Cobalt complexes with tripodal ligands: Implications for the design of drug chaperones

Article abstract of DOI:10.1039/c2dt30727h

Extensive research is currently being conducted into metal complexes that can selectively deliver cytotoxins to hypoxic regions in tumours. The development of pharmacologically suitable agents requires an understanding of appropriate ligand-metal systems

Full text of DOI:10.1039/c2dt30727h

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Cobalt complexes with tripodal ligands. Implications for the design of
drug chaperones†
a
Paul D. Bonnitcha, Byung J. Kim, Rosalie Hocking, Jack K. Clegg Peter Turner, Suzanne M.
a
a
a,b
a
,
a
Neville and Trevor W. Hambley *
a
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Extensive research is currently being conducted into metal complexes that can selectively deliver cytotoxins to hypoxic regions in
tumours. The development of pharmacologically suitable agents requires an understanding of appropriate ligand-metal systems for
chaperoning cytotoxins. In this study, cobalt complexes with tripodal tren (tris-(2-aminoethyl)amine)) and tpa (tris-(2-
pyridylmethyl)amine) ligands were prepared with ancillary hydroxamic acid, β-diketone and catechol ligands and several parameters,
1
0
including: pK , reduction potential and cytotoxicity were investigated. Fluorescence studies demonstrated that only tpa complexes with β-
a
diketones showed any reduction by ascorbate in situ and similarly, cellular cytotoxicity results demonstrated that ligation to cobalt
masked the cytotoxicity of the ancillary groups in all complexes except the tpa diketone derivative [Co(naac)tpa](ClO ) (naac = 1-
4
2
1
5
methyl-3-(2-naphthyl)propane-1,3-dione)). Additionally, it was shown that the hydroxamic acid complexes could be isolated in both the
hydroxamate and hydroximate form and the pK values (5.3-8.5) reveal that the reversible protonation/deprotonation of the complexes
a
occurs at physiologically relevant pHs. These results have clear implications for the future design of prodrugs using cobalt moieties as
chaperones, providing a basis for the design of cobalt complexes that are both more readily reduced and more readily taken up by cells in
hypoxic and acidic environments.
prodrug, with hypoxic cell response (HCR) values between 50
1
4
2
0
Introduction
and 300 for a variety of different cell lines. Other hypoxia-
15
16
selective drugs include aliphatic N-oxides,
quinones,
The remodelling of the vascular system that occurs under hypoxia
is intrinsically linked to the progression of many diseases
including stroke, ischemic heart disease, peripheral vascular
disease, anaerobic infection and, importantly, cancer. Indeed,
hypoxia occurs to a variable extent in most tumours. Its
prominence in cancer is attributable to interrupted, or absent,
endothelial linings and the impaired vascularisation that occur as
a result of uncontrolled cell proliferation.
17
18
5
6
6
7
5
0
5
0
nitroaromatics, and metal complexes. Cobalt complexes are
of particular interest as hypoxia selective prodrugs because of the
marked difference in lability between the higher (III) and lower
II) oxidation states of cobalt. Ware and co-workers have
examined a number of cobalt complexes bearing acetylacetone
derived ancillary ligands with monodentate, bidentate
A number of different nitrogen
mustard complexes with alternate ancillary ligands including
1
(
2
2
3
3
4
4
5
5
0
5
0
5
0
19
20
and
21
tridentate nitrogen mustards.
3
Hypoxia in tumours is generally associated with a poor
treatment prognosis. In chemotherapy, hypoxic cells are less
susceptible to traditional drugs because of the growth arrested
22
23
18
dialkyldithiocarbamate,
tropolonate,
carbonato,
oxalate
18
24
groups as well as cyclen derivatives with hydroxyquinolines
and tpa complexes with hydroxamic acids have been
4
5
state, limited drug penetration and induced resistance
25
investigated.
6
mechanisms of such cells. Similarly, radiotherapy agents are far
less effective in regions of hypoxia, mainly because oxygen is
required to convert the initial free radicals formed by ionisation
into DNA strand breaks.
However, while tumoural hypoxia has been seen as an
impediment to therapy, it has been suggested that it could
exploited as a target for selective activation of prodrugs. One of
the difficulties associated with chemotherapy is that cancer cells,
unlike bacteria and viruses, do not have molecular targets that are
completely foreign to the host which has limited the usefulness of
At this stage however, it is not known whether the biologically
relevant behaviour of these complexes is exclusively limited to
7
certain ligand systems and the relationships between charge, pKa,
8
26-27
lipophilicity and reduction potential are just being unravelled.
The purpose of this study is to examine a variety of cobalt
complexes with tripodal ligands and different bidentate oxygen
co-ligands to ascertain which systems are likely to be the most
biologically effective.
7
5
Abbreviations
4
some chemotherapeutic agents due to toxic side-effects For this
reason differences between tumours and healthy tissue such as
acacH (acetylacetone), ahaH (acetohydroxamic acid), bhaH
(benzohydroxamic acid), bzacH (1-phenyl-1,3-butanedione),
ClacacH (3-chloro-2,4,-pentanedione), haH (generic hydroxamic
acid), HCR (hypoxic cell response), naacH (1-Methyl-3-(2-
9
10
4, 11
pH, tumour specific enzymes, tumour specific antigens,
12
13
hypoxia
and combinations of these have been explored as
potential targets.
3
-Aminobenzotriazine-1,4-di-N-oxide or tirapazamine 80 naphthyl)propane-1,3-dione), nhaH (1-napthylacetohydroxamic
(TPZ) is one of the most well known biologically active aromatic
acid), TAP (tumour activated prodrug), tpa (tris-(2-
pyridylmethyl)amine), TPZ (tirapazamine), tren (tris-(2-
aminoethyl)amine), trp (generic tripodal ligand).
N-oxides and has shown one of the largest differential toxicities
between hypoxic and normoxic cells for any hypoxia activated
This journal is © The Royal Society of Chemistry [year]
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Dalton Transactions
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Fluorescence measurements were performed on a LS 50 B
luminescence spectrometer using a 1 cm × 1 cm silica cuvette.
-
5
4
5
5
6
6
7
5
0
5
0
5
0
Scans were run as 2.0 × 10 M solutions in methanol/water (1:1)
at 100 nm/min. The relative fluorescence intensities were
calculated by comparing the areas under the emission spectra.
Emission spectra for naphthalene hydroxamic acid and catechol
compounds were run between 300 and 500 nm with excitation at
280 nm, while β-diketone complexes were run at 380-600 nm
with excitation at 350 nm.
-
5
Ascorbic acid reduction studies were performed on 1.0 × 10
M solutions of complexes in phosphate buffered (0.1 mM)
methanol/water (1:1) over 24 h after the addition of ascorbic acid
(20 equivalents). Complexes used in reduction study were:
Co(cat)tpa]·ClO4, [Co(cat)tren]·ClO4, [Co(nha-H)tpa]·ClO4,
Co(nha-H)tren]·ClO4, [Co(naac)tpa]·(ClO4)2 and
Co(naac)tren]·(ClO ) .. The equipment and set up used was the
[
[
[
Fig.1 Ligands employed in this study
4
2
same as described for the fluorescence experimentation. The pH
of solution in all cases was 7.0.
Experimental
Materials
Electrochemistry was performed on a BAS100B instrument
using a glassy carbon working electrode, a silver|silver chloride
reference electrode and a platinum auxiliary electrode. Scans
were performed at a scan rate of 100 mV/s unless otherwise
stated and the solutions were degassed with argon for at least ten
minutes prior to scanning. All experiments were performed at
room temperature and were iR compensated. Scans done in DMF
5
0
5
0
5
All solvents used were laboratory grade and were used without
further purification unless otherwise stated. CoCl ·6H O and
2
2
ethane-1,2-diamine were obtained from Merck. Hydrogen
peroxide (30%, v/v) was obtained from Ajax chemicals. All other
chemicals were purchased from Sigma Aldrich. C25 Sephadex
(bead size 40-120 ꢀM) was used for cation exchange
chromatography (column dimensions are given as diameter ×
height).
Many of the procedures involved the preparation of
perchlorate salts of metal complexes. While no issues were
experienced with these compounds, such compounds should also
be handled with care as they are potentially hazardous. Particular
care was taken with the tris(2-methylpyridyl)amine perchlorate
1
1
2
2
and DMF/water (4:1) were performed with 0.1
M
tetrabutylammonium perchlorate (TBAP) as the supporting
electrolyte and the ferrocene/ferrocinium couplewas used as an
internal reference. Aqueous scans were performed with sodium
perchlorate (0.1 M) as the supporting electrolyte and were
referenced to the Ag/AgCl couple.
7
5
Crystallography
(
tpa.nHClO ) ligand as organic perchlorate molecules are
4
potentially explosive.
Data were collected to approximately 56° 2θ using either a
Bruker SMART 1000 diffractometer employing graphite-
DLD-1 human colon carcinoma cells were obtained from
ATCC and used within 6 months of resuscitation. Cells were
maintained in Advanced DMEM (Invitrogen) and supplemented
with 2% FBS and 2 mM Glutamine in a humidified environment
at 37 °C and 5% CO2.
monochromated Mo-Ka radiation generated from a sealed tube
29
(
0.71073 Å)
or
a
Bruker-Nonius APEX2-X8-FR591
8
8
9
9
0
5
0
5
diffractometer employing graphite-monochromated Mo-Kα
radiation generated from a rotating anode (0.71073 Å). Data
integration and reduction were undertaken with either teXsan,
or SAINT and XPREP.
