Mark Tarleton

Library synthesis and cytotoxicity of a family of 2-phenylacrylonitriles and discovery of an estrogen dependent breast cancer lead compound

In our efforts to prevent highly toxic compounds progressing through our anti-parasitic drug development program, we serendipitously discovered a family of 2-phenylacrylonitriles with excellent growth inhibition of a panel of ten human cancer cell lines. Focused library approaches facilitated the identification of a simple pharmacophore, comprising two terminal aromatic moieties linked via a conjugated cyano (acrylonitrile) moiety. Efforts that perturbed this pharmacophore resulted in a significant drop in growth inhibition. Multiple libraries led to the discovery of two key lead compounds. The first, (Z)-2-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)acrylonitrile (31) exhibits broad spectrum growth inhibition with GI50 values of 0.52–3 μM (HT29 and BE2-C cancer cell lines respectively; average = 1.6 μM). Of greater note is (Z)-2-(3,4-dichlorophenyl)-3-(4-nitrophenyl)acrylonitrile (28), a 0.127 ± 0.043 μM growth inhibitor of the estrogen receptor positive (ER+ve) human breast cancer cell line, MCF-7. Analogue 28 displays up to 543 fold selectivity towards MCF-7 cells compared with nine other non-breast derived cancer cell lines. Further screening of 28 against one human, ER−ve breast cancer cell line (MDA-MB231) and one normal non-tumourigenic breast epithelial cell line (MCF-10A) returned poor growth inhibition values of 34 ± 2 and 16 ± 4μM, demonstrating ca. ∼268 and∼126 fold preference for the MCF-7 estrogen dependent breast cancer cells.

Synthesis and anticancer activity of a series of norcantharidin analogues.

Cantharidin (1) and norcantharidin (2) display high levels of anticancer activity against a broad range of tumour cell lines. Synthetic manipulation of norcantharidin yields (3S,3aR,4S,7R,7aS)-3-hydroxyhexahydro-4,7-epoxyisobenzofuran-1(3H)-one (3), which also displays a high level of anticancer activity against tumour cells but interestingly, shows selectivity towards HT29 (colon; GI(50) = 14 μM) and SJ-G2 (glioblastoma; GI(50) = 15 μM) cell lines. Substitution at the hydroxyl group of the cyclic lactone within (3) produces a diasteromeric pair of products that have no difference in cytotoxicity over the cell lines tested. Incorporation of an isopropyl tail at this position (16) produced the most promising compound of this series to date, with strong selectivity towards HT29 (colon; GI(50) = 19 μM) and SJ-G2 (glioblastoma; GI(50) = 21 μM) cell lines but completely void of any activity against the remaining tumour cell lines (GI(50) > 100 μM), as per the parent molecule. We also discovered that the introduction of a terminal phosphate moiety (28) at the same position produced a different trend in cytotoxicity with strong activity in BE2-C (neuroblastoma; GI(50) = 9 μM) cells; suggestive of an alternate mode of action.

Cytotoxic 2-phenyacrylnitriles, the importance of the cyanide moiety and discovery of potent broad spectrum cytotoxic agents

We previously reported the discovery of a simple conjugated cyano pharmacophore which had led to the development of (Z)-2-(3,4-dichlorophenyl)-3-(4-nitrophenyl)acrylonitrile, as a selective inhibitor of oestrogen receptor positive (ER+ve) human breast cancer cell line, MCF-7. Further exploration though modification of the acrylonitrile and aromatic substituents has highlighted key structural components necessary for broad spectrum cytotoxicity. The acrylic acid derivates (Z)-2-(3,4-dichlorophenyl)-3-(4-nitrophenyl)acrylic acid and (Z)-2-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)acrylic acid (9) were inactive; confirming the importance of the cyanide moiety. The most potent 2-phenylacrylonitriles synthesized were (Z)-2-(3,4-dichlorophenyl)-3-(1H-indol-3-yl)acrylonitrile and (Z)-2-(3,4-dichlorophenyl)-3-(1H-indol-5-yl)acrylonitrile (20) with an average GI(50) values of 1.4 and 0.53 μM respectively. Five additional (Z)-2-(3,4-dichlorophenyl)-3-(indolyl)acrylonitriles also displayed average GI(50) values of ≤8.4 μM. In the case of indole, this represents a 32-fold increase in broad spectrum cytotoxicity relative to the lead.

Focused library development of 2-phenylacrylamides as broad spectrum cytotoxic agents.

