Synthetic Makaluvamine Analogs Decrease c-Kit Expression and Are Cytotoxic to Neuroendocrine Tumor Cells.

In an effort to discover viable systemic chemotherapeutic agents for neuroendocrine tumors (NETs), we screened a small library of 18 drug-like compounds obtained from the Velu lab against pulmonary (H727) and thyroid (MZ-CRC-1 and TT) neuroendocrine tumor-derived cell lines. Two potent lead compounds (DHN-II-84 and DHN-III-14) identified from this screening were found to be analogs of the natural product makaluvamine. We further characterized the antitumor activities of these two compounds using pulmonary (H727), thyroid (MZ-CRC-1) and pancreatic (BON) neuroendocrine tumor cell lines. Flow cytometry showed a dose-dependent increase in apoptosis in all cell lines. Induction of apoptosis with these compounds was also supported by the decrease in myeloid cell leukemia-1 (MCL-1) and X-chromosome linked inhibitor of apoptosis (XIAP) detected by Western blot. Compound treatment decreased NET markers chromogranin A (CgA) and achaete-scute homolog 1 (ASCL1) in a dose-dependent manner. Moreover, the gene expression analysis showed that the compound treatment reduced c-Kit proto-oncogene expression in the NET cell lines. Induction of apoptosis could also have been caused by the inhibition of c-Kit expression, in addition to the known mechanisms such as damage of DNA by topoisomerase II inhibition for this class of compounds. In summary, makaluvamine analogs DHN-II-84 and DHN-III-14 induced apoptosis, decreased neuroendocrine tumor markers, and showed promising antitumor activity in pulmonary, thyroid, and pancreatic NET cell lines, and hold potential to be developed as an effective treatment to combat neuroendocrine tumors.

A Novel Marine Natural Product Derived Pyrroloiminoquinone with Potent Activity against Skin Cancer Cells.

Non-melanoma skin cancer is one of the major ailments in the United States. Effective drugs that can cure skin cancers are limited. Moreover, the available drugs have toxic side effects. Therefore, skin cancer drugs with less toxic side effects are urgently needed. To achieve this goal, we focused our work on identifying potent lead compounds from marine natural products. Five lead compounds identified from a class of pyrroloiminoquinone natural products were evaluated for their ability to selectively kill squamous cell carcinoma (SCC13) skin cancer cells using an MTT assay. The toxicity of these compounds was also evaluated against the normal human keratinocyte HaCaT cell line. The most potent compound identified from these studies, C278 was further evaluated for its ability to inhibit cancer cell migration and invasion using a wound-healing assay and a trans-well migration assay, respectively. To investigate the molecular mechanism of cell death, the expression of apoptotic and autophagy proteins was studied in C278 treated cells compared to untreated cells using western blot. Our results showed that all five compounds effectively killed the SCC13 cells, with compound C278 being the most effective. Compound C278 was more effective in killing the SCC13 cells compared to HaCaT cells with a two-fold selectivity. The migration and the invasion of the SCC13 cells were also inhibited upon treatment with compound C278. The expression of pro-apoptotic and autophagy proteins with concomitant downregulation in the expression of survival proteins were observed in C278 treated cells. In summary, the marine natural product analog compound C278 showed promising anticancer activity against human skin cancer cells and holds potential to be developed as an effective anticancer agent to combat skin cancer.

Antibacterial and Antibiofilm Activities of Makaluvamine Analogs.

Streptococcus mutans is a key etiological agent in the formation of dental caries. The major virulence factor is its ability to form biofilms. Inhibition of S. mutans biofilms offers therapeutic prospects for the treatment and the prevention of dental caries. In this study, 14 analogs of makaluvamine, a marine alkaloid, were evaluated for their antibacterial activity against S. mutans and for their ability to inhibit S. mutans biofilm formation. All analogs contained the tricyclic pyrroloiminoquinone core of makaluvamines. The structural variations of the analogs are on the amino substituents at the 7-position of the ring and the inclusion of a tosyl group on the pyrrole ring N of the makaluvamine core. The makaluvamine analogs displayed biofilm inhibition with IC50 values ranging from 0.4 µM to 88 µM. Further, the observed bactericidal activity of the majority of the analogs was found to be consistent with the anti-biofilm activity, leading to the conclusion that the anti-biofilm activity of these analogs stems from their ability to kill S. mutans. However, three of the most potent N-tosyl analogs showed biofilm IC50 values at least an order of magnitude lower than that of bactericidal activity, indicating that the biofilm activity of these analogs is more selective and perhaps independent of bactericidal activity.

