p-Guanidinoethyl calixarene and parent phenol derivatives exhibiting antibacterial activities. Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2009.06.040

The tetra-para-guanidinoethyl-calix[4]arene, its distally-disubstituted ether derivatives involving 2,2′-bithiazolyl- or 2,2′-bipyridyl-methyl groups, as well as the para-guanidinoethylphenol and its analogous derivatives have been synthesized, fully char

Full text of DOI:10.1016/j.bmc.2009.06.040

Bioorganic & Medicinal Chemistry 17 (2009) 5496–5509
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
p-Guanidinoethyl calixarene and parent phenol derivatives exhibiting
antibacterial activities. Synthesis and biological evaluation
Maxime Mourer, Hugues Massimba Dibama, Stéphane Fontanay, Marion Grare, Raphaël E. Duval,
*
Chantal Finance, Jean-Bernard Regnouf-de-Vains
SRSMC, UMR 7565 Nancy Université—CNRS, Groupe d’Etude des Vecteurs Supramoléculaires du Médicament, Faculté de Pharmacie, 5, rue Albert Lebrun, BP 80403,
54001 Nancy Cedex, France
a r t i c l e i n f o
a b s t r a c t
The tetra-para-guanidinoethyl-calix[4]arene, its distally-disubstituted ether derivatives involving 2,20-
bithiazolyl- or 2,20-bipyridyl-methyl groups, as well as the para-guanidinoethylphenol and its analogous
derivatives have been synthesized, fully characterized and evaluated as antibacterial agents towards both
Gram positive and Gram negative reference bacteria. The simple phenolic species showed lower activity
than their calixarene analogues, confirming the hypothesis that a synergistic effect should result from the
spatial organization of guanidinium and heterocycles on a macrocyclic scaffold. Introduction of the
bithiazole and bipyridine substituents enhanced the activity of simple phenol derivatives, reaching, for
the two Staphylococcus aureus strains in particular, the values obtained for their calixarenic parents.
MTT viability assays were carried out to determine selectivity indexes.
Article history:
Received 5 March 2009
Revised 5 June 2009
Accepted 18 June 2009
Available online 25 June 2009
Keywords:
Calix[4]arene
Guanidinium
Phenylethers
Bipyridine
Ó 2009 Elsevier Ltd. All rights reserved.
Bithiazole
Antibacterials
1. Introduction
few reports, essentially in the form of patents, have focused on
their intrinsic therapeutical properties; some of them, hydrophilic,
The emergence of resistance in pathogenic microorganisms to
commercial antibiotics requires the development of specific fields
of research dedicated to the discovery of new drugs.1 In this sense,
we have focused on a promising approach based on a calixarene
platform designed as a molecular drug dispenser with, at the lower
rim, penicillin or quinolone moieties attached via a labile bound,2
as well as on calixarene derivatives displaying intrinsic antimicro-
bial activity. For the latter, among the various approaches that are
under current evaluation in our group, one is devoted to polycat-
ionic calixarene-based podands integrating, for example, guanidi-
nium groups.3 In recent years, the guanidinium derivatives have
been but infrequently investigated as pharmaceutical antimicro-
bial agents.4 Most compounds studied are poly-guanidinium spe-
cies derived from the old antidiabetic and trypanocidal drugs
Synthalin A or B,5 in which the guanidiniums are found at the ends
or along alkyl or polymeric chains, that could be regarded as flex-
ible linear organizing templates.
have shown interesting levels of activity against bacteria,7 fungi,
cancerous cells and viruses,8 enveloped viruses,9 as well as against
thrombosis10 and fibrotic diseases.11 In the mid 50s, the calixarene
derivative ‘Macrocyclon’,12 and more recently some parent spe-
cies,13 were studied in the treatment of tuberculosis and other
mycobacterioses. Functionalized calixarene mimics of vancomycin
has also been studied as antimicrobial agents.14
Given that most bacteria have a negatively charged surface, the
introduction of positive charges on the calixarene core, that should
lead to a constrained and highly organized oligomeric polycation,
could give rise, with regards to the simple phenolic analogue, to
an interesting synergistic effect in ionic interactions with the sur-
face of bacteria, possibly resulting in an antibacterial activity.
Of the few simple organic cations available, guanidinium was
first chosen for its stability over a large range of pH values. Only
few calixarene guanidinium derivatives have been studied so
far.15 Biological studies related to plasmid DNA binding and cell
transfection16a–c or endothelial cell proliferation inhibition16d
properties of upper- or lower-rim guanidylated calixarene deriva-
tives have recently been described.
More rigid, the calixarene species are oligomeric phenolic mac-
rocycles that have demonstrated their excellent organizational
behaviour for a multitude of active functionalities.6 Some but very
Consistent with the anticipated synergistic effect described above,
we have recently shown3 that the tetra-para-guanidinoethyl-
calix[4]arene 4 displayed very interesting antibacterial properties
against various Gram positive and Gram negative reference strains
* Corresponding author. Tel.: +33 03 83682315; fax: +33 03 83682345.
E-mail address: jean-bernard.regnouf@pharma.uhp-nancy.fr (J.-B. Regnouf-de-
Vains).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.040

M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
5497
of bacteria, at levels similar to those required with the well-known
hexamidine 34, but with a lower cytotoxicity. We have also show-
n3a that the constitutive monomer of 4, the para-guanidinoethyl-
phenol 18, does not display any antibacterial activity, confirming
our first hypothesis on the probable role of cyclotetramerization
on the acquisition of activity.
As reference compounds, we have chosen the old anthelminthic
but toxic (pancreas) Synthalin A 33,3,5a having two guanidino
groups attached at the ends of a linear decyl chain, and the well-
known and widely used cutaneous antiseptic 1,6-bis-(para-amidi-
nophenoxy)hexane 34 (hexamidine). The latter, as commercially
available antibacterial compound with the structurally closest io-
nic functionality, is described as an antibacterial for Gram positive
bacteria.
We present here the syntheses of calixarenes species 4, 14, 15
and 16, and of their constitutive monomers 18, 29, 30 and 31, as
well as Synthalin A 33, accompanied by evaluation of their antibac-
terial activity and cellular viability.
In order to enlarge this new family, and as part of our centre of
interest deals with metal chelation that might be involved in bio-
logical activity, we have prepared the chelating podands 14, 15
and 16, incorporating in alternate positions of the lower rim of 4,
two 2,20-bipyridyl-methyl ( -methyl/14, b-methyl/15) or 2,20-
a
bithiazolyl-methyl subunits, respectively. In the cone conforma-
tion of the calixarene, these derivatives should display an amphi-
philic character, strongly orienting the resulting tetracationic
head towards the bacterium surface, and leaving the additional
functionalities externally or internally oriented for further interac-
tions. To evaluate the strength of the synergistic effect generated
by the calixarene platform, we have also prepared the correspond-
ing bipyridyl and bithiazolyl para-guanidinoethylphenyl ethers 29,
30 and 31 (Scheme 1).
2. Chemistry
2.1. Synthalin A and hexamidine
Synthalin A 33 was prepared (Scheme 2) by an adaptation of
the procedure of Elliot et al.,17 based on the reaction of 1,12-diam-
inodecane and 1,3-bis-(tert-butoxycarbonyl)-2-methyl-2-thio-
Scheme 1. Water-soluble guanidinium and amidinium compounds of this study.

5498
M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
Scheme 2. Reagents and conditions: (i) J, NEt3, HgCl2, DMF, Ar, rt, 52%; (j) TFA, CH2Cl2, rt, dialysis, 70%.
pseudourea J, in the presence of HgCl2; the resulting tetra-(Boc)
species 32 was treated with TFA in CH2Cl2 at rt to give after dial-
ysis and lyophilization the double trifluoroacetate salt 33. All
analyses were consistent with the proposed structure, and ele-
mental analysis of each batch confirmed the presence of one mol-
ecule of associated water.
The hexamidine di-isetionate 34 was obtained by evapora-
tion of solvents and lyophilization of the commercial solution,
containing also 0.75 equiv of sodium acetate. The composition
of each batch was monitored by 1H NMR and elemental
analysis.
2.2. Calixarenes
The tetra-para-guanidinoethylcalix[4]arene tetra-trifluoroace-
tate 4 was prepared via a four-step process from the tetra-
para-aminoethyl-calix[4]arene A, obtained as tetra-hydrochloride
salt according to Gutsche et al.18 Following a standard procedure
(Scheme 3), A was then transformed (90% yield) into its tetra-
(N-tert-butoxycarbonyl)- analog 1, easy to purify, and key com-
pound for the development of lower-rim substituted derivatives.
Compound 1 was then deprotected in acidic conditions (TFA,
CH2Cl2) to give the tetra-para-aminoethyl-calix[4]arene, tetra-
Scheme 3. Reagents and conditions: (i) (Boc)2O, NaOH, dioxane, H2O, rt, ca. 12 h, 90%; (j) RCH2Br, K2CO3, MeCN, reflux, Ar, 5–7 h, 5: R = 6-yl-(60-methyl-2,20-bipyridine) (bpy-
), 70%; 6: R = 5-yl-(50-methyl-2,20-bipyridine) (bpy-b), 61.4%; 7: R = 4-yl-(40-methyl-2,20-bithiazole) (btz), 69%; (k) CH2Cl2, TFA, rt, 2: R = H, 92%; 8: R = bpy-
, 91%; 9:
R = bpy-b, 96%; 10: R = btz, 93%; (l) K, NEt3, CH2Cl2, MeOH, rt, 3–5 h, 3: R = H, 65%; 11: R = bpy- , 76%; 12: R = bpy-b, 84%; 13: R = btz, 77%; (m) CH2Cl2, TFA, rt, 4: R = H, 95%,
14: R = bpy- , bpy-b, btz: see offset in Scheme 4).
ꢀTFA, 74%, 15: R = bpy-b, 83%; 16: R = btz, 73%. (bpy-
a
a
a
a
a

M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
5499
trifluoroacetate 2, in 92% yield. The reaction of 2 with N1,N2-(di-
Boc)-N3-triflylguanidine K under the conditions defined by Baker
et al.19 afforded the octa-boc guanidino derivative 3 in 65% yield
after chromatography. Attempts to prepare 3 with the Elliot’s less
expensive process resulted in the formation of multiple products
not easy to separate, leading us to prefer Baker’s procedure.
Acidic hydrolysis of 3 in TFA/CH2Cl2 gave the tetra-guanidino
derivative 4, in 95% yield after a quality normalization process
involving multiple dialysis–lyophilization cycles.
According to the criterions established by de Mendoza and co-
workers,23 13C NMR spectrum of 4 (D2O) indicated that the mole-
cule adopts the cone conformation, showing an Ar-CH2-Ar reso-
nance signal at d 30.96. Nonetheless, the corresponding protons
appear as a broad singlet at 3.82 ppm, indicating that the confor-
mation is quite mobile.3a The incorporation of bipyridyl and
bithiazolyl arms resulted in rigidification of the calixarene plat-
form in the cone conformation, characterized by the appearance
of AB systems (JAB = ca. 13 Hz) for the Ar-CH2-Ar protons in the
1H NMR spectra of 14, 15 and 16, and by corresponding 13C NMR
resonance signals at d ca. 31 ppm. Elemental analysis of com-
pounds 14 and 15 was consistent with the presence of 6 molecules
of CF3COOH, indicating the additional protonation of each bipyri-
dyl group, while 16, that includes the less basic bithiazolyl groups,
contains 4 acid groups. Negative and/or positive mode electrospray
was employed for mass spectrometric analysis of all compounds,
giving in each case the expected information. As already observed
for other poly-ionic calixarene species,24 the amino- and guani-
dino-derivatives generally showed, here in the positive mode, a
succession of groups of signals attributed to mono-, di- and tri-
charged species resulting from the loss of one to four CF3COOH
equilibrated by protonation.
The preparation of the bis-(2,20-bipyridyl) and bis-(2,20-bithiaz-
olyl) guanidino podands 14, 15 and 16 followed a similar strategy,
starting from the tetra-para-(boc-aminoethyl)calix[4]arene 1.
Thus, the reaction of 6-bromomethyl-60-methyl-2,20-bipyridine20a
or 5-bromomethyl-50-methyl-2,20-bipyridine20b,c or 4-bromo-
methyl-40-methyl-2,20-bithiazole21 with 1 in dry MeCN, in the
presence of K2CO3 as base, and following the usual stoichiometries
proposed for base-strength regioselective functionalization of ca-
lix[4]arene,22 afforded the podands 5, 6 and 7, respectively, with
yields of 60–70%. Acidic hydrolysis of amine protecting groups
was performed under the usual conditions (TFA, CH2Cl2), giving
the tetra ammonium trifluoroacetates 8, 9 and 10, respectively,
in almost quantitative yields.
The reaction of 8, 9 and 10 with K under the conditions of Baker
et al.19 afforded in yields of ca. 75% after chromatography the octa-
boc guanidino bipyridyl and bithiazolyl derivatives 11, 12 and 13,
respectively. Attempts to synthesize 11, 12 and 13 by reaction of
bromomethyl heterocycles with the protected guanidino calixa-
rene 3 gave unsatisfactory results, notably in terms of yield and
ease of purification process. Acidic hydrolysis of the Boc-protective
groups in TFA/CH2Cl2 mixture finally gave the corresponding tetra-
guanidinium trifluoroacetates 14, 15 and 16, respectively, in yields
of 75–85%; a quality normalization process involving multiple dial-
ysis–lyophilization cycles was applied in each case.
3. Biological evaluation
The para-guanidinoethylphenol 18, monomer of 4, has been
evaluated as an uptake inhibitor of prazosin by transport-P
system,25 for treating mitochondria-associated diseases,26 non-
insulin-dependent diabetes mellitus and obesity,27 and hypoten-
sion,28 but no antibiotic activity evaluation had been described
until our previous report.3a
In this study, in vitro antibacterial activity [e.g., minimum inhi-
bition concentration (MIC) determination] of compounds 4, 14, 15
and 16 was evaluated and compared to their corresponding mono-
mers 18, 29, 30 and 31. Hexamidine 34 and 1,10-bis-guanidinode-
cane (Synthalin A) 33 were chosen as reference compounds. MICs
were determined for two Gram negative (Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 27853) and three Gram
positive (Staphylococcus aureus ATCC 25923 and ATCC 29213,
Enterococcus faecalis ATCC 29212) reference strains, by broth mic-
rodilution method recommended by the CLSI.29 Due to its presence
in high amounts in calixarenes species, the trifluoroacetate anion
was also evaluated; in fact, sodium trifluoroacetate did not exhibit
2.3. Phenol derivatives
The para-guanidinoethylphenol was prepared as its trifluoro-
acetate salt 18 by acidic hydrolysis of the para-N1,N2-di(Boc)-gua-
nidinoethylphenol 17 in a mixture of TFA and CH2Cl2 at rt.
Compound 17 was obtained from tyramine hydrochloride F in
two different ways: the first one was an adaptation of the proce-
dure of Elliot et al.17 similar to that providing 33, with an interest-
ing yield of 90%; the second one consisted in the reaction of K with
F, adapting the procedure of Baker et al.,19 giving 17 with in 72%
yield. As for the calixarenes, the introduction of the bipyridyl and
bithiazolyl arms was done on an N-protected derivative of tyra-
mine; for consistency in work-up procedures, the boc-protective
group was chosen. The N-Boc-para-aminoethylphenol 19 was pre-
pared by reaction of (Boc)2O and F under standard conditions, with
a yield of ca. 70% after chromatography. The bipyridyl and bithiaz-
olyl derivatives 20, 21 and 22 were prepared in yields of ca. 80%
after chromatography via nucleophilic attack of 6-bromomethyl-
6-methyl-2,20-bipyridine, 5-bromomethyl-50-methyl-2,20-bipyri-
dine or 4-bromomethyl-40-methyl-2,20-bithiazole by 19 in MeCN,
using K2CO3 as base. Standard acidic deprotection of amino groups
of 20, 21 and 22 afforded the trifluoroacetate salts 23, 24 and 25, in
yields of 85%, 96% and 93%, respectively. The formation of the
di(Boc)-guanidino derivatives 26 and 28 was carried out according
to the two procedures mentioned above, with a clearly better yield
when using N,N0-bis(tert-butoxycarbonyl)-N00-triflylguanidine K.
Only this procedure was employed to synthesize 27. The acidic
hydrolysis of Boc-protective groups afforded the bipyridyl deriva-
tives 29 and 30 in yields of 80% and 95%, respectively, and the
bithiazolyl derivative 31 in almost quantitative yield.
any activity at concentrations inferior to 256 l .
g mLꢁ1
Drugs were also tested for their effect on eukariotic cell viability
by MTT assays on MRC-5 cells (human pulmonary embryonic
fibroblasts), or by LDH assays in order to determine IC50 (inhibitory
concentration 50%) and deduce selectivity indexes (SI), at 24, 48
and 168 h exposure to drugs.
As shown in Table 1, the MIC values obtained for the calixarene
species 14, 15 and 16 remain in the micromolar range, close to the
unsubstituted analog 4 and to hexamidine 34, but with a better
homogeneity with regards to the Gram character of the bacterial
strains. The introduction of the bipyridyl and bithiazolyl arms at
the lower rim of the calixarene platform does not result in a strong
modification of the apparent antibacterial behaviour, with regards
to 4.
Passing to the monomeric species 29, 30 and 31, and depend-
ing on the bacterial strain, this behaviour changes drastically.
These compounds display a variable but sometimes very inter-
esting antibacterial activity, notably against the two S. aureus
strains, and, for 30, against E. coli. Compared to the unsubstitut-
ed analog 18, the presence of the biheterocyclic arms appears to
generate an important gain of activity against all of the tested
strains.
1H, 13C NMR, elemental analyses and mass spectrometry were
consistent with the proposed formulas for all new compounds.

