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Synthesis of myrocin G, the putative active form of the myrocin antitumor antibiotics. Christos Economou, Martin Tomanik, and Seth B Herzon J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10891 • Publication Date (Web): 11 Nov 2018 Downloaded from http://pubs.acs.org on November 11, 2018
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Journal of the American Chemical Society
Synthesis of myrocin G, the putative active form of the myrocin antitumor antibiotics. Christos Economou,1 Martin Tomanik,1 and Seth B. Herzon1,2,* 1
Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States. 2Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut 06520, United States. Supporting Information Placeholder
ABSTRACT: The antiproliferative antimicrobial fungal metabolites known as the myrocins have been proposed to cross-link DNA by double nucleotide addition. However, the nature of the DNA-reactive species is ambiguous, as myrocins have been isolated as functionally-distinct 5-hydroxy-γ-lactone and diosphenol isomers. Based on computational studies and literature precedent, we hypothesized that the diosphenol 7 (assigned here the trivial name myrocin G) is the biologically-active form of the representative isolate (+)-myrocin C (1). To probe this, we developed a short enantioselective route to 7. A powerful fragment coupling reaction that forms the central ring of the target in 38% yield and in a single step was developed. In support of our hypothesis, 7 was efficiently transformed to the bis(sulfide) 6, a product previously isolated from reactions of 1 with benzenethiol. This work provides the first direct access to the diosphenol 7, sets the stage for elucidating the mode of interaction of the myrocins with DNA, and provides a foundation for the synthesis of other pimarane diterpenes.
Scheme 1. A. Structures of myrocins A–C (1–3). B. Structure of the bis(sulfide) 6 and the originally proposed mechanism for its formation. C. The diosphenol 7 is 1.4 kcal/mol more stable than 1 and hypothesized to be the biologically-active form of 1. D. Retrosynthetic analysis of 7. A.
CH 3
CH 3
O C
C A CH 3
A
OH H
O O OH
H
CH 3
C
OH
A
O O OH
(–)-myrocin B (2)
(+)-myrocin C (1)
OH O
CH 3 HO
O
O
OH
O
(–)-myrocin A (3)
PhSH, –H 2O CH 3
B.
CH 3
SPh CH 3
PhSH
SPh
O H O OH
O
CH 3 HO
O
O
SPh OH
CH 3 HO
OH
O
OH
6 (63% from 1)
5
4
CH 3
PhS
C.
Efforts to elucidate the mechanism of action of natural products are complicated when the metabolite can adopt two or more functionally-distinct forms. This issue is exemplified by the antiproliferative antimicrobial metabolites myrocins C (1)1 and B (2),2,3 fungal isolates that contain a sensitive 5-hydroxy-γ-lactone residue (Scheme 1A, blue in 1 and 2). Literature indicates4 this substructure undergoes facile ring-opening to the corresponding diosphenol under mildly acidic or basic conditions, raising uncertainty about its fidelity under biological conditions. Consistent with this, the diosphenol isomer of 2, (–)-myrocin A (3), has been identified in fungal cultures.5 Following their landmark total synthesis of (±)-myrocin C (1),6 Danishefsky and Chu-Moyer disclosed that treatment of synthetic (±)-1 with excess thiophenol and triethylamine generated the bis(sulfide) 6 (63%, Scheme 1B).7 The mechanism for formation of 6 was
CH 3
O
CH 3 2 PhSH
(+)-myrocin C (1)
OH CH 3 HO
–H2O
O O
O H
myrocin G (7) putative active form of 1
D.
O R’O
13 9
7
10
A 4
CH 3 RO
C
OH O
I
CH 3
A CH 3 RO
10
O
4
O
+
I
13 9
C
CH 3
O
O 8
9
10
proposed to comprise SN2´ substitution of the tertiary hydroxyl group (1®4), isomerization to the diosphenol 5, and addition to the resulting activated cyclopropane. This reactivity led the authors to speculate that the myrocins cross-link DNA by sequential nucleotide addition reactions.8
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Scheme 2. Synthesis of the fragment coupling partners 9 and 10. TMSE = 2-(trimethylsilyl)ethyl.
