Novel 2,6-disubstituted pyridine hydrazones: Synthesis, anticancer activity, docking studies and effects on caspase-3-mediated apoptosis

Article abstract of DOI:10.1016/j.molstruc.2020.128962

Novel pyridine-based dihydrazones (3a-l) were synthesized by the condensation of appropriate aldehydes and pyridine-2,6-dicarbohydrazide (2) which was obtained by the treatment of dimethyl pyridine-2,6-dicarboxylate (1) with hydrazine hydrate. Structures of all the synthesized compounds were supported by their FTIR, ¹H NMR, ¹³C NMR and microanalytical data. The compounds were screened primarily for their antibacterial activities as well as anticancer activities. None of the synthesized compounds had important antibacterial activity. Among the compounds which were tested against human colon cancer cell line (HT-29), compounds 3f and 3k showed significant activity (IC₅₀=6.78 μM for compound 3f, IC₅₀=8.88 μM for compound 3k). In addition, compound 3g exhibited promising activity against Ishikawa human endometrial cancer cell line (ISH) with an IC₅₀ value of 8.26 μM. At 10 μM, compounds 3f, 3k and 3g caused morphological changes of HT-29 and ISH cells and caspase-3 activation. In addition, these compounds were evaluated against NIH 3T3 mouse embriyonic fibroblast cell line and all synthesized compounds (3a-l) were found to be less toxic than paclitaxel. Moreover, possible inhibition mechanism of compound 3g was evaluated in silico against BRAF kinase enzyme.

Full text of DOI:10.1016/j.molstruc.2020.128962

Journal Pre-proof
Novel 2,6-disubstituted pyridine hydrazones: Synthesis, anticancer
activity, docking studies and effects on caspase-3-mediated
apoptosis
Sevil S¸ enkardes¸ Project administration; Conceptualization; Methodology; Investigation; Formal analysis; Writing-or
Aslı Tu¨re Software; Visualization; Validation; Writing-original draft; Writing-review&editing ,
Sedanur Ekrek Funding acquisition; Investigation ,
˘
Asım Tugrul Durak Funding acquisition; Investigation ,
Mu¨ru¨vvet Abbak Investigation; Methodology; Validation; Visualization ,
¨
Ozge C¸ evik Investigation; Methodology; Validation; Visualization; Writing-original draft; Supervision ,
Banu Kas¸katepe Validation; Investigation;Writing - original draft ,
ú
Ilkay Ku¨c¸u¨kgu¨zel Resources; Supervision; Validation; Writing-original draft ,
S¸ Gu¨niz Ku¨c¸u¨kgu¨zel Resources; Supervision; Validation; Writing - original draft
PII:
S0022-2860(20)31286-2
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128962
MOLSTR 128962
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
30 September 2019
21 July 2020
22 July 2020
Please cite this article as: Sevil S¸ enkardes¸ Project administration; Conceptualization; Methodology; Investigation; F
Aslı Tu¨re Software; Visualization; Validation; Writing-original draft; Writing-review&editing ,
˘
Sedanur Ekrek Funding acquisition; Investigation , Asım Tugrul Durak Funding acquisition; Investigation ,
¨
Mu¨ru¨vvet Abbak Investigation; Methodology; Validation; Visualization , Ozge C¸ evik Investigation; Methodology; Va
Banu Kas¸katepe Validation; Investigation;Writing - original draft , Ilkay Ku¨c¸u¨kgu¨zel Resources; Supervision; Valida
S¸ Gu¨niz Ku¨c¸u¨kgu¨zel Resources; Supervision; Validation; Writing - original draft , Novel 2,6-
ú
disubstituted pyridine hydrazones: Synthesis, anticancer activity, docking studies and ef-
fects on caspase-3-mediated apoptosis, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.128962
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Highlights :
- New 2,6-disubstituted pyridine hydrazone derivatives were synthesized and evaluated for anticancer
activity.
- 3f and 3k showed remarkable cytotoxic activity against HT-29 with IC50 value of 6.78 and 8.88 µM,
respectively.
- Also, 3g showed the best cytotoxic activity against ISH with IC50 value of 8.26 µM.
- 3f, 3g and 3k caused morphological changes of tumor cells and induced apoptosis
- Molecular docking was performed into BRAF kinase binding site

Novel 2,6-disubstituted pyridine hydrazones: Synthesis, anticancer activity, docking
studies and effects on caspase-3-mediated apoptosis
Sevil Şenkardeş1*, Aslı Türe1, Sedanur Ekrek2, Asım Tuğrul Durak2, Mürüvvet Abbak3,
Özge Çevik4, Banu Kaşkatepe5, İlkay Küçükgüzel1, Ş. Güniz Küçükgüzel1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, İstanbul, Turkey;
2Faculty of Pharmacy, Marmara University, İstanbul, Turkey;
3Scientific Technology Research and Application Centre, Adnan Menderes University, Aydın, Turkey;
4Department of Biochemistry, School of Medicine, Adnan Menderes University, Aydın, Turkey.
5Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Ankara University, Ankara, Turkey
*Correspondence: Sevil Şenkardeş
Adress: Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Haydarpaşa 34668 Istanbul, Turkey
Tel: +902164142962
Fax: +902163452952
e-mail: sevil.aydin@marmara.edu.tr
Highlights :
- New 2,6-disubstituted pyridine hydrazone derivatives were synthesized and evaluated for anticancer
activity.
- 3f and 3k showed remarkable cytotoxic activity against HT-29 with IC50 value of 6.78 and 8.88 µM,
respectively.
- Also, 3g showed the best cytotoxic activity against ISH with IC50 value of 8.26 µM.
- 3f, 3g and 3k caused morphological changes of tumor cells and induced apoptosis
- Molecular docking was performed into BRAF kinase binding site
Abstract
Novel pyridine-based dihydrazones (3a-l) were synthesized by the condensation of appropriate
aldehydes and pyridine-2,6-dicarbohydrazide (2) which was obtained by the treatment of dimethyl
pyridine-2,6-dicarboxylate (1) with hydrazine hydrate. Structures of all the synthesized compounds
1
13
were supported by their FTIR, H-NMR, C-NMR and microanalytical data. The compounds were
screened primarily for their antibacterial activities as well as anticancer activities. None of the
synthesized compounds had important antibacterial activity. Among the compounds which were tested
against human colon cancer cell line (HT-29), compounds 3f and 3k showed significant activity
(IC50=6.78 M for compound 3f, IC50=8.88 M for compound 3k). In addition, compound 3g

