Synthesis and antiproliferative activity of monocationic arylthiophene derivatives

Accepted Manuscript Synthesis and antiproliferative activity of monocationic arylthiophene derivatives Mohamed A. Ismail, Magdy M. Youssef, Reem K. Arafa, Shar S. Al-Shihry, Wael M. El-Sayed PII:

S0223-5234(16)31013-3

DOI:

10.1016/j.ejmech.2016.12.007

Reference:

EJMECH 9098

To appear in:

European Journal of Medicinal Chemistry

Received Date: 29 October 2016 Revised Date:

1 December 2016

Accepted Date: 2 December 2016

Please cite this article as: M.A. Ismail, M.M. Youssef, R.K. Arafa, S.S. Al-Shihry, W.M. El-Sayed, Synthesis and antiproliferative activity of monocationic arylthiophene derivatives, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Graphical abstract

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4i; X = H, R1 = H, R2 = Cl, R3 = H

GI50 = 0.20, TGI = 0.37, LC50 = 35.5 µM

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LC50's (µM) = 130.9 (WI38); 146.5 (WISH)

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Synthesis and Antiproliferative Activity of Monocationic Arylthiophene Derivatives

Mohamed A. Ismail,a,b* Magdy M. Youssef, a,b Reem K. Arafa,c Shar S. Al-Shihry,a Wael M. El-

a

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Sayed.d*

King Faisal University, College of Science, Department of Chemistry, Hofuf 31982, Saudi

Arabia b c

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Department of Chemistry, Faculty of Science, Mansoura University, Mansoura 35516, Egypt. Biomedical Sciences Program, University of Science and Technology, Zewail City of Science

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and Technology. Cairo 12588, Egypt

University of Ain Shams, Faculty of Science, Department of Zoology, Abbassia 11566, Cairo,

*

Correspondence to:

Mohamed A. Ismail

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Egypt.

Mansoura University, Faculty of Science, Department of Chemistry, Mansoura 35516, Egypt. Tel: +2-01096849505

Fax: +2-050-2246254

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Email; [email protected]; [email protected]

Wael M. El-Sayed,

University of Ain Shams, Faculty of Science, Department of Zoology, Abbassia, 11566, Cairo, Egypt. Tel: +202/2482-1633

Fax: +202/2684-2123

Email; [email protected], [email protected]

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Abstract: Eleven compounds of substituted 4-(5-arylthiophen-2-yl)benzamidines 4a-k were synthesized from their corresponding mononitriles via treatment with lithium trimethylsilylamide and

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subsequent de-protection with ethanol/hydrogen chloride. In vitro antiproliferative activities of the new monocationic arylthiophenes were evaluated against 60 human cell lines at NCI, USA. This class of compounds displayed promising submicromolar antiproliferative activities with the

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most potent compound being 4i (GI50 and TGI of 0.20 and 0.37 µM, respectively). On the other hand, most of the tested compounds exhibited LC50 at concentrations much higher than those

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they had GI50 at; ~ 10x (for 4b) up to 228x (for 4e) which indicates lower lethality and efficient growth inhibition. Cancer cell lines, HCC-2998 colon, SNB-75 CNS, MDA-MB-435 melanoma, and MCF-7 breast cancer were the most responsive, with GI50s of 0.156, 0.165, 0.163, and 0.168 µM, respectively. The p-chlorophenyl derivatives 4e and 4i discerned themselves with GI50

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values at 0.36 and 0.20 µM, respectively, and LC50 values at ~83 and 36 µM, respectively, but safe to RBCs at 1000 µM. The cytotoxic activity data of these compounds in two normal cell lines; WI38 and WISH proved that they are very safe on normal cells. The plausible mechanism

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of action of the tested monocations was examined by evaluating their antioxidant power, nuclease-like DNA degradation aptitude and tyrosine kinase (TK) inhibition activities. The tested

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monocations showed potent activity in all assays. Compounds 4e and 4i caused 88 and 98%, respectively, inhibition in TK activity at 1 µM and the IC50 for 4i was 13 nM. The tested monocations have selective anticancer activity without insulting normal cells most probably due to inhibition of the key enzyme TK at nanomolar concentrations.

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Keywords: monocationic arylthiophenes, Suzuki coupling, antiproliferative activity, DNA

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affinity, tyrosine kinase inhibition.

1. Introduction

The high incidence of cancer and the high cost of its treatment are important factors

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driving the search for new and effective chemotherapeutic substances with multi-target activity and safe to normal cells. Thiophene containing molecules have been proven as a promising

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chemotype for the design and synthesis of small biologically active molecules possessing a wide spectrum of activities.1-8 In particular, a series of bichalcophene diamidines including asymmetrical bithiophene diamidines, analogue of compound II (Figure 1), showed potent antiprotozoal activity and good DNA binding affinity.3 Moreover, monoamidinic bithiophenes I & II showed good antibacterial,4,5 antimutagenic,6,7 and anticancer activities.8 Recently, a

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number of cationic aromatic compounds, including thiophene based structures have been reported to bind at AT-rich sites of the DNA minor-groove.9-13 It is hypothesized that cationic molecules elicit the observed biological activity by binding to DNA, followed by inhibiting one

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or more of several DNA dependent enzymes.14-16 More recently, a series of substituted phenyl-

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furanylnicotinamidines showed good antibacterial and antiproliferative activities.17 The lack of selectivity and specificity of typical chemotherapy and incapability to

differentiate between the normal rapidly dividing cells and the tumor cells is responsible for the severe side effects. Seeking for novel selective cytotoxic/cytostatic agents that are specific to tumor cells is the central goal of our lab. Since most tumor cells overexpress different types of cytosolic and receptor tyrosine kinases (TK); we designed novel arylthiophen benzamidines and screened them as anticancer agents and as TK inhibitors. Different TKs are involved in many 3

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aspects of cell cycle and many phases of cancer development. TKs are involved in cell proliferation, growth, apoptosis, metastasis, and angiogenesis. Novel anticancer targeted drugs like imatinib, vatalanib, and gefitinib are well known TKIs.18

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In view of the importance of recently reported cationic thiophene derivatives as therapeutic agents and DNA binders, we have synthesized new subclass of compounds I and II, which showed potent antiproliferative activities, by replacing the terminal thiophene ring with

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phenyl, substituted phenyl bioisoteres. Molecular manipulations have also included the positional isomerism technique by altering the position of the fluorine atom from the amidinic phenyl ring

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to the non-amidinic phenyl ring. In this report, DNA nuclease-like ability of this class of compounds was also performed. It is important that these novel compounds exert their cytotoxic activity against cancer cells and pose no or minimal toxicity on normal cells. Therefore, the novel monocationic arylthiophenes were tested for their toxicity in normal human lung fibroblast

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(WI-38) and human amniotic cells (WISH). Finally, we performed the superoxide dismutase

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mimetic activity and erythrocyte hemolysis assay.

Figure 1: Biologically important fluorinated and non-fluorinated thiophene based structures

2. Experimental Section 2.1. Chemistry

All chemicals and solvents were purchased from Sigma-Aldrich Chemical Co. or Fisher Scientific. 4-(5-Bromothiophen-2-yl)-2-fluorobenzonitrile was prepared employing a previously

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reported literature method.6 A Gallenkamp melting-point apparatus was used to record melting points (uncorrected). Infrared (IR) spectra were recorded on a Shimadzu 5800 Fourier transform FT-IR spectrometer using KBr wafer technique. Thin-layer chromatography was made using

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silica gel 60 F254 precoated aluminum sheets and detection was done under ultraviolet light. Mass spectra were performed on a GC-MS (Schimadzu Qp-2010 Plus) spectrometer. 1H and 13C NMR spectra were measured employing a Varian Mercury VX-300 spectrometer and a Bruker Avance

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400MHz spectrometer, and chemical shifts (δ) were in parts per million (ppm) relative to the dimethylsulfoxide-d6 (DMSO-d6) as a solvent. Elemental analyses were measured on a Perkin-

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Elmer 2400 analyzer at the microanalytical laboratories of the Faculty of Science, Cairo University and were within ±0.4 of the theoretical values (Table S1; Supplementary data). 2.1.1. General Procedure for preparation of 4-(5-arylthiophen-2-yl)benzonitriles 3a-k: To a stirred solution of the appropriate bromobenzonitrile (5 mmol), and tetrakis(triphenyl-

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phosphine) palladium (150 mg) in toluene (10 mL) was added 5 mL of a 1.5 M aqueous solution of NaHCO3 followed by phenylboronic acid derivative (6 mmol) in 5 mL of methanol. The vigorously stirred reaction mixture was warmed to 80 oC for 16 hr. The solvent was then

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evaporated to dryness, and the solid residue was partitioned between methylene chloride (250 mL) and an aqueous solution containing 5 mL concentrated ammonia. The target diarylthiophene

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carbonitriles were obtained from the methylene chloride layer upon evaporation to dryness under vacuum.