The isolation of 3 suggests the existence of an analogous diosphenol isomer of 1, “myrocin G (7)” (Scheme 1C). Ring-opening of a lactone structurally related to 1 has been reported to occur under acidic or basic conditions.4b The diosphenol isomer may be stabilized by a strong intramolecular hydrogen bond between the hydroxyl group and the adjacent carbonyl. Collectively, these data suggested to us an alternative order of events for 1®6 wherein ring-opening of 1 to the diosphenol 7 precedes the initial alkylation. Motivated by this analysis, we targeted 7 as the initial entry into this natural product family. In addition, we identified the diosphenol double bond as a strategic locus that could be converted retrosynthetically to the diketone 8 in a redox-neutral fashion (Scheme 1D). Further disconnection of the C9–C10 bond by a fragment coupling reaction9 reveals the α,β-cyclopropylketone 9 and the unsaturated ketone 10 as two precursors of similar complexity. This synthetic strategy features the direct, redox-neutral installation of the C9 alcohol, high modularity, and independent introduction of the peripheral C4 and C13 quaternary centers. The coupling fragments 9 and 10 were prepared in 3– 4 steps from known compounds (Scheme 2). Beginning with the Diels–Alder adduct 11,14 Wittig olefination [potassium bis(trimethylsilyl)amide, methyl triphenylphosophium bromide], tandem enoxysilane hydrolysis and bcarbamate elimination (aqueous hydrochloric acid), and α-dehydroiodination (iodine, pyridine)10 provided the Cring fragment 10 (22% over three steps, Scheme 2A). The A-ring fragment 9 was synthesized from the b-ketoester 12 (Scheme 2B).11 Stereoselective Robinson annulation12 between 12 and acrolein diethylacetal provided the enone 13 (32%, 92% ee). α-Dehydroiodination10 of 13 proceeded in 97% yield. Corey–Chaykovsky cyclopropanation13 (trimethylsulfoxonium iodide, sodium hydride) provided a 2.3:1 mixture
CH3 CHO Bn N CO2CH3
A. TBSO
t-Bu 1.
CH3 CH3 O O TMSE
I 10
22% (3 steps)
NEt2 NH2•HOTf
3-NO2C6H4CO2H
O
O
3. I2, DMAP, pyr.
11 • prepared in four steps, 20% yield, 95% ee
B.
CH3
1. KHMDS, PPh3CH3Br 2. 1 N HCl
CH(OEt)2
CH3 O O TMSE
3. NaH (CH3)3SOI
32%, 92% ee
2.3:1 d.r. 64% (desired)
13
12 • prepared in two steps, 77% yield
I
2. I2, pyr. 97%
O
CH3 O O TMSE
9
of diastereomeric α,β-cyclopropylketones. The major (desired) diastereomer 9 was isolated in 64% yield after recrystallization. The fragment coupling product 14 was obtained by activation of the iodocyclopropane 9 with iso-propylmagnesium chloride–lithium chloride complex,14 followed by addition of the iodoenone 10 (92%, 8.2:1 dr, Scheme 3). The stereoselectivity in the addition was anticipated based on the known stereoelectronic preferences for nucleophilic addition to cycloalkanones15 and consideration of non-bonded interactions in the transition state. Notably, retro-aldol reaction of 14 is prevented by the geometric constraints introduced by the cyclopropane ring. The ring closure precursor 16 was prepared by a fivestep sequence comprising Stille cross-coupling [tetrakis(triphenylphosphine)palladium-(0), copper(I) iodide, cesium fluoride] with tributyl(1-ethoxyvinyl) tin,
Scheme 3. Synthesis of 19. 2.
