .T= 15.8' 6.2 and 2.9 Hz
1.596 IH dd J= 12.5, 7.0 and 5.5 Hz
1.58 (approx.) IH m
1.208 IH ddd-like m
O.822 3H s
504
C-14-H
C-5-H
'C-12-Ha
C-12-Hb
C-3-Ha
C-6-Ha
C-6-Hb
C-3-Hb
C-1-H
C-9-Ha
C-13-H3
Cr2-Ha
C-9-Hb
C-8-Ha
C-8-Hb
C-2-Hb
C-15-H3
彦
要
．＿＿■」
◎つξ
Q
巴ll
妻
N
o
Ql’
一
蓬
雲
蒙
〇
一
番
彗
崩
一
垂
茎
一
一
耳
一’．＿
3
＝＝二二達
サ
4
il
垂
垂
晋
多
ま
妻
↑
一『’
コ
ま
摩
妻
歪一一
董
。
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o～
o
ハ
れ
ρ、
髪
．釜
喰・
｛
oq
垂
o噌
垂
ρ
妻
の
督、
茎
垂
o
ρ
σ
6
四
一
9
多
’ 罫
h
＿し＿＿＿＿＿タ
＿．一・・甲一■
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＿
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←一一一一
ρ
．塁
垂
一一『二’．ボニ，
．三
菱
の
h
茎
毒
富
（⑩
⑩
Q
ξ
擁
毫
蓄
・7→
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ω
い
葺
窒
葺
ρ
φ
N
国
の
ホ
妥
垂
垂
ぎ
o
o
一
寺
・レ
∫
一
重
i
孟
∫
5
一
董
．憂
一石
。
ま
も＝
コ
雪
き
1
｛
チ
ぎ
茎
夢
，耀
4
享
弧
．一
’ゆ
一』郭
垂
茎
｛
二 、
彊
需ゴ
ゐ
σり
一の
茱
鴛
蓋
妄
茎
ぎ
一
」、
層h
凶
更3
ツ、
H
HN
oり
｝
◎り
婁
2
塁
｝
卵
り
「■P
一
孝
3
の
しrく
虫
蚕
妻
Φ
書
‘
｝
蓼
｛
o
一口
り
事
覧
チ
ゴ
Σ
弄
ρ
．，、
‘．．
亨
日
⇒
『．2
」
醤
曝
婆
‘
口
触
1
冨
＜
①
州
州
1
葦
曇
孝
二
．自
⑩
）
垂
雫
3
◎o
し、
r．h
－一一i
505
●
・H
隅
Tab1e 3-119
13
C-IV7VIR chemical shift values of RL-119B
(78 MHz, COM and INEPT , in CDC13, T)IS as an int . std,)
6c
CHx
Assignment
9
CH
C-14
3
CH
C-5
148
7
C
C-4
145
7
C
C-1l
112
2
CH2
C-12
7
C
C-1O
5
CH
C-1
44 4
CH2
C-6
43
3
C
C-7
3
cH2
C-8
1
CH2
C-9
25 o
CH2
C-3
21 8
CH2
C-2
21
4
CH3
C-13
19 4
CH3
C-15
194
.
154
.
86
.
66
.
.
,
39
38
.
.
.
.
.
506
Tab1e 3-120 PhysicochemicaZ properties of hydroxyisdaucenaldehyde
(1 7?
COOH
17
A colorless syrup
Vanillin-H2S04 color: brown
EI-MS m/z (%): 234 (M', 11), 216 (M'-H20, 23), 206 (4.6), 201
(11), 187 (19), 173 (12), 161 (13), 151 (15), 150 (14), '149
'(15), 146 (24), 137 (24), 133 (33), 125 (40), 123 (29), 119
(42), 109 (51), 107 (35), 105 (33), 97 (30), 95 (29), 93
(38), 91 (44), 84 (83), 81 (31), 79 (35), 77 (26), 69
(100), 67 (34), 55 (39), 53 (25), 43 (54), 41 (92).
IH- and 13c-NMR data are shown in Tables 3-118 and 3--119,
respectively.
507
3) Carotane Trienaldehyde Derivative
During the survey of rugosic acid A methyl ester (5), a
compound showing a dull orange color on the peroxide test･ was
detected in the fraction contained 5 (See pp. 402). This compound
denoted RL-PERO-4 was also isolated by PTLC (Fig. 3-313). As the
result, 12.8 mg of a colorless syrup denoted as RL-PERO-4 was
'
obtained. The isolate showing M+ 232 in FD- and EI-MS (Fig. 3-314
' to be a dioxygenated carotanoid. In the IHand 315), was expected
NMR spectrum, it was revealed that RL-PERO-4 posessed an olefinic
bond in the seven-membered ring and the olefinic protons were
vicinally coupled with each other (.J= 12.0 Hz of isolated cis
coupling) (Fig. 3-316, 317 and Tables 3-121, 122).
Informatlon for its oxygenation form was provided by 13C-NMR
analyses (COM and DEPT) (Fig. 3-318 and Table 3-123). Only a
methine earbon 6c 29.0 and two oxygenated carbons (6c 78.3 and 72.3)
were clearly dirrerenciated among 10 sp3 carbons. Especially, the
latter carbons were both non-hydrogen-bearing. These facts were
suggest･ive of the presenee of an epoxy ring on C-1/C-10. A proton
coupling sequence of -CH2-CH2- was assignable to C-8/C-9. However,
there were t,wo possible st･ructures (D and E) for RL-PERO-4 as shown
in Fig. 3-319. As a deshielding effect, of C-14 aldehyde group was
observed on an olefinic proton at 6H ca 6.79 in CDC13 (cf'･ 6H
5.904, br. dd, J= 8.1 and 2.3 E{z, in C6D6), it was suggestive of
the presence of ct,B-unsaturated aldehyde group. Since its UV max
was observed at 270 nm, that olefinic bond was expected to be
further conjugated with this aldehyde group to form a,B;a;B'unsaturated one.
By the NOE experiments, NOE between one of the allylic
methylene proton (6H 1.981 in C6D6) exhibited an NOE with C-15-H3
and C-12-H3. These NOEs are only compatible when the rnethylene
carbon allocates to C-2 (Fig. 3-320). Accordingly, the structure
was presumed to be D. To confirm the proposed structure, chemical
conversion of this compound was carried out.
508
H-EA
,4 :1
o
RL-PERO-4
quenching under
UV 254 nm
de peroxide test: +
N@
iEllil>
Fr-V-3
TL Chromatogram of RL-PERO-4
Fig. 3-313
M+
2
1eez
se.ee
'X'-.
o
dz
i
HZ
t-1
"-'t
Z
se
, ,
- d, ii- - -, t- -- Y s 2se
lse 2ez
1ee
tt+t
va.?e
M!E
:';
1eee
se.ez
-)-
H.
i-l
oo
z'
di
F･
z
f-'t
.?.ee
e
Bez
Fig. 3-314
4ee
3SZ
4se
FD-Mass Spectrum of RL-PERO-4
509
szeM/E
lze
1
3
8Z
129
6Z
4e
43
157 171
115
91
51
63 71
5Z
Fig. 3-315
214
199
77
2Z
189
IZ5
IZZ
15Z
2zl
EI-Ma,ss Spectruln of RL-PERO-4
510
25Z
3ze
レ
’
8
面
掌
8
雨
（6
＄
q⑩
o
8
⊂：）甲．
好
昏註1
・7→
＿：a
“
8
8Σ
噌・
頃
吹F
o
o
め
）
9
寸
I
8
8
め
o
餌
嵐
山
1
丙
繭
8
軸
。
べ
饅．
L
丁
d
φ
ρ
o
8
Φ
南
の
累
8
じ
ご
寓
1
8
φ
国
H
⑩
H
σり
8
「く
．累
1
8
甲6
済
σり
。
一
・F→
国
＄
ド
511
Table 3-121
1
H-N?YR chemical shift }ralues foi' RL-PERO-4
(5C)C M'Hz, in C6D6, TMS as an lnt. std,)
6H
9.107
7.042
5.904
5.537
2.447
1.981
1.710
1.592
1.544
1.212
O.987
O.923
O.718
O.603
Coupling Assignment
(for 18)
'
IH
C-5-H
IH d .J= 12.0 Hz
IH dd .T= 8.1 and 2.3 Hz C-3-H
IH
IH dd J= 18.5 and 2.3 Hz C-2-Ha
IH dd J= 18.5 and 8.1 Hz C-2-Hb
IH dd .J= 13.6 and 8.2 Hz C-9-Ha
IH
IH ddd J= 13.6, 10.5 and 8.0 Hz C-9-Hb
IH br. ddd .T= 11.9, 10.5 and 8.2 Hz C-8-Ha
3H
IH dd .f= 11.9 and 8.0 Hz C-8-Hb
3H
3H
d J= 12.0 Hz C-4-H
sept J= 6.9 Hz C-11-H
d J= 6.9 Hz C-12-H3
d J= 6.9 Hz CH13hH3
512
（』，
2
9
r、；
窮
5
9Qつ
囲
面
o
r「｝
∩
o
雪
詩
丙
9・r→
ロ
臼
の
「N
o
o．
〔．）
一⊂）
1
α〕
費
ま
済
8
ｰゆ
一『・・）
」⊃L
可
iO
ぐ〉ζ』
国
Cl
ω臼
⊂⊃．一．．
占
．o詮’
餉
o
丙q．
r〕臼
・『コ
【臼
ρ
o
①
の
零国
臼
z
l
’ ． F→
φ
・・，芝＝
器
ω
雰
卜
σ；F弓
9
oり
1
め
σり
9鉾
9山
513
TabZe 3-re2 IH-IVMR cheJnical shift values
(500 TvlHz, in CDCI3, TMS as an ip.t. std.
'
6H Coupling
'
fO 1'
RL-PERO-4
)
AssignIIIent
(for 18)
'
C-14-H
C-5-H
9.397 IH s
6.79 (approx.) IH in
6.784 IH d J= 12.2 Hz
5.705. IH d J= 12.2 Hz
2.757 IH dd .J= 18.6 and
2.619 IH dd J= 18.6 and
C-3-II
2
8
1.916 IH m
1.904 IH m
1.806 IH sept .l= 6.9 Hz
1.313 IH m
1.308 IH m
1.111 3H d .T= 6.9 Hz
1.009 3H d J= 6.9 Hz
O.884 3H s
514
.
6
Hz
o
Hz
C-4-H
C-2-Ha
C-2-Hb
C-9-Ha
C-9-Hb
C-11-H
C-8-Ha
C-8-Hb
C-12-H3
C-13-H3
C-15-H3
L
k
Q
Qり
寓国
oo
σq
o
臼
臼
（
白
山
国
。
・窮
穿麗
．⊂）芝
L「1
0O
・5Qつ
の
δ
一臼8
9・7一｛
“
N
9謁
ゆ
。σq
＿（＝，F→
一轟）
ぐ
．ヨ1
一・
国
五
一屋舶
。，Ω→
一21
o
．冑雲
ぢ
ぷ巴
の
鵡餌
一ω：Σ1
1
6Q
一“
．一
u＼⊂つ
一’
．零
@H
oo
一
◎り
潔σつ
1
bO
（＝つ ・州
N山
一く＝）
515
Table 3-123
13C-AUlfR chemical shift values of RL-PERO-4
'
,(78 MHz, COM and INEPT, in CDCI3, TMS as an int, stde)
'
6c CHx Assignment*
193.5 CH
153.8 CH
138.6' C
127.2 CH
125.5 CH
C-14
C-3
C-4
C-5
C-12
78.3 C C-1
72.3 C C-10
42.1 C C-7
38.8 CH2 C-8
33.3 CH2 C-9
29.0 CH C-11
24.0 CH2 ･ C-2
20.7 CH3 C-15
19.0 CH3 C-12
18.4 CH3 . C-13
* The assigmnent for RL-PERO-4 is according to the proposed
structure 18.
516
By tveatment･ of RL-PERO-4 with thiourea/MeOH, a polar product
possessing MW 232 was obtained in a 34 % yield (fine prisms, 2.3 ing
from 6.8 mg, Rf O.26 in C-M 50:2) (Fig. 3-321). As the
'
spectroscopic data were indicative o£ 10-hydroxycarota-1,3,5-triene
aldehyde, the product was introduced an olefinic bond through the
epoxy ring cleavage (18a) (Fig. 3-322, 323 and Tables 3-124, 125).
The olefinic bond formation shoiild occur by electron transformation
via B-hydrogen transferation (C-2) to result in the epoxy clevage
(Ckl-O) (Scheme 3-34). Accordingly, the structure of RL-PERO-4 was
proved to be 18.
s-
zfil cHo XN cHo
--- :
Fig. 3-319 Two Possible Structure for RL--PERO-4
. /.. CH,
H
<---c ,,,
H
CHO
'
N
t
't
.
"-
o
H
iii[iiij7
H3C
CH3
Fig. 3-320 NOEs Observed on RL-PERO-4
517
H-EA 3:1
O
:'i':;1/'t':::'
@
quenching under UV 254 nm
vanillin-H2S04 test: +
@
Reaction
Std.18,
MIX.
Fig. 3-321
TL Chr'omatogram of RL-PERO-4-TU Obt,ained by
Treat,ment of RL--PERO-4 wit,h Thiourea
s･- :
/ CHO
o
thiourea
N･= :
HO
Scheme 3-34
N
1 CHO
Conversion of RL-PERO-4 into a hydroxyl derivative
518
1e2e
M+
l
1
2
-(･,e
,ee
l
'
ttt
･>･
tr,
o'
5
7'
"" e
'' 'I
t
1ee
S2
>ez ' '`i's'ti'
1SZ
'lh
.e ,ze
M./E.
p,;
1eze
-(ie.ee
-)
tI'
oz
hi
t''
-'-"
t
o.?e
e
35e
3ee
4se
4-ze
5eeM/E
FD-Mass Spectrum of RL-PERO-4-TU
Fig . 3-322
IZZ
1
3
8Z
128
6Z
43
4Z
115
91.
2e
157
77
51
63 71
5Z
Fig. 3-323
171
189
214
19S
232
---L---r---"-------------*IZ,Z
IZ5
IZZ
15Z
2ZZ 25Z
EI-Mass Spectrum of RL-PERO-4-TU
519
3ze
！：
8
8
面
累
（
σり
H
80
・8
器
臼
累Σ
・7→
一島
の
鰍
宰飼
ゴΣ1
9Σ
謁
ま
留
ω
8
話
純
の
oo
ゆ
8r
）
一，‘・．＝⊃
の「乙
ｸ岡
1
寸
l
o
〔⊃国
ﾏ→
一。⊃
9
臼
べ
ω
ω1
口
輌
o
の
一『露
r・5
9
rく
臼
べ
ρ
o
①
q
（⊃の
＿（コ
面山
1
8州
σ；rr
N
1
8弱
σり
σり
9蔵
．520
Tab1e 3-124
1
H-Al7itrR
chemical shift va1ues
fOl-
RL-PERO-4-TU
(500 MHz , in CDC!3, TMS as an int. std.)
6H
Coupling
Assignment･
(for 18)
9
7
6
6
5
2
2
2
1
567
IH
.
219
.
640
.
478
.
487
.
118
.
056
041
d J= 6.7 Hz
IH d J= 10.2 Hz
IH d J= 6.7 Hz
IH d J= 10.2 Hz
IH ddd J= 12 .7, 6.4 and 6.1 Hz
IH dd J= 12. 7, 6.5 Hz
IH sept. J= 6.8 Hz
IH ddd J= 12 .9, 7.3 and 7 2 Hz
IH ddd J= 12 .8, 7.8 and 7 4 Hz
3H d J= 6.8 Hz
3H d J= 6.8 Hz
664
3H
･
.
119
1
o
023
ss3
541
1
1
C-14-H
.
.
s
IH
.
s
521
C--5-H
C-3-H
C-6-H
C-2-H
C-9-Ha
C-8-Ha
C-11-H
C-8-Hb
C-9-Hb
C-12-H3
C-13-H3
C-15-H3
・”
＿＿＿＿
cつ
c9
巨＝霞鵠
盾?Q
s』
9
9臼
山
国
窩
∩
℃
9
O
o
掌
◎り
同
o
o
o
呂
尺ε
“
8、遷
ゆ
目
）
㌃㌣
9マO
炉4
国
日餌
一 1
口
触
o
鶏
＿ 臼
9邸
ぢ
量．
の
妄言
〒
臼8
H
一器
ゆ
鍔
8あ
bD
N8唄
匡1
522
Table 3-125
13
C-IVIYR chemical shift values of RL-PERO-4-TU
(78 M'Hz, COM and INEPr-x, in CDC13, Trvl'S as an
int. std.)
'
'
6c CHx Assignment*
193.3
l61.6
'
'
CH C-14
C C-1
142,7 CH
138.5 C
136.7 CH
120.5 CH
111.8
'
C-3
C-4
C-5
C-6
CH C-2
85.5 C C-10
45.1 C C-7
38,7 CH2 C-8
34.8 CH2 C-9
33.7 CH C-11
20.3 CH3 C-15
18.2 CH3 C-12
17.0 CH3 C-13
* The assignment･ for RL-PERO-4 is according
structure 18. '
523
t,o t,he
proposed
Tab1e 3-126 Physicochemical pi'operties of isotrienecat･oanal
epoxide (18)
'- ,,,
/ CHO
o
18
A colorless oil
Vanillin-H2S04 color: brown
UVX,M,89H: 270 nm
FI-MS m/z (%): 233 (M++1, 24), 232 (M+, 100)
EI-MS m/z (%): 232 Ol", 13), 2!4 (4.9), 189 (3.8), 176 (6.2), 175
(14), 162 (5.8), 149 (12), 147 (9.0), 136 (14), 135 (12),
133 (12), 121 (17), 109 (17), 107 (25), 105 (16), 95 (12),
94 (15), 93 (27), 91 (26), 81 (16), 79 (28), 77 (16), 68
(100), 67 (31), 55 (22), 53 (20), 44 (37), 41 (34), 40 (48)
IH- and l3c-NMR data are shown in Tables 3-124 and 3-125,
respectively.
524
.
Tab1e 3-127 Physicochemical properties of RL-PERO-4-TU (18a)
CHO
18a
A colorless prism from n-hexane, mp. 145-1460C
Vanillin-H2S04 color: brown
FI-MS m/z (%): 232 (M+, 100)
EI--MS m/z (%): 232 (M', 13), 203 (4.9), 189 (3.8), 176 (6. 2), 175
(14), 162 (5,8), 149 (12), 147 (9.0), 136 (14), 135 (12),
133 (12), 121 (17), 109 (17), 107 (25), 105 (16), 95 (12),
94 (15), 93 (27), 91 (26), 81 (16), 79 (28), 77 (16) , 68
(100), 67 (31), 55 (22), 53 (20), 44 (37), 41 (34), 40 (48)
IH- and 13c-NMR data are shown in Tables 3-153, 3-154 and 3-155,
respectively.
525
3-7-5
Conelusion
As the result of a survey of carotanoids in Rosa rugosa
leaves, it was found that some carotane aldehydes and their
oxygenated derivatives were also contained. In particular, simple
carotane aldehydes, daucenaldehyde (10), isodaueenaldehyde (12) and
11,12-dehydrodaucenaldehyde (16) are in the position as precursors
of further oxygenated minor carotanoids mentioned above. Those
isomers and the triene aldehyde are speeulated to be formed as a
by-product in the biosynthesis of carota-1,4-dienaldehyde (3)
pathway, since the contents of those compounds are markedly lower
than that of 3. The author eannot discuss the significance o£
those minor carotanoids; however, they provided several informat,ion
about the spectroseopic properties of carotanoids. The proposed
biosynthetie relations amoung isolated compounds are shown in
schemb 3-3s.
526
o
工
。
グ
三
●匿。
工
O
o
o
o
O
o
o
工
O
．多7
o
o
o
工
。
’．，＼ 器
グ
ク’
グ
工
o
工
”9’・’呑1
＼
り’
＼呈／
茶（吉
§／
グ
グ
＼
一
§
甲→
・F→
o
舌
吉8
7、》て詳．
グ
、エ＼
ち
o
工
O
工
。
グ
工
o
ρ，’り。
Nこ
o
。
「
o自
一
o工
’，
’ヘミミ
エ
o
o
’，
ち
グ
1睡工
殴
工
o
工o
oo
工
o
O
o
O
．【→
○
o
二’「’o
’
ρ
o
o
鏑
。
の
・P唖
①
①
o
＿乳＿ノ
。1－1
β囲
め
のI
グ
ノ
。
工
。
佳
ノ。一一
Qり
o
O
コ＝
グ／
／一＿＿一一・
，＼ミ
”o
oり
工
工
o
o
o
Q
o
o
o
傷囑・’詳’”δミ
～軌・・壱→側昭
527
Φ
自
①
‘
o
の
･3-8 Bisabolanoids of Rosa rugosa
3-8-1
Introduction
Rugosa rose (Rosa rugosa) leaves were revealed to contain
various kinds of carotane sesquiterpenoids, which were regiospeeifically oxygenated at the C-14 earbon to yield an ct,B;
unsaturated aldehyde, an a,B-unsaturated carboxyl or an a,Bunsaturated methoxycarbonyl part structure. On the other hand,
carotanoids of Umberifellae and Compositae are mostly not
oxygenated at C-14, wit,h only a exception [43]. Umberifellae and
'
Coinpositae are also known as terpenoid-rich plants. Especially,
'
both families contain some species as an abundant source of
bisabolanoid [55,56] whose biosynt,hetic pathway is partly
overlapped with that of carotanoid. The presence of bisaboianoids
in Compositae and Umberifellae may be recognizable as an evidence
for relationships of bisabolanoids to carotanoids in their
biosynthesis. Indeed, for both sesquiterpenoids cis-transfarnesyl-pyrophosphate pathway is proposed [57].
In Rosa rugosa leaves, some sesquiterpenes of non-carotane
class are also found. During a survey of constituents in rugosa
rose (Rosa rugosa) leaves, those non-carotane sesquiterpenes were
obt･ained and elucidated their structures to be bisabolanoids. In
tt
this
section, the author describes those Rosa rugosa bisabolanoids
'
and discusses on the relationship between carotanoids and
bisabolanoids in this plant.
528
3-8-2 Bisaborosaol A
1) Isolation
During the author's initial experiments, the fresh and nondamaged leaves of Rosa rugosa were soaked in aq. 3 mM CuCl2
solution to survey some stress compounds, which were expected to be
diffusable into the water layer [136]. Although no stress compound
was detected in the preliminary experiment, a noticeable quenching
spot showing a reddish purple color with vanillin-H2S04 reagent was
found as one o£ the major extractives. The focused compound
denoted RL-116 was detectable both in the CuC12 solution and in the
fresh water (control experiment). RL-116 was suceessively aimed to
isolate and elucidate t･he structure. To obtain the eompound,
2.0 kg of fresh leaves of Rosa rugosa were collected (Sample I) and
soaked in 3 mly[ CuC12 solution (25 liters) for 19 hr and
successively in the same volume of tap water for another 24 hr.
The former and the latter layers water were respectively extracted
with EtOAc (1000 ml water/600 ml x 2), and the organie layer was
dried over Na2S04 and concentrated. As the results, total 3.6 g of
extractives (2.1 g and 1.5 g, respectively) were obtained (Scheme
3-36).
Since these extracts showed almost indistinguishable pattern
in TLC, those were combined and coated on 40 ml o£ silica gel. The
gel was put onto the Wako gel column settled i,n n-hexane (gel
volume, 150 ml), and the constituents were eluted with Et20/hexane
mixtures (Table 3-i28 and Fig. 3-326).
Another 4.0 kg of leaves (sample I) was also soaked in 30
liters of 3 mM CuC12 solution and successively in 30 liters of tap
water. The total 60 liters of water layers were extracted wtth
EtOAc as described above to obtain 9.5 g of constituents which were
also fractionated over silica gel c,olumn (volume 500 ml) with
Et20/n-hexane mixtures as eluting solvent (Table 3-129 and Fig. 3327).
529
Fresh Leaves 2,O k
soaked in 3 mM of u'"uCl2 soln. (25 liters)
standing for 19 hr.
Residue
soaked in tap water (25 liters)
standing for 24 hr.
)WYA!,9-L!.t{t L
Water La er
filtrated
extracted with EtOAc (each 30 liters)
dried over Na2S04
concentrated
lst Extractives 1.9 .
2nd Extractives 1.5
mixed (3.4 g)
siliea gel column chromatography
Fraetions Containin RRL-116
(--- Fractions from another 4 kg leaves
combined (1.4 g)
re-columnchromatograhy
RL-116 ca 200 m
Scheme 3-36 Fractions containing RL-116 obtained by coIumn
chromatography of the Sample I (TLC pattern is shown in Fig. 3309)
Tab1e 3-128
Fraction
Fr-Ia-1
Fr-Ia-2
Fr-Ia-3
Fr-Ia-4
Fr-Ia-5
Fr-Ia-6
Fr-Ia-7
Fr-Ia-8
Fr-Ia-9
Fractions obtained by silica gel column chromatograph.v
of extractives from Sample l
Eluting solvent
10
10
10
20
35
35
50
50
100
%
%
%
%
%
%
%
%
%
Et20/hexane
Et20/hexane
Et20/hexane
Et20/hexane
Et20/hexane
Et20/hexane
Et20/hexane
Et20/hexane
Et20/hexane
･530
Volume
100
200
200
200
ml
ml
ml
ml
150 inl
150
150
150
150
ml
ml
ml
ml
H-EA 3:1'
C> o' o
O
ooo
98gg
quenching under
UV 254 nm
&
sie
o
o
ao
1
2
3
4
5
6
7
8
9
Fig. 3`326 TL Chromatogram of Fractions Obtained by Silica Gel
Column Chromatography of Exudates from Sample I
A fraction containing RL-116 in the second extractives (FrIa-8 and Fr-Ib-8) was combined with the former one (total 1.4 g) to
rechromatograph over silica gel column (volume 150 ml). The
'
constituents were suceessively eluted with EtOAc/n-hexane mixtures
as fo]lows: FFr-1; washings with 25 % EtOAe/hexane (100 ml), FFr-2;
eluted with 25 % EtOAe/hexane (50 inl), FFr-3; eluted with 25 %
EtOAc/hexane (50 ml). The focused substance appearing as a single
spot on TL plate was contained in FFr-2. The pure colorless syrup
(ea 200 mg of a colorless syrup) was finally obtained by PTLC in
hexane-acetone 4:l (Rf O.28). For the spectroscopic analyses, RL116 was further purified by PTLC in hexane-EtOAc 4:1. FFr-3 also
contained marked amount of RL-116 (Fig. 3-328).
531
TabZe'3-129 Second fractions obtained by silica ge1 co1umn
chro]natography of extractives from Sample l (4 kg)
Fraction
Volume
Eluting solvent
30 % Et20/hexane
30 % Et20/hexane
50 % Et20/hexane
50 % Et20/hexane
55 % Et20/hexane
Et20
Et20
EtOAc
EtOAc
Fr-Ib-1
Fr-Ib-2
Fr-Ib-3
Fr-Ib-4
Fr-Ib-5
Fr-Ib-6
Fr-Ib-7
Fr-Ib-8
Fr-Ib-9
500
200
200
200
ml
ml
ml
ml
200 lnl
200 ml
200 ml
200 ml
200 ml
H-EA 2:1
o <g
b
o
/ RRL-116
8
9
a
o
1
Fig. 3-327
2
･3
4,
5
6
7
8
9
TL Chromatogram of Fractions Obtained by Silica Gel
Column Chromatography of Exudates from Sample I (4 kg)
532
H--EA 2;1
O
RL-116 tr'- (]])>
quenching under
UV 254 nm
(1>
e
o
o
ObOO
o
o
8
o
1
Fig. 3-328
2
3
4
5
6
7
TL Chroinatogram of Fractions Obtained by Re-column
Chromatography
533
2) Structure Elucidation by Spectroscopic Methods
RL-116 showed the parent ion M+ at m/z 266 (100 %) in FI-MS
(Fig. 3-329) and the EI-HR-rvlS vevealed its molecular formula
C16H2603 (M+ 266･184, ealcd･ 266.188). The EI-MS fragments at m/z
266 (M+, 1.1 %), 248 (M+-H20, 23 %) and 217 (M+-H20riOCH3, 5･2 %)
indicated the presence of an -OH group (Fig. 3-330). The UV
spectrum showed the absorption maximum at 218 nm due to an ct,Bunsaturated hydroxycarbonyl group. Its infrared spectrum (KBr) was
also indicative of the presence of hydroxyl (3500 cm-1 , broad),
carbonyl (172ocm-1, sharp) and ether (126ocra-1, sharp) moieties
'
(Fig. 3-331).
