Designing new iridium(iii) arene complexes of naphthoquinone derivatives as anticancer agents: a structure-activity relationship study

Article abstract of DOI:10.1039/C6DT04339A

A series of iridium(iii) arene complexes of naphthoquinone derivatives of the formula [Ir^III(η⁶-L1)(L2)(3,5-(NO₂)₂pcyd)](PF₆) (L1 = p-methylphenyl)ethynylferrocene; L2 = Lap: lapachol, 1, Plum: plumbagin, 2, Law: lawsone, 3, and Jug: juglone, 4; 3,5-(NO₂)₂pcyd = 3,5-dinitrophenylcyanamide) have been synthesized and investigated for their suitability as potential anticancer drugs. The DNA-binding interactions of the complexes with calf thymus DNA have been studied by absorption, emission, and viscosity measurements. Their cytotoxicity against the cancer cell lines including colon adenocarcinoma (HT-29), liver hepatocellular carcinoma (HepG-2), breast (MCF-7), colon carcinoma (HCT-8), and ovary (A2780) is reported. Remarkably, almost all complexes exhibit significant cytotoxic effects towards HepG-2, MCF-7, and HCT-8 cancer cell lines and complex 1 emerged as the most cytotoxic derivative in comparison with other complexes. The complexes 1-4 increase the production of reactive oxygen species (ROS) in MCF-7 cells. The new compounds also inhibit the enzyme thioredoxin reductase activity at nanomolar concentrations. Furthermore, the complexes induce major levels of cancer cell death by apoptosis that is in correlation with activity in cytotoxicity studies.

Full text of DOI:10.1039/C6DT04339A

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Designing new Iridium(III) arene complexes of naphthoquinone derivatives as anticancer
agents: A structureꢀactivity relationship study
Leila Tabrizi*a,b, Hossein Chiniforoshan *b
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a School of Chemistry, National University of Ireland, Galway, University Road, Galway, Ireland
b Department of Chemistry, Isfahan University of Technology, Isfahan 84156ꢀ83111, Iran
Corresponding author: Leila Tabrizi; Email: LEILA.TABRIZI@nuigalway.ie
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Corresponding author: H. Chiniforoshan; Email: Chinif@cc.iut.ac.ir
Abstract
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A series of Iridium(III) arene complexes of naphthoquinone derivatives of formula [IrIII(η6ꢀ
L1)(L2)(3,5ꢀ(NO2)2pcyd)](PF6) (L1= pꢀmethylphenyl)ethynylferrocene; L2= Lap: lapachol,1,
Plum: plumbagin, 2, Law: lawsone, 3, and Jug: juglone, 4; 3,5ꢀ(NO2)2pcyd= 3,5ꢀdinitro
phenylcyanamide) have been synthesized and investigated for their suitability as potential
anticancer drugs. The DNAꢀbinding interactions of the complexes with calf thymus DNA have
been studied by absorption, emission, and viscosity measurements. Their cytotoxicity against the
cancer cell lines including colon adenocarcinoma (HTꢀ29), liver hepatocellular carcinoma
(HepGꢀ2), breast (MCFꢀ7), colon carcinoma (HCTꢀ8), and ovary (A2780) are reported.
Remarkably, almost all complexes exhibit significant cytotoxic effects towards HepGꢀ2, MCFꢀ7,
and HCTꢀ8 cancer cell lines and complex 1 emerged as the most cytotoxic derivative in
comparison with other complexes. The complexes 1ꢀ4 increase the production of Reactive
oxygen species (ROS) in MCFꢀ7 cells. The new compounds also inhibit enzyme thioredoxin
reductase activity at nanomolar concentrations. Furthermore, the complexes induce major levels
of cancer cell death by apoptosis that is in correlation with activity in cytotoxicity studies.
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Keywords: Ir(III) complex, Naphthoquinone, Phenylcyanamide, Cytotoxicity, Apoptosis.
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1. Introduction
The compound cisplatin and its derivatives carboplatin and oxaliplatin are routinely used in the
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field of chemotherapy. However, Ptꢀbased chemotherapeutics still have a few side effects such
2,3
as a significant toxicity against healthy cells and the increase of platinumꢀresistant tumors.
These disadvantages highlight the vital need for the use of other metals in chemotherapeutics.
The potential of different metal based anticancer agents widely studied in drug discovery such as
organometallic and halfꢀsandwich of Iridium complexes. 4−15 Their hydrophobic arene ligand is
assumed to enable the diffusion via the cell membrane, whereas the coordinating sites can be
varied with diverse ligands to improve biological and pharmacological properties. 10,16
Organometallic iridium complexes have recently attracted great attention due to their rich
synthetic chemistry, variable oxidation states that are available under physiological conditions
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and kinetically stable.
Moreover, several organometallic iridium have been found to bind
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DNA through intercalation. Remarkably, most organometallic ligands of the reported iridium
anticancer complexes are metallocenes, halfꢀsandwich, carbeneꢀ , COꢀ , or πꢀligands. 20ꢀ28 To the
best of our knowledge, there is no report in the literature devoted to the synthesis of Ir(III) arene
complexes as neutral arene ligands do not stabilize Ir(III).
Ferrocenes can be used as biologically active ligands. The ferrocene derivatives are nontoxic,
stable in a biological medium and exhibit antitumor, antimalarial, and antifungal properties. Due
to the reversible redox property of ferrocene and its easy functionalization, these compounds
have found suitable for designing ferroceneꢀbased bioorganometallic species for therapeutic
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applications.
The functionalized ferrocenes show enhanced antitumor activity by
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incorporation of the ferrocenyl group into an appropriate carrier. Many synthetic approaches
have been used to covalently link ferrocenes to other metal centres in order to achieve synergistic
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effects between the two metals. The association of two active metals can lead to cytotoxicities
that are superior to the sum of the individual components. 34ꢀ36
Naphthoquinones constitute a significant class of natural and synthetic compounds extensively
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investigated due to their various pharmacological applications.
methylbutꢀ2ꢀenꢀ1ꢀyl)naphthaleneꢀ1,4ꢀdione), plumbagin
Lapachol (2ꢀhydroxyꢀ3ꢀ(3ꢀ
(5ꢀhydroxyꢀ2ꢀmethylꢀ1,4ꢀ
naphthoquinone), lawsone (2ꢀhydroxyꢀ1,4ꢀnaphthoquinone) and juglone (5ꢀhydroxyꢀ1,4ꢀ
naphthalenedione) used in medicinal chemistry as 5ꢀmembered ring coordination motif of O,Oꢀ
bidentate anionic ligands. 41
In continuation our work on Ruthenium(II) pꢀcymene complexes of naphthoquinone
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derivatives and phenylcyanamide ligands
here we report the synthesis, characterization,
and cytotoxicity properties of complexes [IrIII(η6ꢀL1)(L2)(3,5ꢀ(NO2)2pcyd)](PF6) (L1= pꢀ
methylphenyl)ethynylferrocene; L2= Lap: lapachol,1, Plum: plumbagin, 2, Law: lawsone, 3, and
Jug: juglone, 4; 3,5ꢀ(NO2)2pcyd= 3,5ꢀdinitro phenylcyanamide).
