Discovery, synthesis and molecular substantiation of N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides as anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2019.103171

The effort was taken to develop a series of benzothiazole and quinoline fused bioactive compounds obtained through a four-step synthetic route using a range of substituted acetoacetanilides. Achieved N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides (6a-l) were produced up to 96% of yield while the eco-friendly p-TSA used as a catalyst. Further, the anticancer activity of these compounds was determined using a range of cancer cell lines starting from MCF-7 (Breast cancer), HCT-116 (Colon cancer), PC-3 & LNCaP (Prostate) and SK-HEP-1 (Liver cancer). Present study compounds were also testified for antioxidant properties prior to anticancer studies since the Reactive Oxygen Species (ROS) being vital in cancer development. To determine the cell membrane stability effects of the compounds, human red blood cells (HRBC) based membrane protection assay was determined. In the results, compounds 6a-l were able to produce a dominated result values over PC3 cell lines (Prostate cancer) than the other cell lines used in this study. Since the connectivity of human germ cell alkaline phosphatase (hGC-ALP) in the development of prostate cancer is known, the most active compounds were evaluated for the hGC-ALP inhibition in order to ensure a mechanism of anticancer action of these compounds. The mode of interaction and binding affinity of these compounds was also investigated by a molecular docking study. In the results, 6d, 6i, 6k, and 6l were found with least IC₅₀ values <0.075 μM and highest relative activity of 92%, 90%, and 96% respectively. The need for further animal model evaluation and pre-clinical studies recognized.

Full text of DOI:10.1016/j.bioorg.2019.103171

Journal Pre-Proof
Discovery, synthesis and molecular substantiation of N-(benzo[d]thiazol-2-
yl)-2-hydroxyquinoline-4-carboxamides as anticancer agents
B. Bindu, S. Vijayalakshmi, A. Manikandan
PII:
S0045-2068(19)30905-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.103171
YBIOO 103171
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
5 June 2019
24 July 2019
30 July 2019
Please cite this article as: B. Bindu, S. Vijayalakshmi, A. Manikandan, Discovery, synthesis and molecular
substantiation of N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides as anticancer agents, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103171
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JOURNAL PRE-PROOF
Discovery, synthesis and molecular substantiation of N-(benzo[d]thiazol-2-yl)-
2-hydroxyquinoline-4-carboxamides as anticancer agents
B. Bindu1, S.Vijayalakshmi1,**, Manikandan A2*
1Dept. of Chemistry, Government Arts College, Coimbatore-641018, Tamil Nadu, India.
2Dept. of Biotech, School of Biosciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India.
Corresponding authors: * mailtomicromani@gmail.com; ** vijisomu@rediffmail.com
Abstract
The effort was taken to develop a series of benzothiazole and quinoline fused bioactive compounds
obtained through a four-step synthetic route using a range of substituted acetoacetanilides.
Achieved N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides (6a-l) were produced up
to 96 % of yield while the eco-friendly p-TSA used as a catalyst. Further, the anticancer activity
of these compounds was determined using a range of cancer cell lines starting from MCF-7 (Breast
cancer), HCT-116 (Colon cancer), PC-3 & LNCaP (Prostate) and SK-HEP-1 (Liver cancer).
Present study compounds were also testified for antioxidant properties prior to anticancer studies
since the Reactive Oxygen Species (ROS) being vital in cancer development. To determine the
cell membrane stability effects of the compounds, human red blood cells (HRBC) based membrane
protection assay was determined. In the results, compounds 6a-l were able to produce a dominated
result values over PC3 cell lines (Prostate cancer) than the other cell lines used in this study. Since
the connectivity of human germ cell alkaline phosphatase (hGC-ALP) in the development of
prostate cancer is known, the most active compounds were evaluated for the hGC-ALP inhibition
in order to ensure a mechanism of anticancer action of these compounds. The mode of interaction
and binding affinity of these compounds was also investigated by a molecular docking study. In
the results, 6d, 6i, 6k, and 6l were found with least IC50 values < 0.075 µM and highest relative
activity of 92 %, 90 %, and 96 % respectively. The need for further animal model evaluation and
pre-clinical studies recognized.
Keywords: Alkaline phosphatase (ALP); Anticancer; Benzothiazole; Carboxamide; Drug design;
Molecular docking

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1. Introduction
Recognizing medicinally important molecules is an art in developing novel drugs against various
diseases. Combinatorial approach is one of the effective way to screen most efficacious drug
candidate from a range of derivatives designed from it. This study aimed to synthesis a family of
13 quinoline derivatives by using substituted acetoacetanilides to develop them as anticancer
agents. Cancer is one of the most threatening diseases of this universe. Discovering effective drug
is one of the most need of the hour. The present study aimed to prepare drug candidates in a
[1-3]
[4-6]
combination with benzothiazole and quinoline. Numerous benzothiazole
and quinoline
based compounds and their derivatives are widely synthesized and testified for their biological
[7-9]
activity potentials. Quinolines have demonstrated with a wide range of anticancer activities
.
2-phenylquinoline and 2-phenylquinolone derivatives have already been established as potent
antitumor agents [10,11]. In their derivatives, quinoline ring takes significant responsibility in the
mechanism of actions which are highly involving in the cancer developments like seizing cell
cycle, cell migration distribution, angiogenesis inhibition, apoptosis, and in the nuclear receptor
responsiveness modulations [12-14]
.
Cl
Br
F
OH
O
O
O
O
N
N
S
S
N
S
N
S
HN
HN
HN
HN
N
N
HO
HO
N
HO
N
HO
6a
6b
6c
6d
HO
O
O
O
H2N
O
S
O
O
O
O
O
O
N
N
S
S
N
S
N
S
HN
6e
HN
HN
HN
N
N
HO
HO
N
HO
N
HO
6f
6g
6h
Cl
O
F
O
Cl
O
O
O
O
N
N
S
S
N
S
N
S
HN
HN
HN
HN
N
N
HO
HO
N
HO
N
HO
6i
6j
6k
6l
Figure 1. Achieved final structures of present study compound N-(benzo[d]thiazol-2-yl)-2-
hydroxyquinoline-4-carboxamides and relevant derivatives (6a-l)

