Toward the selective delivery of chemotherapeutics into tumor cells by targeting peptide transporters: Tailored gold-based anticancer peptidomimetics

Article abstract of DOI:10.1021/jm201480u

Complexes [Au^IIIX₂(dtc-Sar-AA-O(t-Bu))] (AA = Gly, X = Br (1)/Cl (2); AA = Aib, X = Br (3)/Cl (4); AA = l-Phe, X = Br (5)/Cl (6)) were designed on purpose in order to obtain gold(III)-based anticancer peptidomimetics that might specifically target two peptide transporters (namely, PEPT1 and PEPT2) upregulated in several tumor cells. All the compounds were characterized by means of FT-IR and mono- and multidimensional NMR spectroscopy, and the crystal structure of [Au^IIIBr₂(dtc-Sar-Aib-O(t- Bu))] (3) was solved and refined. According to in vitro cytotoxicity studies, the Aib-containing complexes 3 and 4 turned out to be the most effective toward all the human tumor cell lines evaluated (PC3, DU145, 2008, C13, and L540), reporting IC₅₀ values much lower than that of cisplatin. Remarkably, they showed no cross-resistance with cisplatin itself and were proved to inhibit tumor cell proliferation by inducing either apoptosis or late apoptosis/necrosis depending on the cell lines. Biological results are here reported and discussed in terms of the structure-activity relationship.

Full text of DOI:10.1021/jm201480u

Article
pubs.acs.org/jmc
Toward the Selective Delivery of Chemotherapeutics into Tumor
Cells by Targeting Peptide Transporters: Tailored Gold-Based
Anticancer Peptidomimetics
Morelle Negom Kouodom,† Luca Ronconi,† Marta Celegato,‡ Chiara Nardon,†,⊥ Luciano Marchio,§
̀
Q. Ping Dou,⊥ Donatella Aldinucci,‡ Fernando Formaggio,† and Dolores Fregona*,†
†Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, Padova 35131, Italy
‡Division of Experimental Oncology 2, Centro di Riferimento Oncologico (CRO-IRCCS), Via F. Gallini 2, Aviano (PN) 33081, Italy
§Department of Chemistry, University of Parma, Parco Area delle Scienze 17A, Parma 43100, Italy
⊥The Developmental Therapeutics Program, Departments of Oncology, Pharmacology and Pathology, Barbara Ann Karmanos
Cancer Institute, School of Medicine, Wayne State University, 540.1 HWCRC, 4100 John R Road, Detroit, Michigan 48201, United
States
S
* Supporting Information
ABSTRACT: Complexes [AuIIIX2(dtc-Sar-AA-O(t-Bu))] (AA = Gly, X
= Br (1)/Cl (2); AA = Aib, X = Br (3)/Cl (4); AA = L-Phe, X = Br (5)/
Cl (6)) were designed on purpose in order to obtain gold(III)-based
anticancer peptidomimetics that might specifically target two peptide
transporters (namely, PEPT1 and PEPT2) upregulated in several tumor
cells. All the compounds were characterized by means of FT-IR and
mono- and multidimensional NMR spectroscopy, and the crystal
structure of [AuIIIBr2(dtc-Sar-Aib-O(t-Bu))] (3) was solved and refined.
According to in vitro cytotoxicity studies, the Aib-containing complexes
3 and 4 turned out to be the most effective toward all the human tumor
cell lines evaluated (PC3, DU145, 2008, C13, and L540), reporting IC50
values much lower than that of cisplatin. Remarkably, they showed no
cross-resistance with cisplatin itself and were proved to inhibit tumor
cell proliferation by inducing either apoptosis or late apoptosis/necrosis depending on the cell lines. Biological results are here
reported and discussed in terms of the structure−activity relationship.
natural candidates as potential alternatives to clinically
established platinum drugs. In fact, their antiarthritic activity
arises from the well-known immunosuppressive and antiin-
flammatory actions, thus establishing, at least in principle, a
connection between the two therapies.7
INTRODUCTION
■
Platinum drugs are well-established as effective anticancer
agents, the forerunner being cisplatin.1 Although their
therapeutic effectiveness is severely hindered by some adverse
side-effects, such as neuro- and nephrotoxicity and tumor
resistance (either intrinsic or acquired during cycles of
therapy),2 the unquestionable clinical success of cisplatin and
its second- and third-generation analogues has triggered the
development of several alternative metal-based potential
anticancer agents.3,4
Overall, gold compounds are currently gaining ground as a
new class of chemotherapeutics owing to their strong tumor
cell growth inhibiting effect, generally achieved by exploiting
noncisplatin-like pharmacodynamic and pharmacokinetic prop-
erties and mechanisms of action.5 In particular, gold(III)
complexes share with platinum(II) derivatives some key
chemical features, such as the preference for square-planar
geometry and the typical d8 electronic configuration, making
them attractive for testing as antineoplastic drugs. Furthermore,
due to their traditional use in medicine for the treatment of
rheumatoid arthritis (chrysotherapy),6 gold(III) derivatives are
In this context, we have previously reported on some
gold(III)−dithiocarbamato derivatives of the type
[AuIIIX2(dtc)] (X = Cl, Br; dtc = various dithiocarbamates)
designed in such a way to reproduce very closely the main
features of cisplatin. The choice of dithiocarbamato ligands is
not accidental; in fact, dithiocarbamates have been successfully
used as chemoprotectants to modulate cisplatin nephrotoxicity
without decreasing its antitumor activity.8 These gold(III)
compounds were shown to exert outstanding in vitro
cytotoxicity, even toward human tumor cell lines intrinsically
resistant to cisplatin, and no cross-resistance to the reference
platinum drug.9,10 In this regard, for the first time, we have
identified the proteasome as a major in vitro and in vivo
Received: November 2, 2011
Published: February 6, 2012
© 2012 American Chemical Society
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Chart 1. Dipeptides and the Corresponding Gold(III)−Dithiocarbamato Complexes Studied in this Work
target,11,12 thus suggesting a different mechanism of action
compared to that of platinum drugs. Remarkably, their
chemotherapeutic properties have been confirmed in vivo,11,13
together with negligible acute toxicity and almost lack of
nephrotoxic side-effects,14 thus supporting the rationale of our
strategy.
ceftibuten), and some angiotensin-converting enzyme (ACE)
inhibitors, such as fosinopril, captopril and enalapril, are
recognized by either PEPT1 or PEPT2 as substrates because
of their resemblance to di- or tripeptides.16 Therefore, peptide
transporters represent excellent targets for the delivery of
pharmacologically active compounds because their substrate
binding site can accommodate a wide range of molecules with
different size, hydrophobicity, and charge.17
On account of these considerations, the rationale of our
research was to design gold(III) complexes of the type
[AuIIIX2(dpdtc)] (X = Cl, Br; dpdtc = dipeptidedithiocarba-
mate) which could both preserve the antitumor properties and
reduce toxic and nephrotoxic side-effects of the previously
reported gold(III) analogues,9,10 together with an enhanced
bioavailability and tumor selectivity due to the dipeptide-
mediated cellular internalization provided by PEPTs. Accord-
ingly, we report here on the synthesis, chemical character-
ization, and in vitro anticancer activity of the gold(III)−
dipeptidedithiocarbamato derivatives [AuIIIX2(dtc-Sar-AA-O(t-
Bu))] (X = Cl, Br; AA = Gly, Aib, L-Phe). Biological results are
discussed in terms of the structure−activity relationship,
focusing on the potential outcome of this novel class of
metal-based peptidomimetics.
Subsequent to the positive achieved outcomes, we have
extended our research toward “second-generation” gold(III)−
dithiocarbamato derivatives of dipeptides as improved intra-
cellular drug transfer and delivery systems supported by
transport proteins. A major problem of therapeutic effective-
ness relies on the crossing of the cell membrane. Cellular
uptake of therapeutics is still a challenging task because of the
plasma membrane, which constitutes an impermeable barrier
for most molecules. In order to overcome this issue, several
carrier-mediated delivery strategies have been investigated.
Among them, much attention has been recently given to
peptide-based delivery systems. Peptide transporters are
integral plasma membrane proteins that mediate the cellular
uptake of di- and tripeptides and peptide-like drugs (i.e.,
peptidomimetics). Two peptide transporters, namely PEPT1
and PEPT2, have been identified in mammals. They are present
predominantly in epithelial cells of the small intestine, bile duct,
mammary glands, lung, choroid plexus, and kidney but are also
localized in other tissues (pancreas, liver, gastrointestinal tract)
and, intriguingly, seem to be overexpressed in some types of
tumors.15 A unique feature is their capability for sequence-
independent transport of most possible di- and tripeptides
inside the cells. These transporters are stereoselective toward
peptides containing L-enantiomers of amino acids. Apart from
degradation products of nutritional proteins, some peptidomi-
metic (e.g., bestatin, L-dopa), nonpeptidic (e.g., valacyclovir,
AZT, 5-aminolevulinic acid) drugs and prodrugs, β-lactam
antibiotics (e.g., cefadrine, cefadroxil, cyclacillin, cefixime,
RESULTS
■
Chemistry. Syntheses. Three tert-butyl-esterified dipep-
tides were prepared as hydrochlorides (HCl·H-Sar-AA-O(t-
Bu), AA = Gly (P1), Aib (P2), L-Phe (P3)) and used to
synthesize the corresponding gold(III)−dithiocarbamato de-
rivatives [AuIIIX2(dtc-Sar-AA-O(t-Bu))] (X = Cl, Br; AA = Gly,
Aib, L-Phe (1−6)) (Chart 1).
Compounds P1−P3, being short peptides, were prepared by
classical solution (not on solid support) strategies,18−21 in order
to obtain large amounts of the final products. With reference to
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Scheme 1, the first step was the tert-butyl esterification of the Z-
protected amino acid (Z-AA-OH) through the acid-catalyzed
solution is subsequently reacted with 0.5 equiv of K[AuIIIX4]
(X= Br, Cl) in water, leading to the sudden precipitation of the
expected gold(III) complex in good yields (70−90%).
Characterization. The starting dipeptides and the corre-
sponding gold(III)−dithiocarbamato derivatives were charac-
terized by means of several techniques in order to assess the
structure as well as to investigate their stability in solution.
The proposed stoichiometry of the gold complexes was
confirmed by both elemental and thermogravimetric analyses,
the latter indicating a good correlation between calculated and
found weight loss values to metallic gold (Table S1 in the
Supporting Information).
Scheme 1. Synthetic Scheme for the Preparation of
Dipeptides HCl·H-Sar-AA-O(t-Bu) (AA = Gly (P1), Aib
a
(P2), L-Phe (P3))
IR spectroscopy was proved useful to identify the synthesized
complexes. With reference to Table 1, by comparison with the
spectra recorded for the starting dipeptide ester hydrochlorides
P1−P3, the disappearance of the broad band at 2680−2780
25
cm−1 (νa/s(NH2 )) together with the detection of two new
absorption bands at around 1560 (ν(N−CSS), somewhat
overlapped with amide II) and at ca. 1000 cm−1 (νa(S−C−S))
in the spectra of the corresponding gold derivatives 1−6, is
consistent with the formation of gold(III)−dithiocarbamato
complexes in which the NCSS moiety chelates the metal center
in a symmetrical bidentate mode.22 Such coordination is also
supported by the presence of two bands in the range 380−420
cm−1 associated with the νa/s(S−Au−S).26 Finally, the two
remaining coordination sites are taken up by cis-halides (either
Br or Cl), as confirmed by the appearance of two new bands at
315−360/215−255 cm−1 related to the νa/s(X−Au−X) (X =
Cl, Br, respectively).27,28 Remarkably, the other main
frequencies originated by the dipeptide chains seem not to be
affected by the formation of the dithiocarbamate and the
subsequent metal coordination, except for a significant increase
of the ν(N−H) together with a shift of the amide II band
toward lower wavenumbers.
+
a
(a) ZOSu, TEA, CH3CN/H2O, r.t., 4 d; (b) isobutene, H2SO4 (cat.),
CH2Cl2, −60 °C/r.t., 7 d; (c) 10% Pd−C, H2, MeOH; (d) NMM,
isobutyl chloroformiate, THF/CHCl3, −15 °C to r.t., overnight; (e)
HCl/Et2O.
reaction with isobutene, followed by the removal of the Z-
protecting group by catalytic hydrogenation. The dipeptides H-
Sar-AA-O(t-Bu) were then obtained by condensation of Z-Sar-
OH with H-AA-O(t-Bu) and subsequent catalytic hydro-
genation. The resulting purified products were finally isolated
as hydrochlorides by slow addition of 1 equiv of dilute HCl in
diethyl ether. This procedure avoided the hydrolysis of the tert-
butyl ester and, consequently, increased yields as well as
reduced the occurrence of side products.
