Isothiocyanate sesquiterpenes from a sponge of the genus Axinyssa

Article abstract of DOI:10.1021/np070593s

The chemical study of a sponge of the genus Axinyssa collected in the Gulf of California has led to the isolation of the new bicyclic sesquiterpenes axinisothiocyanates A-L (1-12) together with the known compounds (1R,6S,7S,10S)-10-isothiocyanato-4-amorphene (13), (4R*,5R*, 7S*,10R*)-4-isocyanoeudesm-11-ene, (-)-epipolasin A, and (+)-aristolone. The structures of the new metabolites have been established by spectroscopic techniques, including the analysis of pyridine-induced ¹H NMR chemical shifts. The cytotoxic activity has been tested against three human tumor cell lines.

Full text of DOI:10.1021/np070593s

608
J. Nat. Prod. 2008, 71, 608–614
Isothiocyanate Sesquiterpenes from a Sponge of the Genus Axinyssa
Eva Zubía,*,† María J. Ortega,† Claudia J. Hernández-Guerrero,†,‡ and J. Luis Carballo§
Departamento de Química Orgánica, Facultad de Ciencias del Mar y Ambientales, UniVersidad de Cádiz, Apartado 40, 11510-Puerto Real
(Cádiz), Spain, and Instituto de Ciencias del Mar y Limnología, UNAM, Apartado 811, Mazatlán 82000, Sinaloa, Mexico
ReceiVed October 23, 2007
The chemical study of a sponge of the genus Axinyssa collected in the Gulf of California has led to the isolation of the
new bicyclic sesquiterpenes axinisothiocyanates A-L (1-12) together with the known compounds (1R,6S,7S,10S)-10-
isothiocyanato-4-amorphene (13), (4R*,5R*,7S*,10R*)-4-isocyanoeudesm-11-ene, (-)-epipolasin A, and (+)-aristolone.
The structures of the new metabolites have been established by spectroscopic techniques, including the analysis of
pyridine-induced 1H NMR chemical shifts. The cytotoxic activity has been tested against three human tumor cell lines.
Sesquiterpenes containing isocyano, isothiocyanate, or formamide
functionalities are secondary metabolites frequently found in
sponges of the orders Axinellida and Halichondrida and their
associated opisthobranch mollusks.1–4 From a structural point of
view, these sesquiterpenes exhibit an array of skeletal types, and
with a few exceptions, the nitrogenous-containing group is the only
hetero function present in the molecule. Active research on the
biosynthesis of terpene isocyanides and related compounds in
sponges has demonstrated the incorporation of cyanide and thio-
cyanate ions into various isocyano, isothiocyanate, and thiocyanate
metabolites, although the biochemical source of the cyanide/
thiocyanate employed by sponges has not yet been disclosed.3
Biological activities such as antihelmintic,5 antimicrobial,5,6 and
cytotoxic7,8 properties have been ascribed to some of these nitrogen-
containing sesquiterpenes, although the most significant results have
been described in the antifouling9,10 and antimalarial11 areas.
As a part of our project aimed on searching for new bioactive
metabolites from sponges, we have investigated specimens of a
sponge of the genus Axinyssa collected in the Gulf of California
(Mexico). Herein we report the isolation, structure determination,
and cytotoxic activity of the new cadinane-type sesquiterpenes
axinisothiocyanates A-L (1-12). These metabolites, characterized
as possessing an isothiocyanate group and various oxygenated
functions, extend the uncommon group of nitrogen-containing
sesquiterpenes that exhibit additional hetero functions.5,10,12,13
1
with the H NMR signals of two methyl groups [δ 1.43 (s, Me-
14), δ 1.31 (s, Me-15)] and an isopropyl group [δ 1.04 (3H, d, J )
6.8 Hz, Me-13), 1.01 (3H, d, J ) 6.8 Hz, Me-12), 3.07 (1H, sept,
J ) 6.8 Hz, H-11)] suggested a sesquiterpenoid framework. The
NMR spectra included signals corresponding to a tetrasubstituted
double bond [δC 142.5 (C, C-7) and 127.9 (C, C-6)] and two carbons
linked to oxygenated functions [δC 72.5 (CH, C-5)/δH 4.47 (1H, s,
H-5) and δC 71.6 (C, C-4)], whereas the resonance at δC 62.9 (C,
C-10) was assigned to a fully substituted carbon bearing the
isothiocyanate group. Diagnostic HMBC correlations were those
of the oxymethine proton (δ 4.47, H-5) with the methylene at δC
32.0 (C-3), the methyl at δC 26.0 (Me-15), the oxygenated carbon
at δC 71.6 (C-4), the methine at δC 41.7 (C-1), and the olefinic
carbon at δC142.5 (C-7), which in turn correlated with the methyls
of the isopropyl group [δH 1.04 (Me-13) and 1.01 (Me-12)]. These
data defined the presence of the moiety -CH2-C(CH3)(OH)-
CH(OH)-C(CH)dC(i-Pr)- in the molecule. The olefinic carbon
Results and Discussion
Freeze-dried specimens of Axinyssa sp. were extracted with
acetone/MeOH (1:1), and the resulting residue was partitioned
between H2O and Et2O. The organic extract was subjected to
column chromatography eluted with hexanes/Et2O mixtures of
increasing polarities, then CHCl3/MeOH mixtures, and finally
MeOH. Repeated separation of fractions eluted with hexanes/Et2O
mixtures afforded the new sesquiterpenoids 1-12 along with the
known compounds (1R,6S,7S,10S)-10-isothiocyanato-4-amorphene
(13),5 (4R*,5R*,7S*,10R*)-4-isocyanoeudesm-11-ene,7 (-)-epipo-
lasin A,14 and (+)-aristolone.15
Axinisothiocyanate A (1) possessed the molecular formula
C16H25NO2S, determined by HRCIMS. The presence of an isothio-
cyanate function was deduced from a strong IR absorption at 2103
cm-1 and a broad signal in the 13C NMR spectrum at δ 131.5 (Table
1). The remaining 15 resonances of the 13C NMR spectrum together
bearing the isopropyl group was further connected to
a
* To whom correspondence should be addressed. Tel: +34 956016021.
Fax: +34 956016193. E-mail: eva.zubia@uca.es.
† Universidad de Cádiz.
-CH2-CH2-C(CH3)(NCS)- moiety based on the HMBC cor-
relations of the carbon at δ 142.5 (C-7) with two methylene protons
at δ 2.08 (H-8) and 1.99 (H-9); the latter one also correlated with
the carbon bearing the isothiocyanate group (δC 62.9, C-10) and
the remaining methyl of the molecule (δC 27.3, Me-14). All these
‡ On leave from Centro Interdisciplinario de Ciencias Marinas (CICI-
MAR), Apdo. 592, La Paz, Baja California Sur, 23090 Mexico.
§ UNAM.
10.1021/np070593s CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/21/2008

Isothiocyanate Sesquiterpenes from Axinyssa
Journal of Natural Products, 2008, Vol. 71, No. 4 609
Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) for
Compound 1a
position
δC
δH (J in Hz)
HMBC
1
2
41.7
23.5
2.39 br d (12.5)
1.87 dddd
C-3
(13.4, 4.5, 4.5, 2.7)eq
1.50 m ax
1.92 ddd
C-1, C-3, C-10
C-1, C-2
3
32.0
(13.7, 13.4, 4.5)ax
1.60 m eq
C-4
4
5
6
7
8
71.6
72.5
127.9
142.5
20.9
4.47 s
C-1, C-3, C-4, C-7, C-15
2.24 dddd
(18.1, 9.4, 5.3, 2.7)ax
2.08 dddd
(18.1, 5.3, 5.1, 1.5)eq
1.99 ddd
(13.1, 5.3, 5.1)eq
1.63 ddd
C-6, C-7, C-9, C-10
C-1, C-7, C-8, C-10, C-14
C-1, C-7, C-8,
Figure 1. Selected NOESY correlations for compounds 1 and 3.
9
34.7
described for 1, indicating that both compounds had the same
relative configuration at C-1, C-5, and C-10. However, the Me-15
in 2 showed NOESY correlations with H-2ax and H-3eq, which
indicated the ꢀ-axial orientation of Me-15. Further support for the
relative configuration of 2 was obtained from the comparison of
(13.1, 9.4, 5.3)ax
10
11
12
13
14
15
-NCS
62.9
28.7
21.6
21.0
27.3
26.0
131.5
3.07 sept (6.8)
1.01 d (6.8)
1.04 d (6.8)
1.43 s
C-6, C-7, C-12, C-13
C-7, C-11, C-13
C-7, C-11, C-12
C-1, C-9, C-10
C-3, C-5
1
the H NMR data recorded in CDCl3 and C5D5N. In particular,
H-1 was shifted by 0.30 ppm in C5D5N relative to CDCl3, in
agreement with a 1,3-diaxial relationship between H-1 and the
hydroxyl at C-5. On the contrary, there was not a significant
difference between the chemical shifts of H-2ax in CDCl3 and
C5D5N, as expected from the equatorial orientation of the hydroxyl
group at C-4. All these data defined that axynisothiocyanate B (2)
was the C-4 epimer of compound 1. With regard to compound 3,
the NOESY spectrum exhibited the correlations H-1/H-2eq, H-3ax,
Me-15/H-2ax, H-3eq, and H-5/Me-15, H-11, which defined the
R-axial orientation of H-1, the ꢀ-axial orientation of Me-15, and
the ꢀ-equatorial orientation of H-5, respectively (Figure 1).
