Skip to main content
NCBI home page
JACS Au logoLink to JACS Au
. 2022 Oct 19;2(11):2561–2570. doi: 10.1021/jacsau.2c00435

[5]-Helistatins: Tubulin-Binding Helicenes with Antimitotic Activity

James L Rushworth †, Aditya R Thawani †, Elena Fajardo-Ruiz ‡, Joyce C M Meiring §, Constanze Heise ‡, Andrew J P White †, Anna Akhmanova §, Jochen R Brandt †, Oliver Thorn-Seshold ‡, Matthew J Fuchter †,*
PMCID: PMC9709948  PMID: 36465552

Abstract

graphic file with name au2c00435_0006.jpg

Helicenes are high interest synthetic targets with unique conjugated helical structures that have found important technological applications. Despite this interest, helicenes have had limited impact in chemical biology. Herein, we disclose a first-in-class antimitotic helicene, helistatin 1 (HA-1), where the helicene scaffold acts as a structural mimic of colchicine, a known antimitotic drug. The synthesis proceeds via sequential Pd-catalyzed coupling reactions and a π-Lewis acid cycloisomerization mediated by PtCl2. HA-1 was found to block microtubule polymerization in both cell-free and live cell assays. Not only does this demonstrate the feasibility of using helicenes as bioactive scaffolds against protein targets, but also suggests wider potential for the use of helicenes as isosteres of biaryls or cis-stilbenes—themselves common drug and natural product scaffolds. Overall, this study further supports future opportunities for helicenes for a range of chemical biological applications.

Keywords: cell cycle, DNA, helicene, mitosis, tubulin, antiproliferative, cancer

Introduction

Helicenes are polycyclic molecules consisting of ortho-fused aromatic or heteroaromatic rings that are angularly arranged to give rise to a helically shaped conformation.1 The synthetic challenge of assembling such helically chiral aromatics has generated much interest, with new synthesis strategies and methodologies regularly reported.2−13 Furthermore, the coupling of a fully conjugated framework to a helical structure has led to a wide variety of applications for helicenes, including asymmetric catalysis,14,15 molecular switches,16−18 dyes,19−22 circularly polarized light emitters,23,24 spintronics,25,26 and organic electronic devices,27−37 to name a few. Despite the increasing number of applications for the use of helicenes in synthesis and materials science, these scaffolds are understudied in the area of chemical biology. Given their extended conjugation, there are several examples where the photophysical properties of helicenes have been exploited to enable them to act as dyes for cell imaging or photodynamic activity.19,20,38−40 Alternatively, and in analogy to more conventional polyaromatic hydrocarbons (PAHs), the cellular cytotoxicity of helicenes has been investigated in a few examples.41−44 However, there are limited examples of the helicene scaffold interacting specifically and directly with therapeutically relevant biological targets.

The best examples of helicenes designed for medicinal chemistry aim to address DNA topologies, including selective binding of helicenes to B/Z-DNA45,46 and G-quadruplex DNA.40,41,47 It is worth noting, however, that despite simple analogies between the helical form of both DNA and the helicenes, there is a large mismatch in helix dimensions: The helical pitch of [n]-helicenes with n = 6–11 is 3.2148 vs 33.2 Å for B-DNA.49 As such, helicene recognition of DNA is likely based on conventional mechanisms of binding, such as intercalation, rather than more specific shape complementarity. Herein, we show, to the best of our knowledge, the first helicene specifically designed to interact with a non-DNA (protein) target, tubulin.

Microtubules (MTs) are intracellular protein scaffolds used by all eukaryotic cells to support mechanical processes, including cell motility, serving as tracks for cargo transport by motor proteins, and separating chromosomes in cell division.50 Structurally, MTs are giant noncovalent polymers of the α/β-tubulin ‘monomer’ protein, and in cells, they are continuously remodeled through regulated cycles of polymerization and depolymerization to fulfill their functions.51,52 Tubulin-binding drugs that disrupt MT de/polymerization rates therefore inhibit MT-dependent cellular processes: both MT stabilizers (taxanes, epothilones) and depolymerizers (colchicine, vinca alkaloids) are potent in vivo-active drugs.53 In cell culture, such compounds characteristically suppress MT polymerization dynamics and block cell proliferation.54,55

A myriad of MT inhibitors are known to bind at the so-called colchicine-domain of β-tubulin. The prototypical colchicine-domain inhibitor (CDI) pharmacophore, shared by colchicine and similar compounds such as stilbene combretastatins,56 is a 3,4,5-trimethoxyaryl “A” ring, held twisted near a mono-methoxyaryl “B” ring (Figure 1A). The B ring can tolerate small polar substituents (OH, F, NH2) on the outer edge, whereas substituents on the inner edge entirely block binding.57,58 “Bridge” rings and annulations to the B ring are well tolerated, although annulations at the A ring lower potency.59 Combretastatin A-4 (CA4), isolated from the African willow tree Combretum caffrum, has been found to possess particularly promising anti-tubulin activity.60CA4 binds to the colchicine-binding site of β-tubulin and has a higher level of bioactivity in its cis-form;61 however, CA4 is prone to configurational instability and can easily isomerize to the inactive trans isomer in the presence of light, heat, or protic media.62−64 While several conformationally restricted analogues of CA4 have been developed,65−70 we envisaged that the angular disposition of the A and B rings, coupled with the need to lock them in a cisoid configuration, would be well suited to a [5]-helicene.

Figure 1.

Figure 1

Open in a new tab

(A) Structures of colchicine and CA4. An SAR overview for CA4 is provided. (B) Syntheses of HA-1 and HA-2, both structural analogues of CA4, are reported in this work. HA-1 was designed as the active tubulin inhibitor and HA-2, where the ortho electron-donating −OH is located on the inner groove of the helicene, was designed to act as a negative control.

Herein, we report our first designs of helicene analogues of the combretastatins: the helistatins (Figure 1B). Our design (HA-1), partly informed by our synthetic strategy (vide infra), is based on an analogue of CA4, originally developed by Cushman and co-workers, whereby the nonessential meta-hydroxyl group in CA4 was removed.71 We coupled our active design with a suitable negative control (HA-2), with a substituent on the inner groove to reduce activity.72 Siles and co-workers showed that the presence of two electron-withdrawing groups (i.e., NO2) ortho to the methoxy diminished tubulin inhibition (IC50) from 1.2 μM (CA4) to 7.4 μM. This effect was exacerbated when the NO2 groups were converted to electron-donating anilines (IC50 > 40 μM).73 Based on this rationale, we added a closely matching electron-donating phenol to this position. We find that HA-1 blocks microtubule polymerization in both cell-free and live cell assays and, as such, represents the first example of a specific protein-targeting helicene.

Results and Discussion

To gauge the general suitability of [5]-helistatins for interaction with the colchicine-binding site of tubulin, we conducted computational docking experiments.74−76 The tubulin-colchicine stathmin-like domain complex (PDB code: 1SAO) was chosen as the model target (Figure 2). There is a good three-dimensional (3D) overlap between the energy-minimized structure of (P)-HA-1 and both a colchicine analogue (DAMA-colchicine, Figure 2A)77 and CA4 (Figure 2B). Overall, the docking studies show excellent complementarity for the colchicine-binding site; both polar and nonpolar interactions were retained. For completeness, we also modeled HA-1 against colchicine in the tubulin-colchicine complex (PDB code: 4O2B),78 and we found the same level of complementarity (Figure S1). The full three-dimensional fit of these molecules can be visualized in Movies S1–S4.

Figure 2.

Figure 2

Open in a new tab

Molecular docking of (P)-HA-1 against (A) DAMA-colchicine77 and (B) CA4 in the tubulin-colchicine stathmin-like domain complex (PDB code: 1SAO). There is excellent overlap in both polar and nonpolar interactions, and the helical pitch provides the correct conformation for binding. See Movies S1–S4 to visualize the full three-dimensional fit.

