Semisynthesis and biological evaluation of abietane-type diterpenes. Revision of the structure of rosmaquinone

Article abstract of DOI:10.1021/np900047p

The new aromatic diterpenes 7β-O-benzylrosmanol (3), 7β-O-benzyl-11,12-di-O-methylrosmanol (4), and 7α-thiophenylcarnosic acid (5) have been obtained by partial synthesis from carnosol (1), an abundant natural diterpene present in Salvia species. The stru

Full text of DOI:10.1021/np900047p

J. Nat. Prod. 2009, 72, 1385–1389
1385
Semisynthesis and Biological Evaluation of Abietane-Type Diterpenes. Revision of the Structure
of Rosmaquinone
Joaqu´ın G. Marrero,† Laila Moujir,‡ Luc´ıa S. Andre´s,*,† Nayely P. Montan˜o,‡ Liliana Araujo,‡ and Javier G. Luis†
Instituto UniVersitario de Bio-Orga´nica “Antonio Gonza´lez”, Departamento de Qu´ımica Orga´nica, UniVersidad de La Laguna, AVenida Astrof´ısico
Francisco Sa´nchez, 2, 38206 La Laguna, Tenerife, Canary Islands, Spain, and Departamento de Microbiolog´ıa y Biolog´ıa Celular, Facultad de
Farmacia, UniVersidad de La Laguna, AVenida Astrof´ısico Francisco Sa´nchez, s/n, 38206 La Laguna, Tenerife, Canary Islands, Spain
ReceiVed January 30, 2009
The new aromatic diterpenes 7ꢀ-O-benzylrosmanol (3), 7ꢀ-O-benzyl-11,12-di-O-methylrosmanol (4), and 7R-
thiophenylcarnosic acid (5) have been obtained by partial synthesis from carnosol (1), an abundant natural diterpene
present in SalVia species. The structures of these compounds were established from their physical and spectroscopic
data. The known diterpenes sagequinone methide A (6), 7ꢀ-O-methylrosmanol (7), 7-O-methylrosmanol (8), and
rosmaquinone B (9) were obtained from rosmanol (2). The spectroscopic data of these semisynthetic diterpenes were
identical to data reported in the literature. In addition, the new semisynthetic isorosmaquinone (10) was obtained from
isorosmanol (12). The proton resonances of rosmaquinone (11) are reassigned based on 2D NMR spectroscopy. These
compounds, as well as eight known analogues, were evaluated for cytotoxic and antimicrobial activities.
The genus SalVia (Lamiaceae) consists of approximately 500
species found worldwide. SalVia species are used as traditional
medicines for the treatment of a variety of diseases. Studies of their
chemical constituents have revealed the presence of a large variety
of diterpenoids with antibacterial,1-4 antioxidant,5 antidiabetic,6,7
antitumor,8,9 leishmanicidal,10 and antiplasmodial10 properties. Most
of these diterpenes are often present in very low quantities. We
have reported an efficient partial synthesis of some of these
compounds from the abundant diterpene carnosol (1).11 Moreover,
we have obtained new abietane diterpene derivatives with a seco
C-ring12 and new quinone derivatives.13
Scheme 1
In the course of our work on the partial synthesis of biologically
interesting diterpenes, we have prepared four new derivatives of
rosmanol (2) and now describe a semisynthetic approach to the
known diterpene sagequinone methide A (6). This is the first
semisynthesis of this natural product. Furthermore, these compounds
and eight known analogues12,13 were assayed for their cytotoxic
and antimicrobial activities.
Results and Discussion
Following the procedure previously reported,11 a solution of
carnosol (1) in acetone was treated with benzyl alcohol and
potassium carbonate to give, after purification, compound 3 (Scheme
1). The low-resolution mass spectrum of 3 showed a molecular
ion [M]+ at m/z 436 (C27H32O5 by HREIMS). The IR spectrum
protons as a multiplet (δ 7.26-7.43) were assigned to the benzyl
group. Correlation between H-7 and H-5R in the ROESY experi-
ment confirmed that the substituent at C-7 is ꢀ-oriented. The above
data were in accordance with the structure of 7ꢀ-O-benzylrosmanol
for 3.
exhibited characteristic absorption bands for phenol (3500 cm-1
)
Methylation of 3 with dimethyl sulfate in acetone gave the
and lactone (1770 cm-1) groups. In the 1H NMR spectrum, signals
for a proton heptuplet (δ 3.05) together with two methyl groups as
doublets at δ 1.12 and 1.16 indicated the presence of an aromatic
isopropyl group. Two methyl groups at δ 0.91 and 1.00 were also
observed. The H-5 proton appeared as a singlet at δ 2.33, and H-6
as a doublet at 4.66 (J ) 3.0 Hz) coupled with another proton
doublet at δ 4.47 (J ) 3.0 Hz) assigned to H-7. This indicated that
the lactone closure was at C-6, and the bridgehead proton and H-6
form a 90° angle. In the low-field region of the spectrum, two broad
singlets at δ 5.83 and 6.30 (interchangeable with D2O) were
assigned to hydroxy groups, and one aromatic proton singlet at δ
6.63 was assigned to H-14. An AB system as two one-proton
doublets (δ 4.77 and 4.89; J ) 11.8 Hz) together with five aromatic
dimethyl ether 4 (Scheme 1), which showed a molecular ion [M]+
1
at m/z 464 (C29H36O5 by HREIMS), while the H NMR spectrum
displayed two additional methoxy groups at δ 3.75 and 3.77.
Treatment of carnosol (1) with thiophenol and potassium carbonate
in acetone (Scheme 1) yielded 5. The low-resolution mass spectrum
of 5 showed a molecular ion [M - 1]+ at m/z 439 corresponding
to the molecular formula C26H31O4S by HREIMS. The IR spectrum
showed absorption bands for phenol (3400 cm-1) and carboxylic
acid (1682 cm-1) groups, while the lactone group absorption was
absent. In the 1H NMR spectrum, a six-proton doublet at δ 1.21 (J
) 7.0 Hz) was assigned to the C-16 and C-17 methyls, and two
singlets at δ 0.88 and 0.83 were assigned to the C-18 and C-19
methyl groups, respectively. In the low-field region, a one-proton
singlet at δ 6.91 was assigned to the aromatic H-14 and a doublet
at δ 4.71 (J ) 3.1 Hz) to H-7; signals at δ 7.28 (3H, m) and 7.48
(2H, d, J ) 7.5 Hz) were assigned to the aromatics protons of the
thiophenyl group. The 13C NMR spectrum showed signals for 26
* To whom correspondence should be addressed. Tel: +34-922-318575.
