Novel guanidine derivatives targeting leukemia as selective Src/Abl dual inhibitors: Design, synthesis and anti-proliferative activity.

A new series of benzene-sulfonamide derivatives 3a-i was designed and synthesized via the reaction of N-(pyrimidin-2-yl)cyanamides 1a-i with sulfamethazine sodium salt 2 as dual Src/Abl inhibitors. Spectral data IR, 1H-, 13C- NMR and elemental analyses were used to confirm the structures of all the newly synthesized compounds 3a-i and 4a-i. Crucially, we screened all the synthesized compounds 3a-i against NCI 60 cancer cell lines. Among all, compound 3b was the most potent, with IC50 of 0.018 µM for normoxia, and 0.001 µM for hypoxia, compared to staurosporine against HL-60 leukemia cell line. To verify the selectivity of this derivative, it was assessed against a panel of tyrosine kinase EGFR, VEGFR-2, B-raf, ERK, CK1, p38-MAPK, Src and Abl enzymes. Results revealed that compound 3b can effectively and selectively inhibit Src/Abl with IC500.25 µM and Abl inhibitory activity with IC500.08 µM, respectively, and was found to be more potent on these enzymes than other kinases that showed the following results: EGFR IC500.31 µM, VEGFR-2 IC500.68 µM, B-raf IC500.33 µM, ERK IC501.41 µM, CK1 IC500.29 µM and p38-MAPK IC500.38 µM. Moreover, cell cycle analysis and apoptosis performed to compound 3b against HL-60 suggesting its antiproliferative activity through Src/Abl inhibition. Finally, molecular docking studies and physicochemical properties prediction for compounds 3b, 3c, and 3 h were carried out to investigate their biological activities and clarify their bioavailability.

Design, Synthesis and Biological Evaluation of Novel Anticancer Amidinourea Analogues via Unexpected 1,3,5-Triazin-2-one Ring Opening.

Amidinoureas are an understudied class of molecules with unique structural properties and biological activities. A simple methodology has been developed for the synthesis of aliphatic substituted amidinoureas via unexpected cycle opening of benzothiazolo-1,3,5-triazine-2-ones and transamination reaction of N-(N-(benzo[d]thiazol-2-yl)carbamimidoyl)aniline-1-carboxamide in good yields. A novel series of amidinoureas derivatives was designed, synthesized, and evaluated for its antiproliferative activity on an aggressive metastatic melanoma A375 cell line model. This evaluation reveals antiproliferative activities in the low micromolar range and establishes a first structure-activity relationship. In addition, analogues selected for their structural diversity were assayed on a panel of cancer cell lines through the DTP-NCI60, on which they showed effectiveness on various cancer types, with promising activities on melanoma cells for two hit compounds. This work paves the way for further optimization of this family of compounds towards the development of potent antimelanoma agents.

A novel aryl-guanidinium derivative, VP79s, targets the signal transducer and activator of transcription 3 signaling pathway, downregulates myeloid cell leukaemia-1 and exhibits preclinical activity against multiple myeloma.

AIMS: We have recently described a novel guanidinium-based compound, VP79s, which induces cytotoxicity in various cancer cell lines. Here, we aim to investigate the activity of VP79s and associated mechanisms of action in multiple myeloma (MM) cells in vitro and ex vivo. MAIN METHODS: The effects of VP79s on cell viability and induction of apoptosis was examined in a panel of drug-sensitive and drug-resistant MM cell lines, as well as ex vivo patient samples and normal donor lymphocytes and platelets. Cell signaling pathways associated with the biological effects of VP79s were analysed by immunoblotting and flow cytometry. Gene expression changes were assessed by quantitative real-time PCR analysis. KEY FINDINGS: VP79s was found to rapidly inhibit both constitutively active and IL-6-induced STAT3 signaling with concurrent downregulation of the IL-6 receptors, CD130 and CD126. VP79s induced a rapid and dose-dependent downregulation of anti-apoptotic Bcl-2 family member, myeloid cell leukaemia-1 (MCL-1). VP79s enhanced bortezomib induced cell death and was also found to overcome bone marrow stromal cell induced drug resistance. VP79s exhibited activity in ex vivo patient samples at concentrations which had no effect on peripheral blood mononuclear cells, lymphocytes and platelets isolated from healthy donors. SIGNIFICANCE: As VP79s resulted in rapid inhibition of the key IL-6/STAT3 signaling pathway and downregulation of MCL-1 expression with subsequent selective anti-myeloma activity, VP79s may be a potential therapeutic agent with a novel mechanism of action in MM cells.

