Novel betulin derivatives inhibit IFN-γ and modulates COX-2 expression

Article abstract of DOI:10.1080/14786419.2018.1528581

Betulin (BE) is a pentacyclic triterpenes, obtained from natural sources and with several biological activities described, such as anti-tumoral and anti-inflammatory activities. The BE esterification at hydroxyl group (C-3 and C-28) resulted in five new ester derivatives with different numbers of carbons or halogens (chlorine and fluorine). Among these BE derivatives, two (2a e 2c) were able to significantly decrease IFN-g (*p = 0.0391; **p = 0.0156) and 2c modulated the expression of COX-2 better than Dexamethasone (DEXA). Regarding to cytotoxic assay, the best results were obtained for BE without modifications, with emphasis on tumoral cell lines Raji and MCF-7. The derivatives 2a and 2c showed immunomodulation activity (for the cytokines IFN-g). The presence of chorine in BE seems to be important for the ability of modulate COX-2 expression, since the ester chloride derivative 2c at 100 μM is more powerful inhibitor of COX-2 than DEXA.

Full text of DOI:10.1080/14786419.2018.1528581

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Novel betulin derivatives inhibit IFN-γ and
modulates COX-2 expression
Sayonara Maria Calado Gonçalves, Glória Najara Silva, Ivan da Rocha Pitta,
Moacyr Jesus Barreto de Melo Rêgo, Simone Cristina Baggio Gnoato & Maira
Galdino da Rocha Pitta
To cite this article: Sayonara Maria Calado Gonçalves, Glória Najara Silva, Ivan da Rocha Pitta,
Moacyr Jesus Barreto de Melo Rêgo, Simone Cristina Baggio Gnoato & Maira Galdino da Rocha
Pitta (2018): Novel betulin derivatives inhibit IFN-γ and modulates COX-2 expression, Natural
Product Research, DOI: 10.1080/14786419.2018.1528581
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1528581
Novel betulin derivatives inhibit IFN-c and modulates
COX-2 expression
a
b
a
ꢀ
Sayonara Maria Calado Gonc¸alves , Gloria Najara Silva , Ivan da Rocha Pitta ,
a
b
^
Moacyr Jesus Barreto de Melo Rego , Simone Cristina Baggio Gnoato and
Maira Galdino da Rocha Pittaa
aLaboratory of Immunomodulation and New Therapeutic Approaches (LINAT), Research Center
Innovation Therapeutics Suely Galdino (NUPIT-SG), Federal University of Pernambuco, Recife, Brazil;
bOrganic Synthesis Laboratory of Phytochemistry, Federal University of Rio Grande do Sul, Porto
Alegre, Brazil
ABSTRACT
ARTICLE HISTORY
Received 2 May 2018
Accepted 22 September 2018
Betulin (BE) is a pentacyclic triterpenes, obtained from natural
sources and with several biological activities described, such as
anti-tumoral and anti-inflammatory activities. The BE esterification
at hydroxyl group (C-3 and C-28) resulted in five new ester deriva-
tives with different numbers of carbons or halogens (chlorine and
fluorine). Among these BE derivatives, two (2a e 2c) were able to
KEYWORDS
Betulin; derivatives;
cytokines; cyclooxygenase-2;
cytotoxicity
ꢀ
ꢀꢀ
p ¼ 0.0156) and 2c
significantly decrease IFN-g ( p ¼ 0.0391;
modulated the expression of COX-2 better than Dexamethasone
(DEXA). Regarding to cytotoxic assay, the best results were
obtained for BE without modifications, with emphasis on tumoral
cell lines Raji and MCF-7. The derivatives 2a and 2c showed
immunomodulation activity (for the cytokines IFN-g). The pres-
ence of chorine in BE seems to be important for the ability of
modulate COX-2 expression, since the ester chloride derivative 2c
at 100 lM is more powerful inhibitor of COX-2 than DEXA.
