Ultrapotent Vinblastines in Which Added Molecular Complexity Further

Ultrapotent vinblastines in which added molecular INAUGURAL ARTICLE complexity further disrupts the target tubulin dimer–dimer interface

Daniel W. Carneya,b, John C. Lukesh IIIa,b, Daniel M. Brodya,b, Manuela M. Brütscha,b, and Dale L. Bogera,b,1

aDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; and bThe Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2014.

Contributed by Dale L. Boger, July 14, 2016 (sent for review June 1, 2016; reviewed by Erick Carreira and Mark Searcey) Approaches to improving the biological properties of natural alkenes through use of a wider range of free-radical traps (17, products typically strive to modify their structures to identify the 18) beyond O2 (air) and was explored specifically for the purpose of essential pharmacophore, or make functional group changes to providing the late-stage, divergent (19) preparation of vinblastine improve biological target affinity or functional activity, change analogs that bear alternative C20′ functionality at a site previously physical properties, enhance stability, or introduce conformational inaccessible for systematic exploration (Fig. 2) (17). In addition to constraints. Aside from accessible semisynthetic modifications of the alternative free-radical traps that were introduced, the broad existing functional groups, rarely does one consider using chemical alkene substrate scope was defined, the addition regioselectivity synthesis to add molecular complexity to the natural product. In was established, the outstanding functional group tolerance was part, this may be attributed to the added challenge intrinsic in the demonstrated, a range of Fe(III) salts and initiating hydride sources synthesis of an even more complex compound. Herein, we report were shown to support the reaction, its underlying free-radical re- synthetically derived, structurally more complex vinblastines in- action mechanism was refined, and mild reaction conditions (0–25 °C,

