Anticancer potential of new 3-nitroaryl-6-(N-methyl)piperazin-1,2,4-triazolo[3,4-a]phthalazines targeting voltage-gated K+ channel: Copper-catalyzed one-pot synthesis from 4-chloro-1-phthalazinyl-arylhydrazones

Article abstract of DOI:10.1016/j.bioorg.2020.104031

A series of six 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo[3,4-a]phthalazines were prepared through a facile and efficient one-pot copper-catalyzed procedure from 4-chloro-1-phthalazinyl-arylhydrazones with relatively good yields (62–83percent). The one-pot copper-catalytic procedure consists of two simultaneous reactions: (i) a direct intramolecular dehydrogentaive cyclization between ylidenic carbon and adjacent pyrazine nitrogen to form 1,2,4-triazolo ring and, (ii) a direct N-amination on carbon-chlorine bond. Then, an in vitro anticancer evaluation was performed for the synthesized compounds against five selected human cancer cells (A549, MCF-7, SKBr3, PC-3 and HeLa). The nitro-derivatives were significantly more active against cancer strains than against the rest of tested compounds. Specifically, compound 8d was identified as the most promising anticancer agent with significant biological responses and low relative toxicities on human dermis fibroblast. The cytotoxic effect of compound 8d was more significant on PC3, MCF-7 and SKBr3 cancer cells with low-micromolar IC₅₀ value ranging from 0.11 to 0.59 μM, superior to Adriamycin drug. Mechanistic experimental and theoretical studies demonstrated that compounds 8d act as a K⁺ channel inhibitor in cancer models. Further molecular docking studies suggest that the EGFR Tyrosine Kinase enzyme may be a potential target for the most active 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo[3,4-a]phthalazines.

Full text of DOI:10.1016/j.bioorg.2020.104031

Journal Pre-proofs
Anticancer potential of new 3-nitroaryl-6-(N-methyl)piperazin-1,2,4-triazo-
lo[3,4-a]phthalazines targeting voltage-gated K+ channel: Copper-catalyzed
one-pot synthesis from 4-chloro-1-phthalazinyl-arylhydrazones
Angel H. Romero, Felipe Sojo, Francisco Arvelo, Christian Calderón, Alvaro
Morales, Simón E. López
PII:
S0045-2068(20)31328-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.104031
YBIOO 104031
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
1 August 2019
12 June 2020
17 June 2020
Please cite this article as: A.H. Romero, F. Sojo, F. Arvelo, C. Calderón, A. Morales, S.E. López, Anticancer
potential of new 3-nitroaryl-6-(N-methyl)piperazin-1,2,4-triazolo[3,4-a]phthalazines targeting voltage-gated K+
channel: Copper-catalyzed one-pot synthesis from 4-chloro-1-phthalazinyl-arylhydrazones, Bioorganic
Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104031
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1
Anticancer potential of new 3-nitroaryl-6-(N-methyl)piperazin-1,2,4-
triazolo[3,4-a]phthalazines targeting voltage-gated K+ channel: Copper-
catalyzed one-pot synthesis from 4-chloro-1-phthalazinyl-arylhydrazones
Angel H. Romero,a* Felipe Sojo,b,c Francisco Arvelo,b,c Christian Calderón,d Alvaro Moralese,
Simón E. Lópezf*
aCátedra de Química General, Facultad de Farmacia, Universidad Central de Venezuela, Los Chaguaramos,
Caracas 1041-A, Venezuela.
bFundación Institutos de Estudios Avanzados-IDEA, Área Salud.
cLaboratorio de Cultivo de Tejidos y Biología de Tumores, Instituto de Biología Experimental-IBE, Facultad de
Ciencias-UCV, Bello Monte, Caracas, Venezuela.
dLaboratorio de Fisiología y Biofísica, Centro de Biología Celular, Instituto de Biología Experimental-
IBE, Facultad de Ciencias, UCV, Bello Monte, Caracas, Venezuela.
eLaboratorio de Biotecnología Clínica Santa María, Cevalfes, Valencia, Venezuela.
fDepartment of Chemistry, University of Florida, Gainesville, Florida.
*Corresponding Author E-mail: angel.ucv.usb@gmail.com (A.H. Romero), simonlopez@chem.ufl.edu (S.E.
López)
Abstract: A series of six 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo[3,4-a]phthalazines were prepared
through a facile and efficient one-pot copper-catalyzed procedure from 4-chloro-1-phthalazinyl-
arylhydrazones with relatively good yields (62-83%). The one-pot copper-catalytic procedure consists of
two simultaneous reactions: (i) a direct intramolecular dehydrogentaive cyclization between ylidenic
carbon and adjacent pyrazine nitrogen to form 1,2,4-triazolo ring and, (ii) a direct N-amination on carbon-
chlorine bond. Then, an in vitro anticancer evaluation was performed for the synthesized compounds
against five selected human cancer cells (A549, MCF-7, SKBr3, PC-3 and HeLa). The nitro-
derivatives were significantly more active against cancer strains than against the rest of tested compounds.
Specifically, compound 8d was identified as the most promising anticancer agent with significant
biological responses and low relative toxicities on human dermis fibroblast. The cytotoxic effect of
compound 8d was more significant on PC3, MCF-7 and SKBr3 cancer cells with low-micromolar IC50
value ranging from 0.11 to 0.59 μM, superior to Adriamycin drug. Mechanistic experimental and
theoretical studies demonstrated that compounds 8d act as a K+ channel inhibitor in cancer models.
Further molecular docking studies suggest that the EGFR Tyrosine Kinase enzyme may be a potential
target for the most active 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo[3,4-a]phthalazines.
Keywords: C-H oxidative cyclization, 1,2,4-triazolo-phthalazine, anticancer activity, potassium-channel,
molecular docking, nitro-substitution.

