Letter Cite This: Org. Lett. 2018, 20, 3788−3792
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Synthesis and Biological Evaluation of Hippolachnin A Analogues Jacob C. Timmerman and John L. Wood* Department of Chemistry and Biochemistry, Baylor University, One Bear Place 97348, Waco, Texas 76798, United States
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S Supporting Information *
ABSTRACT: The marine polyketide hippolachnin A (1) has attracted considerable attention from synthetic chemists due to both its intricate core structure and attractive biological activity against virulent fungi. Described herein are recent efforts to employ our recently developed synthesis in structure−activity relationship studies of 1. These studies have revealed that, in contrast to initial reports, 1 and several structural analogues lack activity against pathogenic fungi.
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thereby evoking, in a relative short period of time, the development of numerous total syntheses.6 The first total synthesis of 1, completed by Carreira and co-workers in 2014,6a was followed shortly thereafter by a second, developed collaboratively by the Wood and Brown groups. In this latter effort, a quadricyclane [2σ + 2σ + 2π] cycloaddition and subsequent C−H oxidation were employed to rapidly construct the core ring system (Scheme 1); late-stage condensation and transesterification then completed the overall six-step sequence to deliver 1. Although several other
ystemic fungal infections are a pervasive source of morbidity and mortality, particularly among immunocompromised populations, and the clinical need for new antifungal therapies has outpaced their development.1−3 As a result, the identification of new antifungal agents possessing unique structures or modes of action are of considerable interest. Based on this growing need, it is understandable that the 2013 report by Lin and co-workers of a new and potently active antifungal agent,4 hippolachnin A (1, Figure 1), garnered
Scheme 1. Wood and Brown Collaborative Total Synthesis of (±)-Hippolachnin A (1)
Figure 1. Hippolachnin A (1).
considerable interest from the synthetic community. Biological assays reported in this original study were said to have demonstrated that 1, which had been isolated from the marine sponge Hippospongia lachne, possesses potent activity against a variety of virulent fungi, in particular, C. neoformans, T. rubrum, and M. gypseum, with MIC values of 0.41 μM; 1 likewise was reported to have activity against C. glabrata and C. albicans with MICs of 1.63 and 13.1 μM, respectively. This data, taken together with its unique structure, suggested that 1 potentially represented a new class of antifungal agents.5 As alluded to above, the interesting biological activity of 1, along with its unique and highly congested octahydro-4oxacyclobtua[cd]pentalene core ring system (Figure 1, highlighted in red), piqued the attention of several research groups © 2018 American Chemical Society
Received: May 1, 2018 Published: June 19, 2018 3788
DOI: 10.1021/acs.orglett.8b01381 Org. Lett. 2018, 20, 3788−3792
Letter
Organic Letters groups have since reported completed syntheses of 1,6c,d the Wood/Brown effort remains the shortest described to date.6b Having completed the total synthesis, we began to consider the biological potential of 1 and possibilities for accessing a range of analogues. The concise synthesis outlined in Scheme 1 was envisioned as amenable to the rapid production of analogues for both structure−activity relationship studies and for use in efforts to define the biological mode of action. Herein, we describe recent efforts toward the former that, in contrast to the original report, reveal that neither 1 nor several structurally related compounds exhibit antifungal activity against C. neoformans or C. albicans. In considering potential analogs, we recognized that 1 can be divided into two hemispheres, a polar region possessing the vinylogous carbonate moiety, and a nonpolar hemisphere comprising four syn-facial ethyl groups arranged on the octahydro-4-oxacyclobuta[cd]pentalene core (Figure 2). We