30
31
2
9-30
Instrumentation
Subsequent computations were
31
carried out using either teXsan or the WinGX-32 graphical user
Microanalysis (C, H, N) was conducted by Chemical &
MicroAnalytical Services Pty Ltd or the Campbell
Microanalytical Laboratory, Department of Chemistry at the
University of Otago.
32
interface. Structures were solved by direct methods using
2
3
SHELXL-86 or SIR97. Multi-scan empirical absorption
corrections, when applied, were applied to the data set using the
30
35
40
33
program SADABS. Data were refined and extended with
SHELXL-93 or SHELXL-97. In general, non-hydrogen atoms
1
13
H and C NMR spectra were collected at 300 K on a Bruker
00 MHz spectrometer using commercially available deuterated
34
3
with occupancies greater than 0.5 were refined anisotropically.
Carbon-bound hydrogen atoms were included in idealised
positions and refined using a riding model. Oxygen and Nitrogen
bound hydrogen atoms were first located in the difference Fourier
map before refinement with bond length restraints. Where these
hydrogen atoms could not be located, they were not modelled.
Disorder was modelled using standard crystallographic methods
including constraints and restraints where necessary. Structural
data are summarised in Table 1 and specific details (where
necessary) pertaining to the refinement is given below.
solvents. TSP (sodium (3-(trimethylsilyl)propionate) was used as
an internal reference in D O. In all other solvents isotopic
2
2
8
impurities were used as internal reference signals.
The mass spectrometry was performed using Electro-Spray
Ionisation on a Finnigan LCQ-8 spectrometer.
UV-visible measurements were performed on a Cary 1E UV-
Visible spectrophotometer using a 1 cm × 1 cm quartz cuvette.
Solutions for pH titrations for hydroxamic acid derivatives were
performed at approximately 1 mM in DMF/water (1:1) with 20
mM buffer (citrate/phosphate/triethylamine).
1
00
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

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[
Co(naac)tren]·0.5H O·2ClO . Two of the perchlorate anions
esterification techniques. A schematic diagram of the ligands
used in this study is shown in Figure 1.
2
4
in the structure are disordered and modelled each over two
positions with occupancies of 0.875 and 0.125 respectively.
[
Co(acac)tpa](ClO ) . Acetylacetone (acacH) (1.4 mmol,
4
2
5
[Co(acac)tren]·2H O·2Cl. This data set proved to be twinned
2
6
5
140 mg, 136 ꢀL) was added to a dispersion of [CoCl tpa]ClO
2 4
520 mg, 1.0 mmol) in methanol (5.0 mL). After 5 minutes,
triethylamine (1.2 mmol, 120 mg, 170 ꢀL) was added and the
solution was refluxed for 1 hour at 50 ºC. The solution was
cooled on an ice bath, diethyl ether (25 mL) was added and the
by a two-fold rotation about c. Accordingly no absorption
correction was applied. The water-hydrogen atoms could not be
located in the difference Fourier map and were not modelled.
(
1
1
2
0
5
0
[Co(Clacac)tpa]·0.5H O·2ClO . The structure crystallised in
2
4
the monoclinic space group Cc with β very close to 90 ° 70 precipitate collected. The crude product was dispersed in an
(
90.172(7) °) and a and b approximately equal, proving to be a
aqueous NaClO solution (0.10 M, 5.0 mL) and acetone added
until the complete dissolution of the product. Slow evaporation of
this solution yielded red square crystals of [Co(acac)tpa](ClO4)2
Yield: 52 %, 337 mg, 0.52 mmol). Crystals suitable for X-ray
analysis were taken directly from this mixture.
DMSO): δ 9.14 (d, H, pyridyl), 8.24 (d, 2H, pyridyl), 8.16 (t,
2H, pyridyl), 7.99 (t, H, pyridyl), 7.79 (d, 2H, pyridyl), 7.74 (t,
H, pyridyl), 7.60 (t, 2H, pyridyl), 7.40 (d, H, pyridyl), 5.88 (s,
H, γ-hydrogen of acac), 5.54 (half of AB system, 2H, CH N),
2
5.27 (s, 2H, CH N), 5.03 (half of AB system, 2H, CH N), 2.75
4
twin emulating tetragonal symmetry with an additional general
twining component. The twinning was accounted for by the use
of the appropriate twin law{Cooper, 2002 #20286} resulting in a
significant decrease in the R factor. In addition, one of the two
complexes in the asymmetric unit is rotationally disorder and was
modelled over two positions with occupancies of 0.75 and 0.25.
The thermal parameters of the lower occupancy component were
refined isotropically with a shared value. Two of the perchlorate
anions are also modelled over two positions with a total
occupancy of 1 each. The water-hydrogen atoms could not be
located in the difference Fourier map and were not modelled.
(
1
75
H NMR
(
1
80
2
2
13
(
s, 3H, CH3),
2.00 (s, 3H, CH3).
C NMR ((DMSO): δ
1
1
2
91.84, 191.64, 163.26, 162.22, 148.84, 147.09, 141.58, 140.94,
26.52, 125.97, 124.73, 122.24, 99.62, 69.78, 68.435, 27.33,
6.24. Mass Spec (ESI positive ion): m/z: 547
2
5
[Co(nha-H)tpa]·EtOH·2H O·ClO . The napthalene group,
water solvents and perchlorate anions are all disordered.
2
4
+
2+
[
Co(cat)tpa]·ClO4. One of the perchlorate anions is 85 ([Co(acac)tpa](ClO
4
) , 224 ([Co(acac)tpa] . Anal. Required for
disordered and modelled over two positions.
CoH C N O Cl : C, 42.67%, H, 3.89 %, N, 8.66 %. Found: C,
25
23
4
10
2
3
0
42.63 %, H, 3.91 %, N, 8.71%.
Cytotoxicity Studies
[
Co(Clacac)tpa](ClO ) . [Co(Clacac)tpa](ClO ) (ClacacH
4
2
4 2
5
IC50 values were determined by seeding 1 × 10 cells/mL into
each well of a 96-well plate and incubating under standard
culturing conditions. Compounds were prepared as 5 mM
90
= 3-chloro-2,4,-pentanedione) was synthesised in an analogous
manner to [Co(acac)tpa](ClO4)2 (Yield: 60 %, 408 mg,
.60 mmol). Slow evaporation of a solution of the product
0
3
4
4
5
5
0
5
0
solutions in 10% DMSO except [Co(naac)tpa](ClO4) and
2
yielded red square crystals of [Co(Clacac)tpa](ClO ) suitable for
4
2
1
[
Co(naac)tren](ClO4) which were prepared as 5 mM solutions in
2
X-ray analysis. H NMR (DMSO): δ 9.12 (d, H, pyridyl), 8.26
(d, 2H, pyridyl), 8.19 (t, 2H, pyridyl), 8.01 (t, H, pyridyl), 7.82
(d, 2H, pyridyl), 7.74 (t, H, pyridyl), 7.63 (t, 2H, pyridyl), 7.39
2
0% DMSO, naacH was prepared as a 13 mM solution in 75%
95
DMSO, and catH2 was prepared as a 5 mM solution in 50%
DMSO solution prior to the assay. Concentrated drug samples
were prepared to ensure the solvent systems were not toxic to the
DLD-1 cells. All solutions were then filtered through a 25 mm
Millex Syringe Filters (Millipore). The dose volume of the
compound solutions was kept below 40 µL and diluted in culture
medium to be added to triplicate wells, spanning a 4-log range of
final concentrations. The cells were then incubated for 72 hrs
before 1.0 mM MTT was added to each well and further
incubated for 4 hrs. The media was removed and 150 µL of
DMSO was added to each well. The absorbance of each well was
(
d, H, pyridyl), 5.60 (half of AB system, 2H, CH N), 5.25 (s,
2
2
H, CH N), 5.02 (half of AB system, 2H, CH N), 2.99 (s, 3H,
2
2
1
3
CH3), 2.27 (s, 3H, CH3).
C NMR ((DMSO): δ 189.45,
1
1
1
00 162.99, 162.78, 149.51, 146.76, 141.46, 140.77, 126.45, 125.82,
24.59, 122.08, 106.11, 69.58, 68.20, 28.14, 27.22. Mass Spec
1
+
(
ESI positive ion) m/z:, 581 ([Co(Clacac)tpa](ClO ) , 241
4
2+
(
[Co(Clacac)tpa] . Anal. Required for CoH C N O Cl : C,
24 23 4 10 3
4
0.52%, H, 3.55 %, N, 8.22 %. Found: C, 40.41 %, H, 3.61 %, N,
05 8.25 %.
3
then measured at 600 nm after shaking for 1 min in a Victor V
[
Co(bzac)tpa](ClO4)2. 1-phenyl-1,3-butanedione (bzacH)
microplate reader (Perkin Elmer). IC50 values were determined
as the compound concentration that reduced the absorbance to
(1.4 mmol, 227 mg) was added to a dispersion of [CoCl tpa]ClO
2
4
(
520 mg, 1.0 mmol) in methanol (5.0 mL). After 5 minutes,
5
0 % of that in untreated control wells.
