Antimycobacterial metabolites from Plectranthus: Royleanone derivatives against Mycobacterium tuberculosis strains

Article abstract of DOI:10.1002/cbdv.200900099

The antimycobacterial activities of eight diterpenes, 1-8, isolated previously from Plectranthus and eleven esters, 9-19, of 7α-acetoxy- 6β,12-dihydroxyabieta-8,12-diene-11,14-dione (5) were evaluated against the MTB strains H₃₇Rv and MDR. Only

Full text of DOI:10.1002/cbdv.200900099

922
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Antimycobacterial Metabolites from Plectranthus: Royleanone Derivatives
against Mycobacterium tuberculosis Strains
by Patrꢀcia Rijoa), M. Fa´tima Simo˜es*a), A. Paula Franciscoa), Rosario Rojasb), Robert H. Gilmanb),
Abraham J. Vaisbergb), Benjamꢀn Rodrꢀguezc), and Cristina Moiteirod)
a
´
) Faculdade de Farmacia da Universidade de Lisboa, iMed.UL, Av. Prof. Gama Pinto,
PT-1649-003 Lisboa (phone: þ351-21-7946400; fax: þ351-21-7946470; e-mail: fasimoes@ff.ul.pt)
b
´
) Laboratorios de Investigacion y Desarrollo, Facultad de Ciencias y Filosofꢀa, Universidad Peruana
´
Cayetano Heredia, Lima, Peru
c
´
) Instituto de Quꢀmica Organica-CSIC, c/Juan de la Cierva 3, ES-28006 Madrid
d
˜
) Instituto Nacional Engenharia, Tecnologia e InovaÅao, I.P., DTIQ, Estrada do PaÅo do Lumiar 22,
PT-1649-038 Lisboa
The antimycobacterial activities of eight diterpenes, 1–8, isolated previously from Plectranthus and
eleven esters, 9–19, of 7a-acetoxy-6b,12-dihydroxyabieta-8,12-diene-11,14-dione (5) were evaluated
against the MTB strains H37Rv and MDR. Only diterpenoids with a quinone framework revealed anti-
MTB activity. Abietane 5 and its 6,12-dibenzoyl, 12-methoxybenzoyl, 12-chlorobenzoyl, and 12-
nitrobenzoyl esters, 9, 11, 12, and 13, respectively, showed potent activities against the MDR strain with
MIC values between 3.12 and 0.39 mg/ml. Cytotoxic activities towards 3T3 and Vero cells were also
evaluated. Compound 11, with the best selectivity index, may be a suitable lead for further chemical
modifications. The complete structural elucidation of the new esters, 9–14, 16, 18, and 19, as well as the
NMR data of known derivatives 15 and 17 are reported.
Introduction. – Tuberculosis (TB) is an old human infectious disease that still
remains a leading cause of death worldwide, both in developing and developed
countries. Mycobacterium tuberculosis (MTB) is the most important mycobacterium
causing human TB. Nowadays, with the emergence of drug- and multidrug-resistant M.
tuberculosis strains (MDR TB, XDR TB) treatment of this disease is of great concern.
Though most people with normal immune systems resist to be infected, those with
weakened immune systems, e.g., caused by other diseases (such as HIV), by
malnutrition, or by inadequate treatment, are seriously infected [1]. A large number
of newly infected people emerge every year, mostly in Asia and in sub-Saharan Africa
with TB being the fifth cause of mortality in South Africa [2].
References to natural products with anti-TB activity are increasingly reported, and
thus it is expected that these compounds will enlarge the diversity of novel scaffolds for
the antimycobacterial drug development [1][3]. Plectranthus species (Labiatae) are
largely distributed in tropical and subtropical Africa, and are traditionally used for the
treatment of respiratory complaints [2][4]. P. barbatus and P. bojeri are indicated for
the treatment of pneumonia, and P. amboinicus revealed anti-MTB activity [4].
Moreover P. aegypticus, P. ambiguus, P. caninus, P. edulis, P. elegans, P. glandulosus, P.
ꢁ 2010 Verlag Helvetica Chimica Acta AG, Zꢂrich

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
923
hadiensis, P. lanceolatus, P. laxiflorus, P. madagascarensis, P. mollis, P. montanus, and P.
stolzii are used to relieve several respiratory diseases [4].
The constituents of Plectranthus plants are mainly diterpenoids which belong to
abietane, kaurane, and labdane classes, royleanone abietanes being a well-represented
subclass [5]. In a few royleanone abietanes, antimycobacterial activities were found,
e.g., in derivatives of carnosic acid, isolated from Salvia africana-lutea [2], and in
horminone, obtained from S. reptans [6] and S. multicaulis [7]. These last abietanes
resemble royleanones isolated from P. grandidentatus and P. hereroensis, which
revealed anti-methicillin-resistant Staphylococcus aureus (MRSA) properties [8].
Royleanones are hydroquinonic abietanes with a 12-hydroxy-11,14-dioxo-quinone
moiety in C ring. Several natural quinone metabolites showed antimycobacterial
activity [3], namely naphthoquinones and benzoquinones such as 7-methyljuglone [9]
and primin [10], respectively.
Pursuing our research efforts to find antimicrobial metabolites from Plectranthus
spp., we report here for the first time the antimycobacterial study of eight natural
diterpenes, 1–8, obtained previously from Plectranthus spp., and a set of eleven
hemisynthetic royleanone derivatives, 9–19, against two MTB strains. Cytotoxicity
assays against eukaryotic murine (3T3) and mammalian (Vero) cell lines are also
reported.
Results and Discussion. – 1. Natural Diterpenoids. Natural diterpenes were
previously isolated from P. fruticosus (three ent-kaurenes: ent-kaur-15-en-19-oic acid
(1), ent-12b-acetoxykaur-16-en-19-oic acid (2), and ent-7b-hydroxy-15b,16b-epoxy-
kauran-19-oic acid (3)) [11], from P. ornatus (one labdane: rhinocerotinoic acid (4))
[12] and from P. grandidentatus (four royleanone abietanes: 7a-acetoxy-6b-hydroxy-
royleanone (5), 6b,7a-dihydroxyroyleanone (6), horminone (7), 6,7-dehydroroylea-
none (8)) [8][12][13] (Fig. 1). Preliminary results highlighted that only royleanone
Fig. 1. Diterpenes Isolated from Plectranthus spp.

924
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
abietanes were active against the assayed MTB strains 7a-acetoxy-6b-hydroxyroylea-
none (5), isolated from P. grandidentatus being the most potent of them, suggesting that
it could be a lead for future drug development.
2. Hemisynthetic Diterpenoids. Assuming to keep the chromophoric system of the
lead royleanone 5 (tautomeric 6-O-hydroxy-7-O-acetoxy-12-O-hydroxy-11,14-dioxo-
quinone motif), eleven royleanone derivatives, 9–19, were prepared (Fig. 2).
Regarding the complex lipophilic cell envelope of mycobacterium, modulation of
lipophilicity was achieved by esterification at C(6) and C(12).
Fig. 2. Ester Derivatives 9–19 of 7a-acetoxy-6b-hydroxyroyleanone (5)
Aromatic and alkylic ester derivatives of 5 were synthesized according to a known
methodology [6]. First, by reaction with the corresponding aromatic acyl chloride, two
highly lipophilic diesters, 9 and 10, [14] (Table) were prepared (Fig. 2), followed by the
hemisynthesis of the aromatic monoesters 11–14 (Fig. 2; with medium log Pꢀ5.00
[14]; Table). To study the effect of the alkylic esterification on the activity of 5, a set of
further five derivatives 15–19 (Fig. 2) was prepared [6].
3. Structure Elucidation. The structures of diterpenoids 9–14, 16, 18, and 19 were
elucidated on the basis of their spectroscopic data (see Exper. Part), mainly the 1H- and
13C-NMR spectra, and COSY, HSQC, and HMBC experiments, in addition to
comparison with literature data [12][13][15–17]. The attachment of the acyl groups at
C(6) and/or C(7) was firmly established by the correlations observed in the HMBC
spectra, whereas the esterification of the OH group at C(12) was evident by the absence
1
of the signal of the H-atom of 12-OH moiety in the H-NMR spectra of 9–13 and 15
and 16.
