Solid-phase synthesis of peptide-platinum complexes using platinum-chelating building blocks derived from amino acids

Abstract

Amino acids have been employed as precursors in the synthesis of platinum-chelating solid-phase building blocks. These chelating molecules were subsequently successfully used in the solid-phase synthesis of peptide-platinum complexes. The newly introduced functionality in the chelating part, as well as the nature of the pendant peptide, was shown to have an important influence on the anticancer activity of the complexes.

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Introduction

The use of combinatorial chemistry complemented by high-throughput screening has developed into a well-established protocol in drug discovery. The development of an efficient synthetic procedure for platinum complexes would significantly facilitate the implementation of such a protocol in the search for novel anticancer platinum drugs. In this respect, we have demonstrated the compatibility of solid-phase synthetic strategies with platinum chemistry by the synthesis of a variety of cisplatin-like complexes and even dinuclear platinum complexes.¹ Intrigued by the finding that amino acid residues and peptides can be successfully employed as site-specific DNA-interacting elements conjugated to metal complexes,² we recently constructed a library of dichloroplatinum(II) tripeptide complexes, 1, by an automated parallel solid-phase synthesis protocol (Fig. 1).^1c

Fig. 1 Peptide-platinum complexes 1 and 2 and platinum-chelating building blocks 3 and 4.

Dichloroplatinum(II) tripeptide complexes 1 were designed on the basis of the assumption that the use of dipeptides B conjugated to a dichloroplatinum moiety A (from building block 3) would not only improve targeting to the DNA but also provide specific, possibly favorable additional interactions with the platinated DNA.^2–4 The modular nature of structure 1 allowed for the facile introduction of a high degree of diversity by varying the amino acids in the glycine-tethered dipeptide (B).

Inspection of the structure of complexes 1 indicates that introduction of additional functionalities in the platinum chelating building block (A) itself would give complexes 2, thereby further increasing the diversity of accessible platinum structures. An appealing strategy therefore involves the construction of analogs of the platinum-chelating ethylenediamine derivative 3 (Fig. 1) from amino acids as versatile key building blocks to give ligands 4. Incorporation of 4 to give 2 would lead to the introduction of chiral centers near to the platinum complex and potential DNA-interacting moieties close to the DNA platination site. In this paper we report the preparation of platinum-chelating building blocks 4 (see Fig. 1) and their application in the solid-phase assembly of tripeptide-platinum complexes 2. The effect of the functionalities close to the platinum core, as well as the appended functionalities (e.g. arginine), on the anticancer properties of the complexes was investigated.

Results and discussion

The solid-phase assembly of the projected platinum complexes 2 commences with the solution-phase synthesis of platinum-chelating building block 4, using amino acids as starting compounds. One approach to the synthesis of target compounds 4 (Scheme 1) comprises the reductive amination of Boc–aminoaldehyde (6) with amino acids 5,⁵ and subsequent protective group manipulation, thereby yielding target compounds 4. Reductive amination of Boc–aminoethanal 6 with tert-butyl protected alanine (5a) or phenylalanine (5b), using NaBH₃CN as reducing agent, gave tert-butyl esters 7a,b, respectively. The secondary nitrogen in 7a,b was protected with a Fmoc group, affording compounds 8a,b. Subsequent acidolysis of the Boc and tert-butyl groups, followed by Fmoc protection of the primary amine in 9a,b, resulted in bis-Fmoc protected building blocks 4a and 4b.

Scheme 1 Synthesis of alanine- and phenylalanine-derived building blocks 4a and 4b. Reagents and conditions: (a) 1.3 equiv. NaBH₃CN, 3% AcOH in MeOH; (b) 2 equiv. DiPEA, 1.1 equiv. Fmoc–OSu, DCM; (c) DCM–TFA (1∶1); (d) 3 equiv. DiPEA, 1.1 equiv. Fmoc–OSu, dioxane; (e) 1.1 equiv. NaBH₃CN, MeOH; (f) 1 equiv. DiPEA, 1.75 equiv. BTSA, DCM, 1.1 equiv. Fmoc–Osu.