10 triethylamine (1.2 mmol, 121 mg, 170 ꢀL) was added and the
solution was refluxed for 1 hour at 60 ºC. The solution was
cooled on an ice bath and the precipitate was collected. The
crude product was washed several times with diethyl ether before
Synthesis
3
6
36
37
25
5
5
[CoCl tren]Cl·H O, [CoCO tren]ClO ·0.5H O, tpa·nHClO4,
2
2
3
4
38
2
being dispersed in an aqueous NaClO solution (0.10 M, 5.0 mL).
4
3
7
[
Co(NO ) tpa]ClO , [CoCl tpa]ClO ,
[Co(aha-H)tpa]ClO4,
2
2
4
2
4
115 Acetone was added until complete dissolution of the product was
achieved. Slow evaporation of this solution yielded red needles of
2
5
[
Co(bha-H)tpa]ClO
and 1-methyl-3-(2-naphthyl)propane-1,3-
4
3
9
dione (naacH),
were all synthesised
using published
[
Co(bzac)tpa](ClO4)2 (Yield: 35 %, 248 mg, 0.35 mmol).
H NMR (DMSO): δ 9.20 (d, H, pyridyl), 8.33 (d, 2H, pyridyl),
experimental procedures.
Methyl-2-(naphthalen-1-yl)acetate
1
6
0
was prepared from 2-(naphthalen-1-yl)acetic acid using standard
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

Dalton Transactions
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g, 2.7 mmol) in 2.5 M HCl (4 mL) and stirred for half an hour
.81-7.77 (m, 3H, pyridyl), 7.63-7.54 (m, 3H, (2H pyridyl, H 60 until effervescence had ceased. BzacH (620 mg, 3.9 mmol) was
8
7
.14 (t, 2H, pyridyl), 8.03 (t, H, pyridyl), 7.87 (d, 2H, phenyl),
phenyl), 7.49-7.40 (m, 3H, (2H Phenyl, 1H Pyridyl)) 5.60 (half
of AB system, 2H, CH N), 5.34 (s, 2H, CH N), 5.14 (half of AB
added to this solution followed by an aqueous sodium hydroxide
solution (0.50 M, 16 mL) and the mixture was stirred at 80 ºC for
3 hours. The solution was cooled to room temperature, filtered,
and solid KI was added to the filtrate until the precipitation of
2
2
1
3
5
0
5
0
5
0
5
0
5
0
5
system, 2H, CH N), 2.93 (s, 3H, CH3).
C NMR ((DMSO):
2
δ 194.44, 181.52, 163.35, 163.12, 149.78, 147.10, 141.60,
1
1
41.04, 134.32, 133.29, 128.85, 127.72, 126.62, 126.01, 124.96, 65 orange/red [Co(bzac)tren]I2 was achieved. The product was
22.31, 96.60, 70.08, 68.60, 28.10. Mass Spec (ESI positive ion):
washed once with ethanol and several times with diethyl ether
before being recrystallised from water (Yield: 51 %, 930 mg, 1.5
+
2+
m/z: 609 ([Co(bzac)tpa](ClO ) ); 255 ([Co(bzac)tpa] ). Anal.
Required for CoH C N O Cl : C, 47.41 %, H, 3.84 %, N, 7.90
%
4
1
1
2
2
3
3
4
4
5
5
mmol). Crystals suitable for X-ray analysis were formed by slow
2
7
28
4
10
2
1
. Found: C, 47.37 %, H, 3.96 %, N, 7.88 %.
evaporation of the filtrate. H NMR (D O): δ 7.99 (d, 2H,
2
70
phenyl), 7.66 (t, H, phenyl), 7.55 (t, 2H, phenyl), 6.50 (s, 1H,
γ-hydrogen of acac), 3.75, (td, 2H, tren), 3.50 (td, 2H, tren),
3.35 (t, 2H, tren), 3.16 (dd, 2H, tren), 2.97 (t, 2H, tren), 2.89
[
Co(naac)tpa](ClO ) . [Co(naac)tpa](ClO ) was synthesised
4
2
4 2
in an analogous manner to [Co(bzac)tpa](ClO ) (Yield: 42 %,
319 mg, 0.42 mmol). Slow evaporation of an acetone/water
solution containing the product yielded crystals suitable for X-ray
analysis. H NMR (DMSO): δ 9.24 (d, H, pyridyl), 8.52 (s, H, 75 63.53, 47.52, 45.98, 29.77 . Mass Spec (ESI positive ion): m/z:
4
2
13
(dd, 2H, tren)
2.30 (s, 3H, CH of acac) C NMR (D O): δ
3 2
197.86, 184.60, 138.83, 135.50, 131.62, 130.25, 98.67, 64.25,
1
2+
naphthalene),
6H pyridyl, 4H naphthalene)) 7.65-7.57 (m, 4H, (2H pyridyl,
2H napthalene), 7.46 (d, H, pyridyl) 6.92 (s, 1H, γ-hydrogen of
acac), 5.72 (half of AB system, 2H, CH N), 5.38 (s, 2H,
CH N), 5.21 (half of AB system, 2H, CH N), 2.99 (3H, s, CH ). 80 perchloric acid (0.1 M) instead of solid KI. The NMR was as
8.37 (d, 2H, pyridyl), 8.17-7.82 (m, 10H,
183 ([Co(bzac)tren] ). Anal. Required for CoH C N O I :
27 16 4 2 2
(
C, 30.99 %, H, 4.39 %, N, 9.04 %. Found: C, 31.03 %, H, 4.42
%, N, 9.02 %. [Co(bzac)tren](ClO ) could also be prepared by a
4
2
small modification of the above synthesis by addition of dilute
2
2
2
3
1
3
C NMR ((DMSO): δ 194.23, 181.45, 163.36, 163.18, 149.58,
described for [Co(acacphen)tren]I2. Anal. Required for
1
47.14, 141.60, 141.05, 134.72, 131.97, 131.67, 129.58, 128.94,
CoH C N O Cl : C, 34.00 %, H, 4.81 %, N, 9.91 %. Found:
27
16
4
10
2
128.76, 128.43, 127.41, 126.95, 126.62, 126.01, 125.00, 123.58,
22.31, 96.96, 70.09, 69.00, 28.20. Mass Spec. (ESI positive ion):
C, 34.02 %, H, 4.81 %, N, 9.72 %.
1
+
2+
m/z:, 659 ([Co(naac)tpa](ClO ) ); 280 ([Co(naac)tpa] ) Anal.
Required for CoH C N O Cl : C, 50.61 %, H, 3.85 %, N, 7.38
%
85
[Co(naac)tren](ClO ) . NaacH (1.0 g, 5.0 mmol) was added
4
4 2
to a dispersion of [CoCl tren]Cl·H O (1.0 g, 3.0 mmol) in MeOH
29
32
4
10
2
2
2
. Found: C, 50.68 %, H, 3.91 %, N, 7.43 %.
(40 mL). The solution was stirred for five minutes before
triethylamine (4.5 mmol) was added and the solution was
refluxed for 48 hours. The solution was dried over a nitrogen
Co(acac)tren]Cl ·2H O. [Co(acac)tren]Cl ·2H O
was
2
2
2
2
prepared by a modification of the procedure used for synthesising 90 line, water (100 mL) was added and the solution was filtered. The
4
0
[
Co(acac)tren]I2 by Nakano et al.. [CoCl tren]Cl·H O (1.0 g,
filtrate was then loaded onto a Sephadex column, washed with
water, and the major product was eluted with 0.30 M NaClO4.
Slow evaporation of the eluent solution caused the precipitation
of red crystals of [Co(naac)tren](ClO ) . (Yield: 25 %, 461 mg,
2
2
3
.0 mmol) was dissolved in water (20 mL) and acacH (480 mg,
495 ꢀL, 4.8 mmol) added. After 10 minutes an aqueous sodium
hydroxide solution (1.0 M, 5.0 mL) was added and the solution
was stirred for 3 hours at 60 ºC. After the mixture was cooled to 95 0.75 mmol) H NMR (D O): δ 8.56 (s, 1H, naphthyl), 8.12-8.03
room temperature, water (75 mL) was added and the solution was
loaded onto a Sephadex column (Na form, 3.5 x 20 cm). The
column was washed with water and 0.01 M NaCl and the product
was eluted with 0.3 M NaCl. Once the excess salt was removed
4
2
1
2
(m, 4H, naphthyl), 7.75-7.65 (m, 2H, naphthyl), 6.67 (s, 1H,
γ-hydrogen of acac), 3.86 (td, 2H, tren), 3.56 (td, 2H, tren), 3.39
(t, 2H, tren), 3.19 (dd, 2H, tren), 3.03 (t, 2H, tren), 2.95 (dd,
+
1
3
2H, tren),
2.32 (s, 3H, CH3). C NMR (D O): δ 197.79,
2
(
with considerable product loss), the compound was dissolved in 100 184.46, 137.74, 136.36, 135.15, 132.14, 131.46, 131.32, 131.13,
a
minimal amount of water, and deep red crystals of
130.63, 130.11, 126.60, 98.99, 64.35, 63.56, 47.56, 46.00,
29.80. Mass Spec (ESI positive ion): m/z: 515
[
Co(acac)tren]Cl ·2H O were precipitated upon the slow
2 2
+
2+
diffusion of ethanol (Yield: 48 %, 616 mg 1.5 mmol). Crystals
([Co(naac)tren](ClO ) ); 208 ([Co(naac)tren] ). Anal. Required
4
suitable for X-ray analysis were taken directly from this crop.
for CoH C N O Cl : C, 39.04 %, H, 4.75 %, N, 9.11 %.