Z
1eos
-gs.ee
･:>-
tr,
oz
I!i
z e
F--
rTTTTTTrilTMTITTrrMTTTrTTpmTTITrlnTTTTTTTVM-TMTITtTrTTTttTMttttTrPitTITTtltTTtrTTMTftITTTTirlTiii-FTTTIin,ITrrTlrttrr
se
15e
1ee
M+
1eze
va.ee
2eOM!E
2
--4s.ee
iii
eil
i
-z
He 2ee
2se 3ee
Fig. 3-329
FI-Mass Spectrum of RL-116.
534
.ze
S5eM!E
lze
r69
8Z
6e
14Z
266
163
4Z
ss 79
ISI '
2Z
i89 2Z5
119
?
2.6k
1!
l-
1l･
;
I.
T
[
1 .:,1
'Tl
i
l
i t
,
11
4
t
p.
5
t'l1t
}
I
l± ±[
l
f
l
l.
h![I
'rb i:
Ee
irl
i'
l.
Iu,i[
t?
L
t'
,[ ,V
il j, 1,
l l-
L"s l!
I:LIt
ilAi
".,-
l!"
ili"J l,i
'i-IE,.
ii
9
lili illitlll
,JiL･,J
:
:ll
:s･
11 i
Ni;
O'
VI I･
: t+ ::
i
iI1.
il ll･ :]II
l･ltl
i'li
I l･ I'1
l'
'-rm
l' ii
l
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g
11r
i
[
+:"' iT
ii'
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-l't/i.i-i,i
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't
5
ll･･
:-LL/
't'
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1
8
li
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IT il ":,.
T1Fl
l [
i
8P
35Z
gze
i
nll･
I
1'
l
I･
25Z
EI-Mass Spectrum of RL-116
'I''
-1･
i
90
e･
216 233
15Z 2ZZ
Fig. 3-330
10D
178
IZZ
5Z
*2z.e
a4B
99
.ii
,
l-
'1
[
6
5'
n
l-,
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]
l
l
li
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v
:f"
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i
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4.
'i i'
:
i
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to
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l.
o
4e EL' ?tAvENutlBE3Rs.oocM",-
Fig.
l
tt/1
boeo
3--331
bso6
.-
;
'
2000 'iSOO'
t l'
'lr
'rago''
'"'
IR Spectrum of RL--116
535
i
:1-. l,.
l4no'
i200
ioob'
aoo
'6od
-'400
In IH-NMR spectrum (in CDCI3: 500 MHz), two isolated olefinic
protons were detected at 6H 7.005 'and 5.125 (Fig. 3-332 and Table
3-13o). The 13c-NMR spectruin revealed four sp2 carbons [6c 139･8
(C), 132.0 (CH), 129.9 (CH) and 124.3 (C)] attributable to two' -'
C=CH- moieties (Fig. 3-333 and Table 3-131). Two allylinethyl
groups (6H 1.696, br. s, and 1.665, br.d, .T= 1.0 Hz) and an ,.
olefinic prot,on (6H 5.125, IH, multiply divided triplet, J= 7.2 and
l.O Hz) were all characteristic of protons of 3,3-dlmethylallyl
group. In COSY-NMR spectrum, the proton network system
(substructure A) including this 3,3-dimethylallyl group was
revealed (Fig. 3-334 and 335). The olefinic proton showed a cross
peak. with a pair of methylene protons (multiplet, 2H around
6H 2.0), in addition to a cross peak with one of the allylmethyl
signals due to an allyl coupling. Furthermore, the methylene '
'
proton (6H 2.0) indicated another coupling
with a triplet signa} at
6H 1･536 (2H, J= 8.3) attributable to a pair of equivalent
methylene protons. The proton-prot,on coupling sequence was closed
there to give a side chain [(CH3)2C=CHCH2CH2-INI. Substructure A
was also confirmed by the two point-homodecoupling experiment
irradiated at 5.125 ppm and 1.536 ppm. As a result, the nonequivalent methylene protons (6H 2.090 and 2.029 geminally coupled
with J= 14.6 Hz) became feasible (Fig. 3-336).
On the other hand, another substructure involving the
conjugation system was also yaised by the IH-NMR spectrum. A
methyl proton assignable to a methoxycarbonyl group was detected at
6H 3.750 (3H, s). The presence of the methoxycarbonyl group was
also proved by 13 C-NMR detection of two carbons at 6c 51･5 (CH3)
and 167.9 (C) both attributable to the -COOMe carbon. Since two
sp2 carbons out of four have had been attributed to substrueture A,
the remaining two had to be assigned to the olefinic bond to form a
conjugation with the carbonyl group. The geometry of the
conjugated C,C-double bond was dedueed to be E (cis regarding the COOMe and an olefinic proton at 6H 7.005) configuration since the
536
Lノ
工
俄
“
円一
一、r
．F
（
◎う
Ho
∩
o
・r→
響
N
Σ
ooゆ
）
⑩
H－
．
創
1
口
舶
。
⇒
ρ
o
Φ
q
の
o
■
乞
1
一
N
h
σり
Qり
1
◎り
●
bO
・7→
」
537
Tab1e 3-130
z
H-IVMR chemical shift values
of RL-116
(500 MHz , in CDCI3, TMS as an int . std.)
.O05
2 .337
2 .087
1
.610
1
.907
1
.249
2 .525
2 .168
3 .729
1
.536
1
.160
2 .090
2 .029
5 .135
1
.692
1
.626
7
Assignment
Coupling
6H
IH
IH
IH
IH
IH
IH
IH
IH
3H
2H
3H
IH
IH
IH
3H
3H
br. m
m-dvid, d*
m-dvid. dd
.J=
18.6 Hz
J= 18.6 , ca 15 Hz
C-4-H
C-5-Ha
m
m
dddd J= 12. 7, 12.2 , 12.2, 5.4 Hz
m-dvid. d
m-dvid. d
C-2-H
C-3-Ha
C-3-Hb
J= 17.6 Hz
J= 17.6 Hz
s
C-5-Hb
C-6-Ha
C-6-Hb
C-7'-H3
s
c-9-H2
C-1O-H3
ddd J= 14 6 , 8.3, 7.3 Hz
ddd J= 14,6 , 8.3, 7.3 Hz
C-11-Ha
C-11-Hb
m-dvid. dt J= 7.3, 1.0 Hz
d .1= 1.0 Hz
C-12-H
C-14-H3
br. s
c-15-H3
t .J== 8.3 Hz
* multiply divided doublet
538
顕講f
く．）o o
婁し．
1．
一日
に1－
／．
3．ε
国
諸
H
で
．錦 自
．138
い
σり
・R
囲
oQ
o
L
鵠
・「→
51 “
N
．8
…註｝
L‘
r⊃
ゆ
ひq ・
一N
）頃
⑩
．臼
一◎o
州⑩
1
臼＋⊇
lll
｝
，・
？
累
1
く⊃
．し9
餌¢
q司の
。Φ
鐸コ
5の
臼の
→一）Φ
o日
Φ
Ω璽の
の邸
▼，
．汗
・
Σ←
寓ρ司
i国
Q窺
州
σつ トー司
3
σり
一3
Qり
Qり
1
¶●
◎り
〔．）
しコ
ビ
b幻
・rl
幽
539
TabZe 3-131 i3C-IWlfR chemical shift valties of RL-116 and
comparisons of the shifts with those of a-bisabolol (g4?
(125 MHz, in CDC13, TMS as an int. std.)
'
6c Property
Assignment 94 [137]
167.9 -COO- C-7 23.15
139.8
=CHC-2
120.53
132.0 =C- C-1 (or C-13) 134.00
129.9 =C- C-13 (or C-1) 131.54
51.5
42.5
39.5
26.8
124.3 =CH- C-12 124.58
74.1 -C-O C-8 74.21
-O-CH3 C-7' CH C-4 ･ 42.94
CH2 C-3
CH2
C-9
40.08
(or C-5, C-6, C-11) 26.88
25.7 CH2 C-6 (or C-3, C-5, C-11) 31.00
23.8
23.3
25.2 CH3
C-14
25.65
CH3
C-10･
23.28
CH2 C-5 (or C-3, C-6, C-11) 22.02
22･3 CH2 C-11 (or C-3, C-5, C,-6) 23.25
17.7
CH3 C-15
17.59
* Hydrogenation degree are elucidated by INEPT experiment
-540
u-
i
i
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e
:
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t t-tLt- ttt"-ttt
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e
7.0 6. {i 6.0 5.5 5.0 4.5
Fig.
Il
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11
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ii
l.ttt ttfi-tt-tfi-lt thtttrrrtt
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- 6.0
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6. [{
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ll5 1]o !s olo "P!'1
3-334a HH-COSY Spectrum of RL-116 (500 MHz, in CDC13)
541
" 3,.[]
- i/- - 3. ts
i
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oo li
tmttt-t-rtrm.. r..t.tt. ...-t.;.ttLttt. .tJ.
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l
l
l
l
!
l
I
...,{IIi -ii
l
I
tltt--tt'ftt-t'- t "ttt
i
i
l
l
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ll
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tr-ttt t tt tt ttmtn t- ttl r't
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i"
.--
l/
t
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di
i'
l
i
l
i
., .l. ..
il
I/ll
l
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-t
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ll
ti
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ll
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liitI
l･
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"-"''-
--mh'
1･
l
l
l
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l
il
tttaLt.."t-tvt-
1.,5
.w 2.0
- 2.5
I
- 3.0
il
tt
l
l.t
oee l
o
Il
d
l
i
o
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::
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ll
li
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ii!,
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.
e
o
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tt-t-t.
ll -- 3.[5
i
l
I
..4ttt- -rm tit
"
Continued (Magnified in High Magnetic Field)
542
.- 4.0
!r..-,･H
H
H
Hi
CH3
'
N
COOCH3
H
H
HH
CH3
substructure A
substructure
H
B
CH3
S /OH
/cx
substructure
Fig. 3-335
c
'
Substructures (A, B and C} of RL-116 Elucidated by
Proton Coupling Sequence
M
xN."vtl
2
Fig･ 3--336
'
Proton Signals Changed .by Irradition of 6H 5･125 and
1.536
543
chemical shift value of the olefinic proton on the B-position was
detected at a markedly lower field, due to a deshielding effect of
the ' 6arbonyl group.
The HH-COSY, CH-COSY and spin-spin decoupling experiments also
proved the presence of a proton coupling network for substructure B
(Fig. 3-337 and Table 3-132). The olefinic proton resonat･ed at･ 6H
7.005 showed a vicinal coupling with a pair of methylene protons at
6H 2.337 and 2.087 geminally coupled each other with .1= 18.6 Hz
(cross peak with C-3 methylene carbon at 6c 26.8). In the HH-COSY,
this coupling sequence showed a further extension to a proton
signal resonating at 6H 1.610 (IH, overlapped) that afforded a
cross peak with C-4 methine'carbon at 6c 42.5 in the CH-COSY
speet,rum. When the olefinic proton was irradiated, together with
those vicinal methylene prototis (6H 2.337 and 2.087), another pair
of methylene protons at 6H 2.525 (br. d, .I=17.6 Hz) and 2.168 (m)
both assignable t,o methylene carbon 6c 25.7 (C-6) were changed
their signal patterns. The lattey fact was suggestive o£ the
presenee of an allyl coupling between them. These allylic
methylene protons were remarkably deshielded to show their chemical
shifts in a downfield by the carbonyl group, and further coupled
with a pair of methylene protons at 6H 1.907 (IH, br. d, J= 12.7
Hz) and 1.249 (IH, dddd, J= 12.7, 12.2, 12.2 and 5.4 Hz) revealing
' at 6c 23.3 (C-5). Coupling
a cross peak wit,h a methylene carbon
' CH=C-CH2-CH2 side was further extended to the
sequence of the
methine proton at 6H 1.610 that was coupled with the methylene
protons at 6H 2.337 and 2.087. Consequeritly, t,he substructure B
was revealed to be a 1,4-disubstituted 1-eyclohexene ring (See Fig.
3-335). The only sp3 methine carbon at 6c 42.5 which showed a
eross peak with the proton at 6H 1.610 in CH-COSY was reasonably
assigned to the C-4 methine carbon.
544
c
1
,
,
l
f,
4
li
t"
i''
c.)
G
f'
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ti:
t)
t'
tb
;l
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1
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[
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td
E
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l
:
"ttt 'tr 'i ''Tt'.'t7..t. L
ii .L･
i
ri･
c)
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I
1i
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i
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c)
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i.L.. tL...t
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to
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l
-
.
IJ.
'J
.=,
r'1'r''
1
/
i
/
!
l
Ii
-- tt
1-
l
tr'=-'-
iti
IJ i'T
..-."-t-"--r-
:
1
t"
1
l
I
i
''' '
:
l
t/
ll
Ii
il
L+l'
'---l
t)
'J
t)
Cl-
t)'
tr:
-'
l
i'
;
11
/1
l
1/
i'
t/
l
1'
lll
j'
t/
/]
ld
;/t
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Ji
u-.--.L-Lh
1
l
i
l･
'
i
i
1
I
d
I
1
l
I
l
]
l
E
'
-]"-..
: '" r'-i't "'- - 1 -･.- -- .-..rt.
'
n"L t't"t..tr
.t
/'t"-tI
l･
l i.. -d- i･,i
i
lt
I
]
J
'
l
/
't""'""'
-I
l
i
r
ll
il
tl
I.
I
1
i
s
1
a;
rl
i:
i
11
:
1
G
.-..;t-"ut--ttt
lt-tJtt'
-tt-tutt
;
t
i
t
I
tt
l
I
I
t
!
5
l
11''ti' 'L+tL-I
'+'TtL
i
Tm"T'"tr
1
l
l
II
al
--i
tttt'iutt r
l
i
-t.
i
l'
l
i
!
I
I
ttttt..tJ.tt.
l
tt
t/
II
ji
/1
ll
j
ll
11
-T' mtT17
r li
1
i'
l
it
1
el
!
1
/:
1
//
/:
I
l･
Li
I
+l
11
i
!,
11
l
II
,T
II
L,
l
'I
l
,l
l
i`t
T al
--･
1
il
,
1
1
i
1
l
l
1
1
Hl,
'1/ '
;
l
!
,
!
li･
l
l
j
l
i
l
l
i
:
L
1
'
l
1I
'
/1
ll
ll
/l
t/
/e
1I
l['
a
...
g
J
-l
l
i
I
+
7
i
l
Il
1/
tt'
,
l
, t'Ttr'7t''t
'fi
1'
'
I
l
d
1i
l/I
tt
1
d-
l
t-
t
(}
t--
:)
t.J.
t-
l
r'T''T'T'I'i'r'rr''T'TTT'-'1"T'-rrT-T''rl-T-Lr'T'rT"rT---r'rlr'r-TrlLrrT-rm rr'J--1-r'rr-r'i-T-T-r-T-ITrTrrrlrl--rLr-'LT'1
-T--
x.1 en m en
W･ Jl C) ut
Fig. 3-337a
m
b
'
.
c)
LJI
Ln o
-S-v NN
U {N LN
.o ut "
e
'
ut
'
o
o
-.
¢o
CH-COSY Spectrum of RL-116 (500 and 270 MHz, in CDC13)
545
II
r
F'
tr
tr
I-
tH't i'-t t-ttt t-
i
l
i･
'lt-"T'-"t--'-'vFtt-t-t"t------."-t-t.-L.-tt.
t
il
n-
'---'-t-Hu-''"m-'----"----m-'mu-t4--'
ji
'T-"""'nd''
L
#
F
;--
t!
i･ e
[
+
l
,l H.--･e"- --- -' ""'-'-+--r,
,ii-iIl
t-
:
L
L
L
L
'
I
l--
..- ttt- tt "-t-t -t-t-t-Vt.tt-HNrt'-vttH----t= t--
l
l
L
L
'
I
l -..-...
T
1'"'-'r
{-.H
L
L
F
l
l･
1
1
'I
i
]
s
1
l
--A+f'-'-"-+-'---"'-r'mh
F
'
-. -tH.t t-+t-t..tt-.t
h4
Jt--tttt"--.
+
-
t--
L
'
s,a
K [-;
Ll1)
1
f
i
--
-q
L. J
T"'
r
r
...-- .-. ......."-- --･ -T
2,a
L
'
l
IS
L
'
t
1..e
F
t
ull
,5
t-'
i
l
i:･ e
i
i4.r-------...--v.-.".-. .-.
"""-'-"--.;
i
`
iS i
:
,
F
.... - -...u'.-..-.---..-.--m---.--
l
l
l
I
i
:
i
t
l
i
il
lI
'l
`
o,o
F
li..-
.H..-"n..t+.-.+--...---t-.--t--L..H-t-.tt-. 't-'r't-t.'.. -. .'. -,. t.- .- t.- t-. i' '--.' '" " "''
F
a,e
;･
:
r"" r"-"""-p
""-.--r.'"-. "-."ijr."-e "v"-r,-"v. e"..-r'". "'.
'-"-"t
,
.
Ce
Fig. 3-337b
:'
=e
JJ
;c･
:-,LZ
:
2･;
:v-t
Continued (Magnified in High Magnetic Field)
546
.
v
･i l./r
-+
'J .-
Tab1e 3-132
Relation between carbon and proton signa1s by CH-COSY
of RL-116
(500 and 125 MHz , in CDC13, TMS as an int . std.)
Carbon
(6c)
139
.
124
.
51
.
42
.
39
.
26
.
25
.
25
.
23
.
Proton
Assignment
(6H)
7
7.005
C-2
3
5.135
C-12,
5
3.729
C-7'
5
1.610
C-4
5
2.090, 2. 029
C-9
8
2.337, 2. 087
C-3
7
i.692
C-14
3
2.525, 2. 168
C-6
8
1.160
23 4
1.907, 1. 249
C-10
C-5
23
.
1
1.536
C-11
17
.
7
1.626
C-15
.
547
The methyl group detected at 6H 1.160 as an isolated signal ln
the IH-NMR was fqvovably allocated to a hydroxylated tertlary
carbon (C-8) resonating at 6c 74.1 (C) (See Fig. 3-335), and this
methyl group on the tertiarily hydroxylated carbon was deduced to
be the third substrueture (C). Thus, all the carbons of total i6
were characterized.
To give a planar structure for the compound, substructures A
and B should be eombined through the tertiary hydroxylated carbon
in the substructure C. Then, these three substructures forin a
. T/
bisabolane skeleton known as one o£ typical sesquiterpenoid
skeleton. Both ct-carbons to the hydroxylated one gave a reasonable
chemical shift values (6c 39.5, in substructure A, and 6c 42.5, in
substructure B), whieh were compatible well with the proposed
planar structure 19 (Fig. 3-338).
10
OH
3
8
9
2
ll
4
l
5
1
6
13
15
7'
7
ooMe
14
Fig. 3-338 Pianar Structure of RL-116
548
Aecording to the literature search, it was found that this
compound had been reported by Harwood et al. as an epimeric byproduet (9sa + 95b) during an alkyl cation reaction of (-)-methyl
perillate (96) (Scheme 3-37) [1381, and its EI-MS, IR and IH-NMR
(100 MHz) spectra of 95a + 95b were approximat,ely agreeable with
those of i9. However, isolation of this compounds as a natural
product was the first example. Therefore, 19 was given a trivial
naine "bisaborosaol A". From Rosacea, bisabolane class
sesquiterpene is the first report, so far as the author's
investigation. Even though some C-7 oxygenated compounds are known
among naturally occurring bisabolanoids, those are quite rare [139145) (Fig. 3-339)･
cooMe
+ HCOOH.
'h, ,,,
ti
OH
cheme 3-37
H
s"N
H
96
S'
HO
cooMe
95
Allyl radical addition to yield a bisabolane
sesquiterpene t138]
549
Tab1e 3-133
PhysicocheJnical properties of bisaborosaol A (19?
HO.ttt
H
:
'
l
COOCH3
i9
A colorless syrup
Vanillln-H2S04 color: reddish purple
[ct]D: + 78 O (c O.06 in acetone)
UVX,IX!ilil.OH: 218 nrn (e 174OO)
FI-l-IS m/z (%): 266 (M+, 1OO)
EI-HR-MS: 266.184 (C16H2603, calcd. 266.188)
EI-MS m/z (%): 266 Oi+, 1.1), 248 (M'-H20, 23), 233 (M'-H20-CH3,
4.4), 217 (M'-H20-CH30. 5.2), 216 (M'-H20-CH30H, 6.3), 205
(17), 192 (6.9), 189 (M'-H20-COOCH3, 16), 188 (8.0), 178
(6.2), 164 (15), 163 (48), 151 (25), 140 (57), 139 (11),
137 (22), 110 (11), 109 (100), 108 (12), 107 (12), 105
(17), 93 (18), 91 (12), 83 (10), 82 (29), 81 (19), 80
(13), 79 (29), 77 (13), 71 (17), 69 (87), 67 (26), 59
(10), 57 (10), 55 (27), 53 (13), 45 (28), 43 (97), 41
(72).
IH-NMR and 13c-NMR data are shown in Tables 3-131 and 3-132,
respectively.
550
工
閃
1－3・
σq
ハ
H
oω
ω
1
）
ω
ω
口
⑩
O
工
〇
ミ
一
ε
ヨ
彷
轟
包
膳
○
工
。
ω
o
ヨ
0工
①
z
6
Gり
く
α
δ
σq
Ω．
α
o◎
び
。
ト
。
卜』・
α
ω
○
×
σq
Φ
8
Φ
望．
00
0工
口⑳
ωΩ．
⑩α
一
o
工
o
○
≦
←」・
一 o
）
○
H・
工
1川工
（
⑩
司
Hト
o
ぐQ．
δ
o
ε＿
口
巴
o
o
0
工
O
ド
。
戯
）
グ＼o
工
ハ
H
oド
ー
ミ
ロ
＜
、
⊆
川に＝
（D
口＿
轟」く◎
口
9QO
心
凸
0
0
0工
O⊃
工
）
工
⊂
＿ Oo
O
O工
ζ
Φ
口
○
幽
工
O
随
警
oN
⊂
＜
口
蒔
巴
o工
0
⊆
o
⊃
（D
ロー
夢・ ⑩
巳¢
1
）
刈
551
o阻；1工
≦⊇．
）
P
ユ＝
ミ ど
ド
げ
Q－ （D
Oo
Oζ
Φ ・
3) Stereochemistry
To elucidate the absolute stereostructure of bisaborosaol A
(19), chemical modification of 19 at the C-7 methoxy carbonyl group
into allylic methyl group was conducted. The expected product
would be directly compared wtth authentic 4S,8S-(-)-ct-bisabolol
(g4) in 13c-NMR and [q]D. According to the conversion scheme
(Scheme 3-38), the methoxycarbonyl group of 19 was reduced with
'
LiAIH4
as the first step.
To 24.4 mg of 19 dissolved in 1.5 ]nl of CHC13 was added an
excess amount of LiAIH4 (ca 50 mg), and the reaction mixture was
stir.red overnight at room temp. After that, 1 ml of EtOAc was
added under cooling in an ice bath to stand for further 3 hr, and
the mixture was then diluted with 15 ml'of E'tOAc. The resulting
'
solution
was washed with a saturated NaCl solution containing
O.2 N HCI. The organic layer was dried over Na2S04, eoncentrated,
and then chromatographed (PTLC) in hexane-EtOAc 1:1. Together with
unchanged material (6.4 mg, 29 %, Rf O.71), a non-quenching product
(RL-116-LAH, 19a) at Rf O.32 showing pinkish blue with vanillin' 13.lmg in a yield
H2S04 reagent was isolated as a colorless syrup
of 60 % (Fig. 3-340).
The product･ was confirmed its structure by FI- and EI-MS, and
1
H- and 13 C-NMR analyses (Fig. 3-341, 342, 343, 344 and Table 3134). In addition to the molecular weight 238, the C-7 carbinol
group was detected a't 6H 4.026 and 3.994 (each d, J= 11.1 Hz, C!!20H) in IH'NMR, and at･ 6c 67.4 (-gH20H) in 13C-NMR, respectively.
552
HO
LiAIH4
HO
cooMe
CH20H
TsCl
4･
LiAIH4 i7C
.
CH20-Ts
CH3
Reduction of bisaborosaol A with LiAIH4 and further
scheme for converslon into or-bisabolol
Scheme 3-38
H-EA 1:1
(>
RL-116-LAH
O
o
::
i.:･t'il.::
quenching under UV 254 nm
vani11in-H 2S04 test: +
sl･li"',"'
i'
;.i--
Reaction
MIX.
Fig. 3-340
Std.19
TL Chromatogram of Reduction Product Obtained by
Treatment of Bisaborosaol A
553
M+
1eee
Z
B.ee
t
l
-)
H
go
hZ
i
-z
He
idLb1ee
1
sz
Lz.ee
Tt
1SZ
l
2sz t
2ee
M!E
2
s.ee
1eee
'M
-H
o
z
Hz
H
hi
e
ii
ldi 1-st, 1--
3ee
3EJj2
Fig. 3-341
1ZZ
Le.zo
t l-It ld tt -t
F'D-Mass Spectrum
5ZeM/E
45e
4-ze
of RL-116-LAH
4
69
8Z
IZ9
6Z
7g 93
4Z
55
2Z
135
2
119
5Z
Fig. 3-342
IZZ
147 159
15Z
177 189 2Z2
22Z
2ZZ
EI-Mass Spectrum of RL-116-LAH
554
25D
1．
（5
8
器
8
，」
3
臼
80
．κ，
ロごつ
w
N
一・
錆
⊂⊃
丙
9
・H
“
．『誠
ぐ⊃ N
「Σ1
o
o
日
匪。
当
．3
（
の
d
∩
）ｮ
⊂〕▽・
・（
w己．寓
q
ω血
姦；
一『一一一一
黶D
8
ρ
噌
ゐ
ご
8山
ゆζビ
鯛
。
9
ぜ
賃
8籍
rく＋⊃
o
Φ
ω
⊂⊃α自
．oΣ
06z
l
＄
5
9
め
臼
Lδ
爲
H
8專
I
σ；cつ
Qり
面
β霞
9
の
555
曇
一（⊃
Cつ （N
餌巨＝
韻⇔
・9
oo
一詔
へ
臼
ご国
げ）o
で
・雪器
Σ
oo
．いく）
い
の
段IH
o
∩
o
臼
甲→
N
ゆ
NH
⊂つ
〔D
い
・9
（〕
｝
に＝1
＜
oニ：1
コ
實
一日マ
日
．．
メCτ
「．
覇哨
o
o邸
一噌臼
ぢ
3践
一の
．8望
Io
一：乙
oぐつ
一「㌔
一？→
器寸
一r野
oり
［
．3α∋
“
0 甲イ
…G）匡8
N
556
Tab1e 3-134
13 C-NMR cheinicaZ shift va1ue of RL-116-LAH (1 9a)
(125 MHz , in CDC13, COM and DEPT, TMS as an int . std.)
