Enhanced cytotoxicity of benzimidazole carbamate derivatives and solubilisation by encapsulation in cucurbit[n]uril

Article abstract of DOI:10.1039/c003732j

The albendazole derivatives (2-methoxyethyl) 5-propylthio-1H-benzimidazole- 2-yl carbamate (MEABZ), N1-(2-methoxyethoxycarbonyl)-2-amino-5- propylthiobenzimidazole and N1-(2-methoxyethoxycarbonyl)-2-amino-6- propylthiobenzimidazole (MEABZ isomers A and B) and (2-hydroxyethyl) 5-propylthio-1H-benzimidazole-2-yl carbamate (HEABZ) have been synthesised. The cytotoxicity of these compounds was evaluated against a human colorectal cancer cell line (HT-29) and a human prostate cancer cell line (PC-3). The results demonstrate MEABZ, a new benzimidazole, is up to ten times more cytotoxic than the parent drug albendazole, whereas the MEABZ isomers A and B and HEABZ show no activity. A comparison of the cytotoxicity of these compounds, relative to ABZ, provides structure-activity data for this important class of anticancer agents. The aqueous solubilities of MEABZ encapsulated in Q[n] have been determined by ¹H NMR spectroscopy. The aqueous solubility of MEABZ at a physiologically relevant pH increased by 1200-fold by encapsulation in Q[8], from 8 μM to 9.4 mM, while Q[6,7] encapsulation substantially increased the solubility to more than 2 mM. Encapsulation in Q[7] and Q[8] induced significant upfield shifts for the MEABZ propyl and benzimidazole resonances. The upfield shifts indicate that the propyl and benzimidazole protons are located within the Q[7] and Q[8] cavity upon encapsulation. By contrast, encapsulation in Q[6] induced large upfield shifts for the ¹H resonances from the carbamate functional group, indicating that MEABZ associates with Q[6] at its portals, with only the carbamate group binding within the cavity. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c003732j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Enhanced cytotoxicity of benzimidazole carbamate derivatives and
solubilisation by encapsulation in cucurbit[n]uril†
Yunjie Zhao,‡a Mohammad H. Pourgholami,b David L. Morris,b J. Grant Collins*a and Anthony I. Day*a
Received 4th March 2010, Accepted 14th May 2010
First published as an Advance Article on the web 4th June 2010
DOI: 10.1039/c003732j
The albendazole derivatives (2-methoxyethyl) 5-propylthio-1H-benzimidazole-2-yl carbamate
(MEABZ), N1-(2-methoxyethoxycarbonyl)-2-amino-5-propylthiobenzimidazole and
N1-(2-methoxyethoxycarbonyl)-2-amino-6-propylthiobenzimidazole (MEABZ isomers A and B) and
(2-hydroxyethyl) 5-propylthio-1H-benzimidazole-2-yl carbamate (HEABZ) have been synthesised. The
cytotoxicity of these compounds was evaluated against a human colorectal cancer cell line (HT-29) and
a human prostate cancer cell line (PC-3). The results demonstrate MEABZ, a new benzimidazole, is up
to ten times more cytotoxic than the parent drug albendazole, whereas the MEABZ isomers A and B
and HEABZ show no activity. A comparison of the cytotoxicity of these compounds, relative to ABZ,
provides structure–activity data for this important class of anticancer agents. The aqueous solubilities
of MEABZ encapsulated in Q[n] have been determined by 1H NMR spectroscopy. The aqueous
solubility of MEABZ at a physiologically relevant pH increased by 1200-fold by encapsulation in Q[8],
from 8 mM to 9.4 mM, while Q[6,7] encapsulation substantially increased the solubility to more than
2 mM. Encapsulation in Q[7] and Q[8] induced significant upfield shifts for the MEABZ propyl and
benzimidazole resonances. The upfield shifts indicate that the propyl and benzimidazole protons are
located within the Q[7] and Q[8] cavity upon encapsulation. By contrast, encapsulation in Q[6] induced
large upfield shifts for the 1H resonances from the carbamate functional group, indicating that MEABZ
associates with Q[6] at its portals, with only the carbamate group binding within the cavity.
