Synthesis of novel triazoloquinoxaline-pyrazole hybrids as antiproliferatives, EGFR inhibitors, and apoptosis inducers

Article abstract of DOI:10.1002/jhet.4144

Novel triazoloquinoxaline-pyrazole hybrids have been developed and synthesized. All derivatives' anticancer activity has been evaluated using Sulforhodamine-B (SRB) assay for cancer cell lines MCF-7, HepG-2, and HCT-116. Compound 12b was 2-fold more cytotoxic than Doxorubicin, while 12a,c demonstrated comparable cytotoxicity to the reference Doxorubicin. Further investigations on the most active derivatives 12a-c were done to study their inhibitory activity on two EGFR subtypes wild EGFR and mutant EGFR (L858R) tyrosine kinases in MCF-7 cell lines. Compound 12b exhibited potent inhibitory activity toward wild EGFR (IC₅₀: 0.98 μM) when compared to Gefitinib (IC₅₀:18.07 μM). 12b also possessed a marked inhibition against mutant EGFR (L858R-TK) exhibiting (IC₅₀:27.45 μM) in comparison to Lapatinib (IC₅₀: 61.06 μM). Compound 12b improved the active Caspase-3 value and the BAX/Bcl-2 reference. Furthermore, 12b showed G2/M cell cycle arrest induced apoptosis in cell line MCF-7. In addition, the most active derivatives have been orally bioavailable as shown in the in silico determination of the ADME characters. The binding pattern of compound 12b was also studied by molecular docking.

Full text of DOI:10.1002/jhet.4144

Synthesis of novel triazoloquinoxaline-pyrazole hybrids asantiproliferatives, EGFR
inhibitors and apoptosis inducers
Hoda S. El Saeed1, Ashraf H. Bayoumi2, Marwa T. Sarg1,* Adel H. Ghiaty2
1Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt.
2Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Boys) Al-Azhar University, Cairo, Egypt.
*Corresponding author: E-mail address:hodasamir85@gmail.com, hodasamir85@gmail.com or
HodaSamir.1030.el@azhar.edu.eg(Hoda S. El Saeed)
ARTICLE INFO
Key words:
ABSTRACT
Novel triazoloquinoxaline-pyrazole hybrids have been developedandsynthesized.All
derivatives' anticancer activity has been evaluated using SRB assay for cancer cell
lines MCF-7, HepG-2, and HCT-116. Compound 12b was two-fold more cytotoxic
than Doxorubicin, while 12a,cdemonstrated comparable cytotoxicity to the reference
Doxorubicin. Further investigations on the most active derivatives 12a-c were done to
study their inhibitory activity on two EGFR subtypes wild EGFR and mutant EGFR
(L858R) tyrosine kinases in MCF-7 cell lines. Compound 12b exhibited
potentinhibitory activity toward wild EGFR (IC50: 0.98µM) when compared
toGefitinib(IC50:18.07µM).12b also possessed a marked inhibition againstmutant
EGFR (L858R-TK) exhibiting (IC50:27.45µM) in comparison to Lapatinib (IC50:
61.06 µM).Compound 12b improved the active Caspase-3 value and the BAX/Bcl-2
reference. Furthermore 12b showed G2/M cell cycle arrest induced apoptosis in cell
line MCF-7. In addition, the most active derivatives have been orally bioavailable as
shown in the in silicodetermination of the ADME characters. The binding pattern of
compound12b was also studied by molecular docking.
Triazoloquinoxaline
Antiproliferative
Caspase-3
Docking
EGFRinhibitors
1.
Introduction
Cancer is possessing high death rate cause in the world, and new treatments and solutions to this disease
need a great deal of attention[1,2].Cells are regulated by proteolytic enzymes called caspases that induce cell
death or apoptosis.The activation of apoptotic process is initiated by either extracellular or intracellular death
signals, which cause activation of procaspases and caspases[3].In the last 10 years, the oncogenic mutations
that change the apoptosis leading to tumor initiation , development, or metastasis have been thought to have
triggered initiation of the cancer. Apoptosis offers a strong basis for understanding the relationship between
cancer genetics and cancer therapy. For this reason, the analysis of the genes and pathways that make up the
central machinery of the pathway of apoptosis offered new insights into tumor biology and enhanced new
therapeutic benefits strategies in cancer treatment[4]. BAX and Bcl-2, which are the important members of
the Bcl-2 family for their influential roles in the development of tumors and the pronostics of human
malignancies, have also gained anenourmous deal of interest in recent years. In response to various cell
pressures, BAX enhances cell death. In contrast, Bcl-2 abandons apoptosis by inhibiting the activity of
BAX[5].Moreover, The transmembrane glycopritein with tyrosine-kinasa (EGFR) is the epidermal growth
factor receptor which are overexpressed in many cancer cells such as (breast, ovarian and colon human
cancers)[6].EGFR family overexpression leads to autophosphorylation of several tyrosine residues within
the COOH-terminal tail of the receptors which initiates a certain cascade resulting in cell proliferation,
differentiation and anti-apoptosis. So, inhibition of EGFR family identified to possess significant role in the
development of targeted chemotherapeutic agents[6].
Literature survey clarified thattriazoloquinoxalineswere known asanticancer agents[7,8].In 2018,Mohamed
Alswahet
al.,reported
that
1-(4-(1-ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-ylamino)phenyl)-3-
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(substitutedphenyl)prop-2-en-1-one A had antiproliferative characteristics with EGFR kinase inhibition
(IC50=0.039µM)[2].Noteworthy,pyrazoles were proved to possess potent anticancer activity[9 ,10, 11].One
of the reported anticancer pyrazoleswhich induced apoptosis via Caspase-3 activation dependent pathway
wasthe reported phenyldiazenyl-1Hpyrazoles-3,5-diamine derivative B[10].
Based on these findings we found that it is interesting to hybridize triazoloquinoxaline scaffold by pyrazole
moieties (c.f.figure 1)hoping to establish more potent, and safer novel anticancer agent.
.
Figure1: General design of novel triazoloquinoxaline-pyrazoles hybrids
2. Results and discussion:
2.1.Chemistry
The key intermediates 2 and 7 were prepared according to reported procedure[2]. The nucleophilic
displacement reaction of the 4-chlorotriazoloquinoxaline derivative 2 with 4-aminoantipyrine or with the
aminopyrazole derivatives afforded compound 3 and diazinyl pyrazoles 4a-c; respectively. In order to
introduce pyrazole moiety at position 4 through longer linker, the intermediate cyanoacetohydrazide 5 was
prepared from the reaction of 2-cyanoacetohydrazide with compound 2, then the product 5 was further
13
reacted with formylpyrazole derivative to yield the acrylohydrazide 6 (scheme 1). The C NMR chart of
3was characterized by a signal at 162.51 due to C=O carbon. In addition, the 1H NMR spectrum of 3 showed
two singlet signals at δ 2.25 and δ3.16 ppm corresponding to 2CH3 protons.The infrared spectrum of 4b
showed bands at a stretching frequencies (υ) 3375, 3290 cm−1 and 3205 cm−1 corresponding to the NH2 and
NH functions; respectively. Furthermore, the 1H NMR spectrum of 4b revealed a singlet signal at δ 2.32 due
to CH3 protons. The shifted value of carbonitrile stretching frequency (υ) from 2264 cm−1in 5 into 2214
cm−1proved the structure of 6.When the hydrazine derivative 7 was condensed with pyrazole carbaldehyde
derivatives in ethanol/acetic acid mixture, the expected Schiff's bases were not achieved, instead, the
diacetylated hydrazide 8 was obtained. However, the target Schiff's bases 9a,b were furnished when the
same reaction was performed in DMF/ piperidine mixture. On the other hand, to synthesize
triazoloquinoxaline hybrids with directly attached pyrazoles, the hydrazino derivative 7was reacted with 2-
(ethoxymethylene) malononitrile or 2-(bis(methylthio)-methylene)malononitrile, Michael addition of NH2
on ylidenic bond resulting in an acyclic intermediates formation, which were cyclized by nucleophilic
addition of NH on cyanocarbon to afford the [1,2,4]triazolo[4,3-a]quinoxaline-3-amino-pyrazole-4-
2
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carbonitriles compounds 10 and 11; respectively. The infrared spectrum of diacetyl hydrazide 8 represented
1
a band at 1716 cm−1for C=Oin addition to two singlet signals at δ1.91 and δ3.06 ppm in the H NMR
1
spectrum corresponding 2CH3 protons. While, The H NMR of the Schiff's bases 9a,b exhibited the singlet
signal indicating the imine hydrogenN=CH at δ 9.67 ppm. In addition, the Infrared spectrum of 11 exhibited
1
an absorption band at frequency (υ) 2210 cm−1represented C≡N function group. Additionally, the H NMR
of compound11showed a signal which appeared as a singlet at δ2.64 ppm attributed to SCH3 protons
(scheme 2). Moreover, cycloaddition of binucleophile hydrazine 7 on diazinyl malononitriles afforded the 1-
([1,2,4]triazolo[4,3-a]quinoxalin-4-yl)-4-(aryl)-1H-pyrazole-3,5-diamines 12a-c. Finally, the reaction of
chalcone derivatives with hydrazine type 7in basic medium assumed to be started with Michael addition
1
followed by condensation to yield the diaryl pyrazoles 13a,b.The H NMR of the diaminopyrazole12b
showed characteristic two singlets at δ 6.91and δ 9.30 ppm representative to the 2NH2 protons and another
singlet signal at δ2.36ppm for CH3 protons. Also, the IR of 13a&b lacked any bands due to NH or
NH2.Besides,the 1H NMR for derivative13ashowed a singlet signal at δ3.86 ppm attributed to OCH3 protons
(scheme 3). The 13C NMR revealed signals of all the compounds in spectra are determined to be consistent
with the expected signals for those structures. Additionally, mass spectrometry indicated molecular ion
peaks at M+.
