Novel Cassane and Cleistanthane Diterpenes from Myrospermum frutescens: 
Absolute Stereochemistry of the Cassane Diterpene Series 
Daniel Torres-Mendoza,* Luis David Urena Gonzalez,* Eduardo Ortega-Barria,* Phyllis D. Coley,1^ 
Thomas A. Kursar,^ Todd L. Capson,? Kerry McPhail," and Luis Cubilla-Rios*>t 
Laboratory of Tropical Bioorganic Chemistry, Faculty of Natural, Exact Sciences and Technology, Apartado 0824-10835, 
University of Panama, Panama City, Republic of Panama, Institute for Advance Scientific Investigation and Technology 
Services, National Secretariat of Science and Technology, Clayton, Ancon, Republic of Panama, Department of Biology, 
University of Utah, Salt Lake City, Utah, Smithsonian Tropical Research Institute, Balboa, Ancon Republic of Panama, 
and College of Pharmacy, Oregon State University, Corvallis, Oregon 97331 
Received March 22, 2004 
Four new diterpenes (1?4) were isolated from the leaves of Myrospermum frutescens as minor constituents. 
Chagresnol (1), 6/3,18-diacetoxycassan-13,15-diene (2), and chagreslactone (3) possess cassane skeletons, 
while chagresnone (4) exhibits a cleistanthane skeleton. Molecular structures and their relative 
stereochemistries were elucidated using NMR spectroscopy in combination with UV, IR, and MS spectral 
data. Although compound 2 was previously reported as a synthetic product, we report its first isolation 
as a natural product. Derivative products (10?13) were obtained to test their activities against Chagas's 
disease. In addition, the absolute stereochemistry of the previously isolated cassane diterpene 5 from M. 
frutescens is presented. 
As part of the Panama-ICBG Project, which uses ecologi- 
cal criteria for drug discovery,1 we previously described five 
novel cassane diterpenes isolated from Myrospermum 
frutescens (Fabaceae).2 These have activity against Trypa- 
nosoma cruzi, the parasite responsible for Chagas's disease. 
Continuing with our study of M. frutescens, here we report 
the isolation and structural elucidation of four additional 
new diterpenes, chagresnol (1), 6/3,18-diacetoxycassa-13,- 
15-diene (2), chagreslactone (3), and chagresnone (4), from 
the EtOAc?MeOH extract of mature leaves. Although we 
previously obtained diacetate 2 as a synthetic product,2 we 
report its first isolation as a natural product here. Cassane 
diterpenes 1?3 and cleistanthane 4 derive from the pima- 
rane diterpene skeleton by migration of a methyl group 
(cassane) or an ethyl group (cleistanthane) from C-13 to 
C-14.3 These compounds were assayed for activity against 
extra- and intracellular forms of T. cruzi together with the 
five previously isolated cassanes (5?9) and deacetylated 
or oxidized derivatives (10?14) of compounds 2, 4, 5, and 
6. A comparison of structure and activity is presented here. 
Determination of the absolute stereochemistry of 5 using 
the Mosher ester method is also presented. 
Results and Discussion 
Liquid?liquid partitioning followed by column chroma- 
tography of the MeOH?EtOAc crude extract of M. frute- 
scens leaves produced a fraction active against T. cruzi, 
which yielded compounds 1 and 3 after successive reversed- 
phase HPLC and preparative TLC. Chagresnol (1) showed 
an HRCIMS ion of mlz 362.2424, consistent with the 
formula C22H34O4 and 6 degrees of unsaturation. Inspection 
of the m NMR spectrum for 1 revealed signals closely 
similar to those for 6/3-hydroxy-18-acetoxycassan-13,15- 
diene (6) isolated previously.2 Noticeable differences in the 
* To whom correspondence should be addressed. Tel:   (507) 681 5371. 
Fax:  (507) 264 4450. E-mail: lucr@ancon.up.ac.pa. 
* University of Panama. 
* Institute for Advance Scientific Investigation and Technology Services. 
1
 University of Utah. 
5
 Smithsonian Tropical Research Institute. 
1
 Oregon State University. 
r?H N17 
R4 
18.** 
R1 
" \ H T 
F?2  R3 
R1 R2 R3 R4 
1 OAc CH3 OH OH 
2 OAc CH3 OAc H 
5 OH CH3 OH H 
6 OAc CH3 OH H 
7 OH CH3 H H 
8 OAc COOH H H 
10 OH CH3 OAc H 
12 OAc CH3 =0 H 
14 OH CH3 =0 H 
I      ?' OAc K2 OAc 
R1 R? 
3 4 OAc OH 
11 OH OH 
13 
spectrum for 1 included the absence of a vinyl methyl 
singlet (<5 1.73, Hg-17 for 6), the appearance of a deshielded 
10.1021/np049890c CCC: $27.50 ? xxxx American Chemical Society and American Society of Pharmacognosy 
Published on Web 00/00/0000 PAGE EST: 4.6 
B    Journal of Natural Products Torres-Mendoza et al. 
