Semisynthetic Analogues Research Articles

From Bacteria to Antineoplastic: Epothilones A Successful History

Malignancies are a major cause of morbidity and mortality worldwide. Cancer is a cell disease, characterized by a deviation of the control mechanisms of proliferation and differentiation of cells. Among the treatments available, chemotherapy is often the first choice. Epothilones are a new class of anticancer drugs that act by interacting with cellular microtubules interrupting the proliferation of cancer cells. Many synthetic and semi-synthetic analogues of epothilones have been prepared aiming improvement in effectiveness and tolerability, based on QSAR studies. These analogues have been effective for treatment of tumors resistant to first-line treatments. Six new epothilones are being subjected to clinical trials. Ixabepilone (Ixempra®) was approved by FDA in 2007, patupilone is in phase III clinical trial for ovarian and peritoneum cancer. Sagopilone, desoxiepothilone and KOS-1584 are in phase II clinical trials, for the treatment of recurrent glioblastoma and advanced metastatic breast cancer, metastasic breast cancer and metastatic pulmonary cancer, respectively. Desoxiepothilone reached only phase II trials and BMS-310705 reached phase III/IV trials, but were not approved for clinical use due to adverse effects such as neurotoxicity and severe diarrhea, which were dose-limiting. Furthermore, the low t1/2 (40h) in comparison with other class analogues, does not recommend the clinical use of this derivative. Some other synthetized epothilones presented antineoplastic activity in vitro, but are not yet submitted to clinical studies. Neuropathies and diarrhea are adverse effects presented by some substances of this class of anticancer drugs.

Protective activity of (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol analogues against diisopropylfluorophosphate neurotoxicity: Preliminary structure–activity relationship and pharmacophore modeling

Diisopropylfluorophosphate (DFP) is an organophosphorous insecticide used as a surrogate for the more toxic chemical warfare nerve agent sarin. DFP produces neurotoxicity in vivo and irreversibly decreases the area of population spikes recorded from the CA1 region of acute hippocampal slices. (1S,2E,4R,6R,7E,11E)-2,7,11-Cembratriene-4,6-diol (1) is a neuroprotective natural cembranoid that reverses DFP-induced damage both in vivo and in the hippocampal slice. Cembranoid 1 acts by noncompetitive inhibition of the α7 nicotinic acetylcholine receptor. This study aims at establishing a preliminary structure–activity relationship to define the neuroprotective cembranoid pharmacophores using the hippocampal slice assay and pharmacophore modeling. Fourteen natural, semisynthetic, or biocatalytic cembranoid analogues 2–15 related to 1 were tested for their capacity to protect the population spikes from DFP-induced damage and intrinsic toxicity. Twelve cembranoids caused significant reversal of DFP toxicity; only 3 active analogues displayed minor intrinsic toxicity at 10μM. The C-4 epimer of 1 (2) and the 4-O-methyl ether analogue of 1 (3), were totally devoid of neuroprotective activity. The results suggested a model for cembranoid binding where the hydrophobic ring surface binds to a hydrophobic (Hbic) patch on the receptor molecule and an electronegative atom (oxygen or sulfur) in proper spatial relationship to the ring surface interacts with an electropositive group in the receptor binding site. A pharmacophore model consisting of 1 hydrogen bond acceptor (HBA), 2 Hbic, and 10 exclusion spheres was established using HipHop-REFINE and supported the above mentioned pharmacophoric hypothesis.

Spongian Diterpenoids Inhibit Androgen Receptor Activity

Androgen receptor is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiologic ligands for androgen receptor ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit androgen receptor transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semisynthetic analogues (T2 and T3) revealed that these diterpenoids have antiandrogen properties that include inhibition of both androgen-dependent proliferation and androgen receptor transcriptional activity by a mechanism that involved competing with androgen for androgen receptor LBD and blocking essential N/C interactions required for androgen-induced androgen receptor transcriptional activity. Structure-activity relationship analyses revealed some chemical features of T1 that are associated with activity and yielded T3 as the most potent analogue. In vivo, T3 significantly reduced the weight of seminal vesicles, which are an androgen-dependent tissue, thereby confirming the on-target activity of T3. The ability to create analogues of diterpenoids that have varying antiandrogen activity represents a novel class of chemical compounds for the analysis of androgen receptor ligand-binding properties and therapeutic development.

