Triazole-Pyridine Dicarbonitrile Targets Phosphodiesterase 4 to Induce Cytotoxicity in Lung Carcinoma Cells

Article abstract of DOI:10.1002/cbdv.201900234

Phosphodiesterase 4 (PDE4) is a key enzyme involved in the hydrolysis of cyclic adenosine monophosphate (cAMP) and widely expressed in several types of cancers. The inhibition of PDE4 results in an increased concentration of intracellular cAMP levels that imparts the anti-inflammatory response in the target cells. In the present report, two series of triazolo-pyridine dicarbonitriles and substituted dihydropyridine dicarbonitriles were synthesized using green protocol (TBAB in refluxed water). We next evaluated the title compounds for their cytotoxicity towards lung cancer (A549) cells and identified 7′-[4-(methylsulfonyl)phenyl]-5′-oxo-1′,5′-dihydrospiro[cyclohexane-1,2′-[1,2,4]triazolo[1,5-a]pyridine]-6′,8′-dicarbonitrile (5h) and 7′-(1-methyl-1H-imidazol-2-yl)-5′-oxo-1′,5′-dihydrospiro[cyclohexane-1,2′-[1,2,4]triazolo[1,5-a]pyridine]-6′,8′-dicarbonitrile (5j) as lead analogs with the IC₅₀ values of 15.2 and 24.1 μm, respectively. Furthermore, all the new compounds were tested for PDE4 inhibitory activity and 5j showed relatively good inhibitory activity towards PDE4 with inhibition of 50.9 % at 10 μm. In silico analysis demonstrated the favorable interaction of the title compounds with the target enzyme. Taken together, the present study introduces a new scaffold for the development of novel PDE4 inhibitors to fight against inflammatory diseases.

Full text of DOI:10.1002/cbdv.201900234

Title: Triazole-Pyridine Dicarbonitrile Targets Phosphodiesterase 4 to
Induce Anticancer Activity in Lung Carcinoma Cells
Authors: Kanchugarakoppa Rangappa, Hosadurga K Keerthy,
Surender Mohan, Basappa Basappa, Shobith Rangappa,
H Bharathkumar, Fredrick Svensson, Andreas Bender,
Chakrabhavi D Mohan, and Rakesh Bhatnagar
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900234
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900234

10.1002/cbdv.201900234
Chemistry & Biodiversity
Triazole-Pyridine Dicarbonitrile Targets Phosphodiesterase 4 to Induce
Anticancer Activity in Lung Carcinoma Cells
Hosadurga
K
Keerthya,b,#
,
Surender Mohanc,#, Basappad,e,#, Hanumantharayappa
Bharathkumard, Shobith Rangappaf, Fredrick Svenssonb, Andreas Benderb, Chakrabhavi
Dhananjaya Mohang,*, Kanchugarakoppal S Rangappah,* Rakesh Bhatnagarc,*
aDepartment of Chemistry, Centre for Post Graduate Studies and Research, St. Agnes College,
Bendur, Mangalore, 575002, India
bCentre for Molecular Informatics, Department of Chemistry, University of Cambridge,
Cambridge CB2 1EW, United Kingdom
cLaboratory of Molecular Biology and Genetic Engineering, School of Biotechnology,
Jawaharlal Nehru University, New Delhi 110067, India
dLaboratory of Chemical Biology, Department of Chemistry, Bangalore University, Central
College campus, Palace Road, Bangalore-560001, India
eLaboratory of Chemical Biology, Department of Studies in Organic Chemistry, University of
Mysore, Manasagangotri, Mysore-570006, India
fAdichunchanagiri Institute for Molecular Medicine, BG Nagara-571448, Nagamangala
Taluk, Mandya District, India
gDepartment of Studies in Molecular Biology, University of Mysore, Manasagangotri, Mysore-
570006, India
hInstitution of Excellence, Vijnana Bhavan, University of Mysore, Manasagangotri, Mysore-
570006, India
#These authors contributed equally to this work.
*Corresponding authors
Correspondence to:
rangappaks@gmail.com
rakeshbhatnagar@mail.jnu.ac.in
cd.mohan@yahoo.com
Abstract
Phosphodiesterase 4 (PDE4) is a key enzyme involved in the hydrolysis of cyclic adenosine
monophosphate (cAMP) and widely expressed in several types of cancers. The inhibition of
PDE4 results in an increased concentration of intracellular cAMP levels that imparts the anti-
inflammatory response in the target cells. In the present report, two series of triazolo-pyridine
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10.1002/cbdv.201900234
Chemistry & Biodiversity
dicarbonitriles and substituted dihydropyridine dicarbonitriles were synthesized using green
protocol (TBAB in refluxed water). We next evaluated the new compounds for their
cytotoxicity towards lung cancer (A549) cells and identified 7-(4-(methylsulfonyl)phenyl)-5-
oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile
(5h) and 7-(1-methyl-1H-imidazol-2-yl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5j) as lead analogs with the IC50 value of 15.2
and 24.1 µM respectively. Furthermore, all the new compounds were tested for PDE4
inhibitory activity and 5j showed relatively good inhibitory activity towards PDE4 with
inhibition of 50.9% at 10 µM. In silico analysis demonstrated the favorable interaction of the
title compounds with the target enzyme. Taken together, the present study introduces a new
scaffold for the development of novel PDE4 inhibitors to fight against inflammatory diseases.
Keywords: Phosphodiesterase 4, Anticancer, Synthetic small molecule, Chemotherapy.
Introduction
PDE4 catalyzes the hydrolysis of cyclic adenosine monophosphate (cAMP), an important
second messenger involved in the regulation of various biological functions such as apoptosis,
inflammation and lipid metabolism.[1] The relay of signals from the activated membrane
spanned G-protein coupled receptors lead to the stimulation of adenylyl cyclase which
generates intracellular cAMP from adenosine triphosphate. The cAMP, in turn, activates
protein kinase A that phosphorylates multiple transcription factors which are involved in the
modulation of expression of the cAMP target genes.[2] PDE4 hydrolyzes and reduces the cAMP
level and counterbalance its cellular effects.[3] PDE4 inhibitors increase the levels of
intracellular cAMP which possess a wider spectrum of anti-inflammatory effects on the
effector cells associated with inflammatory bowel disease, chronic obstructive pulmonary
diseases, psoriasis and asthma.[4-6] Studies have demonstrated that PDE4 is widely expressed
in cancers of brain, lung, colorectal, prostate and promote their growth. Moreover, PDE4
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10.1002/cbdv.201900234
Chemistry & Biodiversity
inhibitors suppressed cell proliferation and induced tumor regression in the tested preclinical
cancer models.[7-11] Taken together, these evidences suggested that PDE4 as a potential
molecular therapeutic target for cancer therapy.