I I CH 3 O O TMSE
9
1. i-PrMgCl•LiCl
O
then 10 92%, 8.2:1 dr
CH 3 O O TMSE
OH O
CH 3
AllocO
CH 3
SnBu 3 O
OEt , CuI Pd(PPh 3)4, CsF
CH 3
3. 1 N HCl CH 3 O O TMSE
90% (2 steps) 14
9
OH O
O
4. TMSOTf, Et 3N 5. m-CPBA
CH 3 OTMS O
6. AllocCl, pyr. 41% (3 steps)
CH 3 O O TMSE
15
16 7. NaOt-Bu, THF, 0 °C
CH 3
CH 3 CH 3
9
9
OH
OR
CH 3 OR O O OH TMSE 20 R = TMS 15%
O
CH 3 OH O O TMSE
TMSEO2C
O
O
CH 3 TMSEO2C CH 3
CH 3 AllocO
–O –O
O
O TMS
18
19 R = TMS 64%
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O
O O
O
TMS OAllyl
17
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hydrolysis of the resulting vinyl ether (aqueous hydrochloric acid), tandem alcohol silylation–enoxysilane formation (trimethylsilyl trifluoromethanesulfonate, triethylamine), Rubottom oxidation (3-chloroperoxybenzoic acid), and conversion of the primary alcohol to an allyl carbonate (allyl chloroformate, pyridine, 37% overall). The structure of the intermediate α-hydroxyketone was confirmed by X-ray analysis.16 After much experimentation, we found that treatment of the allyl carbonate 16 with sodium tert-butoxide in tetrahydrofuran at 0 °C generated the diosphenol 19 (64%). Mechanistic studies suggest that 19 is formed via aldol addition (16→17), carbonate migration (17→18), and βelimination. The silyl migration product 20 was isolated separately in 15% yield. This cyclization cascade provides expedient access to a protected form of 7. After some consideration, we recognized that the fragment coupling–ring closure cascade could potentially be carried out in one flask by embedding a latent enolate nucleophile in the C-ring electrophile. Toward this end, we prepared the enoxysilane 24 by the sequence shown in Scheme 4. Beginning with the α-iodoenone 10, ketalization (ethylene glycol, p-toluenesulfonic acid) followed by lithium–halogen exchange and addition of the Weinreb amide17 21 provided the α,β-unsaturated ketone 22 (70%). Removal of the silyl ether (tetra-n-butylammonium fluoride), installation of the allyl carbonate (allyl chloroformate, pyridine), and removal of the acetal (aqueous hydrochloric acid) generated the βdiketone 23. Site-selective deprotonation of 12 (lithium hexamethyldisilazide) and trapping of the resulting enolate with chlorotrimethylsilane provided the target enoxysilane 24. Attempts to effect the fragment coupling of the enoxysilane 24 with the organomagnesium reagent derived from 9 were unsuccessful. We found, however, that lithium–halogen exchange (n-butyllithium, –78 °C), followed by immediate addition of the enoxysilane 24 and warming to 0 °C provided the fully annulated Scheme 4. Synthesis of the enoxysilane 24. CH3
CH3
1. (HOCH2)2, PTSA, (EtO)3CH
O O O
2. n-BuLi, 21
O
I
70% (two steps)
OTBS 22
10 O TBSO
21
CH3
N OCH3
3. TBAF 4. AllocCl, pyr. 5. 1 N HCl 46% (three steps)
6. TMSCl, LiHMDS
OAlloc
24
1. n-BuLi CH 3 O O TMSE
9
OAlloc
23
+
O
O O TMS
OAlloc
25
24
CH 3
CH 3 TMSEO2C
TMSEO2C
O O O – OTMS Alloc
O CH 3 O O O – TMS Alloc
CH 3
27
26 CH 3
CH 3
TMSEO2C
TMSEO2C
CH 3 –O
O O
O
CH 3 AllocO
O
TMS OAllyl
O TMS
O
–O
17
18 CH 3
CH 3 9
2. TBAF
OH CH 3 O H
O
64%
OH
O 7
OR O
CH 3 OH O O TMSE 19 R = TMS 38%
product 19 in 38% yield (Scheme 5). The modest yield of this transformation is offset to some extent by the rapid increase in molecule complexity achieved. Deprotection of 18 (tetra-n-butylammonium fluoride) then provided the target 7 (64%). Subjecting synthetic 7 to the conditions disclosed by Danishefsky and Chu-Moyer7 generated the bis(sulfide) 6 (74%, Scheme 6). This result provides support for the our hypothesis and indicates that the diosphenol 7 is a competent intermediate in the double nucleophilic addition of thiols. We reasoned that (+)-myrocin C (1) itself might be accessible by temporarily disrupting the diosphenol hydrogen bond in 19 or 7, thereby making lactonization thermodynamically favorable. However, exploratory experiments toward this end have thus far been unsuccessful. In summary, we have developed a concise, enantioselective synthesis of “myrocin G” (7), the putative active Scheme 6. Synthesis of the bis(sulfide) from the diosphenol 7. CH3
CH3
PhS PhSH (10 equiv) Et3N (10 equiv)
OH O
O
Li
O
CH 3 O O TMSE
O
59%
TMS
CH 3 I
CH3
CH3 O
Scheme 5. One-step synthesis of 19 from 9 and 24.
