Conjugates of degraded and oxidized hydroxyethyl starch and sulfonylureas: Synthesis, characterization, and in vivo antidiabetic activity

Article abstract of DOI:10.1021/bc500509a

Orally administered drugs usually face the problem of low water solubility, low permeability, and less retention in bloodstream leading to unsatisfactory pharmacokinetic pro file of drugs. Polymer conjugation has attracted increasing interest in the pharmaceutical industry for delivering such low molecular weight (M_w) drugs as well as some complex compounds. In the present work, degraded and oxidized hydroxyethyl starch (HES), a highly biocompatible semisynthetic biopolymer, was used as a drug carrier to overcome the solubility and permeability problems. The HES was coupled with synthesized N-arylsulfonylbenzimidazolones, a class of sulfonylurea derivatives, by creating an amide linkage between the two species. The coupled products were characterized using GPC, FT-IR, ¹H NMR, and ¹³C NMR spectroscopy. The experiments established the viability of covalent coupling between the biopolymer and N-arylsulfonylbenzimidazolones. The coupled products were screened for their in vivo antidiabetic potential on male albino rats. The coupling of sulfonylurea derivatives with HES resulted in a marked increase of the hypoglycemic activity of all the compounds. 2,3-Dihydro-3-(4-nitrobenzensulfonyl)-2-oxo-¹H-benzimidazole coupled to HES₁₀₁₀₀ was found most potent with a 67% reduction in blood glucose level of the rats as compared to 41% reduction produced by tolbutamide and 38% by metformin. (Chemical Equation Presented).

Full text of DOI:10.1021/bc500509a

Article
pubs.acs.org/bc
Conjugates of Degraded and Oxidized Hydroxyethyl Starch and
Sulfonylureas: Synthesis, Characterization, and in Vivo Antidiabetic
Activity
Muhammad Azhar Abbas,†,‡ Shahid Hameed,*,† Muhammad Farman,† Jorg Kressler,‡
̈
and Nasir Mahmood‡
†Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan
‡Department of Chemistry, Martin-Luther University Halle-Wittenberg, D-06114 Halle (Saale), Germany
S
* Supporting Information
ABSTRACT: Orally administered drugs usually face the problem of
low water solubility, low permeability, and less retention in blood-
stream leading to unsatisfactory pharmacokinetic profile of drugs.
Polymer conjugation has attracted increasing interest in the
pharmaceutical industry for delivering such low molecular weight
(Mw) drugs as well as some complex compounds. In the present work,
degraded and oxidized hydroxyethyl starch (HES), a highly
biocompatible semisynthetic biopolymer, was used as a drug carrier
to overcome the solubility and permeability problems. The HES was
coupled with synthesized N-arylsulfonylbenzimidazolones, a class of
sulfonylurea derivatives, by creating an amide linkage between the two
species. The coupled products were characterized using GPC, FT-IR,
1H NMR, and 13C NMR spectroscopy. The experiments established
the viability of covalent coupling between the biopolymer and N-arylsulfonylbenzimidazolones. The coupled products were
screened for their in vivo antidiabetic potential on male albino rats. The coupling of sulfonylurea derivatives with HES resulted in
a marked increase of the hypoglycemic activity of all the compounds. 2,3-Dihydro-3-(4-nitrobenzensulfonyl)-2-oxo-1H-
benzimidazole coupled to HES10100 was found most potent with a 67% reduction in blood glucose level of the rats as compared
to 41% reduction produced by tolbutamide and 38% by metformin.
The use of less soluble medicaments is also often associated
with toxic side effects because of their deposition in organs such
as liver and/or kidney.2
INTRODUCTION
■
The problem of solubility for orally administered drugs is a
major challenge for formulation scientists. Reasonable solubility
of the drugs is necessary to have good bioavailability through
complete absorbance. Different technological approaches have
been tried to solve the problem during pharmaceutical product
development. The concept of using biopolymers as carrier, and
to increase the water solubility of synthesized drugs, is definitely
a right and proper way to improve the pharmacokinetics and
bioavailability of such compounds. As a number of drugs have
low water solubility, the concept of drug−polymer conjugation,
and solubility properties of such systems, is prone to become
more important in the future. As a result of polymer
conjugation, the water solubility and stability of newly
synthesized drugs, in general, increases along with the drug
volume. The increased drug volume may help protect the drug
from enzymatic and hydrolytic degradation. The polymer may
also direct the drug polymer conjugate to specific sites in the
body.1
An approach followed in recent times for tackling these
problems consists of coupling such less soluble substances to
readily soluble biocompatible polymers. It is possible through
coupling, on one hand, to increase the molecular weight above
the renal threshold so that the plasma residence time of small
molecules may be drastically increased, and on the other hand,
the solubility in aqueous medium may be improved by the
hydrophilic polymer portion.3 Most modifications to date have
been carried out with polyethylene glycol (PEG) or dextran,
PEG being generally preferred because it yields simpler
products.2,3 Dextran conjugates often show high allergenicity,
low metabolic stability, and in many cases low yields of the
coupling reactions.4 Because of the polyfunctional nature of the
molecule, the coupling usually yields a complex and
heterogeneous mixture of conjugates creating complications
and in some cases even death of the patient.5 There have
There are a large number of low-molecular-weight substances
of commercial interest, especially active pharmaceutical
ingredients, with limited or even prevented use because of
low solubility in aqueous medium and short residence time.