accessible from the natural product itself that are a stunning 100- CHEMISTRY 5–30 min, H O/cosolvent) were developed that are remarkably fold more active (IC values, 50–75 pM vs. 7 nM; HCT116), and that 2 50 forgiving of the reaction parameters (17, 18). are now accessible because of advances in the total synthesis of Although the vinblastine C20′ site and its hydroxyl substituent the natural product. The newly discovered ultrapotent vinblas- ′ tines, which may look highly unusual upon first inspection, bind were known to be important, the prior exploration of C20 substituent effects had been limited to a handful of alcohol ac- tubulin with much higher affinity and likely further disrupt the ′ tubulin head-to-tail α/β dimer–dimer interaction by virtue of the ylation reactions, the removal of the C20 hydroxyl group, and a strategic placement of an added conformationally well-defined, specialized set of superacid-mediated functionalizations (3). Our ′ rigid, and extended C20′ urea along the adjacent continuing pro- studies permitted systematic changes at C20 where we initially tein–protein interface. In this case, the added molecular complex- demonstrated that incorporation of a C20′ azide (5) or its re- ity was used to markedly enhance target binding and functional duced amine (6) provided compounds 100-fold less potent than biological activity (100-fold), and likely represents a general ap- vinblastine, but that the conversion of the amine 6 to a C20′ urea proach to improving the properties of other natural products tar- (7) provided a compound with cell growth inhibition activity geting a protein–protein interaction. Significance vinblastine | drug design | chemical synthesis | tubulin | protein–protein interaction Vinblastine is a clinical drug used in frontline combination therapies for treatment of cancer. It acts by inhibition of mi- inblastine and vincristine are superb clinical drugs success- tosis through binding tubulin and disrupting microtubule for- Vfully used in combination therapies for treatment of cancer mation. Because of advances in its total synthesis, we report (Fig. 1) (1–4). Vinblastine is used in frontline therapies for previously inaccessible and unusual modifications to vinblas- treatment of Hodgkin’s disease, testicular cancer, ovarian cancer, tine that improve potency a remarkable 100-fold. These ultra- breast cancer, head and neck cancer, and non-Hodgkin’slym- potent vinblastines display much higher tubulin binding affinities phoma, whereas vincristine is used in the curative treatment reg- and likely further disrupt the tubulin head-to-tail dimer–dimer imens for childhood lymphocytic leukemia and Hodgkin’s disease. interaction by strategic placement of an added rigid, extended Originally isolated from the leaves of Catharanthus roseus (L) group along the adjacent continuing protein–protein interface. G. Don (periwinkle) (5–8), vinblastine and vincristine were among Significantly, the ultrapotent vinblastines are accessible by chem- the initial small molecules shown to bind tubulin and to inhibit ical synthesis in three steps from commercially available materials microtubule formation and mitosis, defining an oncology drug (catharanthine, $16/g; vindoline, $36/g) based on newly in- target central to one of the most successful mechanisms of action troduced synthetic methodology and are inaccessible by nat- still pursued today (9). As a result, they continue to be extensively ural product derivatization, late-stage functionalization, or studied due to interest in their complex dimeric alkaloid struc- biosynthetic methods. tures, their role in the discovery of tubulin as an effective oncology – Author contributions: D.W.C., J.C.L., and D.L.B. designed research; D.W.C., J.C.L., D.M.B., drug target, and their clinical importance (10 13). and M.M.B. performed research; D.W.C., J.C.L., D.M.B., M.M.B., and D.L.B. analyzed data; In the development of a total synthesis of vinblastine and and D.W.C. and D.L.B. wrote the paper. vincristine, we introduced an Fe(III)/NaBH4-mediated free- Reviewers: E.C., Eidgenössische Technische Hochschule; and M.S., University of East radical oxidation of the anhydrovinblastine trisubstituted alkene Anglia. for penultimate installation of the C20′ tertiary alcohol found in The authors declare no conflict of interest. the natural products (14–16). This now-powerful hydrogen atom 1To whom correspondence should be addressed. Email: [email protected]. transfer (HAT)-initiated free-radical reaction was subsequently This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. developed to provide a general method for functionalization of 1073/pnas.1611405113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1611405113 PNAS | August 30, 2016 | vol. 113 | no. 35 | 9691–9698 Downloaded by guest on September 25, 2021 of compounds modified at C20′ that are now a stunning 100- fold more potent than vinblastine and that may initially look unusual in their structure. We also show that this increase in potency correlates directly with enhanced target tubulin binding affinity. Significantly, the remarkable potency of the compounds (IC50 values as low as 50–75 pM) suggest that it is not likely, or even possible, that their cellular functional activity is derived from stoichiometric occupancy of the relatively large number of intracellular tubulin binding sites, but rather impli- cates effective substoichiometric or catalytic occupancy of candidate binding sites sufficient to disrupt tubulin dynamics or assembly during mitosis. We suggest that the newly added linear and rigid C20′ urea substituents on vinblastine extend into and create a narrow channel adjacent to the vinblastine binding site, bind across this adjacent region of the protein–protein interaction defined by the tubulin dimer–dimer interface, further disrupt the tubulin α/β head-to-tail dimer–dimer interaction, and extend across the full protein–protein interface contacting solvent on the distal side of the binding site. This further disruption of the target protein–protein interaction by an added structural element to an already complex natural product (added molecular complexity) represents a unique but rational approach to substantially improve the functional properties of such molecules and likely represents a general approach to improving the properties of other natural products that target protein–protein interactions. Results Synthesis and Activity of Vinblastine C20′ Ureas. Two C20′ ureas 8 and 9 were prepared in our earlier studies that served as our starting points. The first of these is the pyrrolidine urea 8, which exhibited improved cell growth inhibition relative to vinblastine. Thus, a series of functionalized or substituted pyrrolidines were incorporated into the C20′ urea through reaction of 20′-amino- vinblastine (6), derived from reduction of the hydroazidation product 5, with either p-nitrophenyl chloroformate (1.5 equiv 4-NO2PhOCOCl, 10 equiv Et3N, THF, 23 °C, 4 h) or triphosgene (0.4 equiv, 2.4 equiv i-Pr2NEt, CH2Cl2, 0 °C, 15 min) followed by treatment with a secondary amine to afford the product C20′ ureas in good yields (Fig. 3). The latter procedure that uses triphosgene represents an improvement in our original conditions (20), avoiding the purification challenges of removing residual p-nitrophenol from the reaction products. Both set of conditions generate the same intermediate C20′ isocyanate that in turn reacts with the added secondary amine to provide the product ureas. For the comparisons reported herein, the compounds were Fig. 1. Natural product structures and earlier results. examined for cell growth inhibition against L1210 (mouse leukemia), HCT116 (human colon cancer), and HCT116/VM46

