Stereoselective synthesis, characterization, and antibacterial activities of novel isosteviol derivatives with D-ring modification

Article abstract of DOI:10.1002/hlca.201000046

Considerable interests have been attracted by isosteviol and its derivatives because of their large variety of bioactivities. In this project, a series of novel 15- and 16-substituted isosteviol derivatives were stereoselectively prepared by means of func

Full text of DOI:10.1002/hlca.201000046

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Stereoselective Synthesis, Characterization, and Antibacterial Activities of
Novel Isosteviol Derivatives with D-Ring Modification
by Ya Wu a)b), Cong-Jun Liua), Xu Liua), Gui-Fu Daia), Jin-Yu Dua), and Jing-Chao Tao*a)
a) Department of Chemistry, New Drug Research & Development Center, Zhengzhou University,
Zhengzhou 450052, P. R. China (phone/fax: þ 86-371-67767200; e-mail: jctao@zzu.edu.cn)
b) School of Pharmacy, Henan College of Traditional Chinese Medicine, Zhengzhou 450008, P. R. China
Considerable interests have been attracted by isosteviol and its derivatives because of their large
variety of bioactivities. In this project, a series of novel 15- and 16-substituted isosteviol derivatives were
stereoselectively prepared by means of functional interconversions in ring D of the tetracyclic diterpene
isosteviol. All compounds synthesized were characterized by analysis of NMR, IR, HR-MS data, and the
configurations of 33 and 37 were confirmed by X-ray crystallographic analysis. The antibacterial
activities in vitro of these isosteviol derivatives were investigated; the synthetic compounds were more
active against Gram-positive than Gram-negative bacteria, and were especially active against Bacillus
subtilis. Among them, compound 27 (MIC ¼ 1.56 mg/ml) exhibited the highest antibacterial activity and
thus may be exploitable as a lead compound for the development of potent antibacterial agents.
Introduction. – Bacterial infections such as food poisoning, rheumatism, salmo-
nellosis, and diarrhea are caused by multidrug-resistant Gram-positive and Gram-
negative pathogens. Sixty million people are infected, and 20,000 deaths are recorded
every year caused by Staphylococcus aureus, Streptococcus pyogenes, Salmonella
typhimurium, and Escherichia coli. Amoxicillin, norfloxacin, and ciprofloxacin are the
principal drugs of choice for treatment of bacterial infections, since they are effective
against extraintestinal and intestinal wall infection, but these are associated with
several side-effects such as nausea, metallic taste, dizziness, hypertension, etc. [1][2].
Therefore, there is an urgent need to discover new compounds with potent antibacterial
activities for developing new drugs. Moreover, the importance of heterocyclic
compounds has been recognized in this field, and it is well-known that a number of
polycyclic compounds containing heterocycle fragments exhibited a wide variety of
biological activities [3 – 9].
Isosteviol (¼ent-16-oxobeyeran-19-oic acid; 1) is a tetracyclic diterpenoid with a
beyerane skeleton, obtained by acid hydrolysis of stevioside [10][11]. In recent years,
isosteviol derivatives have attracted increasing attention because of their remarkably
broad spectrum of biological activities including anti-inflammatory [12], glucocorticoid
agonist [13], antihypertension [14], antitumor [15], antiproliferation [16], and
inhibition of ent-kaurene synthase [17]. Especially, Lin et al. reported that isosteviol
amide dimers had favorable antibacterial effects and cytotoxicity [18], which prompted
us to study new isosteviol derivatives with hydrophilic functional groups to develop
novel stronger antibacterial agents for therapeutic use. Some novel compounds
containing OH and CH2OH group, and heteroatom-containing frameworks fused with
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 93 (2010)
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isosteviol structure have been prepared in our laboratory [19]. So, hydrophilic
functional groups attached to ring D in isosteviol skeleton may also provide some
possibilities for the construction of heterocycle in the isosteviol precursor.
In view of these reports and in continuation of our previous work [20], a series of
novel compounds containing indole, pyrazoline, and isoxazolidine rings fused with the
isosteviol framework have been designed and synthesized. The in vitro antibacterial
activities of these new isosteviol derivatives were investigated, which would aid in
designing and synthesizing novel stronger antibacterial agents.
Results and Discussion. – All the isosteviol derivatives mentioned below were
synthesized by different methods. Initial efforts were focused on structural modifica-
tions at C(15) and C(16) of isosteviol (1). The synthetic routes are outlined in Scheme 1.
Treatment of isosteviol (1), obtained by acid hydrolysis of stevioside, with EtBr and
KOH in DMSO afforded the corresponding ethyl ester 2 in 96% yield [21]. In addition,
the Fischer indole reaction has remained an extremely important and useful method for
the synthesis of a variety of indole intermediates and biologically active compounds
[22 – 24]; so, indole isosteviol derivatives 5 and 6 were obtained from 1 and 2,
respectively, using AcOH saturated with gaseous HCl as catalyst via Fischer reaction in
good yields (80 and 91%, resp.) [19][25].
Compounds 7 and 8 were stereoselectively synthesized via a one-pot Tollensꢂ (aldol-
Cannizzarro) reaction in good yield (95 and 90%, resp.). The products were
characterized by HR-MS, IR, and NMR, and the configuration of compound 7 was
confirmed by X-ray crystallographic analysis [26]. Compounds 9 and 10 were obtained
in good yields by reduction of 1 and 2, respectively, with NaBH4 in EtOH at 08 [27]. The
configuration of compound 10 was established by X-ray crystallographic analysis [20].
Treatment of 10 with acrylic acid (¼ prop-2-enoic acid) in CH2Cl2 in the presence of
DCC and DMAP furnished the main product 11 [19] and by-product 12. In addition, 12
could be also obtained via Michael addition from 11 with acrylic acid. So, compound 11
and 12 could be selectively synthesized in CH2Cl2 from compound 10 in good yield (85
and 75%, resp.) by controlling the amount of acrylic acid.
Compounds 13 and 14 were selectively synthesized in the presence of Na2CO3 from
compound 8 by controlling the amount of nicotinoyl chloride in good yield (81 and
88%, resp.), as shown in Scheme 2. In addition, compound 15 was obtained by selective
oxidation of 8 with PCC in CH2Cl2. Meanwhile, treatment of 1,3-diol 8 with HNO3/
H2SO4 in CH2Cl2 gave the corresponding dinitro derivative 16 in 80% yield. Treatment
of compound 8 with 4-methylphenylsulfonyl chloride (TsCl) in pyridine furnished com-
pound 17 (75%), which was further converted to the ring-opening product 18 in 96%
yield via Grob fragmentation of compound 17 in the presence of NaOH in MeCN [28].
The electrophile-promoted cyclization of w-substituted alkenes is an increasingly
important method for the synthesis of tetrahydropyrans and six-membered lactones,
which are essential components of a wide range of interesting, biologically active
natural products [29 – 32]. In this regard, with compound 18 as starting material, some
experiments were carried out for the structural modification and functional-group
conversion at the aldehyde group in order to probe the effect of the newly introduced
substituents. So, the corresponding carboxylic acid, amine, alcohol, lactone, and
methyltetrahydropyran derivatives of 18 were synthesized as depicted in Scheme 3.

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Scheme 2
i) 1 equiv. nicotinoyl chloride, Na2CO3, CHCl3, 608, 1 h; 81%. ii) 2 equiv. nicotinoyl chloride, Na2CO3,
CHCl3, 608, 3 h; 88%. iii) Pyridinium chlorochromate (PCC), CH2Cl2, r.t., 1 h; 82%. iv) HNO3/H2SO4,
CH2Cl2, 3 h; 80%. v) TsCl, pyridine, r.t., 18 h; 75%. vi) NaOH, MeCN, r.t., 3 h; 96%.
Compound 18 was oxidized with the Jones reagent (8n) in acetone, resulting in the
carboxylic acids 19 (90%), which was further converted to the d-lactone 20 in the
presence of BF3 · OEt2 in 75% yield. In the NOESY experiment of 20, the correlation
of d(H) 4.32 – 4.20 (HꢀC(15)) with Me(20) (d(H) 0.74) indicated that MeꢀC(15) was
b-oriented. Treatment of 18 in presence of H2O2 and NaOH in MeOH via Baeyer –
Villiger oxidation furnished compound 21 in 75% yield. Reduction of 18 with NaBH4
in EtOH at 08 led to the corresponding hydroxy derivative 23 (96%), the subsequent
BF3 · OEt2-initiated cyclization afforded methyltetrahydropyran 24. In the NOESY
experiment of 24, the correlation of d(H) 3.63 – 3.56 (HꢀC(15)) with Me(20) (d(H)
0.68) indicated that MeꢀC(15) was also b-oriented. Compound 23 was further
converted to the tolyloxymethyl derivative 25 in 85% yield by esterification of 23 with
TsCl in pyridine. Treatment of 25 with NaN3 under basic conditions gave the
corresponding azide, which was further converted to the amino derivative 26 with Ph3P
in H2O at 658 (85%).
As shown in Scheme 4, reaction of 18 with HONH2 · HCl in presence of NaHCO3 in
EtOH gave only one of the two possible geometric isomers of the corresponding
aldoxime 27 (90%). Compound 27 was catalytically tautomerized with BF3 · OEt2 in

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Scheme 3
i) Jones reagent (8n), acetone, 08, 2 h; 90%. ii) MeOH, NaOH, H2O2, 658, 4 h; 75%. iii) NaBH4, EtOH,
08, 10 min; 96%. iv) BF3 · OEt2, CH2Cl2, reflux, 30 h; 74 – 75%. v) TsCl, pyridine, r.t., 12 h; 85%. vi) 1.
NaN3, DMF, 808, 3 h; 80%; 2. Ph3P, H2O, 658, 3 h; 85%.
boiling toluene into its nitrone form, which then intramolecularly cyclized to the fused
isoxazolidine 28 in high yield (96%) [33]. The NOESY spectrum of the product 28
indicated the a-orientation of the H-atoms at C(15) and C(16). Treatment of 28 with
MeI in presence of NaH in DMF at 508 afforded N-methylisoxazolidine 29 in 85%
yield. In addition, treatment of compound 27 with H2SO4 in acetone gave the
dehydration product 30.
The condensation of 18 with PhNHNH2 was carried out in EtOH at 108 to give
phenylhydrazone 31a, and BF3 · OEt2-induced cycloaddition of 31a was also accom-
plished to give pyrazole 32 (84%) [34]. Meanwhile, the reaction of 18 with 4-
nitrophenylhydrazine in EtOH at 108 gave the corresponding 4-nitrophenylhydrazone
31b, which readily cyclized, after purification in the presence of a catalytic amount of
BF3 · OEt2, to afford a single 4,5-dihydro-1H-pyrazole 33 in 75% yield [34]. The
stereostructure of 33 was confirmed through X-ray crystallographic analysis (Fig. 1).
The 2,4-dinitrophenylhydrazone 31c was obtained from aldehyde 18 with 2,4-
dinitrophenylhydrazine in EtOH at 108, but the 2,4-dinitrophenylhydrazone 31c