CONTACT Maira Galdino da Rocha Pitta
mgrpitta@gmail.com
Laboratory of Immunomodulation and New
^
Therapeutic Approaches (LINAT), Federal University of Pernambuco (UFPE), Av. Morais Rego s/n, Campus
ꢀ
Universitario, Recife, PE, 50670-901, Brazil.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
S. M. C. GONC¸ALVES ET AL.
1. Introduction
The compounds of natural origin have been widely studied and used in current ther-
apy due to their great chemical diversity in which can cover a wide range of diseases
(Yogeeswari and Sriram 2005; Yadav et al. 2010). Betulin (BE) (lup-20(29)-ene-3b,28-
diol) is a pentacyclic triterpene, found in abundance in nature. It can be extracted
from different plant sources, such as from the bark of Betula papyrifera (Betulaceae)
popularly known as white oak (white birch) and it can also be obtained by conversion
of betulinic acid BA (Laszczyk 2009). A wide spectrum of biological and pharmaco-
logical activities have been reported for BE and its derivatives, including antitumor,
antibacterial, anti-inflammatory, immunomodulation and antiviral (HIV) activities
ꢀ
(Boryczka et al. 2013; Ding et al. 2013; Bebenek et al. 2015; Dra˛g-Zalesinska et al. 2015;
Laavola et al. 2016).
The literature shows some studies where modified triterpenes, as BE, have had their
action improved. Such as the study described by Zhao et al. (2016) that evaluated the
renal septic effect of BE in mice. They observed that BE inhibited the nuclear factor
kappa-B (NF-kB) signal, reduced serum creatinine and urea levels, and decreased secre-
tion of proinflammatory cytokines (TNF-c, IL-1b and IL-6) in septic mice. In the same
ꢀ
year, Dra˛g-Zalesinska et al. (2015) evaluated the cytotoxic properties of five new BE
derivatives against squamous cell carcinoma. They found that the highest cytotoxicity
activity of these derivatives occurred in the two compounds containing a side chain of
lysine (IC50 ¼ 7 mM) and ornithine (IC50 ¼ 10 mM).
In this sense, exploring the anti-inflamtory, immunomodulation and cytoxicity
activity of BE and its derivatives is potential way to obtain a new medicine. In add-
ition, the investigation of these activities together is based on Rudolf Virchow’s
research (1856), since he indicated that inflammation is closely linked to cancer, and
showed for the first time, the inflammation as a predisposing factor for several can-
cers. Nowadays, the data suggest that at least one in seven malignant tumors is a
result of inflammatory processes. Inflammatory cells such as macrophages and fibro-
blasts, transcription factors, enzymes such as cyclooxygenase-2 (COX-2) and cyto-
kines such as IFN-c, IL-6 and IL-17A are components present in the leucocyte
infiltrate of the tumor microenvironment. The COX-2, a central molecule of the
inflammatory process, is also involved in carcinogenesis (Xiong, Liu, and Yi 2014;
Yang et al. 2015; Khan 2015). Previous studies have shown that this enzyme is
found in high concentrations in several solid tumors, performing the function of
promoting angiogenesis, tumor invasion, metastasis, as well as resistance to apop-
tosis (Harizi 2015; Qu and Liu 2015). For instance, there are some selected forms of
chronic inflammation that predispose cancer, such as infection by Helicobacter pilori
(Porta et al. 2009).
In this context, this article evaluated the immunomodulation, anti-inflammatory
and cytotoxicity activity of the five new semisynthetic BE derivatives. In this purpose,
we assessed the ability of these novel derivatives in modulating inflammatory cyto-
kines IFN-c, IL-6 and IL-17A and inhibiting COX-2 expression. After, we evaluated the
cytotoxicity of the novel derivatives in four tumoral cell lines and one non-
tumoral cell.

NATURAL PRODUCT RESEARCH
3
2. Results and discussion
This article reports for the first time the synthesis of five new derivatives BE with sub-
stituents in C-3 and C-28. In this present research, we evaluated the immunomodula-
tion, anti-inflammatory and cytotoxicity activities of BE and its derivatives. We sought
to evaluate the immunomodulation activity by using cytokines IFN-c, IL-6 and IL-17A
and anti-inflammatory potential through the inhibition of COX-2. We also analyzed its
anti-tumoral activity against the Hep-G2 (well-differentiated hepatocellular carcinomas),
Raji (Burkitts lymphoma), MCF-7 (breast cancer) and DU-145 (Prostate cancer).
