Synthesis, characterization and anti-diabetic therapeutic potential of novel aminophenol-derivatized nitrilotriacetic acid vanadyl complexes

Article abstract of DOI:10.1016/j.jinorgbio.2015.07.012

In the present work, we synthesized three novel aminophenol-derivatized nitrilotriacetic acid vanadyl complexes (VOohpada, VOmhpada, VOphpada) using the strategy of rational incorporation of antioxidant groups in ligand in order to balance the side effects with the therapeutic properties. The complexes were characterized by IR, UV-VIS, ESI-MS and elemental analysis. The biological evaluations in vitro revealed that the position of the hydroxyl group of aminophenol moiety regulated the antioxidant activity of the complexes as well as the cytotoxicity on HK-2 cells. The vanadyl complex of p-hydroxyl aminophenol derivative (VOphpada) exhibited better antioxidant activity and lower cytotoxicity than other analogs. In type II diabetic db/db mice, VOphpada (0.1 mmol/kg/day) effectively reduced blood glucose level, improved glucose tolerance, and alleviated stresses induced by hyperglycemia and hyperlipidemia. VOphpada treatment significantly increased expression of PPARα and γ, activated Akt, and inactivated JNK in muscle and adipose tissues. The insulin enhancement effects of VOphpada were observed more potent than BMOV. Moreover, VOphpada decreased the level of kidney injury molecule-1 marker (KIM-1), suggesting a potentially lower renal toxicity. In overall, the present results suggest VOphpada as a novel hypoglycemic agent with improved efficacy-over-toxicity index.

Full text of DOI:10.1016/j.jinorgbio.2015.07.012

Synthesis, characterization and anti-diabetic therapeutic potential of novel
aminophenol-derivatized nitrilotriacetic acid vanadyl complexes
Na Wang, Ziwei Wang, Xia Niu, Xiaoda Yang
PII:
DOI:
Reference:
S0162-0134(15)30040-4
doi: 10.1016/j.jinorgbio.2015.07.012
JIB 9765
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
28 March 2015
3 July 2015
15 July 2015
Please cite this article as: Na Wang, Ziwei Wang, Xia Niu, Xiaoda Yang, Synthesis,
characterization and anti-diabetic therapeutic potential of novel aminophenol-derivatized
nitrilotriacetic acid vanadyl complexes, Journal of Inorganic Biochemistry (2015), doi:
10.1016/j.jinorgbio.2015.07.012
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ACCEPTED MANUSCRIPT
Synthesis, characterization and anti-diabetic therapeutic potential of novel
aminophenol-derivatized nitrilotriacetic acid vanadyl complexes
Na Wang, Ziwei Wang, Xia Niu, and Xiaoda Yang*
State Key Laboratories of Natural and Biomimetic Drugs and Department of Chemical
Biology, School of Pharmaceutical Sciences, Peking University Health Science Center,
Beijing 100191, P. R. China
*
Corresponding author: Xiaoda Yang
Fax: +86-10-82805611, E-mail: xyang@bjmu.edu.cn.
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ABSTRACT
In the present work, we synthesised three novel aminophenol-derivatized nitrilotriacetic
acid vanadyl complexes (VOohpada,VOmhpada,VOphpada) using the strategy of rational
incorporation of antioxidant groups in ligand in order to balance the side effects with the
therapeutic properties. The complexes were characterized by IR, UV-VIS, ESI-MS and
elemental analysis. The biological evaluations in vitro revealed that the position of the
hydroxyl group of aminophenol moiety regulated the antioxidant activity of the complexes as
well as the cytotoxicity on HK-2 cells. The vanadyl complex of p-hydroxyl aminophenol
derivative (VOphpada) exhibited better antioxidant activity and lower cytotoxicity than other
analogs. In type II diabetic db/db mice, VOphpada (0.1 mmol/kg/day) effectively reduced
blood glucose level, improved glucose tolerance, and alleviated stresses induced by
hyperglycemia and hyperlipidemia. VOphpada treatment significantly increased expression of
PPARα and γ, activated Akt, and inactivated JNK in muscle and adipose tissues. The insulin
enhancement effects of VOphpada were observed more potent than BMOV. Moreover,
VOphpada decreased the level of kidney injury molecule-1 marker (KIM-1), suggesting a
potentially lower renal toxicity. In overall, the present results suggest VOphpada as a novel
hypoglycemic agent with improved efficacy-over-toxicity index.