2
1. Introduction
Cancer is one of the main causes of death in the world, registering approximately 14 million
new cancer cases and 8 million of deaths annually. More than 60% of global new annual cases
occur in Africa, Asia and Central and South America, and the number of new cases is expected to
rise by about 70% over the next 2 decades [1a]. The treatment of cancer has resulted in a
significant challenge for the medicinal chemistry by two important reasons: (i) poor efficacy of
the standard therapy against metastatic cancers, and (ii) significant toxicity associated to
metabolized cancer chemotherapeutic agents [1b-e]. This situation obligates to the design and
synthesis of novel active and non-toxic anticancer leads. In last decades, a variety of new
chemical systems based on phthalazine ring have emerged as potential anticancer agents. In
particular, the Vatalanib (PTK-787), a VEGFR inhibitor, is a promising anticancer based-
phthalazine that is currently in clinical trials against different types of cancer [2]. From Vatalanib,
a variety of 1,4-substituted phthalazines have been designed and synthesized with significant
efficacy against human cancer model [3]. Substitution of the bioactive pharmacophores at
positions 1 and 4 of the phthalazine core, such as substituted aniline or N-substituted piperazinyl
moieties, generated derivatives with an improved inhibitory activity toward VEGFR-2 and cancer
cells [3f-k]. More optimization of 1,4-substituted phthalazines has lead the construction of 1,2,4-
triazolo[3,4-a]phthalazine as a potential anticancer agent [3i]. This type of system is a known
multi-target agents that possess a wide spectrum of pharmacological activities, including anti-
inflammatory agents [4], bromo-domain inhibitors [5], GABA receptors [6], anticonvulsants [7]
and antimicrobials [8]. Recently, Liu and co-workers evaluated the cytotoxic effect of a series of
6-(N-a1kylamino)-1,2,4-triazolo[3,4-a]phthalazine containing a phenyl or methyl group at 3-
position of 1,2,4-triazole core [9] (1a in Figure 1). A structure-activity relationship put in
evidence that the incorporation of a piperazine or a N-substituted piperazine moiety at 6-position
as well as a phenyl ring at 3-position of the 1,2,4-triazolo[3,4-a]phthalazine core are crucial to
increase the anticancer activity of 1a. In particular, the role of the aryl-substitution attached at 3-
position of 1,2,4-triazolo[3,4-a]phthalazine ring received a little attention in the mentioned report.
For a analogue system like 6-(N-alkylamino)-pyrrolo[2,1-a]phthalazine, the incorporation of an
electron-rich group such as 4-methoxyphenyl at the 3-position of fused-heterocyclic core lead a
decrease in anticancer activity compared to the derivatives bearing alkyl substitutions such as
methyl or ethyl [10], which suggests that the selection of aryl-substitutions for this type of fused-

3
heterpocyclic systems is not trivial. Then, with this principles in mind, we designed and prepared
a series of six 3-aryl-6-(N-methylpiperazine)-1,2,4-triazolo[3,4-a]phthalazines bearing a selected
aryl/heteroaryl R1 group at 3-position of the 1,2,4-triazolo core (1b in Figure 1) as potential
anticancer agents against five selected human cancer cells (A549, MCF-7, SKBr3, PC3 and
HeLa). The selection of aryl-substitutions was mainly focused on nitro-substitutions due to its
known relevance in the design of anticancer drugs [11]. There are a number of anticancer drugs
containing a nitro-aryl moiety such as Venetoclax, Niclosamide, which are in clinical III phase
[11a]. Other compounds bearing nitro-aryl substitution such as pretomanid, nitroCBI,
tarloxotinib, nitrobenzyl-phosphoramide mustards, CH-01, KS-119 and O6-benzylguanine
prodrugs have demonstrated excellent preclinical profiles with low toxicity and, they are
undergoing clinical trials for diverse tumor types [11a]. Other substitutions such as fluorine atom
and furyl ring also were considered in the design of our selected number of derivatives due to
their well known reputations in medicinal chemistry [12-13].
N
N
N
N
R1
N
Y(NO2)
N
N
Pharmacophores
N
N
N
Y= aryl/heteroaryl
N
H
N
N
R3
O
1b
anticancer lead core 1a
Figure 1. General structure of the potential anticancer 3-aryl-6-(N-methyl piperazin)-1,2,4-
triazolo[3,4-a]phthalazine (1b) designed from the anticancer 1,2,4-triazolo-phthalazine lead (1a).
Other goals of the present report is the preparation of the 3-aryl-6-(N-alkylamine)-1,2,4-
triazolo-phthalazine from 4-chloro-1-phthalazinyl-arylhydrazones in a one-pot reaction, which
represents an attractive and competitive strategy compared to reported synthetic procedure.
Traditionally, the preparation of this type of systems requires two or more reaction steps (see
protocols in Scheme 1) [5, 14-15]: (i) the first of them is the formation of 3-aryl-1,2,4-triazolo-
phthalazine 3 core from 1-chloro-4-hydrazinephthlzine 2 or 1,4-dichlorophthalazine 4 using
expensive or sophisticated reagents (e.g. benzoyl-halide, trimethyl orthoformate, benzoyl-
hydrazine or bromide oxidizing) and, (ii) the second step involves the N-amination on carbon-
chloride bond at 4-position of phthalazine core (Scheme 1). Recently, strategies based on metal-