(Scheme 2, left). Condensation of lactone 6 with tert-butyl acetate and subsequent hydrogenation led to the isolation of (Z)-tert-butyl vinylogous carbonate 7 (Scheme 2, right).11 In a manner similar to our previous report, transesterification (Z)-7 with methanol led to the isolation of both hippolachnin A (1) and its (E)-alkene isomer (8) in quantities sufficient for various biological assays (Scheme 2, bottom right). Transesterification could also be accomplished using longer chain primary alcohols, such as n-butanol (Scheme 3, left); Scheme 3. Synthesis of Vinylogous Carbonic Acid 10 and nButylhippolachnin 11
however, attempts to similarly introduce more complex alcohols led to intractable mixtures (see Supporting Information for details). Upon treatment with trifluoroacetic acid, vinylogous carbonate 7 (1:1.4 E/Z mixture) could be hydrolyzed to reveal vinylogous carbonic acid 10 as a single isomer (Scheme 3, right). Acid 10 could serve as a positive control for cellular hydrolysis of 1 and likewise serve as a progenitor to other analogs through amide coupling or similar transformations. Further manipulations of the polar region were envisioned that involved substitution of the vinylogous carbonate moiety with nitrogen-containing motifs, such as carbamates or a vinylogous cyanate. To this end, reaction of lactone 6 with acetonitrile anion led to isolation of vinylogous cyanates 12b and 12c in a 1:1.7 ratio, respectively, and in 35% combined yield (Scheme 4). Surprisingly, the major component of this reaction mixture was identified as hemiacetal 12a, which was isolated in 47% yield as a 11:1 mixture of diastereomers.
Figure 2. Topological dissection of 1.
viewed both regions as important, with the former serving as a possible point of hydrolysis or covalent attachment to the target,7,8 and the latter providing the hydrophobicity needed for effective diffusion into the cell or other noncovalent binding interactions.9 Thus, our approach was to independently vary these regions of the molecule. As illustrated in Scheme 2, we began our investigations by targeting analogues of 1 with altered vinylogous carbonate Scheme 2. Synthesis of tert-Butyl Vinylogous Carbonate 7, Saturated Lactone 9, Synthetic 1, and (E)-Hippolachnin A (8)
Scheme 4. Synthesis of Hemiacetal 12 and Vinylogous Cyanates 12a and 12b
The vinyl moieties in 12a could be hydrogenated over prolonged reaction times employing THF as a solvent to produce saturated hemiacetal 13 (Scheme 5), which was isolated as a single diastereomer; the relative configuration of 13 was confirmed by NOE analysis. Hydrogenation of 12a in methanol led to both reduction of the vinyl moieties and concomitant dehydration of the hemiacetal, ultimately leading to near quantitative isolation of vinylogous cyanates 14a and 14b in a 1.1:1 ratio, respectively (Scheme 5a, bottom). Vinylogous cyanate 14a could be selectively hydrolyzed to vinylogous carbamate 15 under basic conditions (Scheme 5b). With the intent to perform target identification studies of 1, we installed an alkyne linker at the vinylogous carbamate by treating acid 10 with amine linker 16 under amide-coupling
moieties. To this end, we employed an early synthetic approach to prepare multiple hundred-milligram quantities of tricyclic lactone 6 (see the Supporting Information for details).10 The three syn-facial vinyl groups of lactone 6 could be reduced yielding saturated lactone 9 in excellent yield 3789
