Synthesis of 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazole derivatives: Their evaluation as potential PDE 4B inhibitors possessing cytotoxic properties against cancer cells

Article abstract of DOI:10.1016/j.bioorg.2013.12.002

The 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazole derivatives were designed as potential inhibitors of PDE4B. These compounds were synthesized via a multi-step sequence consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step in aqueous media. The required alkynes were prepared from nimesulide via N-propargylation and then nitro group reduction followed by a CAN mediated modified Skraup reaction of the resulting amine. All the synthesized compounds showed PDE4B inhibitory properties in vitro at 30 μM with two compounds showing >50% inhibition that were supported by the in silico docking results of these compounds at the active site of PDE4B. Three of these PDE4 inhibitors showed promising cytotoxic properties against A549 human lung cancer cells in vitro with IC₅₀ ～8-9 μM.

Full text of DOI:10.1016/j.bioorg.2013.12.002

Accepted Manuscript
Synthesis of 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazol de-
rivatives: Their evaluation as potential PDE 4B inhibitors possessing cytotoxic
properties against cancer cells
K.S.S. Praveena, Shylaprasad Durgadas, N. Suresh, Surekha Akkenapally, C.
Ganesh Kumar, Girdhar Singh Deora, N.Y.S. Murthy, Khagga Mukkanti,
Sarbani Pal
PII:
S0045-2068(13)00099-0
DOI:
http://dx.doi.org/10.1016/j.bioorg.2013.12.002
YBIOO 1693
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
3 October 2013
Please cite this article as: K.S.S. Praveena, S. Durgadas, N. Suresh, S. Akkenapally, C. Ganesh Kumar, G.S. Deora,
N.Y.S. Murthy, K. Mukkanti, S. Pal, Synthesis of 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazol
derivatives: Their evaluation as potential PDE 4B inhibitors possessing cytotoxic properties against cancer cells,
Bioorganic Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bioorg.2013.12.002
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Synthesis of 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazol derivatives:
Their evaluation as potential PDE 4B inhibitors possessing cytotoxic properties against
cancer cells
K. S. S. Praveena,a Shylaprasad Durgadas,b N. Suresh,c Surekha Akkenapally,d C. Ganesh
Kumar,d Girdhar Singh Deora,e N. Y. S. Murthy,f Khagga Mukkantic Sarbani Pala,*
aDepartment of Chemistry, MNR Degree & PG College, Kukatpally, Hyderabad-500085, India.
bMSN Pharmachem Pvt. Ltd, Plot No. 212/A,B,C,D, APIICL, Phase-II, Pashamylaram,
Patancheru (M), Medak District 502 307, Andhra Pradesh, India
cCentre for Chemical Sciences and Technology, Institute of Science & Technology, Jawaharlal
Nehru Technological University Hyderabad, Kukatpally, Hyderabad-500085, India.
dChemical Biology Laboratory, Medicinal Chemistry and Pharmacology Division,
Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007.
eThe University of Queensland, School of Pharmacy, Brisbane, QLD 4072, Australia.
fDepartment of Chemistry, School of Engineering, Anurag Group of Institutions, R.R.District-
501301, Andhra Pradesh, India
Abstract: The 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazol derivatives were
designed as potential inhibitors of PDE4B. These compounds were synthesized via a multi-step
sequence consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step in
aqueous media. The required alkynes were prepared from nimesulide via N-propargylation and
then nitro group reduction followed by a CAN mediated modified Skraup reaction of the
resulting amine. All the synthesized compounds showed PDE4B inhibitory properties in vitro at
30 µM with two compounds showing > 50% inhibition that were supported by the in silico
docking results of these compounds at the active site of PDE4B. Three of these PDE4 inhibitors
showed promising cytotoxic properties against A549 human lung cancer cells in vitro with IC50 ~
8-9 µM.
Keywords: nimesulide, 1,2,3-triazole, cycloaddition, PDE4B, cytotoxic activities
-----------------------------
*Corresponding author: Tel.: +91 40 23041488; fax: +91 040 23067380
E-mail: sarbani277@yahoo.com
1