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For example, imidazoquinoline skeleton in the 1,4-disubstituted imidazo[4,5-c]quinolines
were found with excellent anti-cancer activity [15]. 2-phenyl quinoline comprising [(2-aminoethyl)
aminomethyl] showed the capability of interpolation into double-stranded DNA which is an
indispensable intention for cytotoxicity [16]. Initially, various substituted acetoacetanilides were
treated with concentrated sulfuric acid to obtain corresponding quinolines. After two more steps,
benzo[d]thiazol-2-amine was fused to achieve the target compounds N-(benzo[d]thiazol-2-yl)-2-
hydroxyquinoline-4-carboxamides and relevant derivatives (6a-l) (Fig. 1). The successive
bioactivity prediction was executed using the online bioactivity prediction tools such as
Molinspiration (molinspiration.com) and PASSonline (pharmaexpert.ru/passonline/). As per the
results displayed in Fig. 2, all compounds were showed a possible biological activity and the
enzyme & kinase inhibition potential was the most favorable biological activity. As the prediction
suggested, we aimed to develop the present study compounds as the protein-tyrosine kinase
inhibitors and so as the anticancer agents.
Figure 2. Bioactivity prediction results of compounds 6a-l
2. Materials and methods
Chemistry: All organic chemicals and solvents were procured from Sigma-Aldrich, Merck and
were used as received without further purification. Thin-Layer chromatography (TLC) was carried
out using pre-coated silica plates (Merck-silica gel 60 F254). Merck silica gel 60 (230-400 mesh)
was used for flash column chromatography. Melting point (mp) were checked using an OptiMelt
automated melting point system and are uncorrected. 1H and 13C NMR spectra were recorded on
Bruker spectrometer (400 & 100 MHz respectively) using CDCl3 as the solvent. Tetramethylsilane
was used as the internal standard. HRMS device (MS JOEL GC mate) used a secondary electron

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multiplier (Agilent Technologies 6890N Network GC system for gas chromatography) was used
to record the MASS.
2.1.
General procedure for the preparation of N-(benzo[d]thiazol-2-yl)-2-
hydroxyquinoline-4-carboxamides and relevant derivatives (6a-l)
Substituted 3-oxo-N-phenylbutanamides (1) (0.012 mol) was added to concentrated sulfuric acid
(0.001 mol) in portions on 75 °C to obtain substituted 4-methyl quinoline-2-ol (2) in an affordable
yield range of 92-98 %. To 1.5g (0.0094 mol) of 4-methyl quinoline-2-ol (2) in water, 2.84g (0.018
mol) of alk. KMnO4 was added with stirring for 4 hours and kept on the water bath for 2 hours till
the color of the mixture changes to brown. The reaction was then filtered and neutralized with 1:1
hydrochloric acid. The precipitate formed was washed with water and dried and recrystallized from
hot water to obtain 2-hydroxyquinoline-4-carboxylic acid (3).
To 1g (0.0053 mol) of 2-hydroxyquinoline-4-carboxylic acid (3), 0.077 mL (0.016 mol) oxalyl
chloride was added and refluxed on a water bath. After the reaction excess oxalyl chloride was
removed by co-distillation with benzene to obtain 2-hydroxyquinoline-4-carbonyl chloride (4). To
0.0054 mol of 2-hydroxyquinoline-4-carbonyl chloride (4) in ethanol 0.00464 mol of 2-
aminobenzothiazole (5), 0.0082 mol and triethylamine were added and refluxed on a water bath
for 6-8 hours to obtain the title compound N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-
carboxamide (6).
2.2.
Characterization
carboxamides (6a-l)
of
N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-
N-(benzo[d]thiazol-2-yl)-6-chloro-2-hydroxyquinoline-4-carboxamide (6a): Chalky white
powder; Melting point: 124-126 0C: IR (KBr): 3242, 3066, 3098, 3028, 2915, 2874, 2333, 1738,
1
1586, 1464, 1430, 1387, 1265, 1212, 1118, 890, 754, 696, 674, 668, 642 cm-1: H NMR (400
MHz, CDCl3) δ ppm: 12.91 (s, 1H), 11.58 (s, 1H), 9.02 (s, 1H), 8.36-8.20 (m, 3H), 7.93-7.91 (d,
13
J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.70-7.61 (m, 2H); C NMR (100 MHz, CDCl3) δ ppm: 178.6,
172.4, 168.0, 158.0, 150.4, 146.0, 143.3, 138.4, 136.8, 129.3, 128.7, 128.6, 128.4, 127.7, 127.5,
126.8, 126.7, 124.9, 123.9, 118.2, 115.5; Elemental Analysis, Calculated: C, 57.39; H, 2.83; Cl,
9.96; N, 11.81; S, 9.01; Obtained: C, 57.38; H, 2.84; Cl, 9.95; N, 11.82; S, 9.02. HRMS for
C17H10ClN3O2S Calculated [M+] m/z 355.7960, Found 355.7962.