N
N
N
CH3
H2N
O
N CH3
N
Ph
N
NH
(ii)
H3C
O
N N
3
Dioxane/ TEA/ reflux/ 12h
H3C
Ph
N
N
NHNH2
Cl
1
N
N
Ar
N
N
N
(i)
NH2
CH(C2H5O)3
N
N
N
NH
NH
NH
N
N
H2N
Ar
(iii)
N
N
N
Dioxane/ TEA/ reflux/ 12h
H2N
N
Cl
a, Ar=C6H5
b, Ar=4-C6H4-CH3
c, Ar=4-C6H4-Br
4 a-c
2
N
N
N
N
N
N
Ph
O
N
N
N
HN
H2N
O
C
H
N
NH
Ph
(v)
Ph
N
NH
NH
(iv)
HN
O
N
DMF/ reflux/ 12h
DMF/ pip/ reflux/ 18h
N
N
C
O
N
H
Ph
5
6
Reagents and conditions:i) Triethoxymethane(excess)/ reflux; ii) 4-Amino-1,5-dimethyl-2-phenyl-1H -pyrazol-3(2H)-
one/ TEA/ dioxane/ reflux; iii) 4-((4-Substitutedphenyl)diazenyl)-1H-pyrazole-3,5-diamines / TEA/ dioxane/ reflux;
iv) / 2-Cyanoacetohydrazide /TEA/ dioxane/ reflux; v)1,3-diphenyl-1H -pyrazole-5-carbaldehyde /DMF/pip/ reflux
3
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Scheme 1: synthesis oftriazolo-quinoxalin compounds 1-8
N
N
N
N
O
NHN
Ar
8
O
N
O
C
N
Ph
(ii)
N
N
H
N
Ar
ethanol/ acetic acid/
reflux/6h
N
N
N
N
N
C
H
Ph
N
N
H
Ar
N
9a,b
N
N
(iii)
O
a, Ar= C6H5-
b,Ar= 4-CH3-C6H4-
C
Ph
H
N
2
Cl
DMF/ pip/ reflux/ 18h
NH2NH2/ ethanol
/ reflux/ 6h
(i)
CN
N
N
N
N
N
N
NC
N
N
N
N
C H
C2H5O
N
N
(iv)
N
NHNH
N
DMF/ pip/ reflux/
13h
H
NHNH2
H2N
10
NC
CN
7
CN
N
N
N
N
CN
NC
N
N
N
C SCH3
H3CS
N
(v)
N
N
NHNH
SCH3
SCH3
DMF/ pip/ reflux/
10h
H2N
11
NC
CN
CN
Reagents and conditions: i) NH2NH2/ absolute ethanol/ reflux; ii) 1-Phenyl-3-(4-
substitutedphenyl)-pyrazolecarbaldehydes /absolute ethanol/ CH3COOH/ reflux; iii) 1-
Phenyl-3-(4-substitutedphenyl)-pyrazolecarbaldehydes / pip/ DMF / reflux; iv) 2-
Ethoxymethylenemalononitrile
TEA/
DMF/
reflux;
v)
2-
(Bis(methylthio)methylene)malononitrile /TEA/ DMF/ reflux
Scheme 2: synthesis of triazolo-quinoxalin compounds 7-12
4
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N
N
N
N
CN
CN
DMF/ TEA/reflux/ 20h
N
N
Ar
N
N
(i)
N
NH2
H2N
N
Ar
N
N
12a-c
N
N
a, Ar=C6H5-
b, Ar=4-CH3-C6H4-
c, Ar=4-Br-C6H4-
NHNH2
O
N
N
7
N
N
Ar
N
Cl
Cl
(ii)
N
Ar
DMF/ TEA/reflux/ 16h
Cl
Cl
a, Ar= 4-OCH3-C6H4-
b, Ar= 2-thienyl
13a,b
Reagents and conditions: i) 2-(4-Substituted-phenyldiazenyl)malononitrile /
TEA/DMF/reflux; ii) 1-Aryl-3-(2,4-dichlorophenyl)prop-2-en-1-ones / DMF/
TEA/reflux
Scheme 3: synthesis of triazolo-quinoxalin compounds 12&13
2.2. Biology
2.2.1.Screening of anti-proliferative activity
The newly synthesized hybrids wereassessedin-vitro against MCF-7, HepG-2 and HCT-116 cell lines (Table 1). Four
of the sixteen tested compounds 12a-c&4b showed high cytotoxic activities (IC50s ranging from 2.79–10.77 µM). 1-
([1,2,4]triazolo[4,3-a]quinoxalin-4-yl)-4-(substituted-phenyldiazenyl)-1H-pyrazole-3,5-diamine analogues12bshowed
about twice the Doxorubicin potency(IC50= 2.79, 3.07 and 3.64 μM) and 12a (IC50 = 6.25, 6.17 and 8.50µM) was more
potent cytotoxic than the reference Doxorubicin (IC50= 4.65,7.46 and8.29 μM) respectively, against the tested cell
lines.
Comparing cytotoxic results of 4a-c&12a-c, it was found that 12a-cwere potent anticancer agents while their
analogues having NH linkers 4a-c demonstrated good to poor activity. On the other hand, compounds 13a, b did not
show any cytotoxicity, which recommend the importance of aryldiazenyldiaminopyrazole for cytotoxic activity.
Up on the abovefindings, it appeared that, the hybrids of triazoloquinoxaline should be directly attached to pyrazole
scaffold(linker X=0). Also,from our findings, the presence of aryldiazenylpyrazolediamine moiety is essential for
cytotoxic activity. In addition,The cytotoxic activity evaluation of the most active compounds on the normal cells
human diploid lung fibroblasts cell line WI-38 was done owing to study their toxicity and selectivity (Table 1). The
5
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results showed that, these compounds are inactive against this normal cell exerting IC50=83.21 , 78.34 & 80.75 µM for
compounds 12a-c; respectively. So, the most active compounds exhibited no cytotoxic activity against normal cells, a
considerable safety margin and selectively toward the cancerous cell lines as idicated by their IC50 values.Due to this,
compounds 12a-c were selected for further biological investigation.
Table 1: Anti-proliferative screening of the 16 newly synthesized compoundsagainst MCF-7, HepG-2,
HCT-116 and WI-38cell lines
Cpd.