Introduction
Albendazole (ABZ), methyl 5-propylthio-1H-benzimidazole-2-yl
carbamate (Fig. 1), is a highly efficacious well known antiparasitic
drug, with minor toxicities.1–3 More recently, ABZ has been shown
to exhibit significant activity against a range of tumours, includ-
ing hepatocellular,4 colorectal,5 ovarian cancers6 and paclitaxel
resistant leukaemic cells.7 The drug has also been shown to inhibit
vascular endothelial growth factor and thus suppress angiogenesis
and malignant ascites formation.8
While ABZ has huge potential in the treatment of cancer, its
clinical application as an anticancer agent has been limited by its
aqueous solubility. In our previous study,10 the aqueous solubility
of ABZ at pH 6.6 was found to be 3 mM, consistent with an
earlier report,9 and 17 mM at pH 3.0. We also demonstrated that
encapsulation of ABZ in cucurbit[n]uril significantly increased the
aqueous solubility of ABZ.10 Cucurbit[n]uril, Q[n], (see Fig. 1) is
aSchool of Physical, Environmental and Mathematical Sciences, University
College, University of New South Wales, Australian Defence Force Academy,
Northcott Drive, Campbell, ACT, 2600, Australia. E-mail: a.day@
adfa.edu.au, g.collins@adfa.edu.au
bUniversity of New South Wales Department of Surgery, St. George Hospital
Fig. 1 (A) albendazole (ABZ) and (B) cucurbit[n]uril.
(SESIAHS), Sydney, NSW, 2217, Australia
1
† Electronic supplementary information (ESI) available: H NMR spec-
trum of ABZ-amine; 1H NMR spectrum of HEABZ; Determination of the
pKa of free and Q[7]- and Q[8]-bound MEABZ; An example of the product
distribution of HEABZ and ABZ-amine that occurs in the synthesis of
HEABZ; Figure showing the solvolysis reaction of the MEABZ isomer B
with methanol to slowly form ABZ-amine. See DOI: 10.1039/c003732j
a family of macrocyclic molecules made from the condensation
of glycoluril and formaldehyde with hydrophobic cavities and
electronegative carbonyl rimmed portals.11 Q[n] can be isolated
in a range of cavity sizes, with portal dimensions ranging from 2.4
to 11.0 A for Q[5] to Q[10].12–15 Inclusion complexes can be formed
˚
with a large variety of drug molecules.16–18 For ABZ, encapsulation
‡ Current Address: School of Pharmacy, Wenzhou Medical College,
College Town, Wenzhou, Zhejiang 325035, P.R. China
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in Q[7] and Q[8] was found to significantly increase the aqueous
solubility of the drug, with the Q[7,8] cavity accommodating the
benzimidazole moiety of ABZ, with the more polar carbamate
‘tail’ projecting out of the portal.10 Such a binding mode would be
stabilized by hydrophobic forces and van der Waals interactions
within the cavity and dipole–ion (assuming a protonated ABZ
structure) and hydrogen bonding at the portals, thereby dramat-
ically increasing the aqueous solubility by 2000-fold from mM
to mM concentrations.10
Another approach to increasing the aqueous solubility of ABZ
is through the formation of derivatives of the parent compound.19
As ABZ binds cucurbit[n]uril with the benzimidazole moiety in
the cavity and the carbamate group projecting out of the portal,
it was proposed to modify the methoxy moiety of the carbamate
to form ABZ derivatives that were more water soluble, but still
capable of strong binding to Q[7,8]. Consequently, it was hoped
that through encapsulation in cucurbit[n]uril in combination with
a more soluble ABZ analogue, a highly active water soluble ABZ-
type anticancer formulation could be obtained. The synthesis,
Q[n] binding and cytotoxicity of four ABZ derivatives were
explored. One compound, (2-methoxyethyl) 5-propylthio-1H-
benzimidazole-2-yl carbamate (MEABZ), was found to be slightly
more water soluble than ABZ, both in the free and Q[7,8]-bound
forms, and significantly more cytotoxic than ABZ in a range of
cancer cell lines.
which are sparingly soluble in water, were added to MEABZ and
D2O in solid form. All samples were shaken using a vortex shaker,
sonicated and then left standing overnight before analysis by NMR
spectroscopy. MEABZ concentrations in solution were measured
1
by comparing the integrals of the MEABZ H resonances with
those of known concentrations of ethanol in D2O, typically
3 mM. The results were corrected for the different spin–lattice
(T1) relaxation times between the ethanol and MEABZ protons.
T1 times were determined using the standard 180◦-t-90◦ pulse
sequence. The concentration ratio of MEABZ to cucurbituril was
determined by comparing the 1H resonance integrals of MEABZ
with those of the Q[n].
