p-TSA-promoted syntheses of 5H-benzo[h]thiazolo[2,3-b]quinazoline and indeno[1,2-d]thiazolo[3,2-a]pyrimidine analogs: Molecular modeling and in vitro antitumor activity against hepatocellular carcinoma

Article abstract of DOI:10.2147/DDDT.S136692

In our efforts to address the rising incidence of hepatocellular carcinoma (HCC), we have made a commitment to the synthesis of novel molecules to combat Hep-G2 cells. A facile and highly efficient one-pot, multicomponent reaction has been successfully de

Full text of DOI:10.2147/DDDT.S136692

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O r i g i n a l r e s e a r c h
p-Tsa-promoted syntheses of 5h-benzo[h]
thiazolo[2,3-b]quinazoline and indeno[1,2-d]
thiazolo[3,2-a]pyrimidine analogs: molecular
modeling and in vitro antitumor activity against
hepatocellular carcinoma
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
29 May 2017
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amit K Keshari1
ashok K singh1
Vinit raj1
Abstract: In our efforts to address the rising incidence of hepatocellular carcinoma (HCC), we
have made a commitment to the synthesis of novel molecules to combat Hep-G2 cells. A facile
and highly efficient one-pot, multicomponent reaction has been successfully devised utilizing a
p-toluenesulfonic acid (p-TSA)-catalyzed domino Knoevenagel/Michael/intramolecular cycliza-
tion approach for the synthesis of novel 5H-benzo[h]thiazolo[2,3-b]quinazoline and indeno[1,2-d]
thiazolo[3,2-a]pyrimidine analogs bearing a bridgehead nitrogen atom. This domino protocol
constructed one new ring by the concomitant formation of multiple bonds (C–C, C–N, and C=N)
involving multiple steps without the use of any metal catalysts in one-pot, with all reactants effi-
ciently exploited. All the newly synthesized compounds were authenticated by means of Fourier
transform infrared spectroscopy, liquid chromatography–mass spectrometry, proton nuclear
magnetic resonance spectroscopy, and carbon-13 nuclear magnetic resonance spectroscopy,
together with elemental analysis, and their antitumor activity was evaluated in vitro on a Hep-G2
human cancer cell line by sulforhodamine B assay. Computational molecular modeling studies
were carried out on cancer-related targets, including interleukin-2, interleukin-6, Caspase-3,
and Caspase-8. Two compounds (4A and 6A) showed growth inhibitory activity comparable
to the positive control Adriamycin, with growth inhibition of 50% ,10 μg/mL. The results of
the comprehensive structure–activity relationship study confirmed the assumption that two or
more electronegative groups on the phenyl ring attached to the thiazolo[2,3-b]quinazoline system
showed the optimum effect. The in silico simulations suggested crucial hydrogen bond and π–π
stacking interactions, with a good ADMET (absorption, distribution, metabolism, excretion, and
toxicity) profile and molecular dynamics, in order to explore the molecular targets of HCC which
were in complete agreement with the in vitro findings. Considering their significant anticancer
activity, 4A and 6A are potential drug candidates for the management of HCC.
amit rai1
Prakruti Trivedi2
Balaram ghosh2
Umesh Kumar3
atul rawat3
Dinesh Kumar3
sudipta saha1
1Department of Pharmaceutical
sciences, Babasaheb Bhimrao
ambedkar University,Vidya
Vihar, raebareli road, lucknow,
Uttar Pradesh, 2Department of
Pharmacy, Birla institute of Technology
& science Pilani, hyderabad campus,
hyderabad,Telangana state, 3centre
of Biomedical research (cBMr),
sanjay gandhi Post-graduate institute
of Medical sciences campus, raebareli
road, lucknow, Uttar Pradesh, india
Keywords: thiazolo[3,2-a]pyrimidine and thiazolo[2,3-b]quinazoline, hepatocellular
carcinoma, domino reactions, interleukins, caspases, molecular docking, ADMET, dynamics,
multi-component reactions, metal-free
correspondence: sudipta saha
Department of Pharmaceutical sciences,
Babasaheb Bhimrao ambedkar University,
Vidya Vihar, raebareli road, lucknow
226025, Uttar Pradesh, india
Introduction
One in six human deaths globally is due to multifaceted disease, such as cancer.
Liver cancer accounted for 788,000 deaths in 2015 and is the second most common
cause of cancer-related deaths, worldwide. The most prevalent primary liver cancer
Tel +91 80 9074 7008
email sudiptapharm@gmail.com
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Keshari et al
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with poor prognosis is hepatocellular carcinoma (HCC), attention as privileged scaffolds comprising a vital class of
which has potentially lethal human malignacy worldwide, heterocyclic structures possessing exciting and varied phar-
particularly in Asia and Africa.1,2 In men, HCC is the fifth macologic activities,22–24 such as: being potential inhibitors of
most commonly diagnosed cancer and the second leading cyclin-dependent kinase,25 CDC25B phosphatase enzymes,26
cause of cancer deaths; in women, it is the seventh most IspF proteins,27 and YycG histidine kinase,28 antibacterial,29
frequently diagnosed cancer, and worldwide, overall, it is the analgesic, and anti-inflammatory activities,30 acting as a
sixth commonest cause of cancer-related deaths.3 In spite of corticotropin-releasing factor receptor antagonist,31 hypo-
continuous efforts to develop novel therapeutic strategies to lipidemic activity,32 antiviral including anti-HIV effects,
treat HCC, it remains a challenge. As a consequence, there- anticancer,33,34 cardiotonic, and inotropic effects,35 acting
fore, increased attention has been paid, especially in the field as potent A2A adenosine receptor inverse agonists with
of liver cancer therapy, to the discovery and development antinociceptive activity,36 and so on.
of safe and novel anticancer agents, together with improved
cytotoxicity toward cancerous cells.4
To the best of our knowledge, among the earlier reported
methods devoted to the synthesis of especially 5H-benzo[h]
Domino reactions involving simultaneous formation of thiazolo[2,3-b]quinazolines,37,38 only a few are described, and
C–C and C–heteroatom multiple bonds in a single flask are metal-free multicomponent domino reactions (MDRs)39,40 are
a means to achieving economical methods for the manufac- still rare. In this regard, most of the reported methods have
ture of especially unusual, fused heterocyclic, medicinally suffered from various drawbacks, for example, harsh reac-
privileged scaffolds without any separation of intermediates tion conditions, multistep synthetic routes, costly reagents/
throughout the process. Moreover, domino reactions, being catalysts, prolonged reaction periods, tedious workups after
attractively appealing, are frequently connected with savings each step, and poor availability of starting materials. There
in terms of energy and reaction periods, are highly convergent, is no route for the direct construction of the indeno[1,2-d]
have fewer environmental impacts, and show atom economy thiazolo[3,2-a]pyrimidine moiety through domino reactions,
in a single-step process starting from multiple reactants.5,6
and a literature survey revealed only one report to be avail-
Conventional multistep chemical reactions suffer from able, which includes the synthesis of 5-phenylindeno[1,2-d]
various limitations compared with the one-pot domino tech- thiazolo[3,2-a]pyrimidine-3,6(2H,5H)-dione and 5-phenyl-
nique, as they require a large number of synthetic operations, 2,3-dihydro indeno[1,2-d]thiazolo[3,2-a]pyrimidin-6(5H)-
including isolation and purification of the products of each one in two steps.41
individual step. The multistep synthetic approach, therefore,
has led to synthetic inefficiency, is time-consuming, or con- synthetic strategy for the construction of these two vital
tains side reactions generating large amounts of waste.7
structural elements, while at the same time avoiding several
In order to arrive at a highly proficient and convergent
A key challenge in recent drug development processes is limitations of earlier reports, and in our venture toward
to design a rapid, versatile, and efficient synthesis that pro- the development of a modern synthetic approach, we first
vides target molecules containing structural complexity and report herein an operationally simple and straightforward
diversity with a choice of fascinating biologic activities.8
metal-free, one-pot MDR for obtaining a panel of novel
Thiazole-based heterocycles are central to modern 5H-benzo[h]thiazolo[2,3-b]quinazoline (1A–15A) and
chemical synthesis due to their synthetic feasibility and their indeno[1,2-d]thiazolo[3,2-a]pyrimidine (1B–15B) analogs
incorporation into various types of therapeutically useful using the reactions of highly substituted α-tetralone or
agents and are of paramount interest in the development of α-indanone with some aromatic aldehydes and distinc-
important pharmacophores in the drug discovery endeavor.9,10 tive 2-aminothiazoles in ethanol (EtOH) in the presence
Functionalized quinazoline and fused quinazolines have of catalytic amounts of p-toluenesulfonic acid (p-TSA;
long been of increasing interest in the field of synthetic 20 mol%).7,42,43 Readily available and cheap starting mate-
organic and medicinal chemistry on account of the wide rials, together with an environmentally benign and mild
range of biologic activities,11–14 including anticancer,15 acidic catalyst, were employed to achieve these diversely
anti-inflammatory,16 antituberculosis,17 anticonvulsant,18 decorated skeletons in impressive yields. They have also
antimalarial,19 antihypertensive,20 antidiabetic,21 and so on. been synthesized by conventional two-step reactions and
Furthermore, suitably substituted thiazolo[2,3-b]quinazolines compared with the preferred MDRs described above.23,44–46
also serve as versatile building blocks in synthetic chemistry. Additionally, all of the newly synthesized compounds were
Nowadays, bridgehead (ring junction) nitrogen atom- evaluated for their antitumor activity in vitro on a Hep-G2
containing thiazolo[3,2-a]pyrimidines command much human cancer cell line by sulforhodamine B (SRB) assay
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
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and processed in TopSpin 2.1. Chemical shifts are expressed
in parts per million (ppm) downfield from tetramethylsilane as
the internal standard. Coupling constants are expressed in Hz.
All of the compounds were analyzed for mass data using a liq-
uid chromatography–mass spectrometry (LCMS)-2020 mass
spectrometer (Schimadzu, Tokyo, Japan). All the compounds
were dissolved in 1:1 (v/v) mixtures of acetonitrile:methanol,
and 10 μL of the resulting solution was injected to acquire the
data. The analysis was performed in electrospray ionization
mode using a capillary column at a flow rate of 0.2 mL/min
with a 50% water/methanol (1:1) mixture for 120 s. Data
analysis was performed using lab solution LCMS data pro-
cessing software. Reaction progress as well as the purity of
the compounds were evaluated with thin layer chromatog-
raphy (TLC) plates by using ethylacetate:n-hexane (3:7) as
the eluent, and the developed chromatogram was visualized
under ultraviolet light and iodine vapors.
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general experimental procedures for
characterization of the synthesized
compounds
One-pot efficient synthesis of substituted
Scheme 1 One-pot efficient synthetic route to the titled compound (1A–15A).
Note: substituted α-tetralone (a), substituted aromatic aldehydes (b) and distinctive
2-aminothiazoles (c).
