Synthesis and in vivo biological activity of large-ringed calixarenes against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.tet.2010.11.034

A series of large-ringed calix[6,7,8]arene analogues have been synthesised and their affect against Mycobacterium tuberculosis in vivo established. In general, when p-phenylcalixarenes and tert-butylcalixarenes were not functionalised at the lower rim, low biological activities were observed. However on going from partially to fully lower rim pegylated calixarenes the anti-mycobacterial properties improved. The addition of cyanopropoxy groups at the lower rim gave rise to low activities, whereas the addition of acetate moieties interestingly had pro-TB effects. Two upper rim sulfonated calixarenes showed promising properties. In the course of this work, a high yielding procedure to synthesise p-phenylcalix[7]arene was also established. Copyright

Full text of DOI:10.1016/j.tet.2010.11.034

Tetrahedron 67 (2011) 373e382
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and in vivo biological activity of large-ringed calixarenes against
Mycobacterium tuberculosis
a
b
ꢀ
ꢀ a
ꢀ
ꢀ a
Kerry J. Goodworth , Anne-Cecile Herve , Evangelos Stavropoulos , Gwenaelle Herve ,
Isabel Casades a b, Alison M. Hill c, Gordon G. Weingarten d, Ricardo E. Tascon b, M. Joseph Colston b
,
,
,
y
Helen C. Hailes a
,
*
a Department of Chemistry, University College London, 20 Gordon Street, London, WC1H OAJ, UK
b The National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA, UK
c School of Biosciences, Hatherly Laboratories, Prince of Wales Road, University of Exeter, Exeter, EX4 4PS, UK
d GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 August 2010
Received in revised form 15 October 2010
Accepted 8 November 2010
Available online 13 November 2010
A series of large-ringed calix[6,7,8]arene analogues have been synthesised and their affect against My-
cobacterium tuberculosis in vivo established. In general, when p-phenylcalixarenes and tert-butylcalix-
arenes were not functionalised at the lower rim, low biological activities were observed. However on
going from partially to fully lower rim pegylated calixarenes the anti-mycobacterial properties improved.
The addition of cyanopropoxy groups at the lower rim gave rise to low activities, whereas the addition of
acetate moieties interestingly had pro-TB effects. Two upper rim sulfonated calixarenes showed prom-
ising properties. In the course of this work, a high yielding procedure to synthesise p-phenylcalix[7]arene
was also established.
Keywords:
Calixarenes
Tuberculosis
Anti-mycobacterial
p-Phenylcalixarene
Calixarene alkylation
Pegylation
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
1 exhibited chemotherapeutic activity against experimental TB in
the guinea pig6 and mouse.6e8 Macrocyclon, also referred to as HOC
Mycobacterium tuberculosis is still a major cause of death
worldwide and infects one-third of the world’s population. Recent
estimates are that there were 9.3 million new cases in 2007 and 1.7
million deaths from tuberculosis (TB), with 23% of the total deaths
among HIV-positive TB cases.1 In addition, the increase in multi-
drug-resistant (MDR) TB cases has led to interest in new drug
therapies and mechanisms of intervention.
The chemistry of calixarenes, and use in a wide range of appli-
cations, has received much attention in recent years due to their
multi-functional capabilities and use as building blocks for host
molecules. For example, they have been used as enzyme mimics,2
gene and drug delivery reagents,3 as sensors4 and in catalysis.5
One particularly interesting application has been as anti-myco-
bacterial agents and the synthesis of these compounds has been
explored.6e17 Early work in the 1950s established that macrocyclon
12.5 EO, was prepared from a tert-octylphenol derived calix[8]
arene (known as HOC 2, Fig. 1) and ethylene oxide (EO) under basic
conditions, resulting in the formation of polyethyleneglycol (PEG)
chains of on average 12.5 units attached to the calixarene lower
rim.8 At the time it was believed that HOC was the p-tert-octyl-calix
[4]arene, although later work established that macrocyclon was
comprised of the larger p-tert-octyl-calix[8]arene possessing eight
PEG chains at the lower rim of approximately 12.5 EO units.9
An analogue of macrocyclon, HOC-60 EO 3, was also noted as
having a pro-TB effect, enhancing the tuberculosis infection.8 Over
several years the heterogeneous mixture, macrocyclon, was used in
a range of experiments: control of a Leishmania donovani infection
in vivo was observed,10 and it was used in chemotherapeutic trials
in pulmonary tuberculosis and leprosy where no serious toxicities
were noted.11,12 Studies also indicated that 1 acted directly, mod-
ifiying host function, and may be stored in macrophages, possibly
associated with lysosomes.13 In addition, experiments established
that 1 inhibited and 3 stimulated triacylglycerol lipase from mac-
rophage extracts14 and phospholipase A2 activity in a model
system.15
* Corresponding author. Tel.: þ44 (0)20 7679 4654; fax: þ44 (0)20 7679 7463;
e-mail address: h.c.hailes@ucl.ac.uk (H.C. Hailes).
y
Deceased 20th February 2003.
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.11.034

374
K.J. Goodworth et al. / Tetrahedron 67 (2011) 373e382
investigated together with non-pegylated compounds and those
with alternative groups at the upper rim.
R
R
R
R
R
OR'
2. Results and discussion
2.1. Chemistry
OR'
OR'
OR'
R'O
R
R'O
R'O
R
Several calixarenes were selected for synthesis and biological
evaluation in vivo against M. tuberculosis infection in mice. These
included p-tert-butylcalixarenes with short PEG chains such as
PEG3-OMe and partial functionalisation with PEG chains, since
previous studies with fully PEG6-OH pegylated compounds, p-tert-
butyl-calix[6,8]arenes 5 and 4, gave comparable activities to mac-
rocyclon. p-tert-Butylcalix[8]arene 6 and p-tert-butylcalix[6]arene
7 were converted to the PEG3-OMe analogue 8 and alternate
pegylated calixarenes 9e12 using potassium carbonate as a base
and the corresponding brominated PEG chains as previously de-
scribed.16 The biological activity of these compounds could then be
compared to the fully pegylated analogues 4, 5 and 13. For com-
parison purposes p-tert-butylcalix[4]arene 14 was also used in bi-
ological screens.
OR'
R
t
1 macrocyclon (HOC 12.5 EO); R = C8H17, R' =
(CH2CH2O)nH where n is avg. of 12.5
t
2 HOC; R = C8H17, R' = H
t
3 HOC 60 EO; R = C8H17, R' = (CH2CH2O)nH
where n is avg. of 60
4 R = C4H9, R' = (CH2CH2O)6H
t
Fig. 1. Macrocyclon and structures of compounds 2e4.
