With the acceleration of social urbanization and industrialization process, environmental pollutantsand the new chemical substances are increasing all the time. Combined with the impact of unhealthylifestyles and behavior, malignant cancer has become the number one killer of human health. Althoughthere are a large number of anticancer drugs continuously listed, efficacy of these drugs has always beennot recognized due to poor selectivity and ease of anticancer drugs tolerability. Therefore, researching anddeveloping of the new structure of the anti-cancer lead compounds becomes a major challenging issue inthe field of pharmaceutical chemistry and of great significance. The mechanism of quinolones onanti-tumor is similar with sterilization mechanism. The targets of these drugs in the cells are two kinds ofDNA topoisomerases: DNA gyrase and topoisomerase IV. According to this principle, Scientists havesynthesized a large number of antitumor quinolones compounds.At present, people focus on the structural modification of the quinolone compounds are concentratedin the N-1and C-7of the quinoline ring, there are less structural modification in Other location. But thesedrugs failed to enter clinical evaluation because of low bioavailability, In vivo toxicity and the body is easyto metabolic inactivation.In order to find effective ways of structural modification of antibacterial quinolone to thetransformation of the antitumor quinolone and get a certain anti-tumor activity of new quinolones, we usethe electronic isostere of heterocyclic or fused heterocyclic to replace the C-3position.1. Design and synthesis of target compoundsIn this paper, we use biological isostere and activity of splicing design principle to design andsynthetize26new compounds with ofloxacin and levofloxacin, and get the correspondingly targetcompounds. C-3carboxyl-bit of quinoline ring has been insteaded by triazole sulfide orthiazolo[3,2-b][1,2,4] triazole. The structures of new compounds syn-thesized had been characterized byMS、1H-NMR and IR. 2. Evaluation of antitumor activity in vitroThe inhibitory activity in vitro of cancer cell against A549cells, Bel-7402cells and HCT-8cells hasbeen evaluated by MTT assay respectively. The results show that the target compounds triazole sulfide andthiazolo [3,2-b][1,2,4] triazole have potential growth inhibitory activity on A549cells, Bel-7402cells andHCT-8cells3. ConclusionStructures of21target compounds were confirmed by spe-ctral data, the results of antitumor activityin vitro show that:10,12和21have very good growth inhibitory activity on A549cells and HCT-8cells.Therefore, quinolone C-3carboxylic is not necessary groups for antitumor activity, the modified structuresin the quinolone C-3carboxylic maybe developed into poten-tial anticancer drugs.
Development of Brucine-loaded Microsphere/Thermally Responsive Hydrogel Combination System for Intra-articular Administration
Intra-articular drug delivery system could directly delivery drug to an affected joint and offer thepossibility of reaching high drug concentrations at the site of action with limited systemic toxicity.However, depending on their chemical structure, some active compounds were rapidly cleared from thejoint, thus requiring numerous injections, which could cause infection or joint disability. To control therelease behavior for prolonged time periods, a novel biologically based drug delivery vehicle was designedfor intra-articular using microsphere/thermally responsive hydrogel combination system in this paper. Andthe brucine was the test drug.Brucine is the active ingredient extracted from Strychnos nux-vomica L. Nux vomica, also known asstrychnine, first recorded in Compendium of Materia Medica, and recorded in each China Pharmacopoeia,is the dried ripe seeds of the Strychnos nux-vomica L. It has effects of activating meridians to stop pain andsubsiding swelling, especially for treating disease like RA and OA.It has been used clinical in traditionalChinese medicine for the treatment of rheumatoid paralysis, numbness, bruises and other diseases afterbeen concocted.Brucine is the active ingredient of nuxvomica, has the effect of analgesic,anti-inflammatory, anti-immune, anti-tumor, anti-arrhythmia, and antagonize the NO inhibitory effect onchondrocyte proliferation. The process of Osteoarthritis （OA） is mainly in the degration of chondrocytes;brucine can effectively promote the proliferation of chondrocytes, hence repair the damage cartilage. Oneof the pathogenesis of Rheumatoid Arthritis （RA） is self-immune, the effect of anti-immune and stop paincan alleviate distress of RA patients.The system was constructed by disperse the brucine microspheres which was prepared by using aspray-drying method in a thermally responsive biopolymer hydrogel contained of chitosan-glycerol-borax.The hydrogel can block the burst release of microspheres, and the microspheres were incorporated inhydrogel as drug depot.The duplexing release system could effectively prolong the release behavior in thejoint cavity, increase the efficiency of brucine for a extended period of time.The microspheres were prepared by Switzerland Buchi B290spray-drying apparatus.Firstlydetermines the craft factors of outlet temperature, Q-flow, pump speed. The outlet temperature was120℃,Q-flow was40m3/h, Pump speed of20%·min-1. Then, investigate the influence of type of chitosan,chitosan