Bioactive gold(i) complexes with 4-mercaptoproline derivatives

Article abstract of DOI:10.1039/c6dt02000c

Unprecedented gold(i) bioconjugates bearing non-proteinogenic amino acid 4-mercaptoproline species as bioorganic ligands have been prepared. Firstly, the synthesis of Boc-Pro(SH)-OMe (1) has been accomplished by standard procedures. The subsequent reaction of 1 with [AuCl(PR₃)] gives complexes Boc-Pro(SAuPR₃)-OMe (PR₃ = PPh₃ (2), PPh₂Py (3)). Starting from complex 2 several structural modifications have been performed, in addition to the incorporation of a different phosphine in 3, such as the formation of the acid Boc-Pro(SAuPPh₃)-OH (4), the synthesis of a dipeptide derivative by coupling the amino acid glycine tert-butyl ester Boc-Pro(SAuPPh₃)-Gly-O^tBu (5), or the coordination of another gold phosphine fragment to the sulfur atom as in [Boc-Pro(SAuPPh₃)₂-OMe]OTf (6). The cytotoxic activity in vitro of these complexes has been evaluated against three different tumor human cell lines, A549 (lung carcinoma), Jurkat (T-cell leukaemia) and MiaPaca2 (pancreatic carcinoma). All the complexes displayed excellent cytotoxic activity with IC₅₀ values in the low μM range and even in the nM range in some cases. Structure-Activity Relationships (SAR) observed from this family of complexes opens the possibility of designing more potent and selective promising gold(i) anticancer agents.

Full text of DOI:10.1039/c6dt02000c

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Bioactive Gold(I) Complexes with 4-Mercaptoproline Derivatives
Alejandro Gutiérrez,a Carlos Cativiela,b Antonio Laguna,a M. Concepción Gimeno,*,a
Received 00th January 20xx,
Accepted 00th January 20xx
Unprecedented gold(I) bioconjugates bearing non-proteinogenic amino acid 4-mercaptoproline species as bioorganic
ligands have been prepared. Firstly, the synthesis of Boc-Pro(SH)-OMe (1) has been accomplished by standard procedures.
The subsequent reaction of 1 with [AuCl(PR3)] gives complexes Boc-Pro(SAuPR3)-OMe (PR3 = PPh3 (2), PPh2Py (3)). Starting
from complex 2 several structural modifications have been performed, in addition to the incorporation of a different
phosphine in 3, such as the formation of the acid Boc-Pro(SAuPPh3)-OH (4), the synthesis of a dipeptide derivative by
coupling the amino acid glycine tert-butyl ester Boc-Pro(SAuPPh3)-Gly-OtBu (5), or the coordination of another gold
phosphine fragment to the sulfur atom as in [Boc-Pro(SAuPPh3)2-OMe]OTf (6). The cytotoxic activity in vitro of these
complexes has been evaluated against three different tumor human cell lines, A549 (lung carcinoma), Jurkat (T-cell
leukaemia) and MiaPaca2 (pancreatic carcinoma). All the complexes displayed excellent cytotoxic activity with IC50 values
in the low ꢀM range and even in the nM range in some cases. Structure-Activity Relationships (SAR) observed from this
family of complexes opens the possibility to design more potent and selective promising gold(I) anticancer agents.
DOI: 10.1039/x0xx00000x
www.rsc.org/
thiolates (auranofin analogues),5 phosphines,6 carbenes,7
among others, have been reported. Mechanistic studies
showed that these compounds exert their therapeutic effect
over different targets than cisplatin,8 thus opening up the way
to the synthesis of new drugs, overcoming the aforementioned
limitations.
Introduction
Metal-based drugs are currently successfully used in the
chemotherapeutic treatment of certain types of cancer. For
instance, cisplatin and other platinum-based drugs are
clinically used in the treatment of testicular and ovarian cancer
with a high therapeutic success,1 meanwhile some ruthenium
complexes are under clinical trials.2 However, for platinum
based drugs the effectiveness is still hindered by several
clinical problems, such as a limited spectrum of activity,
development of resistance and undesired toxicity,3 which
makes highly desirable the research of new metal complexes
with better pharmacological profile, such as activity, selectivity
and low side effects.
In this context, amino acids and peptides are two important
classes of biomolecules that form proteins and enzymes in the
organism and display a wide range of biological activities.9
They could also be employed as useful biocompatible ligands
to deliver the gold(I) atom to its target. Moreover, due to the
fact that tumor cells over-express amino acid receptors and
have more requirements of nutrients, complexes with amino
acids could be more selective to abnormal cells.10
Among all the proteinogenic amino acids, proline is the only
one possessing a cyclic pyrrolidine moiety in its structure. This
important fact determines the properties, structure, function
and biological activities of the peptides and proteins that
incorporate this fragment. An important proline derivative is 4-
hydroxyproline which is found in the organism forming
collagen, conjunctive and bone issue. The introduction of
different functional groups at the 4 position has led to the
amine, chloride or fluoride derivatives. The replacement of the
hydroxide with the thiol group originates the non-
proteinogenic amino acid 4-mercaptoproline, which can be
considered as a hybrid of proline and homocysteine (Figure 1).