More recent studies involved the use of macrocyclon and p-tert-
butyl-calix[6,8]arene analogues with defined n-ethylene glycol
units, referred to as polyethyleneglycol (PEG) chains at the lower
rim, and significant anti-mycobacterial activities were ob-
served.16,17 Notably, the PEG6-OH analogue 4 (Fig. 1) had similar
anti-mycobacterial activities in vivo to macrocyclon, as did the
equivalent calix[6]arene compound 5 (Fig. 2).17 It was also estab-
lished that the effect of macrocyclon was independent of the action
of CD4þ and CD8þ T cells. Further experiments showed that mac-
rocyclon-induced anti-mycobacterial activity was partly mediated
In addition, the p-phenylcalixarene series were of interest due to
their reported extended hydrophobic cavities. If the biological ac-
tivity of the calixarenes is related to their ability to complex small
molecules or interact with hydrophobic membranes it was envis-
aged that a larger hydrophobic cavity may result in interesting
biological properties. Accordingly, the synthesis of large-ringed p-
phenylcalixarenes was investigated. Gutsche has reported the
synthesis of p-phenylcalix[8]arene 15 utilising an adapted Zin-
keeCornforth procedure with aqueous formaldehyde in 14%
yield.18 Modification of the reaction conditions with increased base
afforded p-phenylcalix[6]arene 16 in 10% yield.18 Application of the
one-step Petrolite procedure using paraformaldehyde gave 15 in 4%
yield together with what was suggested to be a cyclic heptamer 15,
but no pure material could be isolated.18 More recently, the syn-
thesis of p-phenylcalix[4,5,6,8]arenes were described utilising
a modified Petrolite procedure with paraformaldehyde in tetralin,
but also varying the concentration of potassium hydroxide or so-
dium hydroxide used.19 A higher molar ratio of base gave a mixture
by an L-arginine-dependent mechanism, and inducible nitric oxide
synthase (iNOS) activity was required.17
In current work, a series of calixarene analogues were prepared
to establish the importance of functional groups at the upper and
lower calixarene rim for anti-mycobacterial properties. Here we
describe the synthesis and biological activities of selected calixar-
enes with a view to enhancing our understanding regarding
structure and activity of these interesting compounds. Notably
compounds with full and partial pegylations at the lower rim were
R
Ph
R
R
R
R
Ph
Ph
R
R
R
R
OR'
OH
OR''
R''O
OH
OH
HO
OR'
OH
OH
OR'' R''O
R
HO
OH
R
R
OR'
OR''
R'O
R''O
R
HO
OR' R'O
OR''
Ph
Ph
OH HO
OR'
R
R
R
Ph
Ph
R
R
t
t
t
6 R = C4H9, R' = R'' = H
18
14 R = Bu
17 R = Ph
5 R = C4H9, R' = R'' =
t
8 R = C4H9, R' = (CH2CH2O)3Me, R'' = R'
(CH2CH2O)6H
t
t
9 R = C4H9, R' = (CH2CH2O)6H, R'' = H
7 R = C4H9, R' = R'' = H
t
t
11 R = C8H17, R' = (CH2CH2O)6H, R'' = H
10 R = C4H9, R' = (CH2CH2O)6H,
R'' = H
t
12 R = C4H9, R' = (CH2CH2O)12H, R'' = H
t
13 R = C4H9, R' = (CH2CH2O)12H, R'' = R'
16 R = Ph, R' = R'' = H
t
15 R = Ph, R' = R'' = H
19 R = Ph, R' = R'' = Ac
21 R = C4H9, R' = R'' = (CH2)3CN
24 R = Ph, R' = R'' = (CH2)3CN
t
20 R = C4H9, R' = R'' = Ac
t
22 R = C4H9, R' = R'' = (CH2)3CN
23 R = Ph, R' = R'' = (CH2)3CN
Fig. 2. Structures of compounds 5e24.

K.J. Goodworth et al. / Tetrahedron 67 (2011) 373e382
375
of ring sizes, including the octamer 15 (38% yield) and hexamer 16
(7% yield) (Table 1, entry 1).19 Subsequently, a ‘solventless’ approach
was described utilising aqueous formaldehyde: use of potassium
hydroxide gave the hexamer 16 in 50% yield (Table 1, entry 2),
highlighting the sensitivity of these procedures to reaction condi-
tions.20 With a view to generating p-phenylcalix[8]arene as the
major isomer, with little smaller-ringed calixarenes produced to
enhance ease of purification, several reaction conditions were
investigated.
standard conditions, using sodium hydride and acetyl chloride to
give 19 in 46% isolated yield. The corresponding p-tert-butylcalix[8]
arene acetate 20 was also synthesised as previously described, for
comparison purposes.25 The p-tert-butyl-octakis(cyanopropoxy)
calix[6,8]arenes 21 and 22 had previously been prepared for testing
as representative non-pegylated analogues,16 and therefore the
corresponding cyanopropoxy derivatives of octamer 15 and hep-
tamer 16 were also synthesised using equivalent procedures to
generate 23 and 24, respectively (Fig. 2). Compounds 23 and 24
Table 1
Base and formaldehyde used, molar ratio of base to p-phenylphenol, and resulting yields of p-phenylcalix[n]arenes
Entry
Base
Molar ratio (base/phenol)
17 (n¼4) yield (%)
10
10
7
1
0
2
0
0.5
3
16 (n¼6) yield (%)
7
50
4
4
0.2
8
1
1.5
0.7
18 (n¼7) yield (%)
0
0
0
0
0
60
0
0
0
15 (n¼8) yield (%)
38
0
16
13
27
0
28
34
21
1a,19
2b,20
3a
KOH (15 M)
KOH
0.45
0.05
0.06
0.03
0.03
0.07
0.07
0.06
0.02
NaOH (50%)c
NaOH (40%, 10 M)c
KOH (40%, 10 M)c
KOH (50%)c
4a
5a
6a
7a
CsOH (50%)c
CsOH (50%)c
CsOH (50%)c
8a
9a
a
Paraformaldehyde.
Aqueous formaldehyde.
Aqueous solution (wt/vol).
b
c
Use of the Petrolite procedure with paraformaldehyde (Table 1,
were formed in 9% and 18% yield and for reactions with octamer 15
to 23, higher equivalents of base and electrophile were required
compared to the previous reaction conditions reported to generate
21 and 22. This highlighted that functionalisation at the lower rim
required more forcing reaction conditions, perhaps due to steric or
conformational effects.
To compare biological activities to the pegylated tert-butylca-
lixarenes, several pegylated p-phenylcalixarenes were also syn-
thesised using the same method as that used to generate the
corresponding tert-butylcalixarenes 4, 5, 8e12. The approach was
to achieve tetra-alkylation using potassium carbonate as base with
the electrophile, and a second procedure with sodium hydride as
base to the fully alkylated products. Accordingly, octamer 15 was
entry 3) gave the p-phenylcalix[4]arene 17, together with 16 and 15
in 7%, 4%, and 16% yields, respectively. Gutsche’s reaction condi-
tions in our hands gave the cyclic products in equally low yields
(entry 4).18 When the same reaction conditions were utilised, but
with potassium hydroxide as the base, which may via the tem-
plating effect increase the proportion of the larger ringed calixar-
enes formed, the octamer 15 was formed in 27% yield with
negligible 16 and no 17 (entry 5). To enhance the proportion of 15
formed, similar reaction conditions to those used in entry 3, but
using potassium hydroxide were used (entry 6). Initially it was
believed that the octamer 15 was formed in high yield, however MS
analysis revealed that the heptamer 18 had been generated in 60%
yield with hexamer 16 and tetramer 17 in a combined yield of 10%.