Fax: +34-922-318571. E-mail: landrest@ull.es.
† Instituto Universitario de Bio-Orga´nica.
‡ Departamento de Microbiolog´ıa y Biolog´ıa Celular.
10.1021/np900047p CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/15/2009

1386 Journal of Natural Products, 2009, Vol. 72, No. 8
Marrero et al.
Scheme 2
Scheme 3
Table 1. HMBC Data of Compound 11 in CDCl3
proton
three-bond correlation
two-bond correlation
H-1R
C-9, C-20
C-3
C-4, C-10
C-2, C-10
C-10
C-1
H-1ꢀ
H-2
H-5
H-6
H-14
C-4, C-9, C-18, C-20
C-4, C-8, C-10, C-20
C-12, C-15
C-10
H-15
H-16 and -17
H-18 and -19
C-12, C-16, C-17
C-13, C-15
C-3, C-4
C-13
carbon atoms, including a signal at δ 181.9 (s) assigned to the
carboxylic acid group (C-20) and signals at δ 141.8 (s, C-12) and
142.7 (s, C-11) assignable to two oxygen-bearing aromatic carbons.
The above data are in accordance with the structure of 7R-
thiophenylcarnosic acid for 5.
We were also interested in obtaining 7ꢀ-O-methylrosmanol (7)
and isorosmaquinone (10) from 2 and 12, respectively, in order to
test their biological activity. Rosmanol (2) was obtained from
carnosol (1) as previously reported.11 In our initial attempts, we
carried out the protection of phenolic groups, followed by trans-
formation of the C-7 hydroxy group into a good leaving group,
followed by substitution with a methoxy group by an SN2 reaction.
Treatment of rosmanol (2) with acetyl chloride, solid sodium
hydroxide, and tetrabutylammonium hydrogen sulfate in dioxane
(Scheme 2)14 yielded 6, which showed physical data identical to
natural sagequinone methide A.15
HMBC, and HMQC experiments of rosmanol (2), which confirmed
that H-7 always appeared downfield from H-6. However, in the
case of rosmaquinone B (9) this is reversed, prompting us to revise
the proton assignment of rosmaquinone (11), which was prepared
by oxidation of rosmanol (2) with silver oxide (Scheme 3).18 HMBC
correlations (H-6/C-20, C-8) established the lactone ring closure
1
at C-6 for 11. A H-1H COSY correlation between H-6 [δ 4.52
(d, J ) 3.3 Hz)] and H-7 [δ 4.40 (d, J ) 3.3 Hz)] together with
the 13C NMR, HSQC, and HMBC (Table 1) experiments allowed
the full NMR assignment of rosmaquinone (11), which unambigu-
ously established H-6 as being downfield compared to H-7.
Oxidation of isorosmanol (12) with freshly prepared Ag2O in
diethyl ether yielded 10 (Scheme 3). The mass spectrum of 10
showed a molecular ion [M]+ at m/z 344 (C20H24O5 by HREIMS).
The IR spectrum displayed characteristic absorption bands for
lactone (1760 cm-1) and o-quinone (1668 cm-1) groups. In the 1H
NMR spectrum, signals for the six-proton methyl doublet (6H, δ
1.11, J ) 7.0 Hz), two geminal methyls singlets (δ 0.86 and 1.05),
and an aromatic proton at δ 6.73 (H-14) were observed. The
δ-lactone required closure at C-7, which is in accordance with the
lower chemical shift of H-7 (δ 5.12, J ) 4.5 Hz) compared to H-6
(δ 4.48, J ) 4.5 Hz). The 13C NMR spectrum showed signals for
20 carbons, including a signal at δ 172.4 (s) assigned to the lactone
carbonyl and signals at δ 69.2 (d), 77.9 (d), 176.3 (s), and 179.3
(s) corresponding to four oxygen-bearing carbons, confirming the
structure of isorosmaquinone as 10.
The antimicrobial activity of compounds 1-4, 6-9, and 11-20
(Figure 1) was weak (Table 2), showing no activity against Gram-
negative bacteria and Candida albicans (MICs > 40 µg/mL), except
for 1 (MIC 20 µg/mL).
Compounds 1-20 can be categorized into three groups based
on their chemical features. Compounds 1, 10, and 12-15 are C-7
γ-lactones, among which isorosmanol (12) and carnosol (1) showed
marginal activity.3 Accordingly, we speculate that two free hydroxy
groups in the C-ring (1 versus 13) increase the antibiotic activity,
but an additional hydroxy group at C-6 (1 versus 12) decreases it.
Compounds 2-4, 6-9, 11, and 16 are C-6 γ-lactones. In this case,
the activity on Staphylococcus aureus depends on the presence of
Treatment of rosmanol (2) with sulfuric acid in methanol at room
temperature yielded a mixture of three products, which were
separated by preparative TLC and identified as 7ꢀ-O-methylros-
manol (7), 7-O-methylrosmanol (8), and quinone (9) (Scheme 2).
Compound 9 showed a molecular ion [M]+ at m/z 358 (C21H26O5
by HREIMS). The IR spectrum displayed characteristic bands for
lactone (1786 cm-1) and o-quinone (1667 cm-1) groups. The
presence of the o-quinone group was confirmed by UV absorption
(340 and 270 nm).16 In the H NMR spectrum, signals for a six-
1
proton doublet for two methyls (δ 1.12, J ) 7.0 Hz) and two three-
proton singlets at δ 0.89 and 1.01 for two geminal methyl groups
were observed. An AB spin system of two one-proton doublets at
δ 3.88 and 4.64 was assigned to H-7 and H-6, respectively, and a
one-proton singlet at δ 6.62 to the vinylic proton H-14. A three-
proton singlet assigned to a methoxy group appears at δ 3.68. The
13C NMR spectrum showed signals for 20 carbon atoms, including
a signal at δ 175.6 (s) assigned to the lactone carbonyl and signals
at δ 59.6 (q), 72.5 (d), 76.9 (d), 179.6 (s), and 180.0 (s) assignable
to five oxygen-bearing carbon atoms.
