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A Proline-Based AminophosphinicAcid Ligand and It’s Vanadyl Complex:Synthesis, Characterization and In VitroInhibitory Effects on α-Amylase And α-GlucosidaseBabak Kaboudin a , Khavar Moradi a , Elham Safaei a , HosseinDehghan b & Peyman Salehi ba Department of Chemistry, Institute for Advanced Studies in BasicSciences, Gava Zang, Zanjan, 45137-66731, Iranb Department of Phytochemistry, Medicinal Plants and DrugsResearch Institute, Shahid Beheshti University, G. C, Evin,1983963113, Tehran, IranPublished online: 10 Sep 2012.

To cite this article: Babak Kaboudin , Khavar Moradi , Elham Safaei , Hossein Dehghan & PeymanSalehi (2012): A Proline-Based Aminophosphinic Acid Ligand and It’s Vanadyl Complex: Synthesis,Characterization and In Vitro Inhibitory Effects on α-Amylase And α-Glucosidase, Phosphorus, Sulfur,and Silicon and the Related Elements, 187:12, 1521-1527

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Phosphorus, Sulfur, and Silicon, 187:1521–1527, 2012Copyright C© Taylor & Francis Group, LLCISSN: 1042-6507 print / 1563-5325 onlineDOI: 10.1080/10426507.2012.692133

A PROLINE-BASED AMINOPHOSPHINIC ACID LIGANDAND IT’S VANADYL COMPLEX: SYNTHESIS,CHARACTERIZATION AND IN VITRO INHIBITORYEFFECTS ON α-AMYLASE AND α-GLUCOSIDASE

Babak Kaboudin,1 Khavar Moradi,1 Elham Safaei,1

Hossein Dehghan,2 and Peyman Salehi21Department of Chemistry, Institute for Advanced Studies in Basic Sciences, GavaZang, Zanjan 45137-66731, Iran2Department of Phytochemistry, Medicinal Plants and Drugs Research Institute,Shahid Beheshti University, G. C, Evin, 1983963113, Tehran, Iran

GRAPHICAL ABSTRACT

N

O

PO H

OV

O

OMe

MeOON

O

OH

PO H

OH

+ VO(acac)2H2O, pH=8-9

reflux, 1h

Abstract A proline-based aminophosphinic acid ligand and it’s vanadium complex have beensynthesized and characterized by spectroscopic techniques. The inhibitory activity on pancre-atic α-amylase and Baker’s yeast α-glucosidase has been examined in vitro. The novel complexshowed more inhibitory potency against pancreatic α-amylase and Baker’s yeast α-glucosidasecompared to acarbose as an antidiabetic drug.

Supplemental materials are available for this article. Go to the publisher’s online edition ofPhosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.

Keywords Vanadyl complex; anti-diabetes; aminophosphinic acid; α-amylase; α-glucosidase

INTRODUCTION

In recent years, the synthesis of vanadyl compounds has attracted significant atten-tion, due to their biological activities with broad application as hyperglycemia and aberrantglucose homeostasis in animal models of insulin-dependent diabetes mellitus (type 1) and

Received 10 April 2012; accepted 6 May 2012.The authors gratefully acknowledge support by the Institute for Advanced Studies in Basic Sciences (IASBS)

Research Council under grant No. G2011IASBS120. The authors thank Dr. Mohammad Rafee, Institute forAdvanced Studies in Basic Sciences (IASBS) for his help in cyclic voltammetry analysis.

Address correspondence to Babak Kaboudin, Department of Chemistry, Institute for Advanced Studies inBasic Sciences, Gava Zang, Zanjan 45137-66731, Iran. E-mail: kaboudin@iasbs.ac.ir

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1522 B. KABOUDIN ET AL.

noninsulin-dependent diabetes mellitus (type 2).1–3 In addition, these compounds haveshown substantial promise as cancer chemoprevention agents.4 Although, simple vanadylsalts such as VOSO4 and NaVO3 have been effective as pharmaceutical agents loweringblood glucose levels in rats and humans, potential metal toxicity have limited the develop-ment of these compounds.5 Recently, numerous studies have shown that organic vanadylcomplexes are less toxic and more effective than vanadium salts in lowering blood glucoselevels.6 A number of vanadium complexes, neutral V(IV) complexes, have been synthe-sized and examined for their insulin mimetic activity. Orvig et al. have reported somevanadium complexes with their potency in lowering blood glucose levels.7–10 One of thesecomplexes, bis(ethylmaltolato)oxovanadium (IV) (BEOV) was in phase II clinical trial,however, a potential side effect, i.e., kidney changes, prevented further applications.