No.
IC50 µM
MCF-7
35.11
32.86
10.75
42.36
63.48
51.49
34.22
85.37
82.16
45.26
38.47
6.25
HePG-2
47.23
57.42
11.66
49.18
98.48
47.35
50.11
79.43
71.19
67.89
45.09
6.17
HCT-116WI-38
45.57
-
-
-
-
-
-
-
-
-
-
-
3
64.19
4a
10.89
4b
60.86
4c
72.38
5
55.28
6
44.95
8
82.38
9a
82.46
9b
60.22
10
59.16
11
8.583.21
3.6478.34
9.5380.75
70.64
12a
12b
2.79
3.07
12c
8.37
10.77
72.44
84.37
7.46
55.17
65.38
4.65
-
-
-
13a
92.14
13b
Doxorubicin
8.29
2.2.2. EGFR inhibition assay
EGFR inhibitory assay for new representative active compounds 12a-c was evaluated using MCF-7 cancer
cells and the results obtained in table 2 showed that, compound12b exhibited reasonableinhibitoryagainstwt-
EGFR expression showing IC50 value= 0.975 µMwhile the two other aryldiazenyl-pyrazole derivatives12c
and 12a exhibited less inhibitory activity towards the same enzymewithIC50 value= 1.35and 1.57 µM;
respectively comparable to Gefitinib (IC50 value= 18.07µM)(Table2). In addition, the potent compounds
12a-crepresentedgood activity on mutant type of EGFR (L858R-TK) with IC50 values (27.45-
40.95µM)higher than Lapatinib(IC50 value=61.06µM)(Table3).
6
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Table 2:IC50 of the hit anticancer compounds, 12a-con EGFR Wild type in MCF-7
Cpd.
No.
IC50 EGFR Wild type (μM)
1.547
0.975
1.35
12a
12b
12c
0.046
18.07
Lapatinib
Gefitinib
Table 3:IC50 of the hit anticancer compounds12a-con EGFR mutant (L858R-TK)in MCF-7
Cpd.
No.
IC50 EGFR mutant (L858R-TK)
(μM)
32.72
27.45
40.95
61.06
12a
12b
12c
Lapatinib
2.2.2.Apoptosis detection results
2.2.2.1. Effect on the active Caspase-3 level
The best understood mammalia Caspases is Caspase-3 in terms of specificity and the roles of apoptosis. The
activation of Caspase-3 is a mark of appoptosis and its testing is used to study the most active hit 12b was
evaluated in the Caspase-3 cascade death pathway. Our findings of the investigation indicated the elevated
level of active Caspase-3 across 9 folds than reference drug, which is considered an apoptotic
marker(Table4).
2.2.2.2. Effect on mitochondrial apoptosis pathway proteins BAX and Bcl-2
BAX and Bcl-2 are the essential mitochondrial protein memberships of the Bcl-2 gene family. Apoptotic
pathway regulates the balance between the two signature proteins. Hence, BAX is pro-apoptotic protein,
which enhances the apoptosis.Several new BAX activators identified as targeted anticancer agents to hoping
to overcome the resistance of other chemotherapeuticagents. The most promising compound 12b was
assessed for its effect on BAX, and Bcl-2, as apoptosis key markers. The high BAX/Bcl-2 ratio indicates the
apoptosis susceptibility in the cell. Lower levels of that ratio can lead to resistance of apoptosis in
7
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humancancer cell.Therefore, the findings clarified the higher BAX/Bcl-2 ratios that regulating apoptotic
mechanism.(Table4).
Table 4:The effect of compound 12b on assessment of some apoptotic markers
Cpd.
No.
Caspase-3
pg/mL
BAX pg/mL
Bcl-2 pg/mL
475.62
51.27
391.56
46.85
1.687
6.165
12b
Cont.
2.2.3. Cell cycle analysis
The cell cycle analysis is used as a basis of the DNA content assessment for determining the cell proportion
in each step of the cell cycle. Incubation with compound 12b generated a shift in population from G0/G1
phase to G2/M phase and increase in apoptotic cells being represented by % of pre-G1population is
seen.Cell cycle arrest on cancer cells with MCF-7, the obtained results are represented in (Figure
2&3),revealing that,apoptosis induced by compound 12b at Pre-G1 phase, the percentage of death of the
cellat Pre-G1 phase is (19.25 %). Also, MCF-7 cells accumulated at G2/M phase, the percent is (37.82 %)
upon treating cells with compound12b. It is to be noted that this increment is accomponiedwitha decrease in
the percentage of S phase (Table 5). The findings from Pre-G1 and G2/M phase indicate that compound
12bis apoptoticinducer and can arrest the G2/M phase in the cell cycle.
Table 5: Effect of 12b on the cell cycle of MCF-7
Cpd.
No.
%G0-G1
%S
%G2/M %Pre-G1
Comment
PreG1apoptosis &
Cell growth arrest
atG2/M
36.51
53.71
25.67
37.67
37.82
8.62
19.25
2.15
12b/MCF-7
Cont. MCF-7
8
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Figure 2: Cell cycle analysis of MCF-7 treated with 12b
Figure 3:Cell cycle analysis of MCF-7 treated with DMSO (negative control)
2.2.5. Annexin V-FITC apoptosis determination
Annexin V assay labeled fluorescent used to detect phosphatidylserine (PS) [12] that was transfered during
early apoptosis from the inner to outer side of the cell membrane [13]. Furthermore, the florescent propidium
iodide (PI) material was needed to detect the percentage of cells that died from necrosis. For this reason,
Annex V / propidium iodide (PI) double staining assay was used to differentiate between apoptosis and
necrosis. The early apoptosis ratio (lower right cytogram quadrant) increases from 0.36 to 10.25 per cent
when MCF7-cells treated with the tested compound 12b for 24 hours; it causes increment in the latter
apoptosis ratio (higher right cytogram quadrants) ) from 1.37% to 6.46%. This means that compound 12b
was induced in comparison with control at approximately 19.25 (total) in both early and late apoptosis
(Table 6)(Figure 4). Compound 12b induced apoptosis rather than necrotic pathway by the programmed cell
death pathways.
9
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Table 6: Effect of 12b and DMSO(control) on the percentage of annexin V-FITC-positive staining in MCF-
7 cells
Apoptosis
Cpd.
No.
Total
Early
Late
Necrosis
19.25
2.15
10.25
0.36
6.46
1.37
2.34
0.42
12b/MCF-7
Cont. MCF-7
(A)
(B)
Figure 4:Effect of 12b(A) and DMSO(control) (B) on the percentage of annexin V-FITC-positive staining
in MCF-7
10
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2.3. In silico evaluation of physicochemical and ADME
Lipinski's rule were previously reported in literature to give an idea about the expected physicochemical properties
of the promising target compounds[14].
Computational study was performed for the derivatives which possess promising anticancer activity, 4b and
12a-c to assesstheir physicochemical and ADME properties using SwissADME[15]. With respects to
physicochemical properties (Table 7), the most active synthesized compounds havenoviolation for Lipinski’s
rule for oral drugs (M log P less than 2.40) so it is orally bioavailable compounds. All the topological polar
surface areas (TPSA) are ranging from (134.53- 137.66) Å 2. Also, absorption (% ABS) was determined by
applying the equation % ABS = 109 – (0.345 × TPSA)[16], found that the calculated % ABS of the four
compounds ranged between 61.51% and 62.59.the four compounds do not pass blood brain barrier.
Table 7: Physicochemical properties of the hit compounds 4b and12a-cbased on Lipinski’s rules, TPSA, %
ABS and number of rotatable bonds
Lipinski’s parameters
No of
rotatable
bonds
Cpd.
No.