2-(4-Cyano-3-fluorophenyl)-5-phenylthiophene (3a): A pale yellow solid in 69% yield, mp 148-149 oC (EtOH). Rf = 0.69, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3121, 3080, (C-H), 2228 (CN), 1614, 1556, 1539 (C=C). MS (EI) m/e (rel.int.); 279 (M+, 100), 245 (5).

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H NMR; δ (ppm) 7.21-7.24 (m, 1H), 7.34-7.49 (m, 3H), 7.64 (d, J = 3.9 Hz, 1H), 7.66-7.74 (m,

2H), 7.83-7.97 (m, 3H). Anal. (C17H10FNS) C, H, N. 2-(4-Cyano-3-fluorophenyl)-5-(4-methoxyphenyl)thiophene (3b): A yellow solid in 72%

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yield, mp 201-202.5 oC. Rf = 0.48, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3095, 2977, 2937 (C-H), 2227 (CN), 1612, 1557, 1544 (C=C). MS (EI) m/e (rel.int.); 309 (M+, 100), 294 (62). 270 (26). 1H NMR; δ (ppm) 3.79 (s, 3H), 7.02 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 3.9 Hz,

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1H), 7.64 (d, J = 8.7 Hz, 2H) 7.79-7.91 (m, 4H). Anal. (C18H12FNOS) C, H, N.

2-(4-Cyano-3-fluorophenyl)-5-(3,5-dimethoxyphenyl)thiophene (3c): A yellowish-green solid

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in 67% yield, mp 142-143 oC. Rf = 0.41, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3093, 3047, 3008, 2978, 2947 (C-H), 2230 (CN), 1619, 1590, 1560 (C=C). MS (EI) m/e (rel.int.); 339 (M+, 100), 310 (16). 1H NMR; δ (ppm) 3.81 (s, 6H), 6.51 (s, 1H), 6.84 (s, 2H), 7.65-7.71 (m, 2H), 7.83-7.96 (m, 3H). Anal. (C19H14FNO2S) C, H, N.

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2-(4-Cyano-3-fluorophenyl)-5-[(4-dimethylamino)phenyl]thiophene (3d): A brown-yellow solid in 75% yield, mp 217.5-219 oC. Rf = 0.55, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3075, 2920, 2901 (C-H), 2232 (CN), 1607, 1565, 1554 (C=C). MS (EI) m/e (rel.int.); 322

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(M+, 100), 306 (17). 1H NMR; δ (ppm) 2.96 (s, 6H), 6.77 (d, J = 9.0 Hz, 2H), 7.39 (d, J = 3.9 Hz,

H, N.

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1H), 7.54 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 8.4 Hz, 1H), 7.78-7.92 (m, 3H). Anal. (C19H15FN2S) C,

2-(4-Cyano-3-fluorophenyl)-5-(4-chlorophenyl)thiophene (3e): A brown-yellow solid in 82% yield, mp 226-228 oC. Rf = 0.71, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3081, 3046 (C-H), 2228 (CN), 1613, 1559, 1541 (C=C). MS (EI) m/e (rel.int.); 313, 315 (M+, 92, 37: chlorine isotopes), 277 (12). 80 (100). 1H NMR; δ (ppm) 7.50 (d, J = 8.7 Hz, 1H), 7.63-7.73 (m, 5H), 7.83 (d, J = 3.9 Hz, 1H), 7.87-7.95 (m, 2H). Anal. (C17H9ClFNS) C, H, N. 6

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2-(4-Cyanophenyl)-5-(4-methoxyphenyl)thiophene (3f): A golden-yellow solid in 73% yield, mp 160-161 oC, Lit.19 mp 158 oC. Rf = 0.60, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3072, 3052, 2963, 2936 (C-H), 2227 (CN), 1601, 1572, 1541 (C=C). MS (EI) m/e (rel.int.);

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291 (M+, 52), 276 (41), 80 (100).

2-(4-Cyanophenyl)-5-(3,5-dimethoxyphenyl)thiophene (3g): A brown-yellow solid in 81% yield, mp 145-146 oC. Rf = 0.52, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3069,

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2973, 2941 (C-H), 2226 (CN), 1589, 1556, 1538 (C=C). MS (EI) m/e (rel.int.); 321 (M+, 100), 307 (19), 292 (18). 1H NMR; δ (ppm) 3.81 (s, 6H), 6.51 (s, 1H), 6.84 (s, 2H), 7.65 (d, J = 3.9 Hz,

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1H), 7.76 (d, J = 3.9 Hz, 1H), 7.88 (s, 4H). Anal. (C19H15NO2S) C, H, N.

2-(4-Cyanophenyl)-5-[(4-dimethylamino)phenyl]thiophene (3h): A brown-yellow solid in 70% yield, mp 139-140 oC, Lit.19 mp 138 oC. Rf = 0.62, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3080, 2892, 2857 (C-H), 2222 (CN), 1599, 1554, 1541 (C=C). MS (EI) m/e

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(rel.int.); 304 (M+, 100), 289 (15).

2-(4-Cyanophenyl)-5-(4-chlorophenyl)thiophene (3i): A golden-yellow solid in 84% yield, mp 80-81 oC, Lit.19 mp 80 oC. Rf = 0.70, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3049

isotopes), 259 (12).

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(C-H), 2222 (CN), 1601, 1555, 1539. MS (EI) m/e (rel.int.); 295, 297 (M+, 100, 42: chlorine

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2-(4-Cyanophenyl)-5-(3,5-dichlorophenyl)thiophene (3j): A golden-yellow solid in 85% yield, mp 199-200 oC (EtOH/DMF). Rf = 0.79, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3136, 3100 (C-H), 2232 (CN), 1602, 1592, 1553 (C=C). MS (EI) m/e (rel.int.); 330, 331, 332, 333 (M+, 18, 100, 23, 53: chlorine isotopes), 299 (9), 295 (9). 1H NMR; δ (ppm) 7.52 (d, J = 3.9 Hz, 1H), 7.71-7.78 (m, 4H), 7.83-7.87 (m, 4H). Anal. (C17H9Cl2NS) C, H, N.

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2-(4-Cyanophenyl)-5-(4-fluorophenyl)thiophene (3k): A golden-yellow solid in 80% yield, mp 161-162 oC, Lit.19 mp 162 oC. Rf = 0.70, petroleum ether (60-80 oC)-EtOAc (8:2). IR (ν’ cm-1 ); 3071, 3053 (C-H), 2222 (CN), 1600, 1556, 1543 (C=C). MS (EI) m/e (rel.int.); 279 (M+, 88),

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260 (3), 80 (100).

