Design and Synthesis of Proteolysis Targeting Chimeras for Inducing BRD4 Protein Degradation

In this paper, we synthesized a series of proteolysis targeting chimeras(PROTACs) using VHL E3 ligase ligands for BRD4 protein degradation. One of the most promising compound 19g exhibited robust potency of BRD4 inhibition with IC₅₀ value of (18.6±1.3) nmol/L, respectively. Furthermore, compound 19g potently inhibited cell proliferation in BRD4-sensitive cell lines RS4;11 with IC₅₀ value of (34.2±4.3) nmol/L and capable of inducing degradation of BRD4 protein at 0.4—0.6 µmol/L in the RS4;11 leukemia cells. These data show that compound 19g is a highly potent and efficacious BRD4 degrader.     

Discovery of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new bromodomain-containing protein 4 (BRD4) inhibitors

Targeting bromodomain-containing protein 4(BRD4) has been proved to be an effective strategy for cancer therapy.To date,numerous BRD4 inhibitors and degraders have been identified,some of which have advanced into clinical trials.In this work,a focused library of new [1,2,4]triazolo [1,5-a]pyrimidine derivatives were discovered to be able to inhibit BRD4.WS-722 inactivated BRD4(BD1/BD2),BRD2(BD1/BD2) and BRD3(BD1/BD2) broadly with the IC₅₀ values less than 5 μmol/L.Besides,WS-722 inhibited growth of THP-1 cells with an IC₅₀ value of 3.86 μmol/L.Like(+)-JQ1,WS-722 inhibited BRD4 in a reversible manner and enhanced protein stability.Docking studies showed that WS-722 occupied the central acetyl-lysine(Kac) binding cavity and formed a hydrogen bond with Asn140.In THP-1 cells,WS-722 showed target engagement to BRD4.Cellular effects of WS-722 on THP-1 cells were also examined,showing that WS-722 could block c-MYC expression,induce G0/G1 phase arrest and p21 up-regulation,and promote differentiation of THP-1 cells.BRD4 inhibition by WS-722 resulted in cell apoptosis and upregulated expression of cleaved caspased-3/7 and PARP in THP-1 cell lines.The [1,2,4]triazolo[1,5-a]pyrimidine is a new template for the development of new BRD4 inhibitors.     

Identification of highly efficacious PROTACs targeting BRD4 against acute myeloid leukemia: Design,synthesis, and biological evaluations

The abnormal activation of BRD4 accelerates the progression of acute myeloid leukemia (AML), developing more precise therapeutics to intervene BRD4 promise to be an excellent opportunity to avoid current limitations of chemotherapy in clinic. Herein, a range of small-molecule PROTACs with the privileged 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one scaffold were rationally designed, which harbored different carbon or ethylenedioxy chains to degrade BRD4 mediated by the E3 ubiquitin ligase CRBN. Among them, the most potential B24 exhibited remarkable BRD4 degradation and excellent anti-proliferative activities in MV4-11 cells, with values of DC₅₀ and IC₅₀ for 0.75 nmol/L and 0.4 nmol/L, respectively, which were better than the BRD4 inhibitor (+)-JQ-1. Notably, this compound could time-dependently degrade the target protein in the BRD4-, CRBN-, and proteasome-dependent manner. Besides, B24 dramatically decreased the level of proto-oncogene c-Myc, and induced cell apoptosis by arresting the cell cycle in G0/G1 phase, down-regulating Bcl-2 and up-regulating Bax to amplify apoptotic effectors. This proof-of-concept study also highlighted the feasibility of BRD4-based PROTACs as a more powerful strategy against AML.     

Design,Synthesis and Biological Evaluation of Novel Selective Thiol-based Histone Deacetylase(HDAC) VI InhibitorsBearing Indeno[1,2-c]pyrazole or Benzoindazole Scaffold

A series of thiol-based indeno[1,2-c]pyrazoles and benzoindazole compounds was designed and synthesized according to the structural specificity of histone deacetylase VI(HDAC6) and the structural characteristics of HDAC inhibitors. The inhibitory activities of the target compounds against HDAC6 and HDAC1 were screened by fluorescence analysis. Most of the target compounds showed moderate inhibitory activity against HDAC6(IC₅₀=44—598 nmol/L). Among them, compound A-4 displayed the highest selectivity against HDAC6 and similar inhibitory activity(IC₅₀=44 nmol/L) to that of the positive drug SAHA(IC₅₀=41 nmol/L) against HDAC6.     

