Platinum- oxazoline complexes as anti-cancer agents: syntheses, characterisation and initial biological studies

Paras N. Yadav; Ramsay E. Beveridge; Jonathan Blay; Alaina R. Boyd; Maja W. Chojnacka; Andreas Decken; Ankur A. Deshpande; Michael G. Gardiner; Trevor W. Hambley; Michael J. Hughes; Leslie Jolly; Jacquelyn A. Lavangie; Timothy D. MacInnis; Sherri A. McFarland; Elizabeth J. New; Robert A. Gossage

doi:10.1039/C0MD00211A

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DOI: 10.1039/C0MD00211A (Concise Article) Med. Chem. Commun., 2011, 2, 274-277

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Platinum-oxazoline complexes as anti-cancer agents: syntheses, characterisation and initial biological studies†‡

Paras N. Yadav§ ^a, Ramsay E. Beveridge¶ ^a, Jonathan Blay ^b, Alaina R. Boyd¶ ^a, Maja W. Chojnacka¶ ^c, Andreas Decken|| ^d, Ankur A. Deshpande¶ ^a, Michael G. Gardiner** ^e, Trevor W. Hambley ^f, Michael J. Hughes¶ ^a, Leslie Jolly¶ ^e, Jacquelyn A. Lavangie¶ ^a, Timothy D. MacInnis¶ ^a, Sherri A. McFarland *^a, Elizabeth J. New ^f and Robert A. Gossage *^ac
^aDepartment of Chemistry, Acadia University, Wolfville, NS B4P 2R6, Canada. E-mail: sherri.mcfarland@acadiau.ca; Fax: +1-902-5851114
^bDepartment of Pharmacology, Sir Charles Tupper Medical Building, Dalhousie University, 5850 College Street, Halifax, NS B3H 1X5, Canada
^cRyerson University, Department of Chemistry & Biology. 350 Victoria Street, Toronto, ON M5K 2B3, Canada. E-mail: gossage@ryerson.ca; Fax: +1-416-9795044
^dDepartment of Chemistry, University of New Brunswick, Fredericton, NB E4L 1G8, Canada
^eSchool of Chemistry, The University of Tasmania, Hobart, TAS 7001, Australia
^fSchool of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia

Received 10th November 2010 , Accepted 14th December 2010

First published on 24th January 2011

Abstract

The syntheses, characterisation and biological activities (IC₅₀; DNA binding) of four mononuclear Pt oxazoline complexes are reported. These materials are the compounds cis-[PtCl₂(NH₃)(Etox-κ¹N)] (1: Etox = 2-ethyl-2-oxazoline), [PtCl₂(anilox-κ²N,N′)] (2: anilox = 4,4-dimethyl-2-[o-anilinyl]-2-oxazoline), cis-[PtCl₂(Etox-κ¹N)₂] (3) and [PtCl(pyox-κ³N,N′,N′)] (4: pyoxH = pyridine-2-carboxyanil-[o-{4,4-dimethyl-2-oxazolinyl}-ide]) and all four are shown to have slightly lower cytotoxicities in vitro when compared to cisplatin against the A2780 ovarian cancer cell line. These new materials all appear to be bio-active via the formation of DNA adducts. Complexes 1 and 2 have been further characterised in the solid-state by X-ray diffraction methods.

Introduction

Platinum compounds containing N-donor ligands have played a unique role in the development of both coordination and medicinal chemistry. The serendipitous discovery (circa 1965) and later clinical application of the simple inorganic complex cisplatin (i.e., cis-[PtCl₂(NH₃)₂]: Fig. 1) as an anti-cancer agent marks¹ an important milestone in the development of inorganic chemotherapy agents.^1,2 Since that time, a number of 2nd and 3rd generation Pt-containing reagents have been designed, tested and/or entered clinical use.^3,4 Modifications of the original cisplatin scaffold have been undertaken to increase clinical efficacy and/or to reduce the (toxic) side-effect profile. Synthetically, this has focused on three main areas: (i) application of Pt complexes with higher formal oxidation state(s) at the metal centre (primarily Pt⁴⁺) such as Satraplatin (a.k.a. JM216: Fig. 1);⁵ (ii) the use of non-halide formal anions within the metal coordination sphere (e.g., Carboplatin; Fig. 1)^3,4 and/or (iii) replacement of one or more ammine ligands with other N-donor fragments, such as the substituted pyridine ligand found in Picoplatin (i.e., ZD0473: Fig. 1).³ These modifications come from the very early establishment of the observed Structure–Activity Relationship (SAR) within the general class of bio-active Pt species.^1,4


	Fig. 1 Some current clinically used Pt-based drugs and the compounds (1–4) used in this study.

