Synthesis and Mechanistic Studies of Novel Antitumor Transition Metal Complexes
by
Hyunsuk Yoo
B.S., Chemistry
Stanford University, 2012
SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTERS IN INORGANIC CHEMISTRY
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
MASSACHUSETTS IN1TE
OF TECHNOLOGY
June 2014
JUN 30 2014
©Massachusetts Institute of Technology, 2014
All rights reserved
Signature of Author:
LIBRARIES
Signature redacted
Department of Chemistry
February 14a", 2014
Certified by:
ignature redacted
-I
Accepted by:
Stephen J. Lippard
Arthur Amos Noyes Professor of Chemistry
Thesis Supervisor
Signature redacted
Robert W. Field
Haslam and Dewey Professor of Chemistry
Chairman, Departmental Committee for Graduate Students
2
Synthesis and Mechanistic Studies of Novel Antitumor Transition Metal Complexes
by
Hyunsuk Yoo
Submitted to the Department of Chemistry on February 14 th, 2014, in partial fulfillment of
the requirements for the degree of Masters.
Abstract
In order to overcome side effects and drug resistance associated with conventional Pt(II)
drugs, our lab has developed novel platinum complexes. One of the new platinum complexes
developed in our lab is the monofunctional platinum anti-cancer compound phenanthriplatin. We
have found that by binding to sulfur complexes, phenanthriplatin undergoes changes in its kinetic
and cytotoxic properties. Sulfur adducts of phenanthriplatin were synthesized to study the complex
roles sulfur compounds serve in the cellular action of the monofunctional compound.
In addition, we have examined how Pt(IV) chemistry can be successfully applied to
increase the efficacy of Pt(II) compounds. We conjugated hydrophobic chains to trans[Pt(NH3)2C2] (TDDP) through isocyanate couplings and successfully transformed TDDP into an
active compound. We demonstrated that Pt(IV) chemistry can be applied to transform even inactive
trans compounds into active complexes that can potentially be used in chemotherapy.
Finally, we examined the anticancer properties of the dinuclear osmium(VI) nitrido
complex [NBu4]2[(OsNC14)2(pyz)]. We studied its cellular activity in the hope of discovering
interesting and unexpected properties. We found that the compound has moderate cytotoxicity and
leads to DNA damage and apoptosis.
Thesis supervisor: Stephen J. Lippard
Title: Arthur Amos Noyes Professor of Chemistry
3
ForMy Mother and Father
4
Acknowledgments
I would like to thank my advisor, Professor Steve Lippard, for giving me the opportunity
to work in his lab. The two years I've spent in his lab were invaluable for developing my research
skills and gaining further insights into chemistry. I am grateful for the support he has given me,
even after I decided to change my career plan.
I would also like to thank my mentor Yao-Rong Zheng. He taught me many crucial lab
skills and was supportive even as I struggled with the most basic syntheses in the beginning. He
has been a great teacher who encouraged me and gave me new ideas when I got stuck. I am also
thankful to Rama for suggesting I work on the dinuclear osmium project as well as teaching me
many biochemistry techniques. I am also thankful to the whole Lippard lab, and especially the
former and current platinum subgroup members Justin, Tim, Ga-Young, Sammy, Malay and
Imogen. They gave brilliant ideas that led me to delve deeper into my projects.
I am grateful to the professors in the Chemistry Department at MIT for expanding my
chemistry knowledge. They truly exposed me to the frontiers of chemistry research. I would also
like to thank the professors who taught me at Stanford University. I am especially thankful to
Professor Waymouth, who was a great research advisor and mentor during my three years in
Stanford, and to Professor Boxer, who taught me to think critically and scientifically about the
knowledge I encounter in textbooks and in papers.
I will always be indebted to my mother and father for their unconditional love and support
they gave me throughout my life. I would have never made it this far had it not for the belief they
had in me. The phone calls I had with my parents always encouraged me to proceed when I was
5
facing difficulties. I would also like to thank my sister Hyunseo for visiting me often from NYC
and giving me emotional support as I struggled to change my career. I am blessed to have my
family.
I would like to thank the Samsung Foundation of Culture for supporting me throughout
graduate school. I am also grateful to the Koch Institute for supporting my Pt(IV) project. Finally,
I am thankful to the friends I was fortunate to meet in MIT. They made Boston feel like home.
6
Table of Contents
PAGE
Abstract
2
Dedication
3
Acknowledgments
4
Chapter 1. The Effect of Sulfur Compounds on the CellularAction of Monofunctional Platinum
Antitumor Compound Phenanthriplatin
Abstract
9
Introduction
9
Experimental
12
Results and Discussion
20
Conclusion
30
References
30
Chapter2. Utilizing Pt(IV) Chemistry to Improve the Cytotoxicity of the Inactive Pt(II) Compound
trans-[Pt(NH)2Cl2]
Abstract
33
Introduction
33
Experimental
35
Results and Discussion
45
7
Conclusion
52
References
53
Chapter 3. Investigation of the Cellular Activity of the Dinuclear Os(VI) Nitrido Complex
[NBU4]2[(OSNC
4 )2(pyZ)]
Abstract
56
Introduction
56
Experimental
58
Results and Discussion
62
Conclusion
68
References
68
Biographical Note
69
8
Chapter 1.
The Effect of Sulfur Compounds on the Cellular Action of
Monofunctional Platinum Antitumor Compound Phenanthriplatin
9
Abstract
By binding to sulfur-donor ligands, the monofunctional platinum antitumor
compound phenanthriplatin undergoes changes in its kinetic and cytotoxic properties. The
present study delineates the complex roles that sulfur compounds may play in the cellular
action of phenanthriplatin.
Introduction
Despite the ongoing efforts to treat the disease, cancer remains one of the leading
causes of death worldwide.' Platinum-based drugs such as cisplatin, oxaliplatin, and
carboplatin are among the most effective and widely used anticancer drugs. The platinumbased complexes have been successfully used to treat ovarian, bladder, head and neck, and
testicular cancers. 2 Patients treated with classic bifunctional compounds do, however,
suffer from side effects such as neurotoxicity, ototoxicity and nephrotoxicity, 23- which
limits the administered dose of Pt drugs. Furthermore, resistance to Pt drugs can occur as a
result of increased levels of drug detoxification by enhanced replication bypass of platinumDNA adducts, increased DNA damage tolerance, and the failure of cell death pathways.' 5
In order to address these shortcomings, our lab has worked to devise novel Pt-based
drugs having higher potency, a spectrum of activity different from those of the approved
compounds, and possibly reduced side effects and the ability to overcome drug resistance.
The discovery that cis-[Pt(NH 3) 2(pyridine)Cl]+ (pyriplatin) can inhibit transcription while
eluding repair mechanisms led us to investigate monofunctional compounds in depth.6 The
10
X-ray structure of RNA polymerase II (Pol II) stalled at a site-specific pyriplatin-DNA
adduct revealed that steric interactions in the complex may be the source of its ability to
inhibit transcription. Based on this new insight, we investigated larger N-heterocyclic
ligands that can better perturb RNA chain elongation, the result of which led to the
rationally
designed monofunctional
Pt compound
cis-[Pt(NH 3) 2 (phenanthridine)Cl]'
(phenanthriplatin), which exhibited greater activity than conventional bifunctional Pt-drugs
and had a totally distinct spectrum of activity from cisplatin.
8
A detailed examination of
the properties of phenanthriplatin showed that it has increased cellular uptake, improved
DNA binding kinetics, and effective transcription inhibition compared to traditional
bifunctional Pt complexes. 8
In order to understand the unique activity of phenanthriplatin, it is essential to know
its intra- and extracellular metabolism. Sulfur-containing molecules, such as human serum
albumin (HSA) and metallothionein (MT), are ubiquitous in the blood and cancer cells.
Owing to their sulfur-donor atoms, which have a strong preference for binding to Pt(II)
complexes, such molecules are intimately involved in the cellular processing of platinum
drugs. 9
10
These proteins are believed to impede the therapeutic efficacy of the Pt-drugs.10'1 1
The strong, irreversible binding of Pt to sulfur rich proteins leads to Pt-drug inactivation
and ultimately to drug resistance.10 For example, glutathione-S-transferase (GST), a protein
that catalyzes the conjugation of glutathione to platinum in vivo, is overexpressed in
cisplatin-resistant cell lines.12
11
In contrast to the foregoing reports about the role of sulfur compounds in platinum
drug chemotherapy, a recent study by Ma and co-workers reported that methionine binding
actually facilitates DNA platination for pyriplatin.13 The authors proposed that, prior to
binding of this monofunctional platinum complex to DNA inside the cell, it is initially
activated by sulfur donors that lead to labilization of a trans ammine ligand, enhancing the
ability of the resulting monofunctional complex to bind DNA. 1 3
In the present work, we investigated the effect of sulfur complexes on the
metabolism of phenanthriplatin. We monitored how sulfur binding can affect the DNA
binding kinetics and cytotoxicity of the resulting derivative (Scheme 1).
cell membrane
NH 2
NH3
H3N-Pt-N
HO2C
S
HN-t -N
met-phenPt
phenanthriplatin
NH
2
7
+
H02C-'
H3N-Pt-N
NH 310
Pt-N
NH 3
Scheme 1. The reaction of phenanthriplatin with methionine (above) and with cysteine
(below).
12
Experimental
Synthesis of Phenanthriplatin
The triflate salt of phenanthriplatin was prepared following a literature procedure.14 To a
solution of cisplatin (500 mg, 1.66 mmol) dissolved in 30 mL DMF, a solution of silver triflate
(257 mg, 0.6 equiv) dissolved in 3 mL DMF was added dropwise, and the reaction was stirred
under protection from light at room temperature. The next day, AgCl precipitate was filtered. To
the supernatant, phenanthridine (300 mg, 1.0 equiv) was added and the reaction was stirred for 5
h at 50 *C. The reaction mixture was evaporated under reduced pressure at 60 'C. Unreacted
cisplatin was removed by filtration and 6 mL acetone was added to the concentrated DMF. The
filtrate was transferred to a separate vial and excess ether was layered on top of the filtrate to a
volume of 20 mL to slowly precipitate out phenanthriplatin. After three days, phenanthriplatin was
collected as large white crystals. The ESI-MS and NMR data obtained for phenanthriplatin were
in good agreement with the reported data for phenanthriplatin (Figure 1).
cis-[Pt(NH3)2(phenanthridine)Cl](SO3CF3). White solid. Yield: 52.5%. ESI-MS m/z calculated
(M'): 444.1, found: 444.0. 1H NMR (400 MHz, DMSO-d 6 ): 8 9.94 (1H, s), 9.77 (1H, d), 8.95 (2H,
q), 8.46 (1H, d), 8.16 (1H, t), 8.03 (1H, t), 7.93 (2H, q), 4.56 (3H, broad), 4.43 (3H, broad).
