Synthetic Analogues of the Marine Bisindole Deoxytopsentin: Potent

Article pubs.acs.org/jnp

Synthetic Analogues of the Marine Bisindole Deoxytopsentin: Potent Selective Inhibitors of MRSA Pyruvate Kinase Clinton G. L. Veale,*,† Roya Zoraghi,‡ Ryan M. Young,† James P. Morrison,‡ Manoja Pretheeban,‡ Kevin A. Lobb,† Neil E. Reiner,‡ Raymond J. Andersen,§ and Michael T. Davies-Coleman⊥ †

Department of Chemistry, Rhodes University, Grahamstown, South Africa Division of Infectious Diseases, Department of Medicine, and §Departments of Chemistry and of Earth and Ocean Sciences, University of British Columbia, Vancouver, BC, Canada ⊥ Department of Chemistry, University of the Western Cape, Bellville, South Africa ‡

S Supporting Information *

ABSTRACT: As part of an ongoing study to elucidate the SAR of bisindole alkaloid inhibitors against the evolutionary conserved MRSA pyruvate kinase (PK), we present here the synthesis and biological activity of six dihalogenated analogues of the naturally occurring sponge metabolite deoxytopsentin, including the naturally occurring dibromodeoxytopsentin. The most active compounds displayed potent low nanomolar inhibitory activity against MRSA PK with concomitant significant selectivity for MRSA PK over human PK orthologues. Computational studies suggest that these potent MRSA PK inhibitors occupy a region of the small interface of the enzyme tetramer where amino acid sequence divergence from common human PK orthologues may contribute to the observed selectivity.

bacterial “hub” proteins may prove to be suitable antibiotic targets for MRSA antibiotic development, as they are highly conserved and extensively connected to other proteins through a complex protein interaction network (PIN). Arising from their connectivity, hub proteins are consequently less prone to mutation, thus limiting the potential for the inadvertent development of antibiotic-resistant bacterial strains.9,11 Accordingly, a large-scale, proteomic investigation of the PIN consisting of 608 proteins from a hospital-acquired strain of MRSA-252 conducted by Zoraghi et al. identified pyruvate kinase (PK) as the most highly connected evolutionary conserved hub protein in the MRSA interactome.9,14,15 PK catalyzes the rate-limiting, final step in glycolysis involving the irreversible conversion of phosphoenolpyruvate (PEP) into pyruvate with the subsequent phosphorylation of ADP into ATP.9,15 Both the products and substrates of PK are involved in a number of additional biological pathways, therefore providing a critical intervention point to disrupt whole cell bacterial metabolism. Additionally, MRSA PK has been shown by gene disruption experiments to be essential for bacterial viability, displaying a high level of enzymatic activity during the exponential growth phase of S. aureus.15

he approval for clinical use of the β-lactam antibiotics in the early 1940s for the first time allowed Staphylococcus aureus infections to be effectively treated and ushered in the antibiotic era.1 The emergence of methicillin-resistant S. aureus (MRSA),2−4 despite its widespread distribution, was initially not considered a major threat, because vancomycin and related glycopeptide antibiotics were effective for nearly two decades against serious MRSA infections.5,6 Unfortunately, as a result of vancomycin’s limited distribution within tissues, coupled with suboptimal dosing regimens, vancomycin-intermediate S. aureus (VISA) and vancomycinresistant S. aureus (VRSA) strains have emerged across the globe.5−7 The resulting drug-resistant bacteria pandemic first reported in global public health care systems8−11 has spread not only into the general community but also into livestock.10,12,13 The annual mortality of 18 000 deaths attributed to MRSA13 now exceeds that of AIDS in the USA,14 necessitating a concerted effort to find new targets and strategies to combat drug-resistant bacteria such as MRSA.9,15 Antibiotic drug discovery has traditionally relied on screening libraries of natural products and synthetic compounds against whole cells.9 However, advances in genomics, proteomics, target identification, and assay development have greatly improved target-based approaches toward drug discovery.16 Selecting bacterial-specific protein targets for antibiotic drug discovery has the disadvantage of potentially exerting selective pressures on the pathogen with the inevitable emergence of resistance.9,11 However, Reiner and co-workers suggested that

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© XXXX American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of William Fenical Received: September 26, 2014

