一個(gè)基于N′-(3-溴-5-氯-2-羥基苯亞甲基)-3-羥基-4-甲氧基苯甲酰肼的甲基麥芽酚配位的氧釩配合物：合成、晶體結(jié)構(gòu)及其胰島素增強(qiáng)活性

李鹿曦　孫瑩　謝青　孫宇冰　李坤華　李巍　由忠錄＊,

(1遼寧師范大學(xué)化學(xué)化工學(xué)院，大連116029) (2大連醫(yī)科大學(xué)附屬第二醫(yī)院放射科，大連116023）

一個(gè)基于N′-(3-溴-5-氯-2-羥基苯亞甲基)-3-羥基-4-甲氧基苯甲酰肼的甲基麥芽酚配位的氧釩配合物：合成、晶體結(jié)構(gòu)及其胰島素增強(qiáng)活性

李鹿曦1孫瑩1謝青2孫宇冰2李坤華2李巍2由忠錄＊,1

(1遼寧師范大學(xué)化學(xué)化工學(xué)院，大連116029) (2大連醫(yī)科大學(xué)附屬第二醫(yī)院放射科，大連116023）

制備了1個(gè)芳酰腙化合物：N′-(3-溴-5-氯-2-羥基苯亞甲基)-3-羥基-4-甲氧基苯甲酰肼(H2L)，并利用元素分析、紅外、紫外、高分辨質(zhì)譜、核磁共振氫譜和碳譜對其進(jìn)行了表征。利用H2L與甲基麥芽酚和VO(acac)2在甲醇中反應(yīng)得到了1個(gè)甲基麥芽酚配位的氧釩配合物[VO(mat)L]·MeOH。通過元素分析、紅外和紫外光譜對其進(jìn)行了表征，同時(shí)還研究了配合物的熱穩(wěn)定性。通過單晶X射線衍射進(jìn)一步確認(rèn)了H2L和配合物的結(jié)構(gòu)。通過對C2C12肌細(xì)胞的胰島素模擬實(shí)驗(yàn)，表明該配合物能顯著促進(jìn)細(xì)胞對葡萄糖的利用，其細(xì)胞毒性僅為0.07 g·L-1。

氧釩配合物；甲基麥芽酚；芳酰腙；晶體結(jié)構(gòu)；胰島素增強(qiáng)活性

Since1980s,inorganicvanadiumsaltsand vanadium complexes with various ligands have been reported to possess potent pharmacological effects of insulin-mimetic activity[1-4].Studiesindicated that vanadium compounds improve not only hyperglycemia in human subjects and animal models of typeⅠdiabetes but also glucose homeostasis in typeⅡdiabetes[5-6].However,the inorganic vanadium salts are considered as less active and more toxic.In order to reduce the side effects of inorganic vanadium salts, vanadium complexes with various organic ligands have received particular attention and demonstrated to be effective[7-9].Among the complexes,bis(maltolato)oxovanadium(BMOV)[10],synthesized by simple metathesis of vanadyl sulfate trihydrate and maltol(3-hydroxy-2-methyl-4-pyrone),has important and interesting insulin-enhancing activity[11-12].Yet,there are some side effects of BMOV,principally diarrhea[13]. Schiff bases play important role in biological chemistry.Several vanadium complexes derived from Schiff bases have shown to normalize blood glucose level with high efficiency and low toxicity,even at low concentration[14-15].Schiff bases with hydrazone type are of particular interest due to their biological properties[16-20].In view of the increasing importance of vanadium complexes with hydrazone type Schiff bases, we report herein the synthesis,characterization,and insulin-enhancing activity of a maltolato-coordinated oxovanadiumcomplexwiththearoylhydrazone compoundN′-(3-bromo-5-chloro-2-hydroxybenzylidene) -3-hydroxyl-4-methoxybenzohydrazide(H2L,Scheme1).