1. Introduction
The dysregulation of 3’,5’-cyclic adenosine mono-phosphate (cAMP) metabolism in oncogenesis
was speculated more than 50 years ago at the time of discovery of cAMP. In his Noble address,
the 1971 noble prize winner in Physiology or Medicine Dr. Earl Sutherland commented that,
defective cAMP formation might be involved in the growth of tumors [1]. Indeed, the relevance
of disordered cAMP metabolism to the genesis of multiple cancers has been confirmed recently.
These studies suggested that the mechanism might involve altered expression and activity of
phosphodiesterases (PDEs) [2,3], a superfamily of enzymes that degrade cAMP and cGMP.
According to their specificity for cAMP or cGMP, PDEs can be subdivided into 11 different
groups or isozymes (PDE1-PDE11) whereas the cAMP specific PDE4 isozymes are encoded by
four genes (A-D) that give rise to four isoforms, e.g., PDE4A-D [4]. Since PDE4 specifically is
widely expressed in tumor cells hence, inhibition of PDE4 in cancerous cells might be a new
therapeutic target for cancer. For example, PDE4 inhibitors have been reported to (i) inhibit brain
tumor cell growth [5,6], (ii) reduce proliferation and angiogenesis of lung cancer cell lines[7] and
(iii) cause selective apoptosis of malignant cells without affecting the normal cells [8].
Recently, we have synthesized [9] a library of compounds based on a template A (Fig. 1)
designed by incorporating the structural features of 2,2,4-substituted 1,2-dihydroquinolines and
nimesulide [10], a well known anti-inflammatory drug available for patient’s use for the
treatment of pain. In continuation of this research we became interested in further structural
elaboration of A that could lead to the identification of PDE4 inhibitors as potential anticancer
agents. Accordingly, in view of PDE4 inhibiting [11] and anticancer [12] properties of triazole
class of compounds we introduced a 1,2,3-triazole moiety to A to design a new template B (Fig.
1). Our anticipation that compounds related to B may inhibit PDE4 was further supported by the
docking studies of a representative molecule e.g. N-m-tolyl-2-(4-{[N-(2,2,4-trimethyl-7-
phenoxy-1,2-dihydroquinolin-6-yl)methylsulfonamido]methyl}-1H-1,2,3-triazol-1-yl)acetamide
(C) into PDE4B protein in silico which showed good interactions (Glide score -8.24) through
three H-bonds with Tyr-233, Hie-234 and Hie-278 residues of PDE4B (Fig. 2). Both, methyl
substituted phenyl and the triazole ring were involved in π-π stacking with Tyr-233 and Hie-234
respectively. In addition, a metal coordination was observed between Mg-1002 and SO2 group of
the molecule C. Due to our continuing interest on NCEs related to nimesulide [13-18], and novel
PDE4 inhibitors [19,20] we now report the synthesis, in vitro PDE4 inhibition and cytotoxic
2

effects of a series of novel 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazol B
derived from nimesulide. To the best of our knowledge synthesis and pharmacological
evaluation of this class of compounds is not known in the literature. Additionally, evaluation of
novel PDE4 inhibitors for the potential treatment of cancer is not common in the literature.
Insert Fig. 1 here
Insert Fig. 2 here.
Insert Scheme 1 here.
2. Materials and methods
2.1. Materials
2.2. Cells and Reagents
Sf9 cells were obtained from ATCC (Washington D.C., USA) and were routinely maintained in
Grace’s supplemented medium (Invitrogen) with 10% FBS. cAMP was purchased from SISCO
Research Laboratories (Mumbai, India). PDElight HTS cAMP phosphodiesterase assay kit was
procured from Lonza (Basel, Switzerland). PDE4B1 clone was procured from OriGene
Technologies (Rockville, MD, USA). PDE4D2 enzyme was purchased from BPS Bioscience
(San Diego, CA, USA).
2.3. General methods
Melting points were determined by open glass capillary method on a Cintex melting point
apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer spectrometer using
KBr pellets. 1H and 13C NMR spectra were recorded on a Bruker ACF-300 machine or a Varian
3

300 or 400 MHz spectrometer using CDCl3 or DMSO-d6, with reference to tetramethylsilane as
an internal reference. Mass spectra were recorded on a Jeol JMC D-300 instrument by using
Electron ionization at 70 ev. All reactions were monitored by TLC (thin layer chromatography)
on pre-coated silica gel plates. Column chromatography was performed by using silica gel (100-
200 mesh, SRL, India) [10-20 times (by weight) of the crude product].
3. Experimental procedure
3.1. General procedure for the synthesis of 5
A mixture of azide (1 mmol), an appropriate terminal alkyne (1 mmol), copper sulphate (0.25
mmol) and sodium ascorbate (200 mg) in 7:1 aqueous DMF (2 mL) was stirred vigorously for
10-30 min. The progress of the reaction is monitored by checking TLC at a regular interval.
After completion of the reaction, the reaction mixture was quenched in crushed ice. The solid
separated was filtered, dried and purified by column chromatography on silica gel using
chloroform / ethyl acetate to give the desired product.
3.1.1. N-((1-((2-chloro-6-methoxyquinolin-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-N-(2,2,4-
trimethyl-7-phenoxy-1,2-dihydroquinolin-6-yl)methanesulfonamide (5a)
Pale yellow solid; yield 95%; mp 94-96 °C; Rf 0.82 (CHCl3: EtOAc 1:1); MS m/z 645.2 (M+1,
1
100%); IR : 3369, 2960, 1613, 1497, 1488, 1336, 1218, 1148. H NMR (CDCl3, 400 MHz) δ
8.00 (d, J 7.8 Hz, 1H), 7.80 (s, 1H), 7.71 (s, 1H), 7.32-7.41 (m, 3H), 6.97- 7.17 (m, 4H), 6.81 (s,
1H), 5.74 (s, 2H), 5.72 (s, 1H), 5.15 (s, 1H), 4.95 (s, 2H), 3.88 (s, 3H), 3.62 (bs, 1H, NH), 3.04
13
(s, 3H), 1.70 (s, 3H), 1.20 (s, 6H); C NMR (CDCl3, 100 MHz) δ 158.6, 155.6, 154.7, 146.0,
145.6, 144.7, 143.5, 137.1, 130.0, 129.7, 128.3, 127.2, 127.1, 124.2, 124.0, 123.9, 119.7, 116.9,
116.8, 105.1, 101.1, 55.7, 52.3, 51.2, 46.7, 39.4, 31.6, 29.7.
3.1.2.
N-((1-((2-chloro-6-methylquinolin-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-N-(2,2,4-
trimethyl-7-phenoxy-1,2-dihydroquinolin-6-yl)methanesulfonamide (5b)
Pale yellow solid; yield 86%; mp 102-104 °C; Rf 0.67 (CHCl3: EtOAc 3:1); MS m/z 629.2 (M+1,
1
100%); IR : 3354, 2964, 2919, 1612, 1588, 1488, 1333, 1148. H NMR (CDCl3, 400 MHz) δ
7.90 (d, J 7.8 Hz, 1H), 7.76 (s, 1H), 7.73 (s, 1H), 7.59 (dd, J 8.2, 2.0 Hz, 1H), 7.48 (s, 1H), 7.34
4

