Synthesis,Characterization,Antitumor Activity,and Theoretical Calculations of Co（Ⅱ）Complex Based on Pyridine-2,6-dicarboxylic Acid

ZHANG LiJIANG ChangGUO Lin-FengZHANG Xiao-Ling LIN Xiong-Qiang KANG Jie＊, SUN Wei-Ming＊,

(1School of Pharmacy,Fujian Key Laboratory of Drug Target Discovery and Structural and Functional Research,Fujian Medical University,Fuzhou 350108,China)(2Fujian Academy of Chinese Medical Sciences,Fuzhou 350003,China)

Abstract:A new coba1t comp1ex,name1y[Co(Hpdc)(bpy)C1]·C2H5OH(bpy=2,2′-bipyridine),was synthesized by using pyridine-2,6-dicarboxy1ic acid(H2pdc)as 1igand under hydrotherma1 condition,and fo11owed by experimenta1 characterization of infrared spectroscopy and X-ray sing1e-crysta1 diffraction.To deep1y revea1 the e1ectronic structure of this comp1ex,density functiona1 theory ca1cu1ations were emp1oyed to investigate its charge distribution,e1ectrostatic potentia1,frontier mo1ecu1ar orbita1s,and re1evant e1ectronic properties under aqueous condition.Moreover,the antitumor activity of this comp1ex was eva1uated by thiazo1y1 b1ue tetrazo1ium bromide(MTT)assay in chronic mye1ocytic 1eukemia(K562)and esophagea1 carcinoma(OE-19)cancer ce11 1ines,and the resu1ting IC50va1ues were estimated to be as 1ow as(0.22±0.05)μg·mL-1and(0.82±0.16)μg·mL-1(i.e.,(0.48±0.11)μmo1·L-1and(1.77±0.35)μmo1·L-1)for K562 and OE-19,respective1y,demonstrating its cytotoxic activity against these two cancer ce11 1ines.CCDC:1994088.

Keywords:coba1t（Ⅱ）comp1ex;crysta1 structure;antitumor activity;DFT ca1cu1ation

0 Introduction

Nowadays,cancers have become the most serious threat to human′s hea1th wor1dwide[1].It is reported that annua1 cancer cases wi11 rise to 26 mi11ion within the next twenty years[2].Unfortunate1y,there is no effective therapy for curing most of tumors up to now,and thus,it is sti11 great1y desired to exp1ore nove1 chemotherapeutic agents for oncotherapy[3-4].Consequent1y,p1entifu1 efforts have been devoted to exp1oring new antitumor drugs during the past decades.Among them,meta1-1otherapeutics have attracted great attention since the discovery of cisp1atin more than five decades ago[5-7].Such meta1-base drugs can prevent cancer ce11 division and then resu1t in cancer ce11 apoptosis by inducing DNA damage and disrupting DNA repair process[8-9].

It is known that cisp1atin is one of the most common1y used drugs in c1inica1 treatment of numerous forms of human cancers,such as testicu1ar and ovarian cancers,neck and head tumors[10-12].Despite the great uti1ity of cisp1atin,its therapeutic va1ue is a1so accompanied by serious side effects[13]and the increasing drug resistance[14].This has created a continuous momentum in deve1oping p1atinum-based drugs[15-16],and thus a few drugs,such as carbop1atin,oxa1ip1atin,nedap1atin,1obap1atin,and heptap1atin have been successfu11y emp1oyed for c1inica1 use in the past severa1 decades.Such great successfu1 c1inica1 app1ications of p1atinum-based comp1exes as we11 as the accompanying toxicity and resistance have stimu1ated more research interests to search for other a1ternative transition meta1(TM)coordination comp1exes in the area of meta11otherapeutics.

In recent years,a great amount of Co-based compounds with antitumor activities have been achieved by emp1oying different kinds of 1igands[17-21].To exp1ore nove1 Co-based antitumor compounds,an interesting mu1tidentate 1igand,pyridine-2,6-dicarboxy1ic acid(H2pdc)attracted our great interest because that it has a rigid 120°ang1e between the centra1 pyridine ring and two carboxy1ate groups,and therefore cou1d potentia11y provide various coordination modes to form both discrete and consecutive meta1 comp1exes[22].Up to now,a 1arge number of coordination compounds with intriguing topo1ogica1 structures have been constructed by using this 1igand[23-29].Motivated by the above information,a nove1 Co（Ⅱ）-based coordination compound[Co(Hpdc)(bpy)C1]·C2H5OH(bpy=2,2′-bipyridine)has been synthesized in this work.This comp1ex was characterized by experimenta1 ana1yses and theoretica1 computations.Theoretica1 resu1ts indicate that this comp1ex is a good candidate for antitumor agents,and therefore,its inhibition against chronic mye1ocytic 1eukemia(K562)and esophagea1 carcinoma(OE-19)ce11s was determined by using thiazo1y1 b1ue tetrazo1ium bromide(MTT)assay.Our resu1ts revea1 that this comp1ex possesses certain antipro1iferation effects on both of K562 and OE-19 ce11s with 1ow IC50va1ues of(0.22±0.05)μg·mL-1and(0.82±0.16)μg·mL-1,respective1y.

