Synthesis and characterization of ruthenium compounds incorporating keto-amine ligands. The applications of catalytic transfer hydrogenation and cancer cell inhibition

Article abstract of DOI:10.1016/j.jorganchem.2016.01.029

A series of keto-amine bidentate precursors 1–5, OCCH₃CHCCH₃NHR (where 1, R = C₆H₃-2,6-ⁱPr₂; 2, R = C₆H₂-2,4,6-Me₃; 3, R = C₆H₄-2-^tBu; 4, R = C₆H₄-2-OMe; 5, R = C₆H₄-2-OMe-5-Me) were synthesized and combined with [Ru(?⁶-p-cymene)Cl₂]₂ to generate the monomeric arene-Ru derivatives, [Ru(?⁶-p-cymene)(OCCH₃CHCCH₃NR)Cl] (where 6, R = C₆H₃-2,6-ⁱPr₂; 7, R = C₆H₂-2,4,6-Me₃; 8, R = C₆H₄-2-^tBu; 9, R = C₆H₄-2-OMe; 10, R = C₆H₄-2-OMe-5-Me) in moderate yield. The ruthenium derivatives effectively catalyzed the conversion rate in transfer hydrogenation of substituted acetophenone. The molecular structures of 2, 6–10 were determined by single crystal X-ray diffractometry in the solid state, revealing a four-coordination environment around the Ru atom. The potential anti-cancer activity of ruthenium derivatives against human hormone-refractory metastatic prostate cancer (HRMPC) cell lines was also studied.

Full text of DOI:10.1016/j.jorganchem.2016.01.029

Accepted Manuscript
Synthesis and characterization of ruthenium compounds incorporating keto-amine
ligands. The applications of catalytic transfer hydrogenation and cancer cell inhibition
Tzung-Han Lin, Kuheli Das, Amitabha Datta, Wohn-Jenn Leu, Hung-Chang Hsiao,
Chia-Her Lin, Jih-Hwa Guh, Jui-Hsien Huang
PII:
S0022-328X(16)30027-4
10.1016/j.jorganchem.2016.01.029
JOM 19380
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 15 October 2015
Revised Date: 20 January 2016
Accepted Date: 21 January 2016
Please cite this article as: T.-H. Lin, K. Das, A. Datta, W.-J. Leu, H.-C. Hsiao, C.-H. Lin, J.-H. Guh, J.-
H. Huang, Synthesis and characterization of ruthenium compounds incorporating keto-amine ligands.
The applications of catalytic transfer hydrogenation and cancer cell inhibition, Journal of Organometallic
Chemistry (2016), doi: 10.1016/j.jorganchem.2016.01.029.
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Synopsis for the Graphical Abstract
Synthesis and characterization of ruthenium compounds
incorporating keto-amine ligands. The applications of catalytic
transfer hydrogenation and cancer cell inhibition
a
a
a
c
Tzung-Han Lin , Kuheli Das , Amitabha Datta , Wohn-Jenn Leu ,
Hung-Chang Hsiao a, Chia-Her Lin b, Jih-Hwa Guh c, Jui-Hsien Huang a,*
A series of ruthenium compounds containing keto-amine bidentate
ligands were synthesized and their potential anti-cancer activity and transfer
hydrogenation activity were also studied.