An alternative approach⁶ uses unprotected amino acids (Scheme 1). Reductive amination of a suspension of phenylalanine (10b) in methanol afforded insoluble 11b. Dissolution of 11b in dichloromethane was mediated by in situ silylation of the carboxylic acid and the secondary amine by bis(trimethylsilyl)acetamide.⁷ Subsequently, the secondary amine was Fmoc-protected, giving 12b. Acidolysis of the Boc group, providing 9b, and Fmoc-protection of the resulting primary amine, furnished the phenylalanine-based building block 4b in an improved overall yield compared to the approach starting from 5.

To investigate the effect on anticancer activity of an appended potential DNA-binding guanidinium function, as well as methyl and benzyl moieties near to the platinum core, the obtained building blocks 4a and 4b and unfunctionalized 3 (R³ = H) were applied in the solid phase construction of peptide-platinum complexes 18a–f (Scheme 2). To this end, immobilized dipeptides glycine–glycine and glycine–arginine (14), prepared via a standard peptide synthesis protocol, were elongated with building blocks 3 and 4a,b to furnish the six immobilized tripeptides 15a–f (Scheme 2). The incorporation of the two new building blocks 4a and 4b proceeded successfully, as ascertained by determining the loading of 15d and 15f. Unmasking of the ethylenediamine moiety in 15, followed by treatment with K₂PtCl₄ (∼6 eq.) in DMF–H₂O (9∶1) for 48 h in the dark, provided immobilized dichloroplatinum complexes 17a–f. Treatment of 17a–f with TFA–H₂O (95∶5) resulted in removal of the arginine-protecting Pbf group and concomitant cleavage from the solid support, yielding crude 18a–f. Purification of these complexes with gel permeation chromatography (HW-40, 1% aq. AcOH) was troublesome, due to unwanted adsorption onto the column material. In response to this, the elution solvent was changed to 1% HCl in MeOH–H₂O, which countered this effect and furnished homogeneous complexes 18a–f in 14–58% yield. The identity of tripeptide-platinum complexes 18a–f was fully ascertained by ¹⁹⁵Pt NMR, ¹H NMR and mass spectroscopy. In this respect it is of interest that 18b–f occur as diastereomers, due to the chiral coordinating nitrogen. The diastereomers of complexes 18c–f could be observed separately with LCMS (18c–e), ¹H NMR (18c and 18d) and ¹⁹⁵Pt NMR (18d and 18f). However, ¹⁹⁵Pt and ¹H NMR failed to detect separate diastereomers of complex 18b and insufficient column retention precluded further investigation of 18b with LCMS. The marked difference between 18b and 18c–f can be explained by the lack of the additional chiral center in the platinum chelating part of 18b. Thus, the chiral coordinating nitrogen in 18b is too far removed from the next chiral center in the molecule, the αCH of arginine, to significantly impose diastereomeric characteristics.

Scheme 2 Solid-phase synthesis of peptide-platinum complexes 18a–f. Reagents and conditions: (a) piperidine; (b) Fmoc–AA–OH, BOP, HOBt, DiPEA, NMP; (c) BOP, HOBt, DiPEA, NMP; (d) K₂PtCl₄, DMF–H₂O (9∶1); e) TFA–H₂O (19∶1).

Having complexes 18a–f in hand, the cytotoxicity of each of them was evaluated in the human ovarian carcinoma A2780 (cisplatin sensitive) and A2780cisR (cisplatin resistant) cell lines. IC₅₀ is defined as the concentration of the drug that results in 50% growth inhibition. The cytotoxicity results are summarized in Table 1.

Table 1 IC₅₀ values (µM) determined in A2780 and A2780cisR cell lines using the MTT assay⁸

Complex	A2780	A2780cisR
18a	24.1	203
18b	131	≫152
18c	123	≫195
18d	136	≫154
18e	186	≫170
18f	31.8	105
Cisplatin	0.37	2.40

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These results reveal that the peptide-platinum complexes 18a–f have a reduced activity compared to cisplatin and are also cross-resistant with cisplatin. Unfunctionalized 18a displayed the highest activity, closely followed by phenyalanine-derived and arginine-tethered 18f. Complexes 18b–d have similar activities and 18e was shown to be the least potent. Even though the pendant dipeptide, as well as the incorporated amino acid in the chelate backbone, seem to have a tremendous effect on the anticancer activity of these platinum complexes, the determined sequence of activity of this small set of peptide-platinum complexes does not yet allow to draw a clear structure-activity relationship.

