Synthesis and biological investigations of nitric oxide releasing nateglinide and meglitinide type II antidiabetic prodrugs: In-vivo antihyperglycemic activities and blood pressure lowering studies

Article abstract of DOI:10.1021/jm300997w

A new group of hybrid nitric oxide-releasing type II antidiabetic drugs possessing a 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (13 and 18), 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (14 and 19), or nitrooxyethyl (15 and 20) moiety attached to the car

Full text of DOI:10.1021/jm300997w

Article
pubs.acs.org/jmc
Synthesis and Biological Investigations of Nitric Oxide Releasing
Nateglinide and Meglitinide Type II Antidiabetic Prodrugs: In-Vivo
Antihyperglycemic Activities and Blood Pressure Lowering Studies
Jatinder Kaur,† Atul Bhardwaj,† Zhangjian Huang,† Deepak Narang,‡ Ting-Yueh Chen,‡ Frances Plane,‡
and Edward E. Knaus*,†
†Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada
‡Department of Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
S
* Supporting Information
ABSTRACT: A new group of hybrid nitric oxide-releasing type
II antidiabetic drugs possessing a 1-(pyrrolidin-1-yl)diazen-1-
ium-1,2-diolate (13 and 18), 1-(N,N-diethylamino)diazen-1-
ium-1,2-diolate (14 and 19), or nitrooxyethyl (15 and 20)
moiety attached to the carboxylic acid group of the type II
antidiabetic drugs nateglinide and meglitinide were synthesized.
These prodrugs, based on the beneficial properties of nitric
oxide (NO), were designed to reduce the risk of adverse
cardiovascular events in diabetic patients. Ester prodrugs (13−
15 and 18−20) exhibited appreciable oral antihyperglycemic
activity comparable to the parent drugs in nonfasted diabetic rats. Systolic and diastolic blood pressure profiles validated the
beneficial hypotensive properties of these prodrugs. These prodrugs released NO (1.3−72.2% range) upon incubation with either
phosphate buffer solution at pH 7.4 or in the presence of serum. This new type of hybrid NO donor prodrug represents an
attractive approach for the rational design of type II antidiabetic drugs with a reduced risk of contraindicated cardiovascular
events.
the best pharmacological approach. The concept of developing
a hybrid multitargeted prodrug that will release each
component of the hybrid in vivo, to furnish the desired
pharmacological effects at the same prodrug dose, warrants
further exploration. In this regard, a NO-glibenclamide
prototype has been reported where the hypoglycemic effect is
enriched by additional NO-vasorelaxing and antiplatelet effects
that are potentially useful to circumvent diabetes-related
cardiovascular disorders.16 More recently, in vitro insulin
release, antiaggregatory, and vasorelaxant properties of some
NO-tolbutamide analogues have been described.17
NO is an efficient vasodilation agent that induces vascular
smooth muscle relaxation, and it reduces the risk of blood clot
formation (thrombosis) by inhibiting platelet aggregation and
adhesion.18 A slow release of NO can provide NO to
circumvent the endogenous NO deficiency (endothelial
dysfunction) in diabetic patients. In view of the beneficial
properties of NO, modification of type II antidiabetic drugs by
incorporating a NO donor moiety offers an attractive drug
design strategy to simultaneously reduce cardiovascular side
effects observed in diabetic patients being prescribed type II
antidiabetic drugs. A hybrid prodrug, or designed multiple
ligand (DML), can provide a variety of potential advantages
INTRODUCTION
■
Diabetes mellitus is a disease in which there is either inadequate
insulin production (type I) or inability to effectively use insulin
(type II). The rising prevalence of type II diabetes, and
associated adverse cardiovascular risks, is now considered a
major public health challenge. In this regard, diabetic patients
are at high risk for macrovascular complications such as
myocardial ischemia, stroke, adverse cerebrovascular events,
and hypertension.1−8 Ultimately, >65% of diabetic patients die
from heart disease or stroke. In diabetic patients, critical
cardiovascular complications9−12 are often attributed to
endothelial dysfunction13,14 that is associated with a deficient
biosynthesis of beneficial vasodilator substances such as NO
and prostacyclin (PGI2). It is widely accepted that a reduction
in the bioavailability of NO is the central mechanism involved
in endothelial dysfunction. This subphysiological level of NO in
diabetic individuals increases the prevalence of severe
thrombosis, atherosclerosis, cardiac inflammation, hyperten-
sion, and remodeling.8,14 Accordingly, in order to reduce
adverse cardiovascular risks, diabetic patients are regularly
prescribed additional drugs targeting hypertension, platelet
aggregation, and dyslipidaemia (ACE-inhibitors, calcium
channel blockers, aspirin, statins, etc.).8,15,16
The use of multitargeted drugs represents an attractive
medicinal chemistry drug design strategy that offers an
alternative to using a multiple drug regimen that may not be
Received: July 10, 2012
Published: August 23, 2012
© 2012 American Chemical Society
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Chart 1. Design of NO-Releasing Type II Antidiabetic Hybrid Prodrugs
relative to the administration of two individual drugs having the
same pharmacological actions. For example, (i) different
relative metabolic rates between patients can result in highly
complex pharmacokinetic (PK) and pharmacodynamic (PD)
relationships for multicomponent drugs leading to unpredict-
able variability among patients that requires extensive and
expensive clinical monitoring of each drug,19 (ii) there is a
lower risk of drug−drug interactions in comparison to cocktails
or multicomponent drugs,20 (iii) patient compliance may be
better, and (iv) there is a potential to enhance efficacy and
safety. Accordingly, coupling a NO donor moiety provides an
opportunity to tune the absorption, distribution, metabolism,
excretion (ADME), and PK and PD properties of a type II
diabetic agent where the deficiency of NO is the cause of
contraindicated side effects associated with diabetes.