concerntration, glutaral ratio, ratio of brucine to chitosan on the entrapment efficiency （EE） and in vitro drug release behavior of brucine micropheres. By single-factor test chose three factors, by orthogonaldesign optimization prescription, selected the best prescription for Chitosan0.75%, glutaral ratio of1:15,the ratio of brucine to chitosan is1:5.The Drug loading（DL%） was15.57%±0.07%and Entrapmentefficiency（EE%） was98.61%±0.42%.The brucine microspheres were characterized by SEM, XRD, and FTIR, spherical as evidenced by thescanning electron microscopy （SEM） photographs. With an average size of2.45μm, in range of0.9–4.5μm,agree with normal distribution. Fourier transforms infrared （FT-IR） spectroscopy and X-ray diffraction（XRD） revealed the absence of drug-polymer interaction and amorphous nature of entrapped drug.The thermosensitive converse hydrogel was constructed using chitosan-glycerol-borax as matrix,taking gelation time and gelation condition as index, investigate the influence of concerntraion of chitosan,ratio of chitosan to glycerol,pH on physical properties of hydrogel. The best prescription was CS2%, CS:glycerol10:1, pH6.7.Taking tube method, investigated the in vitro drug release of brucine microsphere（BM）, brucinehydrogel（BH）,brucine microsphere hydrogel（BMH）, the release behavior of each preparation was evaluatedby first-order, Higuchi, Hixon-Crowell; Peppa’s and Double exponential biphasic kinetics equation. Threepreparations were all fit Double exponential biphasic kinetics equation.Investigated the biocompatibility of BM, BH, BMH to rats’ synovium, results showed that thepreparations appeared to be generally biocompatible with synovium and hence it might be suitable for thedevelopment of treatment strategies for arthritis diseases.In vivo FX imaging technology was employed to determine the release behavior of preparation in vivo.Encapsulated infrared dye LF NIRD-15into microspheres instead of brucine; visually investigate theretention time of drugs in the joint cavity. The release behavior of microsphere and microsphere hydrogelwere investigated, results showed that by dispersing the microspheres into hydrogel, the burst wereobviously retarded, and could delivery drug for7days above.The pharmacokinetics of BMH in rats were preliminarily studied.Firstly using RP-HPLC establish themethod to analyse brucine in vivo.Rats were administrated with BS, BM, BMH respectively, using3p97software calculated the pharmacokinetic parameters, results indicated that BMH has a MRT1.93times ofBM,3.38times of BS, could meet the request of design of our topic. In conclusion, the system significantly prolonged the release of brucine in cavity for more than7days,decrease the frequency of injection and improve the compliance of patients, hence it might be suitable forthe development of treatment strategies for rheumatic diseases.
Synthesis and Biological Evaluation of New2-(Benzylthio)-5-aryloxadiazole Derivatives
Epidermal growth factor receptor (EGFR) plays a critical role in regulating cell proliferation, differentiation, and migration. The up-regulated activity of EGFR correlates with many human tumors, including lung, breast, bladder, prostate, and kidney cancers. It has been also observed the overexpression of EGFR in breast cancer, ovarian cancer and so on. Therefore, EGFR tyrosine kinase represents an attractive target for the development of novel anti-cancer agents. As this kind of inhibitors, gefitinib and erlotinib have been approved by US FDA for treatment of patients with non small-cell-lung cancer (NSCLC).Oxadiazole ring as a building element can be seen in many natural products. Compounds containing one or more oxadiazole ring have various activities such as hypoglycemic, anti-fungal, anti-inflammatory, anti-bacterial and anti-tumor activities. The previous literatures few involve in the anti-tumor drug target for this oxadiazole derivatives when they describe their anti-tumor activities.For further study of the interaction with the probable target of this derivatives and to obtain leading compounds with better bioactivities, we design and synthesize twenty2-(benzylthio)-5-aryl-1,3,4-oxadiazole derivatives. Thirteen compounds are completely new compounds. The result of anti-proliferative in vitro indicates that this5-aryloxadiazole derivatives display more potent anti-proliferative activity in MCF-7cell line than the other two tumor cell lines. In particular, 2-(5-(benzylthio)-1,3,4-oxadiazol-2-yl)aniline, compound3e displays the most potent anti-proliferative activity with IC50of1.09μM, superior to the positive control gefitinib. Further EGFR inhibitory activity experiment can demonstrate that this5-aryloxadiazole derivatives could play their role by inhibiting EGFR.In addition, the result of molecular docking also shows that compound3e could bind to ATP binding site of EGFR kinase. The hydrogen bond and Pi-Pi interaction stabilize the enzyme-ligand complex, which are formed with MET769and LYS721, respectively. The formation mode of hydrogen bond as well as the trend of compound’s conformation in ATP binding site is similar to the known EGFR inhibitor Erlotinib. This finding further demonstrates this5-aryloxadiazole derivatives can be able to perform their antiproliferation via inhibiting the function of EGFR. As a result, it explains a possible mode of this derivatives interacting with EGFR from molecular aspect.
