Design and Synthesis of Cyclolipopeptide Mimics of Dysoxylactam A and Evaluation of the Reversing Potencies against P-Glycoprotein-Mediated Multidrug Resistance.

Inspired by the structure of dysoxylactam A (DLA) that has been demonstrated to reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) effectively, 61 structurally simplified cyclolipopeptides were thus designed and synthesized via an effective method, and their reversing P-gp-mediated MDR potentials were evaluated, which provided a series of more potent analogues and allowed us to explore their structure-activity relationship (SAR). Among them, a well-simplified compound, 56, with only two chiral centers that all derived from amino acids dramatically reversed drug resistance in KBV200 cells at 10 µM in combination with vinorelbine (VNR), paclitaxel (PTX), and adriamycin (ADR), respectively, which is more promising than DLA. The mechanism study showed that 56 reversed the MDR of tumor cells by inhibiting the transport function of P-gp rather than reducing its expression. Notably, compound 56 effectively restored the sensitivity of MDR tumors to VNR in vivo at a dosage without obvious toxicity.

SIBP-03, a novel anti-HER3 antibody, exerts antitumor effects and synergizes with EGFR- and HER2-targeted drugs.

HER3 (human epidermal growth factor receptor 3) acts through heterodimerization with EGFR (epidermal growth factor receptor) or HER2 to play an essential role in activating phosphoinositide 3-kinase (PI3K) and AKT signaling-a crucial pathway that promotes tumor cell survival. HER3 is a promising target for cancer therapy, and several HER3-directed antibodies have already entered into clinical trials. In this study we characterized a novel anti-HER3 monoclonal antibody, SIBP-03. SIBP-03 (0.01-10 µg/mL) specifically and concentration-dependently blocked both neuregulin (NRG)-dependent and -independent HER3 activation, attenuated HER3-mediated downstream signaling and inhibited cell proliferation. This antitumor activity was dependent, at least in part, on SIBP-03-induced, cell-mediated cytotoxicity and cellular phagocytosis. Importantly, SIBP-03 enhanced the antitumor activity of EGFR- or HER2-targeted drugs (cetuximab or trastuzumab) in vitro and in vivo. The mechanisms underlying this synergy involve increased inhibition of HER3-mediated downstream signaling. Collectively, these results demonstrated that SIBP-03, which is currently undergoing a Phase I clinical trial in China, may offer a new treatment option for patients with cancers harboring activated HER3, particularly as part of a combinational therapeutic strategy.

DCZ5248, a novel dual inhibitor of Hsp90 and autophagy, exerts antitumor activity against colon cancer.

Hsp90 is a potential therapeutic target for tumor, as it maintains the stability of a variety of proteins related to tumor development and progression. Autophagy is a self-degradation process to maintain cellular homeostasis and autophagy inhibitors can suppress tumor growth. In this study, we identified DCZ5248, a triazine derivative, was a dual inhibitor of both Hsp90 and late-autophagy with potent antitumor activity against colon cancer cells in vitro and in vivo. We showed that DCZ5248 (0.1-10 µM) induced dose-dependent degradation of Hsp90 client proteins (AKT, CDK4, CDK6 and RAF-1) in HCT 116 colon cancer cells through a proteasome-dependent pathway. Meanwhile, DCZ5248 (0.3 µM) induced cytoplasmic vacuole formation, LC3 II conversion, p62 protein upregulation, and inhibited autophagy at the late stage in the colon cancer cell lines tested. We further revealed that the inhibition of autophagy was achieved by impairing lysosomal functions through induction of lysosomal acidification and attenuation of lysosomal cathepsin activity. The modulation of autophagy by DCZ5248 was independent of Hsp90 inhibition as the autophagy inhibition was not blocked by Hsp90 knockdown. Importantly, inhibition of both Hsp90 function and autophagy by DCZ5248 induced G1-phase cell cycle arrest, apoptosis, and exerted potent antitumor activity against colon cancer cells both in vitro and in vivo. These findings demonstrate that DCZ5248 is a novel dual inhibitor of Hsp90 and autophagy with potential for colon cancer therapy.