Total synthesis of zyzzyanones A-D.

Zyzzyanones A-D is a group of biologically active marine alkaloids isolated from Australian marine sponge Zyzzya fuliginosa. They contain a unique bispyrroloquinone ring system as the core structure. The first total synthesis of all four zyzzyanones is described here. The synthesis of these alkaloids started from a previously known 6-benzylamino indole-4,7-quinone derivative and involves 6-7 steps. The key step in the synthesis involves the construction of a pyrrole ring in one step using a Mn(OAc)3 mediated oxidative free radical cyclization reaction of a 6-benzylamino indole-4,7-quinone derivative with 4-benzyloxyphenyl acetaldehyde diethyl acetal in CH3CN.

Identification of the ZAK-MKK4-JNK-TGFß signaling pathway as a molecular target for novel synthetic iminoquinone anticancer compound BA-TPQ.

Identification and validation of molecular targets are considered as key elements in new drug discovery and development. We have recently demonstrated that a novel synthetic iminoquinone analog, termed [7-(benzylamino)- 1,3,4,8-tetrahydropyrrolo [4,3, 2-de]quinolin-8(1H)-one] (BA-TPQ), has significant anti-breast cancer activity both in vitro and in vivo, but the underlying molecular mechanisms are not fully understood. Herein, we report the molecular studies for BA-TPQ's effects on JNK and its upstream and downstream signaling pathways. The compound up-regulates the JNK protein levels by increasing its phosphorylation and decreasing its polyubiquitination-mediated degradation. It activates ZAK at the MAPKKK level and MKK4 at the MAPKK level. It also up-regulates the TGFß2 mRNA level, which can be abolished by the JNK-specific inhibitor SP600125, but not TGFß pathway-specific inhibitor SD-208, indicating that both JNK and TGFß signaling pathways are activated by BA-TPQ and that the JNK pathway activation precedes TGFß activation. The pro-apoptotic and anti-growth effects of BA-TPQ are significantly blocked by both the JNK and TGFß pathway inhibitors. In addition, BA-TPQ activates the ZAK-MKK4-JNK pathway in MCF7 cells, but not normal MCF10A cells, demonstrating its cancer-specific activities. In conclusion, our results demonstrate that BA-TPQ activates the ZAK-MKK4-JNK-TGFß signaling cascade as a molecular target for its anticancer activity.

Preclinical evaluation of anticancer efficacy and pharmacological properties of FBA-TPQ, a novel synthetic makaluvamine analog.

We have recently designed and synthesized a novel iminoquinone anticancer agent, 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) and initiated its preclinical development. Herein we investigated its efficacy, safety, and pharmacokinetics in in vitro and in vivo models of human pancreatic cancer. Our results demonstrated that FBA-TPQ inhibited pancreatic cancer cell growth, induced apoptosis, and caused cell cycle arrest in vitro. It inhibited the growth of xenograft tumors with minimal host toxicity. To facilitate future preclinical and clinical development of the agent, we also developed and validated a Rapid Resolution Liquid Chromatography (RRLC) method for quantitative analysis of FBA-TPQ in plasma and tissue samples. The method was found to be precise, accurate, and specific. Using this method, we carried out in vitro and in vivo evaluations of the pharmacological properties of FBA-TPQ, including stability in plasma, plasma protein binding, metabolism by S9 enzymes, plasma pharmacokinetics, and tissue distribution. Our results indicate that FBA-TPQ is a potential therapeutic agent for pancreatic cancer, providing a basis for future preclinical and clinical development.