新型双杂环修饰的酰胺硫醚衍生物的合成和生物活性

首次设计并合成了16个新型1,2,4-三唑与1,3,4-噻二唑双杂环修饰的酰胺硫醚衍生物,并对其进行了结构表征。分别评价了目标分子对蛋白酪氨酸磷酸酶1B(PTP1B)和细胞分裂周期25磷酸酶B(Cdc25B)抑制活性,结果发现:16个目标分子对PTP1B具有良好的抑制活性,其中8-C-d和8-D-c的抑制作用最佳,半抑制浓度(IC_(50)值)分别为(1.19±0.22)mg/L和(1.08±0.09)mg/L,优于阳性参照物齐墩果酸(IC_(50)=(1.27±0.19)mg/L),有望作为抗糖尿病药物先导物;对Cdc25B抑制活性测试中,11个目标分子表现出良好的活性,其中8-A-d、8-C-d和8-D-c抑制活性的IC_(50)值分别为(0.97±0.05)、(1.06±0.03)和(0.94±0.11)mg/L,低于阳性参照物Na_3VO_4(IC_(50)=(1.25±0.14)mg/L),有望作为抗肿瘤药物先导物。     

Atomistic insights into the selective therapeutic activity of 6-(2,4-difluorophenoxy)-5-((ethylmethyl)pyridine-3-yl)-8-methylpyrrolo[1,2-a]pyrazin-1(2H)-one towards bromodomain-containing proteins

Cross-target effect has been one of the major mechanisms of drug toxicity, this has necessitated the design of inhibitors that are specifically tailored to target particular biomolecules. 6-(2,4-difluorophenoxy)-5-((ethylmethyl)pyridine-3-yl)-8-methylpyrrolo[1,2-a] pyrazin-1(2H)-one (Cpd38) is an inhibitor possessing high inhibition rate and tailored specificity towards bromodomain-containing protein 4 (BRD4). In this research, we used an array of computational techniques to provide insight at the atomistic level the specific targeting of BRD4 by Cpd38 relative to the binding of Cpd38 with E1A binding protein P300 (EP300); another bromodomain-containing protein (BCP). Comparatively, binding of Cpd38 improved the conformational stability and compactness of BRD4 protein when compared to the Cpd38 bound EP300. Also, Cpd38 induced a conformational change in the active site of BRD4 that facilitated a complementary pose between Cpd38 and BRD4 suitable for effective atomistic interactions. Expectedly, thermodynamic calculations revealed that the Cpd38-BRD4 system had higher binding energy (−36.11 Kcal/mol) than the Cpd38-EP300 system with a free binding energy of −15.86 Kcal/mol. Noteworthy is the opposing role Trp81 (acting as hydrogen bond acceptor) and Pro1074 (acting as hydrogen bond donor) found on the WPF and LPF loops respectively play in maintaining Cpd38 stability. Furthermore, the hydrogen bond acceptor/donator ratio was approximately 4:1 in Cpd38-BRD4 system compared with 2:1 in Cpd38-EP300 system. Taken together, atomistic insights and structural perspectives detailed in this report supplements the experimental report supporting the improved selectivity of Cpd38 for BRD4 ahead of other BCPs while providing leeway for the future design of BET selective agents with better pharmacological profile.     

Synthesis of novel β-propanamides to inhibit cholesteryl ester transfer protein(CETP)

A novel series of β-propanamide derivatives as inhibitors of cholesteryl ester transfer protein(CETP)were synthesized.Previously,H3(IC_(50) 2 μmol/L) was observed to inhibit CETP moderately(Xie et ah,2016).Structural modifications based on H3 led to discovery of the successful CETP inhibitor,known as 1-methyl-4-arylpyrazole.Using a similar approach,compound Q08 was identified as a highly potent CETP inhibitor with an IC_(50) of 490 nmol/L in vitro.     

Identification of Cinobufagin and Resibufogenin as Inhibitors of Enterovirus 71 Infection

In this paper, cinobufagin and resibufogenin were found to inhibit enterovirus 71（EV71） infection in vitro in cell viability and plaque reduction assays. The 50% inhibitory concentrations（lCs0） of einobufagin and resibufoge- nin were （10.94±2.36） and （218±31） nmol/L, respectively, the 50% cytotoxic concentrations（CCs0） of them were （1277±223） and （1385±254） nmol/L, respectively, and the anti-EV71 selectivity index（SI50） of cinobufagin was 116.7, which are promisingly developed into drug. Using a VP1 detection assay and a constructed reporter luciferase, we found that cinobufagin and resibufogenin disrupted the synthesis of EV71 protein. However, neither of them inhibited EV71 RNA replication. Our study suggests that cinobufagin and resibufogenin are the promising candidates that should he fllrther investigated for the treatment of EV71 caused disease.     

