Stepwise assembly of platinum-folic acid conjugates

Article abstract of DOI:10.1016/j.ica.2007.09.020

Folates and folate-like molecules represent a class of vectors for drug targeting and delivery. The folate receptor (FR) is overexpressed in many human cancers, therefore folate-cytotoxic drug conjugates can deliver therapeutic agents specifically to FR positive tumours. We have linked, directly through one of the carboxylate functionalities of the folic acid (α or γ), two different chelating arms for a cytotoxic platinum moiety, namely a diamine and a dicarboxylate. Five different derivatives were synthesised by multi-step procedures, purified and characterised; unfortunately, they resulted barely soluble in water, thereby precluding any biochemical and biological tests.

Full text of DOI:10.1016/j.ica.2007.09.020

Available online at www.sciencedirect.com
Inorganica Chimica Acta 361 (2008) 1447–1455
www.elsevier.com/locate/ica
Stepwise assembly of platinum–folic acid conjugates
a
a
a
a
Elisabetta Gabano , Mauro Ravera , Claudio Cassino , Samuele Bonetti ,
b
a,
*
Giovanni Palmisano , Domenico Osella
a
`
Dipartimento di Scienze dell’Ambiente e della Vita, Universita del Piemonte Orientale ‘‘A. Avogadro’’, Via Bellini 25/G, I-15100 Alessandria, Italy
b
`
Dipartimento di Scienze Chimiche e Ambientali, Universita dell’Insubria, Via Valleggio 11, 22100 Como, Italy
Received 5 September 2007; accepted 17 September 2007
Available online 21 September 2007
Abstract
Folates and folate-like molecules represent a class of vectors for drug targeting and delivery. The folate receptor (FR) is overexpressed
in many human cancers, therefore folate-cytotoxic drug conjugates can deliver therapeutic agents specifically to FR positive tumours. We
have linked, directly through one of the carboxylate functionalities of the folic acid (a or c), two different chelating arms for a cytotoxic
platinum moiety, namely a diamine and a dicarboxylate. Five different derivatives were synthesised by multi-step procedures, purified
and characterised; unfortunately, they resulted barely soluble in water, thereby precluding any biochemical and biological tests.
ꢀ 2007 Elsevier B.V. All rights reserved.
Keywords: Folic acid; Platinum complexes; Drug targeting; Kaminski’s reaction; Coupling agents
1. Introduction
In recent years, the ‘‘drug targeting and delivery’’
approach has been developed with the aim to reduce che-
motherapy-related systemic side-effects by using vectors
that selectively deliver the cytotoxic agent to tumour cells,
thus sparing healthy cells. Such vectors include bioactive
substances, such as specific amino acids and sugars (that
enter to a larger extent tumour cells by virtue of their very
active metabolism) or folates and estrogen analogues (that
are selectively vehiculated by the corresponding receptors
often overexpressed in cancer cells). Alternatively, non-
toxic, non-immunogenic and non-pyrogenic macromolecu-
lar vectors could be used for this purpose. This strategy
exploits the so-called EPR (enhanced permeability and
retention) effect, based on the peculiar features of tumour
blood and lymphatic vessels, resulting in increased macro-
molecular diffusion out of the bloodstream into tumour tis-
sue and inefficient drug drainage from the tumour.
While cytotoxic agents have dominated the development
of clinical cancer chemotherapy in the last half-century,
growing interest is arising nowadays on alternative cyto-
static drugs able to alter the cellular signals that determine
cancer progression and diffusion, with the goal to core (if
possible) or stabilize and treat chronically the disease.
The cytotoxic approach to fight cancer suffers from severe
side effects, due to the poor specificity. In particular, the
hydrolysis of platinum-based drugs generates cationic com-
plexes having electrophylic properties able to target geno-
mic DNA (genotoxic action). The specificity of these
drugs is based only on the higher proliferation index of
tumour with respect to healthy cells: during mitosis,
DNA is totally exposed to the electrophylic attack of the
Pt-drugs in the S-phase of cycle. However, several healthy
tissues have physiologically large population doubling
index, such as bone mallow and gastro-intestinal tract,
and their platination causes heavy side effects [1].
The biological (active targeting) or macromolecular
(passive targeting) vector for the cytotoxic Pt-moiety can
act either as a carrier ligand or leaving group. In both
cases, a spacer must be synthesized, binding the vector
and ending in a coordinating arm able to link the PtX2-
unit. When the vector acts as a leaving group in the final
*
Corresponding author. Tel.: +39 0131 360266; fax: +39 0131 360250.
E-mail address: domenico.osella@mfn.unipmn.it (D. Osella).
0020-1693/$ - see front matter ꢀ 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2007.09.020

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E. Gabano et al. / Inorganica Chimica Acta 361 (2008) 1447–1455
complex, the coordinating functionalities are generally
dicarboxylate groups. In this way, a [Pt(NH3)2]2+ moiety
can be delivered to DNA. The exact rate of dissociation
of platinum from the pharmacophore is crucial to the over-
all cytotoxic properties of the conjugates. Differences in pH
between healthy and tumour tissues, enzymatic activities
and exchange with the carbonate/bicarbonate system con-
cur to define the hydrolysis rate. When the vector acts as
a carrier group in the final complex, it will remain coordi-
nated to the drug upon cell penetration and DNA binding,
significantly modifying the chemical-physical properties of
the resulting DNA adducts (especially the steric bulk) [2].
In this context, folates and folate-like molecules repre-
sent a class of vectors for drug targeting and delivery.
Folate is a generic term describing various water-soluble
vitamins of the B group containing the pteroic acid nucleus
in various chemical forms, including folic acid (1). Folates
are important cofactors in C1 transfers and are involved in
two key metabolic pathways in mammalian and plant cells:
DNA biosynthesis, in particular in purine and pyrimidine
synthesis, and in the methylation cycle [3]. There are two
well-known active transport mechanisms for folates: the
reduced folate carrier (RFC) and the folate receptor
(FR), respectively. The RFC has a higher affinity for L-
methylfolate (reduced folate), whereas the folate receptor
has a higher affinity for folic acid. The FR is overexpressed
in many human cancers: for instance, in about 90% of
ovarian carcinomas; moreover brain, endometrial, kidney
cancers and mesothelioma are estimated to be FR-positive
[4]. For these reasons folate-targeted cancer therapies can
deliver therapeutic drugs specifically to FR-positive
tumour cells with nanomolar affinity (Fig. 1). After the
binding, the cell membrane invaginates the resulting
adduct, forming an endosome. As the endosomal compart-
ment acidifies, the drug may be released from the adduct
into the cytosol [5].
to PEG3000 followed by its chelation with Pt(II) via a
modified dicarboxylate ligand [8]. The folate-PEG-Pt
compound showed a significantly higher uptake com-
pared to non-targeted PEG-Pt when incubated with
FR-positive M109 cells. Unfortunately, this increased
Pt-content in the cells did not cause a parallel increment
in Pt-DNA adducts. Moreover, cytotoxicity data further
showed that the folate-PEG-Pt conjugate was 1.7-fold
less effective than PEG-Pt. In fact, the folate-PEG-Pt
conjugates were not freely released into the cytosol but
were directed to acidic cytoplasmic endosomes and lyso-
somes. The release from these vesicles to the cytoplasm
could be slow and inefficient. Since studies of folate-tar-
geted pH-sensitive liposomes point to a major increase in
cytosol delivery and biological activity, folate receptor
guessed mediated endocytosis (FRME) may lead to an
acidic vesicular compartment with reduced access to
other cell compartments unless the delivery system is
unstable at low pH. Therefore, the authors suggested
that folate-PEG-Pt conjugates might remain trapped in
acidic vesicles following FRME, thus preventing their
cytotoxic activity.