29
20
4
10
2
1
H NMR (D O): δ 5.78 (s, 1H, γ-hydrogen of acac), 3.62 (td, 2H, 105 Found: C, 38.94 %, H, 4.79 %, N, 8.98 %.
2
tren), 3.44 (td, 2H, tren), 3.30 (t, 2H, tren), 3.14 (dd, 2H, tren),
2
acac), 2.14, (s, 3H, CH of acac).
1
Spec (ESI positive ion): m/z: 152 [Co(acac)tren]
Required for CoH C N O Cl : C, 32.13%, H, 7.11%, N,
1
.97 (t, 2H, tren), 2.79 (dd, 2H, tren),
2.23, (s, 3H, CH of
[Co(aha)tren](ClO ) ·H O. Aqueous sodium hydroxide (1.0
M, 3.5 mL) was added dropwise slowly to a solution of aceto-
hydroxamic acid (ahaH) (285 mg, 3.8 mmol) and
3
4 2
2
1
3
C NMR ((D O): δ 195.45,
3
2
93.63, 101.58, 63.78, 63.32, 47.44, 45.87, 29.43, 28.95. Mass
2+
Anal. 110 [CoCl tren]Cl·H O (1.0 g, 3.0 mmol) in water (30 mL) and the
2
2
mixture stirred at 70 °C for 6 hours. The solution was cooled, the
solvent removed over nitrogen and ethanol (20 mL) added to the
solid residue. The solution was filtered and a saturated solution of
sodium percholrate in ethanol was added to the filtrate. The
2
9
11
4
4
2
3.63%. Found: C, 32.07%, H, 7.07 %, N, 13.70 %.
[
Co(bzac)tren]I . [Co(bzac)tren]I was prepared by an
2
2
adaptation of the procedure used for synthesising [Co(acac)tren]I 115 resulting precipitate was collected, washed with ethanol,
2
4
0
by Nakano et al.. A mixture of [CoCO tren]ClO ·0.5H O (1.0
isopropanol and diethyl ether (Yield: 48%, 688 mg, 1.4 mmol).
3
4
2
4
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

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Crystals were grown from an ethanol/water solution of the
1
product. H NMR (D O): δ 3.45-3.30 (m, 4H, tren), 3.23-3.15
60
2-(naphthalen-1-yl)hydroxamic acid.
A
suspension of
2
(
m, 4H, tren), 3.00-2.92 (m, 4H, tren), 2.05 (s, 3H, CH3)
hydroxylammonium chloride (10 g, 0.14 mol) in MeOH (10 mL)
was added dropwise to a solution of KOH (12 g, 0.21 mol) in
water (20 mL), with the temperature kept below 5 ºC using an ice
bath. The solution was filtered and the filtrate was added to a
13
C NMR (D O): δ 173.13, 65.66, 62.97, 47.24, 46.21, 16.97.
2
+
5
0
5
0
5
0
5
0
5
0
5
Mass Spec (ESI positive ion): m/z: 277 ([Co(aha-H)tren] ). Anal.
Required for CoH C N O Cl : C, 19.37 %, H, 4.65 %, N, 14.01
24
8
5
11
2
%
. Found: C, 19.37 %, H, 4.88 %, N, 14.12 %.
65 solution of methyl-2-(naphthalen-1-yl)acetate (3.0 g, 15 mmol) in
methanol (10 mL) and stirred for 4 hours at 5 ºC. The volume
was reduced to 20 mL over a stream of nitrogen before
concentrated HCl was added dropwise until the formation of
nhaH was apparent as a white precipitate. This was collected at
Co(aha-H)tren](ClO4).
[Co(aha-H)tren](ClO4)
was
1
1
2
2
3
3
4
4
5
5
synthesised by dissolving [Co(aha)tren](ClO ) ·H O (200 mg,
4
2
2
0
.42 mmol) in ethanol (5 mL) and adding one equivalent of
sodium hydroxide (1 M, 1.5 equivalents). The solution was 70 the pump, washed with generous amounts of water and diethyl
allowed to sit in a fridge overnight, over which time violet
crystals of [Co(aha-H)tren](ClO ) precipitated out of solution
(Yield: 52 %, 82 mg, 0.22 mmol). NMR experiments performed
in DMSO confirmed the removal of the NH proton on the
hydroxamic acid. The complex crystallised as two isomers in 75 129.66, 128.87, 127.25, 126.77, 126.50, 124.73, 38.11. Mass
approximately 3:2 ratio. Major Isomer: H NMR (D O): δ 3.64-
ether, and dried under vacuum (Yield: 78 %, 2.75 g, 11.7 mmol).
1
H NMR (MeOD): δ 8.04 (d, H, naphthalene), 7.73-7.62 (m, 2H,
4
naphthalene), 7.55-7.42 (m, 4H, naphthalene), 3.91 (s, 2H,
13
CH2). C NMR (MeOD): δ 170.86, 135.30, 133.74, 132.25,
1
+
Spec (ESI positive ion): m/z: 224 ([nhaH + Na] ). Mass Spec
2
1
3
-
2
.85 (m, 12 H, tren), δ1.88 (s, 3H, CH3). C NMR (D O): δ
(ESI negative ion): m/z: 200 ([nha] ). Anal. Required for
2
168.15, 62.85, 59.12, 44.51, 43.90, 14.51
Minor Isomer:
H C NO : C, 71.62 %, H, 5.51 %, N, 6.96 %. Found: C, 71.74
11
12
2
1
3
3
1
.64-2.85 (m, 12 H, tren), 1.82 (s, 3H, CH3).
67.46, 62.68, 60.01, 44.12, 43.69, 14.19. Mass Spec (ESI
C NMR (D O):
%, H, 5.61 %, N, 6.94 %.
2
80
+
positive ion): m/z: 277 ([Co(aha-H)tren] ).
[Co(nha-H)tpa]ClO ·H O. Aqueous sodium hydroxide (1.0
4
2
M, 2.5 mL) was added dropwise to
a dispersion of
[Co(bha-H)tren](ClO4).
[Co(bha-H)tren](ClO4)
was
[CoCl tpa]ClO (520 mg, 1 mmol) and nhaH (240 mg, 1.2 mmol)
2
4
synthesised by an adaption of the procedure used for
in methanol (40 mL). After 1 hour the solution was reduced in
Co(aha)tren](ClO ) but with aqueous sodium hydroxide (1.0 M, 85 volume to approximately 30 mL, water was added (200 mL), the
[
4
2
1
0 mL), benzo hydroxamic acid (bhaH) (520 mg, 3.8 mmol) and
was solution filtered and the filtrate was loaded onto a Sephadex
+
[
CoCl tren]Cl·H O (1.0 g, 3.0 mmol) in water (30 mL). [Co(bha-
column (Na form, 3.5 x 20 cm). The column was washed with
2
2
H)tren](ClO ) was collected as a purple solid (Yield: 64 %, 840
mg, 1.9 mmol). The complex crystallised as two isomers in a
ratio approximately ratio 3:2. Major Isomer: H NMR (D O): δ 90 saturated NaClO solution were added and the solution was left to
water and the major green band eluted with 0.1 M NaCl. The
eluent was reduced in volume over a nitrogen line, a few drops of
4
1
2
4
7
3
.92 (d, 2H, phenyl), 7.70 (m, 3H, phenyl), 3.91 (m, 2H, tren),
.64-2.85 (m, 10 H, tren).
stand in the refridgerator overnight. The resulting precipitate of
1
3
C NMR (D O): δ 128.65, 127.44,
[Co(nha-H)tpa]ClO ·H O was collected, washed with water and
2
4
2
126.10, 124.00, 59.51, 56.25, 42.32, 41.58.
Minor Isomer:
H NMR (D O): δ 7.75 (d, 2H, phenyl, 7.70 (m, 3H, phenyl),
diethyl ether and air dried (Yield 30 %, 200 mg, 0.3 mmol). The
complex crystallised as two isomers in 1:1 ratio. Crystals suitable
1
2
1
3
3
1
.55 (m, 2H, tren) 3.64-2.85 (m, 10 H, tren). C NMR (D O): δ 95 for X-ray diffraction were prepared by slow evaporation of an
28.62, 127.44, 126.31, 124.19, 59.99, 57.44, 42.18, 41.01.
2
1
ethanol/water (95:5) solution containing the product. H NMR
(DMSO): δ 9.07 (d, H, pyridyl), 9.02 (d, H, pyridyl), 8.48 (d,
2H, pyridal), 8.39 (d, H, pyridal), 7.99-7.74 (m, 10H), 7.56-
7.26 (m, 19H), 6.97 (t, 3H) 6.56 (d, H), 6.46 (d, H, pyridyl),
The carbon resonances for the C=O quaternary carbon were not
observed in either isomer. Mass Spec (ESI positive ion): m/z: 340
+
([Co(bha-H)tren] ) Anal. Required for C H ClCoN O :
1
3
23
5
6
C, 35.51 %, H, 5.27 %, N, 15.93 %. Found: C, 35.42 %, H, 5.27 100 5.19-4.88 (m, 12H, CH N), 4.06 (s, 2H, CH CONH) 3.58 (s,
2
2
13
%, N, 15.80 %.
