Synthesis and in vitro enzyme hydrolysis of trioxadiaza- and tetraoxadiaza-crown ether-based complexing agents with disposable ester pendant arms

Article abstract of DOI:10.1055/s-2005-918442

New disposable ester derivatives of the complexing agents N,N′-bis(carboxymethyl)piperazine, -homopiperazine, -1,7-diaza-15-crown-5 and -1,10-diaza-18-crown-6 were synthesized with a variety of synthetic methods and fully characterized. Hydrolytic properties of the pendant arms were studied under different pH conditions as well as in the presence and absence of porcine liver esterase enzyme and approximate hydrolysis half lives were determined by ¹H NMR technique. In vitro studies on pig liver cell homogenates and living thin chicken liver slices proved that the selected double ester 4b can penetrate liver tissues spontaneously, liberate the free complexing agent N,N′-bis(carboxymethyl)-18-ane-N₂O₄ (ODDA) inside the cells and are potentially capable of removing lead and other toxic metal ions from the liver. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2005-918442

PAPER
3555
Synthesis and in vitro Enzyme Hydrolysis of Trioxadiaza- and Tetraoxadiaza-
Crown Ether-Based Complexing Agents with Disposable Ester Pendant Arms
S
ynthesisof Cr
o
í
w
n
E
t
m
w
ithDisposabl
e
e
E
ster Pe
n
a
dant
A
rms Iványi, István Lázár*
University of Debrecen, Department of Inorganic and Analytical Chemistry, Egyetem tér 1, Debrecen 4032, Hungary
Fax +36(52)489667; E-mail: lazar@delfin.unideb.hu
Received 23 February 2005; revised 27 June 2005
Dedicated to Professor Ernő Brücher on the occasion of his 70th birthday.
derivative (16-carboxymethyl-1,4,10,13-tetraoxa-7,16-
Abstract: New disposable ester derivatives of the complexing
agents N,N¢-bis(carboxymethyl)piperazine, -homopiperazine, -1,7-
diaza-15-crown-5 and -1,10-diaza-18-crown-6 were synthesized
with a variety of synthetic methods and fully characterized. Hydro-
diazacyclooctadec-7-yl)acetic acid (ODDA)11a (Figure 1)
forms even stronger complexes with lead and cadmium12
ions (Table 1) and can be potentially used for their effi-
lytic properties of the pendant arms were studied under different pH cient complexation in the presence of essential cations
conditions as well as in the presence and absence of porcine liver es-
like zinc, magnesium and calcium in living organisms.
terase enzyme and approximate hydrolysis half lives were deter-
However, therapeutic application of ODDA has not been
1
mined by H NMR technique. In vitro studies on pig liver cell
homogenates and living thin chicken liver slices proved that the se-
lected double ester 4b can penetrate liver tissues spontaneously, lib-
erate the free complexing agent N,N¢-bis(carboxymethyl)-18-ane-
N2O4 (ODDA) inside the cells and are potentially capable of remov-
ing lead and other toxic metal ions from the liver.
reported so far, supposedly because of lack of the proper
formulation and administration technique, without which
disubstituted [18]aneN2O4 derivatives have been fairly
toxic with a LD50 value of 5–50 mg/kg in mice.11a
Key words: crown compounds, complexes, esters, enzymes, hy-
drolyses
O
O
O
O
O
O
O
O
O
O
O
HO
HO
HO
OH
OH
N
N
N
N
OH
O
O
O
ODDM
ODDA
Acute and chronic lead poisonings, a number of deaths, as
well as temporary extinction of fishes in the river Tisza
were the consequences of food adulteration,1 long-term
environmental pollution2 and two industrial disasters that
hit Hungary in the last decade.3 Removal of Pb2+ ions
from the human body is most commonly accomplished by
open-chained polyamino polycarboxylates like ethylene-
diamine tetraacetic acid (EDTA), diethylenetriamine pen-
taacetic acid (DTPA), etc. However, this therapy have
serious side effects like depletion of zinc ions from the
body, bone marrow depression, kidney damages or terato-
genicity, especially when EDTA was administered in
large doses.4 Most recently other types of chelating agents
have been tested, including 2,3-dimercaptopropanol, D-
penicillamine, glutathione, 1,2-dimercaptosuccinic acid,
2,3-dimercaptopropane-1-sulfonate, and monoisoamyl
2,3-dimercaptosuccinate5 as well as the non-toxic macro-
cyclic derivatives, 1,4,8,11-tetraazacyclotetradecane-
1,4,8,11-tetraacetic acid6 and 2-(16-dicarboxymethyl-
1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-yl)malonic
acid (ODDM)7,8 (Figure 1). ODDM, in the form of its cal-
cium complex, was the first substance which, due to its
unique selectivity for Sr2+ ions over Ca2+ ions effectively
removed radioactive 90Sr2+ ions from experimental pigs,
offering the possibility to be used as an emergency medi-
cal treatment in cases of personal contaminations or nu-
clear power station accidents.9,10 Another crown ether
Figure 1
Table 1 Stability Constants of Selected Complexing Agents with
Essential and Toxic Metal Ions
Metal ion
logKML
EDTA
8.83
ODDM
ODDA
not analyzed
8.39
Mg2+
Ca2+
Sr2+
2.53
7.54
10.61
8.74
9.79
8.29
Zn2+
Pb2+
Cd2+
16.44
17.88
16.36
6.28
8.42
13.03
10.27
13.55
11.07
The excellent and highly specific complexing properties
of the macrocyclic complexing agent ODDA and the lack
of its therapeutic applications inspired us to synthesize
and in vitro examine a series of enzymatically hydrolyz-
able new ester derivatives of two polyoxa-diaza crown
ether-based complexing agents. The new esters as pro-
drugs could be used for the selective removal of lead ions
from the living organisms. So far only the methyl and eth-
yl esters of ODDA were prepared but none was tested in
enzyme studies.11b–e The analogous piperazine and ho-
mopiperazine derivatives served as models for the syn-
SYNTHESIS 2005, No. 20, pp 3555–3564
x
x.
x
x
.
2
0
0
5
Advanced online publication: 27.10.2005
DOI: 10.1055/s-2005-918442; Art ID: T02305SS
© Georg Thieme Verlag Stuttgart · New York

3556
T. Iványi, I. Lázár
PAPER
O
thetic and NMR studies and possible targets for
complexation of smaller cations.
O
HO
H2/10% Pd–C
N
n
N
1f or 2f
3f or 4f
O
OH
r.t., MeOH, 5–15 h
O
Prodrugs with disposable ester groups are widely used to
improve uptake properties of drugs that contain free car-
boxylic groups in their physiologically active form. These
esters enter the tissues more efficiently and hydrolyze in-
side the cells by the esterase enzymes liberating the free
carboxylic drug in situ.13 Under certain conditions the hy-
drolytic process can be advantageously examined on tis-
n = 0 1h (35%)
(40%)
1 2h
O
O
O
O
/10% Pd–C
H2
HO
O
N
N
r.t., MeOH, 5–15 h
O
OH
O
m
O
m = 0 3h (33%)
(38%)
1
sue homogenates or cell suspensions by H or 31P NMR
4h
1
techniques.14 Esterification was used for the open-chained
complexing agent hydroxyethyl(ethylenediamine)triace-
tic acid which, in its lactone form, significantly improved
the excretion of 144Ce and 91Y from the liver.15a In addition
to esterification, attachment of longer alkyl groups to the
open-chained complexing agents can also change biodis-
tribution profile and help removing the toxic metal ions
from the human body.15b
O
X
O
i
ii
-
iii
O NR'"+
O
4
1
N
N
2
2
1l
1m
R"' = Me
Et
(82%)
(80–90%)
X = H 1j (64–97%)
Me 1k (88%)
O
N
R"
O
2
R" = CH2COOBn 1c (69%)
O
O
(39%)
(94%)
1d
1i
CH2CN
Scheme 2 Reagents and conditions: i) for 1j: BrCH2CO2H, NaOH/
H2O, r.t., then pH set to 2, for 1k: ClCH2CO2Me, i-Pr2EtN, MeCN, re-
flux; ii) Me4NOH or Et4NOH for 1l or 1m, respectively; iii) for 1c and
1d: BrCH2CO2Bn and BrC4H5O2, respectively, anhyd MeCN, reflux,
8 h, for 1i: ClCH2CN, NaI, anhyd MeCN, reflux, 5 h
which was purified by dry column vacuum chromatogra-
phy.18
When reactions shown on Scheme 1 were applied in pre-
liminary experiments to 1,4,7-triazacyclononane and
ROH, HCl,
O
Scheme 1 Reagents and conditions: i) for compounds a,b,c,e,f, and
g: 8, 9, 10, 12, 13 and benzyl bromide, respectively, anhyd MeCN, ex-
cess i-Pr2EtN, reflux, 4–8 h
reflux
HOCH2COOH
HO
OR
R = Et 5 (42%)
Bn 6 (27%)
Disposable esters 1a–g, 2a–c, 2f–g, 3a–c, 3f–g, 4a–c, 4f–
g were best prepared in one step from stoichiometric
amount of 8–13 and the diamines 1–4 in an anhydrous di-
polar aprotic solvent (preferably in acetonitrile) in the
presence of i-Pr2NEt as a non-nucleophilic base
(Scheme 1). Reaction of bromoacetic acid hydroxyethyl
ester with the diamines 1–4 gave rapid and so far uniden-
tified side reactions, therefore compound 10 was reacted
with amines 1–4 yielding compounds 1f–4f from which
benzyl protecting groups were removed by catalytic hy-
drogenation on 10% Pd/C in methanol at room tempera-
ture giving 2-hydroxyethyl esters 1h–4h in high yields
(Scheme 2).