The gold(III)−dithiocarbamato complexes 1−6 were pre-
pared as previously reported.22 The main synthetic route to
dithiocarbamates is based on the base-catalyzed nucleophilic
attack of a primary or secondary amine to carbon disulfide.23
This process can even take place without using a strong base,
but, in this case, the yield is much lower and the reaction
slower.24 All attempts to isolate the dithiocarbamato derivatives
of dipeptides P1−P3 without causing their degradation during
the separation/purification process were proved unsuccessful,
so the gold(III) complexes 1−6 were obtained by a template
reaction involving two steps. With reference to Scheme 2,
during the first step, the dithiocarbamic acid of a dipeptide
forms in solution owing to the reaction in water of the starting
dipeptide hydrochloride with carbon disulfide and sodium
hydroxide in equimolar ratio, the pH consequently turning
from 10 to approximately 6 as the reaction proceeds. The
The NMR characterization was carried out by means of
mono- (1H, Table 2) and multidimensional ([1H,13C] HMBC,
Table 3) techniques. Compared with the spectra of the starting
dipeptide ester hydrochlorides P1−P3, those recorded for the
corresponding gold complexes 1−6 show a general shift of all
the signals toward higher δ values. The downfield shift increases
with the closeness to the NCSS moiety, the larger variation (ca.
1 ppm) involving the methyl and the methylene groups directly
bound to the dithiocarbamato nitrogen atom. Only for the
amide proton peaks lower chemical shift values (ca. 1 ppm)
+
were recorded. Moreover, the disappearance of the NH2
proton signal, together with the detection of a new 13C signal
at about 200 ppm (assigned to the dithiocarbamato carbon
atom),29 is consistent with the formation of the gold(III)−
peptide derivatives depicted in Chart 1. Interestingly, in all the
a
Scheme 2. . Template Reaction Involved in the Preparation of the Complexes [AuIIIX2(dtc-Sar-AA-O(t-Bu))]
a
(X/AA ⇒ Br/R1 = R2 = H: Gly (1); Cl/R1 = R2 = H: Gly (2); Br/R1 = R2 = CH3: Aib (3); Cl/R1 = R2 = CH3: Aib (4); Br/R1 = H, R2 = CH2Ph: L-
Phe (5); Cl/R1 = H, R2 = CH2Ph: L-Phe (6)).
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Table 1. Selected IR Frequencies of the Starting Dipeptides P1−P3 and the Corresponding Gold(III)−Dithiocarbamato
Derivatives 1−6
vibrational mode [cm−1
]
+
compound
ν NH
νa/s NH2
ν CO amide I ν N−CSS amide II amide III
νa/s SCS
νa/s SAuS
νa/s XAuX
P1
1
3307
3352
3349
3292
3362
3365
3300
3331
3341
2777/2709
1732
1736
1737
1736
1734
1733
1737
1731
1733
1669
1673
1672
1689
1690
1691
1666
1683
1684
−
1576
1258
1252
1253
1259
1259
1252
1254
1259
1256
−
−
−
a
a
b
−
1568
1561
1006/557
1006/558
−
996/545
996/547
−
418/387
418/384
−
411/383
412/383
−
252/217
c
2
−
2744/2683
−
358/322
P2
3
−
1563
1531
1534
1543
−
b
1560
1563
−
252/223
c
4
−
347/316
P3
5
2775/2704
−
a
a
b
1558
1559
997/562
408/381
252/220
c
6
999/563
408/383
359/326
a
b
c
ν(N−CSS) and amide II overlapped. X = Br. X = Cl.
1
Table 2. H NMR Spectral Data of the Starting Dipeptides P1−P3 (HCl·H-Sar-AA-O(t-Bu), AA = Gly, Aib, L-Phe) and the
Corresponding Gold(III)−Dithiocarbamato Derivatives 1−6 ([AuIIIX2(dtc-Sar-AA-O(t-Bu))], X = Cl, Br; AA = Gly, Aib, L-Phe)
δ(1H) [ppm]
Sar
+
compound
(t-Bu)O
AA
CH3N
CH2N
NH2
AA = Gly
3.82 (CH2)
a
P1
1.41 ((CH3)3C)
1.45 ((CH3)3C)
1.45 ((CH3)3C)
2.53
3.72
4.71/4.75
4.75
8.99
−
8.91 (NH)
b
1
3.95/3.97 (CH2)
7.93 (NH)
3.53/3.57
3.56/3.57
b
2
3.95/3.97 (CH2)
7.92 (NH)
−
AA = Aib
a
P2
1.38 ((CH3)3C)
1.44 ((CH3)3C)
1.44 ((CH3)3C)
1.35 ((CH3)2C)
8.84 (NH)
2.52
3.64
4.63/4.66
4.66
9.05
−
b
3
1.45/1.46 ((CH3)2C)
7.90 (NH)
3.51/3.54
3.54/3.55
b
4
1.45/1.46 ((CH3)2C)
7.88 (NH)
−
AA = L-Phe
a
P3
1.33 ((CH3)3C)
1.43 ((CH3)3C)
1.43 ((CH3)3C)
2.89−3.04 (CH2Ph)
4.40−4.46 (CH)
7.25−7.30 (Ph)
8.92 (NH)
2.44
3.45/3.49
3.49
3.62
4.65/4.70
4.70
8.80
−
b
5
2.98−3.20 (CH2Ph)
4.67−4.74 (CH)
7.25−7.33 (Ph)
7.91 (NH)
b
6
2.98−3.20 (CH2Ph)
4.67−4.74 (CH)
7.25−7.33 (Ph)
7.92 (NH)
−
a
b
DMSO-d6. Acetone-d6.
complexes, the sarcosine residue (C(O)CH2N(CH3)CSS)
bond lengths and angles are reported in Table S2 and Table S3,
respectively, in the Supporting Information.
1
gives rise to two sets of signals (both in H and 13C spectra),
The gold(III) complex adopts a distorted square-planar
geometry achieved through the bidentate dithiocarbamato
ligand and the two cis-bromides. The four donor atoms and
the metal center lie on a plane, and the distortion from the ideal
geometry is a consequence of the small bite angle of the
dithiocarbamato moiety (ca. 75°). The Au−S distances
(2.316(2) and 2.313(3) Å) are approximately 0.1 Å shorter
than those of Au−Br (2.439(2) and 2.440(1) Å). The
dithiocarbamato nitrogen atom is functionalized with two
suggesting the coexistence of two different forms, in agreement
with data previously reported for analogous compounds.22,30
Intriguingly, one interconverts into the other over time, and the
rate of such a conversion strongly depends on the solvent, that
is, it occurs in acetone but not in DMSO (see Discussion).
X-ray Crystal Structure. The X-ray crystal structure of
[AuIIIBr2(dtc-Sar-Aib-O(t-Bu))] (3) is shown in Figure 1. The
corresponding crystallographic data as well as a list of selected
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Table 3. 13C NMR Spectral Data of the Starting Dipeptides P1−P3 (HCl·H-Sar-AA-O(t-Bu), AA = Gly, Aib, L-Phe) and the
Corresponding Gold(III)−Dithiocarbamato Derivatives 1−6 ([AuIIIX2(dtc-Sar-AA-O(t-Bu))], X = Cl, Br; AA = Gly, Aib, L-Phe)
δ(13C) [ppm]
Sar
compound
(t-Bu)O
AA
CH3N
CH2N
CO
CSS
AA = Gly
41.1 (CH2)
a
P1
27.5 ((CH3)3C)
80.8 ((CH3)3C)
28.7 ((CH3)3C)
82.6 ((CH3)3C)
28.7 ((CH3)3C)
82.7 ((CH3)3C)
32.4
48.6
55.1/59.0
55.6
165.6
165.1/165.4
165.1
−
168.4 (CO)
43.1 (CH2)
b
1
40.1/41.1
40.7/41.1
197.7/200.5
195.5/200.6
169.9 (CO)
43.2 (CH2)
b
2
169.8 (CO)
AA = Aib
a
P2
27.3 ((CH3)3C)
79.9 ((CH3)3C)
25.4 ((CH3)2C)
55.8 ((CH3)2C)
172.2 (CO)
25.6 ((CH3)2C)
58.4 ((CH3)2C)
173.7 (CO)
25.5 ((CH3)2C)
58.2 ((CH3)2C)
173.6 (CO)
AA = L-Phe
32.4
48.6
55.3/56.2
55.7
164.2
164.0
163.8
−
b
3
28.6 ((CH3)3C)
81.9 ((CH3)3C)
40.2/41.2
40.6/41.0
196.6/200.3
195.1/200.2
b
4
28.5 ((CH3)3C)
81.9 ((CH3)3C)
a
P3
27.5 ((CH3)3C)
81.1 ((CH3)3C)
36.9 (CH2Ph)
54.3 (CH)
32.8
39.7/40.7
40.5
49.2
54.7/55.6
55.3
165.8
−
126−137 (Ph)
170.2 (CO)
38.5 (CH2Ph)
55.2 (CH)
b
5
28.1 ((CH3)3C)
82.6 ((CH3)3C)
164.1/164.3
164.0/164.1
196.1/199.3
194.5/199.6
128−138 (Ph)
170.8 (CO)
38.3 (CH2Ph)
55.0 (CH)
b
6
27.9 ((CH3)3C)
82.3 ((CH3)3C)
127−138 (Ph)
170.9 (CO)
a
b
DMSO-d6. Acetone-d6.
Figure 1. X-ray crystal structure with atom numbering scheme for
[AuIIIBr2(dtc-Sar-Aib-O(t-Bu))] (3), with thermal ellipsoids drawn at
30% probability.
distinct residues, that is, the N-methyl group (C(2)) and the
dipeptide chain (comprising an amide and a tert-butyl ester
function). The conformation adopted by the latter is such that
the planar amide and the ester groups form an angle of
approximately 74°, and the tert-butyl group is spatially close to
the sarcosine N-methyl moiety. The projection of the crystal
packing along the c crystallographic axis (Figure 2A) reveals the
presence of two distinct zones defined by two types of weak
interactions, the first characterized by the antiparallel super-
imposition of the coordination planes and the second
associated with the hydrophobic interaction between the
peripheral tert-butyl substituents. In addition, couples of
Figure 2. (A) Crystal packing of [AuIIIBr2(dtc-Sar-Aib-O(t-Bu))] (3)
projected along the c crystallographic axis. (B) Depiction of the
hydrogen bonds formed between two symmetry-related molecules
(N(2)−Br(2)′ = 3.791(8) Å).
symmetry-related molecules form two weak hydrogen bonds
between the amide group (N(2)) and one gold(III)-bound
bromide belonging to an adjacent molecule (Br(2)′).
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Table 4. Growth Inhibition of Human Prostate Cancer (PC3 and DU145), Ovarian Adenocarcinoma Cisplatin-Sensitive (2008)
and -Resistant (C13), and Hodgkin’s Lymphoma (L540) Cells by Gold(III)−Dipeptidedithiocarbamato Derivatives 1−6 and
Comparison with That by Reference Drugs Cisplatin and [AuIIIBr2(dtc-Sar-OEt)] (over 72 h)
IC50 [μM]
compound
cisplatin
PC3
DU145
2008
C13
L540
3.3 0.3
1.3 0.1
1.6 0.1
0.8 0.1
1.1 0.1
16.8 1.7
16.5 1.6
0.4 0.1
4.5 0.1
4.5 0.9
2.5 0.3
1.4 0.1
2.2 0.1
13.2 1.2
15.3 1.5
1.1 0.2
19.4 1.2
18.0 1.6
13.2 1.1
4.5 0.2
4.7 0.2
41.2 4.0
43.4 3.8
4.5 0.9
117.2 9.1
11.5 1.2
15.9 1.3
3.7 0.3
5.1 0.4
17.2 1.8
21.5 1.5
2.8 0.4
2.5 0.1
2.1 0.2
3.4 0.2
1.5 0.2
1.7 0.3
16.4 1.5
7.3 0.5
−
1
2
3
4
5
6
a
a
b
b
[AuIIIBr2(dtc-Sar-OEt)]
a
b
Reference 13. Reference 22.
Biological Activity. In Vitro Cytotoxicity. As a
same experimental conditions (except for a weak effect on PC3
cells by complexes 1 and 2).
preliminary screening, the in vitro cytotoxic activity of
complexes 1−6 was investigated toward the human androgen
receptor-negative prostate cancer PC3 and DU145 cells,
ovarian adenocarcinoma 2008 cells and the parent cisplatin-
resistant C13 cell line, and Hodgkin’s lymphoma L540 cells
(Table 4). The inhibition of cell proliferation was evaluated
over 72 h by means of the trypan blue dye exclusion test for the
floating L540 cells, and the BrdU ELISA assay (allowing cell
proliferation to be assessed by quantifying DNA synthesis
during cells activation and proliferation) for adherent prostate
and ovarian cancer cell lines. Results are expressed in terms of
IC50 values and compared to the results from reference drug
cisplatin as well as from the gold(III)−dithiocarbamato
analogue [AuIIIBr2(dtc-Sar-OEt)] previously reported,13,22
tested under the same experimental conditions.
