Synthesis and toxicology of p-phosphonic acid calixarenes and O-alkylated analogues as potential calixarene-based phospholipids

Article abstract of DOI:10.1002/cplu.201100081

A series of p-phosphonic acid calix[n]arenes 1a-g bearing hydroxy groups, n=4 and 5, or alkyl chains of varying lengths at their lower rim, n=4, were synthesised, and in vitro assessments of their effect on cell viability in rat PC12 cells and primary cultures of mixed retinal cells were conducted. Compounds 1a and 1g, bearing hydroxy groups at their lower rim, displayed lower toxicity towards PC12 cells than their alkylated counterparts; concentrations of up to 1 mgmL^-1 did not affect the number of viable cells. Mixed retinal cultures showed a higher susceptibility towards toxic effects for all calixarenes tested. Compound 1b self-assembles into nanofibres from a toluene solution, and 1c assembles into micelles from an aqueous solution, with these nanoscale architectures showing potential applications for various biological roles, such as solubilising p-nitrophenol and curcumin.

Full text of DOI:10.1002/cplu.201100081

DOI: 10.1002/cplu.201100081
Synthesis and Toxicology of p-Phosphonic Acid
Calixarenes and O-Alkylated Analogues as Potential
Calixarene-Based Phospholipids
Adam D. Martin,[a] Emma Houlihan,[b] Natalie Morellini,[b] Paul K. Eggers,[a] Eliza James,[a]
Keith A. Stubbs,[c] Alan R. Harvey,[d] Melinda Fitzgerald,[b] Colin L. Raston,*[a] and
Sarah A. Dunlop*[b]
A series of p-phosphonic acid calix[n]arenes 1a–g bearing hy-
droxy groups, n=4 and 5, or alkyl chains of varying lengths at
their lower rim, n=4, were synthesised, and in vitro assess-
ments of their effect on cell viability in rat PC12 cells and pri-
mary cultures of mixed retinal cells were conducted. Com-
pounds 1a and 1g, bearing hydroxy groups at their lower rim,
displayed lower toxicity towards PC12 cells than their alkylated
counterparts; concentrations of up to 1 mgmLÀ1 did not affect
the number of viable cells. Mixed retinal cultures showed
a higher susceptibility towards toxic effects for all calixarenes
tested. Compound 1b self-assembles into nanofibres from a tol-
uene solution, and 1c assembles into micelles from an aque-
ous solution, with these nanoscale architectures showing po-
tential applications for various biological roles, such as solubil-
ising p-nitrophenol and curcumin.
Introduction
Calix[n]arenes are versatile macrocyclic compounds comprised
of phenolic units linked by methylene bridges,[1] where ‘n’ can
vary from 4 to 20.[2] These compounds have been used in
a wide variety of applications including sensor technology,[3]
stabilisation of nanoparticles,[4] phase-transfer catalysts,[5] and
guest encapsulation.[6,7] The widespread use of calixarenes re-
lates in part to the ease at which their upper and lower rims
can be modified with a wide range of different functionali-
ties.[8,9]
dipeptides and bis(hydrxoymethyl)phosphonic acid calix[4]ar-
enes.[16] The ability of calixarenes to effectively encapsulate a di-
verse range of molecules, coupled with their documented low
toxicity, makes them attractive candidates for a wide range of
drug delivery systems. The biochemistry of various calix[n]ar-
enes, including their complexation towards biologically active
molecules, toxicity, and use in biosensors has already been
covered by an in-depth Review on sulfonated calixarenes,[17]
and
enes.[18,19]
Recently a high yielding synthesis of p-phosphonic acid cal-
a more general Review concerning anionic calixar-
Calix[n]arenes have also recently featured in a variety of ex-
amples in the field of biotechnology owing to their apparent
low toxicity. For instance, parent tert-butylcalix[4]arenes at-
tached to human antibodies do not provoke an immune re-
sponse in test subjects.[10] Solid-lipid nanoparticles (SLNs) fabri-
cated using amphiphilic calix[4]arenes bearing long alkyl
chains incorporating either hydroxyl or phosphonate groups
have no haemolytic properties.[11] This is thought to be due to
the small cavity size of the calix[4]arenes, given that p-sulfona-
