Glycosylated Platinum(IV) Complexes as Substrates for Glucose Transporters (GLUTs) and Organic Cation Transporters (OCTs) Exhibited Cancer Targeting and Human Serum Albumin Binding Properties for Drug Delivery

Article abstract of DOI:10.1021/acs.jmedchem.7b00433

Glycosylated platinum(IV) complexes were synthesized as substrates for GLUTs and OCTs for the first time, and the cytotoxicity and detailed mechanism were determined in vitro and in vivo. Galactoside Pt(IV), glucoside Pt(IV), and mannoside Pt(IV) were highly cytotoxic and showed specific cancer-targeting properties in vitro and in vivo. Glycosylated platinum(IV) complexes 5, 6, 7, and 8 (IC₅₀ 0.24-3.97 μM) had better antitumor activity of nearly 166-fold higher than the positive controls cisplatin (1a), oxaliplatin (3a), and satraplatin (5a). The presence of a hexadecanoic chain allowed binding with human serum albumin (HSA) for drug delivery, which not only enhanced the stability of the inert platinum(IV) prodrugs but also decreased their reduction by reductants present in human whole blood. Their preferential accumulation in cancer cells compared to noncancerous cells (293T and 3T3 cells) suggested that they were potentially safe for clinical therapeutic use.

Full text of DOI:10.1021/acs.jmedchem.7b00433

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Article
Glycosylated Platinum(IV) Complexes as Substrates for Glucose Transporters
(GLUTs) and Organic Cation Transporters (OCTs) Exhibited Cancer
Targeting and Human Serum Albumin Binding Properties for Drug Delivery
Jing Ma, Qingpeng Wang, Zhonglv Huang, Xiande Yang,
Quandeng Nie, Wenpei Hao, Peng George Wang, and Xin Wang
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017
Downloaded from http://pubs.acs.org on June 12, 2017
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Glycosylated Platinum(IV) Complexes as
Substrates for Glucose Transporters (GLUTs)
and Organic Cation Transporters (OCTs)
Exhibited Cancer Targeting and Human Serum
Albumin Binding Properties for Drug Delivery
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†,‡,&
†,*,&
Jing Ma
Qingpeng Wang,
Zhonglv Huang,† Xiande Yang,† Quandeng Nie,† Wenpei
Hao,† Peng George Wang†,* and Xin Wang†, *
† College of Pharmacy, Tianjin Key Laboratory of Molecular Drug Research, State Key
Laboratory of Elementoꢀorganic Chemistry, Nankai University, Tianjin 300071 (P.R. China).
‡ Pharmaceutical College, Henan University, Kaifeng 475004, Henan, China
* Institute of Biopharmaceutical Research, Liaocheng University, Liaocheng 252059, P. R. China
&The authors contribute equally to this work.
KEYWORDS: antitumor, glycosylation, platinum, cancer
ABSTRACT Glycosylated platinum(IV) complexes were synthesized as substrates for GLUTs
and OCTs for the first time, and the cytotoxicity, and detailed mechanism were determined in
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vitro and in vivo. Galactoside Pt(IV), glucoside Pt(IV) and mannoside Pt(IV) were highly
cytotoxic and showed specific cancerꢀtargeting properties in vitro and in vivo. Glycosylated
platinum(IV) complexes 5, 6, 7 and 8 (IC50 0.24 ꢁMꢀ3.97 ꢁM) had better antitumor activity of
nearly 166ꢀfold higher than the positive controls cisplatin (1a), oxaliplatin (3a) and satraplatin
(5a). The presence of a hexadecanoic chain allowed binding with human serum albumin (HSA)
for drug delivery, which not only enhanced the stability of the inert platinum(IV) prodrugs but
also decreased their reduction by reductants present in human whole blood. Their preferential
accumulation in cancer cells compared to noncancerous cells (293T and 3T3 cells) suggested
that they were potentially safe for clinical therapeutic use.
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INTRODUCTION
The FDAꢀapproved platinum(II) drugs, such as cisplatin, carboplatin, and oxaliplatin, are widely
used in all firstꢀline clinical cancer treatments, including ovarian, lung, head, neck and colorectal
cancer. They induce apoptosis in cancer cells primarily via DNA damage.1a,1b However, their
drawbacks, such as toxicity, tumor recurrence and deactivation, remain deadly and problematic
and originate from a lack of tumor selectivity and poor stability.2,3a,3b Recently, platinum
anticancer drug candidates were designed as inert platinum(IV) prodrugs that can be activated by
intracellular reduction following cellular uptake.4 Reductants such as ascorbic acid and
glutathione occur at higher concentrations in tumor cells than in the blood and normal tissues,
which makes them good targets. Satraplatin, the most promising platinum(IV) prodrug, is orally
absorbed via passive diffusion and has greater potential because of its better stability, lower
toxicity, and higher bloodꢀcirculation time than cisplatin. The two functional “axial ligands” of
platinum(IV) prodrugs can be used to tune the physical and chemical properties of the complex
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and impart targeting and additional biological activity without altering the structure of the active
pharmacophore.5a,5b,5c,5d,5e,5f,5g,5h,5i
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The potential of glycoconjugation for diagnosis and therapy has been realized, and it has
become an appealing strategy for the targeted delivery of anticancer drugs.6a,6b,6c Based on the
“the Warburg effect”, glucose transporters (GLUTs) and sodiumꢀdependent glucose transporters
(SGLTs) are widely overexpressed in many human cancers.7a,7b,7c Our group and others recently,
developed glycoconjugation by platinum(II).8a,8b,8c,8d,8e,8f,8g Our group also first designed and
synthesized a series of glycosylated Pt(IV) complexes protected by acetyl groups.8h,8i To explore
whether the transformation of glycosylated Pt(IV) complexes was associated with GLUTs.