exhibited promising activity against Ishikawa human endometrial cancer cell line (ISH) with an IC50
value of 8.26 M. At 10 M, compounds 3f, 3k and 3g caused morphological changes of HT-29 and
ISH cells and caspase-3 activation. In addition, these compounds were evaluated against NIH 3T3
mouse embriyonic fibroblast cell line and all synthesized compounds (3a-l) were found to be less toxic
than paclitaxel. Moreover, possible inhibition mechanism of compound 3g was evaluated in silico
against BRAF kinase enzyme.
Keywords: 2,6‐Disubstituted pyridine, hydrazone, BRAF, apoptosis, anticancer, molecular modeling.
1. Introduction
Endometrial cancer (EC) is one of the the most common gynecologic cancers in developed countries,
which affects female reproductive organs [1–3]. Endometrial cancer is mostly unexpected clinically
or pathologically in women <45 years of age because cases usually happen in patients over 50 [4].
Hormonal factors, obesity, family medical history, diabetes and diet importantly affect the risk of
developing endometrial cancer [5]. It has also been reported that women with endometrial cancer
have a higher risk of colon cancer [6,7]. Fornasarig et al. found that, 13.5% of endometrial cancer
cases had colorectal cancers in their family history [8].
Protein kinase family is one of the largest protein families in the human genome. Protein kinases are
responsible for maintaining cellular functions and biological balance [9]. There are 538 protein kinase
enzymes identified in the body that are responsible for maintaining biological processes [10]. The fact
remains that abnormal activity of kinase proteins is linked with several diseases including cancer [11].
After the introduction of imatinib to the clinic in the year 2001, the number of kinase inhibitors as well
as efforts to generate novel kinase inhibitors are increasing day by day [12]. Today, there are 52 kinase
inhibitor compounds in the clinic approved by the FDA [13].
BRAF is a serine threonine kinase which is considered as a critical therapeutic target in different types
of cancer [14]. Especially, V600E mutant form of BRAF is found in many human cancers including
lung cancer [15], colorectal cancer [16,17], melanoma [18] and endometrial adenocarcinoma [19].
In medicinal chemistry, compounds bearing hydrazide-hydrazone moiety have received much
attention due to their potent biological value in the development of novel antimicrobials, analgesic and
antiinflammatory and especially anticancer agents [20]. The compound PAC-1 (Figure 1), bearing
hydrazone moiety, was reported to enhance the activity of procaspase-3 in vitro and induce apoptosis
in tumor cells in vivo [21]. Picolinoylhydrazone derivative (1) is a potent growth inhibitor of lung,
leukemia and ovarian cancer cell lines with with IC50 values below 10 μM [22]. A bis-isatin
hydrazones of imidazolidine-2,4-dione (2) showed good antiproliferative activity against the human
colon cancer cell line HCT-116 (Figure 1). Another bis-isatin hydrazone derivative (3) displayed a

promising growth inhibitory activity against several distinct cancer cell lines [23]. New 1,4-
dihydropyridine-3,5-dicarbohydrazide (4) was reported as potential anticancer agents in hepatic
HepG2 cancer cell line [24]. Also, our earlier investigations have provided evidence of antitumoral
and apoptosis inducing properties of hydrazide-hydrazone derivatives [25–27].
Figure 1. Rational design of novel 2,6-disubstituted pyridines.
Based on their aforementioned biological importance, combining pyridine nucleus with hydrazone
pharmacophore has been envisaged to generate new pyridine-2,6-dicarbohydrazide derivatives with
anticancer activity. In addition, effects on apoptosis and caspase-3 activation against HT-29 human
colon cancer cell line and ISH Ishikawa human endometrial cancer cell line were also investigated for
the most active derivatives. Considering the recent reports on BRAF inhibitory activity of hydrazone
derivatives [28,29], additional efforts have been made regarding computational studies which include
in silico docking studies to the target BRAF enzyme.
2. Experimental
2.1. Chemistry
1
Melting points were determined on an Electrothermal IA9300 melting point apparatus. H NMR and
13C NMR spectra were obtained on a BRUKER 300 MHz Ultrashield TM spectrometer, DMSO-d6
was used as a solvent, and TMS was used as an internal standard. Chemical reactions were monitored
by thin-layer chromatography under ultraviolet light of 254 nm. The liquid chromatographic system
consists of an Agilent Technologies 1100 series instrument equipped with a quaternary solvent
delivery system and a model Agilent series G1315 A photodiode array detector. Chromatographic data
were collected and processed using Agilent Chemstation Plus software. Chromatographic separation
was performed at ambient temperature using a reversed phase Zorbax C8 (4.0×250 mm) column. All

experiments were performed using ACN:H2O (v/v, 70/30) mobile phase with UV detection at 230 nm.
All the reagents and solutions were of analytical reagent grade.
2.1.1. Synthesis of compounds 1 and 2
Dimethyl pyridine-2,6-dicarboxylate 1 was prepared as previously described in the literature [30,31].
Pyridine-2,6-dicarbohydrazide 2 was prepared by heating hydrazine-hydrate (80%, 5 eq.) and 1 in
methanol. The reaction mixture was heated at reflux for 4 h and allowed to cool to RT, filtered, dried
and recrystallized from methanol to obtain pure product 2 [31].
2.1.2. General procedure for the synthesis of N'2,N'6-Bis(substitutedarylidene)pyridine-2,6-
dicarbohydrazides (3a-l)
A mixture of compound 2 (2.5 mmol) and substituted aldehydes (5.0 mmol) in 50 mL absolute ethanol
was refluxed for 6-12 h. After cooling, the solid precipitate was filtered off, washed with ethanol,
dried, and crystallized from ethanol to give compounds 3a-l [32,33].
N'2, N'6-bis {[4-(Trifluoromethyl)phenyl]methylidene}pyridine-2,6-dicarbohydrazide (3a)
Yield 75%; m.p. 324-326°C; HPLC tR (min.): 3.74; FT-IR: ν/cm−1: 3190 (NH), 1683, 1668, 1618,
(C=O and C=N); 1H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.88 (d, 4H, J= 8.4 Hz, Ar-H), 8.05
(d, 4H, J= 8.1 Hz, Ar-H), 8.29-8.42 (m, 3H, pyridine-H), 8.84 (s, 2H, CH=N), 12.52 (s, 2H, CONH-
N=). Anal. calcd for C23H15F6N5O2 (507.388): C, 54.44; H, 2.98; N, 13.80; Found: C, 54.40; H, 3.04;
N, 13.79.
N'2,N'6-bis{[3-(Trifluoromethoxy)phenyl]methylidene}pyridine-2,6-dicarbohydrazide (3b)
Yield 81%; m.p. 247°C; HPLC tR (min.): 6.17; FT-IR: ν/cm−1: 3228 (NH), 1668, 1579 (C=O and
1
C=N); H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.47-7.86 (m, 8H, Ar-H), 8.28-8.40 (m, 3H,
13
pyridine-H), 8.81 (s, 2H, CH=N), 12.50 (s, 2H, CONH-N=); C NMR (75MHz) (DMSO-d6/TMS) δ
ppm: 119.13 (2CH), 120.54 (2OCF3, d, J=255Hz), 123.21 (2CH), 126.17 (2CH), 127.07 (2CH),
131.59 (2CH), 137.06 (2C), 140.56 (CH), 148.54 (2C), 148.70 (2CH=N), 149.30 (2C), 160.11
(2C=O). Anal. calcd for C23H15F6N5O4.1/2H2O (548.391): C, 50.37; H, 2.94; N, 12.77; Found: C,
50.05; H, 2.78; N, 12.38.
N'2,N'6-bis [(4-Fluoro-3-phenoxyphenyl)methylidene]pyridine-2,6-dicarbohydrazide (3c)
Yield 89%; m.p. 280°C; HPLC tR (min.): 7.82; FT-IR: ν/cm−1: 3190 (NH), 1680, 1658, 1583 (C=O
1
and C=N); H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.07-7.66 (m, 16H, Ar-H), 8.24-8.34 (m,
3H, pyridine-H), 8.71 (s, 2H, CH=N), 12.34 (s, 2H, CONH-N=); 13C NMR (75MHz) (DMSO-
d6/TMS) δ ppm: 118.15 (2CH), 118.43 (2CH), 119.10 (2CH), 124.42 (2CH), 125.20 (2CH), 125.30

