Semisynthesis and acetylcholinesterase inhibitory activity of stemofoline alkaloids and analogues

Article abstract of DOI:10.1021/np100137h

Semisynthesis of the known Stemona alkaloids oxystemofoline (7) and methoxystemofoline (8) has been achieved starting from (11Z)-1′,2′- didehydrostemofoline (6), which confirmed their structures and absolute configurations. The synthesis of (1′R)-hydroxys

Full text of DOI:10.1021/np100137h

J. Nat. Prod. 2010, 73, 935–941
935
Semisynthesis and Acetylcholinesterase Inhibitory Activity of Stemofoline Alkaloids and
Analogues
Kwankamol Sastraruji,† Thanapat Sastraruji,† Stephen G. Pyne,*,† Alison T. Ung,*,§ Araya Jatisatienr,‡ and Wilford Lie†
School of Chemistry, UniVersity of Wollongong, Wollongong, New South Wales, 2522, Australia, Department of Chemistry and Forensic
Science, UniVersity of Technology Sydney, Sydney, 2007, Australia, and Department of Biology, Chiang Mai UniVersity,
Chiang Mai 50202, Thailand
ReceiVed March 3, 2010
Semisynthesis of the known Stemona alkaloids oxystemofoline (7) and methoxystemofoline (8) has been achieved starting
from (11Z)-1′,2′-didehydrostemofoline (6), which confirmed their structures and absolute configurations. The synthesis
of (1′R)-hydroxystemofoline (9) helped establish this compound as a natural product from Stemona aphylla. (1′S)-
Hydroxystemofoline (10) and a number of related analogues were also prepared. In a TLC bioautographic assay, 9 was
found to be the most active acetylcholinesterase inhibitor, but it was not as active as galanthamine.
The Stemona family of more than 80 alkaloids has been classified
by Pilli into eight different structural groups.1 The pyrrolo[1,2-
a]azepine (5,7-bicyclic A,B ring system) nucleus is common to all
compounds in six of these groups, while a pyrido[1,2-a]azepine
A,B ring system (6,7-bicyclic A,B ring system) is found in the more
recently discovered stemocurtisine group of Stemona alkaloids.1,2
A miscellaneous group comprising five alkaloids has also been
identified.1 Greger has classified the Stemona alkaloids into three
skeletal types based on their proposed biosynthetic origins.3 We
recently reported the semisynthesis of (3′R)-stemofolenol (1), (3′S)-
stemofolenol (2), methylstemofoline (3), and (3′S)-hydroxystemo-
foline (4) and the unnatural analogue (3′R)-hydroxystemofoline (5)
from (11Z)-1′,2′-didehydrostemofoline (6). This study allowed for
the first access to diastereomerically enriched samples of these
compounds in quantities sufficient to allow testing of their acetyl-
cholinesterase (AChE) inhibitory activities.4 This paper reports the
semisynthesis of the known Stemona alkaloids oxystemofoline (7)5
and methoxystemofline (8),5 which confirmed their structures and
resolved the controversy about their absolute configurations.5,6 We
also disclose the synthesis of (1′R)-hydroxystemofoline (9), which
we have now discovered is a natural product, (1′S)-hydroxystemo-
foline (10), and a number of related analogues. The inhibitory
activities of these compounds against AChE is also reported.
crystallographic analysis.8 A Stemona alkaloid named parviste-
moninol, which had the same specific rotation as oxystemofoline,
was reported to have the structure enantiomeric to 7.6 It is now
clear that parvistemoninol and oxystemofoline are the same, and
we suggest that the name parvistemoninol no longer be used.
Synthesis of methoxystemofoline (8) (Scheme 3) was achieved
using a method similar to that used for synthesis of 7 except that
the sulfone 25 (Scheme 2) was used for the first step. The yield of
the E-alkene 26 was very low (E/Z ) >99:<1).
Results and Discussion
For the synthesis of oxystemofoline (7) (Scheme 1), (11Z)-1′,2′-
didehydrostemofoline (6) was converted to the known aldehyde
17 as we described previously.4 In order to form the trans-alkene
18, a modified Julia olefination reaction7 was employed using the
sulfone 22 (Scheme 2). However, while the E-selectivity was high
(E/Z ) >99:<1) the yield of 18 was low (33%) due to the high
sensitivity of aldehyde 17 to the strongly basic conditions. TBS-
deprotection of compound 18 gave the homoallylic alcohol 19,
which was then regioselectively hydrogenated to give 7. The specific
rotation of 7 ([R]D22 +297.8 (c 0.52, CH3OH); lit.5 [R]2D0 +106.0 (c
0.1, CH3OH)) was of the same sign but larger in magnitude than
that reported for the natural product. The 1H and 13C NMR data of
Scheme 1. Synthesis of Oxystemofoline (7)
7 proved to be identical to the natural product,5 except for the 13
C
NMR signals for C-6 and C-1′, which were originally incorrectly
assigned. Thus, this synthesis confirmed the proposed structure of
the natural product and established its absolute configuration since
that of the stemofoline · HBr salt has been established by X-ray
* To whom correspondence should be addressed. Tel: +61-24221-3511.
Fax: +61-24221-4287. E-mail: spyne@uow.edu.au.
† University of Wollongong.
§ University of Technology Sydney.
‡ Chiang Mai University.
10.1021/np100137h  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/23/2010

936 Journal of Natural Products, 2010, Vol. 73, No. 5
Scheme 2. Synthesis of Sulfones 22 and 25
Sastraruji et al.
its synthesis from 17 using the chiral borane reagent lIpc2Ball, which
is generally stereoselective for the (R)-homoallylic alcohol prod-
uct.10 This reaction gave a mixture of 27 and 28 in a ratio of 9:1,
in a yield of 77%. When dIpc2Ball was employed, a mixture of 27
and 28 was obtained in a ratio of 14:86, in a yield of 69%. The
pure alcohols 27 and 28 were hydrogenated to give (1′R)-
hydroxystemofoline (9) and (1′S)-hydroxystemofoline (10) in
relatively low yields (36-42%) due to the formation of side
products arising from reduction of the C-11-C-12 double bond
(Scheme 5). Surprisingly, (1′R)-hydroxystemofoline (9) was identi-
cal by NMR spectroscopy to the alkaloid that we had isolated
previously from the root extracts of Stemona aphylla and which
we had incorrectly reported as (2′S)-hydroxystemofoline.11 Thus,
(1′R)-hydroxystemofoline is also a natural product.
Scheme 5. Synthesis of (1′R)-Hydroxystemofoline (9) and
(1′S)-Hydroxystemofoline (10)
Scheme 3. Synthesis of Methoxystemofoline (8)
Dihydroxylation of stemofoline (11),4 using catalytic
K2OsO4 ·2H2O and stoichiometric NMO, gave the diol 31, which
was a single diastereomer by NMR analysis. Oxidative cleavage
of diol 31 with freshly prepared NaIO4 on silica gel4 gave the A,B,C
ring core structure 32 (Scheme 6).
Hydrogenation of 26 gave methoxystemofoline (8) (Scheme 3).
The specific rotation of 8 ([R]2D5 +247 (c 0.29, CH3OH); lit.5 [R]2D1.6
+75.6 (c 0.037, CH3OH)) was of the same sign but much larger in
magnitude compared to that reported for the natural product. Such
alkaloids typically have specific rotations around 200.1 The 1H and
13C NMR data of 8 agreed with those of the natural product
Scheme 6. Synthesis of the A,B,C Ring Core Structure 32
methoxystemofoline5 except for the incorrect assignment of the 13
C
NMR signals for C-6 and C-1′.
Allylation of 174 using indium powder and allyl bromide, with
sonication,9 gave an inseparable mixture of the diastereomeric
alcohols 27 and 28 in a ratio of 65:35 (Scheme 4). Their acetate
derivatives (29 and 30), however, were readily separated. When
the acetate groups were removed under transesterification conditions
using MeOH/Na2CO3, methanol Michael addition products at
C-11-C-12 were also formed. Hydrolysis using LiOH in aqueous
THF was more successful in providing the desired alcohols 27 and
28 (Scheme 4). The configuration of 27 at C-1′ was assigned from
The Wittig reaction was utilized for synthesis of enal 33a from 17 using
(triphenylphosphoranylidene)acetaldehyde.12 This reaction also gave dienal
33b and trienal 33c as a result of consecutive Wittig reactions. These
aldehydes were difficult to separate from each other and the triphenylphos-
phine oxide byproduct. Thus, the mixture was reduced with NaBH4/MeOH
to give a mixture of alcohols 34a-c, which was separated by PTLC to
give pure samples for biological testing (Scheme 7).