PhCH2Br, KOH
r.t. to 127 °C
O
OH
HO
HO
Bn
7 (53%)
R'OH, CH2Cl2
O
BrCH2C(O)Br
Br
OR'
CH CO Et
5
6
7
8 (36%)
9 (56%)
R' =
2
2
CH2CO2Bu
CH2CO2Bn
10 (30%)
O
O
Glycolic acid esters 5, 6 and 2-benzyloxyethanol (7) were
synthesized by published methods.16,17 a-Bromo esters 8–
13 as starting materials were then prepared from bro-
moacetyl bromide and a hydroxyl group-containing sub-
stance in anhydrous dichloromethane according to
Scheme 3. All esters were distilled in vacuo except for 11,
11 (8%)
CH2CF3
12 (47%)
CH2CH2OBn
(32%)
13
Scheme 3
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York

PAPER
Synthesis of Crown Ethers with Disposable Ester Pendant Arms
3557
1,4,7,10-tetraazacyclododecane, both of them produced philicity of the carboxylate anion, tetraalkylammonium
many side products most likely due to the extremely large salts 1l and 1m were prepared from 1j by neutralization
differences in the basicities of the secondary amino with tetraalkylammonium hydroxides, or by direct cou-
groups (7 orders of magnitude) and their capabilities to pling of piperazine and bromoacetic acid in the presence
form bicyclic and/or polymeric structures through sec- of tetraalkylammonium hydroxide as a strong base, or by
ondary transamidation processes. Column chromatogra- hydrolysis of the methyl ester 1k with tetraalkylammoni-
phy of the crude products on silica resulted in rapid um hydroxide (Scheme 2). Tetraethylammonium salt 1m
hydrolysis, thus other starting materials were considered gave better yields than tetramethylammonium salt 1l in
to find a generally applicable synthetic procedure for all the alkylation reactions due to its higher solubility. The re-
aza-macrocycles.
N,N¢-Bis(carboxymethyl)piperazine activity of 2-chloroethanol and chloroacetonitrile has
(1j) (PIDA) and its methyl ester (1k) were selected as been increased by adding sodium iodide to the reaction
model compounds for the synthetic studies. Direct alkyla- mixture, which gave the more reactive iodoethanol and io-
tion of the carboxylate groups of 1j with ethyl bromoace- doacetonitrile in situ.
tate was examined in the presence of i-Pr2NEt in
The spectral data of compounds 1–13 prepared are assem-
acetonitrile, acetone and DMF. No reaction took place af-
bled in Table 2.
ter several days even at or near reflux temperature with 1j,
very likely due to its very low solubility in the reaction
media. In order to increase the solubility and the nucleo-
Table 2 Characteristic Analytical Data of Compounds 1a–13
Producta
1H NMR (CDCl3)
13C NMR (CDCl3)
MALDI-TOF MS
d, J (Hz)
d
m/z
1a
1.28 (t, 3JH,H = 7.4, 6 H, CH3), 2.72 (s, 8 H, ring
13.9 (CH3), 52.5 (ring CH2), 58.9 (NCH2), 60.6 and M + H+ found
CH2), 3.36 (s, 4 H, NCH2), 4.22 (q, 3JH,H = 7.4, 4 H, 61.3 (OCH2), 167.4 and 169.5 (C=O)
(calcd): 375.24
(375.18)
OCH2], 4.66 (s, 4 H, OCH2)
1b
0.93 (t, 3JH,H = 7.4, 6 H, CH3), 1.32–1.42 (m, 4 H,
13.5 (CH3), 18.9 and 30.4 (CH2), 52.4 (ring CH2),
58.7 (NCH2), 60.7 and 65.8 (OCH2), 167.5 and
M + H+ found
(calcd): 431.11
(431.24)
CH2), 1.58–1.66 (m, 4 H, CH2), 2.73 (s, 8 H, ring
CH2), 3.36 (s, 4 H, NCH2), 4.16 (t, 3JH,H = 6.8, 4 H, 169.4 (C=O)
OCH2), 4.66 (s, 4 H, OCH2)
1c
2.64 (s, 8 H, ring CH2), 3.31 (s, 4 H, CH2), 4.71 (s, 4 52.5 (ring CH2), 58.9 (NCH2), 60.7 and 67.1
M + H+ found
(calcd): 499.43
(499.21)
H, OCH2), 5.18 (s, 4 H, OCH2), 7.26–7.38 (m, 10
Harom
(OCH2), 128.4, 128.5 and 128.6 (CHarom), 134.8
(Carom), 167.3 and 169.4 (C=O)
)
1d
2.29–2.41 (m, 2 H, CH2), 2.69–2.78 (m, 2 H, CH2), 28.3 (CH2), 51.9 (ring CH2), 58.1 (NCH2), 65.0
–
2.75 (s, 8 H, ring CH2), 3.40 (d, 3JH,H = 5.8, 4 H,
NCH2), 4.29–4.37 (m, 2 H, OCH2), 4.47–4.53 (m, 2
H, OCH2), 5.46–5.52 (m, 2 H, 2 OCH)
(OCH2), 67.6 (OCH), 168.4 and 172.3 (C=O)
1e
1f
2.68 (s, 8 H, ring CH2), 3.35 (s, 4 H, NCH2), 4.51 (q, 52.5 (ring CH2), 58.5 (NCH2), 59.5, 59.9, 60.35 and M+ found (calcd):
3JH,H = 8.4, 4 H, OCH2)
60.75 (OCH2), 118.2, 121.2, 124.3 and 127.4 (CF3), 367.38 (367.11)
168.6 (C=O)
2.61 (s, 8 H, ring CH2), 3.21 (s, 4 H, NCH2), 3.62– 52.6 (ring CH2), 59.1 (NCH2), 63.5 (CH2), 67.6 and M + H+ found
3.65 (m, 4 H, CH2), 4.25–4.28 (m, 4 H, OCH2), 4.51 72.9 (OCH2), 127.5, 127.6 and 128.3 (CHarom),
(s, 4 H, OCH2), 7.26–7.37 (m, 10 Harom 137.6 (Carom), 170.0 (C=O)
(calcd): 471.47
(471.25)
)
1g
2.65 (s, 8 H, ring CH2), 3.26 (s, 4 H, NCH2), 5.16 (s, 52.7 (ring CH2), 59.2 (NCH2), 66.3 (OCH2), 128.3 M+ found (calcd):
4 H, OCH2), 7.35 (m, 10 Harom and 128.5 (CHarom), 135.6 (Carom) 170.0 (C=O) 383.17 (383.20)