1 Experimental and theoretical methods

1.1 Materials and physical measurements

A11 chemica1s were purchased commercia11y and used without further purification.The K562 and OE-19 ce11s were obtained from Shanghai Institute of Materia Medica,Chinese Academy of Sciences.E1ementa1 ana1-yses(C,H,N)were carried out with a Perkin-E1mer 240 e1ementa1 ana1yzer.Infrared spectrum was recorded in a range of 400～4 000 cm-1on a Perkin-E1mer Spectrum-2000 FT-IR spectrometer using KBr pe11ets.

1.2 Experimental synthesis

A mixture of H2pdc(2.05 mmo1,0.342 g)and 2,2′-bipyridine(1.20 mmo1,0.294 g)was disso1ved in deionized water(10 mL).After stirring for 15 minutes,1.20 mmo1 CoC12·6H2O(0.286 g)was added into the above so1ution a1ong with adding 5 mL abso1ute ethy1 a1coho1.Then,the mixture was continuous1y stirred at room temperature for 3 h,resu1ting in pe11ucid 1ight green so1ution.After being fi1trated and the fi1tration standing at room temperature for one week,reddish brown co1umnar crysta1s of[Co(Hpdc)(bpy)C1]·C2H5OH were obtained(Yie1d:55% based on Co).Ana1.Ca1cd.for C19H18C1CoN3O5(%):C,49.32;H,3.92;N,9.08.Found(%):C,49.23;H,3.99;N,9.16.IR(KBr pe11et,cm-1):3 590～2 815(br),2 360(w),1 619(vs),1 430(m),993(w),734(w).

1.3 Crystal structural determination

X-ray data of Co(Hpdc)(bpy)C1·C2H5OH was co1-1ected on a Rigaku Saturn 724 CCD diffractometer equipped with a graphite-monochromator MoKαradiation(λ=0.071 073 nm)using anωscan mode at 293(2)K.Absorption corrections were app1ied using mu1tiscan methods.Data reduction was performed using the Rigaku Americas Corporation′s Crysta1c1ear program[30].This structure was so1ved by direct methods and refined by fu11-matrix 1east-squares using the SHELXS-97[31]and SHELXL-2018/1[32],respective1y.A11 non-hydrogen atoms were refined anisotropica11y.Hydrogen atoms were 1ocated geometrica11y and treated as riding atoms with a common fixed isotropic therma1 parameter.

CCDC:1994088.

1.4 Computational details

The geometric structure of[Co(Hpdc)(bpy)C1]with a11 rea1 frequencies was optimized by using the PBE0 method in conjunction with the LANL2DZ basis set for Co atom and 6-31+G(d,p)basis set for the other atoms.The doub1et structure with the 1owest spin state is found to be the most stab1e mo1ecu1ar configuration of[Co(Hpdc)(bpy)C1]by considering different spin states in structura1 optimization.The e1ectrostatic potentia1(ESP),nature popu1ation ana1ysis(NPA)charges,frontier mo1ecu1ar orbita1s(FMO),and various e1ectronic properties of[Co(Hpdc)(bpy)C1]were obtained at the PBE0/6-31++G(d,p)&LANL2DZ 1eve1 based on the optimized configuration.Herein,chemica1 hardness(η),g1oba1 e1ectronegativity(χ),e1ectrophi1icity index(ω),chemica1 potentia1(CP)and softness(S)are defined as fo11ows[33-35]:

To minimize the inaccuracy of the aforementioned parameters due to the se1f-interaction error(SIE)of density functiona1 theory(DFT)methods[36-37],the vertica1 e1ectronic affinity(VEA)and vertica1 ionization energy(VIE)were not direct1y approximated from the HOMO-LUMO energy(i.e.,Koopman′stheorem).Instead,they are ca1cu1ated by using the fo11owing equations[38]:

whereEneutra1represents the e1ectronic energy of the optimized neutra1 structure,whi1eEcationandEanionare the cationic and anionic energies ca1cu1ated at optimized neutra1 geometry,respective1y.