Scheme 4. a
Scheme 7. Synthesis of Alcohols 34a, 34b, and 34c
a Reagents and conditions: (a) (i) indium, allyl bromide, THF/aq NH4Cl (5:
l
2), sonication, 2-3 h, 27:28 ) 65:35; (ii) Ipc2Ball, THF, 0 °C, 2 h, 27:28 )
9:1, 77% yield; (iii) dIpc2Ball, THF, 0 °C, 2 h, 27:28 ) 14:86, 69% yield; (b)
Ac2O, pyridine, rt, 4 h, 29: 44% yield (2 steps), 30: 28% yield (2 steps); (c)
LiOH, THF/H2O (2:1), rt, 16 h, 27: 61% yield, 28: 73% yield.

Semisynthesis of Stemofoline Alkaloids
Journal of Natural Products, 2010, Vol. 73, No. 5 937
Table 1. Minimum Amount of Sample Found to Inhibit AChE
as Indicated by a White Zone of Inhibition
substituted stemofoline derivatives 7, 8, 19, and 26 showed
relatively weak activities. Of the truncated side-chain derivatives,
the hydroxymethyl derivative 14 showed relatively high activity
(10 ng), while the methyl derivative 3 and the 3′-hydroxy-1-
propenyl derivative 34a had much reduced activities (100 and 50
ng, respectively).
In summary, semisynthesis of the known Stemona alkaloids
oxystemofoline (7) and methoxystemofline (8) has been achieved
starting from (11Z)-1′,2′-didehydrostemofoline (6), which confirmed
their structures and absolute configurations. The synthesis of (1′R)-
hydroxystemofoline (9) helped to establish this compound as a
natural product from S. aphylla. (1′S)-Hydroxystemofoline (10) and
a number of related analogues were also prepared. In an assay as
AChE inhibitors, 9 was the most active. In general, analogues with
an OH at C-1′ or C-2′ were more active than analogues with an
OH at C-3′ or C-4′, although the C-3′ hydroxy compound 4 was
an exception. The configuration of the carbinol center was also
important for activity, except for the C-2′ epimeric pair of
compounds, 12 and 13, which were equipotent. Studies are
continuing on the insecticidal activity of these alkaloids on insects
of importance to the agricultural industry.
Experimental Section
General Experimental Procedures. These were as described
previously.16 All 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra
were determined in CDCl3 solution unless otherwise indicated. 1H NMR
assignments were achieved with the aid of gCOSY and, in some cases,
NOESY experiments. 13C NMR assignments were based upon DEPT,
gHSQC, and gHMBC experiments. All compounds were homogeneous
by TLC analysis and judged to be of >95% purity based upon 1H NMR
analysis.
Plant Material. The known starting material (11Z)-1′,2′-didehy-
drostemofoline (6) was isolated from the unidentified Stemona species
that we reported earlier.16 Roots of this Stemona species were collected
at Amphur Mae Moh, Lampang, Thailand, in November 2007. The
plant material was identified by Mr. James Maxwell (Department of
Biology, Chiang Mai University) as the same species that we had
studied previously.16 Voucher specimen number 25375 was deposited
at the Herbarium of the Department of Biology, Chiang Mai University.
Extraction and Isolation. The dry, ground root of the Stemona
species (935 g) was extracted with 95% EtOH (4 × 3000 mL) over 4
days at rt. The ethanolic solution was evaporated to give a dark brown
residue (148 g). The extract was partitioned between MeOH/H2O (1:
1) and CH2Cl2. The organic extract was dried over MgSO4 and
concentrated in vacuo to give a dark brown residue (20 g). A portion
of this material (2.50 g) was chromatographed on silica gel (100 mL)
with gradient elution from CH2Cl2 to CH2Cl2/MeOH (95:5) to give
(11Z)-1′,2′-didehydrostemofoline (6)16 as a yellow-brown gum (1.48
g, 59% w/w).
The insecticidal activity shown by the root extracts of Stemona
plants has been associated with the acetylcholinesterase (AChE)
inhibitory activities of their alkaloid components.13,14 Compounds
7-16, 19, 26-32, and 34a-c were therefore screened by TLC
bioautography for their AChE inhibitory activities using the
qualitative method of Hostettmann et al.15 and galanthamine as a
positive control. The results are shown in Table 1. The inhibitory
activities of the previously tested compounds 1-64 are included.
In our earlier study (11Z)-1′,2′-didehydrostemofoline (6) was the
most active inhibitor, with a minimum inhibitory requirement of 5
ng (0.013 nmol).4 In this study (1′R)-hydroxystemofoline (9)
showed a slightly higher inhibitory activity at 5 ng (0.012 nmol),
while its 3′,4′-didehydro deriviative (27) and its (1′S)-epimer (10)
were less active (minimum inhibitory requirements of 10 ng, Table
1). Compound 9 was the most active of the compounds reported
here. Stemofoline (11) was slightly less active than 6 and 9, while
its 11,12-dihydroxy derivative (31) and the tricyclic derivative (32),
which is missing the γ-butyrolactone ring found in 11, were 50
and 10 times less active, with minimum inhibitory requirements of
500 and 100 ng, respectively. Other compounds with activity similar
to that of 11 (which all had a minimum inhibitory requirement of
10 ng) included the trien-ol 34c, the dien-ol 34b, the C-1′ and C-2′
alcohols 12, 13, 14, and 27, and the 1′,2′-diol 15. The C-3′ hydroxy
analogues, (3′S)-4 and its 3′-epimer, (3′R)-5, showed a 10-fold
difference in activities with minimum inhibitory requirements of
10 and 100 ng, respectively. The C-4′ hydroxy- and methoxy-
(11Z)-1′r,2′r- and (11Z)-1′ꢀ,2′ꢀ-Dihydroxystemofoline (15 and 16).
Compound 16 was a minor component from the synthesis of the known
diol 154 using AD-mix-R (4.55 g, Aldrich), methanesulfonamide (617
mg, 6.49 mmol), and 6 (1.25 g, 3.25 mmol). The crude product was
purified by column chromatography (CC) with gradient elution from
CH2Cl2 to CH2Cl2/MeOH (9:1) to give 154 (889 mg, 2.12 mmol, 65%
yield) as a white solid and 16 (28 mg, 0.07 mmol, 2% yield). These
compounds were also prepared from a similar method using AD-mix-ꢀ
(474 mg, Aldrich), methanesulfonamide (64 mg, 0.68 mmol), and 6
(130 g, 0.339 mmol) to give 15 (9.9 mg, 0.024 mmol, 7% yield) and
1
16 (68.9 mg, 0.164 mmol, 48% yield). The H and 13C NMR spectra
of 15 from both methods agreed with those previously reported.4 16:
colorless gum, [R]2D3 +251 (c 1.0, CHCl3); IR νmax 3380, 2960, 2919,
1
2873, 1741, 1680 cm-1; H NMR δ 4.68 (s, 1H, H-2), 4.13 (s, 3H,
O-CH3), 3.75 (t, J ) 7.5 Hz, 1H, H-2′R), 3.52 (s, 1H, H-1′R), 3.49 (br
s, 1H, H-9a), 3.10 (d, J ) 6.5 Hz, 1H, H-7), 3.12-3.07 (m, 1H, H-10),
3.03-2.94 (m, 2H, H-5), 2.05 (s, 3H, H-16), 2.02-1.95 (m, 1H, H-6a),
1.93 (d, J ) 12.5 Hz, 1H, H-1a), 1.88-1.84 (m, 1H, H-6b), 1.82 (dd,
J 9.5 Hz, 2.5 Hz, 1H, H-9), 1.78 (d, J ) 12.5 Hz, 1H, H-1b), 1.67-1.50
(m, 2H, H-3′), 1.36 (d, J ) 6.5 Hz, 3H, H-17), 0.95 (t, J ) 7.5 Hz,
3H, H-4′); 13C NMR δ 170.1 (C-15), 163.1 (C-13), 148.8 (C-11), 128.0
(C-12), 112.5 (C-8), 98.6 (C-14), 85.8 (C-3), 76.4 (C-2), 72.3 (C-1′),
69.9 (C-2′), 61.5 (C-9a), 59.0 (O-CH3), 48.9 (C-7), 48.7 (C-5), 47.8

938 Journal of Natural Products, 2010, Vol. 73, No. 5
Sastraruji et al.
(C-9), 34.7 (C-10), 33.0 (C-1), 28.2 (C-3′), 26.9 (C-6), 18.4 (C-17),
10.4 (C-4′), 9.2 (C-16); ESIMS m/z 420.0 (100%) [M + H]+, 421.2
(15%), 422.1 (5%); HRESIMS m/z 420.2008 [M + H]+, calcd for
C22H30NO7 420.2022.