Key words: vanadium; diabetes; aminophenol; PPARα; PPARγ
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1. Introduction
Vanadium compounds are promising anti-diabetic agents [1, 2]. In previous efforts to
improve pharmacokinetic properties and eliminate gastrointestinal stress, a great deal of
vanadium complexes with organic ligands have been synthesized and evaluated for their
insulin mimetic activity, e.g. bis(maltolato)oxovanadium (BMOV) and its ethyl analog BEOV
by Orvig et al. [1, 3], bis(allixinato)-oxovanadium(IV) and bis(picolinato)oxovanadium(IV)
by Sakurai et al. [4-7] and vanadium (III, IV, V)-dipicolinate by Crans et al. [8-10]. Since the
failure of BEOV in phase II clinical trial due to renal side effect [11], a great challenge has
emerged as how to modulate vanadium metal toxicity to develop novel vanadium complexes
with good balance between the therapeutic properties and the side effects [2, 12].
Although the mechanism of the renal toxicity of BMOV/BEOV has not been elucidated,
many studies have suggested that vanadium-induced oxidative stress due to interaction with
mitochondria could be one major mechanism for vanadium toxicity [13, 14]. In addition,
vanadium-induced inflammatory responses could also be involved [15], amongst which
induction of cyclooxygenase-1,2 (COX-1,2) has been proposed as the main source of
pro-inflammatory factors upon vanadium treatment [16, 17].
Natural and synthetic antioxidants were shown to effectively decrease vanadium toxicity
[18-20]. Therefore, incorporation of antioxidant groups in the structure of vanadium
complexes could be a feasible strategy in designing novel anti-diabetic vanadium complexes
with lower toxicity. Flavonoids are excellent natural occurring antioxidants. A series of
vanadyl flavonoid complexes [21-30] have been synthesized. These complexes were shown to
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be non-toxic and reverse metabolic changes in diabetic mice [22, 23]. Using a different
strategy, we prepared a salicylic acid-derivatized kojic acid vanadyl complexes (BSOV) [31,
32]. BSOV exhibited a similar effect in reducing the blood glucose level and a lower acute
toxicity associated with the higher antioxidant activity [31] .
In the present paper, we report the synthesis and evaluation of novel aminophenol-
derivatized nitrilotriacetic acid vanadyl complexes. In design of the ligand, nitrilotriacetate
was chosen as the metal binding moiety in order to reduce molecular size and improve the
solubility of the complexes. The moiety of acetylaminophenol, a specific inhibitor of COX-1,
2, and 3 [33] , was incorporated in the ligand in order to provide antioxidative and
anti-inflammatory activity. The experimental results showed that the vanadyl complex of
p-aminophenol derivative showed improved therapeutic potency over BMOV.
2. Experimental section
2.1. Materials and Instrumentation
Vanadyl sulfate (VOSO4•xH2O, x=3 to 5) was purchased from Sigma-Aldrich (St. Louis,
MO, USA), Vanadyl acetylacetonate (VO(acac)2) was from Fluka (Steinheim, Germany).
Antibodies for PPARγ, AKT, phosphorylated JNK (p-JNK) and GAPDH were from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for PPARα were from Cell Signaling
Technology (Cell Signaling Technology, Inc. Beverly, MA, US). BSA (fraction V) and
3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were from
Sigma-Aldrich (Milwaukee, WI, USA). L-Ascorbic acid was from Beijing Chemical Reagent
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Company (Beijing, China). All other chemicals and solvents were reagent grade and used
without further purification unless otherwise specified. Bis(maltolato)oxovanadium (BMOV)
was prepared and purified according to the published method [34].
The synthesized ligands and their complexes were characterized by infrared (IR)
spectroscopy, UV-VIS spectroscopy, mass spectrometry, elemental analysis, and NMR
1
spectroscopy. Where appropriate, H NMR was used for further characterization. Infrared
spectra were recorded as KBr disks in the range 4000–400cm−1 on a Nexus-470 FTIR
spectrophotometer (Nicolet Instruments, USA). UV-VIS spectra were performed on a
Techcomp 3100 spectrophotometer (Techcomp Ld., China). Mass spectra (Negative ion mode)
were obtained with a QSTAR quadrupole time-of-flight (TOF) hybrid mass spectrometer
(Applied Biosystems, USA).
2.2. Synthesis
The synthesis route is illustrated in Scheme 1:
[Scheme 1]
Nitrilotriacetic anhydride (2). Compound 2 was prepared according to a previously
reported method [35].
N-(hydroxyphenylcarbamoylmethyl) iminodiacetic acid (H2hpada)(3a,b,c). Under
the protection of nitrogen, compound 2 (20 mmol) and (p,o,m-)aminophenol (20 mmol) were
dissolved into 50 mL of N,N-dimethylformamide (DMF). After stirring for 10 min, 6 mL of
pyridine was added. The mixture was heated with stirring for 24 h in an oil bath at 70 °C.
Then the solvent was evaporated under reduced pressure. The residue was dissolved in 100
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mL water, and the solution was adjusted to pH 9.18 with 2 M sodium hydroxide solution and
extracted with ethyl acetate. The water phase was then adjusted to pH 2.85 with 6 M
hydrochloride. The crude product was collected by filtration and further purified by
recrystallization in DMF.