ACCEPTED MANUSCRIPT
Pictogram for the Graphical Abstract
Synthesis and characterization of ruthenium compounds
incorporating keto-amine ligands. The applications of catalytic
transfer hydrogenation and cancer cell inhibition
a
a
a
c
Tzung-Han Lin , Kuheli Das , Amitabha Datta , Wohn-Jenn Leu ,
Hung-Chang Hsiao a, Chia-Her Lin b, Jih-Hwa Guh c, Jui-Hsien Huang a,*

ACCEPTED MANUSCRIPT
Synthesis and characterization of ruthenium compounds
incorporating keto-amine ligands. The applications of catalytic
transfer hydrogenation and cancer cell inhibition
a
a
a
c
Tzung-Han Lin , Kuheli Das , Amitabha Datta , Wohn-Jenn Leu ,
Hung-Chang Hsiao a, Chia-Her Lin b, Jih-Hwa Guh c, Jui-Hsien Huang a,*
a
Department of Chemistry, National Changhua University of Education,
Changhua, Taiwan 50058
b
Department of Chemistry, Chung-Yuan Christian University, Chun-Li 320,
Taiwan
c School of Pharmacy, National Taiwan University, Taipei, Taiwan 100
*Corresponding author: 886-4-7232105 ext 3512, E-mail address:
juihuang@cc.ncue.edu.tw
Keywords: transfer hydrogenation; keto-amine; ruthenium; Hormone-refractory
Prostate Cancer;
1

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ABSTRACT
A series of keto-amine bidentate precursors 1-5, OCCH3CHCCH3NHR (where
1, R = C6H3-2,6-iPr2; 2, R = C6H2-2,4,6-Me3; 3, R = C6H4-2-tBu; 4, R
=C6H4-2-OMe; 5, R = C6H4-2-OMe-5-Me) were synthesized and combined with
[Ru(ɳ6-p-cymene)Cl2]2 to generate the monomeric arene-Ru derivatives,
[Ru(ɳ6-p-cymene)(OCCH3CHCCH3NR)Cl] (where 6, R = C6H3-2,6-iPr2; 7, R =
C6H2-2,4,6-Me3; 8, R = C6H4-2-tBu; 9, R = C6H4-2-OMe; 10, R =
C6H4-2-OMe-5-Me) in moderate yield. The ruthenium derivatives effectively
catalyzed the conversion rate in transfer hydrogenation of substituted
acetophenone. The molecular structures of 2, 6-10 were determined by single
crystal X-ray diffractometry in the solid state, revealing a four-coordination
environment around the Ru atom. The potential anti-cancer activity of
ruthenium derivatives against human hormone-refractory metastatic prostate
cancer (HRMPC) cell lines was also studied.
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Introduction
The chemistry of η6-arene ruthenium compounds has received
considerable attention in recent years, since a large number of applications in
supramolecular [1-5] and medicinal chemistry [6-11] have been developed with
excellent or promising results. Ruthenium arene-chloride dimeric complexes,
[(ɳ6-arene)RuCl2]2 [12-14], are good starting materials that could generate a
variety of ruthenium derivatives to be used as anti-cancer drugs [15-24] and
homogeneous catalysts [25-27]. The distinctive features of the ruthenium
arene compounds include (i) the variability of ɳ6-arene fragments, (ii)
substitution of the chloride with mono- or multi-dentate anionic ligands, and (iii)
the ability to adjoin mono- or multi-dentate ligands to arene ruthenium
compounds as shown in Scheme 1. The ɳ6-arene fragment in ruthenium
compounds includes alkyl substituted arenes [28], tethered amines,
phosphines, and NHC carbine arenes [29-30], etc. Similarly, mono- and
multi-dentate anionic ligands as well as neutral ligands have been applied to
form new organo-ruthenium derivatives (see Scheme 1).
Catalytic asymmetric transfer hydrogenation of ketones has recently
emerged as a viable means for synthesizing chiral alcohols [31]. Due to its
operational simplicity, the easy availability of reductants, and the high
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enantioselectivities, the catalytic enantioselective reduction of ketones has
been
extensively
studied
during
the
last
decade.
The
[Ru(η6-arene)-(chelating-ligand)Cl]-type compounds exhibit the characteristic
“piano stool” structure, with the unreactive arene as a “spectator ligand” in the
metal coordination sphere and the chloride as a suitable “leaving group” [32].
These structural features seem favorable to afford sequential reactions
involved in catalysis. Recently, Williams et al. [33] reported the synthesis of
water-soluble aminosulfonamide ligands and their applications in the
Ru(II)-catalyzed enantioselective transfer hydrogenation of aromatic ketones.
In the search for new organo-ruthenium compounds, we developed a
series of bidentate keto-amine ligands and have subjected these precursors
into [Ru(ɳ6-p-cymene)Cl2]2 to form new monomeric ruthenium derivatives. By
changing the sterically hindered alkyl substituents on the keto-amine moiety,
we are able to tune the stereogeometries of the ruthenium compounds and to
study the Ru-catalyzed transfer hydrogenation. We also investigated the
potential of the Ru derivatives as anticancer agents against HRMPC PC-3 and
DU-145 cells.