Phlorizin was employed in the present study as a GLUT inhibitor to study its impact on their
antitumor activities. Unfortunately, GLUT inhibition did not affect the IC50 values of the tested
complexes, indicating that the tested glycosylated platinum(IV) complexes were not transferred
by glucose transporters. However, the unprotected glycosylated platinum(II) conjugates not only
exploited both GLUTs and OCTs, but compared with normal epithelial cells in vitro, the
conjugates also accumulated in and killed the cancer cells, demonstrating the potential of
glycoconjugation for the selective destruction of cancer cells by platinum compounds.8f In
addition, the cellular uptake, cytotoxicity profile and glucose transporter 1 (GLUT1) specificity
can be affected by variation in the position of Dꢀglucose substitution in the unprotected
glycosylated platinum(II) conjugates.8g However, the labile platinum(II) prodrugs would react
with nucleophiles in human whole blood and never reach the tumor, which limits their
bioavailability and precludes oral administration.9a,9b The reasons that the bioavailability is
limited are its reactivity and lack of selectivity. The design of platinum(IV) prodrugs is
predicated on the assumption that the low spin octahedral d6 Pt(IV) complexes will not undergo
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hydrolysis or ligand substitutions in the blood but will be activated in cells by reductive
elimination, releasing the cytotoxic squareꢀplanar Pt(II) drug. Consequently, a series of cancerꢀ
targeting unprotected glycosylated Pt(IV) complexes were designed and synthesized.
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Liposomes,10a
polymers,10b
inorganic
nanoꢀmaterials,10c,10d,10e,10f
metal–organic
frameworks,10g and calcium phosphate (CaP) nanoparticles10h have been recently used to
synthesize platinum(IV) prodrugs for drug delivery. It was also reported that the platinum(IV)
prodrugs that contain a hexadecyl chain can interact with HSA to form a platinumꢀprotein
complex for drug delivery and that the interaction was nonꢀcovalent. Extraction with octanol can
completely remove the prodrug from an aqueous solution of HSA.11 As an extension of our study
on novel potent antitumor agents, it was of great interest for us to design and synthesize a series
of glycosylated Pt(IV) analogues that contain a hexadecanoic chain to produce a dualꢀtargeting
mechanism. In addition to interaction with HSA during drug delivery, the hexadecanoic chain
can also enhance the lipophilicity of the molecule, which can affect the ability of a platinum(IV)
prodrug to enter a tumor cell.
We designed, synthesized and biologically evaluated a novel series of dualꢀfunctionalized
glycosylated platinum(IV) complexes as antitumor agents for the first time. The details of the
mechanism of glycosylated platinum(IV) complexes 1ꢀ8 (Figure 1a) were also studied. It was
revealed that the targeting and activity can be improved by the incorporation of glycuronic acid
into an antitumor agent.12a Therefore, we designed the Pt(IV) compounds 1ꢀ3. Then, we also
designed the glycoside compounds 4ꢀ8, which incorporated variable aliphatic chains of different
lengths, to determine whether varying the substitution position or the chain length could alter the
cellular uptake, cytotoxicity profile and the specificity. We selected the clinically widely used
oxaliplatin as the platinum core. These serial complexes contained different glycosyl moieties,
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including glucose, galactose and mannose, and their impact on the antitumor potency could be
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evaluated in vitro and in vivo.
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Figure 1. a). Structures of 1ꢀ8 and 1aꢀ5a as positive control. Docking studies of 6 with HSA and
GLUT1. b). 6 interacting with a variety of amino acid residues of GLUT1 (pdb code 4PYP). c).
The lowest energy structure and 6 buried beneath the protein surface (HSA pdb code 1E7H). d).
6 interacting with a variety of amino acid residues of HSA (pdb code 1E7H).
RESULTS AND DISCUSSION
Synthesis of target compounds
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To the best of our knowledge, the synthetic route of 1ꢀ8 given in detail in the supporting
information has not been previously reported. The oxidation of cisplatin and oxaliplatin by
hydrogen peroxide in water afforded 2a and 4a. The synthetic route of the target compounds 1
and 4 is shown in Scheme 1. Compounds 2 and 3 were prepared according to the procedure
described for compound 1 and 5ꢀ8 according to the procedure described for compound 4. The
asymmetrically functionalized Pt(IV) compounds 1ꢀ8 can be obtained by the reaction of a
solution of 10b, 12b, 14b, 27b, 29b, 31b, 33b and 35b in anhydrous DCM. BCl3 (1 M) was
added at –78 for 2 h. Then, the DCM was removed and purified by column chromatography.
Characterization.
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The Pt(IV) compounds 1ꢀ8 were fully characterized by (1H and C) NMR spectroscopy and
electrospray ionization mass spectrometry (ESIꢀMS). The [M+NH4]+ parent ions of compounds 1,
2 and 3 and the [M+H]+ parent ions of compounds 4, 5, 6, 7 and 8 were readily observed using
ESIꢀMS. Characteristic resonances could be observed at approximately 1.18 ppm ꢀ1.30 ppm
1
(CH2) and 0.79 ppm – 0.92 ppm (CH3) in H NMR and were assigned to the hexadecanoic acid
ligand. Resonances at 3.01 ppm ꢀ 2.65 ppm (1), 2.95 ppm and 2.79 ppm (2), 2.94 ppm ꢀ 2.65 (3),
2.78 ppm (4), 2.80 ppm (5), 2.89 ppm ꢀ 2.66 ppm (6), 2.71 ppm and 1.18 ppm (7) and 2.78 ppm
1
(8) of (CH)2 in H NMR were assigned to the oxaliplatin core. We could observe characteristic
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resonances by C NMR at approximately 183.11 ppm, 178.11 ppm, 176.63 ppm and 165.69
ppm (1), 182.78 ppm, 178.31 ppm, 166.07 ppm and 129.46 ppm (2), 183.17 ppm, 178.05 ppm
and 165.50 ppm (3), 183.21 ppm, 182.66 ppm, 165.43 ppm and 128.02 ppm (4), 183.18 ppm,
182.61 ppm and 165.44 ppm (5), 183.19 ppm, 182.63 ppm and 165.40 ppm (6), 183.10 ppm,
182.81 ppm and 165.38 ppm (7) and 183.19 ppm, 183.06 ppm and 165.27 ppm (8) were assigned
to the carbonyl group.
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Docking studies of the dual targets of platinum(IV) pro-drugs.
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First, we conducted docking studies of the dual targets of the platinum(IV) prodrugs to
investigate the interaction between HSAꢀ612b (pdb code 1E7H) (Figure 1b and 1c) and GLUT1ꢀ
612c (pdb code 4PYP) (Figure 1d). 6 can nonꢀcovalently interact with both HSA and GLUT1.
Hydrogenꢀbonding interactions occurred with ASNꢀ415, ASNꢀ288, TYRꢀ292, THRꢀ30 and
GLNꢀ282 by ligand exchange to form glycosylated acid moieties because it occurred in the
interaction between GLUT1 (pdb code 4PYP) and 6. TRPꢀ388 could nonꢀcovalently interact
with the carboxylate platinum ligand center.
