Synthesis and biological activities of chlorin e6-based conjugate of fluorouracil as dual-mode antitumor photosensitizer / 药学实践杂志

Objective To design and synthesize the conjugate (compound 1) of chlorin e6 (compound 3) with fluorouracil (5-Fu) as novel pH-responsive dual-mode antitumor photosensitizer by acyl hydrazone bond coupling, based on literature reports that combination of 5-Fu and photosensitizer possess synergistic anti-tumor effect, and investigate its photodynamic antitumor activity and mechanism. Methods Lead compound 3 was obtained by alkali degradation with 25% KOH-CH3OH on pheophorbide a (compound 4) which was prepared through acid hydrolysis of chlorophyll a in crude chlorophyll extracts from silkworm excrement. Reflux reaction of 5-Fu with P2S5 in pyridine formed crude 4-thio-5-fluorouracil which was followed to react with hydrazine hydrate (N2H4·H2O) in CH3OH to give 5-fluorouracil-4-hydrazone (compound 2). Then, treatment of compound 3 i.e. acid alkali degradation product of chlorophyll a in silkworm excrement with EDC·HCl generated its 171- and 152 cyclic anhydride which was followed to directly react with intermediate compound 2 to successfully get title compound 1. In addition, its pH-responsive 5-Fu release and photodynamic antitumor activity and their mechanisms in vitro were investigated. Results Compound 1 could responsively release 5-Fu at pH 5.0, with a cumulative release rate of 60.3% within 24 h. It exhibited much higher phototoxicity against melanoma B16-F10 and liver cancer HepG2 cells than talaporfin and its precursor compound 3, with IC50 value being 0.73 μmol/L for B16-F10 cells and 0.90 μmol/L for HepG2 cells, respectively. Upon light irradiation, it also could significantly induce cell apoptosis and intracellular ROS level and block cell cycle in S phase. Its structure was confirmed by UV, 1H-NMR, ESI-MS and elemental analysis data. Conclusion The conjugate compound 1 of compound 3 and 5-Fu has the advantages of strong PDT anticancer activity, high therapeutic index (i.e. dark toxicity/phototoxicity ratio) and responsively release 5-Fu at pH 5.0 etc. which shows “unimolecular” dual antitumor effects of PDT and chemotherapy and is worthy of further research and development.

Design, synthesis and antitumor activities of chlorin p6 ether derivatives as photosensitizer for photodynamic therapy / 药学实践杂志

Objective To designe, synthesize a series of chlorin p6 ether photosensitizers and preliminarily investigate their photodynamic antitumor activity based on previous research results that alkoxyl ether derivatives of 3-vinyl on chlorin f exhibited stronger photosensitive antitumor activity than parent compound. Methods Purpurin-18 (4) was obtained by oxidative degradation with air and alkali on pheophorbide a (5) which was prepared through acid hydrolysis of chlorophyll a from crude chlorophyll extracts in Chinese traditional herb named Silkworm excrement. Then, chlorin p6 trimethylester (2) were formed via basic hydrolysis of internal anhydride ring for lead compound 3 and following immediately methylation with CH2N2. The intermediate 2 reacted with 33% HBr, following nucleophilic substitution with various alkoxyl alcohol to get six title compounds (1). All title compounds were subjected to photodynamic antitumor activity screening for melanoma B16-F10 cell in vitro. Results All title compounds showed much higher phototoxicity against melanoma B16-F10 cells than talaporfin and verteporfin. Their structures were confirmed by 1H-NMR, 13C-NMR, ESI-MS and ESI-HRMS spectra. Conclusion Chlorin p6 ether compounds were promising candidate photosensitizers for PDT applications due to theirs high dark toxicity/phototoxicity ratio and excellent phototoxicity, which were worthy of further research and development.

Design and synthesis of photosensitizer,13,15-cycloimides chlorin p6 / 药学实践杂志

Objective To design and prepare 13 ,15-cycloimides chlorin p6 (1) ,a class of chlorin related antitumor photo-sensitizers ,which contain a more stable six-membered cyclic imide comparing to the exocyclic anhydride ring of purpurin-18 (2) .Compounds (1) exhibit strong absorption at long wavelengths near λmax 700 nm to take full advantage of greater tissue penetration .Methods Pheophorbide a (3) was obtained by acid hydrolysis of chlorophyll a ,which was from crude chlorophyll extracts of Chinese traditional herb named Silkworm excrement .Purpurin-18 (2) was prepared by air oxidation and alkali open loop simultaneously on five-membered beta-keto carboxylic ester ring of pheophorbide a (3) .Finally ,the target compounds 1a~1j were synthesized via condensation of its anhydride ring with various amines including carboxyl-protected amino acids . Results Target compounds 1a~1j were successfully synthesized in yields ranged from 32 .6% to 65 .2% .Their structures were confirmed by elemental analysis ,ESI-MS and 1 H NMR spectra .Conclusion Treatment of purpurin-18 (2) with amines can produce target compounds 1a~1j .The starting raw material was inexpensive and readily available .The reaction conditions were mild and workup was convinient .