equal to vinblastine (Fig. 1) (17). In subsequent studies, we identified the key structural features of such ureas that contrib- ute to their activity, including the importance of the H-bond donor site on the C20′ nitrogen substituent (20). We additionally defined a trend in activity where substitution of the urea terminal nitrogen improves the differential in activity of the derivatives against matched sensitive and resistant tumor cell lines (NR2 > NHR > NH2), discovered a series of potent disubstituted C20′ ureas (e.g., 8 and 9) that displayed further improved activity against resistant tumor cell lines, and established that sterically demanding C20′ ureas were surprisingly well tolerated (20, 21). The target of vinblastine is the tubulin α/β dimer–dimer interface where its binding destabilizes microtubulin assembly derived from the repetitive head-to-tail tubulin binding (9, 22). This disruption of a protein–protein interaction by vinblastine is often overlooked in discussions of such targets as candidate, but challenging, biological targets to address with small molecules perhaps because the target identification preceded the Fig. 2. Hydrogen atom transfer (HAT) free-radical functionalization of contemporary interest (23–27). Herein, we report the discovery unactivated alkenes.

9692 | www.pnas.org/cgi/doi/10.1073/pnas.1611405113 Carney et al. Downloaded by guest on September 25, 2021 3-fluoropyrrolidine urea (17 and 18). As interesting as these

latter observations are, these compounds only approach (14) INAUGURAL ARTICLE or slightly surpass (17 and 18)theactivityof8, which bears no substituent. As a consequence, and rather than ascribing pro- ductive roles to the substituents, it appears that most detract from the properties of the parent compound 8 and a few (14, 17,and18) constitute benign substitutions. More significant was the impact of adding unsaturation (compound 22). Whereas incremental decreases in cell growth activity were seen with increasing ring size of the urea terminal cyclic amine (8 vs. 23–25,5< 6or7< 8) or with the incorporation of the pyrrolidine in an azabicyclo[2,2,1]heptane (26), the introduction of π-unsaturation in either the form of a 3,4-double bond (22)ora 3,4-fused phenyl ring (27 and 9) provided compounds that enhanced cell growth inhibition and improved the differential in activity between the sensitive and resistant HCT116 cell lines (Fig. 5). It is possible that this improved activity is derived in part from alterations in the basicity of the pyrrolidine, reductions in desta- bilizing steric interactions surrounding the pyrrolidine 3,4 position, or through acquisition of additional stabilizing interactions with tubulin, and it is most likely the result of a different combination of such features for 22/9 vs. 27. Finally, replacement of the fused benzene in isoindoline 9 with fused pyridines (28 and 29) was not only permitted, but the change provided a further small enhancement in the cell growth inhibition potency. It is especially notable Fig. 3. Three-step synthesis of vinblastine C20′ ureas. that 22, 9, 28,and29 all exhibit cell growth inhibition activity in the – vinblastine-sensitive tumor cell lines with IC50 values of 400 600 CHEMISTRY pM and that their activity against the vinblastine-resistant tumor (a matched resistant human colon cancer) tumor cell lines, the cell line (HCT116/VM46) now matches the activity that vin- latter of which exhibits resistance (100-fold) to vinblastine blastine displays against the matched sensitive HCT116 cell line through the clinically observed overexpression of P-glycoprotein (IC50 value, 6.8 nM). (Pgp). In initial studies (20), we had established that the elec- Of these, further modifications of the isoindoline urea 9 were tron-rich nature of the urea was responsible in part for their pursued for systematic examination, recognizing the potential for enhanced activity relative to their corresponding carbamate or further disruption of the tubulin dimer–dimer interface (Fig. 6). amide counterparts. As a consequence, we systematically probed Substitution of the isoindoline was well tolerated and the series the pyrrolidine urea 8 to establish whether β-substituents on the displayed a trend where electron-donating substituents (30, 35– pyrrolidine (10–21) or systematic changes in its structure (22–27) 39) proved slightly more potent than compounds bearing elec- mightimpactitsbindingtotubulinandcellgrowthinhibition tron-withdrawing substituents (31–34). Within the former series, properties much like their impact on the pyrrolidine basicity further alkylation (37–39) or acylation (40–53) of the aniline 36 itself. Nearly all such substituted pyrrolidine ureas were less was well tolerated. Simple acetylation to provide the acetamide 8 potent than itself, most showed little preference for the sub- 40 provided a derivative with IC50 values of 470–500 pM in the stituent stereochemistry (R vs. S), and there did not appear to be vinblastine-sensitive tumor cell lines and N-benzoylation main- a direct correlation of the substituent electronic properties with tained this activity with 50 displaying IC50 values of 400 pM. its impact on the cell growth inhibition activity (Fig. 4). The The site proved remarkably tolerant of steric bulk with even exceptions to these generalizations are the behavior of 14, the pivoloyl amide 44 displaying potent activity, and it proved bearing the (R)-3-methoxypyrrolidine, and the two isomers of the capable of accommodating extended rigid acyl groups (e.g., 49).

