Synthesis and Structure-Activity Relationship

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RESEARCH ARTICLE

Synthesis and Structure-Activity Relationship; Exploration of Some Potent Anti-Cancer Phenyl Amidrazone Derivatives Almeqdad Y. Habashneh1, Mustafa M. El-Abadelah1, Sanaa K Bardaweel2 and Mutasem O. Taha2,3* 1

Faculty of Science, Chemistry Department, The University of Jordan, Amman, Jordan; 2Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan; 3 Drug Discovery Unit, Faculty of Pharmacy, The University of Jordan, Amman, Jordan Abstract: Background: Amidrazones have been reported to have significant anti-tumor properties against several cancer cell lines. ARTICLE HISTORY Received: June 23, 2017 Revised: October 25, 2017 Accepted: November 22, 2017 DOI: 10.2174/1573406414666171204143157

Objectives: The current project aims to profile the structure-anticancer activity relationship of phenyl-amidrazons. Methods: Fifteen phenyl-amidrazone-piperazine derivatives were prepared and tested against four cancer cell lines (leukemia, prostate, breast and colon cancers). Results: Six compounds illustrated low micromolar anticancer IC50 values, while the remaining compounds were either inactive or of moderate potencies. All compounds were virtually nontoxic against normal fibroblast cells. Conclusion: Docking into the oncogenic kinase bcr/abl illustrated the critical importance of (i) phalogen substituent on the ligand's phenyl ring and (ii) the presence of positive ionizable moiety at the ligand's piperazine fragment for anticancer activity.

Keywords: Aryl-amidrazones, anticancer bioactivity, docking, SAR, electrostatic attraction, hydrogen bonding. 1. INTRODUCTION Amidrazone derivatives have received considerable attention due to their structural diversity and biological properties. They were reported to exhibit anti-microbial [1-6], antitubercular [7], insecticidal [8], anti-corticotrophin-releasing factor [9], anti-thrombotic [10-12] and anticancer properties [13]. A particularly interesting class of anti-tumor amidrazones is depicted in Fig. (1). The amidrazone core in this group bridges aromatic ring system from one side with piperazine, piperidine, morpholine or thiomorpholine substitution from the other side. Among this group, the p-chlorophenyl derivatives 1 and 2 [13], the thiophenyl analogue 3 [14] and the flavonyl derivatives 6 and 7 [15, 16] exhibited excellent anticancer properties and reasonable toxicities against normal fibroblast cells. However, in contrast, amidrazones bearing coumarin and 2-methylchromone moieties, e.g., compounds 4 and 5 (in Fig. 1), respectively, showed inferior cytotoxic potencies [10,17].

*

Address correspondence to these authors at the Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan and Drug Discovery Unit, Faculty of Pharmacy, The University of Jordan, Amman, Jordan; Tel: 00962777424750, E-mail: [email protected] 1573-4064/18 $58.00+.00

The excellent anti-cancer properties of 1 and 2 combined with their low toxicities against normal fibroblast cells prompted us to explore their structure-activity relationship (SAR) profile. We commenced by preparing 15 different phenyl-amidrazone analogues as shown in Scheme (1) and Table 1. Subsequently, we tested the anticancer properties of the resulting compounds against 4 cancer cell lines, namely, human prostate adenocarcinoma (PC-3), human leukemia (K562), human breast adenocarcinoma (MCF-7) and human epithelial colorectal adenocarcinoma (HCT). Several derivatives illustrated excellent anticancer properties with the best one (10c) yielding IC50 value of 1.9
M against HCT cells, as shown in Table 1. Interestingly, the prepared compounds were virtually nontoxic against normal fibroblast cells. Docking studies provided clear understanding of the corresponding SAR profile. 2. EXPERIMENTAL PART 2.1. General and Materials The following chemicals were purchased from Aldrich and were used as received: aniline, p-chloroaniline, pbromoaniline, p-fluoroaniline 3-chloropentane-2,4-dione, piperazine, N-methylpiperazine, N-ethylpiperazine, Nphenylpiperazine, N-(o-fluorophenyl)piperazine, N-(pfluorophenyl)piperazine, morpholine, thiomorpholine, and © 2018 Bentham Science Publishers

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Habashneh et al.

Fig. (1). Amidrazone derivatives of reported anti-tumor properties.

Scheme 1. Synthesis of targeted phenyl-amidrazone-piperazine derivatives 9-17.

piperidine. Melting points were determined on a Stuart scientific melting point apparatus in open capillary tubes. 1 H and 13C NMR spectra were recorded on a 500 MHz spectrometer (Bruker AVANCE-III). Chemical shifts are expressed in
units; 1H–1H, 19F-13C and 19F–1H coupling constants are given in Hertz. High resolution mass spectra (HRMS) were acquired by electrospray ionization (ESI) technique with the aid of Bruker APEX-2 instrument. The samples were dissolved in acetonitrile, diluted in spray solution (methanol/water 1:1 v/v + 0.1% formic acid), and infused using a syringe pump with a flow rate of 2 mL/min. External calibration was conducted using arginine cluster in the mass range m/z 175–871. Microanalyses data (for C, H, N) were determined on a Euro Vector elemental analysis model EA 3000, and the results agreed with the calculated percentage values within the experimental error (± 0.4 %).