In this study, we link, directly through one of the car-
boxylate functionalities of the folic acid (a or c), two differ-
ent chelating arms able to coordinate a cytotoxic platinum
moiety, namely a diamine and a dicarboxylate. The first is
an example of a carrier conjugate (cisplatin-like conjugate),
while the second represents the model of a leaving conju-
gate (carboplatin-like conjugate) (Fig. 2).
HO
O
O
α
OH
γ
OH
N
H
O
N
N
Many interesting applications of the folate-drug conju-
gates have been reported, in particular containing toxins,
low molecular weight radio- and chemo-therapeutics,
immuno-therapeutic agents, liposomes with entrapped
drugs, etc. [5,6].
Surprisingly, to the best of our knowledge, only two
reports dealt with the application of a platinum-folate
conjugate. In the first example a positive charged plati-
num-tetrahydrofolate complex was synthesised. This
complex was a reasonably good inhibitor of L1210 dihy-
drofolate reductase and of the folate transport system
(50% inhibition at ca. 200 lM) of L1210 cells [7]. A sec-
ond reported conjugate was prepared by coupling folate
N
H
N
H2N
N
O-
O-
1
NH2
H2N
O-
NH2
(1)
(1)
O-
dicarboxylic conjugates
NH2
diamine conjugates
+ "PtCl2"
+
"[Pt(NH2R)]2+
"
O
NH2
NH2R
NH2R
Cl
Pt
(1)
Pt
Drug
Folate
Linker
CB
(1)
N
O
Cl
H2
R = H or CH3
Fig. 1. Structural design of a folate-drug conjugate containing: the carrier
element (Folate), the Linker, possibly with a cleavable bond (CB), and the
therapeutic agent (Drug).
Fig. 2. Sketch of folic acid (1) and a general scheme of the experimental
work.

E. Gabano et al. / Inorganica Chimica Acta 361 (2008) 1447–1455
1449
2. Experimental
Organic phases were joined, anhydrified with anhydrous
Na2SO4 and evaporated to get a colourless oil. Washing
with hexane resulted in a white soft powder. Yield:
850 mg, 59%. 1H NMR (acetone-d6) 6.10–6.20 (m, 2H,
2 · NH), 4.26 (m, 1H, CH, en), 3.40–3.50 (m, 2H, CH2,
en), 1.40 (s, 18H, CH3, Boc) ppm; 13C NMR (acetone-d6)
171.89 (COOH), 156.49 and 156.43 (2 · C(O), Boc), 78.52
and 78.38 (2 · Cquat, Boc), 54.54 (CH, en), 41.91 (CH2,
en), 27.75 (CH3, Boc) ppm.
2.1. General procedures
K2[PtCl4] (Johnson Matthey and Co.) and all other
chemicals (Aldrich) were used without further purification.
All the ligands and the low molecular weight Pt(II) com-
plexes were characterised by multinuclear NMR spectros-
copy [9]. The folic acid–Pt(II) complexes were
characterised by means of thermogravimetric analysis
(TGA) and inductively coupled plasma-mass spectrometry
(ICP-MS). The NMR spectra for the ligands and corre-
sponding Pt complexes were measured on a JEOL Eclipse
Plus spectrometer operating at 400 MHz (1H), 100.5 MHz
(13C) and 85.9 MHz (195Pt). 1H NMR and 13C NMR chem-
ical shifts were reported in parts per million referenced to
residual solvent resonances. 195Pt NMR spectra were
recorded in DMSO-d6, using a solution of K2[PtCl4] in
aqueous KCl as the external reference. The shift for
K2[PtCl4] was adjusted to ꢀ1628 ppm from Na2[PtCl6]
(d = 0 ppm). The thermograms were run in air atmosphere
from r.t. to 1000 ꢁC (heating speed = 10 ꢁC/min) using a
Mettler Toledo TGA/STDA851. The ICP-MS measure-
ments were performed by means of an X5 Series ICP-MS
instrument from Thermo Optek (Cinisello Balsamo, Italy).
Instrument settings were optimized in order to yield maxi-
mum sensitivity for platinum. Mineralized platinum-con-
taining material was dissolved in 2 mL of 2% HNO3 with
the addition of indium (used as the internal standard).
The most abundant isotopes of platinum and indium were
measured at m/z 195 and 115, respectively. Raman spectra
were recorded in the 3500–50 cmꢀ1 region on a Bruker
RFS 100 FT-Raman Spectrophotometer with 1064 nm
radiation from a Nd:YAG laser as the source of excitation.
(ii) Synthesis of 2-Boc. To stirred DMF (50 ml) at 80–
90 ꢁC folic acid 1 (0.750 g, 1.699 mmol) was slowly added
and then 50 ml of DMF were added to complete the disso-
lution of 1. The mixture was cooled at r.t. and CDMT
(0.304 g, 1.733 mmol) was added. The mixture was cooled
at 0 ꢁC and N-methylmorpholine (187 ll, 1.699 mmol)
was added dropwise. After 4 h mono(Boc)-ethylenediamine
(0.278 g, 1.733 mmol) and N-methylmorpholine (187 ll,
1.699 mmol) were added. After 2 h at 0 ꢁC the temperature
was brought to r.t. and the reaction stirred for 14 h. Evap-
oration of the solvent resulted in a thick oil that was tri-
tured and washed several times with cold water to
remove unreacted 1 and get a yellow powder of 2-Boc.
