Combretastatin A4 Analogues Research Articles

3,4-Diarylisoxazoles-Analogues of Combretastatin A-4: Design, Synthesis, and Biological Evaluation In Vitro and In Vivo.

Focusing on the molecular docking results, a series of 3,4-diarylisoxazoles, analogues of Combretastatin A4, bearing various substituents at the fifth position of the isoxazole ring and pharmacophore groups bioisosteric to methoxy substituent at ring B, were synthesized in good yields and high regioselectivity. Depending on the substituent at C5, three approaches were chosen for the construction of isoxazole ring, including nitrosation of gem-dihalocyclopropanes, nitrile oxide synthesis, and difluoromethoxylation of isoxazolone to afford 5-haloisoxazoles, 5-unsubstituted isoxazoles, and 5-difluoromethoxyisoxazoles, respectively. Isoxazoles 43 and 45 showed selective cytotoxicity and antitubulin inhibition properties in vitro, with pharmacodynamic profiles closely related to that of CA-4. Both of them slow down tumor growth (66-74%) in mouse xenografts and slightly exceed in effectiveness Combretastatin A4-phosphate itself.

Design, stereoselective synthesis, and antitumoral activity of combretastatin A-4 analogs

Combretastatin A-4 (CA-4) is a microtubule targeting agent, and it has a great advantage when compared to other compounds with a similar mechanism of action, such as vinca alkaloids, colchicine, podophillotoxin, and paclitaxel: its simpler structure. In this work, the stereoselective synthesis and antiproliferative activity of stable CA-4 analogs, obtained from the cross-coupling reaction between vinyl tellurides and potassium aryltrifluoroborates is described. The method is simple, fast, and general, allowing further applications for the synthesis of more complex compounds.

Design, Synthesis, in Silico and in Vitro Evaluation of New Combretastatin A-4 Analogs as Antimitotic Antitumor Agents.

Combretastatin A-4 (CA-4) binds β-tubulin at the colchicine-binding site preventing tubulin from polymerizing into microtubules. CA-4 and cis combretastatin analogs isomerize to the trans form resulting in decreased cytotoxicity and anti-tubulin activity. However, the excellent anti-cancer potential and relatively simple molecular structure of CA-4, provide an encouraging starting point for the development of new, more stable and more potent anti-tubulin compounds. To synthesize a new series of 4-(3,4,5-trimethoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione derivatives (compounds 10-12) as analogs of CA-4, with different alkyl, and aryl side chain substituents at the triazole moiety (B-ring), resulting in permanent cis form. The newly synthesized CA-4 analogs were characterized by FT-IR, 1HNMR, 13CNMR, and HR-MS(ESI) techniques. Molecular docking studies including docking score (G) ADMET, DFT, and mlocular similarities were performed. The anti-proliferative activity of the new compounds against three human cancer cell lines was evaluated using the MTT assay, and their ability to inhibit tubulin polymerization, and consequently their effects on cell cycle progression and induction of apoptois were assessed. Molecular docking studies showed that compounds 11b and 11d exhibited the highest docking scores (-13.30 and -14.01 Kcal/mol, respectively) into colchicine-binding site, scores very close to the reference drug colchicine (-13.50 Kcal/mol), and that hydrogen bonding and hydrophobic interaction are essential for binding. The most active cytotoxic compound 11b had potent IC50 values ranging from 3.83 to 10.67 µM against the three human cancer cell lines, while exhibiting low cytotoxicity against non-cancer human WI-38, suggesting that compound 11b targets rapidly growing cancer cells. Moreover, compound 11b exhibited potent anti-tubulin activity which was comparable to CA-4. Targeting microtubules caused cell cycle arrest at the G2/M phase resulting in induction of apoptosis. These findings indicate that compound 11b is a promising β-tubulin-binding compound with antimitotic action that has the potential to treat cancer.