Analogously, compounds 3 and 4 were proved the most
effective toward human ovarian adenocarcinoma cells. Remark-
ably, the induction of apoptosis was shown to be the major
mechanism of cell death on the cisplatin-resistant C13 cell line,
whereas on the corresponding sensitive parent cell line (2008),
a higher percentage of cells underwent late apoptosis/necrosis
(as confirmed by the massive amount of FITC-Annexin-V/PI
stained cells). Again, the other gold(III) complexes, as well as
cisplatin, induced negligible effect on this type of tumor cells.
Finally, the L540 cell line was the most sensitive to the
treatment. In particular, compounds 1−4 were proved to
inhibit cell proliferation mainly by inducing late apoptosis/
necrosis, whereas the effect of complexes 5 and 6 and cisplatin
was negligible.
Exposure of L540 cells to increasing concentrations of
complexes 1−4 resulted in a remarkable inhibition of cell
growth, with IC50 values (1.5−3.4 μM) comparable to that
recorded for cisplatin (2.5 μM). The same gold(III) derivatives
exhibited more potent cytotoxicity on both prostate cancer cell
lines, with IC50 values up to 4- and 3-fold lower than cisplatin in
PC3 and DU145 cells, respectively. The ovarian adenocarcino-
ma 2008 cells appeared to be less sensitive to the treatment
than prostate cancer and lymphoma cells. Nonetheless,
compounds 1−4 induced a massive dose-dependent inhibition
of cell proliferation, in particular complexes 3 and 4, for which
IC50 values of 4.5 and 4.7 μM, respectively, were observed (ca.
four times as active as cisplatin). Conversely, the gold(III)
derivatives 5 and 6 did not exhibit significant cytotoxicity.
When tested on the cisplatin-resistant C13 cell line, the
gold(III) complexes reported here (including 5 and 6) were
proved much more active than the reference drug, the best
performers being complexes 3 and 4, for which IC50 values are
up to 31-fold lower than that for cisplatin.
Induction of Apoptosis. The capability to induce apoptosis
was then tested by incubating the various cancer cell lines with
a single cytotoxic dose (10 μM for PC3/DU145/L540 cells, 30
μM for 2008/C13 cells) of compounds 1−6 or cisplatin for 24
h. With reference to Figure 3, treatment of both PC3 and
DU145 prostate cancer cells with complexes 3 and 4 resulted in
a substantial phosphatidylserine exposure (as evident from the
much higher amount of FITC-Annexin-V stained cells
compared to FITC-Annexin-V/PI stained ones), supporting
apoptosis as the primary mechanism involved in cell growth
inhibition. Conversely, cisplatin and the other gold(III)
derivatives showed modest or even no activity under the
DISCUSSION
■
A major success story during the past 40 years of metal-
lopharmaceuticals relates to cisplatin and its second generation
derivatives carboplatin and oxaliplatin.31 Since the 1970s,
platinum-based chemotherapeutics have had a major impact
as anticancer drugs and are still widely used today, particularly
for genitourinary cancers.1 However, their use is restricted by
severe toxicity as well as by intrinsic or acquired resistance.32
Accordingly, a massive number of other metal compounds has
been extensively investigated over the years, aimed at
overcoming these drawbacks. In this regard, some gold(III)
complexes have shown interesting biological properties and are
believed to operate through mechanisms of action different
from those of platinum drugs in clinical use.33
Recently, the emphasis in cancer drug development has been
shifting from cytotoxic nonspecific chemotherapies to molec-
ularly targeted rationally designed drugs promising greater
effectiveness and less side-effects.34 The “magic bullet” concept
(as first proposed by Ehrlich)35 may be summarized in one
word: targeting. Targeting and delivery are two aspects of drug
design that have been receiving much attention in recent years
because of the potential for improving efficacy and reducing
unwanted side-effects.36 Tumor cells express many biomarkers
and receptors that can be specifically targeted by cytotoxic
agents. Conjugated drugs incorporating a tumor targeting
group and a cytotoxic “smart bomb” are emerging as promising
therapies for cancer treatment. In particular, this approach has
been successfully used for peptide-mediated delivery of metal
complexes into cells.37
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the tripeptide Trp-Trp-Trp.39 Peptides consisting solely of L-
amino acids are preferred over D-analogues, and those
consisting of D-stereoisomers only are not transported.38,40
When xenobiotics are taken into account as possible
substrates (i.e., peptidomimetics), the following essential and
affinity-determining features have to be identified for their
recognition:15
(1) A peptide bond is not required but, when present, it must
be in trans conformation.
(2) The N- and the C-terminus have to be separated by
500−635 pm.
(3) A free N-terminal amino group is preferred for
recognition by PEPT2 (but not required by PEPT1),
whereas the C-terminal carboxylic group is not essential
and can be replaced by a hydrophobic function.
(4) The more hydrophobic the side chain, the higher the
affinity to PEPT2; conversely, charged side chains
provide higher affinity for PEPT1.
Although sharing the same physiological functions and
having similar substrate specificity, PEPT1 and PEPT2
essentially differ in the overall transport characteristics, the
former representing a low affinity but high transport capacity
system whereas the latter is a high affinity but low capacity
transporter.17 Moreover, peptidomimetics can act as either
substrates to be internalized or “competitors” recognized but
not transported inside the cell, just inhibiting further uptake of
other di- and tripeptides.16 For instance, PEPT1 and PEPT2
were shown to have high and medium affinity (in terms of
affinity constant Ki), respectively, toward the antiviral prodrug
valganciclovir, which acts as a substrate to be transported in the
first case but as an inhibitor in the latter.41
So far, peptide symporters PEPT1 and PEPT2 have been
localized in some mammalian tissues/organs, such as small
intestine, kidney, pancreas, bile duct, liver, mammary glands,
and lung.16 On the other hand, cancer cells require larger
amounts of peptide-bound amino acids for growth and
metabolism, and consequently peptide transporters might be
upregulated.42 In this regard, it was recently reported that
PEPTs are ubiquitously overexpressed in various types of
tumor cells but not in the corresponding healthy tissues,43,44
thus representing a suitable specific target for the delivery of
pharmacologically active peptidomimetics.
On account of these assumptions, we have been developing
some gold(III)−dithiocarbamato peptidomimetics that can
combine both the anticancer activity and the favorable
toxicological profile of the gold(III) analogues previously
studied,45 with an improved tumor selectivity by targeting
PEPT1 and/or PEPT2 via the dipeptide chain. The basic idea
relies on the potential recognition of the whole metal complex
that, once recognized and transported by PEPTs and delivered
inside the tumor cell, could exert its anticancer activity without
affecting healthy tissues.
Design and Structure of the Complexes. According to
the objectives outlined above, we designed three dipeptides of
the type HCl·H-Sar-AA-O(t-Bu) (AA = Gly, Aib, L-Phe; P1−
P3) and successfully synthesized the corresponding gold(III)−
dipeptidedithiocarbamato derivatives [AuIIIX2(dtc-Sar-AA-O(t-
Bu))] (X = Cl, Br; 1−6) depicted in Chart 1.
Figure 3. FACS analysis of cells after 24 h incubation at 37 °C with
either complexes 1−6 or cisplatin (10 μM for PC3/DU145/L540
cells, 30 μM for 2008/C13 cells) and double stained with FITC-
Annexin-V and PI.
In this context, the proton-coupled peptide transporters
PEPT1 and PEPT2 are currently under intense investigation.
Owing to their capability to promote the cellular uptake of
physiologically occurring di- and tripeptides, they can internal-
ize into the cells suitable peptidomimetic substrates with
therapeutic properties or precursors of pharmacologically active
agents.15−17 Notwithstanding the exceptionally broad substrate
specificity, these membrane proteins are unable to transport
free amino acids or larger peptides, and some specific
constraints must be fulfilled by substrates to be recognized.
Most of all possible natural di- and tripeptides can be accepted,
excluding those containing N-terminal proline residues38 and
The choice of the dipeptides was not accidental. We first
decided to maintain the sarcosinedithiocarbamato moiety to
resemble our “first generation” gold(III) analogues.22 More-
over, the sarcosine-containing dipeptide Gly-Sar is often used as
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reference substrate for peptide transport studies because of its
good affinity toward both PEPT1 and PEPT2.46 The terminal
amino acids were chosen to evaluate how flexibility (Gly),
rigidity (Aib), chirality and hydrophobicity (L-Phe) could
modulate the biological activity of the various complexes.
Finally, esterification of the C-terminus was proved necessary
because it was previously shown that analogues containing a
free carboxylic function are not active.29 Additionally, although
conferring less water solubility, the bulky tert-butyl group is
unlikely to undergo easy hydrolysis (occurring only in strongly
acidic conditions), thus providing stability to the final
compounds and, consequently, avoiding intramolecular cycliza-
tion of the dipeptide chain.
IR spectroscopy provided insights into both the structural
characterization and the coordination mode of complexes 1−6
(Table 1). Peptide bonds C(O)N(H) give rise to several
vibrational modes that may be regarded as diagnostic for some
structural features. In particular, those referred to as amide I
and amide II show relatively strong infrared absorptions and are
very sensitive to the formation of hydrogen bonds.47 The amide
I mode is mainly assigned to the in-plane peptide carbonyl
stretching and is generally observed between 1620 and 1660
cm−1 for H-bonded carbonyls or at higher frequencies (1660−
1710 cm−1) for “free” CO groups.48 Conversely, the major
contribution to amide II band is given by the in-plane N−H
bending, and its detection in the range 1500−1520 cm−1 is
consistent with “free” N−H groups, whereas the presence of
hydrogen bonds induces a shift toward higher wavenumbers
(1540−1570 cm−1).49 As concerns the starting dipeptides P1−
P3, the position of the amide I bands is consistent with non-
hydrogen-bonded peptide carbonyl groups, and this feature is
maintained in the corresponding gold(III)−dithiocarbamato
derivatives. On the contrary, in all cases the amide N−H groups
seem to be involved in the formation of hydrogen bonds,
although their strength decreases when moving from free
dipeptides to the gold(III) counterparts (as confirmed by the
shift of the amide II band of the complexes toward lower
frequencies). This is also supported by the behavior of the
broad band at ca. 3300 cm−1 assigned to the stretching of trans
peptide N−H groups, which shifts toward slightly higher
wavenumbers in the complexes (compared to the associated
free dipeptides), accounting for less strong hydrogen bonds.50
Bands related to the other groups located in the dipeptide
backbones farther from the metal center seem to be negligibly
affected by the formation of the metal complexes.
This structural hypothesis was confirmed by the X-ray crystal
structure of complex 3 (Figure 1) whose square-planar
geometry around the gold(III) center with two cis-halides is
fully consistent with IR data, as well as the occurrence of
hydrogen bonds involving the peptide amide group (Figure
2B).
NMR Spectroscopy and Solution Chemistry. The
synthesized dipeptides P1−P3 and the complexes 1−6 were
characterized by 1H (Table 2) and 13C (Table 3) NMR
spectroscopy. A general downfield shift of the proton signals is
observed for the dipeptide backbone upon formation of the
corresponding gold(III)−dithiocarbamato derivatives, the
greater variation involving sarcosine N-methyl and N-
methylene groups. On moving away from the NCSS moiety,
the chemical shift change diminishes until being negligible for
the tert-butyl ester group. This trend, in agreement with data
previously reported,22 is also maintained for 13C signals. In this
regard, the most significant carbon-13 peaks are those recorded
at ca. 200 ppm and attributed to the dithiocarbamic carbon
atoms. The δ(N13CSS) values are normally detected in the
range 190−215 ppm, and it is generally assumed that they are
strongly dependent on both the type of the dithiocarbamate−
metal bonding and the oxidation state of the metal center.52 For
high oxidation state transition metal derivatives such as
gold(III), 13C peaks are found at 193−201 ppm.53 There is a
strong empirical correlation between δ(N13CSS) values and the
carbon−nitrogen stretching vibrations in the IR spectra. Higher
ν(N−CSS) frequencies are associated with higher degrees of
carbon−nitrogen double bond character, which correlate well
with lower δ(N13CSS) values owing to lower total π-bond
orders.52 These considerations are consistent with experimental
results reported here for all the compounds 1−6.
