Next-generation of BBQ analogues that selectively target breast cancer.

We previously reported on the interaction of 10-chloro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (10-Cl-BBQ) with the Aryl hydrocarbon Receptor (AhR) and selective growth inhibition in breast cancer cell lines. We now report on a library of BBQ analogues with substituents on the phenyl and naphthyl rings for biological screening. Herein, we show that absence of the phenyl Cl of 10-Cl-BBQ to produce the simple BBQ molecule substantially enhanced the growth inhibitory effect with GI50 values of 0.001-2.1 µM in select breast cancer cell lines MCF-7, T47D, ZR-75-1, SKBR3, MDA-MB-468, BT20, BT474 cells, while having modest effects of 2.1-7 µM in other cell lines including HT29, U87, SJ-G2, A2780, DU145, BE2-C, MIA, MDA-MB-231 or normal breast cells, MCF10A (3.2 µM). The most potent growth inhibitory effect of BBQ was observed in the triple negative cell line, MDA-MB-468 with a GI50 value of 0.001 µM, presenting a 3,200-fold greater response than in the normal MCF10A breast cells. Additions of Cl, CH3, CN to the phenyl ring and ring expansion from benzoimidazole to dihydroquinazoline hindered the growth inhibitory potency of the BBQ analogues by blocking potential sites of CYP1 oxidative metabolism, while addition of Cl or NO2 to the naphthyl rings restored potency. In a cell-based reporter assay all analogues induced 1.2 to 10-fold AhR transcription activation. Gene expression analysis confirmed the induction of CYP1 oxygenases by BBQ. The CYP1 inhibitor α-naphthoflavone, and the SULT1A1 inhibitor quercetin significantly reduced the growth inhibitory effect of BBQ, confirming the importance of both phase I and II metabolic activation for growth inhibition. Conventional molecular modelling/docking revealed no significant differences between the binding poses of the most and least active analogues. More detailed DFT analysis at the DSD-PBEP86/Def-TZVPP level of theory could not identify significant geometric or electronic changes which would account for this varied AhR activation. Generation of Fukui functions at the same level of theory showed that CYP1 metabolism will primarily occur at the phenyl head group of the analogues, and substituents within this ring lead to lower cytotoxicity.

Novel piperazine-1,2,3-triazole leads for the potential treatment of pancreatic cancer.

From lead 1, (N-(4-((4-(3-(4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl)sulfonyl)-phenyl)acetamide), a S100A2-p53 protein-protein interaction inhibitor based on an in silico modelling driven hypothesis, four focused libraries were designed and synthesised. Growth inhibition screening was performed against 16 human cancer cell lines including the pancreatic cell lines MiaPaCa2, BxPC3, AsPC-1, Capan-2, HPAC, PANC-1 and the drug resistant CFPAC1. Modification of 1's phenylacetamide moiety, gave Library 1 with only modest pancreatic cancer activity. Modification of the 3-OCH3Ph moiety (Library 2) gave 4-CH3 (26), 4-CH2CH3 (27), 4-CF3 (31) and 4-NO2 (32) with sterically bulky groups more active. A 4-CF3 acetamide replacement enhanced cytotoxicity (Library 3). The 4-C(CH3)336 resulted in a predicted steric clash in the S100A2-p53 binding groove, with a potency decrease. Alkyl moieties afforded more potent analogues, 34 (4-CH3) and 35 (CH2CH3), a trend evident against pancreatic cancer: GI50 3.7 (35; BxPC-3) to 18 (40; AsPC-1) µM. Library 4 analogues with a 2-CF3 and 3-CF3 benzenesulfonamide moiety were less active than the corresponding Library 3 analogues. Two additional analogues were designed: 51 (4-CF3; 4-OCH3) and 52 (4-CF3; 2-OCH3) revealed 52 to be 10-20 fold more active than 51, against the pancreatic cancer cell lines examined with sub-micromolar GI50 values 0.43 (HPAC) to 0.61 µM (PANC-1). MOE calculated binding scores for each pose are also consistent with the observed biological activity with 52. The obtained SAR data is consistent with the proposed interaction within the S100A2-p53 bonding groove.

