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1-Azahomocubane has been prepared 56 years after the parent hydrocarbon. Introduction of a nitrogen atom into this constrained polycyclic environment resulted in minimal changes to the framework geometry, with s-character of the nitrogen lone pair increasing due to strain.

A 2-azabicyclo[4.2.0]octa-4,7-diene derivative was unexpectedly isolated from the photochemical irradiation of 2-vinyl-1,2-dihydropyridine. A cascading 6π–8π–4π electrocyclic rearrangement has been proposed as a possible mechanistic pathway.

Synthesis of the 6-aza[1.0]triblattane skeleton and the unexpected construction of the 7-azatetracyclo[4.2.1.0

A series of 1-benzyloxy-5-phenyltetrazole derivatives and similar compounds were synthesized and evaluated for their in vitro inhibitory activity against androgen-receptor-dependent (22Rv1) and androgen-receptor independent (PC3) prostate cancer cells. The most active compounds had in vitro IC

Background Vitamin K antagonists (VKAs), such as warfarin, have remained the cornerstone of oral anticoagulation therapy in the prevention and treatment of thromboembolism for more than half a century. They function by impairing the biosynthesis of vitamin K-dependent (VKD) clotting factors through the inhibition of vitamin K epoxide reductase (VKOR). The challenge of VKAs therapy is their narrow therapeutic index and highly variable dosing requirements, which are partially the result of genetic variations of VKOR. Objectives The goal of this study was to search for an improved VKA that is tolerant to the genetic variations of its target enzyme. Methods A series of vitamin K derivatives with benzyl and related side-chain substitutions at the 3-position of 1,4-naphthoquinone were synthesized. The role of these compounds in VKD carboxylation was evaluated by mammalian cell-based assays and conventional in vitro activity assays. Results Our results showed that replacing the phytyl side-chain with a methylene cyclooctatetraene (COT) moiety at the 3-position of vitamin K1 converted it from a substrate to an inhibitor for VKD carboxylation. Strikingly, this COT-vitamin K derivative displayed a similar inhibition potency in warfarin-resistant VKOR mutations whose warfarin resistance varied more than 400-fold. Further characterization of COT-vitamin K for the inhibition of VKD carboxylation suggested that this compound targets multiple enzymes in the vitamin K redox cycle. Importantly, the anticoagulation effect of COT-vitamin K can be rescued with high doses of vitamin K1 . Conclusion We discovered a vitamin K analogue that functions as a VKA and is tolerant to genetic variations in the target enzyme.

An efficient and scalable enone-promoted method for the decarboxylation of trans-4-hydroxy-proline has been developed in flow to provide access to (R)-pyrrolidin-3-ol hydrochloride using biomass-derived isophorone. The advantages of deploying flow in this setting were reinforced by direct comparsion to batch. This method lays a foundation for fossil fuel and ammonia free alternative access to amines on scales relevant to manufacturing.

The replacement of one chemical motif with another that is broadly similar is a common method in medicinal chemistry to modulate the physical and biological properties of a molecule (i.e., bioisosterism). In recent years, bioisosteres such as cubane and bicyclo[1.1.1]pentane (BCP) have been used as highly effective phenyl mimics. Herein, we show the successful incorporation of a range of phenyl bioisosteres during the open-source optimization of an antimalarial series. Cubane (19) and closo-carborane (23) analogues exhibited improved in vitro potency against Plasmodium falciparum compared to the parent phenyl compound; however, these changes resulted in a reduction in metabolic stability; unusually, enzyme-mediated oxidation was found to take place on the cubane core. A BCP analogue (22) was found to be equipotent to its parent phenyl compound and showed significantly improved metabolic properties. While these results demonstrate the utility of these atypical bioisosteres when used in a medicinal chemistry program, the search to find a suitable bioisostere may well require the preparation of many candidates, in our case, 32 compounds.

