ZHANG Jin-Chao WANG Li-WeiLI Lu-WeiZHANG Fang-Fang MA Li-LiLI Xiao-Liu

(College of Chemistry&Environmental Science,Chemical Biology Key Laboratory of Hebei Province,Hebei University,Baoding,Hebei 071002,China)

Synthesis,Crystal Structure and Cytotoxicity of Palladium（Ⅱ）Complexes with N-(4-methylbenzoyl)-L-valine Dianion and Aromatic Diimine

ZHANG Jin-Chao＊WANG Li-WeiLI Lu-WeiZHANG Fang-Fang MA Li-LiLI Xiao-Liu

(College of Chemistry&Environmental Science,Chemical Biology Key Laboratory of Hebei Province,Hebei University,Baoding,Hebei071002,China)

Two novel palladium（Ⅱ）complexes,[Pd(bipy)(4-CH3Bzval-N,O)](1)and[Pd(phen)(4-CH3Bzval-N,O)](2) (bipy=2,2′-bipyridine,phen=1,10-phenanthroline,4-CH3Bzval-N,O=N-(4-methylbenzoyl)-L-valine dianion)have been prepared and structurally characterized,the cytotoxicity in vitro has also been investigated by MTT and SRB assays.The complex 2 crystallizes in the hexagonal system,space group P21/n with cell parameters a=1.162 92(8) nm,b=1.07403(7)nm,c=1.82114(12)nm,V=2.2328(3)nm3and Z=4.The complexes(1 and 2)presented cytotoxic effects and selectivity,but were less active than cisplatin against HL-60,BGC-823,Bel-7402 and KB cell lines. CCDC:761046.

N-acylated-L-valine dianion;Pd（Ⅱ）complexe;crystal structure;cytotoxicity

0 Introduction

Now cisplatin and its analogues are some of the most effective chemotherapeutic agents in clinical use as the first line of treatment in testicular and ovarian cancers[1]. Unfortunately, they have several major drawbacks.Commonproblemsincludecumulative toxicities,the serious side effects and inherent ortreatment-induced resistant tumor cells[2].These drawbacks have provided the motivation for alternative chemotherapeutic strategies.

Metals,in particularly,transition metals offer potential advantages over the more common organicbased drugs.The notable analogy between the coordination chemistry of platinum（Ⅱ）and palladium（Ⅱ）complexes has advocated studies of Pd（Ⅱ）complexes as antitumor drugs.The hydrolysis of the leaving ligands in palladium complexes is too rapid.They dissociate readily in solution leading to very reactive species that are unable to reach their pharmacological targets.This implies that if an antitumor palladium drug is to be developed,it must somehow be stabilized by a strongly coordinated nitrogen ligand and a suitable leaving group.Amino acid,bipyridine and phen or their derivatives have been widely used tosynthesize palladium anticancer complexes because amino acid ligands do not dissociate easily in aqueous solution and bipyridine or phen has the ability to participate as DNA intercalators[3-4].Some mixed-ligand palladium（Ⅱ）complexes of 2,2′-bipyridine and amino acids have been synthesized[5].These complexes have also shown growth inhibition against L1210 lymphoid leukemic,P388 lymphocytic leukemic,Sarcoma 180,and Ehrlich ascitic tumor cells.Mital et al.reported the synthesis and cytotoxicity of nine paIladium（Ⅱ）complexes of type [Pd(phen)(AA)]+(where AA is an anion of glycine,L-alanine,L-leucine,L-phenylalanine,L-tyrosine,L-tryptophan,L-valine,L-proline,or L-serine).They are found to exhibit growth inhibition of P388 lymphocytic leukemic cells[6].We previously reported the synthesis and cytotoxicity of a novel palladium （Ⅱ） complex [Pd(Phen)(TsserNO)]·H2O(Phen=1,10-phenanthroline; TsserNO=4-toluenesulfonyl-L-serinate dianion),its cytotoxicity is equal to that of cisplatin against BGC-823 and Bel-7402 cells lines,however it is less potent than cisplatin against HL-60 and KB cell lines[7].Until now,the cytotoxicity of mixed-ligand palladium（Ⅱ）complexes with N-acylated-L-amino acid dianion and aromatic diimine has not been reported.In the present work,we present the synthesis,characterization and cytotoxicity of two novel mixed-ligand palladium（Ⅱ） complexes with N-(4-methylbenzoyl)-L-valine dianion and aromatic diimine for the first time.