2H, CH CONH). C NMR ((DMSO): δ 162.98, 162.48, 162.24,
2
1
61.62, 161.49, 149.34, 149.25, 149.15, 147.63, 139.65, 139.50,
[Co(bha)tren](ClO ) . [Co(bha)tren](ClO ) was synthesised
139.30, 135.43, 134.90, 133.29, 132.25, 132.06, 131.35, 128.24,
128.00, 127.44, 126.78, 126.01, 125.54, 125.36, 125.20, 125.06,
4
2
4 2
by dispersing [Co(bha-H)tren](ClO ) (200 mg, 0.46 mmol) in
water (5.0 mL) and adding 1.1 equivalents of perchloric acid (0.1 105 124.90, 124.74, 124.19, 123.89, 123.05, 122.89, 121.27, 68.91,
M). The solution was allowed to sit in a fridge overnight, over
which time violet crystals of [Co(bha)tren](ClO ) precipitated
(Yield: 55 %, 135 mg, 0.25 mmol). NMR experiments performed
in DMSO confirmed the appearance of the NH proton on the
hydroxamic acid at approximately 12.0 ppm. H NMR (D O): δ 110
4
68.54, 68.04, 67.72, 32.81, 31.76. Mass Spec (ESI positive ion):
+
m/z:
548
([Co(nha-H)tpa] ).
Anal.
Required
for
4
CoH C N O Cl: C, 54.10 %, H, 4.39 %, N, 10.52 %. Found: C,
29
30
5
7
53.98 %, H, 4.47 %, N, 10.50 %.
1
2
7
3
.78 (t, 2H, phenyl), 7.68 (t, H, phenyl), 7.57 (t, 2H, phenyl),
.48 (m, 4H, tren), 3.25 (m, 4H, tren), 3.03 (m, 4H, tren).
C NMR (D O): δ 169.78, 133.05, 129.46, 127.09, 126.30,
[Co(nha)tpa](ClO ) . [Co(nha)tpa](ClO ) was synthesised by
4
2
4 2
dispersing [Co(nha-H)tpa](ClO4) (200 mg, 0.31 mmol) in
ethanol/water (4:1, 5 mL) and adding 1.1 equivalents of
perchloric acid (0.1 M). The volume was reduced until the
1
3
2
6
4
3.30, 60.00, 44.86, 43.84. Mass Spec (ESI positive ion): m/z:
39 ([Co(bha)tren]ClO ), 340 ([Co(bha-H)tren] ), 170 115 desired complex first precipitated, at which point the solution was
+
+
4
2+
([Co(bha)tren] ).
allowed to sit in a fridge overnight, over which time violet
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

Dalton Transactions
Page 6 of 14
View Online
crystals of [Co(nha)tren](ClO ) precipitated out of solution
naphthyl) 8.10-7.91 (m, 4H, napthyl), 7.72-7.50 (m, 9H, napthyl),
4
(
Yield: 75 %, 173 mg, 0.23 mmol). The complex crystallised as
4.27 (s, 2H, CH ), 4.21 (s, 2H, CH ), 3.41-2.75 (m, 24H, tren)
2
2
13
two isomers in 5:2 ratio. NMR experiments performed in DMSO 55 C NMR (D O): δ 174.14, 172.32, 136.09, 135.99, 134.01,
2
confirmed the appearance of the NH proton on the hydroxamic
acid acid at approximately 12.5 ppm. Major Isomer H NMR
133.83, 131.97, 131.93, 131.50, 131.33, 131.04, 130.91,
130.85, 129.41, 129.10, 128.89, 128.29, 128.20, 126.34, 125.48,
65.08, 64.23, 62.41, 61.60, 46.75, 46.09, 45.97, 45.87, 34.85,
34.79. Mass Spec (ESI positive ion): m/z: No ions observed.
1
5
0
5
0
5
0
5
0
5
0
(
DMSO): δ 13.20 (s, NH ), 8.60 (d, H, pyridyl), 8.45 (d, 2H,
pyridyl), 8.10 -7.20 (m, 16H, pyridyl and naphthyl), 5.19-4.95
m, 6H, CH N), 3.96 (s, 2H, CH CONH) Minor Isomer
H NMR (DMSO): 13.20 (s, NH ) 8.85 (d, H, pyridyl), 8.45
(d, 2H, pyridyl), 8.10 -7.20 (m, 16H, pyridyl and naphthyl) 5.19-
(
60
2
2
1
[Co(cat)tpa]ClO . 2,3-napthyldiol (catH ) (1.2 mmol, 190
4
2
1
1
2
2
3
3
4
4
5
mg) was added to a dispersion of [CoCl tpa]ClO (520 mg, 1.0
2 4
mmol) in methanol (5.0 mL). After 5 minutes, triethylamine (2.4
1
3
4
.95 (m, 6H, CH N) 4.50 (s, 2H, CH CONH). C NMR (D O):
2
2
2
δ 170.45, 170.40, 163.98, 163.24, 162.88, 159.85, 149.30,
mmol, 242 mg, 340 ꢀL) was added and the solution was refluxed
1
1
48.91, 148.75, 148.28, 141.50, 141.12, 141.00, 139.84, 133.49, 65 for 1 hour at 60 ºC. The solution was cooled on an ice bath,
33.45, 131.82, 131.39, 131.01, 131.40, 128.88, 128.03, 126.98,
diethyl ether added and the precipitate was collected. The crude
product was washed several times with diethyl ether before being
dispersed in an aqueous NaClO4 solution (0.10 M, 5.0 mL).
Acetone was added until complete dissolution of the product was
70 achieved. Slow evaporation of this solution yielded green needles
126.90, 126.81, 126.74, 126.69, 126.61, 126.51, 126.39, 126.19,
1
6
5
26.08, 125.54, 124.92, 124.30, 123.67, 122.60, 69.81, 69.65,
9.24, 69.18, 32.85, 31.96. Mass Spec (ESI positive ion): m/z
48[Co(nha)tpa] , 274 [Co(nha)tpa] .
+
2+
of [Co(cat)tpa]ClO (Yield: 82 %, 500 mg, 0.82 mmol). Crystals
4
[Co(nha-H)tren]ClO ·H O. [Co(nha-H)tren]ClO ·H O was
suitable for X-ray diffratction were taken directly from this
4
2
4
2
1
prepared using
a
derivation of the synthesis of
mixture. H NMR (DMSO): δ 9.31 (d, H, pyridyl), 8.43 (d, 2H,
[
Co(nha-H)tpa]ClO ·H O described above. Aqueous sodium
pyridyl), 8.00 (m, 3H, pyridyl, napthyl), 7.70 (m, 3H, pyridyl,
4
2
hydroxide (1.0 M, 6.0 mL) was added dropwise to a dispersion of 75 naphthyl), 7.47 (t, 2H, pyridyl), 7.40 (t, 2H, pyridyl), 7.25 (s,
CoCl tren]Cl·H O (660 mg, 2 mmol) and nhaH (500 mg, 2.5 H, napthyl), 7.17 (d, H, pyridyl), 6.90, (m, 2H, naphthyl), 6.47
[
2
2
1
3
mmol) in methanol (150 mL). After 24 hours the solution was
reduced in volume to approx 30 mL, water (100 mL) was added,
(s, H, naphthyl), 5.43, (d, 2H, CH ,), 5.15 (m, 4H, CH x 2). C
2
2
NMR (DMSO): δ 163.91, 163.83, 163.74, 165.02, 151.71,
149.49, 141.20, 141.82, 129.75, 129.54, 126.59, 126.28, 125.01,
the solution was filtered and the filtrate was loaded onto a
+
Sephadex column (Na form, 3.5 × 20 cm). The column was 80 124.89, 124.58, 122.47, 121.28, 121.26, 109.77, 108.92, 69.97,
+
washed with water and the major maroon band was eluted with
0.2 M NaClO . Small crystals of [Co(nha-H)tren]ClO ·H O
69.48. Mass Spec (ESI positive ion): m/z: 507 ([Co(cat)tpa] ).
Anal. Required for C H ClCoN O : C, 55.41 %, H, 3.99 %, N,
4
4
2
28 24
4
6
formed upon slow evaporation of the eluent. (Yield 42 %, 436
9.23 %. Found: C, 55.36 %, H, 4.03 %, N, 9.21 %.
1
mg, 0.84 mmol) H NMR (D O): δ 8.13 (d, 1H, naphthyl), 8.00
2
(
d, 1H, naphthyl), 7.92 (d, 1H, naphthyl), 7.67-7.47 (m, 4H,
85
[Co(cat)tren]ClO ·1.5H O. catH (1.2 mmol, 190 mg) in
4
2
2
naphthyl), 4.01 (s, 2H, CH CONH), 3.64-2.85 (m, 12H, tren).
MeOH (20 mL) was added to a solution of [CoCl tren]Cl (330
2
2
1
3
C NMR (D O): δ 171.70, 136.39, 136.01, 134.51, 131.70,
mg, 1.0 mmol) in water (5 mL). After 5 minutes, triethylamine
(2.4 mmol, 242 mg, 340 ꢀL) was added and the solution was
stirred at room temperature. After 24 hours the solution was
2
1
4
30.54, 129.40, 129.19, 128.71, 127.10, 126.28, 65.09, 62.56,
7.09, 45.87, 35.35. Mass Spec (ESI positive ion): m/z: 404
+
[
Co(nha-H)tren] Anal. Required for CoH C N O Cl: C, 41.43 90 reduced in volume to approx 5 mL, water (100 mL) was added,
2
9
18
5
7
%, H, 5.60 %, N, 13.42 %. Found: C, 41.41 %, H, 5.75 %, N,
the solution was filtered and the filtrate was loaded onto a
+
13.41%.