Received: November 6, 2014
Revised: December 4, 2014
Published: December 5, 2014
© 2014 American Chemical Society
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Article
likewise been reports of unpleasant or hazardous side effects,
such as pruritus, hypersensitivity, and pancreatitis, on the use of
PEG conjugates.6 In addition, the biological activity of the
active ingredients is greatly reduced in some cases after PEG
coupling.7 Moreover, the metabolism of degradation products
of PEG conjugates is still substantially unknown and possibly
represents health risks.4
There is still a need for physiologically well tolerated
alternatives to dextran or PEG conjugates to improve the
solubility of poorly soluble low-molecular-weight substances.
The biopolymer hydroxyethyl starch (HES) is a good choice
for such coupling reactions.8 A substantial advantage of HES is
that it has already been approved by the authorities as a
biocompatible plasma expander and is employed clinically on
large scale.4
with HES, and antidiabetic activity of the resultant conjugates,
in line with our previous studies.22−24 The activity of the neat
molecules was also determined and is being compared with that
of the conjugates.
RESULTS AND DISCUSSION
■
The synthesis of the drug−HES conjugates was accomplished
in three steps. The first step involved the preparation of
antidiabetic drugs from benzimidazolones. Second, coupling of
the drug and the polymer was carried out through an amide
linkage. Finally, the synthesized drugs and their coupled
products were screened for their in vivo antidiabetic activity.
Synthesis of N-Arylsulfonylbenzimidazolones (14−
23). A particular problem observed in the preparation of
benzimidazolone derivatives is the selective functionalization of
a single ureido nitrogen atom. The direct monoalkylation of
one of the nitrogen atoms of benzimidazolone is not
feasible,25,26 particularly with unsymmetrical benzimidazolones.
However, a single alkoxycarbonyl moiety could be easily and
selectively introduced directly to these cyclic urea derivatives.27
An alkoxycarbonyl group, such as a tert-butoxycarbonyl group,
is an attractive and potentially useful protecting group for this
purpose, since it effectively combines reasonable chemical
stability with ready removal under either mild alkaline28 or
acidic conditions.29 The protecting group facilitates function-
alization of the other degenerate nitrogen atom of the
heterocyclic ring under mild conditions (Scheme 1).
To be used as drug carrier, the HES must have a certain size
of the molecule (or Mw) to be eliminated through the kidney.
The large molecules of HES may not be eliminated through the
kidney and accumulate causing serious problems. Therefore,
HES needs to be degraded to lower Mw fractions to meet the
removal requirements. The pharmacokinetic behavior of
medium and low Mw starches has been investigated by many
researchers. It has been observed that excellent degradability of
low Mw HES has not only no quantifiable reduction in clinical
efficacy,9,10 but also improved safety profile due to more rapid
elimination.11 The pharmacokinetic studies have confirmed that
low Mw starch is safe not only in healthy human subjects,12 but
also in patients with mild to severe renal impairment.13
In addition, HES needs to be chemically modified to
generate suitable functional groups for coupling with the drug.
For this purpose, the successful selective degradation and
oxidation of HES to generate carboxylic acid group at the chain
ends has recently been reported from this laboratory.14
Most of the marketed antidiabetic drugs are the sulfonyl
derivatives of urea. Benzimidazolones (1,3-dihydro-2H-benzi-
midazol-2-one) are a unique class of cyclic urea derivatives and
have shown remarkable activity as hypoglycemic agents.15 In
addition, a wide variety of other biochemical and pharmaco-
logical properties of these compounds have also been
demonstrated. They antagonize neurotransmitters,16 inhibit
aldose reductase,17 show antiulcer and antisecretory proper-
ties,18 enhance pulmonary surfactant secretion, and modulate
ion channels.19 Several of these compounds show activity
against leukemia.20 The conversion of these cyclic urea
derivatives to their sulfonyl derivatives may result in a molecule
having enhanced antidiabetic activity. In addition, the
benzimidazolone core may act as aldose reductase inhibitor,
and hence help to prevent cataract formationa complication
associated with diabetes. The coupling of these sulfonyl cyclic
urea derivatives with HES is supposed to increase their water
solubility, and hence, the concentration of the drug outside the
cell that may result in higher penetration into the cell. Second,
hydroxyethylation of starch reduces its in vivo degradation
rate.21 Unsubstituted anhydroglucose units are more prone to
enzymatic degradation by α-amylase. Thus, hydroxyethylation
may restrict the rate of enzymatic degradation of the HES
molecule and prolong intravascular retention time. The
polymer may also direct the drug−polymer conjugate to
specific sites in the body.1 The amide linkage created by the
coupling of sulfonyl cyclic urea derivatives with HES may
undergo in vivo hydrolysis to release the drug molecule.