Analytical HPLC: 99.6 % purity (Figure 2).
13
JILL
(C)
(A)
Cal.: 444.1 tM]
H3N, ,CI
Pt
'N
H3N
a
6
ilL
h
9
Found: 444.0 [M]*
-~-f
bI
C
d
36
180.1
I
(B)
b.6
'
011
I
.
.00
600
60
0.055 0.050
0,045
0.040
I
h&e
0.030
,00 '0*
0.025
z
44 &f
b
C
'I~d
0.035
0
03
1 00099
0.020
0.015
0.010
0.005
0
00 1.03
ULi
2.05
L.A
1.05 1,07 10D4 2-O
L.A
U
LA
6.10
5 i
Chemical Shift (ppn)
I-T
4
....
-4 ...... -T - '
I
Figure 1. NMR and ESI-MS spectra of phenanthriplatin: (a) chemical structure of phenanthriplatin;
(b) 'H NMR spectrum of phenanthriplatin in DMSO-d6; (c) ESI-MS spectrum of phenanthriplatin.
14
Column: CI 8-Zorbax
Temp: Room Temperature
Flow rate: ImL/min
Detection: UV, 240 nm
Mobile Phase:
A: 0.5 % TFA in H204
B: 0.1 % TFA in Acetonitrile
Time (min)
%B
0
2
10
10
35
75
37
75
39
40
10
10
mAU1000
500
0
0
20
40 min
Figure 2. HPLC conditions and HPLC chromatogram for the analysis of phenanthriplatin.
Synthesis of met-phenPt
The mixed triflate (50 %) and chloride (50 %) salt of met-phenPt was synthesized as
follows. cis-[Pt(NH3)2(phenanthridine)Cl](SO 3CF 3) (51.3 mg, 0.087 mmol) was mixed with Lmethionine (11.6 mg, 0.9 equiv) in 2 mL 100 mM HCI, and the reaction was stirred for 2 h at 50 *C.
The reaction mixture was evaporated under reduced pressure at 40 *C and excess acetone was
subsequently added to precipitate out the product. The crude product was collected and washed
twice with acetone, then isolated by centrifugation and dried in vacuo. The final product was
characterized by ESI-MS and NMR spectroscopy (Figure 3).
15
cis-[Pt(NH3)(Cl)(S-L-Met)(phenanthridine)]+.
Off-white solid. Yield: 77 %. Mp 187 'C (dec).
ESI-MS m/z calculated (M'): 576.1, found: 576.1. 'H NMR (400 MHz, D2 0): 6 9.77 (lH, s), 9.51
(1H, broad), 8.59 (2H, t), 8.16 (1H, broad), 7.98 (2H, q), 7.83 (1H, t), 7.78 (1H, t), 3.81 (1H, broad),
2.70 (2H, broad), 2.23 (5H, broad). 13C NMR (400 MHz, D2 0): 6 172.4, 171.3, 160.2, 141.4, 135.2,
132.4, 130.4, 129.8, 129.4, 126.1, 123.8, 122.4, 52.4, 29.5, 29.2, 14.5. 195Pt NMR (400 MHz, D2 0):
6
-2854.
Analytical
HPLC:
93.1
% purity
(Figure
4).
Analysis
calculated
Cis.5 H 23Cl1. 5Fj. 5N 3 O3.5PtS1 .5: C, 33.25; H, 3.47; N, 6.29; found: C, 33.50; H, 3.64; N, 6.28.
for
16
(A)
(C)
j a
H NH2k
H0 2 C 1
S
H3N-Pt-N
Ik z
hII//IiIJ
(B)
e
091213_METPHENPT.001 .001.1 R.ESP
0.0060
0.0055
0.0050
0.0045
0.0040
0.0035
0.0030
0.0020(
0.0015
b,
m
100-002172515-0313
M
M
h, e
1.000
120
10
2
00 196
3
Et 0
120
k
7
1303
4.3
Chenca Sit(ppm )
((E)
30
Cal.: 576.1 [M}l
-2853.99 ppm
Found:
576.1
[MI*
03
Figure 3. NMR and ESI-MS spectra of met-phenPt: (A) chemical structure of met-phenPt; (B)
'H NMR spectrum of met-phenPt in D 20; (C)
13 C
NMR spectrum of met-phenPt in D 20; (D)
NMR spectrum of met-phenPt in D 2 0; (E) ESI-MS spectrum of met-phenPt.
195
Pt
17
Column: Cl 8-Zorbax
Temp: Room Temperature
Flow rate: ImL/min
Detection: UV, 240 nm
Mobile Phase:
A: 0.5 % TFA in H204
B: 0.1 % TFA in Acetonitrile
mAU
2000
15007
c
1500
1000-
i
'
CD
5000
--
0
0
20
40
60
min
Figure 4. HPLC conditions and HPLC chromatogram for the analysis of met-phenPt.
DNA Binding Kinetics
DNA binding kinetics of cisplatin, phenanthriplatin, and met-PhenPt were determined
following a modified literature protocol as follows.1 5 40 pM cisplatin, phenanthriplatin, and met-
phenPt were prepared in 3 mL Tris-HCl (5 mM, pH 7) buffer. 100 pL of 3.03 mM calf thymus
DNA (2.4 equiv) purchased from Sigma-Aldrich was added to each solution and the three solutions
were then incubated at 37 *C. 250 pL aliquots were taken at defined time points (0 min, 40 min, 1
h 40 min, 2 h 40 min, 4 h 40 min, 6 h 40 min, 18 h 30 min) and 5 pL sat. NaCl and 1 mL of ethanol
were added to each aliquot to quench the reaction. The aliquots were than centrifuged at 14,000
rpm for 40 min at 4 *C. Following centrifugation, the platinum content of the supernatant was
measured by GFAAS (graphite furnace atomic absorption spectroscopy). Three independent DNA
18
precipitation experiments were carried out for each compound to obtain standard deviations.
Reaction of Phenanthriplatinwith 5'-GuanosineMonophosphate
4 mM phenanthriplatin and met-phenPt solutions were prepared in D 20. The Pt(II)
solutions were then treated with 40 mM (10 equiv) of 5'-guanosine monophosphate (GMP) and
the reaction was monitored by 1H NMR spectroscopy at room temperature. Deuterated 3(trimethylsilyl)propionic acid sodium salt was used as an internal standard. NMR spectra were
collected on a Varian 400 spectrometer.
Reaction of Activated Phenanthriplatinwith N-Acetyl Cysteine
7.7 mM of phenanthriplatin in D 2 0 was combined with 0.95 equiv of silver nitrate and the
solution was stirred in the dark overnight to activate the platinum compound. Silver chloride was
then
removed
by
filtration
through
a
0.5
pM
syringe
filter.
The
resulting
cis-
[Pt(NH3)2(phenanthridine)(OD2)]* was treated with 1 equiv N-acetyl cysteine and the reaction was
monitored by 'H NMR spectroscopy at room temperature. After the completion of the reaction
was confirmed by NMR, the reaction was lyophilized and the product stored at 4 *C for future
cytotoxicity studies. 1,4-dioxane was used as an internal standard. NMR spectra were collected on
a Varian 400 spectrometer.
Reaction of Phenanthriplatinwith Glutathione
cis-[Pt(NH3)2(phenanthridine)Cl](SO3CF 3) (51.3 mg, 0.087 mmol) was mixed with
19
reduced glutathione (GSH) (24.0 mg, 0.9 equiv) or oxidized glutathione (GSSG) (47.9 mg, 0.9
equiv) in 2 mL 100 mM HCI, and the reaction was stirred for 2 h at 50 *C. The reaction mixture
was evaporated under reduced pressure at 40 'C and excess acetone was subsequently added to
precipitate out the product. After overnight drying, NMR spectra of the products were collected
on a Varian 400 spectrometer.
Cellular Uptake of Platinum Complexes in A549 Cells
Approximately 106 A549 cells were seeded in a 60 mm diameter Petri dish in triplicate
and were incubated overnight in DMEM. The cells were then treated with 5 pM of cisplatin,
phenanthriplatin or met-phenPt at 37 'C in 5% C02 for 5 h. After incubation, medium was removed
and the cells were washed with 1 mL PBS two times. The cells were then detached using 1 mL
trypsin and transferred to a centrifuge tube using an additional 0.5 mL PBS. The cell pellets were
collected by centrifugation at 1800 rpm for 15 min at 4 'C. The pellets were resuspended in 1 mL
PBS and centrifuged again at the same condition. The resulting cell pellets were then suspended
in 200 pL of 70% HNO 3 and digested at 70 'C for 2 h. The platinum content was then measured
through GFAAS.
MITAssay
Cells (A549, HeLa, or PC3) were seeded on a 96 well plate (2000 cells per well) in 100
pL medium and incubated for 24 h. Following incubation, solutions containing platinum
compounds of interest were prepared in DMEM (A549, HeLa) or RPMI (PC3) and the
20
4
concentration verified by GFAAS. The cells were then treated with 100 p L of platinum compounds
at different concentrations and incubated for 72 h at 37 *C in 5% CO 2 . After 72 h incubation, the
cells were treated with 200 iL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (0.8 mg/mL) dissolved in DMEM (A549, HeLa) or RPMI (PC3). Following 3 h treatment,
the medium was removed and 200 pL of DMSO was added to the cells. The absorbance was then
measured at 570 nm using a BioTek Synergy HT multi-detection microplate plate reader. Each
compound was tested in triplicate for each cell line.
