Synthesis of podophyllotoxin-glycosyl triazoles via click protocol mediated by silver (I)-N-heterocyclic carbenes and their anticancer evaluation as topoisomerase-II inhibitors

Article abstract of DOI:10.1080/14786419.2019.1610958

Herein we report the regioselective synthesis of podophyllotoxin-Glycosyl triazole hybrids catalysed by Ag(I)-N-heterocyclic carbene (Ag(I)-NHC) in a short reaction time (～30 min) at ambient conditions. In principle, it is the first report of Click alkyne-azide cycloaddition catalysed by Ag(I)-NHC catalyst and moreover, this new methodology yielded good results when compared with traditional CuAAC in terms of reaction time and selectivity. The synthesised compounds were further explored for in vitro anticancer activity against four human cancer cell lines Du145, HeLa, A-549, and MCF-7 and found that these synthesised compounds possess significant anticancer activity. Further, the compounds 5a and 5e were identified as promising leads due to their better activity across all cell lines than that of the standard drug etoposide. Molecular docking studies of 5a & 5e with DNA Topoisomerase-II were revealed that the free energy calculations of active compounds were in good agreement with observed IC₅₀ values.

Full text of DOI:10.1080/14786419.2019.1610958

Natural Product Research
Formerly Natural Product Letters
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Synthesis of podophyllotoxin-glycosyl triazoles via
click protocol mediated by silver (I)-N-heterocyclic
carbenes and their anticancer evaluation as
topoisomerase-II inhibitors
Srinivas Nerella, Shravankumar Kankala & Brahmeshwari Gavaji
To cite this article: Srinivas Nerella, Shravankumar Kankala & Brahmeshwari Gavaji (2019):
Synthesis of podophyllotoxin-glycosyl triazoles via click protocol mediated by silver (I)-N-
heterocyclic carbenes and their anticancer evaluation as topoisomerase-II inhibitors, Natural
Product Research, DOI: 10.1080/14786419.2019.1610958
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1610958
Synthesis of podophyllotoxin-glycosyl triazoles via click
protocol mediated by silver (I)-N-heterocyclic carbenes
and their anticancer evaluation as topoisomerase-
II inhibitors
Srinivas Nerellaa,b , Shravankumar Kankalaa and Brahmeshwari Gavajia
b
aDepartment of Chemistry, Kakatiya University, Warangal, India; Department of Chemistry, Pingle
Government College for Women, Kakatiya University, Warangal, India
ABSTRACT
ARTICLE HISTORY
Received 10 January 2019
Accepted 10 April 2019
Herein we report the regioselective synthesis of podophyllotoxin-
Glycosyl triazole hybrids catalysed by Ag(I)-N-heterocyclic carbene
(Ag(I)-NHC) in a short reaction time (ꢀ30 min) at ambient condi-
tions. In principle, it is the first report of Click alkyne-azide cyclo-
addition catalysed by Ag(I)-NHC catalyst and moreover, this new
methodology yielded good results when compared with trad-
itional CuAAC in terms of reaction time and selectivity. The syn-
thesised compounds were further explored for in vitro anticancer
activity against four human cancer cell lines Du145, HeLa, A-549,
and MCF-7 and found that these synthesised compounds possess
significant anticancer activity. Further, the compounds 5a and 5e
were identified as promising leads due to their better activity
across all cell lines than that of the standard drug etoposide.
Molecular docking studies of 5a & 5e with DNA Topoisomerase-II
were revealed that the free energy calculations of active com-
pounds were in good agreement with observed IC50 values.
KEYWORDS
Podophyllotoxin; click
chemistry; Ag (NHC)
catalyst; podophyllotoxin-
glycosyl triazoles;
anticancer studies
CONTACT Srinivas Nerella
nerellasrinivas1@gmail.com
Department of Chemistry, Pingle Government College
for Women, Kakatiya University, Warangal, Telangana 506370, India
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1610958
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
S. NERELLA ET AL.
1. Introduction
Plants of Podophyllum species were known for their curative properties for many
centuries. In folklore medicine, the plant parts and its crude extracts were used as
cathartic, purgative and also to treat genital warts and molluscum contagiosum
(Teillac-Hamel et al. 1996). Later on it was identified that Podophyllotoxin (1), is the
main component present in Podophyllum species which is responsible for its healing
properties. Podophyllotoxin is a naturally occurring cyclolignan that can be extracted
from roots and rhizomes of Podophyllum species such as Podophyllum peltatum and
Podophyllum hexandrum (You 2005).
Podophyllotoxin and its analogues exhibit very interesting and diverse pharmaco-
logical activities such as insecticidal, antiviral, antifungal, antitumor, anti-inflammatory,
antioxidative and antispasmogenic (Gordaliza et al. 2004; Lv and Xu 2011). However,
its severe gastrointestinal side effects restricted its use as therapeutic agent (Pettit
et al. 2016). To overcome these side effects, several analogues were synthesised and
reported by various groups. Among all the activities, its antitumor activity (Cheng
et al. 2014) attracted much attention after the success of its semi synthetic derivatives
etoposide and teniposide as effective anticancer drugs (Gupta et al. 2006). Some of
these semi synthetic derivatives differ significantly from their parent compound i.e.,
podophyllotoxin, in their mechanism of action. Podophyllotoxin is a Tubulin inhibiting
agent (Kamal et al. 2014) whereas etoposide and its analogues are inhibitors of DNA
Topoisomerase-II (Mariani et al. 2015).
DNA Topoisomerases are set of enzymes which operate on topology of DNA and
essential for unwinding of DNA strands during replication (Rybenkov et al. 1997). DNA
strands must be separated during replication process, but super coiled nature of DNA
doesn’t allow this. At this juncture, Topoisomerase–II binds to DNA and causes breaks
and seals to strands to ease the torsional strain leading to separation of DNA strands.
Drugs which target Topoisomerase-II inhibit this action leading to stoppage of DNA
replication and eventually leading to arrest of cell proliferation. Etoposide, a glycoside
of podophyllotoxin also works on these lines (Byl et al. 2001). Hence we would like to
synthesise etoposide like glycosides by reacting O-propargyl podophyllotoxin with gly-
cosyl azides. On the other hand, triazoles also exhibit a broad spectrum of biological
activities like Podophyllotoxin (Yempala et al. 2014). Triazole ring is a dynamic pharma-
cophore containing high dipole moment and thus able to participate actively in
hydrogen bond formation as well as in dipole-dipole interactions with biological tar-
gets (Whiting et al. 2006). Especially Triazole moiety is prevalent in natural products
and also in potential pharmaceutical compounds like carboxyamidotriazole (CAI),
Tazobactum and cefatrizine. In addition to this, even glycosyl triazoles have their own
significance in medicinal chemistry as chemical therapeutics (Zhang et al. 2005).
N-Heterocyclic Carbenes (NHCs) were already well established as versatile organo
and organometallic catalysts and found diverse and myriad applications in organic
transformations (Rafet and Naim 2017). Amidst diverse properties of NHCs, their cata-
lytic property is the most significant and studied elaborately. We reported previously
cooperative effect between Ag (I) and organo-NHCs in catalyzing a multicomponent
one-pot reaction (Kankala et al. 2015). Literature survey revealed that the organo and
organometallic NHCs have not yet been explored extensively for alkyne-azide