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Scheme 1. Synthetic route of target compounds 1 and 4. Conditions and reagents: (a)
BnOH,CH3COCl 50 °C, 2 h. (b) TrtCl, DMAP Py, 50 °C (c) BnBr, NaH, DMF, rt, overnight (d)
HBr/AcOH 10 °C (e) K2Cr2O7, H2SO4, acetone, 55 °C, 2 h (f) C2O2Cl2 DMF(cat.) (g) Dry
acetone rt, 2d (h) BCl3, DCM ꢀ78 °C, 2 h (i) Ac2O, AcONa 80 °C, 6 h (j) pꢀtoluenethiol BF3
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Et2O, DCM (k) MeONa, MeOH rt, overnight (l) BnBr, NaH, DMF, overnight (m) TMSOTf,
NIS, CH3CN, 2 h (n) NaOH (1 M), 24 h.
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Hydrogenꢀbonding interactions of 6 with HSA (pdb code 1E7H) were observed in the pocket,
located in the subdomain IIA (known as Sudlow’s site I) with a model of the lowest energy
structure. ARGꢀ222 and LYSꢀ199 of HSA were found to nonꢀcovalently interact with a platinum
ligand. Hydrogen bonding interactions of glycosylated acid moieties occurred with PROꢀ447,
ASPꢀ451, GLUꢀ292 and ASNꢀ295 of HSA using nonꢀcovalent bonds. The lipophilic C16 tail of
fatty acid moieties of 6 is coiled into a hydophobic channel lined by Tyr150, Leu238, Leu219,
Leu234, Phe223, Ile264, Ala261, Ile290, and Tyr150 using nonꢀcovalent bonds.
The above results indicated stable and lowꢀenergy interactions of HSAꢀ6 and GLUT1ꢀ6, which
contributed greatly to the dual target of a pyranosideꢀconjugated platinum(IV) complex. We can
also deduce that once the target compound entered the human blood, it would bind with HSA
during drug delivery, target the tumor tissue, and then preferentially accumulate in the cancer
cells because of “the Warburg effect”.
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Antitumor activities in vitro
The cytotoxicity of compounds 1–8 and the positive controls 1aꢀ5a (Figure 1) were evaluated
in seven human cancer cell lines using the MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide) assay. The inhibitory effects (IC50 in µM) are summarized in
Table 1.
The cytotoxicity of compounds 1ꢀ8 show a rather broad antitumor spectrum and are greater
than that of cisplatin, oxaliplatin and satraplatin. Furthermore, the introduction of the pyranoside
to Pt(IV) complexes prominently enhances the activity, in contrast to the oxoplatins 2a and 4a
precursors. The HeLa (IC50 = 0.44–12.74 µM) and MCFꢀ7 (IC50 = 0.22–15.62 µM) cell lines are
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more sensitive to compounds 1ꢀ8 than the others. We can see that different structural
carbohydrates, different linkers and different pyranoside positions also have a great effect on the
bioactivity of the target compounds. Except for compound 4, the C1ꢀmodified glycoside
compounds (5ꢀ8) are more potent than the C6ꢀmodified pyranoside acids (1ꢀ3). In addition, the
glycosylated Pt(IV) compounds almost fully overcome the drug resistance of A549R cells,
especially the compounds 1 and 2 with RF 0.66 and 0.45, respectively.
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Of the C6ꢀmodified pyranoside acids derivatives (1ꢀ3), compound 1 effectively inhibits the
growth of all the tested cell lines with IC50 values of 1.07ꢀ8.81 µM. The introduction of glucose
and mannose is more potent than of galactose (1 > 2 > 3). It is worth mentioning that galactoside
derivative 3 shows better inhibition effects in HeLa cells than in the other cancer cell lines,
indicating the targeting properties to HeLa. Additionally, prostatic carcinoma (LNCaP and PC3)
shows more sensitivity to compounds 1 and 2.
At the same time, for the C1ꢀsubstituted glycoside compounds (4ꢀ8), when the lengths of the
linkage change from 5 (8), 4 (7) to 3 (4), the antitumor activities dramatically decrease, which is
opposite to the result with the glucoside platinum(II) prodrugs.8fꢀ8g This may result from the
significant different spatial structure of Pt(II) and Pt(IV) or the combination of transport and EPR
effects. Compared with compound 4, the glucoside (6) and galactoside (5) Pt(IV) complexes
show better cytotoxicity to a panel of cancer cells, which is also different from the C6ꢀsubstituted
pyranoside acid derivatives (1ꢀ3).
Above all, compounds 5, 6, 7 and 8 show an especially better antitumor activity with an IC50
of 0.24 µM to 3.97 µM, which is almost 166ꢀfold higher than cisplatin, oxaliplatin and
satraplatin especially to A549R, LNCaP and PC3 cells, which have been reported to be resistant
to cisplatin and oxaliplatin.
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All of the above results indicate that Pt(IV) prodrugs containing different sugars such as
glucose, mannose and galactose show significant cytotoxicity to cancer cells. It is also worth
mentioning that they show different cytotoxicity profiles for different cancer cells. The chain
length and positional isomers also have effects. These properties suggest that these compounds
show great potential for further investigation.
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Table 1. Cytotoxicity profiles of glycosylated Pt(IV) complexes (IC50 in µM) in seven human
carcinoma cell lines. (a RF: Resistant factor = IC50(A549R)/IC50(A549).)