6c
Assignment
H
137.5
C
C-1
132.1
c
C-13
124.6
CH
C-12
i22.9
CH
C-2'
74.5
c
C-8
67.4
CH2
C-7
43.6
CH
C-4
39.6
CH2
C-9
26.8
CH2
C-3
26.0
CH3
C-10
25.9
CH2
C-6
24.2
CH3
C-14
23.8
cH2
C-5
22.5
CH2
C-11
17.9
CH3
C-15
557
Tab1e 3-135
Physicochemical properties of RL-116-LAH (19a)
HO,
ttt
H
CH20H
19a
A .colorless syrup
Rf; O.32 (H-EA 1:1)
Vanillin-H2S04 color: pinkish blue
UvAiMtgg?H: featureless above 210 nm
FI-MS m/z (%): 238 (1OO)
EI-MS m/z (%): 220 (M'-H20, 4.1), 202 (M'-2H20, 3.2), l89
(3.4), 187 (2.2), 177 (2.6), 159 (4.0), 147 (3.0), 135
(24), 133 (10), 119 (8.1), 109 (66), 107 (14), 105
(16), 95 (13), 94 (24), 93 (40), 91 (18), 82 (20), 81
(12), 79 (39), 71 (18), 69 (84), 67 (21), 55 (21), 44
(79), 43 (100), 41 (55).
IH-NMR 6TCMD sC13(500 MHz): ca 5.71 (IH, br. C-2-H), 5,l37 (IH,
br. t, .T=7.1 Hz, C-11-H), 4.026 (IH, d, J=11.1 Hz, C-7Ha), 3.994 (IH, d, J=11.1 Hz, C-7-Hb), ca 2.15 (2H, d
x 2, C-3-Ha and C-6-Ha), ca 2.07 (IH, overlapped, C-3-
Hb), 2.062 (2H, m, C-10-H2), 1.937 (IH, m, C-6-Hb),
1.881 (IH, m, C-3-Hb), 1.693 (3H, br d, J=O.7 Hz, C-14H3), 1.629 (3H, br s, C-13-H3), 1.612 (IH, m, C-4-H),
1.527 (2H, t, J=8.1 Hz, C-9-H2), 1.283 (IH, dddd,
J=12.2, 12.2, 12.1 & 5.5 Hz, C-5-Hb), 1.154 (3H, s, C15-H3)･
13c-NMR data are shown in Table 3-172･
558
On the other hand, 4S,8S-(-)-7-hydroxy-a-bisabolol (101) having
the .same planar structure with that of RL-116-LAH has been isolated
as a naturally occurring sesquiterpene from Vanillosmopsis arborea
(Compositae) by Matos et al. [144]. As the absolute stereostructure
of 101 has also been determined by a chemicat1 correlation method
(Scheme 3-39), 19a was compared its physicochemical properties with
those of 101. In their paper, however, [odD and 13C-NMR chemical
shi£t values were recorded only for the 7-acetyl derivative (101a)･
The diol 19a (8.2 mg) was therefore acetylated in acetic anhydride/
pyridine [1 ml (1:1, v/v), at 80 OC for 1.5 hr]. From the reaction
mixt･ure diluted with excess amount of toluene was removed all the
solvent. The main product RL-.116-LAH-AC (16b) was obtained by PTLC
(hexane-EtOAc 3:1, Rf O.43) as a colorless syrup (7.0 mg, yield 73
%) (Fig. 3-345). The struc'ture was confirmed by EI-ry{S and IH-NMR
(Fig. '3-346 and 347).
HOttbttt
H
Ac20/pyridine
HOttlt
ett
H
N
'
CH20H
t
CH20Ac
. H2
HO
lltttttt
HO.
H2
ttltl
lt
H
Scheme 3-38 Conversion scheme to elucidate the absolute
configuration at C-8 of compound 80
559
H-EA 3:1
t-:i-trt:t;
-}
':-::.n.
RL-116-LAH-AC
vanillin-H2S04
test: posltive
t:.:1::s7 :,
1;';f
V' ;:'r-
-. "'::t;"t
t-tt: :-;-d
Reaction
Std. 19a
MIX.
TL Chromatogram of RL-116--LAH-AC
Fig. 3-345
1Z2
4
8Z
69
6Z
IZ9
4Z
22Z 26e
93
"rtlt:-nt-JJL-e--lhr-----------
79
2Z
*IZ.Z
55
119133
6Z
5Z
1ze
Fig. 3-346
159
177
2za
2ze
15e
25e
3ze
EI-Mass Spectrum of RL-116-LAH-AC
560
35e
L
l∴
1．
8
3
”r這
レ
ヒ
じ1
A
ぷ
98
め。
・r→
“
き国
’ Σ1
oo
ト
需
コ
ゆ
）
曾
〔）＿
¢Σo
・ll』〈
L」1
奮
く
9
l
o⑩
lH
⊂⊃ 甲→
し。
呂
口
畑
。
8
⊂）Σ＝
一へ巳．
r＿の［二・
卜、
ρ
o
①
1
電
＿：
、詳
9
一
9
5
8
面
卜
臼
臼
二
d
8の
φ1と
z
畳
H
8
σ；卜
寸
oり
1
◎う
8 ・
一⊂，
爲
一面
二∫這
561
6碧
函
Tab1e 3-136 Physicochemical properties of 116-LAH-AC (19b)
HO,
ttt
H
CH20Ac
19b
A colorless syrup
Vanillin-H2S04 color: pinkish blue
[alD; + 33 O (c= O.1 in EtOH)
UVXiM.ng9H: £eatureless above 21o nm
EI-MS m/z (%); 262 (M', O.5), 220 (M'-CH2CO, O.6), 202 (M'-
CH3COOH, 5.3), 187 (3.4), 177 (5.6), 159 (7.0), 133 (13),
119 (10), 109 (45), 105 (12), 94 (22), 93 (36), 91 (22), 79
(26), 69 (66), 55 (16), 43 (100), 41 (46).
IH-NMR 6glil8i3(soo MHz): s.76s (IH, br, c-2-H), s.13i (IH, br t,
.J= 6.9 Hz, C-12-H), 4.456 (2H, s, C-7-H2), 2.158 (IH, br
d, J= 18.0 Hz, C-6-Ha), ca 2.15 (IH, d, J= 19.8 Hz, C-3Ha), ca 2.08 (IH, overlapped, C-6-Hb), 2.073 (3H, s, C-7'COCH3), ca 2.06 (2H, overlapped, C-11-H2), 1.948 (IH, br.
t-like m, C-3-Hb), 1.868 (IH, br d, J= 12.4 Hz, C-5-Ha),
1.691 (3H, br s, C-14-H3), 1.627 (3H, br s, C-15-H3), ca
.1.61 (IH, overlapped, C-4-H), 1.519 (2H, t, J=8.3 Hz, C-9H2), 1.292 (IH, dddd, .T= 12.5, 12.4, 12.4 & 5.7 Hz, C-5Hb), 1.148 (3H, s, C-10-H3).
13c-NMR data are shown in Table 3-135･
562
compound lgb was, in the 13c-NMR, quite similar to (4s, 8s)(-)-7-hydroxybisabolol 7-monoacetate (iOia) prepared from 101 by
Matos et al (Fig. 3-348 and Table 3-137) [143]. In 13c-NMR, the
difference of a chemical shift value between two corresponding
carbons in 19b and 101a was approximately within ± 1.0 ppm, except
the C-2 carbon. This result suggested that 19b has at least the
same planar strueture with that of 101a. However the optieal
properties of 19b was opposite to that of 80a. Contrary to 101a
showing laevorotatory (fa]D - 48 O, c 1.0 in EtOH), 19b was
dexorotatory with [a]D + 33 O (c O.1, in EtOH).
0n the other hand, the 4S,8-epi-7-keto-7-methoxy-a-bisabolol
mixture (95a + 95b) synthesized by Harwood et al. indicated
laevorotately ([ct]D - 68.0 o). Moreover, they prepared two
stereolsomers, 4S,8S- and 4-S,8R-7-keto-a-bisabolols (aldehyde
group at C-7) (105a and 105b) which were successfully isolated by
a chromatographic method. The latter isomers both showed
laevorotatory with close values (105a: - 68.2 O, 105b: - 71.3 O>
[137]. This fact was suggestive that optical rotations of those
or-bisabolQl derivatives are mostly affected by the absolute
configuration at C-4. Furthermore, it was expected that the C-8
chiral carbon provides an only small contribution to the optical
rotatory values (Fig. 3-349). To prove this speculation, direct
comparison with an authentic compound was requested. As it was
impossible to purchase 7-hydroxy-ct-bisabolol (94), the initial
conversion reaction (Scheme 3-54) was applied to 19a. However,
this plan was failed since the tosylation of 19a was unexpect,edly
unsuccessful. Therefore, another method was applied to the
determination of absolute eonfiguration.
563
ρり
国＝l
Q O
卜
N
o
頃．
9
9
需
℃
薯
oo
累
“
ぷ
呂 呂
o
R ・H
N
呂 頃
Σ
8 ゆN
H
8
一歪
江
9
H
自
一
韮
）
o
＜
1
＜
日
1
⑩
H
→
1
目
口
一
8
H
舶
o
ρ
富
o
Φ
の
呂
H
Σ
R
o
一
l
の
州
器
一
OQ
哩
σり
呂
l
円
oり
周
N
564
℃n
・r－i
鳳
Table 3-137 15C-AififR chemical shift va1ties of RL-116-LAH-AC and
comparisons of its chemical shifts with those of 7-acetoxy
bisabolol (101a?, (-)-a-bisaboloJ (94? and bisaborosaol A (19?
C-No
RL-116-LAH-AC (19b)
101a
94
[143]
[137]
19
1
132.7
(C)
132.75
134'
.OO
132.0
2
126.2
(CH)
124.33
120.53
139.8
3
26.9
(cH2)
26.68
26.88
26.8
4
43.1
(CH)
42.59
42.9tl
42.5
5
23.5
(CH2)
22.61
23.3
6
25.9
(CH2)
7
68.4
(CH2)
26.50
68.27
22.02
25.65
23.15
167.9
8
74.2
(c)
73.91
74.21
74.1
9
39.4
(CH2)
39.89
39.5
10
24.0
(CH3)
23.00
11
22.3
(CH2)
12
124.4
(CH)
21.89
124.33
40.08
23.28
23.25
124.58
124.3
13
131.9
(c)
131.50
129.9
14
25.7
(CH3)
25.53
131.54
31.00
15
17.7
(CH3)
17.50
17.59
17.7
5i.5
7'
171.0
7'
21.0
(C)
(CH3)
170.91
20.91
565
25.2
23.8
22.3
25.7
HO
HO,.,
H
H
bttll
CHO
CHO
[a]D; - 71.30
'
[or]D; - 68.20
105a
105b
Fig. 3-549 Two Epirneric lsemers of a
Synthesized by Harwood et al.: These
alkyl cation reaction of an isoprene
HCOOH. These products were isolated
chromatographic method.
Bisabolane Aldehyde
derivatives were obtained by
+ (-)-S--perillaldehyde in
f`rom each other by
SSSL;Ncr
ts ,""gege"e'ajssSf
tn t
1 Vb
s" ･
"q".,
;.
.cS2}ki{/-<3)/:
,
.cxittAl A pmD T;AsrE t
.
"
XNoGIMiige'l.illll?, .'
1;sSls> )--t.-.-
---"
NIERBZZ70re .Z7V&Ec78
ieuoo s4 recuscE
566
Doth PREue
4) Determination of Absolute Configuration at C-4
Since two diastereomers 105a and 105b (both S at C-4) were
reported their [ct]D values, bisaborosaol A (19) ean be determine
its stereostructure at C-4 after the compound is converted to the
corresponding aldehyde derivative. The desired derivative was
successiv'ely prepared from 19a by oxidation with active manganese
dioxide as shown in Scheme 3-40. The produet RL-116-LAH-MO
detectable with UV 254 nm and DNPH reagent on TLC was obt,ained as a
colorless syrup (Fig. 3-350).
,RL-116-LAH
19a
4.5 m
in CHC13.)--
added ca 50 mg of active Mn02
stirred overnight at room temp.
diluted with 20 ml o£ EtOAc
washed with 15 ml of distilled water
dried and concentrated
Rea etlon Mixture
PTLC (Rf O.17 in C-M 50:1)
RL-116-LAH-MO O.8 m
ield 18 %
Scheme 3-40
Preparation of RL-116-LAH-MO
Structure of RL-116-LAH-MO (19c) was confirmed by
EI-Ms, IH- and 13 C-NMR (Fig. 3-35i, 352 and 353), and the optical
rotation [a]D + 77 O (c O.1 in MeOH) clearly indicated the
stereocheinistry of C-4 in 19c to be R.
567
C-M
ou"
o
'`
guenching under
UV 254 nm
xt
vanillin-H2S04 test: +
DNPH test: +
o
RL-U6-LAH-MO
e!:'i･::･",'i･:,
:-:.tr-:;
:f::,':i':`
-----liiae:ny-..-..-=;:,:
Reaction Std.19a
mx.
Fig. 3-350 TL Chrematogram of RL-116-LAH-MO
IZZz
1
69
z
9
43
6Zz
a18
2Z3
4Zz
93
2Zz
55
*5 .e
e2
135
12114816e175lgz
5Z
Fig. 3･-351
1ZZ
15e
ael 2sz
E!-Mass Spectrum of RL-116-LAH･-MO
568
3ZZ
弓
CJ
8
8
9i壼＿1【L
面
oo
o
「刈
N
．
C
’
｝ 5
8
Pずコ
A¢
Ho
∩
o
・r→
n
8
r，
N
属
Σ
oo
ur）．
）
〔．．
j．一．．
．（つ1；．
O
5t・．
l
＜
o、o＿：
o噌，N
目
1
8
o
己
へ
い
甲1
⑩
H
州
1
日
感
触
o
oの ・N
@（o
決黶F
o
8
ド
ρ
o
Φ
r o
@r，
@ ■
@“】
1卜 ．c⊃ 悶 卿 α
8
の
く6
@ v
マ
註： α一
「
ｼ》
．8
0；
ρr
【L、
H
Nゆ
Qり
ｩ．
l
om．o》
oり
8
6
甲 →
ω
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569
■
bf〕
・H
函
L。
｝
、Lo
に
ぐ・」
一需
一9
蔀
A．
σう
，r・→
o
o
∩
£
・F｛
雫
一9
藁
8
“
N
ゆ
NH
）
○
Σ
。
φ
1
．8
一濠
a．
〈
日
1
．ヨ
⑩
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自
臼
o
N
講
HH
l
日
蜘
o
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謹
写
q．
ρ
o
⊂）
一．
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Φ
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の
名
o
－J
N
I
臼
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σり
州
o
N
．器
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ゆ
一（5
σり
1
8
σり
●
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570
bO
．8
N
・F1
口
Table 3-138 Physicochemica2 properties of RL-116-LAH-MO (19c?
HO,ttt
H
CHO
19c
A colorless syrup
'
Vanillin-H2S04
color: pinkish blue
'
[edD; + 77 e (c O.1 in MeOH)
EI-MS Jn/z (%); 218 (M", 3.4), 203 (Tvl'-CHsO, 2.0), 190 (M'-CO,
' b.O), 175 (5.3), 162 (4.5), 148 (5.0), 135 (7.8), 133
(7.6), 110 (28), 109 (100), 93 (32), 82 (24), 79 (21), 69
(93), 67 (23), 55 (21), 43 (80), 41 (66).
IH-N>m 6Eilii813(soo MHz): g.442 (IH, s, c-14-H), 6.s4s (IH, br. m,
C--2-H), 5.143 (IH, br t, J= 7.0 Hz, C-12-H), 'ca 2.53 (IH,
overlapped, C-6-Ha), 2.520 (IH, m-divid d, J= 20.0 Hz, C-3Ha), 2.237 (IH, t-like m, C-6-Hb), 2.075 (2H, m, C-11-H2),
ca 2.04 (IH, m, C-3-Hb), 1.938 (IH, d-like m, C-5-Ha),
1.698 (3H, s, C-14-H3), ca 1.68 (IH, overlapped, C-4-H),
1.633 (3H, s, C-15-H3), 1.564 (2H, overlapped with H20, C9-H2), 1.218 (IH, dddd, J= 12.4, 12.4, 12.1 & 5.4 Hz, C-5Hb), 1.182 (3H, s, C-10-H3)･
13c-NMR 6TC"?sCI3(12s MHz): lg3.g (c-7,), 151.3 (C-2), 141.3 (C-1),
134.5 (C-13), 124.2 (C-12), 74.1 (C-8), 43.1 (C-4), 39.6
(C-9) 27.･5 (C-3), 25.7 (C-6), 23.8 (C-10), 22.7(C-14 22.3
(C-5), 22.2 (C-11), 17.7 (C-15).
571
5) Determination of Absolute Configuration at C-8
As Matos et al. has reported [144], C-7-monoacetate (101a and
19b) can be converted into a mixture of cis- and trans- 1-methyl4(1'-hydroxy-1'-methyl-5'-methylhexa)-cyclohexanes by hydrogenation
catalyzed with platinum black or palladium black. As (-)-qbisabolol (94; 4S, 8S) [144] was also convertible to the same
products, the planar structure o£ bisaborosaol A (19) can be ･
confirmed in speetroscopic comparisons of those reduetion products.
If the yield rate of cis and trans was approximately constant,
furthermore, not only the planar but also stereostructure at C-8
can be deduced on 19b (namely C-8 of 19) by the direct comparison
of tPe optical data with those of 94-derived reduction product
(Scheme 3-4i).
Hydrogenation of 19b was accordingly performed to yield a
major 'and a minor products (Scheine 3-42).
HO
H: HO.,,
, Ot,, H
-t
CH20Ac
platinum black
or palladium black
/H2
HO.
HO..b
ttttt
(?)
Scheme 3'41 Catalytic hydrogenation of RL-116-LAH-AC <19b) and
(-)-or-bisabolol (82) to tetrahydrobisabolol
572
RL-116-LAH-AC 19.5 m
in 2 ml of MeOH
added 6.3 mg of platinum black
bubbled H2 gas
stirred for 5 hr . at room temp.
added further 6 mg of platinum black
stirred for 5 hr
Reaction Mixture
.
£iltrated
concentrated
PTLC (H-EA 6:1)
Ma'or Product RL-116-LAC-PLH1
Minor Produet RL-116-LAC-PLH2
Scheme 3-42
Rf O.59 8.9m
Rf O.26 3.1 m
ield 57 %
ield 16 %
Hydrogenation of RL-116-LAH-AC with H2/platinum black
The'minor product RL-116-LAC-PLH2 obtained by PTLC (Fig. 3354) was, by FI-MS and IH-NMR analyses, identified to be a mixture
of cis- and trans-1-acethylmethyl-4-(l'-hydroxy-i'-methyl-5'methylhexa)-cyclohexanes (19d + 19e) (Fig. 3-355, 356 and Table
3-139).
HLEA 6:1
D vaniilindH2S04
test: posltlve
RL- 1 1 6 -- LAC-PLHI -pt 0
RL-116-LAC--PLH2
Ko
Q
Reaction Std. 19b
mx.
Fig. 3-354
TL Chroinatograrn of RL-116-LAH-AC Reduction
573
Produc 't s
2
azze
2e.ee
M+
>-
tr,
ll?.-
hi
;:
;;
z
se
1ze
Fig. 3-355
1se
'
2ee
-,
2sg 3ee
FI-Mass Spectrum of RL-116-LAC-PLH2
574
-t,
.? .?o'
35eM/E.
ヨ
c）
ε＿．
の
9
8
8
面
（
ぐり
H
o
8 Q
o
9
，0
窪
・7→
い
N
Σ
’ o
o
ゆ
》
8
窪
「
N
5ご畠
lo
＜
自
雰罫日
愛
日
1
¢
8州
ω一
1
日
8触
0
蕊
8
H
巳
臼
べ
（5
ρ
o
Φ
8の
面国
二
ま
二
1
：z；
8
8固
『d
c；くD
ゆ
σり
σり
強
＆
－’
1
U bD
8・
・r→
函
575
Table 3-139
Physicochemical properties of RL-U6-LAC-PLH2 (1 9d +
19e)
HO t ttt
HO
tt
19d
t t'
+
CH20Ac
CH20Ac
19e
'
A colorless syrup
Rf: O.26 in H-EA 6:1
Vanill'in-H2S04 color: pinkish blue
FI-MS m/z (%); 285 (M'+1, 11), 284 (M', 15) , 267 (25)
155 (15), 129 (100).
D
(soo ryfHz ) : 4. og3 (d, J=
iH- NMR 6 CT r,igi3
7.8 Hz) , 3.885 (d
Hz), 2.062 (s), 2.054 (s), 1. 160 (d, J= 7.3 Hz)
1.091 (s).
576
'
'
'
199 (71),
J= 7.8
1.100 (s),
On the other hand, RL-116-LAC-PLHI was confirmed as mixture
of the desired products (19f + 19g) by FI-MS, iH- and i5c-NMR
tttt
analyses (Fig. 3-357, 358, 359 and Table 3-140). In the IH-NMR
'
spectrum, these geometric isomers 19f and 19g showed thg C-7-H3
protons as quite different siganls in their chemical shift values
each other. It seemed possible to calculate the relative ratio of
cis to trans form.
:";
1e?e
-(ie.??
>F-t
oz.
M+
hi
be
z
-t
el
5Z
Fig. 3-357
1oe
1se
2ee 2se
FI-Mass Spectrum of RL-116-LAH-PLH2
577
2.ee
M/E
コ
o
噌6
8
8
．8表
eJ州
o
∩
o
竃’H
9
臼
N
P∫〕 い
Σ
o
，目9
）
N
一ゴ
9
日
．o＿1
．o ｱ：コ＝
ば；鼠・ぐ
日
め
コ
個
個£
．是
8篤
し。
輻
。
呂
8臼
Q
Φ
rく＋⊃
臼
qQ
8
8Σ
・z
1
くY）
州
8
oo
8ゆ
Qり
1
。；
8
丙’
0り
●
◎β
・r→
．8函
一6
578
ト
…
『
i巴
9
呂
需
鴇
Aの
冒
呂
Ho
8
・H
o
o
『
9
“
R
『
鴇
ゆ
N
一
お
）
『
冒
N
呂
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幽
8
1
一護
丁こし
⊂⊃」一
住
誘
「
0〈
口
1
ヨ
ー
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9
舘
一
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㊤
州
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l
二
上
。
一
目
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3
尋
←
ρ
o
Φ
鵠
『
1：
一
の
呂
Σ
1
臼
一
に
器
一
σり
H
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9
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579
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Tab1e 3-140
Physicochemical properties of RL-U6-LAC-PLHI (1 9f +
1 9g?
HO
tt
ttt
19f + 19g
A colorless syrup
Vanillin-H2S04 eolor: pinkish blue
EI-MS m/z (%); 226 (M', 7.0), 209 (4.6), 208 Ol+-H20, 3.6), 141
(27), 130 (14), 129 (100), 97 (11)
IH-NMR' 6gMDCsl3(soo MHz): 1.og7 (d, J= 6.3 Hz),
O.950 (d, J= 6.1
Hz), O.887, O.885, O.880, O.873, O .866
.
.
i3c-NMR 6gR8i3(i2s ryfHz): 74.6, 74.s, 47.6 , 46 .9, 40.2, 40.1, 39
35.5, 35.4, 32.8, 32.1, 32.1, 28.0 ,27 .4, 26.9, 26.7, 24
24.1, 22.7, 22.7, 22.6, 21.4, 21.1 , 20 .7, 17.4.
580
7
.
1
'
'
As carried out for 19b, (-)-or-bisabolol (94) was also
hydrogenated. In this ease, palladium black (30 ing) was used as a
catalyst instead of platinum black. After 5 hr stirring, the 82.5
mg of starting material completely disappeared and a major product
was detectable on TLC, whose response to vanillin-H2S04 test was
slightly but clearly distinguishable from g4. By the PTLC
separation, 59.6 mg of the product was obtained (BS-PDH, yield 71
%)･ In the speetroscopic comparisons of FI-Ms, IH- and 13c-NMR,
BS-PLH were completely identical to RL-116-LAC-PLHI (19f + 19g)･
The result also supported the proposed planar structure of
bisaborosaol A (19) (Fig. 3-360, 361, 362 and 363). Since relative
ratio of cis and trans C-7 methyl proton signal in BS-PLH also
showed a good aceordance to that of RL-116-LAC-PLHI, both optical
rotation was measured. As mentioned above, the optical rotation of
these derivatives were expected to be quite small; t･herefore, ORD
spectra of BS-PDH and RL-116-LAC-PLHI were taken. As shown in Fig.
3-364, both showed dexorotatory at 386 nm, which indicated that the
C-4 stereoehemistry of 19f and 19g was R being equal to that of 94.
Consequently, absolute configuration of 19 was established as C-4S,
C-8R.
581
H-EA 6:1
O vanillin-H2S04 test: +
(2))
c>
o
Reaction Std.
MIX.
Fig. 3-360
94
TL Chromatogram of a-Bisabolol Reduction Product
'
i(;
1eee
3s.e?
>fr
I"'t
oZ:
M+
di
Hz:
-i
e
lt,'U"'`S
sg
1i
ltll li .
I- tll-i- l-
1ee
Fig. 3-361
F l-
-ll
1sa
.? .ee
2ez
FI-Mass Spectrurn of BS-PDH
582
2S2
M/E
ll
『
o
自
」
8
面
罰
「
8＿
P6ぐつ
Ho
∩
o
呂混
い
Σ
G）o
o謹。
φ江【Ω
）
Q
幽
書
ぜ
8の
く6自q
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。
§
β§
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「＼
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西そ
，
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8⑩
1
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6 σう
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583
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l
0
6
Qり
●
じ、
加
・H
函
oL6
冒、
0
6
ω
o語
ω
（⊃
6
「＼
585
Tab1e 3-141 Physicochemical properties of'BS-PDfl " 9f + 19g]
HO,
HO,
11/lz
ttly
/ttll
19f + 19g
A .colorless syrup
Vanillin-H2S04 color: pinkish blue
UVAlrIneaOxH: featureless above 210 nm
FI-MS m/z (%); 226 (lvl', 7.8), 209 (5.6), 208 ()I+-H20, 9.4), 141
(35), 130 (13), 129 (100), 97 (13).
IH-N}vlR6CTDMCsl3(soo MHz): 1.096 (d, .J= 6.3 Hz), O.950 (d, J= 7.2
Hz), O.885, O.879, O.872, O.866.
13c-NMR 6TC MD 813(12s MHz): 74.6 (c), 74.5 (C), 47 .6 (CH) , 46 9 (CH),
40.2 (CH2), 40.1 (CH2), 39.7 (CH2), 39 .7 (CH2), 35. 5 (CH2),
35.4, (CH2), 32.8 (CH), 32.1 (CH2), 32 .1 (CH2), 28. O (CH),
27.4 (CH2), 26.9 (CH), 26.7 (CH2), 24. 1 (CH3), 24.1 (CH3),
22.7 (CH3), 22.7 (CH3), 22.6 (CH3), 21 .4 (CH2), 21. 1 (CH2),
21.1 (CH2), 20.7 (CH2), 17.5 (CH3).
