Combretum Caffrum Research Articles

Novel Combretastatin A-4 Analogs-Design, Synthesis, and Antiproliferative and Anti-Tubulin Activity.

Combretastatins isolated from the Combretum caffrum tree belong to a group of closely related stilbenes. They are colchicine binding site inhibitors which disrupt the polymerization process of microtubules in tubulins, causing mitotic arrest. In vitro and in vivo studies have proven that some combretastatins exhibit antitumor properties, and among them, combretastatin A-4 is the most active mitotic inhibitor. In this study, a series of novel combretastatin A-4 analogs containing carboxylic acid, ester, and amide moieties were synthesized and their cytotoxic activity against six tumor cell lines was determined using sulforhodamine B assay. For the most cytotoxic compounds (8 and 20), further studies were performed. These compounds were shown to induce G0/G1 cell cycle arrest in MDA and A549 cells, in a concentration-dependent manner. Moreover, in vitro tubulin polymerization assays showed that both compounds are tubulin polymerization enhancers. Additionally, computational analysis of the binding modes and binding energies of the compounds with respect to the key human tubulin isotypes was performed. We have obtained a satisfactory correlation of the binding energies with the IC50 values when weighted averages of the binding energies accounting for the abundance of tubulin isotypes in specific cancer cell lines were computed.

Discovery, synthesis, activities, structure-activity relationships, and clinical development of combretastatins and analogs as anticancer drugs. A comprehensive review.

Covering: 1982 to up to the end of 2022Bioassay guided purification of the extracts of Combretum caffrum led to the discovery of six series of combretastatins A-D with cytotoxic activities ranging from sub nM to >50 μM ED50's against a wide variety of cancer cell lines. Of these, cis-stilbenes combretastatins A-4 and A-1 were the most potent, exhibiting in vivo efficacy against a wide variety of tumor types in murine models. These antimitotic agents inhibited tubulin polymerization by reversibly binding to the colchicine binding sites. They inhibited tumor growth by a novel antivascular and antineogenesis mechanism in which they stopped blood flows to the blood vessels causing necrosis. Over 20 clinical trials of the phosphate prodrugs of combretastatin A-4 (CA4P) and A-1 (CA1P) showed objective and stable responses against many tumor types, with increased survival times of many patients along with the confirmed cure of certain patients inflicted with anaplastic thyroid cancers. Medicinal chemistry efforts led to the identification of three new leads (AVE8062, BNC105P, SCB01A) with improved in vitro and in vivo potency and an often-improved cellular spectrum. Unfortunately, these preclinical improvements did not translate clinically in any meaningful way. Objectively, CA4P remained the best compound and has garnered many Orphan drug designations by FDA. Clinical trials with tumor genetic mapping, particularly from previous responders, may help boost the success of these compounds in future studies. A comprehensive review of combretastatin series A-D, including bioassay guided discovery, total syntheses, and structure-activity relationship (SAR) studies, biological and mechanistic studies, and preclinical and clinical evaluations of the isolated combretastatins and analogs, along with the personal perspective of the author who originated this project, is presented.

Combretastatin A-4 suppresses the invasive and metastatic behavior of glioma cells and induces apoptosis in them: in-vitro study.

The most common primary brainmalignancy, glioblastoma multiforme, is tremendously resistant to conventionaltreatmentsdue to its potency for metastasis to surrounding brain tissue. Temozolomide is a chemotherapeutic agent that currently is administrated during the treatment procedure. Studies have attempted to investigate new agents with higher effectiveness and fewer side effects. Combretastatin A-4 (CA-4), a natural compound derived from Combretum caffrum, has been recently considered for its potent antitumor activities in a wide variety of preclinical solid tumor models. Our findings have shown that CA-4 exerts potent anti-proliferative and apoptotic effects on glioma cells, and ROS generation may be involved in these cellular events. CA-4 has imposed G2 arrest in U-87 cells. We also observed that CA-4 significantly reduced the migration and invasion capability of U-87 cells. Furthermore, the gene expression and enzyme activity of MMP-2 and MMP-9 were significantly inhibited in the presence of CA-4. We also observed a considerable decrease in PI3K and Akt protein expression following treatment with CA-4. In conclusion, our findings showed significant apoptogenic and anti-metastatic effects of CA-4 on glioma cells and also suggested that the PI3K/Akt/MMP-2/-9 and also ROS pathway might play roles in these cellular events.

