Sesquiterpenes from the sponge Axinyssa isabela

Article abstract of DOI:10.1021/np800465n

Further research on the constituents of the sponge Axinyssa isabela collected in the Gulf of California has led to the isolation of nine new sesquiterpenes, the eudesmanes axinisothiocyanates M and N (1, 2), the bisabolane axinythiocyanate A (3), and the aristolane derivatives axinysones A-E (4-8) and axinynitrile A (9), together with four known sesquiterpenoids (10-13). The structures of the new metabolites have been established by spectroscopic techniques. The absolute configuration of axinysones A (4) and B (5) has been assigned after esterification with (R)- and (S)-MPA acids. In addition, the unusual nitrile-containing sesquiterpene 9 has been synthesized from (+)-aristolone (14). The cytotoxic activity of the compounds isolated has been tested against three human tumor cell lines.

Full text of DOI:10.1021/np800465n

2004
J. Nat. Prod. 2008, 71, 2004–2010
Sesquiterpenes from the Sponge Axinyssa isabela
Eva Zub´ıa,*,† Mar´ıa J. Ortega,† and J. Luis Carballo‡
Departamento de Qu´ımica Orga´nica, Facultad de Ciencias del Mar y Ambientales, UniVersidad de Ca´diz, Apartado 40, 11510-Puerto Real
(Ca´diz), Spain, and Instituto de Ciencias del Mar y Limnolog´ıa, UNAM, Apartado 811, Mazatla´n 82000, Sinaloa, Mexico
ReceiVed July 24, 2008
Further research on the constituents of the sponge Axinyssa isabela collected in the Gulf of California has led to the
isolation of nine new sesquiterpenes, the eudesmanes axinisothiocyanates M and N (1, 2), the bisabolane axinythiocyanate
A (3), and the aristolane derivatives axinysones A-E (4-8) and axinynitrile A (9), together with four known
sesquiterpenoids (10-13). The structures of the new metabolites have been established by spectroscopic techniques.
The absolute configuration of axinysones A (4) and B (5) has been assigned after esterification with (R)- and (S)-MPA
acids. In addition, the unusual nitrile-containing sesquiterpene 9 has been synthesized from (+)-aristolone (14). The
cytotoxic activity of the compounds isolated has been tested against three human tumor cell lines.
Chemical studies on sponges of the genus Axinyssa (order
Halichondrida, family Halichondriidae) have shown that the
secondary metabolism of these organisms is dominated by the
presence of sesquiterpenes exhibiting a wide range of carbon
skeletons and a nitrogenous functionality such as an isocyano,
isothiocyanate, thiocyanate, or formamide group.1,2 In a few
instances, these nitrogen-containing metabolites have been described
to be accompanied by sesqui- and diterpenes without nitrogenous
functionality.3 In addition, a new family of compounds displaying
a sesquiterpene residue linked to a nitrogen-containing fragment,
likely of amino acid origin, has recently been described from A.
aplysinoides.4 On the other hand, terpenoids from Axinyssa species
have shown a wide range of biological activities, which include
antimicrobial, antifouling, antihelmintic, antimalarial, and cytotoxic
properties.1,2a-c,3d,5
In a previous account we described the isolation of new cadinane-
related metabolites possessing an isothiocyanate group and various
oxygenated functions from a sponge of the genus Axinyssa collected
in the Gulf of California.5 Meanwhile, the sponge has been classified
as the new species Axinyssa isabela.6 Further research on the minor
constituents of this sponge has yielded the new sesquiterpenes
axinisothiocyanates M (1) and N (2), axinythiocyanate A (3),
axinysones A-E (4-8), and axinynitrile A (9), together with the
known compounds acanthene B (10),7 11,8 12,9 and 3-isocyano-
theonellin (13).10
Results and Discussion
Freeze-dried specimens of A. isabela were extracted with acetone/
signals of the 13C NMR spectrum were those of the double bond
MeOH (1:1), and the resulting residue was partitioned between H2O
of the isopropenyl group [δ 151.8 (C-11), δ 109.4 (C-13)] and two
and Et2O. The organic extract was subjected to column chroma-
resonances at δ 74.4 (C, C-7) and 64.9 (C, C-4) that were assigned
tography eluting with hexanes/Et2O mixtures of increasing polarity,
to two fully substituted sp3 carbons linked to the hydroxyl and the
then CHCl3/MeOH, and finally MeOH. Repeated separation of
isothiocyanate groups, respectively. The COSY and HMBC cor-
fractions eluted with hexanes/Et2O mixtures afforded the new
relations indicated that 1 was a eudesmane sesquiterpene bearing
sesquiterpenoids 1-9 and the known compounds 10-13.
the isothiocyanate at C-4 and the hydroxyl group at C-7. Thus, the
Axinisothiocyanate M (1) possessed the molecular formula
carbon linked to the isothiocyanate function (δC 64.9, C-4) showed
C16H25NOS, determined by HRCIMS. The presence of hydroxyl
HMBC correlations with the methyl group at δH 1.31 (Me-15) and
and isothiocyanate functions was established from the IR absorp-
with the methine at δH 2.08 (H-5), the carbon of which at δC 47.4
(C-5) showed correlations with the methyl group at δH 0.90 (Me-
14). On the other hand, the location of the hydroxyl function at
C-7 was supported by HMBC correlations of the hydroxylated
carbon (δC 74.4, C-7) with the olefinic protons and the methyl group
of the isopropenyl substituent. The relative configuration of
1
tions at 3466 and 2082 (broad) cm-1, respectively. The H NMR
spectrum displayed the resonances of an isopropenyl unit [δH 5.07
(br s, H-13a), 4.85 (dq, J ) 1.3, 1.3 Hz, H-13b), 1.85 (br s, Me-
12)] and two methyl groups [δH 1.31 (s, Me-15) and 0.90 (s, Me-
14)] that suggested a sesquiterpene framework. The most distinctive
compound 1 (4R*,5R*,7S*,10R*) was deduced from the NOESY
* To whom correspondence should be addressed. Tel: +34 956016021.
spectrum and modeled using MM2 for energy minimization (Figure
Fax: +34 956016193. E-mail: eva.zubia@uca.es.
† Universidad de Ca´diz.
‡ UNAM.
1). The NOE interactions of Me-15 with Me-14 and H-6ax indicated
the 1,3-diaxial relationship between Me-14 and Me-15 and the
10.1021/np800465n CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/14/2008

Sesquiterpenes from the Sponge Axinyssa isabela
Journal of Natural Products, 2008, Vol. 71, No. 12 2005
the bisabolane skeleton. The location of the remaining double bond
in the molecule at C-2 was supported by the HMBC correlations
of the olefinic proton at δH 5.37 (H-2) with the methyl group at δC
23.1 (Me-13) and the methine at δC 42.5 (C-6). The 12.1 Hz
coupling between H-5ax (δH 1.38) and H-6 indicated the axial
orientation of H-6, while the configuration at C-7 remains
undetermined.
