Computational planning of the synthesis of complex natural products.

Training algorithms to computationally plan multistep organic syntheses has been a challenge for more than 50 years1-7. However, the field has progressed greatly since the development of early programs such as LHASA1,7, for which reaction choices at each step were made by human operators. Multiple software platforms6,8-14 are now capable of completely autonomous planning. But these programs 'think' only one step at a time and have so far been limited to relatively simple targets, the syntheses of which could arguably be designed by human chemists within minutes, without the help of a computer. Furthermore, no algorithm has yet been able to design plausible routes to complex natural products, for which much more far-sighted, multistep planning is necessary15,16 and closely related literature precedents cannot be relied on. Here we demonstrate that such computational synthesis planning is possible, provided that the program's knowledge of organic chemistry and data-based artificial intelligence routines are augmented with causal relationships17,18, allowing it to 'strategize' over multiple synthetic steps. Using a Turing-like test administered to synthesis experts, we show that the routes designed by such a program are largely indistinguishable from those designed by humans. We also successfully validated three computer-designed syntheses of natural products in the laboratory. Taken together, these results indicate that expert-level automated synthetic planning is feasible, pending continued improvements to the reaction knowledge base and further code optimization.

Synthesis, characterization, and fungicidal activity of novel Fangchinoline derivatives.

A series of new Fangchinoline derivatives with the carbamate moiety (compounds 1a-1l, 2a-2l) were synthesized by utilizing phenolic hydroxyl to react with isocyanate. The potential fungicidal activity of all the target compounds has been screened against six species of plant pathogen fungi, including Pp. adianticola (Phomopsis adianticola), A. adianticola (Altermaria adianticola), C. fructicola (Colletotrichum fructicola), P. theae (Pestalotiopsis theae), P. adianticola (Phoma adianticola), and G. zeae (Gibberella zeae). Almost all the derivatives showed better fungicidal activity than Fangchinoline. Compounds 1f, 1g, 1h, and 1k exhibited obviously better activity against G. zeae, and Pp. adianticola than Azoxystrobin. Especially compounds 1k displayed high fungicidal activity against G. zeae, Pp. adianticola, and P. theae.

Characterization of Cepharanthin Nanosuspensions and Evaluation of Their In Vitro Activity for the HepG2 Hepatocellular Carcinoma Cell Line.

BACKGROUND: Conventional cancer therapeutics has enormous toxicity and severe side effects that generate multi-drug resistance. Therefore, an urgent need exists for new alternative therapeutic agents for cancer treatment. Cepharanthin (CEP) has anti-cancer potential but has poor aqueous solubility, which limits its clinical use. Nanosuspensions (NS) are attractive as insoluble drug delivery systems. OBJECTIVES: In this study, we used D-alpha Tocopherol acid Polyethylene Glycol Succinate (TPGS), Polyvinylpyrrolidone (PVP) VA64, and Croscamellose Sodium (CCS) as stabilizers to produce TPGS-CEP-NS, PVP VA64-CEP-NS, and CCS-CEP-NS by wet-milling technology, and then characterized the NS and evaluated their functional activities in vitro. METHODS: CEP Nanosuspensions (CEP-NS) were prepared by the wet-milling method. The prepared NS were characterized by particle size distribution, zeta potential, morphology, surface properties, and molecular interactions. The NS were evaluated for their effects on HepG2 cells in vitro. The evaluations included assessment of cellular cytotoxicity, cellular apoptosis, NS uptake by cells, and mitochondrial membrane potential changes. RESULTS: CEP-NS showed an appropriate particle size and were physically stable. All CEP-NS exhibited HepG2 enhanced anti-proliferative effects by reducing cell viability, enhanced cellular uptake, induced cellular apoptosis, and mitochondrial membrane potential loss. CONCLUSIONS: CEP-NS may be effective therapeutic agents for the treatment of hepatocellular carcinoma.

Fangchinoline derivatives induce cell cycle arrest and apoptosis in human leukemia cell lines via suppression of the PI3K/AKT and MAPK signaling pathway.