In the present paper, we report the synthesis of a number of
N-arylsulfonyl derivatives of benzimidazolones, their coupling
Scheme 1. Synthesis of N-Arylsulfonylbenzimidazolones
(14−23)
2(3H)-Benzimidazolones were synthesized by the reaction of
o-phenylenediamine with urea in the presence of acetic acid as
catalyst.30 The selective protection of a single nitrogen atom of
benzimidazolones was successfully achieved in high yield under
inert conditions of argon in dry DMF using di-tert-
butyldicarbonate. In the case of 5-nitro-2(3H)-benzimidazolone
as starting material, two isomers were obtained, which were
separated by flash column chromatography using silica gel as an
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adsorbent and ethyl acetate:n-hexane (1:2) as eluent. The
major regioisomer (2) was used for further derivatization. The
synthesis of mono-protected benzimidazolones (1−3) was
indicated in the IR spectra by the appearance of two strong
carbonyl absorptions: one for amide and the other for ester
linkage. The confirmation of synthesis was witnessed in the 1H
NMR spectra by the presence of only one downfield signal for
NH proton in the range 11.50−9.75 ppm. A singlet in the range
of 1.59−1.36 ppm confirmed the presence of nine protons of
the t-butyl group. In 13C NMR spectra, the ester carbonyl
carbons resonated in the range of 152.0−148.0 ppm. The
formation of the products was further confirmed by EIMS
analysis where a low intensity molecular ion peak was observed
at m/z = 234 for compound 1 and at m/z = 279 for compound
2. The base peak in both compounds appeared due to the loss
of tert-butyloxycarbonyl group. The loss of CO2 molecule from
the molecular ion was also observed.
Scheme 2. Synthesis of Coupled Products (24−43) from
Oxidized HES17500 and HES10100
preactivation of the polymeric acid to acid chloride, and the
second using a direct approach without preliminary activation
of oxidized HES. It was observed that there is no specific
advantage in a two-step reaction in terms of yield of the
reaction. Therefore, all the coupling reactions were carried out
using the direct approach.
The sulfonation of mono-protected benzimidazolones (1 and
2) with arylsulfonyl chlorides was carried out in dry
dichloromethane using triethylamine as a base and dimethyla-
minopyridine as a catalyst. In FT-IR spectra of the synthesized
sulfonylated benzimidazolones (4−13), a band in the region
1198−1190 cm−1 for asymmetric and a second band in the
range of 1360−1345 cm−1 for symmetric stretching vibrations
of the sulfonyl group was observed. The confirmation of
For structure elucidation of coupled products (24−43),
GPC, FTIR, and NMR spectroscopic techniques were used.
The increase in relative molecular weight of conjugates and
change in their elution times, as measured by GPC, supported
the formation of coupled products. The coupling of N-
arylsulfonylbenzimidazolones with oxidized HES was also
indicated in the IR spectra of the products by the presence
of a band in the range 1670−1645 cm−1 attributed to the
stretching vibrations of the CO functionality of the product.
In nitro-group containing conjugates, the bands for NO2
stretchings were observed in the spectral regions of 1585−
1
synthesis was achieved in H NMR spectra by the appearance
of additional signals in the aromatic region and absence of
−NH proton signals observed in the starting materials.
Additional signals in the aromatic region were also observed
in 13C NMR spectra of these compounds. The structures of
sulfonylated products (4−13) were further confirmed by EIMS
analysis. The mass spectra exhibited a molecular ion peak with
low intensity for all the compounds. The base peak resulted by
the loss of −COOCMe3 fragment from the molecular ion. The
loss of SO2 molecule from the molecular ion was also observed.
The mono-protected sulfonylated benzimidazolones (4−13)
were successfully deprotected on treatment with concentrated
hydrochloric acid in diethyl ether at room temperature to give
N-arylsulfonylbenzimidazolones (14−23). The formation of N-
arylsulfonylbenzimidazolones (14−23) was witnessed by the
re-emergence of the −NH band in the region of 3365−3375
cm−1 in the IR spectra. The formation of products was
confirmed in 1H NMR spectra by the reappearance of
downfield signal for the −NH proton in the range 9.98−9.20
ppm. The absence of a singlet at around 1.6 ppm for nine
protons of the t-butyl group further confirmed the formation of
products. In 13C NMR spectra, the signal for only one carbonyl
carbon in the range of 150.1−165.5 ppm also confirmed the
synthesis. The formation of N-arylsulfonylbenzimidazolones
(14−23) was further confirmed by EIMS analysis. The
molecular ion peak was observed in all cases. The loss of the
Ar-SO2 group resulted in base peaks. The loss of SO2 from the
molecular ion also gave a prominent fragment.