Result & Discussion
Synthesis of met-phenPt
The methionine phenanthriplatin adduct [Pt(NH 3 )(Cl)(S-L-met)(phenanthridine)]+
(met-phenPt), where S-L-Met refers to L-Met bound to Pt through S, was prepared by the
synthetic protocol in Scheme 2. By treating phenanthriplatin with methionine under mildly
acidic condition (100 mM HC1, 50 *C), we were able to replace one of the ammine ligands
with methionine.
100 mM HCI
0.9 equiv L-Met ,,H02C
50 0C
N
NH3
H3 N-Pt-N
CI
Scheme 2. Synthetic route for preparing met-phenPt
NH 2
11
s1
H3N-Pt-N
C
21
Kinetics of the Reaction of Met-PhenPtwith DNA
The interaction of phenanthriplatin and met-phenPt with DNA was studied by
treating the two Pt(II) compounds with excess 5'-guanosine monophosphate (GMP) and
monitoring the changes in the 1D 1H NMR spectrum.8 From an analysis of the kinetics of
this reaction, we measured a trans labilization effect of chloride from the complex
analogous to that previously reported for the pyriplatin methionine adduct.13 Whereas the
reaction of 4 mM phenanthriplatin with 10 equiv of GMP proceeded with a
t1/2 of
8.2 h, the
equivalent reaction with 4 mM met-phenPt led to the formation of the GMP adduct in less
than 5 min (Figure 5, Figure 6). Immediately after adding GMP to met-phenPt, peaks in the
NMR spectrum corresponding to the Pt complex were replaced by those of the GMP adduct.
ESI-MS suggested that the Cl ligand trans to methionine is replaced with GMP while the
remaining ligands are unaffected (Figure 7).
22
(A) 4 mM phenanthriplatin + 40 mM GMP
~Jk.
0m
5m
100
3 8
9
9
91
99
99
6
93
9.4
92
9
9t
90
9
33
92
91
9
V7
as
&
&4
93
92
99
33
79
7
881a3 7
a8
89
4
84
3
5'2
9
a3
7,9
79
88
"9
8a.9
9
3
?1
*
I's
1
99
S4
740
98
9
94
93
s3
93
92
99.344s8
89
98
97
76
9
93
84
S2
30
&1
79
77
78
7.9
76
h
3 h
4u4
19 h
333
go
947
ga
9o
93fj
92
11.
90
8.o
98,
V7
1
9-6
84
93
S2
94
S9
7P7
97.
7
(B) 4 mM met-phenPt + 40 mM GMP
0m
Chiem"e
S&M r)
98C
99
99
8
98A
100
9.
90
a
a
9.9
5 m
1 h
C9
e Shi9ft
9Will
3 h
100
gs
00
a
to0
26 h
Figure 5. ID 'H NMR spectra of the reaction between (A) phenanthriplatin, (B) met-phenPt and
GMP under pseudo first order conditions at room temperature in D 20. The peaks that were used
for integration are marked with a red box. The reaction time at which each NMR spectrum was
obtained is labeled on the right.
23
In([phenPt)
In([phenanthriplatin]) vs. time
(mM)
1.60
1.40
1.20
y =-0.0854x+ 1.4002
R2 =0.9759
1.00
PhenPt
.8-
0.60
0.40
0.20
0.00
10.00
8.00
6.00
4.00
2.00
0.00
time (h)
tm h
Figure 6. A pseudo first order kinetic analysis was conducted for the reaction of phenanthriplatin
with excess GMP. The calculated rate constant (k, 25 C) was 0.085 h- 1 and half life (t1 I2, 25 *C)
8.2 h.
Intensity
Intens.
x10
7
-
570.1
3HO 2 C.
-NH
O0,N liNH3
2
NH 3
Cl-Pt-S.
2-
9022
NH 3
s-Pt-N
..
H2 N
HO 2
N
N
C
~\
9~o
OH
H02C
0'
1-
HO
04
180.1
0~-
I.
200
Figure 7.
GMP.
443.5
L L.
400
873.1
A.
00
-14L.
800
1000
m/z
1200
1400
ESI-MS spectrum showing the product formed from the reaction of met-phenPt and
24
Our finding that met-phenPt reacts more rapidly than phenanthriplatin with a DNA
nucleobase was further corroborated by monitoring the kinetics of the reaction between 40 pM
solutions of various Pt(II) complexes with calf-thymus DNA. Figure 8 shows the plot of the ratio
of bound platinum per nucleotide (rb) versus time (h) following addition of the Pt complex to 2.4
equiv of ct-DNA. Although phenanthriplatin and cisplatin showed similar DNA binding kinetics
with t5o%, the time at which the binding reaches 50 %, of approximately 1.5 h, met-phenPt had t5o%
value 5 30 min. As stated above, the difference arises from the presence of methionine trans to the
chloride ligand.16 The strong trans labilizing effect of sulfur facilitates the binding of met-phenPt
to DNA.
Rs
Rb
vs. time
0.35
0.30
0.2!
0.1
-O-
pheWt
0.10
0.05
20.00
Time (hours)
Figure 8. Kinetics of the binding of cisplatin, phenanthriplatin and met-phenPt to ct-
DNA.
Reaction of Phenanthriplatinwith N-Acetyl Cysteine
In contrast to the synthesis of met-phenPt, construction of the related cysteine
25
phenanthriplatin adduct proved to be synthetically challenging. Owing to multiple binding sites
present on cysteine as well as the lability of the Pt thiol adduct, we were unable to obtain a pure
adduct. To minimize the number of products formed, we used N-acetyl cysteine rather than
cysteine. In addition, we activated phenanthriplatin by treating the compound with silver nitrate
prior to addition of N-acetyl cysteine. The reaction between 7.7 mM of phenanthriplatin activated
in this manner, cis-[Pt(NH3)2(phenanthridine)(OD2)]+ (phenPt-OD2) and one equiv of N-acetyl
cysteine was monitored by 1D 'H NMR spectroscopy. Kinetic analysis of the data supported a first
order reaction with k = 0.025 min-' and ti1 2 = 28 min (Figure 10). After one hour, the reaction was
complete and the 'H NMR spectrum showed peaks corresponding to the multiple products that
were formed (Figure 9).
~~~1+
H3Nt OD2
H3N' 'N
a
b
-'
h
~
e
I
9
f
/d
F
C
iI
100
7.7 mM phenPt + 1 equiv N-acetyl cysteine
a
d
h, e
95
0m
0
9.0
Chefica
b CA9 f
Shift (ppm)
1ot
9.5
90
.5
0
10
9.5
9.0
85
80
7.5
30 m
S0AA
mm
9.9 (p5 95
60 m
10.1
. .. ..
95
90
.
. . .. .0
85
60
75
26
Figure 9. The reaction of N-acetyl cysteine with [Pt(NH3)2(phenanthridine)(OD 2)].' After the
addition of N-acetyl cysteine, NMR spectrum of the reaction mixture was obtained at defined time
points. The peaks that were used for integration to create Fig. 3 are marked with a blue box.
In([phenPt-OD2]) vs. time
In([phe nPt-OD2])
(I mM)
2.502.00
i
= -0.025
Sy
1.50 -
x+ 2.15
R2=0.97
1.00-
0.50
0.00
time (min)
0
20
20
40
40
60
60
80
80
Figure 10. Kinetic analysis was conducted for the reaction of N-acetyl cysteine with
[Pt(NH3)2(phenanthridine)(OD 2)].* The calculated rate constant (k, 25 *C) was 0.025 min' and
half life (t 1 2, 25 C) 28 min.
An ESI-MS spectrum of the reaction mixture obtained after the completion of the reaction
is shown in Figure 11. Data suggest that as soon as cysteine displaces chloride to form the
platinum-cysteine adduct, the product undergoes cyclization to form a chelate ring by internal
displacement of additional ligand phenanthridine or NH 3. Peaks corresponding to such chelate
compounds were observed in the ESI-MS spectrum. The different reactivity of cysteine and
27
methionine to phenanthriplatin is illustrated in Scheme 1.
N
hnt-D-t-aey
2
-
N-stee
OH
,00
,, 0
Figure 11. ESI-MS spectrum showing the formation of cyclic compounds upon reacting
phenPt-0D 2 with N-acetyl cysteine.
Cytotoxicity of Platinum-SulfurAdducts
The cytotoxicity of the platinum-sulfur adducts was measured and compared to that
of phenanthriplatin. Table 1 shows IC5o values for each of the three cell lines and standard
deviations obtained from three independent experiments. Despite the enhanced kinetic
properties of met-phenPt, it was less cytotoxic than the parent compound phenanthriplatin.
The cytotoxicity of phenanthriplatin is still 10
-
70 times greater than met-phenPt. A
possible explanation for this observation is that the enhanced kinetic properties of metphenPt not only facilitates the binding to DNA but also the binding to cellular proteins,
which leads to the deactivation. In addition, owing to the hydrophilicity of methionine, metphenPt is taken up less by the cell (Figure 12). The cellular uptake of met-phenPt is
approximately half of that of phenanthriplatin. However, the cellular uptake of met-phenPt
28
was still more than four times greater than cisplatin, perhaps explaining why met-phenPt
was still effective against cancer cells.
Table 1. IC50 values of phenanthriplatin, met-phenPt, and Pt-thioethers were measured in three
different cell lines. Pt-thioethers refer to a collection of compounds obtained from lyophilizing the
products formed from the reaction of phenPt-OH2 and N-acetyl cysteine
unit pM
phenPt
met-phenPt
Pt-thioethers
A549 (Lung) HeLa (Cervix)
0.059 0.009 0.17 0.03
4.10 1.57
4.69 0.74
0.310 0.009
1.78 0.23
PC3 (Prostate)
0.18 0.01
1.57 0.09
1.07 0.11
Cellular Uptake
(pmol/1M cells)
800
700
600
500
400
300
200
100
78
0
McisPt
a phenanthriplatin
U met-phenPt
Figure 12. Cellular uptake of Pt(II) complexes in A549 cell line.