methylene multiplet (8 4.26), and relatively deshielded 
terminal double bond signals at <5 6.85 (H-15, dd; <5 6.77 
for 6), 5.08 (H-16a, d; 6 4.94 for 6), and 5.24 (H-16b, d; 6 
5.10 for 6). Similarly, inspection of the 13C NMR spectrum 
for 1 showed an additional methylene resonance at <5 59.3 
and the absence of a third upfield methyl signal (8 16.3, 
C-17 for 6). These data suggested that 1 possessed a 
hydroxylated CH3-17, which was consistent with HMBC 
data and with a molecular mass increase of 16 for 1 
compared to 6. In a 2D NOESY experiment, reciprocal 
correlations between H-15 and both H-16a and H^-l7 (<5 
4.26) were observed, as well as a correlation of H-16b (8 
5.24) with H-12b (<5 2.43). These correlations defined a 
iraws-conjugated diene in 1, as in the previously isolated 
cassanes,2 which were unfortunately misrepresented as cis- 
conjugated dienes. Hydroxymethylene H-18a (<5 3.72) and 
H-18b (<5 4.03) correlated with H3-19 (<5 1.25) and a-oriented 
H-5 (6 1.23). Methine H-6 (<5 4.31) was also correlated to 
H-5 (8 1.23), signifying a /3-OH at C-6. Hence chagresnol 
(1) was identified as 6/?,17-dihydroxy-18-acetoxycassan-13,- 
15-diene. 
6/3,18-Diacetoxycassan-13,15-diene (2) was isolated after 
successive column chromatography, reversed-phase HPLC, 
and further column chromatography of the hexanes parti- 
tion fraction obtained from liquid?liquid partitioning 
(hexanes?10% aqueous MeOH) of the crude leaf extract. 
The identity of this compound was deduced by comparison 
of its XH NMR and optical rotation data with that of 
chagresnol (1) and of the known synthetic compound.2 
HREIMS data (jnlz 464.2402) for chagreslactone (3) were 
consistent with a molecular formula of CggHggOg, implying 
8 degrees of unsaturation. The XH NMR spectrum for 3 
showed signals characteristic of a cassane diterpene with 
an acetylated geminal methyl group at C-4, similar to 
compounds 1 and 2. A methoxy methyl singlet (8 3.20) and 
a second acetate methyl singlet (<5 2.11) were also evident. 
This second acetate group could be positioned at C-6 on 
the basis of an HMBC correlation from H-6 (<5 5.58) to the 
acetate carbonyl carbon (8 172.6). In addition, an ABC spin 
system in 3 was delineated by a double doublet at 8 3.65 
(H-7, J = 2, 11 Hz) COSY-coupled to the H-6 doublet (J = 
2 Hz) and a multiplet at 8 1.80 (H-8). This was consistent 
with placement of a hydroxyl substituent at C-7 (<5c 72.3). 
Two olefinic (8 116.3 and 170.8) and three carbonyl (8 
170.1, 171.0, and 172.6) carbon signals in the 13C NMR 
spectrum for 3 accounted for 4 of the 8 degrees of unsat- 
uration implied by the molecular formula, thus suggesting 
a tetracyclic metabolite. Quatenary 13C shifts at <5c 170.8, 
170.1, 116.3, and 107.8 were consistent with a fused a,/3- 
butenolide moiety, and both 13C and XH shifts for ring C 
and D atoms were closely similar to those reported for 12,- 
16-epoxy-5a-hydroxy-12a-methoxycassa-13-(15)-en-16- 
one.4 The proposed structure was confirmed by HMBC 
correlations from olefinic H-15 (<5 5.86) to C-13 (<5 170.8), 
C-14 (<5 32.2), C-16 (6 170.1), and C-12 (<5 107.8), in 
combination with correlations from <5 1.80 (H-8) to C-7, C-9, 
C-14, and C-17, and a three-bond HMBC correlation from 
the methoxy methyl singlet (8 3.20) to hemiacetal C-12. 
The relative stereochemistry of 3 was deduced from 2D 
NOESY and ID NOE difference experiments. In ID NOE 
difference experiments, irradiation of H-6 (8 5.58) produced 
enhancements in H-5 (<5 1.38) and H2-18 (6 3.74 and 3.93) 
signals, while irradiation of H-7 (8 3.65) enhanced H-5 and 
H-9 (<5 1.53) resonances. These data were confirmed by 20 
NOESY data and established ^-orientations for both the 
acetate at C-6 and the lactone ring, with an a-acetoxym- 
ethylene    (C-18).     The    methoxy    and    CH3-17    me- 
thyls were assigned a-orientations on the basis of NOESY 
correlations between these two singlets and also between 
H-14 (<5 3.38) and H-8 (<5 1.80). Therefore, chagreslactone 
(3) was characterized as 6/3,18-diacetoxy-(13),15-ene-7/3- 
hydroxy-12-methoxycassan-12,16-olide. This structure re- 
sembles the neocaesalpin5 and dypteryxic acid6 type com- 
pounds. 
Chagresnone (4) was assigned a molecular formula of 
C22H34O4 on the basis of an HREIMS ion at m/z 362.2462 
and 22 resonances in the 13C NMR spectrum. Two IR 
absorptions at 1735 and 1682 cm-1 were consistent with 
13C resonances for acetyl (<5 170.9) and cycloketone (8 210.7) 
moieties, respectively, which accounted for 2 of the 6 
degrees of unsaturation implicit in the molecular formula. 