1 Experimental

1.1 Materials and instruments

4-Methylbenzoyl chloride,K2[PdCl4]and all reagents were of chemical grade,1,10-phenanthroline (phen)and L-valine were of analytical grade.RPMI-1640 medium,trypsin and fetal bovine serum were purchased from Gibco.MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), SRB (sulforhodamine B),benzylpenicillin and streptomycin were from Sigma.Four different human carcinoma cell lines:HL-60 (immature granulocyte leukemia),Bel-7402(liver carcinoma),BGC-823(gastrocarcinoma)and KB (nasopharyngeal carcinoma)were obtained from American Type Culture Collection.

Elemental analysis were determined on a Elementar Vario ELⅢelemental analyzer.IR spectra were recorded in the solid state (KBr pellets)in the range 4000～400 cm-1using a Perkin-Elmer Model-683 spectrophotometer.The1H NMR spectra were measured on a Bruker AVⅢ600 NMR spectrometer in dimethyl sulfoxide-d6with solvent peaks as references.X-ray single crystal structure was performed on a Bruker SMART APEXⅡ CCD diffractometer.The optical density(OD)at 570 nm was measured on a microplate spectrophotometer(Bio-Rad Model 680,USA).

1.2 Preparation of complexes

N-(4-methylbenzoyl)-L-valine(4-CH3BzvalH2),[Pd (bipy)Cl2]and[Pd(phen)Cl2]were synthesized by the reported procedures[8-9].

The complex 1 was prepared as follows:[Pd(bipy) Cl2](15 mg,0.045 mmol)was added to a 3 mL CH3OH/ H2O (volume 1∶1)solution of 4-CH3BzvalH2(21 mg, 0.090 mmol)when the solution temperature was heated to 48℃,the mixture was adjusted to pH=8～9 by NaOH solution,then stirred for 2 h.The solution was heated in vacuo and concentrated it to about 80%of the original volume.

The complex 1 was separated from the solution after a few days,but the crystal suitable for X-ray diffraction was not obtained(Scheme 1).

Scheme 1 Synthetic routines of the complexe 1 and 2

Elemental analysis calc.for C23H23N3O3Pd(%):C, 55.71;H,4.68;N,8.47.Found(%):C,55.66;H,4.57; N,8.51.IR (KBr,cm-1):1 547;1 635;1 388;547; 466.

1H NMR(600 MHz,DMSO-d6,ppm)δ:8.58～8.49 (m,2H),8.42～8.32(m,2H),8.18(d,J=7.9 Hz,2H), 8.11～8.03(m,1H),7.88～7.80(m,1H),7.55(d,J=5.4 Hz,1H),7.22～7.15(m,1H),6.86(d,J=7.9 Hz,2H), 4.57 (d,J=6.4 Hz,1H),2.43～2.36 (m,1H),2.05(s, 3H),1.25(d,J=6.7 Hz,2H),1.18(d,J=6.7 Hz,2H).

The synthesis of the complex 2 was carried out in an identical manner to the complex 1 starting from [Pd(phen)Cl2](20.00 mg,0.06 mmol)and 4-CH3BzvalH2(34.00 mg,0.12 mmol)(Scheme 1).By evaporating the filtered solutions at room temperature,the yellow crystal suitable for X-ray diffraction was obtained after a few days.

Elemental analysis calc.for C25H23N3O3Pd(%):C, 57.76;H,4.46;N,8.08.Found(%):C,57.56;H,4.37; N,8.12.IR(KBr,cm-1):1542;1634;1392;556;460.

1H NMR(600 MHz,DMSO-d6,ppm)δ:8.97(d,J= 8.1 Hz,1H),8.87(d,J=5.7 Hz,1H),8.69(d,J=8.3 Hz, 1H),8.27～8.22(m,2H),8.18(d,J=8.8 Hz,1H),8.16～8.12(m,1H),7.79(d,J=5.0 Hz,1H),7.55～7.50(m, 1H),7.26 (d,J=7.8 Hz,1H),6.80 (d,J=7.4 Hz,2H), 4.62 (d,J=6.0 Hz,1H),2.40～2.36 (m,1H),2.00(s, 3H),1.28(d,J=6.7 Hz,2H),1.21(d,J=6.6 Hz,2H).