4
catalyzed dehydrogenative cyclization have permitted the formation of C-N or C-O bonds via
activation of C-H bond in vicinal vinyl, ylidenic or aldehyde moieties to form heterocyclic
systems [16]. Specifically, H. Ge and co-workers reported a copper-catalyzed dehydrogenative
cyclization of N,N-disubstituted hydrazones through Csp3-H functionalization for the synthesis of
substituted pyrazoles [16c]. Dehydrogenative cyclization is usually catalyzed by transition metals
such as copper (I), copper (II), palladium (II), iron (II), cobalt (II) and silver (I) species [17].
Then, from these bibliographic evidences, we proposed an copper-catalyzed intramolecular
dehydrogenative cyclization to obtain a series of 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo[3,4-
a]phthalazines 6 from 4-chloro-1-phthalazinyl-arylhydrazones 5 (Scheme 1).
a) Strategy using 1-(4-chlorophthalazin-1-yl)hydrazine 2
NH2
N
N
N
HN
N
R1
R1
HNR2R3
N
N
N
a, b or c
N
N
N
NR2R3
Cl
Cl
4
2
3
Conditions: (a) R1-COCl, 76-79% yields; (b) CH(OEt)3, 82% yields; (c) R1COOH, 32-42% yields.
b) Strategy using 1,4-dichlorophthalazine 4
N
N
N
Cl
Cl
N
R1
R1
HNR2R3
N
N
N
N
d
N
N
R1-CONHNH2
41-90%
NR2R3
Cl
4
5
3
c) Strategy using 4-chloro-1-arylhydrazones 6
N
N
N
N
N
HN
H
R1
R1
HNR2R3
N
N
N
f
N
N
N
Br2
63-89%
NR2R3
Cl
Cl
6
4
3
d) This work, a one-pot C-H activation/C-N formation and N-amination
N
N
N
HN
Cl
i) "C-H activation/
C-N formation"
H
R1
N
N
N
N
ii) N-amination
NR2R3
6
4

5
Scheme 1. General synthetic methods for the preparation of 3-aryl-6-(N-methylpiperazin)-1,2,4-
triazolo[3,4-a]phthalazines 4.
2. Results and discussion
2.1. Chemistry
The optimization of reaction conditions initiated using 4-chloro-phthalazinyl-arylhydrazone 7a
and N-methylpiperazine as model substrates. These starting phthalazin-hydrazones were
prepared following our reported procedure [18]. Initially, some typical parameters involving in
the metal-catalyzed intramolecular cyclization such as reaction time ranging from 24 to 48 h,
high temperatures (usually more than 100 ºC), use of polar solvents and oxidant agents (air in
our case) [19-28] were considered in optimization studies. We decided to use copper (I) iodide as
potential catalyst, which already has been used on the oxidative intramolecular cyclization of
N,N-disubstituted hydrazones[16c]. Optimization conditions are summarized in Table 1. Under
standard reaction conditions including the presence of CuI/L-proline (catalyst/ligand) (15%/30%)
and K3PO4 (3 eq.) under aerobic conditions at 100 °C for 36 h, a significant influence of the
solvent was observed for the progress of the reaction (see Table 1, entries 2-5). In particular,
DMF was found as the most convenient solvent, affording an optimal reaction yield (70 %) of
the desired product (Table 1, entry 3). Interestingly, a good yield of the desired product was also
obtained using an excess of N-methylpiperazine in absence of solvent, acting under this
conditions as both solvent and reactant. A decrease of reaction yield (46 %) was detected when
the reaction was carried out in toluene (Table 1, entry 4), while no desired product was found in
THF as media (Table 1, entry 5). To determine the relevance of aerobic conditions in the
reaction, we examined two separated experiments: one of them under aerobic conditions and
other under Ar atmosphere (Table 1, entries 2 and 9). Results showed that no product was
detected when the reaction was carried out under Ar (see Table 1, entry 9), which demonstrated
the important role of oxygen in the synthesis of 8a. With regards to the catalyst system, the use
of CuI/L-proline (catalyst/ligand) increased significantly the reaction yield compared to no-
catalyst conditions from 11 to 73 % (see entries 2 and 11), requiring at least 15% mol of copper
salts. The increase of temperature from 80 ºC to 100 ºC improved significantly the reaction yield;
while higher temperatures (i.e. 130 ºC) did not improve the preliminary results (compare entries
2, 6 and 7 in Table 1). Finally, results showed that both K3PO4 and K2CO3 salts were suitable

6
bases for the preparation of compound 8a (entry 8, Table 1).
Table 1. Reaction optimization conditions (8a).
N
N
N
N
N
H
NH
Catalyst
N
N
Base, Solvent
T (ºC), time (h)
N
Cl
N
7a
8a
Base
Entries
Catalysts
Solventc T(ºC)
% Yieldd
1
2
CuI/L-proline (10%/20%)
NMPz
100
K3PO4
K3PO4
63
CuI/L-proline (15%/30%)
NMPz
100
73
3
4
CuI/L-proline (15%/30%)
CuI/L-proline (15%/30%)
CuI/L-proline (15%/30%)
CuI/L-proline (15%/30%)
CuI/L-proline (15%/30%)
CuI/L-proline (15%/30%)
CuI/L-proline (15%/30%)
No Catalyst
DMF
Toluene
THF
100
100
90
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K2CO3
K3PO4
K3PO4
K3PO4
70
46
0
5
6
NMPz
NMPz
NMPz
NMPz
DMF
80
53
65
70
9
7
130
100
100
100
100
8
9b
10
11
10
No Catalyst
NMPz
11
aReaction conditions: 7a (1eq., 50 mmol), Catalyst (CuI/L-proline), Base (3 eq., 150 mmol), NMPz (3 eq.),Solvent (5 mL). All
b
c
reactions were carried out in presence of air for 36 h. Reaction under Argon. NMPz (N-methylpiperazine). bIsolated yield of
pure product after purification.
With optimized reaction conditions in hand (entry 2, Table 1), different 3-aryl-6-piperazin-
1,2,4-triazolo[3,4-a]phthalazines were prepared from the corresponding phthalazinyl-
arylhydrazone 7a-g (Table 2). All starting phthalazin-hydrazones were prepared following the
reported procedure [18a-b]. As shown in Table 2, the reaction was favoured when substituent Y
was an electron-deficient group such as 4-nitrophenyl or furyl, providing the corresponding
substituted 1,2,4-triazolo[3,4-a]phthalazines with the best reaction yields, 83 and 79%,
respectively (see entries 3 and 6 in Table 2). Other electron-deficient Y substituent, such as 4-
fluorophenyl, 4-chlorophenyl or 3-nitrophenyl provided reasonable yields of 62 and 70 %,
respectively. Unfortunately, substrate containing a 5-nitrofuryl generated a complex mixture
containing at least four compounds according to TLC analysis, which was very difficult to purify.