2.1.2. General method for the preparation of 4-(5-arylthiophen-2-yl)benzamidines 4a-k:

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Substituted 4-(5-arylthiophen-2-yl)benzamidines 4a-k were prepared from their corresponding carbonitriles 3a-k, employing a typical methodology recently described.17

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2-(4-Amidino-3-fluorophenyl)-5-phenylthiophene hydrochloride salt (4a): A golden-yellow solid in 60% yield, mp 297-298.5 oC. IR (ν’ cm-1 ); 3350, 3233 (NH, NH2), 3125, 3011 (C-H), 1657, 1622, 1566, 1552 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.); 296 (M+, 100), 280 (55), 279 (24). 1H NMR; δ (ppm) 7.34-7.49 (m, 3H), 7.64 (d, J = 3.9 Hz, 1H), 7.69-7.87 (m,

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6H), 9.52 (br s, 4H, exchangeable with D2O). Anal. (C17H13FN2S-1.0HCl-0.5H2O) C, H, N. 2-(4-Amidino-3-fluorophenyl)-5-(4-methoxyphenyl)thiophene hydrochloride salt (4b): A yellow solid in 64% yield, mp 276-278 oC. IR (ν’ cm-1 ); 3366, 3230 (NH, NH2), 3115, 2967,

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2931 (C-H), 1671, 1618, 1572, 1551 (C=N, C=C stretch , NH bend). MS (EI) m/e (rel.int.); 326 (M+, 100), 311 (48), 309 (18). 1H NMR; δ (ppm) 3.80 (s, 3H), 7.03 (d, J = 8.7 Hz, 2H), 7.50 (d, J

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= 3.9 Hz, 1H), 7.64-7.85 (m, 6H), 9.46 (br s, 4H, exchangeable with D2O). Anal. (C18H15FN2OS1.0HCl-1.0H2O) C, H, N.

2-(4-Amidino-3-fluorophenyl)-5-(3,5-dimethoxyphenyl)thiophene hydrochloride salt (4c): A yellow solid in 69% yield, mp 287-289 oC. IR (ν’ cm-1 ); 3350, 3239 (NH, NH2), 3092, 2996, 2963 (C-H), 1666, 1619, 1597, 1535 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.); 356 (M+, 100), 339 (98), 310 (47). 1H NMR; δ (ppm) 3.82 (s, 6H), 6.52 (s, 1H), 6.86 (s, 2H), 7.67 (d,

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J = 3.9 Hz, 1H), 7.72-7.88 (m, 4H), 9.47, 9.51 (br s, 4H, exchangeable with D2O). Anal. (C19H17FN2O2S-1.0HCl) C, H, N. 2-(4-Amidino-3-fluorophenyl)-5-[(4-dimethylamino)phenyl]thiophene hydrochloride salt

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(4d): A brown-yellow solid in 70% yield, mp 291-293 oC. IR (ν’ cm-1 ); 3439, 3171 (NH, NH2), 3050, 3025, 2986, 2901 (C-H), 1675, 1621, 1565, 1535 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.); 339 (M+, 41), 322 (100), 307 (10). 1H NMR; δ (ppm) 2.97 (s, 6H), 6.86 (m, 2H),

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7.41 (d, J = 3.9 Hz, 1H), 7.57 (d, J = 8.7 Hz, 2H), 7.66-7.82 (m, 4H), 9.46 (br s, 4H, exchangeable with D2O). Anal. (C19H18FN3S-2.0HCl-0.5H2O) C, H, N.

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2-(4-Amidino-3-fluorophenyl)-5-(4-chlorophenyl)thiophene hydrochloride salt (4e): A yellow solid in 75% yield, mp 297-299 oC. IR (ν’ cm-1 ); 3360, 3250 (NH, NH2), 3059 (C-H), 1666, 1618, 1572, 1552 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.); 330, 332 (M+, 100, 39: chlorine isotopes), 313 (97). 1H NMR; δ (ppm) 7.52 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 3.9

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Hz, 1H), 7.70-7.76 (m, 4H), 7.84-7.88 (m, 2H), 9.52 (br s, 4H, exchangeable with D2O). Anal. (C17H12ClFN2S-1.0HCl) C, H, N.

2-(4-Amidinophenyl)-5-(4-methoxyphenyl)thiophene hydrochloride salt (4f): A yellow solid

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in 63% yield, mp 290-292 oC, Lit.20 melting point not reported (no details). IR (ν’ cm-1 ); 3355, 3260 (NH, NH2), 3091, 2965 (C-H), 1666, 1606, 1574, 1533 (C=N, C=C stretch, NH bend). MS

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(EI) m/e (rel.int.); 308 (M+, 100), 291 (77), 276 (62). 1H NMR; δ (ppm) 3.81 (s, 3H), 7.03 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 3.9 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 3.9 Hz, 1H), 7.907.93 (m, 4H), 9.18 (br s, 2H, exchangeable with D2O), 9.41 (br s, 2H, exchangeable with D2O). C NMR (DMSO-d6); δ 55.3, 114.6, 124.1, 124.9, 125.8, 125.9, 126.8, 127.3, 129.1, 138.6,

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139.2, 144.8, 159.3, 164.9. Anal. (C18H16N2OS-1.0HCl-0.5H2O) C, H, N.

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2-(4-Amidinophenyl)-5-(3,5-dimethoxyphenyl)thiophene hydrochloride salt (4g): A goldenyellow solid in 82% yield, mp 284-285.5 oC. IR (ν’ cm-1 ); 3427, 3351, 3237 (NH, NH2), 3129, 3065, 2962 (C-H), 1669, 1605, 1587, 1551 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.);

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338 (M+, 61), 323 (28), 322 (24), 259 (100). 1H NMR; δ (ppm) 3.82 (s, 6H), 6.51 (s, 1H), 6.85 (s, 2H), 7.66 (d, J = 3.9 Hz, 1H), 7.79 (d, J = 3.9 Hz, 1H), 7.93 (s, 4H), 9.28 (br s, 2H, exchangeable with D2O), 9.42 (br s, 2H, exchangeable with D2O). 13C NMR (DMSO-d6); δ 55.4,

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100.1, 103.6, 125.1, 125.9, 126.3, 127.2, 129.1, 135.0, 138.4, 140.5, 144.5, 161.0, 164.9. Anal. (C19H18N2O2S-1.0HCl-0.5H2O) C, H, N.

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2-(4-Amidinophenyl)-5-[(4-dimethylamino)phenyl]thiophene hydrochloride salt (4h): A brown-yellow solid in 73% yield, mp 287-289 oC. IR (ν’ cm-1 ); 3356, 3201 (NH, NH2), 3131, 2956, 2924 (C-H), 1673, 1606, 1531 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.); 321 (M+, 47), 304 (100), 289 (18). 1H NMR; δ (ppm) 2.99 (s, 6H), 7.02-7.06 (m, 2H), 7.46 (d, J = 3.9

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Hz, 1H), 7.63 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 3.9 Hz, 1H), 7.86-7.93 (m, 4H), 9.24 (br s, 2H, exchangeable with D2O), 9.43 (br s, 2H, exchangeable with D2O). Anal. (C19H19N3S-2.0HCl2.5H2O) C, H, N.

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2-(4-Amidinophenyl)-5-(4-chlorophenyl)thiophene hydrochloride salt (4i): A golden-yellow solid in 66% yield, mp 309.5-312 oC. IR (ν’ cm-1 ); 3409, 3262 (NH, NH2), 3067 (C-H), 1664,

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1606, 1533 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.); 312, 314 (M+, 72, 32: chlorine isotopes), 295 (100), 259 (15). 1H NMR; δ (ppm) 7.50 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 3.9 Hz, 1H), 7.79 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 3.9 Hz, 1H), 7.93 (s, 4H), 9.28 (br s, 2H, exchangeable with D2O), 9.47 (br s, 2H, exchangeable with D2O).

C NMR (DMSO-d6); δ 125.1, 126.0,

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126.2, 126.9, 127.4, 129.08, 129.12, 132.0, 132.5, 138.2, 140.9, 143.1, 164.8. Anal. (C17H13ClN2S-1.0HCl-0.5H2O) C, H, N. 10

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2-(4-Amidinophenyl)-5-(3,5-dichlorophenyl)thiophene hydrochloride salt (4j): A yellow solid in 74% yield, mp 329-331.5 oC. IR (ν’ cm-1 ); 3360, 3283 (NH, NH2), 3074 (C-H), 1666, 1606, 1587, 1562 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.); 346, 347, 348, (M+, 37,

4H), 7.94 (s, 4H), 9.31 (br s, 4H, exchangeable with D2O).

