Betulinic acid derivatives: a new class of α-glucosidase inhibitors and LPS-stimulated nitric oxide production inhibition on mouse macrophage RAW 264.7 cells

Article abstract of DOI:10.1080/14786419.2018.1462182

Chemical manipulation studies were conducted on betulinic acid (1), twenty-one new rationally designed analogues of 1 with modifications at C-28 were synthesized for their evaluation of inhibitory effects on α-glucosidase and LPS-stimulated nitric oxide production in mouse macrophage RAW 264.7 cells. Compound 2 (IC₅₀ = 5.4 μM) exhibited an almost 1.4-fold increase in α-glucosidase inhibitory activity on yeast α-glucosidase while analogues 5 (IC₅₀ 16.4 μM) and 11 (IC₅₀ 16.6 μM) exhibited a 2-fold enhanced inhibitory activity on NO-production than betulinic acid.

Full text of DOI:10.1080/14786419.2018.1462182

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Betulinic acid derivatives: a new class of α-
glucosidase inhibitors and LPS-stimulated nitric
oxide production inhibition on mouse macrophage
RAW 264.7 cells
Narayanarao Gundoju, Ramesh Bokam, Nageswara Rao Yalavarthi, Rajaram
Azad & Mangala Gowri Ponnapalli
To cite this article: Narayanarao Gundoju, Ramesh Bokam, Nageswara Rao Yalavarthi, Rajaram
Azad & Mangala Gowri Ponnapalli (2018): Betulinic acid derivatives: a new class of α-glucosidase
inhibitors and LPS-stimulated nitric oxide production inhibition on mouse macrophage RAW 264.7
cells, Natural Product Research, DOI: 10.1080/14786419.2018.1462182
To link to this article: https://doi.org/10.1080/14786419.2018.1462182
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2018.1462182
Betulinic acid derivatives: a new class of α-glucosidase
inhibitors and LPS-stimulated nitric oxide production
inhibition on mouse macrophage RAW 264.7 cells
Narayanarao Gundojua, Ramesh Bokama, Nageswara Rao Yalavarthia, Rajaram Azadb
and Mangala Gowri Ponnapallia
aNatural Products chemistry division, Indian Institute of chemicaltechnology, hyderabad, India; bdepartment
of animal Biology, university of hyderabad, hyderabad, India
ABSTRACT
ARTICLE HISTORY
received 10 February 2018
accepted 3 april 2018
Chemical manipulation studies were conducted on betulinic acid (1),
twenty-one new rationally designed analogues of 1 with modifications
at C-28 were synthesized for their evaluation of inhibitory effects on
α-glucosidase and LPS-stimulated nitric oxide production in mouse
macrophage RAW 264.7 cells. Compound 2 (IC50 = 5.4 μM) exhibited an
almost 1.4-fold increase in α-glucosidase inhibitory activity on yeast
α-glucosidase while analogues 5 (IC50 16.4 μM) and 11 (IC50 16.6 μM)
exhibited a 2-fold enhanced inhibitory activity on NO-production than
betulinic acid.
KEYWORDS
Sonneratia caseolaris;
betulinic acid; α-glucosidase
inhibition; lPs-stimulated
nitric oxide production
inhibition
1. Introduction
Betulinic, oleanolic, ursolic, and boswellic acids are abundant pentacyclic triterpenoids that
occur in many edible fruits and vegetables. Among various pentacyclic triterpenoids, betu-
linic acid derivatives are widely studied group of compounds of natural origin due to their
CONTACT Mangala Gowri Ponnapalli
pmgowri@yahoo.com, mangala@iict.res.in
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1462182.
© 2018 Informa uK limited, trading as taylor & Francis Group

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N. GUNDOJU ET AL.
broad spectrum of biological profiles (Koehn and Carter 2005). Several triterpenoids are
known to possess significant α-glucosidase (AGH) inhibitors and anti-inflammatory agents
(Yu et al. 2014). As part of our ongoing program on Indian mangrove flora, we reported the
α-glucosidase inhibitory and antihyperglycemic activity of the crude methanol extract and
their metabolites from the fresh fruits of Sonneratia caseolaris (Ashok Kumar et al. 2010). A
careful reexamination of the same extract led to the isolation of betulinic acid (Chao-Min
et al. 2016) in substantial quantities. Herein, we report the isolation of betulinic acid from
the underutilized fruits of S. caseolaris and semi synthesis of 21 new rationally designed
N, O, S containing heterocyclic derivatives for their inhibitory effects on α-glucosidase and
LPS induced NO inhibition on mouse macrophage RAW 264.7 cell lines.
2. Results and discussion
The structural modifications described in this paper were focused on the introduction of
various nitrogen, oxygen and sulfur containing heterocyclic substituted methyl esters as
new scaffolds (Schemes 1–3) at the C-28 region of the betulinic acid, which was isolated as
major metabolite from the fruits of Sonneratia caseolaris. Prior pharmacological studies on
Scheme 1. synthesis of heterocyclic amide derivatives on betulinic acid.
Scheme 2. synthesis of phenyl acetylene derivatives on betulinic acid.