M.Wt
H-
H-bond MLOGP Lipinski
TPSA
%
ABS
bond
donors
acceptor
s
violation
370.37
3
6
2.11
0
134.53 62.59
4
4b
370.37
384.40
449.27
2
3
2
6
6
6
1.71
1.94
2.32
0
0
0
137.66 61.51
134.53 62.59
137.66 61.51
3
4
3
12a
12b
12c
2.3.3. Docking study on EGFR
The minor binding energy comparable to the reference, Erlotinib (score-6.28 and-5.35 Kcal / mol) was
shown on the docking of 12b on an active site of EGFR. Owing to its two bulky aryl rings, the orientation of
12b pyrazolo triazoloquinoxaline was parallel to the hinge area. The hydrogen bond accepter (distance:
3,23Å) with the backbone of NH Met769 in the hinge region was nitrogen from position 2 of
triazoloquinoxaline of 12b. (Figure 5).
11
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Figure 5:The 12b expected binding mode docked in the active site of EGFR. A and B exertingthe 2D and 3D ligand-
receptor interactions (C atoms are colored green, N blue)
3. Conclusion
In this analysis, new hybrids of triazoloquinoxaline pyrazoles was produced and synthesized. Tests have
shown that four hits have triggered cytotoxic activity of HCT-116, HepG-2, and MCF-7 cell lines.However,
The most potent compounds 12a-c showed no cytotoxic effect toward the normal cellline WI-38.Two forms
of the inhibitory activity of EGFR kinase (normal and mutant) were tested for 12a-c, where 12b were found
to be strong inhibitory against the EGFR wild. In order to assess the pro-apoptotic ability of our compounds,
apoptosis studies for the 12b most active compound have been performed. The results showed that 12b
increased active Caspase-3 value and the BAX / Bcl-2 ratiowhen compared to control level. In addition, 12b
has been identified as arresting MCF7 cells at G2 / M and increasing the pre-G1 cell percentage to 8.95
times.
12
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4. Materials and Methods
4.1 Chemistry
Solvents and reagents were bought from specific commercial suppliers and used without further purification.
The yields reported are for the purified products. All reactions were regularly tested using a Thin Layer
Chromatography (TLC) on Merck Silica Gel 60 F254 (0.25 mm thick) and a UV lamp simulation took place.
Melting points measurement was done in the capillary tubes using Stuart SMP3 device. A Simadzu FT / IR
1650 (Perkin Elmer) spectrometer was used for the test of IR spectra (KBr). The Bruker Advance-400 (400
1
MHz for 1H and 100 MHz for 13C) was captured with H and 13C spectra in DMSO-d6. Chemical shifts (δ)
are seen in ppm relative to the TMS as a standard, or to the solvent used in the spectrum. A Shimadzu 2010
GS / MS-QP plus spectrometer with 70 eV was used to perform the mass spectrum.Anticancer research was
conducted on local strain found in the Regional Center for Genetic Engineering, Faculty of Science (Boys),
University of Al-Azhar and the findings were within ± 0.4 percent of the measured outcome.
Compounds4-(phenyldiazenyl)-1H-pyrazole-3,5-diamine derivatives[17], 2-cyanoacetohydrazide[18], 1,3-
diphenyl-1H-pyrazole-5-carbaldehydes[19], ethoxymethylenemalononitrile[20,21],bis(methylthio)-methy-
lenemalononitrile[22,21], 2-(phenyldiazenyl)malononitrileanalogues[23, 24] and 3-(2,4-dichlorophenyl)-1-
(aryl)prop-2-en-1-one derivatives[25, 26] were prepared according to reported procedures.
4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one(3)
Triazoloquinoxaline2 (2.05g, 0.01mol.) and 4-aminoantipyrine (2.03g, 0.2mL, 0.01 mol.) washeated under
reflux for 12 h in dioxane(20 mL)containing 3drops of TEA. The solid product was filtered, dried and
crystallized from ethanol to afford 3.
White powder, yield: (46.3%), m.p. 2230C;IR (KBr, cm-1): 3379 (NH); 3082 (C-H aromatic); 2985(C-H
aliphatic); 1660 (C=O); 1589 (C=N); 1539 (C=C);1H NMR (DMSO-d6, δ ppm): 2.25 (s, 3H, pyrazole-C5-
CH3); 3.16(s, 3H, pyrazole-N1-CH3); 7.36(t, 1H, J=8Hz,N-C6H5-C4-H); 7.38-7.7.50 (m, 4H, N-C6H5-C2,3,5,6
-
H); 7.51-7.60 (m, 3H, triazoloquinoxaline-C7,8,9-H); 8.23 (d, 1H, J=8Hz, triazoloquinoxaline-C6-H); 9.29 (s,
1H, NH, D2O exchangeable); 10.04 (s, 1H, quinoxaline-C1-H);13CNMR (DMSO-d6): 11.06 (CH3-C),
36.51(N-CH3-C), 108.32, 116.63, 122.79, 123.90, 124.56, 126.63, 127.03, 128.03, 129.59, 135.64, 136.79,
138.47, 138.72(Ar-C), 145.41, 153.98(C=N), 162.51(C=O);MS m/z (relative intensity %): 371 (M.+, 26.71);
262(65.97); 143(42.21); 75(62.15); 56.30 (100); Anal. form: C20H17N7O. Calcd.(%) C, 64.68; H, 4.61; N,
26.40; O, 4.31. Found (%): C,64.91; H, 4.85; N, 26.64.
General procedure for preparation of:
N5-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-4-(aryldiazenyl)-1H-pyrazole-3,5-diamine(4a-c)
Chlorotriazoloquinoxaline2 (2.05 g, 0.01 mol.) and 4-(aryldiazenyl)-1H-pyrazole-3,5-diamines were heated
under refluxin dioxane (15mL) containing 3 drops of TEA for 12 h.The solid product was filtered, dried and
crystallized from ethanol to afford the different 1H-pyrazole-3,5-diamine derivatives 4a-c.
N5-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-4-(phenyldiazenyl)-1H-pyrazole-3,5-diamine(4a)
Reddish brown powder, yield: (56%), m.p. ˃3000C.IR (KBr, cm-1): 3375, 3294 (NH2&NH); 3070 (C-H
aromatic); 2904 (C-H aliphatic); 1624 (C=N); 1508 (C=C); 1462 (N=N);1H NMR (DMSO-d6,δ
ppm):4.10(br.s, 2H, NH2, D2O exchangeable); 7.34 (t, 1H, J=7.3Hz, C6H5-C4-H) 7.47 (t, 2H, J=7.3Hz,C6H5-
C3,5-H); 7.60-7.78 (m, 2H, C6H5-C2,6-H ); 7.88 (d, 2H, J=7.7Hz, triazolquinoxaline- C9-H); 8.15-8.23 (m,
13
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2H, triazoloquinoxaline- C7,8-H); 8.42-8.55 (m, 1H, triazoloquinoxaline-C6-H); 8.86 (s, 2H, two NH, D2O
13
exchangeable); 10.38 (s, 1H, triazoloquinoxaline-C1-H); CNMR (DMSO-d6, δ ppm): 113.29, 116.82,
117.26, 120.00, 121.58, 123.67, 123.89, 128.57, 128.76, 128.85,129.17, 129.49, 133.82, 137.79, 138.52,
139.23(Ar-C), 141.69, 153.15(C=N); MS m/z (relative intensity %): 370 (M, 36.72); 367 (M-3, 34.10); 343
(100). Anal. form: C18H14N10. Calcd.(%) C, 58.37; H, 3.81; N, 37.82. Found (%): C,58.48; H, 4.39; N, 26.28.
N5-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-4-(p-tolyldiazenyl)-1H-pyrazole-3,5-diamine(4b)
Reddish brown powder, yield: (52%), m.p.˃3000C.IR (KBr, cm-1): 3375, 3290 (NH&NH2); 3074 (C-H
aromatic); 2978 (C-H aliphatic); 1620 (C=N); 1566 (C=C); 1462 (N=N);1H NMR (DMSO-d6,δ ppm): 2.37
(s, 3H, CH3); 6.31(s, 2H, NH2, D2O exchangeable); 7.28 (d, 2H,J=8.2Hz, 4-CH3-C6H4-C3,5-H); 7.66-7.71 (m,
2H, triazolquinoxaline-C7,8-H); 7.76 (d, 2H, J=8.2Hz, 4-CH3- C6H4-C2,6-H); 8.10-8.18 (m, 1H,
triazoloquinoxaline-C9-H); 8.37-8.46 (m, 1H, triazoloquinoxaline-C6-H); 8.59 (s, 2H,two NH, D2O
exchangeable); 10.21 (s, 1H, triazoloquinoxaline-C1-H); 13CNMR (DMSO-d6): 21.23 (CH3-C), 68.23, 73.15,
121.36, 123.08, 128.34, 128.73, 130.07, 138.32(Ar-C), 143.13, 151.32, 165.12(C=N); MSm/z (relative
intensity %): 384 (M, 12.45); (356, 56.69); (330, 75.06); (254, 85.83);(240, 76.85); (110, 85.02); 111 (100).