The structure of 9 was confirmed by Ag2O oxidation of 7-O-
methylrosmanol (8) (Scheme 2), which yielded a product identical
to 9, therefore establishing 9 as rosmaquinone B.17
In previous work18 the assignment of protons H-6 and H-7 in
rosmaquinone (11) was made on the basis of 1H-1H COSY,

ReVision of the Structure of Rosmaquinone
Journal of Natural Products, 2009, Vol. 72, No. 8 1387
6-mercaptopurine.19 It is important to emphasize that 6, 9-11, and
18 show selective cytotoxicity, as demonstrated by their IC50 values
against the nontumoral mammalian Vero cells (IC50 > 60 µM).
The structure-activity relationship also revealed that the closing
position of the lactone ring does not affect cytotoxic activity, but
the o-quinone group (10 versus 12) and the C-6 hydroxy group
(10 versus 13) increase the activity. On the contrary, the benzyl
group leads to a loss of activity (3 versus 6), and the hydroxy group
is preferable to an o-quinone in the C-ring (2 versus 11). With
respect to diterpenes with an epoxy group, they show only moderate
activity when possessing a p-quinone C-ring and with no double
bond at C-15 and C-16 (18 versus 19).
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 automatic polarimeter in CHCl3 at 20 °C, and
the [R]D values are given in 10-1 deg cm2 g-1. UV spectra were obtained
in absolute EtOH on a JASCO V-560 instrument. IR spectra were
obtained on a Bruker IFS 28/55 (FTIR) spectrometer. The NMR spectra
were recorded on Bruker Avance 300 MHz and Bruker Avance 400
MHz spectrometers in CDCl3, unless otherwise noted. Chemical shifts
are given in ppm with TMS as the internal standard. Low-resolution
mass spectra were run on a VG Micromass ZAB-2F and high-resolution
mass spectra on a VG Micromass ZAB-2F at 70 eV. Merck silica gel
(0.063-0.2) was used for column chromatography. Analytical thin-
layer chromatography (TLC) and preparative TLC were carried out on
precoated Schleicher and Schu¨ll plates.
Plant Material. A voucher specimen of SalVia canariensis L. (No.
25252) was deposited in the herbarium of the Department of Botany,
Faculty of Biology, Universidad de La Laguna. Carnosol (1) and
isorosmanol (12) were isolated from a dried extract of stems and leaves
of S. canariensis L.11
Figure 1
7ꢀ-O-Benzylrosmanol (3). Carnosol (1) (109.8 mg, 0.32 mmol),
benzyl alcohol (0.2 mL, 1.93 mmol, 6.1 equiv), and solid K2CO3 (267.0
mg, 1.93 mmol, 6.09 equiv) in acetone (10 mL) were stirred at room
temperature under Ar for 24 h, followed by evaporation of the solvent
under reduced pressure. The reaction mixture was acidified with 5%
HCl, extracted with EtOAc (3 × 20 mL), washed with brine, and dried
over anhydrous Na2SO4. The residue was purified by silica gel column
chromatography eluting with n-hexane/acetone (9:1), yielding 3 (32.8
mg, 22.6%): [R]20D +7.4 (c 0.09, CHCl3); UV (EtOH) λmax (log ε) 289
(3.68) nm; IR (film) νmax 3500, 2958, 2927, 2870, 1770, 1455, 1395,
1372, 1355, 1327, 1303, 1269, 1217, 1175, 1070, 1016, 964, 909, 757
Table 2. Antimicrobial Activity of 1-4, 6-9, and 11-20 (MIC
in µg/mL)
compounda b S. aureus S. epidermidis B. subtilis B. cereus C. albicans
,
1
2
3
6
7
8
9
11
12
30-25
25-20
>40
10-5
20-10
>40
6-4
10-5
20
20-10
40-20
20-10
40-20
>40
40-20
20-10
20-10
40-20
40-20
40-20
20-10
40-20
10
>40
>40
>40
>40
>40
>40
>40
>40
ntd
40-20
>40
40
40-20
40-20
>40
40
40-20
2.5
>40
40
20
40
1
cm-1; H NMR (300 MHz) δ 0.91 (3H, s, Me-19), 1.00 (3H, s, Me-
40-20
8
35-30
8
18), 1.12 (3H, d, J ) 7.0 Hz, Me-16), 1.16 (3H, d, J ) 7.0 Hz, Me-
17), 1.20 (1H, overlap, H-3R), 1.44 (1H, bd, J ) 13.3 Hz, H-3ꢀ), 1.62
(2H, m, H-2), 1.96 (1H, td, J1 ) 5.4 Hz, J2 ) 13.5 Hz, H-1R), 2.33
(1H, s, H-5), 3.05 (1H, hept, J ) 7.0 Hz, H-15), 3.17 (1H, bd, J )
13.7 Hz, H-1ꢀ), 4.47 (1H, d, J ) 3.0 Hz, H-7), 4.66 (1H, d, J ) 3.0
Hz, H-6), 4.77 (1H, d, J ) 11.8 Hz, -CH2-Ph), 4.89 (1H, d, J ) 11.8
Hz, -CH2-Ph), 5.83 (1H, bs, OH-11), 6.30 (1H, bs, OH-12), 6.63 (1H,
s, H-14), 7.26-7.43 (5H, m, H-2′, H-3′, H-4′, H-5′, H-6′); 13C NMR
(75 MHz) δ 19.0 (t, C-2), 21.9 (q, C-19), 22.0 (q, C-16), 22.1 (q, C-17),
27.1 (d, C-15), 27.1 (t, C-1), 31.4 (q, C-18), 31.5 (s, C-4), 38.0 (t,
C-3), 47.1 (s, C-10), 51.0 (d, C-5), 72.8 (t, -CH2-Ph), 75.0 (d, C-7),
75.4 (d, C-6), 120.8 (d, C-14), 124.5 (s, C-9), 127.0 (d, C-4′), 127.4
(s, C-8), 128.3 (d, C-3′ and C-5′), 128.8 (d, C-2′ and C-6′), 134.8 (s,
C-13), 137.8 (s, C-1′), 141.7 (s, C-12), 142.7 (s, C-11), 179.2 (s, C-20);
EIMS m/z 436 [M]+ (14), 300 (12), 284 (24), 268 (15), 239 (13), 215
(44), 91 (100), 79 (22); HREIMS m/z 436.2275 [M]+ (calcd for
C27H32O5, 436.2250).