Though compounds 15 [15] and 17 [17] were already known, the 1H-NMR data of
15 are now complete and its 13C-NMR spectrum is reported here for the first time. The
1H- and 13C-NMR data of 17 were in good agreement with those reported in literature
[17], except for the assignments of C(20) and the Me C-atom of the 6b-AcO group
which must be reversed. Moreover, several J(H,H) values for 17, not determined
previously, are now reported.
4. Antimycobacterial Activity. Antimycobacterial assays [18] carried on ent-kaurene
and labdane diterpenes, 1–3 and 4, respectively, showed that they have no activity
against MDR and H37Rv strains (MIC values >25 mg/ml) (Table). In contrast, the
royleanones, which bear a p-benzoquinone C ring, i.e., 6b,7a-dihydroxyroyleanone (6),

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
925
Table. In vitro Antimycobacterial and Cytotoxic Activities of Royleanone Diterpenoids
Compounds
Mycobacteria
(MIC [mg/ml]a))
Eukaryotic cell lines
SIc)
log P [14]
(GI50 [mg/ml]b))
H37Rv
MDR
3T3
Vero
3T3
Vero
5
9
10
11
12
13
14
15
16
17
18
19
6
7
8
25
>25
>25
3.12
1.56
>25
0.39
0.78
3.12
25
12.50
6.25
12.96
1.44
ndd)
1.58
2.42
4.90
2.34
4.30
4.00
10.30
4.90
9.92
nd
12.80
2.52
nd
4.15
0.92
nd
4.10
1.62
nd
3.16ꢂ0.62
7.02ꢂ1.05
8.07ꢂ1.29
4.92ꢂ0.83
5.52ꢂ0.77
4.88ꢂ0.67
5.13ꢂ0.75
4.01ꢂ0.69
4.87ꢂ0.91
3.60ꢂ0.56
4.18ꢂ0.64
4.59ꢂ0.73
2.44ꢂ0.52
3.52ꢂ0.52
4.52ꢂ0.58
nd
3.12
2.58
4.42
2.70
2.86
3.20
2.70
10.54
2.90
5.94
nd
4.05
3.10
1.57
0.09
0.34
0.64
0.82
0.78
0.79
nd
6.62
5.67
0.87
0.11
0.26
0.43
0.84
0.46
0.48
nd
25
>25
25
25
25
>25
>25
25
>25
25
12.50
6.25
12.50
12.50
12.50
ꢁ12.50
4
18.58
nd
nd
16.66
nd
nd
1.49
nd
nd
1.33
nd
nd
>25
Isoniazide)
Rifampicine)
0.125
0.063
16
nd
nd
nd
nd
nd
a) MIC Value is the minimum inhibitory concentration of the tested compounds effecting 100% of
inhibition. b) GI50 Value is the concentration of the tested samples resulting in 50% inhibition of cell
growth. c) SI¼GI50 values (3T3 or Vero cells)/MIC value (MDR strain). d) nd¼Not determined.
e) Positive controls.
horminone (7), and 6,7-dehydroroyleanone (8), showed mild activities against the
MDR strain (MIC values ꢁ12.5 mg/ml), and, distinctively, 7a-acetoxy-6b-hydroxy-
royleanone (5) exhibited the more potent antimycobacterial activity against the MDR
strain with a MIC value of 3.12 mg/ml, and against H37Rv strain with a MIC value of
25 mg/ml (Table). These results may suggest that the presence of the 7a-AcO group at B
ring is essential to increase the MDR-MTB activity. As already emphasized, these
results were not unexpected due to the p-benzoquinone framework, present on C ring
of royleanones, responsible for the antimycobacterial activities of several quinonic
compounds [10]. Moreover, it was observed that both acetone extract of P.
grandidentatus and the chromatographic fraction containing these active four
royleanones were inactive against both MTB strains (MIC 12.50–25 mg/ml), indicating
that activity is only exerted by pure metabolites.
Since abietane 5 exhibited the best MIC value, and aiming at achieving compounds
with a improved anti-TB activities, novel abietane derivatives 9–19 were tested.
Of the more lipophilic aromatic derivatives 9 and 10 only the dibenzoyl ester 9
showed slightly higher potency than 5 against the MDR strain (MIC 1.56 mg/ml; Table).
The less lipophilic aromatic 12-esters 11–13 were particularly active against the MDR
strain, but only compound 11 (with a methoxybenzoyl substituent) showed a promising
antimycobacterial activity against both MDR and H37Rv MTB strains with MIC values
of 0.39 and of 3.12 mg/ml, respectively. It is also interesting to note that chlorobenzoyl

926
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
derivative 12 was fourfold more active, against MDR (MIC 0.78 mg/ml) than 13, which
has a nitrobenzoyl substituent (MIC 3.12 mg/ml against MDR). In contrast, the only
assayed 6-aromatic ester (14, an isomer of 13) was inactive. Lead royleanone 5, and its
esters 9, and 11–13 are more active than the first-line antituberculous drugs isoniazid
(MIC 4 mg/ml) and rifampicin (MIC 16 mg/ml) against the MDR-MTB strain. In these
tested derivatives, 9–14, there is no correlation between lipophilicity and activity;
however, the presence of a p-MeO group in the benzoyl moiety (i.e., 11) afforded a
more potent anti-MTB derivative (Table).
Apolar alkyl esters of 5 are less active than the parent diterpene 5, the aromatic
esters 9, 11–13, and isoniazid, the positive control (Table). Neither diesters of 5, i.e.,
6,12-O-diacetyl and 6,12-O-dipropanoyl derivatives, 15 and 16, respectively, nor 6-O-
acetyl, 6-O-propanoyl, and 6-O-butanoyl derivatives, 17, 18, and 19, respectively,
showed significant antimycobacterial-activity enhancement, even though the 6-O-
derivatives of 5 were slightly active against the MDR strain (MIC values ranging from
6.25 to 12.5 mg/ml). These results show that the insertion of an acyloxy chain at C(6) did
not provide active compounds (Table), nevertheless the propanoyl group seems to
increase the activity against MDR strain (MIC 6.25 mg/ml).
Surprisingly, all the royleanones are much more potent against MDR than against
sensitive H37Rv strains, with the exception of the diterpenoid 11.
5. In vitro Cytotoxicity. In accord with the purpose of searching for potential
antimycobacterial lead diterpenes, the cytotoxic bioactivity against the two eukaryotic
3T3 and Vero cell lines were also investigated, according to the recommendations of the
Tuberculosis Drug Screening Program [18]. In this work, we found similar toxicities for
all compounds against both cell lines.
Abietanes 5, 9, 10–19, and horminone (7) did not show good in vitro cytotoxic
profiles either in 3T3 cells (GI50 range 1.44 to 18.58 mg/ml) or against Vero cells (GI50
range 2.58 to 16.6 mg/ml), thus revealing high-to-moderate cytotoxicity [19]. It can be
suggested that the chromophore present in the rings B and C of these royleanones
contributes both to anti-MDR activity and cytotoxicity, once the more potent anti-
MTB abietanoids exhibited also the higher cytotoxicities.
Finally, the anti-TB selectivity (therapeutic) indexes for both MTB assayed strains
were determined towards both 3T3 and Vero cell lines. The majority of the diterpenoids
were non-selective (SI<1), and only 5 and its derivatives 11 and 12 exhibited
selectivity (SI 3.2 and 6.62, resp.) for both the tested MDR strain vs. both cell lines
(Table).
Even though the mode of action of the quinonic diterpenoids mentioned above was
not studied, it should be quite complex, targeting different biological systems of both
prokaryotic and eukaryotic cells [10]. In the literature [20] it is mentioned that the
biological activity of some similarly substituted quinones, mostly 1,4-naphthoquinones,
is related with the ability to accept electrons to forming radical anions and further to
generate reactive oxygen species (ROSs) causing cell damage. Cellular damage has
also been attributed to alkylation, whenever quinones are activated inside the cell, by
binding to cellular nucleophiles (proteins, DNA) forming Michael adducts [10][20].
Other mechanisms related with extrusion pumps or cell-penetration abilities have also
been described [20], as well as the interaction with the potential M. tuberculosis target
Mtr (the NADPH-dependent enzyme mycothiol disulfide reductase) [9].