Experimental therapy of ovarian cancer with synthetic makaluvamine analog: in vitro and in vivo anticancer activity and molecular mechanisms of action.

The present study was designed to determine the biological effects of novel marine alkaloid analog 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) on human ovarian cancer cells for its anti-tumor potential and the underlying mechanisms as a novel chemotherapeutic agent. Human ovarian cancer cells (A2780 and OVCAR-3), and Immortalized non-tumorigenic human Ovarian Surface Epithelial cells (IOSE-144), were exposed to FBA-TPQ for initial cytotoxicity evaluation (via MTS assay kit, Promega). The detailed in-vitro (cell level) and in-vivo (animal model) studies on the antitumor effects and possible underlying mechanisms of action of the compounds were then performed. FBA-TPQ exerted potent cytotoxicity against human ovarian cancer A2780 and OVCAR-3 cells as an effective inhibitor of cell growth and proliferation, while exerting lesser effects on non-tumorigenic IOSE-144 cells. Further study in the more sensitive OVCAR-3 cell line showed that it could potently induce cell apoptosis (Annexin V-FITC assay), G2/M cell cycle arrest (PI staining analysis) and also dose-dependently inhibit OVCAR-3 xenograft tumors' growth on female athymic nude mice (BALB/c, nu/nu). Mechanistic studies (both in vitro and in vivo) revealed that FBA-TPQ might exert its activity through Reactive Oxygen Species (ROS)-associated activation of the death receptor, p53-MDM2, and PI3K-Akt pathways in OVCAR-3 cells, which is in accordance with in vitro microarray (Human genome microarrays, Agilent) data analysis (GEO accession number: GSE25317). In conclusion, FBA-TPQ exhibits significant anticancer activity against ovarian cancer cells, with minimal toxicity to non-tumorigenic human IOSE-144 cells, indicating that it may be a potential therapeutic agent for ovarian cancer.

A novel synthetic iminoquinone, BA-TPQ, as an anti-breast cancer agent: in vitro and in vivo activity and mechanisms of action.

Herein, we report our examination of the anti-breast cancer activity of a novel synthetic compound, 7-(benzylamino)-1, 3, 4, 8-tetrahydropyrrolo [4, 3, 2-de]quinolin-8(1H)-one (BA-TPQ). This agent is an analog of a naturally occurring marine compound, and was found to be the most active out of more than 40 related compounds. We investigated the in vitro activity of BA-TPQ on the survival, proliferation, and apoptosis of breast cancer cells using the MTT and BrdUrd assays, and Annexin/Annexin-PI staining and flow cytometry. The in vivo anti-cancer effects of BA-TPQ were evaluated in xenograft models of breast cancer. Finally, the mechanisms of action of the compound were also assessed by cDNA microarrays, RT-PCR and Western blotting. In a dose-dependent manner, BA-TPQ inhibited cell growth and induced apoptosis and cell cycle arrest in human MCF-7 and MDA-MB-468 breast cancer cells in vitro, and showed in vivo efficacy in mice bearing MCF-7 or MDA-MB-468 xenograft tumors. We demonstrated that BA-TPQ modifies the expression of numerous molecules involved in cell cycle progression and apoptosis. Similar changes in protein expression were observed in vitro and in vivo, as determined by examination of cells and excised xenograft tumors. Our preclinical data indicate that BA-TPQ is a potential therapeutic agent for breast cancer that has multiple hormone-, Her2-, and p53-independent mechanisms of action, providing a basis for further development of the compound as a novel anticancer agent.

FBA-TPQ, a novel marine-derived compound as experimental therapy for prostate cancer.

We recently synthesized a series of novel makaluvamine compounds, and found that the most potent was FBA-TPQ. The effects of FBA-TPQ on human (LNCaP and PC3) and murine (TRAMP C1) prostate cancer cells were evaluated. Potential mechanisms of action of the compound were also determined. FBA-TPQ exhibited dose-dependent cytotoxicity in the low micromolar range, inhibited proliferation, caused cell cycle arrest, and induced apoptosis in prostate cancer cell lines. The compound also decreased the expression of the androgen receptor and PSA. The results presented herein support the further development of FBA-TPQ as a novel agent for prostate cancer.