HeLa
MCFꢀ7
LNCaP
PC3
HepGꢀ2
A549
A549R
RFa
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3
4
5
6
7
8
1.09±1.89
2.27±1.87
1.59±0.67
6.36±1.44
0.88±1.87
13.33±1.67 38.10±0.78 >100
8.81±0.38
12.87±0.89 2.77±0.49
6.33±1.45
1.22±1.58
3.47±0.49
14.83±1.67 10.55±1.49 >100
1.63±0.39
24.51±1.78 10.91±0.48 0.45
1.07±1.90
0.66
/
12.74±0.78 15.62±0.98 >100
63.69±0.78 >100
12.05±0.48 48.45±1.76 4.02
0.89±0.56
0.44±0.45
1.57±0.78
0.74±0.65
0.25±0.44
0.19±0.47
0.31±0.45
0.22±0.78
2.28±0.56
3.03±0.12
2.31±1.67
3.97±0.45
2.94±0.45
0.43±1.56
0.24±0.56
0.53±1.89
0.24±1.56
0.72±0.79
0.98±0.89
0.44±0.78
0.40±1.31
0.31±1.98
10.85±1.98 35.78±1.69 3.3
13.40±2.57
10.06±1.20 39.67±0.69 3.94
3.52±0.67
1.10±1.69
0.76±1.69
0.65±0.69
3.59
2.5
1.9
2.1
0.79±1.79
1.70±1.46
1.68±1.79
1a 0.80±0.93
23.83±0.89 22.85±1.45 3.94±0.39
2a 11.36±1.56 27.01±0.34 50.78±0.79 61.42±0.67 50.00±1.79 >100
3a 3.96±0.75 5.22±2.78 16.78±0.34 39.74±0.76 6.64±1.79
4a 15.78±0.34 >100 22.97±0.89 >100 >100
5a 3.10±0.89 8.01±0.56 5.23±1.77 16.77±1.56 6.70±1.89
/
>100
2.17±1.68
29.67±0.64
9.96±0.57
/
4.59
The effect of incubation time of compounds 6, 7, 8 and 5a on the IC50 values for HeLa and
MCF-7 cells.
Based on previous reports, if compounds 1ꢀ8 exhibit cancerꢀtargeting abilities based on
proteinꢀmediated transport, which become saturated at longer time scales, the initial rate of
accumulation of compounds 1ꢀ8 in cells would be more rapid than that for compound 5a with
passive uptake, which is slower but does not become saturated. We subsequently investigated the
effect of the incubation time on the outcomes of these assays. We incubation the MTT assay for
8 h, 24 h, 30 h and 48 h with HeLa or MCFꢀ7 cells.
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The results revealed that the initial rate of accumulation of compounds 6, 7 and 8, was faster
than that for compound 5a (Figures S1a and S1b). Almost all of the compounds were observed
intracellularly after 8 h incubation in the MTT assay for compounds 6, 7 and 8. The rapid rate of
accumulation in the cell indicates the possibility that the glycosylated Pt(IV) compounds are
transported by the targeted transporters.
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Effects of EDG(1), phloretin(2), Ctd.(3), and the mixture of EDG and Ctd.(4) on the IC50
values of MCF-7 cells.
To understand whether the targeted transporters worked, we investigated the mechanism of
compounds 1 and 6 in MCFꢀ7 cells, which were coꢀincubated with the GLUT1 inhibitors 4,6ꢀ
oethylideneꢀaꢀdꢀglucose (EDG) (1) and phloretin (2), the OCT2 inhibitor Ctd. (3), and a
combination of EDG and Ctd. (4). Both the GLUT1 and OCT2 are overexpressed in MCFꢀ7 cells
8f. These results suggested that the cytotoxicity of 6 (IC50 = 0.57 µM and 0.68 µM) with EDG
and phloretin were regulated via the glucose transporters because IC50 values were elevated
compared with the 48 h IC50 of 6 (IC50 = 0.19 µM)) with no inhibitor. However, the glucuronic
acidꢀmodified compound 1 with an IC50 of 6.36 µM had respective cytotoxicity values of 5.98
µM and 5.89 µM with EDG and phloretin, but it showed no relationship with GLUT1. The
magnitude of the cytotoxicity in the presence of EDG and phloretin was in the order 6 > 1
(Figure 2a), which was also observed for the inhibition of cellular uptake (Figure 2c) in a breast
carcinoma cell line (MCFꢀ7) that had a high level of GLUT1 expression. The results remind us
that when we design sugarꢀplatinum(IV) complexes, the C1ꢀmodified prodrugs can show a close
relationship with the tranporters. EDG and phloretin do not affect compounds 1a and 3a, as
evidenced by their IC50 values, which were not greatly different.
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Furthermore, in the presence of Ctd. and a combination of EDG and Ctd., if IC50 and cellular
uptake were greatly different, the energyꢀdependent organic cation transporters (OCTs)
contributed. Figure 2a shows that the IC50 value of 6 (IC50 = 5.95 µM) in the presence of a
combination of EDG and Ctd. was 31ꢀfold higher than that in the 48 h incubation (IC50 = 0.19
µM) in the cytotoxicity assay and demonstrated greater sensitivity to the OCTs than the C6ꢀ
substituted pyranoside acids compound (1). The same results in the cellular uptake inhibition
also occurred with Ctd. (Figure 2d) It has been reported that the organic cation transporter 2
(OCT2), which is a primary factor responsible for the sensitivity of colorectal cancer to
oxaliplatin, plays a considerable role in the cellular accumulation and consequent cytotoxicity of
platinum complexes containing the ((1R, 2R)ꢀcyclohexaneꢀ1,2ꢀdiamine (DACH) ligand.13a,13b In
our study, the IC50 values for oxaliplatin with Ctd. (IC50 =10.89 µM) are 2.09ꢀfold higher than
that without Ctd.(IC50 = 5.22 µM) and cellular uptake are 1.38ꢀfold higher than that with Ctd.,
indicating that OCT2 also affects the consequent cytotoxicity and cellular uptake of oxaliplatin
(Figure 2b and 2d). However, OCT2 did not contribute to compounds 5, 7 and 8 (Figure 2b).
The reason may be that OCT2 had a close relationship with the glucoside Pt(IV) prodrugs but not
with the mannoside and galactoside complexes. As a result, compound 6 significantly exploits
both glucose and organic cation transporters, both of which are widely overexpressed in cancer
cells. Different sensitivities of GlcꢀPt(IV), GalꢀPt(IV) and ManꢀPt(IV) to the OCTs also occur.
The above observations indicate that the C1ꢀmodified glycoside compounds are superior to the
C6ꢀmodified pyranoside acids derivatives for targeting transport. This phenomenon may be
because of the differences in the spatial structure of the squareꢀplanar platinum(II) and the
octahedral platinum(IV). The above results also suggest that the pyranoside conjugated
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platinum(IV) complexes have important cancerꢀtargeting properties and provide new insight into
the potential of the glycosylated Pt(IV) anticancer prodrugs.
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without Ctd.
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Figure 2. a). Effects of EDG(1), phloretin(2), Ctd.(3), and a mixture of EDG and Ctd.(4) on the
IC50 values of compounds 1, 6 and 1a in MCFꢀ7 cells. b). Effects of Ctd.(3) on the IC50 values of
on the compounds 5, 7, 8 and 3a in MCFꢀ7 cells. c). Effects of EDG on the cellular uptake of
compounds 1, 6 and 3a in MCFꢀ7 cells. d). Effect of Ctd. on the cellular uptake of compounds 1,
6 and 3a in MCFꢀ7 cells.