Thus compound 4 was assigned a tetracyclic carbon 
skeleton. The 1H NMR spectrum for 4 was similar to those 
for compounds 1 and 3. A comparatively shielded pair of 
H2-I8 doublets [<5 3.42 (J = 11 Hz) and 3.73 (1H, d, J = 11 
Hz)] could be attributed to the presence of a hydroxy- 
methylene (IR 3462 cm1) rather than the acetoxymethyl- 
ene found at C-4 in 1 and 3. Two comparatively shielded 
methyl singlets in the spectrum for 4 (8 0.76, CH3-20 and 
0.96, CH3-I9) were consistent with the absence of the 
oxygenated substituent at C-6 in 1 and 3. An acetate 
methyl singlet at <5 2.05 and a pair of double doublets at <5 
3.82 (1H, J = 6, 12 Hz) and 4.36 (1H, J = 8, 12 Hz) were 
assigned to an acetoxymethylene moiety at C-15 on the 
basis of HMBC data. The presence of a cyclopropane ring 
was indicated by HMBC correlations from H-15 to C-8, 
C-12, C-14, and C-17 and also from H2-16 to C-13, C-14, 
and C-15. These correlations, together with a correlation 
from H3-I7 to carbonyl C-12, established the cleistanthane 
skeleton of 4 and were also consistent with the results of 
^H decoupling experiments. The NOESY spectrum for 4 
showed correlations from both H2-I6 protons to H-14 (<5 
1.02), H-15 (8 1.56), and H3-17. A correlation was also 
apparent from H3-17 to H-14, but not to H-15. These data 
supported a cis cyclopropyl ring with an /3-acetoxymethyl- 
ene substituent. Remarkably, H2-18 showed a NOESY 
correlation to H3-20 as well as to H3-19, which in turn 
showed an intense correlation to H-6b (<5 1.80) and a 
weaker one to H-6a (<5 1.35). These data suggest that the 
relative configuration at C-4 is the opposite of that found 
in cassanes 1?3. Indeed, an axial hydroxymethylene is 
consistent with the relatively shielded chemical shift of 
C-18 (6c 65.1 compared to 8 73.0 and 72.3 for 1 and 3, 
respectively). Thus, chagresnone (4) was assigned as 18- 
hydroxy-16-acetoxy-12-oxocleistanthane. 
We previously isolated and reported cassane diterpenes 
from M. frutescens, including 5?9, which are active against 
T. cruzi. We have obtained the absolute configuration at 
C-6 in 5 using a modified Mosher ester method.78 Both the 
(R)- and (S)-methoxyphenyl acetic acid (MPA) esters of 
cassane 5 were prepared and purified by simple column 
chromatography. All of the protons of these derivatives 
were assigned from 2D NMR data (HSQC, HMBC). The 
A<5 (<5R ? (5s) values obtained (Figure 1) were consistent 
with an S configuration at C-6. The other chiral centers in 
the molecules were determined from their relative configu- 
rations. 
To investigate structure?activity relationships, we pre- 
pared derivatives of 6/3,18-diacetoxycassa-13,15-diene (2), 
chagresnone (4), and cassane 6. Alkaline hydrolysis of 2 
yielded monoacetate 10, as evidenced by the absence of a 
second acetate methyl singlet and the comparatively 
shielded pair of H2-18 doublets (6 3.53, J = 11 Hz; 3.14, J 
= 11 Hz) in the ^H NMR spectrum for 10, which was 
Cassane Diterpenes from Myrospermum frutescens Journal of Natural Products    C 
+0.49 
-0.21 
Table 1. 
MHz) 
XH NMR Data for Compounds 1, 3, and 4 (CDC13, 300 
<3H ppm (mult., J/Hz) 
09 
+0.26 
+0.04 
+0.06 
1.4 
O-MPA 
O-MPA 
Figure 1.  Selected A<5fls values for MPA derivatives of compound 5. 
otherwise very similar to that for 2. Alkaline hydrolysis of 
chagresnone 4 yielded diol 11 (IR 3674, 3403, and 1674 
cm1). Comparison of the ^H NMR spectra for 11 and 4 
revealed the absence of an acetate methyl singlet and a 
comparatively shielded pair of Hg-16 double doublets (<5 
3.79, J = 6, 11 Hz; 3.55 J = 8, 11 Hz) in the spectrum for 
11, consistent with the loss of the acetyl group at C-16. 
Oxidation of compound 6 with Jones reagent yielded a 
mixture of products from which ketone 12 was isolated. 
Two carbonyl IR absorptions (1725 and 1710 cm x) for 12 
were consistent with resonances for acetyl (<5 171.0) and 
cyclohexanone (<5 210.8) carbonyl carbons in the 13C NMR 
spectrum for 12 and also the absence of an H-6 multiplet 
(6 4.27) in its % NMR spectrum. Aldehyde 13 was isolated 
from the same mixture. The XH and 13C NMR spectra for 
13 also showed the presence of a cyclohexanone moiety. 
However, no olefinic proton signals were evident in the XH 
NMR spectrum for 13. Rather, a sharp singlet at <5 10.15 
and a comparatively deshielded double-bond methyl singlet 
(6 2.11) indicated the presence of an aldehyde functionality, 
which was supported by 13C NMR data. 
The antitrypanosomal activities of diterpenes 1?4 and 
derivatives 10?13 are presented in Table 3. Compounds 
6, 9, and 12 were more active against the extracellular or 
infectious form of the parasite (IC50 11, 16, and 17 fiM., 
respectively), while compounds 5 and 7 were more active 
against the intracellular form of the parasite (IC50 16 and 
17 ^M, respectively). For the extracellular form, the acetyl 
group at C-18 appeared to be responsible for the greater 
activity in 6, 9, and 12 relative to compounds with a C-18 
hydroxyl group such as 5 (IC50 56 fiM), 7 (IC50 48 fiM), and 
149 (IC50 36 /iM). The presence of the acetate group at C-6 
in 2 (IC50 59 ftM) and 10 (56 ^M) may decrease activity 
due to steric effects. Modifications of the conjugated diene 
system slightly decreased the activity of 1 and 13 with 
respect to 6 (IC50 11 /(M) and 12. On the other hand, in 
the intracellular bioassay, the hydroxyl group on C-18 
produced an increase in activity, although modifications 
at C-6 decreased the activity (10, 12, 13) relative to the 
active compounds 5 and 7. Modifications in the diene 
system appeared to decrease the cytotoxicity of these 
compounds to Vero cells. For example, conjugated diene 1 
(IC50 156 fiM) is more toxic than fully saturated compound 
4 (IC50 448 fiM). 