2. Experimental
2.1. General procedures, materials and physical measurements
All manipulations were carried out under nitrogen atmosphere using standard Schlenk
techniques and vacuumꢀline systems. Workꢀup procedures were performed in air. All materials
used were received from commercial sources unless stated otherwise. 3,5ꢀdinitro
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phenylcyanamide was prepared according to literature methods. All solvents were dried by
standard procedures and freshly distilled prior to use.
Fourier transform infrared spectra were recorded on a PerkinElmer Spectrum 400 (FTꢀIR/FTꢀ
NIR spectrometer). 1H, 13C {1H} nuclear magnetic resonance (NMR) spectra were recorded on a
Brukerꢀ400 MHz spectrometer at ambient temperature in DMSOꢀd6. Elemental analyses were
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performed with an EA 3000 CHNS. Electronic absorption spectra were recorded on a JASCO
7580 UVꢀvisꢀNIR doubleꢀbeam spectrophotometer. Emission intensity measurements were
carried out using a Hitachi F 4500 spectrofluorometer.
2.2. Synthesis of the ligand and complexes
2.2.1. Synthesis of pꢀmethylphenyl)ethynylferrocene (L1)
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The compound was synthetized using a modified literature method. To a solution of pꢀ
methylphenyl iodide (62.4 mg, 0.3 mmol) and ethynylferrocene (63.0 mg, 0.3 mmol) in
diisopropylamine (iPr2NH) (15 mL) was added catalytic amounts (0.2 mol %) of Pd(PPh3)2Cl2
and CuI. The resulting mixture was refluxed under argon atmosphere at 80 ºC for 2 h. Solvent
was removed under vacuum and the crude product purified by column chromatography on SiO2
with a hexane/CH2Cl2 (2:2) eluent to give orange solids of L1 (58.80 mg, 65.3%, and 0.3 mmol).
Anal. Calc. (%) for C19H16Fe (300.1753): C, 76.02; H, 5.37; Found (%): C, 75.95; H, 5.32. IR: ν
3153 (w), 3075 (w), 2981 (m), 2207 (s, C≡C), 1571 (m), 1553 (w), 1467 (m), 1371 (m), 1265
+
(m), 1163 (w), 1042 (w), 886 (m), 673 (w) cmꢀ1. TOFꢀMS: 301 [M+H] . 1H NMR (DMSOꢀd6):
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δ 7.18 (d, 2H, Hꢀ2d or 6d, J 8.0), 7.07 (t, 2H, Hꢀ3d or 5d, J 8.0), 4.50 (t, 2H, HꢀCp, J 7.6),
4.38 (s, 5H, HꢀCp), 4.23 (t, 2H, HꢀCp, 3J 7.6), 1.91 (s, 3H, Hꢀ7d). 13C NMR (DMSOꢀd6): δ 132.0
(Ar), 129.0 (Ar), 127.0 (Ar), 124.9 (Ar), 87.1 (C≡C), 85.0 (C≡C), 73.0 (Cp), 71.0 (Cp), 69.2
(Cp), 65.0 (Cp), 20.8 (Cꢀ7d).
2.2.2. Synthesis of [IrIII(η6ꢀL1)(Lap)(3,5ꢀ(NO2)2pcyd)](PF6), 1
IrCl3·xH2O (0.3 mmol, 95.0 mg) was dissolved in degassed methanol (10 mL). To this
solution, L1 (0.3 mmol, 90.0 mg) in degassed methanol (10 mL) was added dropꢀwise and stirred
at room temperature under an N2 atmosphere for 2 h. Then, Lap (lapachol) (0.3 mmol, 72.7 mg)
and 3,5ꢀ(NO2)2pcyd (3,5ꢀdinitro phenylcyanamide) (0.3 mmol, 67.0 mg) in 20 mL of degassed
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absolute methanol was added to the solution. Triethylamine was added dropwise to the solution
until the pH value was ~7. The solution was stirred at reflux temperature under an N2 atmosphere
for 10 h. Ammonium hexafluorophosphate (0.3 mmol, 48.9 mg) was added. Then the solution
was left in a fridge for 12 h. Dark red colored solids were obtained and were collected by
filtration, washed with cold ethanol and diethyl ether, and dried under vacuum (232.2 mg, 71.3%
yield, and 0.3 mmol). Anal. Calc. (%) for C41H32F6FeIrN4O7P (1085.7415): C, 45.36; H, 2.97; N,
5.16; Found (%): C, 45.31; H, 2.91; N, 5.09. IR: ν 2993 (w), 2848 (m), 2192 (s, C≡C), 2079 (vs,
NCN), 1605 (s), 1573 (m), 1492 (s, NO2), 1466 (m), 1425 (w), 1361 (m), 1317 (m), 1265 (w),
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1229 (w), 1174 (w), 1039 (m), 854 (w), 732 (w) cmꢀ1. TOFꢀMS: 941 [MꢀPF6] . H NMR
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(DMSOꢀd6): δ 8.18 (s, 1H, Hꢀ4f), 8.00 (d, 1H, Hꢀ6, J 7.6), 7.89 (d, 1H, Hꢀ9, J 7.6), 7.70 (s,
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2H, Hꢀ2f, 6f), 7.63 (t, 1H, Hꢀ7, J 7.6), 7.52 (t, 1H, Hꢀ8, J 7.6), 5.60 (d, 1H, Hꢀ Ph (L1), J
7.2), 5.42 (d, 1H, Hꢀ Ph (L1), 3J 7.2), 5.31 (d, 1H, Hꢀ Ph (L1), 3J 7.2), 5.20 (d, 1H, Hꢀ Ph (L1), 3J
7.2), 5.01 (t, 1H, Hꢀ12, 3J 7.2), 4.41 (t, 2H, HꢀCp, 3J 7.6), 4.28 (s, 5H, HꢀCp), 4.10 (t, 2H, HꢀCp,
3J 7.6), 2.86 (d, 2H, Hꢀ11, 3J 8.0), 1.86 (s, 3H, Hꢀ7d), 1.69 (s, 3H, Hꢀ14 or Hꢀ15), 1.50 (s, 3H, Hꢀ
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14 or Hꢀ15). C NMR (DMSOꢀd6): δ 181.1 (Cꢀ4), 178.9 (Cꢀ1), 150.0 (Cꢀ2), 138.8 (Ar), 136.9
(Ar), 134.9 (Ar), 132.9 (Ar), 131.0 (Ar), 129.9 (Ar), 126.9 (Ar), 124.9 (Ar), 123.0 (Ar), 121.1
(Ar), 108.0 (NCN), 88.0 (C≡C), 84.2 (C≡C), 74.9 (Cp), 71.8 (Cp), 68.0 (Cp), 66.0 (Cp), 23.7,
21.2, 19.9, 17.0.
