Synthesis of copper and zinc 2-(pyridin-2-yl)imidazo[1,2-a]pyridine complexes and their potential anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2016.10.041

A small library of novel copper and zinc imidazo[1,2-a]pyridine complexes have been synthesized. Their structures were confirmed by X-ray diffraction crystallography and a selection of these compounds was tested against five cancer cell lines originating from breast cancer (MCF-7 and MDA-MB-231), leukemia (K562 and HL-60) and colorectal cancer (HT-29). The imidazo[1,2-a]pyridines and their zinc complexes showed poor anticancer activity, while the copper complexes were active against the cancer cell lines with IC₅₀values comparable to and lower than camptothecin. For example, copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate 21 had an IC₅₀value lower than 1 μM against the HT-29 cells. Fluorescence microscopy with acridine orange, Hoechst 33342 and ethidium bromide, used in a preliminary investigation to evaluate morphological changes showed that copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate 21 caused both apoptosis, necrosis and paraptosis in the MCF-7 and HL-60 cells. A select group of copper N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amines (26, 27, 29 and 31) induced apoptosis, paraptosis and deformed nuclei in MCF-7 cells.

Full text of DOI:10.1016/j.ejmech.2016.10.041

Accepted Manuscript
Synthesis of copper and zinc 2-(pyridin-2-yl)imidazo[1,2-a]pyridine complexes and
their potential anticancer activity
Jean Dam, Zeenat Ismail, Taurai Kurebwa, Nadia Gangat, Leonie Harmse, Helder M.
Marques, Andreas Lemmerer, Moira L. Bode, Charles B. de Koning
PII:
S0223-5234(16)30903-5
10.1016/j.ejmech.2016.10.041
EJMECH 9006
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 September 2016
Revised Date: 17 October 2016
Accepted Date: 18 October 2016
Please cite this article as: J. Dam, Z. Ismail, T. Kurebwa, N. Gangat, L. Harmse, H.M.
Marques, A. Lemmerer, M.L. Bode, C.B. de Koning, Synthesis of copper and zinc 2-(pyridin-2-
yl)imidazo[1,2-a]pyridine complexes and their potential anticancer activity, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.10.041.
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ACCEPTED MANUSCRIPT
Graphical Abstract:
A small library of novel copper and zinc 2-(pyridin-2-yl)imidazo[1,2-a]pyridine complexes synthesized showed
significant activity against cancer cell lines originating from breast cancer leukemia (K562 and HL-60) and colorectal
cancer (HT-29).

ACCEPTED MANUSCRIPT
Synthesis of copper and zinc 2-(pyridin-2-yl)imidazo[1,2-
a]pyridine complexes and their potential anticancer
activity
Jean Dam,a Zeenat Ismail,b Taurai Kurebwa,b Nadia Gangat,b Leonie Harmse,b Helder M.
Marques,a Andreas Lemmerer,a# Moira L. Bodea and Charles B. de Koninga*
a Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, P.O Wits,
2050, South Africa; b Division of Pharmacology, Department of Pharmacy and Pharmacology,
Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193,
South Africa
Email
addresses:
Charles.deKoning@wits.ac.za;
t.Kurebwa@gmail.com;
Helder.Marques@wits.ac.za;
Jean.Dam@students.wits.ac.za;
Nadia.Gangat@students.wits.ac.za;
Andreas.Lemmerer@wits.ac.za;
Zeenat.Ismail@students.wits.ac.za;
Leonie.Harmse@wits.ac.za;
Moira.Bode@wits.ac.za.
Corresponding Authors:
Charles de Koning
Charles de Koning, Molecular Sciences Institute, School of Chemistry, University of the
Witwatersrand, P.O Wits, 2050, South Africa; Email: Charles.deKoning@wits.ac.za; Tel: +27
11 7176724; Fax: +27 11 7176749.
Abstract A small library of novel copper and zinc imidazo[1,2-a]pyridine complexes have been
synthesized. Their structures were confirmed by X-ray diffraction crystallography and a
selection of these compounds was tested against five cancer cell lines originating from breast
cancer (MCF-7 and MDA-MB-231), leukemia (K562 and HL-60) and colorectal cancer (HT-29).
The imidazo[1,2-a]pyridines and their zinc complexes showed poor anticancer activity, while
the copper complexes were active against the cancer cell lines with IC50 values comparable to
and lower than camptothecin. For example, copper 6-bromo-N-cyclohexyl-2-(pyridin-2-
yl)imidazo[1,2-a]pyridin-3-amine acetate 21 had an IC50 value lower than 1 µM against the HT-
29 cells. Fluorescence microscopy with acridine orange, Hoechst 33342 and ethidium
bromide, used in a preliminary investigation to evaluate morphological changes showed that
copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate 21 caused
both apoptosis, necrosis and paraptosis in the MCF-7 and HL-60 cells. A select group of
copper N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amines (26, 27, 29 and 31)
induced apoptosis, paraptosis and deformed nuclei in MCF-7 cells.
Keywords: Copper and zinc imidazo[1,2-a]pyridines, multicomponent coupling reactions,
breast cancer (MCF-7 and MDA-MB-231), leukemia (K562 and HL-60) and colorectal cancer
(HT-29), apoptosis, necrosis and paraptosis, fluorescence microscopy.
1. Introduction
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Imidazo[1,2-a]pyridines are known to display a wide range of biological activities. The most
notable clinical application of imidazo[1,2-a]pyridines is in the clinical use of zolpidem 1 for the
management of insomnia [1]. Related imidazo[1,2-a]pyridines include the previously used anti-
anxiety medication alpidem 2 [1], and the closely related compounds necopidem 3 [2] and
saripidem 4 [2] (Figure 1) which display sedative and anxiolytic effects. In another therapeutic
class the imidazo[1,2-a]pyridine, GSK812397 5, is currently under investigation for the
treatment of HIV [3]. In addition, imidazo[1,2-a]pyridines show significant activity as anti-
infectious agents, receptor ligands and as enzyme inhibitors [4]. Hence, imidazo[1,2-
a]pyridines are often termed “privileged scaffolds”.
{Figure 1. Biologically active imidazo[1,2-a]pyridines}
One of the most popular methods [5] for the synthesis of imidazo[1,2-a]pyridines is the
Groebke-Blackburn-Bienaymé multicomponent coupling reaction. The use of this method
allows for the formation of functionalized imidazo[1,2-a]pyridines in a single step from
substituted 2-aminopyridines, isocyanides and aldehydes as starting materials, typically in the
presence of an acid catalyst (such as scandium triflate [6], zinc(II) chloride or Montmorillonite
K-10 clay [7]) as shown in Scheme 1 [8].
{Scheme 1. General synthesis of imidazo[1,2-a]pyridines using Groebke-Blackburn-
Bienaymé methodology}
Using this synthetic strategy a library of imidazo[1,2-a]pyridines were assembled in our
laboratory and a small subset of 6-substituted imidazo[1,2-a]pyridines were previously shown
to induce apoptosis in vitro in HT-29 and Caco-2 colorectal cancer cell lines. The IC50 values
ranged from 6-22 µM for HT-29 and 6-20 µM for Caco-2 while camptothecin, showed an IC50
value of about 10 µM for both cell lines. Camptothecin was selected as a positive control due
to its ability to induce apoptosis in colorectal cell lines and because it is the parent compound
of irinotecan which is used in the treatment of metastatic colorectal cancer. In addition, these
imidazo[1,2-a]pyridines were of significant interest as they showed low toxicity towards
unstimulated normal human peripheral leucocytes [9].
As a result of these promising results we thought that the formation of metal-imidazo[1,2-
a]pyridines complexes, in a similar manner to the synthesis of analogues of cisplatin [10,11]
(e.g. carboplatin or oxaliplatin), would result in metal-imidazo[1,2-a]pyridines complexes with
significantly higher, and possibly even selective anticancer activity. The expense, solubility and
toxicity (most notably nephrotoxicity and myelosuppression) of currently used platinum based
chemotherapies [10,11], as well as the rapid development of resistance of certain cancers to
these treatments [12,13], has prompted extensive cancer research into the use of alternative
metals and their complexes.
Copper has become one such metal that has been investigated for this purpose. Copper
occurs naturally as part of the catalytic site in a variety of enzymes in humans [14], because of
the redox potential of copper enabling it to undergo single electron transfer during enzymatic
reactions [15]. However, there is strict regulation of copper levels in the body to prevent any
unwanted radical-type reactions from occurring [16]. Recently, it has been found that cancer
cells contain significantly higher levels of copper than their healthy cell equivalents, suggesting
that cancer cells do not regulate intracellular copper levels as strictly [15,17]. This unique
selectivity is something that could be taken advantage of when designing potential new
compounds to treat cancer [14].
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Copper-based organic ligand complexes have been extensively investigated as anticancer
agents, showing promising activity in a wide variety of cancer cell lines including breast [18-
23], colorectal [18,19], leukaemic [20,23,24], ovarian [24], stomach [23], cervical [25] and
fibrosarcoma [26] amongst others. While the exact mode of action for copper in such
complexes is not yet known, the ligand has been shown to play a vital role in the overall
activity of the copper complex as the coordination of the copper needs to be strong enough to
deliver the copper to its target, but weak enough to release the copper once it is delivered [17].
Also, it has been noted that complexes in which copper is coordinated to planar ligands are
more likely to make the complex act as DNA intercalators [23,24,27].
In this paper we report our results on the synthesis of a series of imidazo[1,2-a]pyridines
coordinated to zinc and copper and their in vitro anticancer activity against cell lines originating
from breast, colorectal and leukemic cancers. While a few metal-imidazo[1,2-a]pyridine
complexes have been investigated for their use in fluorescent materials and LED applications
[28-30], to our knowledge this is the first report on the anticancer activity of metal-imidazo[1,2-
a]pyridine complexes, and in particular those containing zinc and copper.
2. Results and Discussion
2.1. Chemistry
2.1.1 Synthesis
In order to allow for an uncomplicated method for the assembly of both zinc and copper
complexes of the biologically active imidazo[1,2-a]pyridines we had previously synthesized,
(for example 6, Figure 2) substituting the benzaldehyde in the Groebke multicomponent
coupling reaction with picolinaldehyde, as shown in Scheme 2, appeared to be the simplest
option.
{Figure 2. An example of the imidazo[1,2-a]pyridines previously synthesized}
{Scheme 2. General synthesis of pyridyl imidazo[1,2-a]pyridines [9]}
Pyridyl imidazo[1,2-a]pyridines (7-15) were synthesised by refluxing substituted 2-
aminopyridines and isocyanides with 2-picolinaldehyde (1:1:1 mol equivalents) in ethanol over
18 h. The imidazo[1,2-a]pyridines synthesized using this method are shown in Figure 3.
Montmorillonite K-10 clay was used as the catalyst in an equal mass to the 2-aminopyridine as
1
previously described [9]. Characterization of these ligands was performed using H and 13C
NMR spectroscopy, as well as single crystal X-ray diffraction (SCXRD).