This was unexpected: the cyclic heptamers have not been obtained
in comparable yields to the major calixarenes, for example, opti-
mised yields for the generation of p-tert-butylcalix[7]arene have
been reported in the range 11e17%.21 The formation of calix[7]
arenes with p-methyl and p-ethyl substituents has also been de-
scribed in low yield.22 Gutsche and co-workers had mentioned
what they believed to be p-phenylcalix[7]arene 18 as a trace
compound formed in an inseparable mixture using the Petrolite
procedure. This paper referred to a Japanese patent that claimed
formation of the compound, although only sparse characterisation
data was presented.18,23 The use of CsOH as a base (Table 1, entries
7e9) gave higher yields of 15, no heptamer 18, and small amounts
of hexamer 16 and tetramer 17.
reacted
with
17-tetrahydropyranyloxy-3,6,9,12,15-pentaox-
aheptadecyl (25) using potassium carbonate as base to give
a pegylated analogue. By analogy to the same procedure used with
tert-butylcalix[8]arene and from MS data, where the molecular ion
and loss of THP groups were observed, this was assigned to be the
tetra-substituted PEG6-OTHP derivative 26.16 It was also tentatively
assigned as the 1,3,5,7-symmetrically substituted product, al-
though line broadening and poor relaxation of the aromatic 13C
NMR signals meant that this could not be confirmed. Full charac-
terisation was carried out on the THP protected analogues due to
the ability of the deprotected calixarenes to complex several metal
ions and fragment under MS conditions where it became more
difficult to observe the molecular ions. This had been observed
previously with other pegylated calixarenes.16 Compound 26 was
then deprotected as described previously for the pegylated tert-
butylcalix[8]arene 9 and NMR spectroscopy confirmed removal of
the protecting group to give 27.16 The protected intermediate 26
was further reacted with bromide 25 using sodium hydride as base
to effect the full pegylation, giving 28 in 20% yield, which was
confirmed by MALDI-TOF MS.16 In addition, a shift in the UV
maxima from 290 nm in to 255 nm was observed, consistent with
alkylation of the calixarenes.26 Deprotection of 28 produced the
fully substituted PEG6-OH analogue 29 (Fig. 3). Again, the reaction
yields when functionalizing the p-phenylcalix[8]arene were lower
than for the comparable tert-butylcalix[8]arene.
The spectral characterisation of heptamer 18 revealed several
interesting features. Notably the dOH NMR resonance in CDCl3 was
observable at 10.54 ppm, similar to the value for tert-butylcalix[7]
arene (dOH at 10.32).21 Also the methylene bridge protons were
observed as a broad singlet at
spectrum revealed the corresponding carbons at
d
3.95 ppm, while the 13C NMR
32.6 ppm. This
d
suggested that the aryl groups were in a syn orientation by appli-
cation of the ‘de Mendoza rule’, which has been reliably applied to
the calix[4,5,6]arenes.24 Solid state 13C NMR measurements cor-
roborated the solution state conformation, with the methylene
signal observable at approximately
position of CeOH at 149.1 ppm.
d 32.6 ppm and revealed the
d
Heptamer 18 was then functionalised with PEG chains. Reaction
with 1-(2-bromoethoxy)-2(2-methoxyethoxy)ethane 30 (8 equiv)
and potassium carbonate as base (16 equiv) readily gave the fully
alkylated product 31 in 31% isolated yield, in contrast to the
With the focus on large-ringed calixarenes, selected derivati-
sations of the p-phenyl octamer 15 and heptamer 18 were explored.
Initially, the fully substituted acetate of 15 was prepared under

376
K.J. Goodworth et al. / Tetrahedron 67 (2011) 373e382
R
R
R
R
R
R
R
R
OR'
OR'
R'O
OR''
OR'
OR''
R''O
O
O
OR'
OR'
25
30
Br
Br
I
OTHP
OMe
5
R
R'O
R''O
R
R'O
OR' OR'
R
R
2
O
OR'
34
OTHP
11
R
R
R
26 R = Ph, R' = (CH2CH2O)6THP, R'' = H
27 R = Ph, R' = (CH2CH2O)6H, R'' = H
28 R = Ph, R' = R'' = (CH2CH2O)6THP
29 R = Ph, R' = R'' = (CH2CH2O)6H
37 R = SO3H, R' = R'' = H
31 R = Ph, R' = (CH2CH2O)3Me
32 R = Ph, R' = (CH2CH2O)6THP
33 R = Ph, R' = (CH2CH2O)6H
35 R = Ph, R' = (CH2CH2O)12THP
36 R = Ph, R' = (CH2CH2O)12
H
38 R = H, R' = R'' = C12H25
39 R = SO3H, R' = R'' = C12H25
Fig. 3. Structures of compounds 25e39.
partially alkylated products observed for the hexamer 5 and
octamers 2 and 616 under similar conditions, and fewer equivalents
of base and electrophile were also required. Full alkylation was
confirmed by the shift in the UV absorption band from 310 nm in 18
to 260 nm in 31.26 In recently reported work, Hii and co-workers
have described pegylations at the lower rim of calixarenes where
cesium carbonate was used to effect full alkylation, though higher
equivalents of reagents were used and it was noted that potassium
carbonate gave products in much lower yields.27 The ease with
which the etherification of 18 was observed was surprising. Func-
tionalisation with a longer ethylene glycol chain 25 confirmed the
ease of addition to the heptamer, generating 32 in 49% isolated
yield. Characterisation was confirmed by NMR and MALDI-TOF MS,
where the molecular ion was observed complexed to sodium (m/z
3746). Finally, deprotection of 32 yielded heptamer 33, bearing
hexamethylene glycol chains, in 96% yield. Using an analogous
procedure 18 was also reacted with THP protected PEG12-iodide 34
to give the fully alkylated p-calix[7]arene 35. Again, compound
characterisation was more readily achieved on 35 with the THP
protected PEG chain present, and deprotection as previously gave
36. The reason why the heptamer affords fully pegylated com-
pounds so readily is unclear. However tert-butylcalix[7]arene has
been reported to be more conformationally flexible than particu-
larly the tert-butylcalix[4,8]arenes where favourable intra-
molecular hydrogen bonding exists.28 It is possible that by analogy
the phenyl heptamer ring is significantly more conformationally
Table 2
Calix[n]arenes tested for biological activity in vivo
Compound number
Ring size
R
R0
R0
Chemotherapeutic activitya
1
8
8
6
8
6
8
8
6
8
8
8
4
8
7
8
8
6
8
8
8
7
7
7
8
8
tC8H17
tC4H9
tC4H9
tC4H9
tC4H9
tC4H9
tC4H9
tC4H9
tC8H17
tC4H9
tC4H9
tC4H9
Ph
(CH2CH2O)nH (n avg 12.5)
(CH2CH2O)6H
(CH2CH2O)6H
H
R0
þþþ
416
516
616
716
816
916
1016
1116
1216
1316
14
R0
þþþ
17
17
R0
H
H
R0
H
H
H
H
R0
H
H
þþþ
þ
H
None17
(CH2CH2O)3Me
(CH2CH2O)6H
(CH2CH2O)6H
(CH2CH2O)6H
þþþ
þ
þ
þ
(CH2CH2O)12
(CH2CH2O)12
H
H
H
þ
17
þþþ
None
15
18
19
20
H
H
þ
Ph
Ph
H
ꢀ
COCH3
COCH3
(CH3)2CN
(CH3)2CN
(CH2CH2O)6H
(CH2CH2O)6H
(CH2CH2O)3Me
(CH2CH2O)6H
COCH3
COCH3
(CH3)2CN
(CH3)2CN
Pro-TB effectb
tC4H9
tC4H9
tC4H9
Ph
Pro-TB effect
2116
2216
27
ꢀ
ꢀ
H
ꢀ
29
31
33
35
37
39
Ph
Ph
Ph
Ph
SO3H
SO3H
R0
d
d
d
R0
R0
ꢀ
ꢀ
þ
(CH2CH2O)12
H
H
þþþ
þþ
þþ
OC12H25
All activities measured from M. tuberculosis growth in mice spleens and lungs, as determined by CFU bacterial counts of infection. Groups of 3e5 mice were treated with
macrocyclon, control and the new calixarene compounds. All calixarenes were tested in athymic nu/nu mice with the exception of compounds 14 and 20, which were tested in
BALB/c mice. Compounds 1, 6 and 7 were also tested in BALB/c mice with similar results. The activity is calculated from the median change in colony counts of log10 CFUs/
tissue, from the spleen and lung, compared to macrocyclon (100% reduction) and control (0% reduction).
a
None, no detectable anti-TB activity; ꢀ0e20% activity of macrocyclon compared to control; þ 20e50% activity of macrocyclon compared to control; þþ 50e80% activity of
macrocyclon compared to control; þþþ 80e100% activity of macrocyclon.
b
Experiments terminated due to severity of infection.