Axinysone A (4) possessed the molecular formula C15H22O2
determined by HRCIMS. The NMR data (Table 1) were related to
those of (+)-aristolone (14),11 which was the major metabolite of
the sponge.5 Thus, the NMR spectra of 4 exhibited the signals of
an enone [δC 196.5 (C, C-8) and 120.5 (CH, C-9)/δH 6.20 (dd, J )
2.1, 1.2 Hz, H-9) and δC 168.5 (C, C-10)], two methine protons of
a cyclopropane ring [δH 1.75 (dd, J ) 8.0, 1.2 Hz, H-7) and 1.37
(d, J ) 8.0 Hz, H-6)], and four methyl groups [δH 1.23 (s, Me-13),
1.21 (s, Me-12), 1.17 (s, Me-14), and 1.07 (d, J ) 6.8 Hz, Me-
15)]. The most significant difference between the NMR spectra of
4 and (+)-aristolone (14)11 was the absence in 4 of the resonances
due to the allylic methylene at C-1, showing those of an oxymethine
at δC 69.0/δH 4.38 (br d, J ) 12.0 Hz) instead. Moreover, the
oxygenated function was identified as a hydroxyl group from the
IR absorption at 3404 cm-1. The location of the hydroxyl group at
C-1 was further supported by the HMBC correlations of the
oxymethine carbon (δC 69.0, C-1) with the olefinic proton H-9 (δH
6.20) and the methylene protons at C-2 and C-3 [δH 2.15 (H-2eq),
1.39 (H-2ax), 1.62 (H-3eq), and 1.48 (H-3ax)]. The NOESY
correlations Me-15/H-3eq, H-3ax defined the ꢀ-equatorial orienta-
tion of Me-15, while the ꢀ-axial orientation of Me-14 was supported
by the correlation Me-14/H-3ax (Figure 1, energy minimized using
MM2). The R-orientation of the cyclopropane ring was deduced
from the NOE interactions H-6/Me-14 and Me-13/H-4. Finally, the
NOESY correlations of H-1 with H-2eq, H-3ax, and Me-14
established the ꢀ-axial orientation of H-1 and, therefore, the
R-equatorial orientation of the hydroxyl group. On the basis of
biogenetic grounds axinysone A (4) was expected to belong to the
same enantiomeric series as the co-occurring (+)-aristolone (14).11
This proposal was confirmed by assignment of the absolute
configuration of 4. Treatment of 4 with (R)- and (S)-R-methoxy-
R-phenylacetic (MPA) acids yielded the diastereomeric esters 4r
and 4s, respectively. Positive chemical shift differences (∆δ ) δR
- δS) were observed for H-9, H-7, H-6, and Me-14 (+0.37, +0.06,
+0.05, and +0.04 ppm, respectively), whereas negative ∆δ values
were obtained for H-2eq, H-2ax, H-3eq, and H-3ax (-0.26, -0.23,
-0.07, and -0.07 ppm, respectively). These data indicated an S
configuration12 for C-1 and, therefore, an absolute configuration
1S,4S,5S,6R,7S for axinysone A (4).
Figure 1. Selected NOESY correlations for compounds 1 and 4.
trans-fusion of rings. This assignment was further supported by
the NOESY correlations Me-14/H-1eq, H-2ax, H-6ax, H-8ax, H-9eq
and H-5/H-1ax, H-6eq. Finally, the NOESY correlation of the
olefinic proton H-13a with H-6ax indicated the ꢀ-equatorial
orientation of the isopropenyl group at C-7 and, therefore, the
R-axial orientation of the hydroxyl function.
The molecular formula of axinisothiocyanate N (2), C16H25NO2S,
together with the broad IR absorption at 2090 cm-1 indicated that
it was also an isothiocyanate sesquiterpene. The NMR spectra of 2
were similar to those of 1 except for the downfield shift of the
oxygenated carbon at δC 85.2 and the presence of a proton
resonance at δH 7.32, devoid of correlation in the HSQC spectrum.
These data indicated that compound 2 differed from 1 by the
presence of a hydroperoxy function. Furthermore, the HMBC
correlations of the carbon attached to the isothiocyanate group [δC
64.9 (C-4)] with Me-15 [δH 1.31 (s)] and H-5 [δH 1.97 (dd, J )
13.1, 2.5 Hz)] and those of the oxygenated carbon [δC 85.2 (C-7)]
with the protons of the isopropenyl group confirmed the location
of the isothiocyanate and hydroperoxy functions at C-4 and C-7,
respectively. A series of 1D-NOESY experiments revealed the NOE
interactions Me-14/Me-15, H-1eq, H-2ax, H-6ax, H-8ax, H-9eq,
H-5/H-1ax, H-6eq, and H-13a/H-6ax, H-6eq, which indicated that
compound 2 possessed the same relative configuration as 1.
The molecular formula C16H25NS of axinythiocyanate A (3) was
determined by HRCIMS analysis. The presence of a thiocyanate
function was deduced from the sharp IR absorption at 2148 cm-1
and the 13C NMR resonance at δC 112.2. The remaining 15
resonances of the 13C NMR spectrum, together with four methyl
groups in the 1H NMR spectrum at δ 1.69 (d, J ) 0.8 Hz, Me-12),
1.65 (br s, Me-13), 1.63 (br s, Me-15), and 1.51 (s, Me-14), were
attributable to a sesquiterpene framework. The NMR spectra
included the signals of two trisubstituted double bonds [δC 134.1
(C-3) and 119.7 (C-2)/δH 5.37 (br s, H-2); δC 132.8 (C-11) and
122.7 (C-10)/δH 5.09 (tsept, J ) 7.2, 1.4 Hz, H-10)], whereas the
resonance at δC 63.7 (C, C-7) was assigned to the carbon bearing
the thiocyanate group. As these functional groups accounted for
four of the five unsaturations deduced from the molecular formula,
compound 3 had to be monocyclic. The COSY and HMBC
correlations indicated that 3 possessed a bisabolane framework and
defined the positions of the functional groups mentioned above.
One of the double bonds was located at C-10 based on the allylic
coupling of the olefinic proton at δ 5.09 (H-10) with two methyl
groups [δ 1.69 (Me-12) and 1.63 (Me-15)]. The HMBC correlations
of the carbon bearing the thiocyanate group [δC 63.7 (C-7)] with
the methyl group at δH 1.51 (Me-14) and the methine at δH 1.88
(H-6) defined the attachment of the thiocyanate function to C-7 of
The HRCIMS analysis of axinysone B (5) indicated that it was
an isomer of 4. Furthermore, the COSY and HMBC correlations
defined for compound 5 a planar structure identical to that of 4.