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Fig. 1. X-Ray structure of compound 33
containing two electron-withdrawing NO2 groups exhibited great stability against both
thermal and Lewis acid-catalyzed cycloaddition.
Treatment of compound 18, after reduction with NaBH4 with m-chloroperoxyben-
zoic acid (m-CPBA) in CH2Cl2 at 08 afforded the epoxy intermediate 34, which was
then intramolecularly converted to the (hydroxymethyl)tetrahydropyran 35
(Scheme 5). In the NOESY experiment of compound 35, the correlation of d(H)
3.22 (HꢀC(15)) with Me(20) (d(H) 0.66) indicated a-orientation of the H-atom at
C(15). Meanwhile, treatment of compound 18 with NaIO4 and NaBr in AcOH at 908
led stereoselectively to 1,2-dihydroxy derivative 36, which was further converted to
acetal 37 (84%) [35]. The configuration of compound 37 was confirmed by X-ray
crystallographic analysis (Fig. 2). In addition, treatment of 18 with aniline in CH2Cl2 in
the presence of molecular sieves (4 ꢃ) afforded imino intermediate 38, which was
converted to a reduction product 39 with NaBH4.
In further studies, all of the synthetic compounds were tested for their antibacterial
activities against Bacillus subtilis, Staphylococcus aureus, and Shigella flexneri strains.
In our studies, none of the compounds exhibited antibacterial activities against
Sigella, Gram-negative bacterial strains as shown in the Table. In general, the synthetic
compounds were more active against Gram-positive than Gram-negative bacteria, and
were especially active against Bacillus subtilis. The minimum inhibitory concentrations
(MICs) of these compounds against Bacillus subtilis CMCC(B)63501 are collected in
the Table. Nicotinates and nitrates exhibited much higher antibacterial activities than
the precursor 1,3-diol 8 (i.e., 13, 14, 16 vs. 8). In addition, the ring-opened derivatives
containing OH, NH2, and oxime groups were more potent than ring-opened product 18
(i.e., 19, 20, 22, 23, 27 vs. 18). Especially, compound 27 (MIC ¼ 1.56 mg/ml) was the most
potent of these tested compounds against Bacillus subtilis, which may be exploitable as
a lead compound for the development of potent bacteriostat. Meanwhile, the results
indicated that these isosteviol derivatives were capable of inhibiting Staphylococcus
aureus CMCC(B)26003 with moderate activities and had no inhibiting activities against
Shigella flexneri 626.

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Fig. 2. X-Ray structure of compound 37
Table. Antibacterial Activities of Isosteviol Derivatives against Bacillus subtilis, Staphylococcus aureus,
and Shigella flexneri Strains
Compound
Bacillus subtilis
Saphylococcus aureus
Sigella flexneri
1
2
5
6
7
100a)
100a)
> 100a) (17%)b)
> 100a) (17%)b)
> 100a) (3%)b)
> 100a) (18%)b)
> 100a) (16%)b)
> 100a) (66%)b)
> 100a) (15%)b)
> 100a) (4%)b)
> 100a) (84%)b)
> 100a) (77%)b)
> 100a) (32%)b)
> 100a) (58%)b)
> 100a) (47%)b)
> 100a) (75%)b)
> 100a) (15%)b)
> 100a) (63%)b)
> 100a) (52%)b)
NI
NIc)
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
> 200a)
200a)
100a)
200a)
8
9
> 200a)
> 200a)
> 200a)
> 200a)
12.5a)
3.125a)
12.5a)
12.5a)
> 200a)
12.5a)
3.125a)
12.5a)
6.25a)
> 200a)
> 200a)
1.56a)
> 200a)
NI
10
11
12
13
14
15
16
18
19
20
22
23
24
26
27
28
29
30
32
33
35
37
39
> 100a) (59%)b)
> 100a) (53%)b)
NI
> 100a) (40%)b)
100a)
> 100a) (5%)b)
> 100a) (7%)b)
NI
> 100a) (8%)b)
> 100a) (71%)b)
> 100a) (21%)b)
NI
NI
NI
NI
6.25a)
> 200a)
> 200a)
a) MIC [mg/ml]. b) Inhibition [%] determined at 100 mg/ml concentration of compound. c) NI ¼ No
inhibition at 100 mm.

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Conclusions. – In summary, a series of novel isosteviol derivatives containing OH
and HOCH2 groups, and heteroatom-containing frameworks have been successfully
synthesized in high yields; especially some new compounds containing pyrazoline,
pyrazole, and isoxazolidine rings fused with isosteviol structure were stereoselectively
synthesized from compound 8 via Grob fragmentation and subsequent intramolecular
1,3-dipolar cycloaddition. The in vitro antibacterial activities of these new isosteviol
derivatives were investigated, and some of them showed noteworthy activities. Among
all the derivatives, compound 27 showed the highest antibacterial activity against
Bacillus subtilis, and thus may be exploitable as potentially potent antibacterial agents
for therapeutic use. Further efforts aiming at developing potent bacteriostats based on
appropriately modified D-ring fused heterocyclic analogues are continuing in our
laboratory, and they will be reported in due course.
Experimental Part
General. All reagents and solvents were obtained from commercial suppliers. All reactions were
monitored by TLC. M.p.: Beijing Keyi XT5 apparatus; not corrected. IR Spectra: as KBr pellets on a
1
Thermo Nicolet IR200 spectrometer. H- and 13C-NMR spectra: Bruker DPX-400 spectrometer at 400
and 100 MHz, resp., with TMS as internal standard. MS: Waters Q-Tof micro mass spectrometer. X-Ray
analysis: Rigaku RAXIS-IV.
ent-16b-Hydroxy-15a-(hydroxymethyl)beyeran-19-oic Acid (¼(15b,16a)-16-Hydroxy-15-(hydroxy-
methyl)beyeran-18-oic Acid; 7) [26]. To a stirred soln. of isosteviol (1; 0.318 g, 1 mmol) and NaOH
(0.08 g, 2 mmol) in EtOH (20 ml) was added 37% aq. HCHO soln. (2 ml). After stirring for 1 h at 608, the
mixture was concentrated under vacuum, and extracted with CHCl3 and H2O. The org. layer was washed
with sat. aq. NaCl soln., dried (MgSO4), and concentrated under vacuum to give 7 (0.332 g, 95%). White
1
powder. M.p. 233 – 2358. IR (KBr): 3462, 2945, 2927, 2846, 1696, 1456, 1072, 1052. H-NMR (400 MHz,
(D6)acetone): 3.83 (dd, J ¼ 10.4, 5.2, 1 H); 3.62 (d, J ¼ 4.8, 1 H); 3.50 (t, J ¼ 9.6, 1 H); 3.30 (s, 2 H); 2.12 –
1.99 (m, 2 H); 1.95 – 1.70 (m, 6 H); 1.56 – 1.51 (m, 1 H); 1.44 – 1.34 (m, 2 H); 1.17 (s, 3 H); 1.15 – 0.90 (m,
6 H); 0.88 (s, 3 H); 0.87 (s, 3 H). 13C-NMR (100 MHz, (D6)acetone): 179.0; 82.3; 62.4; 57.8; 56.7; 54.5;
50.2; 43.1; 42.6; 40.5; 39.1; 38.2; 38.0; 34.9; 33.8; 29.1; 25.6; 22.3; 19.4; 19.0; 13.4. HR-ESI-MS: 373.2358
([M þ Na]þ, C21H34NaOþ4 ; calc. 373.2355).
Ethyl ent-16b-Hydroxy-15a-(hydroxymethyl)beyeran-19-oate (¼ Ethyl (15b,16a)-16-Hydroxy-15-
(hydroxymethyl)beyeran-18-oate; 8) [26]. To a stirred soln. of 2 (0.346 g, 1 mmol) and EtONa (0.136 g,
2 mmol) in EtOH (20 ml) was added 37% aq. HCHO soln. (2 ml). After stirring for 3 h at 608, the
mixture was concentrated under vacuum, and extracted with CHCl3 and H2O. The org. layer was washed
with sat. aq. NaCl soln., dried (MgSO4), and concentrated under vacuum to give 8 (0.34 g, 90%). White
1
powder. M.p. 181 – 1828. IR (KBr): 3435, 2940, 2838, 1720, 1458, 1378, 1234, 1179, 1153,1123. H-NMR
(400 MHz, CDCl3): 4.09 (q, J ¼ 7.2, 2 H); 3.98 (dd, J ¼ 9.7, 5.0, 1 H); 3.63 (d, J ¼ 4.7, 1 H); 3.56 (t, J ¼ 10.2,
1 H); 2.16 (d, J ¼ 13.0, 1 H); 2.06 – 2.04 (m, 1 H); 1.83 – 1.56 (m, 9 H); 1.43 – 1.37 (m, 2 H); 1.26 (t, J ¼ 7.2,
3 H); 1.22 – 1.19 (m, 1 H); 1.16 (s, 3 H); 1.08 – 0.95 (m, 4 H); 0.94 (s, 3 H); 0.88 – 0.86 (m, 1 H); 0.78 (s,
3 H). 13C-NMR (100 MHz, CDCl3): 177.4; 86.7; 64.9; 60.0; 57.5; 57.0; 54.2; 50.2; 43.6; 42.4; 40.8; 39.6;
38.1; 37.9; 34.8; 33.0; 28.9; 25.0; 22.1; 19.5; 18.8; 14.1; 13.2. HR-ESI-MS: 401.2664 ([M þ Na]þ,
C23H38NaOþ4 ; calc. 401.2668).
ent-16b-Hydroxybeyeran-19-oic Acid (¼(16a)-16-Hydroxybeyeran-18-oic Acid; 9) [27]. A soln. of 1
(0.318 g, 1 mmol) and NaBH4 (0.057 g, 1.5 mmol) in dry EtOH (10 ml) was stirred at 08 for 1 h. Then, the
mixture was concentrated under vacuum, and extracted with CHCl3 and H2O. The org. layer was washed
with sat. aq. NaCl soln., dried (MgSO4), and concentrated under vacuum to give 9 (0.294 g, 92%). White
powder. M.p. 168 – 1698. IR (KBr): 3475, 2990, 2943, 2896, 2841, 1653, 1453, 1371, 1187, 1056, 998, 621.
1H-NMR (400 MHz, CDCl3): 3.61 – 3.56 (m, 1 H); 2.01 (d, J ¼ 12.8, 1 H); 1.76 – 1.62 (m, 5 H); 1.59 – 1.52
(m, 3 H); 1.45 – 1.41 (m, 2 H); 1.31 – 1.17 (m, 3 H); 1.09 (s, 3 H); 1.06 – 0.86 (m, 6 H); 0.82 (s, 3 H); 0.75 (s,
3 H). HR-ESI-MS: 321.2425 ([M þ H]þ, C20H33O3þ ; calc. 321.2430).