Standard ethanol solution
To 3.5 mg (0.0287 mmol) of benzoic acid dissolved in 5.00 mL
D2O, 1.59 mg (0.015 mmol) of sodium carbonate was added, giving
a 5.73 mM sodium benzoate solution. Absolute ethanol (21 mL,
density = 0.789 g mL-1) was dissolved in 2.00 mL D2O. 10 mL of the
ethanol solution was added to 0.60 mL of the sodium benzoate
solution. The ethanol concentration in solution was measured
by comparing the integrals of the ethanol proton peaks with the
known concentration of sodium benzoate.
Determination of pKa for MEABZ
A typical preparation involved adding an excess of MEABZ to
1.0 mL of water at ~ pH 3.5, the suspension was sonicated for
90 min and then left standing overnight at 20 ◦C. The suspension
was then filtered through a 0.5 mm PETE syringe filter. To 50 mL
of the solution, exactly 5.0 mL of water (pH 2.0) was added, the
pH was obtained with 5.0 M HCl. The temperature of the solution
was carefully controlled at 25 ◦C during the titration. The solution
was titrated with 0.1 M KOH to pH 6.5. The pH was measured
using a calibrated pH meter and UV spectra were collected after
each pH adjustment. The UV spectrum was scanned from 200–
400 nm. The pKa of each sample was taken from the mid-point of
the inflection of the pH versus UV reading titration curve.
Experimental
Materials
ABZ was purchased from the Sigma Chemical Company, while
D2O was obtained from Cambridge Isotope Laboratories. Q[6],
Q[7], Q[8] were synthesized as previously described.20,21 All sol-
vents were used as provided and aqueous solutions were made
using Milli-Q water, coming from a Millipore four-stage water
purification unit.
NMR spectroscopy
Preparation of MEABZ encapsulated in Q[n] (MEABZ@Q[n])
for in vitro testing
NMR spectra were recorded on a Varian Unityplus-400 spectrom-
eter operating at 400 MHz for the H nuclei. One-dimensional
1
1H NMR spectra were recorded over a spectral width of 5000 Hz
using 256 transients. All spectra were referenced to DSS (0 ppm)
at 25 ◦C, using the residual HDO resonance at 4.78 ppm as an
internal reference signal.
A typical preparation involved mixing solid MEABZ (3.1 mg,
10 mmol) and solid Q[7] (14.3 mg, 10 mmol) in H2O (pH 2.0,
1.0 mL). Using a vortex shaker the mixture was homogenized,
sonicated for 2 h and then left to stand overnight at 20 ◦C. Filtra-
tion through a 0.5 mm PETE syringe filter gave clear solutions of
the MEABZ@Q[7] complex. The solid product was obtained by
lyophilization. The mole ratio of MEABZ to Q[7] was determined
by 1H NMR. The exact mole ratio for each preparation was
determined before use. Samples dissolved in D2O were compared
using peak integrals against additions of 10 mL of standardised
ethanol (185 mM) in D2O. The MEABZ samples for in vitro
cytotoxicity were prepared in saline solutions in accordance with
the determined MEABZ@Q[7] ratios of the above lyophilized
preparations.
MEABZ aqueous solubility
An excess of the drug was added to 150 mL of water at either
pH 3.5 or 6.6, the suspension was sonicated for 90 min and then
left standing overnight at 20 ◦C. The suspension was then filtered
through a 0.5 mm PETE syringe filter, lyophilized and then re-
dissolved in 50 mL of methanol. The concentration of the drug was
determined from the absorption at 295.7 nm against a standard
curve.
Cucurbit[n]uril titrations of MEABZ
Cell proliferation assay of MEABZ
Q[7] was dissolved in D2O to a specific concentration and then
mixed with known weights of the MEABZ, whereas, Q[6], Q[8]
The cell lines HT-29 (human colorectal cancer cell line) and PC-3
(human prostate cancer cell line) used in this study were originally
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obtained from the American Type Culture Collection (ATCC) and
maintained according to the supplier’s instructions.
(q Bz), 148.01 (carbonate), 154.7 (imidazole); m/z, 309 (M+, 23%),
233 (97), 204 (27), 191 (100), 160 (31), 45 (83); umax 3384 (NH),
The sulforhodamine B (SRB) assay22 was used to examine the
antiproliferative efficacy of ABZ and its derivatives. Cells plated
in 96-well Corning tissue culture dishes at densities of 2,000–
3,000 cell/well were left for 24 h at 37 ◦C under a humidified
atmosphere containing 5% CO2. Following attachment, cells were
treated with cell culture medium (RPMI-1640 plus 5% fetal calf
serum) containing various concentrations (0.01–5 mM) of ABZ or
its derivatives. At the end of the treatment period (72 h), cells were
fixed in 10% (w/v) trichloroacetic acid for 30 min at 4 ◦C followed
by tap water washing (5¥) and staining with 100 ml of 0.4% (w/v)
SRB dissolved in 1% acetic acid. Unbound dye was removed by
five washes with 1% acetic acid before air drying. Bound SRB
was solubilized with 100 ml of 10 mM Tris base (pH 10.5) and the
absorbance read at 570 nm. Each experiment was repeated twice.