(d, J 7.4 Hz, 1H), 7.33 (d, J 7.3 Hz, 1H), 7.15 (d, J 7.3 Hz, 1H), 7.02 (d, J 8.8 Hz, 2H), 6.81 (s,
1H), 5.74 (s, 2H), 5.72 (s, 1H), 5.15 (s, 1H), 4.92 (s, 2H), 3.63 (bs, 1H, NH), 3.04 (s, 3H), 2.51
13
(s, 3H), 1.75 (s, 3H), 1.20 (s, 6H); C NMR (CDCl3, 100 MHz) δ 155.6, 154.8, 147.9, 146.1,
145.6, 144.7, 137.8, 137.7, 133.5, 130.0, 128.2, 128.0, 127.3, 127.1, 127.0, 126.7, 126.6, 124.2,
123.8, 119.8, 116.9, 116.7, 101.1, 52.3, 51.2, 46.7, 39.4, 31.6, 29.7, 20.2.
3.1.3. N-((1-((2-chloroquinolin-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-N-(2,2,4-trimethyl-7-
phenoxy-1,2-dihydroquinolin-6-yl)methanesulfonamide (5c)
Pale yellow solid; yield 84%; mp 180-182 °C; Rf 0.72 (CHCl3: EtOAc 3:1); MS m/z 615.2
1
(M+1, 100%); IR : 3370, 2958, 2921, 2852, 1614, 1487, 1321, 1215. H NMR (CDCl3, 400
MHz) δ 8.01 (d, J 7.8 Hz, 1H), 7.78 (s, 1H), 7.77-7.62 (m, 3H), 7.59-7.55 (m, 1H), 7.34 (d, J 7.4
Hz, 1H), 7.33 (d, J 7.3 Hz, 1H), 7.12 (d, J 7.3 Hz, 1H), 6.96 (d, J 8.8 Hz, 2H), 6.80 (s, 1H), 5.76
(s, 2H), 5.74 (s, 1H), 5.20 (s, 1H), 4.94 (s, 2H), 3.93 (bs, 1H, NH), 3.04 (s, 3H), 1.75 (s, 3H),
13
1.20 (s, 6H); C NMR (CDCl3, 100 MHz) δ 155.6, 154.8, 148.8, 147.5, 145.7, 144.7, 138.3,
131.2, 130.0 (2C), 128.3, 128.2, 127.8, 127.7, 127.3, 127.1, 127.0, 126.9, 124.3, 123.9, 119.8
(2C), 116.8, 116.7, 101.1, 52.3, 51.2, 46.7, 39.4, 31.6, 29.5.
3.1.4.
N-((1-((2-chloro-7-methylquinolin-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-N-(2,2,4-
trimethyl-7-phenoxy-1,2-dihydroquinolin-6-yl)methanesulfonamide (5d)
Pale yellow solid; yield 83%; mp 180 °C; Rf 0.64 (CHCl3: EtOAc 3:1); MS m/z 629.2 (M+1,
100%); IR : 3369, 2964, 2924, 1612, 1589, 1505, 1488, 1335, 1216, 1149. 1H NMR (CDCl3, 400
MHz) δ 7.83-7.80 (m, 3H), 7.59 (d, J 8.2 Hz, 1H), 7.40-7.28 (m, 3H), 7.15 (d, J 7.3 Hz, 1H),
7.00 (d, J 8.8 Hz, 2H), 6.78 (s, 1H), 5.77 (s, 2H), 5.75 (s, 1H), 5.15 (s, 1H), 4.92 (s, 2H), 3.65
(bs, 1H, NH), 3.04 (s, 3H), 2.51 (s, 3H), 1.75 (s, 3H), 1.20 (s, 6H); 13C NMR (CDCl3, 100 MHz)
δ 158.4, 155.8, 155.5, 154.7, 145.4, 144.8, 134.5 (2C), 130.1, 130.0, 129.5 128.3, 127.3, 127.1,
125.2, 124.6, 124.3, 120.2, 120.1, 119.9, 119.8, 116.7, 101.1, 53.4, 52.3, 46.6, 39.5, 31.6, 29.7,
20.9.
3.1.5.
N-phenyl-2-(4-((N-(2,2,4-trimethyl-7-phenoxy-1,2-dihydroquinolin-6-
yl)methylsulfonamido)methyl)-1H-1,2,3-triazol-1-yl)acetamide (5e)
5