cefotaximec 2.5-1.25
a Diterpenes 4 and 13-20 were inactive against all microorganisms
(MIC
>
40 µg/mL). b All compounds were inactive against M.
smegmatis, E. faecalis, and Gram-negative bacteria (MIC > 40 µg/mL).
c Cefotaxime was used as positive control. d nt: not tested.
a hydroxy group at C-7 (2 and 11), since a 7ꢀ-benzyl or a methoxy
group in this position leads to a loss of the activity (3 and 8).
Moreover, the MIC values of 2 and 11 against Bacilus subtilis
indicate that a carbonyl group in the C-ring increases the antibiotic
activity. These results reinforce the structure-activity relationships
as previously reported.3 Within the third type, 17-20 are character-
ized by a C-7 γ-lactone group and a 12,16-epoxy group with no
activity against all microorganisms assayed, indicating that the
epoxy group leads to a loss of activity.
The cytotoxicity of the diterpenes was studied in Vitro against
A-549, HeLa, Hep-2, and MCF-7 human tumor and Vero cells lines,
using 6-mercaptopurine as a positive control (Table 3). Diterpenes
5 and 6 were the most active against HeLa and Hep-2 cell lines
after 48 h of continuous exposure; however, the cytotoxicity of 5
decreased and that of 6 increased when the compounds were
incorporated in the log or exponential growth phase. Similarly, the
activity increased considerably in the case of 6, 9, 11, and 18 against
different cancer cells, which indicates that the mechanism of these
compounds could be related to a biosynthetic process as for
7ꢀ-O-Benzyl-11,12-di-O-methylrosmanol (4). Anhydrous K2CO3
(44.5 mg, 0.3 mmol, 13.1 equiv) was suspended in acetone (5 mL),
and 7-O-benzylrosmanol (3) (10.7 mg, 0.0245 mmol) added. The
solution was treated with Me2SO4 (0.1 mL, 1.05 mmol, 42.9 equiv).
After 12 h, the reaction mixture was acidified with 5% HCl, the acetone
was evaporated under reduced pressure, and the product was extracted
with EtOAc (3 × 15 mL), washed with brine, and dried over anhydrous
Na2SO4. The crude reaction product was chromatographed over silica
gel using CH2Cl2 as eluent to yield 4 (10.9 mg, 95.6%): [R]20D +4.2 (c
0.04, CHCl3); UV (EtOH) λmax (log ε) 280 (3.58); IR (film) νmax 2955,
2925, 2854, 1778, 1456, 1411, 1395, 1375, 1353, 1328, 1306, 1226,
1173, 1109, 1071, 1049, 1034, 957, 944 cm-1; 1H NMR (300 MHz) δ

1388 Journal of Natural Products, 2009, Vol. 72, No. 8
Marrero et al.
Table 3. Cytotoxicity (IC50 µM) of 1-20 against Human Cancer and Vero Cell Lines
compounda
HeLab
HeLac
Hep-2b
Hep-2c
A-549b
A-549c
MCF-7b
MCF-7c
Verob
Veroc
1
2
5
6
7
8
9
10
11
12
13
14
16
17
18
19
43.9
20.2
4.8
>60
51.4
11.1
9.5
nte
nt
15.2
32.0
nt
nt
nt
18.9
19.2
nt
>60
43.4
nt
>60
35.6
42.5
>60
nt
47
27.7
nt
40.9
17.1
nt
46.0
23.3
37.5
43.3
nt
39.5
54.3
nt
>45
>60
>60
33.0
>58.8
1.4
44.5
15.3
nt
26.8
41.1
55.6
22.1
nt
>60
36.1
nt
>45
42.6
>60
18.4
>58.8
5.8
>60
19.9
6.1
51.5
36.4
21.1
>60
33.9
47.2
>60
>60
>60
>60
nt
>45
>60
>60
>58.4
>58.8
118.6
14.0
40.8
42.2
36.0
25.0
>60
>60
>60
33.8
>60
56.1
50.3
>58.8
2.9
26.5
50.8
>60
25.4
nt
28.8
46.0
nt
>60
36.7
42.2
>60
>60
>60
>60
nt
>45
>60
>60
>58.4
>58.8
67.5
>60
>60
33.8
30.8
>60
>60
40.2
40.5
>60
37.8
17.5
>58.8
4.1
nt
nt
42.5
39.5
nt
>60
39.2
nt
>60
>60
nt
nt
nt
>60
>45
>60
>60
>58.4
nt
>60
>45
>60
>60
39.8
nt
nt
nt
>60
>60
>58.4
48.8
47
39.4
51.5
40.4
>58.8
49.3
6-mercaptopurined
431
317.8
a Compounds 3, 4, 15, and 20 were inactive against all cell lines used. b Compounds were added in lag phase of growth. c Compounds were added in
log or exponential phase of growth. d 6-Mercaptopurine was used as positive control. e nt: not tested.