Results were normalized to vehicle treated cells (100% growth).
2960-2445, 1707 (C O), 1635, 1589, 1458, 1261 cm .
-1
=
HEABZ (2-hydroxyethyl) 5-propylthio-1H-benzimidazole-2-yl
carbamate. Ethanediol was dried by azeotropic distillation. THF
was added to the diol and continuously distilled through a column
of activated molecular sieves overnight. This process was repeated
twice with freshly activated molecular sieves. Finally the THF was
removed by distillation.
0.45 g (1.7 mmol) of albendazole dried in an oven at 120 ◦C
overnight, 5 mL of dried ethanediol and 0.02 g (0.12 mmol) of
dried toluene-4-sulfonic acid were added into a 25 mL round flask,
stirred at 135 ◦C in an oil bath for 9 h. The mixture developed a
brown colour. The ethanediol was removed in vacuo at 95 ◦C, 3 mm
Hg. The crude material contained 50% HEABZ. The residue was
extracted with 30 mL of hot water five times. The hot solution
was combined and cooled to RT. The white precipitate that
developed was collected by filtration. The product was dissolved
in a minimum volume of methanol and was loaded onto a
column of alumina (2 cm ¥ 15 cm). The product was eluted with
methanol. M◦ethanol was removed in vacuo. Yield 30 mg, 6%, m.p
Synthesis
ABZ-amine
(5-(propylthio)-1H-benzimidazol-2-amine). To
500 mg (1.88 mmol) of albendazole, 10 mL (0.18 mol) of
1,2-ethanediol and 20 mg of toluene-4-sulfonic acid were added,
and stirred at 120 ◦C in an oil bath for 24 h. The mixture gave
a yellow solution. The 1,2-ethanediol was removed in vacuo.
10 mL of dichloromethane was added and stirred at RT for
several hours. The solid was filtered and dried in a drying pistol at
50 ◦C for one day to remove traces of ethanediol. The filtrate was
evaporated to a quarter of the original volume. White crystals
were collected by filtration and combined with the solid previously
collected. Yield 235 mg (60%). Anal. Calc. for C10H13N3S, C,
57.94%; H, 6.32%; N, 20.27%; Found: C, 57.58%; H, 6.65%; N,
20.08%, 1H NMR (CD3OD) d (ppm) 0.99 (t, J 7.4 Hz, 3H, CH3),
1.58 (Sextet, J 7.4 Hz, 2H, CH2), 2.80 (t, J 7.4 Hz, 2H, CH2),
7.05 (dd, J 8.1, 1.5 Hz, H, benz CH5), 7.09 (d, H, benz CH6),
7.24 (d, J 1.5 Hz, H, CH9); 13C NMR (CD3OD) d (ppm) 13.5
(CH3CH2CH2S), 22.5 (CH3CH2CH2S), 36.9 (CH3CH2CH2S),
112.3 (Ar), 113.8 (Ar), 124.5 (Ar), 127.9 (q Ar), 133.0 (q Ar),
134.8 (q, Ar), 153.3 (imidazole); and m/z 208 (M+1); umax 3390
(NH), 3143 and 3072 (NH), 2963, 1668, 1558, 1442, 1269 cm-1.
1
107.1~108.1 C; H NMR (CD3OD) d (ppm) 0.99 (t, J 7.4 Hz,
3H, CH3), 1.58 (sextet, J 7.4 Hz, 2H, CH2) 2.83 (t, J 7.4 Hz,
2H, CH2), 3.85 (t, J 5.1 Hz, 2H, CH2), 4.10 (t, J 5.1 Hz, 2H,
CH2), 7.14 (d, J 1.6 Hz, H, Bz) 7.14 (d, J 0.8 Hz, 1H, Bz), 7.30
(s-broad, 1H, Bz); m/z, 296 (M++1, 98%), 290 (41), 274 (100);
Found (HRMS): (M-C2H4O)+, 251.1090. Calc. for C13 H17N3O3S:
(M-C2H4O)+, 251.1092. umax 3340 (NH), 3228, 2958, 1650, 1635,
1541, 1458, 1474, 1076 and 1062 (C–O) cm-1.