Absorbed dose simulation of meta-211At-astato-benzylguanidine using pharmacokinetics of 131I-MIBG and a novel dose conversion method, RAP.

OBJECTIVE: We aimed to estimate in vivo 211At-labeled meta-benzylguanidine (211At-MABG) absorbed doses by the two dose conversion methods, using 131I-MIBG biodistribution data from a previously reported neuroblastoma xenograft model. In addition, we examined the effects of different cell lines and time limitations using data from two other works. METHODS: We used the framework of the Monte Carlo method to create 3200 virtual experimental data sets of activity concentrations (kBq/g) to get the statistical information. Time activity concentration curves were produced using the fitting method of a genetic algorithm. The basic method was that absorbed doses of 211At-MABG were calculated based on the medical internal radiation dose formalism with the conversion of the physical half-life time of 131I to that of 211At. We have further improved the basic method; that is, a novel dose conversion method, RAP (Ratio of Pharmacokinetics), using percent injected dose/g. RESULTS: Virtual experiments showed that 211At-MABG and 131I-MIBG had similar properties of initial activity concentrations and biological components, but the basic method did not simulate the 211At-MABG dose. Simulated 211At-MABG doses from 131I-MIBG using the RAP method were in agreement with those from 211At-MABG, so that their boxes overlapped in the box plots. The RAP method showed applicability to the different cell lines, but it was difficult to predict long-term doses from short-term experimental data. CONCLUSIONS: The present RAP dose conversion method could estimate 211At-MABG absorbed doses from the pharmacokinetics of 131I-MIBG with some limitations. The RAP method would be applicable to a large number of subjects for targeted nuclide therapy.

α-ω Alkenyl-bis-S-Guanidine Thiourea Dihydrobromide Affects HeLa Cell Growth Hampering Tubulin Polymerization.

Cancer is going to be the first cause of mortality worldwide in the 21th century. It is considered a multifactorial disease that results from the combined influence of many genetic aberrations, leading to abnormal cell proliferation. As microtubules are strongly implicated in cellular growth, they represent an important target for cancer treatment. The well-known microtubule-targeting agents (MTAs) including paclitaxel, colchicine and vinca alkaloids are commonly used in the treatment of various cancers. However, adverse effects and drug resistance are major limitations in their clinical use. To find new candidates able to induce microtubule alteration with reduced toxic effects or drug resistance, we studied a small new series of derivatives that present imidazolinic, guanidinic, thioureidic and hydrazinic groups (1-9). All the compounds were tested for their antitumor activity against a panel of six tumoral cell models. In particular, compound 8 (nonane-1,9-diyl-bis-S-amidinothiourea dihydrobromide) showed the lowest IC50 value against HeLa cells, together with a low cytotoxicity for normal cells. This compound was able to induce the apoptotic mitochondrial pathway and inhibited tubulin polymerization with a similar efficacy to vinblastine and nocodazole. Taken together, these promising biological properties make compound 8 useful for the development of novel therapeutic approaches in cancer treatment.

Sulfonylguanidine Derivatives as Potential Antimelanoma Agents.

Sulfonylguanidines are interesting bioactive compounds with a broad range of applications in the treatment of different pathologies. 2-Aminobenzazole-based structures are well employed in the development of new anticancer drugs. Two series of novel N-benzazol-2-yl-N'-sulfonyl guanidine derivatives were synthesized with the sulfonylguanidine in either an extra- or intracyclic frame. They were evaluated for their antiproliferative activity against malignant melanoma tumor cells, thus allowing structure-activity relationships to be defined. Additionally, NCI-60 screening was performed for the best analogue to study its efficiency against a panel of other cancer cell lines. The stability profile of this promising compound was then validated. During the synthetic process, an unexpected new deamidination of the sulfonylguanidine towards sulfonamide function was also identified.