This unusual amino acid puts together the properties of both
amino acids that are the nucleophilic character and reducing
ability of the thiol group and the conformational restrictions
corresponding to proline. The employment of non-
proteinogenic amino acids in the design of new compounds
In this context, gold complexes occupy a relevant position and
have been postulated as an interesting class of anticancer
agents. Gold compounds have been traditionally used in
medicine, and some gold(I) drugs, like auranofin, are being
employed clinically in the treatment of rheumatoid arthritis
since last century. However, in the last decades gold
complexes have attracted great attention because of their
excellent anticancer properties in vitro and in vivo, superior in
many cases to cisplatin.4 Consequently, a large number of
gold(I) complexes bearing several types of ligands such as
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1
with better pharmacological properties has been extensively leaving group.16c,17 The H NMR spectrum of 1c shows a new
used. For instance, incorporation of conformationally resonance as a singlet at δ = 3.05 ppm corresponding to the
restricted amino acids (cyclic or quaternary amino acids) often introduced methanesulfonate. This intermediate was reacted
leads to the preparation of compounds with better hydrolytic immediately with potassium thioacetate according to
resistance or receptor recognition.11
previously described conditions yielding the thioester
derivative 1d 16
Note that displacement of the
.
methanesulfonate by the thioacetate group is accompanied by
inversion of configuration, which is characteristic of a SN2
reaction type. The 1H NMR spectrum clearly shows the
disappearance of the signals belonging to the
methanesulfonate and the appearance of a new singlet
Fig 1. Structures of proline (a), 4-hydroxyproline (b), and 4-mercaptoproline (c).
resonance at
The 4-mercaptoproline ester
of the thioester 1d. As an important feature, the band of the
δ
= 2.32 ppm corresponding to the thioacetate.
1
was obtained by methanolysis
The synthesis of the first 4-mercaptoproline derivatives dates
back to 1970,12 and although some of the them showed
interesting biological activities,13 to the best of our knowledge
only a nickel complex has been reported with a proline
derivative substituted in 4 position with a sulfur fragment, but
in any case the ligand is coordinated to the metal as thiolate.14
Our research has been focusing on the synthesis of gold(I)
derivatives with biologically-relevant ligands, and we have
previously reported on the preparation of some gold(I)
complexes with amino acids and peptides with interesting
biological activities.15
thiol group can be observed as a doublet at
the 1H NMR spectrum. The IR spectrum also shows the
absorption corresponding to the thiol group at 2554 cm-1.
δ = 1.76 ppm in
Here we describe the synthesis of unprecedented gold(I)
bioconjugates with N-protected 4-mercaptoproline ester
derivatives. Several structural modifications, such as change of
the type of phosphine ligand or change in the number of
gold(I) atoms per molecule have been performed in order to
establish some Structure-Activity Relationships (SAR) for this
class of compounds. The cytotoxicity of the new complexes has
been studied in vitro against three different human tumor cell
lines. All the complexes exhibit strong cytotoxicity with IC50
Scheme 1. Synthesis of the N-protected 4-mercaptoproline ester compound 1.
values in the ꢀM range, even in the nM in some cases.
This modified thio-proline molecule can be coordinated to a
gold(I) centre by reaction with [AuCl(PPh3)] in the presence of
an excess of K2CO3 to give the desired complex
2). The 1H NMR spectrum of
2 (see Scheme
Results and Discussion
2
shows all the expected
resonances for the thioaminoester and triphenylphosphine
ligands. Remarkably, the complex appears as a mixture of
rotamers in a 1:0.3 ratio. The resonance belonging to the thiol
disappears after complex formation, whereas the proton
corresponding to Cγ appears strongly downfield shifted
(around 0.5 ppm) compared to the starting thiol derivative.
The protons of both methylene groups corresponding to
carbons Cβ and Cδ are diastereotopic, and appear at different
chemical shifts due to the cyclic structure and the presence of
two chiral centres in the molecule. In the 13C{1H} NMR
spectrum all the signals belonging to the pyrrolidine ring
Synthesis and Characterisation
The synthesis of the N-protected 4-mercaptoproline ester
species
1, used as starting material to prepare the gold
complexes, was achieved following the synthetic route showed
in Scheme 1. Firstly, the suitable 4-mercaptoproline protected
ligand
1 was prepared starting from commercially available 4-
hydroxyproline aminoester chlorohydrate 1a in several steps.
The introduction of the amino Boc (tert-butyloxycarbonyl)
protecting group was performed according to standard
methods using Boc2O in the presence of DIPEA (N,N-
diisopropilethylamine),16 and the success of the reaction was
confirmed by the appearance of a signal in the 1H NMR
appear strongly downfield shifted
coordination to gold(I) except in the case of Cγ. The 31P{1H}
NMR spectrum shows a singlet at = 36.5 ppm characteristic
(ꢁδ ≈ 5 ppm) after
δ
spectrum of compound 1a at
δ = 1.41 ppm corresponding to
of gold(I) complexes with thiolate and phosphine as ligands.
The IR and MS data also confirms the expected stoichiometry.
All the signals could be correctly assigned performing 2D NMR
experiments.
the alkyl protons of the protecting group. The IR spectrum also
shows the disappearance of the absorption band belonging to
the amine, whereas the new carbamate absorption is observed
at 1672 cm-1. The hydroxyl group of compound 1b can be
derivatised to a methanesulfonate which is an excellent
In order to evaluate the effect of the phosphine ligand in the
final cytotoxicity of the complex, we have replaced
2 | J. Name., 2012, 00, 1-3
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triphenylphosphine with 2-pyridyl diphenylphosphine. spectrum clearly shows the disappearance of the ester band
Complex
3 was prepared in a similar way to that described and the appearance of the bands corresponding to the
. In this case, [AuCl(PPh2Py)] was carboxylic acid at 3300-3100 and 1694 cm-1. The HRMS(ESI+)
previously for complex
2
employed as starting gold(I) complex. The H and 13C{1H} NMR data also agree with the proposed structure.
1
spectra are very similar with those of complex
exception of the signals belonging to the pyridyl ring of the carboxylic acid of
phosphine ligands, that could be properly assigned by carrying dipeptide . The coupling reaction was carried out according to
2, with the only The coupling of glycine tert-butyl ester through the free
4
gave the desired complex with the
5
out 2D NMR experiments. Although both phosphines possess standard solution peptide synthesis techniques, using PyBOP
similar electronic and sterical properties, they could shows (benzotriazolyloxy-tris[pyrrolidino]-phosphonium
differences in the lipophilicity character, and the pyridyl ring hexafluorophosphate) as activating agent and DIPEA as a base.
could interact or form hydrogen bonds with different Complex 5 was obtained in good yield and in pure form after
1
biologically relevant molecules, thus influencing in the activity chromatographic purification. The H NMR spectrum shows all
of the complex.
the expected resonances for the dipeptide and phosphine
ligands. Complex appears as a rotamers mixture (ratio 1:0.6).