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dx.doi.org/10.1021/np500755v | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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The indole moieties of 2 were also shown to interact hydrophobically with the relevant amino acid residues between Ile361 and His365, while the two bromine residues appear to each occupy a hydrophobic pocket at each end of the binding site formed by Thr353, Ser354, Ala358, and Leu370.15 Sequence alignment between MRSA and human PK isoforms revealed sequence divergence in the C domain10,15 including the entrance to the hamacanthin binding pocket, where differences of three amino acid residues per subunit including His365 result in obstruction of the binding pocket in human PK orthologues (Figure 2).10 Marine bisindole alkaloids19 and halogenated alkaloids20 exhibit a wide range of biological activities, and a substantial body of work has been dedicated to exploring the role of these compounds as potential chemotherapeutic agents against a variety of targets. Members of both the hamacanthin and topsentin classes of bisindole alkaloids have exhibited in vitro activity against a variety of Gram-positive and negative bacteria, and deoxytopsentin (5) proved to be either equally or more active than oxacillin against three strains of MRSA tested in the antimicrobial bioassay.21 Having established the importance of the central imidazole moiety to the deoxytopsentin pharmacophore22 and provided with details of the cis-3,4-dihydrohamacanthin B binding site from X-ray analysis, we were curious as to whether the primary reason for differences in activity between 1 and 2, assuming the same binding site, was due to C-6 bromination on each of the respective indole moieties. The presence of this dibromo substitution pattern may enable the ligands to better occupy the hydrophobic pocket identified in the crystal structure (Figure 1). Recently, a series of halogenated bisindoles modeled on the scaffold of 2 were synthesized and were found to potently inhibit MRSA PK,23 thus supporting our hypothesis that halogenated indole rings are a critical feature of the pharmacophore. The potent activity of the dibrominated cis-3,4-dihydrohamacanthin B alerted us to the possibility of halogen bonding being a key feature contributing to the binding of 2 to MRSA PK. However, investigation of possible bonding distances and angles between the halogen and possible halogen bond acceptors (e.g., O, N, and S) in the cis-3,4-dihydrohamacanthin B MRSA PK cocrystal structure failed to reveal any of the hallmark features of halogen bonding, which are highly directional in nature with a maximum angle range of 140− 180° and a bond length smaller than the van der Waal’s radii of the participating atoms (3.0−3.5 Å depending on the halogen).24,25 Ligand−receptor docking experiments conducted with AutoDock Vina26 against two MRSA PK crystal structures (PDB accession number 3T0T 10 and 3T07 15) placed deoxytopsentin analogues (5−10) in the hamacanthin binding site in both local and global searches for binding sites within the target protein (Figure 1). Due to the static nature of this docking method, it must be noted that binding modes obtained from this technique are speculative. However, docking experiments indicated the possibility of histidine/imidazole and histidine/indole π-stacking interactions, in addition to hydrogen bond interactions between binding site Ser362 and the indole NH. Ligand halogens orientated toward the hydrophobic binding pockets, as observed for the hamacanthin inhibitor (Figure 1). We therefore proposed a structure-based drug design of several pseudosymmetrical deoxytopsentin analogues 5−11,

The identification of evolutionary conserved protein allows structural differences between prokaryotes and eukaryotes to be exploited to introduce selectivity between hosts and pathogens, and MRSA PK was accordingly found to have several structural features that make it distinct from mammalian PK orthologues. X-ray crystallography revealed that PK exists as a homotetramer of four identical subunits consisting of between three and four domains, namely, the A, B, and C domains and the N-terminal domain, whose quaternary structure forms through interaction of the A and C domains of adjacent monomers to form an A−A and a C−C interface referred to as the large and small interface, respectively.9,15,17 The active site for kinase activity is situated at the interface of the A and B domains in each of the respective subunits, while the allosteric effector site is located in the C domain.9,15,17 In a study to identify marine natural products that inhibit MRSA PK, a library of 968 extracts from marine invertebrates and microbes collected from several different and widely dispersed regions of the world was screened against purified recombinant MRSA PK. Only the methanolic extract of the sponge Topsentia pachastrelloides collected on the Aliwal Shoal off the South African east coast was deemed active in the screening program.15 Purification of the extract yielded four known bisindole alkaloids, viz., bromodeoxytopsentin (1), cis3,4-dihydrohamacanthin B (2), spongotine A (3), and bromotopsentin (4), of which only compounds 1 and 2 were found to potently inhibit MRSA PK with IC50 values of 60 and 16 nM, respectively, with 166−600-fold selectivity for the bacterial PK over four human PK isoforms (M1, M2, R, and L). Importantly both compounds 1 (MIC 6.25 μg/mL) and 2 (MIC 12.5 μg/mL) exhibited equal in vitro antibacterial activity against both methicillin-susceptible (RN 4220) and methicillinresistant (MRSA 252) strains of S. aureus,15 indicating that the mechanism of these compounds is not susceptible to current MRSA 252 resistance mechanisms.