In this study, we modified the type II antidiabetic drugs
nateglinide (1, Nat) and meglitinide (2, Meg), which belong to
a family of drugs that unlike sulfonylureas stimulate first-phase
insulin release in a glucose-sensitive manner that theoretically
reduces the risk of hypoglycemic events.21−27 As part of our
ongoing program to design improved drugs, we now describe
the synthesis, in vivo antihyperglycemic activity, and blood
pressure studies of a new group of prototypical NO-releasing
type II antidiabetic hybrid prodrugs that retain the pharmaco-
logical activity of the parent antidiabetic component but also
confer the beneficial biological actions of NO (Chart 1). This
new group of hybrid prodrugs 13 (Nat-PYR), 14 (Nat-DEA),
15 (Nat-NOE), 18 (Met-PYR), 19 (Met-DEA), and 20 (Met-
NOE) were prepared by conjugating the carboxyl group
present in Nat and Meg to NO releasing 1-(pyrrolidin-1-
yl)diazen-1-ium-1,2-diolate (PYR), 1-(N,N-diethylamino)-
diazen-1-ium-1,2-diolate (DEA), or nitrooxyethyl (NOE)
moieties.
Scheme 1. The first type II antidiabetic drug (2R)-2-(trans-4-
isopropylcyclohexanecarboxamido)-3-phenylpropanoic acid
(nateglinide, 1) was synthesized using a methodology similar
to that reported in ref 30 (Scheme 1). Thus, the reaction of
trans-4-isopropylcyclohexanecarboxylic acid (11) with para-
toluenesulphonyl chloride in the presence of Et3N afforded the
mixed anhydride 12, which without isolation using a one-pot
reaction was condensed with D-phenylalanine to furnish Nat (1,
79% yield). The (R)-antipode of Nat was selected for use in
this study since it is reported to be a 70-fold more potent
hypoglycemic agent than the (S)-antipode.24 The second type
II antidiabetic drug 4-(2-(5-chloro-2-methoxybenzamido)-
ethyl)benzoic acid (meglitinide, 2) was prepared by elaboration
of 5-chloro-o-anisic acid (16) to the acid chloride 17.31
Subsequent reaction of 17 with 4-(2-aminoethyl)benzoic acid
hydrochloride in the presence of NaOH afforded Meg26 (2,
57% yield) as illustrated in Scheme 1.
The stable target NO donor ester prodrugs (13, 14, 18, and
19) were synthesized by converting Nat and Meg to the
respective sodium salt by treatment with Na2CO3 in the polar
aprotic solvent hexamethylphosphoramide (HMPA) at 25 °C.
The sodium salt of each drug obtained, upon reaction with a
O2-chloromethyldiazen-1-ium-1,2-diolate 9 or 10 in HMPA,
furnished the respective NO donor ester pro-drug (13, 14, 18,
or 19) in good yield (61−80% range). The corresponding 2-
nitrooxyethyl ester hybrid prodrugs (15 and 20) were
synthesized by reaction of the carboxyl group of either Nat
(1) or Meg (2) with 2-nitrooxyethyl bromide in the presence of
K2CO3 in DMF as illustrated in Scheme 1.
Antihyperglycemic Activity. The in vivo antihyperglyce-
mic activities for the hybrid NO-donor prodrug derivatives of
Nat 13−15 and Meg 18−20 were determined using
streptozotocin (STZ) induced nonfasting diabetic rats (n = 4
per group) for comparison with the parent drugs using an oral
dose of 63.01 μmol/kg. The blood glucose level (BGL) was
measured at 1, 3, 6, and 24 h postdrug administration, and the
% reduction in the blood glucose level (BGLred) relative to the
initial BGL at 0 h just prior to drug administration was
RESULTS AND DISCUSSION
■
Chemistry. The syntheses of O2-chloromethyl-1-(pyrroli-
din-1-yl)diazen-1-ium-1,2-diolate (9) and O2-chloromethyl-1-
(N,N-diethylamino)diazen-1-ium-1,2-diolate (10) were carried
out according to the reported procedures28,29 as illustrated in
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a
Scheme 1. Synthesis of NO-Ester Prodrugs
a
Reagents and conditions: (a) NO oxide (40 psi), NaOMe, MeOH, ether, 25 °C; (b) ClCH2SCH3, DMF/THF, Na2CO3, KI, 25 °C, argon
atmosphere, 48 h; (c) SO2Cl2, DCM, 25 °C, 30 min; (d) p-toluene sulfonyl chloride, Et3N, DCM, 25 °C, 5 h; (e) D-phenylalanine, 25 °C, 12 h; (f)
Na2CO3, compound 9 or 10, HMPA, 25 °C, 65 h for compound 13 and 48 h for compound 14; (g) 2-nitrooxyethyl bromide, K2CO3, DMF, 25 °C,
24 h; (h) thionyl chloride, catalytic amount of dry DMF, 25 °C, 30 min; (i) 4-(2-aminoethyl)benzoic acid hydrochloride, NaOH, acetone, 2 h; (j)
Na2CO3, compound 9 or 10, HMPA, 25 °C, 33 h for compound 18 and 31 h for compound 19; (k) 2-nitrooxyethyl bromide, K2CO3, DMF, 25 °C,
12 h.
calculated (see data in Figures 1 and 2, and Table S1
(Supporting Information)).
In the Nat group of compounds 13−15, Nat-PYR having a 1-
(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate NO-donor moiety
istration is not known, and it is distinctly different from all
other compounds 14−15, 18−20, Nat, Meg, and vehicle alone
evaluated in this study, which did not induce a hyperglycemic
or hypoglycemic effect at 24 h. In comparison, Nat-DEA having
a 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate NO-donor
moiety showed a longer duration of action like that of Nat
showed a lower reduction in the BGL at 1 h (BGLred
=
8.1%; p < 0.05) and 3 h (BGLred = 22.6%; p < 0.05) compared
to the parent drug Nat (BGLred = 13.8% at 1 h; 26.4% at 3 h;
30.6% at 6 h) as illustrated in Figures 1a and b. In this regard,
Nat-PYR did not reduce the BGL at 6 h since it showed a BGL
similar to that of the control BGL at time 0. Nat-PYR induced a
significant hyperglycemic effect at 24 h postdrug administration
where the BGL was increased 48.7% relative to the BGL at time
0 prior to drug administration. The mechanism for this
unexpected hyperglycemic effect at 24 h postdrug admin-
lasting 6 h and provided a greater reduction in BGLs (BGLred
=
21.1% at 1 h; 23.5% at 3 h; 34.1% at 6 h; p < 0.05) than Nat.
Nat-NOE, a 2-nitrooxyethyl ester pro-drug, exhibited a longer 6
h duration of action like Nat-DEA and Nat, but Nat-NOE was
less potent (BGLred = 8.2, 13.4 and 20.5% at 1, 3, and 6 h,
respectively) than either Nat-DEA or Nat.