2.2. Experimental Procedures 2.2.1. Synthesis of Hydrazonoyl Chlorides (8a-d) Compounds 8a-d (Scheme 1) were prepared via the Japp–Klingemann reaction [18-20] which involves coupling of the respective p-halophenylene diazonium chloride with 3-chloropentane-2,4-dione according to literature procedures [6,13,19,21]. The physical and spectral data of the prepared compounds 8a-d were identical to those described in the literature. 2.2.2. Synthesis of Amidrazones (9-17) A solution of the appropriate cyclic amine (3.1 mmol) in THF (10 mL) was added dropwise to a stirred solution of particular hydrazonoyl chloride 8a-d (0.57 mmol) and triethylamine (2 mL) in 20 mL of THF at low temperature (-10 to

SAR Exploration of Some Anti-Cancer Amidrazones

Table 1.

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The IC50 values (
M) of prepared compounds against human cancer cell lines. Values are expressed as mean ± SD of three experiments. IC50 (
M)

Compound PC-3

K562

MCF-7

HCT

9a

>100

>100

>100

>100

10a

4.8±0.8

3.2±0.6

4.1±0.5

3.4±0.6

10b

2.1±0.5

3.1±0.5

2.5±0.3

2.8±0.8

10c

3.9±1.0

3.5±0.5

2.7±0.6

1.9±0.7

10d

>100

>100

>100

>100

11a

>100

>100

>100

>100

12a

62.0±3

71.0±5

58.0±4

67.0±2

13a

8.2±2

7.9±1

8.1±2

8.2±1

13b

5.6±0.8

7.1±0.9

5.8±1.0

6.2±1.0

13c

5.2±1.0

4.2±0.8

4.9±1.0

3.1±0.9

13d

>100

>100

>100

>100

14a

15.0±2

19.0±2

13.0±1

17.0±1

15a

>100

>100

>100

>100

16a

>100

>100

>100

>100

17a

>100

>100

>100

>100

Doxorubicin*

3.7±0.8

0.009±.001

0.56±0.07

0.25±0.04

PC-3: Human prostate adenocarcinoma; K562: Human Leukemia cell line; MCF-7: Human breast adenocarcinoma; HCT: Human epithelial colorectal adenocarcinoma. * Positive control.

0°C). The mixture was stirred continually at 0–5°C for 2–4 h, and then at ambient temperature for additional 2-8 h. The reaction mixture was then poured into water (50 mL) and then the product was extracted by CHCl3 (20 mL). The organic layer was separated, washed with 2N aqueous hydrochloric acid (2 x 20mL), water (20 mL) and dried over sodium sulfate. The solvent CHCl3 was then evaporated and the resulting crude solid product was purified by recrystallization from CHCl3/ pet. ether to give pure amidrazones 9-17.

H2-3'+ H2-5' ), 7.06 (pseudo t, 2H, H-2+ H-6), 7.16–7.19 (m, 2H, H-3+ H-5), 9.29 (s, 1H,N–H,); 13C NMR (125 MHz, CDCl3),
(ppm): 25.7 (O=C-CH3), 46.7 (C-3'+ C-5'), 49.2 (C-2' + C-6'), 115.2 (d, 3JC-F = 7.7 Hz, C–2+ C-6), 116.0 (d, 2 JC-F = 22.8 Hz, C–3+C-5), 138.9 (d, 4JC-F = 1.8 Hz, C–1), 143.5 (C=N-N), 158.3 (d, 1JC-F = 238.7 Hz, C-4), 195.1 (C=O); HRMS(ESI) m/z: Calcd for C13H18FN4O [M + H] + 265.14592; found 265.14610. Anal. Calcd for
C13H17FN4O: C, 59.08; H, 6.48; N, 21.20. Found: C, 59.35; H, 6.36; N, 20.96.

1-[(4-Fluorophenyl)hydrazono]-1-morpholin-4-yl-propan2-one (9a)

1-[(4-Fluorophenyl)hydrazono]-1-thiomorpholin-4-ylpropan-2-one (11a)

84.6 % yield, mp: 120–122 °C; 1H NMR (500 MHz, CDCl3),
(ppm): 2.42 (s, 3H, CH3), 3.08 (m, 4H, H2-2'+H2 6'), 3.84 (m, 4H, H2-3'+H2-5'), 7.03–7.06 (2H, m, Ar), 7.16– 7.17 (m, 2H, Ar), 9.19 (s, 1H, N–H, exchangeable with D2O); 13C NMR (125 MHz, CDCl3),
(ppm): 25.7 (O=CCH3), 48.1 (C-2'+ C-6'), 67.5 (C-3'+ C-5'), 115.3 (d, 3JC-F = 7.7 Hz, C–2+ C-6), 116.1 (d, 2 JC-F = 22.8 Hz, C–3+ C-5), 138.8 (d, 4JC-F = 1.8 Hz, C–1), 142.7 (C=N-N), 158.4 (d, 1JCF = 240.0 Hz, C-4), 194.8 (C=O); HRMS(ESI) m/z: Calcd for C13H15FN3O2 [M - H]- 264.11538; found 264.11488.