K.J. Goodworth et al. / Tetrahedron 67 (2011) 373e382
377
Fig. 4. Representative anti-mycobacterial data for compounds 4, 8, 12, 13, 35, 37, 39 (Table 2) compared to saline controls and macrocyclon. All activities were determined by
measuring M. tuberculosis growth in mice spleens and lungs, as determined by CFU bacterial counts of infection as previously described.17 Groups of 3e5 mice were treated with
macrocyclon, control and the new calixarene compounds. All calixarenes shown in Fig. 4 were tested in athymic nu/nu mice. The activity was calculated from the median change in
colony counts of log10 CFUs/tissue, from the spleen and lung.
flexible than the octamer, so that the free hydroxyls are not as
sterically hindered by adjacent PEG chains as they are in the
octamer.
Several calixarene derivatives were then available for screening
bearing tert-butyl and phenyl groups at the upper rim, which could
also be compared to the previously synthesised calixarenes with
pegylation at the lower rim. Also butyronitriles and acetates at the
lower rim, and a couple of other calixarenes were selected for syn-
thesis, based upon the fact that sulfonatocalixarenes have well
precedented water solubilities. Firstly, calix[8]arene-p-octasulfonic
acid 37 (Fig. 3), which has good water solubility, as do the pegylated
calixarenes, was prepared as previously described from calix[8]

378
K.J. Goodworth et al. / Tetrahedron 67 (2011) 373e382
arene in 90% yield.29 In addition an analogue of 37, alkylated with
dodecyl chains at the lower rim was prepared, to give a calixarene
with a hydrophobic and hydrophilic region comparable to the
pegylated calixarene 4. Accordingly, calix[8]arene was alkylated at
the lower rim using an excess of sodium hydride and 16 equiv of
bromododecane. The product 38 was formed in 68% yield and was
characterised by NMR spectroscopy using deuterated pyridine as
solvent. This was then readily sulfonated in refluxing sulfuric acid,
using an identical proceduretothat used toprepare 37 toafford39 in
76% yield. With these compounds in hand (Figs. 2 and 3 and Table 2)
the biological activities in vivo were determined.
compounds could be readily accessed molecular probes to use in
future experiments for identifying the mode of pro-TB activity, also
reported for HOC-60 when HOC is conjugated to approximately
PEG-60 chains at the lower rim.
The two (cyanopropoxy)calix[6,8]arenes 21 and 22 gave rise to
detectable but low anti-mycobacterial properties, and so the other
(cyanopropoxy)calixarenes 23 and 24 were not tested. Neverthe-
less, it did highlight that substituted but non-pegylated calixarenes
did retain some measure of activity. Next, the upper rim p-phe-
nylcalix[7,8]arenes were examined. The partially PEG6-OH
substituted calix[8]arene 27 showed only slight anti-TB properties,
consistent with the data for compounds 9e12 bearing tert-butyl or
tert-octyl groups at the upper rim. Interestingly however, the fully
PEG6-OH substituted calix[8]arene 29, similarly only had slight
levels of activity. Moving to the pegylated p-phenylcalix[7]arenes,
with PEG3-OMe (31), PEG6-OH (33), or PEG12-OH (35) sub-
stitutions, increase of the PEG chain length increased the anti-
mycobacterial activity markedly, and this was a much clearer trend
than for the comparable tert-butyl series. In general, the large-
ringed tert-butylcalixarenes had higher activities than for the
phenylcalixarenes, with the same PEG chain lengths. For example,
p-phenylcalix[7]arene required PEG12-OH chains at the lower rim
to achieve similar levels of activity to that of macrocyclon and tert-
butylcalix[8]arene with PEG6-OH (Fig. 4aed). This could reflect
requirements for a shorter aryl cavity or hydrophobic region, or
a certain balance between hydrophobicity and hydrophilicity that
is needed for better biodistribution and transportation through cell
membranes.
Finally, the two highly water soluble sulfonated calixarenes 37
and 39 were tested, and whether non-functionalised, or alkylated
at the lower rim, the activity was comparable, and in both cases
approximately 70% of that of macrocyclon 1 (Fig. 4e and f). These
results highlighted that for good anti-mycobacterial activity larger
ringed calixarenes are required with, ideally, good water solubil-
ities. The advantage of compounds such as 4 and 37 exhibiting good
anti-TB activity is that they are synthetically readily available for
further modification and also the preparation of labelled molecular
probes.
2.2. Biological results
Previous work had established that macrocyclon 1 acted by
modifying host function, and for this reason experiments were
carried out in vivo against M. tuberculosis infection using either
BALB/c mice or in most cases athymic nu/nu mice as previously
described.17 Mice were treated with the calixarenes 2 d prior to
intravenous infection, followed by a second identical dose of cal-
ixarene 2e3 d after infection. Compounds were screened against
macrocyclon 1 and controls (treatment with saline solution) and
from this the relative activity of the calixarenes determined
(Table 2). Compound toxicities of selected calixarenes were in ac-
cordance with those previously reported for macrocyclon, indeed at
the concentrations used no toxicities were noted.8,26
Previous work had established that the unsubstituted tert-
butylcalix[6,8]arenes, compounds 7 and 6, respectively, had either
a low anti-mycobacterial effect (7), or in the case of the hexamer no
activity.17 Further studies established that the tetramer 14 was in-
active, that the p-phenylcalixarene 15 had some moderate activity
and that heptamer 18 exhibited low levels of activity. This sug-
gested that with no functionalisation at the lower rim, the calix[8]
arenes had some low anti-mycobacterial activity, but that the
smaller ring sizes, p-phenylcalix[7]arene and tert-butylcalix[4,6]
arenes, had less or no activity, respectively. In addition, different
bacterial colony counts were measured in the spleen compared to
the lung, suggesting the low solubility of these compounds in
aqueous media (typically 0.25 mg/mL in water) resulted in poor
biodistribution. It was for these reasons that functionalisation of
the calixarenes was focused on the larger calix[6,7,8]arene ring
sizes.
2.3. Conclusion
In summary, a range of calixarenes have been prepared for
preliminary screening as anti-TB compounds. Several novel pegy-
lated large-ringed calixarenes were prepared, including derivati-
sation with a phenyl group at the upper rim. In the course of this
work we also identified a new procedure to p-phenylcalix[7]arene
in high yield that was particularly amenable to derivatisation with
activated PEG chains in comparatively high yield and one step only.