The antitumor efficacy of triazoles and pyridines have been well-documented in several
preclinical cancer studies.[12-17] Furthermore, the antitumor potential of triazole conjugated
pyridine has also been established in various malignancies.[18-21] Some of the also reports
indicated that triazolo-pyridine conjugates target PDE4 to induce their biological effect.[22, 23]
Specifically, synthesis of a PDE4 inhibitor, 5,6-Dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-
triazolo[4,3-a]pyridine was described.[23] In another study, the substitution at the 3-position of
5,6-Dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-triazolo[4,3-a]pyridine tricycle led to a 2-thienyl
analog (Tofimilast) which is a potent PDE4 inhibitor with low oral bioavailability.[24] Apart,
there are limited number of reports on the preparation of triazolo pyridine dicarbonitriles and
many of them employ the use of strong base, hazardous organic solvents, expensive catalysts
and tedious laboratory procedures.[25] Considering these drawbacks, we used environmentally
benign conditions such as water as solvent and tetrabutyl ammonium bromide (TBAB) as a
mild water-soluble catalyst for the synthesis of triazolo-pyridine dicarbonitriles and
dihydropyridine carbonitriles. In our effort to explore the pharmacological properties of
synthetic small molecules,[26-30] new spiro[1,2,4]triazolo[1,5-a]pyridine dicarbonitriles were
synthesized and evaluated for their possible anticancer effect towards lung cancer cells and
PDE4 inhibitory activity.
Results and Discussion
Chemistry
We reported the synthetic methodologies for the preparation of various bioactive heterocyclic
moieties.[31-34] In the present study, the green procedure for the synthesis of triazolo-pyridine
dicarbonitriles and dihydropyridine dicarbonitriles catalyzed by TBAB in water has been
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10.1002/cbdv.201900234
Chemistry & Biodiversity
reported. In a similar eco-friendly approach, the synthesis of highly functionalized pyridines,
1,2,4-triazolo[1,5-a]pyridines, and 1,2,4-triazolo[1,5-a]isoquinolines were reported earlier [35,
36]. The reaction between 4-bromo benzaldehyde (1), malononitrile (2), cyanoacetohydrazide
(3) and cyclohexanone (4) was considered to optimize the reaction conditions using various
solvents and catalysts (Table 1).
Initially, we attempted the reaction in refluxed water with organic bases such as piperidine (2
eq) and triethylamine (2 eq) which resulted in no product formation even after refluxing for 8h.
The usage of inorganic catalysts such as sodium silicate, p-TSA in refluxed water resulted in
relatively low yield of 7-(4-bromophenyl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5a). The reaction with TBAB in organic
solvents including ethanol, ethyl acetate, and toluene resulted in a low yield. TBAB in an equal
ratio of refluxed ethanol and water gave 5a with a yield of 72%. Finally, TBAB (1.5 eq) in
refluxed water resulted in the formation of 5a in relatively higher yield (92%). Thus, the TBAB
(2 eq) in refluxed water was considered for further reactions with another aryl/heteroaryl
aldehydes containing electron-withdrawing and electron donation groups to prepare new series
of spiro[1,2,4]triazolo[1,5-a]pyridine dicarbonitriles (5a-l) (Table 2).
We next employed this protocol to synthesize substituted dihydropyridine dicarbonitriles (6a-
i) (Table 3). The formation of (6a-i) from a variety of aryl and heteroaryl aldehydes bearing
electron-withdrawing and electron donation groups resulted in high yields of products within
15-20 min. Hence, the present protocol can be applied to a broad choice of differently
substituted aldehydes. Though we have not performed the mechanistic studies, we propose the
theoretical possible mechanism in the formation of spiro[1,2,4]triazolo[1,5-a]pyridine
dicarbonitriles and substituted dihydropyridine dicarbonitriles. The compound 1 and
malononitrile condense to form arylidinemalononitrile which combines with hydrazone of
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10.1002/cbdv.201900234
Chemistry & Biodiversity
cyanoacetohydrazide and cyclohexanone resulting in the formation of 5a-i. Similarly,
arylidinemalononitrile reacts with cyanoacetohydrazide to yield 6a-i.
Spiro[1,2,4]triazolo[1,5-a]pyridine dicarbonitriles elicit cytotoxicity towards lung carcinoma
cells
The newly synthesized triazolo-pyrimidines were investigated for their cytotoxic potential
towards lung carcinoma (A549) cells using in vitro cytotoxicity assay [37, 38]. Among the new
compounds, 7-(4-(methylsulfonyl)phenyl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (compound 5h) and 7-(1-methyl-1H-imidazol-
2-yl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-a]pyridine-2,1'-cyclohexane]-6,8-
dicarbonitrile (compound 5j) displayed good anticancer activity with the IC50 value of 15.2 and
24.1 µM respectively. The other derivatives presented the moderate growth inhibitory activity
with the IC50 values ranging between 26 to 36 µM (Table 3). These inhibitory values of title
compounds indicate that the electron withdrawing polar functional groups are effective when
compared to electron releasing groups.
Pharmacology
Compound 5j inhibits PDE4 enzyme activity in vitro
All the newly synthesized compounds were tested for PDE4 inhibitory activity at the
concentration of 10 µM (Table 3). Compounds 5j showed relatively good inhibitory activity
towards PDE4 compared to the other structural analogs with inhibition of 50.9% at 10 µM.
Moreover, most of the other compounds displayed moderate to marginal inhibition ranging
between 10 to 44% at 10 µM. The compounds with <10% inhibition were considered as
inactive. Rolipram was used as the positive control which showed inhibition of 87.3%. This
result supports the compound 5j that showed anti-cancer activity by targeting PDE4 enzyme
of human lung cancer cells.
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Chemistry & Biodiversity
In silico analysis
For nine out of the twelve compounds docking identified a common binding mode. The
predicted binding mode of the best-scored compound is shown in Figure 1A. Docking predicts
hydrogen bond interactions with His-234 and a water molecule anchored to Asp-392 and Asn-
395. The compound also forms a face to face stacking interaction with Phe-446 and an edge to
face interaction with Phe-414. Comparing the binding mode to that of the co-crystallized ligand
it is similar with a large volume overlap. Both the hydrogen bond to water and the pi-stacking
interactions are formed by the co-crystallized ligand as well (Figure 1B).
Experimental Section
All chemicals used were of reagent grade and used without further purification. Thin layer
chromatography (TLC) was performed on 0.25 mm silica gel plates (60F254, Merck). Column
chromatography separations were obtained on silica gel (200-400 mesh). 1H NMR spectra were
13
recorded on Bruker NMR 400 MHz instrument in DMSO-d6 solvent [39]. C NMR spectra
were obtained on Agilent NMR instrument at 100 MHz in CDCl3/DMSO-d6 solvent. Chemical
shifts are expressed in ppm downfield relative to TMS. LC-MS analysis was performed on
Agilent LC-MS with electron ionization (ESI) +ve and –ve mode. Elemental analyses were
recorded using PerkinElmer CHNS analyzer.
General procedure for the synthesis of spiro[[1,2,4]triazolo[1,5-a]pyridine dicarbonitriles
In a 50 mL round bottom flask, 2-cyano acetohydrazide (1.0 eq) and cyclohexanone (1.0 eq)
were stirred at 90 ºC in tetrabutyl ammonium bromide solution (8 mL, 10 mol%) for 20 min.