1
1450 and 1420−1375 cm−1. The H NMR spectra confirmed
the formation of coupled products (24−43) by the presence of
additional signals for aromatic protons. The absence of the
downfield signals for −NH (of benzimidazolone nucleus) and
carboxylic acid (of the oxidized HES) protons also provided the
evidence for coupling. The methyl and methoxy protons of N-
arylsulfonylbenzimidazolones were not assignable in most cases
due to overlapping with HES signals. The signal for carbonyl
carbon of the amide group appeared in the range of 165.1−
175.5 ppm in 13C NMR spectra of the coupled products (24−
43).
Solubility of the Coupled Products. The solubility of
conjugated products was determined and compared with that of
the respective drugs. A comparison of the solubility of the
conjugated products and the neat drugs reveals that the
solubility of drugs increases many-fold after its coupling with
HES (Table 1). When the drugs were coupled to oxidized
HES10100, the maximum increase in solubility (566-fold)
observed was that of the drug 18 followed by 540-fold for
compound 17. The minimum increase in the solubility
observed was 61-fold for compound 21.
In Vivo Hypoglycemic Activities. The in vivo hypo-
glycemic activities of N-arylsulfonylbenzimidazolones (14−23)
and their conjugates with HES (24−33) were tested on male
albino rats of the Dawley-Sprague family according to well
established methods.22
The in vivo hypoglycemic activities of N-arylsulfonylbenzi-
midazolones (14−23) were measured at two different
concentrations, i.e., 20 and 40 mg per kg of the rats’ body
weight. It was observed that all the compounds were active, in
general, at a dose of 20 mg of the test compound. However, the
activity was more pronounced at a dose of 40 mg per kg of the
rats’ body weight. The activities of all the compounds at a dose
Coupling of N-Arylsulfonylbenzimidazolones with
HES. The carboxylic acid group in oxidized HES and the free
N−H in the arylsulfonylbenzimidazolones were made to react
for coupling of the drug with HES. The oxidized HES of Mw
17 500 and 10 100 g/mol were used for coupling purposes to
afford the products 24−33 and 34−43, respectively. The
reaction was performed in the presence of dicyclohexyl
carbodiimide (DCC) as an activator in a mixture of DMF
and water (Scheme 2). Two strategies were tried for the
coupling reactions: the first using a two-step reaction with a
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Table 1. Solubility of Drugs (14−23) and their coupled products with HES10100
a
a
drug
solubility (mol × 10−4/5 mL)
solubility of the drug bound to HES10100 (mol × 10−4/5 mL)
increase in solubility (fold)
14
15
16
17
18
19
20
21
22
23
0.034
0.041
0.021
0.062
0.070
0.021
0.027
0.014
0.025
0.030
5.65
9.53
166
232
74
1.56
33.51
39.64
2.74
540
566
131
113
61
3.04
0.861
4.71
188
258
7.73
a
Averages are means of three determinations SD. Values are significantly different (p < 0.05).
of 40 mg demonstrated a remarkable decrease (p < 0.0001) in
the blood glucose level and were comparable to both the
standard drugs, glucophage and tolbutamide, used at a dose of
150 mg per kg of the rats’ body weight (Figure 1). The most
reduction in the blood glucose levels of the rats, respectively.
The least active compounds (26 and 31) were with a bromine
substituent in the arylsulfonyl part of the compounds. Again,
the compounds substituted by a nitro group at position 5 of the
nucleus were found less potent than their unsubstituted
counterparts.
The N-arylsulfonylbenzimidazolones (14−23) were also
coupled to HES10100 to study and compare hypoglycemic
activities. On coupling to HES10100 the activities of the coupled
products (34−43) were even higher (p < 0.0001) as depicted
in Figure 2. This higher activity of the products (34−43)
Figure 1. Comparison of hypoglycemic activity of the commercial
drugs and compounds 14−23 in diabetic rats’ model. p < 0.0001.
Sample size: 5 male adult rats. Statistical Analysis: Tukey HSD test.
active compounds in the series were 17 and 18 with a reduction
in the blood glucose level of 54% and 51%, respectively. These
compounds have a nitro and methoxy substituent, respectively,
on the phenylsulfonyl part of the products. However, it is
interesting to note that when a nitro substituent is present on
the benzene ring of the benzimidazolone nucleus, it has adverse
effects on the hypoglycemic activity of the compounds as
witnessed in compounds 19−23. The substituent effects in the
two series of compounds (14−18 and 19−23) were found
parallel to one another as can be observed from Table 2.