Whole cell uptake studies and DNA platination for HeLa and MCF-7 cells.
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Subsequently, the intracellular distribution of compounds 5, 6, 8, 3a and 5a that have potent
cytotoxicity in HeLa and MCFꢀ7 cells was quantified by inductively coupled plasma mass
spectrometry (ICPꢀMS) using 196Pt detection as shown in Figure S2.
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After incubation with compounds 5, 6 and 8, the accumulation of platinum in MCFꢀ7 cells
was 5.11ꢀ to 17.39ꢀfold and 1.41ꢀ to 4.79ꢀfold higher than that of 3a and 5a, respectively. In
HeLa cells, the accumulation was 7.25ꢀ to 30.96ꢀfold and 1.41ꢀ to 6.07ꢀfold higher than that of
3a and 5a. Platinum in the DNA isolated from HeLa and MCFꢀ7 cells was also measured and
was 1.91ꢀ to 8.34ꢀfold and 1.99ꢀ to 8.69ꢀfold higher than that with 3a and 5a, respectively.
Nuclear DNA was considered as a potential subcellular target and a correlation between the
intracellular platinum accumulation and antitumor activity was confirmed. An annexin V/PI
coupled flow cytometric analysis (Figures S4) clearly indicated that compounds 6 and 7
effectively induced cell damage in A549 cells.
Viability of the cancer cells such as breast cancer MCF-7, breast cancer HeLa, and normal
cells such as 293T and 3T3 in a 48 h incubation assay
An ideal anticancer compound should be selective for cancer cells over normal healthy cells,
thereby mitigating undesired toxic side effects associated with chemotherapy. Platinum(IV) itself
always shows less toxicity to normal cells than platinum(II) drugs.
Therefore, we determined the viability of breast cancer MCFꢀ7 cells, breast cancer HeLa cells,
normal kidney epithelial 293Tcells and mouse embryo 3T3 fibroblasts in a 48 h incubation assay
to evaluate their selectivity for cancer cells to mitigate undesired toxic side effects associated
with chemotherapy. The pyranoside conjugated platinum(IV) complexes 5ꢀ8 show less
cytotoxicity to 3T3 and 293T cells compared with the more sensitive HeLa and MCFꢀ7 cells.
(Figures 3 and S3) For example, the cytotoxicity of 6 for 3T3 cells (IC50 = 2.54 µM) was 13.37ꢀ
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fold higher than that for MCFꢀ7 cells (IC50 = 0.19 µM). Compared to 1a and 3a in the clinic,
they are also even safer for normal cells.
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We can deduce that this phenomenon may contribute to the part of pyranoside with cancerꢀ
targeting properties and the platinum(IV) itself regaining its cytotoxicity via reductants such as
ascorbic acid and glutathione presenting higher concentrations in the tumor cells than in the
blood and normal tissues. It is worth mentioning that these properties cannot be realized by Pt(II)
drugs.
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Figure 3. Viability of MCFꢀ7 cells, HeLa cells, 293T cells and 3T3 cells in a 48 h incubation
assay.
Stability of 6 in RPMI 1640 and the whole human blood
One of most important advantages of inert Pt(IV) complexes is that they are more stable than
Pt(II) prodrugs. This is also a key point for the facilitated intravenousꢀtoꢀoral switch of
satraplatin. As previously reported for the activation of structurally similar platinum compounds
by nucleophiles, the stability of 6 in RPMI 1640 medium and human whole blood was evaluated.
The results are shown in Figure 4.
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In RPMI medium, no significant decomposition of compound 6 was up to 12 h, and 66% of 6
remained unchanged even after 48 h, and a halfꢀlife (t1/2) of up to 170 h in RPMI 1640 medium
suggested that it was highly stable in biological media, which makes it superior to platinum(II)
drugs. (Figure 4a and S5). Compound 6 was more stable in human whole blood, with a t1/2 of
nearly 20 h, compared with 21.6 min and 6.3 min for cisplatin and satraplatin respectively. This
suggested a significant potential for a facilitated intravenous to oral switch of glycosylated
platinum(IV) prodrugs with cancerꢀtargeting properties.
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The stability of glycosylated platinum(IV) complexes 6 is more advantageous than the
platinum(II) prodrugs, which may contribute greatly to the development of platinum(IV)
prodrugs for an intravenous to oral switch.
a.
b.
Stability of 6 in RPMI 1640
Stability of 6 in whole human blood
120%
100%
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Time (h)
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Figure 4. Stability of 6 in whole human blood (a) and RPMI 1640 (b).
Binding studies of 6 with albumin using RP-HPLC
We previously assumed that the significant stability of compound 6 in human blood may have
resulted from the HSA binding properties of the hexadecanoic chain. Based on these
observations, we also determined the HSA binding properties of 6. The results showed that the
pyranoside conjugated platinum(IV) complex 6 containing a hexadecanoic acid chain as an axial
ligand preferably interacted with HSA. (Figure 5a). Conversely, oxaliplatin did not bind HSA
(Figure S7). Docking studies have shown that the interaction is the combination of nonꢀcovalent
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interactions of the hexadecyl chain and hydrogenꢀbond interactions of the pyranoside, suggesting
that the prodrug is buried below the surface of the protein.
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We can also presume from the results that the rate of reduction of 6 by ascorbate can be
reduced as a result of the interaction with HSA during drug delivery. Consequently, the reduction
and DNA binding properties of 6 with and without HSA was studied as shown in Fig. 5b. At 24
h, 48 h, 96 h and 144 h, the remaining compound 6 for the groups that contained HSA was
always less than those without HSA. In addition, the remaining HSA also gradually decreased.
The smaller reduction of 6 by ascorbate in human blood as the result of the interaction with HSA
during drug delivery can further enhance the stability of platinum(IV) prodrugs and their
accumulation in cancer cells.
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Figure 5. a) Binding studies of 6 with HSA after 0 h, 24 h, 48 h and 196 h. b) Reduction of 6 by
ascorbate with and without HSA.