Design, Synthesis, and Biological Testing of Pyrazoline Derivatives of Combretastatin-A4: A Quest for Anticancer, Anti-Inflammatory, and Antioxidant Agents

Three groups of novel analogs of combretastatin-A4 (CA-4), viz., the N 1-phenyl-pyrazoline (5a–e), N 1-alkyl acetylated pyrazoline (6a–c), and N 1-phenyl acetylated pyrazoline (7a–g) were designed, and synthesized in good yield. The structure of the compounds was confirmed by spectroscopic techniques. All the compounds were evaluated for their in vitro anticancer (MCF-7 cell line), antioxidant (DPPH, NO, SOR, and H2O2), and anti-inflammatory activity. Compounds 5d, 7g, 7f, 7e, 7c, 5b, 6a, 7b, and 7a showed excellent potency with GI50 ranging from 0.1 to 10.9 µM against the MCF-7 cell line. Compounds 7f, 7g, 5c, 5d, 5b, 7e, and 6a exhibited good anti-inflammatory activity. Encouraged by these results, all the compounds were also tested for their antioxidant potency. Compounds 6a, 6c, 7b, 7c, 7f, and 7g were found to be excellent scavengers of all four free radicals (DPPH, NO, SOR, and H2O2).

Assessing the Antiproliferative Potential of a Novel Combretastatin A4 Derivative via Modulating Apoptosis, MAPK/ERK and PI3K/AKT Pathways in Human Breast Cancer Cells.

Breast cancer is the most predominant tumor in women. Even though current medications for distinct breast cancer subtypes are available, the non-specificity of chemotherapeutics and chemoresistance imposes major obstacles in breast cancer treatment. Although combretastatin A-4 (CA-4) has been well-reported to have potential anticancer activity, in vivo studies of CA-4 reveal a decrease in its activity. In this respect, a series of CA-4 analogues have been designed, from which one analog [(1-(3-chloro-4-fluorophenyl)-N-(2methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide, C25H22ClFN4O5] showed drastic cytotoxicity against breast cancer cells. Therefore, this research focused on investigating the in vitro molecular mechanism underlying the cytotoxicity of the CA-4 analogue, particularly the MAPK/ERK as well as PI3K/AKT pathways as attractive therapeutic targets in breast cancer. The cell viability of MCF-7, MDA-MB231, and MDA-MB453 was assessed after treatment with the CA-4 analogue, and apoptosis was analyzed via Annexin V-FITC/PI dual staining. MAPK/ERK and PI3K/AKT were thoroughly assessed using western blotting. Real-time PCR was used to estimate apoptosis-related markers, including the P53, Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl2) genes. The CA-4 analogue reduced the survival of all cancerous cells in a concentration-dependent manner and induced apoptosis through the mitochondrial pathway (39.89 ± 1.5%, 32.82 ± 0.6%, and 23.77 ± 1.1% in MCF-7, MDA-MB231, and MDA-MB453 cells), respectively. The analogue also attenuated the expression of pMEK1/2/t-MEK1/2, p-ERK1/2/t-ERK1/2, p-PI3K/t-PI3K, and p-AKT/t-AKT proteins in all three cancer cell lines in a time-dependent manner. Furthermore, the CA-4 analogue upregulated the expression of the P53 gene and dramatically increased the ratio of Bax/Bcl2 genes. The enhanced cytotoxicity can be attributed to substituting the hydroxyl group in CA-4 with chlorine in the meta-position of ring B, substituting the para-methoxy group in CA-4 with fluorine in the analogue, and lastly, introducing an extension to the compound's structure (ring C). Therefore, CA-4 analogue can attenuate the proliferation of human breast cancer cells by inducing apoptosis and simultaneously suppressing the MAPK/ERK and PI3K/AKT pathways.

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.

Synthesis of tetraalkoxyaryldihydrobenzo[g]indazoles from metabolites of dill and parsley seeds essential oil

An efficient method for the conversion of available plant allylpolyalkoxybenzenes into unsubstituted and arylsubstituted polymethoxylated aryldihydrobenzo[g]indazoles, analogues of combretastatin A4, has been developed. The selective demethylation of target compounds resulted in indazoles with different position of OH-group. Their structure was confirmed by X-ray analysis. A sea urchin embryo assay revealed the lack of tubulin-related antimitotic activity of aryldihydrobenzo[g]indazoles with systemic toxicity of a compound featuring OH group and 1-unsubstituted pyrazole ring.