Results and discussion
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Synthesis and characterization of compounds 1-10
A series of keto-amine ligands 1-5, OCCH3CHCCH3NHR (where 1, R =
C6H3-2,6-iPr2; 2, R = C6H2-2,4,6-Me3; 3, R = C6H4-2-tBu; 4, R = C6H4-2- OMe; 5,
R = C6H4-2-OMe-5-Me) were synthesized according to a modified procedure
[34] by the reaction between an arylamine and 2,4-pentanedione in methanol
with a small amount of formic acid (Scheme 2). The amine NH protons for
compounds 1-5 were involved in intramolecular hydrogen bonding with the
1
keto (C=O) oxygen to form a six-membered chelate ring and the H NMR
spectra were relatively similar, showing broad NH signals at ca. δ 12.0. The
methine protons of the keto-amine backbone for compounds 1-5 all displayed
1
characteristic sharp singlets at ca. δ 4.7~5.1. In the H NMR spectrum, the
chemical shift of the methine proton is usually monitored to screen for the
complexation of keto-amine ligands with metals. The keto-amine ligands 1-5
were converted to their corresponding lithium salts by adding one equivalent of
LinBu and then were subjected to a THF solution of [Ru(ɳ6-p-cymene)Cl2]2 [35]
at 0 °C. After workup, complexes [Ru(ɳ6-p-cymene)(OCCH3CHCCH3NR)Cl]
(where 6, R = C6H3-2,6-iPr2; 7, R = C6H2-2,4,6-Me3; 8, R = C6H4-2-tBu; 9, R =
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C6H4-2-OMe; 10, R = C6H4-2-OMe-5-Me) were isolated in moderate yield (see
Scheme 2) and characterized by 1H and 13C NMR spectra. The characteristic
methine proton for the keto-amine backbone of complexes 6-10 showed the
1
typical H NMR signal at ca. δ 4.66~4.77, a more up-field shifted signal than
observed for the corresponding keto-amine ligands. This upfield shift was
presumably due to the electron shielding from the ruthenium atom. It is worth
noting that the steric hindrance between the cymene isopropyl group [36] and
the substituted aryl group of the keto-amine ligands resulted in a slow rotation
of the amine-aryl C-N bond as shown in Scheme 3. This criterion is quite
distinct for larger steric hindrance among aryl rings of the keto-amine for
compounds 6 and 7. At room temperature, i.e., the slow limit, compound 6
showed two septets (δ 3.78 and 3.09) and four doublets (δ 1.42, 1.31, 1.29,
and 1.27) for the two corresponding isopropyl fragments. Compound 7 likely
displayed two singlets (δ 2.37 and 2.14) for two methyl groups at ortho
positions of the 2,4,6-trimethylphenyl fragment. These resonances were
broadened when the 1H NMR spectra were taken at elevated temperature (see
supporting information). The energy barriers for C-N bond rotation of
compounds 6 and 7 were estimated at 60.9 and 59.8 KJ/mol, respectively [37].
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For compounds 8-10, the 1H and 13C NMR spectra corresponded well with the
predicted molecular geometries.
Molecular geometries of compounds 2 and 6-10
The summary of X-ray crystal data collection and the selected bond
lengths and angles for compounds 2 and 6-10 are shown in Table 1 and Table
2, respectively. The crystals of organic keto-amine compound 2 were obtained
from a saturated methylene chloride solution at -20 °C and its molecular
geometry is depicted in Figure 1. Compound 2 showed a planar keto-amine
backbone architecture with conjugated bond lengths [38-39]. The
corresponding bond lengths for C=O and C-N were 1.2438(15) and 1.3409(15)
Å, respectively. Crystals of compounds 6-10 were obtained from concentrated
toluene solutions at -20 °C. The molecular geometri es of 6-10 are depicted in
Figures 2-6 and could be best described as three-legged piano stool
structures with the cymene as the base and the oxygen and nitrogen atoms
from the keto-amine frame and the chloride serving as the three legs.