Abbreviations: etOh, ethanol; p-Tsa, p-toluenesulfonic acid.
and structure–activity relationship prediction on the basis
of in vitro findings. Furthermore, computational molecular
modeling studies with ADMET (absorption, distribution,
metabolism, excretion, and toxicity) profiling and molecular
dynamic (MD) simulation were carried out on cancer-related
targets, including interleukin (IL)-2, IL-6, Caspase-3, and
Caspase-8 receptor sites, to identify the potential modes of
action of the named compounds.
5h-benzo[h]thiazolo[2,3-b]quinazolines (1a–15a)
A mixture of substituted tetralone (1 mmol), appropriate aro-
matic aldehydes (1 mmol), and distinctive 2-aminothiazoles
(1 mmol) in EtOH (5.0 mL) in the presence of 20 mol%
p-TSA was heated under reflux for 3–4 h. The reaction
mixture was poured into ice-cold water. A solid product
was obtained, which was filtered, washed thoroughly with
distilled water, and recrystallized from EtOH. Pure crystals
were obtained (Scheme 1). The progress of the reaction was
monitored by TLC on precoated silica gel-G plates using 30%
ethylacetate:n-hexaneasthesolventsystem.TLCrevealedjust
Materials and methods
general information
All of the reactions described below were carried out with a single spot, which proved the presence of a single product.
commercially available chemicals of reagent grade that
were used as received without further purification unless Two-step synthesis of substituted 5h-benzo[h]
otherwise noted. Reagents were purchased from Merck and thiazolo[2,3-b]quinazolines (1a–15a)
Sigma-Aldrich chemical companies. Melting points (m.p.) These compounds were synthesized as follows.
were determined on an m.p. apparatus and are uncorrected.
Compounds were named following the International Union step i
of Pure and Applied Chemistry rules as applied by Advanced A mixture of substituted tetralones (1 mmol), appropriate
Chemistry Development/ChemSketch. Elemental analyses aldehydes (1 mmol), piperidine (0.1 mmol), and glacial
were performed with a Euro EA 3000 Elemental Analyzer acetic acid (0.1 mmol) in EtOH (5.0 mL) was heated under
for C, H, N, and the results were within ±0.4% of the theoreti- reflux for 10–12 h. The reaction mixture was cooled to
cal values. Infrared (IR) spectra were recorded on a Thermo room temperature for a few minutes and then poured into
Scientific Nicolet 6700 Fourier transform IR spectroscopy ice-cold water. A solid product, substituted benzylidene-
spectrometer. Nuclear magnetic resonance (NMR) spectra tetralones, was obtained, which was filtered, washed thor-
were obtained on a Bruker 800 MHz NMR spectrometer (1H oughly with distilled water, and dried. The dry residue was
13
800 MHz, C 200 MHz) (Bruker, Rheinstetten, Germany) recrystallized from EtOH. The progress of the reaction was
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Keshari et al
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monitored by TLC on precoated silica gel-G plates using [M−H]-. Anal. calcd. for C20H15BrN2S: C, 60.77; H, 3.82;
40% ethylacetate:n-hexane as the solvent system.
N, 7.09; Anal. found: C, 60.87; H, 3.83; N, 7.07.
7-(4-bromophenyl)-3-methoxy-10-methyl-6,7-
step ii
dihydro-5h-benzo[h]thiazolo[2,3-b]quinazoline (2a)
Yellow crystals; yield: 82%; m.p. 200°C–202°C; Rf=0.58
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
3,013.1 (C–H, aromatic), 2,945.5 (C–H), 1,663.7 (C=N),
1,596.9 (C=C), 1,332.5 (C–N), 1,263.9 (C–O), 1,070.0
A solution of distinctive 2-aminothiazoles (1 mmol) and
appropriate benzylidene-tetralones in glacial acetic acid
(2.0 mL) was heated under reflux for 18–20 h. The solvent
was evaporated under vacuum and then the residue obtained
was dissolved in chloroform, washed with water, and the
organic layer separated, dried, and evaporated. The result-
ing solid obtained was recrystallized from EtOH to yield the
target compounds (1A–15A; Scheme 2).
1
(C–Br), 761.9 (C–S). H NMR (DMSO-d6, 800 MHz)
δ ppm: 2.92 (t, 2H, J=8 Hz), 3.03 (t, 2H, J=8 Hz), 3.55 (s, 4H),
3.86 (s, 3H), 6.93 (t, 2H, J=8 Hz), 7.48 (d, 2H, J=8 Hz), 7.62
(t, 3H, J=8 Hz), 7.94 (d, 1H, J=8 Hz). 13C NMR (DMSO-d6,
200 MHz) δ ppm: 27.16, 28.67, 39.76, 39.86, 39.97, 40.07,
40.18, 56.06, 112.81, 114.31, 122.39, 126.65, 130.44,
131.99, 132.35, 134.08, 135.08, 136.84, 146.54, 163.84,
185.75. LCMS (m/z): 437.20 [M−H]-. Anal. calcd. for
C22H19BrN2OS: C, 60.14; H, 4.36; N, 6.38. Anal. found: C,
60.25; H, 4.35; N, 6.40.
7-(4-bromophenyl)-6,7-dihydro-5h-benzo[h]
thiazolo[2,3-b]quinazoline (1a)
Yellow crystals; yield: 77%; m.p. 190°C–192°C; Rf=0.55
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
3,067.7 (C–H, aromatic), 2,944.7 (C–H), 1,665.8 (C=N),
1,595.0 (C=C), 1,295.8 (C–N), 1,068.4 (C–Br), 740.7
1
(C–S). H NMR (dimethyl sulfoxide [DMSO]-d6, 800
MHz) δ ppm: 2.95 (t, 2H, J=8 Hz), 3.07 (t, 3H, J=8 2-methoxy-7-(3,4,5-trimethoxyphenyl)-6,7-dihydro-
Hz), 7.40–7.44 (m, 2H), 7.50 (d, 2H, J=8 Hz), 7.59 (t, 5h-benzo[h]thiazolo[2,3-b]quinazoline (3a)
1H, J=8 Hz), 7.67 (d, 4H, J=8 Hz), 7.97 (d, 1H, J=8 Yellow crystals; yield: 83%; m.p. 214°C–216°C; Rf=0.67
Hz). 13C NMR (DMSO-d6, 200 MHz) δ ppm: 27.12, (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
28.27, 39.76, 39.87, 39.97, 40.07, 40.18, 122.61, 127.54, 3,002.4 (C–H, aromatic), 2,935.5 (C–H), 1,661 (C=N),
127.90, 129.07, 132.03, 132.43, 133.21, 134.13, 134.83, 1,578.2 (C=C), 1,324.1 (C–N), 1,283.9, 1,248.4 (C–O),
134.91, 136.66, 143.93, 187.08. LCMS (m/z): 393.25 760.5 (C–S). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 2.88
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Scheme 2 Two-step synthetic route to the titled compound (1a–15a).
Note: substituted α-tetralone (a), substituted aromatic aldehydes (b) and distinctive 2-aminothiazoles (c).
Abbreviation: gaa, glacial acetic acid.
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
(t, 2H, J=8 Hz), 3.13 (t, 2H, J=8 Hz), 3.72 (s, 3H), 3.83 1,659 (C=N), 1,598.4 (C=C), 1,336.1 (C–N), 1,258.6
(s, 10H), 6.86 (s, 3H), 7.18 (dd, 1H, J=8 Hz and J=8 Hz), (C–O), 769.2 (C–S). 1H NMR (DMSO-d6, 800 MHz) δ ppm:
7.32 (d, 1H, J=8 Hz), 7.44 (d, 1H, J=8 Hz), 7.69 (s, 1H). 2.92 (t, 2H, J=8 Hz), 3.12 (t, 2H, J=8 Hz), 3.71 (s, 3H), 3.82
13C NMR (DMSO-d6, 200 MHz) δ ppm: 27.44, 27.54, 39.76, (s, 7H), 3.86 (s, 3H), 6.84 (s, 3H), 6.93 (s, 1H), 6.96 (s, 1H),
13
39.86, 39.97, 40.07, 40.18, 55.77, 56.46, 60.59, 108.04, 7.64 (s, 1H), 7.93 (d, 1H, J=8 Hz). C NMR (DMSO-d6,
110.59, 121.34, 130.34, 131.18, 134.21, 135.25, 136.32, 200 MHz) δ ppm: 27.27, 28.76, 39.73, 39.84, 39.94, 40.05,
136.70, 138.58, 153.24, 158.65, 187. LCMS (m/z): 437.20 40.15, 56.04, 56.45, 60.58, 107.93, 112.79, 114.15, 126.79,
[M+H]+. Anal. calcd. for C24H24N2O4S: C, 66.03; H, 5.54; 130.38, 131.34, 135.49, 135.86, 138.43, 146.46, 153.23,
N, 6.42. Anal. found: C, 66.20; H, 5.52; N, 6.45.
163.73, 185.88. LCMS (m/z): 435.15 [M−H]-. Anal. calcd.
for C24H24N2O4S: C, 66.03; H, 5.54; N, 6.42. Anal. found: C,
66.21; H, 5.56; N, 6.41.
2-methoxy-4-(2-methoxy-6,7-dihydro-5h-benzo[h]
thiazolo[2,3-b]quinazolin-7-yl)phenol (4a)
4-(6,7-dihydro-5h-benzo[h]thiazolo[2,3-b]
Yellow crystals; yield: 82%; m.p. 218°C–220°C; Rf=0.50
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): 3,451
(–OH), 3,020.2 (C–H, aromatic), 2,937.2 (C–H), 1,659.8
(C=N), 1,572.5 (C=C), 1,326.4 (C–N), 1,248.9 (C–O),
744 (C–S). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 2.86 (t,
2H, J=8 Hz), 3.10 (t, 2H, J=8 Hz), 3.82 (s, 7H), 6.87 (d, 1H,
J=8 Hz), 7.05 (d, 1H, J=8 Hz), 7.14–7.18 (m, 3H), 7.31
(d, 1H, J=8 Hz), 7.43 (s, 1H), 7.68 (s, 1H), 9.57 (s, 1H). 13C
NMR (DMSO-d6, 200 MHz) δ ppm: 27.41, 39.76, 39.86,
39.97, 40.07, 55.75, 56.12, 110.61, 115.01, 115.97, 121.03,
124.23, 127, 130.22, 132.93, 134.43, 136.07, 137.28, 147.94,
148.35, 158.62, 186.89. LCMS (m/z): 391.10 [M−H]-. Anal.
calcd. for C22H20N2O3S: C, 67.33; H, 5.14; N, 7.14. Anal.
found: C, 67.44; H, 5.16; N, 7.13.
quinazolin-7-yl)phenol (7a)
Light brown crystals; yield: 70%; m.p. 190°C–192°C;
Rf=0.45 (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr,
cm-1): 3,204.2 (–OH), 2,951.5 (C–H, aromatic), 2,895.1
(C–H), 1,639.9 (C=N), 1,556.1 (C=C), 1,370.2 (C–N),
740.3 (C–S). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 2.93
(t, 2H, J=8 Hz), 3.09 (t, 3H, J=8 Hz), 6.86 (d, 2H, J=8 Hz),
7.37–7.44 (m, 5H), 7.56 (t, 1H, J=8 Hz), 7.67 (s, 1H), 7.94
(d, 1H, J=8 Hz), 9.98 (s, 1H). 13C NMR (DMSO-d6, 200
MHz) δ ppm: 27.18, 28.29, 39.76, 39.86, 39.97, 40.07, 40.17,
116, 126.54, 127.40, 127.75, 128.88, 132.65, 132.79, 133.58,
133.73, 136.83, 143.63, 158.91, 187.06. LCMS (m/z): 331.05
[M−H]-. Anal. calcd. for C20H16N2OS: C, 72.26; H, 4.85;
N, 8.43. Anal. found: C, 72.45; H, 4.87; N, 8.39.