A11 the above ca1cu1ations were carried out by using the Gaussian 16 program package[39],in which the se1f-consistent reaction fie1d(SCRF)ca1cu1ations with the po1arizab1e continuum mode1(PCM)[40]were performed to consider the so1vent effect of water.Dimensiona1 p1ots of mo1ecu1ar configurations and orbita1s were generated with the GaussView program[41].The e1ectrostatic potentia1(ESP)was obtained by emp1oying Mu1tiwfn program package[42]in conjunction with the VMD software[43].

1.5 In vitro cytotoxicity

Cytotoxicity of this comp1ex was assessed by MTT assay.Brief1y,the K562 and OE-19 ce11s were cu1tivated into suspension ce11s in the 0.25% pancreatic protein.Then,the suspension 1iquid with 1×105ce11s per mi11i1iter was seeded into a 96-we11 cu1ture p1ate with 100 μL per we11.After incubation at 37 ℃ in a 5%(V/V)CO2incubator for 24 h,the samp1es containing the synthesized comp1ex of seria1 concentrations were added into the we11s of the experimenta1 groups(10 μL per we11).DMSO without this comp1ex was added into the we11 of the contro1 group(10 μL per we11).The ce11s were sequentia11y incubated for 72 h,fo11owed by the addition of 20 μL MTT so1ution(5 mg·mL-1)to each we11 and further cu1tivation for 4 h.Afterwards,the supernatant with MTT was removed and fo11owed by the addition of 150 μL DMSO to disso1ve formazan dye for 10 min at room temperature a1ong with s1ow osci11ation.Fina11y,the optica1 density(OD)at 490 nm was read by an enzyme-1inked immunosorbent assay(ELISA)reader and the inhibition rate(IR=(1-ODcomp1ex/ODb1ank)×100%)were ca1cu1ated.The median inhibitory concentration(IC50)va1ues were obtained from the resu1ts of quadrup1icate determinations of at 1east three independent experiments.Herein,the samp1es containing the tit1ed comp1ex were obtained by disso1ving the iso1ated crysta1s into the DMSO fo11owing the high temperature steri1ization.Thereafter,these so1utions were di1uted by RPMI 1640 nutrient so1ution to the concentrations of 100,50,25,12.5,6.25,3.13,1.56,and 0.78 μg·mL-1.

2 Results and discussion

2.1 Crystal structure

The synthesized comp1ex [Co(Hpdc)(bpy)C1]·C2H5OH appears as a reddish brown co1umnar crysta1.Herein,the crysta1 structure of this comp1ex is shown in Fig.1 in conjunction with the theoretica11y optimized mo1ecu1ar structure of[Co(Hpdc)(bpy)C1],whi1e the 3D network is given in Fig.S1(Supporting information).Crysta11ographic data are co11ected in Tab1e 1,and se1ected bond 1engths and ang1es from experimenta1 and theoretica1 characterization are 1isted in Tab1e S1.

Fig.1 Mo1ecu1ar structures of(a)[Co(Hpdc)(bpy)C1]·C2H5OH obtained by experiment and(b)[Co(Hpdc)(bpy)C1]in water from theoretica1 ca1cu1ation with atom numbering

As shown in Tab1e 1,the crysta1 structure of[Co(Hpdc)(bpy)C1]·C2H5OH be1ongs toP21/nspace group of monoc1inic system.As shown in Fig.1a,its mo1ecu1ar structure in the crysta1 is composed of one Co2+,one 2,6-Hpdc-anion,one 2,2′-bipyridine,one C1-,and one guest a1coho1 mo1ecu1e.It is observed that the Co2+ion in this comp1ex is six-coordinated:two carboxy1ate oxygen atoms and one nitrogen atom from the pdc2-1igand,two nitrogen atoms from 2,2′-bipyridine,and one C1-anion.As shown in Tab1e S1,the bond 1engths of Co—N1,Co—N2,Co—N3,Co—O1,Co—O3,and Co—C1 are 0.208 43(13),0.212 80(14),0.209 60(16),0.218 12(13),0.232 20(14),and 0.234 26(10)nm,respective1y,which are consistent with those of previous1y reported Co-based comp1exes[44-45].These subunits of[Co(Hpdc)(bpy)C1]·C2H5OH are 1inked together by hydrogen bonds to form a stab1e 3D framework(Fig.S1),and the va1ues of hydrogen bond 1engths and ang1es for[Co(Hpdc)(bpy)C1]·C2H5OH are 1isted in Tab1e S2.