Experimental Section 
General Experimental Procedures. Optical rotations 
were measured on a JASCO DIP-370 digital polarimeter. IR 
spectra were recorded on a Shimadzu FTIR-8300 spectropho- 
tometer or a Perkin-Elmer FTIR Spectrum-1000. The NMR 
spectra were recorded on a Bruker Avance 300 spectrometer 
with TMS as an internal standard. Irradiation experiments 
were recorded on a Bruker AMX-500 instrument. HREIMS 
data were recorded on a UG-Autospec instrument, and the 
HRCIMS were recorded on a Kratos MS50TC instrument. 
HPLC was carried out on a Waters LC system, including a 
atom no. chagresnol (1) chagreslactone (3) chagresnone (4) 
1 0.90 m 0.99 m 0.89 m 
1.72 m 1.78 m 1.61m 
2 1.52 m 1.57 m 1.48 m 
1.70 m 
3 1.73 m 1.37 m 0.92 m 
1.30 m 
5 1.23 m 1.38 m 1.05 m 
6 4.31m 5.58 d (2) 1.35 m 
1.80 m 
7 1.29 m 3.65 dd (2, 11) 2.05 m 
2.24 dt (3, 13) 0.84 m 
8 2.67 m 1.80 m 1.67 m 
9 0.98 m 1.53 m 1.14 m 
11a 1.81m 1.36 d (3) 1.84 m 
lib 2.47 dd (3, 13) 2.20 dd (1.7, 4.1) 
12a 2.18 m 
12b 2.43 m 
13 
14 3.38 m 1.02 m 
15 6.85 dd (11, 17) 5.86 s 1.56 m 
16a 5.08 d (11) 3.82 dd (6, 12) 
16b 5.24 d (17) 4.36 dd (8, 12) 
17 4.26 m 1.21 d (7.3) 1.22 s 
18a 3.72 d (11) 3.74 d (11) 3.42 d (11) 
18b 4.03 d (11) 3.93 d (11) 3.73 d (11) 
19 1.25 s 1.12 s 0.96 s 
20 1.22 s 1.03 s 0.76 s 
CH3O-I2 3.20 s 
CH3CO-6 2.11s 
CH3CO-I6 2.05 s 
CH3CO-I8 2.06 s 2.09 s 
Table 2. 13C NMR Data for Compounds 1, 3, and 4 (<5C ppm, 
CDCI3, 75 MHz) 
atom no.      chagresnol (1)   chagreslactone (3)   chagresnone (4) 
1 40.6 (CH2) 41.4 (CH2) 38.0 (CH2) 
2 18.4 (CH2) 18.0 (CH2) 18.2 (CH2) 
3 38.0 (CH2) 37.5 (CH2) 35.5 (CH2) 
4 37.4(C) 37.4(C) 38.4 (C) 
5 51.4 (CH) 48.0 (CH) 55.1 (CH) 
6 67.9 (CH) 72.8 (CH) 22.1 (CH2) 
7 41.1 (CH2) 72.3 (CH) 35.5 (CH2) 
8 33.5 (CH) 42.2 (CH) 36.7 (CH) 
9 54.5 (CH) 43.6 (CH) 56.9 (CH) 
10 37.4 (C) 37.6 (C) 37.2 (C) 
11 21.3 (CH2) 37.2 (CH2) 36.4 (CH2) 
12 27.0 (CH2) 107.8 (C) 210.7(C) 
13 134.3 (C) 170.8 (C) 33.4 (C) 
14 138.7 (C) 32.2 (CH) 38.5 (CH) 
15 134.4 (CH) 116.3 (CH) 33.2 (CH) 
16 113.6 (CH2) 170.1 (C) 63.8 (CH2) 
17 59.3 (CH2) 11.3 (CH3) 14.3 (CH3) 
18 73.0 (CH2) 72.3 (CH2) 65.1 (CH2) 
19 19.9 (CH3) 19.6 (CH3) 26.9 (CH3) 
20 17.3 (CH3) 17.9 (CH3) 14.7 (CH3) 
CH3O-I2 51.0 (CH3) 
CH3CO-6 172.6 (C) 
CH3CO-6 21.1 (CH3) 
CH3CO-I6 170.9 (C) 
CH3CO-I6 20.9 (CH3) 
CH3CO-I8 171.3 (C) 171.0 (C) 
CH3CO-I8 21.0 (CH3) 21.7 (CH3) 
600 pump and a 996 photodiode array detector. Melting points 
were determined using an Electrothermal 9100 apparatus and 
are uncorrected. 
Plant Material. Mature leaves of M. frutescens were 
collected and stored as described previously.2 
Extraction and Isolation. Fractionation on silica gel 60 
(37?75 fim, Geduran) of the MeOH partition derived from 
liquid?liquid partitioning (hexanes?10% aqueous MeOH) of 
D    Journal of Natural Products Torres-Mendoza et al. 