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
927
Conclusions. – Screening antimycobacterial activities of 7a-acetoxy-6b-hydroxyr-
oyleanone (5) and its derivatives led to the conclusion, considering potency and
selectivity observed, that 12-O-(4-methoxybenzoyl) derivative 11 may become a valid
lead for further modifications to obtain compounds with better potency, lower
cytotoxicity, and better selectivity. Similarly, 12-O-(4-chlorobenzoyl) derivative 12 and
surely metabolite 5 may be suitable to prepare more derivatives with a valid
therapeutic profile. Certainly, further chemical transformations are required to obtain,
at least, other derivatives with adequate SI values (>10) [18], seeking to determine
their possible inhibition of MTB in intracellular macrophage infection [18]. Besides
this, there is a need to extend antimycobacterial and cytotoxicity studies, namely testing
other resistant Mycobacterium strains, evaluating toxicity on prokaryotic Mycobacter-
iae, and analyzing selectivity differences of MDR- vs. H37Rv-sensitive strains. The final
goal will be to propose derivatives of these natural compounds, i.e., royleanones from
Plectranthus spp., as a new class of antimycobacterial agents.
ˆ
We would like to thank the Portuguese FundaÅa˜o para a Ciencia e a Tecnologia (FCT-MCES (I&D
n8 8/94), POCTI (QCAIII) and FEDER, Ph.D. grant No. SFRH/BD/19250/2004), to the Spanish
´
Comision Interministerial de Ciencia y Tecnologꢀa (CICYT, grant No. CTQ2006-15279-C03-02), and to
the Iberoamerican Program for Science and Technology (CYTED, Project X.11: PIBATUB) for financial
support.
Experimental Part
General. M.p.: Kofler block; uncorrected. Optical rotations: in CHCl3 soln., Perkin-Elmer 241 MC
polarimeter. IR: in KBr, Perkin-Elmer Spectrum One spectrophotometer. 1H- and 13C-NMR spectra: at
r.t.; Varian INOVA-400 spectrometer, equipped with a 5-mm gradient inverse detection probe, operating
at 399.90 and 100.55 MHz, resp.; chemical shifts with respect to the residual CHCl3 signal (d 7.25) and to
the solvent signals (d 77.00), resp.; all the 1H- and 13C-NMR assignments were in agreement with COSY,
HSQC, and HMBC experiments. MS: positive EI mode, 70 eV, Hewlett-Packard 5973 spectrometer.
¤
Plant Material. Natural diterpenes extracted from Plectranthus fruticosus LꢃHerit: ent-kaur-15-en-
19-oic acid (1), ent-12b-acetoxykaur-16-en-19-oic acid (2), and ent-7b-hydroxy-15b,16b-epoxykauran-19-
oic acid (3) [11]; from P. ornatus Codd.: rhinocerotinoic acid (4; (13E)-7-oxolabda-8,13-dien-15-oic acid)
[12]; and from P. grandidentatus Gˇrke: 7a-acetoxy-6b-hydroxyroyleanone (5), 6b,7a-dihydroxy-
royleanone (6), horminone (7), and 6,7-dehydroroyleanone (8) [8][12][13]. Compound 5, used as
starting material for the esterification reactions, was re-isolated from an acetone extract of P.
grandidentatus, which was kept in the Faculty of Pharmacy Hortum.
Hemisynthesis of Royleanone Derivatives 9–19. A soln. of 5 (¼(6b,7a)-6,12-dihydroxy-11,14-
dioxoabieta-8,12-dien-7-yl acetate) in pyridine (Py) and eventually CH2Cl2, and the suitable benzoyl
chloride or appropriate alkyl anhydride was allowed to stand under adequate conditions. The mixture
was then diluted with CH2Cl2 and successively washed with H2O and NaOH soln. (5%), and the org.
layer was dried (anh. Na2SO4). The residue was purified by prep. TLC (silica gel; CH2Cl2/MeCOMe
98 :2) to afford 9–19 [6]. The conditions to obtain each one of the derivatives were as follows. 30.0 mg of
5, BzOCl (2 ml), Py (0.5 ml), r.t., 3 d, gave 9 (22.4% yield); 20.0 mg of 5, 4-chlorobenzoyl chloride
(32 ml), Py (2 ml), CH2Cl2 (2 ml), r.t., 7 d, gave 10 (66.4%, less polar compound) and 12 (27.9%); 32.2 mg
of 5, 4-methoxybenzoyl chloride (27 ml), Py (2 ml), CH2Cl2 (2 ml), r.t, 24 h, yielded 96.6% of 11; 30.3 mg
of 5, 4-nitrobenzoyl chloride (141 mg), Py (2 ml), CH2Cl2 (2 ml), r.t., 40 min, gave 13 (16.8%, most polar
compound) and 11.9% of 14; 30.6 mg of 5, Ac2O (2 ml), Py (2 ml), CH2Cl2 (1 ml), r.t., 24 h, yielded 15
(65.6%) and 8.5% of 17 (most polar compound); 40.5 mg of 5, propanoic anhydride (1 ml), Py (1 ml), 08,
7 d, gave 73.5% of 16 and 18 (8.6%, most polar compound); 15.0 mg of 5, butanoic anhydride (1 ml), Py
(1 ml), 08, 24 h afforded 35.2% of 19.
7a-Acetoxy-6b-benzoyloxy-12-O-benzoylroyleanone (¼(6b,7a)-7-(Acetyloxy)-11,14-dioxoabieta-
8,12-diene-6,12-diyl Dibenzoate; 9). Yellow quadrangular plates (AcOEt/pentane). M.p. 238–2418.

928
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
[a]1D8 ¼ ꢃ29.2 (c ¼ 0.24, CHCl3). IR (KBr): 3062, 2962, 2933, 2867, 1755, 1738, 1726, 1666, 1612, 1601,
1
1452, 1371, 1315, 1269, 1220, 1177, 1138, 1093, 1061, 1016, 932, 887, 808, 754, 712. H-NMR (400 MHz,
CDCl3): 8.15 (dt, J(2’,3’)¼7.6, J(2’,4’)¼2.0, HꢃC(2’), HꢃC(6’) of BzOꢃC(6)); 7.99 (dt, J(2’,3’)¼7.2,
J(2’,4’)¼1.2, HꢃC(2’), HꢃC(6’) of BzOꢃC(12)); 7.69–7.40 (m, HꢃC(3’), HꢃC(4’), HꢃC(5’) of
BzOꢃC(6), BzOꢃC(12)); 5.90 (dd, J(7b,6a)¼2.0, J(7b,5a)¼0.9, HbꢃC(7)); 5.77 (dd, J(6a,7b)¼2.0,
J(6a,5a)¼1.6, HaꢃC(6)); 3.17 (sept., J(15,16(17))¼7.1, H ꢃC(15)); ca. 2.58 (overlapped, HbꢃC(1)), 2.10
(s, MeCOO); ca. 1.78 (overlapped, HbꢃC(2)); 1.77 (s, Me(20)); 1.69 (br. d, J(5a,6a)¼1.6, HaꢃC(5));
1.58 (overlapped, HaꢃC(2)); 1.49 (ddd, J(3b,3a)¼13.2, J(3b,2a)¼3.6, J(3b,2b)¼2.8, HbꢃC(3)); 1.29
(td, J(1a,1b)¼J(1a,2b)¼13.2, J(1a,2a)¼3.8, HaꢃC(1)); ca. 1.38 (overlapped, HaꢃC(3)); 1.21 (d,
J(17,15)¼7.1, Me(17)); 1.19 (d, J(16,15)¼7.1, Me(16)); 1.06 (s, Me(18)); 0.99 (s, Me(19)). 13C-NMR
(100 MHz, CDCl3): 185.3 (C(14)); 180.0 (C(11)); 168.1 (MeCOO); 165.3 (C6H5COOꢃC(6)); 164.0
(C(7’) of C6H5COOꢃC(12)); 152.5 (C(9)); 150.0 (C(12)); 140.0 (C(13)); 136.2 (C(8)); 134.3 (HꢃC(4’) of
BzOꢃC(12)); 133.1 (C(4’) of BzOꢃC(6)); 130.5 (C(3’), C(5’) of BzOꢃC(12)); 129.8 (C(3’), C(5’) of
BzOꢃC(6)); 129.7 (C(1’) of BzOꢃC(6)); 128.8 (C(2’), C(6’) of BzOꢃC(12)); 128.5 (C(2’), C(6’) of
BzOꢃC(6)); 127.9 (C(1’) of BzOꢃC(12)); 68.4 (C(6)), 65.2 (C(7)); 49.3 (C(5)); 42.5 (CH2(3)); 38.8
(C(10)); 38.4 (CH2(1)); 33.8 (C(4)), 33.3 (Me(18)); 25.2 (C(15)), 23.2 (Me(19)); 22.2 (Me(20)), 20.9
(MeCOO); 20.4 (Me(16)), 20.0 (Me(17)), 18.8 (CH2(2)). EI-MS: 539 (1, [MꢃAcO]þ ), 510 (1), 451 (2),
434 (3, [MꢃAcOꢃC6H5CO]þ ), 406 (20), 391 (5), 313 (7), 284 (6), 269 (12), 105 (100, [C6H5CO]þ ), 77
(15).
