Synthesis, characterization and cytotoxicity studies of 1,2,3-triazoles and 1,2,4-triazolo [1,5-a] pyrimidines in human breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2018.05.020

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is essential for physiological functions of tissues and neovasculature. VEGFR signaling is associated with the progression of pathological angiogenesis in various types of malignancies, making it an attractive therapeutic target in cancer treatment. In the present work, we report the synthesis of 1,4-disubstituted 1,2,3-triazoles and 1,2,4-triazolo[1, 5-a]pyrimidine derivatives via copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and screened for their anticancer activity against MCF7 cells. We identified 1-(2′-ethoxy-4′-fluoro-[1,1′-biphenyl]-4-yl)-4-phenyl-1H-1,2,3-triazole (EFT) as lead cytotoxic agent against MCF7 cell lines with an IC₅₀ value of 1.69 μM. Further evaluation revealed that EFT induces cytotoxicity on Ishikawa, MDA-MB-231 and BT474 cells with IC₅₀ values of 1.97, 4.81 and 4.08 μM respectively. However, EFT did not induce cytotoxicity in normal lung epithelial (BEAS-2B) cells. Previous reports suggested that 1,2,3-triazoles are the inhibitors of VEGFR1 and therefore, we evaluated the effect of EFT on the expression of VEGFR1. The results demonstrated that EFT downregulates the expression of VEGFR1 in MCF7 cells. In summary, we identified a potent cytotoxic agent that imparts its antiproliferative activity by targeting VEGFR1 in breast cancer cells. The novel compound could serve as a lead structure in developing VEGFR1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2018.05.020

Accepted Manuscript
Synthesis, Characterization and Cytotoxicity Studies of 1, 2, 3-triazoles and 1,
2, 4-triazolo [1, 5-a] pyrimidines in Human Breast Cancer Cells
Maryam Gilandoust, Kachigere B. Harsha, Chakrabhavi Dhananjaya Mohan,
Ainiah Rushdiana Raquib, Shobith Rangappa, Vijay Pandey, Peter E. Lobie,
Basappa, Kanchugarakoppal S. Rangappa
PII:
S0960-894X(18)30420-7
https://doi.org/10.1016/j.bmcl.2018.05.020
BMCL 25837
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 January 2018
5 May 2018
9 May 2018
Please cite this article as: Gilandoust, M., Harsha, K.B., Mohan, C.D., Raquib, A.R., Rangappa, S., Pandey, V.,
Lobie, P.E., Basappa, Rangappa, K.S., Synthesis, Characterization and Cytotoxicity Studies of 1, 2, 3-triazoles and
1, 2, 4-triazolo [1, 5-a] pyrimidines in Human Breast Cancer Cells, Bioorganic & Medicinal Chemistry Letters
(2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, Characterization and Cytotoxicity Studies of 1, 2, 3-triazoles and
1, 2, 4-triazolo [1, 5-a] pyrimidines in Human Breast Cancer Cells
Maryam Gilandoust,a,# Kachigere B. Harsha,a,# Chakrabhavi Dhananjaya Mohan,b,# Ainiah
Rushdiana Raquib,c Shobith Rangappa,d Vijay Pandey,c Peter E. Lobie,c Basappa,e
Kanchugarakoppal S Rangappaa,*
aDepartment of studies in Chemistry, University of Mysore, Manasagangotri, Mysuru-
570006, India.
bDepartment of studies in Molecular Biology, University of Mysore, Manasagangotri,
Mysuru-570006, India.
cCancer Science Institute of Singapore, National University of Singapore, Singapore-117599.
dAdichunchangiri Institute for Molecular Medicine (AIMM), BG Nagara, Nagamangala
Taluk, Mandya district-571448, India.
eDepartment of studies in Organic Chemistry, University of Mysore, Manasagangotri,
Mysuru-570006, India.
#These authors contributed equally to this work.
*Corresponding author
Abbreviation:
EFT: 1-(2'-ethoxy-4'-fluoro-[1,1'-biphenyl]-4-yl)-4-phenyl-1H-1,2,3-triazole
ABSTRACT
Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is essential for
physiological functions of tissues and neovasculature. VEGFR signaling is associated with
the progression of pathological angiogenesis in various types of malignancies, making it an
attractive therapeutic target in cancer treatment. In the present work, we report the synthesis
of 1,4-disubstituted 1,2,3-triazoles
and 1,2,4-triazolo[1, 5-a]pyrimidine derivatives via
copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and screened for their
anticancer activity against MCF7 cells. We identified 1-(2'-ethoxy-4'-fluoro-[1,1'-biphenyl]-
4-yl)-4-phenyl-1H-1,2,3-triazole (EFT) as lead cytotoxic agent against MCF7 cell lines with
an IC50 value of 1.69 µM. Further evaluation revealed that EFT induces cytotoxicity on
Ishikawa, MDA-MB-231 and BT474 cells with IC50 values of 1.97, 4.81 and 4.08 µM