Table 3.  Compound Activities against Trypanosoma cruzia 
compound 
extracellular 
IC50 C"M) 
intracellular 
ic50 dm 
cytotoxicity6 
IC50 (fiM) 
1 38.8 ? 5.99 76.0 ? 3.84 156 ? 4.64 
3 75.0 ? 13.0 NDC 225 ? 14.1 
4 56.9 ? 1.02 95.5 ? 3.25 448 ? 15.6 
10 56.7 ? 5.76 76.5 ? 2.57 178 ? 4.90 
11 ND ND 433 ? 34.1 
12 17.7 ? 12.7 79.1 ? 15.9 238 ? 13.4 
13 40.4 ? 7.99 61.4 ?0.12 416 ? 89.2 
amphotericin B 1.0 ND ND 
nifurtimox ND 11.0 ND 
" Results show the IC50 value ? the SD (re = 3). b Experiments 
performed with Vero cells. c ND = not determined. 
the MeOH?EtOAc extract of leaves of M. frutescens yielded 
four main fractions (1?4). Fraction 2 was again chromato- 
graphed on silica gel and eluted with a stepped gradient of 
hexanes?EtOAc to yield six fractions (2a?2f) as was previ- 
ously described.2 
Fraction 2d (516 mg) was subjected to silica gel (37?75 /an) 
column chromatography and eluted with 1.6 L of CHCI3, 1.0 
L of 98:2 CHCl3-acetone, 200 mL of 97:3 CHCl3-acetone, 100 
mL of 95:5 acetone-MeOH, 200 mL of 50:50 acetone-MeOH, 
and 100 mL of MeOH to yield compound 4 (83 mg). 
Part of fraction 2 (207 mg) was fractionated by semi- 
preparative reversed-phase HPLC (YMC-Pack ODS-AQ, 85 
fim, 12 nm, 150 x 10 mm) using isocratic elution (flow 1.5 mL/ 
min 85:15 MeOH?H2O). Four fractions were obtained (frac- 
tions 2g?2j). Fraction 2g (121 mg) was subjected to prepara- 
tive TLC (Whatman PK5F silica gel 150 A plates) using 75:25 
CH2Cl2-EtOAc to yield compound 3 (12 mg). Fraction 2h (60 
mg) was subjected to preparative TLC (Whatman PK5F silica 
gel 150 A plates) and eluted with 60:40 hexanes-EtOAc, 
yielding compound 1 (12 mg). 
The hexanes partition fraction (11.3 g), from liquid?liquid 
partitioning (hexanes?10% aqueous MeOH) of the crude 
extract, was chromatographed on silica gel (230?400 mesh) 
eluted sequentially with 75:25 hexanes?EtOAc, 60:40 
hexanes-EtOAc, 1:1 hexanes-EtOAc, 40:60 hexanes-EtOAc, 
EtOAc, and MeOH. The fractions were combined according to 
their TLC profiles into nine fractions (1?9). Fraction 2 (2.54 
g) was fractionated by isocratic preparative reversed-phase 
HPLC (Prep Nova Pack HR Ci8, 6 /<m, 60 A, 25 x 100 mm; 
4.5 mL/min 90:10 MeOH-H20). Four fractions were obtained 
(2a?2d). Fraction 2c yielded 905 mg of compound 6, which was 
used to prepare derivatives 12 and 13. Fraction 2d was 
subjected to silica gel chromatography (7GF) and eluted with 
150 mL of 1:1 hexane?CH2CI2 to yield compounds 7 (38.4 mg) 
and 2 (24.6 mg). 
Chagresnol (1): white solid, [a]24D -63.8 (c 0.072, CHC13); 
IR (KBr) iw 3445, 2925, 1716, 1384, 1250, 1034 cm"1; *H 
NMR data, see Table 1; 13C NMR data, see Table 2; LRCIMS 
(CH4) m/z (%) 363 [M + 1]+ (6), 362 [M+] (11), 347 (25), 321 
(74), 303 (100), 285 (43), 267 (28), 243 (9), 200 (6), 175 (13), 
160 (14), 122 (24), 108 (17), 94 (15); HRCIMS (CH4) m/z 
362.2424 [M+] (calcd for C22H34O4, 362.2457). 
Chagreslactone (3): [a]24D -57.1 (c 0.14, CHC13); IR (KBr) 
iW 3446, 2932, 1738, 1654, 1368, 1249, 1058 cm"1; :H NMR 
data, see Table 1; 13C NMR data, see Table 2; LREIMS m/z 
(%) 464 [M+] (7), 414 (39), 344 (90), 316 (100), 312 (52), 301- 
(46), 285 (31), 271 (25), 257 (19), 203 (19), 189 (12), 161 (5), 
121 (6); HREIMS m/z 464.2402 [M+] (calcd for CgsHgeOg, 
464.2410). 
Chagresnone (4): amorphous powder, mp 138?140?C; 
[a]24D +47.8 (c 0.23, CHC13); IR (CHC13) vmax 3462, 2928, 2849, 
1736, 1682, 1367, 1029 cm"1; JH NMR data, see Table 1; 13C 
NMR data, see Table 2; LREIMS m/z (%) 362 [M+] (5), 302 
(100), 271 (18), 229 (3), 192 (4), 149 (9), 121 (6), 108 (12), 81 
(2); HREIMS m/z 362.2462 [M+] (calcd for C22H34O4, 362.2457). 
