One-pot sequential alkynylation and cycloaddition: Regioselective construction and biological evaluation of novel benzoxazole-triazole derivatives

Article abstract of DOI:10.1021/co500135z

Individually, benzoxazole and triazole moieties are of significant biological interest owing to their importance in drugs and pharmaceuticals. To assess their combined biological impact when woven into one molecule, we designed a novel, regioselective, multicomponent, one-pot (MCOP) approach for the construction of benzoxazole-linked triazoles. The synthesis has been achieved in two sequential steps involving copper-catalyzed alkynylation of benzoxazole followed by a 1,3-dipolar cycloaddition reaction. By combination of these two bioactive units into one core, a series of new benzoxazole-triazole scaffolds has been synthesized and subjected to in vitro antibacterial and anticancer evaluation. Tests against clinical isolates of Staphylococcus aureus and Escherichia coli showed potent Gram-negative activity for compounds 4{1,1,1}, 4{1,1,4}, and 4{1,2,1}. The cytotoxicity of the synthesized library was determined against three cancer cell lines: HeLa, SKBr3, and Hep G2. Compound 4{2,2,2} showed significant cytotoxicity against all the cell lines. These preliminary bioassay evaluations strongly suggest the promise and scope of these novel molecules as therapeutic agents in medical science.

Full text of DOI:10.1021/co500135z

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Article
One-pot sequential alkynylation and cycloaddition:
Regioselective construction and biological
evaluation of novel benzoxazole-triazole derivatives
Ananya Srivastava, Leena Aggarwal, and Nidhi Jain
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/co500135z • Publication Date (Web): 14 Nov 2014
Downloaded from http://pubs.acs.org on November 18, 2014
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Oneꢀpot sequential alkynylation and cycloaddition:
Regioselective construction and biological
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evaluation of novel benzoxazoleꢀtriazole derivatives
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Ananya Srivastava, Leena Aggarwal and Nidhi Jain*
Department of Chemistry, Indian Institute of Technology, New Delhiꢀ110016
Email: njain@chemistry.iitd.ac.in
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KEYWORDS: copper(I) iodide, CꢀH activation, cycloaddition, MCOP (multicomponent oneꢀ
pot), cytotoxic
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ABSTRACT: Individually, benzoxazole and triazole moieties are of significant biological
interest owing to their importance in drugs and pharmaceuticals. To assess their combined
biological impact when woven into one molecule, we designed a novel, regioselective, multi
component, one pot (MCOP) approach for the construction of benzoxazoleꢀlinked triazoles. The
synthesis has been achieved in two sequential steps involving copper catalyzed alkynylation of
benzoxazole followed by a 1, 3ꢀdipolar cycloaddition reaction. By combining these two bioactive
units into one core, a series of new benzoxazoleꢀtriazole scaffolds has been synthesized and
subjected to in vitro antibacterial and anticancer evaluation. Tests against clinical isolates of
Staphylococcus aureus and Escherichia coli showed potent gramꢀnegative activity for
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compounds 4{1,1,1}, 4{1,1,4}, and 4{1,2,1}. The cytotoxicity of the synthesized library was
determined against three cancer cell lines: HeLa, SKBr3, and Hep G2. Compound 4{2,2,2}
showed significant cytotoxicity against all the cell lines. These preliminary bioassay evaluations
strongly suggest the promise and scope of these novel molecules as therapeutic agents in medical
science.
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INTRODUCTION
A triazole possessing three nitrogen atoms in a five membered heterocyclic ring is an
important pharmacophore and shows various clinical properties such as: antiꢀHIV,(1,2)
antimicrobial,(3ꢀ7) antiparasitic,(8) antiꢀinflammatory,(9) anticancer,(10,11) antimalarial,(12) and
antiviral.(13) Another clinically important substructure is benzoxazole, which is widely used as a
starting material for the synthesis of bioactive structures and forms a part of important drugs
such as flunoxaprofen and zoxazolamine. The benzoxazole derivatives have been investigated
for their inhibitory activity on eukaryotic DNA topoisomerase,(14) and are known to exhibit
antibacterial,(15,16) antitubercular,(17) anticancer,(18) antiparasitic,(19) and antiꢀHIV(20) activity
(Figure 1). In view of such therapeutic significance of these compounds, several methods
targeting their individual synthesis as well synthesis in combined form have been reported.(21ꢀ25)
These methods, however, suffer from drawbacks of being carried out in multiple steps; generate
a large number of byproducts, long reaction time, use of harsh reagents and conditions, and low
overall yield of the final product. With a view of harnessing the interesting biological properties
of triazole and benzoxazole molecules, we attempted designing a simple synthetic route to
construct these scaffolds.