To this reaction mixture, an aromatic/heterocyclic carbaldehyde (1.0 eq) and malononitrile (1.2
eq) were added and stirred further for 1-2.5 h at 100 ºC (scheme 1). The reaction was monitored
by TLC using n-hexane and ethyl acetate eluent. After completion of the reaction, solid was
filtered through Whatman filter paper, air dried and purified by column chromatography.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Synthesis of (7-(4-bromophenyl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-a]pyridine-
2,1'-cyclohexane]-6,8-dicarbonitrile) (5a)
The product 5a was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min. To
this reaction mixture, 4-bromobenzaldehyde (1.0 eq) and malononitrile (1.2 eq) were added
o
and stirred further for 45 min at 100 C. This compound was obtained as light brown color
1
solid with 92% yield. IR (KBR ν in cm-1): 3415, 3360, 2220, 2225, 1660; H NMR (DMSO-
d6, 400 MHz δ in ppm): 7.75-7.71 (d, 2H, Ar-H), 7.52 (s, 1H, C-NH), 7.35-7.31 (d, 2H, Ar-H),
2.19 (s, 1H, N-NH), 1.82-1.71 (m, 4H, Ali-H), 1.42-1.31 (m, 6H, Ali-H); 13C NMR (CDCl3,
100 MHz δ in ppm) 170.12, 161.54, 160.25, 130.76, 128.41, 121.53, 110.20, 114.61, 85.15,
76.62, 33.11, 23.53, 22.11; Anal. Calcd. for C19H16BrN5O: C, 55.62; H, 3.93; N, 17.07; found
C, 55.58; H, 3.90; N, 17.10 % , LCMS (MM:ES+APCI) (M+H)+ 410.
Synthesis of 7-(3-bromo-4-fluorophenyl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5b)
The product 5b was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min. To
this reaction mixture, 3-bromo-4-fluoro benzaldehyde (1.0 eq) and malononitrile (1.2 eq) were
o
added and stirred further for 1 hour at 100 C. This compound was obtained as yellow solid
with 90% yield. IR (KBR ν in cm-1): 3420, 3350, 2221, 2219, 1635; 1H NMR (DMSO-d6, 400
MHz δ in ppm): 7.69 (s, 1H, Ar-H), 7.41 (s, 1H, C-NH), 7.14-7.04 (m, 2H, Ar-H), 2.14 (s, 1H,
N-NH), 1.72-1.60 (m, 4H, Ali-H), 1.39-1.23 (m, 6H, Ali-H);13C NMR (CDCl3, 100 MHz δ in
ppm) 170.31, 165.56, 161.45, 129.59, 128.38, 126.11, 118.19, 115.81, 110.80, 87.12, 78.23,
34.95, 26.16, 24.21; Anal. Calcd. for C19H15BrFN5O: C, 53.29; H, 3.53; N, 16.35; found C,
53.32; H, 3.49; N, 16.31 %; LCMS (MM:ES+APCI) (M+H)+ 428.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Synthesis of 7-(3-bromo-4-methoxyphenyl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5c)
The product 5c was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min.
To this reaction mixture, 3-bromo-4-methoxy benzaldehyde (1.0 eq) and malononitrile (1.2 eq)
o
were added and stirred further for 45 min at 100 C. This compound was obtained as yellow
1
solid with 94% yield. IR (KBR ν in cm-1): 3420, 3380, 2219, 2220, 1645; H NMR (DMSO-
d6, 400 MHz δ in ppm): 7.35-7.25 (m, 3H, Ar-H), 7.04 (s, 1H, C-NH), 3.52 (s, 3H, -OCH3),
2.57 (s, 1H, N-NH), 1.92-1.70 (m, 4H, Ali-H), 1.44-1.21 (m, 6H, Ali-H); 13C NMR (CDCl3,
100 MHz δ in ppm) 167.28, 158.04, 157.13, 154.02, 131.19, 130.26, 128.07, 114.97, 113.32,
110.81, 87.90, 74.08, 56.19, 34.01, 26.21, 23.05; Anal. Calcd. for C20H18BrN5O2: C, 54.56; H,
4.12; N, 15.91; found C, 54.50; H, 4.15; N, 15.88 %; LCMS (MM:ES+APCI) (M+H)+ 440.
Synthesis of
7-(3,4-dimethoxyphenyl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5d)
The product 5d was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min. To
this reaction mixture, 3, 4-dimethoxy benzaldehyde (1.0 eq) and malononitrile (1.2 eq) were
o
added and stirred further for 45 min at 100 C. This compound was obtained as white solid
1
with 93% yield. IR (KBR ν in cm-1): 3390, 3356, 2220, 2224, 1650; H NMR (DMSO-d6, 400
MHz δ in ppm): 7.42-7.46 (m, 3H, C-NH), 7.15 (s, 1H, Ar-H), 6.92-6.90 (m, 2H, Ar-H), 3.71
13
(s, 6H, -CH3), 2.12 (s, 1H, N-NH), 1.55-1.49 (m, 4H, Ali-H), 1.36-1.29 (m, 6H, Ali-H); C
NMR (CDCl3, 100 MHz δ in ppm) 19.51, 160.54, 157.26, 134.88, 130.90, 128.41, 115.21,
114.93, 113.21, 85.90, 79.32, 56.24, 32.65, 26.81, 21.93; Anal. Calcd. for C21H21N5O3: C,
64.44; H, 5.41; N, 17.89; found C, 64.40; H, 5.38; N, 17.92 %; LCMS (MM:ES+APCI)
(M+H)+ 392.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Synthesis
of
5-oxo-7-(4-propoxyphenyl)-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8- dicarbonitrile (5e)
The product 5e was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min. To
this reaction mixture, 4-propoxy benzaldehyde (1.0 eq) and malononitrile (1.2 eq) were added
and stirred further for 45 min at 100 oC. This compound was obtained as white solid with 92%
1
yield. IR (KBR ν in cm-1): 3410, 3375, 2220, 2215, 1650; H NMR (DMSO-d6, 400 MHz δ
in ppm): 7.84-7.79 (d, 2H, Ar-H), 7.52 (s, 1H, C-NH), 7.16-7.11 (d, 2H, Ar-H), 6.42-6.35 (q,
2H, -CH2-), 2.17 (s, 1H, N-NH), 1.76-1.70 (m, 2H, -CH2-), 1.60-1.52 (m, 4H, Ali-H), 1.33-
1.21 (m, 6H, Ali-H); 13C NMR (CDCl3, 100 MHz δ in ppm) 168.39, 158.18, 156.27, 153.69,
128.81, 123.42, 116.30, 115.27, 113.18, 84.01, 75.35, 70.42, 32.01, 24.00, 22.46, 21.84, 10.52;
Anal. Calcd. for C22H23N5O2: C, 67.85; H, 5.95; N, 17.98; found C, 67.81; H, 5.98; N, 18.00
%; LCMS (MM:ES+APCI) (M+H)+ 390.
Synthesis of 7-(4-nitrophenyl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-a]pyridine-
2,1'-cyclohexane]-6,8-dicarbonitrile (5f)
The product 5f was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min.
To this reaction mixture, 4-nitro benzaldehyde (1.0 eq) and malononitrile (1.2 eq) were added
and stirred further for 1 hour at 100 oC. This compound was obtained as yellow solid with 89%
1
yield. IR (KBR ν in cm-1): 3395, 3382, 2221, 2220, 1648; H NMR (DMSO-d6, 400 MHz δ
in ppm): 8.39-8.28 (m, 2H, Ar-H), 7.64-7.56 (m, 2H, Ar-H), 7.41 (s, 1H, C-NH), 2.27 (s, 1H,
N-NH), 1.70-1.66 (m, 4H, Ali-H), 1.28-1.18 (m, 6H, Ali-H); 13C NMR (CDCl3, 100 MHz δ in
ppm) 167.03, 159.26, 157.64, 148.47, 140.21, 130.35, 122.18, 115.8 114.13, 88.50, 74.25,
34.42, 27.18, 25.29; Anal. Calcd. for C19H16N6O3: C, 60.63; H, 4.28; N, 22.33; found C, 60.59;
H, 4.30; N, 22.30 %; LCMS (MM:ES+APCI) (M+H)+ 377.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Synthesis of 5-oxo-7-(4-(trifluoromethyl)phenyl)-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5g)
The product 5f was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min.