With our lead compound (E)-3-(4-chlorophenyl)-2-(1H-pyrrole-2-carbonyl)acrylonitrile (1) inducing 50% growth inhibition of 11 cancer cell lines at 27-61 μM, potency enhancements were rapidly established through the synthesis of a series of focused compound libraries. Six highly focused libraries (46 compounds in total) were synthesised. Each library allowed the identification of a new lead compound, viz Library A identified (E)-3-(pentafluorophenyl)-2-(1H-pyrrole-2-carbonyl)acrylonitrile (11) and (E)-3-(1H-indol-3-yl)-2-(1H-pyrrole-2-carbonyl)acrylonitrile (13) as inhibitors with improved cytotoxicity. Synthesis of discrete libraries of amidoacrylamide analogues (Ar-CC(CN)-Ar✠Ar-CC(CN)-C(O)NH)-Ar) resulted in a series of analogues significantly more potent that the lead, 1. Three furan three analogues: (E)-3-(5-chlorofuran-2-yl)-2-cyano-N-(4-methoxybenzyl)acrylamide (33), (E)-3-(5-bromofuran-2-yl)-2-cyano-N-(4-methoxybenzyl)acrylamide (34) and (E)-2-cyano-3-(furan-3-yl)-N-(4-methoxybenzyl)acrylamide (37) returned broad spectrum growth inhibition (GI(50) values of 5-16 μM). Replacement of the furan moiety with simple aromatics gave an additional three analogues: (E)-2-cyano-N-(4-methoxybenzyl)-3-phenylacrylamide (39), (E)-3-(4-chlorophenyl)-2-cyano-N-(4-methoxybenzyl)acrylamide (41) and (E)-2-cyano-N-(4-methoxyphenyl)-3-(naphthalen-1-yl)acrylamide (45) with GI(50) values of 7-24 μM. The final library retained the aromatic substituents but introduced a 3,4-dichlorbenzylamine moiety to afford the 1-naphthyl substituted 52, which was the most potent broad spectrum cytotoxic analogue produced here in with an average GI(50)=8.6 μM. This represents a fivefold potency enhancement relative to 1 and a new cytotoxic scaffold suitable for further development.

Chemoselective flow hydrogenation approaches to isoindole-7-carboxylic acids and 7-oxa-bicyclio[2.2.1]heptanes

Two libraries of highly decorated norcantharidin analogues were accessed via a series of sequential chemoselective flow hydrogenations and solvent-free transformations. Utilising a 10% Pd/C catalyst, modifications to reaction parameters (H2 pressure, temperature and flow rate conditions) allowed facile access to effect selective direct reductive aminations and olefin reductions in the presence of furan, benzyl and nitrile moieties were established. The use of 20% Pd(OH)2/C; Pd tetrakis; 5% Pt/C (sulfided) gave mixtures of furan and olefin (both reduced) and olefin reduced products. RuO2; 0.5% Re/C and Re2O7 resulted in no reduction. Concurrent olefin and nitrile reduction was achieved in the presence of furan moieties by employing a RANEY® nickel catalyst. In total, 31 reaction conditions were examined using less than 200 mg of reagents allowing optimised conditions to be efficiently determined. These optimised hydrogenation conditions afforded desired analogues in near quantitative yields thus removing the requirements of reaction workup and chromatography.

Discovery of acrylonitrile-based small molecules active against Haemonchus contortus

We report the discovery of a series of acrylonitrile-containing molecules and α-amino amides which cause 99–100% lethality in H. contortus. Of the 22 acrylonitrile analogues investigated, the most active were 2-cyano-3-[1-(3-dimethylaminopropyl)-2-methyl-1H-indol-3-yl]-N-hexylacrylamide (13a), 2-cyano-3-[1(2-dimethylaminoethyl)-2-methyl-1H-indol-3-yl]-N-hexylacrylamide (13b), 2-cyano-3-{4-[3-(dimethylamino)propoxy]phenyl}-N-octylacrylamide (21), and 2-cyano-3-{1-[3-(dimethylamino)propyl]-1H-pyrrol-2-yl}-N-octylacrylamide (22) with each displaying LD50 values <15 μM whilst the α-amino amide methyl-2-[2-(2-benzoylphenylamino)-2-(4-methoxyphenyl)acetamido]acetate (12a) had an LD50 value of 10 μM. A cytotoxicity screen of the acrylonitrile analogues (13a, 13b, 21 and 22) against nine cancer cell lines indicated modest to high cytotoxicity. In contrast, the α-amino amide 12a displayed very low cytotoxicity, with a maximum of ∼30% cell death at 25 μM (A2780, an ovarian carcinoma derived cell line) and with a mean of 11% cell death across all cell lines evaluated. Thus, 12a is considered a promising lead candidate for the development of a new anthelmintic.

