Synthesis and Evaluation of the Antiproliferative Properties of a Tethered Tubercidin-Platinum(II) Complex

Article abstract of DOI:10.1002/ejoc.201500998

Herein, the synthesis of a nucleoside platinum(II) complex in which a cisplatin-like unit is joined to 7-deazaadenosine through an amino alkyl chain installed at the C6 position of purine was explored. The capability of the new complex to react with DNA p

Full text of DOI:10.1002/ejoc.201500998

FULL PAPER
DOI: 10.1002/ejoc.201500998
Synthesis and Evaluation of the Antiproliferative Properties of a Tethered
Tubercidin–Platinum(II) Complex
Stefano D’Errico,[a] Giorgia Oliviero,*[a] Nicola Borbone,[a] Elena Di Gennaro,[b]
Andrea Ilaria Zotti,[b] Alfredo Budillon,[b] Vincenzo Cerullo,[c] Fabrizia Nici,[a]
Luciano Mayol,[a] Vincenzo Piccialli,[d] and Gennaro Piccialli[a,e]
Keywords: Platinum / Nucleosides / Antitumor agents / Medicinal chemistry
Herein, the synthesis of a nucleoside platinum(II) complex
in which a cisplatin-like unit is joined to 7-deazaadenosine
through an amino alkyl chain installed at the C6 position of
purine was explored. The capability of the new complex to
react with DNA purine bases was confirmed by a model reac-
tion with deoxyguanosine monophosphate, whereas its anti-
proliferative activity against A549 and Cal27 human cancer
cell lines was studied by sulforhodamine B assay in compari-
son with its unplatinated precursor and cisplatin.
cols that ameliorate the quality of life of patients, even if
they do not resolve the problems of drug resistance.[6]
Introduction
More than 50 years after its discovery,[1] cisplatin (1, Fig-
ure 1) is still one of the most effective drugs for the treat-
ment of several types of tumors, including testicular, ovar-
ian, bladder, cervical, and lung cancer and head and neck
carcinomas.[2] The mechanism of action of cisplatin involves
DNA as the final target. In fact, cisplatin essentially binds
the guanine bases at the N7 position and forms stable intra-
strand adducts or interstrand crosslinks that cause severe
local changes in the secondary structure of DNA.[3] This
DNA distortion hampers its binding with crucial cellular
proteins and is recognized as DNA damage that eventually
leads to cell death by apoptosis.[4] The clinical use of cispla-
tin is partially limited by intrinsic resistance of many tu-
mors and by the insurgence of acquired resistance during
treatments.[2,5] Carboplatin (2) and oxaliplatin (3), second-
and third-generation platinum-based antineoplastic agents,
respectively, have dosages and clinical administration proto-
[a] Dipartimento di Farmacia, Università degli Studi di Napoli
Federico II,
Figure 1. Structures of platinum drugs 1–3 and bis-platinated nu-
cleoside complexes 4 and 5 carrying purine nucleoside scaffolds.
Via Domenico Montesano 49, 80131 Napoli, Italy
E-mail: giorgia.oliviero@unina.it
http://www.unina.it
[b] Experimental Pharmacology, Istituto Nazionale Tumori
Fondazione G. Pascale – IRCCS,
Via Mariano Semmola 52, 80131 Napoli, Italy
[c] Division of Biopharmaceutics and Pharmacokinetics, Faculty
of Pharmacy, University of Helsinki,
Viikinkaari 5 E, 00790 Helsinki, Finland
[d] Dipartimento di Scienze Chimiche, Università degli Studi di
Napoli Federico II,
Via Cinthia 4, 80126 Napoli, Italy
[e] Institute of Protein Biochemistry, National Research Council
of Italy,
For these reasons, research in this field is still very active,
proposing multinuclear platinum complexes[7–10] and com-
posite molecules in which the platinum complex is conju-
gated to biologically important substances, drugs, and carri-
ers.[11–18] Furthermore, thanks to the synergistic effect that
may result, tethering between biologically active molecules
and platinum units could afford novel complexes with im-
proved drug-targeting and drug-delivery properties.[12]
Nucleosides and nucleotides (NNs) and their analogues
play a key role in the regulation of cellular processes and in
signal transduction.[19–22] Furthermore, NN analogues are
Via Pietro Castellino 111, 80131 Napoli, Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500998.