Yield: 0.870 g, 86%. The characterisation indicated that
no diadducts between 1 and mono(Boc)-ethylenediamine
were formed, whereas there were only monoadduct either
1
on a or c carboxylic group. H NMR (DMSO-d6) 11.44
(br. s, 1H, H20), 8.64 (s, 1H, H7), 8.21 (d, 1H, H18), 8.00–
7.80 (m, 2H, H24 and H27), 7.65 (d, 2H, H13 and H15),
6.94 (t, 1H, H10), 6.80–6.70 (m, 3H, H2 and H4), 6.62 (d,
2H, H12 and H16), 4.49 (d, 2H, H9), 4.31 (m, 1H, H19),
3.09–2.96 (m, 4H, H25 and H26,), 2.32–1.85 (m, 4H, H21
and H22), 1.34 (s, 9H, CH3, Boc), ppm; 13C NMR
(DMSO-d6) 174.40 and 174.66 (C20, double signal due to
a and c functionalizations), 172.26 and 172.39 (C23, double
signal due to a and c functionalizations), 166.88 (C17),
161.50 (C2), 157.25 (C4), 156.17 (C(O), Boc), 154.33
(C8a), 151.33 (C11), 149.50 and 149.21 (C6 and C7),
129.56 (C13 and C15), 128.51 (C4a), 121.89 (C14), 111.75
(C12 and C16), 78.18 and 78.23 (Cquat, Boc), 52.54 and
53.32 (C19, double signal due to a and c functionaliza-
tions), 46.48 (C9), 39.88 and 40.30 (C25 and C26), 31.07
and 32.51 (C22, double signal due to a and c functionaliza-
tions), 28.76 (CH3, Boc), 27.14 and 27.49 (C21, double sig-
nal due to a and c functionalizations) ppm.
The laser power was 50 mW and the resolution was 2 cmꢀ1
IR spectra were recorded on a Bruker FTIR Equinox 55
spectrometer in the range 4000–400 cmꢀ1
.
.
2.2. Synthesis of 2-{4-[(2-amino-4-hydroxy-pteridin-6-
ylmethyl)-amino]-benzoylamino}-4-(2-tert-
butoxycarbonylamino-ethylcarbamoyl)-butyric acid (2-Boc)
(i) Protection of 2,3-diaminopropionic acid with di-tert-butyl
dicarbonate: synthesis of 2,3-bis-tert-butoxycarbonylamino-
propionic acid. 2,3-Diaminopropionic acid monohydro-
chloride (1.005 g, 7.149 mmol) was dissolved in 20 ml of
water and then an aqueous solution of NaHCO3 (2.401 g,
28.580 mmol, 20 ml) was added. The mixture was stirred
and cooled at 5 ꢁC, then di-tert-butyl dicarbonate (Boc2O,
2.003 g, 9.182 mmol) dissolved in 10 ml of dioxane was
added. The mixture was kept under stirring and nitrogen
steam and cooled at 0 ꢁC. After 1 h the mixture was
brought at r.t. and kept under stirring overnight. The mix-
ture was washed twice with ethyl acetate and then the
organic phase was washed twice with a saturated solution
of bicarbonate. Aqueous phases were joined and acidified
with 10% HCl (pH 1) and then extracted with ethyl acetate.
2.3. Synthesis of 2-{4-[(2-amino-4-hydroxy-pteridin-6-
ylmethyl)-amino]-benzoylamino}-4-[2-(2,3-bis-tert-
butoxycarbonylamino-propionylamino)-ethylcarbamoyl]-
butyric acid (3)
(i) Deprotection of 2-{4-[(2-amino-4-hydroxy-pteridin-
6-ylmethyl)-amino]-benzoylamino}-4-(2-tert-butoxycarbon-
ylamino-ethylcarbamoyl)-butyric acid (2-Boc). 2-{4-[(2-
Amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}
-4-(2-tert-butoxycarbonylamino-ethylcarbamoyl)-butyric
acid (0.850 g, 1.456 mmol) was dissolved at r.t. in 6.7 ml of
trifluoroacetic acid (TFA). After 1 h stirring at r.t. in the

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E. Gabano et al. / Inorganica Chimica Acta 361 (2008) 1447–1455
dark, the solution was evaporated to dryness resulting in a
thick brown oil that was tritured with diethyl ether to get a
yellow powder of 2 (as CF3COOH salt). Yield: 0.792 g,
91%. 1H NMR (DMSO-d6) 1.89–2.32 (m, 4H, H21 and
H22), 2.83–3.32 (m, 4H, H25 and H26), 4.35 (m, 1H, H19),
4.55 (d, 2H, H9), 6.66 (d, 2H, H12 and H16), 7.67 (d, 2H,
H13 and H15), 7.69–8.25 (m, 5H, H2, H4, H10 and H18),
8.71 (s, 1H, H7), 11.17 (br. s, 1H, H20) ppm; 13C NMR
(DMSO-d6) 174.33 and 174.64 (C23, double signal due to
a and c functionalizations), 173.09 and 173.02 (C20, double
signal due to a and c functionalizations), 167.10 (C17),
159.23 and 159.58 (C2 and C4), 153.67 (C8a), 151.30
(C11), 148.70 and 148.66 (C6 and C7), 129.65 (C13 and
C15), 128.51 (C4a), 121.89 (C14), 111.80 (C12 and C16),
52.26 and 53.48 (C19, double signal due to a and c func-
tionalizations), 46.36 (C9), 31.01 and 32.34 (C22, double
signal due to a and c functionalizations), 36.96 and 39.21
(C25 and C26), 26.97 and 27.06 (C21, double signal due to
a and c functionalizations) ppm.
carbamoyl]-butyric acid, 3 (0.748 g, 0.972 mmol) was trea-
ted at r.t. with 4.4 ml of TFA. After 3 h stirring at r.t., the
solution was evaporated to dryness resulting in a thick
brown oil that was tritured with diethyl ether to get a
yellow powder of 2-{4-[(2-amino-4-hydroxy-pteridin-6-
ylmethyl)-amino]-benzoylamino}-4-[2-(2,3-bis-tert-butoxy-
carbonylamino-propionylamino)-ethylcarbamoyl]-butyric
acid di-trifluoroacetate (L1). Yield: 0.770 g, 99%. 1H NMR
(DMSO-d6) 11.41 (br. s, 1H, H20), 8.69 (s, 1H, H7), 8.25–
7.10 (m, 11H, H2, H4, H10, H18, H24, H27, H31 and H37),
7.68 (d, 2H, H13 and H15), 6.65 (d, 2H, H12 and H16),
4.53 (s, 2H, H9), 4.33 (m, 1H, H19), 4.03–2.84 (m, 7H,
H25, H26, H29 and H30), 2.32–1.91 (m, 4H, H21 and H22)
ppm; 13C NMR (DMSO-d6) 174.32 and 174.64 (C20, dou-
ble signals due to a and c functionalizations), 173.12 and
173.02, (C23, double signals due to a and c functionaliza-
tions), 172.81 (C28), 165.92 (C17), 159.17 and 159.56 (C2
and C4), 151.36 (C8a), 151.36 (C11), 149.00 and 148.78
(C6 and C7), 129.58 (C13 and C15), 128.50 (C4a), 121.85
(C14), 111.80 (C12 and C16), 53.48 (C29), 52.30 and 50.98
(C19, double signal due to a and c functionalizations),
46.40 (C9), 36.96, 37.03 and 38.38 (C25, C26 and C30),
31.01 and 32.33 (C22, double signal due to a and c func-
tionalizations), 27.06 and 27.08 (C21, double signal due to
a and c functionalizations) ppm. 19F NMR (DMSO-d6)
ꢀ74.26 ppm.