The solution chemistry of the gold(III)−dipeptidedithiocar-
bamato complexes was investigated in-depth in order to
1
evaluate their stability. As examples, the H NMR spectra of
complex 1 in acetone-d6 (Figure 4) and in DMSO-d6 (Figure 5)
IR spectra also provide useful information about the metal
coordination. Complexes 1−6 exhibit a characteristic strong
absorption at around 1560 cm−1 (primarily associated with the
“thioureide” band due to the ν(N−CSS)), consistent with
chelating dithiocarbamato ligands coordinated to a gold(III)
metal center.22
The detection of a single band at ca. 1000 cm−1 in all the
gold(III) complexes (commonly attributed to the νa(S−C−S))
is a clear indication for the symmetrical bidentate coordination
of the dithiocarbamato moieties to the metal center, as based
on the Bonati−Ugo criterion.51
New bands, absent in the IR spectra of the free dipeptides
P1−P3, were observed in the range 380−420 cm−1 and
assigned to gold(III)−sulfur stretching vibrations, in agreement
with data previously reported.22 Finally, other informative
bands were recorded at lower wavenumbers (215−360 cm−1),
attributed to the metal−halide stretching modes for terminal
cis-chlorides/bromides.22,26
1
Figure 4. H NMR spectra of complex 1 in acetone-d6 over time.
over time are discussed. First of all, after 17 h, the 1H signal of
the amide proton is not detectable anymore in acetone-d6,
leading to the subsequent simplification of the multiplicity of
the vicinal glycine α-methylene protons. Such an unexpected
experimental evidence is likely to be due to the possible
hydrogen/deuterium exchange with solvent. Peptide amides are
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of N-methyl and N-methylene sarcosine groups. Two forms
have been observed also in DMSO-d6 but, remarkably, there is
no interconversion of one into the other over time (Figure 5),
confirming the crucial role of the solvent in the solution
behavior. The major species in solution is likely to be the
expected gold(III)−dithiocarbamato derivative 1 depicted in
Chart 1, whereas the identification of the second species is still
uncertain, although this behavior has been previously reported
and discussed for some analogues.22 The same results were
obtained for all the other complexes 2−6, no matter what the
dipeptide chain or the halide type (data not shown).
Effects of Gold Complexes on Cancer Cell Prolifer-
ation. Complexes 1−6 were tested for in vitro cytotoxicity
toward human prostate cancer (PC3 and DU145), Hodgkin’s
lymphoma (L540), and ovarian adenocarcinoma cisplatin-
sensitive (2008) and -resistant (C13) cell lines over 72 h. For
comparison purposes, cisplatin was also evaluated as a
reference.
1
Figure 5. H NMR spectra of complex 1 in DMSO-d6 over time.
So far, the peptide transporters PEPT1 and PEPT2 have not
been localized in either the normal genitourinary apparatus or
healthy lymphocytes.16 Therefore, according to our designing
strategy (see above), we first evaluated their expression in the
selected tumor cells by incubation with rabbit polyclonal IgG
PEPT1- or PEPT2-specific antibodies, followed by FITC-
conjugated antirabbit IgG. Remarkably, both peptide trans-
porters were shown to be expressed to a good extent in all five
tumor cell lines (as assessed by FACS analysis, Figure 7), thus
well-known to easily undergo hydrogen exchange reactions
with protic deuterated solvents (e.g., D2O and methanol-d4),54
whereas, to the best of our knowledge, it is not reported for
aprotic deuterated solvents. On the other hand, it is apparent
that such an exchange reaction occurs for complex 1 in acetone-
d6 (Figure 4) and that it is clearly solvent-dependent because it
does not take place in DMSO-d6 (Figure 5). As far as we are
aware, a similar behavior has been reported only once for a
zinc(II) complex coordinated to an amide-like-containing
ligand.55
It is also worth noting that two sets of signals are detected for
N-methyl and N-methylene functions belonging to the
sarcosine residue. With reference to Figure 4, soon after
dissolution of complex 1 in acetone-d6, two 1H signals at 3.53/
3.57 and 4.71/4.75 ppm are observed, for sarcosine NCH3 and
NCH2 groups respectively, at approximately 1:1 ratio.
Intriguingly, the downfield peaks interconvert into their
highfield counterparts over time, progressively decreasing in
intensity and reaching a sort of equilibrium within 85 h (the
final ratio being ca. 1:3). The coexistence of two different forms
of compound 1 is also confirmed by the presence of two signals
for the NCSS carbon atom in the [1H,13C] HMBC spectrum
(Figure 6), each giving rise to long-range couplings with one set
Figure 7. FACS analysis of PEPT1 and PET2 surface expression in
human prostate cancer (PC3 and DU145), ovarian adenocarcinoma
cisplatin-sensitive (2008) and -resistant (C13), and Hodgkin’s
lymphoma (L540) cells after incubation with rabbit polyclonal IgG
PEPT1- or PEPT2-specific antibodies (1:100 dilution), followed by
FITC-conjugated AffiniPure F(ab′)2 fragment donkey antirabbit IgG
(H+L) (1:50 dilution). Dotted lines indicate background fluorescence
of cells, as determined by isotype-matched immunoglobulins or
autofluorescence. X- and Y-axes indicate the logarithms of the relative
intensity of fluorescence and relative cell number, respectively.
Figure 6. [1H,13C] HMBC spectrum of complex 1 in acetone-d6.
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making them suitable for preliminary screening of the
anticancer activity of our gold(III)−dipeptidedithiocarbamato
derivatives.
With reference to Table 4, compounds 5 and 6 were less
active, showing IC50 values higher than cisplatin. Apparently, an
aromatic or too hydrophobic moiety next to sarcosine appears
to be detrimental to the activity of the complexes.
parameter to distinguish by flow cytometry between cells
undergoing either necrosis or late apoptosis.
On account of these remarks, focusing on the most active
complexes 3 and 4, apoptosis was shown to be the major
mechanism of cell death for prostate cancer PC3 and DU145
cells and for ovarian adenocarcinoma cisplatin-resistant C13
cells. Conversely, for the parent cisplatin-sensitive 2008 cells as
well as for the Hodgkin’s lymphoma L540 cell line, the majority
of dead cells appeared to undergo late apoptosis/necrosis over
24 h. As previously mentioned, owing to the fact that these
complexes might exert their cytotoxic activity within the first 24
h or even less (see above), we cannot rule out the hypothesis
that cells first undergo early apoptosis but, as the process goes
on, after 24 h it is not possible to distinguish between late
apoptotic and necrotic cells. The other gold(III) complexes, as
well as cisplatin, were proved less or negligibly effective in
inducing apoptosis except for a weak effect on PC3 and L540
cells by complexes 1 and 2.
The remaining gold(III) complexes were generally more
effective than the reference drug, the derivative 3 turning out to
be the best performer on all the investigated tumor cell lines. In
particular, it was proved about 30-fold more active than
cisplatin in inhibiting cell proliferation of cisplatin-resistant
ovarian adenocarcinoma C13 cells, ruling out the occurrence of
cross-resistance phenomena.
The capability of the most active complexes 3 and 4 to
inhibit cell proliferation was also evaluated after shorter
exposure times (i.e., 24 h) on prostate cancer PC3 and
DU145 cells (data not shown). Remarkably, for both
compounds, IC50 values turned out to be fully comparable to
those here reported after 72 h, thus confirming that they exert
their cytotoxic activity within the first 24 h or even less.
Conversely, as expected, cisplatin was much less active after 24
h exposure, with IC50 values 5- and 3-fold higher toward PC3
and DU145 cells, respectively (compared to data obtained after
72 h).
The evidence that Aib-containing complexes 3 and 4 showed
a higher cytotoxic effect is not surprising. The α-amino-
isobutyric acid is a natural noncoded amino acid and is
abundant in a class of peptide antibiotics (namely, antibiotic
peptaibols) exhibiting antiviral and anticancer activity.56,57 It
was recently suggested that Aib plays a crucial role in the
biological activity of peptaibols because it forces the peptide
backbone to fold into helical arrangements, providing a relevant
capability to cross and/or perturb cell membranes.58 Therefore,
the presence of an Aib residue in compound 3 might increase
its bioavailability and, consequently, the biological activity.
Remarkably, its corresponding IC50 values are comparable to
those reported for the “first generation” analogue [AuIIIBr2(dtc-
Sar-OEt)] toward the same tumor cell lines (Table 4), thus
confirming that the favorable anticancer properties are
preserved notwithstanding the modification of the organic
chain.
All together, these preliminary results confirm that the most
active gold(III)−dipeptidedithiocarbamato derivatives exert
their cancer cell growth-inhibitory activity by exploiting
different mechanisms of action compared to the reference
drug cisplatin and are able to overcome the onset of acquired
resistance.
CONCLUSIONS
■
Killing cancer cells is easy, but achieving this goal without
affecting healthy cells and without impairing the normal
physiological body functions is not trivial. Cancer cells are
well-known to overexpress some specific biomarkers and
receptors needed for carcinogenesis and tumor growth;
therefore, target chemotherapies aim to block cancer cell
proliferation by interfering with such specific upregulated
molecules. In this regard, membrane peptide transporters
PEPT1 and PEPT2 were proved to be upregulated in several
tumor types and, owing to their capability to promote the
cellular uptake of potentially all physiologically occurring di-
and tripeptides, they can also internalize peptide-like chemo-
therapeutics resembling the main structural features of small
peptides (i.e., peptidomimetics). Thus, they represent an
excellent target for the site-specific delivery of pharmacologi-
cally active substrates acting as “Trojan horses”.
Induction of Apoptosis. Although the mechanism of
action of the object gold(III) derivatives is still under
investigation, we tested their capability to inhibit human
tumor cell proliferation by inducing apoptosis using the
Annexin-V/PI double staining assay over 24 h (Figure 3).
One of the earliest features of apoptosis is the translocation of
the plasma membrane phospholipid phosphatidylserine (PS)
from the inner to the outer leaflet of the membrane itself,
thereby exposing PS to the external environment. The
phospholipid binding protein Annexin-V specifically binds to
PS exposed on the cell surface and, once detected by flow
cytometry, it allows the identification of early stage apoptotic
cells. Remarkably, the relative integrity of the membrane of cells
undergoing early apoptosis prevents internalization of PI. On
the other hand, because of extensive membrane damage,
necrotic cells are quickly stained by both Annexin-V and PI,
thus allowing the differentiation between early apoptotic and
necrotic cells. Anyway, a major drawback of this assay is that as
apoptosis progresses and the plasma membrane becomes
compromised (i.e., late apoptotic cells) PI is also internalized.
Therefore, for longer exposure times there is no clear-cut
In order to develop an innovative metal-based target
chemotherapy, in the present work we explore the rational
design of some gold(III)−dipeptidedithiocarbamato peptido-
mimetics of the type [AuIIIX2(dtc-Sar-AA-O(t-Bu))] (X = Cl,
Br; AA = Gly, Aib, L-Phe; 1−6, Chart 1) designed on purpose
to combine the well-known anticancer properties of the
gold(III) metal center and the intrinsic chemoprotective
function of coordinated dithiocarbamates, with an enhanced
bioavailability and tumor selectivity through the dipeptide-
mediated cellular internalization by exploiting membrane
transporters PEPT1 and PEPT2.
Complexes 1−6 were fully characterized both in solid state
and in solution, and the X-ray crystal structure of derivative 3
was obtained and discussed. Preliminary in vitro cytotoxicity
tests were carried out on human prostate (PC3 and DU145),
ovarian adenocarcinoma (2008), and Hodgkin’s lymphoma
(L540) cells, some of them showing promising anticancer
activity with IC50 values up to 5-fold lower than that of the
reference drug cisplatin toward all the investigated cell lines.
When cisplatin-resistant ovarian adenocarcinoma C13 cells
were tested, activity levels comparable to those observed on the
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chromatic reaction as appropriate. Flash column chromatography was
performed on MercK Kiesegel 60 silica gel (40−63 μm, 230−400
mesh) as stationary phase.
Polarimetric Measurements. Optical rotations were measured at
546 nm in spectrophotometric grade methanol at 20 °C using a
Perkin-Elmer 241 polarimeter equipped with a Haake D thermostat
and a cell with an optical pathway of 10 cm.
Elemental Analysis. CHNS elemental analyses were carried out on
either a Fisons EA-1108 CHNS-O or a Perkin-Elmer 2400 CHN
microanalyzer.
IR Spectroscopy. FT-IR spectra were recorded in nujol on a Nicolet
Nexus 870 spectrophotometer (1000 scans, resolution 2 cm−1) for the
range 50−600 cm−1 and in solid KBr on either a Nicolet 55XC or a
Perkin-Elmer 580B spectrophotometer (32 scans, resolution 2 cm−1)
for the range 400−4000 cm−1. Data processing was carried out using
OMNIC version 5.1 (Nicolet Instrument Corporation).
parent sensitive 2008 cells were detected for all the gold(III)
derivatives, thus ruling out the occurrence of cross resistance
with cisplatin (Table 4). Overall, the Aib-containing derivatives
3 and 4 turned out to be the best performers and, remarkably,
apoptosis was the major mechanism involved in the growth
inhibition of PC3, DU145 and cisplatin-resistant C13 cells.
Conversely, they exerted their cytotoxic effect on L540 and
cisplatin-sensitive 2008 cells mainly by inducing late apoptosis/
necrosis over 24 h (Figure 3). All together, these results are
likely to account for a different mechanism of action compared
to that of clinically established platinum drugs.