X-ray analysis of 2 cocrystallized with MRSA PK (PDB accession number 3T07)15 revealed the ligand to be a noncompetitive PK inhibitor binding neither at the active nor effector sites as originally expected, but rather in a small symmetrical hydrophobic binding pocket situated at the small interface formed through the antiparallel interaction of two identical α-helices 357−370 from two different subunits (Figure 1).15 The small interface was reported to be a region crucial for establishing tetramer rigidity and therefore ensuring efficient catalytic activity.15 Zoraghi et al. also noted that prominent His365 residues undergo side chain rearrangement in the presence of the ligand, forming π-stacking interactions with the indole moieties, thus anchoring the piperazine moiety into the binding site, while the respective Ser362 residues from each subunit form hydrogen bonds with the indole NH’s. B



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Figure 1. X-ray cocrystal structure of 2 (green) bound to a hydrophobic symmetrical binding pocket on MRSA PK located at the small interface constructed from the antiparallel arrangement of two identical α-helices from individual subunits colored red for clarity (PDB accession number 3T07). Overlaid is the highest scoring docked orientation of natural product 6 with the bromine atoms, displayed as CPK models occupying the hydrophobic pockets. π-Stacking interactions occur between the His365 residues and the indole moieties of both 2 and 6 and between these amino acid residues and the imidazole ring of 6. A hydrogen bond interaction between the indole NH of compound 6 and Ser362, analogous to that seen for compound 2, is observed.

Figure 2. Left: Portion of the X-ray crystal structure of human M2 PK (PDB accession number 4FXF)18 highlighting the hamacanthin binding pocket (red), obstructed by two arginine residues (Arg399 and 400) and one glutamic acid (Glu418) highlighted in green, from each of the two protein subunits. Right: Portion of the X-ray crystal structure of apo MRSA PK (PDB accession number 3T05).15 Highlighted in green are amino acid residues (Thr348, Lys349, and His365) in the same relative position as those blocking the cis-3,4-dihydrohamacanthin B pocket in the human M2 PK. The obstructed binding site of M2 PK and lack of π-stacking potential with histidine presumably contribute toward selectivity for bacterial over human PK orthologues.10

including the naturally occurring compounds 527 and 628 and the unusual diiododeoxytopsentin (9), to test whether the activity of the privileged bromodeoxytopsentin scaffold could be modified in order to improve both MRSA PK inhibitory activity and selectivity over human PK isoforms. The structure of the deoxytopsentin/cis-3,4-dihydrohamacanthin B MRSA PK binding site suggested, therefore, the importance of C-6 halogenation on both the indole rings of the proposed deoxytopsentin ligands. 5-Dibromodeoxytopsentin (10) and 7-chlorodeoxytopsentin (11) were synthesized to either support or refute this assumption.

cyclocondensation in EtOH at room temperature31 to yield the desired bisindole imidazoles (33−37, Scheme 1). A similar approach was used to prepare the nonhalogenated 5 and the C5 dibromo and C-7 dichloro analogues. While 3-acetylindole (12) was commercially available, the halogenated 3-acetyl indoles (13−18) required prior preparation from the respective halogenated indole precursors (19− 24) via a cosolvent Friedel−Crafts acylation method developed by Ottoni et al.32 in which SnCl4 was added to a stirred solution of the indole precursor in dry CH2Cl2 under argon at 0 °C. The brightly colored aggregates thus formed were dissolved in nitromethane prior to the addition of the acetyl chloride acylating agent. After workup, the brown tarry solid products were crystallized via slow evaporation from acetone to yield the required halogenated 3-acetylindoles in variable yield. A method adapted from Yamada et al.33 was used to prepare 6iodoindole 23 from 6-nitroindoline over five steps, with the addition of sulfamic acid improving the yield for the final iodination step.