Design, Synthesis, and Anti-tumor Activity of Novel 2-Aryl Benzimidazole Compounds.

Combretastatin A-4 (CA-4) is a natural product isolated from the bark of the South African bush willow tree Combretum caffrum, which exerts tubulin inhibition, but its clinical application is limited due to poor stability and water solubility. 2-aryl benzimidazoles are excellent pharmacological skeletons with many activities, especially in tumor inhibition, and better pharmacokinetic properties. Several scaffold CA-4 analogs have been synthesized to date possessing antitumor activities. The benzimidazole was applied as the core moiety to replace the B ring and unstable linkage of CA-4, and the 5-aryl acetenyl group was introduced to improve the antitumor activity. MCF-7, A549, Caco-2, Siha, and Eca-109 tumor cell lines were used to study inhibition by these agents in vitro. The benzimidazole structure was constructed from the oxidation of o-nitroaniline and aldehyde and the following schemes, and the structural characterization was carried out. The antitumor effects were evaluated in vitro through MTT assay, cell cycle arrest, and apoptosis assay. Molecular docking with tubulin (Protein ID: 1SA0) was analyzed for the structure-activity relationship. Among these derivatives, 4a-4h series (with 6-methoxy group) compounds inhibited the tumor cell lines much stronger than the CA-4 and cisplatin, especially compound 4f showed prominently inhibitory activity in Siha cell with IC50 value as 0.61 μmol/L. The further assay showed that the cell cycle was arrested at the G0/G1 phase as well verified in apoptosis assay. Molecular docking indicated that 4f had stronger affinity energy and hydrogen bond than CA-4. The compound 4f has the potency to be used as an anti-tubulin agent and the 2-trimethoxyphenyl benzimidazole skeleton deserves further study as an antitumor structure.

A review on the secretory structures, bioactive compounds and biological activities of selected Combretum species

Species of Combretum are highly valued in Africa due to the plethora of traditional medicinal uses they may offer. Species such as Combretum molle, Combretum erythrophyllum, Combretum album, Combretum apiculatum, Combretum caffrum and Combretum quadrangulare are commonly utilized and are known to contain medicinally important phytochemicals. Traditionally, they are used to treat bacterial infections, venereal diseases, abdominal pain, sores, infertility, and labour pains, while displaying, anti-viral, anti-parasitic, cytotoxic, and mutagenic activity. There have been numerous studies released on the presence, isolation and bioactivity of phytochemicals. However, the transformation of the isolate into a drug of pharmacological importance has yet to be appraised on a large scale. Therefore, further evaluation of this genus and particularly the transformation of the isolates needs to be explored. This review aimed to collate current and prudent information pertaining to the presence of secretory structures in relation to potential medicinal properties, ethnopharmacological applications and bioactive compounds present within selected Combretum species.

Synthesis and antimitotic evaluation of Schiff bases derivatives based on 2-amino-combretastatin

Combretastatins A, B, and D are a group of stilbenes, dihydrostilbenes, phenanthrenes, and macrocyclic lactones that may be found in the bark of the Combretum caffrum tree. Preclinical studies have shown that the lead chemical, combretastatin A-4, is highly cytotoxic and inhibits tubulin polymerisation by interacting with the microtubule's colchicine binding site. The anti-cancer activity of a new series of Schiff bases derived from 2-amino-combretastatin as analogues of combretastatin A-4 (CA-4) was investigated. The structures of the synthesised derivatives were proved using 1 H and 13 C NMR, I.R. spectroscopy and elemental analysis. As tubulin polymerisation inhibitors, several drugs demonstrated excellent anti-proliferative activity. IC 50 values for compounds 8a, 8d, 8c and 8i were 1.6, 1.9, 1.9 and 1.3 µM, respectively. In addition, prepared Schiff bases exhibited anti-proliferative action in several human cell lines derived from various organs, namely, HT-29, MCF-7, and A-549, as well as the non-tumor cell line HEK-273. The Schiff bases substituent at the 2-position of ring A in the combretastatin chemical skeleton may play a significant role in the bioactivity of this group of drugs.