174 (54 mg, 0.150 mmol), LiHMDS (0.16 mL of 1 M in THF), and
sulfone 25 (51 mg, 0.180 mmol) to give 26 (9 mg, 0.022 mmol, 15%
yield) as a yellow gum after purification by CC elution with EtOAc:
Rf ) 0.23 in MeOH/EtOAc (1:4); [R]2D5 +206.2 (c 0.71, CHCl3); IR
1
1′,2′-Didehydro-4′-(tert-butyldimethylsiloxyl)stemofoline (18). A
mixture of sulfone 22 (157 mg, 0.424 mmol) in dry DMF (10 mL)
under a N2 atmosphere was cooled to -60 °C, LiHMDS (0.39 mL of
1 M in THF) was added dropwise, and the solution was stirred at
-60 °C for 2 h. The mixture was transferred via a cannula to a flask
containing a solution of the aldehyde 174 (127 mg, 0.353 mmol) in
dry DMF (10 mL) at -60 °C under N2. The reaction warmed to rt
over 20 h before addition of a saturated aqueous solution of NaHCO3
(10 mL). The mixture was extracted with diethyl ether (3 × 20 mL),
and the extract was washed with brine and dried (MgSO4). The
concentrated residue was purified by CC using gradient elution from
CH2Cl2 to CH2Cl2/MeOH (98:2) to give the trans-alkene product 18
(61 mg, 0.117 mmol, 33% yield): Rf ) 0.50 in MeOH/EtOAc (1:4);
[R]2D2 +179.6 (c 1.0, CHCl3); IR νmax 2955, 2924, 2883, 2852, 1746,
1621 cm-1; 1H NMR δ 5.74 (dt, J ) 15.5 Hz, 7.0 Hz, 1H, H-2′), 5.58
(d, J ) 15.5 Hz, 1H, H-1′), 4.21 (br s, 1H, H-2), 4.14 (s, 3H, O-CH3),
3.64 (t, J ) 7.0 Hz, 2H, H-4′), 3.50 (br s, 1H, H-9a), 3.13-3.07 (m,
2H, H-5a, H-10), 3.00-2.95 (m, 1H, H-5b), 2.86 (d, J ) 6.0 Hz, 1H,
H-7), 2.28 (q, J 7.0 Hz, 2H, H-3′), 2.07 (s, 3H, H-16), 1.95 (d, J 12.5
Hz, 1H, H-1a), 1.88-1.84 (m, 2H, H-6a, H-9), 1.84-1.76 (m, 2H,
H-1b, H-6b), 1.38 (d, J ) 6.5 Hz, 3H, H-17), 0.88 (s, 9H, O-Si
(CH3)2C(CH3)3), 0.04 (s, 6H, O-Si (CH3)2C(CH3)3); 13C NMR δ 169.8
(C-15), 162.9 (C-13), 148.5 (C-11), 129.7 (C-1′), 128.4 (C-2′), 128.0
(C-12), 112.9 (C-8), 98.7 (C-14), 83.2 (C-3), 80.8 (C-2), 62.8 (C-4′),
61.0 (C-9a), 59.0 (O-CH3), 51.4 (C-7), 48.2 (C-5), 47.8 (C-9), 36.1
(C-3′), 34.7 (C-10), 33.0 (C-1), 27.1 (C-6), 26.1 (O-Si(CH3)2C(CH3)3),
18.5 (C-17, O-Si(CH3)2C(CH3)3), 9.3 (C-16), -5.1 (O-Si
(CH3)2C(CH3)3); ESIMS m/z 516.3 (100%) [M + H]+, 517.3 (30%),
518.3 (10%); HRESIMS m/z 516.2768 [M + H]+, calcd for
C28H42NO6Si 516.2781.
1′,2′-Didehydro-4′-hydroxystemofoline (19). To a solution of 18
(32.7 mg, 0.064 mmol) in MeOH (2.0 mL) at rt was added 10% aqueous
HCl (1.0 mL), and the solution was left to stir for 30 min. The reaction
mixture was evaporated to a white residue, which was partitioned
between saturated aqueous NaHCO3 and CH2Cl2. The layers were
separated and the aqueous layer was extracted with CH2Cl2 (3 × 10
mL). The CH2Cl2 extract was washed with brine and dried (MgSO4).
The concentrated residue was purified by CC using gradient elution
from EtOAc to EtOAc/MeOH (90:10) to give the alcohol 19 (22.2 mg,
0.055 mmol, 87% yield): Rf ) 0.11 in MeOH/EtOAc (1:4); [R]2D3 +240.8
(c 1.0, CHCl3); IR νmax 3740, 2960, 2919, 2847, 1743, 1683, 1618
cm-1; 1H NMR (300 MHz) δ 5.73 (dt, J ) 15.3 Hz, 6.6 Hz, 1H, H-2′),
5.62 (d, J ) 15.9 Hz, 1H, H-1′), 4.21 (br s, 1H, H-2), 4.13 (s, 3H,
O-CH3), 3.64 (t, J ) 6.6 Hz, 2H, H-4′), 3.49 (br s, 1H, H-9a), 3.10-3.03
(m, 2H, H-5a, H-10), 3.02-2.97 (m, 1H, H-5b), 2.84 (d, J ) 5.7 Hz,
1H, H-7), 2.32 (q, J ) 6.3 Hz, 2H, H-3′), 2.05 (s, 3H, H-16), 1.94 (d,
J ) 12.0 Hz, 1H, H-1b), 1.88-1.84 (m, 2H, H-6a, H-6b), 1.83-1.79
(m, 1H, H-9), 1.79-1.74 (m, 1H, H-1a), 1.36 (d, J ) 6.3 Hz, 3H,
H-17); 13C NMR (75 MHz) δ 169.9 (C-15), 162.9 (C-13), 148.4 (C-
11), 130.6 (C-1′), 128.0 (C-12), 127.9 (C-2′), 112.8 (C-8), 98.7 (C-
14), 83.2 (C-3), 80.6 (C-2), 61.9 (C-4′), 61.0 (C-9a), 59.0 (O-CH3),
51.4 (C-7), 48.1 (C-5), 47.7 (C-9), 35.9 (C-3′), 34.7 (C-10), 33.0 (C-
1), 27.0 (C-6), 9.2 (C-16); ESIMS m/z 402.2 (100%) [M + H]+, 403.2
(20%), 404.2 (10%); HRESIMS m/z 402.1898 [M + H]+, calcd for
C22H28NO6 402.1917.
νmax 2957, 2929, 2868, 1745, 1621, 1117 cm-1; H NMR δ 5.75 (dt,
J ) 15.5 Hz, 7.0 Hz, 1H, H-2′), 5.60 (d, J ) 16.0 Hz, 1H, H-1′), 4.22
(br s, 1H, H-2), 4.13 (s, 3H, O-CH3), 3.51 (br s, 1H, H-9a), 3.41 (t,
J ) 6.5 Hz, 2H, H-4′), 3.33 (s, 3H, 4′-O-CH3), 3.12-3.06 (m, 2H,
H-5a, H-10), 3.00-2.95 (m, 1H, H-5b), 2.86 (d, J ) 6.0 Hz, 1H, H-7),
2.34 (q, J ) 7.0 Hz, 2H, H-3′), 2.07 (s, 3H, H-16), 1.95 (d, J ) 12.0
Hz, 1H, H-1a), 1.83-1.77 (m, 4H, H-1b, H-6, H-9), 1.37 (d, J ) 6.5
Hz, 3H, H-17); 13C NMR δ 169.8 (C-15), 163.0 (C-13), 148.5 (C-11),
129.6 (C-1′), 128.2 (C-2′), 128.1 (C-12), 112.9 (C-8), 98.8 (C-14), 83.3
(C-3), 80.7 (C-2), 72.2 (C-4′), 61.0 (C-9a), 59.0 (O-CH3), 58.7 (4′-O-
CH3), 51.4 (C-7), 48.2 (C-5), 47.8 (C-9), 32.8 (C-3′), 34.7 (C-10), 33.0
(C-1), 27.0 (C-6); ESIMS m/z 415.8 (100%) [M + H]+, 416.9 (20%),
417.9 (5%); HRESIMS m/z 416.2089 [M + H]+, calcd for C23H30NO6
416.2073.
Methoxystemofoline (8). Compound 8 was prepared using a method
similar to the synthesis of 7, from compound 26 (10 mg, 0.025 mmol)
and PdCl2 (3 mg, 30% w/w) over a 1 h period. The crude product was
purified by CC with gradient elution from EtOAc to EtOAc/MeOH
(95:5) to give 8 (4.4 mg, 0.010 mmol, 43% yield) as a yellow gum:
Rf ) 0.16 in MeOH/EtOAc (1:4); [R]2D5 +247.4 (c 0.29, CH3OH); lit.5
[R]2D1.6 +75.6 (c 0.037, CH3OH). The NMR data agree with those of
the natural product5 except for the assignment of the 13C NMR signals
for C-6 and C-1′, which were incorrectly assigned. ESIMS m/z 417.9
(100%) [M + H]+, 418.9 (25%), 419.9 (10%); HRESIMS m/z 418.2233
[M + H]+, calcd for C23H32NO6 418.2230.