Development of bis-thiobarbiturates as successful urease inhibitors and their molecular modeling studies

Bis-thiobarbiturate derivatives 1–15 have been synthesized, characterized by1 HNMR and EI-MS and screened for urease inhibition. All compounds showed various degree of urease inhibitory activity with IC_(50) values ranging 7.45 0.12 74.24 0.81 mmol/L while the standard thiourea behaved normally(IC_(50) = 21.10 0.12). Compounds 1(IC_(50) = 7.45 0.12 μmol/L), 9(IC_(50) = 18.17 1.03 μmol/L) and 13(IC_(50) = 8.61 0.45 μmol/L) showed excellent urease inhibitory activity in the series. Molecular modeling studies were performed to understand the binding site with the bimetallic nickel center of the enzyme.Structure-activity relationship has also been established for these compounds. This study identified bisthiobarbiturate as a novel class of urease inhibitors.     

新型细胞分裂周期25磷酸酯酶B和蛋白酪氨酸磷酸酶1B抑制剂三唑并噻二唑-均三嗪的合成

以苯亚氨基为桥,设计合成了18个含有三唑并噻二唑和均三嗪双杂环的新型分子(4a~4i和5a~5i),并利用红外光谱、核磁共振谱和高分辨质谱等技术手段对其进行了结构表征。将吗啉和四氢吡咯分别与三聚氯氰发生双取代反应合成三嗪衍生物(1A和1B),然后将1A和1B分别与对氨基苯甲酸反应,合成重要中间体(2A和2B)。通过熔融法将8种脂肪酸与二氨基硫脲缩合得1,2,4-三唑衍生物3a~3h,最后将2A和2B在三氯氧磷和四丁基溴化铵催化下分别与3a~3h反应得目标产物。为了进一步比较3-脂肪基和3-苯基对药效活性的影响,利用相同方法设计合成了目标产物4i和5i。评价了目标产物对细胞分裂周期25磷酸酯酶B(Cdc25B)和蛋白酪氨酸磷酸酶1B(PTP1B)抑制活性。结果发现:所有目标分子对Cdc25B均表现出良好的抑制活性,半抑制浓度(IC_(50)值)在2.40~0.31 mg/L之间,目标分子4a~4f和5a~5i的IC_(50)值均低于阳性参照物Na_3VO_4[(1.25±0.14)mg/L],有望成为潜在的Cdc25B抑制剂;在PTP1B测试中,14个目标分子具有优良的抑制活性,IC_(50)值在0.98~0.37 mg/L之间,低于阳性参照物齐墩果酸[(1.19±0.27)mg/L],有望成为潜在的PTP1B抑制剂。     

Synthesis,Docking and Biological Evaluation of Isoquinolonic Acid Derivatives

A series of isoquinolonic acid derivatives（4a-4o） was synthesized via one-pot synthesis for their anti-tumor activity. The structures of all the targeted compounds were confirmed by IH nuclear magnetic resonance （IH NMR） spectrometry and mass spectrometry（MS）. The anti-tumor activities of compounds 4a-4o against MG63（human osteosarcoma cells） and B16-F10（mouse melanoma cells） were examined. To evaluate the antitumor effect of the as-synthesized compounds, we compared the half maximal inhibitory concentration（1C50） of compounds 4a--4o to that of camptothecin（CPT） which appeared to be active against a broad range of human cancers. Among all the compounds, compound 41 shows the most potent biological activity against MG63 cells[IC50=（2.16i0.26） μmol/L] and B16-F10 cells[IC50=（6.95±0.24）μmol/L], thus providing useful information for the antitumor activity and potential practical use of isoquinolonic acid compounds. In addition, we screened out an efficient compound（41） that shows potential inhibit activity against Topoisomerase 1（Topo 1） by docking simulation.     