Observations on the Effects of Residualization and Dehalogenation on the Utility of N-Succinimidyl Ester Acylation Agents for Radioiodination of the Internalizing Antibody Trastuzumab.

Trastuzumab is an antibody used for the treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancers. Since trastuzumab is an internalizing antibody, two factors could play an important role in achieving high uptake and prolonged retention of radioactivity in HER2-positive tumors after radioiodination-residualizing capacity after receptor-mediated internalization and susceptibility to dehalogenation. To evaluate the contribution of these two factors, trastuzumab was radiolabeled using the residualizing reagent N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB) and the nonresidualizing reagent N-succinimidyl-3-[*I]iodobenzoate ([*I]SIB), both of which are highly dehalogenation-resistant. Paired-label uptake and intracellular retention of [125I]SGMIB-trastuzumab and [131I]SIB-trastuzumab was compared on HER2-expressing BT474 human breast carcinoma cells. Tumor uptake and normal tissue distribution characteristics for the two labeled conjugates were assessed in mice bearing BT474M1 xenografts. The internalization and intracellular retention of initially-bound radioactivity in BT474 cells was similar for the two labeled conjugates up to 4 h, but were significantly higher for [125I]SGMIB-trastuzumab at 6 and 24 h. Similarly, [*I]SGMIB labeling resulted in significantly higher uptake and retention of radioactivity in BT474M1 xenografts at all studied time points. Moreover, tumor-to-tissue ratios for [125I]SGMIB-trastuzumab were consistently higher than those for [131I]SIB-trastuzumab starting at 12 h postinjection. Thus, optimal targeting of HER2-positive breast cancers with a radioiodinated trastuzumab conjugate requires an acylation agent that imparts residualizing capacity in addition to high stability towards dehalogenation in vivo.

Self-assisted membrane-penetrating helical polypeptides mediate anti-inflammatory RNAi against myocardial ischemic reperfusion (IR) injury.

Anti-inflammatory RNA interference (RNAi) provides a promising paradigm for the treatment of myocardial ischemia reperfusion (IR) injury. To overcome the membrane barriers against intracardial siRNA delivery, various guanidinated helical polypeptides with potent and aromaticity-assisted membrane activities were herein developed and used for the delivery of siRNA against RAGE (siRAGE), a critical regulator of the pro-inflammatory cascade. Aromatic modification of the polypeptide led to notably enhanced trans-membrane siRNA delivery efficiencies, and more importantly, allowed more siRNA cargoes to get internalized via non-endocytosis, an effective pathway toward gene transfection. Subsequently, benzyl-modified polypeptide (P-Ben) was identified as the top-performing material with the highest RAGE silencing efficiency yet lowest cytotoxicity in H9C2 cells. Intracardial injection of the P-Ben/siRAGE polyplexes at 150 µg siRNA per kg led to remarkable RAGE knockdown by ∼85%, thereby attenuating the inflammatory cytokine release and reducing the cardiomyocyte apoptosis as well as myocardium fibrosis to recover the cardiac function after IR injury. This study therefore provides an effective strategy for the design of membrane-penetrating gene delivery materials, and may provide a promising addition to the anti-inflammatory treatment of myocardial IR injury.

Monanchoxymycalin C with anticancer properties, new analogue of crambescidin 800 from the marine sponge Monanchora pulchra.

A new pentacyclic guanidine alkaloid, monanchoxymycalin C (1) was isolated from a new collection of marine sponge Monanchora pulchra along with the known monanchoxymycalin A (2). The structure of 1 was elucidated on the basis of spectroscopic data. Monanchoxymycalin C exhibits cytotoxic activity against human cancer HeLa cells at low micromolar concentrations, induces apoptosis-related death of malignant cells and inhibits cancer cell colony formation. In addition, synergistic and additive effects have been observed in combination with cisplatin.

Investigating Cell Uptake of Guanidinium-Rich RAFT Polymers: Impact of Comonomer and Monomer Distribution.