Fangchinoline, a bisbenzylisoquinoline alkaloid extracted from Stephania tetrandra S. Moore, is known to exert anti-cancer activity. A series of new fangchinoline derivatives have been synthesized and evaluated for their anti-cancer activity. In cell viability assay, these fangchinoline derivatives displayed higher proliferation inhibitory activity on leukemia and breast cancer cell lines than the parental compound. Among them, 3e exhibited strongest cell growth inhibitory activity in a dose- and time-dependent manner on leukemia cell line HEL through induction of G0/G1 cell cycle arrest and apoptosis. Treatment of HEL cells with 3e also resulted in significant suppression of the MAPK and PI3K/AKT pathway as well as c-MYC downregulation, which may responsible for induction of apoptosis and cell cycle arrest. In docking analysis, high affinity interaction between 3e and Akt1 was responsible for drastic kinase inhibition by the compound. This derivative compound may be useful as a potent anti-cancer agent for treatment of leukemia.

Design, synthesis and in vitro evaluation of fangchinoline derivatives as potential anticancer agents.

The isolation and modification of natural products play an important role in the synthesis of anti-tumor drugs for the treatment of cancer. The present study was designed to evaluate the effects of fangchinoline derivatives against cancer cells. In vitro cytotoxicity of all derivatives against five cancer cell lines (A549, Hela, HepG-2, MCF-7 and MDA-MB-231 cell lines) and HL-7702 normal cells was assessed using the CCK-8 assay, and the results showed that most of the synthesized compounds displayed better cytotoxic effects on all the tested cells compared to that of the parent fangchinoline. In particular, compound 3i had the strongest inhibitory effect on cell proliferation, with an IC50 value of 0.61 µM against A549 cells. Compared with fangchinoline and HCPT (hydroxycamptothecine), the anti-proliferative activity of compound 3i was significantly increased. More interestingly, compound 3i had slight toxic side effects on normal cells, with an IC50 value of 27.53 µM. Moreover, the cell viability and cell cycle assays revealed that compound 3i inhibited A549 cell proliferation and arrested A549 cells at the G2/M-phase. The apoptosis-inducing effects of compound 3i and the associated molecular mechanisms were assessed using flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR and Western blot analysis. These results suggested that compound 3i induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that compound 3i is a promising candidate for future development as an anti-tumor drug.

Synthesis of 5-Alkynyltetrandrine Derivatives and Evaluation of their Anticancer Activity on A549 Cell Lines.

BACKGROUND: Lung cancer is one of the most prevalent malignancies and thus the development of novel therapeutic agents for managing lung cancer is imperative. Tetrandrine, a bis-benzyltetrahydroisoquinoline alkaloid isolated from Stephania tetrandra S. Moore, has been found to exert cytotoxic effects on cancerous cells. METHODS: A series of 5-alkynyltetrandrine derivatives was synthesized via the Sonogashira cross-coupling reactions and evaluated as potential anti-tumor agents. The anti-tumor activities of 12 compounds on lung cancer cells (A549) were evaluated using the MTT method. The population of apoptotic cells was measured using a TUNEL assay. Real-time PCR quantified the gene expression levels of Bcl-2, Bax, survivin and caspase-3. The content of Cyt-C was detected using a Human Cyt-C ELISA kit. RESULTS: Most of these compounds exhibited better activities than tetrandrine itself on A549 cells. Among them, compound 7 showed the highest cytotoxicity among the tested compounds against human lung adenocarcinoma A549 cells with an IC50 of 2.94 µM. Preliminary mechanistic studies indicated that compound 7 induced apoptosis of human lung cancer A549 cells and increased the level of the proapoptotic gene Bax, release of Cyt-C from mitochondria and activation of caspase-3 genes. CONCLUSION: The results suggest that compound 7 exerts its antitumor activity against A549 cells through the induction of the intrinsic (mitochondrial) apoptotic pathway. These findings will contribute to the future design of more effective anti-tumor agents in lung cancer therapy.

Synthesis and Biological Evaluation of Fangchinoline Derivatives as Anti-Inflammatory Agents through Inactivation of Inflammasome.

Twenty eight 7-substitued fangchinoline analogues, of which twenty two were novel, were synthesized and evaluated for their effect to inhibit lipopolysaccharide/nigericin (LPS/NIG)-induced IL-1ß release at both cell and protein levels at the concentration of 5 µM. Among them, compound 6 exhibited promising inhibitory potency against IL-ß activation with an IC50 value of 3.7 µM. Preliminary mechanism study revealed that 6 might target NLRP3 protein, and then block ASC pyroptosome formation with-NLRP3, rather than acting on the activation of the NLRP3 inflammasome (NF-κB and MAPK pathways) or caspase-1 protein. Our current study supported the potential role of compound 6 against IL-ß activation, and provided powerful information for developing fangchinoline derivatives into a novel class of anti-inflammatory agents.

