Catalytic transfer hydrogenation and anticancer activity of arene-ruthenium compounds incorporating bi-dentate precursors

Article abstract of DOI:10.1039/c5dt01310k

Ruthenium based organometallic compounds are presently a subject of great attention as anticancer drugs and appear to work reasonably well on tumor cells. We develop a series of mononuclear arene-ruthenium compounds incorporating N,O and N,N bidentate lig

Full text of DOI:10.1039/c5dt01310k

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Synopsis for the Graphical Abstract
Catalytic transfer hydrogenation and anticancer activity of
areneꢀruthenium compounds incorporating biꢀdentate precursors
a
b
a
a
YuꢀHsiang Chang, WohnꢀJenn Leu, Amitabha Datta, HungꢀChang Hsiao,
c
b,
a,
ChiaꢀHer Lin, JihꢀHwa Guh, * JuiꢀHsien Huang *
A series of areneꢀRu compounds were synthesized and their catalytic transfer
hydrogenation and anticancer activity towards human hormoneꢀrefractory
prostate cancer were investigated.
1

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Pictogram for the Graphical Abstract
Catalytic transfer hydrogenation and anticancer activity of
areneꢀruthenium compounds incorporating biꢀdentate precursors
a
b
a
a
YuꢀHsiang Chang, WohnꢀJenn Leu, Amitabha Datta, HungꢀChang Hsiao,
c
b,
a,
ChiaꢀHer Lin, JihꢀHwa Guh, * JuiꢀHsien Huang *
2

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Catalytic transfer hydrogenation and anticancer activity of
areneꢀruthenium compounds incorporating biꢀdentate precursors
a
b
a
a
YuꢀHsiang Chang, WohnꢀJenn Leu, Amitabha Datta, HungꢀChang Hsiao,
c
b,
a,
ChiaꢀHer Lin, JihꢀHwa Guh, * JuiꢀHsien Huang *
a
Department of Chemistry, National Changhua University of Education,
Changhua, Taiwan 50058
b
School of Pharmacy, National Taiwan University, Taipei, Taiwan 100
c
Department of Chemistry, Chung-Yuan Christian University, Chun-Li 320,
Taiwan
Keywords: Catalytic transfer hydrogenation; anticancer activity; ruthenium;
pyrrole; ketoꢀamine ligands
*
Corresponding authors: JHH, 886-4-7232105 ext 3512, E-mail address:
juihuang@cc.ncue.edu.tw;
address:jhguh@ntu.edu.tw
JHG,
886-2-33937561,
E-mail
3

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Abstract
Ruthenium based organometallic compounds are presently an object of great
attention as anticancer drugs and appear to work reasonably well on tumor
cells. We develop a series of mononuclear areneꢀruthenium compounds
incorporating N,O and N,N bidentate ligands and their activity as anticancer
drugs against human hormoneꢀrefractory metastatic prostate cancer
(HRMPCs) cell lines are governed. The ruthenium compounds also indulge as
effective catalysts in the transfer hydrogenation of ꢀC=Oꢀ
→
ꢀCH(OH)ꢀ system.
t
Three types of ligands, sodium glutamate, C
4
H
3
NH(2ꢀCH
2
NH Bu), and
6
C
4
H
3
NH(2ꢀCH=NR) are separately coupled with [(
η
ꢀcymene)RuCl
2
]
2
(1)
(cymene = 4ꢀisopropyltoluene) to synthesize five Ruꢀderivatives, such as
6
2
[
(
η
ꢀcymene)RuCl(
κ
ꢀ
N,OꢀOOCCHNH
2
CH
2
CH
2
COOH)]
(2),
(3),
(4),
and
6
6
6
6
t
{
{
{
{
(
η
η
η
η
ꢀcymene)RuCl[C
ꢀcymene)RuCl[C
4
H
H
3
3
N(2ꢀCH
2
NH Bu)]}
(
(
(
4
N(2ꢀCH=NCH
2
Ph)]}
(C
ꢀcymene)RuCl{C
4
H
3
N[2ꢀCH=NCH
2
4
H
7
O)]}}
(5)
n
ꢀcymene)RuCl[C
4
H
3
N(2ꢀCH BuNHCH
2
(C
4
H
7
O))]} (7). To the best of our
knowledge, the aforementioned Ru compounds are not only characterized by
1
13
H and C NMR spectroscopy but first time established by single crystal Xꢀray
diffractometry. Compound 4 induces a concentrationꢀdependent apoptosis in
4

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PCꢀ3 cells and initiates the conversion rate in transfer hydrogenation.
Introduction
1
Platinum, a precious metal, is widely used in organic and medicinal
2
3
chemistry. Regarding the metalꢀbased anticancer drugs, cisplatin is the most
wellꢀknown drug for chemotherapy; however, several concerns have raised the
attention of using cisplatin due to (i) the platinum production limitation that
4
5
makes the platinum price increasing dramatically, (ii) drug resistance, and (iii)
6
side effects including nausea, vomiting, diarrhea, and hair loss. Therefore, the
development of other metalꢀbased anticancer agents is currently an interesting
approach for inorganic and bioinorganic chemists. In searching for
metalꢀbased anticancer agents with less side effects, rutheniumꢀbased
compounds have shown promising results presumably due to (i) the variable
oxidation states and higher coordination number, (ii) slower ligand exchange
rates, and (iii) easier reducing capacity from Ru(III) to Ru(II), i.e. less active
and nonꢀtoxic Ru(III) compounds that can be activated at the site of the
7
cancerous cells. Interest in Ru anticancer drugs has been growing rapidly
8
9
10
11
12
since NAMIꢀA, KP1019, RM175, RATPA and DW1 as shown in Scheme
1
have either entered into phase I or II clinical trial or shown highly potential for
5