NF-κB Down–regulation and PARP Cleavage by novel 3-α-butyryloxy-β-boswellic Acid Results in Cancer Cell Specific Apoptosis and in vivo Tumor Regression

The present study relates to the induction of apoptosis thereof cytotoxicity and anti-cancer activity displayed by semi-synthetic analog of Boswellic acid i.e. 3-α-Butyryloxy-β-boswellic acid (BOBA). The cytotoxicity data revealed the differential sensitivity of cancer cell lines towards BOBA which may display its impact against different types of cancers. Considering the inhibitory potential of BOBA, we further sought to understand the target for BOBA deciphering the mechanism of action leading to apoptotic cell death and it was for the first time reported about the triterpenoid ring especially the β-boswellic acid derivative is targeting PI3K pathway. Our data revealed that BOBA treatment provides evidence about the apoptotic nature showing the potential of targeting mitochondria dependent pathways during apoptosis in HL-60 cells. BOBA induced hypo-diploid sub-G(1) DNA population in HL-60 cells as was also evident from the pattern of DNA fragmentation and mitochondrial membrane potential (ΛΨm) loss. Morphological analysis under fluorescent and scanning electron microscopy displayed typical features such as cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. These events paralleled with the down-regulation of NF-κB and induced PARP cleavage. Furthermore, it is noteworthy that BOBA also depicted significant growth inhibition in Ehrlich Ascitic Tumour (EAT), Ehrlich Ascitic Carcinoma (EAC) and Sarcoma- 180 tumour models. Taken together, BOBA treatment may represent as potential agent to the currently available anticancer agents in both prophylactic and/or therapeutic applications. Also, our findings may open up a new perspective in the construction of novel anticancer agents based on boswellic acids that will facilitate the development of these agents for anticancer therapeutics.

Ginseng saponin metabolite 20(S)-protopanaxadiol inhibits tumor growth by targeting multiple cancer signaling pathways

Plant-derived active constituents and their semi-synthetic or synthetic analogs have served as major sources of anticancer drugs. 20(S)-protopanaxadiol (PPD) is a metabolite of ginseng saponin of both American ginseng (Panax quinquefolius L.) and Asian ginseng (Panax ginseng C.A. Meyer). We previously demonstrated that ginsenoside Rg3, a glucoside precursor of PPD, exhibits anti-proliferative effects on HCT116 cells and reduces tumor size in a xenograft model. Our subsequent study indicated that PPD has more potent antitumor activity than that of Rg3 in vitro although the mechanism underlying the anticancer activity of PPD remains to be defined. Here, we investigated the mechanism underlying the anticancer activity of PPD in human cancer cells in vitro and in vivo. PPD was shown to inhibit growth and induce cell cycle arrest in HCT116 cells. The in vivo studies indicate that PPD inhibits xenograft tumor growth in athymic nude mice bearing HCT116 cells. The xenograft tumor size was significantly reduced when the animals were treated with PPD (30 mg/kg body weight) for 3 weeks. When the expression of previously identified Rg3 targets, A kinase (PRKA) anchor protein 8 (AKAP8L) and phosphatidylinositol transfer protein α (PITPNA), was analyzed, PPD was shown to inhibit the expression of PITPNA while upregulating AKAP8L expression in HCT116 cells. Pathway-specific reporter assays indicated that PPD effectively suppressed the NF-κB, JNK and MAPK/ERK signaling pathways. Taken together, our results suggest that the anticancer activity of PPD in colon cancer cells may be mediated through targeting NF-κB, JNK and MAPK/ERK signaling pathways, although the detailed mechanisms underlying the anticancer mode of PPD action need to be fully elucidated.

Abstract LB-158: Phase I study of pazopanib and ixabepilone in patients with solid tumors.