to calix[8]arenes with their significantly larger cavity display
a much higher haemolytic effect than the calix[4 and 6]arene
analogues.[12] Cellular uptake of fluorescent, water soluble cal-
ix[4]arene-based probes has also been demonstrated using
a nonspecific uptake mechanism not linked to either of the
main endocytic pathways, resulting in accumulation of the
probe within the cell cytoplasm.[13] Calixarenes have also
proved to be effective in the encapsulation of various biologi-
cally relevant molecules, including the encapsulation and sub-
sequent extraction of cytochrome c by carboxylic functional-
ised p-tert-octylcalix[6] and [8]arenes,[14] encapsulation of the
model photosensitiser chlorine e6 by micelles of PEGylated cal-
ix[4]arene,[15] and the formation of stable complexes between
ix[n]arenes was established, as a six-step procedure.[20,21] This
new class of calixarene has tuneable properties; examples in-
clude the binding of calcium ions in the solid state[22] and the
[a] Dr. A. D. Martin, Dr. P. K. Eggers, Dr. E. James, Prof. C. L. Raston
Centre for Strategic Nano-Fabrication, The University of Western Australia
35 Stirling Hwy, Crawley 6009 (Australia)
Fax: (+61)864888683
E-mail: colin.raston@uwa.edu.au
[b] E. Houlihan, Dr. N. Morellini, Asst. Prof. M. Fitzgerald, Prof. S. A. Dunlop
School of Animal Biology, The University of Western Australia
35 Stirling Hwy, Crawley 6009 (Australia)
Fax: (+61)864887527
E-mail: sarah.dunlop@uwa.edu.au
[c] Dr. K. A. Stubbs
School of Biomedical, Biomolecular and Chemical Sciences
The University of Western Australia
35 Stirling Hwy, Crawley 6009 (Australia)
[d] Prof. A. R. Harvey
School of Anatomy and Human Biology, The University of Western Australia
35 Stirling Hwy, Crawley 6009 (Australia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201100081.
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Biochemistry of Calixarenes
encapsulation of the potent anticancer agent carboplatin.[23]
p-Phosphonic acid calixarenes bearing long C18 alkyl chains at
their lower rim have also been synthesised and shown to self-
assemble into uniform nanofibres.[24] The phosphonate ester of
calix[4]arene O-alkylated with C18 chains is of particular interest
as it shows a bilayer arrangement close to the size of cellular
phospholipid bilayers.[25] As such, these phosphonated calixar-
enes may be useful for a range of biological applications, in-
cluding the delivery of therapeutic agents to an injury site. In
an effort to understand the biological role of the molecules
and to gain insight into any potential toxicity, we prepared
a series of p-phosphonic acid calix[n]arenes (where n=4, 5),
bearing hydroxyl groups or alkyl chains at their lower rim, and
have investigated their self-assembly prowess. The effects that
these compounds have on cell viability in vitro on both an im-
mortalised cell line and freshly isolated primary cultures have
also been assessed.
tained in overall yields of 50–60%.[20,25] Compounds 1a–g are
soluble in water at high pH (>10) and in DMSO. Only com-
pounds 1a–c and 1g are readily soluble at neutral pH. Com-
pounds 1a–f were prepared with the molecules locked in the
symmetrical C4v cone conformation, with the conditions used
for the alkylation step predetermining the formation of the
cone conformer.
Effects of p-phosphonic acid calix[n]arenes on cell viabilities
To determine whether or not the calix[n]arenes are in any way
toxic, the viability (using a LIVE/DEAD cell kit) of PC12 and
mixed retinal cultures was assessed at a range of calix[n]arene
concentrations: 0.001, 0.01, 0.1, 0.3, and 3 mgmLÀ1. Both the
concentrations at which viability was significantly reduced and
the approximate concentrations at which viability was reduced
by 50% (IC50 values) are displayed in Table 1, with representa-
Results and Discussion
Table 1. Concentrations (mgmLÀ1) where a significant decrease in cell vi-
ability is observed and (in parentheses) where viability is inhibited by ap-
proximately 50%, for calixarenes 1a–g in both PC12 and mixed retinal
cells.