To this reaction mixture, 4-trifluoromethyl benzaldehyde (1.0 eq) and malononitrile (1.2 eq)
o
were added and stirred further for 1 hour at 100 C. This compound was obtained as yellow
1
solid with 88% yield. IR (KBR ν in cm-1): 3390, 3350, 2220, 2222, 1650; H NMR (DMSO-
d6, 400 MHz δ in ppm): 8.39-8.28 (m, 2H, Ar-H), 7.64-7.56 (m, 2H, Ar-H), 7.41 (s, 1H, C-
NH), 2.27 (s, 1H, N-NH), 1.70-1.66 (m, 4H, Ali-H), 1.28-1.18 (m, 6H, Ali-H); 13C NMR
(CDCl3, 100 MHz δ in ppm) 167.03, 159.26, 157.64, 148.47, 140.21, 130.35, 122.18, 115.8
114.13, 88.50, 74.25, 34.42, 27.18, 25.29; Anal. Calcd. for C20H16F3N5O: C, 60.15; H, 4.04;
N, 14.27; found C, 60.11; H, 4.07; N, 14.23 %; LCMS (MM:ES+APCI) (M+H)+ 400.
Synthesis of 7-(4-(methylsulfonyl)phenyl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5h)
The product 5h was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min.
To this reaction mixture, 4-(methylsulfonyl) benzaldehyde (1.0 eq) and malononitrile (1.2 eq)
were added and stirred further for 1.5 hours at 100 oC. This compound was obtained as white
1
solid with 90% yield. IR (KBR ν in cm-1): 3418, 3345, 2222, 2218, 1640; H NMR (DMSO-
d6, 400 MHz δ in ppm): 8.05-7.90 (m, 4H, Ar-H), 7.32 (s, 1H, C-NH), 3.84 (s, 3H, -CH3), 2.30
13
(s, 1H, N-NH), 1.69-1.57 (m, 4H, Ali-H), 1.31-1.19 (m, 6H, Ali-H); C NMR (CDCl3, 100
MHz δ in ppm) 170.20, 161.93, 160.24, 139.85, 136.27, 129.29, 127.51, 116.22, 114.34, 87.56,
78.11, 48.50, 34.83, 25.16, 22.17; Anal. Calcd. for C18H15BrN6O: C, 52.57; H, 3.68; N, 20.44;
found C, 52.52; H, 3.70; N, 20.25 %; LCMS (MM:ES+APCI) (M+H)+ 411.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Synthesis
of
7-(6-bromopyridin-3-yl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5i)
The product 5i was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min.
To this reaction mixture, 4-bromopyrine-3-carbaldehyde (1.0 eq) and malononitrile (1.2 eq)
were added and stirred further for 1.5 hours at 100 oC. This compound was obtained as white
1
solid with 87% yield. IR (KBR ν in cm-1): 3395, 3348, 2220, 2219, 1650; H NMR (DMSO-
d6, 400 MHz δ in ppm): 8.35 (s, 1H, Py-CH), 7.83-7.80 (d, 1H, Ar-H), 7.56-7.53 (d, 1H, Ar-
H), 7.40 (s, 1H, C-NH), 2.49 (s, 1H, N-NH), 1.75-1.65 (m, 4H, Ali-H), 1.30-1.18 (m, 6H, Ali-
13
H); C NMR (CDCl3, 100 MHz δ in ppm) 168.32, 159.61, 158.09, 150.04, 140.19, 136.67,
130.79, 122.81, 116.02, 88.08, 74.21, 35.76, 26.85, 22.11; Anal. Calcd. for C20H19N5O3S: C,
58.67; H, 4.68; N, 17.10; found C, 58.60; H, 4.70; N, 17.12 %; LCMS (MM:ES+APCI)
(M+H)+ 410.
Synthesis of 7-(1-methyl-1H-imidazol-2-yl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5j)
The product 5j was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min.
To this reaction mixture, 1-methyl-1H-imidazole-2-carbaldehyde (1.0 eq) and malononitrile
o
(1.2 eq) were added and stirred further for 2 hours at 100 C. This compound was obtained as
1
off-white solid with 90% yield. IR (KBR ν in cm-1): 3390, 3345, 2222, 2221, 1638; H NMR
(DMSO-d6, 400 MHz δ in ppm): 7.74 (s, 1H, C-NH), 7.25-7.22 (m, 2H, Imi-H), 3.87 (s, 3H, -
13
CH3), 2.05 (s, 1H, N-NH), 1.64-1.60 (m, 4H, Ali-H), 1.37-1.31 (m, 6H, Ali-H); C NMR
(CDCl3, 100 MHz δ in ppm) 170.75, 160.22, 159.93, 136.01, 127.99, 120.48, 119.98, 102.04,
88.07, 74.01, 36.06, 33.12, 25.21, 22.52; Anal. Calcd. for C17H17N7O: C, 60.88; H, 5.11 N,
29.24; found C, 60.85; H, 5.07; N, 29.35 %; LCMS (MM:ES+APCI) (M+H)+ 336.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Synthesis of 7-(1H-indol-3-yl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-a]pyridine-
2,1'-cyclohexane]-6,8-dicarbonitrile (5k)
The product 5k was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min.
To this reaction mixture, 1H-indole-3-carbaldehyde (1.0 eq) and malononitrile (1.2 eq) were
o
added and stirred further for 2 hours at 100 C. This compound was obtained as brown solid
1
with 91% yield. IR (KBR ν in cm-1): 3410, 3385, 2218, 2220, 1645; H NMR (DMSO-d6, 400
MHz δ in ppm): 10.50 (s, 1H, Ind-NH), 7.88 (s, 1H, Ind-CH), 7.67 (s, 1H, C-NH), 7.44-7.40
(m, 4H, Ar-H), 2.16 (s, 1H, N-NH), 1.63-1.54 (m, 4H, Ali-H), 1.48-1.39 (m, 6H, Ali-H); 13C
NMR (CDCl3, 100 MHz δ in ppm) 170.12, 159.51, 158.18, 138.27, 130.11, 125.61, 121.78,
120.91, 115.63, 110.84, 106.45, 86.21, 74.17, 35.21, 26.42, 23.41; Anal. Calcd. for
C21H18N6O: C, 68.09; H, 4.90; N, 22.69; found C, 68.11; H, 4.85; N, 22.72 %; LCMS
(MM:ES+APCI) (M+H)+ 371.
Synthesis of 7-(2-methyl-1H-indol-3-yl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-
a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile (5l)
The product 5l was obtained by stirring 2-cyano acetohydrazide (1.0 eq) and cycloxexanone
(1.0 eq) at 90 oC in 8 mL of 10 mol% of tetrabutyl ammonium bromide solution for 20 min.