Design,Synthesis and Study on the Antitumor Activity of Benzamide HDAC Inhibitors Containing Fused Heterocyclic Groups
Following cardiovascular disease, cancer is currently the second majorcause of death in the world. Because of lacking of the selectivity to the drugtarget,traditional chemotherapeutic drugs can kill many normal cells anddamage to normal tissue, even to some serious adverse reactions when they killthe cancer cells,so it has not yet reached satisfactory results. In the clinicaltreatment , resistance of multidrug in tumors has also become the main reasonof chemotherapy failure.Transcriptional regulation of gene is the premise which maintains normalfunction of the heathy cells. When the transcriptional regulation is dysfunctional,the cell may become cancerous. The acetylation status of histone play animportant regulatory role in gene transcription, so it has a close relationshipwith the generation and proliferation of the tumor and expression levels ofoncogene and tumor suppressor gene. Histone acetylation status is decided bythe two types of enzymes: histone acetyltransferase activity (HAT) and histone deacetylase (HDAC). Under normal physiological conditions, the regulation oftwo types of enzymes for histone acetylation is in a state of equilibrium. Whenthe transformation occurred, HDAC activities servously increased which makesHDAC and HAT out of balance and impact cell proliferation and cell cycleregulation molecule expression imbalance and cell deterioration.Histone deacetylase inhibitors because they can effectively inhibit tumorcells proliferation and promote differentiation, induce apoptosis, avoid tumorresistance and have the advantages of high selectivity, specificity, efficiency andlow toxicity, is a new direction for cancer treatment. Histone deacetylaseinhibitors contain two categories: hydroxamic acid and benzamide. Benzamidehave the advantage of high selectivity, low toxicity and good tolerabilityadvantages compared with the hydroxamic acids . Some benzamide drugs haveentered into clinical phase, such as CI994, MS-275,CS055 and MGCD0103.MGCD0103 is another Histone deacetylase inhibitor with oral bioavailabilityand has high anti-cancer activities and selectivity, but its bioavailability for thedog and rat are 1-90% and 47%,Coefficient of variation is big.Based on MGCD0103 as the leading compound, we have used theprinciple of non-classical electronic emission to transformat the structure ofMGCD0103. According to the previous studies in our work, we replacepyrazine, substituted phenyl, benzothiophene or their derivative to pyridinering.,but their water-solubility and biological activities is bad, pharmacokineticresults are not satisfactory too.The heterocyclic groups can be used asstress-activated protein kinase, interleukin-1 receptor-associated kinase,cyclin-dependent kinase, phosphatidylinositol 3 – kinase inhibitors ,benzodiazepine receptors and benzodiazepine ligands, so they are contained in the head of some drugs, they are also good hydrophobic groups and have goodpotential medicinal value. So we took heterocyclic groups or substitutedheterocyclic groups instead of pyridyl group of MGCD0103;improved thehydrophobicity of the enzyme binding sit, increased the combination with thetargets, and maintained the structure of the connection area of the planarstructure or increased the length of the connection area and kept its active siteN-(2 – aminophenyl) benzamide in the enzyme inhibition; pulled fluorine atomsin meta position or ortho position of 2′-Amino; enhance the condensationbetween the amino and carboxylic acid, to improve the stability of the aminoand to examine its impact on the activity. We designed and synthesized 12 newcompounds, and their structures had been verified by 1H-NMR.We used MCF-7 to evalute the activities of target compounds in vitro . Theresults showed that eight of them have some inhibition activity. There are seventarget compounds showed better anti-activity in vitro than MGCD0103, especia-lly compound 2,3 showed the best activity, and their activities are almost theMGCD0103 six times.