Therefore, compound 3 displayed the same relative configuration
as 2 at C-1, C-4, and C-5. Consequently, both compounds had to
differ by their configuration at C-10. The upfield shift of H-2ax (δ
1.02) and the downfield shift of H-1 (δ 2.66) in 3 with respect to
2 [δ(H-2ax) 1.37, δ(H-1) 2.42] were consistent with an R-orientation
of the isothiocyanate group. The NOESY correlations Me-14/H-1,
H-2ax, H-2eq, H-9ax, H-9eq and H-2ax/H-9ax supported this
proposal and also indicated that ring B in 3 adopts a conformation
different from that of 1 and 2, which allows the ꢀ-oriented Me-14
to be equatorial (Figure 1).
1.31 s
a Assignments aided by COSY, HSQC, HMBC, and NOESY experi-
ments.
data indicated that axinisothiocyanate A (1) was a cadinane-type
sesquiterpene containing hydroxyl groups at C-4 and C-5, a dou-
ble bond at C-6, C-7, and the isothiocyanate group at C-10. The
NOESY correlations of Me-15 with H-3eq and H-3ax and those of
Me-14 with H-9eq and H-9ax indicated an equatorial orientation
of both Me-15 and Me-14, whereas the correlations of H-1 with
H-3ax and H-9ax indicated an axial orientation of H-1 (Figure 1,
energy minimized using MM2 calculations). The proton H-5
exhibited NOEs only with Me-15 and the isopropyl group,
suggesting an equatorial orientation of H-5 and, therefore, an axial
orientation of the hydroxyl group at C-5. This assignment was
confirmed from the pyridine-induced solvent shifts in the 1H NMR
spectrum. Pyridine interaction with hydroxyl groups through
hydrogen bonding and collision complex associations causes a
significant deshielding of protons occupying positions geminal,
vicinal, and 1,3-diaxial to a hydroxyl function.16,17 In particular,
protons 1,3-diaxial to a hydroxyl group have been described to
experience deshielding effects on the order 0.2–0.4 ppm relative
Axinisothiocyanate D (4) also possessed the molecular formula
C16H25NO2S, determined by HRCIMS. The IR spectrum showed
the presence of hydroxyl (3416 cm-1) and isothiocyanate (2120
cm-1) functions. The NMR data (Table 2) were consistent with a
cadinane-related sesquiterpene containing a trisubstituted double
bond [δC 139.0 (C) and δC 131.2 (CH)/δH 5.94 (dd, J ) 1.5, 1.5
Hz)] and three fully substituted sp3 carbons, two of them linked to
hydroxyl groups [δC 74.5 (C) and 69.0 (C)] and the remaining one
bearing the isothiocyanate function [δC 65.5 (C)]. In the HMBC
spectrum, the olefinic proton (δ 5.94, H-5) exhibited correlations
with the methyl group at δC 28.2 (Me-15), with the methine at δC
43.5 (C-1), and with the hydroxylated carbon at δC 74.5 (C, C-7),
which in turn was correlated with the methyl groups of the isopropyl
unit [δH 0.96 (Me-13) and 0.77 (Me-12)]. On the other hand, the
olefinic methine carbon (δ 131.2, C-5) exhibited HMBC correlations
with the methine proton at δ 2.08 (m, H-1), which in turn was
correlated with the carbon bearing the isothiocyanate group (δ 65.5,
C-10). These and the remaining correlations defined the location
of the double bond at C-5,C-6, the hydroxyl groups at C-4 and
C-7, and the isothiocyanate function at C-10 of the bicyclic
to chloroform.16 The H NMR spectrum of 1 in C5D5N exhibited
1
the H-1 resonance at δ 2.76, that is, 0.37 ppm downfield shifted
with respect to the H-1 resonance in CDCl3. These data indicated
that the hydroxyl at C-5 was 1,3-diaxial to H-1. Similarly, the H-2ax
resonance was 0.58 ppm downfield shifted in C5D5N relative to
CDCl3, in agreement with the axial orientation of the hydroxyl
group at C-4. The absolute configuration of 1 was assigned by
derivatization with (R)- and (S)-R-methoxy-R-phenylacetic (MPA)
acids to yield the diastereomeric esters 1r and 1s, respectively.18
Positive chemical shift differences (∆δ ) δR - δS) were observed
for Me-15, H-3eq, and H-3ax (+0.37, +0.11, and +0.07 ppm,
respectively), whereas negative ∆δ values were obtained for H-1,
Me-14, H-9ax, H-9eq, H-8ax, and H-8eq (-0.55, -0.30, -0.34,
-0.15, -0.10, and -0.12 ppm, respectively). These data indicated
an S configuration18 for C-5 and therefore an absolute configuration
1R,4S,5S,10S for axinisothiocyanate A (1).
The molecular formula C16H25NO2S determined by HRCIMS
analysis of axinisothiocyanates B (2) and C (3), together with the
similarities of their NMR spectra with those of 1, suggested that 2
and 3 were stereoisomers of 1. The correlations observed for H-1,
H-5, and Me-14 in the NOESY spectrum of 2 were similar to those
1
framework. The overlap of signals in the H NMR spectrum in
CDCl3, and in particular of those corresponding to Me-14 and Me-
15, complicated unambiguous assignment of the NOESY correla-

610 Journal of Natural Products, 2008, Vol. 71, No. 4
Zubía et al.
Table 2. NMR Spectroscopic Data for Compounds 4, 7, 8, and 11a
4b
7c
8d
11b
position
1
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
43.5
2.08 m
72.6
45.1
2.08 ddd
45.4
1.85 br d (12.8)
(9.2, 6.6, 2.0)
2.02 m eq
1.72 dddd
2
21.7
36.0
1.99 dddd
(13.4, 5.7, 5.7, 3.4)eq
1.72 m ax
31.1
34.6
2.19 ddd
(14.0, 14.0, 3.4)ax
2.09 ddd
(14.0, 4.4, 3.8)eq
2.37 m ax
2.00 dddd
22.2
36.5
19.8
30.9
1.57 m eq
1.50 m ax
(13.5, 13.5, 9.2, 3.3)ax
3
1.92 m eq
1.51 ddd
1.97 m eq
1.56 m ax
2.00 m
(12.3, 12.3, 3.4)ax
(12.3, 4.4, 3.4, 1.5)eq
4
5
6
69.0
131.2
139.0
69.1e
134.2
137.9
69.2
133.1
133.6
133.5
124.2
35.2
5.94 dd (1.5, 1.5)
5.95 dd (1.8, 1.5)
5.87 dd (2.0, 1.7)
5.57 dq (5.6, 1.3)
2.26 ddd
(11.0, 5.6, 5.6)
1.27 dddd
(11.3, 11.0, 2.8, 2.8)
1.50 m eq
7
8
74.5
33.2
41.9
22.2
2.45 dddd
(12.9, 4.5, 3.4, 1.8)
1.59 dddd,
86.1
25.0
43.5
20.3
2.08 m eq
2.51 ddd
1.63 ddd
(14.0, 14.0, 3.4)ax
(13.1, 4.5, 4.0, 3.0)eq
1.48 dddd
(14.2, 14.2, 4.3)ax
1.95 m eq
1.37 m ax
(13.1, 13.1, 12.9, 3.5)ax
2.35 ddd
(13.5, 13.1, 4.0)ax
1.72 ddd
9
36.7
1.92 m eq
1.69 m ax
35.4
36.2
2.02 m eq
1.68 ddd
(14.2, 14.2, 3.6)ax
34.3
1.77 br d (13.0)eq
1.48 m ax
(13.5, 3.5, 3.0)eq
10
11
12
13
14
15
-NCS
-OH
-OOH
65.5
29.7
16.3
15.7
26.8
28.2
130.5
69.2e
26.9
22.1
17.6
22.1
64.5
31.8
16.6
16.3
26.5
28.2
nd
65.7
26.6
21.5
15.3
27.5
23.4
130.0
1.90 m
1.97 sept d (6.7, 3.4)
0.80 d (6.7)
0.86 d (6.7)
1.46 s
1.95 sept (6.9)
0.86 d (6.9)
1.01 d (6.9)
1.39 s
2.00 m
0.77 d (6.9)
0.96 d (6.9)
1.41 s
0.90 d (6.9)
0.87 d (6.9)
1.39 s
1.41 s
28.3
129.8
1.71 s
1.46 s
1.65 br s
6.88 (s)
7.21 s
a Assignments aided by COSY, HSQC, HMBC, and NOESY experiments. b Recorded in CDCl3 at 400 MHz. c Recorded in C5D5N at 600 MHz.