Synthesis of p-phosphonic acid calixarenes
p-Phosphonic acid calix[n]arenes 1a and 1g, Scheme 1, were
prepared according to a six-step published procedure from
readily available starting materials.[20] The new lower rim
O-alkyl analogues 1b–f, Scheme 1, were prepared by a variation
Calixarene
PC12 cells
Mixed retinal cells
24h
72h
24h
72h
1a
1b
1c
1d
1e
1 f
>3.0 (>3.0)
0.3 (1.0–3.0)
1.0 (1.0)
0.3 (1.0)
1.0 (1.0)
3.0 (1.0–3.0)
0.1 (0.3)
1.0 (1.0)
1.0 (1.0)
1.0 (1.0)
1.0 (1.0)
0.1 (0.3)
1.0 (1.0)
0.3 (0.3)
1.0 (1.0)
3.0 (1.0)
0.1 (0.1)
0.01 (0.01)
0.3 (0.3)
0.1 (0.3)
1.0 (0.3–1.0)
0.01 (0.1)
0.01 (0.01)
1.0 (0.3–1.0)
1.0 (1.0)
3.0 (>3.0)
0.1 (0.1)
3.0 (1.0–3.0)
1g
tive data in Figure 1. We have established that both the length
of the alkyl chain and the number of phenolic units in the
macrocycle play an important role in the toxicity profiles of
these compounds towards rat pheochromocytoma neural pro-
genitor (PC12) cells. Relatively high concentrations (1 mgmLÀ1
)
of both 1a and 1g were tolerated by PC12 cells, whereas 1b
and 1 f displayed an increased toxicity (toxic at 0.1 mgmLÀ1).
Compounds 1c, 1d, and 1e, with intermediate alkyl chain
lengths, were less toxic towards PC12 cells than for the shorter
and longer chain lengths. Concentrations of up to 1.0 mgmLÀ1
were needed to cause a significant reduction in the number of
viable cells, as summarised in Figure 1 and Table 1. Another
trend observed was the decrease in the number of live cells as
the calixarene concentration increased, 3 mgmLÀ1 being toxic
for all calixarenes tested.
Scheme 1. p-Phosphonic acid calix[n]arenes used in the toxicology studies,
along with their associated synthetic procedure.
Mixed retinal cells were generally more susceptible than
PC12 cells to the toxic effects associated with 1a–g. Com-
pounds 1a and 1g, which showed the least toxicity towards
PC12 cells, nevertheless resulted in a significant decrease in
the number of viable cells in the mixed retinal cultures at con-
centrations as low as 0.01 and 0.1 mgmLÀ1 for 1a and 1g, re-
spectively. The addition of alkyl chains to the calixarenes had
little effect on their toxicity towards the mixed retinal cells,
of the protocol for other O-alkyl compounds, where R=CH3,
n-C4H9, n-C18H37.[22] This variation shortens the synthetic proto-
col to five steps, replacing the acetylation step with an alkyla-
tion, thereby eliminating the deacetylation step. The presence
of alkyl chains also slightly lowers the overall yields relative to
the pristine calixarenes, R=H, with all compounds being ob-
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309

C. L. Raston, S. A. Dunlop et al.
bilayer arrangements present in the solid state, whereas com-
pound 1g forms molecular capsules in solution and nanofibres
in the solid state, with a controlled encapsulation and release
of carboplatin drug molecules.[20,23] The amphiphilic calixar-
enes, 1b–f, were designed as phospholipid mimics. They have
potential for forming micelles and/or vesicles with the phos-
phonate groups pointing either towards the solvent or to-
wards themselves, depending on the polarity of the solvent
and concentration effects.
Atomic force microscopy (AFM) established that com-
pound 1b assembles into nanofibres from toluene, their
height varying between 5–8 nm and 45–55 nm, Figure 2. The
smaller nanofibres, Figure 2b, are bridged by circular struc-
tures, with a residual solvent layer visible around the smaller
fibres. Nanofibre formation is not observed in areas devoid of
Figure 1. Effects of compounds 1a--f on PC12 cell viabilities (meanÆSEM)
after 72 h in culture and representative images of untreated and treated
PC12 cells. Statistically significant reductions in viability of cultures treated
with calixarene preparations compared to untreated cells are indicated by *
(pꢀ0.05), scale bar=100 mm.
with toxic effects generally observed following treatment be-
tween 0.1 and 0.01 mgmLÀ1 after 72 hours (Table 1). An excep-
tion to this was 1d, where no decrease in the number of
viable cells was recorded at a concentration of 0.1 mgmLÀ1
after 24 hours and at a concentration of 0.3 mgmLÀ1 after
72 hours (Table 1). Although some preparations showed a de-
crease in the concentration required for reduced viability as
time passed, there was no consistent effect to indicate a time-
dependent toxicity. However, longer term studies are impor-
tant to address the possibility of chronic toxicity.