3a (p-H2hpaida):White; Yield 60.75%; IR (cm-1): 3309 cm-1(V(O-H)); 1580 cm-1,1399 cm-1 ,
808 cm-1 (ring VC=C);1661 cm-1 (Vas(COO));1355 cm-1 (Vs(COO)); 1H NMR (δ):10.02
(s,1H);7.39-7.41 (d,J = 7.2 Hz,2H); 6.70-6.72 (d,J = 7.2Hz,2H); 3.53 (s,4H); 3.44 (s,2H);
ESI-MS:m/z 281 [M-1]¯, MW calc. (C12H14O6N2) 282.25.
3b (m-H2hpada): light yellow; Yield 61.49%; IR (cm-1):3433 cm-1 (V(O-H） ); 1600
1
cm-1 ,1489 cm-1 (ring VC=C); 1637 cm-1 (Vas(COO)); 1417 cm-1 (Vs(COO)); H NMR (δ): 10.13
(s,1H); 7.23 (s,1H); 7.08 (t, J = 8.0Hz,1H); 6.95 (d, J =8.0Hz,1H); 6.47 (d,J = 8.5Hz,1H); 3.54
(s,4H); 3.45 (s,2H); ESI-MS: m/z 281 [M-1]¯, MW calc. (C12H14O6N2) 282.25.
3c (o-H2hpada): White; Yield 64.72%; IR (cm-1): 3346 cm-1 (V(O-H));1617 cm-1,1502 cm-1,
814 cm-1,747 cm-1 ( ring VC=C); 1659 cm-1 (Vas(COO)); 1468 cm-1 (Vs(COO)); 1H NMR (δ):10.15
(s,1H);7.24 (t, J = 2.0Hz,1H); 7.09 (t, J = 8.0Hz,1H); 6.95 (d, J= 8.0,2.0 Hz,1H); 6.48 (d, J =
8.0,2.0 Hz,1H); 3.46 (s, 4H); 3.54 (s, 2H); ESI-MS: m/z 281 [M-1]¯, MW calc. (C12H14O6N2)
282.25.
Vanadyl N-(hydroxyphenylcarbamoylmethyl) iminodiacetate (VOhpada) (4a,b,c).
Compound 3a,b,c (9.52 mmol) was dissolved in 25 mL deionized waters. Then freshly
prepared vanadyl (IV) hydroxide (2.02 g) was added and the solution was left with stirring for
2 h at room temperature. The unreacted vanadyl (IV) hydroxide was removed by filtration.
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Finally, the product was obtained by recrystallization.
4a (VOphpada): Dark blue-gray; Yield 63.99%; IR (cm-1): 3342 cm-1 (V(O-H));1635 cm-1,
1399 cm-1, 808 cm-1 (ring VC=C); 1635 cm-1 (Vas(COO)); 1370 cm-1(Vs(COO)); 974 cm-1(VV=O );
UV-VIS (H2O,pH 5) (λ(nm) (ε (M-1∙cm-1))): 800(94), 597(55); ESI-MS (negative ion mode):
m/z 364 [M-1]¯; Elemental Analysis (%): C 39.58;N 7.6; Anal. Calc. VO(H2O)L:C 39.58; N
7.69.
4b (VOmhpada): Dark blue-gray; Yield 72.61%; IR (cm-1): 3434 cm-1 (V(O-H)); 1617 cm-1,
1480 cm-1 (ring VC=C); 1632 cm-1 (Vas(COO)); 1417 cm-1 (Vs(COO)); 974 cm-1(VV=O); UV-VIS
(H2O,pH 5) (λ(nm) (ε (M-1∙cm-1))): 846(102), 625(56); ESI-MS (negative ion mode): m/z 364
[M-1]¯; Elemental Analysis (%): C 39.58;N 7.6; Anal. Calc. VO(H2O)L: C 39.5;N 7.78.
4c (VOohpada): Dark blue-gray; Yield 76.76%; IR (cm-1): 3434 cm-1 (V(O-H)); 1600 cm-1,
1480 cm-1,779 cm-1 (ring VC=C); 1632 cm-1 (Vas(COO)); 1453 cm-1 (Vs(COO)); 971(VV=O);
UV-VIS (H2O,pH 5) (λ(nm) (ε (M-1∙cm-1))): 826(107), 597(55); ESI-MS (negative ion mode):
m/z 364 [M-1]¯; Elemental Analysis (%): C 39.25; N 7.93, Anal. Calc. VO(H2O)L: C 39.5;
N 7.78.