Anal. form: C19H16N10. Calcd.(%) C, 59.37; H, 4.20; N, 36.44. Found (%): C,59.53; H, 4.31; N, 36.28.
N5-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-4-((4-bromophenyl)diazenyl)-1H-pyrazole-3,5-diamine(4c)
Reddish brown powder, yield: (45%), m.p.˃3000C.IR (KBr, cm-1): 3390, 3290 (NH &NH2); 3070 (C-H
aromatic); 2970 (C-H aliphatic); 1624 (C=N); 1566 (C=C); 1462(N=N); 1064 (p-substituted-phenyl);1H
NMR (DMSO-d6,δ ppm): 7.66 (d, 2H, J=8Hz, 4-Br- C6H4-C2,6-H); 7.68-7.76 (m, 2H, triazoloquinoxaline-
C7,8-H); 7.84 (d, 1H, J=8Hz, 4-Br- C6H4-C3,5-H); 8.11 (d, 1H, J=8Hz, triazoloquinoxaline-C9-H); 8.44 (d,
1H, J=8Hz, triazoloquinoxaline-C6-H); 8.85 (br.s, 2H, twoNH, D2O exchangeable); 10.28 (s, 1H,
triazoloquinoxaline-C1-H);13CNMR (DMSO-d6): 74.51, 123.54, 128.60, 129.62, 132.36(Ar-C), 141.11,
151.91, 162.25(C=N); MS m/z (relative intensity %): 449 (M, 33.45);275.76(66.13); 224.12 (88.25); 205
(100); (114.02, 78.81). Anal. form: C18H13BrN10. Calcd.(%) C, 48.12; H, 2.92; Br, 17.79; N, 31.18. Found
(%): C,48.31; H, 3.16; N; 31.40.
N'-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-2-cyanoacetohydrazide(5)
Equivalnts of triazoloquinoxaline 2 (2.05 g, 0.01mol.)and 2-cyanoacetohydrazide (0.99gm, 0.01 mol) was
heated under reflux in DMF (10 mL) for 12 h. The reaction mixture was trated with ice cold water (20 mL),
and the solid mass was filtered, dried and crystallized from dioxane to afford the target compound5.
Light brown powder, yield( 84%), m.p. ˃3000C.IR (KBr, cm-1): 3323, 3213 (NH2); 3105 (C-H aromatic);
2908, 2839(C-H aliphatic); 2264 (C
N) 1740 (C=O); 1620 (C=N); 1550 (C=C);1H NMR (DMSO-d6,δ
ppm): 3.91 (s, 2H, CH2CN); 7.32-7.64(m, 2H, triazoloquinoxaline-C7-H& NH, D2O exchangeable); 7.68-
7.73 (m, 1H,triazolo quinoxaline-C8-H); 8.31-8.42 (m, 1H, triazoloquinoxaline-C9-H); 8.41-8.45 (m, 1H,
triazoloquinoxaline-C6-H),10.02 (s, 1H, triazoloquinoxaline-C1-H); 10.20 (s, 1H, NH, D2O exchangeable);
13CNMR (DMSO-d6):24.64(CH2-C),116.23(C
N), 116.80, 118.33, 128.24, 128.42, 138.50(Ar-C), 138.88,
139.53(C=N), 153.12(C=O); MS m/z (relative intensity %): 267 (M, 38); 264 (74.14); 215 (86.23); 121
(100). Anal. form: C12H9N7O.Calcd(%):C, 53.93; H, 3.39; N, 36.69; O, 5.99. Found (%): C,54.14; H, 3.54;
N, 36.51.
N'-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-2-cyano-3-(1,3-diphenyl-1H-pyrazol-5-yl)acrylohydrazide(6)
14
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Cyanoacetohydrazide analogue 5(2.67g, 0.01 mol.) and1,3-diphenyl-1H-pyrazole-5-carbaldehyde (2.48g,
0.01mol.) was heated under reflux in DMF (10mL) as solvent and piperidine (3drops) as basic catalyst for 18
hours. The reaction mixture was concentrated and treated with ice cold water and then the obtained
precipitate was filtered, dried, and crystallized from DMF to afford the acrylohydrazide 6.
Yellow powder, yield(57%) ,m.p.(above 3000C).IR (KBr, cm-1): 3394 (NH) ; 3062 (C-H aromatic); 2947 (C-
1
H aliphatic); 2214 (C N); 1700 (C=O); 1631 (C=N); 1597 (C=C); H NMR (DMSO-d6, δ ppm): 7.16-8.18
(m, 12H, C=CH, pyrazole- C4-H, pyrazole-N1-C6H5-C2,3,4,5,6-H, pyrazole-C3-C6H5-C3,4,5–H&
triazoloquinoxaline-C7,8,9-H,); 8.38-8.53 (m, 3H, triazoloquinoxaline-C6-H, pyrazoleC3-C6H5-C2,6 -H ); 9.61
(s, 1H, NH, D2O exchangeable); 10.09 (s, 1H, triazoloquinoxaline-C1-H);. 10.11(s, 1H, NH, D2O
exchangeable). MS m/z (relative intensity %): 498(M+1, 23.83); 497 (M, 26.83); 62 (100). Anal. form:
C28H19N9O. Calcd(%) C, 67.60; H, 3.85; N, 25.34; O, 3.22.Found (%): C,67.49; H, 4.02; N, 25.57.
N'-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-N-acetylacetohydrazide(8)
An equimolar mixture of 4-Hydrazino triazoloquinoxaline 7 (2.05 g, 0.01mol.) and1-phenyl-3-aryl-1H-
pyrazole-4-carbaldehydeanalogueswere heated under reflux in ethanol/ acetic acid (15mL/10 drops) for
6hours. The
solid product was filtered, dried and crystallized from ethanol to afford N-
acetylacetohydrazide8.
Yellow powder, yield (55%), m.p.˃3000C.IR (KBr, cm-1):3286 (NH); 3059 (C-H aromatic); 2947 (C-H
aliphatic); 1716 (C=O); 1643 (C=N); 1581 (C=C). 1H NMR (DMSO-d6,δ ppm): 1.91 (s,3H,COCH3); 3.06 (s,
3H, COCH3); 7.71(t, 2H, triazoloquinoxaline-C7,8-H,) ; 8.31 (d, 1H, J=8Hz, triazoloquinoxaline-C9-H); 8.46
(d, 1H, J=8Hz, triazoloquinoxaline-C6-H);10.01 (s, 1H, triazoloquinoxaline-C1-H); 11.99 (s, 1H, NH, D2O
exchangeable). 13CNMR (DMSO-d6):15.27, 21.53, 118.41, 118.45, 123.05, 124.32, 128.05, 128.43, 138.67,
149.23, 172.58.MS m/z (relative intensity %): 284 (M, 25.91); 283 (M-1, 44.58); 229(54); 169 (100);
69(98). Anal. form: C13H12N6O2. Calcd. (%) C, 54.93; H, 4.25; N, 29.56; O, 11.26.Found (%): C,55.19; H,
4.38; N, 29.78.
General procedures for synthesis of (9a,b):
Compound7 (2.05g, .0.01 mol.) and 1,3-diaryl-1H-pyrazole-4-carbaldehyde derivativeswasheated under
reflux for 18 hours in dioxane / DMF (10mL/5mL) containing catalytic amount of piperidinefor 12 h. The
obtained solid was filtered, washed with ethanol, dried and crystallized from dioxane to yield the
triazoloquinoxalines 9a, b.