Off white solid; yield 90%; mp 200-202 °C; Rf 0.54 (CHCl3: EtOAc 1:1); MS m/z 573.2 (M+H+,
1
100%); IR : 3345, 2956, 2922, 2919, 1698, 1488, 1332, 1150. H NMR (CDCl3, 400 MHz) δ
7.87 (s, 1H), 7.41-7.31 (m, 5H), 7.16-6.85 (m, 8H), 6.84 (s, 1H), 5.75 (s, 1H), 5.15 (s, 1H), 5.12
(s, 2H), 4.83 (bs, 1H, NH), 3.05 (s, 3H), 1.79 (s, 3H), 1.25 (s, 6H); 13C NMR (CDCl3, 100 MHz)
δ 164.5, 155.9, 155.6, 145.6, 143.9, 138.8, 130.4, 129.3, 127.9, 127.2, 126.8, 125.9, 124.6, 124.2,
120.4, 119.5, 115.7, 115.3, 100.3, 52.6, 51.8, 45.1, 31.9, 18.5.
3.1.6.
N-p-Tolyl-2-(4-((N-(2,2,4-trimethyl-7-phenoxy-1,2-dihydroquinolin-6-
yl)methylsulfonamido) methyl)-1H-1,2,3-triazol-1-yl)acetamide (5f)
Pale yellow solid; yield 88%; mp 98-100 °C; Rf 0.5 (C9HCl3: EtOAc 1:1); MS m/z 587.3
1
(M+H+, 100%); IR : 3347, 2921, 2852, 1691, 1610, 1487, 1332, 1213, 1147. H NMR (CDCl3,
400 MHz) δ 8.01 (s, 1H), 7.87 (s, 1H), 7.39-7.30 (m, 5H), 7.14-7.02 (m, 6H), 6.84 (s, 1H), 5.79
(s, 1H), 5.15 (s, 1H), 5.12 (s, 2H), 5.10 (bs, 1H, NH), 3.09 (s, 3H), 2.29 (s, 3H), 1.79 (s, 3H),
1.25 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 164.5, 155.5, 154.7, 145.4, 144.8, 134.6, 130.0
(2C), 129.5 (2C), 128.3, 127.9, 125.2, 124.6, 124.3, 120.2 (2C), 119.9 (2C), 116.8, 116.7, 116.5,
101.1, 53.4, 52.3, 46.6, 39.5, 31.6 (2C), 29.4.
3.1.7.
N-m-Tolyl-2-(4-((N-(2,2,4-trimethyl-7-phenoxy-1,2-dihydroquinolin-6-
yl)methylsulfonamido)methyl)-1H-1,2,3-triazol-1-yl)acetamide (5g)
Pale yellow solid; yield 95%; mp 186-188 °C; Rf 0.59 (CHCl3: EtOAc 1:1); MS m/z 587.2
1
(M+H+, 100%); IR : 3347, 2921, 2852, 1691, 1610, 1487, 1332, 1213, 1147. H NMR (CDCl3,
400 MHz) δ 8.01 (s, 1H), 7.98 (s, 1H), 7.20-7.02 (m, 11H), 6.84 (s, 1H), 5.79 (s, 1H), 5.16 (s,
13
1H), 5.12 (s, 2H), 4.97 (bs, 1H, NH), 3.06 (s, 3H), 2.31 (s, 3H), 1.79 (s, 3H), 1.26 (s, 6H); C
NMR (CDCl3, 100 MHz) δ 163.1, 155.6, 154.7, 145.4, 144.8, 138.9, 137.0, 130.0, 128.8, 128.3,
127.3, 127.1, 125.7, 125.2, 124.6, 124.3, 120.8, 119.7, 117.2, 116.8, 101.1, 53.4, 52.3, 46.6, 39.5,
31.6, 29.7, 21.4;
3.1.8.
N-(4-Methoxyphenyl)-2-(4-((N-(2,2,4-trimethyl-7-phenoxy-1,2-dihydroquinolin-6-
yl)methylsulfonamido) methyl)-1H-1,2,3-triazol-1-yl)acetamide (5h)
Pale yellow solid; yield 95%; mp 88-90 °C; Rf 0.66 (CHCl3: EtOAc 1:1); MS m/z 603.2 (M+H,
1
100%); IR : 3341, 2920, 2852, 1694, 1610, 1510, 1487, 1332, 1233, 1215, 1145. H NMR
(CDCl3, 400 MHz) δ 8.01 (s, 1H), 7.36-7.31 (m, 8H), 7.18-7.02 (m, 2H), 6.85- 6.81 (m, 3H),
6