NATURAL PRODUCT RESEARCH
3
Figure 1. Synthesis of Podophyllotoxin-glycosyl triazoles (5a–l).
cycloaddition. Therefore we would like to employ diverse NHCs for the click 1,
3-dipolar cyclisation reaction to find out the efficacy of these catalysts and the best
catalyst among them.
2. Results and discussion
This work describes a facile route for the regioselective synthesis of 1, 4- disubstituted
podophyllotoxin-glycosyl triazoles via catalytic 1, 3-dipolar cycloaddition of sugar
azides and 4b-O-propargyl podophyllotoxin. We have made 4b-O-propargylated podo-
phyllotoxin to react with pre-synthesised sugar azides in cycloaddition to obtain podo-
phyllotoxin-glycosyl triazole hybrids. The 4b-O-propargyl podophyllotoxin (3) was
obtained from podophyllotoxin (1) and propargyl alcohol (2) in presence of Boron tri-
floride etherate in a single step synthesis (Figure 1). The synthesised compound (3)
was then treated with sugar azides (4a–l) in the presence of Ag (I)-NHC catalyst to
obtain podophyllotoxin-glycosyl triazole hybrids (5a–l).
Initially we chose to study 1, 3-dipolar cycloaddition reaction between 4b-O-prop-
argyl podophyllotoxin (3) and galactose azide (4e) in ethanol. We would also like to
explore the efficacy of a variety of potential catalysts in catalyzing 1, 3-dipolar cyclo-
addition as depicted in Table S1. There was no cycloaddition observed in the absence
of catalyst (Table S1, entry 1). We are also interested to know the progress of click
reaction employing widely used copper salts like CuI, CuSO4 and Cu(OAc)2, but
obtained products in trace amounts after 24 h at reflux conditions (Table S1, entries
2–4). When silver salts (AgOAc and AgOTf) and zinc salts (Zn (OAc)2 and Zn (OTf)2)
were employed as catalysts there was also no progress in the above cycloaddition
reactions at reflux temperature (Table S1, entries 5–8).
However, when Ag(I)-NHC (NHC precursor ¼ 1,3-Bis (2,4,6-trimesitylphenyl) imidazo-
lium chloride) was employed as catalyst, the results were remarkable yielding podo-
phyllotoxin-glycosyl triazole hybrid (5e) selectively just in 30 minutes with better yields
(94%, Table S1, entry 9). On the other hand, previous reports revealed that alkyne-
azide cycloaddition reaction employing catalysed and uncatalysed methodlogies was
accomplished only under refluxing conditions with longer reaction times. The out
come of these results clearly demonstarted the efficiency of Ag(I)-NHC as a potential
catalyst for 1,3-dipolar cycloaddition.

