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Discovery of a dual-targeting organometallic ruthenium complex with high activity inducing early stage apoptosis of cancer cells.
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Metallomics
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J. Du, E. Zhang, W. Zheng, Y. Zhang, Z. Wang, Q. Luo, K. Wu and Y. Lin, Metallomics, 2015, DOI:
10.1039/C5MT00122F.
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Page 1 of 12 Metallomics
1 Journal Name
RSCPublishing
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5 ARTICLE
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Discovery of A Dual-Targeting Organometallic
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Ruthenium Complex With High Activity Inducing Early
11
Cite this: DOI: 10.1039/x0xx00000x
12 Stage Apoptosis of Cancer Cells
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Jun Du,a Erlong Zhang,a, b Yao Zhao,b* Wei Zheng,b Yang Zhang,b Yu Lin,b t
15 p
Received 00th January 2015, Zhaoying Wang,b Qun Luo,b Kui Wu,b Fuyi Wangb*
16
Accepted 00th January 2015 i
17 r
c
18 DOI: 10.1039/x0xx00000x Abstract: Ruthenium based complexes are promising antitumour candidates due to their lower toxicity
19 and better water-solubility compared to the platinum antitumour complexes. Epidermal growth factor s
www.rsc.org/
20 receptor (EGFR) has been found to be overexpressed in a large set of tumour cells. In this work, a series u
21 of organoruthenium complexes containing EGFR-inhibiting 4-anilinoquinazoline pharmacophores were n
22
synthesised and characterised. These complexes were shown excellent inhibitory activity against EGFR a
23
and high affinity to interact with DNA via minor groove binding, featuring dual-targeting property. In
M
24
vitro screening demonstrated that the as-prepared ruthenium complexes are anti-proliferating towards
25
a series of cancer cell lines, in particular the non-small-cell lung cancer cell line A549. Fluorescence-
26 d
activated cell sorting analysis and fluorescence microscopy revealed that the most active complex 3
27 induced much more early-stage cell apoptosis than its cytotoxic arene ruthenium analogue and the e
28
EGFR-inhibiting 4-anilinoquinazolines, verifying the synergetic effect of the two mono-functional t
29 p pharmacophores.
30
e
31
c
32
Introduction Among the large library of non-platinum antitumour c
33
complexes, ruthenium based complexes have attracted A
34
Cancer is a severe and still growing threat to human health. increasing interest due to their high efficacy, good water
35
36 Since the first discovery of cisplatin as an effective antitumour solubility and low toxicity.8-19 Two RuIII complexes, NAMI-A s
agent in late 1960s, various platinum analogues have been and KP1019 are now under phase II clinical trials,20, 21 and the
37 c
extensively studied and some of them have been successfully organometallic half-sandwich ruthenium(II) complexes in type
38 i
used in clinic such as carboplatin and oxaliplatin.1 However, of [(η6-arene)Ru(X)(Y)(Z)] have been extensively studied in m
39
the administration of cisplatin and its analogues in clinic is recent years.22-26 This kind of complexes adopt an octahedral
40
limited by their poor water solubility, severe dose-limiting side geometry, of which three coordination sites are occupied by o
41
effects such as nephrotoxicity, neurotoxicity and arene ligands, stabilizing the ruthenium centre in +2 oxidative l
42
43 myelosuppression, and inherent or acquired resistance.2, 3 In status, and other three sites offer possibilities for coordination a l
44 recent years, other metal-based antitumour agents such as with a variety of ligands to tune their biological activities, such t
45 ruthenium, osmium, and iridium, have shown promising as hydrophobicity, cellular uptake, reactivity and selectivity e
46 anticancer effects, providing good alternative to platinum-based towards biological targets.6-8, 27 For instance, the {(η6-arene)Ru} M
47 drugs.4-11 (arene = cyclopentadienyl, benzene, p-cymene, biphenyl, etc.)
48 units can coordinate with ethylenediamine, imidazole, 2-
49 a College of Chemistry and Materials Science, Key Laboratory of (aminomethyl)pyridine, derived enzyme inhibitors to get a
Functional Molecular Solids, The Ministry of Education, Anhui
50 Laboratory of Molecular-Based Materials, Anhui Normal University, series of novel complexes with diverse biological activity and
51 Wuhu 241000, P. R. China. improved antitumour activity,24, 28-33 indicating the possibility
52 b Beijing National Laboratory for Molecular Sciences; CAS Key Laboratory of organometallic ruthenium(II) arene complexes for the
53 of Analytical Chemistry for Living Biosystems; Beijing Centre for Mass development of multi-functional antitumour drugs.
Spectrometry; Institute of Chemistry, Chinese Academy of Sciences, Beijing
54 The epidermal growth factor receptor (EGFR), which
100190, P. R. China. Email: fuyi.wang@iccas.ac.cn; yaozhao@iccas.ac.cn
55 belongs to receptor tyrosine kinase (RTK) family, plays an
† Electronic Supplementary Information (ESI) available. See
56 important role in the cellular signal transduction and thus
DOI: 10.1039/b000000x/
57 regulates cell growth. EGFR is overexpressed in a broad type of
58
59
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human cancer cells such as squamous carcinoma, cervical and elemental analysis, and complex 4 was further
2
cancer, lung cancer cells,34 which make it an attractive target characterised with X-ray crystallography analysis. Their
3
for developing novel antitumour drugs. It was found that biological activities have been evaluated using enzyme-linked
4
blocking the ATP binding site of EGFR can inhibit its tyrosine immunosorbent assay (ELISA), anti-proliferation assay and
5
kinase activity, so as to inhibit the growth of tumours.35 In light molecular modelling analysis.
6
of this discovery, many kinds of ATP-competitive EGFR
7
Experimental Section
8 inhibitors have been developed as antitumour agents. To date,
9 4-anilinoquinazoline derivatives have shown good potency in Materials
10 inhibiting EGFR and EGF-stimulated growth of a certain type
The staring material 6-hydroxy-4-(3'-chloro-4'-fluoroanilino)-7-
11 of cancer cells. Some of them, for instance, gefitinib and
methoxy-quinazoline was purchased from Shanghai FWD
12 erlotinib, have been widely used in clinic for the therapy of
13 non-small-cell lung cancer and squamous carcinoma.34 Chemical Co, [(η6-p-cymene)RuCl 2 ] 2 from TCI (Shanghai)
Development Co., Ltd. (China), 1, 2-dibromoethane and 1, 3-
14 Compared to the traditional cytotoxic antitumour drugs, this
dibromopropane from Beijing Ouhe Technology Co. (China), t
15 kind of drugs has much less toxicity towards normal tissues p
ethylenediamine from Beijing Xingjin Chemicals Co. (China).
