Antitubercular activity increase in labdane diterpenes from Copaifera oleoresin through structural modification

Article abstract of DOI:10.21577/0103-5053.20160268

The labdane diterpenes copalic acid, 3β-acetoxy-copalic acid, 3β-hydroxy-copalic acid and ent-agathic acid were isolated from Copaifera langsdorffii oleoresin. These four compounds were submitted to structural modifications by reduction with hydrogen/palladium, esterification with diazomethane, esterification with methanol/sulfuric acid and conversion into sodium salt, furnishing 15 compounds. All compounds were assayed in vitro against Mycobacterium tuberculosis (H37Rv, ATCC 27294). The four compounds displayed minimum inhibitory concentration (MIC) value of 125 μg mL^-1, and were not considered active. A methylated derivative of compound 3β-hydroxy-copalic acid, and a sodium salt of copalic acid displayed MIC values of 25 μg mL^-1 (71.7 μM) and 6.25 μg mL^-1 (19.2 μM), respectively. The sodium salt of copalic acid stood out by displaying similar activity in comparison with streptomycin (MIC 6.25 μg mL^-1) and a better activity compared to μM value of pyrazinamide (MIC 3.12 μg mL^-1; 25.34 μM). Therefore, the methylated derivative of compound 3β-hydroxy-copalic acid and the sodium salt of copalic acid should be considered for further studies.

Full text of DOI:10.21577/0103-5053.20160268

http://dx.doi.org/10.21577/0103-5053.20160268
J. Braz. Chem. Soc., Vol. 28, No. 6, 1106-1112, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
Antitubercular Activity Increase in Labdane Diterpenes from Copaifera Oleoresin
through Structural Modification
Aline N. Silva,a Ana Carolina F. Soares,a Mirela M. W. Cabral,a Alex R. P. de Andrade,a
Marilza B. M. da Silva,a Carlos H. G. Martins,a Rodrigo C. S. Veneziani,a
Sérgio R. Ambrósio,a Jairo K. Bastosb and Vladimir C. G. Heleno*,a
aNúcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca,
14404-600 Franca-SP, Brazil
bFaculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,
14040-903 Ribeirão Preto-SP, Brazil
The labdane diterpenes copalic acid, 3β-acetoxy-copalic acid, 3β-hydroxy-copalic acid and
ent-agathic acid were isolated from Copaifera langsdorffii oleoresin. These four compounds
were submitted to structural modifications by reduction with hydrogen/palladium, esterification
with diazomethane, esterification with methanol/sulfuric acid and conversion into sodium salt,
furnishing 15 compounds.All compounds were assayed in vitro against Mycobacterium tuberculosis
(H37Rv, ATCC 27294). The four compounds displayed minimum inhibitory concentration (MIC)
value of 125 µg mL-1, and were not considered active. A methylated derivative of compound
3β-hydroxy-copalic acid, and a sodium salt of copalic acid displayed MIC values of 25 µg mL-1
(71.7 µM) and 6.25 µg mL-1 (19.2 µM), respectively. The sodium salt of copalic acid stood out
by displaying similar activity in comparison with streptomycin (MIC 6.25 µg mL-1) and a better
activity compared to µM value of pyrazinamide (MIC 3.12 µg mL-1; 25.34 µM). Therefore, the
methylated derivative of compound 3β-hydroxy-copalic acid and the sodium salt of copalic acid
should be considered for further studies.
Keywords: Copaifera oilresin, labdane diterpenes, structural modification, antitubercular
activity, Mycobacterium tuberculosis
Introduction
leading to a ratio of mortality above 39%. If we further
consider that one third of the world population is infected
with the tubercle bacillus,4 this gives us the dimension of
this public health problem.
Although preventable,5 TB causes substantial morbidity,
being one of the major causes of infection morbidity,6
that leads to human suffering and present a negative
socioeconomic impact.7 Prevention is carried out mostly by
the use of BCG (Bacille Calmette-Guérin) vaccine, the only
one used to prevent TB.6 Nevertheless, it is not intensively
used in several countries.6
Thus, it is clearly noted that TB is a serious world health
problem that requires some serious measures.A promising
way to do that is the search for novel therapeutics, mainly
for drug resistant strains of TB.8 This can be provided by
the search for new drugs to fight this disease,5 and the use
of natural product is certainly an interesting option.9
The complex mixture which presents diverse
composition, known as copaiba resin-oil, is widely used
Tuberculosis (TB) is a severe respiratory disease
which causes deaths counted in millions every year.1,2 This
contagious and airborne disease is caused by mycobacteria
from the Mycobacterium tuberculosis complex and,1,2 since
it is transmitted through air, it is certainly hard to control.3
Most active TB cases (75%) are pulmonary, but one fourth
of them affect other systems.3 The burden of TB involves
estimated 9.6 million people infected and 1.5 million dead
in 2014. Considering death from a single infectious agent,
only human immunodeficiency virus (HIV) kills more
than TB.2
It is evaluated by the World Health Organization
(WHO)2 that 480 thousand people developed multidrug-
resistant TB (MDR-TB) in 2014. Moreover, 190 thousand
deaths from MDR-TB were estimated for the same year,
*e-mail: vladimir.heleno@unifran.edu.br