4
S. NERELLA ET AL.
Figure 2. A plausible mechanism for the formation of 1, 4-disubstituted triazoles catalyzed by Ag
(I)-NHC.
We have also interested to investigate the above cycloaddition reaction using other
co-catalysts (CuI & Zn(OTf)2) along with Ag(I)-NHC and the results were comparable
with Ag(I)-NHC (Table S1, entries 10 and 11). We have also studied the same reaction
using simple organo-NHC as a catalyst and found that the reaction was relatively slow
and the yields of triazoles were moderate (Table S1, entry 12). However, we have got
better yields when Silver salt (AgOTf) was introduced during the reaction course into
the organo-NHC mediated cycloaddition (Table S1, entry 13) than the organo-NHC
alone mediated 1,3-dipolar cycloaddition.
Employing the same optimised conditions (RT, 5 mol% Ag(I)-NHC, 30 min, ethanol),
we investigated the scope of the reaction with a variety of structurally diverse sugar
azides (4a–l) for the cycloaddition reaction and the results are depicted in Table S2.
The structures of the podophyllotoxin-glycosyl triazole hybrids (5a–l) were established
on the basis of elemental analysis and spectral data (1H NMR, 13C NMR and Mass).
The literature reports reveal that the synthesis of regioselective Podophyllotoxin-tri-
azole hybrids from 4b-azidopodophyllotoxin and terminal alkynes was easy to achieve.
However, the selective synthesis of these hybrids from 4b-O-propargylpodophyllotoxin
and azides (simple or sugar) is still remain as a challenge to accomplish. All of the ear-
lier efforts were not so successful and produced mixture of regioisomers (Hong et al.
2011; Mahesh et al. 2014). The results and conditions depicted in Table S2 for the
regioselective synthesis of podophyllotoxin-glycosyl triazole hybrids (5a–l) suggest
that our yields and reaction time are comparable or even better than those for the
closely related synthesis of podophyllotoxin-triazole hybrids in earlier reports (Bhat
et al. 2008; Reddy et al. 2011).
Based on the results obtained in the regioselective synthesis of 1, 4-disubstituted-1,
2, 3-triazoles a plausible mechanism has been deduced as depicted in Figure 2. The
formation of triazole could occur in a domino fashion in cycloaddition. According to
Figure 2, the organo-NHC catalyst will interact first with the 4b-O-Propargylated podo-
phyllotoxin to form a zwitterion. The reactive zwitterionic species will now interact
with sugar azide through nucleophilic attack and form another zwitterion via C-N

NATURAL PRODUCT RESEARCH
5
bond formation. This undergoes finally to C-N heterocyclisation to produce regioselec-
tively the corresponding 1, 4-disubstituted-1, 2, 3-triazole.
2.1. In vitro anticancer activity
The anticancer activity of podophyllotoxin-glycosyl triazole hybrids (5a–l) was eval-
uated by MTT assay against human tumor cell lines Du145, HeLa, A-549 and MCF-7
and compared with etoposide as working standard, and the final inhibitory concentra-
tion to an extent of 50% was calculated using the reported methods (Mosmann 1983).
The in vitro cytotoxicity values depicted in Table S3 indicate that the podophyllotoxin-
glycosyl triazole hybrids (5a–l) show promising anticancer activity against all the
tested cancer cell lines.
Among the synthesised compounds, 5a and 5e have shown more potent antitumor
activity across all cell lines than the standard drug etoposide. The compound 5a has
shown the best activity against Prostate cancer cell lines (Du 145) with IC50 value
1.02 0.02 lM whereas 5e has shown the best activity against breast cancer cell lines
(MCF-7) with IC50 value 1.25 0.02 lM. As far as the structure–activity relationship is
concerned, some correlations can be drawn from the data to support the observed
cytotoxic activity based on the type of the sugar substituents present on the triazole
ring. The podophyllotoxin-glycosyl triazole hybrids bearing both protected and unpro-
tected sugars have shown potential anticancer activity. However, the compounds with
acetyl protected sugars particularly 5a and 5e have exhibited better anticancer activity
than the unprotected sugars. This can be attributed to the presence of acetyl groups
which have high binding affinity with the enzyme as supported by the docking stud-
ies. This effective drug-receptor interaction might be one of the promising reasons for
the improvement in the activity of acetyl protected podophyllotoxin-glycosyl tri-
azole hybrids.
2.2. Molecular docking studies
Docking studies of 5a and 5e were performed with Auto Dock software to reveal the
interactions of these compounds with the active sites of Topoisomerase-II enzyme
with PBD ID 1ZXN (Figure S1). The results were impressive showing high binding affin-
ity of these compounds towards the enzyme and a free energy of ꢁ9.2 and ꢁ9.3 was
observed for 5a and 5e respectively.
Docking studies of 5a revealed that the molecule bound well with the enzyme
through various types of interactions such as Hydrogen bonding, alkyl, pi-alkyl and
carbon hydrogen interactions. Glycosyl moiety was shown strong binding affinity with
the enzyme through hydrogen bonding with amino acids like Aspargine C92,
Threonine C184, Arginine C-70, and Serine C-120. Triazole ring was interacted with
Arginine C70 via pi-alkyl interactions. Other notable interactions of 5a include the pi-
alkyl interaction between aromatic rings (B and E) of Podophyllotoxin and
Isoleucine D5.
The compound 5e has shown strong interactions with the enzyme and formed
hydrogen bonds with amino acid residues like ARG C70, SER C121, SER C120 and ASN