7-(4-bromophenyl)-3-methoxy-6,7-dihydro-5h-
benzo[h]thiazolo[2,3-b]quinazoline (5a)
7-(4-chlorophenyl)-2-methoxy-9-methyl-6,7-dihydro-
5h-benzo[h]thiazolo[2,3-b]quinazoline (8a)
Yellow crystals; yield: 78%; m.p. 194°C–196°C; Rf=0.52
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
3,012.8 (C–H, aromatic), 2,945.7 (C–H), 1,663.2 (C=N),
1,597 (C=C), 1,332.6 (C–N), 1,263.4 (C–O), 1,070
Yellow crystals; yield: 65%; m.p. 196°C–198°C; Rf=0.40
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
3,067.6 (C–H, aromatic), 2,941.2 (C–H), 1,660.7 (C=N),
1,589.1 (C=C),1,322.7(C–N),1,251.5(C–O),1,031.5(C–Cl),
1
(C–Br), 762.5 (C–S). H NMR (DMSO-d6, 800 MHz)
1
743.9 (C–S). H NMR (DMSO-d6, 800 MHz) δ ppm:
δ ppm: 2.92 (t, 2H, J=8 Hz), 3.03 (t, 2H, J=8 Hz), 3.86
(s, 4H), 6.93–6.98 (m, 2H), 7.48 (d, 2H, J=8 Hz), 7.62
(s, 1H), 7.66 (d, 3H, J=8 Hz), 7.94 (d, 1H, J=8 Hz). 13C
NMR (DMSO-d6, 200 MHz) δ ppm: 27.15, 28.67, 39.74,
39.84, 39.95, 40.05, 40.15, 56.06, 112.81, 114.31, 122.39,
126.64, 130.44, 131.99, 132.35, 134.08, 135.07, 136.84,
146.55, 163.84, 185.76. LCMS (m/z): 425.15 [M+]. Anal.
calcd. for C21H17BrN2OS: C, 59.30; H, 4.03; N, 6.59.
Anal. found: C, 59.19; H, 4.04; N, 6.61.
2.88 (t, 4H, J=8 Hz), 3.04 (t, 4H, J=8 Hz), 3.36 (s, 3H),
7.19 (dd, 1H, J=16 Hz and J=16 Hz), 7.32 (d, 1H, J=16 Hz),
7.45(d, 1H, J=8Hz), 7.53–7.58(m, 4H), 7.69(s, 1H). 13CNMR
(DMSO-d6, 200 MHz) δ ppm: 27.30, 27.46, 39.78, 39.88,
39.99, 40.09, 40.19, 55.79, 110.61, 121.49, 129.10, 130.43,
132.19, 133.83, 134.06, 134.59, 134.83, 136.39, 136.56,
158.68, 186.92. LCMS (m/z): 397.20 [M+2]. Anal. calcd.
for C22H19ClN2OS: C, 66.91; H, 4.85; N, 7.09. Anal. found:
C, 66.79; H, 4.87; N, 7.12.
3-methoxy-7-(3,4,5-trimethoxyphenyl)-6,7-dihydro-
7-(4-chlorophenyl)-3-methoxy-9-methyl-6,7-dihydro-
5h-benzo[h]thiazolo[2,3-b]quinazoline (6a)
5h-benzo[h]thiazolo[2,3-b]quinazoline (9a)
Light brown crystals; yield: 86%; m.p. 214°C–216°C; Yellow crystals; yield: 68%; m.p. 196°C–198°C; Rf=0.42
Rf=0.66 (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR(KBr, cm-1):3,050.8
(KBr, cm-1): 3,050.6 (C–H, aromatic), 2,944.8 (C–H), (C–H, aromatic), 2,943.9 (C–H), 1,660.5 (C=N), 1,587.9
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(C=C), 1,336.3 (C–N), 1,274.7 (C–O), 1,030.8 (C–Cl), (DMSO-d6, 800 MHz)δppm: 2.97 (t, 2H, J=8Hz),3.09(t,3H,
760.5 (C–S). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 2.92 J=8 Hz), 7.40–7.44 (m, 2H), 7.54–7.61 (m, 6H), 7.70 (s, 1H),
(t, 4H, J=8 Hz), 3.04 (t, 4H, J=8 Hz), 3.43 (s, 3H), 6.93–6.97 7.98 (d, 1H, J=8 Hz). 13C NMR (DMSO-d6, 200 MHz)
(m, 2H), 7.52 (dd, 4H, J=24 Hz and J=24 Hz), 7.64 (s, 1H), δ ppm: 27.11, 28.28, 39.78, 39.88, 39.99, 40.09, 40.19,
7.94 (d, 1H, J=8 Hz). 13C NMR (DMSO-d6, 200 MHz) 127.53, 127.89, 129.06, 129.10, 132.20, 133.23, 133.85,
δ ppm: 27.14, 28.68, 39.78, 39.88, 39.99, 40.09, 40.19, 134.11, 134.58, 134.76, 136.60, 143.92, 187.07. LCMS (m/z):
56.06, 112.81, 114.30, 126.67, 129.06, 130.43, 132.10, 353.0 [M+2]. Anal. calcd. for C20H15ClN2S: C, 68.46; H, 4.31;
133.65, 134.01, 136.78, 146.53, 163.84, 185.73. LCMS N, 7.98. Anal. found: C, 68.53; H, 4.34; N, 7.95.
(m/z): 395.25 [M+]. Anal. calcd. for C22H19ClN2OS: C, 66.91;
H, 4.85; N, 7.09. Anal. found: C, 66.75; H, 4.86; N, 7.11.
7-(4-chlorophenyl)-2-methoxy-10-methyl-6,7-
dihydro-5h-benzo[h]thiazolo[2,3-b]quinazoline (13a)
Yellow crystals; yield: 74%; m.p. 198°C–200°C; Rf=0.46
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
7-(4-chlorophenyl)-9-methyl-6,7-dihydro-5h-
benzo[h]thiazolo[2,3-b]quinazoline (10a)
Yellow crystals; yield: 64%; m.p. 188°C–190°C; Rf=0.44 3,067.9 (C–H, aromatic), 2,940.4 (C–H), 1,660.7 (C=N),
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): 1,591 (C=C), 1,324.7 (C–N), 1,256.8 (C–O), 1,033.4 (C–Cl),
3,067.7 (C–H, aromatic), 2,944.7 (C–H), 1,668.1 (C=N), 748.3 (C–S). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 2.88
1,596.4 (C=C), 1,317.4 (C–N), 1,027.7 (C–Cl), 743 (C–S). (t, 4H, J=8 Hz), 3.04 (t, 4H, J=8 Hz), 3.47 (s, 3H), 7.19 (d, 1H,
1H NMR (DMSO-d6, 800 MHz) δ ppm: 2.95 (t, 4H, J=8 Hz), J=8 Hz), 7.32 (d, 1H, J=8 Hz), 7.45 (s, 1H), 7.53–7.58 (m, 4H),
3.07 (t, 4H, J=8 Hz), 7.39–7.44 (m, 2H), 7.54–7.61 (m, 5H), 7.68 (s, 1H). 13C NMR (DMSO-d6, 200 MHz) δ ppm:
7.70 (s, 1H), 7.97 (s, 1H). 13C NMR (DMSO-d6, 200 MHz) 27.30, 27.46, 39.78, 39.88, 39.99, 40.09, 40.19, 55.78,
δ ppm: 27.11, 28.28, 39.78, 39.88, 39.99, 40.09, 40.20, 110.61, 121.48, 129.09, 130.42, 132.18, 133.83, 134.06,
127.53, 127.89, 129.11, 132.20, 133.23, 133.85, 134.12, 134.59, 134.82, 136.38, 136.54, 158.67, 186.91. LCMS (m/z):
134.58, 134.76, 136.61, 143.92, 187.07. LCMS (m/z): 393.25 [M−H]-. Anal. calcd. for C22H19ClN2OS: C, 66.91; H,
365.20 [M+]. Anal. calcd. for C21H17ClN2S: C, 69.12; H, 4.70; 4.85; N, 7.09. Anal. found: C, 66.83; H, 4.87; N, 7.10.
N, 7.68. Anal. found: C, 69.25; H, 4.71; N, 7.67.
7-(4-chlorophenyl)-3-methoxy-10-methyl-6,7-
7-(4-chlorophenyl)-2-methoxy-6,7-dihydro-5h-
benzo[h]thiazolo[2,3-b]quinazoline (11a)
Yellow crystals; yield: 70%; m.p. 194°C–196°C; Rf=0.49 (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): 3,051.0 (C–H, aromatic), 2,938.8 (C–H), 1,653.8 (C=N),
3,067.8 (C–H, aromatic), 2,940.6 (C–H), 1,660.6 (C=N), 1,602.6 (C=C), 1,339 (C–N), 1,266.3 (C–O), 1,091.8 (C–Cl),
1,589.3 (C=C),1,323.0(C–N),1,252.2(C–O),1,031.6(C–Cl), 764.1 (C–S). H NMR (DMSO-d6, 800 MHz) δ ppm:
744.8 (C–S). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 2.88 2.94 (t, 3H, J=8 Hz), 3.06 (t, 4H, J=8 Hz), 3.86 (s, 4H),
(t, 2H, J=8 Hz), 3.04 (t, 2H, J=8 Hz), 3.82 (s, 4H), 7.20 (d, 1H, 6.93–6.98 (m, 2H), 7.53–7.56 (m, 4H), 7.64 (s, 1H), 7.95
dihydro-5h-benzo[h]thiazolo[2,3-b]quinazoline (14a)
Light brown crystals; yield: 76%; m.p. 198°C–200°C; Rf=0.48
1
13
J=8 Hz), 7.33 (d, 1H, J=8 Hz), 7.45 (s, 1H), 7.53–7.58 (d, 1H, J=8 Hz). C NMR (DMSO-d6, 200 MHz) δ ppm:
(m, 5H), 7.69 (s, 1H). 13C NMR (DMSO-d6, 200 MHz) 27.14, 28.68, 39.78, 39.88, 39.99, 40.09, 40.19, 56.06,
δ ppm: 27.30, 27.46, 39.78, 39.88, 39.99, 40.09, 40.19, 55.79, 112.81, 114.30, 126.67, 129.07, 130.43, 132.11, 133.65,
110.62, 121.50, 129.10, 130.44, 132.19, 133.83, 134.07, 134.01, 134.74, 136.79, 146.54, 163.84, 185.74. LCMS (m/z):
134.83, 136.56, 158.69, 186.98. LCMS (m/z): 381.25 [M+]. 393.25 [M−H]-. Anal. calcd. for C22H19ClN2OS: C, 66.91; H,
Anal. calcd. for C21H17ClN2OS: C, 66.22; H, 4.50; N, 7.35. 4.85; N, 7.09. Anal. found: C, 66.79; H, 4.86; N, 7.07.