Reduction and DNA binding properties of 6 and oxaliplatin with and without ascorbic acid
We also investigated the reduction and DNA binding properties of 6 and 3a with and without
ascorbic acid for the inherent proficiency of the Pt prodrugs to bind at the N7 position of guanine
bases, thus leading to DNA damage and inducing cell death.14a,14b,14c 5’ꢀdGMP was used as a
model for DNA. Adducts can only be observed in the presence of the reductant ascorbic acid for
RPꢀHPLC analysis at 37 °C in Fig. S6. The fractions that contained unknown peaks were
separated and identified by ESIꢀMS analysis observing the same products with 3a. The results
showed that the bisꢀsubstituted products of 6 originating from the combination with 5’ꢀdGMP of
3a, were generated after the reduction by a reductant such as ascorbic acid.
Biodistribution of 5, 6, 8, and 5a in vivo
To determine whether pyranoside conjugated platinum(IV) complexes can exhibit cancerꢀ
targeting in vivo, the biodistribution of 5, 6, 8, and 5a, a nonꢀtargeted platinum complex used as
the positive control in MCFꢀ7 bearing animals was assessed by ICPꢀMS at 9 h, and 24 h post
administration (i.v.).
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The in vitro IC50 values of 5, 6, 8 and 5a were respectively 0.25 µM, 0.19 µM, 0.22 µM and
8.01 µM, which were not greatly different. Surprisingly, the accumulation Pt of 6 in the tumor
tissue after 24 h was approximately 6.2, 7.9 and 2.2ꢀfold higher than 5, 8 and 5a as shown in
Figure 6A-D and 6F, which were considerably different from the in vitro results. Additionally,
the accumulation of 5 was higher in tumors and the heart, liver, spleen, and lungs than 5a after 9
h. However, 5a and 8 did not show in vivo cancerꢀtargeting properties (Figure 6A and 6D).
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Figure 6. AꢀD. Biodistribution of 5, 6, 8, and 5a, as the positive control in MCFꢀ7 bearing
animals was assessed by ICPꢀMS at 9 h, and 24 h post administration (i.v.). EꢀF. Biodistribution
of 5, 6, 8, and 5a at 9 h and 24 h.
The results in vivo indicated that 6 exhibited enhanced cancerꢀtargeting abilities and
decreased toxicity for MCFꢀ7ꢀbearing animals compared with 5, 8 and the positive control 5a.
The encouraging selectivity of 6 to MCFꢀ7 cells after 24 h in vivo prompted the adventurous
assumption that different cancer cells might have different sensitivities to different glycosylated
platinum(IV) prodrugs.
Effects of EDG(1), phloretin(2), Ctd.(3), and a mixture of EDG and Ctd.(4) on the IC50 values
of the Madine-darby canine kidney (MDCK) cells.
It is well known that MDCK cells are widely used to establish an intestinal epithelial model
for the evaluation of the oral administration of a drug, and can also be used to screen substrates
and inhibitors in vitro. The most important goal regarding Pt(IV) prodrugs is to find a prodrug
with an enhanced potential for oral administration. To determine whether targeted transport
works in MDCK cells, we also investigated the mechanism of compounds 6 and 5a, which were
also coꢀincubated with the GLUT1 inhibitor 4, 6ꢀoꢀethylideneꢀaꢀdꢀglucose (EDG) and phloretin,
the OCT2 inhibitor Ctd., and a combination of EDG and Ctd. as shown in Figure 7.
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The results show that IC50 with inhibitors are higher than in the 48 h MTT assays, indicating a
5
6
close relationships of 6 with the transporters in MDCK cells. As GLUT and OCT substrates in
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MDCK cells, compound 6 would contribute greatly to the intravenous to oral switch for Pt(IV)
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prodrugs.
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Figure 7. Effects of EDG(1), phloretin(2), Ctd.(3), and the mixture of EDG and Ctd.(4) on the
IC50 values of compounds 6 and 5a in MDCK cells.
In vivo efficacy of 6 and 3a.
To further investigate the antitumor potential of the glycosylated Pt(IV) complexes,
compounds with potent in vitro activity were selected for further evaluation in vivo, using a
mouse model of GLUT1 expressing breast cancer (MCFꢀ7) in Balb/c mice. Intraperitoneal
3
injections of 5 mg Pt/kg of 6 or 3a were begum when the tumors reached ~80ꢀ150 mm for three
successive treatments one week. Treatment groups included 6, 3a, and a control group. The
antitumor efficacy was evaluated by measuring the tumor volume over a period of 14 days. The
toxic side effects of the platinum agents under investigation were assessed daily by a thorough
examination of physical condition of the animals, including body weight, lethargy, loss of
appetite, and general behavior.
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The tumor treatment revealed that 6 effectively reduced the disease burden by significantly
delaying the tumor growth (Figure 8A). Moreover, 3a treatment showed no statistically
significant difference compared to the 6ꢀtreated group. Notably, tumors treated with 6, or 3a
were 2ꢀfold smaller compared to control group tumors, indicating excellent efficacy. Importantly,
the bodyweight of mice in Figure 8B indicates that the glycosylated Pt(IV) compounds 6
exhibited reduced toxicity compared with 3a. Images of the tumors at the end of the experiment
are shown in Figure 8C. The results reveal that the glycosylated Pt(IV) complexes had lower
toxicity and higher dose tolerance in vivo with comparative tumor control rates to platinum(II)
drugs such as 3a.
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Figure 8. In vivo antitumor activities of compounds GlcꢀPt(IV) 6 and oxaliplatin in MCFꢀ7. (A)
Tumor growth as a function of time. (B) The bodyweight of mice during the treatments. (C) The
images of tumors at the end of the experiment.
CONCLUSIONS
Platinum anticancer drug candidates are designed as inert platinum(IV) prodrugs that can be
activated following cellular uptake by intracellular reducing agents that are abundant in cancer
cells. For the first time, we synthesized and determined the cytotoxicity, and the detailed
mechanism of pyranoside conjugated platinum(IV) complexes in vitro and in vivo. Pyranosideꢀ
conjugated platinum(IV) complexes are GLUT and OCT substrates and contributed greatly to the
targeted therapy. Previously reported glycosylated Pt(IV) complexes protected by acetyl groups
8h,8i
did not have such properties. The cellular uptake and target engagement among these
glycosylated Pt(IV) complexes is variable. A major reason is offꢀtarget effects due to the
presence of glucose.