Synthesis and in vitro anti-lung cancer activity of novel 1, 3, 4, 8-tetrahydropyrrolo [4, 3, 2-de]quinolin-8(1H)-one alkaloid analogs.

The high mortality rate and lack of effective therapies make lung cancer an ideal target for novel therapeutic agents. The present study was designed to implement a novel chemical synthesis pathway and to determine the biological activities of synthetic makaluvamine analogs in human lung cancer. Seventeen compounds were synthesized and purified, and their chemical structures were elucidated on the basis of physicochemical constants and NMR spectra. Their in vitro activity was determined in human lung cancer cell lines. Based on initial screens, compound Ic was found to be the most potent, and was therefore used as a model for further studies in lung cancer cells. Ic induced both apoptosis and S-phase cell cycle arrest. Furthermore, it activated p53 and induced cleavage of PARP and caspases 8 and 9. Our preclinical data indicate that the makaluvamine analogs are potential therapeutic agents against lung cancer, providing a basis for further development of Ic (and perhaps other analogs) as a novel anti-cancer agent.

In vitro and in vivo anticancer activity of novel synthetic makaluvamine analogues.

PURPOSE: The present study was designed to determine biological structure-activity relationships for four newly synthesized analogues of natural compounds (makaluvamines). The compounds, 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ); 7-(phenethylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (PEA-TPQ); 7-(3,4-methylenedioxyphenethylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (MPA-TPQ); and 7-(3,4-dimethoxyphenethylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (DPA-TPQ), were synthesized and purified, and their chemical structures were elucidated on the basis of physicochemical constants and nuclear magnetic resonance spectra. EXPERIMENTAL DESIGN: The structure-activity relationship of the compounds was initially evaluated by comparing their in vitro cytotoxicity against 14 human cell lines. Detailed in vitro and in vivo studies were then done in MCF-7 and MDA-MB-468 breast cancer cell lines. RESULTS: The in vitro cytotoxicity was compound, dose, and cell line dependent. Whereas all of the compounds exerted some activity, FBA-TPQ was the most potent inducer of apoptosis and the most effective inhibitor of cell growth and proliferation, with half maximal inhibitory concentration values for most cell lines in the range of 0.097 to 2.297 mumol/L. In MCF-7 cells, FBA-TPQ exposure led to an increase in p53/p-p53, Bax, ATM/p-ATM, p-chk1 and p-chk2, and p-H2AX; and cleavage of poly(ADP)ribose polymerase, caspase-3, caspase-8, and caspase-9. It also decreased the levels of MDM2, E2F1, Bcl-2, chk1/2, and proteins associated with cell proliferation [cyclin-dependent kinase (Cdk)2, Cdk4, Cdk6, cyclin D1, etc.]. Moreover, FBA-TPQ inhibited the growth of breast cancer xenograft tumors in nude mice in a dose-dependent manner. Western blot analysis ofthe xenograft tumors indicated that similar changes in protein expression also occur in vivo. CONCLUSION: Our preclinical data indicate that FBA-TPQ is a potential therapeutic agent for breast cancer, providing a basis for the development of the compound as a novel anticancer agent.

A facile synthesis of bispyrroloquinone and bispyrroloiminoquinone ring system of marine alkaloids.

Bispyrroloquinone and bispyrroloiminoquinone are two important polycyclic ring systems present in biologically active marine alkaloids such as Zyzzyanones, tsitsikammamines and wakayin. A facile synthesis of these two ring systems starting from a 6-benzylamino indole-4,7-quinone or 6-benzylamino pyrroloiminoquinone is described here. This chemistry involves the construction of a pyrrole ring in a single step by treatment of the starting reagents with ethyl acetoacetate or phenylbutane-1,3-dione in the presence of ceric ammonium nitrate in MeOH/CH2Cl2 solvent.