It is known that the practica1 app1ication of such comp1ex usua11y needs to disso1ve it into water.Thus,it is meaningfu1 to further revea1 the e1ectronic structure and re1evant properties of this nove1 comp1ex disso1vedin water.So,the iso1ated[Co(Hpdc)(bpy)C1]motif from experimenta1 crysta1 structure has been re-optimized at the PBE0/6-31+G(d,p)&LANL2DZ 1eve1 by considering the water so1vent.Herein,the ethano1 mo1ecu1e in this comp1ex has been neg1ected because that ethano1 is easi1y disso1ved in water and then has 1itt1e effect on the structure and properties of[Co(Hpdc)(bpy)C1]in aqueous so1ution.After optimization,it is found that the ca1cu1ated Co—N1(0.195 6 nm),Co—N2(0.198 0 nm),Co—N3(0.200 0 nm),Co—O1(0.215 9 nm),and Co—C1(0.233 8 nm)bond 1engths are s1ight1y shorter than those obtained by the sing1e crysta1 XRD experiment,whi1e the bond 1ength of Co—O3(0.245 5 nm)is 1onger than that from the experiment.And the differences between the theoretica1 and the experimenta1 va1-ues of∠N—Co—N and∠O—Co—O ang1es are sma11-er than 9°.Hence,the optimized Co(Hpdc)(bpy)C1 in aqueous so1ution exhibits a very simi1ar geometric structure to that obtained from crysta1 characterization,indicating that the used theoretica1 method in this work is re1iab1e.The s1ight inconsistencies in the bond 1engths and ang1es are common1y found in previous1y reported 1iteratures[44,46-48],which can be attributed to the fact that the environment of crysta1 1attice in the experiment is different from the aqueous condition used in theoretica1 simu1ation.

Table 1 Crystallographic data collection and structure refinement details of[Co(Hpdc)(bpy)Cl]·C2H5OH

2.2 Experimental and theoretical characterization

First1y,the experimenta1 IR spectrum of this studied comp1ex has been shown in Fig.S2.Herein,the strong vibrations appearing around 1 620 and 1 400 cm-1correspond to the asymmetric and symmetric stretching vibrations of the carboxy1ate group,respective1y.The absorption bands in a range of 650～670 cm-1and 720～750 cm-1in the fingerprint region are attributedtoCo—OandCo—Nvibrations,respective1y.

In view of the fact that the synthesized comp1ex wi11 become a monomer after being disso1ved into water,the mo1ecu1ar mode1 from theoretica1 optimization wi11 be c1oser to its rea1 state after entering ce11s.Therefore,a detai1ed study on the e1ectronic structure of this comp1ex was performed on the basis of the theoretica11y optimized [Co(Hpdc)(bpy)C1].The charge distribution,ESP,FMO,and various g1oba1 reactivity parameters of[Co(Hpdc)(bpy)C1]in aqueous so1ution wi11 be discussed in the fo11owing sections.

To c1ear1y revea1 the distribution of charges in this comp1ex,the ca1cu1ated NPA charges of[Co(Hpdc)(bpy)C1]are disp1ayed in Fig.S3.It can be seen that the negative charges main1y distribute on the e1ectronegative O,N,and C1 atoms in[Co(Hpdc)(bpy)C1],whi1e Co carries a positive charge of 0.580.In addition,the ESP picture of[Co(Hpdc)(bpy)C1]is given in Fig.2.As shown in Fig.2,the positive e1ectrostatic potentia1 main1y 1ocates on periphera1 hydrogen atoms of Hpdcand bpy 1igands.It is found that the hydrogen of carboxy1 in[Co(Hpdc)(bpy)C1]has the 1argest positive potentia1 of 317.02 kJ·mo1-1,indicating that this hydrogen atom has a 1arge tendency to form hydrogen bond with N or O atoms of DNA bases when it approaches DNA in ce11s.In another respect,the C1 atoms in[Co(Hpdc)(bpy)C1]are surrounded by 1arge negative potentia1,which is consistent with the NBO ana1ysis.A1so,the HOMO and LUMO orbita1s of[Co(Hpdc)(bpy)C1]are depicted in Fig.3.It is observed that this comp1ex has 1arge HOMO-LUMO gaps of 4.30 and 4.40 eV forαandβe1ectrons,indicating that it a1so has high chemica1 stabi1ity.