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Ethyl ent-16b-Hydroxybeyeran-19-oate (¼ Ethyl (16a)-16-Hydroxybeyeran-18-oate; 10). A soln. of
2 (0.346 g, 1 mmol) and NaBH4 (0.057 g, 1.5 mmol) in dry EtOH (10 ml) was stirred at 08 for 1 h. Then,
the mixture was concentrated under vacuum, and extracted with CHCl3 and H2O. The org. layer was
washed with sat. aq. NaCl soln., dried (MgSO4), and concentrated under vacuum to give 10 (0.334 g,
96%). White powder. M.p. 152 – 1538. IR (KBr): 3533, 2978, 2939, 2880, 2837, 1700, 1460, 1374, 1318,
1231, 1178, 1151, 1049. 1H-NMR (400 MHz, CDCl3): 4.09 (q, J ¼ 7.2, 2 H); 3.85 (q, J ¼ 4.8, 1 H); 2.16 (d,
J ¼ 13.2, 1 H); 1.81 – 1.51 (m, 11 H); 1.26 (t, J ¼ 7.2, 3 H); 1.23 – 1.18 (m, 1 H); 1.16 (s, 3 H); 1.04 – 0.93 (m,
4 H); 0.90 (s, 3 H); 0.88 – 0.86 (m, 1 H); 0.74 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.6; 80.6; 59.9; 57.1;
55.8; 55.2; 43.7; 42.8; 42.0; 41.7; 39.9; 38.1; 38.0; 33.7; 29.0; 24.9; 21.7; 20.4; 18.9; 14.1; 13.3. HR-ESI-MS:
371.2554 [M þ Na]þ, C21H34NaOþ4 ; calc. 371.2562).
Ethyl ent-16b-Acryloxybeyeran-19-oate (¼ Ethyl (16a)-16-[(Prop-2-enoyl)oxy]beyeran-18-oate; 11)
[19]. A mixture of 10 (0.348 g, 1 mmol), prop-2-enoic acid (0.792 g, 1.1 mmol), DCC (0.412 g, 2 mmol),
and DMAP (0.024 g, 0.2 mmol) was stirred at r.t. After stirring for 12 h, the mixture was filtered, and the
filtrate was concentrated. The residue was purified by CC (SiO2; petroleum ether (PE)/AcOEt 6 :1) to
give 11 (0.341 g, 85%). IR (KBr): 3101, 2950, 2847, 1723, 1625, 1455, 1405, 1378, 1194, 1151, 1060, 981, 811.
1H-NMR (400 MHz, CDCl3): 6.38 (d, J ¼ 17.2, 1 H); 6.13 (dd, J ¼ 17.2, 10.4, 1 H); 5.81 (d, J ¼ 10.4, 1 H);
4.80 (q, J ¼ 4.8, 1 H); 4.15 – 4.06 (m, 2 H); 2.17 (d, J ¼ 13.6, 1 H); 1.92 – 1.68 (m, 7 H); 1.61 – 1.33 (m, 7 H);
1.25 (t, J ¼ 7.2, 3 H); 1.15 (s, 3 H); 1.09 – 094 (m, 4 H); 0.90 (s, 3 H); 0.87 – 084 (m, 1 H); 0.70 (s, 3 H).
13C-NMR (100 MHz, CDCl3): 177.5; 166.4; 130.2; 128.9; 81.7; 59.9; 57.0; 55.7; 54.8; 43.7; 42.4; 41.6; 41.5;
40.6; 39.9; 38.5; 38.0; 34.6; 28.9; 24.9; 21.7; 20.2; 18.9; 14.1; 13.2. HR-ESI-MS: 403.2835 ([M þ H]þ,
C25H39Oþ4 ; calc. 403.2848).
Treatment of 10 with Prop-2-enoic Acid. A mixture of 10 (0.348 g, 1 mmol), prop-2-enoic acid
(1.584 g, 2.2 mmol), DCC (0.412 g, 2 mmol), and DMAP (0.024 g, 0.2 mmol) was stirred at r.t. After
stirring for 16 h, the mixture was filtered, and the filtrate was concentrated. The residue was purified by
CC (SiO2; PE/AcOEt 6 :1) to give ethyl (16a)-16-({3-[(prop-2-enoyl)oxy]propanoyl}oxy)beyeran-18-
oate (12; 0.355 g, 75%). IR (KBr): 2948, 2848, 1728, 1634, 1456, 1407, 1388, 1180, 1117, 1058, 980, 809.
1H-NMR (400 MHz, CDCl3): 6.41 (dd, J ¼ 17.2, 1.2, 1 H); 6.09 (dd, J ¼ 17.2, 10.4, 1 H); 5.81 (dd, J ¼ 10.4,
1.6, 1 H); 4.78 (q, J ¼ 4.8, 1 H); 4.44 (t, J ¼ 6.4, 2 H); 4.15 – 4.04 (m, 2 H); 2.71 (t, J ¼ 6.4, 2 H); 2.15 (d, J ¼
13.6, 1 H); 1.92 – 1.68 (m, 7 H); 1.61 – 1.33 (m, 7 H); 1.24 (t, J ¼ 7.2, 3 H); 1.15 (s, 3 H); 1.04 – 0.92 (m,
4 H); 0.90 (s, 3 H); 0.86 – 0.84 (m, 1 H); 0.69 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.5; 170.7; 165.8;
131.1; 128.0; 82.1; 60.2; 59.9; 57.0; 55.6; 54.7; 43.6; 42.3; 41.5; 41.4; 40.5; 39.9; 38.2; 38.0; 34.6; 34.2; 28.9;
24.8; 21.6; 20.1; 18.9; 14.1; 13.2. HR-ESI-MS: 497.2868 ([M þ Na]þ, C28H42NaO6þ ; calc. 497.2879).
Ethyl ent-16b-Hydroxy-15a-[(nicotinoyloxy)methyl]beyeran-19-oate (¼ Ethyl (15b,16a)-16-
Hydroxy-15-{[(pyridin-3-ylcarbonyl)oxy]methyl}beyeran-18-oate; 13). A mixture of 8 (0.378 g, 1 mmol)
and nicotinoyl chloride (0.141 g, 1 mmol) in dry CHCl3 (10 ml) was stirred at 608 in the presence of
Na2CO3. After stirring for 1 h, the mixture was extracted with aq. Na2CO3, brine, and H2O successively.
The CHCl3 phase was dried (Na2SO4), filtered, and concentrated to give a crude product, which was
crystallized from CHCl3 to give 13 (0.391 g, 81%). M.p. 88.1 – 88.98. IR (KBr): 3422, 2945, 2848, 1722,
1592, 1458, 1383, 1282, 1150, 1025, 742, 702. 1H-NMR (400 MHz, CDCl3): 9.26 (s, 1 H); 8.78 (d, J ¼ 4.0,
1 H); 8.41 – 8.34 (m, 1 H); 7.42 (dd, J ¼ 8.0, 4.8, 1 H); 4.68 (dd, J ¼ 10.8, 4.8, 1 H); 4.25 (t, J ¼ 10.8, 1 H);
4.18 – 4.06 (m, 2 H); 3.65 (d, J ¼ 4.8, 1 H); 2.41 – 2.28 (m, 1 H); 2.25 – 2.13 (m, 1 H); 2.05 (d, J ¼ 19.2,
1 H); 1.86 – 1.40 (m, 11 H); 1.25 (t, J ¼ 7.2, 3 H); 1.17 (s, 3 H); 1.07 – 0.95 (m, 5 H); 0.94 (s, 3 H); 0.92 –
0.85 (m, 1 H); 0.80 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.3; 165.3; 153.0; 150.6; 137.5; 126.4;
123.5; 85.8; 67.5; 60.0; 57.5; 57.0; 54.1; 47.3; 43.6; 42.8; 41.1; 39.6; 38.2; 37.9; 35.0; 33.1; 28.9; 25.0; 22.1;
19.5; 18.9; 14.1; 13.2. HR-ESI-MS: 484.3051 ([M þ H]þ, C29H42NO5þ ; calc. 484.3063).
Ethyl ent-16b-(Nicotinoyloxy)-15a-[(nicotinoyloxy)methyl]beyeran-19-oate (¼ Ethyl (15b,16a)-16-
[(Pyridin-3-ylcarbonyl)oxy]-15-{[(pyridin-3-ylcarbonyl)oxy]methyl}beyeran-18-oate; 14). A mixture of
8 (0.378 g, 1 mmol) and nicotinoyl chloride (0.282 g, 2 mmol) in dry CHCl3 (10 ml) was stirred at 608 in
the presence of Na2CO3. After stirring for 3 h, the mixture was extracted with aq. Na2CO3, brine, and
H2O successively. The CHCl3 phase was dried (Na2SO4), filtered, and concentrated to give a crude
product which was crystallized from CHCl3 to give 14 (0.517 g, 88%). IR (KBr): 2950, 2850, 1723, 1590,
1460, 1286, 1127, 1024, 971, 741, 702. 1H-NMR (400 MHz, CDCl3): 9.12 (d, J ¼ 18.4, 2 H); 8.76 (s, 1 H);
8.68 (s, 1 H); 8.23 – 8.15 (m, 2 H); 7.38 (dd, J ¼ 8.0, 4.8, 1 H); 7.25 (dd, J ¼ 8.0, 4.8, 1 H); 5.28 (d, J ¼ 4.8,