(1′R)- and (1′S)-Hydroxy-3′,4′-didehydrostemofoline (27 and 28).
To a solution of aldehyde 174 (94 mg, 0.261 mmol) in THF/saturated
aqueous NH4Cl (5:2, 6 mL) was added indium powder (60 mg, 0.52
mmol) and allyl bromide (135 µL, 1.56 mmol). The reaction flask was
sealed and sonicated for 3 h. The THF was evaporated to give a white
residue, which then was partitioned between CH2Cl2 and aqueous
NaHCO3. The layers were separated and the aqueous layer was extracted
with CH2Cl2 (3 × 10 mL). The CH2Cl2 was washed with brine and
dried (MgSO4). The crude mixture of 27 and 28 (81 mg) was put
through the next step without purification.
(1′R)- and (1′S)-Hydroxy-3′,4′-didehydrostemofoline (27 and 28).
To a solution of aldehyde 174 (35 mg, 0.099 mmol) in dry THF (3
l
mL) at 0 °C under a N2 atmosphere was added Ipc2Ball (0.49 mL of
1 M in pentane, 0.49 mmol) and was left to stir at 0 °C for 2 h. The
reaction mixture was quenched with MeOH (5 mL), and 10% aqueous
HCl (5 mL) was added. The aqueous solution was washed with CH2Cl2
(3 × 10 mL). The aqueous phase was basified with aqueous NaOH
and then extracted with CH2Cl2 (3 × 10 mL). The CH2Cl2 extract was
washed with brine and dried (MgSO4). The concentrated residue was
purified by CC with gradient elution, EtOAc to EtOAc/MeOH (95:5),
to give a mixture of 27 and 28 (dr ) 9:1, 31 mg, 0.076 mmol, 77%
yield) as a white gum.
The compounds were also prepared from 174 (35 mg, 0.099 mmol)
d
using the above procedure except that Ipc2Ball (0.49 mL of 1 M in
pentane, 0.493 mmol) was used. This gave a mixture of 27 and 28
(dr ) 14:86, 27 mg, 0.068 mmol, 69% yield) as a white gum.
(1′R)- and (1′S)-Acetyl-3′,4′-didehydrostemofoline (29 and 30).
A mixture of 27 and 28 (81 mg) was dissolved in pyridine (2 mL),
and acetic anhydride (2 mL) was added at rt. The reaction mixture
was left to stir for 4 h before the addition of saturated aqueous NaHCO3
(5 mL), and the mixture was extracted with CH2Cl2 (3 × 10 mL). The
CH2Cl2 extract was washed with brine, dried (MgSO4), and concentrated
in vacuo. The crude product was purified by CC using gradient elution
(petroleum ether/EtOAc (1:1) to EtOAc) to give 29 (51 mg, 0.115
mmol, 44% yield over 2 steps) as a pale yellow gum as a major product
and 30 (32 mg, 0.073 mmol, 28% yield over 2 steps) as a pale yellow
gum as a minor product. 29: Rf ) 0.46 in MeOH/EtOAc (1:4); [R]D24
+226.5 (c 1.0, CHCl3).; IR νmax 2924, 2858, 1741, 1618, 1372, 1234
Oxystemofoline (7). To a solution of alcohol 19 (10.9 mg, 0.027
mmol) in dry MeOH (2 mL) at rt was added PdCl2 (2.2 mg, 20% w/w),
and the flask was flushed with N2 for 10 min before left to stir under
a H2 atmosphere (balloon) for 18 h. The flask was flushed with N2,
and the solution was filtered through Celite and then washed with
MeOH. The filtrate was dried (MgSO4) and concentrated in vacuo. The
crude product was purified by CC eluting with EtOAc to give
oxystemofoline 7 (7.3 mg, 0.018 mmol, 66% yield): Rf ) 0.39 in
MeOH/CH2Cl2 (1:9); [R]D22 +297 (c 0.52, CH3OH); lit.8 [R]2D0 +106.0
(c 0.1, CH3OH). The NMR data agree with those of the natural product5
except the assignment of the 13C NMR signals for C-6 and C-1′, which
were incorrectly assigned. ESIMS m/z 403.8 (100%) [M + H]+, 404.9
(20%), 405.8 (13%); HRESIMS m/z 404.1977 [M + H]+, calcd for
C22H30NO6 404.2073.
1
cm-1; H NMR δ 5.78-5.70 (m, 1H, H-3′), 5.19 (s, 1H, H-1′), 5.14
(s, 1H, H-(4′E)), 5.08 (t, J ) 12.5 Hz, 1H, H-(4’Z)), 4.45 (br s, 1H,
H-2), 4.13 (s, 3H, O-CH3), 3.46 (br s, 1H, H-9a), 3.26-3.20 (m, 1H,
H-5a), 3.09-3.03 (m, 1H, H-10), 3.01-2.96 (m, 1H, H-5b), 2.85 (d,
J ) 6.0 Hz, 1H, H-7), 2.43-2.35 (m, 1H, H-2′), 2.06 (s, 3H, H-16),
2.03 (s, 3H, 1′-OCOCH3), 1.93 (d, J ) 12.5 Hz, 1H, H-1a), 1.89-1.87
(m, 2H, H-6), 1.83 (ddd, J ) 18.0 Hz, 10.5 Hz, 3.5 Hz, 1H, H-9), 1.63
(d, J ) 12.0 Hz, 1H, H-1b), 1.36 (d, J ) 6.5 Hz, 3H, H-17); 13C NMR
1′,2′-Didehydro-4′-methoxystemofoline (26). Compound 26 was
prepared using a method similar to the synthesis of 18, from aldehyde

Semisynthesis of Stemofoline Alkaloids
Journal of Natural Products, 2010, Vol. 73, No. 5 939
δ 170.5 (1′-OCOCH3), 169.8 (C-15), 162.9 (C-13), 148.2 (C-11), 133.3
(C-3′), 128.1 (C-12), 118.3 (C-4′), 112.7 (C-8), 98.8 (C-14), 85.4 (C-
3), 76.6 (C-2), 70.7 (C-1′), 60.9 (C-9a), 59.0 (O-CH3), 49.4 (C-7), 48.2
(C-5), 47.9 (C-9), 35.6 (C-2′), 34.6 (C-10), 33.2 (C-1), 27.4 (C-6), 21.2
(1′-OCOCH3), 18.4 (C-17), 9.3 (C-16); ESIMS m/z 443.9 (100%) [M
+ H]+, 444.9 (25%), 446.0 (5%); HRESIMS m/z 444.2011 [M + H]+,
calcd for C24H30NO7 444.2022. 30: Rf ) 0.59 in MeOH/EtOAc (1:4);
[R]2D4 +188.0 (c 1.0, CHCl3); IR νmax 2924, 1740, 1629, 1460, 1362,
with MeOH. The filtrate was dried (MgSO4) and concentrated in vacuo.
The crude product was purified by CC [gradient elution from CH2Cl2
to CH2Cl2/MeOH (98:2)] to give the alcohol 9 (4.1 mg, 0.010 mmol,
35% yield) as a yellow gum: Rf ) 0.19 in MeOH/EtOAc (1:4); [R]D23
+298.7 (c 0.34, CHCl3); IR νmax 3462, 2960, 2919, 2868, 1744, 1620
cm-1; 1H NMR (300 MHz) δ 4.47 (br s, 1H, H-2), 4.14 (s, 3H, O-CH3),
3.64-3.59 (m, 1H, H-1′b), 3.54 (br s, 1H, H-9a), 3.23-3.15 (m, 1H,
H-5a), 3.15-3.07 (m, 1H, H-10), 3.07-2.99 (m, 1H, H-5b), 2.80 (br
s, 1H, H-7), 2.07 (s, 3H, H-16), 1.98 (d, J ) 12.3 Hz, 1H, H-1a),
1.91-1.86 (m, 3H, H-6, H-9), 1.70-1.62 (m, 2H, H-1b, H-3′a),
1.55-1.47 (m, 2H, H-2′), 1.44-1.42 (m, 1H, H-3′b), 1.38 (d, J ) 6.6
Hz, 3H, H-17), 0.97 (t, J ) 7.2 Hz, 3H, H-4′); 13C NMR (75 MHz) δ
169.8 (C-15), 162.8 (C-13), 148.2 (C-11), 128.2 (C-12), 112.9 (C-8),
98.8 (C-14), 87.4 (C-3), 75.6 (C-2), 67.9 (C-1′), 61.1 (C-9a), 59.0 (O-
CH3), 48.2 (C-9), 48.1 (C-7), 47.5 (C-5), 34.5 (C-10), 34.2 (C-2′), 34.0
(C-1), 27.4 (C-6), 20.2 (C-3′), 18.5 (C-17), 14.3 (C-4′), 9.3 (C-16);
ESIMS m/z 404.2 (100%) [M + H]+, 405.2 (18%), 406.2 (10%);
HRESIMS m/z 404.2069 [M + H]+, calcd for C22H30NO6 404.2073.