Fig. 4. Substituted pyrrolidine ureas.

Carney et al. PNAS | August 30, 2016 | vol. 113 | no. 35 | 9693 Downloaded by guest on September 25, 2021 Fig. 5. Pyrrolidine replacements.

Only bulky or rigid extended substitution on the para position of entails inhibition of tubulin polymerization upon exposure to the an added N-benzoyl group (51–53) proved to diminish the ac- candidate compounds. However, and as noted by others, this tivity of the now potent series. Even appending a 4-biphenyl acyl method is insensitive, rarely correlates with the functional activity, 51 group to the isoindoline aniline provided a compound ( ) that, and lacks the resolution to discriminate among a series of closely although less potent than either 40 or 50, was still slightly more potent than vinblastine despite being buried in a region of the tubulin binding site thought to be sensitive to steric interactions before our studies. Finally, disubstitution of the isoindoline was found to be well tolerated and, in the case examined (54), provided a compound equipotent with 35 and slightly more potent than the parent isoindoline 9. Even more impressive levels of activity and further improve- ments in the cell growth inhibition were observed when the benzoyl amide of 50 was replaced with heteroaromatic amides (Fig. 7). A clear trend in activity emerged in the series examined (55–67) to confidently indicate the functionality responsible for and needed to provide the additional enhancements in activity. Although all such compounds were exceptionally potent, those with six-membered heteroaromatic groups with nitrogen in the 2-position relative to the acyl carbonyl routinely displayed the more potent activity (e.g., 55 vs. 50, 56, and 57; 58, 60, and 61 vs. 59 and 62), and those with two such nitrogens where at least one of which is in the 2-position displayed stunning potency (58, 60, and 61). A similar trend was observed with the five-membered heteroaromatic substituents. It is likely this reflects an inter- nal hydrogen bond between the heteroaromatic nitrogen at the 2-position and the adjacent amide NH that is observed in solution in the 1H NMR spectra of the compounds. Complementary to this conformational constraint, the C20′ urea also preferentially adopts the carbonyl eclipsed conformation that fixes a preferred orienta- tion of the C20′ urea (Fig. 7). Compounds 58, 60,and61 exhibited IC50 values of 50–75 pM against the vinblastine-sensitive tumor cell lines (L1210 and HCT116), representing increases of 100-fold relative to vinblastine. They also displayed subnanomolar activity against the vinblastine-resistant tumor cell line (HCT116/VM46; IC50 values, 830–880 pM), representing increases of 700-fold relative to vinblastine. They are also 10-fold more potent than the C20′ urea 9 that lacks a substituent. Because of their extraordinary potency, two heterocyclic amides (55 and 60) were further functionalized with an aryl amine, which would permit covalent conjugation with targeting modal- ities that require this ultrapotent activity (e.g., antibodies, folate) for selective delivery to tumors (Fig. 8). In each case, the aniline derivative maintained the activity of the parent analog (69 and 70 vs. 60) or even further improved its potency (68 vs. 55). Clearly, varied opportunities with subtle impacts on activity are available for both the site and appropriate functionalization of such derivatives for the preparation of tumor-targeting conjugates for which the released free drug payload remains extraordinarily potent.

Tubulin Binding. With compounds displaying remarkable functional cellular potency, we sought to determine whether the im- provements in activity correlated with enhanced tubulin binding affinity. The most common method for assessing tubulin binding Fig. 6. Substituted isoindoline ureas.