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8, 25: chlorine isotopes), 329 (100). 1H NMR; δ (ppm) 7.57 (d, J = 3.9 Hz, 1H), 7.77-7.83 (m, C NMR (DMSO-d6); δ 123.7,

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125.3, 126.7, 127.1, 127.5, 127.7, 129.1, 134.9, 136.5, 138.0, 140.9, 142.2, 164.8. Anal.

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(C17H12Cl2N2S-1.0HCl) C, H, N.

2-(4-Amidinophenyl)-5-(4-fluorophenyl)thiophene hydrochloride salt (4k): A golden-yellow

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solid in 72% yield, mp 290-292 oC. IR (ν’ cm-1 ); 3340, 3248 (NH, NH2), 3083 (C-H), 1678, 1658, 1606, 1547 (C=N, C=C stretch, NH bend). MS (EI) m/e (rel.int.); 296 (M+, 100), 279 (50). 1

H NMR; δ (ppm) 7.27-7.33 (m, 2H), 7.59 (d, J = 3.9 Hz, 1H), 7.74-7.79 (m, 3H), 7.93 (s, 4H),

9.26 (br s, 2H, exchangeable with D2O), 9.45 (br s, 2H, exchangeable with D2O). Anal.

2.2. Biology

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(C17H13FN2S-1.0HCl-2.0H2O) C, H, N.

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2.2.1. In vitro anti-proliferative screening:

Nine of the newly synthesized substituted 4-(5-arylthiophen-2-yl)benzamidines were

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selected by NCI, Bethesda, MD, USA for evaluating their antiproliferative activity. The selected arylthiophene derivatives were subjected to a primary in vitro one-dose (10 µM) antiproliferative assay followed by a five-dose screen study against the standard NCI panel of 60 cancer cell lines. Data presented in Table 1 were obtained from the percent growth graphs of treated cells of the primary single high dose screening assay. Growth inhibition 50 (GI50) values against the individual cell lines obtained from the five-dose experiments were presented in Table 2. The median GI50, total growth inhibition (TGI) and lethal concentration 50 (LC50) values listed in 11

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Table 3 were calculated for the tested compounds against all of the tested cell lines as acquired from the mean graph midpoints. Cell viability or growth was estimated adopting the experimental procedure employing a 48h continuous drug exposure protocol employing the

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sulphorhodamine B (SRB) protein assay according to the previously published standard method.21-23

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2.2.2. Cytotoxicity in normal cells

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Human lung fibroblast Human lung fibroblast (WI-38) and human amniotic (WISH) cell lines were obtained from VACSERA, (Cairo, Egypt). All reagents, RPMI-1640 medium, MTT, DMSO and 5-fluorouracil were obtained from Sigma (St. Louis, USA). Fetal Bovine serum was purchased from GIBCO (UK).

This colorimetric assay is based on the conversion of the yellow tetrazolium bromide (MTT) to a

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purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells.24 Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, penicillin and streptomycin at 37 oC in a 5% CO2 incubator. The cells were seeded in a 96-well plate at a

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density of 104 cells/well at 37 oC for 48 h under 5% CO2. After incubation the cells were treated

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with different concentrations of arylthiophene derivatives and incubated for 24 h. After 24 h of drug treatment, 20 µl of MTT solution at 5 mg/ml was added and incubated for 4 h. A 100 µl of DMSO was added to each well to dissolve the purple formazan formed. The absorbance was measured at 570 nm using a plate reader (EXL 800, USA). Control untreated cells in addition to 5-fluorouracil treated cells were performed for comparison. The data are presented as mean ± SEM for three independent experiments (Table 4).

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2.2.3. Erythrocyte hemolysis assay The assay was performed as described elsewhere.25 Briefly, 2% of erythrocyte suspension in PBS (v/v) was prepared. To 200 µl of this suspension, 100 µl of different concentrations of

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arylthiophene derivatives was added (final concentration 0.5-10 mM). A positive control was also performed using 100 µl of 20 mM H2O2 in PBS, pH 7.4. Two more controls were executed; one with saline 0.9% and the other with ascorbate (0.5-10 mM). The tubes were incubated at 37 o

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C for 30 min followed by centrifugation at 900xg for 5 min. In fresh microfuge tubes containing

800 µl of PBS, 200 µl of the supernatant was added. The absorbance of the mixture was

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measured at 540 nm. The hemolysis induced by H2O2 was considered as 100% and the data were expressed as a percent of this hemolysis. The data are presented as mean ± SEM for three independent experiments (Table 5).

2.2.4. SOD mimetic catalytic activity assay

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SOD mimetic catalytic activity of the arylthiophene derivatives was evaluated by NBT/NADH/PMS (Nitroblue Tetrazolium/reduced Nicotinamide/Phenazine methosulphate) to photo generate a reproducible and constant flux of O2•− at pH = 8.3 (phosphate buffer) as

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reported by Bridges et al. (1981).26 A typical methodology has been recently described in brief,17

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where herein arylthiophene compounds (2 µM) were used as test compounds. The data presented in table 6 are the average of triplicate experiments.

2.2.5. Nuclease-like activity assay: DNA cleavage assay of the tested 2,5-diarylthiophene derivatives was carried out as described by Youssef et al. (2016).17 In brief, the tested arylthiophene derivatives were dissolved in DMSO (10 mM) and 0.1, 0.5, 1, 1.5, 2 and 2.5 µM were added individually to 2 µg of E. coli

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DNA. DNA only and DNA with DMSO were used as controls. The reactions were performed at 37 oC for one hour. A solution of 0.06% w/v bromophenol blue, 10% w/v ficol 400 and 0.5% w/v SDS was added to the reaction mixtures former to running the gel. DNA degradation was

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evaluated via agarose gels electrophoresis,27 and presented in Figure 3.

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2.2.6. Tyrosine kinase inhibition study

Human breast cells MCF7 were used. Cells, media, fetal bovine serum, DMSO, and

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trypsin-EDTA were obtained from ATCC (Manassas, VA, USA). Cells were seeded in 75-cm2 tissue culture flasks at 37 oC in a humidified atmosphere (5% CO2) and the medium was renewed every 2-3 days. When confluent, the arylthiophene derivatives were added at 1 µM concentration and incubated for 2 h, cells were then washed twice with ice-cold PBS. Lysis buffer (50 mM HEPES buffer, pH 7.4 containing 0.1% Triton, 10% glycerol, 1 mM dithiothreitol and 1 mM

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activated sodium vanadate pH 10 in order to inhibit the activity of phosphatases) was added and the cell lysate was transferred to microfuge tubes and incubated on ice for 15 min. The tubes were centrifuged at 10,000 x g for 15 min at 4 oC. The supernatant was immediately separated

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and assayed for tyrosine kinase (TK) activity using a kit from Sigma (Cat # PTK101). Each

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arythiophene derivative has been assigned three wells and the experiment was performed twice. Epidermal growth factor receptor (EGFR) was used as a positive control. A standard curve was constructed and the color developed was spectrophotometrically measured at 492 nm. Compounds showing significant inhibitory activity were further analyzed at concentrations 11000 nM to calculate the IC50s. The data are depicted in Figure 4.

2.2.7. Statistical analysis of data 14

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Statistical analyses were performed using ANOVA, followed by Fisher’s protected least significant difference multiple range test. Differences were considered significant at P < 0.05.

