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

Article abstract of DOI:10.1039/b411219a

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.

Full text of DOI:10.1039/b411219a

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P A P E R
Solid-phase synthesis of peptide-platinum complexes using
platinum-chelating building blocks derived from amino acids
Marc S. Robillard, Sophie van Alphen, Nico J. Meeuwenoord, Bart A. J. Jansen,
Gijs A. van der Marel, Jacques H. van Boomw and Jan Reedijk*
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands. E-mail: reedijk@chem.leidenuniv.nl; Fax: þ31-71-527-4671;
Tel: þ31-71-527-4459
Received (in Montpellier, France) 21st July 2004, Accepted 17th September 2004
First published as an Advance Article on the web 8th December 2004
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.
alities (e.g. arginine), on the anticancer properties of the
complexes was investigated.
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 signifi-
cantly 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 com-
plexes.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 re-
cently constructed a library of dichloroplatinum(II) tripeptide
complexes, 1, by an automated parallel solid-phase synthesis
protocol (Fig. 1).1c
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 inter-
actions 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 con-
struction 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 function-
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 comp-
ounds 4 (Scheme 1) comprises the reductive amination of
Boc–aminoaldehyde (6) with amino acids 5,5 and subsequent
protective group manipulation, thereby yielding target com-
pounds 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.
An alternative approach6 uses unprotected amino acids
(Scheme 1). Reductive amination of a suspension of phenyl-
alanine (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 ap-
pended 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 ¼ 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 success-
fully, as ascertained by determining the loading of 15d and 15f.
w Deceased July 31, 2004.
T h i s j o u r n a l i s & T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 5
220
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Fig. 1 Peptide-platinum complexes 1 and 2 and platinum-chelating building blocks 3 and 4.
Unmasking of the ethylenediamine moiety in 15, followed
by treatment with K2PtCl4 (B6 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,
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.
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).
N e w J . C h e m . , 2 0 0 5 , 2 9 , 2 2 0 – 2 2 5
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Table 1 IC50 values (mM) determined in A2780 and A2780cisR cell
Experimental
lines using the MTT assay8
Methods and materials
Complex
A2780
A2780cisR
1H NMR and 13C NMR spectra were recorded using a Bruker
DPX 300 spectrometer. Chemical shifts (d) 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 d ¼ ꢀ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 Econospheres C18 column (AllTech, 4.6 mm ꢁ 250
mm, 5 mm 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
18a
18b
24.1
131
123
203
c152
c195
c154
c170
105
18c
18d
136
186
18e
18f
31.8
0.37
Cisplatin
2.40
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 aCH of arginine, to significantly impose diaster-
eomeric characteristics.
column (26 mm ꢁ 60 cm). The flow speed was 1.5 ml minꢀ1
.
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 lꢀ1) for 5 h, then distilled and stored
over molecular sieves (4 A). MeOH (Rathburn, HPLC grade)
was stored over 3 A 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 techni-
cal 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.
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.
General procedure for solid-phase synthesis of peptides
Peptides were synthesized on an ABI 433A (Applied Biosys-
tems, division of Perkin Elmer) peptide synthesizer employing
a FastMocs peptide synthesis protocol. Generally, after clea-
vage 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-(20,40-di-
methoxyphenyl–Fmoc–aminomethyl) phenoxyacetamidonorleu-
cyl–MBHA; 0.5 mmol gꢀ1] was purchased from NovaBiochem
(Switzerland). The standard Fmoc–amino acids used in the
synthesis were Fmoc–Arg(Pbf)–OH and Fmoc–Gly–OH.
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 dis-
played 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 struc-