Ecust004 Suppresses Breast Cancer Cell Growth, Invasion, and Migration via EMT Regulation.

PurposeErianin is a small chemical compound extracted from Dendrobium chrysotoxum and has excellent antineoplastic effects against a variety of cancers. Combretastatin A-4 (CA4) is the most effective member of natural phenolic stilbene compounds isolated from the African willow tree Combretum caffrum. Ecust004 (Chemical Formula: C18H21NO7S) is a drug candidate optimized from structure–activity relationship studies of the sulfamate derivatives of Erianin and CA4, which has better bioavailability and pharmacokinetic profiles than Erianin and CA4.MethodsTo investigate the antitumor activity of Ecust004 in different types of breast cancer cells, MDA-MB-231 and MCF7 cells were treated with Ecust004. MTT and CCK8 were used to determine the effects of Ecust004 on cell proliferation. Wound-healing and Transwell assays were used to evaluate the migration and invasion level of cells treated with Ecust004. The expression of genes and proteins associated with epithelial–mesenchymal transition was detected by RT-PCR and Western blotting. In vivo studies further clarified the functional effects of Ecust004.ResultsEcust004 treatment decreased the growth and proliferation of MDA-MB-231 and MCF7 cells at a lower dosage than Erianin. In addition, compared to Erianin and CA4, Ecust004 can better inhibit the invasion and migration of MDA-MB-231 and MCF7 cells. Accordingly, the expression of genes associated with epithelial–mesenchymal transition, such as E-cadherin and vinculin, was increased. Finally, compared with Erianin and CA4, Ecust004 exhibited a better anti-tumor activity in vivo.ConclusionEcust004 inhibits the proliferation, invasion, and migration of breast cancer cells, and therefore represents a potential agent for development as an antitumor drug.

Combretastatins: An Overview of Structure, Probable Mechanisms of Action and Potential Applications.

Combretastatins are a class of closely related stilbenes (combretastatins A), dihydrostilbenes (combretastatins B), phenanthrenes (combretastatins C) and macrocyclic lactones (combretastatins D) found in the bark of Combretum caffrum (Eckl. & Zeyh.) Kuntze, commonly known as the South African bush willow. Some of the compounds in this series have been shown to be among the most potent antitubulin agents known. Due to their structural simplicity many analogs have also been synthesized. Combretastatin A4 phosphate is the most frequently tested compounds in preclinical and clinical trials. It is a water-soluble prodrug that the body can rapidly metabolize to combretastatin A4, which exhibits anti-tumor properties. In addition, in vitro and in vivo studies on combretastatins have determined that these compounds also have antioxidant, anti-inflammatory and antimicrobial effects. Nano-based formulations of natural or synthetic active agents such as combretastatin A4 phosphate exhibit several clear advantages, including improved low water solubility, prolonged circulation, drug targeting properties, enhanced efficiency, as well as fewer side effects. In this review, a synopsis of the recent literature exploring the combretastatins, their potential effects and nanoformulations as lead compounds in clinical applications is provided.

Antifungal and anti-mycotoxigenic activity of selected South African medicinal plants species