Hydrolysis of 2. Compound 2 (50 mg) was hydrolyzed with 
5% KOH in MeOH. After 16 h the mixture was neutralized 
with 2 N HC1 and extracted with CHCI3 and then was washed 
with water and dried over Na2S04. Compound 10 (7 mg) was 
isolated from the mixture by silica gel column chromatography, 
eluting with 60:40 hexanes-EtOAc. [a]24D -56.8 (c 0.22, 
CHCI3); IR (KBr) vmax 3446, 2994, 1716, 1374,1257, 1029 cm"1; 
JH NMR data (CDC13, 300 MHz) 6.79 (1H, dd, J = 11, 17 Hz, 
H-15), 5.46 (1H, d, J = 2.6 Hz, H-6), 5.12 (1H, d, J = 17 Hz, 
H-16a), 4.98 (1H, d, J = 11 Hz, H-16b), 3.53 (1H, d, J = 11 
Hz, H-18a), 3.14 (1H, d, J = 11 Hz, H-18b), 2.07 (3H, s, 
CH3COO), 1.69 (3H, s, H3-17), 1.47 (1H, m, H-5), 1.17 (3H, s, 
H3-2O), 1.00 (1H, m, H-9), 0.93 (3H, s, H3-19); 13C NMR data 
(CDCI3, 75 MHz) 170.6 (CH3COO), 135.6 (CH, C-15), 135.4 (C, 
C-14), 129.4 (C, C-13), 110.9 (CH2, C-16), 71.5 (CH2, C-18), 70.3 
(CH, C-6), 54.1 (CH, C-9), 49.2 (CH, C-5), 40.3 (CH2, C-l), 38.1 
(C, C-4), 37.5 (CH2, C-3), 37.3 (C, C-10), 36.7 (CH, C-8), 36.7 
(CH2, C-7), 26.4 (CH2, C-12), 21.9 (CH3, CH3COO), 21.3 (CH2, 
C-ll), 19.1 (CH3, C-19), 18.3 (CH2, C-2), 16.8 (CH3, C-20), 16.0 
(CH3, C-17); LRCIMS (CH4) m/z (%) 347 [M + 1]+ (9), 302 (70), 
288 (100), 259 (32), 227 (26), 200 (29), 167 (38), 148 (76), 108 
(36), 94 (36); HRCIMS (CH4) m/z 347.2219 [M + 1]+ (calcd for 
C22H34O3, 347.2222). 
Hydrolysis of 4. Compound 4 (20 mg) was hydrolyzed with 
10% KOH in MeOH. After 24 h, the mixture was neutralized 
with 2 N HC1, extracted with CHCI3, washed with H20, and 
dried over Na2S04. The organic layer gave compound 11 (20 
mg): white solid, mp 66-72 ?C; [a]24D +37.0 (c 0.7, CHC13); 
IR (KBr) iw 3674, 3403, 2971, 2922, 1674, 1394, 1066 cm"1; 
JH NMR data (300 MHz, CDC13) d 3.79 (1H, dd, J = 6, 11 Hz, 
H-16a), 3.73 (1H, d, J = 11 Hz, H-18a), 3.55 (1H, dd, J = 8, 11 
Hz, H-16b), 3.42 (1H, d, J = 11 Hz, H-18b), 1.68 (1H, m, H-8), 
1.51 (1H, m, H-16), 1.47 (2H, m, H2-2), 1.23 (3H, s, H3-15), 
1.12 (1H, m, H-9), 1.05 (1H, m, H-5), 1.01 (1H, m, H-14), 0.96 
(3H, s, H3-19), 0.76 (3H, s, H3-20); 13C NMR data (75 MHz, 
CDCI3) (5 211.4 (C=0, C-12), 65.1 (CH2, C-18), 62.3 (CH2, C-17), 
57.0 (CH, C-9), 55.2 (CH, C-5), 38.4 (C, C-4), 38.3 (CH, C-14), 
38.0 (CH2, C-l), 37.3 (CH, C-16), 37.2 (C, C-10), 36.8 (CH, C-8), 
36.4 (CH2, C-ll), 35.5 (CH2, C-7), 35.5 (CH2, C-3), 33.4 (C, 
C-13), 26.9 (CH3, C-19), 22.1 (CH2, C-6), 18.2 (CH2, C-2), 14.7 
(CH3, C-20), 14.1 (CH3, C-15); LRCIMS (CH4) m/z (%) 321 [M 
+1]+ (76), 320 [M+] (59), 303 (100), 271 (23), 259 (9), 193 (11), 
175 (14), 160 (14), 122 (16), 107 (8), 94 (4); HRCIMS (CH4) 
m/z 320.2338 [M+] (calcd for C20H32O3, 320.2351). 
