Synthesis of trinorditerpenoid derivatives and evaluation of antimicrobial and cytotoxic activity

Article abstract of DOI:10.1007/s10600-005-0139-2

The synthesis of optically active trinorditerpenes was carried out, and their antimicrobial and antitumor activity was tested. The synthetic derivative 12-hydroxypodocarpa-8,11,13-triene (7) showed GI₅₀ at 6.6 μM against breast cancer MDA-MB-43

Full text of DOI:10.1007/s10600-005-0139-2

Chemistry of Natural Compounds, Vol. 41, No. 3, 2005
SYNTHESIS OF TRINORDITERPENOID DERIVATIVES AND
EVALUATION OF ANTIMICROBIAL AND CYTOTOXIC ACTIVITY
R. Alonso,1* H. Gomis,1 A. Taddei,2 and C. Sajo2
UDC 547.41
The synthesis of optically active trinorditerpenes was carried out, and their antimicrobial and antitumor
µM
7
activity was tested. The synthetic derivative 12-hydroxypodocarpa-8,11,13-triene ( ) showed GI50 at 6.6
against breast cancer MDA-MB-435 (LC50 = 50.9 µM and log10 GI50 = –5.18). The 12-acetyloxypodocarpa-
8,11,13-triene (8) showed GI50 at 12.1 µM against leukemia RPMI-8226 (LC50 = 76.1 µM and
log10 GI50 = –4.92).
Key words: trinorditerpenoid derivatives, antimicrobial and cytotoxic activity.
The trinorditerpenes have attracted the interest of natural product chemists during the past decades. In this context,
simple and sequential modifications were performed on the methyl O-methylpodocarpane molecule (1) because it is a chiral
starting material for the synthesis of diterpenoids with biological activity [1–3]. Our goal was to make this class of
trinorditerpenoids readily available for the investigation of the basic structural requirements to explore the antimicrobial and
antitumor activity.
The compounds synthesized by us were evaluated for their antimicrobial activity in a preliminary screening. We
observed that the compound 7 showed activity against Gram-positive bacteria, Gram-negative bacteria, yeast, and mold (Table
1). This particular antimicrobial ability attracted our attention to the compound 7 since it has a more simple structure than the
other compounds reported here. Of the eight compounds reported here, four were accepted and tested at the National
Institute of Health (NIH), Developmental Therapeutics Program (DTP), to evaluate the antitumor activity, where the
12-hydroxypodocarpa-8,11,13-triene (7) showed GI50 at 11.3 µM against melanoma SK-MEL5 (LC50 = 48.3 µM and
log10GI50= –4.95). The 12-acetyloxypodocarpa-8,11,13-triene (8) showed GI50 at 12.1 µM against leukemia RPMI-8226
(LC50 = 76.1 µM and log10GI50 = –4.92).
OM e
OM e
O M e
12
11
13
14
20
i
ii
iii
1
2
8
6
5
7
4
H
19
O
H
H
O
18
O
1
2
3
iv
O
S
O H
O
O
O M e
O H
O M e
O
v
vii
viii
H
H
H
H
R
6
7
4: R = CHO
5: R = CN
8
vi
i
ii.
iii.
iv.
NaI/Zn, DMF; TPAP, NMO, CH2Cl2, 87%;
. LiAlH4, THF, 89%; Ts-Cl, Py, THF, 90%;
v. vi.
NH2NH2, KOH, 48%; NH2NH2, KOH, Microwave, 25%;
vii. viii.
Py, acetic anhydride, 61%
AlCl3, CH2Cl2, 63%;
1) Centrode Quimica, InstitutoVenezolanode Investigaciones Cientificas (IVIC), Caracas 1020-A, Venezuela, fax+58(0)212-
5041469, e-mail:raalonso@ivic.ve; 2)DepartamentodeBiologiaCelular, UniversidadSimon Bolivar, (USB), Apartado89000,
Caracas1080-A, Venezuela. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 255-259, May-June, 2005. Originalarticle
submitted October 5, 2004.
0009-3130/05/4103-0315 ©2005 Springer Science+Business Media, Inc.