ture-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.
Na-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]alanine
tert-
butyl ester (7a). Aldehyde 6 (1 g, 6.28 mmol) and H–Ala–
OtBu ꢂ HCl (5a; 1.14 g, 6.28 mmol) were dissolved in 3% AcOH
in MeOH (15.7 ml) containing molecular sieves (3 A).
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 þ H]1.
222
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Na-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]phenylalanine
tert-butyl ester (7b). Aldehyde 6 (0.5 g, 3.14 mmol) and H–Phe–
OtBu ꢂ HCl (5b; 0.68 g, 2.62 mmol) were dissolved in 3% AcOH
in MeOH (6.5 ml) containing molecular sieves (3 A). 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 chromatogra-
phy (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): d ¼ 7.27
(m, 5H, Phe arom.), 4.97 (br s, 1H, NH), 3.35 (t, J ¼ 7.1 Hz,
1H, aPhe), 3.15–3.08 (m, 2H, BocNCH2CH2N), 2.87 (d, J ¼
7.1 Hz, 2H, bPhe), 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 þ H]1.
graphy (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 1C): d ¼ 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 þ CH2 Fmoc þ aAla), 3.33–2.90 (br m, 4H, NC2H4N),
1.37 (br m, 3H, bAla); 13C NMR (d4 MeOD, 55 1C): d ¼ 175.4
(C , CO H), 158.6, 157.7 (C , 2 ꢁ C O), 145.2 (C arom., 4 ꢁ
Q
q
2
q
q
Fmoc), 142.6 (Cq 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, aAla), 56.6 (CH, 2 ꢁ Fmoc), 47.1 (CH2, NC2H4N),
40.8 (CH2, NC2H4N), 15.6 (CH3, bAla); ESI-MS: m/z 575
[M ꢀ H]ꢀ.
Na-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-Na-(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
Na-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]-Na-(9-fluorenyl-
methoxycarbonyl)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), afford-
1
4b in 46% (from 8b) yield (0.16 g, 0.25 mmol). H NMR (d4
1
ing 8a in 39% (from 5a) yield (1.24 g, 2.43 mmol). H NMR
MeOD, 55 1C): d ¼ 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, aPhe), 4.14 (m,
2H, CH2 Fmoc), 3.20 (br m, 2H, bPhe), 3.12–2.46 (m, 4H,
NC2H4N); 13C NMR (d4 MeOD, 55 1C): d ¼ 175.8 (Cq,
(CDCl3): d ¼ 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 þ CH Fmoc þ aAla), 3.48–
2.89 (br m, 4H, NC2H4N), 1.41 (br s, 21H, tBu þ Boc þ bAla);
ESI-MS: m/z 511 [M þ H]1, 455 [M ꢀ tBu]1.
CO H), 158.3 (C , C O), 157.3 (C , C O), 145.2, 145.2,
Q
Q
2
q
q
Na-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]-Na-(9-fluorenyl-
methoxycarbonyl)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 1C. After stirring overnight the solvent was
evaporated and the residue purified by column chromatogra-
phy (hexane–EtOAc, 4 : 1 v/v). Product 8b was obtained in 31%
yield (0.32 g, 0.54 mmol). 1H NMR (CDCl3): d ¼ 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, aPhe), 4.66 (m,
1H, CH Fmoc), 4.45 (m, 1H, CH2 Fmoc), 4.27 (m, 1H, CH2
Fmoc), 3.28 (br d, J ¼ 6.1 Hz, 2H, bPhe), 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 þ H]1.
145.2, 145.1, 142.9, 142.6 (Cq arom., Fmoc), 139.6 (Cq arom.,
Phe), 129.9-123.7 (CH arom., 5 ꢁ Phe þ 12 ꢁ Fmoc), 121.0
(CH arom., 4 ꢁ Fmoc), 68.5 (CH2, Fmoc), 67.6 (CH2, Fmoc),
64.7 (CH, aPhe), 57.1 (CH, 2 ꢁ Fmoc), 40.2 (CH2, NC2H4N),
38.7 (CH2, NC2H4N), 35.9 (CH2, bPhe); ESI-MS: m/z 651
[M ꢀ H]ꢀ.
Na-[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, afford-
ing insoluble white solid 11b in 52% yield (200 mg, 0.65 mmol),
which was used without characterization.
Na-[N-(tert-Butyloxycarbonyl)-2-aminoethyl]-Na-(9-fluorenyl-
methoxycarbonyl)phenylalanine (12b). Bis(trimethylsilyl)aceta-
mide (281 ml, 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): d ¼ 7.71 (m,
2H, Fmoc), 7.52 (m, 2H, Fmoc), 7.38 (m, 2H, Fmoc), 7.36–7.07
(m, 7H, Fmoc þ Phe), 4.70 (m, 1H, aPhe), 4.44 (m, 2H, CH
Fmoc), 4.24 (m, 1H, CH2 Fmoc), 4.16 (m, 1H, CH2 Fmoc), 3.31
(m, 2H, bPhe), 3.06 (br m, 2H, BocNCH2CH2N), 2.83 (br m,
2H, BocNCH2CH2N), 1.37 (s, 9H, Boc).
Na-[2-Aminoethyl]-Na-(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.
Na-[2-Aminoethyl]-Na-(9-fluorenylmethoxycarbonyl)phenyl-
alanine (9b). Compound 8b (0.32 g, 0.54 mmol) was dissolved
in DCM (0.34 ml) at 0 1C 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.
Na-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-Na-(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 chromato-
Na-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-Na-(9-
fluorenylmethoxycarbonyl)phenylalanine (4b) from 12b. Com-
pound 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 ml, 1.35 mmol) and
N e w J . C h e m . , 2 0 0 5 , 2 9 , 2 2 0 – 2 2 5
223