Study on preparation, characterization and multidrug resistance reversal of red blood cell membrane-camouflaged tetrandrine-loaded PLGA nanoparticles.

The multidrug resistance in tumor (MDR) is a major barrier to efficient cancer therapy. Modern pharmacological studies have proven that tetrandrine (TET) has great potential in reversing MDR. However, it has a series of medication problems in clinic such as poor water solubility, low oral bioavailability and short half-life in vivo. Aiming at the above problems, red blood cell membrane-camouflaged TET-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (RPTNs) had been developed. The RPTNs had spherical shell-core double layer structure with average particle size of 164.1 ± 1.65 nm and encapsulation efficiency of 84.1% ± 0.41%. Compared with TET-PLGA nanoparticles (PTNs), the RPTNs reduced RAW 264.7 macrophages' swallowing by 32% due to its retention of natural membrane proteins. The cumulative drug release of RPTNs was 81.88% within 120 h. And pharmacokinetic study showed that the blood half-life of RPTNs was 19.38 h, which was 2.95 times of free drug. When RPTNs of 2 µg/mL TET were administered in combination with adriamycin (ADR), significant MDR reversal effect was observed in drug-resistant cells MCF-7/ADR. In a word, the RPTNs hold potential to improve its efficacy and broaden its clinical application.

Endothelium-dependent and endothelium-independent vasorelaxant effects of tiliacorinine 12'-O-acetate and mechanisms on isolated rat aorta.

Tiliacorinine 12'-O-acetate is a modified analog of Tiliacorinine, a major compound in Tiliacora triandra. The present study explored the vasorelaxation property of tiliacorinine 12'-O-acetate and its mechanism in isolated rat aorta using the organ bath technique. Tiliacorinine 12'-O-acetate exhibited concentration-dependent (10-15-10-3.5 M) vasorelaxation in endothelium-intact rings (Emax = 93.53 ± 2.79%) and endothelium-denuded rings (Emax = 74.31 ± 5.09%). The effects of tiliacorinine 12'-O-acetate were attenuated by pre-incubation with N(ω)-nitro-l-arginine methyl ester (L-NAME, endothelium nitric oxide synthase inhibitor) (100 µM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, soluble quanylylcyclase inhibitor) (1 µM), and 4-aminopyridine (1 mM, Kv channel blocker). However, this effect was not impacted by indomethacin (10 µM, cyclooxygenase inhibitor), tetraethylammonium (5 mM, Kca channel blocker), barium chloride (1 mM, KIR channel blocker), or glibenclamide (10 µM, KATP channel blocker). Moreover, pretreatment with tiliacorinine 12'-O-acetate reduced the effect of L-NAME (100 µM) on acetylcholine-induced vasorelaxation. Tiliacorinine 12'-O-acetate showed inhibitory effects on CaCl2-induced contracted rings and reduced the contraction induced by phenylephrine (10 µM) and caffeine (20 mM) in a Ca2+-free solution. The results of this study suggest that tiliacorinine 12'-O-acetate induced endothelium-dependent vasorelaxation through the eNOS/NO/sGC pathway, and also induced endothelium independent vasorelaxation involving the modulation of sGC activity, Kv channels, Ca2+ influx through Ca2+ channels and intracellular Ca2+ release. The data concerning the benefits of tiliacorinine 12'-O-acetate might be further investigated for the application of tiliacorinine 12'-O-acetate as an antihypertensive compound.

A Fluorescent Benzo[g]isoquinoline-Based HIF Prolyl Hydroxylase Inhibitor for Cellular Imaging.

Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.

Novel dauricine derivatives suppress cancer via autophagy-dependent cell death.

Eleven dauricine derivatives were synthesized and evaluated for their anti-cancer effect in different cancer cells and their autophagic activity in HeLa model cell. Among these newly synthesized compounds, carbamates 2a, 2b, carbonyl ester 3a and sulfonyl ester 4a exhibited potent cytotoxic effects on tested cancer cells with IC50 values ranged from 2.72 to 12.53 µM, which were more potent than that of dauricine (higher than 15.53 µM). The above four derivatives are validated to induce autophagy-dependent cell death in HeLa cancer cells. These findings offer us a promising source for generating novel autophagic enhancers for anti-cancer therapy.

Novel synthetic 4-chlorobenzoyl berbamine inhibits c-Myc expression and induces apoptosis of diffuse large B cell lymphoma cells.