Concluding remarks

The results described in this paper present a straightforward method for the synthesis of platinum-chelating building blocks from amino acids. The synthetic strategy is likely to be amendable to the incorporation of a wide range of amino acid residues in various positions on the backbone of the platinum chelating unit. Such modifications in the platinum complex impart structural diversity, which is required to expand the collection of distinct types of platinum structures available through a solid-phase combinatorial or parallel synthesis approach. Chelating ligands 4a and 4b were successfully used in the solid-phase synthesis of a range of peptide-platinum complexes. The newly introduced functionality in the chelating part, as well as the nature of the pendant peptide, was shown to influence the anticancer activity of the complexes. Future studies will deal with larger groups of molecules, to allow the determination of a more quantitative structure-activity relationship.

Experimental

Methods and materials

¹H NMR and ¹³C NMR spectra were recorded using a Bruker DPX 300 spectrometer. Chemical shifts (δ) are given in ppm relative to TMS as an internal standard. ¹⁹⁵Pt spectra were taken on a Bruker DPX 300 spectrometer and were calibrated using K₂PtCl₄ as an external reference at δ = −1614 ppm. Electrospray mass spectra were recorded on a Finnigan MAT TSQ-70 equipped with a custom-made electrospray ionization (ESI) interface. LCMS analysis was performed on a Jasco HPLC system (detection simultaneously at 214 and 254 nm) coupled to a Perkin Elmer Sciex API 165 mass instrument. An analytical Econosphere^® C₁₈ column (AllTech, 4.6 mm × 250 mm, 5 µm particle size) was used. The applied eluents were (A) H₂O; (B) CH₃CN and (C) 0.5% aq. TFA. Gel permeation chromatography was performed using a Fractogel HW-40 column (26 mm × 60 cm). The flow speed was 1.5 ml min⁻¹. The applied eluents were 1% aq. AcOH or 0.01 M HCl in H₂O–MeOH (1∶1 v/v). Detection was performed at 214 nm.

All solvents and reagents were obtained from commercial sources and were used as received unless stated otherwise. DCM (Baker, p.a.) and DiPEA (Biosolve) were dried by refluxing over CaH₂ (5 g l⁻¹) for 5 h, then distilled and stored over molecular sieves (4 Å). MeOH (Rathburn, HPLC grade) was stored over 3 Å molecular sieves.

TLC analysis was conducted on DC Fertigfolien (Schleicher & Schuell, F 1500, LS 254). Compounds were visualized by UV light and by spraying with a ninhydrin solution followed by charring. Eluents for column chromatography were of technical grade and distilled before use. Column chromatography was performed on Baker silica gel 60 (0.063–0.200 mm). All reactions were performed at room temperature unless stated otherwise.

General procedure for solid-phase synthesis of peptides

Peptides were synthesized on an ABI 433A (Applied Biosystems, division of Perkin Elmer) peptide synthesizer employing a FastMoc^® peptide synthesis protocol. Generally, after cleavage of the Fmoc group from the resin with piperidine, a five-fold excess of amino acid was dissolved in NMP and activated by sequential addition of 1 equiv. BOP–HOBt (0.5 M BOP and 0.5 M HOBt in DMF–NMP 1∶1 v/v), and 2 equiv. DiPEA (1.25 M in NMP). The resulting solution was transferred to the reaction vessel, which was then shaken for 1 h. The coupling procedure of the first amino acid was performed twice to ensure a high initial loading. All solvents used in the automated peptide synthesis were of peptide synthesis grade and were purchased from Biosolve. Piperidine, DiPEA and TFA were from Biosolve. BOP and HOBt were from Neosystem Laboratoire (France). Rink Amide MBHA resin [4-(2′,4′-dimethoxyphenyl–Fmoc–aminomethyl) phenoxyacetamidonorleucyl–MBHA; 0.5 mmol g⁻¹] was purchased from NovaBiochem (Switzerland). The standard Fmoc–amino acids used in the synthesis were Fmoc–Arg(Pbf)–OH and Fmoc–Gly–OH.