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N-(benzo[d]thiazol-2-yl)-6-bromo-2-hydroxyquinoline-4-carboxamide (6b): White solid;
Melting point: 128-130 0C: IR (KBr): 3332, 3116, 3028, 2908, 2864, 2333, 1739, 1588, 1466, 1432,
1
1378, 1266, 1234, 1108, 896, 758, 696, 672, 664 cm-1: H NMR (400 MHz, CDCl3) δ ppm: 12.00
(s, 1H), 10.61 (s, 1H), 9.03 (s, 1H), 8.07-7.87 (m, 4H), 7.51 (s, 1H), 7.17-7.00 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ ppm: 178.7, 173.3, 167.7, 152.8, 151.7, 144.0, 143.9, 138.3, 137.9, 137.7,
137.3, 137.0, 128.8, 129.5, 126.0, 125.9, 125.7, 124.9, 122.8, 112.1, 110.1; Elemental Analysis:
C, 51.01; H, 2.52; Br, 19.96; N, 10.50; S, 8.01; Obtained: C, 51.02; H, 2.53; Br, 19.96; N, 10.51;
S, 8.02; HRMS for C17H10BrN3O2S Calculated [M+] m/z 400.2500, Found 400.2500.
N-(benzo[d]thiazol-2-yl)-6-fluoro-2-hydroxyquinoline-4-carboxamide (6c): Pale yellow
powder; Melting point: 120-122 0C: IR (KBr): 3296, 3018, 3002, 2919, 2860, 2342, 1818, 1742,
1
1566, 1430, 1370, 1264, 1234, 1119, 898, 758, 696, 674, 664, 642 cm-1: H NMR (400 MHz,
CDCl3) δ ppm: 12.06 (s, 1H), 10.32 (s, 1H), 8.88 (s, 1H) 8.26-8.25 (d, J = 8.0, 1H), 8.24-8.04 (m,
13
2H), 7.96 (s, 1H), 7.94-7.82 (m, 2H), 7.66-7.63 (t, J = 12, 1H); C NMR (100 MHz, CDCl3) δ
ppm: 178.9, 174.9, 168.9, 154.0, 152.9, 145.2, 145.1, 139.4, 139.1, 138.9, 138.5, 138.2, 131.0,
130.6, 127.2, 127.1, 126.9, 126.1, 124.0, 113.3, 103.4; Elemental Analysis: C, 60.17; H, 2.97; F,
5.60; N, 12.38; S, 9.45; Obtained: C, 60.19; H, 2.98; F, 5.60; N, 12.39; S, 9.46; HRMS for
C17H10FN3O2S Calculated [M+] m/z 339.3444, Found 339.3445.
N-(benzo[d]thiazol-2-yl)-2,6-dihydroxyquinoline-4-carboxamide (6d): Chalky white powder;
Melting point: 124-126 0C: IR (KBr): 3301, 3187, 3023, 2984, 2862, 2318, 1734, 1586, 1464, 1430,
1
1342, 1265, 1224, 1118, 884, 762, 698, 664, 652, 646 cm-1: H NMR (400 MHz, CDCl3) δ ppm:
12.90 (s, 1H), 11.64 (s, 1H), 8.73 (s, 1H), 8.49 (s, 1H), 8.31-8.28 (t, J = 8.0, 1H), 8.20-8.17 (t, J =
8.0 Hz, 1H), 7.91-7.90 (d, J = 8.0, 1H), 7.78-7.68 (m, 3H), 7.32-7.31 (d, J = 8.0, 1H); 13C NMR
(100 MHz, CDCl3) δ ppm: 176.7, 172.7, 167.7, 152.9, 151.7, 144.1, 144.0, 138.3, 137.9, 137.7,
137.4, 128.8, 129.5, 126.0, 125.9, 125.8, 124.9, 122.8, 112.2, 102.2; Elemental Analysis: C, 60.53;
H, 3.29; N, 12.46; S, 9.50; Obtained: C, 60.55; H, 3.29; N, 12.47; S, 9.50; HRMS for C17H11N3O3S
Calculated [M+] m/z 337.3530, Found 337.3533.
N-(benzo[d]thiazol-2-yl)-2-hydroxy-6-methoxyquinoline-4-carboxamide (6e): Ash color
solid; Melting point: 126-128 0C: IR (KBr): 3340, 3186, 3088, 3021, 2918, 2866, 2331, 1734, 1586,
1
1464, 1432, 1387, 1265, 1213, 1118, 894, 754, 696, 674, 664, 641 cm-1: H NMR (400 MHz,

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CDCl3) δ ppm: 12.17 (s, 1H), 11.79 (s, 1H), 8.88 (s, 1H), 8.54-8.49 (t, J = 8.0, 1H), 8.35-8.33 (t,
J = 6.8, 1H), 8.21-8.19 (d, J = 8.0 Hz, 1H), 7.73-7.61 (m, 3H), 7.42-7.40 (d, J = 8.0, 1H), 3.83 (s,
3H); 13C NMR (100 MHz, CDCl3) δ ppm: 179.0, 177.4, 169.9, 164.3, 164.2, 161.9, 161.7, 155.7,
147.0, 146.4, 145.5, 137.6, 130.2, 129.7, 129.0, 126.0, 125.7, 122.1, 116.7, 114.2, 113.7, 112.9,
97.5, 54.8; Elemental Analysis calculated: C, 61.53; H, 3.73; N, 11.96; S, 9.12; Obtained: C, 61.53;
H, 3.72; N, 11.97; S, 9.13; HRMS for C18H13N3O3S Calculated [M+] m/z 351.3800, Found
351.3800.
N-(benzo[d]thiazol-2-yl)-6-ethoxy-2-hydroxyquinoline-4-carboxamide (6f): White powder;
Melting point: 132-134 0C: IR (KBr): 3241, 3064, 3086, 3012, 2976, 2832, 2336, 1748, 1502, 1463,
1
1432, 1387, 1265, 1234, 1118, 890, 754, 692, 678, 646 cm-1: H NMR (400 MHz, CDCl3) δ ppm:
13.23 (s, 1H), 11.14 (s, 1H), 8.55 (s, 1H), 7.94-7.71 (m, 2H), 7.69-7.62 (m, 1H), 7.55-7.54 (d, J =
8.0 Hz, 1H), 7.46-7.38 (m, 2H), 7.17-7.15 (d, J = 8.0, 1H), 3.75-3.70 (m, 2H), 1.80-1.72 (t, J =
13
8.0, 3H); C NMR (100 MHz, CDCl3) δ ppm: 178.2, 173.2, 168.2, 156.0, 147.1, 141.1, 139.7,
138.0, 136.5, 135.7, 129.2, 128.9, 128.8, 128.7, 126.5, 126.3, 125.7, 125.5, 116.0, 112.9, 98.4,
59.6, 14.81; Elemental Analysis, calculated: C, 62.45; H, 4.14; N, 11.50; S, 8.77; Obtained: C,
62.43; H, 4.16; N, 11.50; S, 8.76; HRMS for C19H15N3O3S Calculated [M+] m/z 365.4070, Found
365.4072.
N-(benzo[d]thiazol-2-yl)-2-hydroxy-7-((2-hydroxyethyl)sulfonyl)-6-methoxyquinoline-4-
0
carboxamide (6g): White solid; Melting point: 148-150 C: IR (KBr): 3324, 3192, 3084, 3012,
1
2964, 2827, 2342, 1734, 1574, 1418, 1402, 1386, 1234, 1208, 1107, 894, 758, 696, 664 cm-1: H
NMR (400 MHz, CDCl3) δ ppm: 12.94 (s, 1H), 11.18 (s, 1H), 8.94 (s, 1H), 8.54-8.53 (d, J = 4,
1H), 7.99-7.94 (m, 2H), 7.37-7.26 (m, 3H), 4.37 (s, 1H), 3.75-3.71 (t, J = 8.0, 2H), 3.37 (s, 3H),
3.16-3.02 (t, J = 8.0, 2H); 13C NMR (100 MHz, CDCl3) δ ppm: 177.9, 172.7, 167.7, 155.7, 146.5,
142.5, 140.9, 137.7, 132.8, 132.1, 129.0, 129.0, 128.7, 128.4, 128.0, 127.8, 125.9, 125.6, 116.7,
113.0, 97.8, 59.8, 55.0, 54.7; Elemental Analysis, calculated: C, 52.28; H, 3.73; N, 9.15; S, 13.95;
Obtained: C, 52.29; H, 3.72; N, 9.16; S, 13.96; HRMS for C20H17N3O6S2 Calculated [M+] m/z
459.4910, Found 459.4913.
7-amino-N-(benzo[d]thiazol-2-yl)-2-hydroxy-6-methoxyquinoline-4-carboxamide
(6h):
0
Yellow solid; Melting point: 128-130 C: IR (KBr): 3214, 3065, 2964, 2876, 2345, 1789, 1618,