(ii) Synthesis of 3. 2,3-bis-tert-butoxycarbonylamino-
propionic acid (0.554 g, 1.82 mmol) was dissolved in
DMF (50 ml) at r.t.; CDMT (0.325 g, 1.85 mmol) was
added and the mixture was cooled at 0 ꢁC; after 10 min
N-methylmorpholine (200 ll, 1.82 mmol) was added drop-
wise. After 4 h 2 (1.106 g, 1.85 mmol) and N-methylmor-
pholine (0.184 g, 1.82 mmol) were added. After 2 h at
0 ꢁC the temperature was brought to r.t. and the reaction
stirred for 14 h. Evaporation of the solvent resulted in a
thick oil that was tritured with cold water resulting in a yel-
(ii) Synthesis of (L1)PtCl2. L1 (0.830 g, 1.041 mmol)
was dissolved in water (40 ml) at 65 ꢁC and then a solution
of K2[PtCl4] (0.432 g, 0.306 mmol) in water (5 ml) was
added and the pH value was adjusted to 5–6 with 0.1N
aqueous KOH (the pH of the mixture tended to 1–2
because of the progressing reaction but it had to be kept
from the region of hydroxocomplexes, i.e. at basic pH).
The resulting light brown precipitate was washed with cold
1
low powder. Yield: 0.760 g, 53%. H NMR (DMSO-d6)
11.40 (br. s, 1H, H20), 8.64 (s, 1H, H7), 7.66 (d, 2H, H13
and H15), 8.20–6.70 (m, 9H, H2, H4, H10, H18, H24, H27,
H31, and H37), 6.62 (d, 2H, H12 and H16), 4.48 (d, 2H, H9),
4.33 (m, 1H, H19), 3.94–2.83 (m, 7H, H25, H26, H29 and
H30), 2.31–1.88 (m, 4H, H21 and H22), 1.36 (s, 18H, CH3,
Boc), ppm; 13C NMR (DMSO-d6) 174.38 and 174.65 (C20,
double signals due to a and c functionalizations), 172.28
and 172.41 (C23, double signals due to a and c functionaliza-
tions), 170.76 (C28), 166.90 (C17), 161.41 (2 · C(O), Boc),
156.24 and 156.34 (C2 and C4), 152.37 (C8a), 151.33 (C11),
149.16 and 148.89 (C6 and C7), 129.68 (C13 and C15),
128.50 (C4a), 121.90 (C14), 111.72 (C12 and C16), 78.54 and
78.82 (2 · Cquat, Boc), 55.42 (C29), 52.67 and 53.32 (C19, dou-
ble signal due to a and c functionalizations), 46.47 (C9),
42.41 (C30), 38.74 and 38.89 (C25 and C26), 31.06 and 32.58
(C22, double signal due to a and c functionalizations),
28.72 (CH3, Boc), 27.40 and 27.47 (C21, double signal due
to a and c functionalizations) ppm.
1
water and dried under vacuum (0.227 g, yield 85%). H
NMR (DMSO-d6) 11.55 (br. s, 1H, H20), 8.68 (s, 1H,
H7), 7.68 (d, 2H, H13 and H15), 8.25–6.97 (m, 11H, H2,
H4, H10, H18, H24, H27, H31, H37), 6.56 (d, 2H, H12 and
H16), 4.49 (m, 2H, H9), 4.33 (m, 1H, H19), 4.02–2.80 (m,
7H, H25, H26, H29 and H30), 2.32–1.91 (m, 4H, H21 and
H22), ppm; 13C NMR (DMSO-d6) 174.48 and 174.65
(C20, double signals due to a and c functionalizations),
174.32 (C28), 172.50 and 173.50 (C23, double signals due
to a and c functionalizations), 167.00 (C17), 157.12 and
161.35 (C2 and C4), 154.38 (C8a), 151.36 (C11), 149.00
and 149.28 (C6 and C7), 129.65 (C13 and C15), 128.52
(C4a), 121.72 (C14), 111.75 (C12 and C16), 53.48 (C29),
52.31 and 52.59 (C19, double signal due to a and c func-
tionalizations), 46.47 (C9), 36.95, 38.53 and 50.16 (C25,
C26 and C30), 31.02 and 31.14 (C22, double signal due to
a and c functionalizations), 27.10 and 27.25 (C21, double
signal due to a and c functionalizations) ppm. 195Pt
NMR (DMSO-d6) ꢀ3233 and ꢀ3243 ppm (these signals
correspond to the complex (L1)PtCl(DMSO)). TGA: Pt
and K2O residue 27.71%, calc. Pt and K2O residue
27.72%; ICP-MS: Pt content 22.25%, calc. Pt content
22.33%.
2.4. Synthesis of cis-[(2-(4-((2-amino-4-hydroxy-pteridin-
6-ylmethyl)-amino)-benzoylamino)-4-(2-(2,3-bis-tert-
butoxycarbonylamino-propionylamino)-ethylcarbamoyl)-
butyric acid) dichloride platinum(II)] ((L1)PtCl2)
(i) Deprotection of 3 to give L1. 2-{4-[(2-Amino-
4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-4-[2-
(2,3-bis-tert-butoxycarbonylamino-propionylamino)-ethyl-

E. Gabano et al. / Inorganica Chimica Acta 361 (2008) 1447–1455
1451
2.5. Synthesis of the protected carboxylic ligands
(DMSO-d6) d (ppm): 11.45 (br. s, 1H, H20), 8.65 (H7),
8.22–7.87 (m, 3H, H24, H18 and H10), 7.65 (d, 2H, H13
and H15), 6.95 (m, 3H, NH2 and H4), 6.64 (d, 2H, H12
and H16), 4.49 (d, 2H, H9), 4.37 (m, 1H, H19), 3.18 (t,
1H, H28), 3.01 (m, 2H, H25), 2.33–1.92 (m, 4H, H21
and H22), 1.63 (m, 2H, H27), 1.39–1.38 (m, 20H, H26,
H31, H32, H33, H36, H37, and H38); 13C NMR (DMSO-
d6) d (ppm): 174.62 and 174.40 (C20, double signal due
to a and c functionalizations), 172.12 and 171.96 (C23,
double signal due to a and c functionalizations), 168.81
(C29 and C34), 166.80 (C17), 161.39 (C4), 157.14 (C2),
154.32 (C8a), 151.32 (C11), 149.25 and 149.12 (C6 and
C7), 129.63 (C13 and C15), 128.51 (C4a), 122.00 (C14),
111.74 (C12, C16), 81.28 (C30, C35), 53.32 (C28), 52.80
and 51.31 (C19, double signal due to a and c functional-
izations), 46.49 (C9), 38.55 (C25), 32.64 and 32.62 (C22,
double signal due to a and c functionalizations), 31.12
(C26), 28.08 (C31, C32, C33, C36, C37 and C38), 27.18
and 27.17 (C21, double signal due to a and c functional-
izations), 26.09 (C27).