2.3. In vitro tests of antioxidant activity
The antioxidant activity of vanadium complexes and free ligand was tested by hydroxyl
radical scavenging capacity (HRSC) as previously described [36]. Briefly, antioxidants at
various concentrations were added to the Fenton reaction media containing 0.01 mM
Rhodamine B, 1.0 mM FeSO4, 5.0 mM of cetyl trimethyl ammonium bromide (CTAB), and
20 mM acetic acid (pH 2.8). After addition of 2.0 mM (final concentration) of H2O2, the
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reactions were left for 10 min at room temperature. Then absorbance As at 550 nm was
measured. The antioxidant recovery capacity R was calculated as:
Where A0 is the control (cantioxidant = 0) and Ab is the reagent blank. Then the data was further
fitted to the following equation:
1
1
K
1


R cantixidant k Rmax Rmax
Then Rmax and k/K were obtained using an OriginTM 7.2 program. HRSC index was calculated
by setting the k/K value of ascorbic acid as 1.0.
2.4. Cytotoxicity on HK-2 human kidney cells
The cytotoxicity of vanadium complexes on HK-2 cells were conducted using the MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [37]. Briefly,4~5×103
cells were plated onto 96-well plates (Corning, USA). Upon confluence, the cells were
incubation with different concentrations of vanadium complexes for 24 h at 37 °C. Then,
MTT reagent was added to the cells according to the manufacturer protocol and the
absorbance at 570 nm was measured with a microplate reader (Multiskan Mk3; Thermo
Labsystems, Vantaa, Finland).
2.5. Animal treatments
Male C57BLKS/J diabetic obese (db/db) mice (8 weeks) were from Model Animal
Research Center of Nanjing University and housed under controlled temperature (23 °C) and
lighting (12 h lighting between 6 A.M. and 6 P.M.) with free access to water and standard
mouse diet. All care and handling of animals were performed with the approval of
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Institutional Review Board for Laboratory Animal Care (Approval No. LA2010-052).
When the fasting blood glucose levels of mice reached ≥12 mM, a total of 28 mice
randomly divided into four groups: Group I, Control diabetic mice (n=7); Group II,
BMOV-treated diabetic mice (n=7, 0.1 mmol/kg/day); Group III, Compound 4a (VOphpada)
treated diabetic mice (n=7, 0.1 mmol/kg/day); Group IV, Ligand (Compound 3a,
p-H2hpada)-treated mice (n=7, 0.1 mmol/kg/day). The BMOV-treated group was considered
as the positive control group.
To avoid any irritation by oral gavage, vanadium treatments for db/db mice were
conducted by feeding the animal with pellet food containing desired amounts of vanadyl
complexes as described previously [38]. Throughout the experimental period, the body weight,
food and water intake were monitored. Blood samples were obtained from the tail vein of the
mice and blood glucose levels were determined with an Accu-Chek blood glucose monitor
(Roche Diagnostics GmbH, Mannheim, Germany). At the end of the study, the animals were
euthanized and tissue samples of muscle, fat, livers and kidney were taken off for further
analysis.
2.6. Glucose tolerance tests (OGTT)
At the end of experiment, OGTT was performed. Briefly, the mice were fasted for 12 h,
and glucose (200 mg*mL-1) was delivered by oral gavage at a dose of 2 g*kg-1. Blood samples
were available from the tail vein at 0, 5, 15, 30, 60, 120 and 180 min after glucose loading.
The blood glucose levels were monitored with an Accu-Chek blood glucose monitor (Roche
Diagnostics GmbH, Mannheim, Germany).
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2.7. Serum biochemical parameters test
The mice were fasted for 12 h and serum was prepared from the orbit blood sampling for
determination of biochemical parameters in serum including total cholesterol (TCHO),
triglycerides (TG), alanine aminotransferase (ALT), aspartate transaminase (AST), creatinine
(Cre), blood urea nitrogen (BUN), and albumin (Alb). Since the volume of blood sample from
single mouse was not enough for sample loading, an equivalent volume mixture of all seven
samples in the same treated group were applied to an OLYMPUS AU400 clinical chemistry
analyzer (Olympus, Tokyo, Japan) with the assay kits from Randox (Antrim, UK).
2.8. PCR assay for PPARs expression
Total RNA was separated from mice muscle and fat with a TRIzol extraction kit
(Invitrogen). After quantification of extracted RNA pellets with an BioPhotometer 6131
(Eppendorf, Germany), first-strand complementary DNA synthesis was performed using a
Superscript First Strand Synthesis Kit (Invitrogen). All samples were analyzed with a
quantitative real-time PCR (Eppendorf, Germany) using SYBR Green contained PCR Master
Mix (Applied Biosystem, Inc.USA) according to the manufacturer protocol. The mRNA
expression levels of all genes were normalized to that of GAPDH in the same sample, and
then the relative differences between the diabetic control group and the treatment groups were
calculated and expressed as relative increases, with the control value set to 100%.