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Figure 1. Benzoxazoleꢀtriazole scaffolds with varied biological activity [Ref.21ꢀ25].
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Ubiquitous in organic molecules, unactivated carbonꢀhydrogen (CꢀH) bonds are among the
simplest chemical moieties found in nature.(26) Although CꢀH bonds with dissociation energy in
the range 90ꢀ100 Kcal/mol are typically inert to chemical transformations, directly utilizing them
as reaction partners is highly desirable from the perspective of organic chemistry.(27) In targetꢀ
oriented synthesis, methods to convert CꢀH bonds to new carbonꢀcarbon (CꢀC) or carbonꢀ
heteroatom bonds can expedite the synthesis of architecturally complex intermediates en route to
drugs, agrochemicals, or other biologically active molecules.(28) Similarly in diversityꢀoriented
synthesis, an entire library of analogs can be synthesized directly from a lead compound via late
stage CꢀH functionalization.(29) This is in contrast to the traditional methods that require one to
return to the early stages to generate diversity which is costly in terms of chemical and energy
inputs. Therefore, in view of time and atom economies, transitionꢀmetal catalyzed direct
functionalization has gained enormous attention over the past decade as an effective and
straightforward method. To this end, we have adopted a novel multiꢀcomponent oneꢀpot (MCOP)
approach involving a copper catalyzed CꢀH activation of benzoxazole creating an alkynylꢀ
heteroaryl linkage in-situ; followed by tandem cycloaddition with azides. To the best of our
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knowledge, our method demonstrates the synthesis of an array of new benzoxazole linked
triazole derivatives through direct CꢀH bond functionalization of sp2ꢀhybridized (hetero)aryl
carbon with an spꢀhybridized carbon of an alkyne coupled with 1,3ꢀdipolar cycloaddition with
azide in one pot. The biological efficacy of the synthesized molecules has been evaluated by
carrying out antibacterial tests against the clinical isolates of S. aureus and E. coli, and in vitro
cytotoxic activity against human cervical, breast, and liver cancer cell lines.
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RESULTS AND DISCUSSION
Synthesis of benzoxazole-triazole derivatives
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The synthesis of benzoxazole linked triazoles was rationalized in two sequential steps in one pot.
This involved an in situ generation of alkyne from the corresponding dibromoꢀolefin precursor
via CꢀH activation of benzoxazole at Cꢀ2 position.(30,31) This was followed by 1,3ꢀdipolar
cycloaddition between the alkyne and benzyl azide to form the triazole ring. In the first step,
benzoxazole 1{1} (1.0 equiv.), (2,2ꢀdibromovinyl)benzene 2{1} (1.0 equiv.), CuI (20 mol%),
PPh3 (20 mol%) and LiOtꢀBu (1.0 equiv.) in dioxane were heated at 120 °C for 1h. This was
followed by addition of benzyl azide 3{1} (1.0 equiv.) to the reaction mixture and heating the
contents at 150 °C for another 12 h when the desired product 2ꢀ(3ꢀBenzylꢀ5ꢀphenylꢀ3Hꢀ
[1,2,3]triazolꢀ4ꢀyl)ꢀbenzooxazole 4{1,1,1} was formed. The product was isolated in 42% yield
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and was characterized by H, 13C NMR and mass spectrometry. The HNMR spectrum of
4{1,1,1} (Figure S2) showed 14 protons between 7ꢀ8 ppm corresponding to the protons of the
three benzene rings. The benzylic protons appeared at 6.06 ppm which confirmed the presence of
azide moiety in the product. Further, peaks at 134.98 and 141.01 ppm in 13CNMR confirmed
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formation of triazole ring. The HRMS (ESI) of the compound was found to be 375.1214, which
is in accordance to the mass of 4{1,1,1}.
Encouraged by these preliminary results, a detailed optimization study oriented towards
understanding the influence of solvent, base, ligand, catalyst, time and temperature was taken up
(Table 1). 1{1} was chosen as the substrate for carrying out various optimization reactions.
Screening of solvents such as DMF, acetonitrile, DMSO and dioxane indicated that dioxane was
very specific for this reaction, as the reaction did not take place in all the other solvents tested
(Table 1, entry 1-4). Next, evaluation of the reaction with inorganic bases weaker than LiOtꢀBu
such as KOtꢀBu, NaH, KOH, K2CO3, Cs2CO3, and K3PO4 showed that except LiOtꢀBu, none of
the bases yielded any product in this reaction (Table 1, entry 5-10). Increasing the amount of
LiOtꢀBu to 2.0 and 3.0 equiv. increased the yield of 4{1,1,1} to 49% and 64% respectively.