Dysoxylactam A: A Macrocyclolipopeptide Reverses P-Glycoprotein-Mediated Multidrug Resistance in Cancer Cells.

A 17-membered macrocyclolipopeptide, named dysoxylactam A (1) comprising an unprecedented branched C19 fatty acid and an l-valine, was isolated from the plants of Dysoxylum hongkongense. The challenging relative configuration of 1 was established by means of residual dipolar coupling-based NMR analysis. The absolute configuration of 1 was determined by single-crystal X-ray diffraction on its p-bromobenzoate derivative (2). Compound 1 dramatically reversed multidrug resistance in cancer cells with the fold-reversals ranging from 28.4 to 1039.7 at the noncytotoxic concentration of 10 µM. The mode-of-action study of 1 revealed that it inhibited the function of P-glycoprotein (P-gp), a key mediator in multidrug resistance.

SHR-A1403, a novel c-Met antibody-drug conjugate, exerts encouraging anti-tumor activity in c-Met-overexpressing models.

Emerging evidence demonstrates that a c-Met antibody-drug conjugate (ADC) has superior efficacy and safety profiles compared with those of currently available small molecules or antibody inhibitors for the treatment of c-Met-overexpressing cancers. Here we described both the in vitro and in vivo efficacies of SHR-A1403, a novel c-Met ADC composed of a humanized IgG2 monoclonal antibody against c-Met conjugated to a novel cytotoxic microtubule inhibitor. SHR-A1403 showed high affinity to c-Met proteins derived from human or monkey and potent inhibitory effects in cancer cell lines with high c-Met protein expression. In mice bearing tumors derived from cancer cell lines or patient HCC tissues with confirmed c-Met overexpression, SHR-A1403 showed excellent anti-tumor efficacy. Antibody binding with c-Met contributed to SHR-A1403 endocytosis; the subsequent translocation to lysosomes and cytotoxicity of the released toxin are speculated to be predominant mechanisms underlying the anti-tumor activity of SHR-A1403. In conclusion, SHR-A1403 showed significant anti-tumor activity in cancer cell lines, xenograft mouse models and an HCC PDX model, which all have high c-Met levels. These data provide references for SHR-A1403 as a potential therapy for the treatment of cancers with c-Met overexpression.

Aberrant modulation of ribosomal protein S6 phosphorylation confers acquired resistance to MAPK pathway inhibitors in BRAF-mutant melanoma.

BRAF and MEK inhibitors have shown remarkable clinical efficacy in BRAF-mutant melanoma; however, most patients develop resistance, which limits the clinical benefit of these agents. In this study, we found that the human melanoma cell clones, A375-DR and A375-TR, with acquired resistance to BRAF inhibitor dabrafenib and MEK inhibitor trametinib, were cross resistant to other MAPK pathway inhibitors. In these resistant cells, phosphorylation of ribosomal protein S6 (rpS6) but not phosphorylation of ERK or p90 ribosomal S6 kinase (RSK) were unable to be inhibited by MAPK pathway inhibitors. Notably, knockdown of rpS6 in these cells effectively downregulated G1 phase-related proteins, including RB, cyclin D1, and CDK6, induced cell cycle arrest, and inhibited proliferation, suggesting that aberrant modulation of rpS6 phosphorylation contributed to the acquired resistance. Interestingly, RSK inhibitor had little effect on rpS6 phosphorylation and cell proliferation in resistant cells, whereas P70S6K inhibitor showed stronger inhibitory effects on rpS6 phosphorylation and cell proliferation in resistant cells than in parental cells. Thus regulation of rpS6 phosphorylation, which is predominantly mediated by BRAF/MEK/ERK/RSK signaling in parental cells, was switched to mTOR/P70S6K signaling in resistant cells. Furthermore, mTOR inhibitors alone overcame acquired resistance and rescued the sensitivity of the resistant cells when combined with BRAF/MEK inhibitors. Taken together, our findings indicate that RSK-independent phosphorylation of rpS6 confers resistance to MAPK pathway inhibitors in BRAF-mutant melanoma, and that mTOR inhibitor-based regimens may provide alternative strategies to overcome this acquired resistance.