The primer sequences are listed as follow:
Primer sequences
PPARα
Forward primer 5'-TCACACAATGCAATCCGTTT-3'
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Reverse primer 5'-GGCCTTGACCTTGTTCATGT-3'
Forward primer 5' -GTGAAGTTCAACGCACTGGA-3’
Reverse primer 5'-ATGTCCTCAATGGGCTTCAC-3'
Forward primer 5'-GACTTCAACAGCAACTCCCAC-3'
Reverse primer 5'-TCCACCACCCTGTTGCTGTA-3’
PPARγ
GAPDH
2.9. Western blotting analysis
The frozen tissues were homogenized in a lysis buffer (20 mM Tris-HCl, 150 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% TritonX-100 and protease inhibitor, pH 7.4) and then
centrifuged (12,000 rpm) at 4°C for 25 min to discard tissues debris. The supernatants were
collected and protein concentrations were measured using a BCA Protein Assay kit (Pierce,
Rockford, IL, US). Samples containing 40 μg total proteins were applied to a 10%
SDS-PAGE and then subjected to desire antibodies after transfer to a polyvinylidene
difluoride (PVDF) membrane (Millipore Corp, Atlanta, GA, US). The membrane was blocked
for 1 h with Tris-buffered saline containing 0.1% Tween 20 and 5% bovine serum albumin
and then incubated with desired primary antibodies overnight at 4°C followed by a
horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) at room
temperature for 1 h. The protein bands were developed with a ChemiDoc XRS kit (Bio-Rad,
USA) on an X-ray film (Kodak, Fujian, China) according to the specification of manufacturer.
Protein expression results were analyzed and quantified using a Scion Image Software (Scion,
Frederick, MD).
2.10. Acute oral toxicity of VOphpada in mice
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The acute oral toxicity study was conducted according to the method of Ref. [39]. Briefly,
healthy ICR mice were divided randomly into six groups (n=10). After 12 h fasting, each
group of mice were given were administered as suspensions in double distilled water (ddH2O)
intragastrically at doses of 1735, 1400, 1120, 896, 716.8, 573 mg*kg-1 body weight according
to the dosage ratio of 1:0.8 among groups, with ddH2O as control. After vanadium
administration, the mice were fed normally and observed successively for mortality for 14
days and the LD50 value were calculated.
2.11. Statistical analysis
All data are presented as means±SD. Each experiment was performed at least in triplicate.
The statistical analysis was performed by one-way analysis of variance (ANOVA) followed
by Dunnett's multiple comparison tests using a SPSSTM 13.0 software and an OriginTM 7.2
program. Statistical significance was accepted at the level of p<0.05.
3. Results
3.1. Synthesis
The vanadium complexes (4a~c) were prepared from ligands (3a~c) as described above.
The three complexes are well water soluble. The IR data revealed a peak at ~974 cm-1, which
is in the region (930-1030 cm-1) of characteristic VIV=O stretching frequencies [31, 40, 41],
confirm the formation of vanadyl complexes. The UV-VIS spectra (Fig.1) of three
VIVO-hpada complexes are similar to those of [VO(H2O)nta]¯ [42-44] and its analogs[45] and
are consistent with the tetragonally compressed symmetry commonly associated with spectra
of the vanadyl ion and its complexes [45]. The results of elemental analysis agreed with a
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formula of VIVO(H2O)L for the complexes. In ESI mass spectra, three related peaks were
observed at m/z 346, 363, 364. The peaks at m/z 346 and 363 correspond to the formula of
VIVOL and VVO(OH)L, respectively. While the peak at m/z 364, shown as a shoulder of peak
m/z 364, is the [M-1]¯ molecular ion peak for VIVO(H2O)L. It is possible that in the process of
sampling and/or mass analysis, most complexes may either loose a water molecule or be
oxidized to +5 valence complexes. According to the crystal and solution structures of a series
of mononuclear vanadium (IV) complexes with nitrilotriacetate and iminodiacetate
derivatives [45] , the formula of vanadyl complexes were assigned to be VIVO(H2O)L.
[Fig.1]
3.2. Determination of pKa1(OH) value
The equilibrium constant for dissociation of coordination water (1) is a very important
molecular property for susceptibility and rate of oxidation to VV [42, 46]:
pKa1(OH)

-
+

VO(H O)L
VO(OH)L +H
2
(1)
Thus, the pKa1(OH) values of the three VIVO-hpada complexes 4a~c were determined by
pH-dependent spectrophotometric measurements as described in Materials and Methods. The
pH-dependent spectra and titration curve for VIVO-hpada complexes are shown in Fig. 2. The
pKa1(OH) values were calculated to be 7.4±0.2, 6.5±0.1 and 7.6±0.4. for VOphpada ,
VOmhpada and VOohpada , respectively. These values for VIVO-hpada complexes are close
to that of [VIVO(H2O)nta]¯ (pKa1(OH) =6.9) [46].