Screening of copper and palladium salts as catalyst showed that product formation occurred only
with CuI and CuBr as catalysts (Table 1, entry 4, 12) while no trace of product was seen with
Cu(OAc)2 and Pd(OAc)2 (Table 1, entry 13, 14). To study the effect of the ligand in controlling
the reaction yield, different ligands were used. It was found that in the absence of any ligand, no
product was formed (Table 1, entry 18) and all the starting material was recovered. With
nitrogen and oxygen containing ligands, however, poor yield of product 4{1,1,1} was obtained
(Table 1, entry 15-17). It was with triphenylphosphine that the yield was found to be maximal
(Table 1, entry 4). After deriving the best conditions for the first step, optimization of
temperature for the next step which is a thermal 1,3ꢀdipolar cycloaddition reaction was carried
out. It was found that cyclization proceeded only at or above 150 °C while at a lower temperature
of 120 °C, no product was seen.
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Table 1. Optimization of reaction conditions.a
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Entry
1.
Catalyst
CuI
Ligand
PPh3
PPh3
PPh3
PPh3
Base
Solvent
DMF
Yieldb %
LiOtꢀBu
LiOtꢀBu
LiOtꢀBu
LiOtꢀBu
ꢀ c
2.
CuI
CH3CN
DMSO
Dioxane
ꢀ c
ꢀ c
3.
CuI
4.
CuI
64(61)d(42)e(49)f
5.
6.
CuI
CuI
CuI
CuI
CuI
CuI
CuI
CuBr
PPh3
PPh3
PPh3
PPh3
PPh3
PPh3
PPh3
PPh3
KOtꢀBu
NaH
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
ꢀ c
ꢀc
7.
KOH
ꢀc
8.
K2CO3
Cs2CO3
K3PO4
ꢀ
ꢀc
9.
ꢀc
10.
11.
12.
ꢀc
ꢀc
LiOtꢀBu
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13.
14.
15.
16.
17.
18.
Cu(OAc)2
Pd(OAc)2
CuI
PPh3
PPh3
LiOtꢀBu
LiOtꢀBu
LiOtꢀBu
LiOtꢀBu
LiOtꢀBu
LiOtꢀBu
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
ꢀ c
ꢀ c
LꢀProline
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14
ꢀ c
CuI
2,2’ꢀBipyridine
8ꢀHydroxyquinoline
ꢀ
CuI
CuI
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aReaction conditions: 1{1} (1.0 mmol), 2{1} (1.0 mmol), 3{1} (1.0 mmol), catalyst (10 mol%),
ligand (15 mol%), base (3.0 equiv) in solvent (2 mL) for 1h at 120 ºC and for 12h at 150 ºC.
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bYield of isolated product, reaction was run for 24h, 20 mol% CuI, With 1.0 equiv. of LiOtꢀ
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Bu, fWith 2.0 equiv. of LiOtꢀBu.
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After optimization, the versatility of this protocol was ascertained by synthesizing a variety of
benzoxazoleꢀtriazole scaffolds. Chemset of dibromoolefins 2 (Figure 2) with methoxy, methyl,
bromo, nitro and trifluoromethyl substituents at o/p positions in the benzene ring was prepared in
quantitative yields from the corresponding commercially available aldehydes via the Ramirez
procedure.(32) Chemset of benzyl azides 3 (Figure 2) with various electron withdrawing
substituents such as cyano, nitro, fluoro and difluoro was prepared quantitatively from the
corresponding benzyl bromides by reaction with sodium azide. As shown in Table 2, the MCOP
synthesis attempted thereafter with the synthesized building blocks was quite facile and enabled
construction of a library of benzoxazoleꢀtriazole derivatives 4 in moderate to high yields.
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Benzoxazoles
1
2
Dibromoolefins 2
Br
Br
Br
Br
Br
OCH3
1
4
5
6
3
2
Benzyl azides
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Figure 2. Structure and numbering of the building blocks.
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It was found that of the chemset 2, 2{2} with pꢀmethoxy substitution gave the highest yield of
products with various azides (Table 2, entry 5-9). The reaction conditions were also well
tolerated by ortho-bromo substituted olefin 2{4} and 2ꢀ[3ꢀBenzylꢀ5ꢀ(2ꢀbromoꢀphenyl)ꢀ3Hꢀ
[1,2,3]triazolꢀ4ꢀyl]ꢀbenzooxazole 4{1,4,1} was obtained in moderate yield (Table 2, entry 12).