(2CH), 126.02 (2CH), 130.73 (2CH), 132.17 (2CH), 132.22 (2C), 140.52 (CH), 144.29 (2C), 144.45
(2C), 148.55 (2CH=N), 148.98 (2C), 155.03 (2CF, d, J= 252 Hz), 159.93 (2CO). Anal. calcd for
C33H23F2N5O4.H2O (609.578): C, 65.02; H, 4.13; N, 11.49; Found: C, 65.65; H, 3.78; N, 11.66.
N'2,N'6-bis [(5-Bromo-2-methoxyphenyl)methylidene]pyridine-2,6-dicarbohydrazide (3d)
Yield 76%; m.p. 269°C; HPLC tR (min.): 5.96; FT-IR: ν/cm−1: 3159 (NH), 1681, 1668, 1597 (C=O
1
and C=N); H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 3.93 (s, 6H, -CH3), 7.17 (d, 2H, J= 9 Hz,
Ar-H); 7.63 (dd, 2H, J1= 9 Hz, J2= 2.7 Hz, Ar-H), 8.02 (d, 2H, J= 2.4 Hz, Ar-H), 8.26-8.39 (m, 3H,
13
pyridine-H), 9.00 (s, 2H, CH=N), 12.41 (s, 2H, CONH-N=); C NMR (75MHz) (DMSO-d6/TMS) δ
ppm: 56.61 (2OCH3), 113.04 (2C), 114.99 (2CH), 124.76 (2C), 126.08 (2CH), 128.03 (2CH), 134.45
(2CH), 140.38 (CH), 143.78 (2C), 148.58 (2CH=N), 157.48 (2C), 159.99 (2CO). Anal. calcd for
C23H19Br2N5O4.H2O (607.251): C, 45.49; H, 3.49; N, 11.53; Found: C, 45.77; H, 3.08; N, 11.54.
N'2,N'6-bis{[2-(Trifluoromethoxy)phenyl]methylidene}pyridine-2,6-dicarbohydrazide (3e)
Yield 81%; m.p. 345°C; HPLC tR (min.): 4.98; FT-IR: ν/cm−1: 3192 (NH), 1681, 1666, 1602 (C=O
1
and C=N); H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.51-8.19 (m, 8H, Ar-H), 8.29-8.42 (m,
3H, pyridine-H), 8.94 (s, 2H, CH=N), 12.52 (s, 2H, CONH-N=); 13C NMR (75MHz) (DMSO-
d6/TMS) δ ppm: 118.53 (2CH), 120.55 (2OCF3, q, J= 273Hz), 126.62 (2CH), 128.67 (2CH), 131.66
(2CH), 131.87 (2C), 134.11 (2CH), 134.57 (CH), 143.82 (2C), 148.13 (2CH=N), 156.25 (2CO),
159.70 (2CO). Anal. calcd for C23H15F6N5O4.H2O (557.401): C, 49.56; H, 3.07; N, 12.56; Found: C,
49.68; H, 2.78; N, 12.52.
N'2,N'6-bis(Naphthalen-2-ylmethylidene)pyridine-2,6-dicarbohydrazide (3f)
Yield 76%; m.p. 351°C; HPLC tR (min.): 5.92; FT-IR: ν/cm−1: 3275 (NH), 1678, 1599 (C=O and
1
C=N); H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.61-8.11 (m, 12H, Ar-H), 8.33-8.45 (m, 3H,
pyridine-H), 9.02 (d, 2H, J= 8.4 Hz, Ar-H), 9.48 (s, 2H, CH=N), 12.46 (s, 2H, CONH-N=); 13C NMR
(75MHz) (DMSO-d6/TMS) δ ppm: 120.52 (2CH), 121.20 (2CH), 123.47 (2CH), 124.81 (2CH),
126.17 (2CH), 127.07 (2CH), 129.86 (2CH), 130.54 (2CH), 130.75 (2C), 131.46 (2C), 134.11 (2C),
140.63 (CH), 147.64 (2C), 148.58 (2CH=N), 159.87 (2CO); Anal. calcd for C29H21N5O2.1/2H2O
(480.514): C, 72.49; H, 4.61; N, 14.57; Found: C, 72.61; H, 4.47; N, 14.78.
N'2,N'6-bis[(5-Methylthiophene-2-yl)methylidene]pyridine-2,6-dicarbohydrazide (3g)
Yield 73%; m.p. 260°C; HPLC tR (min.): 3.96; FT-IR: ν/cm−1: 3524 (OH), 3185 (NH), 1672, 1656,
1622 (C=O and C=N); 1H NMR (600 MHz), (DMSO-d6/TMS)  ppm: 2.51 (s, 6H, CH3); 6.89 (d, 2H,
J= 3.6 Hz, thiophene-H); 7.38 (d, 2H, J= 3.0 Hz, thiophene-H); 8.25-8.33 (m, 3H, pyridine-H), 8.94
(s, 2H, CH=N), 12.21 (s, 2H, CONH-N=); 13C NMR (75MHz) (DMSO-d6/TMS) δ ppm: 15.90