Previous researches have showed the immunomodulation and anti-inflamatory
activities for BE and derivativies, such as the formyl and pyrazol groups added to BE
generating derivatives capable of reducing the expression of IL-6 and COX-2 enzyme
in a significant manner (Laavola et al. 2016). However, it is only one of the few studies
in the literature that have evaluated the anti-inflammatory effect of BE and its deriva-
tives, as also few studies that related BE derivatives with modulating activity of some
cytokines (Bernard et al. 2001; Alakurtti et al. 2006; Zhang et al. 2015; Laavola et al.
2016; Zhao et al. 2016). None studies were found in the literature that evaluated the
modulating ability of the IFN-c and IL-17A cytokines by BE and its derivatives.
Therefore, our starting point was to evaluate the ability of the BE and its new deriva-
tives in reducing the production of pro-inflammatory cytokines IFN-c, IL-6 and IL-17A.
According to our results, the changes performedmade to obtain derivatives 2a and
2c made them able to significantly decrease IFN-c and 2c showed anti-inflammatory
activity better than its precursor, BE, and also DEXA (Figure S11). It seems that the
presence of substituents O-acetyl (2a) and the O-dichloroacyl (2c) at C-3 and C-28
positions make BE able to reduce the production of IFN-c and COX-2. For the mol-
ecule inflammatory COX-2 assay, we added BE at concentrations of 1, 10 and 100 lM
to compare the activity of derivatives with its precursor. Since the derivative 2c at con-
centration of 100 lM was a better modulator of COX-2 than its precursor, BE and the
drug already used in clinical practice, DEXA, it indicated that the presence of chorine
is important to increase the anti-inflammatory activity of the BE (Figure S12). There is
no previous studies about synthesized derivatives from BE using these substituents,
which makes the synthesis and activities of our novel derivatives unprecedented.
After evaluating the immunomodulation and anti-inflammatory activities of the
novel BE derivatives, we decided to investigate the cytotoxic action of these deriva-
tives facing tumor cell lines. Studies have shown that the synthesis of derivatives from
BE precursor has generated more cytotoxic and selective compounds for neoplastic
cells (Grivennikov, Greten and Karin 2010; Csuk et al. 2010; Urban et al. 2012; Siewert
et al. 2014).
Our results of the cytotoxicity assay to BE derivatives showed the best result to 2d,
that was more activity against Raji and Hep-G2 strains, specially on Hep-G2
(IC50 ¼ 60.47 mM) and all derivatives were not cytotoxic to the PBMC, non-tumoral cell.
BE without modification was more activity than its derivatives, especially on tumoral
cells Raji and MCF-7 (IC50 ¼ 45 lM). Doxorubicin, a drug used in clinical practice, was
used as a negative control (Table S1). Since the modifications of BE did not improve
the activity on tumor cells, new modifications should be proposed in order to reach
this aim.

4
S. M. C. GONC¸ALVES ET AL.
Figure 1. Chemical structure of Betulin (BE) and substituents that gave the derivatives.
3. Experimental
3.1. Semisynthesis of the BE derivatives
BE was purchased from Sigma – Aldrich (Missouri, EUA). For obtaining of the new BE
derivatives, it was followed the same reaction scheme used for the obtaining of betu-
linic acid derivatives described by Silva and collaborators (2013) [26]. Briefly, the com-
mercial anhydride (1.1 mmol, 5 Eq) and DMAP (0.22 mmol, 1 Eq) were added to BE
(0.22 mmol) in CH2Cl2 (2 mL) and react for one hour. The crude residue was purified in
column chromatography to give the expected pure compounds. Column chromatog-
raphy was carried out using silica gel 60 (Merck). Analytical thin layer chromatography
was performed on silica gel 60 F254 plates (Merck) and spots visualized by spraying
with anisaldehyde/sulphuric. Then, two parts of the BE were modified due to the pres-
ence of two hydroxyl at C-3 and C-28 with the same reactivity. For the structure char-
acterization the High-resolution mass spectra (HR-EI-MS) were obtained on a
Micromass-Waters Q-TOF Ultima spectrometer and Infrared spectra were recorded on
a PerkinElmer FTIR BX spectrometer. Also, 1H and 13C Nuclear Magnetic Resonance
(NMR) spectra were recorded on a Bruker AC 400 spectrometer operating at 400 and
75 MHz, respectively, using tetramethylsilane as the internal standard and chloroform-
d. The chemical shifts (d) were expressed in parts per million (ppm). The purity of the
BE derivatives was also verified by melting points with a Kofler bench. The structure
of BE and their derivatives are disposed in Figure 1.