By contrast, Pt-thioethers, a collection of compounds obtained from lyophilization
of products formed from the reaction of phenPt-OH2 and N-acetyl cysteine, had slightly
29
better cytotoxicity than met-phenPt but were still 5-10 times less cytotoxic than
phenanthriplatin. Our finding suggests that both methionine and cysteine adducts of
phenanthriplatin are still active against the cancer cells tested.
CellularRedox PotentialDetermines the Reactivity of Phenanthriplatin
In addition to methionine and cysteine, we tested the reactivity of reduced
glutathione (GSH) toward phenanthriplatin. Under the same mildly acidic condition used
to synthesize met-phenPt, the reaction of phenanthriplatin and glutathione led to the
formation of multiple products. Phenanthriplatin did not react with glutathione (GSSG)
under the same conditions. 'H NMR spectra of the products from the two reactions are
compared in Figure 13. Simply by oxidizing glutathione, we were able to significantly
reduce the reactivity of its thiols. These results indicate that the cellular redox potential will
be
very
important
in
determining
the
reactivity
of
sulfur
compounds
toward
phenanthriplatin.
phenanthriplatin
phenanthriplatin + GSH
phenanthriplatin + GSSG
Figure 13. 1D 'H NMR showing the different reactivity of reduced glutathione (GSH) and
30
oxidized glutathione
(GSSG) toward phenanthriplatin.
Phenanthriplatin was treated with
glutathione under the same mildly acidic condition used to synthesize met-phenPt.
Conclusion
In conclusion, we studied the effect of sulfur compounds on the kinetic and cellular
properties
of phenanthriplatin.
Our data indicate
that, upon
methionine
binding,
phenanthriplatin binds more rapidly to DNA through trans labilization. However, the
enhanced kinetic properties did not lead to better cytotoxicity, with met-phenPt being
10-70 times less active than phenanthriplatin. On the other hand, the reaction of cysteine
with phenanthriplatin led to a complex reaction mixture that was more active than metphenPt but still less than phenanthriplatin. For met-phenPt, one of the reasons for reduced
cytotoxicity was reduced cellular uptake. We also discovered that the reactivity of sulfur
compounds toward phenanthriplatin can be controlled by changing the redox state of the
reactants. By oxidizing glutathione, we were able to halt its reaction with phenanthriplatin
under mild conditions. Overall, our study suggests that different sulfur compounds have
complex effects on the metabolism of phenanthriplatin.
References
1.
M. Adeli, R. Soleyman, Z. Beiranvand and F. Madani, Chem. Soc. Rev., 2013, 42, 52315256.
2.
L. Kelland, Nat. Rev. Cancer, 2007, 7, 573-584.
31
3.
C. A. Rabik and M. E. Dolan, Cancer Treat. Rev., 2007, 33, 9-23.
4.
D. Wang and S. J. Lippard, Nat. Rev. Drug. Disc., 2005, 4, 307-320.
5.
K. Cheung-Ong, G. Giaever and C. Nislow, Chem. Bio., 2013, 20, 648-659.
6.
K. S. Lovejoy, R. C. Todd, S. Zhang, M. S. McCormick, J. A. D'Aquino, J. T. Reardon, A.
Sancar, K. M. Giacomini and S. J. Lippard, Proc.NatL. Acad. Sci. U. S. A., 2008, 105, 89028907.
7.
D. Wang, G. Zhu, X. Huang and S. J. Lippard, Proc.NatL. Acad. Sci. U. S. A., 2010, 107,
9584-9589.
8.
G. Y. Park, J. J. Wilson, Y. Song and S. J. Lippard, Proc.NatL. Acad. Sci. U. S. A., 2012,
109, 11987-11992.
9.
M. Kuo, H. W. Chen, I.-S. Song, N. Savaraj and T. Ishikawa, CancerMetastasisRev., 2007,
26, 71-83.
10.
X. Wang and Z. Guo, AnticancerAgents Med. Chem., 2007, 7, 19-34.
11.
J. Reedijk, Chem. Rev., 1999, 99,2499-2510.
12.
W. H. Ang, I. Khalaila, C. S. Allardyce, L. Juillerat-Jeanneret and P. J. Dyson, J.Am. Chem.
Soc., 2005, 127, 1382-1383.
13.
G. Ma, Y. Min, R Huang, T. Jiang and Y. Liu, Chem. Commun., 2010, 46, 6938-6940.
14.
T. C. Johnstone and S. J. Lippard, J.Am. Chem. Soc., 2014.
15.
0. Novakova, H. Chen, 0. Vrana, A. Rodger, P. J. Sadler and V. Brabec, Biochemistry,
2003, 42, 11544-11554.
16.
S. S. Zumdahl and R. S. Drago, J.Am. Chem. Soc., 1968, 90, 6669-6675.
32
Chapter 2.
Utilizing Pt(IV) Chemistry to Improve the
Cytotoxicity of the Inactive Pt(II) Compound trans-[Pt(NH3)2C2]
33
Abstract
By attaching long hydrophobic chains to trans-[Pt(NH 3)2Cl2] (TDDP) using Pt(IV)
chemistry, we generated amphiphilic complexes displaying up to a 7.7-fold increase in cellular
uptake and up to a 75-fold increase in cytotoxicity compared to TDDP.
Introduction
Following the serendipitous discovery of the anticancer activity of cisplatin, cisplatin has
successfully been applied in the clinic. Its widespread success led scientists to search for and
discover the second generation platinum-based anticancer agents carboplatin and oxaliplatin.
Currently, these three compounds are used to treat a numerous cancers including ovarian, cervical,
and lung.3-5
A common feature of the three Pt-based anticancer drugs is the presence of two leaving
groups in cis positions. For cisplatin, there are two cis chloride ions, and for carboplatin and
oxaliplatin there are two carboxylate oxygen atoms. The structures of the three compounds
translate into their activities in the cell, which comprise the following four common steps: (a)
cellular uptake through active or passive transport; (b) activation of the Pt(II) complex by
displacement of the two cis chloride ligands or carboxylates; (c) DNA binding; and lastly, (d)
multifactorial cellular response to the damage on the genome. 6' 7 Figure 1 illustrates these four steps
through which cisplatin ultimately triggers apoptosis.
34
[CI-]=104mM
[CI-I=4-lOmM
H2N
H3N
H3 N
C
H3N,
i
Cl
HN"\Cl
~
ctrl, ctr2,
/OH
Pt\
H3N
H3
2+
N
HN;
0
OH
H2
N
NH
N
Pt
' cellular response
NN
passive transport
extracellular matrix
intracellular fluid
Figure 1. Four steps in the cellular action of cisplatin.
In order to form intrastrand cross-links, it is necessary for the platinum complex to have
two leaving groups in cis positions. Because the stereoisomer of cisplatin, trans-[Pt(NH3)2C2]
(TDDP), is inactive, it was long assumed that only cis-substituted platinum complexes would
display anticancer activity. Recently, several active trans platinum compounds have been
discovered that display activity different from that of cisplatin and violating the previously known
structure-activity 'rules' that guided the design of antitumor platinum drug candidates for many
years. 8
In addition to its inability to form intrastrand cross-links in a manner analogous to cisplatin,
the inactivity of TDDP has also been ascribed to stem from kinetic instability, which makes it more
susceptible to deactivation by cellular nucleophiles. '9 Active trans complexes were thus
synthesized using bulky amines or Pt(IV) pro-drugs to decrease kinetic lability (Figure 2).1112
These novel compounds were reported to be as active as cisplatin and to display a totally different
spectrum of activity compared to cisplatin. 13 For example, JM-335 lacks cross-resistance to
cisplatin in cell lines where resistance was ascribed to enhanced DNA repair.10
35
OH
YNOKIN
_N-Pt'N
N
'CI
CI,
-N
/N
Pt,
'C
N::/
O
CI
.N
N. PC CI
CI
CN-:'PINH 3
NNP
I
H2 OH
-C
JM-335
Figure 2. Active trans Pt complexes.
In the present study, we focused on the synthesis of Pt(IV) complexes having trans
stereochemistry that can act as pro-drugs for trans Pt(II) compounds. Pt(IV) chemistry has widely
been used in this manner to attach various targeting moieties to cisplatin while preserving the
stability and activity of the parent drug. 6 In addition, they facilitate the use of nanoparticle carriers
such as PLGA-PEG, gold nanoparticles, or carbon nanotubes for drug delivery.14-16 Following a
recent strategy employing Pt(IV) chemistry to attach hydrophobic tails to improve the stability and
cytotoxicity of cisplatin, we conjugated hydrophobic chains to TDDP, which remarkably conveyed
significant biological activity.' 7
Experimental
Synthesis of t,t,t-[Pt(NH) 2Cl2 (OH)(CO2 CH2 CH2 CO2H)].1.5 DMSO (Compound 4)
t,t,t-[Pt(NH 3) 2C 2(OH)(CO 2CH 2CH 2CO 2H)]-1.5 DMSO (Compound 4) was prepared
following
the literature protocol
used
to
synthesize
analogous
Pt(IV)
complex
c,c,t-
[Pt(NH 3)2C12 (OH)(CO2CH 2CH 2CO 2H)]. 18 To a solution of t,t,t-[Pt(NH 3) 2C 2 (OH) 2] (209 mg,
0.628 mmol) suspended in 16 mL anhydrous DMSO, succinate anhydride (56.5 mg, 0.565 mmol)
was added and the reaction mixture stirred at room temperature for 12 h. The solution was
36
lyophilized and 10 mL acetone was added to precipitate a yellow solid. The solid was washed two
times with additional 10 mL acetone and dried in vacuo. The final product was characterized by
ESI-MS and NMR spectroscopy (Figure 3).
t,t,t-[Pt(NH13)2C2(OH)(CO2CH2CH2CO2H)]-1.5 DMSO. Yellow solid. Yield: 47.0 %. ESI-MS
m/z calculated ([M+H]+): 435.0, found: 434.9. 'H NMR (400 MHz, DMSO-d6): 6 5.75 (6H, broad),
2.34 (4H, m).
DMSO-d6 ):
13
C NMR (400 MHz, DMSO-d6 ): 6 180.0, 174.5, 32.2, 30.6.