)
1hb
3.29 (s, 8 H, ring CH2), 3.81–3.84 (m, 4 H, CH2),
3.90 (s, 4 H, NCH2), 4.31–4.33 (m, 4 H, OCH2)
52.4 (ring CH2), 59.1 (NCH2), 60.2 (CH2OH), 66.1 M + H+ found
(OCH2), 170.2 (C=O)
(calcd): 291.43
(291.16)
1i
2.68 (s, 8 H, ring CH2), 3.35 (s, 4 H, NCH2), 4.77 (s, 48.2 (ring CH2), 52.5 (NCH2), 58.4 (OCH2), 168.6
–
4 H, OCH2)
(C=O)
2a
1.28 (t, 3JH,H = 7.4, 6 H, CH3), 1.84–1.91 (pent,
14.0 (CH3), 28.1 (ring CH2), 53.7 and 54.4 (ring
M + H+ found
3JH,H = 5.8, 2 H, ring CH2), 2,91–2.95 (m, 8 H, ring NCH2), 58.7 (NCH2), 60.5 and 61.3 (OCH2), 167.5 (calcd): 389.23
NCH2), 3.56 (s, 4 H, NCH2), 4.22 (q, 3JH,H = 7.4, 4 and 170.5 (C=O)
(389.19)
H, OCH2), 4.65 (s, 4 H, OCH2)
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York

3558
T. Iványi, I. Lázár
PAPER
Table 2 Characteristic Analytical Data of Compounds 1a–13 (continued)
Producta
1H NMR (CDCl3)
d, J (Hz)
13C NMR (CDCl3)
d
MALDI-TOF MS
m/z
2b
0.93 (t, 3JH,H = 7.4, 6 H, CH3), 1.32–1.42 (m, 4 H,
ring CH2), 1.58–1.66 (m, 4 H, CH2), 1.83–1.90 (m, 53.7 and 54.5 (ring NCH2), 58.7 (NCH2), 60.4
2 H, ring CH2), 2.90–2.94 (m, 8 H, ring NCH2), 3.56 (OCH2), 65.1 (CH2) 167.6 and 170.5 (C=O)
(s, 4 H, NCH2), 4.14–4.18 (q, 3JH,H = 6.8, 4 H, CH2),
13.3 (CH3), 18.8 (CH2), 28.2 (ring CH2), 30.3 (CH2), M + H+ found
(calcd): 445.29
(445.25)
4.64 (s, 4 H, OCH2)
2c
1.79 (pent, 3JH,H = 5.8, 2 H, ring CH2], 2.83–2.87 (m, 27.9 (ring CH2), 53.6 and 54.2 (ring NCH2), 58.6
8 H, ring NCH2), 3.52 (s, 4 H, NCH2), 4.69 (s, 4 H, (NCH2), 60.4 and 67.1 (OCH2), 128.3, 128.4 and
M + H+ found
(calcd): 513.48
(513.22)
OCH2), 5.18 (s, 4 H, OCH2), 7.26–7.36 (m, 10 Harom
)
128.5 (CHarom), 134.8 (Carom), 167.4 and 170.4
(C=O)
2e
2f
1.84 (pent, 3JH,H = 5.8, 2 H, ring CH2], 2.86–2.89 (m, 28.4 (ring CH2), 53.6 and 54.5 (ring NCH2), 58.5
–
8 H, ring NCH2), 3.54 (s, 4 H, NCH2), 4.49 (q,
(NCH2), 59.3, 59.7, 60.1 and 60.5 (OCH2), 118.2,
121.3, 124.4 and 127.4 (CF3), 169.6 (C=O)
3JH,H = 8.4, 4 H, OCH2)
1.79 (pent, 3JH,H = 5.8, 2 H, ring CH2], 2.81–2.85 (m, 28.0 (ring CH2), 53.6 and 54.4 (ring NCH2), 58.8
8 H, ring NCH2), 3.42 (s, 4 H, NCH2), 3.64–3.66 (m, (NCH2), 63.1 (CH2), 67.5 and 72.75 (OCH2), 127.4, (calcd): 485.43
M + H+ found
4 H, CH2), 4.26–4.28 (m, 4 H, OCH2), 4.53 (s, 4 H, 127.45 and 128.1 (CHarom), 137.5 (Carom), 170.9
OCH2), 7.23–7.35 (m, 10 Harom (C=O)
(485.27)
)
2g
1.81 (pent, 3JH,H = 5.8, 2 H, ring CH2), 2.84–2.86 (m, 28.2 (ring CH2), 53.8 and 54.6 (ring NCH2), 59.1
8 H, ring NCH2), 3.45 (s, 4 H, NCH2), 5.14 (s, 4 H, (NCH2), 66.1 (OCH2), 128.2, 128.3 and 128.4
M + H+ found
(calcd): 397.13
(397.21)
OCH2), 7.29–7.36 (m, 10 Harom
)
(CHarom), 135.7 (Carom), 171.0 (C=O)
2hb
2.24 (pent, 3JH,H = 5.8, 2 H, ring CH2), 3.46 (t,
3JH,H = 5.4, 4 H, ring NCH2], 3.63 (s, 4 H, ring
NCH2), 3.83–3.86 (m, 4 H, CH2), 4.12 (s, 4 H,
NCH2), 4.34–4.37 (m, 4 H, OCH2)
22.6 (ring CH2), 51.3 and 54.7 (ring NCH2), 57.3
(CH2OH), 59.6 (NCH2), 67.7 (OCH2), 168.4 (C=O) (calcd): 305.47
(305.17)
M + H+ found
3a
3b
1.28 (t, 3JH,H = 7.4, 6 H, CH3), 2.97–3.01 (m, 8 H,
ring NCH2), 3.58–3.63 (m, 12 H, ring OCH2), 3.65 (NCH2), 61.4 (OCH2), 69.1, 69.9 and 70.4 (ring
(s, 4 H, NCH2), 4.22 [q, 3JH,H = 7.4, 4 H, CH2], 4.63 OCH2), 167.6 and 170.9 (C=O)
(s, 4 H, OCH2)
14.0 (CH3), 54.3 and 55.9 (ring NCH2), 60.4
M + H+ found
(calcd): 507.23
(507.26)
0.93 (t, 3JH,H = 7.4, 6 H, CH3], 1.32–1.42 (m, 4 H,
CH2), 1.58–1.66 (m, 4 H, CH2), 2.99–3.03 (m, 8 H, NCH2), 55.8 (NCH2), 60.4 (OCH2), 65.2 (CH2),
ring NCH2), 3.59–3.63 (m, 12 H, ring OCH2), 3.67 69.1, 69.8 and 70.4 (ring OCH2), 167.6 and 170.7
(s, 4 H, NCH2), 4.16 (t, 3JH,H = 6.8 4 H, CH2), 4.63 (C=O)
13.5 (CH3), 18.9 and 30.4 (CH2), 53.9 and 54.2 (ring M + H+ found
(calcd): 563.29
(563.32)
(s, 4 H, OCH2)
3c
3f
2.92–2.97 (m, 8 H, ring CH2), 3.54–3.59 (m, 8 H,
ring NCH2), 3.61 (s, 4 H, ring NCH2), 3.62 (s, 4 H, 67.0 (OCH2), 69.1, 69.8 and 70.4 (ring OCH2),
53.8 and 54.2 (ring NCH2), 55.9 (NCH2), 60.3 and M + H+ found
(calcd): 631.45
(631.29)
NCH2), 4.67 (s, 4 H, OCH2), 5.18 (s, 4 H, OCH2),
7.31–7.40 (m, 10 Harom
128.3, 128.4 and 128.5 (CHarom), 134.9 (Carom),
167.4 and 170.8 (C=O)
)
2.92–2.96 (m, 8 H, ring NCH2), 3.53 (s, 4 H, ring
53.9 and 54.3 (ring NCH2), 56.2 (NCH2), 63.3
M + H+ found
(calcd): 603.55
(603.33)
OCH2), 3.55–3.59 (m, 8 H, ring OCH2), 3.61 (s, 4 H, (CH2), 67.7 (OCH2), 69.1, 69.8 and 70.4 (ring
NCH2), 3.65–3.67 (m, 4 H, CH2), 4.25–4.28 (m, 4 H, OCH2), 73.0 (OCH2), 127.6, 127.7 and 128.4
OCH2), 4.55 (s, 4 H, OCH2), 7.28–7.37 (m, 10 Harom
