A Precise qNMR Method for the Rapid Quantification of Lot-to-Lot Variations in Multiple Quality Attributes of Pentosan Polysulfate Sodium.

Pentosan polysulfate sodium (PPS) is an orphan drug with anticoagulant activity. PPS is prepared from the chemical processing of xylan extracted from beechwood tree to yield a mixture of 4-6 kDa polysaccharides. The chain is mainly composed of sulfated xylose (Xyl) with branched 4-O-methyl-glucuronate (MGA). During generic drug development, the quality attributes (QAs) including monosaccharide composition, modification, and length need to be comparable to those found in the reference list drug (RLD). However, the range of QA variation of the RLD PPS has not been well characterized. Here, multiple PPS RLD lots were studied using quantitative NMR (qNMR) and diffusion ordered spectroscopy (DOSY) to quantitate the components in the mixture and to probe both inter- and intra-lot precision variability. The DOSY precision assessed using coefficient of variation (CV) was 6%, comparable to PPS inter-lot CV of 5%. The QAs obtained from 1D qNMR were highly precise with a precision CV < 1%. The inter-lot MGA content was 4.8 ± 0.1%, indicating a very consistent botanical raw material source. Other process-related chemical modification including aldehyde at 0.51 ± 0.04%, acetylation at 3.3 ± 0.2% and pyridine at 2.08 ± 0.06%, varied more than MGA content. The study demonstrated that 1D qNMR is a quick and precise method to reveal ranges of variation in multiple attributes of RLD PPS which can be used to assess equivalency with generic formulations. Interestingly, the synthetic process appeared to introduce more variations to the PPS product than the botanical source of the material.

Correction: A novel derivative of betulinic acid, SYK023, suppresses lung cancer growth and malignancy.

[This corrects the article DOI: 10.18632/oncotarget.3701.].

A novel derivative of betulinic acid, SYK023, suppresses lung cancer growth and malignancy.

Herein, we evaluated the anti-cancer effect and molecular mechanisms of a novel betulinic acid (BA) derivative, SYK023, by using two mouse models of lung cancer driven by KrasG12D or EGFRL858R. We found that SYK023 inhibits lung tumor proliferation, without side effects in vivo or cytotoxicity in primary lung cells in vitro. SYK023 triggered endoplasmic reticulum (ER) stress. Blockage of ER stress in SYK023-treated cells inhibited SYK023-induced apoptosis. In addition, we found that the expression of cell cycle-related genes, including cyclin A2, B1, D3, CDC25a, and CDC25b decreased but, while those of p15INK4b, p16INK4a, and p21CIP1 increased following SYK023 treatment. Finally, low doses of SYK023 significantly decreased lung cancer metastasis in vitro and in vivo. Expression of several genes related to cell migration, including synaptopodin, were downregulated by SYK023, thereby impairing F-actin polymerization and metastasis. Therefore, SYK023 may be a potentially therapeutic treatment for metastatic lung cancer.

Perspectives on biologically active camptothecin derivatives.

Camptothecins (CPTs) are cytotoxic natural alkaloids that specifically target DNA topoisomerase I. Research on CPTs has undergone a significant evolution from the initial discovery of CPT in the late 1960s through the study of synthetic small-molecule derivatives to investigation of macromolecular constructs and formulations. Over the past years, intensive medicinal chemistry efforts have generated numerous CPT derivatives. Three derivatives, topotecan, irinotecan, and belotecan, are currently prescribed as anticancer drugs, and several related compounds are now in clinical trials. Interest in other biological effects, besides anticancer activity, of CPTs is also growing exponentially, as indicated by the large number of publications on the subject during the last decades. Therefore, the main focus of the present review is to provide an ample but condensed overview on various biological activities of CPT derivatives, in addition to continued up-to-date coverage of anticancer effects.

Recent progress on C-4-modified podophyllotoxin analogs as potent antitumor agents.