The NOESY correlations observed for Me-15, Me-14, H-4, and
H-6 indicated that compound 5 also possessed the same relative
configuration as 4 at C-4, C-5, C-6, and C-7. However, the 3.0 Hz
coupling of the oxymethine proton H-1 with H-2eq and H-2ax in
5, together with the NOESY correlations of H-1 with H-2eq, H-2ax,
and H-9 established the R-equatorial orientation of H-1 and,
therefore, the ꢀ-axial position of the hydroxyl group. The absolute
1
configuration of 5 was secured through H NMR analysis of the
MPA esters 5r and 5s. Positive chemical shift differences (∆δ )
δR - δS) were observed for H-2eq and H-2ax (+0.15 and +0.11,
respectively), whereas negative ∆δ values were obtained for H-9,
H-7, H-6, and Me-14 (-0.03, -0.07, -0.08, and -0.30 ppm,
respectively). These data indicated an R configuration12 for C-1
and, therefore, an absolute configuration 1R,4S,5S,6R,7S for ax-
inysone B (5). A compound exhibiting the same structure and
relative configuration as 5 has been previously described from the
terrestrial plant Aristolochia debilis.13 Although the optical rotation

2006 Journal of Natural Products, 2008, Vol. 71, No. 12
Zub´ıa et al.
Table 1. NMR Spectroscopic Data (CDCl3) for Compounds 4, 7, 8, and 9a
4b
7b
8c
9b
δH (J in Hz)
2.19 ddddd
position
1
δC
δH (J in Hz)
4.38 br d (12.0)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
69.0
32.2
1.98 m eq
129.9
6.09 s
32.6
1.43 ddd
(13.7, 13.7, 4.1) ax
(14.0, 14.0,4.8, 4.2, 2.3) ax
2.09 dddd
(14.0,4.2, 2.2,1.8) eq
2
3
35.4
28.4
2.15 m eq
1.39 dddd
(13.5, 12.0, 12.0,3.2) ax
1.62 dddd
(13.5, 3.8,3.2, 3.2) eq
1.48 dddd
(13.5, 13.5,12.4, 3.2) ax
1.84 dqd (12.4,
6.8, 3.8)
22.9
29.4
1.66 m eq
1.34 m ax
198.3
42.4
26.3
30.9
1.70 m eq
1.30 m ax
1.49 m eq
1.30 m ax
2.35 m
2.25 m
1.50 m eq
1.35 m ax
4
38.5
34.3
1.95 m
38.3
37.7
1.66 m
5
6
7
39.6
39.8
35.2
42.3
41.6
36.4
37.7
32.1
22.8
36.8
31.5
20.8
1.37 d (8.0)
1.75 dd
1.19 d (8.4)
1.97 d (8.4)
0.81 d (9.0)
1.41 dd (9.0, 2.1)
0.80 d (9.2)
1.13 ddd (9.2, 6.9, 1.3)
(8.0, 1.2)
8
9
196.5
120.5
168.5
24.4
29.7
16.5
208.8
74.6
82.7
30.4
32.0
18.7
17.9
17.0
56.1
56.7
164.6
21.3
29.2
16.2
21.5
14.8
3.65 ddd (4.2, 2.1, 1.0)
3.46 d (4.2)
25.4
112.2
146.1
19.3
29.8
16.1
21.4
15.9
3.68 ddd (6.9, 4.2, 2.5)
5.11 br s
6.20 dd (2.1, 1.2)
4.23 d (2.6)
10
11
12
13
14
15
-OH
1.21 s
1.23 s
1.17 s
1.07 d (6.8)
2.22 br s
1.22 s
1.41 s
1.22 s
1.03 d (6.7)
4.02 d (2.6),
1.72 br s
1.11 s
1.11 s
1.30 s
1.03 d (6.6)
1.11 s
1.33 s
1.03 s
0.96 d (7.0)
23.3
15.9
-CN
121.7
a Assignments aided by COSY, HSQC, HMBC, and NOESY experiments. b Recorded at 600 MHz. c Recorded at 400 MHz.
for that compound was not reported, it likely belongs to the
enantiomeric series of (-)-aristolone, also isolated from the plant.
The molecular formula of axinysone C (6), C15H22O3, was
determined by HRCIMS and indicated that 6 possessed the same
degree of unsaturation as axinysones A (4) and B (5) but with an
additional oxygen atom. The NMR spectra of 6 were related to
those of 5 except for the significant downfield shift of the
oxymethine carbon at δC 85.9 and the presence of a proton
resonance at δH 7.93 (br s). These data suggested that compound
6 was the hydroperoxy analogue of 5. This assignment was
supported by the HMBC correlation of the oxymethine carbon (δC
85.9, C-1) with the olefinic proton H-9 (δH 5.96) and the correlation
of the oxymethine proton (δH 4.47, H-1) with C-5 (δC 39.0). The
2.7 Hz coupling of H-1 with both H-2eq and H-2ax supported the
R-equatorial orientation of H-1 and, therefore, the ꢀ-axial orientation
of the hydroperoxy group.
Axinysone D (7) possessed the molecular formula C15H24O3,
determined by HRCIMS. The presence of hydroxyl and ketone
functions was defined from the IR absorptions at 3436 and 1690
cm-1, respectively. The 1H NMR spectrum displayed the resonances
Figure 2. Selected NOESY (T) and NOESY-1D (f) correlations
for compounds 7 and 9.
of four methyl groups [δ 1.41 (s, Me-13), 1.22 (s, Me-12), 1.22 (s,
Me-14), and 1.03 (d, J ) 6.7 Hz, Me-15)] and those of two methine
protons at δ 1.97 (d, J ) 8.4 Hz, H-7) and 1.19 (d, J ) 8.4 Hz,
H-6) attributable to the protons of the cyclopropane ring of an
aristolane sesquiterpene. The NMR spectra also included the signals
of a ketone carbonyl at δC 208.8 (C-8), an oxymethine at δC 74.6
(C-9)/δH 4.23 (d, J ) 2.6 Hz, H-9), and a fully substituted carbon
linked to a hydroxyl at δC 82.7 (C-10). The HMBC correlations of
the carbonyl carbon with the protons of the cyclopropane ring [δ
1.97 (H-7), 1.19 (H-6)] and with the oxymethine proton (δ 4.23)
defined the location of the carbonyl group at C-8 and the secondary
hydroxyl group at C-9 of the aristolane framework. The location
of remaining hydroxyl group at C-10 was deduced from the HMBC
correlations of the oxygenated carbon at δC 82.7 (C-10) with the
methylene protons at δH 1.98 (H-1eq)/1.43 (H-1ax) and the methine
at δH 1.19 (H-6). The relative configuration of compound 7 was
defined from NOESY and 1D-NOESY data and modeled using
MM2 for energy minimization, as shown in Figure 2. Thus, the
NOESY correlations of Me-15 with H-3ax and H-3eq defined the
ꢀ-equatorial orientation of Me-15, whereas the NOESY cross-peak
between Me-14 and H-3ax supported the ꢀ-axial orientation of Me-
14. On the other hand, the irradiation of H-9 caused NOEs on
H-2ax, H-4, and Me-13. These data indicated the R-orientation of
H-9, the cis-fusion of the six-membered rings, and the R-orientation
of the cyclopropane ring (Figure 2).