) (CHarom), 137.7 (Carom), 171.3 (C=O)
3g
2.93–2.97 (m, 8 H, ring NCH2), 3.54–3.59 (m, 12 H, 54.0 and 54.4 (ring NCH2), 56.55 (NCH2), 66.0
M + H+ found
(calcd): 515.07
(515.28)
ring OCH2), 3.61 (s, NCH2, 4 H), 5.13 (s, 4 H,
OCH2), 7.31–7.37 (m, 10 Harom
(OCH2), 69.4, 70.15 and 70.5 (ring OCH2), 128.2,
128.25 and 128.5 (CHarom), 135.7 (Carom), 171.5
(C=O)
)
3hb
4a
3.28 (s, 4 H, ring NCH2), 3.72–3.76 (m, 12 H, ring 54.6 and 54.9 (ring NCH2), 59.7 (NCH2), 55.7
OCH2), 3.80–3.83 (m, 4 H, 2 CH2OH, 3.94 (s, 4 H, (CH2OH), 59.7 (NCH2), 66.1, 67.3 and 69.8 (ring
M + H+ found
(calcd): 423.12
(423.23)
NCH2), 4.29–4.32 (m, 4 H, OCH2)
OCH2), 169.4 (C=O)
1.28 (t, 3JH,H = 7.4, 6 H, CH3), 3.01 (t, 3JH,H = 5.3, 8 14.0 (CH3), 53.9 (ring NCH2), 55.5 (OCH2), 60.4
H, ring NCH2], 3.61 (s, 8 H, ring OCH2), 3.63–3.66 (CH2), 61.3 (OCH2), 69.8 and 70.4 (ring OCH2),
(t, 3JH,H = 7.4, 8 H, ring OCH2], 3.71 (s, 4 H, CH2), 167.6 and 171.0 (C=O)
4.22 (q, 3JH,H = 7.4, 4 H, CH2), 4.63 (s, 4 H, OCH2)
M + H+ found
(calcd): 551.34
(551.28)
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York

PAPER
Synthesis of Crown Ethers with Disposable Ester Pendant Arms
3559
Table 2 Characteristic Analytical Data of Compounds 1a–13 (continued)
Producta
1H NMR (CDCl3)
13C NMR (CDCl3)
MALDI-TOF MS
d, J (Hz)
d
m/z
4b
0.93 (t, 3JH,H = 7.4, 6 H, CH3), 1.32–1.42 (m, 4 H,
13.5 (CH3), 18.8 (CH2), 30.3 (CH2), 53.8 (ring
M + H+ found
CH2), 1.58–1.66 (m, 4 H, CH2), 3.02 (t, 3JH,H = 5.3, NCH2), 55.3 (CH2), 60.3 (OCH2), 65.1 (CH2), 69.7 (calcd): 607.22
8 H, ring NCH2], 3.61 (s, 8 H, ring OCH2), 3.63–
3.66 (t, 3JH,H = 5.3, 8 H, ring OCH2), 3.73 (s, 4 H,
CH2), 4.16 (t, 3JH,H = 6.8, 4 H, CH2), 4.63 (s, 4 H,
OCH2)
and 70.4 (ring OCH2), 167.6 and 170.9 (C=O)
(607.34)
4c
4f
2.96 (t, 3JH,H = 5.3, 8 H, ring NCH2), 3.58–3.61 (m, 53.9 (ring NCH2), 55.5 (CH2), 60.3 (OCH2), 67.0
16 H, ring OCH2), 3.67 (s, 4 H, CH2), 4.67 (s, 4 H, (CH2), 69.85 and 70.5(ring OCH2), 128.3, 128.45
M + H+ found
(calcd): 675.51
OCH2), 5.18 (s, 4 H, OCH2), 7.31–7.38 (m, 10 Harom
) and 128.5 (CHarom), 135.0 (Carom), 167.5 and 171.1 (675.31)
(C=O)
2.95 (t, 3JH,H = 5.3, 8 H, ring NCH2), 3.56 (s, 4 H,
54.0 (ring NCH2), 56.1 (CH2), 63.2 (OCH2), 67.9
M + H+ found
(calcd): 647.37
(647.35)
CH2), 3.58–3.61 (m, 8 H, ring OCH2), 3.59 (s, 8 H, (CH2), 70.0 and 70.6 (ring OCH2), 73.1 (OCH2),
ring OCH2), 3.65–3.68 (t, 3JH,H = 7.4, 4 H, CH2),
127.6, 127.7 and 128.4 (CHarom), 137.8 (Carom),
171.6 (C=O)
4.28 (t, 3JH,H = 7.4, 4 H, CH2), 4.55 (s, 4 H, CH2),
7.28–7.37 (m, 10 Harom
)
4g
2.96 (t, 3JH,H = 5.3, 8 H, ring NCH2), 3.57–3.61 (m, 54.0 (ring NCH2), 56.1 (CH2), 65.9 (OCH2), 69.9
M + H+ found
(calcd): 559.09
(559.30)
16 H, ring OCH2, CH2), 5.13 (s, 4 H, CH2), 7.28–
7.42 (m, 10 Harom
and 70.5 (ring OCH2), 128.2, 128.25 and 135.8
(CHarom), 135.8 (Carom), 171.5 (C=O)
)
4hb
(D2O): 3.59 (m, 8 H, ring NCH2), 3.74 (s, 8 H, ring (D2O): 54.5 (ring NCH2), 55.5 (NCH2), 59.5
OCH2), 3.86–3.89 (m, 4 H, CH2OH), 3.89–3.91 (m, (CH2OH), 64.3 (OCH2), 68.3 and 69.9 (ring OCH2), (calcd): 467.53
M + H+ found
8 H, ring NCH2), 4.28 (s, 4 H, NCH2), 4.37–4.39 (m, 167.2 (C=O)
4 H, OCH2)
(467.26)
8
9
1.3 (t, 3JH,H = 7.4, 3 H, CH3), 3.96 (s, 2 H, BrCH2), 13.9 (CH3), 24.9 (BrCH2), 61.7 and 61.6 (OCH2),
4.24 (q, 3JH,H = 7.4, 2 H, OCH2), 4.69 (s, 2 H, OCH2) 166.6 and 166.9 (C=O)
–
–
0.94 (t, 3JH,H = 7.4, 3 H, CH3), 1.33–1.43 (m, 2 H,
CH2), 1.60–1.68 (m, 2 H, CH2), 3.95 (s, 2 H,
BrCH2), 4.19 (t, 3JH,H = 6.8, 2 H, OCH2), 4.69 (s, 2
H, OCH2)
13.5 (CH3), 18.8 (CH2), 24.8 (BrCH2), 30.3 (CH2),
61.7 and 65.4 (OCH2), 166.3 and 167.0 (C=O)
10
11
3.93 (s, 2 H, BrCH2), 4.73 (s, 2 H, OCH2), 5.21 (s, 2 24.8 (BrCH2), 61.7 and 67.2 (OCH2), 128.33, 128.35
–
–
H, OCH2), 7.32–7.40 (m, 5 Harom
)
and 128.5 (CHarom), 134.7 (Carom), 166.6 and 166.8
(C=O)
2.31–2.43 (m, 2 H, CH2), 2.72–2.81 (m, 1 H, CH2), 24.9 (BrCH2), 28.3 (CH2), 65.0 (OCH2), 68.9
3.97 (s, 2 H, BrCH2), 4.32–4.39 (m, 1 H, OCH2),
4.42–4.56 (m, 1 H, OCH2), 5.48–5.53 (m, 1 H,
OCH)
(OCH), 166.2 and 171.9 (C=O)
12
13
3.95 (s, 2 H, BrCH2), 4.55 (q, 3JH,H = 8.4, 2 H,
OCH2)
24.4 (BrCH2), 60.8, 61.2, 61.6 and 62.1 (OCH2),
117.9, 120.9, 124.0 and 127.1 (CF3), 165.8 (C=O)
–
–
3.67–3.70 (m, 2 H, CH2), 3.85 (s, 2 H, BrCH2),
4.32–4.35 (m, 2 H, OCH2), 4.56 (s, 2 H, OCH2),
25.6 (BrCH2), 65.2 (CH2), 67.4 and 73.05 (OCH2),
127.6, 127.7, 128.3 (CHarom), 137.6 (Carom), 167.1
(C=O)
7.26–7.38 (m, 5 Harom
)
a Satisfactory microanalyses obtained for new compounds: C 0.45, H 0.38, N 0.30.
b Solvent: D2O.