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1
1543, 1427, 1327, 1266, 1223, 996, 864, 654, 674, 668, 648 cm-1: H NMR (400 MHz, CDCl3) δ
ppm: 12.58 (s, 1H), 11.25 (s, 1H), 8.41-8.39 (m, 1H), 8.09-8.07 (m, 1H), 7.82 (s, 1H), 7.65-7.63
13
(m, 2H), 7.40 (s, 1H), 6.88 (s, 1H), 5.58, (s, 2H), 3.77 (s, 3H); C NMR (100 MHz, CDCl3) δ
ppm: 178.3, 173.7, 168.3, 156.2, 147.1, 143.2, 142.0, 141.2, 140.1, 138.0, 128.8, 128.7, 128.1,
127.7, 126.6, 126.3, 125.9, 125.6, 115.9, 112.9, 98.3, 58.1; Elemental Analysis, calculated: C,
59.01; H, 3.85; N, 15.29; O, 13.10; S, 8.75; Obtained: C, 59.01; H, 3.85; N, 15.29; S, 8.75; HRMS
for C18H14N4O3S Calculated [M+] m/z 366.3950, Found 366.3951.
N-(benzo[d]thiazol-2-yl)-7-chloro-2-hydroxy-6-methoxyquinoline-4-carboxamide
(6i):
0
Yellow solid; Melting point: 124-126 C: IR (KBr): 3256, 3048, 3018, 3002, 2948, 2866, 2334,
1
1724, 1524, 1468, 1432, 1388, 1268, 1234, 1108, 898, 756, 696, 674, 664, 642 cm-1: H NMR
(400 MHz, CDCl3) δ ppm: 12.79 (s, 1H), 11.11 (s, 1H), 8.37 (s, 1H), 8.13 (s, 1H), 8.10-7.90 (m,
13
1H), 7.63 (s, 1H), 7.48-7.38 (m, 3H), 3.80 (s, 3H); C NMR (100 MHz, CDCl3) δ ppm: 177.7,
173.0, 166.7, 154.6, 145.5, 141.5, 140.4, 139.6, 138.5, 136.4, 127.2, 126.0, 125.0, 124.0, 114.3,
111.3, 96.7, 53.2; Elemental Analysis, calculated: C, 56.04; H, 3.14; Cl, 9.19; N, 10.89; S, 8.31;
Obtained: C, 56.05; H, 3.14; Cl, 9.19; N, 10.89; S, 8.32; HRMS for C18H12ClN3O3S Calculated
[M+] m/z 385.8220, Found 385.8222.
N-(benzo[d]thiazol-2-yl)-7-fluoro-2-hydroxy-6-methoxyquinoline-4-carboxamide
(6j):
0
Chalky white solid; Melting point: 136-138 C: IR (KBr): 3167, 3089, 3024, 2976, 2819, 2364,
1
1739, 1584, 1466, 1434, 1388, 1266, 1234, 1120, 898, 764, 686, 678, 664, 646 cm-1: H NMR
(400 MHz, CDCl3) δ ppm: 12.63 (s, 1H), 11.25 (s, 1H), 8.83-8.79 (d, J = 8.0, 1H), 8.43-8.39 (t, J
= 9, 1H), 8.09-8.08 (t, J = 4, 1H), 7.55-7.44 (m, 4H), 3.73 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
ppm: 175.4, 171.1, 166.4, 154.3, 145.2, 141.3, 140.1, 139.3, 138.2, 136.1, 127.0, 126.9, 126.2,
125.8, 124.7, 124.0, 123.7, 114.0, 111.0, 96.4, 57.7; Elemental Analysis, calculated: C, 58.53; H,
3.27; F, 5.14; N, 11.38; S, 8.68; Obtained: C, 58.54; H, 3.28; F, 5.14; N, 11.39; S, 8.69; HRMS
for C18H12FN3O3S Calculated [M+] m/z 369.3704, Found 369.3706.
N-(benzo[d]thiazol-2-yl)-2-hydroxy-6,7-dimethylquinoline-4-carboxamide (6k): Grey color
solid; Melting point: 124-126 °C: IR (KBr): 3248, 3067, 3084, 3002, 2924, 2867, 2327, 1738,
1542, 1426, 1445, 1314, 1234, 1208, 1124, 884, 852, 796, 682, 668, 640 cm-1: 1H NMR (400 MHz,
CDCl3) δ ppm: 12.49 (s, 1H), 11.42 (s, 1H), 8.95 (s, 1H), 8.55-8.48 (m, 1H), 8.40-8.31 (m, 1H),