Compounds 7-15 were used to assemble the zinc complexes (16-20 in Figure 4) and the
copper complexes (21-31 in Figure 5). This was achieved by stirring a diethylether solution of
the relevant imidazo[1,2-a]pyridine in an equimolar ratio with the appropriate metal salts,
ZnCl2, Zn(OAc)2, CuCl2, Cu(OAc)2 and Cu(NO3)2 over 18 h at room temperature. During this
time a powder formed in the solution and this was collected by filtration and washed with
diethylether to remove any unreacted imidazo[1,2-a]pyridine. The impure copper complexes
were then dissolved in acetone, filtered and concentrated in vacuo to remove any unreacted
copper salts. The powder was then crystallized slowly from suitable solvents to afford X-ray
quality crystals.
{Figure 3. Imidazo[1,2-a]pyridines synthesized using the Groebke multicomponent reaction}
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1
The zinc-imidazo[1,2-a]pyridines complexes were characterized using H and 13C NMR
spectroscopy, some elemental analysis and SCXRD. Due to the paramagnetic nature of the
copper complexes NMR spectroscopy could not be used for characterization and so these
compounds were characterised primarily utilizing SCXRD and supplemented by elemental
analysis. Unfortunately, both the zinc and copper complexes could not be characterized by
mass spectroscopy as they were found to be unstable under mass spectroscopic conditions
resulting in only the [M+H]+ of the organic ligand appearing during analysis.
{Figure 4. Zinc imidazo[1,2-a]pyridine complexes}
{Figure 5. The structures of copper imidazo[1,2-a]pyridine complexes}
2.1.2. Characterization
The single crystal X-ray diffraction structures of the pyridyl imidazo[1,2-a]pyridines (7-15)
revealed a twisted conformation to allow for hydrogen bonding between the amine and the
pyridine nitrogen. This hydrogen bonding was present in all the organic ligand crystal
structures and also preserved in the ligand crystals that were grown in hydrobromic acid. For
example, in Figure 6 the X-ray diffraction crystal structures of the pyridyl imidazo[1,2-
a]pyridine 8, shows the hydrogen bonding under discussion, while, for example, the metal
complexes 16 and 21 shows that the pyridine ring rotates in order to form the bidentate ligand-
metal complexes.
{Figure 6. The asymmetric units of metal-imidazo[1,2-a]pyridines 8, 16 and 21.
Displacement ellipsoids are shown at the 50% probability level. Hydrogen atoms not involved
in hydrogen bonding are omitted for clarity.}
To highlight the importance of X-ray crystallography in this study, the copper complexes of
the halogen substituted pyridyl-imidazo[1,2-a]pyridines 11 and 12 were found not to be the
monomeric units as expected given the monomeric nature of all the other complexes. Single
crystal X-ray diffraction revealed surprising dimeric units 30 and 31 that would be
indistinguishable from the monomeric unit utilizing any other method of characterization (given
that the complexes are unstable under mass spectroscopic conditions). Figure 7 shows the
dimeric structures for the copper compounds 30 and 31. These dimeric units would
presumably collapse into two monomeric units in an aqueous environment and behave in a
similar manner to the other copper complexes.
{Figure 7. The asymmetric units of the dimeric copper imidazo[1,2-a]pyridines 30 and 31.
Displacement ellipsoids are shown at the 50 % probability level. Hydrogen atoms are omitted
for clarity.}
Furthermore, crystals suitable for SCXRD of copper complexes 23 and 25 could not be
grown despite numerous attempts. As the definitive structure of these complexes cannot be
determined, copper complexes 23 and 25 have been excluded from the results reported in this
paper.
2.1.3. Complex Stability
Of vital importance to this project is the stability of the metal-pyridyl imidazo[1,2-a]pyridine
complexes (16-31) under physiological conditions. A selection of these compounds were
studied using UV-vis spectroscopy to determine the pK values of the nitrogen atoms in the free
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ligand which are involved in chelation in the metal complexes. The traditional closed cell pK
titration could not be used as the imidazo[1,2-a]pyridines were found to react with the silicone
flow cell tubing. Therefore, titrations were performed using an out-of-cell procedure. Using 2-
(N-morpholino)ethanesulfonic acid (MES) and NaH2PO3 as buffers at a 1 mM concentration in
a 50 % MeOH/H2O solution, the compounds were diluted to an appropriate concentration. The
solutions were allowed to equilibrate at 23 °C over night before being divided into individual 10
mL aliquots. Each aliquot was adjusted individually to the desired pH using negligible volumes
of concentrated HCl and NaOH to span a pH range of approximately 2 to 11 for the
imidazo[1,2-a]pyridines and their zinc complexes and 2 to 10 for the copper-containing
complexes. The absorption spectra for the solutions were obtained using UV-vis spectroscopy
between 200 and 600 nm. The total absorbance at the monitoring wavelength AT, is given by
Eq. 1 (if there is a single pK) or Eq. 2 (for two pKas) where Ai is the absorbance due to the ith
species.
ꢃ
ꢅꢆ
ꢀꢂ ꢄ + ꢀꢇꢈꢂ
ꢀꢁ =
(Eq. 1)
ꢃ
ꢅꢆ
ꢈꢂ + ꢄ
ꢃ
ꢅꢆꢇ
ꢃ
ꢅꢆ
+ ꢀꢇꢈꢂ ꢄ + ꢀꢉꢈꢂꢈꢇ
ꢀꢂ ꢄ
ꢀꢁ =
(Eq. 2)
ꢃ
ꢅꢆꢇ ꢅꢆ
ꢃ
+ ꢈꢂ ꢄ + ꢈꢂꢈꢇ
ꢄ
The absorbance change as a function of pH was fitted to Eq. 1 or 2 as appropriate as
objective function with Ai and Ki as variables using non-linear least squares methods in
SigmaPlot. The results are presented in Table 1.
Interestingly, the uncoordinated imidazo[1,2-a]pyridines (8, 13, 14, 15) displayed only one
pK value. This is most likely explained by the significant hydrogen bonding seen in the
imidazo[1,2-a]pyridines of the crystal structures as previously discussed (Figure 7) where only
the imidazo[1,2-a]pyridine is protonated under acidic conditions. The pK values for these
compounds are between 3.9 and 4.1. The zinc compounds (16-20) showed significantly large
spectroscopic changes over the entire pH range of 2-11 and the data were adequately fitted
with a two pK model (Eq. 2) with pK1 ranging between 2.8 and 3.5 and pK2 between 6.3 and
7.5. The copper complexes (21-24) were more difficult to monitor as the spectroscopic
changes for both pK’s were not always visible at the particular wavelength being monitored.
Copper complexes 21 and 22 could be fitted using the two pK model (Eq. 2) however the
individual pK’s of complex 24 had to be modelled separately using the single pK model (Eq. 1)
due to the relatively small spectroscopic changes of the compound. The individual pK values
were calculated only from the wavelengths monitored where a significant spectroscopic
change was observed at the individual pK ranges. Similar pK1 values (3.5-3.6) were observed
for 21 and 22; however, complex 24 was significantly higher (4.4). The pK2 values for
compounds 22 and 24 were similar (5.2-5.6). The pK2 of 21 was the highest of all the
compounds monitored (8.8). Figure 8 is an example of the spectroscopic changes seen as the
pH changes, as well as the SigmaPlot fit to the data. At the concentration required to monitor
the changes in the ligand the d-d copper absorption band was not visible. However on
increasing the concentration of this solution this band was visible at around 670 nm.
Combining the pK results for the free ligands (8, 13, 14 and 15) with the results obtained
from the complexes we can conclude that in solution where the pH is above pK2 the complex
can be expected to remain intact. When the pH of the solution is lowered to below pK2 of the
complex, the pyridyl nitrogen becomes protonated thereby breaking the first coordinative bond.
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Further lowering of the solution’s pH to below the pK1 of the complex, the imidazo[1,2-
a]pyridine nitrogen becomes protonated, effectively destroying the metal complex. The data of
Table 1 show that the complexes are expected to be stable especially under physiological
conditions (approximately pH 7.35) and that while pK2 of some of these compounds are very
close to (and in one case above) physiological conditions at least the first coordinate bond to
the imidazo[1,2-a]pyridine core, can be expected to remain intact.
{Figure 8. An example of the changes observed in the absorbance spectra at varying pH’s
(on the left) and the change in the absorbance spectra at a particular wavelength (on the right)
for compounds 13 (A), 17 (B) and 22 (C).}
{Table 1. pKa values for selected imidazo[1,2-a]pyridines and their copper and zinc
complexes}
2.2 Pharmacology
2.2.1 Evaluation of Anticancer Activity
The imidazo[1,2-a]pyridines and their corresponding metal complexes were evaluated to
determine their potential to inhibit proliferation of human cancer cell lines originating from
breast and colorectal carcinomas, as well as from leukemia. The HT-29 cell line represented
colorectal cancer and was previously utilized in our laboratories [9]. In addition, the
compounds were tested against two breast cancer cell lines (MCF-7 and MDA-MB-231) and
two leukemic cell lines (K562 and HL-60).
Initial testing involved exposing the relevant cancer cells to the imidazo[1,2-a]pyridine ligands
as well as the zinc and copper complexes thereof, at a final concentration of 100 µM at 37 °C
for 48 h. Cell viability was measured with the tetrazolium based MTT assay [31]. Compounds
were considered to be active when the viability of the cells had decreased by 80% when
compared to the negative control. These compounds were further tested to determine the 50%
inhibitory concentration, (IC50 values), which is an indication of the relative potency of a
compound. These data are summarized in Table 2. The inhibitory effect of these compounds
was compared to that of camptothecin, an inhibitor of topoisomerase I and a known inducer of
apoptosis. Camptothecin is also the parent compound of irinotecan and topotecan, anticancer
agents used in the management of metastatic colorectal, ovarian and small cell lung cancers
[32].
The 2-pyrido-imidazo[1,2-a]pyridine ligands showed poor activity towards the cancer cell
lines tested in this study and only the zinc-adamantyl imidazo[1,2-a]pyridine complex, 19,
showed any significant activity against the HT-29 colorectal cancer cell line with an IC50 value
of 13.2 µM. The copper complexes on the other hand showed particularly excellent activity
across the five cancer cell lines tested in this study. In addition some complexes displayed
selective inhibition of specific cell lines as described below.
Proliferation of the progesterone positive breast cancer cell line, MCF-7 was most affected by
copper complex 21, with an IC50 value less than 1 ꢀM and 26, 27 and 29 showing good activity
with IC50 values below 2 ꢀM. Copper complexes 22 and 28 had IC50 values less than 5 ꢀM and
two copper complexes, 30 and 31, showed activity close to 10 µM. The triple negative receptor
breast cancer line MDA-MB-231 proved to be less sensitive to the copper complexes with IC50
values ranging between 19 and 49 µM for most imidazo[1,2-a]pyridine copper complexes, with
the exception of the 5-substituted copper imidazo[1,2-a]pyridine 29 with an IC50 value of 2.4
µM.
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The HT-29 colorectal cell line was the most sensitive to the 6-subtituted copper imidazo[1,2-
a]pyridines 21 and 27 with IC50 values below 1 µM, while 24 and 29 have IC50 values below 3
µM. Six-substituted copper imidazo[1,2-a]pyridines 22, 26, 28 as well as 31 showed IC50
values comparable to that of camptothecin, below 10 µM.