However, the reaction failed completely with dibromoolefins having strongly deactivating
groups like nitro 2{5} and trifluoromethyl 2{6} (Table 2, entry 17, 18). Screening of the chemset
3 showed that those with electron withdrawing substituents such as fluoro, cyano and nitro gave
moderate to high yield of products in contrast to those with electron donating substituent like
methyl 3{6} which did not yield any product (Table 2, entry 10). By and large, the electronics
on benzyl azide influenced the product yield and it followed the trend: cyano (Table 2, entry 3,
7, 15) > nitro (Table 2, entry 4, 8, 16) > difluoro (Table 2, entry 2, 6, 14) > fluoro (Table 2,
entry 9). Moreover, it was found that the reaction with 5ꢀmethyl derivative of benzoxazole 1{2}
gave higher yield of products (Table 2, entry 13-16) compared to its unmethylated counterpart.
Interestingly, it was observed that of the two isomeric cycloaddition products possible, only
one regioisomer of benzoxazoleꢀtriazole derivatives 4 was formed selectively. Since 1HNMR and
13CNMR spectra could not conclusively distinguish between the two isomers, the structural
assignment of the regioisomeric triazoles was based on NOESY analysis.
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Table 2. Substrate scope of reaction.a
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entry
1
product
yielda (%)
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4{1,1,1}
2
3
4{1,1,2}
4{1,1,3}
4{1,1,4}
4{1,2,1}
4{1,2,2}
4{1,2,3}
4{1,2,4}
4{1,2,5}
4{1,2,6}
4{1,3,2}
4{1,4,1}
4{2,2,1}
4{2,2,2}
4{2,2,3}
4{2,2,4}
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4{1,5,1}
4{1,6,1}
0
0
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aReaction conditions: 1 (1.0 mmol), dibromoolefin 2 (1.0 mmol), azide 3 (1.0 mmol), CuI (10
mol%), PPh3 (15mol%), LiOtꢀBu (3.0 equiv) in dioxane (2 mL) for 1h at 120 ºC and 12h at 150
ºC. bYield of isolated product.
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Figure 3 shows the NOESY HNMR spectrum of the isomer 4{1,2,1} formed from the
reaction between 1{1}, 2{2} and 3{1}. As is evident from the spectrum, crossꢀpeaks are obtained
due to interaction between benzyl protons (Hb) and orthoꢀaryl protons (Hc) of benzyl azide
possible with both the isomers 4{1,2,1} and 4{1,2,1}’. However, absence of crossꢀpeaks due to
interaction between Hb and phenyl protons of the dibromoolefin fragment (Ha) suggested the
regioisomer to be 4{1,2,1} (see the encircled area in (Figure 3). If the product was 4{1,2,1}’,
then a cross peak should have appeared due to the spatial vicinity between Ha and Hb. The
distance between Ha and Hb in 4{1,2,1} is way too large to observe any cross peak. The reason
behind the regioselective formation of single isomer can be rationalized based on the fact that
benzoxazole acts as a powerful electronꢀwithdrawing group thereby making the conjugated
alkyne considerably electronꢀdeficient and polarized.(33) This then directs the dipolar
cycloaddition with azide in a specific controlled manner leading to a single regioisomer. Since
chromatographic separation of regioisomeric triazoles is tedious, routes which can selectively
yield a particular single isomer are attractive. In this regard, the approach offers a regiospecific
transformation for synthesis of benzoxazole linked triazoles.
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Figure 3. Two possible regioisomers (4{1,2,1} and 4{1,2,1}’) and partial NOESY HNMR
spectrum.
173 Mechanism
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An overview of the proposed mechanistic cycle with copper(I) as the active catalytic species (A)
is shown in figure 4. The first step involves deprotonation of benzoxazole by LiOtꢀBu followed
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by lithiumꢀcopper transꢀmetallation to generate a three coordinated copper(I) intermediate B.(34)
This is followed by generation of alkynyl bromide from 1,1ꢀdibromoolefin by
dehydrobromination effected by LiOtꢀBu. The alkyne gets added onto B via oxidative addition
resulting a four coordinated copper(III) complex C.(35ꢀ37) D is thereafter expelled from complex
C via reductive elimination and the catalytic copper(I) species A is regenerated back. Formation
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of D was confirmed by isolating it at this stage and characterizing by H and 13C NMR
spectroscopy. The next step is the tandem [3+2] cycloaddition of D with benzyl azide under
thermal conditions to yield the benzoxazoleꢀtriazole derivative (E). The regioselectivity of E can
be explained by considering the polarity of the triple bond conjugated with the benzoxazole
moiety (partial positive charge is localized on βꢀcarbon) and the dipole of the benzyl azide as
depicted in figure 4. Due to this localization of charges, one regioisomer is selectively obtained.
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Figure 4. Proposed mechanism.