The compounds prepared and previous calixarenes synthesised
in our group were used in preliminary in vivo experiments against
M. tuberculosis in mice. These indicated that when not pegylated at
the lower rim, p-tert-butylcalix[8]arene and p-phenylcalix[8]arene
were more active than the p-tert-butylcalix[4,6]arenes and p-phe-
nylcalix[7]arene, and that the highly water soluble sulfonate 37
showed activities approaching those of macrocyclon. When deri-
vatising with PEG chains at the lower rim, this readily enhanced the
anti-mycobacterial properties of the tert-butylcalix[6,8]arenes, but
for the p-phenylcalix[7]arenes longer PEG chains were required to
achieve this effect. For the tert-butylcalix[6,8]arenes, full pegyla-
tions gave rise to higher anti-mycobacterial properties, than the
corresponding partial pegylations. Lower rim substitution with
acetate groups gave rise to pro-TB activities, and lower rim func-
tionalisation with non-PEG chains such as cyanopropoxy gave
compounds with low activities. Further experiments with selected
compounds will now be performed to probe the mode of action of
this interesting family of anti-mycobacterial compounds.
Previous work had established that the tert-butylcalix[6,8]are-
nes, compounds 5 and 4, with full pegylation with PEG6-OH at the
lower rim had comparable activities to that of macrocyclon 1, so the
effect of the pegylation was investigated in more detail.17 First
compound 8, with PEG3-OMe groups at all eight phenolic positions
was compared to analogue 4 and macrocyclon 1. Levels of anti-
mycobaterial activity were approximately 80% that of 1 and 4
(Fig. 4a and b), suggesting that shorter n-ethylene oxide chains
could be tolerated. Partially pegylated compounds 9e12 were also
tested and compared to macrocyclon 1 and the fully pegylated
compounds such as 13. Interestingly, the compounds all had less
anti-mycobacterial activity than the fully pegylated calixarenes
(Fig. 4c and d), regardless of ring size and length of PEG chain (6 or
12). In addition, the group at the upper rim, whether it was tert-
octyl or tert-butyl made little difference, and PEG-OH chains greater
than six repeat units did not improve the activity further as had
been observed previously with the fully alkylated analogues 5 and
13.17 Other lower rim fully substituted calixarenes were then
investigated.
The fully acetylated calix[8]arenes 19 and 20 gave interesting
data in that pro-TB activity was detected in both cases. It is unclear
why this is the case, particularly because the phenolic acetates are
likely to be hydrolysed in vivo and both non-functionalised calix-
arenes, 15 and 6, demonstrated anti-TB properties. However, these

K.J. Goodworth et al. / Tetrahedron 67 (2011) 373e382
379
3. Experimental
3.4. 5,11,17,23-Tetraphenyl-25,26,27,28-tetrahydroxycalix[4]
arene (p-phenylcalix[4]arene)29 17
3.1. General chemical experimental
A
mixture of p-phenylphenol (8.50 g, 0.050 mol), para-
Unless otherwise noted, solvents and reagents were reagent
grade from commercial suppliers and used without further purifi-
cation. THF was dried by distillation from a sodium/benzophenone
suspension under a dry N2 atmosphere. CH2Cl2 was dried by dis-
tillation from CaH2 under a dry N2 atmosphere. All moisture-sen-
sitive reactions were performed under a nitrogen atmosphere using
oven-dried glassware. Reactions were monitored by TLC on Kie-
selgel 60 F254 plates with detection by UV, or permanganate, nin-
hydrin (for ureas) and phosphomolybdic acid stains. Flash column
chromatography was carried out using silica gel (particle size
formaldehyde (3.00 g, 0.100 mol) and sodium hydroxide (0.20 mL,
0.003 mol, 50% aqueous solution) in xylene (50 mL) was heated at
130 ꢁC, under nitrogen for 4 h with the azeotropic removal of water.
The resulting precipitate was collected by filtration and washed
with water (20 mL), ethyl acetate (20 mL) and acetone (20 mL). The
product was purified by flash chromatography (hexane/ethyl ace-
tate, 3:1) to give 17 as a white powder (0.640 g, 7%). Rf 0.93 (ethyl
acetate/hexane, 2:1); nmax/cmꢂ1 (KBr) 3084, 3045, 748; dH
(300 MHz; DMSO-d6) 4.91 (8H, s, ArCH2Ar), 7.38e7.72 (28H, m,
ArH); dC (100 MHz; acetone-d6) 32.6 (CH2), 126.2, 126.6, 127.2,
128.7, 129.7, 132.2 (C-5), 141.7 (C-6), 149.6 (CeOH); m/z (HRFABþ)
found (Mþ) 728.2899; C52H40O4 requires 728.2927.
40e63 m
m). Melting points are uncorrected. 1H NMR and 13C NMR
spectra were recorded in CDCl3 at the field indicated. Compounds,1,
4e14, 20, 25, 28 and 30, were prepared as previously de-
scribed.16,25,29 Selected spectra and the biological screening data
are in Supplementary data.
3.5. 5,11,17,23,29,35,41,47-Octaphenyl-49,50,51,52,53,54,55,56-
octakis(acetyl)calix[8]arene18 19
The reaction was carried out under anhydrous conditions. To
a suspension of sodium hydride (720 mg, 0.018 mol) in THF/DMF
(4:1, 40 mL), p-phenylcalix[8]arene (874 mg, 0.600 mmol) was
added in portions. Following stirring for 15 min acetyl chloride
(750 mg, 9.55 mmol) was added dropwise. The resulting solution
was heated at 70 ꢁC for 24 h. The reaction mixture was quenched
with iced water (20 mL), the crude productextracted into chloroform
(3ꢃ50 mL) and washed with saturated lithium chloride solution
(2ꢃ20 mL). Drying (MgSO4), filtration and evaporation in vacuo
affordedacrudeoil, whichupon flashchromatography(hexane/ethyl
acetate, 3:1) afforded 19 as colourless crystals (490 mg, 46%). Mp
235e239 ꢁC (ethyl acetate/hexane)(lit. 230e265 ꢁC dec;chloroform/
ethanol);30 nmax/cmꢂ1 (KBr) 2954, 2854, 1759, 1597, 1555; dH
(300 MHz; CDCl3) 1.85 (24H, s, Me), 3.78 (16H, s, ArCH2Ar), 7.23e7.81
(56H, m, ArH); dC (100 MHz; CDCl3) 21.2 (CH3), 31.0 (ArCH2Ar),125.5,
126.2 (signals superimposed), 127.9, 133.4, 141.0 (signals super-
imposed), 152.9 (CeO), 168.0 (C]O); m/z (FABþ) 1793 (Mþ, 1%).
3.2. 5,11,17,23,29,35,41,47-Octaphenyl-49,50,51,52,53,54,55,56-
octahydroxycalix[8]arene (p-phenylcalix[8]arene)18 15
A
mixture of p-phenylphenol (8.50 g, 0.050 mol), para-
formaldehyde (3.00 g, 0.10 mol) and caesium hydroxide (0.85 mL,
2.83 mmol; 50% aqueous solution) in xylene (50 mL) was heated at
130 ꢁC for 4 h under nitrogen, with the azeotropic removal of water.
The resulting precipitate was collected by filtration in vacuo and
washed with water (20 mL), ethyl acetate (20 mL) and acetone
(20 mL). The mixture was purified using flash silica chromatography
(hexane/ethyl acetate, 3:1) to yield 15 as a white powder (3.10 g,
34%). Rf 0.25 (ethyl acetate/hexane, 2:1); lmax/nm (KOH/MeOH) 230,
290; nmax/cmꢂ1 (KBr) 3200, 2920, 2960e2850; dH (300 MHz; DMSO-
d6) 4.01 (16H, br s, ArCH2Ar), 7.20e7.71 (56H, m, ArH); dC (100 MHz;
acetone-d6) 33.5 (CH2), 126.8, 127.2, 127.9, 129.4, 130.4, 132.8 (C-5),
142.3 (C-6), 154.0 (CeOH); m/z (ESþ) 1479 (MNaþ, 5%).