New Combretastatin Analogs as Anticancer Agents: Design, Synthesis, Microtubules Polymerization Inhibition, and Molecular Docking Studies.

A new series of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol derivatives were synthesized as analogs for the anticancer drug combretastatin A-4 (CA-4) and characterized using FT-IR, 1 H-NMR, 13 CNMR, and HR-MS techniques. The new CA-4 analogs were designed to meet the structural requirements of the highest expected anticancer activity of CA-4 analogs by maintaining ring A 3,4,5-trimethoxyphenyl moiety, and at the same time varying the substituents effect of the triazole moiety (ring B). In silico analysis indicated that compound 3 has higher total energy and dipole moment than colchicine and the other analogs, and it has excellent distribution of electron density and is more stable, resulting in an increased binding affinity during tubulin inhibition. Additionally, compound 3 was found to interact with three apoptotic markers, namely p53, Bcl-2, and caspase 3. Compound 3 showed strong similarity to colchicine, and it has excellent pharmacokinetics properties and a good dynamic profile. The in vitro anti-proliferation studies showed that compound 3 is the most cytotoxic CA-4 analog against cancer cells (IC50 of 6.35 μM against Hep G2 hepatocarcinoma cells), and based on its selectivity index (4.7), compound 3 is a cancer cytotoxic-selective agent. As expected and similar to colchicine, compound 3-treated Hep G2 hepatocarcinoma cells were arrested at the G2/M phase resulting in induction of apoptosis. Compound 3 tubulin polymerization IC50 (9.50 μM) and effect on Vmax of tubulin polymerization was comparable to that of colchicine (5.49 μM). Taken together, the findings of the current study suggest that compound 3, through its binding to the colchicine-binding site at β-tubulin, is a promising microtubule-disrupting agent with excellent potential to be used as cancer therapeutic agent.

Advances in antitumor research of CA-4 analogs carrying quinoline scaffold

Combretastatin A-4 (CA-4) is a potent inhibitor of tubulin polymerization and a colchicine binding site inhibitor (CBSI). The structure-activity relationship study of CA-4 showed that the cis double bond configuration and the 3,4,5-trimethoxy group on the A ring were important factors to maintain the activity of CA-4. Therefore, starting from this condition, chemists modified the double bond and also substituted 3,4,5-trimethoxyphenyl with various heterocycles, resulting in a new generation of CA-4 analogs such as chalcone, Flavonoid derivatives, indole, imidazole, etc. Quinoline derivatives have strong biological activity and have been sought after by major researchers for their antitumor activity in recent years. This article reviews the research progress of novel CA-4 containing quinoline analogs in anti-tumor from 1992 to 2022 and expounds on the pharmacological mechanisms of these effective compounds, including but not limited to apoptosis, cell cycle, tubulin polymerization inhibition, immune Fluorescence experiments, etc., which lay the foundation for the subsequent development of CA-4 containing quinoline analogs for clinical use.

Synthesis, Characterisation and Mechanism of Action of Anticancer 3-Fluoroazetidin-2-ones.

The stilbene combretastatin A-4 (CA-4) is a potent microtubule-disrupting agent interacting at the colchicine-binding site of tubulin. In the present work, the synthesis, characterisation and mechanism of action of a series of 3-fluoro and 3,3-difluoro substituted β-lactams as analogues of the tubulin-targeting agent CA-4 are described. The synthesis was achieved by a convenient microwave-assisted Reformatsky reaction and is the first report of 3-fluoro and 3,3-difluoro β-lactams as CA-4 analogues. The β-lactam compounds 3-fluoro-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxy phenyl)azetidin-2-one 32 and 3-fluoro-4-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one) 33 exhibited potent activity in MCF-7 human breast cancer cells with IC50 values of 0.075 µM and 0.095 µM, respectively, and demonstrated low toxicity in non-cancerous cells. Compound 32 also demonstrated significant antiproliferative activity at nanomolar concentrations in the triple-negative breast cancer cell line Hs578T (IC50 0.033 μM), together with potency in the invasive isogenic subclone Hs578Ts(i)8 (IC50 = 0.065 μM), while 33 was also effective in MDA-MB-231 cells (IC50 0.620 μM). Mechanistic studies demonstrated that 33 inhibited tubulin polymerisation, induced apoptosis in MCF-7 cells, and induced a downregulation in the expression of anti-apoptotic Bcl2 and survivin with corresponding upregulation in the expression of pro-apoptotic Bax. In silico studies indicated the interaction of the compounds with the colchicine-binding site, demonstrating the potential for further developing novel cancer therapeutics as microtubule-targeting agents.