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190 Antibacterial activity
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The chemset 4 was screened for their in vitro antibacterial activity against the representative
Gramꢀpositive (Staphylococcus aureus) and Gramꢀnegative (Escherichia coli) clinical isolates
and the results are summarized in table 3. A standard technique(38) based on the optical detection
of the bacterial suspension at 620 nm (OD620) was applied to evaluate the antibacterial activity of
the test compounds. The antibacterial activity was evaluated by subtracting the OD for each
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replicate at T0 from the OD for each replicate at T24. Cultures were incubated at 37 °C in an
orbital shaker at 70 rpm to prevent adherence and clumping of cells. The antibacterial activity
(i.e. growth inhibition) for the test sample was determined using the formula:
% Inhibition = [1ꢀ (OD test sample/OD of control)] x 100.
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Table 3. Antibacterial activity of chemset 4 against Staphylococcus aureus and Escherichia coli.
Staphylococcus aureus
Escherichia coli
Entry
no.
Compound
MIC ± SD
(mg/mL)
IC50 ± SD
(mg/mL)
MIC ± SD
IC50 ± SD
(mg/mL)
(mg/mL)
1.
2.
4{1,1,1}
4{1,1,2}
4{1,1,3}
4{1,1,4}
4{1,2,1}
4{1,2,2}
4{1,2,3}
4{1,2,4}
4{1,2,5}
4{1,3,2}
4{1,4,1}
4{2,2,1}
4{2,2,2}
4{2,2,3}
4{2,2,4}
4.1 ± 0.16
ND
8.2 ± 0.28
0.8 ± 0.03
2.1 ± 0.07
2.7 ± 0.08
3.2 ± 0.09
1.8 ± 0.05
1.2 ± 0.04
2.1 ± 0.07
3.1 ± 0.08
2.2 ± 0.07
2.7 ± 0.06
2.2 ± 0.06
2.4 ± 0.06
3.1 ± 0.07
2.3 ± 0.06
2.7 ± 0.08
2.6 ± 0.07
ND
ND
0.9 ± 0.04
1.1 ± 0.05
0.4 ± 0.03
0.2 ± 0.01
1.1 ± 0.05
1.3 ± 0.06
0.9 ± 0.02
1.4 ± 0.06
1.6 ± 0.06
0.9 ± 0.03
1.3 ± 0.05
1.4 ± 0.06
1.2 ± 0.04
0.9 ± 0.03
3.
ND
4.
3.6 ± 0.14
4.9 ± 0.20
ND
5.2 ± 0.19
6.1 ± 0.23
ND
5.
6.
7.
ND
ND
8.
ND
ND
9.
>11 ± 0.40
ND
>15 ± 0.57
ND
10.
11.
12.
13.
14.
15.
ND
ND
3.8 ± 0.15
>10 ± 0.39
ND
>20 ± 0.76
>15 ± 0.51
ND
> 19 ± 0.43
> 16 ± 0.58
16.
17.
Ampicillin
Gentamicin
0.42 ± 0.01
ND
0.78 ± 0.03
ND
ND
ND
0.09 ± 0.003
0.17 ± 0.006
202
203
204
205
206
207
Minimum Inhibitory Concentration (MIC): Exponentially growing bacterial cells were treated with different
concentrations of test compounds for 24 h and cell growth inhibition was analyzed through the antimicrobial assay.
IC50 is defined as the concentration at which a 50% decrease in cell number occurs compared to the control cultures
in the absence of an inhibitor. The values represent the mean ± S.D. of three individual observations. Mean per cent
decreases in cell number of three independent experiments were used to calculate the linear regression equation. ND
= not determined
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The antibacterial detection limits for each compound expressed as Minimum Inhibitory
Concentration (MIC) and Inhibitory Concentration 50% (IC50) were also determined. It was
found that while most of the synthesized compounds showed little or no antibacterial activity,
4{1,2,1} was reasonably effective against E. coli with the MIC and IC50 values of 0.2 ± 0.01
mg/mL and 1.2 ± 0.04 mg/mL respectively. This was followed by compound 4{1,1,4} with MIC
and IC50 values ranging between 0.4 ± 0.03 mg/mL and 1.8 ± 0.05 mg/mL respectively. The
antibacterial activity against S. aureus in general, was less compared to E. coli isolates; possibly
due to its resistance against the test compounds. To verify that the observed activity was only
due to benzoxazoleꢀtriazole moiety, control experiments were performed in which S. aureus and
E. coli cultures were treated with the carrier solution under same experimental conditions in the
absence of test compounds. No antibacterial activity was seen in all these cases indicating that
the effect was purely due to the test compounds in carrier solution. The antibacterial activity of
4{1,2,1} with dose dependence was examined by evaluating the % inhibition against variable
concentrations of 4{1,2,1}. As can be seen in Figure 5, the % inhibition increased with increase
in concentration from 0.2 mg/mL to 4.0 mg/mL. On further increasing the concentration, no
significant increase in % inhibition was observed. Moreover, the inhibitory effect was more
prominent against E. coli compared to S. aureus. A similar trend was also observed with
compounds 4{1,1,4} and 4{1,1,1} against E. coli (data not shown). The % inhibition data
followed the trend 4{1,2,1}>4{1,1,4}>4{1,1,1} which is also in agreement with the results
obtained for the IC50 values. The MIC and IC50 values for positive control of S. aureus was
found to be 0.42 ± 0.01 mg/mL and 0.78 ± 0.03 mg/mL respectively (Table 3, entry 16). For the
positive control of E.coli, the MIC and IC50 values were found to be 0.09 ± 0.003 mg/mL and
0.17 ± 0.006 mg/mL respectively (Table 3, entry 17).