3.1.1. 3,28-O-acetylbetulin (2a)
The compound 2a was prepared by using acetic anhydride. White pow-
der, yield ¼ 90%.
1H NMR (400 MHz, CDCl3), d (ppm): 1.03 (s, 15 H, CH3-23; CH3-24; CH3-25; CH3-26
and CH3-27); 1.06 (m, 1 H CH-13); 1.09 (t, 1 H, CH-18); 1.12 (t, 2 H, CH2-1); 1.24 (t, 2 H,
CH2-16); 1.36 (t, 2 H, CH2-15); 1.39 (m, 4 H, CH2-2 and CH2-21); 1.43 (t, 4 H, CH2-11 and
CH2-22); 1.50 (t, 2 H, CH2-6); 1.51 (m, 2 H, CH2-12); 1.56 (t, 2 H, CH2-7); 1.62 (t, 1 H, CH-
5); 1.79 (br s, 3 H, CH3-30); 1.84 (m, 2 H, CH2-2); 1.86 (t, 1 H, CH-9); 2.07 (s, 6 H, CH3-32

NATURAL PRODUCT RESEARCH
5
and CH3-34); 2.44 (ddd, 1 H, CH-19); 4.24 (dd, 2 H, CH2-28); 4.46 (dd, 1 H, CH-3); 4.58 (s,
1 H, CH-29b); 4.68 (s, 1 H, CH-29a).13C NMR (75 MHz, CDCl3), d (ppm): 16.15 (C-25);
16.48 (C-26 and C-27); 18.15 (C-6); 19.09 (C-34); 20.78 (C-32); 21.06 (C-30); 21.32 (C-11);
25.12 (C-2); 26.90 (C-12); 23.68 (C-23 and C-24); 27.93 (C-15); 29.55 (C-21); 29.71 (C-16
and C-22); 35.53 (C-7); 37.03 (C-10); 37.53 (C-13); 37.77 (C-4); 38.36 (C-1); 40.86 (C-8);
42.66 (C-14); 48.74 (C-19); 46.28 (C-18); 47.69 (C-17); 50.25 (C-9); 55.35 (C-5); 62.78 (C-
28); 80.89 (C-3); 109.88 (C-29); 150.09 (C-20); 170.99 (C-31); 171.60 (C-33).
HRMS (ESI-MS, m/z); [M þ Na]þ calculated for C34H54O4Na: 549.7793; found:
549.2620. Melting point: 210–214 ꢁC.
3.1.2. 3,28-O-trifluoracylbetulin (2b)
The compound 2 b was prepared by using trifluoroacetic anhydride. White powder,
yield 80%.
1H NMR (400 MHz, CDCl3), d (ppm): 0.83 (t, 2 H, CH2-16); 0.99 (m, 1 H, CH-13); 1.06 (s,
15 H, CH3-23, CH3-24, CH3-25, CH3-26 and CH3-27); 1.08 (t, 1 H, CH-18); 1.15 (t, 2 H,
CH2-1); 1.30 (m, 2 H, CH2-12); 1.33 (t, 2 H, CH2-15); 1.42 (m, 2 H, CH2-21); 1.43 (t, 4 H,
CH2-11 and CH2-22); 1.45 (t, 2 H, CH2-6); 1.62 (t, 1 H, CH-5); 1.64 (t, 2 H, CH2-7); 1.78 (br
s, 3 H, CH3-30); 1.80 (m, 2 H, CH2-2); 1.84 (t, 1 H, CH-9); 1.99 (ddd, 1 H, CH-19); 4.15 (dd,
2 H, CH2-28); 4.58 (dd, 1 H, CH-3); 4.62 (s, 1 H, CH-29b); 4.71 (s, 1 H, CH-29a). 13C NMR
(75 MHz, CDCl3), d (ppm): 15.98 (C-26); 16.14 (C-27); 16.24 (C-25); 18.07 (C-6 and C-30);
19.08 (C-11); 20.77 (C-23 and C-24); 23.25 (C-2); 25.04 (C-12); 26.92 (C-15); 27.77 (C-21);
29.33 (C-16); 34.00 (C-22); 34.21 (C-7); 37.04 (C-10); 37.74 (C-13); 38.07 (C-4); 38.20 (C-
1); 40.88 (C-8); 42.74 (C-14); 46.61 (C-18); 47.53 (C-17); 48.76 (C-19); 50.19 (C-9); 55.23
(C-5); 66.84 (C-28); 86.23 (C-3); 110.30 (C-29); 116.05 (C-32); 116.11 (C-34) 149.50 (C-20);
157.74 (C-31); 158.16 (C-33).