Antidiabetic (hypoglycemic) structure−activity data were
also acquired for a similar group (Meg-PYR; Meg-DEA, Meg-
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Figure 1. (a) Percentage reduction of BGL in STZ induced nonfasting diabetic rats after oral administration (63.01 μmol/kg) of compounds 13, 14,
and 15 at 1, 3, 6, and 24 h postdrug administration. (b) Change in BGL in STZ induced nonfasting diabetic rats after oral administration (63.01
μmol/kg) of compounds 13, 14, and 15 at 1, 3, 6, and 24 h postdrug administration. Values are the mean % changes (n = 4 animals in each group).
The level of significance represents the difference in the BGL at time 0 prior to drug administration, relative to that at the indicated postdrug
administration times, for each animal (the difference is significant when *p < 0.05).
Figure 2. (a) Percentage reduction of BGL in STZ induced nonfasting diabetic rats after oral administration (63.01 μmol/kg) of compounds 18, 19,
and 20 at 1, 3, 6, and 24 h postdrug administration. (b) Change in BGL in STZ induced nonfasting diabetic rats after oral administration (63.01
μmol/kg) of compounds 18, 19, and 20 at 1, 3, 6, and 24 h postdrug administration. Values are the mean % changes (n = 4 animals in each group).
The level of significance represents the difference in the BGL at time 0 prior to drug administration, relative to that at the indicated postdrug
administration times, for each animal (the difference is significant when *p < 0.05).
NOE) of hybrid Meg NO-donor prodrugs (see data in Figures
2a and b). Meg-PYR, Meg-DEA, and Meg-NOE showed a
lower reduction in BGLs (BGLred in the 20.0 to 28.3% range)
than the parent drug Meg (BGLred = 30.5%) at 1 h but a larger
reduction in BGLs (BGLred in the 12.5 to 17.0% range) than
Meg (BGLred = 8.8%) at 3 h postdrug administration. Meg-
PYR, Meg-DEA, Meg-NOE, and Meg exhibit a short duration
of action since weak (Met-DEA and Met-NOE) hypoglycemic
activity was observed at 6 h, and no hypoglycemic activity was
observed (Met-Pyr, Met-DEA, Met-NOE, Meg, and vehicle
control) at 24 h postdrug administration. Table S1 listing the
minimum of three consecutive measurements were made for
each mouse at each time interval and the mean value is the
average of these three measurements. The data obtained for
BPsys, BPdias, BPmean (average of BPsys and BPdia) and HR are
illustrated in Figure 3a−d. Table S2 listing numerical data
standard deviation is provided as Supporting Information.
All of the NO-donor Meg-PYR (90.3), Meg-DEA (96.8),
Meg-NOE (105.8), Nat-PYR (108.4) and Nat-DEA (113.0)
prodrugs showed a significant reduction (*p < 0.05) in BPsys
(mm Hg), except for Nat-NOE (129.9), relative to the control
group (126.5) at 1 h postdrug administration (see data in
Figure 3a). In comparison, only the Nat-PYR (106.2) and Nat-
DEA (98.1) conjugates showed a significant reduction in BPsys
relative to the control group (118.3 mmHg)) at 3 h postdrug
administration. The mechanism responsible for these transient
changes in BPsys at 3 h is not known since the BPsys profile
changed at 6 h postdrug administration where BPsys for Nat-
NOE, Meg-PYR, Meg-DEA and Meg-NOE (103.4 to 106.9
mmHg range), but not that for Nat-PYR (134.3) or Nat-DEA
structure−activity data described (numerical data
deviation) is provided in Supporting Information.
standard
Blood Pressure Studies. Systolic blood pressure (BPsys,
mm Hg), diastolic blood pressure (BPdia, mm Hg), and heart
rate (HR, beats min−1) were measured at 1, 3, and 6 h time
intervals following oral administration of either the vehicle
alone (control group, n = 6) or NO donor prodrugs 13−15 and
18-20 (63.01 μmol/kg po, n = 3) to C57 black mice. A
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Figure 3. BPsys, BPdia, BPmean, and HR in conscious mice following oral administration of either vehicle alone (control group, mean SD, n = 6) or
test compounds 13−15 and 18−20 (63.01 μmol/kg po; n = 3). Data are presented as the group mean SD. Statistical significant differences
between control groups and drug treatment groups at each time interval are denoted as *p < 0.05.
(134.4), was significantly lower (*p < 0.05) than that for the
control group (126.2 mmHg).
presence of rat serum that contains nonspecific esterases. The
indirect quantitation of NO as nitrite anion using the Griess
reaction (see data in Table 1) was determined for incubation
The BPdia structure−activity data acquired for the Nat and
Meg hybrid NO-donor conjugates (13−15, 18−20) presented
in Figure 3b indicates that the BPdia profile for this group of
compounds follows a very similar profile to that previously
described for BPsys at 1, 3, and 6 h postdrug administration
(compare profiles in Figure 3a and Figure 3b for each
respective hybrid NO-donor conjugate). The only difference
observed is for the Nat-PYR conjugate 13 which shows a
significant reduction (*p < 0.05) in BPsys, but not BPdia (*p >
0.05), at 3 h postdrug administration. Accordingly as expected,
BPmean (average of BPsys and BPdia) follows a similar profile (see
Figure 3c) to that observed for BPsys (Figure 3a) and BPdia
(Figure 3b).