Vol. 28, No. 6, 2017
Silva et al.
1107
and applied in research works dealing with its reported
biological activities.10-13 The trees which exude those resin-
oils are from the Copaifera genus, which includes several
species.14,15 Those medicinal plants are also commonly used
in Brazilian folk medicine due to their pharmacological
applications proved by popular use.13,14 These oils may
present diverse composition depending on their botanical
source. This turns them an interesting source of natural
products to be isolated and assayed against different
pathological agents.16-18
Figure 1. Structures of the natural diterpenes, precursors in this work.
Synthetic structural modifications
Terpenoids are one of the largest classes of natural
products,12 and some of them are widely used as starting
materials in natural products synthesis.16 Some diterpenes
displayed a remarkable antitubercular activity against
M. tuberculosis, despite the fact that only a limited number
of them were assayed against this pathogenic agent.4
Moreover, according to Rawat and Rawat,3 there are some
cases where the semi-synthetic diterpenes were more active
than the natural precursor and some of them with high
activity against M. tuberculosis.3
As presented in most of the papers dealing with
biological activities, one important goal is to reach
a considerable number of structures to be assayed.
Commonly, a slight difference in the compound
structure leads to a considerable difference in biological
activity,4 which makes structural modification a
promising method to achieve more active and less toxic
compounds.
Structural modifications were performed by submitting
the isolated compounds to four different protocols:
reduction with hydrogen/palladium, esterification with
diazomethane, esterification with methanol/sulfuric acid
and conversion into sodium salt. Scheme 1 summarizes
these transformations.
Catalytic hydrogenation of 1 to 4 produced compounds 5
to 8 in good yields. Both double bonds present in the
starting compounds were reduced. The reduction of the
terminal methylene (C8-C17) probably occur with high
stereoselectivity: only the α-methyl products may be
obtained. This is what should be expected due to the presence
of the bulky α-substituent in C9 and due to the relatively huge
size of Pd particle, allowing the catalyst approach to occur
only in the other surface of the double bond.
Treatment of 1 to 4 with diazomethane resulted in
compounds 9, 10, 13 and 14. As expected, all carboxylic
groups were converted into the corresponding methyl
esters. However, in compound 3, it also took place
a less usual conversion of the alcoholic OH group
into the corresponding methyl ether, thus resulting in
product 14.
Considering the potential of this class of compounds
and our research interests in terpenoids,19-21 we decided to
submit the previously isolated labdane diterpenes (1-4)
to structural modifications searching for more active
compounds against M. tuberculosis.
The esterification with methanol/sulfuric acid is
expected to produce essentially the same products obtained
with diazomethane but, as it has been noted previously,21
some minor differences may occur resulting in additional
derivatives. In fact, while starting compounds 1 and 2
led to the same products as the obtained by reacting with
diazomethane, namely compounds 9 and 10, compounds 3
and 4 were converted into new derivatives 11 and 12,
respectively. The alcoholic OH group of compound 3 was
not transformed into methyl ether and, for compound 4,
the neopentylic carboxyl group at C4 was not converted
into methyl ester.
Compound 15 was obtained by the conversion of the
acid diterpene 1 into a sodium salt. This was initially
undertaken according to a procedure described in the
literature.25 Nevertheless, it was verified that this method
was not totally effective for our substrates. The products
were either afforded in extremely low yielding, or could
Results and Discussion
Isolation of natural diterpenes
The oleoresin sample was purchased from “Apis-Flora
Comercial e Industrial”, a Brazilian herbal company
located in the city of Ribeirão Preto, state of São Paulo,
under the register: lot 0790310, manufactured 09/2010.
The natural diterpenes copalic acid (1), 3β-acetoxy-
copalic acid (2), 3β-hydroxy-copalic acid (3) and
ent-agathic acid (4) were isolated as described earlier by
our research group.18 The isolated diterpenes 1 to 4 had
their nuclear magnetic resonance (NMR) data compared
to literature.22-24 Purity of the isolated compounds was
estimated to be between 95-98% by NMR. All structures
of natural and semi-synthetic diterpenes of this work are
shown in Figure 1 and Scheme 1.