Synthesis and antitumor activity of a series of lactone-opened camptothecin derivatives.

A series of E-ring lactone-opened camptothecin (CPT) derivatives bearing with terminal aza-heterocyclic groups were synthesized, and their antitumor activity was evaluated both in vitro and in vivo. Hydroxyl-amide analogues with morpholin-4-yl displayed excellent antitumor activity in vitro and efficient inhibition on tumor xenograph model in nude mice. Ester-amide compounds acted less active in vitro cytotoxicity and lower inhibition activity in vivo. Substitutions at 7- and 10- positions favored the antitumor activity.

Pharmacokinetics and disposition of anlotinib, an oral tyrosine kinase inhibitor, in experimental animal species.

Anlotinib is a new oral tyrosine kinase inhibitor; this study was designed to characterize its pharmacokinetics and disposition. Anlotinib was evaluated in rats, tumor-bearing mice, and dogs and also assessed in vitro to characterize its pharmacokinetics and disposition and drug interaction potential. Samples were analyzed by liquid chromatography/mass spectrometry. Anlotinib, having good membrane permeability, was rapidly absorbed with oral bioavailability of 28%-58% in rats and 41%-77% in dogs. Terminal half-life of anlotinib in dogs (22.8±11.0 h) was longer than that in rats (5.1±1.6 h). This difference appeared to be mainly associated with an interspecies difference in total plasma clearance (rats, 5.35±1.31 L·h-1·kg-1; dogs, 0.40±0.06 L·h-1/kg-1). Cytochrome P450-mediated metabolism was probably the major elimination pathway. Human CYP3A had the greatest metabolic capability with other human P450s playing minor roles. Anlotinib exhibited large apparent volumes of distribution in rats (27.6±3.1 L/kg) and dogs (6.6±2.5 L/kg) and was highly bound in rat (97%), dog (96%), and human plasma (93%). In human plasma, anlotinib was predominantly bound to albumin and lipoproteins, rather than to α1-acid glycoprotein or γ-globulins. Concentrations of anlotinib in various tissue homogenates of rat and in those of tumor-bearing mouse were significantly higher than the associated plasma concentrations. Anlotinib exhibited limited in vitro potency to inhibit many human P450s, UDP-glucuronosyltransferases, and transporters, except for CYP3A4 and CYP2C9 (in vitro half maximum inhibitory concentrations, <1 µmol/L). Based on early reported human pharmacokinetics, drug interaction indices were 0.16 for CYP3A4 and 0.02 for CYP2C9, suggesting that anlotinib had a low propensity to precipitate drug interactions on these enzymes. Anlotinib exhibits many pharmacokinetic characteristics similar to other tyrosine kinase inhibitors, except for terminal half-life, interactions with drug metabolizing enzymes and transporters, and plasma protein binding.

Unusual Cytotoxic Steroidal Saponins from the Gorgonian Astrogorgia dumbea.

Three steroidal saponins, including astrogorgiosides A (1) and B (2) bearing acetamido-glucose moieties, and astrogorgioside C (3) with a 19-nor and bearing an aromatized B ring steroid aglycone, together with a known major saponin dimorphoside A (4), were obtained from the gorgonian Astrogorgia dumbea collected near Dongshan Island in East China Sea. Structures of these compounds were elucidated by in-depth spectral and chemical methods, including 2D-NMR, HR-ESI-MS spectra, and acidic hydrolysis. For the first time, acetamido-glucose moiety is being reported from a gorgonian. The B-ring aromatized steroid aglycone of compound 3 is also rare in marine natural products. Compounds 1-3 exhibited moderate cytotoxic activity with IC50 values of 26.8-45.6 µM against human tumor cells Bel-7402 and K562.