Abstract Background: Pazopanib (P) is a TKI targeting multiple receptors including VEGF, PDGF, and C-KIT and acts by inhibiting tumor angiogenesis. Ixabepilone (I) is a semi-synthetic analog of epothilone B and promotes microtubule stabilization inducing tumor cell apoptosis. The purpose of this study was to determine the optimal tolerated regimen (OTR) of combination P and I in patients with advanced, previously treated solid tumors. Methods: I was administered intravenously on day 1 of a 21-day cycle. Dose escalation started at 32 mg/m2. P was administered orally once daily beginning on day 1 at a starting dose of 400mg and escalating at 200mg increments up to 800mg. Disease assessment was done every 2 cycles. A correlative study was conducted to evaluate the changes in levels of VEGF, FGF basic, HGF, IL-8, CXCL-10, MCP-1, M-CSF, MIP 1alpha,MIP-1 beta, PDGF-BB, sE-selectin, TNF-alpha, SAA, PIGF-1, SICAM-1,SDF-1 from baseline to pre-cycle 2 using the Luminex™ system. Replicates for levels were averaged and differences in averages were used in proportional hazards models to predict progression-free survival. Results: Thirty one patients (11 female) were enrolled from December 2009 to September 2011.The median age was 58 years (range 30-72). Primary tumor sites included lung (8), colon (9), skin (2), gallbladder (1), cholangiocarcinoma (1), neuroendocrine (1), head and neck (6), esophagus (1), small cell cancer (1), ovary (1) and leiomyosarcoma (1). A total of 120 cycles of chemotherapy was administered with a median number of cycles per patient of 4 (range 1-13). Three patients had dose-limiting toxicities (DLT) including grade 4 neutropenia and fatigue at dose level 2 (I at 40 mg/m2 and P at 400 mg). Dose level 2a was added to the study to confirm the safety of the proposed I dose (32 mg/m2) before escalating the dose of P to levels 3 and 4. One patient had DLT at dose level (I at 32 mg/m2 and P at 800 mg) due to grade 4 thrombocytopenia. Dose level 3 was determined to be the OTR (P at 600 mg and I at 32 mg/m2). Grade 3-4 hematologic toxicities included neutropenia (5), febrile neutropenia (1), thrombocytopenia (1), and anemia (1). Grade 3-4 non-hematologic toxicities included diarrhea (1) and abdominal pain (1). Disease stabilization was seen in 13 patients and partial response in 8 patients. Progression of disease was seen in 9 patients. There was a significant increase in CXCL10, SAA, and SICAM-1 whereas sE-selectin decreased after the first cycle, but only decrease in sE-selectin was associated with a progression free survival benefit (HR 0.401; 95% CI: 0.168-0.959]. Conclusions: We were able to establish the OTR for combination of P and I. Activity of this regimen was seen in patients with a variety of previously treated solid tumors and it warrants further investigation. We were able to demonstrate significant changes in cytokine levels and correlation of sE-selectin decrease with progression free survival benefit. Citation Format: Chitra Ganesan, Sri J. Obulareddy, Robin L. Bliss, Arkadiusz Z. Dudek. Phase I study of pazopanib and ixabepilone in patients with solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-158. doi:10.1158/1538-7445.AM2013-LB-158

Abstract 4061: A novel CXCR4-mediated mechanism of docetaxel resistance in prostate cancer cells.