2.2.3. Synthesis of [IrIII(η6ꢀL1)(Plum)(3,5ꢀ(NO2)2pcyd)](PF6), 2
Complex 2 was prepared in a similar way to 1 with the use of ligand of plumbagin (Plum) (0.3
mmol, 56.4 mg) instead of lapachol. Dark red colored solids were obtained and were collected by
filtration, washed with cold ethanol and diethyl ether, and dried under vacuum (215.4 mg, 69.6%
yield, and 0.3 mmol). Anal. Calc. (%) for C37H26F6FeIrN4O7P (1031.6511): C, 43.08; H, 2.54; N,
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5.43; Found (%): C, 43.00; H, 2.47; N, 5.36. IR: ν 2988 (w), 2868 (m), 2194 (s, C≡C), 2081 (vs,
NCN), 1608 (s), 1568 (m), 1495 (s, NO2), 1459 (m), 1431 (w), 1355 (m), 1321 (m), 1271 (w),
+
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1231 (w), 1176 (w), 1041 (m), 857 (w), 743 (w) cmꢀ1. TOFꢀMS: 887 [MꢀPF6] . H NMR
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(DMSOꢀd6): δ 8.07 (s, 1H, Hꢀ4f), 7.69 (s, 2H, Hꢀ2f, 6f), 7.51 (t, 2H, Hꢀ7a, 8a, J 7.6), 7.10 (d,
1H, Hꢀ6a, 3J 7.6), 6.74 (s, 1H, Hꢀ2a), 5.69 (d, 1H, Hꢀ Ph (L1), 3J 7.2), 5.49 (d, 1H, Hꢀ Ph (L1),
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3J 7.2), 5.39 (d, 1H, Hꢀ Ph (L1), J 7.2), 5.21 (d, 1H, Hꢀ Ph (L1), J 7.2), 4.39 (t, 2H, HꢀCp, J
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7.6), 4.25 (s, 5H, HꢀCp), 4.11 (t, 2H, HꢀCp, J 7.6), 2.11 (s, 3H, Hꢀ11a), 1.83 (s, 3H, Hꢀ7d). 13C
NMR (DMSOꢀd6): δ 189.0 (Cꢀ1a), 184.7 (Cꢀ4a), 158.9 (Cꢀ9a), 140.1 (Ar), 138.7 (Ar), 136.0
(Ar), 134.0 (Ar), 131.9 (Ar), 128.9 (Ar), 126.6 (Ar), 124.2 (Ar), 122.1 (Ar), 120.8 (Ar), 109.3
(NCN), 87.1 (C≡C), 85.5 (C≡C), 74.1 (Cp), 71.0 (Cp), 69.2 (Cp), 67.1 (Cp), 20.0, 17.0.
2.2.4. Synthesis of [IrIII(η6ꢀL1)(Law)(3,5ꢀ(NO2)2pcyd)](PF6), 3
Complex 3 was prepared in a similar way to 1 with the use of ligand of lawsone (Law) (0.3
mmol, 52.2 mg) instead of lapachol. Dark red colored solids were obtained and were collected by
filtration, washed with cold ethanol and diethyl ether, and dried under vacuum (266.8 mg, 87.4%
yield, and 0.3 mmol). Anal. Calc. (%) for C36H24F6FeIrN4O7P (1017.6245): C, 42.49; H, 2.38; N,
5.51; Found (%): C, 42.41; H, 2.32; N, 5.45. IR: ν 2994 (w), 2877 (m), 2190 (s, C≡C), 2082 (vs,
NCN), 1610 (s), 1565 (m), 1493 (s, NO2), 1451 (m), 1435 (w), 1359 (m), 1324 (m), 1276 (w),
+
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1235 (w), 1172 (w), 1044 (m), 854 (w), 738 (w) cmꢀ1. TOFꢀMS: 873 [MꢀPF6] . H NMR
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(DMSOꢀd6): δ 8.22 (d, 1H, Hꢀ6b or 9b, J 7.6), 8.10 (d, 1H, Hꢀ6b or 9b, J 7.6), 7.99 (s, 1H, Hꢀ
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4f), 7.90 (s, 2H, Hꢀ2f, 6f), 7.81 (t, 1H, Hꢀ7b or 8b, 3J 7.6), 7.65 (t, 1H, Hꢀ7b or 8b, J 7.6), 6.36
(s, 1H, Hꢀ3b), 5.73 (d, 1H, Hꢀ Ph (L1), 3J 7.2), 5.44 (d, 1H, Hꢀ Ph (L1), 3J 7.2), 5.32 (d, 1H, Hꢀ
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Ph (L1), 3J 7.2), 5.21 (d, 1H, Hꢀ Ph (L1), 3J 7.2), 4.42 (t, 2H, HꢀCp, J 7.6), 4.29 (s, 5H, HꢀCp),
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4.11 (t, 2H, HꢀCp, J 7.6), 1.85 (s, 3H, Hꢀ7d). C NMR (DMSOꢀd6): δ 180.9 (Cꢀ1b), 178.9 (Cꢀ
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4b), 156.0 (Cꢀ2b), 139.9 (Ar), 138.0 (Ar), 136.2 (Ar), 133.0 (Ar), 131.6 (Ar), 129.9 (Ar), 127.6
(Ar), 124.8 (Ar), 122.1 (Ar), 120.9 (Ar), 106.2 (NCN), 88.1 (C≡C), 85.9 (C≡C), 74.9 (Cp), 72.0
(Cp), 70.0 (Cp), 68.0 (Cp), 20.0.