16 while being high active against tumour tissues, and is described
i
17 as “Molecular Targeting Drugs”.36, 37 Organic solvents including absolute methanol, absolute ethanol, r
absolute ether, acetonitrile, dichloromethane and DMSO were c
18 Since cancer is a multigenic disease, the drugs that can act
19 on two or more targets may achieve better therapeutic effect all analytical grade and used directly without further s
20 than the mono-functional drugs. In the previous works of our purification. Column chromatography silica gel and thin layer u
21 group, we utilized the “pharmacophore conjugation” strategy to chromatography silica gel were purchased from Qingdao Jiyida n
22 design and synthesise a series of dual-functional ruthenium Silica Reagent Manufacture (China). All 1H NMR and 13C
a
23 antitumour complexes. We modified the 6-position of 4- NMR were recorded on an Avance III 400 spectrometer
24 anilinoquinazoline pharmacophore with either ethylenediamine (Bruker). M
25 or imidazole so that the modified anilinoquinazolines could be
Synthesis of 6-(2-(2-(pyridin-2-yl-methylamino))ethoxy)-4-(3'-
26 coordinated to RuII or RuIII to give a series of novel ruthenium d
chloro-4'-fluoroanilino)-7-methoxy-quinazoline (L1)
27 complexes. An important character of these complexes is that e
28 6-hydroxy-4-(3'-chloro-4'-fluoroanilino)-7-methoxy-
there are two different pharmacophores in one molecule: one is t
29 the {(arene)RuII} group30 or the {RuIIICl (DMSO) ]} quinazoline (319 mg, 1 mmol) and potassium carbonate (552 p
n m
30 fragment38 which is likely responsible for inducing DNA mg, 4 mmol) were added to DMF (30 mL) and stirred at e
31 ambient temperature for 0.5 h. Then 1, 2-dibromoethane (0.34
damage; the other one is the 4-anilinoquinazoline group which c
32 mL, 4 mmol) was added and the resulting mixture was heated at
acts as the EGFR inhibitor. Those two pharmacophores endue c
33 80 °C for 4 h. After cooling to room temperature, the solid was
the ruthenium complexes dual-targeting property. Among them,
A
34 a RuIII complex and a RuII complex, of which both contain the filtered off. The filtrate was concentrated to 3 mL in vacuum,
35 and the resulting residue was purified by flash chromatography
4-anilinoquinazoline ligand 6-(2-(2-aminoethylamino)ethoxy)-
36 on Silica gel using ethyl acetate/petroleum (5:3) as eluent s
4-(3'-chloro-4'-fluoroanilino)-7-methoxy-quinazoline (L0),
37 giving L′1 as white powder (254 mg, 60%). Then 6-(2-(2- c
exhibited excellent and selective antiproliferative activity
38 bromoethoxy)ethoxy)-4-(3'-chloro-4'-fluoroanilino)-7- i
towards the EGF (epidermal growth factor) stimulated growth m
39
methoxy-quinazoline (L′1) (424 mg, 1 mmol) and 2-
of human MCF-7 breast cancer with better ability inducing
40
apoptosis than the mono-functional EGFR-inhibiting (aminomethyl)pyridine (393 µL, 4 mmol) were added to DMF o
41
gefitinib.30, 38 However, the complex [RuIIICl (DMSO)(L3)] (10 mL) and the mixture was heated at 80 °C for 4 h. After l
42 4
concentrating, cooling to room temperature, the mixture was l
43 (L3 = 6-(2-(2-(1H-imidazol-1-yl))ethoxy)-4-(3'-chloro-4'- a
poured into water (40 mL), and the forming precipitate was
44 fluoroanilino)-7-methoxy-quinazoline, Scheme 1) exhibited t
collected by centrifugation, purified by flash chromatography
45 little cytotoxicity towards MCF-7 cancer cell line (IC 50 > 100 e
on silica gel using chloroform/methanol (20:1) as eluent giving
46 µM), though it is more active against the enzyme activity of M
47 EGFR than the complex [RuIIICl 3 (DMSO)(L0)].38 Since RuII 6-(2-(2-(pyridin-2-yl-methylamino))ethoxy)-4-(3'-chloro-4'-
48 arene complexes usually possess higher cytotoxicity than RuIII fluoroanilino)-7-methoxy-quinazoline (L1) as yellow powder
49 complexes,39 in order to improve the antitumour activity of Ru- (272 mg, 60%). ESI-MS (m/z): 454.146 ([M + H]+,
50 L3 complexes, in this work, two complexes, [(η6-p- C 23 H 22 N 5 O 2 ClF requires 454.141); m. p. 93 – 96 °C. 1H NMR
51 cymene)RuII(L3)Cl ] (3) and [(η6-p-cymene)RuII(en)L3]2+ (4) (DMSO-d 6 , 400 MHz) δ (ppm): 8.51 (s, 2H); 8.14 (dd, 1H);
2
7.85 (s, 1H); 7.84 – 7.80 (m, 1H); 7.76 (td, 1H); 7.49 – 7.42 (m,
52 were prepared. In addition, to examine the structure activity
2H); 7.25 (dd, 1H); 7.21 (s, 1H); 4.25 (t, 2H); 3.95 (s, 3H) 3.93
53 relationships of this kind of dual targeting antitumour agents
(s, 2H); 3.04 (t, 2H). Anal. calcd. (%) for C H ClFN O : C,
54 and thus further explore the mechanisms of their antitumour 23 21 5 2
60.86; H, 4.66; N, 15.43; Found: C, 60.44; H, 4.64; N, 15.78.
55 activity, three analogues were also prepared (complexes 1, 2,
56 and 5) using imidazole and 2-(aminomethyl)pyridine as the
Synthesis of 6-(2-(3-(pyridin-2-yl-methylamino))propoxy)-4-(3'-
57 coordination ligands to {(p-cymene)RuII} fragment. These
chloro-4'-fluoroanilino)-7-methoxy-quinazoline (L2)
58 complexes were synthesised and characterised by NMR, MS
59
60
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4-(3'-chloro-4'-fluoroanilino)-6-hydroxy-7-methoxy- bromoethoxy)propoxy)-4-(3'-chloro-4'-fluoroanilino)-7-
2
quinazoline (319 mg, 1 mmol) and potassium carbonate (552 methoxy-quinazoline (L′2) (554.0 mg, 1.21 mmol), the reaction
3
mg, 4 mmol) were added in acetone (60 mL) and stirred at was continued for 4 h. After concentrating and cooling to room
4
ambient temperature for 0.5 h. Then 1, 3-dibromopropane (0.34 temperature, the mixture was poured into water (20 mL) and
5
mL, 4 mmol) was added and the resulting mixture was heated at ethyl acetate (10 mL). A white solid precipitation was got by
6
70 °C for 7 h. Then cooling to room temperature, The solid was filter, washed by water and ethyl acetate (362.5 mg, 70%). M.
7
8 filtered off, and after concentrating, the residue was p. 201 – 203 °C. ESI-MS (m/z): 428.128 ([M + H]+,
9 chromatographed by flush chromatography on Silica gel using C 21 H 19 ClFN 5 O 2 requires 428.128); 1H NMR (DMSO-d 6 , 400
10 ethyl acetate/petroleum (5:3) as eluent to give L′2 as yellow MHz) δ (ppm): 9.51 (s, 1H); 8.50 (s, 1H); 8.10 (dd, 1H); 7.78
11 powder (284.7 mg, 65%). Then we used 6-(2-(3- (s, 1H); 7.76 (m, 1H); 7.64 (s, 1H); 7.43 (t, 1H); 7.23 (s, 2H);
12 bromoethoxy)propoxy)-4-(3'-chloro-4'-fluoroanilino)-7- 6.91 (s, 1H); 4.20 (t, 2H); 4.08 (t, 2H); 3.97 (s, 3H); 2.30(m,
13 methoxy-quinazoline (L′2) (438 mg, 1 mmol) and 2- 2H). 13C NMR (DMSO-d 6 , 100.6 MHz) δ (ppm): 156.5, 155.1,
14 (aminomethyl)pyridine (393 µL, 4 mmol) were added in DMF 153.2, 148.5, 147.6, 137.8, 129.1, 123.9, 122.7, 119.8, 119.3,
t
15 (10 mL) and the resulting mixture was heated at 80 °C for 4 h. 119.2, 117.1, 116.8, 109.2, 107.9, 103.5, 66.2, 56.4, 43.4, 30.6. p
16 After concentrating solution, cooled to room temperature, the Anal. calcd. (%) for C H ClFN O : C, 56.57; H, 4.75; N,
21 21 5 3 i
17 mixture was poured into water (40 mL), the obtained deposit 15.71; Found: C, 56.51; H, 4.61; N, 15.46. r
c
18 was got by centrifugation, and the residue was
19 chromatographed by using column chromatography on silica General method for preparation of complexes 1 – 3 (see s
20 gel using chloroform/methanol (20:1) as eluent to give 6-(2-(3- Scheme 1). Ligand (0.4 mmol) and [(η6-p-cymene)RuCl] (0.2 u
2 2
21 (pyridin-2-yl-methylamino))propoxy)-4-(3'-chloro-4'- mmol) were added to 20 mL methanol and the resulting mixture n
22 fluoroanilino)-7-methoxy-quinazoline (L2) as yellow powder was heated at 65 °C for 4 h, then ammonium
a
23 (269 mg, 60%). ESI-MS (m/z): 468.161 ([M + H]+, hexafluorophosphate (0.8 mmol) was added, and stirred for 1 h.