Abstract Docetaxel (DTX), a semi-synthetic analog of paclitaxel, has emerged as the standard of care for chemotherapy of hormone-resistant prostate cancer. DTX confers its anti-neoplastic activity by inhibiting microtubule depolymerization, which leads to G2/M cell cycle arrest and subsequent apoptosis. However, most patients treated with DTX ultimately develop resistance to the drug and succumb to the disease. Therefore, understanding the mechanisms underlying DTX resistance is a priority area in prostate cancer research. Increasingly, it is being suggested that cancer cells may use common pathways for disease aggressiveness/metastatic spread and chemotherapeutic resistance. Therefore, we investigated the role of CXCL12/CXCR4 signaling, which is known to promote invasion and metastasis, in DTX-resistance of prostate cancer cells. Our data demonstrated that CXCL12 treatment rescued the prostate cancer cells from DTX-induced cytotoxicity and G2/M mitotic arrest. Furthermore, the cytoprotective effect of CXCL12 was abolished upon pretreatment of prostate cancer cells with AMD3100 (a small molecule antagonist of CXCR4) or siRNA-mediated silencing of CXCR4, thus confirming the role of CXCR4 in DTX-resistance. Immunofluorescence analyses revealed enhanced polymerization of microtubules in DTX-treated prostate cancer cells. In accordance with this, immunoblot analyses demonstrated increased levels of detyrosinated (glu-) and acetylated (ace-) tubulins, specific markers of the polymerized tubulin, in prostate cancer cells treated with DTX. Co-treatment of CXCL12 abrogated DTX-induced stabilization of microtubules in the prostate cancer cells, an effect that was diminished when the cells were pre-treated with AMD3100. Altogether, these initial findings demonstrate the role of CXCL12/CXCR4 signaling axis in DTX-resistance of prostate cancer cells and thus could be an attractive target for therapeutic enhancement of DTX. Citation Format: Arun Bhardwaj, Sanjeev K. Srivastava, Sumit Arora, Stephen J. Hyde, Joel F. Andrews, Steven McClellan, Seema Singh, Ajay P. Singh. A novel CXCR4-mediated mechanism of docetaxel resistance in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4061. doi:10.1158/1538-7445.AM2013-4061

Ixabepilone and eribulin mesylate: two novel agents approved for the chemotherapeutic treatment of metastatic breast cancer

While the majority of breast cancer patients present with early stage disease, 40% of these patients will eventually progress to metastatic disease. Resistance to existing chemotherapeutic agents continues to pose a challenge in the management of these patients. This review will analyze the two most recently approved chemotherapy drugs for the treatment of breast cancer; ixabepilone and eribulin mesylate. Ixabepilone is a semisynthetic analog of epothilone B, and is thought to overcome taxane resistance via disruption to microtubule homeostasis. Two Phase III studies, one by Rugo et al. and the other by Thomas et al., showed improvement in progression-free survival with the combination of ixabepilone and capecitabine to approximately 6 months compared with 4.2 months in the capecitabine-alone group in both trials. These resulted in ixabepilone being approved for use alone or in combination with capecitabine for the treatment of locally advanced or metastatic breast cancer after failure of an anthracycline and a taxane in either the adjuvant or the metastatic setting. The drug has been shown to have activity even in heavily pretreated patients who have received at least two prior chemotherapy regimens for the treatments of metastatic disease. The main adverse events were noted to be fatigue, cytopenias and peripheral sensory neuropathy, all of which were manageable and reversible. Eribulin mesylate is another novel agent that has been approved for the treatment of metastatic breast cancer. It functions by inhibiting mitotic-spindle formation and has demonstrated efficacy against various taxane-resistant tumor cell lines. Median progression-free survival was approximately 3 months and overall survival ranged from 9 to 13 months amongst the various trials. Eribulin mesylate is approved to treat patients with metastatic breast cancer who have received at least two prior chemotherapy regimens for metastatic disease, including both anthracycline- and taxane-based chemotherapy regimens. The most common toxicities observed were fatigue, nausea, cytopenias and peripheral neuropathy. In conclusion, both drugs provide additional options for patients who have progressed on other agents.

Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality

Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant Phe(B24), one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [D-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the L-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [D-HisB24]-insulin is more flexible, and the reverse chirality of the B24C(α) atom swayed the D-His(B24) side chain into the solvent. Furthermore, the pocket vacated by Phe(B24) is filled by Phe(B25), which mimics the Phe(B24) side and main chains. The B25→B24 downshift results in a subsequent downshift of Tyr(B26) into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic L-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.

Orally active fumagillin analogues: transformations of a reactive warhead in the gastric environment.