Sephadex column (Na form, 3.5 x 20 cm). The column was
washed with water and the major maroon band was eluted with
0.20 M NaClO . Small crystals of [Co(cat)tren]ClO ·H O formed
[
Co(nha)tren](ClO ) . [Co(nha)tren](ClO ) was synthesised
4
2
4 2
4
4
2
by dispersing [Co(nha-H)tren](ClO ) (200 mg, 0.39 mmol) in 95 upon slow evaporation of the eluent. (Yield 36 %, 130 mg, 0.36
4
1
ethanol (5.0 mL) and adding one equivalent of perchloric acid
(0.10 M). The volume was reduced until the desired complex first
precipitated, at which point the solution was allowed to sit in a
fridge overnight, over which time red crystals of
mmol). H NMR (DMSO): δ 7.30 (m, 2H, naphthyl), 6.81,(m,
3H, naphthyl) 6.65 (s, H, naphthyl), 4.99 (s, 2H, amine), 4.45 (s,
1
3
2H, amine), 3.98( s, 2H, amine), 2.80 (m, 12H, tren). C NMR
(DMSO): δ 165.9, 163.3, 128.9, 128.6, 123.4, 123.3 119.9,
[
Co(nha)tren](ClO ) precipitated out of solution (Yield: 68 %, 100 119.4, 108.3, 107.0, 61.1, 58.7, 43.9, 42.8. Mass Spec (ESI
4
2
+
1
63 mg, 0.27 mmol). The complex crystallised as two isomers in
positive ion): m/z: 362 ([Co(cat)tren] ). Anal. Required for
a 1:1 ratio. NMR experiments performed in DMSO confirmed
C H ClCoN O C, 39.24 %, H, 5.56 %, N, 11.44 %. Found: C,
1
6
27
4
7.5
the appearance of the NH proton on the hydroxamic acid acid at
39.17 %, H, 5.50 %, N, 11.46 %.
1
approximately 13.1 ppm. H NMR (D O): δ 8.28 (d, 1H,
2
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 14
Dalton Transactions
Table 1 Crystallographic Data
Compound
[Co(naac)tren] [Co(bha)tren]·
[Co(aha)tren] [Co(bzac)tren]·0.6I· [Co(acac)tren]· [Co(nha)tren]·
[Co(Clacac)tpa]·0.5H O· [Co(nha-H)tpa]·EtOH·
2
·
0.5H O·2ClO
2ClO4
·2ClO4
1.4ClO4
2H O·2Cl
2ClO4
2ClO4
2H O·ClO4
2
4
2
2
Formula of
C
H121Cl Co N
80 8 4 1
C
13 2 5
H24Cl CoN O
C
8
H22Cl
2
CoN
5
C
H27C11.4CoI0.6N O
16 4 7.
C
11 2 4
H25Cl CoN O
C
18 2 5
H28Cl CoN O
C
H50Cl Co N O
46 6 2 8 21
80 8 4 16 42.5
C H121Cl Co N O
Refinement Model
Molecular Weight
Crystal System
6
O
42.50
10
O
478.13
Triclinic
10
6
4
10
2506.25
Monoclinic
540.20
Orthorhombic
581.72
Orthorhombic
407.18
Triclinic
604.28
Monoclinic
1381.50
Monoclinic
2506.25
Monoclinic
Space Group
a / Å
b / Å
c / Å
P2 /c
1
P2
1
2
1
2
1
P1¯
8.455(3)
9.447(3)
12.232(4)
98.584(6)
101.007(6)
112.488(6)
859.2(5)
1.848
P2
1
2
1
2
1
P1
7.779(3)
7.864(3)
8.045(3)
100.627(6)
106.855(6)
102.071(6)
444.5(3)
1.521
P2 /n
1
Cc
17.281(6)
17.298(6)
18.867(6)
P2 /c
1
13.3978(8)
28.7543(18)
26.4339(17)
9.1320(6)
13.5840(9)
16.711(1)
10.139(2)
14.370(3)
15.118(3)
8.2091(3)
16.7587(7)
17.1486(7)
13.3978(8)
28.7543(18)
26.4339(17)
α
β
γ
/ °
/ °
/ ° 3
93.677(4)
93.560(2)
90.172(7)
93.677(4)
V / Å
Dc / g cm
Z
10162.5(11)
1.638
4
2073.0(2)
1.731
4
2202.7(8)
1.754
4
2354.65(16)
1.705
4
5640(3)
1.627
4
10162.5(11)
1.638
4
-3
2
1
Crystal Size / mm
0.28x0.20x0.20
0.2x0.2x0.1
0.2x0.2x0.02
0.32x0.19x0.15
0.339x0.255x0.1 0.30x0.28x0.25
0.46x0.31x0.25
0.28x0.25x0.20
2
9
Crystal Colour
Crystal Habit
Temperature / K
red
block
150(2)
red
red
plate
273(2)
0.71073
red
red
plate
150(2)
0.71073
red
red
green
block
150(2)
0.71073
prism
273(2)
0.71073
prism
150(2)
0.71073
block
150(2)
0.71073
prism
150(2)
0.71073
0
.71073
λ
ꢀ
(
MoKα
/mm
)
-1
0.951
0.766, 0.827
1.149
0.8046, 0.9015
1.372
0.283766,
1.838
0.649, 0.759
1.286
N/A
1.022
0.6688, 0.7457
0.957
0.670, 0.787
0.951
0.766, 0.827
T(Empirical)min,max
1
.000000
2
θmax / °
63.60
56.02
55.88
56.74
55.90
60.00
56.70
63.60
hkl range
-19 18, -42 42, -
9 39
-12 12, 0 17, 0
22
-11 10, -12 12,
0 16
-13 13, -18 18, -20 19
-10 10, -10 10, -
10 10
-11 11, -23 23, -
24 23
-23 22, -22 23, -25 24
-19 18, -42 42, -39 39
3
N
N
obs (Nvar
ind (Rmerge
)
218559 (1507)
34567 (0.0320)
4887 (280)
2802 (N/A)
2975(235)
3992 (N/A)
22247(308)
5347 (0.0383)
4102 (212)
3778 (0.0640)
38930 (325)
6851 (0.0477)
27035 (835)
13195 (0.0736)
218559 (1507)
34567 (0.0320)
)
N
obs - (I > 2σ(I))
23279
4569
2097
4869
3772
5628
12072
23279
R1 - (I > 2σ(I)),
wR2 - (all)
GoF
0.0346, 0.1054
0.0425, 0.1219
0.0720, N/A
0.0308, 0.0777
0.0557, 0.1505
0.0335, 0.1215
0.0836, 0.2292
0.0343, 0.1047
1.038
1.048
2.510
1.051
1.054
1.132
1.014
1.039
Residual Extrema / -0.480, 0.628
e Å
-0.372, 1.082
-1.26, 1.48
-0.331, 0.609
-0.940, 0.769
-0.619, 0.648
-0.589, 0.756
-0.463, 0.610
-
-3
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Table 1 (Contd.)
Compound
[Co(bzac)tpa]·2ClO4,
CoN
[Co(cat)tpa]·ClO4
[Co(naac)tpa]·2ClO4
CoN
[Co(acac)tpa]·H O·2ClO
2 4
Formula of Refinement Model
C
28
H
27Cl
2
4
O
10
C
56
H48Cl
2
Co
2
N
8
O
12
C
32
H
29Cl
2
4
O
10
23 2 4 11
C H27Cl CoN O
Molecular Weight
Crystal System
Space Group
a / Å
b / Å
c / Å
709.37
Monoclinic
P2 /n
12.011(2)
18.474(3)
13.884(3)
1213.78
Monoclinic
P2 /c
21.553(3)
10.8220(14)
22.239(3)
759.42
Monoclinic
P2 /n
12.836(5)
17.758(7)
14.707(6)
665.32
Triclinic
P2 /c
1
15.9070(8)
8.9780(4)
19.1400(9)
1
1
1
α
β
γ
/ °
/ °
/ °
106.166(6)
93.066(9)
104.636(7)
97.115(3)
3
V / Å
Dc / g cm
Z
2958.9(9)
1.592
4
5179.7(12)
1.556
4
3244(2)
1.555
4
2712.4(2)
1.629
4
-
3
Crystal Size / mm
Crystal Colour
Crystal Habit
0.28x0.20x0.13
red
prism
0.2x0.15x0.10
green
plate
0.345x0.267x0.198
red
prism
0.30x0.25x0.20
red
block
Temperature / K
90(2)
150(2)
150(2)
90(2)
λ
ꢀ
(MoK
α
)
0.71073
0.826
0.71073
0.818
0.71073
0.759
0.71073
0.897
-
1
(MoKα
) /mm
T(Empirical)min,max
max / °
0.6793, 0.7458
57.44
-16 16, -24 24, -18 18
26163 (407)
0.6688, 0.7457
50.00
-25 25, -12 12, -26 26
52547 (721)
0.737, 1.00
56.16
-16 16, -23 22, -19 19
31644 (443)
7718 (0.0813)
0.6640, 0.7460
60.10
-22 22, -12 12, -26 26
30220 (378)
2
θ
hkl range
N
obs (Nvar
)
N
ind (Rmerge
)
7646 (0.0711)
9100 (0.1921)
7922 (0.0536)
N
obs - (I > 2σ(I))
5192
0.0516, 0.1453
4288
0.0635, 0.1303
5676
0.0407, 0.0995
6203
0.0420, 0.1189
R1 - (I > 2σ(I)),
wR2 - (all)
GoF
1.033
0.937
1.205
1.028
-
-3
Residual Extrema / e Å
-0.524, 0.905
-0.647, 0.744
-0.459, 0.663
-0.727, 1.113
Results and Discussion
Crystal structures
5
0
5
0
5
The bond lengths and angles observed in the solid state structures
of the complexes (Table 2) are commensurate with those of
4
1 21, 42 25
43
similar compounds described in the literature.
representations of [Co(naac)tpa](ClO4)2
Co(naac)tren](ClO4)2 are shown in Figure 2. For all the tpa
ORTEP
and
[
1
1
2
2
complexes with the exception of [Co(Clacac)tpa](ClO4)2, the
bond distance from the tertiary amine to the metal centre is longer
(
1.9421(17) – 1.958(5) Å) than the Co-N lengths associated with
the pyridyl moeites (1.915(5) – 1.939(3) Å). In contrast, the Co-N
bond lengths of the tertiary and equatorial amines of the tren
ligands (1.925(3) – 1.9521(16) Å) are generally shorter than the
Co-N lengths for the axial amines (1.9509(11) – 1.9669(16) Å).