Antifungal and anti-mycotoxigenic activity of 25 acetone leaf extracts of South African medicinal plants with potent antioxidant activity were investigated against three phytopathogenic fungal strains. The extracts exhibited varying degrees of in vitro anti-mycotoxigenic effect against Fusarium verticillioides, Aspergillus flavus and Aspergillus ochraceous. Markhamia obtusifolia (Baker) Sprague exhibited the lowest minimum inhibitory concentration (MIC) values as low as 0.08 mg/ml against Aspergillus flavus and Furasium verticilloides at both 24 and 48 hr incubation period, while Curtisia dentata exhibited similar MIC value against Aspergillus ochraceous. Curtisia dentata further yielded the highest total activity of 1583 ml/g against Aspergillus ochraceous at 24 and 48 hr incubation period. In the mycelial growth inhibition (MGI) evaluation, Fusarium verticilloides was more sensitive to plants extracts, while Kirkia wilmsii exhibited highest MGI of 50.08% against Fusarium verticilloides on the 6th day of incubation. Five acetone extracts from Acokanthora oppositifolia, Bauhinia galpinii, Combretum caffrum, Ricinus communis and Solanum aculeastrum exhibited lowest IC50 value of 0.01 mg/ml against (2,2’-azinobis-3-ethylbenzthiazoline-6-suphonic acid (ABTS). Curtisia dentata and Markhamia obtusifolia extracts were further subjected to gas chromatography mass-spectrophotometry (GC-MS) analysis. Curtisia dentata revealed the presence of triterpenoid compounds, β-amyrin (53.30%) and α-amyrin (6.42%), while Markhamia obtusifolia yielded the presence of neophytadiene (4.38%) and palmitic acid (3.61%) The results suggest that natural products from plants may well be used as possible substitutes for synthetic fungicides. Given the antifungal and antioxidant potential of the selected plants, they may have potential as possible leads for the development of biofungicides that may well prevent oxidation related food spoilage.

Abstract CT067: A Phase I study of Fosbretabulin in combination with everolimus in neuroendocrine tumors (Grades 1-3) that have progressed after at least one prior regimen for metastatic disease

Abstract Background: Based on the SEER database, the incidence of gastroenteropancreatic neuroendocrine tumors (GEPNETs) has risen six-fold in the last four decades. At present, more than 150,000 GEPNET patients exist in the United States. Fosbretabulin, a synthetic, water-soluble, phosphorylated prodrug of the natural product combretastatin A4 (CA4P), initially isolated from the bark of the South African bush willow, Combretum caffrum, is the lead compound in a class of agents termed vascular disrupting agents (VDAs) and has shown activity as a single agent in ovarian cancer and GEPNETs. Everolimus, an mTOR inhibitor, is FDA approved for the management of well-differentiated NETs. A Phase I trial combining fosbretabulin and everolimus to determine the recommended Phase II trial dose (RP2D) and safety data in metastatic GEPNET patients was conducted. Method: An investigator-initiated, single center, open-label, phase I study involving GEPNETs incorporated partial order continual reassessment method (PO-CRM) to define the dose escalation. The primary objective was to establish the maximum tolerated dose (MTD) of the combination of everolimus and fosbretabulin in NETs that have progressed after at least one prior regimen for metastatic disease. Secondary objective included identifying the safety profile of the combination using NCI CTCAE4 reporting criteria. Patients received daily oral everolimus (2.5 mg, 5 mg, 7.5 mg, and 10 mg). Fosbretabulin was administered IV 60 mg/m2 either q3 weekly or q weekly based on PO-CRM. Patients were treated for 12 weeks with all combinations. Results: Of the 17 patients enrolled, 16 completed the 12-week trial. One patient was not evaluable due to noncompliance with the treatment regimen. No DLTs were observed at day 21. The highest dose of 10 mg daily oral everolimus in combination with weekly 60mg/m2 IV fosbretabulin is the RP2D. No grade 4 or 5 toxicities were noted. Grade 3 toxicities were seen in 5 patients that include increased abdominal pain and hyperglycemia (not related to study drug), fatigue (possibly related), decreased lymphocyte count and anemia (related). Several patients had delay in treatment due to grade 2 AE’s (GI symptoms, rash, thrombocytopenia) and one patient was unable to complete treatment due to pneumonitis. Only one patient had radiologic progression at the first q 3 monthly CT scan of chest, abdomen, and pelvis. A detailed table with all grade toxicities will be presented at the meeting. Conclusion: Ten mg PO daily everolimus plus 60 mg/m2 fosbretabulin IV weekly is the RP2D. ClinicalTrials.gov Identifier: NCT0301429 Citation Format: Aman Chauhan, Susanne Arnold, John Wu, Stacey Slone, Emily Dressler, Heather Flynn, Val Adams, Heidi Weiss, Lowell Anthony. A Phase I study of Fosbretabulin in combination with everolimus in neuroendocrine tumors (Grades 1-3) that have progressed after at least one prior regimen for metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT067.