5500
M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
Scheme 4. Reagents and conditions: (i) (Boc)2O, NaOH, dioxane, H2O, rt, ca. 12 h, 73%; (j) RCH2Br, K2CO3, MeCN, reflux, Ar, 5 h, 20: R = 6-yl-(60-methyl-2,20-bipyridine) (bpy-
), 82%; 21: R = 5-yl-(50-methyl-2,20-bipyridine) (bpy-b), 80%; 22: R = 4-yl-(40-methyl-2,20-bithiazole) (btz), 85%; (k) CH2Cl2, TFA, rt, 23: R = bpy-
ꢀ1/2 TFA, 86%; 24: R = bpy-b,
96%; 25: R = btz, 95%; (l) way 1: K, NEt3, CH2Cl2, MeOH, rt, 2–4 h, 17: R = H, 72%; 26: R = bpy- , 85%; 27: R = bpy-b, 84%; 28: R = btz, 95%; way 2: J, NEt3, HgCl2, DMF, rt, Ar, 17:
, 64%; 28: R = btz, 60%; (m) CH2Cl2, TFA, rt, 18: R = H, 90%; 29: R = bpy-
ꢀ1/2 TFA, 82%; 30: R = bpy-b, 95%; 31: R = btz, 98%.
a
a
a
R = H, 90%; 26: R = bpy-
a
a
Table 1
MIC values (l
g mLꢁ1 and (10ꢁ6 mol Lꢁ1)) obtained by broth microdilution method, according to CLSI guideline
E. coli
P. aeruginosa
S. aureus
S. aureus
E. faecalis
ATCC 25922
ATCC 27853
ATCC 25923
ATCC 29213
ATCC 29212
Minimum inhibitory concentration (MIC) in
l
g mLꢁ1 (10ꢁ6 mol Lꢁ1
)
4 (1239.0)
4 (3.2)
512 (1750.0)
16 (8.6)
128 (240.0)
32 (17)
8 (16.8)
32 (26.0)
>512 (1750.0)
16 (8.6)
128 (240.0)
32 (17)
128 (270)
16 (9.5)
128 (262.5)
128 (256.8)
32 (47.9)
8 (6.5)
8 (6.5)
32 (26.0)
>512 (1750.0)
16 (8.6)
64 (120.2)
8 (4.2)
64 (135)
8 (4.7)
64 (131.3)
256 (513.6)
2 (3.0)
18 (293.24)
14 (1867.53)
29 (532.47)
15 (1885.56)
30 (475.46)
16 (1695.49)
31 (487.52)
33 (498.45)
34 (668.22)
512 (1750.0)
16 (8.6)
16 (30.0)
8 (4.2)
8 (16.8)
8 (4.7)
16 (33.0)
64 (128.4)
4 (6.0)
512 (1750.0)
16 (8.6)
8 (15.0)
8 (4.2)
4 (8.4)
16 (9.5)
16 (9.5)
8 (16.4)
16 (32.0)
<1 (1.5)
64 (131.3)
64 (128.4)
8 (12.0)
Molecular weights in italic.
The analysis of molar or massic MIC ratios (Table 2; monomer
vs calixarene) confirms the smaller impact of the calixarene struc-
ture on the antibacterial activity, for the group of the substituted
species.
As shown in Table 3, the bipyridyl and bithiazolyl analogs 14,
16, 29, 31 and, to a lesser extent, 15 and 30, have an impact on
MRC-5 cells viability stronger than 4 and 18, reaching the IC50 of
hexamidine 34 and Synthalin A 33 at 24 h.
Table 2
Massic (mr) and molar (Mr) MIC ratios between calixarene species and their
corresponding monomers
18/4
29/14
30/15
31/16
mr
Mr
mr
Mr
mr
Mr
mr
Mr
E. coli ATCC 25922
128
>16
64
547
>67
269
269
>67
8
8
1
28
28
3.5
1.7
14
0.25
4
1
16
4
4
8
2
14
28
7
P. aeruginosa ATCC 27853
S. aureus ATCC 25923
S. aureus ATCC 29213
E. faecalis ATCC 29212
1
The calculated selectivity indexes SI, that is, IC50 at 24 h/MIC re-
ported in Table 4, thus appear less interesting for the substituted
species in the calixarene family, and sometimes better for the
64
0.5
4
0.5
8
2
32
0.5
8
1.7
28
>16

M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
5501
Table 3
IC50 values obtained with viability assays (MTT; a:LDH)
4
18
293.2
14
1867.5
29
532.5
15a
1885.5
30a
475.5
16
1695.5
31
487.5
33
498.5
34
668.2
1239.0b
IC50 (l )
g mLꢁ1
24 h
48 h
168 h
256<
256<
128–256
256<
256<
32-64
16–32
16–32
16–32
32–64
16–32
2–4
>128
>128
nd
>128
>128
nd
16–32
16–32
16–32
64–128
32–64
8–16
128–256
8–16
<1
32–64
16–32
4–8
Compounds were added at concentrations from 256 to 1
l
g mLꢁ1 for 24, 48 and 168 h. Each value is representative of three independent determinations. nd: not determined.
bMolecular weights in g molꢁ1
.
Table 4
this sense, such properties could be involved in the structure–
activity relationships of the calixarene family, but seem only trans-
lated by a gain in cellular toxicity.
Selectivity indexes calculated for the five reference strains, after 24 h exposure of
MRC-5 cells to the compounds of the study
E. coli
ATCC 25922
P. aeruginosa
ATCC 27853
S. aureus
ATCC 25923
S. aureus
ATCC 29213
E. faecalis
ATCC 29212
The development of new derivatives and investigations devoted
to the mode of antibacterial action of these two new families of
compounds are now under way.
Selectivity indexes IC50 24 h/MIC
4
>64
>0.5
1–2
0.25–0.5
>4
>8
0.5
1–2
0.25–0.5
>4
>32
>0.5
1–2
2–4
>16
>16
2–4
4–8
2–4
8–16
>32
>0.5
1–2
4–8
>16
>32
1–2
8–16
8–16
32–64
>8
0.5
18
14
29
15
30
16
31
33
34
5. Experimental
5.1. Chemistry
1–2
0.5–1
>16
>2
2–4
1–2
0.5–1
16–32
16
>1
1–2
1–2
2–4
4–8
1–2
0.5–1
1–2
1–2
5.1.1. General remarks
Melting points (°C, uncorrected) were determined on an Elec-
trothermal 9200 in Capillary apparatus. 1H and 13C spectra were
recorded on a Bruker DRX 400 (chemical shifts in ppm). Mass spec-
tra (electronic ionization—EI, and electrospray—ES) were recorded
on a Micromass Platform II apparatus, at the Service Commun de
Spectrométrie de Masse Organique, Nancy. Infrared measurements
monomers (cases of 30 and 31). Nevertheless, the bipyridyl calixa-
rene 15 exhibits a relatively pronounced selectivity towards Gram
positive strains, with SI >16.
It is interesting to note (Table 3) that the toxicity of all calixarene
species remains stable at 48 and 168 h, while the monomers, Synth-
alin A and hexamidine become more toxic, resulting for the latter in
lower selectivity indexes. This fact can be of great importance when
considering differences in eukaryotic and prokaryotic cell growth
kinetics, or when considering long-lasting decontamination
processes. This point is the subject of current specific investigations.
were performed on a Vector 22 Bruker FT apparatus (KBr,
cmꢁ1) and UV spectra were recorded a SAFAS UV mc2 apparatus,
kmax in nm,
in dm3 molꢁ1 cmꢁ1. Elemental analyses were per-
m in
e
formed at the Service de Microanalyse, Nancy. Merck TLC plates
were used for chromatography analysis (SiO2, ref 1.05554; Al2O3,
ref 1.05581). All commercially available products were used with-
out further purification unless otherwise specified.
5.1.2. 5,11,17,23-Tetra-(Boc-aminoethyl)-25,26,27,28-
calix[4]arene tetrol 1
4. Conclusion
1 M aqueous NaOH (14 mL, 14 mmol) was added to a solution of
A (0.83 g, 1.05 mmol) in a mixture of dioxane and H2O (40 and
24 mL, respectively). After stirring during 10 min, di-tert-butyl-
dicarbonate (Boc2O) (0.97 g, 4.44 mmol) was added. The resulting
solution was stirred overnight at rt and under Ar (TLC monitoring:
Al2O3, CH2Cl2/MeOH, 98:2). The solution was concentrated under
vacuum, and AcOEt (40 mL) was added; the pH was brought to 8
with 1 M HCl, and the organic phase was recovered. The aqueous
phase was extracted with AcOEt (2 ꢂ 30 mL), and the combined or-
The results obtained in this study show that the introduction of
two bipyridyl or bithiazolyl arms at the lower rim of the tetra-
para-guanidinoethyl-calix[4]arene platform does not greatly
modify the antibacterial properties of the lead compound 4, main-
taining the micromolar activity levels encountered for commercial
products for both the reference Gram positive and negative strains.
These new calixarene compounds have a stronger impact on
eukariotic cell viability at 24 h, thus diminishing their selectivity
indexes; nevertheless, the fact that this toxicity remains stable
for calixarenes 4, 14, 15 and 16 at 48 and 168 h, a contrario to ref-
erence compounds, reinforces the general interest of these struc-
tures for the building of a new family of antibacterial agents.
As for the initially-studied unsubstituted derivatives, but to a
lesser extent, our results tend to confirm the organizational role
of the calixarene core, that tethers close together and arrays at
its upper rim four guanidinium groups, for the genesis of an anti-
bacterial activity. At this stage of the study, and according to pre-
vious results, this seems indicative of a synergistic effect in ionic
interactions with the membrane targets. Nevertheless, the intro-
duction of the biheterocyclic substituents on the single phenol unit
(monomers) generates an antibacterial activity unforeseen with
the unsubstituted analog. This may be attributable to either the
amphiphilic character of the new monomeric species, or to a spe-
cific activity of the water-solubilized biheterocyclic subunits. In
ganic phases were dried over Na2SO4, and evaporated to dryness.
The resulting oily material was chromatographed (Al2O3, CH2Cl2/
MeOH, 99:1) to give 1 (0.93 g, 90%). White solid. Mp: 131–
131 °C. IR (KBr): 3351.6 (OH); 1701.3 (CO); 1509.6 (NH). UV–vis
(CH2Cl2): 280 (31,186). 1H NMR (400 MHz, CDCl3): 1.27 (m, 36H,
Me3C); 2.59 (t, J = 6.6 Hz, 8H, CH2CH2N); 3.27 (br s, 8H, CH2CH2N);
3.44–4.21 (AB, JAB = 13.6 Hz; 8H, ArCH2Ar); 6.86 (s, 8H, ArH); 10.26
(s, 4H, OH). 13C NMR (100 MHz, CDCl3): 28.64 (Me3C); 32.34
(ArCH2Ar); 35.73 (CH2CH2N); 42.17 (CH2CH2N); 79.54 (Me3C);
128.57 (Co); 129.91 (Cm); 132.96 (Cp); 147.75 (Cipso); 156.40 (CO).
Anal. Calcd for C56H76O12N4 (997.31): C, 67.44; H, 7.68; N, 5.61.
Found: C, 67.43; H, 7.35; N, 5.61. ES-MS (pos. mode): 1019.08
[M+Na+]+. ES-MS (neg. mode): 995.15 [MꢁH+]ꢁ.
5.1.3. Compounds 5, 6 and 7—general procedure
A mixture of 1 (1 equiv) and K2CO3 (1 equiv) in dry MeCN was
refluxed under Ar during 0.5 h. The bromoalkyl reactant (2 equiv)