9694 | www.pnas.org/cgi/doi/10.1073/pnas.1611405113 Carney et al. Downloaded by guest on September 25, 2021 INAUGURAL ARTICLE

Fig. 7. Ultrapotent heterocyclic amides of the C20′ isoindoline urea.

related compounds. Methods that measure competitive binding ultrapotent vinblastines (58, 60,and61), each 100-fold more potent at the vinblastine tubulin binding site are more predictive, and than vinblastine, displaced 100% of the BODIPY–-vinblastine, in- these typically involve measurement of the competitive dis- dicating that these ultrapotent C20′ ureas possess a much stronger placement of radiolabeled vinblastine or fluorescently labeled binding affinity for tubulin than vinblastine, 10′-fluorovinblastine, vinblastine probes. We have previously used the competitive dis- and 28. Although it is not possible to rule out the impact of other placement of 3H-vinblastine to assess the tubulin binding of selected features or even introduction of an unrecognized second mecha- members of the vinblastine C20′ ureas (20, 21). This assay involves nism of action, this direct correlation of functional cell growth filtering coincubation solutions through DE-81 filter disks, which sequester tubulin and its bound ligands from solution (28, 29). CHEMISTRY However, the assay is not ideal due to both the cost associated with using 3H-vinblastine and the technical challenges of quantita- tively reproducing the results of the assay, which uses repeated rinses of the sequestered proteins containing reversibly bound compounds. This is further complicated by the fact that the manufacture of DE-81 filter paper has been discontinued. As a consequence, we elected to examine a commercially available boron-dipyrromethene (BODIPY)–vinblastine fluorescent probe to assess tubulin binding and to develop and generalize its use in a direct competitive binding assay (30–34). Although introduced and used principally to visualize protein driven efflux from cells, BODIPY–vinblastine exhibits a significant increase in fluorescence intensity (FI) upon binding to tubulin. This behavior of BODIPY–vinblastine allows fluorescent measurements of coincubation solutions to establish the percentage displacement of probe by a competitive binding site ligand. Tubulin was incubated with BODIPY–vinblastine and a representative range of competitive ligands including vinblastine, 10′-fluorovinblastine [71, a previously reported vinblastine analog with 10-fold improved functional activity (35)], the potent compound 28,and three ultrapotent C20′ urea analogs (58, 60,and61) reported here. At the concentration tested, vinblastine displaced 31% of the BODIPY–vinblastine bound to tubulin (Fig. 9). 10′-Fluo- rovinblastine and 28, which are roughly 10-fold more potent than vinblastine, displayed a higher tubulin affinity, displacing 48–49% of the BODIPY–vinblastine. Remarkably, the three

Fig. 9. Tubulin (0.1 mg/mL, 0.91 μM) was incubated with BODIPY–vinblastine (BODIPY–VBL) (1.8 μM) and a vinblastine analog (18 μM) at 37 °C in PEM (80 mM

PIPES-K pH 6.8, 2 mM MgSO4, 0.5 mM EGTA pH 6.8) buffer containing 850 μM GTP. The BODIPY–VBL fluorescence intensity (FI) [excitation (ex), 480 nm; emis- sion (em), 514 nm] of 100-μL aliquots from each incubation were measured in a fluorescence microplate reader at 37 °C. Control experiments were performed with BODIPY–VBL (control 1) in the absence of a competitive ligand (maximum FI enhancement due to tubulin binding) and (control 2) in the absence of tubulin (no FI enhancement due to tubulin binding). Percentage BODIPY–VBL displacement was calculated by the formula (control 1 FI – experiment FI)/(control 1 FI – Fig. 8. Further functionalized derivatives. control 2 FI) × 100. Reported values are the average of four measurements ± SD.

Carney et al. PNAS | August 30, 2016 | vol. 113 | no. 35 | 9695 Downloaded by guest on September 25, 2021 inhibition activity with target tubulin binding affinity and the relative magnitude of the effects suggest that the properties of the potent and ultrapotent C20′ ureas are derived predominately, if not exclusively, from on-target effects on tubulin.