0.94 (3H, s, Me-19), 1.01 (3H, s, Me-18), 1.11 (3H, d, J ) 7.0 Hz,
Me-16), 1.16 (3H, d, J ) 7.0 Hz, Me-17), 1.48 (4H, m, H-2 and H-3),
1.91 (1H, td, J1 ) 5.5 Hz, J2 ) 12.2 Hz, H-1R), 2.35 (1H, s, H-5),
3.16 (1H, bd, J ) 12.2 Hz, H-1ꢀ), 3.23 (1H, m, H-15), 3.75 (3H, s,
Ar-OCH3), 3.77 (3H, s, Ar-OCH3), 4.48 (1H, d, J ) 2.8 Hz, H-7),
4.66 (1H, d, J ) 2.8 Hz, H-6), 4.78 (1H, d, J ) 11.7 Hz, -CH2-Ph),
4.90 (1H, d, J ) 11.7 Hz, -CH2-Ph), 6.80 (1H, s, H-14), 7.35-7.46
(5H, m, H-2′, H-3′, H-4′, H-5′, H-6′); EIMS m/z 464 [M]+ (64), 358
(63), 314 (42), 299 (23), 287 (30), 271 (83), 243 (27), 201 (29), 111
(30), 91 (100), 69 (73); HREIMS m/z 464.2511 [M]+ (calcd for
C29H36O5, 464.2563).
7r-Thiophenylcarnosic Acid (5). Carnosol (1) (22.6 mg, 0.07
mmol) in acetone (5 mL) was treated with thiophenol (0.05 mL, 0.49
mmol, 7.2 equiv) and solid K2CO3 (104.0 mg, 0.75 mmol, 11.0 equiv),
and the mixture was stirred at room temperature under Ar for 6 h, after
which the acetone was evaporated under reduced pressure. The reaction
mixture was acidified with 5% HCl, extracted with EtOAc (3 × 15
mL), washed with brine, and dried over anhydrous Na2SO4. The residue
was purified by silica gel column chromatography eluting with CH2Cl2,
yielding 5 (24.1 mg, 80.6%): [R]20D -3.1 (c 0.04, CHCl3); UV (EtOH)
H-6), 6.84 (1H, s, H-14), 6.96 (1H, d, J ) 5.9 Hz, H-7), 7.63 (1H, bs,
Ar-OH); 13C NMR (75 MHz) δ 18.8 (t, C-2), 21.4 (q, C-16 and C-17),
22.3 (q, C-19), 26.9 (d, C-15), 27.4 (t, C-1), 32.3 (q, C-18), 32.4 (s,
C-4), 38.4 (t, C-3), 48.9 (s, C-10), 60.9 (d, C-5), 72.9 (d, C-6), 115.8
(s, C-8), 133.8 (s, C-9), 134.8 (d, C-14), 139.9 (d, C-7), 143.8 (s, C-13),
145.8 (s, C-11), 176.2 (s, C-20), 180.4 (s, C-12); EIMS m/z 328 [M]+
(12), 300 (13), 284 (44), 269 (13), 228 (11), 215 (100), 149 (11), 69
(11), 57 (20); HREIMS m/z 328.1698 [M]+ (calcd for C20H24O4,
328.1674).
Treatment of Rosmanol (2) with H2SO4/MeOH. To a round
bottoned flask with molecular sieves under Ar was added a solution of
rosmanol (2) (96.8 mg, 0.28 mmol) in MeOH (10 mL) and concentrated
H2SO4 (0.03 mL). After 24 h, the reaction mixture was filtered over
Celite, the solvent was evaporated under reduced pressure, and the
product was extracted with EtOAc (3 × 15 mL), washed with H2O
and brine, and dried over anhydrous Na2SO4. The crude reaction product
was chromatographed by preparative TLC using CH2Cl2/Me2CO (96:
4) as eluent to yield 7ꢀ-O-methylrosmanol (7) (25.9 mg, 25.7%), 7-O-
methylrosmanol (8) (33.1 mg, 32.8%), and rosmaquinone B (9) (1.3
mg, 1.3%).
λ
max (log ε) 287 (3.82), 232 (4.46) nm; IR (film) νmax 3400, 2961, 2870,
7ꢀ-O-Methylrosmanol (7): [R]20D +4.5 (c 0.02, CHCl3); UV (EtOH)
1682, 1583, 1439, 1424, 1391, 1367, 1326, 1292, 1217, 1165, 1129,
λ ;
max (log ε) 289 (3.33), 220 (4.08) nm; IR (film) νmax 3500, 1750 cm-1
1086, 1025, 987, 951, 756, 692, 667 cm-1; H NMR (300 MHz) δ
1H NMR (300 MHz) δ 0.96 (3H, s, Me-19), 1.00 (3H, s, Me-18), 1.10,
1.17 (3H each one, d, J ) 7.0 Hz, Me-16 and Me-17), 1.96 (1H, s,
H-5), 3.01 (1H, hept, J ) 6.8 Hz, H-15), 3.21 (1H, bd, J ) 14.2 Hz,
H-1ꢀ), 3.58 (3H, s, -OCH3), 4.42 (1H, d, J ) 2.0 Hz, H-6), 4.93 (1H,
d, J ) 2.0 Hz, H-7), 5.69 (1H, bs, Ar-OH), 5.95 (1H, bs, Ar-OH), 6.83
(1H, s, H-14); EIMS m/z 361 [M + 1]+ (7), 347 (21), 300 (21), 271
(60), 255 (74), 246 (66), 231 (50), 201 (46), 177 (40), 137 (59), 128
(44), 105 (37), 69 (100); HREIMS m/z 360.1951 [M]+ (calcd for
C21H28O5, 360.1937).