Recent Trends in Tubulin-Binding Combretastatin A-4 Analogs for Anticancer Drug Development.

Although significant progress over several decades has been evidenced in cancer therapy, there remains a need for the development of novel and effective therapeutic strategies to treat several relapsed and intractable cancers. In this regard, tubulin protein has become one of the efficient and major targets for anticancer drug discovery. Considering the antimitotic ability, several tubulin inhibitors have been developed to act against various cancers. Among various tubulin inhibitors available, combretastatin-A4 (CA-4), a naturally occurring lead molecule, offers exceptional cytotoxicity (including the drugresistant cell lines) and antivascular effects. Although CA-4 offers exceptional therapeutic efficacy, several new advancements have been proposed, in terms of structural modification via A and B rings, as well as cis-olefinic bridging, which provide highly efficient analogs with improved tubulin-binding efficiency to meet the anticancer drug development requirements. This review systematically emphasizes the recent trends and latest developments in the anticancer drug design and discovery using CA-4 analogs as the tubulin inhibiting agents by highlighting their structure-activity relationships (SAR) and resultant pharmacological efficacies.

Recent Advances in Combretastatin A-4 Inspired Inhibitors of Tubulin Polymerization: An Update.

Cancer is one of the leading causes of fatality and mortality worldwide. Investigations on developing therapeutic strategies for cancer are supported throughout the world. The massive achievements in molecular sciences involving biochemistry, molecular chemistry, medicine, and pharmacy, and high throughput techniques such as genomics and proteomics have helped create new potential drug targets for cancer treatment. Microtubules are very attractive targets for cancer therapy because of the crucial roles they play in cell division. In recent years, lots of efforts have been put into the identification of new microtubule-targeting agents (MTAs) in anticancer therapy. Combretastatin A-4 (CA-4) is a natural compound that binds to microtubules' colchicine binding site and inhibits microtubule polymerization. Due to CA-4's structural simplicity, many analogs have been synthesized. This article summarises the new molecule development efforts to reach CA-4 analogues by pharmacophore group modifications, which have been reported since 2015.

New Heterocyclic Combretastatin A-4 Analogs: Synthesis and Biological Activity of Styryl-2(3H)-benzothiazolones.

Here, we describe the synthesis, characterization, and biological activities of a series of 26 new styryl-2(3H)-benzothiazolone analogs of combretastatin-A4 (CA-4). The cytotoxic activities of these compounds were tested in several cell lines (EA.hy926, A549, BEAS-2B, MDA-MB-231, HT-29, MCF-7, and MCF-10A), and the relations between structure and cytotoxicity are discussed. From the series, compound (Z)-3-methyl-6-(3,4,5-trimethoxystyryl)-2(3H)-benzothiazolone (26Z) exhibits the most potent cytotoxic activity (IC50 0.13 ± 0.01 µM) against EA.hy926 cells. 26Z not only inhibits vasculogenesis but also disrupts pre-existing vasculature. 26Z is a microtubule-modulating agent and inhibits a spectrum of angiogenic events in EA.hy926 cells by interfering with endothelial cell invasion, migration, and proliferation. 26Z also shows anti-proliferative activity in CA-4 resistant cells with the following IC50 values: HT-29 (0.008 ± 0.001 µM), MDA-MB-231 (1.35 ± 0.42 µM), and MCF-7 (2.42 ± 0.48 µM). Cell-cycle phase-specific experiments show that 26Z treatment results in G2/M arrest and mitotic spindle multipolarity, suggesting that drug-induced centrosome amplification could promote cell death. Some 26Z-treated adherent cells undergo aberrant cytokinesis, resulting in aneuploidy that perhaps contributes to drug-induced cell death. These data indicate that spindle multipolarity induction by 26Z has an exciting chemotherapeutic potential that merits further investigation.