2.2.5. Synthesis of [IrIII(η6ꢀL1)(Jug)(3,5ꢀ(NO2)2pcyd)](PF6), 4
Complex 4 was prepared in a similar way to 1 with the use of ligand of juglone (Jug) (0.3
mmol, 52.2 mg) instead of lapachol. Dark red colored solids were obtained and were collected by
filtration, washed with cold ethanol and diethyl ether, and dried under vacuum (242.4 mg, 79.4%
yield, and 0.3 mmol). Anal. Calc. (%) for C36H24F6FeIrN4O7P (1017.6245): C, 42.49; H, 2.38; N,
5.51; Found (%): C, 42.44; H, 2.30; N, 5.47. IR: ν 2979 (w), 2888 (m), 2193 (s, C≡C), 2086 (vs,
NCN), 1609 (s), 1562 (m), 1495 (s, NO2), 1445 (m), 1428 (w), 1362 (m), 1333 (m), 1282 (w),
+
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1241 (w), 1181 (w), 1056 (m), 861 (w), 741 (w) cmꢀ1. TOFꢀMS: 873 [MꢀPF6] . H NMR
(DMSOꢀd6): δ δ 8.05 (s, 1H, Hꢀ4f), 7.89 (s, 2H, Hꢀ2f, 6f), 7.71 (t, 1H, Hꢀ7c, 3J 7.6), 7.33 (d, 1H,
Hꢀ6c or 8c, 3J 7.6), 7.17 (d, 1H, Hꢀ6c or 8c, 3J 7.6), 7.01 (d, 1H, Hꢀ2c or 3c, 3J 7.6), 6.85 (d, 1H,
Hꢀ2c or 3c, 3J 7.6), 5.61 (d, 1H, Hꢀ Ph (L1), 3J 7.2), 5.48 (d, 1H, Hꢀ Ph (L1), 3J 7.2), 5.33 (d, 1H,
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Hꢀ Ph (L1), J 7.2), 5.21 (d, 1H, Hꢀ Ph (L1), J 7.2), 4.42 (t, 2H, HꢀCp, J 7.6), 4.29 (s, 5H, Hꢀ
Cp), 4.11 (t, 2H, HꢀCp, 3J 7.6), 1.86 (s, 3H, Hꢀ7d). 13C NMR (DMSOꢀd6): δ 188.1 (Cꢀ1c), 183.5
(Cꢀ4c), 157.9 (Cꢀ9c), 138.6 (Ar), 136.9 (Ar), 134.9 (Ar), 132.0 (Ar), 130.1 (Ar), 128.5 (Ar),
126.1 (Ar), 124.3 (Ar), 122.0 (Ar), 121.0 (Ar), 108.0 (NCN), 87.0 (C≡C), 84.1 (C≡C), 74.9
(Cp), 72.0 (Cp), 70.8 (Cp), 68.9 (Cp), 21.0.
2.3. DNA binding studies
The complexes were dissolved in 10 mM Tris–HCl buffer (pH 7.2). The concentration of CT
DNA was determined from the UV absorption intensity at 260 nm with ε = 6600 Mꢀ1 cmꢀ1. 48 The
calf thymus DNA (CTꢀDNA) solution in Tris–HCl buffer gave an absorbance ratio at 260 and
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280 nm (A260/A280) of about 1.8ꢀ1.9 indicating that the CT DNA was sufficiently free of
protein. The DNA concentration was varied between 0 and 100 µM, while the complex
concentration was kept constant at 5.0 µM at room temperature.
Viscosity measurements were carried out using an Ubbelohde viscometer, maintained at
constant temperature (37 ± 0.1 °C) in a thermostat. The DNA concentration was kept constant at
50 µM in all samples, while the complex concentration was increased from 50 to 500 µM (10
mM TrisꢀHCl buffer, pH 7.2). Mixing of the solution was achieved by bubbling nitrogen gas
through the viscometer. The flow time was measured using a digital stopwatch. The average
values of three replicated measurements were used to evaluate the viscosity of the samples. Data
are presented as (η/η0)1/3 versus the ratio of the concentration of complex to DNA, where η is the
viscosity of DNA in the presence of complex and η0 the viscosity of DNA alone. Viscosity
values were calculated as η = (t – t0)/ t0, where t is the flow time of CT DNA solution in the
presence or absence of the complex and t0 that of the buffer alone.
2.4. Stability studies
The stability of the complexes in plasma was assessed using a procedure analogous to that
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recently reported. An aliquot (25 mL) of a solution containing the respective complexes 1ꢀ4
(final concentration 20 ꢁM) and diazepam (final concentration 10 ꢁM) in dimethyl sulfoxide
(DMSO) was added to human plasma solution (975 mL) to give a total volume of 1000 mL. The
resulting mixture was incubated for 72 h at 37 ˚C with continuous and gentle shaking (ca. 300
rpm). The reaction was stopped by adding acetonitrile (2 mL), and the mixture was centrifuged
for 45 min at 1000 g at 4 ˚C. The acetonitrile was evaporated, and the residue was suspended in
acetonitrile/H2O (1:1, v/v; 200 mL). The suspension was filtered and the filtrate was analyzed by
high performance liquid chromatographyꢀultraviolet (HPLCꢀUV). A 0.1 mL aliquot of the
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solution was injected into an HPLC system (Thermo, USA) connected to a UV/Vis
spectrophotometer. A Hypersil Gold Dim (100 × 2.1 mm, Thermo, USA) reversedꢀphase column
ꢀ1
was used at a flow rate of 0.5 mL min . The runs were performed with a linear gradient of A
(acetonitrile (SigmaꢀAldrich HPLCꢀgrade) and B (distilled water containing 0.1% HCOOH): t =
0ꢀ3 min, 20% A; t = 7 min, 50% A; t = 20 min, 90% A; t = 23 min, 100% A; t = 25 min, 100%
A; t = 28 min, 20% A. 51
Recoveries of diazepam and complexes 1ꢀ4 were determined by comparing the peak area ratios
of the spiked drugꢀfree plasma to the peak area ratios obtained by direct injection of the solutions
of the same concentration of complexes 1ꢀ4 or diazepam (20 ꢁM for complexes 1ꢀ4 and 10 ꢁM
for diazepam).
2.5. Determination of lipophilicity
Water or phosphateꢀbuffered saline (PBS) was stirred with octanol for 24 h, and then the
mixture was centrifuged for 5 min to obtain octanolꢀsaturated water and waterꢀsaturated octanol.
1ꢀ4 were dissolved in the octanolꢀsaturated water (or PBS for cisplatin) at a typical concentration
of 0.05 to 5 mM, and then they were mixed with waterꢀsaturated octanol in volumetric ratios of
1:1, 1:2, and 2:1. All mixtures were prepared in duplicate. The mixtures were vortexed for 0.5 h,
and then they were centrifuged for 5 min. The layers were separated carefully using a fineꢀgauge
needle. The Ir content in the layers was analyzed by graphite furnace atomic absorption
spectroscopy (GFAAS). The partition coefficient was taken as the ratio of the concentration of Ir
in the octanol layer to that in the aqueous layer (P = coct/cwater). The reported error is the standard
deviation of triplicate measurements obtained according to this protocol.