(2CH3), 125.76 (2CH), 127.01 (2CH), 132.52 (2CH), 137.16 (2CH=N), 140.48 (CH), 144.04 (2C),
146.06 (2C), 148.67 (2C), 159.70 (2CO). Anal. calcd for C19H17N5O2S2.3/2H2O (438.512): C, 52.02;
H, 4.59; N, 15.96; S,14.62; Found: C, 51.58; H, 4.57; N, 15.69, S,14.67.
N'2,N'6-bis[(3-Fluorophenyl)methylidene]pyridine-2,6-dicarbohydrazide (3h)
Yield 79%; m.p. 275°C; HPLC tR (min.): 3.20; FT-IR: ν/cm−1: 3526 (OH), 3371 (NH), 1668, 1595
1
(C=O and C=N); H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.30-7.69 (m, 8H, Ar-H), 8.28-8.40
13
(m, 3H, pyridine-H), 8.78 (s, 2H, CH=N), 12.45 (s, 2H, CONH-N=); C NMR (75MHz) (DMSO-
d6/TMS) δ ppm: 113.66 (2CH, d, J= 22Hz), 117.69 (2CH, d, J= 22 Hz), 124.14 (2CH), 126.11 (2CH),
131.54 (2CH), 137.13 (2CH, d, J= 7.5 Hz), 140.52 (CH), 148.56 (2C), 149.15 (2CH=N), 160.05
(2CO), 162.91 (2C-F, d, J=243 Hz). Anal. calcd for C21H15F2N5O2.2H2O (443.403): C, 56.88; H, 4.32;
N, 15.79; Found: C, 56.52; H, 4.29; N, 15.60.
N'2,N'6-bis[(4-Fluoro-3-methoxyphenyl)methylidene]pyridine-2,6-dicarbohydrazide (3i)
Yield 93%; m.p. 304°C; HPLC tR (min.): 4.72; FT-IR: ν/cm−1: 3159 (NH), 1666, 1649, 1589 (C=O
1
and C=N); H NMR (600 MHz), (DMSO-d6/TMS)  ppm: 3.95 (s, 6H, CH3), 7.34-7.62 (m, 6H, Ar-
H), 8.29-8.38 (m, 3H, pyridine-H), 8.77 (s, 2H, CH=N), 12.45 (s, 2H, CONH-N=); 13C NMR
(75MHz) (DMSO-d6/TMS) δ ppm: 56.48 (2CH3), 111.36 (2CH), 116.85 (2CH, d, J= 18.75 Hz),
121.70 (2CH, d, J=6.75 Hz), 126.03 (2CH), 131.60 (2C, d, J= 3.75 Hz), 140.53 (CH), 148.05
(2CH=N), 148.67 (2C), 149.73 (2C),149.96 (2C-F, d, J= 251 Hz), 159.94 (2CO). Anal. calcd for
C23H19F2N5O4.1/3H2O (473.429): C, 58.35; H, 4.19; N, 14.79; Found: C, 58.66; H, 4.24; N, 14.70.
N'2,N'6-bis[(4-Bromothiophen-2-yl)methylidene]pyridine-2,6-dicarbohydrazide (3j)
Yield 78%; m.p. 297°C; HPLC tR (min.): 4.72; FT-IR: ν/cm−1: 3636 (OH), 3375 (NH), 1668, 1600
(C=O and C=N); 1H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.61 (d, 2H, J=1.5 Hz, thiophene-H);
7.82 (dd, 2H, J1= 1.3 Hz, J2= 0.7 Hz, thiophene-H); 8.26-8.36 (m, 3H, pyridine-H), 8.99 (s, 2H,
CH=N), 12.39 (s, 2H, CONH-N=); 13C NMR (75MHz) (DMSO-d6/TMS) δ ppm: 109.97 (2C), 126.04
(2CH), 127.18 (2CH), 133.37 (2CH=N), 140.60 (2C), 140.92 (2CH), 144.23 (CH), 148.46 (2C),
159.97 (2CO). Anal. calcd for C17H11Br2N5O2S2.2H2O (577.270): C, 35.37; H, 2.62; N, 12.13; S,11.11;
Found: C, 35.77; H, 2.49; N, 12.07, S,11.06.
N'2,N'6-bis[(4-Phenylthiophen-2-yl)methylidene]pyridine-2,6-dicarbohydrazide (3k)
Yield 83%; m.p. 360°C; HPLC tR (min.): 4.89; FT-IR: ν/cm−1: 3257 (NH), 1693, 1658,1599 (C=O and
1
C=N); H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.32-8.07 (m, 14H, Ar-H); 8.24-8.39 (m, 3H,
13
pyridine-H), 9.09 (s, 2H, CH=N), 12.39 (s, 2H, CONH-N=); C NMR (75MHz) (DMSO-d6/TMS) δ
ppm: 124.68 (2CH), 125.95 (4CH), 126.44 (4CH), 128.04 (2CH), 129.46 (2CH), 130.52 (2CH),

134.85 (2CH=N), 136.56 (2C), 140.22 (CH), 142.34 (2C), 145.42 (2C), 148.60 (2C), 159.86 (2CO).
Anal. calcd for C29H21N5O2S2.1/3H2O (541.644): C, 65.03; H, 3.95; N, 13.07; S,11.97; Found: C,
64.66; H, 4.01; N, 13.38, S,11.90.
N'2,N'6-bis{[4-Fluoro-2-(trifluoromethyl)phenyl]methylidene}pyridine-2,6-dicarbohydrazide (3l)
Yield 79%; m.p. 344-346°C; HPLC tR (min.): 6.35; FT-IR: ν/cm−1: 3525 (OH), 3188 (NH), 1674,
1
1656, 1622 (C=O, C=N); H NMR (300 MHz), (DMSO-d6/TMS)  ppm: 7.69 -8.41 (m, 9H, Ar-H),
9.02 (s, 2H, CH=N), 12.62 (s, 2H, CONH-N=); Anal. calcd for C23H13F8N5O2.3H2O (597.415): C,
46.24; H, 3.21; N, 11.72; Found: C, 46.36; H, 3.35; N, 11.71.
2.2. Biological Screening
2.2.1. Cell culture and cell viability assay
Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
Human colon cancer cell line (HT-29 cells) and Ishikawa human endometrial cancer cell line (ISH
cells) and mouse fibroblast cell line (NIH 3T3 cells) were maintained in Dulbecco's modified Eagle's
medium (DMEM, Invitrogen) or RPMI-1640 supplemented with 10% FBS, 2ꢀmM l‐glutamine,
100ꢀU/mL penicillin, and 100ꢀμg/mL streptomycin and kept in a humidified atmosphere at 37°C
incubator with 5% CO2 in air. To determine the cell viability cells were plated onto 96‐well plates
(1ꢀ×ꢀ104 cells/well). The cells were treated with different concentrations (0, 0.1, 1, 10, 100, and
1000ꢀμM) of synthesized compounds incubated for 24ꢀh. After the incubation cells were washed with
PBS and added to 100ꢀμL DMEM/RPMI-1640. A total of 10ꢀμL of the MTT (3‐(4,5‐dimethylthiazol‐2‐
yl)‐2,5‐diphenyltetrazolium bromide) (Vybrant, Invitrogen) labeling reagent was added to each well
and incubated for 4 h in a humidified atmosphere at 37°C incubator with 5% CO2 in the air. After the
incubation, 100ꢀμL of the SDS buffer was added into each well for the solubilization of formazan
precipitate. Then absorbance was measured by a microplate reader at 570ꢀnm and was carried out in
triplicate of each assay [34]. Observation of morphological changes of apoptotic cells was performed
according to the methods [35,36] with slight modifications.
2.2.2. Caspase 3 activity assay
The caspase activity was measured after cell incubation for 24 h with compounds 3f, 3g and 3k (10
M), using a Caspase-3 Activity Assay Kit (C10427, Invitrogen), as reported previously [36].
2.2.3. Antibacterial activity test
All synthesized compounds were tested for in vitro antibacterial activity against Escherichia coli
ATCC 25922, Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus
ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 6633 with using micro