5.75 (s, 1H), 5.16 (s, 1H), 5.10 (s, 2H), 4.92 (bs, 1H, NH), 3.78 (s, 3H), 3.05 (s, 3H), 2.18 (s,
3H), 1.25 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 162.5, 156.8, 155.5, 154.7, 145.4, 144.8,
130.1, 128.3, 127.3, 127.1, 125.2, 124.3, 122.0, 121.9, 119.7, 116.8, 116.7, 114.1, 101.1, 55.5,
53.4, 52.3, 46.6, 39.5, 31.8, 29.4;
3.1.9.
N-(4-(methylsulfonamido)-3-phenoxyphenyl)-2-(4-((N-(2,2,4-trimethyl-7-phenoxy-1,2-
dihydroquinolin-6-yl)methylsulfonamido)methyl)-1H-1,2,3-triazol-1-yl)acetamide (5i)
Off white solid; yield 83%; mp 220-222 °C; Rf 0.52 (CHCl3: EtOAc 1:1); MS m/z 758.2 (M+H+,
100%); IR : 3341, 2926, 2855, 1700, 1613, 1589, 1487, 1330, 1213, 1147. 1H NMR (CDCl3, 400
MHz) δ 7.80 (s, 1H), 7.36-7.31 (m, 2H), 7.18-7.02 (m, 15H), 6.85- 6.81 (s, 1H), 5.76 (s, 1H),
5.17 (s, 1H), 5.10 (s, 2H), 4.96 (bs, 1H, NH), 3.07 (s, 3H), 2.95 (s, 3H), 2.20 (s, 3H), 1.25 (s,
6H); 13C NMR (CDCl3, 100 MHz) δ 164.6, 156.3, 155.9, 155.6, 151.4, 145.6, 137.8, 130.5,
128.3, 127.9, 127.2, 126.8, 124.6, 124.4, 123.6, 120.4, 119.6, 116.8, 115.7, 115.3, 114.4, 109.3,
100.3, 52.4, 51.8, 46.1, 40.8, 31.9, 18.4.
3.2. In vitro assay of compounds for the inhibition of PDE4B enzyme
3.2.1. PDE4B protein production and purification
PDE4B1 cDNA was sub-cloned into pFAST Bac HTB vector (Invitrogen) and transformed into
DH10Bac (Invitrogen) competent cells. Recombinant bacmids were tested for integration by
PCR analysis. Sf9 cells were transfected with bacmid using Lipofectamine 2000 (Invitrogen)
according to manufacturer’s instructions. Subsequently, P3 viral titer was amplified, cells were
infected and 48 h post infection cells were lysed in lysis buffer (50 mM Tris-HCl pH 8.5, 10
mM 2-Mercaptoethanol, 1 % protease inhibitor cocktail (Roche), 1 % NP40). Recombinant His-
tagged PDE4B protein was purified as previously described elsewhere (Wang et al., 1997).
Briefly, lysate was centrifuged at 10,000 rpm for 10 min at 4˚C and supernatant was collected.
Supernatant was mixed with Ni-NTA resin (GE Life Sciences) in a ratio of 4:1 (v/v) and
equilibrated with binding buffer (20 mM Tris-HCl pH 8.0, 500 mM-KCl, 5 mM imidazole, 10
mM 2-mercaptoethanol and 10 % glycerol) in a ratio of 2:1 (v/v) and mixed gently on rotary
shaker for 1 hour at 4˚C. After incubation, lysate-Ni-NTA mixture was centrifuged at 4,500 rpm
for 5 min at 4˚C and the supernatant was collected as the flow-through fraction. Resin was
washed twice with wash buffer (20 mM Tris-HCl pH 8.5, 1 M KCl, 10 mM 2-Mercaptoethanol
7

and 10% glycerol). Protein was eluted sequentially twice using elution buffers (Buffer I: 20 mM
Tris-HCl pH 8.5, 100 mM KCl, 250 mM imidazole, 10 mM 2-mercaptoethanol, 10% glycerol,
Buffer II: 20 mM Tris-HCl pH 8.5, 100 mM KCl, 500 mM imidazole, 10 mM 2-
mercaptoethanol, 10% glycerol). Eluates were collected in four fractions and analyzed by SDS-
PAGE. Eluates containing PDE4B protein were pooled and stored at -80˚C in 50% glycerol until
further use.
3.2.2. PDE4 enzymatic assay
The inhibition of PDE4 enzyme was measured using PDE light HTS cAMP phosphodiesterase
assay kit (Lonza) according to manufacturer’s recommendations. Briefly, 10 ng of in house
purified PDE4B1 enzyme was pre-incubated either with DMSO (vehicle control) or compound
for 15 min before incubation with the substrate cAMP (5 µM) for 1 hour. The reaction was
halted with stop solution and reaction mix was incubated with detection reagent for 10 minutes
in dark. Luminescence values (RLUs) were measured by a Multilabel plate reader (Perklin
Elmer 1420 Multilabel counter).The percentage of inhibition was calculated using the following
formula:
3.3. MTT assay for cytotoxicity
The viability of the cells was assessed by MTT [3,4,5-dimethylthiazol-2-yl)-2-5-
diphenyltetrazolium bromide] assay, which is based on the reduction of MTT by the
mitochondrial dehydrogenase of intact cells to a purple formazan product. Doxorubicin, a known
anticancer drug was used as a reference compound in this assay. Cells (1 × 104) were plated in a
96-well plate. After 24 h, they were treated with different concentration (0 – 10 µM) of different
test compounds diluted appropriately with culture media for 48 h. Cells grown in media
containing equivalent amount of DMSO served as positive control and cells in medium without
any supplementation were used as negative control. After the treatment, media containing
compound were carefully removed by aspiration. 100 µL of 0.4 mg/mL MTT in PBS was added
to each well and incubated in the dark for 4 h. 100 µL of DMSO was added to each well and kept
in an incubator for 4 h for dissolution of the formed formazan crystals. Amount of formazan was
determined by measuring the absorbance at 540 nm using an ELISA plate reader. The data were
8