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Figure 5. Histogram showing the dose dependent activity of 4{1,2,1} towards E. Coli and S.
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234 Cytotoxicity evaluation
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The cytotoxicity of chemset 4 was assessed using MTT [3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide] assay which relies on the conversion of a tetrazolium compound
(MTT) to purple colored formazan crystals by the activity of mitochondrial dehydrogenase
present in cells. Cell viability was calculated using the equation: % Viability = [(OD compound
treated cultures) / (OD untreated cell control cultures)] × 100. To begin with, the cytotoxicity of
chemset 4 was determined at a concentration of 6.0 µg/mL against HeLa cells as shown in figure
6. For all the compounds except 4{2,2,2}, we observed almost no cytotoxicity recovering greater
than 80% of cells at all concentrations tested compared to untreated controls. Interestingly
however, compound 4{2,2,2} displayed moderate cytotoxicity against HeLa cells and was
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compared with the positive control daunomycin (figure 6). While the % viability of cancer cells
was 22 ± 0.86 % for daunomycin, for compound 4{2,2,2} the % viability was 45 ± 1.84%.
Encouraged by this result, we decided to carry out further cytotoxicity evaluation on other cell
lines like SKBr3, and Hep G2 using 4{2,2,2}.
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Figure 6. Cytotoxicity evaluation of chemset 4 on HeLa cell line using MTT assay at
concentration 6.0 µg/mL. C represents the control experiment. The results shown are mean ±
SD of five individual observations.
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The IC50 values of compound 4{2,2,2} were found to be 6.8 ± 0.34 µg/mL, 7.1 ± 0.41 µg/mL and
11.2 ± 0.64 µg/mL against HeLa, SKBr3 and Hep G2 cancer cells, respectively. A dose
dependent cytotoxicity analysis of 4{2,2,2} indicated that increasing the concentration of
4{2,2,2} from 5 – 30 µg/mL led to decrease in the % viability of the three cancer cell lines as
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shown in figure 7A indicating the potential applicability of this compound as a cancer
therapeutic agent. These results suggested a potential anticancer activity vested in this new class
of benzoxazoleꢀtriazole scaffolds.
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Figure 7. (A) Dose dependent cytotoxicity of 4{2,2,2} on HeLa, SKBr3, Hep G2 cell lines using
the MTT assay. The results shown are mean ± SD of five individual observations. (B) Light
Micrographs of untreated and treated HeLa cancer cells. Left panel corresponds to untreated
cells, middle panel represents the 4{2,2,2} treated cells, and the right panel corresponds to
daunomycin treated cells. Concentration of 4{2,2,2} and daunomycin was 10 µg/mL. Cells were
incubated at 37 °C in a 5% CO2 humidified incubator.
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To corroborate the observations of MTT assay, cells were also examined microscopically after
48 h of treatment with 4{2,2,2}. It was found that the cells not only underwent a marked
reduction in viability but also showed a change in their morphology as depicted in figure 7B.
Cells appeared to be round and more granular which signifies some kind of apoptotic change in
the cellular cytoplasm. As expected, the cell morphology with standard anticancer drug,
daunomycin suffered a much greater disintegration and collapsed.
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273 Conclusions
In conclusion, we report a regioselective synthesis and biological evaluation of a new class of
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benzoxazoleꢀtriazole derivatives. The compounds have been synthesized in moderate yields via
facile and efficient multicomponent one pot synthetic approach. This has been realized by
invoking a copper catalyzed direct CꢀH activation of benzoxazole followed by tandem thermal
cycloadditon between alkyne and azide. The synthesized compounds have been evaluated for
their antibacterial and anticancer activity. While most of the compounds were found to be weak
antibacterials, 4{1,1,1}, 4{1,1,4}, and 4{1,2,1} showed good gramꢀnegative activity. Compound
4{2,2,2} has been identified to have potent cytotoxic effect against cancer cell lines: HeLa,
SKBr3, and Hep G2 and is comparable to the control daunomycin. Our work is the first report on
the synthesis and application of this class of compounds as efficient antiꢀcancer agents. Future
efforts shall be directed towards modifying the benzoxazoleꢀtriazole scaffold and developing
promising lead structures through SAR as anticancer agents and establish their mechanism of
action.