Research on Controlled Release Drug Delivery System of Biomaterial and Ceramic Porous Scaffolds
In clinic,it has been difficult to repair the large critical size defects arising from trauma,infection,tumor resection.Autologous bone is still the gold standard in bone reconstructive surgery because of its osteoconductivity, osteoinductivity,osteogenic potential and lack of immunogenicity as well.However,this standard auto graft has its own share of problems like inadequate amount,at least10%morbidity and potential complications in bone surgery.And it will take a long time to creep substitution. The bone allograft and xenograft has antigenicity, especially in large bone graft, often fail in the graft due to severe immune rejection, and has danger in the spread of pathogens as well. Therefore, in recent years with the emergence of tissue engineering, people began to use the principles and techniques of tissue engineering to plant the cells which have the osteogenic potential in the scaffold after differentiation, proliferation. And at last the engineered artificial bone has been made and can promote a large segmental bone defect repair. In cultivating tissue engineered bone in vitro, specially in large tissue engineered bone, the currently used bone substitutes still face many unsolved problems including incomplete formation of new bone tissue, failure of neovascularization, and slow growth of the capillary network. Meanwhile, drug is ofen used for better bone healing in clinical.However, due to bone tissue, such as in the biological density, low blood flow, permeability and poor specificity, the traditional methods of drug administration is difficult to arrive at the ideal site of the lesion, and thus its efficacy is low.Hydroxyapatite (HA) has very similar composition and structure to natural bone and with good biocompatibility, bone osteoconductivity and osteoinductivity.It has been widely used as bone substitute materials. HA can absorb chemical molecules and biological macromolecules such as proteins, so as a drug carrier has been widely studied. But it has problems with adsorption of impurities, low drug loading and burst release.A novel scaffold with large size and controlled porous structure has been designed and prepared for the aim of massive tissue engineered bone in previous experiment of our group. It was composed of HA spherules and porous HA tube.HA spherules can easily compound with biological active substance before being filled into the HA tubes. This study was designed to load the biological active substances promoting bone growth, neovasculariztion on the scaffold and get a new drug delivery system.This system can release the biological active substances in a sustained and effective manner in order to promote bone growth and healing in the repair of bone defects.HA spherules were prepared by the method of sol-gel and water/oil emulsification technology and the porous structures were characterized. Sal B,the major water-soluble components of the traditional Chinese medicine radix salviae miltiorrhizae, was investigated on osteoblast proliferation and differentiation and was loaded on the HA spherules.Then the drug-loaded spherules were wrapped with several different concentrations of PLA and several different concentrations of chitosan. The case of drug release and the best package substance with the right concentration were investigated. Finally,the influence of Sal B-loaded HA sustained-release spherules on the activity of osteoblasts cultured in vitro was studied.The main conclusions were obtained as follows:1. The HA spherules,prepared by the method of sol-gel and water/oil emulsification technology,possessed the porous structures can meet the organization growth.2. Sal B has been proven to be effective in promoting osteoblast proliferation and differentiation. SalB has the potential toameliorate bone healing by stimulating both the total metabolic activity and ALP activity of osteoblastic cells. It was suggested that Sal B at a mass concentration=160μg/mL significantly contributed to osteoblast proliferation. Moreover, the higher mass concentration indicated more obvious proliferation. However, Sal B at a mass concentration>160μg/mL had toxic effect on ostaoblasts. It showed that Sal B can promote bone growth and healing as the biological active substances used in the repair of bone defects, and the best effective concentration was160μg/mL.3. The results showed that PLA can form film but can not be attached to surface of HA sperules due to its hydrophobicity.So it was easy to come off and wrappd poor.However,it were explored that chitosan has good hydrophilicity,good film-formed and good cell compatibility,which was used to wrap on the surface of the HA spherules to get the Sal B-loaded HA sustained release spherules and2%concentration percentage was choosen in this study.4.The chitosan-coated Sal B-loaded HA sustained-release spherules prepared in this study can release Sal-B in vitro for a long period, effectively promoted osteoblast proliferation, maintained biological activity of osteoblasts, contributed to osteoblast differentiation and proliferation, increased utilization rate of drug in local region, decreased adverse reaction, and persistently contributed to bone formation. It is helpful for construction and repair of bioceramic scaffolds and is of high clinical value in treatment of large segmental bone defects.