Despite the promise of this design, the [5]-helistatins proved to be extremely challenging to prepare with respect to many of the conventional methods used for helicene synthesis. The highly functionalized, electron-rich, and asymmetric nature of the terminal rings often prevented the formation of the desired product at, or close to, the final step of numerous attempted routes. For completeness, these unsuccessful synthesis strategies are outlined in the Supporting Information. Briefly, initial efforts were focused on formation of the helicene via oxidative photocyclization; however, the final photoisomerization step afforded a furan (Scheme S1) instead of the desired helicene.79 Various protecting group strategies were employed, although these only resulted in decomposition. Oxidative cyclization was then examined (Scheme S2), although this also led to the formation of decomposition products.80−82 We then employed radical methodology83 and successfully synthesized the target phenanthrene/stilbene intermediate (Scheme S3); however, attempts at performing the final ring closure resulted in either dehalogenation or decomposition. Finally, we turned our attention to cycloisomerization methodology, first via a [2 + 2 + 2] cycloisomerization (Scheme S4),84 and finally, a π-Lewis acid cycloisomerization mediated by PtCl2, which is the methodology described herein.

Through knowledge gained in our extensive survey of relevant chemistry, we ultimately managed to achieve the successful synthesis of [5]-helistatins HA-1 and HA-2 in a modular and semi-two directional fashion via common intermediate 5 (Scheme 1A). Initially, 1,2-dibromobenzene was subjected to a regiospecific ortho-lithiation protocol with the subsequent aryl lithium being trapped in situ with TMSCl to generate the bis-silylated species 2. The identity of 2 was confirmed by X-ray crystallography. The protocol developed for this reaction, a modification of earlier work by Serwatowski and Lulinski, does not generate any monosilylated product.85 Iodo-desilylation of 2 provided 3 in 63% yield. The subsequent Sonogashira coupling, however, proved capricious with significant batch-to-batch variations in product yield. These issues were traced to elemental sulfur contamination, confirmed via X-ray crystallography (Figure S2), stemming from the Na2S2O4 quench utilized in the iodo-desilylation step. It was noted that, at room temperature, a PhMe/DIPA solution of 3 would rapidly turn black when brought into contact with the Cu(I) utilized in the Sonogashira coupling. We presume that this is the result of CuxSy species being formed in situ, which in turn do not participate in the catalytic cycle. From this key observation, we devised a procedure to purify the precursor material using acid-activated Cu(0) turnings to sequester any elemental sulfur. This operationally simple purification ensured that the Sonogashira coupling was both robust and reproducible. As an aside, all steps up to this point in the synthesis do not require involved chromatographic purification; instead purification can be achieved primarily by re-crystallizations and/or silica plugs. With access to multigram quantities of 4, derivatization into the two helicene precursors 10 and 11 proceeded via sequential Suzuki–Miyaura cross-couplings. To obtain 11, the trifluoroborate salt 9 was utilized, featuring a cyclopropyl methyl ether (CME) protecting group (Scheme 1B,C, see Figure S5 for crystal structure). Our choice of this rather uncommon protecting group was guided by the need for it to be sufficiently stable to lithiation conditions while not interfering with the final Pt-mediated cyclization step and simultaneously to be easy to remove (vide infra). The bis-alkynes 10 and 11 are, thus, primed for a PtCl2-mediated cyclization to the desired helicenes (Scheme 1C). Pt(II) and Pt(IV) complexes have been used in the analogous syntheses of substituted phenanthrenes and helicenes through judicious use of similar alkyne handles.30,86 Thus, 10 was converted to a complex mixture from which only HA-1 could be obtained. It is likely that this mixture contained compounds that result from both incomplete ring closure and ring closure at an undesired position, forming structures such as azulenes87 and fluorene derivatives,88 and hence the relatively low yield. Similarly, PtCl2-mediated cyclization of 11 should, theoretically, enable access to HA-2 and its regioisomer 13; however, a complex mixture from which only HA-2 was obtained: regioisomer 13 could not be isolated. We propose that, due to steric interactions with the CME group, cyclization to yield 13 is sterically disfavored. Instead, cyclization at the less-hindered position is preferred and leads to 12. Deprotection of the CME group on 12 proceeded smoothly using 12 M HCl in MeOH/iPrOH and provided HA-2.

Scheme 1. (A) Reaction conditions: (a) LDA, TMSCl, and THF, −78–0 °C, 24 h; (b) 2, ICl and CH2Cl2, −10–0 °C, 18 h; (c) 3, TIPS-Acetylene, CuI (10 mol %), Pd(dppf)Cl2 (5 mol %), and PhMe/DIPA (2:1), 70 °C, 6 h; (d) 4, (3,4,5-trimethoxyphenyl)boronic acid, Pd(dppf)Cl2 (12 mol %), K2CO3 and 1,2-Dimethoxyethane/H2O (10:1), 85 °C, 18 h. (B) Reaction conditions: (a) 6, cyclopropyl methyl bromide, K2CO3, THF, 70 °C, 18 h; (b) 7, nBuLi, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, −78–0 °C, 24 h; (c) KF (10 M aq.) MeCN/MeOH (1:1), 21 °C, 1 min, then l-(+)-tartaric acid in THF, 2–5 min, then filter. (C) Reaction conditions: (a) 4-methoxyphenylboronic acid or 9, Pd(dppf)Cl2 (10 mol %), K2CO3 or Cs2CO3, and 1,2-dimethoxyethane/H2O (10:1), 85 °C, 18 h; (b) TBAF, THF, r.t., 2 h; (c) PtCl2 (40 mol %), PhMe, 80 °C, 18 h; (d) 12 M HCl, MeOH/iPrOH (1:2), 60 °C, 18 h.

Scheme 1

Open in a new tab

MT inhibitors exert antiproliferative activity by blocking cell division (mitosis). We evaluated the antiproliferative effects of HA-1 in a HeLa human cervical cancer cell line, assessing cell viability after 40 h. HA-1 was found to suppress ca. 40% of typical cell proliferation at low micromolar concentrations with a well-formed sigmoidal dose–response curve, featuring a Hill slope typical for tubulin-depolymerizing agents (Figure 3A). The plateauing response above 10–20 μM is typical for lipophilic compounds applied near their solubility limits. HA-2 was entirely inactive up to this concentration (Figure 3A). This observation supports our initial hypothesis that SAR trends for colchicine-derived inhibitors are translatable to [5]-helistatins, including that the addition of the −OH on the inner groove of HA-2 diminishes its activity, as predicted. To overcome the limitations with solubility, a prodrug approach akin to those used with CA4 might be appropriate. This could include conversion of the phenol to the phosphate salt,89 or functionalization with an aminohydroxy moiety.90

Figure 3.

Figure 3

Open in a new tab

Cellular bioactivity and mechanism of action of HA-1. (A) Antiproliferative activity (HeLa cells, 40 h incubation; one experiment representative of three independent experiments is shown). (B) Suppression of tubulin polymerization in cell-free conditions. Docetaxel is a positive control for polymerization enhancement, roughly indicating the magnitude of a meaningful difference from DMSO-only control behavior. HA-1 at 10 μM shows meaningful polymerization inhibition, while HA-2 up to 20 μM and colchicine or HA-1 at the substoichiometric concentration of 2 μM are all insignificantly inhibiting conditions (turbidimetric assay; greater absorbance indicates a greater degree of polymerization). (C, D) Live cell EB3-GFP “comets”, indicating growing MTs during HA-1/HA-2 treatments (HeLa cells). Cells were first imaged in media for baseline MT dynamics, then treated with 1% DMSO and imaged again, then 20 μM HA-1/HA-2 was applied, and the same cells were again imaged, which allows longitudinal comparisons over each treatment (10/12 cells acquired for HA-1/HA-2). (C) Each cell’s comet count was normalized to that in the first five frames of the no-cosolvent control before statistics (Wilcoxon) calculated for groups; mean ± SD with data points overlaid. (D) Stills from representative movies, Movies S5 and S6, showing that HA-1 suppresses polymerizing microtubule count, but HA-2 does not.

To test the mechanism of HA-1 bioactivity in a cell-free system, we examined its ability to suppress polymerization of purified tubulin protein. HA-1 was a strong polymerization suppressor at 10 μM (whereas HA-2 was entirely inactive up to 20 μM). This both matches the cellular activity data and confirms the ability of HA-1 to inhibit tubulin protein directly (Figure 3B).

Finally, we examined whether HA-1 maintains its MT-inhibitory activity in cells. We imaged live cells transfected with the fluorescently labeled MT end binding protein EB3-GFP, which marks the polymerizing plus ends of MTs as dynamic “comets”, by confocal microscopy.91 EB3 imaging sensitively and quantitatively reveals inhibition of typical MT dynamics by inhibitors.92 Cells were first imaged upon treatment with DMSO alone, to establish baseline MT dynamics; then HA-1 was applied at 20 μM, significantly suppressing the polymerization of MTs (to 60% of control; Figure 3C, D and Movie S5). Controls with HA-2 under the same conditions showed no inhibition, as expected (Figure 3C and Movie S6). These assays show that helicene HA-1 is an effective inhibitor of microtubule dynamics in live cells, and that its effect is not a result of the nonspecific properties of the helicene scaffold but is due to its specifically designed pharmacophore.