Although the tetraalkylammonium process was satisfacto- recommended to synthesize disposable ester derivatives
ry considering its yields and simplicity for the piperazine of polyazamacrocyclic polycarboxylates like 1,4,7-tricar-
derivatives, complete removal of the co-product tetraalkyl- boxymethyl-1,4,7-triazacyclononane and 1,4,7,10-tetra-
ammonium halide was in several cases impossible when carboxymethyl-1,4,7,10-tetraazacyclododecane.
the procedure was applied to diaza-crown ethers, due to
strong molecular complex formation between tetraalkyl-
ammonium ion and the crown ether ring. In summary, it
Strong molecular complexation has been found in the
MALDI-TOF MS spectrum for all derivatives of 3 and 4
with the matrix compound 2,5-dihydroxybenzoic acid
can be stated that simple nucleophilic substitution
plus potassium ions. The phenomenon seems to be a gen-
(Scheme 1) worked best for the crown ether derivatives,
eral property of the diaza-crown ethers, as unsubstituted
macrocycle 3 and 4 showed the same effect. Although
while the tetraalkylammonium process (Scheme 2) can be
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York

3560
T. Iványi, I. Lázár
PAPER
both rings preferred the larger sized potassium ions, the hydrolysis of the ‘outer’ ester group was significantly
smaller [15]aneN2O3 ring also gave a lower intensity com- higher and NMR resonances belonging to a new labile
plex peak with the smaller sodium ion plus matrix too.
complexing agent with carboxymethoxycarbonylmethyl
pendant arms have been clearly identified. Double esters
hydrolyzed enzymatically with a half-life of 3–4 hours,
which was considered the most advantageous for mild ad-
ministration. Other esters hydrolyzed either too reluctant-
ly (hydroxyethyl ester 1h, t1/2 >16 h) or more rapidly
(cyanomethyl ester, 1i, t approx. 45 min).
Simple 1H NMR spectra as well as chemical and structural
similarities of the piperezine derivatives made them the
model of choice for studying the chemical and enzyme
hydrolysis of the pendant arms by 1H NMR spectroscopy
assuming that the ring size has a negligible effect on the
rate and mechanism. Very acidic (pH <1), moderately
acidic (pH 3, simulated gastric juice), physiological (pH In order to check the in vitro hydrolysis and uptake prop-
7.4) and very basic (pH >12) conditions were used and 1H erties, 4b was tested on homogenized pig liver tissues and
NMR spectra of the hydrolysis mixtures recorded as a on incubated fresh thin chicken liver slices as the latter
function of time. Hydrolysis half lives were calculated or, technique is widely used to test drug metabolism in the
in case of rapid reactions, estimated from the correspond- liver.19 Experiments with pig liver homogenate showed
ing integral ratios and are listed in Table 3. All esters (1a, hydrolysis rate of 4b similar to the one measured with iso-
1b, 1e, 1i, 1h) hydrolyzed in very basic solutions almost lated porcine liver esterase enzyme for 1b. However,
instantaneously and proved to be fairly stable against hy- these results did not answer the question whether macro-
drolysis at pH 3. Esters 1e and 1h were practically stable cycle 4b had or had not been able to enter intact liver cells.
as concluded from the negligible amount of the liberated
alcohol in the samples after five days. Cyanomethyl ester
In the thin liver slice experiments ODDA and 4b were
tested simultaneously. Calcium complexes of both ODDA
1i showed slow hydrolysis in a few days, but that can be
and 4b were synthesized and used as NMR standards un-
considered insignificant from a practical point of view.
der identical conditions. ODDA or macrocycle 4b was
dissolved in Hank’s Balanced Salt Solution (HBSS) add-
ed to liver slices in a submerged shaken vessel, incubated
Table 3 Approximate Hydrolysis Half Lives (t) of Selected Piper-
azine Derivatives with Disposable Pendant Arms in the Absence and
Presence of Porcine Liver Esterase Enzymea
for 30 minutes at 36 ºC under a saturated carbogen (5%
CO2, 95% O2) atmosphere, after which the slices were
carefully flushed several times with fresh HBSS, freezed,
homogenized in phosphate buffer, centrifuged and the su-
Media
1a
1b
1e
1h
16 h
1i
with es-
teraseb
3–4 h
3–4 h
n.d.
45 min
1
pernatant was examined with H NMR. Longer than 30
e.i.
minutes incubation and monitoring times were not al-
lowed to avoid necrosis of the liver tissues. Recorded
spectra clearly showed that both ODDA and 4b were able
to enter liver cells, but the influx was significantly higher
for 4b. Figure 2 indicates that the entire amount of ODDA
was in the calcium complex form inside the cells, no
peaks of the free ODDA could be observed. In contrast to
ODDA, double ester 4b (Figure 3) showed no intracellu-
lar complexation with the calcium ions. Instead of that,
peaks of non-hydrolyzed 4b and the first hydrolysis prod-
uct n-butanol could be clearly identified. In animal studies
co-administration of calcium and zinc ions may be re-
quired for the 4b treatment in order to maintain the intra-
cellular ionic balance.
without
>1 week >1 week >1 week very slow 2–3 h
esteraseb
pH 3c
st.
st.
st.
st.
st.
pH <1c
pH >12c
n.d.
£10 s
20 min
£10 s
st.
st.
slow
£10 s
<1 min
£10 s
a st. = stable, n.d. = not determined, e.i. enzyme inhibition.
b pH 7.4, 36 °C.
c At 25 °C.
Double esters 1a and 1b hydrolyzed fairly rapidly under
very acidic conditions and some differences in the rate of
hydrolysis of the inner and outer ester groups were ob-
served. When hydrolysis was completed only the free
complexing agent PIDA, glycolic acid and the corre-
sponding ethanol or butanol were observed in the hydro-
lysis mixture. Under physiological conditions (phosphate
buffer, pH 7.4, 36 °C, without esterase enzyme) all esters
except for the cyanomethyl ester 1i were virtually stable,
their hydrolysis half-lifes were greater than approximate-
ly one week. Compound 1i hydrolyzed much more rapidly
with a half-life of 2–3 hours. Simultaneous identification
of both 1i and the partially hydrolyzed monoester was
possible.
In summary hydrolysis half lives and in vitro liver tissue
permeation and hydrolysis studies clearly showed that
double ethyl glycolic ester 4a or butyl glycolic ester 4b
derivatives of macrocyclic complexing agent ODDA can
be considered as a potential candidate for the removal of
lead or other toxic metal ions from living organisms.
Solvents of reagent grade were purchased from Spectrum-3D Kft.
(Debrecen, Hungary) and used without further purification except
for MeCN, which was dried with and distilled from CaCl2, and
CH2Cl2, which was dried with and distilled from P2O5 and stabilized
with amylene. i-Pr2NEt was obtained from Aldrich and distilled
from over solid KOH pellets and then from dry zinc powder. Re-
agents were purchased from Aldrich, Fluka and Merck and used
without further purification. Silica gel 60 (15–40 mm) for column
In the presence of porcine liver esterase enzyme under
physiological conditions all esters hydrolyzed at signifi-
cantly higher rate. For double esters 1a and 1b the rate of
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York

PAPER
Synthesis of Crown Ethers with Disposable Ester Pendant Arms
3561
MestRe-C 2.3 and 3.99. NMR samples were dissolved in CDCl3 and
1
D2O, respectively. H NMR chemical shift references were tetra-
methylsilane and DSS as 0 ppm for CDCl3 and D2O, respectively.
For the 13C NMR spectra, center of the CDCl3 triplet as 77.0 ppm
and methyl peak of CH3CN as 0.3 ppm for D2O was set.
TLC examinations were performed on Silica 60 F254 plates (Merck)
using various MeOH–CHCl3 compositions as eluents to which a
few drops of HNO3 was added in some cases to improve the sepa-
ration and the shape of the spots. For visualization of the compo-
nents 254 nm UV light and the Dragendorff’s reagent was used,
consecutively. Analysis indicated by the symbols of the elements or
functions were within 0.4% of the theoretical values.
Dry Column Vacuum Chromatography
The procedure described in the paper of Pedersen et al.18 was used
with the following modifications. A 20 cm tall glass filter funnel
with 7 mm inside diameter was filled to 5.5–6 cm height with dry
silica gel 60 (0.015–0.040 mm, Merck Cat. No. 115111) and the
surface of the gel was pressed firmly with a flat-ended glass rod.
Hexane (5 mL) was poured onto the surface and sucked through by
vacuum to make a compact column packing. In a typical separation
300–700 mg sample could be purified with this column. The sample
was evaporated onto the surface of a small amount of silica gel to
obtain a dry powder, which was then applied onto the top of the col-
umn and pressed firmly to form a thin layer. In general CHCl3–
MeOH mixtures with increasing MeOH content were poured onto
the column in 10 mL portions. Each portion was sucked through the
column by vacuum completely to get a dry column again and each
eluted fraction was collected separately. As a practical guide, 0%,
1%, 2%, 3%, 5%, 10%, etc. of increasing MeOH contents were used
in the consecutive steps and each fraction was checked by TLC.