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Properties of a Tethered Tubercidin–PtII Complex
Scheme 1. Reagents and conditions: (i) N,O-Bis(trimethylsilyl)acetamide (BSA), trimethylsilyl trifluoromethanesulfonate (TMSOTf),
CH3CN, 2 h, 80 °C, 60%; (ii) 9, EtOH, 5 h, reflux, 80%; (iii) trifluoroacetic acid (TFA)/CH2Cl2 (1:1), 1 h, r.t., 99%; (iv) 12, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA), 16 h, r.t., 83%;
(v) MeOH, AcONa, H2/Pd/C, 2 h, r.t., 99%; (vi) MeONa, MeOH, 1 h, r.t., 95%; (vii) K2PtCl4, H2O/MeOH (1:1), 16 h, r.t., in the dark,
70%. Bz = benzoyl, Boc = tert-butoxycarbonyl.
known to inhibit the growth of several cancer lines[23–26] involving exclusively the exocyclic 1,2-diamine function of
and many of them are clinically used as antiviral the linker. The capability of 17 to mimic the reactivity of
drugs.[27–29] Despite this, only a few platinum complexes cisplatin with the DNA bases was assessed by monitoring
carrying nucleoside scaffolds have been proposed.[30–33] In a model reaction of 17 with deoxyguanosine 5Ј-monophos-
principle, a platinum complex conjugated to a biologically phate (dGMP) by 1H NMR spectroscopy.[40,41] The prelimi-
active nucleoside could combine the reactivity of the plati- nary information on the antiproliferative activity of 17 and
num center with the pharmacological properties of the nu- its unplatinated precursor 16 against Cal27 and A549
cleoside analogue (e.g., antimetabolite activity or active tumor cell lines are also reported.
transmembrane transport). However, the properties of such
a composite molecule are not predictable because each moi-
Results and Discussion
ety of the complex can strongly influence the chemical and
pharmacological behavior of the other. In this context, we
recently focused our attention on the development of new
nucleoside dinuclear platinum(II) complexes.
Synthesis and Characterization
Our synthetic route started with the construction of bis-
In particular, we synthesized bis-platinated nucleoside halogenated 7-deazapurine-β-d-riboside 8 (Scheme 1). The
complexes 4 and 5 (Figure 1)[34,35] carrying two monofunc- nucleoside scaffold was prepared according to a procedure
tional platinum(II) centers, the first coordinating the N7 by Seela and Ming,[42] which performs the glycosylation
position of the purine system and the second coordinating step by using 7-bromo-6-chloro-7-deazapurine 6 as the ag-
the terminal amino group of an alkyl chain installed on the lycon moiety. As outlined in that paper, the presence of the
purine base. These substances showed moderate (or low) electron-withdrawing bromine atom at the C7 position of
antiproliferative activities upon testing against A278, HeLa, the purine atom makes the formation of the glycosidic bond
A549, and MCF7 cell lines.
with suitably protected ribose moiety 7 possible in good
In this paper, we describe the synthesis and the prelimi- yields and with complete β stereoselectivity. Nucleoside 8
nary biological evaluation of a novel nucleoside platinum was then treated with mono-Boc-protected 1,3-diamino-
complex (i.e., 17, Scheme 1) carrying a neutral cisplatin unit propane unit 9 to afford derivative 10. After quantitative
conjugated to the 1,2-diamine moiety of 7-deazaadenosine removal of the Boc group (TFA in CH2Cl2), resulting nucle-
(7-deaza-A) analogue 16. 7-Deaza-A (also known as tuber- oside 11 was treated with bis-Boc-protected 2,3-diamino-
cidin) is an interesting antimetabolite endowed with antibi- propanoic acid (racemic mixture, 12)[43] by using EDC/
otic and antitumor properties.[36,37] In particular, tubercidin HOBt as the activating agents of the carboxylic function
shows significant in vitro cytotoxicity against the murine to give 13 (83% yield) as a mixture of diastereoisomers.
P388 and the human lung adenocarcinoma A549 cell Compound 13 was then subjected to hydrogenolysis in a
lines.[38,39] In our context, the absence of the N7 purine Parr apparatus by using Pd/C as the catalyst to remove the
atom in 7-deaza-A avoids the issue of the fast reactivity of C7 bromine. It was reported that the presence of a base
N7 with platinum during the coordination reaction, which (AcONa) in the reaction mixture is indispensable to neu-
thus allows the construction of a monoplatinated complex tralize HBr that is formed during the reaction.[44] The
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hydrogenolysis proceeded very cleanly to give 14 quantita-
The reactivity of the cis-dichloroplatinum moiety of 17
tively after a standard workup procedure. The benzoate towards dGMP was confirmed by the appearance of down-
protecting groups were then removed from 14 with a solu- field-shifted aromatic signals (δ = 8.4–9.0 ppm), attributable
tion of MeONa in MeOH to give 15 after chromatographic to H8 of dGMP complexed with 17, already in the first
purification (95% yield). Finally, the Boc protecting groups hour of incubation (Figure 2). Because of the asymmetry of
in 15 were removed by TFA treatment, and the crude was the diaminoalkyl moiety coordinated with the platinum(II)
treated with a concentrated aqueous solution of NH4OH to center, up to six aromatic signals belonging to H8 of dGMP
affording 7-deaza-A derivative 16, as the free diamine al- complexed with 17 were expected [two signals for each of
most quantitatively (98%). The reaction of 16 with K2PtCl4 the two complexes 7-deaza-A-Pt(dGMP)(Cl) and 7-
in a H2O/MeOH (1:1) solution gave platinum complex 17 deaza-A-Pt(dGMP)(H2O); two signals for 7-deaza-A-Pt-
as a pale-yellow solid that precipitated from the reaction (dGMP)2]. This spectral complexity was observed in the
mixture in 70% yield.