586
（
口
／
）
o
o
⑩
oゆ
①
o
頃
。7→
の
Φ
ρT
鵠目
山の
1，Ω・
・・
oo
o
oゆ
のの鴫
Φω
℃漆邸
℃
煽9
邸
いΦ
P｛〉｝¢
1 臼・H
国。日
トコギ）臼
ρ食
○
ゆ
．・
ｮ
q→σ5（P
l＋♪ρ
ooΦ
《臼で
日0
1×の
⑩①邸
Hて5武
州
oo
守
1でOD
卜4q）1
国≧o
o
くH，二1＋）
oの而
o
LΩ田のり
ζつ＝①H
臼〉一
ρ・F→
（
り
＼
o
l
o←）目
Φ而α＝
日
Ql＞
の・ア→q→
の
角o
o餌て5⊆1
ooΦ
σつO
。Φ
●r→
Qq（d
u
）
α司
l
Qり
qq
4÷∂
マ＋∂㊦
り
1’
NbooO
函
、H
lQ
鵠の
一（口
・H〆⊃く♪
1・詐
。 ．
o
o
N
＜o
io
日
oH
H
｝臼
餌
ど＋
臼
◎つ
・富rl
IOb鵡
訴コ噛
oゆ
・＋のq
①
トユ）
山
層9』
o
⑩
σつ偏葺●
＼
o
霞
q
①・H
（
く）OQ
o
H
o
i
587
Thus, the absolute configuration of bisaborosaol A (19) was
established. This stereostrueture, espeeially R at C-4 was,
comparatively rare among naturally occurring bisabolanoids.
Bisabolanoids whose stereochemistry has already been established
are listed in Fig. 3-365 with their plant sources [146-150].
HO/1/t
H
H
H
cooMe
o
juvabione (gs)
(-)-a-bisabolol
[146]
HO1/lz
[147]
HO
H11/
(+)-hernandulcin (106)
[149, 150]
Fig. 3-365
(94)
HO
1 -bisabolone (1O7)
[148]
Some of Stereochemically Established Bisabolane
Derivatives
588
-3-8-3 Bisaborosaic Acid A
1) Isolation o£ an Acid Corresponding to Bisaborosaol A
During a survey of acidic 6onstituents in Rosa rugosa, a
'
substance showing a reddish purple
color, being similar to the
'
response of bisaborosaol A (19), was detected by vanillin-H2S04
test. To isolate the focused substance, acidic constituents were
prepared from the diffusates of damaged leaves (11 kg) through
extraction of the crude EtOAc-solubles with 5 % NaHC03 solution
followed by re-extraetion of the aqueous layer with EtOAc after
being acidified. The acidic extractives were coated on 80 ml of
silica gel which was put on a silica gel column (gel volume, 1500
ml), and successively fractionated as follows: Fr-A-1; washings
with 40 % EtOAc/n-hexane (1500 ml), Fr-A-2, 3 and 4; eluates with
' 60 % EtOAc/n-hexane
(each 500 ml), Fr-A-5; eluates with 100 %
EtOAc/n-hexane (500 ml). Fr-A-2 and 3 mainly contained the focused
substance, and Fr-A-4 and 5 also involved it in small amounts (Fig.
3-366). From a part of Fr-A-3 (ca 1/5), the focused acid was
isolated by PTLC developed in hexane-EtOAc-HCOOH 25:25:1 (Rl' O.44)
to give ca 50 mg of a colorless syrup. This acid was also detected
in an acidie fraction from the MeOH extract obtained of unwounded
Rosa rugosa leaves (Sample V).
589
2) Structure Elucidation
The isolate RL-123A showing M+ 252 in FD-MS (Fig. 3-367)
afforded quite similar spectra to those of bisaborosaol A (16) in
EI-Ms, IH- and 13C-NMR (Fig,. 3-368, 36g, 37o and Tables 3-142,
143). The results suggested that the isolate was an acid
corresponding to 19. Aceordingly, hydrocleavage of 19 was carried
out as shown in Scheme 3-43, and the reaction product RL-116-HYD
was successfully obtained.
H--EA 3:1
A
A
Aa8
Bisaborosaic Acid A
a
(IC5
dAA)
(b
O
2
Fig. 3-366
7
TL Chromatogram of Coulmn Fractions of Acidic
Coristituents from Sample IV
590
M+
2
leg. e
, 7 Cl VI M
iV'.l
--- .t
->-
}<o
H
z
{"
Hz
-'
e
Le.ee
se
lse '
1ee
29.e
2se
seeM/E
z
1eze
2s.ee
･"
t-
oZ
F--l
ui
lt
z
--
e
see
4ee
st..-ie
gz
1
de.ee
see
･55eM/E
FD-Mass Spectrum of RL-123A
Fig. 3-367
zz
,
4 rJ' ro
9
69
43
'
'
6Z
4Z
2Z
82
55
59
5Z
Fig･ 3--368
123
93
IZZ
149
164177191234
lsz2zz･2sz3ez
EI-Mass Spectrum of RL-123A
591
日
’1．
P
聖＝）
9
」
3
蘇
13
rj
留
3
8
（
○り
μ，
耐
。
∩
o
ρ
・H
8
“
N
’－
9
Σ
荒1
叱．
8
o
o
j》． し○
叱で“ 》
旺
げ｝1．・
く
ρり
N祠
1
乏適
日
餌
しP
伯
◎
づ
零
90
「、
Φ
の
r「
？
餌
8 Σ
1∫；
卜
！ξ
婿 そ
∫
言．
’‘
目
亨．
ﾆ∫；
3
①
⑩
σり
q；
1
σり
■
ぬ。
2
．c；
1
592
・「→
山
Tab1e 3-142
1H-AilSfR
chemical shift vralues of RL-123A
(500 MHz , in CDC13, TMS as an int . std.)
6H
Coupling
Assigmnent
7
.145
5
.133
IH s-like br. m
IH t-like m J= 7 .O Hz
2
.520
IH br.d J= 17.5
Hz
IH m-dvid. d* J= 18.8 Hz
2 .165
IH br. t-like m
2 .12 (approx.) IH m
2
.377
2
.063
1
.916
1
.692
1
.626
2H m
IH m-dvid.d J=
3H br. s
3H br. s
11.3 Hz
(approx.) IH m
1 .542
IH t .J= 8.2 Hz
1
.62
1
.251
1
.167
C-2-H
C-12-H
C-6-Ha
C-3-Ha
C-6-Hb
C-3-Hb
C-11-H2
C-5-Ha
C-14-H3
C-15-H3
C-4-H
,C-9-H2
IH dddd J= 12.4, 12.3' 12.3 and 5.4 Hz C-11-Hb
3H s
CtlO-H3
* multiply divided doublet
593
董
cつ
ぐ“
＝寓
。（．）
董
r，
出
o
〔2
臼
身1
一r：1
．R
（σり
o
目
o
o
2
・r弓
信
’N
撃撃
oN
州
．8
）
i4
＜
oう
．＝i三
eq
H
I
日
一臼
．畠
餉
o
ρo
．f？
ぶ
・・
①
の
@
α竃
．〔己 墨
・・
@ o
州
σり
一「二
〇
卜1
．需
い． oつ
σり
．二；1
℃の
・H
口
．8
L
f」
594
Tab1.e
3-143
i 3c-iwsfR
chemical shift values of RL-123A
(125 MHz , in CDC13, TMS as an int . std.)
6c
Property
Assignment
172 4
-coo-
C-7
142
.
3
=CH-
C-2
132
1
.
=C-
C-13
129
.
5
=c-
C-1
124
.
2
=CH-
C-12
2
-c-o
C-8
3
CH
C-.4
39 4
CH2
C-9
27
.
o
cH2
C-6
25
.
7
CH3
C-14
24
.
8
CH2
C-3
23
･
8
CH3
C-10
23
･
2
CH2
C-5
22
･
2
CH2
C-11
17
.
7
CH3
C-15
.
74
42
:
.
595
Bisaborosaol A 19.8 m
Dissolved in 2 ml of EtOH
added 2 ml of 1 N NaOH solution
standing overnight at room temperature
diluted with 20 ml of a satd. NaCl
acidified to pH 3. O with 2 N HCI
extracted with 15 ml of EtOAc
EtOAc La er
dried over Na2S04
rernoved the.solvent dn vacuo
PTLC (C-M-F 100:5:2)
14.5 m of the Main Product RL-116-}IYD l9h
Rf O.35
77 % ield
Scheme 3-43
Alkali hydrocleavage of bisaborosaol A
, The product (RL-116-HYD) was completely indistinguishable from
RL-123A in TLC, EI-MS and IH-NMR (Fig. 3-371, 372 and 373). RL123A was thus characterized to be 20. 0n the other hand, the
methylation product of 20 with CH2N2, RL-123A-ME also revealed to
be ident,ical to 19 in EI-Ms, IH- and 13C-NMR (Fig. 3-374, 375 and
376). The methyl ester (RL-123A-ME) was further agreeable with 19
in optical votation ([a]D + 68 e, c O.1 in acetone, cL 19; + 78 O,
c O.06 in MeOH). This naturally occurring bisabolane acid (20) was
aecordingly proved to be a carboxylic acid derivative stereocheinically corresponding to 19 and was named bisaborosaic acid A.
596
H-EA-F 25:25:1
O quenching under UV 254 nm
O O
Reaction
Std. 20
MIX.
TL Chromatograms of RL-116-HYD Obtained by
Hydrolysis of Bisaborosaol A {19}.
Fig. 3-371
IZZ
ez
1
43
9
69
6Z
4Z
59
2Z
55
B2
95
126
149
191
164 178
5Z
lez
15e
219
2ZZ
234
25Z
3ze
Fig. 3-372 EI-Mass Spectrum of RL-116-HYD: The fragmentation
pattern is well agreeable with that of bisaborosaic acid A {20)
597
1
1
．＝≧
1．
日
！
〔．
j
で．；
嵩
8
8
ρ；
εミ
A
σり
8
3
Pり
Ho
q
Q
一
甲→
ρ
A
8
ぜ
oo
3．
9
l」⇔
e．こ
8ぎ
8
曾
溢‘志
》
∩
ρ」
「．1
聾
⑩
耐
o
3一＿
零
丙
茜
“，
1驚
H
q
「
伯
臼
。
丙
田
臼
κ
ρ
o
①
の
←
8
9
（「．｝
α〕
乙パ
」く
膣
H
臼
2
8
6
監∫；
卜．
b
oり
ト
σり
i
◎り
8
曾
1
598
b分
・F｛
函
zz
1
9
69
8Z
6Z
43
4Z
2S6
2Z
e2 93
119
59
se
Fig. 3-374
*IZ.Z
14Z
55
1ZZ
163
151
ISZ
EI --Mass
lsg2e524e
178217233
2ZZ25Z31Z
Spectrum of RL-123A-ME
599
1
皿一
LR
『
c⊃
鞭
．一
L
一
一
コくつ
一吋
P
鶉
／
Sl
，二
R
き
ぐ
Aの
Ho
o
o
臼
♂り
、
・r－1
“
N
？
Σ
8
2
享
～
三
d
印
「
国
1．語
1ρ
の
N
r』
州
1
日
1．l
u、
セぐう
ー「
舶
。
づ
N
『，心
1
・段 く
8
噌Fl
㍉
．5
殉▼．．r1・
臼
｝＿
≧
｝
ギ
『d
）
－ ，q．
＿ミン
臼
σこ
ぎ
巳。
の
01y
葵
需
oo
’1
3
。
①
r．
の
ン
3
～～曽
「
1・：
卜
α；
．；、｝・
■1
「
1
鵠
例
ゆ
ト
2
｛，
○り
l
oり
rη
●
bO
・r→
8
，；
監「
600
iつ
・『
・8
・爾
一響
・繋
｛oり
・拙
Ho
o
1？
・州
員
・器
・8
一9
－3
a
一1う
頃
c9
州
）
国
1
，ぐ
φう
、臼
N
回
6
日
島1
幅
o
r1
ρ
o
Φ
．ε1
の
．畠
I
忘；
Q
の
，3
d
o卜
Qり
3
ド
ξ
oり
。
もめ
．8
ぐ5
601
・ドi
Table 3-144 Physicochemica1 properties of hisaborosaic acid A (20?
HO,
ttt
H
=
:
COOH
A eolorless syrup
Rf O.35 (C-M-F 100:5:2)
20
Vanillin-H2S04 test: pinkish red
FI-MS m/z (%): 253 (M++1, 30), 252 (M+, 100)
EI-MS m/z (%): 234 (M'-H2, 4.5), 2i9 (1.3), 191 (5.6),
189 (2 .7)
178 (2.0), 177 (2.6), 164 (2.6), 151 (6.4), 150 (5.3)
(11), 126 (7.8), 123 (9.0), 110 (10), 109 (100), 105
93 (7.9), 82 (22), 81 (12), 79 (17), 71 (13), 69 (80)
(19), 55 (14), 45 (20), 43 (77), 41 (51). " '
IH- and 13c-NMR spectra data are shown in Tables 3-142 and
3-143, respeetively, '
602
'
149
(8 .5)
'
'
67
'
Tab1e 3-145 Physicochemical properties of RL-116-HYZ) (19h)
HOtttt
H
COOH
19h
A colorless syrup
Vanillin-H2S04 color: pinkish red
EI-MS m/z (%): 234 Ol'-H20, 5.3), 219 (1.9), 191 (7.7), 189
(2.2), 179 (1.9), 178 (2.1), 164 (2.6), 151 (6.8), 149
(11), 126 (8.5), 123 (8.0), 110 (10), 109 (100), 95 (16),
82 (27),81 (14), 79 (18), 71 (16), 69 (77), 67 (17), 59
(28), 55 (15), 45 (19), 43 (78), 41 (51).
IH-NMR6CTDMCsl3(soo ryfHz): 7.144 (IH, br, s, C-2-H), 2.372 (IH, mdivid. d, J= 18.6 Hz, C-3-Ha, ca 2.12 (IH, m, C-3-Hb),
1.624 (IH, m, C-4-H), 1.917 (IH, m-divid. d, J= 12.9 Hz, C5-Ha), 1.250 (IH, dddd, J= 12.5, l2.4, 12.2 & 5.2 Hz, C-5Hb), 2.521 (IH, br. d, J= 17,7 Hz, C-6-Ha), 2.162 (IH, m,
C-6-Hb), 1.541 (2H, t, J= 8.2 Hz, C-9-H2), 1.166 (3H, s, C10-H3), 2.062 (2H, m, C-11-H2), 5.133 (IH, m-like t, .T= 7.1
Hz, C-12-H), 1,692 (3H, br. s, C-14-H3), 1.626 (3H, br. s,
C-15-H3)･
603
Tab1e '3-146 Physicochemical properties of RL-123A-ME (19]
HOtttl
H
COOCH3
19
A colorless syrup
[or]D: t 68 O (c O.1 in acetone)
Vanillin-H2S04 color: pinkish red
EI-MS m/z (%); 266 (M', O.5), 248 (M+-H20, 7.4), 233 (2.4), 217
(2.5), 216 (2.2), 205 (8.7), 189 (6.3), 163 (19), 151
(9.6), 140 (22), 137 (14), 119 (13), 109 (100), 105 (16),
93 (24), 82 (24), 79 (22), 69 (83), 67 (20), 55 (17), 45
(18), 43 (60), 41 (50).
iH-NMR 6[iili)/8I3(soo MHz): 7.oo6 (iH, ddd, .T= s.s, 2.s & 2.7 Hz, c-2-
-H), 2.337 (IH, m-divid. d, .J= 19.0 Hz, C-3-Ha), ca 2.09
(IH, m, C-3-Hb), 1.612 (IH, m, C-4-H), 1.907 (IH, m-divid.
d, .r= 12.7 Hz, C-5-Ha), 1.241 (IH, dddd, J= 12.4, 12.3,
12.3 & 5.1 Hz, C-5-Hb), 2.526 (iH, br. d, J= l7.7 Hz, C-6Ha), 2,171 (IH, m, C-6-Hb), 1.536 (2H, t, J= 8.2 Hz, C-9H2), 1.161 (3H, s, C-10-H3), 2.062 (2H, m, C-11-H2), 5.136
(IH, m-like t, .J= 7.2 Hz, C-12-H), 1.694 (3H, br. s, C-14H3), 1,628 (3H, br. s, C-15-H3), 3.731 (3H, s, C-7'-H3).
13c-NMR 69MDCsl3(12s MH,): 16s.o (c-7), 13g.g (c-2), 132,3 (C-1),
130.2 (C-13), 124.5 (C-12), 74.3 (C-8), 51.7 (C-7'), 42.7
(C-4), 39.6 (C-9), 26.9 (C-3), 25.9 (C-5), 25.4 (C-10),
24.0 (C-14), 23.5 (C-5), 22.4 (C-11), 17.9 (C-15).
604
3-8-4 )lonoxygenated Drivative of Bisaborosaol A
1) Isolation and Speetroscopie Analyses
During the isolation of bisaborosaol A (19), two compounds
showing a clear yellow color with vanillin-H2S04 reagent on TL
plates were detected under the spot of 19 (in Fr-I-8), and these
were tentative.Iy named RL-117B and RL-ll8B (Rf O.38 and O.34,
respectively, cf. O.47 in 19). By the re-column chroniatography,
RL-117B was eluted in FFr-I-3 and successive FFr-I-4 (25 %
EtOAc/hexane 50 ml). The latter fraction also contalned RL-118B as
the major substance. With the guidance of the characteristic
'
'
response to vanillin-H2S04 reagent, each
compound was isolated by
PTLC (H-EA 3:1) (Fig. 3-377).
Both of them showed M+ 282 in FI-MS (Fig. 3-378 and 379),
although the parent ions were undetectable in EI-MS of the each
compound (Fig. 3-380 and 381). The IH-NMR spectra were both
similar to that of 19; however, two methyl signals were observed
upfield to be assignable to a methyl group allocat,ed not to an
olefinic but to an oxygenated carbon (Fig. 3-382 and 383, and
Tables 3-147 and 148). Furthermore, each double-doublet, methine
proton (6H 3.415 in RL-117B, and 3.553 in RL-118B, respectively)
was newly detected instead of the C-12 olefinic proton of 19. The
results suggested that the olefinic bond on the side chain was
monoxygenated to contend the molecular weight 282 (16 + O). An
exchangeable and broad singlet proton was also detectable in both
of the IH-NMR spectra･
sinee each 13c-NMR spectrum of the compounds was quite
similar, RL-117B and RL-118B were eonsidered to be epimerie to each
other (Fig. 3-384, 385 and Tables 3-149, 150). Although from 19
three monoxygenated forms (D, E and F) were possible to draw (Fig.
3-386), D having an epoxy group was neglected due to the chemical
605
sift values of the oxygenated earbOns. These carbon shifts [e.g.
6c 86.4 (CH), in RL-117B] were too low as a methine carbon in an
epoxide ring. As a matter of fact, if an epoxy group was induced
to the olefinic bond, it will be cyclized by intramolecular
alcoholysis wit,h the C-8-OH, as known in 1-linalool (68) and (-)-abisabolol (94), for example [151,152]. Epoxidation of 19 was
therefore carried out.
H-EA 3:1
0
quenching under UV 254 nm
:L,1i,l7,:.'s--l-.8
8
FFr-I-4
s
Fig. 3-377
TL Chromatogram of FFr-I-4 Containing RL-117B and
RL-118B
606
1eee
Z
]
eeez
i
y
h
oz
hi
Hz
F-l
He
I
-l H
l
bl
se
.ee
t
2eeM!E
1se
1ee
Z
1eee
g.ee
M++1
M+
'M
k
Heo
Z
u
Hz
H
e
l'
i-+ tn
l
2ee
Fig. 3-378
2ZZ
l,
, gkVrTTT-trnrtrnTtrrrfihTtTnTritrhpfTri
'ird
'l bp
t
.ee
t
3ee
2se
35eM/E
FI-Mass Spectrum of RL-117B
4
143
8Z
264
191
6Z
S9
4Z
55
2Z
85
79
67
91
5Z
*25.e
le7
7
1
251
125
71
IZZ
Fig. 3-379
119
!37
181
2Z5
173
15Z
223235
2ZZ
asz
EI-Mass Spectrum of RL-1l7B
607
t
3ZZ
-
35Z
2
jeee
Je.e?
>
Ho
z
z e
F,
ur
le
Lft,ee
,
50
1
1se
1OCI
M!E
Z
1ge?
M++1
>
h
H
oz
hi
Hz
F--t
je.e2
M+
hN,.
"i
e
,.ee
2SO.
20¢
Fig. 3-380
zz
sec
M!E
FI-Mass Spectrum of RL-118B
1
3
191
26'4'
ez
251
6Z
4S
59
S5
4Z
125
71
79
2Z
181
IZ5
93
163
173
2]5
.
5Z
`
IZZ
Fig. 3-381
15e
*25. e
2Z5
25e 3ZZ
2ZZ
'
EI-Mass Spectrum of RL-118B
608
35Z
4ZZ
ll
r，
c」
8
9
9
臼
8
9
面
零
po A
⑩
q
⑩
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曾
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d
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@k）
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9
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面
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臼
ζ：⊃窄一
卜左：
＆
＿：L
9マ
面 日
綱
o
N
。
腔
器
8彗
hこ 臼
ρ
o
Φ
の
L
コ
卜二・．
卜・・1：．
＄
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ひ1
β
配
〔て； Σ＝
lz
じ
鵠
H
ト
li：
．8 N
c； OD
oり
I
oり
L
●
8
掌
609
bの
・目
国
Tab1e 3-147
1
H-AijSIR
'chemical shift values of RL-U 7B
(500 MHz, in CDC13, TMS as an int . std.)
6H
Coupling
Assignment
7
057
IH
br. m
3
481
3H
s
3
405
IH
dd .1= 10.3 and 5.2 Hz
2
655
IH
br. d J= 15.0 Hz
.
192
IH
.
075
IH
1
975
IH
t-like m
m-dvid. d J= 19.3 Hz
br. s (exchangeable)
1
719
IH
m
1
682
IH
m
1
559
IH
d-like m
2
2
.
43 (approx.)
.
403
.
381
1
1
1
1
249
1
221
.
074
o
.
930
o
.
834
1
IH
IH
IH
3H
3H
IH
3H
C-2-H
C-7'-H3
IH t-like m
dd .I= 9.8 and 8.6 Hz
m
dd .T= 9.8 and 9..O Hz
s
s
dddd J= 12.5, 12.4, 12.4 and 5.1 Hz
s
610
C-12-H
C-6-Ha
C-6-Hb
C-3-Ha
C-13-OH
C-11-Ha
C-3-Hb
C-5-Ha
C-4-H
C-9-Ha
C-11-Hb
C-12-H
C-l4-H3
C-15-H3
C-5-Hb
C-1O-H3
．『
o
8
8
，こ
8
8
9
A
Nつ
ぷ
＿＿ニメ，
8
爲
零q
竹 。H
」
」
NA
臼
9
－OqO
ω〔LO
」
」
8
蜜
」
」
需
⊂りr
匡Ω
）
田
3二
蓬
ω 1
口
【L
8
＄
二
」
触
。
目
べ
9＿ノ
3
面
1三；
附
と冒
詞
o
①
の
め
Q．
爲
呂
l
丙
po
1
9
H
6 0つ
o◎
ト
σり
1
5
82
cq
・匿
ど＼
611
0り
9
－6
●
も。
・H
h
Tab1e 3-148 IH-ALMR chemical shift values of RL-118B
(500 MHz, in CDCI3, TMS as an int. std.)
6H Coupling Assignment
7
3 .552 IH dd J= 8.0 and 6.0 Hz C-12-H3
3
2 .588 IH br.d .f= 17.8 Hz C-6-Ha
2
2 .106 IH m-dvid. d* ,J= l9.8 Hz C-2-Ha
1
1
1 .620'
1
1 .488 IH dddd J= 17.7, 8.7, 6.8 and 5.4 Hz C-11-Hb
.138 IH t-likem C-6-Hb
.788 IH s(exchangeable) C-13-OH
.753. IH t-likem C-2-Hb
.553 IH d-likem C-5-Ha
1 .408 IH ddd J= 12.2, 9.7 and 5.4 Hz C-9-Ha
1
1 .237 IH ddd J= 12.2, 8.6 and 7.2 Hz C-9-Hb
1
1
o .945 IH dddd J= 12.4, 12.4, 12.3 and 5.1 Hz C-5-Hb
o
* multiply divided doublet
.40 (approx.)m C-4-H
612
聾
語｝ぎ
｝
9
雲
層9
鴇「
9
（
現
豊
曾
国
∩
零 て5
露
需
Σ
○
9
（．）
＄
㊤
ミ
忠
8 口
o
⑩
o
・H
お
N
8
8
§
oo
㊤
）
蔓
qq
聲
卜
州
目
1
§
日
配
皇
奨も
貫
屋
ρ
o
呈
①
甲
の
屋
oN
唖
ヨ〒
o
舅の
州
四
型
頃
N
罠
寸
◎O
Qり
1
σり
婁
o
罠
oう
田
613
bO
・P→
蟹
函
Table 3-149
(68 rvlHz,
6c
167.3
139.5
130.4
86.4
84.6
70,1
51.1
43.3
35.8
27.9
27.7
26.4
25.7
24.8
24,1
23.4
13C-AUS:tR chemical shdft values of RL-11 7B
in CDC13, TMS as an int. std., COM and DEPT)
Property
-coo=CH=C-
-CH-O
-c-o
-c-o
-o-CH3
CH
CH2
CH3
CH2
CH2
CH2
CH3
CH2
CH3
Assigmnen't
C-7
C-2
C-1
C-12
C-8 or C-13
C-8 or C-13
C-7'
C-4
C-9
C-10
C-11 or C-3
C-11 or C-3
C-6
C-14
C-5
C-15
614
遷
ト
ｼ⊃
・一
萎
Lヨ
臼
購
A、
o臼
「∫Ω→
国
q
ロ
9
一ωΣ
oo
”しつで
こ
一詩“
⑩
㊤
・詔Q
8・H
直
．8．．Σ：
一邑
亀ゆ
ぐ噸
。） r4
’＝：＿
ρQ
⊂）OQ
一「4
州
州
9
「．二司
．．
x）駕
鏑
o
一写
一日
角
⊂】十」
一の 〇
一Φ
oの
一ω
一α＝
じ
窯
O
l
…（．）
σり
州
。
－r∫り
ゆ
OQ
o σ）
リへ
一 1
σり
Oぐつ
rヨ
・州
函
615
bO
3-150
Ta b1e
Z3c-swR .chemica1 shift values of RL-118B
(68 MHz , in CDC13, TMS as int . std.)
6c
Property
167
.
139
3
-coo-
C-7
5
=CH-
C-2
=C-
C-1
7
-CH-O
C-12
5
-c-o
C-8
9
-c-o
-o-CH3
C-13
130 4
.
84
84
.
70
.
5i
･
Assignment
1
C-7'
5
CH
C-4
8
CH2
C-9
27
7
cH2
C-il
27
5
CH3
C-10
2
CH2
C-3
5-
CH2
C-6
1
CH3
C-14
5
CH2
C-5
7
CH3
C-15
43
35
26
.
.
25
25
.
24
.
21
616
HO
cooMe
o
o
HO
cooMe
B
A
o
cooMe
OH
C
Fig. 3-386
Three Possible Monoxigenated Structures for RL-117B and
RL-118B
617
2) Epoxidation of Bisaborosaol A
m-CPBA was used to obtain a monoxygenated product of
bisaborosaol A (19). In 2 ml of ice-cold CHC13, 11.5 mg of 19 and
10.0 mg of m-CPBA were dissolved, and the mixture was stirred for
1 hr, at which point, 25 ml of EtOAc was added to the reaction
mixture. Then the mixt,ure was washed with 5 % aq. Na2C03 solution
(25 ml x 2). From the organic layer, two major products 116-CPBA-1
and 116-CPBA-2 which were respeetively agreeable with RL-117B and
RL-118B on TL plates (Rf values and responses to vanillin-H2S04
reagent) were obtained by PTLC (116-CPBA-1: 5.2 mg in 43 %, and
116-CPBA-2: 4.5 mg in 37 % yield, respectively) (Fig. 3-387).