7a-Acetoxy-6a-[(4-chlorobenzoyl)oxy]-12-O-(4-chlorobenzoyl)royleanone (¼(6b,7a)-7-(Acetyl-
oxy)-11,14-dioxoabieta-8,12-diene-6,12-diyl Bis(4-chlorobenzoate); 10). Yellow amorphous powder.
[a]2D2 ¼ ꢃ40.8 (c¼0.262, CHCl3). IR (KBr): 3074, 3097, 2961, 2932, 2867, 1751, 1726, 1669, 1594, 1488,
1402, 1371, 1268, 1216, 1173, 1137, 1092, 1064, 1012, 931, 887, 847, 757, 732. 1H-NMR (400 MHz, CDCl3):
8.08 (d, Jo ¼8.6, HꢃC(2’), HꢃC(6’) of 4-ClꢃC6H4COOꢃC(12)); 7.92 (d, Jo ¼8.6, HꢃC(2’), HꢃC(6’) of
4-ClꢃC6H4COOꢃC(6)); 7.51 (d, Jo ¼8.6, HꢃC(3’), HꢃC(5’) of 4-ClꢃC6H4COOꢃC(12)); 7.39 (d, Jo ¼
8.6, HꢃC(3’), HꢃC(5’) of 4-ClꢃC6H4COOꢃC(6)); 5.88 (d, J(7b,6a)¼1.6, HbꢃC(7)); 5.76 (t,
J(6a,7b) ¼J(6a,5a)¼1.6, HaꢃC(6)); 3.17 (sept., J(15,16(17))¼7.0, H ꢃC(15)); 2.58 (overlapped,
HbꢃC(1)); 2.10 (s, MeCOO); 1.79 (qt, J(2b,1a)¼J(2b,2a)¼J(2b,3a)¼14.0, J(2b,1b)¼J(2b,3b)¼3.7,
HbꢃC(2)); 1.74 (s, Me(20)); 1.70 (d, J(5a,6a)¼1.6, HaꢃC(5)); 1.60 (dquint., J(2a,2b)¼14.0, J(2a,1a)¼
J(2a,1b)¼J(2a,3a)¼J(2a,3b)¼3.6, HaꢃC(2)); 1.48 (dtd, J(3b,3a)¼13.4, J(3b, 2b)¼3.7, J(3b,2a)¼3.6,
J(3b,1b)¼1.0, HbꢃC(3)); 1.30 (overlapped, HaꢃC(1)); 1.28 (overlapped, HaꢃC(3)); 1.21 (d, J(16,15)¼
7.0, Me(16)); 1.19 (d, J(17,15)¼7.0, Me(17)); 1.06 (s, Me(18)); 0.97 (s, Me(19)). 13C-NMR (100 MHz,
CDCl3): 185.2 (C(14)); 179.7 (C(11)); 168.1 (MeCOO); 164.5 (4-ClꢃC6H4COOꢃC(6)), 163.2 (4-
ClꢃC6H4COOꢃC(12)); 152.4 (C(9)); 149.5 (C(12)); 141.0 (C(4’) of 4-ClꢃC6H4COOꢃC(12)); 139.8
(C(13)); 139.4 (C(4’) of 4-ClꢃC6H4COOꢃC(6)); 135.7 (C(8)); 131.8 (C(2’), C(6’) of 4-
ClꢃC6H4COOꢃC(12)); 131.2 (C(2’), C(6’) of 4-ClꢃC6H4COOꢃC(6)); 129.2 (C(3’), C(5’) of 4-
ClꢃC6H4COOꢃC(12)); 128.9 (C(3’), C(5’) of 4-ClꢃC6H4COOꢃC(6)); 128.1 (C(1’) of 4-
ClꢃC6H4COOꢃC(6)); 126.4 (C(1’) of 4-ClꢃC6H4COOꢃC(12)); 68.7 (C(6)); 65.2 (C(7)); 49.3 (C(5));
42.5 (C(3)); 38.8 (C(10)); 38.4 (C(1)); 33.8 (C(4)); 33.3 (Me(18)); 25.2 (C(15)); 23.2 (Me(19)); 22.2
(Me(20)); 20.4 (Me(16)); 20.2 (Me(17)); 18.8 (CH2(2)). EI-MS: 607 (3, [MꢃAcO]þ ), 468 (3, [Mꢃ
AcOꢃOCꢃC6H5Cl]þ ), 440 (9), 313 (6), 269 (9), 139 (100, [4-ClꢃC6H4ꢃCO]þ ), 111 (16).
7a-Acetoxy-6b-hydroxy-12-O-(4-methoxybenzoyl)royleanone (¼(6b,7a)-7-(Acetyloxy)-6-hydroxy-
11,14-dioxoabieta-8,12-dien-12-yl 4-Methoxybenzoate; 11). Yellow fine needles (AcOEt/pentane). M.p.
215–2188. [a]1D8 ¼ þ43.9 (c¼0.353, CHCl3). IR (KBr): 3474, 2962, 2931, 2867, 1745, 1724, 1668, 1607,
1581, 1512, 1463, 1371, 1256, 1221, 1168, 1141, 1100, 1063, 1019, 1002, 963, 936, 898, 843, 816, 759, 746.
1H-NMR (400 MHz, CDCl3): 8.09 (d, J(2’,3’(6’,5’))¼8.9, HꢃC(2’), HꢃC(6’)); 6.98 (d, J(3’,2’(5’,6’))¼8.9,
HꢃC(3’), HꢃC(5’)); 5.67 (d, J(7b,6a)¼1.7, HꢃC(7b)); 4.32 (dd, J(6a,5a)¼1.9, J(6a,7b)¼1.7,
HaꢃC(6)); 3.89 (s, MeO); 3.17 (sept., J(15,16(17))¼7.0, HꢃC(15)); 2.49 (br. m, HbꢃC(1)); 2.06 (s,
MeCOO); 1.79 (qt, J(2b,1a)¼J(2b,2a)¼J(2b,3a) ¼ 13.7, J(2b,1b)¼J(2b,3b)¼3.4, HbꢃC(2)); 1.62 (s,
Me(20)); 1.53 (dquint., J(2a,2b)¼13.7, J(2a,1a)¼J(2a,1b)¼J(2a,3a)¼J(2a,3b)¼3.7, HaꢃC(2)); 1.45
(ddd, J(3b,3a)¼13.2, J(3b,2a)¼3.7, J(3b,2b)¼3.4, HbꢃC(3)); 1.36 (d, J(5a,6a)¼1.9, HaꢃC(5)); 1.23
(overlapped, HaꢃC(1)); 1.22 (d, J(17,15)¼7.1, Me(17)); 1.21 (s, Me(19)); 1.19 (overlapped, HaꢃC(3));
1.19 (d, J(16,15)¼7.0, Me(16)); 0.94 (s, Me(18)). 13C-NMR (100 MHz, CDCl3): 185.9 (C(14)); 179.9

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
929
(C(11)); 169.7 (MeCOO); 164.4 (C6H4CO); 163.7 (C(4’)); 153.0 (C(9)); 149.9 (C(12)); 139.5 (C(13));
135.5 (C(8)); 132.7 (HꢃC(2’), HꢃC(6’)); 120.2 (C(1’)); 114.1 (HꢃC(3’)), HꢃC(5’)); 68.9 (C(7)); 67.3
(C(6)); 55.6 (MeO); 49.8 (C(5)); 42.3 (C(3)); 38.9 (C(10)); 38.3 (C(1)); 33.7 (Me(18)); 33.5 (C(4)); 25.1
(C(15)); 23.8 (Me(19)); 21.7 (Me(20)); 20.9 (MeCOO); 20.4 (Me(16)); 20.2 (Me(17)); 18.9 (C(2)). EI-
MS: 524 (0.5, Mþ ), 496 (1, [MꢃCO]þ ), 464 (0.5, [MꢃAcOH]þ ), 436 (3, [MꢃAcOHꢃCO]þ ), 137 (2),
136 (17), 135 (100, [MeOC6H4CO]þ ), 93 (1), 92 (3), 91 (1), 79 (1), 78 (1), 77 (6), 55 (1).