d Recorded in CDCl3 at 600 MHz. e Signals marked with the same letter in the same column may be interchanged.
configuration at C-4 and possesses the Me-15 R-equatorially
oriented. Unfortunately the close chemical shifts of Me-15 and
H-3ax in C5D5N, and also in CDCl3, precluded the observation of
the expected NOE between Me-15 and H-3ax in the NOESY and
1D-NOESY experiments. Nevertheless, the higher chemical shift
of H-2ax in C5D5N relative to CDCl3 by 0.29 ppm fully supported
the axial orientation of the hydroxyl at C-4 and therefore the
equatorial position of Me-15. On the other hand, the NOESY
spectrum of 6 indicated that it was the C-10 epimer of 4 exhibiting
the correlations Me-14/H-2ax, H-8ax, H-9eq that supported the
ꢀ-axial orientation of Me-14.
Figure 2. Selected NOESY correlations for compound 4.
Axinisothiocyanate G (7) was the seventh isomer of this series
of sesquiterpenes of molecular formula C16H25NO2S. The NMR
tions. However, the 1H NMR spectrum recorded in C5D5N exhibited
a better dispersion of signals, and the NOESY spectrum in this
solvent was analyzed to define the relative configuration of 4. The
correlations H-11/H-1, H-9ax and H-1/H-9ax indicated 1,3-diaxial
relationships among the isopropyl group, H-1, and H-9ax (Figure
2, energy minimized using MM2 calculations). The R-equatorial
orientation of Me-14 was defined from the NOESY correlations
Me-14/H-1, H-2eq, H-9eq, H-9ax, whereas the correlations Me-
15/H-2ax, H-3eq indicated the ꢀ-axial orientation of Me-15. All
these data defined the relative configuration 1R*,4R*,7S*,10S* of
axinisothiocyanate D (4).
The HRCIMS analysis of axinisothiocyanates E (5) and F (6)
established that both compounds were isomeric with those above-
described. In particular, the COSY and HMBC correlations
indicated that 5 and 6 possessed a planar structure identical to that
of compound 4. Similar to 4, the NOESY spectrum of 5 in C5D5N
showed correlations consistent with an R-axial orientation of H-1
and the isopropyl group (H-11/H-1, H-9ax and H-1/H-9ax) and with
an R-equatorial orientation of Me-14 (Me-14/H-1, H-2eq, H-9ax,
H-9ec). Therefore, compound 5 had to differ from 4 by the
spectra of 7 recorded in CDCl3 and also in C5D5N (Table 2) were
related to those of compounds 4-6, displaying the resonances of
a trisubstituted double bond [δC 137.9 (C) and δC 134.2 (CH)/δH
5.95 (dd, J ) 1.8, 1.5 Hz) in C5D5N], and those attributable to two
hydroxylated carbons and one linked to the isothiocyanate group
[δC 72.6 (C), 69.2 (C), and 69.1 (C)]. The location of the double
bond at C-5,C-6 of the cadinane framework was inferred from the
allylic coupling of the olefinic proton [δH 5.95 (dd, J ) 1.8, 1.5
Hz)] with a methine proton at δ 2.45 (dddd, J ) 12.9, 4.5, 3.4, 1.8
Hz, H-7) whose corresponding carbon [δC 41.9 (CH, C-7)] exhibited
HMBC correlations with the methyl groups of the isopropyl unit
[δ 0.86 (Me-13) and 0.80 (Me-12)]. On the other hand, the
hydroxylated carbon at δ 72.6 (C, C-1) showed HMBC correlations
with the olefinic proton H-5 and with a methyl group at δH 1.46 (s,
Me-14), which additionally exhibited a weak four-bond correlation
with the carbon atom of the isothiocyanate group (δC 129.8). These
data unambiguously defined the location of the isothiocyanate group
at C-10 and of the hydroxyl group at C-1. The remaining hydroxyl
function of the molecule had therefore to be placed at C-4, geminal
to the methyl at δH 1.71 (s, Me-15). The large coupling constant

Isothiocyanate Sesquiterpenes from Axinyssa
Journal of Natural Products, 2008, Vol. 71, No. 4 611
(12.9 Hz) between H-7 and H-8ax indicated the R-axial orientation
of H-7 and, therefore, a ꢀ-equatorial orientation of the isopropyl
group. This assignment was further supported by NOESY correla-
tions H-11/H-5, Me-12/H-8eq, and Me-13/H-5, H-8ax. The NOESY
correlations Me-14/H-2eq, H-9ax, H-9eq and Me-15/H-2ax, H-3eq
defined the R-equatorial orientation of Me-14 and the ꢀ-axial
orientation of Me-15, respectively. The relative configuration at
C-1 was deduced from the NOESY correlation of the proton of
the hydroxyl group at C-1 [δ 6.88 (s)] with H-9ax and H-2eq. This
assignment was further supported by the pyridine-induced shifts
of protons 1,3 with respect to the hydroxyl at C-1. Thus, H-7 and
H-9ax were deshielded in C5D5N relative to CDCl3 by 0.31 and
0.24 ppm, respectively, in agreement with the R-axial orientation
of the hydroxyl at C-1. All these data defined the configuration
1S*,4R*,7S*,10S* for axinisothiocyanate G (7).
Figure 3. Selected NOESY correlations for compound 11.
had been previously described from the sponge Axinyssa fenestra-
tus.5 Compound 14 was proposed to possess Me-14 and Me-15
R-equatorial on the basis of 13C NMR chemical shifts, while the
configuration at C-1 and C-7 was not determined. The 13C NMR
data reported for 14 (in CDCl3) are almost identical to those of 10
(except for signals assigned in ref 5 to C-11 and Me-15, C-1 and
C-7, which could be interchanged), suggesting that 14 should be
revised to 10, for which the relative configuration has been
unambiguously defined. Surprisingly, the partial 1H NMR data
(C6D6) reported for 14 are significantly different from those recorded
for 10 in C6D6, and we therefore cannot conclude if compound 14
isolated from A. fenestratus is identical or not to axinisothiocyanate
J (10).
Axinisothiocyanate K (11) possessed the molecular formula
C16H25NS, determined by HRCIMS. The IR absorption at 2096
cm-1 and a broad 13C NMR signal at δ 130.0 confirmed the
presence of an isothiocyanate group. The remaining 15 resonances
of the 13C NMR spectrum (Table 2) were attributable to a cadinane-
type sesquiterpene containing a trisubstituted double bond [δ 133.5
(C) and 124.2 (CH)] and a fully substituted carbon linked to the
isothiocyanate function [δ 65.7 (C)]. In particular, the analysis of
the COSY and HMBC correlations indicated that 11 possessed the
same planar structure as compound 13 from A. fenestratus5 and
two cadinanes from Stylissa sp.4c and Acanthella caVernosa,19
respectively. The relative configuration 1R*,6S*,7R*,10R* of
compound 11 was determined from the analysis of the coupling
constants and the NOESY data. Thus, the NOESY correlation H-1/
H-6 together with the JH-1,H-6 coupling constant of 5.6 Hz indicated
the cis (equatorial-axial) orientation of these protons (Figure 3,
energy minimized using MM2). The 11.0 Hz coupling between H-6
and H-7 defined the trans-1,2-diaxial relationship between these
protons and, therefore, the equatorial orientation of the isopropyl
group at C-7. These assignments were also supported by the
NOESY correlations H-6/H-8ax and H-6/Me-13. Furthermore, the
spatial arrangement deduced for the cis-fused bicycle of 11 (Figure
3) exhibits a dihedral angle between H-5 and H-6 of 45°, which is
consistent with the observed coupling constant JH-5,H-6 of 5.6 Hz.
It is worth noting that this spatial arrangement is different from
that of the cis-fused rings of compound 13 (H-6 1,3-diaxial to H-2ax
and H-1 1,3-diaxial to H-9ax), where the dihedral angle H-5,H-6
is close to 90° and, consequently, the coupling constant JH-5,H-6 is
almost zero. Finally, the NOESY correlations of Me-14 with H-1,
H-2eq, and H-9eq defined the ꢀ-equatorial orientation of Me-14.
The molecular formula of axinisothiocyanate H (8), C16H25NO3S,
indicated that 8 possessed the same degree of unsaturation as the
compounds described above but with an additional oxygen atom.