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antitumor activity. The diversity of modes of action of Ru anticancer drugs is
also likely to enhance the anticancer activities and to reduce the potential to
develop tumor resistance.
The catalytic hydrogenation of polar bonds and the asymmetric versions
of these are key reactions in fine chemical and pharmaceutical synthesis.
Ruthenium complexes display favorable reactivity and selectivity, since it has a
2
strong tendency to perform a heterolytic activation of H instead of oxidative
13
addition to make a metal dihydride. Ruthenium compounds have remarkable
catalytic activity and easy accessibility in asymmetric transfer hydrogenation of
14
ketones, a convenient route of enantiomerically pure alcohols. In past years,
some papers dealing with Ru(II) complexes containing N,O donor ligands have
15
been published, in order to compare their structure and reactivity with those
of similar ruthenium derivatives, which show promising catalytic activity. Noyori
16
et al.
before reported the halfꢀsandwich (arene)Ru(II) complex that
undergoes to catalytic transformations, such as, for example, the highly
efficient asymmetric hydrogen transfer on C=O containing substrates. We also
17
checked some articles on the synthesis and reactivity of areneꢀruthenium
18
derivatives as well as N,O donor ligands.
From literature survey it is clear that ruthenium complexes have
6

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interesting anticancer properties in vivo and they might be a good alternative
of platinumꢀbased drugs for anticancer therapy. Besides this, we have put our
attention to furnish supportive information on Ruꢀcatalyzed asymmetric
transfer hydrogenation. We prefer the Ru derivatives incorporating bidentate
N,O or N,N donor ligands to put less steric hindrance around ‘[RuꢀH]’ motif that
initiates the conversion rate of ketone to alcohol. As well our approach is to
check the potentiality of Ru derivatives as drugs in anticancer effect against
human hormoneꢀrefractory metastatic prostate cancers (HRMPCs), one of the
most commonly diagnosed cancers and top leading cause of cancerꢀrelated
deaths in various countries.
Results and Discussion
Synthesis and characterization of compounds 2ꢀ7. A series of ruthenium
compounds mediating bidentate N,O or N,N ligands are synthesized, as
6
shown in Scheme 2 and 3. The initial reactant, [(
η
ꢀcymene)RuCl
O and phellandrene, is
refluxed with two equivs of sodium glutamate in methanol to produce the new
2 2
] (cymene =
19
4
3 2
ꢀisopropyltoluene), (1), obtained from RuCl .nH
6
2
2 2 2
ruthenium compound [(η ꢀcymene)RuCl(κ ꢀN,OꢀOOCCHNH CH CH COOH)]
(2), in moderate yield (Scheme 2). Compound 2 is insoluble in organic solvents
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like toluene, diethyl ether, methylene chloride, and THF but soluble in highly
1
polar solvents like methanol and H
2
O. On H NMR spectroscopy, the methyl,
methylene, and methine protons of 2 show resonances between
δ 1.1 ~ 3.5
and the aromatic protons of cymene ring belong in the range, 5.5 ~6.0.
δ
Similarly, reacting 1 with two equivs of lithium pyrrolyl amine ligand,
t
Li[C
4
H
3
N(2ꢀCH
2
NH Bu)]
in
toluene
generates
compound
6
t
{(η
ꢀcymene)RuCl[C
4
H
3
N(2ꢀCH
2
NH Bu)]} (3) in satisfactory yield. Compound 3
is soluble in methylene chloride, toluene, and THF but only has partial
1
solubility in diethyl ether. The H NMR spectrum of 3 shows two doublets of
t
doublet for the methylene protons of C
H
2
NH Bu at
δ
3.67 and 3.81. The amino
t
proton of CH
2
N
H
Bu fragment is observed at
δ
3.29 as a broad multiplet due to
the coupling with adjacent methylene protons, subsequently confirmed by
1
homoꢀnuclear decoupled H NMR spectra. The asymmetrical environment
around the Ru center can be affirmed from the four arene protons of cymene
moiety showing two multiplets at
δ 5.34 and 5.49.
Further reaction of 1 with lithiated pyrrole imine system features new
organoꢀruthenium derivatives, as shown in Scheme 3. Reacting pyrroleꢀimine,
4 3 2
C H NH(2ꢀCH=NCH Ph), with nꢀBuLi in methylene chloride generates a
pyrrolyl lithium salt, Li[C
4
H
3
2
N(2ꢀCH=NCH Ph)], which afterwards precipitates
8