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General experimental details:
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Chemicals were either purchased or purified by standard techniques without special instructions.
1
The products were purified by column chromatography on silica gel. H NMR and 13C NMR
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spectra were measured on a Bruker DPXꢀ 300 MHz spectrometer (1H 300 MHz, C 75 MHz)
using CDCl3 as the solvent with tetramethylsilane (TMS) as the internal standard at room
temperature. All 13C NMR spectra were obtained with complete proton decoupling. Chemical
shifts are given in δ relative to TMS, the coupling constants J are given in Hz. The ESIꢀMS was
performed on MICROTOFꢀII mass instrument. Data are reported as follows: chemical shift (in
ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad),
coupling constant (in Hz), and integration. Flash column chromatography was performed on
silica gel (230–400 mesh) and analytical thinꢀlayer chromatography was carried out using 250
µm commercial silica gel plates. Visualization of the developed chromatogram was performed
by UV absorbance.
General procedure for the synthesis of 1,1-dibromoolefins 2:
1,1ꢀdibromoolefins were synthesized using the Ramirez procedure starting from commercially
available aldehydes. To a solution of benzaldehyde (10.0 mmol, 1.0 equiv.) and carbon
tetrabromide (10.5 mmol, 1.5 equiv.) in dichloromethane (25 mL) at 0 °C, a solution of PPh3
(21.0 mmol, 2.0 equiv.) in dichloromethane (10 mL) was added slowly. The reaction mixture
was stirred until complete conversion of aldehyde was observed before being concentrated. The
resulting crude was purified by flash chromatography over SiO2 with pure hexane to afford
compound 2{1} as a colorless liquid (84% yield).
(2,2-dibromovinyl)benzene 2{1}:
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1H NMR (CDCl3, 300MHz): δ 7.59 (d, J=6.3 Hz, 2H), 7.54 (s, 1H), 7.43 (d, J=6.6 Hz, 2H); C
NMR (CDCl3, 75 MHz): δ 136.90, 135.32, 128.61, 128.47, 89.75. The 1H and 13C NMR spectra
of remaining members of chemset 2 are essentially similar to the reported data. (39ꢀ41)
General procedure for the synthesis of azides 3:
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Following a reported procedure, a solution of sodium azide (1.5 equiv.) in water (8 mL) was
added dropwise over 1 h to a solution of 2,5ꢀdifluorobenzyl bromide (1.0 equiv.) in acetone (20
mL) at 0°C. The reaction was allowed to warm up to 23 °C and stirred for 16 h. Acetone was
removed under reduced pressure at 25 °C and the reaction mixture was extracted with hexane.
Thereafter, the combined organic layers were put together and dried over Na2SO4 and the solvent
was removed under reduced pressure to afford benzyl azide as colorless oil (100% yield).
2-azido-1,4-difluorobenzene 3{2}:
1H NMR (CDCl3, 300MHz): δ 6.97ꢀ6.87 (m, 3H), 4.26 (s, 2H); C NMR (CDCl3, 75 MHz): δ
13
160.28, 158.27, 157.06, 155.05, 124.67, 116.83, 48.04. The H and 13C NMR spectra of
1
remaining members of chemset 3 are essentially similar to the reported data. (42,43)
General procedure for one pot synthesis of benzoxazole-triazole derivatives 4:
A dried round bottom flask was charged with benzoxazole (0.840 mmol, 1.0 equiv.), CuI (0.084
mmol, 10 mol%), PPh3 (0.126 mmol, 15 mol%), LiOtBu (2.521 mmol, 3.0 equiv.). 2,2ꢀ
dibromovinylbenzene (0.840 mmol, 1.0 equiv.) was diluted into dioxane (10 mL) and the
solution was added to the R.B. flask. The reaction mixture was then refluxed with water
condenser in a preꢀheated oil bath at 120 °C for 1h. After this 2,5ꢀdifluorobenzyl azide (1.0
o
equiv.) was added and the contents were heated up to 150 C and stirred for 12 h.The reaction
mixture was diluted with EtOAc and water was added. This mixture was extracted with EtOAc
and the combined organic layers were put together and dried upon Na2SO4. Solvents were
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removed under reduced pressure and the crude was purified by column chromatography on silica
gel to afford 4{1,1,2} as a white solid.