1.3 Crystal structure determination

The single crystal of the complex with approximate dimensions of 0.45 mm×0.33 mm×0.33 mm was selected for X-ray diffraction analysis.Data collection was performed on a Bruker SMART APEXⅡ CCD diffractometer equipped with a graphite monochromatized Mo Kα radiation (λ=0.071 073 nm)at 296(2) K.A total of 11 067 reflections were collected in the range of 1.92°≤θ≤28.22°for the complex,of which 3939(Rint=0.0133)reflections were unique,and reflections were considered as observed (I＞2σ(I)).The maximum andminimum transmission factorsare 0.763 8 and 0.695 3,respectively.Multi-scan absorption corrections were applied using the SADABS program.The structure was solved by the direct method using the SHELXS-97program.Refinementson F2wereperformed using SHELXL-97 by the full-matrix least-squares method with anisotropic thermal parameters for all nonhydrogen atoms.The hydrogen atoms of the ligand were generated geometrically,while the H atoms of the coordination watermolecules were located from difference Fourier synthesis and refined with restraint parameters.A summary of crystallographic data and refinement parameters is given in Table 1.

CCDC:761046.

Table 1 Crystallographic data for complex 2

1.4 Cell culture

Four human carcinoma cell lines were used for cytotoxicity determination:HL-60,Bel-7402,BGC-823 and KB.They were cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum,100 units· mL-1of penicillin and 100 μg·mL-1of streptomycin.Cells were maintained at 37 ℃ in a humidified atmosphere of 5%CO2in air.

1.5 Cytotoxicity analysis

The cells harvested from exponential phase were seeded equivalently into a 96-well plate,complexes were then added tothewellstoachieve final concentrations.Control wells were prepared by addition of culture medium.Wells containing culture medium without cells were used as blanks.The plates were incubated at 37℃in a 5%CO2incubator for 48 h.The MTT assay was performed as described by Mosmann[10].Upon completion of the incubation,stock MTT dye solution (20 mL,5 mg·mL-1)was added to each well.After 4 h incubation,2-propanol(100 mL)was added to solubilize the MTT formazan.The OD of each well was then measured on a microplate spectrophotometer at a wavelength of 570 nm.The SRB assay was performed as previously described[11].Upon completion of the incubation,the cells were fixed in 10%trichloroacetic acid (100 mL)for 30 min at 4℃,washed five times in tap water and stained with 0.1%SRB in 1%acetic acid (100 mL)for 15 min.The cells were washed four times in 1% acetic acid and air-dried.The stain was solubilized in 10 mmol·L-1unbuffered Tris base(100 mL)and OD was measured at 540 nm as above.The IC50value was determined from plot of%viability against dose of compounds added.

2 Results and discussion

2.1 Chemical characterization

The elemental analysis data of the complexes 1 and 2 are in good agreement with the calculated values.This provides support for the suggested composition of the complexes.

In IR spectra,the amide group of 4-CH3BzvalH2has a sharp and strong νNHin 3 313 cm-1region.This peak disappears for both complexes,indicating that the amide group has been deprotonated.The amide group deprotonated and coordinating to metal ion is also indicated by the amide（Ⅱ） shifting from ～1 611 to～1543 cm-1and the disappearance of the amide（Ⅱ）from～1546 cm-1region.The carboxylate group of the complexes 1 and 2 shows two bands,an intense antisymmetric carboxylate stretchingand a symmetric carboxylate stretching,at about 1 630 and 1 385 cm-1, respectively.The values ofof the complexes are in the range 240～250 cm-1,which is greater thanof the corresponding sodium carboxylates,so the carboxylate group may be monodentate coordinated through oxygen atoms.This is further confirmed by the appearance of the peaks of νPd-O.

4-CH3BzvalH2show a doublet at δ=6.63,which is associated with the proton of the amide group,but these peaks disappear for the complexes,which showing that the amide group has been deprotonated.The methylene1H resonances(L-valine)shifted to the down field as a result of deprotonated amide nitrogen coordinating to Pd（Ⅱ）.The β-hydrogen of 4-CH3BzvalH2appeared as a dd quartet,but in the complexes this proton appeared as a doublet,which also confirmed the deprotonation of amide group.