The new amide bond appears at = 6.81 and = 6.50 ppm for
each rotamer. In the 13C{1H} NMR spectrum, the new amide
bond appears at = 168.8 ppm, strongly upfield shifted
5
R3P
Au
S
Ph3P
Ph3P
δ
δ
Au
Au
S
S
HCl—HꢀGlyꢀOtBu
δ
O
LiOH
COOMe
N
COOH
N
N
COOtBu
PyBOP
DIPEA
MeCN
(almost 10 ppm) compared to the starting free carboxylic acid.
However, the signals of Cα and Cβ appear downfield-shifted (3-
4 ppm). In the IR spectrum, the disappearance of the
carboxylic acid band and the appearance of the new amide
bond at 1742 cm-1 are also observed. All the signals could be
correctly assigned by the realisation of 2D NMR experiments.
Complexes with more than one gold(I) atom per molecule are
expected to exhibit higher cytotoxic activity, as we previously
have observed,15 and in addition species with lipophilic cations
have attracted a lot of attention due to their ability to cross
cell membranes. For these reasons, we decided to synthesise
MeOH
Boc
Boc
Boc
HN
PR3 = PPh3 (2), PPh2Py (3)
4
5
[Au(OTf)(PPh3)]
Ph3P
K2CO3
OTf
[AuCl(PR3)]
PPh3
Au
Au
HS
S
COOMe
N
COOMe
Boc
N
Boc
1
6
Scheme 2. Synthesis of gold(I) complexes from N-Boc protected
mercaptoproline.
the dinuclear cationic complex. The reaction of
[Au(OTf)(PPh3)] generated in situ afforded the desired
dinuclear complex in high yield and pure form after
2 with
The preparation of the dipeptide ligand containing
mercaptoproline and glycine tert-butyl ester by using a similar
procedure to that described in Scheme 1 failed in the thioester
acetate introduction. The introduction of the second amino
acid ester group was accomplished with success, but the step
of the nucleophilic substitution of the methanesulphonate
group for the thioacetate produced a mixture of compounds,
probably due to the higher steric hindrance of the dipeptide
molecule compared to the amino ester 1c, what prevented the
SN2 reaction. Therefore, we decided to follow a different route
6
1
recrystallisation (Scheme 2). The H NMR spectrum shows all
the resonances expected for the molecule, slightly downfield
shifted (around 0.2 ppm) compared to their analogues in
complex 2. In this case, the aromatic protons belonging to the
triphenylphosphine integrate for 30H instead of 15H ppm
because in this case two [Au(PPh3)]+ fragments coordinate to
the thiolate ligand. In the 13C{1H} NMR spectrum, the most
notable feature is the resonance corresponding to Cγ, that
appears strongly downfield shifted (4 ppm) compared with
that starts from complex
first step, basic hydrolysis of the amino ester moiety in
complex , employing LiOH as a base, gave the desired amino
acid-containing complex . Note that although the Au-S and
2, as is shown in Scheme 2. In the
complex
single signal at
3-4 ppm) compared to
2
. Remarkably, the 31P{1H} NMR spectrum shows a
= 34.2 ppm, strongly upfield shifted (around
, and characteristic for this type of
δ
2
2
4
dinuclear complexes. The HRMS(ESI+) shows [M]+ = 1178.2186
m/z with the expected isotopic pattern and the IR spectrum
also agree with the proposed structure.
Au-P bonds show high stability in basic media this type of
complexes quickly decompose in acidic media (pH < 3) as we
previously described. So during the reaction workup, careful
acidification must be carried out. The spectroscopic data are
Cytotoxic activity
very similar to complex 2, with the notable exception of the
disappearance of the methyl ester signals in the 1H NMR
The cytotoxic activity of complexes 2-6 was tested against
spectrum. Again, complex 4 appears as a rotamers mixture in
1:1 ratio. In the 13C{1H} NMR spectrum, in addition to the
disappearance of the methyl resonance, the new signal
three different human tumor cell lines: A549 (lung carcinoma),
Jurkat (T-cell leukaemia) and MiaPaca2 (pancreatic carcinoma)
and compared with that of cisplatin.
corresponding to the carboxylic acid is observed at
δ = 177.5
Compounds 2-6 are not soluble in water, but they are soluble
ppm, strongly downfield-shifted (around 4.5 ppm) compared
to the ester. The 31P{1H} NMR spectrum shows a single
in DMSO and in the DMSO/water mixtures used in the tests,
which contain a small amount of DMSO. We did not observe
any precipitation of the complexes or metallic gold while
resonance similar to that observed for complex
2. The IR
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performing the tests. Their colourless DMSO-d6 solutions are cytotoxicity. Then, the amino ester ligand plays an important
very stable at room temperature, as shown in the 1H NMR role in the activity of the complex, and complexes with an
spectra in which the signals remain the same for weeks. Cells ester instead of carboxylic acid moiety yield more active
were exposed to different concentrations of each compound compounds. Most probably, the higher lipophilic character of
for a total of 24 h. Using the colorimetric MTT viability assay, the ester allows the complex to better penetrate into the cell,
(MTT
= 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium or another possibility is that the carboxylic acid moiety could
bromide), the IC50 values (final concentration < 0.5 % DMSO) further react with other biological molecules preventing the
were calculated from dose–response curves obtained by non- complex to reach their final targets.
linear regression analysis. IC50 values are concentrations of a To get a better insight in the relationship between lipophilicity
drug required to inhibit tumour cell proliferation by 50%, and hydrophilicity in these complexes we have measured such
compared to the control cells treated with DMSO alone. The relationship in terms of logD7.4 (n-octanol/water partition
IC50 values for complexes 2-6 are collected in Table 1. These coefficient under physiological conditions). Complexes 2 and 3
values can be compared with those reported for cisplatin bearing the phosphine ligands PPh3 and PPh2py have a clear
dissolved in water: IC50 at 24 h in A549, MiaPaca2 and Jurkat lipophilic character with log logD7.4 values of 0.75 and 0.68,
cells are 114.2 µM, 76.5 µM and 10.8 µM, respectively.18
respectively, whereas the acid derivative 4 has a value of 0.41,
which represent a slightly more balanced relationship between
the lipophilic and hydrophilic character but still being mainly
lipophilic.