Clinical efficacy and toxicity data on phase I study of fosbretabulin in combination with everolimus in neuroendocrine tumors.

4114 Background: Fosbretabulin, a synthetic, water-soluble, phosphorylated prodrug of the natural product combretastatin A4 (CA4P), initially isolated from the bark of the South African bush willow, Combretum caffrum, is the lead compound in a class of agents termed vascular disrupting agents (VDAs). Everolimus, an mTOR inhibitor, is FDA approved for the management of well-differentiated NETs. A Phase I trial combining fosbretabulin and everolimus to determine the recommended Phase II trial dose (RP2D), safety data and early clinical efficacy in metastatic GEPNET patients was conducted. Methods: An investigator-initiated, single center, open-label, phase I study involving GEPNETs incorporated partial order continual reassessment method (PO-CRM) to define the dose escalation. The primary objective was to establish the maximum tolerated dose (MTD) of the combination of everolimus and fosbretabulin in NETs that have progressed after at least one prior regimen for metastatic disease. Secondary objective included identifying the safety profile of the combination using NCI CTCAE4 reporting criteria. Patients received daily oral everolimus (2.5 mg, 5 mg, 7.5 mg, and 10 mg). Fosbretabulin was administered IV 60 mg/m2 either q3 weekly or q weekly based on PO-CRM. Patients were treated for 12 weeks with all combinations. RECIST 1.1 was used to evaluate radiological responses at 3 month. Results: Of the 17 patients enrolled, 16 completed the 12-week trial. One patient was not evaluable due to noncompliance. No DLTs were observed at day 21. The highest dose of 10 mg daily oral everolimus in combination with weekly 60mg/m2 IV fosbretabulin is the RP2D. No grade 4 or 5 toxicities were noted. Grade 3 toxicities were seen in 5 patients; abdominal pain and hyperglycemia (not related to study drug), fatigue (possibly related), decreased lymphocyte count and anemia (related). Several patients had delay in treatment due to grade 2 AE’s (GI symptoms, rash, thrombocytopenia) and one patient was unable to complete treatment due to pneumonitis. All evaluable patients except one had stable disease at 3 months. One patient showed SD but non target lesion demonstrated PD. One patient had > 30% decrease in tumor size but overall sum of lesions showed SD. A detailed table with all grade toxicities and waterfall plot of RR will be presented at the meeting. Conclusions: Ten mg PO daily everolimus plus 60 mg/m2 fosbretabulin IV weekly is the RP2D. Early clinical data suggests clinical activity and stable disease in all but one patient at 3 months. Clinical trial information: NCT0301429.

Cytotoxic Stilbenes and Derivatives as Promising Antimitotic Leads for Cancer Therapy

The growing incidence of cancer, the toxic side-effects associated with conventional chemotherapeutic agents and the development of multidrug resistance (MDR) drive the search for novel and more effective drugs with multi-target activity and selectivity towards cancer cells. Stilbenes are a group of naturally occurring phenolic compounds of plant origin derived from the phenylpropanoid pathway that may exist as cis- or trans-isomers. Although the trans-isomer is the more common and stable configuration, resveratrol being a representative compound, cis-stilbenes are potent cytotoxic agents that bind to and inhibit tubulin polymerization, destabilizing microtubules. This review summarizes the chemistry and biological evaluation of cytotoxic stilbenes and their synthetic derivatives as promising antimitotic leads for cancer therapy, focusing on the most potent compounds, the combretastatins. Combretastatins isolated from the South African bushwillow Combretum caffrum are among the most potent antimitotic and vascular disrupting agents (VDAs) of natural origin. Preclinical studies have demonstrated their potent antitumor effects in a wide variety of tumors, both in vitro and in vivo, being currently under evaluation in phase 2 and phase 3 clinical trials for several types of solid tumors. Topics covered herein include synthetic medicinal chemistry, modes of action, structure-activity relationships (SAR), preclinical and clinical studies as VDAs in cancer therapy, either as single agents or in combination with cytotoxic anticancer drugs, antiangiogenic agents, or radiation therapy, and development of appropriate formulations based on nanocarriers (e.g., liposomes, nanoemulsions, polymeric, lipid and ceramic nanoparticles, carbon nanotubes) for improved bioavailability and targeted delivery of combretastatins to the tumor vasculature.