Axinysone E (8) possessed the molecular formula C15H20O2,
determined by HRCIMS measurement. The 1H NMR spectrum
exhibited the signals of four methyl groups [δ 1.30 (3H, s, Me-
14), 1.11 (6H, s, Me-12 and Me-13), 1.03 (3H, d, J ) 6.6 Hz,
Me-15)] and two methine protons [δ 1.41 (dd, J ) 9.0, 2.1 Hz,
H-7) and 0.81 (d, J ) 9.0 Hz, H-6)] indicative of an aristolane
sesquiterpene. The NMR signal of a carbonyl at δC 198.3 (C-2)
together with those of a trisubstituted double bond at δC 129.9 (C-

Sesquiterpenes from the Sponge Axinyssa isabela
Journal of Natural Products, 2008, Vol. 71, No. 12 2007
Scheme 1. Synthesis of Axinynitrile A (9) from (+)-Aristolone
(14)
Hz)] showed a NOESY correlation with Me-13 [δ 1.08 (s)]. The
substitution of the hydroxyl group by a nitrile function was achieved
by treatment of the alcohols 15a/15b with TMSCN and BF3 ·Et2O15
to yield the cyano derivative 9 (77.9% yield) together with the
elimination product 1(10),8-aristoladiene14 (11.8% yield). Com-
pound 9 exhibited optical rotation and spectroscopic data identical
to those of the natural axinynitrile A.
The new sesquiterpenes 1-9 and the known compounds 10, 11,
13, and 14 isolated from A. isabela were tested in cytotoxicity assays
against the human tumor cell lines MDA-MB-231 (breast adeno-
carcinoma), A-549 (lung adenocarcinoma), and HT-29 (colon
adenocarcinoma). Compounds 2, 8, and 13 were mildly active. In
particular, compound 2 inhibited the growth of MDA-MB-231 and
A-549 cell lines with GI50 values of 33.3 and 32.6 µM, respectively.
Compound 8 was active against A-549 and HT-29 with GI50 values
of 38.7 and 38.3 µM, respectively. Finally, compound 13 exhibited
growth inhibitory activity with GI50 values of 27.0, 36.4, and 33.4
µM against MDA-MB-231, A-549, and HT-29, respectively. The
remaining compounds were inactive at the highest concentration
tested (10 µg/mL).
1)/δH 6.09 (s, H-1) and δC 164.6 (C-10) established the presence
of an R,ꢀ-unsaturated ketone. Two methines at δC 56.1 (C-8)/δH
3.65 (ddd, J ) 4.2, 2.1, 1.0 Hz, H-8) and δC 56.7 (C-9)/δH 3.46 (d,
J ) 4.2 Hz, H-9), together with the remaining oxygen atom of the
molecular formula, were accommodated in an oxirane ring. In the
HMBC spectrum, the carbonyl carbon (δC 198.3, C-2) was
correlated with the methylene protons at δH 2.35 (m, H2-3), the
carbon of which at δC 42.4 (C-3) showed a correlation with the
methyl group at δH 1.03 (d, J ) 6.6 Hz, Me-15). These data
indicated the location of the ketone at C-2 and, consequently, of
the conjugated double bond at C-1,C-10 of the aristolane skeleton.
On the other hand, the location of the epoxy function at C-8,C-9
was supported by the COSY correlation between the oxymethine
proton at δ 3.65 (H-8) and the bridgehead proton H-7 (δ 1.41).
The NOESY correlations indicated that compound 8 possessed the
same relative configuration as compounds previously described at
C-4, C-5, C-6, and C-7, while the ꢀ-orientation of the oxirane ring
was proposed from the NOESY correlations of H-8 and H-9 with
Me-13.
The molecular formula of axinynitrile A (9), C16H23N, was
determined by HRCIMS. The NMR spectra featured resonances
attributable to an aristolane sesquiterpene containing a trisubstituted
double bond [δC 146.1 and 112.2/δH 5.11 (br s)]. This unsaturation
was located at C-9 from the HMBC correlations of the olefinic
carbons at δC 112.2 (C-9) and 146.1 (C-10) with the bridgehead
protons at δH 1.13 [(ddd, J ) 9.2, 6.9, 1.3 Hz, H-7)] and 0.80 [(d,
J ) 9.2 Hz, H-6)], respectively. In addition to the 15 resonances
of the sesquiterpene skeleton, the 13C NMR spectrum exhibited a
signal at δ 121.7 (C) that together with the weak IR absorption at
2236 cm-1 and the nitrogen atom of the molecular formula was
assigned to a nitrile group. This function had to be linked to the
methine which gave rise to the signals at δC 25.4/δH 3.68 (ddd, J
) 6.9, 4.2, 2.5 Hz). This methine was identified as C-8 of the
aristolane skeleton from the COSY correlations of the proton at δ
3.68 (H-8) with the cyclopropyl proton H-7 and the HMBC
correlation of the carbon at δC 25.4 (C-8) with the cyclopropyl
proton H-6. The NOESY correlations Me-15/H-3eq, H-3ax and Me-
14/H-1ax, H-3ax supported the ꢀ-equatorial orientation of Me-15
and the ꢀ-axial orientation of Me-14, respectively (Figure 2, energy
minimized using MM2). The NOESY correlation H-6/Me-14
defined the ꢀ-orientation of the cyclopropyl protons. Finally a weak
NOESY correlation between H-8 and Me-14 suggested the ꢀ-ori-
entation of H-8 and, therefore, the R-orientation of the nitrile group.
In order to confirm the unusual presence of the nitrile functionality
and to assign the absolute configuration of the molecule, compound
9 was synthesized from (+)-aristolone (14), also isolated from the
sponge (Scheme 1). Luche reduction of 14 led to a 3:1 mixture of
the epimeric alcohols 15a and 15b. Attempts to separate both
isomers by chromatography were unfruitful since the allylic alcohols
were readily transformed into 1(10),8-aristoladiene14 through an
acid-catalyzed 1,4-elimination. Nonetheless, the analysis of the
NMR spectra of the mixture allowed the full assignment of the
NMR data of 15a and partial assignment of those corresponding
to the minor isomer 15b. In particular, the configuration at C-8 for
each isomer was assigned from the NOESY spectrum. In the major
isomer 15a the proton geminal to the hydroxyl [δ 4.51 (ddd, J )
7.1, 3.8, 2.5 Hz, H-8)] exhibited a weak NOE interaction with Me-
14 [δ 0.92 (s)], whereas in 15b the proton H-8 [δ 4.13 (d, J ) 4.4
This study, taken together with our preceding results,5 has shown
that the sponge A. isabela is a prolific source of sesquiterpenoids,
which exhibit diverse skeletal types and comprise derivatives with
and without nitrogenous functionalities. Among the nitrogen-
containing metabolites herein described, compounds 3 and 9 exhibit
unusual features. Compound 3 adds to the uncommon class of
thiocyano-substituted marine terpenoids, for which only seven
representatives have been previously reported.1c,2a Moreover,
axinythiocyanate A (3) is the first account of a bisabolane
sesquiterpene bearing a thiocyanate group. On the other hand, the
nitrile functionality has been found in a small number of terrestrial
and marine natural products exhibiting very diverse structures.16
Among these metabolites, cyanopuupehenol and cyanopuupehenone
from a Verongid sponge17 together with compound 9 herein
described from a Halichondrid sponge are, to the best of our
knowledge, the only examples of natural terpenoids bearing a cyano
group. Moreover, the presence of the cyano substituent in axinyni-
trile A (9) represents a departure from the wide array of nitrogen-
containing terpenoids so far described from sponges of the order
Halichondrida, usually containing isocyano, isothiocyanate, or
formamide groups and less frequently thiocyanate or isocyanate
functionalities.1,2 From a biosynthetic point of view, the nitrile
function in 9 could arise from a cyanide ion, which has been
demonstrated to be a precursor of the isocyano, isothiocyanate, and
thiocyanate substituents present in terpenoids of sponges.1c With
regard to the non-nitrogenous sesquiterpenes herein described from
A. isabela, all of them fall in the aristolane class. In spite of the
vast array of sesquiterpenes so far described from marine organisms,
only a few compounds feature the aristolane skeleton.18 Compounds
4-9 and 14 from A. isabela, together with (+)-9-aristolene from
Acanthella caVernosa,18e appear to be the only aristolane sesquit-
erpenoids described from sponges.