C-Myc expression is associated with poor prognosis and aggressive progression of diffuse large B cell lymphoma (DLBCL), and the development of drug-like c-Myc inhibitors remains challenging. In this study, we report a novel berbamine derivative termed 4-chlorobenzoyl berbamine (CBBM) that potently induced the apoptosis of c-Myc-overexpressing DLBCL cells but spared normal blood cells. The compound showed IC50 values ranging from 1.93 to 3.89 µmol/L in DLCBL cells and exhibited a 4.75- to 9.64-fold increase in anti-tumor activity compared to berbamine. Additionally, CBBM inhibited the proliferation of the DLBCL line OCI-Ly3 cells through G0/G1 cell-cycle arrest and induced apoptosis. Further studies have shown that CBBM treatment leads to the proteasome-dependent degradation of c-Myc protein in OCI-Ly3 cells. Interestingly, we found that the inhibitory effect of CBBM was positively correlated with basal levels of CaMKIIγ, which is a key inducer of c-Myc expression in DLBCL cells. We also observed that CBBM inhibits the JAK2/STAT3 pathway, leading to reduced c-Myc transcription. Collectively, these findings suggest that CBBM could be a promising lead compound for treatment of c-Myc-driven DLBCL.

Synthesis, spectroscopic, physicochemical and structural characterization of tetrandrine-based macrocycles functionalized with acridine and anthracene groups: DNA binding and anti-proliferative activity.

In this work, we report on the synthesis of two new mono-alkylated tetrandrine derivatives with acridine and anthracene units, MAcT and MAnT. The compounds were fully characterized by physicochemical techniques and single-crystal X-ray diffraction analysis. In addition, both derivatives were studied as nucleotide receptors and double-stranded DNA binders in aqueous phosphate buffer at pH = 7.2 using UV-vis and fluorescence spectroscopy. According to the molecular recognition studies, MAcT and MAnT exhibit high affinity (K ∼ 105 M-1) and selectivity for ds-DNA, presumably in an intercalation mode. Finally, the anti-proliferative effects of the tetrandrine derivatives on different cancer cell lines were explored, revealing promising activities. Particularly, the mono-anthracene tetrandrine derivative MAnT showed an IC50 of 2.74 µg/mL on the HeLa cervical cancer cell line, representing a value 3.3 times smaller than that obtained for unsubstituted tetrandrine. Examination of the cytotoxic effects on the HeLa cell line by inverted microscopy suggests that the cell death mechanism consists basically in apoptosis. The molecular modelling of three ds-DNA-MAcT complexes, suggested that the macrocycles may use an intercalation binding mode towards DNA. MAcT is predicted to bind into the major groove of the ds-DNA providing non-covalent interactions such as electrostatic, van der Waals and hydrophobic interactions that lead to selectivity. Overall experimental data supports the mode of action of MAnT and MAcT as cytotoxic compounds against cancer cell lines via a DNA interaction mechanism.

Design, Synthesis and Anticancer Evaluation of Fangchinoline Derivatives.

Twenty fangchinoline derivatives were synthesized from the natural product fangchinoline, and their anticancer activities on human breast cancer MDA-MB-231 cell line, human prostate cancer PC3 cell line, human melanoma WM9 cell line and human leukaemia HEL and K562 cell lines were evaluated. The biological result showed that those derivatives exhibited potent activities on inhibiting cancer cell growth, and the structure-activity relationships were investigated. Among them, compound 4g, which was protected by benzoyl group in 7-phenolic position and nitrified in 14-position, showed impressive inhibition on all 5 cancer cell lines, especially WM9 cell line, with an IC50 value of 1.07 µM. Further mechanistic studies demonstrated that compound 4g may induce cancer cell death by apoptotic means. These research results suggested that compound 4g could be a lead for the further development toward an anticancer agent against human melanoma WM9 in the future.

Green Routes for the Production of Enantiopure Benzylisoquinoline Alkaloids.

Benzylisoquinoline alkaloids (BIAs) are among the most important plant secondary metabolites, in that they include a number of biologically active substances widely employed as pharmaceuticals. Isolation of BIAs from their natural sources is an expensive and time-consuming procedure as they accumulate in very low levels in plant. Moreover, total synthesis is challenging due to the presence of stereogenic centers. In view of these considerations, green and scalable methods for BIA synthesis using fully enzymatic approaches are getting more and more attention. The aim of this paper is to review fully enzymatic strategies for producing the benzylisoquinoline central precursor, (S)-norcoclaurine and its derivatives. Specifically, we will detail the current status of synthesis of BIAs in microbial hosts as well as using isolated and recombinant enzymes.