(1′S)-Hydroxystemofoline (10). Compound 10 was prepared via a
method similar to the synthesis of 9, using alcohol 28 (17 mg, 0.042
mmol) and Pd/C (1.7 mg, 10% w/w) to give alcohol 10 (7 mg, 0.017
mmol, 40% yield) as a white gum: Rf ) 0.28 in MeOH/EtOAc (1:4);
[R]2D3 +219.0 (c 0.58, CHCl3); IR νmax 3183, 2924, 2854, 1744, 1615,
982 cm-1; 1H NMR δ 4.36 (br s, 1H, H-2), 4.14 (s, 3H, O-CH3), 3.73
(d, J ) 9.5 Hz, 1H, H-1′a), 3.58 (br s, 1H, H-9a), 3.23 (br s, 1H, H-5a),
3.12-3.10 (m, 1H, H-10), 3.07-3.03 (m, 1H, H-5b), 3.00 (d, J ) 6.0
Hz, 1H, H-7), 2.07 (s, 3H, H-16), 2.10-2.02 (m, 1H, H-6a), 1.99 (d,
J ) 12.5 Hz, 1H, H-1a), 1.91-1.87 (m, 2H, H-6b, H-9), 1.78-1.76
(m, 1H, H-1b), 1.65-1.57 (m, 2H, H-2′b, H-3′b), 1.38 (d, J ) 6.5 Hz,
3H, H-17), 1.41-1.30 (m, 2H, H-2′a, H-3′a), 0.95 (t, J ) 6.5 Hz, 3H,
H-4′); 13C NMR δ 169.8 (C-15), 162.9 (C-13), 148.2 (C-11), 128.2
(C-12), 112.8 (C-8), 98.8 (C-14), 87.0 (C-3), 77.4 (C-2), 70.9 (C-1′),
62.0 (C-9a), 59.0 (O-CH3), 49.4 (C-5), 47.9 (C-9), 47.7 (C-7), 34.6
(C-10), 35.2 (C-2′), 33.1 (C-1), 27.2 (C-6), 19.9 (C-3′), 18.4 (C-17),
14.1 (C-4′), 9.3 (C-16); ESIMS m/z 404.2 (100%) [M + H]+, 405.2
(20%), 406.2 (5%); HRESIMS m/z 404.2064 [M + H]+, calcd for
C22H30NO6 404.2073.
1
1234 cm-1; H NMR δ 5.76-5.68 (m, 1H, H-3′), 5.10 (s, 1H, H-1′),
5.05 (dd, J ) 16.5 Hz, 8.0 Hz, 2H, H-4′), 4.48 (br s, 1H, H-2), 4.13 (s,
3H, O-CH3), 3.48 (br s, 1H, H-9a), 3.18-3.12 (m, 1H, H-5a), 3.09-3.06
(m, 1H, H-10), 3.04-2.98 (m, 1H, H-5b), 2.71 (d, J ) 6.0 Hz, 1H,
H-7), 2.62 (ddd, J ) 14.0 Hz, 3.0 Hz, 1.5 Hz, 1H, H-2′a), 2.14-2.09
(m, 1H, H-2′b), 2.08 (s, 3H, 1′-OCOCH3), 2.06 (s, 3H, H-16),
2.05-2.01 (m, 1H, H-6b), 1.98 (d, J ) 12.5 Hz, 1H, H-1a), 1.85-1.80
(m, 1H, H-6a), 1.82 (dd, J ) 10.5 Hz, 4.5 Hz, 1H, H-9), 1.67 (d, J )
12.0 Hz, 1H, H-1b), 1.37 (d, J ) 7.5 Hz, 3H, H-17); 13C NMR δ 170.9
(1′-OCOCH3), 169.7 (C-15), 162.8 (C-13), 148.2 (C-11), 134.0 (C-3′),
128.1 (C-12), 117.9 (C-4′), 112.5 (C-8), 98.8 (C-14), 84.9 (C-3), 75.8
(C-2), 69.7 (C-1′), 61.2 (C-9a), 59.0 (O-CH3), 48.6 (C-7, C-5), 47.8
(C-9), 35.0 (C-2′), 34.6 (C-10), 33.5 (C-1), 26.7 (C-6), 21.0 (1′-
OCOCH3), 18.5 (C-17), 9.3 (C-16); ESIMS m/z 443.9 (100%) [M +
H]+, 444.9 (25%), 445.9 (5%); HRESIMS m/z 444.2015 [M + H]+,
calcd for C24H30NO7 444.2022.
(1′R)-Hydroxy-3′,4′-didehydrostemofoline (27). To a solution of
acetate derivative 29 (24 mg, 0.053 mmol) in THF/H2O (2:1, 3.0 mL)
was added LiOH (21 mg of 53% assay, 0.265 mmol) at rt, and the
reaction mixture was left to stir for 16 h. Water was added (5 mL),
and the mixture was extracted with CH2Cl2 (3 × 10 mL). The CH2Cl2
extract was first washed with saturated aqueous NaHCO3 solution and
then brine and dried (MgSO4). The concentrated residue was purified
by CC using gradient elution [EtOAc to EtOAc/MeOH (98:2)] to give
the alcohol 27 (13 mg, 0.032 mmol, 61% yield) as a pale yellow gum:
Rf ) 0.23 in MeOH/EtOAc (1:4); [R]2D4 +308.0 (c 1.0, CHCl3); IR
ν
max 3446, 2965, 2919, 2847, 1743, 1621 cm-1; 1H NMR δ 5.98-5.88
(m, 1H, H-3′), 5.18 (d, J ) 18.0 Hz, 1H, H-(4′Z)), 5.12 (d, J ) 10.0
Hz, 1H, H-(4′E)), 4.48 (br s, 1H, H-2), 4.13 (s, 3H, O-CH3), 3.68 (dd,
J ) 9.5 Hz, 3.5 Hz, 1H, H-1′b), 3.51 (br s, 1H, H-9a), 3.18-3.12 (m,
1H, H-5a), 3.10-3.05 (m, 1H, H-10), 3.05-3.00 (m, 1H, H-5b), 2.82
(d, J ) 5.0 Hz, 1H, H-7), 2.39-2.34 (m, 1H, H-2′b), 2.32-2.26 (m,
1H, H-1′a), 2.06 (s, 3H, H-16), 1.97 (d, J ) 12.5 Hz, 1H, H-1a),
1.94-1.89 (m, 2H, H-6), 1.89-1.84 (m, 1H, H-9), 1.64 (d, J ) 13.0
Hz, 1H, H-1b), 1.37 (d, J ) 6.0 Hz, 3H, H-17); 13C NMR δ 169.8
(C-15), 162.9 (C-13), 148.2 (C-11), 135.0 (C-3′), 128.1 (C-12), 117.7
(C-4′), 112.6 (C-8), 98.8 (C-14), 87.0 (C-3), 75.6 (C-2), 67.9 (C-1′),
61.0 (C-9a), 59.0 (O-CH3), 48.2 (C-7), 48.1 (C-9), 47.6 (C-5), 36.7
(C-2′), 34.5 (C-10), 33.9 (C-1), 27.4 (C-6), 18.4 (C-17), 9.3 (C-16);
ESIMS m/z 402.2 (100%) [M + H]+, 403.2 (20%); HRESIMS m/z
402.1912 [M + H]+, calcd for C22H28NO6 402.1917.
11,12-Dihydroxystemofoline (31). To a solution of 1118 (40 mg,
0.104 mmol) in 2:1 acetone/H2O (3.0 mL) at rt was added 4-methyl-
morpholine-N-oxide (23 mg, 0.193 mmol) and K2OsO4 ·2H2O (2 mg,
0.005 mmol), respectively. The reaction was left to stir at rt for 16 h,
sodium sulfite (50 mg) was added, and stirring was continued for 1 h.