Wiskostatin and other carbazole scaffolds as off target inhibitors of dynamin I GTPase activity and endocytosis.

Wiskostatin (1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol) (1) is a carbazole-based compound reported as a specific and relatively potent inhibitor of the N-WASP actin remodelling complex (S-isomer EC50 = 4.35 µM; R-isomer EC50 = 3.44 µM). An NMR solution structure showed that wiskostatin interacts with a cleft in the regulatory GTPase binding domain of N-WASP. However, numerous studies have reported wiskostatin's actions on membrane transport and cytokinesis that are independent of the N-WASP-Arp2/3 complex pathway, but offer limited alternative explanation. The large GTPase, dynamin has established functional roles in these pathways. This study reveals that wiskostatin and its analogues, as well as other carbazole-based compounds, are inhibitors of helical dynamin GTPase activity and endocytosis. We characterise the effects of wiskostatin on in vitro dynamin GTPase activity, in-cell endocytosis, and determine the importance of wiskostatin functional groups on these activities through design and synthesis of libraries of wiskostatin analogues. We also examine whether other carbazole-based scaffolds frequently used in research or the clinic also modulate dynamin and endocytosis. Understanding off-targets for compounds used as research tools is important to be able to confidently interpret their action on biological systems, particularly when the target and off-targets affect overlapping mechanisms (e.g. cytokinesis and endocytosis). Herein we demonstrate that wiskostatin is a dynamin inhibitor (IC50 20.7 ± 1.2 µM) and a potent inhibitor of clathrin mediated endocytosis (IC50 = 6.9 ± 0.3 µM). Synthesis of wiskostatin analogues gave rise to 1-(9H-carbazol-9-yl)-3-((4-methylbenzyl)amino)propan-2-ol (35) and 1-(9H-carbazol-9-yl)-3-((4-chlorobenzyl)amino)propan-2-ol (43) as potent dynamin inhibitors (IC50 = 1.0 ± 0.2 µM), and (S)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8a) and (R)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8b) that are amongst the most potent inhibitors of clathrin mediated endocytosis yet reported (IC50 = 2.3 ± 3.3 and 2.1 ± 1.7 µM, respectively).

In Vitro Activity of Robenidine Analogues NCL259 and NCL265 against Gram-Negative Pathogens.

Multidrug-resistant (MDR) Gram-negative pathogens, especially Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Enterobacter spp., are recognized by the World Health Organization as the most critical priority pathogens in urgent need of drug development. In this study, the in vitro antimicrobial activity of robenidine analogues NCL259 and NCL265 was tested against key human and animal Gram-negative clinical isolates and reference strains. NCL259 and NCL265 demonstrated moderate antimicrobial activity against these Gram-negative priority pathogens with NCL265 consistently more active, achieving lower minimum inhibitory concentrations (MICs) in the range of 2−16 µg/mL. When used in combination with sub-inhibitory concentrations of polymyxin B to permeabilize the outer membrane, NCL259 and NCL265 elicited a synergistic or additive activity against the reference strains tested, reducing the MIC of NCL259 by 8- to 256- fold and the MIC of NCL265 by 4- to 256- fold. A small minority of Klebsiella spp. isolates (three) were resistant to both NCL259 and NCL265 with MICs > 256 µg/mL. This resistance was completely reversed in the presence of the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PAßN) to yield MIC values of 8−16 µg/mL and 2−4 µg/mL for NCL259 and NCL256, respectively. When NCL259 and NCL265 were tested against wild-type E. coli isolate BW 25113 and its isogenic multidrug efflux pump subunit AcrB deletion mutant (∆AcrB), the MIC of both compounds against the mutant ∆AcrB isolate was reduced 16-fold compared to the wild-type parent, indicating a significant role for the AcrAB-TolC efflux pump from Enterobacterales in imparting resistance to these robenidine analogues. In vitro cytotoxicity testing revealed that NCL259 and NCL265 had much higher levels of toxicity to a range of human cell lines compared to the parent robenidine, thus precluding their further development as novel antibiotics against Gram-negative pathogens.