We report a relay cross metathesis (ReXM) reaction for the construction of terpenoids in an iterative protocol. The protocol features the cross metathesis of a relay-actuated Δ6,7-functionalized C10-monoterpenoid alcohol with C10-monoterpenoid citral to form a C15-sesquiterpene. Subsequent functional group manipulation allows for the method to be repeated in an iterative fashion. The method is used for the synthesis of a diterpene-benzoate macrolide of biogenetic relevance to the bromophycolide family of natural products.

The cubane phenyl ring bioisostere paradigm was further explored in an extensive study covering a wide range of pharmaceutical and agrochemical templates, which included antibiotics (cefaclor, penicillin G) and antihistamine (diphenhydramine), a smooth muscle relaxant (alverine), an anaesthetic (ketamine), an agrochemical instecticide (triflumuron), an antiparasitic (benznidazole) and an anticancer agent (tamibarotene). This investigation highlights the scope and limitations of incorporating cubane into bioactive molecule discovery, both in terms of synthetic compatibility and physical property matching. Cubane maintained bioisosterism in the case of the Chagas disease antiparasitic benznidazole, although it was less active in the case of the anticancer agent (tamibarotenne). Application of the cyclooctatetraene (COT) (bio)motif complement was found to optimize benznidazole relative to the benzene parent, and augmented anticancer activity relative to the cubane analogue in the case of tamibarotene. Like all bioisosteres, scaffolds and biomotifs, however, there are limitations (e.g. synthetic implementation), and these have been specifically highlighted herein using failed examples. A summary of all templates prepared to date by our group that were biologically evaluated strongly supports the concept that cubane is a valuable tool in bioactive molecule discovery and COT is a viable complement.

D-Ring-seco-limonoids (tetranortriterpenoids), such as gedunin and xylogranin B display anti-cancer activity, acting via inhibition of Hsp90 and/or associated chaperon machinery (e.g., p23). Despite this, these natural products have received relatively little attention, both in terms of an enabling synthetic approach (which would allow access to derivatives), and as a consequence their structure-activity relationship (SAR). Disclosed herein is a generally applicable synthetic route to the BCD ring system of the seco-D-ring double bond containing limonoids. Furthermore, cell based assays revealed the first skeletal fragment that exhibited inhibition of the p23 enzyme at a level which was equipotent to that of gedunin, despite being much less structurally complex.

We thank the Shirley Boyde Trust for their generous support through a Shirley Boyde Foundation Grant to J.A.T. and acknowledge Institut Pasteur funding to M.H., A.H., P.R., and F.L.-A.. J.A.T. acknowledges The University of Hong Kong Outstanding Young Researcher Award 2015-16, The University of Hong Kong Outstanding Research Student Supervisor Award 2016-2017, and The University of Hong Kong Seed Fund for Basic Research grants 201711159185 and 201611159269. We thank the staff of the platform for production and purification of recombinant proteins of Institut Pasteur for technical assistance during the expression and purification of the proteins steps and the staff of the crystallography platform at Institut Pasteur for carrying out robot-driven crystallization screening. We acknowledge synchrotron SOLEIL (Saint-Aubin, France) for granting access to their facility and the staff of Proxima1 for helpful assistance during the data collection.; International audience; for the overall study design and data interpretation. Y.W.C. performed all cubamer selections, characterized the cubamers and oversaw all application experiments. A.W.C.W., Y.L., and A.B.K. were involved in aspects of cubamer characterization and in assay design. F.L.-A. expressed and purified the proteins and performed the biochemical characterization of the triphosphate. P.R. performed all chemical syntheses and was involved in aspects of experimental design of cubamer structural analysis. P.R. was also significantly involved in all data interpretation. G.P.S. was involved in steps of synthesis relating to cubanes and advised on study design.