Scheme 1　Structure of H2L

1　Experimental

1.1Materials and measurements

Starting material,reagents and solvents were purchased from commercial suppliers and used as received.Elemental analyses were performed on a Perkin-Elmer 240C elemental analyzer.IR spectra were recorded on a Jasco FT/IR-4000 spectrometer as KBr pellets in the 4 000～400 cm-1region.Electronic spectra were recorded on a Lambda 10 spectrometer. Absorbance was recorded on a Bio-Tek model ELx800 96-well plate reader.HRMS data was obtained with ESI(electrospray ionization)mode.1H NMR and13C NMR were recorded on Bruker 300 MHz and 75 MHz spectrometer,respectively.X-ray diffraction was carried out on a Bruker SMART 1000 CCD diffractometer.

1.2Preparation of H2L

To a methanolic solution(20 mL)of 3-bromo-5-chlorosalicylaldehyde(0.470 g,2.0 mmol)was added a methanolic solution(20 mL)of 3-hydroxyl-4-methoxybenzohydrazide(0.364 g,2.0 mmol)with stirring.The mixture was stirred for 10 min at room temperature and filtered.Upon keeping the filtrate in air for a few days,colorless block-shaped crystals of the compound,suitable for X-ray crystal determination, were formed at the bottom of the vessel on slow evaporation of the solvent.The crystals were isolated, washed three times with MeOH and dried in a vacuum desiccator containing anhydrous CaCl2.Yield: 78%.Anal.Calcd.for C15H12BrClN2O4(%):C,45.08; H,3.03;N,7.01.Found(%):C,44.92;H,3.13;N, 7.12.IR data(KBr,cm-1):3 429 m,3 206 w,1 641 s, 1 605 s,1 569 m,1 526 w,1 443 s,1 362 w,1 316 m, 1257s,1217s,1167m,1132w,1025w,976w,947 w, 870 w,822 w,763 w,699 m,633 w,562 w,480 w. UV-Vis(methanol,λ/nm(ε/(L·mol-1·cm-1))):295 (18 725),308(20 150),337(12,750),407(1 153). HRMS(ESI):m/z Calcd.for C15H11BrClN2O4[M+] 398.974 2;Found:398.973 9.1H NMR(300 MHz, DMSO-d6):δ 12.83(s,1H,OH),12.34(s,1H,NH), 9.38(s,1H,OH),8.50(s,1H,CH=N),7.69(dd,1H, J=12.7,2.5 Hz,ArH),7.66(s,1H,ArH),7.47(s,1H, ArH),7.44(dd,1H,J=12.5,5.0 Hz,ArH),7.08(d,1H, ArH),3.85(s,3H,CH3).13C NMR(75 MHz,DMSO-d6): δ 162.59,153.23,151.24,146.33,132.76,129.16, 124.40,123.17,120.43,119.70,114.95,111.41, 110.73,55.73.

1.3Preparation of the complex

A methanolic solution(30 mL)of VO(acac)2(0.27 g,1.0 mmol)was added to a methanolic solution(20 mL)of H2L(0.40 g,1.0 mmol)and maltol(0.13 g,1.0 mmol)with stirring.The mixture was stirred at room temperature for 30 min to give deep brown solution. The resulting solution was allowed to stand in air for a few days until three quarters of the solvent was evaporated.Brown block-shaped single crystals of the complex,suitable for X-ray single crystal diffraction were formed at the bottom of the vessel.The crystals were isolated by filtration,washed three times with cold methanol and dried in a vacuum desiccator containing anhydrous CaCl2.Yield:53%.Anal.Calcd. for C22H19BrClN2O9V(%):C,42.50;H,3.08;N,4.51. Found(%):C,42.65;H,3.22;N,4.38.IR data(KBr, cm-1):3 456 w,1 619 m,1 587 s,1 531 m,1 500 m, 1 451 m,1 428 m,1 366 m,1 338 w,1 259 s,1 236 w, 1202s,1185s,1131m,1091w,1022m,974s,946m, 927 w,850 m,834 m,812 w,758 m,733 s,711 m, 637m,604m.UV-Vis(acetonitrile,λ/nm(ε/(L·mol-1· cm-1))):280(27 360),352(15 333),437(8 130).