The HR of mice was determined and the data are shown in
Figure 3d (complete data is provided in Table S2 as Supporting
Information). The HR at 1 h (454−568 range), 3 h (458−631
range) and 6 h (435−566 range) was generally higher than that
for control mice (449, 448, and 439 beats min−1 at 1, 3, and 6 h
postdrug administration, respectivcly). One plausible explan-
ation for the increase in HR, for those compounds in which an
elevated HR occurred, may be a homeostatic mechanism to
Table 1. Percentage (%) of NO Released from Compounds
13−15 and 18−20
a
% NO released
PBS (pH 7.4)
PBS (pH 7.4) + Serum
compd 1 h
4 h
6 h
24 h
1 h
3 h
6 h
24 h
13
14
15
18
19
20
6.7
4.4
6.7
5.1
4.2
4.1
6.8
5.1
7.5
5.7
6.0
7.2
52.7
58.8
7.7
59.9
61.3
9.2
40.2
44.9
1.3
47.2
50.2
1.9
50.1
57.2
3.9
65.3
72.2
6.3
7.2
8.9
37.6
54.5
2.1
51.7
55.9
2.3
59.2
61.5
2.6
60.5
63.6
3.8
6.1
6.5
7.7
9.4
a
Percentage of NO released (mean value, n = 3) relative to a
theoretical maximum release of 2 mol of NO/mol of test compounds
(13, 14, 18, and 19) and 1 mol of NO/mol of test compounds (15
and 20) was determined using the Griess reaction. Variation from the
mean % value was ≤ 0.02%. Incubations were carried out for 1, 4, 6,
and 24 h in 2.4 mL of phosphate buffer solution (PBS, pH 7.4) or for
1, 3, 6, and 24 h in PBS buffer (2.4 mL) containing 90 μL of rat serum
at 37 °C.
compensate for the reduction in BPsys, BPdia and BPmean
.
Nitric oxide Release. The diazen-1-ium-1,2-diolate and
nitrooxy moieties used to prepare the hybrid prodrug
conjugates of Nat 13−15 and Meg 18-20 are well established
NO donor groups. Therefore, it was of interest to determine
the percentage of NO released from the hybrid ester prodrugs
13−15 and 18−20 upon incubation in either phosphate-
buffered saline (PBS at pH 7.4) or PBS at pH 7.4 in the
times of 1, 3, 4, 16, and 24 h. The rate of NO release from
diazen-1-ium-1,2-diolates can be controlled by chemical
modification such as attachment of an alkyl substituent to the
O2-position.32 These O2-substituted-diazen-1-ium-1,2-diolates
are stable compounds that hydrolyze slowly even in acidic
solution.33 When compounds 13, 14, 18, and 19 were
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incubated in PBS buffer (pH 7.4), the percentage of NO
released varied from a low of 4.1% to 6.7% at 1 h up to a
maximal release of 59.9% (Nat-Pyr), 61.3% (Nat-DEA), 8.9%
(Meg-Pyr), and 6.5% (Meg-DEA) for a 24 h incubation. In this
regard, the Meg-Pyr and Meg-DEA conjugates, like Nat-NOE
and Meg-NOE, release a much lower % of NO. In contrast,
incubation of Meg-Pyr and Meg-DEA in PBS at pH 7.4 in the
presence of rat serum that contains nonspecific esterases
increased the amount of NO released. Accordingly, the extent
of NO release from compounds 13, 14, 18, and 19 was
substantially higher (37.6−54.5% range) at 1 h reaching
maximal release (60.5−72.2% range) for 24 h incubations.
This increase in % NO release indicates that nonspecific
esterases present in rat serum cleave the hybrid prodrug esters
more effectively than PBS at pH 7.4. A plausible mechanism for
the ester hydrolysis of hybrid ester prodrugs containing a
diazen-1-ium-1,2-diolate moiety was described in an earlier
study.34 In contrast to compounds 13, 14, 18, and 19
containing a diazen-1-ium-1,2-diolate NO-donor moiety, the
extent of NO released upon incubation of the hybrid
nitrooxyethyl ester prodrugs (Nat-NOE, 6.7 to 9.2% range;
Meg-NOE, 4.1 to 9.4% range) was lower for incubations of 1, 3,
4, 6, and 24 h in both PBS buffer at pH 7.4 and in PBS buffer
containing rat serum (Nat-NOE, 1.3−6.3% range; Meg-NOE,
2.1−3.8% range).
Vascular Relaxation. Nat-DEA (1 μM) caused rapid and
complete reversal, and Nat-NOE (1 μM) caused less rapid but
near complete reversal of phenylephrine-induced tone in
isolated rat mesenteric arteries, which was abolished by ODQ
(10 μM; n = 3), a selective inhibitor of soluble guanylyl
cyclase.35 Representative traces of the effects of Nat-DEA and
Nat-NOE are shown in Figure 4. The ability of Nat-DEA and
Nat-NOE to reverse phenylephrine-induced tone in isolated
mesenteric arteries demonstrates that these compounds can act
directly on vascular smooth muscle cells to cause relaxation
(vasodilation) of resistance vessels. Abolition of the response by
ODQ indicates that Nat-DEA acts via activation of soluble
guanylyl cyclase and supports the contention that Nat-DEA
reduces blood pressure via the generation of NO.
Figure 4. Vascular relaxation (panel A) by Nat-DEA (1 μM) and
(panel B) by Nat-NOE (1 μM) of phenylephrine-induced tone in rat
mesenteric arteries in the presence and absence of the selective
inhibitor of soluble guanylyl cyclase inhibitor ODQ (10 μM).
rational design of type II antidiabetic drugs with a reduced risk
of contraindicated cardiovascular events frequently observed in
diabetic individuals.
CONCLUSIONS
■
A new group of hybrid NO-releasing type II antidiabetic ester
prodrugs possessing a PYR, DEA, or NOE moiety attached to
the carboxylic acid group of the type II oral antidiabetic drugs
Nat and Meg were synthesized. In vivo biological studies
indicated that this group of hybrid prodrugs (i) exhibit
antihyperglycemic activity with durations of action up to six
hours that compares favorably to that of the parent antidiabetic
drug Nat or Meg and (ii) reduce BPsys, BPdia, and BPmean for up
to six hours postdrug administration. In vitro NO release
studies showed that the PYR and DEA moieties are effective
NO-donors in phosphate buffer at pH 7.4 in the presence of rat
serum. The hybrid Nat-DEA prodrug 14 was identified as a
potential lead-compound that (i) exhibited an appreciable
reduction in the BGL (BGLred in the 21.1−34.1% range) of
diabetic rats up to six hours postdrug administration, (ii)
showed a significant reduction in blood pressure up to three
hours postdrug administration, and (iii) induced vascular
relaxation of mesenteric arteries, which was blocked by ODQ,
indicative of soluble guanylyl cyclase activation supporting the
contention that Nat-DEA reduces blood pressure via the
generation of NO. Accordingly, this new type of hybrid NO
donor prodrug constitutes an attractive approach for the
EXPERIMENTAL SECTION
■
General. Melting points were measured in capillaries using a
Thomas-Hoover capillary apparatus and are uncorrected. H and 13C
1
NMR spectra were recorded on a Bruker Avance III 600 MHz or AM-
300 300 MHz NMR spectrometer using CDCl3 or D2O as solvent.