respectively. However, EFT did not induce cytotoxicity in normal lung epithelial (BEAS-2B)
cells. Previous reports suggested that 1,2,3-triazoles are the inhibitors of VEGFR1 and
therefore, we evaluated the effect of EFT on the expression of VEGFR1. The results
demonstrated that EFT downregulates the expression of VEGFR1 in MCF7 cells. In
summary, we identified a potent cytotoxic agent that imparts its antiproliferative activity by
targeting VEGFR1 in breast cancer cells. The novel compound could serve as a lead structure
in developing VEGFR1 inhibitors.
Keywords: 1,2,3-triazole; 1,2,4-triazole; Angiogenesis; VEGFR1; Anticancer agent.
Angiogenesis is the process of formation of a new blood vessel from the pre-existing
vasculature and vascular endothelial cells. Angiogenesis is essential for nutrient supply,
disposal of metabolic wastes, growth, and development [1]. Angiogenesis is a key player in
several pathological conditions including cancer, wound healing, inflammation, rheumatoid
arthritis, ischemic cardiovascular diseases and age-related macular degeneration [2].
However, aberrant angiogenesis is one of the fundamental processes responsible for the
transformation of the dormant tumor to malignancy [3]. Initially, Judah Folkman postulated
that angiogenesis is essential for tumor proliferation and tumor releases proangiogenic factors
called tumor angiogenesis factors (TAF) which are essential for neovasculature [4]. Further
studies on angiogenesis revealed that vascular endothelial growth factor (VEGF) is the
pivotal proangiogenic mitogen that stimulates the growth of new blood vessels by interacting
with vascular endothelial growth factor receptors (VEGFR) [5]. VEGFR belongs to receptor
tyrosine kinase superfamily with three major subtypes namely, VEGFR1, VEGFR2, and
VEGFR3. The binding of VEGF to VEGFR results in the receptor dimerization and
activation of an intracellular signaling cascade that leads to the expression of genes
responsible for cell survival, proliferation, migration, angiogenesis, and vascular permeability