315

TABLE 1. Antimicrobial Activity of Compounds 2-8
Microorganisms
2
3
4
5
6
7
8
(C)
Pseudomonas aeruginosa
Escherichia coli
Staphylococcus aureus
Bacillus subtilis
Candida tropicalis
Aspergillus niger
-
-
13
12
13
12
10
-
-
-
-
-
-
-
-
-
-
-
9
-
-
10
-
-
-
8
15
11
-
12
10
17
20
14
16
7
9
-
-
-
20
22
20
30
31
25
-
-
______
Inhibition diameters in mm at 2 mg/mL, – = no activity, paper discs ( = 5 mm, 10 µL), (C) = antibiotic control: amikacine,
nalidilixic acid, vancomicine, and nistatine. Note: In the case of the inhibition zone around of the paper filter of 7–8 mm,
positive disc of inhibition is considered very small, which indicates that the different compounds are not very interesting. The
inhibition zone of9–14mmisinteresting;neverthelessthe compounds are still not important. Important inhibition zones appear
from 15 mm, and it is important to mention that those compounds can be considered as an alternative when problems of
resistance appear on the test.
TABLE 2. Primary Anticancer Assay of 3-Cell Line Panel
Growth percentage
Compound
Concentration
Breast
MCF7
N-S Cell Lung
NCI-H460
CNS
SF-268
2
5
7
8
1.00 E-04 M
1.00 E-04 M
1.00 E- 04 M
1.00 E-04 M
178
195
4
145
176
0
115
115
1
0
5
1
All these synthetic compounds 2, 4, 6, and 7 had physical and spectroscopic data identical to those reported by R.
Cambie and co-worker [1, 3, 4].
The starting material for preparation of compound 8 was methyl O-methyl podocarpane (1) (Scheme 1) which was
reduced with lithium aluminium hydride in THF to afford the alcohol compound 2 in 89% yield; then the protection of the
alcohol group using p-toluenesulfonyl chloride produced the compound 3 in 90% yield. In 1960, Matsumoto and co-workers
[5] reported the deoxygenation of the alcohol using p-toluenesulfonyl ether and the NaI/Zn system, but this method failed to
produce the compound 6. The aldehyde 4 was obtained in 87% yield by oxidation of the alcohol compound 2 using TPAP
reagents with NMO [6]. Wolff-Kishner reduction of the aldehyde 4 with partial demethylation affored the phenol 7 in 27.2%
yield [10] and compound 6 in 48% yield. R. Cambie and co-worker [7] reported identical spectroscopic data for compound 6;
we used here a Huang Minlon [8] modification of the Wolff-Kishner reduction process to increas the yield of compound 6 up
to 60%. Anpai Li and co-workers [9] reported similar spectroscopy data for compound 7 synthesized by us. We search another
method to reduce the C-19 aldehyde, and finally we used microwave-induced synthesis of hydrazones and Wolff-Kishner
reduction of compound 4, as reported by Jagir S. Sandhu and co-workers [10], but this method failed; perhaps we obtained the
compound 5 in 25% yield. Therefore, cleavage of the 12-methoxy group of compound 6 using AlCl conditions affords the
3
compound 7 in 63% yield. Acetylation of compound 7 produced a stable compound 8 in 61% yield.
Antimicrobial. These synthetic trinorditerpenoids 2, 6, and 7 were significant to continue exploring the
chemotherapeutic potential as an antitumor agent.
Antitumor. The evaluation of potential chemotherapeutic activity was performed on compounds 2, 5, 7, and 8 at DTP
against a panel of 3 cell lines MCF7 (breast), NCI-H460 (lung), and SF-268 (CNS) according to the standard protocol [11]
(Table 1). Both compounds 7 and 8 that have passed DTP criteria for activity in this assay were scheduled automatically for
evaluation against the full panel of 60 tumor cells derived from nine cancer types (leukaemia, non-small cell lung, colon, CNS,
melanoma, ovarian, renal, prostate, and breast) according to the standard protocol [12] (Table 2).