Helvetica Chimica Acta – Vol. 93 (2010)
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1 H); 4.75 (dd, J ¼ 10.8, 4.8, 1 H); 4.42 – 4.30 (m, 1 H); 4.20 – 4.08 (m, 2 H); 2.68 – 2.62 (m, 1 H); 2.19 (d,
J ¼ 12.8, 1 H); 1.91 – 1.71 (m, 10 H); 1.47 – 1.28 (m, 2 H); 1.26 (t, J ¼ 7.2, 3 H); 1.18 (s, 3 H); 1.15 – 1.04 (m,
3 H); 1.01 (s, 3 H); 0.99 – 0.87 (m, 2 H); 0.85 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.1; 165.1; 164.6;
153.3; 153.1; 150.6; 150.5; 137.0; 137.0; 126.1; 125.9; 123.4; 123.1; 85.6; 66.3; 60.0; 57.4; 56.9; 54.0; 45.2;
43.6; 43.3; 41.6; 39.6; 38.2; 37.8; 34.8; 34.2; 28.8; 24.8; 22.0; 19.4; 18.8; 14.0; 13.2. HR-ESI-MS: 611.3081
([M þ Na]þ, C35H44N2NaOþ6 ; calc. 611.3097).
Ethyl ent-15a-(Hydroxymethyl)-16-oxobeyeran-19-oate (¼ Ethyl (15b)-15-(Hydroxymethyl)-16-oxo-
beyeran-18-oate; 15) [10]. A mixture of 8 (0.378 g, 1 mmol) and PCC (0.236 g, 1.1 mmol) was stirred at
r.t. for 1 h. Then, the mixture was filtered, and the filtrate was concentrated. The residue was purified by
CC (SiO2; PE/AcOEt 7:1) to give 15 (0.308 g, 82%). M.p. 155 – 1578. IR (KBr): 3534, 2958, 2857, 1735,
1721, 1462, 1151. 1H-NMR (400 MHz, CDCl3): 4.16 – 4.08 (m, 2 H); 3.95 – 3.88 (m, 1 H); 3.70 (t, J ¼ 10.4,
1 H); 2.56 – 2.18 (m, 1 H); 2.54 – 2.48 (m, 1 H); 2.19 (d, J ¼ 13.3, 1 H); 1.89 – 1.69 (m, 8 H); 1.42 – 1.29 (m,
4 H); 1.27 (t, J ¼ 7.2, 3 H); 1.19 (s, 3 H); 1.18 – 1.10 (m, 2 H); 0.98 (s, 3 H); 0.97 – 0.80 (m, 2 H); 0.75 (s,
3 H). 13C-NMR (100 MHz, CDCl3): 226.1; 177.2; 60.4; 60.1; 57.1; 56.7; 52.9; 52.5; 48.4; 43.6; 40.5; 39.6;
38.2; 37.8; 37.0; 35.2; 28.9; 21.6; 19.8; 19.6; 18.8; 14.1; 13.3. HR-ESI-MS: 399.2514 ([M þ Na]þ,
C23H36NaOþ4 ; calc. 399.2511).
Ethyl (15b,16a)-16-(Nitrooxy)-15-[(nitrooxy)methyl]beyeran-18-oate (16). To a stirred soln. of 8
(0.378 g, 1 mmol) in CH2Cl2 (20 ml) at 08 was added a mixture of HNO3 (0.13 ml) and H2SO4 (0.49 ml)
for 15 min. After stirring at r.t. for 3 h, the mixture was extracted with CH2Cl2 and H2O. The org. layer
was washed with sat. aq. NaCl soln., dried (MgSO4), and the filtrate was concentrated. The residue was
purified by CC (SiO2; PE/AcOEt 6 :1) to give 16 (0.374 g, 80%). M.p. 120.7 – 122.28. IR (KBr): 2943,
2852, 1721, 1625, 1467, 1384, 1279, 1180, 977, 853. 1H-NMR (400 MHz, CDCl3): 5.01 (d, J ¼ 4.8, 1 H); 4.75
(dd, J ¼ 10.8, 5.2, 1 H); 4.49 – 4.37 (m, 1 H); 4.10 (q, J ¼ 7.2, 2 H); 2.56 – 2.48 (m, 1 H); 2.19 – 2.15 (m,
1 H); 1.88 – 1.56 (m, 7 H); 1.45 – 1.28 (m, 4 H); 1.26 (t, J ¼ 7.2, 3 H); 1.16 (s, 3 H); 1.15 – 1.04 (m, 3 H); 1.02
(s, 3 H); 0.99 – 0.83 (m, 3 H); 0.74 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.1; 92.5; 73.2; 60.1; 57.1; 56.6;
53.8; 43.5; 43.4; 42.8; 41.7; 39.4; 38.1; 37.8; 34.8; 33.6; 28.8; 24.8; 21.7; 19.1; 18.7; 14.0; 13.0. ESI-HR-MS:
491.2372 ([M þ Na]þ, C23H36N2NaOþ8 : calc. 491.2369).
Ethyl ent-16b-Hydroxy-15a-{[(4-toluenesulfonyl)oxy]methyl}beyeran-19-oate (¼ Ethyl (15b,16a)-
16-Hydroxy-15-({[(4-methylphenyl)sulfonyl]oxy}methyl)beyeran-18-oate; 17). A mixture of 8 (0.378 g,
1 mmol) and TsCl (0.238 g, 1.1 mmol) in pyridine (5 ml) was stirred at r.t. for 18 h. Then, the mixture was
filtered, and the filtrate was extracted with CH2Cl2 and aq. HCl soln. (5m). The org. layer was washed
with sat. aq. NaCl soln., dried (MgSO4), and the filtrate was concentrated. The residue was purified by
CC (SiO2; PE/AcOEt 4 :1) to give 17 (0.399 g, 75%). IR (KBr): 3541, 2950, 2928, 2851, 1718, 1598, 1458,
1
1361, 1177, 1151, 1097, 1020, 948, 924, 816, 779, 665, 555. H-NMR (400 MHz, CDCl3): 7.82 (d, J ¼ 8.2,
2 H); 7.37 (d, J ¼ 8.2, 2 H); 4.32 (dd, J ¼ 9.8, 3.6, 1 H); 4.07 (q, J ¼ 7.1, 2 H); 3.96 (t, J ¼ 9.8, 1 H); 3.47 (d,
J ¼ 7.5, 1 H); 3.46 (s, 3 H); 2.37 – 2.30 (m, 1 H); 2.22 – 2.14 (m, 2 H); 1.80 – 1.28 (m, 8 H); 1.23 (t, J ¼ 7.1,
3 H); 1.19 – 1.11 (m, 1 H); 1.16 (s, 3 H); 1.10 – 0.93 (m, 5 H); 0.88 (s, 3 H); 0.86 – 0.79 (m, 3 H); 0.67 (s,
3 H). 13C-NMR (100 MHz, CDCl3): 177.2; 144.8; 132.9; 129.8; 129.8; 127.7; 127.7; 84.9; 72.8; 59.9; 57.4;
56.8; 53.8; 47.6; 43.5; 42.8; 40.9; 39.5; 38.0; 37.8; 34.6; 33.0; 28.8; 24.8; 21.9; 21.6; 19.3; 18.7; 14.0; 12.9. HR-
ESI-MS: 555.2742 ([M þ Na]þ, C30H44NaO6Sþ; calc. 555.2757).
Product 18 of Ring Opening. A mixture of 17 (0.532 g, 1 mmol) and NaOH (0.048 g, 1.1 mmol) in dry
MeCN (5 ml) was stirred at r.t. for 3 h. Then, the mixture was filtered, the filtrate was concentrated, and
the residue was extracted with CH2Cl2 and H2O. Then, the org. layer was washed with sat. aq. NaCl soln.,
dried (MgSO4), and the filtrate was concentrated. The residue was crystallized from CHCl3 to give ethyl
(5b,8a,9b,10a,13a)-8-ethenyl-13-formyl-13-methylpodocarpan-15-oate (18; 0.345 g, 96%). M.p. 116.5 –
117.88. IR (KBr): 3072, 2937, 2796, 2704, 1716, 1458, 1384, 1238, 1183, 1029, 912, 704. 1H-NMR
(400 MHz, CDCl3): 9.27 (d, J ¼ 1.6, 1 H); 5.95 (dd, J ¼ 17.6, 10.8, 1 H); 5.12 (d, J ¼ 10.0, 1 H); 5.08 (d, J ¼
3.2, 1 H); 4.06 – 3.95 (m, 2 H); 2.32 – 2.24 (m, 1 H); 2.16 – 2.10 (m, 2 H); 1.88 – 1.39 (m, 9 H); 1.26 (d, J ¼
13.2, 1 H); 1.20 (t, J ¼ 7.2, 3 H); 1.14 (s, 3 H); 1.10 – 0.89 (m, 5 H); 0.88 (s, 3 H); 0.58 (s, 3 H). 13C-NMR
(100 MHz, CDCl3): 205.2; 177.3; 142.5; 113.6; 59.9; 57.7; 57.5; 55.2; 46.1; 43.7; 40.8; 40.1; 39.6; 38.1; 38.0;
32.3; 28.7; 24.6; 20.0; 19.1; 17.4; 14.0; 13.2. HR-ESI-MS: 383.2560 ([M þ Na]þ, C23H36NaO3þ ; calc.
383.2562).