The initial step in the hydrolysis of starch to maltose and finally to absorbable glucosecatalyzed by pancreatic α-amylase as a key enzyme in the digestive system.11 Althougha number of vanadium complexes have been synthesized and examined for antidiabetesactivities, the synthesis and screening of novel inhibitors of carbohydrate digestive enzymesof vanadium complexes still remains as an active ongoing research area.

A phosphinic moiety (-PO2-CH2-) is considered a mimic of the tetrahedral transitionstate of peptide bond hydrolysis and due to this, phosphinates have attracted considerableinterest providing a wide range of potent inactivators of proteolytic enzymes, particularlymetalloproteases.12 1-aminoalkylphosphinic acids, the phosphinic acid analogues of 1-amino carboxylic acids, are an important class of compounds that exhibit a variety ofinteresting and useful properties.13

In contrast to extensive studies on the synthesis of vanadium complex,7–10 to the bestour knowledge, there is no report in the literature for the synthesis of vanadium complexcontaining an aminoalkylphosphinic acid as a ligand. To develop antidiabetic vanadiumagents with desirable efficacy and safety, we have designed and synthesized a prolinederivative of aminoalkylphosphinic acid ligand and it’s vanadyl complex (Scheme 1).

N

O

PO H

OV

O

OMe

MeOO

Scheme 1

RESULTS AND DISCUSSION

The aminophosphinic acid ligand 1 was synthesized via one-step Mannich-type re-action using a modified literature procedure described previously (Scheme 2).14 The freeligand exists in the zwitterionic form. Novel aminophosphinic acid vanadyl complex 2 wassynthesized by a simple reaction of VO(acac)2 and aminophosphinic acid ligand 1. Whena mixture of ligand 1 and VO(acac)2 in a molar ratio of 1:1 in pH = 8–9 (using sodiumhydroxide) was stirred in air for 1 h at reflux, a solid precipitated after cooling the mixtureto room temperature. In our experiment, the M/L ratio of starting materials was 1:1; hence,it was not surprising that the complex had an M/L ratio of 1:1 (Scheme 2).

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PROLINE-BASED AMINOPHOSPHINIC ACID LIGAND 1523

NH

O

OH

L-proline

+ CH2O + H3PO2

N

O

OH

PO H

OH

complex 2,LVO(acac)

N

O

PO H

OV

O

OMe

MeOON

O

OH

PO H

OH

+ VO(acac)2H2O, pH=8-9

reflux, 1h

(excess)

EtOH

r.t. 24 h

ligand 1

ligand 1

Scheme 2

The prepared complex was characterized by UV-visible IR absorption and massspectroscopic techniques.

In the IR spectrum of the complex, the vibration bands of the free ligands wereobserved with some shift, proving the coordination of ligand to the metal.15 The IR (KBrdisk) of complex 2 showed one strong absorption band at 959 cm−1 attributable to thevanadyl group (V = O).

The electronic spectra of LVO(acac) complex recorded in H2O are presented insupplementary material. The band with moderate intensity observed around 931 nm isassigned to a ligand to metal charge transfer. Comparing the electronic spectrum of thiscomplex with one for VO(acac)2 shows that this band shift to lower energy. It can attributeto the electron-withdrawing effect of the ligand on stabilizing the (lowest unoccupiedmolecular orbital) orbitals.

The electron-impact–mass spectrometry spectrum gave a relatively intense ion peakat m/z 359, indicating the formation of 1:1 complex of LVO(acac) between vanadyl andligand. Other ion peaks with lower intensity appeared in a fragmentation pattern that beganwith the loss of acetyl acetonate fragments, then ligand followed by loss of vanadyl. Theoriginal spectrum is shown in the online Supplementary Materials (Figure S8).

The cyclic voltammetric behavior of complex 2 was studied in water at room tem-perature. Figure 1 shows the results of cyclic voltammetry (CV) analysis on VO(acac)2

and the synthesized complex 2 (LVO(acac)). The voltammetric behavior of VO(acac)2 ischaracterized by one oxidation peak in this medium at a glassy carbon (Figure 1). The peakpotential is about 0.7 V (versus Ag|AgCl). No reduction peak appears in the reverse scan inthe applied potential range (0–1). The synthesized complex 2 undergoes oxidation at 0.5 V(versus Ag|AgCl) which occurs at 0.2 V less than VO(acac)2. These results indicate thatthe oxidation of VO(acac)2 and the complex 2 take place differently that may be due toincreasing electron density on the vanadyl atom in the complex 2.

Recently, the synthesis of new chemical compounds as potential pancreatic α-amylaseinhibitors has attracted significant attentions.15 A recent study has demonstrated that organicvanadium compounds are much safer as antidiabetic agents than inorganic vanadium salts.Screening of inhibitors of carbohydrate digestive enzymes has been used for alternativeprevention and treatment of type 2 diabetes mellitus. Pancreatic α-amylase is an enzyme

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1524 B. KABOUDIN ET AL.