In the leukemic cell line K562, 21, 27, and 28 showed inhibition of proliferation comparable to
that of camptothecin (2 to 3 µM), while the rest of the imidazo[1,2-a]pyridine copper complexes
were active with IC50 values below 10 µM. None of the compounds investigated in this study
compared with the significant potency of the camptothecin (IC50 = 0.005 µM) in the HL-60 cell
line. However, several compounds (21, 27, and 29) showed good activity of less than 3 µM
and two compounds (22 and 28) showed activity of less than 10 µM.
Of great interest is the identification of two copper imidazo[1,2-a]pyridines with unique
selectivity towards specific cell lines. MCF-7 cells were highly sensitive to 26 with an IC50 value
less than 2 µM compared to the other cell lines which displayed IC50 values higher than 6 µM.
In the HT-29 cells, the adamantyl containing copper imidazo[1,2-a]pyridine, 24, showed
significant activity with an IC50 value of 2.7 µM compared to no activity in the other cell lines.
Based on these observations we can conclude that in general, copper imidazo[1,2-a]pyridine
complexes that contained a cyclohexyl amine showed greater anticancer activity than those
possessing a benzyl, ester and adamantyl substituent. It was also not advantageous to place a
halogen (bromine or chlorine) on the 2- pyridyl ring instead of the imidazo[1,2-a]pyridine core.
The MDA-MB-231 cell line required a bromine atom at the 5-position to show any significant
activity. The cytotoxic effect on the other cancer cell lines appeared to be more tolerant of
changes to the imidazo[1,2-a]pyridine core, whereas there was little difference in activity
regarding the presence of a bromine atom or whether this bromine was located at the 5- or 6-
position of the imidazo[1,2-a]pyridine. Interestingly, it was only in the K562 cell line where a
chlorine atom in the 6-position of the imidazo[1,2-a]pyridine showed any comparable activity to
those possessing a bromine atom (or absence thereof).
The anticancer activity of metal salt precursors (Cu(OAc)2, CuCl2, Zn(OAc)2, ZnCl2) were also
investigated and they were found to be inactive against the cancer cell lines used in this study.
Considering that the pyrido-imidazo[1,2-a]pyridines also show poor activity, this supports the
supposition that the activity seen for the copper complexes is a result of the copper 2-pyrido-
imidazo[1,2-a]pyridine complexes rather than the ligand or the copper salt.
{Table 2. IC50 values for active compounds (µM)}
One of the major advantages of the imidazo[1,2-a]pyridines we previously reported for their
anticancer activity was their apparent lack of toxicity towards unstimulated normal human
peripheral leucocytes.[9] We wondered whether this was also be the case for the imidazo[1,2-
a]pyridine copper complexes (21-31). Leucocytes prepared from human blood were treated
with a selection of imidazo[1,2-a]pyridines and copper compounds at 20 µM for 24 h as shown
in Figure 9. Compounds causing a greater than 50% decrease in leukocyte viability after 24
hrs were considered to be potentially toxic. The imidazo[1,2-a]pyridine ligands 7, 9 and 10
confirmed the low toxicity that was noted previously. This is however a preliminary observation
and more assays utilizing normal cells representing other tissues are required. It must be
noted that many cancer chemotherapeutic agents, for example cisplatin, are used in the
treatment of cancer despite their toxicity towards normal cells and that a decrease in the white
blood cell count is a common feature of cancer chemotherapeutic regimens.
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Figure 9 indicates that compound 21 displayed potentially serious toxicity towards
leucocytes at 20 µM. The other active compounds 26, 27, 28 and 29 displayed a more
favourable effect with leucocyte viability ranging between 60 and 70%. The two less potent
compounds against the cancer cell lines, 30 and 31 also displayed lower toxicity towards the
leucocytes showing viability between 80 and 90%. It is encouraging that the most potent
compounds (26, 27, 29 and 31), with the exception of 21 show leucocyte availability greater
than 50 % when exposed to the compounds at 20 µM.
Given the low IC50 values of 21 against the different cell lines we wondered if 20 µM was not
an excessive concentration to test against leucocytes. To test this we exposed leucocytes to
various concentrations of 21 guided by IC50 values obtained for a set of cell lines. Figure 10
shows that lower concentrations of 21 results in significantly higher leucocyte viability.
{Figure 9. Leucocyte cell viability when treated with 20 µM of selected imidazo[1,2-
a]pyridines and copper complexes for 24 h}
{Figure 10. Leucocyte viability when treated with copper imidazo[1,2-a]pyridine 21 at varying
concentrations for 24 h. Leucocyte viability improves with lower concentrations of 21.}
2.2.2. Effects on Cancer Cell Morphology
The morphology of the cells were evaluated using the vital stains, acridine orange (AO),
Hoechst 33342 (HO) and ethidium bromide (EB). Changes to the cell morphology making use
of epifluorescent microscopy provided valuable clues regarding the mechanism of cell death.
Acridine orange stains healthy nuclei and cell cytoplasm an emerald green colour and in
addition also function as a lysosomotropic agent which makes it useful to detect acidic
vesicular organelles within a cell which are stained red [33,34,35]. Acridine orange is used in
this context as a as a preliminary indicator of autophagy [35]. Ethidium bromide enters cells
only when membranes are damaged and stains nuclei a bright orange-red detectable using
the same UV filter cube used for Hoechst stain, indicating necrotic cell death [33] or late
apoptosis depending on the staining pattern of the nuclei. Hoechst 33342 is a specific stain for
nuclei and can distinguish between apoptotic and non-apoptotic nuclei, showing an increase in
fluorescent intensity and specific changes to the structure of the nucleus which include
chromatin condensation and nuclear fragmentation [33]. Figure 11 shows the changes
induced by the copper-imidazo[1,2-a]pyridine 21 when MCF-7 and HL-60 cells were exposed
to a concentration of the test compound equivalent to an IC75 value for the respective cell lines.
There was evidence of both apoptotic and necrotic changes in both cell lines with late
apototic/necrotic changes being more prominent in the HL-60 cells. A purely necrotic nucleus
stains a uniform red/orange colour (Figure 11 panel d, labelled NN) whereas a late apoptotic
nucleus will show evidence of fragmentation as seen in Figure 11 panel j, (labelled LAN).
Many MCF-7 cells partially detached from the growth surface and assumed a rounded shape
while remaining anchored to the growth surface by filopodia, indicated with an F, (Figure 11,
panels b and f). Some of these detached, rounded cells showed a fragmented nucleus which
is consistent with apoptosis (AN) while others showed necrotic changes (NN) as shown in
Figure 11, panel d. The HL-60 cells (Figure 11, panels k-l), showed that some cells were in a
late apoptotic stage as evidenced by the globular nature of the Hoechst and/or acridine orange
stained nuclei.
{Figure 11. The effect of imidazo[1,2-a]pyridine 21 on cell and nuclear morphology on MCF-
7 and HL-60 cells after a 48 h exposure period indicates apoptotic and necrotic cell death.
MCF-7 and HL-60 cells were treated with 21 at a concentration of 1.5 µM and 2.0 µM,
8

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respectively. Images were captured at a 400 x magnification and scale bars represent 20
micron. Legend: N = normal nucleus; NN = necrotic nucleus; AN = apoptotic nucleus; LAN =
late apoptotic nucleus}
The effects of the copper imidazo[1,2-a]pyridines, 26, 27 and 29 on the morphology of MCF-
7 cells are shown in Figure 12. MCF-7 cells were exposed to a concentration of 10 µM of each
compound for 24 hours. The cells were the stained with a combination of Hoechst 33342,
acridine orange and ethidium bromide as described above. None of the cells showed the
characteristic red/orange staining of the nuclei viewed using the UV filter cube that is observed
with ethidium bromide entering the cells, indicating that the cell membranes were intact and
that there was no necrosis present.
The untreated MCF-7 cells (Figure 12, panels a,b,c,d) stained in a typical manner, showing
the somewhat rounded morphology of these cells and a tendency of the cells to grow on top of
each other, accounting for the cells on the image that are not in sharp focus.
A prominent feature of the three test compounds (26, 27 and 29) was the formation of
cytoplasmic vacuoles (V) in the perinuclear space (Figure 12, panels e,f,i,j). Moreover, cells
treated with both 26 and 27 caused the appearance of acidic granules (AG) which did not co-
localize with the cytoplasmic granules (Figure 12, panels k and g) but appears to co-exist in
the perinuclear space as shown in Figure 13 where MCF-7 cells were treated with 10 µM of
21 for 24 hours. The extensive formation of cytoplasmic vacuoles has been associated with
endoplasmic reticulum stress and paraptosis. Paraptosis is a type of programmed cell death
that is associated with extensive cytoplasmic vacuolisation, expression of phosphatidylserine
on the cell membrane (annexin V positive) but no activation of caspases, or the formation of
apoptotic bodies [26]. Indeed, it was shown by Tardito et. al. that a thioxotriazole copper(II)
complex has caused paraptotic cell death in a number of cell lines [26]. In addition, the
formation of the acidic granules detected with the acridine orange indicate the potential of the
compounds to induce autophagy which may lead to autophagic cell death.
Cells treated with 29 (Figure 12, panels m and n) showed rounding of the cells and
detachment from the growth surface. Some cells remain attached to the growth surface by
filopodia (F). Although not clearly visible due to the rounded shape of the cells, 29 also
induced cytoplasmic vacuole formation, and at a lower dose (5 µM), shown in Figure 14, there
is a clear indication of the formation of acidic granules.
Hoechst 33342 stain revealed profound changes in the typical nuclear morphology of the
treated cells. The copper imidazo[1,2-a]pyridines, 27 and 29 caused a high percentage of
nuclei to become deformed indicating the likelihood that the nucleus is the target organelle of
these compounds with some early indicators for apoptosis. MCF-7 cells treated with a lower
concentration of 29 (5 µM) appears to favour fragmentation off the nucleus indicating that 29 at
a lower concentrations cause apoptosis (Figure 14). The lower concentration also caused the
formation of acidic granules in a high percentage of cells which were not evident when the
cells were treated with 10 µM (Figure 12 and 14). Cells treated with 27 also showed the
presence of apoptotic nuclei (Figure 12, panel h and p). Compound 26 showed the presence
of contracted nuclei (CN) and a loss of the normal round or oval shaped nuclei (block arrow) in
Figure 12, panel l, which is indicative of pre-apoptotic changes. It must be emphasized that
this is a preliminary observation and requires further investigation using more specific
indicators for apoptosis.