Anal. found: C, 66.32; H, 4.52; N, 7.33.
7-(4-chlorophenyl)-10-methyl-6,7-dihydro-5h-
7-(4-chlorophenyl)-6,7-dihydro-5h-benzo[h]
thiazolo[2,3-b]quinazoline (12a)
benzo[h]thiazolo[2,3-b]quinazoline (15a)
Yellow crystals; yield: 79%; m.p. 186°C–188°C; Rf=0.56
Yellow crystals; yield: 61%; m.p. 184°C–186°C; Rf=0.43 (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR(KBr, cm-1):3,069.4 3,069.7 (C–H, aromatic), 2,944.8 (C–H), 1,668.1 (C=N),
(C–H, aromatic), 2,944.7 (C–H), 1,668.1 (C=N), 1,596.4 1,596.1 (C=C), 1,317.3 (C–N), 1,089.4 (C–Cl), 742.3 (C–S).
(C=C), 1,317.4 (C–N), 1,020.1 (C–Cl), 742.7 (C–S). 1H NMR 1H NMR (DMSO-d6, 800 MHz) δ ppm: 2.97 (t, 4H,
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
J=8 Hz), 3.09 (t, 4H, J=8 Hz), 7.40–7.44 (m, 2H), 7.55–7.61 step i
(m, 5H), 7.70 (s, 1H), 7.98 (d, 1H, J=8 Hz). 13C NMR A mixture of substituted indanones (1 mmol), appropriate
(DMSO-d6, 200 MHz) δ ppm: 27.11, 28.29, 39.79, 39.89, aldehydes (1 mmol), piperidine (0.1 mmol), and glacial
39.99, 40.09, 40.20, 127.54, 127.90, 129.07, 129.11, acetic acid (0.1 mmol) in EtOH (5.0 mL) was heated under
132.21, 133.24, 133.85, 134.13, 134.59, 134.76, 136.61, reflux for 10–12 h. The reaction mixture was cooled to room
143.93, 187.08. LCMS (m/z): 367.30 [M+2]. Anal. calcd. temperature for a few minutes and then poured into ice-cold
for C21H17ClN2S: C, 69.12; H, 4.70; N, 7.68. Anal. found: water. A solid product, substituted benzylidene-indanones,
C, 69.23; H, 4.72; N, 7.66.
was obtained, which was filtered, washed thoroughly with
distilled water, and dried. The dry residue was recrystal-
lized from EtOH. The progress of the reaction was moni-
tored by TLC on precoated silica gel-G plates using 40%
ethylacetate:n-hexane as the solvent system.
One-pot efficient synthesis of substituted
indeno[1,2-d]thiazolo[3,2-a]pyrimidines (1B–15B)
A mixture of substituted α-indanone (1 mmol), appropriate
aromaticaldehydes(1mmol),anddistinctive2-aminothiazoles
(1 mmol) in EtOH (5.0 mL) in the presence of 20 mol% step ii
p-TSA was heated under reflux for 3–4 h. The reaction A solution of distinctive 2-aminothiazoles (1 mmol) and
mixture was then poured into ice-cold water. A solid product appropriate benzylidene-indanones in glacial acetic acid
was obtained, which was filtered, washed thoroughly with (2.0 mL) was heated under reflux for 18–20 h. The solvent
distilled water, and recrystallized from EtOH. Pure crystals was evaporated under vacuum and then the residue obtained
were obtained (Scheme 3). The progress of the reaction was was dissolved in chloroform, washed with water, and the
monitored by TLC on precoated silica gel-G plates using organic layer separated, dried, and evaporated. The result-
30% ethylacetate:n-hexane as the solvent system. TLC ing solid obtained was recrystallized from EtOH to yield the
revealed just a single spot, which proved the presence of a target compounds (1B–15B; Scheme 4).
single product.
5-(4-bromophenyl)-5,6-dihydroindeno[1,2-d]
thiazolo[3,2-a]pyrimidine (1B)
Two-step synthesis of substituted indeno[1,2-d]
thiazolo[3,2-a]pyrimidines (1B–15B)
These compounds were synthesized as follows.
Yellow crystals; yield: 75%; m.p. 186°C–188°C; Rf=0.54
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
3,023.4 (C–H, aromatic), 2,928.9 (C–H), 1,693.2 (C=N),
1,620.8 (C=C), 1,325.1 (C–N), 733.9 (C–S), 1,070.9 (C–Br).
1H NMR (DMSO-d6, 800 MHz) δ ppm: 4.14 (s, 3H),
7.50–7.54 (m, 2H), 7.70–7.82 (m, 8H). 13C NMR (DMSO-d6,
200 MHz) δ ppm: 32.32, 39.76, 39.86, 39.97, 40.07, 40.17,
123.83, 124.19, 127.21, 128.28, 132.01, 132.47, 133.11,
134.61, 135.59, 136.38, 137.56, 150.54, 193.80. LCMS (m/z):
381.25 [M+]. Anal. calcd. for C19H13BrN2S: C, 59.85; H, 3.44;
N, 7.35. Anal. found: C, 59.97; H, 3.42; N, 7.34.
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Yellow crystals; yield: 66%; m.p. 178°C–180°C; Rf=0.41
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
3,064.6 (C–H, aromatic), 2,922.5 (C–H), 1,697.1 (C=N),
1,621.3 (C=C), 1,326.1 (C–N), 740.4 (C–S), 1,037.4 (C–Cl).
1H NMR (DMSO-d6, 800 MHz) δ ppm: 4.14 (s, 3H),
7.49–7.53 (m, 4H), 7.63 (d, 1H, 1H, J=8 Hz), 7.68 (d, 1H, J=8
Hz), 7.74 (t, 1H, J=8 Hz), 7.83 (s, 1H), 7.98 (d, 1H, J=8 Hz).
13C NMR (DMSO-d6, 200 MHz) δ ppm: 31.84, 39.76,
39.86, 39.97, 40.07, 40.17, 124.32, 127.24, 128.33, 130.56,
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Scheme 3 One-pot efficient synthetic route to the titled compound (1B–15B).
Note: substituted α-indanone (a′), substituted aromatic aldehydes (b) and
distinctive 2-aminothiazoles (c).
Abbreviations: etOh, ethanol; p-Tsa, p-toluenesulfonic acid.
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Scheme 4 Two-step synthetic route to the titled compound (1B–15B).
Note: substituted α-indanone (a′), substituted aromatic aldehydes (b) and distinctive 2-aminothiazoles (c).
Abbreviation: gaa, glacial acetic acid.
130.97, 131.70, 132.96, 135.39, 135.76, 137.43, 138.14, (s, 5H), 4.08 (s, 4H), 7.05 (d, 1H, J=8 Hz), 7.20 (s, 1H), 7.44
150.80, 193.62. LCMS (m/z): 339.20 [M+2]. Anal. calcd. (s, 1H), 7.71–7.75 (m, 5H). 13C NMR (DMSO-d6, 200 MHz)
for C19H13ClN2S: C, 67.75; H, 3.89; N, 8.32. Anal. found: δ ppm: 32.39, 39.76, 39.86, 39.97, 40.07, 40.18, 56.32,
C, 67.65; H, 3.91; N, 8.30.
110.65, 115.99, 123.49, 126.06, 130.67, 130.88, 132.42,
132.89, 134.77, 136.98, 153.47, 165.53, 191.97. LCMS (m/z):
425.20 [M+]. Anal. calcd. for C21H17BrN2OS: C, 59.30; H,
4.03; N, 6.59. Anal. found: C, 59.20; H, 4.04; N, 6.61.
5-(4-bromophenyl)-2-methyl-5,6-dihydroindeno
[1,2-d]thiazolo[3,2-a]pyrimidine (3B)
Yellow crystals; Yield: 80%; m.p. 192°C–194°C; Rf=0.57
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): 5-(4-bromophenyl)-3-methyl-5,6-dihydroindeno
3,024.1 (C–H, aromatic), 2,926.7 (C–H), 1,693.3 (C=N), [1,2-d]thiazolo[3,2-a]pyrimidine (5B)
1,621.2 (C=C), 1,325.3 (C–N), 733.5 (C–S), 1,070.4 Yellow crystals; yield: 78%; m.p. 192°C–194°C; Rf=0.54
1
(C–Br). H NMR (DMSO-d6, 800 MHz) δ ppm: 4.14 (s, (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
6H), 7.50–7.54 (m, 2H), 7.69–7.78 (m, 6H), 7.81 (d, 1H, 3,025.1 (C–H, aromatic), 2,928.1 (C–H), 1,693.1 (C=N),
J=8Hz). 13C NMR (DMSO-d6, 200 MHz) δ ppm: 34.53, 1,622.1 (C=C), 1,324.7 (C–N), 732.9 (C–S), 1,070.1 (C–Br).
41.97, 42.08, 42.18, 42.29, 42.39, 126.04, 126.40, 129.42, 1H NMR (DMSO-d6, 800 MHz) δ ppm: 4.14 (s, 6H),
130.49, 134.23, 134.67, 135.31, 136.82, 137.79, 138.59, 7.51–7.54 (m, 2H), 7.70–7.77 (m, 6H), 7.82 (d, 1H, J=8 Hz).
139.77, 152.75, 196. LCMS (m/z): 396.95 [M+H]+. Anal. 13C NMR (DMSO-d6, 200 MHz) δ ppm: 32.32, 39.76,
calcd. for C20H15BrN2S: C, 60.77; H, 3.82; N, 7.09. Anal. 39.86, 39.97, 40.07, 40.18, 123.83, 124.19, 127.21, 128.28,
found: C, 60.85; H, 3.81; N, 7.08.
132.01, 132.46, 133.10, 134.61, 135.59, 136.38, 137.56,
150.54, 193.79. LCMS (m/z): 395.00 [M+]. Anal. calcd.
for C20H15BrN2S: C, 60.77; H, 3.82; N, 7.09. Anal. found:
C, 60.89; H, 3.80; N, 7.11.