Semisynthetic analogues of fumagillin, 1, inhibit methionine aminopeptidase-2 (MetAP2) and have entered the clinic for the treatment of cancer. An optimized fumagillin analogue, 3 (PPI-2458), was found to be orally active, despite containing a spiroepoxide function that formed a covalent linkage to the target protein. In aqueous acid, 3 underwent ring-opening addition of water and HCl, leading to four products, 4-7, which were characterized in detail. The chlorohydrin, but not the diol, products inhibited MetAP2 under weakly basic conditions, suggesting reversion to epoxide as a step in the mechanism. In agreement, chlorohydrin 6 was shown to revert rapidly to 3 in rat plasma. In an ex vivo assay, rats treated with purified acid degradants demonstrated inhibition of MetAP2 that correlated with the biochemical activity of the compounds. Taken together, the results indicate that degradation of the parent compound was compensated by the formation of active equivalents leading to a pharmacologically useful level of MetAP2 inhibition.

Electrochemical synthesis of biflavonoids

Biologically active biflavonoids have consistently attracted the attention of researchers [1]. However, only a few of these compounds have a broad spectrum of biological activity. This has prompted scientists to prepare their semi-synthetic and synthetic analogs [2]. Thus, several approaches including metal-catalyzed cross coupling, oxidative coupling, Ulmann condensation with nucleophiles, etc. have been used to synthesize biflavonoids [2]. We hypothesized that it would be possible to use an electrochemical method to prepare novel compounds based on flavonoids [3–5]. The flavonols isorhamnetin and kaempferide that were isolated earlier by us from Alhagi pseudalhagi (M. Bieb.) Fisch. were used for the synthesis. The flavonol (1 mmol) was dissolved in CH3CN (100 mL). The electrolysis was carried out in the presence of LiClO4 (0.1 M) in a cell with a diaphragm and a Pt anode of working surface 2 cm 2 and current density 5 mA/cm2 for 3 h. When the electrolysis was finished, about 90% of the solvent was distilled off. The remainder was used to separate the products by column chromatography over silica gel. In both instances, the principal products were recrystallized from acetone to afford light-brown powders in 0.22 ± 0.05 g yields. Their physicochemical and spectral characteristics were determined by known methods. Compound 1. C32H22O14, mp 285 ± 3°C, [ ]D 20 +39.7° (c 0.57, MeOH). UV spectrum (MeOH, max, nm): 251, 325, 356. IR spectrum (KBr, max, cm –1): 3430, 3370 (OH), 1681, 1600 (C=O), 1607, 1570 (aromatic C=C). Mass spectrum (EI, 70 eV), m/z (Irel, %): 630 (M +, 100), 615 (13), 315 (17). PMR spectrum (400 MHz, CDCl3, , ppm): 3.96 (6H, d, 3 , 3 -OCH3), 6.83 (2H, s, H-6, 6 ), 7.23 (2H, s, H-5 , 5 ), 7.52 (4H, s, H-2 , 6 , 2 , 6 ), 9.35 (2H, s, OH-4 , 4 ), 10.12 (2H, s, OH-7, 7 ), 10.82 (2H, s, OH-3, 3 ), 12.10 (2H, s, OH-5, 5 ). Compound 2. C32H20O12, mp 291 ± 3°C, [ ]D 20 +38.4° (c 0.52, MeOH). UV spectrum (MeOH, max, nm): 210, 271, 368. IR spectrum (KBr, max, cm –1): 3423 (OH), 1590 (C=O), 1520, 1460 (aromatic C=C). Mass spectrum (EI, 70 eV), m/z (Irel, %): 596 (M +, 100), 581 (12), 298 (22). PMR spectrum (400 MHz, CDCl3, , ppm): 3.83 (6H, d, OCH3-4 , 4 ), 6.83 (2H, s, H-6, 6 ), 7.36 (4H, s, H-3 , 3 , 5 , 5 ), 7.50 (4H, s, H-2 , 6 , 2 , 6 ), 10.16 (2H, s, OH-7, 7 ), 10.76 (2H, s, OH-3, 3 ), 12.17 (2H, s, OH-5, 5 ).