presented as percent dead cells, whereas absorbance from non-treated control cells was defined
as 100% live cells. The percent dead cells was plotted (Y-axis) against concentration (X-axis) of
compounds, where IC50 values could be interpolated from the graph.
3.4. Docking Studies
3.4.1. Docking Method
The docking analysis of molecules was performed using Maestro, version 9.2 [21] implemented
from Schrödinger molecular modeling suite. Molecules were sketched in 3D format using build
module of maestro and LigPrep module was used to produce low-energy conformers. The
structural coordinates of Phosphodiesterase 4B (PDB ID: 1XMY) [22] were obtained from the
protein data bank (PDB). PBD protein was prepared by giving preliminary treatment like adding
hydrogen, adding missing residues, refining the loop with prime and finally minimized by using
OPLS-2005 force field. The grid for molecular docking was generated with bound co-
crystallized ligand. Molecules were docked using Glide in extra-precision mode, with up to three
poses saved per molecule. The ligands were kept flexible, whereas the receptor was kept rigid
throughout the docking studies. The lowest energy conformations were selected and, the ligand
interactions (H-Bond and Hydrophobic interactions) with target protein were determined.
4. Results and discussion
4.1. Chemistry
The target compounds 5 (or B) were synthesized by adopting a multi-step sequence as outlined
in Scheme 1. The synthesis of 1,2,3-triazoles are generally carried out by using click chemistry
approach which involves copper (I)-catalyzed 1,3-cycloaddition reaction between a terminal
alkyne and an azide [23]. Thus, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) in
aqueous media was used as a key step in our synthesis. A mixture of CuSO4 and sodium
ascorbate was used as a precatalyst system which generated the Cu(I) species in situ. The
required alkyne was prepared from nimesulide (1) that was reacted with propargyl bromide to
give the compound 2. The reduction of nitro group of 2 followed by ceric ammonium nitrate
(CAN) catalyzed reaction of the resulting amine with acetone afforded the 2,2,4-trimethyl-1,2-
dihydroquinoline 3 via a modified Skraup reaction [9]. The alkyne (3) was then coupled with a
9

number of azides (4) in the presence of CuSO4 and sodium ascorbate smoothly under mild
conditions in water to afford the desired compound (5) in 83-95% yield.
4.2. Pharmacology
To assess their PDE4B inhibitory potential, all the synthesized compounds were tested using an
enzyme based in vitro assay [24]. A well known PDE4 inhibitor Rolipram was used as a
reference compound in this assay. While all these compounds showed significant inhibition of
PDE4 when tested at 30 µM (Table 1) two of them e.g. 5e and 5g showed promising inhibition
(> 50%). Notably, nimesulide did not show any inhibition at the same concentration whereas the
compound A (Fig. 1) was found to be a moderate inhibitor of PDE4 (inhibition < 40%). To
understand the nature of interactions of this class of heterocycles with the PDE4B protein
docking studies were performed using compound 5e in addition to 5g (or compound C as shown
in Fig. 2) that showed ~ 50% inhibition of PDE4B. The dock scores of compounds 5e and 5g
after docking into the PDE4B protein are presented in Table 2. The molecular interactions of 5e
are presented in Fig. 3 which indicated that 5e interacted with Hie-234 through a H-bond and
with Phe-446 through π-π stacking.
Insert Table 1 here.
Insert Table 2 here.
Insert Fig. 3 here.
In view of encouraging PDE4B inhibitory properties of 2,2,4-trimethyl-1,2-dihydroquinolinyl
substituted 1,2,3-triazol derivatives we then decided to perform in vitro cytotoxic evaluation of
these compounds. Being the second leading cause of death worldwide cancer especially lung,
prostate, cervical and liver cancers have attracted enormous attention and development of
suitable agents to treat these types of cancers is highly desirable. We have used a number of
human cancer cell lines e.g. A549 (lung adenocarcinoma epithelial cell line), DU145 (prostate
10