2.2 Crystal structure

Complex 2 crystallizes in the monoclinic system and space group P21/n.A diagram of the crystal structure of complex 2 is presented in Fig.1.the Pd2+ion is coordinated by two nitrogen atoms of phen,one deprotonated amide nitrogen and one carboxylic oxygen.The deprotonated ligand 4-CH3BzvalH2acts as a bidentate ligands combining with Pd2+ion through one carboxyl oxygen atom and one deprotonated amide nitrogen atom,which leads to a five member chelating cycle.The angle between planar N(2)-Pd(1)-N(3)and planar O(1)-Pd(1)-N(1)is 8.459(51)°which indicates that the Pd(1)-O(1)-N(1)-N(2)-N(3)plane is slightly distorted.The Pd-N (deprotonated amide)bond length (0.199 35(15)nm)is similar to the Pd-N(phen)bond lengths(0.200 48(16)and 0.202 01(15)nm),while it is longer than Pd-O (carboxylic oxygen)bond length (0.19814(13)nm)(Table 2).

Fig.1 Molecular structure of complex 2,showing displacement ellipsoids at 30%probability level and the atom numbering scheme

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

2.3 Cytotoxic studies

As listed in Table 3,The complexes 1 and 2 exerted cytotoxic effects against HL-60,BGC-823,Bel-7402 and KB cell lines with a lower IC50value(＜50 μmol·L-1),but they were less active than cisplatin.Complex 1 displayed the better cytotoxicity than complex 2 against the tested carcinoma cell lines.It suggests that aromatic diimine has important effect on cytotoxicity,the palladium（Ⅱ）complexes with bipy have better cytotoxicity than the corresponding palladium（Ⅱ）complexes with phen.

Table 3 Cytotoxicity of complexes in vitro(n=5)

3 Conclusions

In summary,two novel palladium（Ⅱ） complexes, [Pd(bipy)(4-CH3Bzval-N,O)](1) and [Pd(phen)(4-CH3Bzval-N,O)] (2)have been synthesized and structurally characterized.Crystal structure of the complex 2 has been determined by X-ray diffraction analysis.The Pd2+ion is coordinated by two nitrogen atoms of phen,one deprotonated amide nitrogen atom and one carboxylic oxygen atom.Cytotoxic data indicate that two complexes display cytotoxic effects against HL-60,BGC-823,Bel-7402 and KB cell lines,moreover, the palladium（Ⅱ） complexes with bipy have better cytotoxicity than the corresponding palladium （Ⅱ）complexes with phen.This suggests that it may be a new class metal-based anticancer drugs.

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芳香亞胺與N-(4-甲基苯甲酰)-L-纈氨酸雙陰離子合鈀（Ⅱ）配合物的合成、晶體結構及體外抗腫瘤活性

張金超＊王立偉 李路偉 張芳芳 馬麗麗 李小六

(河北大學化學與環(huán)境科學學院，河北省化學生物學重點實驗室，保定 071002)

本文首次報道了2個鈀（Ⅱ）的配合物[Pd(bipy)(4-CH3Bzval-N，O)](1)和[Pd(phen)(4-CH3Bzval-N，O)](2)(bipy=2，2′-聯(lián)吡啶，phen=1，10-菲咯啉，4-CH3Bzval-N，O=N-(4-甲基苯甲酰)-L-纈氨酸雙陰離子)的合成及晶體結構，利用MTT法和SRB法研究了配合物的體外抗腫瘤活性。配合物2屬單斜晶系P21/n空間群，其中a=1.162 92(8)nm，b=1.074 03(7)nm，c=1.821 14(12)nm，V= 2.2328(3)nm3，Z=4。結果顯示：2個配合物對HL-60,BGC-823，Bel-7402和KB 4種人的腫瘤細胞表現出一定的活性和選擇性，但其活性均小于順鉑。

N-?；?L-纈氨酸雙陰離子；鈀（Ⅱ）配合物；單晶結構；抗腫瘤活性

O614.82+3

A

1001-4861(2011)03-0565-06

2010-09-06。收修改稿日期：2010-10-25。

國家“重大新藥創(chuàng)制”科技重大專項(No.2009ZX09103-139)，973計劃前期研究專項(No.2010CB534913)，科技部“科技人員服務企業(yè)行動項目”(No.2009GJA20025)，河北省應用基礎研究計劃重點基礎研究項目(No.08966415D)和河北省高等學?？茖W技術研究重點項目(No. ZD2010142)資助。

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