5502
M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
was then added, and reflux was maintained during ꢂ h (TLC mon-
itoring, Al2O3, CH2Cl2/MeOH 98:2). After cooling to rt, the solvent
was evaporated to dryness, and the solid residue was dissolved
in CH2Cl2, filtered, then chromatographed to give the pure podand.
UV–vis (CH2Cl2): 291 (23,731); 330 (34,366). 1H NMR (400 MHz,
CDCl3): 1.46 (m, 36H, Me3C); 2.48 (br t, J = 7.1 Hz, 4H, CH2CH2N
of B); 2.54 (s, 6H, Mebtz); 2.67 (br t, J = 6.9 Hz, 4H, CH2CH2N of
A); 3.14 (br t, J = 6.5 Hz, 4H, CH2CH2N of B); 3.30 (m, 4H, CH2CH2N
of A); 3.32–4.30 (AB, JAB = 13.1 Hz, 8H, ArCH2Ar); 4.55 (br s, 2H, NH
of A or B); 4.66 (br s, 2H, NH of A or B); 5.23 (s, 4H, OCH2 btz); 6.73
5.1.3.1. 5,11,17,23-Tetra-(Boc-aminoethyl)-25,27-bis(6-methyl-
eneoxy-60-methyl-2,20-bipyridyl)-26,28-dihydroxycalix[4]arene
5. From 1 (0.1 g, 0.1 mmol), K2CO3 (0.014 g, 0.1 mmol), MeCN
0
(s, 4H, ArH of B); 6.87 (s, 4H, ArH of A); 7.00 (s, 2H, H5 btz); 7.70 (br
s, 2H, OH); 7.96 (s, 2H, H5 btz). 13C NMR (100 MHz, CDCl3): 17.54
(Me btz); 28.89 (CMe3); 31.91 (ArCH2Ar); 35.67 (CH2CH2N of A);
36.17 (CH2CH2N of B); 42.21 (CH2CH2N of B); 42.56 (CH2CH2N of
(10 mL),
6-bromomethyl-60-methyl-2,20-bipyridine
(0.053 g,
0.2 mmol); 5 h (TLC monitoring, Al2O3, CH2Cl2/MeOH 98:2); chro-
matography: Chromatotron, Al2O3, CH2Cl2; 5 (0.09 g, 70%). White
powder. Mp: 114–115 °C. IR (KBr): 3367.5 (OH); 1707.9 (CO). UV–
vis (CH2Cl2): 290 (47737). 1H NMR (400 MHz, CDCl3): 1.41–1.43
(m, 36H, Me3C); 2.43 (m, 4H, CH2CH2N of B); 2.61 (s, 6H, Me bpy);
2.69 (m, 4H, CH2CH2N of A); 3.11 (m, 4H, CH2CH2N of B); 3.33 (m,
4H, CH2CH2N of A); 3.34–4.38 (AB, JAB = 13.2 Hz, 8H, ArCH2Ar);
4.50–4.62 (m, 4H, NH); 5.21 (s, 4H, OCH2bpy); 6.72 (s, 4H, ArH of
0
A); 74.57 (OCH2btz); 79.50 (CMe3); 116.40 (C5 btz); 118.28 (C5
btz); 128.23 (Cp of A); 129.40 (Cm of A); 129.88 (Cm of B); 129.99
(Co of B); 133.55 (Co of A); 136.45 (Cp of B); 150.93 (Cipso of B);
0
152.03 (Cipso of A); 154.02 (C2 btz); 154.75 (C2 btz); 156.24 (COO
0
of B); 156.39 (COO of A); 160.76 (C4 btz); 162.27 (C4 btz). Anal.
Calcd for C72H88O12N8S4ꢀ2CH2Cl2 (1552.45): C, 57.13; H, 5.96; N,
7.20; S, 8.25. Found: C, 56.97; H, 5.49; N, 7.27; S, 8.53. ES-MS
(pos. mode): 1406.6 [M+Na+]+.
0
B); 6.89 (s, 4H, ArH of A); 7.04 (d, J = 7.5 Hz, 2H, H5 bpy); 7.54 (t,
0
J = 7.5 Hz, 2H, H4 bpy); 7.61 (d, J = 7.5 Hz, 2H, H4 bpy); 7.76 (br s,
0
2H, OH); 8.04 (d, J = 7.6 Hz, 2H, H5 bpy); 8.16 (d, J = 7.8 Hz, 2H, H3
5.1.4. Compounds 2, 8, 9 and 10—general procedure
A solution of tetra-boc aminoethyl calixarene derivative in dry
CH2Cl2 and TFA was stirred at rt under Ar during ca. 3 h. The sol-
vent were evaporated, and the residual TFA was eliminated by five
dissolution in CH2Cl2–evaporation cycles. The semi-solid residual
material was dried under high vacuum, then triturated in dry
Et2O to give the corresponding salt.
bpy); 8.35 (d, J = 7.8 Hz, 2H, H3 bpy). 13C NMR (100 MHz, CDCl3):
25.00 (Me bpy); 28.89 (CMe3); 31.98 (ArCH2Ar); 35.69 (CH2CH2N of
A); 36.13 (CH2CH2N of B); 42.24 (CH2CH2N of B); 42.63 (CH2CH2N
of B); 79.16 (OCH2bpy); 79.47 (CMe3); 118.70 (C3 bpy); 120.51 (C3
bpy); 121.51 (C5 bpy); 123.75 (C5 bpy); 128.27 (Co of A); 129.37
0
0
(Cm of A); 129.77 (Cp of A); 129.86 (Cm of B); 133.48 (Co of B);
0
136.26 (Cp of B); 137.45 (C4 bpy); 128.50 (C4 bpy); 151.19 (Cipso of
0
B); 152.26 (Cipso of A); 155.68 (C6 bpy); 156.22 (C6 bpy); 156.39
5.1.4.1. 5,11,17,23-Tetra-(aminoethyl)-25,26,27,28-calix[4]arene
tetrol, tetra-trifluoroacetate salt 2. From 1 (0.53 g, 0.54 mmol),
CH2Cl2 (40 mL), TFA (10 mL); 2 (0.53 g, 92%). White precipitate. Mp:
>200 °C (dec). IR (KBr): 3144.7 (OH); 1686.6 (COO). UV–vis (H2O):
284 (9675). 1H NMR (400 MHz, DMSO-d6): 2.59 (t, J = 7.9 Hz, 8H,
CH2CH2N), 2.92 (br s, 8H, CH2CH2N); 3.67 (br s, 8H, ArCH2Ar), 6.91
(s, 8H, ArH), 7.84 (br s, 12H, NH3). 1H NMR (400 MHz, D2O): 2.66 (t,
J = 7.1 Hz, 8H, CH2CH2N); 3.04 (t, J = 7.3 Hz, 8H, CH2CH2N); 3.83 (br
s, 8H, ArCH2Ar); 6.97 (s, 8H, ArH). 13C NMR (100 MHz, D2O): 31.01
(Ar-CH2-Ar); 32.25 (ArCH2CH2N); 40.83 (ArCH2CH2N); 116.78 (q,
J = 291.97 Hz, CF3COOH); 128.98 (Cm); 129.32 (Co); 130.62 (Cp);
147.80 (Cipso); 163.07 (q, J = 35.21 Hz, CF3COOH). Anal. Calcd for
C36H44O4N4ꢀ4CF3COOHꢀH2O (1070.86): C, 49.35; H, 4.70; N, 5.23.
Found: C, 49.31; H, 4.85; N, 5.30. ES-MS (pos. mode): 711.00
[Mꢁ3CF3COOH+H+]+. ES-MS (neg. mode): 1050.60 [MꢁH+]ꢁ, 936.76
[MꢁCF3COOHꢁH+]+, 822.99 [Mꢁ2CF3COOHꢁH+]+, 709.03 [Mꢁ3CF3
COOHꢁH+]+, 595.19 [Mꢁ4CF3COOHꢁH+]+.
0
(COO of A and B); 156.68 (C2 bpy); 158.23 (C2 bpy). Anal. Calcd for
C80H96O12N8ꢀ0.5H2O (1370.68): C, 70.10; H, 7.13; N, 8.17. Found:
C, 68.82; H, 6.93; N, 8.12. ES-MS (pos. mode): 681.5 [M+2H]2+/2
,
,
692.9 [M+H+Na]2+/2
, ,
703.5 [M+2Na]2+/2 711.4 [M+Na+K]2+/2
1361.9 [M+H]+, 1384.0 [M+Na]+, 1400.7 [M+K]2+
.
5.1.3.2. 5,11,17,23-Tetra-(Boc-aminoethyl)-25,27-bis(5-methyl-
eneoxy-50-methyl-2,20-bipyridyl)-26,28-dihydroxycalix[4]arene
6. From 1 (0.3 g, 0.3 mmol), K2CO3 (0.105 g, 0.8 mmol), MeCN
(30 mL),
5-bromomethyl-50-methyl-2-20-bipyridine
(0.158 g,
0.6 mmol); 4 h (TLC monitoring, SiO2, CH2Cl2/MeOH 98:2); chro-
matography: Al2O3, CH2Cl2; 6 (0.251 g, 61.4%). White powder.
Mp: 127–128 °C. IR (KBr): 3365.02 (OH); 1708.98 (CO). UV–vis
(CH2Cl2): 290 (68,214). 1H NMR (400 MHz, CDCl3): 1.42 (br s,
36H, Me3C); 3.13 (m, J = 5.8 Hz; 4H, CH2CH2N of B); 3.27 (m, 4H,
CH2CH2N of A); 3.26–4.20 (AB, JAB = 13.08 Hz, 8H, ArCH2Ar); 5.14
(s, 4H, OCH2bpy); 6.72 (s, 4H, ArH of B); 6.83 (s, 4H, ArH of A);
0
7.51 (dd, J = 7.2 Hz, 2H, H4 bpy); 7.61 (br s, 2H, OH); 8.12 (dd,
5.1.4.2. 5,11,17,23-Tetra-(aminoethyl)-25,27-bis(6-methylene-
oxy-60-methyl-2,20-bipyridyl)-26,28-dihydroxycalix[4]arene,
hexa-trifluoroacetate salt 8. From 5 (0.15 g, 0.119 mmol), CH2Cl2
(15 mL), TFA (5 mL); 8 (0.17 g, 91%). White precipitate. Mp: 100–
101 °C. IR (KBr): 3413.3 (OH); 2934.8 (NH3þ); 1677.9 (COO). UV–
vis (H2O): 286 (28,048). 1H NMR (400 MHz, D2O): 2.53 (s, 6H, Meb-
py); 2.62 (t, J = 7.3 Hz, 4H, CH2CH2N of A); 2.88 (t, J = 7.5 Hz, 4H,
CH2CH2N of B); 3.03 (t, J = 7.5 Hz, 4H, CH2CH2N of A); 3.21 (t,
J = 7.5 Hz, 4H, CH2CH2N of B); 3.56–4.33 (AB, JAB = 13.1 Hz, 8H,
ArCH2Ar); 5.13 (s, 4H, OCH2 bpy); 6.98 (s, 4H, ArH of B); 7.19 (s,
4H, ArH of A); 7.58 (d, J = 8.3 Hz, 2H, H5 bpy); 7.85 (d, J = 8.8 Hz,
0
J = 8.04 Hz, 2H, H3 bpy); 8.23 (d, J = 8.04 Hz, 2H, H4 bpy); 8.38 (d,
0
J = 7.6 Hz, 2H, H3 bpy); 8.39 (s, 2H, H6 bpy); 8.87 (s, 2H, H6 bpy).
13C NMR (100 MHz, CDCl3): 18.72 (Mebpy); 28.86 Me3C; 32.14
(ArCH2Ar); 35.64, 36.18 (CH2CH2N); 42.25, 42.52 (CH2CH2N);
76.09 (OCH2bpy); 79.45 (Me3C); 121.14, 121.27, 136.95, 137.72,
0
0
0
148.82, 149.83 (C3, C3 , C4, C4 , C6, C6 , of bpy); 129.27, 129.89 (Cm
of Ar); 128.26, 132.20, 133.68, 136.39, 150.90 (Co and Cp of Ar, C5,
C5 , of bpy); 151.96, 153.56 (Ci of Ar); 156.25 (COO); 156.40,
0
0
156.52 (C2, C2 , of bpy). Anal. Calcd for C80H96N8O12ꢀ0.25CH2Cl2
(1444.66): C, 69.70; H, 7.03; N, 8.10. Found: C, 69.87; H, 6.98; N,
8.09. ES-MS (pos. mode): 1361.5 [M+H]+, 1383.5 [M+Na]+, 1389.2
0
2H, H5 bpy); 7.91 (t, J = 7.8 Hz, 2H, H4 bpy); 8.16 (d, J = 7.8 Hz, 2H,
[3M+3H+CH2Cl2]3+/3
.
H3 bpy); 8.27 (d, J = 8.1 Hz, 2H, H3 bpy); 8.48 (t, J = 7.8 Hz, 2H, H4
0
0
bpy). 1H NMR (400 MHz, DMSO-d6): 2.54 (s, 6H, CH3 bpy); 2.60 (t,
J = 8.3 Hz, 4H, CH2CH2N of A or of B); 2.68 (t, J = 8.0 Hz, 4H, CH2CH2N
of A or B); 2.96 (m, 8H, CH2CH2N of A or B); 3.44–4.31 (AB,
JAB = 12.8 Hz, 8H, ArCH2Ar); 5.14 (s, 4H, OCH2 bpy); 6.99 (s, 4H,
ArH of A or B); 7.06 (s, 4H, ArH of A or B); 7.26 (d, J = 7.6 Hz, 2H, H
bpy); 7.66 (t, J = 7.8 Hz, 2H, H bpy); 7.70 (t, J = 7.8 Hz, 2H, H bpy);
7.77 (d, J = 7.5 Hz, 2H, H bpy); 7.88 (br s, 12H, NH); 8.19 (d,
J = 7.8 Hz, 2H, H bpy); 8.32 (d, J = 7.8 Hz, 2H, H bpy); 8.40 (s, 2H,
5.1.3.3. 5,11,17,23-Tetra-(Boc-aminoethyl)-25,27-bis(4-methyl-
eneoxy-40-methyl-2,20-bithiazolyl)-26,28-dihydroxycalix[4]arene
7. From 1 (0.33 g, 0.33 mmol), K2CO3 (0.046 g, 0.33 mmol), MeCN
(15 mL),
4-bromomethyl-40-methyl-2,20-bithiazole
(0.182 g,
0.66 mmol); 7 h (TLC monitoring, Al2O3, CH2Cl2/MeOH 98:2); chro-
matography: Al2O3, CH2Cl2; 7 (0.28 g, 69%). Yellow powder. Mp:
128–129 °C. IR (KBr): 3370.9 (OH); 1704.7 (CO); 1509.8 (NH).

M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
5503
OH). 13C NMR (100 MHz, D2O): 19.44 (Mebpy); 30.92 (ArCH2Ar);
32.31 (CH2CH2N of A); 32.36 (CH2CH2N of B); 40.57 (CH2CH2N of
A); 41.03 (CH2CH2N of B); 77.12 (OCH2bpy); 116.71 (q,
5.1.5. Compounds 3, 11, 12 and 13—general procedure
The calixarene tetra-ammonium salt, then Et3N were added to a
solution of N,N0-bis(tert-butoxycarbonyl)-N00-triflylguanidine K in
dry CH2Cl2 and MeOH. The mixture was stirred at rt under Ar
(TLC monitoring; SiO2, CH2Cl2/MeOH 99:1), and the solvent were
evaporated to dryness. The solid residue was dissolved in CH2Cl2,
washed with 2 M NaHSO4 then with saturated NaHCO3. The aque-
ous phases were washed with CH2Cl2 and the combined organic
phases were dried over Na2SO4 or MgSO4, filtered and concen-
trated. The residue was chromatographed to give the expected tet-
ra-(di-boc-guanidinoethyl) calixarene derivative.
J = 291.8 Hz, CF3COOꢁ); 122.37 (C3 bpy); 122.48 (C3 bpy); 125.13
0
0
(C5 bpy); 128.43 (C5 bpy); 129.02 (Cp of B); 129.11 (Co of A or B);
129.68 (Cm of A); 129.85 (Cm of B); 134.57 (Co of A or B); 134.75 (Cp
0
of A); 140.13 (C4 bpy); 146.55 (C6 bpy); 147.18 (C4 bpy); 147.54
0
0
(C6 bpy); 150.75 (Cipso of B); 151.20 (Cipso of A); 155.19 (C2 bpy);
156.99 (C2 bpy); 163.21 (q, J = 35.2 Hz, CF3COOꢁ). Anal. Calcd for
C60H64O4N8ꢀ6CF3COOHꢀ4H2O (1717.38): C, 50.35; H, 4.57; N, 6.52.
Found: C, 50.39; H, 4.43; N, 6.76. ES-MS (pos. mode): 1189.97
[Mꢁ2CF3COOH+H+]+, 1074.93 [Mꢁ3CF3COOH+H+]+, 481.47
5.1.5.1. 5,11,17,23-Tetra-[(N,N0-di-Boc)guanidinoethyl]-25,26,27,28-
calix[4]arene tetrol 3. From A (0.24 g, 0.32 mmol) or 2 (0.34 g;
0.32 mmol), Et3N (0.53 mL, 3.81 mmol), K (0.50 g, 1.28 mmol) in
CH2Cl2/MeOH (30 mL; 1:1). 3 h (TLC monitoring; SiO2, CH2Cl2/MeOH
99:1); washings: 2 M NaHSO4 (30 mL), satd NaHCO3 (30 mL), CH2Cl2
(10 mL). Drying: MgSO4. Chromatography (SiO2, from CH2Cl2 to
CH2Cl2/MeOH 99:1). 3 (0.33 g, 65%). White solid. Mp: 134–135 °C. IR
(KBr): 3336.8 (OH); 2978.8 (CH3); 1721.2 (COO); 1638.7 (NH). UV–vis
(CH2Cl2): 280 (11,703). 1H NMR (400 MHz, CDCl3): 1.50 (s, 9H, Me3C);
1.52 (s, 9H, Me3C); 2.69 (t, J = 7 Hz, 8H, CH2CH2N); 3.47–4.22 (AB,
JAB = 12.8 Hz, 8H, ArCH2Ar); 3.63 (q, J = 6.5 Hz, 8H, CH2CH2N); 6.92 (s,
8H, ArH); 8.41 (s, 4H, CH2NH); 10.17 (s, 4H, OH); 11.50 (s, 4H, CNH).
13C NMR (100 MHz, CDCl3): 28.47 (Me3C); 28.72 (Me3C); 32.12 (ArCH2-
Ar); 34.84 (ArCH2CH2NH); 42.59 (ArCH2CH2NH); 79.75 (Me3C); 83.47
(Me3C); 128.69 (Co); 129.75 (Cm); 132.50 (Cp); 147.80 (Cipso); 153.57
(CO); 156.46 (C guan); 163.89 (CO). Anal. Calcd for C80H116O20N12
(1565.85): C, 61.36; H, 7.46; N, 10.73. Found: C, 61.27; H, 7.35; N,
10.68. ES-MS (neg. mode): 1562.5 [MꢁH+]ꢁ, ES-MS (pos. mode):
1587.66 [M+Na+]+.
[Mꢁ4CF3COOH+2H+]2+/2, 321.51 [Mꢁ4CF3COOH+3 H+]3+/3
.
5.1.4.3. 5,11,17,23-Tetra-(aminoethyl)-25,27-bis(5-methylene-
oxy-50-methyl-2,20-bipyridyl)-26,28-dihydroxycalix[4]arene, hexa-
trifluoroacetate salt 9. From 6 (0.2 g, 0.15 mmol), CH2Cl2 (20 mL),
TFA (7 mL); 9 (0.199 g, 96%). Slightly pink solid. Mp: 129–130 °C. IR
(KBr): 3400.2 (OH); 2900.8 (NH3þ); 1660.1 (COO). UV–vis (H2O): 286
(38,304). 1H NMR (400 MHz, D2O) 2.41 (s, 6H, Mebpy); 2.65 (t,
J = 7.42 Hz, 4H, CH2CH2NH of A); 2.87 (t, J = 7.3 Hz, 4H, CH2CH2N of B);
3.04 (t, J = 7.54 Hz, 4H, CH2CH2N of B); 3.22 (t, J = 7.3 Hz, 4H, CH2CH2N
of B); 3.55–4.21 (AB, JAB = 13.6 Hz, 8H, ArCH2Ar); 5.23 (s, 4H, OCH2
0
bpy); 6.97 (s, 4H, ArH of B); 7.16 (s, 4H, ArH of A); 7.82–7.89 (m, 2H4
0
0
and 4H3 ,3 of bpy); 8.26 (s, 2H, H6 bpy); 8.37 (d, J = 7.8 Hz, 2H, H4 bpy);
9.03 (s, 2H, H6 bpy). 13C NMR (100 MHz, D2O): 17.74 (Mebpy); 30.93
(ArCH2Ar); 32.25, 32.29 (CH2CH2N); 40.47, 41.00 (CH2CH2N); 74.47
(OCH2bpy); 116.71 (q, J = 291.8 Hz, CF3COOꢁ); 122.00, 122.31, 129.55,
0
0
0
129.93, 137.78, 142.94, 145.55, 146.68 (C3, C3 , C4, C4 , C6, C6 , of bpy
and Cm of Ar); 128.11, 128.70, 133.79, 135.00, 135.58, 137.69, 147.10,
0
0
149.05, 150.26, 151.30 (C2, C2 , C5, C5 of bpy; Cp, Co and Ci of Ar);
163.21 (q, J = 35.2 Hz, CF3COOꢁ). Anal. Calcd for C60H64O4N8ꢀ6CF3
COOHꢀ1.5H2O (1671.48): C, 51.71; H, 4.40; N, 6.70. Found: C, 51.69; H,
4.19; N, 6.87. ES-MS (pos. mode): 1075.3 [Mꢁ3CF3COOH+H+]+, 961.3
5.1.5.2. 5,11,17,23-Tetra-[(N,N0-di-Boc)guanidinoethyl]-25,27-
bis(6-methyleneoxy-60-methyl-2,20-bipyridyl)-26,28-dihydroxy-
calix[4]arene 11. From 8 (0.28 g, 0.163 mmol), CH2Cl2 (20 mL),
[Mꢁ4CF3COOH+ H+]+, 481.29 [Mꢁ4CF3COOH+2H+]2+/2
.
MeOH (some drops), Et3N (0.33 mL, 2.37 mmol),
K (0.31 g,
0.79 mmol). 3 h (TLC monitoring: Al2O3, CH2Cl2). CH2Cl2 (30 mL);
washings: 2 M NaHSO4 (30 mL), satd NaHCO3 (30 mL). Drying:
Na2SO4. Chromatography (Al2O3, CH2Cl2). 11 (0.24 g, 76%). White
powder. Mp: 138–139 °C. IR (KBr): 3336.5 (OH), 1721.7 (CO);
1639.77 (NCO, NHCO). UV–vis (CH2Cl2): 288 (54,477). 1H NMR
(400 MHz, CDCl3): 1.42–1.50 (m, 72H, Me3C); 2.59 (t, J = 7.2 Hz,
4H, CH2CH2N of B); 2.63 (s, 6H, CH3 bpy); 2.77 (t, J = 7.0 Hz, 4H,
CH2CH2N of A); 3.36, 4.37 (AB, JAB = 13.2 Hz, 8H, ArCH2Ar); 3.53 (q,
J = 6.9 Hz; 4H, CH2CH2N of B); 3.64 (q, J = 6.8 Hz; 4H, CH2CH2N of
A); 5.20 (s, 4H, OCH2bpy); 6.79 (s, 4H, ArH of B); 6.93 (s, 4H, ArH of
5.1.4.4. 5,11,17,23-Tetra-(aminoethyl)-25,27-bis(4-methylene-
oxy-40-methyl-2,20-bithiazolyl)-26,28-dihydroxycalix[4]arene,
tetra-trifluoroacetate salt 10. From 7 (0.7 g, 0.56 mmol), CH2Cl2
(40 mL), TFA (15 mL); 10 (0.75 g, 93%). Pale yellow powder. Mp:
90–91 °C. IR (KBr): 3412.3 (OH); 2929.6 (NH3þ); 1678.5 (NH3þ).
UV–vis (D2O): 289 (8521); 330 (21,231). 1H NMR (400 MHz, D2O):
2.66 (s, 6H, CH3 btz); 2.58 (t, J = 7.2 Hz, 4H, CH2CH2N of A or B);
2.88 (t, J = 7.4 Hz, 4H, CH2CH2N of A or B); 3.01 (t, J = 7.3 Hz, 4H,
CH2CH2N of A or B); 3.25 (t, J = 7.3 Hz, 4H, CH2CH2N of A or B);
3.49–4.35 (AB, JAB = 13.6 Hz, 8H, ArCH2Ar); 4.98 (s, 4H, OCH2btz);
0
0
A); 7.12 (d, J = 7.6 Hz, 2H, H5 bpy); 7.60 (t, J = 7.8 Hz, 4H, H4–4
bpy); 7.95 (s, 2H, OH); 8.09 (d, J = 7.5 Hz, 2H, H5 bpy); 8.18 (d,
0
6.85 (s, 4H, ArH of B); 7.10 (s, 6H, ArH of A and H5 btz); 7.69 (s, 2H,
0
J = 7.8 Hz, 2H, H3 bpy); 8.33 (m, 4H, CH2NH of B and H3 bpy); 8.41
H5 btz). 1H NMR (400 MHz, DMSO-d6): 2.46 (s, 6H, CH3 btz); 2.62
(t, J = 7.7 Hz, 4H, CH2CH2N of B); 2.69 (t, J = 7.6 Hz, 4H, CH2CH2N of
A); 2.97 (CH2CH2N of A and B) 3.42–4.27 (AB, JAB = 12.8 Hz, 8H,
ArCH2Ar); 6.99 (s, 4H, ArH of B); 7.06 (s, 4H, ArH of A); 7.49 (s, 2H,
(t, J = 6.5 Hz, 2H, CH2NH of A); 11.47 (s, 2H, NH of A or B); 11.48 (s,
2H, NH of A or B). 13C NMR (100 MHz, CDCl3): 25.03 (Me bpy);
28.43, 28.51; 28.73 (CMe3); 31.93 (ArCH2Ar); 34.90 (CH2CH2N of
A); 35.19 (CH2CH2N of B); 42.65 (CH2CH2N of B); 43.01 (CH2CH2N
H5 btz); 7.98 (m, 12H, H5 btz and NH3); 8.15 (s, 2H, OH). 13C NMR
0
0
of A); 79.14 (OCH2bpy); 79.65, 83.35 (Me3C); 118.73 (C3 bpy);
(100 MHz, DMSO-d6): 17.02 (Me btz); 30.94 (ArCH2Ar); 32.55
(CH2CH2N of A); 32.90 (CH2CH2N of B); 40.36, 40.42 (CH2CH2N of
0
120.44 (C3 bpy); 121.46 (C5 bpy); 123.76 (C5 bpy); 128.18 (Co of
A); 129.17 (Cp of A); 129.42 (Cm of B); 130.13 (Cm of A); 133.42 (Co
0
A and B); 117.90 (C5 Btz); 119.88 (C5 btz); 127.89 (Cp of A); 128.08
0
of B); 135.78 (Cp of B); 137.52 (C4 bpy); 138.58 (C4 bpy); 151.18 (Cipso
(Co of B); 129.09 (Cm of A); 129.58 (Cm of B); 134.21 (Cp of B);
134.84 (Co of A); 151.16 (Cipso of B); 151.84 (Cipso of A); 153.36 (C2
of B); 152.47 (Cipso of A); 153.53 (CO); 156.43 (C guan of B); 156.50 (C
0
0
guan of A); 153.54, 155.73, 156.83, 158.23 (C2, C2 , C6, C6 of bpy);
163.98 (CO). Anal. Calcd for C104H136O20N16, (1930.3): C, 64.71; H,
7.10; N, 11.61. Found: C, 64.65; H, 6.86; N, 11.34. ES-MS (pos. mode):
1828.99 [Mꢁboc+2H+]+, 1951.98 [M+Na+]+.
0
0
btz); 154.20 (C2 btz); 159.75 (C4 Btz); 161.51 (C4 Btz); no visible
TFA. Anal. Calcd for C52H56O4N8S4ꢀ4CF3COOHꢀ4H2O, (1512.45): C,
47.66; H, 4.53; N, 7.40; S, 8.48. Found: C, 47.64; H, 4.28; N, 7.27; S,
8.57. ES-MS (pos. mode): 1213.24 [Mꢁ2CF3COOH+H+]+, 1098.44
[Mꢁ3CF3COOH+H+]+, 984.61 [Mꢁ4CF3COOH+H+]+, 493.23 [Mꢁ4CF3
5.1.5.3. 5,11,17,23-Tetra-[(N,N0-di-Boc)guanidinoethyl]-25,27-
bis(5-methyleneoxy-50-methyl-2,20-bipyridyl)-26,28-dihydroxy-
calix[4]arene 12. From 9 (0.205 g, 0.144 mmol), CH2Cl2 (20 mL),
COOH+2H+]2+/2
,
329.35 [Mꢁ4CF3COOH+3H+]3+/3
. ES-MS (neg.
mode): 1439.22 [MꢁH+]ꢁ, 1325.32 [MꢁCF3COOHꢁH+]ꢁ, 1210.28
[Mꢁ2CF3COOHꢁH+]ꢁ.
MeOH (some drops), Et3N (241 lL, 1.735 mmol), K (0,226 g,