CH3 HO
O O
OH
myrocin G (7)
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SPh
CH2Cl2, 23°C 74%
CH3 HO
OH O
OH 6
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form of the antiproliferative antimicrobial metabolite myrocin C (1). Key to the success of this approach was the development of a powerful annulation strategy that forges the central ring of the target in a single step from two synthetic precursors of similar complexity. With access to 7, we are now in position to probe its antiproliferative activity and determine its biological target. The fragment coupling reaction we have developed should be amenable to the synthesis of other pimarane diterpenes. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. The Supporting Information file contains detailed experimental procedures and characterization data for all new compounds.
AUTHOR INFORMATION Corresponding Author
[email protected] Funding Sources
No competing financial interests have been declared. Financial support from the National Institutes of Health (Ruth L. Kirschstein National Research Service Award F31CA213964-03 to C.E.) and Yale University is gratefully acknowledged.
REFERENCES 1. (a) Hsu, Y. H.; Hirota, A.; Shima, S.; Nakagawa, M.; Nozaki, H.; Tada, T.; Nakayama, M. Structure of Myrocin C, A New Diterpene Antibiotic Produced by a Strain of Myrothecium sp. Agric. Biol. Chem. 1987, 51, 3455. (b) Nakagawa, M.; Hsu, Y. H.; Hirota, A.; Shima, S.; Nakayama, M. MYROCIN C, A NEW DITERPENE ANTITUMOR ANTIBIOTIC FROM MYROTHECIUM VERRUCARIA. I. TAXONOMY OF THE PRODUCING STRAIN, FERMENTATION, ISOLATION AND BIOLOGICAL PROPERTIES. J. Antibiot. 1989, 42, 218. (c) Hsu, Y. H.; Hirota, A.; Shima, S.; Nakagawa, M.; Adachi, T.; Nozaki, H.; Nakayama, M. MYROCIN C, A NEW DITERPENE ANTITUMOR ANTIBIOTIC FROM MYROTHECIUM VERRUCARIA. II. PHYSICOCHEMICAL PROPERTIES AND STRUCTURE DETERMINATION. J. Antibiot. 1989, 42, 223. 2. (a) Hsu, Y. H.; Nakagawa, M.; Hirota, A.; Shima, S.; Nakayama, M. Structure of Myrocin B, A New Diterpene Antibiotic Produced by Myrothecium verrucaria. Agric. Biol. Chem. 1988, 52, 1305. (b) Lehr, N.-A.; Meffert, A.; Antelo, L.; Sterner, O.; Anke, H.; Weber, R. W. S. Antiamoebins, myrocin B and the basis of antifungal antibiosis in the coprophilous fungus Stilbella erythrocephala (syn. S. fimetaria). FEMS Microbiol. Ecol 2006, 55, 105. 3. For a review of pimarane diterpenes, see: Wang, X.; Yu, H.; Zhang, Y.; Lu, X.; Wang, B.; Liu, X. Bioactive Pimarane-type
Diterpenes from Marine Organisms. Chem. Biodiversity 2018, 15, e1700276. 4. (a) Langschwager, W.; Hoffmann, H. M. R. Ring-Chain Tautomerism Provides a Route to 7a-Hydroxy-3a-methyl-2,7dioxoperhydrobenzofuran. Synthesis of the Hydroxy γ-Lactone Substructure of Myrocin and Other Bioactive Natural Products. Liebigs Ann. 1995, 797. (b) Zander, N.; Langschwager, W.; Hoffmann, H. M. R. The Enol Lactone Approach to Protected Hydroxy γ-Lactones (5Hydroxy-dihydro-furan-2-ones). Synth. Commun. 1996, 26, 4577. 