1
These data together with the H NMR resonance at δH 7.21 (1H,
s), devoid of correlations in the HSQC spectrum, indicated that 8
contained a hydroperoxide function. Furthermore, the 13C NMR
resonance at δC 86.1 (C) was attributed to a fully substituted carbon
bearing the hydroperoxy group. The COSY and HMBC correlations
defined for 8 a planar structure identical to that of compounds 4-6,
except for the presence of the hydroperoxy group at C-7. In
particular, this assignment was supported by the HMBC correlations
of the carbon at δ 86.1 (C, C-7) with the olefinic proton (δ 5.87,
H-5) and the methyl groups of the isopropyl unit [δ 1.01 (Me-13)
and 0.86 (Me-12)]. The NOESY correlations of H-1 with Me-14
and H-11, of Me-15 with H-2ax and H-3eq, and of H-5 with Me-
15 and the hydroperoxide proton indicated that 8 possessed the
same relative configuration as compound 4.
The NMR spectra of axinisothiocyanate I (9) also exhibited the
resonances of a hydroperoxide function [δH 7.05 (1H, br s)] linked
to a fully substituted carbon [δC 85.6 (C, C-7)] as well as those of
a hydroxylated carbon [δC 70.0 (C, C-4)], a carbon bearing the
isothiocyanate group [δC 60.1 (C, C-10)], and a trisubstituted double
bond [δC 138.2 (CH, C-5)/δH 5.92 (br s, H-5) and δC 132.0 (C,
C-6)]. The COSY and HMBC correlations defined for 9 the same
planar structure as 8. Furthermore, the NOESY correlations H-1/
H-2eq, H-9ax, Me-15/H-2ax, H-3eq, and Me-14/H-1, H-2eq, H-9ax,
H-9eq confirmed that 9 possessed the same relative configuration
as 8 at C-1, C-4, and C-10, respectively. Therefore, the stereo-
chemical difference between 9 and 8 had to be at C-7. In fact, the
NOESY correlation between the olefinic proton H-5 and H-11
supported the ꢀ-equatorial orientation of the isopropyl group.
The molecular formula of axinisothiocyanate J (10), C16H25NOS,
was determined by HRCIMS. The most distinctive difference
between the NMR spectra of 10 and those of compounds 4-6
described above was the absence of the signal due to the
hydroxylated carbon C-7, showing the signals of a methine at δC
47.4 (CH)/δH 1.69 (m) instead. Further confirmation was obtained
from the HMBC correlations of this methine carbon with the olefinic
proton H-5 [δ 5.57 (d, J ) 1.0 Hz)] and the methyl groups of the
isopropyl unit [δ 0.98 (d, J ) 6.8 Hz) and 0.92 (d, J ) 6.8 Hz)].
The relative configuration 1R*,4R*,7S*,10S* of compound 10 was
assigned from NOESY data. Due to the overlapping of signals, the
NOESY spectra in CDCl3 and in C5D5N were analyzed. The
NOESY spectrum in C5D5N exhibited correlations of H-1 with H-7
and H-9ax, which indicated 1,3-diaxial relationships among H-1,
H-7, and H-9ax. The ꢀ-equatorial orientation of the isopropyl group
was further supported from the correlations H-11/H-5 and Me-13/
H-8ax observed in the NOESY in CDCl3. The correlations Me-
14/H-1, H-2eq, H-9eq, H-9ax in C5D5N defined the R-equatorial
orientation of Me-14, whereas the ꢀ-axial orientation of Me-15 was
deduced from the correlation Me-15/H-2ax observed in the NOESY
spectrum in CDCl3. The sesquiterpene 14, closely related to 10,
The molecular formula of axinisothiocyanate L (12), C16H23NOS,
together with the IR absorption at 2088 cm-1 suggested that 12
was also an isothiocyanate sesquiterpene. In addition to a fully
substituted carbon bearing the nitrogenous function [δC 64.3 (C,
C-10)], the NMR spectra showed the resonances of a carbonyl [δC
197.6 (C)] and a trisubstituted double bond at δC 135.2 (C) and δC
149.3 (CH)/δH 6.96 (dq, J ) 6.4, 1.4 Hz), that together with a
vinylic methyl group at δH 1.81 (dd, J ) 1.4, 1.0 Hz) indicated the
presence of an R-methyl-R,ꢀ-unsaturated ketone moiety. The
olefinic proton showed a COSY correlation with the methine proton

612 Journal of Natural Products, 2008, Vol. 71, No. 4
Zubía et al.
common in vertical walls, small caves, and overhangs at depths between
4 and 25 m from Isabel Island (Nayarit, Mexico, 21°50′33′′ N
105°53′10′′ W), the only locality where the species has been found. A
voucher has been deposited in the Sponge Collection of the UNAM
under the code LEB-ICML-UNAM-56.
at δ 2.62 (H-6) and an HMBC correlation with a methine at δC
44.8 (C-1), whose corresponding proton (δ 2.41, H-1) was
correlated with the carbonyl carbon. These data supported the
presence of the cyclohexenone ring depicted in formula 12. The
remaining NMR data were accommodated in a six-membered ring
bearing the isothiocyanate function and the isopropyl group at the
same positions as in the previously described compounds. The
coupling constants of H-6 with H-1, H-5, and H-7 (4.3, 6.4, and
10.2 Hz, respectively) together with the NOESY correlations Me-
14/H-1, H-2eq, H-9eq indicated that 12 possessed the same relative
configuration as 11. Among the different stereochemical classes
of the cadinane-related sesquiterpenoids, compounds 11 and 12
belong to the muurolane class, characterized by possessing H-1,
H-6, and the isopropyl group cis-oriented.
Compounds 1-8 and 10–13 isolated from Axinyssa sp. were
tested in cytotoxicity assays against the human tumor cell lines
MDA-MB-231 (breast adenocarcinoma), A-549 (lung adenocarci-
noma), and HT-29 (colon adenocarcinoma). Compounds 2 and
10-13 showed moderate activity, whereas compounds 1 and 3-8
were not active at the highest concentration tested (10 µg/mL). In
particular, the most active compound was axinisothiocyanate J (10),
which exhibited GI50 values of 10.4, 11.8, and 11.8 µM and TGI
values of 14.3, 19.0, and 14.0 µM against MDA-MB-231, A-549,
and HT-29 cell lines, respectively. Compounds 2, 11, and 12
exhibited growth inhibitory activity of A-549 and HT-29 cells with
GI50 values ranging from 21.0 to 34.5 µM [GI50 (µM) for 2: 25.7
(A-549) and 21.0 (HT-29); for 11: 34.5 (A-549) and 22.8 (HT-
29); for 12: 31.4 (A-549) and 31.4 (HT-29)]. Finally, compound
13 inhibited the growth of the A-549 cell line with a GI50 value of
33.4 µM.
Extraction and Isolation. Freeze-dried specimens of the sponge
Axinyssa sp. (280.5 g) were repeatedly extracted with acetone/MeOH
(1:1, 5 × 3 L) at room temperature. After filtration, the solution was
evaporated under reduced pressure to obtain a residue that was
partitioned between H2O and Et2O. The organic layer was evaporated
to dryness to give an extract (18.1 g), which was chromatographed on
a SiO2 column using solvents of increasing polarities from hexanes/
Et2O (95:5) to Et2O, then CHCl3/MeOH (80:20 and 50:50), and finally
MeOH. The fraction eluted with hexanes/Et2O (95:5) was chromato-
graphed over a silica gel column using hexane and hexanes/Et2O
mixtures (99:1 to 80:20) as eluants. Repeated purifications of selected
fractions by normal-phase HPLC using hexanes and hexanes/EtOAc
(99:1) yielded (-)-epipolasin A14 (110.5 mg, 3.9 × 10-2 % dry wt),
compound 11 (30.0 mg, 1.1 × 10-2 % dry wt), compound 13 (85.0
mg, 3.0 × 10-2 % dry wt), and 4-isocyanoeudesm-11-ene7 (70.0 mg,
2.5 × 10-2 % 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, 92:8) afforded compound 12 (27.0 mg, 9.6 × 10-3
% dry wt). The fraction of the general chromatography eluted with
hexanes/Et2O (80:20) was subjected to column chromatography eluted
with hexanes/Et2O mixtures (90:10 to 50:50) to yield (+)-aristolone15
(440 mg, 0.157% 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 compounds 5 (11.0 mg, 3.9 × 10-3
%
dry wt) and 10 (21.0 mg, 7.4 × 10-3 % dry wt). The fractions of the
general chromatography eluted with hexanes/Et2O (50:50 and 30:70)
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 compounds 1 (8.0 mg, 2.9 × 10-3 % dry
wt) and 3 (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 (acetone/
H2O, 75:25, MeOH/H2O, 80:20 or 65:35) to obtain compounds 8 (11.0
mg, 3.9 × 10-3 % dry wt), 4 (27.0 mg, 9.6 × 10-3 % dry wt), 2 (15.5
mg, 5.5 × 10-3 % dry wt), 7 (1.5 mg, 5.3 × 10-4 % dry wt), 6
(2.5 mg, 8.9 × 10-4 % dry wt), and 9 (1.0 mg, 3.6 × 10-4 % dry wt).