Successively, each product was confirmed to be identical t,o the
authentic eompound obtained as a naturally oceurring sesquiterpene,
in EI-Ms, IH- and 13c-NMR and the optical property (Fig･ 3-3ss,
389, 390, 391, 392 and 393).
H--EA 3:1
O quenching under
UV 254 nm
RL-116-CPBA-1
･`!･･･
(I!iil:l)
'tii`k vaniliin'H2P04
･k !.
(ill])
7
@
test: pos1t1ve
RL-116-CPBA-2
Std .21
Fig. 3-387
Reaction std,22
MIX.
TL Chromatogram of Reaction Product Obtained by m-CPBA
Oxidation of Bisaborosaol A (19)
618
1ez
4
8Z
6e
125
143
4Z
i91
71
59
85
2Z
55
79
le7
91
67
137
119
IS3
97
5Z
1IZ
2Z5
264
223 aS5
25Z
2ZZ
15Z
IZZ
Fig. 3-388
17e
3ze
EI-Mass Spectrum of RL-116-CPBA-1
4
se
6Z
59
4Z
125
71
55
67
191
85
79
2Z
143
137
91
IZ5
119
163
5Z
Fig. 3-389
IZZ
15Z
178
2Z5
223 235
2ZZ
264
25Z
EI-Mass Spectrum of RL-116-CPBA-2
619
3Ze
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8
8
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8
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9
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o
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1d
V
臣
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β例
州
．ω1
8
＆
秩ｨ
。
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零
二【L
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8
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z
l
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620
目・
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9
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621
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0；
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ぐq
①
d
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1
81
1くl1
σり
零
呂
●
～
d
．t
く．
622
三．i
℃o
B
山
！・
1．．
L
lN
lG
N．
A
臼
幽
国
。
j
℃
rl白
髭，
芝
O
Q
1口
“
⑩
Ll
q
⑫
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・r→
し ドス
』
い
N
頃
F8
ト
1．
8
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IN
州
｝
N
l
＜
〔と
ρq
dl
Q
量
F
．臼
零
E
⑩
州
祠
1
日
鋸
L r、
Lg
綱
o
ρ
．一
〔⊃
o
ﾙ、
Φ
の
．〔乙
に
F
r‘
類
z
Q
の
l
H
．1｝1
｝：；；
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●
．8
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L
623
℃の
。H
函
RL-117B and RL-118B were therefore considered to be eyclizat,ion produet.s derived from the epoxy intermediate. Since m-CPBA
was presumed to attack the olefinic bond in the sidechain nonstereoselect,ively to give the epoxy int,ermediate, these compounds
produced via diastereo-epoxides were regarded as the diast,ereoisomeric at the newly formed C-12 chiral center (Scheme 3-44).
Usually, 1-hydroxy-3-monoene derivatives, for exsample, linalool
(68) or a-bisabolol (94), give tetrahydrofuran derivatives by
epoxtdation with m-CPBA in CHC13, through cyclization between the
epoxy group and t,he Y-hydroxyl group as the result of intramolecular alcoholysis [1511. The tetrahydrofuran type struct,ure
for RL-117B or RL-118B was thus quite reasonable. The tetraydrofuran ring structure for each coinpound was furt,her supported by
the presence of EI-MS fragments at, m/z 223 OI+-59) and m/z 59
(C3H70+) as shown in Fig, 3-394). Consequently, the
tetrahydrofuran st･ructures were proposed for t,hese compounds.
cooMe
m/z 59
(C3H70')
Fig. 3-394
EI-MS Fragmentation of 'the Tetrahydrofuran Derivative
624
Φ
Φ
Σ
o
o
o
o
O
o
＝：
o
ユ＝
o
。
ー
コ＝
ぐ
○
qα
’，
’，
工
Q
l
嬉
ρ
・H
匡
＜
HO
の
O
o
£
ω
・ →
qa
Σ
o
悩
o
o
o
o
・H
一
Φ
Σ
o
o
o
一
ρ
で
・「→
ンぐ
o
＜
田
？
∋←
∈ブ．o
／
。
窪
8
H
留
＝
8
L＿＿＿＿＿＿＿＿」h
8
甲
8
＊
oエ
625
国
ぢ
寸
ザ1
σり
Φ
鐸
Φ
40
の
Tab1e 3-151
Physicochemical properties of bisaborosaol Bl (20
H
;
;
t
o
COOCH3
OH
21
A ¢olorless syrup
'
Vanillin-H2S04 eolor: yellow
[a]D + 63 O (c O,19 in MeOH)
uv x"l,ligOxH: 2is nm (e ii3oo)
FI-MS m/z (%): 283 (M'+1, 81), 282 (M+, 42), 265 (21), 223
(50), 143 (82), 139 (3･O, 133 (22), 112 (l8), 59 (100).
EI-MS m/z (%): 267 (1.2), 264 (M'-H20, 1.5), 251 (O.9), 235 (M'H20-OCH3, 5.4), 224 (3.7), 223 (M'-COOCH3,-4.5), 205 (M+H20-COOCH3, 14), 192 (14), 191 (61), 181 (11), 173 (7.2),
163 (10), 145 (19), 143 (89), 137 (13), 125 (50), 119 (12)
107 (23), 105 (20), 93 (15), 91 (16), 85 (43), 83 (15), 81
(21), 79 (25), 77 (11), 71 (43), 69 (18), 67 (l8), 59 (48)
57 (11), 55 (25), 53 (18), 43 (100), 41 (37).
IH- and 13c-NMR data are shown in Tables 3-147 and 3-149,
respectively,
626
Tab1e 3-152 Physicochemical properties of bisaborosaol B2 (18)
H
;
;
l
o
COOCH3
:
:
-
OH
22
A eolorless syrup
Vanillin-H2S04 color: yellow
[ctID + 68 O (c O.2 in MeOH)
MeOH : 218 nm (e 12200)
UV Mnax
FI-MS m/z (%): 283 (M'+1, 66), 282 (M', 23), 265 (11), 223
(59), 143 (100), 139 (56), 59 (69).
EI-MS m/z (%): 267 (i.8), 264 (M'-H20, 2.1), 251 (1.4), 235 (M'H20-OCH3, 9.4), 233 (3.9), 224 (8.0), 223 (tyI'-COOCH3, 8.9),
205 (M+-H20-COOCH3, 23), 192 (28), 191 (95), 181 (21), 147
(20), 145 (24), 143 (100), 125 (39), 107 (19), 105 (20), 93
(16), 91 (15), 85 (42), 81 (18), 79 (23), 71 (28), 59 (45),
55 (18), 53 (16), 43 (58), 4i (27).
IH- and 13c-NMR data are shown in Tables 3-148 and 3-150,
respectively.
627
Tab1e 3-i53 Physicochemical properties of 116-CPBA-1
(= bisaborosaol Bl, 21)
v
H
'=
r
;.
b
COOCH3
OH
21
A colorless syrup
Vanillin-H2S04 color: yellow
[ct]D: + 69 O (c O.2 in MeOH)
EI-MS m/z (%): 264 (M+-H20, 3.0), 235 ()vCE+-H20-OCH3, 2.8), 233
(2.1), 223 (M'-COOCH3, 2.0), 205 (M'-H20-COOCH3, 8.0),
192 (6.6), 191 (34), 181 (5.2), 178 (9.1), 163 (5.7),
145 (10), 143 (46), 137 (19), 125 (55), 119 (12), 107
(15), 105 (13), 91 (12), 85 (26), 81 (11), 79 (17), 71
(34), 67 (12), 59 (26), 55 (13), 53 (12), 43 (100), 41
(22).
IH-NMR 6S･}TfhE}6 (soo MHz): 7.oss (IH, br. s, c-2-H), 2.078 (IH,
multiply divided d, .7i= 19.1 Hz, C-3-Ha), 1.685 (IH, m, C-3Hb), 1.429 (IH, dddd, J= 12.2, 11.8, 4.9 and 2.5 Hz, C-4H), 1.561 (IH, d-like m, .T= 12.4 Hz, C-5-Ha), O.933 (IH,
dddd, J= 12.4, 12.4, 12.4 and 5.2 Hz, C-5-Hb), 2.655 (IH,
br. d, .T= 17.7 Hz, C-6-Ha), 2.193 (IH, t-like m, C-6-Hb),
1.406 (IH, dd, J= 9.3 and 7.5 Hz, C-9-Ha), 1.252 (IH, dd,
.T= 9.3 and 7.2 Hz, C-9-Hb), O.836 (3H, s, C-10-H3), ca l.73
(IH, m, C-11-Ha), 1.378 (IH, ddd, .T= 8.6, 7.7 and 3.3 Hz,
C-11-Hb), 3.408 (IH, dd, .I= 10.4 and 5.1 Hz, C-12-H), 1;223
(3H, s, C-14-H3), 1.075 (3H, s, C-15-H3), 1.966 (IH, br. s,
C-13-O!!, 3.483 (3H, s, C-7'-H3)･
13c-NMR 6TC 1Ds6(12s MHz): 13o.4 (c-1), 139.6 (C-2), 26.4 (C-3),
ty6
43.3 (C-4), 24.1 (C-5), 25.7 (C--6), 167.4 (C-7), 84.7 (C8), 35.8 (C-9), 27.9 (C-10), 27.7 (C-11), 86.4 (C-12), 70,2
(C-13), 24.8 (C-14), 23.4 (C-15), 51.1 (C-7').
628
Tab1e 3-154
Physicochemical properties of U6-CPBA-2
(= bisaborosaol B2, 22)
1 ,,
Tn
=
.=
=o
COOCH3
OH
22
A colorless syrup
Vanillin-H2S04 color: yeHow
[ct]D: + 65 O (c O.2 in MeOH)
EI-MS m/z (%); 264 (M'-H20, 2.4), 235 (M+-H20-OCH3, 3.4), 233
(2.'2), 223 (M+-COOCH3, 2.6) , 205 (M'-H20-COOCH3, 12), 192
(10), l91 (47), 181 (8.1), 178 (11), 163 (7.9), 145 (15),
143 (48), 137 (25), 125 (47) , 119 (16), 107 (17), 105 (18)
93 (13), 91 (17), 85 (39), 81 (16), 79 (26), 77 (13), 71
(41), 67 (18), 59 (46), 55 (20), 53 (20), 43 (100), 41
(32).
iH-NMR 6El&Ds6 (soo MHz): 7.oio (IH, br. s, C-2-H); 2,108 (IH,
'
multiply divided d, J= 19. O Hz, C-3-Ha), 1.755 (IH, m, C-3Hb), ca 1.41 (IH, m, C-4-H) , 1.557 (IH, d-like m, C-5-Ha),
O.947 (IH, dddd, .1= 12.4, 12.4, 12.3 and 5.2 Hz, C-5-Hb),
2.595 (IH, br. d, J= 17.8 Hz, C-6-Ha), 2.141 (IH, t-like m,
C-6-Hb), ca 1.42 (IH, m, C-9-Ha), 1.238 (IH, ddd, J= 12.2,
8.5 and 6.5 Hz, C-9-Hb), O. 864 (3H, s, C-10-H3), 1.622 (IH,
dddd, J= 12.3, 9.7, 8.1 and 7.6 Hz, C-11-Ha), 1.489 (IH,
dddd, J= 12.3, 8.6, 6.8 and 5.4 Hz, C-11--Hb), 3.553 (IH,
dd, .T= 8.1 and 6.8 Hz, c-12-H), 1.217 (3H, s, C-14-H3),
1.071 (3H, s, C-15-H3), 1. 780 (IH. br. s, C-13-O!l), 3.480
(3H, s, C-7'-H3)･
13c-NMR 6!iiRIDs6 o2s MHz): 130.4 (C-1), 139.5 (C-2), 26 .2 (C-3),
43.5 (C-4), 24.5 (C-5), 25. 5 (C-6), 167.3 (C-7) , 84.7 (C8), 35.8 (C-9), 27.5 (C-10) , 27.7 (C-11), 84.5 (C-12), 70,9
(C-13), 25.1 (C-14), 21.8 (C-15), 51.1 (C-7').
629
3) Stereochemical Analysis and Further Note
While the pianar structures of RL-117B and RL-118B were thus
proposed, the relative and absolute stereostructure on the
tetrahydrofuran ring was elucidated by NOE experiments applied to
these two compounds. When the C-9 methyl proton was each
irradiated, NOE on C-12 methine proton was observed only in RL118B. On the other hand, RL-117B showed an NOE bet･ween C-9-H3 and
C-13-OH (Ftg. 3-395). The relative configuration was, according to
the results, elueidated as trans in RL-117B (21) and cis in RL-118B
(22). Since the absolute configuration of bisaborosaol A (19) has
already been determined to be 4-R, 8-S, as mentioned above, the
absolute configurations of those derivatives were also solved as R
in 21 and S in 22, respectively. As both eompounds were novel
bisabolanoids, they were named bisaborosaol Bl and bisaborosaol B2.
Unlike 21 found as one of major constituents in MeOH extracts
of Rosa rugosa leaves, 22 was detected only as a minor compound on
TLC in the MeOH extract. Furthermore, the amount of 19 diffused
into wat,er was greatly reduced when the leaves were injured or
treated with CuC12. These faets allowed a speculation that 19 is
convertible to 22 by an oxigenase-like enzyme aetivat,ed during the
leaves being damaged physieally or chemically.
Currently, Jaenseh and his colleagues isolated a bisabolanoid
possessing the same planar structure as 19 from Podolepis rugata
[153]. From the source, the monoxygenated tertahydrofuran derivative is also found, as well as Rosa rugosa. The latter compound was
considered to be the trans form because the chemical shift value of
C-12-H was resonated to 6H 3.68, higher than C-7'-OC!!3 (6H 3,83)
[153]. Furthermore, this compound showed laevorotatry ([a]D - 29e,
c 1 in CHC13), indicating that the C-4 chiral center of those
isolates was R (structure 95 and lq8), contrary to 19 and 21.
Thus, the bisabolanoids of Rosa rugosa were confirmed to be
stereochemically different group from those C-7 oxygenated
630
bisabolanoids originated in Compositae plants (Fig. 3-396)
t144,145,153,154].
When (-)-ct-bisabolol (94) was metabolized by a fungus
Aspergillus niger, only the cis type of tetrahydrofuran derivative
(110) can be obtained as the initial metabolite [137]. Although
this report may be contradictory to the fact that only trans form
(21) was detectable in the methanol ext,racts of fresh Rosa rugosa
leaves and 10s in the Podolepis rugata was the trans form, this
fungal metabolism can be a model of the selective epoxidation and
suecessive non-biochemical eyelization,
CH3
CH3
H
.=
=
--
H
.=
.=-
'o"
o
COOCH3
"NN
H
H=
OH
COOCH3
4
OH
RL-117B (21)
'
RL-118B (22)
Fig. 3-395 NOEs Observed in the Two Compounds , and the
Estabilsed Stereostructure
631
1
Hq I
'
H
o
cooMe
"NN
OH
H
95
cooMe
108
PodoZepis rugata
[153]
HO
H
o
CHO
1OO
OH
CHO
109
PZeiotaxis rugosa
[154]
H
HO
o
H-
OH
110
94
.
(--)-or--bisabolol (94) metabolite by AspergiUus nlger
'
Fig. 3-396 Some Tetrahydrofuran Derivatives
of Bisabolanoids and
Their Precursors Found as Naturally Occurring. Bisabolahoids or
Fungal Metabolites
632
4) Dehydration
To obtain a chemical proof for the presence of tetrahydrofuran
ring in bisaborosaol Bl and B2 (21 and 22), dehydration of 21 was
earried out [155]. By the chemical eonversion, the dehydration
produet containing an exomethylene protons was expected (Scheme 345). In 1 ml of pyridine, 12.7 mg of 21 was dissolved, and t,hen to
the mixture '60 1 of POCI3 was added. The mixture was kept in a
freezer for 24 hr and successively left at room t.emperature for 1
hr. After the reaction, to the mixture was poured 5 ml of ice
water, and the resulting solution was extracted with 3 ml of Et20.
The Iess polar and quenching product RL-117B-H20 (2･la) was isolated
' mg of a colorless syrup in
by PTLC in hexane-EtOAc 10:1 to give 3.2
'
a yield of 26 % (reeovered 21, 1.0 mg, 8 %) (Fig. 3-397). This
product affording M+ 264 in EI-MS (Fig. 3-398) showed the
exomethylene part (proton: 6H 5.169 and 4.836, carbon: 6c 109.9,
=cH2)in the IH- and 13c-NMR spectra (Fig. 3-399 and 4oo).
Consequently, structure of RL-117B-H20 was formulated as 21a, which
provided concreteevidence for the tetrahydrofuran strueture 21.
Since bisaborosaol B2 (22) has given some line of evidence as a
diastereomer of 21, the structure of 21 and 22 were finally
established.
H
; -:
t
o
"
e
'
POC13
. --.m")
cooMe
H
: -:
:
o
:
:
1
cooMe
OH
'
Scheme 3-45 Dehydration of bisaborosaol Bl (20): Dhehydration
product is expected to show a pair of exomethylene protons at C-14
m`1 H-NMR spectrum.
633
H-･EA 10:1
O
quenching under
UV 254 nm
RL-117B-H20 <>
<gF-------(IIF------
---
Std. 21
Reaction
MIX.
Fig.
IZZ
3-397 TL Chromatogram of Dehydration Product from RL-117B
4
125
BZ
17e
6Z
163
4Z
233
ISI
2S4
*IZ.Z
IZ7
2Z
2Z5217
67 79
53
5Z
91
119
IZZ
137
15Z
2ZZ
,
Fig. 3-398
EI-Mass Spectrum of RL-117B-H20
634
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636
Table 3-155 Physicochemical properties of RL-117B-H20 (21a?
H
c. ?
.=
o
COOCH3
2la
A colorless syrup
Rf: O.56 (H-EA 10:1)
EI-MS m/z (%): 261 (M+, 1.2), 219 (O.2), 233 Ol+-OCH3, 2.2), 217
(O.2), 207 (O.6), 205 (O.7), 204 (O.7), 178 (3.1), 161
(2.8), 137 (7.7), 126 (10), 125 (100), 107 (28), 91 (11),
81 (11), 79 (23), 77 (13), 67 (15), 55 (10), 53 (13), 43
(90), 41 (22).
IH-NMR 6CT6MDs6: 7.061 (IH, br. s-like m, C-2-H), 2.190 (IH, multiply
divided d, J= 19.8 Hz, C-3-Ha), 1.749 (IH, t-Iike m, C-3Hb), 1.499 (IH, m, C-4-H), 1.614 (IH, m, C`5-Ha), O.976
(IH, dddd, J= 12.3, 12.2, 12.1 and 5.3 Hz, C-5-Hb), 2.660
(IH, br. d, J= 17.0 Hz, C-6-Ha), 2.218 (IH, t-like m, C-6Hb), 1.422 (IH, m, C-9-Ha), 1.294 (IH, ddd, J= 12.0, 7.8
and 2.6 Hz, C-9-Hb), O.964 (3H, s, C-10-H3), ca 1.64 (IH,
m, C-11-Ha), 1.538 (IH, m, C-11-Hb), tl.130 (IH, dd, J= 9.6
and 5.9 Hz, C-12-H), 5.169 (IH, br, s, C-14-}Ia), 4.836 (IH,
br. s, C-14-Hb), 1.668 (3H, s, C-15-H3), 3.471 (3H, s, C7'-H3)･
13c-NMR 6!i]fiDs6: 13o.4 (c, c-1), 139.7 (CH, C-2), 27.9 (CH2, C-3),
-l3.4 (CH, C-4), 24.2 (CH2, C-5), 25.7 (CH2, C-6), 167.4 (C,
C-7), 84.8 (C, C-8), 35.8 (CH2, C-9), 23,7 (CH3, C-10),
31.6 (CH2, C-11), 82.8 (CH, C-12), 146.6 (C, C-13), 109,9
(CH2, C-14), 18,2 (CH3, C-15), 51.5 (CH3, C-7').
637
3-8-5 Exoperoxy Bisabolanoids
1) Isolation
During a search for peroxy constituents in the Rosa rugosa
leaves, a neutral fraction (Fr-VIIH-12, recovered with EtOAc)
obtained as described (pp. 290) was found to eontain some
compounds clearly positive to the peroxide test. Those substances
were more polar than rugosal A (1) and showed properties as a
neutral compound. Isolation of the compounds was accordingly
carried out wit･h the guidance of the positive responce to the
peroxide reagent. As the result of successive PTLCs (H-EA 1:1, C-M
50:4 and multiple development PTLC in B-EA 25:10), were isolated
three peroxides all showing properties as bisabolane exoperoxides.
RL-PERO-5, -6 and -7 [Rf O.25, O.23 and O.17 in B-EA 25:10] (Fig.
3-401), all showed the same molecular weight (M+ 298 in FD-MS),
which suggested that those were isomeric peroxides.
'
2) Structure Elucidation of RL-PERO-5 and -6
RL-PERO-5, obtained as a colorless syrup (2.1 mg) from ca 1/3
of Fr-VI-12 showed m/z 298 (M+, 45 %) and 265 (M+-OOH, 81 %) in FIMS, and the latter fragment was indicative of -OOH partial
structure for the isolate (Fig. 3-402). In EI-MS of the isolate,
neither the parent ion nor the fragment due to -OOH fission was
detectable (Fig. 3-403). On the other hand, its IH-NMR spectrum
showed some signals similar to those of bisaborosaol A (19) [e.g.
6H 7.012 (IH, br. s-like m) and 2.587 (IH, br. d), corresponding to
C-2 and C-6 protons of 19, respectively)]. This fact indicated
that RL-PERO--5 was a relative of 19.
As characteristic proton signals, a pair of exomethylene
protons (6H 4.967 and 4.892), an oxygenated methine proton (6H
4.219, triplet, J= 6.6 Hz) and an exchangeable proton (6H 7.550,
singlet) were recognizable (Fig. 3-404 and Table 3-156).
638
H-EA 3:l
Q
bQ
Qo
oo
o6
o
g
t tttl
r'
(,jhj,ii
o
tftV
x
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Ll
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fi
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i
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t,,, .I
p
t-t.
k
12
34567
tn t
i il :')
Ci
S･/
std 8 9 10 11 12 13
19
o
･::11::
B-EA 25:10
B-EA 5:1x3
jlz;?,
quenching under
UV 254 nm
vanillin-H2S04
test: +
peroxide test: +
th".':I'-;E,:g-nyz
x
RL-PERO-7
a
RL-PERO de
A
------------ -
peroxide test: + RL-PERO
Fig. 3-401 TL Chromatograms of RL-PERO-5
639
N7
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1eee
S,?D
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iI,
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tL
g
e
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29en!E
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,-
?
-f '
Fig. 3-402
5S?M/El
3e2
2se
2?a
IZZ
?.??
l, l ti l
't'
FI-Mass Spectrum of RL-PERO--5
4
8Z
79
6Z
137
91
69
4Z
le5
12S
119
55
2Z
l),
ii
'
Fig.
163
IZZ
3-403
192
2za
ll1,
,1I
,1
l
t)
5Z
liiill
IL
ll
kl
177
1
2ZZ
15Z
23Z
a46 262
25Z
'
EI-Mass Spectrum･ of RL-PERO-5
640
3ZZ
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国
Tab1e 3-ls6 1ll-NMR chemical shift values of RL-l])ERO-5
(500 MHz, in CDC13, TMS as an int. std.)
6H
Coupling
Assignment
7.550
7.012
III s
IH br, s-like
4,967
IH d J= O,9 Hz
4.892
IH dd ,J= 1.0 and O.9 Hz
III t J= 6.6 Hz
4.219
3.473
3Hs
br. d .1= 18.0 Hz
2.587
IH
2.112
1. 965
IH br. t-like m
IH m-dvid. d J= 18.9 Hz
1.711
IH br. t.-like m
1.646
3Hs
1.55 (approx.) 2H m
1.54 (approx.) 1}I m
O.900
1}I ddd J= 13.8, 10.8 and 6.4 Hz
IH ddd .J= 13.8, 11.3 and 5.0 Hz
IH br, t-like m
IH dddd J= 12.6, 12.4, 12.4 and 5.2 Hz
O.752
3H s
1.390
1.278
1.221
642
C-13-OOH
C-2-H
C-14-Ha
C-14-Hb
C-12-H
C-7'-H3
C-6-Ha
C-6-Hb
C-3-Ha
C-3-Hb
C-15-H3
C-11-H2
C-5-Ha
C-9-Ha
C-9-Hb
C-4-H
C-5-Hb
C-1O-H3
Disappearance of signais attributable to the 3,3-dimethylallyl
'
moiety of 19 suggested that hydroperoxidation
at C-12 of 19
foliowed by the coneerted olefjnic bo,nd tra-nslocatfon to C-13/C-14
yieided 23 as a proposed structure for RL-PERO-5. In addition to a
coupling sequence on the 1,4-disubstituted cyclohexene moiety,
HH-COSY spectrum of the compound (Fig. 3-405) clearly indicated a
sequence MFC(OOH)H-CH2-CH2- attributable t･o C-9, 11 and 12 (Fig.
' The peroxylation on the sidechain was further
3-406, 407).
supported by IH-NMR deteetion of an allylic methyl group at 6H
1.646 assignable to the C-15. Furthermore, Fl-MS fragment at m/z
159 (100 %), which results from fission of the sidechain, was also
indicative of the structure (Fig. 3-4e7).
13 C-NMR analyses of the compound eventually proved this
structure (Fig. 3-408 and Table 3-157). Two oxygenated sp3 carbons
at 6c 89.5 (CH) and 73.1 (C) were assignable to C-12 and C-8,
respeetiVely, and the former carbon resonating in a comparatively
lower field should be reasonably assigned to a hydroperoxylated
earbon as discussed in Section 4 of this chapter (See pp. 283). In
addition, carbons of the sidechain part were approximately
agreeable in their chemical shift values wit･h those of
isoaminobisabolenol (111) found in a sponge, Theonella sp. by
Kitagawa el al. (Table 3-158) [156].
The second peroxide, RL-PERO-6 showed almost, the same mass
fragmentation patterns with those of 23 in FI- and EI-MS (Fig. 3409 and 410). However, the proton signals assignable to C-9-H2, C-
11-H2 and C-12-OOH, were slightly but clearly different from those
of 23 (Fig. 3-411, 412 and Table 3-159). As the 13C-NMR spectrum
was almost indistinguishable £rom that of 23 (Fig. 3-413 and Table
3-160), RL-PERO-6 was easily characterized to be an epiiner of 23 at
C-12 (structure 24). The stereostructure of RL-PERO-5 and RL-PERO6 remained unsolved. However, the absolute configuration at C-12
will be determined, since the benzoate chirality methods is
'
applicable
to the compounds of this type after reduction to the
diols f35,157].
643
,
1
l
,
-
'
'
.
D.D
-
.
s
.f
g
fe
r
e.
g
o
e
v"
j
a
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8e
-ct
.
2.0
0
D
o
.
bo
im
e
va
3.0
'
'
o
.
.
4.0
"
D
e
5.0
o
t
6.0
i
-
l
1
?.o
'
i
l
i
L-T-r-r-T-
7.5
7.0
6.5
6.D
Fig. 3-405
T
5.5
'
5.0
4.5
4.0
PPM
3.5
3.0
2.S
2,O
1.5
1.0
.s
o.o
HH-COSY Spectrum of RL-PERO-5 (500 MHz, in C6D6)
644
PpPM
anmnniindg
nnA
fnnnd tn hp A nAvQl
Thege t,wn
iiu T v J-nnmnn"nrl
Vv"-rv L-i-" : L.-i-U
v .. v Tv--- =v --- v- ny We 1'A
were named bisaborosao1 CI and bisaborosaol C2, respect,ively.