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in addition of heptane. Interestingly, conjugation of two equivs of
Li[C
4
H
3
N(2ꢀCH=NCH
2
Ph)] and
1
in toluene originates compound
6
1
{(η
ꢀcymene)RuCl[C
4
H
3
N(2ꢀCH=NCH
2
Ph)]} (4) in 42% yield. The H NMR
spectrum of compound 4 shows four doublets for the four arene protons of
cymene and two doublets for the methyl groups of CHMe fragment, indicating
its asymmetrical arrangement around the Ru center. The chemical shift of
methylene protons of NC Ph is observed at 5.13, overlapping with the
resonance of one arene protons. Similarly, the coupling of 1 and another
2
H
2
δ
bidentate pyrroleꢀimine ligand,
4 3 2 4 7
C H NH[2ꢀCH=NCH (C H O)] is also
4 3 2 4 7
investigated. While two equivs of C H NH[2ꢀCH=NCH (C H O)] is directly
added to the THF solution of 1 at room temperature, compound
6
{(η
ꢀcymene)RuCl{C
4
H
3
N[2ꢀCH=NCH
2
(C
4
H
7
O)]}} (5) is initiated along with the
elimination of two equivs of hydrogen chloride. However, the low yield (32%)
could not be raised by adding strong bases like NaOH or NEt to remove
3
hydrogen chloride. Compound 5 is also obtained from the reaction of 1 with
lithiated ligand, Li{C N[2ꢀCH=NCH (C O)]}, a product of ꢀBuLi and
4
H
3
2
4
H
7
n
4 3 2 4 7
C H NH[2ꢀCH=NCH (C H O)] in methylene chloride as similar yield. After
repeating reꢀcrystallization of compound 5 from diethyl ether, the imine
fragment (CH=N) shows a pair of characteristic proton and carbon resonances,
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at
tetrahydrofuranyl fragment of 5 may flip away or belong towards the Ru atom
see Scheme 4), subjecting the appearance as diastereoisomers to achieve
δ 7.50 & 7.60 and at δ 160.0 & 159.7, respectively. It seems that the
(
two sets of signals. They have chirality at the metal center, and an additional
asymmetric carbon in the ligand.
n
Moreover, a lithiated compound Li[C
4
H
3
N(2ꢀCH BuNHCH
2
(C
4
H
7
O))] (6) is
4 3 2 4 7
isolated while aggregating C H NH[2ꢀCH=NCH (C H O)] and nꢀBuLi in
1
13
heptane. The H and C NMR spectra of 6 supports the insertion of anionic
butyl group into the C=N double bond to result a lithiated pyrrolyl amino
1
derivative. The H NMR spectrum of compound 6 shows the corresponding
singlet of N
H
proton at
a
δ 9.74. Treating compound 1 with 6 in toluene
generates
ruthenium
pyrrole
compound,
6
n
{(η
ꢀcymene)RuCl[C
4
H
3
N(2ꢀCH BuNHCH
2
(C
4
H
7
O))]} (7) in moderate yield.
1
13
The H and C NMR spectra of 7 are not pure due to the overlapping of the
butyl, tetrahydrofuranyl, methyl, and isopropyl moieties (see supporting
information).
Molecular structure of compounds. Crystals of compounds 2ꢀ5 and 7,
suitable for single crystal Xꢀray diffraction study, are obtained from different
10

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organic solvents. The summary of data collection and selected bond lengths &
angles are listed in Table 1 and 2, respectively. Inserting monosodium
20
glutamate in binding metals is reported; however, there is no survey of crystal
structures concerning glutamatoꢀbased metal derivative till date. To the best of
our knowledge, compound 2 is the first Ruꢀbased glutamate derivative,
supported by Xꢀray diffraction analysis. The asymmetric unit consists of two
independent molecules of 2. However, these two molecules are quite similar;
so one molecular unit is presented in Figure 1. Compound 2 shows a threeꢀleg
21
piano stool geometry with the Ru to the center of cymene at ca. 1.6 Å. The
glutamate moiety binds to Ru through the amine nitrogen and one of
carboxylate oxygen atoms forming a fiveꢀmembered chelate ring. The bond
lengths of Ru to glutamate are Ru(1)ꢀO(1) 2.080(14) and Ru(1)ꢀN(1) 2.128(16)
Å. Interestingly, it is observed that the corresponding bond lengths of the
coordinate carboxylate group of the glutamate shows neither double bond nor
single bond character [1.22(2) and 1.33(3) Å] since it involves in resonance
while coordinating to Ru atom (Scheme 5), but the uncoordinated carboxylate
unit displays a regular double bond mode [1.18(3) Å]. Compound 2 exhibits
both intraꢀ and interꢀmolecular hydrogen bonding network (Figure 2 and Table
22
S2), to strengthen the molecular stability as reported earlier.
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The molecular geometries of compounds 3ꢀ5 and 7 are quite similar and
depicted in Figures 3ꢀ6. The unit cells for compounds 3ꢀ5 all contain two
molecular units where compound 3 shows an intraꢀmolecular Cl———H hydrogen
bonding with the bond distance about 2.5570(4) Å. Compound 3ꢀ5 and 7 show
threeꢀleg piano stool geometries with the bond lengths of Ru to the center of
cymene and to the Cl atoms, ranging from 1.6751(4)~1.6843(5) Å and
2.4094(13)~2.4210(16) Å, respectively. Two types of substituted asymmetric
pyrrole ligands are involved in coordination with compounds 3, 4, 5, and 7.
Besides RuꢀNpyrrole bond, a pyrroleꢀamine bond is incorporated in compounds
3
and 7, whereas there exists a pyrroleꢀimine bond in compounds 4 and 5. The
RuꢀNpyrrole bond lengths for compounds 3, 4, 5, and 7 are very similar, ca.
.042(4)~2.066(4) Å, despite different ligand modes. However, RuꢀNpyrroleꢀamine
2
and RuꢀNpyrroleꢀimine bond lengths are slight different, RuꢀNpyrroleꢀamine bond
lengths (2.215(4) Å for 3 and 2.170(4) Å for 7) are much longer than those of
RuꢀNpyrroleꢀimine bond lengths (2.114(5) Å for 4 and 2.119(4) Å for 5), presumably
due to the steric hindrance of the bulkier pyrroleꢀamine ligands.
Anticancer activity in HRMPC cells. The sulforhodamine B (SRB) assay is
developed to measure drugꢀinduced antiꢀproliferative activity and cytotoxicity
12