The reaction mixture was filtered through a pad of cotton. A saturated
aqueous solution of NaHCO3 was added, and the mixture was extracted
with CH2Cl2 (3 × 10 mL). The CH2Cl2 extract was washed with brine
and dried (MgSO4). The concentrated residue was purified by CC
[CH2Cl2 to CH2Cl2/MeOH (90:10)] to give 31 (25 mg, 0.058 mmol,
56% yield) as a pale yellow gum: Rf ) 0.40 in MeOH/CH2Cl2 (1:9);
[R]2D5 -8.4 (c 1.55, CHCl3); IR νmax 3282, 2954, 2931, 2871, 1671,
1327, 1022 cm-1; 1H NMR (300 MHz, CD3OD) δ 4.25 (br s, 1H, H-2),
4.14 (s, 3H, O-CH3), 3.40 (br s, 1H, H-9a), 3.14-3.08 (m, 3H, H-5,
H-9), 2.86 (br, 1H, H-10), 2.44 (d, J ) 5.7 Hz, 1H, H-7), 2.05-1.93
(m, 1H, H-1a), 1.96 (s, 3H, H-16), 1.91-1.78 (m, 2H, H-6), 1.65-1.60
(m, 1H, H-1b), 1.60-1.55 (m, 2H, H-1′), 1.45-1.28 (m, 4H, H-2′,
H-3′), 1.08 (d, J ) 6.9 Hz, 3H, H-17), 0.94 (t, J ) 11.0 Hz, 3H, H-4′);
13C NMR (75 MHz, CD3OD) δ 175.1 (C-15), 171.4 (C-13), 112.3 (C-
8), 109.1 (C-11), 104.1 (C-12), 100.4 (C-14), 84.2 (C-3), 79.2 (C-2),
62.5 (C-9a), 59.8 (O-CH3), 51.4 (C-7), 48.3 (C-5), 45.3 (C-9), 37.2
(C-10), 33.6 (C-1), 32.4 (C-1′), 28.3 (C-2′), 26.6 (C-6), 24.2 (C-3′),
14.3 (C-4′), 12.7 (C-17), 8.2 (C-16). The NMR spectra were also
determined in CDCl3; however, the signal for C-12 could not be
observed. 1H NMR (CDCl3) δ 4.25 (br s, 1H, H-2), 4.12 (s, 3H, O-CH3),
3.45 (br s, 1H, H-9a), 3.14-3.08 (m, 1H, H-5a), 3.02-2.96 (m, 1H,
H-5b), 2.73-2.66 (m, 1H, H-10), 2.51 (d, J ) 6.0 Hz, 1H, H-7), 2.01
(s, 3H, H-16), 1.98 (d, J ) 12.5 Hz, 1H, H-1a), 1.91 (d, J ) 10.0 Hz,
1H, H-9), 1.86-1.81 (m, 1H, H-6a), 1.79-1.73 (m, 1H, H-6b), 1.70
(d, J ) 12.5 Hz, 1H, H-1b), 1.54 (d, J ) 9.5 Hz, 2H, H-1′), 1.42-1.36
(m, 1H, H-2′), 1.35-1.31 (m, 2H, H-3′), 1.28-1.20 (m, 1H, H-2′),
1.13 (br s, 2H, 11-OH, 12-OH), 1.03 (d, J ) 6.5 Hz, 3H, H-17); 0.90
(t, J ) 7.0 Hz, 3H, H-4′); 13C NMR δ 172.7 (C-15), 168.0 (C-13),
111.3 (C-8), 101.9 (C-11), 100.0 (C-14), 83.2 (C-3), 78.7 (C-2), 61.0
(C-9a), 59.1 (O-CH3), 49.8 (C-7), 47.5 (C-5), 46.0 (C-9), 36.0 (C-10),
32.9 (C-1), 31.5 (C-1′), 27.4 (C-2′), 26.2 (C-6), 23.2 (C-3′), 14.1 (C-
4′), 12.6 (C-17), 8.7 (C-16); ESIMS m/z 422.0 (100%) [M + H]+, 423.1
(1′S)-Hydroxy-3′,4′-didehydrostemofoline (28). Compound 28 was
prepared via a method similar to the synthesis of 27, using acetate
derivative 30 (15 mg, 0.034 mmol) and LiOH (14 mg of 53% assay,
0.170 mmol) to give alcohol 28 (10 mg, 0.025 mmol, 73% yield) as a
white gum: Rf ) 0.36 in MeOH/EtOAc (1:4); [R]2D4 +380.0 (c 0.41,
1
CHCl3); IR νmax 3286, 2957, 2924, 2854, 1744, 1615 cm-1; H NMR
δ 5.90-5.82 (m, 1H, H-3′), 5.19 (d, J ) 11.5 Hz, 1H, H-(4′Z)), 5.16
(s, 1H, H-(4′E)), 4.40 (s, 1H, H-2), 4.13 (s, 3H, O-CH3), 3.71 (d, J )
10.5 Hz, 1H, H-1′a), 3.49 (br s, 1H, H-9a), 3.17-3.13 (m, 1H, H-9),
3.13-3.06 (m, 1H, H-10), 3.03 (d, J ) 5.5 Hz, 1H, H-5a), 3.04-2.98
(m, 1H, H-7), 2.53 (dd, J ) 14.0 Hz, 5.5 Hz, 1H, H-2′a), 2.07 (s, 3H,
H-16), 2.02 (d, J ) 14.5 Hz, 1H, H-2′b), 2.00 (d, J ) 11.5 Hz, 1H,
H-6b), 1.97 (d, J ) 13.0 Hz, 1H, H-1a), 1.90-1.88 (m, 1H, H-6a),
1.85 (d, J ) 7.5 Hz, 1H, H-5b), 1.69 (d, J ) 12.5 Hz, 1H, H-1b), 1.37
(d, J ) 6.0 Hz, 3H, H-17); 13C NMR δ 169.8 (C-15), 162.9 (C-13),
148.5 (C-11), 135.2 (C-3′), 128.1 (C-12), 118.6 (C-4′), 112.9 (C-8),
98.8 (C-14), 86.0 (C-3), 77.0 (C-2), 69.8 (C-1′), 61.7 (C-9a), 59.0 (O-
CH3), 49.3 (C-7, C-9), 48.0 (C-5), 37.8 (C-2′), 34.6 (C-1, C-10), 27.3
(C-6), 18.4 (C-17), 9.3 (C-16); ESIMS m/z 402.2 (100%) [M + H]+,
403.2 (20%), 404.2 (10%); HRESIMS m/z 402.1903 [M + H]+, calcd
for C22H28NO6 402.1917.
(1′R)-Hydroxystemofoline (9). To a solution of alcohol 27 (12 mg,
0.029 mmol) in dry MeOH (2 mL) at rt was added Pd/C (1.2 mg, 10%
w/w), and the flask was flushed with N2 for 10 min before being left
to stir under a H2 atmosphere (balloon) for 45 min. The flask was
flushed with N2, and the solution was filtered through Celite and washed

940 Journal of Natural Products, 2010, Vol. 73, No. 5
Sastraruji et al.
(20%), 424.1 (5%); HRESIMS m/z 422.2166 [M + H]+, calcd for
C22H32NO7 422.2179.
388.0 (100%) [M + H]+, 389.1 (20%); HRESIMS m/z 388.1762
[M + H]+, calcd for C21H25NO6 388.1760. 34b: Rf ) 0.13 in MeOH/
CH2Cl2 (1:9); [R]D25 +229.9 (c 0.77, CHCl3); IR νmax 3288, 3007,
(2S,2aR,6S,7aS,7bS,8R,9S)-7b-Butylhexahydro-9-methyl-4H-2,2,6-
(epoxy[1]propanyl[3]ylidene)furo[2,3,4-gh]pyrrolizin-10-one (32). To
a stirred mixture of silica gel (1.67 g) suspended in diethyl ether (1
mL) at rt was added NaIO4 (16 mg, 0.076 mmol) in water (1 mL).
Then a solution of 31 (25 mg, 0.058 mmol) in CH2Cl2 (2 mL) was
added, and the mixture was left to stir for 1 h at rt. The reaction mixture
was filtered through a pad of cotton, and the filtrate was dried (MgSO4).