Intracellular interactions of serum-derived C3 in human B cells – Regulation of gene transcription

For the first time combretastatins were isolated from African willow tree Combretum Caffrum. Subsequent studies have shown the impact of combretastatin A4 phosphate, a water-soluble prodrug, on endothelial cells in tumor vascular system. The same effect was not observed in the vascular system. This selectivity is associated with combretastatins mechanism of action: binding to colchicine domain of microtubules, which affects the cytoskeleton functionality of immature endothelial cells. At the same time, combretastatins directly induce cell death via apoptosis and/or mitotic catastrophe pathways. The combination of both elements makes combretastatin an anticancer compound of high efficiency.The cis-configuration is crucial for its biological activity. To date, many derivatives were synthesized. The attempts to resolve spontaneous isomerization to less active trans-stilbene derivative are still in progress. This issue seems to be overcome by incorporation of the ethene bridge with heterocyclic moiety in combretastatins structure. This modification retains the cis-configuration and prevents isomerization. Nevertheless, combretastatin A4 phosphate disodium is still the most potent compound of this group.The combination therapy, which is the most effective treatment, includes combretastatin A4 phosphate (CA4P) and conventional chemotherapeutics and/or radiotherapy. CA4P is relatively well tolerated giving adverse events of moderate severity, which includes: nausea, vomiting, headache, and tumor pain. The aforementioned effects subside on the day of drug administration or on the following day.

Synthesis and biological evaluation of new 3-amino-2-azetidinone derivatives as anti-colorectal cancer agents

Several synthetic combretastatin A4 (CA-4) derivatives were recently prepared to increase the drug efficacy and stability of the natural product isolated from the South African tree Combretum caffrum. A group of ten 3-amino-2-azetidinone derivatives, as combretastatin A4 analogues, was selected through docking experiments, synthesized and tested for their anti-proliferative activity against the colon cancer SW48 cell line. These molecules, through the formation of amide bonds in position 3, allow the synthesis of various derivatives that can modulate the activity with great resistance to hydrolytic conditions. The cyclization to obtain the 3-aminoazetidinone ring is highly diastereoselective and provides a trans biologically active isomer under mild reaction conditions with better yields than the 3-hydroxy-2-azetidinone synthesis. All compounds showed IC50 values ranging between 14.0 and 564.2 nM, and the most active compound showed inhibitory activity against tubulin polymerization in vitro, being a potential therapeutic agent against colon cancer.

Computational design and synthesis of novel fluoro-analogs of combretastatins A-4 and A-1

Combretastatin A-1 (CA-1) and combretastatin A-4 (CA-4) isolated from the African bush willow Combretum caffrum are highly potent tubulin polymerization inhibitors, possessing strong antitumor activities because of their vascular disrupting properties. Extensive SAR studies have been done for CA-4 analogs. Because of poor solubility, water-soluble prodrugs of CA-4 and CA-1 have been developed, which are currently in human clinical trials. Fluorine plays an important role in the current drug discovery and development due to its unique properties. Thus, several fluorine-containing analogs of CA-4/CA-1 have been studied. However, no analogs, which have a CF3O-, CF2HO- or CF3- group instead of the 4′-methoxy group in the B ring, have been investigated. Therefore, we set out to design and synthesize those novel fluoro-analogs of CA-4/CA-1. For the design of the new analogs, we took a structure-based design approach based on the X-ray crystal structure of colchicine-tubulin complex (PDB: 4O2B) and computational docking analysis using the AutoDock Vina program. A library of novel fluoro-analogs of CA-4/CA-1 was generated and their docking energy scores obtained. It was found that those novel fluoro-analogs exhibited better docking energy scores than CA-4/CA-1. Also, docking poses of all of these fluoro-analogs were virtually superimposable and very good fit to the colchine binding site. Among 15 compounds designed and analyzed, we have synthesized 5 compounds and evaluated their cytotoxicity against drug-sensitive and multidrug-resistant cancer cell lines. All fluoro-analogs exhibited strong cytotoxicity even against multidrug-resistant cell line. However, the critical activity of this class of compounds is its vascular disrupting activity. Thus, further biological evaluations are warranted for those novel fluoro-analogs of CA-4/CA-1.