2.5.1. Synthesis of 2-(4-{4-[(2-amino-4-oxo-3,4-dihydro-
pteridin-6-ylmethyl)-amino]-benzoylamino}-4-carboxy-
butyrylamino)-malonic acid diethyl ester (4)
Folic acid 1 (0.906 g, 2.052 mmol) was dissolved in 80 ml
DMF in a 100 ml flask, under stirring at 90 ꢁC. After dis-
solution of folic acid, the mixture was cooled at r.t. and
CMDT (0.353 g, 2.012 mmol) was added. The mixture
was cooled to 0 ꢁC and N-methylmorpholine (226 ll,
2.052 mmol) was added. The mixture reacted under stirring
at 0 ꢁC for 4 h and then 2-amino-malonic acid diethyl ester
(0.426 g, 2.012 mmol) and N-methylmorpholine (226 ll,
2.052 mmol) dissolved in 10 ml DMF were added. The
mixture reacted under stirring at 0 ꢁC for 2 h and then
for 14 h at r.t. to get a yellow mixture. DMF was removed
under reduced pressure and the residue was washed with
water (to remove unreacted folic acid), ethanol and diethyl
1
ether. Yield: 0.503 g, 41%. H NMR (DMSO-d6) d (ppm):
11.44 (s, 1H, H20), 8.81–8.69 (m, 2H, NH24 and NH18),
8.65 (s, 2H, H7), 8.17–8.08 (m, 2H, NH10 and H3), 7.65
(d, 2H, H13 and H15), 6.96 (m, 3H, H2 and H4), 6.64 (d,
2H, H12 and H16), 5.06 (d, 1H, H25), 4.49 (d, 2H, H9),
4.35 (m, 1H, H19), 4.19 (m, 4H, H27 and H30), 2.32 (m,
2H, H22), 1.89 (m, 2H, H21), 1.18 (t, 6H, H28 and H31);
13C NMR (DMSO-d6) d (ppm): 174.65 and 174.34 (C20,
double signal due to a and c functionalizations), 172.73
and 172.64 (C23, double signal due to a and c functional-
izations), 167.00 (C26 and C29), 166.92 (C17), 161.40 (C4),
157.16 (C2), 154.28 (C8a), 151.40 (C11), 149.26 and 149.07
(C6 and C7), 129.63 (C13 and C15), 128.52 (C4a), 121.86
(C14), 111.74 (C12 and C16), 62.30 (C27 and C30), 56.74
(C25), 52.79 and 52.72 (C19, double signal due to a and c
functionalizations), 46.48 (C9), 32.01 and 30.99 (C22, dou-
ble signal due to a and c functionalizations), 27.42 and
27.05 (C21, double signal due to a and c functionaliza-
tions), 14.41 (C28 and C31).
2.6. Synthesis of Pt(II) complexes of the carboxylic ligands
2.6.1. Synthesis of cis-[(NH2R)2 PtI2] (R = H, Me)
K2PtCl4 (0.415 g, 1.000 mmol) was dissolved in water
(4 ml) and heated over a sand bath at 40 ꢁC under stirring.
An aqueous solution of KI (0.996 g, 6.000 mmol of KI in
2 ml of water) was added to the mixture in the dark. After
30 min, the mixture was filtered and an aqueous solution
(3.330 mmol, 2 M) of NH3 or NH2CH3 was added to the
filtrate; this induced the immediate precipitation of fine yel-
low crystals of cis-[(NH3)2PtI2]. The precipitate was filtered
and washed with water, ethanol and diethyl ether. Yields:
90–96%.
2.6.2. Synthesis of cis-[diammino(potassium 2-{4-[(2-
amino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-amino]-
benzoylamino}-4-(dicarboxymethyl-carbamoyl)-
butyrato)platinum(II)], (L2)Pt(NH3)2
(i) Deprotection of 4. 2-(4-{4-[(2-Amino-4-oxo-3,4-dihy-
dro-pteridin-6-ylmethyl)-amino]-benzoylamino}-4-carboxy-
2.5.2. Synthesis of 2-[3-(4-{4-[(2-Amino-4-oxo-3,4-
dihydro-pteridin-6-ylmethyl)-amino]-benzoylamino}-4-
carboxy-butyrylamino)-propyl]-malonic acid di-tert-butyl
ester (5)
butyrylamino)-malonic acid diethyl ester
4 (0.379 g,
Folic acid 1 (0.602 g, 1.364 mmol) was dissolved in
70 ml DMF in a 100 ml flask, under stirring at 90 ꢁC.
After dissolution of 1, the mixture was cooled at r.t.
and CMDT (0.245 g, 1.394 mmol) was then added. The
mixture was cooled to 0 ꢁC and N-methylmorpholine
(150 ll, 1.364 mmol) was added. The mixture reacted
under stirring at 0 ꢁC for 4 h and then 2-(3-amino-pro-
pyl)-malonic acid di-tert-butyl ester (0.381 g, 1.394 mmol)
and N-methylmorpholine (150 ll, 1.364 mmol) dissolved
in 5 ml DMF were added. The mixture reacted under
stirring at 0 ꢁC for 2 h and then for 14 h at r.t.. After
that time the mixture was pale yellow. DMF was
removed under reduced pressure and the residue was
washed with water (to remove unreacted folic acid), eth-
anol and diethyl ether. Yield: 0.808 g, 85%. 1H NMR
0.634 mmol) was dissolved in 20 ml water by adding a
stoichiometric amount of KOH (0.071 g, 1.268 mmol)
and getting L2 as potassium salt.
(ii) Synthesis of (L2)Pt(NH3)2. cis-[(NH3)2PtI2] (0.300 g,
0.621 mmol) was added to a solution of AgNO3 (0.207 g,
1.218 mmol) in water (ꢁ30 ml) and the mixture was stir-
red at 40 ꢁC overnight in the dark. It was then filtered
to remove AgI and L2 (as potassium salt) was added to
the filtrate. Immediately the precipitation of an orange
compound started. The precipitate was isolated by centri-
fugation and washed with water, ethanol and diethyl ether
and dried under vacuum. Yield: 0.421 g, 84%. TGA: Pt
and K2O residue 29.86%, calc. Pt and K2O residue
29.97%; ICP-MS: Pt content 24.06%, calc. Pt content
24.15%.