[6]. Even though VEGFR2 has relatively major role than VEGFR1 in VEGF-mediated
signaling, it is noteworthy that VEGFR1 mediates the survival of endothelial cells and
contributes to the progression of cancer [7, 8]. VEGF has been reported to act as an intracrine
survival factor in breast cancer cells through its binding to VEGFR1 and the same study also
revealed that knockdown of VEGFR2 had no effect on the survival of tested cancer cells [9].
In another study, Dales et al. investigated the prognostic value of VEGFRs and reported that
expression of VEGFR1 is positively correlated with the high risk of metastasis and relapse
[10]. They concluded that VEGFR1 may further be considered as a potential tool to evaluate
the aggressiveness of breast cancer [10]. Taken together, these reports support the intimate
relationship between VEGFR1 and cancer progression and encourage the development of
VEGFR1 inhibitors as anticancer therapeutics.
Triazole-based heterocycles were identified as biologically important pharmacophores and
were reported to possess good anticancer potential targeting in multiple types of malignancies
[11]. Several studies have suggested that triazoles target VEGFR to induce their anticancer
activity. Specifically, (1,2,3-triazol-4-yl)benzenamines were identified as potent inhibitors of
VEGFR1 and VEGFR2, and the activity was comparable to Vatalanib in both homogenous
time-resolved fluorescence (HTRF) enzymatic and cellular assays [12]. In another study,
1,2,4-triazol-3-yl-anilines were described as potent ATP-competitive inhibitors of VEGFR1
and 2 [13]. In continuation of our efforts in exploring the anticancer potential of various
heterocycles [14-20], we prepared the series of novel 1,2,3- and 1,2,4-triazoles and
investigated the effect of newly synthesized compounds on breast cancer (MCF7) cells. The
lead compounds 1-(2'-ethoxy-4'-fluoro-[1,1'-biphenyl]-4-yl)-4-phenyl-1H-1,2,3-triazole
(EFT, 3a) and 1-(4-(benzo [b]thiophen-2-yl)phenyl)-4-phenyl-1H-1,2,3-triazole (3j) were
chosen to investigate the possible VEGFR inhibitory activity in breast cancer cells.

Preparation of 1,4 disubstituted 1,2,3 triazoles 3(a-j): The synthesis of the title compounds
was carried out as illustrated in Scheme 1 (Figure 1A). In the present work, 1-(4-
bromophenyl)-4-phenyl-1,2,3-triazole (1) was synthesized via copper (I)-catalyzed azide-
alkyne cycloaddition (CuAAC) between 4-bromophenylazide and phenylacetylene in the
presence of sodium ascorbate and CuSO4.5H2O in tert-butanol and water mixture as the
solvent system. In the next step, 1-(4-bromophenyl)-4-phenyl-1,2,3-triazole was used for
Suzuki-Miyaura cross-coupling reactions. We treated 1-(4-bromophenyl)-4-phenyl-1,2,3-
triazole with various aryl /heteroaryl boronic acids (2) in presence of K2CO3 and Pd(II)
catalyst under a nitrogen atmosphere to yield a novel series of 1,4 disubstituted 1,2,3 triazoles
3(a-j) (Scheme 1) with a yield of 70-92% as summarised in table 1. The structure of all the
synthesized compounds 3(a-j) were confirmed by nuclear magnetic resonance (NMR) and
high-resolution mass spectral (HRMS) analysis. All the final compounds 3(a-j) showed a
characteristic singlet around 8.2 δ corresponding to a proton on the triazole ring. Spectral
analysis data is provided as supplementary information.

Table 1: Structure of 1,4-disubstituted 1,2,3-triazole derivatives 3(a-j) and cytotoxic activity
against breast cancer cells.
Entry
Compound
Yield
(%)
IC50 (µM))
MCF7 BT474
MDA-
Ishikawa
MB-231
1.69
NT
4.08
NT
4.81
NT
1.97
92
82
3a
3b
NT
70
73
<50
<50
NT
NT
NT
NT
NT
NT
3c
3d
90
92
70
<50
<50
<50
NT
NT
NT
NT
NT
NT
NT
NT
NT
3e
3f
3g
3h
90
<50
NT
NT
NT
80
85
<50
NT
NT
NT
NT
NT
3i
3j
14.4
<50
NT: Not Tested