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7.43-7.33 (m, 3H), 7.26 (s, 1H), 2.53 (m, 3H), 2.35 (s, 3H); 13C NMR (100 MHz, CDCl3) δ ppm:
178.7, 172.5, 168.5, 159.1, 156.0, 148.8, 146.5, 137.6, 132.4, 131.8, 129.1, 129.0, 128.2, 127.8,
127.0, 126.9, 118.6, 118.4, 114.7, 110.9, 110.2, 19.9, 17.9; Elemental Analysis, calculated: C,
65.31; H, 4.33; N, 12.03; S, 9.18; Obtained: C, 65.31; H, 4.33; N, 12.03; S, 9.18; HRMS for
C19H15N3O2S Calculated [M+] m/z 349.4080, Found 349.4082.
N-(benzo[d]thiazol-2-yl)-7-chloro-2-hydroxy-6-methylquinoline-4-carboxamide
(6l):
Brownish solid; Melting point: 128-130 0C: IR (KBr): 3298, 3112, 3016, 2984, 2892, 2346, 1764,
1
1538, 1424, 1408, 1342, 1234, 1206, 1122, 898, 756, 698, 676, 664, 646 cm-1: H NMR (400
MHz, CDCl3) δ ppm: 12.66 (s, 1H), 11.11 (s, 1H), 8.99 (s, 1H), 8.13-8.04 (m, 1H), 7.93-7.74 (m,
13
1H), 7.43 (s, 1H), 7.41-7.36 (m, 3H), 2.26 (s, 3H); C NMR (100 MHz, CDCl3) δ ppm: 179.1,
174.5, 169.1, 157.4, 156.0, 154.0, 146.8, 138.5, 130.5, 129.5, 128.8, 128.5, 128.4, 128.2, 127.5,
125.9, 120.6, 119.6, 115.7, 112.6, 111.1, 109.8, 20.3; Elemental Analysis, calculated: C, 58.46; H,
3.27; Cl, 9.59; N, 11.36; S, 8.67; Obtained: C, 58.47; H, 3.28; Cl, 9.59; N, 11.37; S, 8.68; HRMS
for C18H12ClN3O2S Calculated [M+] m/z 369.8230, Found 369.8233.
2.3.
Cell lines preparation
The early passage of MCF-7 (Breast cancer), HCT-116 (Colon cancer), PC-3 & LNCaP (Prostate)
and SK-HEP-1 (Liver cancer) cell lines used in this study were developed and cultured as
previously described[17,18]. All cancer cells were cultured in DMEM (Dulbecco’s Modified Eagle’s
Medium) supplemented with 10% Fetal Bovine Serum (FBS), (100U) 20 μg/ml penicillin, and 100
μg/ml streptomycin. They were sub-cultured by removing existing medium and adding fresh 0.25%
trypsin 0.53 mM EDTA for several minutes, then trypsin was removed and the culture sit at 37°C
for 10 to 15 minutes. Incubation was carried out at 37 °C in an atmosphere of 5% CO2. For the
assay, 1 ml of homogenized cell suspension was poured in each well of a microtiter plate and kept
in a desiccator. After incubated for 48 hours, the cells were observed for any physiological changes
in an inverted microscope. 0.05 ml of test samples was liquefied in 4.95 ml of DMSO to attain a
working concentration of 1 mg/ml.
2.4.
MTT assay
The anticancer activity of N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides (6a-l) on
various cancer cell lines was determined by the MTT (3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl

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tetrazolium bromide) assay as we previously reported [17-19]. About 5000 cells were seeded in 96-
well flat-bottom titer plates and then incubated for 24, 48, and 72 hours in presence 5% CO2
atmosphere and 37 °C temperature. The drug candidates 6a-l in different concentration (10-150
µg/mL) were added into the 96-well titer plate and incubated further at different time periods.
After completion of the incubation, the medium was removed from the wells. The wells were then
washed with PBS; 100 µL of the working MTT dye in DMEM (Dulbecco's Modified Eagle's
Medium) media was added and incubated for another 2 hours. MTT lysis buffer (100 µL) was
added and incubation was continued for at least 4 hrs. The absorbance was measured at 570 nm
and the cell viability was calculated using the following formula,
Cell viability (%) = Mean OD/Control OD x 100%
2.5.
hGC-ALP activity assay
The protocol for executing the hGC-ALP assay was adopted from our earlier report [20]. Generally,
ALP catalyzes the hydrolysis of phosphate esters in an alkaline buffer and generating organic
radical and inorganic phosphate. The changes in alkaline phosphatase quantity and activity are
associated with numerous disease states (especially in the liver and bone). Thus,
transphosphorylation buffers are widely used in ALP inhibition investigations. These are enabling
the “double displacement” reaction mechanism of ALP [21-23]. The ALP assay kit which was used
in this study uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow
(λmax= 405 nm) when dephosphorylated by ALP. Serum and plasma are carefully diluted up to 10
times; to measure the intracellular ALP (isozyme origin: human Germ cell ALP (hGC-ALP)), the
washed cells (1×105) are homogenized in the assay buffer and centrifuged to remove insoluble
substances at 13,000 rpm for 5 minutes. The range of volume of prepared candidate drug samples
was loaded into the 96-well plate wells.
A centralized total volume of the reaction mixture was brought to 100 μl by adding assay
buffer. To attain this, 10-50 µl (5 fold (10 mg/mL) solution) of 6a-l in different concentrations was
added to separate wells, bring volume to 80 µl. and 20 µl stop solution was added and assorted
well to avoid ALP activity in the samples. Then, 75 µl of the 5 mM pNPP solution was added to
each well comprising 6a-l in different concentrations. Due to the colored samples are anticipated
to obstruct with OD. 405 nm readings, distilled water was served as background control.
Consequently, 75 µl distilled water was added to all wells and mixed thoroughly. This was then