As a further extension of our previous research on gold(III)−
dithiocarbamato anticancer agents,59 this work aims to develop
“second generation” gold(III) compounds that are able to
maintain both the outstanding antitumor activity and the
favorable nephrotoxicity and acute toxicity levels of their
previous analogues, together with an improved selectivity
toward tumor cells. The real breakthrough is not simply the use
of gold compounds to treat cancer but the rational design of
gold-based drugs which may be very effective, nontoxic, and
potentially selective toward cancer cells, their enormous
potential impact relying on the possible site-specific delivery
in localized cancers, thus strongly improving the cellular uptake
and minimizing unwanted side-effects. As far as we are aware of,
this appears to be the first time that metal-based peptidomi-
metic anticancer agents are designed specifically to target
peptide transporters. Eventually, this idea appears to be an
original and innovative strategy in order to develop novel and
more selective chemotherapeutic agents that could provide a
valuable alternative to the platinum-based anticancer drugs in
clinical use.
NMR Spectroscopy. All NMR spectra were acquired in the
appropriate deuterated solvent at 298 K on a Bruker Avance
DRX300 spectrometer using a BBI [1H,X] probe-head equipped
with z-field gradients. Data processing was carried out using
MestReNova version 6.2 (Mestrelab Research S.L.). Typical
1
acquisition parameters for 1D H NMR spectra (1H: 300.13 MHz):
16 transients, spectral width 7.5 kHz, using 32 k data points and a
delay time of 5.0 s. Spectra were processed using exponential
weighting with a resolution of 0.5 Hz and a line-broadening threshold
of 0.1 Hz. Typical acquisition parameters for 2D [1H,13C] HMBC
NMR spectra (1H: 300.13/13C: 75.48 MHz): 512 transients of 32
scans/block, spectral width 7.5/18.8 kHz, 2k/2k data points and a
1
delay time of 2.0 s. Sequences were optimized for J(13C,1H) = 145
Hz/nJ(13C,1H) = 5 Hz with no 1H decoupling. Spectra were processed
by using sine-square weighting with a resolution of 1.0/3.0 Hz and a
1
line-broadening threshold of 0.3/1.0 Hz. H and 13C chemical shifts
were referenced to TMS at 0.00 ppm via internal referencing to the
residual peak of the deuterated solvent employed.61
Thermogravimetric Analysis. Thermogravimetric (TG) and differ-
ential scanning calorimetry (DSC) curves were recorded with a TA
Instruments thermobalance equipped with a DSC 2929 calorimeter.
Measurements were carried out in the range 25−1000 °C in alumina
crucibles under air (flux rate 30 cm3 min−1) at a heating rate of 5 °C
min−1, using alumina as a reference.
X-ray Crystallography. Single crystal diffraction data were collected
at 293(2) K with a Bruker Smart APEXII diffractometer using Mo Kα
radiation. The unit cell parameters were obtained using 60 ω-frames of
0.5° width and scanned from three different zones of reciprocal lattice.
Intensity data were integrated from several series of exposure frames
(0.3° width) covering the sphere of reciprocal space.62 Absorption
corrections were applied using SADABS,63 which gave minimum and
maximum transmission factors of 0.573 and 1.000, respectively. The
structure was solved by direct methods (SIR2004)64 and refined on F2
with full-matrix least-squares (SHELXL-97),65 using WinGX software
package.66 Non-hydrogen atoms were refined anisotropically, and
hydrogen atoms were added at calculated positions. Graphical material
was prepared with ORTEP-3 for Windows.67
A summary of data collection and structure refinement for complex
3 is reported in the Supporting Information (Table S2). X-ray
crystallographic data have been deposited at the Cambridge Crystallo-
graphic Data Center and allocated the deposition number CCDC
848402, and the corresponding CIF file can be obtained free of charge
from http://www.ccdc.cam.ac.uk.
Syntheses. The purity of all the synthesized compounds was
≥95% as determined by elemental and thermogravimetric analyses.
Synthesis of Dipeptides and Related Intermediates. Z-Sar-OH, Z-
L-Phe-OH, and Z-AA-O(t-Bu) (AA = Gly, L-Phe, Aib) were prepared
according to literature methods (see Supporting Information for
details).18,19
Complexes 1 and 3 are currently under investigation for their
in vivo anticancer activity as well as for the elucidation of their
mechanism of action. Should they really resemble the behavior
of their previous analogues, we are expecting the proteasome to
be a major biological target, and preliminary outcomes seem to
confirm this hypothesis.
These results represent a solid starting point for the
recognition of this class of gold(III) peptidomimetics as
suitable candidates for further pharmacological testing and,
remarkably, allowed us to file an international patent for their
use in cancer chemotherapy (just extended in several countries
worldwide).60
EXPERIMENTAL SECTION
■
Materials. Sarcosine, L-phenylalanine, N-methylmorpholine, palla-
dium catalyst (10% on activated charcoal), TEA (Fluka), isobutyl
chloroformiate (Lancaster), Z-Gly-OH, Z-Aib-OH, DMSO, DMSO-d6,
acetone-d6, carbon disulfide, propidium iodide, trypan blue (Aldrich),
cisplatin (Pharmacia and Upjohn), isobutene (Siad), ZOSu (Iris
Biotech), potassium tetrachloro- and tetrabromoaurate(III) dihydrate
(Alfa Aesar), RPMI medium, penicillin, streptomycin, L-glutamine
(Cambrex Bio Science), and fetal bovine serum (Gibco) were of
reagent grade or comparable purity and were used as supplied. All
other reagents and solvents were used as purchased without any
further purification.
Instrumentation. Melting Points. Melting points were
determined using a Stuart SMP10 apparatus and were not
corrected.
Chromatography. Thin layer chromatography was performed on
silica gel Merck Kieselgel 60F254 precoated glass plates. Retention
factors (Rf) were measured using either chloroform/ethanol 9:1 (Rf1)
or 1-butanol/acetic acid/water 3:1:1 (Rf2) or toluene/ethanol 7:1 (Rf3)
as eluents. Spots were visualized by direct UV irradiation at 254 nm or
developed by exposure to either iodine vapors or NaClO/ninhydrin
General Synthetic Route to tert-Butyl-Esterified Dipeptide
Hydrochlorides. A THF solution (15−20 mL) of Z-Sar-OH is treated
with NMM (1 equiv), cooled to −15 °C and then treated with IBCF
(1 equiv) under stirring.20 After 10 min, a cold suspension of H-AA-
O(t-Bu) (AA = Gly, L-Phe, Aib, 1 eq; obtained by previous catalytic
2222
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Journal of Medicinal Chemistry
Article
hydrogenation in CH2Cl2 of the corresponding Z-protected
derivative)21 in CHCl3 (10−35 mL) is added and the pH adjusted
to ca. 8 with NMM. After being stirred overnight at room temperature,
the reaction mixture is concentrated under reduced pressure, the
residue taken up with AcOEt, washed with 10% KHSO4, H2O, 5%
NaHCO3, H2O, and dried over Na2SO4. The organic layer is then
concentrated to dryness and the product purified by flash
chromatography using AcOEt/petroleum ether 1:1 as eluent, to
obtain the corresponding Z-Sar-AA-O(t-Bu) (yield = 70−90%).
Each Z-Sar-AA-O(t-Bu) intermediate is then Z-deprotected by
catalytic hydrogenation in MeOH, and an ether solution of the
corresponding H-Sar-AA-O(t-Bu) is dropwise treated with 1 equiv of a
dilute ether solution of HCl under stirring. Eventually, the solvent is
evaporated to dryness, the residue taken up with fresh ether and
filtered, and the solvent removed, yielding the corresponding dipeptide
hydrochloride HCl·H-Sar-AA-O(t-Bu) (AA = Gly, L-Phe, Aib).
HCl·H-Sar-Gly-O(t-Bu) (P1). White powder. Yield: 78%. Mp:
129−130 °C. Rf1(CHCl3/EtOH 9:1): 0.05; Rf2(BuOH/AcOH/H2O
3:1:1): 0.50; Rf3(PhMe/EtOH 7:1): 0.05. Anal. Calcd for
C9H19ClN2O3 (MW = 238.71 g mol−1): C, 45.28; H, 8.02; N,
dried under reduced pressure with P2O5, yielding the corresponding
gold derivative [AuIIIX2(dtc-Sar-AA-O(t-Bu))] (X = Cl, Br; AA = Gly,
Aib, L-Phe).
[AuIIIBr2(dtc-Sar-Gly-O(t-Bu))] (1, dibromido[1-(1,1-dimethy-
lethyl) N-dithiocarboxy-κS,κS′)-N-methylglycylglycinato]gold-
(III)). Dark orange powder. Yield: 77%. Mp: decomposes at 153 °C.
Anal. Calcd for C10H17AuBr2N2O3S2 (MW = 634.16 g mol−1): C,
18.94; H, 2.70; N, 4.42; S, 10.11%; found: C, 19.20; H, 2.78; N, 4.42;
S, 10.25%. TG (air): calcd weight loss to Au(0) −68.9%; found
−68.6%. FT-IR (KBr, υmax/cm−1): 3352 (ν, N−H); 1736 (ν, C
̅
Oester); 1673 (amide I); 1568 (ν, N−CSS + amide II); 1252 (amide
III); 1228 (ν, C−O(t-Bu)); 1161 (ν, O−(t-Bu)); 1006 (νa, S−C−S).
FT-IR (nujol, υmax/cm−1): 557 (νs, S−C−S); 418,387 (νa,s, S−Au−S);
̅
1
252,217 (νa,s, Br−Au−Br). H NMR (acetone-d6, 300.13 MHz, TMS,
δ/ppm): 1.45 (s, 9H, (CH3)3C); 3.53/3.57 (s, 3H, CH3N Sar); 3.95/
3.97 (d, 3J = 5.6 Hz, 2H, CH2 Gly); 4.71/4.75 (s, 2H, CH2N Sar); 7.93
(t br, J = 5.5 Hz, 1H, NH Gly). 13C NMR (acetone-d6, 75.48 MHz,
3
TMS, δ/ppm): 28.7 ((CH3)3C); 40.1/41.1 (CH3N Sar); 43.1 (CH2
Gly); 55.1/59.0 (CH2N Sar); 82.6 ((CH3)3C); 165.1/165.4 (CO
Sar); 169.9 (CO Gly); 197.7/200.5 (CSS).
11.74%; found: C, 45.27; H, 8.18; N, 11.52%. FT-IR (KBr, υmax
/
[AuIIICl2(dtc-Sar-Gly-O(t-Bu))] (2, dichlorido[1-(1,1-dimethy-
lethyl) N-dithiocarboxy-κS,κS′)-N-methylglycylglycinato]gold-
(III)). Yellow ochre powder. Yield: 77%. Mp: decomposes at 155 °C.
Anal. Calcd for C10H17AuCl2N2O3S2 (MW = 545.26 g mol−1): C,
22.03; H, 3.14; N, 5.14; S, 11.76%; found: C, 22.00; H, 3.23; N, 5.08;
S, 11.96%. TG (air): calcd weight loss to Au(0) −63.9%; found
̅
+
cm−1): 3307 (ν, N−H); 2777,2709 (νa,s, NH2 ); 1732 (ν, COester);
1669 (amide I); 1576 (amide II); 1258 (amide III); 1242 (ν, C−O(t-
1
Bu)); 1167 (ν, O−(t-Bu)). H NMR (DMSO-d6, 300.13 MHz, TMS,
δ/ppm): 1.41 (s, 9H, (CH3)3C); 2.53 (s, 3H, CH3N Sar); 3.72 (s, 2H,
3
3
CH2N Sar); 3.82 (d, J = 5.8 Hz, 2H, CH2 Gly); 8.91 (t, J = 5.8 Hz,
1H, NH Gly); 8.99 (s br, 2H, NH2+ Sar). 13C NMR (DMSO-d6, 75.48
MHz, TMS, δ/ppm): 27.5 ((CH3)3C); 32.4 (CH3N Sar); 41.1 (CH2
Gly); 48.6 (CH2N Sar); 80.8 ((CH3)3C); 165.6 (CO Sar); 168.4
(CO Gly).
−63.7%. FT-IR (KBr, υmax/cm−1): 3349 (ν, N−H); 1737 (ν, C
̅
Oester); 1672 (amide I); 1561 (ν, N−CSS + amide II); 1253 (amide
III); 1229 (ν, C−O(t-Bu)); 1162 (ν, O−(t-Bu)); 1006 (νa, S−C−S).