Study on the Molecular Design, Synthesis and Antioxidation Activity of Ferulic Acid Derivatives
Ferulic acid has biological activity in antioxidation, anti-tumor, anti-lipoperoxidation and anti-aging. Ferulic acid exists widely in many medicinal plants with limited resources cause the content is very low, it can not meet the demands of the market, so the synthesis of ferulic acid and its derivatives has become one of research hot spots. The theoretical calculation of quantum chemistry can study the relationship between the structure and antioxidant activities, which can reduce the blindness on screening structures of antioxidant, so it can serve as theoretical guidance on researching and developing new antioxidant.The structure, infrared, raman and1H/13C-NMR spectra of ferulic acid were calculated using density functional theory (B3LYP) and Hartree-Fock method. Based on this, we designed six series(twenty-six compounds) ferulic acid derivatives whose precursor structure was ferulic acid, the optimized geometries of neutral, radical, radical cation forms of ferulic acid and its derivatives were obtained. The two main working mechanism–H atom donation and single-electron trasfer were investigated, and the O-H bond dissociation enthalpy(BDE) and ionization potential(IP) were computed in gas phase. Five compounds of the1st serie were synthetized, and their antioxidant activities were measured experimentally by DPPH. Through the combination of experimental and the theoretical research, structure-activity relationship were analysed and many important conclusions were obtained as follows:(1)The computational results of ferulic acid showed that the structure optimized by B3LYP and HF were in substantial agreement, the main atoms and benzene ring created a conjugated system. The frequencies calculated by HF were10%higher than B3LYP, the vibrational modes of ferulic acid in DFT calculation were assigned and compared with the experimental datas from SDBS database, the calculated results were in good agreement with the experimental results. The caculated and experiment chemical shift of1H/13C-NMR existed some errors, the hydrogen bonding may bring big errors of1H-NMR, and13C-NMR had smaller errors than1H-NMR. Overall, the caculated and experiment results had good consistency, this suggested that DFT(B3LYP) and HF can effectively predict the structure of ferulic acid. (2)The computational results of series of ferulic acid derivatives showed that the introduction of withdrawing groups on the moleculars increased both BDE and IP,the electron-donating groups showed the oppsite effect,however the acetyl group showed opposite qualities in two working mechanism,the order of BDE was FA一3>FA一2> FA一5>FA一1>FA一4>FA一6>FA一7>FA-11. and the order of IP was FA一2>FA一5>FA一3> FA一4>FA-1>FA-7>FA-6>FA-11. BDE and IP decreased with the extension of ester chain. the order of BDE and IP was FA一1>FA一22>FA一23>FA一24>FA一25>FA-26. The BDE and IP increased with the introduction of groups with lone pairs of electrons on benzene ring,the order of BDE was FA一1>FA-8>FA一7>FA一10>FA一9,and the order of IP was FA一1>FA一7>FA一8>FA-9>FA一10. The extended conjugate chain in molecular decreased BDE and IP. the order of BDE and IP was FA-18>FA-1>FA-17. The introduction of hydroxy group, alkyl group and ester group on the end chain decreased BDE and IP,and aldehyde group and carboxyl group increased BDE and IP,the order of BDE and IP was FA-19>FA-l>FA-22>FA-20>FA-21. The introduction of ether on the side-chain decreased BDE and IP,however the length of ether had little influence on BDE and IP. he order of BDE and IP was FA-1>FA-14>FA-15>FA-16>FA-17> FA-18>FA-19.The antioxidant activities increased as the decreasing of BDE and IP.(3)5-bromovanillin and5-nitrovanillin were syntheSized using vanilla as raw material,the knoevenagel condensation of vanilla,5一bromovanillin,5一nitrovanillin, syringaldehyde and3一Methoxybenzaldehyde with malonate acid respectively giving products of corresponding ferulic acid and its derivatives,their production rate were about55％-75％, the structure had been determined by1H-NMR.(4)Antioxidant activities of ferulic acid and its derivatives were determined by DPPH·.The IC50of scavenging DPPH pf FA-1,FA-5,FA-2and FA-11were33.15,55.24,1802,23.66μmol·L-1, their order of capability of scavenging DPPH were FA-11>FA-1>FA-5>FA-2>FA-27,which showed phenolic hydroxyl group was the most important activity groups of antioxidant,the introduction of withdrawing groups on the moleculars decreased the activities of antioxidants,while the electron-donating groups showed the oppsite effect.The experimental results were in agreement with the computational results, which showed DFT can effectively disclose the relationship between molecular structure and antioxidant activities of ferulic acid and its derivatives,and played a significant role in synthesis and perfomance analysis of antioxidant.
Synthesis, Crystal Structure and Biological Activity of a-N-heterocyclic Thiosemicarbazones and Their Main Group Metal Complexes