Table 1. IC50 (24 h) of complexes (2-6) against A549, MiaPaca2 and Jurkat
(ꢀM).
COMPLEX
A549
MiaPaca2
Jurkat
The complex
also exhibits excellent cytotoxic activity whit IC50 values
comparable to those found for complex . The effect of
5, with a dipeptide containing 4-mercaptoproline,
Boc-Pro(SAuPPh3)-OMe (2)
Boc-Pro(SAuPPh2Py)-OMe (3)
Boc-Pro(SAuPPh3)-OH (4)
Boc-Pro(SAuPPh3)-Gly-OtBu (5)
[Boc-Pro(SAuPPh3)2-OMe]OTf (6)
Cisplatin
1.8±0.15
3.8±0.37
>25
3.0±0.19
6.1±0.54
>25
0.8±0.08
3.5±0.32
9.3±0.65
0.6±0.08
0.5±0.07
10.8
2
lengthen the peptide chain from one to two residues does not
seem to have a critical influence in the cytotoxicity of the final
complex, although the dipeptide complex is slightly more
active in the MiaPaca2 and Jurkat cell lines, and slightly less
active in A549 compared to the complex with the amino ester
3.5±0.29
1.9±0.16
114.2
2.3±0.22
1.8±0.17
76.5
2.
Finally, the introduction of an additional [Au(PPh3)]+
fragment gives excellent cytotoxic agents. Complex is slightly
more potent than its mononuclear analogue in all cell lines.
6
Studies of the cytotoxic activity in many gold phosphine
complexes revealed that cytotoxicity is mainly due to the
metal atom, while the ancillary phosphine ligand stabilizes the
gold metallic centre and allow membrane crossing. The role of
thiolates in general and the thiolate amino acid ligand in
particular is also important because these ligands take part in
diverse exchange reactions with other biomolecules in the
organism, which determine facts such as transport or
biodistribution.
2
This complex is charged, highly lipophilic and with more gold(I)
atoms per molecule.
Comparison of the bioactivity with related gold(I) phosphine
complexes bearing sulfur ligands can be made. This complexes
present better activity than the gold thionicotinic acid
derivatives functionalised with amino acid esters15c and similar
to those reported for gold cysteine containing dipeptides
previously described by us.15d The importance of the P-Au-S
unit is remarkable, as similar values were found in different
cell lines for gold species with different sulfur ligands such as
dithiocarbamates.19
In general, the Jurkat cell line was the most sensitive, and the
A549 and MiaPaca2 exhibit more resistance to our complexes.
All the complexes displayed excellent cytotoxicity in all the cell
lines, with IC50 values in the low micromolar range (< 25
even in some cases in the nanomolar range with values as low
as 0.5 M.
The gold-amino ester conjugate
ꢀM)
From these results, we can extract some important Structure-
Activity Relationships (SARs). Gold(I) complexes with thiolates
and phosphines of this type are expected to be good cytotoxic
agents. The functionalisation of the thiolate ligand with an
ester moiety, lengthen the amino acid chain or coordinate
additional gold(I) atoms seem to be suitable approaches in
order to obtain excellent cytotoxic complexes. However, the
change of the ester moiety or triphenylphosphine ligand by a
carboxylic acid moiety or 2-pyridil diphenylphosphine ligands,
respectively, is associated with a decrease in the potency of
the complex.
ꢀ
2
displayed excellent
cytotoxicty much greater than cisplatin, over 100, 23 and 10
times folder in A549, MiaPaca2 and Jurkat cell lines,
respectively. Therefore, the gold(I) complex with the 4-
mercaptoproline ester constitutes a good candidate and an
excellent start point for the design of new gold-peptide
anticancer agents.
The
change
of
triphenylphosphine
by
2-pyridyl
diphenylphosphine
3
as type of phosphine ligand used reduces
Further design of new complexes following the SARs obtained
from this work and further biological studies in order to obtain
information about the mechanism and targets of our
complexes are currently conducted in our laboratories.
by half the cytotoxic activity of the complex. Again, lipophilicity
seems to be a key feature in the final cytotoxicity of the
complex.
The complex with the 4-mercaptoproline acid
4 instead of the
4-mercaptoproline ester results in a drastic decrease of
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2.30 and 2.05 (m, diastereotopic protons, 2H, Cβ,ProH2), 1.41 (s,
9H, CBocH3). HRMS ESI(-) m/z: [M+Na]+ = 268.1155 (calcd),
268.1153 (found). IR (cm-1): 3500-3300 (br, OH), 1746 (s,
COOMe), 1672 (s, OCON), 1155 and 1085 (s, C-O). TLC Rf: 0.4
(AcOEt/hexane 4:6).
Conclusions
The synthesis of an N-protected mercaptoproline derivative
has been developed and the first gold thiolate complexes have
been prepared. The complex bearing a triphenylphosphine
coordinated to the sulfur atom serves as precursors for the
synthesis of other metal thiolates containing different 4-
Synthesis of Boc-Pro(OMs)-OMe (1c). To a solution of 1b
(1.170 g, 4.77 mmol) in dry CH2Cl2 (50 mL) under argon
atmosphere, DIPEA (0.984 mL, 5.75 mmol) was added, and the
mixture was stirred for 5 min. Then, MsCl (0.445 mL, 5.75
mmol) was added at 0 °C dropwise. The reaction mixture was
stirred at room temperature for 2 h, and the solvent was
evaporated, affording 1c in nearly quantitative yield (1.542 g,
4.77 mmol), as an orange oil pure enough to be employed
thioproline derivatives, including
a 4-thioproline-glycine
peptide. These are unprecedented examples of metal
complexes coordinated to the 4-mercaptoproline derivatives in
the form of thiolates.