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3. Results and Discussion 3.1. Chemistry

Synthesis of a series of novel substituted 4-(5-arylthiophen-2-yl)benzamidines 4a-k

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begins with a Suzuki coupling reaction between 4-(5-bromothiophen-2-yl)benzonitrile derivatives6,28 1a,b and the corresponding phenylboronic acids 2a-f to afford the arylthiophene

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benzonitriles 3a-k (Figure 2). The target carbonitriles were then converted to the corresponding monoamidines 4a-k by treatment with lithium trimethylsilylamide, followed by de-protection with ethanol/hydrogen chloride. The newly synthesized diarylthiophenes 4a-k and 3a-k were assigned based on their spectral and elemental analyses. Thus, 1H NMR spectrum of the

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monocationic compound 4b displayed one singlet signal at δ 9.46 (4H) characteristic for the cationic amidine group, in addition to signals corresponding to the 2,5-disubstituted thiophene, 1,4-disubstituted and trisubstituted benzene moieties. Furthermore, its mass spectrum showed a 13

C NMR spectrum of

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peak at m/z 326 corresponding to the molecular ion peak (M+).

compounds 4f/4g and 4i/4j displayed their characteristic amidinic carbon peaks at δ 164.9 and

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164.8, respectively. Identification of the known mononitriles 3f, 3h, 3i and 3k was done on the basis of comparing their melting points to those previously reported,19 and their unreported spectral data. In addition, the yield reported herein (this manuscript) for mononitriles 3f, 3h, 3i and 3k (70-84 %) is much higher than the yield previously reported (42-51 %).19

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Figure 2: Synthesis scheme for the new monoamidinic 2,5-diarylthiophene derivatives.

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3.2. Biology

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Reagents and conditions: (i) Pd(PPh3)4, NaHCO3, 80 oC; (ii) a) LiN(TMS)2, r.t. b) ethanol/hydrogen chloride, r.t.

3.2.1. In vitro anti-proliferative screening

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A series of nine substituted 4-(5-arylthiophen-2-yl)benzamidines was subjected to an in vitro anti-proliferative screening against a panel of 60 cancer cell lines at NCI, USA. The chosen arylthiophenes were evaluated at an initial high dose (10 µM). Where after, all nine chosen compounds, due to showing satisfactorily high mean percent growth inhibition (MPGI), underwent a five dose screen against the full panel of cancer cell lines and their GI50s were determined.

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The mean percent growth inhibition results of the nine arylthiophene monocations of the initial single dose screen against the tested cancer cell lines are presented in table 1. The data indicate that at 10 µM, there is a significant MPGI of 67-84% against the 60 cell lines examined.

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However, at this relatively high concentration, it seems that all monoamidines resulted in almost similar response (plateau or saturation effect).

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Abbreviation: MPGI, mean percent growth inhibition.

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Table 1: In vitro Mean percent growth inhibition of the novel monocationic 2,5-diarylthiophenes against a panel of 60 cell lines at a single dose level (10 µM). Compd # I II 4a 4b 4c 4e 4f 4g 4i 4j 4k -72.5 83.9 -79.2 -80.5 -77.8 -67.3 -70.7 -74.1 -70.5 -70.3 -78.7 MPGI

The individual GI50 values of the nine arylthiophene monocations, promoted for a fivedose screen credit to their high antiproliferative profile in the initial assay, are presented in table 2. Among the tested nine monocations, the most active compound was the non-fluorinated 4-

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chlorophenyl derivative 4i, showing GI50 value less than 0.2 µM against most cancer subpanel cell lines belonging to all tested nine types of cancer. The most responsive to the antiproliferative effect of the tested monocation derivative 4i were cancer cell lines HOP-92 Non-small cell lung,

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SNB-75 CNS, OVCAR-5 Ovarian, RXF 393 Renal and PC-3 Prostate cancer with GI50 values of 0.156, 0.157, 0.169, 0.157, and 0.164 µM, respectively. Interestingly, the only cell line that

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seems irresponsive to 4i was the invasive ductal carcinoma cells BT-549 breast cell line (GI50 at 1.73 µM), a feature that needs further investigation. Coming second in potency was the fluorinated 4-chlorophenyl derivative 4e showing GI50 value less than 0.35 µM against most cancer cell lines belonging to all tested nine types of cancer.

Table 2: In vitro antiproliferative activity of the monocationic 2,5-diarylthiophenes against a panel of 60 cell lines at five dose level.a,b Cancer type/cell line 4a 4b 4c 4e 4f 4g 4i 4j 4k Leukemia 17

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a b

3.11 2.19 2.66 3.26 2.51 3.17

0.306 0.229 0.262 0.326 0.246 0.224

0.194 0.212 0.272 0.243 0.248 0.218

0.231 NT NT NT 0.282 NT

1.88 1.90 0.418 1.65 1.46 0.231

5.10 10.5 1.31 12.9 11.6 6.00 3.69 6.80

4.76 10.4 1.38 14.4 11 3.58 2.43 3.73

0.400 1.61 0.665 1.73 1.43 0.502 0.195 1.20

0.217 1.48 0.589 2.03 1.15 0.386 0.207 0.194

0.226 NT 0.750 2.14 0.665 1.24 0.204 0.251

2.58 1.90 1.75 2.23 1.92 1.69 1.84 1.96

0.202 NT 0.156 0.451 0.190 0.182 0.193 0.186

1.52 1.69 1.71 2.09 1.75 NT 0.375 2.35

2.12 1.58 1.54 2.19 1.58 1.33 0.392 1.87

1.82 2.45 3.26 4.62 3.40 4.41 4.05

1.78 1.85 3.04 2.58 2.79 3.16 1.92

0.208 0.207 0.173 0.196 0.267 0.352 0.212

0.200 0.156 0.171 0.172 0.203 0.226 0.186

0.224 1.33 0.186 0.183 0.196 0.339 0.211

1.33 1.82 1.76 1.22 1.09 1.84 1.95

0.203 0.183 0.170 0.171 0.177 0.196 0.177

0.304 0.634 0.468 0.255 0.379 0.698 0.318

0.204 0.492 1.58 0.252 0.323 1.34 0.386

11.2 NT 4.74 11.7 10.5 4.70

4.85 NT 8.96 9.44 10.7 2.92

1.15 1.59 0.165 1.03 1.30 0.198

0.197 0.691 0.206 0.341 0.165 0.203

1.74 1.27 0.160 0.811 1.71 0.232

1.85 1.82 1.75 1.97 1.58 1.67

0.184 0.171 0.171 0.194 0.157 0.172

1.63 1.38 1.97 1.20 NT 0.254

1.54 1.66 1.62 1.93 1.65 0.457

2.58 3.25 2.12 2.18 4.91 8.23 3.43 2.18 10.3

2.23 2.15 1.9 1.83 2.41 2.31 2.67 1.84 1.82

0.196 0.195 0.184 0.194 1.86 0.176 0.956 1.85 1.44

0.205 0.490 0.179 0.163 1.50 0.205 1.27 1.62 0.218

0.198 2.17 1.41 0.924 2.30 1.55 1.39 2.07 1.41

0.924 1.84 1.73 1.70 1.95 1.79 1.81 2.76 1.87

0.179 0.219 0.172 0.172 0.241 0.176 0.204 0.310 0.178

0.155 NT 1.54 1.52 1.99 1.71 1.60 2.48 1.55

0.164 1.55 1.60 1.66 2.05 1.57 1.60 2.58 1.65

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0.200 0.214 0.202 0.207 0.194 0.249

1.54 1.07 0.225 1.18 0.639 0.284

1.82 0.679 0.268 1.58 0.799 0.307

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3.20 2.14 3.97 4.40 2.78 3.11

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CCRF-CEM HL-60(TB) K-562 MOLT-4 RPMI-8226 SR NSCLC A549/ATCC HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522 Colon cancer COLO 205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 CNS cancer SF-268 SF-295 SF-539 SNB-19 SNB-75 U251 Melanoma LOX IMVI MALME-3M M14 MDA-MB-435 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62

data represent the compounds GI50 in µM against the tested cell lines. NT: not tested.