HRMS (ESI-MS, m/z); [M-H þ 2K]þ calculated for C34H47F6O4K2: 711.2636; found:
711.3934 . Melting point: 175–176 ꢁC.
3.1.3. 3,28-O-dichloroacylbetulin (2c)
The compound 2c was prepared by using chloroacetic anhydride. White powder,
yield, 85%.
1H NMR (400 MHz, CDCl3), d (ppm): 0.99 (s, 15 H, CH3-23, CH3-24, CH3-25, CH3-26
and CH3-27); 1.06 (m, 3 H, CH-13 and CH2-16); 1.06 (t, 1 H, CH-18); 1.13 (t, 2 H, CH2-1);
1.26 (m, 2 H, CH2-21); 1.30 (t, 2 H, CH2-15); 1.34 (m, 2 H, CH2-11); 1.42 (t, 2 H, CH2-7);
1.45 (m, 4 H, CH2-2 and CH2-12); 1.51 (t, 2 H, CH2-6); 1.64 (t, 1 H, CH-5); 1.74 (t, 2 H,
CH2-22); 1.79 (br s, 3 H, CH3-30); 1.81 (t, 1 H, CH-9); 2.00 (ddd, 1 H, CH-19); 4.07 (dd, 2 H,
CH2-28); 4.48 (dd, 1 H, CH-3); 4.61 (s, 1 H, CH-29b); 4.70 (s, 1 H, CH-29a); 5.92 (s, 1 H,
CH-34); 5.96 (s, 1 H, CH-32). 13C NMR (75 MHz, CDCl3) d (ppm): 15.96 (C-25); 16.11 (C-
26); 16.32 (C-27); 18.01 (C-6); 19.08 (C-30); 20.74 (C-11); 23.19 (C-23 and C-24); 25.06
(C-2); 26.95 (C-12); 27.80 (C-15); 29.41 (C-21); 29.48 (C-16); 33.98 (C-7 and C-22); 34.29
(C-10); 37.01 (C-13); 37.66 (C-4); 38.21 (C-1); 40.84 (C-8); 42.70 (C-14); 46.75 (C-18);
47.66 (C-17); 48.80 (C-19); 50.17 (C-9); 55.28 (C-5); 64.43 (C-34); 64.78 (C-32); 66.16 (C-
28); 80.90 (C-3); 109.08 (C-29); 149.70 (C-20); 164.30 (C-31); 164.96 (C-33).
HRMS (ESI-MS, m/z); [M þ H þ 2Na]þ calculated for C34H51Cl4O4Na2: 711.5579;
Found: 711.4124. Melting point: 166–170 ꢁC.

6
S. M. C. GONC¸ALVES ET AL.
3.1.4. 3,28-O-isobutylbetulin (2d)
The compound 2d was prepared by using isobutyric anhydride. White pow-
der, yield ¼ 93%.