Expeditious synthesis,antileishmanial and antioxidant activities of novel 3-substituted-4-hydroxycoumarin derivatives

A series of novel 3-substituted-4-hydroxycoumarin derivatives 6(a–1) were synthesized in high yield using one-pot three component coupling reaction catalyzed by ceric ammonium nitrate. These compounds were evaluated for antileishmanial activity against Leishmania donovani promastigotes and antioxidant activity(DPPH-radical scavenging activity). Two compounds, 6h(IC_(50)= 9.90 μmol/L) and 6i(IC_(50)= 6.90 μmol/L) displayed potent antileishmanial activity when compared with standard antileishmanial agents pentamidine(IC_(50)= 16.15 μmol/L) and miltefosine(IC_(50)= 12.50 μmol/L). Three compounds, 6c(IC_(50)= 10.79 μmol/L), 6h(IC50= 10.60 μmol/L), and 6i(IC_(50)= 10.73 μmol/L) showed significant antioxidant activity favorably with the antioxidant standards butylated hydroxy toluene(IC_(50)= 16.47 μmol/L) and ascorbic acid(IC_(50)= 12.69 μmol/L). A molecular docking study of compounds 6(a–1) suggested a possible mode of binding with the Adenine phosphoribosyltransferase enzyme of L.donovani. ADME properties were predicted in silico and support the potential of 6(a–1) to show favorable drug-like properties.     

Design,Synthesis and Antitumor Activity of Novel Indolin-2-one Derivatives Containing 4-Thiazolidinone Moiety

A series of novel indolin-2-one derivatives containing 4-thiazolidinone moiety(7a―7r) were synthesized and evaluated for their in vitro antitumour activities against MDA-MB-231(human breast cancer), H460(human lung cancer) and HT-29(human colon cancer) cell lines by standard 3-(4,5-dimethylthiazae-2-yl)-2,5-diphenyltetrazo- lium bromide(MTT) assay. Representative compounds(7d, 7k, 7m, 7p) with higher cytotoxicity were further examined against one normal cell line(WI-38, human fetal lung fibroblasts). The preliminary investigation shows that most of the compounds display moderate to excellent potency and high selectivity against different human cancer cell lines. In particular, the most potent compound 7m shows promising cytotoxicity against MDA-MB-231, H460 and HT-29 cell lines with IC₅₀ values of 0.78, 0.056 and 0.018 μmol/L, respectively. The potency is much higher than that of Sunitinib(IC₅₀=3.46 μmol/L against MDA-MB-231, IC₅₀=2.59 μmol/L against H460, IC₅₀=1.50 μmol/L against HT-29) by 4.4, 46.3 and 83.3 fold.     

Synthesis and α-glucosidase inhibitory activity of chrysin,diosmetin, apigenin,and luteolin derivatives

Several derivatives have been synthesized from chrysin, diosmetin, apigenin, and luteolin, which were isolated from diverse natural plants. The α-glucosidase inhibitory activity of these compounds was evaluated. The glucosidase inhibitory activity of all derivatives （IC50 〈 24.396 μmol]L） was higher compared with that of the reference drug, acarbose （IC50=563.601 ±40.492μmol/L）, and 1- deoxynojirimycin （IC50 = 226.912± 12.573 μmol/L）. O3＇,O7-Hexyl diosmetin （IC50 = 2.406 ± 0.101μmol/L） was the most potent inhibitor identified. These compounds showed a higher inhibitory ability compared with their precursors except the luteolin derivatives. In general, the inhibitory activity of the synthetic derivatives was enhanced with long alkyl chains at positions 3＇, 4＇ and 7 of the flavonoid.     

New cyclopiane diterpenes with anti-inflammatory activity from the sea sediment-derived fungus Penicillium sp. TJ403-2

Three new rare cyclopiane diterpenes(1-3),together with thirteen known compounds(4-16),were isolated and identified from a sea sediment-derived fungus Penicillium sp.TJ403-2.The planar and relative structures of compounds 1-3 were elucidated by HRESIMS,one-and two-dimensional NMR analyses,and their absolute configurations were further established by X-ray crystallography experiment.Compounds 1-3 were evaluated for the anti-inflammatory activity against LPS-induced NO production,and compound 1 showed notable inhibitory potency with an IC50 value of2.19±0.25μmol/L,which was three fold lower than the positive control indomethacin(IC50=8.76±0.92μmol/L).Further Western blot and immunofluorescence experiments demonstrated its mechanism of action to be that 1 inhibited the NF-κB-activated pathway,highlighting it as a promising starting point for the development of new anti-inflammatory agents.     