7
Probably, the electron-deficient 5-nitro-furyl in phthalazin-hydrazone 7g is highly sensitive to
ring opening reaction under copper-catalyzed conditions. Recent reports have demonstrated that
five membered rings such as furyl, imidazole and thiophene can suffer ring opening reaction
under the presence of metallic-catalyst (Co, Cu, Sc or Ir) [29-30]. The structures of products 8a-f
were confirmed by IR, NMR and elemental analysis. All experimental synthetic and
characterization details can be found in Supplementary Material.
Table 2. Synthesis of 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo[3,4-a]phthalazines 8a-g.a
N
N
Y
N
N
N
CuI/L-proline
Y
(15%/30%)
N
NH
N
K3PO4 (3 eq.)
N-methylpiperazine
100 °C, 36 h
N
Cl
N
7a-g
8a-g
N
N
N
N
N
N
N
N
N
F
NO2
N
N
N
73 %, 8a
62 %, 8b
83 %, 8c
N
N
N
N
N
N
N
N
N
N
N
O
N
N
N
N
Cl
N
N
N
NO2
79%, 8e
70 %, 8d
65%, 8f
N
N
N
N
N
N
N
N
O
NO2
N
N
traces, 8g
N
N
aReaction conditions: 7a-g (1.0 eq., 50 mmol), CuI (0.15 eq., 7,5 mmol), L-proline (0.30 eq., 15 mmol), K3PO4 (3.0 eq., 150
mmol), N-methylpiperazine (5.0 eq., 250 mmol). bIsolated yield of pure product after purification.

8
Cu+2
Cu+
O2
N
Y
N
Y
NH
N
NH
N
N
Y
N
L
Y
Cu

N
Cu+2
-H+
N
N
H
N
N-amination
NH
N
L
N
N
N
NH
N
C
Cl
Cl
Cl
A
B
D
N
1/2O
+
2
H
N+
Cu OH
Y
N
Cu
N
N
N
N
Y
N
N
E
N
N
N
N
F
Scheme 2. Plausible mechanism.
A putative mechanism of reaction was proposed for the formation of the product 8 (or F) from
the starting material 7 (or A) (Scheme 2) based on bibliographic evidences [17,31] and theoretical
calculations. Theoretical calculation permitted to elucidate the most probable reaction route for
the synthesis of 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo[3,4-a]phthalazines 8 from 4-chloro-
phthalazinyl-arylhydrazones 7 and, their details can be found in Supplementary Material (Figures
S1 and S2). Initially, an intramolecular cyclization between nitrogen 3 of the phthlazine ring and
the ylidene carbon occurs to form the intermediate C, passing by adduct B. This step depends on:
(i) the use of a basic agent (K3PO4 or N-methylpiperazine) to facilitate the nucleophilic attack of
nitrogen 3 to ylidenic carbon and, (ii) the electron-deficient nature of substituent Y that increase
the reactivity of the ylidene carbon for the cyclization process. Next, the carbon-chlorine bond
reacts with N-methylpiperazine under a SNAr reaction to lead the desired intermediate D, which
under aerobic conditions in presence of copper(II) metal may induce the dehydrogenation of
ylidenic carbon to form the adduct E, which released the target product F and regenerated
copper(II) species to complete the catalytic cycle. The action of copper on the arylhydrazone-
phthalazine system was observed qualitatively during the reaction, where the reaction mixture

9
was turned from yellow colour to a blue-green solution colour, which suggests complexation of
copper species with phthalazine-hydrazone. With regards to the N-amination reaction, although
the carbon-chlorine in C is sufficiently reactive for simple N-amination, we think that the
copper(I) specie may facilitate the activation of C-Cl bond via Ullmann condensation, which is
well documented [32]. Thus, copper metal seems to have a dual role in the current reaction as
catalyst either for the dehydrogenative intramolecular cyclization or for the N-amination step.
2.2.Biological evaluation
With all compounds in hand, an in vitro anti-proliferative evaluation of the six synthesized
1,2,4-triazole-phthalazine derivatives 8a-f was performed against five selected human tumor cell
lines such as MCF-7 (breast carcinoma, no over-expressing the HER2/c-erb-2 gene), PC3
(prostate carcinoma), HeLa (cervical epithelial carcinoma), SKBr3 (breast carcinoma, over-
expressing the HER2/c-erb-2 gene) and A549 (lung cancer). Adriamycin (Adr.) and colchicine
(CN) were used as reference drugs. Cytotoxic effect of compounds 8a-f was evaluated using the
MTT assay according to reported protocol [33]. Results are summarized in Table 3. It should be
noted that the majority of synthesized 1,2,4-triazolo-phthalzines 8a-f showed a more remarkable
efficacy on breast cancer cells (either against MCF-7 or SKBr3). Specifically, four compounds
(8b, 8c, 8d and 8e) exhibited good IC50 activities against breast cancer cells below 12 μM.
Interestingly, the therapeutic effect of the synthesized compounds was comparable between the
breast SKBr3 and MCF-7 cancer cells, which is an important result due to that the SKBr3 strain
is a resistant-breast carcinoma containing an over-expression of the HER2/c-erb-2 gene that plays
an important role in the disruption of the normal growth cell and contributes to the progression of
aggressive tumor cells and increase of metastatic processes [34]. Contrary to the breast cancer
cells, lung carcinoma lines A549 resulted to be the most resistant amongst the tested cancer
strains by the action of the 1,2,4-triazolo-phthalzines 8a-f, exhibiting only compounds 8c, 8d and
8e an moderate cytotoxic effect with IC50 values ranging from 13.98 to 20.40 μM. In general,
compound 8d exhibited excellent anticancer activities against PC3, MCF-7 and SKBr3
carcinoma cells with low-micromolar IC50 values ranging from 0.11 to 0.59 μM, while compound
8c showed an appreciable anticancer activity against HeLa lines with an IC50 value of 0.54 μM.
These activities of 8c and 8d were significantly lower from 4- to 6-fold than those found for the
Adriamycin reference drug. On the other hands, 8c also presented acceptable anticancer activities