M
24 C H ClFN O requires 468.160); m. p. 95 – 98 °C. 1H NMR After removing the solvent on a rotary evaporator till 2 – 3 ml,
24 24 5 2
25 (acetone-d , 400 MHz) δ (ppm): 8.55 (s, 1H); 8.50 (d, 1H); 8.24 the residue was purified by column chromatography on silica
6
26 d
(dd, 1H); 7.81 – 7.77 (m, 1H); 7.73 (s, 1H); 7.69 (td, 1H); 7.44 gel using dichloromethane/methanol (30:1) as eluent to give
27 (d, 1H); 7.29 (t, 1H); 7.22 (s, 1H); 7.20 (dd, 1H); 4.26 (t, 2H); complex as yellow powder. e
28
3.98 (s, 3H); 3.91 (s, 2H); 2.88 (t, 2H); 2.08 (m, 2H). Anal. t
29 p calcd. (%) for C H ClFN O : C, 61.60; H, 4.95; N, 14.97; Complex 1[PF ]: 164 mg, 47%. ESI-MS (m/z): found 724.1191
24 23 5 2 6
30 Found: C, 61.56; H, 5.02; N, 14.78. ([M − PF ]+ requires 724.1195). 1H NMR (acetone-d , 400 e
6 6 31
MHz) δ (ppm): 9.17(d, 1H), 8.76 (s, 1H), 8.15 (d, 1H), 8.09 (t, c
32 Synthesis of 6-(2-(2-(1H-imidazol-1-yl))ethoxy)-4-(3'-chloro-4'-
1H), 7.99 (s, 1H), 7.77 (bs, 1H), 7.66 (m, 2H), 7.58 (m, 1H), c
33 fluoroanilino)-7-methoxy-quinazoline (L3)
7.34 (s, 1H), 6.08 – 5.92 (m, 4H), 3.96 (s, 3H), 4.67 (t, 2H),
A
34
Imidazole (138 mg, 2 mmol), tetrabutyl ammonium bromide 4.30 (s, 2H), 3.57 (t, 2H), 2.85 (m, 1H), 2.11 (s, 3H), 1.22 (d,
35
(16 mg, 0.05 mmol), sodium hydroxide (240 mg, 6 mmol) were 3H), 1.12 (d, 3H). 13C NMR (DMSO-d , 100.6 MHz ) δ (ppm):
36 6 s
added in acetonitrile (20 mL) and the resulting mixture was 159.6, 157.5, 155.7, 155.4, 155.0, 152.6, 148.6, 139.9, 125.9,
37 c
heated at 80 °C for 1 h, then 6-(2-(2-bromoethoxy)ethoxy)-4- 125.5, 125.1, 123.9, 122.0, 119.7, 117.4, 117.2, 108.5, 105.8,
38 i
(3'-chloro-4'-fluoroanilino)-7-methoxy-quinazoline (L′1) (517 104.8, 97.1, 85.9, 84.32, 83.38, 82.8, 67.6, 61.0, 56.7, 55.3, m
39
mg, 1.21 mmol) was added, and the reaction continued for 4 h. 31.1, 22.7, 21.3, 17.9. Anal. calcd. (%) for
40
After concentrating and cooling to room temperature, the C H Cl F N O PRu (M + 2H O): C, 43.77; H, 4.34; N, 7.73; o
41 33 39 2 7 5 4 2
mixture was poured into water (20 mL) and ethyl acetate (10 Found: C, 43.34; H, 4.08; N, 7.34. l
42
l
43 mL). After filtering, washing by water and ethyl acetate, white a
44 solid precipitation was got as the product (389 mg, 78%). ESI- Complex 2[PF 6 ]: 159 mg, 45 %. m. p. 158 – 161 °C. ESI-MS t
45 MS (m/z): 414.113 ([M + H]+, C 20 H 18 ClFN 5 O 2 requires (m/z): found 738.1372, ([M − PF 6 ]+ requires 738.1352). 1H e
46 414.109); m. p. 274 – 279 °C. 1H NMR (DMSO-d 6 , 400 MHz) NMR (DMSO-d 6 , 400 MHz) δ (ppm): 9.07 (d, 1H); 8.68 (s, M
47 δ (ppm): 9.54 (s, 1H); 8.48 (s, 1H); 8.07 (dd, 1H); 7.78 (s, 1H); 1H); 8.06 (t, 2H); 7.96 (s, 1H); 7.74 (m, 1H); 7.69 (d, 1H); 7.62
48 7.75 (s, 1H), 7.73 (m, 1H); 7.42 (t, 1H), 7.30 (s, 1H); 7.20 (s, (t, 1H); 7.52 (s, 1H); 7.25 (s, 1H); 5.97 – 5.86 (m, 4H); 4.41 (m,
49 1H); 6.92 (s, 1H); 4.49 (t, 2H); 4.39 (t, 2H); 3.94 (s, 3H). Anal. 4H); 4.28 (td, 2H); 3.84 (s, 3H); 2.63 (m, 1H); 2.20 (m, 2H);
50 calcd. (%) for C 20 H 17 ClFN 5 O 2 : C, 58.05; H, 4.14; N, 16.9; 1.95 (s, 3H); 1.12 (d, 3H); 1.07 (d, 3H). 13C NMR (DMSO-d 6 ,
51 Found: C, 58.04; H, 4.20; N, 16.32. 100.6 MHz ) δ (ppm): 159.7, 157.4, 155.7, 155.5, 153.3, 151.8,
52 149.0, 139.9, 125.8, 125.2, 124.0, 123.9, 122.1, 119.6, 119.5,
Synthesis of 6-(2-(3-(1H-imidazol-1-yl))propoxy)-4-(3'-chloro-4'-
53 119.5, 117.4, 117.2, 108.6, 106.2, 103.4, 96.5, 86.3, 84.74,
fluoroanilino)-7-methoxy-quinazoline (L4)
54 82.77, 81.8, 67.4, 61.9, 56.6, 55.2, 31.1, 27.9, 22.5, 21.8, 17.9.
55 Imidazol (138 mg, 2 mmol), tetrabutyl ammonium bromide (16 Anal. calcd. (%) for C H Cl F N O PRu(M + 4H O): C,
34 45 2 7 5 6 2
56 mg, 0.05 mmol), sodium hydroxide (240 mg, 6 mmol) were 42.73; H, 4.75; N, 7.33; found: C, 42.56; H, 4.08; N, 7.93.
57 added in acetonitrile (20 ml) and the resulting mixture was
58 heated at 80 °C for 1 h, then added 6-(2-(3-
59
60
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Complex 3: 173 mg, 60%. m. p. 177 – 179 °C. ESI-MS (m/z): (Rigaku Corporation, Japan) using graphite monochromated
2
found 684.0895 ([M – Cl]+, requires 684.0884). 1H NMR Mo radiation (λ = 0.71073 Å) on a Rigaku Saturn 724 CCD
3
(DMSO-d , 400 MHz) δ (ppm): 8.49 (s, 1H); 8.39 (d,1H); 8.25 area detector, and the structure analysis was performed using
4 6
(d, 1H); 8.16 (m, 1H); 7.94 (s, 1H); 7.41 (m, 2H); 7.17 (d, 1H); SHELXL (Sheldrick, 2013).
5
5.85 – 5.63 (m, 4H); 4.59 (t, 2H); 4.53 (t, 2H); 3.93 (s, 3H);
6
2.83 (m, 1H); 2.09 (s, 3H); 1.19 (d, 3H); 0.96 (d, 3H). 13C NMR Hydrolysis
7
8 (DMSO-d 6 , 100.6 MHz ) δ (ppm): 156.6, 154.7, 153.3, 147.7, The stock solutions of complexes 1 – 5 (2 mM) were prepared
9 141.4, 137.4, 130.5, 123.8, 122.7, 122.6, 121.6, 116.9, 109.22, in methanol. Then 5 µL of the solution (10 µL for complex 2)
10 107.81, 106.9, 106.3, 102.4, 100.6, 100.4, 86.8, 86.1, 86.0, was diluted to 200 µL with deionized water in a quartz cuvette
11 81.6, 56.5, 47.1, 30.4, 22.3, 22.0, 18.3. Anal. calcd. (%) for and the UV-Vis spectra was recorded by scanning from 200 –
12 C 30 H 33 Cl 3 FN 5 O 3 Ru (M + H 2 O): C, 49.42; H, 4.42; N, 9.61; 500 nm at certain time intervals at 37 °C. The wavelengths
13 found: C, 49.13; H, 4.54; N, 9.62. corresponding to the LMCT band at ca. 330 – 336 nm were
14 selected for kinetic study. The time-dependent absorbance were
t
15 General method for preparation of complexes 4 and 5 (see fitted by Origin 8.0 (OriginLab Corporation, US) to give the p
16 Scheme 1). Ligand (0.4 mmol) and [(η6-p- first order rate constant k, and half reaction time t was
1/2 i
17 cymene)Ru(en)Cl][PF] (0.4 mmol) were added to 20 mL calculated by formula as follows: r
6
18 methanol and the resulting mixture was heated at 65 °C for 4 h, A = C e−kt + A c
0
19 then ammonium hexafluorophosphate (0.8 mmol) was added t = ln2 / k s
1/2
20 and stirred for 1 h. The product was purified by column u
21 chromatography on silica gel using dichloromethane/methanol where A is the absorbance, A 0 and C are constants. The species n
22 (25:1) as eluent to give the product as white powder. before and after hydrolysis for 1 h were analysed by LC-MS as
a
23 described below.