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study on human cellꢀlines. The related data demonstrate that several areneꢀRu
derivatives display higher antiꢀproliferative activity than cisplatin on HRMPC
cell lines, namely PCꢀ3 and DUꢀ145 (see SI, Table S1). The obtained data
show that PCꢀ3 cells are more susceptible to the compoundꢀinduced
cytotoxicity than DUꢀ145 cells using SRB assay. The effect of areneꢀruthenium
compounds on HRMPC cells while fixing with propidium iodide to analyze DNA
content is represented in Figure 7. It is observed that compounds 3, 4 and 7
induce a concentrationꢀdependent apoptosis in PCꢀ3 cells; among these
compound 4 participates significantly as its 30
ꢀM conc. on 48 hrs duration.
The mitochondria, a pivotal organelles in the cytoplasm, participates in
23
potential anticancer strategy to induce cancer cell death. The mitochondrial
membrane potential (∆Ψ ) is determined using flow cytometric analysis of
m
m
JCꢀ1 staining. JCꢀ1 aggregates (red fluorescence) higher ∆Ψ under normal
m
condition. After drugꢀinduced loss of ∆Ψ , JCꢀ1 monomers are dominant with
green fluorescence, the indicator of mitochondrial dysfunction. The data in
Figure 8 demonstrate that both compounds 3 and 4 result in a significant
production of JCꢀ1 monomers (R2 green fluorescence) indicating the loss of
∆
m
Ψ . Thus following JCꢀ1 assay, we observe that compound 4 is well effective
24
for mitochondriaꢀinvolved apoptotic detection.
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Caspases, a family of cysteine proteases, play a crucial role in apoptosis.
Caspaseꢀ9 is an initiator caspase in mitochondrial apoptosis pathway in which
25
the proꢀenzyme of caspaseꢀ9 is cleaved into a 37ꢀkDa active form.
Mitochondrial apoptosis pathway may induce a crosstalk activation of
caspaseꢀ8, a major initiator caspase in extrinsic apoptosis pathway. Recently,
the immune fluorescence microscopic examination and biochemical analysis
show that proꢀcaspaseꢀ8 and active caspaseꢀ8 predominantly coꢀlocalize with
26
the mitochondria mainly on outer mitochondrial membrane.
The
corresponding data (see SI, Figure S1) demonstrate that the most potential
compound 4 significantly induces the activation of caspase cascades,
including the activation of caspaseꢀ9, ꢀ8, ꢀ3 and ꢀ7.
Transfer hydrogenation catalytic study. Homogeneous asymmetric
hydrogenation of olefins and ketones catalyzed by chiral transition metal
complexes provides a powerful means for preparing optically active organic
27
compounds. The reduction of carbonyl [C=O] groups to pure secondary
alcohols is a reaction of fundamental importance, more particularly the
asymmetric reduction of ketone to enantiometric secondary alcohols in modern
synthetic chemistry. For asymmetric hydrogenation, Ru(II) compounds are
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28
efficient catalysts such as in the reaction of ꢀC=Oꢀ
→ ꢀCH(OH)ꢀ in alcoholic
medium maintaining basic pH where H or other reducing agents like N H ,
2
2 4
4 4
LiAlH , NaBH may be used. In hydrogenation, the hydrido metal compounds
(
n
MH(L) ) may participate as catalyst and then can accelerate the reduction of
ketone in the catalytic cycle where reducing equivalentꢀH may be supplied by
the reducing solvent (like isopropanol) or from other reducing agents.
Presence of ancillary ligands to Ru(II) critically control the reaction efficiency
and percentage of yield. Electron donating substrate may reduce the stability
of hydridoꢀRu(II) ‘[RuꢀH]’ motif and hence decreases the rate. Besides the
presence of acidic substituent or labileꢀH in any part of the catalyst or substrate
or solvent also scavenge ‘[RuꢀH]’ and reduces the crop yield. Possible
reduction mechanism is given in Scheme 6. The results of a series of
substituted acetophenone reduced to isopropyl alcohol in the presence of
ruthenium compounds is shown in Table 3. Although the ruthenium
compounds play as catalysts; however, the substituents on the acetophenone
also play an important role in the reduction transfer hydrogenation.
Compounds 4 and 5 have higher reactivity toward the transfer hydrogenation
reactions (entry 3, 4, 9, 10, 15 and 16, Table 3) in comparing other substituted
acetophenones like 2, 3, and 7. Presumably, the pyrroleꢀimine ligand systems
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in compounds 4 and 5 may stabilize RuꢀH system due to πꢀdonating ability of
the chelated unit. Electron withdrawing groups on the aryl ring of ketones such
as 4’ꢀnitroꢀacetophenone dramatically reduce the conversion of ketone to
alcohol (entry 19ꢀ23, Table 3). This result indicates the coordination of C=O
fragments of ketones to the ruthenium atom before forming RuꢀHꢀꢀꢀCꢀO
fourꢀmembered ring (Scheme 6), a critical step for the transfer hydrogenation.
The phenomenon is also verified by the low conversion of sterically bulky
2’,4’,6’ꢀtrimethylacetophenone and isopropyl alcohol (entry 24ꢀ28, Table 3).
Conclusion
In this article we highlight the current interest on ruthenium based
1
13
metallodrugs. Besides the characterization by H and C NMR spectroscopy,
the single crystal Xꢀray diffraction analysis confirms the geometry of Ru atom.
The ruthenium compounds containing pyrroleꢀimine ligand systems show
higher conversion of transfer hydrogenation from ketoneꢀsubstrates to
secondary alcohols. In addition, the aryl ring of acetophenone with electronic
withdrawing and sterically hindered bulky groups defer the transfer
hydrogenation in presence of Ru catalysts. Furthermore, the toxicity of the
compounds is tested on HRMPC cell lines, namely PCꢀ3 and DUꢀ145.
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Compound 4 is the most influential to display effective anticancer activity
against HRMPCs and in apoptotic cell death through mitochondriaꢀinvolved
activation of caspase cascades. Further investigations with Ruꢀderivatives
manifested by newly designed organic precursors and their catalytic approach
on transfer hydrogenation as well as the potentiality on apoptosis in human
cells are under way.
Experimental Section
General procedure: All reactions were performed under a nitrogen
atmosphere using standard Schlenk techniques or in a glove box. Toluene and
2 2
diethyl ether were dried by refluxing over sodium benzophenone ketyl. CH Cl
2 5
was dried over P O . All solvents were distilled and stored in solvent reservoirs
that contained 4ꢀÅ molecular sieves and were purged with nitrogen.
6
19
[
[
[
(
η
ꢀcymene)RuCl
2
]
2
(cymene
=
4ꢀisopropyltoluene),
29
30
LiC
4
H
3
N-(2-CH=NCH
2
-C
4
H
7
O)],
[LiC
4
H
3
N-(2-CH=NCH
2
-C
6
H
5
)]
and
t
31
LiC
4
H
3
Nꢀ(2ꢀCH
2
NH Bu] were prepared according to published procedures.
1
13
H and C NMR spectra were recorded using a Bruker Avance 300
1
13
spectrometer. The chemical shifts for H and C spectra were recorded in ppm
3 6 6
relative to the residual protons of CDCl (δ =7.24, 77.0 ppm) and C D (δ =
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7.16, 128.0 ppm). Elemental analyses were performed using a Heraeus
CHNꢀOS Rapid Elemental Analyzer at the Instrument Center of the NCHU.
6
2
Synthesis of [(
2 2 2
η ꢀcymene)RuCl(κ ꢀN,OꢀOOCCHNH CH CH COOH)] (2). A
250 mL round bottom flask was charged with 1.0 g of 1 (1.6 mmol), excess of
monosodium glutamate (0.52 g, 3.2 mmol), and 100 mL of methanol. The
solution was then refluxed for 4 hours and filtered to avoid unꢀsoluble solid.
The filtrate was reduced to small amount and stored at ꢀ20°C to yield 0.56 g of
final product (41.0%). Crystals suitable for Xꢀray diffraction study were
1
obtained from a methanol solution of at ꢀ20°C. H NMR (
δ, D
2
O): 1.26 (m, 6H,
CHMe
2
), 2.17 (m, 3H, Me), 2.51 (m, 2H, CH
2
CH
2
CHNH
2
CO ), 2.79 (sept, J =
2
HH
2
6.6Hz, 1H, CHMe ), 3.05 (m, 1H), 5.76 (m, 4H). Anal. Calcd. for
15 4
C H22NClO Ru: C, 43.22; H, 5.32; N, 3.36. Found: C, 43.78; H, 5.41; N,
3.35%.
6
t
4 3 2
Synthesis of {(η ꢀcymene)RuCl[C H N(2ꢀCH NH Bu)]} (3). A Schlenk flask
charged with 0.10 g (0.16 mmol) of 1 and 15 mL of toluene was added a 15 mL
t
toluene solution of [LiC
4
H
3
Nꢀ(2ꢀCH
2
NH Bu] (0.052 g, 0.32 mmol) dropwise at
0°C. The mixture was stirred for 6 hours at room temperature and filtered
through celite. The filtrate was concentrated to small amount of volume and
18