195Pt
NMR (400 MHz,
5 956. Analytical HPLC: 96.0 % purity (Figure 4). Analysis calculated for
C7H2 1Cl 2 N2 O6 j5PtSI.5 : C, 15.25; H, 3.83; N, 5.08; found: C, 15.42; H, 3.36; N, 5.19.
37
0
(A)
H3N,,
OH
(C)"
I.,,Cl
CPtN
CI1
I NNH3
17
OH
(B)
L-J.
04
4.0
S.5
50
2.5
Pv-
(E)
(D)
955.72 ppm
Ca .:43&.0
liiiI diA
i Iil i I i I li lmi1I
.
.
.I '
.
Fr
- [M+H\-
1+HJ'
id: 434.9 IM+Hl.
Figure 3. NMR and ESI-MS spectra of 4: (a) chemical structure of 4; (b) 1H NMR spectrum
of 4 in DMSO-d6; (c) 13C NMR spectrum of 4 in DMSO-d6; (d)
DMSO-d6; (e) ESI-MS spectrum of 4.
195Pt
NMR spectrum of 4 in
38
Column: CI 8-Zorbax
Temp: Room Temperature
Flow rate: lmL/min
Detection: UV, 240 nm
Mobile Phase:
A: 0.5 % TFA in H2 0
B: 0.1 % TFA in Acetonitrile
Time (min)
0
5
35
37
38
40
%B
60
60
98
98
5
5
mAU -
2000-
1500-
1000-
500c
C-7
0-
0
10
u
C
Irz
20
30
min
Figure 4. HPLC conditions and HPLC chromatogram for the analysis of compound 4
General Synthesis of 5
Isocyanate moieties were attached to 4 following an analogous literature protocol for
cisplatin.1 7 To a glass vial containing compound 4 (20 mg, 0.046 mmol) dissolved in 2 mL of
anhydrous DMF were added two equiv of isocyanate agents and the reactions were stirred at room
temperature for 3 h. The solvent was then removed under reduced pressure at 65 'C and 5 mL of
diethyl ether was added to precipitate a yellow solid. The solid was washed twice with diethyl
39
ether and dried in vacuo.
Synthesis of ttt-[Pt(NH)2 C12(dodecyl carbamate)(CO2 CH2 CH2CO2H)] (CompoundSa)
Reagents used in the reaction: Compound 4 (20 mg, 0.046 mmol), and dodecyl isocyanate
(19 mg, 0.092 mmol).
t,t,t-[Pt(NH3)2C2(dodecyl carbamate)(CO2CH2CH2CO2H)]. Pale yellow solid. Yield: 27.1 %.
ESI-MS m/z calculated ([M-H1-): 644.0, found: 643.9. 1H NMR (400 MHz, DMF-d7 ): 5 6.56 (7H,
broad), 3.02 (2H, m), 2.48 (4H, broad), 1.45 (2H, m), 1.28 (20H, broad), 0.88 (3H, t). 13C NMR
(400 MHz, DMF-d7): 5 181.1, 174.3, 165.0, 36.4, 32.8, 27.9, 23.5, 14.7.
19 5Pt
NMR (400 MHz,
DMF-d7 ): 5 1048 (Figure 5). Analytical HPLC: 91.8 % purity (Figure 6). Analysis calculated for
C17H3 7 CI2 N30 6Pt: C, 31.63; H, 5.78; N, 6.51; found: C, 33.38; H, 5.77; N, 6.73.
40
OH
a
O
(A)
b
03113'0^"60,520as
(C)
C
H3 N,
'Pt"
NH3
CI ,,4I
b
C HN
HN
d
Ion
f
0
e
~
(B)
0.70
M
260
160
160
110
120
100
80
Cls,,o~8 Shift (pp, 5
60
20
00
0
20
ITPT_C 2_PROCESSED.ESP
0.65 -
0.55
water
0.50
0.45
I
0.40
0.35
0.30
I
0,25
0.20
0.15
Et 2O
0.10
0.05
0-
1
4 b, c
7.49
I
......
...
2,05
L-I
O.
0
sr55
4.5
s.
3pmChemical 56,5 (ppm)
(D)
203 12594 3 00
"- I I
- -
4.35
(
2.0
1E)5
1.0
(E)
-
0.5
rr
0
1
Ca,.: 644.0 [M-H]-
1048.30 ppm
I
Found: 643.9 [M-H]-
I
(M-HJ-06-
z
Figure 5. NMR and ESI-MS spectra of 5a: (A) chemical structure of Sa; (B) 'H NMR
spectrum of 5a in DMF-d7; (C) 13C NMR spectrum of 5a in DMF-d7 ; (D)
5a in DMF-d7 ; (E) ESI-MS spectrum of 5a.
19 5Pt NMR
spectrum of
41
Column: Cl 8-Zorbax
Temp: Room Temperature
Flow rate: ImL/min
Detection: UV, 220 nm
Mobile Phase:
A: 0.5 % TFA in HO
B: 0.1 % TFA in Acetonitrile
Time (min)
%B
0
40
2.5
25
40
98
mAU
1500
1000
31
98
32
40
1
32.5
40
CO
500
0
0
50
Figure 6. HPLC conditions and HPLC chromatogram for the analysis of compound 5a.
Synthesis of t,t,t-[Pt(NH)2C1
2(hexadecyl carbamate)(CO2CH2 CH2CO2H)J (Compound 5b)
Reagents used in the reaction: Compound 4 (20 mg, 0.046 mmol), and hexadecyl
isocyanate (25mg, 0.092 mmol).
t,t,t-[Pt(NH3)2C12(hexadecyl carbamate)(CO2CH2CH 2 CO 2H)]. Pale yellow solid. Yield: 45.8 %.
ESI-MS m/z calculated ([M-H]-): 700.0, found: 700.0. 1H NMR (400 MHz,
DMF-d7 ): 5 6.64 (5H,
42
broad), 6.29 (1H, broad), 3.04 (2H, m), 2.51 (4H, m), 1.43 (2H, m), 1.28 (22H, broad), 0.88 (3H,
t). 13C
95
NMR (400 MHz, DMF-d 7 ): 8 179.9, 173.9, 164.4, 41.5, 31.7, 26.9, 22.5, 13.7. 1 Pt NMR
(400 MHz, DMF-d7): 6 1074 (Figure 7). Analytical HPLC: 89.5 % purity (Figure 8). Analysis
calculated for C2 H45C 2N306Pt: C, 35.95; H, 6.47; N, 5.99; found: C, 34.32, H, 4.33; N, 5.93.
C
(A\
()
OH
Sa
V)
b
41
5
03513
)O
b
030]
0
C
HN
I
04
o
a
g
d
R=
o
j
C16
(B)
z
s
1da
2o
oth'..........
lit
040
a- M
.r..mp
10Og12130cft_yao5.MF.O1.oo1
1.
040
0.35
0.30
025
Et2O
e
0.10
a"
d
0ss
5-02
4
waer
t
bt
050
85
6-i.j
0781.3
Chme.mo
(D)
Si1
f
20
L.A
g
28
L.j
10
L.A
(ppm)
(E)
1074.77 ppm
Cal: 700.0
[M-H]
Found: 700.0 [M-H]-
fM-Hr
43
Figure 7. NMR and ESI-MS spectra of 5b: (A) chemical structure of 5b; (B) 'H NMR
spectrum of 5b in DMF-d7; (C)
13 C
NMR spectrum of 5b in DMF-d7 ; (D) 95Pt NMR spectrum of
5b in DMF-d7 ; (E) ESI-MS spectrum of 5b.
Column: C I8-Zorbax
Temp: Room Temperature
Flow rate:
ImL/min
Detection: UV, 220 nm
Mobile Phase:
A: 0.5 % TFA in H20
B: 0.1 % TFA in Acetonitrile
Time (min)
%B
0
40
2.5
40
25
98
31
98
32
40
32.5
40
mAU
a)
1000
C
1I(N
0
10
20
30
Figure 8. HPLC conditions and HPLC chromatogram for the analysis of compound 5b.
MITAssay
The cytotoxicity profile of TDDP, 5a, and 5b was analyzed against different cancer cell
lines (A549, A2780, A2780CP70). A detailed procedure for the MTT assay can be found under
Chapter 1 experimental section.
44
Cellular Uptake
A detailed procedure for the cellular uptake can be found in the Chapter 1 experimental
section.
DLS Measurements of Nanostructuresformed by TDDP, 5a, and 5b
A 5 mg portion of TDDP, 5a, or 5b was suspended in 10 mL of PBS and ultrasonicated
for one h to reach maximum solubility. The three solutions containing the Pt compounds were then
filtered through a 0.2 prm syringe filter to remove undissolved solid. The concentrations of the
three solutions were then measured by GFAAS and adjusted to be 30 pM. Possible nanostructure
formation was determined using DynaPro NanoStar Light Scatterer. Data were analyzed using the
DYNAMICS 7.1.7.16 program using globular protein Mw-R model.
The Effect of Concentrationon NanostructureFormation
A series of dilutions were made to prepare different concentration of 5a in PBS (57 pM,
9.5 pM, 0.95 pM, 0.095 ptM). The nanostructures formed by these solutions were studied using
DynaPro NanoStar Light Scatter.
TEM Imaging
Two drops of 30 pM of 5a were applied to a mesh copper grid purchased from Electron
Microscopy Sciences. TEM images were then obtained using 200 kV JEOL 200CX General
Purpose TEM at the MIT Center for Materials Science and Engineering (CMSE).
45
DFTCalculations
DFT calculations were performed using the Gaussian-03 software package. The geometry
of carbamate complexes was optimized in the gas phase starting from the structure obtained by
modifying Wilson's X-ray structure of c,c,t-[Pt(NH3) 2Cl 2(O 2 CCF3) 2]' 9 using the B3LYP functional.
LANL2DZ basis set and effective core potential were utilized for platinum atom and 6-3 1G basis
set was used for other elements.