N^α-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]alanine tert-butyl ester (7a). Aldehyde 6 (1 g, 6.28 mmol) and H–Ala–OtBu·HCl (5a; 1.14 g, 6.28 mmol) were dissolved in 3% AcOH in MeOH (15.7 ml) containing molecular sieves (3 Å). NaBH₃CN (0.53 g, 8.48 mmol) was slowly added; the reaction mixture was stirred overnight. EtOAc (125 ml) and 5% aq. NaHCO₃ (50 ml) were added and the aqueous layer was extracted with EtOAc (3 × 60 ml). The combined organic layers were washed with 5% aq. NaHCO₃ (50 ml), brine (50 ml) and dried over MgSO₄. No purification was performed. ESI-MS: m/z 289 [M + H]⁺.

N^α-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]phenylalanine tert-butyl ester (7b). Aldehyde 6 (0.5 g, 3.14 mmol) and H–Phe–OtBu·HCl (5b; 0.68 g, 2.62 mmol) were dissolved in 3% AcOH in MeOH (6.5 ml) containing molecular sieves (3 Å). To this NaBH₃CN (0.22 g, 3.53 mmol) was slowly added. After stirring for 2 h, EtOAc (50 ml) and 5% aq. NaHCO₃ (20 ml) were added. The aqueous layer was extracted with EtOAc (3 × 25 ml) and the combined organic layers were washed with 5% aq. NaHCO₃ (20 ml), brine (20 ml) and dried over MgSO₄. After evaporation, the residue was purified by column chromatography (3% TEA in hexane–EtOAc, 5∶1 → 1∶1 v/v), affording 7b in 66% yield (0.63 g, 1.73 mmol). ¹H NMR (CDCl₃): δ = 7.27 (m, 5H, Phe arom.), 4.97 (br s, 1H, NH), 3.35 (t, J = 7.1 Hz, 1H, αPhe), 3.15–3.08 (m, 2H, BocNCH₂CH₂N), 2.87 (d, J = 7.1 Hz, 2H, βPhe), 2.74 (m, 1H, BocNCH₂CH₂N), 2.56 (m, 1H, BocNCH₂CH₂N), 1.45 (s, 9H, Boc), 1.36 (s, 9H, tBu); ESI-MS: m/z 365 [M + H]⁺.

N^α-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]-N^α-(9-fluorenylmethoxycarbonyl)alanine tert-butyl ester (8a). Crude 7a was dissolved in DCM (30 ml), then DiPEA (2.19 ml, 12.6 mmol) and Fmoc–OSu (2.44 g, 7.22 mmol) were added. After stirring overnight the solvent was evaporated and the residue purified by column chromatography (hexane–EtOAc, 3∶2 v/v), affording 8a in 39% (from 5a) yield (1.24 g, 2.43 mmol). ¹H NMR (CDCl₃): δ = 7.75 (m, 2H, Fmoc arom.), 7.56 (m, 2H, Fmoc arom.), 7.38–7.30 (m, 4H, Fmoc arom.), 4.92 (br s, 1H, NH), 4.54–4.07 (br m, 4H, CH₂ Fmoc + CH Fmoc + αAla), 3.48–2.89 (br m, 4H, NC₂H₄N), 1.41 (br s, 21H, tBu + Boc + βAla); ESI-MS: m/z 511 [M + H]⁺, 455 [M − tBu]⁺.

N^α-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]-N^α-(9-fluorenylmethoxycarbonyl)phenylalanine tert-butyl ester (8b). Compound 7b (0.63 g, 1.73 mmol) was dissolved in DCM (9 ml), then DiPEA (0.59 ml, 3.48 mmol) and Fmoc–OSu (0.67 g, 2.0 mmol) were added at 0 °C. After stirring overnight the solvent was evaporated and the residue purified by column chromatography (hexane–EtOAc, 4∶1 v/v). Product 8b was obtained in 31% yield (0.32 g, 0.54 mmol). ¹H NMR (CDCl₃): δ = 7.78 (m, 2H, Fmoc arom.), 7.69–7.55 (m, 2H, Fmoc arom.), 7.43 (m, 2H, Fmoc arom.), 7.34 (m, 2H, Fmoc arom.), 7.27–7.10 (m, 5H, Phe arom.), 5.12 (br s, 1H, NH), 4.98 (br m, 1H, αPhe), 4.66 (m, 1H, CH Fmoc), 4.45 (m, 1H, CH₂ Fmoc), 4.27 (m, 1H, CH₂ Fmoc), 3.28 (br d, J = 6.1 Hz, 2H, βPhe), 3.08 (m, 1H, NC₂H₄N), 2.87 (m, 1H, NC₂H₄N), 2.66 (m, 1H, NC₂H₄N), 2.46 (m, 1H, NC₂H₄N), 1.50 (s, 9H, Boc), 1.42 (s, 9H, tBu); ESI-MS: m/z 588 [M + H]⁺.