The corresponding bond lengths between ruthenium and the center of the
cymene ring belong at ca. 1.67 Å [40-43]. In general, the keto-amine backbone
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was coordinated with the ruthenium atom to form a six-membered chelated
ring where the ruthenium atom deviated from the keto-amine plane at ca. 0.53
Å. The Ru-Namine and Ru-Oketo bond lengths were at ca. 2.11 and 2.05 Å,
respectively. As showed in Scheme 3, the isopropyl and methyl fragments of
the phenyl rings for compounds 6 and 7 showed steric hindrance with the
cymene isopropyl groups.
Transfer hydrogenation using ruthenium catalysts
The transfer hydrogenation reaction applied to the reduction of ketones
has been explored in detail in recent years. Homogeneous asymmetric
hydrogenation of olefins and ketones catalyzed by chiral transition metal
complexes has provided a powerful means for preparing optically active
organic 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 enantiomeric secondary alcohols in
modern synthetic chemistry. The most commonly used metal catalysts are
ruthenium-based compounds, usually with (+2) as the formal oxidation state of
Ru atom. In hydrogenation, the hydrido metal compounds (MH(L)n) may be
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generated as the catalyst which can accelerate the reduction of ketone in the
catalytic cycle where a reducing equivalent-H may be supplied by the reducing
solvent (like isopropanol) or from other reducing agents. In Scheme 4,
compounds 6-10 were used as catalysts for transfer hydrogenation in the
isopropyl alcohol system, proceeding as -C=O- → -CH(OH)-. The results of the
studies are shown in Table 3. The transfer hydrogenation reactions for
acetophenone and isoproplyl alcohol displayed high conversion yields
regardless of the nature of the substituents on the aryl rings of the catalysts.
When 4-methyl acetophenone was used, the conversion yields were lower
than those obtained when acetophenone was used and no transfer
hydrogenation
reactions
were
observed
while
the
2,4,6-trimethyl-acetophenones were used. All these results showed that the
steric hindrance of the substituted acetophenone is the key factor for the
transfer hydrogenation conversion yield, regardless of the steric geometries of
the ruthenium catalysts.
Anti-proliferative activities of ruthenium compounds
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The discovery of compounds to combat prostate cancer is one of the main
challenges found in clinical oncology. The demand for therapeutic drugs is
particularly high because of the poor prognosis for patients with
hormone-refractory metastatic prostate cancer (HRMPC). The majority of
human prostate cancer cell lines, including the two "classical" cell lines
DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. Due to
this unmet medical need, prostate cancer is the target of a significant
proportion of both basic and clinical research. The anti-proliferative activities of
ruthenium compounds have been examined using sulforhodamine B assay, a
standard screening test used for in vitro anticancer drug screens at the U.S.
National Cancer Institute. The data in Table 4 confirmed that the ruthenium
compounds are effective in inhibiting the cell proliferation in both HRMPC PC-3
and DU-145 cells. We also listed the screening data of cisplatin,
[(η6-p-cymene)RuII(L)Cl]Cl [44], and [Ru(η6 -cymene)Cl(η2-dppm)]PF6 [45] for
their in vitro antitumour activity. The in vitro cytotoxicities (GI50) for PC-3 and
DU-145 cells using compounds 6-10 were all less than 30 µM, ranging from
4.25-19.68 µM and 5.48-20.93 µM, respectively. Compound 7 showed the best
result with values of 4.25 and 5.48 µM for PC-3 and DU-145, respectively.
Comparing the steric hindrance and electronic effects of the substituted phenyl
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keto-amine ligands, compounds with the bulky 2,6-diisoproproylphenyl group
(compound 6) or the strong electron donating 2-methoxylphenyl group
(compounds 9 and 10) displayed lower cytotoxicity than those with the
2,4,6-trimethyl or 2-tbutyl phenyl substituents (compounds 7 and 8). This
results indicate that suitable steric hindrance and electron donating ability are
crucial for the ruthenium compounds to perform as good anti-cancer reagents.
Therefore, fine tuning of the substituents of the phenyl keto-amine ligands and
the use of different ligand systems are the main targets in the search for new
organometallic anticancer reagents.