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E. Gabano et al. / Inorganica Chimica Acta 361 (2008) 1447–1455
2.6.3. Synthesis of cis-[di(methylamine)(potassium 2-{4-
[(2-amino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-amino]-
benzoylamino}-4-(dicarboxymethyl-carbamoyl)-
butyrato)platinum(II)], (L2)Pt(NH2CH3)2
(i) Deprotection of 4. The reaction was carried out as
described for (L2)Pt(NH3)2.
precipitate was isolated by centrifugation and washed with
water, ethanol and diethyl ether and dried under vacuum.
Yield: 0.304 g, 86%. TGA: Pt and K2O residue 27.49%,
calc. Pt and K2O residue 27.59%; ICP-MS: Pt content
22.17%, calc. Pt content 22.22%.
(ii) Synthesis of (L2)Pt(NH2CH3)2. cis-[(NH2CH3)2-
PtI2] (0.161 g, 0.317 mmol) was added to a solution of
AgNO3 (0.105 g, 0.620 mmol) in water (ꢁ30 ml) and the
mixture was stirred at 40 ꢁC overnight in the dark. It was
then filtered to remove AgI and a stoichiometric quantity
of L2 (as potassium salt) was added to the filtrate. Slowly
a suspension of an orange compound started. Water is
removed under reduced pressure and the residue was
washed with water, ethanol and diethyl ether and dried
under vacuum. Yield: 0.425 g, 82%. TGA: Pt and K2O
residue 29.07%, calc. Pt and K2O residue 28.98%; ICP-
MS: Pt content 23.42%, calc. Pt content 23.34%.
3. Results and discussion
3.1. General remarks
Folic acid 1 (also known as pteroylglutamic acid) can be
considered a conjugated pterin therein three subunits can
be recognized: 6-methylpterin, 4-aminobenzoic acid and
glutamic acid. In particular, the distal glutamyl residue
embodies two carboxylates (i.e., a- and c-) which will serve
as handles for further synthetic manipulations (Fig. 2). It
has been suggested that the receptor binding properties of
folic acid are retained when derivatized via its c-carboxyl-
ate, and somewhat weakened when a-carboxylate is
involved [10]. However, this matter is nowadays controver-
sially discussed [11]. Accordingly, recent studies of
99mTc-derivatives of folic acid indicate that the receptor
recognition and the following internalisation of these deriv-
atives are almost the same for both a- and c-derivatized
compounds as well as for pteroic acid conjugates [11b].
Taken together, the foregoing results indicate that the reg-
ioselectivity (a- versus c-functionalization) could represent
an unresolved but not dramatic problem in the preparation
of folate-drug conjugates. In the event, the product profile
for both steps (activation and coupling) is exceedingly com-
plex [12]. Besides a- and c-mono activated derivatives, a
canonical peptide coupling protocol using N,N0-dicyclo-
hexylcarbodiimide (DCC) in the presence of an activator
(N-hydroxysuccinimide, HOSu) generates various by-prod-
uct like bis-activated derivatives and anhydrides. As a con-
sequence, several undesirable side-products are formed in
the subsequent reaction with nucleophiles and coupling
occurring at the a-carboxylate proceeds with complete
racemization. Furthermore, it has been reported that isom-
erization between a-conjugates and c-conjugates frequently
occurs through the agency of DCC [13]. Accordingly, sep-
aration of these mixtures [10a,14] is troublesome and,
indeed, some of the biological results reported in the liter-
ature have been obtained on mixtures of all the possible
compounds. Nevertheless, under optimized conditions,
reaction between folic acid and primary amines gave a mix-
ture of a- or c-conjugates and unreacted folic acid with no
apparent bi-conjugates [10a,15]. In any case, the c-deriva-
tive is claimed to be the main product of the reaction
[10a,14,16].
2.6.4. Synthesis of cis-[diammino(potassium 2-{4-[(2-
amino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-amino]-
benzoylamino}-4-(4,4-dicarboxy-butylcarbamoyl)-
butyrate)platinum(II)], (L3)Pt(NH3)2
(i) Deprotection of 5. 2-[3-(4-{4-[(2-amino-4-oxo-3,4-
dihydro-pteridin-6-ylmethyl)-amino]-benzoylamino}-4-car-
boxy-butyrylamino)-propyl]-malonic acid di-tert-butyl
ester 5 (0.578 g, 0.830 mmol) reacted with trifluoroacetic
acid (TFA, 20 ml) for 1 h. Excess TFA was removed under
reduced pressure and the residue was washed with diethyl
ether. The resulting acid was dissolved in water and
KOH (0.093 g, 1.66 mmol) was added to get L3 (as potas-
sium salt).
(ii) Synthesis of (L3)Pt(NH3)2. cis-[(NH3)2PtI2]
(0.393 g, 0.814 mmol) was added to a solution of AgNO3
(0.263 g, 1.546 mmol) in water (ꢁ20 ml) and the mixture
was stirred at 40 ꢁC overnight in the dark. It was then fil-
tered to remove AgI and L3 (as potassium salt) was added
to the filtrate. Immediately the precipitation of an orange
compound started. The precipitate was isolated by centri-
fugation and washed with water, ethanol and diethyl ether
and dried under vacuum. Yield: 0.602 g, 87%. TGA: Pt and
K2O residue 28.61%, calc. Pt and K2O residue 28.50%;
ICP-MS: Pt content 23.03%, calc. Pt content 22.96%.
2.6.5. Synthesis of cis-[di(methylamine)(potassium 2-{4-
[(2-amino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-amino]-
benzoylamino}-4-(4,4-dicarboxy-butylcarbamoyl)-
butyrate)platinum(II)], (L3)Pt(NH2CH3)2
(i) Deprotection of 5. The reaction was carried out as
described for (L3)Pt(NH3)2.
(ii) Synthesis of (L3)Pt(NH2CH3)2. cis-[(NH2CH3)2-
PtI2] (0.205 g, 0.403 mmol) was added to a solution of
AgNO3 (0.134 g, 0.789 mmol) in water (ꢁ30 ml) and the
mixture was stirred at 40 ꢁC overnight in the dark. It was
then filtered to remove AgI and a stoichiometric quantity
of L3 (as potassium salt) was added to the filtrate. Immedi-
ately the precipitation of an orange compound started. The
We used coordinating arms of different length, either car-
rying (diamine) group or leaving (malonate) group. In par-
ticular, we synthesised one diamine conjugate (L1) to realize
a cisplatin-analogue (L1)PtCl2 able to exploit the folate
receptor-mediated endocytosis (FRME) to deliver platinum
once inside the cells. Then, we synthesised two dicarboxylic
conjugates (L2 and L3), which differ in the length of the

E. Gabano et al. / Inorganica Chimica Acta 361 (2008) 1447–1455
1453
coordinating arm, to realize metal chelates not only able
to take advantage of FRME but also to directly release
platinum moiety after hydrolysis of the folic leaving ligand.