Table 2: In vitro antiproliferative activity of the monocationic 2,5-diarylthiophenes against a panel of 60 cell lines at five dose level.a,b (Continued) Cancer type/cell line 4a 4b 4c 4e 4f 4g 4i 4j 4k Ovarian cancer 18

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b

0.484 0.815 0.361 0.207 1.20 1.59 1.64

0.611 0.268 0.224 0.248 0.983 0.361 1.14

NT 1.73 1.58 0.180 0.321 1.49 1.96

2.11 1.95 2.06 1.90 2.48 1.97 2.08

NT 0.194 0.172 0.169 0.223 0.185 0.246

NT 1.70 NT NT 2.42 1.93 0.454

1.79 1.71 1.63 1.55 2.11 1.77 1.61

6.02 12.8 6.56 6.79 7.01 8.01 10.7 2.68

3.93 6.58 4.24 3.94 4.68 5.97 4.33 3.17

0.174 1.33 0.181 0.233 0.449 0.712 1.78 0.473

0.192 1.38 0.203 0.173 0.187 0.517 1.74 1.72

0.184 1.74 0.359 1.50 1.00 0.189 1.97 1.67

1.87 1.73 1.77 1.81 1.81 1.97 1.97 1.77

0.178 0.191 0.180 0.167 0.157 0.180 0.220 0.175

1.31 NT 1.89 1.51 NT 1.57 1.87 NT

1.47 1.42 1.56 1.67 1.48 1.59 1.85 1.58

4.18 11.3

4.05 6.25

1.30 0.426

0.394 0.430

0.773 0.201

1.80 1.77

0.164 0.185

1.75 0.97

1.59 1.63

3.17 3.34 10.9 10.7 5.13 2.04

2.08 2.75 4.82 5.9 2.92 1.39

0.194 NT 1.47 1.37 NT 0.182

0.168 NT 0.726 1.50 NT 0.215

0.175 0.267 2.14 1.65 0.300 0.192

1.22 2.08 1.99 1.80 1.65 1.68

0.171 0.184 0.200 1.73 0.179 0.173

0.193 NT 2.05 2.05 1.48 1.03

0.323 1.56 2.16 1.65 1.20 1.26

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4.38 9.77 6.42 4.98 8.22 10.1 9.21

data represent the compounds GI50 in µM against the tested cell lines. NT: not tested.

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a

5.29 8.90 10.4 10.9 11.3 14.1 10.2

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IGROV-1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 NCI/ADR-RES SK-OV-3 Renal cancer 786-0 A498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31 Prostate Cancer PC-3 DU-145 Breast Cancer MCF-7 MDA-MB-231/ATCC HS-578T BT-549 T-47D MDA-MB-468

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The most receptive to the antiproliferative influence of the tested monocation 4e were cancer cell lines HCC-2998 Colon, SNB-75 CNS, MDA-MB-435 Melanoma, and MCF-7 Breast

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cancer with GI50 values of 0.156, 0.165, 0.163, and 0.168 µM, respectively. In addition, 3,5dimethoxyphenyl derivative 4c showed potent activity against HCT-116 Colon, SF-539 CNS cancer cell lines with GI50 values of 0.173 µM and 0.165 µM, respectively.

Finally, the median GI50, TGI and LC50 values of the examined arylthiophenes in the five dose screen are depicted in table 3. The most active compounds were found to be in order 4i, 4e,

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4b, 4c, 4f, and 4a displaying sub-micromolar GI50 values of 0.20, 0.36, 0.45, 0.47, 0.61, and 0.74 µM, respectively. All the other tested monocations exhibited GI50s below 1.69 µM. The most potent antiproliferative agent was the non-fluorinated 4-chlorophenyl derivative 4i showing a

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sub-micromolar TGI of 0.37 µM. On the other hand, the most lethal agent was the fluorinated 4methoxyphenyl analog 4b which elicited an LC50 of 4.16 µM. The fluorinated 4-chlorophenyl derivative 4e came second by exhibiting a TGI of 1.02 µM and LC50 of 83.2 µM. In spite of the

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fact that the fluorinated p-chlorophenyl derivative 4e displayed high potency with regards to its growth inhibitory power reflected in its GI50 and TGI values (0.36 and 1.02 µM, respectively)

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yet, it was the least cytotoxic in this series with an LC50 of 83.2 µM. Other tested monocations were potent with regards to the three measured parameters GI50, TGI and LC50 which were less than 1.69, 4.78 and 38.01 µM, respectively.

a

LC50 (µM) for the monocationic 2,5-diarylthiophenes against a panel of

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Table 3: Median GI50, TGI and 60 cell lines at five dose level. I II MG-MIDa 0.63 1.28 GI50b 1.99 3.38 TGIc d 48.9 17.78 LC50

4a 0.74 1.90 22.4

4b 0.45 1.14 4.16

4c 0.47 1.28 7.07

4e 0.36 1.02 83.2

4f 0.61 1.54 15.8

4g 1.69 3.46 25.7

4i 0.20 0.37 35.5

4j 1.02 4.78 72.4

4k 1.17 3.01 38.01

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MG-MID = Mean graph midpoint representing mean sensitivity of all examined cell lines to the test compound. GI50 = Compound concentration causing 50% growth inhibition of tested cells. c TGI = Compound concentration causing 100% growth inhibition of tested cells. d LC50 = Compound concentration causing 50% lethality of tested cells. b

An insight about the structural features associated with the enhanced antiproliferative

activity of this class of monocationic arylthiophenes was concluded from the SAR findings among the tested monocations. First, it was shown that replacing the terminal thiophene ring of either the fluorinated bithiophene derivative I (GI50 = 0.63) or the non-fluorinated bithiophene derivative II (GI50 = 1.28) by the electron donating moiety 4-methoxyphenyl- led to 20

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enhancement of anti-proliferative activity as shown for the isosteres 4b (fluorine presence), 4f (fluorine absence) with GI50 values of 0.45 and 0.61, respectively. Second, replacing the terminal thiophene ring of either the bithiophene derivatives I or II by the electron withdrawing moiety p-

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chlorophenyl- led to an immense enhancement of antiproliferative activity, this was the case for compound 4e (GI50 = 0.36) and compound 4i (GI50 = 0.20). However, adding one more chlorine atom as in case of compound 4j (3,5-dichlorophenyl- derivative) reduced the antiproliferative

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activity. Also, altering the position of the fluorine atom from the amidinic phenyl ring to the nonamidinic phenyl ring had a negative impact on the antiproliferative activity (GI50s: compound 4a

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versus its isostere 4k). Moreover, it was noted that the presence of fluorine atom next to the amidine group led to enhanced antiproliferative activity (GI50s: compounds I versus II; 4b versus 4f; 4c versus 4g), but 4e versus 4i deviated from the previous pattern. It is very crucial that any novel anticancer agent to be selective against only tumor cells

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and therefore, we examined the effect of arylthiophenes in two primary cell types; normal human lung fibroblast (WI-38) and human amniotic (WISH) cells. All tested arylthiohphenes were much less toxic than 5-fluurouracil (5-FU) to normal cells examined. The IC50 values ranged

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from ~53-232 µM. The most toxic compound to normal cells among the tested compounds was 4e with IC50 of ~6x that of 5-FU in WI38 and ~9x that of 5-FU in WISH. The IC50 values of 4i

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which is the most effective arylthiophene derivative against cancer cells were ~ 131-147 µM. This means that all tested arylthiophenes exert their anticancer activity outside the realms of causing any toxicity in normal cells or in plain words, they are safe and selective.

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Table 4: In vitro cytotoxicity of diarylthiophenes against human lung fibroblasts (WI-38) and human amniotic epithelial cells (WISH). WI38 WISH Compd # a IC50 (µM) 5-FUb 9.3 ± 0.3c 7.1 ± 0.4 91.5 ± 1.9 82.2 ± 1.7 4a 104.2 ± 2.3 112.3 ± 2.4 4b 122.7 ± 2.9 115.0 ± 2.6 4c 53.1 ± 1.3 64.2 ± 1.5 4e 192.4 ± 3.6 180.0 ± 3.8 4f 73.9 ± 1.8 87.2 ± 2.0 4g 130.9 ± 2.6 146.5 ± 2.8 4i 137.6 ± 3.2 156.1 ± 3.3 4j 196.8 ± 3.8 232.1 ± 4.6 4k a

IC50 values were determined from the dose-response curves as the mean of two parallel experiments; three wells per every concentration employing the MTT assay. b 5-Fluorouracil (5-Fu) was used as a positive control. c Data are presented as mean ± SEM.