5-(4-bromophenyl)-8-methoxy-3-methyl-5,6-
dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine (4B)
Yellow crystals; yield: 69%; m.p. 198°C–200°C; Rf=0.48
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): 5-(4-bromophenyl)-8-methoxy-5,6-
3,018.7 (C–H, aromatic), 2,903.2 (C–H), 1,687.0 (C=N), dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine (6B)
1,622.9 (C=C), 1,335.3 (C–N), 1,288.7 (C–O), 763.9 (C–S), Yellow crystals; yield: 75%; m.p. 188°C–190°C; Rf=0.61
1,071.3 (C–Br). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 3.91 (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR(KBr, cm-1):3,018.4
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
(C–H,aromatic),2,902.6(C–H),1,686.9(C=N),1,622.6(C=C), 1H NMR (DMSO-d6, 800 MHz) δ ppm: 3.35 (s, 3H), 3.74
1,335.0 (C–N), 1,288.6 (C–O), 763.9 (C–S), 1,071.1 (C–Br). (s, 6H), 3.91 (s, 4H), 4.15 (s, 2H), 7.04 (dd, 1H, J=8 Hz
1H NMR (DMSO-d6, 800 MHz) δ ppm: 3.91 (s, 3H), 4.08 and J=8 Hz), 7.10 (s, 3H), 7.23 (s, 1H), 7.44 (s, 1H), 7.73
13
(s, 3H), 7.06 (d, 1H, J=8 Hz), 7.20 (s, 1H), 7.44 (s, 1H), (d, 1H, J=8 Hz). C NMR (DMSO-d6, 200 MHz) δ ppm:
7.72–7.75 (m, 6H). 13C NMR (DMSO-d6, 200 MHz) δ ppm: 32.22, 39.76, 39.86, 39.97, 40.07, 40.18, 56.31, 56.44,
32.39, 39.76, 39.86, 39.97, 40.07, 40.18, 56.32, 110.65, 60.63, 108.59, 110.41, 116.15, 125.87, 131.03, 132.40,
115.99, 123.49, 126.06, 130.67, 130.88, 132.42, 132.89, 135.15, 139.32, 153.49, 165.38, 192.08. LCMS (m/z): 421.15
134.77, 136.98, 153.47, 165.53, 192.08. LCMS (m/z): 413.25 [M−H]-. Anal. calcd. for C23H22N2O4S: C, 65.38; H, 5.25; N,
[M+2]. Anal. calcd. for C20H15BrN2OS: C, 58.40; H, 3.68; N, 6.63. Anal. found: C, 65.22; H, 5.26; N, 6.64.
6.81. Anal. found: C, 58.31; H, 3.70; N, 6.83.
5-(4-chlorophenyl)-3-methyl-5,6-dihydroindeno
5-(4-bromophenyl)-8-methoxy-2-methyl-5,6-
[1,2-d]thiazolo[3,2-a]pyrimidine (10B)
dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine (7B)
Yellow crystals; yield: 78%; m.p. 182°C–184°C; Rf=0.43
Yellow crystals; yield: 77%; m.p. 198°C–200°C; Rf=0.47 (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): 3,066.8 (C–H, aromatic), 3,030.5 (C–H), 1,695.5 (C=N),
3,018.2 (C–H, aromatic), 2,902.5 (C–H), 1,687.1 (C=N), 1,623.9 (C=C), 1,326.7 (C–N), 729.1 (C–S), 1,090.1 (C–Cl).
1,622.7 (C=C), 1,335.7 (C–N), 1,288.7, 1,246.9 (C–O), 763.5 1H NMR (DMSO-d6, 800 MHz) δ ppm: 4.13 (s, 6H),
1
(C–S), 1,071.9 (C–Br). H NMR (DMSO-d6, 800 MHz) 7.49–7.58 (m, 3H), 7.68 (d, 1H, J=8 Hz), 7.72 (d, 1H,
δ ppm: 3.90 (s, 5H), 4.07 (s, 4H), 7.04 (dd, 1H, J=8 Hz J=8 Hz), 7.80–7.83 (m, 4H). 13C NMR (DMSO-d6, 200 MHz)
and J=8 Hz), 7.19 (d, 1H, J=8 Hz), 7.44 (s, 1H), 7.70–7.75 δ ppm: 32.28, 39.76, 39.86, 39.97, 40.07, 40.18, 124.16,
(m, 5H). 13C NMR (DMSO-d6, 200 MHz) δ ppm: 32.39, 127.19, 128.25, 129.51, 131.90, 132.88, 134.28, 134.93,
39.76, 39.86, 39.97, 40.07, 40.18, 56.31, 110.64, 115.98, 135.55, 136.25, 137.56, 150.52, 193.76. LCMS (m/z): 351.02
123.49, 126.05, 130.66, 130.88, 132.41, 132.89, 134.77, [M+]. Anal. calcd. for C20H15ClN2S: C, 68.46; H, 4.31;
136.97, 153.46, 165.52, 191.96. LCMS (m/z): 425.00 [M+]. N, 7.98. Anal. found: C, 68.63; H, 4.30; N, 7.96.
Anal. calcd. for C21H17BrN2OS: C, 59.30; H, 4.03; N, 6.59.
Anal. found: C, 59.18; H, 4.04; N, 6.60.
5-(4-chlorophenyl)-8-methoxy-3-methyl-5,6-
dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine (11B)
Yellow crystals; yield: 65%; m.p. 186°C–188°C; Rf=0.48
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
5-(3,4,5-trimethoxyphenyl)-5,6-dihydroindeno[1,2-d]
thiazolo[3,2-a]pyrimidine (8B)
Yellow crystals; yield: 72%; m.p. 206°C–208°C; Rf=0.52 3,019.3 (C–H, aromatic), 2,967.8 (C–H), 1,695.5 (C=N),
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): 1,623.9 (C=C), 1,326.7 (C–N), 1,249.1 (C–O), 729.1 (C–S),
1
3,046.1 (C–H, aromatic), 2,942.6 (C–H), 1,690.6 (C=N), 1,090.1 (C–Cl). H NMR (DMSO-d6, 800 MHz) δ ppm:
1,624.7 (C=C), 1,328.0 (C–N), 1,266.5 (C–O), 736.4 (C–S). 3.90 (s, 5H), 4.07 (s, 4H), 7.03 (d, 1H, J=8 Hz), 7.18 (s, 1H),
1H NMR (DMSO-d6, 800 MHz) δ ppm: 3.36 (s, 3H), 3.90 7.44 (s, 1H), 7.56 (d, 2H, J=8 Hz), 7.73–7.79 (m, 3H).
(s,7H),4.20(s,2H),7.14(s,3H),7.49–7.54(m,2H),7.71–7.74 13C NMR (DMSO-d6, 200 MHz) δ ppm: 32.35, 39.77, 39.87,
(m, 2H), 7.80 (s, 1H). 13C NMR (DMSO-d6, 200 MHz) 39.97, 40.08, 40.18, 56.30, 110.63, 115.96, 126.04, 129.47,
δ ppm: 32.08, 39.76, 39.86, 39.97, 40.07, 40.18, 56.49, 60.64, 130.56, 130.89, 132.66, 134.44, 134.61, 136.85, 153.45,
108.84, 124.08, 127.16, 128.18, 130.87, 133.79, 134.58, 165.51, 191.94. LCMS (m/z): 381.20 [M+]. Anal. calcd.
135.37, 137.75, 139.59, 150.56, 153.51, 193.82. LCMS (m/z): for C21H17ClN2OS: C, 66.22; H, 4.50; N, 7.35. Anal. found:
393.25 [M+H]+. Anal. calcd. for C22H20N2O3S: C, 67.33; C, 66.38; H, 4.49; N, 7.34.
H, 5.14; N, 7.14. Anal. found: C, 67.21; H, 5.16; N, 7.16.
5-(4-chlorophenyl)-5,6-dihydroindeno[1,2-d]
8-methoxy-5-(3,4,5-trimethoxyphenyl)-5,6-
thiazolo[3,2-a]pyrimidine (12B)
dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine (9B)
Yellow crystals; yield: 65%; m.p. 176°C–178°C; Rf=0.51
Yellow crystals; yield: 66%; m.p. 208°C–210°C; Rf=0.68 (SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1):
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): 3,067.4 (C–H, aromatic), 3,030.3 (C–H), 1,697.1 (C=N),
3,060.8 (C–H, aromatic), 2,939.2 (C–H), 1,679.6 (C=N), 1,625.0 (C=C), 1,326.7 (C–N), 729.2 (C–S), 1,093.0 (C–Cl).
1,624.6 (C=C), 1,330.4 (C–N), 1,241.5 (C–O), 760.8 (C–S). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 4.13 (s, 3H), 7.50
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(s, 1H), 7.54 (s, 1H), 7.57 (d, 2H, J=8 Hz), 7.68 (d, 1H, (DMSO-d6, 200 MHz) δ ppm: 32.35, 39.66, 39.87, 39.97,
J=8 Hz), 7.72 (d, 1H, J=8 Hz), 7.80–7.83 (m, 4H). 13C NMR 40.08, 40.29, 56.30, 110.64, 115.96, 126.04, 129.47, 130.56,
(DMSO-d6, 200 MHz) δ ppm: 32.28, 39.75, 39.86, 39.96, 130.89, 132.66, 134.44, 134.61, 136.85, 153.45, 165.51,
40.07, 40.17, 124.17, 127.19, 128.26, 129.51, 131.91, 132.88, 191.95. LCMS (m/z): 382.95 [M+2]. Anal. calcd. for C21H17Cl-
134.28, 134.93, 135.55, 136.25, 137.55, 150.52, 193.77. N2OS: C, 66.22; H, 4.50; N, 7.35. Anal. found: C, 66.28;
LCMS (m/z): 339.15 [M+2]. Anal. calcd. for C19H13ClN2S: H, 4.51; N, 7.33.