1108
Antitubercular Activity Increase in Labdane Diterpenes from Copaifera Oleoresin
J. Braz. Chem. Soc.
Scheme 1. Summary scheme of the structural modifications undertaken in this work.
not even be isolated from reaction mixtures. This made
Initially, the results obtained for the hydrogenation
clear the need of a set of test reactions aiming to adapt
conditions for better yields. Due to the availability of the
natural compounds, the tests could be performed only
with copalic acid (1), which is the major component of the
oleoresin. The new procedure for 1 afforded product 15
in 55% yield.
derivatives, compounds 5 to 8, will be considered. Despite
the fact that compounds 6 and 7 showed the same activity
as the natural compounds (inactive), compound 8 became
Table 1. Antitubercular activity results for all compounds and first line
drugs
Purities of the semi-synthetic compounds were
estimated to be between 95 and 97% by NMR.
Compound
Precursor
MIC / (µg mL-1)
125
MIC / µMb
410.6
344.8
390.1
373.8
202.6
341.0
385.2
738.6
392.5
332.0
373.7
358.7
344.8
71.7
1
−
−
−
−
1
2
3
4
1
2
3
4
4
3
1
−
−
−
−
2
125
Anti-tubercular activity
3
125
4
125
The activities of all compounds were established by
assayingthemagainstthecelllineMycobacteriumtuberculosis
H37Rv, ATCC (American Type Culture Collection) 27294,
as described in the Experimental section. Results are given
as minimum inhibitory concentration (MIC) in µg mL-1.
Positive control for those experiments was the widely used
anti-tubercular drug isoniazid. This drug is recommended
and considered essential by the World Health Organization
(WHO).26 Table 1 summarizes the obtained results and,
moreover, presents the activity published in the literature
of three other first line drugs in use for TB: ethambutol,
pyrazinamide and streptomycin.
As seen on Table 1, none of the four isolated compounds
presented activity against M. tuberculosis. They were all
considered inactive, presenting MIC of 125 µg mL-1. This
makes the structural modifications a search for active
compounds.
5
62.5
125
6
7
125
8
250
9
125
10
125
11
125
12
125
13
125
14
25
15
6.25
1.64
3.12
6.25
0.06
19.2
Ethambutol27 a
Pyrazinamide28
Streptomycin28
Isoniazid
7.22
25.34
10.75
0.44
aEthambutol dihydrochloride (EMB.2HCl); bcalculated values in
micromolar. MIC: minimum inhibitory concentration.