[Fig. 2.]
3.3. In vitro antioxidant activity
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The antioxidant activity of vanadium complexes 4a~c and the ligands was estimated as
hydroxyl radical scavenging capacity (HRSC) (Table 1). Compound 4a (VOphpada) gave the
best HRSC index among the complexes and ligands. In addition, VOphpada exhibited
significantly better antioxidant activity than its ligand.
[Table 1]
3.4. Cytotoxicity on kidney cells
The in vitro cytotoxicity of vanadyl complexes 4a~c on HK-2 cells was tested and the
IC50 were calculated in Table 2. The complex 4a (VOphpada) showed significantly lower
toxicity than the other analogs, its parental compound (VOimda) and the reference compound
BMOV.
[Table 2]
3.5. Median lethal dose of VOphpada
The results of acute oral toxicity were listed in Table 3. The oral LD50 of VOphpada was
calculated to be 1045 mg*kg-1. According to Loomis and Hayes classification [47] , chemical
substance with a LD50 within the range of 5,000–15,000 mg*kg-1, 500–5,000 mg*kg-1, 50–500
mg*kg-1, and 1–50 mg*kg-1 is regarded as practically nontoxic, slightly toxic, moderately toxic,
and highly toxic, respectively. Compound 4a (VOphpada) is in the category of slightly toxic
substance.
[Table 3]
3.6. The effects of compound
(VOphpada ) on animal growth of db/db mice
4a
The daily food and water intake of animals were shown in Fig. 3. At the treatment dose,
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both BMOV and VOphpada did not decrease animal’appetite while BMOV and phpada
ligand slightly increased food intake. While the daily water intake for vanadium treated mice
was significantly lower than that of diabetic control, indicating the improvement of glucose
metabolism of the animals. In agreement with the food and water consumption, the body
weight of the treated groups steadily increased (Fig.4) and the treated mice were significantly
heavier than the diabetic control.
[Fig. 3]
[Fig.4]
3.7. Blood glucose level and Glucose tolerance test
Compared with the diabetic control, the blood glucose of db/db mice treated with
vanadium complexes (0.1 mmol/kg/day) decreased gradually to normal range (<12 mM)
(Fig.5). The glucose tolerance was also improved with significance (Fig.6) after 6-weeks
vanadium treatment. Obviously, compound 4a (VOphpada) produced much stable
hypoglycemic effect than BMOV in db/db mice. Interestingly, the ligand 3a (H2hpada) alone
did not decrease glucose level, however, somewhat improved glucose tolerance of the diabetic
animals (Fig.6).
[Fig.5]
[Fig.6]
3.8. Serum biochemical parameters
As shown in Table 4, treatment with vanadium complexes greatly reduced the levels of
AST, ALP, BUN, and Cre, indicating that vanadium treatment alleviated the injuries in liver,
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heart, and kidney caused by hyperglycemia. The effects of compound 4a (VOphpada) is
essentially similar to those of BMOV.
[Table 4]
3.9. Effects of vanadium compounds on PPARs transcription in muscle and fat
As shown in Fig.7, treatment with vanadium compounds increased mRNA levels of
PPARα in muscle and both PPARα and PPARγ in fat. The compound 4a (VOphpada)
exhibited much stronger enhancement than BMOV. While the ligand 3a alone caused
decrease of mRNA levels of PPARα and/or PPARγ.
[Fig.7]
3.10. Effects of vanadium treatment on AKT, PPARα, PPARγ, and pJNK, in muscle and fat
tissues
As shown in Fig.8, vanadium treatment caused elevation of Akt/PKB, PPARα and
PPARγ while decrease phosphorylation activation of JNK (pJNK) in both muscle and fat
tissues. Interestingly, vanadium complexes activated more PPARα in muscle but more PPARγ
in fat. In overall, the effects of compound 4a were significantly stronger than BMOV. In
contrast, the ligand 3a alone did not significantly affect Akt/PKB, PPARα or PPARγ.
[Fig.8]
3.11. The effect of vanadium complexes on kidney injury molecule-1 (TIM-1/KIM-1)
The renal toxicity of vanadium complexes was investigated by measuring the level of
TIM-1/KIM-1 marker. As shown in Fig.9, neither vanadium complexes nor the ligand 3a
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caused increase of TIM-1/KIM-1 expression. However, VOphpada slightly reduced
TIM-1/KIM-1 level with significance.
[Fig.9]
4. Discussion
The vanadyl complexes with derivatives of nitrilotriacetic acid or iminodiacetic acid
have been thoroughly investigated as models of inactive form of vanadium haloperoxidases.
