Synthesis, characterization, and bioactivities of platinum(II) complexes bearing pyridinecarboxaldimines containing aliphatic groups

Article abstract of DOI:10.1139/cjc-2012-0371

Condensation of 2-pyridinecarboxaldehyde with six primary amines containing aliphatic groups gave the corresponding pyridinecarboxaldimines (N-N′R). Addition of these ligands to [PtCl₂(coe)]₂ (coe = cis-cyclooctene) gave complexes of the type cis-PtCl₂(N-N′R) (1: R = propyl, 2: R = hexyl, 3: R = octyl, 4: R = nonyl, 5: R = hexadecyl, and 6: R = octadecyl) in moderate yields. The molecular structure of the hexyl derivative (2) has been confirmed by an X-ray diffraction study. Crystals of 2 were triclinic with a = 8.6858(19) A, b = 8.7567(19) A, c = 9.5080(19) A, α = 76.546(3), β = 87.151(3), and γ = 78.586(3) in the space group P1. All platinum complexes show considerable anti-bacterial and anti-mycobacterial properties.

Full text of DOI:10.1139/cjc-2012-0371

1
31
ARTICLE
Synthesis, characterization, and bioactivities of platinum(II) complexes
bearing pyridinecarboxaldimines containing aliphatic groups
Erin L. Stewart, Alyssa E. Patterson, Taryn O'Neill, Haoxin Li, Andrew Flewelling, Christopher M. Vogels, Andreas Decken, Vett K. Lloyd,
Christopher A. Gray, and Stephen A. Westcott
Abstract: Condensation of 2-pyridinecarboxaldehyde with six primary amines containing aliphatic groups gave the correspond-
ing pyridinecarboxaldimines (N-N=R). Addition of these ligands to [PtCl (coe)] (coe = cis-cyclooctene) gave complexes of the type
2
2
cis-PtCl (N-N=R) (1: R = propyl, 2: R = hexyl, 3: R = octyl, 4: R = nonyl, 5: R = hexadecyl, and 6: R = octadecyl) in moderate yields. The
2
molecular structure of the hexyl derivative (2) has been confirmed by an X-ray diffraction study. Crystals of 2 were triclinic with
a = 8.6858(19) Å, b = 8.7567(19) Å, c = 9.5080(19) Å, ␣ = 76.546(3)°, ␤ = 87.151(3)°, and ␥ = 78.586(3)° in the space group P1. All platinum
complexes show considerable anti-bacterial and anti-mycobacterial properties.
Key words: aliphatic amines, anti-microbial, anti-mycobacterial, pyridinecarboxaldimines, platinum.
Résumé : La condensation du pyridine-2-carboxaldéhyde avec six amines primaires contenant des groupes aliphatiques conduit
a` la formation des pyridinecarboxaldimines correspondantes (N-N=-R). L'addition de ces ligands au [Pt(Cl )(coe)] (coe = cis-
2
2
cyclooctène) conduit a` la formation de complexes de type cis-PtCl (N-N=R) (1: R = propyle, 2: R = hexyle, 3: R = octyle, 4: R = nonyle,
2
5: R = hexadécyle et 6: R = octadécyle), avec des rendements moyens. On a pu confirmer la structure moléculaire du dérivé hexyle
(
2) par des études de diffraction des rayons X. Les cristaux du composé 2 sont tricliniques, dans le groupe d'espace P1, avec
a = 8,6858(19) Å, b = 8,7567(19) Å, c = 9,5080(19) Å, á = 76,546(3)°, â = 87,151(3)° et ã = 78,586(3)°. Tous les complexes de platine
présentent des propriétés antibactériennes et antimycobactériennes importantes.
Mots-clés : amines aliphatiques, antimicrobien, antimicybactérien, pyridinecarboxaldimines, platine.
1
Introduction
side effects associated with cisplatin. Although the anti-cancer
chemistry of cisplatin derivatives has been studied extensively,
the use of platinum compounds to treat other diseases and illness
is an area that has received much less attention. We report
herein our results on the synthesis and initial biological testing of
cis-dichloro(pyridin-2-ylcarboxaldimine)platinum(II) compounds
containing a variety of aliphatic groups, which we propose will
improve lipophilicities and aid in drug delivery.