Fractions containing the pure product were combined and evaporat-
ed under reduced pressure. The spectral data of compounds 1–4 and
8–13 prepared are collected in Table 2 (see above).
Figure 2 Thin liver slice experiments with ODDA at pH 7.4. a)
blank liver, b) liver loaded with ODDA - asterisks indicate presence
of ODDA-calcium complex, c) free ODDA, d) ODDA calcium com-
plex
Rate and Mechanism of the Hydrolysis of Active Esters
Determination of the rate and mechanism of the hydrolysis of active
1
esters were carried out by using a 360 MHz H NMR instrument.
Kinetic hydrolysis experiments were performed with the corre-
sponding piperazine esters in D2O solutions in the NMR tube. The
active ester (50 mg) was dissolved in D2O (700 mL) and the pH was
set to the required value by adding DCl or NaOD solutions. Triton
X-100 was used to solubilize esters that showed limited solubility
in H2Owater. For the enzymatic hydrolysis 1.1 mg of porcine liver
esterase enzyme was added and the pH 7.8 was kept constant with
a phosphate buffer. Solutions were placed into a thermostated water
Figure 3 Thin liver slice experiments with ODDA double ester 4b at
pH 7.4. a) blank liver, b) liver loaded with 4b – single asterisks indi-
cate presence of 4b while double asterisk shows presence the hydro-
lysis product n-butanol, c) n-butanol, d) free 4b, e) calcium complex
1
bath and at appropriate intervals H NMR spectra were recorded.
Hydrolyses under acidic and basic conditions were performed at 20
°C, the acidity of the solutions was set to pH <1 and pH 3 by 15%
and 3% DCl solutions, respectively, while the pH >12 value was ad-
justed by adding 5 M NaOD solution.
chromatographic separations were purchased from Merck. Porcine
liver esterase enzyme was purchased from Sigma.
Both glucose-containing and glucose-free modified Hank’s bal-
anced salt solutions (HBSS) without phenol red were prepared from
analytical grade chemicals and twice deionized H2O and filtered
through a 0.45 m membrane filter disc (chemical composition of
HBSS was as follows (mg/L): NaCl 8000, KCl 400, Na2HPO4·2H2O
44.9, KH2PO4 60, CaCl2 144, MgSO4·7H2O 246, NaHCO3 350, glu-
cose 1000). pH of the HBSS was measured with a Radiometer pH-
meter with combined glass electrode and set to 7.4 after equilibra-
tion with carbogen (5% CO2/95% O2). Food grade pig liver was pur-
chased in a supermarket. Freshly dissected livers of food grade
chickens (1–1.5 kg weight) were purchased from a local producer
(Debrecen-Józsa, Hungary). The livers were carefully rinsed with
glucose-free Hank’s balanced salt solution equilibrated with carbo-
gen at 0–5 °C immediately after their removal from the body and
then stored on ice in glucose-containing HBSS at 0 °C under an
equilibrated carbogen atmosphere for less than 1 h. Liver slices
Mass spectrometry analyses were carried out by Bruker BIFLEX II-
ITM MALDI-TOF mass spectrometer equipped with a TOF analyz-
er. In all cases 19 kV acceleration voltage was used with pulsed ion
extraction (PIETM). The positive ions were detected in the reflec-
tron mode. A nitrogen laser (337 nm, 3 ns pulse width, 106–107 W/
cm2) operating at 4 Hz was used to produce laser desorption and
100–150 shots were summed. Samples were prepared with dihy-
droxybenzoic acid (DHB) matrix dissolved in THF (20 mg/mL).
Bulk solutions of the samples in THF were made in a concentration
of 5 mg/mL. The solutions were mixed in 10:1 v/v ratio (ma-
trix:sample). A volume of 0.5–1 mL of these solutions was deposited
onto the stainless steel sample plate and allowed to air-dry.
1H and 13C NMR spectra were recorded on Bruker AM 360 spec-
trometer, and processed on a PC with the computer program
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York

3562
T. Iványi, I. Lázár
PAPER
(0.5–1 mm thick) were cut manually from the same lobe of the liv-
ers. In simultaneous experiments 2–3 g of liver slices were incubat-
ed at 37 °C in 10 mL of glucose-containing HBSS under carbogen
atmosphere in a shaken vessel for 15 min then 20 mL of HBSS con-
taining either 50 mg of ODDA or 50 mg of 4b was added and shak-
ing was continued for 30 min. After the loading process liver slices
were washed twice with sugar-free HBSS, freezed and powderized
at –80 °C, homogenized in 66 mM pH 7.4 phosphate buffer at r.t.,
sonicated, centrifuged at 2500 g for 15 min, then 400 microliters of
the supernatant was mixed with 300 microliters of D2O and that so-
lution was used for further NMR studies.
Compound 1f
Prepared as described above for 1a, starting form piperazine (1; 104
mg, 1.21 mmol) and 13 (656 mg, 2.40 mmol). The product was fur-
ther purified by dry column vacuum chromatography resulting in
the pure product 1f (299 mg, 0.635 mmol) as a light yellow oil in
53% yield.
Diamine derivatives 2f, 3f and 4f were obtained by this method in
42, 46 and 4% yields, respectively.
Compound 1g
Prepared as described above for 1a, starting from piperazine (1; 183
mg, 2.12 mmol) and benzyl bromide (976 mg, 4.26 mmol); oily sol-
id (460 mg, 57%).
Diamine Derivative 1a; Typical Procedure
Piperazine (1; 80.6 mg, 0.935 mmol) was dissolved in anhyd MeCN
(20 mL) in the presence of an excess of i-Pr2NEt and 8 (425 mg,
1.89 mmol) dissolved in anhyd MeCN (15 mL) was added drop-
wise. After the addition was complete, the mixture was refluxed for
4 h. MeCN and excess of i-Pr2NEt were evaporated under reduced
pressure resulting in an oily solid, which was triturated with Et2O
(20 mL). The i-Pr2NEt·HBr settled was removed by filtration. The
filtrate was concentrated to 5 mL and filtered through a compressed
cotton wool plug. The filtrate was evaporated to give a white solid
which proved to be pure 1a (211 mg, 60%).
Diamine derivatives 2g, 3g and 4g obtained by this method in 71,
72 and 68% yields, respectively.
Compound 1h
Compound 1f (28 mg, 0.060 mmol) was dissolved in MeOH (30
mL) in a two-necked flask under dry N2 and 10% Pd/C (3 mg) was
added. The flask was equipped with a three-way gas inlet stopcock
and a mercury manometer/overpressure protection system and dry
and O2-free H2 was introduced until the overpressure reached ap-
proximately 1 bar. Continuous pressure drop indicated proceeding
of the reaction. H2 was introduced periodically to maintain the re-
quired overpressure. Finally, when constant pressure reading was
achieved, the pressure was dropped and the flask was purged with
N2. The catalyst was removed by filtration under N2 and the filtrate
was evaporated under reduced pressure to yield 1h as a brownish oil
(6 mg, 35%).
Diamine derivatives 2a (76%), 3a (70%) and 4a (89%) obtained by
this method were yellowish oils.
Diamine Derivative 1b–4b
Prepared as described above for compound 1a, starting from piper-
azine (1; 126 mg, 1.46 mmol) and 9 (742 mg, 2.93 mmol) yielding
a light yellowish oil (508 mg, 81%).
Caution! When the catalyst saturated with H2 gets in contact with
the air a fire or explosion may occur spontaneously, therefore all
operations with Pd/C should be performed under an inert atmo-
sphere.
Diamine derivatives 2b, 3b and 4b obtained by this method were
obtained in 79, 83 and 83% yields, respectively.
Diamine Derivative 1c
Procedure A: Compound 1c was prepared as described above for
1a, starting form piperazine (1; 53.8 mg, 0.624 mmol) and 10 (359
mg, 1.25 mmol) yielding 1c as a light yellow solid (211 mg, 68%).