10
against breast MCF-7 and SKBr3 carcinomas with IC50 values of 5.55 and 11.13 μM,
respectively. Compound 8b showed a good anticancer effect against MCF-7 and SKBr3 cells
with IC50 values of 5.85 and 8.48 μM, respectively. Furyl derivative 8e only was active against
SKBr3 cells with an IC50 value of 5.06 μM. Finally, compound 8a and 8f showed from a
moderate to a weak anticancer response against the five selected cancer cells.
From these results, it is possible to establish a structure-activity relationship for the 3-aryl-6-(N-
methylpiperazin)-1,2,4-triazolo[3,4-a]phthalazine. In general, the 3-nitrosubstitution on the aryl
ring seems to be the most convenient pharmacophore because its derivative led the most
significant response against all tested tumor lines. The 4-nitrosubstitution on the aryl moiety
generated compounds with acceptable activity against MCF-7, HeLa and SKBr3. A decrease of
biological activity on the PC3 and HeLa cancer cells was observed when the Y substituent was
replaced by the 4-fluoro-phenyl moiety. While un-substituted phenyl, 4-chlorophenyl or furyl
groups led a loss of the biological activity of the 1,2,4-triazole-phthalazine compounds against
the tested cancer cells. Although it is important to mention that an acceptable IC50 against SKBr3
cell was observed for the furyl-derivative 8e. A comparison with the 3-phenyl-6-(N-substituted-
piperazine)-1,2,4-triazolo[3,4-a]phthalazines reported by Liu [9] (see structure I in Figure 1)
permitted us to identify the nitro-substitution at 3- or 4-position of the phenyl ring as relevant
pharmacophores to increase the therapeutic efficacy of this type of 1,2,4-trizolo-phthalazines by
about 3- to 9-fold against MCF-7 and HeLa strains. Finally, the acyclic form of these compounds,
derivatives 7a-f, were also evaluated on breast cancer strains MCF7-and SKBr3 and these
derivatives exhibited a weak anticancer activity (IC50>20 M), which put in evidence that the
formation of 1,2,4-triazolo is essential for the activity of this type of compounds.
Table 3. In vitro growth inhibitory effects against PC3, MCF-7, HeLa and SKBr3 carcinoma
cells of compounds 8a-f and reference drugs.
N
N
Y
N
N
N
N
8a-f
Entries
Compds
IC50 (μM)

11
PC3
MCF-7
HeLa
SKBr3
A549
1
2
3
4
5
6
7
8
8a
8b
32.60 ± 2.96 41.37 ± 2.91 120.09 ± 3.02
20.72 ± 2.97
233.84±3.54
21.40 ± 2.70
17.58 ± 2.62
0.41 ± 0.11
5.85 + 1.06
5.55 ±1.06
0.11 ± 0.05
28.60 ± 2.95
0.54 ± 0.39
8.58 ± 1.07
22.62 ± 3.08
41.01 ± 3.12
N.D.
8.48 ± 1.05
11.13 ± 1.02
0.59 ± 0.44
5.06 ± 1.08
12.23 ± 1.16
0.04 ± 0.01
>200.00
8c
20.40±2.64
13.98±1.80
17.33±2.01
> 200.00
N.D.
8d
8e
17.66 ± 3.08 12.24 ± 1.03
24.89 ± 1.89 15.23 ± 2.11
8f
CNa
Adr.b
24.98 ± 2.44
0.03 ± 0.01
3.62 ± 0.12
1.66 ± 0.08
2.45 ± 0.37
2.36 ± 0.40
N.D.
a CN: colchicines drug, Adr.: bAdriamycin drug, Marked in bold parameters indicated a pronounced anticancer activity. N.D.: Not determined.
Secondly, the toxicity of the most active derivatives 8b, 8c, 8d and 8e was evaluated on a
primary culture of human dermis fibroblasts following protocol reported for peritoneal
macrophage with a few modifications [35a-d]. The toxicity results are listed in Table 4, together
with their respective selectivity indexes. From active compounds, derivatives 8b and 8d showed
acceptable toxicities on the human dermis fibroblasts with LD50 values more than 250 μM.
Unfortunately, one of the most active derivatives, the 4-nitrophenyl derivative 8c, exhibited a
relative high toxicity against both strains. It may be associated to the higher susceptibility of a
high π-conjugated nitro group placed at 4-position of the phenyl ring compared with 3- or 2-nitro
derivatives to suffer biotransformations mediated by nitroreductase enzymes producing a variety
of chemical toxic intermediates, for which are usually attributed toxicity, mutagenic and
carcinogenic activities [35e]. Probably, other high π-conjugated nitro derivatives such as the
derivative 8g, that was not compatible with our synthetic protocol, could exhibit a high toxicity.
Recently, we found that the acyclic form of 8g, the corresponding phthlazine-hydrazones 7g as
well as the 5-nitrothiophene derivative demonstrated to be highly toxic against macrophage and
Vero cells [18]. Then, from anticancer evaluation and toxicity studies, the compound 8d, a 3-
nitrophenyl derivative less susceptible to biotransformations compared to the high π-conjugated
4-nitrophenyl or 5-nitroheteroaryl partners, emerges as an attractive anticancer candidate for
further assays due to its excellent selectivity indexes (S.I. >250) against PC3, MCF-7, HeLa,
SKBr3 and A549 carcinoma cells, being especially promising against prostate and breast cancer.
It seems that a robust nitro substitution is essential to provide a controlled biotransformation as a
promising anticancer candidate based on 1,2,4-triazolo-phthalazine. Compound 8b represents an
interesting alternative for further assays in breast cancer model.