Cisplatin, cis-[PtCl (NH ) ], and related platinum-based com-
plexes are currently used as anti-cancer agents against testicular
and ovarian malignancies. The primary mechanism of action in
2
3 2
4
1
these platinum drugs is believed to arise from the metal's inter-
action with DNA, where the platinum is believed to bind prefer-
entially with the N-7 positions of the purine bases. There are
severe limitations to platinum therapy, however, such as neural
and kidney toxicity as well as intrinsic and acquired resistance of
tumor cells to the drugs. These complications have provided in-
centive for further research into the development of platinum-
based complexes with increased solubility and enhanced
specificity towards cancer cells. Previous studies have shown that
cis-amminedichloro(2-methylpyridine)platinum(II) (ZD0473) (Fig. 1a)
Experimental
General procedures and methods
Reagents and solvents used were obtained from Aldrich Chem-
icals. Potassium tetrachloroplatinate was purchased from Pre-
cious Metals Online Ltd. [PtCl (␩ -coe)]
previously reported. NMR spectra were recorded on a JEOL JNM-
GSX270 FT NMR spectrometer. H NMR chemical shifts are re-
2
5
was prepared as
2
2
2
has considerable cytotoxicity in cisplatin-resistant cell lines.
1
We have been making ZD0473 analogues by replacing the NH3
group with a pendant imine group. Previous studies have shown
that the platinum complex derived from aniline (Fig. 1b) has
shown considerable activity against the hormone-independent
ported in ppm and are referenced to residual protons in
1
3
deuterated solvent at 270 MHz. C NMR chemical shifts are refer-
enced to solvent carbon resonances as internal standards at
68 MHz. Multiplicities are reported as singlet (s), doublet (d), triplet
(t), quintet (quint), sext (sextet), multiplet (m), and overlapping
(ov). The infrared spectra were obtained using a Mattson Genesis
FT-IR spectrometer and are reported in cm . Melting points were
measured uncorrected with a Mel-Temp apparatus. Elemental
analyses for carbon, hydrogen, and nitrogen were carried out at
3
human mammary carcinoma cell line MDA-MB 231. Varying the
aniline functionality allows us to design compounds with a wide
range of physical and chemical properties. As well, the use of
bidentate ligands prevents trans labilization and undesired dis-
placement of the ligands by sulfur and nitrogen donors in biomol-
ecules, interactions believed responsible for some of the adverse
−
1
Received 25 September 2012. Accepted 26 October 2012.
E.L. Stewart, A.E. Patterson, C.M. Vogels, and S.A. Westcott. Department of Chemistry and Biochemistry, Mount Allison University, Sackville, NB E4L 1G8, Canada.
T. O'Neill, H. Li, A. Flewelling, and C.A. Gray.* Department of Biology, University of New Brunswick, Saint John, NB E2L 4L5, Canada.
†
A. Decken. Department of Chemistry, University of New Brunswick, Fredericton, NB E3B 5A3, Canada.
V.K. Lloyd.‡ Department of Biology, Mount Allison University, Sackville, NB E4L 1G7, Canada.
Corresponding author: Stephen A. Westcott (e-mail: swestcott@unb.ca).
*Corresponding author for anti-microbial and anti-mycobacterium studies (e-mail: cgray@unb.ca).
†Corresponding author for X-ray study (e-mail: adecken@unb.ca).
‡Corresponding author for Artemia toxicity bioassay (e-mail: vlloyd@mta.ca).
Can. J. Chem. 91: 131–136 (2013) dx.doi.org/10.1139/cjc-2012-0371
Published at www.nrcresearchpress.com/cjc on 2 November 2012.

132
Can. J. Chem. Vol. 91, 2013
Fig. 1. Structure of (a) ZD0473 and (b) dichloro-(E)-N-(pyridin-2-ylmethylene)aniline-platinum(II).
Guelph Chemical Laboratories Ltd. (Guelph, Ontario). Anti-
mycobacterial susceptibility tests were performed using modified
Middlebrook 7H9 broth base (BBL MGIT; Becton Dickinson, Mis-
sissauga, Ontario) in non-tissue culture treated, low-binding,
black 96-well microtitre plates sealed with polyester films
1H, HC = N), 8.15 (ov ddd, JHH = 7.7, 7.7, 1.2 Hz, 1H, Ar), 7.80 (d,
JHH = 7.7 Hz, 1H, Ar), 7.63 (ov ddd, JHH = 7.7, 5.7, 1.2 Hz, 1H, Ar), 4.15
(t, JHH = 7.4 Hz, JHPt = 38.6 Hz, 2H, NCH ), 2.01 (sext, J = 7.4 Hz, 2H,
2
HH
13
1
NCH CH ), 0.96 (t, J = 7.4 Hz, 3H, CH CH ); C{ H} ␦: 166.6, 159.3,
2
2
HH
2
3
1
(
1
43.3, 138.1, 128.5, 128.3, 65.5, 43.4, 24.5. IR (Nujol): 2930 (s), 2856
s), 1631 (m, ␯C = N), 1601 (m), 1461 (m), 1378 (m), 1301 (m), 1237 (m),
050 (m). Anal. calcd. for C H N Cl Pt (414.31) (%): C 26.09, H 2.93,
(50 ␮m). Fluorometric readings (in relative fluorescence units)
were recorded using a Molecular Devices Gemini EM dual-
scanning microplate spectrofluorometer with a 530 nm excitation
filter and a 590 nm emission filter operating in top-scan mode and
are the mean values of 30 reads per well. Antibiotic susceptibility
tests were performed using BBL Mueller Hinton II cation adjusted
broth and Difco Sabouraud Dextrose broth (Becton Dickinson,
Mississauga, Ontario) for bacterial and fungal cultures respec-
tively in non-tissue culture treated, clear 96-well microtitre plates.