A range of well-defined guanidinium-rich linear polymers with demonstrable efficiency for cellular internalization were developed. A protected guanidinium-functional acrylamide monomer (di-Boc-guanidinium ethyl acrylamide, GEAdiBoc) was synthesized and then polymerized via RAFT polymerization to yield well-defined homopolymers, which were then deprotected and functionalized with a fluorescein dye to observe and quantify their cellular uptake. The cellular uptake of these homopolymers was first compared to analogous polyarginines, which are commonly used in modern drug delivery. Following this, a range of well-defined guanidinium-rich copolymers were prepared in which the monomer distribution was varied using a convenient one-pot sequential RAFT polymerization approach. Systematic quantification of the cell uptake of these compounds, supported by fluorescent confocal microscopy data, revealed that while the overall hydrophobicity of the resulting copolymers has a direct impact on the amount of copolymer taken up by cells, the distribution of monomers has an influence on both the extent of uptake and the relative extent to which each route of internalization (endocytosis vs direct translocation) is exploited.

Novel Guanidine Compound against Multidrug-Resistant Cystic Fibrosis-Associated Bacterial Species.

Chronic pulmonary infection is a hallmark of lung disease in cystic fibrosis (CF). Infections dominated by non-fermentative Gram-negative bacilli are particularly difficult to treat and highlight an urgent need for the development of new class of agents to combat these infections. In this work, a small library comprising thiourea and guanidine derivatives with low molecular weight was designed; these derivatives were studied as antimicrobial agents against Gram-positive, Gram-negative, and a panel of drug-resistant clinical isolates recovered from patients with CF. One novel compound, a guanidine derivative bearing adamantane-1-carbonyl and 2-bromo-4,6-difluouro-phenyl substituents (H-BDF), showed potent bactericidal activity against the strains tested, at levels generally higher than those exhibited by tobramycin, ceftazimide and meropenem. The role that different substituents exert in the antimicrobial activity has been determined, highlighting the importance of the halo-phenyl group in the guanidine moiety. The new compound displays low levels of cytotoxicity against THP-1 and A549 cells with a selective index (SI) > 8 (patent application PCT/IB2017/054870, August 2017). Taken together, our results indicate that H-BDF can be considered as a promising antimicrobial agent.

Evidence for the involvement of TNF-α, IL-1ß and IL-10 in the antinociceptive and anti-inflammatory effects of indole-3-guanylhydrazone hydrochloride, an aromatic aminoguanidine, in rodents.

BACKGROUND: Indole-3-guanylhydrazone hydrochloride (LQM01) is a new derivative of aminoguanidine hydrochloride, an aromatic aminoguanidine. METHODS: Mice were treated with LQM01 (5, 10, 25 or 50 mg/kg, i.p.), vehicle (0.9% saline i.p.) or a standard drug. The mice were subjected to carrageenan-induced pleurisy, abdominal writhing induced by acetic acid, the formalin test and the hot-plate test. The model of non-inflammatory chronic muscle pain induced by saline acid was also used. Mice from the chronic protocol were assessed for withdrawal threshold, muscle strength and motor coordination. LQM01 or vehicle treated mice were evaluated for Fos protein. RESULTS: LQM01 inhibits TNF-α and IL-1ß production, as well as leukocyte recruitment during inflammation process. The level of IL-10 in LQM01-treated mice increased in pleural fluid. In addition, LQM01 decreased the nociceptive behavior in the acetic acid induced writhing test, the formalin test (both phases) and increased latency time on the hot-plate. LQM01 treatment also decreased mechanical hyperalgesia in mice with chronic muscle pain, with no changes in muscle strength and motor coordination. LQM01 reduced the number of Fos positive cells in the superficial dorsal horn. This compound exhibited antioxidant properties in in vitro assays. CONCLUSIONS: LQM01 has an outstanding anti-inflammatory and analgesic profile, probably mediated through a reduction in proinflammatory cytokines release, increase in IL-10 production and reduction in neuron activity in the dorsal horn of the spinal cord in mice. GENERAL SIGNIFICANCE: Beneficial effects of LQM01 suggest that it has some important clinical features and can play a role in the management of 'dysfunctional pain' and inflammatory diseases.