Binding Model. In contrast to expectations based on the steric constraints of the tubulin binding site surrounding the vinblastine C20′ center depicted in the X-ray cocrystal structure of a tubulin- bound complex (22), large C20′ urea derivatives such as those detailed herein are accommodated, with some exhibiting ultrapotent functional activity in cell-based proliferation assays and remarkable tubulin binding affinity. This raises several key ques- tions that include whether there is room for such substituents in the purported vinblastine binding site. The binding site for vinblastine is created by and lies at the head-to-tail tubulin α/β dimer–dimer

Fig. 11. Models of the ultrapotent vinblastine 60 in the vinblastine binding site of tubulin constructed without altering the proteins found the X-ray crystal structure of tubulin-bound vinblastine (PDB ID code 1Z2B) (22). Two possible binding conformations are shown representing a 180° rotation about the C20′ isoindoline nitrogen bond. In one conformation (Top), the pyrazinoyl group extends over the top of the β-tubulin peptide loop (highlighted in orange) into a region where the α-tubulin (tan) and β-tubulin (orange/blue) subunits are in close contact, thereby further disrupting the protein–protein interaction. In the second conformation (Bottom), the pyrazinoyl group continues to bind along the extended dimer–dimer interface, establishing additional new contacts with α-tubulin.

interface (Fig. 10). Vinblastine is essentially completely buried in the protein binding site, adopting a T-shaped bound conformation with C3/C4 (bottom of T) lying at the solvent interface and the C20′ site (top corner of T) extending deepest into the binding pocket lying at one corner. This binding of vinblastine destabilizes the protein–protein interaction integral to microtubulin assembly. Inspection of the vinblastine–tubulin X-ray structure reveals that the C20′ alcohol extends toward a narrow channel that leads from the buried C20′ site to the opposite face of the protein, representing the continuation of the protein–protein interaction defined by the tubulin dimer–dimer interface. Even without adjusting the proteins found in the vinblastine-bound X-ray structure, the modeled C20′ isoindoline group of 9 extends behind a key peptide loop in β-tubulin that constitutes the top of the vinblastine binding site into this narrow channel, continuing along the tubulin protein–protein interface and presumably is responsible for further disruption of the dimer tubulin–tubulin interaction (Fig. 10). It is likely that the rigid linear C20′ urea of the ultrapotent vinblastines extends into and expands this narrow channel, even Fig. 10. X-ray crystal structure of tubulin-bound vinblastine (PDB ID code α β – α β – further disrupts the tubulin / head-to-tail dimer dimer interface, 1Z2B) (22). (A) Binding site at the tubulin / head-to-tail dimer dimer in- – terface. (B) Binding site comparison of vinblastine (left column, X-ray) and and extends across the full protein protein interface, contacting modeled vinblastine C20′ urea 9 (right column); ribbon structure of binding solvent on the distal side (Fig. 11). Thus, and although the struc- site (Top), corresponding space-filling model (Middle), and side view with ture of the potent C20′ ureas and their optimization may appear the adjacent α-tubulin protein (green) at the dimer–dimer interface re- unusual on a first inspection, they represent rational structural moved. A peptide loop (highlighted in orange) on the β-tubulin protein additions to a specific site on the natural product with appropriate (light blue) covers the top side of the vinblastine velbanamine subunit in- conformational constraints needed to further enhance disruption ′ cluding C20 , creating a deep and largely hydrophobic pocket for ligand of the target protein–protein interaction. binding. Without adjusting the proteins found in the vinblastine-bound More provocatively, the activity of the ultrapotent analogs (IC50 X-ray structure, the isoindoline group of modeled 9 extends behind the loop – peptide into a channel continuing along the tubulin protein–protein in- values, 50 75 pM) suggest that it is not likely or even possible that terface. Aryl amide substituents on the isoindoline such as those found in 50 their cellular functional activity is derived from stoichiometric and 55–67 continue to extend along the tubulin dimer protein–protein in- occupancy of the relatively large number of intracellular tubulin terface (Fig. 11). binding sites. Rather, their potency suggests substoichiometric or

9696 | www.pnas.org/cgi/doi/10.1073/pnas.1611405113 Carney et al. Downloaded by guest on September 25, 2021 INAUGURAL ARTICLE

Fig. 12. Structure of 60, the contributing conformational features of the added molecular complexity, and modeled interaction with tubulin.