NMR spectra recorded at an incubation time Ն3 h. Al-
The structures of all intermediates and of complex 17 though the detailed assignment of all the dGMP H8 signals
were supported by spectroscopic analyses. High-resolution was not accomplished, because it is beyond the scope of
mass spectrometry and CHN analysis further confirmed the this paper, the analysis of the literature data and the relative
structure and purity of final complex 17.
intensity of the NMR signals recorded at increasing incu-
bation times (Figure 2) allowed us to tentatively assign
(1) the signals at δ = 8.55 and 8.62 ppm of the dGMP H8
proton of the complexes 7-deaza-A-Pt(dGMP)(Cl) and 7-
deaza-A-Pt(Cl)(dGMP) (+ in Figure 2); (2) the signal at δ
= 8.99 ppm (visible at incubation times Ն 6 h) of the dGMP
NMR Spectroscopy Study of the Reactivity of 17 towards
dGMP
To assess the capability of 17 to react with the guanine H8 proton of one of the two aqua complexes 7-deaza-
bases of DNA as a bifunctional coordinating agent, such A-Pt(dGMP)(H2O) or 7-deaza-A-Pt(H2O)(dGMP) (᭹);
as cisplatin, we incubated 17 with a fourfold excess amount (3) the signals at δ = 8.52 and 8.65 ppm of the H8 protons
of dGMP in a water buffer containing 50 mm KH2PO4 and of the two dGMP in the 7-deaza-A–Pt(dGMP)2 complex
50 mm KCl in a NMR tube in the dark at 37 °C. It is well (♦). The formation of platinum–d(GMP) complexes during
known that the chemical shifts of H8 and H1Ј of dGMP the incubation time was also confirmed by the appearance
complexed with platinum(II) species are very sensitive to of new upfield-shifted signals for the H2, H7, H8, and H1Ј
the geometry and the composition of the complex.[40] Thus, protons of 17 in the NMR spectra. In particular, the relative
we monitored the reaction by recording the 1H NMR spec- intensity of the NMR signals of H7 and H8 before and
trum every 30 min for the first 6 h and then every 6 h until after coordination with d(GMP) disclosed that more than
no further changes were noticed. 10% [D7]DMF was added 50% of 17 reacted with one or two molecules of d(GMP)
to the buffer to assure the complete water solubility of 17.
at incubation times within 12 and 36 h (Figure 2).
1
Figure 2. Aromatic region of the H NMR spectra recorded at different time points monitoring the reaction between 17 and a fourfold
excess amount of dGMP (50 mm KH2PO4, 50 mm KCl, pH 7.0, 37 °C in H2O/D2O = 9:1 containing 10% [D7]DMF). The signals of 17
and dGMP are labeled by using regular and bold-type fonts, respectively. The H8 NMR signals of dGMP complexed with 17 are labeled
with ᭹, ♦, and + (see also the Results and Discussion section).
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Properties of a Tethered Tubercidin–PtII Complex
Antiproliferative Studies on the A549 and Cal27 Cell Lines
Table 1. Sensitivity of the A549 and Cal27 cell lines to cisplatin,
16, and 17.
Compound 17, its unplatinated precursor 16, and cispla-
tin were examined by sulforhodamine B (SRB) assay[45] for
their capability to inhibit growth of the A549 and Cal27
cell lines. To this end, cisplatin was dissolved in phosphate-
buffered saline (PBS), whereas compound 16 and complex
17 were dissolved in 10% DMSO/PBS. All compounds were
diluted immediately to the final concentrations by using the
cell growth medium. The results showed that, although less
active than cisplatin, both compounds induced dose-de-
pendent growth inhibition in both cell lines after 96 h of
treatment (Figure 3). Unexpectedly, for the tested cell lines,
we observed that the antiproliferative activity of unplati-
nated precursor 16 was 1.5-fold higher than that of plati-
nated complex 17 (Table 1). Further studies are needed to
ascertain whether the lower activity of 17 is due to the
residual “tubercidin-like” activity found for 16 or to the
target “cisplatin-like alkylation” of purine bases by the
dichloroplatinum moiety.
[a]
Compound
IC50 at 96 h [μm] ϮSD
A549
Cal27
Cisplatin
16
17
2.69Ϯ0.80
23.62Ϯ4.83
36.20Ϯ2.72
1.04Ϯ0.20
26.44 Ϯ1.64
37.75 Ϯ5.58
[a] The half-maximal inhibitory concentration (IC50) values were
computed after 96 h of treatment (mean ϮSD from at least three
separate experiments performed in quadruplicate). SD: standard
deviation.