Crambescidin 800, Isolated from the Marine Sponge Monanchora viridis, Induces Cell Cycle Arrest and Apoptosis in Triple-Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is currently the only group of breast cancers without an effective targeted therapy. Marine sponges have historically been a source of compounds with anticancer activity. In this study, we screened extracts from twenty marine sponges collected off the coast of Western Australia for cytotoxic activity against TNBC cells. One very active extract derived from the sponge Monanchora viridis was selected for bioactivity-guided fractionation. Through multiple steps of purification, we isolated a potent cytotoxic compound, which was identified as crambescidin 800 (C800). We found that C800 exhibited cytotoxic potency in a panel of breast cancer cells, of which TNBC and luminal cancer cell models were the most sensitive. In addition, C800 induced cell cycle arrest at the G2/M phase, resulting in a decline in the expression of cyclin D1, CDK4, and CDK6 in TNBC cells. This effect was associated with the inhibition of phosphorylation of Akt, NF-κB, and MAPK pathways, resulting in apoptosis in TNBC cells.

Metformin selectively targets redox control of complex I energy transduction.

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD+ couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD+ couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled.

Permanent antimicrobial cotton fabrics obtained by surface treatment with modified guanidine.

Antimicrobial cotton fabrics received much attention for the demand of health and hygiene fields. In this work, an antimicrobial copolymer was prepared via a reaction between polyhexamethylene guanidine hydrochloride and polypropylene glycol diglycidyl ether. The copolymer has amphiphilic characteristic and excellent antimicrobial properties. When the copolymer was adhered onto cotton fabrics through physical adsorption and chemical bonding using dipping-drying method, the resultant cotton fabrics had excellent and durable antimicrobial properties. The antimicrobial rates against Escherichia coli and Staphylococcus aureus were higher than 99.99% when the adsorption amount of the copolymer was above 35.5mg/g. The antimicrobial cotton fabrics remained the excellent antimicrobial properties even after laundered with detergent solution.

Novel 2-(2-arylmethylthio-4-chloro-5-methylbenzenesulfonyl)-1-(1,3,5-triazin-2-ylamino)guanidine derivatives: Inhibition of human carbonic anhydrase cytosolic isozymes I and II and the transmembrane tumor-associated isozymes IX and XII, anticancer activity, and molecular modeling studies.

A series of novel 2-(2-arylmethylthio-4-chloro-5-methylbenzenesulfonyl)-1-(6-substituted-4-chloro-1,3,5-triazin-2-ylamino)guanidine derivatives 9-20 have been synthesized by substitution of chlorine atom at the 1,3,5-triazine ring in compounds 5-8 with 3- or 4-aminobenzenesulfonamide and 4-(aminomethyl)benzenesulfonamide hydrochloride. All the synthesized compounds were evaluated for their inhibitory activity toward hCA I, II, IX and XII as well as anticancer activity against HeLa, HCT-116 and MCF-7 human tumor cell lines. The investigated compounds showed weak inhibitory potency against the human CA I, while activity toward hCA II was differentiated and depended on structure of inhibitor (KI: 5.4-933.1 nM). Compounds containing the 4-sulfamoylphenyl moiety (9-12) exhibited the strongest inhibitory activity against hCA IX with KI values from 37.1 to 42.9 nM, as well as against hCA XII in range of 31-91.9 nM. The most promising compound 12 (KI = 41 nM) showed the highest selectivity toward hCA IX versus hCA I (hCA I/hCA IX = 18) and hCA II (hCA II/hCA IX = 4). Compound 12 displayed prominent cytotoxic effect selectively toward HeLa cancer cells (IC50 = 17 µM) and did not exhibit toxicity to the non-cancerous HaCaT cells. In silico analysis suggested that despite the lack of a single binding pose, the selective affinity is conferred by specific interactions with an arginine moiety, as well as better-defined binding modes within the active site.

A Systematic Structure-Activity Study of a New Type of Small Peptidic Transfection Vector Reveals the Importance of a Special Oxo-Anion-Binding Motif for Gene Delivery.