Crystal packing is generally dominated by hydrogen-bonding in
the tren-containing species and π-π interactions in the tpa-
containing complexes.
NMR studies indicate that the β-diketone derivatives
synthesised here exist as only one isomeric form in solution
despite two isomers being possible. The crystallographic data
suggests in all cases that the bulkier β-diketone substiuent is cis
to the tertiary amine of the tripodal ligand. While this would be
expected to be the more sterically favoured isomer for the tpa
derivatives, this reasoning can not be extended to the tren
derivatives, where the steric preference between the cis and trans
isomers is unlikely to be of significance.
30
Fig. 2 ORTEP representations of [Co(naac)tpa](ClO ) and
4
2
4 2
[Co(naac)tren](ClO ) .
This journal is © The Royal Society of Chemistry [year]
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Table 2 Bond length (Å) comparison between the cobalt-tren and cobalt- tpa complexes with different ancillary ligands.
Tren complexes
Structure
Co-N(1)
1.9502(11)
Co-N(2)
Co-N(3)
1.9620(12)
1.9656(12)
1.9659(12)
1.9594(12)
1.962(3)
1.964(4)
1.956(8)
1.961(10)
1.9668(16)
Co-(N4)
Co-O(1)
1.8901(9)
1.8850(9)
1.8840(9)
1.8906(9)
1.894(2)
1.906(4)
1.879(6)
1.899(6)
1.9006(14)
Co-O(2)
1.8918(9)
1.8909(9)
1.8877(9)
1.8905(9)
1.903(2)
1.899(3)
1.882(6)
1.902(5)
1.9029(13)
[
Co(naac)tren](ClO
4
)
2
1.9380(11)
1.9465(11)
1.9477(11)
1.9396(11)
1.940(3)
1.945(4)
1.942(7)
1.937(6)
1.9521(16)
1.9509(11)
1.9557(11)
1.9562(11)
1.9516(11)
1.949(3)
1.958(4)
1.954(7)
1.974(10)
1.9669(16)
1
1
1
.9462(11)
.9467(11)
.9482(11)
[
[
[
[
[
Co(bha)tren](ClO
Co(aha)tren](ClO
Co(bzac)tren](ClO
Co(acac)tren](ClO
Co(nha-H)tren]ClO
4
)
)
2
1.925(3)
1.936(4)
1.942(7)
1.927(6)
1.9389(16)
4
2
4
)
2
4
)
2
4
Tpa complexes
Co-N(3)
Structure
Co-N(1)
1.928(11)
Co-N(2)
1.837(9)
1.73(2)
1.939(3)
1.935(2)
1.916(4)
1.918(4)
1.9296(18)
1.9304(17)
Co-(N4)
1.945(8)
1.945(7)
1.921(3)
1.929(3)
1.927(5)
1.926(5)
1.9316(19)
1.9281(18)
Co-O(1)
1.894(7)
1.94(2)
1.854(2)
1.872(2)
1.892(4)
1.872(4)
1.8739(15)
1.8847(14)
Co-O(2)
1.933(8)
2.06(2)
1.867(3)
1.886(2)
1.897(4)
1.891(4)
1.8857(15)
1.9027(14)
[
Co(Clacac)tpa](ClO
4
)
2
1.916(8)
1.908(7)
1.918(3)
1.928(3)
1.915(5)
1.919(5)
1.9211(19)
1.9240(18)
1
.82(2)
[
[
[
Co(nha-H)tpa](ClO
Co(bzac)tpa](ClO
Co(cat)tpa](ClO
4
)
1.956(3)
1.944(2)
1.946(5)
4
)
2
4
)
1
.958(5)
[
[
Co(naac)tpa](ClO
Co(acac)tpa](ClO
4
)
)
2
1.9421(17)
1.9510(17)
4
2
In contrast to the β-diketone derivatives, the NMR data of
several of the hydroxamic acid complexes showed more than one
isomeric form is present in solution. While the Co-O lengths for
the β-diketone moieties are similar in the tpa and tren derivatives,
the substitution of tpa for tren affects these bond lengths for the
hydroxamic acid complexes.
In these derivatives, the Co-O bond lengths are uniformly longer
in the tren hydroxamic acid complexes (1.894(2) – 1.906(4) Å)
than in the tpa hydroxamic acid structures described here
5
0
5
1.0
pH 6.9
pH 9.8
pH 12.0
0.8
0.6
0.4
0.2
0.0
1
(1.854(2)
– 1.867(3) Å). The other bond lengths are
commensurate with previously observed structures, and the
25
carbonyl C=O bond lengths observed in the solid state support
the hydroximate/hydroxamate assignments made from NMR, MS
and elemental data. The CIFs can be found in the ESI†.
1
3
00
350
400
450
500
Wavelength (nm)
pK Studies
a
Fig. 3 Comparison between the fluorescence admission of nhaH at
different pHs. Studies were performed in methanol/water (1:1) with
excitation at 280 nm.
The absorption, excitation and emission spectra of nhaH have
4
4
2
2
3
3
0
5
0
5
been previously described. While the absorption spectra are
unchanged on varying pH, the fluorescence of nhaH was
eliminated when the pH was raised. (Figure 3). These results
suggest the pK of the hydroxamic acid is approximately 9.0,
which is within the range observed for other hydroxamic acids.
4
0
The shift from hydroximate to hydroximate not only changes
the formal charge of the complex, but also greatly influences the
reduction potential (discussed further below). With the correct
choice of ligand and complex charge, this can be potentially
exploited to incorporate two methods of tumour targeting in the
a
45
For the complexes studied here, both the hydroxamate
monoanionic) and hydroximate (dianionic) forms could be
2
7
(
45
one complex, and studies along these lines have begun.
isolated. Interconversion between the two is associated with
addition or removal respectively of the N-H proton which gives
rise to a distinctive colour change that can be followed by UV/vis
spectrometry (Figures 4 and 5). As shown in Table 3, the pKa
values for the cobalt-tpa complexes are significantly lower than
for the equivalent cobalt-tren complexes and the aromatic
a
Table 3 pK values obtained for hydroaximic acids and their complexes
Cobalt-tren
complex
8.5
Cobalt-tpa
complex
7.0
Ligand
Free ligand
46
ahaH
bhaH
nhaH
9.44
6
8.8
9.1
7.7
7.8
5.3
6.2
hydroxamic acids (5.3) had significantly lower pK values than
a
for the aliphatic aha derivatives (6.2 and 7.0 for
[Co(aha)tpa](ClO ) and [Co(nha)tpa](ClO ) respectively).
4
2,
4 2
This journal is © The Royal Society of Chemistry [year]
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Table 4 Reduction Potentials of cobalt complexes. Scans performed in
+
30
DMF and 80% DMF are referenced to Fc/Fc . Those performed in H
2
O
are referenced to Ag/AgCl
800
600
400
200
DMF
DMF (80%)
2
H O
Compound
E
pc
E
pa
E
pc
E
pa
E
pc
E
pa
[
Co(acac)tpa](ClO
4
)
2
-605
-520
-465
-315
-415
-420
-600
-550
-570
-570
-670
-840
-455
-
-
[
Co(Clacac)tpa](ClO
4
)
2
-380
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
[
[
Co(bzac)tpa](ClO
4
4
)
2
2
-560
-400
Co(naac)tpa](ClO
)
-560
-400
a
[
Co(nha)tpa](ClO
Co(nha-H)tpa (ClO
Co(bha)tpa](ClO
Co(bha-H)tpa](ClO
[Co(aha)tpa](ClO
Co(aha-H)tpa](ClO
Co(cat)tpa](ClO
Co(acac)tren](ClO
4
)
2
-740
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
400
450
500
550
600
650
700
[
]
4
)
-1385
-720
-
-
-
-
-
Wavelength (nm)
b
[
4
)
2
nr
nr
Fig. 4 UV/Vis spectra of [Co(bha)tpa](ClO
.0 to 8.0. Studies were performed at approximately 1 mM in DMF/water
1:1) with 20 mM buffer (citrate/phosphate/triethylamine).