4-(2-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-[1,2,4]triazolo-[4,3-a]quinoxaline (9a)
Yellow powder, yield: (35%), m.p.˃3000C).IR (KBr, cm-1): 3425, (NH); 3055 (C-H aromatic); 2947 (C-H
1
aliphatic); 1631 (C=N); 1543 (C=C). H NMR (DMSO-d6,δ ppm): 7.22-7.37 (m, 2H, pyrazole-C3-phenyl-
C4-H & pyrazole-N-C6H5-C4-H); 7.39-7.46 (m, 4H, pyrazole- C3-C6H5- C3,5-H & pyrazole-N-C6H5-C3,5-H);
7.50-7.69 (m, 2H pyrazole-N-C6H5-C2 ,6-H); 7.70-8.25 (m, 3H, triazoloquinoxaline-C7,8,9-H); 8.67-8.69 (m,
2H, pyrazole- C3-C6H5-C2,6-H); 8.86 (d, 1H, J=12Hz, triazoloquinoxaline-C6-H); 9.69 (s, 1H, N=CH); 10.08
(s, 1H, triazoloquinoxaline-C1-H); 10.62 (s,1H, NH, D2O exchangeable). 13CNMR (DMSO-d6): 22.39,
117.04, 118.36, 119.47, 122.95, 127.77, 128.46, 128.93, 129.82, 129.95, 130.14, 130.37, 138.89, 139.57,
146.43, 156.23. MS m/z (relative intensity %): 432 (M+2, 22.48); 430 (M, 34.48), 389 (61.42); 227(100);
189(76). Anal. form: C25H18N8. Calcd. (%) C, 69.75; H, 4.21; N, 26.03Found (%): C,69.61; H, 4.39; N,
26.28.
4-(2-((1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)-[1,2,4]triazolo[4,3-a]quinoxaline
(9b)
15
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Orange yellow powder, yield: (55%), m.p.˃3000C.IR (KBr, cm-1): 3410, (NH); 3082 (C-H aromatic); 2943
(C-H aliphatic); 1631 (C=N); 1543 (C=C);1H NMR (DMSO-d6,δ ppm): 2.42 (s, 3H, CH3); 7.24 (t, 1H,J=8Hz
,N-C6H5-C4-H); 7.35-7.43 (m, 4H, 4-CH3-C6H4-C3,5-H&N-C6H5-C3,5-H); 7.54-7.68 (m, 2H,
triazoloquinoxaline-C7,8-H); 7.71-7.75 (m, 1H,N-C6H5-C2,6-H); 7.86 (d, 1H, J=8Hz, 4-CH3-C6H4-C2,6-H)
7.94 (s, 1H, pyrazole-C5-H); 8.11 (d, 1H, J=8Hz triazoloquinoxaline-C9-H); 8.43-8.47 (m, 1H,
triazoloquinoxaline-C6-H); 8.42-8.48 (m, 1H, triazoloquinoxaline-C6-H); 9.67 (s, 1H, N=CH); 10.05 (s, 1H,
triazoloquinoxaline-C1-H); 11.38 (s,1H, NH, D2O exchangeable); 13CNMR (DMSO-d6): 66.81(CH3), 116.92,
119.29, 119.54, 119.77, 127.76, 128.42, 128.77, 128.89, 129.12, 129.23, 129.40, 129.53, 130.16, 130.31,
134.25, 138.72, 139.12, 139.45, 139.54(Ar-C), 142.06, 149.26, 152.69(C=N).MS m/z (relative intensity %):
445 (M, 53.94); 444 (M-1, 73.82); 257(77); 209(93); 96 (100). Anal. form: C26H20N8. Calcd. (%) C, 70.26;
H, 4.54; N, 25.21. Found (%): C,69.98; H, 4.67; N, 25.47.
1-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-3-amino-1H-pyrazole-4-carbonitrile (10)
Hydrazine analogue7 (2.05g, 0.01 mol) and 2-(ethoxymethylene)malononitrile (1.22g, 0.01mol.) was heated
under reflux in dioxane/ DMF mixture ( 10/ 5mL), containing few drops of TEAfor 13 h. The reaction
mixture was filtered, dried and crystallized from ethanol to afford the carbonitrile 10.
Brown powder, yield (43%), m.p.˃ 3000CIR (KBr, cm-1): 3305, 3170 (NH2); 3080 (C-H aromatic); 2214 (C
1
N); 1635 (C=N); 1539 (C=C). H NMR (DMSO-d6,δ ppm): 7.06 (s, 2H, NH2, D2O exchangeable); 7.19
(s, 1H, pyrazole-C5-H); 7.69-7.74 (m, 2H, triazoloquinoxaline-C7,8-H); 8.41-8.48(m, 1H,
triazoloquinoxaline-C6,9-H); 10.04 (s, 1H, triazoloquinoxaline-C1-H).13C-NMR (DMSO-d6): 82.01,
118.40(C
N), 128.52, 138.90(Ar-C), 147.31, 157.14(C=N).MS m/z (relative intensity %): 276 (M,
52.01); 275 (M-1, 52.27); 200(44.50); 102 (100). Anal. form: C13H8N8. Calcd.(%) C, 56.52; H, 2.92; N,
40.56. Found (%): C,56.76; H, 3.18; N, 40.34.
1-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-3-amino-5-(methylthio)-4,5-dihydro-1H-pyrazole-4-
carbonitrile(11)
An equimolar mixture of 7 (2.05 g, 0.01 mol) and 2-(bis(methylthio)methylene) malononitrile (1.70g,
0.01mol.) was heated under reflux in dioxane/ DMF mixture (10/ 5mL) containing few drops of TEAfor 10
h. The reaction mixture was filtered, dried and crystallized from ethanol to yield the title compound11.
Orange powder, yield (60.6%), m.p.˃ 3000C.IR (KBr, cm-1): 3394, 3294 (NH2); 3116 (C-H aromatic);
2924(C-H aliphatic); 2210 (C
N); 1631 (C=N); 1566 (C=C);1H NMR (DMSO-d6,δ ppm): 2.64 (s, 3H,
pyrazole-C5-S-CH3); 7.73 (t, 1H,J=8Hz, triazoloquinoxaline-C7-H); 7.81 (t, 1H, J=8Hz, triazoloquinoxaline
-C8-H); 8.19 (s, 2H, NH2, D2O exchangeable); 8.25 (d, 1H, J=8Hz, triazoloquinoxaline-C9-H); 8.46 (d, 1H,
13
J=8Hz, triazoloquinoxaline -C6-H),10.25 (s, 1H, triazoloquinoxaline-C1-H); CNMR (DMSO-d6): 13.46(S-
CH3), 72.88(pyrazole-C4), 114.04(C N), 116.81, 124.87, 128.47, 129.57, 129.87, 133.50, 138.58,
139.01(Ar-C), 141.87, 151.89, 155.90(C=N).MS m/z (relative intensity %): 323 (M+1, 17.48); 322 (M,
60.01); 313.25 (100); (254, 77); (212, 78); (66, 69).Anal. form: C14H12N8S. Calcd. (%):C, 51.84; H, 3.73; N,
34.55; S, 9.89. Found (%): C,52.34; H, 3.29; N, 34.53.
General procedure for synthesis of (12a-c):
Hydrazino derivative7 (2.05g, .0.01mol.) and different malononitrile derivatives (0.01mol) was heated under
reflux in DMF (10 mL) containing catalytic amount of TEA for 20 hours. The reaction mixture was then
treated with ice cold water; the precipitated solid was filtered, dried, and crystallized from ethanol to afford
4-(4-substituted-phenyldiazenyl)-1H-pyrazole-3,5-diamine12a-c.