12
Table 4. Toxicity evaluations and selectivity indexes (S.I.) of compounds 8a-f
S.I.c
Fibroblasts
Entries LD50 (μM)a
PC3
> 7.7
11.7
MCF-7
> 6.0
42.7
HeLa
SKBr3
A549
8a
8b
8c
8d
8e
8f
>250.0
>250.0
2.1
12.1
29.4
0.5
≥ 1.0
≥ 1.0
0.3
8.7
10.8
>29.1
0.8
5.83 ± 1.23
> 250.0
0.3
1.1
>423.7
>609.8
~1.0
> 17.9
1.0
> 2273
1.4
17.33±1.25
> 250.0
3.4
> 10
> 16
> 6
> 20
≥ 1
aToxicty on dermis fibroblasts.bToxicity on peritoneal macrophage. cSelectivity indexes: LD50 of firbroblasts/IC50 of specific carcinoma cell and ,
LD50 of peritoneal macrophage/IC50 of specific carcinoma cell in brakets. dResistance ratio: IC50 of parental MCF-7cells/IC50 of resistant SKBr3.
2.3. Mechanistic Studies
A mechanistic study was performed for the most active derivative. Firstly, a qualitative DNA
analysis using agarosa gel was carried out for SKBr3 cancer culture treated with the most active
compounds 8b, 8c and 8d (see Figure 2A). Results put in evidence that compounds 8b and 8d
induced an appreciable DNA degradation on the SKBr3 cell compared with untreated cells,
which suggested that they may act on some phase of cell division including cellular early
apoptosis and cell arrest. Recently, Liu and co-workers demonstrated that their active 3-phenyl-6-
(substituted-piperazine)-1,2,4-triazolo[3,4-a]phthalazines showed the ability to induce cellular
early apoptosis and cell cycle arrest at the G2/M phase in the esophageal carcinoma cell line EC-
9706 for a range of concentrations from 2 to 4.5 µM [9]. Other analogues of our structures such
as 6-(N-alkylamino)-pyrrolo[2,1-a]phthalazine also demonstrated to arrest the cell cycle
progression at the G2/M phase with apoptosis consequences, which seems to be mediated by the
inhibitory action of these compounds on the phosphorylation of VEGFR-2 [10]. All these
evidences motivated to us to focus our mechanistic study on molecular targets involving in cell
processes such as cell cycle arrest at the G2/M phase and early apoptosis. In this sense, potassium
channels, that are involving in different cell processes such as cell proliferation, cell cycle
progression, migration and apoptosis, emerge as a potential molecular target due to its blockades
of K+ channels is involved in the attenuation of cancer progression. These ionic channels are
widely distributed in a great variety of cells including cancer cells. Several voltage-gated K+
channels of the Shaker family (Kv) have been reported on breast cancer tissue or cell lines [36-
37]. Specifically, Kv1.3 and Kv1.5 are widely implicated in the development of different tumors

13
[37j-l]. Experimental evidences have demonstrated that pharmacological blockades of K+
channels lead to cell proliferation inhibition in different cancer cells, being an excellent target to
design new anticancer drugs [37]. The molecules targeting K+ channels affect Kv channel
functions by blocking the ion-conducting pore from external to internal cell. Usually, charged
molecules and coordinating compounds containing alkyl-substituted amine chain side (e.g.
piperazine linker) have been widely identified as K+ channel inhibitors [38]. In this sense, our
1,2,4-triazolo-phthalazin bearing a N-methylpiperazin moiety emerged as a potential K+ channel
inhibitor because this system has the ability to coordinate with any metal specie through the
piperazine moiety. With all this information in mind, we firstly decided to evaluate the ability of
the most active derivative 8d to promote blockade of K+ channels. To evaluate the effect of the
studied 1,2,4-triazolo-phthalazine on K+ channels activity, we measured the current into cancer
cell for a range of voltage from -120 to 70 mV according to our previous report [33]. The
measurement of cell current was performed through the connection of a cancer cell to an
electrical circuit. Three independent experiments were performed: (a) a first untreated cancer cell;
(b) a second cell treated with the most active derivative 8d; and (c) a third cancer cell treated with
a positive control of TRAM-34 (T34) [39], a highly selective calcium-activated potassium
channel (IKCa1) inhibitor. SKBr3 and MCF-7 cancer cells were used to perform this assay.
Results are shown in Figures 2B and 2C for SKBr3 and MCF-7 strains, respectively. The
equilibrium potential found in cell system is placed at -60 mV near to equilibrium potential of
potassium, which indicates that current registered to potential different of -60 mV correspond to
potassium current (see Figure 1). Experimental details are described in the experimental section
of Supplementary Material. As was expected, a reduction of current was appreciated to both
potential extremes (central point is to -60 mV of voltage) when the cell is treated with T34
inhibitor compared to untreated cell. With respect to the effect of our compound 8d on cellular
current, an inhibition of current was observed for the potential extremes compared to untreated
control, which suggested that the compound 8d may be involved in the blockades of the voltage-
gated K+ channels (Kv). The percentages of inhibition, measured at 70 mV, are similar for both
cell lines: (i) 33 % for MCF-7 cell and, (ii) 35 % for SKBr3 cells. It is well documented that the
reduction of the current into cell is associated to the blockades of K+ channels and it may be
involved in the attenuation of cancer progression.