MEABZ regio-isomers
carbonyl)-2-amino-5-propylthiobenzimidazole;
A
and B: N1-(2-methoxyethoxy-
N1-(2-methoxy-
ethoxycarbonyl)-2-amino-6-propylthiobenzimidazole. To 100 mg
(0.48 mmol) of 5-propylthio-1H-benzimidazol-2-amine (ABZ-
amine), 0.458 g of (3.32 mmol) K2CO3, 5 mL of acetone/methanol
and 60 mL (0.48 mmol) of 2-methoxyethyl chloroformate were
added, stirred under N2 at RT for 3 h. The solid was removed by
filtration. The filtrate was collected and the acetone/methanol
was removed in vacuo, leaving the crude product. The crude
product was dissolved in a minimum volume of acetone and
loaded onto a silica gel column (2 cm ¥ 10 cm). The products
were eluted with a 1 : 3 diethyl ether : hexane solution. MEABZ
isomer A eluted first, followed by MEABZ isomer B. MEABZ
isomer A crystallized from the eluant, yield 10 mg, 7%, m.p.
117.6~120.8 ◦C; Anal. Calc. for C14H19N3O3S, C, 54.35%; H,
6.19%; N, 13.58%; S, 10.36% Found:. C, 54.00%; H, 6.07%; N,
13.27%; S, 10.12%; 1H NMR (CD3OD) d (ppm) 0.99 (t, J 7.4 Hz
3H, CH3), 1.60 (sextet, J 7.4 Hz, 2 H, CH2), 2.86 (t, J 7.4 Hz, 2H,
CH2), 3.43 (s, 3H, OCH3), 3.79 (m, 2H, CH2 CH2OCH3), 4.61
(m, 2H, CH2 CH2OCH3), 7.05 (dd, J 8.4, 1.8 Hz, 1H, Bz), 7.21
(d, J 1.8 Hz, 1H, Bz), 7.63 (d, J 8.4 Hz, 1H, Bz); m/z 332 (M +
MEABZ (2-methoxyethyl) 5-propylthio-1H-benzimidazole-2-
yl carbamate. To 100 mg (0.48 mmol) of 5-propylthio-1H-
benzimidazol-2-amine (ABZ-amine), 0.458 g (3.32 mmol) of
K2CO3, 5 mL of DMF and 112 mL (0.90 mmol) of 2-methoxyethyl
chloroformate were added and stirred under N2 at RT for 2 days.
The solid was removed by filtration. The filtrate was collected and
DMF was removed in vacuo, leaving the crude product (75~80%).
The final product was purified by silica gel chromatography. The
product was eluted with a 2 : 1 diethyl ether : hexane solution. Yield
110 mg, 74%, m.p 149.1~150.5 ◦C; Anal. Calc. for C14 H19N3O3S
C, 54.35%; H, 6.19%; N, 13.58%; S, 10.36% Found: C, 54.34%;
1
H, 6.11%; N, 13.42%; S, 10.58%; H NMR (CD3OD) d (ppm)
Na+, 27%), 310 (M++1, 57), 266 (11), 208 (100), 59 (91); umax 3412
-1
=
1.00 (t, J 7.4 Hz 3H, CH3), 1.60 (Sextet, J 7.4 Hz 2H, CH2),
2.86 (t, J 7.3 Hz, 2H, CH2), 3.40 (s, 3H, OCH3), 3.68 (m, 2H, CH2
CH2OCH3), 4.37 (m, 2H, CH2 CH2OCH3), 7.20 (dd, J 8.3, 0.6 Hz,
H, Bz), 7.34 (dd, J 8.3, 1.7 Hz, 1H, Bz), 7.48 (dd, J 1.7, 0.6 Hz,
1H, Bz); 13C NMR (CD3OD) d (ppm) 12.1 (CH3CH2CH2S), 22.3
(CH3CH2CH2S), 37.3 (CH3CH2CH2S), 57.7 (OCH3), 64.5 (ethyl),
70.2 (ethyl), 113.5 (broad, Bz), 116.3 (broad, Bz), 125.2 (Bz), 128.7
(NH), 1734 (C O), 1321 cm .