Preclinical activity of lobaplatin as a single agent and in combination with taxanes for ovarian carcinoma cells.

Lobaplatin, one of the third-generation platinum compounds, has shown encouraging anticancer activity in a variety of tumor types. However, the efficacy of lobaplatin in ovarian cancer has not been systemically evaluated. In this study, lobaplatin as a single agent and in combination with taxanes was investigated in-vitro and in an in vitro model of ovarian carcinoma. Using the sulforhodamine B (SRB) assay, the cytotoxic effects of lobaplatin alone and in combination with taxanes were compared with cisplatin and carboplatin in seven ovarian cancer cell lines. In addition, in-vitro antitumor activities were evaluated with cisplatin-sensitive and cisplatin-resistant human ovarian cancer xenografts in nude mice. The cytotoxicity of lobaplatin was similar to or higher than that of cisplatin and carboplatin, with IC50 values from 0.9 to 13.8 µmol/L in a variety of ovarian cancer cells. The combination of lobaplatin with docetaxel yielded enhanced cytotoxic activity in vitro. In addition, in platinum-sensitive ovarian cancer xenografts, lobaplatin alone showed similar antitumor activity to cisplatin and carboplatin. Furthermore, lobaplatin alone or in combination with docetaxel exhibited significant activity in platinum-resistant ovarian cancer xenografts. These results indicate that the use of lobaplatin alone or in combination with docetaxel might be a rational and novel therapeutic strategy for ovarian cancer. Further clinical development of lobaplatin is clearly warranted.

Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro.

AIM: To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro. METHODS: 32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells. RESULTS: C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC50 at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC50 at low micromole levels. C817 (0.5 or 1 µmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells. CONCLUSION: C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.

Antitumor activity of lobaplatin alone or in combination with antitubulin agents in non-small-cell lung cancer.

OBJECTIVE: Lobaplatin is used to treat patients with breast cancer, small-cell lung cancer, and chronic myelogenous leukemia in China. In this study, we assessed the in-vitro and in-vivo activities of lobaplatin alone or in combination with antitubulin agents against human non-small-cell lung cancer (NSCLC). METHODS: The cytotoxicities of lobaplatin against NSCLC cells were determined by the sulforhodamine B (SRB) assay. Cell cycle analysis and apoptosis were assessed using flow cytometry, and the in-vivo antitumor activities were evaluated in human NSCLC xenografts in nude mice. RESULTS: The cytotoxicity of lobaplatin was similar to or higher than that of cisplatin and carboplatin, with a mean IC(50) of 2.5 µmol/l in a variety of NSCLC cells. In addition, lobaplatin arrested cells in the S phase and triggered apoptosis. The combination of lobaplatin with antitubulin agents yielded synergistic cytotoxic activity in vitro. In NSCLC xenografts, lobaplatin alone showed significant antitumor activity. The combination of lobaplatin with antitubulin agents, especially with paclitaxel, led to significantly enhanced activity, which was superior to that of cisplatin combined with antitubulin agents. CONCLUSION: These data demonstrate that the use of lobaplatin alone or in combination with antitubulin agents might be a rational and novel therapeutic strategy for human NSCLC and warrants further clinical investigation.

A novel and simple hollow-fiber assay for in vivo evaluation of nonpeptidyl thrombopoietin receptor agonists.

Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult because of the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to preclinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for nonpeptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. Cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a nonpeptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signaling transducers and activators of transcription in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.

Structure and cytotoxicity of diterpenoids from Isodon eriocalyx.

A new ent-atisanoid, eriocatisin A (1), six new ent-abietanoids, eriocasins B-E (2-4, 7), 3-acetyleriocasin C (5), and 3ß-acetoxyeriocasin D (6), and seven new ent-kauranoids, maoesins A-F (8, 10-14) and 3α-acetoxy-maoesin A (9), together with 21 known compounds, were isolated from the aerial parts of Isodon eriocalyx. The structures of 1-14 were determined by spectroscopic data interpretation. All compounds isolated were evaluated for their in vitro growth inhibitory activity against the HT-29, BEL-7402, and SK-OV-3 human cancer cell lines. Compounds 17, 18, and 20 showed inhibitory effects for all three tumor cell lines used, with IC(50) values in the range 2.1-7.3 µM.