Podophyllotoxin (PPT), as well as its congeners and derivatives, exhibits pronounced biological activities, especially antineoplastic effects. Its strong inhibitory effect on tumor cell growth led to the development of three of the most highly prescribed anticancer drugs in the world, etoposide, teniposide, and the water-soluble prodrug etoposide phosphate. Their clinical success as well as intriguing mechanism of action stimulated great interest in further modification of PPT for better antitumor activity. The C-4 position has been a major target for structural derivatization aimed at either producing more potent compounds or overcoming drug resistance. Accordingly, numerous PPT derivatives have been prepared via hemisynthesis and important structure-activity relationship (SAR) correlations have been identified. Several resulting compounds, including GL-331, TOP-53, and NK611, reached clinical trials. Some excellent reviews on the distribution, sources, applications, synthesis, and SAR of PPT have been published. This review focuses on a second generation of new etoposide-related drugs and provides detailed coverage of the current status and recent development of C-4-modified PPT analogs as anticancer clinical trial candidates.

Design, synthesis, mechanisms of action, and toxicity of novel 20(s)-sulfonylamidine derivatives of camptothecin as potent antitumor agents.

Twelve novel 20-sulfonylamidine derivatives (9a-9l) of camptothecin (1) were synthesized via a Cu-catalyzed three-component reaction. They showed similar or superior cytotoxicity compared with that of irinotecan (3) against A-549, DU-145, KB, and multidrug-resistant (MDR) KBvin tumor cell lines. Compound 9a demonstrated better cytotoxicity against MDR cells compared with that of 1 and 3. Mechanistically, 9a induced significant DNA damage by selectively inhibiting Topoisomerase (Topo) I and activating the ATM/Chk related DNA damage-response pathway. In xenograft models, 9a demonstrated significant activity without overt adverse effects at 5 and 10 mg/kg, comparable to 3 at 100 mg/kg. Notably, 9a at 300 mg/kg (i.p.) showed no overt toxicity in contrast to 1 (LD50 56.2 mg/kg, i.p.) and 3 (LD50 177.5 mg/kg, i.p.). Intact 9a inhibited Topo I activity in a cell-free assay in a manner similar to that of 1, confirming that 9a is a new class of Topo I inhibitor. 20-Sulfonylamidine 1-derivative 9a merits development as an anticancer clinical trial candidate.

Optimization of 4-(N-cycloamino)phenylquinazolines as a novel class of tubulin-polymerization inhibitors targeting the colchicine site.

The 6-methoxy-1,2,3,4-tetrahydroquinoline moiety in prior leads 2-chloro- and 2-methyl-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)quinazoline (1a and 1b) was modified to produce 4-(N-cycloamino)quinazolines (4a-c and 5a-m). The new compounds were evaluated in cytotoxicity and tubulin inhibition assays, resulting in the discovery of new tubulin-polymerization inhibitors. 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin- 2(1H)-one (5f), the most potent compound, exhibited high in vitro cytotoxic activity (GI50 1.9-3.2 nM), significant potency against tubulin assembly (IC50 0.77 µM), and substantial inhibition of colchicine binding (99% at 5 µM). In mechanism studies, 5f caused cell arrest in G2/M phase, disrupted microtubule formation, and competed mostly at the colchicine site on tubulin. Compound 5f and N-methylated analogue 5g were evaluated in nude mouse MCF7 xenograft models to validate their antitumor activity. Compound 5g displayed significant in vivo activity (tumor inhibitory rate 51%) at a dose of 4 mg/kg without obvious toxicity, whereas 5f unexpectedly resulted in toxicity and death at the same dose.

A-ring modified betulinic acid derivatives as potent cancer preventive agents.

Ten new 3,4-seco betulinic acid (BA) derivatives were designed and synthesized. Among them, compounds 7-15 exhibited enhanced chemopreventive ability in an in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus early antigen (EBV-EA) activation assay in Raji cells. Specifically, analogs with a free C-28 carboxylic acid, including 7, 8, 11, and 13, inhibited EBV-EA activation significantly. The most potent compound 8 displayed 100% inhibition at 1×10(3) mol ratio/TPA and 73.4%, 35.9%, and 8.4% inhibition at 5×10(2), 1×10(2), and 1×10 mol ratio/TPA, respectively, comparable with curcumin at high concentration and better than curcumin at low concentration. The potent chemopreventive activity of novel seco A-ring BAs (8 and 11) was further confirmed in an in vivo mouse skin carcinogenesis assay.