Conclusion
Herein, we have reported the synthesis of a series of arene-ruthenium
compounds prepared from keto-amine bi-dentate precursors and their
1
structures are discussed. All the compounds were well characterized by H
and 13C NMR spectroscopy. The electronic and steric effects of the substituted
phenyl keto-amine ligands on the ruthenium compounds showed minimal
effects in the transfer hydrogenation reduction of acetophenone. However, the
substituents on the acetophenone made significant contributions to catalytic
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reduction. Furthermore, the anti-proliferative activities of Ru-derivatives were
tested on HRMPC cell lines, namely PC-3 and DU-145. Compounds 7 and 8
were the most influential candidates that displayed effective anticancer activity
against HRMPCs. Further investigations with Ru-derivatives prepared from
new bidentate organic precursors and their potential effects on apoptosis in
different human cells are currently being carried out.
Experimental Section
Materials and physical techniques
All reactions were performed under a nitrogen atmosphere using standard
Schlenk techniques or in glove box. Tetrahydrofuran and toluene were dried by
refluxing over sodium benzophenone ketyl. Solvents were distilled and stored
in solvent reservoirs that contained 4-Å molecular sieves and were purged with
13
nitrogen. 1H and C NMR spectra were recorded using a Bruker Avance 300
spectrometer and the chemical shifts were recorded in ppm relative to the
residual protons of CDCl3 (δ = 7.24, 77.0 ppm). Elemental analyses were
performed using a Heraeus CHN-OS Rapid Elemental Analyzer at the
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Instrument Center of the NCHU. Ligands 1-5 and [Ru(ɳ6-p-cymene)Cl2]2 were
prepared using a modified procedure [35].
Synthesis of compounds 1-5, OCCH3CHCCH3NHR.
A general method for synthesis of compounds 1-5 was described as
follows. A round bottom flask charged with 2,4-pentanedione and 30 mL of
methanol was added aryl amine slowly. A small amount of formic acid was
used as catalyst. The solution was stirred for overnight and solvent was
removed over vaccum to yield the final product.
Compound 1:
2,4-pentanedione (5.0 g, 49.9 mmol), 2,6-diisopropylaniline (8.85 g, 49.9
mmol), gave 11.8 g of brown solid product, 93% yield. 1H NMR (δ, CDCl3): 1.05
(d, 6H, CHMe2), 1.11 (d, 6H, CHMe2), 1.53 (s, 3H, Me), 2.00 (s, 3H, Me), 2.95
(sept, 1H, CHMe2), 5.12 (s, 1H, C=CHC), 7.06 (m, 2H, PH), 7.17 (m, 1H,
PH),12.02 (br, 1H,NH). 13C NMR (δ, CDCl3): 18.8, 22.4, 24.3, 28.2, 28.7, 95.4,
123.3, 128.1, 133.3, 146.0, 162.8, 195.5.
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Compound 2:
2,4-pentanedione (10.0 g, 99.8 mmol), 2,4,6-trimethylaniline (13.5 g, 99.8
mmol), gave 18.6 g of yellowish solid product, 86% yield. 1H NMR (δ, CDCl3):
1.57 (s, 3H, Me), 2.04 (s, 3H, Me), 2.10 (s, 6H, Me), 2.22 (s, 3H, Me), 5.15 (s,
1H, C=CHC), 6.88 (s, 2H, Ph), 11.82 (br, 1H, NH). 13C NMR (δ, CDCl3): 17.1,
17.8, 20.0, 27.9, 94.91, 128.1, 133.1, 134.7, 136.1, 162.1, 194.5. Anal. Calcd:
C, 77.50; H, 8.24; N, 6.78. Found: C, 77.37; H, 8.81; N, 6.44%.
Compound 3:
2,4-pentanedione (3.00 g, 29.9 mmol), 2-tbutylaniline (4.47 g, 29.9 mmol),
gave 6.78 g of reddish oily product, 98% yield. 1H NMR (δ, CDCl3): 1.11 (s, 9H,
tBu), 1.50 (s, 3H, Me), 1.81 (s, 3H, Me), 4.92 (s, 1H, C=CHC), 6.66 (m, 1H, Ph),
13
6.88 (m, 2H, Ph), 7.14 (m, 1H, Ph), 12.31 (br, 1H,NH). C NMR (δ, CDCl3):
12.9, 28.2, 29.9, 34.2, 96.4, 125.8, 126.2, 126.3, 129.0, 136.3, 144.9, 160.4,
167.6. Anal. Calcd: C, 77.88; H, 9.15; N, 6.05. Found: C, 78.37; H, 9.76; N,
6.42%.