look for potential solutions, one of the few reagents we
found to be right for the purpose was 2-chloro-4,6-dime-
thoxy-1,3,5-triazine (CDMT). CDMT (also known as
Kaminski’s reagent) [25] has been shown to be a versatile
reagent in terms of stability, mild reaction conditions and
cost. Gratifyingly, it was found that a DMF solution of 1
reacts smoothly (0ꢁ ! r.t./20 h) with mono(Boc)-ethylene-
diamine upon addition of CDMT and N-methylmorpho-
line (NMM). The characterisation (13C NMR) indicated
that no diadducts between 1 and mono(Boc)-ethylenedia-
mine were formed, whereas there were only monoadducts
either on a or c carboxylic group. The resulting Boc-amide
2 (86%) was N-(Boc)-deprotected (neat TFA, r.t., 1 h) and
used directly in the coupling reaction with 2,3-di(Boc)-2,3-
diaminopropionic acid. The successful iteration of Kamin-
ski’s protocol in DMF (0ꢁ ! r.t./20 h) and subsequent
Boc-deprotection of 3 in neat TFA at r.t. for 3 h led to
desired ligand L1 (as trifluoroacetate) (48% over three
steps). The complex cis-(L1)PtCl2 was obtained in good
yield by reaction of K2[PtCl4] and L1 according to Miller’s
procedure for platinum coordination [26]. Thus, K2[PtCl4]
was added to an aqueous solution of L1 (as trifluoroace-
tate) at about 60 ꢁC. During the reaction, the pH was kept
at 6.0 by the dropwise addition of 0.1N KOH. The pH
value is important at this stage of the reaction: it must be
high enough to deprotonate the diamine ligand, thus mak-
ing it suitable for platinum coordination, but not so high as
to cause the formation of inactive hydroxo-Pt species.
Spectral evidence (multinuclear NMR, namely 1H, 13C
and 195Pt NMR) strongly supports the structures assigned
to L1 and (L1)PtCl2. The characterisation was confirmed
with thermogravimetric analysis (TGA) on the solid sam-
ple and inductively coupled plasma mass spectrometry
3.2. Synthesis of the diamine conjugate (L1)
The diamine conjugate L1 was prepared by a four-step
procedure from 1 employing 2,3-diaminopropionic acid
(as a chelating group for platinum) linked to one carboxyl-
ate of 1 (a or c) through ethylenediamine as a spacer
(Scheme 1, where for sake of simplicity the spacer is repre-
sented linked to the c carboxylate only). Aware of the pre-
vious problems connected to DCC, we decided to explore
the coupling reaction between folic acid and mono
(Boc)-ethylenediamine in the presence of a non-carbodiim-
ide coupling agent. The first set of conditions tested was
mixed anhydride formation with isobutyl chloroformate
(IBCF) [17] or pivaloyl chloride [18] but this approach
came to naught. Other protocols via uronium or phospho-
nium salt of a non-nucleophilic anion ðPF6ꢀÞ such as
O-(benzotriazol-1-yl)-N,N,N0,N0-bis(tetramethylene)uronium
hexafluorophosphate (HBPyU) [19], (benzotriazol-1-
yloxy)tripyrrolidinophosphonium
hexafluorophosphate
(PyBOP) [20], (benzotriazol-1-yloxy)tris(dimethylamino)
phosphonium hexafluorophosphate (BOP, Castro’s
reagent) [21] and O-(benzotriazol-1-yl)-N,N,N0,N0-tetram-
ethyluronium hexafluorophosphate (HBTU) [22] were
examined in the attempt to limit by-product formation,
but in all instances, either low yields were obtained or iso-
lation problems occurred. Attempts were made to use other
condensing agents (e.g., diphenylphosphoryl azide (DPPA)
[23] and diethyl cyanophosphonate (DECP) [24]), but none
proved successful. After a concerted literature survey to
O
CO2H
O
CO2H
H
N
OH
O
N
N
H
O
N
N
H
(a) (b)
N
N
O
N
N
O
HN
N
H
HN
N
H
H2N
H2N
H2N
2 (as CF3COOH salt
1
(c)
O
CO2H
O
CO2H
H
H
N
N
O
N
N
H
O
N
H
(b)
N
N
O
O
N
N
O
HN
HN
HN
N
H
HN
N
H
Boc
O
NH2
N
H
H2N
N
H2N
NH2
NH
L1 (as CF3COOH salt)
3
Boc
(d)
°
a) Boc(en), CDMT, NMM, DMF from 0 C to r.t.; b) neat, TFA, r.t.; c) 2,3-di(Boc)-2,3-diaminopropionic acid, CDMT,
NMM, DMF, from 0 C to r.t.; d) NaOH, H2O, then K2PtCl4 at 65 C; overall yield: 35% (over 5 steps)
°
°
(L1)PtCl2
Scheme 1. Sketch of the synthesis of cis-[(2-(4-((2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino)-benzoylamino)-4-(2-(2,3-bis-tert-butoxycarbonylamino-
propionylamino)-ethylcarbamoyl)-butyric acid) dichloride platinum(II)], (L1)PtCl2; Boc(en) = mono(Boc)-ethylenediamine, CDMT = 2-chloro-4,6-
dimethoxy-1,3,5-triazine, DMF = dimethylformamide, NMM = N-methylmorpholine, TFA = trifluoroacetic acid.

1454
E. Gabano et al. / Inorganica Chimica Acta 361 (2008) 1447–1455
(ICP-MS) on the solution of the mineralised sample. In the
TGA experiments, after heating of the sample in air atmo-
sphere from r.t. to 1000 ꢁC, the organic moiety was com-
pletely burnt and the platinum recovered as pure metal
together with K2O [27]. The ICP-MS analysis confirmed
the Pt content in the mineralized sample.
Unfortunately, (L1)PtCl2 showed low solubility in
DMSO and was completely insoluble in water, thereby pre-
cluding any biochemical and biological test.
monoadducts either on a or c carboxylic group. The prod-
ucts were sufficiently pure (NMR) for further reactions.
Thus, ligand L2 (as K+ salt) was prepared in quantitative
yield by exposure to KOH aqueous solutions at r.t.
whereas conversion of 5 to L3 was accomplished under
standard conditions (neat TFA, r.t.), followed by exposure
to KOH aqueous solutions at r.t. (Scheme 2, where for sake
of simplicity the spacer is represented linked to the c car-
boxylate only). The complexation of L2 and L3 was carried
out according to Dhara’s protocol [29] because it is rapid,
easy and efficient. Thus, a solution of K2[PtI4] was reacted
with NH3 or NH2CH3 to produce cis-[(NH2R)PtI2 (R = H
or CH3). The following replacement of iodide ions with
water by means of Ag2SO4 or AgNO3, yielded the corre-
sponding diaqua-complex cis-[(NH2R)Pt(H2O)2]2+. The
treatment of the L2 and L3 (as K+ salts) with a solution
of cis-[(NH2R)Pt(H2O)2]2+ yielded the final complexes
(Scheme 2). We chose to synthesise these Pt-complexes
using two different amines: NH3 in order to get cisplatin-
like complexes, namely (L2)Pt(NH3)2 and (L3)Pt(NH3)2,
and NH2CH3 in order to improve the water solubility of
the resulting compounds, namely (L2)Pt(NH2CH3)2 and
(L3)Pt(NH2CH3)2, even if the methylation is known to be
detrimental for the cytotoxic activity [30].