The metabolic fate of CA4 and its analogues are known to include quinone species, which retain strong inhibitory activity against tubulin polymerization.93 However, the experiments utilizing purified tubulin protein (Figure 3B), where oxidative metabolism is not possible, show that the observed bioactivity of HA-1 is inherently a feature of the helicene scaffold.

Given the intrinsic helical chirality of HA-1, we performed a chiral HPLC separation of (P)- and (M)-HA-1. (P)-HA-1 was found to have an enantiomerization half-life of 1.6 h at 37.5 °C (Figure S6). The judicious addition of methyl groups on the B ring of HA-1 might offer one way to reduce its conformational flexibility.94

Conclusions

In summary, we report the first helicene designed to bind to a defined and therapeutically relevant protein target, with bioactivity at cellularly relevant concentrations and mechanistically validated using a stringent live cell imaging assay. While there is significant room for improvement in the potency of HA-1, this molecule nonetheless acts as a valuable proof of concept example toward helicene-based bioactive agents. We anticipate that our study will motivate further use of helicene-type bioactive reagents for chemical and cell biology, especially where conformationally flexible biaryl/stilbenoid reagents are already known.95

Methods

Docking Methodology

Docking studies were carried out with Flare, version 5, Cresset, Litlington, Cambridgeshire, U.K..74−76 The crystal structure files (PDB: 1SAO and 4O2B) were downloaded directly from the protein data bank. Default protein preparation was carried out to set charge states and tautomers and to define the reference molecules (DAMA-colchicine and colchicine for 1SAO and 4O2B, respectively). The ligands CA4 and (P)-HA-1 were then imported and were subjected to energy minimization to ascertain the most suitable conformers. These energy-minimized structures were then aligned to the reference molecules via molecular field and shape-guided substructure alignment. The pdb file for each ligand is provided in the Supporting Information (Files S7–S10). The ligand coordinates (calculated with PyMOL) are as follows: HA-1 [17.273, 65.702, 42.681]; HA-2 [18.386, 64.438, 42.211]; colchicine [16.981, 65.997, 43.482]; and CA4 [16.956, 65.867, 42.621].

General Synthetic Methodology

All reagents and solvents were purchased from commercial sources and used as supplied unless otherwise indicated. All reactions were carried out under an inert atmosphere, using anhydrous solvents. All reactions were monitored by thin-layer chromatography (TLC) using Merck silica gel 60 F254 plates (0.25 mm). TLC plates were visualized using ultraviolet (UV) light (254 nm) and/or an appropriate TLC stain. Silica column chromatography was performed using Merck Silica Gel 60 (230–400 mesh) treated with a solvent system specified in the individual procedures. Solvents were removed by a rotary evaporator, and the compounds were further dried using high-vacuum pumps. Infrared spectra were recorded neat on an Agilent Cary 630 FTIR. Reported absorptions are in wavenumbers (cm–1). 1H and 13C NMR were recorded on a Bruker Advance 400 spectrometer at 400 and 100 MHz, respectively. Chemical shifts (δ) are quoted in ppm (parts per million) downfield from tetramethylsilane, referenced to residual solvent signals. The following abbreviations are used to designate multiplicity within 1H NMR analysis: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br. = broad signal. High-resolution mass spectra (ESI, APCI) were recorded by the Imperial College London Department of Chemistry Mass Spectroscopy Service using a Micromass Autospec Premier and Micromass LCT Premier spectrometer.

Full synthetic details are outlined in the Supporting Information.

General Procedure for the Final Helicene Forming Step

PtCl2 (1 equiv) was added to a solution of alkyne (2.5 equiv) in degassed PhMe (to give an alkyne concentration of 60 mM). The reaction was heated to 80 °C for 18 h. Subsequently, the reaction was concentrated in vacuo and purified directly via Si column chromatography with various mixtures of Et2O in hexane to afford the title compound.

Resazurin Antiproliferation Assay

HeLa human cervical cancer cells were sourced from ATCC and maintained under standard cell culture conditions in Dulbecco’s modified Eagle’s medium (DMEM; PAN-Biotech: P04-035550) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were grown and incubated at 37 °C in a 5% CO2 atmosphere. Cells were seeded in 96-well plates at 10,000 cells/well and left to adhere for 24 h, before treating with test compounds for 48 h (final well volume 100 μL, 1% DMSO; three technical replicates); the cosolvent control was treated with DMSO only. Cells were then treated with resazurin for 3 h to measure metabolic activity (through reduction to fluorescent resorufin) as a readout for live cell count.96 Fluorescence was measured at 590 nm (excitation 544 nm) using a FLUOstar Omega microplate reader (BMG Labtech). Absorbance data were normalized to viable cell count from the cosolvent control cells as 100% viability, where 0% viability was set to correspond to fluorescence signal from PBS with no cells, treated with resazurin (this underestimates true values corresponding to “no live cells” by ca. 5–15%, but does not affect assay outcomes and interpretation). Three independent experiments were performed. Viability data were plotted against the log of compound concentration (log10([drug]) (M)). One representative HeLa experiment out of three is shown as Figure 3A (one data point per technical replicate). HA-1 shows antiproliferative activity, while HA-2 does not.

Cell-Free Tubulin Polymerization Assay

Tubulin (99% purity) from the porcine brain was obtained from Cytoskeleton Inc. (cat. #T240). Polymerization was performed at 5 mg/mL tubulin, in polymerization buffer BRB80 (80 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) pH = 6.9; 0.5 mM EGTA; 2 mM MgCl2), in a cuvette (120 μL final volume, 1 cm path length) in a Agilent CaryScan 60 with a Peltier cell temperature control unit maintained at 37 °C, with glycerol (10 μL). Tubulin was first incubated for 5 min at 37 °C with the test compound in buffer with 3% DMSO, without GTP. Then GTP was added (1 μL spike, with mixing, final GTP concentration 1 mM) to initiate polymerization, and the change in absorbance at 340 nm due to scattering from the turbid medium was monitored (greater turbidity = more polymerization).

Cellular Microtubule Dynamics Imaging

HeLa cells were transfected with EB3-GFP using FuGENE 6 (Promega) according to the manufacturer’s instructions. Experiments were imaged on a Nikon Eclipse Ti microscope equipped with a perfect focus system (Nikon), a spinning disk-based confocal scanner unit (CSU-X1-A1, Yokogawa), an Evolve 512 EMCCD camera (Photometrics) attached to a 2.0× intermediate lens (Edmund Optics), a Roper Scientific custom-made set with 487 nm (150 mW) laser, ET-GFP filter (Chroma), a motorized stage MS-2000-XYZ, a stage top incubator INUBG2E-ZILCS (Tokai Hit), and lens heating calibrated for incubation at 37 °C with 5% CO2. Microscope image acquisition was controlled using MetaMorph 7.7, and images were acquired using a Plan Apo VC 40× NA 1.3 oil objective. Imaging conditions were initially confirmed to minimize GFP bleaching and phototoxicity in untreated cells. For compound-treated acquisitions, a compound diluted in prewarmed cell medium was applied to cells and incubated on cells for at least 1 min before commencing acquisition. Comet count analysis was performed in ImageJ using the ComDet plugin (Katrukha 2020, ComDet plugin for ImageJ, v0.5.3, Zenodo, doi: 10.5281/zenodo.4281064). Blinding was not performed as assay readout is unbiased (Fiji/ImageJ plugins). Data were analyzed using Prism 9 software (GraphPad).

Acknowledgments

A significant amount of underpinning synthetic chemistry was conducted on this project, and the authors would like to thank the following undergraduate chemistry students at Imperial College London for their efforts and contributions: Jingyi Yu, Fong Kai Lee, Xinjie Pan, Rachel Welsh, and Matthew Wootten. The authors would also like to thank Dr. Joshua Almond-Thynne for his input on the computational docking experiments. The authors thank J. Thorn-Seshold for data assembly and Maximilian Wranik and Zlata Boiarska (Paul Scherrer Institute) for investigating the crystallization of HA-1 with tubulin. The authors are grateful to Henrietta Lacks, now deceased, and to her surviving family members for their contributions to biomedical research.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.2c00435.