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3.2.2. Erythrocyte hemolysis assay

The novel arylthiophene derivatives were tested for their toxicity and the oxidative stress power they may pose on erythrocytes and compared to H2O2 (a strong oxidizing agent). All

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compounds were cytotoxic to RBCs at concentration 10 mM and caused hemolysis (85-163 %). At concentration 5 mM, compounds 4b and 4c were not toxic and did not pose any stress on the

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RBCs membrane (Table 5). Interestingly, these two compounds had median LC50 values against cancer cell lines at 4.16 and 7.07 µM, respectively. This indicates that these compounds had specific toxicity against cancer cells without affecting normal cells at almost 1000x LC50 reported for cancer cells.

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Table 5: Erythrocyte hemolysis assay. Compd # 4a 4b 4c 4e 4f 4g 4i 4j 4k Conc (mM) 87.0 ± 9.2a 69.7 ± 8.6a 109.0 ± 11.9a 134.7 ± 11.7a 85.0 ± 9.3a 164.0 ± 14.1a 137.0 ± 15.2a 138.0 ± 10.6a 203.3 ± 8.9a 10 66.3 ± 4.6a 0.0 ± 0.0 30.0 ± 2.6 105.0 ± 9.8a 54.3 ± 6.5a 88.0 ± 9.1a 75.3 ± 6.4a 112.3 ± 10.5a 122.0 ± 7.8a 5 a a 12.7 ± 1.8 0.0 ± 0.0 16.7 ± 1.5 71.0 ± 5.4 0.0 ± 0.0 71.3 ± 8.1 63.3 ± 5.8a 58.7 ± 6.2a 87.0 ± 9.0a 2.5 a a a a 0.0 ± 0.0 0.0 ± 0.0 10.3 ± 0.9 35.3 ± 4.5 0.0 ± 0.0 53.7 ± 5.4 48.7 ± 5.3 29.7 ± 3.3 65.0 ± 5.1a 1.25 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 36.0 ± 2.6a 28.3 ± 3.1a 0.0 ± 0.0 54.7 ± 4.4a 1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 11.3 ± 0.8 5.0 ± 0.7 0.0 ± 0.0 32.7 ± 3.9a 0.5 a significant hemolysis as compared to negative control (2% erythrocyte incubated with 0.9% saline; 9.68 ± 0.9). The data are expressed as a percent of hemolysis caused by H2O2 (20 mM). The results represent mean ± SEM. The experiment was performed in triplicate.

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Ascorbate 18.3 ± 2.1 9.7 ± 1.2 5.0 ± 0.8 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

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At concentrations 2.5 and 1.25 mM, compounds 4a, 4b, 4c, and 4f were devoid of toxicity in RBCs. All tested compounds except derivative 4k were devoid of toxicity at 0.5 mM and caused no hemolysis. Instead, many compounds had antioxidant/membrane stabilizing

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properties at 1 mM comparable to that of ascorbate. The p-chlorophenyl derivatives 4e and 4i were toxic to RBCs at 1 and 0.5 mM, respectively. The aforementioned derivatives displayed median GI50s against cancer cells at 0.36 and 0.2 µM, respectively, through mechanisms other

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than killing the cells as indicated from lower toxicity they pose on cancer cells; LC50s were 83.2 and 35.5 µM, respectively. The close analysis of these values show that they were only toxic to

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normal cells at concentration 2500x the concentration at which they inhibited cancer cell growth by 50%.. It is evident that the mono-chlorophenyl substitution regardless of the presence or absence of fluorine atom is responsible for the peculiar profile of both compounds 4e and 4i. Compound 4k, isostere of 4a, was the only compound having para-fluoro substituent on the

investigated.

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phenyl ring and was also the only compound causing hemolysis to RBCs at all concentrations

3.2.3. SOD mimetic catalytic activity:

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The formation of free radicals, reactive oxygen species (ROS) and reactive nitrogen

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species (RNS) is unavoidable during the oxidative metabolic process. High cellular concentration of these reactive molecules promote damage and denaturing reactions to many cellular components creating oxidative stress in cells thus resulting in many diseases including cancer. Based on the displayed DNA binding aptitude by the tested monocations, it was

considered worthwhile to study the antioxidant activity of this class of arylthiophenes. Superoxide radical anions (O2•−) are sources for dynamic free radicals that have potential for reacting with biological macromolecules and thus causing cell damage. Inhibition of the 24

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reduction of NBT to formazan by the tested arylthiophenes was employed for detection of their SOD-mimetic superoxide scavenging catalytic activity. In the presence of O2•− and as the reaction proceeds, NBT gets reduced to formazan, the color changes from colorless to blue, with

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associated increase in the absorbance at 560 nm. SOD decreases the superoxide ion concentration and thereby lowers the rate of formazan formation. In the SOD-like activity test,

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the tested compounds compete with NBT for oxidation of the generated superoxide ions.

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Data presented are the average of triplicate experiments

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a

% Inhibitiona ----68.8 54.5 55.0 55.5 56.7 59.0 59.2 57.0 58.0 60.6 62.3 59.6 37.9 35.0 35.3

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Compd # Control Horse radish 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 3a 3b 3e

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Table 6: Superoxide Dismutase mimetic activity of the 2,5-diarylthiophene derivatives as antioxidant agents.

The data presented in table 6, indicates the scavenging efficacy of each arylthiophene

derivative, giving the final concentration that produced efficient quenching of the superoxide radical anion. The halogenated derivatives 4j, 4i and 4k exhibited the highest antioxidant activities with an inhibition percent of 62.3, 60.6 and 59.6, respectively. The other derivatives displayed good SOD like activity with an inhibition percent more than 54%. The effect of

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monocations on superoxide radical anions (O2•−) adds to their merit as anticancer agents where they can contribute to avoid the severe effects of extreme reactive species such as ROS and RNS in living organisms. However, this SOD catalytic activity did not correlate well with the

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antiproliferative power of the tested monoamidines suggesting another mechanism rather than just a mere scavenging activity. In addition, the arylthiophene carbonitriles 3a, 3b and 3e

respectively.

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3.2.4. DNA affinity and DNA cleavage ability

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displayed weak SOD like activity as indicated by an inhibition percent of 37.9, 35.3 and 35.0,

One of the properties of an anticancer agent is to initiate apoptosis and DNA fragmentation. The ability of the novel arylthiophene monocations 4a-k to cleave genomic DNA as one plausible mechanism of action was studied paralleled to that of controls utilizing agarose

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gel electrophoresis. In the current study, the tested monocations displayed DNA degradation effect in a concentration dependent manner which verifies their binding affinity to the DNA (Figure 3). When the genomic DNA was permitted to interact with arylthiophenes at 0.1, 0.5, 1,

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1.5, 2 and 2.5 µM concentrations, the cleavage of the DNA was found to increase by increasing the concentration of the tested monocations. The results indicate that the monocation derivative

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4k exhibited an efficient cleavage ability of DNA at 0.5 µM concentration (Figure 3; B). Furthermore, the monocation derivative 4j demonstrated a good cleavage power of DNA at a concentration of 1 µM (Figure 3; C). While the monocationic derivatives 4e, 4f and 4i exhibited a significant cleavage at 1.5 µM (Figure 3; D). The monocations 4g and 4h displayed an efficient DNA cleavage but at concentration of 2 µM (Figure 3; E). In addition, all the monocations 4a-4k were found to exhibit strong nuclease like activity on the genomic DNA at the high concentrations of 2 and 2.5 µM (Figure 3; E and F). The weak correlation between the 26

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antiproliferative activity of these monocations and their DNA cleavage activity suggests additional mechanism. On the other hand, the weak DNA degradation ability of the tested mononitrile derivatives 3a, 3b and 3e, compared to the corresponding monoamidines may be due

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to the alteration in the pharmacokinetic characters of the carbonitrile group and the amidine group. In fact, the weak DNA cleavage ability of the arylthiophene carbonitriles compared to their corresponding monoamidines were in agreement with our previously reported patterns for

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the bithiophene analogues.8

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Figure 3:Degradation effect of 0.1 µM (A), 0.5 µM (B), 1 µM (C) 1.5 µM (D) 2 µM (E) and 2.5 µM (F) of the novel 2,5 diarylthiophenes (Lanes 3-16) on the genomic DNA isolated from E. coli Lane1 E. coli DNA; lane 2 E. coli DNA + DMSO.