Table 1　Crystallographical and experimental data for H2L and the complex

1.4Crystal structure determination

Diffraction intensities for H2L and the complex were collected at 298(2)K using a Bruker SMART 1000 CCD area-detector diffractometer with Mo Kα radiation(λ=0.071 073 nm).The crystals with dimensions of 0.30 mm×0.27 mm×0.26 mm for H2L and 0.23 mm×0.22 mm×0.18 mm for the complex were mounted on the top of thin glass fibers.The collected data were reduced with SAINT[21],and multi-scan absorption correction was performed using SADABS[22]. Structures of H2L and the complex were solved by direct methods,and refined against F2by full-matrix least-squares methods using SHELXTL[23].All nonhydrogen atoms were refined anisotropically.The amino hydrogen atom in H2L was located from a difference Fourier map and refined isotropically,with N-H distance restrained to 0.090(1)nm.The remaining hydrogen atoms were placed in calculated positions andconstrainedtorideontheirparentatoms. Crystallographic data for H2L and the complex are summarized in Table 1.Selected bond lengths and angles are given in Table 2.

Continued Table 1

Table 2Selected bond lengths(nm)and angles(°)for H2L and the complex

CCDC:1437202,H2L;1437201,the complex.

1.5Cell culture and viable cell counts

The biological assay was determined according to the literature method[14].In general,C2C12 mouse skeletal muscle cells were cultured in Dulbecco modified Eagle′s medium with 4 mmol·L-1L-glutamine adjusted to contain 1.5 g·L-1Na2CO3,4.5 g·L-1glucose,and 10%fetal bovine serum in a humidified atmosphere of 5%CO2and 95%air at 37℃.C2C12 cellsweresub-culturedinlogphaseto70% confluence and seeded at a density of 5000 cells per well into 96-well culture plates.To limit batch-tobatch variation,cell subcultures were limited to 10 passages.After three days culture myotube formation was induced by replacing the fetal bovine serum in the medium with 10%horse serum.All experiments were done in five days when more than 75%of the cells were differentiated morphologically.The cells weresuspendedinatrypanblue(0.1%w/w) phosphate buffered saline solution and the ratio of stained to nonstained cells was determined after 5 min of incubation time.Viable cell counts were performed using a hemocytometer.

1.6Glucose uptake determination

Three hours prior to glucose uptake,cells were incubated in glucose and serum-free media.On the 5th day,the medium was removed and replaced with 50 mL modified Dulbecco modified Eagle′s medium without phenol red,supplemented with 8.0 mmol·L-1glucose and 0.1%bovine serum albumin containing either the complex at concentration of 0.10 g·L-1or the positive control,insulin,or metformin,at 1.0 mmol·L-1were added to the 96-well plate.The plate was then incubated for 2 h at 37℃and 5%CO2. After incubation,4.0 mL media was removed from each well and transferred to a new 96-well plate to which 196 mL deionized water was added in each well.A total of 50 mL of this diluted medium was transferred to a new 96-well plate and 50 mL of the prepared glucose assay reagent was added per welland incubated for 30 min at 37℃.Absorbance was taken at 570 nm on a 96-well plate reader.The glucose concentration per well was calculated from a standard curve.Glucose utilization was determined by subtracting the glucose concentration left inthe medium of the relevant wells following incubation to media not exposed to cells during incubation.All assays were performed in triplicate to minimize the error.

1.7Cytotoxicity assay

MTT(3-((4,5-Dimethylthiazo)-2-yl)-2,5-diphenyltetrazolium bromide)was dissolved in phosphatebuffered saline without phenol red at a concentration of 2.0 g·L-1.Dulbecco modified Eagle′s medium in the 96-well plate was refreshed with 200 mL of fresh media followed by addition of 50 mL of MTT solution to each well.The plate was wrapped in aluminium foil to prevent light and incubated at 37℃for 4 h,after which the media with MTT was removed and replaced with 200 mL DMSO and 25 mL Sorensen′s glycine buffer.Absorbance was read at 570 nm in a plate reader.

2　Results and discussion

2.1Chemistry

The aroylhydrazone compound H2L was readily prepared by the condensation reaction of 3-bromo-5-chlorosalicylaldehydewith3-hydroxyl-4-methoxybenzohydrazide in methanol.Facile reaction of VO(acac)2with H2L and maltol in methanol afforded the oxovanadiumcomplex.Crystals of H2L and the complex are stable in open air at room temperature. Elemental analyses are in good agreement with the chemical formulae proposed for the compounds.