  • Computational simulation of DAMA-colchicine in the stathmin-like domain complex (PDB code: 1SAO),77 overlayed with HA-1, followed by CA4 (Movie S1) (MP4)

  • Computational simulation of DAMA-colchicine in the stathmin-like domain complex (PDB code: 1SAO),77 overlayed with HA-1, followed by CA4 (Movie S2) (MP4)

  • Computational simulation of colchicine in the tubulin-colchicine complex (PDB code: 4O2B),78 overlayed with HA-1, followed by CA4 (Movie S3) (MP4)

  • Computational simulation of colchicine in the tubulin-colchicine complex (PDB code: 4O2B),78 overlayed with HA-1, followed by CA4 (Movie S4) (MP4)

  • Inhibition of microtubule dynamics by HA-1 (data related to Figure 3); no-cosolvent, and 1% DMSO, control phases (baseline dynamics) precede the HA-2 phase (as indicated by captioning; 20 μM HA-1; EB3-GFP transiently transfected HeLa cells) (Movie S5) (MP4)

  • No inhibition of microtubule dynamics by HA-2 (data related to Figure 3); no-cosolvent, and 1% DMSO, control phases (baseline dynamics) the precede HA-2 phase (as indicated by captioning; 20 μM HA-2; EB3-GFP transiently transfected HeLa cells) (Movie S6) (MP4)

  • 4O2B PDB file with HA-1 bound as a ligand (File S7) (PDB)

  • 4O2B PDB file with HA-2 bound as a ligand (File S8) (PDB)

  • 4O2B PDB file with colchicine bound as a ligand (File S9) (PDB)

  • 4O2B PDB file with CA4 bound as a ligand (File S10) (PDB)

  • Crystal structure data (CIF)

  • Molecular docking studies; alternative synthetic routes considered; synthetic methodology and associated analytical data (including NMR and X-ray crystallography data); chiral HPLC purification data of HA-1; and enantiomerization kinetics; biological data including the resazurin antiproliferation assay; cell-free tubulin polymerization assay, and cellular microtubule dynamics imaging (PDF)

Author Present Address

∥ J.L.R. is presently employed at Sixfold Bioscience, Translation & Innovation Hub, 84 Wood Lane, London W12 0BZ, U.K

Author Present Address

⊥ E.F.-R. is presently employed at the Faculty of Biology, Ludwig-Maximilians University of Munich, Munich 81377, Germany.

Author Present Address

# C.H. is presently employed at Cambrium GmbH Max-Urich-Straße 3, Berlin 13355, Germany.

Author Present Address

□ J.R.B. is presently employed at Queen Mary University of London, Department of Chemistry, Mile End Road, London E1 4NS, United Kingdom

Author Contributions

∇ J.L.R. and A.R.T. contributed equally and are co-first authors. M.J.F. oversaw the project and supervised the synthetic investigations. O.T.-S. suggested biological targets, supervised biological experiments, and coordinated biological data assembly. A.A. supervised cellular microtubule dynamics imaging. J.L.R., A.R.T, J.R.B., and M.J.F. jointly devised the synthetic investigations. J.L.R., A.R.T., and J.R.B. performed the synthesis. A.R.T. performed the chiral purification and investigated the enantiomerization half-life. E.F.-R. performed cell viability, tubulin polymerization, and live cell microtubule dynamics assays. J.C.M.M. performed live cell microtubule dynamics assays and coordinated data assembly. C.H. performed cell viability assays and transfections. A.J.P. was responsible for X-ray crystallography studies. All authors contributed to writing the manuscript. CRediT: James Rushworth data curation, formal analysis, methodology, writing-original draft; Aditya Raymond Thawani data curation, formal analysis, methodology, writing-original draft; Elena Fajardo-Ruiz data curation, formal analysis, methodology, writing-review & editing; Joyce C.M. Meiring data curation, formal analysis, methodology, writing-review & editing; Constanze Heise data curation, formal analysis, methodology, writing-review & editing; Andrew J. P. White data curation, formal analysis, methodology, writing-review & editing; Anna Akhmanova supervision; Jochen R Brandt data curation, formal analysis, methodology, writing-review & editing; Oliver Thorn-Seshold data curation, formal analysis, funding acquisition, methodology, supervision, writing-review & editing; Matthew J Fuchter conceptualization, funding acquisition, methodology, project administration, resources, supervision, writing-review & editing.

Funding for this work was provided by the Commonwealth Scientific and Industrial Research Organization (CSIRO), Imperial College London, the EPSRC (EP/L014580/1, EP/R00188X/1), and the German Research Foundation (DFG: Emmy Noether grant no. 400324123 and SFB 1032 project B09 number 201269156 to O.T.S.). J.C.M.M. acknowledges support from an EMBO Long Term Fellowship (ALTF 261-2019). The authors also acknowledge the funding support from “Laboratory for Synthetic Chemistry and Chemical Biology” under the Health@InnoHK Program launched by Innovation and Technology Commission, The Government of Hong Kong Special Administrative Region of the People’s Republic of China.

The authors declare no competing financial interest.

Supplementary Material

au2c00435_si_001.mp4 (27.1MB, mp4)
au2c00435_si_002.mp4 (15.4MB, mp4)
au2c00435_si_003.mp4 (24.9MB, mp4)
au2c00435_si_004.mp4 (12MB, mp4)
au2c00435_si_005.mp4 (3.2MB, mp4)
au2c00435_si_006.mp4 (7.4MB, mp4)
au2c00435_si_007.pdb (5.3MB, pdb)
au2c00435_si_008.pdb (5.3MB, pdb)
au2c00435_si_009.pdb (5.3MB, pdb)
au2c00435_si_010.pdb (5.3MB, pdb)
au2c00435_si_011.cif (1.7MB, cif)
au2c00435_si_012.pdf (21.7MB, pdf)