H
H
H
l
H
H
H
COOCH3
H
Fig. 3-406
H
Proton Sequence on 1 4-Disubstituted 1-Cyclohexene
Moiety of RL-PERO-5
'
in/z 159
in FI-MS
CH3
HO
H
H
H
H
H
HOO
H3C
H
Fig. 3-407
Proton Sequence on the Sidechain Part of RL-PERO-5
645
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3-157
Tab1･e
Z3C-MY[R ehemicaJ shift va1ues of RL-PERO-5
(125 MHz ･, in CDC13, COM and DEPT, TMS as an int . std.)
6c
Property
167
.
144
.
139
.
Assigmnent
3
-coo-
C-7
3
=C-
C-13
5
=CH-
C-2
130 4
=C-
C-1
113
8
.CH2
C-14
89
5
-CH-O
-c-o
C-12
C-8
1
CH3
C-7'
42
9
CH
C-4
35
3
CH2
C-9
8
CH2
C-3
7
CH2
C-6
2
CH2
C-11
6'
cH3
C-1O
23 4
CH2
C-5
17 6
CH3
C-15
.
73
51
26
r
.
.
25
25
.
23
.
1
.
647
Table 3-158
wi.th
C-No
Co]nparison of RL-PERO-5 cai'bon chemical shift･
those of isoaminobisaboleiiol (111) and bisaborosaol A (1 9)
RL-PERO-5
111 [156]
i9
1
130.4
134.0
132.0
2
139.5
120.0
139.8
3
26.8
26.2
4
42.9
42.4
5
23,4
23.7
6
25.7
30.9
26.8
42.5
23.3
25.2
7
167,3
23.2
167.9
8
73,1
57.3
7i4.1
9
35.3
29.2
10
23.6
22.2
11
25.2
33.7
39.5
23.8
22.3
12
89.5
75.6
124.3
13
144.3
147.3
129.9
14
i13.8
110.9
25.7
15
17.6
18.2
17.7
7'
ira1 tt es
51.1
51.5
H2N ,,
11/z
H
HO
isoaJninobisabolenol (111)
648
r/,
poo
:ri
C.' r.1
r.1
s ]?fi
->-
h
b-:
oz:
thrr".,agLe
di
;･
Eev
-t
.
z
'
.ptL-
--
.
f
-t
1ez
tt?･
･? .2?
,d
itt･Z
2e?･in./E
2
1?ee
M+ +1
M+
L.･
F,
sv ?e
N
Lr)
･'
Z
y.
･-
-?"
lt't
?
.e .??
t lli-:T÷,=rH:`rtlt pt
1 H-" hl`
-}- - t-IL l-t--i----t--llt+-d t--. tT-- et-- t''"e
--Gl4-t-i---
;d.-Lt---
2[JO
2?Z
l ･i,,
3S2l,1/Eil
e･z'Le
Fig. 3-409 FI-Mass Spectrum of RL-PERO-6
IZZ
BZ
6Z
79
S9
4Z
91
137
IZ5
119
55
177
145
2Z
163
51
IZZ
Fig. 3-410
2Z2
2RZ
,
15Z
2ez
'
246 26a
25Z
EI-Mass Spectrum of RL-PERO-6
649
sze
3'
5Z
1零
」
」
r3
τり
‘，
o
o
「．つ
．
一
oo
N
でj
，
r．
j
e；
0N
o一
N
●
一
口
r．
o附
o
N
N
C
「「
ぐo
rイ，
の
一
幽
『
Q⑩
o
・r→
⊂）
oq
（一）
い
「」
二
芝
oい
1＝～：・一
一
1・・
Σ
巳
に
ぜ【L
」，【・
くΩ
l
。
¢
｛
「．
m
r．，
〉
o
q⊃Σ
o
国
q
q国
1誠
一
o貸
o
o
lo
）
しし、
，
一
．【L
l
触
c団Ω一
oqD
。
r，
「⊃
コ
回
「、
ρ
o
Φ
～
の
。
㎝
r，
ザ，
8差
q．
ぽ、
べし
餌
Σ
z
l
oo
寓
円
頃
（｝
r．）
n】
州
H
→
l
Qり
累託
hこ【し
‘」
「． D」
℃o
・「咽
で⊃
ミ
650
鳳
Ta bl e
3-159 IH-A[IYR chemical shift values of RL-PERO-6
(500 MHz, in CDC13, TMS as an int,. std.)
6H Coupling Assignment
7 .7
4
4 .894 IH dd J= 1.5 and 1.5 Hz C-14-Hb
4 .183 IH dd .T= 6.9 and 5.6 Hz C-12-H
3
2
2
1 ,949 IH m-dv .i d. d .T= 19.0 Hz C-3-Ha
1
1
1
3
1
1
1 .1 .225 IH dd J= 12,5 and 5.0 Hz C-9-Hb
1
o .906 IH dddd J= 12.5, 12.5, 12.4 and 5.2 Hz C-5-Hb
o
.O19 IH br. s-like C-2-H
.590 IH br.d .T= 17.7 Hz C-6-Ha
.123 IH br. t-likem C-6-Hb
.717 IH br. t-likem C-3-Hb
.65 (approx.) IH in C-11-Ha
.5･-16
IH
in
'
C-5-Ha
.46 (approx.) IH m C-11-Hb
.222 IH br. t-likem C-4-H
' '
651
t
l
t
J
nv
7., ac
nc
s.' oe'
E tlO
,
a.Do
,
3.ne
?.oe
c.e
:･ J:
:
ae
o
o.o
t"'
e
1
o
'b
･:a
.Ifg)
e
;
'
H---Hnvg)
-pt
a
D9
r-
ps.
e
.di
i..
e
-v'
e
-'i
.
g
ge
dy
pt
b
"
e
1.0
@r
2.0
as
I"
a
3.0
pt
-t
eO
.
'
o
e
.-
t
6
e
.
@
ew
--
4.n
e
5.0
.o
'
6.n
.
--
e,･
o
o
be
"t
e
=
4
7.0
-oo
.
1
7.5
Fig.
?,o
6.5
3-412
t
6.0
t
S.5
5.0
4.5
4.0
PPM
3.5
5.0
2.5
2.0
!s
1.0
.5
HH-'COSY Spectrum of RL-PERO-6 (500 MHz , in C6D6)
652
o.o
PPPM
．一一一
鵠 澱
o Q
一く⊃
試
σq
レ
Q
F
卜臼
9
A
臼
自
国
曾
董
∩
℃
3
，パ
N
Σ
話
o
Q
⑩
縞
直
柄
q①
o
・F1
『
日
9
Σ
W
…き一
‘L
o
■o
N例
｝
⑩
l
o
『
爲
．臼
国
氏
1
．紹
日
㎏
o
，曾
ぷ
ρ
o
①
の
。
一しΩ
ρ
1
φう
州
、器
ぐ、
一P
ﾐへ
の
一
面
1
σり
一8
℃o
f馳」
・r→
山
653
C-ALMR chemica1 shift i'alues of RL-PERO-6 and
Table 3-160 13
comparison of the shlft values with those of RL-PERO-5 (23]
'
(68 MHz, in CDC13, TMS as an int. std.)
6c propert,y Assignment
167.3 -COO- C-7
23
167.3
144.4 =C- C-13
14-1 .3
130.4 =C- C-1
130.4
139.4 =CH- C-2
113.8 =CH2 C-14
89.5 -CH-O C-12
73.1 -C-O C-8
139.5
113,8
89.5
73.1
51.1 CH3 C-7'
51.1
35.-l CH2 C-9
35.3
42.7 CH C-4
42.9
26.8 CH2 C-3
25.7 CH2 C-6
25.1 CH2 C-11
23.6 CH3 C-10
23.5 CH2 C- 5,
17.6 CH3 C-15
26.8
654
25.7 ･
25.2
23.6
23.4
17.6
Tab1e 3-161
Physicochemical properties of bisaborosaol Cl
(RL-PERO-5, 23?
COOCH3
HOO
23
A colorless syrup
Vanillin-H2S04 color: pinkish red
FI-MS m/z (%): 299 (M'+1, 57), 298 (M', 45), 281 (29), 265
(81), 183 (40), 159 (100), 141 (41), 139 (76).
EI-MS m/z (%): 262 (4.2), 246 (3.1), 230 (6.4), 202 (5.7), 192
(12), 178 (20), 177 (21), 176 (13), 145 (19), 139 (20),
137 (45), 125 (28), 119 (23), 107 (32), 105 (34), 93 (27)
91 (43), 79 (63), 77 (40), 70 (35), 69 (39J, 55 (23), `13
(100), 41 (85).
IH- and 13c-N>IR data are shown in Tables 3-156 and 3'157,
respectively.
655
Table 3-162
Physicochemical propert･ies of bisaborosaol C2
(RL-PERO-6, 24)
HO
COOCH3
sss
HOO's
24
A colorless sYrup
Vanillin-H2S04 color: pinkish red
FI-rylS ni/z (%): 299 (M++1, 86), 298 (M', 43), 281 (36), 265
(100), 183 (34), 159 (92), 141 (57), 139 (73).
EI-MS in/z (%): 262 (6.1), 260 (3.6), 246 (3.1), 230 (8.5),
202 (7.i), 192 (8.0), 178 (26), 177 (31), 176 (21), 163
(11), 148 (21), 145 (29), 137 (･l7), 125 (29), 119 (32),
107 (32), 105 (42), 93 (31), 91 (51), 79 (56), 77 (38),
(36), 69 (47), 55 (27), 43 (100), 4! (99).
IH- and 13c-NMR data are shown in Table 3F159 and 3-160,
respectively.
656
70
3) Structure Elucidation Of RL-PERO-7
RL-PERO-7 obtained as a golorless syrup (4.1 mg) showed a
weak parent ion at m/z 298 in FI-MS together with the base fragment
at m/z 183+ (Fig, 3-414). In EI-MS, however, the compound,afforded
neither the parent ion nor the fragment at m/z 183 (Fig. 3-415)･
On the other hand, this compound also indicated some signals
characteristic of a bisabolanoid in the IH-NMR and HH-COSY spectra,
where the 1,4-disubstituted cyclohexene structure was vis-ible (Fig.
3-416, 417, 418 and Table 3-163). Furthermore, some signals
attributable to the sidechain were indicative of,its partial
structure on the sidechain. In the IH-NMR speetrum, two olefinic
protpns were detected at 6H 5.690 (IH, ddd, J= 14.8, 7.5 and 7.2
Hz) and 5.516 (IH, br. d, .T= 14.8 Hz) which showed a clear trans
couplipg with each other. Since the £ormer proton was further
coupled vicinally with a pair of methylene protons with J= 7.5 and
7.7 Hz, a substructure I-CH2-CH=CH- became feasible. In addit,ion,
two singlet methyl groups at 6H 1.261 and 1.250 were reasonably
assigned to an oxygenated isopropyl group. The structure of the
sidechain was therefore elucidated as shown in Fig. 3-419.
Cleavage of the sidechain was responsible for the fragment at m/z
183.
By the 13c-NMR speetroseopy, this substrueture was proved
(Fig. 3-420 and Table 3-164). The carbon cheinical shifts for
side chain carbons in RL-PERO-7 showed a good correspondence
those of a bisabolane alkaroid, aminobisabolenol (112) which
also been isolated from the sponge together with isoaminobisabolenol (111) by Kitagawa et al. (Table 3-165) [156].
Furthermore, the clear deshielding effect on the C-13 carbon
PERO-7 was reasonably attributed to hydroperoxylation at the
of RL-PERO-7 was elucidated as 25, and this novel bisabolane
exoperoxide w-as named bisaborosaol D.
657
the
with
has
in RL-
f't
1zee
J5v??
>"
:1
M++1
S･i-
W.
'Zl
?
t-t
dwpmtrh,F"v
"pt
-t
sg
2?-2
'! sg
102
.9 .??
2SO
3. 5e tr1/E
vv,ez
FI-Mass Spectrum of RL-PERO-7
Fig. 3-414
IZZ
l)hrrtvdril･･･rrJ,ErtfrfttTTT
rt,,.l-i,,tgli,4i!tt.T"i"Fl･4LEriitts-r,
4
8Z
6Z
139
79
,
IZ7
4Z
262
rf"4lr----------r
2Z
55 67
391
*5.Z
15Z
119
,
163 17619Z 2z3
5Z
Fig. 3-415
21e
15Z
lez
'
246
231
25Z
EI-Mass Spectrum of RL-PERO-7
658
3ZZ
35Z
1
し
。⊃
帽。；
8
8
べ
8
ド；
へ
⑩
∩
り
Q
8
9
・H
「コ
“
N
し8。
一
Σ
二註
8
8ぎ
【ず；‘L
面
｝
卜
6
臼
二
9
0
＆
8
。
『
国
α
1
、5
日
7・
臼．
O
o
LO
面
駒
。．
8
賃
「・
ρ
L8
o
＄
①
．fq
r、：
ざ
9
d
：r
最
鼠
の
Σ
1
8
6
8
．鼠
8
α；
縫；
H
⑩
H坤
l
ぼ
r・、
Qり
【L．
曾
＄
8
監∫｛
卜
659
●
bO
δ
・r→
，一・
賊
Table 3-163 Physicochen]ical properties of bisaborosaol D (25?
'
(500 MHz, in C6D6, TMS as an int. std.)
6H Coupling Assignment
5.690 IH ddd J= 14.8, 7.5 and 7.2 Hz C-11'H
5.516 IH dd, .T= 14.8 and O.8 Hz C-12-H
2.630 IH br.d J= 15.3 Hz C-6-Ha
2.182
IH
t-likem
C-6-Hb
IH m-dvid. d* J= 19.4 Hz C-3mHa
2.082'
2.019 IH br. dd J= 13.7 and 7,5 Hz C-9-Ha
1.937 IH br. dd J= 13.7 and 7.2 Hz C-9-Hb
1,639 IH m-dvid.d J= 12.3 Hz C-5-Ha
1.396 ,IH br, t-like C-4-H
O.958 IH dddd .J= 12.4, 12.4, 12.4 and 5.1 Hz C-10-H2
* muJtiply divided doublet
660
PE
o一
0．0
o
o
亀
．
8
1』
．
璽0・
亀08
範
“幽。一
2．0
S
9
α90
⑨調
⑨
脇
@匂
3．0
oo
曹
ρ
一
4．0
o
昌．
覆
o
5．0
認
6．0
フ．0
・o
℃
o
‘フ．5
フ。0
6．5
6．0
5．5
5．0
4．5
4．0
3．5
3．0
2．5
2．〔｝
1．5
1．0
．5
0．0
PP岡
Fig・3－417 HH－COSY Spect・um・f RL－PERO－7（500 MH・・‡n C6D6）
661
PP
H
H
H
H
H
COOCH3
H
H
H
Fig. 3-418
Proton Sequence on 1, 4-Disubstituted 1-Cyclohexene
Moiety of RL-PERO-7
m/z 183
ko
CH3
H
ij
H
H
CH3
H3C
OOH
Fig. 3-419
Proton Sequence on the ･Sidechain Part of RL-PERO-7
662
o
一L．
N
Q
σり
QQ
踏国
臼
・驚臼
へ
曾
、E℃
鴇
『
鵠
国
Q
で
蔀
oo
5
い
⑩
∩
⑩
じ〕
萬
『
鴻
℃
鴇
『
溜
一黛
o
．8’H
直
8顕
ゆ
uIH
自N
一・
I」 ）
円
崖
9
卜l
o
口
山
臼
1
口
一島1
触
O
．曾
溶
妻
ﾏc
甲’
の
．5α自
7’
lz
．9
州
か．’
『
穿
認
．辞麟
亀．
『
曽
ｰ
@ζつ
0
σq
ごつ
寸@cつ
一r，・ 1
，璽
o～ 切
・H
一。）
賄
663
Tab1e 3-164
13 C-Nme chemical shift vaZues of RL-PERO-7
(125 MHz, in C6D6, TMS as an int . std., COM and DEPT)
6c
Property
Assignment
167 4
-coo-
C-7
139
6
=CH-
C-2
138 4
=CH-
C-12
130 4
=C-
C-1
126
.
2
=CH-
C-11
81
.
5
-CH-O
C-13
3
-c-o
C-8
1
cH3
C-7'
9
CH
C-4
42
8
CH2
C-9
26
9
CH2
C-3
7
CH2
C-6
7
CH3
C-14
24 4
CH3
C-15
23 8
CH3
C-10
23
CH2
C-5
.
.
.
.
73
51
.
42
.
25
.
24
.
.
.
.
5
664
Tab1e 3-165 Comparison of RL-PERO-7 carbon chemical shift values
with those of aminobisabolenoZ (112? and bisaborosaoZ A (19)
C-No
112 [156]
RL-PERO-7
1
130.4
133.9
2
139.6
119,7
3
26.9
25.9
4
42.9
41.1
5
23.5
23.6
6
25.7
30.7
7
167.4
23.2
8
73.3
59.0
9
42.8
39.4
10
23.8
21.5
11
126.2
119.4
12
138.4
144.7
13
81.5
70.7
14
24.7
29.3
15
24.4
29.3
7'
19
51.1
H2N ,,
/tlz
H
OH
aminobisabolenol (112)
665
Bisaborosaol D (25) is probably formed from bisaborosaol A
(19), as well as bisaborosaol Cl (23) and bisaborosaol C2 (24).
Unlike 23 and 24, the derivative only gave the trans form which
requests much less energy than the cis form. As it has been proved
that t･he radieal reactlon on an olefine having 3,3-dimethylallyl
part indicates neither stereoseleetivity nor position-selectivity
[126], 19 may be convertible to these hydroperoxide isomers (Scheme
3-46). The fact that only the trans form was isolated from the
Rosa rugosa leaf extracts therefore seems to be compatible.
HO
HO
-Hb
Hb
l
COOM
---
cooMe
e:
Ha
l
l'Ha
HO
HO
/'le
---
j
tu
COOM
;-
HO
HOO
cooMe
J
cooMe
h
OOH
HO
HO
i
SNS
HOO
l
cooMe
cooMe
HOO
Scheme 3--46 Froination of the Bisabolane Hydroperoxides (23 - 25)
froin Bisaborosaol A (19>
666
Tab1e 3-166
Phiysicochemical properties of bisaborosaol D
(RL-PERO-7, 25)
HO
H
:
:
t
" ･'･b
COOCH3
OOH
25
A colorless syrup
Vanillin-H2S04 color: pinkish red
' 281 (29), 265
FI-MS m/z (%): 299 (M"+1, 57), 298 (M", 45),
'
(81), 183 (40), 159 (100), 141 (41), 139 (76).
EI-MS m/z (%): 262 (4.2), 246 (3.1), 230 (6.4), 202 (5.7), 192
(12), 178 (20), 177 (21), 176 (13), 145 (19), 139 (20),
137 (45), 125 (28), 119 (23), 107 (32), 105 (34), 93 (27)
91 (43), 79 (63), 77 (40), 70 (35), 69 (39), 55 (23), 43
(100), 41 (85).
IH- and 13c-NMR data are shown in Tables 3-164 and 3-165,
respectively.
667
4) Discussion on the Exoperoxy Bisabolanoids
In the f-ormation routes, these exoproxy bisabolanoids are
considered to be yielded through a radical reaction as shown in
Seheme 3-46. Firstly, hydrogen radieal abstraction oecurs either
at C-14 or C-11. Successive allyl radicals of two type (G and H)
are attacked by 302 moleeule. Since C-12 radical is more stable
than C-14 in G, and C-13 than C-11 in H, those are accordingly
peroxylated at C-12 and C-13, respectively, to yie!d 23, 24 (both
from G) and 25 (from H). As mentioned above, the nonstereoselectivity in the peroxylation at the C-12 and the selective
trans-olefinic bond formation of the C-13-exoperoxide are
compatible with the radical reaction .scheme. In the Rosa rugosa
leaves, these bisabolane peroxides are contained wit,h less amounts
than those of carotane peroxides. eontained in the Rosa rugosa
leaves with less amounts. However, these compounds which are
unexpectedly stable as the isolated state may provide a possibility
that in the leaf tissues some oxidative condition is set up and
unignorable amounts of free radieals are yielded. Since t,hese
peroxybisabolanoids show a stable property, bisaboyosaol A (19)
which'is considered to be their precursor may function as a radical
seavenger.
668
3-8-6 Relation between Bisaboranoids and Carotanoids of
Rosa rugosa
In addition to the carotanoids which are contained as the
major sesquiterpenes in Rosa rugosa, bisabolanoids are also found
as the major part of the constituents. In particular, some of
those bisabolanoids such as bisaborosaol A (19) are involved in the
leaf extractives in a high content (ca 50 - 100 mg/kg f.w.). Since
bisabolanoids and carotanoids are closely related in their
biogenesis, resemblance of those two in their regto-specific
oxygenation at the methyl groups (C-14 and C-7 in the carotanoids
and bisabolanoids, respectively) are reasonable. Significance or
physiological roles of those bisabolanoids has not been known.
However, some facts; (1) several bisabolanoids are contained in
Rosa rugosa leaves and some of them are found with around 50 ppm
coneentration, (2) the oxygenated forms of t,hem are comparatively
stable, and (3) those bisabolanoids were more detectable than the
carotanoids in the diffusate of the unwounded leaves soaked in tap
water, may be indicative of some roles of the bisabolanoids in the
tissues. They may function as a radical scavenger as mentioned
above, or may take a part in an antifungal agent (See Section 10,
pp･ 703) (Scheme 3-47).
669
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3-9 Aeoranoid in Rosa rugosa
3-9-1 Isolation and St,ructure Elucidation of Rosacoranone
In RL fraction containing carota-1,4-dienaldehyde (3), daucenaldehyde (10), isodaucenaldehyde (13), and dehydrodaucenaldehyde
(16), a new compound RL-B was also involved as the fifth
constituent, The fourth peak of RL-fraetion in HPLC (Fig. 3-421),
deteet,ed as a constituent close to isodaucenaldehyde (13) was
roughly isolated and further purified next by HPLC in the same
system to give ca 3 m'g of a colorless syrup. EI-Mass spectrum of
the isolat,e was agreeable with the GC-Mass spectrum of RL-B (M+
218, ClsH220, 218･184 in EI-HR-MS) (Fig. 3-422), Its met,hanolic UV
absorption maximum was exhibited at 228 nm. These physicoehemical
properties･of RL-B suggested its carotane dienaldehyde structure
isomeric to 3. By the IH-NMR spectrbscopy, however, it was
revealed that RL-B possesses no formyl proton. On the other hand,
an exomethylene group (6H 6.130 and 5.028, each d, J= 1.5 Hz), an
allyl methyl group (6H 1.562, 3I{, br. s) and an isbptopyl group (6H
O.721 and O.585, both 3H, d, .J= 6.7 Hz, and 1.516, IH, double
sept., J= 6.7 and ca 6.5 Hz) became feasible to allow a speculation
that RL-B was a non-carot,anoide sesquiterpene (Fig. 3-423 and Table
3-167).
Further decoupling and HH-COSY experiments elucidated two
coupling systems (Fig. 3-424). When the olefinic proton at 6H
5,028 (IH, br. m) was irradiated, a pair of methylene protons at 6H
2,180 and l.617 (eaeh IH, br. d, .J= 17.8 Hz) changed their signal
patterns. In addition, the allylic methyl signal and a broad
signal at 6H 1.727 (2H) became sharpened by the irradition. When
the broad signal was irradiated, a pair of methylene proton at 6H
1.378 and 1.l23 collapsed into isolated doublets (geminal coupling,
-T= 13･O Hz). When the 6H 1.727 signal were regarded as that of an
671
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8Z
68
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176
E
91
6Z
79
133
147
55
4Z
119
+t
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2Z
218
19Z
4
5Z
IZZ
IZZ
15Z
2ZZ
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18
176
8Z
a18
68
6e
41
147
?9
4Z
133
91
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19e
59
161
12Z
2Z
5Z
Fig. 3--422
IZZ
15Z
2ze
25Z
3ZZ
GC-MS <top) and EI-MS (bottom) Spectrum of RL-B
673
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Table 3-167
1H-AuslR
ehemical shift values of RL-B
(500 MHz , in C6D6, TMS as an int . std.)
Coupling
6H
Assignment
6
.130
IH
d J= 1.5 Hz
5
.239
IH
br. s-like m
5
.208
IH
d J= 1.5 Hz
2
.180
IH
2
.165
IH
m-dvid. d* J= 17.8 Hz
dd J= 18.2, 8.1 Hz
1
.953
IH
dd k 18.2, 9.7 Hz
1
.727
2H
br. m
1
.617
IH
m-dvid. d* J= 17.8 Hz
1
.562
3H
br. s
1
.516
IH
d sept Ji 6.7, ca 6.5 Hz
1
.378
IH
m
1
.360
IH
1
.123
IH
o
.721
3H
o
.585
3H
ddd J= 9.7, 8.1, ea 6.5 Hz
ddd .T= 13.0, 5 O, 4.7 Hz
d .T= 6.7 Hz
d J= 6.7 Hz
* multiply divided doublet
675
C-15-Ha
C-3-H
C-15-Hb
C-2-Ha
C-9-Ha
C-9-Hb
C-5-H2
C-2-Hb
C-14-H3
C-11-H
C-6-Ha
C-10-H
C-6-Hb
C-12-H3
C-13-H3
l
e
k.･
i
--...
o
i:l1･lii''lI.-.Iiettttttt
t+.t
ttJt.-.t
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otttttt.....ttttttt.t.t-t.t..t.
IG)
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l
tltttt"tttttt
l'
Il･..tttttt
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[56.
ttttt
l
l
I
ilo t'
y
5'.5
Fig. 3-424a
'
-l･-------
r5'
.t.
5.0
HH -- COSY
d.S
li.o
L
i
r
5.0
,
2.5
rl
2.01. 51.
1
Spectrum of RL-B (500 MHz , in C6D6>
676
{:. ･ /
.t.tt
ttttt
3.5
PPM
t}'t:
l-.-.- tt
,
1
o
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li
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ll.1..1
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Il
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li
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tttttttt
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t
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Fig. 3-424b
LO
,Y,ii,iii11i
i'
'l
l/
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l
ie a - "-`ajI} c((li) -i, o
1 r, 5
. i.--
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2.5
Continued (Magnified in High Magnetic Field)
677
equivalent methylene protons, a substructural unit (A) became
N-"O / rFlq. 3-425>. '
feasib!e
On the other hand, a signal at 6H 1.360 (IH, ddd, .T= 9.7, 8.1
and ca 6.5 Hz) was elearly collapsed by irradiation on the double
septet signal. Therefore, the 6H 1.360 signal must be assigned to
a methine proton geminal to the isopropyl group. Furthermore, it
was revealed by the HH-COSY that a pair of methylene protons each
appearing as a double-doublet (6H 2.165, .J= 18.2 and 8.1 Hz, and
1.953, J= 18.2 and 9.7 Hz) was vicinal to the methine proton.
Consequently, the second part structure (B) was elucidated (See
Fig. 3-425).
Those two units were proved by 13c-NMR analyses (coM,
DEPT and CH-COSY, Fig. 3-426, 427 and Tables 3-168, 169). The
presence of five methylene carbons on RL-B suggested that the
' 6H 1.727 should attributed to one of the met,hylene
protons at
earbons. This assignment was provided a proof by the CH-COSY which
revealed a correlation between 6c 27.1 Tnethylene earbon and the
'
'
1.727 protons.