1
0.83 (3H, s, Me-19), 0.88 (3H, s, Me-18), 1.21 (6H, d, J ) 7.0 Hz,
Me-16 and Me-17), 1.47 (5H, m, H-2, H-3 and H-6R), 1.94 (1H, d, J
) 12.5 Hz, H-5), 2.36 (1H, d, J ) 12.4 Hz, H-6ꢀ), 2.72 (1H, td, J1 )
3.5 Hz, J2 ) 13.5 Hz, H-1R), 3.17 (1H, hept, J ) 7.0 Hz, H-15), 3.30
(1H, bd, J ) 13.7 Hz, H-1ꢀ), 4.71 (1H, d, J ) 3.1 Hz, H-7), 5.90 (2H,
bs, Ar-OH), 6.91 (1H, s, H-14), 7.28 (3H, m, H-3′, H-4′, H-5′), 7.48
(2H, d, J)7.5 Hz, H-2′ and H-6′); 13C NMR (75 MHz) δ 20.3 (t, C-2),
22.0 (q, C-19), 22.3 (q, C-16), 22.5 (q, C-17), 23.9 (t, C-6), 27.3 (d,
C-15), 32.1 (q, C-18), 34.0 (s, C-4), 34.5 (t, C-1), 41.5 (t, C-3), 47.0
(d, C-5), 48.6 (s, C-10), 50.0 (d, C-7), 122.1 (d, C-14), 122.9 (s, C-8),
126.9 (s, C-9), 127.0 (d, C-4′), 128.9 (d, C-3′ and C-5′), 131.8 (d, C-2′
and C-6′), 133.9 (s, C-1′), 136.3 (s, C-13), 141.8 (s, C-12), 142.7 (s,
C-11), 181.9 (s, C-20); EIMS m/z 439 [M - 1]+ (5), 331 (100), 285
(33), 215 (20), 154 (19), 136 (18), 69 (18), 55 (16); HREIMS m/z
439.1968 [M - 1]+ (calcd for C26H31O4S, 439.1943).
7-O-Methylrosmanol (8): [R]20 -3.4 (c 0.02, CHCl3); IR (film)
D
νmax 3580, 3500, 2940, 2920, 2840, 1760, 1450, 1365, 1240, 1080,
1
1035, 955 cm-1; H NMR (300 MHz) δ 0.94 (3H, s, Me-19), 1.02
(3H, s, Me-18), 1.23 (6H, d, J ) 7.0 Hz, Me-16 and Me-17), 2.00
(1H, td, J1 ) 5.6 Hz, J2 ) 13.8 Hz, H-1R), 2.25 (1H, s, H-5), 3.07
(1H, hept, J ) 7.0 Hz, H-15), 3.17 (1H, bd, J ) 14.6 Hz, H-1ꢀ), 3.66
(3H, s, -OCH3), 4.27 (1H, d, J ) 3.2 Hz, H-6), 4.71 (1H, d, J ) 3.2
Hz, H-7), 5.48 (1H, bs, Ar-OH), 6.00 (1H, bs, Ar-OH), 6.80 (1H, s,
H-14); EIMS m/z 360 [M]+ (100), 314 (81), 298 (80), 284 (88), 269
(84), 245 (93), 228 (38), 215 (93); HREIMS m/z 360.1931 [M]+ (calcd
for C21H28O5, 360.1937).
Sagequinone Methide A (6). Anhydrous NaOH (46.2 mg, 1.2 mmol,
2.8 equiv) was suspended in dioxane (10 mL) and treated with solid
rosmanol (2) (144.2 mg, 0.42 mmol), tetrabutylammonium hydrogen
sulfate (4.2 mg, 0.01 mmol, 0.03 equiv), and AcCl (0.14 mL, 2.0 mmol,
4.7 equiv), and the mixture was stirred at room temperature under Ar
for 7 days. The mixture was filtered and the solvent evaporated. The
crude product was chromatographed over silica gel using CH2Cl2/
Rosmaquinone B (9): [R]20 -2.8 (c 0.01, CHCl3); UV (EtOH)
D
λ
max (log ε) 340 (2.02), 270 (2.51) nm; IR (film) νmax 2964, 1786, 1667,
1
1460, 1395, 1350, 1260, 1216, 1170, 1091, 995, 960, 756 cm-1; H
NMR (300 MHz) δ 0.89 (3H, s, Me-19), 1.01 (3H, s, Me-18), 1.12
(6H, d, J ) 7.0 Hz, Me-16 and Me-17), 1.98 (1H, s, H-5), 2.91 (1H,
hept, J ) 7.0 Hz, H-15), 3.20 (1H, bd, J ) 11.1 Hz, H-1ꢀ), 3.68 (3H,
s, -OCH3), 3.88 (1H, d, J ) 2.9 Hz, H-7), 4.64 (1H, d, J ) 2.9 Hz,
H-6), 6.62 (1H, s, H-14); 13C NMR (75 MHz) δ 18.4 (t, C-2), 21.3 (q,
C-16), 21.9 (q, C-19), 22.0 (q, C-17), 25.1 (t, C-1), 27.5 (d, C-15),
31.1 (s, C-4), 31.5 (q, C-18), 38.0 (t, C-3), 45.8 (s, C-10), 50.2 (d,
C-5), 59.6 (q, O-CH3), 72.5 (d, C-6), 76.9 (d, C-7), 133.6 (d, C-14),
Me2CO (99:1) as eluent to give 6 (76.0 mg, 55.6%): [R]20 -25.2 (c
D
0.10, CHCl3); UV (EtOH) λmax (log ε) 287 (3.34) nm; IR (film) νmax
1
3410, 2920, 1760, 1610 cm-1; H NMR (300 MHz) δ 0.93 (3H, s,
Me-19), 1.03 (3H, s, Me-18), 1.13 (6H, d, J ) 7.0 Hz, Me-16 and
Me-17), 1.20 (1H, dd, J1 ) 3.4 Hz, J2 ) 13.5 Hz, H-3R), 1.44 (1H,
bd, J ) 13.1 Hz, H-3ꢀ), 1.57 (2H, m, H-2), 1.96 (1H, td, J1 ) 5.6
Hz, J2 ) 14.0 Hz, H-1R), 2.30 (1H, s, H-5), 3.05 (1H, hept, J ) 8.1
Hz, H-15), 3.17 (1H, bd, J ) 14.6 Hz, H-1ꢀ), 4.87 (1H, d, J ) 5.9 Hz,

ReVision of the Structure of Rosmaquinone
Journal of Natural Products, 2009, Vol. 72, No. 8 1389
138.3 (s, C-9), 145.7 (s, C-8), 150.0 (s, C-13), 175.6 (s, C-20), 179.6
(s, C-11), 180.0 (s, C-12); EIMS m/z 358 [M]+ (24), 314 (30), 284
(11), 269 (26), 258 (60), 245 (100), 243 (23), 215 (31), 69 (10);
HREIMS m/z 358.1741 [M]+ (calcd for C21H26O5, 358.1780).