87.2 % yield, mp: 100-121 oC; 1H NMR (500 MHz, CDCl3),
(ppm): 2.39 (s, 3H, O=C-CH3), 2.74 (m, 4H, H2 3'+ H2-5'), 3.42 (m, 4H, H2-2'+ H2-6'), 7.03 (pseudo t, 2H, H2+H-6), 7.14–7.18 (2H, m, H-3+ H-5), 9.01 (s, 1H, N–H); 13 C-NMR (125 MHz, CDCl3),
(ppm): 24.4 (O=C-CH3), 28.61 (C-3'+ C-5'), 50.24 (C-2'+ C-6'), 115.3 (d, 3JC-F = 7.6 Hz, C–2+ C-6), 116.1 (d, 2JC-F = 22.8 Hz, C–3+ C-5), 138.7 (d, 4JC-F = 1.6 Hz, C–1), 143.9 (C=N-N), 158.4 (d, 1JC-F = 239.3 Hz, C-4), 194.8 (C=O); HRMS(ESI) m/z: Calcd for C13H15FN3OS [M -H]+ 280.09253; found 280.09103.

1-[(4-Fluorophenyl)hydrazono]-1-piperazin-1-yl-propan-2one (10a)

1-[(4-Fluorophenyl)hydrazono]-1-piperidin-1-yl-propan-2one (12a)

57.4% yield, mp: 76-77 oC; 1H NMR (500 MHz, CDCl3),
(ppm): 1.77 (s, 1H, N(4')-H, exchangeable with D2O), 2.43 (s, 3H, O=C-CH3), 2.99 (m, 4H, H2-2'+ H2-6' ), 3.01 (m, 4H,

76.8 % yield, mp: 100-101 oC; 1H NMR (500 MHz, CDCl3),
(ppm): 1.64 (m, 2H, H2-4'), 1.66 (m, 4H, H2-3'+ H2-5'), 2.42 (s, 3H, O=C-CH3), 2.98 (m, 4H, H2-2'+ H2-6' ),

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7.04 (pseudo t, 2H, H-2+H-6), 7.15–7.18 (m, 2H, H-3+H-5), 9.11 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),
(ppm): 24.1 (C-4'), 25.7 (C-3'+ C-5'), 26.7 (O=C-CH3), 49.1 (C-2'+ C-6'), 115.1 (d, 3JC-F = 7.5 Hz, C–2+ C-6), 116.0 (d, 2JC-F = 22.9 Hz, C–3+C-5), 139.1 (d, 4JC-F = 2.0 Hz, C–1), 144.7 (C=N-N), 158.2 (d, 1JC-F = 239.5 Hz, C-4), 195.1 (C=O); HRMS(ESI) m/z: Calcd for C14H19FN3O [M + H] + 264.15065; found 264.14936. 1-[(4-Fluorophenyl)hydrazono]-1-(4-methylpiperazin-1-yl)propan-2-one (13a) 78.9 % yield, mp: 130-131oC; 1H NMR (500 MHz, CDCl3),
(ppm): 2.39 (s, 3H, O=C-CH3), 2.42 (s, 3H, NCH3), 2.57 (m, 4H, H2-3'+ H2-5'), 3.12 (m, 4H, H2-2'+ H2-6'), 7.05 (pseudo t, 2H, H-2+H-6), 7.16–7.19 (2H, m, H-3+H-5), 9.08 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),
(ppm): 25.6 (O=C-CH3), 46.3 (N-CH3), 47.6 (C-2'+ C-6'), 55.7 (C3'+ C-5'), 115.2 (d, 3JC-F = 7.7 Hz, C–2+ C-6), 116.0 (d, 2JC-F = 22.8 Hz, C–3+C-5), 139.0 (d, 4JC-F = 1.9 Hz, C–1), 143.4 (C=N-N), 158.3 (d, 1JC-F = 238.7 Hz, C-4), 195.1 (C=O); HRMS(ESI) m/z: Calcd for C14H20FN4O [M + H] + 279.16157; found 279.15925. Anal. Calcd for C14H19FN4O: C, 60.41; H, 6.88; N, 20.13. Found: C, 60.08; H, 6.66; N, 19.84. 1-(4-Ethylpiperazin-1-yl)-1-[(4-fluorophenyl)-hydrazono]propan-2-one (14a) 82.7 % yield, mp : 98-100 oC; 1H NMR (500 MHz, CDCl3),
(ppm): 1.16 (t, J = 7.2 Hz, 3H, CH3-CH2-), 2.42 (s, 3H, O=C-CH3), 2.53 (q, 2H, J = 7.2 Hz, CH3-CH2-N), 2.61 (m, 4H, H2-3'+ H2-5' ), 3.14 (m, 4H, H2-2'+ H2-6' ), 7.03 (pseudo t, 2H, H-2+H-6), 7.16–7.19 (m, 2H, H-3+H-5), 9.09 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),
(ppm): 11.8 (CH3-CH2), 25.7 (O=C-CH3), 47.6 (C-2'+ C-6'), 52.5 (C-+ C-5'), 53.4 (N-CH2), 115.2 (d, 3JC-F = 7.8 Hz, C–2+ C-6), 116.1 (d, 2JC-F = 22.8 Hz, C–3+ C-5), 139.0 (d, 4JC-F = 1.9 Hz, C–1), 143.4 (C=N-N), 158.3 (d, 1 JC-F = 239.1 Hz, C-4), 194.8 (C=O); HRMS(ESI) m/z: Calcd for C15H22FN4O [M + H]+ 293.17722; found 293.17567. 1-[(4-Fluorophenyl)hydrazono]-1-(4-phenylpiperazin-1-yl)propan-2-one (15a) 88.3 % yield, mp: 129-130oC; 1H NMR (500 MHz, CDCl3),
(ppm): 2.46 (s, 3H, O=C-CH3), 3.27 (m, 4H, H2 3'+ H2-5'), 3.30 (m, 4H, H2-2'+ H2-6'), 6.93 (pseudo t, 1H, H4''), 7.00 (d, 2H, J = 7.9 Hz, H-2''+ H-6''), 7.06 (pseudo t, 2H, H-2+ H-6), 7.17–7.19 (m, 2H, H-3+ H-5), 7.32 (pseudo t, 2H, H-3''+H-5''), 9.17 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),
(ppm): 25.7 (O=C-CH3), 48.0 (C-2'+ C-6'), 50.2 (C-3'+ C-5'), 115.2 (d, 3JC-F = 7.4 Hz, C–2 + C-6), 116.0 (d, 2 JC-F = 22.9 Hz, C–3+ C-5), 116.4 (C-2''+ C-6''), 120.1 (C3''+ C-5''), 129.2 (C-4''), 138.9 (d, 4JC-F = 1.9 Hz, C–1), 143.2 (C=N-N), 151.4 (C-1''), 158.4 (d, 1JC-F = 239.1 Hz, C-4), 194.9 (C=O); HRMS(ESI) m/z: Calcd for C19H22FN4O [M + H]+ 341.17722; found 341.17351. 1-[(4-Fluorophenyl)hydrazono]-1-[4-(4-fluorophenyl)piperazin-1-yl]-propan-2-one (16a) o