3.3. Synthesis of the dicarboxylic conjugates
Also in the case of the synthesis of the dicarboxylic con-
jugates, the use of carbodiimide-based coupling agents such
as DCC [10a,14], and 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide (EDC) [28], as well as diphenylphosphoryl
azide (DPPA) [23] and diethyl cyanophosphonate (DECP)
[24] resulted in a number of by-products and poor yield.
Once again CDMT/NMM proved to be efficient as cou-
pling agent for the functionalization of 1. Therefore, the
conditions chosen for coupling in the synthesis of folic
acid-based dicarboxylic ligands L2 and L3 were those
developed by Kaminski [25]. Pleasingly, we found the same
reactivity pattern. For example, when 1 was reacted with
diethyl aminomalonate hydrochloride with CTDM in
DMF in the presence of NMM (0ꢁ ! r.t.; 20 h), the amide
4 was obtained as the virtually exclusive product (41%)
along with unreacted 1. The same behaviour was observed
when 1 reacted with di(t-butyl)-2(3-aminopropyl)malonate
[8] under similar conditions resulting in the formation of 5.
The characterisation (13C NMR) indicated that no diad-
ducts between 1 and R-malonate [R = 2-amino; 2-(3-ami-
nopropyl)] were formed, whereas there were only
The ligands L2 and L3 are soluble in some polar organic
solvents (i.e., DMF and DMSO) and in aqueous alkaline
solutions and fully characterised by multinuclear NMR
spectroscopy. Unfortunately, the corresponding Pt com-
plexes showed solubility traits similar to the other folic
acid-based (L1)PtCl2 complex (vide supra). This precluded
any multinuclear NMR analyses whereas both TGA and
ICP-MS confirmed the proposed molecular formulas for
these complexes.
O
CO2H
H
N
CO2R
CO2R
(L2)Pt(NH3)2
(c)
(d)
O
N
N
H
(as K+ salt)
(a)
N
N
O
(b)
HN
N
H
L2 (R:H)
(as K+ salt)
H2N
4 (R:Et)
(L2)Pt(NH2CH3)2
(as K+ salt)
O
CO2H
OH
O
N
N
H
N
N
O
HN
N
H
H2N
1 (FA)
CO2R
CO2H
O
H
N
(L3)Pt(NH3)2
c)
d)
O
N
N
H
CO2R
(f) (b)
(e)
N
N
O
HN
N
L3 (R:H)
(as K+ salt)
H
H2N
(L3)Pt(NH2CH3)2
5 (R:t-Bu)
a) diethyl aminomalonate hydrochloride, CDMT, NMM, DMF, from 0° to r.t.; b) KOH, H2O, r.t.; c) cis[(NH3)2Pt(H2O)2]2+, H2O; d)
C
cis[(NH2CH3)2Pt(H2O)2]2+, H2O; e) di(
t-butyl)-2-(3-aminopropyl)malonate, CDMT, NMM, DMF, from 0°C to r.t.; f) TFA
Scheme 2. Sketch of the synthesis of folic acid derivatives and their corresponding Pt-complexes. CDMT = 2-chloro-4,6-dimethoxy-1,3,5-triazine,
DMF = dimethylformamide, NMM = N-methylmorpholine, TFA = trifluoroacetic acid.

E. Gabano et al. / Inorganica Chimica Acta 361 (2008) 1447–1455
1455
[7] K.S. Vitols, Y. Montejano, T. Duffy, L. Pope, G. Grundler, F.M.
Huennekens, Adv. Enzyme Regul. 26 (1987) 17.
[8] O. Aronov, A.T. Horowitz, A. Gabizon, D. Gibson, Bioconjugate
Chem. 14 (2003) 563.
[9] J.A. Lyon, P.D. Ellis, R.B. Dunlap, Biochemistry 12 (1973) 2425.
[10] (a) S. Wang, R.J. Lee, Carla J. Mathias, M.A. Green, P.S. Low,
Bioconjugate Chem. 7 (1996) 56;
(b) W. Guo, G.H. Hinkle, R.J. Lee, J. Nucl. Med. 40 (1999) 1563.
[11] (a) P.W. Wedeking, R.E. Wager, T. Arunachalam, K. Ramalingam,
K.E. Linder, R.S. Ranganathan, A.D. Nunn, N. Raju, M.F. Tweedle,
U.S. Patent 6093382;
In order to further corroborate the structural assign-
ments, Raman and IR spectra were performed. Raman
spectroscopy is useful to evaluate the shift of the stretching
bands of carboxylic groups from free ligands to complexes
and the presence of m(Pt–X) bands of the complexes. As
reported in the literature [31], m(Pt–N) bands are in the
range 600–400 cmꢀ1 while m(Pt–O) between 500 and
400 cmꢀ1. In the free ligands the m(C=O) bands are
between 1800 and 1650 cmꢀ1 [32], whereas in the corre-
sponding complexes m(C=O) bands are in the 1750–
1560 cmꢀ1 region. Unfortunately, all the complexes of
ligands L2 and L3 were fluorescent and the diagnostic
bands in the Raman spectra were hidden by this fluores-
cence. Therefore, we could not evaluate the coordination
of the ligands to platinum by means of Raman spectros-
copy. On the contrary, the IR spectroscopy was useful to
evaluate the shift of m(C=O) of carboxylic groups from free
ligands to complexes, even though the spectra were highly
crowded due to the various functional groups of the mole-
cules. In particular, the spectrum of free malonic acid itself
shows a characteristic strong band at 1725 cmꢀ1, that
moves in the spectrum of the corresponding cis-
[(NH3)2Pt(malonate)] complex to 1625 cmꢀ1. The same sit-
uation holds in the comparison of the spectra of L2 and L3
with those of the corresponding Pt-complexes. Ligands L2
and L3 show a strong band at around 1729 cmꢀ1 that shifts
to 1638 cmꢀ1 in the complexes. These shifts of the vibra-
tional bands, together with the TGA and ICP-MS data,
indicate that complexes (L2,3)Pt(NH2R) (R = H or CH3)
have been successfully synthesized and purified but once
again their low solubility precluded any biochemical and
biological evaluations.
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`
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