Optical densities (OD) were measured using a Molecular Devices
Emax microplate reader with a 600 nm filter.
9
12
2
2
N 6.76; found: C 26.25, H 2.88, N 6.49.
PtCl (N,N=-hexyl) (2)
2
Yield: 86%, mp 149–150 °C. NMR spectroscopic data (in CDCl ):
3
1
H ␦: 9.65 (dd, JHH = 4.9, 1.5 Hz, JHPt = 36.1 Hz, 1H, Ar), 8.66 (s, JHPt
5.7 Hz, 1H, HC = N), 8.15 (ov ddd, JHH = 7.7, 7.7, 1.5 Hz, 1H, Ar), 7.82
d, JHH = 7.7 Hz, 1H, Ar), 7.66 (ov ddd, JHH = 7.7, 4.9, 1.5 Hz, 1H, Ar),
=
9
(
4
2
3
3
N
.15 (t, JHH = 7.2 Hz, JHPt = 38.6 Hz, 2H, NCH ), 1.95 (quint, J = 7.2 Hz,
2 HH
H, NCH CH ), 1.36–1.24 (ov m, 6H, –(CH ) –), 0.86 (t, J = 7.2 Hz,
2
2
2 3
HH
1
3
1
H, CH CH ); C{ H} ␦: 166.7, 157.0, 150.3, 139.4, 128.3, 126.8, 61.4,
Synthesis
2
3
1.3, 31.2, 26.2, 22.6, 14.0. IR (Nujol): 2940 (s), 2857 (s), 1623 (m, ␯C =
), 1597 (m), 1460 (m), 1379 (m), 1297 (m), 1240 (m), 1114 (m). Anal.
General synthesis of iminopyridine ligands
The iminopyridines L1-6 were prepared by modification of a
calcd. for C H N Cl Pt (456.27) (%): C 31.59, H 3.98, N 6.14; found:
6
12 18
2
2
known synthesis. A CH Cl (10 mL) solution of the appropriate
2
2
C 31.77, H 4.12, N 5.87.
amine (1 equivalent) was added to a CH Cl (15 mL) solution of
2
2
2
-pyridinecarboxaldehyde. Activated 4 Å molecular sieves (10 g)
PtCl (N,N=-octyl) (3)
2
were added and the reaction mixture was allowed to stand at RT
for 2 days. Following the removal of the molecular sieves by filtra-
tion, solvent was removed under vacuum to afford the desired
iminopyridines as orange–red oils.
Yield: 82%, mp 126–127 °C. NMR spectroscopic data (in CDCl ):
3
1
H ␦: 9.49 (d, JHH = 5.3 Hz, JHPt = 32.3 Hz, 1H, Ar), 8.76 (s, JHPt = 96.6 Hz,
1
H, HC = N), 8.14 (ov ddd, JHH = 7.9, 7.9, 1.3 Hz, 1H, Ar), 7.93 (d,
JHH = 7.9 Hz, 1H, Ar), 7.63 (ov ddd, JHH = 7.9, 5.3, 1.3 Hz, 1H, Ar), 4.10
(t, JHH = 7.3 Hz, JHPt = 36.3 Hz, 2H, NCH ), 1.90 (quint, JHH = 7.3 Hz,
2H, NCH CH ), 1.29–1.23 (ov m, 10 H, –(CH ) –), 0.84 (t, J = 6.9 Hz,
(E)-N-(Pyridine-2-ylmethylene)hexadecane-1-amine (L5)
2
1
NMR spectroscopic data (in CDCl ): H ␦: 8.62 (d, J = 4.7 Hz, 1H,
3
HH
2
2
2 5
HH
1
3
1
Ar), 8.35 (s, 1H, HC = N), 7.96 (d, JHH = 7.9 Hz, 1H, Ar), 7.72 (ov
ddd, JHH = 7.9, 4.7, 1.5 Hz, 1H, Ar), 7.28 (m, 1H, Ar), 3.65 (t, JHH = 6.9 Hz,
2
3H, CH CH ); C{ H} ␦: 167.7, 157.2, 149.8, 139.6, 128.2, 127.5, 61.2,
2 3
31.8, 31.1, 29.2 (2C), 26.6, 22.7, 14.2. IR (Nujol): 2926 (s), 2856 (s), 1624
(m, ␯C = N), 1597 (m), 1459 (m), 1378 (m), 1299 (m), 1239 (m). Anal.