Imidazole Analogs of Vascular-Disrupting Combretastatin A-4 with Pleiotropic Efficacy against Resistant Colorectal Cancer Models.

Specific targeting of the tumoral vasculature by vascular-disrupting agents (VDA), of which combretastatin A-4 (CA-4) is a main representative, has been considered a new therapeutic strategy against multidrug-resistant tumors. In addition, CA-4 and analogs are tubulin-targeting agents and can exert direct antitumor effects by different mechanisms. Herein, we analyzed a series of synthetic CA-4 analogs featuring N-methylimidazole-bridged Z-alkenes with different halo- or amino-substituted aryl rings in vitro and in vivo, focusing on models of colorectal cancer. Combined in vitro/in vivo structure–activity relationship studies using cell lines and xenograft tumors susceptible to VDA-induced vascular damage demonstrated a clear association of cytotoxic and vascular-disrupting activity with the ability to inhibit tubulin polymerization, which was determined by specific substitution constellations. The most active compounds were tested in an extended panel of colorectal cancer (CRC) cell lines and showed activity in CA-4-resistant and chemotherapy-resistant cell lines. The bromo derivative brimamin was then compared with the known fosbretabulin (CA-4P) by activity tests on DLD-1- (multidrug-resistant) and HT29- (CA-4-resistant) derived xenograft tumors. Treatment did not induce pronounced vascular-disrupting effects in these tumors. Histological analyses revealed distinct tumor substructures and vessel compositions of DLD-1/HT29 tumors, which clearly differed from the tumor models susceptible to VDA treatment. Even so, brimamin effectively retarded the growth of DLD-1 tumors, overcoming their resistance to standard treatment, and it inhibited the outgrowth of disseminated HT29 tumor cells in an experimental metastasis model. In conclusion, combretastatin analogous N-methylimidazoles proved capable of inducing vascular-disrupting effects, comparable to those of CA-4P. In addition, they showed antitumor activities in models of drug-resistant colorectal cancer, independent of vascular-disrupting effects.

Design and discovery of new antiproliferative 1,2,4-triazin-3(2H)-ones as tubulin polymerization inhibitors targeting colchicine binding site

Thirty-five new colchicine binding site inhibitors have been designed and synthesized based on the 1,2,4-triazin-3(2H)-one nucleus. Such molecules were synthesized through a cascade reaction between readily accessible α-amino ketones and phenyl carbazate as a masked N-isocyanate precursor. The synthesized derivatives are cisoid restricted combretastatin A4 analogues containing 1,2,4-triazin-3(2H)-one in place of the olefinic bond, and they have the same essential pharmacophoric features of colchicine binding site inhibitors. The synthesized compounds were evaluated in vitro for their antiproliferative activities against a panel of three human cancer cell lines (MCF-7, HepG-2, and HCT-116), using colchicine as a positive control. Among them, two compounds 5i and 6i demonstrated a significant antiproliferative effect against all cell lines with IC50 ranging from 8.2 − 18.2 µM. Further investigation was carried out for the most active cytotoxic agents as tubulin polymerization inhibitors. Compounds 5i and 6i effectively inhibited microtubule assembly with IC50 values ranging from 3.9 to 7.8 µM. Tubulin polymerization assay results were found to be comparable with the cytotoxicity results. The cell cycle analysis revealed significant G2/M cell cycle arrest of the analogue 5i in HepG-2 cells. The most active compounds 4i, 4j, 5 g, 5i and 6i did not induce significant cell death in normal human lung cells Wl-38, suggesting their selectivity against cancer cells. Also, These compounds upregulated the level of active caspase-3 and boosted the levels of the pro-apoptotic protein Bax by five to seven folds in comparison to the control. Moreover, apoptosis analyses were conducted for compound 5i to evaluate its apoptotic potential. Finally, in silico studies were conducted to reveal the probable interaction with the colchicine binding site. ADME prediction study of the designed compounds showed that they are not only with promising tubulin polymerization inhibitory activity but also with favorable pharmacokinetic and drug-likeness properties.