Chemical shifts are given in parts per million (ppm) with
tetramethylsilane (TMS) as an internal reference. Infrared (IR)
spectra were recorded as films on NaCl plates using a Nicolet 550
Series II Magna FTIR spectrometer. Mass spectra (MS) were recorded
on a Water’s Micromass ZQ 4000 mass spectrometer using the ESI
ionization mode. The purity of the compounds was established using
elemental analysis, which were acquired for C, H, N by the
Microanalytical Service Laboratory, Department of Chemistry,
University of Alberta. Compounds showed a single spot on
Macherey-Nagel Polygram Sil G/UV254 silica gel plates (0.2 mm)
using a low, medium, and highly polar solvent system, and no residue
remained after combustion, indicating a purity >98%. Column
chromatography was performed on a Combiflash Rf system using a
gold silica column. All other reagents, purchased from the Aldrich
Chemical Co. (Milwaukee, WI) and TCI America, were used without
further purification. Compounds 1,30 2,26 5,36 6,37 7,28 8,29 9,28 and
10,29 were prepared according to literature procedures.
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(2R)-2-(((trans)-4-Isopropylcyclohexanecarboxamido)-3-
phenylpropanoyl)oxy)methoxy)-1-(pyrrolidin-1-yl)diazene oxide
(13). The sodium salt of nateglinide was prepared in situ by stirring
1 (0.5 g, 1.57 mmol) with a suspension of Na2CO3 (0.166 g, 1.57
mmol) and HMPA (4.5 mL) for 19 h at 25 °C. A solution of O2-
(chloromethyl)-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (9, 0.282
g, 1.57 mmol) in HMPA (1.5 mL) was added, and the reaction was
allowed to proceed for 46 h at 25 °C. Water (15 mL) was added, the
mixture was extracted with EtOAc (3 × 25 mL), the organic phase was
dried (Na2SO4), and the solvent was removed in vacuo. The residue
obtained was purified by silica gel column chromatography using
EtOAc/hexane (1:1, v/v) as eluant to furnish 13 (0.58 g, 80%) as a
(15 mL). The organic phase was dried (Na2SO4), the solvent was
removed in vacuo, and the residue obtained was purified by silica gel
chromatography using ethyl acetate−hexane (1:1, v/v) as eluent to
furnish compound 15 (0.337 g, 83%) as a light brown solid; mp 114−
115 °C; [α]21.0 = −32.99 (0.500, CHCl3). IR (CHCl3): 3279, 2944,
D
1
2822, 1745, 1682, 1621, 1280 cm−1. H NMR (CDCl3, 600 MHz): δ
0.77 [d, J = 6.6 Hz, 6H, CH(CH3)2], 0.88−0.95 (m, 3H, one
cyclohexyl H-3, one cyclohexyl H-5, cyclohexyl H-4), 1.30−1.34 (m,
3H, one cyclohexyl H-3, one cyclohexyl-5, CHMe2), 1.69−1.71 (m,
2H, one cyclohexyl H-2, one cyclohexyl H-6), 1.72−1.80 (m, 2H, one
cyclohexyl H-2, one cyclohexyl H-6), 1.94−1.99 (m, 1H, cyclohexyl H-
1), 3.04−3.06 (m, 2H, CH2Ph), 4.26−4.35 (m, 2H, CO2CH2), 4.51−
4.53 (m, 2H, CH2ONO2), 4.80−4.83 (m, 1H, NH−CH), 5.77 (d, J =
7.8 Hz, 1H, NH), 7.05 (dd, J = 8.4, 1.8 Hz, 2H, ortho-phenyl
hydrogens), 7.15−7.23 (m, 3H, meta- and para-phenyl hydrogens).
13C NMR (CDCl3, 150 MHz): δ 19.7 (CHMe2), 29.4 and 29.7
(cyclohexyl C-2, C-3, C-5, C-6), 32.7 (CHMe), 37.8 (CH2Ph), 43.2
and 45.4 (cyclohexyl C-4, C-1), 53.2 (NHCHCO), 60.8 (CO2CH2),
70.0 (CH2ONO2), 127.3, 128.7, and 129.1 (phenyl CH), 135.6
(phenyl C-1), 171.5 (CO), 175.7 (CO). ESI-MS: 407 [M + H]+.
Anal. Calcd. for C21H30N2O6: C, 62.05; H, 7.44; N, 6.84. Found: C,
62.13; H, 7.40; N, 6.84.
pale yellow viscous liquid, [α]21.0 = −25.39 (0.500, CHCl3). IR
D
1
(CHCl3): 3282, 2934, 2813, 1734, 1636, 1269, 1183 cm−1. H NMR
(CDCl3, 300 MHz): δ 0.83 [d, J = 7.3 Hz, 6H, CH(CH3)2], 0.90−0.99
(m, 3H, one cyclohexyl H-3 and one cyclohexyl H-5, cyclohexyl H-4),
1.34−1.41 (m, 3H, one cyclohexyl H-3 and one cyclohexyl H-5,
CHMe2), 1.72−1.83 (m, 2H, one cyclohexyl H-2 and one cyclohexyl
H-6) 1.92−1.99 (m, 6H, one cyclohexyl H-2 and one cyclohexyl H-6,
pyrrolidinyl H-3, H-4), 2.00−2.05 (m, 1H, cyclohexyl H-1), 3.11 and
3.17 (two dd J = 14. 1, 5.7 Hz, 1H each, CH2Ph), 3.54−3.59 (m, 4H,
pyrrolidinyl H-2, H-5), 4.91−4.94 (m, 1H, NH−CH), 5.70 (d, J = 7.32
Hz, 1H, OCHH’O), 5.88 (d, J = 7.32 Hz, 1H, OCHH’O), 5.98 (d, J =
7.8 Hz, 1H, NH), 7.07 (dd, J = 7.6, 1.8 Hz, 2H, ortho-phenyl
hydrogens), 7.15−7.29 (m, 3H, meta- and para-phenyl hydrogens).