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 polarimeter. UV spectra were recorded on a
GBC Cintra-101 spectrometer. IR spectra were recorded on a Perkin-
Elmer FT-IR System Spectrum BX spectrophotometer. 1H and 13C NMR
spectra were recorded on a Varian INOVA 600 or on a Varian INOVA
400 spectrometer using CDCl3 or C6D6 as solvents. Chemical shifts
were referenced using the corresponding solvent signals [δH 7.26 and
δC 77.0 for CDCl3, δH 7.15 and δC 128.0 for C6D6]. COSY, HSQC,
HMBC, and NOESY experiments were performed using standard
Varian pulse sequences. Low-resolution mass spectra were recorded
on a Finnigan Voyager GC8000top spectrometer. High-resolution mass
spectra (HRMS) were obtained on a Autospec-Q mass spectrometer.
Column chromatography was carried out on Merck silica gel 60
(70-230 mesh). HPLC separations were performed on a LaChrom-
Hitachi apparatus equipped with LiChrospher Si-60 (Merck) columns

2008 Journal of Natural Products, 2008, Vol. 71, No. 12
Zub´ıa et al.
in normal phase and LiChrosorb RP-18 columns in reversed phase,
using a differential refractometer RI-71. All solvents were spectroscopic
grade or were distilled prior to use.
18.2 (CH3, Me-14), signals marked with an asterisk may be inter-
changed; HRCIMS(+) m/z 295.1597 (calcd for C16H25NO2S, 295.1606).
Axinythiocyanate A (3): colorless oil; [R]25D +39.7 (c 0.1, CHCl3);
UV (MeOH) λmax (log ε) 233 (3.23) nm; IR (film) νmax 2148 cm-1; 1H
NMR (CDCl3, 600 MHz) δ 5.37 (1H, br s, H-2), 5.09 (1H, tsept, J )
7.2, 1.4 Hz, H-10), 2.10 (3H, m, H-1, H2-9), 2.02 (2H, m, H2-4), 1.98
(1H, m, H-1), 1.93 (1H, m, H-5eq), 1.88 (1H, m, H-6), 1.81 (1H, m,
H-8), 1.77 (1H, m, H-8), 1.69 (3H, d, J ) 0.8 Hz, Me-12), 1.65 (3H,
br s, Me-13), 1.63 (3H, br s, Me-15), 1.51 (3H, s, Me-14), 1.38 (1H,
dddd, J ) 12.1, 12.1, 12.1, 5.6 Hz, H-5ax); 13C NMR (CDCl3, 150
MHz) δ 134.1 (C, C-3), 132.8 (C, C-11), 122.7 (CH, C-10), 119.7
(CH, C-2), 112.2 (C, -SCN), 63.7 (C, C-7), 42.5 (CH, C-6), 38.9 (CH2,
C-8), 30.9 (CH2, C-4), 27.2 (CH2, C-1), 25.7 (CH3, Me-12), 24.6 (CH2,
C-5), 23.8 (CH3, Me-14), 23.1 (CH2, C-9), 23.1 (CH3, Me-13), 17.7
(CH3, Me-15); CIMS m/ z 263 (4) [M]+, 205 (78), 204 (27), 189 (9),
69 (100); HRCIMS(+) m/z 263.1694 (calcd for C16H25NS, 263.1708).
Collection and Identification. Specimens of Axinyssa isabela6 (order
Halichondrida, family Halichondriidae) were collected by hand using
scuba in Isla Isabel (Gulf of California, Mexico) and immediately
frozen. A voucher specimen is deposited in the Sponge Collection of
the UNAM under the code LEB-ICML-UNAM-56.
Extraction and Isolation. The extraction of the sponge and the
column chromatography of the resulting extract has been previously
described.5 The fraction of the general chromatography eluted with
hexanes/Et2O (95:5) was chromatographed over a silica gel column
using hexanes and hexanes/Et2O mixtures (99:1 to 80:20) as eluants.
Repeated purifications of selected fractions by normal-phase HPLC
using hexanes or hexanes/EtOAc (99:1) yielded acanthene B (10)7 (5.3
mg, 1.9 × 10-3 % dry wt), 118 (3.6 mg, 1.3 × 10-3 % dry wt), 3 (9.7
mg, 3.5 × 10-3 % dry wt), 129 (5.0 mg, 1.8 × 10-3 dry wt), 1310 (5.5
mg, 2.0 × 10-3 % dry wt), and 9 (4.5 mg, 1.6 × 10-3 % dry wt). The
fraction of the general chromatography eluted with hexanes/Et2O (90:
10) was further separated over a silica gel column using hexanes/Et2O
mixtures (92:8 to 80:20) as eluants. Repeated separations of selected
fractions by normal-phase HPLC (hexanes/EtOAc, 94:6 or 93:7)
afforded compounds 1 (1.2 mg, 4.3 × 10-4 % dry wt) and 2 (3.8 mg,
1.3 × 10-3 % dry wt). The fraction of the general chromatography
eluted with hexanes/Et2O (80:20) was subjected to column chroma-
tography eluted with hexanes/Et2O mixtures (90:10 to 50:50). Separa-
tions of selected fractions by normal-phase HPLC (hexanes/EtOAc,
84:16) afforded compound 8 (2.5 mg, 8.9 × 10-4 % dry wt). The
fraction of the general chromatography eluted with hexanes/Et2O (70:
30) was chromatographed over a silica gel column eluted with hexanes/
Et2O mixtures (85:15 to 60:40). Subsequent purification of selected
fractions by normal-phase HPLC (hexanes/EtOAc, 70:30) yielded
compound 7 (3.5 mg, 1.2 × 10-3 % dry wt). The fractions of the general
chromatography eluted with hexanes/Et2O (30:70, 20:80) and Et2O were
joined and chromatographed over a silica gel column eluted with
hexanes/Et2O mixtures (30:70 and 20:80) and then Et2O. The fraction
eluted with hexanes/Et2O (30:70) was further separated by HPLC in
normal (hexanes/EtOAc, 70:30) and reversed phase (MeOH/H2O, 80:
20) to yield compound 6 (2.0 mg, 7.1 × 10-4 % dry wt). The fraction
eluted with hexanes/Et2O (20:80) was subjected to repeated HPLC
separations in normal-phase (hexanes/EtOAc, 60:40) and reversed-phase
Axinysone A (4): colorless oil; [R]25 +254.0 (c 0.1, CHCl3); UV
D
(MeOH) λmax (log ε) 232 (3.93) nm; IR (film) νmax 3404, 1639 cm-1
;
1H NMR (CDCl3, 600 MHz) see Table 1; 13C NMR (CDCl3, 150 MHz)
see Table 1; EIMS m/ z 234 (50) [M]+, 219 (21), 216 (88), 201 (100);
HRCIMS(+) m/z 235.1696 [M + H]+ (calcd for C15H23O2, 235.1698).