2.5. Cell culture
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The human colon adenocarcinoma (HTꢀ29), liver hepatocellular carcinoma (HepGꢀ2), breast
(MCFꢀ7), colon carcinoma (HCTꢀ8), ovary (A2780) cancer cell lines nonꢀmalignant fibroblasts
HEK293) and nonꢀtumorigenic (LLCꢀPK1) cell line were purchased from Pasture Institute,
,
(
Tehran, Iran. All reagents and cell culture media were purchased from Gibco Company
(Germany). The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% FBS, 100 IU mLꢀ1 of penicillin, 100 µg mLꢀ1 of streptomycin and 2 mM
of Glutamax at 37 ˚C in a humidified incubator at 5% CO2. The adherent cultures were grown as
monolayers and were passaged once in 4ꢀ5 days by trypsinizing them with 0.25% Trypsinꢀ
EDTA.
2.6. Cytotoxicity
The growth inhibitory effect towards tumor cells was evaluated by means of The MTT (3ꢀ(4,5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) tetrazolium reduction assay. 52 Briefly, 8
× 103 cells/well, dependent upon the growth characteristics of the cell line, were seeded in 96ꢀ
well microplates in growth medium (100 ꢁL). Stock solutions of complexes 1–4 in water were
freshly prepared and diluted with cell culture medium to the desired concentrations. After 24 h,
the medium was removed and replaced with a fresh one containing the compound to be studied
at the appropriate concentration. Triplicate cultures were established for each treatment. After 72
h, each well was treated with 10 ꢁL of a 5 mg/mL MTT saline solution, and following 5 h of
incubation, 100 ꢁL of a sodium dodecylsulfate (SDS) solution in HCl 0.01 M were added. After
an overnight incubation, cell growth inhibition was detected by measuring the absorbance of
each well at 570 nm using a BioꢀRad 680 microplate reader. Mean absorbance for each drug
dose was expressed as a percentage of the control untreated well absorbance and plotted vs drug
concentration. IC50 values, the drug concentrations that reduce the mean absorbance at 570 nm to
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50% of those in the untreated control wells, were calculated by four parameter logistic (4ꢀPL)
model. Evaluation was based on means from at least three independent experiments.
2.11. Metal cellular accumulation studies
MCFꢀ7 cancer cells were plated in a sixꢀwell cell culture plate at a density of 106 cells/well and
maintained at 37 °C in a 5% CO2 atmosphere. Complexes 1ꢀ4 were added to cells for 24 h at 2
ꢁM concentration. Cells were harvested from the plate by using ethylenediaminetetraacetic acid
(EDTA)ꢀtrypsin and centrifuged 200 g for 5 min. Cell pellet from each compound was washed
with 1 mL of PBS. Cells were counted, transferred to Eppendorf tubes, and centrifuged at 10000
g for 5 min at 4 °C. The supernatant was discarded, and pellet was digested with 100 ꢁL of
subboiled 65% HNO3 (subboiled suprapur 65% nitric acid, Merck) overnight. Afterward,
intracellular iridium concentrations were measured by flameless atomic absorption spectrometry.
An atomic absorption spectrometer (model 800, PerkinꢀElmer, Shelton, USA) equipped for
Zeemanꢀeffect background correction, with a transversely heated graphite tube atomizer and ASꢀ
800 autosampler, was used. The autosampler was not used, and the samples were pipetted
manually into the atomizer. Pyrolytic graphite platforms in pyrolytically coated tubes were
obtained from the same manufacturer (part number B050ꢀ4033). Argon at 250 mL minꢀ1 was
used as inert gas except during atomization, when it was stopped. Iridium hollow cathode lamp
(PerkinꢀElmer) was used as the radiation sources. Probes were injected at a volume of 20 ꢁL into
regular graphite wall tubes. All the measurements were done in triplicate.
2.9. Apoptosis Experiments.
For apoptosis determination assays, cells were cultured overnight in 6ꢀwell plates and then
exposed to different concentrations (10 µM) of complexes 1ꢀ4 for 24 h. According the
manufacturer’s instructions of an AnnexinVꢀFITC apoptosis detection kit, cells were washed
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with iceꢀcold PBS, detached by trypsinization and then centrifuged at 2000 rpm (4 ºC) for 5 min.
After being resuspended in 1×Annexin binding buffer (200 mL), the cells were harvested and
stained with propidium iodide (5 mL) and Annexin VꢀFITC (2.5 mL) for 15 min at room
temperature. The apoptotic rates of cell samples were acquired using a FACScan flow cytometer
(Becton Dickinson, Franklin Lakes, NJ).
2.10. Intracellular Reactive Oxygen Species (ROS) Determination.
Intracellular ROS were quantified to determine the oxidative stress in MCFꢀ7 cells after the
treatment with 2 ꢁM for 20 h of complexes 1ꢀ4. MCFꢀ7 cells (20 × 103) were seeded on 96ꢀwell
plates and left attached overnight. Untreated cells used in the treatment (0.4%) were used as a
control. The treated cells were loaded with 20 ꢁM dichloroꢀdihydroꢀfluorescein diacetate
(DCFHꢀDA) and incubated for 45 min at 37 °C before added compounds. Fluorescence was
measured on FLUOstar Galaxy spectrophotometer (excitation/emission wavelengths: 490
nm/525 nm). Experiments were repeated in triplicate.
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2.11. TrxR inhibition
Thioredoxin reductases (TrxꢀR) sourced from rat liver was obtained from SigmaꢀAldrich. It is
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a buffered aqueous glycerol solution, ≥ 100 units/mg protein, in 50 mM TrisꢀHCl, pH 7.5, 300
mM NaCl, 1 mM EDTA, and 10% glycerol. The complexes 1ꢀ4 were preincubated for 5 min at
room temperature; the reaction started with 2 mM 5,5′ꢀdithiobis(2ꢀnitrobenzoic acid) (DTNB),
and the increase of the absorbance was monitored at 412 nm over 5 min at 25 °C. The reaction
velocity in the presence of inhibitor was normalized relative to the control to generate percent
activity and plots of percent activity versus concentration were constructed to obtain IC50 values
(that is, the concentration that inhibited 50% of enzyme activity).
3. Results and discussion
3.1. Synthesis and characterization
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The ligand L1 was prepared using a modified literature method (Scheme S1).