Oxidation of 7-O-Methylrosmanol (8) with Ag2O. Freshly prepared
Ag2O (80.7 mg, 0.35 mmol, 4.8 equiv) was added to a solution of 7-O-
methylrosmanol (8) (26.2 mg, 0.07 mmol) in Et2O (10 mL). After being
shaken for 2 h, the solution was filtered, the solvent was evaporated
under reduced pressure, and the product was chromatographed over
silica gel using CH2Cl2 as eluent to yield rosmaquinone B (9) (22 mg,
84.3%).
measuring the increase in optical density at 550 nm (OD550) with a
microplate reader (Multiskan Plus II). All wells with no visible growth
after 24 h, except for M. smegmatis (48 h), in a rotatory shaker at 37
°C were subcultured by transferring in duplicate (0.1 mL) to nutrient,
brain heart infusion, or Sabouraud agar plates. After overnight
incubation, colony counts were performed and the MIC was defined
as the lowest concentration of compound at which growth was inhibited
after 24 h of incubation.
Cytotoxic Activity. HeLa (human carcinoma of the cervix), A-549
(human lung carcinoma), MCF-7 (human breast adenocarcinoma),
Hep-2 (human carcinoma of larynx), and Vero (African green monkey
kidney) cell lines were each grown as a monolayer in Dulbecco’s
modified Eagle’s medium, DMEM (Sigma), supplemented with 5%
fetal calf serum (Gibco) and 1% of penicillin-streptomycin mixture
(10.000 UI/mL). The cells were maintained at 37 °C in 5% CO2 and
98% humidity. Cytotoxicity was assessed using the colorimetric MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] reduc-
tion assay.21 Cell suspensions (0.1 mL of 2 × 104 cells/well) in lag
phase and log phase of growth were incubated in a microtiter well plate
(96-well Iwaki) with the compounds at different concentrations
predissolved in DMSO. After 48 h the optical density was measured
using a microELISA reader (Multiskan Plus II) at 550 nm after
dissolving the MTT formazan with DMSO (150 µL). The percentage
viability (IC50) was calculated from the curve. All the experiments were
repeated three times.
Rosmaquinone (11). Freshly prepared Ag2O (55.9 mg, 0.241 mmol,
2.03 equiv) was added to a solution of rosmanol (7) (41.3 mg, 0.12
mmol) in Et2O (5 mL). After being stirred for 2 h, the solution was
filtered, the solvent was evaporated under reduced pressure, and the
product was chromatographed over silica gel using CH2Cl2 as eluent
1
to yield 11 (37 mg, 90.4%): [R]20 -5.4 (c 0.06, CHCl3); H NMR
D
(300 MHz) δ 0.88 (3H, s, Me-19), 1.02 (3H, s, Me-18), 1.06 (3H, d,
J ) 7.0 Hz, Me-16), 1.08 (3H, d, J ) 7.0 Hz, Me-17), 1.22 (1H, m,
H-3R), 1.45 (3H, overlap, H-1R, H-2ꢀ and H-3ꢀ), 1.60 (1H, m, H-2R),
2.05 (1H, s, H-5), 2.93 (1H, hept, J ) 7.0 Hz, H-15), 3.21 (1H, bd, J
) 10.5 Hz, H-1ꢀ), 4.40 (1H, d, J ) 3.3 Hz, H-7), 4.52 (1H, d, J ) 3.3
Hz, H-6), 6.79 (1H, s, H-14); 13C NMR (75 MHz) δ 18.2 (t, C-2), 21.1
(q, C-16 and C-17), 21.7 (q, C-19), 25.0 (t, C-1), 27.3 (d, C-15), 30.9
(q, C-18), 31.2 (s, C-4), 37.8 (t, C-3), 45.7 (s, C-10), 49.8 (d, C-5),
68.0 (d, C-7), 76.1 (d, C-6), 133.5 (d, C-14), 138.1 (s, C-9), 146.7 (s,
C-8), 150.1 (s, C-13), 175.5 (s, C-20), 179.5 (s, C-11), 179.9 (s, C-12);
EIMS m/z 344 [M]+ (15), 316 (25), 285 (27), 257 (48), 238 (21), 231
(27), 230 (44), 229 (38), 215 (33), 211 (13), 203 (45), 201 (48), 187
(43), 173 (21), 161 (25), 159 (21), 141 (25), 131 (22), 128 (39), 115
(57), 105 (26), 91 (66), 55 (100); HREIMS m/z 344.1586 [M]+ (calcd
for C20H24O5, 344.1548).
Acknowledgment. This work was supported by a Grant from the
Ministerio de Ciencia y Tecnolog´ıa (BQU2006-13376/BQU) and
FICIC-GI no. 05/2008. L.A. and N.P.M. are grateful to the University
of Andes (Me´rida, Venezuela) and to the CajaCanarias, respectively,
for their fellowships.
References and Notes
Isorosmaquinone (10). Freshly prepared Ag2O (235.6 mg, 1.02
mmol, 6.4 equiv) was added to a solution of isorosmanol (12) (56.8
mg, 0.16 mmol) in Et2O (10 mL). After being shaken for 4.5 h, the
solution was filtered, the solvent was evaporated under reduced pressure,
and the product was chromatographed over silica gel using CH2Cl2 as
eluent to yield 10 (51.9 mg, 91.4%): [R]20D -4.6 (c 0.03, CHCl3); UV
(EtOH) λmax (log ε) 420 (2.21), 290 (2.62), 212.5 (3.10) nm; IR (film)
(1) Ikram, M.; Haq, I. Fitoterapia 1980, 51, 231–235.
(2) Gonza´lez, A. G.; Abad, T.; Jime´nez, I. A.; Ravelo, A. G.; Luis, J. G.;
Aguiar, Z.; San Andre´s, L.; Plasencia, M.; Herrera, J. R.; Moujir, L.
Biochem. Syst. Ecol. 1989, 17, 293–296.
(3) Moujir, L.; Gutie´rrez-Navarro, A. M.; San Andre´s, L.; Luis, J. G.
Phytochemistry 1993, 34, 1493–1495.
(4) Moujir, L.; Gutie´rrez-Navarro, A. M.; San Andre´s, L.; Luis, J. G.