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2-[3-(2,5-Difluoro-benzyl)-5-phenyl-3H-[1,2,3]triazol-4-yl]-benzooxazole 4{1,1,2}:
1H NMR (CDCl3, 300 MHz): δ 7.60ꢀ7.58 (m, 3H), 7.31ꢀ7.27 (m, 3H), 7.26 (d, J=1.8 Hz, 1H),
7.23 (d, J=4.2 Hz, 1H), 7.21 (d, J=2.7 Hz, 1H), 7.06 (s, 1H), 6.85ꢀ6.64 (m, 3H), 5.98 (s, 2H); 13C
NMR (CDCl3, 75 MHz): δ 160.29, 157.89, 157.07, 154.69, 152.73, 150.01, 149.01, 140.94,
129.73, 129.22, 128.91, 128.42, 126.46, 125.30, 121.93, 120.67, 116.10, 115.82, 110.96, 47.48;
HRMS (ESI): calcd for C24H14F2N4O [M+H]+ 389.1208, found 389.1215.
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General procedure for in vitro antibacterial activity:
The bacterial cultures of S aureus and E coli were isolated from the clinical samples and
characterized using routine laboratory technique. S aureus and E coli were grown for 24 h on
blood agar plates (5% sheep red blood cells, and tryptic soy agar (TSA)) and macConkey agar at
37 °C, respectively. The cells were suspended in 1 % saline solution and the optical density of
0.6 at 600 nm (OD600) was adjusted to obtain a density of 7 x 105 colony forming units per mL
(CFU/mL), which was confirmed by standard plate count method. The antibacterial test was
performed using an optical method. At first, the stock solution was prepared in DMSO and
further diluted using the sterile PBS. The sample was always freshly prepared and thoroughly
mixed before performing the antibacterial test. The desired concentrations of the samples were
treated with the bacterial cultures aseptically and the samples were incubated for 24 h at 37 °C in
shaker incubator. After incubation, the antibacterial activity was calculated by recording the
OD600 and the percentage bacterial growth inhibition was calculated as formula given below.
% Inhibition = [1ꢀ (OD test sample/OD of control)] x 100.
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Each of the experiments was performed in triplicate. Control experiments were also performed
without the test samples.
General procedure for in vitro anticancer activity:
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The cytotoxicity/anticancer activity of the compounds was assessed using MTT [3ꢀ(4,5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide] assay which relies on the conversion of
a tetrazolium compound (MTT) to purple colored formazan crystals by the activity of
mitochondrial dehydrogenase present in cells. Cell viability was calculated using the equation: %
Viability = [(OD compound treated cultures) / (OD untreated cell control cultures)] × 100. The
analysis method was as follows. Briefly, cell lines were cultured in Tꢀ75 tissue culture flasks
(Nunc, Denmark) at 37 °C in a 5% CO2 humidified incubator in suitable media and supplements.
Cells were seeded in separate wells containing 100 ꢁL medium at a final density of 2 × 104
cells/well, in a 96 well microtiter plates under the similar experimental conditions. After
overnight incubation, the cells were treated with various concentrations of the test compounds
(5–50 ꢁg/mL) or dimethyl sulfoxide (DMSO) (carrier solvent) separately in a final volume of
200 ꢁL in triplicates. After 24 h of incubation, 10 ꢁL of MTT reagent (5 mg/mL) was added to
each well and the plate was incubated at 37 °C in the dark for 4 h. After this, the media along
with MTT was removed and the formazan crystals were solubilised in DMSO (100 ꢁL/well). The
reduction of MTT was measured by recording the absorbance at 570 nm by using a microplate
reader. Effects of the test compounds on cell viability were calculated using cells treated with
DMSO as control.
ACKNOWLEDGMENTS
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This work has been financially supported by the Department of Science and Technology, India
under the project RP02555. A.S. thanks Council of Scientific and Industrial Research, India for
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graduate fellowship. The authors thank DSTꢀFIST for funding the ESIꢀHRMS facility at IIT
Delhi. Our sincere thanks to Ms Ritu Khosla, Institute of Liver and Biliary Sciences, Delhi
(ILBS) and Dr. Kanwaljit Kaur, National Institute of Immunology, Delhi (NII) for helping us
with the cell line experiments.
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SUPPORTING INFORMATION
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1HNMR, 13CNMR, and mass spectra of chemset 4. This material is available free of charge via
the Internet at http://pubs.acs.org.
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ABBREVIATIONS: CuI (copper(I) iodide), DMSO (dimethylsulfoxide), DMF (N,Nꢀ
dimethylformamide), NOESY (Nuclear Overhauser effect spectroscopy).
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AUTHOR INFORMATION
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Corresponding Author
* Phone: 011ꢀ2659ꢀ1562. Eꢀmail: njain@chemistry.iitd.ac.in
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