broth dilution method according to EUCAST recommendation [37]. The micro-dilution method was
performed in cation adjusted Mueller Hinton Broth (CMHB) with serial dilution of compounds in 96
well plates. 100 µL of CMHB with different concentrations of compounds was added into the well.
Bacterial suspension was prepared from a fresh culture of bacteria and adjusted using a nephelometer
to 0.5 McFarland units and diluted at 1/100 ratio. 100 µL bacterial suspension was added in to the
well. Microplates were incubated 35±2°C for 16 to 20 hours. The minimum concentration that inhibits
bacterial growth accepted as minimal inhibition concentration (MIC). Moxifloxacin was used as
control.
2.2.4. Statistical analysis
All assays were performed in triplicate in three independent assays, and the obtained values were
analyzed and expressed as mean ± standard error of the mean (SEM). Statistical analysis was carried
out by analysis of variance (one-way ANOVA) followed by a Tukey test.
2.3. In silico studies
Co-crystal structure of BRAF V600E protein with vemurafenib (PDB ID: 3OG7) was obtained from
Protein Data Bank (https://www.rcsb.org/). Vemurafenib and water molecules were removed from the
structure first and protein, as well as ligands, were prepared by using Biovia Discovery Studio
Visualizer and MGLTools software. AutoDock Vina docking tool was used to estimate possible
binding conformations of compounds. Binding modes were evaluated with Biovia Discovery Studio
Visualizer.
For validation of the docking process, vemurafenib was re-docked into BRAF. Excellent
superimpositioning between co-crystallised structure and re-docked structure was observed. RMSD
value, which is expected to be under 3.000 Å after re-docking process, was found as 0.557.
3. Results and Discussion
3.1. Chemistry
The synthesis of the new compounds is illustrated in Scheme 1. The preparation of compound 1 was
achieved via heating pyridine-2,6-dicarboxylic acid and methanol as previously reported [31].
Compound 2 was obtained by heating compound 1 with hydrazine hydrate [32]. Through the
condensation reaction of compound 2 and various aldehydes in the presence of acetic acid, new
dihydrazone derivatives 3a-l were synthesized.

OCF3
3a
3c
3b
3d
MeOH
CF3
OPh
O
O
HO
OH
OCH3
N
N
conc. H SO4
2
O
O
1
O
O
F
Br
NH2NH2
MeOH
OCF3
3e
3f
F
3g
3i
3h
3j
S
S
NH
NH
NH2
H2N
N
OCH3
F
ArCHO
EtOH
Br
O
O
S
gl.AcOH
2
CF3
Ph
3k
3l
NH
NH
Ar
N
N
N
Ar
F
O
O
3a-l
Scheme 1. Synthetic route to compounds 3a-l.
The IR spectra of compounds 3a-l showed the disappearance of the absorption band of NH2 groups
1
and the appearance of absorption bands at 3190–3375cm–1 attributed to the NH.. Furthermore, the H
NMR spectrum showed a singlet signal at δ 8.71-9.48ꢀppm corresponding to the azomethine protons,
in addition to a singlet signal at δ12.21-12.62 ppm indicating the N-H proton of the hydrazone moiety.
As for the 13C NMR spectra, signals for aliphatic and aromatic groups in compounds were observed at
expected regions [38,39].
The C=N bond can give rise to the formation of E/Z isomers. Based on literature precedence the
isomeric ratio of products was presumed to be mainly E in all cases [40,41]. The presence of a single
set of NMR signals in DMSO-d6 indicates that only one geometric isomer and rotameric form exists in
solution. Structural elucidation was supported by a 2D NMR-NOESY spectrum of compound 3f as
shown in Figure 2. The NOESY spectrum, which showed a spatial interaction between CONH and
CH=N, supported the formation of the E geometrical isomer. Expectedly, a spatial interaction between
the NH and the pyridine hydrogens was not observed which is in good agreement with the crystal
structure of N'2,N'6-bis[(pyridin-2-yl)methylidene]pyridine-2,6-dicarbohydrazide [42]. This is
consistent with multiple literature reports which state that the E-isomer is the predominant form in
solution due to hindered rotation about the imine bond.

Figure 2. 2D NOESY of 3f in DMSO-d6 at 295 K (x- and y-axis, 1H-NMR chemical shift).
3.2. Biological results
Newly synthesized twelve compounds were screened for their in vitro growth inhibitory activities
against two human cancer cell lines namely HT-29 human colon cancer cell line and ISH Ishikawa
human endometrial cancer cell line and mouse embryonic fibroblast cells NIH 3T3 (as healthy cells)
by MTT assay method, taking paclitaxel as reference. As shown in Table 1, these derivatives bearing
pyridine and hydrazone moiety exhibited remarkable anticancer effects. Among them, compound
N'2,N'6-bis(naphthalen-2-ylmethylidene)pyridine-2,6-dicarbohydrazide
3f
and
N'2,N'6-bis[(4-
phenylthiophen-2-yl)methylidene]pyridine-2,6-dicarbohydrazide 3k displayed potent inhibitory
activity (IC50=6.78 M and 8.88 M, respectively) for HT-29 cell line in comparison to the standard
paclitaxel
(IC50=0.35
M).
Taken
altogether,
N'2,N'6-bis[(5-methylthiophene-2-
yl)methylidene]pyridine-2,6-dicarbohydrazide 3g was the most potent compound in this series, having
an IC50 value of 8.26 M in suppressing ISH cell growth.
The synthesized compounds were evaluated against mouse embryonic fibroblast cells NIH 3T3 for
their safety. Their attained IC50 values were 80.06-521.78 μM, showing a safe profile and selectivity
toward the tested cancer cell lines. All of the compounds were found less toxic in the non-cancerous
mouse cell line (NIH 3T3) in vitro, as compared to its toxicity over ISH and HT-29, This result is

encouraging, since the well-known drug paclitaxel, which has a much lower IC50 value in ISH or HT-
29, does not exhibit this property.
Table 1. In vitro cytotoxic activities of compounds 3a-l.
IC50 (μM)a
Comp.
NIH 3T3
184.67
521.78
131.56
112.38
109.71
117.78
80.13
ISH
40.40
29.97
31.77
31.66
82.80
10.23
8.26
HT-29
32.68
132.16
32.03
31.66
31.78
6.78
3a
3b
3c
3d
3e
3f
12.29
31.58
118.85
31.74
8.88
3g
80.06
32.81
31.59
31.56
31.90
107.20
1.025
3h
604.17
585.43
212.74
324.91
74.21
3i
3j
3k
383.75
3l
0.35
Paclitaxel
aIC50 compound concentration required to inhibit tumour cell proliferation by 50%.
At 10 M, compounds 3f, 3g and 3k effectively inhibited the cancer cell viability in a time-dependent
manner (Figure 3).
Figure 3. Effects of compounds on cell survival in NIH 3T3, ISH, HT-29 cancer cells. a) NIH 3T3 cells b) ISH
cells c) HT-29 cells survival measurement with MTT assay after compounds treatment (10 μM) of cell lines at
three different time points. (*p<0.05, **p<0.01, ***p<0.001, compare to control).