Compounds 16 and 21 were the least active compounds in the
series with a reduction in the blood glucose level of 14% and
12%, respectively. Both these compounds have a bromine atom
as a substituent at the phenylsulfonyl part of the molecule.
The N-arylsulfonylbenzimidazolones coupled products (24−
33) with HES17500 were screened for their hypoglycemic activity
at the dose levels of 20 and 40 mg per kg of the rats’ body
weight. The activity of the compounds increased in general and
all the coupled products (24−33) were found more active than
the standard drugs (glucophage and tolbutamide) except
compounds 26 and 31 (Table 3). However, the substituent
effects were found parallel to those of the uncoupled N-
arylsulfonylbenzimidazolones (14−23), the most active com-
pounds being with a nitro (in compound 27) or a methoxy (in
compound 28) group as substituents in the phenylsulfonyl part
of the molecule. These compounds exhibited 57% and 58%
Figure 2. Comparison of hypoglycemic activity of the commercial
drugs and compounds 34−43 in diabetic rats model. p < 0.0001.
Sample size: 5 male adult rats. Statistical Analysis: Tukey HSD test.
coupled with HES10100 as compared to compounds (24−33)
coupled with HES17500 may be attributed to the increased
solubility of the product in water, hence higher availability of
the drug. The most active products were 37 and 38, causing a
reduction in the blood glucose of 67% and 65%, respectively
(Table 4). There is a significant enhancement in the activity
when compared to the values of 54% and 51% reduction
observed for the uncoupled drugs 17 and 18. Although
products 36 and 41 were least active of all these coupled
products, their activity too was higher than that of the
corresponding uncoupled products (16 and 21).
It may be observed from Tables 2−4 that the synthesized
compounds (14−23) are reasonably active when compared to
the standard commercial drugs (glucophage and tolbutamide)
and the coupled products are even more potent than the neat
compounds. This increased activity may be attributed to the
increased solubility of the drugs on coupling with HES. The
continuous increase in the activity with time indicates the
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Table 2. Hypoglycemic Activities of N-Arylsulfonylbenzimidazolones (14−23)
blood glucose (mg/dL)
a
compound
quantity (mg/kg)
3 h
6 h
12 h
% reduction (after 12 h)
NC (with vehicle)
98.34 2.3
306.3 8.6 (after 5 days)
194.71 3.24
183.45 5.62
269.54 2.5
245.02 2.3
235.84 2.9
211.08 1.10
279.48 1.35
266.48 1.54
248.09 1.12
214.43 1.89
251.12 2.67
220.51 3.15
273.15 1.2
252.22 0.9
250.28 1.1
210.48 2.5
282.29 2.2
278.12 2.9
255.28 1.2
251.10 1.6
261.95 2.5
238.15 3.2
DC
200
150
150
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
DGT
DTT
14
196.83 2.98
188.30 5.8
284.71 2.3
270.01 2.8
269.51 1.7
245.02 1.9
290.96 2.1
280.97 2.33
257.25 1.73
251.12 1.24
260.31 1.5
248.07 2.4
290.57 2.5
282.10 1.2
276.12 2.1
241.24 2.6
299.28 3.6
289.64 3.1
268.48 2.8
262.80 2.1
272.42 2.0
258.10 2.7
186.57 3.32
178.32 6.1
220.51 2.3
177.63 1.76
209.45 1.33
169.40 2.76
274.42 1.26
263.98 1.1
199.08 2.7
140.89 1.16
192.95 1.32
150.08 1.74
234.26 3.5
190.65 2.8
241.15 1.5
192.80 2.6
281.90 2.9
272.80 2.1
210.28 1.5
160.85 2.7
211.34 1.9
168.34 1.5
38
41
28
43
31
45
10
14
35
54
37
51
24
39
22
36
08
12
32
47
31
45
15
16
17
18
19
20
21
22
23
a
% change in blood glucose was calculated using formula %change = [(Tc − Tt)/Tc] × 100, where Tc = values before administration of drug, Tt =
values after administration of drug; data are expressed as mean SD; NC = normal control; DC = diabetic control; DGT = diabetic glucophage
treated; DTT = diabetic tolbutamide treated.
increased retention of the drugs in bloodstream. This may be
useful as a daily dose of the medicine. Furthermore, the
compound treated rats were found normal and no toxic
reaction or mortality was observed during this investigation.
MATERIALS AND METHODS
■
General. The reagents used were of high purity grade.