1H NMR (400 MHz, CDCl3), d (ppm): 0.96 (t, 2 H, CH2-16); 1.02 (s, 15 H, CH3-23, CH3-
24, CH3-25, CH3-26 and CH3-27); 1.06 (t, 1 H, CH-18); 1.13 (m, 3 H, CH2-1 and CH-13);
1.17 (d, 12 H, CH3-33, CH3-34, CH3-37 and CH3-38); 1.38 (t, 4 H, CH2-15 and CH2-21);
1.41 (m, 4 H, CH2-2 and CH2-11); 1.49 (m, 4 H, CH2-6 and CH2-12); 1.57 (t, 2 H, CH2-7);
1.60 (t, 1 H, CH-5); 1.67 (t, 2 H, CH2-22); 1.79 (br s, 3 H, CH3-30); 2.42 (m, 1 H, CH-36);
2.53 (m, 1 H, CH-32); 1.82 (t, 1 H, CH-9); 2.03 (ddd, 1 H, CH-19); 2.26 (t, 2 H, CH2-36);
2.38 (t, 2 H, CH2-32); 4.26 (dd, 2 H, CH2-28); 4.44 (dd, 1 H, CH-3); 4.57 (s, 1 H, CH-29b);
4.67 (s, 1 H, CH-29a). 13C NMR (75 MHz, CDCl3) d (ppm): 16.05 (C-25); 16.07 (C-26);
16.59 (C-27); 19.12 (C-33, C-34, C-37 and C-38); 19.25 (C-6); 20.82 (C-11 and C-30);
23.68 (C-23 and C-24); 25.18 (C-2); 26.95 (C-12); 27.96 (C-15); 29.61 (C-21); 29.82 (C-16);
34.12 (C-22); 34.26 (C-36); 34.49 (C-32); 34.57 (C-7); 37.10 (C-10); 37.57 (C-13); 37.97 (C-
1 and C-4); 38.36 (C-8); 40.91 (C-14); 42.73 (C-18); 46.57 (C-17); 48.83 (C-19); 50.29 (C-9);
55.39 (C-5); 62.42 (C-28); 80.35 (C-3); 109.88 (C-29); 150.19 (C-20); 176.82 (C-35); 177.52
(C-31).
HRMS (ESI-MS, m/z); [M-2H þ K]þ calculated para C38H60O4K: 619.9789; found:
619.3745. Melting point: 110–112 ꢁC.
3.1.5. 3,28-O-butanoylbetulin (2e)
The compound 2e was prepared by using butyric anhydride. White pow-
der, yield ¼ 64%.
1H NMR (400 MHz, CDCl3), d (ppm): 0.96 (t, 6 H, CH3-34 and CH3-38); 0.99 (s, 15 H,
CH3-23, CH3-24, CH3-25, CH3-26 and CH3-27); 1.02 (t, 1 H, CH-18); 1.15 (m, 1 H CH-13);
1.36 (t, 2 H, CH2-16); 1.41 (m, 2 H, CH2-12); 1.49 (t, 2 H, CH2-6); 1.52 (t, 2 H, CH2-1); 1.60
(t, 2H; CH2-7); 1.61 (t, 1 H, CH-5); 1.63 (t, 2 H, CH2-15); 1.64 (m, 2 H, CH2-21); 1.67 (t, 2 H,
CH2-11); 1.70 (t, 2 H, CH2-22); 1.74 (m, 2 H, CH2-37); 1.77 (m, 2 H, CH2-33); 1.78 (br s,
3 H, CH3-30); 1.84 (m, 2 H, CH2-2); 1.98 (t, 1 H, CH-9); 2.21 (ddd, 1 H, CH-19); 2.26 (t, 2 H,
CH2-36); 2.38 (t, 2 H, CH2-32); 4.40 (dd, 3 H, CH-3 and CH2-28); 4.52 (s, 1 H, CH-29b);
4.61 (s, 1 H, CH-29a). 13C NMR (75 MHz, CDCl3) d (ppm): 13.71 (C-38); 14.72 (C-35);
16.54 (C-25); 18.14 (C-26); 18.53 (C-27); 18.62 (C-33 and C-37); 19.10 (C-6); 20.77 (C-11
and C-30); 23.76 (C-23 and C-24); 25.13 (C-2); 27.93 (C-12); 29.55 (C-15); 29.76 (C-16
and C-21); 34.08 (C-22); 34.56 (C-36); 36.40 (C-32); 36.75 (C-7); 37.03 (C-13); 37.53 (C-
10); 37.80 (C-4); 38.34 (C-1); 40.86 (C-8); 42.67 (C-14); 46.37 (C-18); 47.70 (C-17); 48.74
(C-19); 50.24 (C-9); 55.34 (C-5); 62.35 (C-28); 80.55 (C-3); 109.83 (C-29); 150.15 (20);
173.54 (C-31); 174.16 (C-35).