NATURAL PRODUCT RESEARCH
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Scheme 3. synthesis of isoxazole derivatives on betulinic acid.
betulinic acid demonstrated that the introduction of nitrogen-containing heterocyclic moi-
eties will be beneficial for the development of new therapeutic agents (Qiu et al. 2009; Urban
et al. 2012; Xu et al. 2012; Dang Thi et al. 2014; Gao et al. 2014). On the basis of promising in
vitro cytotoxicity of piperazine, isoxazole (Jun et al. 2012), and acetylenic derivatives in a
related series of pentacyclic lupane triterpenoids (Boryczka et al. 2013), we incorporated
heterocyclic scaffolds into the betulinic acid template. Methyl ester analogues (2–9) were
synthesized in excellent yields (90–93%) by the reaction between BA and different chloro-
acetyl amides in the presence of K2CO3 in DMF as solvent at room temperature. Chloroacetyl
morpholine, chloroacetyl thiomorpholine, and Chloroacetyl N-substituted piperazines were
synthesized by the reaction of chloroacetyl chloride with morpholine, thiomorpholine, and
N-substituted piperazines in the presence of DCM using TEA as a base. The products were
confirmed by the application of spectroscopic methods. BA was treated with propargyl
bromide in the presence of K2CO3 in DMF solvent at room temperature to afford
propargyl ester (10, 95% yield) as the key intermediate step for new semi synthetic derivatives
(Schemes 2–3). The new series of analogues (11–16) were synthesized by a Sonogashira
coupling (Sonogashira et al. 1975) using terminal alkyne C–H with substituted aryl iodides.
The coupling reaction was catalyzed by Pd (PPh3)2Cl2 and CuI in DMF solvent at room tem-
perature to afford the product in excellent yields (90–98%) (Scheme 2). These products were
easily identified by the absence of terminal methine at δ ~2.43 ppm in the 1H NMR.
Furthermore, new analogues (Scheme 3) of betulinic acid with 3, 5-disubstituted isoxazole
moieties were synthesized since isoxazoles are often encountered medicinally important
pharmacophores (Cicchi et al. 2003; Jager and Colinas 2003; Grunanger and Vita-Finzi 1991).
In general, uncatalyzed 1, 3-dipolar cycloaddition of nitrile oxides with acetylenes have been
known for a long period, but its application to the synthesis of 3, 5-disustituted isoxazoles
are scarce owing to low yields of isoxazole products (Huisgen 1984), side reactions and the
possibility of both regioisomers. We have developed a methodology to improve yields, and
wide scope of functional group on betulinic acid for a regioselective, experimentally con-
venient one-pot, three steps uncatalyzed 1, 3-dipolar cycloaddition of nitrile oxides with
terminal acetylene. Moreover, an aldehyde is first converted into the corresponding aldoxime
by the reaction with hydroxylamine. Without isolation, the aldoxime is transformed to the

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N. GUNDOJU ET AL.
chloroaldoxime. We have standardized the protocol with KHCO3 and EtOAc: H2O (3:1) at
room temperature for 6 h to accomplish compounds (17–23) in good to excellent yields
(88–91%), whereupon nitrileoxides are generated in situ and further reacted without isola-
tion. The formation of 3, 5-disubstituted isoxazole moiety was confirmed by the presence
of characteristic isoxazole proton singlet at δ ~6.66 ppm in the 1H NMR and at δ ~102.0 ppm
in the13C NMR.
The AGH inhibitory effects of BA analogues (2–23) were evaluated according to the mod-
ified method by Tomoyuki et al. (1999) and results are summarized in Table S1. Compound
2 (IC50 = 5.4 μM) exhibited an almost 1.4-fold increase in AGH inhibitory activity compared
to BA (IC50 = 7.8 μM) whereas replacement of morpholine by thiomorpholine in the analogue
8 (IC50 = 42.2 μM) showed eight fold decrease in the AGH inhibitory activity, which strongly
suggested that morpholine is essential for inhibiting α-glucosidase action compared to
thiomorpholine substituent. Compounds 3 (IC50 = 8.9 μM), 15 (IC50 = 9.4 μM), and 23 (IC50
=
10.8 μM) displayed potent AGH inhibition compared to other derivatives. 2-Pyridyl (3),
p-methoxy (15), and p-bromo (23) substituents played a prime role to increase AGH inhibition
compared to other substituents in piperazine, acetylenic, and isoxazole analogues respec-
tively. Compounds 4–14 and 16–22 were found to be less or inactive against AGH inhibition
compared to BA.
The cell viability of BA analogues (1–23) on macrophage RAW 264.7 cell lines were deter-
mined by MTT assay (Figure S94). After 24 h of incubation, these compounds displayed cell
viability > 90%. The anti-inflammatory activity of BA and its derivatives (1–23) were evaluated
against LPS-stimulated NO production in RAW 264.7 cells. As shown Table S2, analogue 5
(IC50 = 16.4 μM) and 11 (IC50 = 16.6 μM) significantly suppress the NO production compared
to that of betulinic acid (IC50 = 31.1 μM). Our results suggested that O-anisyl (5) substituent
in the piperazine amide and m-cyanomethylphenyl acetylene (11) are essential functional-
ities for the mediation of anti-inflammatory activity while other derivatives showed the less
potent or inactive NO-production inhibitory activity compared to BA (1).
3. Conclusions
Our findings demonstrated that BA was encountered as a major metabolite from the
underutilized edible fruits of S. caseolaris. 21 New BA analogues were designed and synthe-
sized for SAR studies. Our study revealed that certain ester functionalities enhanced AGH
inhibition of BA significantly compared to NO production inhibition. Further studies are
required to exploit AGH inhibitory potential of these analogues.
Acknowledgments
We are thankful to UGC for financial support and awarding SRF to NRG and director, Dr S. Chandrasekhar
for constant encouragement and support. This work was supported by the Science and Engineering
Research Board, New Delhi, India through a grant awarded to MGP (Grant number: EMR/2015/002319).
Disclosure statement
No potential conflict of interest was reported by the authors.

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Funding
This work was supported by the UGC; SERB, Government of India [grant number EMR/2015/002319].
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