Hydroxyethyl starch with molar mass of 70 000 g/mol was a
kind gift from Serumwerk, Bernburg, Germany. The oxidizing
agent sodium hypochlorite NaOCl (13%), sodium hydroxide
(3% solution), α-amylase bacterial enzyme (source: Bacillus
licheniformis, EC number 3.2.1.1), calcium chloride solution
(2.5 mM/L Ca2+), N,N-dicyclohexylcarbodiimide (DCC), and
N,N-dimethylaminopyridine (DMAP) were purchased from
Sigma-Aldrich (Germany). Diethyl ether and dichloromethane
were supplied by Lab-scan (Germany). Tetrahydrofuran
(THF), triethylamine (TEA), benzoyl chloride, chloroform,
carbon tetrachloride, and methanol were purchased from
CONCLUSIONS
■
In conclusion, two series of N-arylsulfonylbenzimidazolones
were synthesized using a multistep reaction sequence. The
synthesized N-arylsulfonylbenzimidazolones were successfully
coupled with oxidized HES through an amide linkage. The in
vivo antidiabetic activity of the synthesized compounds and
their coupled products with HES was evaluated and compared
with that of marketed drugs, tolbutamide and glucophage. All
the synthesized compounds were found active in general, but
their coupled products with HES were found even more active,
as expected. 2,3-Dihydro-3-(4-nitrobenzensulfonyl)-2-oxo-1H-
benzimidazolone (17) was found most potent of all the
synthesized compounds with a 54% reduction in blood glucose
level of the rats as compared to 41% reduction produced by
tolbutamide and 38% by glucophage. On coupling with
HES10100, the activity increased further to 67% reduction in
blood glucose level as compared to 54% by the drug itself. The
higher activity of the drug may be attributed to its increased
solubility affecting the availability of drug. However, it was
observed that the compounds substituted with a nitro group in
the aromatic ring of benzimidazolone nucleus were in general
less potent than their unsubstituted counterparts. The
compound treated rats were found normal and no toxic
reaction or mortility was observed during this investigation.
Riedel-deHaen (Germany). Acetone, ethyl acetate, and ethanol
̈
were obtained from commercial sources and distilled before
use. All the reactions were monitored using precoated silicagel-
60 HF254 TLC plates and visualized under UV at 254 nm. Flash
column chromatography was carried out on silica gel (35−70
μm, Grace). Melting points of the compounds were determined
in open capillaries using Gallenkemp melting point apparatus
and are uncorrected. FT-IR spectra were recorded on a Hitachi
model 270−50 spectrophotometer or on a Bruker Tensor 37
spectrophotometer as KBr discs; 1H NMR spectra were
recorded on a Bruker Avance 300 or 400 MHz Gemini 2000
(Varian) NMR spectrophotometer using TMS as an internal
standard. The multiplicities are abbreviated as s = singlet, d =
doublet, dd = doublet of doublet, ad = apparent doublet, t =
triplet, at = apparent triplet, dt = doublet of triplet, td = triplet
of doublet, q = quartet, and m = multiplet. The EIMS were
recorded on a MAT-112-S machine at 70 eV. Determination
and comparison study of Mw and Mn were carried out on
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Table 3. Hypoglycemic Activities of the Coupled Products of N-Arylsulfonylbenzimidazolones with HES17500 (24−33)
blood glucose (mg/dL)
a
compound
quantity (mg/kg)
3 h
6 h
12 h
% reduction (after 12 h)
NC (with vehicle)
98.34 2.3
306.3 8.6 (after 5 days)
194.71 3.24
183.45 5.62
288.45 2.1
269.10 1.53
275.82 1.42
259.14 2.63
284.45 2.2
282.68 2.8
257.12 1.6
259.73 1.2
254.32 2.15
242.32 1.8
272.30 2.7
252.76 2.1
233.18 2.2
204.86 1.6
280.90 1.2
275.10 1.6
250.65 2.7
239.40 3.2
261.95 3.6
228.30 1.7
DC
200
150
150
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
DGT
DTT
24
196.83 2.98
188.30 5.8
299.85 2.4
287.51 2.5
290.45 2.92
278.02 2.14
295.16 2.43
290.1 3.26
272.3 2.1
186.57 3.32
178.32 6.1
217.62 1.35
171.83 1.5
199.65 1.2
158.51 3.3
270.21 1.52
260.92 1.53
192.28 1.52
132.81 1.25
183.52 2.4
129.82 1.24
228.20 0.7
184.62 1.1
232.98 2.7
178.40 1.9
279.98 2.8
261.82 3.1
201.52 1.2
155.25 1.1
204.20 1.6
157.94 2.3
38
41
29
45
35
48
11
15
37
57
41
58
26
41
25
42
10
15
35
51
33
49
25
26
27
28
29
30
31
32
33
268.4 1.6
265.2 2.8
259.21 2.4
288.90 1.4
281.78 2.2
274.96 2.8
238.82 2.1
296.38 2.3
287.75 2.2
262.50 2.5
258.28 1.8
267.90 2.1
256.45 2.5
a
% change in blood glucose was calculated using formula %change = [(Tc − Tt)/Tc] × 100, where Tc = values before administration of drug, Tt =
values after administration of drug; data are expressed as mean SD; NC = normal control; DC = diabetic control; DGT = diabetic glucophage
treated; DTT = diabetic tolbutamide treated.