The
Iridium(III) complexes 1ꢀ4 (Fig.1) were synthesized in excellent yield by reaction of the
naphthoquinone derivatives (lapachol, plumbagin, lawsone and juglone) as bidentate ligands and
3,5ꢀ(NO2)2pcyd (3,5ꢀdinitro phenylcyanamide) as monodentate ligand with IrCl3 and L1 in
methanol (Scheme S2). The complexes 1ꢀ4 are air stable and soluble in soluble in water,
alcohols, acetone, acetonitrile, chlorinated solvents, DMF and DMSO. The complexes 1ꢀ4 were
1
13
characterized using ESIꢀMS, elemental analysis and IR, H and C spectroscopy (Fig. S1ꢀS10).
The formation of the complexes 1ꢀ4 is clearly evidenced by the parent peaks in the mass spectra.
The mass spectra of 1, 2, 3 and 4 show peaks of ([IrIII(η6ꢀL1)(Lap)(3,5ꢀ(NO2)2pcyd)] ꢀ PF6)+,
([IrIII(η6ꢀL1)(Plum)(3,5ꢀ(NO2)2pcyd)]ꢀPF6)+, ([IrIII(η6ꢀL1)(Law)(3,5ꢀ(NO2)2pcyd)]ꢀPF6)+ and
([IrIII(η6ꢀL1)(Jug)(3,5ꢀ(NO2)2pcyd)]ꢀPF6)+ centered at m/z = 941, 887, 873 and 873 respectively
(Fig. S11–S15).
1
The H NMR spectra of complexes 1ꢀ4 display all the expected signals due to the coordinated
arene rings, naphthoquinone ligands (lapachol, plumbagin, lawsone and juglone) and PTA
(Fig.S1ꢀS5, Table S1).
1
In the H NMR spectra of complexes 1ꢀ4 the disappearance of the hydroxyl signal of
naphthoquinone ligands is observed which indicated that the hydroxyl group coordinated to the
Ir(III) ions. The η5ꢀbonded cyclopentadienyl ring protons in complexes 1ꢀ4 are equivalent due to
free rotation in the solution state and appeared as a singlet at range 4.25ꢀ429 ppm. The other Cp
ring protons of the ferrocenyl moiety in complexes 1ꢀ4 appeared as two triplets at ranges 4.10ꢀ
4.11 ppm and 4.39ꢀ4.42 ppm. The less downfield shift of the functionalized Cp ring protons in
complexes 1ꢀ4 indicate the presence of this ligand in coordination to Ir(III). Chiral Iridium(III)
complexes are formed upon coordination of the O,Oꢀchelating naphthoquinone derivatives. This
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is evident by the loss of twoꢀfold symmetry of the arene protons, induced by the stereogenic
metal center. This is observed as four sets of doublets for the aromatic protons of arene in L1.
Upon coordination of phenylcyanamide ligands to Ir(III), the proton chemical shifts decrease
with respect to the free ligands. Also, the NH proton of phenylcyanamide ligands disappeared
due to coordination of the cyanamide group to Ir(III) in complexes 1ꢀ4.
The IR spectra of complexes (1ꢀ4) confirm the presence of the naphthoquinone ligands
coordinated to the metal. The band located at 3340ꢀ3350 cm−1 in the free naphthoquinone ligands
assigned to OH group, disappears upon coordination, as expected. In addition, the IR spectra of
ꢀ1
the solid of the naphthoquinone ligands displays strong band at 1640ꢀ1660 cm attributable to
the free carbonyl which are shifted to lower wavenumbers 1605ꢀ1610 cm ꢀ1 in complexes 1ꢀ4.
The ν (NCN) bands of 3,5ꢀ(NO2)2pcyd ligand are observed in the region 2140ꢀ2145 cmꢀ1, when
the 3,5ꢀ(NO2)2pcyd ligand coordinates to Ir(III), ν (NCN) of complexes 1ꢀ4 is shifted to lower
energies (2079ꢀ2086 cmꢀ1) suggesting the importance of coordination of cyanamide through the
cyano nitrogen.
Fig.1.
3.2. DNA binding studies
The understanding of the interaction between DNA and metal complexes is important because
DNA is a major pharmacological target of many antitumor compounds. 53 The interaction of 1ꢀ4
with CT DNA was studied using viscosity measurements, UVꢀVis absorption and fluorescence
spectroscopy.
Viscosity measurements were performed to study the effect of the complexes on the relative
specific viscosity of the solution containing CT DNA. A classical intercalative mode makes a
significant increase in viscosity of DNA due to an increase in separation of base Pairs at
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intercalation sites, and therefore an increase in overall DNA length occurs. The viscosity of
CT DNA solutions was measured at 37 ˚C after addition of varying amounts of 1ꢀ4. As obvious
from the plots of (η/ηo) 1/3 vs. R = [complex]/[DNA] (Fig. S16) the relative viscosity of CT DNA
reveals a significant increase in the presence of increasing amounts of the complexes. Such
behavior indicates that the complexes are inserted between the DNA bases, hence causing in an
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intercalative mode of binding between DNA and complex. The ability of the complexes to
increase the viscosity of DNA follows the order 4 < 3 < 2 < 1.
Electronic absorption spectroscopy is the most generally used method for exploring the binding
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strengths and binding modes of metal complexes to CT DNA. Fig. 2 showed the absorption
spectra of complexes 1ꢀ4 in the absence and presence of CT DNA. The Kb values were measured
in TrisꢀHCl buffer (pH 7.2) by changing the concentration of DNA from 0 to 100 µM while
keeping the complex concentration constant (5 µM). With the increasing DNA concentration, the
absorption bands of the complexes 1ꢀ4 exhibited hypochromicity and bathochromic shifts that
confirmed DNA intercalative binding mode.
The Kb values were obtained from the ratio of the slope to intercept of the plots of [DNA]/(εaꢀ
εf) versus [DNA] according to the equation:
[DNA] / (εaꢀ εf) = [DNA] / (εbꢀ εf) + 1 / Kb (εbꢀ εf)
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where, ɛa, ɛb, and ɛf are the apparent, bound and free complex extinction coefficients. The Kb
values are 3.31 × 105 Mꢀ1, 2.42 × 105 Mꢀ1, 1.87 × 105 Mꢀ1, and 1.72 × 105 Mꢀ1 for complexes 1ꢀ4,
respectively (Fig. 2, inset) and increase in the order 4 < 3 < 2 < 1.
Fig.2.