Morphological changes in the cancer cells treated with compounds 3f, 3g and 3k using inverted
microscopy showed the apoptosis. As shown in Figure 4, 24 h after exposure to 10 μM of the selected
compounds, a significant number of HT-29 and ISH cells detached from the plate. When compared to
the control, compounds-treated cancer cells began to show cell shrinkage, rounding and fragmentation
that were indicative of apoptosis. Apoptosis can be characterized by morphological changes in the
nucleus, chromatin condensation and cell shrinkage [43]. The results showed that lead compounds
triggered apoptosis as a molecular mechanism. Such morphological changes were not apparent in the
control cells.
Figure 4. Effects of compounds on cell growing and apoptosis in HT-29, ISH and NIH 3T3 cells. Cell
morphological changes with microscobic image (10X). Cells were treated with 10 μM compounds HT-29, ISH
and NIH 3T3 cells at 24 well plates and and stained with crystal violet. Cells growing shown as overview images
(up), crystal violet staining (down) and percentages of cell growing (right). (**p<0.01, ***p<0.001 compare to
control).
Caspase-3 is an effector caspase that has a central role in cell apoptosis. By using DMSO as a
negative control, the ability of compounds 3f, 3g and 3k to activate caspase-3 in cancer cells was
evaluated (Figure 5). Indeed, these compounds appeared to induce caspase-3 activation as compared to
the untreated control cells, as compound-treated cells showed more active caspase-3 expression. This
study has shown that compounds might induce apoptotic cell death through a caspase-dependent
pathway.

Figure 5. Effects of compounds (10 μM) on the caspase-3 activity in a) HT-29 cells b) ISH cells. (**p<0.01,
***p<0.001 compare to control).
Furthermore, compounds 3a-l were investigated for their antibacterial activity against Escherichia coli
ATCC 25922, Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus
ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 6633 with using micro
broth dilution method according to EUCAST recommendation. Moxifloxacin was used as the standard
reference drug. MIC values of all compounds were found >50 µg/mL. Evaluation of the activity of the
synthesized compounds exhibited that, the tested compounds did not exhibit remarkable antibacterial
activity.
3.3. Molecular modeling
Regarding that the synthesized compounds were tested against endometrium and colon cancer cell
lines, molecular modeling studies were designed with BRAF V600E protein since overactivity of
BRAF V600E has been correlated with endometrial and colon cancers [16,17,19]. In this point of
view, docking studies were performed to estimate possible inhibition of BRAF with designed
compounds. Exemplarily, docking analyses of compound 3g, which showed superior anticancer
activity in endometrium cancer cells is presented here.
The binding energy calculated for the compound 3g was found as -8.2 kcal/mol while the binding
energy of vemurafenib -11.4 was found kcal/mol. The E-isomer of the compound was selected for the
docking analysis as E isomers of the compounds were proved with spectral data. According to docking
studies, compound 3g forms two conventional hydrogen bonds in the activation site. One of the H
bonds is formed between Cys532 residue, which places in the hinge region of BRAF protein and
hydrazone NH atom (Figure 6). This interaction is so fundamental as hydrogen bond interaction with
the hinge region is critical for any kinase protein inhibition [44]. The other hydrogen bond is formed
between the carbonyl group and Ser536 residue. Besides, compound 3g makes a carbon-hydrogen
bond with Asn580. As seen in Figures 6 and 7, compound 3g settles properly in the hydrophobic

pocket of BRAF. Both 5-methylthiophene rings of compound 3g contribute hydrophobic interactions
by interacting with Phe468, Val471, Ala481, Lys483 and Leu514 amino acid residues. Sulfur atom of
thiophene ring makes pi-sulfur interaction with Phe583. Van der Waals forces are observed between
compound 3g and Thr529, Gly534, His539, Asn581, Asp594 residues.
Figure 6. The ligand 3g (space filling) is embedded into the kinase domain (left panel). Interaction between
compound 3g and V600E- BRAF, hydrogen bonds are shown as green lines (right panel).

Figure 7. Two-dimentional interaction diagram for compound 3g. Interaction diagram legends include: 1) pink
circles = residues involved in alkyl and pi-alkyl interacitons; 2) light green circles = residues involved in carbon
hydrogen bond and van der Waals interactions; 3) green circles = hydrogen bond interactions; 4) yellow circles =
pi-sulfur interactions.
4. Conclusion
In the present work, a novel series of hydrazide-hydrazones containing pyridine moiety were
synthesized. These compounds were evaluated for their anticancer activity against human colon cancer
cell line (HT-29) and Ishikawa human endometrial cancer cell line (ISH). The results showed that the
majority of compounds had moderate to high inhibition on cancer cells with low cytotoxicity on
normal cells. The hydrazone derivatives 3f, 3g and 3k revealed higher activity than the other
substituted hydrazone derivatives. Our results showed that these compounds are capable of inducing
apoptosis through caspase-3 activation. The molecular modeling displayed that compound 3g
exhibited similar and additional binding interactions when docked into BRAF kinase, which suggests
that 3g may act as BRAF kinase inhibitors and this may contribute to their anticancer activity.