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Helvetica Chimica Acta – Vol. 93 (2010)
Oxidation of 18. A mixture of 18 (0.360 g, 1 mmol) and Jones reagent (8n) in dry acetone (5 ml) was
stirred at 08 for 2 h. Then, the mixture was concentrated, and the residue was extracted with CH2Cl2 and
H2O. The org. layer was washed with sat. aq. NaCl soln., dried (MgSO4), and the filtrate was
concentrated. The residue was purified by CC (SiO2; PE/AcOEt 2 :1) to give (5b,8a,9b,10a,13a)-8-
ethenyl-15-ethoxy-13-methyl-15-oxopodocarpane-13-carboxylic acid (19; 0.338 g, 90%). M.p. 160.2 –
161.88. IR (KBr): 3423, 3084, 2941, 2856, 1722, 1695, 1629, 1462, 1403, 1239, 1183, 1150, 1027, 896.
1H-NMR (400 MHz, CDCl3): 6.11 (dd, J ¼ 17.6, 10.8, 1 H); 5.05 (d, J ¼ 17.6, 1 H); 5.01 (d, J ¼ 11.2, 1 H);
4.08 – 4.02 (m, 2 H); 2.35 (d, J ¼ 22.0, 1 H); 2.21 – 2.06 (m, 3 H); 1.96 – 1.39 (m, 9 H); 1.24 (t, J ¼ 7.2, 3 H);
1.17 (s, 3 H); 1.13 (s, 3 H); 1.10 – 0.84 (m, 5 H); 0.62 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 184.6; 177.3;
140.8; 112.4; 59.8; 58.4; 57.8; 55.3; 45.6; 43.7; 41.0; 40.8; 39.75; 38.2; 38.0; 36.0; 30.0; 28.7; 19.8; 19.1; 18.0;
13.9; 13.4. HR-ESI-MS: 377.2710 ([M þ H]þ, C23H37O4þ ; calc. 377.2692).
Cyclization of 19. Compound 19 (0.376 g, 1 mmol) was dissolved in CH2Cl2 (5 ml), and BF3 · OEt2
(48% soln. in Et2O, 0.47 ml, 1.5 mmol) was added dropwise during stirring of the mixture under reflux for
30 h. The soln. was then diluted with H2O and extracted with CH2Cl2, and the combined org. phases were
dried (Na2SO4), the filtrate was concentrated. The residue was purified by CC (SiO2; PE/AcOEt 5 :1) to
give ethyl (3S,6S,6aS,8aS,9R,12aS,12bS)-dodecahydro-3,6,9,12a-tetramethyl-4-oxo-2H-3,6a-methano-
naphtho[2,1-c]oxocine-9(6H)-carboxylate (20; 0.278 g, 74%). M.p. 118.2 – 119.68. IR (KBr): 2957, 2913,
2843, 1714, 1451, 1381, 1238, 1172, 1025. 1H-NMR (400 MHz, CDCl3): 4.32 – 4.20 (m, 1 H); 4.15 – 4.02 (m,
2 H); 2.19 (d, J ¼ 18.4, 1 H); 2.09 – 1.54 (m, 9 H); 1.46 – 1.37 (m, 3 H); 1.26 (t, J ¼ 7.2, 3 H); 1.17 (s, 3 H);
1.14 (s, 3 H); 1.10 (d, J ¼ 2.8, 3 H); 1.09 – 0.82 (m, 5 H); 0.74 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.0;
175.4; 85.6; 60.5; 57.1; 55.5; 48.7; 44.7; 43.6; 41.8; 39.9; 38.6; 38.4; 37.8; 34.9; 33.6; 28.7; 28.2; 25.6; 19.5;
18.8; 14.5; 13.4. HR-ESI-MS: 399.2534 ([M þ Na]þ, C23H36NaO4þ ; calc. 399.2511).
Treatment of 18 with H2O2. Compound 18 (0.360 g, 1 mmol) was dissolved in MeOH (5 ml), and then
NaOH (0.080 g, 2 mmol) and H2O2 (40%, 0.5 ml) were added. After stirring at 658 for 4 h, the mixture
was concentrated, and the residue was extracted with CH2Cl2 and H2O. Then, the org. layer was washed
with sat. aq. NaCl soln., dried (MgSO4), and the filtrate was concentrated. The residue was purified by
CC (SiO2; PE/AcOEt 3 :1) to give ethyl (5b,8a,9b,10a,13a)-8-ethenyl-13-hydroxy-13-methylpodocarpan-
15-oate (22; 0.261 g, 75%). M.p. 80.1 – 81.28. IR (KBr): 3426, 3080, 2933, 2872, 2843, 1718, 1626, 1453,
1388, 1183, 1153, 1031, 905. 1H-NMR (400 MHz, CDCl3): 6.54 (dd, J ¼ 17.6, 10.8, 1 H); 5.11 (d, J ¼ 11.2,
1 H); 5.06 (d, J ¼ 17.6, 1 H); 4.12 – 4.02 (m, 2 H); 2.18 – 2.08 (m, 2 H); 1.96 – 1.39 (m, 11 H); 1.24 (t, J ¼ 7.2,
3 H); 1.16 (s, 3 H); 1.13 (s, 3 H); 1.10 – 0.86 (m, 5 H); 0.70 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.3;
144.1; 111.7; 70.3; 59.9; 58.2; 58.1; 57.7; 43.7; 40.9; 40.5; 40.1; 39.7; 38.1; 38.1; 31.8; 28.7; 19.6; 19.1; 16.7;
14.0; 13.5. HR-ESI-MS: 371.2561 ([M þ Na]þ, C22H36NaO3þ ; calc. 371.2562).
Reduction of 18. A mixture of 18 (0.360 g, 1 mmol) and NaBH4 (0.057 g, 1.5 mmol) in dry EtOH
(10 ml) was stirred at 08 for 10 min. Then, the mixture was concentrated under vacuum, and extracted
with CHCl3 and H2O. The org. layer was washed with sat. aq. NaCl soln., dried (MgSO4), and
concentrated under vacuum to give ethyl (5b,8a,9b,10a,13a)-8-ethenyl-13-(hydroxymethyl)-13-methyl-
podocarpan-15-oate as a white powder (23; 0.337 g, 96%). M.p. 115.1 – 116.78. IR (KBr): 3441, 3069,
2952, 2922, 2847, 1715, 1450, 1381, 1190, 1153. 1H-NMR (400 MHz, CDCl3): 6.51 (dd, J ¼ 17.6, 10.8, 1 H);
5.13 (d, J ¼ 17.6, 1 H); 5.08 (d, J ¼ 11.2, 1 H); 4.12 – 4.02 (m, 2 H); 3.56 (d, J ¼ 12, 1 H); 3.08 (d, J ¼ 12,
1 H); 2.14 – 2.06 (m, 2 H); 1.84 – 1.39 (m, 11 H); 1.22 (t, J ¼ 7.2, 3 H); 1.15 (s, 3 H); 1.13 – 0.85 (m, 5 H);
0.84 (s, 3 H); 0.66 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.1; 145.2; 110.7; 67.7; 59.6; 58.1; 57.4; 53.1;
43.4; 41.6; 40.5; 39.2; 37.8; 36.4; 34.9; 30.7; 29.0; 28.4; 19.7; 18.8; 17.0; 13.7; 13.3. HR-ESI-MS: 385.2709
([M þ Na]þ, C23H38NaOþ3 ; calc. 385.2719).
Cyclization of 23. Compound 23 (0.362 g, 1 mmol) was dissolved in CH2Cl2 (5 ml), and BF3 · OEt2
(48% soln. in Et2O; 0.47 ml, 1.5 mmol) was added dropwise during stirring of the mixture under reflux for
30 h. The soln. was then diluted with H2O and extracted with CH2Cl2, and the combined org. phases were
dried (Na2SO4), and then the filtrate was concentrated. The residue was purified by CC (SiO2; PE/
AcOEt 5 :1) to give ethyl (3S,6S,6aS,8aS,9R,12aS,12bS)-dodecahydro-3,6,9,12a-tetramethyl-2H-3,6a-
methanonaphtho[2,1-c]oxocine-9(6H)-carboxylate (24; 0.271 g, 75%). M.p. 158.2 – 159.78. IR (KBr):
2958, 2921, 2845, 1699, 1626, 1466, 1448, 1239, 1190, 1153, 921, 623. 1H-NMR (400 MHz, CDCl3): 4.11 –
4.01 (m, 2 H); 3.76 (d, J ¼ 11.2, 1 H); 3.63 – 3.56 (m, 1 H); 3.18 (d, J ¼ 11.2, 1 H); 2.18 – 1.89 (m, 3 H);
1.82 – 1.31 (m, 11 H); 1.22 (t, J ¼ 7.2, 3 H); 1.16 (s, 3 H); 1.08 (d, J ¼ 6.8, 3 H); 1.05 – 0.83 (m, 4 H); 0.81 (s,