3.2.5. Tyrosine kinase (TK) inhibition Vatalanib and Gefitinib are well-known FDA-approved anticancer drugs, they work through

inhibiting

many

receptor

tyrosine 27

kinases

especially

VEGF

and

EGFR,

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respectively.18 The structural similarity (terminal chlorophenyl group and amidine-like group) between previous drugs and arylthiophene derivatives 4e and 4i encouraged us to investigate the

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tyrosine kinase inhibition properties of arylthiophene derivatives. 1.6 1.4 1.2

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1 A492 0.8 0.6

* 0.2

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0.4

***

***

0 EGFR

4a

4b

4c

4e

4f

4g

4i

4j

4k

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Figure 4: The inhibition activity of the novel arylthiophene derivatives at 1 µM and 2h incubation against tyrosine kinase. The matrix was MCF-7 cells, each compound has been assigned three wells and the experiment was performed twice. Epidermal growth factor receptor (EGFR) was used as a positive control. A standard curve was constructed and the color developed was spectrophotometrically measured at 492 nm. Data are expressed as mean ± SEM.

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The ability of arylthiophene monocations to inhibit tyrosine kinase was tested at 1µM and compared to EGFR. Some compounds 4a, 4f, 4g, 4j, and 4k showed moderate inhibition

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(19-36%, Figure 4) while compounds 4b, 4c, and 4e have significant inhibitory activities of 66, 74, and 88%, respectively. Compound 4i, which had the best antiproliferative activity with GI50 value of 0.2 µM, had the strongest inhibition activity of 98%. This order correlates very well with the antiproliferative activity of the tested monocations suggesting that these compounds act mainly through inhibiting the tyrosine kinases. Compounds showing strong inhibition activity were further examined to calculate the IC50 values. The IC50 values for compounds 4b and 4c were 920 and 877 nM, respectively. The IC50 value for compound 4e was 107 nM, while 4i had 28

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IC50 value at 13 nM. Inhibition of TK is considered as a promising approach in cancer therapy since it differentiates between cancer and normal cells resulting in fewer side effects known for conventional chemotherapy. Therefore, development of novel anticancer TK inhibitors is of

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central importance since cancer cells rapidly develop resistance to TKI.18 4. Conclusion

In conclusion, compounds 4b and 4c with methoxyphenyl-substitution have similar

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signature with GI50 values at ~ 0.46 µM were more toxic to cancer cells with LC50 value at ~4-7 µM but safe to RBCs at 5000 µM and resulted in ~70% inhibition in TK activity. Presence of

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methoxy groups in 4b and 4c enhanced the activity as compared to the parent compound 4a. Compounds 4e and 4i with p-chlorophenyl-substitution discerned themselves with GI50 values at ~0.20-0.36 µM were more toxic to cancer cells with LC50 values at ~36-83 µM but safe to RBCs at 1000 µM and resulted in 88-98% inhibition in tyrosine kinase activity. The chlorophenyl

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substitution boosted the activity to maximum as in 4e and 4i but presence of fluorine atom next to the amidine group "fluorinated chlorophenyl derivative, 4e" reduced the antiproliferative activity as well as the toxicity. Addition of one more chlorine atom weakened the activity (4j

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versus 4i). Replacement of chlorine atom with fluorine atom had a negative impact on the antiproliferative activity (4k versus 4i). The current results suggest that the electron withdrawing

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hydrophobic substitutions are more in favor of activity than the electron donating hydrophilic ones. This may be explained in terms of the halogen (F and/or Cl) substitution leading to the seemingly orthogonal effects of increasing local polarity and molecular hydrophobicity and consequently change in the pharmacokinetic properties.29-31 In general, this study presents monocationic arylthiophenes scaffolds as promising leads for further optimization as specific cytotoxic to cancer cells and tyrosine kinase inhibitors at nanomolar concentrations.

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Conflict of interests The authors declare no conflict of interest.

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Acknowledgment We thank Deanship of Scientific Research, King Faisal University, KSA for the financial support of Project No. 150123. The in vitro anticancer screening was made at the National Cancer Institute, USA.

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Tables legends: Table 1: In vitro Mean percent growth inhibition of the novel monocationic 2,5-diarylthiophenes against a panel of 60 cell lines at a single dose level (10 µM). MPGI: mean percent growth inhibition.

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Table 2: In vitro antiproliferative activity of the monocationic 2,5-diarylthiophenes against a panel of 60 cell lines at five dose level.a,b a data represent the compounds GI50 in µM against the tested cell lines. b NT: not tested.

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Table 3: Median GI50, TGI and LC50 (µM) for the monocationic 2,5-diarylthiophenes against a panel of 60 cell lines at five dose level. a MG-MID = Mean graph midpoint representing mean sensitivity of all examined cell lines to the test compound. b GI50 = Compound concentration causing 50% growth inhibition of tested cells. c TGI = Compound concentration causing 100% growth inhibition of tested cells. d LC50 = Compound concentration causing 50% lethality of tested cells. Table 4: In vitro cytotoxicity of diarylthiophenes against human lung fibroblast (WI-38) and human amniotic (WISH)

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The viability of the cells was measured after 48h of incubation with different concentrations of the investigated compounds by means of an MTT assay. The IC50 was determined from the doseresponse curves as the mean of two parallel experiments; three wells per every concentration. 5Fluorouracil (5-Fu) was used as a positive control. Data are expressed as mean ± SEM. Table 5: Erythrocyte hemolysis assay a

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significant hemolysis as compared to negative control (2% erythrocyte incubated with 0.9% saline; 9.68 ± 0.9). The data are expressed as a percent of hemolysis caused by H2O2 (20 mM). The results represent mean ± SEM. The experiment was performed in triplicate.

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Table 6: Superoxide Dismutase mimetic activity of the 2,5-diarylthiophene derivatives as antioxidant agents. The data presented in table 6 are the average of triplicate experiments Figure legends

Figure 1: Biologically important non-fluorinated and fluorinated thiophene based structures Figure 2: Synthesis scheme for the new monoamidinic 2,5-diarylthiophene derivatives. Reagents and conditions: (i) Pd(PPh3)4, NaHCO3, 80 oC; (ii) a) LiN(TMS)2, r.t. b) ethanol/hydrogen chloride, r.t.

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Figure 3: Degradation effect of 0.1 µM (A), 0.5 µM (B), 1 µM (C) 1.5 µM (D) 2 µM (E) and 2.5 µM (F) of the novel 2,5 diarylthiophenes (Lanes 3-16) on the genomic DNA isolated from E. coli Lane1 E. coli DNA; lane 2 E. coli DNA + DMSO.

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Figure 4: The inhibition activity of the novel arylthiophene derivatives at 1 µM and 2h incubation against tyrosine kinase. The matrix was MCF-7 cells, each compound has been assigned three wells and the experiment was performed twice. Epidermal growth factor receptor (EGFR) was used as a positive control. A standard curve was constructed and the color developed was spectrophotometrically measured at 492 nm. Data are expressed as mean ± SEM.

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ACCEPTED MANUSCRIPT Highlights • Presence of methoxy groups in 4b and 4c enhanced the anticancer activity • Compounds 4e and 4i with p-chlorophenyl-substitution were the most active anticancer agents with 88-98% inhibition in tyrosine kinase activity.

than the electron donating hydrophilic ones.

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• The electron withdrawing hydrophobic substitutions are more in favor of activity • Novel monocationic arylthiophenes are tyrosine kinase inhibitors at nanomolar

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concentrations.