1-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-4-(4-phenyldiazenyl)-1H-pyrazole-3,5-diamine (12a)
16
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Reddish brown powder, yield: (47%), m.p.˃300 C. R ( Br, cm-1): 3394, 3275 (NH2); 3097, 3062 (C-H
aromatic); 1616 (C=N); 1504 (C=C); 1477(|N=N);1H NMR (DMSO-d6,δ ppm): 6.96 (s, 2H, NH2, D2O
exchangeable); 7.31 (t, 1H, J=7.2Hz, C6H5-C4-H); 7.37 (t, 2H, C6H5-C3-H); 7.43-7.53 (m, 2H, , C6H5-C2, 6-
H); 7.67-7.75 (m, 2H, triazoloquinoxaline-C7,8 -H); 8.01 (d, 1H, J=7.6Hz, triazoloquinoxaline-C9-H); 8.42-
8.47 (m, 1H, triazoloquinoxaline-C6-H); 9.40 (s, 2H, NH2, D2O exchangeable); 10.03 (s, 1H,
triazoloquinoxaline-C1-H); 13CNMR (DMSO- d6): 108.10, 118.31, 121.26, 122.16, 122.76, 128.45, 128.65,
129.60, 129.65, 138.86, 139.46 (Ar-C), 152.64, 152.71, 153.56(C=N). MS m/z (relative intensity %): 372
(M+2, 17.78); 370 (M, 17.00) ; (335, 100); (314, 78); (304, 84); (104, 47); Anal. form: C18H14N10. Calcd.
(%) C, 58.37; H, 3.81; N, 37.82. Found (%): C,58.59; H, 3.94; N, 37.59.
1-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-4-(p-tolyldiazenyl)-1H-pyrazole-3,5-diamine(12b)
Reddish brown powder, yield: (44%), m.p.˃300 C. R ( Br, cm-1): 3390, 3271 (NH2); 3101 (C-H aromatic);
1
2912, 2839 (C-H aliphatic); 1604(C=N); 1504 (C=C);1473 (N=N). H NMR (DMSO- d6,δ ppm): 2.36 (s,
3H, CH3); 6.91 (s, 2H, NH2, D2O exchangeable); 7.29 (d, 2H, J=8.4Hz, 4-CH3- C6H5-C3,5-H); 7.59 (d, 1H,
J=8Hz, 4-CH3- C6H5-C2-H); 7.70-7.72 (m, 3H, triazoloquinoxaline-C7,8,9-H); 7.91 (d, 1H, J=8Hz, 4-CH3-
C6H5-C6-H); 7.86 (d, 1H, J=8Hz, 4- CH3- C6H5-C5-H); 8.44 (d, 1H, J=8Hz, triazoloquinoxaline-C6-H); 9.30
(s, 2H, NH2, D2O exchangeable); 10.03 (s, 1H, triazoloquinoxaline-C1-H);13CNMR (DMSO- d6): 21.32
(CH3-C), 118.29, 121.16, 122.08, 122.76, 128.62, 130.09, 130.14, 138.85, 139.30(Ar-C), 139.45,
150.69(C=N).MS m/z (relative intensity %): 384 (M ,76.50); 382 (M-2, 62.13) ; 284(66); 224(71); 174
(78.50). Anal. form: C18H14N10. Calcd.(%) C, 58.37; H, 3.81; N, 37.82. Found (%):C, 59.54; H, 4.36; N,
36.21.
1-([1,2,4]Triazolo[4,3-a]quinoxalin-4-yl)-4-((4-bromophenyl)diazenyl)-1H-pyrazole-3,5-diamine(12c)
Reddish brown powder, yield: (44%), m.p.˃300 C. R ( Br, cm-1): 3379, 3298 (NH2); 3070 (C-H aromatic);
1
1620 (C=N); 1566 (C=C); 1473(N=N). H NMR (DMSO-d6, δ ppm): 7.00 (s,2H, NH2, D2O exchangeable);
7.53 (d, 2H, J=8Hz, 4-Br-C6H4-C2,6-H); 7.66-7.70 (m, 2H, triazoloquinoxaline-C7,8-H); 7.76 (d, 1H, J=8Hz,
triazoloquinoxaline-C9-H); 7.82 (d, 1H, J=8Hz, 4-Br-C6H4-C3-H); 7.85 (s,2H, NH2, D2O exchangeable);
7.86 (d, 1H, J=8Hz, 4-Br-C6H4-C5-H); 8.40 (d, 1H, J=8Hz, triazoloquinoxaline-C6-H); 10.21 (s, 1H,
triazoloquinoxaline -C1-H).13C-NMR (DMSO- d6): 116.70, 118.05, 120.83, 123.38, 124.12, 124.30, 128.37,
132.33, 132.70(Ar-C), 133.87, 142.91, 152.69(C=N).MS m/z (relative intensity %): 449 (M,34.50);
421(38.59), 315(68); 81(73); 175 (100). Anal. form: C18H13BrN10. Calcd.(%) C, 48.12; H, 2.92; Br, 17.79;
N, 31.18. Found (%): C,48.31; H, 3.20; N, 31.37.
General procedure for synthesis of (13a,b)
Equimolar amounts of 7(2.05g, .0.01 mol.) andproper chalcone derivative (0.01 mol) were dissolved in
dioxane/ DMF mixture (10mL/5mL) containing 3drops of TEA. The reaction mixture was heated at reflux
for 16 h, the solid product was then filtered, dried and crystallized from dioxane to afford derivatives 13a,b.
4-(5-(2,4-Dichlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-1-yl)-[1,2,4]triazolo[4,3-
a]quinoxaline(13a)
Orange powder. Yield: (35%), m.p.˃300 C. R ( Br, cm-1): 3086, 3062 (C-H aromatic); 2931, 2897 (C-H
aliphatic); 1620 (C=N); 1504 (C=C); 1033(p-substituted phenyl);1H NMR (DMSO-d6,δ ppm): 3.86 (s, 3H,
OCH3); 7.09 (d, 2H, J=8Hz, 4-OCH3-C6H4-C3,5-H); 7.17 (s, 1H, pyrazole-C4-H); 7.36-7.41 (m, 1H,
triazoloquinoxaline-C7-H); 7.54, 7.56 (dd, 1H, J=8, 4Hz, triazoloquinoxaline-C8-H); 7.66-7.72 (m, 1H,
triazoloquinoxaline-C9-H); 7.73 (d, 2H, J=8Hz, 2,4-(Cl)2-C6H3-C5-H ); 7.76 (d, 2H,J=8Hz, 4-OCH3- C6H4-
C2,6-H); 7.82 (d, 2H, J=8Hz, 4-OCH3- C6H4-C5-H); 7.93 (d, 1H, J=8Hz, 2,4-(Cl)2- C6H3-C6-H); 8.00 (s, 1H,
17
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2,4-(Cl)2-C6H3-C3-H ); 8.08 (d, 1H, J=8Hz, triazoloquinoxaline-C6-H); 8.46; 9.38 (s, 1H, triazoloquinoxaline
13
-C1-H); CNMR (DMSO-d6): 55.78(OCH3-C), 114.18, 118.41, 118.46, 123.07, 128.04, 128.40, 128.60,
129.12, 129.75, 131.46, 134.13(Ar-C), 138.68, 140.81, 160.95(C=N);MS m/z (relative intensity %): 487 (M,
18.80); (416, 81.17); (128, 80.09); 118 (100). Anal. form: C25H16Cl2N6O. Calcd.(%) C, 61.61; H, 3.31; Cl,
14.55; N, 17.24; O, 3.28. Found (%): C,61.50; H, 3.48; N, 17.51.