calcd. for C H N Cl Pt (484.46) (%): C 34.71, H 4.59, N 5.78; found:
H, NCH ), 1.70 (m, 2H, –CH –), 1.30–1.23 (ov m, 26H, –(CH ) –),
2 2 2 13
1
13
0
.85 (t, JHH = 7.4 Hz, 3H, CH CH ); C{ H} ␦: 161.7, 154.8, 149.4,
2
3
1
4
22
2
2
136.5, 124.6, 121.2, 61.7, 32.0, 30.8, 29.8 (7C), 29.7, 29.5, 29.4, 27.4,
C 34.44, H 4.55, N 5.71.
2
2.7, 14.2. IR (Nujol): 1650 (␯C = N).
PtCl (N,N=-nonyl) (4)
2
General synthesis of platinum complexes
1
Yield: 91%, mp 126–128 °C. NMR spectroscopic data (in CDCl ): H
3
A CH Cl (5 mL) solution of the appropriate iminopyridine li-
2
2
␦
: 9.43 (d, JHH = 5.4 Hz, JHPt = 31.6 Hz, 1H, Ar), 8.88 (s, JHPt = 93.7 Hz,
gand (0.80 mmol) was added dropwise to a stirred CH Cl (5 mL)
2
2
1
H, HC = N), 8.14 (ov ddd, JHH = 7.7, 7.7, 1.2 Hz, 1H, Ar), 8.03 (d, JHH
=
=
2
solution of [PtCl (␩ -coe)] (300 mg, 0.40 mmol). The reactions
2
2
7.7 Hz, 1H, Ar), 7.63 (ov ddd, JHH = 7.7, 5.4, 1.2 Hz, 1H, Ar), 4.09 (t, JHH
were allowed to proceed at RT for 18 h at which point hexane
5 mL) was added and the solutions stored at 5 °C. The resulting
6.8 Hz, JHPt = 38.8 Hz, 2H, NCH ), 1.89 (quint, J
= 6.8 Hz, 2H,
2
HH
(
NCH CH ), 1.27–1.21 (ov m, 12H, –(CH ) –), 0.83 (t, J = 6.2 Hz, 3H,
2
2
2 6
HH
orange precipitates were collected by suction filtration to afford
the desired platinum compounds.
1
3
1
CH CH ); C{ H} ␦: 168.3, 157.3, 149.6, 139.8, 128.2, 128.0, 61.0, 31.9,
2
3
31.1, 29.5, 29.3 (2C), 26.6, 22.7, 14.2. IR (Nujol): 2923 (s), 2853 (s),
PtCl (N,N=-propyl) (1)
1625 (m, ␯C = N), 1598 (m), 1460 (m), 1377 (m), 1295 (m), 1240 (m).
2
Yield: 84%, mp 208–210 °C. NMR spectroscopic data (in CDCl ):
Anal. calcd. for C H N Cl Pt (498.49) (%): C 36.14, H 4.86, N 5.62;
3
15 24
2
2
1
H ␦: 9.71 (d, JHH = 5.7 Hz, JHPt = 37.1 Hz, 1H, Ar), 8.65 (s, JHPt = 96.7 Hz,
found: C 36.36, H 5.03, N 5.38.
Published by NRC Research Press

Stewart et al.
133
Fig. 2. Synthesis of platinum complexes 1–6.
PtCl (N,N=-hexadecyl) (5)
Centre, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223
336033 or e-mail: deposit@ccdc.cam.ac.uk).
2
1
Yield: 92%, mp 137–140 °C. NMR spectroscopic data (in CDCl ): H ␦:
3
1
H ␦: 9.67 (d, JHH = 6.4 Hz, JHPt = 38.3 Hz, 1H, Ar), 8.65 (s, JHPt = 94.5 Hz,
Biological testing
1H, HC = N), 8.14 (ov ddd, JHH = 7.7, 7.7, 1.5 Hz, 1H, Ar), 7.81 (d, JHH
=
7
.7 Hz, 1H, Ar), 7.66 (ov ddd, JHH = 7.7, 6.4, 1.5 Hz, 1H, Ar), 4.16
Artemia toxicity bioassay
(t, JHH = 7.4 Hz, JHPt = 37.8 Hz, 2H, NCH ), 1.95 (quint, J = 7.4 Hz,
2
HH
Dehydrated Artemia salina (brine shrimp) eggs were obtained
from Ward's Scientific and hatched following the manufacturer's
guidelines. Thirty nauplii were then transferred to a test tube
containing 5 mL of stock salt solution with a final concentration
of either 10, 100, or 1000 ppm of a test compound or the positive
2
3
6
H, NCH CH ), 1.32–1.23 (ov m, 26H, –(CH ) –), 0.86 (t, J = 6.9 Hz,
2 2 2 13 HH
1
3
1
H, CH CH ); C{ H} (50 °C) ␦: 167.7, 157.3, 149.8, 139.5, 128.1, 127.5,
2
3
1.1, 31.9, 31.1, 29.7 (4C), 29.6, 29.5, 29.4, 29.3, 29.2, 26.6, 23.8, 22.6,
14.0. IR (Nujol): 2922 (s), 2852 (s), 1624 (m, ␯C = N), 1587 (m), 1469
(
(
m), 1377 (m), 1299 (m), 1238 (m). Anal. calcd. for C22H38N Cl Pt
596.70) (%): C 44.28, H 6.43, N 4.70; found: C 44.57, H 6.18, N 4.54.