Compound 4:
2,4-pentanedione (5.0 g, 49.9 mmol), 2-methoxyaniline (6.85 g, 49.9 mmol),
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1
gave 10.61 g of brown oily product, 98% yield. H NMR (δ, CDCl3): 1.52 (d,
3H, Me), 1.65 (d, 3H, Me), 3.18 (s, 3H, OMe), 4.75 (s, 1H, C=CHC), 6.45 (m,
2H, PH), 6.67 (m, 2H, PH), 12.00 (br, 1H, NH). 13C NMR (δ, CDCl3): 18.6, 27.9,
54.4, 96.8, 110.3, 119.3, 123.6, 125.1, 126.9, 151.5, 158.9, 194.2.
Compound 5:
2,4-pentanedione (5.0 g, 49.9 mmol), 2-methoxy-5-methylaniline (6.85 g, 49.9
mmol), gave 10.38 g of dark black oily product, 94% yield. 1H NMR (δ, CDCl3):
1.72 (d, 3H, Me), 1.84 (s, 3H, Me), 2.01 (s, 3H, Me), 3.53 (s, 3H, OMe), 4.94 (s,
1H, C=CHC), 6.55 (m, 1H, Ph), 6.69 (m, 2H, Ph), 12.11 (br, 1H, NH). 13C NMR
(δ, CDCl3): 18.9, 19.7, 28.1, 54.8, 96.8, 124.9, 125.9, 126.8, 128.9, 150.0,
150.7, 194.5.
Synthesis of compounds 6-10
A general method for synthesis of compounds 6 - 10 was described as
follows. A Schlenk flask charged with keto-imine and 10 mL of THF was cooled
to 0°C and added Li nBu slowly. The resulting solution was added to another
flask which contains [Ru(ɳ6-p-cymene)Cl2]2 and 10 mL of THF at room
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temperature. After stirring for 3 hours, THF was removed and the residue was
extracted. The extraction was filtered through celite and the filtrate was
concentrated to a small amount and stored at -20°C to yield the final product.
{Ru(ɳ6-p-cymene)[OCCH3CHCCH3N(C6H3-2,6-iPr2)]Cl} (6)
Compound 1 (0.40 g, 1.54 mmol), LinBu (2.5 M in hexane, 0.61 mL, 1.54
mmol), and [Ru(ɳ6-p-cymene)Cl2]2 (0.47 g, 0.77 mmol) were used and the final
product was obtained from a saturated toluene solution to yield 0.473 g of
1
deep red crystals (57%). H NMR (δ, CDCl3): 1.03 (br, 6H, CHMe2), 1.31 (br,
12H, CHMe2), 1.56 (s, 3H, Me), 1.77 (s, 3H, Me), 1.97 (s, 3H, Me), 2.62 (sept,
1H, CHMe2), 3.16 (br, 1H, CHMe2), 3.84 (br, 1H, CHMe2), 4.05 (br, 1H,
cymene), 4.75 (s, 1H, CCHC), 4.81 (br, 1H, cymene), 5.26 (br, 1H, cymene),
5.31 (br, 1H, cymene), 7.21 (d, 3H, Ph). 13C NMR (δ, CDCl3): 17.8, 21.5, 23.3,
25.0, 25.3, 25.5, 26.1, 27.5, 27.9, 30.6, 82.0, 84.2, 86.8, 95.0, 97.4, 103.5,
123.4, 125.0, 126.4, 142.0, 144.0, 151.5, 166.2, 177.6. Anal. Calcd: C, 61.41;
H, 7.06; N, 2.65. Found: C, 61.77; H, 7.29; N, 2.92%.