A retrosynthetic disconnection-reconnection analysis of epoxypolyenes – substrates that can undergo cyclization to podocarpane-type tricycles – reveals relay-actuated 6,7-functionalized monoterpenoid alcohols for ruthenium benzylidene catalyzed olefin cross metathesis with homoprenyl benzenes. Successful implementation of this approach provided several epoxypolyenes as expected (E:Z, ca. 2-3:1). The method is further generalized for the cross metathesis of pre-existing trisubstituted olefins in other relay-actuated 6,7-functionalized monoterpenoid alcohols with various other trisubstituted alkenes to form new trisubstituted olefins. Epoxypolyene cyclization of an enantiomerically pure, but geometrically impure, epoxypolyene substrate provides an enantiomerically pure, trans-fused, podocarpane-type tricycle (from the E-geometrical isomer).

A retrosynthetic disconnection-reconnection analysis of epoxypolyenes – substrates that can undergo cyclization to podocarpane-type tricycles – reveals relay-actuated Δ6,7-functionalized monoterpenoid alcohols for ruthenium benzylidene catalyzed olefin cross metathesis with homoprenyl benzenes. Successful implementation of this approach provided several epoxypolyenes as expected (E:Z, ca. 2-3:1). The method is further generalized for the cross metathesis of pre-existing trisubstituted olefins in other relay-actuated Δ6,7-functionalized monoterpenoid alcohols with various other trisubstituted alkenes to form new trisubstituted olefins. Epoxypolyene cyclization of an enantiomerically pure, but geometrically impure, epoxypolyene substrate provides an enantiomerically pure, trans-fused, podocarpane-type tricycle (from the E-geometrical isomer).

Despite the difficulty in administering a safe dose regimen and reports of emerging resistance, warfarin (1) remains the most widely-used oral anticoagulant for the prevention and treatment of thrombosis in humans globally. Systematic substitution of the warfarin phenyl ring with either 1,3,5,7-cyclooctatetraene (COT) (2), cubane (3), cyclohexane (4) or cyclooctane (5) and subsequent evaluation against the target enzyme, vitamin K epoxide reductase (VKOR), facilitated interrogation of both steric and electronic properties of the phenyl pharmacophore. The tolerance of VKOR to further functional group modification (carboxylate 14, PTAD adduct 15) was also investigated. The results demonstrate the importance of both annulene conferred π-interactions and ring size in the activity of warfarin.

A series of prenyl-containing malonates are kinetically benchmarked against the standard allyl-containing congeners using a ruthenium benzylidene precatalyst for ring-closing metatheses. The prenyl grouping is found to be a superior acceptor olefin compared to an allyl group in RCM processes with ruthenium alkylidenes derived from terminal alkenes. The prenyl group is also found to be a highly competent acceptor for a ruthenium alkylidene derived from a 1,1-disubstituted olefin in a RCM process.

The widely occurring tetranortriterpene natural product gedunin has a well-established range of important biological properties. Although this molecule has received substantial semi-synthetic attention from medicinal chemists, it has not received devoted de novo synthetic attention since it was first isolated in 1960. A key feature of the approach presented herein was the decision to utilize a Robinson annulation reaction, which facilitated the installation of the challenging 7-acetoxy functional group and the completion of the fully functionalized ABC ring system.

The highly strained cubylmethyl radical undergoes one of the fastest radical rearrangements known (reported k = 2.9 × 1010 s-1 at 25 °C) through scission of two bonds of the cube. The rearrangement has previously been used as a mechanistic probe to detect radical-based pathways in enzyme-catalyzed C-H oxidations. This paper reports the discovery of highly selective cytochrome P450-catalyzed methylcubane oxidations which notionally proceed via cubylmethyl radical intermediates yet are remarkably free of rearrangement. The bacterial cytochrome P450 CYP101B1 from Novosphingobium aromaticivorans DSM 12444 is found to hydroxylate the methyl group of a range of methylcubane substrates containing a regio-directing carbonyl functionality at C-4. Unlike other reported P450-catalyzed methylcubane oxidations, the designed methylcubanes are hydroxylated with high efficiency and selectivity, giving cubylmethanols in yields of up to 93%. The lack of cubane core ring-opening implies that the cubylmethyl radicals formed during these CYP101B1-catalyzed hydroxylations must have very short lifetimes, of just a few picoseconds, which are too short for them to manifest the side reactivity characteristic of a fully equilibrated P450 intermediate. We propose that the apparent ultrafast radical rebound can be explained by a mechanism in which C-H abstraction and C-O bond formation are merged into a dynamically coupled process, effectively bypassing a discrete radical intermediate. Related dynamical phenomena can be proposed to predict how P450s may achieve various other modes of reactivity by controlling the formation and fate of radical intermediates. In principle, dynamical ideas and two-state reactivity are each individually able to explain apparent ultrashort radical lifetimes in P450 catalysis, but they are best considered together.