5504
M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
0,578 mmol), 16 h (TLC monitoring, Al2O3, CH2Cl2). CH2Cl2 (50 mL);
washings: H2O (2 ꢂ 30 mL). Drying: Na2SO4. Chromatography
(Al2O3, CH2Cl2); 12 (0,234 g, 84%). Slightly pink solid. Mp: 125–
126 °C. IR (KBr): 3334.06 (OH); 1721.1 (COO); 1638.76(NCO, NHCO).
UV–vis (CH2Cl2): 289 (52,921). 1H NMR (400 MHz, CDCl3) 1.39–1.51
(m, 72H, Me3C); 2.34 (s, 6H, CH3 bpy); 2.57 (t, J = 7.17 Hz, 4H,
CH2CH2N of B); 2.72 (t, J = 7.18 Hz, 4H, CH2CH2N of A); 3.27–4.22
(AB, JAB = 13.1 Hz, 8H, ArCH2Ar); 3.52 (q, J = 6.86 Hz, 4H, CH2CH2N
of B); 3.6 (q, J = 6.79 Hz; 4H, CH2CH2N of A); 5.13 (s, 4H, OCH2bpy);
6.78 (s, 4H, ArH of B); 6.87 (s, 4H, ArH of A); 7.47 (dd, J = 8,3 Hz,
high vacuum, then triturated in dry Et2O to give a precipitate. The latter
was dissolved inH2O, dialyzed (Float-A-Lyser, cellulose acetate, MWCO
100D) then lyophilized to give the expected guanidinium salt.
5.1.6.1. 5,11,17,23-Tetra-(guanidinoethyl)-25,26,27,28-calix[4]-
arene tetrol, tetra-trifluoroacetate salt 4. From
3 (0.19 g,
0.12 mmol), CH2Cl2 (11 mL), TFA (4 mL); 1 h. 4 (0.14 g, 95%). White
cotton. Mp: 215 °C (dec). IR (KBr): 3365 (OH); 3185 (@NH); 1671
(NH2). UV–vis (H2O): 285 (3460). 1H NMR (400 MHz, D2O): 2.56
(t, J = 6.4 Hz; 8H, CH2CH2N); 3.23 (t, J = 6.4 Hz; 8H, CH2CH2N);
3.82 (br s, 8H, ArCH2Ar), 6.94 (s, 8H, ArH). 1H NMR (400 MHz,
DMSO-d6): 2.51 (m, 8H, CH2CH2N); 3.22 (q, J = 6.5 Hz, 8H,
CH2CH2N); 3.55 (br s, 8H, ArCH2Ar); 6.94 (s, 8H, ArH); 7.56 (t,
J = 4.9 Hz, CH2CH2NH). 13C NMR (100 MHz, D2O): 30.96 (ArCH2Ar);
33.65 (CH2CH2N); 42.77 (CH2CH2N); 129.12 (Co); 129.44 (Cm);
132.34 (Cp); 147.73 (Cipso); 156.99 (C guan); no visible TFA. Anal.
Calcd for C40H52O4N12ꢀ4CF3COOHꢀH2O (1239.02): C, 46.53; H,
4.71; N, 13.56. Found: C, 46.50; H, 4.71; N, 13.13. ES-MS (ESꢁ):
1332.54 [M+CF3COOꢁ]ꢁ, 1218.68 [MꢁH+]ꢁ. ES-MS (pos. mode):
1106.72 [MꢁCF3COOꢁ]+, 992.86 [Mꢁ2CF3COOꢁ+H+]+, 878.99
[Mꢁ2CF3COOꢁ+2H+]+, 765.19 [Mꢁ4CF3COOꢁ+3H+]+.
0
2H, H4 bpy); 7.76 (s, 2H, OH); 8.14 (dd, J = 8.06 Hz, 2H, H4 bpy);
0
8.21 (d, J = 8.06 Hz, 2H, H3 bpy); 8.29 (t, J = 6 Hz, 2H, CH2NH of B
or A); 8.35–8.41 (m, 6H, 2H3, 2H6 of bpy and 2 CH2NH of B or A);
8.89 (dd, J = 1.51 Hz, 2H, H6 of A); 11.46 (s, 4H, NH guan). 13C NMR
(100 MHz, CDCl3): 18.74 (Me bpy); 28.41, 28.48, 28.72 (Me3C);
32.06 (ArCH2Ar); 34.85, 35.15 (CH2CH2N); 42.55, 42.89 (CH2CH2N);
76.17 (OCH2 bpy); 79.58, 79.63, 83.29, 83.34 (CMe3); 121.08, 121.25,
0
0
0
128.15, 130.16, 136.69, 137.55, 148.66, 149.88 (C3, C3 , C4, C4 , C6, C6 ,
of bpy and Cm of Ar); 128.18, 129.33, 132.28, 133.50, 133.61, 135.92,
137.59, 150.96, 152.22, 153.52, 153.73, 156.43, 156.49, 156.59 (C2,
0
0
C2 , C5, C5 of bpy; Cp, Co and Ci of Ar; C guan, CO); 153.57, 164.03
(CO). Anal. Calcd for C104H136O20N16ꢀCH2Cl2 (2013.94): C, 62.62; H,
6.90; N, 11.12. Found: C, 62.85; H, 7.03; N, 11.36. ES-MS (pos; mode):
1930.7 [M+H+]+, 1831.0 [MꢁBoc+H+H+]+; 965.5 [M+2H+]2+/2, 915.6
5.1.6.2. 5,11,17,23-Tetra-(guanidinoethyl)-25,27-bis(6-methyl-
eneoxy-60-methyl-2,20-bipyridyl)-26,28-dihydroxycalix[4]arene,
hexa-trifluoroacetate salt 14. From 11 (0.35 g, 0.18 mmol),
CH2Cl2 (20 mL), TFA (7.5 mL); 3 h. 14 (0.25 g, 74%). White cotton.
Mp: 94–95 °C. IR (KBr): 3386.2 (OH); 1676.2 (NH). UV–vis (H2O):
286 (26,644). 1H NMR (400 MHz, D2O): 2.47 (t, J = 4.8 Hz, 4H,
CH2CH2N of A); 2.49 (s, 6H, Me bpy); 2.78 (t, J = 6.4 Hz, 4H,
CH2CH2N of B); 3.17 (t, J = 6.0 Hz, 4H, CH2CH2N of A); 3.41
(t, J = 6.5 Hz, 4H, CH2CH2N of B); 3.51–4.30 (AB, JAB = 13.3 Hz, 8H,
ArCH2Ar); 5.09 (s, 4H, OCH2 bpy); 6.90 (s, 4H, ArH of B); 7.16 (s,
4H, ArH of A); 7.49 (d, J = 7.8 Hz, 4H, H5 bpy); 7.68 (d, J = 7.8 Hz,
[MꢁBoc+H+2H+]2+/2; 644.5 [M+3H+]3+/3, 610.8 [MꢁBoc+H+3H]3+/3
,
577.4 [Mꢁ2Boc+2H+3H]3+/3, 544.1 [Mꢁ3Boc+3H+3H]3+/3, 510.51
[Mꢁ4Boc+4H+3H]3+/3, 477.4 [Mꢁ5Boc+5H+3H]3+/3, 444.0 [Mꢁ6Boc+
6H+3H]3+/3
3H]3+/3
,
410.6 [Mꢁ7Boc+7H+3H]3+/3
,
377.4 [Mꢁ8Boc+8H+
.
5.1.5.4. 5,11,17,23-Tetra-[(N,N0-di-Boc)guanidinoethyl]-25,27-
bis(4-methyleneoxy-40-methyl-2,20-bithiazolyl)-26,28-dihy-
droxycalix[4]arene 13. From 10 (0.45 g, 0.30 mmol), CH2Cl2
0
(40 mL), MeOH (some drops), Et3N (522
l
L, 3.75 mmol),
K
4H, H5 bpy); 7.83 (t, J = 7.8 Hz, 4H, H4 bpy); 8.10 (d, J = 7.8 Hz,
0
(0.49 g, 1.24 mmol). 5 h (TLC monitoring: Al2O3, CH2Cl2). CH2Cl2
(50 mL); washings: 2 M NaHSO4 (40 mL), satd NaHCO3 (40 mL).
Drying: Na2SO4. Chromatography (Al2O3, CH2Cl2/Hex 90:10); 13
(0.48 g, 77%). White powder. Mp: 147–178 °C. IR (KBr): 3335.9
(OH), 1721.1 (CO); 1638.8 (NCO, NHCO). UV–vis (CH2Cl2): 291
(12,133); 330 (27,720). 1H NMR (400 MHz, CDCl3): 1.44–149 (m,
72H, Me3C); 2.53 (s, 6H, CH3 btz); 2.58 (t, J = 7.0 Hz, 4H, CH2CH2N
of A or B); 2.75 (t, J = 7.0 Hz, 4H, CH2CH2N of A or B); 3.33–4.30
(AB, JAB = 13.1 Hz, 8H, ArCH2Ar); 3.52 (q, J = 6.5 Hz, 4H, CH2CH2N
of A or B); 3.63 (q, J = 6.5 Hz, 4H, CH2CH2N of A or B); 5.22 (s,
4H, OCH2btz); 6.78 (s, 4H, ArH of A or B); 6.91 (s, 4H, ArH of A or
B); 6.98 (s, 2H, OH); 7.80 (s, 2H, H btz); 7.97 (s, 2H, H btz); 8.30
(t, J = 4.6 Hz, 2H, CH2CH2NH of A or B); 8.40 (t, J = 4.6 Hz, 2H,
CH2CH2NH of A or B); 11.47 (s, 2H, NH of A or B); 11.48 (s, 2H,
NH of A or B). 13C NMR (100 MHz, CDCl3): 17.59 (Me btz); 28.45,
28.51, 28.72 (Me3C); 31.85 (ArCH2Ar); 34.90 (CH2CH2N of A);
35.19 (CH2CH2N of B); 42.56 (CH2CH2N of B); 42.93 (CH2CH2N of
4H, H3 bpy); 8.16 (d, J = 8.1 Hz, 4H, H3 bpy); 8.30 (t, J = 7.5 Hz,
4H, H4 bpy). 1H NMR (400 MHz, DMSO-d6): 2.55 (s, 6H, Me bpy);
0
2.63 (t, J = 7.0 Hz, 8H, CH2CH2N); 3.26 (m, 8H, CH2CH2N); 3.41–
4.37 (AB, JAB = 13.0 Hz, 8H, ArCH2Ar); 5.14 (s, 4H, OCH2 bpy); 7.03
(s, 4H, ArH); 7.08 (s, 4H, ArH); 7.26 (d, J = 7.4 Hz, 4H, H bpy);
7.64 (t, J = 7.6 Hz, 4H, H bpy); 7.69 (t, J = 7.6 Hz, 4H, H bpy); 7.79
(d, J = 7.4 Hz, 4H, H bpy); 8.20 (d, J = 7.8 Hz, 4H, H bpy); 8.33 (d,
J = 7.8 Hz, 4H, Hbpy); 8.37 (s, 2H, OH). 13C NMR (100 MHz, D2O):
19.57 (Me bpy); 30.93 (ArCH2Ar); 33.73 (CH2CH2N of B); 33.82
(CH2CH2N of A); 42.39 (CH2CH2N of A); 42.98 (CH2CH2N of B);
77.25 (OCH2 bpy); 116.72 (q, J = 291.9 Hz, CF3COOꢁ); 122.16 (C3
bpy); 122.38 (C3 bpy); 125.07 (C5 bpy); 128.22 (C5 bpy); 128.98
(Co of A or B); 129.73 (Cm of A); 129.83 (Cm of B); 130.72 (Cp of
B); 134.44 (Co of A or B); 136.38 (Cp of A); 140.01 (C4 bpy);
146.69 (C4 bpy); 146.87 (C6 bpy); 147.78 (C6 bpy); 150.42 (Cipso
of B); 150.93 (Cipso of A); 155.26 (C2 bpy); 156.84 (C2 bpy and C
0
0
0
0
0
guan of A); 157.12 (C guan of B); 163.17 (q, J = 35.5 Hz, CF3COOꢁ).
Anal. Calcd for C64H72O4N16ꢀ6CF3COOHꢀ4H2O (1867.53): C, 48.87;
H, 4.53; N, 12.00. Found: C, 48.71; H, 4.35; N, 11.86.
ES-MS (neg. mode): 1697.56 [M+CF3COOHꢁH+]ꢁ. ES-MS (pos.
mode): 1470.74 [MꢁCF3COOH+H+]+, 1356.85 [Mꢁ2CF3COOH+H+]+,
1243.08 [Mꢁ3CF3COOH+H+]+, 1129.00 [Mꢁ4CF3COOH+H+]+,
0
A); 79.62 (OCH2btz); 83.34 (Me3C); 116.33 (C5 btz); 118.22 (C5
btz); 128.17 (Co of A); 129.43 (Cm of A); 130.13 (Cm of B); 133.50
(Co of B); 135.99 (Cp of B); 150.90 (Cipso of B); 152.23 (Cipso of A);
0
153.56 (Cp of A); 154.11 (C2 btz); 154.75 (C2 btz); 156.44 (C guan
0
of A); 156.50 (C guan of B); 160.85 (C4 btz); 162.20 (C4 btz);
164.00 (CO of B); 164.04 (CO of A). Anal. Calcd for
C96H128O20N16S4ꢀ1.5CH2Cl2 (2074.28): C, 56.17; H, 6.29; N, 10.80.
Found: C, 56.32; H, 6.30; N, 10.82. ES-MS (pos. mode): 1952.34
[M+H+]+, 1852.50 [MꢁCOOC(CH3)3+H+]+.
565.39 [Mꢁ4CF3COOH+2H+]2+/2, 377.46 [Mꢁ4CF3COOH+3H+]3+/3
.
5.1.6.3. 5,11,17,23-Tetra-(guanidinoethyl)-25,27-bis(5-methyl-
eneoxy-50-methyl-2,20-bipyridyl)-26,28-dihydroxycalix[4]arene,
hexa-trifluoroacetate salt 15. From 12 (0.2 g, 0.104 mmol),
CH2Cl2 (20 mL), TFA (6 mL); 15 (0.163 g, 83%). Slightly pink solid.
Mp: 120–121 °C. IR (KBr): 3357.88 (OH); 1673.88 (NH). UV–vis
(H2O): 287 (40,098). 1H NMR (400 MHz, D2O). 2.38 (s, 6H, Me
bpy); 2.56 (t, J = 6.42 Hz, 4H, CH2CH2N of A); 2.75 (t, J = 6.5 Hz, 4H,
CH2CH2N of B); 3.23 (t, J = 6.42 Hz, 4H, CH2CH2N of A); 3.39
5.1.6. Compounds 4, 14, 15 and 16—general procedure
A solution of Boc-guanidino calixarene derivative in a mixture of
CH2Cl2 and TFA was stirred at rt under Ar. The solvent were evaporated,
and the residual TFA was eliminated by five dissolution in CH2Cl2–
evaporation cycles. The semi-solid residual material was dried under