Preparation of [1, 2, 4] triazolo [1, 5-a] pyrimidine derivatives 7(a-k) [21]: The synthesis of
the title compounds was carried out as illustrated in Scheme 2 (Figure 1B). Initially, starting
from the transition metal free coupling reaction between 2-chloro-5-bromopyrimidine (4)
with hydrazine hydrate to produce the corresponding hydrazine (5) which was condensed
with formaldehyde to afford hydrazones. Further, hydrazone intermediate was in situ cyclized
in the presence of IPh(OAc)2 to yield 5-bromo-[1,2,4]-triazolo[1,5-a]pyrimidine (6).
Similarly, scaffold (6) was crossed-coupled with various aryl/heteroaryl boronic acids under
inert (nitrogen) atmosphere in the presence of K2CO3 and palladium(II) catalyst to obtain
novel series of [1,2,4]triazolo[1,5-a]pyrimidines 7(a-k) in 65-90% yield as shown in table 2.
Table 2: Structure of [1,2,4]-triazolo [1,5-a] pyrimidine derivatives 7(a-k) and cytotoxic
activity against breast cancer cells.
Entry
Compound
Yield
(%)
IC50 (µM)
MCF7
MDA-
MB-231
90
93.8
50.3
7a
73
70
>100
NT
>100
>100
7b
7d
7e
7g
85
80
>100
NT
>100
>100

90
88
>100
3.91
>100
>100
7h
7i
65
>100
>100
7k
NT: Not Tested
1,2,3-triazoles elicit growth inhibitory effect on breast cancer cells: Initially, we investigated
the growth inhibitory effect of 1,2,3-triazoles against MCF7 cells and 1,2,4-triazolo-
pyrimidines against MCF7 and MDA-MB-231 cells using the AlamarBlue assay [22, 23].
The detailed methodology is provided in the supplementary information. Among the tested
compounds, 1-(2'-ethoxy-4'-fluoro-[1,1'-biphenyl]-4-yl)-4-phenyl-1H-1,2,3-triazole (EFT, 3a)
and 1-(4-(benzo[b]thiophen-2-yl)phenyl)-4-phenyl-1H-1,2,3-triazole (3j) were identified as
lead anticancer compounds with an IC50 values of 1.69 and 14.4 µM for MCF7 cells
respectively. Further, we evaluated the effect of EFT on Ishikawa, MDA-MB-231 and BT474
cells and IC50 values were found to be 1.97, 4.81 and 4.08 µM respectively. In addition, we
also evaluated the effect of new compounds on the viability of MCF7 cells at different time
points (24, 48 and 72 h) at 25 µM. The results revealed that EFT significantly decreases the
cell viability of MCF7 in a time-dependent manner (Figure 2A). However, 1,2,4-triazolo-
pyrimidines did not induce significant growth inhibition in the tested cells at 25 µM for 72 h.
Further, we tested the effect of EFT on normal lung epithelial (BEAS-2B) cells at 100 µM for
72 h using MTT assay as described earlier [24, 25]. The lead compound, EFT, did not induce
significant cytotoxicity against the tested non-diseased cells (Figure 2B).

EFT reduces the expression of VEGFR1 in breast cancer cells: Kiselyov and co-workers
described triazole derivatives as potent and ATP competitive inhibitors of VEGFR1 [12].
Therefore, we evaluated the effect of EFT and 3j on the expression of VEGFR1 in MCF7
cells by immunoblotting as described previously [26, 27]. The treatment with EFT
significantly downregulated the expression of VEGFR1 in a dose-dependent manner (Figure
3A and B). We also observed a marginal reduction in the expression of VEGFR1 on
treatment with compound 3j. VEGFR signaling activates a cascade of downstream pathways
including P38-MAPK, JNK, ERK, Akt/PKB and NF-κB [28]. Among them, Akt signaling
leads to an increased production of the hypoxia-inducible factor α (HIF1α and HIF2α)
transcription factors [29] and VEGFR1 (FLT-1) was reported as one of the target genes of