13C NMR (CDCl3, 75 MHz): δ 19.6 (CHMe2), 22.9 (pyrrolidinyl C-3,
C-4), 28.9 and 29.2 (cyclohexyl C-2, C-3, C-5, C-6), 32.6 (CHMe2),
37.3 (CH2Ph), 43.1 and 45.3 (cyclohexyl C-4, C-1), 50.6 (pyrroldinyl
C-2, C-5), 52.5 (NHCHCO), 87.9 (OCH2O), 127.0, 128.3, and 129.3
(phenyl CH), 135.4 (phenyl C-1), 173.6 (CO), 176.2 (CO). ESI-
MS: 483 [M+Na]+. Anal. Calcd. for C24H36N4O5: C, 62.59; H, 7.88;
N, 12.16. Found: C, 62.56; H, 7.93; N, 12.22.
2-(((4-(2-(5-Chloro-2-methoxybenzamido)ethyl)benzoyl)oxy)-
methoxy)-1-(pyrrolidin-1-yl)diazene oxide (18). The sodium salt of
meglitinide was prepared in situ by stirring meglitinide (2, 0.334 g, 1
mmol) in a suspension of Na2CO3 (0.105 g, 1 mmol) and HMPA (4.5
mL) for 19 h at 25 °C. A solution of O2-(chloromethyl)-1-(pyrrolidin-
1-yl)diazen-1-ium-1,2-diolate (9, 0.216 g, 1.2 mmol) in HMPA (1.5
mL) was added, and the reaction was allowed to proceed for 14 h at 25
°C. Water (15 mL) was added to the reaction mixture, which was
extracted with EtOAc (3 × 25 mL). The organic phase was dried
(Na2SO4), the solvent was removed in vacuo, and the residue obtained
was purified by silica gel column chromatography using EtOAc/hexane
(1:1, v/v) as eluent to furnish compound 18 (0.372 g, 78%) as a white
solid; mp 110−111 °C. IR (CHCl3): 3280, 2987, 2846, 1710, 1612,
(3R)-3-Benzyl-10-ethyl-1-((trans)-4-isopropylcyclohexyl)-1,4-
dioxo-5,7-dioxa-2,8,9,10-tetraazadodec-8-en-9-oxide (14). The so-
dium salt of nateglinide was prepared in situ by stirring 1 (0.5 g, 1.57
mmol) with a suspension of Na2CO3 (0.166 g, 1.57 mmol) and
HMPA (4.5 mL) for 19 h at 25 °C. A solution of O2-(chloromethyl)-1-
(N,N-diethylamino)diazen-1-ium-1,2-diolate (10, 0.284 g, 1.57 mmol)
in HMPA (1.5 mL) was added, and the reaction was allowed to
proceed for 29 h at 25 °C. Water (15 mL) was added and the mixture
was extracted with EtOAc (3 × 15 mL). The combined EtOAc
fractions were dried (Na2SO4), the solvent was removed in vacuo, and
the residue obtained was purified by silica gel column chromatography
using EtOAc/hexane (1:1, v/v) as eluent to furnish compound 14
1
1255, 1145 cm−1. H NMR (CDCl3, 600 MHz): δ 1.95−197 (m, 4H,
two pyrrolidinyl H-3, H-4), 3.01 (t, J = 6.9 Hz, 2H, NHCH2CH2),
3.57−3.61 (m, 4H, pyrrolidinyl H-2, H-5), 3.76 (t, J = 6.6 Hz, 2H,
NHCH2), 3.79 (s, 3H, OCH3); 6.02 (s, 1H, OCH2O), 6.88 (d, J = 9.0
Hz, 1H, MeO-phenyl H-3), 7.35−7.39 (m, 3H, benzoate H-3, H-5, Cl-
phenyl H-4), 7.84 (broad s, 1H, NH), 8.05 (d, J = 8.4 Hz, 2H,
benzoate H-2, H-6), 8.17 (d, J = 2.4 Hz, 1H, Cl-phenyl H-6). 13C
NMR (CDCl3, 150 MHz): δ 22.9 (pyrrolidinyl C-3, C-4), 35.6
(NHCH2CH2), 40.6 (NHCH2), 50.7 (pyrrolidinyl C-2, C-5), 56.2
(OCH3), 87.9 (OCH2O), 112.8 (MeO-phenyl C-3), 122.7 (MeO-
phenyl C-1), 126.7 (Cl-phenyl C-5), 127.6 (benzoate C-1), 129.0,
130.3, 131.9, and 132.3 (Cl-phenyl C-6 and C-4, benzoate C-2, C-3, C-
5, C-6), 145.5 (benzoate C-4), 155.9 (MeO-phenyl C-2), 164.0 (C
O), 165.0 (CO). ESI-MS: 477, 479 [M + H]+. Anal. Calcd. for
C22H25ClN4O6: C, 55.40; H, 5.20; N, 11.75. Found: C, 55.40; H, 5.33;
N, 11.49.
(0.444 g, 61%) as a white solid; mp 125−126 °C; [α]21.0 = −13.00
D
(0.500, CHCl3). IR (CHCl3): 3263, 2916, 2835, 1740, 1682, 1242,
1
1136 cm−1; H NMR (CDCl3, 600 MHz): δ 0.86 [d, J = 6.6 Hz, 6H,
CH(CH3)2], 0.98−1.08 (m, 3H, one cyclohexyl H-3, one cyclohexyl
H-5, cyclohexyl H-4), 1.13 (t, J = 7.2 Hz, 6H, two CH2CH3), 1.37−
1.42 (m, 3H, one cyclohexyl H-3, one cyclohexyl-5, CHMe2), 1.77−
1.79 (m, 2H, one cyclohexyl H-2, one cyclohexyl H-6), 1.83−1.90 (m,
2H, one cyclohexyl H-2, one cyclohexyl H-6), 1.99−2.01 (m, 1H,
cyclohexyl H-1), 3.11 and 3.21 (dd, J = 14.1, 5.7 Hz, 1H each,
CH2Ph), 3.26 (q, J = 7.2 Hz, 4H, two NCH2), 4.94−4.98 (m, 1H,
NH−CH), 5.81 (d, J = 7.2 Hz, 1H, OCHH’O), 5.83 (d, J = 7.2 Hz,
1H, OCHH’O), 5.98 (d, J = 6.6 Hz, 1H, NH), 7.10 (dd, J = 8.4, 1.8
Hz, 2H, ortho-phenyl hydrogens), 7.25−7.30 (m, 3H, meta- and para-
phenyl hydrogens); 13C NMR (CDCl3, 150 MHz): δ 11.4 (CH2CH3),
19.7 (CHMe2), 28.9 and 29.4 (cyclohexyl C-2, C-3, C-5, C-6), 32.7
(CHMe2), 37.3 (CH2Ph), 43.1 and 45.4 (cyclohexyl C-4, C-1), 47.9
(CH2CH3), 52.4 (NHCHCO), 87.9 (OCH2O), 127.2, 128.6, and
129.4 (phenyl CH), 135.3 (phenyl C-1), 170.4 (CO), 175.7 (C
O). ESI-MS: 463 [M + H]+. Anal. Calcd. for C24H38N4O5: C, 62.31;
H, 8.28; N, 12.11. Found: C, 62.39; H, 8.35; N, 12.19.