Axinisothiocyanate A (1): white solid; [R]25D -33.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 243 (3.13) nm; IR (film) νmax 3416, 2103,
1656 cm-1; 1H NMR (CDCl3, 600 MHz) see Table 1; 1H NMR (C5D5N,
600 MHz) δ 6.23 (br s, OH), 4.89 (1H, s, H-5), 3.22 (1H, sept, J )
6.9 Hz, H-11), 2.76 (1H, br d J ) 12.5 Hz, H-1), 2.27 (1H, ddd, J )
13.5, 13.5, 4.3 Hz, H-3ax), 2.15 (1H, m, H-8), 2.08 (1H, dddd, J )
13.5, 12.5, 12.5, 3.7 Hz, H-2ax), 1.89 (2H, m, H-8, H-9), 1.87 (1H, m,
H-2eq), 1.80 (1H, br ddd, J ) 13.5, 3.7, 2.8 Hz, H-3eq), 1.69 (3H, s,
Me-15), 1.48 (1H, m, H-9), 1.29 (3H, s, Me-14), 1.00 (3H, d, J ) 6.9
Hz, Me-12), 0.90 (3H, d, J ) 6.9 Hz, Me-13); 13C NMR (CDCl3, 150
MHz) see Table 1; EIMS m/z 295 (6) [M]+, 280 (3), 252 (42), 236
(36), 218 (20), 179 (44), 175 (78), 107 (75), 79 (74), 55 (100);
HRCIMS(+) m/z 295.1602 (calcd for C16H25NO2S, 295.1606).
Axinisothiocyanate B (2): white solid; [R]25D -2.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 238 (3.03) nm; IR (film) νmax 3392, 2097,
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, C5D5N, or C6D6 as solvent. 1H and 13
C
chemical shifts were referenced using the corresponding solvent signals
[δH 7.26 and δC 77.0 for CDCl3, δH 7.55 and δC 135.5 for C5D5N, and
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 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 in normal phase and LiChrosorb
RP-18 columns in reversed phase, using a RI-71 differential refracto-
meter. All solvents were spectral grade or were distilled prior to use.
Collection and Identification. Specimens of Axinyssa sp. (family
Halichondriidae, order Halichondrida) were collected by hand using
scuba at the Gulf of California and immediately frozen. The sponge is
an undescribed species whose morphological features are typical of
the genus Axinyssa. It is an incrusting to massive globular amorphous,
sometimes lobate, yellow sponge, with a fleshy and compressible
consistency in life, but crumbly when dry. The largest specimen
measured 17 cm long by 12 cm wide and 5 cm high. The surface in
some parts is smooth, but generally is conulose-microconulose or hispid
due to the terminal part of the choanosomal fibers that form brushes of
spicules on the surface. Oscules are from 1 to 5 mm in diameter and
scattered irregularly on the surface. Aquiferous canals are visible around
some oscula. The ectosome is thin, membranous, partly detachable,
and without a specialized skeleton, but with some spicules arranged
criss-cross or paratangentially to the surface. The choanosomal skeleton
is largely confused, with some vague ascending single spicules or tracts
ending at the surface in bouquets. The spicules are curved oxeas,
stylotes, and some strongyles in a wide range of sizes but difficult to
divide into categories. The oxeas are 320-(619.5)-720 × 5-(10)-15 µm,
strongyles measure 475-(526)-625 × 9-(9.6)-10 µm, and stylotes
measure 480-(520)-590 × 6-(8.8)-10 µm. The species is relatively
1
1660 cm-1; H NMR (CDCl3, 600 MHz) δ 4.52 (1H, s, H-5), 3.04
(1H, sept, J ) 6.7 Hz, H-11), 2.42 (br d, J ) 12.5 Hz, H-1), 2.22 (1H,
dddd, J ) 18.3, 10.3, 5.3, 3.0 Hz, H-8ax), 2.07 (1H, dddd, J ) 18.3,
5.3, 3.8, 1.6 Hz, H-8eq), 1.98 (1H, ddd, J ) 13.1, 13.1, 4.4, H-3ax),
1.98 (1H, m, H-9eq), 1.92 (1H, dddd, J ) 13.1, 4.4, 4.4, 2.8 Hz, H-2eq),
1.63 (1H, m, H-3eq), 1.63 (1H, ddd, J ) 13.0, 10.3, 5.3 Hz, H-9ax),
1.42 (3H, s, Me-14), 1.37 (1H, m, H-2ax), 1.20 (3H, s, Me-15), 1.02
1
(3H, d, J ) 6.7 Hz, Me-12), 1.02 (3H, d, J ) 6.7 Hz, Me-13); H
NMR (C5D5N, 600 MHz) δ 4.81 (1H, s, H-5), 3.07 (1H, sept, J ) 6.9
Hz, H-11), 2.72 (1H, br d, J ) 12.5 Hz, H-1), 2.38 (1H, ddd, J )
13.4, 13.4, 4.3 Hz, H-3ax), 2.12 (1H, dddd, J ) 17.9, 9.7, 5.2, 2.8 Hz,
H-8ax), 1.89 (2H, m, H-2eq, H-8eq), 1.79 (1H, ddd, J ) 12.8, 5.2, 5.0
Hz, H-9eq), 1.75 (1H, m, H-3eq), 1.46 (2H, m, H-2ax, H-9ax), 1.37

Isothiocyanate Sesquiterpenes from Axinyssa
Journal of Natural Products, 2008, Vol. 71, No. 4 613
(3H, s, Me-15), 1.22 (3H, s, Me-14), 0.93 (3H, d, J ) 6.9 Hz, Me-13),
0.87 (3H, d, J ) 6.9 Hz, Me-12); 13C NMR (CDCl3, 150 MHz) δ 140.8
(C, C-7), 130.6 (C, NCS), 127.7 (C, C-6), 72.7 (C, C-4), 72.6 (CH,
C-5), 62.8 (C, C-10), 42.1 (CH, C-1), 35.2 (CH2, C-9), 32.8 (CH2, C-3),
28.8 (CH, C-11), 27.3 (CH3, Me-14), 24.7 (CH2, C-2), 23.7 (CH3, Me-
15), 21.5 (CH3, Me-12), 20.6 (CH2, C-8), 20.6 (CH3, Me-13); EIMS
m/z 295 (15) [M]+, 280 (11), 277 (12), 262 (11), 252 (72), 236 (83),
234 (27), 219 (25), 218 (51), 179 (79), 175 (100); HRCIMS(+) m/z
295.1604 (calcd for C16H25NO2S, 295.1606).
21.4 (CH3, Me-14), 17.1 (CH3, Me-12), 16.3 (CH3, Me-13); CIMS m/z
295 (1) [M]+, 277 (2), 252 (100), 236 (47), 219 (31), 204 (59), 193
(96), 175 (97); HRCIMS m/z 295.1601 (calcd for C16H25NO2, 295.1606).
Axinisothiocyanate G (7): colorless oil; [R]25D +69.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 240 (3.66) nm; IR (film) νmax 3390, 2101,
1660 cm-1; 1H NMR (C5D5N, 600 MHz) see Table 2; 1H NMR (CDCl3,
600 MHz) δ 5.61 (1H, br s, H-5), 2.14 (1H, m, H-7), 2.11 (2H, m,
H-2, H-9ax), 2.08 (1H, m, H-11), 1.87 (2H, m, H-2, H-3eq), 1.80 (1H,
ddd, J ) 13.8, 3.5, 3.3 Hz, H-9eq), 1.71 (1H, m, H-3ax), 1.69 (1H, m,
H-8eq), 1.45 (1H, dddd, J ) 13.2, 13.2, 13.2, 3.8 Hz, H-8ax), 1.40
(3H, s, Me-15), 1.39 (3H, s, Me-14), 0.99 (3H, d, J ) 6.8 Hz, Me-12),
0.91 (3H, d, J ) 6.8 Hz, Me-13); 13C NMR (C5D5N, 150 MHz) see
Table 2; EIMS m/z 295 (3) [M]+, 280 (4), 252 (19), 236 (89), 234
(34), 219 (15), 201 (15), 193 (56), 183 (56), 175 (48), 165 (100);
HRCIMS(+) m/z 296.1668 [M + H] (calcd for C16H26NO2S, 296.1684).