316

TABLE 3. In Vitro Antitumor Screening for Compounds 1 and 2
Compound
Cell line
7
8
G, %
GI50
LC50
Log10; GI50
Leukemia
G, %
GI50
LC50
Log10; GI50
CCRF-CEM
46
1
87.4
34.7
40.0
30.9
12.1
>100
>100
>100
>100
>100
76.1
-4.06
-4.46
-4.40
-4.51
-4.92
>-4.00
HL-60(TB)
K-562
-33
-61
-11
-65
6
16.0
10.9
11.7
12.3
8.72
>100
79.9
>100
75.5
>100
-4.80
-4.96
-4.93
-4.91
-5.06
14
21
-65
61
MOLT-4
RPMI-8226
SR
>100
N-SC Lung
A549/ATCC
EKVX
-77
-94
19.0
21.5
70.4
62.7
-4.72
-4.67
-22
-27
-93
-66
-70
-76
-84
-4
24.7
26.0
15.9
21.7
18.5
19.8
13.5
27.5
24.2
>100
>100
57.7
80.9
75.7
71.7
59.9
>100
>100
-
4.61
-4.59
-4.80
-4.6
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
-78
-78
-92
-57
47
18.2
17.3
18.3
18.6
21.5
68.7
68.0
60.8
89.9
>100
-4.74
-4.76
-4.74
-4.73
-4.67
-4.73
-4.70
-4.87
-4.56
-18
Colon
COLO 205
HCT-116
HCT-15
HT29
-48
24
20.4
42.2
17.1
2.32
14.1
13.8
>100
>100
72.6
82.2
69.0
60.9
-4.69
-4.37
-4.77
-4.63
-4.85
-4.86
-35
-96
-76
-1
21.9
18.4
19.5
31.7
19.2
28.2
>100
58.7
-4.66
-4.74
-4.71
-4.50
-4.72
-4.55
-72
-65
-73
-83
71.2
>100
>100
>100
KM-12
-47
8
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
-77
-52
-93
-78
-81
-91
16.4
20.6
18.3
17.0
12.4
14.6
67.9
96.8
59.8
67.7
61.0
58.0
-4.79
-44
-26
20.9
19.1
>100
>100
-4.68
-4.72
-4.69
-4.74
-4.77
-4.91
-4.84
-66
-92
19.6
19.9
79.5
61.8
-4.71
-4.70
Melanoma
LOX IMVI
M14
-78
6
16.9
28.2
20.1
19.6
11.3
19.6
13.1
67.5
>100
91.8
70.0
48.3
66.0
50.8
-4.77
-4.55
-4.70
-4.71
-4.95
-4.71
-4.88
-95
-88
4
17.7
18.2
29.8
19.3
30.9
18.2
17.6
58.3
62.6
-4.75
-4.74
-4.53
-4.71
-4.51
-4.74
-4.75
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
-56
-78
-100
-84
-100
>100
>100
>100
>100
61.2
-37
-18
-48
-89
317

TABLE 3. (continued)
Cell line
Compound
7
8
G, %
GI50
LC50
Log10; GI50
Ovarian
G, %
GI50
LC50
Log10; GI50
IGROV1
-74
-76
-77
-93
-70
20.4
17.0
16.6
14.0
21.7
73.5
69.2
68.1
55.5
77.8
-4.69
-4.77
-4.78
-4.85
-4.66
27
49.4
15.8
>100
>100
-4.31
-4.80
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
-47
-75
-55
22.3
18.8
73.8
91.9
-4.65
-4.73
Renal cancer
786-0
-11
-100
-88
27.3
16.2
18.1
19.5
16.0
17.5
18.2
19.9
>100
54.5
62.2
66.0
76.6
55.9
62.6
81.4
-4.56
-4.79
-4.74
-4.71
-4.80
-4.76
-4.74
-4.70
-94
18.1
59.3
-4.74
A 478
ACHN
CAKI-1
RXF 393
SN 12C
TK-10
UO-31
-97
7
19.1
28.0
41.2
18.6
58.8
>100
>100
58.8
63.5
>100
-4.72
-4.55
-4.39
-4.73
-84
-67
-10
-96
-78
-12
-100
-88
-65
31.3
20.1
-4.50
-4.70
Prostata cancer
PC-3
-92
-95
15.8
16.4
57.9
57.0
-4.80
-4.79
-71
75.7
DU-145
Breast cancer
MCF7
-48
-83
-95
-83
-88
-100
13.3
16.5
16.2
15.6
6.65
19.4
>100
63.9
57.0
62.9
50.9
58.0
-4.88
-4.78
-4.79
-4.81
-5.18
-4.71
34.6
26.0
17.8
24.7
16.8
36.5
37.5
>100
95.7
65.8
>100
95.5
>100
>100
-4.46
-4.59
-4.75
-4.61
-4.77
-4.44
-4.43
NCI/ADR/RES
MDA-MB 231
HS 578T
-53
-82
-34
-53
1
MDA-MB-435
BT-549
T-47D
10
______
G, % = Growth percentage at 1.00 E - 4.0 molar concentration, GI50 at µM concentration, LC50 at µM concentration, means
log10 GI50, and empty space = ND or not determined.