Results & Discussion
Synthesis and Characterizationof TDDP-BasedPt(IV) Carbamates
TDDP-based Pt(IV) carbamates were prepared by modifying an analogous literature
protocol for cisplatin (Scheme 1).17 TDDP was first oxidized by 30 % H 2 0 2 to form 3. A succinate
moiety was then attached to one of the hydroxyl groups of 3 to generate 4. Isocyanate reagents (RN=C=O), where R refers to dodecyl and hexadecyl group, were then attached to the remaining
hydroxyl group, resulting in the formation of two amphiphilic Pt(IV) carbamates 5a and 5b.
0
C.
cIa
Ha
3N.
Pf"
NH3
CI
H3N
1
70.3%
CI
PI
2
TDDP
HA..,, OH.C
1 0
C
O"Pt
NH 3
cI I N NH 3
OH
73.5%
3
47.0%
b
A 1 I",
CI
OH
0
OH
dd
I NNH3
OH
4
C16:45.8 %
C27%
C12: 27.1I %
A " I Ic
Pt,
CI* I "NH3
0
HN
o
H
5a
R=
C12
C16
Scheme 1. Synthetic scheme used to synthesize TDDP-based Pt(IV) carbamates.
Sb
46
Cytotoxicity Study of 5a and 5b
The cytotoxic properties of 5a and 5b were measured in three cancer cell lines (A549,
A2780, A2780CP70) and compared to that of TDDP (Table 1). By attaching long hydrophobic
chains to TDDP, we were able to dramatically increase its cytotoxicity. The largest increase
occurred for 5b in A549 lung cancer cell lines; compared to TDDP, 5b had a nearly 75-fold
increase in cytotoxicity. In addition, 5b was 50-fold more cytotoxic in A2780 (ovarian cancer)
cells and 40-fold more lethal in the A2780CP70 (ovarian cancer resistant to cisplatin) cell line. For
comparison, 5b was approximately 2-5 times more cytotoxic than 5a. These results are consistent
7
with observations made previously by us using cisplatin-derived carbamates.1
Table 1. IC5o values of TDDP, 5a and 5b measured in three different cell lines.
unit: pM
TDDP
5a
5b
A549 (Lung)
423.36 ± 21.67
14.67 ± 1.34
5.64 ± 0.80
A2780
76.98
8.34
1.55
(Ovarian)
± 13.03
± 0.89
± 0.23
A2780CP70
24.3
414.3
45.57 3.45
0.99
10.21
The improved cytotoxicity of TDDP-based Pt(IV) carbamates is explained by a large
increase in cellular uptake; the cellular uptake of 5b is 7.7 times greater than that of TDDP and 5a
is taken up 4.1 times better than TDDP (Figure 9). Increases in cellular uptake promote
cytotoxicity.
20
47
Cellular Uptake
(pmol/ IM cels)
250M0
200.00
150.00
100.00
50.00
0.00
U transplatin
U5a
U 5b
Figure 9. Cellular uptake of Pt(II) complexes in A549 cell line.
NanostructureFormation
Our interest in understanding how the TDDP-based Pt(IV) carbamates are delivered to
cancer cells prompted us to investigate potential nanostructures formed by 5a and 5b. When the
carbamates are suspended in PBS, they spontaneously assemble, forming supramolecular
constructs. DLS measurements of 30 ptM solutions of 5a and 5b revealed that 67.2 % of 5a exists
as r = 5.24 nm micelles and 27.8 % as r = 128.66 nm lipid bilayers, whereas 5b exists solely as
3.92 nm micelles (Figure 10). By contrast, TDDP alone suspended in PBS produces no evidence
for the formation of nanostructures (Figure 11). DFT calculations suggest that the ability of 5a and
5b to form nanostructures comes from an increase in the dipole moments of these complexes
compared to that of TDDP. Introducing succinate and carbamate group to TDDP increases the
dipole moment of 5b from 0.18 to 6.20 Debye (Figure 12).
48
(a)
micelle
Pt(IV) bilayer
(b)
Figure 10. DLS measurement of 30 pM of 5a and 5b. (a) 5a suspended in PBS. 67.2 % of 5a
exists as r = 5.24 nm micelles and 27.8 %as r = 128.66 nm lipid bilayers. (b) 5b suspended in PBS.
100 %of 5b exists as 3.92 nm micelles.
49
eas 8
1.03
1
02
1.01
100
099
0.10
1.00
10.00
100 0
1.0E+3
1.0E4
1 OE.S
I
0E+6
1.0E-7
Tnw (PS)
(b)-
Mea, 3
12
0.10
t
1000
too00
1 0-3
Ti"
1.OE.4
1.OE.5
1"E4
10E-7
(PS)
Figure 11. Autocorrelation functions of DLS measurements corresponding to (a) TDDP (b) 5a and
(c) 5b. Autocorrelation functions show that while TDDP does not form any defined nanostructures,
50
5a and 5b do.
(a)
(c)
Dipole Moment
(Debye)
0.1755
6.2009
(b)
Figure 12. (a) DFT optimized structure of TDDP. (b) DFT optimized structure of 5b. (c) Table
showing the dipole moment of TDDP and 5b computed from the corresponding DFT optimized
structures.
The formation of nanostructures by 5a was furthered verified by transmission electron
microscopy (TEM) imaging (Figure 13). The images are consistent with the DLS data. TEM
images taken at 50-K magnification clearly show large constructs consistent with lipid bilayer
formation, whereas 400-K magnification images reveal smaller micelle structures.
51
(a)
(b)
Figure 13. TEM images of 30 pM 5a suspended in PBS. (a) TEM image taken at 50k X
magnification. (b) TEM image taken at 400k X magnification.
Based on the amphiphilic character of 5a and 5b, we initially surmised that both
carbamates were able to form platinum bilayers in PBS. Further studies revealed, however, that
only 5a aggregates into r > 100 nm lipid bilayer structures. The slight increase in chain length
going from 5a to 5b leads to a dramatic increase in the thermodynamic stability of micelle
compared to lipid bilayer. Additional DLS characterization of 5a revealed that the size of the
nanostructure also depends on the concentration of the platinum compound, with the larger bilayer
construct favored at higher concentrations while lower concentrations of 5a only give micelles
(Figure 14).
52
Average NP size
(nm)
70
60
so
40
20
10
-1.5
-1
-0.5
0
0.5
1
1.5
2
log ([5a]) (pM)
Figure 14. Average nanoparticle size of 5a as a function of log ([5a]). Average nanoparticle sizes
were calculated from DLS measurements.
Conclusion
In summary, utilization of Pt(IV) chemistry to attach hydrophobic chains to TDDP
generates cytotoxic complexes displaying significant anticancer activity. Compound 5b displays
up to a 7.7-fold increase in cellular uptake in A549 cells compared to TDDP, thus generating a 75fold improvement in cytotoxicity. Further investigations revealed that both 5a and 5b form
nanostructures when suspended in PBS medium. We attribute this behavior to the increase in
dipole moment of the trans Pt(IV) complexes compared to that of TDDP. To better understand the
molecular and cellular basis for the activity of the new trans Pt complexes, further studies are
needed to determine the structure of TDDP bound to DNA and the ensuing cellular responses.
53
References
1.
B. Rosenberg, L. VanCamp and T. Krigas, Nature, 1965, 205, 698-699.
2.
B. Rosenberg, L. VanCamp, J. E. Trosko and V. H. Mansour, Nature, 1969, 222,385-386.
3.
H. M. Keys, B. N. Bundy, F. B. Stehman, L. I. Muderspach, W. E. Chafe, C. L. Suggs, J.
L. Walker and D. Gersell, N. Engl. J. Med., 1999, 340, 1154-1161.
4.
M. Morris, P. J. Eifel, J. Lu, P. W. Grigsby, C. Levenback, R. E. Stevens, M. Rotman, D.
M. Gershenson and D. G. Mutch, N. Engl. J.Med., 1999, 340, 1137-1143.
5.
L. Kelland, Nat. Rev. Cancer,2007, 7, 573-584.
6.
D. Wang and S. J. Lippard, Nat.Rev. Drug. Disc., 2005, 4, 307-320.
7.
P. Heffeter, U. Jungwirth, M. Jakupec, C. Hartinger, M. Galanski, L. Elbling, M. Micksche,
B. Keppler and W. Berger, Drug Resist. Update, 2008, 11, 1-16.
8.
M. N. Coluccia, G., AnticancerAgents Med. Chem., 2007, 7, 111-123.
9.
E. Wong and C. M. Giandomenico, Chem. Rev., 1999, 99, 2451-2466.
10.
L. R. B. Kelland, C. F. J.; Mellish, K. J.; Jones, M.; Goddard, P. M.; Valenti, M.; Bryant,
A.; Murrer B. A.; Harrap, K. R., Cancer Res., 1994, 54, 5618-5622.
11.
N. Farrell, T. T. B. Ha, J. P. Souchard, F. L. Wimmer, S. Cros and N. P. Johnson, J. Med.
Chem., 1989, 32, 2240-2241.
12.
N. Beusichem. M. V.; Farrell, Inorg. Chem., 1992, 31, 634-639.
13.
J. M. Perez, M. A. Fuertes, C. Alonso and C. Navarro-Ranninger, Crit. Rev. Oncol.
Hematol., 2000, 35, 109-120.
14.
S. Dhar, W. L. Daniel, D. A. Giljohann, C. A. Mirkin and S. J. Lippard, J.Am. Chem. Soc.,
54
2009, 131, 14652-14653.
15.
S. Dhar, F. X. Gu, R. Langer, 0. C. Farokhzad and S. J. Lippard, Proc. Natl. Acad. Sci.
U.S.A., 2008, 105, 17356-17361.
16.
S. Dhar, Z. Liu, J. Thomale, H. Dai and S. J. Lippard, J. Am. Chem. Soc., 2008, 130, 1146711476.
17.
Y-R. Zheng, K. Suntharalingam, T. C. Johnstone, H. Yoo, W. Lin, J. G. Brooks and S. J.
Lippard, UnpublishedResults, 2013.
18.
M. D. Hall, C. T. Dillon, M. Zhang, P. Beale, Z. Cai, B. Lai, A. P. Stampfl and T. W.
Hambley, J. Biol. Inorg. Chem., 2003, 8,726-732.