Optimization of synthesis process of photosensitizer chlorin f / 药学实践杂志

Objective To improve the synthesis process of chlorine f (1).Methods A "one-pot"method was applied to prepare Photosensitizer component (1),using pheophorbide a (3) as raw material by oxidating and cracking of the E-ring of (3) with bubbling oxygen in alcoholic solution of potassium hydroxide at 0 ℃ followed by refluxing in nitrogen atmosphere.In order to obtain the optimal synthetic procedure,the orthogonal experimental design of L9 (34 ) was adopted to investigate three different levels of four main factors i.e.ring opening reaction time,alcoholic variety,alkali concentration and refluxing reaction time.Results The target compound (1) was optimizedly synthesized through treatment of raw material (3) with bubbling oxy-gen in 25% ethanol solution of potassium hydroxide at 0℃ for 30 min,followed by refluxing in nitrogen atmosphere for 20 min in yield of 40.8%.Conclusion The procedure developed has some advantages of simple and safty operation,and high synthetic yield.

Optimization of synthesis of photosensitizer chlorin e6-C15 monomethyl ester by the orthogonal design / 药学实践杂志

Objective To optimize the synthesis of photosensitizer chlorine e6‐C15 monomethyl ester (1) .Methods Tar‐get compound 1 was synthesized through E ring cracking of pheophorbide a (3) in methanol solution of potassium hydroxide followed by adding H2 O for ester hydrolysis with“one‐pot” process .Four main factors that influencing the synthetic yield of target compound 1 are ring cleavage reaction time (A) ,reflux hydrolysis reaction time (B) ,alkali concentration (C) and weight ratio of material 3 to alkali (D) .Each of the four factors was chosen at three levels and evaluated by the orthogonal ex‐perimentaldesignofL9(34).Results Theoptimizationtermsofthesynthesisoftargetcompound1wereB1C2A2D1 .Theyield of compound 1 was raised from 43 .0% to 56 .5% .Conclusion The developed process has the advantages of simple operation , good safety and high reaction yield ,and is suitable for industrial production .

Establishment of recombinant Mycobacterium smegmatis secreting IL-2 / 第三军医大学学报

Objective To establish a recombinant Mycobacterium (M.) smegmatis secreting interliukin-2 (IL-2) for the prevention and treatment of human bladder cancer. Methods The M. tuberculosis HSP70 promotor, ?-antigen signal peptide gene, and human IL-2 cDNA were amplified from plasmid pY6013, pIJK-1, and pHIG53 respectively by PCR. The products were cloned into plasmid pRR3 to construct a mycobacterial shuttle-expressing plasmid pR-a-IL-2 secreting human IL-2. After confirming the construction was correct by enzyme digestion, plasmid pR-?-IL-2 was transduced into M. smegmatis mc 2 155 by electroporation. The stability of the recombinant mycobacteria was evaluated and the activity of IL-2 secreted by the bacteria was assayed. Results Structure of the pR-a-IL-2 was correct and it was effectively transduced into M. smegatis mc 2 155. The recombinant mycobacteria stably expressed IL-2. The IL-2 activity in the medium was 118.5 U/ml. Conclusion The successful establishment of recombinant M. smegmatis can provide the basis for the research of biotherapy and prevention of the recurrence of bladder cancer.

SYNTHESIS, PRELIMINARY ANTIGASTRELCOSIS ACTIVITY AND THE PROTECTIVE EFFECT ON ACUTE LIVER INJURY OF ZINC CHLORIN E4 / 药学学报

AIM　To study the synthesis of zinc chlorin e4 (1), its experimental antigastrelcosis activity as well as the protection against acute liver injuries. METHODS　Chlorin e6 (3) was prepared through acidic and alkaline oxidative degradation using silkworm excrement crude chlorophyll extracts as starting material. Compound 1 was synthesized via Zn(OAc)2 complex action with Chlorin e4 (2) which was prepared by refluxing 3 in pyridine. Gastric ulcers were induced by abdominal injection of 0.2% indomethacin at 20 mg.kg-1 in rats. The ulcer indexes and ulcer numbers in gastric mucosa were determined. Acute liver injuries were induced by abdominal injection of 0.3% thioacetamide (TAA) or 0.3% CCl4 at 20 mg.kg-1 in mice, and activities of SGPT in mice were determined. RESULTS　Compound 1 is previously unknown. Compared with control group, abdominal administration of 1 at 100 mg.kg-1 reduced significantly the gastric ulcer index (P<0.001) and the number of ulcer (P<0.001) induced by indomethacin in rats. Abdominal administration of 1 at 100 mg.kg-1×3 exhibited marked inhibitory effects on elevated activities of SGPT induced by TAA (P<0.02) or CCl4 (P<0.01) in mice. CONCLUSION　These results show that 1 has significant protective effect against indomethacin-induced gastric lesion in rats and TAA or CCl4 induced acute liver injuries in mice. It is suggested that 1 may be a promising new drug candidate for antigastrelcosis and liver injury protection.

Synthesis and Antifungal Activity of Sulconazole and Its Analogues / 第二军医大学学报

In order to search for a more potent and less toxic antifungal agent, five novel sulconazole analogues were synthesized by changing 1-imidazolyl and p-chlorobenzyl in sulconazole structure. The results of preliminary biological tests show that analogues Ⅰ and Ⅱ possess more potent antifungal activities and wider antifungal spectra than sulconazole.