In order to study further evidence for the interaction mode, the binding of the complexes 1ꢀ3 to
CT DNA has been considered by a competitive binding fluorescence assay using ethidium
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bromide (EB). Upon binding of a metal complex to DNA the fluorescence of the EB bound to
DNA can be remarkably quenched. 57
The fluorescence spectra of the EB–DNA system on addition of increasing amounts of
complexes 1ꢀ4 are shown in Fig. 3. The intensity of the emission band at 590 nm of the EB–
DNA system decreases with increasing concentrations of 1ꢀ4.
The observed linearity in the plot of Io/I vs. the concentration ratio of the complexes to DNA is
in good agreement with the linear Stern–Volmer equation: 58
I0/I= 1+ Ksv [Q]
where, I0 and I are the fluorescence intensities exhibited in the absence and presence of the
compounds, respectively; [Q] corresponds to the concentration ratio of the compound to DNA.
The slope of the plot of I0/I versus [Q] gives Ksv (Fig. 3, inset). The Ksv values that were obtained
from the experiments are 8.21 × 104 Mꢀ1, 6.56 × 104 Mꢀ1, 3.24 × 104 Mꢀ1, and 2.89 × 104 Mꢀ1 for
complexes 1ꢀ4, respectively.
These results confirm that the complexes 1ꢀ4 have an intercalative mode of interaction with CT
DNA 54 and the binding affinity increase in the order 4 < 3 < 2 < 1.
Fig.3.
3.3. Biological activity
3.3.1. Stability of the complexes in solution
It is essential to study the stability of complexes in aqueous solutions in considering of them as
anticancer drugs. Therefore the stability of complexes 1ꢀ4 was investigated in aqueous buffers
under physiologicalꢀlike conditions (in Tris buffer, 1 M, pH 7.2, T = 37 º C). The changes in the
spectral profiles were monitored over 72 h for all complexes (Fig. S17). The results did not
reveal any considerable change in either the intensity or the position of the absorption bands.
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Furthermore, there was no change in the absorption spectra recorded even after a period of 72 h
indicating their stability under physiological conditions.
3.3.2. Stability of the complexes in human plasma
Anticancer compounds must remain stable under physiological conditions in plasma to ensure
their effective transport to cancer cells and to perform their pharmacological function. Before
considering the biological behavior of 1ꢀ4, their stabilities in plasma were evaluated by HPLC.
As clearly evident from the liquid chromatographyꢀUV (LCꢀUV) traces (Fig. S18ꢀS21, Table
S2), 1ꢀ4 do not undergo any noticeable decomposition in plasma within 3 days, suggesting that
they should be stable under physiological conditions.
3.3.3. Hydrophobicity (log P)
The octanol/water partition coefficients (log P) for the complexes 1ꢀ4 were determined since
lipophilicity correlates with cytotoxic potency for some reported series of metallodrugs. 59 The
determined values are listed in Table 1. The most lipophilic complex is 1, with a log P value of
4.65 ± 0.05. The log P values increase in the order 4 < 3 < 2 < 1, which corresponds to the trend
in cytotoxicity.
Table 1.
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3.3.4. In vitro cytotoxicity
The cytotoxicity of the new complexes 1ꢀ4 and of the corresponding uncoordinated ligands
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(3,5ꢀ(NO2)2pcyd, L1, Lap, Plum, Law and Jug) was evaluated toward a panel of human cancer
cell lines, including HTꢀ29 colon adenocarcinoma, HepGꢀ2 liver hepatocellular carcinoma,
MCFꢀ7 breast, HCTꢀ8 colon adenocarcinoma, A2780 ovary cells as well as versus HEK293 nonꢀ
malignant fibroblasts. The cytotoxicity parameters, in terms of IC50 obtained after 72 h exposure
by the MTT assay, are listed in Table 2. The cytotoxicity of cisplatin was evaluated under the
identical conditions for comparison. The cell growth inhibitory potency of all the complexes 1ꢀ4
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was noticeably much higher than their parent ligands. Complexes 1ꢀ4 exhibited an important in
vitro antitumor activity with IC50 values in the low micromolar range that the activity follows the
trend 1 > 2 > 3 > 4. Complex 1 emerged as the most cytotoxic derivative with IC50 values better
than those calculated for cisplatin against HepGꢀ2, MCFꢀ7, and HCTꢀ8 cell lines. Against nonꢀ
tumor cells in rapid proliferation (human embryonic kidney HEK293 cells), complex 4 was
found the least cytotoxic (Table 2). Moreover, the selectivity index values (SI = quotient of the
average IC50 toward nonꢀmalignant cells divided by the average IC50 for the malignant cells)
determined for complexes 1ꢀ4 were approximately 3ꢀ4 times higher than that determined with
the reference drug, indicating to a special cytotoxicity of complexes 1ꢀ4 versus neoplastic cells.
Furthermore, the cytotoxicity of 1ꢀ4 against normal renal LLCꢀPK1 cells was studied and
compared with that of cisplatin (Table 3). The complexes were three times less toxic to LLCꢀ
PK1 cells than cisplatin. However, the activity against cancer cells follows the trend 1 > 2 > 3 >
4, 1 displays the lowest toxicity in normal renal cells and 4 has the highest toxicity. The free
ligands have no evaluable cytotoxicity (IC50 > 100 ꢁM) against LLCꢀPK1 cells.
McGowan and coꢀworkers have shown that the Ir–Cp* complexes of the napthoquinone
conjugate exhibited about a fourꢀfold increase in antitumor effects against HTꢀ29 cell lines in
comparison with the free ligand or the metal source [IrCp*Cl2]2 alone. 60
Table 2.
Table 3.
3.3.5. Accumulation studies
The cellular accumulation of the complexes 1ꢀ4 in MCFꢀ7 cancer cell lines was considered to
study the potential association between cellular uptake and cytotoxicity. Cellular concentrations
were determined by AAS after 24 h of exposure to 1ꢀ4 at 2 ꢁM. The results are shown in Fig. 4.
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The accumulation of complexes 1ꢀ4 in the MCFꢀ7 cells increases in the order 4 ˂ 3 < 2 < 1 that
displays a direct relationship between cellularꢀiridium accumulation in MCFꢀ7 cancer cell lines
and cytotoxicity.
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Fig.4.
3.3.6. Apoptosis Studies
Apoptotic studies of the complexes 1ꢀ4 were carried out with MCFꢀ7 cell lines (Fig. 5).