MEABZ isomer B crystallized from the eluant, yield 8 mg, 5%,
m.p. 122.8~125.2 ◦C; Anal. Calc. for C14H19N3O3S, C, 54.35%; H,
6.19%; N, 13.58%; S, 10.36% Found: C, 54.35%; H, 5.91%; N,
13.27%; S, 10.21%.; 1H NMR (CD3OD) d (ppm) 0.99 (t, J 7.4 Hz
3H, CH3), 1.59 (sextet, J 7.4 Hz, 2 H, CH2), 2.84 (t, J 7.4 Hz,
2H, CH2), 3.47 (s, 3H, OCH3), 3.81 (m, 2H, CH2 CH2OCH3), 4.62
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Table 1 Microanalysis results for MEABZ and the MEABZ isomers A
(m, 2H, CH2 CH2OCH3), 7.13 (d, J 8.2 Hz, 1H, Bz), 7.25 (dd, J
8.2 Hz, 1.8 Hz, 1H, Bz), 7.85 (d, J 1.8 Hz, 1H, Bz); m/z 332 (M +
and B
Na+, 16%), 310 (M++1, 34), 266 (10), 208 (100), 59 (59); umax 3426
Found
-1
=
(NH), 3109-2808, 1735 (C O), 1627, 1458, 1381, 1328, 1219 cm .
Required
Elements
Calculated
C14H19N3O3S
MEABZ
(A)
(B)
Results
C
H
N
S
54.35
6.19
13.58
10.36
54.34
6.11
13.42
10.58
54.00
6.07
13.27
10.12
54.35
5.91
13.27
10.21
Synthesis of HEABZ and ABZ-amine
The amount of water in the ethanediol, the reaction temperature
and the time were some of the variables responsible for the
proportion of hydrolysis versus transesterification, leading to
different ratios of HEABZ to ABZ-amine (Scheme 1).
Compared to the parent molecule ABZ-amine, the product
MEABZ has a methoxyethyl carbamate functional group. This
main difference is seen in the 1H and 13C NMR spectra in CD3OD,
where the chemical shifts are d 3.40 and 3.68 ppm for the ethylene
protons and 4.37 ppm for methoxyl protons in the 1H NMR
and d 57.7 ppm for methoxy, 64.5 and 70.2 ppm for ethylene
and 148.01 ppm for the carbamate in the 13C NMR. In support
of the structure of MEABZ, the 13C NMR spectrum shows a
characteristic feature for the aromatic ring of ABZ – the peaks
at 118 ppm and 120 ppm are broad due to the relatively slow
exchange of the proton from the N1 to the N3 of the imidazole
ring. These exchange broadened resonances are observed in the
13C NMR spectrum of MEABZ with the peaks at 113.5 and
116 ppm, but not in the spectra of the MEABZ isomers A and
B. Significantly, the M+ ion, at m/z 309 in the mass spectrum and
a peak at 1707 cm-1 for the carbamate stretch in the IR spectrum
both support the MEABZ structure. In addition, the microanalysis
agrees with the calculated empirical formula, shown in Table 1.
A series of experiments were carried out to determine the
effect of water in the reaction. Under wet conditions, the reaction
mixture mainly contained ABZ-amine as determined by 1H NMR
in D2O. The ratio of HEABZ was determined by comparing
the integrals of the hydroxyl ethyl proton peaks at 3.62 ppm
and 4.31 ppm against the propylthio methylene proton peaks
at 0.94 ppm, 1.54 ppm and 2.80 ppm. The proportion of
HEABZ can be optimized by careful drying of the ethanediol,
the ABZ, and by using anhydrous toluene-4-sulfonic acid and a
dry nitrogen atmosphere. The highest proportion of HEABZ that
could be obtained was 50% (see Supplementary Information†).
The amount of water in the reaction system determines the
balance between the HEABZ and ABZ-amine. However, under
anhydrous conditions, the ethanediol could also be responsible
for the formation of ABZ-amine, through the formation of an
alkylcarbonate. This was not tested, but observations made with
changes in temperature also point to this conclusion.
Synthesis of MEABZ
Synthesis of MEABZ isomers A and B
The relative reactivity of the three nucleophilic nitrogen substitu-
tion positions of ABZ-amine was affected by the polarity of the
solvent. Only MEABZ was observed when DMF was the solvent
(Scheme 2).
Two MEABZ regio-isomers, A and B, were observed when acetone
was used as the solvent (Scheme 3). The H NMR (in CD3OD),
microanalysis, IR and mass spectral data of isomers A and
B indicated that they are isomeric with MEABZ. The major
1
Scheme 1 Synthesis of HEABZ and ABZ-amine.
Scheme 2 Synthesis of MEABZ.
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