Compound 1i
Large excess of chloroacetonitrile (233 mg, 3.09 mmol) was added
in one portion to a reaction mixture of 1l and Me4NBr (1l content:
249 mg, 0.715 mmol, total weight of the mixture: 433 mg) dissolved
in anhyd MeCN (20 mL). After the addition of NaI (10 mg, 0.06
mmol), the solution was stirred and refluxed for 5 h. Tetramethyl-
ammonium halides were removed by filtration and the filtrate was
evaporated under reduced pressure to a brownish oily solid. Et2O
was added to precipitate the product as a solid, which was collected
by filtration, washed with Et2O and dried yielding 1i (189 mg) as a
white solid in 94% yield. As estimated from the integral ratios of the
1H NMR spectrum, this product still contained approximately 4% of
tetramethylammonium halide.
Diamine derivatives 2c, 3c and 4c obtained by this method were ob-
tained 69, 81 and 83% yields, respectively.
Procedure B: Bromoacetic acid benzyl ester (287 mg, 1.25 mmol)
was added to the reaction mixture containing 1m (288 mg, 0.62
mmol) and Et4NBr (263 mg, 1.25 mmol) dissolved in anhyd MeCN
(20 mL) and refluxed for 8 h. Evaporation under reduced pressure
gave a oily solid, which was triturated with Et2O. The solid was col-
lected by filtration, washed with Et2O and dried yielding a mixture
of 1c (216 mg, 26 mass% in the mixture, 69% yield) and Et4NBr (74
mass% in the mixture), as estimated from the 1H NMR spectrum.
Compound 1j
Compound 1d
Procedure A: The title compound 1j was prepared by the process
described for 1l (see below) with the following modifications: Pip-
erazine (1; 1.15 g, 13.4 mmol) and bromoacetic acid (3.74 g, 26.9
mmol) were reacted and a total volume of 10.26 mL of aq NaOH so-
lution (5.224 mol/dm3, 53.6 mmol) was added. After the reaction
was complete, the acidity of the reaction mixture was set to pH 2
with HCl and then the mixture was concentrated under reduced
pressure until crystal formation started. After 10 h at r.t., the crystals
were collected by filtration, washed with a few drops of H2O and
dried under reduced pressure to constant weight yielding 1j as a
white microcrystalline solid (2.35g) in 64% yield.
To the reaction mixture of 1m and Et4NBr (503 mg, containing
0.629 mmol of 1m) partially dissolved in anhyd MeCN (20 mL)
was added 2-bromo-g-butyrolactone (210 mg, 1.26 mmol) in one
portion and the solution was refluxed for 3 h. Et4NBr was filtered
off and the filtrate was evaporated under reduced pressure to a dark
brown oily solid. Et2O was added and the precipitated solid was col-
lected by filtration, washed with Et2O and dried yielding 1d (90 mg,
0.24 mmol) as a dark brown solid in 39% yield.
Compound 1e
Prepared as described above for 1a, starting from piperazine (1; 185
mg, 2.15 mmol) and 12 (963 mg, 4.35 mmol); oily solid (354 mg,
45%).
Procedure B: PIDA methyl ester 1k (9.99 g, 43.4 mmol) was dis-
solved in HCl (150 mL, 6 mol/dm3) and the solution was brought to
boiling. MeOH and HCl were distilled off as long as MeOH or the
methyl ester could have been found in the hydrolysis mixture by
NMR. After hydrolysis was complete, the solution was evaporated
to dryness yielding 1j (11.65 g) as a white powder in 97% yield.
Homopiperazine derivative 2e was obtained from 2a similarly in
40% yield.
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York

PAPER
Synthesis of Crown Ethers with Disposable Ester Pendant Arms
3563
Compound 1k
lution. After cooling to r.t., H2O (30 mL) was added and the two-
phased system was extracted with several portions of Et2O. The or-
ganic phases were combined, dried (Na2SO4), filtered and concen-
trated under reduced pressure to a dark brown liquid, which was
then purified by vacuum distillation at 4 mm Hg. Fraction with a
boiling range of 101–107 °C contained pure 7 (5.19 g) as a colorless
liquid in 53% yield.
Prepared and worked up by the method used for 1a starting from
piperazine (1; 1.07 g, 12.4 mmol) and chloroacetic acid methyl ester
(2.70 g, 24.8 mmol). After evaporation of the ethereal filtrate 1k,
was obtained as a white solid (2.51 g) in 88% yield.
Compound 1l; Typical Procedures
Procedure A: Piperazine (1; 334 mg, 3.8 mmol) and bromoacetic
acid (1.14 g, 8.4 mmol) were dissolved in H2O (5 mL) and Me4NOH
(5.90 mL of 2.66 mol/dm3 solution, 15.7 mmol) was added. The so-
lution was heated to 80 °C and kept at this temperature. Then the pH
was set to 9–10 and kept in this range by adding Me4NOH solution
as the reaction proceeded. When constant pH reading was reached,
the mixture was evaporated under reduced pressure on a rotary
evaporator. The residue was suspended in absolute EtOH and evap-
orated again to remove traces of H2O. The obtained brownish solid
was triturated with Et2O, collected by filtration and dried under N2
using Schlenk-type inert atmosphere technique to yield a white
powder which proved to be a mixture of 1l and Me4NBr (total
weight: 2.25 g, 1l content: 1.14 g). The yield calculated for 1l was
approx. 86% as estimated from the 1H NMR spectrum. This mixture
was used for the following reactions without separation of the com-
ponents.
Bromoacetic Acid Ethoxycarbonylmethyl Ester (8)
Ethyl glycolate (5; 4.33 g, 41.5 mmol) was dissolved in anhyd
CH2Cl2 (30 mL) and bromoacetyl bromide (8.39 g, 41.5 mmol) was
added dropwise. The mixture was refluxed for 4 h, then CH2Cl2 was
removed by a rotary evaporator. The residual brownish liquid was
purified by distillation under reduced pressure (122–128 °C/10 mm
Hg) resulting in 8 (3.32 g) as a colorless liquid in 36% yield.
Bromoacetic Acid Butoxycarbonylmethyl Ester (9)
Prepared as described for 8 from butyl glycolate (4.60 g, 34.8
mmol) in anhyd CH2Cl2 (30 mL) and bromoacetyl bromide (7.03 g,
34.8 mmol). The product 9 was obtained as a colorless liquid after
distillation at reduced pressure (129 °C/8 mm Hg, 4.929 g) in 56%
yield.
Bromoacetic Acid Benzyloxycarbonylmethyl Ester (10)
Prepared similar to compounds 8 and 9, starting from 6 (4.13 g, 24.8
mmol) dissolved in anhyd CH2Cl2 (30 mL) and bromoacetyl bro-
mide (5.01 g, 24.8 mmol) yielding 10 (2.165 g) as a moderately vis-
cous, yellowish liquid in 30% yield; bp 168 °C/5 mm Hg.
Procedure B: Compound 1j (978 mg, 3.55 mmol) was dissolved in
H2O and neutralized with Me4NOH solution (5.35 mL, 2.66 mol/
dm3, 14.3 mmol). Work-up was performed according to Procedure
A. The product was a mixture of 1l and Me4NCl (total weight: 1.89
g), the amount of pure 1l in the mixture was 1.01 g (2.90 mmol, 82%
yield) as estimated from the 1H NMR spectrum.
Bromoacetic Acid 2-Oxotetrahydrofuran-3-yl Ester (11)
This ester was prepared by the method used for compound 8, start-
ing from 3-hydroxy-dihydrofuran-2-one (2.46 g, 24.1 mmol) dis-
solved in anhyd CH2Cl2 (30 mL) and bromoacetyl bromide (4.87 g,
24.1 mmol). Crude 11 was distilled off from the reaction mixture at
158 °C/4 mm Hg. Further purification was performed by dry col-
umn vacuum chromatography giving pure 11 (430 mg) as a light
yellow liquid in very low (8%) yield.
Procedure C:
Compound 1k (827 mg, 3.59 mmol) and Me4NOH (1.42 g, 15.6
mmol) were dissolved in H2O and the solution was heated at 90 °C
for 1 h. The mixture was evaporated to dryness under reduced pres-
sure, co-evaporated twice with absolute EtOH to remove traces of
H2O, triturated with CH2Cl2, and collected by filtration. The solid
was washed with Et2O and finally dried under N2 yielding pure 1l
(1.02 g) as a white solid in 82% yield.