7a-Acetoxy-6b-hydroxy-12-O-(4-chlorobenzoyl)royleanone (¼(6b,7a)-7-(Acetyloxy)-6-hydroxy-
11,14-dioxoabieta-8,12-dien-12-yl 4-Chlorobenzoate; 12). Yellow fine needles (AcOEt/pentane). M.p.
221–2238. [a]1D8 ¼ þ44.8 (c¼0.355, CHCl3). IR (KBr): 3485, 2962, 2929, 2867, 1749, 1732, 1667, 1596,
1461, 1371, 1252, 1220, 1141, 1094, 1066, 1008, 896, 844, 749. 1H-NMR (400 MHz, CDCl3): 8.07 (d, Jo ¼8.5,
HꢃC(2’), HꢃC(6’)); 7.49 (d, Jo ¼8.5, HꢃC(3’), HꢃC(5’)); 5.68 (dd, J(7b,6a)¼2.0, J(7b,5a)¼0.6,
HbꢃC(7)); 4.33 (dd, J(6a,7b)¼2.0, J(6a,5a)¼1.6, HaꢃC(6)); 3.17 (sept., J(15,16(17))¼7.1, H ꢃC(15));
2.49 (m, HbꢃC(1)); 2.06 (s, (MeCOO); 1.92 (br., OH); 1.80 (qt, J(2b,1a)¼J(2b,2a)¼J(2b,3a)¼13.6,
J(2b,1b)¼J(2b,3b)¼3.6, HbꢃC(2)); 1.62 (s, Me(20)); 1.54 (dquint., J(2a,2b)¼13.6, J(2a,1a) ¼
J(2a,1b) ¼J(2a,3a)¼J(2a,3b)¼3.7, HaꢃC(2)); 1.45 (dtd, J(3b,3a)¼13.3, J(3b,2a)¼3.7, J(3b,2b)¼
3.6, J(3b,1b)¼0.9, HbꢃC(3)); 1.36 (dd, J(5a,6a)¼1.6, J(5a,7b)¼0.6, HaꢃC(5)); 1.24 (s, Me(19)); 1.24
(overlapped, HaꢃC(1)); 1.21 (d, J(16(17),15)¼7.1, Me(16), Me(17)); 1.20 (overlapped, HaꢃC(3)); 0.94
(s, Me(18)). 13C-NMR (100 MHz, CDCl3): 185.7 (C(14)); 179.6 (C(11)); 169.7 (MeCOO); 163.2
(C6H4CO); 152.5 (C(9)); 149.8 (C(12)); 140.9 (C(4’)); 139.5 (C(13)); 135.7 (C(8)), 131.8 (C(2’), C(6’));
129.2 (C(3’), C(5’)); 126.5 (C(1’)); 68.9 (C(7)); 67.2 (C(6)); 49.8 (C(5)); 42.3 (C(3)); 38.9 (C(10)); 38.3
(C(1)); 33.7 (C(4)); 33.5 (Me(18)); 25.2 (HꢃC(15)); 23.8 (Me(19)); 21.7 (Me(20)); 20.9 (MeCOO); 20.4
(Me(16)); 20.2 (Me(17)); 18.9 (C(2)). EI-MS: 486 (1, [MꢃCH2¼C¼O]þ ), 470 (3, [MꢃAcOH]þ ), 468
(7, [MꢃAcOH]þ ), 329 (9), 302 (8), 301 (8), 283 (6), 269 (7), 141 (43), 140 (10), 139 (100), 111 (14), 91
(2), 77 (1).
7a-Acetoxy-6b-hydroxy-12-O-(4-nitrobenzoyl)royleanone (¼(6b,7a)-7-(Acetyloxy)-6-hydroxy-
11,14-dioxoabieta-8,12-dien-12-yl 4-Nitrobenzoate; 13). Yellow rectangular plates (AcOEt/pentane).
M.p. 217–2198. [a]D18 ¼ þ36.2 (c¼0.174, CHCl3). IR (KBr): 3452, 3109, 3056, 2956, 2933, 2867, 1752,
1733, 1668, 1608, 1530, 1462, 1369, 1347, 1320, 1252, 1218, 1140, 1101, 1070, 1011, 966, 932, 897, 869, 854,
712. 1H-NMR (400 MHz, CDCl3): 8.37 (d, J(2’,3’(6’,5’))¼9.0, HꢃC(2’), HꢃC(6’)); 8.32 (d,
J(3’,2’(5’,6’))¼9.0, HꢃC(3’), HꢃC(5’)); 5.68 (d, J(7b,6a)¼1.7, HbꢃC(7)); 4.34 (dd, J(6a,5a)¼1.9,
J(6a,7b)¼1.7, HaꢃC(6)); 3.18 (sept., J(15,16(17))¼7.1, H ꢃC(15)); 2.49 (m, HbꢃC(1)); 2.07 (s,
MeCOO); 1.81 (qt, J(2b,1a)¼J(2b,2a)¼J(2b,3a)¼13.6, J(2b,3b)¼3.4, J(2b,1b)¼3.2, HbꢃC(2)); 1.62
(s, Me(20)); 1.55 (dquint., J(2a,2b)¼13.6, J(2a,3b)¼3.8, J(2a,1a)¼J(2a,1b)¼J(2a,3a)¼3.7,
HaꢃC(2)); 1.46 (ddd, J(3b,3a)¼13.4, J(3b,2a)¼3.8, J(3b,2b)¼3.4, HbꢃC(3)); 1.37 (d, J(5a,6a)¼1.9,
HaꢃC(5)); ca. 1.23 (overlapped, HaꢃC(1)); ca. 1.22 (overlapped, Me(16), Me(17)); 1.22 (s, Me(19)); ca.
1.20 (overlapped, HaꢃC(3)); 0.95 (s, Me(18)). 13C-NMR (100 MHz, CDCl3): 185.5 (C(14)); 179.2
(C(11)); 169.6 (MeCOO); 164.6 (C6H4CO); 152.9 (C(9)); 151.2 (C(4’)); 149.7 (C(12)); 139.8 (C(13));
135.9 (C(8)); 133.4 (C(1’)); 131.6 (C(2’), C(6’)); 123.9 (C(3’), C(5’)); 68.8 (C(7)); 67.2 (C(6)); 49.8 (C(5));
42.2 (C(3)); 39.0 (C(10)); 38.4 (C(1)); 33.7 (Me(18)); 33.5 (C(4)); 25.3 (C(15)); 23.8 (Me(19)); 21.7
(Me(20)); 20.9 (MeCOO); 20.4 (Me(16), Me(17)); 18.9 (C(2)). EI-MS: 539 (1, Mþ ), 521 (6, [MꢃH2O]þ ),
506 (2, [MꢃH2OꢃMe]þ ), 497 (14), 479 (15, [MꢃAcOH]þ ), 450 (10), 329 (18), 283 (15), 150 (100,
[O2NꢃC6H4CO]þ ), 120 (63), 92 (13), 76 (8), 55 (7).