References

  1. Shen Y.; Chen C. Helicenes: Synthesis and Applications. Chem. Rev. 2012, 112, 1463–1535. 10.1021/cr200087r. [DOI] [PubMed] [Google Scholar]
  2. Hartung T.; Machleid R.; Simon M.; Golz C.; Alcarazo M. Enantioselective Synthesis of 1,12-Disubstituted [4]Helicenes. Angew. Chem., Int. Ed. 2020, 59, 5660–5664. 10.1002/anie.201915870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dhawa U.; Tian C.; Wdowik T.; Oliveira J. C. A. A.; Hao J.; Ackermann L. Enantioselective Pallada-Electrocatalyzed C–H Activation by Transient Directing Groups: Expedient Access to Helicenes. Angew. Chem., Int. Ed. 2020, 59, 13451–13457. 10.1002/anie.202003826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Guo S.-M.; Huh S.; Coehlo M.; Shen L.; Pieters G.; Baudoin O. A C-H Activation-Based Enantioselective Synthesis of Lower Carbo[n]Helicenes (n = 4–6). ChemRxiv 2022, 10.26434/chemrxiv-2022-gvp7b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crassous J.; Stará I. G.; Starý I.. Helicenes - Synthesis, Properties, and Applications, 1st ed.; Crassous J.; Stará I. G.; Starý I. Eds.; Wiley: Weinheim, 2022. [Google Scholar]
  6. Redero P.; Hartung T.; Zhang J.; Nicholls L. D. M.; Zichen G.; Simon M.; Golz C.; Alcarazo M. Enantioselective Synthesis of 1-Aryl Benzo[5]Helicenes Using BINOL-Derived Cationic Phosphonites as Ancillary Ligands. Angew. Chem., Int. Ed. 2020, 59, 23527–23531. 10.1002/anie.202010021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Pelliccioli V.; Hartung T.; Simon M.; Golz C.; Licandro E.; Cauteruccio S.; Alcarazo M. Enantioselective Synthesis of Dithia[5]Helicenes and Their Postsynthetic Functionalization to Access Dithia[9]Helicenes. Angew. Chem., Int. Ed. 2022, 61, e202114577 10.1002/anie.202114577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. González-Fernández E.; Nicholls L. D. M.; Schaaf L. D.; Farès C.; Lehmann C. W.; Alcarazo M. Enantioselective Synthesis of [6]Carbohelicenes. J. Am. Chem. Soc. 2017, 139, 1428–1431. 10.1021/jacs.6b12443. [DOI] [PubMed] [Google Scholar]
  9. Wang Q.; Zhang W. W.; Zheng C.; Gu Q.; You S. L. Enantioselective Synthesis of Azoniahelicenes by Rh-Catalyzed C-H Annulation with Alkynes. J. Am. Chem. Soc. 2021, 143, 114–120. 10.1021/jacs.0c11735. [DOI] [PubMed] [Google Scholar]
  10. Li G.; Matsuno T.; Han Y.; Wu S.; Zou Y.; Jiang Q.; Isobe H.; Wu J. Fused Quinoidal Dithiophene-Based Helicenes: Synthesis by Intramolecular Radical–Radical Coupling Reactions and Dynamics of Interconversion of Enantiomers. Angew. Chem., Int. Ed. 2021, 60, 10326–10333. 10.1002/anie.202100606. [DOI] [PubMed] [Google Scholar]
  11. Reger D.; Haines P.; Amsharov K. Y.; Schmidt J. A.; Ullrich T.; Bönisch S.; Hampel F.; Görling A.; Nelson J.; Jelfs K. E.; Guldi D. M.; Jux N. A Family of Superhelicenes: Easily Tunable, Chiral Nanographenes by Merging Helicity with Planar π Systems. Angew. Chem., Int. Ed. 2021, 60, 18073–18081. 10.1002/anie.202103253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Stará I. G.; Starý I. Helically Chiral Aromatics: The Synthesis of Helicenes by [2 + 2 + 2] Cycloisomerization of π-Electron Systems. Acc. Chem. Res. 2020, 53, 144–158. 10.1021/acs.accounts.9b00364. [DOI] [PubMed] [Google Scholar]
  13. Full J.; Panchal S. P.; Götz J.; Krause A. M.; Nowak-Król A. Modular Synthesis of Organoboron Helically Chiral Compounds: Cutouts from Extended Helices. Angew. Chem., Int. Ed. 2021, 60, 4350–4357. 10.1002/anie.202014138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Demmer C. S.; Voituriez A.; Marinetti A. Catalytic Uses of Helicenes Displaying Phosphorus Functions. C. R. Chim. 2017, 20, 860–879. 10.1016/j.crci.2017.04.002. [DOI] [Google Scholar]
  15. Hasan M.; Borovkov V. Helicene-Based Chiral Auxiliaries and Chirogenesis. Symmetry 2018, 10, 10 10.3390/sym10010010. [DOI] [Google Scholar]
  16. Isla H.; Crassous J. Helicene-Based Chiroptical Switches. C. R. Chim. 2016, 19, 39–49. 10.1016/j.crci.2015.06.014. [DOI] [Google Scholar]
  17. Günther K.; Grabicki N.; Battistella B.; Grubert L.; Dumele O. An All-Organic Photochemical Magnetic Switch with Bistable Spin States. J. Am. Chem. Soc. 2022, 144, 8707–8716. 10.1021/jacs.2c02195. [DOI] [PubMed] [Google Scholar]
  18. Brandt J. R.; Pospíšil L.; Bednárová L.; da Costa R. C.; White A. J. P.; Mori T.; Teplý F.; Fuchter M. J. Intense Redox-Driven Chiroptical Switching with a 580 MV Hysteresis Actuated through Reversible Dimerization of an Azoniahelicene. Chem. Commun. 2017, 53, 9059–9062. 10.1039/C7CC04903J. [DOI] [PubMed] [Google Scholar]
  19. Babič A.; Pascal S.; Duwald R.; Moreau D.; Lacour J.; Allémann E. [4]Helicene–Squalene Fluorescent Nanoassemblies for Specific Targeting of Mitochondria in Live-Cell Imaging. Adv. Funct. Mater. 2017, 27, 1701839 10.1002/adfm.201701839. [DOI] [Google Scholar]
  20. Bauer C.; Duwald R.; Labrador G. M.; Pascal S.; Lorente P. M.; Bosson J.; Lacour J.; Rochaix J. D. Specific Labeling of Mitochondria of: Chlamydomonas with Cationic Helicene Fluorophores. Org. Biomol. Chem. 2018, 16, 919–923. 10.1039/c7ob02906c. [DOI] [PubMed] [Google Scholar]
  21. Li H.; Duwald R.; Pascal S.; Voci S.; Besnard C.; Bosson J.; Bouffier L.; Lacour J.; Sojic N. Near-Infrared Electrochemiluminescence in Water through Regioselective Sulfonation of Diaza [4] and [6]Helicene Dyes. Chem. Commun. 2020, 56, 9771–9774. 10.1039/D0CC04156D. [DOI] [PubMed] [Google Scholar]
  22. Saal F.; Zhang F.; Holzapfel M.; Stolte M.; Michail E.; Moos M.; Schmiedel A.; Krause A.-M.; Lambert C.; Würthner F.; Ravat P. [N]Helicene Diimides (n = 5, 6, and 7): Through-Bond versus Through-Space Conjugation. J. Am. Chem. Soc. 2020, 142, 21298–21303. 10.1021/jacs.0c11053. [DOI] [PubMed] [Google Scholar]
  23. Chen Y. Circularly Polarized Luminescence Based on Small Organic Fluorophores. Mater. Today Chem. 2022, 23, 100651 10.1016/j.mtchem.2021.100651. [DOI] [Google Scholar]
  24. Wade J.; Brandt J. R.; Reger D.; Zinna F.; Amsharov K. Y.; Jux N.; Andrews D. L.; Fuchter M. J. 500-Fold Amplification of Small Molecule Circularly Polarised Luminescence through Circularly Polarised FRET. Angew. Chem., Int. Ed. 2021, 60, 222–227. 10.1002/anie.202011745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kiran V.; Mathew S. P.; Cohen S. R.; Delgado I. H.; Lacour J.; Naaman R. Helicenes-A New Class of Organic Spin Filter. Adv. Mater. 2016, 28, 1957–1962. 10.1002/adma.201504725. [DOI] [PubMed] [Google Scholar]
  26. Kettner M.; Maslyuk V. V.; Nürenberg D.; Seibel J.; Gutierrez R.; Cuniberti G.; Ernst K.-H.; Zacharias H. Chirality-Dependent Electron Spin Filtering by Molecular Monolayers of Helicenes. J. Phys. Chem. Lett. 2018, 9, 2025–2030. 10.1021/acs.jpclett.8b00208. [DOI] [PubMed] [Google Scholar]