M
24 Complex 4 [PF ] : 171.8 mg, 43%. m. p. 123 – 125 °C. ESI-
6 2 High performance liquid chromatography (HPLC)
25 MS (m/z): found 822.1742 ([M − 2PF + CF COO]+, requires
6 3
26 822.1732). 1H NMR (DMSO-d , 400 MHz) δ (ppm): 8.53 (s, A Rheodyne sample injector, an Agilent Eclipse XDB-C18 d
6
27 1H); 8.34 (s, 1H); 8.14 (d, 1H); 7.90 (s, 1H); 7.80 (s, 1H); 7.61 reversed-phase column (150 × 4.6 mm, 5 µm, USA) and an e
28 Agilent 1200 series HPLC system were used to analyse the
(s, 1H); 7.45 (t, 1H); 7.26 (s, 1H); 7.22 (s, 1H); 6.37 (bs, 4H); t
29 hydrolytic mixtures of complexes 1 – 5. The mobile phases p 5.64 (s, 4H); 4.59 – 4.47 (m, 4H); 3.98 (s, 3H) ; 2.46 (m, 1H);
30 2.27 (bs, 2H); 2.07 (s, 3H); 1.87 (bs, 2H); 1.00 (d, 6H). 13C were water containing 0.1% TFA (A), and acetonitrile e
31 containing 0.1% TFA (B). The gradient was increased from
NMR (DMSO-d , 100.6 MHz ) δ (ppm):156.8, 155.1, 155.0, c
6
32 10% to 80% B during 20 min, with a flow rate of 1 mL/min.
152.9, 152.7, 148.0, 146.3, 142.4, 136.9, 130.4, 124.3, 123.1, c
33
123.1, 122.6, 119.4, 119.2, 117.1, 116.9, 109.0, 107.1, 106.5,
34 Electrospray ionization mass spectroscopy (ESI-MS) A
103.7, 98.5, 83.6, 83.0, 68.0, 56.6, 47.4, 44.8, 30.3, 22.5, 17.5.
35 The positive-ion ESI mass spectra were obtained with an Xevo
Anal. calcd. (%) for C H ClF N O P Ru (M + 6H O): C,
36 32 51 13 7 8 2 2 G2 Q-TOF mass spectrometer (Waters, USA) equipped with a s
34.71; H, 4.68; N, 8.86; found: C, 34.69; H, 4.03; N, 8.96.
37 Masslynx 4.1 workstation for data analysis. The spray voltage c
38 and the cone voltage were 3.3 kV and 5 V, respectively. The i
Complex 5 [PF ] : 162.2 mg, 40%. m. p. 138 – 141 °C. ESI- m
39 6 2
MS (m/z): found 836.1872 ([M − 2PF + CF COO]+, requires desolvation temperature was 350 °C and the source temperature
40 6 3
836.1888). 1H NMR (DMSO-d , 400 MHz) δ (ppm): 8.72 (s, 373 K. Nitrogen was used as both cone gas and desolvation gas o
41 6
with a flow rate of 50 L/h and 800 L/h, respectively. The
1H); 8.25 (s, 1H); 8.02 (dd, 1H); 7.98 (s, 1H); 7.73 – 7.71 (m, l
42
spectra were acquired in the range of 300 – 1000 Da. l
43 1H); 7.62 (s, 1H); 7.52 (t, 1H); 7.29 (s, 1H); 7.23 (s, 1H); 6.36 a
44 (bs, 4H); 5.63 (q, 4H); 4.47 (bs, 2H); 4.27 (t, 2H); 4.20 (t, 2H); DNA interaction t
45 4.02 (s, 3H) ; 2.41 (m, 1H); 2.28 (bs, 2H); 2.10 (s, 3H); 1.89 – e
46 1.87 (bs, 2H); 1.05 (d, 6H). 13C NMR (DMSO-d 6 , 100.6 MHz ) Hoechst 33342 (20 µM) and CT DNA (200 µM) were dissolved M
in Tris-HCl buffer (pH = 7.2) and incubated at ambient
47 δ (ppm): 157.8, 156.2, 156.1, 153.7, 151.3, 149.2, 142.0, 135.6,
temperature for 4 h. Then complex 1 or 3 (0 – 16 mM) were
48 130.5, 125.9, 124.6, 124.5, 122.3, 119.8, 119.6, 117.5, 117.3,
added to this solution to make the final concentrations from 0 to
49 108.3, 106.4, 104.1, 103.8, 103.8, 98.6, 83.5, 83.1, 66.4, 56.9,
160 µM. After incubating for 12 h, the resulting mixture was
50 45.2, 44.9, 30.3, 29.8, 22.6, 17.6. Anal. calcd. (%) for
measured on an F-4500 fluorescence spectrophotometer
51 C H Cl F N O P Ru (M + 3H O + 2CH Cl ): C, 33.98; H,
35 51 5 13 7 5 2 2 2 2
(HITACHI) with excitation wavelength at 370 nm and emission
52 4.16; N, 7.93; found: C, 33.55; H, 3.85; N, 8.34.
spectra from 400 to 650 nm. Modified Stern-Volmer plot40 was
53
X-ray crystallography employed to evaluate the affinity of complexes with DNA.
54
Different F /F values recorded at 490 nm with different
55 The single crystals of complex 4 suitable for X-ray analysis was 0
56 obtained by slowly diffusion of ethyl ether into the methanol concentrations of complex 1 or 3 was fitted by Origin 8.0
57 solution at ambient temperature. X-ray diffraction analysis was (OriginLab Corporation, US) and K sv was calculated by
58 carried out at 173K on a Rigaku Saturn 724+ diffractometer equations as follows:
59
60
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Journal Name ARTICLE
1
F /F = 1 + K [Q] the docking pocket was generated at the ATP binding cleft
2 0 sv
where F and F are the fluorescence intensities of Hoechst-CT automatically. Then complexes 1 – 5 (in which 1 – 3 are in the
3 0
DNA complex recorded before and after adding complex 1 or hydrolysed form) were successively docked into the pocket,
4
3, respectively. [Q] is the concentration of complex 1 or 3. and the molecular models corresponding to the constringent