9

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{Figure 12. The effect of the copper imidazo[1,2-a]pyridines 26, 27 and 29 on the cell and
nuclear morphology of MCF-7 cells shows the formation of cytoplasmic vacuoles, acidic
granules, deformed- and apoptotic nuclei. Cells were treated with 10 µM of the test
compounds for 24 h. Images were captured at a 400 x magnification and scale bars represent
20 micrometers. Legend: N = nucleus; CN = contracted nucleus; AN = apoptotic nucleus; DN
= deformed nucleus; AG = acidic granules: F = filopodia; V = vacuoles}
{Figure 13. The formation of cytoplasmic vacuoles do not co-localize with the acidic
granules. MCF-7 cells were treated with 10 µM of 21 for 24 hours. Images were captured at a
400 x magnification and scale bars represent 20 micrometers. Legend: AG = acidic granules,
V = Cytoplasmic vacuoles, N = nucleus}
{Figure 14. The effect of 29 on MCF-7 cells at a concentration of 5 µM for 24 hours indicates
that the formation of apoptotic nuclei and acidic granules is a dose dependent effect. Images
were captured at a 400 x magnification and scale bars represent 20 micrometers. Legend: AN
= apoptotic nucleus; AG = acidic granules}
3. Conclusion
Following on from a previous study, the synthesis of a small library of coordination
complexes of copper and zinc pyridine-substituted imidazo[1,2-a]pyridines was accomplished.
The zinc complexes (16-20) were found to have poor activity (IC50 values greater than 10 µM)
in the cancer cell lines tested. The copper complexes (21, 26, 27, 29 and 31) were found to
show significant cytotoxicity to breast, colorectal and leukemic cancer cell lines. Of great
promise is the apparent selectivity of some complexes for specific cell lines. For example, the
copper-imidazo[1,2-a]pyridine complex 29 showed activity against the progesterone positive
MCF-7 and MDA-MB-231 receptor triple negative breast cancer cell lines. In fact, the copper
complex 29 was the only compound that showed potent inhibitory activity against the MDA-
MB-231 cells.
Preliminary morphological analysis indicated changes in cell morphology that included
apoptosis, paraptosis and autophagy. Specific effects on DNA morphology indicated that the
nucleus may be a target organelle. The effect on normal leucocytes is encouraging indicating
a potential sparing effect on normal cells.
The apparent selectivity of specific copper containing imidazo[1,2-a]pyridines for cell lines
originating from different cancers hold promise for the future development of these complexes.
This study has revealed potential lead compounds that could be further investigated and
developed as potential new treatments for cancer. Investigations to elucidate their specific
molecular targets and the exact mechanism of cell death, caused by each of the copper
complexes are on-going.
4. Experimental Procedures
4.1. Chemistry
4.1.1. General Experimental Procedures
All the reagents and chemicals were purchased from Sigma-Aldrich, Merck or ACE
Chemicals and used without further purification unless otherwise noted. 1H NMR and 13C NMR
spectra were recorded using Bruker Advance-500 and Bruker Advance-300 spectrometers
operating at 500 MHz and 300 MHz respectively. All spectra were recorded in CDCl3 or MeOD
10

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with all chemical shift values reported in parts per million referenced against TMS as an
internal standard. Coupling constants are given in Hertz (Hz). Infrared spectra were recorded
on a Bruker Tensor 27 standard system spectrometer. The Groebke multicomponent coupling
reactions were monitored by TLC which was performed on aluminium-backed
MachereyeNagel Alugram Sil G/UV254 plates pre-coated with 0.25 mm silica gel 60.
Compounds (7-15) were purified by means of recrystallization or by flash column
chromatography using Merck silica gel (particle size 0.035-0.070 mm) with various EtOAc and
hexane mobile phase mixtures. Purity of the compounds as determined by elemental analyses
were carried out using a CHNS analyzer and most were found to have a purity of >95%.
However some of these results are outside the range viewed as establishing analytical purity,
they are provided to illustrate the best values obtained to date. HRMS was performed on a
Waters Synapt G2 and quoted in relative abundance (m/z).
4.1.2. General Procedure for the Groebke Multicomponent Reaction (7-15)
The general procedure for the synthesis of imidazo[1,2-a]pyridines involved the combination
of the appropriate 2-aminopyridine, isocyanide and pyridinecarbaldehyde in equal mole ratio in
a round bottomed flask as previously reported.[9] To this Montmorrillonite K-10 Clay was
added (same mass as the 2-aminopyridine used) followed by ethanol as the solvent. The
mixture was stirred under reflux conditions for 4-48 h, in some cases with additional stirring at
room temperature for a further 12 h. The clay was filtered off through celite and washed with
hot EtOAc. The filtrate was then concentrated in vacuo and the product purified by
recrystallization or by silica gel column chromatography.
4.1.2.1. N-Cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine (7)
2-Aminopyridine (1.00 g, 10.63 mmol), 2-pyridinecarboxaldehyde (1.14 g, 10.63 mmol, 1.00
eq), cyclohexyl isocyanide (1.16 g, 10.63 mmol, 1.00 eq) and Montmorrillionite K-10 Clay (1.20
g) were combined and the general procedure followed. The resulting mixture was purified by
recrystallization from an ether/CH2Cl2 mixture to afford 7 as yellow crystals (2.31 g, 74 %). Rf =
0.53 (50% EtOAc/Hexane), M.p. = 123-124 ˚C, 1H NMR (300 MHz, CDCl3): δH = 8.54 (1H, m),
8.18 (1H, dt, J = 8.1 and J = 1.1), 8.01 – 7.94 (1H, m), 7.73 (1H, td, J = 7.7 and J = 1.7), 7.52
(1H, d, J = 9.1), 7.18 – 6.98 (2H, m), 6.73 (1H, td, J = 6.7 and J = 1.1), 6.25 (1H, s), 3.10 (1H,
d, J = 9.9), 1.89 (2H, dd, J = 10.2 and J = 4.5), 1.77 – 1.65 (2H, m), 1.55 (1H, d, J = 3.9), 1.39
– 1.14 (5H, m). 13C NMR (75 MHz, CDCl3): δC = 155.1, 148.3, 140.7, 136.4, 131.2, 130.2,
123.2, 123.0, 121.0, 120.2, 117.8, 111.6, 55.5, 34.0, 25.8, 25.0. IR (νm.cm-1) = 3262, 2925,
2848, 1590, 1555, 1477, 1403, 1346, 1229, 1095, 1073, 892, 792, 760, 734, 694, 639. EIS-MS
(m/z) calculated for C18H21N4, (M+H), 293.1766, found 293.1765 [37].
4.1.2.2. 6-Bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine (8)
2-Amino-5-bromopyridine (2.00 g, 11.56 mmol), 2-pyridinecarboxaldehyde (1.24 g, 11.56
mmol, 1.00 eq), cyclohexyl isocyanide (1.26 g, 11.56 mmol, 1.00 eq) and Montmorrillionite K-
10 Clay (2.00 g) were combined and the general procedure followed. The resulting mixture
was purified by recrystallization from an ether/methanol mixture to afford 8 as yellow needles
1
(1.4 g, 33 %). Rf = 0.50 (50% EtOAc/Hexane), M.p. = 135 - 136 ˚C, H NMR (500 MHz,
CDCl3): δH = 8.55 (1H, m), 8.15 (1H, dt, J = 8.1 and 1.1), 8.09 (1H, dd, J = 1.9 and 0.9), 7.75
(1H, td, J = 7.8 and 1.8), 7.41 (1H, dd, J = 9.5 and 0.9), 7.17 – 7.09 (2H, m), 6.21 (1H, d, J =
9.9), 3.07 (1H, m), 1.93 – 1.84 (2H, m), 1.76 – 1.66 (2H, m), 1.57 (1H, dd, J = 9.7 and 4.4),
1.37 – 1.17 (5H, m). 13C NMR (126 MHz, CDCl3): δC = 154.8, 148.5, 139.2, 136.7, 131.5,
131.4, 126.7, 123.1, 121.5, 120.5, 118.6, 106.7, 55.6, 34.2, 25.9, 25.0. IR (νm.cm-1) = 3338,
2925, 2918, 2858, 2856, 1612, 1554, 1506, 1456, 1390, 1303, 1298, 1247, 1205, 1159, 1126,
11

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1085, 1024, 912, 887, 844, 779, 736, 702, 667, 634. EIS-MS (m/z) calculated for C18H20BrN4,
(M+H), 371.0871, found 371.0874. Anal. Calcd (%) for C18H19N4Br: C 58.23, H 5.16, N 15.09.
Found: C 58.54, H 5.10, N 14.86.
4.1.2.3. 6-Chloro-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine (9)
Following the general procedure, 2-amino-5-chloropyridine (1.00 g, 7.78 mmol), 2-
pyridinecarboxaldehyde (0.833 g, 7.78 mmol, 1.00 eq), cyclohexyl isocyanide (0.857 g, 7.78
mmol, 1.00 eq) and Montmorrillionite K-10 Clay (1.00 g) were combined in ethanol. Column
chromatography was used to purify the compound with 20% EtOAc/Hexane as an eluent to
afford a yellow powder, 9 (2.27 g, 89 %). Rf = 0.57 (50% EtOAc/Hexane), M.p. = 118-119 ˚C,
1H NMR (300 MHz, CDCl3): δH = 8.60 – 8.52 (1H, m), 8.15 (1H, dt, J = 8.0 and J =, 1.1), 7.98
(1H, dd, J = 2.0 and J = 0.9), 7.74 (1H, dd, J = 8.7 and J = 6.8), 7.46 (1H, d, J = 9.5), 7.13 (1H,
m), 7.08 – 6.91 (1H, m), 6.21 (1H, s), 3.07 (1H, s), 1.88 (2H, d, J = 11.1), 1.78 – 1.62 (2H, m),
1.56 (1H, d, J = 6.0), 1.45 – 1.13 (5H, m).13C NMR (75 MHz, CDCl3): δC = 154.7, 148.3, 139.0,
136.5, 131.48, 131.46, 124.5, 121.3, 120.7, 120.3, 120.0, 118.2, 55.4, 34.0, 25.8, 24.9. IR
(νm.cm-1) = 3305, 2930, 2853, 1590, 1476, 1400, 1324, 1287, 1232, 1201, 1184, 1115, 1085,
1046, 943, 891, 807, 786, 740, 702, 626. EIS-MS (m/z) calculated for C18H20ClN4, (M+H),
327.1376, found 327.1369. Anal. Calcd (%) for C18H19N4Cl: C 66.15, H 5.86, N 17.14. Found:
C 66.62, H 5.69, N 16.95.
4.1.2.4. 5-Bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine (10)
2-Amino-6-bromopyridine (2.00 g, 11.56 mmol), 2-pyridinecarboxaldehyde (1.238 g, 11.56
mmol, 1.00 eq), cyclohexyl isocyanide (1.262 g, 11.65 mmol, 1.00 eq) and Montmorrillionite K-
10 Clay (1.00 g) were combined and the general procedure followed. The resulting mixture
was purified by column chromatography with 20% EtOAc/Hexane as an eluent to afford a
1
viscous golden oil, 10 (2.976 g, 69 %). Rf = 0.68 (50% EtOAc/Hexane), H NMR (300 MHz,
CDCl3): 8.57 (1H, m), 8.18 (1H, dt, J = 8.1, 1.1), 7.82 – 7.70 (1H, m), 7.49 (1H, dt, J = 8.6,
1.0), 7.21 – 7.12 (1H, m), 6.98 – 6.84 (2H, m), 6.28 (1H, s), 3.08 (1H, s), 1.91 (2H, s), 1.69
(2H, d, J = 23.3 Hz), 1.56 (1H, s), 1.19 – 1.08 (5H, m). 13C NMR (75 MHz, CDCl3): 154.6,
148.2, 142.9, 136.5, 133.2, 123.6, 121.5, 121.0, 118.8, 117.3, 112.3, 60.3, 32.8, 26.0, 25.6. IR
(νm.cm-1) = 3268, 2928, 2852, 1589, 1552, 1470, 1428, 1400, 1298, 1225, 1144, 1074, 976,
891, 768, 721. EIS-MS (m/z) calculated for C18H20BrN4, (M+H), 371.0871, found 371.0866.