7a-Acetoxy-6b-(4-nitrobenzoyl)oxyroyleanone (¼(6b,7a)-7-(Acetyloxy)-12-hydroxy-11,14-dioxo-
abieta-8,12-dien-6-yl 4-Nitrobenzoate; 14). Red amorphous solid. [a]1D8 ¼ ꢃ42.5 (c¼0.113, CHCl3). IR
(KBr): 3383, 3109, 3080, 3056, 2962, 2931, 2867, 1753, 1732, 1643, 1609, 1531, 1462, 1374, 1347, 1270, 1216,
1168, 1146, 1098, 1016, 981, 873, 756, 719. 1H-NMR (400 MHz, CDCl3): 8.27 (d, J(2’,3’(6’,5’))¼9.0,
HꢃC(2’), HꢃC(6’)); 8.14 (d, J(3’,2’(5’,6’))¼9.0, HꢃC(3’), HꢃC(5’)); 7.19 (s, OH); 5.87 (d, J(7b,6a)¼
1.6, HbꢃC(7)); 5.78 (t, J(6a,5a)¼J(6a,7b)¼1.6, HaꢃC(6)); 3.15 (sept., J(15,16(17))¼7.0, HꢃC(15)),
2.75 (br. d, J(1b,1a)¼12.9, HbꢃC(1)); 2.09 (s, MeCOO); 1.84 (qt, J(2b,1a)¼J(2b,2a)¼J(2b,3a)¼14.0,
J(2b,1b)¼J(2b,3b)¼3.5, HbꢃC(2)); 1.73 (s, Me(20)); 1.70 (d, J(5a,6a)¼1.6, HaꢃC(5)); 1.64 (dquint.,
J(2a,2b)¼14.0, J(2a,1a)¼J(2a,1b)¼J(2a,3a)¼J(2a,3b)¼3.4, HaꢃC(2)); 1.52 (br. d, J(3b,3a)¼13.7,
HbꢃC(3)); 1.28 (overlapped, HaꢃC(3)); 1.25 (overlapped, HaꢃC(1)); 1.21 (d, J(16,15)¼7.0, Me(16));
1.17 (d, J(17,15)¼7.0, Me(17)); 1.05 (s, Me(19)); 0.98 (s, Me(18)). 13C-NMR (100 MHz, CDCl3): 185.2

930
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
(C(14)); 183.1 (C(11)); 168.1 (MeCOO); 163.6 (C6H4CO); 150.9 (C(4’)); 150.6 (C(12)); 149.2 (C(9));
136.7 (C(8)); 135.0 (C(1’)); 130.9 (C(2’), C(6’)); 125.0 (C(13)); 123.7 (C(3’), C(5’)); 69.4 (C(6)); 64.9
(C(7)); 49.2 (C(5)); 42.4 (C(3)); 38.5 (C(1)); 38.4 (C(10)); 33.7 (Me(18)); 33.3 (C(4)); 24.2 (C(15)); 23.3
(Me(19)); 22.0 (Me(20)); 20.8 (MeCOO); 19.8 (Me(16)); 19.6 (Me(17)); 18.9 (C(2)). EI-MS: 539 (0.5,
Mþ ), 497 (1, [MꢃCH2CO]þ ), 479 (7, [MꢃAcOH]þ ), 372 (1, [MꢃHOOCꢃPhꢃNO2]þ ), 330 (43,
[MꢃCH2COꢃHOOCꢃPhꢃNO2]þ ), 314 (100), 298 (46), 283 (20), 271 (23), 245 (68), 232 (84), 213
(25), 201 (18), 187 (24), 167 (54).
6b,7a-Diacetoxy-12-O-acetylroyleanone (¼(6b,7a)-11,14-Dioxoabieta-8,12-diene-6,7,12-triyl Triace-
1
tate; 15). M.p., [a]D, IR, and MS data identical to those reported in [15]. H-NMR (400 MHz, CDCl3):
5.68 (d, J(7b,6a)¼2.0, HbꢃC(7)); 5.48 (dd, J(6a,7b) ¼ 2.0, J(6a,5a)¼1.6, HaꢃC(6)); 3.09 (sept.,
J(15,16(17))¼7.0, HꢃC(15)); 2.52 (br. d, J(1b,1a)¼12.5, HbꢃC(1)); 2.34 (s, MeCOOꢃC(12)); 2.04 (s,
MeCOOꢃC(6)); 2.03 (s, MeCOOꢃC(7)); 1.78 (qt, J(2b,1a)¼J(2b,2a)¼J(2b,3a)¼14.0, J(2b,1b)¼
J(2b,3b)¼3.7, HbꢃC(2)); 1.59 (s, Me(20)); 1.56 (overlapped, HaꢃC(2)); 1.52 (d, J(5a,6a)¼1.6,
HaꢃC(5)); 1.45 (dddd, J(3b,3a)¼13.4, J(3b,2b)¼3.7, J(3b,2a)¼3.3, J(3b,1b)¼1.4, HbꢃC(3)); 1.24
(overlapped, HaꢃC(1)); 1.23 (overlapped, HaꢃC(3)); 1.18 (d, J(16,15)¼7.0, Me(16)); 1.17 (d, J(17,15)¼
7.0, Me(17)), 0.98 (s, Me(18)); 0.97 (s, Me(19)). 13C-NMR (100 MHz, CDCl3): 185.4 (C(14)); 179.7
(C(11)); 169.0 (MeCOOꢃC(6)); 168.3 (MeCOOꢃC(12)); 168.2 (MeCOOꢃC(7)); 152.2 (C(9)); 149.3
(C(12)); 139.4 (C(13)); 135.7 (C(8)); 67.2 (C(6)); 65.2 (C(7)); 49.0 (C(5)); 42.4 (C(3)); 38.9 (C(10)); 38.3
(C(1)); 33.6 (C(4)); 33.2 (Me(18)); 25.2 (C(15)); 22.9 (Me(19)); 21.4 (Me(20)); 21.3 (MeCOOꢃC(7));
20.8 (MeCOOꢃC(6)); 20.4 (MeCOOꢃC(7)); 20.2 (Me(16)); 20.2 (Me(17)); 18.8 (C(2)).
7a-Acetoxy-6b-(propanoyloxy)-12-O-propanoylroyleanone (¼(6b,7a)-7-(Acetyloxy)-11,14-dioxo-
abieta-8,12-diene-6,12-diyl Dipropanoate; 16). Yellow rectangular plates (AcOEt/pentane). M.p. 137–
1398. [a]1D8 ¼ þ29.9 (c¼0.257, CHCl3). IR (KBr): 2962, 2940, 2873, 1776, 1764, 1744, 1668, 1612, 1462,
1375, 1275, 1210, 1172, 1136, 1113, 1079, 1027, 931, 893, 804, 750. 1H-NMR (400 MHz, CDCl3): 5.68 (dd,
J(7b,6a)¼2.0, J(7b,5a)¼0.5, HbꢃC(7)); 5.49 (br. dd, J(6a,7b)¼2.0, J(6a,5a)¼1.6, HaꢃC(6)); 3.09
(sept., J(15,16(17))¼7.1, H ꢃC(15)); 2.66 (dq, J(2’A,2’B)¼17.2, J(2’A,3’)¼7.6, MeCHAHBCOOꢃC(12));
2.61 (dq, J(2’B,2’A)¼17.2, J(2’B,3’)¼7.6, MeCHAHBCOOꢃC(12)); 2.51 (br. d, J(1a,1b)¼12.4,
HbꢃC(1)); 2.32 (dq, J(2’A,2’B)¼16.8, J(2’A,3’)¼7.6, MeCHAHBCOOꢃC(6)); 2.25 (q, J(2’B,2’A)¼
16.8, J(2’B,3’)¼7.6, MeCHAHBCOOꢃC(6)); 2.04 (s, MeCOO); 1.77 (dddt, J(2b,2a)¼14.4, J(2b,1a)¼
J(2b,3a)¼13.8, J(2b,1b) ¼J(2b,3b)¼3.6, HbꢃC(2)); 1.59 (s, Me(20)); 1.55 (dquint., J(2a,2b)¼14.4,
J(2a,1a)¼J(2a,1b)¼J(2a,3a)¼J(2a,3b)¼3.6, HaꢃC(2)); 1.53 (dd, J(5a,6a)¼1.6, J(5a,7b)¼0.5,
HaꢃC(5)); 1.44 (dtd, J(3b,3a)¼13.8, J(3b,2a)¼J(3b,2b)¼3.6, J(1b,3b)¼1.6, HbꢃC(3)); 1.27 (t,
J(3’’,2’’A)¼J(3’’,2’’B)¼7.6, MeCH2COOꢃC(12)); 1.24 (overlapped, HaꢃC(1)); 1.23 (overlapped,
HaꢃC(3)); 1.17 (d, J(16,15)¼7.1, Me(16)); 1.16 (d, J(17,15)¼7.1, Me(17)); 1.11 (t, J(3’,2’A)¼
J(3’,2’B)¼7.6, MeCH2COOꢃC(6)); 0.98 (s, Me(18)); 0.96 (s, Me(19)). 13C-NMR (100 MHz, CDCl3):
185.4 (C(14)); 179.7 (C(11)); 172.5 (MeCH2COOꢃC(6)); 171.7 (MeCH2COOꢃC(12)); 168.1 (Me-
COO); 152.3 (C(9)); 149.4 (C(12)); 139.3 (C(13)); 135.6 (C(8)); 67.2 (C(6)); 65.3 (C(7)); 49.0 (C(5));
42.4 (C(3)); 38.8 (C(10)); 38.3 (C(1)); 33.6 (C(4)); 33.2 (Me(18)); 27.8 (MeCH2COOꢃC(6)); 27.2
(MeCH2COOꢃC(12)); 25.1 (HꢃC(15)); 23.0 (Me(19)); 21.5 (Me(20)); 20.7 (MeCOO); 20.2 (Me(16));
20.1 (Me(17)); 18.8 (C(2)), 8.9 (MeCH2COOꢃC(12)); 8.8 (MeCH2COOꢃC(6)). EI-MS: 502 (0.3, Mþ ),
460 (1, [MꢃCH2¼C¼O]þ ), 443 (2, [MꢃCMeOO]þ ), 428 (0.5, [MꢃCMeCH2OOH]þ ), 386 (34, [M ꢃ
CH2COꢃCMeH2CO2H]þ ), 330 (100), 312 (28), 302 (27), 269 (26), 57 (48).