5
energy gradient (0.05 kcal/mol) were gained. The docking
6
EGFR inhibition assay
scores are given as −logK , which represent the dissociation
7 D
8 Enzyme-linked immunosorbent assay (ELISA) was used to constants of the EGFR-inhibitor complexes.
9 evaluate the inhibition of compounds against the activity of
Secondary ion mass spectrometry images
10 EGFR. Various concentrations of the tested complexes in water
11 containing 1% DMSO were added to 4.37 µL DTT buffer and SIMS (secondary ion mass spectrometry) analysis and imaging
12 0.13 µL 188 ng/µL EGFR, after 5 min at room temperature, 25 was conducted using a TOF-SIMS V mass spectrometer
13 µL PTP1B (Tyr66), 0.36 µL ATP and 4.14 µL D 2 O were added. (IONTOF GmbH, Munster, Germany). Dual-beam depth
14 The mixture was incubated at 37 °C for 1 h, and 18 µL EDTA profiling strategy was used. A 10 keV argon cluster ion beam
t
15 was added to stop the reaction. Aliquot (25 µL) of the reaction (Ar n +) was used as a sputter beam, which scanned on a 300 × p
16 mixture and 75 µL D O were transferred to 96-well 300 µm2 area across the A549 cell surface. The current of the
2 i
17 streptavidin-coated plate and incubated at 37 °C for 1 h. Then Ar + was ~ 2 nA with lead-off time 60 µs. A 30.0 keV Bi + r
n 3
c
18 the solution was poured out and the plate was washed three beam with a 200 pA DC current, 100 ns pulse width and
19 times with 200 µL PBS/T. Then 100 µL primary antibody (P- repetition rate 5 kHz was applied as an analysis beam, which s
20 Tyr-100, 1:1000 in PBS/T with 1.5% BSA) was added and scanned on a 100 × 100 µm2 area at the centre of the Ar + crater u
n
21 incubated at 37 °C for 1 h. After washing three times with 200 by 256 × 256 pixels with the highest resolution of 500 nm. n
22 µL PBS/T, 100 µL secondary antibody (IgG (H+L), 1:1000 in Positive spectra were recorded and calibrated by H+, CH + and
3 a
23 PBS/T with 1.5% BSA) was added and incubated at 37 °C for 1 C H +. The signal intensities were displayed on a colour scale,
2 5 M
24 h. After washing three times with 200 µL PBS/T, 100 µL TMB which were directly related to the concentration of ions of
25 (1 mg/mL TMB : Citric acid-phosphate buffer : 30% H O = interest.
2 2
26 d
100 : 900 : 1) was added. After 15 min, 100 µL of 2 M H SO
2 4
27 was added and the plate was recorded at 450 nm on the ELISA Fluorescence microscopy e
28 plate reader (SpectraMax M5 Molecular Devices Corporation). A549 cells (3 × 105 per well) were plated to laser scanning t
29 p confocal petri dish and grew in the absence of EGF for 24 h.
30 Anti-proliferation assay Then the cells were exposed to each complex at 37 °C for 24 h. e
31
The ruthenium complexes were evaluated for anti-proliferative The fluorescent dyer Hoechst 33342 (2.5 mg) was dissolved in c
32
activity against five human cancer cell lines: cervical cancer 1 mL deionised water, and then diluted to 25 µg/mL by c
33
(HeLa), non-small-cell lung carcinoma (A549), breast cancer medium. After removing the cell culture medium and washing
A
34
(MCF-7), prostate cancer (PC3), and squamous cell carcinoma once with PBS, 1 mL 1 µg/mL Hoechst 33342 was added and
35
(A431). The A431 cells was maintained in F12K and others the cells were incubated at 37 °C for 10 min and washed three
36 s
were in DMEM medium supplemented with 10% FBS, 1% PS times with 1 mL PBS. The cells were maintained by minimal
37 c
at 37 °C with 5% CO . Aliquot (100 µL) of each cell line in colourless medium. Fluorescence images were obtained by a
38 2 i
medium was placed in 96-well plates at the following density, FV1000-IX81 confocal laser scanning microscope m
39
A431 80000/mL, A549 50000/mL, MCF-7 70000/mL, PC-3 (OLYMPUS), at excitation wavelength of 405 nm and emission
40
50000/mL, HeLa 50000/mL, and incubated in the absence or wavelength of 425 – 500 nm. o
41
presence of 100 ng/mL epidermal growth factor (EGF, Sigma, l
42
Fluorescence-activated cell sorting (FACS) analysis l
43 USA) for 24 h. Then the medium was removed and 200 µL a
44 fresh medium with complexes of various concentrations were A549 cells were seeded in a density of 2 × 105 per well in a 6- t
45 added. After incubated at 37 °C for 48 h, the cells were washed well plate and cultured for 24 h, then the cells were exposed to e
46 with PBS twice and measured by MTT assay. different tested compounds at 37 °C for 24 h. The supernatant M
47 was removed, and the cells were washed with PBS and
Docking analysis
48 detached by trypsin digestion. After washing again with PBS,
49 All docking studies and molecular modelling were carried out the cells were transferred to FACS tubes and centrifuged at
50 by Surflex-Dock module of Sybyl X 1.1 program, running on 1000 rpm for 3 min. After re-suspension in 0.5 mL binding
51 Dual-core Intel(R) E5300 CPU 2.60 GHz, RAM Memory 2 GB buffer, the cells were incubated with 5 µL Annexin-V conjugate
52 under the Windows XP system. The crystal structure of the for 5 min, followed by the addition of 5 µL 7-AAD in the dark
53 EGFR-erlotinib complex from PDB (1M17)41 was used as the prior to the FACS analysis. The FACS assays were performed
54 leading structure to build the corresponding structures of the on a Calibur Flow Cytometer (BD, Franklin Lakes, New Jersey,
55 complexes with EGFR in this work. All the water molecules in US), of which the FL2 channel was used to record the intensity
56 the EGFR-erlotinib crystal was eliminated except H O10 as it of Annexin V-PE staining and FL3 channel for 7-AAD
2
57 plays an important role for the hydrogen bonds between staining. The data were analysed by Sell Quest software (BD,
58 erlotinib and EGFR.42 After extracting the erlotinib molecule, Franklin Lakes, New Jersey, US).
59
60
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ARTICLE Journal Name
1
Results and discussion The structure of complex 4 was further characterised by X-
2
ray crystallography (CCDC deposit number 1061312), as
3
Synthesis and characterisation
shown in Fig. 1. The structure of complex 4 adopts a triclinic
4
To develop desired 4-anilinoquinazoline ligands for space group. Ru centre forms π coordination bonds with the p-
5
6 coordinating to {(η6-p-cymene)RuII} pharmacophore, 4-(3'- cymene group to construct the typical “piano stool” structure,12,
7 chloro-4'- fluoroanilino)-6-hydroxy-7-methoxyquinazoline was 15, 32, 46 the rest of coordination sites of Ru occupied by three N
8 reacted with imidazole or 2-(aminomethyl)pyridine to give atoms (N5, N6, N7) from ethylenediamine and L3. The details
9 ligands L1 – L4, respectively (Scheme 1) following a of data collection and structure refinement are listed in Table
10 published method.38, 43 Then the arene-ruthenium(II) dimer S1 and selected bond lengths (Å), angles and torsions (°) in
11 complex [(η6-p-cymene)RuCl 2 ] 2 was reacted with ligands L1 or Table S2 in the supporting information. In solution, two
12 L2 to produce complexes 1 or 2, respectively, and [(η6-p- methylene between the quinazoline and the imidazole groups
13 cymene)Ru(en)Cl][PF] was reacted with L3 or L4 to produce are quite flexible, and distance between the gefitinib activation
6
14 complexes 4 or 5, respectively.30, 44 While complex 3 was group and Ru activation group is long enough for both moieties
t
15 produced by reacting [(η6-p-cymene)RuCl] with L3, as shown to exert their effects. However, in the EGFR enzyme inhibitory p
2 2
16 in Scheme 1. These target complexes were synthesised with activity experiments below, it was found that the Ru groups in
i
17 moderate yields and their structures were characterised with 1H, complexes 1, 2, 4 and 5 still hinder the binding of the r
18 13C NMR, MS, and elemental analysis. The details are given in quinazoline unit to the ATP binding pocket of EGFR. c
19 the experimental section. s
20 In the 1H NMR spectra of complex 1 – 5, the resonances u
21 between 9.2 and 7.2 ppm are assigned to the aromatic protons n
22 of the 4-(3’-chloro-4’-fluoroanilino)-7-methoxy-quinazoline
a
23 and 2-(aminomethyl)pyridine (10H) or imidazole groups (9H).