4.1.2.5. 2-(5-Bromopyridin-2-yl)-N-cyclohexylimidazo[1,2-a]pyridin-3-amine (11)
2-Aminopyridine (0.253 g, 2.688 mmol), 5-bromo-2-pyridinecarboxaldehyde (0.500 g, 2.688
mmol, 1.00 eq), cyclohexyl isocyanide (0.293 g, 2.688 mmol, 1.00 eq) and Montmorrillionite K-
10 Clay (0.500 g) were combined and the general procedure followed. The resulting mixture
was purified by column chromatography with 20% EtOAc/Hexane as an eluent to afford a
yellow solid, 11 (0.816 g, 82 %). Rf = 0.63 (50% EtOAc/Hexane), M.p. = 144 ˚C, 1H NMR (300
MHz, CDCl3): δH = 8.60 (1H, dt, J = 2.4 and 0.8), 8.08 (1H, dt, J = 8.6 and 0.8), 7.96 (1H, m),
7.85 (1H, m), 7.50 (1H, m), 7.09 (1H, m), 6.76 (1H, m), 5.93 (1H, d, J = 9.8), 3.14 – 3.00 (1H,
m), 1.89 (2H, d, J = 11.7), 1.72 (2H, q, J = 5.2), 1.57 (1H, s), 1.40 – 1.15 (5H, m). 13C NMR (75
MHz, CDCl3): δC = 153.6, 149.3, 141.0, 139.1, 131.3, 129.5, 123.6, 123.1, 121.6, 117.9, 117.4,
111.8, 55.6, 34.1, 25.8, 25.0. IR (νm.cm-1) = 3302, 2922, 2848, 1580, 1537, 1471, 1444, 1413,
1359, 1292, 1265, 1230, 1197, 1139, 1120, 1086, 1047, 1002, 922, 889, 841, 752, 702, 636,
617. EIS-MS (m/z) calculated for C18H20BrN4, (M+H), 371.0871, found 371.0855. Anal. Calcd
(%) for C18H19N4Br: C 58.23, H 5.16, N 15.09. Found: C 58.23, H 5.29, N 14.88.
4.1.2.6. 2-(5-Chloropyridin-2-yl)-N-cyclohexylimidazo[1,2-a]pyridin-3-amine (12)
12

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The
combination
of
2-aminopyridine
(0.332
g,
3.53
mmol),
5-chloro-2-
pyridinecarboxaldehyde (0.500 g, 3.53 mmol, 1.00 eq), cyclohexyl isocyanide (0.386 g, 3.53
mmol, 1.00 eq) and Montmorrillionite K-10 Clay (0.500 g) according to the general procedure
resulted in the formation of 12 (0.997 g, 86 %) as a yellow powder following purification by
column chromatography with a 20% EtOAc/Hexane eluent. Rf = 0.54 (50% EtOAc/Hexane),
M.p. = 139-140 ˚C, 1H NMR (300 MHz, CDCl3): δH = 8.50 (1H, dt, J = 2.5 and 0.8), 8.13 (1H,
dt, J = 8.6 and 0.8), 7.96 (1H, m), 7.71 (1H, m), 7.50 (1H, m), 7.09 (1H, m), 6.75 (1H, m), 5.93
(1H, d, J = 9.8), 3.06 (1H, m), 1.94 – 1.83 (2H, m), 1.73 (2H, d, J = 7.8), 1.57 (1H, s), 1.40 –
1.15 (5H, m). 13C NMR (75 MHz, CDCl3): δC = 153.5, 147.3, 141.0, 136.4, 131.4, 129.7, 129.0,
123.7, 123.2, 121.3, 118.0, 111.9, 55.8, 34.3, 25.9, 25.1. IR (νm.cm-1) = 3303, 2925, 2850,
1579, 1541, 1471, 1446, 1363, 1346, 1288, 1230, 1199, 1139, 1107, 1089, 1008, 923, 891,
842, 798, 752, 738, 704, 638, 621. EIS-MS (m/z) calculated for C18H20ClN4, (M+H), 327.1376,
found 327.1379. Anal. Calcd (%) for C18H19N4Cl: C 66.15, H 5.86, N 17.14. Found: C 66.85, H
6.07, N 16.89.
4.1.2.7. N-Benzyl-6-bromo-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine (13)
Combining 5-bromo-2-aminopyridine (2.304 g, 13.317 mmol), benzyl isocyanide (1.56 g,
13.317 mmol, 1.00 eq) and pyridinecarbaldehyde (1.43 g, 13.317 mmol, 1.00 eq) with
Montmorrillionite K-10 Clay (2.30 g) and following the general procedure with EtOH as the
solvent resulted in complex 13 (2.037g, 40 %), which was collected as a yellow powder after
1
recrystallization from ether. Rf = 0.57 (50% EtOAc/Hexane), M.p. = 121 ˚C, H NMR (300
MHz, Chloroform-d) δ 8.53 – 8.49 (1H, m), 8.19 – 8.07 (2H, m), 7.74 (1H, td, J = 7.8, 1.8), 7.48
13
– 7.23 (6H, m), 7.19 – 7.10 (2H, m), 6.54 (1H, t, J = 7.0), 4.24 (2H, d, J = 6.8). C NMR (75
MHz, CDCl3) δ 154.5, 148.6, 139.2, 138.9, 136.6, 131.9, 131.3, 128.5, 128.1, 127.5, 126.9,
122.8, 121.5, 120.3, 118.5, 106.8, 51.4. IR (νm.cm-1) = 3302, 3028, 2930, 2856, 1589, 1516,
1477, 1429, 1396, 1352, 1317, 1290, 1251, 1226, 1110, 1037, 995, 931, 790, 733, 798, 663,
628, 617. EIS-MS (m/z) calculated M+H for C17H19N3Br 379.0560, found 379.0544. Anal.
Calcd (%) for C19H15N4Br: C 60.17, H 3.99, N 14.77. Found: C 60.44, H 5.33, N 14.58.
4.1.2.8. Ethyl 2-[(6-bromo-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-yl)amino]acetate (14)
2-Amino-5-bromopyridine (1.835 g, 10.609 mmol), 2-pyridinecarboxaldehyde (1.137 g, 10.61
mmol, 1.00 eq), ethyl isocyanoacetate (1.20 g, 10.61 mmol, 1.00 eq) and Montmorrillionite K-
10 Clay (1.80 g) were combined and the general procedure followed. The resulting mixture
was purified by recrystallization from a CH2Cl2/hexane mixture to afford 14 as mustard flakes
1
(1.763 g, 44 %). Rf = 0.23 (50% EtOAc/Hexane), M.p. = 142 - 144 ˚C, H NMR (500 MHz,
Chloroform-d) δ 8.61 – 8.57 (1H, m), 8.27 – 8.21 (1H, m), 8.15 (1H, dt, J = 7.9, 1.1), 7.76 (1H,
td, J = 7.7, 1.8), 7.43 (1H, dd, J = 9.5, 0.9), 7.21 – 7.14 (2H, m), 6.49 (1H, s), 4.18 (2H, q, J =
13
7.1), 3.88 (2H, s), 1.22 (3H, t, J = 7.1). C NMR (126 MHz, CDCl3) δ 170.6, 154.2, 148.8,
139.3, 136.7, 131.9, 130.5, 127.1, 122.8, 121.7, 120.4, 118.6, 107.0, 61.3, 48.6, 14.1. IR
(νm.cm-1) = 3199, 3087, 2976, 2927, 2904, 1735, 1591, 1548, 1481, 1431, 1396, 1371, 1340,
1317, 1238, 1207, 1110, 1097, 1024, 995, 958, 871, 794, 746, 696, 632. EIS-MS (m/z)
calculated for C21H24N3O2Br, (M+H), 375.0458, found 375.0449. Anal. Calcd (%) for
C16H15N4Br: C 51.22, H 4.03, N 14.93. Found: C 51.02, H 4.65, N 14.65.
4.1.2.9.
N-[(1S,3S)-Adamantan-1-yl]-6-bromo-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine
(15)
The general procedure was followed using 2-amino-5-bromopyridine (0.322 g, 1.863 mmol),
2-pyridinecarboxaldehyde (0.199 g, 1.863 mmol, 1.00 eq), adamantyl isocyanide (0.300 g,
1.863 mmol, 1.00 eq) and Montmorrillionite K-10 Clay (0.500 g). The resulting mixture was
13

ACCEPTED MANUSCRIPT
purified by flash column chromatography using 30-50 % EtOAc/Hexane as an eluent. The 15
was collected as a yellow powder (0.738 g, 94 %). Rf = 0.50 (50% EtOAc/Hexane), M.p. =
159-162 ˚C, 1H NMR (500 MHz, CDCl3): δH = 8.57 (1H, m), 8.44 (1H, dd, J = 1.9 and J = 0.9),
8.16 – 8.12 (1H, m), 7.76 (1H, td, J = 7.7 and J = 1.8), 7.40 (1H, dd, J = 9.5 and J = 0.9), 7.18
(1H, m), 7.14 (1H, dd, J = 9.5 and J = 1.9), 5.39 (1H, s), 2.00 – 1.96 (3H, m), 1.71 (6H. d, J =
2.9), 1.58 (3H, d, J = 12.7), 1.55 – 1.49 (3H, m). 13C NMR (126 MHz, CDCl3) δC = 154.7,
148.3, 134.0, 136.5, 135.7, 128.2, 127.2, 124.4, 121.9, 121.3, 118.1, 106.2, 57.7, 43.5, 36.2,
29.8. IR (νm.cm-1) = 3303, 2904, 2849, 1591, 1568, 1476, 1425, 1395, 1321, 1288, 1229,
1199, 1103, 1088, 1045, 920, 842, 787, 743, 696, 603. EIS-MS (m/z) calculated for
C21H24N3O2Br, (M+H), 423.1186, found 423.1180. Anal. Calcd (%) for C22H23N4Br: C 62.42, H
5.48, N 13.23. Found: C 62.74, H 7.24, N 13.09.
4.1.3. General Procedure for the synthesis of metal-imidazo[1,2-a]pyridines (16-20)
The general procedure for metal-complex formation was loosely based on a procedure by
Steffen and Palenik [36]. In a round bottom flask, the relevant imidazo[1,2-a]pyridine was
dissolved in diethyl ether. The metal salt (1.00 eq) was added to the solution and the reaction
stirred for 18 h at room temperature. Over time a precipitate formed and after 24 h was
collected by gravity filtration and washed with diethyl ether and recrystallized using an
appropriate method to determine the absolute configuration of the complex. The copper-
imidazo[1,2-a]pyridine complexes were dissolved in acetone after filtration and the solution
filtered again to remove any unreacted metal salts. The filtrate was concentrated in vacuo to
afford a fine powder. This method was used unless otherwise stated.