Conclusions
In this paper, we described the synthesis of a new nucleo-
side platinum(II) complex (i.e., 17) in which a cisplatin-like
unit is joined to 7-deazaadenosine through an amino alkyl
chain installed at the C6 position of purine. The cisplatin-
like capability of 17 to react with the purine bases of DNA
was confirmed by monitoring a model reaction with dGMP,
whereas preliminary antiproliferative data on 17 and on un-
platinated precursor 16 demonstrated that both com-
pounds, although less potent than cisplatin, were able to
inhibit the proliferation of the A549 and Cal27 human cell
lines in a dose-dependent manner. Interestingly, the anti-
proliferative activity of free diamine 16 was 1.5-fold higher
than that of platinated complex 17 on both cell lines. Stud-
ies are ongoing in our laboratories to assess the suitability
of compound 16 to chelate other metals involved in cancer
research and to construct novel unprecedented tubercidin
derivatives with polyaminoalkyl chains at the C6 position
of purine.
Experimental Section
General Methods: All reagents and solvents for the chemical syn-
theses were obtained from commercial sources and were used with-
out further purification. All cell culture media, serum, antibiotics,
and glutamine were purchased from LONZA (Basel, Switzerland).
Sulforhodamine B (SRB) was from ICN Biomedicals (Irvine, CA,
1
USA). H NMR and 13C NMR spectra were acquired with Varian
Mercury Plus 400 MHz and Varian UNITY Inova 500 MHz in-
struments by using CD3OD, (CD3)2SO, or C6D6 as solvents. A to-
tal of 450 μL of a 50 mm KH2PO4, 50 mm KCl, pH 7 buffer in H2O/
D2O (9:1) was used to record the 1H NMR spectra required to
monitor the reaction of 17 (dissolved in 50 μL of [D7]DMF) with
dGMP. A double pulsed gradient spin echo (DPFGSE) module
was included in the NMR pulse sequences to suppress the water
signal.[46,47] Chemical shifts are reported in parts per million (δ)
relative to the residual solvent signal [1H: CD2HOD δ = 3.31 ppm,
(CD3)(CD2H)SO δ = 2.54 ppm, C6D5H δ = 7.15 ppm; 13C: CD3OD
δ = 49.0 ppm, (CD3)2SO δ = 40.4 ppm, C6D6 δ = 128.6 ppm] and
were assigned by 2D NMR experiments. UV spectra were recorded
with a Jasco V-530 UV spectrophotometer. IR spectra were re-
corded with a Jasco FTIR 430 spectrophotometer. Optical rota-
tions were determined with a Jasco polarimeter by using a 1 dm
cell at 25 °C; concentrations are expressed in g100 mL–1. High-res-
olution MS spectra were recorded with a Thermo Orbitrap XL
mass spectrometer by using the electrospray ionization (ESI) tech-
Figure 3. Antiproliferative effect of cisplatin, 16, and 17 in the
A549 (top) and Cal27 (bottom) cell lines. Cell growth assessment
was performed by sulforhodamine B colorimetric assay (see the Ex-
perimental Section) and is expressed as the percentage of control
for each time point. Values are the mean ϮSD from at least three
independent experiments performed in quadruplicate.
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nique in positive mode. Elemental analyses were performed with a
Thermo Finnigan Flash EA 1112 CHN analyzer.
Compound 13: Racemic acid 12 (177 mg, 0.58 mmol) was dissolved
in dry DMF (4 mL) and then DIPEA (0.4 mL, 2.3 mmol), EDC
(222 mg, 1.2 mmol), and HOBt (157 mg, 1.2 mmol) were added.
The mixture was stirred for 30 min at room temperature. Com-
pound 11 (200 mg, 0.24 mmol), dissolved in dry DMF (2 mL), was
added to the previous solution, and the mixture was stirred for 16 h
at room temperature (TLC monitoring: n-hexane/AcOEt, 4:6). The
solvents were removed under reduced pressure, and the crude mate-
rial was diluted with AcOEt (20 mL) and extracted with brine (3ϫ
20 mL). The organic layer was separated, dried (Na2SO4), filtered,
and concentrated under reduced pressure. The crude material con-
taining compound 13 was purified by chromatography (silica gel,
EtOAc in n-hexane, gradient up to 70%). The fractions containing
the product were collected and concentrated to afford pure 13 as a
mixture of diastereomers. Amorphous white solid (200 mg, 83%).