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RESULTS AND DISCUSSION Our proposed pseudosymmetrical synthetic targets (6−9) featuring a C-6 halogen substituent on each indole ring were prepared via a one-pot selenium dioxide-mediated oxidation29,30 of appropriate 3-acetylindoles to the corresponding indolyl-3-glyoxals. In the presence of ammonium acetate, two molecules of indolyl-3-glyoxal then undergo dehydrative C



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Scheme 1a

Reagents and conditions: (a) SnCl4, CH2Cl2, AcCl, MeNO2, 0 °C to rt, Ar, 2 h. (b) Boc2O, DMAP, MeCN, 0 °C to rt, Ar, 3 h. (c) SeO2, H2O, 1,4dioxane, 75 °C, 6 h. (d) NH4OAc, EtOH, rt, 5 h. (e) 180 °C, Ar, 5 min.

a

topsentins (32−38) proceeded smoothly to yield the desired fluorescent deoxytopsentin pigments, which were subjected to RP-HPLC purification prior to bioassay. All compounds were assayed for their ability to inhibit enzymatic activity against purified recombinant MRSA and human PKs. Halogenated deoxytopsentin analogues 6−10 were found to be potent inhibitors of MRSA PK (Table 1).

Initial attempts at SeO2-mediated oxidation of 12 resulted in over-oxidation to indolyl-3-glyoxylic acid. We surmised that the influence of the indole nitrogen on ring activity could be reduced through formation of an electron-withdrawing N-Boc carbamate ester, which subsequently resulted in a noticeable reduction in glyoxylic acid formation. We identified the oxidation of N-Boc-protected 3-acetylindoles as a critical juncture to optimize over all product yields and accordingly conducted a series of optimization experiments on N-Boc carbamate ester 25, varying reaction time, temperature, and reagent equivalents. Our initial concerns revolved around the possible influence of reduced selenium species in spurious side reactions as reported by Sharpless and co-workers34 including the formation of organo-selenium byproducts, e.g., from Se(II) addition to susceptible olefins.34 While initial attempts to lower SeO2 equivalents through the use of an additional oxidizing agent, tert-butyl hydroperoxide (TBHP), were unsuccessful, we observed a delicate balance between the relative equivalence of H2O and SeO2, with slight increases in H2O equivalence aiding glyoxal formation and subsequent yield of 32 with a fortuitous improvement in the recovery of 25. Interestingly, the halogenated analogues (26−31) were successfully oxidized at higher yields over two steps when subjected to higher equivalents of SeO2 prior to cyclization than in the case of 25. Due to limited starting material availability, reactions of compounds 29 and 30 were conducted on a smaller scale, with disappointingly low yields, but gratifyingly significant proportions of recoverable starting materials that could be recycled. Thermolytic cleavage35 of Boc-protected

Table 1. Summary of the IC50 Values of Compounds Tested against MRSA PK

a

compound

IC50 (nM) ± SD

1 5 6 7 8 9 10 11

60a 240 ± 49 2.1 ± 0.1 24 ± 3 1.5 ± 0.5 3.3 ± 1.7 6.4 ± 1.6 26 ± 3b

Assayed on a separate occasion. bIC50 (μM).

On the basis of our rationale for synthesis of dihalogenated topsentin analogues, we were not surprised that nonhalogenated 5 was an order of magnitude less active than the original bromodeoxytopsentin inhibitor. Interestingly, all of the C-6-halogenated analogues 6−9 exhibited potent low nanomolar inhibition of MRSA PK, displaying activity greater than bromodeoxytopsentin and broadly similar activity to cis-3,4D



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Figure 3. Percent inhibition data of topsentin analogues 5−11 against MRSA PK and four mammalian orthologues. *Percent inhibition data at 10 μM.

dihydrohamacanthin B, providing further evidence that halogenation of both indole rings is a critical feature of the pharmacophore. Interestingly, the inhibition of MRSA PK was apparently unaffected by bromination at C-5 (i.e., 10), while chlorination at C-7 (i.e., 11) resulted in a dramatic loss of activity. Compounds 6, 8, and 9 were further found to completely inhibit MRSA enzymatic activity at a concentration of 5 μM, while simultaneously exhibiting zero inhibition of human PK orthologues M1 and M2 and less than 40% inhibition of human PK orthologues R and L at the same concentration, a result potentially attributed to sequence divergence in the binding site. A slight drop in inhibitory activity was observed with the C-5-halogenated analogue 10, while also displaying a similar pattern of human PK inhibition (Figure 3). The compounds featuring the smallest substituents, namely, the nonhalogenated (5) and difluorinated (7) analogues, were less active, displaying moderate inhibition at 10 μM with significant selectivity for MRSA PK, in preference to the four human PK orthologues. At the same concentration 11 displayed minimal inhibitory activity and as such was not assayed for selectivity.