To this reaction mixture, 2-methyl-1H-indole-3-carbaldehyde (1.0 eq) and malononitrile (1.2
o
eq) were added and stirred further for 2.5 hours at 100 C. This compound was obtained as
1
brown solid with 85% yield. IR (KBR ν in cm-1): 3390, 3360, 2221, 2222, 1646; H NMR
(DMSO-d6, 400 MHz δ in ppm): 8.35 (s, 1H, Ind-NH), 7.42-7.38 (m, 2H, Ar-H), 7.26 (s, 1H,
C-NH), 7.08-7.00 (m, 2H, Ar-H), 2.22 (s, 3H, -CH3), 2.01 (s, 1H, N-NH), 1.43-1.39 (m, 4H,
Ali-H), 1.24-1.21 (m, 6H, Ali-H); 13C NMR (CDCl3, 100 MHz δ in ppm) 169.41, 157.23,
156.45, 133.11, 128.23, 124.25, 117.86, 114.86, 110.11, 86.93, 79.90, 34.20, 24.51, 21.91;
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Anal. Calcd. for C22H20N6O: C, 68.73; H, 5.24; N, 21.86; found C, 68.77; H, 5.22; N, 21.80
%; LCMS (MM:ES+APCI) (M+H)+ 385.
General procedure for the synthesis of 1,6-diamino-2-oxo-4-phenyl substituted-1,2-
dihydropyridine-3,5-dicarbonitriles
In a 50 mL round bottom flask, aldehyde (1.0 eq), malononitrile (1.0 eq), tetra butyl ammonium
bromide solution was taken and stirred for 10 min at 50ºC, followed by addition of 2-
cyanoacetohydrazide (1.0 eq) and stirring at 70ºC for 10 min (Scheme 2). The progress of the
reaction was monitored using TLC and after completion of the reaction, solid was filtered,
dried and purified by column chromatography.
Synthesis of 1,6-diamino-2-oxo-4-phenyl-1,2-dihydropyridine-3,5-dicarbonitrile (6a)
Melting point 238-240oC.[40]
Synthesis of 1,6-diamino-4-(4-bromophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile
(6b)
The product 6b was obtained from 4-bromobenzaldehyde (1.0 eq), malononitrile (1.0 eq) and
2-cyanoaceto hydrazide (1.0 eq). This compound was obtained as light brown color solid with
1
95% yield. Melting point: 232-234 oC IR (KBR ν in cm-1): 3406, 2244, 1674; H NMR (400
MHz, DMSO-d6, δ in ppm): 5.63(s, 2H, N-NH2), 7.48-7.51 (m, 2H, Ar-H), 7.57-7.62 (m, 2H,
13
Ar-H), 8.47 (s, 2H, C-NH2); C NMR (100 MHz, DMSO-d6, δ in ppm): 159.98, 159.89,
159.75, 156.98, 130.33, 130.29, 124.98, 116.89, 116.33, 115.76, 86.20, 74.40; Anal. Calcd.for
C13H8BrN5O: C, 47.29; H, 2.44; N, 21.21; found C, 47.19; H, 2.58; N, 21.28%; LCMS
(MM:ES+APCI) [M-1]- 330.
Synthesis of 1,6-diamino-2-oxo-4-(4-propoxyphenyl)-1,2-dihydropyridine-3,5-dicarbonitrile
(6c)
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10.1002/cbdv.201900234
Chemistry & Biodiversity
The product 6c was obtained from 4-propoxybenzaldehyde (1.0 eq), Malononitrile (1.0 eq) and
2-cyanoaceto hydrazide (1.0 eq). This compound was obtained as light brown color solid with
1
95% yield. Melting point: 226-228 oC, IR (KBR ν in cm-1): 3395, 2250, 1678; H NMR (400
MHz, DMSO-d6, δ inppm): 0.99 (t, 3H, CH3), 1.72 (m, 2H, CH2-CH3), 3.99 (t, 2H, CH2-
CH2), 5.60 (s, 2H, N-NH2), 7.03 (d, 2H, J = 8.8, Ar-H), 7.38 (d, 2H, J = 9.2, Ar-H), 8.35 (s,
13
2H, C-NH2); C NMR (100 MHz, DMSO-d6, δ in ppm): 159.77, 158.23, 157.29, 157.07,
132.99, 129.93, 128.29, 116.94, 115.92, 113.04, 112.79, 110.72, 86.94, 74.84, 69.92, 22.74,
11.84; Anal. Calcd.for C16H15N5O2: C, 62.13; H, 4.89; N, 22.64 ; found C, 61.99; H, 4.68; N,
22.78% ; LCMS (MM:ES+APCI) [M-1]- 308.
Synthesis
of
1,6-diamino-4-(3,4-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3,5-
dicarbonitrile (6d)
Melting point 291-292 oC.[36]
Synthesis of 1,6-diamino-4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile
(6e)
Melting point 3359- 361 oC.[36]
Synthesis of 1,6-diamino-4-(4-(methylsulfonyl)phenyl)-2-oxo-1,2-dihydropyridine-3,5-
dicarbonitrile (6f)
The product 6f was obtained from 4-(methylsulfonyl)benzaldehyde (1.0 eq), Malononitrile (1.0
eq) and 2-cyanoaceto hydrazide (1.0 eq). This compound was obtained as light brown color
solid with 97% yield., IR (KBR ν in cm-1): 3390, 2245, 1676; 1H NMR (400 MHz, DMSO-
d6, δ in ppm): 3.29 (s, 3H, -CH3), 5.66 (s, 2H, N-NH2), 7.74-7.77 (m, 2H, Ar-H), 8.07-8.10
13
(m, 2H, Ar-H), 8.53(s, 2H, C-NH2); C NMR (100 MHz, DMSO-d6, δ in ppm): 159.77,
159.68, 157.09, 150.75, 148.69, 126.90, 126.11, 117.08, 116.22, 86.08, 74.59, 45.16; Anal.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Calcd.for C14H11N5O3S: C, 51.06; H, 3.37; N, 21.27 ; found C, 50.99; H, 3.48; N, 21.38%;
LCMS (MM:ES+APCI) [M-1]- 328.
Synthesis of 1,6-diamino-4-(3-bromo-4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3,5-
dicarbonitrile (6g)
The product 6g was obtained from 3-bromo-4-methoxy benzaldehyde (1.0 eq), malononitrile
(1.0 eq) and 2-cyanoaceto hydrazide (1.0 eq). This compound was obtained as brown solid with
1
93% yield. IR (KBR ν in cm-1): 3394, 2242, 1669; H NMR (400 MHz, DMSO-d6, δ in ppm):
3.91 (s, 3H, -OCH3), 5.63 (s, 2H, N-NH2), 7.47-7.50 (m, 2H, Ar-H), 7.71 (s, 1H, Ar-H), 8.40
(s, 2H, C-NH2); 13C NMR (100 MHz, DMSO-d6, δ in ppm): : 159.62, 158.14, 157.19, 157.02,
132.89, 129.83, 128.29, 116.84, 115.97, 113.03, 112.89, 110.87, 86.84, 74.84, 56.37; Anal.
Calcd.for C14H10BrN5O2: C, 46.69; H, 2.80; N, 19.44 ; found C,46.50; H,2.71; N, 19.29%;
LCMS (MM:ES+APCI) [M-2]- 358.
Synthesis of 1,6-diamino-4-(4-hydroxyphenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile
(6h)
The product 6h was obtained from 4-hydroxy benzaldehyde (1.0 eq), Malononitrile (1.0 eq)
and 2-cyanoaceto hydrazide (1.0 eq). This compound was obtained as light brown solid with
1
94% yield.IR (KBR ν in cm-1): 3401, 2235, 1668; H NMR (400 MHz, DMSO-d6, δ in ppm):
5.62 (s, 2H, N-NH2), 5.81 (s, 1H, O-H), 7.02-7.04 (m, 2H, Ar-H), 7.07-7.09 (m, 2H, Ar-H),
13
8.36 (s, 2H, C-NH2); C NMR (100 MHz, DMSO-d6, δ in ppm): 160.01, 159.82, 159.75,
157.10, 130.39, 130.25, 125.24, 117.14, 116.26, 115.67, 86.16, 74.56; Anal. Calcd.for
C13H9N5O2: C, 58.43; H, 3.39; N, 26.21 ; found C,58.50; H,3.25; N, 26.29%; LCMS
(MM:ES+APCI) [M-1]- 266.