  27. Sahasithiwat S.; Mophuang T.; Menbangpung L.; Kamtonwong S.; Sooksimuang T. 3,12-Dimethoxy-7,8-Dicyano-[5]Helicene as a Novel Emissive Material for Organic Light-Emitting Diode. Synth. Met. 2010, 160, 1148–1152. 10.1016/j.synthmet.2010.02.039. [DOI] [Google Scholar]
  28. Yang Y.; da Costa R. C.; Fuchter M. J.; Campbell A. J. Circularly Polarized Light Detection by a Chiral Organic Semiconductor Transistor. Nat. Photonics 2013, 7, 634–638. 10.1038/nphoton.2013.176. [DOI] [Google Scholar]
  29. Dhbaibi K.; Abella L.; Meunier-Della-Gatta S.; Roisnel T.; Vanthuyne N.; Jamoussi B.; Pieters G.; Racine B.; Quesnel E.; Autschbach J.; Crassous J.; Favereau L. Achieving High Circularly Polarized Luminescence with Push–Pull Helicenic Systems: From Rationalized Design to Top-Emission CP-OLED Applications. Chem. Sci. 2021, 12, 5522–5533. 10.1039/D0SC06895K. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weimar M.; Da Costa R. C.; Lee F. H.; Fuchter M. J. A Scalable and Expedient Route to 1-Aza[6]Helicene Derivatives and Its Subsequent Application to a Chiral-Relay Asymmetric Strategy. Org. Lett. 2013, 15, 1706–1709. 10.1021/ol400493x. [DOI] [PubMed] [Google Scholar]
  31. Shi L.; Liu Z.; Dong G.; Duan L.; Qiu Y.; Jia J.; Guo W.; Zhao D.; Cui D.; Tao X. Synthesis, Structure, Properties, and Application of a Carbazole-Based Diaza[7]Helicene in a Deep-Blue-Emitting OLED. Chem. - Eur. J. 2012, 18, 8092–8099. 10.1002/chem.201200068. [DOI] [PubMed] [Google Scholar]
  32. Brandt J. R.; Wang X.; Yang Y.; Campbell A. J.; Fuchter M. J. Circularly Polarized Phosphorescent Electroluminescence with a High Dissymmetry Factor from PHOLEDs Based on a Platinahelicene. J. Am. Chem. Soc. 2016, 138, 9743–9746. 10.1021/jacs.6b02463. [DOI] [PubMed] [Google Scholar]
  33. Ward M. D.; Wade J.; Shi X.; Nelson J.; Campbell A. J.; Fuchter M. J. Highly Selective High-Speed Circularly Polarized Photodiodes Based on π-Conjugated Polymers. Adv. Opt. Mater. 2022, 10, 2101044 10.1002/adom.202101044. [DOI] [Google Scholar]
  34. Wan L.; Shi X.; Wade J.; Campbell A. J.; Fuchter M. J. Strongly Circularly Polarized Crystalline and B-Phase Emission from Poly(9,9-dioctylfluorene)-Based Deep-Blue Light-Emitting Diodes. Adv. Opt. Mater. 2021, 9, 2100066 10.1002/adom.202100066. [DOI] [Google Scholar]
  35. Wade J.; Hilfiker J. N.; Brandt J. R.; Liirò-Peluso L.; Wan L.; Shi X.; Salerno F.; Ryan S. T. J.; Schöche S.; Arteaga O.; Jávorfi T.; Siligardi G.; Wang C.; Amabilino D. B.; Beton P. H.; Campbell A. J.; Fuchter M. J. Natural Optical Activity as the Origin of the Large Chiroptical Properties in π-Conjugated Polymer Thin Films. Nat. Commun. 2020, 11, 6137 10.1038/s41467-020-19951-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yang Y.; Rice B.; Shi X.; Brandt J. R.; da Costa R. C.; Hedley G. J.; Smilgies D.-M.; Frost J. M.; Samuel I. D. W.; Otero-de-la-Roza A.; Johnson E. R.; Jelfs K. E.; Nelson J.; Campbell A. J.; Fuchter M. J. Emergent Properties of an Organic Semiconductor Driven by Its Molecular Chirality. ACS Nano 2017, 11, 8329–8338. 10.1021/acsnano.7b03540. [DOI] [PubMed] [Google Scholar]
  37. Josse P.; Favereau L.; Shen C.; Dabos-Seignon S.; Blanchard P.; Cabanetos C.; Crassous J. Enantiopure versus Racemic Naphthalimide End-Capped Helicenic Non-Fullerene Electron Acceptors: Impact on Organic Photovoltaics Performance. Chem. - Eur. J. 2017, 23, 6277–6281. 10.1002/chem.201701066. [DOI] [PubMed] [Google Scholar]
  38. Latterini L.; Galletti E.; Passeri R.; Barbafina A.; Urbanelli L.; Emiliani C.; Elisei F.; Fontana F.; Mele A.; Caronna T. Fluorescence Properties of Aza-Helicenium Derivatives for Cell Imaging. J. Photochem. Photobiol., A 2011, 222, 307–313. 10.1016/j.jphotochem.2011.06.013. [DOI] [Google Scholar]
  39. Li M.; Feng L. H.; Lu H. Y.; Wang S.; Chen C. F. Tetrahydro[5]Helicene-Based Nanoparticles for Structure-Dependent Cell Fluorescent Imaging. Adv. Funct. Mater. 2014, 24, 4405–4412. 10.1002/adfm.201400199. [DOI] [Google Scholar]
  40. Summers P. A.; Thomas A. P.; Kench T.; Vannier J.-B.; Kuimova M. K.; Vilar R. Cationic Helicenes as Selective G4 DNA Binders and Optical Probes for Cellular Imaging. Chem. Sci. 2021, 12, 14624–14634. 10.1039/d1sc04567a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shinohara K.-i.; Sannohe Y.; Kaieda S.; Tanaka K.; Osuga H.; Tahara H.; Xu Y.; Kawase T.; Bando T.; Sugiyama H. A Chiral Wedge Molecule Inhibits Telomerase Activity. J. Am. Chem. Soc. 2010, 132, 3778–3782. 10.1021/ja908897j. [DOI] [PubMed] [Google Scholar]
  42. Zhou Y.; Gan F.; Zhang Y.; He X.; Shen C.; Qiu H.; Liu P. Selective Killing of Cancer Cells by Nonplanar Aromatic Hydrocarbon-Induced DNA Damage. Adv. Sci. 2019, 6, 1901341 10.1002/advs.201901341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhao H.; Xu X.; Zhou L.; Hu Y.; Huang Y.; Narita A. Water-Soluble Nanoparticles with Twisted Double [7]Carbohelicene for Lysosome-Targeted Cancer Photodynamic Therapy. Small 2022, 18, 2105365 10.1002/smll.202105365. [DOI] [PubMed] [Google Scholar]
  44. He X.; Gan F.; Zhou Y.; Zhang Y.; Zhao P.; Zhao B.; Tang Q.; Ye L.; Bu J.; Mei J.; Du L.; Dai H.; Qiu H.; Liu P. Nonplanar Helicene Benzo[4]Helicenium for the Precise Treatment of Renal Cell Carcinoma. Small Methods 2021, 5, 2100770 10.1002/smtd.202100770. [DOI] [PubMed] [Google Scholar]
  45. Honzawa S.; Okubo H.; Anzai S.; Yamaguchi M.; et al. Chiral Recognition in the Binding of Helicenediamine to Double Strand DNA: Interactions between Low Molecular Weight Helical Compounds and a Helical Polymer. Bioorg. Med. Chem. 2002, 10, 3213–3218. 10.1016/S0968-0896(02)00175-X. [DOI] [PubMed] [Google Scholar]
  46. Tsuji G.; Kawakami K.; Sasaki S. Enantioselective Binding of Chiral 1,14-Dimethyl[5]Helicene-Spermine Ligands with B- and Z-DNA. Bioorg. Med. Chem. 2013, 21, 6063–6068. 10.1016/j.bmc.2013.07.022. [DOI] [PubMed] [Google Scholar]
  47. Lousen B.; Pedersen S. K.; Răsădean D. M.; Pantoş G. D.; Pittelkow M. Triggering G-Quadruplex Conformation Switching with [7]Helicenes. Chem. - Eur. J. 2021, 27, 6064–6069. 10.1002/chem.202004990. [DOI] [PubMed] [Google Scholar]
  48. Ravat P. Carbo[n]Helicenes Restricted to Enantiomerize: An Insight into the Design Process of Configurationally Stable Functional Chiral PAHs. Chem. - Eur. J. 2021, 27, 3957–3967. 10.1002/chem.202004488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Nekludova L.; Pabo C. O. Distinctive DNA Conformation with Enlarged Major Groove Is Found in Zn-Finger-DNA and Other Protein-DNA Complexes. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6948–6952. 10.1073/pnas.91.15.6948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Akhmanova A.; Steinmetz M. O. Tracking the Ends: A Dynamic Protein Network Controls the Fate of Microtubule Tips. Nat. Rev. Mol. Cell Biol. 2008, 9, 309–322. 10.1038/nrm2369. [DOI] [PubMed] [Google Scholar]