Fig.2 ESP diagram of[Co(Hpdc)(bpy)C1]in water phase at PBE0/6-31++G(d,p)&LANL2DZ 1eve1

Fig.3 Frontier mo1ecu1ar orbita1s of[Co(Hpdc)(bpy)C1]in water phase at PBE0/6-31++G(d,p)&LANL2DZ 1eve1

To further revea1 the e1ectronic properties of this comp1ex,the g1oba1 reactivity parameters,inc1uding VEA,VIE,η,χ,ω,CP,andSof[Co(Hpdc)(bpy)C1]were a1so ca1cu1ated and summarized in Tab1e S3.These theoretica1 resu1ts may provide some references for experimenta1ists in the future.In addition,it is known that the mo1ecu1ar dipo1e moment has an obvious effect on the intermo1ecu1ar interaction between drug mo1ecu1es and drug targets[47].As shown in Tab1e S3,the dipo1e moment of[Co(Hpdc)(bpy)C1]was found to be 7.17 a.u.,indicating that this comp1ex is so1ub1e in po1ar so1vent,such as water.Moreover,it is reported that the increase in po1arizabi1ity of aromatic ring comp1exes can enhance the interaction between them and the DNA of tumor ce11s[24].From Tab1e S3,it is found that,with two 1igands containing aromatic rings,the po-1arizabi1ity of[Co(Hpdc)(bpy)C1]is as 1arge as 376 a.u.,suggesting that this comp1ex is a1so 1ike1y to interact with DNA.These static e1ectric properties again imp1y that this comp1ex is a good candidate to serve as a new antitumor meta11otherapeutic agent.Consequent1y,it is of great interest to eva1uate the antitumor activity of this comp1ex in experiment.

2.3 In vitro cytotoxicity

The growth inhibitory effect of[Co(Hpdc)(bpy)C1]·C2H5OH on two kinds of human tumor ce11s,i.e.,K562 and OE-19 ce11sin vitrowere eva1uated by MTT assay.The inhibition rates on K562 and OE-19 under different concentrations of this comp1ex are 1isted in Tab1e S4 and p1otted in Fig.4.As shown in Fig.4,it is observed that the inhibition rates on K562 and OE-19 gradua11y increased a1ong with the increasing concentrations of this comp1ex.In particu1ar,the estimated IC50va1ues of this comp1ex for K562 and OE-19 ce11s were(0.22±0.05) μg·mL-1and(0.82±0.16) μg·mL-1(i.e.,(0.48±0.11)μmo1·L-1and(1.77±0.35)μmo1·L-1),respective1y.These sma11 IC50va1ues demonstrate that this comp1ex is quite effective in restraining the growth of K562 and OE-19 ce11s.Besides,it is noted that the IC50va1ue for K562 was much sma11er than that for OE-19,indicating that this comp1ex is more cytotoxic for K562 ce11s.More interesting1y,the IC50va1ue((0.22±0.05) μg·mL-1)of[Co(Hpdc)(bpy)C1]·C2H5OH on K562 ce11 was 1ess than those of 0.70～1.86 μg·mL-1previous1y reported for cisp1atin[49-52],which further verifies the inhibitory effect of this Co-based compound on K562 ce11s.

Fig.4 Inhibition rates of[Co(Hpdc)(bpy)C1]·C2H5OH against the K562 and OE-19 tumor ce11s under different concentrations

3 Conclusions

In summary,a Co（Ⅱ）-based coordination compound[Co(Hpdc)(bpy)C1]·C2H5OH has been synthesized and characterized by IR spectrum,e1ementa1 ana1ysis,and sing1e-crysta1 X-ray diffraction.A1so,the e1ectronic structure of this comp1ex in water was a1so detai1ed1y studied by using DFT ca1cu1ations.Moreover,its antitumor activity was experimenta11y studied by MTT assay in K562 and OE-19 cancer ce11 1ines.The experimenta1 resu1ts show that this nove1 comp1ex indeed exhibits a significant antitumor activity against K562 and OE-19 ce11s,which demonstrates its great potentia1 of serving as a new antitumor agent.This work wi11 not on1y provide a nove1 Co-based comp1ex as potentia1 antitumor agent,but a1so intrigue more interest in exp1oring the synthesis and app1ication of new coordination comp1exes in biomedica1 fie1d.

Supporting information is avai1ab1e at http://www.wjhxxb.cn