Synthesis of 1,6-diamino-4-(1H-indol-3-yl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile
(6i)
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10.1002/cbdv.201900234
Chemistry & Biodiversity
The product 6i was obtained from indole-3-carbaldehyde (1 eq), Malononitrile (1.0eq) and 2-
cyanoaceto hydrazide (1eq). This compound was obtained as brown solid with 94% yield. IR
1
(KBR ν in cm-1): 3402, 2240, 1670; H NMR (400 MHz, DMSO-d6, δ in ppm): 5.60 (s, 2H,
N-NH2), 7.08-7.20 (m, 2H, Ar-H), 7.44-7.48 (m, 2H, Ar-H), 7.78 (s, 1H, Ar-H), 8.25 (s, 2H,
C-NH2), 11.81(s, 1H, Ind-NH); 13C NMR (100 MHz, DMSO-d6, δ in ppm): 160.12, 157.94,
157.34, 157.17, 133.04, 132.84, 130.03, 129.77, 128.29, 116.97, 115.84, 113.03, 112.72,
111.03, 87.3, 74.96; Anal. Calcd.for C15H10N6O: C, 62.06; H, 3.47; N, 28.95 ; found C,62.15;
H,3.35; N, 28.79%, LCMS (MM:ES+APCI) [M-1]- 289.
Pharmacology
MTT assay
The antiproliferative effect of triazolo-pyridines towards lung cancer cells were investigated
by the MTT dye uptake method as described earlier.[41, 42] Briefly, A549 cells (2.5X104/ml)
were incubated in triplicate in a 96-well plate in the presence or absence of the indicated
concentrations of compounds in a final volume of 0.2 ml for different time intervals at 37°C.
Thereafter, 20 µl of MTT solution (5 mg/ml in PBS) was added to each well. After 2h of
incubation at 37°C, 0.1 ml of lysis buffer (20% SDS, 50% dimethylformamide) was added and
incubated 1h at 37 °C, and subsequently, the optical density was measured at 570 nm.
PDE4 assay
The PDE4 inhibition assay was performed at Prof. Manojith Pal’s laboratory, Institute of Life
Sciences, University of Hyderabad, Hyderabad, India. The PDE4 inhibitory activity of newly
synthesized compounds as described previously.[43]
In silico analysis
A crystal structure of PDE4 (PDB ID 3D3P) was retrieved from the Protein Data Bank [44]
[45]
[46]
and prepared for docking using the Protein Preparation Wizard
in Maestro
of default
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10.1002/cbdv.201900234
Chemistry & Biodiversity
settings and keeping only water molecule with at least three hydrogen bond interactions. The
docking was done using Glide SP at default settings.[47-49]
Conclusions
The library of new triazole-pyridine conjugates was synthesized as anticancer agents targeting
PDE4. All the new compounds were investigated for anticancer activity against lung cancer
cells.
Compound
5h
(7-(4-(methylsulfonyl)phenyl)-5-oxo-3,5-dihydro-1H-
spiro[[1,2,4]triazolo[1,5-a]pyridine-2,1'-cyclohexane]-6,8-dicarbonitrile) and compound 5j (7-
(1-methyl-1H-imidazol-2-yl)-5-oxo-3,5-dihydro-1H-spiro[[1,2,4]triazolo[1,5-a]pyridine-2,1'-
cyclohexane]-6,8-dicarbonitrile) showed good cytotoxicity. Further investigation revealed that
5j displayed good inhibition in vitro towards PDE4 at a single dose (10 µM). In silico analysis
conclusively supported the in vitro results and docking predicted hydrogen bond interactions
with His-234 and a water molecule anchored to Asp-392 and Asn-395. Overall, this is a
preliminary report that presents compound 5j as an inhibitor of PDE4 and further chemical
modifications of the lead structure may yield a potent inhibitor.
Author Contributions
HKK, SM, HB, SR, and FS performed the experiments. AB, CDM, B, KSR, and RB designed
the experiments and provided the resources. CDM, B, and KSR wrote the paper.
References
[1]
G. Schett, V. S. Sloan, R. M. Stevens, P. Schafer, ‘Apremilast: a novel PDE4 inhibitor in the
treatment of autoimmune and inflammatory diseases’, Ther. Adv. Musculoskelet. Dis. 2010, 2, 271-
278.
[2]
K. Tasken, E. M. Aandahl, ‘Localized effects of cAMP mediated by distinct routes of protein
kinase A’ Physiol. Rev. 2004, 84, 137-167.
[3]
V. K. Raker, C. Becker, K. Steinbrink, ‘The cAMP Pathway as Therapeutic Target in
Autoimmune and Inflammatory Diseases’, Front. Immunol. 2016, 7, 123.
[4] B. J. Lipworth, ‘Phosphodiesterase-4 inhibitors for asthma and chronic obstructive
pulmonary disease’, The Lancet 2005, 365, 167-175.
[5] C. P. Page, D. Spina, 'Phosphodiesterase Inhibitors in the Treatment of Inflammatory
Diseases', Eds. S. H. Francis, M. Conti and M. D. Houslay, Springer Berlin Heidelberg, Berlin,
Heidelberg, 2011.
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900234
Chemistry & Biodiversity
[6]
M. D. Houslay, P. Schafer, K. Y. J. Zhang, ‘Keynote review: Phosphodiesterase-4 as a
therapeutic target’, Drug Discov. Today 2005, 10, 1503-1519.
[7]
P. Goldhoff, N. M. Warrington, D. D. Limbrick, Jr., A. Hope, B. M. Woerner, E. Jackson, A.
Perry, D. Piwnica-Worms, J. B. Rubin, ‘Targeted inhibition of cyclic AMP phosphodiesterase-4
promotes brain tumor regression’, Clin. Cancer Res. 2008, 14, 7717-7725.
[8]
S. S. Pullamsetti, G. A. Banat, A. Schmall, M. Szibor, D. Pomagruk, J. Hanze, E. Kolosionek, J.
Wilhelm, T. Braun, F. Grimminger, W. Seeger, R. T. Schermuly, R. Savai, ‘Phosphodiesterase-4
promotes proliferation and angiogenesis of lung cancer by crosstalk with HIF’, Oncogene 2013, 32,
1121-1134.
[9]
L. Yang, E. Jackson, B. M. Woerner, A. Perry, D. Piwnica-Worms, J. B. Rubin, ‘Blocking CXCR4-
mediated cyclic AMP suppression inhibits brain tumor growth in vivo’, Cancer Res. 2007, 67, 651-
658.
[10]
T. Tsunoda, T. Ota, T. Fujimoto, K. Doi, Y. Tanaka, Y. Yoshida, M. Ogawa, H. Matsuzaki, M.
Hamabashiri, D. R. Tyson, M. Kuroki, S. Miyamoto, S. Shirasawa, ‘Inhibition of Phosphodiesterase-4
(PDE4) activity triggers luminal apoptosis and AKT dephosphorylation in a 3-D colonic-crypt model’,
Mol. Cancer 2012, 11, 46.