6b,7a-Diacetoxyroyleanone (¼(6b,7a)-12-Hydroxy-11,14-dioxoabieta-8,12-diene-6,7-diyl Diacetate;
17). Yellow amorphous solid. [a]1D8 ¼ ꢃ10.7 (c¼0.056, CHCl3). IR and MS data identical to those
described in [17], whereas the 13C-NMR spectrum is also identical to that described in [17], but the
signals at d(C) 21.3 (Me) and 20.8 (Me) must be assigned to Me(20) and MeCOOꢃC(6), resp. 1H-NMR
(500 MHz, CDCl3): 7.17 (br. s, OH); 5.70 (d, J(7b,6a)¼1.7, HbꢃC(7)); 5.50 (dd, J(6a,7b) ¼ 1.7,
J(6a,5a)¼1.5, HaꢃC(6)); 3.16 (sept., J(15,16(17))¼7.1, H ꢃC(15)); 2.67 (dddd, J(1b,1a)¼12.9,
J(1b,2b)¼3.1, J(1b,2a)¼2.7, J(1b,3b)¼1.2, HbꢃC(1)); 2.04 (s, MeCOOꢃC(6)); 2.03 (s,
MeCOOꢃC(7)); 1.82 (qt, J(2b,1a)¼J(2b,2a)¼J(2b,3a)¼14.0, J(2b,1b)¼J(2b,3b)¼3.1, HbꢃC(2));
ca. 1.60 (overlapped, HaꢃC(2)); 1.59 (s, Me(20)); 1.56 (d, J(5a,6a)¼1.5, HaꢃC(5)); 1.49 (dt, J(3b,3a)¼
13.4, J(3b,2b)¼J(3b,2a)¼3.1, HbꢃC(3)); 1.24 (overlapped, HaꢃC(1)); 1.24 (overlapped, HaꢃC(3));
1.22 (d, J(16,15)¼7.1, Me(16)); 1.19 (d, J(17,15)¼7.1, Me(17)); 0.99 (s, Me(18)); 0.98 (s, Me(19)).

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
931
7a-Acetoxy-6b-propanoyloxyroyleanone (¼(6b,7a)-7-(Acetyloxy)-12-hydroxy-11,14-dioxoabieta-
18
8,12-dien-6-yl Propanoate; 18). Yellow amorphous solid. [a] ¼ ꢃ25 (c¼0.044, CHCl3). 1H-NMR
546
(400 MHz, CDCl3): 7.17 (br., HOꢃC(12)); 5.70 (br. d, J(7b,6a)¼2.0, J(7b,5a)<0.4, HbꢃC(7)); 5.51 (dd,
J(6a,7b)¼2.0, J(6a,5a)¼1.6, HaꢃC(6)); 3.16 (sept., J(15,16(17))¼7.1, H ꢃC(15)); 2.66 (dtd, J(1b,1a)¼
12.9, J(1b,2b)¼3.6, J(1b,2a)¼3.4, J(1b,3b)¼0.8, HbꢃC(1)); 2.29 (m, CH2(2’)), 2.04 (s, MeCOO); 1.82
(qt, J(2b,1a)¼J(2b,2a)¼J(2b,3a)¼14.0, J(2b,1b)¼J(2b,3b)¼3.6, HbꢃC(2)); 1.59 (s, Me(20)); 1.58
(overlapped m, HaꢃC(2)), 1.54 (br. d, J(5a,6a)¼1.6, J(5a,7b)<0.4, HaꢃC(5)); 1.48 (dtd, J(3b,3a)¼
13.6, J(3b,2a)¼J(3b,2b)¼3.6, J(3b,1b)¼0.8, HbꢃC(3)); 1.22 (overlapped, HaꢃC(1), HaꢃC(3)); 1.22
(d, J(16,15)¼7.1, Me(16)); 1.19 (d, J(17,15)¼7.1, Me(17)); 1.12 (t, J(3’,2’A)¼J(3’,2’B)¼7.6, MeCH2-
COO); 0.99 (s, Me(18)); 0.97 (s, Me(19)). 13C-NMR (100 MHz, CDCl3): 185.4 (C(14)); 183.2 (C(11);
172.5 (MeCH2COO); 168.0 (MeCOO); 150.8 (C(l2)); 149.3 (C(9)); 137.1 (C(8)); 124.8 (C(13)); 67.1
(C(6)); 65.2 (C(7)); 49.0 (C(5)); 42.4 (C(3)); 38.7 (C(10)); 38.4 (C(1)); 33.6 (C(4)); 33.2 (Me(18)); 27.8
(MeCH2COO); 24.2 (C(15)); 23.0 (Me(19)); 21.4 (Me(20)); 20.8 (MeCOO); 19.8 (Me(16)); 19.7
(Me(17)); 18.9 (C(2)); 8.9 (MeCH2COO). EI-MS: 446 (0.4, Mþ ), 404 (1, [MꢃCH2CO]þ ), 386 (4, [Mꢃ
AcOH]þ ), 372 (2, [MꢃMeCH2CO2H]þ ), 348 (4), 330 (100, [MꢃMeCH2CO2HꢃCH2CO]þ ), 315 (20),
298 (12), 287 (10), 261 (21), 260 (15), 248 (20), 245 (13), 232 (12), 57 (18, [C2H5C¼O]þ ).
7a-Acetoxy-6b-butanoyloxyroyleanone (¼(6b,7a)-7-(Acetyloxy)-12-hydroxy-11,14-dioxoabieta-
8,12-dien-6-yl Butanoate; 19). Amorphous solid. [a]1D8 ¼ þ12.1 (c¼0.19, CHCl3). IR (KBr): 3378,
2962, 2931, 2873, 1755, 1659, 1638, 1612, 1462, 1374, 1282, 1220, 1168, 1144, 1103, 1030, 959, 900, 756.