Oxidation of 6. Compound 6 (170 mg) was dissolved in 
acetone (previously distilled over KMn04) and Jones reagent 
added in the usual manner. The products were purified by 
column chromatography on silica gel 60, yielding ketone 12 
(14.5 mg): [a]24D -36.5 (c 0.23, CHC13); IR (KBr) vmax 3445, 
2931,1725,1710, 1464, 1380, 1037 cm"1; *H NMR partial data 
(CDCI3, 300 MHz) 6 6.78 (1H, dd, J = 11, 17 Hz, H-15), 5.16 
(1H, d, J = 17 Hz, H-16a), 5.02 (1H, d, J = 11 Hz, H-16b), 
4.05 (1H, d, J = 11 Hz, H-18a), 3.77 (1H, d, J = 11 Hz, H-18b), 
1.70 (3H, s, H3-17), 1.23 (3H, s, H3-19), 0.86 (3H, s, H3-20); 
13C NMR data (CDC13, 75 MHz) d 210.8 (C=0, C-6), 171.0 
(MeCO-18), 135.0 (CH, C-15), 134.3 (C, C-14), 129.4 (C, C-13), 
112.0 (CH2, C-16), 71.9 (CH2, C-18), 60.2 (CH, C-5), 53.6 (CH, 
C-9), 47.7 (CH2, C-7), 42.8 (CH, C-8), 41.3 (C, C-10), 38.2 (CH2, 
C-l), 36.3 (CH2, C-3), 35.3 (C, C-4), 26.4 (CH2, C-12), 21.5 (CH2, 
C-ll), 21.0 (CH3CO-I8), 17.8 (CH3, C-19), 17.8 (CH2, C-2), 16.0 
(CH3, C-20), 15.5 (CH3, C-17); LREIMS m/z (%) 345 [M + 1]+ 
(5), 287 (75), 271 (46), 257 (42), 203 (10), 167 (100), 149 (91), 
123 (78), 109 (76), 91 (38); HREIMS m/z 345.2048 [M + 1]+ 
(calcd for C22H23O3, 345.2073); and aldehyde 13 (7.0 mg): [a]24D 
-23.4 (c 0.32, CHCI3); IR (KBr) vmax 3446, 2933, 2361, 1730, 
1711,1380,1038 cm"1; XH NMR partial data (CDC13, 300 MHz) 
10.15 (1H, s, CHO), 4.09 (1H, d, J = 11 Hz, H-18a), 3.78 (1H, 
d, J = 11 Hz, H-18b), 211 (3H, s, H3-I6), 2.04 (3H, s, CH3CO- 
18), 1.25 (3H, s, H3-19), 0.88 (3H, s, H3-20); 13C NMR data 
(CDCI3, 75 MHz) 209.4 (C=0, C-6), 191.2 (CHO), 170.9 
(CH3CO-18),155.4 (C, C-13), 134.4 (C, C-14), 71.9 (CH2, C-18), 
60.3 (CH, C-5), 52.9 (CH, C-9), 46.3 (CH2, C-7), 43.6 (CH, C-8), 
41.1 (C, C-10), 38.4 (CH2, C-l), 36.1 (CH2, C-3), 35.7 (C, C-4), 
23.9 (CH2, C-12), 20.9 (CH3CO-I8), 20.8 (CH, C-ll), 17.9 (CH3, 
C-17), 17.7 (CH2, C-2), 16.5 (CH3, C-19), 14.6 (CH3, C-16); 
LRCIMS (CH4) m/z (%) 347 [M + 1]+ (7), 303 (31), 287 (100), 
257 (27), 245 (9), 229 (6), 204 (7), 175 (6), 167 (13), 148 (11), 
123 (22), 108 (18), 80 (9); HRCIMS (CH4) m/z 346.2137 [M]+ 
(calcd for C21H30O4, 346.2144). 
Cassane Diterpenes from Myrospermum frutescens PAGE EST: 4.6 Journal of Natural Products    E 
Assignment of Absolute Stereochemistry. The MPA 
esters of the diol 5 were prepared by treatment with the 
corresponding (R)- and (S)-MPA in the presence of DCC and 
DMAP in CH2C12. The reaction mixture was filtered to remove 
the dicyclohexylurea and the ester purified by flash chroma- 
tography on silica gel eluting with hexanes?EtOAc. The 
spectra of the resulting MPA esters were recorded, the signals 
assigned, and the A(5BS values calculated (Figure 1). The model 
designed by the Riguera group was used for configuration 
assignment of the secondary alcohols.8 
6/?,18-Di[(R)-methoxyphenyl acetate]cassan-13,15-di- 
ene: [a]24D -87.8 (c 1.03, CHC13); XH NMR data (CDC13, 500 
MHz) 6.83 (1H, dd, J = 11, 17 Hz, H-15) 5.16 (1H, d, J = 17 
Hz, H-16a), 5.01 (1H, d, J = 11 Hz, H-16b), 4.30 (1H, d, J = 
11 Hz, H-18a), 3.93 (1H, s, H-6), 3.42 (1H, d, J = 1 Hz, H-18b), 
1.80 (1H, m, H-7a), 1.64 (3H, s, H3-17), 1.10 (3H, s, H3-19), 
1.04 (3H, s, H3-2O), 0.69 (1H, m, H-5), 0.46 (1H, m, H-7b); 13C 
NMR data (CDC13, 125 MHz) 136.4 (C, C-14), 135.4 (CH, C-15), 
128.8 (C, C-13), 110.6 (CH2, C-16), 72.5 (CH2, C-18), 67.7 (CH, 
C-6), 53.9 (CH, C-9), 50.0 (CH, C-5), 40.4 (CH2, C-l), 40.1 (CH2, 
C-7), 37.7 (CH2, C-3), 36.7 (C, C-10), 35.8 (CH, C-8), 32.6 (C, 
C-4), 26.5 (CH2, C-12)21.1 (CH2, C-ll), 19.6 (CH3, C-19), 18.2 
(CH2, C-2), 17.0 (CH3, C-20), 15.9 (CH3, C-17); EIMS mlz 434 
[M - C9H10O3]+ (10), 268 (34), 253 (14), 239 (11), 183 (8), 121 
(100), 105 (12), 91 (14). 