The crude product was purified by CC [CH2Cl2 to CH2Cl2/MeOH (95:
5)] to give 32 (10 mg, 0.035 mmol, 60% yield) as a clear yellow gum:
Rf ) 0.37 in MeOH/EtOAc (1:4); [R]2D5 +26.3 (c 0.21, CHCl3); IR
1
2937, 2872, 1726, 1613, 1005 cm-1; H NMR δ 6.34 (dd, J ) 14.5
Hz, 10.5 Hz, 1H, H-3′), 6.30 (dd, J ) 15.5 Hz, 10.5 Hz, 1H, H-2′),
5.87 (dt, J ) 14.5 Hz, 5.5 Hz, 1H, H-4′), 5.76 (d, J ) 14.5 Hz, 1H,
H-1′), 4.23 (s, 1H, H-2), 4.20 (d, J ) 6.0 Hz, 2H, H-5′), 4.14 (s,
3H, O-CH3), 3.52 (br s, 1H, H-9a), 3.14-3.06 (m, 2H, H-5a, H-10),
3.03-2.97 (m, 1H, H-5b), 2.88 (d, J ) 5.5 Hz, 1H, H-7), 2.08 (s,
3H, H-16), 1.96 (d, J ) 12.5 Hz, 1H, H-1a), 1.90-1.82 (m, 3H,
H-6, H-9), 1.78 (d, J ) 12.5 Hz, 1H, H-1b), 1.59 (br s, 1H, 5′-OH),
1.38 (d, J 6.5 Hz, 3H, H-17); 13C NMR δ 169.8 (C-15), 162.9 (C-
13), 148.4 (C-11), 132.8 (C-4′), 131.7 (C-1′), 130.6 (C-2′), 130.0
(C-3′), 128.1 (C-12), 112.9 (C-8), 98.8 (C-14), 83.5 (C-3), 80.8 (C-
2), 63.4 (C-5′), 61.1 (C-9a), 59.0 (O-CH3), 52.1 (C-7), 48.4 (C-5),
47.8 (C-9), 34.7 (C-10), 33.0 (C-1), 27.0 (C-6), 18.5 (C-17), 9.3
(C-16); ESIMS m/z 414.0 (100%) [M + H]+, 415.0 (20%);
HRESIMS m/z 414.1902 [M + H]+, calcd for C23H27NO6 414.1917.
34c: Rf ) 0.27 in MeOH/CH2Cl2 (1:9); [R]2D5 +174.2 (c 0.15, CHCl3);
1
νmax 2945, 2921, 2868, 1797, 970 cm-1; H NMR δ 4.32 (br s, 1H,
H-2), 3.41 (br s, 1H, H-9a), 3.19-3.12 (m, 1H, H-5b), 3.05-2.98 (m,
1H, H-5a), 2.77 (dq, J ) 11.5 Hz, 7.5 Hz, 1H, H-10), 2.65 (d, J ) 6.0
Hz, 1H, H-7), 1.98 (d, J ) 12.5 Hz, 1H, H-1a), 1.96-1.92 (m, 1H,
H-9), 1.92-1.88 (m, 1H, H-6a), 1.84-1.78 (m, 1H, H-6b), 1.75 (dt, J
) 12.5 Hz, 3.5 Hz, 1H, H-1b), 1.62-1.50 (m, 2H, H-1′), 1.46-1.38
(m, 1H, H-2′), 1.35 (q, J ) 7.0 Hz, 2H, H-3′), 1.26 (d, J ) 7.0 Hz, 3H,
IR νmax 3378, 2962, 2921, 2851, 1742, 1618 cm-1 1H NMR δ
;
10-CH3), 1.28-1.20 (m, 1H, H-2′), 0.92 (t, J ) 7.0 Hz, 3H, H-4′); 13
C
6.38-6.33 (m, 1H, H-2′), 6.31-6.26 (m, 1H, H-5′), 6.26-6.23 (m,
2H, H-3′, H-4′), 5.87 (dt, J ) 14.5 Hz, 5.5 Hz, 1H, H-6′), 5.77 (d,
J ) 15.0 Hz, 1H, H-1′), 4.24 (br s, 1H, H-2), 4.21 (d, J ) 5.5 Hz,
2H, H-7′), 4.14 (s, 3H, O-CH3), 3.52 (br s, 1H, H-9a), 3.14-3.06
(m, 2H, H-5b, H-10), 3.04-2.88 (m, 1H, H-5a), 2.88 (d, J ) 5.5
Hz, 1H, H-7), 2.07 (s, 3H, H-16), 1.96 (d, J ) 12.0 Hz, 1H, H-1a),
1.91-1.86 (m, 1H, H-9), 1.86-1.77 (m, 2H, H-6), 1.78 (d, J )
12.5 Hz, 1H, H-1b), 1.75 (br s, 1H, 7′-OH), 1.38 (d, J 6.5 Hz, 3H,
H-17); 13C NMR δ 169.8 (C-15), 162.9 (C-13), 148.4 (C-11), 132.9
(C-6′), 132.5 (C-4′), 132.2 (C-3′), 131.8 (C-1′), 131.2 (C-5′), 130.7
(C-2′), 128.1 (C-12), 112.9 (C-8), 98.8 (C-14), 85.6 (C-3), 80.8 (C-
2), 63.5 (C-7′), 61.1 (C-9a), 59.0 (O-CH3), 52.1 (C-7), 48.4 (C-5),
47.8 (C-9), 34.7 (C-10), 33.0 (C-1), 27.0 (C-6), 18.5 (C-17), 9.3
(C-16); ESIMS m/z 440.1 (100%) [M + H]+, 441.2 (5%); HRESIMS
m/z 440.2049 [M + H]+, calcd for C25H29NO6 440.2073.
Bioautography Procedure. TLC bioautography was performed
using the method described by Hostettmann et al.15 TLC plates were
prepared for bioautography by washing with acetone and then
thoroughly dried. Samples were applied to the plates in varying
quantities and sprayed with AChE enzyme stock solution (prepared
from acetylcholinesterase (EC 3.1.1.7, 906 U/mg) as described in the
literature15). The plates were incubated at 37 °C for 20 min and then
sprayed with freshly prepared indicator solution (from 1-naphthyl
acetate and Fast Blue B salt prepared according to the literature15) to
give the plate a purple coloration after 1-2 min. A white spot indicated
inhibition of AChE by the sample.
NMR δ 178.5 (C-11), 109.26 (C-8), 83.2 (C-3), 78.9 (C-2), 61.2 (C-
9a), 50.2 (C-7), 47.7 (C-5), 45.7 (C-9), 35.9 (C-10), 32.9 (C-1), 31.9
(C-1′), 27.3 (C-2′), 26.6 (C-6), 23.2 (C-3′), 14.1 (C-4′), 13.4 (10-CH3);
ESIMS m/z 278.2 (100%) [M + H]+, 279.2 (20%); 280.2 (3%);
HRESIMS m/z 278.1679 [M + H]+, calcd for C16H24NO6 278.1756.
(2S,2aR,6S,7aS,7bS,8R,9S,10Z)-Tetrahydro-10-(3-methoxy-4-methyl-
5-oxo-2(5H)-furanylidene)-9-methyl-4H-2,2,6-(epoxy[1]propanyl[3]ylidene)-
furo[2,3,4-gh]pyrrolizine-7b(6H)-(2E)-2-propenal (33a), (2S,2aR,6S,7aS,
7bS,8R,9S,10Z)-Tetrahydro-10-(3-methoxy-4-methyl-5-oxo-2(5H)-fura-
nylidene)-9-methyl-4H-2,2,6-(epoxy[1]propanyl[3]ylidene)furo[2,3,4-gh]-
pyrrolizine-7b(6H)-(2E,4E)-2,4-pentadienal (33b), and (2S,2aR,6S,7aS,
7bS,8R,9S,10Z)-Tetrahydro-10-(3-methoxy-4-methyl-5-oxo-2(5H)-fura-
nylidene)-9-methyl-4H-2,2,6-(epoxy[1]propanyl[3]ylidene)furo[2,3,4-gh]-
pyrrolizine-7b(6H)-(2E,4E,6E)-2,4,6-septatrienal (33c). To a solution of
174 (56 mg, 0.157 mmol) in dry toluene (3 mL) at rt under a N2
atmosphere was added (triphenylphosphoranylidene)acetaldehyde (95
mg, 0.314 mmol). Then the reaction mixture was heated to 80 °C under
N2 for 2 days. The reaction was quenched with saturated aqueous
NaHCO3 solution and extracted with CH2Cl2 (3 × 10 mL). The CH2Cl2
extract was washed with brine and dried over MgSO4. After evaporation
the crude product mixture (114 mg) was obtained as a yellow gum.
This mixture was taken through the next step without further purification.
(5Z)-5-[(2S,2aR,6S,7aS,7bS,8R,9S)-Hexahydro-7b-[(1E)-3-hydroxy-
butenyl]-9-methyl-4H-2,2,6-(epoxy[1]propanyl[3]ylidene)furo[2,3,4-gh]-
pyrrolizin-10-ylidene]-4-methoxy-3-methyl-2(5H)-furanone (34a), (5Z)-
5-[(2S,2aR,6S,7aS,7bS,8R,9S)-Hexahydro-7b-[(1E,3E)-5-hydroxy-
1,3-pentadienyl]-9-methyl-4H-2,2,6-(epoxy[1]propanyl[3]ylidene)furo[2,3,4-
gh]pyrrolizin-10-ylidene]-4-methoxy-3-methyl-2(5H)-furanone (34b), and
(5Z)-5-[(2S,2aR,6S,7aS,7bS,8R,9S)-Hexahydro-7b-[(1E,3E,5E)-7-hydroxy-
1,3,5-septatrienyl]-9-methyl-4H-2,2,6-(epoxy[1]propanyl[3]ylidene)furo[2,3,4-
gh]pyrrolizin-10-ylidene]-4-methoxy-3-methyl-2(5H)-furanone (34c). To
a solution of 33a-c (114 mg) in dry MeOH (2 mL) was added NaBH4
(12 mg) at rt. The reaction mixture was left to stir at rt for 45 min.