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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).
(br m, 1H, NC2H4N) 2.60–2.35 (br m, 2H, NC2H4N); 195Pt
NMR (0.1 M DCl in D2O): d ¼ ꢀ2403; ESI-MS: m/z 587 [M þ
H]1; RP HPLC: Rt ¼ 10.37 and 10.73 min.
N-[2-Aminoethyl]phenylalanine–Gly–Arg–NH2 dichloroplati-
num 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 gꢀ1. A part (1/2) of
16f (0.122 g, 50 mmol) was reacted with 0.05 M K2PtCl4 in
DMF–H2O (9 : 1 v/v, 8 ml), affording 18f in 14% (from 13)
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 mmol). 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 mmol).
1H NMR (D2O): d ¼ 4.06 (m, 1H, CH2), 4.03 (AB, 2H, aGly),
3.95 (s, 2H, aGly), 3.77 (m, 1H, CH2), 3.05–2.86 (br m, 1H,
NC2H4N), 2.82–2.52 (br m, 3H, NC2H4N); 195Pt NMR (D2O):
d ¼ ꢀ2376; ESI-MS: m/z 497 [M þ H]1.
1
yield (5.1 mg, 7.1 mmol). H NMR (0.1 M DCl in D2O): d ¼
7.37–7.24 (m, 5H, Phe), 4.38–4.17 (m, 2H, aPhe þ aArg), 3.78
(AB, 2H, aGly), 3.38–3.10 (m, 5H, bPhe þ dArg þ NC2H4N),
2.97–2.78 (br m, 1H, NC2H4N), 2.73–2.45 (m, 2H, NC2H4N),
1.85 (m, 2H, bArg), 1.64 (m, 2H, gArg); 195Pt NMR (0.1 M
DCl in D2O): d ¼ ꢀ2368, ꢀ2383; ESI-MS: m/z 686 [M þ H]1;
RP HPLC: Rt ¼ 14.39 min.
Cytotoxicity studies
A2780 (human ovarian carcinoma) and A2780cisR (cisplatin-
resistant) cell lines were grown as monolayers in DMEM
(Gibco BRLt, Invitrogen Corporation, Netherlands) supple-
mented with 10% fetal calf serum (Gibco, Paisley, Scotland),
penicillin (100 units mlꢀ1; Duchefa, Netherlands) and strepto-
mycin (100 mg mlꢀ1; Duchefa, Netherlands) in a humidified 6%
CO2, 94% air atmosphere, 37 1C. 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 ml growth medium. The cells were
incubated for 24 h prior to drug testing to allow cell adhesion.
Stock solutions (1 mg mlꢀ1 complete millipore) of the com-
pounds 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 mlꢀ1. Each concentration
was tested in quadruplicate, using 100 ml per well added to the
100 ml of complete medium in the well. In the control group,
100 ml of complete medium was added. The plates were
incubated for 72 h, after which MTT (5 mg mlꢀ1, 50 ml) 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 ml). 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 Prisms software
(version 3.05, 2000).
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
mmol). 1H NMR (0.1 M DCl in D2O): d ¼ 4.28 (q, J ¼ 7.0 Hz,
aAla), 4.07 (m, aAla), 3.92 (AB, 2H, aGly), 3.89 (AB, 2H,
aGly), 3.04 (br m, 1H, NC2H4N), 2.68–2.50 (br m, 3H,
NC2H4N), 1.42 (d, J ¼ 7.1 Hz, bAla), 1.31 (d, J ¼ 7.0 Hz,
bAla); 195Pt NMR (0.1 M DCl in D2O): d ¼ ꢀ2387; ESI-MS:
m/z 511 [M þ H]1; RP HPLC: Rt ¼ 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 tripep-
tide 15d on the resin was 0.260 mmol gꢀ1. Yellow powder, 31%
1
(from 13) yield (20.2 mg, 31.2 mmol). H NMR (0.1 M DCl in
Acknowledgements
D2O): d ¼ 4.21 (m, 1H, aArg), 4.21, 3.99 (2 ꢁ m, 1H, aAla),
3.88 (m, 2H, aGly), 3.10 (m, 2H, dArg), 2.90 (br m, 1H,
NC2H4N), 2.72–2.51 (br m, 3H, NC2H4N), 1.78 (m, 2H, bArg),
1.71–1.42 (m, 5H, gArg þ bAla); 195Pt NMR (0.1 M DCl in
D2O): d ¼ ꢀ2371, ꢀ2386; ESI-MS: m/z 610 [M þ H]1; RP
HPLC: Rt ¼ 10.70 and 11.78 min.
This research was supported by the Council for Chemical
Sciences of The Netherlands Organization for Scientific Re-
search (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.
N-[2-Aminoethyl]phenylalanine–Gly–Gly–NH2 dichloroplati-
num (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 pow-
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