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stored at ꢀ20°C to yield 0.071 g of brownꢀred final product (51.0%). Crystals
suitable for single crystal Xꢀray diffraction analysis were obtained using
1
diffusion method from a methylene chloride/heptane combined solvent. H
NMR (δ, CDCl
3
): 0.99 (d,
J
HH = 6.9 Hz, 3H, CHMe
2
), 1.11 (d,
J
HH = 6.9 Hz, 3H,
t
CHMe
2
), 1.46 (s, 9H, Bu), 2.09 (s, 3H, Me), 2.52 (sept,
J
HH = 6.9 Hz, 1H,
C
H
Me
arene C
), 6.85 (m, 1H, pyr C
0.7, 49.7, 57.1, 75.1, 82.1, 82.6, 86.0, 96.7, 101.1, 104.4, 109.8, 128.7, 133.7.
2
), 3.29 (br, 1H, N
H
), 3.67 (dd, 1H, C
H
2
), 3.81 (dd, 1H, C
), 6.19 (m, 1H, pyr
3
H). C{ H} NMR (δ, CDCl ): 18.2, 20.8, 23.2, 29.2,
2
H ), 5.34 (m, 2H,
H
), 5.49 (m, 2H, arene C
H
), 5.90 (m, 1H, pyr C
H
13
1
C
H
3
29 2
Anal. Calcd. for C19H N ClRu: C, 54.08; H, 6.93; N, 6.64. Found: C, 54.05; H,
6.92; N, 6.50%.
6
Synthesis of {(
η
ꢀcymene)RuCl[C
4
H
3
N(2ꢀCH=NCH
2
Ph)]} (4). Similar
procedure as for synthesizing compound 3 is adopted. 1 (0.20 g, 0.32 mmol)
4 3 2 6 5
and [LiC H Nꢀ(2ꢀCH=NCH ꢀC H )] (0.13 g, 0.64 mmol) were used. The filtrate
was concentrated to small amount of volume and stored at ꢀ20°C to yield 0.12
g of yellowish final product (41.7%). Crystals suitable for single crystal Xꢀray
diffraction analysis were obtained using diffusion method from a methylene
chloride/heptane combined solvent. 1H NMR ( , CDCl
δ
3
): 0.83 (d, JHH = 6.9 Hz,
3
H, CHMe ), 1.05 (d, HH = 6.9 Hz, 3H, CHMe ), 2.07 (s, 3H, Me), 2.50 (sept,
2
J
2
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J
HH = 6.9 Hz, 1H, C
.13 (m, 3H, NC
H
Me
2
), 4.81 (m, 1H, arene C
H
), 4.96 (m, 1H, arene C
), 6.27 (m, 1H, pyr C
), 7.50 (s, 1H, C =NCH
2
H
),
),
).
5
6
H
2
+ arene C
H
), 5.43 (m, 1H, arene C
H
H
.65 (m, 1H, pyr C
H
), 7.41 (m, 6H, phenyl + pyr C
H
H
1
3
1
C{ H} NMR (
δ, CDCl
3
): 18.8, 21.6, 22.7, 30.9, 80.3, 81.6, 82.6, 83.9, 99.0,
RuCl:
1
01.3, 112.9, 116.1, 138.3, 138.5, 139.2, 159.4. Anal. Calcd. for C22
H
25
N
2
C, 58.21; H, 5.55; N, 6.17. Found: C, 57.24; H, 5.48; N, 5.88%.
6
Synthesis of {(
η
ꢀcymene)RuCl[C
4
H
3
N(2ꢀCH
=
NCH
2
(C
4
H
7
O))]} (5). A flask
4 3 2 4 7
charged with 1 (0.20 g, 0.32 mmol) and {C H NH[2ꢀCH=NCH (C H O)]} (0.115
g, 0.64 mmol) was added 15 mL of methanol. The solution was stirred for 3 hrs
at room temperature and an excess of NaOH was added. The solution was
then stirred for another 3 hrs and solvent was removed under vacuum. The
residue was extracted with diethyl ether and filtered through celite. The filtrate
was concentrated to small volume and stored at ꢀ20°C to yield 0.092 g of final
product (32%). Crystals suitable for Xꢀray diffraction study were obtained from
1
8
a methylene chloride solution of at ꢀ20°C. H NMR (
δ,
d
ꢀTHF): 0.88ꢀ1.05 (m,
), 2.51 (sept, 1H, C Me ),
N), 5.22ꢀ5.60 (m, 4H, C ), 6.04 (m, 1H,
), 7.32 (s, 1H, pyr C ), 7.50 + 7.60 (two s, 1H,
). Anal. Calcd. for C20 OClRu: C, 53.62; H, 6.07; N, 6.25. Found:
6
H, CHMe
2
), 1.39ꢀ1.80 + 2.09 (m, 7H,C
H
2
+ C
H
3
H
2
3
.57ꢀ4.30 (m, 5H, C
H
2
O + C O + C
H
H
2
6 4
H
pyr C
H
), 6.43 (t,1H, pyr C
H
H
C
H=NCH
2
27 2
H N
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C, 54.07; H, 5.62; N, 6.72%.
n
Synthesis of Li[C
4
H
3
N(2ꢀCH BuNHCH
2
(C
4
H
7
O))] (6). A flask charged with
n
C
4
H
3
NH[2ꢀCH BuNHCH
2
(C
4
H
7
O)] (0.50 g, 2.80 mmol) and 10 mL of THF was
n
slowly added a heptane solution (10 mL) of BuLi (2.5 M, 1.12 mL, 2.80 mmol)
at 0°C and stirred for 3 hrs. The suspension was filtered and the resulted solid
was reꢀcrystallized from a saturated THF solution at ꢀ20°C to yield 0.464 g of
1
8
final product (68.4%). H NMR (δ, d ꢀTHF): 0.86 (m, 3H, Me), 1.27ꢀ1.80 (m,
1
5
1
0H, C
H
2
), 2.48 (m, 2H, NC
H
2
), 3.55ꢀ3.76 (m, 4H, C
H
O + C
H
2
O + C
H
BuN),
13
8
.83 (m, 2H, pyr), 6.60 (m, 1H, pyr), 9.74 (s, 1H, N
H
). C NMR (
δ,
d
ꢀTHF):
4.50, 23.7, 26.5, 29.6, 29.8, 37.6, 59.0, 68.2, 79.7, 106.2, 107.3, 119.3. Anal.
Calcd. for C14
N, 11.81%.
23 2
H N OLi: C, 69.40; H, 9.57; N, 11.56. Found: C, 69.80; H, 9.12;
6
n
4 3 2 4 7
Synthesis of {(η ꢀcymene)RuCl[C H N(2ꢀCH BuNHCH (C H O))]} (7). A
flask containing 1 (0.20 g, 0.32 mmol) and 15 mL of toluene was added
dropwise a toluene solution (15 mL) of 6 (0.16 g, 0.64 mmol) at 0°C. The
solution was stirred for 4 hrs at room temperature and filtered through celite.
The filtrate was concentrated to small volume and stored at ꢀ20°C to yield 0.33
g of final product (64.0%).Crystals suitable for Xꢀray diffraction study were
obtained from a methylene chloride solution of at ꢀ20°C. Anal. Calcd. for
21