Design, synthesis and biological activities of tetrandrine and fangchinoline derivatives as antitumer agents.

The isolation and modification of natural products is always a very important resources to anti-tumor drugs. Therefore, a novel series of tetrandrine and fangchinoline derivatives were designed and synthesized, and their antiproliferative activities against HepG2, MCF-7 cells were evaluated and described. From the activity result obtained, high to very high activity in vitro has been found, one of the tested compounds (compound 5d) exhibited the most significant cytotoxic effects. Compound 5d increased 29.2, 7.37 times anti-proliferative activity for HepG2 cells and MCF-7 cells compared to sunitinib (IC50=16.06µM and 25.41µM). Finally flow cytometry determined that compound 5d could indeed inhibit the proliferation of HepG2 cells via inducing apoptosis.

Design and synthesis of novel tetrandrine derivatives as potential anti-tumor agents against human hepatocellular carcinoma.

Tetrandrine, a lead anti-tumor compound with a bis-benzyltetrahydroisoquinoline skeleton isolated from medicinal plant Stephania tetrandra. In order to obtain active anti-tumor agents and evaluate their structure-activity relationships, a series of novel tetrandrine derivatives were designed and synthesized in this study. Their anti-tumor activities against human hepatocellular carcinoma cell lines (HMCC97L and PLC/PRF/5) were also evaluated. The bioassay results showed that the derivatives exhibited moderate to strong inhibition against the two cell lines. Among them, compound 31 showed prominent cytotoxicity with IC50 = 1.06 µM (15.8 folds than that of tetrandrine, and 30.3 folds than that of Sorafenib). Further studies on the mechanisms demonstrated that the in vitro anti-tumor activity of compound 31 was predominantly due to the inducement of apoptosis of HCC cells. Compound 31 was capable of initiating endoplasmic reticulum stress-associated apoptotic cell death, and the activation of JNK as well as caspase pathways were probably involved. Our results suggest that compound 31, a new 14-position substituted amide tetrandrine derivative, might be a potential candidate for developing novel anti-HCC drugs in the coming future.

Synthesis and evaluation of benzylisoquinoline derivatives for their inhibition on pancreatic lipase and preadipocyte proliferation.

The present study was designed to synthesize and evaluate a series of benzylisoquinoline derivatives. These compounds were synthesized by Bischler-Napieralski cyclization to yield 1-benzyl-3,4-dihydroisoquinolines, and the products were obtained by reductions. All these compounds were identified by MS, (1)H NMR and (13)C NMR. The inhibitory activities on pancreatic lipase and preadipocyte proliferation for the synthesized compounds and alkaloids from Nulembo nucifera were assessed in vitro. Most of the compounds showed inhibitory activities on both pancreatic lipase and preadipocyte proliferation. Particularly, compounds 7p-7u and 9d-9f exhibited significant inhibitory activity on pancreatic lipase while compounds 7c, 7d, 7f, 7g, 7i, and 7j potently inhibited the proliferation of 3T3-L1 preadipocytes. Our results provided a basis for future evaluation and development of these compounds as leads for therapeutics for human diseases.

Design and synthesis of new tetrandrine derivatives and their antitumor activities.

A series of tetrandrine derivatives were designed and synthesized using Suzuki coupling reaction. Eleven targeted compounds with over 50% inhibition against HL60 and A549 human cancer cell lines at 10 µM were further evaluated for the in vitro antitumor activities by MTT or SRB assay. The biological results revealed that some compounds exhibited potent antitumor activities. Thiophene derivative 6 and acetylphenyl derivative 5 were the most active ones against HL60 and A549 cell lines, with IC50 values less than 5 µM, which thus could be considered as useful candidate for further development of new antitumor agents.

A divergent approach to benzylisoquinoline-type and oxoaporphine alkaloids via regioselective direct ring metalation of alkoxy isoquinolines.

Methoxy- and benzyloxy-substituted isoquinolines are regioselectively metalated at C-1 with the Knochel-Hauser base, subsequent trapping with aromatic aldehydes gives aryl(isoquinolin-1-yl)carbinols as building blocks for divergent syntheses of different types of benzylisoquinoline alkaloids. Photochemical cyclization of ortho-bromo analogues under reductive conditions gives oxoaporphine alkaloids. Nine benzylisoquinoline alkaloids and two oxoaporphine alkaloids were obtained in two or three steps from appropriate isoquinolines.