2.2Structure description of H2L

Fig.1 gives perspective view of H2L together with the atomic labeling system.The molecule of the compound adopts E configuration with respect to the methylidene unit.The distance of the C(7)-N(1)methylidene bond(0.127 6(4)nm)confirms it as a typical double bond.The shorter distance of the C(8)-N(2) bond(0.134 9(4)nm)and the longer distance of the C(8)-O(2)bond(0.122 8(3)nm)for the-C(O)-NH-unit than usual,suggest the presence of conjugation effect in the molecule.The bond lengths in the compound are withinnormal values[20,24].The dihedral angle between the two benzene rings is 34.0(5)°.The crystal structureofthecompound is stabilized by intermolecular hydrogen bonds,to form two-dimensional sheets along the bc plane(Table 3,Fig.2).

Fig.1　Molecular structure of H2L

Fig.2　Crystal packing of the complex viewed along the b axis

2.3Structure description of the complex

Fig.3gives perspective view of the complex together with the atomic labeling system.The asymmetric unit of the compound contains a mononuclear vanadiumcomplexandamethanolmoleculeof crystallization.The V atom in the complex is in an octahedral coordination,with the phenolate O,imino N,and enolate O atoms of L,and the hydroxy O atom of the maltolate ligand defining the equatorial plane, and with one oxo O and the carbonyl O atom of the maltolate ligand locating at the axial positions.The V atom deviates from the least-squares plane defined by the equatorial atoms by 0.028 0(1)nm.The coordinate bond lengths in the complex are similar to those observed in vanadium complexes with aroylhydrazoneligands[25-27].Distortion of the octahedral coordination can be observed from the coordinate bond angles, ranging from 74.51(7)°to 107.11(7)°for the perpendicular angles,and from 150.55(8)°to 173.49(8)°for the diagonal angles.The dihedral angle between the two benzene rings of the aroylhydrazone ligand is 14.8(3)°.During coordination,the C(7)-N(1),N(1)-N(2) and C(8)-O(2)bonds of the complex are longer than those of the free aroylhydrazone,while the N(2)-C(8) bond of the complex is shorter than that of the free aroylhydrazone.The crystal structure of the compound is stabilized by intermolecular hydrogen bonds,to form two-dimensional sheets along the ab plane(Table 3,Fig.4).

Table 3Hydrogen bond distances(nm)and bond angles(°)for H2L and the complex

Fig.3　Molecular structure of the complex

Fig.4　Crystal packing of the complex,viewed along the a axis

2.4IR and electronic spectra

The medium and broad absorption centered at 3 429 cm-1in the spectrum of H2L and 3 456 cm-1in thespectrumofthecomplexsubstantiatesthe presence of phenol groups.The sharp band indicative of the N-H vibration of H2L is located at 3 206 cm-1, and the intense band indicative of the C=O vibration is located at 1 641 cm-1in the spectrum of H2L, which are absence in the complex,indicating the enolisation of the amide functionality and subsequent protonreplacementbytheVatom.Thestrong absorption bands at 1 605 cm-1for H2L and 1619 cm-1for the complex are assigned to the azomethine ν(C= N)[28].The typical absorption at 974 cm-1of the complex can be assigned to the V=O vibration[29].

Electronic spectra of H2L and the complex were recorded with 10-5mol·L-1in methanol and acetonitrile,respectively,in the range of 200～600 nm.In the UVVis region the complex show band centered at 352 nm and weak band at 437 nm.The weak band is attributed to intramolecular charge transfer transitions from the pπ orbital on the nitrogen and oxygen to the empty d orbitals of the metal[30].The intense bands observed at 280 nm for the complex and 295 nm for H2L are assigned to intraligand π-π*transitions[30].