Acknowledgements
The authors would like to thank TÜBİTAK [2209-A] for partially providing financial support (Project
No: 1919B011800585).
References
[1]
[2]
[3]
[4]
P. Morice, A. Leary, C. Creutzberg, N. Abu-rustum, E. Darai, P. Vi, Endometrial cancer,
Lancet. 387 (2016) 1094–1108. doi:10.1016/S0140-6736(15)00130-0.
L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, Global cancer statistics,2012, CA Cancer J. Clin. 65
(2015) 87–108. doi:10.3322/caac.21262.
T. Van Nyen, C.P. Moiola, E. Colas, D. Annibali, F. Amant, Modeling Endometrial Cancer:
past,present,and future, Int. J. Mol. Sci. 19 (2018) 2348. doi:10.3390/ijms19082348.
G.P. Pellerin, M.A. Finan, Endometrial cancer in women 45 years of age or younger:A
clinicopathological analysis, Am. J. Obstet. Gynecol. 193 (2005) 1640–1644.
doi:10.1016/j.ajog.2005.05.003.
[5]
S.G.O. Clinical, P. Endometrial, C. Working, W.M. Burke, J. Orr, M. Leitao, E. Salom, P.
Gehrig, A.B. Olawaiye, M. Brewer, D. Boruta, T.J. Herzog, F. Abu, G. Oncology, C. Practice,
Endometrial cancer:A review and current management strategies:Part II, Gynecol. Oncol. 134
(2014) 393–402. doi:10.1016/j.ygyno.2014.06.003.
[6]
[7]
[8]
K. Bergfeldt, K. Institutet, S. Einhorn, K. Institutet, P. Hall, K. Institutet, Increased risk of
second primary malignancies in patients with gynecological cancer:A swedish record-linkage
study, Acta Oncol. 34 (1995) 771–777. doi:10.3109/02841869509127185.
D.S. Weinberg, C.J. Newschaffer, A. Topham, Risk for colorectal cancer after gynecologic
cancer, Ann. Intern. Med. 131 (1999) 189–193. doi:10.7326/0003-4819-131-3-199908030-
00005.
M. Fornasarig, E. Campagnutta, R. Talamini, S. Franceschi, G. Boz Carlo Scarabelli Cristina
Maria Andreaus Giovanni Scozzari Maurizio Valentini Risk factors for endometrial cancer
according to familial susceptibility, Int. J. Cancer. 32 (1998) 29–32. doi:10.1002/(sici)1097-
0215(19980703)77:1<29::aid-ijc6>3.0.co;2-1.
[9]
G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase
complement
of
the
human
genome,
Science.
298
(2002)
1913–1934.
doi:10.1126/science.1075762.
[10] D. Fabbro, S.W. Cowan-jacob, H. Moebitz, Ten things you should know about protein
kinases:IUPHAR review 14, Br. J. Pharmacol. 172 (2015) 2675–2700. doi:10.1111/bph.13096.
[11] K.S. Bhullar, N.O. Lagarón, E.M. McGowan, I. Parmar, A. Jha, B.P. Hubbard, H.P.V.
Rupasinghe, Kinase-targeted cancer therapies:progress, challenges and future directions, Mol.
Cancer. 17 (2018) 48. doi:10.1186/s12943-018-0804-2.
[12] A. Türe, D.C. Kahraman, R. Cetin-Atalay, S. Helvacıoğlu, M. Charehsaz, İ. Küçükgüzel,
Synthesis,anticancer activity,toxicity evaluation and molecular docking studies of novel
phenylaminopyrimidine-(thio) urea hybrids as potential kinase inhibitors, Comput. Biol. Chem.
78 (2019) 227–241. doi:10.1016/j.compbiolchem.2018.12.003.
[13] R. Roskoski Jr, Properties of FDA-approved small molecule protein kinase inhibitors,
Pharmacol. Res. 152 (2020) 104609. doi: 10.1016/j.phrs.2019.104609.
[14] S.R. Whittaker, G.S. Cowley, S. Wagner, F. Luo, D.E. Root, L.A. Garraway, Combined Pan-
RAF and MEK inhibition overcomes multiple resistance mechanisms to selective RAF
inhibitors, Mol. Cancer The. 14 (2015) 617–632. doi:10.1158/1535-7163.MCT-15-0136-T.
[15] J.G.B. Alvarez, G.A. Otterson, Agents to treat BRAF-mutant lung cancer, Drugs Context. 8
(2019) 31–35. doi:10.7573/dic.212566.

[16] D. Barras, BRAF mutation in colorectal cancer:An update, Biomark Cancer. 7 (2015) 9–12.
doi:10.4137/BIC.S25248.
[17] Y. Nakamura, N. Hattori, N. Iida, S. Yamashita, A. Mori, K. Kimura, T. Yoshino, T. Ushijima,
Targeting of super-enhancers and mutant BRAF can suppress growth of BRAF-mutant colon
cancer cells via repression of MAPK signaling pathway, Cancer Lett. 402 (2017) 100–109.
doi:10.1016/j.canlet.2017.05.017.
[18] P.A. Ascierto, J.M. Kirkwood, J.-J. Grob, E. Simeone, A.M. Grimaldi, M. Maio, G. Palmieri,
A. Testori, F.M. Marincola, N. Mozzillo, The role of BRAF V600 mutation in melanoma, J.
Transl.Med. 10 (2012) 85. doi:10.1186/1479-5876-10-85.
[19] M. He, V. Breese, S. Hang, C. Zhang, J. Xiong, C. Jackson, BRAF V600E mutations in
endometrial
adenocarcinoma,
Diagn.
Mol.
Pathol.
22
(2013)
35–40.
doi:10.1097/PDM.0b013e31826c7fe0.
[20] S. Rollas, S.G. Küçükgüzel, Biological activities of hydrazone derivatives, Molecules. 12
(2007) 1910–1939. doi:10.3390/12081910.
[21] Q.P. Peterson, D.R. Goode, D.C. West, K.N. Ramsey, J.J.Y. Lee, P.J. Hergenrother, PAC-1
activates procaspase-3 in vitro through relief of zinc-mediated inhibition, J. Mol. Biol. 388
(2009) 144–158. doi:10.1016/j.jmb.2009.03.003.
[22] D. Zhang, Y. Ma, Y. Liu, Z. Liu, Synthesis of sulfonylhydrazone-and acylhydrazone-
substituted 8-ethoxy- 3-nitro-2H-chromenes as potent antiproliferative and apoptosis inducing
agents full paper synthesis of sulfonylhydrazone-and acylhydrazone-substituted 8-ethoxy-3-
nitro-2H-chromene,
Arch.
Pharm.
(Weinheim).
347
(2014)
576–588.
doi:10.1002/ardp.201400082.
[23] H.S. Ibrahim, S.M. Abou-seri, N.S.M. Ismail, M.M. Elaasser, M.H. Aly, H.A. Abdel-aziz, Bis-
isatin hydrazones with novel linkers:Synthesis and biological evaluation as cytotoxic agents,
Eur. J. Med. Chem. 108 (2016) 415–422. doi:10.1016/j.ejmech.2015.11.047.
[24] S.M. Gomha, A.A.M. El-reedy, Intramolecular ring transformation of bis-oxadiazoles to bis-
thiadiazoles and investigation of their anticancer activities, J. Heterocycl. Chem. 55 (2018)
2360–2367. doi:10.1002/jhet.3300.
[25] Ş.G. Küçükgüzel, D. Koç, P. Çıkla-Süzgün, D. Özsavcı, Ö. Bingöl-Özakpınar, P. Mega, O.
Orun, P. Erzincan, S. Sağ-Erdem, F. Şahin, Synthesis of tolmetin hydrazide-hydrazones and
discovery of a potent apoptosis inducer in colon cancer cells, Arch. Pharm. (Weinheim).348
(2015) 730–742. doi:10.1002/ardp.201500178.
[26] S. Şenkardeş, N. Kaushik-Basu, İ. Durmaz, D. Manvar, A. Basu, R. Atalay, Ş.G. Küçükgüzel,
Synthesis of novel diflunisal hydrazide hydrazones as anti-hepatitis C virus agents and
hepatocellular carcinoma inhibitors, Eur. J. Med. Chem. 108 (2016) 301–308.
doi:10.1016/j.ejmech.2015.10.041.
[27] P. Çıkla, D. Özsavcı, Ö. Bingöl-Özakpınar, A. Şener, Ö. Çevik, S. Özbaş-Turan, J. Akbuğa, F.
Şahin, Ş.G. Küçükgüzel, Synthesis, cytotoxicity, and pro-apoptosis activity of etodolac
hydrazide derivatives as anticancer agents, Arch. Pharm. (Weinheim). 346 (2013) 367–379.
doi:10.1002/ardp.201200449.
[28] K.M. Amin, O.M. El-Badry, DE Abdel Rahman, U.M. Ammar, M.M. Abdalla, Design,
synthesis, anticancer evaluation and molecular docking of new V600E BRAF inhibitors
derived from pyridopyrazinone, Eur. J. Chem. 7 (2016) 19–29. doi:10.5155/eurjchem.7.1.19-
29.1346.
[29] S.A. Abdelatef, M.T. El-saadi, N.H. Amin, A.H. Abdelazeem, H.A. Omar, K.R.A. Abdellatif,
Design, synthesis and anticancer evaluation of novel spirobenzo [h] chromene and
spirochromane derivatives with dual EGFR and B-RAF inhibitory activities, Eur. J. Med.
Chem. 150 (2018) 567–578. doi:10.1016/j.ejmech.2018.03.001.
[30] P. Kapoor, A. Pathak, P. Kaur, P. Venugopalan, R. Kapoor, Steric control of coordination:
Unusual coordination mode of dimethylpyridine-2,6-dicarboxylate in a new dinuclear copper
(II) complex [(dmpc)(Cl)(µ-Cl) Cu2 (µ-Cl)(Cl)(dmpc)] and reversal of the coordination mode,
Transit. Met. Chem. 29 (2004) 251–258. doi:10.1023/B:TMCH.0000020351.16762.f2.