In this formally gold(I) thiolate-amino acid or thiolate-
dipeptide phosphine complexes with the general formula
[Au(SR)(PR3)] different structural modifications, such as change
in the type of phosphine, the use of an ester, acid or amino
without further purification. 1H NMR (CDCl3, 300 MHz,
δ
(ppm), J (Hz)): rotamers mixture (ratio 1:0.7): 5.25 (m, 1H,
Cγ,ProH2), 4.43 (td, 1H, J = 16.0 and 7.8, Cα,ProH), 3.82 (m, 2H,
Cδ,ProH2), 3.74 (s, 3H, OCH3), 3.05 (s, 3H, CH3SO3), 2.61 and 2.26
(m, diastereotopic protons, 2H, Cβ,ProH2), 1.46 and 1.42 (s, 9H,
+
acid in the carboxylic side chain, or the number of AuPPh3
fragments coordinated to the sulfur centre were introduced, in
order to evaluate their influence in the biological activity of the
final complexes. The cytotoxic activity in vitro of these
complexes has been evaluated against different tumor human
cell lines (A549, MiaPaca2 and Jurkat). The complexes show
excellent cytotoxic activity with IC50 values in the very low
CBocH3). HRMS ESI(+) m/z: [M+Na]+
= 346.0930 (calcd),
346.0927 (found). IR (cm-1): 1747 (s, COOMe), 1701 (s, CON),
1364 and 1159 (w, SO3), 1159 (s, C-O). TLC Rf: 0.5
(AcOEt/hexane 3:7).
micromolar range, as low as 0.5 ꢀM. The structural changes
Synthesis of Boc-Pro(SAc)-OMe (1d). To a solution of 1c (1.170
g, 4.77 mmol) in dry DMF (20 mL) under argon atmosphere,
AcSK (0.953 g, 8.35 mmol) was added. The reaction mixture
was stirred at 65 °C under 24 h. Then, the mixture was diluted
with Et2O (100 mL) and washed with water (3x50 mL). The
organic phase was dried over anhydrous MgSO4, filtered off
and evaporated, and the reaction crude was purified by
performed in the parent compound have led to the synthesis
of the most effective compound in all the cell lines, which is
the complex with two AuPPh3+ fragments coordinated to the 4-
mercaptoproline methyl ester ligand.
Experimental
column chromatography using as eluent
AcOEt/hexane (2:8) affording 1d (yield = 70.2 %) as a brown oil
(1.015 g, 3.35 mmol). 1H NMR (CDCl3, 300 MHz,
(ppm), J
a mixture of
Instrumentation. Mass spectra were recorded on a BRUKER
ESQUIRE 3000 PLUS, with the electrospray (ESI) technique and
on a BRUKER MICROFLEX (MALDI-TOF). 1H, 13C{1H}, 31P{1H} and
19F NMR, including 2D experiments, were recorded at room
temperature on a BRUKER AVANCE 400 spectrometer (1H, 400
MHz, 13C{1H}, 100.6 MHz) or on a BRUKER AVANCE II 300
spectrometer (1H, 300 MHz, 13C{1H}, 75.5 MHz), with chemical
δ
(Hz)): rotamers mixture (ratio 1:0.85): 4.33 (dt, 1H, J = 25.6 and
7.9, Cα,ProH), 3.97 (m, 1H, Cγ,ProH2), 3.96 and 3.34 (m,
diastereotopic protons, 2H, Cδ,ProH2), 3.47 (s, 3H, OCH3), 2.70
and 1.96 (m, diastereotopic protons, 2H, Cβ,ProH2), 2.32 (s, 3H,
CH3COS), 1.45 and 1.41 (s, 9H, CBocH3). HRMS ESI(-) m/z:
[M+Na]+ = 326.1033 (calcd), 326.1051 (found). IR (cm-1): 1750
(s, COOMe), 1691 (s, OCON and CH3COS), 1154 (s, C-O). TLC Rf:
0.5 (AcOEt/hexane 3:7).
shifts (δ, ppm) reported relative to the solvent peaks of the
deuterated solvent.20
Starting Materials. [AuCl(PPh3)] and [AuCl(PPh2Py)] were
prepared according to published procedures.21 All other
reagents were commercially available. Solvents were used as
received without purification or drying.
Synthesis of Boc-Pro(SH)-OMe (1). To a solution of 1d (0.918
g, 3.03 mmol) in MeOH (30 mL), NaOMe (0.164 g, 3.03 mmol)
was added. The reaction mixture was stirred at room
temperature for 20 min. Then, the solvent was evaporated and
the crude was dissolved in AcOEt (50 mL) and washed with
water (3x50 mL). The organic phase was dried over anhydrous
Synthesis of Boc-Pro(OH)-OMe (1b). To a solution of HCl·H-
Pro(OH)-OMe 1a (1.090 g, 6 mmol) in CH2Cl2 (12 mL), Boc2O
(1.44 g, 6.6 mmol) and DIPEA (1.54 mL, 9 mmol) were added.
