Regioselective synthesis of 6-aryl-benzo[h][1,2,4]-triazolo[5,1-b] quinazoline-7,8-diones as potent antitumoral agents

Article abstract of DOI:10.1016/j.bmcl.2013.06.040

Three-component coupling of aldehyde, 2-hydroxy-1,4-naphthoquinone and 3-amino-1,2,4-triazole has been achieved using a catalytic amount of sulfamic acid under solvent free conditions to produce a novel series of 6-aryl-benzo[h][1,2,4]-triazolo[5,1-b]quinazoline-7,8-dione derivatives in good yields and with high regioselectivity. These compounds are found to exhibit potent antitumoral properties.

Full text of DOI:10.1016/j.bmcl.2013.06.040

Accepted Manuscript
Regioselective synthesis of 6-aryl-benzo[h][1,2,4]-triazolo[5,1-b] quinazo‐
line-7,8-diones as potent antitumoral agents
Liqiang wu, Chong Zhang, Weilin Li
PII:
S0960-894X(13)00761-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.06.040
BMCL 20604
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 January 2013
13 May 2013
13 June 2013
Please cite this article as: wu, L., Zhang, C., Li, W., Regioselective synthesis of 6-aryl-benzo[h][1,2,4]-triazolo[5,1-
b] quinazoline-7,8-diones as potent antitumoral agents, Bioorganic & Medicinal Chemistry Letters (2013), doi:
http://dx.doi.org/10.1016/j.bmcl.2013.06.040
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Graphical Abstract
Regioselective synthesis of 6-aryl-benzo[h][1,2,4]-triazolo[5,1-b] quinazoline-7,8-
diones as potent antitumoral agents
Liqiang wu,a,* Chong Zhang,a Weilin Lia
O
O
O
Ar
N
H
N
O
N
N
N
N
+
sulfamic acid
neat, 110 °C
+
NH2
ArCHO
N
OH
IC50= 1.31-59.19 μM against SGC7901
IC50= 1.19-24.93 μM against HepG2

Regioselective
synthesis
of
6-aryl-benzo[h][1,2,4]-triazolo[5,1-b]
quinazoline-7,8-diones as potent antitumoral agents
Liqiang wu,* Chong Zhang, Weilin Li
School of Pharmacy, Xinxiang Medical University, Xinxiang 453003, China
ABSTRACT Three-component coupling of aldehyde, 2-hydroxy-1,4-naphthoquinone and 3-amino-
1,2,4-triazole has been achieved using a catalytic amount of sulfamic acid under solvent free
conditions to produce a novel series of 6-aryl-benzo[h][1,2,4]-triazolo[5,1-b]quinazoline-7,8-dione
derivatives in good yields and with high regioselectivity. These compounds are found to exhibit potent
antitumoral properties.
Keywords: Sulfamic acid; 2-Hydroxy-1,4-naphthoquinone; Aldehydes; Ortho-naphthoquinone;
Antitumor; Solvent-free
*Corresponding author. Tel.: +86 371 3029879; fax: +86 371 3029879; e-mail: wliq1974@163.com

Naphthoquinones constitute a major class of naturally occurring compounds, and have wide range
of biological and therapeutic properties such as antioxidant,1 antifungal,2 anti-inflammatory,3
antiallergic,4 antiviral,5 and anticancer activity.6 A variety of naturally occurring and synthetic
substituted 1,2-naphthoquinones received a great deal of attention for their anticancer activity. Many
derivatives of ortho-naphthoquinone are cytotoxic and have been used as antitumor drugs. Among
them, some naturally occurring 1,2-naphthoquinones include Lantalucratins,7 Biflorin,8
Rhinacanthone,9 β-lapachone10 and Mansonones,11 and synthetic 1,2-naphthoquinones include Salvicin
(Figure 1).12 One of these quinones was β-lapachone, a 1,2-napthaquinone derivative and a natural
product, which inhibited tumor growths in rats implanted with W-256 carcinoma.13 The compound was
found to be cytotoxic to many human cancer cell lines through inhibition of DNA repair enzymes.14
Frydman et al.15 have recently demonstrated that several 1,2-napthoquinone derivatives can induce
DNA-TOPO II-mediated cleavages of DNA in an ATP-independent manner and this effect may be
crucial for the cytotoxicity of these compounds. Considering the above reports, development of new
and simple synthetic methods for efficient preparation of new ortho-naphthoquinones are therefore an
interesting challenge.
< Figure 1>
Multi-component reactions (MCRs) have received considerable attention because of their wide
range of applications in pharmaceutical chemistry for generation of structural diversity and
combinatorial libraries for drug discovery.16 The convergent character, atom economy, ease of
one-pot operation, access to structurally diverse libraries of compounds render MCRs greatly
advantageous over linear multistep synthesis.17 Further, it has become imperative both in academia and
industry to design catalyst- and solvent-free MCRs, as these processes are rendered green with