It is important to note that mixed retinal cultures are com-
prised of several cell types including retinal ganglion cells
(neurons) and astrocytes. Although retinal ganglion cells will
not replicate in culture, astrocytes may undergo several popu-
lation doublings over a 72 hours culture period. As such, the
decreased number of viable retinal cells may be a composite
of increased proliferation of astrocytes and loss of retinal gan-
glion cells and perhaps other neurons. Accordingly, the varia-
tion between the different calix[n]arenes in terms of greater or
less toxicity after 24 or 72 hours in culture may be due to dif-
ferential effects on the balance of astrocyte proliferation and
neuronal loss. Nevertheless, the increased toxicity of some of
the calix[n]arene preparations towards mixed retinal cultures
indicates that the preparations cannot all necessarily be
deemed safe for in vivo use.
Figure 2. AFM micrographs of nanofibres of 1b formed from a toluene solu-
tion, (a) and (b), and nanocrystals formed from an aqueous solution (c),
along with their powder
X-ray diffraction pattern for the compound in (c) and (d). AFM scale bars are
500 nm, 1 mm, and 500 nm for (a), (b) and (c), respectively.
this layer. The appearance of fibre ends on the larger fibres,
Figure 2a, suggests that the larger nanofibres have a stepwise
growth mechanism similar to crystal growth, with each “step”
corresponding to a height of 2–3 nm. This step-wise growth
was also seen in the formation of nanocrystals of 1b, Fig-
ure 2c, from an aqueous solution of the compound deposited
onto a mica substrate. Repeated attempts to form large crys-
tals of 1b were unsuccessful, however, the powder X-ray dif-
fraction pattern of 1b, Figure 2d, shows some crystalline mate-
rial offset by the amorphous material presumably associated
with inefficient packing of alkyl chains, as disordered species.
This is one line of evidence indicating that the alkane chains
are in the fluid state at room temperature and is supported by
the ease at which 1c forms micelles at room temperature. Mi-
celles of 1c readily form by dissolving the compound in an
aqueous solution at pH>9. Decreasing the pH to 7 or lower
produces micelles that have a stability of greater than weeks
at concentrations as high as 30 mgmLÀ1 and as low as
Self-assembly of p-phosphonic acid calix[n]arenes
Two different classes of p-phosphonated calixarenes were pre-
pared, the amphiphilic calix[4]arenes with n-alkane chains, 1b–
f, and calix[4,5]arenes devoid of such chains, 1a and 1g,
Scheme 1. Accordingly, different self-assembly outcomes are
inevitable. In this context, we have previously shown that the
pristine calix[4,5]arenes themselves, 1a and 1g, assemble into
distinctly different structures. Compound 1a forms nanorafts in
solution with a bilayer arrangement of calixarenes, with similar
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Biochemistry of Calixarenes
0.3 mgmLÀ1. Micelles and vesicles generally require the alkane
chains of the amphiphiles to be fluid or “above main lamellar
chain-melting phase transition temperature” in order for the
amphiphiles to form a close packed nonpolar system to the ex-
clusion of water,[26]
The micelles of 1c formed at basic pH in the presence of p-
nitrophenol result in a close to colourless solution. This obser-
vation indicates that 1c does not encapsulate the solution it
was formed in, as this would be indicated by the acidified solu-
tion maintaining the bright yellow colour of the original basic
solution, owing to the p-nitrophenolate anion. Additional evi-
dence for the formation of micelles without encapsulating any
solvent comes from the toxicology data shown below.