the aromatic imidazole of deoxytopsentin. While these results disclose important SAR information about the potential of deoxytopsentin MRSA PK inhibition and related selectivity, whole cell MIC data against MRSA strains, other bacterial strains, and uninfected human cells would be useful to elucidate further important information about their SAR, especially in the context of the reported susceptibility of bisindoles to cellular efflux.23

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined using either a Reichert hot stage microscope or a Stuart SMP30 (Bibby Scientific Ltd.) and are uncorrected. IR spectra were recorded on a PerkinElmer Spectrum 2000 FT-IR. NMR spectra were acquired on a Bruker 600 MHz Avance II spectrometer. Chemical shifts are reported in ppm, referenced to residual solvent resonances (CDCl3 δH 7.26, δC 77.0; DMSO-d6 δH 2.50, δC 39.50; CD3OD δH 3.31, δC 49.00 ppm).36 High-resolution mass spectrometry was performed on a Waters Synapt G2 TOF instrument with an ESI source. Flash chromatography was performed using Kieselgel 60 (230−400 mesh) silica gel. Purity of all tested compounds was >95%, determined by semipreparative RP-HPLC, with an Onyx Monolithic C18 column, 100 × 10 mm, on an Agilent 1100 Series quad pump and an Agilent 1100 diode array detector. General Procedure for the Synthesis of 3-Acetylindoles (13−18). To a stirred solution of indole 19−24 (500 mg, 3.70 mmol, 1 equiv) in dry CH2Cl2 (7.5 mL) under argon at 0 °C was added SnCl4 (519.5 μL, 1.2 equiv). The ice bath was removed, and the reaction suspension was allowed to stir for a further 30 min. Acetyl chloride (3.70 mmol 1 equiv) was added dropwise to the reaction mixture, followed by nitromethane (4.5 mL). After 4 h the reaction was quenched with ice and water. Organic material was extracted with EtOAc (100 mL), washed with H2O (2 × 20 mL) and saturated brine (2 × 30 mL), and dried over anhydrous MgSO4. Solvent was removed in vacuo to afford a brown, tarry solid, which was dissolved in cold acetone. The acetone was allowed to slowly evaporate over several days, affording brown prisms, which were washed with cold CHCl3 to yield 13−18. For spectroscopic data of compounds 13−16 and 18 see the Supporting Information. Compounds 13,37 14,38 15, and 1639 have been reported previously. 1-(6-Iodo-1H-indol-3-yl)ethanone (17): white crystals from acetone; yield 64%; mp 189−192 °C; IR (cm−1) 3106 2980 2301 1703 1630 1572 1381 1268 923; 1H NMR (600 MHz, CD3OD) δ 8.09 (1H, s, H-2), 8.00 (1H, d, J = 8.3 Hz, H-4), 7.81 (1H, s, H-7), 7.48 (1H, d, J = 8.2 Hz, H-5), 2.51 (3H, s, H-2′); 13C NMR (150 MHz, CD3OD) δ 196.3 (C, C-1′), 139.7 (C, C-7a), 135.8 (CH, C-2), 131.9, (CH, C-5), 126.2 (C, C-3a), 124.5 (CH, C-4), 122.0 (CH, C7), 118.5 (C, C-3), 87.6 (C, C-6), 27.2 (CH3, C-2′) ppm; ESMS m/z

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CONCLUSION This paper emphasizes the important role that marine natural products continue to play in revealing new protein/smallmolecule binding sites in key enzymes of medical significance. The combination of a bioactive natural product chemical structure coupled with the topography of a new binding site revealed by X-ray analysis of a protein-bound natural product provides a powerful platform from which to launch a successful SAR study. Accordingly, a cohort of new and known imidazolecontaining analogues of a naturally occurring marine bisindole were synthesized. The MRSA PK inhibitory activities of these synthetic compounds revealed the privileged nature of the imidazole-containing topsentin scaffold, which displayed both potent inhibition of, and high selectivity for, MRSA PK over human PK isoforms. The enhanced MRSA PK inhibitory activity of analogues containing large halogen substituents was attributed to the symmetrical nature of the binding pocket, which features two large hydrophobic binding pockets at either end. Docking studies suggest that the synthetic deoxytopsentin bisindole analogues occupy the same hydrophobic pocket as the bisindole hamacanthin, with the possibility of an additional π-stacking interaction between the binding site histidines and E