2
2
(
cisplatin) or negative (distilled water) controls. The test tubes
were incubated at 30 °C for 24 h after which the number of sur-
viving brine shrimp was counted. Triplicates tests were per-
formed for each compound and control. The LC50 values were
calculated from the linear trendline log10 of concentration of com-
pound and number of surviving brine shrimp after 24 h.
PtCl (N,N=-octadecyl) (6)
2
Yield: 90%, mp 139–142 °C. NMR spectroscopic data (in CDCl ):
3
1
H ␦: 9.72 (d, JHH = 6.2 Hz, JHPt = 39.1 Hz, 1H, Ar), 8.64 (s, JHPt = 98.4 Hz,
1
H, HC = N), 8.14 (ov ddd, JHH = 7.7, 7.7, 1.5 Hz, 1H, Ar), 7.78 (d,
JHH = 7.7 Hz, 1H, Ar), 7.67 (ov ddd, JHH = 7.7, 6.2, 1.5 Hz, 1H, Ar), 4.18
t, JHH = 7.4 Hz, JHPt = 38.0 Hz, 2H, NCH ), 1.97 (quint, J = 7.4 Hz, 2H,
Anti-mycobacterial susceptibility screening assay
Anti-mycobacterial activity against Mycobacterium tuberculosis
strain H37Ra (ATCC 25177) was evaluated using a microplate resa-
zurin assay modified from Collins and Franzblau. Stock solu-
tions (5.0 mg/mL) of test compounds and rifampin (10 ␮g/mL,
positive control) were prepared with sterile-filtered DMSO and
stored at 4 °C. Antibiotic and test compound solutions were used
within 1 month and 1 week of preparation, respectively. Immedi-
ately prior to use, stock solutions of test compounds and rifampin
(
2
HH
NCH CH ), 1.32–1.23 (ov m, 30H, –(CH ) –), 0.86 (t, J = 7.0 Hz, 3H,
CH CH ); C{ H} (50 °C) ␦: 167.4, 157.3, 149.9, 139.4, 128.1, 127.3,
2
2
2 15
HH
1
3
1
2
3
10
6
1.1, 31.9, 31.1, 29.7 (7C), 29.6, 29.5, 29.4, 29.3, 29.2, 26.6, 22.7, 14.0.
IR (Nujol): 2924 (s), 2851 (s), 1624 (m, ␯C = N), 1597 (m), 1468 (m), 1377
m), 1300 (m), 1241 (m). Anal. calcd. for C24H42N Cl Pt (624.76) (%):
(
2
2
C 46.14, H 6.79, N 4.48; found: C 45.88, H 6.62, N 4.77.
X-ray crystallography
(40 ␮L) were diluted with modified Middlebrook 7H9 broth (960 ␮L)
Crystals of 2 were grown from a saturated solution of chlo-
roform at 5 °C. Single crystals were coated with Paratone-N oil,
mounted using a polyimide MicroMount, and frozen in the cold
nitrogen stream of the goniometer. A hemisphere of data was
collected on a Bruker AXS P4/SMART 1000 diffractometer using ␻
and ␪ scans with a scan width of 0.3° and 10 s exposure times. The
detector distance was 5 cm. The data were reduced (SAINT) and
corrected for absorption (SADABS). The structure was solved by
direct methods and refined by full-matrix least squares on F
and the resulting solutions (100 ␮L) were transferred to non-
peripheral wells of a 96-well microtitre plate and inoculated with
suspensions of M. tuberculosis (100 ␮L, cell density 2.0 × 10 cells/mL).