Treatment of PC12 cells with 1 mgmLÀ1 of 1c in micelle
form resulted in only an approximately 25% decrease in cell vi-
ability compared to an approximately 75% decrease following
treatment with 1 mgmLÀ1 of 1c in monomeric form (Figure 1
and Figure 4). This finding indicates that the toxicity of the pre-
formed micelles is lower than for 1c in monomeric form, as
seen in Figure 1. If 1c encapsulated some of the basic pH sol-
vent it was formed in, then an increase in toxicity would be ex-
pected, not a decrease. This apparent decrease in toxicity may
also be a result of the preformed micelles of 1c having a lower
probability of integrating themselves into and subsequently
disrupting the bilayer arrangement of the cells than monomer-
ic 1c.
Micelle formation
As mentioned in the previous section, it appears that 1c spon-
taneously forms micelles at a pH>9. Producing micelles from
amphiphilic calix[4]arenes by controlling the pH value is
a known technique and there are a number of examples
where this class of compound has been shown to form mi-
celles.[27] Further, the compounds in this paper were designed
as phospholipid mimics and thus the formation of the micelles
was expected. Herein, we give evidence that 1c forms micelles
without encapsulating the high-pH and hence toxic solvent.
The size of the micelles was independent of both the adjust-
ed final pH value and concentration and had mean diameter
between 4.2 and 5.6 nm, as measured by dynamic light scatter-
ing (DLS). This correlates with slightly larger than twice the
length between the phosphonate group and the terminal
methyl group of a straightened alkane chain of 1c (1.9 nm as
estimated by using Chem3D). Thus, the DLS data give a size
which only allows the superstructures formed by 1c to be mi-
celles.
The colorimetric test gives evidence that there is no solvent
stored in a pocket within the superstructures of 1c. As shown
by Figure 3a, p-nitrophenol forms a bright yellow solution at
basic pH but is colourless at acidic pH. Figure 3b shows the
solutions of p-nitrophenol alone, p-nitrophenol plus 1c in mi-
cellular form (dissolved in a basic solution of water and p-nitro-
phenol), and 1c in micellular form alone at a pH<3.
Figure 4. Effects of preformed micelles of 1c on PC12 cell viabilities
(meanÆSEM) after 72 h in culture and representative images of untreated
and treated PC12 cells (objective 40ꢁ). Statistically significant reductions in
viability of cultures treated with calixarene preparations compared to un-
treated cells are indicated by * (pꢀ0.05). Scale bar=100 mm.
The novelty of the micelles of 1c is their stability at low pH.
Compound 1c is only sparingly soluble in water at a pH<7.
However, once the micelles are formed they maintain their sta-
bility in water to pH<1. The advantage of this is the similarity
in the solubility profile to known antioxidants such as curcu-
min. This compound is known to be soluble under basic condi-
tions but precipitates at low pH. Therefore, if a basic solution
of 1c and curcumin is prepared and the pH subsequently de-
creased to pH 7, the antioxidant is forced to intercalate into
the micelle, as illustrated in Figure 3c. This solution of curcu-
min and 1c displays remarkable stability. Both the curcumin
and 1c remained soluble for several months whereas solutions
of curcumin alone were only sparingly soluble at pH<9. The
stability of this system has many possible advantages, such as
Figure 3. p-Nitrophenol (12 mgmLÀ1) at pH 14 (a) and at a pH 3 after mi-
celle-formation (b), from left to right solutions of p-nitrophenol (12 mgmLÀ1
)
alone, p-nitrophenol (12 mgmLÀ1) plus 1c (7 mgmLÀ1) and 1c (7 mgmLÀ1
)
alone and at pH 6.5 after micelle formation (c), left curcumin (1 mgmLÀ1
alone and right curcumin (1 mgmLÀ1) plus 1c (7 mgmLÀ1).
)
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C. L. Raston, S. A. Dunlop et al.