Axinysone B (5): colorless oil; [R]25 +108.0 (c 0.1, CHCl3); UV
D
(MeOH) λmax (log ε) 232 (3.96) nm; IR (film) νmax 3400, 1641 cm-1
;
1H NMR (CDCl3, 600 MHz) δ 5.86 (1H, d, J ) 1.3 Hz, H-9), 4.38
(1H, dd, J ) 3.0, 3.0 Hz, H-1), 1.99 (1H, dddd, J ) 14.2, 3.0, 3.0, 3.0
Hz, H-2eq), 1.84 (1H, dddd, J ) 13.2, 12.9, 12.9, 3.0 Hz, H-3ax),
1.79 (1H, dd, J ) 7.9, 1.3 Hz, H-7), 1.77 (1H, m, H-4), 1.58 (1H,
dddd, J ) 14.2, 13.2, 3.6, 3.0 Hz, H-2ax), 1.42 (1H, d, J ) 7.9 Hz,
H-6), 1.40 (1H, m, H-3eq), 1.36 (3H, s, Me-14), 1.21 (3H, s, Me-12),
1.19 (3H, s, Me-13), 1.09 (3H, d, J ) 6.6 Hz, Me-15); 13C NMR
(CDCl3, 150 MHz) δ 197.2 (C, C-8), 165.2 (C, C-10), 127.3 (CH, C-9),
73.2 (CH, C-1), 40.3 (CH, C-6), 39.0 (C, C-5), 38.8 (CH, C-4), 36.4
(CH, C-7), 32.7 (CH2, C-2), 29.8 (CH3, Me-12), 25.4 (C, C-11), 24.9
(CH2, C-3), 24.6 (CH3, Me-14),16.2 (CH3, Me-15), 16.1 (CH3, Me-
13); EIMS m/z 234 (16) [M]+, 219 (14), 216 (40), 201 (72), 173 (36),
149 (57), 105 (80), 91 (100); HRCIMS(+) m/z 235.1685 [M + H]+
(calcd for C15H23O2, 235.1698).
Axinysone C (6): colorless oil; [R]25 +139.0 (c 0.1, CHCl3); UV
D
(MeOH) λmax (log ε) 232 (3.94) nm; IR (film) νmax 3382, 1643 cm-1
;
1H NMR (CDCl3, 600 MHz) δ 7.93 (1H, br s, OOH), 5.96 (1H, d, J )
1.3 Hz, H-9), 4.47 (1H, dd, J ) 2.7, 2.7 Hz, H-1), 2.16 (1H, m, H-2eq),
1.83 (1H, dd, J ) 7.9, 1.3 Hz, H-7), 1.80 (1H, m, H-4), 1.62 (2H, m,
H-2ax, H-3ax), 1.44 (1H, d, J ) 7.9 Hz, H-6), 1.40 (1H, m, H-3eq),
1.31 (3H, s, Me-14), 1.22 (3H, s, Me-12), 1.22 (3H, s, Me-13), 1.08
(3H, d, J ) 6.6 Hz, Me-15); 13C NMR (CDCl3, 150 MHz) δ 196.7 (C,
C-8), 159.8 (C, C-10), 130.6 (CH, C-9), 85.9 (CH, C-1), 40.1 (CH,
C-6), 39.0 (C, C-5), 38.6 (CH, C-4), 36.3 (CH, C-7), 29.8 (CH3, Me-
12), 29.4 (CH2, C-2), 25.4 (C, C-11), 23.4 (CH2, C-3), 23.3 (CH3, Me-
14), 16.2 (CH3, Me-13), 16.2 (CH3, Me-15); EIMS m/z 251 (1) [M +
H]+, 217 (3), 109 (40), 59 (100); HRCIMS(+) m/z 251.1644 [M +
H]+ (calcd for C15H23O3, 251.1647).
(MeOH/H2O, 65:35) to obtain compounds 5 (16.2 mg, 5.8 × 10-3
%
dry wt) and 4 (16.4 mg, 5.8 × 10-3 % dry wt).
Axinisothiocyanate M (1): colorless oil; [R]25D -40.8 (c 0.1, CCl4);
UV (MeOH) λmax (log ε) 243 (3.09) nm; IR (film) νmax 3466, 2082
cm-1; 1H NMR (CDCl3, 600 MHz) δ 5.07 (1H, br s, H-13a), 4.85 (1H,
dq, J ) 1.3, 1.3 Hz, H-13b), 2.08 (1H, dd, J ) 13.0, 2.9 Hz, H-5),
2.03 (1H, dddd, J ) 13.2, 3.3, 3.3, 1.5 Hz, H-3eq), 1.85 (3H, br s,
Me-12), 1.82 (1H, ddd, J ) 13.2, 13.2, 4.8 Hz, H-3ax), 1.76 (1H, ddd,
J ) 14.0, 13.8, 4.2 Hz, H-8ax), 1.73 (1H, dd, J ) 13.4, 13.0 Hz, H-6ax),
1.64 (1H, m, H-9ax), 1.61 (1H, m, H-6eq), 1.55 (2H, m, H2-2), 1.48
(1H, dddd, J ) 14.0, 3.8, 2.7, 2.7 Hz, H-8eq), 1.43 (1H, m, H-1eq),
1.31 (3H, s, Me-15), 1.23 (2H, m, H-1ax, H-9eq), 0.90 (3H, s, Me-
14); 13C NMR (CDCl3, 150 MHz) δ 151.8 (C, C-11), 109.4 (CH2, C-13),
74.4 (C, C-7), 64.9 (C, C-4), 47.4 (CH, C-5), 41.9 (CH2, C-3), 40.0*
(CH2, C-1), 39.9* (CH2, C-9), 34.4 (C, C-10), 32.9 (CH2, C-6), 31.8
(CH2, C-8), 22.1 (CH3, Me-15), 19.0 (CH3, Me-12), 18.9 (CH2, C-2),
18.1 (CH3, Me-14), signals marked with an asterisk may be inter-
changed; CIMS m/ z 279 (6) [M]+, 221 (36), 220 (20), 203 (100);
HRCIMS(+) m/z 279.1635 (calcd for C16H25NOS, 279.1657).
Axinysone D (7): white solid; [R]25D -39.5 (c 0.1, CHCl3); IR (film)
1
νmax 3436, 1690 cm-1; H NMR (CDCl3, 600 MHz) see Table 1; 13C
NMR (CDCl3, 150 MHz) see Table 1; EIMS m/z 252 (3) [M]+, 237
(2), 234 (2), 219 (2), 139 (13), 127 (49), 126 (88), 111 (100);
HRCIMS(+) m/z 252.1719 (calcd for C15H24O3, 252.1725).