Our particular interests lie in the arena of oxazole (and 4,5-dihydro-1,3-oxazole: i.e, 2-oxazoline)^6,7 coordination chemistry and in ligand design strategies involving these heterocyclic frameworks. Oxazoles (ox) are found in a wide variety of both natural and synthetic products and have found a number of diverse applications (e.g., coordination chemistry, catalysis, etc.).⁶ Despite these advances, relatively few transition metal oxazol(in)e compounds have been investigated as anti-cancer agents and even fewer contain Pt as the metal centre.⁸ The work described herein details the synthesis of representatives of four classes of Pt-ox compounds: (a) ox analogues (1) of ZD0473 (i.e., cis-[PtCl₂(NH₃)(2-picoline)]: Fig. 1); (b) relatives of Oxaliplatin which contain an N,N′-chelating ox ligand (2); (c) bis-ox Pt-compounds devoid of an N–H bond (3) and finally (d) a Pt complex containing a tridentate ligand of our own design which incorporates both a pyridine and an ox fragment (4).⁹ These four compounds are shown schematically in Fig. 1. Hence, the work described herein addresses the latter of the three main areas of Pt chemotherapy design, namely the replacement of one or more ammine ligands (vide supra). These four model compounds will serve as indicators as to the efficacy of the four general molecular designs (Fig. 1).

Results and discussion

Syntheses and structural characterisation

Compound 1 is obtained by employing previously established protocols⁸ in which cisplatin is first converted in situ to Pt(NH₃)₂(NO₃)₂, followed by ligand metathesis with 2-ethyl-2-oxazoline (Etox) to give Pt(Etox)(NH₃)(NO₃)₂, and finally treatment with excess KCl to yield 1.^10,11 Compound 2 can be obtained by the addition of solid 4,4-dimethyl-2-(2′-anilinyl)-2-oxazoline^7b,9 to a stoichiometric aq. solution of K₂PtCl₄ containing a trace of c. H₂SO₄ (as ligand transfer agent). Subsequent stirring yields 2 in the form of a yellow-coloured powder.^12,13 The treatment of a methanolic suspension of the inorganic polymer [PtCl₂]_n with excess Etox and stirring overnight at room temperature (RT) yielded grey-coloured 3,^14,15 and 4 was prepared as reported previously.⁹

Compounds 1 and 2 have been further characterised in the solid-state by single crystal X-ray diffraction methods.^11,13,15 Both materials are found to be mononuclear Pt complexes as expected and exhibit the desired cisoidal oriented chlorido ligands. Molecular representations of a molecule of 1 or 2 that is found in the unit cell can be found below in Fig. 2 and 3, respectively.


	Fig. 2 ORTEP ¹⁶ representation of a molecule of complex 1 showing the atomic numbering scheme (thermal ellipsoids are shown at the 50% probability level).


	Fig. 3 ORTEP ¹⁶ representation of a molecule of complex 2 showing the atomic numbering scheme (thermal ellipsoids are shown at the 50% probability level).

These two materials contain a Pt atom that is in essentially a square planar coordination environment.^11,13 The Pt–Cl and Pt–N bond lengths are typical for a formally Pt(II) metal centre and are otherwise unsurprising.¹⁷

Biological screening

The results of the biological screening of complexes 1–4 can be found in Table 1; the results for cisplatin are also given.¹⁸ The latter four materials show slightly lower cytotoxicity values when compared with cisplatin. However, all are active within an order of magnitude. These results suggest that the four representatives of the potential classes of 2nd generation cisplatin analogues (i.e., Fig. 1) are all worthy of further investigations. Hence, the syntheses, screening and establishment of more detailed Structure–Activity Relationships for a larger series of each class represented by 1–4 is warranted.