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incubated for 75 min at 22-25 ºC in dark. OD was measured at every 15 minutes up to eight gradual
intervals. ALP activity of the test samples can then be calculated:
ALP activity (U/ml) = Amount of pNPP generated by sample (μmol)/Sample Volume (mL)/
Reaction Time (minutes)
2.6.
Molecular docking studies
Docking study performed in order to get more insight into the binding mode of the PQPDs into
the binding pockets of PI3 Kinase. Autodock4.2.6 and Autodock Tools (ADT)1.5.6. and the
Arguslab version 4.0.1 was used for the docking studies. All strategy and measures for docking
studies were followed and customized accordingly to our previous reports [24-26]. Their 3D atomic
coordinates of N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides (6a-l) were created
using ACD/Labs – Chemsketch 12.0 software. Compound 6a-l geometries were cleaned and
generated as the corresponding pdb. Ligand files. 3D crystal structure of human placental alkaline
phosphatase (ALP) (they are also known as germ cell like ALP) (PDB ID: 1EW2) retrieved from
the protein data bank (PDB) (Source: www.rcsb.org/pdb/). Statistical mechanical analysis for the
ligands 6a-l was analyzed and the lowest binding energy, ligand efficiency, and the inhibitory
constant (ki) values were extracted. Molecular interactions like hydrogen bonding, π-π interaction
and π-cation interaction results were analyzed and validated for structure-activity relationships.
2.7.
Statistical analysis
All results were expressed as the percentage decrease with respect to control values and compared
by one-way ANOVA with Dunnett’s post test was performed. GraphPad Prism version 6.07 for
Windows, GraphPad Software, San Diego California USA, www.graphpad.com was used for
statistical analysis. A difference was considered statistically significant if p≤0.05.
3. Results and discussions
3.1.
Synthesis of N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides
(6a-l)
In the present study, the chemical reaction using different Lewis acids entry and bifunctional
organocatalysts were used to obtain novel N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-
carboxamides (6a-l) in higher yield using substituted acetoacetanilides (Scheme 1). Among the

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range of solvents used, the use of p-TSA.H2O enhanced the reactivity, a lesser amount of reaction
time and higher amount of yield deprived of purification over tiresome column chromatography
technique. The maximum quantity of treatment of p-TSA.H2O essential for this reaction was
adjusted (15 mol %) in order to reduce excess catalyst usage. However, inappropriately,
diminishing the catalyst amount reduced the yield of compounds 6j, 6k, and 6l. Later, the use of
15 mol percentage of p-TSA.H2O in water was able to provide satisfaction with a high reaction
yield of 6f (98 %). Furthermore, the influence of different solvent usage was also examined in
order to optimize the product yield in lesser reaction time. Quite a few numbers of organic solvents
such as methanol (MeOH), CH3CN and showed no significant to ethanol (EtOH) that in turn has
also no imperative to P-TSA.H2O, has given high reaction yield of 6l (95 %).
Initially, the reaction of 2-hydroxyquinoline-4-carbonyl chloride (4) was started with
substituted acetoacetanilides having electron donating entities like -4Br; -4OMe; -4OEt; -4Me
groups that are having solid electron-withdrawing potency were reacted in quicker rates of
chemical association and dissociation processes after the addition of different catalysts compared
with that substituted acetoacetanilide reaction in the usual solvent conditions. In general, all
compounds were able to synthesize in presence different catalyst using eco-friendly ethanol
(EtOH) as a solvent. While loading 10 % catalyst (SnCl2.H2O, p-TSA.H2O) to 6b and 6i were not
able to synthesize in higher yields. Thus, we increased the catalyst addition up to 15-20 % produced
a significant yield of the compounds 6b, 84 % and 6i, 98 % in the presence of p-TSA.H2O.
O
1
CH3
2
3
COOH
30 minutes 95o C
R
Alk. KMnO4
4 Hr Stirring
2 Hr Reflux
N
H
O
conc. H2SO4
N
OH
N
OH
-4Br; -4OMe; -4OEt; -4Me
Oxalyl Chloride
24 Hrs RT
OH
N
H
S
Cl
O
N
Benzo[d]thiazol-2-amine (5)
N
O
EtOH pTSA.H2O
N
OH
6
4
Scheme 1. Route of synthesis of N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides
(6a-l)
Compounds 6c, 6j was synthesized with a catalyst percentage range of 5 - 20 % mol AlCl3
(20%), p-TSA.H2O (15 %) given a moderate to good yield of compounds 6c (76 %) and 6j (96 %)
and 6d, 6k FeCl3 (20%) p-TSA.H2O (15 %) produced a remarkable yield of 92 % and 96 %