Compound 2 showed activity against Gram-positive bacteria, yeast, and mold (Table 1) but failed the primary
anticancer assay at DTP (Table 2).
The results of cytostatic and cytotoxic activity of both 7 and 8 are shown in Table 3. The synthetic trinorditerpene
compound 7 inhibited cell proliferation at 11.3 µM against melanoma SK-MEL5, and was active against all cell lines, with
mean log10 GI50 values ranging from –4.55 (melanoma M14) to –5.06 (leukemia SR). Cytotoxic effects, evaluated by the LC50
parameter, became visible at somewhat higher concentrations, i.e., 70.4 µM for the 60 cell line panels. The compound 8 is an
acetyl derivative of compound 7 and inhibited cell proliferations with GI50 at 12.1 µM against leukemia RPMI-8226. It was
active against all cell lines, with mean log10 GI50 values ranging from –4.31 (ovarian cancer IGROVI) to –4.92 (leukemia
RPMI-8226) (Table 3).
In fact, this work was an initial biological evaluation of these synthetic trinorditerpenoids. Both 7 and 8 had promising
activity against human cancer cell lines and are targets for future investigation in vivo in our laboratory. Further studies will
be required to elucidate the cell targets as antitumor.
318

EXPERIMENTAL
Melting points were measured on a Kofler hot-stage apparatus and are uncorrected. IR spectra were recorded with a
Nicolet 5DXC FT-IR spectrometer. NMR spectra were obtained for solutions in CDCl3 on a Bruker Avance-300 and 500
spectrometer. The assignments of carbon signals were made by means of 2D NMR 1H and 13C single bond and multiple bond
correlation studies. Mass spectra were determined on a Kratos MS 25 RFA spectrometer at 70 eV using a direct inlet system.
Rotations were measured in chloroform solutions at 25°C with a Perkin-Elmer instrument 341 polarimeter. Elemental analysis
was performed at Atlantis Microlab. For column chromatography, silica gel 60 (Merck, 70–230 mesh) was used, thin layer
chromatograms were prepared on silica gel G or silica gel GF254 60 (Merck) and the spots were observed byexposure to iodine
vapor or UV light. All organic extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure below
60°C. Ether refers todiethyl ether. Tetrahydrofuran, dichloromethane, and pyridine were freshlydistilled before used. All other
solvents and reagents were obtained from commercial suppliers and were used without further purification.
19-Hydroxymethyl-12-methoxypodocarpa-8,11,13-triene (2). A solution of lithium aluminium hydride (1.88 g,
49.0 mmol) was added tocompound 1 (5 g, 16.5 mmol) in tetrahydrofuran (15 mL) and the contents were stirred under nitrogen
at 5°C for 24 hours. The excess reagent was decomposed with water and after addition of 5% aqueous H2SO4 the product was
extracted with ether. Evaporation of the ether extract and subsequent purification by column chromatography (hexane–ether
10:1) gave 4.5 g (89%) of compound 2 as a white crystal (hexane), mp 105°C, lit., [13] 90–91.5°C; νmax (liquid film)/cm-1 3373
(OH); 1H NMR (500 MHz, δ, J/Hz, CDCl3): 1.03 (3H, s, 20-CH3), 1.16 (3H, s, 18-CH3), 1.37–1.48 (2H, m, 3-H), 1.62 (1H,
m, 1α-H), 1.61–1.65 (2H, m, 2-H), 1.72–1.83 (2H, m, 6-H), 1.89 (1H, m, 5-H), 2.26 (1H, m, 1β-H), 2.69–2.90 (2H, m, 7-H),
3.70 (2H, dd, J = 81, J = 9, 19-CH2), 3.75 (3H, s, O-CH3), 6.64 (1H, dd J = 9, J = 3, 13-H), 6.85 (1H, d, J = 2.4, 11-H), 6.93
(1H, d, J = 9, 14-H); 13C NMP (75.45 MHz, δ, CDCl3): 19.76, 20.03, 22.84, 27.80, 27.98, 35.72, 37.60, 37.90, 39.13, 50.04
(C-5), 55.26 (Ar-OCH3), 71.70 (C-19), 110.00, 111.20, 125.20, 130.08, 150.20, 157.85; anals. C 73.96%, H 9.60%, calcd for
C18H26O2, C 73.93%, H 9.65%.