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(rel int) 144 [M + H]+ (100), 116 (15), 89 (4); HRESMS m/z 285.9732 [M + H]+ (calcd for C10H9INO 285.9729). General Procedure for the Synthesis of N-tert-Butoxycarbonyl-Substituted 3-Acetylindoles (25−31). Boc2O (2020 mg, 1.5 equiv) and DMAP (77 mg, 0.1 equiv) were added to a stirring solution of 12 (1000 mg, 6.3 mmol, 1 equiv) in HPLC grade MeCN (15 mL) at 0 °C under an atmosphere of argon. After 3 h the MeCN was removed in vacuo and the solid recrystallized from MeOH to yield 25 (1565 mg, 6.04 mmol, 96%) The same procedure was applied to varying quantities of 3-acetylindoles 13−18 depending on availability using the same equivalents and solvent ratios as above. For spectroscopic data of compounds 25, 26, and 28−31 see the Supporting Information. Compounds 2540 and 2941 have been reported previously. 6-Chloro-3-acetyl-1-(tert-butoxycarbonyl)indole (27): white needles from MeOH; yield 94%; mp 180−182 °C; IR (cm−1) 2970 2162 1732 1665 1548 1361 1272 1150; 1H NMR (600 MHz, CDCl3) δ 8.28 (1H, d, J = 8.4 Hz, H-4), 8.18 (1H, s, H-2), 8.15 (1H, s, H-7), 7.32 (1H, dd, J = 8.5, 1.7 Hz, H-5), 2.55 (3H, s, H-2′), 1.71 (9H, s, H-3″); 13 C NMR (150 MHz, CDCl3) δ 193.6 (C, C-1′), 148.7 (C, C-1″), 135.9 (C, C-7a), 132.5 (CH, C-2), 131.5, (C, C-6), 125.8 (C, C-3a), 124.9 (CH, C-5), 123.5 (CH, C-4), 120.3 (C, C-3), 115.3 (CH, C-7), 85.9 (C, C-2″), 28.0 (3 × CH3, C-3″), 27.6 (CH3, C-2′) ppm; ESMS m/z (rel int) 238 [M + H]+ (5), 194 (7), 152 (17), 117 (100), 89 (10), 57 (15); HRESMS m/z 294.0889 [M + H]+ (calcd for C15H1735ClNO3 294.0897). Synthesis of N-tert-Butoxycarbonyl-Substituted Deoxytopsentin (32). Freshly sublimed SeO2 (286 mg, 2.59 mmol, 2.7 equiv) was added to a mixture of 1,4-dioxane (7 mL) and H2O (200 μL, 11 equiv), after which the reaction mixture was heated to 60 °C to allow all the SeO2 to dissolve. To this was added 25 (250 mg, 0.96 mmol, 1 equiv), and the temperature increased to 75 °C. After 6 h the crude reaction mixture was filtered through Celite, which was washed with CH2Cl2 (20 mL). The combined fractions were washed with H2O (2 × 10 mL) and saturated brine (2 × 10 mL) and dried over anhydrous MgSO4, and the organic fraction was concentrated in vacuo to yield an orange oil (287 mg). The crude reaction mixture was dissolved in EtOH (7 mL) followed by the addition of NH4OAc (374 mg, 5 equiv) and allowed to stir for 5 h. The bright orange solution was concentrated in vacuo and redissolved in CH2Cl2 (15 mL). The organic solution was once again washed with H2O (2 × 10 mL) and saturated brine (2 × 10 mL) and dried over anhydrous MgSO4 to yield a yellow, amorphous solid (248 mg). Purification via Si flash chromatography (hexane/EtOAc, 5:1) yielded 32 (142 mg, 0.27 mmol, 53% over two steps). (N1′,N1″-Di-tert-butoxycarbonyl)indol-3-yl[5-(indol-3-yl)-1H-imidazole-2-yl]-methanone (32): yellow, amorphous solid; yield 53%; IR (cm−1) 2980, 1741, 1667, 1370, 1150, 749; 1H NMR (600 MHz, DMSO-d6) δ 13.62 (1H, s, NH-1), 9.69 (1H, s, H-2″), 8.45 (1H, d, J = 7.8 Hz, H-4″), 8.38 (1H, d, J = 7.7 Hz, H-4′), 8.19 (1H, d, J = 8.1 Hz, H-7″), 8.14 (2H, m, H-2′, H-7′), 8.09 (1H, s, H-4), 7.42 (4H, m, H-5′, H-5″, H-6′, H-6″), 1.69 (9H, s, H-11″), 1.68 (9H, s, H-10′); 13C NMR (150 MHz, DMSO-d6) δ 176.2 (C, C-8″), 149.1 (C, C-9″), 148.6 (C, C-8′), 144.8 (C, C-2), 136.6 (CH, C-2″), 136.4 (C, C-5), 135.1 (C, C7a″), 134.7 (C, C-7a′), 127.9 (C, C-3a″), 127.8 (C, C-3a′), 125.5 (CH, C-6″), 124.7 (CH, C-6′), 124.4 (CH, C-5″), 123.1 (CH, C-5′), 122.3 (CH, C-4″), 122.0 (CH, C-2′), 121.1 (CH, C-4′), 118.6 (CH, C-4), 116.6 (C, C-3″), 116.5 (CH, C-3′), 114.9 (CH, C-7″), 114.8 (CH, C-7′), 85.4 (C, C-10″), 84.1 (C, C-9′), 27.7 (3 × CH3, C-11″), 27.6 (3 × CH3, C-10′) ppm; ESMS m/z (rel int) 415 [M + H]+ (9), 371 (10), 254 (50), 228 (5), 210 (10), 182 (15), 155 (100), 144 (18), 116 (5); HRESMS m/z 527.2308 [M + H]+ (calcd for C30H31N4O5 527.2294). General Procedure for the Synthesis of N-tert-Butoxycarbonyl-Substituted Deoxytopsentins (33−37). Freshly sublimed SeO2 (248 mg, 2.25 mmol, 6.3 equiv) was added to a mixture of 1,4dioxane (4 mL) and H2O (101 μL, 15 equiv), after which the reaction mixture was heated to 60 °C to allow all the SeO2 to dissolve. To this was added 26 (100 mg, 0.36 mmol, 1 equiv), and the temperature increased to 75 °C. After 6 h the crude reaction mixture was filtered