14
(B)
(A)
(C)
Figure 2. (A) Electrophoresis, Agarosa gel 3%. (1) 100 pb MW Invitrogen; (2) Control, SKBr3;
(3) SKBr3+8c; (4) SKBr3+ 8d; (5) SKBr3+ 8b; (6) Low Mass MW Invitrogen. (B) Effect of the
3-(3-nitrophenyl)-6-(N-methylpiperazine)-1,2,4-triazolo[3,4-a]phthalazine 8d on current of K+
channels in SKBr3 cell. Untreated control (black curve), cell exposed at 0.5 µM of 8d (red
curve), cell exposed at T34 (green curve). (C) Effect of the 3-(3-nitrophenyl)-6-(N-
methylpiperazine)-1,2,4-triazolo[3,4-a]phthalazine 8d on current of K+ channels in MCF-7cell.
Untreated control (black curve), cell exposed at 0.5 µM of 8d (red curve).
In order to understand more about the inhibitory response of the most active compound 8d as
potassium channel blockade, a tentative molecular docking study was performed on KscA
protein. This protein represents a useful potassium-channel protein template to estimate the
potassium blockade capacity of a specific compound [40a-c]. Crystal structure of the KscA
complex is available in Protein data Bank under PDB code: 2BOB for Streptomyces lividans
[40d]. Tetrabutylammonium (TBA) is co-crystallized as a competitive inhibitor into crystal KscA

15
protein. This potassium channel from Streptomyces lividans is an integral membrane protein with
sequence similarity to all known K+ channels, particularly in the pore region. Arguslab 4.0
software [41] was used to perform the molecular docking of the most active 3-phenyl-6-(N-
methylpiperazine)-1,2,4-triazolo[3,4-a]phthalazine 8d on the mentioned enzyme. The geometry
of the derivative 8d was optimized using the B3LYP method [42]. The molecular docking
showed that the compound 8d (Edocking= -4.14 kcal/mol) exhibited a lower affinity with TBA-
active site compared to TBA inhibitor energy (-5.73 kcal/mol); however, it is important to
mention that compound 8d showed a similar disposition into active site compared to TBA
inhibitor. Two important hydrogen bonding interactions were identified for compound 8d with
485-Thr residue (1.67 and 2.37 Aº). This finding reveals that compound 8d may be localized into
potassium-channel gated similar to TBA inhibitor, affecting the potassium ion exchange between
the exterior and interior of channel, which permits to explain the significant experimental
inhibitory potassium-channel blockade response found for compound 8d. Interestingly, other
derivatives such as 8b, that not exhibiting H-bonding interactions and lower affinity energy of -
3.13 kcal/mol, presented a different orientation compared to the compound 8d into potassium-
channel KscA active site, which suggests that the H-bonding of 1,2,4-triazolo ring with 485-Thr
residue may be crucial in the inhibitory activity of derivative 8d as potassium-channel blocker.
See details of superimposition between 8b and 8d in Figure S3B of Supplementary Material.
C)
A)
B)
Figure 3. Molecular docking of most active compound 8d on TBA-active site of KscA protein.
(A) Visualization of potassium-channel gated into KscA protein with TBA-active site (in gray
ribbons), TBA (yellow color) and compound 8d (orange color). (B) Illustration of TBA-active

16
site (gray ribbons), thallium (pink ribbons), TBA inhibitor (yellow color) and compound 8d
(orange color). (C) TBA-active site (in cylinder white lines), TBA inhibitor (yellow color) and
compound 8d (orange color).
A)
C)
B)
Figure 4. Molecular docking of most active compound 8d on EGFR Tyrosine Kinase protein.
(A) Visualization of the EGFR Tyrosine Kinase active site (in gray ribbons) with compound 8d
(orange color) into full protein. (B) EGFR Tyrosine Kinase-8d complex and AQ ligand (yellow
color) into active site (in gray ribbons). (C) Superimposition between compound 8d (orange
color) and AQ ligand (yellow color) and their H-bonding interactions in red lines.
In order to obtain more information about the mechanism of action of the most active
compound 8d, an extra molecular docking was performed on an EGFR tyrosine kinase template.
The inhibition of the phosphorylation of EGFR tyrosine kinase enzymes is associated to cell
cycle arrest at the G2/M phase and early apoptosis [10,44]. The EGFR tyrosine kinase has
demonstrated to be an important target for anticancer phthalazine [3f-k,43a-d] as well as for