FT-IR (nujol, υmax/cm−1): 558 (νs, S−C−S); 418,384 (νa,s, S−Au−S);
̅
1
358,322 (νa,s, Cl−Au−Cl). H NMR (acetone-d6, 300.13 MHz, TMS,
HCl·H-Sar-Aib-O(t-Bu) (P2). White powder. Yield: 84%. Mp:
156−157 °C. Rf1(CHCl3/EtOH 9:1): 0.10; Rf2(BuOH/AcOH/H2O
3:1:1): 0.60; Rf3(PhMe/EtOH 7:1): 0.05. Anal. Calcd for
C11H23ClN2O3 (MW = 266.78 g mol−1): C, 49.53; H, 8.69; N,
δ/ppm): 1.45 (s, 9H, (CH3)3C); 3.56/3.57 (s, 3H, CH3N Sar); 3.95/
3.97 (d, 3J = 5.9 Hz, 2H, CH2 Gly); 4.75 (s br, 2H, CH2N Sar); 7.92 (s
br, 1H, NH Gly). 13C NMR (acetone-d6, 75.48 MHz, TMS, δ/ppm):
28.7 ((CH3)3C); 40.7/41.1 (CH3N Sar); 43.2 (CH2 Gly); 55.6 (CH2N
Sar); 82.7 ((CH3)3C); 165.1 (CO Sar); 169.8 (CO Gly); 195.5/
200.6 (CSS).
10.50%; found: C, 49.53; H, 8.47; N, 10.37%. FT-IR (KBr, υmax
/
̅
+
cm−1): 3292 (ν, N−H); 2744,2683 (νa,s, NH2 ); 1736 (ν, COester);
1689 (amide I); 1563 (amide II); 1259 (amide III); 1247 (ν, C−O(t-
[AuIIIBr2(dtc-Sar-Aib-O(t-Bu))] (3, dibromido[1-(1,1-dimethy-
lethyl) N-dithiocarboxy-κS,κS′)-N-methylglycyl-2-
methylalaninato]gold(III)). Dark orange powder. Yield: 76%. Mp:
decomposes at 167 °C. Anal. Calcd for C12H21AuBr2N2O3S2 (MW =
662.21 g mol−1): C, 21.76; H, 3.20; N, 4.23; S, 9.68%; found: C, 21.73;
H, 3.33; N, 4.34; S, 10.37%. TG (air): calcd weight loss to Au(0)
1
Bu)); 1152 (ν, O−(t-Bu)). H NMR (DMSO-d6, 300.13 MHz, TMS,
δ/ppm): 1.35 (s, 6H, (CH3)2C Aib); 1.38 (s, 9H, (CH3)3C); 2.52 (s,
3H, CH3N Sar); 3.64 (s, 2H, CH2N Sar); 8.84 (s, 1H, NH Aib); 9.05
+
(s br, 2H, NH2 Sar). 13C NMR (DMSO-d6, 75.48 MHz, TMS, δ/
ppm): 25.4 ((CH3)2C Aib); 27.3 ((CH3)3C); 32.4 (CH3N Sar); 48.6
(CH2N Sar); 55.8 ((CH3)2C Aib); 79.9 ((CH3)3C); 164.2 (CO
Sar); 172.2 (CO Aib).
−70.3%; found −70.2%. FT-IR (KBr, υmax/cm−1): 3362 (ν, N−H);
̅
1734 (ν, COester); 1690 (amide I); 1560 (ν, N−CSS); 1531 (amide
HCl·H-Sar-L-Phe-O(t-Bu) (P3). White powder. Yield: 90%. Mp:
80−81 °C. Rf1(CHCl3/EtOH 9:1): 0.20; Rf2(BuOH/AcOH/H2O
II); 1252 (amide III); 1214 (ν, C−O(t-Bu)); 1144 (ν, O−(t-Bu)); 996
(νa, S−C−S). FT-IR (nujol, υmax/cm−1): 545 (νs, S−C−S); 411,383
20
3:1:1): 0.65; Rf3(PhMe/EtOH 7:1): 0.10. [α]546 = +7.8° (c 0.5,
̅
(νa,s, S−Au−S); 252,223 (νa,s, Br−Au−Br). 1H NMR (acetone-d6,
300.13 MHz, TMS, δ/ppm): 1.44 (s, 9H, (CH3)3C); 1.45/1.46 (s, 6H,
(CH3)2C Aib); 3.51/3.54 (s, 3H, CH3N Sar); 4.63/4.66 (s, 2H, CH2N
Sar); 7.90 (s, 1H, NH Aib). 13C NMR (acetone-d6, 75.48 MHz, TMS,
δ/ppm): 25.6 ((CH3)2C Aib); 28.6 ((CH3)3C); 40.2/41.2 (CH3N
Sar); 55.3/56.2 (CH2N Sar); 58.4 ((CH3)2C Aib); 81.9 ((CH3)3C);
164.0 (CO Sar); 173.7 (CO Aib); 196.6/200.3 (CSS).
MeOH). Anal. Calcd for C16H25ClN2O3 (MW = 328.83 g mol−1): C,
58.44; H, 7.66; N, 8.52%; found: C, 58.61; H, 7.78; N, 8.55%. FT-IR
+
(KBr, υmax/cm−1): 3300 (ν, N−H); 2775,2704 (νa,s, NH2 ); 1737 (ν,
̅
COester); 1666 (amide I); 1543 (amide II); 1254 (amide III); 1240
1
(ν, C−O(t-Bu)); 1163 (ν, O−(t-Bu)). H NMR (DMSO-d6, 300.13
MHz, TMS, δ/ppm): 1.33 (s, 9H, (CH3)3C); 2.44 (s, 3H, CH3N Sar);
2.89−3.04 (m, 2H, CH2Ph L-Phe); 3.62 (s, 2H, CH2N Sar); 4.40−4.46
(m, 1H, CH L-Phe); 7.25−7.30 (m, 5H, Ph L-Phe); 8.80 (s br, 2H,
[AuIIICl2(dtc-Sar-Aib-O(t-Bu))] (4, dichlorido[1-(1,1-dimethy-
lethyl) N-dithiocarboxy-κS,κS′)-N-methylglycyl-2-
methylalaninato]gold(III)). Yellow ochre powder. Yield: 81%. Mp:
decomposes at 167 °C. Anal. Calcd for C12H21AuCl2N2O3S2 (MW =
573.31 g mol−1): C, 25.14; H, 3.69; N, 4.89; S, 11.19%; found: C,
25.10; H, 3.84; N, 4.84; S, 11.37%. TG (air): calcd weight loss to
+
3
NH2 Sar); 8.92 (d, J = 7.1 Hz, 1H, NH L-Phe). 13C NMR (DMSO-
d6, 75.48 MHz, TMS, δ/ppm): 27.5 ((CH3)3C); 32.8 (CH3N Sar);
36.9 (CH2Ph L-Phe); 49.2 (CH2N Sar); 54.3 (CH L-Phe); 81.1
((CH3)3C); 126.6 (p-CH L-Phe); 128.3 (m-CH L-Phe); 129.3 (o-CH
L-Phe); 137.0 (CH2-C L-Phe); 165.8 (CO Sar); 170.2 (CO L-
Phe).
Au(0) −65.6%; found −65.4%. FT-IR (KBr, υmax/cm−1): 3365 (ν, N−
̅
H); 1733 (ν, COester); 1691 (amide I); 1563 (ν, N−CSS); 1534
General Synthetic Route to Gold(III)−Dipeptidedithiocarbamato
Complexes. An aqueous solution of HCl·H-Sar-AA-O(t-Bu) (AA =
AA = Gly, L-Phe, Aib) is dropwise treated under stirring with CS2 (1
equiv) and, subsequently, with NaOH (1 equiv). After 2−3 h, pH
turns from 10 to ca. 6 according to the reaction pathway depicted in
Scheme 2, and the solution is slowly added under stirring to an
aqueous solution of K[AuIIIX4] (0.5 equiv; X = Cl, Br), leading to the
sudden precipitation of a solid. The residue is then filtered off, washed
with cold water, purified by recrystallization from acetone/water, and
(amide II); 1252 (amide III); 1214 (ν, C−O(t-Bu)); 1146 (ν, O−(t-
Bu)); 996 (νa, S−C−S). FT-IR (nujol, υmax/cm−1): 547 (νs, S−C−S);
̅
412,383 (νa,s, S−Au−S); 347,316 (νa,s, Cl−Au−Cl). 1H NMR
(acetone-d6, 300.13 MHz, TMS, δ/ppm): 1.44 (s, 9H, (CH3)3C);
1.45/1.46 (s, 6H, (CH3)2C Aib); 3.54/3.55 (s, 3H, CH3N Sar); 4.66
(s, 2H, CH2N Sar); 7.88 (s, 1H, NH Aib). 13C NMR (acetone-d6,
75.48 MHz, TMS, δ/ppm): 25.5 ((CH3)2C Aib); 28.5 ((CH3)3C);
40.6/41.0 (CH3N Sar); 55.7 (CH2N Sar); 58.2 ((CH3)2C Aib); 81.9
2223
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Journal of Medicinal Chemistry
Article
((CH3)3C); 163.8 (CO Sar); 173.6 (CO Aib); 195.1/200.2
(CSS).
Just before the experiments, calculated amounts of drug solutions were
added to RPMI medium and filter sterilized, to a final DMSO amount
lower than 0.5% (v/v). All the tested gold(III) compounds were
proved by 1H NMR to be stable in DMSO over 48 h (Figure S1 in the
Supporting Information).
[AuIIIBr2(dtc-Sar-L-Phe-O(t-Bu))] (5, dibromido[1-(1,1-dime-
thylethyl) N-dithiocarboxy-κS,κS′)-N-methylglycyl-L-
phenylalaninato]gold(III)). Dark orange powder. Yield: 72%. Mp:
decomposes at 124 °C. Anal. Calcd for C17H23AuBr2N2O3S2 (MW =
721.92 g mol−1): C, 28.19; H, 3.20; N, 3.87; S, 8.85%; found: C, 28.14;
H, 3.11; N, 3.87; S, 8.73%. TG (air): calcd weight loss to Au(0)
Cell Proliferation Assay. Amounts of 2.5 × 103 PC3 or DU145
cells, and 4.0 × 103 2008 or C13 cells, were seeded in 96-well flat-
bottomed microplates in RPMI medium (100 μL) and incubated at 37
°C in 5% CO2 atmosphere for 24 h (to allow cell adhesion) before
drug testing. The medium was then removed and replaced with fresh
medium containing the compounds to be tested (previously dissolved
in DMSO, see above) at increasing concentrations (0.3, 0.6, 1.25, 2.5,
5, 10, 20, 40, 80 μM) at 37 °C for 72 h. Each treatment was performed
in triplicate. Cell proliferation for cell lines was assayed using the BrdU
Cell Proliferation ELISA kit (Roche Diagnostics, Germany), according
to the manufacturer’s protocol.70 Alternatively, 2.0 × 105 L540 cells
mL−1 were seeded in 24-well plates and treated as previously
described. After the treatment, the viable cells were evaluated by the
trypan blue dye exclusion test.71 Negative exponential dose−response
curves were obtained by linear regression and IC50 values (i.e., the half
maximal inhibitory concentration representing the concentration of a
substance required for 50% inhibition in vitro) calculated.
−72.7%; found −72.9%. FT-IR (KBr, υmax/cm−1): 3331 (ν, N−H);
̅
1731 (ν, COester); 1683 (amide I); 1558 (ν, N−CSS + amide II);
1259 (amide III); 1214 (ν, C−O(t-Bu)); 1155 (ν, O−(t-Bu)); 997
(νa, S−C−S). FT-IR (nujol, υmax/cm−1): 562 (νs, S−C−S); 408,381
̅
(νa,s, S−Au−S); 252,220 (νa,s, Br−Au−Br). 1H NMR (acetone-d6,
300.13 MHz, TMS, δ/ppm): 1.43 (s, 9H, (CH3)3C); 2.98−3.20 (m,
2H, CH2Ph L-Phe); 3.45/3.49 (s, 3H, CH3N Sar); 4.65/4.70 (s, 2H,
CH2N Sar); 4.67−4.74 (m, 1H, CH L-Phe); 7.25−7.33 (m, 5H, Ph L-
3
Phe); 7.91 (d, J = 7.9 Hz, 1H, NH L-Phe). 13C NMR (acetone-d6,
75.48 MHz, TMS, δ/ppm): 28.1 ((CH3)3C); 38.5 (CH2Ph L-Phe);
39.7/40.7 (CH3N Sar); 54.7/55.6 (CH2N Sar); 55.2 (CH L-Phe); 82.6
((CH3)3C); 127.9 (p-CH L-Phe); 129.4 (m-CH L-Phe); 130.4 (o-CH
L-Phe); 137.7 (CH2-C L-Phe); 164.1/164.3 (CO Sar); 170.8 (CO
L-Phe); 196.1/199.3 (CSS).