N-aryl-6-methoxy-1,2,3,4-tetrahydroquinolines: a novel class of antitumor agents targeting the colchicine site on tubulin.

Structural optimizations of the prior lead 1a led to the discovery of a series of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinoline derivatives as a novel class of tubulin polymerization inhibitors targeted at the colchicine binding site. The most active compound 6d showed extremely high cytotoxicity against a human tumor cell line panel (A549, KB, KBvin, and DU145) with GI50 values ranging from 1.5 to 1.7 nM, significantly more potent than paclitaxel, especially against the drug-resistant KBvin cell line, in the same assays. Analogs 5f, 6b, 6c, and 6e were also quite potent, with a GI50 range of 0.011-0.19 µM. In further studies, active compounds 6b-e and 5f significantly inhibited tubulin assembly, with IC50 values of 0.92-1.0 µM and strongly inhibited colchicine binding to tubulin, with inhibition rates of 75-99% (at 5 µM), comparable with or more potent than combretastatin A-4 (IC50 0.96 µM). Current studies included design, synthesis, and biological evaluations of 24 new compounds (series 3-6). Related SAR analysis, molecular modeling, and evaluation of essential drug-like properties, i.e. water solubility, log P, and in vitro metabolic stability, were also performed.

Design, synthesis and potent cytotoxic activity of novel podophyllotoxin derivatives.

Twenty new acyl thiourea derivatives of podophyllotoxin and 4'-demethylepipodophyllotoxin were prepared and screened for their cytotoxicity against four human tumor cell lines, A-549, DU-145, KB, and KBvin. With IC50 values of 0.098-1.13 µM, compounds 13b, 13c, and 13o displayed much better cytotoxic activity than the control etoposide. Most importantly, 13b and 13o exhibited promising cytotoxicity against the drug resistant tumor cell line KBvin with IC50 values of 0.098 and 0.13 µM, respectively, while etoposide lost activity completely. Structure-activity relationship (SAR) correlations of the new derivatives have been established. Compounds 13b and 13o merit further development as a new generation of epipodophyllotoxin-derived antitumor clinical trial candidates.

Synthesis and biological evaluation of novel spin labeled 18ß-glycyrrhetinic acid derivatives.

Eighteen novel spin-labeled 18ß-glycyrrhetinic acid (GA) derivatives were designed, synthesized, and evaluated for cytotoxicity against four human tumor cell lines (A-549, DU-145, KB and KBvin). Most of the derivatives showed more significant cytotoxicity than that of the parent compound GA. The best compound, 6j, with a tryptophan amino moiety and piperidine nitroxyl radical showed GI(50) values of 13.7-15.0 µM, and was fivefold more potent than GA. In a mechanism of action study, compound 7a was confirmed as a 20S proteasome inhibitor in both in vitro and cell-based assays. These findings support further optimization efforts based on 18ß-GA as a lead compound to develop potential anticancer drug candidates.

Design, synthesis, and preclinical evaluations of novel 4-substituted 1,5-diarylanilines as potent HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) drug candidates.

Twenty-one new 4-substituted diarylaniline compounds (DAANs) (series 13, 14, and 15) were designed, synthesized, and evaluated against wild-type and drug resistant HIV-1 viral strains. As a result, approximately a dozen new DAANs showed high potency with low nano- to subnanomolar EC(50) values ranging from 0.2 to 10 nM. The three most promising compounds 14e, 14h, and 15h exhibited high potency against wild-type and drug-resistant viral strains with EC(50) values at the subnanomolar level (0.29-0.87 nM) and were comparable to or more potent than the new NNRTI drug riplivirine (2) in the same assays. Druglike physicochemical property assessments revealed that the most active DAANs (EC(50) < 10 nM) have better aqueous solubility (>1-90 µg/mL at pH 7.4 and pH 2) and metabolic stability in vitro than 2, as well as desirable log P values (<5) and polar surface areas (PSA) (<140 Å(2)). These promising results warrant further development of this novel compound class as potential potent anti-AIDS clinical trial candidates.

Design and synthesis of diarylamines and diarylethers as cytotoxic antitumor agents.