1H NMR (400 MHz, CD3OD): δ = 8.19 (s, 2 H, 2ϫ2-H), 8.12–
8.07 (m, 4 H, arom.), 8.02–7.97 (m, 4 H, arom.), 7.91–7.86 (m, 4
H, arom.), 7.66–7.33 (complex signal, 20 H, 2ϫ8-H and arom.),
6.61 (d, J = 5.5 Hz, 2 H, 2ϫ1Ј-H), 6.25–6.19 (m, 2 H, 2ϫ2Ј-H),
6.18–6.12 (m, 2 H, 2ϫ3Ј-H), 4.93–4.78 (complex signal partially
covered by residual solvent signal, 4 H, 2ϫ4Ј-H and 2ϫ5Ј-Ha),
4.67 (dd, J = 3.6, 12.2 Hz, 2 H, 2ϫ5Ј-Hb), 4.15–4.08 (m, 2 H,
2ϫCH), 3.71–3.54 (m, 4 H, 2ϫCH2NH), 3.41 (dd, J = 4.7,
14.1 Hz, 2 H, 2ϫCHaNHBoc), 3.36–3.26 (2 H, 2ϫCHbNHBoc,
Cell Culture and Proliferation Assay: Human lung-cancer-derived
A549 cells and human tongue squamous-carcinoma-derived Cal27
cells were grown in adhesion in Dulbecco’s modified Eagle’s me-
dium. The medium was supplemented with 10% heat-inactivated
fetal bovine serum, penicillin (50 UmL–1), streptomycin
(500 μgmL–1), and glutamine (4 mmolL–1) in a humidified atmo-
sphere of 95% air and 5% CO2 at 37 °C. Cell proliferation of A549
and Cal27 was evaluated in the presence and absence of increasing
concentrations of the compounds by colorimetric assay with sulfo-
rhodamine B (SRB, ICN Biomedicals, Irvine, CA, USA). In detail,
1000cellswell–1 were seeded in 96-multiwell plates (Falcon, Becton
Dickinson Labware, Franklin Lakes, NJ, USA) in quadruplicate.
After 24 h, the cells were treated with the indicated compounds for
96 h and then the SRB assay was performed as described before.[45]
The growth was evaluated as percentage compared to untreated
cells.
Compound 10: A mixture of compound 8 (300 mg, 0.44 mmol), tert-
butyl (3-aminopropyl)carbamate (9; 383 mg, 2.2 mmol), and Et3N
(61 μL, 0.44 mmol) was heated at reflux in EtOH (12 mL). During
the reaction, a colorless solid, identified as compound 10, precipi-
tated. After 5 h (TLC monitoring: CH2Cl2/MeOH, 98:2), the sys-
tem was cooled to room temperature, and the solid was then fil-
tered, washed with cold EtOH, and dried. Colorless amorphous
solid (287 mg, 80%). [α]D = –122.1 (c = 0.1, CH2Cl2). 1H NMR
(400 MHz, C6D6): δ = 8.46 (s, 1 H, 2-H), 8.31–8.24 (m, 2 H, arom.),
8.02–7.92 (complex signal, 4 H, arom.), 7.20–6.81 (complex signal
partially covered by residual solvent signal, 10 H, 8-H and arom.),
6.75 (d, J = 5.6 Hz, 1 H, 1Ј-H), 6.37–6.31 (m, 1 H, 2Ј-H), 6.25–
6.16 (complex signal, 2 H, 3Ј-H and NH), 4.74–4.65 (m, 1 H, 4Ј-
H), 4.64–4.56 (br. t, J = 6.1 Hz, 1 H, NH), 4.40–4.32 (m, 2 H, 5Ј-
covered by residual solvent signal), 3.24–3-14 (m,
4 H,
2ϫCH2NHCO), 1.83–1.72 (m, 4 H, 2ϫCH2), 1.41 (s, 9 H, Boc),
1.39 (s, 9 H, Boc), 1.37 (s, 9 H, Boc), 1.36 (s, 9 H, Boc) ppm. 13C
NMR (100 MHz, CD3OD): δ = 173.2, 167.5, 166.7, 166.4, 158.9,
157.9, 157.7, 153.9, 150.4, 134.8, 134.7, 134.5, 130.9, 130.8, 130.7,
130.3, 129.9, 129.7, 129.6, 129.5, 122.5, 103.4, 89.7, 87.7, 81.4, 80.9,
80.5, 75.4, 72.9, 64.9, 57.4, 43.1, 38.4, 37.0, 30.7, 28.7 ppm. IR
(KBr pellet): ν = 3330, 2923, 1723, 1597, 1262, 1160, 1117,
˜
708 cm–1. UV (MeOH): λmax = 275 nm. HRMS (ESI): m/z =
H
a,b), 3.41–3.31 (m, 2 H, CH2NH), 2.96–2.87 (m, 2 H,
1000.3085 [M + H]+ (C48H55BrN7O12 requires 1000.3092).
CH2NHBoc), 1.44 (s, 9 H, Boc), 1.37–1.26 (m, 2 H, CH2) ppm.