4-(5-(2,4-Dichlorophenyl)-3-(thiophen-2-yl)-1H-pyrazol-1-yl)-[1,2,4]triazolo[4,3-a]quinoxaline(13b)
Brick red powder, yield: (44%), m.p˃3000C. IR (KBr, cm-1): 3105 (C-H aromatic); 1647 (C=N); 1535
(C=C); 1327, 1049(C-S-C);1H NMR (DMSO-d6,δ ppm): 7.12-7.22 (m, 1H, thiophene- C4-H); 7.30-7.40 (m,
1H, thiophene-C3-H ); 7.42-7.50(m, 1H, thiophene-C5-H);7,56 (d, 1H, J=7.9Hz, 2,4-(Cl)2-C6H3-C5-H) ;
7.62-7.75 (m, 3H, triazoloquinoxaline-C7,8-H& pyrazole-C4-H); 7.91 (s, 1H, 2,4-(Cl)2-C6H3-C3-H ); 8.10 (d,
J=7.9Hz, 1H, 2,4-(Cl)2-C6H3-C6-H ); 8.30-8.41(m, 2H, triazoloquinoxaline-C6,9-H); 9.92 (s, 1H,
triazoloquinoxaline-C1-H); 13CNMR (DMSO-d6): 116.62, 118.35, 122.54, 122.93, 123.66, 126.07, 127.98,
128.47, 138.38(Ar-C), 138.65, 138.89, 139.55, 147.65 (C=N);MS m/z (relative intensity %): 463 (M, 48.69);
(415, 64.86); (294, 51.46); 92 (100). Anal. form: C22H12Cl2N6S. Calcd.(%) C, 57.03; H, 2.61; Cl, 15.30; N,
18.14; S, 6.92. Found (%): C,57.31; H, 2.87; N, 18.28.
4.2. Biological evaluation
4.2.1. Antiproliferative screening
The newly synthesized compounds underwent cytotoxicity in this study. All the chemicals used in this
analysis are highly analytical in nature. The American Type Culture Collection (ATCC) is the source of the
selected cancer cell lines; hepatocellular carcinoma cell lines (HepG-2), human breast adenocarcinoma cell
lines (MCF-7), human colon carcinoma cell lines (HCT-116) and human diploid lung fibroblasts WI-38; which
were obtained frozen in liquid nitrogen (-180 ° C) and maintained at the National Type Culture Collection
(NTCC). Doxorubicin has been used as a standard reference drug, for comparison.
The cytotoxicity on human cancer cell line (HepG-2), MCF-7, HCT-116 and the normal cell lineWI-38 was
assessed in vitro using Sulforhodamine-B stains (SRB) assay following the Skehan process. [27] Cells were
cultured for 24 hours before treatment with the compounds in 96 multi well plates allowing the cells to
fasten the plate wall. To the cell monolayer is applied the following concentrations of the compound under
examination (0, 6.25, 12.5, 25, 50 and 100 μg / mL)
Triplicate wells have been prepared for any single dose. Monolayer cells were cultured at 37oC for 48 hours
with the compounds and 5 percent CO2 in the atmosphere. After couple of days the cell was fixed, rinsed and
stained with Sulforhodamine B stain. To get rid of the extra stain, it was washed with acetic acid, and stain
attached with Tris EDTA buffer was retrieved. In an ELISA reader, color intensity was assayed. The
relationship between surviving part of cells and concentration of drugs was plotted and Sigmaplot software
calculated IC50 (the concentration needed for 50 per cent cell viability inhibition) for every compound.
4.2.2.EGFRwt-TK assay
Using the EnzyChromTM Kinase Assay Kit (EKIN-400) Kinase Reaction [28], the inhibiton of compounds
12a-c on EGFRwt-TK activities was determined. Users can offer their own enzyme, ultra-pure ATP (e.g.
Sigma # A7699), and substrates. Place a 20 μL reaction mixture containing the kinase, ATP, and substrate in
the assay buffer given (pH 7.0) or any appropriate kinase buffer. Perform a Blank Control containing ATP
and substrate, but no enzyme. Incubate for the targeted time period e.g. 30 min at desired temperature.
Principles: Prepare 900 μL 10 μM ADP Premix by making a mixture of the standard 3 μL 3 mM and the
18
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distilled water 897 μL. Dilute so as to be below normal. Transfere 20 μL standards to separate ADP
Detection Plate wells. Prepare adequateworking reagent by combining 25 μL Reagent A and 25 μL Reagent
B for every well. Add 40 μL Working Reagent well to every assay. Tap mixing pad. Incubate these pad for
10 min at room temperature. the 530 nm platewas readed, and the activity of kinasewas measured. Dilute
standard according to the following procedure. Add 20 µL standards inside separate wells of the plate.ADP
Determination: Prepare adequateworking reagent for every well by blending 25 µL Reagent A and 25 µL
Reagent B. Add 40 µL Working Reagent to each assay well. Tap a plate to insure mixing. Incubate at room
temperature for 10 min. Read plat at 530nm and the kinase activity was calculated.
4.2.3.EGFRL858R-TK assay
The assay EGFRL858R-TK was done in the kinase reaction buffer (50 mM HEPES, 10 mM MgCl2, 2 mM
DTT, 1 mM EGTA, and 0.01 percent Tween 20). EGFRL858R-TK (the final concentration is 0.09 ng / mL)
and various concentrations of 12a-c or reference drug compounds (final concentration which is 2000e0.488
nM) were placedin each well and incubated for two hours, then pre-mixed ATP (final concentration: 5 mM)
and ULight-poly GT (final concentration was 0.1 mM) were placed inside each well. After 120 minutes of
incubation at room temperature (r.t.), EDTA was supplied and incubated for another five minutes with a
final concentration of 10 mM.
4.2.4. Cell cycle analysis
MCF-7 cells were seeded at a concentration of1x105 cells per well in a 6-well plate, after which they
incubated for one day. The cells were subjected toa vehicle (0.1 percent DMSO) or 2.04 μM of 12b
compound for 24 h. After this, cells were collected and fixed for 12 hours using ice-cold 70 percent ethanol
at 4C. Ethanol was eleminated and the cells were rinsed with cold PBS followed by incubation in 0.5 mL
of PBS which contain 1 mg / mL Rnase for 30 min at 37C auxiliary cells. The cells were subjected to stain
in the dark using propidium iodide for 30 min. Thereafter,Flow cytometer was used to measure the contents
of DNA[29].
4.2.5. Apoptosis detection studies
4.2.5.1. Annexin V-FITC assay
MCF-7 cells were cultured in a 6-well plate (1 X105 cell / well), incubated for one day (24 hours), then it
was subjected to a vehicle (0.1% DMSO) or 2.04 μM 12b compoundfor 24hours.Upon adding annexin V-
FITC and PI to the binding buffer (10 mM of HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at pH= 7.4), the
cells were then collected and washed using PBS then it were stained for 15 min at room temperature in the
dark depending on the flow cytometer apparatus [30].
4.2.5.2. Determination of the active Caspase-3
Quantikine-Human active Caspase-3 Immunoassay (R&D Systems, Inc. Minneapolis, USA)was applied to
detect the active Caspase-3 level. The cells were washed with PBS, collected and lysed through the addition
of the protease-inhibiting extraction buffer (1 mL per 1 x 107 cells), then the lysate was diluted
instantelybefore the study. Upon using a microplate reader set at 450 nm, the optical density of each well
was measured within 30 min when the assay has finished.
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4.2.5.3. Determination of the gene expression of some apoptosis keymarkers (BAX and Bcl-2)
Cells were obtained from the American Type Culture Collection and the cells were cultured in RPMI 1640
containing 10 percent bovine fetal serum at 37 C inducted with BAX or Bcl-2 validated compound and
lysed using the Cell Extraction Buffer. Standard Diluent Buffer which was used to dilute this lysate along
the assay and measured in comparable to human active BAX or Bcl-2 content. Cells are plated at a density of
1.2 – 1.8x 10,000 cells / well in a volume of 100 μL full growth medium plus 100 μL of the compound tested
per well in a 96-well plate for 24 hours before measurement for human active BAX or Bcl-2.
4.3. In silico physicochemical and ADME propertiesstudy
Using Chemdraw 12.0 the chemical structures were translated to SMILES databank. Then, owing to predict
the physicochemical descriptors, lipophilicity, pharmacokinetics characters, ADME parameters, and
medicinal chemistry friendliness, these SMILES were inserted in SwissADME website.
4.5.Molecular modeling study
Molecular Operating Environment (MOE) 2015.10 was utilized to perform all the molecular modeling
calculations and docking simulation studies. Protein Data Bank (PDB) was utilized to getEGFR (PDB code:
1M17) 3D X-ray structures.
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