[AuIIICl2(dtc-Sar-L-Phe-O(t-Bu))] (6, dichlorido[1-(1,1-dime-
thylethyl) N-dithiocarboxy-κS,κS′)-N-methylglycyl-L-
phenylalaninato]gold(III)). Light brown powder. Yield: 82%. Mp:
decomposes at 138 °C. Anal. Calcd for C17H23AuCl2N2O3S2 (MW =
635.38 g mol−1): C, 32.14; H, 3.65; N, 4.41; S, 10.09%; found: C,
32.21; H, 3.65; N, 4.40; S, 9.95%. TG (air): calcd weight loss to Au(0)
Evaluation of Apoptosis. Amounts of 2.5 × 105 PC3 or DU145 or
2008 or C13 cells, and 2.0 × 105 L540 cells mL−1 were incubated in
six-well Petri dishes with RPMI medium supplemented with 10% FBS
as is (control) or containing 10 μM (30 μM for both cisplatin-sensitive
and -resistant human ovarian adenocarcinoma cells) of either gold
complexes 1−6 or cisplatin at 37 °C for 24 h. Cells were then
harvested and resuspended in 50 μL of binding buffer (10 mM
HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), incubated
with 5 μL of FITC-conjugated Annexin-V (Pharmigen, USA) in the
dark for 15 min, and assayed after the addition of 0.3 mL of binding
buffer and 10 μL of propidium iodide (10 μg mL−1 in binding buffer)
to each sample. Apoptosis induction was assessed by flow cytometry
detection of Annexin-V-labeled cells according to their forward and
right-angle scattering, electronically gated and analyzed on a
FACScalibur flow cytometer (Becton-Dickinson), using CellQuest
software (Becton-Dickinson), as described elsewhere.72
−69.0%; found −69.1%. FT-IR (KBr, υmax/cm−1): 3341 (ν, N−H);
̅
1733 (ν, COester); 1684 (amide I); 1559 (ν, N−CSS + amide II);
1256 (amide III); 1213 (ν, C−O(t-Bu)); 1155 (ν, O−(t-Bu)); 999
(νa, S−C−S). FT-IR (nujol, υmax/cm−1): 563 (νs, S−C−S); 408,383
̅
(νa,s, S−Au−S); 359,326 (νa,s, Cl−Au−Cl). 1H NMR (acetone-d6,
300.13 MHz, TMS, δ/ppm): 1.43 (s, 9H, (CH3)3C); 2.98−3.20 (m,
2H, CH2Ph L-Phe); 3.49 (s, 3H, CH3N Sar); 4.70 (s, 2H, CH2N Sar);
4.67−4.74 (m, 1H, CH L-Phe); 7.25−7.33 (m, 5H, Ph L-Phe); 7.92 (d,
3J = 7.8 Hz, 1H, NH L-Phe). 13C NMR (acetone-d6, 75.48 MHz, TMS,
δ/ppm): 27.9 ((CH3)3C); 38.3 (CH2Ph L-Phe); 40.5 (CH3N Sar);
55.0 (CH L-Phe); 55.3 (CH2N Sar); 82.3 ((CH3)3C); 127.5 (p-CH L-
Phe); 129.2 (m-CH L-Phe); 130.4 (o-CH L-Phe); 137.7 (CH2-C L-
Phe); 164.0/164.1 (CO Sar); 170.9 (CO L-Phe); 194.5/199.6
(CSS).
Statistical Analyses. All values are given as means SD of three
measurements from three independent experiments. Statistical
comparisons were drawn using Student’s t test. Differences were
considered significant where p < 0.05.
Biological Activity. Cell Lines and Culture Conditions.
Hodgkin’s lymphoma (L540) and human androgen-resistant
prostate cancer (PC3 and DU145) cell lines were obtained
from the German Collection of Microorganisms and Cell
Cultures (Braunschweig, Germany). Human ovarian adenocar-
cinoma (2008) cell line was kindly provided by Prof. G.
Marverti (University of Modena, Italy). The parent cisplatin-
resistant subclone (C13) was obtained by weekly treatment of
2008 cells with 1 μM cisplatin.68 All cells were cultured at 37
°C in 5% CO2 and moisture-enriched atmosphere in RPMI
medium supplemented with 10% heat-inactivated FBS, 0.1%
ASSOCIATED CONTENT
* Supporting Information
■
S
Thermal data (Table S1), crystallographic data for complex 3
(Tables S2 and S3), spectroscopic characterization of the
reaction intermediates, and a stability check in DMSO over
time of complex 3 as the model compound (Figure S1). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(w/v) L
-glutamine and antibiotics (0.2 mg mL−1 penicillin and
streptomycin).
AUTHOR INFORMATION
Corresponding Author
*Phone: +39-(0)49-8275159, fax: +39-(0)2-700500560, e-mail:
dolores.fregona@unipd.it.
■
Evaluation of Peptide Transporter Expression. PEPT1 and PEPT2
expression in the human tumor cell lines investigated here (PC3,
DU145, L540, 2008, C13) were incubated with rabbit polyclonal IgG
PEPT1-specific (H-235, 200 μg mL−1) or PEPT2-specific (H-245, 200
μg mL−1) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at a
dilution of 1:100, followed by FITC-conjugated AffiniPure F(ab′)2
fragment donkey antirabbit IgG (H+L) (1.5 mg mL−1, Jackson
ImmunoResearch Europe, UK) at a dilution of 1:50, according to the
manufacturer’s protocol.69 Viable antibody-labeled cells were identified
according to their forward and right-angle scattering, electronically
gated and analyzed on a FACScalibur flow cytometer (Becton-
Dickinson, Franklin Lakes, NJ), using CellQuest software (Becton-
Dickinson).
ACKNOWLEDGMENTS
■
This work was supported by the European Union (Marie Curie
European Re-Integration Grant 204828-PEPMIDAS to L.R.),
Fondazione Cassa di Risparmio di Padova e Rovigo (www.
fondazionecariparo.it), and Fondazione ABO (www.
fondazioneabo.org). D.A. thanks Associazione Italiana per la
Ricerca sul Cancro (AIRC) and Ministero della Salute (Ricerca
Finalizzata FSN-IRCCS) for funding. The authors gratefully
acknowledge Dr. Loris Calore for elemental analyses, Mrs.
Cinzia Borghese and Dr. Naike Casagrande for assistance with
Samples Preparation. Before use, gold(III) complexes 1−6 and
cisplatin were dissolved in DMSO, aliquoted, and stored at −80 °C.
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Article
Anticancer Agents: Activity, Toxicology and Histopathological Studies
in Rodents. Int. J. Cancer 2011, 129, 487−496.
in vitro experiments, and colleagues of Centro Interdiparti-
mentale di Ricerca “Ruolo degli Ioni Metallici nelle Scienze
(15) Rubio-Aliaga, I.; Daniel, H. Peptide Transporters and their
Roles in Physiological Processes and Drug Disposition. Xenobiotica
2008, 38, 1022−1042.
́
Biomediche” (BIOMETION) and of Consorzio Interuniversi-
tario di Ricerca in Chimica dei Metalli nei Sistemi Biologici
(CIRCMSB) for stimulating discussions.
(16) Brandsch, M.; Knutter, I.; Bosse-Doenecke, E. Pharmaceutical
̈
and Pharmacological Importance of Peptide Transporters. J. Pharm.
Pharmacol. 2008, 60, 543−585.
ABBREVIATIONS USED
■
(17) Brandsch, M. Transport of Drugs by Proton-Coupled Peptide
Transporters: Pearls and Pitfalls. Expert Opin. Drug Metab. Toxicol.
2009, 5, 887−905.
AA, amino acid; Aib, α-aminoisobutyric acid (α-methylalanine);
BrdU, bromodeoxyuridine; DMSO, methyl sulfoxide; dpdtc,
dipeptidedithiocarbamate; DSC, differential scanning calorim-
etry; dtc, dithiocarbamate; ELISA, enzyme-linked immunoad-
sorbent assay; FACS, fluorescence-activated cell sorting; FBS,
fetal bovine serum; FITC, fluorescein isothiocyanate; HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HMBC,
heteronuclear multiple bond correlation; IBCF, isobutyl
chloroformiate; IgG, immunoglobulin G; NMM, N-methyl-
morpholine; PEPT, peptide transporter; PI, propidium iodide;
PS, phosphatidylserine; Sar, sarcosine (N-methylglycine); TEA,
triethylamine; TG, thermogravimetry; THF, tetrahydrofuran;
TMS, tetramethylsilane; Z-, benzyloxycarbonyl-; ZOSu, N-
(benzyloxycarbonyloxy)succinimide
(18) Valle, G.; Formaggio, F.; Crisma, M.; Bonora, G. M.; Toniolo,
C.; Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.
Linear Oligopeptides. Part 147. Chemical and Crystallographic Study
of the Reaction between Benzyloxycarbonyl Chloride and α-Amino-
isobutyric Acid. J. Chem. Soc., Perkin Trans. 2 1986, 1371−1376.
(19) McGahren, W. J.; Goodman, M. Synthesis of Peptide
Oxazolones and Related Compounds. Tetrahedron 1967, 23, 2017−
2030.
(20) Vaughan, J. R. Jr.; Osato, R. L. Preparation of Peptides Using
Mixed Carboxylic Acid Anhydrides. J. Am. Chem. Soc. 1951, 73, 5553−
5555.
(21) Green, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis; Wiley-Interscience: New York, 1999; pp 736−739.
(22) Ronconi, L.; Giovagnini, L.; Marzano, C.; Bettìo, F.; Graziani,
R.; Pilloni, G.; Fregona, D. Gold Dithiocarbamate Derivatives as
Potential Antineoplastic Agents: Design, Spectroscopic Properties, and
in Vitro Antitumor Activity. Inorg. Chem. 2005, 44, 1867−1881.
(23) Shkaraputa, L. N.; Kononov, A. V.; Polackov, A. D. Preparation
of Dithiocarbamates. Ukr. Chem. J. 1991, 9, 979−989.
(24) Vasiliev, A. N.; Polackov, A. D. Synthesis of Potassium (1,1-
Dioxothiolan-3-yl)-dithiocarbamate. Molecules 2000, 5, 1014−1017.
(25) Colthup, N. B.; Daly, L. H.; Wiberley, S. E. Introduction to
Infrared and Raman Spectroscopy; Academic Press: New York, 1990.
(26) Forghieri, F.; Preti, C.; Tassi, L.; Tosi, G. Preparation,
Properties and Reactivity of Gold Complexes with some Heterocyclic
Dithiocarbamates as Ligands. Polyhedron 1988, 7, 1231−1237.
(27) Beurskens, P. T.; Blaauw, H. J. A.; Cras, J. A.; Steggerda, J. J.
Preparation, Structure, and Properties of Bis(N,N-
dibutyldithiocarbamato)gold(III) Dihaloaurate(I). Inorg. Chem. 1968,
7, 805−810.
(28) Coates, G. E.; Parkin, C. Tertiary Phosphine Complexes of
Trimethylgold: Infrared Spectra of Complexes of Gold and some
Other Metals. J. Chem. Soc. 1963, 421−429.
(29) Ronconi, L.; Maccato, C.; Barreca, D.; Saini, R.; Zancato, M.;
Fregona, D. Gold(III) Dithiocarbamate Derivatives of N-methylgly-
cine: an Experimental and Theoretical Investigation. Polyhedron 2005,
24, 521−531.
(30) Giovagnini, L.; Ronconi, L.; Aldinucci, D.; Lorenzon, D.; Sitran,
S.; Fregona, D. Synthesis, Characterization and Comparative In Vitro
Cytotoxicity Studies of Platinum(II), Palladium(II) and Gold(III)
Methylsarcosinedithiocarbamate Complexes. J. Med. Chem. 2005, 48,
1588−1595.
REFERENCES
■
(1) Kelland, L. The Resurgence of Platinum-Based Cancer
Chemotherapy. Nat. Rev. Cancer 2007, 7, 573−584.
(2) Kostova, I. Platinum Complexes as Anticancer Agents. Recent Pat.
Anti-Cancer Drug Discovery 2006, 1, 1−22.
(3) Sun, R. W. -Y; Ma, D. -L.; Wong, E. L. -M; Che, C. -M. Some
Uses of Transition Metal Complexes as Anti-Cancer and Anti-HIV
Agents. Dalton Trans. 2007, 4884−4892.
(4) Bruijnincx, P. C. A.; Sadler, P. J. New Trends for Metal
Complexes with Anticancer Activity. Curr. Opin. Chem. Biol. 2008, 12,
197−206.
(5) Nobili, S.; Mini, E.; Landini, I.; Gabbiani, C.; Casini, A.; Messori,
L. Gold Compounds as Anticancer Agents: Chemistry, Cellular
Pharmacology, and Preclinical Studies. Med. Chem. Res. 2010, 30,
550−580.
(6) Ott, I. On the Medicinal Chemistry of Gold Complexes as
Anticancer Drugs. Coord. Chem. Rev. 2009, 253, 1670−1681.
(7) Tiekink, E. R. T. Anti-Cancer Potential of Gold Complexes.
Inflammopharmacology 2008, 16, 138−142.