M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
5505
(t, J = 6.67 Hz, 4H, CH2CH2N of B); 3.56–4.25 (AB, JAB = 13.34 Hz, 8H,
ArCH2Ar); 5.24 (s, 4H, OCH2 bpy); 6.96 (s, 4H, ArH of B); 7.13 (s, 4H,
(400 MHz, CDCl3): 1.441 (s, 9H, Me3C); 2.703 (t, J = 7.9 Hz; 2H,
CH2CH2N); 3.329 (m, 2H, CH2CH2N); 4.602 (br s, 1H, NH or OH);
6.023 (br s, 1H, NH or OH); 6.774 (d, J = 8.3 Hz; 2H; ArH); 7.013
(d, J = 8.3 Hz; 2H; ArH). 13C NMR (100 MHz, CDCl3): 28.84
(Me3C); 35.64 (CH2CH2N); 42.49 (CH2CH2N); 80.15 (Me3C);
115.97 (Cm); 130.18 (Co); 130.50 (Cp); 155.38 (Cipso); 156.84 (CO).
Anal. Calcd for C13H19O3N (237.29): C, 65.80; H, 8.07; N, 5.90.
Found: C, 65.77; H, 8.09; N, 5.90. EI-MS: 181 [Mꢁ(Me)3-
COꢁH+Na].+; 107 [Mꢁ(Me)3COC(O)NHCH2]+.
0
0
ArH of A); 7.80 (s, 4H, H4 , 4 bpy); 7.84 (d, J = 8.31 Hz, 2H, H3 bpy);
0
8.24 (s, 2H, H6 bpy); 8.35 (d, J = 9.32 Hz, 2H, H3 bpy); 8.99 (s, 2H,
H6 bpy). 13C NMR (100 MHz, DMSO-d6): 18.10 (Me bpy); 31.20
(ArCH2Ar); 33.96 (CH2CH2N of B); 34.36 (CH2CH2N of A); 42.24
(CH2CH2N of A); 42.91 (CH2CH2N of B); 75.84 (OCH2 bpy); 117.65
(q, J = 298.6 Hz, CF3COOꢁ); 120.61, 120.65, 136.71, 138.40, 148.33,
0
0
0
148.98 (C3, C3 , C4, C4 , C6, C6 of bpy); 127.66 (Co of Ar B); 129.40
(Cm of Ar A); 129.51 (Cp of B); 129.84 (Cm of Ar B); 133.94 (Co of Ar
A); 136.08 (Cp of Ar A); 150.90, 151.37 (Ci of Ar A and Ar B);
5.1.8. Compounds 20, 21 and 22—general procedure
0
0
133.02, 134.23, 151.81, 154.56 (C2, C2 , C5, C5 of bpy); 157.25 (C
guan); 163.17 (q, J = 35.5 Hz, CF3COOꢁ). Anal. Calcd for C64H72O4N16-
6CF3COOHꢀ4H2O (1885.56): C, 48.41; H, 4.60; N, 11.89. Found: C,
48.82; H, 4.21; N, 11.98. ES-MS (pos. mode): 1471.4 [Mꢁ2CF3-
COOHꢁCF3COOꢁ]+, 1357.3 [Mꢁ3CF3COOHꢁCF3COOꢁ]+, 1243.4
[Mꢁ4CF3COOHꢁCF3COOꢁ]+, 1129.3 [Mꢁ5CF3COOHꢁCF3COOꢁ]+,
K2CO3 (1 equiv) was added to a solution of 19 (1.1 equiv) in
anhydrous MeCN. The mixture was refluxed under Ar during
30 min, and the bromomethyl reactant (1 equiv) was added in
one portion. Reflux was continued during 5 h (TLC monitoring),
and the solvent was evaporated to dryness. The residue was tritu-
rated in CH2Cl2, then filtered. The filtrate was concentrated and
chromatographed to give the expected ether.
565.3 [Mꢁ6CF3COOH+2H+]2+/2, 377.3 [Mꢁ6CF3COOH+3H+]3+/3
.
5.1.6.4. 5,11,17,23-Tetra-(guanidinoethyl)-25,27-bis(4-meth-
yleneoxy-40-methyl-2,20-bithiazolyl)-26,28-dihydroxycalix[4]
arene, tetra-trifluoroacetate salt 16. From 13 (0.23 g,
0.111 mmol), CH2Cl2 (20 mL), TFA (7 mL); 2 h. 16 (0.15 g, 73%).
White cotton. Mp: 110–111 °C. IR (KBr): 3366.1 (OH); 2179.5
(NH3þ); 1671.1 (NH3þ). UV–vis (H2O): 289 (10,595); 330
(24414). 1H NMR (400 MHz, D2O): 2.29 (s, 6H, CH3 btz); 2.44 (t,
J = 5.6 Hz, 4H, CH2CH2N of A or B); 2.76 (t, J = 6.4 Hz, 4H, CH2CH2N
of A or B); 3.17 (t, J = 6.4 Hz, 4H, CH2CH2N of A or B); 3.39 (t,
J = 7.0 Hz, 4H, CH2CH2N of A or B); 3.40–4.25 (AB, JAB = 13.0 Hz,
8H, ArCH2Ar); 5.03 (s, 4H, OCH2 btz); 6.80 (s, 4H, ArH of A or B);
7.00 (s, 2H, H btz); 7.05 (s, 4H, ArH of A or B); 7.63 (s, 2H, H
btz). 1H NMR (400 MHz, DMSO-d6): 2.46 (s, 6H, CH3 btz); 2.55
(m, 4H, CH2CH2N of B); 2.62 (t, J = 6.1 Hz, 4H, CH2CH2N of A);
3.26 (m, 8H, CH2CH2N of A and B); 3.38–4.29 (AB, JAB = 13.1 Hz,
8H, ArCH2Ar); 5.17 (s, 4H, OCH2 btz); 6.60–7.60 (br signal, 16H,
NH guanidine); 7.01 (s, 4H, ArH of B); 7.06 (s, 4H, ArH of A);
5.1.8.1. N-Boc-Aminoethyl-4-[6-methyleneoxy-60-methyl-2,20-
bipyridyl]-benzene 20. From K2CO3 (0.04 g, 0.33 mmol), 19
(0.09 g, 0.38 mmol), MeCN (20 mL), 6-bromomethyl-60-methyl-
2,20-bipyridine (0.09 g, 0.33 mmol). TLC monitoring: Al2O3; CH2Cl2.
Chromatography: Al2O3; CH2Cl2/Hex 90:10. 20 (0.12 g, 85%). White
solid. Mp: 81–82 °C. IR (KBr): 3353.5 (–CONH–); 1685.6 (CO). UV–
vis (CH2Cl2): 289 (19,189). 1H NMR (400 MHz, CDCl3): 1.43 (s, 9H,
Me3C); 2.64 (s, 3H, Mebpy); 2.73 (t, J = 6.8 Hz, 2H, CH2CH2NH);
3.34 (m, 2H, CH2CH2NH); 4.52 (br s, 1H, NH); 5.27 (s, 2H, OCH2bpy);
6.96 (d, J = 8.3 Hz, 2H, ArH); 7.11 (d, J = 8.3 Hz, 2H, ArH); 7.18 (d,
J = 7.5 Hz, 2H, H bpy); 7.52 (d, J = 7.8 Hz, 2H, H bpy); 7.71 (t,
J = 7.7 Hz, 2H, H bpy); 7.82 (t, J = 7.7 Hz, 2H, H bpy); 8.20 (d,
J = 7.5 Hz, 2H, H bpy); 8.32 (d, J = 7.5 Hz, 2H, H bpy). 13C NMR
(100 MHz, CDCl3): 25.09 (Mebpy); 28.83 (Me3C); 35.70, 42.34
(CH2CH2NH and CH2CH2NH); 71.34 (Me3C); 79.54 (OCH2bpy);
0
0
115.37 (Co or Cm); 118.63, 120.40, 121.35, 123.72 (C3, C3 , C5 and C5
0
of bpy); 130.22 (Co or Cm); 131.90 (Cp); 137.46, 138.01 (C4, C4 of
0
0
0
7.49 (s, 2H, H5 btz); 7.61 (m, CH2CH2NH of A or B); 7.69 (m,
bpy); 155.88, 156.30, 156.36, 157.27, 157.53,158.37 (C2, C2 , C6, C6
CH2CH2NH of A or B); 7.97 (s, 2H, H5 btz); 8.10 (s, 2H, OH). 13C
NMR (100 MHz, DMSO-d6): 17.03 (Me btz); 31.00 (ArCH2Ar);
33.98 (CH2CH2N of A); 34.37 (CH2CH2N of B); 42.24, 42.92
(CH2CH2N of A and B); 73.74 (OCH2btz); 117.45 (q, J = 298.5 Hz,
of bpy, Cipso, CO). Anal. Calcd for C25H29O3N3 (419.52): C, 71.57; H,
6.96; N, 10.01. Found: C, 71.52; H, 6.84; N, 9.94. EI-MS: 421 [M+H+]+.
5.1.8.2. N-Boc-Aminoethyl-4-[5-methyleneoxy-50-methyl-2,20-
bipyridyl]-benzene 21. From K2CO3 (0.12 g, 0.843 mmol), MeCN
(30 mL), 19 (0.2 g, 0.843 mmol), 5-bromomethyl-50-methyl-2-20-
bipyridine (0.222 g, 0.843 mmol); 23 h (TLC monitoring, SiO2,
CH2Cl2/MeOH 98:2) addition of NEt3 (2 drops); chromatography:
SiO2, CH2Cl2/MeOH 99.4:0.6. 21 (0.281 g, 80%). White solid. Mp:
134–135 °C. IR (KBr): 3378.32 (–CONH–); 1689.06 (CO). UV–vis
(CH2Cl2): 289 (26,079). 1H NMR (400 MHz, CDCl3): 1.43 (s, 9H,
Me3C); 2.39 (s, 3H, Mebpy); 2.73 (t, J = 6.8 Hz, 2H, CH2CH2NH);
3.34 (br s, 2H, CH2CH2NH); 4.53 (br s, 1H, NH); 5.11 (s, 2H, OCH2b-
py); 6.93 (d, J = 8.56 Hz, 2H, ArH); 7.12 (d, J = 8.6 Hz, 2H, ArH); 7.26
(dd, J = 8.06 Hz, 1H, H bpy); 7.87 (dd, J = 8.31 Hz, 1H, H bpy); 8.28
(d, J = 8.06 Hz, 1H, H bpy); 8.38 (d, J = 8.06 Hz, 2H, H bpy); 8.50
(s, 1H, H bpy); 8.71 (dd, J = 1.26 Hz, 1H, H bpy). 13C NMR
(100 MHz, CDCl3): 18.78 (Mebpy); 28.82 (Me3C); 35.71
(CH2CH2NH); 42.33 (CH2CH2NH); 68.00 (Me3C); 79.59 (OCH2bpy);
CF3COOꢁ); 117.92 (C5 btz); 119.73 (C5 btz); 127.72 (Co of A);
0
129.19 (Cp of A); 129.30 (Cm of A); 129.69 (Cm of B);134.04 (Co
of B);135.84 (Cp of B); 150.92 (Cipso of B); 151.56 (Cipso of A);
0
153.43 (C2 btz); 154.22 (C2 btz); 157.14, 157.19 (C guan);
159.28 (q, J = 30.8 Hz, CF3COOꢁ); 159.75 (C4 btz); 161.51 (C4
0
btz). Anal. Calcd for C56H64O4N16S4ꢀ4CF3COOHꢀ3H2O, (1695.49):
C, 45.35; H, 4.39; N, 13.20. Found: C, 45.33; H, 4.39; N, 13.21.
ES-MS (pos. mode): 1494.22 [MꢁCF3COOH+H+]+, 1380.29 [Mꢁ2
CF3 COOH+H+]+, 1266.54 [Mꢁ3CF3COOH+H+]+, 1152.49 [Mꢁ4
CF3COOH+H+]+, 577.20 [Mꢁ4CF3COOH+2H+]2+/2, 385.29 [Mꢁ4
CF3COOH+3H+]3+/3
.
5.1.7. N-Boc-tyramine 19
A solution of NaOH (0.46 g, 11,5 mmol) in H2O (15 mL) was
added to
a solution of tyramine hydrochloride F (1.00 g,
0
5.76 mmol) in dioxane/H2O (25/12 mL). The mixture was stirred
at rt under Ar during 10 min, and di-tert-butyl-dicarbonate (Boc2O)
(1.26 g, 5.76 mmol) was added. The solution was stirred overnight
at rt, then was concentrated. AcOEt (40 mL) was added and pH was
brought to 7–8 with 1 M HCl. The aqueous phase was collected and
washed with AcOEt (2 ꢂ 15 mL). The combined organic phases
were dried over Na2SO4, filtered and evaporated to dryness. The
raw material was chromatographed (Al2O3, CH2Cl2/Hex, 85:15) to
give 19 (0.98 g, 73%). Mp: 61–62 °C. IR (KBr): 3378.9 (–OH;
–CONH–); 1686.6 (CO). UV–vis (CH2Cl2): 277 (1803). 1H NMR
115.40, 130.27 (Co and Cm of Ar); 121.00, 121.04 (C3 , of bpy);
3
0
0
132.18 (Cp of Ar); 136.64, 137.89, 148.73, 150.09 (C4, C4 , C6, C6 of
0
0
bpy); 132.66, 133.95, 156.28, 156.50 (C2, C2 , C5, C5 of
bpy);153.72 (Ci of Ar);157.46 (COO). Anal. Calcd for C25H29O3N3
(419.52): C, 71.57; H, 6.97; N, 10.02. Found: C, 71.65; H, 6.88; N,
9.93. ES-MS (pos. mode): 420.1 [M+H+]+, 442.2 [M+Na+]+.
5.1.8.3. N-Boc-Aminoethyl-4-[4-methyleneoxy-40-methyl-2,20-
bithiazolyl]-benzene 22. From K2CO3 (0.23 g, 1.64 mmol), 19
(0.45 g, 1.89 mmol), MeCN (40 mL), 4-bromomethyl-40-methyl-2,