6
To reduce evaporation from the plates, sterile water (200 ␮L) was
added to perimeter wells. In addition to the rifampin positive
controls, negative controls (4% DMSO in modified Middlebrook
7H9 broth (100 ␮L) inoculated with suspensions of M. tuberculosis
(100 ␮L)) and blanks (2% DMSO in modified Middlebrook 7H9
broth (200 ␮L) and test solutions (100 ␮L) with modified Middle-
brook 7H9 broth (100 ␮L)) were included in each plate. All controls
and test compounds were tested in triplicate. Plates were incu-
7
8
2
9
(SHELXTL). All non-hydrogen atoms were refined using anisotro-
pic displacement parameters. Hydrogen atoms were included in
calculated positions and refined using a riding model. Crystallo-
graphic information has also been deposited with the Cambridge
Crystallographic Data Centre (CCDC 882438). Copies of the data
can be obtained free of charge from www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the Cambridge Crystallographic Data
bated (37 °C, 5% CO ) for 3 days in a humid environment before a
2
solution of resazurin (62.5 ␮g/mL) in 5% aqueous Tween 80 (50 ␮L)
was added to all wells. Plates were then incubated for a further
24 h, sealed with an adhesive polyester film, and mycobacterial
Published by NRC Research Press

134
Can. J. Chem. Vol. 91, 2013
Fig. 3. Molecular structure of 2 with ellipsoids drawn at the 50% probability level and hydrogen atoms omitted for clarity. Selected bond
distances (Å): Pt(1)–N(2) 2.003(6), Pt(1)–N(1) 2.031(6), Pt(1)–Cl(2) 2.2833(18), Pt(1)–Cl(1) 2.3001(19), N(1)–C(5) 1.327(10), N(1)–C(1) 1.348(10), N(2)–C(6)
o
1
1
.278(9), N(2)–C(7) 1.483(9). Selected bond angles ( ): N(2)–Pt(1)–N(1) 80.6(2), N(2)–Pt(1)–Cl(2) 94.72(18), N(1)–Pt(1)–Cl(2) 175.35(18), N(2)–Pt(1)–Cl(1)
75.64(17), N(1)–Pt(1)–Cl(1) 95.01(18), Cl(2)–Pt(1)–Cl(1) 89.62(7), C(5)–N(1)–C(1) 119.3(6), C(6)–N(2)–C(7) 121.3(6).
Table 1. Crystallographic data collection parameters for complex 2.
Table 2. Artemia toxicity data.
Complex
Chemical formula
Formula mass
Crystal dimensions (mm )
Crystal system
Space group
Z
a (Å)
b (Å)
2
LC50 (ppm)
C H Cl N Pt
456.27
0.30×0.10×0.05
Triclinic
P1
Compound
(Artemia assay)a
12
18
2 2
1
2
3
4
5
6
32.1
4.33
4.96
5.16
3.77
3.51
5.17
3
2
8.6858 (19)
8.7567 (19)
9.5080 (19)
76.546 (3)
87.151 (3)
78.586 (3)
689.4 (3)
2.198
198 (1)
MoK␣ (␭ = 0.71073 Å)
10.544
4518
2949
Cisplatin
c (Å)
aDue to the compounds' insolubility in wa-
ter, DMSO was used as a vehicle.
0
␣
␤
␥
( )
0
( )
0
( )
3
Volume (Å )
dcalcd (mg/m )
T (K)
against Candida albicans (ATCC 14053) were evaluated using a mi-
crobroth dilution antibiotic susceptibility assay modified from
McCulloch et al. Stock solutions of test compounds (5.0 mg/mL)
were prepared with sterile-filtered DMSO, stored at 4 °C, and used
within 1 week of preparation. Immediately prior to use, stock
solutions (40 ␮L) were diluted with the appropriate nutrient broth
3
11
Radiation
−
1
␮
(mm )
Total reflections collected
Independent reflections
(
960 ␮L) and the resulting test solutions (100 ␮L) were transferred
No. of variables
155
2.20–27.50
1.063
to non-peripheral wells of a 96-well microtitre plate in triplicate.
Wells were then inoculated with suspensions of the appropriate
0
␪
( )
2
GoF on F
6
microbial strain (100 ␮L, cell density 1.0 × 10 CFU/mL), and to
R1 (I > 2␴(I))
0.0408
reduce evaporation from the plates, sterile water (200 ␮L) was added
to perimeter wells. Positive controls consisted of one column con-
taining a twofold serial dilution of antibiotic (50–1.56 ␮g/mL, 100 ␮L
per well, kanamycin for S. aureus and P. aeruginosa and nystatin for
C. albicans) as 4% DMSO solutions in the appropriate nutrient broth
inoculated with suspensions of the appropriate pathogen (100 ␮L).
Additionally, each plate contained six untreated controls (4% DMSO
in the appropriate nutrient broth (100 ␮L) inoculated with suspen-
sions of the appropriate pathogen (100 ␮L)) and six un-inoculated
blanks (200 ␮L of 2% DMSO in the appropriate nutrient broth). The
optical densities of the wells were measured before and after a
wR (all data)
Largest diff. peak and hole (Å)
0.1073
4.250 and −4.030
= (⌺[w(F 2 4])1/2, where w = 1/[␴ (F 2) +
࿣/⌺ |F |. wR − F ) ]/⌺[F
c o 2 o c o o
2
2 2
2
Note: R
1
= ⌺࿣F
o
| – |F
(
0.0803P) ] and P = (max(Fo , 0) + 2Fc2)/3.