{Ru(ɳ6-p-cymene)[OCCH3CHCCH3N(C6H2-2,4,6-Me3)]Cl} (7)
Compound 2 (0.40 g, 1.84 mmol), LinBu (2.5 M in hexane, 0.73 mL, 1.84
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mmol), and [Ru(ɳ6-p-cymene)Cl2]2 (0.56 g, 0.92 mmol) were used and the final
product was obtained from a saturated toluene solution to yield 0.33 g of red
crystals (36.7%). 1H NMR (δ, CDCl3): 1.20 (br, 6H, CHMe2), 1.47 (s, 3H, Me),
1.96 (s, 3H, Me), 2.00 (s, 3H, Me), 2.14 (br, 3H, Me), 2.32 (s, 3H, Me), 2.39 (br,
3H, Me), 2.74 (sept, 1H, CHMe2), 3.74 (br, 1H, cymene), 4.73 (br, 1H, cymene),
4.76 (s, 1H, CCHC), 5.03 (br, 1H, cymene), 5.27 (br, 1H, cymene), 6.93 (s, 2H,
Ph). 13C NMR (δ, CDCl3): 17.8, 20.9, 22.4, 22.9, 26.2, 30.9, 92.5, 97.4, 103.7,
134.7, 152.4, 152.4, 164.6, 178.4. Anal. Calcd: C, 59.19; H, 6.62; N, 2.88.
Found: C, 58.82; H, 6.75; N, 2.65%.
{Ru(ɳ6-p-cymene)[OCCH3CHCCH3N(C6H4-2-tBu)]Cl} (8)
Compound 3 (0.30 g, 1.28 mmol), LinBu (2.5 M in hexane, 0.51 mL, 1.28
mmol), and [Ru(ɳ6-p-cymene)Cl2]2 (0.39 g, 0.64 mmol) were used and the final
product was obtained from a saturated toluene solution to yield 0.33 g of red
crystals (51.2%). 1H NMR (δ, CDCl3): 1.19 (m, 6H, CHMe2), 1.45 (s, 9H, tBu),
1.54 (s, 3H, Me), 1.80 (s, 3H, Me), 1.95 (s, 3H, Me), 2.65 (sept, 1H, CHMe2),
4.06 (d, 1H, cymene), 4.69 (s, 1H, CCHC), 5.00 (d, 1H, cymene), 5.07 (d, 1H,
cymene), 5.41 (d, 1H, cymene), 7.15 (m, 2H, Ph), 7.45 (d, 1H, Ph), 7.63 (d, 1H,
Ph). 13C NMR (δ, CDCl3): 17.36, 21.20, 22.85, 25.02, 25.57, 30.48, 33.57,
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36.34, 74.55, 81.37, 87.74, 88.83, 91.47, 96.84, 105.22, 125.51, 126.91,
128.89, 129.58, 140.23, 154.83, 167.13, 177.24. Anal. Calcd: C, 59.93; H, 6.84;
N, 2.80. Found: C, 60.13; H, 6.49; N, 2.99%.
{Ru(ɳ6-p-cymene)[OCCH3CHCCH3N(C6H4-2-OMe)]Cl} (9)
Compound 4 (0.40 g, 1.95 mmol), LinBu (2.5 M in hexane, 0.78 mL, 1.95
mmol), and [Ru(ɳ6-p-cymene)Cl2]2 (0.60 g, 0.975 mmol) were used and the
final product was obtained from a saturated toluene solution to yield 0.44 g of
1
red crystals (47.2%). H NMR (δ, CDCl3): 1.16 (d, 6H, CHMe2), 1.57 (s, 3H,
Me), 1.92 (s, 3H, Me), 1.98 (s, 3H, Me), 2.59 (sept, 1H, CHMe2), 3.58 (d, 1H,
cymene), 3.91 (s, 3H, OMe), 4.72 (s, 1H, CCHC), 5.05 (d, 1H, cymene), 5.11
(d, 1H, cymene), 5.28 (d, 1H, cymene), 6.95 (m, 2H, Ph), 7.14 (d, 1H, Ph), 7.69
(d, 1H, Ph). 13C NMR (δ, CDCl3): 18.25, 20.86, 23.26, 23.28, 26.32, 30.40,
55.20, 54.57, 77.35, 77.75, 85.96, 95.13, 96.06, 100.76, 110.26, 121.49,
126.41, 127.78, 146.61, 151.67, 164.78, 176.87. Anal. Calcd: C, 53.59; H, 6.13;
N, 2.84. Found: C, 53.70; H, 6.04; N, 2.97%.