2.5Thermal stability

Thermal gravimetric analysis was conducted to examine the stability of the complex(Fig.5).The first step started at 130℃and completed at 163℃,which corresponds to the loss of the methanol molecule.The observed weight loss of 5.1%is in accordance with the calculated value.The second step,from 163 to 345℃,corresponds to the loss of the maltolate ligand except for one O atom.The observed weight loss of 17.4%is in accordance with the calculated value (17.5%).The third step,at 345℃,corresponds to the loss of aroylhydrazone ligand except for the O1,O2, N1,N2 and C8 atoms.The observed weight loss of 52.0%is in accordance with the calculated value (52.5%).The last step,from 345 to 454℃,corresponds to the loss of the remaining part of the ligands,and formation of V2O5.The total observed weight loss of 86.3%is in accordance with the calculated value of 85.4%.

Fig.5TG curve of the complex

2.6Glucose uptake in the presence of the complex

Glucose level is a key diagnostic parameter for many metabolic disorders.Biovision glucose assay kit provides direct measurement of glucose in various biologicalsamples.Theglucoseenzymemix specifically oxidizes glucose to generate a product, which reacts with a dye to generate color.The generated color is proportional to the glucose amount. The method is rapid,simple,sensitive,and suitable for high throughput[14].The insulin-like activity of vanadium compounds is usually related to their ability to lower the blood glucose level by activating the glucose transport into the cell of the peripheral tissues.In this study,we have investigated the in vitro glucose uptake by C2C12 muscle cells following exposure to the complex.The results are given in Table 4.

Table 4　Glucose uptake in C2C12 cell line results*%

*Results show the uptake of glucose from the culture media containing 8.0 mmol·L-1glucose by C2C12 cells over one 1 h; C2C12 cells were pre-exposed to the compounds,in glucose and serum-free media for 3 h before the glucose uptake experiments; Basal glucose uptake for solvent vehicle only(DMSO)is represented as 100%and the subsequent increase or decrease induced by the compounds is reflected as±100%.

Insulin-mimetic testonC2C12musclecells indicates that the complex significantly stimulated cell glucose utilization with cytotoxicity at 0.07 g·L-1.In general,the insulin enhancing activity of the complex issimilartothereferencedrugsInsulinand Metformin.So,it is a promising vanadium-based insulin-like material.

References:

[1]Pillai S I,Subramanian S P,Kandaswamy M.Eur.J.Med. Chem.,2013,63:109-117

[2]Smee J J,Epps J A,Ooms K,et al.J.Inorg.Biochem., 2009,103:575-584

[3]Sanna D,Micera G,Garribba E.Inorg.Chem.,2013,52: 11975-11985

[4]He L,Wang X S,Zhao C,et al.Metallomics,2014,6:1087-1096

[5]Crans D C,Trujillo A M,Pharazyn P S,et al.Coord.Chem.Rev.,2011,255:2178-2192

[6]Sheela A,Roopan S M,Vijayaraghavan R.Eur.J.Med. Chem.,2008,43:2206-2210

[7]Zhang Y,Yang X D,Wang K,et al.J.Inorg.Biochem., 2006,100:80-87

[8]Dornyei A,Marcao S,Pessoa J C,et al.Eur.J.Inorg.Chem., 2006,18:3614-3621

[9]Haratake M,Fukunaga M,Ono M,et al.J.Biol.Inorg.Chem., 2005,10:250-258

[10]McNeill J H,Yuen V G,Hoveyda H R,et al.J.Med.Chem., 1992,35:1489-1491

[11]Yuen V G,Orvig C,McNeill J H.Can.J.Physiol.Pharmacol., 1993,71:263-269

[12]Yuen V G,Orvig C,Thompson K H,et al.Can.J.Physiol. Pharmacol.,1993,71:270-276

[13]Fujimoto S,Fujii K,Yasui H.J.Clin.Biochem.Nutr.,1997, 23:113-129

[14]Nejo A A,Kolawole G A,Opoku A R,et al.J.Coord.Chem., 2009,62:3411-3424

[15]Xie M J,Yang X D,Liu W P,et al.J.Inorg.Biochem., 2010,104:851-857

[16]Amir M,Ali I,Hassan M Z,et al.Arch.Pharm.,2014,347: 958-968

[17]Rajitha G,Prasad K V S R G,Umamaheswari A,et al.Med. Chem.Res.,2014,23:5204-5214

[18]El-Sayed M A A,Abdel-Aziz N I,Abdel-Aziz A A M,et al. Bioorg.Med.Chem.,2011,19:3416-3424

[19]Horiuchi T,Chiba J,Uoto K,et al.Bioorg.Med.Chem.Lett., 2009,19:305-308

[20]Zhang M,Xian D M,Li H H,et al.Aust.J.Chem.,2012, 65:343-350

[21]SMART and SAINT,Bruker AXS Inc.,Madison,2002.