3.3. 5,11,17,23,29,35-Hexaphenyl-37,38,39,40,41,42-
hexahydroxycalix[6]arene (p-phenylcalix[6]arene)18 16 and
5,11,17,23,29,35,41-heptaphenyl-43,44,45,46,47,48,49-
heptahydroxycalix[7]arene (p-phenylcalix[7]arene) 18
3.6. 5,11,17,23,29,35,41,47-Octaphenyl-49,50,51,52,53,54,55,56-
octakis-(cyanopropoxy)calix[8]arene 23
The reaction was carried out under anhydrous conditions. To
a solution of 15 (100 mg, 0.07 mmol) in acetonitrile (50 mL), po-
tassium carbonate (606 mg, 4.39 mmol) was added and the re-
action mixture stirred at 40 ꢁC for 15 min. 4-Bromobutyronitrile
(0.44 mL, 4.39 mmol) was added and the reaction mixture stirred at
80 ꢁC for 14 d. Water (10 mL) was added, the solution dried
(MgSO4) and evaporated in vacuo to afford the crude product as
a yellow oil. Distillation under reduced pressure then flash silica
chromatography (ethyl acetate/hexane, 2:1) afforded 23 as a yellow
oil (12 mg, 9%). nmax/cmꢂ1 (film) 2950, 2890, 2246, 1076; dH
(300 MHz; CDCl3) 1.84e2.02 (16H, m, 8ꢃOCH2CH2), 2.38e2.51
(16H, m, 8ꢃCH2CN), 3.43e3.95 (32H, m, 8ꢃOCH2, 8ꢃArCH2Ar),
7.18e7.72 (56H, m, ArH); dC (100 MHz; CDCl3) 14.8 (OCH2CH2), 25.8
(CH2CN), 31.0 (ArCH2Ar), 71.3 (CH2O), 117.8 (CN), 126.8, 128.9, 129.4,
134.7, 136.4, 141.9, 157.1 (COCH2); m/z (FABþ) 1993 (Mþ, 15%).
A
mixture of p-phenylphenol (8.50 g, 0.050 mol), para-
formaldehyde (3.00 g, 0.100 mol) and potassium hydroxide
(0.40 mL, 3.60 mmol; 50% aqueous solution) in xylene (50 mL) was
heated at 130 ꢁC for 4 h under nitrogen, with the azeotropic re-
moval of water. The resulting precipitate was collected by filtration
in vacuo and washed with water (20 mL), ethyl acetate (20 mL) and
acetone (20 mL). Purification by flash silica chromatography (hex-
ane/ethyl acetate, 3:1) yielded 16 (0.730 g, 8%) and 18 as a white
powder (5.46 g, 60%). Data for 16: Rf 0.85 (ethyl acetate/hexane,
2:1); nmax/cmꢂ1 (KBr) 3068, 3040; dH (300 MHz; DMSO-d6) 4.10
(12H, s, ArCH2Ar), 7.21e7.64 (42H, m, ArH); dC (100 MHz; acetone-
d6) 33.4 (CH2), 126.7, 127.1, 127.8, 129.2, 130.3, 132.7 (C-5), 142.2 (C-
6), 154.0 (CeOH); m/z (ESþ) 1093 (MHþ, 1%). Data for 18. Mp
>300 ꢁC (ethyl acetate/hexane); lmax/nm (KOH/MeOH) 275; nmax
/
cmꢂ1 (KBr) 3190, 2980; dH (500 MHz; CDCl3) 4.10 (14H, br s,
ArCH2Ar), 7.28 (7H, t, J 7.5 Hz, Ph-para H), 7.38 (14H, t, J 7.5 Hz, Ph-
meta H), 7.44 (14H, s, Ar-meta H), 7.49 (14H, d, J 7.5 Hz, Ph-ortho H),
10.54 (7H, br s, OH); dC (125 MHz; acetone-d6) 32.6 (CH2), 126.6,
126.8 (CH), 127.7, 128.9 (CH), 129.3, 133.5, 141.6; dC (75 MHz;
CPMAS) 32.7 (CH2), 128.2 (signals superimposed), 134.1 (C-5), 140.1
(C-6), 149.1 (CeOH); m/z (FABþ) 1298 (MNaþ, 1%), 1276 (Mþ, 1), 648
(10); m/z (HRFABþ) found (MHþ) 1275.5235; C91H71O7 requires
1275.5200.
3.7. 5,11,17,23,29,35,41-Heptaphenyl-43,44,45,46,47,48,49-
heptakis-(cyanopropoxy)-calix[7]arene 24
The reaction was carried out under anhydrous conditions. To
a suspension of 18 (100 mg, 0.08 mmol) in acetonitrile (50 mL) po-
tassium carbonate (345 mg, 2.50 mmol) was added and the reaction
mixture stirred for 15 min at 40 ꢁC. 4-Bromobutyronitrile (0.25 mL,
2.50 mmol) was added and the reaction mixture stirred vigorously
for 14 d at 80 ꢁC. Water (10 mL) was added, the solution dried

380
K.J. Goodworth et al. / Tetrahedron 67 (2011) 373e382
(MgSO4) and evaporated in vacuo to afford the crude product as
a yellow oil. Distillation under reduced pressure then flash silica
chromatography (ethyl acetate/hexane, 2:1) afforded 24 as a yellow
oil (24 mg, 18%). nmax/cmꢂ1 (film) 2910, 2329, 1076; dH (300 MHz;
CDCl3) 1.65e1.90 (14H, m, 7ꢃOCH2CH2), 2.45 (14H, t, J 7.1, 7ꢃCH2CN),
3.74e4.00 (28H, t, J 5.9, 7ꢃOCH2, 7ꢃArCH2Ar), 7.08e7.63 (49H, m,
ArH); dC (75 MHz; CDCl3) 16.4 (OCH2CH2), 28.6 (CH2CN), 31.3
(ArCH2Ar), 71.2 (CH2O), 119.7 (CN), 127.1, 128.3, 129.2, 134.3, 137.4,
140.7, 154.6 (COCH2); m/z (FABþ) 1745 (MHþ, 12%), 1676
(Mþꢂ(CH2)3CN, 10), 1608 (Mþꢂ2[(CH2)3CN], 8), 1540 (Mþꢂ3
[(CH2)3CN], 11).
8ꢃTHP(CH2)3), 3.41e3.83 (approx. 220H, m, 8ꢃTHPeOCH2,
8ꢃArCH2Ar, 96ꢃPEGeOCH2), 4.58 (8H, t, J 2.6, 4ꢃTHPeOCH),
7.24e7.78 (56H, m, ArH); dC (100 MHz; CDCl3) 19.2 (CH2), 25.1
(CH2), 30.1 (CH2), 61.9 (CH2), 66.9 (CH2), 70.2e70.4 (signals super-
imposed), 98.6 (CHOCH2), 126.8 (CH), 127.6, 128.5 (CH), 134.2, 136.3,
140.7, 154.3 (COCH2); m/z (MALDI-TOF) 1001.6 ([MNaꢂ3[THP]]4þ
/
4); m/z (ESþ) found 1791.83 (MNaꢂ8THPꢂH)2þ/2; [C210H294NaO56]/
2 requires 1791.88. To a solution of hydrochloric acid (5 mL; 18 M)
and dichloromethane (5 mL), 28 (50 mg, 0.01 mmol) was added
and the resulting solution stirred at rt for 2 h. Evaporation in vacuo
afforded a brown oil, which was suspended in a 10% aqueous so-
lution of sodium hydroxide (10 mL) and the solution was stirred
vigorously for 30 min. The product was extracted into dichloro-
methane (10 mL), dried (MgSO4) and evaporated to afford 29 as
a viscous yellow oil (36 mg, 86%). nmax/cmꢂ1 (film) 3140, 2908,1454,
1118; dH (300 MHz; CDCl3) 3.52e4.10 (approx. 210H, m, 8ꢃArCH2Ar,
96ꢃPEGeOCH2), 7.45e7.77 (56H, m, ArH).