Figure 1 Cyclic voltammogram of 0.001 M of VO(acac)2 and LVO(acac) with 0.15 M NaCl in water on glassycarbon electrode, v = 0.1 V s−1, (versus Ag|AgCl electrode) (Color figure available online).

that catalyses the initial step in hydrolysis of starch to oligosaccharides, trisaccharides,and disaccharides. Afterward, α-glucosidase enzymes in the brush border of the smallintestines, hydrolysis this mixture of saccharides to monosaccharides, specially glucose.Control of postprandial hyperglycemia via modulation of pancreatic α-amylase or intestinalα-glucosidase to delay carbohydrate absorption by dietary antidiabetic agents is an attractivestrategy to control or prevent of hyperglycemia and diabetes mellitus.

The pancreatic α-amylase and Baker’s yeast α-glucosidase inhibition assays of com-pound 2 were performed according to the literature procedures with slightly modifications.16

The α-amylase activity was determined using starch as a substrate in a colorimetric reactionusing 3,5-dinitrosalicylic acid.

The inhibitory activity of the compound 2 against yeast α-glucosidase was deter-mined by measuring the formation of p-nitrophenol by α-glucosidase after reaction withp-nitrophenyl-α-D-glucopyranoside (PNP) substrate in the presence and absence of com-pound 2.17 The obtained results are summarized in Table 1. Compound 2 was found topossess significant α-amylase (96.58 ± 8.23%) and α-glucosidase (83.83 ± 5.36%) in-hibitory activity.

Table 1 The percentage of enzyme inhibition of VO(acac)2, LVO(acac), and acarbose on α-amylase and α-glucosidasea

α-amylase α-glucosidaseCompound 20 µg mL−1 0.1 µg mL−1

VO(acac)2 24.94 ± 7.53 59.39 ± 8.89LVO(acac) (2) 96.58 ± 8.23 83.83 ± 5.36Acarbose 82.09 ± 5.93 85.18 ± 4.27

aResults are expressed as mean ± S.E.M., n = 3.

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PROLINE-BASED AMINOPHOSPHINIC ACID LIGAND 1525

Acarbose, as a positive control for this assay, showed 82.09 ± 5.93% inhibitionactivity against α-amylase and 85.18 ± 4.27 against α-glucosidase. On the other hand,VO(acac)2 showed low to moderate inhibition activity against α-amylase and α-glucosidase.The obtained results showed that compound 2 was found to possess significant α-amylaseand α-glucosidase inhibitory activity. Powerful synthetic α-glucosidase inhibitors (i.e.,voglibose) are available, but may case hepatic disorders at high doses. The α-amylaseinhibitor from aminophosphinic acid, analogues of 1-amino carboxylic acids, is potentiallypowerful, and therefore may be a preferred alternative for modulation of carbohydratedigestion and control of glycemic index of food products.

In summary, proline-based aminophosphinic acid ligand and it’s vanadium complexhas been synthesized, characterized, and it’s inhibitory activity on pancreatic α-amylaseand Baker’s yeast α-glucosidase has been examined in vitro. Novel α-amylase and α-glucosidase inhibitors offer an attractive therapeutic approach to the treatment of postpran-dial hyperglycemia by decreasing glucose release from starch and delaying carbohydrateabsorption. The novel complex showed more inhibitory potency against on pancreaticα-amylase and Baker’s yeast α-glucosidase.

EXPERIMENTAL

All solvents and reagents were purchased from commercial sources and used withoutfurther purification. All melting points were taken on a Yanagimoto and Buchi 510 apparatusand are not corrected. Mass spectra were recorded on an Agilent Technology using electronimpact ionization (EI) techniques. NMR spectra were obtained on a Bruker Avance 400NMR spectrometer (1H NMR: 400 MHz, 13C NMR: 100 MHz, 31P NMR: 162.0 MHz).Analytical TLC was carried out with Merck KGaA (Darmstadt, Germany) plates precoatedwith silica gel 60 F254 (0.25 mm thick). CV was carried out in a three-electrode systemusing a computer-controlled potentiostat (Zenium Zahner, Germany).