[22]Sheldrick G M.SADABS,University of G?ttingen,Germany, 1996.

[23]Sheldrick G M.Acta Crystallogr.,2008,A64:112-122

[24]Dinda R,Sengupta P,Ghosh S,et al.J.Chem.Soc.Dalton Trans.,2002:4434-4439

[25]REN Jin-Qi(任晉琦),JIAO Qing-Zhu(焦慶祝),WANG Yi-Nuo(王藝諾),et al.Chinese J.Inorg.Chem.(無機(jī)化學(xué)學(xué)報(bào)),2014,30(3):640-648

[26]ZHAO Yue(趙月),HAN Xiao(韓笑),ZHOU Xiao-Xia(周曉霞),et al.Chinese J.Inorg.Chem.(無機(jī)化學(xué)學(xué)報(bào)),2013,29 (4):867-874

[27]HE Miao(賀淼),JIAO Qing-Zhu(焦慶祝),CHEN Xiang-Fei (陳相飛),et al.Chinese J.Inorg.Chem.(無機(jī)化學(xué)學(xué)報(bào)), 2015,31(8):1590-1596

[28]You Z L,Xian D M,Zhang M.CrystEngComm,2012,14: 7133-7136

[29]Sangeetha N R,Kavita V,Wocadlo S.J.Coord.Chem., 2000,51:55-66

[30]Asgedom G,Sreedhara A,Kivikoski J,et al.J.Chem.Soc., Dalton Trans.,1996,1:93-97

A Maltolato-Coordinated OxovanadiumComplex Derived from N′-(3-Bromo-5-chloro-2-hydroxybenzylidene)-3-hydroxyl-4-methoxybenzohydrazide: Synthesis,Crystal Structure,and Insulin-Enhancing Activity

LI Lu-Xi1SUN Ying1XIE Qing2SUN Yu-Bing2LI Kun-Hua2LI Wei2YOU Zhong-Lu＊,1
(1Department of Chemistry and Chemical Engineering,Liaoning Normal University,Dalian，Liaoning 116029,China) (2Department of Radiology,The Second Hospital of Dalian Medical University,Dalian,Liaoning 116023,China)

An aroylhydrazone compound,N′(3-bromo-5-chloro-2-hydroxybenzylidene)-3-hydroxyl-4-methoxybenzohydrazide(H2L),was prepared and characterized by elemental analysis,IR,UV,HRMS,1H NMR and13C NMR spectrum.Reaction of H2L,maltol(3-hydroxy-2-methyl-4-pyrone)and VO(acac)2in methanol afforded a maltolatocoordinated oxovanadiumcomplex,[VO(mat)L]·MeOH.The complex was characterized by elemental analysis, IR and UV spectra.Thermal analysis was also performed.Structures of H2L and the complex were further confirmed by single crystal X-ray diffraction.Insulin-mimetic tests on C2C12 muscle cells indicate that the complex significantly stimulated cell glucose utilization with cytotoxicity at 0.07 g·L-1.CCDC:1437202,H2L; 1437201,the complex.

oxovanadium complex;maltol;aroylhydrazone;crystal structure;insulin-enhancing activity

O614.51+1

A

1001-4861(2016)02-0376-08

10.11862/CJIC.2016.040

2015-11-18。收修改稿日期：2015-12-21。

遼寧省自然科學(xué)基金(No.2015020673)、遼寧省高校優(yōu)秀人才項(xiàng)目(No.LR2014032)和遼寧省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練項(xiàng)目(No.201510165079)資助。

＊通信聯(lián)系人。E-mail：youzhonglu@126.com