The reaction mixture was stirred overnight at room
temperature and, then, was washed with an aqueous
saturated solution of NaCl (3x25 mL). The organic phase was
dried over anhydrous MgSO4, filtered off and evaporated. The
crude was purified by column chromatography of silicagel
using as eluent a mixture of AcOEt/hexane (4:6) affording 1b
(yield = 79.6 %) as a colorless oil (1.170 g, 4.77 mmol). 1H NMR
MgSO4, filtered off and evaporated, affording 1 (yield = 74.4 %)
as a green oil (0.589 g, 2.25 mmol) pure enough to be
employed without further purification. 1H NMR (CDCl3, 300
MHz,
δ (ppm), J (Hz)): rotamers mixture (ratio 1:0.7): 4.22 (td,
1H, J = 21.3 and 8.0, Cα,ProH), 3.89 and 3.22 (m, diastereotopic
protons, 2H, Cδ,ProH2), 3.69 (s, 3H, OCH3), 3.23 (m, 1H, Cγ,ProH2),
2.65 and 1.87 (m, diastereotopic protons, 2H, Cβ,ProH2), 1.76 (d,
1H, J = 7.5, SH), 1.40 (B) and 1.35 (A) (s, 9H, CBocH3). 13C{1H}
(CDCl3, 300 MHz, δ (ppm), J (Hz)): 4.50 (m, 1H, Cγ,ProH2), 4.39 (t,
1H, J = 8.1, Cα,ProH), 3.73 (s, 3H, OCH3), 3.56 (dd, diastereotopic
NMR (CDCl3, 300 MHz,
δ (ppm), J (Hz)): rotamers mixture:
172.7 (A) and 172.5 (B) (COOMe), 153.6 (B) and 152.9 (A)
(CONPro), 80.4 (B) and 80.3 (A) (C, CBoc), 58.9 (A) and 58.5 (B)
protons, 2H, J = 11.7 and 4.2 and J = 30.9 and 4.2, Cδ ,ProH2),
,
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J. Name., 2013, 00, 1-3 | 5
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ARTICLE
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(Cα,ProH), 55.7 (B) and 55.2 (A) (Cδ,ProH2), 52.1 (B) and 52.0 (A) Ar), 1151 and 1099 (s, C-O), 746, 709 and 691 (w, Ar). TLC Rf:
(OCH3), 41.1 (A) and 40.3 (B) (Cβ,ProH2), 35.1 (B) and 34.4 (A) 0.5 (AcOEt/hexane 1:1).
(Cγ,ProH) and 28.2 (A) and 28.0 (B) (CBocH3). HRMS ESI(+) m/z: Synthesis of Boc-Pro(SAuPPh3)-OH (4). To a suspension of
[M+Na]+ = 284.0927 (calcd), 284.0944 (found). IR(cm-1): 2554 complex
2 (0.719 g, 1 mmol) in methanol (5 mL) was added
(w, SH), 1747 (s, COOMe), 1694 (s, OCON), 1154 (s, C-O).
Synthesis of Boc-Pro(SAuPPh3)-OMe (2). To a solution of
LiOH (0.120 g, 5 mmol). The mixture was stirred at room
temperature for 24 h. The reaction is followed by thin layer
1
(0.123 g, 0.47 mmol) in acetone (10 mL), K2CO3 (0.130 g, 0.94 chromatography and is complete when the starting compound
mmol) and [AuCl(PPh3)] (0.232 g, 0.47 mmol) were added and is strongly retained at the origin of the thin layer
the reaction mixture was stirred at room temperature for 24 h. chromatography (Rf = 0 (acetone/hexane 1:1)). At this point,
After this time, the reaction mixture was filtered over celite the methanol is evaporated and the product dissolved in
and the clear solution evaporated under reduced pressure. water. The resulting white solution is acidified dropwise with a
Complex
using as eluent a mixture of acetone/hexane (3:7), affording the solution was extracted three times with dichloromethane
pure complex (0.334 g, 0.46 mmol) as an orange solid (Yield and the organic phase was dried over anhydrous MgSO4,
= 98.8 %). H NMR (CDCl3, 300 MHz, (ppm), J (Hz)): rotamers filtered off and evaporated to yield the desired complex
mixture (ratio 1:0.3): 7.48 (m, 15H, Ar), 4.20 (m, 1H, Cα,ProH), (yield = 91.2 %) as a green solid (0.644 g, 0.91 mmol). H NMR
4.01 and 3.34 (m, diastereotopic protons, 2H, Cδ,ProH2), 3.79 (CDCl3, 300 MHz, (ppm), J (Hz)): rotamers mixture (ratio 1:1):
2 was purified by column chromatography on silicagel saturated solution of KHSO4 until a slightly acid pH (3-4). Then,
2
1
δ
4
1
δ
(m, 1H, Cγ,ProH2), 3.71 (B) and 3.63 (A) (s, 3H, OCH3), 2.77 and 7.49 (m, 15H, Ar), 4.31 and 4.21 (m, 1H, Cα,ProH), 4.06 (A,B) and
1.96 (m, diastereotopic protons, 2H, Cβ,ProH2), 1.41 (B) and 1.37 3.38 (A) and 3.23 (B) (m, diastereotopic protons, 2H, Cδ,ProH2,),
(A) (s, 9H, CBocH3). 13C{1H} NMR (CDCl3, 300 MHz,
δ
(ppm), J 3.80 (m, 1H, Cγ,ProH2), 2.80 (A, B) and 2.31 (A) and 2.06 (B) (m,
(Hz)): rotamers mixture: 173.1 (A) and 172.9 (B) (COOMe), diastereotopic protons, 2H, Cβ,ProH2), 1.44 (B) and 1.38 (A) (s,
153.7 (B) and 152.9 (A) (CONPro), 134.0 (d, CH, J = 13.7, C2), 9H, CBocH3). 13C{1H} NMR (CDCl3, 300 MHz,
(ppm), J (Hz)):
δ
131.7 (d, CH, J = 2.3, C4), 129.1 (d, CH, J = 11.6, C3), 129.0 (d, C, rotamers mixture: 177.5 (COOH), 155.3 (CONPro), 134.0 (d, CH,
J = 58.8, C1), 80.4 (B) and 79.7 (A) (C, CBoc), 59.7 (A) and 59.1 J = 13.7, C2), 131.8 (d, CH, J = 2.3, C4), 129.2 (d, CH, J = 11.6,
(B) (Cα,ProH), 59.0 (B) and 58.5 (A) (Cδ,ProH2), 51.8 (B) and 51.7 C3), 129.0 (d, C, J = 58.7, C1), 81.0 and 80.3 (C, CBoc), 59.7 (A)
(A) (OCH3), 45.5 (A) and 45.0 (B) (Cβ,ProH2), 39.1 (B) and 38.6 (A) and 59.4 (B) (Cα,ProH), 58.6 (Cδ,ProH2), 45.5 (B) and 44.3 (A)
(Cγ,ProH) and 28.3 (B) and 28.2 (A) (CBocH3). 31P{1H} NMR (CDCl3, (Cβ,ProH2), 38.9 (Cγ,ProH) and 28.1 (CBocH3). 31P{1H} NMR (CDCl3,
300 MHz,
δ δ
(ppm), J (Hz)): 37.7. HRMS ESI(+) m/z: [M+Na]+ = 300 MHz, (ppm), J (Hz)): 36.3. HRMS ESI(-) m/z: [M-H]- =
742.1426 (calcd), 742.1455 (found). IR (cm-1): 1746 (s, 704.1293 (calcd), 704.0564 (found). IR (cm-1): 3300-3100 (br,
COOMe), 1693 (s, OCON), 1479 and 1435 (w, Ar), 1152 and COOH), 1709 (s, OCON), 1694 (s, COOH), 1479 and 1435 (w,
1099 (s, C-O), 746, 709 and 691 (w, Ar). TLC Rf: 0.5 Ar), 1153 and 1099 (s, C-O), 746, 710 and 691 (w, Ar).