19.
J. J. Wilson and S. J. Lippard, Inorg. Chem., 2011, 50, 3103-3115.
20.
M. R. Reithofer, A. K. Bytzek, S. M. Valiahdi, C. R. Kowol, M. Groessl, C. G. Hartinger,
M. A. Jakupec, M. Galanski and B. K. Keppler, J. Inorg. Biochem., 2011, 105, 46-5 1.
55
Chapter 3.
Investigation of the Cellular Activity of the
Dinuclear Os(VI) Nitrido Complex [NBu4]2[(OsNCl4)2(pyz)]
56
Abstract
We report the cellular activity of the dinuclear osmium(VI) nitrido complex
[NBu4]2[(OsNC14)2(pyz)] (pyz = pyrazine) (complex 1). The cytotoxicity toward different cancer
cell lines ranged from 13 to 69 pM. Despite the negative charge of the dinuclear osmium complex,
a significant amount of 1 was taken up by cells, half of which was found in the nucleus. Gel
electrophoresis and DNA binding kinetics experiment suggested that complex 1 binds efficiently
to DNA. In addition, flow cytometric analysis illustrated that 1 exerts its cytotoxic effects through
apoptosis.
Introduction
In order to address the shortcomings associated with the platinum-based drugs' used in
anticancer therapy, researchers have actively searched for other transition metal anticancer
complexes with novel properties. Transition metal complexes of ruthenium 2 , titanium 3 , and
osmium4 have shown some promising in vitro and in vivo anti-tumor properties. Ruthenium has
been spotlighted as it showed some interesting anticancer properties. 5 Two ruthenium compounds
have entered clinical trials and were identified to be less toxic and capable of overcoming the
resistance induced by platinum drugs in cancer cells. 5
Although analogous organometallic Os and Ru complexes have similar 3D structures, 4
osmium containing compounds have not yet been fully explored due to the widely held belief that
all Os compounds are toxic. 6 However, recent research has shown that certain half-sandwich
organometallic iodido Os arene complexes and Os(VI) nitrido complexes can have interesting
57
antitumor activity.4 In fact, our lab has developed a series of Os(VI) nitrido complexes whose
7
cellular responses are tunable by subtle ligand modifications. One of the newly developed Os(VI)
nitrido complexes was shown to be the first known osmium compound to induce ER stress in
cancer cells.7
Our discovery implies that osmium chemistry has much potential in the design of novel
therapeutic agents that may overcome drawbacks associated with platinum therapy. In the hope of
discovering unique anti-tumor activity of novel osmium structures, we investigated the cellular
properties of the negatively charged dinuclear osmium(VI) nitrido complex. Many dinuclear
osmium (VI) complexes have previously been synthesized and their electronic properties
explored. 8 - 0 However, to the best of our knowledge, no cellular study has yet been conducted on
these negatively charged dinculear Os complexes. We here report the cellular activity of the
dinuclear osmium(VI) nitrido complex [NBu 4 ]2[(OsNC14)2(pyz)] (pyz = pyrazine) (complex 1).8
N
Cl-,,
C1 I
[NBu4] 2
,.Cl
N
CN
CI
N
C
Cl,
CEI*0P ,1FCI
N
1
Figure 1. Structure of the osmium(VI) nitrido complex under investigation.
58
Experimental
Materialsand Methods
The dinuclear Os complex 1 was kindly synthesized and donated by Dr. K.
Suntharalingam.
Cell Culture Conditions
A2780 ovarian cancer cell lines were cultured in RPMI media supplemented with 10 %
fetal bovine serum and 1 % penicillin/streptomycin. A549, HeLa, and MRC5 cell lines were
cultured in DMEM media supplemented
with 10 % fetal bovine serum and 1
%
penicillin/streptomycin. The cells were grown at 37 'C in a humidified atmosphere containing 5 %
CO 2.
MTTAssay
Complex 1 was prepared as a 10 mM solution in DMSO and diluted using RMPI (A2780)
or DMEM (A549, HeLa, and MRC5). The final concentrations of DMSO in each well was 0.5 %.
The rest of the procedure for the MTT assay can be found in the Chapter 1 experimental section.
Gel Electrophoresis
Plasmid DNA (pUC 18) was kindly donated by Dr. J. J. Wilson. Solutions containing 82.5
pg of DNA and 0, 12.5, 25, 50, 100, 250, 500 and 1000 pM of complex 1 with a total reaction
volume of 18 ptL were incubated at 37 "C for 24 h. Following incubation, 5 pL of loading buffer
59
(0.25 % bromophenol blue, 0.25 % xylene cyanol and 60 % glycerol) was added and the reaction
mixtures were loaded onto a 1% agarose gel. Tris-acetate EDTA (TAE) was used as the running
buffer. The agarose gel was then run for 2.5 h at 80 V. Finally, the gels were stained in the TAE
solution containing ethidium bromide (1.0 ptg/mL) overnight. The DNA bands were analyzed using
a Flor-S reader under UV light (BioRad).
DNA Binding Kinetics
The DNA binding kinetics of 1 were determined and compared to cisplatin. 25 pM of
cisplatin and complex 1 were prepared in 2.79 mL of Tris-HCI (5 mM, pH 7) buffer. 210 pL of
1.78 mM calf thymus DNA (5 equiv) purchased from Sigma-Aldrich was added to the three
solutions to a total volume of 3 mL and the solutions were incubated at 37 *C. 250 pL aliquots
were taken at defined time points (0, 0.5, 1.5, 3, 5, 7, 16 h) and 5 pL sat. NaCl and 1 mL of ethanol
were added to the aliquots to quench the reaction. The rest of the procedure can be found in the
Chapter 1 DNA precipitation experiment.
Cellular Uptake
Approximately 5x10 6 A2780 cells were seeded in a 60 mm diameter Petri dish in triplicate
and were incubated for overnight in RPMI. The cells were then treated with 5 pM of complex 1
and incubated at 37 *C in 5% C02 for 12 h. The rest of the procedure can be found in the Chapter
1 cellular uptake experiment.
60
CellularDistribution
Cell pellets treated with 5 pM of complex 1 were collected following the same procedure
used to collect cell pellets for the cellular uptake experiment. The Thermo Scientific NE-PER
Nuclear and Cytoplasmic Extraction Kit was used to extract separate cytoplasmic, nuclear and
membrane fractions. The fractions were then suspended in 200 pL of 70% HNO 3 and digested at
70 'C for 2 h. The Os content was then analyzed using graphite furnace-atomic absorption
spectroscopy (GFAAS).
Nuclear DNA Osmium Content
Nuclear fractions of A2780 cells treated with 5 pM of complex 1 for 12 h were collected
following the same procedure used to collect nuclear fractions in the cellular distribution
experiment. The resulting nuclear extract was treated with equal volume (200 p L) of DNAzol. The
reaction mixture was then vortexed for 1 min and left on ice for 15 min. 1 mL of ethanol was then
added and the reaction mixture vortexed for 1 min and centrifuged for 20 min. The mixture was
left again on ice for another 15 min and centrifuged for 20 min. The resulting supernatant was
removed and the pellet re-dissolved in 200 ptL of water. The DNA concentration was determined
by UV-visible spectroscopy, and osmium quantified by GFAAS.
Cell Cycle Assay
A2780 cells were incubated with and without 15 pM of complex 1. Following 24 h, 48 h,
or 72 h incubation, cells were harvested from the culture media by trypsinization. Following
61
centrifugation, cells were washed with PBS and fixed with 1 mL of 70 % ethanol in PBS. Before
flow cytometry studies, the fixed cells were collected by centrifugation at 2500 rpm for 5 min and
washed with 1 mL PBS. The cell pellets were resuspended in 400 tL of 50 ptg/mL propidium
iodide (Sigma) in PBS and treated with 10 pL of 100 ptg/mL RNaseA (Sigma). DNA content was
measured on a FACSCalibur-HTS flow cytometer (BD Biosciences) using laser excitation at 488
nm and 20,000 events per sample were acquired. Cell cycle profiles were analyzed using the
ModFit software.
Apoptosis Assay
105 A2780 cells were incubated with doxorubicin (2 pM for 24 h), cisplatin (25 PM for
72 h), complex 1 (25 pM for 72 h) or without any treatment (72 h) and harvested from cultures by
trypsinization. Following centrifugation and removal of the media, an Annexin V-FITC Early
Apoptosis Detection Kit was used to check for apoptosis. After the cell pellets were suspended in
a Ix annexin binding buffer (96 L) (10 mM HEPES, 140 mM NaCl, 2.5 mM CaC2, pH 7.4), 1
pL FITC annexin V and 12.5 pL PI (10 pg/mL) were added to each sample and the samples
incubated on ice for 15 min. After incubation, 150 pL of additional binding buffer was added to
each sample. The samples were read on the FACSCalibur-HTS flow cytometer (BD Biosciences)
and 20,000 events per sample were acquired. Cell populations were then analyzed using the
FlowJo software (Tree Star).
62
Results & Discussion
Cytotoxicity Assay
Cytotoxicity profile of complex 1 was evaluated against a panel of human cancer cell lines
using the MTT assay (Table 1). In the cell lines tested, 1 displayed micromolar IC50 values ranging
from 13 pM in the A2780 ovarian cancer cell line to 69 pM in the A549 lung cancer cell. In all
four cell lines tested, complex 1 showed moderate toxicity that was in all cases, worse than
cisplatin. As a measure of therapeutic potential, we conducted cytotoxicity studies with healthy
lung fibroblast MRC5 cells. 1 was as toxic to MRC5 as it was to A549 cells, indicating
nonselective toxicity of complex 1.
Table 1. Cytotoxicity profile of complex 1 against a panel of human cell lines. The values reported
are an average of three independent experiments.
uni: pM A549 (Lung) HeLa (Cervical)
36 6
69 6
1
3
0.1
4 1
cisplatin
A2780 (Ovarian)
13 0.4
1 0.4
MRC5 (Lung Nornal)
69 ±1
11 ±1
Cellular Uptake/Distribution
The cellular uptake and distribution of complex 1 were studied in the A2780 ovarian
cancer cell line (Figure 2). Our study shows that despite the negative charge on complex 1, a
significant amount (214 pmol/1x 106 cells) of the dinuclear osmium complex was taken up by cells.