2
2
growth assessed fluorometrically at 37 °C. Fluorescence values
were corrected for any background fluorescence of the media and
test compounds by subtracting the mean fluorescence readings of
the appropriate blanks from the mean fluorescence readings of the
control and test compound wells. The percent inhibition of myco-
bacterial growth was then defined as [1 − (mean test or positive con-
trol fluorescence/mean negative control fluorescence)] × 100.
24 h incubation period (37 °C), and the change in OD (⌬OD) was
calculated by subtracting the initial OD from the final OD of cor-
responding wells. ⌬OD values were corrected for background ab-
sorbance of the media by subtracting the mean ⌬OD readings of
the blanks from the mean ⌬OD readings of the control and test
Antibiotic susceptibility screening assay
Anti-bacterial activity against Staphylococcus aureus (ATCC 29213)
and Pseudomonas aeruginosa (ATCC 10145) and anti-fungal activity
Published by NRC Research Press

Stewart et al.
135
Table 3. Anti-microbial and anti-mycobacterial testing of platinum compounds 1–6.
Mycobacterium
Candida albicans
Staphylococcus aureus
tuberculosis
Compound IC50 (␮g/mL)a MIC (␮g/mL)b IC50 (␮g/mL) MIC (␮g/mL) IC50 (␮g/mL) MIC (␮g/mL)
1
2
3
4
5
6
Inactivec
Inactive
81.5
49.2
Inactive
Inactive
Inactive
Inactive
100
100
Inactive
Inactive
58.7
61.2
25.7
13.9
Inactive
40.2
100
100
75
75
Inactive
75
1.65
1.91
1.52
0.70
1.78
51.3
75.00
25.00
12.50
6.25
12.50
>200.00c
a
b
IC50 values estimated by probit analysis.
MIC is defined as the lowest concentration that showed over 90% inhibition.
c
Less than 50% inhibition in initial screening (at 100 ␮g/mL) is considered to be inactive.
compound wells. The percent inhibition of bacterial or fungal
growth was then defined as [1 − (mean test or positive control
ligand to the metal. Similar trends are observed for the pyridine
hydrogen alpha to the nitrogen atom as the chemical shift
changes from ␦ 8.62 to 9.71 ppm (JHPt = 37 Hz). Similar trends are
observed for all platinum complexes 1–6.
Complex 2 has also been characterized by an X-ray diffraction
study (Fig. 3) with crystallographic data given in Table 1. The
nitrogen–platinum bonds of 2.003(6) Å (imine) and 2.031(6) Å (pyri-
⌬OD/mean negative control ⌬OD)] × 100.
Determination of minimum inhibitory concentrations (MIC) and
median lethal concentrations (IC50
)
MIC and IC50 values were determined for test compounds that
exhibited >50% growth inhibition at a concentration of 100 ␮g/mL
in the initial screening assays. Assays were performed as de-
scribed previously on twofold serial dilutions of active com-
pounds in triplicate. All compounds were tested at a minimum of
dine) are similar to those reported in other iminopyridine plati-
26–28
num systems.
The imine C(6)–N(2) distance is 1.278(9) Å and in
29
the range of accepted carbon–nitrogen double bonds. The N(2)–
Pt(1)–N(1) angle is 80.6(2)° and is considerably less than 90 , as is
o
10 and maximum of 20 concentrations obtained from two dilution
often observed in related complexes owing to the bidentate na-
ture of the iminopyridine ligand. The platinum chloride distances
are Pt(1)–Cl(2) 2.2833(18) Å and Pt(1)–Cl(1) 2.3001(19) Å, with a typi-
cal angle between these three atoms of Cl(2)–Pt(1)–Cl(1) 89.62(7)°.
Interestingly, compound 2 forms ␲-stacked dimers around an in-
version center with a “normal” platinum–platinum distance of
series (400.0–0.781 and 300.0–0.586 ␮g/mL). The MIC of a com-
pound was considered to be the lowest assay concentration at
which it inhibited mycobacterial growth by more than a mean
1
1
value of 90%. For most of the compounds, reliable estimates of
their absolute IC50 values could not be obtained through four-
parameter logistic regression of the microbial growth data (in
3.4 Å.
12
relative fluorescence units or OD units). Therefore, IC50 values
were obtained by probit analysis1
3,14
performed by fitting mean
Bioactivities
percent inhibition values calculated from the growth data to the
probit model by the maximum likelihood method1 using SPSS
Statistics 19 (IBM).