(8) Shimada, H.; Takahashi, K.; Funakoshi, T.; Kojima, S. Protective
Effects of Dithiocarbamates Against Toxicity of cis-Diamminedichlor-
oplatinum in Mice. Biol. Pharm. Bull. 1993, 16, 368−371.
(9) Ronconi, L.; Fregona, D. The Midas Touch in Cancer
Chemotherapy: from Platinum- to Gold-Dithiocarbamato Complexes.
Dalton Trans. 2009, 10670−10680.
(10) Ronconi, L.; Aldinucci, D.; Dou, Q. P.; Fregona, D. Latest
Insights into the Anticancer Activity of Gold(III)-Dithiocarbamato
Complexes. Anti-Cancer Agents Med. Chem. 2010, 10, 283−292.
(11) Milacic, V.; Chen, D.; Ronconi, L.; Landis-Piwowar, K. R.;
Fregona, D.; Dou, Q. P. A Novel Anticancer Gold(III) Dithiocarba-
mate Compound Inhibits the Activity of a Purified 20S Proteasome
and 26S Proteasome in Human Breast Cancer Cell Cultures and
Xenografts. Cancer Res. 2006, 66, 10478−10486.
(31) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. The Status of
Platinum Anticancer Drugs in the Clinic and in Clinical Trials. Dalton
Trans. 2010, 39, 8113−8127.
(32) Boulikas, T. Clinical Overview on Lipoplatin: a Successful
Liposomal Formulation of Cisplatin. Expert Opin. Invest. Drugs 2009,
18, 1197−1218.
(12) Zhang, X.; Frezza, M.; Milacic, V.; Ronconi, L.; Fan, Y.; Bi, C.;
Fregona, D.; Dou, Q. P. Inhibition of Tumor Proteasome Activity by
Gold Dithiocarbamato Complexes via both Redox-Dependent and
-Independent Processes. J. Cell. Biochem. 2010, 109, 162−172.
(13) Cattaruzza, L.; Fregona, D.; Mongiat, M.; Ronconi, L.; Fassina,
A.; Colombatti, A.; Aldinucci, D. Antitumor Activity of Gold(III)-
Dithiocarbamato Derivatives on Prostate Cancer Cells and Xenografts.
Int. J. Cancer 2011, 128, 206−215.
(33) Berners-Price, S. J; Filipovska, A. Gold Compounds as
Therapeutic Agents for Human Diseases. Metallomics 2011, 3, 863−
873.
(34) de Bono, J. S.; Ashworth, A. Translating Cancer Research into
Targeted Therapeutics. Nature 2010, 467, 543−549.
(35) Strebhardt, K.; Ulrich, A. Paul Herlich’s Magic Bullet Concept:
100 Years of Progress. Nat. Rev. Cancer 2008, 8, 473−480.
(36) Harper, B. W.; Krauser-Heuer, A. M.; Grant, M. P.; Manohar,
M.; Garbutcheon-Singh, K. B.; Aldrich-Wright, J. R. Advances in
Platinum Chemotherapeutics. Chem.Eur. J. 2010, 16, 7064−7077.
(14) Marzano, C.; Ronconi, L.; Chiara, F.; Giron, M. C.; Faustinelli,
I.; Cristofori, P.; Trevisan, A.; Fregona, D. Gold(III)-Dithiocarbamato
2225
dx.doi.org/10.1021/jm201480u | J. Med. Chem. 2012, 55, 2212−2226

Journal of Medicinal Chemistry
Article
(37) Noor, F.; Wustholz, A.; Kinscherf, R.; Metzler-Nolte, N. A
(56) Brown, K. L.; Hancock, R. E. W. Cationic Host Defense
(Antimicrobial) Peptides. Curr. Opin. Immunol. 2006, 18, 24−30.
(57) Johnstone, S. A.; Gelmon, K.; Mayer, L. D.; Hancock, R. E.;
Bally, M. B. In Vitro Characterization of the Anticancer Activity of
Membrane-Active Cationic Peptides. I. Peptide-Mediated Cytotoxicity
and Peptide-Enhanced Cytotoxic Activity of Doxorubicin against Wild-
Type and p-Glycoprotein Overexpressing Tumor Cell Lines. Anti-
Cancer Drug Des. 2000, 15, 151−160.
̈
Cobaltocenium-Peptide Bioconjugate Shows Enhanced Cellular
Uptake and Directed Nuclear Delivery. Angew. Chem., Int. Ed. 2005,
44, 2429−2432.
(38) Biegel, A.; Knutter, I.; Hartrodt, B.; Gebauer, S.; Theis, S.;
̈
Luckner, P.; Kottra, G.; Rastetter, M.; Zebisch, K.; Thondorf, I.;
Daniel, H.; Neubert, K.; Brandsch, M. The Renal Type H+/Peptide
Symporter PEPT2: Structure-Affinity Relationships. Amino Acids 2006,
31, 137−156.
(58) Toniolo, C.; Bruckner, H. Peptaibiotics; Wiley-VCD/VHCA:
̈
Weinheim/Zurich, 2009.
̈
(39) Biegel, A.; Gebauer, S.; Hartrodt, B.; Brandsch, M.; Neubert, K.;
Thondorf, I. Three-Dimensional Quantitative Structure−Activity
Relationship Analyses of β-Lactam Antibiotics and Tripeptides as
Substrates of the Mammalian H+/Peptide Cotransporter PEPT1. J.
Med. Chem. 2005, 48, 4410−4419.
(40) Nielsen, C. U.; Andersen, R.; Brodin, B.; Frokjaer, S.; Taub, M.
E.; Steffansen, B. Dipeptide Model Prodrugs for the Intestinal
Oligopeptide Transporter. Affinity for and Transport via hPepT1 in
the Human Intestinal Caco-2 Cell Line. J. Controlled Release 2001, 76,
129−138.
(41) Sugawara, M.; Huang, W.; Fei, Y. J.; Leibach, F. H.; Ganapathy,
V.; Ganapathy, M. E. Transport of Valganciclovir, a Ganciclovir
Prodrug, via Peptide Transporters PEPT1 and PEPT2. J. Pharm. Sci.
2000, 89, 781−789.
(59) Fregona, D.; Ronconi, L.; Marzano, C. Dithiocarbamate
Complexes of Gold(III) and their Use as Antitumor Agents. Ital.
Pat. IT1347835B1, 2008.
(60) Fregona, D.; Ronconi, L.; Formaggio, F.; Dou, Q. P.; Aldinucci,
D. Gold(III) Complexes with Oligopeptides Functionalized with
Sulfur Donors and Use thereof as Antitumor Agents. PCT Int. Appl.
WO2010105691A1, 2010.
(61) Fulmer, G. R.; Miller, A. J. M.; Sherden, N. H.; Gottlieb, H. E.;
Nudelman, A.; Stoltz, B. M.; Bercaw, J. E.; Goldberg, K. I. NMR
Chemical Shifts of Trace Impurities: Common Laboratory Solvents,
Organics, and Gases in Deuterated Solvents Relevant to the
Organometallic Chemist. Organometallics 2010, 29, 2176−2179.
(62) SMART (Control) and SAINT (Integration) Software for CCD
Systems; Bruker AXS Inc., Madison, WI, 1994.
(42) Rubio-Aliaga, I.; Daniel, H. Mammalian Peptide Transporters as
Targets for Drug Delivery. Trends Pharmacol. Sci. 2002, 23, 434−440.
(43) Mitsuoka, K.; Miyoshi, S.; Kato, Y.; Murakami, Y.; Utsumi, R.;
Kubo, Y.; Noda, A.; Nakamura, Y.; Nishimura, S.; Tsuji, A. Cancer
Detection Using a PET Tracer, 11C-Glycylsarcosine, Targeted to H+/
Peptide Transporter. J. Nucl. Med. 2008, 49, 615−622.
(44) Landowski, C. P.; Vig, B. S.; Song, X.; Amidon, G. L. Targeted
Delivery to PEPT1-Overexpressing Cells: Acidic, Basic, and Secondary
Floxuridine Amino Acid Ester Prodrugs. Mol. Cancer Ther. 2005, 4,
659−667.
(63) Area-Detector Absorption Correction; Siemens Industrial
Automation Inc.:, Madison, WI, 1996.
(64) Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.;
Cascarano, G. L.; De Caro, L.; Giacovazzo, C.; Polidori, G.; Spagna, R.
SIR2004: an Improved Tool for Crystal Structure Determination and
Refinement. J. Appl. Crystallogr. 2005, 38, 381−388.
(65) Sheldrick, G. M. SHELX-97. Programs for Crystal Structure
Analysis (Release 97-2); University of Gottingen, Gottingen, Germany,
̈
̈
1997.
(66) Farrugia, L. J. WinGX Suite for Small-Molecule Single-Crystal
Crystallography. J. Appl. Crystallogr. 1999, 32, 837−838.
(67) Farrugia, L. J. ORTEP-3 for Windows - A Version of ORTEP-III
with a Graphical User Interface (GUI). J. Appl. Crystallogr. 1997, 30,
565−568.
(45) Aldinucci, D.; Ronconi, L.; Fregona, D. Groundbreaking
Gold(III) Anticancer Agents. Drug Discovery Today 2009, 14, 1075−
1076.
(46) Geissler, S.; Zwarg, M.; Knutter, I.; Markwardt, F.; Brandsch, M.
̈
The Bioactive Dipeptide Anserine is Transported by Human Proto-
Coupled Peptide Transporters. FEBS J. 2010, 277, 790−795.
(47) Picquart, M.; Abedinzadeh, Z.; Grajcar, L.; Baron, M. H.
Spectroscopic Study of N-Acetylcysteine and N-Acetylcystine/Hydro-
gen Peroxide Complexation. Chem. Phys. 1998, 228, 279−291.
(68) Andrews, P. A.; Murphy, M. P.; Howell, S. B. Differential
Potentiation of Alkylating and Platinating Agent Cytotoxicity in
Human Ovarian Carcinoma Cells by Glutathione Depletion. Cancer
Res. 1985, 45, 6250−6253.
(69) Santa Cruz Biotechnology, http://www.scbt.com/protocols.
html.
́
(48) Fillaux, F.; De Loze, C. Spectroscopic Study of Monosubstituted
(70) Profit, S.; Unteregger, G. Quantitative Measurement of Cell
Proliferation Using the BrdU ELISA: a Comparison between
Colorimetric and Chemiluminescent Detection. Biochemica 2001, 4,
33−35.
(71) Stroben, W. Trypan Blue Exclusion Test of Cell Viability. In
Current Protocols in Immunology; Coligan, J. E., Bierer, B., Margulies, D.
H., Shevach, E. M., Strober, W., Coico, R., Eds.; John Wiley & Sons:
New York, 2001; appendix 3B.
(72) Aldinucci, D.; Lorenzon, D.; Stefani, L.; Giovagnini, L.;
Colombatti, A.; Fregona, D. Antiproliferative and Apoptotic Effects
of Two New Gold(III) Methylsarcosinedithiocarbamate Derivatives
on Human Acute Myeloid Leukemia Cells In Vitro. Anti-Cancer Drugs
2007, 18, 323−332.
Amides. II. Rotation Isomers in Amides Substituted by Aliphatic Side-
Chain Models. Biopolymers 1972, 11, 2063−2077.
́
(49) Fillaux, F.; De Loze, C. Structure of Matrix Isolated N-
Methylacetamide. Chem. Phys. Lett. 1976, 39, 547−551.
(50) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G.
Infrared and Raman Characteristic Frequencies of Organic Molecules;
Academic Press: London, 1991; pp 144−145.
(51) Bonati, F.; Ugo, R. Organotin(IV) N,N-Disubstituted
Dithiocarbamates. J. Organomet. Chem. 1967, 10, 257−268.
(52) van Gaal, H. L. M.; Diesveld, J. W.; Pijpers, F. W.; van der
Linden, J. G. M. 13C NMR Spectra of Dithiocarbamates. Chemical
Shifts, Carbon-Nitrogen Stretching Vibration. Inorg. Chem. 1979, 18,
3251−3260.
(53) Cras, J. A.; Willemse, J. Dithiocarbamates and Related Ligands.
In Comprehensive Coordination Chemistry. The Synthesis, Reactions,
Properties and Applications of Coordination Compounds; Wilkinson, G.;
Gillard, R. D.; McCleverty, J. A., Eds.; Pergamon Press: Oxford, 1987;
pp 579−593.
(54) Dempsey, C. E. Hydrogen Exchange in Peptides and Proteins
Using NMR Spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 2001,
39, 135−170.
(55) Bakir, M.; Conry, R. R.; Green, O. X-Ray Crystallographic and
Molecular Sensing Properties of [ZnCl2(κ2-N,N’-dpknph)] (dpknph =
di-2-pyridylketone-p-nitrophenyl hydrazone). J. Mol. Struct. 2008, 889,
35−44.
2226
dx.doi.org/10.1021/jm201480u | J. Med. Chem. 2012, 55, 2212−2226