C, 67.75; H, 3.89; N, 8.32. Anal. found: C, 67.61; H, 3.90;
N, 8.33.
srB assay
The Hep-G2 cells derived from human liver carcinoma were
purchased from the National Cell Repository NCCS, Pune,
India and later cultured in the lab of ACTREC, Tata Memorial
5-(4-chlorophenyl)-8-methoxy-5,6-dihydroindeno
[1,2-d]thiazolo[3,2-a]pyrimidine (13B)
Light brown crystals; yield: 60%; m.p. 184°C–186°C; Rf=0.47 Centre, Navi Mumbai, India for in vitro testing purposes. As
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): per the instructions, the cells were grown in Roswell Park
3,058.1, 3,019.1 (C–H, aromatic), 2,943.5 (C–H), 1,688.0 Memorial Institute medium 1640 containing 10% fetal bovine
(C=N), 1,622.1 (C=C), 1,304.0 (C–N), 1,249.4 (C–O), 765.2 serum. Then, 100 μL cells containing media were seeded into
1
(C–S), 1,088.4 (C–Cl). H NMR (DMSO-d6, 800 MHz) 96-well plates at a density of 5×103 cells/well and incubated at
δ ppm: 3.90 (s, 3H), 4.06 (s, 3H), 7.03 (d, 1H, J=8 Hz), 7.18 37°C in a humidified atmosphere containing 5% CO2 for 24 h
(s, 1H), 7.44 (s, 1H), 7.56 (d, 2H, J=8 Hz), 7.73–7.79 (m, 4H). prior to addition of the experimental drugs. Initially, all the
13C NMR (DMSO-d6, 200 MHz) δ ppm: 32.36, 39.77, 39.87, experimental drugs were solubilized in DMSO at 100 mg/mL
39.98, 40.09, 40.19, 56.30, 110.63, 115.96, 126.03, 129.47, and further diluted to 1.0 mg/mL using water and stored
130.55, 130.87, 132.65, 134.43, 134.61, 136.85, 153.44, frozen prior to use. Aliquots of 100 μL (containing 90 μL of
165.50, 191.93. LCMS (m/z): 339.05 [M+2]. Anal. calcd. medium) of various dilutions were added to appropriate wells,
for C20H15ClN2OS: C, 65.48; H, 4.12; N, 7.64. Anal. found: resulting in the required drug concentrations of 10, 20, 40,
C, 65.63; H, 4.10; N, 7.63.
and 80 μg/mL, and were maintained at standard conditions for
48 h. For each of the experiments, a well-known anticancer
drug Adriamycin (ADR) was used as a positive control at
concentrations of 10, 20, 40, and 80 μg/mL. The assay was
5-(4-chlorophenyl)-2-methyl-5,6-dihydroindeno
[1,2-d]thiazolo[3,2-a]pyrimidine (14B)
Yellow crystals; yield: 68%; m.p. 180°C–182°C; Rf=0.51 terminated by the gentle addition of 50 μL of cold 30% (w/v)
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): trichloroacetic acid and the mixture was incubated for 60 min
3,031.5 (C–H, aromatic), 2,923.5 (C–H), 1,695.5 (C=N), at 4°C. The supernatant was discarded and the plates were
1,623.4 (C=C), 1,326.9 (C–N), 729.2 (C–S), 1,089.7 (C–Cl). washed five times using tap water and then air dried. Next,
1H NMR (DMSO-d6, 800 MHz) δ ppm: 4.13 (s, 6H), SRB solution (50 μL) at 0.4% (w/v) in 1% acetic acid was
7.49–7.59 (m, 3H), 7.68–7.73 (m, 2H), 7.80–7.84 (m, 4H). added to each well and incubated for 20 min at room tem-
13C NMR (DMSO-d6, 200 MHz) δ ppm: 32.28, 39.65, perature. After staining, the unbound dye was recovered and
39.86, 39.96, 40.07, 40.28, 124.17, 127.19, 128.26, 129.5, the residual dye was removed by washing five times with 1%
131.9, 132.88, 134.28, 134.93, 135.55, 136.25, 137.55, acetic acid and then air dried. Subsequently, the bound stain
150.52, 193.77. LCMS (m/z): 349.0 [M−H]-. Anal. calcd. was eluted with 10 mM trizma base and the absorbance was
for C20H15ClN2S: C, 68.46; H, 4.31; N, 7.98. Anal. found: examined at a wavelength of 540 nm on an enzyme-linked
C, 68.69; H, 4.32; N, 7.99.
immunosorbent assay plate reader.
The growth inhibition of 50% (GI50) was calculated using
the formula [(Ti-Tz)/(C-Tz)] ×100%, where Tz represents
time zero growth, C is the control growth, and Ti is the test
5-(4-chlorophenyl)-8-methoxy-2-methyl-5,6-
dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine (15B)
Yellow crystals; yield: 81%; m.p. 188°C–190°C; Rf=0.66 growth at four concentration levels in the presence of the
(SiO2, ethylacetate:n-hexane, 3:7, v/v). IR (KBr, cm-1): drug.47,48
3,019.2 (C–H, aromatic), 2,904.3 (C–H), 1,688.0 (C=N),
1,620.9 (C=C), 1,289.4 (C–N), 1,248.5 (C–O), 765.6 (C–S), Molecular docking
1,088.7 (C–Cl). 1H NMR (DMSO-d6, 800 MHz) δ ppm: 3.90 The structures of the ligands were designed by Chem Draw
(s, 5H), 4.07 (s, 4H), 7.05 (d, 1H, J=8 Hz), 7.19 (s, 1H), 7.45 Ultra 12.0, and the geometry was optimized six times through
(s, 1H), 7.57 (d, 2H, J=8 Hz), 7.73–7.80 (m, 3H). 13C NMR GaussView 5.0 (trial version). In addition, the National
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
Centre for Biotechnology Information49 and the Protein the simulation cell boundary. Then, the MD simulation was
Data Bank50 were utilized as chemical sources to obtain the run for 3,000 ps at a temperature of 298 K and 1 bar pres-
established four homological cancer protein targets, that is, sure to obtain snapshot (sim) trajectories. Finally, the sim
IL-2 (1Z92), IL-6 (1IL6), Caspase-3 (1QX3), and Caspase-8 trajectories were analyzed and the resulting data were plotted
(1IBC). DS Visualizer software51 was used to remove the using Sigma Plot 11.0 tools.
co-crystal with the assigned target. Next, the active site
Results and discussion
Design
was identified using the CASTp database.52 For a particular
system, validation of the docking protocol was performed
using re-docking studies, where we found excellent agree-
ment between the localization of ligands upon docking, and
from the crystal structure of the assigned targets. We dem-
onstrated the reliability and quality of the docking method
in reproducing experimentally observed binding-mode-
assigned targets. In addition, the docking analyses of the test
set were carried out using Autodock4.1,53 together with the
Lamarkian genetic algorithm for automated flexible ligand
docking, and the binding energy was estimated as negative
kcal/mol. In addition, probable H-bonds and π-bonds were
also assessed.54−56
These two core structural motifs are formed by the fusion
of three biodynamic privileged heterosystems in such a way
that one nitrogen atom occupies a bridge head position,
therefore being common to both the heterocyclic rings, that
is, the thiazole and the pyrimidine rings, and possessing
unique structural diversity. Thiazolo[2,3-b]quinazoline
and thiazolo[3,2-a]pyrimidine, as heterocyclic skeletons,
exhibit a diverse range of pharmacologic activities. In view
of the great medicinal importance associated with these
remarkable structures, we are interested in studying MDRs
with the aim of evolving an efficient and highly convergent
synthetic procedure for the library of densely functional-
ized 5H-benzo[h]thiazolo[2,3-b]quinazoline (1A−15A) and
indeno[1,2-d]thiazolo[3,2-a]pyrimidine (1B−15B) analogs
in a single operation.
Prediction of aDMe properties
The ADME and drug-like properties of selected ligands were
predicted by employing MedChem Designer and QikProp
tools. Chemical structures were optimized with LigPrep.
Additionally, the percentage absorbance (% ABS) and Lip-
inski’s violation were evaluated for this study.57
We initiated our study of the proposed method with
10 mol% p-TSA as the acidic catalyst (Scheme 5) by investi-
gating the conditions for the reaction comprising 5-methoxy-
2,3-dihydro-1H-inden-1-one (1 mmol), 4-bromobenzaldehyde
(1 mmol), 4-methylthiazol-2-amine (1 mmol), and EtOH
MD simulation
The nature of the inhibitor used on the active site domain of (5.0 mL) at 60°C to yield the corresponding product (4B,
IL-6 was investigated by means of MD simulation studies. 51% yield) as a model in 4.5 h (entry 1). With this finding
The energies of dock configurations were minimized to in hand, when the acidic catalyst was changed from 10% to
eliminate the unfavorable atomic contacts as starting con- 20% and the temperature from 60°C to 80°C, the product
formations for dynamic simulation in Elmar Krieger MD yield increased to 69% after 3 h reaction time (entry 4). We
simulation tools (trial version).58 An AMBER03 force examined the reaction in different organic solvents, including
field was assigned to execute a real-time MD simulation.59 EtOH, methanol (MeOH), ethylacetate (EtOAc), acetonitrile
The complex was solvated through an HOH model at (CH3CN), and toluene (PhCH3) under varied temperature con-
density =0.997 g/L inside the 10Å simulation cell boundary ditions (Table 1). The proposed reaction is optimized for the
and then adjusted to the physiologic pH at 7.4. Moreover, amount of acidic catalyst required, as well as for temperature,
physiologic NaCl solution with 0.9% mass fraction Na+ and to improve the yields of the desired molecules (entries 1–8),
Cl- ion concentration was used to maintain and neutralize and the optimum amount was found to be 20 mol% p-TSA in
+
2
6
2
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1
1
E
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ꢁ
6
+ꢂ1
+ꢀ&2
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Scheme 5 Optimization of the model reaction.
Note: substituted α-indanone (a′), substituted aromatic aldehydes (b) and distinctive 2-aminothiazoles (c).
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Table 1 Optimization of catalytic amount of p-Tsa, nonaqueous
solvents, and temperature in one-pot synthesis of the model
reactiona
α-tetralone or α-indanone with different aromatic aldehydes,
piperidine and glacial acetic acid in EtOH. This reaction
proceeds in two steps with a comparatively longer reaction
time (18–20 h) and giving moderate yields (40%–50%).
Entry
p-TSA
Solvents
Temperature
Time
(h)
Yield
(%)b
(mol%)
(°C)
1
2
3
4
5
6
7
8
9
10
10
20
20
10
10
20
20
20
20
20
etOh
etOh
etOh
60
80
60
80
60
80
60
80
4.5
4.0
3.5
3.0
5.0
4.5
4.0
3.5
5.0
5.0
5.0
51
56
63
69
49
54
54
60
33
29
27
Plausible mechanism
These reactions presumably proceed through a Knoev-
enagel condensation between substituted α-tetralone or
α-indanone and some appropriate aromatic aldehydes in
the first step to construct α,β-unsaturated ketones, respec-
tively, which undergo a Michael-type addition approach
with the nucleophilic endocyclic nitrogen of the distinctive
2-aminothiazole under the maintained reaction conditions.
Then, successive intramolecular cyclization occurred
with the loss of a water molecule to give 5H-benzo[h]
thiazolo[2,3-b]quinazolines (1A−15A) and indeno[1,2-d]
thiazolo[3,2-a]pyrimidines (1B−15B). In this setting, the
domino approach and the reaction sequence of Knoevenagel
EtOH
MeOh
MeOh
MeOh
MeOh
etOac
ch3cn
Phch3
Reflux
Reflux
Reflux
10
11
Notes: areaction conditions: 5-methoxy-2,3-dihydro-1h-inden-1-one (1 mmol),
4-bromobenzaldehyde (1 mmol), 4-methylthiazol-2-amine (1 mmol), solvent (5.0 ml),
and p-Tsa. bIsolated pure yield. Bold entry signifies the optimized reaction conditions.