Antigiardial Activity of Novel Guanidine Compounds.

From four focused compound libraries based on the known anticoccidial agent robenidine, 44 compounds total were synthesised and screened for antigiardial activity. All active compounds were counter-screened for antibiotic and cytotoxic action. Of the analogues examined, 21 displayed IC50 <5 µM, seven with IC50 <1.0 µM. Most active were 2,2'-bis{[4-(trifluoromethoxy)phenyl]methylene}carbonimidic dihydrazide hydrochloride (30), 2,2'-bis{[4-(trifluoromethylsulfanyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (32), and 2,2'-bis[(2-bromo-4,5-dimethoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (41) with IC50 =0.2 µM. The maximal observed activity was a 5 h IC50 value of 0.2 µM for 41. The clinically used metronidazole was inactive at this timepoint at a concentration of 25 µM. Robenidine off-target effects at bacteria and cell line toxicity were removed. Analogue 41 was well tolerated in mice treated orally (100 mg/kg). Following 5 h treatment with 41, no Giardia regrowth was noted after 48 h.

3,5-Bis(trifluoromethyl)phenylsulfonamides, a novel pancreatic cancer active lead. Investigation of the terminal aromatic moiety.

Virtual screening identified N-(6-((4-bromobenzyl)amino)hexyl)-3,5-bis(trifluoromethyl)benzenesulfonamide (1) a lead compound that bound to the S100A2-p53 binding groove. S100A2 is a Ca2+ binding protein with implications in cell signaling and is known to be upregulated in pancreatic cancer. It is a validated pancreatic cancer drug target. Lead 1, inhibited the growth of the MiaPaCa-2 pancreatic cancer cell line (GI50 = 2.97 µM). Focused compound libraries were developed to explore the SAR of this compound class with 4 libraries and 43 compounds total. Focused library (Library 1) development identified lipophillic sulfonamides as preferred for MiaPaCa-2 activity, with -CF3 and -C(CH3)3 substituents well tolerated (MiaPaCa-2 GI50 < 6 µM). Contraction of the hexylamino spacer to ethyl (Library 2) and propyl (Library 3) proved beneficial to activity against a broad spectrum panel of cancer cell lines: HT29 (lung), MCF-7 (breast), A2780 (ovarian), H460 (colon), A431 (skin), Du145 (prostate), BE2-C (neuroblastoma), U87 and SJ-G2 (glioblastoma) (cohort-1); and a pancreatic cancer cell line panel: MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, HPAC and PANC-1 (cohort-2). With a marked preference for a propyl linker the observed GI50 values ranged from 1.4 to 30 µM against cohort-1 and 1.4-30 µM against cohort-2 cell lines. In Library 4 the terminal aromatic moiety was explored with 4-substituted analogues preferred (with activity of 48 (4-Cl) > 47 (3-Cl) > 46 (2-Cl)) against the cell lines examined. The introduction of bulky aromatic moieties was well tolerated, e.g. dihydrobenzo[b][1,4]dioxine (51) returned cohort-2 GI50 values of 1.2-3.4 µM. In all instances the observed docked binding poses and binding scores were consistent with the observed cytotoxicity. This in turn supports, but does not prove, that these analogues function via S100A2-p53 binding groove inhibition.

Dynole 34-2 and Acrylo-Dyn 2-30, Novel Dynamin GTPase Chemical Biology Probes.