HIF-1 [30]. Moreover, in another similar study, Li et al. reported a new small molecule that
inhibited VEGFR2 tyrosine kinase activity and also reduced the expression of VEGFR2 [31].
Therefore, we speculate that EFT downregulates the expression of VEGFR1 via modulating
the activity of Akt and HIF-1 and further investigation is essential to decipher the possible
mechanism. No significant change was observed in the levels of β-Actin.
In conclusion, VEGFR family proteins have been emerged as potential drug targets to treat
various malignancies and several attempts have been made in implementing them as
therapeutic targets. Many antibodies and small-molecule based drugs targeting VEGFR
family proteins are approved for the treatment of several cancers. Sunitinib, sorafenib, and
pazopanib are the multikinase inhibitors that inhibit VEGFR1/2/3, PDGFR, c-Kit and
approved for the treatment of renal cell cancer and for gastrointestinal stromal tumors; renal
cell cancer and hepatocellular carcinoma; and advanced renal cell cancer respectively [32].
Several VEGFR inhibitors are in the different phases of clinical trials. These reports
emphasize the role of VEGFR family proteins as potential drug targets in the treatment of
cancers. 1,2,3- and 1,2,4-triazoles were identified as pharmacologically important scaffolds
because of their medicinal properties. Several research groups have developed various

methodologies for the preparation of triazoles and their conjugation with a broad range of
heterocyclic derivatives. Multiple reports have been demonstrated the action of triazole
derivatives on various cellular targets. In this report, we prepared series of 1,2,3- and 1,2,4-
triazole derivatives and identified EFT as a lead antiproliferative agent against breast cancer
cells. Further evaluation demonstrated that EFT decreases the expression of VEGFR in breast
cancer cells and these results are in agreement with the available literature. We hypothesize
that the reduction in VEGFR1 levels is due to the modulation of Akt and HIF-1 signaling that
needs to be verified in future studies. This is a report presenting triazoles as VEGFR1
inhibitors and the further comprehensive investigation is essential to dissect the detailed
molecular mechanism of lead compound and its off-targets.
Acknowledgments
This research was supported by University Grants Commission to KSR.
Conflict of interest
None declared
References and Notes
[1] J.A. Nagy, L. Benjamin, H. Zeng, A.M. Dvorak, H.F. Dvorak, Vascular permeability, vascular
hyperpermeability and angiogenesis, Angiogenesis, 11 (2008) 109-119.
[2] H. Dvorak, Angiogenesis: update 2005, Journal of Thrombosis and Haemostasis, 3 (2005) 1835-
1842.
[3] D. Xu, T.L. Wang, L.P. Sun, Q.D. You, Recent progress of small molecular VEGFR inhibitors as
anticancer agents, Mini reviews in medicinal chemistry, 11 (2011) 18-31.
[4] J. Folkman Tumor Angiogenesis: Therapeutic Implications, New England Journal of Medicine, 285
(1971) 1182-1186.
[5] R. Roopashree, C.D. Mohan, T.R. Swaroop, S. Jagadish, B. Raghava, K.S. Balaji, S. Jayarama,
Basappa, K.S. Rangappa, Novel synthetic bisbenzimidazole that targets angiogenesis in Ehrlich ascites
carcinoma bearing mice, Bioorganic & medicinal chemistry letters, 25 (2015) 2589-2593.
[6] S. Masoumi Moghaddam, A. Amini, D.L. Morris, M.H. Pourgholami, Significance of vascular
endothelial growth factor in growth and peritoneal dissemination of ovarian cancer, Cancer
metastasis reviews, 31 (2012) 143-162.
[7] M. Shibuya, Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1),
The international journal of biochemistry & cell biology, 33 (2001) 409-420.
[8] J. LeCouter, D.R. Moritz, B. Li, G.L. Phillips, X.H. Liang, H.P. Gerber, K.J. Hillan, N. Ferrara,
Angiogenesis-independent endothelial protection of liver: role of VEGFR-1, Science (New York, N.Y.),
299 (2003) 890-893.