17
1,2,4-triazolo[3,4-a]phthalazine [3i,10,43e] derivatives. In addition, a large number of
quinazoline, an isostere of phthalazine ring, have frequently been identified as EGFR and VEGFR
kinase inhibitors [44]. To perform molecular docking, a X-ray EGFR crystallographic structure under
PDB ID code 1M17 [45] was used as platform for the molecular docking of compounds 8b and 8d.
From molecular docking on EGFR tyrosine kinase (Figure 4), it was found that derivatives 8b and 8d
presented lower binding energies of -8.97 and 10.17 kcal/mol compared to AQ (4-aniline-
quinazoline) ligand (-7.74 kcal/mol), which suggests that the tested 1,2,4-triazolo-phthalazines may
act as tyrosine kinase inhibitor. Furthermore, the most active derivative exhibited a similar orientation
compared to AQ into active site with a barely superimposition of their corresponding fused-
heterocyclic rings and with a particular orientation of nitrogen H-acceptor atoms to 769-Met residue
(Figure 4B and 4C), an important residue in the tyrosine kinase inhibitory activity [45]. Derivative 8d
exhibited three H-bonding interactions through nitro moiety with the 721-Lys (2.99 Aº), 830-Thr
(2.28 Aº) and 831-Asp (2.99 Aº) residues. Derivative 8b showed a H-bonding interaction with
769-Met residue through a nitrogen of 1,2,4-triazolo ring. See comparative disposition of 8b and
8d into active site of EGFR protein can be found in Figure S3A of Supplementary Material. In
summary, the experimental and theoretical findings suggest that the degradation of DNA in
cancer cell treated with active 1,2,4-triazolo-phthalazines is associated to the blockade of potassium
channels and inhibition of EGFR tyrosine kinase enzyme.
As last topic, an in silico drug-likeness study was performed on the tested compounds using
Chemdraw Ultra suite software [46]. Then, the parameters of Lipinski´s ruler, which can predict
oral bioavailability and membrane permeability of synthetic drugs as well as to describe the
molecular properties related to the pharmacokinetic aspects in human body such as absorption,
distribution, metabolism and excretion (ADME) [47], were calculated for the five derivatives and
Adriamycin reference drug. The results are summarized in Table 5. In general, according to these
criteria, all tested 3-substitutedphenyl-6-(N-methylpiperazine)-1,2,4-triazolo[3,4-a]phthalazine
derivatives displayed good bioavailability properties, with a molecular weight < 500 g/mol, a Log
P < 5, acceptor and donor hydrogen bond interactions below 10 and 5. Curiously, the most active
derivatives presented the lowest Log P, which indicates that the activity of these compounds may
be compromised by their absorption properties [48]. In this sense, the compounds with the lowest
Log P should have a better bioavailability, which is directly reflected in their anticancer activity.
This fact confirmed the importance of this parameter in the biological effect of these 1,2,4-
triazolo 8a-f. To complement our in silico evaluation, we calculated other molecular descriptors

18
such as the topological polar surface area and the absorption percentage (% Abs). This study
reflected the potential intestinal absorption, bioavailability and hematoencephalic barrier
permeation of our compounds according to Veber’s rules (TPSA < 140 A2) [49], with TPSA
ranging from 46.80 to 98.61 A2 and % absorption between 74 and 92%.
Table 5. Calculated theoretical parameters of the Lipinski´s ruler for the 1,2,4-triazole-
phthalazine 8a-f.
Entries Compds M.W. (g/mol)
%
Log P
TPSA
HA HD ROTB
Lipinski´s rule
violations
Absorption
1
2
3
4
5
6
8a
8b
8c
8d
8e
8f
344.17
362.17
389.16
389.16
334.15
344.17
92.86
92.86
74.98
74.98
89.67
92.96
4.07
4.23
2.91
2.91
2.69
4.07
46.80
46.80
98.61
98.61
56.03
46.80
6
7
8
8
7
6
0
0
0
0
0
0
9
9
9
9
9
9
0
0
0
0
0
0
Percentage of absorption is calculated by % Absorption = 109 - (0.345 × TPSA). TPSA, topological polar surface area, Log P (partition
coefficient) calculated by Chemdraw program package, HA hydrogen bond acceptors, HD hydrogen bond donors, ROTB number of rotatable
bonds. Adr.: Adr.adriamycin and CN:colchicine.
3. Conclusion
In summary, a series of 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo-phthalazines were
synthesized as potential anticancer agents. A copper-catalyzed aerobic oxidative C-H/N-H
coupling of phthalazin-based on aryl/heteroarylhydrazinyl was developed by utilizing CuI as catalyst
and air as oxidant. The protocol permitted the further addition of N-methylpiperazine at 4-
poistion by chlorine atom in a one pot-reaction. Compared with the traditional 3-aryl-6-
piperazine-1,2,4-triazolo[3,4-a]phthalazines derivatives syntheses from phthalazin-based on
aryl/heteroarylhydrazinyl, this protocol provided a more attractive approach by its simplicity,
economy and one-pot reaction procedure. With regards to anticancer evaluation, the majority of
compounds showed excellent anticancer activities against PC-3, MCF-7, and SKBr3 cancer cells,
very in particular for the derivative 8d, which presented a good anticancer profile with low
toxicities and acceptable selectivity indexes. A structure-activity relationship study suggests that
the formation of fused triazolo-phthalazine and nitro-substitution are crucial pharmacophores in
the biological activity of this kind of compounds. Mechanism of action studies indicated that the

19
K+ channel blockade was identified as one of the possible modes of action of the most active
compound 8d against cancer cells (MCF-7 and SKBr3). Finally, the results demonstrated that
compound 8d represents a promising anticancer candidate and further genotoxicity evaluation,
mechanistic assays, experiments against resistant cells and in vivo assays are still required to
estimate its real therapeutic potential.
Acknowledgments
Authors thank to Decanato de Investigacion y Desarrollo (DID, Universidad Simon Bolivar),
for its financial support. A.R. thanks to Decanato de Estudios de Postgrado, Departamento de
Quimica (Universidad Simon Bolivar) for a Doctoral Fellowship. Authors thank to Lic. Noelani
Ciguela for the NMR collection data as well as Instituto de Biología Experimental-IBE and
Facultad de Farmacia (Universidad Central de Venezuela) for research resources.
Supplementary material
1
13
Full experimental details, H-NMR and C-NMR spectra associated with the new compounds
obtained herein can be found in the online version of the Supplementary material, at science
direct website.
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Confict of Interest
Authors declared not conflict of interest.
Highlights
1. Synthesis of
a
new series of 3-aryl-6-(N-methyl)piperazin-1,2,4-triazolo[3,4-
a]phthalazines
2. Nitro-derivatives exhibited a significant anticancer activity against five human cancer
cell.
3. Moreover, the cytotoxic profile of the newly synthesized compounds was checked.
4. The most active compounds were targeted to potassium channels.
5. Molecular docking and MD simulations were performed