4 2
) as the pH is increased from
[
4
)
-1350
-800
- 850
- 680
-880
-770
-520
-835
-
2
(
4 2
)
[
4
)
2
-1400
-1120
-978
[
4
)
-910 -1040
610
600
590
580
570
560
550
540
530
[
4
)
2
-
-
-
-
-
-
-
-
-
-
-940
-910
-685
-510
-450
-570
-860
-530
-830
-620
-930
-780
[
[
Co(bzac)tren](ClO
4
4
)
2
2
-960
Co(naac)tren](ClO
)
-952
-910
[
Co(nha)tren](ClO
Co(nha-H)tren](ClO
Co(bha)tren](ClO
Co(bha-H)tren](ClO
Co(aha)tren](ClO
Co(aha-H)tren](ClO
[Co(cat)tren](ClO
-‘indicates no peak observed
4
)
2
-1150
-1650
-1130
-1630
-1210
-1720
-1420
-1040
-1380
-970
[
[
[
4
)
)
[
4 2
)
4
-1370
-1050
-1400
-1240
[
4 2
)
2
3
4
5
6
7
8
4
)
pH
5
4
)
Fig. 5 pH titration curve (pH vs λmax) for [Co(bha)tpa](ClO
4
)
2
. Studies
a
‘
‘
were performed at approximately 1 mM in DMF/water (1:1) with 20 mM
buffer (citrate/phosphate/triethylamine).
b
nr’ indicates results were not reproducible
35
Within a particular tripodal ligand class, the reduction
10
Electrochemistry
potentials of the complexes corresponded strongly with the
ancilliary oxygen ligand in the order β-diketone > hydroxamate >
catechol > hydroximate. This is consistent with the doubly
deprotonated ligands conferring a greater electron density onto
The electrochemistry results for all compounds are shown in
Table 4. The reduction potentials of all tren compounds are
significantly more negative than for the comparable tpa
complexes of the same ancillary ligands subtype. This stems from 40 the metal centres than the comparable monoanionic oxygen
15
20
25
the differences in bonding of the pyridyl and amine nitrogen
donors with the metal centres. While amines can only undergo σ
interactions with metal centres, pyridyl rings are capable of acting
bidentates.
The presence of an electron withdrawing chloride group in the
[Co(Clacac)tpa](ClO4)2, derivative made the reduction potential
significantly more positive. This is commensurate with previous
45 studies of cobalt(III) β-diketonato complexes which found direct
correlations between reduction potentials and the Hammett σ
4
7
as π-acceptors
capabilities and reduction potential is well established.
and the relationship between π-acceptor
4
8
The greater irreversibility observed for the tren complexes is
also related to their more negative reduction potentials (Figure 6).
Previous studies have qualitatively revealed that an increased rate
of solvolysis and thus hinderance of the reoxidation wave occurs
5
1
parameters of the substituents on the β-diketone ligands.
The pH of the solutions used for the electrochemistry scans
also influenced the cyclic voltametry results, particularly for the
4
9
with decreased reduction potential. In addition, the π back- 50 cobalt-hydroxamic acid complexes. Increasing the pH above the
bonding interactions between the metal centre and tpa potentially
pKa values of the hydroxamate complexes resulted in
deprotonation and hence formation of the hydroximate in situ.
This corresponded with a shift to the more negative reduction
potentials associated with the doubly deprotonated (hydroximate)
5
0
result in decreased ligand lability, which may contribute to the
greater reversibilty observed in the reduction of the tpa
complexes.
55
ligands.
The reduction potentials of cobalt-hydroximate
complexes prepared in situ by this manner were indistinguishable
from those of freshly prepared cobalt-hydroximate solutions. For
1
0
| Journal Name, [year], [vol], 00–00
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biological environments. The naacH free ligand was shown to be
potential was observed with altered pH. However, an increase in 45 mildly cytotoxic under both hypoxic and normoxic conditions
the catechol and diketone derivatives, no shift in reduction
the current magnitude in the anodic return wave was observed at
higher pH values, presumably because high pH maintains as
deprotonated the free ancillary ligand that forms under reduction
in a deprotonated, which facilitates reoxidation of the cobalt
complexes.
(IC50 74 ± 3 and 60 ± 1.6 µM respectively) and there wss no
statistically significant difference between the cytotoxicity of the
free ligand and the cytotoxicity of the respective tpa complex
([Co(naac)tpa](ClO ) (IC 74 ± 4 and 66 ± 3 µM under hypoxic
5
4
2
50
50 or
normoxic
condition
respectively).
In
contrast
[
Co(naac)tren](ClO ) has far lower cytotoxicity than free naacH
4
2
under both hypoxic and normoxic conditions. The above results
indicated that the cytotoxicity of the naacH ligand is masked
upon binding to the cobalt-tren derivative while the behaviour of
Reduction with ascorbic acid
1
1
2
2
0
5
0
5
As the naphthyl moieties are fluorescent, all the complexes
contained naphthyl derivatives were tested for reduction with 55 the cobalt-tpa derivative is indistinguishable from that of free
ascorbate by fluorimetry (full details in experimental data).
For all cobalt complexes the fluorescence intensity was less
than 1% of that observed for the equivalent concentration of free
ligand, which could be related to deprotonation of the ligands
ligand in solution. This is despite both compounds showing no
degredation by NMR over 48h in aqueous solution.
Unfortunately, the [Co(naac)tpa](ClO ) complex did not show
4
2
any hypoxic selectivy and it was is not possible to ascertain
upon binding to cobalt, or more directly to the presence of the 60 whether any of the complexes are hypoxia selective due to their
cobalt which has previously been reported to quench fluorescence
by electron or energy exchange mechanisms.
When ten equivalents of ascorbic acid were added to the
solution and the fluorescence was measured at a variety of time
points over 24 h, only [Co(naac)tpa](ClO ) , demonstrated any
increase in fluorescence after 24 h, which is consistent with the
reduction potentials for most of the complexes being too negative
to allow reduction by ascorbate. In contrast, the
[Co(naac)tpa](ClO4)2 solution showed a steady increase in
fluorescence such that after 24 h it was approximately 40% of the
intensity of the comparable reference solution of the ligand which
is consistent with reduction and ligand dissociation under these
conditions.
low cytotoxicity.
5
2
Table 5 IC50 (µM) of the compounds used in this study in human DLD-1
colon carcinoma cells.
Compound
tren
Normoxia
>200
Hypoxia
>200
4
2
tpa
5 ± 0.3
>200
5 ± 0.2
>200
nhaH
naacH
60 ± 1.6
127 ± 7
>200
74 ± 3
113 ± 4
>200
catH
2
[CoCl
2
2
tren]ClO
4
[
[
CoCl
tpa]ClO
4
>200
>200
Co(nha-H)tren]ClO
4
>200
>200
[
Co(acac)tpa](ClO4)2
Co(acac)tren](ClO4)2
0.03
0.02
0.01
0.00
[Co(nha-H)tpa]ClO
4
>200
>200
[
[
[
[
[
Co(naac)tren](ClO
Co(naac)tpa](ClO
Co(cat)tren]ClO
Co(cat)tpa]ClO
4
)
2
>200
>200
4
)
2
66 ± 3
>200
74 ± 4
>200
4
4
>200
>200
-
-
0.01
0.02
6
5
Conclusions
Cobalt complexes that are selectively reduced under hypoxic
conditions have been investigated as chaperones for prodrugs. In
this study, a clear correlation between reduction potential, ligand
dissociation under ascorbic acid challenge, and cytotoxicity was
0
-500
-1000
-1500
-2000
+
Potential (mV vs Fc/Fc )
3
3
4
0
5
0
Fig. 6 Comparison between the cyclic voltammograms of
Co(acac)tpa](ClO and [Co(acac)tren](ClO . Scans were performed in
DMF at 100 mVs .
[
4
1
)
2
4 2
)
-
70 observed. Specifically, the free naacH ligand and
Co(naac)tpa](ClO ) show comparable cytotoxicities, both under
[
4
2
hypoxic and normoxic conditions, while all other complexes
demonstrate higher IC50 values for the cobalt complexes when
compared to their respective free ligands. The cyotoxicity data
supports the notion that [Co(naac)tpa](ClO ) quickly dissociates
Cytotoxicity
As with the fluorescence work, all naphthyl derived ligands and
their associated compounds were tested for cytotoxicity. None of
the complexes or their ligands were particularly cytotoxic (Table
7
5
4
2
and
loses
its
naacH
ligand
in
solution
while
[
Co(naac)tren](ClO ) , and the other complexes investigated, do
4 2
5
) with the exception of the free tpa ligand. Similar to what has
not. This is commensurate with [Co(naac)tpa](ClO4)2 having a
previously been observed, the cytotoxicity of the free tpa ligand
significantly less negative reduction potential (-560 mV E in
pc
53
+
c
was far greater than that of any of the tpa complexes, which
suggests that the toxicity of the tpa ligand is masked by
coordination to a metal centre, and this is maintained in
80
DMF vs F /F ) than any of the other complexes examined in
cytotoxicity studies (-970 to -1650 mV in DMF vs F /F ), as well
c
+
c
c
as being the only complex to display appreciable reduction by
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Dalton Transactions
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ascorbic acid over 24h. Together, these results support the
correlation between reduction potential and ligand dissociation.
The ability to manipulate formal complex charge to influence
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0
5
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Alterations of ancilliary ligands significantly modifies pKa and electrochemical behaviour in model
cobalt drug chaperones