M
24
The three protons of the 7-methoxy group of the quinazoline
25
show typical sharp singlet at 3.9 – 4.0 ppm. The aromatic
26 d
protons of the p-cymene group show resonances at ca. 5.9 ppm
27 for complexes 1 – 2 , and ca. 5.6 ppm for complexes 3 – 5. The e
28
coordination to ruthenium shifts the resonances of arene t
29 p protons to the higher field (5.8 – 6.3 ppm). The proton on the
30
tertiary carbon of the isopropyl group in p-cymene shows a Fig. 1. X-ray crystal structure of the cation of complex 4. The hydrogen atoms, e
31
typical quintet at 2.4 – 2.8 ppm.
ethyl acetate, methanol and PF6 − groups are omitted for clarity.
c
32
Complexes 1 – 5 have a derived quinazoline group which is c
33 Hydrolysis of Ru complexes
the active site of gefitinib. Meanwhile, they have a half- A
34
sandwich ruthenium moiety, which is broadly regarded The hydrolysis of halide leaving groups in {(η6-arene)RuII}
35
36 cytotoxic.32, 45 A flexible two/three-carbon chain connect the complexes is broadly considered as an essential step to activate s
above two fragments. This design enable the two active sites the complexes towards biomolecules.12, 23 In this work, the
37 c
exert their individual effects but have minimal influence to each hydrolysis reactions of complexes 1 – 5 were carried out in in
38 i
other. methanol-water (1 : 40) at 37 °C and were followed by UV-Vis m
39
spectrophotometry and characterized by HPLC-ESI-MS before
40
F and after hydrolysis for 1 h. The time-dependent UV-Vis o
41
HN Cl
42 HO N F F + spectra of the hydrolysis of 1 – 3 are shown in Fig. 2a, c and e. l l
43 O N N H HN Cl NRu Cl HN Cl By comparison, no obvious changes in the absorption spectra of a
44 N()n O N N H ()n O N 4 and 5 were observed, and are therefore not shown here. t
4 4 5 6 F L L 1 2 , , n n = = 2 3 O N 1 2 , , n n = = 2 3 O N H Th P e L C m -E on S o I- /d M i- S a , q u a a s s s h p o ec w ie n s in o f F 1 ig . – S 1 3 – w e S r 3 e . c T h h a e r a i c o te n r s i z m ed /z b a y t M e
HN Cl
47 Br()n O N 2+ 688.1400, 702.1608, 684.0895 and 648.1098 were observed
4 4 8 9 L L O 1 2 ' ' , , n n = = 2 3 N N N()n O HN N C F l H2N N R H u 2 N N()n O HN N C F l a c n ym d e c n o e r ) r R e u sp L o 1 n (H de 2 d O ) to − H th 2 e O h − y H dr ] o + l y re ti q c u i s r p es e c m ie / s z 6 1 8 -H 8. 2 1 O 43 4 ([ ) ( η (F 6- i p g - .
50 O N O N S1), 2-H O ([(η6-p-cymene)RuL2(H O) − H O − H]+ requires
L3,n=2 4,n=2 2 2 2
51 L4,n=3 5,n=3 m/z 702.1591) (Fig. S2), 3-H O ([(η6-p-cymene)RuClL3(H O) −
2 2
52 H O]+ requires m/z 684.0884) and 3-2H O ([(η6-p-
F 2 2
53 HN Cl cymene)RuL3(H 2 O) 2 − 2(H 2 O) − H]+ requires m/z 648.1121)
54 Cl Ru C N l N O O N N (Fig. S3) respectively.
55
3
The plots of the hydrolysis kinetics of 1 – 3 are displayed in
56 Scheme 1. Synthesis of 4-anilinoquinazoline derivatives L1 – L4 and complexes 1 Fig. 2b, d and f. The rate constants (k) and half-times (t ) of 1
1/2
57 – 5. – 3 were fitted according to first order reaction kinetics,45 as
58 shown in Table 1. Notably, complex 3 containing two chloride
59
60
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ligands undergoes two-step hydrolysis, but the second chloride
leaves as soon as the first one hydrolyse. For easy
demonstration, the hydrolysis of 3 was treated as a single step
first order reaction in calculating the kinetics. Di-aqua species
3-2H O was observed as the major product after hydrolysis for 2
only 1 h, as shown in the LC-MS study in Fig. S3 in the
supporting information.
The hydrolysis of complex 3 is the fastest (t = 3.3 min), 1/2
compared to 34 min for complex 1 and 32 min for complex 2.
The hydrolysis kinetics of complexes 1 and 2 is similar to the
complexes of general formula [Ru(η6-arene)Cl(N-N)],8, 45 and 2.0
the pseudo-simultaneous hydrolysis of the two chlorides in 3 0 5 mim
1.5 10 has also been reported for Ru arene PTA complex (RAPTA, 15
30 PTA = 1,3,5-triaza-7-phosphaadamantane).47 The stability of 1.0 4 6 5 0
75
the EGFR inhibiting group with Ru centre may benefit the dual- 90 0.5 105
targeting feature of these complexes. 115
0.0
200 300 400 500 600
DNA interaction
Ruthenium complexes [(η6-arene)RuII(en)Cl]PF might exert
6
their anticancer activities by covalently and non-covalently
interaction with DNA.7, 48 The hydrolysis of leaving groups
lead to covalently bound to DNA base, in particular guanine, accompanied by the intercalation of the arene ligands into DNA
bases.23, 25, 46, 49 Moreover, interaction of ruthenium complexes
with DNA via groove binding is also a very important
mechanism for their anti-proliferation activity.50 Here, the
nucleus staining reagent Hoechst 33342 and CT DNA (calf
Fig. 2. Time-dependent UV-Vis spectra for the aquation of 1 (a), 2 (c) and 3 (e). thymus DNA) were used to study the interaction of complexes The right panel (b, d and f) are the plots of the changes in the absorbance (black
1 and 3 with DNA. Free Hoechst 33342 has weak fluorescence square) at selected wavelength (330 nm for 1, 333 nm for 2 and 336 nm for 3
(aquation of two Cl−), respectively) vs time (squares) and fittings according to
which can be enhanced when it binds to DNA at the minor
first-order reaction kinetics (lines).
groove.40 Thereafter, if other DNA minor groove binder is
added, Hoechst 33342 can be competitively replaced, resulting
Table 1. The hydrolysis rate constants (k) and half-times (t ) of complexes 1 1/2
in decreased fluorescence intensity. When complex 1 or 3 (from – 5.
0.5 to 160 µM) was added to the solution of Hoechst 33342-CT Complex 1 2 3 (1st & 2nd ) 4 5
DNA complex and incubated for 12 h, the fluorescence k (10 -4 s-1) 3.6 5 3.9 0 39 .7 - a -
emission were found to decrease with the increased amount of t (min) 34.0 32.0 3.3 - -
1/2
Ru complex added, as shown in Fig. 3. This result suggests that
a No substantial hydrolysis observed.
complex 1 and 3 can bind to the minor groove of CT DNA. The
Stern-Volmer constant40 (K , quenching constant) was
sv
employed to evaluate the binging affinity of the complexes with
DNA and the linear Stern-Volmer plot was shown in Fig. S4 in
the supporting information. The K values of complexes 1 and sv
3 are 10.3 × 104 and 4.3 × 104 µM−1, respectively, indicating
that the minor groove binding of 1 with CT DNA is stronger
than that of 3.
A DNA replication inhibition experiment was also carried
out to examine if the ruthenium complexes bound DNA leads to
Fig. 3. The fluorescence spectra of Hoechst 33342-CT DNA (20:200 μM) complex
the inhibition of the replication by polymerase. As shown in Fig. reacting with various concentration of complex 1 (a) or 3 (b). The excitation
S5, the binding of the ruthenium complex 3 to a Homo Sapiens wavelength (λ ex) was 370 nm; r 1 or r 3 is the molar ratio of corresponding complex
to Hoechst 33342.
High Mobility Group Box 1 sequence DNA can substantially
inhibit its replication.