We report the design and implementation of a relay crossmetathesis (ReXM) reaction for the ascent of the terpenoids in an iterative protocol. The method features the reaction of naturally occurring terpenoid building blocks – with preexisting trisubstituted olefins – which combine to construct a new trisubstituted olefin resulting in a five-carbon unit homologation. Subsequent functional group manipulation allows for the method to be repeated in aniterative fashion. The method is used for the synthesis of a diterpenebenzoate macrolide of biogenetic relevance to the bromophycolide family of natural products.

The first enantioselective synthesis of (R)-2-cubylglycine, an analogue of (R)-2-phenylglycine in which the phenyl ring has been replaced by cubane, is disclosed. The key step was a telescoped Strecker reaction using (S)-2-amino-2-phenylethanol as a chiral auxiliary. Exploration of an alternative synthetic approach resulted in unprecedented cubane C–H insertion.

Cubane was recently validated as a phenyl ring (bio)isostere, but highly strained caged carbocyclic systems lack π character, which is often critical for mediating key biological interactions. This electronic property restriction associated with cubane has been addressed herein with cyclooctatetraene (COT), using known pharmaceutical and agrochemical compounds as templates. COT either out performed or matched cubane in multiple cases suggesting that versatile complementarity exists between the two systems for enhanced bioactive molecule discovery.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.

In this Communication, a La Trobe University affiliation is missing for the author David A. Winkler. The penultimate author address entry must read: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052 (Australia) and La Trobe Institute for Molecular Science, La Trobe University, Bundoora, 3084 (Australia).

An efficient nitro-Mannich type direct α-C(sp(3))-H functionalisation of N-aryl-1,2,3,4-tetrahydroisoquinolines catalysed by simple iron salts in combination with O2 as the terminal oxidant is described. The use of a Teflon AF-2400 membrane Tube-in-Tube reactor under continuous flow conditions allowed for considerable process intensification to be achieved relative to previous batch methods.

Functional polymer tubing with an OD of 1/16 or 1/8 in. was fabricated by a simple polymer coextrusion process. The tubing was made of an outer impervious polypropylene layer and an inner layer, consisting of a blend of a functional polymer, polyethylene-co-methacrylic acid, and a sacrificial polymer, polystyrene. After a simple solvent leaching step using common organic solvents, the polystyrene was removed, leaving behind a porous inner layer that contains functional carboxylic acid groups, which could then be used for the immobilization of target molecules. Solution-phase reactions using amines or isocyanates have proven successful for the immobilization of a series of small molecules and polymers. This flexible multilayered functional tubing can be easily cut to the desired length and connected via standard microfluidic fittings.

An improved solid-phase synthesis of the neuroprotective tripeptide, glycyl-prolyl-glutamic acid, bearing a hydrophobic tail to create an amphiphile is described. The new solid-phase synthesis was adapted to prepare several analogues bearing differing hydrophobic tails.