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83.1% yield, mp: 151-152 C; H NMR (500 MHz, CDCl3),
(ppm): 2.46 (s, 3H, O=C-CH3), 3.23 (m, 4H, H2 3'+H2-5'), 3.26 (m, 4H, H2-2'+H2-6'), 6.95 (d, 2H, J = 4.6 Hz,

Habashneh et al.

H-2''+H-6''), 7.01 (pseudo t, 2H, H-3''+H-5''), 7.06 (pseudo t, 2H, H-2+H-6), 7.17–7.19 (m, 2H, H-3+H-5), 9.16 (s, 1H, N– H); 13C NMR (125 MHz, CDCl3),
(ppm): 25.7 (O=C-CH3), 48.0 (C-2'+ C-6'), 50.2 (C-3'+ C-5'), 115.2 (d, 3JC-F = 7.4 Hz, C–2 + C-6), 115.6 (d, 2JC-F = 22.0 Hz, C-3''+ C-5''), 116.1 (d, 2 JC-F = 22.9 Hz, C–3+ C-5), 118.3 (d, 3JC-F = 7.7 Hz, C-2''+ C-6''), 138.9 (d, 4JC-F = 1.9 Hz, C–1), 143.2 (C=N-N), 148.1 (d, 4JC-F = 2.2 Hz, C-1''), 157.2 (d, 1JC-F = 237.7 Hz, C-4''), 158.4 (d, 1JC-F = 239.4 Hz, C-4), 194.9 (C=O); HRMS(ESI) m/z: Calcd for C19H19F2N4O [M - H]+ 357.15324; found 357.14923. 1-[(4-Fluorophenyl)hydrazono]-1-[4-(2-fluorophenyl)piperazin-1-yl]-propan-2-one (17a) 77.5 % yield, mp: 90-92 oC; 1H NMR (500 MHz, CDCl3),
(ppm): 2.49 (s, 3H, O=C-CH3), 3.23 (m, 4H, H2 3'+ H2-5' ), 3.29 (m, 4H, H2-2'+ H2-6' ), 7.47-7.49 (m, 6H, H2+ H-6+H-3''+ H-4''+ H-5''+H-6''), 7.17–7.19 (m, 2H, H3+H-5), 9.30 (s, 1H, N-H); 13C NMR (125 MHz, CDCl3),
(ppm): 25.7 (O=C-CH3), 48.1 (C-2'+C-6'), 51.4 (C-3'+ C-5'), 115.2 (d, 3JC-F = 7.8 Hz, C–2+ C-6), 116.1 (d, 2JC-F = 22.8 Hz, C–3+C-5), 116.3 (d, 2JC-F = 20.6 Hz, C-3''), 119.1 (d, 4JC3 F = 2.7 Hz, C-5''), 122.7 (d, JC-F = 7.9 Hz, C-4''), 124.3 (d, 3 JC-F = 3.5 Hz, C-6''), 139.0 (d, 4JC-F = 1.9 Hz, C–1), 139.9 (d, 2JC-F = 19.7 Hz, C-1''), 143.3 (C=N-N), 155.8 (d, 1JC-F = 244.8 Hz, C-2''), 158.4 (d, 1JC-F = 239.4 Hz, C-4), 194.8 (C=O); HRMS(ESI) m/z: Calcd for C19H19F2N4O [M - H] + 357.15324; found 357.14902. 1-[(4-Chlorophenyl)hydrazono]-1-piperazin-1-yl-propan-2one (10b) 64.5 % yield, mp = 226-228 oC; 1H NMR (500 MHz, CDCl3),
(ppm): 1.62 (s, 1H, N(4')-H), 2.50 (s, 3H, O=CCH3), 3.13 (m, 8H, H2-2'+ H2-3'+ H2-5'+ H2-6'), 7.03 (d, J = 8.7 Hz, 2H, H-2+H-6), 7.28 (d, J = 8.7 Hz, 2H, H-3+H-5), 9.22 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),
(ppm): 25.9 (O=C-CH3), 47.9 (C-3'+ C-5'), 48.7 (C-2'+ C-6'), 115.5 (C–2+ C-6), 127.2 (C–1), 129.5 (C–3+ C-5), 141.0 (C=N-N), 143.4 (C-4), 195.1 (C=O); HRMS(ESI) m/z: Calcd for C13H18ClN4O [M + H] + 281.11691; found 281.11558. Anal. Calcd for C13H17ClN4O: C, 55.61; H, 6.10; N, 19.96. Found: C, 55.36; H, 5.95; N, 19.64. 1-[(4-Chlorophenyl)hydrazono]-1-(4-methylpiperazin-1-yl)propan-2-one (1, Fig. 1) 81.2 % yield, mp: 119-120oC; 1H NMR (500 MHz, CDCl3),
(ppm): 2.16 (s, 3H, O=C-CH3), 2.22 (s, 3H, NCH3), 2.50 (m, 4H, H2-3'+ H2-5'), 3.06 (m, 4H, H2-2'+ H2-6'), 7.09 (d, J = 8.7 Hz, 2H, H-3+H-5), 7.24 (d, 2H, H-3+H-5), 9.05 (s, 1H, N–H); 13C NMR (125 MHz CDCl3),
(ppm): 25.8 (O=C-CH3), 46.4 (N-CH3), 47.7 (C-2'+ C-6'), 55.8 (C3'+ C-5'), 115.3 (C–2+ C-6), 126.8 (C-4), 129.4 (C–3+C-5), 141.3 (C=N-N), 143.8(C–1), 195.0 (C=O); HRMS(ESI) m/z: Calcd for C14H20ClN4O [M + H]+ 295.13256; found 295.13276. Anal. Calcd for C14H19ClN4O: C, 57.04; H, 6.50; N, 19.01. Found: C, 56.73; H, 6.52; N, 18.90. 1-[(4-Bromophenyl)-hydrazono]-1-piperazin-1-yl-propan2-one (10c) 68.2 % yield, mp: 235-237 oC; 1H NMR (500 MHz, CDCl3),
(ppm): 1.62 (s, 1H, N(4')-H), 2.39 (s, 3H, O=C-