Structural characterization and cytotoxicity studies of different forms of a combretastatin A4 analogue

It is well known that combretastatin A4 (CA-4), which is a natural stilbene isolated from Combretum caffrum, is used to inhibit angiogenesis. However, depending on the dose administered to the patient, it can cause some side-effects. Herein, we present the synthesis and structural characterization of a novel N-acylhydrazone derivative – LASSBio-1735 – a CA-4 analogue. LASSBio-1735 has displayed in vitro antiproliferative activity against HL-60 (human leukemia), SF-295 (human glioblastoma), MDA-MB435 (melanoma) and HCT-8 (ileocecal adenocarcinoma) tumor cells. We found different hydration levels in two batches of the as-synthesized compound. As a consequence, we could successfully determine the crystal structures – by using X-ray powder diffraction data and a simulated annealing procedure – of the anhydrous and hydrated forms. The effects on cell viability of anhydrous and hydrated forms of LASSBio-1735 were comparatively evaluated in different tumor cell lines, and the hydrated form exhibited higher cytotoxicity in human leukemia K562 cells. These findings lead us to perform a quantitative phase analysis on one of the samples and may shed some light on the search for possible new solvates and/or hydrates.

Combretastatins: In vitro structure-activity relationship, mode of action and current clinical status.

For the first time combretastatins were isolated from African willow tree Combretum Caffrum. Subsequent studies have shown the impact of combretastatin A4 phosphate, a water-soluble prodrug, on endothelial cells in tumor vascular system. The same effect was not observed in the vascular system. This selectivity is associated with combretastatins mechanism of action: binding to colchicine domain of microtubules, which affects the cytoskeleton functionality of immature endothelial cells. At the same time, combretastatins directly induce cell death via apoptosis and/or mitotic catastrophe pathways. The combination of both elements makes combretastatin an anticancer compound of high efficiency. The cis-configuration is crucial for its biological activity. To date, many derivatives were synthesized. The attempts to resolve spontaneous isomerization to less active trans-stilbene derivative are still in progress. This issue seems to be overcome by incorporation of the ethene bridge with heterocyclic moiety in combretastatins structure. This modification retains the cis-configuration and prevents isomerization. Nevertheless, combretastatin A4 phosphate disodium is still the most potent compound of this group. The combination therapy, which is the most effective treatment, includes combretastatin A4 phosphate (CA4P) and conventional chemotherapeutics and/or radiotherapy. CA4P is relatively well tolerated giving adverse events of moderate severity, which includes: nausea, vomiting, headache, and tumor pain. The aforementioned effects subside on the day of drug administration or on the following day.

The Use of Stilbene Scaffold in Medicinal Chemistry and Multi- Target Drug Design.

The stilbene scaffold is a basic element for a number of biologically active natural and synthetic compounds, and it is considered as a privileged structure. Stilbenes exemplified by resveratrol, combretastatin A-4 and pterostilbene are of significant interest for drug research and development because of their potential in therapeutic and preventive application. Resveratrol, present in grapes and other food products, plays a role in the prevention of several human pathological processes and has been suggested as an anticancer agent. Moreover, recent evidence has revealed its potential effect on the aging process, diabetes and neurological dysfunction. Combretastatin A-4, from the bark of South African bush willow Combretum caffrum, also shows significant antitumor activity. Pterostilbene is closely related to resveratrol, sharing the same unique therapeutic potential as anti-inflammatory, antineoplastic and antioxidant agent. Therefore, research and development of stilbene-based medicinal chemistry have become rapidly evolving and increasingly active topics covering almost the whole range of therapeutic fields. In the present review, we provide an overview of the role of stilbenes in medicinal chemistry. In this context, we highlight the chemical methodologies adopted for the synthesis of stilbene derivatives, and outline the successful design of novel stilbene based hybrids in the field of cancer, Alzheimer's and other relevant diseases. This information may be useful in further design of stilbene-based molecules as new leads for the development of novel agents with clinical potential or as effective chemical probes to dissect biological processes.