Amino acid derivatives of pyropheophorbide-α ethers as photosensitizer: Synthesis and photodynamic activity

Ten new water-soluble amino acid conjugates of pyropheophorbide-α ethers 4a-4j were synthesized and investigated for their in vitro photodynamic antitumor activity. The results showed that all compounds exhibited higher phototoxicity and lower dark toxicity against three kinds of tumor cell lines than BPD-MA. In particular, themost phototoxic compound 4d and 4j individually showed IC₅₀ values of 41 nmol/L and 33 nmol/L against HCT116 cell, which represented 7.8- and 9.7-fold increase of antitumor potency compared to BPD-MA, respectively, suggesting that they were promising photosensitizers for PDT applications because of their strong absorption at long wavelength (λ_max>650 nm), high phototoxicity, low dark cytotoxicity and good water-solubility.     

Synthesis and Anti-leukemia Evaluation of Tetrahydro-4H-pyrano[3,2-c]pyridines and Corresponding Anti-CD14 Monoclonal Antibody Conjugates

A series of novel tetrahydro-4H-pyrano[3,2-c]pyridines（3a-3p） were synthesized and found to possess potent antiproliferative activity against leukemia K562 cells in vitro. Preliminary bioassay indicates that compounds 3a and 3e afford the best activity, the IC50 values of them were 6.93 and 7.51μg/mL, respectively, which were lower than that of the anticancer drug 5-FU（1C50=8.56μg/mL）. To reduce the toxicity of compounds 3a-3p to the prolife- ration of normal bematopoietic cells, a tumor targeted CD14 monoclonal antibody（McAb） was used in conjugation with compounds 3a--3p to get conjugates 4a--4p, respectively. The inhibitory activities of conjugates 4a--4p toward K562 cells were discovered to approach those of compounds 3a--3p. In the presence of CD14 McAb, tumor cells were found to be much more susceptible to conjugates 3a--3p than normal hematopoietic cells. Therefore, the toxicity of conjugates 4a--4p to normal hematopoietic cells declined obviously. For example, as for the toxicity of compound 3a compared with that of compound 4a, the value of ICs0 increased from 35.90 μmol/L to 39.52 μmol/L.     

嘧啶并嘧啶酮类化合物作为L3MBTL1选择性络合剂的设计与合成

用嘧啶并嘧啶酮替换Lethal 3 malignant brain tumor 1(L3MBTL1)小分子络合剂UNC669分子中的芳香部分,合成了一系列嘧啶并嘧啶酮类化合物.采用同质邻近发光放大法(AlphaScreen^®)测试了其活性,得到IC₅₀值为1.21 μmol/L的化合物8a;通过对其5位基团进行改造,最终获得了3个选择性L3MBTL1络合剂8g, 8o与8p,它们仅对L3MBTL1有活性,对其同源蛋白L3MBTL3在内的其它甲基化识别蛋白则无活性.     

N-(2-吡啶甲基)-L-丝氨酸铜配合物的合成、晶体结构及抑制蛋白酪氨酸磷酸酶活性

在甲醇溶液中, 还原希夫碱HL[N-(2-吡啶甲基)-L-丝氨酸]与CuCl₂·2H₂O以摩尔比1∶1反应, 得到1个新的中性单核铜配合物[CuLCl(H₂O)](Ⅰ). 通过X射线单晶衍射、 元素分析、 红外光谱、 电喷雾质谱和粉末X射线衍射分析等对其进行了表征. 晶体结构分析表明, 在该配合物中还原希夫碱以三齿双螯合环配位到中心铜离子, 同时氯离子和溶剂水分子也参与配位, 形成1个具有四方锥构型的五配位铜(Ⅱ)配合物, 该配合物通过分子间弱相互作用连接成二维超分子结构. 生物活性测试结果表明, 配合物Ⅰ能有效抑制蛋白酪氨酸磷酸酶1B(PTP1B)和T细胞蛋白酪氨酸磷酸酶(TCPTP), IC₅₀值分别为0.32和0.45 μmol/L.     

Synthesis and biological evaluation of novel 1, 2, 3-benzotriazin-4-one derivatives as leukotriene A4 hydrolase aminopeptidase inhibitors

A series of novel 1,2,3-benzotriazin-4-one derivatives were designed,synthesized and their inhibitory activities against leulcotriene A_4 hydrolase aminopeptidase in vitro were evaluated.Many compounds showed moderate to good activities at the concentration of 10 μmol/L.Among them,compound Ⅳ-16 exhibited the highest inhibitory activity up to 80.6% with an IC_(50) of 1.30 ± 0.20 μmol/L The compound Ⅳ-16 was also tested the proliferation inhibitory activities in THP1 human AML cell line and its binding model with LTA_4H enzyme by molecular docking was studied.It indicated that 1,2,3-benzotriazin-4-one was a promising scaffold for further study.The relationship between structure and inhibitory activity was also preliminarily discussed.