The MeOH was then evaporated to give a white residue. A saturated
aqueous solution of NaHCO3 (5. mL) was added, and the mixture
was extracted with CH2Cl2 (3 × 10 mL). The CH2Cl2 extract was
washed with brine and dried (MgSO4). Evaporation gave the crude
product as a yellow gum (112 mg). This was purified by PTLC using
10% MeOH/EtOAc to give 34a (7.1 mg, 0.018 mmol, 12% yield
over 2 steps), 34b (17.5 mg, 0.042 mmol, 27% yield over 2 steps),
and 34c (3.3 mg, 0.008 mmol, 5% yield over 2 steps). 34a: Rf )
0.06 in MeOH/CH2Cl2 (1:9); [R]2D5 +280.1 (c 1.14, CHCl3); IR νmax
Acknowledgment. We thank the Australian Research Council and
the University of Wollongong for financial support.
1
Supporting Information Available: Copies of the H NMR and
13C NMR spectra of all compounds and full experimental procedures
for Scheme 2. This material is available free of charge via the Internet
at http://pubs.acs.org.
References and Notes
(1) Pilli, R. A.; Rosso, G. B.; de Oliveira, M. C. F. The Alkaloids; Cordell,
G. A., Ed.; Elsevier: San Diego, 2005; Vol. 62, Chapter 2, pp 77-
173.
(2) (a) Mungkornasawakul, P.; Pyne, S. G.; Jatisatienr, A.; Supyen, D.;
Lie, W.; Ung, A. T.; Skelton, B. W.; White, A. H. J. Nat. Prod. 2003,
66, 980–982. (b) Kaltenegger, E.; Brem, B.; Mereiter, K.; Kalchhauser,
H.; Kahlig, H.; Hofer, O.; Vajrodaya, S.; Greger, H. Phytochemistry
2003, 63, 803–816. (c) Mungkornasawakul, P.; Pyne, S. G.; Jatisatienr,
A.; Supyen, D.; Jatisatienr, C.; Lie, W.; Ung, A. T.; Skelton, B. W.;
White, A. H. J. Nat. Prod. 2004, 67, 675–677. (d) Mungkornasawakul,
P.; Matthews, H.; Ung, A. T.; Pyne, S. G.; Jatisatienr, A.; Lie, W.;
Skelton, B. W.; White, A. H. ACGC Chem. Res. Commun. 2005, 19,
30–33. (e) Lin, L.-G.; Tang, C.-P.; Dien, P.-H.; Xu, R.-S.; Ye, Y.
Tetrahedron Lett. 2007, 48, 1559–1561. (f) Wang, Y.-Z.; Tang, C.-
P.; Dien, P.-H.; Ye, Y. J. Nat. Prod. 2007, 70, 1356–1359.
(3) Greger, H. Planta Med. 2006, 72, 99–113.
1
3329, 2933, 2921, 2864, 1752, 1621, 1003 cm-1; H NMR δ 5.94
(dt, J ) 15.0 Hz, 5.0 Hz, 1H, H-2′), 5.81 (d, J ) 15.5 Hz, 1H,
H-1′), 4.24 (s, 1H, H-2), 4.19 (br s, 2H, H-3′), 4.14 (s, 3H, O-CH3),
3.51 (br s, 1H, H-9a), 3.13-3.07 (m, 2H, H-5a, H-10), 3.02-2.97
(m, 1H, H-5b), 2.88 (d, J ) 5.5 Hz, 1H, H-7), 2.07 (s, 3H, H-16),
1.96 (d, J ) 12.0 Hz, 1H, H-1a), 1.92-1.86 (m, 2H, H-6), 1.86-1.82
(m, 1H, H-9), 1.78 (d, J ) 12.0 Hz, 1H, H-1b), 1.70 (br s, 1H,
3′-OH), 1.38 (d, J ) 6.5 Hz, 3H, H-17); 13C NMR δ 169.8 (C-15),
162.9 (C-13), 148.4 (C-11), 130.5 (C-2′), 129.1 (C-1′), 128.1 (C-
12), 112.9 (C-8), 98.8 (C-14), 83.1 (C-3), 80.6 (C-2), 63.0 (C-3′),
61.1 (C-9a), 59.0 (O-CH3), 51.6 (C-7), 48.3 (C-5), 47.8 (C-9), 34.7
(C-10), 32.9 (C-1), 27.0 (C-6), 18.4 (C-17), 9.3 (C-16); ESIMS m/z
(4) Baird, M. C.; Pyne, S. G.; Ung, A. T.; Lie, W.; Sastraruji, T.;
Jatisatienr, A.; Jatisatienr, C.; Dheeranupattana, S.; Lowlam, J.;
Boonchalermkit, S. J. Nat. Prod. 2009, 72, 679–684.
(5) Lin, W.-H.; Xu, R.-S.; Zhong, Q.-X. Acta Chim. Sin. 1991, 49, 1034–
1037.

Semisynthesis of Stemofoline Alkaloids
Journal of Natural Products, 2010, Vol. 73, No. 5 941
(6) Xu, R.-S.; Tang, Z.-J.; Feng, S.-C.; Yang, Y.-P.; Lin, W.-H.; Zhong, Q.-
X.; Zhong, Y. Mem. Inst. Oswaldo Cruz 1991, 86 (Suppl. II), 55–59.
(7) Ramachandran, P. V.; Srivastava, A.; Hazra, D. Org. Lett. 2007, 9,
157–160.
(14) Wang, P.; Liu, A.-L.; An, Z.; Li, Z.-H.; Du, G.-H.; Qin, H.-L. Chem.
BiodiVersity 2007, 4, 523–530.
(15) Marston, S.; Kissling, J.; Hostettmann, K. Phytochem. Anal. 2002,
13, 51–54.
(8) Irie, H.; Masaki, N.; Ohno, K.; Osaki, K.; Taga, T.; Uyeo, S. J. Chem.
Soc., Chem. Commun. 1970, 1066.
(9) Kong, W.; Fu, C.; Ma, S. Org. Biomol. Chem. 2008, 6, 4587–4592.
(10) (a) Racherla, U. S.; Brown, H. C. J. Org. Chem. 1991, 56, 401–404.
(b) Racherla, U. S.; Liao, Y.; Brown, H. C. J. Org. Chem. 1992, 57,
6614–6617. (c) Felpin, F. X.; Lebreton, J. J. Org. Chem. 2002, 67,
9192–9199.
(11) Mungkornasawakul, P.; Chaiyong, S.; Sastraruji, T.; Jatisatienr, A.;
Jatisatienr, C.; Pyne, S. G.; Ung, A. T.; Korth, J.; Lie, W. J. Nat.
Prod. 2009, 72, 848–851.
(12) Izquierdo, I.; Plaza, M. T.; Franco, F. Tetrahedron: Asymmetry 2004,
15, 1465–1469.
(16) Sastraruji, T.; Jatisatienr, A.; Pyne, S. G.; Ung, A. T.; Lie, W.;
Williams, M. C. J. Nat. Prod. 2005, 68, 1763–1767.
(17) Compound 13 was isolated from the roots of Stemona aphylla, which
were collected from Lampang, Thailand, in April 2009. The species
of plant was identified by Mr. James F.Maxwell from the Department
of Biology, Chiang Mai University, and found to be identical to the
plant materials in ref 16.
(18) Sastraruji, K.; Pyne, S. G.; Ung, A. T.; Mungkornasawakul, P.; Lie,
W.; Jatisatienr, A. J. Nat. Prod. 2009, 72, 316–318.
(19) Thompson, C. M.; Quinn, C. A.; Hergenrother, P. J. J. Med. Chem.
2009, 68, 117–125.
(20) Dale, J.; Kristiansen, P. O. Acta Chem. Scand. 1972, 26, 1471–1478.
(21) Dale, J.; Fredriksen, S. B. Acta Chem. Scand. 1985, 39B, 511–512.
(13) Mungkornasawakul, P.; Pyne, S. G.; Jatisatienr, A.; Lie, W.; Ung,
A. T.; Issakul, K.; Sawatwanich, A.; Supyen, D.; Jatisatienr, C. J.
Nat. Prod. 2004, 67, 1740–1743.
NP100137H