Abbreviations: ch3cn, acetonitrile; etOac, ethylacetate; etOh, ethanol; MeOh,
methanol; Phch3, toluene; p-Tsa, p-toluenesulfonic acid.
EtOH at 80°C. The best results were obtained for 20 mol% condensation/Michael-type addition/intramolecular cycl-
p-TSA in 3 h at 80°C (entry 4, 69% yield). ization were done in a single step in a one-pot procedure
In order to explore the scope of these conditions, we in EtOH. The plausible mechanism for the reaction is
embarked on the synthesis of a panel of novel 5H-benzo[h] delineated in Scheme 6.
thiazolo[2,3-b]quinazoline (1A–15A) and indeno[1,2-d]
thiazolo[3,2-a]pyrimidine (1B–15B) analogs bearing a in vitro antitumor screening
bridgehead nitrogen atom from a range of substrates by A library of 30 5H-benzo[h]thiazolo[2,3-b]quinazoline and
the advancement of facile and efficient MDRs of highly indeno[1,2-d]thiazolo[3,2-a]pyrimidine derivatives were
substituted α-tetralone (a) or α-indanone (a’) with some screened against Hep-G2 cells. The inhibition activities (GI50)
aromatic aldehydes (b) and distinctive 2-aminothiazoles of the synthetic compounds 1A–15A, 1B–15B, and ADR on
(c) in EtOH in the presence of 20 mol% p-TSA. The Hep-G2 cells are summarized in Table 2A and B. The effects
α-tetralone, 6-methoxy-1-tetralone, 7-methoxy-1-tetralone of treatment with the most active members (4A and 6A)
or α-indanone, 5-methoxy-1-indanone, and the substituted of this study and with ADR on Hep-G2 are shown by the
benzaldehydes with 4-bromo, trimethoxy, 3-hydroxy-4- plotted growth curve (Figure 1) and the microscopic images
methoxy (vanillin), 4-chloro were reacted with distinctive (Figure 2).
2-aminothiazole, 4-methylthiazol-2-amine, 5-methylthiazol-
2-amine to afford a library of proposed target molecules, that B) revealed that the presence of –OH and –OCH3 groups in
is, (1A–15A) and (1B–15B), respectively. the phenyl ring, preferably at the R1, R2, and R3 positions,
Close examination of the activity data (Table 2A and
The one-pot, three-component reaction went smoothly in conjugation with the introduction of the –OCH3 group in
in EtOH in the presence of p-TSA and gave the targeted the tetralone ring at X1 or X2 is crucial for inhibitory activity.
compounds (1A–15A) and (1B–15B) in impressive yields up Substitution of 3-methoxy-4-hydroxy on the phenyl ring and
to 86%. The various substitutions of synthesized derivatives 6-methoxy on the tetralone ring led to compound 4A, while
are given in Table 2A and B. All the newly synthesized com- substitutions of 3,4,5-trimethoxy on the phenyl ring and
pounds were authenticated on the basis of Fourier transform 7-methoxy on the tetralone ring led to compound 6A with
infrared spectroscopy, LCMS, and 1H and 13C NMR spectral better cytotoxicity (GI50 ,10 μg/mL). In contrast, substitu-
and elemental analyses.
tions with –Br, –Cl, and –OH groups on the phenyl ring at
Desired compounds have also been synthesized by a mul- the R2 position were ineffective against Hep-G2 cells. The
tistep synthesis involving the reaction of distinctive 2-ami- methyl substitution on the thiazole ring did not contribute to
nothiazoles with the appropriate benzylidene-tetralones/ producing inhibitory activity. Replacement of the tetralone
indanones achieved by the reaction of highly substituted ring with the indanone ring system, that is, 1B–15B, reduced
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
Table 2 Various substitutions and in vitro cytotoxicity data of synthesized derivatives against human hepatoma (hep-g2) cell lines:
A (1a–15a) and B (1B–15B)
Comp code
X1
X2
R1
R2
R3
Y1
Y2
*GI50 (µg/mL)
LC50 (µg/mL)
TGI (µg/mL)
A
1a
2a
3a
4A
5a
6A
7a
8a
9a
10a
11a
12a
13a
14a
15a
–h
–Och3
–h
–h
–h
–Och3
–Och3
–h
–h
–h
–Och3
–h
–h
–Och3
–h
–Och3
–h
–h
–h
–h
–Och3
–Och3
–h
–Och3
–h
–h
–h
–h
–h
–h
–h
–h
–h
–Br
–Br
–h
–h
–Och3
–h
–h
–Och3
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–ch3
–ch3
–ch3
–h
–h
–h
–h
–ch3
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–ch3
–ch3
–ch3
.80
ne
ne
ne
ne
ne
.80
.80
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
.80
.80
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
–Och3
–Oh
–Br
–Och3
–Oh
–cl
–cl
–cl
–cl
–cl
–h
,10
.80
,10
76.1
.80
.80
.80
.80
.80
.80
.80
.80
–Och3
–Och3
–h
–h
–Och3
–h
–h
–h
–h
–Och3
–h
–cl
–cl
–cl
–h
–h
Comp code
X
R1
R2
R3
R4
Y1
Y2
*GI50 (µg/mL)
LC50 (µg/mL)
TGI (µg/mL)
B
1B
2B
3B
4B
5B
6B
7B
8B
–h
–h
–h
–Och3
–h
–Och3
–Och3
–h
–Och3
–h
–Och3
–h
–Och3
–h
–h
–cl
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–h
–Och3
–Och3
–h
–h
–h
–h
–h
–h
–Br
–h
–Br
–Br
–Br
–Br
–Br
–Och3
–Och3
–cl
–cl
–cl
–cl
–cl
–cl
–h
–h
–h
–h
–h
–h
–h
–Och3
–Och3
–h
–h
–h
–h
–h
–h
–h
–h
–h
–ch3
–ch3
–h
–h
–h
–h
–h
–ch3
–h
–h
–h
–ch3
–h
–h
–h
–h
–h
–h
–ch3
–ch3
.80
.80
.80
.80
.80
.80
.80
.80
.80
.80
.80
.80
.80
.80
.80
,10
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
ne
,10
9B
–h
10B
11B
12B
13B
14B
15B
ADR
–ch3
–ch3
–h
–h
–h
–Och3
–h
Notes: *gi50 value of #10 μg/ml (or 1 μM) is considered to demonstrate activity in case of pure compounds (synthetic compound). Bold values indicate the active
compounds.
Abbreviations: aDr, adriamycin, positive control compound; gi50, concentration of drug causing 50% inhibition of cell growth; lc50, concentration of drug causing 50%
cell kill; ne, “not effective” even at the concentration .80 μg/ml; Tgi, concentration of drug causing total inhibition of cell growth.
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Scheme 6 Plausible scenario to account for the formation of (1a–15a) and same for the preparation of (1B–15B).
Note: substituted α-tetralone (a), substituted aromatic aldehydes (b) and distinctive 2-aminothiazoles (c).
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Figure 1 growth curve of (1a–15a and 1B–15B): human hepatoma cell line (hep-g2).
Abbreviation: aDr, adriamycin.
the anticancer activity against Hep-G2 human liver cancer lead to the death of cancerous cells while minimizing that
cell lines. of normal cells.
The growth curve of the in vitro findings suggested that
the percentage growth inhibition values of the potent com- Molecular docking studies
pounds 4A and 6A was #50% at 10 μg/mL concentration, Computational analysis was performed using various
but they did not move toward a negative value. Therefore, well-known liver cancer targets, specifically IL-2, IL-6,
it might be expected in future that both compounds could Caspase-3, and Caspase-8, through Autodoc 4.1, together
Figure 2 Microscopic pictures showing the effect of treatments with the active compounds (A) 4a and (B) 6a and the reference drug (C) aDr on hep-g2 human liver
cancer cell line.
Note: scale bar 50 μm.
Abbreviation: aDr, adriamycin.
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
with Lamarkian genetic algorithm for automated docking which indicates the interactions of amino acids with ligands,
of flexible ligands. Molecular docking poses of both the H-bonds, and π-bonds as well as the bond lengths. The
potent compounds 4A and 6A with the related targets IL-2, docking affinities (kcal/mol), numbers of H-bonds and
IL-6, Caspase-3, and Caspase-8 are presented in Figure 3 π-bonds, and the interactions of amino acids of the potent
Figure 3 Docking images of active compounds (A, C, E, G) 4a and (B, D, F, H) 6a for caspase-3, caspase-8, il-2 and il-6.
Abbreviation: il, interleukin.
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members with them are given in Table 3. Compounds 4A partition coefficients (QPlogPo/w), and Lipinski’s violation.
and 6A exhibited strong affinity (binding interaction energy Interestingly, it was found that the % ABS obtained for all
ranged from -6.1 to -7.87 kcal/mol), while others showed members was 100% and the QPlogPo/w prediction was found
good binding affinity with preferred molecular targets. to be within the accepted range of -2.0 to 6.5. Moreover, all
Compound 4A displayed excellent affinity with IL-6 members followed the violated Lipinski parameters.
(-7.87 kcal/mol, 2H and 4π-bonds), IL-2 (-7.68 kcal/mol,
5π-bonds), Caspase-3 (-6.69 kcal/mol, 1H and 7π-bonds),
and Caspase-8 (-6.48 kcal/mol, 1π-bond). A similar pattern
was observed for compound 6A with IL-2 (-7.63 kcal/mol,
1H and 1π-bonds), IL-6 (-7.43 kcal/mol, 2H and 4π-bonds),
and Caspase-8 (-6.76 kcal/mol, 1H and 5π-bond), but with
less affinity for Caspase-3 (-6.14 kcal/mol, 1H and 6π-bonds).