13C NMR (100 MHz, C6D6, 40 °C): δ = 166.0, 165.3, 165.2, 156.9,
156.4, 153.7, 150.4, 133.3, 133.2, 130.4, 130.1, 129.8, 129.5, 128.8,
128.6, 128.5, 120.6, 103.0, 89.1, 86.9, 80.6, 78.6, 75.0, 72.3, 64.1,
Compound 14: Compound 13 (150 mg, 0.15 mmol) was dissolved
in MeOH (10 mL) in a Parr reactor; NaOAc (13 mg, 0.16 mmol)
and Pd/C (10% w/w, 42 mg, 0.04 mmol) were added and air was
removed by insufflating H2. The apparatus was then charged with
H2 (1.0 MPa), and the system was stirred for 2 h (TLC monitoring:
n-hexane/AcOEt, 3:7) at room temperature. H2 was removed, and
the mixture was filtered through a Celite 545 pad that was then
washed with MeOH (3ϫ 10 mL). The solvent was evaporated un-
37.5, 30.7, 28.6 ppm. IR (KBr pellet): ν = 3411, 3382, 2980, 2936,
˜
1728, 1687, 1602, 1562, 1513, 1451, 1270, 1175, 1127, 1027,
711 cm–1. UV (CH2Cl2): λmax = 235, 280 nm. HRMS (ESI): m/z =
836.1911 [M + Na]+ (C40H40BrN5O9Na requires 836.1907).
Compound 11: A solution of compound 10 (250 mg, 0.29 mmol) in
CH2Cl2 (1.0 mL) was cooled to 0 °C. TFA (1.0 mL) was added in der reduced pressure, and the crude was diluted with AcOEt
one portion. The mixture was then warmed to room temperature
and stirred for 1 h (TLC monitoring: CH2Cl2/MeOH, 8:2). Solvents
were evaporated under reduced pressure, and the crude material
containing compound 11 as its TFA salt was used for the next
step without purification. Oil (238 mg, 99%). [α]D = –76.8 (c = 0.5,
(20 mL) and extracted with brine (3ϫ 20 mL). The organic layer
was separated, dried (Na2SO4), filtered, and concentrated under
reduced pressure. The crude material containing compound 14 was
used for the next reaction step without purification. Oil (137 mg,
99%). 1H NMR (400 MHz, CD3OD): δ = 8.21 (s, 2 H, 2ϫ2-H),
CH2Cl2). 1H NMR (400 MHz, CD3OD): δ = 8.18 (s, 1 H, 2-H), 8.16–8.08 (m, 4 H, arom.), 8.04–7.97 (m, 4 H, arom.), 7.92–7.83
8.10–8.05 (m, 2 H, arom.), 8.01–7.96 (m, 2 H, arom.), 7.85–7.89
(m, 2 H, arom.), 7.65–7.30 (complex signal, 10 H, 8-H and arom.),
6.61 (d, J = 5.3 Hz, 1 H, 1Ј-H), 6.26–6.19 (m, 1 H, 2Ј-H), 6.16–
(m, 4 H, arom.), 7.66–7.34 (complex signal, 18 H, arom.), 7.23 (d,
J = 3.7 Hz, 2 H, 2ϫ8-H), 6.67 (d, J = 5.9 Hz, 2 H, 2ϫ1Ј-H), 6.58
(d, J = 3.7 Hz, 2 H, 2ϫ7-H), 6.28–6.20 (m, 2 H, 2ϫ2Ј-H), 6.19–
6.11 (m, 1 H, 3Ј-H), 4.95–4.80 (complex signal partially covered by 6.12 (m, 2 H, 2ϫ3Ј-H), 4.93–4.77 (complex signal partially covered
residual solvent signal, 2 H, 4Ј-H and 5Ј-Ha), 4.67 (dd, J = 11.7, by residual solvent signal, 4 H, 2ϫ4Ј-H and 2ϫ5Ј-Ha), 4.71–4.64
3.5 Hz, 1 H, 5Ј-Hb), 3.71 (t, J = 6.7 Hz, 2 H, CH2NH), 3.01 (t, J (m, 2 H, 2ϫ5Ј-Hb), 4.15–4.08 (m, 2 H, 2ϫCH), 3.70–3.45 (m, 4
= 7.2 Hz, 2 H, CH2NH3+), 2.08–1.97 (m, 2 H, CH2) ppm. 13C
NMR (100 MHz, CD3OD): δ = 167.5, 166.7, 166.4, 161.2 (q, J =
37.7 Hz), 156.6, 151.5, 149.8, 134.9, 134.6, 130.9, 130.8, 130.7,
130.2, 129.8, 129.6, 123.9, 115.8, 103.4, 89.8, 88.1, 81.5, 75.4, 72.8,
H, 2ϫCH2NH), 3.44–3.36 (m, 2 H, 2ϫCHaNHBoc), 3.36–3.26 (2
H, 2ϫCHbNHBoc, covered by residual solvent signal), 3.22–3.11
(m, 4 H, 2ϫCH2NHCO), 1.83–1.73 (m, 4 H, 2ϫCH2), 1.40 (s, 9
H, Boc), 1.38 (s, 9 H, Boc), 1.36 (s, 9 H, Boc), 1.35 (s, 9 H, Boc)
ppm. 13C NMR (100 MHz, CD3OD): δ = 173.0, 167.5, 166.7,
166.5, 158.9, 158.3, 157.7, 153.1, 150.8, 134.8, 134.7, 134.5, 130.9,
64.8, 38.5, 37.9, 28.7 ppm. IR (neat): ν = 3060, 2884, 1722, 1682,
˜
1599, 1448, 1269, 1204, 1127, 711 cm–1. UV (MeOH): λmax
277 nm. HRMS (ESI): m/z 736.1390 [M
(C35H32BrN5NaO7 requires 736.1383).