(AcOEt/hexane 1:1).
Synthesis of Boc-Pro(SAuPPh3)-Gly-OtBu (5). To a suspension
of (0.705 g, 1 mmol) in acetonitrile (5 mL) was added DIPEA
Synthesis of Boc-Pro(SAuPPh2Py)-OMe (3). The reaction of
1
4
(0.052 g, 0.2 mmol), K2CO3 (0.0552 g, 0.4 mmol) and (0.377 mL, 2.2 mmol). The mixture was stirred for 5 min at
[AuCl(PPh2Py)] (0.991 g, 0.2 mmol) following a procedure room temperature and then PyBOP added (0.624 g, 1.2 mmol).
similar to
2
afforded
3
(0.134 g, 0.19 mmol) as an orange solid The resulting solution was stirred for an additional 45 min. To
(ppm), J (Hz)): this solution at 0 °C was added dropwise a solution of HCl·H-
(Yield = 96.3 %). 1H NMR (CDCl3, 300 MHz,
δ
rotamers mixture (ratio 1:0.5): 8.75 (d, 1H, J = 4.8, H1), 7.87 (t, Gly-OtBu (0.201 g, 1.2 mmol) and DIPEA (0.205 mL, 1.2 mmol)
1H, J = 7.5, H4), 7.80-7.73 (m, 1H, H3), 7.67-7.62 and 7.50-7.40 in acetonitrile (5 mL). After the addition the mixture was
(m, 10H, Ar), 7.38-7.33 (m, 1H, H2), 4.26-4.16 (m, 1H, Cα,ProH), stirred for 48 h at room temperature. The acetonitrile was
4.12-3,97 and 3.39-3.32 (m, diastereotopic protons, 2H, evaporated and dichloromethane (40 mL) was added. This
Cδ,ProH2), 3.85-3.76 (m, 1H, Cγ,ProH2), 3.71 (B) and 3.64 (A) (s, solution was washed with a saturated NaHCO3 solution in
3H, OCH3), 2.84-2.71 and 2.00-1.92 (m, diastereotopic protons, water (3 x 15 mL) and a saturated solution of NaCl (3 x 15 mL).
2H, Cβ,ProH2), 1.41 (B) and 1.37 (A) (s, 9H, CBocH3). 13C{1H} NMR The organic phase was dried over anhydrous MgSO4, filtered
(CDCl3, 300 MHz,
and 173.0 (B) (COOMe), 154.4 (C, d, J = 80.8, C5), 152.9 purified by column chromatography on silicagel using as eluent
(CONPro), 151.2 (CH, d, J = 15.4, C1), 136.6 (d, CH, J = 10.7, C3), a mixture of AcOEt/hexane (1:1), affording (yield = 76.8 %) as
134.4 (d, CH, J = 13.7, C7), 131.8 (d, CH, J = 2.5, C9), 131.2 (d, a green oil (0.629 g, 0.77 mmol). H NMR (CDCl3, 300 MHz,
δ (ppm), J (Hz)): rotamers mixture: 173.2 (A) off and evaporated to dryness. The crude of the reaction was
5
1
δ
CH, J = 32.0, C4), 129.0 (d, CH, J = 7.2, C8), 128.8 (d, C, J = 58.9, (ppm), J (Hz)): rotamers mixture (ratio 1:0.6): 7.46 (m, 15H,
C6), 125.2 (d, CH, J = 2.4, C2), 80.5 (B) and 79.8 (A) (C, CBoc), Ar), 6.87 (B) and 6.58 (A) (m, 1H, CONHGly), 4.12 (m, 1H,
59.7 (A) and 59.1 (B) (Cα,ProH), 58.5 (B) and 53.7 (A) (Cδ,ProH2), Cα,ProH), 4.12 and 3.25 (m, diastereotopic protons, 2H,
51.9 (B) and 51.7 (A) (OCH3), 45.5 (A) and 45.1 (B) (Cβ,ProH2), Cδ,ProH2,), 3.93 and 3.83 (dd and m, ABX system, 2H, J = 18.3
39.1 (B) and 38.6 (A) (Cγ,ProH) and 29.2 (B) and 28.3 (A) (CBocH3). and 5.1, Cα,GlyH2), 3.72 (m, 1H, Cγ,ProH2), 2.76 and 2.10 (m,
31P{1H} NMR (CDCl3, 300 MHz,
ESI(+) m/z: [M+H]+ = 721.1558 (calcd), 721.1725 (found). IR 13C{1H} NMR (CDCl3, 300 MHz,
δ
(ppm), J (Hz)): 35.7. HRMS diastereotopic protons, 2H, Cβ,ProH2,), 1.40 (s, 18H, CBocH3).