EGFR inhibitory activity
These results indicates that complexes can interact with
DNA so strongly that the function of DNA can be inhibited. Epidermal growth factor (EGF) stimulates cell growth through
Considering the subcellular distribution of Ru complexes, as binding to its receptor (EGFR) and initiating a series of cellular
shown below, the binding to DNA can be substantial in vitro. signal transduction pathways for proliferation and
differentiation. Therefore, inhibiting the activity of EGFR
This journal is © The Royal Society of Chemistry 2012 J. Name., 2012, 00, 1-3 | 7
ecnabrosbA
wavelength / nm
2.0
0 mim
5
1.5 10 15
30
1.0 4 6 5 0
75 90 0.5 120
150
0.0
200 300 400 500 600
333 nm
ecnabrosbA
wavelength / nm
0.73
0.72
330 nm
0.71
0.70
0.69
0 30 60 90 120 150
mn
033
ta ecnabrosbA
time / min
0.99
0.97
0.95
0.93
0.91
0 30 60 90 120
mn
333
ta ecnabrosbA
time / min
2.0
1.5 0 min
5 10 1.0 15 30
45 0.5 60
0.0 200 300 400 500 600
ecnabrosbA
wavelength / nm
0.75
336 nm 0.70
0.65
0.60
0.55
0.50
0.45 0 10 20 30 40 50 60
mn 633
ta ecnabrosbA
(a) (b)
(c) (d)
(e) (f)
time / min
2500 r 3
0
2000 0.5 1
2 1500 3
4 1000 5
6 500 8
0
400 450 500 550 600 650
Wavelength / nm
u.a
/ ytisnetnI
2500
2000
1500
1000
500
0
400 450 500 550 600 650
u.a
/ ytisnetnI
Page 7 of 12 Metallomics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
t 15 p
16 i
17 r
c 18
19 s
20 u
21 n
22 a
23 M 24
25
26 d
27 e
28
t
29 p
30
e
31
c
32
c
33
A 34
35
36 s
37 c
38 i
m
39
40
o
41
(a) r (b) 1 l
42 0 l
43 0 1 .5 a
2 44 3 t
4 e 45 5
46 6 M 8
47
48
Wavelength / nm
49
50
51
52
53
54
55
56
57
58
59
60
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ARTICLE Journal Name
1
kinase can effectively suppress the EGF stimulated malignance. proliferation activity towards A549, PC-3, MCF-7 and A431
2
L3 has been previously shown highly active to inhibit the cell lines in the absence of EGF, and towards A549 and A431
3
EGFR activity with half maximal inhibitory concentration cells in the presence of EGF. Complex 5 showed high/moderate
4
(IC ) value of 60.2 nM.38 Herein, enzyme-linked anti-cancer activity towards A549, HeLa, MCF-7 and A431 cell
5 50
immunosorbent assay (ELISA) was used to measure the lines in the absence of EGF, and moderate activity against
6
inhibition efficacy of 1 – 5 towards EGFR with gefitinib as a A549 and A431 cell lines in the presence of EGF.
7
8 reference. The IC 50 values are listed in Table 2 and the dose- It is usually believed that EGF can cause the conformational
9 dependent inhibition curves shown in Fig. S3 in the supporting change of EGFR, which fully activates the protein kinase and
10 information. All of the synthesised complexes are highly active subsequently transduces the signal of cell growth. As a result, a
11 EGFR inhibitors with IC 50 values at nano-molar level. It should small molecule EGFR inhibitor should become more potent in
12 be noticed that for complex 3, its activity (IC 50 = 66.1 nM) is inhibiting the cell growth when exogenous EGF is added. In
13 very close to its ligand L3, and much higher than the other this work, as shown in Table 2, only 2 and 3 show EGF
14 complexes (1, 2, 4, and 5) and even higher than gefitinib (IC 50 dependent anti-proliferation activity against A431 cancer cell t
15 = 94.0 nM). This result suggests that introducing line, the IC 50 values being 53/>100 and 22/32 µM in the p
16 organometallic Ru group can keep the inhibitory activity of the presence/absence of EGF. But for the rest of the cases, when
i
17 4-anilinoquinazoline ligands towards EGFR and the existing of EGF was added to the medium, the IC values are higher than r
50
c
18 the small leaving group, chloride, in this class of complexes or close to those in the absence of EGF. These results indicate
19 may be better than the en group for maintaining their EGFR that besides the EGFR inhibition, complexes 1 – 5 may exert s
20 inhibitory activity. their antitumour activity partially by means of other u
21 mechanisms such as DNA interaction as mentioned above, n
22 In vitro anti-proliferation activities which however may not be efficient enough to counteract the
a
23 The anti-proliferation activities of complexes 1 – 5 towards five effect of adding EGF.
M
24 human cancer cell lines: cervical cancer (HeLa), non-small-cell The overall antitumour activity of complex 3 is higher than
25 lung carcinoma (A549), breast cancer (MCF-7), prostate cancer 1, 2, 4, 5. Specifically, in the absence of EGF, the anti-
26 d
(PC-3), and squamous cell carcinoma (A431) were evaluated. proliferation activity of complex 3 towards A549 cells is even
27 This study was performed either in the presence or in the better than RM116, gefitinib or the combined use of RM116 e
28
absence of EGF (100 ng/mL), in order to evaluate the and gefitinib. Moreover, in the presence of EGF, 3 is still t
29 p contribution of blocking the signal transduction of EGF to the comparable to RM116, gefitinib or the combined use of them
30
inhibitory potency of the tested complexes towards the growth against the growth of A549 cells. Those results indicate that the e
31
of tumour cells. The well-established antitumour drug cisplatin possible hydrolysis of two chloride ligands in complex 3 may c
32
was used as a reference in the absence of EGF, and gefitinib in contribute to the enhanced anticancer activity of this complex. c
33
the presence of EGF. In addition, ruthenium complex RM11622 Compared to the previously reported Ru complex
A
34
was also used as a “(arene)Ru-moiety-only” control against [RuIIICl (DMSO)(L3)],38 having the same EGFR inhibiting 4-
35 4
A549 cancer cell line. The IC values of the tested complexes anilinoquinazoline group (L3), complex 3 has similar potency
36 50 s
against each cancer cell line were listed in Table 2. in inhibiting EGFR, but higher anti-proliferation activity
37 c
Complex 1 exhibited least activity among these complexes, towards MCF-7 cell line in the absence of EGF. More evidence
38 i
showing only low activity against MCF-7 cell line. Complex 2 based on the interaction of 3 with EGFR and DNA can be m
39
showed moderate anti-proliferation activity towards A549 cell found in docking analysis and mass spectrometry imaging (vide
40
line in the absence of EGF, and towards A431 cell line in the infra). o
41
presence of EGF. Complex 3 and 4 showed high/moderate anti- l
42
l
43 a
44 t
e
45
46 M
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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1 Journal Name
RSCPublishing
2
3
4
5 ARTICLE
6
7
8
9 Table 2. The half maximal inhibitory concentrations (IC ) against A549, HeLa, PC-3, MCF-7 and A431 cell lines (µM), IC towards EGFR (nM) and in
50 50
10 silico docking scores (minus logarithm of the disassociation constants) to EGFR ATP binding pocket of complexes 1 – 5 and reference compounds.
11
A549 (µM) HeLa (µM) PC-3 (µM) MCF-7 (µM) A431 (µM)
12 EGFR (nM) Docking
scores
13 +EGF −EGF +EGF −EGF +EGF −EGF +EGF −EGF +EGF −EGF
14
t
1 >100 >100 >100 >100 >100 >100 >100 83 ± 4 >100 >100 180 ± 12 7.1
15 p
16
2 98 ± 3 50 ± 6 >100 >100 >100 >100 >100 88 ± 6 54 ± 7 >100 347 ± 29 6.9 i
17 r
c
18 3 31 ± 6 15 ± 2 >100 >100 >100 64 ±9 >100 54 ± 4 22 ± 2 32 ± 7 66 ± 11 8.6
19 s
20 4 52 ± 4 24 ± 4 >100 >100 >100 78 ± 6 >100 73 ± 3 67 ± 6 31 ± 6 145 ± 28 7.4 u
21
n
5 68 ± 6 34 ± 8 >100 48 ± 3 >100 >100 >100 77 ± 4 85 ± 11 29 ± 5 217 ± 14 7.3
22
a
23 gefitiniba 20 ± 2 31 ± 3 16 ± 0.5 -b 34 ± 3 - 34 ± 2 - 6 ± 1 - 94 ± 3 8.4
M
24
25 cisplatina - 11 ± 1 - 12 ± 1 - 13 ± 3 - 16 ± 1 - 11 ± 1 - -
26 d
[RuIIICl(DMSO)(L3)]38 - - - - - - >100 >100 - - 60.8 ± 3.5
27 4 e
28 RM116c 33 ± 4 31 ± 8 - - - - - - - - - - t
29 p
30 RM116 + gefitinib 21 ± 2 25 ± 3 - - - - - - - - - - e
31
32 a Cisplatin was used as reference in the absence of EGF, and gefitinib in the presence of EGF c
c
33 b Not tested/applicable.