through Celite, which was washed with CH2Cl2 (15 mL). The combined fractions were washed with H2O (2 × 10 mL) and saturated brine (2 × 10 mL) and dried over anhydrous MgSO4, and the organic fraction was concentrated in vacuo to yield an orange oil (118 mg). A portion of the crude reaction mixture (46 mg) was dissolved in EtOH (2 mL) followed by the addition of NH4OAc (60 mg, 5 equiv) and allowed to stir (5 h). The bright orange solution was concentrated in vacuo and redissolved in CH2Cl2 (10 mL). The organic solution was once again washed with H2O (2 × 10 mL) and saturated brine (2 × 10 mL) and dried over anhydrous MgSO4 to yield a yellow, amorphous solid (40 mg). Purification via Si flash chromatography (hexane/ EtOAc, 5:1), yielded 33 (20 mg, 0.035 mmol, 51% over two steps). The same procedure was applied to varying quantities of N-Bocprotected 3-acetylindoles 27−30 depending on availability using the same equivalents and solvent ratios above. For spectroscopic data of compounds 33−37 see the Supporting Information. General Procedure for the Thermal Cleavage of N-tertButoxycarbonyl-Substituted Deoxytopsentins (5−10). Compounds 32−37 were heated to 180 °C as dry solids under an inert atmosphere of argon. Once the desired temperature was achieved, gas bubbles were generated and subsided after 5 min, at which time the heat source was removed and the compounds were allowed to reach room temperature, yielding compounds 5−10 in quantitative yield. All compounds were routinely subjected to RP-HPLC (MeOH/H2O, 3:1), to deliver compounds suitably pure for bioassay. For spectroscopic data of compounds 5−7, 9, and 10 see the Supporting Information. Compounds 527 and 628 have been reported previously as marine natural products. 6″-Chloro-1H″-indol-3-yl[5-(6′-chloro-1H′-indol-3-yl)-1H-imidazole-2-yl]methanone (6′,6″-dichlorodeoxytopsentin) (8): yellow, amorphous solid; yield 100%; IR (cm−1) 2880 1672 1519 1445 1241 1137; 1H NMR (600 MHz, DMSO-d6, 1 equiv TFA) δ 12.26 (1H, s, NH-1″), 11.54 (1H, s, NH-1′), 9.19 (1H, s, H-2″), 8.35 (1H, d, J = 8.5 Hz, H-4″), 8.06 (1H, d, J = 8.4 Hz, H-4′), 8.03 (1H, s, H-2′), 7.79 (1H, s, H-4), 7.62 (1H, d, J = 1.6 Hz, H-7″), 7.51 (1H, d, J = 1.6 Hz, H-7′), 7.29 (1H, dd, J = 8.5, 1.8 Hz, H-5″), 7.14 (1H, dd, J = 8.5, 1.7 Hz, H-5′); 13C NMR (150 MHz, DMSO-d6, 1 equiv TFA) δ 175.2 (C, C-8″), 144.3 (C, C-2), 138.0 (CH, C-2″), 136.8 (C, C-7a″), 136.7 (C, C-7a′), 129.8 (C, C-6″), 128.3 (C, C-6′), 127.6 (C, C-3a″), 126.4 (C, C-3a′), 125.3 (C, C-5), 124.8 (CH, C-5″), 123.4 (CH, C-5′), 122.7 (CH, C-4″), 122.4 (CH, C-4′), 122.3 (CH, C-2′), 121.2 (CH, C-4), 120.0 (C, C-3″), 113.6 (CH, C-3′), 112.2 (CH, C-7″) 111.5 (CH, C-7′) ppm; ESMS m/z (rel int) 244 [M + H]+ (5), 191 (22), 189 (100), 181 (18), 177 (12), 149 (8); HRESMS m/z 395.0455 [M + H]+ (calcd for C20H1335Cl2N4O 395.0466). Synthesis of 7′,7″-Dichlorodeoxytopsentin (11). Compound 11 was synthesized as per the method described above for halogenated topsentin analogues 6−10 with the exception of the benchtop purification step, which was omitted in favor of deprotection of the crude reaction mixture. Routine RP-HPLC (MeOH/H2O, 3:1) delivered compound 11 as a yellow, amorphous solid (14 mg, 0.035 mmol, 35% over three steps). For spectroscopic data see the Supporting Information. Ligand−Receptor Docking Studies. All modeled ligands were constructed using Discovery Studio 3.5 Visualizer,42 and docking experiments were performed using AutoDock Vina26 against crystal structures of MRSA pyruvate kinase (PDB accession number 3T0T10 and 3T0715). The crystal structures are in complex with known inhibitors. Protein crystal structures were prepared for docking by removing the ligand present in the active site, followed by the addition of polar hydrogens in AutoDock tools. Atoms were assigned by the AutoDock 443 typing rules, and electrostatic charges were calculated as Gasteiger charges. Docking coordinates were defined by X, Y, and Z coordinates obtained from the originally cocrystallized ligand. The search space was confined to a cube of 30 Å with docking repeated eight times in the case of local searches and 80 Å with docking repeated 64 times in the case of global searches. Our method was validated by redocking the crystal structure ligands using our defined criteria. Docking success was evaluated by the lowest relative binding energy and visual inspection of ligand−receptor interaction. F