N^α-[2-Aminoethyl]-N^α-(9-fluorenylmethoxycarbonyl)alanine (9a). Compound 8a (1.24 g, 2.43 mmol) was dissolved in DCM (1.6 ml) and TFA (9 ml) was added. After stirring for 2 h, the solvent was co-evaporated with toluene, resulting in a brown oil of 9a.

N^α-[2-Aminoethyl]-N^α-(9-fluorenylmethoxycarbonyl)phenylalanine (9b). Compound 8b (0.32 g, 0.54 mmol) was dissolved in DCM (0.34 ml) at 0 °C and TFA (2 ml) was added. After stirring for 1 h, the solvent was co-evaporated with toluene, resulting in a brown oil of 9b.

N^α-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-N^α-(9-fluorenylmethoxycarbonyl)alanine (4a). The crude oil 9a was dissolved in dioxane (30 ml), then DiPEA (1.27 ml, 7.29 mmol) and Fmoc–OSu (0.90 g, 2.67 mmol) were added and the reaction mixture was stirred overnight. EtOAc (30 ml) was added and the solution was washed with 1 M HCl (20 ml), H₂O (20 ml) and brine (20 ml), and dried over MgSO₄. After evaporation, the product was purified by column chromatography (1% AcOH in EtOAc–hexane, 1∶1 v/v). The product-containing fraction was washed with 5% aq. Na₂CO₃, H₂O, brine and dried over MgSO₄. Product 4a was obtained as a white powder in 58% (from 8a) yield (0.815 g, 1.41 mmol). ¹H NMR (d⁴ MeOD, 55 °C): δ = 7.78 (m, 4H, Fmoc), 7.60 (m, 4H, Fmoc), 7.39–7.29 (m, 8H, Fmoc), 4.48–4.17 (m, 7H, CH Fmoc + CH₂ Fmoc + αAla), 3.33–2.90 (br m, 4H, NC₂H₄N), 1.37 (br m, 3H, βAla); ¹³C NMR (d⁴ MeOD, 55 °C): δ = 175.4 (C_q, CO₂H), 158.6, 157.7 (C_q, 2 × C=O), 145.2 (C_q arom., 4 × Fmoc), 142.6 (C_q arom., 4 × Fmoc), 128.8, 128.1, 126.0, 120.9 (CH arom., 16 × Fmoc), 68.6 (CH₂, Fmoc), 67.5 (CH₂, Fmoc), 57.6 (CH, αAla), 56.6 (CH, 2 × Fmoc), 47.1 (CH₂, NC₂H₄N), 40.8 (CH₂, NC₂H₄N), 15.6 (CH₃, βAla); ESI-MS: m/z 575 [M − H]⁻.

N^α-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-N^α-(9-fluorenylmethoxycarbonyl)phenylalanine (4b) from 8b. To crude oil 9b in dioxane (7 ml) was added a solution of DiPEA (284 ml, 1.6 mmol) and Fmoc–OSu (202 mg, 0.60 mmol) in dioxane (2 ml). After stirring overnight, EtOAc (7 ml) was added and the solution was washed with 1 M HCl (7 ml), H₂O (7 ml) and brine (7 ml). After evaporation, the product was purified by column chromatography (EtOAc–hexane, 1∶1 v/v), furnishing 4b in 46% (from 8b) yield (0.16 g, 0.25 mmol). ¹H NMR (d⁴ MeOD, 55 °C): δ = 7.72 (m, 4H, Fmoc arom.), 7.56–7.46 (br m, 4H, Fmoc arom.), 7.33 (m, 4H, Fmoc arom.), 7.24 (m, 4H, Fmoc arom.), 7.21–7.01 (m, 5H, Phe), 4.42 (br m, 2H, CH Fmoc), 4.28 (m, 2H, CH₂ Fmoc), 4.21 (m, 1H, αPhe), 4.14 (m, 2H, CH₂ Fmoc), 3.20 (br m, 2H, βPhe), 3.12–2.46 (m, 4H, NC₂H₄N); ¹³C NMR (d⁴ MeOD, 55 °C): δ = 175.8 (C_q, CO₂H), 158.3 (C_q, C=O), 157.3 (C_q, C=O), 145.2, 145.2, 145.2, 145.1, 142.9, 142.6 (C_q arom., Fmoc), 139.6 (C_q arom., Phe), 129.9-123.7 (CH arom., 5 × Phe + 12 × Fmoc), 121.0 (CH arom., 4 × Fmoc), 68.5 (CH₂, Fmoc), 67.6 (CH₂, Fmoc), 64.7 (CH, αPhe), 57.1 (CH, 2 × Fmoc), 40.2 (CH₂, NC₂H₄N), 38.7 (CH₂, NC₂H₄N), 35.9 (CH₂, βPhe); ESI-MS: m/z 651 [M − H]⁻.