12-Methoxy-19-tosyloxypodocarpa-8,11,13-triene(3). Asolution ofp-toluenesulfonyl chloride(3.6mL, 0.0184mol)
in 20 mL dry THF was added to a solution of compound 2 (3.9 g, 0.142 mol) and pyridine (6.61 g 0.184 mol) in the same
solvent (20 mL) under nitrogen atmosphere over 20 min with cooling. After another hour, the mixture was warmed to room
temperature during 3 hours. The solution was then washed with 10% HCl (150 mL) and water (300 mL), then extracted with
chloroform (2 × 200 mL), dried (Na2SO4), and evaporated in vacuum to give a yellow solid. This crude was purified further by
column chromatographyon silica gel(hexane–ether, 10:2)toafford5.26 g (90%) ofa yellowsolid (methanol–hexane) mp 74°C;
[α]D20 +1° (c 1.08, CHCl3); 1H NMR (500 MHz, δ, J/Hz, CDCl3): 0.99 (3H, s, 18-CH3), 1.01 (3H, s, 20-CH3), 1.90–2.19 (2H,
m, 1-H), 1.43–1.58 (2H, m, 2-H), 1.32–1.40 (2H, m, 3-H), 1.90 (1H, m, 5-H), 1.60–1.75 (2H, m, 6-H), 2.69–2.75 (2H, m, 7-H),
3.73 (3H, s, ArO-CH3), 4.10 (2H, dd J = 12, J = 9, 19-CH2), 6.71 (1H, d J = 3, 11-H), 6.63 (1H, dd J = 9, J = 3, 13-H), 6.90
(1H, d, J = 9, 14-H), 7.53–7.93 (4H, m, SO2–C6H4–CH3); 13C NMP (75.45 MHz, δ, CDCl3): 18.67, 18.80, 24.90, 26.44, 29.39,
34.90, 37.07, 37.31, 38.59, 50.39 (Ar-OCH3), 50.59 (C-5), 78.88, 109.80, 110.68, 126.33, 127.44, 128.74, 128.74, 129.35,
133.19, 133.19, 135.69, 149.85, 157.38; anals. C 70.74%, H 7.61%, calcd for C25H32O4S, C 70.06%, H 7.53%.
12-Methoxy-19-carbaldehyde-podocarpa-8,11,13-triene (4). Compound 2 (0.123 g, 0.40 mmol) was dissolved in
dichloromethane (5 mL) containing both 4 Å molecular sieves (0.279 g, 0.55 mmol) and N-methylmorpholine N-oxide
(0.070 g, 0.60 mmol). After stirring the mixture for 5 min, tetra-n-propylammonium perrutherate (0.007 g, 0.02 mmol) was
added and the reaction followed by TLC until complete. The reaction mixture was filtered through silica gel (hexane–ether,
20
10:2) and afforded 0.096 g (87%) compound 4 as a white solid (methanol–hexane) mp 137°C, lit., [7] 130–132°C; [α]D +54°
1
(c 2.25, CHCl3); H NMR (500 MHz, δ, J/Hz, CDCl3): 1.09 (3H, s, 18-CH3), 1.12 (3H, s, 20-CH3), 1.69 (1H, m, 1α-H), 2.02
(1H, m,1β-H), 1.42–1.47 (2H, m, 2-H), 1.23–1.28 (2H, m, 3-H), 1.67–1.81 (2H, m, 6-H), 2.26 (1H, m, 5-H), 2.85 (1H, m, 7-H),
2.96 (1H, dd J = 17.5, J = 10, 7-H), 3.80 (3H, s, O-CH3), 6.71 (1H, d, J = 8.5, 14-H), 6.83 (1H, bs, 11-H), 7.01 (1H, d J = 8.5,
13-H) and 9.86 (1H, s, CHO); 13C NMP (75.45 MHz, δ, CDCl3): 18.91, 19.18, 24.00, 24.17, 30.35, 33.80, 38.23, 38.30, 48.61,
51.87 (C-5), 55.22 (Ar-OCH3), 110.64, 111.26, 126.88, 129.91, 148.78, 157.79, 205.66 (C-19); anals. C 79.83%, H 9.08%,
calcd for C18H24O2, C 79.37%, H 8.88%.