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correspondingly. Meanwhile, compounds 6e and 6l were synthesized using the L-proline,
SnCl2.H2O (25 % mol) as catalyst was also produced a moderate to the respectable yield of 70 and
98 % respectively. In the end, compounds 6j, 6g, 6l, and 6n were synthesized using p-TSA.H2O (
catalyst range of 5 - 20 mol %) produced a maximum yield of 96 % (6j). In the investigation of
reaction, properties/effect of different organic solvents like MeOH and CH3CN showed no
dominance to EtOH. In the meantime, the same reactions accomplished in the presence of water
produced tremendous yield within 2-6 hours.
3.2.
Evaluation of Alkaline Phosphatase (ALP) inhibitory potentials
Initially, through a competitive ELISA colorimetric assay, the essential diagnostic screen of
N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides (6a-l) against hGC-ALP assessed.
Not only to unveil the hGC-ALP inhibition prospective of 6a-l, but the main purpose of hGC-ALP
inhibition assay is to segregate the most effective compounds among hydroxyquinoline-
carboxamides (6a-l) in order to develop them as anti-prostate cancer drugs. Because of the straight
association between hGC-ALP and prostate cancer already revealed. Therefore, screening the
utmost and appropriate candidate drugs was deliberated here, not only for limiting the essential
research interlude but also to keep an economical way of drug development processes. Fig. 3A-C
illustrates the results of hGC-ALP inhibition assay.
Based on the results the best compounds were screened for further anticancer studies by
comparing with standard (Estradurin). Obviously, the results were found in a dose-dependent
manner. For authorizing or screening the most effective hGC-ALP inhibitors among 6a-l, a relative
% activity along with the IC50 values was considered and according to the values the compounds
were ranked from best to worst. In the assessments, 6d, 6i, 6k, and 6l were found with the smallest
amount of IC50 values < 0.075 µM (Fig. 3A) with a highest relative activity of 92 %, 90 %, and 96
% respectively (Fig. 3B). The IC50 of compounds 6h, 6i, and 6k found between 0.05-0.15 µM
ranges. 6d and 6g-l were showed excellent inhibition potential than the standard, Estradurin (82 %
of the relative activity with an IC50 1.5 µM).
Fig.3C illustrates the dose-response inhibition activity of for the most potent compounds 6l
and the standard Estradurin. As 6l started to show maximum inhibition from 10-4 and lasted up to
10-7 (µg/mL), its stability was recognized while Estradurin showed maximum activity only at the
concentrations 10-5 and 10-6 (µg/mL). Among all, 6a-c and 6f found with lesser or no hGC-ALP

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inhibition activity. Simultaneously, we have screened compounds 6d and 6g-l for further anti-
prostate cancer evaluations as they exhibited excellent hGC-ALP inhibition as well as firm
interaction to the human Placental ALP (hP-ALP) binding pocket (Fig. 3D). Both ALP are highly
resemble structurally and functionally.
Figure 3. Results of hGC-ALP inhibition by 6a-l. Results are the average of duplicate or triplicate
values. Figure 3C. Dose-response curve of 6l, and Estradurin. Results are the average of duplicate
or triplicate values
3.3.
Molecular docking studies – assessment of Receptor-Ligand interaction
To get more insight into the molecular level interaction of present study N-(benzo[d]thiazol-2-yl)-
2-hydroxyquinoline-4-carboxamides (6a-l), these compounds were docked into the active sites of
human placental ALP (PDB ID: 1EW2) which is highly relevant to Human Germ Cell ALP.
Molecular mechanistic values were retrieved from best-docked poses such as lowest binding
affinity, least binding energy, and ligand efficiency. Molecular interactions by means of non-
covalent bonding such as π-π interaction and π-cation interaction along with hydrogen bonding
interactions were verified. In the results, expected binding free energy values of ALP was

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established in -6.28 to -10.79 kcal/mol and inhibitory constant (ki) were found in the range of 0.005
to 5 µM (Fig. 4A-D).
As emphasized in yellow (Fig. 4A), compounds 6d, and 6g-l were showed the most prominent
results. There were a correlation between the molecular docking (in silico) values to ALP
inhibition (in vitro), thus the progress of the present research was evenly poised and given away
for screening pre-eminent compounds for evaluating anti-prostate cancer studies were screened.
Furthermore, for the comparison purpose, molecular docking was conducted for also to Estradurin,
the standard drug used for the ALP assay. Among 6a-l, except 6e, remaining compounds showed
dominated molecular mechanistic values as displayed in Fig. 4A.
Figure 4A-D. Molecular mechanistic values of 6a-l to Human Placental ALP (PDB ID: 1EW2)
(values are the average of three repeated docking processes). Yellow highlight in Fig. 4A indicates
the most desirable docking values. Yellow rectangle highlighted indicates the key amino acid
comparison with compound 6k (Fig. 4B), 6l (Fig. 4C) and Estradurin (Fig. 4D).
3.3.1. Receptor-Ligand interface assessments by molecular docking simulations
Docking simulation is the process of assessment of interactions between Ligand & Receptor. As
displayed in Fig 4B-C, these two compounds found to have all expected interactions such as non-
covalent bonding (π-π interaction) and hydrogen bonding. All hydrogen bonding were established

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within 1.5Å to the 2.1Å range. Estradurin, the standard ALP inhibitor established hydrogen bonds
with Arg166 (1.587Å), Asp42 (2.063Å), His153, 432 (2.188Å), Ser92 (1.693Å) (Fig. 4D). As
displayed in Fig. 4B, compound 6k found to have both hydrogen bond with key amino acid
residues (Arg166 (2.145Å); His432 (1.923Å) and Glu428) and non-covalent bond (π-π interaction
between quinoline aryl ring to His153). Compound 6l also established with as many hydrogen
bonds along with the anticipated key amino acid residues (Arg166 (2.017Å); His153 (2.058Å) and
His432 (1.887Å)) as well as with few others (His317 (2.083Å) and His320 (1.761Å)). In the
plausible mechanism, His432 probably donates an electron to the quinoline carboxylic oxygen
end, where the -O- is present in compound 6k and 6l. Hence, a conceivable –NH2-O- complex
could be recognized which enables the ALP inhibition promising.
Figure 5A-D. Anticancer effect of 6i and 6l against PC-3 cell lines. PC-3-a control treated
(Distilled water); PC-3-b standard (Estradurin) treated; PC-3-c compound 6i treated and PC-3-d
compound 6l treated. Yellow circle mark indicates the necrosis generated due to the
drug/compound action
3.4.
Anticancer activity evaluations
The anti-prostate cancer drug efficacy of 6k and 6l through ALP inhibition mechanism was
recognized through in silico and in vitro studies. In the activity, among all the cell lines used,