N^α-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]phenylalanine (11b). Phenylalanine 10b (205 mg, 1.24 mmol) was suspended in MeOH (2 ml) and 6 (297 mg, 1.87 mmol) was added, followed by NaBH₃CN (86 mg, 1.37 mmol). The mixture was stirred overnight, filtered and washed with MeOH, affording insoluble white solid 11b in 52% yield (200 mg, 0.65 mmol), which was used without characterization.

N^α-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]-N^α-(9-fluorenylmethoxycarbonyl)phenylalanine (12b). Bis(trimethylsilyl)acetamide (281 µl, 1.14 mmol) and DiPEA (0.11 ml, 0.65 mmol) were added to 11b (0.20 g, 0.65 mmol) suspended in DCM (1.3 ml). The mixture was stirred until it was clear, after which Fmoc–OSu (0.23 g, 0.68 mmol) was added. After stirring overnight, the solution was co-evaporated with toluene and the residue purified by column chromatography [hexane–EtOAc (2∶1 v/v) → AcOH–EtOAc (1∶99 v/v)], producing 12b in 74% yield (0.25 g, 0.48 mmol) as a colorless oil. ¹H NMR (CDCl₃): δ = 7.71 (m, 2H, Fmoc), 7.52 (m, 2H, Fmoc), 7.38 (m, 2H, Fmoc), 7.36–7.07 (m, 7H, Fmoc + Phe), 4.70 (m, 1H, αPhe), 4.44 (m, 2H, CH Fmoc), 4.24 (m, 1H, CH₂ Fmoc), 4.16 (m, 1H, CH₂ Fmoc), 3.31 (m, 2H, βPhe), 3.06 (br m, 2H, BocNCH₂CH₂N), 2.83 (br m, 2H, BocNCH₂CH₂N), 1.37 (s, 9H, Boc).

N^α-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-N^α-(9-fluorenylmethoxycarbonyl)phenylalanine (4b) from 12b. Compound 12b (0.24 g, 0.45 mmol) was dissolved in TFA–DCM (1∶1 v/v, 6 ml) and stirred for 2 h. The solvent was co-evaporated with toluene and the resulting colorless oil 9b was dissolved in dioxane (5.7 ml). DiPEA (237 µl, 1.35 mmol) and Fmoc–OSu (0.17 g, 0.50 mmol) were added and the reaction mixture was stirred overnight. EtOAc (10 ml) was added to the mixture, which was washed with 1 M HCl (10 ml), H₂O (10 ml) and brine, dried over MgSO₄, filtered and evaporated. The resulting oil was purified by column chromatography [hexane–EtOAc (1∶1 v/v) → AcOH–EtOAc (1∶99 v/v)], furnishing 4b as a white powder in 73% yield (0.216 g, 0.33 mmol).