Effects of sesquiterpene, flavonoid and coumarin types of compounds from Artemisia annua L. on production of mediators of angiogenesis

BackgroundIn addition to recognized antimalarial effects, Artemisia annua L. (Qinghao) possesses anticancer properties. The underlying mechanisms of this activity are unknown. The aim of our experiments was to investigate the effects of distinct types of compounds isolated from A. annua on the immune-activated production of major mediators of angiogenesis playing a crucial role in growth of tumors and formation of metastasis. MethodsIncluded in the study were the sesquiterpene lactones artemisinin and its biogenetic precursors arteannuin B and artemisinic acid. The semi-synthetic analogue dihydroartemisinin was used for comparative purposes. The flavonoids were represented by casticin and chrysosplenol D, the coumarin type of compounds by 4-methylesculetin. Their effects on the lipopolysaccharide (LPS)-induced in vitro production of nitric oxide (NO) were analyzed in rat peritoneal cells using Griess reagent. The LPS-activated production of prostaglandin E2 (PGE2) and cytokines (VEGF, IL-1β, IL-6 and TNF-α) was determined in both rat peritoneal cells and human peripheral blood mononuclear cells using ELISA. ResultsAll sesquiterpenes (artemisinin, dihydroartemisinin, artemisinic acid, arteannuin B) significantly reduced production of PGE2. Arteannuin B also inhibited production of NO and secretion of cytokines. All NO, PGE2 and cytokines were suppressed by flavonoids casticin and chrysosplenol D. The coumarin derivative, 4-methylesculetin, was ineffective to change the production of any of these factors. ConclusionsThe inhibition of immune mediators of angiogenesis by sesquiterpene lactones and flavonoids may be one of the mechanisms of anticancer activity of Artemisia annua L.

Metabolites of PPI-2458, a Selective, Irreversible Inhibitor of Methionine Aminopeptidase-2: Structure Determination and In Vivo Activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only ∼10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ([(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

Olive secoiridoids and semisynthetic bioisostere analogues for the control of metastatic breast cancer

(−)-Oleocanthal (1) and ligstroside aglycone (2) are common bioactive olive oil secoiridoids. Secoiridoid 1 has been previously reported as a c-MET inhibitor. Chemically, (−)-oleocanthal is the elenolic acid ester of the common olive phenolic alcohol tyrosol. Therefore, several analogues (4–13) were synthesized by esterification and carbamoylation of tyrosol using diverse phenolic naturally occurring in olive and heterocyclic acids as elenolic acid bioisosteres to assess the effect of replacing the acid moiety of (−)-oleocanthal. Their c-MET inhibitory activity as well as their antiproliferative, antimigratory, and anti-invasive activities against the highly metastatic human breast cancer cell line MDA-MB231 has been assessed. Ligstroside aglycone (2) showed the best antimigratory activity. Generally, tyrosol esters showed better activities versus carbamate analogues. Tyrosol sinapate (5) showed the best c-MET phosphorylation inhibitory activity in Z′-LYTE™ kinase assay. Both 1 and 5 competitively inhibited the ATP binding into its pocket in the c-MET catalytic domain. Compound 5 showed selective activities against tumor cells without toxicity to the non-tumorigenic human breast MCF10A epithelial cell line. Tyrosol esters with a phenolic acid containing hydrogen bond donor and/or acceptor groups at the para-position have better anticancer and c-MET inhibitory activities. Olive oil secoiridoids are excellent scaffolds for the design of novel c-MET inhibitors.