{Ru(ɳ6-p-cymene)[OCCH3CHCCH3N(C6H2-2-OMe-5-Me)]Cl} (10).
Compound 5 (0.40 g, 1.80 mmol), LinBu (2.5 M in hexane, 0.72 mL, 1.80
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mmol), and [Ru(ɳ6-p-cymene)Cl2]2 (0.55 g, 0.90 mmol) were used and the final
product was obtained from a saturated toluene solution to yield 0.35 g of red
crystals (40.4%). 1H NMR (δ, CDCl3): 1.20 (m, 6H, CHMe2), 1.63 (s, 3H, Me),
1.97 (s, 3H, Me), 2.02 (s, 3H, Me), 2.30 (s, 3H, Me), 2.63 (sept, 1H, CHMe2),
3.67 (d, 1H, cymene), 3.93 (s, 3H, OMe), 4.66 (br, 2H, H2O) 4.76 (s, 1H,
CCHC), 5.03(d, 1H, cymene), 5.16 (d, 1H, cymene), 5.33 (d, 1H, cymene),
6.87 (m, 2H, Ph), 6.98 (m, 1H, Ph), 7.55 (d, 1H, Ph). 13C NMR (δ, CDCl3): 18.1,
20.5, 20.7, 23.2, 26.3, 30.3, 55.2, 55.2, 77.1, 84.7, 86.3, 86.8, 94.3, 95.9,
101.0, 110.9, 126.6, 128.0, 146.2, 149.4, 164.5, 176.6. Anal. Calcd: C, 56.44;
H, 6.18; N, 2.86. Found: C, 56.77; H, 6.46; N, 3.05%.
Cell lines and cell culture
Human hormone-refractory prostate cancer cell lines PC-3 and DU-145
were collected 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 µg/ml). Cultures were maintained in a humidified
incubator at 37 oC in 5% CO2/95% air.
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Sulforhodamine B (SRB) assays
Cells were seeded in 96-well plates in medium with 5% FBS. After 24
hours, cells were fixed with 10% TCA to represent cell population at the time of
drug addition (T0). After additional incubation of DMSO or the compound for 48
hours (PC-3 cells) or 72 hours (DU145 cells), 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 (T0), control
growth (C), and cell growth in the presence of the compound (Tx), the
percentage growth was calculated at each of the compound concentrations
levels. Percentage growth inhibition was calculated as: [1-(Tx-T0)/(C-T0)] 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. Data are expressed as mean±SEM of
three to five determinations.
X-ray crystallography
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Suitable crystals of compounds 2, 6-10 were attached to a fine glass fiber
with paratone oil and mounted in goniostat for data collection. Data collections
were performed at 150 K under liquid nitrogen vapor for all complexes. Data
were collected on a Bruker SMART CCD diffractometer with graphite
monochromated Mo-Kα radiation. No significant crystal decay was found. Data
were corrected for absorption empirically by means of
ψ scans. All
non-hydrogen atoms were refined with anisotropic displacement parameters.
For all 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. An absorption correction was
performed with the program SADABS [46] and the structures of both
complexes were determined by direct methods procedures in SHELXS [47]
and refined by full-matrix least-squares methods, on F2’s, in SHELXL [48]. All
the relevant crystallographic data and structure refinement parameters are
summarized in Table 1. Crystallographic data for the structures reported in this
paper have been deposited with the Cambridge Crystallographic Data Centre
as supplementary publications nos. CCDC-1056020 (2), CCDC-1056021 (6),
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CCDC-1056022 (7), CCDC-1056023 (8), CCDC-1056024 (9) and
CCDC-1056025 (10). Copies of the data can be obtained free of charge on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
+44-1223-336-033;
e-mail:
deposit@ccdc.cam.ac.uk
or
www:
http://www.ccdc.cam.ac.uk).
Acknowledgements
The authors express their appreciation to the Ministry of Science and
Technology, Taiwan for financial assistance (NSC 102-2738-M-018-001). We
also thank the National Changhua University of Education for supporting the
facility of X-ray diffractometer and NMR spectrometer.
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