5506
M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
20-bithiazole (0.45 g, 1.64 mmol). TLC monitoring: Al2O3; CH2Cl2.
Chromatography: Al2O3, CH2Cl2/Hex 85:15. 22 (0.60 g, 85%). White
solid. Mp: 126–127 °C. IR (KBr): 3333.4 (–CONH–); 1684.2 (CO).
UV–vis (CH2Cl2): 330 (6979). 1H NMR (400 MHz, CDCl3): 1.46 (s,
9H, Me3C); 2.54 (s, 3H, Mebtz); 2.77 (t, J = 6.8 Hz, 2H, CH2CH2NH);
3.37 (m, 2H, CH2CH2NH); 4.54 (br s, 1H, NH); 5.26 (s, 2H, OCH2btz);
6.97 (d, J = 8.5 Hz, 2H, ArH); 7.02 (s, 1H, H btz); 7.15 (d, J = 8.3 Hz,
2H, ArH); 7.42 (s, 1H, H btz). 13C NMR (100 MHz, CDCl3): 17.54
(Mebtz); 28.82 (Me3C); 35.72 (CH2CH2NH); 42.30 (CH2CH2NH);
66.70 (Me3C); 79.57 (OCH2btz); 115.31 (Cm); 116.27, 118.04 (C5 and
C
20H21ON3ꢀCF3COOHꢀ0.5H2O (442.17): C, 59.72; H, 5.24; N, 9.50.
Found: C, 59.77; H, 5.20; N, 9.57. ES-MS (pos. mode): 320.0
[MꢁCF3COOꢁ]+.
5.1.9.3. Aminoethyl-4-[4-methyleneoxy-40-methyl-2,20-bithiaz-
olyl]-benzene, trifluoroacetate salt 25. From TFA (10 mL), 22
(0.55 g, 1.27 mmol), CH2Cl2 (40 mL). TLC monitoring: Al2O3,
CH2Cl2. 25 (0.55 g, 95%). White powder. Mp: 184–185 °C. IR
(KBr): 3093.2, 1675.4, 1515.15 (NH3þ). UV–vis (H2O): 331
(13649). 1H NMR (400 MHz, D2O): 2.41 (s, 3H, Mebtz); 2.94 (t,
J = 7.3 Hz, 2H, CH2CH2N); 3.24 (t, J = 7.3 Hz, 2H, CH2CH2N); 5.17
(s, 2H, OCH2btz); 7.05 (d, J = 8.3 Hz, 2H, ArH); 7.27 (d, J = 7.3 Hz,
2H, ArH); 7.27 (s, 1H, H btz); 7.66 (s, 1H, H btz). 1H NMR
(400 MHz, DMSO-d6): 2.44 (s, 3H, Mebtz); 2.80 (t, J = 7.7 Hz, 2H,
CH2CH2N); 3.02 (br s, 2H, CH2CH2N); 5.21 (s, 2H, OCH2btz); 7.04
(d, J = 7.55 Hz, 2H, ArH); 7.20 (d, J = 7.55 Hz, 2H, ArH); 7.52 (s,
1H, H btz); 7.80 (br s, 2H, NH2); 7.89 (s, 1H, H btz). 13C NMR
(100 MHz, D2O): 17.03 (Mebtz); 32.58 (CH2CH2N); 40.52
(CH2CH2N); 65.49 (OCH2btz); 115.29 (Co or Cm); 117.66, 120.72
0
C5 of btz) 130.24 (Co); 132.14 (Cp); 154.31 (Cipso); 154.70, 156.27,
0
0
157.37, 160.79 (C2, C2 , C4, C4 of btz); 162.19 (CO). Anal. Calcd for
C21H25O3N3S2 (431.57): C, 58.44; H, 5.83; N, 9.73. Found: C, 57.90;
H, 5.73; N, 9.52. EI-MS: 431 [M+H+]+.
5.1.9. Compounds 23, 24 and 25—general procedure
TFA was added to a solution of boc-aminoethyl derivative in
CH2Cl2. The mixture was stirred at rt under Ar during ca. 2 h (TLC
monitoring). The solvents were evaporated, and the residual TFA
was eliminated by successive dissolution in CH2Cl2–evaporation
cycles, until a white solid was obtained (ca. four cycles). The latter
was dried under high vacuum, triturated in Et2O, filtered and
rinsed with Et2O to give the expected ammonium salt.
0
(C5, C5 of btz); 130.02 (Cp); 130.13 (Co or Cm); 153.56, 154.11,
0
0
157.30 (C2, C2 , C4, C4 of btz; Cipso); no visible TFA. Anal. Calcd for
C16H17ON3S2ꢀCF3COOHꢀ0.5H2O (454.48): C, 47.57; H, 4.21; N,
9.24; S, 14.11. Found: C, 47.48; H, 4.13; N, 9.13; S, 13.66. ES-MS
(pos. mode): 332.33 [MꢁCF3COOH+H+]+, 195.28 [CH3ꢁbtzꢁCH2]+.
5.1.9.1. Aminoethyl-4-[6-methyleneoxy-60-methyl-2,20-bipyri-
dyl]-benzene, sesqui-trifluoroacetate salt 23. From TFA
(10 mL), 20 (0.52 g, 1.24 mmol), CH2Cl2 (30 mL). TLC monitoring:
SiO2, CH2Cl2/MeOH 99:1. 23 (0.52 g, 86%). White powder. Mp:
130–131 °C. IR (KBr): 3029.6 (–NH3þ); 1674.2 (COO); 1515.88 (–
NH3þ). UV–vis (H2O): 287 (11,399). 1H NMR (400 MHz, D2O):
2.73 (s, 3H, Mebpy); 2.93 (t, J = 7.5 Hz, 2H, CH2CH2N); 3.22 (t,
J = 7.3 Hz, 2H, CH2CH2N); 5.34 (s, 2H, OCH2bpy); 7.08 (d,
J = 8.6 Hz, 2H, ArH); 7.27 (d, J = 8.6 Hz, 2H, ArH); 7.63 (d,
J = 7.5 Hz, 1H, H bpy); 7.69 (d, J = 6.8 Hz, 1H, H bpy); 8.03 (m, 3H,
H bpy); 8.18 (m, 1H, H bpy). 1H NMR (400 MHz, DMSO-d6): 2.57
(s, 3H, Mebpy); 2.79 (t, J = 8.0 Hz, 2H, CH2CH2N); 3.01 (m, 2H,
CH2CH2N); 5.27 (s, 2H, OCH2bpy); 7.04 (d, J = 8.3 Hz, 2H, ArH);
7.20 (d, J = 8.6 Hz, 2H, ArH); 7.34 (d, J = 7.5 Hz, 1H, H bpy); 7.57
(d, J = 7.5 Hz, 1H, H bpy); 7.77 (br s, 3H, NH3); 7.85 (t, J = 7.7 Hz,
1H, H bpy); 7.98 (t, J = 7.8 Hz, 1H, H bpy); 8.19 (d, J = 7.8 Hz, 1H,
H bpy); 8.32 (d, J = 7.8 Hz, 1H, H bpy). 13C NMR (100 MHz,
DMSO-d6): 24.57 (Mebpy); 32.57 (CH2CH2N); 40.51 (CH2CH2N);
70.78 (OCH2bpy); 115.35 (Co or Cm); 118.07, 119.86, 122.08,
5.1.10. Compounds 17, 26, 27 and 28—general procedure way 1
A suspension of the ammonium salt in CH2Cl2 was solubilized
by addition of the minimum amount of MeOH. NEt3, then K were
added, and the solution was stirred at rt under Ar. The reaction
was followed by TLC. When all the amine was consumed, the sol-
vent were evaporated to dryness. The residue was solubilized in
CH2Cl2, and the solution was washed with 2 M aqueous NaHSO4,
satd aqueous NaHCO3, then satd aqueous NaCl. After drying over
Na2SO4 or MgSO4, filtration and evaporation, the resulting material
was chromatographed to give the expected di-boc guanidinoethyl
derivative. General procedure way 2: A solution of ammonium salt
(1 equiv), NEt3 (ca. 4 equiv)), 1,3-bis(tert-butoxycarbonyl)-2-
methyl-2-thiopseudourea J (1.1ꢁ1.2 equiv)) in dry DMF (dstd over
CaH2, or conserved over anhydrous CaSO4) was cooled at 4 °C un-
der Ar. HgCl2 was added in one portion, and stirring was continued
at rt under Ar to give a white suspension. The solvent was evapo-
rated to dryness under high vacuum, and the solid residue was trit-
urated in AcOEt or CH2Cl2, and filtered over Celite. The filtrate was
evaporated to dryness, and residual J was eliminated by trituration
of the residue with heptane, and filtration. The resulting solid was
finally chromatographed to give the expected di-boc guanidinoethyl
derivative.
0
0
124.05 (C3, C3 , C5, C5 of bpy); 129.99 (Cp); 130.17 (Co or Cm);
0
137.91, 138.43 (C4, C4 of bpy); 154.64, 155.28, 156.89, 157.42,
0
0
157.95 (C2, C2 , C6, C6 of bpy; Cipso); no visible TFA. Anal. Calcd for
C20H21ON3ꢀ1.5CF3COOH (490.43): C, 56.32; H, 4.92; N, 8.56. Found:
C, 56.09; H, 4.25; N, 8.56. ES-MS (pos. mode): 320.35
[MꢁCF3COOH+H+]+, 184.31 [CH3ꢁbpyꢁCH2+]+.
5.1.10.1. 4-[(N,N0-Di-Boc)guanidinoethyl]-phenol 17. Way 1:
From tyramine hydrochloride F (0.21 g, 1.20 mmol), K (0.47 g,
1.20 mmol), NEt3 (0.66 mL, 0.48 g, 4.80 mmol), CH2Cl2 (10 mL),
MeOH (10 mL). 2 h, TLC monitoring, SiO2, CH2Cl2, and the solvent
were evaporated to dryness. Washings: CH2Cl2 (20 mL), 2 M NaH-
SO4 (30 mL), satd NaHCO3 (30 mL), satd NaCl (40 mL). Chromatog-
raphy: SiO2 deactivated with NEt3; CH2Cl2). 17 (0.32 g, 72%). White
5.1.9.2. Aminoethyl-4-[5-methyleneoxy-50-methyl-2,20-bipyri-
dyl]-benzene, trifluoroacetate salt 24. From TFA (6 mL), 21
(0.25 g, 0.6 mmol), CH2Cl2 (20 mL), TLC monitoring: Al2O3, CH2Cl2.
24 (0.255 g, 96%). Slightly pink solid. Mp: 169–170 °C. IR (KBr):
3007 (NH3þ); 1676 (COO); 1554 (C@N). UV–vis (H2O): 288
(27,679), 240 (22,009), 228 (20,464). 1H NMR (400 MHz, D2O)
2.36 (s, 3H, Mebpy); 2.92 (t, J = 7.17 Hz, 2H, CH2CH2N); 3.22 (t,
J = 7.17 Hz, 2H, CH2CH2N); 5.19 (s, 2H, OCH2bpy); 7.04 (d,
J = 8.6 Hz, 2H, ArH); 7.25 (d, J = 8.31 Hz, 2H, ArH); 7.78 (d,
J = 8.56 Hz, 1H, H bpy); 7.89 (d, J = 7.81 Hz, 1H, H bpy); 7.98 (s,
2H, H bpy); 8.41 (s, 1H, H bpy); 8.61 (s, 1H, H bpy). 13C NMR
(100 MHz, DMSO-d6): 18.18 (Mebpy); 32.56 (CH2CH2N); 40.52
(CH2CH2N); 67.08 (OCH2bpy); 115.38, 130.16 (Co andCm of Ar);
powder. Way
2: from tyramine hydrochloride F (0.50 g,
2.87 mmol), NEt3 (0.74 mL, 10.07 mmol), DMF (15 mL), J (0.92 g,
3.16 mmol), HgCl2 (0.86 g, 3.16 mmol). 18 h. Trituration in AcOEt.
Chromatography: SiO2, CH2Cl2/MeOH, 99:1. 17 (0.98 g; 90%).
White solid. Mp: 142–143 °C. IR (KBr): 3447.9 (OH); 1724.7
(COO); 1624.5 (C@N). UV–vis (CH2Cl2): 276 (1762). 1H NMR
(400 MHz, CDCl3): 1.48 (s, 9H, Me3C); 1.49 (s, 9H, Me3C); 2.77 (t,
J = 7.4 Hz, 2H, CH2CH2NH); 3.60 (q, J = 7.0 Hz, 6H, CH2CH2NH);
5.82 (br s, 1H, NH); 6.75 (d, J = 8.5 Hz, 2H, ArH); 7.00 (d,
J = 8.3 Hz, 2H, ArH); 8.43 (s, 1H, OH); 11.44 (s, 1H, NH). 13C NMR
(100 MHz, CDCl3): 28.42, 28.59 (Me3C); 34.75 (CH2CH2N); 43.13
0
0
120.24, 120.38, 137.03, 137.99, 148.93, 149.92 (C3, C3 , C4, C4 , C6,
0
0
0
C6 of bpy); 133.10, 134.10, 152.82, 155.36 (C2, C2 , C5, C5 of bpy);
130.02 (Cp of Ar); 157.30 (Ci of Ar); no visible TFA. Anal. Calcd for