Bromoacetic Acid 2,2,2-Trifluoroethyl Ester (12)
The synthesis of 12 was similar to the one used for 5, starting from
2,2,2-trifluoroethanol (7.21 g, 72.1 mmol) in anhyd CH2Cl2 (30 mL)
and bromoacetyl bromide (7.18 g, 35.6 mmol). Pure 12 was ob-
tained after atmospheric distillation (bp 139 °C) as a colorless liquid
(3.74 g, 47%).
Compound 1m
Prepared by each of the methods used for 1l. Product 1m was ob-
tained as a very hygroscopic white solid which, depending on the
procedure, was in pure form or contaminated with Et4NBr or
Et4NCl. Isolated or calculated yields were in the 80–90% range.
Bromoacetic Acid 2-Benzyloxyethyl Ester (13)
Ethyl Glycolate (5)
Prepared by the method of Fisher.16 Glycolic acid (20.04 g, 0.2635
mol) was dissolved in absolute EtOH (70 mL) and HCl (0.5 mL,
37%) was added. The solution was refluxed for 4 h, and then frac-
tionated by distillation (157 °C/760 mm Hg). Ethyl glycolate (11.47
g, 0.110 mmol) was obtained as a colorless liquid in 42% yield.
This compound was obtained by the process used for compound 8
starting from 2-benzyloxyethanol (5.19 g, 34.1 mmol) dissolved in
anhyd CH2Cl2 (30 mL) and bromoacetyl bromide (6.89 g, 34.1
mmol). After distillation at reduced pressure (154–158 °C/5 mm
Hg) compound 13 was obtained as a colorless liquid (3.01 g) in 32%
yield.
Benzyl Glycolate (6)
Prepared from glycolic acid (10.03 g, 0.132 mol) and benzyl alcohol
(30 mL) in the presence of 37% HCl (0.40 mL). The mixture was
refluxed for 6 h and then purified by vacuum distillation (132 °C/5
mm Hg). Benzyl glycolate was obtained as a colorless liquid (5.996
g) in 27% yield.
Acknowledgment
Financial support from the Hungarian Research Foundation (OTKA
T-032100) is gratefully acknowledged. István Lázár thanks the Bo-
lyai János Research Scholarship (BO/00452/99) and the Széchenyi
István Scholarship (SzÖ 303/2003) of the Hungarian Academy of
Sciences for financial support. The authors would like to thank Dr.
Sándor Kéki and Dr. János Török (Department of Applied Chemi-
stry, University of Debrecen) for recording MALDI-TOF MS spec-
tra.
2-Benzyloxyethanol (7)
Prepared with a modification of the method published by Butler et
al.17 To a vigorously stirred ethane-1,2-diol (10.01 g, 0.161 mol) so-
lution of KOH (3.60 g, 64.2 mmol) was added benzyl bromide
(11.03 g, 64.4 mmol) dropwise in 2 h, then the temperature was in-
creased to and kept at 127 °C for 2 h resulting in a dark brown so-
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York

3564
T. Iványi, I. Lázár
PAPER
(11) (a) Kulstad, S.; Malmsten, L. A. Acta Chem. Scand. 1979,
B33, 469. (b) Gatto, V. J.; Arnold, K. A.; Viscariello, A. M.;
Miller, S. R.; Morgan, C. R.; Gokel, G. W. J. Org. Chem.
1986, 51, 5373. (c) White, B. D.; Arnold, K. A.; Gokel, G.
W. Tetrahedron Lett. 1987, 28, 1749. (d) Tsukube, H.;
Adachi, H.; Morosawa, S. J. Chem. Soc., Perkin Trans. 1
1989, 89. (e) White, B. D.; Mallen, J.; Arnold, K. A.;
Fronczek, F. R.; Gandour, R. D.; Gehrig, L. M. B.; Gokel, G.
W. J. Org. Chem. 1989, 54, 937.
(12) Chang, A. C.; Rowland, M. E. Inorg. Chem. 1983, 22, 3866.
(13) (a) Ariens, E. J. Drug Design; Academic Press: New York,
1971. (b) Rautio, J.; Nevalainen, T.; Taipale, H.;
Vepsäläinen, J.; Gynther, J.; Lainen, K.; Järvinen, T. Eur. J.
Pharm. Sci. 2000, 11, 157. (c) Kozak, A. US Patent 6 077
837, 2000; Chem. Abstr. 2000, 133, 63944.
(14) (a) Mahfouz, N. M.; Aboul-Fadl, T.; Diab, A. K. Eur. J.
Med. Chem. 1998, 33, 675. (b) Ferretti, A.; Knijn, E.; Iorio,
E.; Pulciani, S.; Giambenedetti, M.; Molinari, A.; Meschini,
S.; Stingaro, A.; Calcabrini, A.; Freitas, I.; Strom, R.;
Arancia, G.; Podo, F. Biochim. Biophys. Acta 1999, 1438,
329. (c) Changani, K. K.; Fuller, B. J.; Bryant, D. J.; Bell, J.
D.; Ala-Korpela, M.; Taylor-Robinson, S. D.; Moore, D. P.;
Davidson, B. R. J. Hepatology 1997, 26, 336. (d) Grivet, J.-
P.; Delort, A.-M.; Porais, J.-C. Biochimie 2003, 85, 823.
(15) (a) Catsch, A. Federation Proc., Suppl. 10, Part II 1961, 20,
206. (b) Miller, S. C.; Bruenger, F. W. US Patent 5403862,
1995; Chem. Abstr. 1995, 123, 106741.
References
(1) LEAD Action News Vol. 3, No. 3 Winter 1995; Adulteration
of Paprika in Hungary http://www.zip.com.au/~djrat/
lanv3n3/lanv3n3-6.html.
(2) (a) Selypes, A.; Bánfalvi, S.; Bokros, F.; Győry, E.; Takács,
S. Bull. Environm. Contamin. Toxicol. 1997, 58, 408.
(b) Schweizer, K. Hungary still counting cost of pollution
legacy, http://www.planetark.com/dailynewsstory.cfm/
newsid/12588/newsDate/1-Oct-2001/story.htm.
(3) Lakatos, Gy.; Fleit, E.; Mészáros, I. Toxicology Lett. 2003,
140-141, 333.
(4) (a) Domingo, J. L. Reprod. Toxicol. 1998, 12, 499.
(b) http://www.pdrhealth.com/content/natural_medicine/
chapters/200120.shtml.
(5) Aposhian, H. V.; Maiorino, R. M.; Gonzalez-Ramirez, D.;
Zuniga-Charles, M.; Xu, Z.; Hurlbut, K. M.; Junco-Munoz,
P.; Dart, R. C.; Aposhian, M. M. Toxicology 1995, 97, 23.
(6) Ma, D.; Lu, F.; Overstreet, T.; Milenic, D. E.; Brechbiel, M.
W. Nucl. Med. Biol. 2002, 29, 91.
(7) (a) Brücher, E.; Győri, B.; Emri, J.; Solymosi, P.; Sztanyik,
L. B.; Varga, L. J. Chem. Soc., Chem. Commun. 1993, 574.
(b) Brücher, E.; Győri, B.; Emri, J.; Jakab, S.; Kovács, Z.;
Solymosi, P.; Tóth, I. J. Chem. Soc., Dalton Trans. 1995,
3353.
(8) Varga, L.; Törőcsik, M.; Brücher, E.; Emri, J.; Győri, B.;
Sztanyik, B. L.; Juhász, I. L.; Király, Ö. Hungarian Patent
209389, Chem. Abstr. 1991, 115, 35724.
(9) Varga, L. P.; Sztanyik, L. B.; Rónai, E.; Bodó, K.; Brücher,
E.; Győri, B.; Emri, J.; Kovács, Z. Int. J. Radiat. Biol. 1994,
66, 399.
(10) Emri, J.; Győri, B.; Brücher, E.; Sztanyik, L. B.; Varga, L.;
Király, Ö.; Kanyár, B. PCT Int. Appl. WO 9110655, Chem.
Abstr. 1991, 115, 183388.
(16) Fischer, E.; Speier, A. Ber. Dtsch. Chem. Ges. 1895, 28,
3252.
(17) Butler, C. L.; Renfrew, A. G.; Clapp, M. J. Am. Chem. Soc.
1938, 60, 1472.
(18) Pedersen, D. S.; Rosenbohm, C. Synthesis 2001, 2431.
(19) Lerche-Langrand, C.; Toutain, H. J. Toxicology 2000, 153,
221.
Synthesis 2005, No. 20, 3555–3564 © Thieme Stuttgart · New York