OMA1520 and OMA1774, novel 1,2,4-triazole bearing analogs of combretastatin A-4, inhibit hepatocellular carcinoma: Histological and immunohistochemical studies

Combretastatin A-4 (CA-4) received significant interest as a potential anticancer agent in recent years. Several CA-4 analogs were synthesized and investigated to enhance the activity or solve the in vivo decreased activity of CA-4. AimThe present study aims to investigate the chemotherapeutic and the antiproliferative effects of the mono and the dual therapy of the newly synthesized CA-4 analogs OMA1520 and OMA1774 against hepatocellularcarcinoma (HCC) induced in male adult rats by N-methylnitrosourea (MNU). Methods50 male rats were divided into 5 groups of 10 animals in each group. Group I: normal healthy control; group II: MNU treated group, group III: MNU animals treated by OMA1520, group IV: MNU animals treated by OMA1774, and group V: MNU animals treated by both OMA1520 and OMA1774. The rats were assessed for liver cancer progression or inhibition by evaluating the histopathological, immunohistochemical, biochemical, and antioxidant enzyme status. ResultsThe present work indicated that OMA1520 and OMA1774 possessed substantial chemotherapeutic efficiency against HCC. The histological and immunohistochemical examinations of liver tissues confirmed the biochemical sera data. Also, they diminished the cytotoxic effects of MNU and restored the normal histological hepatic architecture. Both analogs restored the normal levels of liver enzymes and functions and revealed potential antioxidant effects. OMA1520 and OMA1774 reduced the inflammatory and tumor markers' elevated expressions in serum. ConclusionSubstantial evidence in our results suggests that both CA-4 analogs could be possible alternative anticancer agents, and their co-administration provides a synergistic activity.

Light-driven photoswitching of quinazoline analogues of combretastatin A-4 as an effective approach for targeting skin cancer cells.

A novel quinazoline series of photoswitchable combretastatin A-4 (CA-4) analogues were synthesized and their photochemical properties and antiproliferative activity against A431 epidermoid carcinoma cells were studied. It was found that quinazoline analogues, in contrast to the majority of the known CA-4, exhibit high antiproliferative activity in the E-form as well. Photoswitching of the E-form to the Z-form resulted in a multiple (9-fold) increase in antiproliferative activity. 1H NMR monitoring showed that these compounds are very resistant to UV (λ = 365 nm) or sunlight irradiation and do not undergo photodegradation with a loss of antiproliferative activity that is inherent in heterocyclic analogues of CA-4. Similar photoswitching and an increase in antiproliferative activity are observed on exposure to sunlight. A selected compound (1a-Z51) in sub-micromolar concentrations induced apoptosis in A431 cells, while rad50/ATM/p53 were not involved in cell death. The growth of A431 cells was significantly inhibited after combination treatment with compound 1a-Z51 and chemotherapy drugs (cisplatin or 5-fluorouracil). In summary, the quinazoline analogues of CA-4 represent a promising strategy to achieve a photoswitchable potency for the treatment of cancers, including the development of combination therapies.

Synthesis, biological evaluation, and modeling studies of 1,3-disubstituted cyclobutane-containing analogs of combretastatin A4

With the aim of circumventing the adverse cis/trans-isomerization of combretastatin A4 (CA4), a naturally occurring tumor-vascular disrupting agent, we designed novel CA4 analogs bearing 1,3-disubstituted cyclobutane moiety instead of the cis-stilbene unit of the parent compound. The corresponding cis and trans cyclobutane-containing derivatives were prepared as pure diastereomers. The structure of the target compounds was confirmed by X-ray diffraction study. The title compounds were evaluated for their cytotoxic properties in human cancer cell lines HepG2 (hepatocarcinoma) and SK-N-DZ (neuroblastoma), and the overall activity was found in micromolar range. Molecular docking studies and molecular dynamics simulation within the colchicine binding site of tubulin were in good agreement with the obtained cytotoxicity data.