Axinisothiocyanate H (8): colorless oil; [R]25D +42.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 241 (3.64) nm; IR (film) νmax 3386, 2102
Axinisothiocyanate C (3): white solid; [R]25D +57.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 239 (3.05) nm; IR (film) νmax 3406, 2091
cm-1; 1H NMR (CDCl3, 600 MHz) δ 4.55 (1H, s, H-5), 3.00 (1H, sept,
J ) 6.8 Hz, H-11), 2.66 (1H, br d, J ) 12.7 Hz, H-1), 2.22 (1H, dddd,
J ) 18.1, 10.1, 5.8, 2.0 Hz, H-8ax), 2.08 (1H, dddd, 18.1, 5.5, 4.1, 1.3
Hz, H-8eq), 1.97 (1H, ddd, J ) 13.3, 13.3, 4.4 Hz, H-3ax), 1.80 (1H,
dddd, J ) 13.1, 5.8, 4.1, 1.0 Hz, H-9eq), 1.75 (1H, dddd, J ) 12.4,
4.4, 4.4, 2.9 Hz, H-2eq), 1.64 (1H, dddd, J ) 13.3, 4.0, 2.9, 1.0 Hz,
H-3eq), 1.58 (1H, ddd, J ) 13.1, 10.1, 5.5 Hz, H-9ax), 1.34 (3H, s,
Me-14), 1.12 (3H, s, Me-15), 1.06 (3H, d, J ) 6.8 Hz, Me-12), 1.02
(1H, m, H-2ax), 0.99 (3H, d, J ) 6.8 Hz, Me-13); 13C NMR (CDCl3,
150 MHz) δ 139.3 (C, C-7), 128.5 (C, C-6), 73.0 (CH, C-5), 72.7 (C,
C-4), 62.5 (C, C-10), 42.9 (CH, C-1), 34.6 (CH2, C-3), 32.2 (CH2, C-9),
28.8 (CH, C-11), 25.9 (CH2, C-2), 25.5 (CH3, Me-14), 23.7 (CH3, Me-
15), 21.4 (CH3, Me-12), 21.0 (CH2, C-8), 21.0 (CH3, Me-13);
HRCIMS(+) m/z 295.1592 (calcd for C16H25NO2S, 295.1606).
Axinisothiocyanate D (4): colorless oil; [R]25D +90.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 242 (3.19) nm; IR (film) νmax 3416, 2120
1
cm-1; H NMR (CDCl3, 600 MHz) see Table 2; 13C NMR (CDCl3,
150 MHz) see Table 2; HRCIMS(+) m/z 311.1564 (calcd for
C16H25NO3S, 311.1555).
Axinisothiocyanate I (9): colorless oil; [R]25D +34.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 238 (3.07) nm; IR (film) νmax 3376, 2084
cm-1; 1H NMR (CDCl3, 600 MHz) δ 7.05 (1H, br s, OOH), 5.92 (1H,
br s, H-5), 2.64 (1H, sept, J ) 6.9 Hz), 2.42 (1H, ddd, J ) 9.5, 6.3,
2.1 Hz, H-1), 2.05 (1H, dddd, J ) 13.5, 6.3, 5.0, 3.5 Hz, H-2eq), 1.96
(dddd, J ) 12.9, 5.0, 3.2, 1.2 Hz, H-3eq), 1.85 (2H, m, H-8, H-9),
1.74 (2H, m, H-8, H-9), 1.70 (1H, dddd, J ) 13.5, 12.9, 9.5, 3.2 Hz,
H-2ax), 1.60 (1H, ddd, J ) 12.9, 12.9, 3.5 Hz, H-3ax), 1.44 (3H, s,
Me-15), 1.37 (3H, s, Me-14), 1.00 (3H, d, J ) 6.9 Hz, Me-13), 0.99
(3H, d, J ) 6.9 Hz, Me-12); 13C NMR (CDCl3, 150 MHz) δ 138.2
(CH, C-5), 132.0 (C, C-6), 85.6 (C, C-7), 70.0 (C, C-4), 60.1 (C, C-10),
43.1 (CH, C-1), 36.3 (CH2, C-3), 34.4 (CH2, C-9), 27.9 (CH3, Me-15),
27.7 (CH, C-11), 26.4 (CH3, Me-14), 23.2 (CH2, C-8), 21.9 (CH2, C-2),
18.9 (CH3, Me-12), 16.2 (CH3, Me-13); EIMS m/z 311 (1) [M]+, 295
(3), 253 (5), 183 (14), 85 (100); HRCIMS(+) m/z 278.1559 [M + H
- H2O2]+ (calcd for C16H24NOS, 278.1579).
1
1
cm-1; H NMR (CDCl3, 400 MHz) see Table 2; H NMR (C5D5N,
600 MHz) δ 6.67 (1H, dd, J ) 1.8, 1.2 Hz, H-5), 5.89 (s, OH), 2.16
(1H, ddd, J ) 13.7, 3.5, 3.2 Hz, H-8eq), 2.14 (1H, m, H-1), 2.04 (1H,
dddd, J ) 12.6, 4.9, 3.6, 1.2 Hz, H-3eq), 1.94 (1H, m, H-2eq), 1.94
(1H, ddd, J ) 14.2, 13.7, 3.8 Hz, H-8ax), 1.89 (1H, sept, J ) 6.9 Hz,
H-11), 1.84 (1H, ddd, J ) 12.6, 12.6, 3.6 Hz, H-3ax), 1.74 (1H, ddd,
J ) 14.2, 3.8, 3.2 Hz, H-9eq), 1.72 (1H, m, H-2ax), 1.64 (1H, ddd, J
) 14.2, 14.2, 3.5 Hz, H-9ax), 1.63 (3H, s, Me-15), 1.32 (3H, s, Me-
14), 1.09 (3H, d, J ) 6.9 Hz, Me-13), 0.97 (3H, d, J ) 6.9 Hz, Me-
12); 13C NMR (CDCl3, 125 MHz) see Table 2; EIMS m/z 295 (3) [M]+,
252 (22), 234 (28), 218 (27), 203 (26), 202 (49), 175 (100);
HRCIMS(+) m/z 295.1600 (calcd for C16H25NO2S, 295.1606).
Axinisothiocyanate E (5): colorless oil; [R]25D +30.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 237 (3.50) nm; IR (film) νmax 3407, 2092
Axinisothiocyanate J (10): colorless oil; [R]25 +118.0 (c 0.1,
D
CHCl3); UV (MeOH) λmax (log ꢀ) 241 (3.46) nm; IR (film) νmax 3384,
2090, 1659 cm-1; 1H NMR (CDCl3, 600 MHz) δ 5.57 (1H, d, J ) 1.0
Hz, H-5), 2.15 (1H, sept d, J ) 6.8, 3.3 Hz, H-11), 2.05 (1H, ddd, J
) 13.5, 3.2, 3.2 Hz, H-9eq), 2.00 (1H, m, H-1), 1.97 (1H, m, H-2eq),
1.92 (1H, dddd, J ) 12.4, 5.5, 3.4, 1.0 Hz, H-3eq), 1.73 (1H, m, H-8eq),
1.69 (2H, m, H-2ax, H-7), 1.61 (1H, ddd, J ) 13.5, 13.2, 3.7 Hz,
H-9ax), 1.53 (1H, ddd, J ) 12.4, 12.4, 3.2 Hz, H-3ax), 1.44 (1H, m,
H-8ax), 1.41 (3H, s, Me-15), 1.38 (3H, s, Me-14), 0.98 (3H, d, J )
1
cm-1; H NMR (CDCl3, 400 MHz) δ 6.05 (1H, dd, J ) 1.4, 1.4 Hz,
H-5), 2.11 (1H, m, H-8), 2.08 (1H, m, H-1), 1.94 (3H, m, H2-2, H-9),
1.89 (2H, m, H-3, H-11), 1.68 (2H, m, H-8, H-9), 1.41 (3H, s, Me-
14), 1.39 (1H, m, H-3), 1.32 (3H, s, Me-15), 0.98 (3H, d, J ) 6.8 Hz,
Me-13), 0.71 (3H, d, J ) 6.8 Hz, Me-12); 1H NMR (C5D5N, 600 MHz)
δ 6.60 (1H, dd, J ) 1.6, 1.4 Hz, H-5), 2.23 (1H, dddd, J ) 13.0, 12.6,
9.1, 3.5, H-2ax), 2.16 (1H, ddd, J ) 13.4, 3.3, 3.3 Hz, H-8eq), 2.06
(1H, m, H-3eq), 2.04 (1H, ddd, J ) 9.1, 6.4, 1.6 Hz, H-1), 1.98 (1H,
ddd, J ) 14.0, 13.4, 3.3 Hz, H-8ax), 1.89 (1H, sept, J ) 6.8 Hz, H-11),
1.80 (1H, dddd, J ) 13.0, 6.4, 5.5, 3.5, H-2eq), 1.74 (1H, ddd, J )
14.0, 3.8, 3.3 Hz, H-9eq), 1.66 (1H, ddd, J ) 14.0, 14.0, 3.5 Hz, H-9ax),
1.47 (3H, s, Me-15), 1.46 (1H, ddd, J ) 13.0, 12.6, 3.5 Hz, H-3ax),
1.35 (3H, s, Me-14), 1.11 (3H, d, J ) 6.8 Hz, Me-13), 0.94 (3H, d, J
) 6.8 Hz, Me-12); 13C NMR (C5D5N, 150 MHz) δ 141.1 (C, C-6),
131.5 (CH, C-5), 130.9 (C, NCS), 74.0 (C, C-7), 66.6 (C, C-10), 66.2
(C, C-4), 44.0 (CH, C-1), 36.7 (CH2, C-9), 36.5 (CH2, C-3), 33.2 (CH2,
C-8), 31.5 (CH3, Me-15), 30.4 (CH, C-11), 27.1 (CH3, Me-14), 21.0
(CH2, C-2), 17.0 (CH3, Me-12), 16.3 (CH3, Me-13); EIMS m/z 277 (3)
[M - H2O]+, 234 (49), 175 (100); HRCIMS(+) m/z 295.1597 (calcd
for C16H25NO2S, 295.1606).