[9] T.H. Lee, S. Seng, M. Sekine, C. Hinton, Y. Fu, H.K. Avraham, S. Avraham, Vascular endothelial
growth factor mediates intracrine survival in human breast carcinoma cells through internally
expressed VEGFR1/FLT1, PLoS medicine, 4 (2007) e186.
[10] J.P. Dales, S. Garcia, P. Bonnier, F. Duffaud, S. Carpentier, A. Djemli, O. Ramuz, L. Andrac, M.
Lavaut, C. Allasia, C. Charpin, [Prognostic significance of VEGF receptors, VEGFR-1 (Flt-1) and VEGFR-
2 (KDR/Flk-1) in breast carcinoma], Annales de pathologie, 23 (2003) 297-305.
[11] N. Ashwini, M. Garg, C.D. Mohan, J.E. Fuchs, S. Rangappa, S. Anusha, T.R. Swaroop, K.S. Rakesh,
D. Kanojia, V. Madan, Synthesis of 1, 2-benzisoxazole tethered 1, 2, 3-triazoles that exhibit
anticancer activity in acute myeloid leukemia cell lines by inhibiting histone deacetylases, and
inducing p21 and tubulin acetylation, Bioorganic & medicinal chemistry, 23 (2015) 6157-6165.
[12] A.S. Kiselyov, M. Semenova, V.V. Semenov, (1,2,3-Triazol-4-yl)benzenamines: synthesis and
activity against VEGF receptors 1 and 2, Bioorganic & medicinal chemistry letters, 19 (2009) 1344-
1348.
[13] A.S. Kiselyov, E. Piatnitski, D. Milligan, X. Ouyang, 1H-1,2,4-triazol-3-yl-anilines: novel potent
inhibitors of vascular endothelial growth factor receptors 1 and 2, Chemical biology & drug design,
69 (2007) 331-337.
[14] C.P. Baburajeev, C. Dhananjaya Mohan, H. Ananda, S. Rangappa, J.E. Fuchs, S. Jagadish, K.
Sivaraman Siveen, A. Chinnathambi, S. Ali Alharbi, M.E. Zayed, J. Zhang, F. Li, G. Sethi, K.S. Girish, A.
Bender, Basappa, K.S. Rangappa, Development of Novel Triazolo-Thiadiazoles from Heterogeneous
"Green" Catalysis as Protein Tyrosine Phosphatase 1B Inhibitors, Scientific reports, 5 (2015) 14195.
[15] C.P. Baburajeev, C.D. Mohan, S. Rangappa, D.J. Mason, J.E. Fuchs, A. Bender, U. Barash, I.
Vlodavsky, Basappa, K.S. Rangappa, Identification of Novel Class of Triazolo-Thiadiazoles as Potent
Inhibitors of Human Heparanase and their Anticancer Activity, BMC cancer, 17 (2017) 235.
[16] S. Anusha, C.D. Mohan, H. Ananda, C.P. Baburajeev, S. Rangappa, J. Mathai, J.E. Fuchs, F. Li, M.K.
Shanmugam, A. Bender, G. Sethi, Basappa, K.S. Rangappa, Adamantyl-tethered-biphenylic