(ppm), J (Hz)): rotamers
δ
(cm-1): 1748 (s, COOMe), 1694 (s, OCON), 1479 and 1436 (w, mixture: 172.5 (A) and 168.8 (B) (COOtBu), 157.4 (A) and 154.0
(B) (CONPro), 134.0 (d, CH, J = 13.7, C2), 131.5 (d, CH, J = 2.4,
6 | J. Name., 2012, 00, 1-3
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ARTICLE
i
C4), 129.7 (C, C1), 129.1 (d, CH, J = 11.5, C3), 81.9 (B) and 80.3 were incubated for 1-3 h at 37 ºC. Finally, 100 µl/well PrOH
(A) (C, CBoc), 62.1 (Cα,ProH), 59.3 (B) and 59.0 (A) (Cδ,ProH2), 49.7 (0.05 M HCl) was added. The optical density was measured at
(B) and 46.0 (A) (Cβ,ProH2), 42.0 (Cα,GlyH2), 38.7 (B) and 38.2 (A) 490 nm using a 96-well multiscanner autoreader (ELISA). The
(Cγ,ProH) and 28.1 (B) and 27.9 (A) (CBocH3). 31P{1H} NMR (CDCl3, IC50 was calculated by non-linear regression analysis using
300 MHz,
δ
(ppm), J (Hz)): 38.3. HRMS ESI(+) m/z: [M+H]+
=
Origin software (Origin Software, Electronic Arts, Redwood
819.2290 (calcd), 819.2311 (found). IR (cm-1): 3320 (br, CONH), City, California, USA).
1742 (s, COOMe), 1684 (s, CONH), 1479 and 1435 (w, Ar), 1151 Distribution Coefficients (log D7.4
)
and 1099 (s, C-O), 744 and 691 (w, Ar). TLC Rf: 0.5 The n-octanol-water coefficients of the complexes were
(AcOEt/hexane 6:4). determined as previously reported using shake-flask
Synthesis of [Boc-Pro(SAuPPh3)2-OMe]OTf (6). To a solution method.22 Buffered-saline distilled water (100 mL, phosphate
of complex
(0.072 g, 0.1 mmol) in CH2Cl2 (5 mL) was added buffer [PO43-] = 10 µM, [NaCl] = 0.15 M, pH 7.4) and n-octanol
a
2
[Au(OTf)(PPh3)] (0.061 g, 0.1 mmol) freshly prepared. The (100 mL) were shaken together for 72 h to allow saturation of
mixture was stirred for 2 h at room temperature. Then, the both phases. Approximately 1 mg of the complexes was
yellow solution resultant was filtered on Celite. The solution dissolved in 5 mL of the aqueous phase and 5 mL of the
was concentrated to ca. 5 mL and the addition of hexane (20 organic phase were added, mixing for 10 minutes. The
mL) afforded
6
(yield = 97.4 %) as a brown solid (0.126 g, 0.09 resultant emulsion was centrifuged to separate the phases.
(ppm), J (Hz)) rotamers The concentration of the compounds in each phase was
mmol). 1H NMR (CDCl3, 300 MHz,
δ
:
mixture (ratio 1:0.2): 7.46 (m, 30H, Ar), 4.59 (B) and 4.28 (A) determined using UV absorbance spectroscopy. LogD7.4 was
(m, 1H, Cα,ProH), 4.59 (B) and 4.27 (A) (m, 1H, Cγ,Pro), 4.08 and defined as log{[compound(organic)]/[compound(aqueous)]}.
3.75 (m, diastereotopic protons, 2H, Cδ,ProH2), 3.71 (B) and 3.66
(A) (s, 3H, OCH3), 2.98 and 2.11 (A) and 2.87 and 2.47 (B) (m,
2H, Cβ,ProH2) and 1.37 (s, 9H, CBocH3). 13C{1H} NMR (CDCl3, 300
Acknowledgments
MHz,
δ (ppm), J (Hz)): rotamers mixture: 172.8 (A) and 172.7
Authors thank the Ministerio de Economía y Competitividad
(MINECO/FEDER CTQ2013-48635-C2-1-P, CTQ2015-70371-
REDT and CTQ2013-40855-R) and Gobierno de Aragón-Fondo
Social Europeo (E77 and E40) for financial support. A. Gutiérrez
thanks to CSIC for a JAE-Predoc fellowship. We gratefully thank
Dr. Isabel Marzo for the cytotoxicity measurements.
(B) (COOMe), 166.4 (B) and 152.8 (A) (CONPro), 134.0 (d, CH, J =
13.6, C2), 132.5 (d, CH, J = 2.6, C4), 129.7 (d, CH, J = 11.9, C3),
127.6 (d, C, J = 61.2, C1), 114.4 (d, C, J = 305.6, CF3), 80.8 (C,
CBoc), 58.9 (B) and 59.5 (A) (Cα,ProH), 58.0 (B) and 57.6 (A)
(Cδ,ProH2), 52.3 (B) and 52.2 (A) (OCH3), 44.0 (A) and 43,3 (B)
(Cβ,ProH2), 42.4 (A) and 41.8 (B) (Cγ,ProH) and 28.2 (CBocH3).
31P{1H} NMR (CDCl3, 300 MHz,
δ
(ppm), J (Hz)): 34.2. 19F NMR
(CDCl3, 300 MHz,
δ
(ppm), J (Hz)): -78.0. HRMS ESI(-) m/z: [M]+
References
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1100 (s, C-O), 744, 711 and 690 (w, Ar).
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6
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DOI: 10.1039/C6DT02000C
ARTICLE
Journal Name
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Dalton Transactions
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DOI: 10.1039/C6DT02000C
Graphical Abstract
Bioactive Gold(I) Complexes with 4-Mercaptoproline Derivatives
Alejandro Gutiérrez, Carlos Cativiela, Antonio Laguna, M. Concepción Gimeno
Unprecedented gold(I) bioconjugates bearing non-proteinogenic amino acid 4-mercaptoproline species as
bioorganic ligands have been prepared. The complexes displayed excellent cytotoxic activity with IC50 values in the
low µM range and even in the nM range.