N-[2-Aminoethyl]glycine–Gly–Gly–NH₂ dichloroplatinum (18a). The peptide synthesis of bis-Fmoc protected tripeptide 15a was carried out as described above starting from resin 13 (200 mg, 100 µmol). Upon cleavage of the Fmoc group in 13 the following building blocks were sequentially coupled: (1) Fmoc–Gly–OH (5 equiv.), (2) Fmoc–Gly–OH (5 equiv.), (3) 3 (5 equiv.). After the assembly was complete, the Fmoc groups in 15a were removed and 16a was reacted with 0.05 M K₂PtCl₄ in DMF–H₂O (9∶1 v/v, 10 ml) for 48 h. The resin was filtered and washed with H₂O, DMF, DCM and treated with TFA–H₂O (95∶5 v/v, 2.5 ml) for 1.5 h. After filtration, the resin was washed with TFA and the combined TFA fractions were precipitated with Et₂O. The solid was washed with Et₂O, taken up in H₂O and lyophilized. Gel permeation chromatography (HW 40, 0.01 M HCl in MeOH–H₂O, 1∶1 v/v) afforded 18a as a yellow powder in 58% (from 13) yield (28.6 mg, 57.5 µmol). ¹H NMR (D₂O): δ = 4.06 (m, 1H, CH₂), 4.03 (AB, 2H, αGly), 3.95 (s, 2H, αGly), 3.77 (m, 1H, CH₂), 3.05–2.86 (br m, 1H, NC₂H₄N), 2.82–2.52 (br m, 3H, NC₂H₄N); ¹⁹⁵Pt NMR (D₂O): δ = −2376; ESI-MS: m/z 497 [M + H]⁺.

N-[2-Aminoethyl]glycine–Gly–Arg–NH₂ dichloroplatinum AcOH (18b; mixture of diasteriomers). 18b was prepared as described previously.^1a

N-[2-Aminoethyl]alanine–Gly–Gly–NH₂ dichloroplatinum (18c; mixture of diasteriomers). As described for 18a with: (1) Fmoc–Gly–OH (4 equiv.), (2) Fmoc–Gly–OH (4 equiv.), (3) 4a (4 equiv.), 0.05 M K₂PtCl₄ in DMF–H₂O (9∶1 v/v, 12.3 ml) for 24 h. Yellow powder, 28% (from 13) yield (14.2 mg, 27.8 μmol). ¹H NMR (0.1 M DCl in D₂O): δ = 4.28 (q, J = 7.0 Hz, αAla), 4.07 (m, αAla), 3.92 (AB, 2H, αGly), 3.89 (AB, 2H, αGly), 3.04 (br m, 1H, NC₂H₄N), 2.68–2.50 (br m, 3H, NC₂H₄N), 1.42 (d, J = 7.1 Hz, βAla), 1.31 (d, J = 7.0 Hz, βAla); ¹⁹⁵Pt NMR (0.1 M DCl in D₂O): δ = −2387; ESI-MS: m/z 511 [M + H]⁺; RP HPLC: R_t = 2.25 and 2.55 min.

N-[2-Aminoethyl]alanine–Gly–Arg–NH₂ dichloroplatinum HCl (18d; mixture of diasteriomers). As described for 18a with (1) Fmoc–Arg(Pbf)–OH (4 equiv.), (2) Fmoc–Gly–OH (4 equiv.), (3) 4a (4 equiv.), (4) 0.05 M K₂PtCl₄ in DMF–H₂O (9∶1 v/v, 14.5 ml). The loading of bis-Fmoc protected tripeptide 15d on the resin was 0.260 mmol g⁻¹. Yellow powder, 31% (from 13) yield (20.2 mg, 31.2 µmol). ¹H NMR (0.1 M DCl in D₂O): δ = 4.21 (m, 1H, αArg), 4.21, 3.99 (2 × m, 1H, αAla), 3.88 (m, 2H, αGly), 3.10 (m, 2H, δArg), 2.90 (br m, 1H, NC₂H₄N), 2.72–2.51 (br m, 3H, NC₂H₄N), 1.78 (m, 2H, βArg), 1.71–1.42 (m, 5H, γArg + βAla); ¹⁹⁵Pt NMR (0.1 M DCl in D₂O): δ = −2371, −2386; ESI-MS: m/z 610 [M + H]⁺; RP HPLC: R_t = 10.70 and 11.78 min.

N-[2-Aminoethyl]phenylalanine–Gly–Gly–NH₂ dichloroplatinum (18e; mixture of diasteriomers). As described for 18a with (1) Fmoc–Gly–OH (4 equiv.), (2) Fmoc–Gly–OH (4 equiv.), (3) 4b (4 equiv.), 0.05 M K₂PtCl₄ in DMF–H₂O (9∶1 v/v, 12.1 ml). Crude 18e was lyophilized from 3% aq. AcOH. Yellow powder, 24% (from 13) yield (14.3 mg, 24.3 µmol). ¹H NMR (0.1 M DCl in D₂O): δ = 7.24–7.09 (m, 5H, Phe), 4.15 (m, αPhe), 3.83 (m, 1H, αGly), 3.79 (s, 2H, αGly), 3.44 (AB, 1H, αGly), 3.29 (br m, 1H, NC₂H₄N), 3.03 (d, J = 8.1 Hz, 2H, βPhe), 2.68 (br m, 1H, NC₂H₄N) 2.60–2.35 (br m, 2H, NC₂H₄N); ¹⁹⁵Pt NMR (0.1 M DCl in D₂O): δ = −2403; ESI-MS: m/z 587 [M + H]⁺; RP HPLC: R_t = 10.37 and 10.73 min.