diminution of waste, time, manpower and cost.
In recent years, the use of solid acidic catalysts has offered important advantages in organic
synthesis, for example, operational simplicity, environmental compatibility, nontoxic, low cost, and
ease of isolation. A tremendous upsurge of interest in various chemical transformations processes by
catalysts under heterogeneous conditions has occurred. One of those heterogeneous catalysts is
sulfamic acid (SA), which makes reaction processes convenient, more economic, and environmentally
benign. Owing to the numerous advantages associated with the cheap and nonhazardous catalyst, SA
has been explored as a powerful catalyst for various organic transformations.18 We now report a highly
regioselective procedure for the preparation of 6-aryl-benzo[h][1,2,4]-triazolo[5,1-bquinazoline-7,8-
diones along with their antitumor activities against the SGC7901 (stomach cancer cell line) and HepG2
(Human hepatocellular liver carcinoma cell line) using SA as an efficient and versatile catalyst under
solvent-free conditions (Scheme 1).
< Scheme 1>
Initially, to achieve suitable conditions for the synthesis of 6-aryl-benzo[h][1,2,4]- triazolo[5,1-b]
quinazoline-7,8-diones, we tested the three-component reaction of benzaldehyde, 2-hydroxy-1,4-
naphthoquinone, and 3-amino-1,2,4-triazole as a simple model system at 110 °C under solvent-free
conditions using various catalysts (Table 1). As could be seen in Table 1, the best result was obtained
with 10 % mol of SA as the catalyst at 110 °C under solvent-free conditions (Table1, entry 3). Using
less catalyst resulted in lower yields, whereas higher amounts of catalyst did not affect reaction times
and yields. When this reaction was carried out without SA, or InCl3, FeCl3 the yield of the expected
product was low (Table 1, entry 1, 6, 7). In the presence of p-TsOH, silica sulfuric acid the product was
obtained in moderate yield (Table 1, entry 8, 9).
< Table 1>

In order to extend the above reaction (Scheme 1) to a library system, various kinds of
arylaldehydes 1 (Table 2) were subjected to react with 2-hydroxy-1,4-naphthoquinone 2 and
3-amino-1,2,4-triazole 3 to give the corresponding 6-aryl-benzo[h][1,2,4]-triazolo[5,1-b]quinazoline-
7,8-diones, and representative examples are shown in Table 2. All of 1 gave the expected products in
high yields, either bearing electron-withdrawing groups (such as halide, nitro) or electron-donating
groups (such as alkyl group) under the same reaction condition. To further demonstrate the scope and
limitation of the substrates, aliphatic aldehydes, such as phenylacetaldehyde, propionaldehyde,
n-butylaldehyde and n-heptaldehyde, were used as reactants to react with 2-hydroxy-1,4-
naphthoquinone and 3-amino-1,2,4-triazole. However, the desired products were not found and
1
obtained successfully. The structure of the ortho-quinone structures 4 is in full agreement with IR, H
NMR, 13C NMR, MS and elemental analysis as illustrated below for a representative example
(compound 4c). The ESI-MS spectrum of 4c displayed a peak at m/z 361 for [M + H]+, and gave two
very close carbonyl resonances at δ 178.6 and 178.2 ppm, which was the very close chemical shifts for
the carbonyl 13C NMR signals suggested an ortho-quinone structure (in the para-quinone 5 the C-7
signal was more deshielded, about appearing about at 182 ppm, and C-12 resonating about at 179
ppm).19 Two peak at 1685 and 1598cm-1 were observed for C=O stretching in the IR spectrum as a
1
broad peak, which was more easily attributable to the ortho-quinone moiety. In its HNMR spectrum,
H-9 and H-12 occur as doublets, respectively, at 8.98 (J = 8.0 Hz) and 8.25 (J = 7.6 Hz) ppm. The H-9
occur about at 9 ppm, more downfield than expected of aromatic protons. This is explicable by the
close proximity of these protons to the lone pairs of the neighbouring nitrogens and the consequent
anisotropic and van de Waals deshielding. These spectral data are consistent with the structure of 4c.
The formation of isomeric systems 4 and 5 is possible in the reaction (Figure 2). So, we considered it
desirable to obtain independent chemical evidence for the presence of ortho- or para-quinone units in