  51. Glotzer M. The 3Ms of Central Spindle Assembly: Microtubules, Motors and MAPs. Nat. Rev. Mol. Cell Biol. 2009, 10, 9–20. 10.1038/nrm2609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Kapitein L. C.; Hoogenraad C. C. Building the Neuronal Microtubule Cytoskeleton. Neuron 2015, 87, 492–506. 10.1016/j.neuron.2015.05.046. [DOI] [PubMed] [Google Scholar]
  53. Jordan M. A. Mechanism of Action of Antitumor Drugs That Interact with Microtubules and Tubulin. Curr. Med. Chem-Anti-Cancer Agents 2012, 2, 1–17. 10.2174/1568011023354290. [DOI] [PubMed] [Google Scholar]
  54. Steinmetz M. O.; Prota A. E. Microtubule-Targeting Agents: Strategies To Hijack the Cytoskeleton. Trends Cell Biol. 2018, 28, 776–792. 10.1016/j.tcb.2018.05.001. [DOI] [PubMed] [Google Scholar]
  55. Peterson J. R.; Mitchison T. J. Small Molecules, Big Impact. Chem. Biol. 2002, 9, 1275–1285. 10.1016/S1074-5521(02)00284-3. [DOI] [PubMed] [Google Scholar]
  56. Nguyen T. L.; McGrath C.; Hermone A. R.; Burnett J. C.; Zaharevitz D. W.; Day B. W.; Wipf P.; Hamel E.; Gussio R. A Common Pharmacophore for a Diverse Set of Colchicine Site Inhibitors Using a Structure-Based Approach. J. Med. Chem. 2005, 48, 7917–7917. 10.1021/jm058275i. [DOI] [PubMed] [Google Scholar]
  57. Gaspari R.; Prota A. E.; Bargsten K.; Cavalli A.; Steinmetz M. O. Structural Basis of Cis- and Trans-Combretastatin Binding to Tubulin. Chem 2017, 2, 102–113. 10.1016/j.chempr.2016.12.005. [DOI] [Google Scholar]
  58. Borowiak M.; Nahaboo W.; Reynders M.; Nekolla K.; Jalinot P.; Hasserodt J.; Rehberg M.; Delattre M.; Zahler S.; Vollmar A.; Trauner D.; Thorn-Seshold O. Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015, 162, 403–411. 10.1016/j.cell.2015.06.049. [DOI] [PubMed] [Google Scholar]
  59. Kraus Y.; Glas C.; Melzer B.; Gao L.; Heise C.; Preuße M.; Ahlfeld J.; Bracher F.; Thorn-Seshold O. Isoquinoline-Based Biaryls as a Robust Scaffold for Microtubule Inhibitors. Eur. J. Med. Chem. 2020, 186, 111865 10.1016/j.ejmech.2019.111865. [DOI] [PubMed] [Google Scholar]
  60. Ganina O. G.; Daras E.; Bourgarel-Rey V.; Peyrot V.; Andresyuk A. N.; Finet J. P.; Fedorov A. Y.; Beletskaya I. P.; Combes S. Synthesis and Biological Evaluation of Polymethoxylated 4-Heteroarylcoumarins as Tubulin Assembly Inhibitor. Bioorg. Med. Chem. 2008, 16, 8806–8812. 10.1016/j.bmc.2008.09.003. [DOI] [PubMed] [Google Scholar]
  61. Zhou P.; Liu Y.; Zhou L.; Zhu K.; Feng K.; Zhang H.; Liang Y.; Jiang H.; Luo C.; Liu M.; Wang Y. Potent Antitumor Activities and Structure Basis of the Chiral β - Lactam Bridged Analogue of Combretastatin A - 4 Binding to Tubulin. J. Med. Chem. 2016, 59, 10329–10334. 10.1021/acs.jmedchem.6b01268. [DOI] [PubMed] [Google Scholar]
  62. Tron G. C.; Pirali T.; Sorba G.; Pagliai F.; Busacca S.; Genazzani A. A. Medicinal Chemistry of Combretastatin A4: Present and Future Directions. J. Med. Chem. 2006, 49, 3033–3044. 10.1021/jm0512903. [DOI] [PubMed] [Google Scholar]
  63. Griggs J.; Metcalfe J. C.; Hesketh R. Targeting Tumour Vasculature: The Development of Combretastatin A4. Lancet Oncol. 2001, 2, 82–87. 10.1016/S1470-2045(00)00224-2. [DOI] [PubMed] [Google Scholar]
  64. Lu Y.; Chen J.; Xiao M.; Li W.; Miller D. D. An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site. Pharm. Res. 2012, 29, 2943–2971. 10.1007/s11095-012-0828-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Kaur R.; Kaur G.; Gill R. K.; Soni R.; Bariwal J. Recent Developments in Tubulin Polymerization Inhibitors: An Overview. Eur. J. Med. Chem. 2014, 87, 89–124. 10.1016/j.ejmech.2014.09.051. [DOI] [PubMed] [Google Scholar]
  66. Zheng S.; Zhong Q.; Mottamal M.; Zhang Q.; Zhang C.; Lemelle E.; McFerrin H.; Wang G. Design, Synthesis, and Biological Evaluation of Novel Pyridine-Bridged Analogues of Combretastatin-A4 as Anticancer Agents. J. Med. Chem. 2014, 57, 3369–3381. 10.1021/jm500002k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Chaudhary V.; Venghateri J. B.; Dhaked H. P. S.; Bhoyar A. S.; Guchhait S. K.; Panda D. Novel Combretastatin-2-Aminoimidazole Analogues as Potent Tubulin Assembly Inhibitors: Exploration of Unique Pharmacophoric Impact of Bridging Skeleton and Aryl Moiety. J. Med. Chem. 2016, 59, 3439–3451. 10.1021/acs.jmedchem.6b00101. [DOI] [PubMed] [Google Scholar]
  68. Romagnoli R.; Baraldi P. G.; Prencipe F.; Oliva P.; Baraldi S.; Tabrizi M. A.; Lopez-Cara L. C.; Ferla S.; Brancale A.; Hamel E.; Ronca R.; Bortolozzi R.; Mariotto E.; Basso G.; Viola G. Design and Synthesis of Potent in Vitro and in Vivo Anticancer Agents Based on 1-(3′,4′,5′-Trimethoxyphenyl)-2-Aryl-1H-Imidazole. Sci. Rep. 2016, 6, 26602 10.1038/srep26602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Madadi N. R.; Penthala N. R.; Howk K.; Ketkar A.; Eoff R. L.; Borrelli M. J.; Crooks P. A. Synthesis and Biological Evaluation of Novel 4,5-Disubstituted 2H-1,2,3-Triazoles as Cis-Constrained Analogues of Combretastatin A-4. Eur. J. Med. Chem. 2015, 103, 123–132. 10.1016/j.ejmech.2015.08.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wang Z.; Yang Q.; Bai Z.; Sun J.; Jiang X.; Song H.; Wu Y.; Zhang W. Synthesis and Biological Evaluation of 2,3-Diarylthiophene Analogues of Combretastatin A-4. MedChemComm 2015, 6, 971–976. 10.1039/c5md00028a. [DOI] [Google Scholar]
  71. Cushman M.; Nagarathnam D.; Gopal D.; He H. M.; Lin C. M.; Hamel E. Synthesis and Evaluation of Analogues of (Z)-1-(4-Methoxyphenyl)-2-(3,4,5-Trimethoxyphenyl)Ethene as Potential Cytotoxic and Antimitotic Agents. J. Med. Chem. 1992, 35, 2293–2306. 10.1021/jm00090a021. [DOI] [PubMed] [Google Scholar]
  72. Monk K. A.; Siles R.; Hadimani M. B.; Mugabe B. E.; Ackley J. F.; Studerus S. W.; Edvardsen K.; Trawick M. L.; Garner C. M.; Rhodes M. R.; Pettit G. R.; Pinney K. G. Design, Synthesis, and Biological Evaluation of Combretastatin Nitrogen-Containing Derivatives as Inhibitors of Tubulin Assembly and Vascular Disrupting Agents. Bioorg. Med. Chem. 2006, 14, 3231–3244. 10.1016/j.bmc.2005.12.033. [DOI] [PubMed] [Google Scholar]
  73. Siles R.; Ackley J. F.; Hadimani M. B.; Hall J. J.; Mugabe B. E.; Guddneppanavar R.; Monk K. A.; Chapuis J.-C.; Pettit G. R.; Chaplin D. J.; Edvardsen K.; Trawick M. L.; Garner C. M.; Pinney K. G. Combretastatin Dinitrogen-Substituted Stilbene Analogues as Tubulin-Binding and Vascular-Disrupting Agents. J. Nat. Prod. 2008, 71, 313–320. 10.1021/np070377j. [DOI] [PubMed] [Google Scholar]