Viscotek 270 dual detector GPC/SEC and Waters GPC
equipped with a Knauer pump, two PSS columns, and RI
detector (W410), using a solution of NaNO3 in water
(concentration 1−2%) as eluent. Polyethylene glycol calibra-
tion was used to calculate the molar masses. EBA 21 Hettich
Zentrifuge was used with disposable centrifuge tubes for
centrifugation.
General Method for the Synthesis of tert-Butyl 2,3-
dihydro-2-oxobenzimidazole-1-carboxylates (1−3). To a
solution of benzimidazolone (0.015 mol) in dry N,N-
dimethyformamide (5.0 mL) was added sodium hydride
(0.015 mol) under argon.27 After stirring for 40 min, di-tert-
butyldicarbonate (0.015 mol) was added dropwise from a
dropping funnel and the mixture stirred for 24 h at 20 °C. The
N,N-dimethyformamide was removed in vacuo, and the residue
was treated with saturated ammonium chloride solution and
extracted with ethyl acetate (3 × 50 mL). The solvent was
removed in vacuo and the product purified by flash column
chromatography using ethyl acetate:n-hexane (1:4) as eluent. In
case of 5-nitro-2(3H)-benzimidazolone two isomers were
obtained, which were separated by flash column chromatog-
raphy using ethyl acetate:n-hexane (1:2) as eluent.
General Method for the Synthesis of 2,3-Dihydro-2-
oxo-3-benzenesulfonyl-1H-benzimidazole-1-carboxylic
acid, 1,1-Dimethylethyl esters (4−13). To the mono-
protected benzimidazolone (0.01 mol) dissolved in dry
dichloromethane (50.0 mL) was added triethylamine (0.01
mol) as a base and dimethylaminopyridine (1−2 crystals) as a
catalyst. To this well-stirred solution, arylsulfonyl chloride (0.11
mol) was added portionwise and stirring continued at 20 °C for
3 h. The mixture was diluted with 1 N HCl (10 mL) and
extracted with dichloromethane (3 × 30 mL). The solvent was
removed in vacuo and the crude product recrystallized from
chloroform:n-hexane (1:3).
General Method for Synthesis of N-Arylsulfonylben-
zimidazolones (14−23). In a 100 mL round-bottom flask, 1.3
mmol of monoprotected sulfonylated benzimidazolone was
dissolved in acetonitrile (5.0 mL) and conc. hydrochloric acid
(2.5 mL), separately dissolved in diethyl ether (6.0 mL) was
added dropwise. The mixture was stirred at 20 °C for 3 h,
concentrated on a rotary evaporator, and triturated with diethyl
ether to give analytically pure monosulfonylated benzimidazo-
lones.
General Method for Coupling of Arylsulfonylbenzi-
midazolones with Oxidized HES (24−43). Oxidized HES14
(HES17500 or HES10100; 0.00015 mol) was taken in 3.0 mL of
DMF/distilled water (equal volumes) under magnetic stirring
(5 rpm) at room temperature. After 30 min, DCC (0.002 mol)
and DMAP (0.001 mol), dissolved separately in 5.0 mL water,
were added slowly to the reaction mixture from a dropping
funnel. After another 30 min, arylsulfonylbenzimidazolone
(0.002 mol) dissolved in DMF/distilled water (3.0 mL), was
added slowly under constant stirring. The reaction mixture was
left for 24 h under moderate stirring. Thereafter, the solvent
was evaporated under vacuum, the product taken in water,
washed with ethyl acetate (2 × 5.0 mL), and precipitated using
acetone. The product was filtered, washed with methanol, and
air-dried. The solid product was dissolved in 50.0 mL of water
and further purification was achieved by dialysis against distilled
water for 48 h in a dialysis membrane. The solution was finally
lyophilized, and the product was analyzed by GPC.