Moreover, both the active members of the present study have
MD simulation
MD simulation was performed on the active inhibitor 4A
with IL-6 to explore the binding poses in depth. Compound
4A displayed high binding affinity (low docking energy) and
stable complex for IL-6 receptor. Therefore, we decided to
investigate the influence of compound 4A on the active site
of IL-6 receptor. The stability of the system under simulation
strong affinity binding to IL-2, Caspase-3, Caspase-8, and the
was assessed using the root-mean-square deviation (RMSD)
binding energies, particularly for the IL-6 receptor site, are
of atomic positions of the backbone atoms, relative to the
predominantly high (from -7.43 to -7.87 kcal/mol). Accord-
ingly, it might be assumed that the promising cytotoxic
starting structures. The binding energy versus time plot indi-
cates the change in conformation and, therefore, the binding
properties of these compounds, which were indicated by the
energy, with respect to time. As shown in Figures 4 and 5, the
in vitro antitumor activity on Hep-G2 cells, might be better
RMSD, the binding energy, and the potential energy of IL-6
mediated through an IL-6-dependent mechanism (Table 3).
with compound 4A, including the complex, were computed
using MD trajectory frames. Furthermore, we monitored
Prediction of aDMe properties
the structural stability of the backbone structure through-
A study of pharmacokinetic parameters was carried out utiliz- out the process through a graphic profile. We observed a
ing QikProp version 4.5 tools to predict the ADME proper- dramatic fluctuation in RMSD at 1,000 ps (time), whereas no
ties of both series (1A–15A) and (1B–15B) and the ranges significant fluctuations were observed after 1,500 ps (time),
for the calculated properties of all members, along with and it achieved an almost steady state, indicating the stabil-
their average values are summarized in Table 4. In addition, ity of the backbone structure with the ligand at about 1,500
we also calculated % ABS, number of H-bond acceptors ps time in the MD simulation. The binding energy and the
(n–OH), number of H–bond donors (n-OHNH), octanol/water potential energy of the complex were estimated as a function
Table 3 Docking affinity of active compounds with assigned anticancer receptors
Ligands Receptors Binding affinity Amino acids involved in interaction
H-bonds π-bonds
(kcal/mol)
4a
il-2
-7.680
-7.871
-6.695
arg a 38 Thr a 41 asP B 6 ala B 13 Thr B 14 Phe B 15 lYs B 16 TYr B 20
glU B 106 Thr B 115 glU B 116 arg B 117 ile B 118 TYr B 119 Phe B 121
asn a 62 leU a 63 PrO a 66 lYs a 67 MeT a 68 ala a 69 Phe a 75 leU a
166 arg a 169 ser a 170 glU a 173 Phe a 174 ser a 177
Thr a 62 ser a 65 TYr a 204 TrP a 206 arg a 207 ser a 209 Phe a 250
ser a 251 Phe a 256 hOh a 645 hOh a 684 hOh a 696 hOh a 708 hOh
a 733 hOh a 736
0
2
1
5
4
7
il-6
caspase-3
caspase-8
il-2
-6.480
-7.630
-7.438
-6.142
ser a 65 TYr a 204 TrP a 206 arg a 207 asn a 208 ser a 209 TrP a 214
glU a 248 ser a 249 Phe a 250 ser a 251
0
1
0
1
1
1
3
6
6a
arg a 38 Thr a 41 asP B 6 glU B 9 Phe B 15 lYs B 16 ala B 17 TYr B 20
glU B 113 Thr B 115 glU B 116 arg B 117 ile B 118 TYr B 119 Phe B 121
glU a 43 Thr a 44 lYs a 47 ser a 48 MeT a 50 arg a 105 Phe a 106 glU
a 107 ser a 108 gln a 157 TrP a 158 asP a 161 Thr a 164
ser a 65 TYr a 204 TrP a 206 arg a 207 asn a 208 ser a 209 TrP a 214
ser a 249 Phe a 250 ser a 251 Phe a 256 hOh a 645 hOh a 665 hOh a
684 hOh a 696 hOh a 736
il-6
caspase-3
caspase-8
-6.760
ser a 63 arg a 64 ser a 65 TYr a 204 ser a 205 TrP a 206 arg a 207
asn a 208 ser a 209 TrP a 214 glU a 248 ser a 249 Phe a 250 ser a 251
1
5
Abbreviation: il, interleukin.
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
Table 4 Pharmacokinetic parameters important for oral bioavailability and protein binding parameters of synthesized compounds
Comp
Rule
% ABS
MW
Volume
Donor HB
Acceptor HB
Lipinski’s violation
QPlogPo/w
.80% is high,
,25% is poor
,500
500.0–2,000.0
,5
,10
#1
-2.0 to 6.5
1a
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
395.316
439.369
436.525
392.472
425.342
436.525
332.419
394.92
1,041.638
1,168.198
1,261.8
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6.493
6.799
6.007
5.196
6.476
6.006
5.108
6.493
6.724
6.576
6.402
6.419
6.724
6.724
6.741
6.036
5.672
6.374
6.247
6.264
6.018
6.357
5.516
5.445
6.191
6.174
5.961
5.943
6.3
2a
1.75
4
3a
4a
1,138.921
1,105.697
1,261.747
1,011.802
1,041.638
1,159.867
1,065.077
1,097.431
1,033.256
1,159.867
1,159.815
1,095.69
3.25
1.75
4
5a
6a
7a
1.75
1
8a
9a
394.92
1.75
1
10a
11a
12a
13a
14a
15a
1B
364.892
380.891
350.865
394.918
394.918
364.892
381.289
336.838
395.316
425.342
395.316
411.315
425.342
392.472
422.498
350.865
380.891
336.838
366.864
350.865
380.891
1.75
1
1.75
1.75
1
972.937
1
2B
942.479
1
3B
1,037.84
1
4B
1,080.745
1,016.635
1,037.038
1,101.954
1,122.39
1.75
1
5B
6B
1.75
1.75
3.25
4
7B
8B
9B
1,177.838
1,008.558
1,072.663
964.548
10B
11B
12B
13B
14B
15B
1
1.75
1
1,028.656
1,029.454
1,093.555
1.75
1
1.75
6.282
Abbreviations: ABS, absorbance; QPlogPo/w, octanol/water partition coefficients; MW, molecular weight; HB, H-bonds.
of time, which indicated that the potential energy (kJ/mol)
did not show further fluctuations after 100 ps, whereas the
average complex binding energy was observed to be about
-0.9 kg/mol. The fluctuations in the residue of the backbone
structure are illustrated in Figure 4. In summary, we exam-
ined these data, where we found the structural stability of
compound 4A with the active site domain of IL-6 receptor.
Conclusion
We have successfully devised a highly proficient and opera-
tionally simple metal-free, one-pot MDR for obtaining a series
of novel, hitherto unreported, 5H-benzo[h]thiazolo[2,3-b]
quinazoline (1A–15A) and indeno[1,2-d]thiazolo[3,2-a]
pyrimidine (1B–15B) analogs displaying potent anticancer
activity against Hep-G2 cells as an alternative approach
for the treatment of HCC. Considering this library of novel
compounds, we concluded that 5H-benzo[h]thiazolo[2,3-b]
Figure 4 Docking complex of 4a with the il-6 receptor.
Notes: structural conformational changes (A) before MD simulation and (B) after
MD simulation: backbone of the active site domain complex, which shows the
contraction of ligand with amino acids residue.
Abbreviations: il, interleukin; MD, molecular dynamic.
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Figure 5 The stability profile of ligand and protein complex under the MD simulation.
Notes: (A) average rMsD versus time graph that shows the convergence of simulated structure toward an equilibrium state with respect to the reference structure (initial
structure). (B) Potential energy of complex versus time graph that shows the stability of ligand and protein complex and (C) binding energy of complex versus time graph
that also shows the stability of ligand and protein complex.
Abbreviations: MD, molecular dynamic; rMsD, root-mean-square deviation.
quinazoline, along with a substituted phenyl ring (hydro- computational approaches demonstrated effective oral
phobic side chain) establishes an important pharmacophoric absorption and protein binding. These compounds, therefore,
structure, and the R1, R2, and R3 positions of the phenyl ring, might be stable in some form of pharmaceutical dosage.
as well as the X1 and X2 positions of the tetralone ring system, Additionally, MD simulation supported our hypothesis
are the key reactive sites that could be modified with various regarding the stability of compound 4A with IL-6 protein
groups to elicit greater antitumorigenic potential.
during the simulation run. The results from the MD simula-
We have also demonstrated that substitutions with more tion run of the active inhibitor showed very less fluctuation
electronegative groups (–OH, –OCH3) on the hydrophobic with the active site domain of IL-6 and it achieved an almost
side chain directly linked to thiazolo[2,3-b]quinazoline steady state. We concluded that the compounds would be
led to active members 4A and 6A, eliciting enhanced anti- bound stably to IL-6. Further studies on the biologic activities
tumorigenic activity, with GI50 ,10 μg/mL, which was of the potent compounds are underway in our laboratory.
confirmed by docking analyses. Additionally, 3-methoxy-4-
This approach, using a one-pot, multicomponent reaction
hydroxyphenyl–substituted 5H-benzo[h]thiazolo[2,3-b] sequence involving a domino Knoevenagel condensation/
quinazoline led to 4A, which displayed excellent antitum- Michael-type addition followed by an intramolecular cycliza-
origenic activity among a library of 30 novel synthesized tion, where the desired molecules are obtained in a one-flask
compounds with values of GI50 ,10 μg/mL. Various domino manner with atom and step economy in impressive
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p-Tsa-promoted syntheses of drug candidates to prevent hcc
8. Kuramoto M, Sakata Y, Terai K, et al. Preparation of leukotriene B(4)
inhibitory active 2- and 3-(2-aminothiazol-4-yl)benzo[b]furan deriva-
tives and their growth inhibitory activity on human pancreatic cancer
cells. Org Biomol Chem. 2008;6(15):2772–2781.
9. Misra RN, Xiao HY, Kim KS, et al. N-(cycloalkylamino)acyl-2-
aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-
dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecar
box-amide (BMS-387032), a highly efficacious and selective antitumor
agent. J Med Chem. 2004;47(7):1719–1728.
10. Patel AB, Chikhalia KH, Kumari P. An efficient synthesis of new
thiazolidin-4-one fused s-triazines as potential antimicrobial and anti-
cancer agents. J Saudi Chem Soc. 2014;18(5):646–656.
11. Rahman MU, Jeyabalan G, Saraswat P, Parveen G, Khan S, Yar MS.
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Commun. 2016;47(5):379–408.
12. Monchaud D, Allain C, Teulade-Fichou MP. Development of a
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yields (up to 86%) from readily available and low-priced
starting materials, is a resource-effective and desirable route.
Additionally, the target compounds were also synthesized
by conventional two-step reactions, which were compared
with this novel approach on the basis of obtained yields
(40%–50%). Finally, we conclude that this newly developed
method is both more useful and more profitable in each and
every aspect, compared with the conventional route. Due to
the importance of these two core structural motifs, that is,
5H-benzo[h]thiazolo[2,3-b]quinazoline and indeno[1,2-d]
thiazolo[3,2-a]pyrimidine, especially in the areas of phar-
maceutical and medicinal chemistry, we suggest that the
protocols that we have outlined should open up new avenues
of investigation, with enormous implications for achieving
diversity in chemical synthesis.
Acknowledgments
The authors would like to express their gratitude to Babasaheb
Bhimrao Ambedkar University (BBAU), Lucknow, India for
providing the support and research facilities. The authors also
express their sincere gratitude to the Centre of Biomedical
Research (CBMR), Lucknow for providing the NMR facili-
ties and the Advanced Centre for Treatment, Research and
Education in Cancer (ACTREC), Tata Memorial Centre, Navi
Mumbai, India for providing the in vitro SRB assay for anti-
cancer screening of drugs. The authors also acknowledge Mr
Pranesh Kumar for helping them during docking analysis.
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activity of Mannich bases of 2-methyl-3H-quinazolin-4-ones. Indian
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Disclosure
The authors report no conflicts of interest in this work.
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