Vol. 28, No. 6, 2017
Silva et al.
1109
even less effective and compound 5 became more active,
showing that some slight difference in structure is capable
to change activity. The activity displayed by compound 5
can be considered a moderate activity. First line drugs for
TB are, at least, ten times more active.
Despite being widely used, µg mL-1 is not the only unit to
express MIC values. The concentration in µM (micromolar)
is also an important parameter, or even a better one, as it takes
into account the molar quantity of the active substance. If
a comparison of the µM values is undertaken, compound 5
becomes relatively more active. The first line anti-tubercular
drug pyrazinamide presents, in this new comparison, only
eight fold the activity of the modified diterpene.
In all cases of hydrogenation, the only improvement
of activity achieved was due to the hydrogenated product
compound 5, which bears only one functional group, as
seen on Scheme 1. On the other hand, in all other cases,
there are two functional groups in each molecule, as also
seen on Scheme 1 for compounds 6 to 8. Therefore, despite
the reduction of the double bonds in all cases, it seems
that the presence of two functional groups in the molecule
hampers the increase of activity. If both functional groups
are carboxylic acid, as seen in compound 8, the activity
becomes even worse with the hydrogenation.
The methyl esters prepared from the natural compounds
by reaction with diazomethane or by the methanol-sulfuric
acid condition were also assayed against M. tuberculosis.
Due to the similarity of the functional groups present in
the derivatives 9-14, their biological activity results will be
discussed as a single group.
acetoxyl groups in position 3 enhanced the activity,
regardless the presence of another functional group in
the molecule.
Finally, the evaluation of our sodium salt derivative (15)
led to the most promising result in this work. Compound 15
reached a MIC value of 6.25 µg mL-1, which is similar to
streptomycin. Moreover, this derivative was more active
(1.32 times) than pyrazinamide when considering µM
values.
This gives rise to some future considerations in the
search for active compounds against M. tuberculosis,
especially among diterpenes. The activity of the salt
indicates a possibility of water soluble compounds to be
interesting for this kind of activity. Probably, the increased
solubility of this compound allows this diterpene skeleton
to reach some regions in the mycobacterial structure that is
not achieved by the other less soluble derivatives, leading
to this expressive activity.
Actually, only two kinds of structural feature showed
to be relevant to anti-tubercular activity for this type of
skeleton in this work: the presence of a methoxyl group in
position 3; and a sodium salt prepared from the carboxylic
acid, yielding a considerably more soluble structure.
This can give rise for future works in the search for
anti-tubercular agents.
Conclusions
The search for active compounds against M.
tuberculosis, starting from natural labdane diterpenes,
was successful. Simple structural modifications led to
eleven derivatives, three of them were more active than
the natural products. The best derivative displayed an
activity comparable to two of the first line drugs for
TB treatment. Due to the relevance of the results here
presented, the natural diterpenes can be considered as
promising for further investigations of the antitubercular
activity, both as potential natural source or as prototypes
for new active compounds.
Five from six derivatives obtained, named compounds 9
to 13, displayed the same low activity as the natural
compounds, thus being considered inactive. On the other
hand, compound 14 reached an activity that could be
considered significant, displaying a MIC of 25 µg mL-1.
The activity of streptomycin, one of the first line drugs,
is only four times more active than this. Nevertheless, the
comparison of µM activity value for this compound with the
value for pyrazinamide, shows that the commercial drug is
less than three times more active than compound 14. Thus,
a considerable activity was reached with this compound
and it should be further investigated.
Experimental
Comparing structures, one can roughly say that methyl
esters cannot display better activities than the carboxylic
acids against M. tuberculosis in those structures.
Perhaps it can be further concluded, in a comparison
of compounds 1-4 with compounds 9-13, that it makes
no difference to have either methyl ester or carboxylic
acid for this kind of activity. Nevertheless, compound 14
shows that a methoxyl group in position 3 can be crucial
to express a much better activity. Neither hydroxyl nor
Isolation of compounds
The four natural diterpenes (1-4) were isolated from
a sample of Copaifera langsdorffii oleoresin as described
previously by our research group.18 A sample of 20.0 g
of the oilresin furnishes 450.0 mg of 1, 230.0 mg of 2,
130.0 mg of 3 and 150.0 mg of 4. The oleoresin was
purchased fromApis Flora Company in Ribeirão Preto-SP,
Brazil, lot 0790310.