RL-B also exhibited a pair of exomethylene protons at 6H 6.130
and 5.280 (each d, .T= 1.5 Hz). As one of the proton was resonated
in a markedly lower field, it was expected that the exomethylene
proton were deshielded by something. On the other hand, the
presence of a carbonyl earbon (6c 204.3) and a quarternary carbon
(6c 44.5) were also revealed in the 13c-NMR spectrum. The former
carbon contributed to the conjugation system (uv XmMEt£Hx 22s nm) to
form an a,B-unsaturated ketone group with one of olefinic bonds on
t,he molecule. As substructure A involves the non-conjugated
olefinic bond, the exomethylene part should contribute to the
conjugation system to form partial structure C. Subsequently,
these three substructures were eonnected each other via the
quarternary carbon at 6c 44.5 (See Fig. 3-425).
678
-/:i
KA
CH3 H
.,......xx･
;.-Nx
x
-CH2
"/"'
c
c
H-.--･
Xi
H
A
krgi
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ossc
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7
H-'--NH
2
CH3
,
CH3
B
CH2
1
T
¥'
s"c-s
c
s
ta
C
Fig. 3-425
Two Proton Sequence of RL-B.Elucidated by Decoupling
and cosy Experiments and other Partial Structures
679
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A
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b£
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N 函
680
TabZe 3-168
(125
13C-Ai]etlR chemical shift values of RL-B
MHz' in
6c
C6D6, TMS as an int . std.)
Property
204
.
154
.
3
c=o
Assignment
C-8
8
=C-
C-7
o
=C-
C-4
3
=CH-
C-3
115 o
.CH2
C-15
8
CH
C-10
5
C
C-1
7
cH2
C-9
3
CH2
C-2
7
CH
C-11
1
CH2
C-5
2
CH2
C-6
5
CH3
C-12
23 4
CH3
C-14
20 4
CH3
C-15
134
120
･
.
47
.
44
38
.
37
27
.
27
･
25
23
.
.
.
681
Q
l,
t.nt-..t.t
ttt
'.Ht.N
o.o
-
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-LLLnt-t.
.ttttt-.-
ttt..ttttt
=''---.'t'..-..
t.t+.t.+mttrtttttt
t"t'tt't'tttt
t't'''ttTtttt'tttt't't't
tt...
t.ttu.t.7.t...tt..trt
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twu-.
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.. ttt .ttfttt--.ttt
t.t±ttT..t7t-tt.t
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tt.tttttt
2.5
d.t....t
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t--.tttttm-ttttt..tt.t.
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-t..u..tt..tt
.-tt.-.T+..ttt.tttt
Ttttt.Tm
3.0
-
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t-.-tt.-.rr.Ttt-ttt
-ttttttttmt.tttt..
tmtt.-tt
u...rt.tttt
'ttt''ant''-ttt
ttt.ttt
tt-tttttt-t.tt-tt-tt
t--t.-.
tttt..u..t.t.t..
.tttLt--tt
.Tt.mN-tt-"t.t
.tT.
t.t-..t.t. t7t-ttt
tttt.t..t
t.t..
..
ttt-tt-tJttt
lttt.nt
.Jttt.tt..
ttt
''
3.5
ttttumtt-tL''tt
trr
t.tttttL--t.ttt-.
'
ttt
7nT."t--.ttt
4.5
-ttt-u.
.ttt..ttt
.t..t
4.0
tu.t
.-
5.0
tttVttL-'-t-Tml
t.ntt"t=T.t.Tt.
tt
tttt.t..
.tt.rTt.m
T-t
.-+tttt
tt..-.
--ttt-tt-t.t- tt.ttt"-ttJ-tt...t
t.
tt-tt-ttt.t-..Tttrr
,t4et2o
ttttTtttt-tt
...
tt-tutt--t+.-.-tt-
"
,40
ttt..nnv-
t.tTmttt
r-
-
+
5. ti
6.0
PP
PPM
Fig. 3-427a CH-COSY NMR Spectrum RL-B (125 and 500 MHz, in C6D6)
682
Cttttttm.t7.t.t-.tt
... t...
.t--+ttr
tt.ttttt-ttvt.t
L.
.t......t
.ttnrtt..ttt.tt.
rt-tt..
ttttttttttt
.tt
tttt.tt.
-t.tt.ttt-ttt-.
t-llttttt.ttt.tt
tt.t.tt.ttttt
tt.tt
t.t
..
t....t..-
tt......t.t...tt
.--
-J
.5
.8
d-.
+1
Ttt+.tttrtlt.. tt...t.tt
t-tt-.tt..t
...tt.
tt
......
+.t...t.t
..ll..ttttt
..tttttt-t.
.ttt-.
tt.tttt-
t.t
t.tttttttt
t.t
tt
tttttttttttttt
Lt.t. -t-llt.ttt
t.t...tt-
ttt-tttttLtt
1.0
ttrtttTt
ttttttttttttt
.tt-L.tttttt
L2
-t"t"tt
d-ttt.
trJ-mt"Tt.
-
.tt.-.
ttttt
t.t
tttrtttt
-.tttt.tt.tt
ttttt.-tt.ttt
tt.tt.
t.,..,........
tt.t
'ttttt.t.-
'
ttt
tttttttt
tt.T ttt-.
t..t..
tt
tJ.-t.
Ld
.t
tttt
L6
rr...
tt"tt
H
1.8
.
i
tttt-ttt
tl
tt.t-.
29.028.0
Fig. 3-427b
l27.026.02t5.024.025.022.021
i.O20.019
PPM
Continued (Magnified in High Magnetic Field)
683
2.0
Tab1e 3-169 Correlation betvfeen carbon and proton signa1s by CH'cosy of RL-B
(500 and 125 MHz, !n C6D6, TMS as an int . std.)
Carbon
Position
Proton
(6c)
(6H)
3
C-2
2
3
C-4
5
1
C-5
1
25 2
C-6
1
38
.
7
C-9
2
47
'
8
C-1O
1
27
.
7
C-11
1
23
.
5
C-12
o
20 4
C-13
o
23 4
C-14
1
C-15
6
37
'
12o
.
27
.
.
.
115
o
180, 1. 617
･
239
727
.
378, 1. 123
.
165, 1. 953
.
360
.
516
.
721
585
.
562
.
130, 5. 028
684
From the unsaturation number calculated as 5, the molecule
11! -- A-1 t !- -.
, De Dleycnc. As Lne quarzernary carDon ls supposea to
musL
destribute its single bonds (x 4) to each part. structure, RL-B must
form a spiro-ring structure through the quarternary earbon. When
the third unit (C) was tentativeiy fixed to combine with the
quarternary earbon which is compatible with an isolated geminal
coupling of the exomethylene protons, 3,8- or 5,6-bicyclic
structures are aceordingly given (Fig. 3-428). In the case of 3,8bicyclic structure, the methylene prot.ons on its cyclopropane ring
should have a geminal coupling around .T= 4-5 Hz [158,159].
However, the methylene protons (6H 2.165 and 1.953) which would be
assignable to this position possessed a coupling constant J= 18.2
Hz as geminal coupling. The skeleton of the compound was therefore
allowed to be 5,7-bicyelic. The five-membered ring can take four
different structures (st,rueture D, E, F and G as shown in Fig. 3428). The latter structure F and G may be rejected because they do
not cQmply with the isoprene rule.
To identify the substitution pattern, reduction of t,he
carbonyl group was earried out. It was expected by this experiment
that not only position of the isopropyl group but also the
substitution pattern around an unsaturated earbonyl group would
become feasible (Scheme 3-48). Accordingly, 3.2 ing of RL-B was
treated with excess LiAIH4 t,o give 2.2 mg of a non-quenching
product, RL-B-LAH (Scheme 3-49) (TLC in Fig. 3-429).
685
R2
Rl
Rs
R2
/
Rt= CH2, R2= O
3,8-bicyclic structure
or Rt= O, R2= CH2
Rl R2
R2
Rl
N
N<i,,i
5,6-bicyclic structure
A
Rl== CH2, R2= O
C
Rl= CH2, R2= O
B
RI= O, R2= CH2
D
RI= O, R2= CH2
Fig. 3-428 Possible Structures for RL-B
686
o
CH2
CH2
o
･N(i)k
N<k)
B
`
HO
A
1
CH2
CH2
OH
"
x<"
,Li,tt
B/
o
CH2
CH2
o
x<"i
x<ii
D
s
HO
c
`
CH2
CH2
OH
"
x<ii
Ci
D!
Scheme 3-48 Newly Formed Hydroxyl Methine Proton as the Result of
Reduction of the Carbonyl Group: In case the methine proton was
observed as a double doublet, the structure A will be proved.
687
H--AT 4:1
(z)
o
t.,:i}.･F.
quenching under
UV 254 nm
vanillin-H2S04
test: posltive
4tEISII･:･;;
Reaction Std. 26
MIX.
Fig . 3-429
TL Chromatogram of Reduction Product Obtained by
Treatment of RL-B with LiAIH4
RL-B (3.2 mg dissolved in 2 ml of CHC132added 20 mg of LiAIH4
stirred 2 hr. at room temp.
eooled and added 5 ml of EtOAc
diluted with 15 ml of sat. NaCl soln.
extracted with 20 ml of EtOAc
dried over Na2S04
concentrated in vacuo
PTLC (developed in H-AT 4:1)
Product (RL-B-LAH, Rf O.45, 2.2 mg o£
syrup, 68 % yield)
Scheme 3-49
Reduction of RL-B with LiAIH4
688
a eolorless
RL-B-LAH, showing M+ 220 in FI-MS (Fig. 3-430), was confirmed
to be not.a stereoisomeric mixture but a pure product in IH-rN'rii'
spectrum, suggesting the stereo-selective reduction at the carbonyl
group (Fig. 3-431 and 432), The new methine proton on the
hydroxylated carbon was observed at 6H 4.449 as a broad doubledoublet. Since the methylene protons on the five-membered ring,
originally detected as two double-doublets, changed into two
double-double-doublets on the IH-N)IR spectrum (6H 2.224, J= 12･7,
8.0 and 6,5 Hz, and 1.444, J= 12.7, 12.3 and'7.7' Hz), the reduction
product must be At (26a). Accordingly, RL-B was elucidated as
structure A (26). Other proton sequences are shown in Fig. 3-433･
Thus, planar structure of RL-B was elucidated as 26 whose ske].eton
has been known as an acorane, and this novel acoranoid was named
rosacoranone A. Interestingly, acoranoids are closely related to
carota' ndids as well as bisabolanoids, and some conversion schemes
to obtain modefied carotanoids from simple carot,anoids via
aeoranoids are known, in the area of organic synthesis (Scheme 350) I158,159].
*
13
C-NMR Spectra of RL-B--LAH are shown in Fig. 3-434.
689
+
}I
f`'
1z2,e
Bg,?e･
'-'-
H
oZ
z
I-±
hi
･,-t
e -v
--
'1' v･ -
1--- l--- i -- t
se
.2 ,?e
- t- 1 t 1-- Iip- v - V' il ' 'l
.b ,
2?e
1se
1ee
2if･ertl/E
k
-Bo.?e
1zee
>ke
ltt
O
z
-Z
LJ･
t-l
?
Bg2
Fig. 3-430
1ee
,t -l
B::,e
'I'' t l,
l- -,
I,
2r.n.
FD-Mass Spectrum of
t'I'
'l'l
42e
t'
.D .?e
4E･eFl/El.
RL--B--LAH
9
se
159
6Z
ae
119
41
Sl
55
2Z
le5
131
67
ae2
145
177
187
22Z
5Z
Fig. 3--431
lez
15Z
E!-Mass Spectrum of RL-B-- LAH
690
2ZZ
2se
｝1
o
o
8
」
8
d
需
曾
9
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呂
8
二
面
蓬
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9
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二
8＾
雨ぷ
8
0
呂●H
ぜ“
N
Σ
0
8Σo
》
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日
露
8ρq
めム
。α一uつ
げ1自L
厚
隠
d
需
d
鯛
。
§1
量
8
器隻
そ
芝
3
6
冒
α
げ
N
σり
寸
1
◎り
ξ
に
8℃o
ゴ嵐
鼠
り
691
H∩
H
HO
c；
H
、
、
㌔
、，
，！
、
CH3
／てへ
〆
ノ’
@．
，’
℃，＼H
Hゆ，／’
HもH）
CH3
Fig． 3－433
Proton Sequences and Carbon Assignments of RL－B－LAH
692
σつ
OO
．畿寓
c団
ド
L，
＝；
o
臼
穿1
A
り臼
ヨQ司
国
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oo
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ぐニ
ハ
D ζつ
H
Q
く）q
．・Y｝Q
・H
…R
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N
c〕＝1
B）Σ1
〔エぱ）
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m
．，祠
@）
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．旧く
＿」1
I
qq
－2q
身も
亡⊃ 1
くづ
一ロ19
…o
践
曼の
望
口l
o
。：乙
oり
⊂）ア→
一（o
岬
…I
Qり
σり
．呂lrr
．8。
Nも幻
・r→
隔
693
Tab1e 3-170
Physicochemical propert-ies of rosacoranone (= RL-B,
26)
o
N
26
A. colorless syrup
VaniUin-H2S04 color: brown
FI-MS m/z (%): 219 (M+, 19), 218 (M+, 100)
'
EI-HR-MS: 218.184 (ClsH220, calcd. 218.188)
GC-MS m/z (%): 219 OI++1, 13), 218 (M', 75), 203 (37),
190 (27),
177 (16), 176 (89), 175 (77), 161 (22), 147 (51), 133
(43), 120 (20), 108 (100), 107 (45), 105 (48), 93 (22), 92
(21), 91 (39), 80 (26), 79 (43), 77 (39), 68 (72) , 67
(31), 55 (25), 53 (25), 43 (43), 41 (54).
EI-MS m/z (%): 218 Ol', 34), 203 (32), 190 (22),
176 (69), 175 (67), 161 (23), 147 (52), 133 (48),
(21), 119 (34), 108 (100), 107 (44), 105 (75), 93
(25), 91 (62), 80 (32), 79 (56), 77 (52), 68 (84)
(44), 55 (41), 53 (34), 43 (60), 41 (79).
IH- and 13c-NtyiR data are shown in Table 3-167 and 3-168
120
(34), 92
'
'
respectively.
694
67
TabZe 3-1 71
Physicochemical properties of RL-B-LAH (26a]
HO
N
26a
A colorless syrup
Rf: O.45 (H-EA 4:1)
Vanillin-H2S04 color: pinkish purple
EI-MS m/z (%): 218 (M+, 34), 203 (32), 190 (22),
176 (69), 175 (67), 161 (23), 147 (52), 133 (48), 120
'(21), 119 (34), 108 (100), 107 (44), 105 (75), 93 (34), 92
(25), 91 (62), 80 (32) , 79 (56), 77 (52), 68 (84), 67
(44), 55 (41), 53 (34) , 43 (60), 41 (79).
IH-NMR 6CT6MDs6 (soo rviHz): 1.6o8 (IH, ddd, .T= 12.9, 10.8 and 5.3 Hz,
C-2-Ha), ca 1.53 (IH, partly overlapped, C-2-Hb), ca 2.02
(IH, br. m, C-3-Ha), 1 .913 (IH, br. d, J= ca 19 Hz, C-3Hb), 5.375 (IH, br. in, C-5-H), 2.345 (IH, br. d, .T= 17.9
Hz, C-6-Ha), 1.720 (IH , d, J= 17.9 Hz, C-6-Hb), 4.449 (IH,
br. t-like, J= ca 8 Hz , C-8-H), 2.224 (IH, ddd, J= 12.7,
8.0 and 6.5 Hz, C-9-Ha), 1.444 (IH, ddd, J= 12.7, 12.1 and
7,7 Hz, C-9-Hb), 1.338 (IH, ddd, .J= 12.1, 6.5 and ca 6 Hz,
C-10-H), 1.784 (IH, d sept, J= 6.8 and ca 6 Hz, C-11-H),
O.961 (3H, d, .T='6,8 Hz, C-12-H3), O.897 (3H, d, .T= 6.8 Hz
C-13-H3), 1.653 (3H, br. s, C-14-H3), 5.072 (IH, d, J= 1.8
Hz, C-15-Ha), 5.069 (IH, d, J= 1.8 Hz, C-15-Hb).
695
'
N")
i
1
I
'"i)
'
.-.-------pp
--
ss
.-･r :
OH
l
l
'
,
,
1
+
.., s
+
..rf:
1
l
OR
RO
Naegeli and Kaizer [159]
I
Zalkow et al. [158]
OH
ss
"
..･c :
Scheme 3-50
H
Some examples for chemieal conversions
carotane-acorane rerationships
696
to' ' show
3-9-2 Relative Configuration of Rosacoranone
Acoranoids are known to be difficult to determine their
absolute or even relative configurations.' When a NOESY experiment
was carried out on rosacoranone (26), some NOEs were observed (Fig.
3-435). However, those were not informative in relation to the
relative configuration of 26. RL-B-LAH (26a) was therefore used
for the stereostructure determination. In CsDsN, prot･ons coplanar
to the C-8 hydroxyl group are expected to be deshielded [160]. As
a matter of fact, C-8-H, C-15-Ha and C-9-Hb were strongly (A6H
O.4-O.3), and C-15-Hb, C-3-Ha and C-2-Hb were slightly (A6E{
O.2-vO.1) deshielded in CsDsN (Fig. 3-436). As C-10 methine proton
was utterly unchanged its chemical shift, it was considered as
ant,iplanar (Table 3-172), NOEs between C-12-H3 and C-2-Ha and C13-H3 and C-9-Hb were, together with the deshielding of C-2-Hb and
C-3-Hb, suggestive their coplanar position. Consequently, relat･ive
configurations of 26a and 26 were elucidated as shown in Fig. 3437.
H(>
H
o
HHK H
N
H
CH3
H
H
H3C
<
ili.//
CH3
Fig. 3-435 NOEs Observed in RL-B (NOESY in C6D6, 500-MHz)
697
1．
’6
8
蜜
0
8
．i
cJ
A
⊂二←
u）紀．
9
d
『〔よ
に．
臼
8【◎
po κ）
’鵠
Q
尉
丙
・H
wN
⊂， “
器
穿
丙
「＝＝1
o
o
ゆ
9
。．一一）
ロリ
ニ註
．の＝1
＜
q
臼
『講
ぜ
I
ρQ
l
o日
一¢
ｿミ
ω
呂
身
ぜ
駒
o
目角
一べぢ
a
L曾
濱
の
。Σ1
『：乙
【Ol
州
塁』
濱
⑩
80つ
’6．
臼
済
◎り
需
。H
●
bO
占
698
o国
9
Table 3-172
DeshieJding effect of RL-B-LAH in CsDsN
in CsDsN
C-No
2 .369 br. d (17.9)
l .828 br. d (17.9)
.445 br. m
2 .19 (approx.) m
1 .87 (approx.) m
5
1
.547 ddd (12.6, 11
.65 (approx.) m
4 .773 br. dd (8.1,
2 .300 ddd (12.5, 7.
.o
, 5.9)
1
.732
1 .340
1 .704
o .925
o .883
1 .664
5 .464
5 .233
1
6
7. 9)
6,
6.8)
ddd (12.3, 12 .3 , 8.4)
ddd (12.0, 6. o, 6.0)
d sept (6.8, ca 6)
d (6.8)
d (6.8)
br, s'
d (1.8)
d (1.8)
.36 (approx.) br
*A6= 6HCT51,1?;5N
2Ha
2Hb
3H
5Ha
5Hb
6Ha
6Hb
8H
9Ha
9Hb
10H
1IH
699
A6 H (ppm) *
2
.345
+
O.02
1
.720
+
O.09
5
.375
+
O.07
2
.02
+
O.17
1
.913
+
O.04
1
.608
1
.53
+
O.12
4
.449
+
O.32
2
.224
+
O.08
1
.444
+
O.31
1
.338
±
o
1
.784
12H3
o
.961
O.08
O.03
13H3
o
.897
O.Ol
14H3
1
.653
+
o.oi
15Ha
5
.072
+
O.39
15Hb
5
.069
+
O.16
8-OH
6,C,D,,C,i3
in CDCl3
O.06
HO
o
N
N
RL-B
RL-B-LAH
3-437 Relative Stereostructures RL-B and Its Derivative
rel .i R* , 10 R, '* for RL-B and rel.1R* , 8R* , 10R * for RL-B-LAH
Fig.
700
3-9-3 'Relation between Bisaborosaol.A and Rosacoranone
Acoranoids are found in some monocots as their common
sesquiterpenoids [161.162]. On the other hand, in dicots the
sesquiterpenoids are comparatively rare, and those sources are
mostly primitive dicot, such as Chloranthtis plants [163] (Fig. 3438). Thus, the presenee of an acoranoid in Rosaceae which
belonging to a rather evolved dicot is an interesting fact from the
view point of chemotaxonomy. Although acoranoids are biochemically
synthesized from cis-trans-farnesylpyrophosphate via bisabolanoids
[63], 26 has a different oxygenation pattern from that of
bisabolanoids originating in Rosa rugosa. Carotanoids from Rosa
rugosa are, without exeeptions, resio-specificaUy oxygenated at C14 as well as the bisabolanoid C-7 carbon which is biogenet,ically
equivalent to C-14 in caro,tanoids and acoranoids. In Rosa rugosa
leaves, sesquiterpenoid is considered to take the biogenesis as
shown in'Scherne 3-51. Significance and function of this minor
sesquiterpene are not, known.
,, 7-
N
N
ts-･
113 [161]
114 [161]
OH
115 [163]
Fig. 3-438 Some Tipical Naturaliy Oceurring Acoranoids
701
l
OPP
l
N
t
t
,
o
HO,
tllttt
H
N
･･ :
Scheme 3-51
Acoranoid
cooMe
Biochemical Relationship between Bisaboranoids and
702
3-10 Bioassay
3-10-l Antimicrobial Activity of Rugosal A
1) TLC Bioautography
The antifungal activity of rugosal A (1) was tested by TLC
bioautography using Cladosporium herbarum HU 9262. Pure
crystallines of 1 were dissolved in acetone to be 1000 ppm (10.0
mg/10.0 ml), and 15 vl of the solution was taken by a 50 ul
microsyringe and charged onto silica gei thin-layer plates (O.25 mm
thickness) to ' form a circular zone ca 10 mm in diameter (ca O.8
cm2). Suceessively, 1 ml of the original solution was steadily
'
taken in a test tube by measuring pipette, and an equal volume of
fresh acetone was added to the solution. The diluted ,solution (500
ppm) was similarly charged on the plate. Accordingiy, several
solutions ranged from 1000 ppm to 15 ppm of 1 was prepared as shown
in Scheme 3-52. ･The amount of 1 charged on the area was calculated
as listed on Table 3-173. Charges of the test solution and acetone
as a control experiment were duplicated.
703
m solution of 1 10.0m 10.0 ml of acetone
･charged
on TLC (15 vl x 2)
taken 1 ml by measuring pipet.te
added 1 ml of fresh acetone
shaken we1l
500
taken o .3 ml
m solution
charged on. TLC x2
added 2.35 ml of acetone
taken 1 ml
shaken well
added 1 ml of acetone
60mofsolution
shaken
chargedonTL x2
250
taken 1 ml
m solution
charged on TLC x 2
added 1 ml of acetone
taken 1 ml
shaken
30
added 1 ml of acetone
mofsolution
shaken
chargedonTL x2
ke n1ml
I.L!!JL2.9i}
125
take
solution
.L-as!RiLSS
charged on TLC x 2
added 1 ml of acetone
shaken
1000
1ate :-M-------
L
uat5 soluti
[
removed the organic solvent in vacuo
inoculated the £ungal spore [87]
incubated
Scheme 3-52 Procedures for TLC-bioautography
Tab1e 3-173
concn. (ppm)
weight (ug)
Prepared concentrations of rugosal A solution and
each ca1cu1ated weight/charged area
1000
500
250
125
60
30
15
o
15
7.5
3.8
1.9
O.9
O.5
O.2
o
704
After the chqrged TLC plates were dried enough in
vacuo, the spore suspension (See Chapter 2-1) was uniformly
sprayed on the test plate, which were kept at 25 OC in the dark
under a moistured condition. After 3 days incubation, 1
exhibited clear inhibitory zones in the range £rom
15 ug to O.9 ug/ O.8 cm2, and even with O.45 ug (15 vl of 15 ppm
solution), this substance showed a marked retardation of,
the fungal growth (Fig. 3-43g and Table 3-174).
Tab1e 3-174 Antifungal activlty of' rugosaZ A in TLC
bioautography
weight (ug)
activity*
15
7,5
3.8
1.9
O.9
O.5
+++
+++
++
+
+
+
O.2
o'
* +++ .
. a severe inhibition of growth (the antifungal zone ls
wider than the charged area), ++ : a clear inhibition of growth
(the fungal area is corresponded to the chargeds area) , + : a
clear retardation of growth, ± : ambiguous retardation of growth,
-: negative effeet.
2) Paper Disc Method
Successively, the antimicrobial activity of rugosal A (1) was
tested by t･he paper disc method, using AspergilZus flavus,
C2adosporium herbarum, Saccharomyces cereviciae, Staphylococcus
aureus and Echerichia coZi as test microorganisms. The result was,
as indicated in Table 3-175, not clear. It was obvious that, under
a nutrient-rich conditlon 1 is not toxic against Echericha coli.
On the basis of this consideration, agar plates only containing
defatted extracts of Rosa rugosa fruits were prepared, and
antimicrobial assay was again tested using this rugosa rose-medium.
705
As shown in Table 3-176, the growth inhibition of the test microbe
w a s m a r k e d] y h e i g h fv e n e d .
In this series of assay, a gram-positive bacterium
Staphylococcus aureus showed a clear but only thin inhibitory zone
on PYG-agar culture (glucose:pepton:yeast extract= 5:1:O.1).
However, in another assay carried out in Japan Tobacco Industry
K.K., 1 showed a clear toxicity against BacilZus subtilis in the
concentration of 25 pg.
3) Spore Germination Assay
According to the resuit of antimicrobial test against several
phytopathogenic microbials (Table 3-177 by Hokko Kagaku Kogyo
K.K.), 1 exhibited remarkable toxicity against the following
fungus; Pyricularia oryzae, Botrytis cinerea, Colletotrichum
lagenariull? and Aspergillus niger, and bacterium Xanthomonas
campestris pv. or/zae.
I H- +H 1-- n--- 4p.-"-- T- - - - -t r -- -r - " .- -7r =t -T :trmmxanMswmmmauaNmrmrmmepa
lll･lll"#.Illit1¥'¥"IZS""'"'ii2if,lj'ik'g･',",,'.:,,,iiill/l//k/,mklli,ll"il･llll,i,gg"lii,igi,,1,3,,i'2;#kS'ksi,g",le;g$s,,",,k",ts
t
i
i
{
1 15.0 7.5 3.8 1.9 O.9 O.5 O.2 O (,.)
m.ss,,ts/tsi,pm,,g.wikesrree/ggaswa･ee,k'eekew
t
'
l
i
!t
l
l
Fig. 3-435 TLC-bioautography for Several Concentrations of Rugosal
A
706
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≡超冒麗胡超冒器胡紹
708
Table 3-177
lnhibitory activity of rugosal A on spore germination
of several phy-topathogens
spore germination
MIC
1･ss (ppa)
50
os
12.5
6.3
3.1
Alternaria kikuchiana
+
+ .v-
H-t･
･H-}･
+-H･
･H-l･
Aspergi1lus niger
-
-
--v±
±
+ .v"
botrytis cinerea
e
±
±.v+
+ .vH
-"vH+
-
±
±
±-v+
<Fungi>
bercospora beticola
Ciadosporium cuoumeriruim
Coch:iobo1us miyabeanus
Co"etotricham 1agenarium
Fusarium oxysporum f. sp. oucumerinum
F.roseuN
C;omere;:a cingulata
Gibberel1a fujikuroi
Mycosphaere11a me1onis
ts
HHf
>50
･t-H
50
+
-H-f･
･t･H
-H-i･
･H-l･
>so
±Av+
+.H･
･HNt.