Axinysone E (8): colorless oil; [R]25 +84.3 (c 0.07, CHCl3); UV
D
(MeOH) λmax (log ε) 242 (3.87) nm; IR (film) νmax 1670 cm-1; 1H NMR
(CDCl3, 400 MHz) see Table 1; 13C NMR (CDCl3, 100 MHz) see Table
1; CIMS m/z 233 (89) [M + H]+, 217 (47), 203 (45), 161 (84), 91
(100); HRCIMS m/z 233.1529 [M + H]+ (calcd for C15H21O2,
233.1541).
Axinisothiocyanate N (2): colorless oil; [R]25 -75.7 (c 0.05,
D
Axinynitrile A (9): white solid; [R]25 +70.0 (c 0.1, CHCl3); IR
CHCl3); UV (MeOH) λmax (log ε) 244 (3.21) nm; IR (film) νmax 3416,
D
1
2090 cm-1; H NMR (CDCl3, 400 MHz) δ 7.32 (br s, -OOH), 5.07
(film) νmax 2236 cm-1; H NMR (CDCl3, 600 MHz) see Table 1; 13C
1
(1H, br s, H-13a), 5.03 (1H, br s, H-13b), 2.18 (1H, ddd, J ) 13.7,
2.5, 2.5 Hz, H-6eq), 2.03 (1H, dddd, J ) 13.1, 3.2, 3.2, 1.5 Hz, H-3eq),
1.97 (1H, dd, J ) 13.1, 2.5 Hz, H-5), 1.85 (1H, m, H-8eq), 1.85 (3H,
br s, Me-12), 1.81 (1H, ddd, J ) 13.1, 13.1, 4.6 Hz, H-3ax), 1.64 (1H,
ddd, J ) 13.9, 13.9, 4.2 Hz, H-8ax), 1.62 (1H, dd, J ) 13.7, 13.1 Hz,
H-6ax),1.57 (2H, m, H2-2), 1.52 (1H, m, H-9ax), 1.44 (1H, m, H-1eq),
1.31 (3H, s, Me-15), 1.21 (2H, m, H-1ax, H-9eq), 0.91 (3H, s, Me-
14); 13C NMR (CDCl3, 100 MHz) δ 148.0 (C, C-11), 112.1 (CH2, C-13),
85.2 (C, C-7), 64.9 (C, C-4), 47.5 (CH, C-5), 41.9 (CH2, C-3), 39.9*
(CH2, C-1), 39.8* (CH2, C-9), 34.5 (C, C-10), 28.1 (CH2, C-8), 27.3
(CH2, C-6), 21.7 (CH3, Me-15), 18.8 (CH2, C-2), 18.7 (CH3, Me-12),
NMR (CDCl3, 150 MHz) see Table 1; EIMS m/ z 229 (36), 214 (47),
186 (95), 130 (100); HRCIMS(+) m/z 229.1825 (calcd for C16H23N,
229.1830).
Synthesis of the (R)-MPA Ester 4r. A solution of 4 (0.8 mg, 3.4
× 10-3 mmol) in 0.25 mL of CH2Cl2 was treated with CH2Cl2 solutions
of N,N′-dicyclohexylcarbodiimide (2.2 mg, 0.011 mmol in 0.25 mL),
N,N-dimethylaminopyridine (0.9 mg, 7.4 × 10-3 mmol in 0.25 mL),
and (R)-R-methoxy-R-phenylacetic acid (1.8 mg, 0.011 mmol in 0.25
mL) and stirred at room temperature for 5 h. The reaction mixture was
purified over a preparative TLC (hexanes/EtOAc, 1:1) to obtain 1.2
1
mg of the (R)-MPA ester 4r: H NMR (CDCl3, 600 MHz; selected

Sesquiterpenes from the Sponge Axinyssa isabela
Journal of Natural Products, 2008, Vol. 71, No. 12 2009
data, assignments aided by COSY and NOESY experiments) δ 5.94
(1H, dd, J ) 1.9, 1.2 Hz, H-9), 5.57 (1H, ddd, J ) 12.5, 5.6, 2.1 Hz,
H-1), 1.87 (1H, dddd, J ) 12.3, 5.6, 3.3, 3.3 Hz, H-2eq), 1.81 (1H,
dqd, 12.5, 6.9, 4.0, H-4), 1.77 (1H, dd, J ) 8.1, 1.2 Hz, H-7), 1.55
(1H, m, H-3eq), 1.46 (1H, m, H-3ax), 1.38 (1H, d, J ) 8.1, H-6), 1.23
(3H, s, Me-13), 1.22 (1H, m, H-2ax), 1.22 (3H, s, Me-14), 1.21 (3H,
s, Me-12), 1.05 (3H, d, J ) 6.9 Hz, Me-15).
(CH, C-9), 63.2 (CH, C-8), 32.0 (CH, C-6), 29.9 (CH3, Me-12), 28.2
(CH, C-7), 22.7 (CH3, Me-14), 16.1 (CH3, Me-15), 15.9 (CH3, Me-
13).
Reaction of Alcohols 15a and 15b with TMSCN. To a cooled (-10
°C) solution of 15a/15b (15 mg, 0.068 mmol) in CH2Cl2 (1.5 mL) was
added TMSCN (60 µL, 0.48 mmol). Then 700 µL of a 0.1 M solution
of BF3 ·Et2O in CH2Cl2 was added in small portions during 1 h. The
mixture was further stirred at -10 °C for 30 min and then at 0 °C for
30 min. The reaction was quenched with saturated aqueous NaHCO3
solution (12 mL), stirred for 20 min, and extracted with CH2Cl2 (3 ×
15 mL). The organic layer was washed with brine and dried over
MgSO4. After filtration and solvent evaporation under reduced pressure,
the residue was chromatographed over a SiO2 column eluted with
hexanes and hexanes/Et2O (95:5) to obtain 1(10),8-aristoladiene17 (1.6
mg, 0.008 mmol, 11.8% yield) and axinynitrile A (9, 12.2 mg, 0.053
mmol, 77.9% yield).
Synthesis of the (S)-MPA Ester 4s. A solution of 4 (1.2 mg, 5.1 ×
10-3 mmol) in 0.25 mL of CH2Cl2 was treated with CH2Cl2 solutions
of N,N′-dicyclohexylcarbodiimide (2.0 mg, 9.7 × 10-3 mmol in 0.25
mL), N,N-dimethylaminopyridine (1.0 mg, 8.2 × 10-3 mmol in 0.25
mL), and (S)-R-methoxy-R-phenylacetic acid (1.5 mg, 9.0 × 10-3 mmol
in 0.25 mL) and stirred at room temperature for 3 h. The reaction
mixture was purified over preparative TLC (hexanes/EtOAc, 1:1) to
1
obtain 1.8 mg of the (S)-MPA ester 4s: H NMR (CDCl3, 600 MHz;
selected data, assignments aided by COSY and NOESY experiments)
δ 5.57 (1H, dd, J ) 2.0, 1.3 Hz, H-9), 5.52 (1H, ddd, J ) 12.1, 5.6,
2.1 Hz, H-1), 2.13 (1H, m, H-2eq), 1.85 (1H, m, H-4), 1.71 (1H, dd,
J ) 8.1, 1.2 Hz, H-7), 1.62 (1H, m, H-3eq), 1.53 (1H, m, H-3ax), 1.45
(1H, m, H-2ax), 1.33 (1H, d, J ) 8.1, H-6), 1.23 (3H, s, Me-13), 1.20
(3H, s, Me-12), 1.18 (3H, s, Me-14), 1.06 (3H, d, J ) 6.7 Hz, Me-15).