Considering the structural homology of vanadyl nitrilotriacetate to vanadyl dipicolinate
complexes, we proposed that vanadyl complexes with nitrilotriacetic acid derivatives could
exhibit good hypoglycemic effect. In addition, vanadyl nitrilotriacetate exhibits high water
solubility and small molecular size. Therefore, we designed and synthesized the novel
aminophenol-derivatized nitrilotriacetic acid vanadyl complexes. Spectroscopic
characterization gave the formula of VIVO(H2O)L as expected. Nevertheless, fully
characterization of the solution chemistry of the complexes (e.g. pH-metric chemical
speciation, redox potential and plasma protein binding, etc.) would be appropriate for further
works. It has been suggested that the solution stability, the ability to interact with cellular
redox reactions, and the capacity to remain intact upon binding to serum albumin may be
crucial for the insulin-enhancing activity of Vdipic complexes [48] .
The toxicity of the complexes was firstly evaluated in vitro on HK-2 kidney cells (Table
2) along with their antioxidant capacity (Table 1). It is noted that: (i) the cytotoxicity of
VOphpada (4a) is lower than BMOV; (ii) similar to the acute toxicity of BSOV analogs [31],
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the toxicity of three aminophenol-derivatized nitrilotriacetic acid vanadyl complexes
(4b>4c>4a) again show an inversed relationship with their antioxidant activities (4b<4c<4a);
(iii) the p-aminophenol derivative VOphpada showed an increased antioxidant activity over
its ligand p-H2phpada while its cytotoxicity was significantly lower than the parental complex
(VOimda). In contrast, complexes 4b and 4c showed decreased antioxidant activities over
their ligands and their cytotoxicity was significantly higher than the reference. This suggests
that the position of the hydroxyl group could exert significant impact on the redox behaviors
of the complexes. It is also possible that vanadyl complexes with antioxidant activity higher
than their ligands normally give lower cytotoxicity and vice versa. Nevertheless, the exact
structural effects and underlying mechanisms need to be investigated in further works.
Based on the above evaluations of complex susceptibility, antioxidant activity and renal
cytotoxicity, VOphpada was chosen to test its anti-diabetic therapeutic potential and animal
toxicity. Herein, the vanadium complexes was administrated in pellet food to avoid any
gastrointestinal irritation under a dose (0.1 mmol/kg/day) without any feeding suppression.
Therefore, mice in treated groups consumed equal amount of more food than the diabetic
control (Fig.3). Both BMOV and VOphpada significantly reduced blood glucose level (Fig.5)
and thus water consumption (Fig.3B). However, VOphpada was obviously more effective
than BMOV in improving glucose tolerance (Fig.6). Interestingly, the ligand (p-H2hpada)
alone also showed a weak capacity of ameliorating blood glucose status (Fig.5&6), possibly
due to antioxidant activity like that of resveratrol [49] . Consequently, the animals in all three
treated groups generated more body growth than the diabetic control.
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The levels of serum biomarkers in Table 4 revealed the changes of health status during
the treatment. Although db/db mice exhibited hyperglycemia and hyperlipidemia (Chol and
TG), the mice maintained fairly normal liver (ALT, AST, and ALP) and kidney (Cre)
functions compared with the healthy C57BL/6 mice. However, the elevated BUN level and
reduced serum albumin (Alb) indicated significant high level of protein degradation, a typical
sign of stress caused by hyperglycemia and/or hyperlipidemia. Treatment with BMOV or
VOphpada ameliorated blood glucose (Fig.5&6) and lipid levels, and reduced BUN level
(especially the BUN/Cre ratio) to normal ranges indicating the effective relief of
hyperglycemic stress upon vanadium treatment. But vanadium treatment did not improve
much on Alb level. Previously, we observed that vanadyl complexes reduced insulin synthesis
on β-cells upon high glucose stimulation through induction of unfolded protein responses [39].
It is possible that vanadium treatment may restrict general protein synthesis via a similar
mechanism. Considering the role of albumin in maintaining the serum colloid osmotic
pressure, long term low serum albumin could alter renal function. However, it was also
observed that Alb levels in groups with increased food consumption (BMOV and p-H2hpada
groups) were notably improved, suggesting that dietary modification (e.g. addition of leucine
or its metabolite β-hydroxy-β-methylbutyrate) to promote protein synthesis may be a potential
solution of this side effects of vanadium treatment for the future studies.
PCR and western blot analysis (Fig.7&8) revealed that both BMOV and VOphpada
exhibited hypoglycemic effect through insulin enhancing mechanism: (i) both vanadyl
complexes caused significant elevation of Akt, indicating activation of insulin signal
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transduction; (ii) both treatment increased expression of PPARα and γ, primarily in muscle
and adipose tissue, respectively. Different from in vitro s tudies [50, 51] that vanadium
compounds elevated PPARγ expression exclusively by suppressing protein degradation,
vanadyl complexes here in animals acted at both transcriptional and post-translational levels.