1
6.8 Hz, Me-12), 0.92 (3H, d, J ) 6.8 Hz, Me-13); H NMR (C5D5N,
600 MHz) δ 5.92 (s, OH), 5.87 (br s, H-5), 2.03 (1H, m, H-3eq), 2.00
(1H, m, H-11), 1.90 (1H, m, H-1), 1.87 (1H, m, H-2eq), 1.82 (2H, m,
H-3ax, H-9eq), 1.68 (3H, s, Me-15), 1.63 (1H, m, H-2ax), 1.56 (1H,
br d, J ) 11.4 Hz, H-7), 1.50 (1H, m, H-8eq), 1.42 (1H, m, H-9ax),
1.37 (1H, m, H-8ax), 1.18 (3H, s, Me-14), 0.84 (3H, d, J ) 6.9 Hz,
Me-12), 0.83 (3H, d, J ) 6.9 Hz, Me-13); 1H NMR (C6D6, 600 MHz)
δ 5.49 (1H, br s, H-5), 1.98 (1H, sept d, J ) 6.8, 2.5 Hz, H-11), 1.72
(1H, dddd, J ) 12.3, 5.5, 3.0, 1.1 Hz, H-3eq), 1.51 (1H, m, H-9eq),
1.46 (1H, m, H-2), 1.42 (1H, m, H-2), 1.42 (3H, s, Me-15), 1.35 (1H,
m, H-1), 1.38 (1H, m, H-3ax), 1. 32 (3H, m, H-7, H2-8), 0.91 (1H, m,
H-9ax), 0.87 (3H, d, J ) 6.8 Hz, Me-12), 0.86 (3H, d, J ) 6.8 Hz,
Me-13), 0.80 (3H, s, Me-14); 13C NMR (CDCl3, 125 MHz) δ 137.2
(C, C-6), 130.2 (CH, C-5), 69.3 (C, C-4), 66.0 (C, C-10), 47.4 (CH,
C-7), 46.9 (CH, C-1), 40.4 (CH2, C-9), 36.2 (CH2, C-3), 28.2 (CH3,
Me-15), 26.8 (CH, C-11), 26.7 (CH3, Me-14), 22.5 (CH2, C-8), 22.1
(CH3, Me-12), 21.6 (CH2, C-2), 17.5 (CH3, Me-13); EIMS m/z 279 (5)
[M]+, 264 (21), 261 (3), 236 (5), 220 (8), 149 (38), 55 (100);
HRCIMS(+) m/z 279.1658 [M]+ (calcd for C16H25NOS, 279.1657).
Axinisothiocyanate F (6): colorless oil; [R]25D -73.0 (c 0.1, CHCl3);
UV (MeOH) λmax (log ꢀ) 240 (3.05) nm; IR (film) νmax 3390, 2080,
1
1668 cm-1; H NMR (C5D5N, 600 MHz) δ 6.56 (1H, d, J ) 1.8 Hz,
H-5), 5.60 (s, OH), 2.49 (1H, ddd, J ) 7.0, 7.0, 1.8 Hz, H-1), 2.14
(1H, ddd, J ) 13.9, 3.6, 3.6 Hz, H-8eq), 2.09 (1H, dddd, J ) 13.9,
7.0, 7.0, 4.4 Hz, H-2eq), 2.00 (1H, sept, J ) 6.8 Hz, H-11), 1.94 (1H,
ddd, J ) 13.9, 13.3, 3.6 Hz, H-9ax), 1.85 (1H, m, H-3), 1.82 (1H, m,
H-3), 1.76 (1H, ddd, J ) 13.3, 3.6, 3.6 Hz, H-9eq), 1.69 (1H, dddd, J
) 13.9, 9.6, 7.0, 4.4 Hz, H-2ax), 1.56 (1H, ddd, J ) 13.9, 13.9, 3.6
Hz, H-8ax), 1.43 (3H, s, Me-15), 1.25 (3H, s, Me-14), 1.09 (3H, d, J
) 6.8 Hz, Me-13), 0.96 (3H, d, J ) 6.8 Hz, Me-12); 13C NMR (C5D5N,
150 MHz) δ 138.8 (C, C-6), 133.1 (CH, C-5), 74.1 (C, C-7), 67.5 (C,
C-4), 66.0 (C, C-10), 43.9 (CH, C-1), 37.8 (CH2, C-9), 36.9 (CH2, C-3),
33.0 (CH2, C-8), 30.3 (CH, C-11), 29.5 (CH3, Me-15), 21.6 (CH2, C-2),
Axinisothiocyanate K (11): colorless oil; [R]25 +26.3 (c 0.1,
D
CHCl3); UV (MeOH) λmax (log ꢀ) 242 (3.38) nm; IR (film) νmax 2096
1
cm-1; H NMR (CDCl3, 400 MHz) see Table 2; 13C NMR (CDCl3,
125 MHz) see Table 2; EIMS m/z 263 (36) [M]+, 248 (8), 205 (94),
161 (82), 149 (58), 121 (97), 95 (100); HRCIMS (+) m/z 263.1703
(calcd for C16H25NS, 263.1707).
Axinisothiocyanate L (12): colorless oil; [R]25 -14.7 (c 0.1,
D
CHCl3); UV (MeOH) λmax (log ꢀ) 240 (4.05) nm; IR (film) νmax 2088,
1673 cm-1; 1H NMR (CDCl3, 600 MHz) δ 6.96 (1H, dq, J ) 6.4, 1.4
Hz, H-5), 2.62 (1H, ddd, J ) 10.2, 6.4, 4.3 Hz, H-6), 2.41 (1H, dddd,
J ) 15.1, 4.3, 3.9, 1.0 Hz, H-1), 2.40 (1H, dd, J ) 16.4, 3.9 Hz, H-2eq),

614 Journal of Natural Products, 2008, Vol. 71, No. 4
Zubía et al.
2.34 (1H, dd, J ) 16.4, 15.1 Hz, H-2ax), 1.92 (1H, hept d, J ) 7.2,
2.6 Hz, H-11), 1.87 (1H, m, H-9eq), 1.81 (3H, dd, J ) 1.4, 1.0 Hz,
Me-15), 1.59 (2H, m, H-8eq, H-9ax), 1.50 (2H, m, H-7, H-8ax), 1.36
(3H, s, Me-14), 0.96 (3H, d, J ) 7.2 Hz, Me-13), 0.94 (3H, d, J ) 7.2
Hz, Me-12); 13C NMR (CDCl3, 150 MHz) δ 197.6 (C, C-3), 149.3
(CH, C-5), 135.2 (C, C-4), 132.0 (C, NCS), 64.3 (C, C-10), 44.8 (CH,
C-1), 42.7 (CH, C-7), 36.3 (CH, C-6), 35.8 (CH2, C-2), 33.8 (CH2,
C-9), 27.7 (CH, C-11), 26.8 (CH3, Me-14), 21.2 (CH3, Me-12), 20.2
(CH2, C-8), 15.9 (CH3, Me-15), 15.6 (CH3, Me-13); CIMS m/z 277
(7) [M]+, 219 (100); HRCIMS m/z 277.1496 [M]+ (calcd for
C16H23NOS, 277.1500).
PharmaMar. C.J.H.-G. acknowledges financial support from CICIMAR-
COTEPABE and a fellowship from COFAA (Mexico).
1
Supporting Information Available: H and 13C NMR spectra of
compounds 1–12 and a photograph of the sponge Axinyssa sp. This
information is available free of charge via the Internet at http://
pubs.acs.org.