Phytother. Res. 1996, 10, 172–174.
(5) Inatani, R.; Nakatani, N.; Fuwa, H. Agr. Biol. Chem. 1983, 47, 521–
528.
ν
max 3462, 2963, 2876, 1760, 1668, 1462, 1329, 1219, 1110, 757, 612
1
cm-1; H NMR (300 MHz) δ 0.86 (3H, s, Me-19), 1.05 (3H, s, Me-
18), 1.11 (6H, d, J ) 7.0 Hz, Me-16 and Me-17), 1.26 (1H, bd, J )
13.9 Hz, H-3ꢀ), 1.49 (1H, bd, J ) 13.9 Hz, H-3R), 1.61 (1H, bd, J )
13.9 Hz, H-2ꢀ), 1.79, 1.83 (1H, dt, J1 ) 3.3 Hz, J2 ) 13.8 Hz, H-2R),
2.28 (1H, td, J1 ) 4.8 Hz, J2 ) 13.6 Hz, H-1R), 2.62 (1H, bd, J )
13.6 Hz, H-1ꢀ), 2.93 (1H, hept, J ) 7.0 Hz, H-15), 4.48 (1H, d, J )
4.5 Hz, H-6), 5.12 (1H, d, J ) 4.5 Hz, H-7), 6.73 (1H, s, H-14); 13C
NMR (75 MHz) δ 18.1 (t, C-2), 20.5 (q, C-19), 21.2 (q, C-16), 21.6
(q, C-17), 26.9 (t, C-1), 27.8 (d, C-15), 32.5 (q, C-18), 34.2 (s, C-4),
40.3 (t, C-3), 48.4 (s, C-10), 56.1 (d, C-5), 69.2 (d, C-6), 77.9 (d, C-7),
131.3 (d, C-14), 135.6 (s, C-9), 150.3 (s, C-13), 151.9 (s, C-8), 172.4
(s, C-20), 176.3 (s, C-11), 179.3 (s, C-12); EIMS m/z 344 [M]+ (17),
300 (57), 287 (50), 270 (49), 255 (100), 231 (47), 215 (27), 165 (10),
115 (15), 69 (15); HREIMS m/z 344.1619 [M]+ (calcd for C20H24O5,
344.1624).
(6) Hitokoto, H.; Morozumi, S.; Wauke, T.; Saiki, S. Appl. EnViron.
Microbiol. 1980, 39, 818–822.
(7) Han, Y. M.; Oh, H.; Na, M.; Kim, B. S.; Oh, W. K.; Kim, B. Y.;
Jeong, D. G.; Ryu, S. E.; Sok, D.-E.; Ahn, J. S. Biol. Pharm. Bull.
2005, 28 (9), 1795–1797.
(8) Ulubelen, A.; Topcu, G.; Tan, N.; Lin, L. J.; Cordell, G. A.
Phytochemistry 1992, 31, 2419–2421.
(9) Dat, N. T.; Jin, X.; lee, J.-H.; Lee, D.; Hong, Y.-S.; Lee, K.; Kim,
Y. H.; Lee, J. J. J. Nat. Prod. 2007, 70, 1093–1097.
(10) Sairafieangpour, M.; Christensen, J.; Stæ, D.; Budwik, B. A.; Kharazmi,
A.; Bagherzadeh, K.; Jaroszewski, J. W. J. Nat. Prod. 2001, 64, 1398–
1403.
(11) Marrero, J. G.; San Andre´s, L.; Luis, J. G. J. Nat. Prod. 2002, 65,
986–989.
(12) Marrero, J. G.; San Andre´s, L.; Luis, J. G. Synlett 2002, 1517–1519.
(13) Marrero, J. G.; San Andre´s, L.; Luis, J. G. Chem. Pharm. Bull. 2005,
53, 1524–1529.
(14) Illi, V. O. Tetrahedron Lett. 1979, 26, 2431–2432.
(15) Tada, M.; Hara, T.; Hara, C.; Chiba, K. A. Phytochemistry 1997, 45
(7), 1475–1477.
(16) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric
Identification of Organic Compounds, 4th ed.; John Wiley & Sons:
New York, 1981.
(17) Mahmoud, A. A.; Al-Shihry, S. S.; Son, B. W. Phytochemistry 2005,
66, 1685–1690.
(18) Gonza´lez, A. G.; San Andre´s, L.; Herrera, J. R.; Luis, J. G.; Ravelo,
A. G. Can. J. Chem. 1989, 67, 208–212.
Antimicrobial Activity. Antimicrobial activity was determined
against Gram-positive (Bacillus subtilis ATCC 6051, B. cereus ATCC
21772, Staphylococcus aureus ATCC 6538, S. epidermidis ATCC
14990, Enterococcus faecalis ATCC 29212, Mycobacterium smegmatis
ATCC 19420) and Gram-negative (Escherichia coli ATCC 9637,
Proteus mirabilis CECT 170, Pseudomonas aeruginosa AK 958,
Salmonella sp CECT 4569) bacteria and the yeast Candida albicans
CECT 1039.
The bacteria cultures were developed in nutrient broth (NB) or brain
heart infusion broth (for E. faecalis, and M. smegmatis containing 0.06%
Tween 80), and the yeast was cultured in Sabouraud liquid medium at
37 °C. All media were purchased from Oxoid.
The minimal inhibitory concentration (MIC) was determined for each
compound in triplicate, by the broth microdilution method as previously
described.20 The compounds were dissolved in DMSO, and wells with
the same proportions of DMSO were used as controls and never
exceeded 1% (v/v). The starting microorganism concentration was
approximately (1-5) × 105 cfu/mL, and growth was monitored by
(19) Deininger, M. In Cell Cycle and Growth Control. Biomolecular
Regulation and Cancer, 2nd ed.; Stein, G. S., Pardee, A. B., Eds.;
John Wiley & Sons: NJ, 2004; Part V: Chapter 20, pp 667-703.
(20) De Leo´n, L.; Beltra´n, B.; Moujir, L. M. Planta Med. 2005, 71, 313–
319.
(21) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
NP900047P