125
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Bioconjugate Chemistry
Article
Table 4. Hypoglycemic Activities of the Coupled Products of N-Arylsulfonylbenzimidazolones with HES10100 (34−43)
blood glucose (mg/dL)
a
compound
quantity (mg/kg)
3 h
6 h
12 h
%reduction (after 12 h)
NC (with vehicle)
98.34 2.3
306.3 8.6 (after 5 days)
194.71 3.24
183.45 5.62
266.47 2.2
251.09 1.62
214.37 1.76
186.79 2.45
269.48 2.21
266.45 2.5
241.9 1.76
223.52 1.35
248.12 1.65
186.83 2.7
272.30 2.7
252.76 2.1
233.18 2.2
204.86 1.6
280.90 1.2
275.10 1.6
250.65 2.7
239.40 3.2
261.95 3.6
228.30 1.7
DC
200
150
150
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
DGT
DTT
34
196.83 2.98
188.30 5.8
278.29 2.2
266.41 2.92
266.51 3.12
186.57 3.32
178.32 6.1
199.10 1.23
155.63 1.9
168.65 1.7
149.40 3.4
264.42 1.62
244.98 1.72
159.08 1.43
101.13 1.14
153.15 1.62
107.23 1.84
221.20 0.7
173.62 1.1
223.98 2.7
178.40 1.9
277.98 2.8
252.82 3.1
190.84 1.2
142.25 1.1
191.20 1.6
145.94 2.3
38
41
35
49
45
52
15
20
48
67
50
65
29
45
28
43
11
19
38
54
39
54
35
36
37
38
39
40
41
42
43
238.9 1.86
281.76 2.56
284.81 3.42
251.17 1.57
248.08 2.3
254.25 2.6
241.88 2.1
288.90 1.4
281.78 2.2
274.96 2.8
238.82 2.1
296.38 2.3
287.75 2.2
262.50 2.5
258.28 1.8
267.90 2.1
256.45 2.5
a
% change in blood glucose was calculated using formula %change = [(Tc − Tt)/Tc] × 100, where Tc = values before administration of drug, Tt =
values after administration of drug; data are expressed as mean SD; NC = normal control; DC = diabetic control; DGT = diabetic glucophage
treated; DTT = diabetic tolbutamide treated.
The spectroscopic data for all the synthesized compounds
and coupled products is available in the Supporting
Information.
to induce diabetes. Three days later the blood glucose was
monitored and rats showing blood glucose levels above 300
mg/dL were taken as diabetic rats for further experiments.
The test compounds were administrated orally to diabetic
rats at a varying dose of 20 and 40 mg/kg bodyweight to
compare the glucose-lowering potential. An appropriate vehicle
control (DMSO) was also run. Blood samples were collected
from the tail vein by making a snip with a scalpel or sharp
scissors, just prior to drug administration and then at regular
intervals of 3 h up to 12 h for blood glucose analysis using
Accu-check blood glucose analyzer (Roche). The reduction in
blood glucose level (mg/dL) at interval of 3 h was calculated as
a difference from the values before drug administration. The
percent decrease in the sugar level of normal as well as
alloxanized rats was calculated using the formula
Determination of Solubility. Solubility of all the samples
of HES (degraded as well as oxidized) was tested using an
aqueous suspension of 500 mg/5 mL distilled water. The
suspension was stirred for 15 min at 40 °C, transferred to a
centrifuge tube and centrifuged at 1000 rpm for 15 min. The
centrifuge tube was removed from the centrifuge machine; the
supernatant cleared by centrifugation was carefully transferred
into a preweighed crucible and dried in a vacuum oven at 70 °C
to a constant weight. The difference in the weight after drying
the supernatant gave the weight of the soluble material and was
used to calculate the percentage solubility. In the case of
coupled products, the increase in the weight of HES was used
to calculate the solubility.
Determination of Bioactivity. Healthy growing male
albino rats of the Dawley-Sprague family weighing 150−170 g
were housed in clean well-ventilated cages at a temperature of
22 1 °C, relative humidity of 40−50%, a constant 12 h light
and 12 h dark cycles, free access to standard laboratory animal
diet and clean drinking water. The rats were divided into five
groups.
%Reduction in blood glucose = [(T − T)/T] × 100
c
t
c
where Tc = values before administration of drug; Tt = values
after administration of drug.
Statistical Analysis. All values presented are means SD
values. Statistical comparisons of the differences were
performed using one way analysis of variance (ANOVA)
followed by Tukey’s post hoc test, using STATISTICA 5.5. p-
Values below 0.05 were considered statistically significant.
Group 1: Normal rats receiving DMSO as vehicle
Group 2: Alloxan induced diabetic control
Group 3: Glucophage treated reference
Group 4: Tolbutamide treated reference
Group 5: Test compound treated rats
ASSOCIATED CONTENT
■
S
* Supporting Information
The fasting rats (12 h) were given an intraperitoneal (i.p.)
injection of alloxan monohydrate freshly dissolved in citrate
buffer (pH 4.5) at a dose of 200 mg/kg body weight of the rat
All the spectral/characterization data and Representative NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Bioconjugate Chemistry
Article
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AUTHOR INFORMATION
Corresponding Author
*E-mail: shameed@qau.edu.pk; shahidhameed@daad-alumni.
de. Tel.: +92 51 9064 2133. Fax: +92 51 9064 2241.
■
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Present Address
Nasir Mahmood, Department of Chemical Engineering,
COMSATS Institute of Information Technology, Lahore-
54000, Pakistan.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are thankful to the Higher Education Commission
(HEC) of Pakistan for financial support through indigenous
scholarship scheme. Mr. Azhar Abbas is grateful to HEC for a
6-month fellowship at Martin Luther University under
international research support initiative program (IRSIP).
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