Before carrying out time-consuming and expensive anti-cancer
studies, we decided to conduct a preliminary evaluation on com-
pounds 1–6 to see if these new platinum species displayed any
biological activity. Previous studies have shown that bioactive
compounds are often toxic to brine shrimp (A. salina) larvae and
5
Results and discussion
that LC values can be directly correlated with these toxicity
Synthesis and structure
The addition of a primary amine to commercially available
50
0–33
3
studies.
Indeed, the brine shrimp lethality assay has been
2-pyridinecarboxaldehyde to afford the corresponding pyridine-2-
used to prescreen compounds for anti-cancer properties and good
ylcarboxaldimine ligand is a well-known route to a versatile class
correlations have been found between the in vivo and in vitro
of bidentate ligands.16–19
tests.34,35 We have found that the LC50 data suggest that the pro-
Variation of the amine allows for the
facile design of ligands and metal complexes with different
physical and chemical properties. The ligands L1-6 were pre-
pyl derivative 1 is not overly toxic; however, the other compounds
were on par, if not more toxic, than cisplatin (see Table 2). Fur-
thermore, it generally appears that increasing the length of the
hydrocarbon tail increases the toxicity and thus, potentially, the
biological activity. This increase in activity could be due to im-
proved cell penetration due to greater lipophilicity.
pared via
a simple addition of the primary amine to
2
-pyridinecarboxaldehyde. Although L1-4 and L6 have all been
6
made previously, the hexadecyl derivative L5 has not yet been
reported. The corresponding platinum complexes 1–6 (Fig. 2)
were prepared by addition of the pyridinecarboxaldimine ligands
Encouraged by these initial results, we decided to investigate
any potential anti-microbial properties of compounds 1–6. Al-
though none of the analogues showed significant activity against
the Gram-negative bacterium P. aeruginosa in our initial antibiotic
screening (growth inhibition ranged from 5% to 25% at 100 ␮g/mL),
some compounds did show weak anti-fungal activity and moder-
ate activity against the Gram-positive bacterium S. aureus (see
Table 3). Of particular interest, however, is the anti-mycobacterial
activity exhibited by the complexes. With the exception of the
octadecyl analogue 6, the analogues showed good activity against
M. tuberculosis H37Ra and compound 4 in particular exhibited ac-
tivity that is comparable with some currently used tuberculosis
drugs. The octyl and nonyl derivatives 3 and 4 were consistently
the most active in our antibiotic assays, whilst the low solubilities
of the hexadecyl and octadecyl compounds (5 and 6) were proba-
bly responsible for their reduced activity in the assays. These re-
sults are remarkable in that only a handful of platinum complexes
2
20
to CH Cl solutions of [PtCl (␩ -coe)] (coe = cis-cyclooctene).
2
2
2
2
Complexes 1–6 contain long chain side groups that are of par-
ticular interest, as a number of biologically relevant natural prod-
2
1
ucts, such as capsaicin, also possess a hydrocarbon tail. Using
lipophilic groups to deliver platinum complexes is a rapidly grow-
ing area of interest.2
2–25
As such, we have decided to look at the
synthesis and initial bioactivities of platinum complexes contain-
ing long chain aliphatic hydrocarbons bound to an iminopyridine
ligand. In this study, complexes 1–6 have been characterized by a
number of physical methods, including multinuclear NMR spec-
1
troscopy. A significant downfield shift in the H NMR is observed
2
for the imine sp proton upon coordination of the ligand to the
metal center. For instance, the singlet at ␦ 8.35 ppm for the free
ligand derived from 2-pyridinecarboxaldehyde and hexylamine
shifts to 8.65 ppm in complex 1. Platinum satellites are also ob-
served for this resonance (JHPt = 97 Hz) upon complexation of the
Published by NRC Research Press

136
Can. J. Chem. Vol. 91, 2013
have previously been examined for anti-mycobacterial activity.36–40
Our results suggest that lipophilic platinum complexes show consid-
erable promise for the treatment of tuberculosis.
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Conclusion
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Pyridinecarboxaldimines derived from the condensation of
2-pyridinecaboxaldehyde and aliphatic amines have been pre-
pared in high yields and used as ligands for platinum. The result-
ing metal complexes have been examined for their potential anti-
microbial properties and these complexes have shown incredible
promise as lead compounds for tuberculosis drug development.
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Acknowledgments
Thanks are gratefully extended to the Natural Sciences and En-
gineering Research Council of Canada, Mount Allison University,
the University of New Brunswick, the Canada Research Chair Pro-
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and the Harrison McCain Foundation (C.A.G.) for financial sup-
port. We also thank Dan E. Durant for his expert technical assis-
tance, John A. Johnson (Department of Biology, University of New
Brunswick, St. John) for assistance with the C. albicans, P. aerugi-
nosa, and S. aureus bioassays, Duncan Webster (Horizon Health) for
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