1H-NMR (400 MHz, CDCl3): 7.18 (br. s, OH); 5.69 (dd, J(7b,6a)¼2.1, J(7b,5a)¼0.6, HbꢃC(7)); 5.50 (t,
J(6a,7b)¼J(6a,5a)¼2.1, HaꢃC(6)); 3.16 (sept., J(15,16(17))¼7.1, H ꢃC(15)); 2.66 (ddd, J(1b,1a)¼
13.0, J(1b,2a)¼3.3, J(1b,2b)¼2.9, HbꢃC(1)); 2.28 (dt, J(2’A,2’B)¼15.9, J(2’A,3’)¼7.1, MeCH2CHAHB-
COO); 2.20 (dt, J(2’B,2’A)¼15.9, J(2’B,3’)¼8.0, MeCH2CHAHBCOO); 2.04 (s, MeCOO); 1.82 (qdd,
J(2b,1a) ¼J(2b,2a)¼J(2b,3a) ¼ 13.8, J(2b,1b)¼J(2b,3b)¼2.9, HbꢃC(2)); 1.63 (overlapped,
MeCH2CH2COO); 1.60 (overlapped, HaꢃC(5)); 1.59 (s, Me(20)); 1.58 (overlapped, HaꢃC(2)); 1.47
(ddd, J(3b,3a)¼13.0, J(3b,2a)¼3.6, J(3b,2b) ¼2.9, HbꢃC(3)); 1.23 (overlapped, HaꢃC(1), HaꢃC(3));
1.22 (d, J(16,15)¼7.1, Me(16)); 1.19 (d, J(17,15)¼7.1, Me(17)); 0.99 (s, Me(18)); 0.98 (s, Me(19)); 0.91 (t,
J(4’,3’)¼7.4, MeCH2CH2COO). 13C-NMR (100 MHz, CDCl3): 185.3 (C(14)); 183.2 (C(11)); 171.7
(MeCH2CH2COO); 168.1 (MeCOO); 150.8 (C(12); 149.3 (C(9)); 137.1 (C(8)); 124.8 (C(13)); 67.0
(C(6)); 65.2 (C(7)); 49.0 (C(5)); 42.4 (C(3)); 38.7 (C(10)), 38.4 (C(1)), 36.4 (MeCH2CH2COO), 33.6
(C(4)); 33.2 (Me(18)); 24.1 (C(15)); 23.0 (Me(19)); 21.3 (Me(20)); 20.8 (MeCOO); 19.8 (Me(16)); 19.7
(Me(17)); 18.9 (C(2)); 18.1 (MeCH2CH2COO); 13.7 (MeCH2CH2COO). EI-MS: 460 (0.3, Mþ ) 418 (1,
[MꢃCH2¼C¼O]þ ), 401 (5, [MꢃAcO]þ ), 348 (5), 330 (100, [MꢃAcOꢃMeCH2CH2CO]þ ), 315 (24,
[MꢃAcOꢃMeCH2CH2COꢃMe]þ ), 297 (11), 287 (12), 261 (26), 248 (24), 232 (14), 217 (7), 201 (8),
187 (6), 83 (8), 71 (13), 55 (5).
Antimycobacterial Activity in vitro. Bioassays were conducted on sensitive H37Rv ATCC 27294
(American Type Culture Collection) and multidrug-resistant (clinical isolate, strain 02TBDM039EP097)
Mycobacterium tuberculosis strains. For the preparation of the inoculums, a suspension of MTB was
prepared by mixing growth from slants (20–30-d-old) with 100 ml of Tween 80 into 0.2% (BSA; Sigma
Chemical Co., St. Louis, MO). Turbidity of the suspension was then adjusted to a McFarland standard
No. 1 (3ꢄ107 CFU/ml) by adding Tween 80 and BSA. The bacterial suspension (300 ml) was further
transferred to 7.2 ml of 7 H9GC broth (4.7 g of Middlebrook 7 H9 broth base (Difco, Detroit, MI), 20 ml
of 10% glycerol, 1 g of Bacto Casitone (Difco), 880 ml of distilled H2O, 100 ml of OADC (oleic acid,
albumin, dextrose, catalase; Remel, Lenexa, KS)). For the bioassay, the compounds were resuspended in
DMSO at a concentration of 1 mg/ml (stock soln.). These stock solns. were further diluted with
appropriate volumes of 7 H9GC broth to yield final concentrations of 0.1 to 50 mg/ml. Final drug
concentration ranges of standard antibiotics used as positive controls were 0.125 to 32 mg/ml for isoniazid
and 0.063 to 16 mg/ml for rifampicin (Sigma Chemical Co., St. Louis, MO). The standard drugs or
compounds (100 ml) were mixed in the wells with 100 ml of bacterial inoculum, resulting in a final
bacterial concentration of ca. 1.2 ꢄ106 CFU/ml. Solvent (DMSO) was included in every experiment as a
negative control. The plates were sealed in plastic bags and then incubated at 378 for 5 d. On day 5, 50 ml
of the MTT/Tween 80 mixture (1.5 ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide; Aldrich Chemical Co., Milwaukee, WI) at a dilution of 1 mg/ml in absolute EtOH and 1.5 ml of

932
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
10% Tween 80) was added to the wells, and the plate was incubated at 378 for 24 h. After this incubation
period, the growth of the microorganism was visualized by the change in color of the dye from yellow to
purple. The tests were carried out in triplicate. MIC Value is defined as the lowest drug concentration that
prevents the aforementioned change in color.
Cell Growth-Inhibition Assay. The percent inhibition of cell growth relative to the negative control
(solvent) was evaluated colorimetrically using a sulforhodamine B dye, according to a published
procedure [19], by comparison to the control. The GI50 value was defined as the concentration of test
sample resulting in a 50% reduction of absorbance as compared with untreated controls that received a
serial dilution of the solvent in which the test samples were dissolved, and was determined by linear
regression analysis.
REFERENCES
[1] Y. L. Janin, Bioorg. Med. Chem. 2007, 15, 2479.
[2] L. J. McGaw, N. Lall, J. J. M. Meyer, J. N. Eloff, J. Ethnopharmacol. 2008, 119, 482.
[3] B. R. Copp, A. N. Pearce, Nat. Prod. Rep. 2007, 24, 278.
[4] C. W. Lukhoba, M. S. J. Simmonds, A. J. Paton, J. Ethnopharmacol. 2006, 103, 1.
˜
[5] C. Gaspar-Marques, M. F. Simoes, M. L. Valdeira, B. Rodrꢀguez, Nat. Prod. Res. 2008, 22, 167.
´
´
[6] M. Martꢀnez-Vazquez, P. Miranda, N. A. Valencia, M. L. Torres, R. Miranda, J. Cardenas, M.
´
Salmon, Pharm. Biol. 1998, 36, 77.
[7] A. Ulubelen, G. Topcu, C. B. Johansson, J. Nat. Prod. 1997, 60, 1275.
˜
[8] C. Gaspar-Marques, P. Rijo, M. F. Simoes, M. A. Duarte, B. Rodriguez, Phytomedicine 2006, 13, 267.
[9] A. Mahapatra, S. P. N. Mativandlela, B. Binneman, P. B. Fourie, C. J. Hamilton, J. J. M. Meyer, F.
van der Kooy, P. Houghton, N. Lall, Bioorg. Med. Chem. 2007, 15, 7638.
[10] D. Tas¸demir, R. Brun, V. Yardley, S. G. Franzblau, P. Rꢂedi, Chem. Biodiversity 2006, 3, 1230.
˜
[11] C. Gaspar-Marques, M. F. Simoes, B. Rodrꢀguez, J. Nat. Prod. 2004, 67, 614.
˜
[12] P. Rijo, C. Gaspar-Marques, M. F. Simoes, M. L. Jimeno, B. Rodrꢀguez, Biochem. Syst. Ecol. 2007, 35,
215.
˜
[13] A. P. Teixeira, O. Batista, M. F. Simoes, J. Nascimento, A. Duarte, M. C. de la Torre, B. Rodrꢀguez,
Phytochemistry 1997, 44, 325.
[14] I. V. Tetko, J. Gasteiger, R. Todeschini, A. Mauri, D. Livingstone, P. Ertl, V. A. Palyulin, E. V.
Radchenko, N. S. Zefirov, A. S. Makarenko, V. Y. Tanchuk, V. V. Prokopenko, J. Comput.-Aided
Mol. Des. 2005, 19, 453; VCCLAB, Virtual Computational Chemistry Laboratory, http://
www.vcclab.org.
[15] M. Hensch, P. Rꢂedi, C. H. Eugster, Helv. Chim. Acta 1975, 58, 1921.
[16] B. Rodrꢀguez, Magn. Reson. Chem. 2003, 41, 741.
[17] C.-I. Chang, M.-H. Tseng, Y.-H. Kuo, Chem. Pharm. Bull. 2005, 53, 286.
[18] Tuberculosis Drug Screening Program, ꢄSearch for new drugs for treatment of Tuberculosisꢃ,
Antimicrob. Agents Chemother. 2001, 45, 1943.
[19] J. C. Aponte, A. J. Vaisberg, R. Rojas, L. Caviedes, W. H. Lewis, G. Lamas, C. Sarasara, R. H.
Gilman, G. B. Hammond, J. Nat. Prod. 2008, 71, 102.
[20] A. Mital, V. S. Negi, U. Ramachandran, Med. Chem. 2008, 4, 492.
Received March 27, 2009