SAR Exploration of Some Anti-Cancer Amidrazones

CH3), 3.13 (m, 8H, H2-2'+ H2-3'+ H2-5'+ H2-6'), 6.86 (d, J = 8.7 Hz, 2H, H-2+H-6), 7.25 (d, 2H, H-3+H-5), 9.22 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),  (ppm): 25.9 (O=CCH3), 46.1 (C-3'+ C-5'), 48.5 (C-2'+ C-6'), 114.3 (C-4), 114.5(C–2+ C-6), 129.5 (C–3+ C-5), 141.5 (C=N-N), 143.4 (C–1), 195.1 (C=O); HRMS(ESI) m/z: Calcd for C13H1879BrN4O [M + H]+ 325.06640; found. 325.06662, Calcd for C13H1881BrN4O [M + H]+ 327.06435; found. 327.06457. Anal. Calcd for C13H17BrN4O: C, 48.01; H, 5.27; N, 17.23. Found: C, 48.18; H, 5.17; N, 17.05. 1-[(4-Bromophenyl)-hydrazono]-1-(4-methylpiperazin-1yl)-propan-2-one (13c) 85.7 % yield, mp: 121-122 oC; 1H-NMR (500 MHz, CDCl3),  (ppm): 2.32 (s, 3H, O=C-CH3), 2.37 (s, 3H, NCH3), 2.50 (m, 4H, H2-3'+ H2-5'), 3.05 (m, 4H, H2-2'+ H2-6'), 7.04 (d, J = 8.7 Hz, 2H, H-2+H-6), 7.38 (d, 2H, H-3+H-5), 9.02 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),  (ppm): 25.7 (O=C-CH3), 46.4 (N-CH3), 47.7 (C-2'+ C-6'), 55.8 (C3'+ C-5'), 114.1 (C-4), 115.7 (C–2+C-6), 132.3 (C–3+C-5), 141.8 (C=N-N), 143.8 (C–1), 194.8 (C=O); HRMS(ESI) m/z: Calcd for C14H2079BrN4O [M + H] + 339.08205; found 339.0792, Calcd for C13H1881BrN4O [M + H]+ 341.08000; found. 341.08023. Anal. Calcd for C14H19BrN4O: C, 49.57; H, 5.65; N, 16.52. Found: C, 49.61; H, 5.64; N, 16.35. 1-(Phenylhydrazono)-1-piperazin-1-yl-propan-2-one (10d) 59.6 % yield, mp: 194-196 oC; 1H NMR (500 MHz, CDCl3),  (ppm): 1.62 (s, 1H, N(4')-H), 2.48 (s, 3H, O=CCH3), 3.15 (m, 8H, H2-2'+ H2-3'+ H2-5'+ H2-6'), 6.96 (pseudo t, 1H, H-4), 7.25 (d, J = 7.3 Hz, 2H, H-2+H-6), 7.53 (pseudo t, 2H, H-3+H-5), 9.29 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),  (ppm): 25.8 (O=C-CH3), 48.7 (C-3'+ C-5' + C-2' + C-6'), 114.5 (C–2+ C-6), 122.4(C-4), 129.6 (C–3+C-5), 142.3 (C=N-N), 142.4 (C–1), 195.4 (C=O); HRMS(ESI) m/z: Calcd for C13H19N4O [M + H]+ 247.15589; found. 247.15605. 1-(4-Methylpiperazin-1-yl)-1-(phenylhydrazono)-propan-2one (13d) 78.4 % yield, mp: 77—79oC; 1H NMR (500 MHz, CDCl3),  (ppm): 2.35 (s, 3H, O=C-CH3), 2.38 (s, 3H, NCH3), 2.54 (m, 4H, H2-3'+ H2-5'), 3.08 (m, 4H, H2-2'+ H2-6'), 6.96 (pseudo t, 1H, H-4), 7.17 (d, J = 7.3 Hz, 2H, H-2+H-6), 7.29 (pseudo t, 2H, H-3+H-5), 9.07 (s, 1H, N–H); 13C NMR (125 MHz, CDCl3),  (ppm): 25.7 (O=C-CH3), 46.2 (NCH3), 47.5 (C-2' + C-6'), 55.7 (C-3'+ C-5'), 114.1 (C–2+ C6), 122.1 (C-4), 129.4 (C–3+C-5), 142.6 (C=N-N), 143.3 (C– 1), 194.9 (C=O); HRMS(ESI) m/z: Calcd for C14H21N4O [M + H]+ 261.17154; found 261.17171. 2.2.3. Cell Line and Culture Conditions Cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Invitrogen, USA) containing 10% heat inactivated fetal bovine serum (HI-FBS) (Invitrogen), 2 mmol L–1 of L-glutamine, 50 U mL–1 of penicillin and 50 μg mL–1 of streptomycin. All cells were maintained in an atmosphere of 5 % CO2 with 95 relative humidity at 37 °C.