12-Hydroxypodocarpa-8,11,13-triene (7). A solution of compound 6 (0.150 g, 0.47 mmol) in anhydrous
dichloromethane (7 mL) and aluminum trichloride (0.183 g, 1.78 mmol) was stirred at –10°C for 15 min. After the reaction
was stirred overnight at room temperature, the mixture was poured into ice-water and extracted with dichloromethane. The
combined organic layer was successively washed with saturated NaHCO3 and brine solution, and then with dried (Na2SO4),
319

filtered, and evaporated under reduced pressure togive crude. The crude was purified bycolumn chromatography(hexane–ether
10:1) to afford 0.088 g (63%) of compound 7 as a white crystal (hexane–methanol) m.p. 140°C, [α]D20 +66.8° (c 3.76, CHCl3);
lit., [3] 139–142°C, [α]D +72° (c 0.86, CHCl3); 1H NMR (500 MHz; J/Hz, CDCl3): δH = 0.90 (3H, s, 19-CH3), 0.92 (3H, s,
20
18-CH3), 1.15 (3H, s, 20-CH3), 1.20 (1H, dd J = 12, J = 4, 3α-H), 1.29 (1H, dd, J = 13, J = 2.3, 3β-H), 1.37 (1H, m, 2-H), 1.45
(1H, bd, J = 15, 1β-H), 1.58 (1H, m, 2-H), 1.61 (1H, m, 6-H), 1.68 (1H, m, 6β-H), 1.82 (1H, m, 5-H), 2.17 (1H, bd, J = 10,
1α-H), 2.76 (1H, m, 7-H), 2.84 (1H, dd, J = 6, J = 3, 7-H), 4.47 (1H, bs, OH), 6.53 (1H, dd, J = 7.5, J = 5, 13-H), 6.70 (1H, d,
J = 5, 11-H) and 6.88 (1H, d J = 8, 14-H); 13C NMP (75.45 MHz, δ, CDCl3): 19.10, 19.27, 21.63, 24.71, 29.56, 33.28, 33.46,
+
37.88, 38.78, 41.63, 50.22 (C-5), 110.94, 112.52, 127.49, 129.95, 151.70, 153.54; GC/MS m/z 244 (100, M ), 228 (74), 147
(75), 91 (10), 69 (20), 41 (12); anals. C 77.82%, H 9.99%, calcd for C17H24O, C 77.78%, H 9.98%.
12-Methoxypodocarpa-8,11,13-triene (6). A mixture of compound 4 (6.6 g, 24.2 mmol), 8.9 mL of 90% hydrazine
hydrate (13.2 g, 410 mmol), diethylenglycol (50 mL), and potassium hydroxide pellets (8.14 g, 145 mmol) in a 250 mL two-
necked round-bottomed flask was put under reflux conditions until most of the potassium hydroxide had dissolved, then heated
at a 112°C under reflux conditions for 1 hour. After that we removed the reflux condenser and fitted a still-head and condenser
on it for downward distillation. We distilled it until the temperature of the liquid rose to 216°C and kept it at that temperature
for 3.5 hours. After that the mixture was cooled down, the solution was then washed with 5% HCl (150 mL) and water
(300 mL), extracted with ether (3 × 200 mL), dried (Na2SO4), and evaporated in vacuum to give a yellow oil as a crude product;
it was then purified by column chromatography (hexane–ether, 10:1) to afford 1.7 g (27.2%) of compound 7 and the desired
20
compound 6 as a white solid (hexane–methanol): 3 g (48%), mp 34°C, lit., [3] 31–32°C, [14] 30–31°C; [α]D +65.5° (c 6.6,
CHCl3); 1H NMR (500 MHz, δ, J/Hz, CDCl3): 0.90 (3H, s, 19-CH3), 0.92 (3H, s, 20-CH3), 1.16 (3H, s, 18-CH3), 1.28 (1H,
bd J = 13.2, 3β-H), 1.38 (1H, td, J = 12.9, J = 3.8, 1α-H), 1.40 (1H, td, J = 13.2, J = 4.2, 3α-H), 1.55 (1H, m, 2-H), 1.66 (1H,
m, 6-H), 1.78 (1H, m, 2-H), 1.82 (1H, m, 6-H), 1.85 (1H, dd, J = 12.3, J = 2.2, 5-H), 2.23 (1H, br d, J = 12.9, 1β-H), 2.77 (1H,
m, 7-H), 2.87 (1H, dd, J = 17, J = 1.6, 7-H), 3.76 (3H, s, O-CH3), 6.78 (1H, d, J = 2.5, 11-H), 6.63 (1H, dd, J = 8.3, J = 2.3,
13-H), 6.93 (1H, d, J = 8.5, 14-H); 13C NMP (75.45 MHz, δ, CDCl3): 19.18, 19.34, 21.69, 24.74, 29.44, 33.03, 33.35, 38.00,
38.8, 41.70, 50.34 (C-5), 55.23 (O-CH3), 110.15 (C-11), 110.71 (C-13), 127.47, 129.76 (C-14), 151.45, 157.66; GC/MS m/z
+
258 (100, M ), 243 (61), 161 (58), 115 (7), 91 (3), 69 (7), 41 (6); anals. C 83.62%, H10.13%, calcd for C18H26O, C 83.67%,
H 10.14%.