2922 (s), 1594 (m), 1464 (m), 1404 (w), 1273 (m), 1128 (m), 983
remaining viable at a pH value lower than that of the stomach
and higher pH value than the intestines, with implications in
oral delivery methods. However, the activity of the antioxidant
carried within these micelles and the location where 1c accu-
mulates has yet to be tested but is the subject of ongoing re-
search, along with other polyphenol antioxidants such as re-
sveratrol.[28]
1
(m) cm-1; H NMR ([D6]DMSO, 258C, 400 MHz) d=6.96 (s, 8H, ArH),
6.27 (br. s, 8H, OH), 4.29 (d, J=13.5 Hz, 4H, ArCH2 Ar), 3.77 (t, J=
7.5 Hz, 8H, OCH2), 3.22 (d, J=13.5 Hz, 4H, ArCH2 Ar), 1.77 (m, 8H,
CH2), 1.26–1.40 (br. s, 56H, CH2), 0.78 ppm (t, J=7.6 Hz, 12H, CH3);
13C NMR ([D6]DMSO, 258C, 400 MHz) d=158.56, 134.19, 130.75,
74.89, 31.54, 30.56, 29.79, 29.52, 29.11, 29.08, 26.17, 22.19,
13.55 ppm; 31P NMR ([D6]DMSO, 258C, 300 MHz) d=16.85 ppm;
HRMS (FAB): m/z calcd for C68H108O16P4 [M+H+]: 1275.4521; found:
1275.4537.
Conclusion
5, 11, 17, 23-tetra(dihydroxyphosphoryl) 25, 26, 27, 28-tetrado-
decoxycalix[4]arene (1e): m.p.>3008C (decomp); IR (KBr) 3411 (s),
2922 (s), 1605 (m), 1466 (m), 1405 (w), 1273 (m), 1127 (m), 995
We have synthesised a series of new amphiphilic calix[4]arenes
bearing p-phosphonic acid groups at their upper rim and alkyl
chains at their lower rim. The toxicology of these phospholipid
mimics has been investigated, with mixed retinal cells proving
to be more susceptible to the toxic effects of 1a--g than PC12
cells. In the presence of hydroxy groups at the calixarene
lower rim, toxicity is decreased and concentrations of at least
1.0 mgmLÀ1 can be tolerated in PC12 cells for 1a or 1g. Fur-
thermore, the self-assembly of these phospholipid mimics was
studied, with 1c forming micelles of 4–5 nm diameter which
are stable over a large range of pH values and are also able to
intercalate curcumin, as a model antioxidant. These micelles
also show an apparent decrease in toxicity towards PC12 cells
compared to their monomeric analogues, thus making them
possible candidates for targeted drug delivery.
1
(m) cm-1; H NMR ([D6]DMSO, 258C, 400 MHz) d=6.95 (s, 8H, ArH),
6.19 (br. s, 8H, OH), 4.30 (d, J=13.4 Hz, 4H, ArCH2 Ar), 3.87 (t, J=
7.2 Hz, 8H, OCH2), 3.24 (d, J=13.4 Hz, 4H, ArCH2 Ar), 1.80 (m, 8H,
CH2), 1.20–1.31 (br. s, 72H, CH2), 0.79 ppm (t, J=7.5 Hz, 12H, CH3);
13C NMR ([D6]DMSO, 258C, 400 MHz) d=158.54, 134.21, 130.76,
74.89, 31.55, 30.47, 29.80, 29.48, 29.12, 29.07, 26.15, 22.21,
13.65 ppm; 31P NMR ([D6]DMSO, 258C, 300 MHz) d=17.02 ppm;
HRMS (FAB): m/z calcd for C76H124O16P4 [M+H+]: 1417.7918, found:
1417.7990.
5, 11, 17, 23-tetra(dihydroxyphosphoryl) 25, 26, 27, 28-tetrate-
tradecoxy-calix[4]arene (1 f): m.p.>3008C (decomp); IR (KBr) 3412
(s), 2922 (s), 1617 (m), 1466 (m), 1405 (w), 1273 (m), 1127 (m), 999
1
(m) cm-1; H NMR (CDCl3, 258C, 400 MHz) d=7.57 (s, 8H, ArH), 6.30
(br. s, 8H, OH), 4.28 (d, J=13.6 Hz, 4H, ArCH2 Ar), 3.76 (t, J=7.4 Hz,
8H, OCH2), 3.09 (d, J=13.6 Hz, 4H, ArCH2 Ar), 1.69 (m, 8H, CH2),
1.12–1.38 (br. s, 88H, CH2), 0.74 ppm (t, J=7.3 Hz, 12H, CH3);
13C NMR (CDCl3, 258C, 400 MHz) d=158.56, 134.23, 130.77, 74.91,
31.75, 30.49, 29.72, 29.66, 29.62, 29.54, 29.22, 22.49, 13.94 ppm;
31P NMR (CDCl3, 258C, 300 MHz) d=18.93 ppm; HRMS (FAB): m/z
calcd for C92H156O16P4 [M+H+]: 1642.0422; found: 1642.0380.