3.8. 5,11,17,23,29,35,41,47-Octaphenyl-49,51,53,55-tetrakis-
(17-hydroxy-3,6,9,12,15-pentaoxaheptadecyloxy)-50,52,54,56-
tetrahydroxycalix[8]arene 27
The reaction was carried out under anhydrous conditions. To
a suspension of 13 (500 mg, 0.343 mmol) and potassium carbonate
(758 mg, 5.49 mmol) in acetonitrile (60 mL) at rt, mono-THP-
mono-bromo-hexaethyleneglycol 25 (1.18 g, 2.75 mmol) in aceto-
nitrile (10 mL) was added and the reaction mixture heated at reflux
for 4 d. Water was added (30 mL), the crude product extracted into
dichloromethane (50 ml), dried (phase separator) and evaporated
in vacuo to afford the crude product as a yellow oil. Purification
using flash silica chromatography (ethyl acetate/hexane, 2:1, then
ethyl acetate/methanol, 10:1) afforded 26 as a viscous yellow oil
(196 mg, 20%). nmax/cmꢂ1 (film) 2868, 1456, 1122; lmax/nm (KOH/
MeOH) 230, 255; dH (300 MHz; CD2Cl2) 1.67e1.98 (24H, m, 4ꢃTHP
(CH2)3), 3.55e4.00 (approx. 120H, m, 4ꢃTHPeOCH2, 8ꢃArCH2Ar,
48ꢃPEGeOCH2), 4.76 (4H, br s, 4ꢃTHPeOCH), 7.45e7.77 (56H, m,
ArH); dC (100 MHz; CDCl3) 18.9 (CH2), 24.9 (CH2), 29.8 (CH2), 30.0
(CH2), 61.0 (CH2), 66.1 (CH2), 70.0e70.8 (signals superimposed),
98.3 (CHOCH2), 126.2 (CH), 126.7 (CH), 127.1 (CH), 127.5 (CH), 128.1,
133.3, 140.2, 140.4, 157.9 (COCH2); m/z (MALDI-TOF) 2872 (MNaþ,
40%), 2849 (Mþ, 30), 2786 (MNaþꢂC5H10O, 45), 2680 (Mþꢂ2
[C5H10O], 35). p-Phenycalix[8]arene-tetra-hexaethyleneglycolTHP
26 (50.0 mg, 0.02 mmol) was added in portions to a solution of
hydrochloric acid (5 mL; 18 M) and dichloromethane (5 mL) at rt.
The resulting solution was stirred at rt for 2 h. Removal of solvents
afforded a brown oil, which was suspended in a 10% aqueous so-
lution of sodium hydroxide (10 mL) and stirred vigorously for
30 min. The product was extracted into dichloromethane (5 mL),
dried (MgSO4) and evaporated to afford 27 as a yellow viscous oil
(43 mg, 85%). nmax/cmꢂ1 (film) 3240, 2868, 1456, 1122; dH
(300 MHz; CD2Cl2) 3.55e4.00 (approx. 110H, m, 8ꢃArCH2Ar,
48ꢃPEGeOCH2), 7.45e7.77 (56H, m, ArH); dC (100 MHz; CDCl3)
62.2 (CH2), 70.0e73.2 (signals superimposed), 125.4, 126.2, 127.7,
127.9, 128.0, 134.2, 140.2, 156.5 (COCH2); m/z (ES-TOF) found
2514.2686 (MHþ); C152H177O32 requires 2514.2223.
3.10. 5,11,17,23,29,35,41-Heptaphenyl-43,44,45,46,47,48,49-
heptakis-(9-methoxy-3,6-dioxadecyloxy)-calix[7]arene 31
The reaction was carried out under anhydrous conditions. To
a solution of p-phenylcalix[7]arene (500 mg, 0.39 mmol) in aceto-
nitrile (100 mL) at rt, potassium carbonate (879 mg, 6.33 mmol) was
added and the reaction mixture stirred at 40 ꢁC for 1 h. 1-(2-Bro-
moethoxy)-2(2-methoxyethoxy)ethane 30 (709 mg, 3.12 mmol) in
acetonitrile (10 mL) was added dropwise and the reaction mixture
was stirred at 80 ꢁC for 4 d. Water (10 mL) was added and the crude
product extracted into dichloromethane (30 mL) then dried
(MgSO4) and evaporated in vacuo to afford the crude product as
a brown oil. Flash silica chromatography (ethyl acetate/hexane, 2:1,
then ethyl acetate/methanol, 10:1) afforded the product, which was
stirred in brine (20 mL) overnight. Extraction into dichloromethane
(10 mL), drying (hydrophobic frit) and evaporation in vacuo afforded
the title compound as an orange oil (281 mg, 31%). nmax/cmꢂ1 (film)
2870, 2322, 1438, 1119; lmax/nm (KOH/MeOH) 230, 260; dH
(300 MHz; CDCl3) 3.38 (21H, s, Me), 3.48e3.93 (approx. 100H, m,
42ꢃPEGeOCH2, 7ꢃArCH2Ar), 7.25e7.60 (49H, ArH); dC (100 MHz;
CDCl3) 30.1 (CH2), 58.8 (OCH3), 70.3e71.7 (signals superimposed),
126.6 (CH), 128.4 (signals superimposed), 133.6, 136.6, 140.3, 154.5
(COCH2); m/z (ESþ) 2320 (MNaþ, 30%), 1172 ([MNa2]2þ/2, 60).
3.11. 5,11,17,23,29,35,41-Heptaphenyl-43,44,45,46,47,48,49-
heptakis-(17-hydroxy-3,6,9,12,15-pentaoxaheptadecyloxy)
calix[7]arene 33
The reaction was carried out under anhydrous conditions. To
a suspension of p-phenylcalix[7]arene (127 mg, 0.10 mmol) in
acetonitrile (40 mL) at rt, potassium carbonate (224 mg,1.62 mmol)
was added and the reaction mixture stirred at 40 ꢁC for 15 min.
Compound 25 (342 mg, 0.80 mmol) in acetonitrile (10 mL) was
added dropwise and the resulting solution stirred at 80 ꢁC for 4 d.
Water was added (10 mL) and the product extracted into chloro-
form (2ꢃ30 mL), dried (MgSO4) and evaporated in vacuo to afford
crude 32 as a yellow oil. Purification via flash silica chromatography
(ethyl acetate/hexane, 2:1, then ethyl acetate/methanol, 10:1) gave
32 as a viscous yellow oil (181 mg, 49%). nmax/cmꢂ1 (film) 2869,
1456, 1123; lmax/nm (KOH/MeOH) 232, 258; dH (400 MHz; acetone-
d6) 1.64e1.70 (42H, m, 7ꢃTHP(CH2)3), 3.62e3.93 (approx. 200H, m,
96ꢃPEGeOCH2, 7ꢃArCH2Ar), 4.78 (7H, t, J 3.3, 7ꢃTHPeOCH),
7.47e7.89 (49H, m, ArH); dC (100 MHz; CDCl3) 19.4 (CH2), 25.4
(CH2), 30.1 (CH2), 30.6 (CH2), 62.1 (OCH2), 66.7 (OCH2), 70.0e72.0
(signals superimposed), 98.9 (CHOCH2), 126.3, 126.7, 127.6, 128.5,
134.5, 137.6, 141.0, 155.2 (COCH2); m/z (TOF) found 3734.9 (MNaþ);
C210H294NaO56 requires 3735.0.