catalytic occupancy of candidate binding sites is sufficient to dis- the underlying structure in a way that requires chemical synthesis rupt tubulin dynamics or assembly during mitosis. The question of a modified natural product. In part, this may be attributed to these ultrapotent vinblastines pose is whether substoichiometric the added challenge that might accompany the more complex binding site occupancy is sufficient to either trap/cap polymerizing compound synthesis. The synthetically derived ultrapotent vin- microtubulin in nonproductive conformations or disrupt microtubulin blastines that we detailed, which are 100-fold more active than the

dynamics, or whether such compounds serve catalytically to ac- natural product, represent vinblastine analogs accessible by chem- CHEMISTRY tively disassemble microtubulin. Although we cannot yet answer ical synthesis in three steps from commercially available and rela- this question, it is a focus of current efforts. tively inexpensive materials (catharanthine, $16/g; and vindoline, $36/g) based on methodology we introduced and are presently Discussion inaccessible by classical natural product derivatization, late-stage In recent studies, we reported concise 12-step total syntheses of functionalization, or biosynthetic methods. The newly discovered vinblastine and related natural products (14–16) based in part ultrapotent vinblastines, which display much higher tubulin bind- on introduction of a powerful single-pot two-step diastereoselective ing affinities, likely further disrupt the target tubulin head-to-tail Fe(III)-promoted vindoline/catharanthine coupling and sub- α/β dimer–dimer interaction by virtue of the strategic placement of sequent Fe(III)-NaBH4/air–mediated free-radical C20′ oxidation the added rigid and extended C20′ urea along the adjacent con- that was extended for use with alternative free-radical traps (17, tinuing protein–protein interface (Fig. 12). The stunning success 18). This methodology, inspired by the structure of vinblastine, has with vinblastine suggests the approach of adding molecular com- permitted systematic studies of the effects of deep-seated plexity to enhance target binding and functional biological activity structural changes within either the lower vindoline or upper for other natural products targeting a protein–protein interaction velbanamine subunits (13). One general observation made in may represent an appealing and general approach to achieving these studies is that, although modifications to the key struc- such objectives. tural features of vinblastine typically result in reductions in biological activity, addition of structural features can sub- Materials and Methods stantially improve them (20, 21, 35, 36). This includes not only Synthesis of Vinblastine C20′ Ureas. Full details of the synthesis, purification, our demonstration of the impact of the addition of an indole C10′ and characterization of all compounds reported herein, including copies of fluorine substituent (35) and the incorporation of the C20′ ethyl the 1H NMR of all tested compounds, are provided in SI Appendix. All re- group into a benign more complex cis-fused C15′-C20′ six-mem- agents were obtained from commercial sources unless noted otherwise. bered ring (36), but also the discovery of the improved activity of C20′ urea replacements for the tertiary alcohol (20, 21). Herein, we Cell Growth Inhibition Assays. Full details of the cell growth inhibition assays (L1210, HCT116, and HCT116/VM46) are provided in SI Appendix. Compounds reported the discovery of compounds in this latter series that are were tested in duplicate (n = 2−18 times) at six concentrations between now a stunning 100-fold more potent than vinblastine. Such com- 0–1,000 nM, or 0 and 10,000 nM. The IC values reported represent of the 60 – 50 pounds including exhibited IC50 values of 50 75 pM against the average of 4−36 determinations (SD ± 10%). vinblastine sensitive tumor cell lines (e.g., HCT116), representing increases of 100-fold relative to vinblastine, and even subnanomolar Tubulin Binding Assay. Full details of the tubulin binding assay are provided in ± activity against a resistant tumor cell line (HCT116/VM46; IC50 SI Appendix. Reported values are the average of four measurements SD. values, 830–880 pM) insensitive to vinblastine by virtue of the clinically observed Pgp overexpression, representing increases Supplementary Information. Full experimental details and copies of 1H NMR of 700-fold relative to vinblastine (Fig. 12). spectra are provided. The supplementary data associated with this article Approaches to improving the biological properties of natural can be found in SI Appendix. products typically involve efforts to simplify their structures or to ACKNOWLEDGMENTS. We especially thank Katharine K. Duncan for the modify their structures through semisynthetic transformations, biological assessments of early samples in the series reported in refs. 20 and diverted total synthesis (37), or late-stage functionalizations (38, 21. We gratefully acknowledge the financial support of National Institutes 39). Rarely does one consider adding molecular complexity to of Health Grants CA115526 and CA042056.

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