6/?,18-Di[(S)-methoxyphenyl acetate]cassan-13,15-di- 
ene: [a]24D -29.6 (c 0.5, CHC13); :H NMR data (CDC13, 500 
MHz) 6.80 (1H, dd, J = 11, 17 Hz, H-15), 5.33 (1H, s, H-6), 
5.10 (1H, d, J = 17 Hz, H-16a), 4.95 (1H, d, J = 11 Hz, H-16b), 
3.81 (1H, J = 11 Hz, H-18a), 3.64 (1H, J = 11 Hz, H-18b), 
1.58 (3H, s, H3-17), 1.54 (1H, m, H-7a), 0.92 (3H, s, H3-20), 
0.88 (1H, m, H-5), 0.42 (1H, m, H-7b), 0.28 (3H, s, H3-19); 13C 
NMR data (CDCI3, 125 MHz) 136.8 (C, C-14), 135.3 (CH, C-15), 
128.8 (C, C-13), 111.1 (CH2, C-16), 71.2 (CH2, C-18), 70.1 (CH, 
C-6), 53.7 (CH, C-9), 48.6 (CH, C-5), 40.5 (CH2, C-l), 40.2 (CH2, 
C-7), 37.1 (CH, C-8), 36.9 (C, C-10), 36.6 (C, C-4), 36.3 (CH2, 
C-3), 26.4 (CH2, C-12), 22.7 (CH2, C-ll), 18.1 (CH3, C-19), 17.3 
(CH2, C-2), 16.7 (CH3, C-20), 15.8 (CH3, C-17); EIMS mlz 434 
[M - C9H10O3]+ (37), 268 (90), 185 (11), 132 (8), 121, (100), 
105 (8), 91 (9). 
T. cruzi Bioassay. The assay is based on inhibition of the 
parasites by added compound or extract.2 A /3-galactosidase- 
expressing transgenic T. cruzi (Tulahuen strain, clone C4) was 
used. Growth of the intracellular form of T. cruzi was 
determined from the cleavage of chlorophenol red-/3-D-galac- 
toside (CPRG, 570 nm). 
Cytotoxicity Assay. Vero cells adhering to 96-well plates 
were used to evaluate the toxicity of the compounds purified 
from M. frutescens on the basis of reduction of 3-(4,5-dimethyl- 
thiazol-2-yl)-2,5-diphenyltetrazolium bromide10 (MTT, Sigma). 
After treatment with the test compound and 4 h incubation 
at 37?, cell viability was evaluated in an ELISA reader at 570 
nm. 
Acknowledgment. The authors extend thanks to Dr. R. 
Riguera, R. Garcia, and F. Freire from the Department of 
Organic Chemistry of the Universidad de Santiago de Com- 
postela for assistance with the establishment of the absolute 
configuration of cassanes and some of the MS. Funding comes 
from the International Cooperative Biodiversity Groups Pro- 
grams (grant number #1U01 TW01021-01 to P.C.) from the 
NIH, the National Science Foundation, and the U.S. Depart- 
ment of Agriculture. 
Supporting Information Available: ID and 2D NMR data for 
chagreslactone (3) and chagresnone (4). This material is available free 
of charge via the Internet at http://pubs.acs.org. 
References and Notes 
(1) Coley, P. D.; Heller, M. V.; Aizprua, R.; Araiiz, B.; Flores, N.; Correa, 
M.; Gupta, M.; Soils, P. N.; Ortega-Barria, E.; Romero, L.; Gomez, 
B.; Ramos, M.; Cubilla-Rios, L.; Capson, T. L.; Kursar, T. A. Front 
Ecol. Environ. 2003, 1, 421-428. 
(2) Torres-Mendoza, D.; Urena Gonzalez, L. D.; Ortega-Barria, E.; 
Capson, T. L.; Cubilla-Rios, L. J. Nat. Prod. 2003, 66, 928-932. 
(3) Sam, N.; Arreguy San-Miguel, B.; Taran, M.; Delmond, B. Tetrahe- 
dron 1991, 47, 9187-9194. (b) Pegel, K. H.; McGarry, E. J.; Phillips, 
L.; Waight, E. S. J. Chem Soc. (C) 1971, 5, 904-909. (c) Ellestad, G. 
A.; Kunstmann, M. P.; Morton, G. O. J. Chem. Soc., Chem. Commun. 
1973, 312-313. 
(4) Roach, J. S.; McLean, S.; Reynolds, W. F.; Tinto, W. T. J. Nat. Prod. 
2003, 66, 1378-1381. 
(5) Kinoshita, T.; Kaneko, M.; Noguchi, H.; Kitagawa, I. Heterocycles 
1996, 43, 409-414. (b) Kinoshita, T. Chem. Pharm. Bull. 2000, 48, 
1375-1377. 
(6) Jang, D. S.; Park, E. J.; Hawthorne, M. E.; Schunke, J.; Graham, J. 
G.; Cableses, F.; Santarsiero, B. D.; Mesecar, A. D.; Fong, H. H. S.; 
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(7) Seco, J. M.; Latypov, S.; Quinoa, E.; Riguera, R. Tetrahedron 1997, 
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(8) Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron: Asymmetry 2001, 
12, 2915-2925. 
(9) The oxidation of compound 5 to yield 14 is described in ref 2. 
(10) Chiari, E.; Camargo, E. P. In Genes and Antigens of Parasites. A 
Laboratory Manual; Morel, C. M., Ed.; Fundacao Oswaldo Cruz: Rio 
de Janeiro, Brazil, 1984; pp 23-28. 
NP049890C