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Antitubercular Activity Increase in Labdane Diterpenes from Copaifera Oleoresin
J. Braz. Chem. Soc.
General methods for compounds analysis
procedures. In all cases, chromatographic methods, mainly
open classic column chromatography, were applied to
purify the obtained derivatives.
NMR spectra were recorded on a Bruker Avance DRX
400 spectrometer (400 MHz for 1H and 100 MHz for 13C).
Samples were dissolved in CDCl3 or methanol-d4, always in
the concentration range from 8 to 16 mg mL-1. All samples
were analyzed at 298 K and TMS (tetramethylsilane) was
used as internal reference.
Hydrogenation
The natural diterpene (0.05 g) was initially dissolved
in 20 mL of absolute ethanol and then, a catalytic portion
of Pd/C was added. The reactional mixture was placed in
a pressure vessel and kept under stirring in a hydrogen
atmosphere of 2 atm, at room temperature, for two hours.
After this, the mixture was filtered in Celite© and the solvent
was removed under reduced pressure. The products were
all obtained as light yellow oils.
Spectral identification of obtained compounds
The natural compounds were fully identified by NMR
spectral data, mainly by comparison to previously obtained
data.18 The characterization of obtained compounds was
1
easily undertaken with comparison of both H NMR
Esterification with diazomethane
data, product and starting material. For the hydrogenation
products, all signals are practically the same, except initially
for the ones peculiarly related to olefinic hydrogens, which
were all absent, confirming the hydrogenation of both
double bonds in all cases. An extra signal for a methyl
group was always noted in the products spectra, together
with a shielding effect of the CH3 in position 16, endorsing
the structures of compounds 5-8. For the other derivatives,
the structural determination was even easier, where the only
difference is the presence of methoxyl groups’ signals.
Once the carboxylic group’s hydrogen was not observed
for any starting compound, the new signals were easily
observed and led surely to the determination of the chemical
structures of compounds 9-14.
In a round bottom 125 mL flask, 2.4 g of diazogen
(N-methyl-N-nitroso-p-toluenesulfonamide) were dissolved
in anhydrous ethylic ether. The flask was cooled to 0 °C and
a solution of 0.4 g of KOH in 10 mL of anhydrous ethylic
ether was added dropwise to the mixture.After the addition,
a distilling system was prepared with this flask and it was
heated to 100 °C. Produced diazomethane was distilled and
collected in an Erlenmeyer placed in an ice bath (0 °C) as
a solution in anhydrous ethylic ether. Immediately after its
preparation, the diazomethane solution was added to the
solution containing the acid diterpene, 50 mg in 10 mL of ethyl
ether, under stirring, until the gas (N2) formation was ceased.
The reaction mixture had the solvent removed under reduced
pressure. All products were obtained as light yellow oils.
The last structural confirmation needed was for
compound 15. The extreme similarity between this
product and its precursor (compound 1) could lead to the
most difficult clarification. Instead of that, the structural
confirmation was very clear. Most of the signals are exactly
the same in compound 15, compared to the same signals
in compound 1. The only difference, noted in the 1H NMR
spectrum, was the chemical shift of signals from H-12
(d 2.00 for 15 and 2.13 for 1) and CH3-16 (d 2.16 for 15
and 2.28 for 1). These results are expected, because the
carboxylic group induces a greater deshielding than
the sodium carboxylate group. Moreover, the obtained
product was not soluble in chloroform like compound 1.
Deuterated methanol was needed for NMR analysis of
the compound 15, corroborating its chemical structure, as
previously described.29
Esterification with methanol
In a round bottom 15 mL flask, a solution of the
diterpene (0.05 g) in methanol (5 mL) was prepared. Then,
4 drops of concentrated sulfuric acid were slowly added
and the final mixture was kept under stirring at room
temperature for 18 h. The reaction mixture was poured into
an extraction funnel containing 40 mL of water. The final
mixture was extracted with 3 portions (20 mL each) of ethyl
acetate. The organic layer was dried under MgSO4 and the
solvent was removed under reduced pressure.
Sodium salt formation
In a 25 mL Erlenmeyer flask, 0.05 g of copalic acid (1)
was dissolved in 5 mL of n-hexane. To this mixture, 2.5 mL
of a NaOH 0.5 M solution was added and the final mixture
was allowed to stir vigorously at room temperature for
3 minutes.After, the mixture was filtered in a cold Büchner
funnel and the retained solid was washed once with cold
methanol and once with cold water. The residue was left
to dry under 50 °C in filter paper, from which the product,
as a white solid, was then collected.
Structural modification experiments
All experiments are described only once for all
compounds, because the natural diterpene 1-4, used
as starting materials, were submitted to quite similar

Vol. 28, No. 6, 2017
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Antitubercular potential assays
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The obtained compounds were assayed against
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FAPESP has sponsored the publication of this article.