This protocol describes the chemical synthesis of the dynamin inhibitors Dynole 34-2 and Acrylo-Dyn 2-30, and their chemical scaffold matched partner inactive compounds. The chosen active and inactive paired compounds represent potent dynamin inhibitors and very closely related dynamin-inactive compounds, with the synthesis of three of the four compounds readily possible via a common intermediate. Combined with the assay data provided, this allows the interrogation of dynamin in vitro and potentially in vivo.

Synthesis of Phthaladyn-29 and Naphthalimide-10, GTP Site Directed Dynamin GTPase Inhibitors.

Herein we describe the detailed synthesis of the dynamin inhibitors Phthaladyn-29 and Napthaladyn-10, and their chemical scaffold matched partner inactive compounds. Combined with the assay data provided, this allows the interrogation of dynamin in vitro and potentially in vivo.

Pyrimidyn-Based Dynamin Inhibitors as Novel Cytotoxic Agents.

Five focused libraries of pyrimidine-based dynamin GTPase inhibitors, in total 69 compounds were synthesised, and their dynamin inhibition and broad-spectrum cytotoxicity examined. Dynamin plays a crucial role in mitosis, and as such inhibition of dynamin was expected to broadly correlate with the observed cytotoxicity. The pyrimidines synthesised ranged from mono-substituted to trisubstituted. The highest levels of dynamin inhibition were noted with di- and tri- substituted pyrimidines, especially those with pendent amino alkyl chains. Short chains and simple heterocyclic rings reduced dynamin activity. There were three levels of dynamin activity noted: 1-10, 10-25 and 25-60 µM. Screening of these compounds in a panel of cancer cell lines: SW480 (colon), HT29 (colon), SMA (spontaneous murine astrocytoma), MCF-7 (breast), BE2-C (glioblastoma), SJ-G2 (neuroblastoma), MIA (pancreas), A2780 (ovarian), A431 (skin), H460 (lung), U87 (glioblastoma) and DU145 (prostate) cell lines reveal a good correlation between the observed dynamin inhibition and the observed cytotoxicity. The most active analogues (31 a,b) developed returned average GI50 values of 1.0 and 0.78 µM across the twelve cell lines examined. These active analogues were: N2 -(3-dimethylaminopropyl)-N4 -dodecyl-6-methylpyrimidine-2,4-diamine (31 a) and N4 -(3-dimethylaminopropyl)-N2 -dodecyl-6-methylpyrimidine-2,4-diamine (31 b).

Amino alcohol acrylonitriles as broad spectrum and tumour selective cytotoxic agents.

We have identified specific dichlorophenylacrylonitriles as lead compounds in the development of novel anticancer compounds, notably, (Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide (1) and ANI-7 (2). Herein we specifically probe the SAR associated with the terminal aromatic ring and associated cytoxicity in a broad range of human cancer cell lines. Synthesis of three focused libraries revealed a poor tolerance for electron withdrawing and donating moieties (Library A). A clear preference for hydrophobic substituents on a terminal piperazine moiety (Library B) with good levels of broad spectrum cytotoxicity, e.g. 13a (GI50 2.5-6.0 µM), as did the introduction of a methylene spacer with 13i (4-CH3PhCH2; GI50 1.5-4.5 µM). Removal of the aromatic moiety and installation of simple hydrophobic groups (Library C), in particular an adamantyl moiety, afforded highly active broad spectrum cytotoxic agents with GI50 values ranging from 1.7 µM (14k; 1-adamantyl) to 5.6 µM (14i; pyrrolidine). Within these libraries we note lung cancer selectivity, relative to normal cells, of 13h (fluoro substituted acrylonitrile, GI50 1.6 µM, 9.3-fold selective); the colorectal selectivity of 14h (methylpiperidine analogue, GI50 0.36 µM, 6.9-fold selective) and the breast cancer selectivity of 13f (nitrile substituted acrylonitrile, GI50 2.3-6.0 µM, up to 20-fold selective). The latter was confirmed as a novel AhR ligand and a CYP1A1 activating compound, that likely induces cell death following bioactivation; a phenomenon previously described in breast cancer cell populations.