  74. Cheeseright T.; Mackey M.; Rose S.; Vinter A. Molecular Field Extrema as Descriptors of Biological Activity: Definition and Validation. J. Chem. Inf. Model. 2006, 46, 665–676. 10.1021/ci050357s. [DOI] [PubMed] [Google Scholar]
  75. Bauer M. R.; Mackey M. D. Electrostatic Complementarity as a Fast and Effective Tool to Optimize Binding and Selectivity of Protein–Ligand Complexes. J. Med. Chem. 2019, 62, 3036–3050. 10.1021/acs.jmedchem.8b01925. [DOI] [PubMed] [Google Scholar]
  76. Kuhn M.; Firth-Clark S.; Tosco P.; Mey A. S. J. S.; Mackey M.; Michel J. Assessment of Binding Affinity via Alchemical Free-Energy Calculations. J. Chem. Inf. Model. 2020, 60, 3120–3130. 10.1021/acs.jcim.0c00165. [DOI] [PubMed] [Google Scholar]
  77. Ravelli R. B. G.; Gigant B.; Curmi P. A.; Jourdain I.; et al. Insight into Tubulin Regulation from a Complex with Colchicine and a Stathmin-like Domain. Nature 2004, 428, 198–202. 10.1038/nature02393. [DOI] [PubMed] [Google Scholar]
  78. Prota A. E.; Danel F.; Bachmann F.; Bargsten K.; Buey R. M.; Pohlmann J.; Reinelt S.; Lane H.; Steinmetz M. O. The Novel Microtubule-Destabilizing Drug BAL27862 Binds to the Colchicine Site of Tubulin with Distinct Effects on Microtubule Organization. J. Mol. Biol. 2014, 426, 1848–1860. 10.1016/j.jmb.2014.02.005. [DOI] [PubMed] [Google Scholar]
  79. Liu L.; Yang B.; Katz T. J.; Poindexter M. K. Improved Methodology for Photocyclization Reactions. J. Org. Chem. 1991, 56, 3769–3775. 10.1021/jo00012a005. [DOI] [Google Scholar]
  80. Kita Y.; Gyoten M.; Ohtsubo M.; Tohma H.; Takada T. Non-Phenolic Oxidative Coupling of Phenol Ether Derivatives Using Phenyliodine(III) Bis(Trifluoroacetate). Chem. Commun. 1996, 12, 1481 10.1039/cc9960001481. [DOI] [Google Scholar]
  81. Murase M.; Kotani E.; Okazaki K.; Tobinga S. Application of Iron(III) Complexes, Tris(2,2′-Bipyridyl)Iron(III) Perchlorate and Some Iron(III) Solvates, for Oxidative Aryl-Aryl Coupling Reactions. Chem. Pharm. Bull. 1986, 34, 3159–3165. 10.1248/cpb.34.3159. [DOI] [Google Scholar]
  82. Jin Z.; Wang Q.; Huang R. Intramolecular Biaryl Oxidative Coupling of Stilbenes by Vanadium Oxytrichloride (VOCl3): Facile Synthesis of Substituted Phenanthrene Derivatives. Synth. Commun. 2004, 34, 119–128. 10.1081/SCC-120027245. [DOI] [Google Scholar]
  83. Harrowven D. C.; Guy I. L.; Nanson L. Efficient Phenanthrene, Helicene, and Azahelicene Syntheses. Angew. Chem., Int. Ed. 2006, 45, 2242–2245. 10.1002/anie.200504287. [DOI] [PubMed] [Google Scholar]
  84. Songis O.; Míšek J.; Schmid M. B.; Kollárovič A.; Stará I. G.; Šaman D.; Císařová I.; Starý I. A Versatile Synthesis of Functionalized Pentahelicenes. J. Org. Chem. 2010, 75, 6889–6899. 10.1021/jo1013977. [DOI] [PubMed] [Google Scholar]
  85. Luliński S.; Serwatowski J. Regiospecific Metalation of Oligobromobenzenes. J. Org. Chem. 2003, 68, 5384–5387. 10.1021/jo0340511. [DOI] [PubMed] [Google Scholar]
  86. Mamane V.; Hannen P.; Fürstner A. Synthesis of Phenanthrenes and Polycyclic Heteroarenes by Transition-Metal Catalyzed Cycloisomerization Reactions. Chem. - Eur. J. 2004, 10, 4556–4575. 10.1002/chem.200400220. [DOI] [PubMed] [Google Scholar]
  87. Usui K.; Tanoue K.; Yamamoto K.; Shimizu T.; Suemune H. Synthesis of Substituted Azulenes via Pt(II)-Catalyzed Ring-Expanding Cycloisomerization. Org. Lett. 2014, 16, 4662–4665. 10.1021/ol502270q. [DOI] [PubMed] [Google Scholar]
  88. Fürstner A.; Mamane V. Flexible Synthesis of Phenanthrenes by a PtCl 2 -Catalyzed Cycloisomerization Reaction. J. Org. Chem. 2002, 67, 6264–6267. 10.1021/jo025962y. [DOI] [PubMed] [Google Scholar]
  89. Grisham R.; Ky B.; Tewari K. S.; Chaplin D. J.; Walker J. Clinical Trial Experience with CA4P Anticancer Therapy: Focus on Efficacy, Cardiovascular Adverse Events, and Hypertension Management. Gynecol. Oncol. Res. Pract. 2018, 5, 1 10.1186/s40661-017-0058-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. Hori K.; Saito S.; Nihei Y.; Suzuki M.; Sato Y. Antitumor Effects Due to Irreversible Stoppage of Tumor Tissue Blood Flow: Evaluation of a Novel Combretastatin A-4 Derivative, AC7700. Jpn. J. Cancer Res. 1999, 90, 1026–1038. 10.1111/j.1349-7006.1999.tb00851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Roostalu J.; Thomas C.; Cade N. I.; Kunzelmann S.; Taylor I. A.; Surrey T. The Speed of GTP Hydrolysis Determines GTP Cap Size and Controls Microtubule Stability. eLife 2020, 9, e51992 10.7554/eLife.51992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. Gao L.; Meiring J. C. M.; Kraus Y.; Wranik M.; Weinert T.; Pritzl S. D.; Bingham R.; Ntouliou E.; Jansen K. I.; Olieric N.; Standfuss J.; Kapitein L. C.; Lohmüller T.; Ahlfeld J.; Akhmanova A.; Steinmetz M. O.; Thorn-Seshold O. A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton. Cell Chem. Biol. 2021, 28, 228–241.e6. 10.1016/j.chembiol.2020.11.007. [DOI] [PubMed] [Google Scholar]
  93. Aprile S.; Grosso E. D.; Tron G. C.; Grosa G. In Vitro Metabolism Study of Combretastatin A-4 in Rat and Human Liver Microsomes. Drug Metab. Dispos. 2007, 35, 2252–2261. 10.1124/dmd.107.016998. [DOI] [PubMed] [Google Scholar]
  94. Rajan B.; Bedekar A. V. Effect of Methyl Substituent in the Fjord Region on the Conformational Stability of Aza[5]Helicenes. J. Mol. Struct. 2021, 1234, 130178 10.1016/j.molstruc.2021.130178. [DOI] [Google Scholar]
  95. Wranik M.; Weinert T.; Slavov C.; Masini T.; Furrer A.; Gaillard N.; Gioia D.; Ferrarotti M.; James D.; Glover H.; Carrillo M.; Kekilli D.; Stipp R.; Skopintsev P.; Brünle S.; Mühlethaler T.; Beale J.; Gashi D.; Nass K.; Ozerov D.; Johnson P. J. M.; Cirelli C.; Bacellar C.; Braun M.; Wang M.; Dworkowski F.; Milne C.; Cavalli A.; Wachtveitl J.; Steinmetz M. O.; Standfuss J. Molecular Snapshots of Drug Release from Tubulin over Eleven Orders of Magnitude in Time. bioRxiv 2022, 10.1101/2022.02.17.480857. [DOI] [Google Scholar]
  96. Gao L.; Meiring J. C. M.; Varady A.; Ruider I. E.; Heise C.; Wranik M.; Velasco C. D.; Taylor J. A.; Terni B.; Weinert T.; Standfuss J.; Cabernard C. C.; Llobet A.; Steinmetz M. O.; Bausch A. R.; Distel M.; Thorn-Seshold J.; Akhmanova A.; Thorn-Seshold O. In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M. J. Am. Chem. Soc. 2022, 144, 5614–5628. 10.1021/jacs.2c01020. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

au2c00435_si_001.mp4 (27.1MB, mp4)
au2c00435_si_002.mp4 (15.4MB, mp4)
au2c00435_si_003.mp4 (24.9MB, mp4)
au2c00435_si_004.mp4 (12MB, mp4)
au2c00435_si_005.mp4 (3.2MB, mp4)
au2c00435_si_006.mp4 (7.4MB, mp4)
au2c00435_si_007.pdb (5.3MB, pdb)
au2c00435_si_008.pdb (5.3MB, pdb)
au2c00435_si_009.pdb (5.3MB, pdb)
au2c00435_si_010.pdb (5.3MB, pdb)
au2c00435_si_011.cif (1.7MB, cif)
au2c00435_si_012.pdf (21.7MB, pdf)

Articles from JACS Au are provided here courtesy of American Chemical Society

RESOURCES