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24 37 2
C H N ClORu: C, 56.96; H, 7.37; N, 5.53. Found: C, 57.05; H, 7.27; N,
5.58%.
Xꢀray structure determination. All of the crystals for compounds 2ꢀ5 and 7
were mounted on a glass fiber using epoxy resin and transferred to a goniostat.
The data were collected on a Bruker SMART CCD diffractometer using
graphite monochromated MoꢀK
room temperature and no significant crystal decay was found. The data were
corrected for absorption empirically via scans. All nonꢀhydrogen atoms were
α
radiation. The crystal data were collected at
ψ
refined using anisotropic displacement parameters. For all of the structures,
the hydrogen atom positions were calculated, and they were constrained to
idealized geometries and treated as riding where the H atom displacement
parameter was calculated from the equivalent isotropic displacement
parameter of the bound atom. The structures were determined using
32
directꢀmethod procedures in SHELXS
and refined using fullꢀmatrix
2
33
leastꢀsquares methods on
F
’s in SHELXL. All the relevant crystallographic
data and structure refinement parameters are summarized in Table 1. The
crystallographic data for the structures reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre as supplementary
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publications nos. CCDCꢀ1034394 (2), CCDCꢀ1034395 (3), CCDCꢀ1034396 (4),
CCDCꢀ1034397 (5) and CCDCꢀ1034398 (7). These data can be obtained free
of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Transfer Hydrogenation. A flask was charged with Ru compound (0.005
mmol, ~0.002 g), KOH (1 mmol) and 2ꢀpropanol (5 mL). The solution was
stirred at 82
°C for 10 mins and cooled to room temperature. Substituted
acetophenone (1 mmol, 0.116 mL) was added and the resulting solution was
stirred at 82°C for 3 hrs. The volatiles then were removed under vacuum and
1
residues were characterized by H NMR spectroscopy.
Cell lines and cell culture. Human HRMPC cell lines PCꢀ3 and DUꢀ145 were
provided from American Type Culture Collection (Rockville, MD). Cells were
cultured in RPMI 1640 medium with 10% FBS (v/v) and penicillin (100
U/mL)/streptomycin (100 ꢁg/mL). Cultures were maintained in a humidified
o
incubator at 37 C in 5% CO
2
/95% air.
SRB assays. Cells were seeded in 96ꢀwell plates in medium with 5% FBS.
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After 24 hours, cells were fixed with 10% TCA to represent cell population at
the time of drug addition (T ). After additional incubation of DMSO or the
0
compound for 48 hours, cells were fixed with 10 % TCA and SRB at 0.4% (w/v)
in 1% acetic acid was added to stain cells. Unbound SRB was washed out by
1
% acetic acid and SRB bound cells were solubilized with 10 mM Trizma base.
The absorbance was read at a wavelength of 515 nm. Using the following
absorbance measurements, such as time zero (T ), control growth (C), and
0
cell growth in the presence of the compound (T
x
), the percentage growth was
calculated at each of the compound concentrations levels. Percentage growth
0 0
inhibition was calculated as: [1ꢀ(TxꢀT )/(CꢀT )] x 100%. Growth inhibition of
50% (IC50) is determined at the compound concentration which results in 50%
reduction of total protein increase in control cells during the compound
incubation.
Flow cytometric assay of DNA content. After the treatment, the cells were
o
harvested by trypsinization, fixed with 70% (v/v) alcohol at 4 C for 30 min and
washed with PBS. After centrifugation, cells were incubated in 0.1 mL of
4
phosphateꢀcitric acid buffer (0.2 M NaHPO , 0.1 M citric acid, pH7.8) for 30
min at room temperature. Then, the cells were centrifuged and reꢀsuspended
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with 0.5 mL PI solution containing Triton Xꢀ100 (0.1% v/v), RNase (100 ꢀg/mL)
and PI (80 ꢀg/mL). DNA content was analyzed with FAC Scan and Cell Quest
software (Becton Dickinson, Mountain View, CA).
m
Measurement of mitochondrial membrane potential (∆Ψ ). JCꢀ1, a
mitochondrial dye staining mitochondria in living cells in a membrane
potentialꢀdependent fashion, was used to determine ∆Ψ . Cells were treated
m
with or without compound. Thirty minutes before the termination of incubation,
the cells were incubated with JCꢀ1 (final concentration of 2 µM) at 37°C for 30
min. The cells were finally harvested and the accumulation of JCꢀ1 was
determined using flow cytometric analysis.
Acknowledgements
This work is supported by the Ministry of Science and Technology of Taiwan.
We also thank the National Changhua University of Education for supporting
the Xꢀray diffractometer and NMR spectrometer. The financial support of the
Conquest software and Web CSD from National Center for Highꢀperformance
Computing of Taiwan is also acknowledged.
References
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1
(a) Q. A. Acton, “Platinum Compounds—Advances in Research and
Application: 2013 Edition: Scholarly Brief”, Scholarly Ed., 2013; (b) L.
Varennikov and E. Yedemsky, Ed. “Platinum: Compounds, Production and
Applications”, 2013; (c) V. G. Torgov and F. A. Kuznetsov, Ed., “Noble
Metals: Chemistry and Technology: Collection of Scientific Papers”,
1989.
2
3
4
(a) H. M. Pinedo and J. H. Schornagel, “Platinum and Other Metal
Coordination Compounds in Cancer Chemotherapy 2”, Springer Science
&
Business Media, 1996; (b) L. R. Kelland and N. P. Farrell, Eds.
“PlatinumꢀBased Drugs in Cancer Therapy”, Humana Press Inc., Totowa
NJ, 2000.
(a) B. Lippert, Ed.; “Cisplatin: Chemistry and Biochemistry of a Leading
Anticancer Drug”, WileyꢀVCH, Weinheim, 1999; (b) R. D. Wood and S. S.
Lange, Proc. Natl. Acad. Sci. USA, 2014, 111, 2864ꢀ2865; (c) S. Dasari
and P. B. Tchounwou, Eur. J. Pharmacol., 2014, 740, 364ꢀ378; (d) E.
Shaili, Sci. Prog., 2014, 97, 20ꢀ40.
(a) M. Galanski (2006), Recent developments in the Weld of anticancer
platinum complexes. Recent Pat Anticancer Drug Discov 1:285ꢀ295; (b) V.
Brabec, J. Kasparkova (2005), Modi Wcations of DNA by platinum
26