Based on a shared structural core of diarylamine in several known anticancer drugs as well as a new cytotoxic hit 6-chloro-2-(4-cyanophenyl)amino-3-nitropyridine (7), 30 diarylamines and diarylethers were designed, synthesized, and evaluated for cytotoxic activity against A549, KB, KB-vin, and DU145 human tumor cell lines (HTCL). Four new leads 11e, 12, 13a, and 13b were discovered with GI(50) values ranging from 0.33 to 3.45µM. Preliminary SAR results revealed that a diarylamine or diarylether could serve as an active structural core, meta-chloro and ortho-nitro groups on the A-ring (either pyridine or phenyl ring) were necessary and crucial for cytotoxic activity, and the para-substituents on the other phenyl ring (B-ring) were related to inhibitory selectivity for different tumor cells. In an investigation of potential biological targets of the new leads, high thoughput kinase screening discovered that new leads 11e, 12 and 13b especially inhibit Mer tyrosine kinase, a proto-oncogene associated with munerous tumor types, with IC(50) values of 2.2-3.0µM. Therefore, these findings provide a good starting point to optimize a new class of compounds as potential anticancer agents, particularly targeting Mer tyrosine kinase.

Optimization of 2,4-diarylanilines as non-nucleoside HIV-1 reverse transcriptase inhibitors.

The current optimization of 2,4-diarylaniline analogs (DAANs) on the central phenyl ring provided a series of new active DAAN derivatives 9a-9e, indicating an accessible modification approach that could improve anti-HIV potency against wild-type and resistant strains, aqueous solubility, and metabolic stability. A new compound 9e not only exhibited extremely high potency against wild-type virus (EC(50) 0.53 nM) and several resistant viral strains (EC(50) 0.36-3.9 nM), but also showed desirable aqueous solubility and metabolic stability, which were comparable or better than those of the anti-HIV-1 drug TMC278 (2). Thus, new compound 9e might be a potential drug candidate for further development of novel next-generation NNRTIs.

Antitumor agents 292. Design, synthesis and pharmacological study of S- and O-substituted 7-mercapto- or hydroxy-coumarins and chromones as potent cytotoxic agents.

Thirty-five S- and O-substituted 7-mercaptocoumarin (9-23) and 7-hydroxy- or 7-mercapto-chromone (24-43) analogs were designed, synthesized and evaluated in vitro against four human tumor cell lines [KB (nasopharyngeal), KB-vin (vincristine-resistant subline), A549 (lung) and DU145 (prostate)] with paclitaxel as the positive control. Many of the synthesized compounds exhibited potent cytotoxic activity. Among them, compounds 10 and 18 showed broad spectrum activity with GI(50) values ranging from 0.92 to 2.11 µM and 2.06-14.07 µM, respectively. However, 33, a 3-brominated compound, displayed significant and selective inhibition against MDR KB-vin with a GI(50) of 5.84 µM. Regardless of the size of the 7-alkoxy group, 2-α-bromoethyl-8-bromomethyl compounds (40-43) exhibited increased cytotoxicity compared with 2-ethyl-8-bromomethyl compounds (36-39). Moreover, in a preliminary pharmacological study, 10 not only remarkably increased cellular apoptosis in a concentration-dependent manner, but also clearly induced A549 cell cycle arrest at the G2/M phase. Thus, these coumarin derivatives merit investigation as novel potential antitumor agents with further structural modification to produce an optimal lead compound and elucidate the detailed pharmacological mechanism(s).

Bis-chalcone analogues as potent NO production inhibitors and as cytotoxic agents.

Chalcones have a distinctive 1,3-diarylpropenone skeleton and exert numerous biological effects. Using a one-step Claisen-Schmidt condensation, we synthesized eleven bis-chalcones (3-13) and three acetyl chalcones (14-16) from substituted aldehydes and diacetylresorcinol. The compounds were tested for in vitro cytotoxic activity against four human cancer cell lines (A549, DU145, KB, and KB-VIN) and inhibition of NO production in lipopolysaccharide (LPS)-activated microglial cells. Among them, four compounds (3, 5, 6, and 13) showed significant cytotoxic activity with EC(50) values ranging from 1.57 to 5.14 µM, and seven compounds (3, 5-8, 10, and 13) displayed potent anti-inflammatory activity by inhibiting NO production with IC(50) values ranging from 0.95 to 8.65 µM. A mechanism of action study of active compounds 6 and 7 discovered that these compounds down-regulated iNOS expression by inhibiting p65 NF-κB activation/nuclear translocation due to prevention of IκBα degradation. Structure-activity relationship (SAR) findings are also discussed.