N-[2-Aminoethyl]phenylalanine–Gly–Arg–NH₂ dichloroplatinum HCl (18f; mixture of diasteriomers). As described for 18a with: (1) Fmoc–Arg(Pbf)–OH (4 equiv.), (2) Fmoc–Gly–OH (4 equiv.), (3) 4b (4 equiv.). The loading of bis-Fmoc protected tripeptide 15f on the resin was 0.244 mmol g⁻¹. A part (1/2) of 16f (0.122 g, 50 µmol) was reacted with 0.05 M K₂PtCl₄ in DMF–H₂O (9∶1 v/v, 8 ml), affording 18f in 14% (from 13) yield (5.1 mg, 7.1 µmol). ¹H NMR (0.1 M DCl in D₂O): δ = 7.37–7.24 (m, 5H, Phe), 4.38–4.17 (m, 2H, αPhe + αArg), 3.78 (AB, 2H, αGly), 3.38–3.10 (m, 5H, βPhe + δArg + NC₂H₄N), 2.97–2.78 (br m, 1H, NC₂H₄N), 2.73–2.45 (m, 2H, NC₂H₄N), 1.85 (m, 2H, βArg), 1.64 (m, 2H, γArg); ¹⁹⁵Pt NMR (0.1 M DCl in D₂O): δ = −2368, −2383; ESI-MS: m/z 686 [M + H]⁺; RP HPLC: R_t = 14.39 min.

Cytotoxicity studies

A2780 (human ovarian carcinoma) and A2780cisR (cisplatin-resistant) cell lines were grown as monolayers in DMEM (Gibco BRL™, Invitrogen Corporation, Netherlands) supplemented with 10% fetal calf serum (Gibco, Paisley, Scotland), penicillin (100 units ml⁻¹; Duchefa, Netherlands) and streptomycin (100 µg ml⁻¹; Duchefa, Netherlands) in a humidified 6% CO₂, 94% air atmosphere, 37 °C. Cells were passed after trypsinization when the plates were 80–90% full.

Cell growth inhibition by the platinum compounds was determined using the MTT assay.⁸ After trypsinization, cells were divided into 96-wells plates at concentrations of 3–5 × 10³ cells per well in 100 µl growth medium. The cells were incubated for 24 h prior to drug testing to allow cell adhesion. Stock solutions (1 mg ml⁻¹ complete millipore) of the compounds were freshly prepared. The dilutions (5 step dilutions) were prepared in complete medium. The concentrations used were 0.2, 0.1, 0.02, 0.01, 0.002 mg ml⁻¹. Each concentration was tested in quadruplicate, using 100 µl per well added to the 100 µl of complete medium in the well. In the control group, 100 µl of complete medium was added. The plates were incubated for 72 h, after which MTT (5 mg ml⁻¹, 50 µl) was added to each well, followed by incubation for 2 h. The medium was discarded and the blue formazan crystals were dissolved in DMSO (100 µl). Optical density of the wells was measured at 590 nm using a Biorad 550 microplate reader. The IC₅₀ values (drug concentration that results in 50% reduction of cell growth with respect to the untreated control) were determined graphically using GraphPad Prism^® software (version 3.05, 2000).

Acknowledgements

This research was supported by the Council for Chemical Sciences of The Netherlands Organization for Scientific Research (CW–NWO) and by The Netherlands Foundation for Technical Sciences (STW). Support and sponsorship by COST Action D8/00097 (Biocoordination Chemistry) and D20/0003 (Metal Compounds in the Treatment of Cancer) is kindly acknowledged. The authors wish to thank Johnson & Matthey (Reading, UK) for their generous loan of K₂PtCl₄.

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Footnote

† Deceased July 31, 2004.

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