PPARα and γ are well recognized targets for insulin enhancers [52], e.g. rosiglitazone.
Increase of PPARα and γ activity can significantly improve lipid and glucose metabolism
[52] ; (iii) as the consequence of PPARγ activation [52] , vanadium treatment significantly
reduced phosphorylation of JNK (c-Jun NH2-terminal protein Kinase). JNK belongs to the
stress-activated protein kinase family [38, 53]. Phosphorylation of IRS1 (insulin receptor
substrate-1) by JNK will cause down-regulation of insulin signaling; this has been regarded as
one major mechanism for insulin resistance [53]. Thus, inactivation of JNK by vanadium
treatment should play an important role in the insulin enhancement action of vanadyl
complexes. Again, VOphpada exhibited more potent effects than BMOV.
Toxicity, especially renal side effect, is currently the most concern for anti-diabetic
vanadyl complexes. As in Table 3, VOphpada is a slightly toxic substance, exhibiting a
similar cute oral toxicity (LD50 = 1045 mg*kg-1) to that of BMOV (LD50 = 902 mg*kg-1) [31].
The six-week treatment with BMOV and VOphpada (Fig.9) did not cause significant
elevation of the kidney injury molecule-1 (TIM-1/KIM-1); while VOphpada even slightly
reduced TIM-1/KIM-1 level, suggesting potentially lower long term toxicity of VOphpada.
However, considering the potential of vanadium-induced inflammatory responses (not
investigated herein) and possible consequences of low serum albumin (if last ever), the long
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term renal toxicity of VOphpada needs to be carefully investigated in future works.
5. Conclusion
In the present work, we reported design and synthesis of novel aminophenol-derivatized
nitrilotriacetic acid vanadyl complexes. The position of the hydroxyl group was observed
significantly influence the antioxidant activity and cytotoxicity of the vanadyl complexes;
while VOphpada exhibited a high antioxidant activity and fairly low cytotoxicity. In a type II
diabetic model, VOphpada exhibited significantly more potent insulin enhancement effects
than BMOV while showing similar short term toxicity. Further works on long term renal
toxicity would be appropriate for the development of VOphpada as novel hypoglycemic agent.
Nonetheless, the present results suggest that proper incorporation of antioxidant groups in
ligand could lead to vanadyl complexes with improved balance between the therapeutic
properties and the side effects.
Abbreviations
BMOV
BEOV
BSOV
bis(maltolato)oxovanadium
bis(ethylmaltolato)oxovanadium (IV)
bis((5-hydroxy-4-oxo-4H-pyran-2-yl)methyl2-hydroxybenzoatato)ox
ovanadium(IV)
H2hpada
N-(hydroxyphenylcarbamoylmethyl) iminodiacetic acid
Vanadyl N-(hydroxyphenylcarbamoylmethyl) iminodiacetate
VOhpada
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TG
triglycerides
ALT
AST
Cre
alanine aminotransferase
aspartate transaminase
creatinine
BUN
Alb
blood urea nitrogen
albumin
TCHO
OGTT
total cholesterol
glucose tolerance test.
PPAR-γ
PPAR-a
AKT(PKB)
pJNK
peroxisome proliferator-activated receptor-γ
peroxisome proliferator-activated receptor-a
Protein kinase B
PhosphoJun N-terminal kinase
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
Acknowledgements
Project (No. 21271012 ) supported by NNSFC.
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Table 1. Hydroxyl radical scavenging capacity (HRSC) of vanadium complexes and the ligands.
Compounds
Rmax
k/K
r2
HRSC index
1
L-ascorbic acid
2.30
0.404
0.9904
ligand
3a
0.466
0.912
30.7
36.8
0.9753
0.9037
76.0
91.2
Complex
4a
(VOphpada)
ligand
3b
Complex
0.678
1.75
15.8
2.45
13.9
2.71
0.9475
0.9357
0.9700
0.9399
39.1
6.08
34.5
6.71
4b
ligand
3c
0.670
1.60
Complex
4c
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Table 2. Half inhibition concentration (IC ) of vanadium complexes
on HK2 cells.
4a~c
50
Compounds
IC50 (μM)
470 ± 47
20 ± 2
(VOphpada)
4a
4b
4c
71 ± 15
BMOV
333 ± 29
136 ± 27
Vanadyl iminodiacetate (VOimda)
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Table 3. Acute toxicity results of (VOphpada) in mice
4a
Mice
Dosage
Rate of death
(%)
Groups
1
Log(dosage)
3.24
Number of death
10
-1
*
number
(mg kg )
10
1,735
100
2
3
4
5
6
10
10
10
10
10
1,400
1,120
896
3.15
3.05
2.95
2.86
2.76
9
7
5
2
0
90
70
50
20
0
717
573
29