Helvetica Chimica Acta – Vol. 93 (2010)
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3 H); 0.68 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.1; 76.5; 71.7; 59.4; 57.8; 56.4; 53.3; 44.4; 42.6; 40.5;
39.8; 37.9; 36.1; 35.2; 31.7; 29.4; 28.4; 22.3; 19.7; 18.9; 17.5; 14.8; 14.3. HR-ESI-MS: 385.2731 ([M þ Na]þ,
C23H38NaOþ3 ; calc. 385.2719).
Treatment of 23 with TsCl. A mixture of 23 (0.362 g, 1 mmol) and TsCl (0.238 g, 1.1 mmol) in
pyridine (5 ml) was stirred at r.t. for 12 h. Then, the mixture was filtered, and the filtrate was extracted
with CH2Cl2 and aq. HCl soln. (5m). The org. layer was washed with sat. aq. NaCl soln., dried (MgSO4),
and the filtrate was concentrated. The residue was purified by CC (SiO2; PE/AcOEt 5 :1) to give ethyl
(5b,8a,9b,10a,13a)-8-ethenyl-13-methyl-13-({[(4-methylphenyl)sulfonyl]oxy}methyl)podocarpan-15-
oate (25; 0.438 g, 85%). IR (KBr): 3076, 2933, 2872, 2849, 1720, 1598, 1455, 1363, 1180, 959, 844, 666.
1H-NMR (400 MHz, CDCl3): 7.76 (d, J ¼ 8.0, 2 H); 7.34 (d, J ¼ 8.0, 2 H); 6.07 (dd, J ¼ 17.6, 10.8, 1 H);
4.99 – 4.93 (m, 2 H); 4.12 – 4.01 (m, 2 H); 3.95 (d, J ¼ 9.6, 1 H); 3.57 (d, J ¼ 9.2, 1 H); 2.92 (d, J ¼ 28.0,
1 H); 2.44 (s, 3 H); 2.13 (d, J ¼ 13.2, 1 H); 1.99 (d, J ¼ 13.2, 1 H); 1.83 – 1.39 (m, 11 H); 1.25 (t, J ¼ 7.2,
3 H); 1.12 (s, 3 H); 1.08 – 0.93 (m, 6 H); 0.81 (s, 3 H); 0.59 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.3;
144.5; 143.0; 133.0; 130.2; 129.7; 127.9; 127.0; 112.5; 59.9; 58.4; 57.6; 54.9; 43.6; 42.1; 40.4; 39.5; 38.0; 37.9;
35.2; 34.6; 29.7; 28.7; 28.2; 21.6; 19.9; 19.0; 16.9; 14.0; 13.5. HR-ESI-MS: 539.2802 ([M þ Na]þ,
C30H44NaO5Sþ; calc. 539.2807).
Treatment of 25 with NaN3. A mixture of 25 (0.516 g, 1 mmol) and NaN3 (0.130 g, 2 mmol) in DMF
(5 ml) was stirred at 808 for 3 h. Then, the mixture was extracted with CH2Cl2 and H2O. The org. layer
was concentrated, and the residue was dissolved in THF (5 ml), and then the Ph3P (0.524 g, 2 mmol) and
H2O (0.05 ml) were added. After stirring at 658 for 3 h, the mixture was concentrated, and the aq. HCl
soln. (0.5M) was added to attain pH < 3. The H2O layer was extracted with Et2O, the org. layer was
washed with sat. aq. NaCl soln., dried (MgSO4), and the filtrate was concentrated. The residue was
purified by CC (SiO2; PE/AcOEt 1:1) to give ethyl (5b,8a,9b,10a,13a)-13-(aminomethyl)-8-ethenyl-13-
methylpodocarpan-15-oate (26; 0.245 g, 68%). IR (KBr): 3356, 3075, 2948, 2921, 2845, 1721, 1456, 1381,
1191, 1081. 1H-NMR (400 MHz, CDCl3): 6.28 (dd, J ¼ 17.6, 10.8, 1 H); 5.10 (d, J ¼ 17.6, 1 H); 5.07 (d, J ¼
11.2, 1 H); 4.06 – 3.96 (m, 2 H); 3.26 (d, J ¼ 10.8, 1 H); 2.88 (d, J ¼ 10.8, 1 H); 2.18 – 1.74 (m, 4 H); 1.70 –
1.31 (m, 9 H); 1.25 (t, J ¼ 7.2, 3 H); 1.16 (s, 3 H); 1.13 – 0.83 (m, 5 H); 0.78 (s, 3 H); 0.67 (s, 3 H). 13C-NMR
(100 MHz, CDCl3): 177.4; 146.1; 111.7; 62.7; 59.8; 58.4; 57.1; 54.1; 42.4; 41.1; 39.8; 39.2; 36.8; 35.4; 34.4;
31.2; 29.4; 27.1; 20.2; 18.7; 17.4; 14.3; 13.1. HR-ESI-MS: 362.3041 ([M þ H]þ, C23H40NO2þ ; calc.
362.3059).
Treatment of 18 with HONH2 · HCl. A mixture of 18 (0.360 g, 1 mmol) and HONH2 · HCl (0.103 g,
1.5 mmol) in EtOH was stirred in presence of NaHCO3 at 608 for 2 h. Then, the mixture was
concentrated under vacuum, and extracted with CH2Cl2 and H2O. The org. layer was washed with sat. aq.
NaCl soln., dried (MgSO4), and concentrated under vacuum to give ethyl (5b,8a,9b,10a,13a)-8-ethenyl-
13-[(E)-(hydroxyimino)methyl]-13-methylpodocarpan-15-oate (27; 0.363 g, 97%). White powder. M.p.
148.5 – 149.68. IR (KBr): 3439, 3070, 2954, 2921, 2855, 1699, 1627, 1448, 1377, 1239, 1190, 1153, 1025, 943,
628. 1H-NMR (400 MHz, CDCl3): 7.13 (s, 1 H); 6.10 (dd, J ¼ 17.6, 10.8, 1 H); 5.05 (d, J ¼ 10.8, 1 H); 4.98
(d, J ¼ 17.6, 1 H); 4.09 – 4.02 (m, 2 H); 2.24 (dd, J ¼ 13.2, 9.6, 1 H); 2.15 – 2.09 (m, 2 H); 1.89 – 1.40 (m,
7 H); 1.26 (d, J ¼ 13.2, 1 H); 1.20 (t, J ¼ 7.2, 3 H); 1.14 (s, 3 H); 1.10 – 0.97 (m, 5 H); 0.94 (s, 3 H); 0.93 –
0.83 (m, 2 H); 0.61 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.4; 159.3; 143.5; 111.2; 59.8; 58.4; 58.2; 57.7;
43.7; 40.9; 39.8; 39.6; 38.1; 38.0; 36.7; 35.4; 29.8; 28.7; 19.8; 19.0; 17.1; 13.9; 13.3. HR-ESI-MS: 376.2848
([M þ H]þ, C23H38NOþ3 ; calc. 376.2852).
Cyclization of 27. To a soln. of 27 (0.375 g, 1 mmol) in toluene (5 ml), 48% BF3 · OEt2 (48% soln. in
Et2O, 0.05 ml, 0.17 mmol) was added dropwise, and the mixture was heated under N2 for 2 h at 1188. H2O
(10 ml) was added to the mixture, which was then neutralized with NaHCO3, and the org. phase was
separated and dried (Na2SO4). After evaporation in vacuo, the crude product was purified by CC (PE/
AcOEt 2 :1) to give ethyl (3S,3aS,6aS,6bS,8aS,9R,12aS,12bS)-hexadecahydro-3,9,12a-trimethyl-3,6b-
methanonaphtho[2’,1’:3,4]cyclohepta[1,2-c][1,2]oxazole-9-carboxylate (28; 0.356 g, 95%). White pow-
der. M.p. 136.5 – 138.18. IR (KBr): 3026, 2956, 2934, 2872, 2855, 1704, 1467, 1382, 1238, 1178, 1149, 1052,
1
1029, 976, 859. H-NMR (400 MHz, CDCl3): 4.16 – 4.04 (m, 2 H); 3.84 – 3.78 (m, 1 H); 3.74 – 3.70 (m,
1 H); 3.30 (d, J ¼ 7.2, 1 H); 2.90 (q, J ¼ 6.4, 1 H); 2.17 (d, J ¼ 13.2, 1 H); 1.83 – 1.43 (m, 8 H); 1.36 – 1.28
(m, 4 H); 1.25 (t, J ¼ 7.2, 3 H); 1.17 (s, 3 H); 1.10 (dd, J ¼ 12.0, 2.0, 1 H); 1.09 – 0.97 (m, 2 H); 0.95 (s, 3 H);
0.93 – 0.80 (m, 2 H); 0.79 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.3; 73.0; 72.7; 59.9; 57.6; 56.3; 52.0;