compounds induce apoptosis in cancer cells by targeting Bcl homologs, Bioorganic & medicinal
chemistry letters, 26 (2016) 1056-1060.
[17] C.D. Mohan, V. Srinivasa, S. Rangappa, L. Mervin, S. Mohan, S. Paricharak, S. Baday, F. Li, M.K.
Shanmugam, A. Chinnathambi, M.E. Zayed, S.A. Alharbi, A. Bender, G. Sethi, Basappa, K.S. Rangappa,
Trisubstituted-Imidazoles Induce Apoptosis in Human Breast Cancer Cells by Targeting the
Oncogenic PI3K/Akt/mTOR Signaling Pathway, PloS one, 11 (2016) e0153155.
[18] N.C. Anilkumar, M.S. Sundaram, C.D. Mohan, S. Rangappa, K.C. Bulusu, J.E. Fuchs, K.S. Girish, A.
Bender, Basappa, K.S. Rangappa, A One Pot Synthesis of Novel Bioactive Tri-Substitute-Condensed-
Imidazopyridines that Targets Snake Venom Phospholipase A2, PloS one, 10 (2015) e0131896.
[19] V. Srinivas, C.D. Mohan, C.P. Baburajeev, S. Rangappa, S. Jagadish, J.E. Fuchs, A.Y. Sukhorukov,
Chandra, D.J. Mason, K.S. Sharath Kumar, M. Madegowda, A. Bender, Basappa, K.S. Rangappa,
Synthesis and characterization of novel oxazines and demonstration that they specifically target
cyclooxygenase 2, Bioorganic & medicinal chemistry letters, 25 (2015) 2931-2936.
[20] K.S. Rakesh, S. Jagadish, T.R. Swaroop, C.D. Mohan, N. Ashwini, K.B. Harsha, F. Zameer, K.S.
Girish, K.S. Rangappa, Anti-Cancer Activity of 2,4-Disubstituted Thiophene Derivatives: Dual
Inhibitors of Lipoxygenase and Cyclooxygenase, Medicinal chemistry (Shariqah (United Arab
Emirates)), 11 (2015) 462-472.
[21] M. Gilandoust, K.B. Harsha, S. Madan Kumar, K.S. Rakesh, N.K. Lokanath, K. Byrappa, K.S.
Rangappa, 5-Bromo-1,2,4-triazolo[1,5-a]pyrimidine, IUCrData, 1 (2016) x161944.
[22] N.B. Sulaiman, C.D. Mohan, S. Basappa, V. Pandey, S. Rangappa, H. Bharathkumar, A.P. Kumar,
P.E. Lobie, K.S. Rangappa, An azaspirane derivative suppresses growth and induces apoptosis of ER-
positive and ER-negative breast cancer cells through the modulation of JAK2/STAT3 signaling
pathway, International journal of oncology, 49 (2016) 1221-1229.
[23] C.P. Baburajeev, C.D. Mohan, G.S. Patil, S. Rangappa, V. Pandey, A. Sebastian, J.E. Fuchs, A.
Bender, P.E. Lobie, Basappa, K.S. Rangappa, Nano-cuprous oxide catalyzed one-pot synthesis of a