1-(((4-(2-(5-Chloro-2-methoxybenzamido)ethyl)benzoyl)oxy)-
methoxy)-3,3-diethyltriaz-1-ene 2-oxide (19). The sodium salt of
meglitinide was prepared in situ by stirring 2 (0.334 g, 1 mmol) with a
suspension of Na2CO3 (0.105 g, 1 mmol) in HMPA (4.5 mL) for 19 h
at 25 °C. A solution of O2-(chloromethyl)-1-(N,N-diethylamino)-
diazen-1-ium-1,2-diolate (10, 0.218 g, 1.2 mmol) in HMPA (1.5 mL)
was then added, and the reaction was allowed to proceed for 12 h at 25
°C. Water (15 mL) was added, the mixture was extracted with EtOAc
(3 × 25 mL), and the combined EtOAc extracts were dried (Na2SO4).
Removal of the solvent in vacuo gave a residue that was purified by
silica gel column chromatography using EtOAc/hexane (1:1, v/v) to
afford 19 (0.335 g, 70%) as a white solid; mp 90−92 °C. IR (CHCl3):
1
3199, 2977, 2854, 1725, 1622, 1252, 1132 cm−1. H NMR (CDCl3,
600 MHz): δ 1.02 (t, J = 7.2 Hz, 6H, two CH2CH3), 2.92 (t, J = 6.9
Hz, 2H, NHCH2CH2), 3.12 (q, J = 7.2 Hz, 4H, NCH2CH3), 3.68 (t, J
= 6.0 Hz, 2H, NHCH2CH2), 3.70 (s, 3H, OCH3); 6.0 (s, 1H,
OCH2O), 6.79 (d, J = 9.0 Hz, 1H, MeO-phenyl H-3), 7.26−7.31 (m,
3H, benzoate H-3, H-5, Cl-phenyl H-4), 7.74 (broad s, 1H, NH), 7.95
(d, J = 8.4 Hz, 2H, benzoate H-2, H-6), 8.09 (d, J = 2.4 Hz, 1H, Cl-
(2R)-2-Nitrooxyethyl 2-((trans)-4-isopropylcyclohexanecarboxa-
mido)-3-phenylpropanoate (15). A solution of 2-nitrooxyethyl
bromide (204 mg, 1.2 mmol), nateglinide (317 mg, 1 mmol) and
K2CO3 (310 mg, 1.5 mmol) in dry DMF (5 mL) was stirred at 25 °C
for 24 h. Water (15 mL) was added, the mixture was extracted with
EtOAc (3 × 20 mL), and the EtOAc extracts were washed with brine
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Journal of Medicinal Chemistry
Article
phenyl H-6). 13C NMR (CDCl3, 150 MHz): δ 11.3 (CH2CH3), 35.6
(NHCH2CH2), 40.5 (NHCH2CH2), 48.1 (CH2CH3), 56.1 (OCH3),
87.8 (OCH2O), 112.7 (MeO-phenyl C-3), 122.6 (MeO-phenyl C-1),
126.7 (Cl-phenyl C-5), 127.3 (benzoate C-1), 129.0, 130.2, 131.9, and
132.3 (Cl-phenyl C-6 and C-4, benzoate C-2, C-3, C-5, C-6), 145.6
(benzoate C-4), 155.8 (MeO-phenyl C-2), 163.9 (CO), 164.7 (C
O). ESI-MS: 479, 481 [M + H]+. Anal. Calcd. for C22H27ClN4O6: C,
55.17; H, 5.68; N, 11.70. Found: C, 55.20; H, 5.68; N, 11.76
Nitric Oxide Release Assay. In vitro nitric oxide release was
estimated by quantification of nitrite produced by the reaction of nitric
oxide with oxygen and water using the Griess reaction upon incubation
of the test compound (2.4 mL of 5.0 × 10−2 mM) in phosphate buffer
solution (PBS) at pH 7.4 (incubation times of 1, 4, 6, and 24 h) and in
2.4 mL of PBS to which 90 μL of serum was added (incubations times
of 1, 3, 6, and 24 h) at 37 °C. The amount of nitric oxide released at
each time interval was determined for test compounds 13−15 and
18−20 using the reported procedure.39
Vascular Relaxation (Wire Myography). Third order branches of
the rat superior mesenteric artery were mounted in a Mulvany-Halpern
myograph (model 400A, J.P. Trading, Denmark) for recording of
changes in isometric tension as previously described.40 The endothelial
cell layer was removed by gently rubbing the intimal surface of the
arteries with a hair prior to mounting. Successful removal of the
endothelium was confirmed by the absence of relaxation to
acetylcholine (10 μM).
2-Nitrooxyethyl 4-(2-(5-chloro-2-methoxybenzamido)ethyl)-
benzoate (20). A solution of 2-nitooxyethyl bromide (204 mg, 1.2
mmol), meglitinide (2, 333 mg, 1 mmol) and K2CO3 (310 mg, 1.5
mmol) in dry DMF (5 mL) was stirred at 25 °C for 12 h. Water (15
mL) was added, and the mixture was extracted with EtOAc (3 × 20
mL) and washed with brine (20 mL). The EtOAc fraction was dried
(Na2SO4), the solvent was removed in vacuo, and the residue obtained
was purified by silica gel column chromatography using ethyl acetate−
hexane (1:1, v/v) as eluent to furnish compound 20 (0.359 g, 85%) as
a cream colored solid; mp 100−102 °C. IR (CHCl3): 3269, 2955,
The ability of Nat-DEA (1 μM) and Nat-NOE (1 μM) to cause
vascular relaxation via activation of soluble guanylyl cyclase was
assessed by adding the drugs to tissues in which tone was raised to
75% of the maximal by phenylephrine (1−3 μM) in the absence and
presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10
μM).