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2.2.4. In vitro Cytotoxicity All cells were seeded at 5x103 cells per well in 96-well plates and incubated to allow adhesion for 24 h. Compounds under investigation were dissolved in dimethylsulfoxide (DMSO) and subsequently diluted in culture media (test sample). The final DMSO concentration was maintained constant at 1% v/w. For each tested compound, three triplicates of each concentration were evaluated in three independent assays for a total of 9 triplicates. DMEM samples were employed as negative controls while doxorubicin was used as a positive control. Test samples were allowed to incubate for 24 h at 37 °C in a 5 % CO2 incubator. At the end of the exposure period, MTT assay was carried out as outlined in the manufacturer’s protocol. Briefly, viable cell count was determined using the 3-(4,5dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. The yellow tetrazolium dye [MTT, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] was reduced by metabolically active cells into an intracellular purple formazan product. The quantity of formazan product, as determined by the absorbance at 490 nm, is directly proportional to the number of viable cells in the culture. Cell viability was calculated based on the measured absorbance relative to the absorbance of the cells exposed to the negative controls, which represented 100% cell viability. 2.2.5. Docking Experiments The chemical structures of compounds 9a, 10c, 10d, 11a, 13a, 13c, 13d, and 15a were sketched in Chemdraw ultra (version 7.01) and saved in MDL SD file format. The binding site was generated from imatinib co-crystallized within bcr/abl kinase domain (PDB code: 1IEP, resolution = 2.10 ). Hydrogen atoms were added to the protein utilizing Discovery Studio 2.5 templates for protein residues. The protein structure was utilized in subsequent docking experiments without energy minimization. Explicit water molecules were kept in the structure. Docking was performed using LigandFit docking engine. LigandFit considers the flexibility of the ligand and treats the receptor as rigid. LigandFit docking is composed of few steps ([22]): (i) Conformational search of flexible ligands employing Monte Carlo randomized search. (ii) Selection of ligand poses and conformations based on shape similarity with the binding site. (iii) Candidate conformers/poses are enrolled in calculation of the dock (enthalpic) energies. (iv) Each docked conformation/pose is further fitted into the binding pocket through a number of rigid-body minimization iterations. (v) Docked conformers and poses that have docking energies below certain user-defined threshold are subsequently clustered according to their RMS similarities. Representative conformers and poses are then selected, further energy-minimized within the binding site, and saved for subsequent scoring ([22]). In the current research the following LigandFit settings were implemented: (i) number of Monte Carlo search trials 30000, search step for torsions with polar hydrogens = 30 degree. (ii) The Root Mean Square Difference (RMS) threshold for ligand-to-binding site shape match was set to 4.0 Å employing a maximum of 1.0 binding site partitions.

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Fig. (2). Comparison between the docked pose of imatinib (red) as produced by docking simulation and the crystallographic structure of this inhibitor within bcr/abl kinase domain (blue, PDB code: 1IEP).