Three new 18-oxygenated ent-kaurane diterpenoids from Isodon leucophyllus.

Three new 18-oxygenated ent-kaurane diterpenoids, isoleuconins A-C (1-3) and ten known diterpenoids were isolated from the aerial parts of Isodon leucophyllus. The structures were elucidated by 1D and 2D NMR spectroscopic analysis. All of the compounds were evaluated for their cytotoxicity. Rabdokunmin A (13) showed significant cytotoxicity against HT-29 cells, with an IC50 value of 6.2 microM.

Synthesis and structure-activity relationship studies of cytotoxic anhydrovinblastine amide derivatives.

A series of 3-demethoxycarbonyl-3-amide methyl anhydrovinblastine derivatives (5b-24b) was designed, synthesized, and evaluated for their proliferation inhibition activities against two tumor cell lines (A549 and HeLa). Most of the amide anhydrovinblastine derivatives exhibited potent cytotoxicity, with the size of the introduced substituents being the foremost factor in determining the resultant cytotoxic activity. Test results in vivo against sarcoma 180 of three potent compounds (6b, 12b, and 24b) indicated that the introduction of an amide group at the 22-position of anhydrovinblastine (1e) improved both potency and toxicity.

Design, synthesis and biological evaluation of novel fluorinated docetaxel analogues.

A series of novel fluorinated docetaxel analogues have been synthesized and evaluated in vitro and in vivo. Incorporated one, two or three fluorine atom(s) either at both meta position on C-2 benzolate and 3'-N-tert-butyloxyl group or only at 3'-N-tert-butyloxyl group has resulted in potent analogues which have comparable or superior in vitro and in vivo cytotoxicity to docetaxel. Among them, compounds 14d and 14e have displayed more potent cytotoxicity than docetaxel both in human cancer cell line SK-OV-3 in vitro and in human non-small cell lung cancer A549 xenografts in vivo. Preliminary data show that compound 14a has reduced acute animal toxicity in mice compared with docetaxel.

Kadcoccilactones A-J, triterpenoids from Kadsura coccinea.

Ten new highly oxygenated triterpenoids, kadcoccilactones A-J (1-10), and two known triterpenoids, kadsuphilactone A and micrandilactone B, were isolated from the stems of the evergreen climbing plant Kadsura coccinea. The structures of the new compounds were elucidated by spectroscopic evidence, with that of 1 confirmed by single-crystal X-ray diffraction analysis. This is the first report on nortriterpenoids (2, 4-6, 8-10) from the genus Kadsura.

Synthesis, cytotoxic activity, and SAR analysis of the derivatives of taxchinin A and brevifoliol.

Twenty-one derivatives of taxchinin A (1) and brevifoliol (2) were synthesized and evaluated for cytotoxicity against human non-small lung cancer (A549) cell line. Nine derivatives showed potent activity with IC(50) values from 0.48 to 6.22microM. 5-Oxo-13-TBDMS-taxchinin A (11) and 5-oxo-13,15-epoxy-13-epi-taxchinin A (15) are the most potent derivatives, with IC(50) at 0.48 and 0.75microM, respectively. The structure-activity relationship (SAR) of these compounds established that exocyclic unsaturated ketone at ring C is the key structural element for the activity, while the alpha,beta-unsaturated ketone positioned at ring A has no effect for the activity. The significant cytotoxicity of derivatives 11 and 15 may be due to the conformational change in the taxane rings. The 3D-QSAR study was conducted on this series of compounds, which provided optimal predictive comparative molecular field (CoMFA) model with cross-validated r(2) (q(2)) value of 0.64.