･H-+･
as
±･
+
-
-H-f-
･H-t･
H-+
+･H･
>50
-N±
+Av-
+･H･
H-l･
･H-･
H-}･
>50
-H-t･
HNt.
-H-l･
-
>50
+･H}
-H-l･
H.i.
･H-t･
>50
+-H･
-
HNf
･H-t･
>50
･IH
･H-l･
.+H
-
H
-
±"v+
+
±
.
.
-
･H-･
+
+
H
H-I･
-H-
･H-l
H-t･
P. glumae
+･H･
･H-t･
H-･
P. solanacearum
･i-H
･H･+
P. syringae pv. phaseolicola
HAvH+
H.vlTH
P. syringae pv. Iachrymans
'H'--v'H'+ '
H'vH+
HAv--
Erwinia carotovora subsp. carotovora
++-vH+
-.vH+'
-
±
Pseudosnonas avemae
X. canlpestris pv. oryzae
･H-+･H･
-HH･
Xantbomonas campestris pv. citri
50
-
±
<Bacteria>
･H--
-H-･
±
Rhi2opus nigricans
as
+-.H
PyricuIaria oryzae
Penici11ium digitatum
'l"t"f'
>50
.HAv-NH
'
･H-I･
>so
･H-t･
+H.
>50
H-l-
･H-t･
H-t･
>sc
･H･+
+･H･
･H-}･
.H"t'
)50
･t-H
･H-l･
･i-H･
･HHt
>50
･H-+
dH-t･
･H-･
>50
+byH+
>50
-
-
Corynebacterium michiganense
･HNi.
･H-f
,H-
A. tutaefaciens
･H-i･
++･l-
-HH･
709
>50
-H-f
-
:
-
6.3
+･H
･H-t･
++.v+･H-t･
･H.t.
)se
-.v+H
++Nl-
･H-i･
･i-H
･H-f
>so
H+
･H-t･
･-H-
>50
±
r2.5
3-10-2 Structure-Activity Correlation of Rugosal A
Some derivatives of rugosal A (1) was also tested its
antifungal activity by TLC-bioautography (Table 3-178). As the
result, requirement of the fundamental strUeture of 1 became
feasible. Inactivity of rugosic acid A (2) was suggestive that the
polarity is important in the exhibition of antifungal aetivit.v,
The endoperoxy group seems necessary, since peroxide rearranged one
lost its activity at least the test derivatives. On the other
hand, without a,B-unsaturated aldehyde group, RSA-NBH-i (lc) and
RSA-HCL (lg) were able to exhibit the activity, while RSA-AC-1 (la)
was.almost inactive suggesting C-2-OH is also necessary to the
aetivity. Those results were informative to plan the search of
precugsors and metaboXites.
710
Table 3-178
Antifunga1 actl vi tles of some rugosal A derivatives
:D::fl9ilil':;re--.-.erlvatxve
(Ag)
100
zo
2
-H-
+
"
-
"
-
-+
+
25
--
･ov2 o
-
A OH
Poizf cHo
+
6Ac
-
.･O-d'i cHP,&",3
6H
'
Ovil ,eH
-
/x 6H
CLo'hf COOH
+
+
.
+
"
-H-
+
--
+
.
+
q
"
.
-H-
+
+
6H
--d-
eAc'
1 CHo
crI
OAc
., eH
cr hf 20H
dH
O･dl COOCH3
-
'
Each derivatives dissolved in 15 vl of EtOH was charged on O. 25 mm
silica gel plates.
711
3-10-3 Ant,ifungal Activity of Bisaborosaols
As tested in rugosal A (1), antifungal actvity of bisaborosaol
A (19), Bl (21) and B2 (22) was also examined by the TLCbioautography. The results were showed in Tab!e 3-179. Not only
17 but also 19 and 21 exhibited an antifungal activity; however,
the values of the three were 10 more time lower than that of 1 in
t, his assay .
Tab1e 3-179
l)repared concentrations of bisaborosaol A, Bl and B2
solutions and each antifunga･1 activity
concentration (ppm)
weight (ug)
activity* 19
21
22
c f. 1
6600
3300
1650
830
410
210
o
100
50
25
13
6
3
o
++
++
+
±
±
++
++
+
±
±
++
++
+
±
±
++++
++++
+++
+++
* activity is according to Tab1e 3-174
712
+-+
++
3-10-4 Cytotoxie Assay of Rugosal A and Its Related Compounds
Cytotoxieity of 1 and 2 was also tested, using the P-388 ADRS
mouse cell. IDso of 1 against the cell was O.54 (ug/ml). On the
other hand, 1 exhibited IDso O.21 against another cell line
resistant to adriamycin (P-388r). Those results suggested that 1
possesses non-selective cytotoxicity, although the activity was not
so remarkable compared with some antibiotics. Compound 3 was
comparatively less active.
3-10-5 Antifeedant Assay of Rugosal A
Rugosal A (1) did not showed any acute toxicit･y nor antifeedant activity against test larva (See pp. 43). The test worms
tended to avoid foods containing 1 when they are not starved;
however, under a starved condition they fed the foods and did' not
show any physiological changes. Compound 1 was therefore
considered to be a passive-typed retardant.
713
Chapter 4 Summary and Conclusion
4-1 Summary of the Thesis
Rosa rugosa is known to be a rose strongly resistant to
diseases. From the leaves of Rosa rugosa, an antifungal
sesquiterpene, rugosal A (1) was isolated and elucidated its
structure including the absolute configuration. Rugosal A (1)
initially isolated from damaged leaves of Rosa rugosa exhibited its
structure as a carotane type of sesquit,erpene aldehyde which
possessed a unique 1,4-endoperoxide bridge whose oxygen had
intramoleeular hydrogen-bond with an aHylic hydroxyl group. This
or,B-unsat,urated aldehyde showed a remarkable antifungal activity on
TLC bioautography using Cladosporium herbarum. Several derivatives
of 1 were also prepared. As the related carotane acid, rugosic
acid A (2) was also found. This aeid was accumulated in the
t,issues in a considerable amount, although it was non-fungitoxie.
This acid provided sorne important informations for stvuetural
elucidation of 1. These carotane peroxides showed" some
characteristic chemical rearrangements at the peroxide bonds, under
a basie or an acidie condition. Their reaction mechanisms were
further discussed.
In the course of searching the precursors of 1, earota-1,4dienaldehyde (3) was isolated. This eompound was quite unstable to
air, and gave 1 and 2 through an intermediate, endo-exoperoxy
carotane aldehyde 3e. By the structural analyses of the
int,ermediary compound and some byproducts in the oxygenation
reaction of 3, conversion scheme of 3 into 1 was proposed. On the
other hand, metabolic pathway of 1 through 2 was also examined. As
t･he result, some peroxy-bond rearranged carotane acids were
isolated in the mature leaves, and it was suggested t,hat peroxide
rearrangement of 2 was the main pathway in the metabolism of the
714
carotane peroxides. Furtherinore, several minor carotanoids were
also isolated and their struct.ures were elucidated. 'Thus,
biogenesis of Yhe carotanoids in Rosa rugosa leaves was also
suggested.
Not only earotanoids, but also bisabolanoids were contained in
the leaves. Bisaborosaol A (19) and its oxygenated forms were
found as a major constituents of t,he plant. These bisabolanoids
gave an evidence showing a correlation between bisabolanoids and
carotanoids in the biosynthesis from cis-trans-farnesyl
pyrophosphate. In addition, an acoranoid was also isolated as a
'
minor constituent･ of Rosa rttgosa.
Rugosal A (1) and rugosic aeid A (2) exhibit,ed a drastic
change in amount according to t･he seasons. In the flowering
season, the amount of the carotane peroxides became the highest.
It was calculated that 2 and 1 should exist in the most act,ive
tissues at a coneentration of more than 500 ppm (1, 200 ppm). As
an ant,ifungal activity of 1 against, Cladosporittm herbarum was
clearly visible at the concentration of 125 ppm on TLC plate, it
seemed tha't 1 could function as a defensive agent in Rosa rugosa
leaves.
;..-s(SS>
.-A,.i.At.6vgzgrs
YbU SEE..
HERE ,,
･×
..l
N oitN
k
N
PL,4N7:s ueLaE
rw
715
.DVVZs:L6L.E
;iVFAPO/V
'
5-2 Conclusion
When Rosa rugosa leaf was mechanically damaged (and also
chemically damaged by NaCN solution), a typieal browning is
observed on the damaged part. This brown'ing, due to hypersensitive
cell disruption and accompanying accumulation of polyphenols, is
known to be a defense system for some plants. In the case of Rosa
rugosa, this drastic browning probably eontributes to its
resistance against several pathogens. Indeed, some non-resistant
roses show only a slight browning by these kinds of damaging.
However, the author dearly proposes that the hypothetical disease
resistanee factor of Rosa rugosa is mostly due to rugosal A (1)
which is markedly rich in t,he leaves and exhibits a considerably
antifungal activity. In addition, other sesquiterpenes in the
leaves may also be regarded as the resistance factor.
One of the experimental evidence for the proposal is that
other roses, particularly some rose cultivars, were quite poor in
sesquiterpenoids. In another series of studies, the author
examined sesquiterpenoids of some other Rosa plantS [69]. In TLC,
GC and GC-MS analyses, it revealed that Rosa aciOularis, Rosa
woodsii and Rosa acicularis var. nipponesis contained some
bisabolanoids but no carotanoid, and some other wild roses were
rich in monoterpenoids but very poor in sesquiterpenoids. On the
other hand, cultivated roses examined so far were very poor in
these terpenoids, suggesting that the resistance of wild roses are
due to those ehemicals. Some Rosa rugosa hybrids .examined so far
(such as Hansa) were all eontained rugosal A in a marked amount.
The antifungal activity of the Rosa rugosa leaf could be
found, so far as the author has tested, apparently less than 10 mg
of fresh leaves. When the EtOAc extract from fresh leaves was
diluted in various concentrations and eaeh of the extracts was
developed on TLC in a specific solvent, the compound 1 in the
716
sample showed the clearest antifungal zone on TLC-plate at the
concentration of ca 2 pg/10 mg leaves. On the other hand,
exudates from the damaged leaves (25 mg damaged leaves) exhibited
a clear antifungal zone for 1, but no other antifungal spot. In
eontrast, polyphenolics from the damaged leaves rarely showed any
activity in the fungus test, As the result, it was suggested that
1 was relatively eoneentrated in exudates of the damaged leaves.
Since mechanical damage is considered to eause pathogenic
infections, this effective diffusion of the antifungal agent from
the wounded part may be significant in the defense system of Rosa
'
rugosa.
The antifungal substance was accumulated in the leaf tissues
as the result of oxygenation of carota-1,4-dienaldehyde (3) and
further oxygenated to rugosie acid A (2) to be pooled in the
tissues. As 3, exhibiting an easily oxidized nature, was
present in the leaf tissues in a noticeabld amount, it was
speculated that 3 functioned as an antioxidant in the leaves.
Since Rosa rugosa grows under strong light in a coastal area during
summer season, it may need a protection system against peroxidation
of the thylacoid membrane. For t,he protection, 3 must be used as
an antioxidant. As the plant must store energy by photosynthetie
reaction and regeneration during a short summer season in the
northern area, it may sacrifiee some membrane lipids for the
effective photoreaction. The fact that Rosa rugosa contains a
marked amount of B--carotene, ct-tocopherol and aseorbic acid [79]
(See Fig. 1-3, pp. 43), all known as common antioxidants and
radical scavengers, fully supports this hypothesis. Furthermore,
its large root system also should be taken into account in the
energy resource. Rosa rugosa rnust have a large and deep root
system due to dry and nutrient-poor sandy soil on the coast. To
support this root system, its leaves should have enough glucose
produetion capacity with the photosynthetie reaction during the
717
short summer season. This, alinost overworked photosynthesis may
cause a peroxidation of membrane without such an antioxidant.
Indeed, during the survey of these sesquiterpenoids in Rosa
rugosa, it was unraveled that the leaves are under a quite strong
oxidative condition due to the presence of bisaborosaol Cl, C2 and
D (23 - 25). Moreover, the presence of 6-demethoxy-4'methylcapillarisin (73) in Rosa rttgosa [78] suggests the oxidative
condition. !t is likely that 73 is fromed through hydroperoxide
intermediate to result in oxygen migration between C-2 and C-1'.
This Hock eleavage-like pathway may be possible because in carotane
peroxide, similar reaction are considerable (Scheme 4-1).
Therefore, the author expects as follows. From this oxidative
condition, Rosa rugosa protects its tissues by B-earot,ene, ascorbic
acid, carota-1,4-dienaldehyde (3) and bisaborosaol A (19).
On t,he other hand, when 3 was oxygenated, oxidized metabolites,
mainly 1 were then re-utilized as antifungal substances. This
seems very e£fective because the plant does not consume any other
energy and carbohydrates to produce the defense compounds against
microbial
invasion. ' '
The resist,anee of Rosa rugosa is probably explicable with some
complex factors, one of which is the aptifungal sesquiterpenes as
discussed above. As the other factor, hypersensitive cell
disruption with a severe browning conducting to a severe oxidative
eondition in the tissues induced by a chemical or a mechanical
damage is also proposed. Furthermore, 'surprisingly high contents
of polyphenolics in the rhizome may contribute to the resistance of
Rosa rugosa. Indeed, Rosa rugosa root extvaet shows very clear
antimicrobial activity. Thus, not only the aerial part but also
underground part should contribute to the highly resistant nature
of Rosa rugosa against pathogens. A model of dinamic roles of the
carotanoids in the aerial part are shown in Fig. 4-1.
718
HOxz :×. -Ox ON./ X
Z,,,'.i i V
,,7
HO
,,7
o
l
1
l
OH O
OMe
6--deine't hoxy-4 ' -
tu
1
OMe
methylcapillarisin (73)
:--･
1
OH o
t
HO
,,,-
l
peroxylation
41
o
OMe
tu
OOH
tu
OH o
t
HO
.-'
l
Hock cleavage
OH
o
o
tu
OMe
OH o
t
HO
41
o
dehyd ration
o
tu
OMe
1
OH o
Scheme 4-1
Possible formation pathway for the phenoxychromone, via
peroxylation of a flavonoid
719
The author has been eneounted several physiological aspects of
the carotane peroxides, although these were not deseribed in this
thesis. When a leaf-let of Rosa rugosa were slightly pressed
between two sheets of filter paper which previously got wet with nhexane or MeOH, and after that, the paper was sprayed with the
peroxide reagent, the shape of the leaf-let clearly appeared as a
red color according to the peroxide reaction. As an interesting
observation, only underside of the leaf-let showed the response, as
shown in Fig. 4-2, By this simple experiment, it beeame understandable the localization of the peroxides. In short, it was
suggested that the peroxides localized in the tissue of the
underside of the leaf. In fact, when a flake of tissue on surface
of the upside and underside of the leaf were each separated
carefully and suspended in a sinall volume of a EtOH/peroxide
reagent mixture in test tubes, only the test tube in which
underside tissue was suspended turned a slight pink. With
chlorophyli, the same result was obtained.
To obtain further aspeet, the tissue stained with the peroxide
reagent were'directly observed with a microscope. 'Under the
mieroscopic observation, numerous numbers of trichomes are
recognizable on the underside of the leaf, and some of 'them seemed
to be stained to a red color as shown in Fig. 4-3. Although some
effect･ of red light eannot be excluded, it is likely that the
trichomes contained carotane peroxides in a high concentration as
those of tomato plant which is known as a secretory and storage
cell (or organ) for sesquiterpenoid of the plant [9]. These
observatiQns are an interesting object, as with th'e enhancement of
ratio in diffusion of carotane peroxides from the wounded part.
Further investigation is requested t･o reveal a funetion of
the trichomes and the carotane peroxides in Rosa rugosa.
From aerial part of Rosa rugosa, callus eeln be obtalned
easily. Using a pteee of callus, sesquiterpenes in the callus were
720
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preliminarily examined. Unlike leaf-tissues, callus of this plant
did not contained any marked terpenoid. However, tlssue culturing
is expected to be available in the near future, in studies on
s
'
sesquiterpenes of Rosa rugosa.
Chemotaxonomically, these constituents in Rosa rtigosa are
characteristic among Rosaceae plants. This.sesquiterpene phase in
this Rosaeeae plant is quite similar to those of some Compositae
and Umberifellae plants. Furthermore, the phenoxychromone (73)
whose skeleton is quite rare as a naturally occurring compound was
also reported from a Compositae plant, Artemisia capiZlaris [164].
As shown in Fig, 4-4, Rosaceae and Compositae are, however, not
closely related to each other, while Umberiferae are somehow
close to Rosaceae [165]. Further studies and discussion should also
be carried out in this theme from the chemotaxonomical view point.
Rosa rugosa has suecessfully dispersed its descendants as
' western roses, due to its
important hybrid races to crossbreed with
resistant nature, and will bloom out further as an ancestor of
excellent rose cultivars in the gardens all over the world. Rosa
rugosa is commonly distributed along coastal area bf'Hokkaido and
was chosen as the flower of Hokkaido. We are proud of this
excellent wild rose, and of cause we have responsibility to
conserve its presence for the next generations.
･724
ASTERIDAE
Compositae
ROSIDAE'
Rosaceae
oommrNIDAE
Gramineae
Umberifellae
(Compopetalae)
LIL:IDAE MAGNOLIIDAE .
(qhoripetalae) DILLENIIDAE
Chloranthaceae '
rvlONOCOTYLEDONE
i DICOTYLEDONE
X. BRYOPHYTINA
TRACHEOPHYTINA Hipataceae
Xl,. .i-.c==;ia 2gg.O¥g.Cg:",.otina)
PLAN!IrAE MYCOTA
'
Fig. 4-4 A Possible Genealogical Tree of Plant Kingdom: The
genealogical tree is based on and rearranged from the reference
[165]. Rosaceae and Uinberifellae are on the same branch, while
Compositae forms a branch for another group.
･725
Abstract in Japanese
北海道の沿岸地に自生するハマナス（Rosa rugosa Thunb．）は、その可憐な
花と芳香により、古くから鑑賞用に用いられてきた。しかしながらその園芸的
価値はわが国よりもむしろヨーロッパあるいはアメリカにおいて大きく評価さ
れ、高められた。その理由のひとつに、ハマナスが、西洋の園芸バラで問題と
なっていた幾つかの病害虫に対して顕著な抵抗性を有していたことが挙げられ
る。特にバラ黒一病は鑑賞用品種の商晶価値を落とすことから大きな問題とな
っていた。ハマナスはこの病気に対する抵抗性に優れていたため、ヨーロッパ
へ導入された後台木あるいは交配種として急速に広まり、一部の地域では野生
化するまでに至った。 ハマナスに認められる病虫害抵抗性因子の化学的あるい
は生化学的研究は、1987年にMashchenkoらが羅病性バラとハマナスを含めた
抵抗性バラの葉中cholesterol含量差に注目しこれを抵抗性因子であるとした
報告［81］が出されるまで行われていなかった。
著者は、各種植物の二部を機械的傷害処理したのち水抽出し、その水層の有
機溶媒転溶物の抗菌活性をスクリーニングした結果、本植物傷害葉抽出物中に
極めて強い抗菌活性成分を検出した。次いで活性化合物本体の構造を決定する
目的で、この活性成分の単離精製を試み、各種クロマトグラフィーにより
1．3kgの傷害処理葉から120鵬gの活性化合物を無色針状結晶として得た。こ
の化合物は、M＋266（C15H2204）を示し、各種機器分析により、特徴的な部分
構造として、α，β一不飽和アルデヒド基、1，5位にかかるエンドパーオキシド
架橋、および一方のパーオキシド酸素原子と分子内水素結合したアリル水酸基
が存在することが判り、文献未知のカロタン構造を有するセスキテルペンアル
デヒドであることが推測された。最終的には、本化合物の各種化学変換および
対応するカルボン酸メチルエステルのINADEQUATE法によるC－C相関の測定に
より本平面構造を証明し、これをルゴサールAと命名した。その立体化学につ
いては、NOESY法で相対配置を、励起子キラリティー法［106］で絶対配置を決
定した。
本化合物の骨格であるカロタン型セスキテルペンは、高等植物における分布
がセリ科および小数のキク科に限られており、バラ科では例の無いものであっ
た。また、C－14位における酸化はセリ科あるいはキク科ではほとんど知られて
おらず、これがバラ科由来の幽幽タンにおける特徴のひとつになると考えられ
る。ハマナス中には、更にルゴサールAに対応するカルボン酸（ルゴス酸）が
含まれており、その含有量は最多で500mg／kgにものぼった。ルゴス酸はその
安定性、非抗菌活性および含有量から、ルゴサールAの主解毒代謝産物である
と考えられ、実際に空気酸化によりルゴサールAからルゴス酸が生じることを
確認した。
ル：ゴサールAの生成過程においては、その1，5エンドパーオキシド架橋が、
低分子天然物パーオキシドとして一般に見出される1，3ジエン体と一重項酸素
閉におこるDiels－Alder付加反応型の 1，4エンドパーオキシド架橋と異なり、
1，4一ジエン構造をもつ不飽和脂肪酸の酸化様式（エキソパーオキシドラジカル
体を中間体にとる）をとることが予想された。同様に1，5一パーオキシド架橋を
726
有するハナルピノールの生成経路［100］を比較して予想前駆体を想定しその検
索を行った結果、低極性部よりカロター1，4一ジエンアルデヒドを見い出した。こ
の、前駆体と考えられたセスキテルペンアルデヒドは空気中で極めて不安定な
性質を有し、幾つかのパーオキシド試薬陽性化合物へと酸化された。そのうち
の二化合物は比較的安定な結晶として得られ、それらはその立体化学も含めて
ルゴサールA、ルゴス酸に一致した。これにより、カロター1，4一ジエンアルデヒ
ドはル：ゴサールAの直接の前駆体で有ることを確認した。その酸化経路につい
ては、中間体の捕足による解明を試み、1，5一パーオキシド架橋をもち、C－2位
にヒドロパーオキシドの置換した中間体を高収率で得ることができたことから、
C－3位の活性メチレンから水素の引き抜きを引金として三重項酸素二分子付加
を伴うラジカル反応を提出した。
ハマナス葉組織中におけるカ出撃ンパーオキシドの代謝分解は、それらの葉
の成長熟成に伴うドラスティックな消長から、酵素レベルでの制御が働くと予
想された。ハマナス葉中脳ロタノイドの最大プールであるルゴス酸の代謝産物
はそのパーオキシド架橋の変換、修飾が主な経路であると考えられたため、幾
つかの化学変換物を標準として酸性画面を検索した結果、ルゴス酸の酸処理物、
アルカリ処理物および還元体に対応する成分を天然物として単離することがで
’きた。特に、酸処理で生成するケタール体は完全に老化あるいは黄化した葉に
比較的高濃度で存在し、これが組織中で安定な物であることが示唆された。
ルゴサールA型のパーオキシド（アルデヒド基の還元によるアルコール誘導
体を含む）では、それぞれ酸あるいはアルカリによりエンドパーオキシド基の
変換が容易に起こった。その反応成生物は、従来知られているエンドパーオキ
シドの変換反応と異なり、その主原因はこれらカロタンパーオキシドのコンフ
ォメーションによると考えられた。
ハマナス葉山にはこれらの他に多様なカロタンセスキテルペンが微量成分と
して含まれており、それらはカロター1，4一ジエンアルデヒドの異性体がさまざま
な修飾（酸素化、脱水素等）を受けて生成したもの．と考えられる。
ハマナス葉にはまた、カロタン以外のセスキテルペンも比較的高含有量で含
まれていた。そのなかで100mg／kg以上の含有量を示すピサボラン二二スキテ
ルペンについてその構造を絶対配置を含めて決定し、これをピサボロザオール
Aと命名した。この化合物は、天然物としては珍しいC－7が酸化されたピサボ
ランであり、最近Bohhannらのグループによって相次いで見出されているキ
ク科のピサボランとはC－4位の絶対配置が異なり、従って右旋性を示す。本化
合物は二種の酸化物群を葉中代謝産物として与え、オレフィン側鎖に生じたエ
ポキシ環のγ一位のヒドロキシル基とのアルコーリシスによるテトラヒドロフラ
ン誘導体およびオレフィン部分に一重項酸素分子が攻撃して生成するヒドロバ
ーオキシド誘導体として面長出物中に見出される。特に後者のグループは、生
成量こそ微量ではあるがハマナス組織が強い酸化条件下にあることを示すもの
であった。
さらに、カロタンとピサボランの関係も興味をひいた。これらは共にcis一
727
trans一ファルネシルピロフォスフェートを前駆体として生合成される。ともに
ファルネソールの13位に由来するメチル基が酸化されている事実は、これら
二種のセスキテルペンの密接な関連性を示すものと考えられる。
ピサボラン型に加えて、アコラン型セスキテルペンも見いだされた。この骨
格は、さきのセスキテルペン類と同じく cis－trans一ファルネシルビ。ロフオスフ
ェートから生合成されるが、ファルネソールのC－13メチル由来の炭素が酸化
されておらず、ハマナス成分としては希な修飾様式を示した。
ルゴサールAの抗菌活性は、TLCバイオオートグラフィーあるいはペーパー
ディスク法で詳しく検討した。本化合物は、一部の糸状菌（Cladosporlum
herbarumおよびイネイモチ病菌を含む）、酵母あるいはグラム陽性菌にある程
度の抗菌活性を認め、特にイモチ病菌に対しては市販農薬のEDDPに匹敵する
抗菌活性を示した。細胞毒性はP－398「を使った試験でLD5g諸0．21μg／副を
示した。いくつかの誘導体についての構造活性相関では、パーオキシド架橋お
よびアリル水酸基が活性発現に必須であること、不飽和アルデヒド基は必ずし
も活性発現には必要無いがこれがカルボキシル基へ酸化されると活性が完全に
消失すること、この活性はメチル化によって回復：することがわかった。この結
果は、生体内でのカロタンの代謝、生成および意義を考察するうえで非常に有
用な情報となった。ピサボロザオールAはTLCバイオオートグラフィーでル
ゴサールAの1／10程度の抗菌性を示したにすぎなかった。
一連の化学的知見に加えて、いくつかの生理的知見も得られた。最も活性の
高い開花期には1kgハマナス葉中におよそ700 mgものカロタンパーオキシ
ドが含まれるが、これらのほとんどが葉裏面のトリコーム（突起様構造）に含
まれている実験：事実をいくつか見出している。また、・傷害部から浸出するルゴ
サールAの相対量が著しく高いことも示唆された。これらの観察は、ルゴサー
ルAが実際に葉組織中で糸状菌感染に対する防御因子となっている可能性を示
しており［9］、更に詳しい検証が望まれる。また、カ中門ー1，4一ジエンァルデヒ
ドがハマナス葉組織中、特にシラコイド膜上で抗酸化剤として機能しているこ
とも考えられる。この場合、ルゴサールAは酸化老廃物にしか過ぎず、ある部
分へ集積されたのち糸状菌に対する防御物質として転用されると考えられる。
これは極めて効率の良い仕組みであり、ハマナスが夏季の短い高緯度で自生し
ている事実を考え併せれば、炭化水素の効率的利用型防御機構という点で興味
深い例であるかもしれない。
・0舳δノCHO
・ρ’δノ。。OH
OH
OH
ルゴス酸
ルゴサールA
HO，
＼
臼
’’”
、
＼
5
CHO
8
カロター1，4一ジエンアルデヒド
728
COOMe
ピサボロザオールA
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