HRMS (ESI-MS, m/z); [M þ Na]þ calculated for C38H62O4Na: 605.8563; Found:
605.3057. Melting point: 100–104 ꢁC.
3.2. Enzyme-linked immunosorbent assays (ELISA)
From the culture supernatant at cytokines pro-inflammatory like interleukin IL-6, IL-
17A and Interferon gamma (IFN-g) measured by using enzyme-linked immunosorbent
assay (ELISA) kits (eBiosciences, USA, and BD Biosciences, USA) according to the

NATURAL PRODUCT RESEARCH
7
Table 1. Comparison of the IC50 values (mM) of BE and its new derivatives against tumor cell lines
(Raji (Burkitts lymphoma), MCF-7 (breast cancer), Hep-G2 (well-differentiated hepatocellular carcin-
oma) and DU-145 (prostate cancer)) and non-tumor cell (peripheral blood mononuclear cell
(PBMC)), determined by using MTT assay.
MCF-7
HEP-G2
DU145
IC50
1.38 49.36 13.43
PBMC
RAJI
IC50
Compounds
IS
IC50
IS
IC50
IS
IS
IC50
BE
2a
2b
2c
2d
2e
45.78 0.71 2.18 45.21 31.40
2.21
—
—
—
—
72.23 3.42
88.71 0.00
85.86 0.00
89.27 0.00
60.47 0.00
86.09 0.00
2.02
—
>100
>100
>100
>100
>100
>100
85.10 4.78 1.17
92 3.07 1.08
89.45 7.60 1.11
95.53 0.75 1.04
96.75 3.28 1.03
>100
>100
>100
>100
>100
1.12
1.16
1.12
1.65
1.16
>100
>100
>100
>100
>100
—
—
—
—
—
Doxorubicin 11.91 9.15 8.39
2.71 0.87
36.90 39.22 14.48 2.54
6.88 1.06
14.53 >100
manufacturers’ instructions. The absorbance used the difference between 570 and
450 nm readings. The minimum limits of detection of the ELISA kits used in the experi-
ment were 4.69 pg/mL for IL-6, 3.9 pg/mL for IL-17A, 31.25 pg/mL and 4.68 pg/mL for
IFN-g.
3.3. Western blot analysis
Monocytes (U937) were plated contraction of 106 cells per ml and then stimulated
with Escherichia coli lipopolysaccharide LPS 0111: B4 (10 mg/ml). For negative control
were used non-selective COX, acetylsalicylic acid (ASA) dose of 10 mg/ml as well as
COX-2 inhibitor, 10 lM DEXA. Test group, they were added to the novel 2a and 2c as
well as BE. Analysis of protein expression was performed polyacrylamide gel electro-
phoresis (SDS-PAGE) 10%. Then the proteins contained in the gel were transferred to
Polyvinylidene Fluoride Membranes (PVDF) (Amersham Hybond-P PVDF Membrane).
Membranes incubated overnight with COX-2 and b-actin, used as normalizer. Both
antibodies were diluted at concentrations of 1: 2000. The secondary antibody added
to the membrane and incubated for 2 h in concentration 1:2000. The densitometry
determined by ImageJ software. Final quantification given by the ratio between the
expression of the protein of interest and b-actin, used as normalizer.
3.4. MTT assay
The cytotoxic activities of all the compounds were evaluated using the (3-[4,5-dime-
thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) (Thermofischer, Massachusetts,
EUA). The cells were plated in 96-well plates (1 ꢂ 104 cells/well) and incubated over-
night. The cells were treated with various concentrations of new BE derivatives (1, 10
and 100 lM) for 72 h at 37 ꢁC, in a humidified atmosphere with 5% CO2. Control cells
treated with 0.1% DMSO alone and 0.1mM Doxorubicin was included as a negative
control. After, was added in cells incubated 30 mL MTT solution (5 mg/mL) for 3 h at
37 ꢁC. Then was added the solution Sodium dodecyl sulfate (SDS 20%) for dissolution
of the precipitate. The absorbance was measured at 570 nm using the ELISA plate
reader after 24 h (Table 1).