DOI: 10.1021/acs.orglett.8b01381 Org. Lett. 2018, 20, 3788−3792
Letter
Organic Letters Scheme 5. (a) Hydrogenation of Hemiacetal 12a. (b) Hydrolysis of Vinylogous Cyanate 14a to Vinylogous Carbamate 15
Scheme 8. Total Synthesis of C10-Propylhippolachnin A (23)
conditions to afford functionalized vinylogous carbamate 17 (Scheme 6).12 Scheme 6. Synthesis of 17 via an Amide Coupling of Vinylogous Carbonate 10
reduction of the pseudoequatorial carbomethoxy moiety, ringopening cross metathesis, Wittig methylenation, and subsequent hydrolysis led to carboxylic acid 21, which proved isolable as a single diastereomer by silica gel column chromatography (Scheme 8). Exposure of 21 to C−H oxidation as described for the synthesis of lactone 6 was effective in producing lactone 22 (38% yield). An end-game sequence analogous to that employed in the synthesis of 1 was used to convert 22 to C10-propylhippolachnin A (23). Attempts to apply a similar synthetic sequence employing a variety of benzylidene malonates were met with difficulty at various stages in the sequence. With synthetic 1 in hand, along with several analogues thereof, we began an initial round of biological testing against C. neoformans and C. albicans in collaboration with the Community for Antimicrobial Drug Discovery at the University of Queensland.15 Table 1 summarizes the reported
To explore the importance of the vinylogous carbonate moiety, we sought to reduce 1 to 2,3-dihydrohippolachnin A (18). The double bond of 1 proved unreactive to hydrogenation under a variety of standard conditions. However, in taking precedent from Bartlett13 and Barrett,14 treatment of 1 with rhodium on alumina at 260 PSI of hydrogen led to the formation of ester 18 as a single diastereomer (Scheme 7),
Table 1. Biological Testing Data for Natural (+)-1 and Synthetic (±)-1 against C. neoformans and C. albicans
Scheme 7. Hydrogenation of 1 to Isolated Ester 18
MIC (μM) fungus
natural (+)-1
synthetic (±)-1
C. neoformans C. albicans
0.41 13.2
>104a >104b
a
41.1% of fungal growth inhibited at this concentration. b13.7% of fungal growth inhibited at this concentration.
MICs of natural 1 and those obtained by us for synthetic 1 against both C. neoformans and C. albicans. In the latter, as well as assays against a variety of other microbes and some human cancer cell lines, synthetic 1 showed little to no activity (see the Supporting Information).16 Similarly, synthetic analogues 7, 8, 9, 10, 11, 13, 14a, 15, 17, 18, and 23 were found to be uniformly inactive against C. neoformans and C. Albicans in addition to a broader panel of both microbial and human cell lines.17 As delineated below, while natural 1 was reported to exhibit MIC values of 0.41 and 13.1 μM against C. neoformans and C.
resulting from reduction at the convex face of 1. The relative configuration of ester 18 was confirmed by NOE analysis and by comparison with its C3 diastereomer described by Carreira and Brown.6a,b In order to probe the SAR of the nonpolar hemisphere of 1, we sought to change the substituent pattern with respect to the four syn-facial ethyl groups. To this end, [2σ + 2σ + 2π] quadricyclane cycloaddition of butylidene malonate 19 led to adduct 20 in 57% yield and in 4.1:1 dr (Scheme 8). While separation of this diastereomeric mixture was problematic, 3790
DOI: 10.1021/acs.orglett.8b01381 Org. Lett. 2018, 20, 3788−3792
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Organic Letters