The Preparation of Aminophosphinic Acid Ligand 1. The aminophosphinicacid ligand 1 was synthesized via a Mannich-type reaction using a modified literatureprocedure described previously.14 In a two-necked flask equipped with a reflux condenserand a dropping funnel, L-proline (5 mmol) and paraformaldehyde (5 mmol, calculated asformaldehyde monomer) were suspended in anhydrous ethanol (15 mL). Hypophospho-rous acid (30% aqueous solution, 25 mmol) was added to the reaction mixture and themixture was stirred at ambient temperature for 24 h. Acetone (10 mL) was added to thereaction mixture and a solid formed. The solid product was filtered off and washed withn-hexane/EtOAc (1:1, 3 × 25 mL) and dried on air to give product 1 in 65% yield.

White solid, mp: 220–222 ◦C; 1H-NMR (D2O/TMS-400 MHz): 1.81–1.98 (m,1H), 2.01–2.15 (m, 2H), 2.35–2.50 (m, 1H), 3.15–3.25 (m, 2H), 3.30–3.40 (m, 1H),3.75–3.90 (m, 1H), 4.29 (1H, t, J = 8.0 Hz), 7.08 (1H, d, JHP = 557.5 Hz); 31P-NMR(D2O/H3PO4–162.0 MHz): 10.05 ppm; IR (KBr): 3650–2120 (-OH), 1829 and 1703 (C O),1184 (P O), 1045–1000 (P-O) cm−1; 13C-NMR (D2O-NaOD/TMS-100.6 MHz): 22.4,27.8, 54.2 (d, JCP = 84.0 Hz), 57.1 (d, JCP = 4.0 Hz), 68.9 (d, JCP = 5.0 Hz), 171.2; Anal.Calcd for C6H12NO4P. C, 37.30; H, 6.26; N, 7.25%. Found: C, 37.12; H, 6.15; N, 7.05%.

Synthesis of Complex LVO(acac) (2). Ligand 1 (1 mmol, 0.193 g) was dissolvedin water (35 mL) and 0.1 M NaOH solution was added to bring the solution to pH = 8–9.VO(acac)2. Note that, 2H2O (1 mmol, 0.297 g) was added to the reaction mixture and stirredin air for 1 h at reflux. After evaporation of the solvent, a dark green solid was formed. Theprecipitate was collected and dissolved in small amount of water. Acetone was added to the

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1526 B. KABOUDIN ET AL.

solution and a dark green solid was precipitated. The precipitate was collected and driedin air at room temperature. The prepared complex was characterized by elemental analysisand IR absorption, and mass spectroscopy. IR (cm−–1, KBr disk) data: 3650–2850 cm−1

(νO-H, νC-H); 1696 and 1640 cm−1 (νC O); 1184 and 1026 cm−1 (νP O); 959 cm−1 (νV O).EI-MS data: m/z 359 [M + 1]+. Anal Calcd. For C11H18NO7PV. C, 36.87; H, 5.10; N,3.91%. Found: C, 36.65; H, 5.15; N, 3.75%.

Pancreatic α-Amylase Inhibition Assay

The pancreatic α-amylase inhibition assay was performed according to the literatureprocedure with slight modifications.16 α-amylase (Sigma) was dissolved in 0.1 M phosphatebuffer, pH 6.9. 50 µL of various concentrations of samples (in water) were added to 150µL starch solution (containing 1% starch and 17 mM NaCl). The reaction was initiated byadding 10 µL α-amylase (26 U mL−1) to the reaction mixture. After 30 min, the reactionwas quenched by adding 20 µL of NaOH solution (2 N), then 20 µL of dinitrosalicylicacid (DNS) reagent (44 mM 3,5-dinitrosalicylic acid, 106 mM potassium sodium tartarate,40 mM NaOH) was added. The mixture was heated at 100 ◦C for 20 min. After coolingto room temperature, absorbance (Abs) was recorded at 540 nm using spectrophotometer.Acarbose was used as a positive control for this assay. The percentage inhibition wascalculated by follow the equation.

%Inhibition = [�Abs540(Control) − �Abs540(Sample)]

�Abs540(Control)× 100

Baker’s Yeast α-Glucosidase Inhibitory Activity

The α-glucosidase inhibition assay was performed according to the slightly modifiedmethod of Hwang et al.17 Note that, 40 µL of α-glucosidase (Sigma) solution (1 U mL−1)was added to 1 µL of sample solution (different concentrations in water) and 69 µL of 0.1 Msodium phosphate buffer, pH 7.0. After 15 min incubation in 37 ◦C, 40 µL of substratesolution (5 mM p-nitrophenyl α-D-glucopyranoside) was added to the reaction mixture andincubated at 37 ◦C for 30 min, and terminated by adding 150 µL of 0.1 M Na2CO3. Theabsorbance was determined at 405 nm using a spectrophotometer. The percentage inhibitionof activity was calculated as (A0 – A1)/A0

∗100, here A0 is the absorbance without the sample,and A is the absorbance with the sample. All samples were analyzed in triplicate.

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