5. (a) Klemke, C.; Kehraus, S.; Wright, A. D.; König, G. M. New Secondary Metabolites from the Marine Endophytic Fungus Apiospora montagnei. J. Nat. Prod. 2004, 67, 1058. (b) Tsukada, M.; Fukai, M.; Miki, K.; Shiraishi, T.; Suzuki, T.; Nishio, K.; Sugita, T.; Ishino, M.; Kinoshita, K.; Takahashi, K.; Shiro, M.; Koyama, K. Chemical Constituents of a Marine Fungus, Arthrinium sacchari. J. Nat. Prod. 2011, 74, 1645. 6. Chu-Moyer, M. Y.; Danishefsky, S. J. A Remarkable Cyclopropanation: The Total Synthesis of Myrocin C. J. Am. Chem. Soc. 1992, 114, 8333. 7. Chu-Moyer, M. Y.; Danishefsky, S. J. The Mode of Action of Myrocin C: Evidence For a CC-1065 Connection. Tetrahedron Lett. 1993, 34, 3025. 8. For a review of natural products that damage DNA by nucleotide addition to an activated cyclopropane, see: Wolkenberg, S. E.; Boger, D. L. Mechanisms of in Situ Activation for DNA-Targeting Antitumor Agents. Chem. Rev. 2002, 102, 2477. 9. This disconnection drew inspiration from a related elegant fragment coupling employed by Baran and co-workers en route to (–)maoecrystal V. See: Cernijenko, A.; Risgaard, R.; Baran, P. S. 11-Step Total Synthesis of (−)-Maoecrystal V. J. Am. Chem. Soc. 2016, 138, 9425. 10. Johnson, C. R.; Adams, J. P.; Braun, M. P.; Senanayake, C. B. W.; Wovkulich, P. M.; Uskokovic, M. R. Direct α-Iodination of Cycloalkenones. Tetrahedron Lett. 1992, 33, 917. 11. (a) Ueda, Y.; Roberge, G.; Vinet, V. A simple method of preparing trimethylsilyl- and tert-butyldimethylsilyl-enol ethers of αdiazoacetoacetates and their use in the synthesis of a chiral precursor to thienamycin analogs. Can. J. Chem. 1984, 62, 2936. (b) Meng, Z.; Yu, H.; Li, L.; Tao, W.; Chen, H.; Wan, M.; Yang, P.; Edmonds, D. J.; Zhong, J.; Li, A. Total synthesis and antiviral activity of indolosesquiterpenoids from the xiamycin and oridamycin families. Nat. Commun. 2015, 6, 6096. 12. Xu, C.; Zhang, L.; Luo, S. Asymmetric Enamine Catalysis with β-Ketoesters by Chiral Primary Amine: Divergent Stereocontrol Modes. J. Org. Chem. 2014, 79, 11517. 13. Corey, E. J.; Chaykovsky, M. Dimethyloxosulfonium Methylide ((CH3)2SOCH2) and Dimethylsulfonium Methylide ((CH3)2SCH2). Formation and Application to Organic Synthesis. J. Am. Chem. Soc. 1965, 87, 1353. 14. (a) Ren, H.; Krasovskiy, A.; Knochel, P. Preparation of cyclic alkenylmagnesium reagents via an iodine/magnesium exchange. Chem. Commun. 2005, 543. For a review, see: (b) Klatt, T.; Markiewicz, J. T.; Samann, C.; Knochel, P. Strategies to Prepare and Use Functionalized Organometallic Reagents. J. Org. Chem. 2014, 79, 4253. 15. Bürgi, H. B.; Dunitz, J. D.; Lehn, J. M.; Wipff, G. Stereochemistry of reaction paths at carbonyl centres. Tetrahedron 1974, 30, 1563. 16. See the Supporting Information. 17. Nahm, S.; Weinreb, S. M. N-METHOXY-NMETHYLAMIDES AS EFFECTIVE ACYLATING AGENTS. Tetrahedron Lett. 1981, 22, 3815.
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CH 3
CH 3 I CH 3 O O TMSE
O
+
n-BuLi O
38% O
TMS
OAlloc
(–)-myrocin G R=H
OR CH 3 OH O O TMSE R = TMS
O
TBAF, 64%
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