Experimental Section
Synthesis of p-phosphonic acid calixarenes
Phosphonic acid calixarenes 1b–f were synthesised according to
a modified literature procedure.[17,18] Starting materials were ob-
tained from Sigma–Aldrich and were used without purification. Cal-
ixarenes 1a and 1g have been previously reported.[22]
Cell culture
Rat pheochromocytoma cells (PC12) were obtained from the Mis-
sissippi Medical Centre (Jackson, Mississippi, USA) and grown in
RPMI medium supplemented with 10% horse serum (HS), 5%
foetal bovine serum (FBS), 2 mm L-glutamine, 2 mm penicillin-
streptomycin, 1 mm MEM sodium pyruvate, and 0.1 mm MEM non-
essential amino acids (GIBCO, Invitrogen, Carlsbad, California, USA).
Cells were grown on flasks coated with poly-l-lysine (PLL;
10 mgmLÀ1; Sigma, St Louis, Missouri, USA) and housed in an incu-
bator at 378C, 5% CO2. PC12 cells were seeded at 2ꢁ105 cellsmLÀ1
for experiments assessed at 24 h and 1ꢁ105 cellsmLÀ1 for experi-
ments assessed at 72 h, in 96-well plates coated with PLL as above.
5, 11, 17, 23-tetra(dihydroxyphosphoryl) 25, 26, 27, 28-tetrahex-
oxycalix[4]arene (1b): m.p.>3008C (decomp); IR (KBr) 3437 (s),
2922 (s), 1594 (m), 1460 (m), 1271 (m), 1125 (m), 998 (m) cm-1;
1H NMR ([D6]DMSO, 258C, 400 MHz) d=6.97 (s, 8H, ArH), 6.04 (br.
s, 8H, OH), 4.35 (d, J=13.5 Hz, 4H, ArCH2 Ar), 3.85 (t, J=7.2 Hz, 8H,
OCH2), 3.27 (d, J=13.5 Hz, 4H, ArCH2 Ar), 1.83 (m, 8H, CH2), 1.24–
1.35 (br. s, 24H, CH2), 0.87 ppm (t, J=7.5 Hz, 12H, CH3); 13C NMR
([D6]DMSO, 258C, 400 MHz) d=158.56, 134.21, 130.77, 74.89, 31.57,
31.49, 30.18, 29.80, 25.94, 25.50, 22.35, 13.86 ppm; 31P NMR
([D6]DMSO, 258C, 300 MHz) d=16.17 ppm; HRMS (FAB): m/z calcd
for C52H76O16P4 [M+H+]: 1081.4106; found: 1081.4184.
Mixed retinal cultures were prepared as follows. Rats were main-
tained and treated in accordance with National Health and Medical
Research Council (NHMRC) Australian Code of Practice for the Care
and Use of Animals for Scientific Purposes and as approved by the
University of Western Australia Animal Ethics Committee. Piebald
Virol Glaxo (PVG) hooded rat pups age P0-P3 (Animal Resources
Centre, Murdoch, Western Australia) were terminally euthanized
(by overdose with Euthal). Retinae were isolated and incubated
with papain (165 unitsmLÀ1; Worthington Biochemical Corporation,
Lakewood, New Jersey, USA), l-cysteine (5 mg; Sigma, St Louis,
Missouri, USA), and DNase (10,000UmLÀ1; Sigma, St Louis, Missouri,
USA) in HBSS (10 mL) for 30 min at 378C, gently triturated in Neu-
robasal-A cell media, supplemented with 10% FBS and 2 mm L-glu-
tamine (GIBCO, Invitrogen, Carlsbad, California, USA) using a sterile
Pasteur pipette and filtered through sterile nylon gauze. Mixed reti-
5, 11, 17, 23-tetra(dihydroxyphosphoryl) 25, 26, 27, 28-tetraoc-
toxycalix[4]arene (1c): m.p.>3008C (decomp); IR (KBr) 3413 (s),
2922 (s), 1618 (m), 1460 (m), 1384 (w), 1271 (m), 1125 (m), 1007
1
(m) cm-1; H NMR ([D6]DMSO, 258C, 400 MHz) d=6.96 (s, 8H, ArH),
6.32 (br. s, 8H, OH), 4.31 (d, J=13.3 Hz, 4H, ArCH2 Ar), 3.84 (t, J=
7.6 Hz, 8H, OCH2), 3.26 (d, J=13.3 Hz, 4H, ArCH2 Ar), 1.82 (m, 8H,
CH2), 1.22–1.38 (br. s, 40H, CH2), 0.83 ppm (t, J=7.5 Hz, 12H, CH3);
13C NMR ([D6]DMSO, 258C, 400 MHz) d=158.59, 134.19, 130.74,
74.87, 31.52, 30.22, 29.96, 29.56, 29.22, 29.02, 26.02, 22.21,
13.78 ppm; 31P NMR ([D6]DMSO, 258C, 300 MHz) d=16.21 ppm;
HRMS (FAB): m/z calcd for C60H92O16P4 [M+H+]: 1193.5414; found:
1193.5445.