Exploring Diverse-Ring Analogues on Combretastatin A4 (CA-4) Olefin as Microtubule-Targeting Agents.

Combretastatin-4 (CA-4) as a tubulin polymerization inhibitor draws extensive attentions. However, due to its weak stability of cis-olefin and poor metabolic stability, structure modifications on cis-configuration are being performed. In this work, we constructed a series of novel CA-4 analogues with linkers on olefin containing diphenylethanone, cis-locked dihydrofuran, α-substituted diphenylethanone, cyclobutane and cyclohexane on its cis-olefin. Cytotoxic activity of all analogues was measured by an SRB assay. Among them, compound 6b, a by-product in the preparation of diphenylethanone analogues, was found to be the most potent cytotoxic agents against HepG2 cells with IC50 values of less than 0.5 μM. The two isomers of 6b induced cellular apoptosis tested by Annexin V-FITC and propidium iodide (PI) double staining, arrested cells in the G2/M phase by PI staining analysis, and disrupted microtubule network by immunohistochemistry study in HepG2 cells. Moreover, 6b-(E) displayed a dose-dependent inhibition effect for tubulin assembly in in vitro tubulin polymerization assay. In addition, molecular docking studies showed that two isomers of 6b could bind efficiently at colchicine binding site of tubulin similar to CA-4.

Abstract P3-10-21: Preclinical evaluation of a novel phospholipid drug conjugate with a combretastatin A-4 analogue for improved breast cancer therapy

Abstract Purpose: Breast cancer (BrCa) is the most common invasive cancer in women globally and the second most common cause of death from cancer in women in the US. Combretastatin A-4 (CA4) and associated analogues have been shown to be potent cytotoxins and inhibitors of tumor blood flow. However, CA4 and its analogues have not progressed rapidly through clinical trials. It has been shown that with some of the CA4 analogues, incomplete disruption of the vasculature and/or proangiogenic factors around the tumor result in survival of the cap portion of the tumor and future progression. Additionally, the over expression of MDR-1 gene which encodes for P-glycoprotein (Pgp) efflux pumps which is overexpressed in many cancer cells has been shown to limit the activity of these molecules. Phospholipid-drug conjugates (PDCs) are a modality designed to selectively deliver molecules to the tumor or tumor microenvironment, significantly increasing intracellular or local concentrations. In the current study, we established a series of PDCs using a single CA4 analogue (CA4A) conjugated to our phospholipid ether targeting platform (PDC-CA4A) utilizing different peptide linkers that allow for facile targeting and release of the CA4A payload. In this study, we evaluated the in vitro and in vivo therapeutic potential of different PDC-CA4A molecules. Methods: In vitro assessment of cellular internalization, accumulation and release of the payload was evaluated via mass spectrometry in A549 and normal human dermal fibroblast (NHDFs) cells. Additionally, in vitro assessment of cytotoxicity was performed in A549, MCF7, HCT116 and NHDFs with fifty percent inhibitory concentrations (IC50s) being determined for each. Subcutaneous HCT116 and MCF7 xenograft tumor models were used to evaluate the antitumor efficacy of several PDC-CA4A molecules compared with the free CA4A molecule. Results: All PDC-CA4A molecules showed excellent uptake, accumulation and release of the CA4A payload in the A549 cell line. Uptake was detected at the first time point tested (30 minutes) after exposure and continued through 24 hours. A steady state of release of the payload and new PDC-CA4A molecules entering the cell was reached 3 hours post exposure. Cytotoxicity tests demonstrated IC50s ranging from nanomolar to micromolar concentration depending upon the linker being utilized. In vivo, PDC-CA4A-109 (specific molecule) demonstrated statistically significant benefit over both free CA4A and the positive control group. Conclusion: Our results suggest that PDC-CA4A-109 is a promising antitumor agent to treat both breast cancer and colorectal cancers. Further clinical study is warranted to evaluate the safety and feasibility of PDC-CA4A-109 as a novel targeted therapy. Citation Format: Jarrod Longcor, Randall Hoover, Mark Wolf. Preclinical evaluation of a novel phospholipid drug conjugate with a combretastatin A-4 analogue for improved breast cancer therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-21.