New betulinic acid derivatives as potent proteasome inhibitors.

In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC(50) values of 1.42, 1.56, and 1.80 µM, respectively, which are three to fourfold more potent than the proteasome inhibition controls LLM-F and lactacystin.

New bichalcone analogs as NF-κB inhibitors and as cytotoxic agents inducing Fas/CD95-dependent apoptosis.

A series of novel bichalcone analogs were synthesized and evaluated in lipopolysaccharide (LPS)-activated microglial cells as inhibitors of nitric oxide (NO) and for in vitro anticancer activity using a limited panel of four human cancer cell lines. All analogs inhibited NO production. Compounds 4 and 11 exhibited optimal activity with IC(50) values of 0.3 and 0.5 µM, respectively, and were at least 38-fold better than the positive control. A mechanism of action study showed that both compounds significantly blocked the nuclear translocation of NF-κB p65 and up-regulation of iNOS at 1.0 µM. Compound 4 and three other analogs (3, 20, and 23) exerted significant in vitro anticancer activity GI(50) values ranging from 0.70 to 13.10 µM. A mode of action study using HT-29 colon cancer cells showed that 23 acts by inducing apoptosis signaling.

Bioactive constituents from the roots of Panax japonicus var. major and development of a LC-MS/MS method for distinguishing between natural and artifactual compounds.

Two new saponins, panajaponol (1) and pseudoginsenoside RT1 butyl ester (2), together with 35 known compounds (3-37), were isolated from the roots of Panax japonicus var. major. The structures of 1 and 2 were elucidated on the basis of spectroscopic analysis and chemical methods. Furthermore, a LC-MS/MS method was developed for confirming 2, 3, and 8 as natural compounds containing a butyl ester group. This method should be useful for distinguishing between minor natural and artifactual compounds in Panax species. Moreover, compounds 3, 6, 8, 9, 11, 13, and 15 exhibited strong inhibition of superoxide anion generation and elastase release by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB), with IC(50) values ranging from 0.78 to 43.6 µM. In addition, 1 showed greater than 2- to 3-fold selective cytotoxic activity against KB and DU145 cancer cell lines.

Synthesis, in vitro anti-inflammatory and cytotoxic evaluation, and mechanism of action studies of 1-benzoyl-ß-carboline and 1-benzoyl-3-carboxy-ß-carboline derivatives.

In the present study, various 1-substituted and 1,3-disubstituted ß-carboline derivatives were synthesized by a modified single-step Pictet-Spengler reaction. The compounds were examined for cytotoxicity and anti-inflammatory activity, as measured by the inhibition of prostaglandin E(2) (PGE(2)) production and nitric oxide (NO) production. While only two compounds (28 and 31) showed marginal cytotoxicity against four human cancer cell lines, most of the tested compounds exhibited potent inhibitory activity of both NO and PGE(2) production. Moreover, compounds 6 and 16 significantly reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2), suggesting that ß-carboline analogs can inhibit NO and PGE(2) production at the translational level. In addition, several of the ß-carboline derivatives (1, 2, 4-8, 11, 13, 22, 25, 27, 31, and 41-43) displayed significant inhibitory activity of superoxide anion (O(2)(·-)) generation or elastase release compared to the reference compound, with 6 being the most potent. N-Formyl-L-methionyl-phenylalanine (FMLP)-induced phosphorylation of c-JunN-terminal kinase (JNK) and protein kinase B (AKT) were also inhibited by 6, suggesting that it suppresses human neutrophil functions by inhibiting the activation of JNK and AKT signaling pathways. Therefore, the synthetic 1-benzoyl-3-carboxy ß-carboline analogs may have great potential to be developed as anti-inflammatory agents.