Overall, compounds 4ꢀ8, which were C1ꢀsubstituted glycoside compounds, were more
effective than compounds 1ꢀ3, which were C6ꢀsubstituted pyranoside acids. However,
compounds 1ꢀ3 that contained different pyranosides such as glucose, galactose, mannose also
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showed specific tumor targeting properties in vitro. Different chain lengths of between the sugar
and the platinum also made a great difference, possibly because of a combination of effects of
EPR and transporters. In a MTT assay after 8 h incubation, most of compounds 6, 7 and 8 were
intracellular, indicating that they would rapidly accumulate in cancer cells. They also
demonstrated the potential to exploit the two transporters GLUT1 and OCT2, both of which are
overexpressed on the surface of tumor cells. Their preferential accumulation kills more cancer
cells than noncancerous cells (293T and 3T3 cells) in vitro and indicates a low toxicity for the
antineoplastic chemotherapy of platinum drugs. Binding with HSA during drug delivery also
enhanced the stability of the dual targeted Pt(IV) compounds in human whole blood. A halfꢀlife
of 6 of up to 170 h in RPMI 1640 medium suggested that this compound was highly stable in
biological media, more so than platinum(II) drugs. The biodistribution of 5, 6 and 8 in vivo made
a great difference with the in vitro results. The encouraging selectivity of 6 in vivo in MCFꢀ7
tumor cells may contribute to the assumption that different cancer cells would have different
sensitivities to different glycosylated platinum prodrugs in vivo. Compound 6 is a substrate of
GLUT and OCT in MDCK cells, and it would contribute greatly to the intravenous to oral switch
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for Pt(IV) prodrugs. However, previously reported glycosylated Pt(IV) complexes protected by
8h,8i
acetyl groups
also did not have similar properties. The in vivo antitumor activities revealed
that the glycosylated Pt(IV) complexes exhibited important antitumor activities and reduced
toxicity in vivo with a tumor control rate similar to that of the platinum(II) drug oxaliplatin.
All the results above show great differences with the glucoseꢀplatinum(II) prodrugs and
enhanced of cancerꢀtargeting effects, which will contribute to the design of platinum(IV)
prodrugs that will facilitate an intravenous to oral switch. Eventually, the desirable biological
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performance of the glycosylated Pt(IV) complexes mentioned above could prove to be of prime
importance in the future development of platinum antitumor drugs.
Experimental section.
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HPLC Studies.
The purities of all target compounds were determined by HPLC. The HPLC analyses were also
performed as on a Waters E2695ꢀ2998 equipped with a Venusil MP C18 column (150×4.6 mm,
5 ꢁM). The purity of the platinum complexes (1–8) was confirmed to be ≥95% by analytical
HPLC (in the Supporting Information). The linear gradient was as follow. (Table 2)
Table 2. Methods of the HPLC analyses for the purities of all target compounds.
Time(min) A (water)
90
B(Methol)
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90
0
10
35
45
100
100
0
General Procedure for the Synthesis of Compound 1.
Preparation of 6b.
To a stirred solution of 5b (1.6 g) in 40 mL acetone, K2Cr2O7 (1.49 g) dissolved in H2SO4 (18
mL, 6 mol/L) was added in a dropwise manner at 0 °C under an N2 atmosphere and the resulting
solution was stirred at 55 °C for an additional 2 h. After evaporation under reduced pressure, the
precipitate was then washed with 100 mL (×3) of DCM and dried. The residue obtained was
purified by flash column chromatography to give the desired compound S6 as a white solid
(yield: 1.2 g, 71%).
Preparation of 10b.
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To 6b (200 mg, 0.36 mmol) under N2 atmosphere, C2O2Cl2 (4 mL) was added with catalytic
DMF at 00C. White smoke was observed and then the reaction was stirred for another 30 min at
00C. The resulting solution was stirred at room temperature for 2 h. The C2O2Cl2 was removed to
obtain 9b as a slightly yellow solid. Pt(IV) compounds combined with 4a and hexadecanoic acid
as axial ligands (200 mg, 0.30 mmol) was added along with acetone (20 mL) under an N2
atmosphere. After 2 days, the liquid was evaporated and purified by column chromatography to
give the desired compound 10b as a yellow solid (yield: 166 mg, 46%).
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Preparation of 1.
To a solution of 10b (120 mg, 0.10 mmol) in anhydrous DCM, BCl3 (1 M)（0.80 mL，0.80
mmol）was added at –78 ℃ for 2 h. Then, the DCM was removed and purified by column
1
chromatography to get the white solid 1 (41 mg, 48%). H NMR (400 MHz, CD3OD) δ 5.14 (s,
1H), 4.60 – 4.35 (m, 1H), 4.03 – 3.66 (m, 1H), 3.62 – 3.38 (m, 2H), 3.01 – 2.65 (m, 2H), 2.45 –
13
2.20 (m, 4H), 1.72 – 1.51 (m, 6H), 1.30 (s, 26H), 0.92 (s, 3H). C NMR (100 MHz, CD3OD) δ
183.11, 178.11, 176.63, 165.69, 97.09, 92.95, 75.99, 75.89, 74.54, 73.09, 72.75, 72.43, 71.95,
71.50, 61.92, 61.29, 35.92, 31.71, 31.14, 29.46, 29.31, 29.18, 29.12, 28.92, 25.61, 23.75, 23.61,
22.37, 13.13. HRMS: Calcd. for C30H54N2O13Pt (M+NH4)+: 863.3612, found: 863.3594.
General Procedure for the Synthesis of 2.
Preparation of 7b, 11b and 12b.
Compounds 7b, 11b and 12b were prepared according to the procedure described for compounds
6b, 9b and 10b, respectively.
Preparation of 2.
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To a solution of 12b (120 mg, 0.10 mmol) in anhydrous DCM, BCl3 (1 M) (0.80 mL, 0.80
mmol) was added at –78 ℃ for 2 h. The DCM was removed and purified by column
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chromatography to get the white solid 2 (46 mg, 54%). H NMR (400 MHz, CD3OD) δ 5.24 (s,
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1H), 4.42 – 4.26 (m, 1H), 3.97 – 3.76 (m, 1H), 3.54 – 3.43 (m, 2H), 2.95 (s, 1H), 2.79 (s, 1H),
2.43 – 2.22 (d, J = 20.7 Hz, 4H), 1.70 – 1.52 (m, 6H), 1.30 (s, 26H), 0.92 (s, 3H). 13C NMR (100
MHz, CD3OD) δ 182.78, 178.31, 166.07, 129.46, 96.77, 92.98, 81.72, 72.77, 71.62, 71.01,
70.50, 70.38, 69.11, 68.25, 61.78, 61.67, 35.78, 31.68, 31.15, 31.01, 29.41, 29.26, 29.12, 29.09,
28.88, 26.71, 25.49, 23.75, 23.57, 22.35, 22.27, 13.09. HRMS: Calcd. for C30H54N2O13Pt
(M+NH4)+: 863.3612, found: 863.3593.