=
=
+
Na]+ 130.8, 130.7, 130.3, 129.9, 129.7, 129.6, 129.5, 122.5, 105.2, 101.6,
87.2, 81.2, 80.9, 80.5, 75.3, 73.0, 65.2, 57.5, 43.0, 38.5, 37.2, 30.6,
7554
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 7550–7556

Properties of a Tethered Tubercidin–PtII Complex
28.7 ppm. IR (neat): ν = 3341, 2972, 1723, 1602, 1503, 1262, 1160,
5.50–5.41 (br. s, 8 H, 2ϫPtNH2CH2 and 2ϫPtNH2CH), 5.29
(br. t, 2 H, 2ϫ5Ј-OH), 5.23 (br. d, 2 H, 2ϫ2Ј-OH), 5.09 (br. d, 2
H, 2ϫ3Ј-OH), 4.38–4.45 (m, 2 H, 2ϫ2Ј-H), 4.11–4.04 (m, 2 H,
2ϫ3Ј-H), 3.93–3.85 (m, 2 H, 2ϫ4Ј-H), 3.66–3.40 (complex signal
˜
1119, 708 cm–1. UV (MeOH): λmax = 276 nm. HRMS (ESI) m/z =
922.3989 [M + H]+ (C48H55N7O12 requires 922.3987).
Compound 15: Compound 14 (115 mg, 0.12 mmol) was dissolved
in MeOH (1.5 mL) and then MeONa (32 mg, 0.60 mmol) was
added. The solution was stirred for 2 h at room temperature (TLC
monitoring: AcOEt/MeOH, 9:1). The solution was neutralized with
a few drops of glacial acetic acid; the solvents were removed under
reduced pressure, and the crude was diluted with AcOEt (20 mL)
and extracted with brine (3ϫ 20 mL). The organic layer was sepa-
rated, dried (Na2SO4), filtered, and concentrated under reduced
pressure. The crude material was purified by column chromatog-
raphy (silica gel, MeOH in AcOEt, gradient up to 10%). The frac-
tions containing the product were collected and concentrated to
afford pure 15. Amorphous solid (67 mg, 95%). 1H NMR
(400 MHz, CD3OD): δ = 8.12 (s, 2 H, 2ϫ2-H), 7.25 (d, J = 3.6 Hz,
2 H, 2ϫ8-H), 6.57 (d, J = 3.6 Hz, 2 H, 2ϫ7-H), 5.96 (d, J =
6.6 Hz, 2 H, 2ϫ1Ј-H), 4.71–4.65 (m, 2 H, 2ϫ2Ј-H), 4.30–4.26 (m,
2 H, 2ϫ3Ј-H), 4.18–4.08 (complex signal, 4 H, 2ϫ4Ј-H and
2ϫCH), 3.84 (dd, J = 2.1, 12.4 Hz, 2 H, 2ϫ5Ј-Ha), 3.71 (dd, J =
2.3, 12.4 Hz, 2 H, 2ϫ5Ј-Hb), 3.68–3.48 (m, 4 H, 2ϫCH2NH),
partially covered by residual solvent signal, 14 H, 2ϫ5Ј-Ha,b
,
2ϫCH2NH, 2ϫPtNH2CH2, 2ϫPtNHCH), 3.19–3.11 (m, 4 H,
CH2NHCO), 1.80–1.66 (m, 4 H, 2ϫCH2) ppm. 13C NMR
(100 MHz, [D6]DMSO): δ = 167.0, 157.0, 152.3, 150.1, 123.1,
104.3, 100.1, 88.4, 86.0, 74.6, 71.6, 62.7, 61.9, 38.4, 37.7, 29.7 ppm.