Combretastatin A-4 and Derivatives: Potential Fungicides Targeting Fungal Tubulin

Combretastatin A-4, first isolated from the African willow tree Combretum caffrum, is a tubulin polymerization inhibitor in medicine. It was first postulated as a potential fungicide targeting fungal tubulin for plant disease control in this study. Combretastatin A-4 and its derivatives were synthesized and tested against Rhizoctonia solani and Pyricularia oryzae. Several compounds have EC50 values similar to or better than that of isoprothiolane, which is widely used for rice disease control. Structure-activity relationship study indicated the the cis configuration and hydroxyl group in combretastatin A-4 are crucial to the antifungal effect. Molecular modeling indicated the binding sites of combretastatin A-4 and carbendazim on fungal tubulin are totally different. The bioactivity of combretastatin A-4 and its derivatives against carbendazim-resistant strains was demonstrated in this study. The results provide a new approach for fungicide discovery and fungicide resistance management.

Abstract 1667: Targeting acute myelogenous leukemia with novel combrestatin analogs and development of predictors of response

Abstract Introduction: Most adult acute myelogenous leukemia (AML) patients relapse and die of the disease. AML stem cells (AML-SCs) have been postulated to resist standard chemotherapeutic agents and thus give rise to relapse. Combretastatin A-4 (CA-4), a natural product isolated from the South African tree Combretum caffrum, has been reported to possess anti-leukemic activity in AML cell lines. To increase the repertoire of combretastatin analogs with potent anti-leukemic activity, about 100 CA-4 analogs were tested in AML cells, and studied the mechanism that AML cells resist to such microtubule (MT) inhibitors. Methods: Cell viability/apoptosis was determined by annexin V and 7-AAD staining and flow cytometry. Cellular ROS production was measured by CellROX. Cell cycle was determined by DAPI staining and flow cytometry. Stem cell function was evaluated by colony assay and mouse xenograft assays. Tubulin depolymerization was measured by immunofluorescence or immunoblotting for microtubule fraction. Interaction of tubulin with BTAN or p38 MAPK was determined by pull down assays. Results: One of the most potent analogs, BTAN, was further investigated in a panel of leukemic cell lines and primary AML samples. Similar to CA-4, BTAN inhibited tubulin polymerization in vitro and induced MT depolymerization and cell cycle arrest at G2/M in AML cells. BTAN induced cell death of AML blast, progenitor and stem cells via caspase activation regardless of their proliferating status and level of intracellular reactive oxygen species (ROS). In addition, BTAN impaired the physical contact of AML with stromal cells. To further identify the mechanism of resistance to BTAN, we investigated several signal transduction pathways including AKT, JAK-STAT, ERK1/2, JNK and p38. p38 MAPK inhibitor sensitized leukemia cells to BTAN treatment, and the sensitivity of AML cells to BTAN correlated with the basal level of active/phosphorylated p38 MAPK, suggesting p38 MAPK may interfere with the sensitivity to BTAN in AML. We demonstrated that tubulin interacted with p38 MAPK, especially its phosphorylated form, which was disrupted by treatment with a p38 MAPK inhibitor. In addition, in the presence of p38 inhibitor, the accumulation of BTAN was potentiated in AML cells, suggesting activated p38 may interfere with the interaction between BTAN and its tubulin targets. Conclusion: Our data demonstrated that BTAN is a newly identified anti-AML-SC agent that works by disrupting the MT cytoskeleton and inducing caspase activation. These data suggest that the MT network is involved in the regulation of pro-survival signaling pathways even in quiescent AML-SCs, and that p38 MAPK is a key modulator for the response of AML blasts to microtubule inhibitors and that co-treatment with p38 MAPK inhibitors could be beneficial to microtubule destabilizing agents in AML. Note: This abstract was not presented at the meeting. Citation Format: Hongliang Zong, Narsimha R. Penthala, Vijayakumar N. Sonar, Paraskevi Giannakakou, Gail J. Roboz, Peter A. Crooks, Monica L. Guzman. Targeting acute myelogenous leukemia with novel combrestatin analogs and development of predictors of response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2015-1667