3.9. 5,11,17,23,29,35,41,47-Octaphenyl-49,50,51,52,53,54,55,56-
octakis-(17-hydroxy-3,6,9,12,15-pentaoxaheptadecyloxy)calix
[8]arene 29
The reaction was carried out under anhydrous conditions. To
a suspension of sodium hydride (60%; 44 mg, 1.10 mmol) in THF
(40 mL), 27 (196 mg, 0.069 mmol) in THF (10 mL) and mono-
bromo-mono-THP-hexaethyleneglycol 26 (236 mg, 0.55 mmol) in
THF (10 mL) was added and the reaction mixture heated at reflux
temperature for 4 d. Water was added (10 mL), the product
extracted into dichloromethane (40 mL), dried (phase separator),
and evaporated to afford the crude product as a yellow oil. Purifi-
cation using flash silica chromatography (ethyl acetate/hexane, 2:1,
then ethyl acetate/methanol, 10:1) afforded 28 as a viscous yellow
oil (58 mg, 20%). nmax/cmꢂ1 (film) 3240, 2868, 1456, 1122; lmax/nm
(KOH/MeOH) 230, 262; dH (300 MHz; CDCl3) 1.44e1.66 (48H, m,

K.J. Goodworth et al. / Tetrahedron 67 (2011) 373e382
381
To a solution of hydrochloric acid (18 M; 5 mL) and dichloro-
methane (5 mL), 32 (60 mg, 0.02 mmol) was added and the
resulting solution stirred at rt for 2 h. Evaporation in vacuo afforded
a brown oil, which was suspended in an aqueous solution of so-
dium hydroxide (10%; 10 mL) and the solution was stirred vigor-
ously for 30 min. The product was extracted into dichloromethane
(10 mL), dried (MgSO4) and evaporated in vacuo to afford 33 as
a viscous yellow oil (48 mg, 96%). nmax/cmꢂ1 (film) 3120, 2848, 1447,
1117; lmax/nm (KOH/MeOH) 230, 260; dH (300 MHz; CDCl3)
3.43e3.99 (182H, m, 96ꢃPEGeOCH2, 7ꢃArCH2Ar), 6.97e7.89 (49H,
m, ArH); dC (100 MHz; CDCl3) 30.1 (CH2), 61.5 (OCH2), 69.5e72.4
(signals superimposed), 126.5, 126.7, 127.9, 128.5, 133.6, 137.0, 140.5,
155.1 (COCH2); m/z (ESþ) 3126 (MNaþ, 30%).
Water (30 mL) was added and the solution was neutralised by the
addition of solid potassium carbonate. Following filtration in vacuo,
sodium carbonate was added to the filtrate, and evaporation in
vacuo afforded a pale yellow solid, which was dissolved in water
(30 mL), and the remaining precipitate removed by filtration in
vacuo. Following evaporation the crude solid was recrystallised
from water and methanol to afford 39 as a white solid (430 mg,
76%). Mp >300 ꢁC (THF/water); nmax/cmꢂ1 (KBr) 3400, 2858, 1454,
1376, 1051; dC (100 MHz; D2O) 14.2 (CH3), 20.8 (CH2), 29.2e31.0
(signals superimposed), 33.3 (OCH2CH2), 72.7 (OCH2), 126.5, 128.4,
139.0, 156.2 (COCH2); m/z (ES) 1439 ([Mþ2Na]2þ/2, 18%).
3.15. In vivo experiments
Experiments were performed as previously described.17 Further
details are provided in Supplementary data. Mice were infected
with 2ꢃ105 mycobacterial cells (intravenously) and the level of
infection determined by assessment of infection in the lungs and
spleens.17 In experiments with the calixarenes, macrocyclon and
controls, CFUs were measured 24e35 d after infection. The doses of
3.12. 5,11,17,23,29,35,41-Heptaphenyl-43,44,45,46,47,48,49-
heptakis-(35-hydroxy-3,6,9,12,15,18,21,24,27,30,33-
undecaoxapentatriacontyloxy) calix[7]arene 36
The reaction was carried out under anhydrous conditions. To
a suspension of p-phenylcalix[7]arene (200 mg, 0.16 mmol) and
potassium carbonate (347 mg, 2.51 mmol) in acetonitrile (50 mL) at
rt, 32 (929 mg, 1.26 mmol) in acetonitrile (10 mL) was added drop-
wise. The resulting solution was stirred at 80 ꢁC for 7 d. Water
(20 mL) was added and the crude product extracted into dichloro-
methane (40 mL). Drying (phase separator) and evaporation in
vacuo afforded the crude product as a brown oil. Flash chromatog-
raphy (ethyl acetate/hexane, 2:1, then ethyl acetate/methanol,10:1)
calixarenes in 200 mL of endotoxin-free saline were injected into
each mouse intraperitoneally, in two doses, one 48e72 h before
infection and one 48e72 h after infection.17 Calixarenes were ad-
ministered in doses of 2e3 mg/200 mL.
Acknowledgements
afforded the THPintermediate35as aviscous oil(190 mg, 22%). nmax
/
We are grateful to J.W. Cornforth for providing macrocyclon. The
authors would like to thank the BBSRC (31/B10963 to G.H. and
A.C.H), Glaxo SmithKline (Action TB Program) and MRC for funding.
We also thank Abil Aliev for NMR support.
cmꢂ1 (film) 2873, 1442, 1182; dH (400 MHz; CDCl3) 1.40e1.60 (42H,
m, 7ꢃTHP(CH2)3), 3.14e4.69 (approx. 400H, m, 192ꢃPEGeOCH2,
7ꢃArCH2Ar), 5.01 (7ꢃTHPeOCH), 7.15e7.91 (49H, m, ArH); dC
(100 MHz; CDCl3) 19.5 (CH2), 25.4 (CH2), 30.7 (CH2), 32.2 (CH2), 62.2
(OCH2), 67.7 (OCH2), 69.6e71.9 (signals superimposed), 98.7 (CH)
124.8, 126.5, 126.6, 128.3, 134.0, 136.4, 140.4, 154.8(COCH2); m/z
(MALDI-TOF) 1133.6 ([MNa5]5þ/5, 30%). To a solution of hydrochloric
acid (18 M; 5 mL) and dichloromethane (5 mL), 35 (50 mg,
0.01 mmol) was added and the solution stirred at rt for 2 h. Evapo-
ration in vacuo afforded a brown oil, which was suspended in an
aqueous solution of sodium hydroxide (10%; 10 mL), and the solu-
tion was stirred vigorously for 30 min. The product was extracted
into dichloromethane (10 mL), dried (MgSO4) and evaporated in
vacuo afforded 36 as a brown viscous oil (23 mg, 50%). nmax/cmꢂ1
(film) 2885, 1437, 1165; dH (400 MHz; CDCl3) 3.21e5.22 (74H, m,
192ꢃPEGeOCH2, 7ꢃArCH2Ar), 7.10e7.94 (49H, m, AH).
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2010.11.034.
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