IR (KBr pellet): ν = 3247, 1665, 1610, 1561, 1363, 1311, 1245,
˜
1089, 721 cm–1. UV (H2O): λmax = 274 nm. HRMS (ESI): m/z =
717.1531 [M
– Cl +
DMSO]+ (C19H33ClN7O6PtS requires
717.1549). C17H27Cl2N7O5Pt (675.44): calcd. C 30.23, H 4.03, N
14.52; found C 30.27, H 4.08, N 14.58.
Reaction of 17 with dGMP: Complex 17 (2 mg, 3.0ϫ10–3 mmol)
was dissolved in [D7]DMF (50 μL) and then added to 450 μL of a
solution of dGMP (4 equiv.) in H2O/D2O (9:1) containing 50 mm
KH2PO4 and 50 mm KCl, pH 7. The mixture was transferred to a
NMR tube and was incubated at 37 °C. Water-suppressed
1H NMR spectra (500 MHz, 37 °C) were then recorded as de-
scribed in the general methods section and in the text.
3.46–3.39 (m,
2
H, 2ϫCHaNHBoc), 3.36–3.26 (2 H,
2ϫCHbNHBoc, covered by residual solvent signal), 3.24–3.11 (m,
4 H, 2ϫCH2NHCO), 1.86–1.75 (m, 4 H, 2ϫCH2), 1.42 (s, 18 H,
2ϫBoc), 1.38 (s, 18 H, 2ϫBoc) ppm. 13C NMR (100 MHz,
CD3OD): δ = 173.0, 158.8, 158.3, 157.7, 152.2, 149.5, 124.7, 105.9,
100.0, 91.6, 87.3, 80.9, 80.5, 75.3, 72.7, 63.7, 57.4, 43.0, 38.4, 37.2,
Acknowledgments
This work was supported by POR Campania FESR 2007-2013 –
Rete delle Biotecnolgie in Campania (project “FARMA-
BIONET”).
30.6, 28.7 ppm. IR (KBr pellet): ν = 3307, 2917, 1695, 1607, 1503,
˜
1366, 1256, 1160, 1048 cm–1. UV (MeOH): λmax = 277 nm. HRMS
(ESI): m/z =610.3184 [M + H]+ (C27H44N7O9 requires 610.3201).
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and stirred for 1 h (TLC monitoring: CH2Cl2/MeOH, 8:2). The sol-
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the next reaction step without purification. Oil (33 mg, 99%). H
NMR (400 MHz, CD3OD): δ = 8.12 (s, 2 H, 2ϫ2-H), 7.27 (d, J =
3.7 Hz, 2 H, 2ϫ8-H), 6.58 (d, J = 3.7 Hz, 2 H, 2ϫ7-H), 5.95 (d, [7] L. R. Kelland, N. P. Farrell (Eds.), Platinum-Based Drugs in
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J = 6.6 Hz, 2 H, 2ϫ1Ј-H), 4.69–4.63 (m, 2 H, 2ϫ2Ј-H), 4.29–4.24
(m, 2 H, 2ϫ3Ј-H), 4.13–4.08 (m, 2 H, 2ϫ4Ј-H), 3.84 (dd, J = 2.6,
12.4 Hz, 2 H, 2ϫ5Ј-Ha), 3.72 (dd, J = 2.8, 12.4 Hz, 2 H, 2ϫ5Ј-
Hb), 3.58 (t, J = 6.7 Hz, 4 H, 2ϫCH2NH), 3.33–3.26 (6 H, covered
by residual solvent signal, 2ϫCH and 2ϫCH2NHCO), 2.85 (dd,
J = 5.0, 13.1 Hz, 2 H, 2ϫCHaNH2), 2.85 (dd, J = 6.7, 13.1 Hz, 2
H, 2ϫCHbNH2), 1.91–1.80 (m, 4 H, 2ϫCH2) ppm. 13C NMR
(100 MHz, CD3OD): δ = 176.1, 158.1, 152.0, 149.7, 124.6, 105.9,
100.1, 91.5, 87.3, 75.3, 72.7, 63.7, 57.7, 46.7, 38.9, 37.7, 30.4 ppm.
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˜
UV (MeOH): λmax = 274 nm. HRMS (ESI): m/z = 410.2147 [M +
H]+ (C17H28N7O5 requires 410.2152).
Compound 17: Compound 16 (20 mg, 0.031 mmol) was dissolved
in H2O/MeOH (1:1, 1 mL). K2PtCl4 (13 mg, 0.031 mmol) was
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then dried. H NMR (400 MHz, [D6]DMSO): δ = 8.21 (br. t, 2 H,
2ϫNHCO), 8.12 (s,
2 H, 2ϫ2-H), 7.55–7.50 (m, 2 H,
2ϫNHCH2), 7.33 (br. d, J = 3.6 Hz, 2 H, 2ϫ8-H), 6.60 (br. d, J
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Eur. J. Org. Chem. 2015, 7550–7556
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Received: July 29, 2015
Published Online: November 3, 2015
7556
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Eur. J. Org. Chem. 2015, 7550–7556