Synthesis of the (R)-MPA Ester 5r. A solution of 5 (3.0 mg, 0.013
mmol) in 0.3 mL of CH2Cl2 was treated with CH2Cl2 solutions of N,N′-
dicyclohexylcarbodiimide (8.0 mg, 0.039 mmol in 0.3 mL), N,N-
dimethylaminopyridine (3.0 mg, 0.025 mmol in 0.3 mL), and (R)-R-
methoxy-R-phenylacetic acid (6.0 mg, 0.036 mmol in 0.3 mL) and
stirred at room temperature for 24 h. The reaction mixture was purified
over a preparative TLC (hexanes/EtOAc, 6:4) to obtain 3.0 mg of the
(R)-MPA ester 5r: 1H NMR (CDCl3, 600 MHz; selected data,
assignments aided by COSY and NOESY experiments) δ 5.94 (1H, d,
J ) 1.2 Hz, H-9), 5.54 (1H, dd, J ) 2.5, 2.5 Hz, H-1), 1.95 (1H, m,
H-2eq), 1.73 (1H, m, H-4), 1.72 (1H, dd, J ) 7.8, 1.2 Hz, H-7), 1.60
(2H, m, H-2ax, H-3ax), 1.42 (1H, m, H-3eq), 1.31 (1H, d, J ) 7.8,
H-6), 1.17 (3H, s, Me-12), 1.15 (3H, s, Me-13), 1.03 (3H, d, J ) 6.9
Hz, Me-15), 0.84 (3H, s, Me-14).
Cytotoxicity Assays. Compounds 1-11, 13, and 14 were tested
against the human tumor cell lines MDA-MB-231 (breast adenocar-
cinoma), A-549 (lung adenocarcinoma), and HT-29 (colon adenocar-
cinoma). Doxorubicin was used as positive control (GI50 ) 0.1, 0.1,
and 0.1 µM against MDA-MB-231, A-549, and HT-29, respectively).
A colorimetric assay using sulforhodamine B (SRB) has been adapted
for quantitative measurement of cell growth and viability as described
in the literature.19
Acknowledgment. This research was supported by grants from
Ministerio de Educacio´n y Ciencia (Spain)-FEDER (research project
CTQ2004-02361) and from Junta de Andaluc´ıa (FQM-285). Cytotox-
icity assays were performed through a cooperative agreement with
PharmaMar.
1
Supporting Information Available: H and 13C NMR spectra of
compounds 1-9. This information is available free of charge via the
Internet at http://pubs.acs.org.
Synthesis of the (S)-MPA Ester 5s. A solution of 5 (3.0 mg, 0.013
mmol) in 0.3 mL of CH2Cl2 was treated with CH2Cl2 solutions of N,N′-
dicyclohexylcarbodiimide (8.0 mg, 0.039 mmol in 0.3 mL), N,N-
dimethylaminopyridine (3.0 mg, 0.025 mmol in 0.3 mL), and (S)-R-
methoxy-R-phenylacetic acid (6.0 mg, 0.036 mmol in 0.3 mL) and
stirred at room temperature for 24 h. The reaction mixture was purified
over preparative TLC (hexanes/EtOAc, 6:4) to obtain 2.9 mg of the
(S)-MPA ester 5s: 1H NMR (CDCl3, 600 MHz; selected data,
assignments aided by COSY and NOESY experiments) δ 5.97 (1H, d,
J ) 1.2 Hz, H-9), 5.46 (1H, dd, J ) 2.8, 2.8 Hz, H-1), 1.80 (1H, dddd,
J ) 14.8, 3.0, 3.0, 3.0 Hz H-2eq), 1.79 (1H, dd, J ) 7.8, 1.2 Hz, H-7),
1.71 (1H, m, H-4), 1.49 (1H, m, H-2ax), 1.39 (1H, d, J ) 7.8, H-6),
1.19 (3H, s, Me-12), 1.17 (3H, s, Me-13), 1.14 (3H, s, Me-14), 1.00
(3H, d, J ) 6.9 Hz, Me-15).
References and Notes
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Reduction of (+)-Aristolone (14). To a cooled (-78 °C) solution
of 4 (42.0 mg, 0.19 mmol) in CH2Cl2 (1.5 mL) was added a solution
of CeCl3 ·7H2O (212.0 mg, 0.57 mmol) in MeOH (4 mL). After stirring
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reaction mixture was further stirred at -78 °C for 30 min and then left
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(15 mL), vigorously stirred, and extracted with Et2O (3 × 15 mL).
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1
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yield). Compound 15a: H NMR (C6D6, 600 MHz) δ 5.30 (1H, br s,
H-9), 4.51 (1H, ddd, J ) 7.1, 3.8, 2.5 Hz, H-8), 2.09 (1H, m, H-1ax),
1.97 (1H, m, H-1eq), 1.57 (1H, m, H-4), 1.54 (1H, m, H-2eq), 1.33
(1H, m, H-3eq), 1.27 (3H, s, Me-12), 1.20 (2H, m, H-2ax, H-3ax),
1.13 (1H, ddd, J ) 9.3, 7.1, 1.2 Hz, H-7), 1.02 (3H, s, Me-13), 0.92
(3H, s, Me-14), 0.91 (3H, d, J ) 6.9 Hz, Me-15), 0.74 (1H, d, J ) 9.3
Hz, H-6); 13C NMR (C6D6, 150 MHz) δ 143.3 (C, C-10), 123.6 (CH,
C-9), 65.9 (CH, C-8), 38.6 (CH, C-4), 37.5 (C, C-5), 35.0 (CH, C-6),
32.9 (CH2, C-1), 31.5 (CH2, C-3), 30.8 (CH3, Me-12), 28.4 (CH, C-7),
26.9 (CH2, C-2), 21.6 (CH3, Me-14), 19.6 (C, C-11), 17.8 (CH3, Me-
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1
13), 16.5 (CH3, Me-15). Compound 15b: H NMR (C6D6, 600 MHz)
δ 5.25 (1H, ddd, J ) 4.4, 1.6, 1.6 Hz, H-9), 4.13 (1H, d, J ) 4.4 Hz,
H-8), 1.08 (3H, s, Me-13), 1.07 (3H, s, Me-14), 1.00 (1H, m, H-7),
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2010 Journal of Natural Products, 2008, Vol. 71, No. 12
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NP800465N