[11]
R. Böttcher, D. J. P. Henderson, K. Dulla, D. van Strijp, L. F. Waanders, G. Tevz, M. L. Lehman,
D. Merkle, G. J. L. H. van Leenders, G. S. Baillie, G. Jenster, M. D. Houslay, R. Hoffmann, ‘Human
phosphodiesterase 4D7 (PDE4D7) expression is increased in TMPRSS2-ERG-positive primary prostate
cancer and independently adds to a reduced risk of post-surgical disease progression’, Br. J Cancer
2015, 113, 1502-1511.
[12]
M. Gilandoust, K. B. Harsha, C. D. Mohan, A. R. Raquib, S. Rangappa, V. Pandey, P. E. Lobie,
Basappa, K. S. Rangappa, ‘Synthesis, characterization and cytotoxicity studies of 1,2,3-triazoles and
1,2,4-triazolo [1,5-a] pyrimidines in human breast cancer cells’, Bioorg. Med. Chem. Lett. 2018, 28,
2314-2319.
[13]
N. Ashwini, M. Garg, C. D. Mohan, J. E. Fuchs, S. Rangappa, S. Anusha, T. R. Swaroop, K. S.
Rakesh, D. Kanojia, V. Madan, A. Bender, H. P. Koeffler, Basappa, K. S. Rangappa, ‘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’, Bioorg. Med.
Chem. 2015, 23, 6157-6165.
[14]
C. D. Mohan, H. Bharathkumar, Dukanya, S. Rangappa, M. K. Shanmugam, A. Chinnathambi,
S. A. Alharbi, T. A. Alahmadi, A. Bhattacharjee, P. E. Lobie, A. Deivasigamani, K. M. Hui, G. Sethi,
Basappa, K. S. Rangappa, A. P. Kumar, ‘N-Substituted Pyrido-1,4-Oxazin-3-Ones Induce Apoptosis of
Hepatocellular Carcinoma Cells by Targeting NF-kappaB Signaling Pathway’, Front Pharmacol 2018,
9, 1125.
[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 2017, 17, 235.
[16]
substituted 1,2,4-triazoles: potential antitumor agents’, Bioorg. Med. Chem. 2003, 11, 1701-1708.
[17] C. D. Mohan, S. Hari, H. D. Preetham, S. Rangappa, U. Barash, N. Ilan, S. C. Nayak, V. K.
Y. A. Al-Soud, N. A. Al-Masoudi, A. E.-R. S. Ferwanah, ‘Synthesis and properties of new
Gupta, Basappa, I. Vlodavsky, K. S. Rangappa, ‘Targeting Heparanase in Cancer: Inhibition by
Synthetic, Chemically Modified, and Natural Compounds’, iScience. 2019, 15, 360-390.
[18]
Activities of Novel [1,2,4]triazolo[1,5-a]pyrimidines’, Molecules. 2007, 12, 1136-1146.
[19] M. Fares, S. M. Abou-Seri, H. A. Abdel-Aziz, S. E. S. Abbas, M. M. Youssef, R. A. Eladwy,
X.-L. Zhao, Y.-F. Zhao, S.-C. Guo, H.-S. Song, D. Wang, P. Gong, ‘Synthesis and Anti-tumor
‘Synthesis and antitumor activity of pyrido [2,3-d]pyrimidine and pyrido[2,3-d] [1,2,4]triazolo[4,3-
a]pyrimidine derivatives that induce apoptosis through G1 cell-cycle arrest’, Eur. J. Med. Chem. 2014,
83, 155.
[20]
H. B. El-Nassan, ‘Synthesis and antitumor activity of novel pyrido[2,3-d][1,2,4]triazolo[4,3-
a]pyrimidin-5-one derivatives’, Eur. J. Med. Chem. 2011, 46, 2031-2036.
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900234
Chemistry & Biodiversity
[21]
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 2015, 10,
e0131896.
[22]
Approach’, Int. J Mol. Sci. 2007, 8, 714-722.
[23] F. J. Urban, B. G. Anderson, S. L. Orrill, P. J. Daniels, ‘Process Research and Large-Scale
Z. Yang, P. Sun, ‘3D-QSAR Study of Potent Inhibitors of Phosphodiesterase-4 Using a CoMFA
Synthesis of a Novel 5,6-Dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-triazolo[4,3-a]pyridine PDE-IV Inhibitor’,
Org. Process Res. Dev. 2001, 5, 575-580.
[24]
A. J. Duplantier, E. L. Bachert, J. B. Cheng, V. L. Cohan, T. H. Jenkinson, K. G. Kraus, M. W.
McKechney, J. D. Pillar, J. W. Watson, ‘SAR of a Series of 5,6-Dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-
triazolo[4,3-α]pyridines as Potent Inhibitors of Human Eosinophil Phosphodiesterase’, J. Med. Chem.
2007, 50, 344-349.
[25]
L. Zribi, F. Zribi, J. Marco-Contelles, F. Chabchoub, L. Ismaili, ‘Facile one-pot synthesis of new
[1,2,4]triazolo[1,5-a]pyridine derivatives by ultrasonic irradiation’, Synth. Commun. 2017, 47, 1934-
1939.
[26]
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’, Bioorg. Med. Chem. Lett.
2016, 26, 1056-1060.
[27]
A. C. Nirvanappa, C. D. Mohan, S. Rangappa, H. Ananda, A. Y. Sukhorukov, M. K.
Shanmugam, M. S. Sundaram, S. C. Nayaka, K. S. Girish, A. Chinnathambi, M. E. Zayed, S. A. Alharbi,
G. Sethi, Basappa, K. S. Rangappa, ‘Novel Synthetic Oxazines Target NF-kappaB in Colon Cancer In
Vitro and Inflammatory Bowel Disease In Vivo’, PLoS One 2016, 11, e0163209.
[28]
V. Pandey, B. Wang, C. D. Mohan, A. R. Raquib, S. Rangappa, V. Srinivasa, J. E. Fuchs, K. S.
Girish, T. Zhu, A. Bender, L. Ma, Z. Yin, Basappa, K. S. Rangappa, P. E. Lobie, Proc. Natl. Acad. Sci. U S
A. 2018, 115, E10505-E10514.
[29]
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 2016, 1, 1412-1424.
[30]
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’, Bioorg. Med. Chem. Lett. 2015, 25, 2931-2936.
[31]
K. H. Kumar, S. Paricharak, C. D. Mohan, H. Bharathkumar, G. P. Nagabhushana, D. K.
Rajashekar, G. T. Chandrappa, A. Bender, Basappa, K. S. Rangappa, ‘Nano-MoO3-mediated synthesis
of bioactive thiazolidin-4-ones acting as anti-bacterial agents and their mode-of-action analysis using
in silico target prediction, docking and similarity searching’, New J Chem. 2016, 40, 2189-2199.
[32]
H. K. Keerthy, H. K. Vivek, H. Bharathkumar, S. Rangappa, K. C. Bulusu, L. H. Mervin, J. E.
Fuchs, B. S. Priya, Basappa, N. Swamy S, A. Bender, K. S. Rangappa, ‘MOLPRINT 2D-based
identification and synthesis of novel chromene based small molecules that target PLA2: validation
through chemo- and bioinformatics approaches’, RSC Adv. 2015, 5, 89797-89808.