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Helvetica Chimica Acta – Vol. 93 (2010)
51.1; 44.9; 43.6; 40.3; 39.9; 39.2; 38.2; 37.8; 35.5; 28.9; 21.7; 21.5; 19.2; 18.9; 14.1; 13.7. HR-ESI-MS:
376.2835 ([M þ H]þ, C23H38NOþ3 ; calc. 376.2852).
Treatment of 28 with MeI. To a soln. of 28 (0.375 g, 1 mmol) and NaH (0.026 g, 1.1 mmol) in DMF
(5 ml), MeI (0.156 g, 1.1 mmol) was added dropwise, and the mixture was heated at 508 for 2 h. The
mixture was filtered, and the filtrate was concentrated. The residue was extracted with CHCl3 and H2O.
The org. layer was washed with sat. aq. NaCl soln., dried (MgSO4), and concentrated under vacuum. The
residue was purified by CC (SiO2; PE/AcOEt 6 :1) to give ethyl (3S,3aS,6aS,6bS,8aS,9R,12aS,12bS)-
hexadecahydro-3,4,9,12a-tetramethyl-3,6b-methanonaphtho[2’,1’:3,4]cyclohepta[1,2-c][1,2]oxazole-9-
carboxylate (29; 0.330 g, 85%). M.p. 158.6 – 159.98. IR (KBr): 2957, 2941, 2859, 1714, 1458, 1384, 1239,
1181, 1150, 1039, 978. 1H-NMR (400 MHz, CDCl3): 4.11 – 4.02 (m, 2 H); 3.92 (dd, J ¼ 8.4, 8.0, 1 H), 3.58
(dd, J ¼ 8.4, 3.6, 1 H); 3.13 (d, J ¼ 7.2, 1 H); 2.92 – 2.85 (m, 1 H); 2.78 (s, 3 H); 2.17 (d, J ¼ 12.4, 1 H);
1.83 – 1.43 (m, 8 H); 1.36 – 1.28 (m, 3 H); 1.26 (t, J ¼ 7.2, 3 H); 1.16 (s, 3 H); 1.12 – 0.97 (m, 3 H); 0.89 (s,
3 H); 0.87 – 0.77 (m, 3 H); 0.75 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.5; 79.5; 71.6; 59.8; 57.6; 56.3;
53.2; 52.0; 44.9; 43.6; 41.3; 39.8; 39.2; 38.5; 38.2; 37.6; 34.5; 28.4; 21.9; 20.7; 19.8; 19.2; 13.9; 13.1. HR-ESI-
MS: 412.2814 ([M þ Na]þ, C24H39NNaO3þ ; calc. 412.2828).
Treatment of 27 with H2SO4. To a soln. of 27 (0.375 g, 1 mmol) in acetone (5 ml), a mixture of H2SO4
(0.09 ml) and acetone (5 ml) was added dropwise, and the mixture was heated under N2 for 2 h at 408.
H2O (10 ml) was added to the mixture, which was then neutralized with NaHCO3, and the org. phase was
separated and dried (Na2SO4). After evaporation in vacuo, the crude product was purified by CC (PE/
AcOEt 4 :1) to give ethyl (5b,8a,9b,10a,13a)-13-cyano-8-ethenyl-13-methylpodocarpan-15-oate (30;
0.253 g, 71%). White powder. M.p. 124.5 – 126.18. IR (KBr): 3097, 2978, 2937, 2852, 2226, 1725, 1634,
1452, 1379, 1225, 1148, 1014, 902, 772. 1H-NMR (400 MHz, CDCl3): 6.54 (dd, J ¼ 17.6, 10.8, 1 H); 5.19 (d,
J ¼ 10.8, 1 H); 5.12 (d, J ¼ 17.6, 1 H); 4.12 – 4.01 (m, 2 H); 2.22 – 2.16 (m, 3 H); 1.76 – 1.33 (m, 10 H); 1.29
(s, 3 H); 1.22 (t, J ¼ 7.2, 3 H); 1.15 (s, 3 H); 1.11 – 0.85 (m, 5 H); 0.84 (s, 3 H). 13C-NMR (100 MHz,
CDCl3): 177.2; 141.4; 125.5; 112.3; 59.9; 58.1; 57.8; 57.6; 55.2; 52.9; 43.7; 40.5; 39.7; 39.6; 38.0; 31.5; 29.6;
28.6; 26.2; 19.6; 18.0; 13.9; 13.5. HR-ESI-MS: 380.2558 ([M þ Na]þ, C23H35NNaO2þ ; calc. 380.2566).
Treatment of 18 with PhNHNH2. To a soln. of 18 (0.360 g, 1 mmol) in EtOH (10 ml), PhNHNH2
(0.10 ml, 1.00 mmol) and 2 drops of glacial AcOH were added. The mixture was stirred for 2 h at r.t. The
soln. was then poured into H2O (10 ml), and the white precipitate was filtered, washed with H2O, and
dried. The product was recrystallized from CHCl3/light PE to give ethyl (5b,8a,9b,10a,13a)-8-ethenyl-13-
methyl-13-[(E)-(2-phenylhydrazinylidene)methyl]podocarpan-15-oate (31a; 0.427 g, 95%). M.p. 132.1 –
133.78. IR (KBr): 3297, 3134, 2949, 2925, 1720, 1698, 1601, 1510, 1452, 1396, 1256, 1185, 1114, 749.
1H-NMR (400 MHz, CDCl3): 7.22 (t, J ¼ 8.0, 2 H); 6.98 (d, J ¼ 8.0, 2 H); 6.79 (t, J ¼ 7.2, 1 H); 6.66 (s, 1 H);
6.08 (dd, J ¼ 17.6, 11.2, 1 H); 4.94 (s, 1 H); 4.91 (d, J ¼ 4.4, 1 H); 4.06 – 3.88 (m, 2 H); 2.49 (dd, J ¼ 12.8,
2.0, 1 H); 2.14 – 2.10 (m, 2 H); 1.91 – 1.44 (m, 8 H); 1.33 – 1.26 (m, 2 H); 1.20 (t, J ¼ 7.2, 3 H); 1.14 (s, 3 H);
1.12 – 0.96 (m, 5 H); 0.93 (s, 3 H); 0.89 – 0.84 (m, 1 H); 0.56 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.9;
154.6.0; 146.3; 140.7; 129.6; 129.6; 120.7; 118.5; 118.5; 112.3; 61.9; 59.9; 58.1; 53.7; 43.8; 43.4; 40.2; 40.1;
38.3; 37.9; 37.8; 36.6; 29.1; 22.5; 21.8; 21.3; 19.0; 14.1; 12.5. HR-ESI-MS: 473.3138 ([M þ Na]þ,
C29H42N2NaOþ2 ; calc. 473.3144).
Treatment of 18 with 4-Nitrophenylhydrazine. To a soln. of 18 (0.360 g, 1 mmol) in EtOH (10 ml), 4-
nitrophenylhydrazine (0.154 g, 1.00 mmol), and 2 drops of glacial AcOH were added. The mixture was
stirred for 2 h at r.t. The soln. was then poured into H2O (10 ml), and the white precipitate was filtered,
washed with H2O, and dried. The product was recrystallized from CHCl3/light PE to give ethyl
(5b,8a,9b,10a,13a)-8-ethenyl-13-methyl-13-{(E)-[2-(4-nitrophenyl)hydrazinylidene]methyl}podocarpan-
15-oate (31b; 0.420 g, 85%). M.p. 205.6 – 206.98. IR (KBr): 3312, 3088, 2944, 2847, 1702, 1593, 1319, 1270,
1
1167, 1104, 906, 841. H-NMR (400 MHz, CDCl3): 8.14 (d, J ¼ 9.2, 2 H); 7.61 (s, 1 H); 6.97 (d, J ¼ 9.2,
2 H); 6.81 (s, 1 H); 6.02 (dd, J ¼ 18.0, 10.8, 1 H); 4.98 (s, 1 H); 4.94 (d, J ¼ 3.2, 1 H); 4.06 – 3.98 (m, 2 H);
2.51 – 2.44 (m, 1 H); 2.12 (t, J ¼ 12.4, 2 H); 1.86 – 1.31 (m, 9 H); 1.18 (t, J ¼ 7.2, 3 H); 1.14 (s, 3 H); 1.12 –
0.83 (m, 4 H); 0.96 (s, 3 H); 0.92 – 0.83 (m, 4 H); 0.57 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.3; 153.0;
150.3; 144.3; 139.4; 126.2; 126.2; 111.0; 111.0; 110.8; 59.8; 58.5; 58.1; 57.7; 43.7; 40.9; 39.9; 39.6; 38.1; 37.3;
35.3; 29.8; 29.6; 28.7; 19.8; 19.0; 17.1; 13.9; 13.4. HR-ESI-MS: 518.2996 ([M þ Na]þ, C29H39N3NaO4þ ; calc.
518.2995).

Helvetica Chimica Acta – Vol. 93 (2010)
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Cyclization of 31a. To a soln. of 31a (0.450 g, 1 mmol) in toluene (5 ml), 48% BF3 · OEt2 (48% soln.
in Et2O, 0.05 ml, 0.17 mmol) was added dropwise, and the mixture was heated under a N2 atmosphere for
2 h at 1188. H2O (10 ml) was added to the mixture, which was then neutralized with NaHCO3, and the
org. phase was separated and dried (Na2SO4). After evaporation in vacuo, the crude product was purified
by CC (PE/AcOEt 2 :1) to give ethyl (3S,6bS,8aS,9R,12aS,12bS)-1,3,5,7,8,8a,9,10,11,12,12a,12b-dodec-
ahydro-3,9,12a-trimethyl-5-phenyl-2H-3,6b-methanonaphtho[2’,1’:3,4]cyclohepta[1,2-c]pyrazole-9-car-
boxylate (32; 0.374 g, 84%). White powder. M.p. 61.9 – 63.48. IR (KBr): 3113, 2949, 2847, 1720, 1598,
1
1572, 1506, 1381, 1150, 1033, 948, 756, 690. H-NMR (400 MHz, CDCl3): 7.61 (d, J ¼ 8.0, 2 H); 7.50 (s,
1 H); 7.39 (t, J ¼ 8.0, 2 H); 7.17 (t, J ¼ 7.2, 1 H); 4.20 – 4.08 (m, 2 H); 2.19 (d, J ¼ 13.6, 1 H); 2.06 – 1.92 (m,
4 H); 1.77 – 1.40 (m, 9 H); 1.35 (s, 3 H); 1.30 (t, J ¼ 7.2, 3 H); 1.23 (s, 3 H); 1.20 – 0.86 (m, 4 H); 0.59 (s,
3 H). 13C-NMR (100 MHz, CDCl3): 177.3; 167.0; 140.9; 130.7; 129.2; 129.2; 125.0; 120.7; 118.5; 118.5;
65.3; 59.9; 57.1; 53.5; 43.8; 43.4; 40.2; 40.1; 38.3; 37.9; 37.8; 36.6; 29.1; 22.5; 21.8; 21.3; 19.0; 14.1; 12.5. HR-
ESI-MS: 447.3009 ([M þ H]þ, C29H39N2O2þ ; calc. 447.3012).
Cyclization of 31b. To a soln. of 31b (0.450 g, 1 mmol) in toluene (5 ml), 48% BF3 · OEt2 (48% soln.
in Et2O, 0.05 ml, 0.17 mmol) was added dropwise, and the mixture was heated under N2 for 2 h at 1188.
H2O (10 ml) was added to the mixture, which was then neutralized with NaHCO3, and the org. phase was
separated and dried (Na2SO4). After evaporation in vacuo, the crude product was purified by CC (PE/
AcOEt 2 :1) to give ethyl (3S,6aR,6bS,8aS,9R,12aS,12bS)-1,3,5,6,6a,7,8,8a,9,10,11,12,12a,12b-tetradeca-
hydro-3,9,12a-trimethyl-5-(4-nitrophenyl)-2H-3,6b-methanonaphtho[2’,1’:3,4]cyclohepta[1,2-c]pyrazole-
9-carboxylate (33; 0.406 g, 75%). White powder. M.p. 186.8 – 188.78. IR (KBr): 3298, 3110, 2951, 2849,
1720, 1619, 1590, 1516, 1333, 1275, 1140, 920. 1H-NMR (400 MHz, CDCl3): 10.92 (s, 1 H); 9.11 (d, J ¼ 2.4,
1 H); 8.29 (d, J ¼ 9.6, 1 H); 7.91 (d, J ¼ 9.6, 1 H); 7.45 (s, 1 H); 4.11 – 4.02 (m, 2 H); 2.41 – 2.33 (m, 1 H);
2.01 – 1.62 (m, 9 H); 1.56 – 1.28 (m, 6 H); 1.23 (t, J ¼ 7.2, 3 H); 1.16 (s, 3 H); 1.06 (s, 3 H); 1.04 – 0.83 (m,
4 H); 0.71 (t, J ¼ 7.2, 3 H); 0.69 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.2; 158.9; 145.2; 137.6; 129.9;
128.7; 123.5; 116.4; 59.8; 59.5; 58.1; 49.1; 43.7; 40.5; 38.7; 38.4; 38.0; 38.0; 37.7; 36.4; 30.5; 28.7; 20.8; 19.3;
19.1; 17.4; 14.8; 14.0; 7.7. HR-ESI-MS: 565.3013 ([M þ Na]þ, C29H42N4NaO6þ ; calc. 565.3002).
Treatment of 23 with m-CPBA. A mixture of 23 (0.362 g, 1 mmol) and m-CPBA (0.258 g, 1.5 mmol)
in CHCl3 (5 ml) was stirred at 08 for 5 h, and then the mixture was poured into H2O and neutralized with
aq. NaHCO3 soln. The org. layer was washed with sat. aq. NaCl soln., dried (MgSO4), and concentrated
under vacuum. The residue was purified by CC (SiO2; PE/AcOEt 2 :1) to give ethyl (3S,6R,6aS,8aS,
9R,12aS,12bS)-dodecahydro-6-(hydroxymethyl)-3,9,12a-trimethyl-2H-3,6a-methanonaphtho[2,1-c]oxo-
cine-9(6H)-carboxylate (35; 0.293 g, 78%). M.p. 85.5 – 86.78. IR (KBr): 3576, 2982, 2928, 2846, 1716,
1459, 1380, 1328, 1235, 1179, 1149, 1023, 969. 1H-NMR (400 MHz, CDCl3): 4.16 – 4.04 (m, 2 H); 3.87 (d,
J ¼ 8.4, 2 H); 3.71 (d, J ¼ 9.2, 1 H); 3.28 (dd, J ¼ 10.4, 2.2, 1 H); 3.22 (d, J ¼ 8.0, 1 H); 2.14 (d, J ¼ 13.6,
1 H); 2.09 – 2.04 (m, 1 H); 1.87 – 1.34 (m, 9 H); 1.27 (t, J ¼ 7.2, 3 H); 1.24 – 1.15 (m, 2 H); 1.14 (s, 3 H);
1.08 – 0.80 (m, 5 H); 0.72 (s, 3 H); 0.66 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.1; 89.9; 79.8; 64.7; 59.9;
59.6; 58.1; 57.2; 43.6; 40.5; 39.7; 38.6; 38.0; 36.7; 36.2; 31.9; 28.6; 25.5; 22.3; 21.0; 19.3; 14.1; 14.0. HR-ESI-
MS: 399.2502 ([M þ Na]þ, C23H36NaO4þ ; calc. 399.2511).
Cyclization of Compound 18. A mixture of 18 (0.360 g, 1 mmol), NaIO4 (0.319 g, 1.5 mmol), and
NaBr (0.153 g, 1.5 mmol) in glacial AcOH (10 ml) was stirred at 708 for 8 h, then, the mixture was
concentrated under vacuum, and extracted with H2O and CHCl3. The org. layer was washed with aq.
Na2S2O4 soln. and sat. aq. NaCl soln., dried (MgSO4), and concentrated under vacuum. The residue was
purified by CC (SiO2; PE/AcOEt 4 :1) to give ethyl (3S,4S,7R,7aS,9aS,10R,13aS,13bS)-tetradecahydro-
3,10,13a-trimethyl-2H-4,7-epoxy-3,7a-methanonaphtho[2,1-d]oxonine-10-carboxylate (37; 0.245 g, 65%).
1
M.p. 83.3 – 84.58. IR (KBr): 2945, 2929, 1725, 1464, 1377, 1226, 1156, 989. H-NMR (400 MHz, CDCl3):
4.95 (s, 1 H); 4.74 – 4.66 (m, 1 H); 4.14 – 4.06 (m, 2 H); 3.87 (d, J ¼ 7.2, 1 H); 3.55 – 3.51 (m, 1 H); 2.18 –
2.13 (m, 2 H); 1.88 – 1.44 (m, 11 H); 1.26 (t, J ¼ 7.2, 3 H); 1.16 (s, 3 H); 1.15 – 1.11 (m, 5 H); 0.91 (s, 3 H);
0.76 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.2; 108.1; 78.8; 65.7; 60.0; 57.9; 57.9; 44.6; 43.7; 39.6; 38.1;
37.9; 37.2; 36.7; 35.0; 29.7; 28.8; 24.6; 21.6; 19.7; 19.1; 14.1; 13.3. HR-ESI-MS: 379.2840 ([M þ H]þ,
C23H39Oþ4 ; calc. 379.2848).
Reduction of 38. A mixture of 38 (0.435 g, 1 mmol) and NaBH4 (0.057 g, 1.5 mmol) in dry EtOH
(10 ml) was stirred at 08 for 1 h. Then, the mixture was concentrated under vacuum, and extracted with
CHCl3 and H2O. The org. layer was washed with sat. aq. NaCl soln., dried (MgSO4), and concentrated