(iii) The interaction energies were assessed employing Consistent Force Field (CFF) with a nonbonded cutoff distance of 10.0 Å and distance-dependent dielectric. An energy grid extending 5.0 Å from the binding site was implemented. (iv) Rigid body ligand minimization parameters were: 50 iterations of steepest descend (SD) minimization followed by 100 Broyden–Fletcher–Goldfarb–Shanno (BFGS) iterations applied to every successful orientation of the docked ligands. (v) A maximum of 10 highest-ranking docked conformations/poses of optimal interaction energies were saved for each docked structure. (vi) The saved conformers/poses were further energy-minimized within the binding site for a maximum of 1000 iterations using CHARMm force field. The resulting docked poses were scored employing consensus scoring based on PLP1, PLP2 [23], ligscore1 and ligscore2 [24], PMF [25], and JAIN [26] scoring functions. The docked poses of best consensus scores were analyzed and shown in Figs. (3 and 4). 2.2.6. Calculation of Binding Energy The binding energies of docked ligands (shown in Figs. (3 and 4), i.e., 9a, 10c, 10d, 11a, 13a, 13c, 13d, 15a, and imatinib) were estimated as described by Tirado-Rives and Jorgensen [27] and implemented in Discovery Studio (version 2.5). This approach requires single receptor structure and a set of bound ligand poses to calculate binding energy. The highest ranking 10 docked poses were selected for each docked ligand for this purpose. The generalized Born implicit solvent model was used in the calculations. The results are summarized in Table 2. 3. RESULTS 3.1. Chemistry The hydrazonoyl chlorides 8a-d (Scheme 1) were prepared via direct coupling of the respective phalophenylenediazonium chlorides with 3-chloropentane2,4-dione in aqueous pyridine solution (Japp–Klingemann reaction) [6, 13, 18-21]. The desired amidrazone adducts 917 were obtained by nucleophilic addition of the appropriate cyclic-secondary amine onto the particular nitrile-imine (the reactive 1,3-dipolar species generated in situ from its corresponding hydrazonoyl chloride precursors 8a-d in the presence of trimethylamine) (Scheme 1).

The newly synthesized amidrazone derivatives 9-17 were characterized by MS and NMR spectral data. These data, detailed in the experimental section, are consistent with the suggested structures. Thus, the mass spectra display the correct molecular ion peaks for which the measured high resolution mass spectral (HRMS) data are in good agreement with the calculated values. DEPT 135 and 2D (COSY, HMQC and HMBC) experiments showed correlations that helped in the 1H- and 13C-signal assignments to the different carbons and their attached and/or neighboring hydrogens. 3.2. Biology The new amidrazones were screened against leukemia, prostate, breast and colon cancers. Interestingly, some members exhibited potent cytotoxic bioactivities. Compounds 10a, 10b, 10c, 13a, 13b, 13c and 14a were particularly potent exhibiting IC50 values within low micromolar range, as in Table 1. The remaining compounds were virtually inactive except for the flourobenzene-piperidine amidrazone 12a which exhibited moderate IC50 values ranging between 58 to 71
M, as in Table 1. All compounds were virtually nontoxic to normal fibroblast cells (IC50 values > 100
M). Elemental microanalyses (for C, H and N) indicated satisfactory purities of compounds 10a, 10b, 10c, 13a, 13b, 13c and 14a with percentage purities within experimental error (± 0.4 %). This rules out the possibility that the observed anticancer properties are due to impurities (e.g., of synthetic origin). 4. DISCUSSION The pharmacophoric similarities between aromatic amidrazone derivatives and the central part of the anticancer agent imatinib are well established [15, 16]. In fact, docking and structure-activity relationship (SAR) studies on anticancer flavone derivatives 6 and 7 (Fig. 1) showed they bind and block oncogenic protein tyrosine kinases, particularly bcr/abl, in a comparable fashion to imatinib [15, 16]. Accordingly, we anticipated our new amidrazones to share some pharmacophoric similarities with imatinib, and thus, bind and block bcr/abl. This assumption is further supported by the fact that bcr/abl is over-expressed in leukemia, prostate, breast and colon cancers cell lines [28].

SAR Exploration of Some Anti-Cancer Amidrazones

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Fig. (3). (A) X-ray crystallographic structure of imatinib co-crystallized within bcr/abl kinase domain (PDB code: 1IEP, resolution = 2.10 ), (B), (C), and (D)represent the docked poses of potent compounds 10c, 13a and 13c, respectively, into the same protein. Hydrogen bonding interactions are shown as dotted green lines. Table 2.

Calculated binding energies for docked compounds in Figs. (3 and 4). Compound

Binding Energy (kcal/mol)

9a

-23.58

10c

-29.93

10d

-22.38

11a

-23.52

13a

-29.87

13c

-32.89

13d

-25.75

15a

-21.32

Imatinib*

-50.06

* Docked pose of imatinib as in Fig. (2).

To test this hypothesis we docked the new amidrazones into the ATP binding pocket of bcr/abl and compared the

resulting conformers/poses with the co-crystallographic pose of imatinib (IC50 against bcr/abl = 190 nM) [29] within bcr/abl (PDB code: 1IEP). However, prior to this step it was necessary to validate the docking settings by comparing docked and co-crystallized (experimental) poses of imatinib within bcr/abl, as in Fig. (2). Clearly, our docking settings closely reproduced the bound pose of imatinib with RMSD value of 0.32 Å thus allowing us to confidently proceed to dock the new amidrazones into bcr/abl. Fig. (3) compares the co-crystallized pose of imatinib with the docked poses of potent inhibitors 10c, 13a and 13c (Scheme 1 and Table 1). Although imatinib's pyridinylpyrimidine fragment (present within aromatic cage comprised of Phe382, Tyr253 and Phe317, Fig. 3A) is not represented in the new amidrazones, nevertheless, the hydrogen bonding interactions connecting the central amidic linker of imatinib with the carboxylic acid side chain of Glu286 and the peptidic NH of Asp381 (Fig. 3A) agree with hydrogen-bonding interactions connecting the amidrazone =N
NH
system with same protein atoms

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Fig. (4). Docked poses of inactive compounds: (A) 9a, (B) 11a, (C) 15a, (D) 10d and (E) 13d into the binding pocket of the kinase domain of bcr/abl (PDB code: 1IEP, resolution = 2.10 ).