(Z)-2-(3,4-Dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile exhibits selective anti-tumour activity in breast cancer cell lines via the aryl hydrocarbon receptor pathway

We have previously reported the synthesis and breast cancer selectivity of (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile (ANI-7) in cancer cell lines. To further evaluate the selectivity of ANI-7, we have expanded upon the initial cell line panel to now include the breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468, BT20, MDA-MB-231); normal breast cells (MCF-10A); and cell lines derived from colon (HT29), ovarian (A2780), lung (H460), skin (A431), neuronal (BE2C), glial (U87, SJG2), and pancreatic (MIA) cancers. We now show that ANI-7 is up to 263-fold more potent at inhibiting the growth of breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468) than normal breast cells (MCF-10A) or cell lines derived from other tumor types. Measures of growth inhibition, cell cycle analysis, morphologic assessment, Western blotting, receptor binding, gene expression, small interfering RNA technology, reporter activity, and enzyme inhibition assays were exploited to define the mechanism of action of ANI-7. In this work, we report that ANI-7 mediates its effects via the activation of the aryl hydrocarbon receptor (AhR) pathway and the subsequent induction of CYP1-metabolizing mono-oxygenases. The metabolic conversion of ANI-7 induces DNA damage, checkpoint activation, S-phase cell cycle arrest, and cell death in sensitive breast cancer cell lines. Basal expression of AhR, the AhR nuclear translocator, and the CYP1 family members do not predict for sensitivity; however, inherent expression of the phase II–metabolizing enzyme sulfur transferase 1A1 does. For the first time, we identify (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile as a new AhR ligand.

Crystal Structures of (3R,3aR,4S,7R,7aS)-3-(Allyloxy)hexahydro- 4,7-epoxyisobenzofuran-1(3H)-one and (3S,3aR,4S,7R,7aS)-3-((E)- But-2-en-1-yloxy)hexahydro-4,7-epoxyisobenzofuran-1(3H)-one: Confirmation of NMR Predicted Stereocentre Geometry

Crystal structures of two isomeric norcantharidin derivatives (3R,3aR,4S,7R,7aS)-3-(allyloxy)hexahydro-4,7-epoxyisobenzofuran-1(3H)-one (7b), and (3S,3aR,4S,7R,7aS)-3-((E)-but-2-en-1-yloxy)hexahydro-4,7-epoxyisobenzofuran-1(3H)-one (8a) have been determined. In both instances the equivalent enantiomer was also obtained. The crystal structures of these compounds clarify the stereochemistry inferred only by NMR analysis before.Graphical AbstractTwo norcantharidin analogues have been separated into their two possible diastereomers and the structural identity of each compound has been confirmed from their corresponding crystal structures and NMR.

Abstract A127: A novel class of compounds that selectively target (ER +ve) breast cancer cells.

Estrogens play an important role in breast cancer development, with approximately 60% of premenopausal and 75% of postmenopausal breast cancer patients having estrogen-dependent carcinomas. In recent decades endocrine therapy has emerged for the treatment of estrogen receptor (ER) positive breast cancer, including aromatase inhibitors (anastrozole) and estrogen receptor antagonists (tamoxifen). However, drug resistance is often induced by these therapies, tumor selectivity is sometimes poor with non-tumor populations being affected, and target selectivity is also a problem with “off-target-effects” culminating in adverse toxicities and/or limited efficacy. For the past ten years collaboration between the Calvary Mater Newcastle Hospital and the University of Newcastle (Australia) has seen the development of many small molecule drugs for the treatment of malignancy. Our recent findings have discovered a family of 2-phenylacrylonitriles showing growth inhibition against a panel of human cancer derived cell lines. Focused library approaches facilitated the identification of a simple pharmacophore, comprising two terminal aromatic moieties linked via a conjugated cyano moiety. Multiple libraries led to the discovery of two key compounds, MT-CN-07 and MT-CN-18. Both exhibit broad spectrum growth inhibition with GI50 values of 3.2–46μM and 3.1–69μM, respectively, in a panel of nine human cancer cell lines derived from colon, ovarian, lung, skin, prostate and pancreatic carcinomas, neuroblastoma and glioblastoma1. An initial screen in one breast cancer ER+ve cell line (MCF-7) suggested that these compounds may selectively target estrogen dependent breast cancer cells. Indeed recent studies have shown that MT-CN-07 and MT-CN-18 are up to 645 fold more potent at inhibiting the growth of ER+ve breast cancer cells (ZR-75–1, T47D, MCF-7) with GI50 values of 0.08–0.6μM, and 0.107–2.25μM, respectively, when compared with the nine cell lines derived from other tumor types. Moreover, these compounds are up to 450 fold more potent in ER+ve breast cancer cell lines when compared with the estrogen independent cell line MDA-MB-231 (ER-ve, GI50 = 36μM and 26μM, respectively) and the non-tumorigenic breast epithelial cell line MCF10A (GI50 = 36μM and 37μM, respectively). Screening of these compounds in the drug resistant, etoposide-selected breast cancer cell line, MCF7/VP, which has amplification and over-expression of the ABCC1 gene, showed they were not substrates for this drug resistant mechanism. The ability to specifically target estrogen dependent tumors, while having little or no effect on normal breast cells, or other tumor types is a unique finding. Thus we have identified a novel group of molecules that could be exploited for the treatment of estrogen sensitive breast cancer. Reference: 1. Tarleton M, Gilbert J, Robertson MJ, McCluskey A, Sakoff JA. (2011) Library synthesis and cytotoxicity of a family of 2-phenylacrylonitriles and discovery of an estrogen dependent breast cancer lead compound. Med Chem Comm. 2, 31–37 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A127.