4.1.3. 1. Zinc 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate (16)
Using the general procedure, 8 (0.200 g, 0.539 mmol) and zinc(II) acetate monohydrate
(0.099g, 0.539 mmol, 1.00 eq) were combined with an extra addition of a few drops of EtOH.
After recrystallization by slow evaporation from isopropyl alcohol the structure of the complex
16 was confirmed by single crystal X-ray crystallography and NMR spectroscopy (0.149 g, 50
1
%). H NMR (500 MHz, CDCl3) δH = 8.87 (1H, d, J = 5.1), 8.22 (1H, s), 8.01 (1H, d, J = 8.0),
7.93 (1H, d, J = 9.0), 7.89 (1H, t, J = 7.8), 7.40 (1H, t, J = 6.4), 7.31 (1H, d, J = 9.5), 3.80 (1H,
s), 2.82 (1H, s), 2.10 (6H, s), 1.85 – 1.70 (5H, m), 1.32 (2H, q, J = 11.1), 1.17 (3H, t, J = 9.8).
13C NMR (126 MHz, CDCl3) δC = 180.6, 149.8, 147.2, 139.6, 139.4, 131.9, 130.4, 127.8,
124.3, 123.3, 121.2, 118.5, 109.4, 57.8, 34.4, 25.4, 25.0, 22.1. IR (νm.cm-1) = 3259, 3074,
2923, 2854, 1581, 1406, 1332, 1236, 1092, 1047, 1015, 822, 798, 758, 674, 618. Anal. Calcd
(%) for ZnC22H25N4O4Br: C 47.63, H 4.54, N 10.10. Found: C 47.30, H 5.64, N 9.94.
4.1.3. 2. Zinc N-benzyl-6-bromo-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate (17)
Imidazo[1,2-a]pyridine 13 (0.500 g, 1.319 mmol) and zinc(II) acetate monohydrate (0.290 g,
1.319 mmol, 1.00 eq) were combined according to the general procedure with the addition of a
few drops of EtOH. The metal-imidazo[1,2-a]pyridine 17 was collected as a light yellow
powder. After vapour diffusion recrystallization from MeOD and diethyl ether, the structure of
1
the complex was confirmed by single crystal X-ray crystallography (0.483 g, 65 %). H NMR
(500 MHz, CDCl3) δH = 8.80 (1H, s), 7.90 – 7.80 (2H, m), 7.75 (1H, s), 7.70 (1H, td, J = 7.8
and 1.7), 7.30 (1H, d, J = 6.5), 7.26 – 7.22 (3H, m), 7.16 (3H, td, J = 9.1, 3.5), 4.88 (1H, s),
4.07 (2H, d, J = 6.1), 2.06 (6H, s). 13C NMR (126 MHz, CDCl3) δC = 180.7, 149.5, 146.7,
139.4, 138.3, 131.9 (×3), 129.9, 128.9 (×2), 128.8, 128.1, 124.2, 123.5, 121.0, 118.0, 109.0,
77.6, 52.6, 22.1 (×2). IR (νm.cm-1) = 3271, 3072, 3018, 1577, 1407, 1329, 1232, 1038, 1016,
810, 781, 740, 675, 619. Anal. Calcd (%) for ZnC23H21N4O4Br: C 49.09, H 3.76, N 9.96.
Found: C 48.91, H 3.57, N 9.84.
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4.1.3. 3. Zinc ethyl 2-[(6-bromo-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-yl]amino)acetate acetate
(18)
Zinc(II) acetate monohydrate (0.108 g, 0.108 mmol, 1.10 eq) and 14 (0.200 g, 0.533 mmol)
were combined according to the general procedure with additional EtOH added. Complex 18
was collected as a mustard powder (0.226 g, 76 %). After vapour diffusion recrystallization
from CDCl3 and diethyl ether, the structure of the complex was confirmed by single crystal X-
ray crystallography. 1H NMR (500 MHz, CDCl3) δH = 8.85 (1H, d, J = 5.1), 8.54 – 8.50 (1H, m),
8.08 (1H, d, J = 8.0), 8.01 (1H, s), 7.90 (1H, td, J = 7.8 and 1.7), 7.45 – 7.40 (1H, m), 7.38 (1H,
d, J = 9.5), 4.20 (2H, q, J = 7.2), 3.83 (2H, d, J = 5.8), 2.09 (6H, s), 1.89 (1H, s), 1.25 (3H, t, J
= 7.1). 13C NMR (126 MHz, CDCl3) δ = 180.6, 171.3, 149.5, 146.5, 139.8, 139.6, 132.2,
127.52, 124.5, 124.1, 121.1, 118.3, 109.3, 61.9, 49.1, 22.7, 14.1. IR (νm.cm-1) = 3273, 3080,
2980, 2922, 2845, 1730, 1582, 1474, 1416, 1337, 1296, 1238, 1198, 1105, 1028, 928, 820,
792, 727, 677, 673, 619. Anal. Calcd (%) for ZnC20H21N4O6Br: C 43.00, H 3.79, N 10.03.
Found: C 42.17, H 3.54, N 10.24.
4.1.3. 4. Zinc N-[(1S,3S)-adamantan-1-yl]-6-bromo-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-
amine acetate (19)
Imidazo[1,2-a]pyridine 15 (0.161 g, mmol) and zinc(II) acetate monohydrate (0.077 g, 0.419
mmol, 1.10 eq) were combined following the general procedure with an additional few drops of
EtOH. Complex 19 was collected as a white powder (0.161 g, 70 %). After vapour diffusion
recrystallization from CDCl3 and diethyl ether, the structure of the complex was confirmed by
single crystal X-ray crystallography. 1H NMR (500 MHz, CDCl3) δH = 8.93 – 8.88 (1H, m), 8.43
(1H, d, J = 8.0), 8.37 (1H, s), 8.01 (1H, d, J = 9.5), 7.88 (1H, t, J = 7.8), 7.44 – 7.36 (2H, m),
13
3.34 (1H, s), 2.09 (6H, s), 2.06 (3H, s), 1.77 – 1.72 (6H, m), 1.66 – 1.51 (6H, m). C NMR
(126 MHz, CDCl3) δ = 180.6, 149.8, 147.5, 140.3, 138.9, 133.3, 132.2, 125.0, 124.6, 124.1,
121.9, 118.6, 109.2, 58.2, 44.3, 35.9, 29.7, 22.1. IR (νm.cm-1) = 3271, 3074, 3018, 2907, 2855,
1585, 1530, 1503, 1472, 1412, 1393, 1323, 1234, 1088, 1049, 1013, 922, 824, 797, 760, 706,
671, 617. Anal. Calcd (%) for ZnC24H29N4O4Br: C 51.46, H 4.82, N 9.23. Found: C 51.16, H
5.38, N 9.05.
4.1.3. 5. Zinc 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine dichloride
(20)
Imidazo[1,2-a]pyridine 8 (0.250 g, 0.673 mmol), zinc(II) chloride (0.092g, 0.673 mmol, 1.00
eq) and triethyl orthoformate (0.200 g, 1.346 mmol, 4.00 eq) were combined and refluxed
overnight. Complex 20 was collected as a cream powder. After vapour diffusion
recrystallization from CH2Cl2/hexane, the structure of the complex was confirmed by single
crystal X-ray crystallography (0.283 g, 83 %). IR (νm.cm-1) = 3289, 3092, 2932, 2858, 1742,
1634, 1604, 1528, 1503, 1474, 1450, 1412, 1331, 1240, 1144, 1097, 1047, 1042, 843, 789,
806, 758, 711, 685, 648, 546. Anal. Calcd (%) for ZnC18H19N4BrCl2: C 42.59, H 3.77, N 11.04.
Found: C 42.95, H 3.98, N 10.34.
4.1.3. 6. Copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate
(21)
Imidazo[1,2-a]pyridine 8 (0.200 g, 0.539 mmol) and copper(II) acetate monohydrate (0.106g,
0.539 mmol, 1.00 eq) were combined following the general procedure. The product purified by
recrystallization from CH2Cl2 to afford 21. The structure of the complex was confirmed by
single crystal X-ray crystallography (0.708 g, 99 %). IR (νm.cm-1) = 3444, 3252, 3076, 2925,
2856, 1571, 1500, 1389, 1329, 1245, 1098, 1047, 1020, 784, 755, 674, 619. Anal. Calcd (%)
for CuC22H25N4O4Br: C 47.79, H 4.56, N 10.13. Found: C 47.83, H 4.37, N 10.18.
15

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4.1.3. 7. Copper N-benzyl-6-bromo-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate (22)
The general procedure was followed using 13 (0.200 g, 0.527 mmol) and copper(II) acetate
monohydrate (0.116 g, 0.570 mmol, 1.10 eq). After collection of the green powder, the product
22 (0.320 g, 99 %) was recrystallized by vapour diffusion from MeOD and ether. The structure
of the complex was confirmed by single crystal X-ray crystallography. IR (νm.cm-1) = 3428,
3246, 3084, 3029, 2999, 2925, 1577, 1564, 1526, 1498, 1482, 1391, 1330, 1244, 1204, 1166,
1124, 1096, 1045, 1018, 975, 930, 842, 813, 785, 750, 704, 675, 647, 617. Anal. Calcd (%)
for CuC23H21N4O4Br: C 49.25, H 3.77, N 9.99. Found: C 47.52, H 4.77, N 9.41.
4.1.3. 8. Copper N-((1S,3S)-adamantan-1-yl)-6-bromo-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-
amine acetate (24)
The general procedure was followed using 15 (0.150 g, 0.355 mmol) and copper(II) acetate
monohydrate (0.078 g, 0.390 mmol, 1.10 eq). The product was collected as a turquoise solid
(0.144 g, 67 %) and after recrystallization from MeOD and diethyl ether was found to be 24
from SCXRD. IR (νm.cm-1) = 3257, 3086, 2905, 2845, 1585, 1558, 1530, 1506, 1476, 1412,
1385, 1360, 1327, 1250, 1186, 1151, 1117, 1088, 1047, 1018, 924, 853, 806, 791, 762, 735,
706, 675, 646, 619. Anal. Calcd (%) for CuC24H29N4O4Br: C 51.62, H 4.83, N 9.26. Found: C
51.53, H 4.94, N 9.19.
4.1.3. 9. Copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine nitrate
(26)
The combination of an diethyl ether solution of 8 (0.300 g, 0.808 mmol) with Cu(NO3)2
(0.188g, 0.808 mmol, 1.00 eq) according to the general procedure resulted in the formation of
a lime green powder, 26 (0.439 g, 97 %) that was collected by filtration. A slow evaporation
recrystallization from a MeOH/CH2Cl2 solution provided suitable crystals of 26 for SCXRD
which confirmed the structure. IR (νm.cm-1) = 3327, 3094, 2924, 2854, 1610, 1568, 1479,
1452, 1410, 1302, 1275, 1246, 1161, 1113, 1097, 1069, 1009, 970, 893, 872, 808, 783, 750,
708, 675, 652. Anal. Calcd (%) for CuC18H19N6O6Br: C 38.69, H 3.43, N 15.04. Found: C
38.03, H 3.73, N 14.54.