cancer cell line), HeLa (cervical cancer cell line) and HepG2 (hepatocellular liver carcinoma cell
line) for our in vitro assay. A colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay was used to measure the effect of test compounds along with
a standard drug doxorubicin on call viability and the corresponding IC50 values are presented in
Table 3. While all the compounds except 5i were found to be active against A549 cells,
compound 5a, 5f and 5g showed promising effects comparable to that of doxorubicin. The
compound 5f showed activities against HepG2 cells but was 16 fold less potent than
doxorubicin. All other compounds were found to be either less active or inactive (e.g. 5b, 5d and
5h). The compound 5i showed best activities against Hela cells though found to be 3 fold less
potent than doxorubicin. Among other compounds, 5f and 5g showed IC50 ~ 14-15 µM against
Hela cells. The compound 5b and 5f showed IC50 ~ 10 µM when tested against DU145 cells
whereas 5d, 5h and 5i were found to be inactive. Notably, compounds 5e and 5g though
identified as the most active inhibitors of PDE4 enzyme were not really the most active cytotoxic
compounds. Nevertheless, the compound 5g being promising in both PDE4 and MTT assay
(against A549 cells) seemed to have medicinal value. Thus the present class of PDE4 inhibitors
has potential for the development of new anticancer agents especially for lung cancer.
Insert Table 3 here.
5. Conclusions
In conclusion, novel 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazol derivatives
were designed as potential inhibitors of PDE4. These compounds were synthesized via a multi-
step sequence consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step
in aqueous DMF. The required alkynes were prepared from nimesulide via N-propargylation of
the methanesulfonamide moiety and then nitro group reduction followed by a CAN mediated
modified Skraup reaction of the resulting amine. All the synthesized compounds showed PDE4B
inhibitory properties in vitro at 30 µM with two compounds e.g. 5e and 5g showing > 50%
inhibition. This observation was supported by the docking studies of these two compounds at the
active site of PDE4B enzyme. In vitro cytotoxic evaluation of these compounds were also
performed using four human cancer cell lines e.g. A549, DU145, HeLa and HepG2 in an MTT
assay. Three of these PDE4 inhibitors e.g. 5a, 5f and 5g showed promising cytotoxic properties
11

against A549 human lung cancer cells in vitro with IC50 ~ 8-9 µM. The present class of
compounds therefore represents new templates for the identification and development of novel
PDE4 inhibitors / potential anti-cancer agents.
Acknowledgements. The authors (K. S. S. P. and S. P.) thank Mr. M. N. Raju, the chairman of
M.N.R. Educational Trust for his constant encouragement.
References and notes
[1] E. W. Sutherland, Nobel Prize in physiology or medicine 1971: the action of hormones
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Cancer Biol. Ther. 1 (2002) 268–276.
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R. T. Schermuly, Expert Opin. Investig. Drugs 19 (2010) 117–131.
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Life Sci. 62 (2005) 1198–1220.
[5] P. Goldhoff, N. M. Warrington, D. D. Limbrick, J. A. Hope, B. M. Woerner, E. Jackson,
A. Perry, D. Piwnica-Worms, J. B. Rubin, Clin. Cancer Res. 14 (2008) 7717-7725.
[6] L. Yang, E. Jackson, B. M. Woerner, A. Perry, D. Piwnica-Worms, J. B. Rubin, Cancer
Res. 67 (2007) 651-658.
[7] S. S. Pullamsetti, G. A. Banat, M. Szibor, D. Pomagruk, J. Hänze, E. Kolosionek, J.
Wilhelm, T. Braun, F. Grimminger, W. Seeger, R. T. Schermuly, R. Savai, Oncogene 32
(2013) 1121-1134.
[8] F. Mentz, M. D. Mossalayi, F. Ouaaz, S. Baudet, F. Issaly, S. Ktorza, M. Semichon, J. L.
Binet, H. Merle-Beral, Blood 88 (1996) 2172-2182.
[9] S. Durgadas, V. K. Chatare, K. Mukkanti and S. Pal, Lett. Org. Chem. 7 (2010) 306-310
[10] Rainsford, K. D. Curr. Med. Res. Opin. 22 (2006) 1161-1170.
[11] D. J. Cavalla, M. Chasin, L. J. Dolby, R. W. Frith Phenyl-triazole compounds for PDE-
IV inhibition, US Patent Application No US 6248769 B1, Jun 19, 2001.
[12] S. J. Yan, Y. J. Liu, Y. L. Chen, L. Liu, J. Lin, Bioorg Med Chem Lett. 20 (2010) 5225-
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12

[13] S. Pericherla, J. Mareddy, D. P. G. Rani, P. V. Gollapudi, S. Pal, J. Braz. Chem. Soc. 18
(2007) 384-390.
[14] L. V. Reddy, M. Nakka, A. Suman, S. Ghosh, M. Helliwell, K. Mukkanti, A. K.
Mukherjee, S. Pal, J. Heterocycl. Chem. 48 (2011) 555-562.
[15] S. Durgadas, V. K. Chatare, K. Mukkanti, S. Pal, Appl. Organometal. Chem. 24
(2010) 680-684.
[16] L.V. Reddy, M. Kethavath, M. Nakka, S. S. Beevi, L. N. Mangamoori, K. Mukkanti, S.
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M. Pal, Chem. Commun 47 (2011) 10263-10265.
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13

Figure captions
Fig. 1. Design of novel and potential PDE4 inhibitors (B) as anticancer agents.
Fig. 2. Binding mode and interactions of molecule C at the inhibitor binding site of PDE4B
(HIE: Histidine with hydrogen on the epsilon (E) nitrogen).
Scheme 1. Synthesis of 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazol
derivatives 5 (or B)
Fig. 3. Binding mode and interactions of molecule 5e at the inhibitor binding site of PDE4B
(HIE: Histidine with hydrogen on the epsilon (E) nitrogen).
Figures
Me
R
N
Me
R
Me
Me
Me
Me
SO2Me
N
NHSO2Me
OPh
N
N
N
H
N
H
N
N
N
OPh
1,2,3-triazole
A
B
R = quinolin-3-yl, arylcarbamoyl, 4-(methylsulfonamido)arylcarbamoyl etc
Fig. 1. Design of novel and potential PDE4 inhibitors (B) as anticancer agents.
14