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Helvetica Chimica Acta – Vol. 93 (2010)
under vacuum. The residue was purified by CC (SiO2; PE/AcOEt 4 :1) to give ethyl (5b,8a,9b,10a,13a)-
8-ethenyl-13-methyl-13-[(phenylamino)methyl]podocarpan-15-oate (39; 0.419 g, 96%). M.p. 57.5 – 59.18.
IR (KBr): 3422, 3063, 2938, 2843, 1721, 1595, 1486, 1452, 1379, 1149, 1093, 963, 754, 691. 1H-NMR
(400 MHz, CDCl3): 7.13 (t, J ¼ 8.0, 2 H); 6.65 (t, J ¼ 7.2, 1 H); 6.56 (d, J ¼ 7.6, 2 H); 6.40 (dd, J ¼ 17.6, 11.2,
1 H); 5.10 (d, J ¼ 17.6, 1 H); 5.01 (d, J ¼ 11.2, 1 H); 4.12 – 4.01 (m, 2 H); 2.98 – 2.92 (m, 2 H); 2.17 – 2.11
(m, 2 H); 1.87 – 1.41 (m, 10 H); 1.21 (t, J ¼ 7.2, 3 H); 1.15 (s, 3 H); 1.12 – 0.87 (m, 6 H); 0.85 (s, 3 H); 0.65
(s, 3 H). 13C-NMR (100 MHz, CDCl3): 177.4; 149.2; 144.0; 129.1; 129.1; 116.8; 113.1; 113.1; 111.0; 59.8;
58.8; 57.8; 54.8; 51.0; 43.7; 41.6; 40.8; 39.6; 38.5; 38.1; 34.6; 30.2; 29.6; 28.7; 19.9; 19.1; 17.3; 14.0; 13.6. HR-
ESI-MS: 438.3374 ([M þ H]þ, C29H44NO2þ ; calc. 438.3372).
Antibacterial Activity Assay. The bacteria strains were Staphylococcus aureus CMCC(B)26003,
Bacillus subtilis CMCC(B)63501, and Shigella flexneri 626. All tested bacteria strains were purchased
from Henan Provincial Institute of Food and Drug Control except Shigella flexneri 626, a multidrug-
resistant strain separated from clinical studies. For the determination of the antibacterial susceptility,
yeast extract (Oxoid, USA), tryptone (Oxoid, USA), and Mueller– Hinton Broth (MHB, Beijing
Aoboxing, China) were used.
Minimum Inhibitory Concentration (MIC) Measurements. Each tested compound was dissolved in
DMSO before serial two-fold dilution into the desired testing concentration ranges using sterile liquid
medium. DMSO was used for solvent control test, and the final concentration of DMSO was 2% in all the
tested samples. The seed was cultured in Shigella flexneri 626 in MHB culture medium and others in
Luria – Bertani (LB) culture medium, until containing 109 colony forming units (cfu) per ml. All media,
sterilized by autoclave at 1218 for 20 min, were used to dilute microorganism in the exponential-growth
phase, until the final concentration of microorganism was 105 cfu/ml in the 96-well plates, and then
inoculated to the 96-well plates and mixed with the compound to be tested. Every microplate had a
negative control and a blank without bacterium, and a sample blank. The MIC value was defined as the
lowest concentration of tested compounds, allowing no visible growth of test-strain bacteria after an
incubation at 378 for 6 h. Absorbance was measured by ELISA reader (Bio-Tek Instruments, Microplate
Autoreader, power waveX) at 450 nm.
X-Ray Crystallographic Analysis. X-Ray crystal data of compounds 33 and 37 were collected by a
Rigaku AFC5R diffractometer with graphite-monochromated MoKa radiation (l ¼ 0.71073 ꢃ). The
structure was solved by the direct methods and refined with a full-matrix least-squares method.
Crystal Data for Compound 33. C30H39N2O4, Mr 491.63, orthorhombic, space group P212121, a ¼
7.6600(15), b ¼ 21.257(4), c ¼ 32.780(7), V¼ 5337.6(18) ꢃ3, Z ¼ 8, m(MoKa) ¼ 0.081 cmꢀ1, F(000) ¼
2120, Dc ¼ 1.224 g/cm3, crystal dimensions: 0.20 ꢁ 0.18 ꢁ 0.17 mm. A total of 15417 reflections (5091
unique) were collected using the w-2q scan technique to a maximum 2q value of 558, and 4289 reflections
with I > 2s (I) were used in the structure determination. Final R and Rw values were 0.0619 and 0.1381,
resp. The maximum and minimum peaks in the difference map were 0.195 and ꢀ 0.232 e ꢃꢀ3, resp. The
data have been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication No. CCDC-714716.
Crystal Data for Compound 37. C23H36O4, Mr 376.52, orthorhombic, space group P212121, a ¼
8.9274(18), b ¼ 9.1642(18), c ¼ 25.500(5), V¼ 2086.2(7) ꢃ3, Z ¼ 4, m(MoKa) ¼ 0.080 cmꢀ1, F(000) ¼
824, Dc ¼ 1.199 g/cm3, crystal dimensions: 0.20 ꢁ 0.18 ꢁ 0.17 mm. A total of 6333 reflections (2147
unique) were collected using the w-2q scan technique to a maximum 2q value of 518, and 1891 reflections
with I > 2s (I) were used in the structure determination. Final R and Rw values were 0.0680 and 0.1666,
resp. The maximum and minimum peaks in the difference map were 0.257 and ꢀ 0.259 e ꢃꢀ3, resp. The
data have been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication No. CCDC-705231.
The authors are grateful for the financial support to the National Natural Science Foundation of
China (Project No. 20772113) and the Doctoral Research Fund of Henan Chinese Medicine.

Helvetica Chimica Acta – Vol. 93 (2010)
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Received December 2, 2009