A novel semi-synthetic andrographolide analogue A5 inhibits tumor angiogenesis via blocking the VEGFR2-p38/ERK1/2 signal pathway

The present study is designed to observe the inhibitory effect of compound A5, a semi-synthetic analogue of the natural compound andrographolide, on angiogenesis and its underlying mechanism. Compound A5 is semi-synthesized from natural compound neoandrographolide. Andrographolide, the aglycon of neoandrograoholide, and A5 all inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs) proliferation, and that the inhibition shown by A5 is the best. A5 also inhibited VEGF-induced tube formation in HUVECs in a concentration-dependent manner. VEGF-induced neoangiogenesis in vivo was observed by Matrigel formation assay. The Matrigel picture and CD31 staining results showed that A5 inhibited VEGF-induced neoangiogenesis in vivo. Further, Western-blot results showed that A5 inhibited VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38 kinase. The antitumor effect of A5 was analyzed in a xenograft mouse tumor model inoculated with hepatoma Hep3B cells. The results showed that A5 decreased tumor weight and tumor size without affecting body weight in the xenograft mouse, and A5 also decreased CD31 staining in tumor tissue. Taken together, the present study demonstrates that compound A5 inhibits tumor growth via blocking neoangiogenesis, and the cellular VEGFR2-p38/ERK1/2 signal pathway.

In vitro and In vivo Biological Activities of Nardostachys Jatamansi Roots

Since age old, plants have been the source of medicines for the treatment of various diseases. Regardless of the availability of a wealth of synthetic drugs, plants remain–even in the 21st century–an integral part of the health care in different countries, especially the developing ones. Efforts are being made to develop safe and cost effective anticancer agents from natural sources. As the age of modern medicine, single pure drugs emerged, and plant-derived active principles, their semisynthetic and synthetic analogs have served as a major route to new pharmaceu ticals. Since 1961, plant-derived compounds have been approved for use as anticancer drugs: vinblastine (Velban®), vincristine (Oncovin®), eto poside (VP-16®), taxol (Paclitaxel®), etc [1].

Discovery and development of natural compounds into medicinal products

The landscape of small-molecule drug discovery has undergone substantial changes over the past 20 years, in which the testing of tens to hundreds of compounds in animal models was replaced by the high-throughput screening (HTS) of megalibraries against modified cell lines or isolated biomolecular targets. Natural products were a fruitful source of lead molecules, but their impact waned considerably since their heyday between the 1940s and 1960s. During the 1990s, pharmaceutical companies began to invest heavily in the HTS of combinatorial chemistry libraries that typically contained compounds with non-natural (synthetic) motifs. Incredibly, the contribution by natural products towards lead molecules (chemical starting points) and drugs remained steady despite the diversion of resources and the general decline in discovery. An analysis covering the 30-year period to December 2010 indicated that approximately 39% of all 1,068 small-molecule new chemical entities were either a natural compound, close semi-synthetic analogue or a synthetic analogue of the natural product pharmacophore. The application of concepts, methodologies and knowledge learned during maturation of the high-throughput paradigm to natural compounds has the potential to increase their contribution for the pharmaceutical industry.

Quantification of Acylfulvene– and Illudin S–DNA Adducts in Cells with Variable Bioactivation Capacities

Illudin S and its semisynthetic analogue acylfulvene (AF) are structurally similar but elicit different biological responses. AF is a bioreductive alkylating anticancer agent with a favorable therapeutic index, while illudin S is in general highly toxic. AF toxicity is dependent on the reductase enzyme prostaglandin reductase 1 (PTGR1) for activation to a cytotoxic reactive intermediate. While illudin S can be metabolized by PTGR1, available data suggest that its toxicity does not correspond with PTGR1 function. The goal of this study was to understand how drug cytotoxicity relates to cellular bioactivation capacity and the identity and quantity of AF- or illudin S-DNA adducts. The strategy involved identification of novel illudin S-DNA adducts and their quantitation in a newly engineered SW-480 colon cancer cell line that stably overexpresses PTGR1 (PTGR1-480). These data were compared with cytotoxicity data for both compounds in PTGR1-480 versus normal SW-480 cells, demonstrating that AF forms more DNA adducts and is more cytotoxic in cells with higher levels of PTGR1, whereas illudin S cytotoxicity and adduct formation are not influenced by PTGR1 levels. Results are discussed in the context of an overall model for how changes in relative propensities of these compounds to undergo cellular processes, such as bioactivation, contributes to DNA damage, and cytotoxicity.