Amphiphilic compounds are used in a variety of applications due to their lyotropic liquid-crystalline phase formation, however only a limited number of compounds, in a potentially limitless field, are currently in use. A library of organic amphiphilic compounds was synthesised consisting of glucose, galactose, lactose, xylose and mannose head groups and double and triple-chain hydrophobic tails. A modular, high-throughput approach was developed, whereby head and tail components were conjugated using the copper-catalysed azide–alkyne cycloaddition (CuAAC) reaction. The tails were synthesised from two core alkyne-tethered intermediates, which were subsequently functionalised with hydrocarbon chains varying in length and degree of unsaturation and branching, while the five sugar head groups were selected with ranging substitution patterns and anomeric linkages. A library of 80 amphiphiles was subsequently produced, using a 24-vial array, with the majority formed in very good to excellent yields. A preliminary assessment of the liquid-crystalline phase behaviour is also presented.

The α9β1 and α4β1 integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α4β1 integrin have been thoroughly investigated with respect to HSC function, the role of α9β1 integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α9β1 integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α9β1 integrin with potent cross-reactivity against α4β1 integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α9β1 and α4β1 integrins, which showed faster binding to α4β1 integrin relative to α9β1, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α9β1 and α4β1 integrins; (2) investigating the biochemical properties of α9β1 and α4β1 integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.

A new, chromatography-free synthesis for the preparation of an experimental UV-B absorber is reported. A key step of the process is a one-pot partial reduction of a symmetrical imide with a sequential dehydration step. The synthesis uses several continuous-flow steps to increase sample throughput and was used to prepare sufficient material to support further testing activities in >99% purity.

A scalable process for the preparation of high purity dimethyl 1,4-cubanedicarboxylate (3) is reported. The work described herein builds on previous synthetic work from this and other laboratories, to provide a reliable process that can be used to prepare multigram quantities of 3 in a partially telescoped, 8 step process, with minimal purification of intermediates.

Silvestrol (1) and episilvestrol (2) are protein synthesis inhibitors, and the former has shown efficacy in multiple mouse models of cancer; however, the selectivity of these potent cytotoxic natural products has not been described. Herein, it is demonstrated that eukaryotic initiation factors eIF4AI/II were the only proteins detected to bind silvestrol (1) and biotinylated episilvestrol (9) by affinity purification. Our study demonstrates the remarkable selectivity of these promising chemotherapeutics.

The lyotropic phase behaviour of a library of sugar-based amphiphiles was investigated using high-throughput small-angle X-ray scattering (SAXS). Double unsaturated-chain monosaccharide amphiphiles formed inverse hexagonal and cubic micellar (Fd3m) lyotropic phases under excess water conditions. A galactose-oleyl amphiphile from the library was subsequently formulated into hexosome nanoparticles, which have potential uses as drug delivery vehicles. The nanoparticles were shown to be stable at elevated temperatures and non-cytotoxic up to at least 200μgmL-1.

A small library of amphiphilic compounds was synthesized in an array using the Huisgen 1,3-dipolar cycloaddition of terminal alkynes with azides (CuAAC or click reaction). The self-assembling properties of these compounds were evaluated by polarizing microscopy and synchrotron small-angle X-ray scattering analysis.

An aryl substrate with dual functionality consisting of a nitrile oxide and a pinacolyl boronate ester was prepared by mild hypervalent iodine oxidation (diacetoxyiodobenzene) of the corresponding aldoxime, without decomposition of the boronate functionality. The nitrile oxide was trapped in situ with a variety of dipolarophiles to yield aryl isoxazolines with the boronate ester function intact and available for subsequent reaction.

Cubane is a highly strained unsaturated molecule that was first synthesized in 1964 by Philip E. Eaton. Since cubane’s discovery, it has been researched in pharmaceuticals, explosives, and polymers. Due to its range of uses, we have explored the thermo-stability of a number of cubane derivatives. Some derivatives have revealed its propensity to undergo cage opening/rearrangement. In examining 1-iodocubane-4-carboxaldehyde, we observed that benzoic acid, benzaldehyde, benzyl alcohol, and benzyl benzoate were surprisingly formed in this thermo-decay.