Page 27 of 53
Dalton Transactions
View Article Online
DOI: 10.1039/C5DT01310K
complexes. Relation to resistance of tumors to platinum antitumor drugs.
Drug Resist Updat 8:131ꢀ146; (c) E. S. Antonarakis and A. Emadi, Cancer
Chemother. Pharmacol. 2010, 66, 1ꢀ9.
5
(a) J. Pracharova, L. Zerzankova, J. Stepankova, O. Novakova, N. J.
Farrer, P. J. Sadler, V. Brabec and J. Kasparkova, Chem. Res. Toxicol.,
2012, 1099ꢀ1112; (b) J. Kaspárková, F. S. Mackay, V. Brabec and P. J.
Sadler, J. Biol. Inorg. Chem., 2003, 8, 741ꢀ745; (c) N. Farrell, L. R. Kelland,
J. D. Roberts and M. Van Beusichem, Cancer Res., 1992, 52, 5065ꢀ5072;
(d) N. A. Kratochwil, M. Zabel, K. J. Range and P. J. Bednarski, J. Med.
Chem., 1996, 39, 2499ꢀ2507.
6
7
8
(a) T. Ludwig and H. Oberleithner, Cell Physiol. Biochem., 2004, 14,
431ꢀ440; (b) S. R. McWhinney, R. M. Goldberg and H. L. McLeod, Mol.
Cancer Ther., 2009, 8, 10ꢀ16; (c) C. A. Rabik and M. E. Dolan, Cancer
Treat. Rev., 2007, 33, 9ꢀ23.
(a) E. S. Antonarakis and A. Emadi, Cancer Chem. & Pharmacol., 2010,
66, 1ꢀ9; (b) R. Trondl, P. Heffeter, C. R. Kowol, M. A. Jakupec, W.
Bergerbd and B. K. Keppler, Chem. Sci., 2014, 5, 2925ꢀ2932; (c) A.
Levina, A. Mitra and P. A. Lay, Metallomics, 2009, 458ꢀ470.
(a) J. M. RademakerꢀLakhai, D. van den Bongard, D. Pluim, J. H. Beijnen
27