References and Notes
(1) Chang, C. W. J.; Scheuer, P. J. In Topics in Current Chemistry;
Scheuer, P. J., Ed.; Springer-Verlag: Berlin, 1993; pp 33–75..
(2) Chang, C. W. J. In Progress in the Chemistry of Organic Natural
Products; Herz, W., Falk, H., Kirby, G. W., Moore, R. E., Eds.;
Springer: New York, 2000; pp 1–186..
(3) Garson, M. J.; Simpson, J. S. Nat. Prod. Rep. 2004, 21, 164–179.
(4) (a) Yasman; Edrada, R. A.; Wray, V.; Proksch, P. J. Nat Prod. 2003,
66, 1512–1514. (b) Manzo, E.; Ciavatta, M. L.; Gavagnin, M.; Mollo,
E.; Guo, Y.-W.; Cimino, G. J. Nat. Prod. 2004, 67, 1701–1704. (c)
Mitome, H.; Shirato, N.; Miyaoka, H.; Yamada, Y.; van Soest,
R. W. M. J. Nat. Prod. 2004, 67, 833–837.
(5) Alvi, K. A.; Tenenbaum, L.; Crews, P. J. Nat. Prod. 1991, 54, 71–
78.
(6) Gulavita, N. K.; de Silva, E. D.; Hagadone, M. R.; Karuso, P.; Scheuer,
P. J.; Van Duyne, G. D.; Clardy, J. J. Org. Chem. 1986, 51, 5136–
5139.
(7) Petrichtcheva, N. V.; Duque, C.; Dueñas, A.; Zea, S.; Hara, N.;
Fujimoto, Y. J. Nat. Prod. 2002, 65, 851–855.
(8) Pham, A. T.; Ichiba, T.; Yoshida, W. Y.; Scheuer, P. J.; Uchida, T.;
Tanaka, J.; Higa, T. Tetrahedron Lett. 1991, 32, 4843–4846.
(9) Fusetani, N. Curr. Org. Chem. 1997, 1, 127–152.
(10) Hirota, H.; Okino, T.; Yoshimura, E.; Fusetani, N. Tetrahedron 1998,
54, 13971–13980.
(11) (a) König, G. M.; Wright, A. D.; Sticher, O.; Angerhofer, C. K.;
Pezzuto, J. M. Planta Med. 1994, 60, 532–537. (b) Wright, A. D.;
Wang, H.; Gurrath, M.; König, G. M.; Kocak, G.; Neumann, G.; Loria,
P.; Foley, M.; Tilley, L. J. Med. Chem. 2001, 44, 873–885.
(12) Li, C.-J.; Schmitz, F. J.; Kelly, M. J. Nat. Prod. 1999, 62, 1330–
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(13) Fusetani, N.; Wolstenholme, H. J.; Shinoda, K.; Asai, N.; Matsunaga,
S.; Onuki, H.; Hirota, H. Tetrahedron Lett. 1992, 33, 6823–6826.
(14) Simpson, J. S.; Garson, M. J.; Hooper, J. N. A.; Cline, E. I.;
Angerhofer, C. K. Aust. J. Chem. 1997, 50, 1123–1127.
(15) (a) Harrigan, G. G.; Ahmad, A.; Baj, N.; Glass, T. E.; Gunatilaka,
A. A. L.; Kingston, D. G. I. J. Nat. Prod. 1993, 56, 921–925. (b)
Piers, E.; Britton, R. W.; de Waal, W. Can. J. Chem. 1969, 47, 831–
840.
Synthesis of the (R)-MPA Ester 1r. A solution of 1 (1.5 mg, 5.1
× 10-3 mmol) in 0.3 mL of CH2Cl2 was treated with CH2Cl2 solutions
of N,N′-dicyclohexylcarbodiimide (5.0 mg, 0.024 mmol in 0.4 mL),
N,N-dimethylaminopyridine (1.0 mg, 8.2 × 10-3 mmol in 0.3 mL),
and (R)-R-methoxy-R-phenylacetic acid (4.0 mg, 0.024 mmol in 0.4
mL) and stirred at room temperature for 24 h. The reaction mixture
was purified using preparative TLC (hexane/EtOAc, 7:3) to obtain 1.3
1
mg of the (R)-MPA ester 1r: H NMR (CDCl3, 600 MHz; selected
data, assignments aided by COSY and NOESY experiments) δ 5.58
(1H, s, H-5), 3.18 (1H, sept, J ) 6.9 Hz, H-11), 2.08 (1H, dddd, J )
18.1, 7.9, 5.3, 2.0 Hz, H-8ax), 1.89 (1H, dddd, J ) 18.1, 6.6, 5.2, 1.4
Hz, H-8eq), 1.74 (3H, m, H-2eq, H-3ax, H-9eq), 1.64 (m, H-3eq), 1.50
(1H, m, H-1) 1.38 (1H, m, H-2ax), 1.20 (3H, s, Me-15), 1.19 (1H, m,
H-9ax), 1.00 (3H, s, Me-14), 0.97 (3H, d, J ) 6.9 Hz, Me-12), 0.91
(3H, d, J ) 6.9 Hz, Me-13).
Synthesis of the (S)-MPA Ester 1s. A solution of 1 (1.5 mg, 5.1 ×
10-3 mmol) in 0.3 mL of CH2Cl2 was treated with CH2Cl2 solutions of
N,N′-dicyclohexylcarbodiimide (5.0 mg, 0.024 mmol in 0.4 mL), N,N-
dimethylaminopyridine (1.0 mg, 8.2 × 10-3 mmol in 0.3 mL), and
(S)-R-methoxy-R-phenylacetic acid (4.0 mg, 0.024 mmol in 0.4 mL)
and stirred at room temperature for 24 h. The reaction mixture was
purified over preparative TLC (hexane/EtOAc, 7:3) to obtain 1.4 mg
1
of the (S)-MPA ester 1s: H NMR (CDCl3, 600 MHz; selected data,
assignments aided by COSY and NOESY experiments) δ 5.59 (1H, s,
H-5), 3.20 (1H, sept, J ) 6.8 Hz, H-11), 2.18 (1H, dddd, J ) 18.1,
7.7, 5.5, 2.1 Hz, H-8ax), 2.05 (1H, m, H-1), 2.01 (1H, dddd, J ) 18.1,
6.6, 5.3, 1.4 Hz, H-8eq), 1.89 (1H, ddd, J ) 12.9, 6.6, 5.5 Hz, H-9eq),
1.85 (1H, dddd, J ) 13.1, 4.4, 4.2, 2.6 Hz, H-2eq), 1.67 (1H, ddd, J )
13.9, 13.9, 4.4 Hz, H-3ax), 1.53 (2H, m, H-3eq, H-9ax), 1.46 (1H, m,
H-2ax), 1.30 (3H, s, Me-14), 1.00 (3H, d, J ) 6.8 Hz, Me-12), 0.94
(3H, d, J ) 6.8 Hz, Me-13), 0.83 (3H, s, Me-15).
Cytotoxicity Assays. Compounds 1-8 and 10-13 were tested
against the human tumor cell lines MDA-MB-231 (breast adenocar-
cinoma), A-549 (lung adenocarcinoma), and HT-29 (colon adenocar-
cinoma). A colorimetric assay using sulforhodamine B (SRB) was
adapted for quantitative measurement of cell growth and viability as
described in the literature.20
(16) Demarco, P. V.; Farkas, E.; Doddrell, D.; Mylari, B. L.; Wenkert, E.
J. Am. Chem. Soc. 1968, 90, 5480–5486.
(17) For recent examples of pyridine-induced shifts see: (a) Su, B.-N.;
Misico, R.; Park, E. J.; Santarsiero, B. D.; Mesecar, A. D.; Fong,
H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. Tetrahedron 2002, 58, 3453–
3466. (b) Gu, J.-Q.; Li, W.; Kang, Y.-H.; Su, B.-N.; Fong, H. H. S.;
van Breemen, R. B.; Pezzutto, J. M.; Kinghorn, A. D. Chem. Pharm.
Bull. 2003, 51, 530–539.
(18) (a) Seco, J. M.; Quiñoá, E.; Riguera, R. Chem. ReV. 2004, 104, 17–
117. (b) Seco, J. M.; Quiñoá, E.; Riguera, R. Tetrahedron Asymmetry
2001, 12, 2915–2925.
(19) Clark, R. J.; Stapleton, B. L.; Garson, M. J. Tetrahedron 2000, 56,
3071–3076.
(20) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
J. Natl. Cancer Inst. 1990, 82, 1107–1112.
Acknowledgment. This research was supported by grants from
Ministerio de Educación y Ciencia (Spain)-FEDER (research project
CTQ2004-02361) and from Junta de Andalucía (FQM-285). Cytotox-
icity assays were performed through a cooperation agreement with
NP070593S