(2) (a) Richardson, M. R. J. Antimicrob. Chemother. 2005, 56, S5− S11. (b) Butts, A.; Krysan, D. J. PLoS Pathog. 2012, 8, e1002870. (c) Arendrup, M. C. Clin. Microbiol. Infect. 2013, 20, 42−48. (3) (3) Roemer, T.; Krysan, D. J. Cold Spring Harbor Perspect. Med. 2014, 4, a019703. (4) Piao, S.-J.; Song, Y.-L.; Jiao, W.-H.; Yang, F.; Liu, X.-F.; Chen, W.-S.; Han, B.-N.; Lin, H.-W. Org. Lett. 2013, 15, 3526−3529. (5) Nett, J. E.; Andes, D. R. Infect. Dis. Clin. N. Am. 2016, 30, 51−83. (6) (a) Ruider, S. A.; Sandmeier, T.; Carreira, E. M. Angew. Chem., Int. Ed. 2015, 54, 2378−2382. (b) McCallum, M. E.; Rasik, C. M.; Wood, J. L.; Brown, M. K. J. Am. Chem. Soc. 2016, 138, 2437−2442. (c) Winter, N.; Trauner, D. J. Am. Chem. Soc. 2017, 139, 11706− 11709. (d) Li, Q.; Zhao, K.; Peuronen, A.; Rissanen, K.; Enders, D.; Tang, Y. J. Am. Chem. Soc. 2018, 140, 1937−1944. (e) Datta, R.; Dixon, R. J.; Ghosh, S. A. Tetrahedron Lett. 2016, 57, 29−31. (7) For a review on pharmaceuticals based on covalent mechanisms of action, see: Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. Nat. Rev. Drug Discovery 2011, 10, 307−317. (8) Yang, Y.-.H.; Aloysius, H.; Inoyama, D.; Chen, Y.; Hu, L.-.Q. Acta Pharm. Sin. B 2011, 1, 143−159. (9) Ghannoum, M. A.; Rice, L. B. Clin. Microbiol. Rev. 1999, 12, 501−517. (10) Lactone 6, derived from the Wood route synthesis of 1, was identified as an ideal starting material for derivatization owing to its modularity with respect to the substituent at C4 (hippolachnin A numbering; see the Supporting Information for details). This modularity stems from utilization of an alkylidene malonate in the quadricyclane [2 + 2 + 2] cycloaddition in the Wood route, whereas the Brown route employs a [2 + 2 + 2] cycloaddition with acid 2 that already possesses an ethyl group at C4. (11) Typically, the crude material after hydrogenation contained a ∼ 1:1.4 mixture of (E)-7 and (Z)-7, respectively, and was pure enough for further manipulation. Purification of the crude material by silica gel chromatography led to isolation of (Z)-7, along with (E)-7, which contained unidentified, coeluting impurities. (12) (a) Nomura, D. K.; Dix, M. M.; Cravatt, B. F. Nat. Rev. Cancer 2010, 10, 630−638. (b) Leslie, B. J.; Hergenrother, P. J. Chem. Soc. Rev. 2008, 37, 1347−1360. (13) Bartlett, P. A.; Jernstedt, K. K. Tetrahedron Lett. 1980, 21, 1607. (14) Barrett, A. G. M.; Sheth, H. G. J. J. Org. Chem. 1983, 48, 5017− 5022. (15) Blaskovich, M. A.; Zuegg, J.; Elliott, A. G.; Cooper, M. A. ACS Infect. Dis. 2015, 1, 285−287. (16) Racemic hippolachnin A prepared by Trauner has been found to display a similar lack of activity in assays performed by Prof. Dr. Marc Stadler at the Helmholtz Centre for Infection Research, Braunshweig, Germany. Trauner, D. Personal communication, New York University, 2018. (17) See the Supporting Information for biological assay details. (18) DMax denotes percentage inhibition observed at the maximum concentration tested (104 μM for 1). (19) This analysis assumes that the non-natural enantiomer of 1 displays no inhibitory activity; this scenario establishes an upper limit of inhibitory activity of synthetic 1. (20) Repeated attempts to obtain this information from the corresponding authors of the original isolation study4 were met with no response. (21) It is not clear from the isolation report what culture broth was used for C. neoformans and C. ablicans. Our testing employed yeastnitrogen broth, which has been shown to provide more clinically relevant MIC values: (a) Ghannoum, M. A.; Ibrahim, A. S.; Fu, Y.; Shafiq, M. C.; Edwards, J. E.; Criddle, R. S. J. Clin. Microbiol. 1992, 30, 2881−2886. (b) Cuenca-Estrella, M.; Diaz-Guerra, T. M.; Mellado, E.; Rodriguez-Tudela, J. L. J. Clin. Microbiol. 2001, 39, 525−532. (c) Rex, J. H.; Pfaller, M. A.; Walsh, T. J.; Chaturvedi, V.; Espinel-Ingroff, A.; Ghannoum, M. A.; Gosey, L. L.; Odds, F. C.; Rinaldi, M. G.; Sheehan, D. J.; Warnock, D. W. Clin. Microbiol. Rev. 2001, 14, 643−658.