We discovered a new class of artificial peptidic transfection vectors based on an artificial anion-binding motif, the guanidiniocarbonylpyrrole (GCP) cation. This new type of vector is surprisingly smaller than traditional systems, and our previous work suggested that the GCP group was important for promoting critical endosomal escape. We now present here a systematic comparison of similar DNA ligands featuring our GCP oxo-anion-binding motif with DNA ligands only consisting of naturally occurring amino acids. Structure-activity studies showed that the artificial binding motif clearly outperformed natural amino acids such as histidine, lysine, and arginine. It improved the ability to shuttle foreign genetic material into cells, yet successfully mediated endosomal escape. Also, plasmids that were complexed by our artificial ligands were stabilized against cytosolic degradation to some extent. This resulted in the successful expression of plasmid information (comparable to gold standards such as polyethyleneimine). Hence, our study clearly demonstrates the importance of the tailor-made GCP anion-binding site for efficient gene transfection.

Novel 2-(2-alkylthiobenzenesulfonyl)-3-(phenylprop-2-ynylideneamino)guanidine derivatives as potent anticancer agents - Synthesis, molecular structure, QSAR studies and metabolic stability.

A series of new 2-(2-alkylthiobenzenesulfonyl)-3-(phenylprop-2-ynylideneamino)guanidine derivatives have been synthesized and evaluated in vitro by MTT assays for their antiproliferative activity against cell lines of colon cancer HCT-116, cervical cancer HeLa and breast cancer MCF-7. The obtained results indicated that these compounds display prominent cytotoxic effect. The best anticancer properties have been observed for derivatives 44 (IC50 = 6-18 µM) and 45 (IC50 = 8-14 µM). Very good results of antiproliferative assays have been also shown for compounds 26, 36, and 46 and noticeable anticancer profile has been found for set of derivatives 34-39. Based on results of MTT assays the structure-activity relationships have been drawn. More in-depth biological research revealed that compounds 26, 33, 37, 39, 41 and 43 display cytotoxic effect only against cancer cells and do not inhibit the growth of non-malignant HaCaT cells. Furthermore, the novel series of derivatives have shown good metabolic stability, especially among the pharmacologically active compounds. To obtain a deeper insight into the molecular description of compounds activity the QSAR studies have been applied. Support vector machines (SVM) have been used to developed QSAR models for predicting the anti-proliferative activity of novel derivatives. The obtained SVM models have shown prognostic ability for HCT-116 and HeLa cell lines and as a result these models may be useful for further development of structurally similar derivatives with better biological properties.

Synthesis and Biological Evaluation of Amidinourea and Triazine Congeners as Inhibitors of MDA-MB-231 Human Breast Cancer Cell Proliferation.

A series of novel amidinourea derivatives was synthesized, and the compounds were evaluated as inhibitors of MDA-MB-231 human breast cancer cell proliferation. In addition, a second series of triazine derivatives designed as rigid congeners of the amidinoureas was synthesized, and the compounds were evaluated for their antiproliferative activity. Among the two series, amidinourea 3 d (N-[N-[8-[[N-(morpholine-4-carbonyl)carbamimidoyl]amino]octyl]carbamimidoyl]morpholine-4-carboxamide) emerged as a potent anticancer hit compound with an IC50 value of 0.76 µm, similar to that of tamoxifen.

Marine guanidine alkaloids crambescidins inhibit tumor growth and activate intrinsic apoptotic signaling inducing tumor regression in a colorectal carcinoma zebrafish xenograft model.

The marine environment constitutes an extraordinary resource for the discovery of new therapeutic agents. In the present manuscript we studied the effect of 3 different sponge derived guanidine alkaloids, crambescidine-816, -830, and -800. We show that these compounds strongly inhibit tumor cell proliferation by down-regulating cyclin-dependent kinases 2/6 and cyclins D/A expression while up-regulating the cell cyclin-dependent kinase inhibitors -2A, -2D and -1A. We also show that these guanidine compounds disrupt tumor cell adhesion and cytoskeletal integrity promoting the activation of the intrinsic apoptotic signaling, resulting in loss of mitochondrial membrane potential and concomitant caspase-3 cleavage and activation. The crambescidin 816 anti-tumor effect was fnally assayed in a zebrafish xenotransplantation model confirming its potent antitumor activity against colorectal carcinoma in vivo.Considering these results crambescidins could represent promising natural anticancer agents and therapeutic tools.