4. To this end, we reacted 4c with o-phenylenediamine for 30 min under solvent-free conditions,
affording compound 6c in 99% yield, confirming the ortho-quinone structure (Scheme 2).19 The
structure of 6c was fully characterized by spectroscopic data and elemental analysis, The H-5 and H-8
occur as a multiplet at 9.25-9.31ppm, more downfield than expected of aromatic protons. This is
explicable by the close proximity of these protons to the lone pairs of the neighbouring nitrogens and
the consequent anisotropic and van de Waals deshielding. The lack of any carbonyl signal and the
presence of two imine carbon signals at 154.6 and 154.4 ppm in 13 C NMR spectrum of 6c, and the fact
that 6c is formed by the reaction of one molecule of 4c with one molecule of
o-phenylenediamine clearly support the structure of 6c, which, in turn, further corroborates the
structure of 4 and the regiochemistry of its formation.
<Table 2>
<Figure 2>
<Scheme 2>
Although the detailed mechanism of the above reaction remains to be fully clarified, the
formation of 6-aryl-benzo[h][1,2,4]-triazolo[5,1-b]quinazoline-7,8-diones could be explained by a
reaction sequence presented in Scheme 3. It is conceivable that SA catalyzes the formation of a
carbocation in a reversible reaction with the aromatic aldehyde. The higher reactivity of the
carbocation compared with the carbonyl species is utilized to facilitate Knoevenagel condensation
between arylaldehyde 1 and 2-hydroxy-1,4-naphthoquinone 2 via intermediate 7, and after dehydration
olefin 8 is produced. Subsequent Michael-type addition of 3-amino-1,2,4-triazole 3 to the olefin
followed by intramolecular nucleophilic cyclization, dehydration, and dehydrogenation affords the
corresponding products 4.20
<Scheme 3>

All synthesized compounds were evaluated for their antiproliferative activities against the human
gastric carcinoma cell line SCG7901, hepatoma cell line HepG2, and their IC50 values in micromolar
concentration are represented in Table 3. It is clearly observed that all the tested compounds showed
moderate to good antiproliferative activities against the tested cancer cell lines. The compound 4b, 4g,
4h, 4j showed better antitumor activity against all cancer cell line. The results in Table 3 showed also
some important structure-activity relationships (SARs) for this series of derivatives. First, the
ortho-quinone moiety appeared to have an important effect upon cytotoxicity, compounds 6c was less
potent cytotoxicity, than the corresponding analogues with an ortho-quinone moiety. The wide activity
range observed for compounds 4a-4j (IC50 from 1.19 to >40 μM) indicated that the nature of
substituents at the C-6 position markedly affected the activity profile of these compounds. Substitution
of the electron-rich aromatic ring at the C-6 position confered far greater cytotoxicity in comparison
with the substitution of the electron-rich aromatic ring at the C-6 position.
<Table 3>
In summary, we have developed a novel method for the synthesis of 6-aryl-benzo[h][1,2,4]-
triazolo[5,1-b]quinazoline-7,8-dione derivatives by means of a three-component reaction between
aldehyde, 2-hydroxy-1,4-naphthoquinone and 3-amino-1,2,4-triazole using a catalytic amount of SA
under neat conditions. This method is simple and convenient to prepare a wide range of ortho-quinone
derivatives in a single-step operation which are found to possess interesting antitumor properties.
Acknowledgements
We are pleased to acknowledge the financial support from Xinxiang Medical University

Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://
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O
O
O
O
O
O
O
O
O
O
R2
R1
O
O
Rhinacanthone
Mansonone E
Lantalucratin A: R1=OCH3 R2=H
B: R1=OH R2=H
Biflorin
O
C: R1=H R2=OH
OH
O
O
O
O
OH
O
O
O
β-lapachone
Salvicin
Mansonone F
Figure 1. Structures of potent anticancer 1,2-naphthoquinone analogues.