4.1.3. 10. Copper N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate (27)
Combining imidazo[1,2-a]pyridine 7 (0.300 g, 1.074 mmol) and copper(II) acetate
monohydrate (0.214 g, 1.074 mmol, 1.00 eq) according to the general procedure resulted in
the formation of a green powder (0.455 g, 89 %). After vapour diffusion recrystallization from
MeOD and diethyl ether and characterisation by SXCRD the green powder was found to be
27. IR (νm.cm-1) = 3302, 3265, 3107, 3082, 3049, 2930, 2857, 1582, 1506, 1479, 1381, 1327,
1292, 1247, 1151, 1091, 1047, 1010, 941, 926, 850, 775, 758, 673, 646, 621. Anal. Calcd (%)
for CuC22H26N4O4: C 55.74, H 5.53, N 11.82. Found: C 55.82, H 5.34, N 11.49.
4.1.3. 11. Copper 6-chloro-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate
(28)
Imidazo[1,2-a]pyridine 9 (0.500 g, 1.530 mmol) and copper(II) acetate monohydrate (0.305 g,
1.530 mmol, 1.00 eq) were combined following the general procedure. A dark green powder
precipitated from the reaction and this was collected by gravity filtration and washed with ether
to afford 28. The structure of the complex was confirmed by single crystal X-ray
crystallography (0.789 g, 99 %). IR (νm.cm-1) = 3379, 3282, 3282, 3067, 2930, 2858, 1610,
1568, 1528, 1502, 1477, 1400, 1331, 1292, 1242, 1151, 1128, 1097, 1078, 1047, 1022, 877,
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822, 787, 756, 725, 679, 621. Anal. Calcd (%) for CuC22H25N4O4Cl: C 51.97, H 4.96, N 11.02.
Found: C 51.94, H 4.85, N 10.93.
4.1.3. 12. Copper 5-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate
(29)
The general procedure was followed using 10 (1.776 g, 4.784 mmol) and copper(II) acetate
monohydrate (0.955 g, 4.784 mmol, 1.00 eq) resulting in a green powder of 29. The structure
of the complex was confirmed by single crystal X-ray crystallography (2.238 g, 85 %) following
suitable crystal formation by slow evaporation from MeOD. IR (νm.cm-1) = 3414, 3287, 3080,
2924, 2855, 1576, 1485, 1391, 1334, 1306, 1256, 1159, 1099, 1047, 1022, 964, 930, 785,
758, 679, 648, 621. Anal. Calcd (%) for CuC22H25N4O4Br: C 47.79, H 4.56, N 10.13. Found: C
47.64, H 4.24, N 9.17.
4.1.3. 13. Copper 2-(5-bromopyridin-2-yl)-N-cyclohexylimidazo[1,2-a]pyridin-3-amine acetate
(30)
The combination of 11 (0.300 g, 0.808 mmol) with copper(II) acetate monohydrate (0.161 g,
0.808 mmol, 1.00 eq) according to the general procedure resulted in the formation of a green
solid (0.408 g, 35 %). Following slow evaporation crystallization from isopropyl alcohol, the
structure of the product 30 was confirmed. IR (νm.cm-1) = 3294, 3250, 3105, 3043, 2928,
2855, 1570, 1508, 1479, 1389, 1377, 1331, 1248, 1146, 1105, 1030, 1013, 949, 928, 841,
779, 752, 673, 619. Anal. Calcd (%) for CuC22H25N4O4Br: C 47.79, H 4.56, N 10.13. Found: C
46.12, H 5.67, N 9.47
4.1.3. 14. Copper 2-(5-chloropyridin-2-yl)-N-cyclohexylimidazo[1,2-a]pyridin-3-amine acetate
(31)
The general procedure was followed using imidazo[1,2-a]pyridine 6 (0.300 g, 0.918 mmol)
and copper(II) acetate monohydrate (0.183 g, 0.918 mmol, 1 eq). The resulting green powder
(0.408 g, 50 %) was found to be 31 following crystallization by slow evaporation from MeOD.
IR (νm.cm-1) = 3510, 3221, 3111, 3053, 2930, 2855, 1595, 1504, 1483, 1423, 1375, 1325,
1246, 1146, 1121, 1097, 1069, 1032, 1013, 928, 854, 826, 756, 681, 667, 617. Anal. Calcd
(%) for CuC22H25N4O4Cl: C 51.97, H 4.96, N 11.02. Found: C 51.05, H 4.95, N 10.45.
4.2. Crystal Structure Determination
Suitable crystals of the ligands for single crystal X-ray diffraction were grown from isopropyl
alcohol. Zinc-imidazo[1,2-a]pyridine crystals were grown from deuterated chloroform and the
copper-imidazo[1,2-a]pyridine crystals from deuterated methanol unless otherwise stated.
Following mounting of the crystals on a glass fibre, the intensity data for the crystals were
collected on a Bruker APEX II CCD area detector diffractometer with graphite monochromated
Mo-K radiation (50 kV, 30 mA) at 173K. The collection method involved ω-scans having a
width of 0.5°. Data reduction was carried out using SAINT+ version 6.02.6 software and
SADABS was used to make empirical absorption corrections [38]. The crystal structures were
solved by direct methods using SHELXS-97 [39]. Non-hydrogen atoms were first refined
isotropically followed by anisotropic refinement by full matrix least-squares calculations based
on F2 using SHELXL-97 [38]. C-bound H atoms were first located in the difference map, then
positioned geometrically and allowed to ride on their respective parent atoms, with thermal
displacement parameters 1.2 times of the parent C atom. Where possible, the coordinates and
isotropic displacement parameters of the N-bound and O-bound H atoms involved in hydrogen
bonding interactions were allowed to refine freely. Diagrams and publication material were
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generated using WinGX [39] and ORTEP-3 [41]. Crystallographic data of each crystal are
summarized in the Supplementary Information.
4.3. pK determination of metal complexes
The pK values for selected ligand and metal complexes were determined using UV-vis
spectroscopy. The spectra were recorded on either a Cary 100 UV-vis spectophotometer or
Cary 300 Bio UV-vis spectrophotometer, fitted with cuvette holders for 1 cm path length
cuvettes. Temperature was maintained at 25.0 ± 0.1 °C using a water-circulating bath. Spectra
were recorded with a spectral band width of 2 nm, signal averaging time of 0.1 s, data interval
of 0.5 nm and a scan rate of 300 nm.min-1. Collection of the absorbance spectra was
facilitated by the Scan application of the Cary WinUV software suite.[42] A 250 mL solution of
the selected compounds were made up by dissolving them to appropriate concentrations in 50
% MeOH/H2O solutions containing buffers NaH2PO3 and MES in a final concentration of 1
mM. After 12 h of equilibration at 23 °C, the solut ion was divided into 10 mL aquilots and the
pH of each vial adjusted individually. The baseline was obtained using 50% MeOH/H2O and
this solution remained in the reference cell throughout the experiment. pH measurements were
performed using a Metrohom High-precision 780 pH meter and Metrohm Unitrode combination
pH electrode. The electrode was calibrated using a 3 buffer calibration method with Metrohm
ready-to-use buffer solutions at pH 4, 7 and 9.
4.4. Data Analysis
SigmaPlot [43] was used to analyse the spectroscopic data to determine the pK of the
selected compounds. The free ligand structures were successfully fitted to a one pK model
(Eq. 1) and the zinc complexes were using a two pK model (Eq. 2). Due to the significant
differences between the absorption spectra of pK1 and pK2 of the copper complexes, the
complexes were either fitted with a two pK model or each pK was fitted separately using a
single pK model. The equations used to fit the spectral data can be found in the
Supplementary Information. The pK values were determined using the average K values and
the error calculated from the sum of weighted relative percentage error reciprocals.
4.5. Pharmacology
4.5.1. Evaluation of Anti-cancer Activity
4.5.1.1. Maintenance of cancer cell lines
The five cancer cell lines used in this study originated from breast cancer (MCF-7 and MDA-
MB-231), colorectal cancer (HT-29) and leukemia (K562 and HL-60). With the exception of the
HT-29 cell line, which was obtained from the Fox Chase Cancer Center, all cell lines were
obtained from the NCI and was authenticated by short tandem repeat (STR) analysis by
Inqaba Biotech, South Africa. In addition, screening for mycoplasma and bacterial infections
are routinely performed in our laboratory. Cells are observed daily by phase contrast
microscopy to ensure the absence of microbial contamination. The breast and colorectal
cancer cell lines were maintained as a monolayer in culture flasks whereas the leukemic cell
lines are maintained in suspension culture. MCF-7 and HT-29 cells were maintained in
Dulbecco’s Modified Eagles’s Medium (DMEM) supplemented with 10% (v/v) of foetal bovine
serum (FBS) obtained from Gibco. The MDA-MB-231 breast cancer line was maintained in a
1:1 (v/v) solution of DMEM and HAM-F12 media supplemented with 15% (v/v) FBS. The K562
and HL-60 cells were maintained in RPMI 1640 media supplemented with 10 % (v/v) FBS. All
culture media was obtained from Sigma. Cells were maintained under sterile conditions at 37
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°C in a humidified incubator under a normal atmosph ere supplemented with 5% CO2. All
procedures were carried out under aseptic conditions.
4.5.1.2. Cell viability assay
Cell viability was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide) assay as described by Mosmann et al. [31] with minor modifications.
Briefly, cells were seeded at predetermined cell numbers (dependent on cell type) in a 96 well
plate and allowed to adhere for 18 h before the addition of the test compounds. The K562 and
HL-60 leukaemic cells were exposed immediately to the test compounds. All cells were
exposed to the test compounds for a total of 48 h in a humidified incubator at 37 °C and 5%
CO2. At 46 h, 40 ꢀl of MTT (5 mg/mL in prepared in phosphate buffered saline) was added to
each well and allowed to incubate for 2 h at 37°C a s described above. This was followed by
centrifugation at 400 x g for 5 minutes and aspiration of the growth media. The insoluble
formazan crystals were solubilized by the addition of 200 µl of DMSO per well. The plates
were shaken at 1000 rpm in a plate shaker for 5 mins to ensure complete solubilisation of the
formazan crystals. The absorbance of each well was measured in a Labsystems iEMS micro-
plate reader at 540 nm and a reference wavelength of 690 nm. All data points were obtained in
quadruplicate and each experiment was repeated at least four times. The percentage cell
viability was calculated by expressing the absorbance of treated cells as a percentage of
untreated control cells. IC50 values were calculated by using the GraphPad Prizm version 5
software package.