Table 1 Cytotoxicity of 1–4 and cisplatinvs. the A2780 cancer cell line (72 h incubation) and r_b values with pUC19 plasmid DNA

Compounds	Inhibition IC₅₀, μM^a	Reference	r _b values
a Values are mean for data from at least three independent experiments with quadruplicate readings in each experiment. b cis-isomer; Meox = 2-methyl-2-oxazoline-κ¹N. c n.b ., 96 h exposure.
cisplatin	1.3 (± 0.1)	this work	0.076
1	4.1 (± 0.3)	this work	0.083
2	16.9 (± 0.5)	this work	0.41
3	9.9 (± 1.0)	this work	0.20
4	20.2 (± 1.7)	this work	0.81
[PtCl ₂ (NH ₃ )(Meox)] ^b ^, ^c	0.65 (± 0.1)	8	—
cisplatin ^c	0.2 ± (0.05)	8	—

DNA interaction studies

The cytotoxicity associated with cisplatin and its analogues is generally believed to arise from direct interactions with DNA. Intracellular formation of monoaquated platinum species leads to monofunctional adducts with DNA primarily through attachment to guanineN7 residues. Upon further coordination to DNA, such adducts cyclise to form guanine-guanine and adenine-guanine cross-links. This class of modification of the DNA tertiary structure by Pt compounds can be followed by an electrophoretic mobility shift assay, whereby the unwinding of covalently-closed, supercoiled plasmid DNA is monitored.²⁰ It has been shown previously that cisplatin unwinds supercoiled (Form I) DNA by 13° with a bound drug-to-nucleotide ratio (r_b) of 0.076, where r_b corresponds to the amount of platinum complex that is necessary for complete removal of all supercoils from DNA.²¹ Experimentally, r_b is measured as the coalescence point in a titration of plasmid DNA with a platinum complex, where the rate of migration of Form I decreases until it comigrates with relaxed, nicked DNA (Form II). Fig. 4 shows the DNA unwinding assay for 1 with pUC19 plasmid DNA. The coalescence point in a titration of 120 μM pUC19 occurs with 10 μM 1, corresponding to an r_b value of 0.083, which is only slightly larger than that measured for cisplatin. The degree of distortion in the DNA helix induced by 1 is especially evident at higher concentrations (Lane 7), where the gel bands disappear because the intercalating dye used for imaging can no longer penetrate the helix. In general, the magnitude of r_b for 1–4 parallels the cytotoxicity observed toward the A2780 cell line, with smaller values of r_b translating to smaller IC₅₀ values. As expected, platinum complexes that interact more strongly with DNA are the most cytotoxic. An exception to this trend occurs in the case of 3, underscoring the importance of additional factors in predicting anticancer activity. Together these results indicate that the Pt-oxazoline compounds interact with DNA much like cisplatin but may offer certain advantages, including reduced toxicity toward normal cells or higher selectivity toward cancer cells.


	Fig. 4 DNA unwinding assay for 1 with 800 ng pUC19 plasmid (electrophoresed for 50 min on a 1% agarose gel in 1X TAE at 80 V). Lane 1, DNA only; Lane 2, 5 μM 1; Lane 3, 10 μM 1; Lane 4, 50 μM 1; Lane 5, 100 μM 1; Lane 6, 200 μM 1; and Lane 7, 400 μM 1.

Acknowledgements

This work has been generously supported by funding from Research Corporation (RAG: Cottrell College Science Award). The authors are also indebted to the support of The Canadian Breast Cancer Foundation (RAG: Research Project Grant), NSERC Canada (Discovery Grants: JB, SAM and RAG; USRA: MJH, JAL and MWC), in addition to the support of Acadia and Ryerson Universities. RAG was the recipient of a 2006 J. W. T. Jones Travelling Fellowship, generously supplied by the Royal Society of Chemistry, which enabled this author to carry out some of this research during a sabbatical leave. The technical assistance of Annette Hooper (SAM) is also acknowledged.