carbazole-based STAT3 inhibitor: a facile approach via intramolecular C-N bond formation reactions,
RSC Advances, 6 (2016) 36775-36785.
[24] C.D. Mohan, N.C. Anilkumar, S. Rangappa, M.K. Shanmugam, S. Mishra, A. Chinnathambi, S.A.
Alharbi, A. Bhattacharjee, G. Sethi, A.P. Kumar, Basappa, K.S. Rangappa, Novel 1,3,4-Oxadiazole
Induces Anticancer Activity by Targeting NF-κB in Hepatocellular Carcinoma Cells, Frontiers in
Oncology, 8 (2018).
[25] H.K. Keerthy, C.D. Mohan, K. Sivaraman Siveen, J.E. Fuchs, S. Rangappa, M.S. Sundaram, F. Li,
K.S. Girish, G. Sethi, Basappa, A. Bender, K.S. Rangappa, Novel synthetic biscoumarins target tumor
necrosis factor-alpha in hepatocellular carcinoma in vitro and in vivo, The Journal of biological
chemistry, 289 (2014) 31879-31890.
[26] A. Sebastian, V. Pandey, C.D. Mohan, Y.T. Chia, S. Rangappa, J. Mathai, C.P. Baburajeev, S.
Paricharak, L.H. Mervin, K.C. Bulusu, J.E. Fuchs, A. Bender, S. Yamada, Basappa, P.E. Lobie, K.S.
Rangappa, Novel Adamantanyl-Based Thiadiazolyl Pyrazoles Targeting EGFR in Triple-Negative Breast
Cancer, ACS Omega, 1 (2016) 1412-1424.
[27] C.D. Mohan, H. Bharathkumar, K.C. Bulusu, V. Pandey, S. Rangappa, J.E. Fuchs, M.K.
Shanmugam, X. Dai, F. Li, A. Deivasigamani, K.M. Hui, A.P. Kumar, P.E. Lobie, A. Bender, Basappa, G.
Sethi, K.S. Rangappa, Development of a novel azaspirane that targets the Janus kinase-signal
transducer and activator of transcription (STAT) pathway in hepatocellular carcinoma in vitro and in
vivo, The Journal of biological chemistry, 289 (2014) 34296-34307.
[28] G. Song, Y. Li, G. Jiang, Role of VEGF/VEGFR in the pathogenesis of leukemias and as treatment
targets (Review), Oncology reports, 28 (2012) 1935-1944.
[29] B.D. Manning, L.C. Cantley, AKT/PKB signaling: navigating downstream, Cell, 129 (2007) 1261-
1274.
[30] G.L. Semenza, HIF-1, O₂, and the 3 PHDs, Cell, 107 (2001) 1-3.
[31] Z. Li, B. Wang, L. Tang, S. Chen, J. Li, Quinazoline derivative compound (11d) as a novel
angiogenesis inhibitor inhibiting VEGFR2 and blocking VEGFR2-mediated Akt/mTOR /p70s6k
signaling pathway, Iranian journal of basic medical sciences, 19 (2016) 411-416.
[32] K.L. Meadows, H.I. Hurwitz, Anti-VEGF Therapies in the Clinic, Cold Spring Harbor Perspectives in
Medicine, 2 (2012) a006577.
Figure legends
Figure 1: A. Scheme 1. Synthesis of 1,4 disubstituted 1,2,3 triazole derivatives. Reagents and
condition: i) 4-Bromophenylazide, (+)-Sodium L-ascorbate, CuSO4. 5H2O, H2 t-Bu H
18 h ii -B(OH)2, Dikis, EtOH: H2O: Dioxan (1:1:5), 120° C, 40 min. B. Scheme 2.
Synthesis of [1, 2, 4]triazolo[1,5-a]pyrimidine derivatives. Reagents and condition: i)
NH2NH2·H2 Et H Δ 1.5 h ii CH2O, EtOH, RT, 2 h; and IPh(OAc)2, CH2Cl2 15 h
iii -B(OH)2, Dikis, EtOH: H2O:Dioxan (1:1:5), 120° C, 40 min.
Figure 2: 5000 MCF7 cells were seeded per well (96-well plate) and 25µM of respective
compounds were added after 24h. Cell viability assay was performed using AlamarBlue
reagent with 4h incubation after 24, 48, and 72h. B. 5000 BEAS-2B cells were seeded per

well (96-well plate) and MTT assay was performed to determine the cell viability of BEAS-
2B cells upon treatment with 25, 50 and 100µM of EFT for 24, 48 and 72h. EFT did not
induce a significant cytotoxic effect on BEAS-2B cells. Readings were taken in triplicates
and normalized against DMSO control.
Figure 3: A. 300,000 MCF7 (in RPMI+10%FBS) cells were seeded per 6cm dish. Cells were
allowed to grow for 24h before being subjected to compound treatment (in RPMI+2%FBS).
Cells were collected after 48h and harvested for Western Blot. 40µg of protein was loaded
per lane. Primary antibodies used were against VEGFR1 (ab32152) and β-actin (sc-47778).
B. Quantification and graphical representation of VEGFR1 expression after the treatment
with EFT and 3j in breast cancer cells. A gradual decrease in the expression of VEGFR1 was
observed in a dose-dependent manner.

Highlights
 VEGFR signalling is associated with the progression of various malignancies.
 1,2,3-triazoles and 1,2,4-triazolo-pyrimidines were synthesized via CuAAC reaction.
 1,2,3-triazole derivative (EFT) was identified the lead cytotoxic agent.
 EFT reduced the expression of VEGFR1 in MCF7 cells.