Background: HIV-1 integrase is a clinically validated therapeutic target for the treatment of HIV-1 infection, with one approved therapeutic currently on the market. This enzyme represents an attractive target for the development of new inhibitors to HIV-1 that are effective against the current resistance mutations. Methods: A fragment-based screening method employing surface plasmon resonance and NMR was initially used to detect interactions between integrase and fragments. The binding sites of the fragments were elucidated by crystallography and the structural information used to design and synthesize improved ligands. Results: The location of binding of fragments to the catalytic core of integrase was found to be in a previously undescribed binding site, adjacent to the mobile loop. Enzyme assays confirmed that formation of enzyme–fragment complexes inhibits the catalytic activity of integrase and the structural data was utilized to further develop these fragments into more potent novel enzyme inhibitors. Conclusions: We have defined a new site in integrase as a valid region for the structure-based design of allosteric integrase inhibitors. Using a structure-based design process we have improved the activity of the initial fragments 45-fold.

The 1,3-dipolar cycloaddition reaction of nitrile oxides with carbon dipolarophiles is a versatile and powerful synthetic method to prepare isoxazolines. In particular nitrile oxide cycloaddition reactions with exocyclic methylene or alkylidene compounds generally proceed regioselectively leading to spiro heterocyclic compounds. This review deals with progress in the field of nitrile oxide cycloaddi- tion chemistry for the synthesis of spiro isoxazolines.

Since the initial synthesis of cubane, numerous derivatives have been made with a diverse range of physical, chemical, and biological properties. Some iodinated cubane derivatives have been reported to be thermolytically unstable and/or rearrange in situ. An iodinated cubane-containing, norbornene-based polymer showed rapid thermo-decomposition during TGA studies. Bis-(4-iodocubylmethyl)-dialkoxy disulfide undergoes fragmentation more easily than its non-iodinated counterpart. The synthesis and thermal behaviour of a library of iodinated cubane compounds are herein reported. Most of the iodinated cubane derivatives showed melting/decomposition with no exotherm upon cooling. 4-Iodo-1-vinylcubane was observed to rearrange to 4-vinyl-trans-β-iodostyrene and its cyclooctatetraene intermediate during DSC analysis. TGA studies on 1-iodo-4-(hydroxymethyl)-cubane suggest that this particular iodinated cubane scaffold is mostly prone to rapid thermo-decomposition.

The first reported cubane-based chiral Schiff base ligand has been successfully synthesized. This ligand has been evaluated on the nitro-aldol (Henry) reaction. The reactions were performed in the presence of four different copper salts, using eight different solvents, and five different temperatures. The highest enantioselectivity obtained for this novel ligand was ~39% ee.

Total synthesis of the anticancer 1,4-dioxane containing natural products silvestrol (1) and episilvestrol (2) is described by an approach based on the proposed biosynthesis of these novel compounds. The key steps included an oxidative rearrangement of the protected d-glucose derivative 11 to afford the 1,4-dioxane 12, which could be elaborated to the coupling partner 5 and a photochemical [3 + 2]-cycloadditon between the 3-hydroxyflavone 27 and methyl cinnamate followed by base-induced alpha-ketol rearrangement and reduction to give the cyclopentabenzofuran core 33. The core (-)-6 and 1,4-dioxane fragment 5 were united by a highly stereoselective Mitsunobu coupling with the modified azodicarboxylate DMEAD to afford the axial coupled product 36. Deprotection then gave episilvestrol (2). Silvestrol (1) was synthesized by a coupling between core (-)-6 and the dioxane 44 followed by deprotection. Compound 1 was also synthesized from episilvestrol (2) by a Mitsunobu inversion. In addition, the analogue 4'-desmethoxyepisilvestrol (46) was synthesized via the same route. It was found that 46 and episilvestrol 2 displayed an unexpected concentration-dependent chemical shift variation for the nonexchangeable dioxane protons. Synthetic compounds 1, 2, 38, 46, and 54 were tested against cancer cells lines, and it was found that the stereochemistry of the core was critical for activity. Synthetic analogue 4'-desmethoxyepisilvestrol (46) was also active against lung and colon cancer cell lines.