A
34
c RM116 = [(η6-p-cymene)Ru(en)Cl][PF] 35 6
36 s
Docking analysis complex 3 has a smaller organometallic Ru group and the Cl
37 c
ligands may hydrolyse, leading to formation of an additional H-
38 To further investigate the EGFR inhibiting activity of the newly i
bond between the aqua ligand (H-O-H) and the C=O of Asp831 m
39 synthesised ruthenium complexes and 4-anilinoquinazoline
in EGFR (see Fig. S7a and S7b in the supporting information).
40 derivatives, in silico docking analysis was carried out with
This H-bond compromises the steric hindrance and thus o
41 Surflex-Dock module of Sybyl-X 1.1 program, and the affinity
increases the affinity of 3 to EGFR. l
42 of these compounds with the ATP binding site of EGFR kinase l
43 was evaluated. As complexes 1 – 3 are ready to hydrolyse in a
Subcellular distribution of ruthenium complexes in A549 cells
44 aqueous solutions, their mono-/di-aqua products were used for t
45 docking analysis. The docking scores are given as the minus Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was e
46 logarithm of the disassociation constants, as listed in Table 2. used to determine the subcellular distribution of the Ru M
47 The high docking scores of complexes 1 – 5 suggest the high complexes in A549 cells using 3 as an example. The nucleus
48 affinity of the small molecules towards EGFR, and the DNA and membrane proteins were extracted respectively from
49 inhibition potency against EGFR given from ELISA is in complex 3 treated A549 cells, and the level of Ruthenium was
50 determined by ICP-MS (experimental details in the supporting
consistence with the trend (Table 2), indicating the rationality
51 information). The Ru was 1058 ± 101 ng per mg membrane
of this docking model. Complex 3 has a docking score of 8.6,
52 protein and 26 ± 11 ng per mg nucleus DNA.
which is slightly higher than that of gefitinib (8.4). However,
53 The distribution of Ru in cell level was further determined
the rest of the complexes are substantially less affinitive
54 by secondary ion mass spectrometry (SIMS) to image the
towards EGFR than that of gefitinib. We speculate that the
55 presence of ruthenium complexes in single cells. Mass
bulky organometallic Ru groups in complexes 1, 2, 4 and 5
56 spectrometry images were taken after sputtering with a 10 keV
increases the steric hindrance which destabilizes the binding
57 argon cluster ion beam, so the image only shows the existence
conformation, and therefore reduces their affinity. In contrast,
58 of Ru inside the cells. Fig. 4 displays the distribution maps
59
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obtained from A549 cells treated with complex 1, 2 or 3, 20.00 µm
2 1.0
respectively. Images of the total ions shown in Fig. 4a, c and e (a) (b)
3 200
4 depict the profile of cells; while the images for Ru complexes 0.8
are shown in Fig. 4b, 4d and 4f. Due to sputtering off of the cell 160
5
membrane prior to the SIMS imaging, little complexes 1 and 2 0.6
6 120
were found to localise inside the cells (Fig. 4b and 4d). In
7 0.4
8 contrast, higher level of complex 3 was observed in the central 80
9 region of the cell (Fig. 4f), which may contribute to the higher 20 µµµµm 40 20 µµµµm 0.2
10 anti-proliferation activity of 3 than that of the other complexes.
11 These results indicates that although a large amount of total 0 689.72 u 50.00 µm 0 1 . . 0 0
12 complex 3 tends to bind to the membrane protein as it has an (c) (d)
300
13 EGFR binding moiety, it can still enter A549 cell and exert not 0.8
14 only enzyme inhibition but also DNA binding effect. This again
0.6 t
15 support the dual-targeting anticancer mechanism of this 200 p
16 complex. 0.4 i
17 r
100
18 Cell apoptosis 0.2 c
20 µµµµm 20 µµµµm
19 As complex 3 is the most cytostatic one against the cancer cell s
20 lines among all the metal complexes studied in this work, its total 0 703.751 u0.00 µm 4 0 . . 0 0 u
(e) (f)(b)
21 mechanism of action was further explored. Fluorescence n
200
22 microscopy imaging was used to evaluate the potential of 3.0
a
23 complex 3, gefitinib and RM116 to induce apoptosis of A549
150 M
24 cells without additional EGF. The cells were treated with 2.0
25 different compounds and were then stained with Hoechst 33342. 100
26 d
Their microscopic images are shown in Fig. 5a (blank), 5b 1.0
27 (gefitinib), 5c (RM116), 5d (RM116 + gefitinib) and 5e 10 µµµµm 50 10 µµµµm e
28
29
(complex 3), respectively. Fig. 5b indicates that gefitinib
total 0 649.33 u 0.0 p
t
caused the aggregation of the A549 cell nuclei. By contrast, Fig. Fig. 4. SIMS images obtained from A549 cells treated with complex 1 (a and b), 2
30 5c – e give clear evidence of forming much apoptotic bodies (c and d), and 3 (e and f), respectively. The left panel (a, c and e) are total ion e
31 images; right panel (b, d and f) are images of ions at m/z 689.7 ([1 − Cl − H]+,
with condensed chromatin, indicating that RM116 and 3 exert calcd. 689.2), 703.8 ([2 − Cl]+, calcd. 703.2), and 649.3 ([3 − 2Cl − H]+, calcd. c
32
their effect by inducing apoptosis 649.1), respectively. Fields of view for complexes 1 – 3 are 150 × 150 μm, 150 × c
33 150 μm and 50 × 50 μm, respectively.
FACS was then used to quantitatively measure the ratio of
A
34
apoptosis induced by the above compounds (Fig. 5f – j). The
35
results indicated that complex 3 (Fig. 5j) induced a large ratio
36 s
of A549 cells early-stage apoptosis (56.8%), much higher than
37 c
those caused by gefitinib (7.52%, Fig. 5g), RM116 (10.7%, Fig.
38 i
5h) and combined gefitinib and RM116 (18.6%, Fig. 5i). m
39
Gefitinib is regarded as an EGFR inhibitor which mainly blocks
40
the cell signalling pathway initiated by auto-phosphorylation of o
41
EGFR and have less capacity to induce apoptosis.51 In contrast, l
42
l
43 RM116 containing the cytotoxic organometallic Ru a
44 pharmacophore is anticipated to induce more apoptosis.22 In t
45 this work, the coupling of EGFR inhibiting 4- e
46 anilinoquinazoline group with {(arene)RuII} moiety in complex M
47 3 produces “1 + 1 > 2” effect in inducing A549 apoptosis, in
48 particular early-stage apoptosis (56.8%). These results suggest
49 the success of developing dual-targeting anticancer complexes
50 based on the synergetic effect of mono-functional
51 pharmacophores.
52
53
54
55
56
57
58
59
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Their structures were characterised and the hydrolysis
2
properties were investigated. These complexes exhibited strong
3
EGFR inhibiting activity and high affinity to DNA via minor
4
groove binding. The in vitro screening results indicated that this
5
group of complexes are moderately active to inhibit the growth
6
of A549 cancer cell line, and the anti-proliferative activity of
7
8 the most active complex 3 approaches to that of cisplatin, being
9 higher than those of gefitinib, complex [RuIIICl 4 (DMSO)(L3)]
10 with similar EGFR inhibiting 4-anilinoquinazoline group, arene
11 ruthenium complex RM116, and combined gefitinib and
12 RM116. Intriguingly, complex 3 was shown to induce much
13 higher level of early stage apoptosis of A549 cancer cell line
14 than gefitinib, RM116, and combined gefitinib and RM116.
t
15 These findings validate the dual-targeting features of the p
16 EGFR-inhibiting and DNA-binding organometallic ruthenium
i
17 anticancer complexes developed in this work and provide r
c
18 insights into the future development of more effective and
19 specified antitumour agents. s
20 u
21 Acknowledgment n
22
This work was supported by the National Science Foundation a
23
24 of China (No. 21301181, 21371006, 21135006, 21321003, M
25 21127901, 21275148), the Anhui Provincial Natural Science
26 Foundation (No. KJ2011A153). d
27 e
28 Notes and references
t
29 p
30
e
31
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