8
S. M. C. GONC¸ALVES ET AL.
3.5. Ethical approval
The ethics committee was approved by the research Ethics Committee Involving
Human Beings of the Federal University of Pernambuco CEP-CCS-UFPE: 1.351.357.
3.6. Obtaining and isolation of peripheral blood lymphocytes (PBMC)
Eight healthy volunteers were selected to evaluate the anti-inflammatory activity of
new BE derivatives. Blood samples collected at Immunomodulation and New
Therapeutic Approaches Laboratory (LINAT-UFPE). Informed written consent obtained.
The exclusion criterion was to show no other systemic diseases that could have poten-
tially altered their inflammatory profile functions. PBMCs were isolated from fresh per-
ipheral blood collected in sodium heparin tubes. Briefly, whole blood was laid over
one layer of Ficoll–Paque Plus (density gradient of 1.077 g/L, GE Healthcare, Little
Chalfont, UK) and centrifuged at 400 g for 45 min, cells were washed once in PBS (pH
7.4) (Ebioscience, San Diego, CA, USA).
3.7. Culture of peripheral blood lymphocytes (PBMC)
PBMCs isolated from healthy individuals were plated in 12-well plates (1 ꢂ 106/1000 mL
cells/well) in RPMI-1640 (Gibco, Massachusetts, EUA) with L-glutamine containing 10%
heat inactivated fetal bovine serum (FBS) (Lonza, Valais, Switzerland), 10 nM HEPES
(Gibco, Massachusetts, EUA) and 1% penicillin/streptomycin (Gibco, Massachusetts,
EUA), at 37 ꢁC, in a humidified atmosphere with 5% CO2 for 48 h. Under two condi-
tions: unstimulated cells (None) and stimulated cells with Phorbol 12-myristate 13-
acetate (2 mL) and Ionomycin (5 mL) (P þ I), (Ebioscience, San Diego, CA, USA), and cells
with methylprednisolone in 100 mg/mL (MP) (Sigma-Aldrich, Missouri, EUA). They were
also added BE derivatives at concentrations (1, 10 and 100 mM). After 48 h superna-
tants were collected and stored at 20 degrees for later measurement of cytokines.
3.8. Statistical analysis
Statistical analysis performed using GraphPad Prism version 6 program. The data eval-
uated using the normal test and variance test. Data presented as median maximum
and minimum, and standard deviation Wilcoxon test was used and p < 0.05 was con-
sidered significant. IC50 measured by Origin 8 software.
4. Conclusion
The strategy of adding esters (with different numbers of carbons or halogens) at C-3
and C-28 positions in BE, to generate derivatives with immunomodulation and anti-
inflammatory potential was conquered. After analyzing the five derivatives semisynthe-
sized, two of them (2a and 2c) showed modulatory effect of pro-inflammatory cyto-
kine IFN-g. In addition, BE and 2a and 2c were capable of modulating the expression
of a protein, which is a key component of the inflammatory process, the COX-2. The
derivative 2c at 100 lM is a more powerful inhibitor of COX-2 than drugs used in

NATURAL PRODUCT RESEARCH
9
clinical practice (DEXA), indicating that chloride might be important for improving the
anti-inflammatory activity of BE. However, for the anti-tumor evaluation, the new BE
derivatives did not show promissory cytotoxic activity against tumor cell lines, the bet-
ter result was for BE without modification. Then, other modifications must be per-
formed in order to improve the anti-tumor activity, but the good result obtained
against non-tumor cell must be reproduced.
Acknowledgments
This work was supported by grants and funding from the Brazilian agencies CAPES, CNPq and
INCT-IF. We thank the Post Graduate Programme in Therapeutic Innovation (PPGIT/UFPE) and
Post Graduate Programme in Pharmaceutical Sciences (PPGCF/UFRGS). GNSS received
CAPES Fellowship.
Conflict of interest
The authors declare that there are no conflicts of interest.
Each author contributed to the preparation of article. All authors read and approved the
final manuscript.
Funding
National Institute of science and technology and infrastructure (Inct_if) and Foundation support
science and technology of Pernambuco (FACEPE).
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