Heterocyclic thiosemicarbazones and their metal complexes have generated considerable interest dueto varied coordination environments and marked biological activities, notable for antibacterial, antifungaland antitumor activities. In many cases, their biological properties are often related to the donor sequenceof the ligands and metal ion coordination. The widespread use of bismuth compounds in the medicinalrealm for centuries is coming from their high effectiveness and low toxicity in the therapy of diversemicrobial infections, involving syphilis, diarrhea, gastritis and colitis. In addition to antimicrobial activity,bismuth compounds revealed anticancer activities, Radioisotope212Bi and213Bi compounds have beenapplied as targeted radio-therapeutic agents for cancer therapy, and furthermore they have the ability toreduce the side-effects of cisplatin （cis DDP） in carcinoma treatment. Bismuth is also considerednowadays as the least toxic heavy metal and the bismuth （III） atom with a larger ionic radius has one inertelectron pair （6s2） and forms the complexes of the non-transition metal center with higher coordinationnumbers. Tin complexes are known for their multiple applications as antimicrobials and biocides.Moreover, organotin （IV） compounds are known to exhibit important cytotoxic effects against tumor celllines. Heterocyclic thiosemicarbazones and their main group metal bismuth （III） and gallium （III）complexes are scarce. Although heterocyclic thiosemicarbazones and its organotin （IV） complexes arenumerous, the syntheses of tin complexes with thiosemicarbazones could be a strategy of preparation ofnew drug candidates in which the metal and ligand could act synergistically. The chemists and medicalscientists always devote themself to synthesis and biological evaluation of the better and more practicalthiosemicarbazones and its organotin （IV） complexes. So it is important to study the synthesis, crystalstructures and biological activities of heterocyclic thiosemicarbazones and their main group metal bismuth （III）、 gallium （III） and organotin （IV） complexes in the finding of new drugs against bacteria and cancer.In this paper, we studied the synthesis, single-crystal structures and biological activities ofbis（2acetylpyrazine） thiocarbonohydrazone （H2L1）、2-acetylpyrazine N（4） phenylthiosemicarbazones（HL2）、2-acetylpyrazine N（4）-pyridylthiosemicarbazones （HL3）、2-acetylpyridine N（4）pyridylthiosemicarbazone （HL4）、2-acetylpyridine N（4） pyridylthiosemicarbazones （HL5） and2.6-diacetylpyidine bis（N（4）-methylthiosemicarbazones）（HL6） and their main group metal complexes.Main group metal complexs [Bi（HL1）（NO3）2（H2O）]（1）、[Ga（L1）（HL1）]（2） and[（Ph）2Sn（HL1）（CH3COO）] CH3CH2OH （3） where H12L=bis（2-acetylpyrazine） thiocarbohydrazones;Bismuth（III） complex [Bi（L2）（NO3）2（CH3OH）]（4） and gallium（III） complex [Ga（L2）2]·（NO3）·（H2O）（5）derived from2-acetylpyrazine N（4）-phenylthiosemicarbazones （HL2）; Bismuth（III） complex[Bi（HL3）·（NO3）3]（6）,[Bi（L4）·（NO3）2·（CH3CH2OH）]（7） and [Bi（HL5）·（NO3）3]（8） derived from2acetylpyrazine N（4）-pyridylthiosemicarbazones （HL3）,2acetylpyridine N（4）-phenylthiosemicarbazones（HL4）,2-acetylpyridine N（4）-pyridylthiosemicarbazones （HL5）, respectively, and Bismuth（III） complex[Bi（H2L6）（NO3）2] NO3（9） derived from2.6-diacetylpyidine bis（N（4）-methylthiosemicarbazones）（H2L6）have been synthesized and characterized by elemental analysis, MS, IR spectra and single-crystal X-raydiffraction studies. Biological studies, carried out in vitro against selected bacterias and different cancercell lines, respectively, have shown that the free ligands and theirs corresponding complexes show distinctdifferences in the biological properties.Bismuth （III） complexes show higher activities against Gram positive bacteria Bacillus cereus andGram negative bacteria Salmonella typhimurium than positive control antibiotics ampicillin （Amp）,chloramphenicol （Cm）, kanamycin sulfate （Kan）, respectively. Gallium （III） complexes2、5are inactiveagainst the tested microorganisms. Organotin （IV） complex3only shows excellent activity against Staphylococcus aureus （MIC=15.6μg/mL）.The comparison of cytotoxic activity of the free ligands and their complexes indicates that bismuth （III）complexes1、7、8and9show much lower IC50value than the other compounds. Its possible apoptoticmechanism of complexes1、7、8was evaluated in HepG2cells. For morphologic observation, cells werestained with acridine orange （AO） and ethylene dibromide （EB） and assessed by fluorescence microscopy.For detecting the change of the mitochondria membrane potential （MMP）, Rhodamine123dye combinedthe high content live cell imaging system was used. For analysis of intracellular ROS, theoxidation-sensitive fluorescent probe DCFH DA was used for complexes1and7.Effect of the complex8on Annexin-V FITC/PI double staining and caspase activation was measuredin HepG2cells by indirect immunofluorescence.The acute toxicity and antitumor activity of complex9were evaluated on leukemia P388-bearing mice.The LD50of9is44.7mg/kg in Kunming mice by caudal vein injection （95%confidence interval39～51.2mg/kg）. The tumor inhibition ratio of the complex9is61.6%.