References

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Synthesis of 1. An analogous procedure was used as previously reported⁸ with the substitution of Etoxin lieu of Meox (Yield: 36%). Mp 194 °C (decomp.). ¹H NMR (dmso-d₆): δ = 1.20 (t, 3H, J = 7.6 Hz, –C H₃), 2.83 (q, 2H, J = 7.6 Hz, –CC H₂), 3.78 (t, 2H, J = 9.6 Hz, –NC H₂), 4.14 (s [br], 3H, N H₃), 4.45 (t, 2H, J = 9.6 Hz, –OC H₂). Anal. Calc. (Found): C 15.71 (15.73); H 3.16 (3.08); N 7.33 (7.22)%. MS (EI, 70 eV): m/z = 382.0 (M⁺).
X-Ray diffraction data of 1. Formula: C₅H₁₂N₂OCl₂Pt, M_r: 382.16 g mol⁻¹, crystal size: 0.425 × 0.10 × 0.025 mm³, space group: C2/c, a = 18.924(2), b = 10.8634(7), c = 11.9925(10) Å;, β = 127.540(2)°, V = 1954.8(3) Å³, T = 173(1) K, Z = 8, ρ_c = 2.597 g mL⁻¹, Mo-Kα = 0.71073 Å, F(000): 1408, hkl range: –23/+22, –13/+14, –15/+14, refl. measured: 6568, refl. unique: 2194, R_int = 0.0591, parameters refined: 102, R(F)/w R(F²) (all refl.): 0.0484/0.1109, GoF (F²): 1.024, Δρ_fin (max./min.): 2.709/-2.530 eÅ⁻³. Selected bond lengths (Å) and angles (°): Pt–Cl1 2.305(1), Pt–Cl2 2.299(2), Pt–N1 1.995(4), Pt–N2 2.042(7), N1–C2 1.282(9); Cl1–Pt–Cl2 91.79(6), Cl1–Pt–N1 179.0(2), Cl1–Pt–N2 90.3(2), N1–Pt–N2 88.9(2) Cl1–N2–C7 121.1(6).
Synthesis of 2. A sample of K₂PtCl₄ (0.3760 g: 0.9058 mmol) was dissolved in 7 mL of water in the presence of 8 drops of c. H₂SO₄. To this mixture was added a sample of 2-(2′anilinyl)-4,4-dimethyl-2-oxazoline^{7b,9, 22} (0.1733 g: 0.9109 mmol) in the form of a solid. The mixture was then stirred rapidly at RT (overnight). The resulting precipitate was isolated by filtration and washed with water (2 × 2 mL), 95% aq. EtOH (1 mL) and Et₂O (2 × 2 mL). The yield of yellow-orange powder was 0.2111 g (52%). Mp >200 °C. ¹H NMR (acetone-d₆): δ = 1.47 (s, 6H, –C H₃), 4.42 (s, 2H, –OC H₂), 6.65 (m, 1H, Ar H), 6.82 (m, 1H, Ar H), 7.28 (m, 1H, Ar H), 7.63 (m, 1H, Ar H). Anal. Calc. (Found): C 28.96 (28.44); H 3.09 (2.97); N 6.14 (6.13)%. Crystals suitable for X-ray diffraction work were obtained by recrystallisation of 2 from aq. 95% EtOH at RT.
X-ray diffraction data of 2. Formula: C₁₁H₁₄N₂OCl₂Pt, M_r = 456.22 g mol⁻¹, space groupP2₁/n, a = 6.525(2), b = 11.816(3), c = 17.129(4) Å, β = 99.99(4)°, V = 1300.6(6) Å³, T = 193(2) K, Z = 4, ρ_c = 2.330 g mL⁻¹, Mo-Kα = 0.71073 Å, F(000): 856, hkl range: ±7, ±14, ±20, refl. unique = 2230, R_int = 0.0454, parameters refined = 160, R(F)/w R(F²) (all refl.) = 0.0454/0.1335, Δρ_fin (max./min.) = 0.428/0.313 eÅ⁻³. Selected bond lengths (Å) and angles (°): Pt–Cl1 2.294(3), Pt–Cl2 2.324(3), Pt–N1 2.043(7), Pt–N2 2.06(1), N1–C5 1.28(1); Cl1–Pt–Cl2 88.3(1), Cl1–Pt–N1 174.1(2), Cl1–Pt–N2 90.6(3), N1–Pt–N2 84.3(3) Pt1–N2–C7 110.0(7).
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Footnotes

† Oxazoles XXVI

‡ CCDC reference numbers 800341 and 800342. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c0md00211a

§ Current address: School of Chemistry, Institute of Science & Technology, Tribhuvan University, Kirtipur, Kathmandu, Nepal.

¶ Undergraduate research participant; these authors contributed equally to this research.

|| Author to whom correspondence should be addressed concerning the crystallographic characterisation of complex 1. E-mail: adecken@unb.ca

** Author to whom correspondence should be addressed concerning the crystallographic characterisation of complex 2. E-mail: michael.gardiner@chem.utas.au

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