M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
5507
(CH2CH2N); 80.18, 83.70 (Me3C); 115.98 (Co); 129.76 (Cp); 129.95
(Cm); 153.50 (CO); 155.42 (Cipso); 156.59 (C guan). Anal. Calcd for
C19H29O5N3 (379.45): C, 60.14; H, 7.70; N, 11.07. Found: C, 60.13;
H, 7.65; N, 11.04. ES-MS (pos. mode): 380.37 [M+H+]+.
White powder. Way 2: from 25 (0.74 g, 1.63 mmol), NEt3
(0.80 mL, 5.81 mmol), J (0.53 g, 1.82 mmol), DMF (30 mL), HgCl2
(0.49 g, 1.82 mmol). 5 h. Trituration in CH2Cl2. Chromatography:
Al2O3, CH2Cl2/Hex, 1:1. 28 (0.58 g, 60%). White powder. Mp: 126–
127 °C. IR (KBr): 3347.0 (CONH); 1720.9 (CO); 1647,2, 1617.7
(NCO); 1510.5 (NH). UV–vis (CH2Cl2): 286 (2387). 1H NMR
(400 MHz, CDCl3): 1.48 (s, 9H, Me3C); 1.51 (s, 9H, Me3C); 2.52 (s,
3H, Mebtz); 2.84 (t, J = 6.9 Hz, 2H, CH2CH2N); 3.63 (q, J = 6.5 Hz,
2H, CH2CH2N); 5.24 (s, 2H, OCH2btz); 6.94 (d, J = 8.1 Hz, 2H,
ArH); 6.99 (s, 1H, H btz); 7.15 (d, J = 8.3 Hz, 2H, ArH); 3.39 (s, 1H,
H btz); 8.48 (br s, 1H, NH); 11.47 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3): 17.55 (Mebtz); 28.46, 28.72 (Me3C); 34.80 (CH2CH2N);
42.82 (CH2CH2N); 66.69 (Me3C); 79.66 (OCH2btz); 83.43 (Me3C);
5.1.10.2. [(N,N0-Di-Boc)-guanidinoethyl]-4-[6-methyleneoxy-60-
methyl-2,20-bipyridyl]-benzene 26. Way 1: From 23 (0.25 g,
0.51 mmol), NEt3 (0.24 mL, 1.73 mmol), K (0.23 g, 0.58 mmol),
CH2Cl2 (20 mL), EtOH. 2 h, TLC monitoring: Al2O3, CH2Cl2. Washings:
CH2Cl2 (40 mL), 2 M NaHSO4 (30 mL), satd NaHCO3 (30 mL). Drying:
Na2SO4. Chromatography: Al2O3, CH2Cl2. 26 (0.28 g, 85%). White
powder. Way 2: from 23 (0.64 g, 1.30 mmol), NEt3 (0.72 mL,
5.14 mmol), J (0.47 g, 1.61 mmol), DMF (30 mL). HgCl2 (0.44 g,
1.61 mmol). 5 h. Trituration in CH2Cl2. Chromatography: Al2O3,
CH2Cl2. 26 (0.47 g, 64%). Mp: 132–133 °C. IR (KBr): 3328.4 (CONH);
1734.3 (CO); 1654.9, 1619.0 (NCO); 1511.2 (NH). UV–vis (CH2Cl2):
290 (17,595). 1H NMR (400 MHz, CDCl3): 1.47 (s, 9H, Me3C); 1.50
(s, 9H, Me3C); 2.64 (s, 3H, Mebpy); 2.81 (t, J = 7.3 Hz, 2H, CH2CH2N);
3.63 (q, J = 7.0 Hz, 2H, CH2CH2N); 5.27 (s, 2H, OCH2bpy); 6.95 (d,
J = 8.6 Hz, 2H, ArH); 7.14 (d, J = 8.6 Hz, 2H, ArH); 7.18 (d, J = 7.8 Hz,
1H, H bpy); 7.51 (d, J = 7.5 Hz, 1H, H bpy); 7.71 (t, J = 7.8 Hz, 1H, H
bpy); 7.82 (t, J = 7.8 Hz, 1H, H bpy); 8.20 (d, J = 7.8 Hz, 1H, H bpy);
8.32 (d, J = 7.8 Hz, 1H, H bpy); 8.35 (m, 1H, CH2CH2NH); 11.46 (s,
1H, NH). 13C NMR (100 MHz, CDCl3): 25.02 (Mebpy); 28.46, 28.72
(Me3C); 34.81 (CH2CH2N); 42.85 (CH2CH2N); 71.31 (Me3C); 79.66
(OCH2bpy); 83.42 (Me3C); 115.36 (Cm); 118.69, 120.44, 121.38,
0
115.29 (Co or Cm); 116.25, 118.01 (C5, C5 btz); 130.27 (Co or Cm)
0
131.69 (Cp); 153.56 (Cipso); 154.30, 154.70 (C2, C2 btz); 156.50 (C
0
guan); 157.43 (CO); 160.80, 162.19 (C4, C4 btz); 163.91 (CO). Anal.
Calcd for C27H35O5N5S2ꢀ0.2CH2Cl2 (590.7): C, 54.30; H, 6.04; N,
11.86; S, 10.86. Found: C, 55.41; H, 6.09; N, 11.98; S, 10.85. ES-
MS (pos. mode): 374.2 [Mꢁ2Boc+3H+]+; 474.3 [MꢁBoc+2H+]+;
574.2 [M+H+]+.
5.1.11. Compounds 18, 29, 30 and 31—general procedure
TFA was added to a solution of di-boc-guanidinoethyl deriva-
tives in CH2Cl2. The mixture was stirred at rt under Ar during ca.
2–3 h (TLC monitoring). The solvents were evaporated, and the
residual TFA was eliminated by successive dissolution in CH2Cl2–
evaporation cycles, until a white solid was obtained (ca. four cy-
cles). The latter was dried under high vacuum, triturated in Et2O,
filtered and rinsed with Et2O to give the expected guanidinium salt.
0
0
123.74 (C3, C3 , C5, C5 of bpy); 130.25 (Co); 131.46 (Cp); 137.55,
0
138.01 (C4, C4 of bpy); 153.57 (Cipso); 156.23 (CO); 156.51 (C guan);
0
0
155.85, 157.30, 157.61, 158.34 (C2, C2 , C6, C6 of bpy); 163.97
(CO). Anal. Calcd for C31H39O5N5 (561.67): C, 66.29; H, 6.99; N,
12.47. Found: C, 66.34; H, 7.02; N, 12.38. EI-MS: 345 [Mꢁ(Me)3-
COC(O)NHꢁ(Me)3COC(O)]+; 302 [Mꢁ((Me)3COC(O))2guan]+; 289
[Mꢁ((Me)3COC(O))2guanCH2]+.
5.1.11.1. 4-(Guanidinoethyl)-phenol,trifluoroacetatesalt18. From
17 (0.5 g, 1.31 mmol), CH2Cl2 (30 mL), TFA (10 mL). 2 h, TLCmonitoring:
SiO2, CH2Cl2/MeOH 99:1. 18 (0.35 g, 90%). White solid. Mp: 156–157 °C.
IR (KBr): 3408.8 (OH); 1686.3 (NH). UV–vis (H2O): 274 (1916). 1H NMR
(400 MHz, D2O): 2.80 (t, J = 6.6 Hz, 2H, CH2CH2NH); 3.41 (t, J = 6.6 Hz,
2H, CH2CH2NH); 6.86 (d, J = 7.5 Hz, 2H, ArH); 7.17 (d, J = 7.8 Hz, 2H,
ArH). 1H NMR (400 MHz, DMSO-d6): 2.66 (t, J = 7.3 Hz, 2H, CH2CH2NH);
3.29 (q, J = 6.7 Hz, 2H, CH2CH2NH); 6.70 (d, J = 8.6 Hz, 2H, ArH); 6.90 (br
s, 1H, NH), 7.05 (d, J = 8.3 Hz, 2H, ArH); 7.34 (br s, 2H, NH2); 7.5 (s, 1H,
OH); 9.27 (s, 1H, NH). 13C NMR (400 MHz, D2O): 33.68 (CH2CH2NH);
42.82 (CH2CH2NH); 115.82 (Co); 116.71 (q, J = 291.9 Hz, CF3COOH);
130.59 (Cm); 130.64 (Cp); 154.49 (Cipso); 157.06 (C guan); 163.35 (q,
J = 35.9 Hz, CF3COOH). Anal. Calcd for C9H13ON3ꢀCF3COOH (293.24): C,
45.05; H, 4.81; N, 14.33. Found: C, 44.85; H, 4.90; N 14.03. ES-MS
(pos. mode): 180.38 [MꢁCF3COOH+H+]+. ES-MS (neg. mode): 406.17
[M+CF3COOHꢁH+]ꢁ.
5.1.10.3. [(N,N0-Di-Boc)-guanidinoethyl]-4-[5-methyleneoxy-50-
methyl-2,20-bipyridyl]-benzene 27. From 24 (0.233 g, 0.54 mmol),
NEt3 (0.3 mL, 2.15 mmol), K (0,21 g, 0,54 mmol), CH2Cl2 (25 mL),
MeOH. 23 h (TLC monitoring, SiO2, CH2Cl2/MeOH 98:2 with 2 drops
ofNEt3). CH2Cl2 (50 mL). Washings:H2O(2 ꢂ 30 mL). Drying:Na2SO4.
Chromatography: SiO2, deactivated by CH2Cl2/NEt3 90:10 then
CH2Cl2; 12 (0.234 g, 84%) slightly pink powder. Mp: 148–149 °C. IR
(KBr): 3331.55 (CONH); 1709.01(CO); 1636.81 (NCO); 1512.12
(NH). UV–vis (CH2Cl2): 289 (28,230). 1H NMR (400 MHz, CDCl3) 1.47
(s, 9H, Me3C); 1.50 (s, 9H, Me3C); 2.41 (s, 3H, Mebpy); 2.82 (t,
J = 7.16 Hz, 2H, CH2CH2N); 3.64 (q, J = 7.0 Hz, 2H, CH2CH2N); 5.29 (s,
2H, OCH2bpy); 6.92 (d, J = 8.56 Hz, 2H, ArH); 7.15 (d, J = 8.56 Hz, 2H,
ArH); 7.67 (d, J = 7.8 Hz, 1H, H bpy); 7.9 (dd, J = 8.2 Hz, 1H, H bpy);
8.33 (d, J = 8.08 Hz, 1H, H bpy); 8.37 (br s, 1H, NH); 8.43 (d,
J = 8.04 Hz, 1H, H bpy); 8.52 (s, 1H, H bpy); 8.72 (s, 1H, H bpy);
11.46 (s, 1H, NH guan). 13C NMR (100 MHz, CDCl3): 18.78 (Mebpy);
28.44, 28.71 (Me3C); 34.81 (CH2CH2N); 42.79 (CH2CH2N); 67.99
(Me3C); 79.63 (OCH2bpy); 83.41 (Me3C); 115.37, 130.30 (Co and Cm
5.1.11.2. Guanidinoethyl-4-[6-methyleneoxy-60-methyl-2,20-bi-
pyridyl]-benzene, sesqui-trifluoroacetate salt 29. From 26
(0.23 g, 0.41 mmol), CH2Cl2 (20 mL), TFA (5 mL). 2 h, TLC monitor-
ing: SiO2, CH2Cl2/MeOH 99:1. 29 (0.18 g, 82%). White solid. Mp:
142–143 °C. IR (KBr): 3422.2 (NH2); 3182.0 (NH3þ); 1694.0
(C@O); 1663.5, 1514.7 (NH3þ). UV–vis (H2O): 286 (15,506). 1H
NMR (400 MHz, D2O): 2.66 (s, 3H, Mebpy); 2.84 (t, J = 6.5 Hz, 2H,
CH2CH2N); 3.43 (t, J = 6.5 Hz, 2H, CH2CH2N); 5.34 (s, 2H, OCH2btz);
7.07 (d, J = 8.8 Hz, 2H, ArH); 7.26 (d, J = 8.6 Hz, 2H, ArH); 7.50 (d,
J = 7.5 Hz, 1H, H bpy); 7.65 (d, J = 7.5 Hz, 1H, H bpy); 7.89 (d,
J = 7.8 Hz, 1H, H bpy); 7.97 (t, J = 7.0 Hz, 1H, H bpy); 8.01 (d,
J = 7.8 Hz, 1H, H bpy); 8.02 (t, J = 7.8 Hz, 1H, H bpy). 1H NMR
(400 MHz, DMSO-d6): 2.58 (s, 3H, Mebpy); 2.73 (t, J = 7.0 Hz, 2H,
CH2CH2N); 3.42 (q, J = 5.8 Hz, 2H, CH2CH2N); 5.26 (s, 2H, OCH2btz);
6.70–7.80 (broad signal, 3H, NH guan); 7.03 (d, J = 7.6 Hz, 2H, ArH);
7.21 (d, J = 7.6 Hz, 2H, ArH); 7.35 (d, J = 7.6 Hz, 1H, H bpy); 7.49 (br
s, 1H, CH2CH2NH); 7.55 (d, J = 7.6 Hz, 1H, H bpy); 7.86 (d, J = 7.5 Hz,
1H, H bpy); 7.98 (t, J = 7.3 Hz, 1H, H bpy); 8.49 (d, J = 7.8 Hz, 1H, H
bpy); 8.32 (t, J = 7.8 Hz, 1H, H bpy). 13C NMR (100 MHz, DMSO-d6):
0
0
of Ar); 120.99, 121.05, 136.64, 137.87, 148.73, 150.07 (C3, C3 , C4, C4 ,
0
C6, C6 of bpy); 131.73 (Cp of Ar); 132.67, 133.91, 156.53, 157.55 (C2,
0
0
C2 , C5, C5 of bpy); 153.57, 153.74 (Cipso of Ar, CO); 157.55 (C2 of
bpy); 156.47 (C guan); 164.02 (CO). Anal. Calcd for C31H39N5O5ꢀ0.33-
CH2Cl2 (589.98): C, 63.79; H, 6.78; N, 11.87. Found: C, 63.70; H, 5.57;
N, 11.96. EI-MS (pos. mode): 320.1 [Mꢁ(BocNH–C@NBoc)]+; 342.0
[Mꢁ(BocNH–C@NBoc)+Na+]+; 639.3 [2(Mꢁ(BocNH–C@NBoc))+H+]+.
5.1.10.4. [(N,N0-Di-Boc)-guanidinoethyl]-4-[4-methyleneoxy-40-
methyl-2,20-bithiazolyl]-benzene 28. Way 1: From 25 (0.25 g,
0.55 mmol), NEt3 (0.235 mL, 1.68 mmol), K (0.22 g, 0.56 mmol),
CH2Cl2 (20 mL), MeOH. 4 h, TLC monitoring: Al2O3, CH2Cl2. Wash-
ings: CH2Cl2 (30 mL), 2 M NaHSO4 (20 mL), satd NaHCO3 (20 mL).
Drying: Na2SO4. Chromatography: Al2O3, CH2Cl2. 28 (0.31 g, 95%).