O
Ar
N
O
O
H
N
N
N
N
N
+
SA
+
NH2
ArCHO
neat, 110 oC
N
OH
O
1
2
3
4
Scheme 1. Synthesis of 6-aryl-benzo[h][1,2,4]- triazolo[5,1-b]quinazoline-7,8-diones using sulfamic
acid as a catalyst.

Table 1. Catalyst optimization for the synthesis 7-phenyl-benzo[h][1,2,4]-triazolo[5,1-b]quinazoline-
7,8-dionea
Entry
Catalyst
Mol%
-
Time/ h
Yield/ %b
1
2
3
4
5
6
7
8
9
-
12
2
12
78
90
89
90
18
23
68
67
SA
5
SA
SA
10
15
20
10
10
10
10
1
1
SA
1
InCl3
6
FeCl3
6
p-TsOH
silica sulfuric acid
3
3
a Reaction conditions: benzaldehyde (1 mmol), 2-hydroxy-1,4- naphthoquinone (1 mmol); 3-amino-
1,2,4-triazole (1 mmol); 110 ◦C; neat.
b Isolated yield.

Table 2. Preparation of 6-aryl-benzo[h][1,2,4]- triazolo[5,1-b] quinazoline-7,8-dionesa
Entry
Ar
C6H5
Time/ h
Product
4a
Yield/ %b
1
2
3
4
5
6
7
8
9
10
1
2
90
86
89
83
86
92
90
93
87
86
2,4-Cl2-C6H3
4-Cl-C6H4
4b
4c
1.5
1.5
1.5
1
4-F-C6H4
4d
4e
3-NO2-C6H4
3,4,5-(MeO)3-C6H2
4-MeO-C6H4
2-thiophenyl
4-NO2-C6H4
4-Me-C6H4
4f
1
4g
1
4h
4i
1
1
4j
a Reaction conditions: aldehyde (1 mmol), 2-hydroxy-1,4-naphthoquinone (1 mmol); 3-amino- 1,2,4-
triazole (1 mmol); SA (0.1 mmol); 110 ◦C; neat.
b Isolated yield.

O
O
Ar
N
O
Ar
N
N
N
O
N
N
N
N
5
4
Figure 2. Structures of isomeric systems 4 and 5.

Cl
N
Cl
N
N
O
N
N
N
O
N
NH2
NH2
N
N
N
+
H
8
neat, 100 oC
5
H
6c
4c
Scheme 2. Proof of the ortho-quinone structure of 4c, based on its reaction with o-phenylenediamine.

O
O
O
O
OH
Ar
OH
OH
-H2O, H+
O
OH
H+
2
Ar
H
Ar
H
1
6
H
O
Ar
H O
Ar
N
O
O
N
N
O
O
Ar
N
N
N
N
H
H2N
H+
N
H
O
N
N
3
OH+
H2N
OH
7
OH Ar
O
Ar
O
N
N
N
O
N
N
N
-2H
-H2O
N
N
4
Scheme 3. A plausible mechanistic pathway to explain the SA-catalyzed formation of compounds 4.

Table 3. Cytotoxic activities of 6-aryl-benzo[h][1,2,4]- triazolo[5,1-b]quinazoline-7,8-diones.
IC50 (μM)
Compd
SGC7901
HepG2
2.16 ± 0.04 3.45 ± 0.73
1.83 ± 0.12 2.99 ± 0.10
34.92 ± 2.06 24.93 ± 5.23
6.12 ± 0.13 7.85 ± 0.46
9.88 ± 1.33 15.31 ± 1.41
59.19 ±7.73 17.86 ± 4.33
1.31 ± 0.13 1.71 ± 0.16
1.45 ± 0.24 2.13 ± 0.32
4a
4b
4c
4d
4e
4f
4g
4h
4i
2.65 ± 0.2
1.71 ± 0.06 1.19 ± 0.19
> 200 > 200
4.74 ± 0.72
4j
6c
Doxorubicin 5.72 ± 0.19 1.02 + 0.16