[33]
K. S. Rakesh, S. Jagadish, K. S. Balaji, F. Zameer, T. R. Swaroop, C. D. Mohan, S. Jayarama, K. S.
Rangappa, ‘3,5-Disubstituted Isoxazole Derivatives: Potential Inhibitors of Inflammation and Cancer’,
Inflammation. 2016, 39, 269.
[34]
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’, Med. Chem. 2015, 11, 462-472.
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900234
Chemistry & Biodiversity
[35]
A. H. M. Hussein, ‘Utility of Cyano Acid Hydrazide in Heterocyclic Chemistry: A New Route for
the Synthesis of New l,2,4-Triazolo[l,5-a]pyridines and 1,2,4-Triazolo[l,5-a]isoquinolines’, Zeitschrift
für Naturforschung B 1998, 53, 488-494.
[36]
pyridones via a one-pot three-component reaction’, RSC Adv. 2018, 8, 27131.
[37] H. K. Keerthy, M. Garg, C. D. Mohan, V. Madan, D. Kanojia, R. Shobith, S. Nanjundaswamy, D.
H. Hosseini, M. Bayat, ‘An efficient and ecofriendly synthesis of highly functionalized
J. Mason, A. Bender, Basappa, K. S. Rangappa, H. P. Koeffler, ‘Synthesis and characterization of novel
2-amino-chromene-nitriles that target Bcl-2 in acute myeloid leukemia cell lines’, PLoS One 2014, 9,
e107118.
[38]
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’, J Biol. Chem. 2014, 289, 34296-34307.
[39]
S. Anusha, B. Cp, C. D. Mohan, J. Mathai, S. Rangappa, S. Mohan, Chandra, S. Paricharak, L.
Mervin, J. E. Fuchs, M. M, A. Bender, Basappa, K. S. Rangappa, ‘A Nano-MgO and Ionic Liquid-
Catalyzed 'Green' Synthesis Protocol for the Development of Adamantyl-Imidazolo-Thiadiazoles as
Anti-Tuberculosis Agents Targeting Sterol 14α-Demethylase (CYP51)’, PLoS One 2015, 10, e0139798.
[40]
M. Suresh, P. Lavanya, C. V. Rao, ‘Synthesis and pharmacological evaluation of novel
2H/6Hthiazolo-[3′,2′:2,3][1,2,4]triazolo[1,5-a]pyridine-9-carbonitrile derivatives’, Arabian Journal of
Chemistry 2016, 9, 136-142.
[41]
H. Bharathkumar, C. D. Mohan, S. Rangappa, T. Kang, H. K. Keerthy, J. E. Fuchs, N. H. Kwon,
A. Bender, S. Kim, Basappa, K. S. Rangappa, ‘Screening of quinoline, 1,3-benzoxazine, and 1,3-
oxazine based small molecules against isolated methionyl-tRNA synthetase and A549 and HCT116
cancer cells including an in silico binding mode analysis’, Org. Biomol. Chem. 2015, 13, 9381-9387.
[42]
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’, J Biol. Chem. 2014, 289,
31879-31890.
[43]
R. Adepu, D. Rambabu, B. Prasad, C. L. T. Meda, A. Kandale, G. Rama Krishna, C. Malla
Reddy, L. N. Chennuru, K. V. L. Parsa, M. Pal, ‘Novel thieno[2,3-d]pyrimidines: their design, synthesis,
crystal structure analysis and pharmacological evaluation’, Org. Biomol. Chem. 2012, 10, 5554-5569.
[44]
P. E. Bourne, ‘The Protein Data Bank’, Nucleic. Acids. Res. 2000, 28, 235-242.
[45] G. M. Sastry, M. Adzhigirey, T. Day, R. Annabhimoju and W. Sherman, ‘Protein and ligand
H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov and
preparation: parameters, protocols, and influence on virtual screening enrichments’, J Comput Aided
Mol Des 2013, 27, 221-234.
[46]
[47]
[48]
V. S. Maestro, LLC: New York, NY 2015, 2015.
V. S. Glide, LLC: New York, NY, 2015.
R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J. Klicic, D. T. Mainz, M. P. Repasky,
E. H. Knoll, M. Shelley, J. K. Perry, D. E. Shaw, P. Francis, P. S. Shenkin, ‘Glide: a new approach for
rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy’, J Med. Chem.
2004, 47, 1739-1749.
[49]
T. A. Halgren, R. B. Murphy, R. A. Friesner, H. S. Beard, L. L. Frye, W. T. Pollard, J. L. Banks,
‘Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database
screening’, J Med. Chem. 2004, 47, 1750-1759.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Figure legends
Scheme 1 and 2: Synthetic procedure for the preparation of substituted pyridines.
Figure 1: A. The predicted binding mode of the best-scored compound. B. Docking predicts
hydrogen bond interactions with His-234 and a water molecule anchored to Asp-392 and Asn-
395. The compound also forms a face to face stacking interaction with Phe-446 and an edge to
face interaction with Phe-414.
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10.1002/cbdv.201900234
Chemistry & Biodiversity
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10.1002/cbdv.201900234
Chemistry & Biodiversity
Graphical abstract
Table 1: Optimization of a solvent-catalyst system for the preparation of 5a
Entry
Solvent
Catalyst
Time (h)
Yield a (%)
Water
Piperidine
8
-
-
1
Water
Water
Triethylamine
TBAB
8
1
2
3
4
5
6
7
8
9
92
Water
Sodium Silicate
p-TSA
2.5
3
63
Water
Traces
58
Ethanol
Piperidine
TBAB
3
Ethyl acetate
Toulene
10
10
6
Traces
Traces
72
TBAB
Ethanol:Water (5:5)
TBAB
Table 2: Reaction conditions employed for the synthesis of substituted pyridines
Entry
R
5(a-l)
Time
(H)
6(a-i)
Time
(min)
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10.1002/cbdv.201900234
Chemistry & Biodiversity
1
10a
-
-
-
-
6a
6h
2
3
15
12
1
1.5
1
5a
6b
-
4
5
6
-
5b
5c
15
6g
10a
1
1
6d
6c
5d
5e
7
10
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10.1002/cbdv.201900234
Chemistry & Biodiversity
8
18a
1.5
1.5
6e
-
5d
5f
9
-
10
10
2
2
5h
5i
6f
-
11
12
-
-
-
-
2.5
2.5
5j
13
14
-
5k
20
3
5l
6i
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10.1002/cbdv.201900234
Chemistry & Biodiversity
a Literature reported compounds
Table 3: Substituted pyridines showing IC50 values and % inhibition studies of human lung
cancer cells and PDE4 enzymes.
Compound
A549 Cells
IC50 (µM)
28.1±0.11
Average PDE4
inhibition (%)
22.09
5a
5b
5c
5d
5e
5f
5g
5h
5i
35.5±0.12
26.1±0.15
NA
-0.39
33.86
34.90
-
30.2±0.32
25±0.6
NA
-
-
15.2±0.12
30.2±0.05
24.1±0.03
35.6±0.23
31.2±0.25
NT
27.40
-
5j
50.90
1.81
44.80
-
5k
5l
6a
6b
6c
6d
6e
6f
6g
NT
6.61
10.75
1.67
26.25
-1.36
12.07
NT
NT
NT
NT
NT
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10.1002/cbdv.201900234
Chemistry & Biodiversity
6h
NT
-
9.77
87.30
-
Rolipram
Paclitaxel
0.0046
Paclitaxel represents the positive control and the IC50
value of paclitaxel is taken from our previous
report.[41]
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