5508
M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
24.54 (Mebpy); 33.93 (CH2CH2N); 42.59 (CH2CH2N); 70.77 (OCH2b-
was continued at rt during 20 h, giving a light brown precipitate.
The solvent was evaporated under high vacuum, to give a solid that
was triturated in CH2Cl2, filtered over Celite, concentrated and
chromatographed (Al2O3, CH2Cl2/Hex, 70:30) to give 32 (0.50 g,
52%). Mp: 101–102 °C. IR (KBr): 3336.8 (–NH, –NH2); 1739.3 (–
C@N); 1654.5 (–N–CO). 1H NMR (400 MHz, CDCl3): 1.26 (m, 12H,
H3,4,5); 1.50 (m, 40H, H2, Me3C); 3.39 (q, J = 7.2 Hz, 4H; H1), 8.29
(s, 2H, NH); 11.50 (s, 2H, NH). 13C NMR (100 MHz, CDCl3): 27.21,
29.33, 29.73 (C3, C4, C5); 28.45, 28.69 (Me3C); 29.59 (C2); 41.34
(C1); 79.51, 83.30 (Me3C); 153.70 (CO); 156.78 (C guan); 164.04
(CO). Anal. Calcd for C32H60O8N6ꢀ0.33C6H12 (685.58): C, 59.56; H,
9.51; N, 12.26. Found: C, 59.42; H, 9.17; N, 12.63. EI-MS: 577
[Mꢁ(Me)3COC(O)+Na]+; 577 [Mꢁ(Me)3COC(O)ꢁ(Me)3C+Na]+.
0
0
py); 115.12 (Co or Cm); 118.10, 119.87, 122.09, 124.08 (C3, C3 , C5, C5
0
bpy); 130.27 (Co or Cm); 131.06 (Cp); 137.97, 138.43 (C4, C4 bpy);
156.94 (C guan); 157.14 (Cipso); 154.60, 155.21, 157.20, 157.94
0
0
(C2 and C2 , C6, C6 bpy). Anal. Calcd for C21H23ON5.1.5CF3COOH
(532.47): C, 54.13; H, 4.63; N, 13.15. Found: C, 54.25; H, 4.76; N,
13.03. ES-MS (pos. mode): 365.35 [MꢁCF3COOH+H+]+. ES-MS
(neg. mode): 588.12 [M+CF3COOꢁ]ꢁ.
5.1.11.3. Guanidinoethyl-4-[5-methyleneoxy-50-methyl-2,20-bipyr-
idyl]-benzene, trifluoroacetate salt 30. From 27 (0.244 g,
0.434 mmol), CH2Cl2 (25 mL), TFA (6 mL). 22 h, TLC monitoring:
SiO2, CH2Cl2/MeOH 98:2. 30 (0.195 g, 95%). Slightly pink solid.
Mp: 162–163 °C. IR (KBr): 3392.73 (NH2); 3116.38 (NH3þ);
1673.98 (C@O); 1626.79, 1513.25 (NH3þ). UV–vis (H2O): 288
(19,072), 240 (15,422), 228 (14,836). 1H NMR (400 MHz, D2O):
2.35 (s, 3H, Mebpy); 2.79 (t, J = 6.28 Hz, 2H, CH2CH2N); 3.43 (t,
J = 6.54 Hz, 2H, CH2CH2N); 5.16 (s, 2H, OCH2bpy); 7.00 (d,
J = 8.04 Hz, 2H, ArH); 7.20 (d, J = 8.28 Hz, 2H, ArH); 7.76 (d,
J = 7.04. Hz, 1H, H bpy); 7.87 (d, J = 7.28 Hz, 1H, H bpy); 7.96 (s,
2H, H bpy); 8.40 (s, 1H, H bpy); 8.58 (s, 1H, H bpy). 1H NMR
(400 MHz, DMSO-d6): 2.64 (s, 3H, Mebpy); 2.86 (t, J = 7.16 Hz, 2H,
CH2CH2N); 3.45 (q, J = 4.78H, CH2CH2N); 5.33 (s, 2H, OCH2bpy);
7.14 (d, J = 8.32 Hz, 2H, ArH); 7.34 (d, J = 8.32 Hz, 2H, ArH); 6.9–
7.77 (broad signal, 3H, NH guan); 7.79 (br s, 1H, CH2CH2NH););
7.89; (d, J = 7.8 Hz, 1H, H bpy); 8.12 (d, J = 8.28 Hz, 1H, H bpy);
8.42 (t, J = 8.04 Hz, 1H, H bpy); 8.51 (d, J = 8.08 Hz, 1H, H bpy);
8.66 (s, 1H, H bpy); 8.87 (s, 1H, H bpy). 13C NMR (100 MHz,
DMSO-d6): 18.19 (Mebpy); 33.93 (CH2CH2N); 42.59 (CH2CH2N);
5.1.13. 1,10-Bis-(guanidino]-decane, bis-trifluoroacetate salt
33; Synthalin A
TFA (15 mL) was added to a solution of 32 (1.14 g, 1.7 mmol) in
anhydrous CH2Cl2 (40 mL). The mixture was stirred at rt under Ar
during 3 h, and the solvents were evaporated. The residual TFA was
eliminated by successive dissolution in CH2Cl2–evaporation cycles,
until a white solid was obtained (ca. four cycles). The latter was
dried under high vacuum, triturated in Et2O, filtered and rinsed
with Et2O (0.85 g). This last solid was dialyzed (Float-A-Lyser, cel-
lulose acetate, MWCO 100 D) and lyophilized to give 33 (0.6 g;
70%). White cotton. Mp: 109–110 °C. IR (KBr): 3376.7 (NH, NH2);
1675.9 (C@N). 1H NMR (400 MHz, D2O): 1.28 (m, 12H, H3,4,5);
1.56 (m, J = 6.4 Hz, 4H, H2); 3.15 (t, J = 7.0 Hz, 4H, H1). 1H NMR
(400 MHz, DMSO-d6): 1.26 (m, 12H, H3,4,5); 1.45 (m, 4H, H2); 3.09
(m, J = 6.4 Hz, 4H, H1). 13C NMR (100 MHz, D2O): 26.17, 28.65,
28.89 (C3, C4, C5); 28.20 (C2); 41.53 (C1); 116.67 (q, J = 291.4 Hz,
CF3COOꢁ); 157.05 (C guan); 163.20 (q, J = 35.2 Hz, CF3COOꢁ). Anal.
Calcd for C12H28N6ꢀ2CF3COOHꢀH2O (498.45): C, 38.55; H, 6.47; N,
16.86. Found: C, 38.55; H, 6.50; N, 16.99. ES-MS (pos. mode):
371.41 [MꢁCF3COOH+H+]+; 257.54 [Mꢁ2CF3COOH+H+]+. ES-MS
(neg. mode): 597.25 [M+CF3COOHꢁH+]ꢁ; 483.38 [M+CF3
COOHꢁH+]ꢁ; 369.45 [MꢁCF3COOHꢁH+]ꢁ.
67.08 (OCH2bpy); 115.16, 130.24 (Co or Cm of Ar); 120.22, 120.37,
0
0
0
137.02, 137.98, 148.95, 149.94 (C3, C3 , C4, C4 , C6, C6 of bpy);
0
0
131.09 (Cp of Ar); 133.13, 134.09, 152.84, 155.37 (C2, C2 , C5, C5 of
bpy); 157.10, 157.14 (C guan; Cipso of Ar); 158.85 (q, J = 30.9 Hz,
CF3COOꢁ); CF3COOꢁ not observed. Anal. Calcd for C21H23ON5-
CF3COOH (475.46): C, 58.10; H, 5.09; N, 14.73. Found: C, 57.96;
H, 5.20; N, 14.63. ES-MS (pos; mode): 362.1 [MꢁCF3COOꢁ]+.
5.1.11.4. Guanidinoethyl-4-[4-methyleneoxy-40-methyl-2,20-bithiaz-
5.2. Biology
olyl]-benzene, trifluoroacetate salt 31. From 28 (0.25 g,
0.42 mmol), CH2Cl2 (30 mL), TFA (10 mL). 3 h, TLC monitoring:
Al2O3, CH2Cl2. 31 (0.2 g, 98%). White solid. Mp: 179–180 °C. IR
(KBr): 3367.2 (NH2); 3166.7 (NH3þ); 1674.9 (C@O); 1624.2,
1515.3 (NH3þ). UV–vis (H2O): 331 (12,553). 1H NMR (400 MHz,
D2O): 2.44 (s, 3H, Mebtz); 2.85 (t, J = 6.5 Hz, 2H, CH2CH2N); 3.43
(t, J = 6.5 Hz, 2H, CH2CH2N); 5.22 (s, 2H, OCH2btz); 7.06 (d,
J = 8.1 Hz, 2H, ArH); 7.26 (d, J = 8.1 Hz, 2H, ArH); 7.30 (s, 1H, H
btz); 7.68 (s, 1H, H btz). 1H NMR (400 MHz, DMSO-d6): 2.44 (s,
3H, Mebtz); 2.74 (t, J = 7.3 Hz, 2H, CH2CH2N); 3.33 (q, J = 6.8 Hz,
2H, CH2CH2N); 5.20 (s, 2H, OCH2btz); 6.50–7.80 (broad signal,
4H, NH3þ, NH); 7.02 (d, J = 8.4 Hz, 2H, ArH); 7.21 (d, J = 8.5 Hz,
2H, ArH); 7.51 (s, 1H, H btz); 7.63 (m, 1H, CH2CH2NH); 7.88 (s,
1H, H btz). 13C NMR (100 MHz, DMSO-d6): 17.03 (Mebtz); 33.94
(CH2CH2N); 42.60 (CH2CH2N); 65.48 (OCH2btz); 115.06 (Co or
5.2.1. Bacteriology
Bacteria were grown in Mueller-Hinton broth (Difco, 275730),
or Mueller Hinton agar (Difco, 225250); purity of isolates was
checked at the time of every test by examination of colony mor-
phology and Gram staining. For MIC determination experiments,
bacteria were inoculated in 96-well U shape microtiter plates to
yield a final inoculum of 1 ꢂ 105 colony-forming units (CFU)/mL.
Then various concentrations of the drugs were added. The cultures
were grown for 18–24 h at 35 °C. The resulting bacterial growth
was measured with an ELISA plate reader (Multiskan EX, Thermo
Electron Corporation, France) at a wavelength of 540 nm.
5.2.2. Cell viability
MRC-5 cells (human pulmonary embryonic fibroblasts) were
obtained from BioMerieux (France). The cells were maintained in
modified Eagle’s medium (MEM, Invitrogen 41090) supplemented
with 10% decomplemented fetal bovine serum (FBS, Invitrogen
10270, batch 40Q5150K) without antibiotics at 37 °C, 5% CO2, un-
der a humid atmosphere. Cells were plated at 104 cells/well in
96-well plates (Sarstedt 831835). Forty eight hours after plating,
the growth medium was removed and replaced with the test solu-
0
Cm); 117.67, 120.69 (C5, C5 btz); 130.22 (Co or Cm); 131.08 (Cp);
0
153.60, 154.12 (C2, C2 btz); 157.09, 157.14 (Cipso and C guan);
0
159.85, 161.34 (C4, C4 btz); no visible TFA. Anal. Calcd for
C17H19ON5S2ꢀCF3COOH (487.52): C, 46.81; H, 4.13; N, 14.36; S,
13.15. Found: C, 46.61; H, 4.42; N, 14.39; S, 12.79. ES-MS (pos.
mode): 374.24 [MꢁCF3COOH+H+]+. ES-MS (neg. mode): 600.04
[M+CF3COOꢁ]ꢁ.
tions (100 lL). Viability tests were performed after 24, 48 and
5.1.12. 1,10-Bis-[(N,N0-di-Boc)-guanidino]-decane 32
168 h exposure to drugs; they were carried out using a commer-
cially available cell proliferation reagent MTT [3-(4,5-dimethyl-
thiazolyl-2-yl-2,5-diphenyltetrazolium bromide] (Sigma 1350380)
as described previously.3,30 The assay is based on cleavage of
the tetrazolium salt MTT by active mitochondria to produce an
The 1,10-diaminodecane (0.25 g, 1.45 mmol) was added under
Ar to a solution of NEt3 (1.41 mL, 10.15 mmol) and J (0.93 g,
3.19 mmol) in dry DMF (15 mL). The mixture was cooled to 4 °C,
and HgCl2 (0.86 g, 3.19 mmol) was added in one portion. Stirring

M. Mourer et al. / Bioorg. Med. Chem. 17 (2009) 5496–5509
5509
5. (a) Frank, E.; Nothmann, M.; Wagner, A. Klinische Wochenschrift 1926, 5, 2100;
(b) Frank, E.; Nothmann, M.; Wagner, A. Klinische Wochenschrift 1928, 7, 1996;
(c) Lourie, E. M.; Yorke, W. Ann. Trop. Med. 1937, 31, 435. cf C.A. 31, 8006.9..
6. (a) For example: Gutsche, C. D. Calixarenes, In Stoddart, J. F., Ed.; Monographs
in Supramolecular Chemistry; Royal Society of Chemistry: Cambridge, 1989.;
(b)Calixarenes 2001; Asfari, Z., Böhmer, V., Harrowfield, J., Vicens, J., Eds.;
Kluwer: Dordrecht, The Netherlands, 2001.
7. Yo, T., Fujiwara, K., Otsuka, M. JP 10203906, 1998.
8. (a) Harris, S. J. WO Patent 9519974, 1995.; (b) Harris, S. J. WO Patent 0244121,
2002.
9. Hwang, K. M.; Qi, Y. M.; Liu, S. Y.; Choy, W.; Chen, J. WO Patent 9403164, 1994.
10. Hwang, K. M.; Qi, Y. M.; Liu, S. Y.; Lee, T. C.; Choy, W.; Chen, J. WO Patent
9403165, 1994.
11. Hulmes, D.; Coleman, A.; Aubert-Foucher, E. (CNRS, Fr) WO Patent 0007585,
2000.
insoluble purple formazan salt. Since this conversion only occurs
with viable cells, it directly correlates with cell count. After 24,
48 and 168 h exposure, 10
added to each well and plates were incubated at 37 °C for 4 h.
Then, insoluble purple formazan was dissolved by adding 100
lL of a 5 mg/mL MTT solution were
lL
SDS to each well. The absorbance A540 nm was measured with a ref-
erence wavelength of A690 nm, using an ELISA reader (Multiskan EX,
Thermo Electron Corporation, France). The results were expressed
as the percent absorbance of treated versus untreated control cul-
tures. Eight wells per dose and time point were counted in 3 differ-
ent experiments. Lactate dehydrogenase (LDH) release
experiments: The detection procedures of LDH release were in
accordance with the manufacturer’s instructions (CytoTox 96Ò
Non-Radioactive Cytotoxicity Assay, Promega Corp, Madison, WI).
Briefly, all reagents stocked at 4 °C were slowly warmed up. Then,
12. Cornforth, J. W.; D’Arcy Hart, P.; Nicholls, G. A.; Rees, R. J. W.; Stock, J. A. Br. J.
Pharmacol. 1955, 10, 73.
13. (a) D’Arcy Hart, P.; Armstrong, A. J.; Brodaty, E. Infect. Immun. 1996, 64, 1491;
(b) Colston, M. J.; Hailes, H. C.; Stavropoulos, E.; Herve, A. C.; Herve, G.;
Goodworth, K. J.; Hill, A. M.; Jenner, P.; Hart, P. D.; Tascon, R. E. Infect. Immun.
2007, 72, 6318; (c) Loiseau, F. A.; Hill, A. M.; Hii, K. K. Tetrahedron 2007, 63,
9947.
on appropriate microtiter plate (MTP), 10 lL of lysis solution were
added to ‘control cell’ wells in order to determine 100% release of
14. Casnati, A.; Fabbi, M.; Pelizzi, N.; Pochini, A.; Sansone, F.; Ungaro, R.; Di
Modugno, E.; Tarzia, G. Bioorg. Med. Chem. Lett. 1996, 6, 2699.
15. For example: (a) Auletta, T.; Dordi, B.; Mulder, A.; Sartori, A.; Onclin, S.;
Bruinink, C. M.; Péter, M.; Nijhuis, C. A.; Beijleved, H.; Schönherr, H.; Vancso, G.
J.; Casnati, A.; Ungaro, R.; Ravoo, B. J.; Huskens, J.; Reinhoudt, D. N. Angew.
Chem., Int. Ed. 2004, 43, 369; (b) Mulder, A.; Auletta, T.; Sartori, A.; Del Ciotto,
S.; Casnati, A.; Ungaro, R.; Huskens, J.; Reinhoudt, D. N. J. Am. Chem. Soc. 2004,
126, 6627.
16. (a) Dudic, M.; Colombo, A.; Sansone, F.; Casnati, A.; Donofrio, G.; Ungaro, R.
Tetrahedron 2004, 60, 11613; (b) Sansone, F.; Dudic, M.; Donofrio, G.; Rivetti,
C.; Baldini, L.; Casnati, A.; Cellai, S.; Ungaro, R. J. Am. Chem. Soc. 2006, 128,
14528; (c) Bagnacani, V.; Sansone, F.; Donofrio, G.; Baldini, L.; Casnati, A.;
Ungaro, R. Org. Lett. 2008, 10, 3953; (d) Dings, R. P. M.; Chen, X.; Hellebrekers,
D. M. E. I.; van Eijk, L. I.; Zhang, Y.; Hoye, T. R.; Griffioen, A. W.; Mayo, K. H. J.
Natl. Cancer Inst. 2006, 98, 932.
LDH; and MTP was centrifuged at 2500 g for 4 min to have ‘cell-
free’ supernatants. Subsequently, 25
ferred to a new MTP, and 25 L of freshly prepared Substrate Mix
were added to each well and incubated up to 30 min in the dark at
room temperature. Finally, 50 L of stop solution were added to
lL of each sample were trans-
l
l
each well. The absorbance A490 nm was measured using an ELISA
reader (Multiskan EX, Thermo Electron Corporation, France). The
results were expressed as the percent absorbance of treated versus
untreated control cultures. Eight wells per dose and time point
were counted in 3 different experiments.
17. Elliot, A. J.; Morris, P. E.; Petty, S. L., Jr.; Williams, C. H. J. Org. Chem. 1997, 62,
8071.
18. Gutsche, C. D.; Nam, K. C. J. Am. Chem. Soc. 1988, 110, 6153.
19. Baker, T. J.; Tomioka, M.; Goodman, M.; Mergott, D. G.; Roush, W. R. Org. Synth.
2000, 78, 91.
20. (a) Obtained by reaction of PBr3 on [82740-71-4].; (b) Ziessel, R.; Hissler, M.;
Ulrich, G. Synthesis 1998, 9, 1339; (c) Heck, R.; Dumarçay, F.; Marsura, R. Chem.
Eur. J. 2002, 8, 2438.
21. Pellet-Rostaing, S.; Regnouf-de-Vains, J.-B.; Lamartine, R. Tetrahedron Lett.
1996, 37, 5889.
Acknowledgements
We are grateful to the MRES and CNRS for financial support,
especially M. Mourer for a PhD grant. The authors thank E. Dubs
for preparing some of the chemical intermediates, J.-M. Ziegler, F.
Dupire and S. Adach for mass measurements and elemental analy-
ses, and Mrs. Nicole Marshall for correcting the manuscript.
22. For example: Regnouf-de-Vains, J.-B.; Lamartine, R. Helv. Chim. Acta 1994, 77,
1817. and references cieted therein.
23. (a) Jaime, C.; de Mendoza, J.; Prados, P.; Nieto, P. M.; Sanchez, C. J. Org. Chem.
1991, 56, 3372; (b) Magrans, J. O.; de Mendoza, J.; Pons, M.; Prados, P. J. Org.
Chem. 1997, 62, 4518.
24. (a) Mourer, M.; Regnouf-de-Vains, J.-B. Tetrahedron Lett. 2006, 47, 1895; (b)
Psychogios, N.; Regnouf-de-Vains, J.-B. Tetrahedron Lett. 2002, 43, 7691.
25. Al-Damluji, S.; Kopin, I. J. Brit. J. Pharm. 1998, 124, 693.
26. (a) Ghosh, S.; Davis, R. E. WO 9955321, 1999.; (b) Michel, R.; Truchot, R.; Gully-
Martin, V. Biochem. Pharm. 1971, 20, 2587.
References and notes
1. Leeb, M. Nature 2004, 431, 892.
2. (a) Ben Salem, A.; Regnouf-de-Vains, J.-B. Tetrahedron Lett. 2001, 42, 7033; (b)
Ben Salem, A.; Regnouf-de-Vains, J.-B. Tetrahedron Lett. 2003, 44, 6769.
3. (a) Mourer, M.; Duval, R. E.; Finance, C.; Regnouf-de-Vains, J.-B. Bioorg. Med.
Chem. Lett. 2006, 16, 2960; (b) Grare, M.; Mourer, M.; Regnouf-de-Vains, J.-B.;
Finance, C.; Duval, R. E. Pathol. Biol. 2006, 54, 470; (c) Grare, M.; Mourer, M.;
Fontanay, S.; Regnouf-de-Vains, J.-B.; Finance, C.; Duval, R. E. J. Antimicrob.
Chemother. 2007, 60, 575.
27. Colca, J. R.; Larsen, S. D.; Meglasson, M. D.; Tanis, S. P. WO 9303714, 1993.
28. Goeres, E.; Schwartz, J.; Haeussler, R.; Petsch, G.; Wehl, A. Acta Biol. Med. Ger
1970, 24, 369.
4. (a) Laqua, A.; Holtschmidt, U.; Bertelmann, G. Fr 2,418,789, 1979.; (b)
Wallhaüsser, K. H.; May, A.; Bücking, H.-W. EP 0,003,999, 1979.; (c) Diery, H.;
Wagemann, W.; Bücking, H.-W.; Hille, M.; Wallhaüsser, K. H. U.S. Patent
4,339,459, 1982.; (d) Butlion, M. WO 98/56366, 1998. Amtmann, E. U.S. Patent
0,192,717, 2004.
29. National Committee for Clinical Laboratory Standards. 2001. Methods for
dilution antimicrobial susceptibility tests for bacteria that grow aerobically.
5th ed. Approved standard. NCCLS document M7-A5. National Committee for
Clinical Laboratory Standards. Wayne, PA, USA.
30. Mosmann, T. J. Immunol. Methods. 1983, 65, 55–63.