Article

Biological Assay. MRSA and human PK isoforms were sourced as His-tagged contructs, via a method described previously.9 PK inhibitory activity was determined by measuring UV absorbance changes of NADPH at 340 nm in a continuous assay coupled to rabbit muscle lactate dehydrogenase (L-LDH). Absorbance was measured using a Benchmark Plus microplate spectrophotometer (Bio-Rad). The reaction contained 60 mM Na+-HEPES, pH 7.5, 5% glycerol, 67 mM KCl, 6.7 mM MgCl2, 0.24 mM NADH, 5.5 units L-LDH, 2 mM ADP, and 10 mM PEP. Assayed compounds were dissolved in DMSO with final concentrations of DMSO never exceeding 1% of the assay volume. IC50 values were calculated by curve fitting on a fourparameter dose−response model with variable slope using Graphpad Prism 5.0.44 All values determined represent three measurements, each in triplicate.

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ASSOCIATED CONTENT

S Supporting Information *

1 H and 13C NMR spectra for all assayed compounds, spectroscopic data for all synthetic compounds not present in the main text, concentration−response curves for the inhibition of MRSA pyruvate kinase. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +2746 603 8096. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Rhodes University, the South African Medical Research Council (MRC), Genome Canada, Genome British Columbia, and the Natural Sciences and Engineering Research Council of Canada (NSERC). A postgraduate bursary from Deutscher Akademischer Austauschdienst (DAAD) awarded to R.M.Y. is gratefully acknowledged.

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DEDICATION Dedicated to Dr. William Fenical of Scripps Institution of Oceanography, University of California−San Diego, for his pioneering work on bioactive natural products.

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Synthetic Analogues of the Marine Bisindole Deoxytopsentin: Potent

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