albicans, respectively, these data show synthetic 1 has an MIC > 104 μM for both C. neoformans and C. albicans.4 Extrapolating from DMax values,18 it can be estimated that synthetic 1 is on the order of ca. 125 times less active against C. neoformans than described previously in the literature.5,19 Although a scenario in which only the natural enantiomer of 1 is active could indeed be operative, that alone cannot explain the marked discrepancy in activity of natural and racemic, synthetic 1 against C. neoformans. However, since neither the culture conditions nor the exact varieties of C. neoformans and C. albicans used in the original study were disclosed,20 a caveat remains regarding the observed lack of activity. The data presented here indicate that 1 lacks activity against C. neoformans variety grubii (strain H99, ATCC 208821) and the CLSI standard C. albicans (ATCC 90028).21−23 Exploiting the modularity of our original synthetic route to the total synthesis of 1, we were able to generate a variety of analoges for biological testing. Data from the latter revealed that neither synthetic 1 nor any of the prepared analogues possess the promising biological activity reported in the original disclosure.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01381.
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Experimental procedures, spectroscopic data, and NMR spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
John L. Wood: 0000-0002-9066-5588 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support for this work was provided by The Welch foundation (Chair, AA-006) and the Cancer Prevention Research Institute of Texas (CPRIT, R1309). Antimicrobial screening was performed by CO-ADD (The Community for Antimicrobial Drug Discovery), funded by the Wellcome Trust (UK) and The University of Queensland (Australia). The authors also acknowledge Dr. Kenneth Hull (Baylor University) for helpful discussions and Murray D. Wan for preliminary investigations into the analogue syntheses.
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REFERENCES
(1) (a) Crawford, J.; Dale, D. C.; Lyman, G. H. Cancer 2004, 100, 228−237. (b) Park, B. J.; Wannemuehler, K. A.; Marston, B. J. AIDS 2009, 23, 525−530. (c) Pagano, L.; Akova, M.; Dimopoulos, G.; Herbrecht, R.; Drgona, L.; Blijlevens, N. J. Antimicrob. Chemother. 2011, 66 (Suppl I), 5−14. (d) Lockhart, S. R.; Iqbal, N.; Cleveland, A. A.; Farley, M. M.; Harrison, L. H.; Bolden, C. B.; Baughman, W.; Stein, B.; Hollick, R.; Park, B. J.; Chiller, T. J. Clin. Microbiol. 2012, 50, 3435−3442. (e) Vallabhaneni, S.; Cleveland, A.; Farley, M.; Harrison, L. H.; Schaffner, W.; Beldavs, Z. G.; Derado, G.; Pham, C. D.; Lockhart, S. R.; Smith, R. Open Forum Infect. Dis 2015, 2, ofv163. 3791
DOI: 10.1021/acs.orglett.8b01381 Org. Lett. 2018, 20, 3788−3792
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Organic Letters (22) It has been documented that culture medium can affect susceptibility to antifungal agents: (a) Doern, G. V.; Tubert, T. A.; Chapin, K.; Rinaldi, M. G. J. Clin. Microbiol. 1986, 24, 507−511. (b) Del Poeta, M.; Barchiesi, F.; Morbiducci, V.; Arzeni, D.; Marinucci, G.; Ancarani, F.; Scalise, G. J. J. Chemother. 1994, 6, 173−176. (23) For other examples of synthetic natural products which display contrasting biological activity to their respective isolation reports, see: (a) Hirose, T.; Noguchi, Y.; Furuya, Y.; Ishiyama, A.; Iwatsuki, M.; Otoguro, K.; O̅ mura, S.; Sunazuka, T. Chem. - Eur. J. 2013, 19, 10741−10750. (b) Cernijenko, A.; Risgaard, R.; Baran, P. S. J. Am. Chem. Soc. 2016, 138, 9425−9428. (c) Villaume, M. T.; Sella, E.; Saul, G.; Borzilleri, R. M.; Fargnoli, J.; Johnston, K. A.; Zhang, H.; Fereshteh, M. P.; Dhar, T. G. M.; Baran, P. S. ACS Cent. Sci. 2016, 2, 27−31.
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DOI: 10.1021/acs.orglett.8b01381 Org. Lett. 2018, 20, 3788−3792