5, 11, 17, 23-tetra(dihydroxyphosphoryl) 25, 26, 27, 28-tetrade-
coxycalix[4]arene (1d): m.p.>3008C (decomp); IR (KBr) 3437 (s),
312
www.chempluschem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2012, 77, 308 – 313

Biochemistry of Calixarenes
nal cultures were seeded at 8ꢁ105 cellsmLÀ1 for experiments as-
sessed at 24 h and 5ꢁ105 cellsmLÀ1 for experiments assessed at
72 h, in 96-well plates precoated with PLL (10 mgmLÀ1) and laminin
(10 mgmLÀ1; Invitrogen, Carlsbad, California, USA).
sistance of the Australian Microscopy & Microanalysis Research
Facility at the Centre for Microscopy, Characterisation & Analysis,
which is funded by The University of Western Australia, State and
Commonwealth Governments. M.F. is supported by the Neuro-
trauma Research Program of Western Australia and S.A.D. by the
NHMRC (APP1002347).
Treatment of cells with p-phosphonic acid calix[n]arenes
Compounds 1a–g were assessed by dissolving each calixarene
preparation (3 mgmLÀ1) in DMSO (final maximum concentration of
DMSO in solution 1%) by heating in a thermomixer (Eppendorf,
Crown Scientific Pty Ltd. Minto, NSW, Australia) at 708C, 750 rpm
and serially diluted to give final concentrations of 1, 0.3, 0.1, 0.01,
and 0.001 mgmLÀ1. The effects of each concentration of calixarene
on cell viabilities were assessed in triplicate. Control cultures were
incubated in complete media+1% DMSO.
Keywords: curcumins
phosphonated calixarenes · toxicology
·
micelles
·
phospholipids
·
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The effects of p-phosphonic acid calixarenes on PC12 and mixed
retinal cell viability were assessed following 24 or 72 h treatment,
using the LIVE/DEAD viability/cytotoxicity kit for mammalian cells
(Molecular Probes Inc. Eugene, Oregon, USA). 1 mm calcein-AM and
2 mm Ethidium homodimer-1 (Ethd-1) diluted in PBS was added to
cells and incubated at 378C with 5% CO2 for 30 min. Cells were
photographed at four predetermined locations in each well with
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cultures, by using a fluorescent inverted microscope (Olympus 1ꢁ
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PC12 cells and in two of eight predetermined squares of a grid for
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sessment. Viabilities were represented as the total number of live
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Compound 1c was added to a solution at pH 10 made up from
MilliQ water and 1m NaOH or from MilliQ water, curcumin (ob-
tained from Sigma–Aldrich and used without purification) and 1m
NaOH or a solution at pH 14 made up from MilliQ water, p-nitro-
phenol and 1m NaOH. The mixture was readjusted to pH 10 with
1m NaOH where applicable, stirred until 1c was completely dis-
solved and then the pH was adjusted to the necessary pH value
with 1m HCl.
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Acknowledgements
Received: October 24, 2011
Revised: February 6, 2012
Published online on March 1, 2012
Support of this study by the Australian Research Council is ac-
knowledged, along with the facilities, scientific, and technical as-
ChemPlusChem 2012, 77, 308 – 313
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chempluschem.org
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