Effects of Net Charge and Helicity on the Biological Activities of Membrane Active Peptides
Nowadays, cancer has become a world-wide problem with the incidence of cancerincreasing every year. Both of human health and the safety of life face a huge threatand there are still some greater challenges for present cancer research to go forward.In addition to antibacterial activities of antimicrobial peptides (AMPs), a significantnumber of these bactericidal peptides have shown to have a broad spectrum ofanticancer and antiviral activities. And those cationic antimicrobial peptides withanticancer activities can be called anticancer peptides (ACPs). With low-accessibilityof induction of resistance due to aiming at the membrane of tumor cells, amphipathicα-helical anticancer peptides prove their promising potentials in cancer treatments.Compared with the neutral membrane of human normal cells, anticancer peptides aremore likely to be attracted to the negatively charged membrane of bacterial and cancercells. Thus some of anticancer peptides are more toxic to bacterial and cancer cells,not human normal cells. The plasma membranes of human normal cells carry anoverall neutral charge on their outer surface: primarily due to the fact that the majorlipid components of these membranes are zwitterionic, including:phosphatidylethanolamine, lecithin and sphingomyelin. While the outer membrane ofcancer cells contains only a small amount of phosphatidylserine (PS), being onlyslightly more negatively charged. Although PS is only3%-9%of the totalphospholipides of cancer cell membranes, PS plays an important role in the anticanceractivities of anticancer peptides.We utilized an amphipathic α-helical anticancer peptide A12L/A20L (Peptide P)with significant anticancer activity as the framework, systematically designed a seriesof peptide analogs with different net charges by replacing certain amino acid residues,to study the effects of net charge and the number of positively charged residues on thehydrophilic/polar face of peptide P on biological activity of cationic anticancer peptides. Synthesis of the peptides was carried out by solid-phase synthesis usingFmoc chemistry and purification by reversed-phase high performance liquidchromatography (RP-HPLC) with purification over95%. All the peptides areidentified and analyzed quantitively using ESI-MS and amino acid analysis. Ourresults showed that the ability of anticancer peptides to self-associate decreased as thepositive charge on the polar face of peptides increased and the total hydrophobicitydecreased. CD spectra of the anticancer peptides indicated that high helical structureof all the peptide analogues could be induced in the mimic of the membrane’shydrophobic environment. However, when the net charge of peptide was too high, theelectrostatic repulsions among the intramolecular positive amino acids of peptide willaffect peptide-folding and α-helical stability. We also found that there was an obviousnet charge threshold among the anticancer peptide P analogs. Increases or decreases innet charge beyond the threshold value resulted in a dramatic reduction in bothanticancer activity and therapeutic index. Compared with the neutral membrane ofhuman normal cells, the negatively charged membrane of cancer cells is much moresensitive to net charge changing of the anticancer peptide. As the net charge changes,the hemolytic activity of analogs within the range of concentrations examinedchanged slightly，while the anticancer activity exhibited remarkable differences. Ouramphipathic α-helical anticancer peptide analogs showed good specificity betweencancer cells and human normal cells, which might play a crucial role in makingfurther improvement in target selectivity, and designing anticancer peptide agents withhigher efficacy and lower toxicity.In addition, we altered peptide helicity by replacing L-amino acid using D-aminoacids, to study the effect of helicity on the selectivity of anticancer peptides, and wehave successfully selected two anticancer peptides with high anticancer activity andlow toxicity to the human normal cells. In this paper, we utilized liposomes to mimicprokaryotic membrane and eukaryotic membrane. According to the tryptophanfluorescence emission and the tryptophan quenching data of each peptide, we havefurther identified that rational peptide design by changing helicity can significantly reduce the toxicity of anticancer peptides against liposome mimic eukaryoticmembrane, thus resulting in high selectivity of anticancer peptides, which will make asolid foundation for the research and exploration of anticancer peptides in clinicalpractice.
Preparation of Amphiphilic Centipede-like Copolymer PASP-Na-DDA-PEG and Synthesis of Targeted Nanoprob
Semiconductor QDs are a new class of fluorescent labeling agents and haverecently been used for a broad range of biological applications. This broad interest isdriven by their unique optical and electronic properties such as size tunable lightemission, superior signal brightness, resistance to photobleaching, and simultaneousexcitation of multiple fluorescence colors. Thus, these QDs show promise asfluorescent probes for cell labeling and tracking. However, hydrophobic QDsprepared in organic phase make them insoluble in physiological solution, which to alarge extent limits their application in bioengineering. In this paper, we synthesis anovel class of amphiphilic centipede-like copolymer PASP-Na-DDA-PEG, totransfer hydrophobic octadecylamine–coated CdSe/ZnS QDs into hydrophiphilicones via surface modification.As we all know, aspartic acid is a kind of natural amino acid. The amphiphiliccopolymer we prepare is derivated from aspartic acid. Polymer itself can degradeinto non-toxic molecules that do no harm to environment.The amphiphilic comb copolymer PASP-Na-DDA was synthesized by thermalcondensation of aspartic acid (ASP) and aminolysis by dodecylamine (DDA),followed by hydrolysis of the remaining succinimide units in the copolymerbackbone. We synthesized a series of amphiphilic comb copolymer with differentDDA grafting level. The ratio of hydrophiphilic and hydrophobic chain werecontrolled