6
S. NERELLA ET AL.
C122. It was clearly shown that the sugar moiety of the compound was actively par-
ticipated in hydrogen bonding. The aromatic rings of Podophyllotoxin were interacted
with amino acid residues such as ILE D5 and ARG C70 through pi-alkyl attractions.
Other prominent interactions include pi-donor hydrogen between methyl hydrogens
of E-ring of podophyllotoxin and ILE D5, ARG D4 and ASP C124. The docking studies
and scores of 5a and 5e were in good agreement with observed IC50 values.
3. Experimental
3.1. General procedure for the synthesis of 4b-(prop-2-ynyloxy)
epipodophyllotoxin (3)
Podophyllotoxin (1) and propargyl alcohol (2) was dissolved in dichloromethane
(10 ml) and added Boron trifloride etherate slowly drop wise over a period of
10 minutes while maintaining the temperature at ꢁ20 ꢂC and stirring was continued
for 2 hours at the same temperature. The combined organic layers were washed con-
secutively with cold dilute HCl and brine solution. The extracted organic solution was
dried under reduced pressure to afford a crude product which was subjected to col-
umn to afford pure product (3).
3.2. General procedure for the synthesis of sugar azides (4a-l)
Sugar azides were synthesised by the reported procedures in the literature based on
Bertho’s method. Firstly, Sugars are acetylated, then brominated with HBr in AcOH
(33%) at 0 ꢂC and finally made to react with NaN3 under reflux conditions for 2 h in
the presence of PTC to yield corresponding glycosyl azides, which were subjected to
column chromatography to afford pure products (4a–l)
3.3. General procedure for the synthesis of podophyllotoxin-triazole hybrids
(5a-l)
O-Propargylated podophyllotoxin (3) and Ag (I)-NHC was dissolved in dry EtOH and
added sugar azide (4a–l). The reaction mass was stirred for 30 minutes. The organic
layer was separated and the aqueous layer extracted with dichloromethane. The com-
bined organic layers were dried and evaporated under reduced pressure to afford a
crude product which was subjected to column chromatography to afford pure
products (5a–l).
4. Conclusion
In conclusion, we have developed a simplistic catalytic approach for the regioselective
synthesis of 1, 4-disubstiututed podophyllotoxin-glycosyl triazole hybrids in high yields
and in short period of time. To our knowledge, this is the first methodology devel-
oped, optimised and extensively studied with Ag (I)-NHCs as catalyst for click reaction.
These advantages make this methodology facile, appropriate and can be put into
practice to create diverse compound libraries in future. The synthesised hybrid

NATURAL PRODUCT RESEARCH
7
molecules 5a and 5e have shown impressive in vitro anticancer activity and thus iden-
tified as promising lead compounds for future studies such as in vivo studies. The suc-
cess of Ag (I)-NHCs as versatile catalysts for click reaction opens up entirely new vistas
in this field and paves the way for further design and chemical modification of podo-
phyllotoxin as anticancer agents. These promising findings and the computer model-
ling studies indicate that potent activities may be found in many related
podophyllotoxin derivatives in the future.
Acknowledgement
SN would like to thank UGC for financial assistance and study leave through MRP & Faculty
Development Programme respectively.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the University Grants Commission, Government of India under
Grant number UGC-MRP-5732/15/620.
ORCID
Srinivas Nerella
http://orcid.org/0000-0003-3750-8206
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