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compounds 6k and 6l were found with the selective anti-proliferative activity mostly on both the
cancer cells (PC-3 and LNCaP). In the meantime, there was no activity was found (up to 150
µg/ml) against normal epithelial (MCF-10) cell lines. The IC50 values found for compounds 6k
(0.048±0.002 µM) and 6l (0.018±0.002 µM) designate them as more active than the standard
Bicalutamide (CDX) (1.3±0.08 µM). The relative % activity was 92.26±1.12 %, 94.45±1.08 %,
and 88.24±2.04 % respectively for 6k, 6l and Bicalutamide (CDX). Fig. 5A-D shows the
anticancer effects before (Fig. 5A&B) and after (Fig. 5C&D) treating the compounds 6k and 6l on
PC-3 cell lines. There was significant necrosis found on PC-3 cell lines after treating (2 hours)
6k (Fig. 5C) and 6l (Fig. 5D).
3.5.
Results of HRBC membrane stabilization potential of 6a-l
Compounds 6k and 6l were taken to measure the inflammation preventing capacities through the
inhibition of HRBC membrane lysis on Hypotonicity induced red blood cells. In the results, both
compounds were showed no toxic effect even after the maximum concentration (150 µg/mL) used
in this study. compound 6k showed a maximum protective effect up to 96.12±0.80 % with an IC50
0.022±0.002 µM, and 6l showed 98.34±0.92 %) while the standard Diclofenac showed up to
90.22±1.02 % (IC50 1.0±0.02 µM).
1.1.
Druggability assessments of hydroxyquinoline-4-carboxamides (6a-l)
Table 2 depicts the centralized three repeated predicted values of ADMET properties of 6a-l. There
were no compounds found violating the druggability properties as they showed affordable values.
Especially the potentially identified ALP inhibitors 6k and 6l had affordable MlogP values (2.5
and 2.8 respectively) which ensure less lipophilicity.
Table 2. ADMET assessments data of hydroxyquinoline-4-carboxamides (6a-l)
Name
6a
6b
6c
6d
6e
6f
DiffCoef
0.755
0.746
0.776
0.771
0.741
0.714
0.633
0.722
0.718
0.736
0.722
0.727
MlogP
2.587
2.704
2.469
1.578
1.815
2.048
-0.113
1.567
2.048
1.932
2.552
2.82
RO5
0
RO5_Code
<None>
<None>
<None>
<None>
<None>
<None>
<None>
<None>
<None>
<None>
<None>
<None>
MWt
M_NO
T_PSA
75.11
75.11
75.11
95.34
84.34
84.34
138.71
110.36
84.34
84.34
75.11
75.11
HBDH
355.804
400.26
339.35
337.359
351.386
365.413
459.502
366.4
5
5
5
6
6
6
9
7
6
6
5
5
2
2
2
3
2
2
3
4
2
2
2
2
0
0
0
0
0
6g
6h
6i
6j
6k
6l
0
0
0
0
0
0
385.831
369.376
349.413
369.831

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Notes: MlogP. Moriguchi estimation of logP. HBDH. A number of Hydrogen bond donor protons. M_NO. A total number of Nitrogen and Oxygen
atoms. T_PSA. The topological polar surface area in square angstroms. Ro5 - Rule Of Five. Rule Of Five_Code. Hb = number of Hydrogen bond
donor protons; Mw = molecular weight; NO = number of Nitrogen- and Oxygen-based Hydrogen bond acceptors.
1.2.
Structure-Activity Relationships
In order to recognize the Structure-Activity Relationship (SAR) primarily, effectively synthesized
novel N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides (6a-l) with a wide range of
electron withdrawing and electron discharging groups R1 position of quinoline ring were
synthesized and established as active drug candidates against prostate cancer tested on PC-3 and
LNCaP prostate cancer cell lines. Effective molecular interaction (verified through molecular
docking studies) ALP inhibition (validated through in vitro Human Germ Cell ALP assay),
potential compounds 6k (substituted with –S, –Cl, -CH3, -OH along with carbonyl and amides),
and 6l (substituted with –S, –CH3, -CH3, -OH along with carbonyl and amides) in the inhibition of
alkaline phosphatase were recognized. Fig. 6 exemplifies the plausible mechanism of action of
ALP inhibition by the compound 6l.
High polar groups to enhance drug solubility
ALP
ALP
N
HO
Arginin 166
NH
NH
O
N
H2N
N
H
OH
O
H2N
Histidine 153
OH
NH2
NH
N
6k
6l
Interaction of ALP to
Cation - Pi interaction
Pi - Pi interaction
S
O
Hydrogen bonding
Figure 6. The plausible mechanism proposed for compound 6k – Human Germ Cell ALP
interaction
2. Conclusions
In the present study, novel N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides (6a-l)
were synthesized and their anti-prostate cancer efficacy was recognized through ALP inhibition
potentials. The synthetic reaction carried out in ethanol in the presence of a different bio-friendly
catalyst. In the product development, we used only simple filtration and extraction and remarkably
not the tedious chromatographic techniques. In conclusion, novel N-(benzo[d]thiazol-2-yl)-2-
hydroxyquinoline-4-carboxamides (6a-l) were efficaciously synthesized as ALP inhibitors so as

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the anti-prostate cancer agents by means of ecologically friendly catalytic systems in very good
yields. The therapeutic solicitations of the compounds were examined and attained the following
verdicts
 As the interrelation between Human Germ Cell ALP and prostate cancer was
systematically assessed and related.
 Compounds 6k and 6l were showed potent Human Germ Cell ALP enzyme inhibition
activity and anti-prostate cancer effects.
With these positive results, currently taking forward the in vivo studies in animal models only for
the potentially identified and most efficacious compounds 6k, and 6l.
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Graphical abstract

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Highlights
 Novel hydroxyquinoline-4-carboxamides (6a-l) was synthesis and tested for
druggability
 All synthetic routes were reflected eco-friendly chemistry approach
 Hydroxyquinoline-4-carboxamides (6a-l) were able to bind on catalytic site of alkaline
phosphatase enzyme (ALP)
 Hydroxyquinoline-4-carboxamides (6a-l) exhibited anti-prostate cancer activity
 Compounds 6k and 6l were found as precise ALP inhibitors and prostate cancer
therapeutic agents