General Procedure for the Synthesis of 3.
Preparation of 8b, 13b and 14b.
Compounds 8b, 13b and 14b were prepared according to the procedure described for
compounds 6b, 9b and 10b, respectively.
Preparation of 3
To a solution of 14b (120 mg, 0.10 mmol) in anhydrous DCM, BCl3 (1 M) (0.80 mL, 0.80 mmol)
was added at –78 ℃for 2 h. The DCM was removed and purified by column chromatography to
get the white solid 3 (87 mg, 48%). 1H NMR (400 MHz, CD3OD) δ 5.15 (s, 1H), 4.27 (d, J = 7.9
Hz, 1H), 3.92 (s, 1H), 3.85 – 3.69 (d, J = 14.3 Hz, 2H), 2.94 – 2.83 (m, 1H), 2.83 – 2.65 (m, 1H),
2.43 – 2.20 (m, 4H), 1.70 – 1.46 (m, 6H), 1.28 (s, 26H), 0.90 (t, J = 6.1 Hz, 3H). 13C NMR (100
MHz, CD3OD) δ 183.17, 178.05, 165.50, 94.55, 73.18, 70.95, 70.69, 69.73, 62.01, 61.23, 35.93,
31.71, 31.18, 29.45, 29.41, 29.30, 29.17, 29.12, 28.91, 25.62, 23.76, 23.57, 22.37, 13.11. HRMS:
Calcd. for C30H54N2O13Pt (M+NH4)+: 863.3612, found: 863.3606.
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General Procedure for the Synthesis of 4.
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Preparation of 21b.
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NaOH (1 M) (17 mmol) was added to a solution of 20b (1.1 g, 1.7 mmol) dissolved in dioxane
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17 mL and stirred for 12 h at room temperature. The product 21b was finally purified by column
chromatography as a slightly yellow liquid with a yield of 84% (0.9 g).
Preparation of 26b and 27b.
To 21b (113 mg, 0.18 mmol) under an N2 atmosphere, C2O2Cl2 (2 mL) was added with catalytic
DMF at 00C. White smoke was observed and then the reaction was stirred for another 30 min at
00C. The resulting solution was stirred at room temperature for 2 h. The C2O2Cl2 was removed to
obtain 26b as slightly yellow solid. Pt(IV) compounds combined with 4a and a hexadecanoic
acid as an axial ligand (100 mg, 0.15 mmol) was added along with acetone (10 mL) under an N2
atmosphere. After 2 days, the reaction was evaporated and purified by column chromatography
to give the desired compound 27b as a yellow solid (yield: 120 mg, 63%).
Preparation of 4
To a solution of 27b (120 mg, 0.10 mmol) in anhydrous DCM, BCl3 (1 M) (0.80 mL, 0.80
mmol) was added at –78 ℃ for 2 h. The DCM was removed and purified by column
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chromatography to get the white solid 4 (36 mg, 41%). Data for compound 4. H NMR (400
MHz, MeOD) δ 5.01 (d, J = 3.6 Hz, 1H), 4.02 – 3.63 (m, 3H), 3.54 (t, J = 6.2 Hz, 2H), 2.91 (s,
1H), 2.78 (t, J = 9.3 Hz, 2H), 2.51 – 2.40 (m, 2H), 2.39 – 2.23 (m, 4H), 1.86 – 1.71 (m, 3H), 1.69
– 1.62 (d, J = 8.4 Hz, 2H), 1.60 – 1.46 (m, 5H), 1.28 (s, 26H), 0.89 (t, J = 6.6 Hz, 3H). 13C NMR
(100 MHz, MeOD) δ 183.21, 182.66, 165.43, 128.02, 100.06, 73.23, 71.18, 70.74, 67.29, 66.11,
61.82, 61.49, 60.85, 35.98, 32.58, 31.72, 31.19, 29.46, 29.42, 29.31, 29.17, 29.13, 28.92, 28.28,
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25.62, 23.73, 23.67, 22.38, 13.13. HRMS: Calcd. for C34H62N2O14Pt (M+H)+: 918.3922, found:
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General Procedure for the Synthesis of 5.
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Preparation of 22b.
Compound 22b obtained as a slight yellow solid was prepared according to the procedure
described for compound 21b.
Preparation of 28b and 29b.
To 22b (226 mg, 0.36 mmol) under an N2 atmosphere, C2O2Cl2 (4 mL) was added with catalytic
DMF at 00C. White smoke was observed and then the reaction was stirred for another 30 min at
00C. The resulting solution was stirred at room temperature for 2 h. The C2O2Cl2 was removed to
obtain 28b as slightly yellow solid. Pt(IV) compounds combined with 4a and a hexadecanoic
acid as an axial ligands (200 mg, 0.30 mmol) along with acetone (20 mL) was added under an N2
atmosphere. After 2 days, the liquid was evaporated and purified by column chromatography to
give the desired compound 29b as a yellow solid (yield: 196 mg, 51%).
Preparation of 5.
To a solution of 29b (120 mg, 0.10 mmol) in anhydrous DCM, BCl3 (1 M) (0.80 mL, 0.80
mmol) was added at –78 ℃ for 2 h. The DCM was removed and purified by column
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chromatography to get the white solid 5a (25 mg, 28%). Data for compound 5a. H NMR (400
MHz, MeOD) δ 4.02 – 3.82 (m, 1H), 3.80 – 3.60 (m, 2H), 3.58–3.42 (m, 2H), 2.80 (s, 2H), 2.58
– 2.40 (m, 2H), 2.38–2.22 (m, 4H), 1.99 – 1.73 (m, 2H), 1.72–1.48 (m, 6H), 1.30 (s, 28H), 0.92
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(s, 3H). C NMR (100 MHz, MeOD) δ 183.18, 182.61, 165.44, 103.68, 75.23, 73.52, 71.18,
68.92, 68.56, 61.79, 61.46, 61.12, 60.84, 35.97, 32.56, 31.73, 31.18, 29.47, 29.43, 29.32, 29.19,
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