(COOH of Glu286 and peptidic NH of Asp381, Figs. 3B3D). Similar analogy can be noticed between hydrogen bonding interaction connecting the hydroxyl of Thr315 with the central aniline NH of imatinib (Fig. 3A) compared to putative hydrogen-bonding connecting the aromatic halogens of 10c, 13a and 13c (Figs. 3B-3D) with the same hydroxyl (i.e., of Thr315). Furthermore, the close proximity and apparent hydrophobic stacking of the methylbenzene linker of imatinib against the butylene of Lys271 (Fig. 3A) compares to fitting the halogenated-phenyl rings of our active amidrazones close to the same side chain (Figs. 3B-3D) allowing similar hydrophobic interactions. Similarly, the electrostatic attraction connecting the piperazine ring of imatinib with the carboxylate side chain of Asp381 (Fig. 3A) corresponds to electrostatic attraction connecting the piperazine nitrogens of 10c, 13a and 13c with the same carboxylate group in the binding pocket (Figs. 3B-3D). Moreover, the closer proximity between the carboxylate of Asp381 and the cationic piperazine nitrogen atoms of the new amidrazones (10c, 13a and 13c) should allow stronger

hydrogen-bonding-reinforced electrostatic attraction compared to the equivalent interaction in imatinib's case (cationic piperazine with Asp381 carboxylate). On the other hand, inactive analogs 9a, 11a, and 15a (Figs. 4A to 4C) have either non-ionizable substituents analogous to piperazine (e.g., morpholine in 9a or thiomorpholine in 11a) or have their ionizable piperazine nitrogen atoms conjugated to aromatic rings that significantly weaken their ionizabilities (as in 15a). Accordingly, these derivatives are expected to lose the electrostatic attractive interaction with the carboxylate of Asp381. The docked poses of these analogues support this proposition as in Figs. (4A-4C). Loss of activity in these compounds (9a, 11a, and 15a) indicates the critical significance of ligands' electrostatic attraction with Asp381. Still, the hydrogen-bonding interactions connecting the aromatic halogen substitutents of amidrazone ligands with the hydroxyl of Thr315 (seen in potent analogues 10c, 13a and 13c, Fig. 3B-3D) appear to also have crucial role in ligand-protein affinity. This conclusion is evident from that

SAR Exploration of Some Anti-Cancer Amidrazones

fact that inactive compounds 10d and 13d (Figs. 4D and 4E) lack halogen substituents on their phenyls and therefore lost any hydrogen-bonding propensities with Thr315 hydroxyl. In fact, the moderate potency of 12a (Scheme 1 and Table 1) indicates that this interaction is even more crucial for ligand binding than the electrostatic attraction connecting ligands' piperazine rings with Asp381 carboxylate. Clearly, 12a has piperidine ring incapable of electrostatic attraction with Asp381, still the presence of fluorinated phenyl fragment at close proximity to Thr315 hydroxyl allowed mutual hydrogen-bonding and yielded moderate anticancer properties. Binding energy estimates support our conclusions, i.e., the combined effects of hydrogen bonding to Thr315 and electrostatic attraction to Asp381 promote ligand binding to Bcr/Abl binding site. Table 2 shows the estimated binding energies of docked compounds in Figs. (3 and 4) compared to imatinib. Clearly, potent inhibitors 10c, 13a and 13c showed more negative (exothermic) binding energies (32.89, -29.87 and -29.93 kcal/mol, respectively) compared to inactive inhibitors 9a, 10d, 11a, 13d and 15a (rang from 25.75 to -21.32 kcal/mol) suggesting that potent inhibitors have stronger binding interactions compared to inactive inhibitors. Unsurprisingly, imatinib showed the highest exothermic binding energy (-50.06 kcal/mol), which correlates with high affinity reported for imatinib towards Bcr/Abl [29]. In conclusion, we prepared and tested 15 phenylamidrazone-piperazine derivatives against four cancer cell lines. Six compounds illustrated low micromolar IC50 values against, while the remaining were inactive or of moderate potencies. Docking studies into the oncogenic kinase bcr/abl illustrated the critical importance of ligand's electrostatic and hydrogen-bonding interactions with binding site's Asp381 and Thr315, respectively. ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Medicinal Chemistry, 2018, Vol. 14, No. 00

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

Not applicable. HUMAN AND ANIMAL RIGHTS No Animals/Humans were used for studies that are base of this research.

[13]

[14]

CONSENT FOR PUBLICATION Not applicable.

[15]

CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

[16]

ACKNOWLEDGMENTS

[17]

The authors thank the Deanship of Scientific Research at The University of Jordan for funding the current research. [18]

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