4.5.2. The isolation of leukocytes from human blood
Whole blood was collected from human volunteers in heparin containing blood collection
tubes. Permission to collect 30 ml of whole blood from volunteers was obtained from the
Human Research Ethics Committee (Medical) of the University of the Witwatersrand;
clearance number M120669. White blood cells (leucocytes) were isolated under sterile
conditions from whole blood by differential centrifugation by using the Histopaque -1077
solution (Sigma-Aldrich). Briefly, a volume of blood was carefully layered (without mixing the
two phases) over an equal volume of Histopaque -1077 solution in a 50 mL centrifuge tube
and centrifuged for 30 mins at 250 × g at room temperature. The leucocytes collect at the
plasma/ Histopaque-1077 interphase and erythrocytes and granulocytes sediment to the
bottom of the tube. Following centrifugation the upper phase containing plasma and platelets
were aspirated and discarded. The leukocyte containing interphase was collected and washed
three times with 10 mL of phosphate buffered saline and centrifugation at 250×g for 10 mins at
room temperature. The leukocytes were then re-suspended in 10 mL RPMI 1640 media
containing 20% v/v FBS. This was followed by counting the cells on a haemocytometer after
the addition of trypan blue to determine the number and viability of the cells. The leukocytes
were seeded at a density of 40 000 cells/well and exposed to test compounds for 24 h before
the determination of their viability by the MTT assay as described in the previous section.
4.5.3. Cell morphology
The adherent cell lines (MCF-7, MDA-MB-231 and HT-29) were grown on sterile glass
coverslips which were placed in 6 well culture plates. Cells were seeded at a density of 50 000
cells/coverslip and allowed to grow overnight before the start of treatment. The following day
the media was replaced with fresh media containing the appropriate compound for testing.
Cells were typically incubated for 24 h, unless indicated otherwise, at 37 °C in a humidified
incubator with 5% CO2.
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The cells were viewed under phase contrast microscopy (Olympus CKX41 inverted
microscope) to determine if cells have detached from the glass surface and had to be
harvested from the culture media by centrifugation at 250 × g for separate staining. These
cells were washed twice with PBS before the addition of the staining solution. After the culture
media was removed, the coverslips, with remaining attached cells, were gently irrigated twice
with 3 mL of PBS. The coverslips were then flooded with 300 µl of a vital dye mixture
containing acridine orange (10 µg/ml), ethidium bromide (10 µg/mL) and Hoechst 33342 (5
µg/mL). This was followed by incubation at room temperature in the dark for 30 mins. The
coverslips were then washed with PBS and placed cell side downwards on a microscope slide.
The cells were viewed with an Olympus BX41 epifluorescent microscope under 100 and 400
times magnification using the appropriate filters for acridine orange and Hoechst 33342 and
ethidium bromide. Ethidium bromide that penetrated the cell membrane caused DNA to
fluoresce red under the Hoechst 33342 filter (Olympus U-MWU2) instead of blue. Acridine
orange was observed using the Olympus-UMWIB2 filter cube which allows observation of both
green and red fluorescence. In addition to staining nucleic acids green, acridine orange stains
acidic compartments a bright red colour and is useful for an early indicator for autophagic
vacuole formation [35]. Images were captured in the colour mode with a DP72 digital camera
with an ISO setting of 200 and a fixed exposure time for each fluorochrome (typically 30
milliseconds for the Hoechst 33342 stain and 50 milliseconds for acridine orange). Capturing
images in the colour mode provides a record for the differential staining of cells since the
penetration of ethidium bromide to the nucleus and binding to DNA is visible using the UV filter
cube for Hoechst 33342. Following capturing, the images were optimized for contrast and
brightness using the Olympus Cell Sense software package. In the interest of clarity the
images were cropped to compile Figures 11, 13 and 14. Phase contrast and Hoechst stained
images were combined to obtain merged images using the Olympus Cell Sense software
package. The raw uncropped images are available from the authors.
Acknowledgments
This work was supported by the Medical Research Council (MRC) of South Africa, the Blue
Skies Initiative of the National Research Foundation, Pretoria (South Africa), the Southern
African Biochemistry and Informatics for Natural Products Network (SABINA) and the
University of the Witwatersrand (Science Faculty Research and Health Sciences Faculty
Councils) as well as the McGill bequest to the Pharmacology Division. We also gratefully
acknowledge SABINA for a generous Fellowship to JD. The analytical services of
Stellenbosch University are thanked for providing the HRMS-MS spectroscopy services, as
well as some microanalyses. The University of Johannesburg is also thanked for providing a
microanalysis service.
Supporting Information
1
A PDF containing the H and 13C NMR spectra, basic crystallographic information for
selected compounds and an example of the pK data and calculations are provided in the
Supporting Information.
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SCHEMES
Scheme 1. General synthesis of imidazo[1,2-a]pyridines using Groebke-Blackburn-Bienaymé
methodology.
Scheme 2. General synthetic scheme of pyridyl imidazo[1,2-a]pyridines
FIGURES
Figure 1. Biologically active imidazo[1,2-a]pyridines
Figure 2. An example of the imidazo[1,2-a]pyridines previously synthesized
Figure 3. Imidazo[1,2-a]pyridines synthesized using the Groebke multicomponent reaction
Figure 4. Zinc imidazo[1,2-a]pyridine complexes
Figure 5. The structures of copper imidazo[1,2-a]pyridine complexes
Figure 6. The asymmetric units of metal-imidazo[1,2-a]pyridines 8, 16 and 21. Displacement
ellipsoids are shown at the 50% probability level. Hydrogen atoms not involved in hydrogen
bonding are omitted for clarity.
Figure 7. The asymmetric units of the dimeric copper imidazo[1,2-a]pyridines 30 and 31.
Displacement ellipsoids are shown at the 50% probability level. Hydrogen atoms are omitted
for clarity.
Figure 8. An example of the changes observed in the absorbance spectra at a varying pH’s
(on the left) and the change in the absorbance spectra at a particular wavelength (on
the right) for compounds 13 (A), 17 (B) and 22 (C).
Figure 9. Leucocyte cell viability when treated with 20 µM of selected imidazo[1,2-a]pyridines
and copper complexes for 24 h.
Figure 10. Leucocyte viability when treated with copper imidazo[1,2-a]pyridine 21 at varying
concentrations for 24 h. Leucocyte viability improves with lower concentrations of 21.
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Figure 11. The effect of imidazo[1,2-a]pyridine 21 on cell and nuclear morphology on MCF-7
and HL-60 cells after a 48 h exposure period indicates apoptotic and necrotic cell death. MCF-
7 and HL-60 cells were treated with 21 at a concentration of 1.5 µM and 2.0 µM, respectively.
Images were captured at a 400 × magnification and scale bars represent 20 micron. Legend:
N = normal nucleus; NN = necrotic nucleus; AN = apoptotic nucleus; LAN = late apoptotic
nucleus
Figure 12. The effect of the copper imidazo[1,2-a]pyridines 26, 27 and 29 on the cell and
nuclear morphology of MCF-7 cells shows the formation of cytoplasmic vacuoles, acidic
granules, deformed- and apoptotic nuclei. Cells were treated with 10 µM of the test
compounds for 24 h. Images were captured at a 400 × magnification and scale bars represent
20 micrometers. Legend: N = nucleus; CN = contracted nucleus; AN = apoptotic nucleus; DN
= deformed nucleus; AG = acidic granules; F = filopodia; V = vacuoles.
Figure 13. The formation of cytoplasmic vacuoles do not co-localize with the acidic granules.
MCF-7 cells were treated with 10 µM of 21 for 24 hours. Images were captured at a 400 x
magnification and scale bars represent 20 micrometers. Legend: AG = acidic granules, V =
Cytoplasmic vacuoles, N = nucleus.
Figure 14. The effect of 29 on MCF-7 cells at a concentration of 5 µM for 24 hours indicates
that the formation of apoptotic nuclei and acidic granules is a dose dependent effect. Images
were captured at a 400 × magnification and scale bars represent 20 micrometers. Legend: AN
= apoptotic nucleus; AG = acidic granules
TABLES
Table 1. pKa values for selected imidazo[1,2-a]pyridines and their copper and zinc complexes
Table 2. IC50 values for active compounds (µM)
EQUATIONS
ꢃ
ꢅꢆ
ꢀꢂ ꢄ + ꢀꢇꢈꢂ
ꢀꢁ =
(Eq. 1)
(Eq. 2)
ꢃ
ꢅꢆ
ꢈꢂ + ꢄ
ꢃ
ꢅꢆꢇ
ꢃ
ꢅꢆ
+ ꢀꢇꢈꢂ ꢄ + ꢀꢉꢈꢂꢈꢇ
ꢀꢂ ꢄ
ꢀꢁ =
ꢃ
ꢅꢆꢇ ꢅꢆ
ꢃ
+ ꢈꢂ ꢄ + ꢈꢂꢈꢇ
ꢄ
26

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TABLES
Table 1. pKa values for selected imidazo[1,2-a]pyridines and their copper and zinc
complexes
Compound
pK1
pK2
-
8
3.9 ± 0.1
4.1 ± 0.1
4.00 ± 0.07
3.9 ± 0.1
3.2 ± 0.1
3.5 ± 0.1
2.81 ± 0.08
3.50 ± 0.07
3.5 ± 0.1
3.6 ± 0.1
3.5 ± 0.3
4.4 ± 0.2
13
14
15
16
17
18
19
20
21
22
24
-
-
-
6.76 ± 0.08
6.30 ± 0.06
7.5 ± 0.2
6.35 ± 0.07
6.73 ± 0.08
8.8 ± 0.2
5.6 ± 0.3
5.2 ± 0.3
Table 2. IC50 values for active compounds (µM)
MDA-MB-
231
Compound
HT-29
MCF-7
K562
HL-60
0.33 ±
0.04
Camptothecin
8.0 ± 0.8
0.32 ± 0.01
2.6±0.24 0.005±0.001
8
9
NA
NA
NA
NA
35.4 ± 0.4
23 ± 3
NA
NA
36.20±4.1
NA
NA
29.2 ± 0.7
NA
NA
15
18
49 ± 5
38.3 ± 0.5
26.7 ±2.2
NA
NA
NA

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19
21
13 ± 1
0.79 ±
NA
NA
NA
NA
0.62 ± 0.03
28 ± 2
1.9±0.9
2.2±0.5
0.02
22
24
6.2 ± 0.1
2.7 ± 0.5
6.80 ±
3.7 ± 0.5
NA
30 ± 2
NA
7.7±0.5
NA
6.5±1.0
NA
26
1.1 ± 0.3
19 ± 1
9 ± 1
12 ± 4
0.08
27
28
29
30
31
0.8 ± 0.2
8.1 ± 0.2
1.6 ± 0.3
NA
1.1 ± 0.6
3.8 ± 0.9
1.20 ± 0.08
8.2 ± 0.4
9.2 ± 0.9
NA
3 ± 1
3.0 ± 0.4
6.0 ± 0.4
4 ± 2
3 ± 1
5 ± 2
19 ± 1
2.4 ± 0.4
23 ± 4
18 ± 2
2.1 ± 0.3
13 ± 3
± 2
7.4 ± 0.4
10.6 ± 0.7
NA – IC50 values were not determined as the compounds were not found to be
significantly active at 100 µM