Fig. 2. Binding mode and interactions of molecule C at the inhibitor binding site of PDE4B
(HIE: Histidine with hydrogen on the epsilon (E) nitrogen).
.
15

Fig. 3. Binding mode and interactions of molecule 5e at the inhibitor binding site of PDE4B
(HIE: Histidine with hydrogen on the epsilon (E) nitrogen).
16

Scheme
Br
1. Sn/HCl
NSO2CH3
O
NHSO2CH3
NSO2CH3
O
90 °C, 6 h
O
Yield; 75%
H3C
K2CO3 / acetone
rt
Yield; 85%
2. CAN/Acetone
50-55 °C, 20 h
Yield; 80%
NH
NO2
NO2
H3C CH3
3
R
2
1
N
N
RCH2N3 (4)
MeO2S
N
Me
N
CuSO4.5H2O
Sodium ascorbate
7:1 H2O-DMF, rt
20-30 min
OPh
Me
Me
N
H
5
Me
MeO
PhNHCO-
R =
Me
N
Cl
N
Cl
N
Cl
N
Cl
5e; 90%
5a; 95%
5c; 84%
5d; 83%
5b; 86%
PhO
NHCO-
5i; 83%
p-MeC6H4NHCO-
p-MeOC6H4NHCO-
m-MeC6H4NHCO-
5f; 88%
5g; 95%
5h; 95%
MeO2SHN
Scheme 1. Synthesis of 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazol
derivatives 5 (or B)
17

Tables
Table 1. Inhibition of PDE4B by compound 5 at 30 µM.
Entry
Compound 5
Average % Inhibition
SD
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
5a
38.3
39.3
37.8
36.1
52.7
43.4
58.2
46.4
34.4
90.2
1.3
2.3
2.6
1.9
1.2
2.1
0.9
2.5
1.2
1.1
5b
5c
5d
5e
5f
5g
5h
5i
Rolipram
SD = standard deviation
Table 2. Glide score and contributing XP parameters.
PhobEn
Electro
Compound code
GScore
LipophilicEvdW
HBond
5e
5g
-9.16
-8.24
-5.71
-6.33
-1.57
0.0
-0.67
-0.52
-1.21
-1.39
GScore: glide score
LipophilicEvdW: Chemscore lipophilic pair term and fraction of the total protein-ligand vdw energy
HBond: Rewards for hydrogen bonding interaction between ligand and protein
PhobEn: Hydrophobic enclosure reward
Electro: Electrostatic reward
18

Table 3. In vitro evaluation cytotoxicities of compound 5 against various cancer cell lines.
Entry Compound 5 IC50 values (μM)
A549a
HepG2b
Helac
19.2 0.22
NAe
DU145d
13.3 0.22
10.4 0.21
18.2 0.12
NAe
1
5a
8.4 0.11
11.4 0.12
16.8 0.15
49.7 0.14
11.4 0.22
9.6 0.19
8.7 0.24
18.8 0.18
NAa
16.8 0.13
NAe
2
5b
3
5c
24.5 0.16
NAe
35.86 0.21
NAe
4
5d
6
5e
93.1 0.16
13.1 0.21
94.5 0.11
NAe
24.8 0.21
14.6 0.18
15.8 0.32
NAe
25.2 0.13
10.1 0.11
12.4 0.21
NAe
7
5f
8
5g
9
5h
10
11
5i
20.2 0.11
0.8 0.10
9.7 0.24
3.5 0.21
NAe
Doxorubicin
9.7 0.09
0.5 0.12
aA549 – Lung cancer (CCL-185), bDU145 – Prostate cancer (HTB-81), cHeLa - Cervical cancer
(CCL-2), dHepG2- Liver cancer (HB-8065), eNA = Not active
19

Graphical Abstract
Synthesis of 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted
1,2,3-triazol derivatives: Their evaluation as potential PDE 4B
inhibitors possessing cytotoxic properties against cancer cells
K. S. S. Praveena, N. Suresh, Surekha Akkenapally,
C. Ganesh Kumar, Girdhar Singh Deora, N. Y. S. Murthy,
Sarbani Pal*
Cytotoxic properties
PDE4 inhibition
R
Compound
IC50 (µM)
N
N
A549 cells
8.4 0.11
9.6 0.19
8.7 0.24
MeO2S
NH
MeO2S
N
Me
5a
5f
5g
N
OPh
Nimesulide
OPh
O2N
Me
Me
Doxorubicin 9.7 0.09
N
H
20

Highlights
ꢀ A series of novel 1,2,3-triazol derivatives are synthesized.
ꢀ Their synthesis involved CuAAC in aqueous media as a key step.
ꢀ All the synthesized compounds showed PDE4B inhibitory properties in vitro.
ꢀ Three of them showed promising cytotoxic properties against lung cancer cells.
21