[31] R. Gao, G. Xu, L. Zheng, Y. Xie, M. Tao, W. Zhang, A highly selective and sensitive reusable
colorimetric sensor for Ag + based on thiadiazole-functionalized polyacrylonitrile fiber, J.
Mater. Sci. (2016) 5996–6006. doi:10.1039/c6tc00621c.
[32] R. Kumar, H. Jain, P. Gahlyan, A. Joshi, C.N. Ramachandran, A highly sensitive pyridine-
dicarbohydrazide based chemosensor for colorimetric recognition of Cu2+, AMP2−, F− and
AcO− ions, New J. Chem. (2018) 8567–8576. doi:10.1039/c8nj00918j.
[33] N. Yadav, A.K. Singh, Dicarbohydrazide based chemosensors for copper and cyanide ions via
a displacement approach, New J. Chem. 42 (2018) 6023–6033. doi:10.1039/c8nj00230d.
[34] I. Çoruh, Ö. Çevik, K. Yelekçi, T. Djikic, Ş.G. Küçükgüzel, Synthesis, anticancer activity, and
molecularmodeling of etodolac-thioether derivatives as potent methionine aminopeptidase
(type
II)
inhibitors,
Arch.
Pharm.
(Weinheim).
351
(2018)
e1700195.
doi:10.1002/ardp.201700195.
[35] S. Raghavan, P. Manogaran, B.K. Kuppuswami, G. Venkatraman, K. Kumari, G. Narasimha,
Synthesis and anticancer activity of chalcones derived from vanillin and isovanillin, Med.
Chem. Res. 24 (2015) 4157–4165. doi:10.1007/s00044-015-1453-2.
[36] O. Erdogan, M. Abbak, M. Demirbolat, F. Birtekocak, M. Aksel, S. Paşa, O. Çevik, Green
synthesis of silver nanoparticles via Cynara scolymus leaf extracts:The characterization ,
anticancer potential with photodynamic therapy in MCF7 cells, PloS one (2019) 1–15.doi:
10.1371/journal.pone.0216496
[37] 2006 ISO 20776-1: Clinical Laboratory Testing and In Vitro Diagnostic Test Systems-
Susceptibility Testing of Infectious Agents and Evaluation of Performance of Antimicrobial
Susceptibility Test Devices; ISO: Geneva, Switzerland, INTERNATIONAL STANDARD, n.d.
[38] Ş.G. Küçükgüzel, A. Mazi, F. Şahin, S. Öztürk, J. Stables, Synthesis and biological activities of
diflunisal hydrazide-hydrazones, Eur. J. Med. Chem. 38 (2003) 1005–1013.
doi:10.1016/j.ejmech.2003.08.004.
[39] S. Dória, F. Palace-berl, K. Fernanda, M. Pasqualoto, M. Ishii, A. Kleber, C. Maria, R. Viola,
D. Augusto, L.C. Tavares, Ligand-based design, synthesis, and experimental evaluation of
novel benzofuroxan derivatives as anti-Trypanosoma cruzi agents, Eur. J. Med. Chem. 64
(2013) 200–214. doi:10.1016/j.ejmech.2013.03.053.
[40] A. Özdemir, G. Turan-Ziitouni, Z.A. Kaplancıklı, M.D. Altıntop, The synthesis of some new
hydrazone derivatives containing the benzothiazole moiety, J. Serb. Chem. Soc. 77 (2012)
141–146. doi:10.2298/JSC110321171O.
[41] P. Patorski, E. Wyrzykiewicz, G. Bartkowiak, Synthesis and conformational assignment of N-
(E)-stilbenyloxymethylenecarbonyl-substituted hydrazones of acetone and o-(m-and p-) chloro-
1
(nitro-) benzaldehydes by means of H and 13C NMR spectroscopy, J. spectrosc. (2013)
197475. doi:10.1155/2013/197475.
[42] C. Li, J. Guo, W. Wu, Crystal structure of N’2,N'6-bis(pyridin-2-ylmethyl)-pyridine-2,6-
dicarbohydrazide pentahydrate, C19H25N7O7, Z. Kristallogr. Cryst. Mater. 229 (2014) 245–246.
doi:10.1515/ncrs-2014-0124.
[43] A. Saraste, K. Pulkki, Morphologic and biochemical hallmarks of apoptosis, Cardiovasc. Res.
45 (2000) 528–537. doi:10.1016/s0008-6363(99)00384-3.
[44] M. Tong, M.A. Seeliger, Targeting Conformational Plasticity of Protein Kinases, ACS Chem.
Biol. 10 (2015) 190-200. doi:10.1021/cb500870a.
Sevil Şenkardeş: Project administration, Conceptualization, Methodology, Investigation, Formal
analysis, Writing-original draft, Writing-review&editing, Supervision. Aslı Türe: Software,
Visualization, Validation, Writing-original draft, Writing-review&editing. Sedanur Ekrek: Funding
acquisition, Investigation. Asım Tuğrul Durak: Funding acquisition, Investigation. Mürüvvet
Abbak: Investigation, Methodology, Validation, Visualization. Özge Çevik: Investigation,

Methodology, Validation, Visualization, Writing-original draft, Supervision. Banu Kaşkatepe:
Validation, Investigation, Writing - original draft. İlkay Küçükgüzel: Resources, Supervision,
Validation, Writing-original draft. Ş. Güniz Küçükgüzel: Resources, Supervision, Validation,
Writing - original draft.
Declaration of interests
☐ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Graphical Abstract