Dalton Transactions
Page 28 of 53
View Article Online
DOI: 10.1039/C5DT01310K
and J. H. Schellens, Clin. Cancer Res., 2004, 10, 3717ꢀ3727; (b) G.
Mestroni, E. Alessio and G. Sava, 1998 International Patent PCT C07F
15/00, A61 K 31/28, WO 98/0043.
9
(a) F. Lentz, A. Drescher, A. Lindauer, M. Henke and R. A. Hilger,
Anticancer Drugs, 2009, 20, 97ꢀ103; (b) M. M. Henke, H. Richly, A.
Drescher, M. Grubert and D. Alex, Int. J. Clin. Pharmacol. Ther., 2009, 47,
58ꢀ60.
1
0 (a) R. L. Hayward, Q. C. Schornagel, R. Tente, J. S. Macpherson and R. E.
Aird, Cancer Chemother. Pharmacol., 2005, 55, 577ꢀ583; (b) R. E. Aird, J.
Cummings, A. A. Ritchie, M. Muir and R. E. Morris, Br. J. Cancer, 2002, 86,
1
652ꢀ1657.
1 (a) A. Bergamo, A. Masi, P. J. Dyson and G. Sava, Int. J. Oncol., 2008, 33,
281ꢀ1289; (b) C. Scolaro, A. Bergamo, L. Brescacin, R. Delfino and M.
1
1
Cocchietto, J. Med. Chem., 2005, 48, 4161ꢀ4171; (c) C. A. Vock, C.
Scolaro, A. D. Phillips, R. Scopelliti and G. Sava, J. Med. Chem., 2006, 49,
5552ꢀ5561.
1
2 (a) E. Meggers, G. E. AtillaꢀGokcumen, H. Bregman, J. Maksimoska and
S. P. Mulcahy, Syn. Lett., 2007, 8, 1177ꢀ1189; (b) K. S. Smalley, R.
Contractor, N. K. Haass, A. N. Kulp and G. E. AtillaꢀGokcumen, Cancer
28

Page 29 of 53
Dalton Transactions
View Article Online
DOI: 10.1039/C5DT01310K
Res., 2007, 67, 209ꢀ217.
3 (a) J. Halpern, J. F. Harrod and B. R. James, J. Am. Chem. Soc., 1966, 88,
150ꢀ5155; (b) F. Fache, E. Schulz, M. L. Tommasino and M. Lemaire,
1
5
Chem. Rev., 2000, 100, 2159ꢀ2232; (c) R. Noyori, Angew. Chem. Int. Ed.
Engl., 2002, 41, 2008ꢀ2022; (d) H. ꢀU. Blaser, C. Malan, B. Pugin, F.
Spindler, H. Steiner and M. Studer, Adv. Synth. Catal., 2003, 345,
1
03ꢀ151.
4 (a) B. Zhang, H. Wang, G. ꢀQ. Lin and M. ꢀH. Xu, Eur. J. Inorg. Chem.,
011, 4205ꢀ4211; (b) W. Baratta, M. Ballico, A. D. Zotto, E. Herdtweck, S.
1
2
Magnolia, R. Peloso, K. Siega, M. Toniutti, E. Zangrando and P. Rig,
Organometallics, 2009, 28, 4421ꢀ4430; (c) R. Noyori and T. Ohkuma,
Angew. Chem. Int. Ed. Engl., 2001, 40, 40ꢀ73.
1
5 (a) O. A. Dyachenko, L. O. Atovmyan and S. M. Aldosin, J. Chem. Soc.,
Chem. Commun., 1975, 105ꢀ106; (b) F. Basuli, S. M. Peng and S.
Bhattacharya, Polyhedron, 1998, 18, 391ꢀ402; (c) P. Gupta, S. Dutta, F.
Basuli, S. ꢀM. Peng, G. ꢀH. Lee and S. Bhattacharya, Inorg. Chem., 2006,
45, 460ꢀ467; (d) F. Basuli, S. ꢀM. Peng and S. Bhattacharya, Inorg. Chem.,
2001, 40, 1126ꢀ1133.
16 (a) R. Noyori and S. Hashiguchi, Acc. Chem. Res., 1997, 30, 97ꢀ102; (b)
29