Complex 1 was essentially equally distributed in the cytoplasm and in the nucleus. That a large
portion of the administered osmium complex is found in nucleus encouraged us to investigate the
63
possible interaction of 1 with the DNA.
pmol/1 million cells
250
200
150
" whole cell
0 cytosol
100
" nucleus
membrane
50
0
*fl2
Figure 2. Osmium content in cytosol, nucleus, and membrane fractions isolated from the A2780
cell treated with complex 1 (5 pM for 12 h).
DNA Binding
The osmium content of the nuclear DNA extracted from the A2780 cells was analyzed. A
significant amount of osmium (40.8 pmol Os/pg nuclear DNA) was detected in the nuclear DNA
of the samples treated with 1. The amount of osmium found was more than two times larger than
those of mononuclear osmium nitrido compounds previously synthesized in our lab.7 Such high
amount of osmium per nuclear DNA prompted us to investigate the DNA binding properties of 1.
The interaction of 1 with pUC18 plasmid was studied using gel electrophoresis (Figure 3).
In contrast to the [NBu4]Cl control, as the concentration of 1 increased, there was a clear increase
in the amount of nicked circular DNA and a decrease in the amount of supercoiled DNA. At >250
piM, a band corresponding to nicked DNA appeared and then disappeared. Our result suggests that
64
1 induces conformational changes and degradation of the DNA. Such conclusion is consistent with
the previous report that mononuclear osmium nitrido complexes form stable adduct with GMP."
(12.5 uM - 1000 uM)
(12.5 uM - 1000 uM)
Form 11: Nicked
Fom I: Supercoiled
Form I: Supercoiled
Compound 1
[NBu 4]CI
Figure 3. Agarose gel electrophoresis of pUC18 DNA treated with 1 after 24 h incubation at 37 *C.
Lane 1, 9: DNA only; lanes 2-8: DNA+ 12.5,25,50, 100,250,500 and 1000 pM of 1 or [NBu 4 ]C.
The dynamics of DNA binding were further studied by monitoring the kinetics of the
reaction between 25 pM of complex 1 and 5 equiv ct-DNA (Figure 4) through a DNA precipitation
experiment.12 Our study shows that 1 binds to DNA in a rate comparable to cisplatin. The half-life
of the DNA binding reaction for both cisplatin and 1 was 1.5 h. Our result illustrates the excellent
DNA binding capability of 1.
Rv
0.120
0.100
0.080
-4--cisplatin
0.060
0.040
0.020
0.000
(hours)
-Time
0.00
2.00
400
600
8.00
10.00
12.00
1400
16.00
18.00
Figure 4. Kinetics of the binding of cisplatin (25 pM) and 1 to ct-DNA (125 pM). The ratio of
bound platinum per nucleotide (rb) was calculated at various time points.
65
Flow Cytometry Study
A flow cytometry study was conducted to investigate whether 1 affects the cellular cycle
(Figure 5). It was found that 1 does not induce any change to the cell cycle; changes in phase
populations remained <5 % even after a 72 h incubation.
To understand the cellular response to 1, we monitored whether 1 induces apoptosis
through a dual Annexin V staining/ PI flow cytometry assay. In the event of apoptosis, cells express
13
phosphatidylserine residues on the membrane exterior, which can be detected by Annexin V. We
monitored the occurrence of apoptosis in A2780 cells treated with 25 ptM of complex 1 for 72 h.
We found that complex 1 induces both early- and late-stage apoptosis (Figure 6). The results of
the apoptosis assay, along with those of DNA binding studies, indicates DNA damage is one of the
most important mechanisms responsible for the cytotoxicity of 1.
66
I
Untreated
0om
i..9
om
CIO G
24 h
0
so
09
200
IS
I.
I
M
CI
C1h..00 (FL2,A)
48 h
00
100
100
M0
2M0
0
so
100
C00,o14
100
(FL2-Al
200
20
72 h
LL~
0
Cllo
0102A)
a
00
IN
00
20
0
Figure 5. Histograms representing the different phases of the cell cycle for A2780 cells in the
absence and presence of 1 over the course of 72 h. 24 h untreated: Gi: 43.0 %S: 43.0 % G2/M:
14.0 %.48 h untreated: G1: 44.6 %S: 44.6 %G2/M: 10.8 %.72 h untreated: G1: 65.8 %S: 24.4 %
67
G2/M: 9.9 %. 24 h treated: G1: 42.6 %S: 43.1 %G2/M: 14.4 %.48 h treated: G1: 46.2 %S: 40.2 %
G2/M: 13.6 %. 72 h treated: G1: 66.0 % S: 22.9 % G2/M: 11.2 %.
Untreated
10
4
Doxorubicin
01
02
0.320%
0.547%
10
10
4
01
02
0.708%
2.481A
3
~0
Le
-e
0
0
0
162
10
05
0
10
10-
03
04_
tOo
-
6.23%
2.0%
100
10
102
100
Annexin V-FITC
01
03
10
10
10
10
Annexin V-FITC
Cisplatin
10
-
10
104
103
I
02
01
02
30,01%
4.90%
10
4
01
0.880%
02
1.14%
1013
v)
E)
102
0V
10
78.
47
10
10
03
04
10
0
30.0%
10
10
102
Annexin V-FITC
10
104
0
10
1
109
10
1t2
103
104
Annexin V-FITC
Figure 6. FITC Annexin V/PI binding assay plots of untreated cells, cells treated with doxorubicin
(2 pM for 24 h), cisplatin (25 pM for 72 h), or 1 (25 pM for 72 h).
68
Conclusion
In summary, we explored the antitumor properties of the dinuclear Os complex 1. 1
exhibited moderate cytotoxicity (IC5 o values ranged from 13 to 69 pM) and was found to bind and
induce degradation of DNA. Flow cytometry experiments suggested apoptosis to be one of the
most important mechanisms responsible for the cytotoxicity of complex 1. Our study is an
additional step forward in understanding the anticancer chemistry of osmium compounds. Further
studies need to follow exploring the biological activity of additional osmium constructs.
References
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L. Kelland, Nat. Rev. Cancer, 2007, 7, 573-584.
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Biographical Note
The author was born on March 5', 1990 in Seoul, Korea, the son of Sun Kook Yoo and
Junghwa Hyun. After graduating from Korean Minjok Leadership Academy (KMLA) in 2009, he
attended Stanford University, where he majored in biochemistry and minored in mathematics. He
worked under Professor Waymouth for three years to understand the thermodynamics of ring
opening polymerization of lactones and cyclic carbonates. He graduated from Stanford University
with the S.S. & I.M.F. Marsden Memorial Prize in Chemistry, the most prestigious award for
graduating seniors at Stanford. As a recipient of a Samsung Scholarship and a Koch Institute
CCNE Fellowship, he attended Massachusetts Institute of Technology to work on platinum
chemistry in the laboratory of Professor Stephen J. Lippard. His hobbies include learning foreign
languages, reading fantasies, and playing poker. He is pursuing a medical degree from Seoul
National University in Seoul, Korea.
70
Education
2012-2014
S.M. in Inorganic Chemistry, Massachusetts Institute of Technology, MA
Research Advisor: Stephen J. Lippard
2009-2012
B.S. in Chemistry with Honors, Stanford University, Stanford, CA
Research Advisor: Robert M. Waymouth
Minor in Mathematics, Stanford University, Stanford, CA
2006-2009
Korean Minjok Leadership Academy, Gangwon, Korea
Fellowships and Awards
2013
CCNE Graduate Fellowship
-One year of graduate school tuition support from MIT Koch Institute
2011
S.S. & I.M.F. Marsden Memorial Prize in Chemistry
-Awarded to the top chemistry undergraduate in Stanford along with $1000 in
prize; Most prestigious award for graduating chemistry majors
2011
Samsung Fellowship
-Annual scholarship of $50,000 for 5 years
2010
Bing Undergraduate Summer Research Fellowship
- $5600 stipend for 2011 summer undergraduate research
2006-2008
Three Gold Prizes in Korean Chemistry Olympiad
2008
I" Place Prize in Pohang Chemistry Olympiad
Work Experience
2012-2013
Chemistry teaching assistant at MIT.
2010-2011
Math grader at Stanford University.
2010-2011
Math & chemistry tutor at Stanford University.
2009-2011
Chemistry teaching assistant at KMLA.
2006-2011
Translator for PLAN Korea, an organization dedicated to providing support to
the third world children.
71
Publications and Conference Proceedings
1. Jin, S.; Yoo, H.; Woo, Y; Lee, M.; Vagaska, B.; Kim, J.; Uzawa, M.; Park, J. "Selective
sterilization of Vibro parahaemolyticus from the Bacterial Mixture by Low Amperage Electric
Current." J. Microb. 2009, 6, 537-4 1.
2. Kim, H.; Kim, M.; Lee, M.; Yoo, H.; Park, J. "Cells behavior and characterizations of
PLGA/EGCG films." 2009 BiomaterialsAcademy. 2009, 226.
3. Yoo, H.; Decrisci, A.; Waymouth, R.M. "An Investigation on the Thermodynamic
Characteristics of Ring Opening Polymerization of 6-Valerolactone." NCURS. 2011, 20.
4. Yoo, H.; Decrisci, A.; Waymouth, R.M. "Solvent Effect on the Thermodynamics of RingOpening Polymerization of 6-Valerolactone." Stanford University Honors Thesis. 2012
5. Yoo, H.; Zheng, Y-R.; Lippard, S. J. "Utilizing Pt(IV) Chemistry to Improve the Cytotoxicity
of the Inactive Pt(II) Compound trans-[Pt(NH3) 2C12]." Manuscript in Preparation.
6. Zheng, Y-R.; Suntharalingam, K.; Johnstone, T, C.; Yoo, H.; Lin, W.; Brooks, J. G.; Lippard, S.
J. "A Supramolecular Bioinorganic Hybrid for Delivery of Potent Pt(IV) Prodrugs." Manuscript
in Preparation.