by the regants. Then we studied the effect of grafting level on micellarbehavior in water solution. CMC, hydrodynamic diameter and micellar morphologywere determined. Considering all the factors above, we supposed the optimum polymer used for QDs modification was the one with DDA grafting level of40%.Experiments demonstrated comb-shape copolymer could be used to prepare denseand small nanoparticles.As is well known, PEG is widely used in bioengineering system because it issoluble and biocompatible. So we consider to graft PEG into the comb copolymer, toprepare amphiphilic centipede-like copolymer PASP-Na-DDA-PEG. We found theproperties of QDs nanocomposites, including particle size, optical propertie andmorphology, were affected by PEG chain density of the polymer coating. ComparedHD and PL spectra of QDs nanocomposites, with different PEG grafting level from0~60%. Results indicated that when PEG grafting level was40%, QDsnanocomposites showed the smallest size (105nm), and simultaneously exhibitedsuperior fluorescent properties among all the nanocomposites. These shell/corespherical structural nanoparticles were well dispersed in water, and did not formlarge aggregates. Apparently, polymer with PEG chain density of40%was theoptimum surface coating for QDs. The optical properties of QDs before and afterencapsulation were identical. The QDs nanocomposites in aqueous solutionpresented the same peak shape of absorption as initial QDs in chloroform), whichindicated that this transfer process didn’t destroy the structure of QDs nanocrystal.Finally, we studied the high-quality QDs nanocomposites, with VEGF antibodybinding to, served as a probe for tumor cell targeting and tracking. Anti-VEGF canspecificly recognize VEGF receptor which can be overexpressed by liver cancercells. Then we used the luminescence preporty of QDs to determine the location oftumor. This could improve the precision and sensitivity of cancer detection. PEGchain in the polymer coating limited nonspecific binding of QDs-anti-VEGF andcells and permitted cell receptors to access the antibody on the particle surface withhigh efficiency. PEG could effectively reduce the toxicity of QDs-anti-VEGFnanoprobe, which was demonatrated by Cytotoxicity Test.
The Synthesis and Research of Bis(Indolyl)Methanes and Novel Isoxazole Derivatives
The bisindole methane compounds and their derivatives is a very important class of heterocyclic compounds with high biological and pharmacological activities. It can adjust some of the carcinogenic metabolites, which can prevent cancer. For example, for breast cancer prevention. It also has anti-fungal and anti-tumor effect. Thus it has a very important value in the field of drug research and development. Oxidation of bisindole methane compounds containing conjugated bisindole-based skeleton is also used as a colorimetric sensor. Therefore, the synthesis of bisindole methane compounds has a great value.There are many ways to synthesize the bisindole methane compounds, generally using a protonic acid or Lewis acid catalysis. But it requires adding an excess of acid, the reaction conditions are more stringent, and the post-processing causes environmental pollution. Lewis acid general requirements for anhydrous, and will captured by nitrogen-containing substrates, thus it need to be added more than the stoichiometric. Lithium perchlorate can solve these problems, but it is more expensive, relatively long reaction time. A catalyst containing rare earth metals or ionic liquids are expensive. But the reaction time is longer, the yield is not high.Microwave has been used for chemical synthesis of large numbers of compounds, it can significantly reduce the reaction time with less byproduct, the reaction is very clean. The solvent-free reaction is a technology to avoid harmful solvents in organic reactions. It can shorten the reaction time, the yield is high and it may increase the stereoselectivity of the reaction, and the post-processing is very simple.In our work, we use the solvent-free conditions, silica catalysis, microwave synthesis method. The first10kinds of aromatic aldehydes with indole reaction,85%-98%yield to get aromatic substituted bisindole methane compounds. The yield of the reaction is high and the reaction time is very short, only10minutes. Isoxazole derivatives is very important pharmacologically active pharmacophore.It is often used as useful intermediates for the synthesis of natural products of biological activity. As the further study of the method, we use aldehyde containing isoxazole ring and indole reaction with the same method,94%-99%yield to get the bisindole methane compounds containing isoxazole ring. Isoxazole derivatives with good biological and pharmacological activity, the activity of the bisindole methane compounds containing Isoxazole ring might be better. At the same time, the reaction yield is high, and also proved that the method has universal applicability.1,3-dipolar cycloaddition is the best way to build the isoxazole ring. Our method is double use of1,3-dipolar cycloaddition to synthesize18kinds of new isoxazole derivatives containing isoxazole ring. The second use of1,3-dipolar cycloaddition reaction in our strategy, we use anhydrous zinc chloride as a catalyst, byproduct rarely and yield moderate to good. Center structure of the heterocyclic skeleton are generally drug or class of drug molecules, the isoxazole ring itself is a good active pharmacophore The new isoxazole derivatives synthesized in this paper, containing two isoxazole ring, the heterocyclic skeleton may have very good biological and pharmacological activity. Follow-up can test the biological activity of these compounds, as well as the introduction of the pharmacophore, such as pyridine to the heterocyclic skeleton to synthesize more new isoxazole derivatives with better biological activity.In summary, our work are in the microwave solvent-free conditions, silica catalytic synthesis of16different bisindole methane compounds. And using1,3-dipolar cycloaddition to construct the heterocyclic skeleton containing two isoxazole ring, and synthesized18kinds of new isoxazole derivatives.