Inhibition of Cathepsin D protease activity by Punica granatum fruit peel extracts, isolates, and semisynthetic analogs

Crude extracts of Punica granatum fruit peel, its isolates, and semisynthetic analogs were investigated for their Cathepsin D protease reticence. Hexane extract (HE), chloroform extract (CE), and ethyl acetate extract (EAE) exhibited significant inhibition of Cathepsin D activity. The bioactivity-guided isolation of hexane- and chloroform-soluble fractions yielded piperine and ursolic acid as bioactive constituents. Upon semisynthetic modifications of ursolic acid, two of the analogs (7 and 8) exhibited higher inhibition of Cathepsin D activity: IC50 at 12.87 and 9.40 μM, respectively. P. granatum fruit peel extracts may act as breast cancer chemopreventive agent.

Abstract P1-15-07: Ixabepilone-associated peripheral neuropathy in metastatic breast cancer patients and its effects on the ultrastructure of neurons

Abstract Background: Peripheral neuropathy is a dose-limiting toxicity of most microtubule-stabilizing chemotherapeutic agents. Ixabepilone, a semisynthetic analog of the natural epothilone B, has activity in a wide range of tumors including taxane-resistant disease. In this study, we sought to understand the effect of ixabepilone on the development of peripheral neuropathy both clinically and its effect at the ultrastructural level of the peripheral nerves and circulating factors over time. Parallel studies in animal models of neuropathy were performed at the same time (Proc AACR 2010 Abstract 4184). Methods: This open-label, non-randomized phase II study enrolled 14 patients with metastatic breast cancer. Ixabepilone was administered by 2 schedules: the FDA approved dose of 40 mg/m2 every 3 weeks (q3w) and 16 mg/m2 on day 1, 8, and 15 of a 28-day cycle (weekly). Five controls, 2 with residual taxane-associated peripheral neuropathy and 3 with no prior chemotherapy or peripheral neuropathy, were also accrued. The primary objectives were to characterize the natural history of ixabepilone-associated peripheral neuropathy using the Total Neuropathy Score Clinical (TNSc) assessment tool prior to each cycle and to correlate changes in the ultrastructure of dermal myelinated nerve fibers via a 3 mm punch biopsy of an area 10 cm above the lateral malleolus every 2 cycles with electron microscopy (EM), as well as circulating factors (both inflammatory and neurotrophic) considered to be important in the pathogenesis of chemotherapy-induced peripheral neuropathy. Secondary objectives included progression-free survival (PFS) and non-neurologic toxicity. Results: 14 patients were enrolled and were equally divided between the 2 schedules of ixabepilone chemotherapy. There were no differences in baseline characteristics between the two groups. Mean age was 54 years (range 32–71). Mean number of previous chemotherapy regimens was 3.5 (range 0–8). 57% of patients had received a taxane in the adjuvant setting and 64% in the metastatic setting. The mean neuropathy score (TNSc) at baseline was 4.6 (range 1–11). At a mean cumulative dose of 185 mg/m2, the TNSc with ixabepilone q3w schedule was 3.7 points higher/worse (95% CI: 2.2–5.3, p = 0.03) than the mean score observed in patients on the weekly schedule. The sensory component was most significantly affected, predominantly numbness. In 3 patients, the chemotherapy schedule was changed from every 3 weeks to weekly due to > grade 2 toxicity at a mean cumulative dose of 107 mg/m2, and TNSc decreased/improved by 2.7 points. PFS in patients on q3w ixabepilone was 133 days (range 28–280) and in patients on weekly ixabepilone was 179 days (range 66–336), nonsignificant. Evaluation of EM and circulating factors is ongoing. Conclusions: Weekly ixabepilone appears to have a more favorable neurotoxicity profile compared to the standard q3w schedule. Integration of the EM data and the circulating factor data are underway and will be presented. Ixabepilone-associated peripheral neuropathy may improve in patients switched to weekly ixabepilone without compromising efficacy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-15-07.