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Exposure of phosphatidylserine (PS) to the outer side of the cell membrane, an early sign of
apoptosis, was followed by a flow cytometric assay based on the annexin VꢀFITC, propidium
iodide (PI) method, which also can identify necrotic or late apoptotic cells. The four quadrants
detecting the living (lower left quadrant Q3, not stained cells), the early apoptotic (lower right
quadrant Q4, only the annexinꢀ V stained cells), the necrotic (upper left quadrant Q1, only PI
stained cells), and the late apoptotic (upper right quadrant Q2, cells stained with both fluorescent
dyes) cells appear in these diagrams. As showed in Fig. 5, it is clear that complexes 1ꢀ4 induce a
high incidence of early to late stage apoptosis in MCFꢀ7 cell lines at 24 h without increasing
necrotic population. These results are comparable with the iridium(III) βꢀketoiminato complex
also induced high levels of apoptosis/necrosis, consistent with its much higher IC50 values in
both the HTꢀ29 and A2780 cancer cell lines. 61
Fig.5.
3.3.7. Intracellular Reactive Oxygen Species (ROS) Determination
DCFHꢀDA (2′,7′ꢀDichlorodihydrofluorescein diacetate) was used to study the effect of
complexes 1ꢀ4 for comparative purposes, on the production of ROS by MCFꢀ7 cells. The DCFHꢀ
DA method is considered to provide a highly sensitive, quantifiable, realꢀtime evaluation of ROS
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production.
To consider ROS formation, MCFꢀ7 cells untreated or treated with 1ꢀ4 were
incubated with 20 ꢁM DCFHꢀDA for 30 min in the dark. This cellꢀpermeant nonfluorescent dye
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gets oxidized by cellular ROS to produce 2,7ꢀdichlorofluorescein (DCF), which emits at 525 nm
(λex = 490 nm). The emission intensity of DCF was quantified by using FACS techniques, and it
gave an estimate to the amount of ROS generated within the cells. Treatment of MCFꢀ7 cells
with 1ꢀ4 at 2 ꢁM resulted in an increase of fluorescence, demonstrating the production of ROS
(Fig. 6).
Fig.6.
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3.3.8. TrxR inhibition
To further consider the mode of action of complexes 1ꢀ4, the inhibition of TrxR activity the
synthesized complexes was carried out. Thioredoxin are large homodimeric proteins that show
an important effect in the intracellular redox metabolism, along with a few other biochemical
systems. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin
64ꢀ67
NADPH Reductase (TrxR).
The inhibitory effects of complexes 1ꢀ4 toward TrxR were
evaluated at increasing concentrations, and the doseꢀeffect curves are exhibited in Fig. 7. TrxR
appeared noticeably inhibited by complexes 1ꢀ4 at nanomolar concentrations. Despite different
enzyme inhibition trends, all complexes 1ꢀ4 were able to decrease the TrxR activity by
approximately 75% at a concentration of 93 nM. Therefore, these results may indicate the
selenoenzyme TrxR as a protein target for complexes 1ꢀ4, however it is likely that other
mechanisms may be important. These results are comparable with the iridium(III) picolinamide
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and βꢀketoiminato 61 complexes that inhibit TrxꢀR, with IC50 values in the nanomolar range.
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Fig.7.
4. Conclusions
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We achieved a successful synthesis of novel Iridium(III) arene complexes of naphthoquinone
[IrIII(η6ꢀL1)(L2)(3,5ꢀ(NO2)2pcyd)](PF6)
pꢀ
derivatives
of
formula
(L1=
methylphenyl)ethynylferrocene; L2= Lap: lapachol,1, Plum: plumbagin, 2, Law: lawsone, 3, and
Jug: juglone, 4; 3,5ꢀ(NO2)2pcyd= 3,5ꢀdinitro phenylcyanamide). The DNAꢀbinding interactions
of the complexes show an intercalative mode of interaction with CT DNA. Almost all
investigated compounds show antiproliferative effects against HepGꢀ2, MCFꢀ7, and HCTꢀ8
cancer cell lines that complex 1 was the most effective anticancer compound among the ones
tested. Conversely, against human embryonic kidney noncancerous cells, complexes 1ꢀ4 were 3ꢀ
fold lower than cisplatin treatment, suggesting a preferential cytotoxicity of the new complexes
versus neoplastic cells. Further mechanistic investigation showed that complexes 1ꢀ4 induce
significant cancer cell death by apoptosis. Compounds 1ꢀ4 are able to increase the production of
ROS in MCFꢀ7 cells at 2 ꢁM. Studies on the inhibition of the selenoenzyme TrxR demonstrated
that complexes 1ꢀ4 decrease the TrxR activity, suggesting the selenoenzyme TrxR as a protein
target for new complexes. These results highlight the importance of the structure and
lipophilicity of the ligand for activity. Therefore, the higher activity of the more lipophilic
derivatives in these compounds likely outcomes from a better cellular uptake. Overall, these
novel Ir(III) organometallic compounds have a diversity of interesting biological effects that
make them attractive as potential anticancer drugs.
Acknowledgement
We acknowledge general support from the Department of Chemistry, Isfahan University of
Technology (IUT).
483
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623
Table 1. Octanol/water partition coefficients
Compound
logP a
4.65 ± 0.05
3.38 ± 0.03
2.95 ± 0.02
2.86 ± 0.02
1
2
3
4
624
625
a Errors are calculated from triplicate experiments
626
627
628
629
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Table 2. Results of MTT assay presented as IC50 (µM) values obtained after 72 h treatment. IC50 values are calculated as mean values
obtained from two to three independent experiments and presented with their standard deviations.
Compounds
HTꢀ29
HepGꢀ2
> 100
MCFꢀ7
> 100
HCTꢀ8
A2780
HEK293
> 100
> 100
> 100
> 100
> 100
> 100
SI
3,5ꢀ(NO2)2pcyd
> 100
> 100
> 100
ND
ND
ND
ND
ND
ND
L1
Lap
Plum
Law
Jug
1
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
6.41 ± 0.05
8.22 ± 0.10
13.23 ± 0.20
4.32 ± 0.10
6.45 ± 0.15
7.65 ± 0.20
7.12 ± 0.30
1.53 ± 0.08
3.95 ± 0.20
5.42 ± 0.12
6.32 ± 0.10
3.61 ± 0.20
6.15 ± 0.10
8.75 ± 0.20
9.92 ± 0.10
6.15 ± 0.10
7.88 ± 0.10
9.95 ± 0.10
12.15 ± 0.10
62.43 ± 0.20 14.17
72.45 ± 0.20 10.81
81.20 ± 0.10 8.64
87.10 ± 0.10 6.81
2
3
4
21.15 ± 0.35
Cisplatin
2.43 ± 0.3
8.23 ± 0.20
7.43 ± 0.15
12.30 ± 2.47
3.42 ± 1.11
19.32 ± 2.31 2.86
The sign (>) indicates that IC50 value is not reached at the maximum concentration in the examined range.
ND: not determined
29