1
2823, 1758, 1649, 1619, 1282 cm−1. H NMR (CDCl3, 600 MHz): δ
3.01 (t, J = 6.9 Hz, 2H, NHCH2CH2), 3.77 (t, J = 6.9 Hz, 2H,
NHCH2), 3.79 (s, 3H, OCH3); 4.62−4.63 (m, 2H, CO2CH2), 4.82−
4.84 (m, 2H, CH2ONO2), 6.88 (d, J = 8.4 Hz, 1H, MeO-phenyl H-3),
7.36−7.39 (m, 3H, benzoate H-3, H-5, Cl-phenyl H-4), 7.83 (broad s,
1H, NH), 8.01−8.03 (d, J = 8.4 Hz, 2H, benzoate H-2, H-6), 8.19 (d, J
= 2.4 Hz, 1H, Cl-phenyl H-6). 13C NMR (CDCl3, 150 MHz): δ 35.6
(NHCH2CH2), 40.5 (NHCH2), 56.1 (OCH3), 60.7 (CO2CH2), 70.4
(CH2ONO2), 112.7 (MeO-phenyl C-3), 122.6 (MeO-phenyl C-1),
126.6 (Cl-phenyl C-5), 127.5 (benzoate C-1), 128.9, 129.9, 131.9, and
132.3 (Cl-phenyl C-6 and C-4, benzoate C-2, C-3, C-5, C-6), 145.3
(benzoate C-4), 155.8 (MeO-phenyl C-2), 163.9 (CO), 165.9 (C
O). ESI-MS: 423, 425 [M + H]+. Anal. Calcd. for C19H19ClN2O7: C,
53.97; H, 4.53; N, 6.63. Found: C, 53.86; H, 4.49; N, 6.53.
Animals. Male Sprague−Dawley rats, 300−350 g in weight
purchased from the University of Alberta Health Science Animal
Services Facility, were employed in the streptozotocin-induced diabetic
rat model to determine antihyperglycemic activity. Antihypertensive
blood pressure measurement studies were carried out using male and
female C57BL/6 mice, aged 3−5 months and weighing 22−33 g,
purchased from the Charles River Animal Facility. All animal
experiments were conducted in accordance with guidelines established
by Health Science Laboratory Animal Services (HSLAS), University of
Alberta.
ASSOCIATED CONTENT
■
S
* Supporting Information
In vivo antihyperglycemic activity for compounds 13−15 and
18−20, and the reference drugs nateglinide and meglitinide;
and blood pressure (mm Hg) and heart rate (beats min−1) data
at 1, 3, and 6 h postdrug administration. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 780-492-5993. Fax: 780-492-1217. E-mail: eknaus@
ualberta.ca.
Notes
The authors declare no competing financial interest.
Antihyperglycemic Studies. Male Sprague−Dawley rats, 300−350
g in weight, were employed for the determination of antihyperglycemic
activity in a streptozotocin-induced diabetic rat model. Streptozotocin
was dissolved in citrate buffer (pH 4.5), and a calculated amount of the
fresh solution (65 mg/kg ip dose) was injected intraperitoneally into
nonfasting rats. Blood glucose was measured after 48 h using
glucostrips (One Touch Ultra 2 test strips), and animals showing a
blood glucose level within the range of 20−30 mmol/L, which are
considered to be diabetic, were selected for determination of
antihyperglycemic activity. A solution of the test compound (one of
13−15, 18−20, nateglinide, and meglitinide) dissolved in water
containing 1% methyl cellulose or a control solution (water +1%
methyl cellulose) not containing the test compound was administered
orally to diabetic rats. The reference drugs nateglinide (1), meglitinide
(2) and the test compounds 13−15 and 18−20 were administered at
the same equimolar dose of 63.01 μmol/kg po. Following
administration of the experimental drug or control vehicle a blood
sample was collected by puncture of the tail vein, and the blood
glucose level was measured at 1, 3, 6, and 24 h postdrug administration
using a One Touch Ultra 2 Blood Glucose Meter.
ACKNOWLEDGMENTS
■
We are grateful to the Canadian Institutes of Health Research
(grant no. MOP-14712) for financial support of this research.
ABBREVIATIONS USED
■
NO, nitric oxide; PGI2, prostacyclin; Nat-PYR, (2R)-2-
(((trans)-4-isopropylcyclohexanecarboxamido)-3-
phenylpropanoyl)oxy)methoxy)-1-(pyrrolidin-1-yl)diazene
oxide; Nat-DEA, (3R)-3-benzyl-10-ethyl-1-((trans)-4-isopropyl-
cyclohexyl)-1,4-dioxo-5,7-dioxa-2,8,9,10-tetraazadodec-8-en-9-
oxide; Nat-NOE, (2R)-2-nitrooxyethyl 2-((trans)-4-isopropyl-
cyclohexanecarboxamido)-3-phenylpropanoate; Meg-PYR, 2-
(((4-(2-(5-chloro-2-methoxybenzamido)ethyl)benzoyl)oxy)-
methoxy)-1-(pyrrolidin-1-yl)diazene oxide; Meg-DEA, 1-(((4-
(2-(5-chloro-2-methoxybenzamido)ethyl)benzoyl)oxy)-
methoxy)-3,3-diethyltriaz-1-ene 2-oxide; Meg-NOE, 2-nitro-
oxyethyl 4-(2-(5-chloro-2-methoxybenzamido)ethyl)benzoate;
BGL, blood glucose level; BGLred, reduction in blood glucose
level; BPsys, systolic blood pressure; BPdia, diastolic blood
pressure; BPmean, mean blood pressure; HR, heart rate
In Vivo Blood Pressure Measurement. Noninvasive blood pressure
and heart rate measurements were performed according to a protocol
approved by the Health Sciences Animal Welfare Committee at the
University of Alberta (Edmonton, Canada). Following oral admin-
istration of each test compound (63.01 μmol/kg po dose) dissolved in
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