12-Methoxy-19-carbonitrile-podocarpa-8,11,13-triene (5). A mixture of compound 4 (4.7 g, 17 mmol) and 90%
hydrazine hydrate (9.29 g, 290 mmol) in diethyleneglycol (50 mL) was taken in an Erlenmeyer flask and placed in a commercial
microwave oven operating at 2450 MHz frequency. After irradiation of the mixture for 20 min (monitored by TLC), it was
cooled to room temperature, extracted with chloroform, and dried over anhydrous Na2SO4. Removal of the solvent gave the
respective hydrazone derivative. This hydrazone derivative compound was used without purification in the next steps. For the
Wolff-Kishner reduction, a mixture of hydrazone and potassium hydroxide pellets (5.7 g, 102 mmol) was taken in an
Erlenmeyer flask and placed in a microwave oven. After irradiation for 30 min and the usual work up, we obtained a yellow
oil as a crude product, which was then purified by column chromatography (hexane–ether, 10:2) to afford 1.7 g (27.2%) of
nitrile compound 5 as a white solid (hexane–methanol), mp 99°C; νmax (liquid film)/cm-1 3607.82, 3582.89, 2919.16, 2845.23,
2300.27 (CN), 1605.97; 1H NMR (500 MHz, δ, J/Hz, CDCl3): 1.37 (3H, s, 20-CH3), 1.46 (3H, s, 18-CH3), 1.52 (1H, m, 1-H),
1.82 (1H, d, J = 9 Hz, 5-H), 1.90–2.00 (2H, m, 6-H), 2.09–2.18 (2H, m, 2-H), 2.11–2.19 (2H, m, 3H), 2.36 (1H, d, J = 9, 1-H),
2.88 (1H, m, 7-H), 2.99 (1H, dd, J = 9.6, J = 3, 7-H), 3.81 (3H, s, O-CH3), 6.82 (1H, br s, 11-H), 6.72 (1H, d, J = 10, 13-H) and
7.02 (1H, d, J = 10, 14-H); 13C NMP (75.45 MHz, δ, CDCl3): 19.76, 19.96, 22.75, 27.79, 27.98, 35.32, 37.47, 37.84, 39.08,
+
49.12, 55.25 (O-CH3), 109.85, 111.12, 124.52, 126.61 (C-19), 129.86, 149.08, 157.86; GC/MS m/z 269 (60 M ), 254 (100),
237 (23), 171 (8), 134 (9), 115 (8), 670 (11), 55 (3), 39 (5); anals. C 80.66%, H 8.63%, calcd for C18H13NO, C 80.26%,
H 8.61%.
12-Acetylhydroxypodocarpa-8,11,13-triene (8). A white crystal (hexane–methanol); mp 170°C, GC/MS m/z 286
(6, M ), 244 (100), 147 (75), 229 (30), 147 (31), 115 (5), 69 (2); anals. C 79.78%, H 9.10%, calcd for C19H26O2, C 79.68%,
+
H 9.15%.
ACKNOWLEDGMENT
I would like to thank Dr. Tatsuhiko Nakano for his interest in this work, and M. Sc. M. Gomez and M. Sc. S. Pekerar
for measurements of the mass and NMR spectra at the “Laboratorio Nacional de Analisis Quimico No. Lab. 1998003690.” The
320

authors wish to thank Dr. V. L. Narayanan, Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD,
USA for performing the cytostatic and cytotoxic screening studies.
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