Isolation, Structural Assignment, and Total Synthesis of Barmumycin

Adriana Lorente, Daniel Pla, Librada M. Ca~ nedo, Fernando Albericio,* ) and Mercedes Alvarez* Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, Baldiri Reixac 10, E-08028 Barcelona, Spain, Instituto Biomar, S.A. Parque Tecnol ogico de Le on-M10.4, E-24009 Armunia, Le on, Spain, CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain, Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain, and Laboratory ofOrganic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain. Current address: Memorial Sloan-Kettering Cancer Center, New York, NY 10065. E-mail: plaquerd@mskcc.org


Introduction
Natural products from terrestrial plants and microorganisms have long been a traditional source of drugs; however, over the past few years, marine organisms have garnered ever-increasing attention as a rich bank of new bioactive compounds. 1 Marine actinomycetes have also proven to be an important source of biologically active compounds. 2 Among the marine actinomycetes that our group has studied, those of the genus Streptomyces have clearly shown the most pharmacological potential; however, in many bioactive cultures they have yielded only compounds that are already known. During ongoing research efforts to explore the biosynthetic potential of rare marine microorganisms, we isolated two known compounds, pretomaymycin 3 and oxotomaymycin 4 (Figure 1), plus the previously unknown compound barmumycin from the culture broth of the marine actinomycete Streptomyces sp. BOSC-022A, isolated from a tunicate collected off the Scottish coast. Barmumycin and its diacetate show antitumor activity at micromolar concentrations in all 12 cancer cell lines tested (see Table 1 in the Supporting Information). Herein we report the isolation, total synthesis, and structure elucidation of barmumycin.
Decanolides are chemical entities abundant in terrestrial organisms, though only a few (e.g., modiolides A and B 5 and xestodecalactones A-C 6 ) have been isolated from marine sources. To the best of our knowledge, the aniline moiety within its 10-membered lactone had never been reported in naturally occurring macrocycles.
We sought to synthesize 1 to compare it against an authentic sample of barmumycin in order to assess its structural assignment. Our retrosynthetic analysis of 1 entailed formation of the exocyclic double bond via Wittig reaction; dihydroxylation of a double bond to give the alcohol required for lactonization; and finally, introduction of a functionalized five-carbon chain onto the nitrogen of methyl 2-amino-5-methoxybenzoate (Scheme 1).
The functionalized five-carbon chain on the aniline nitrogen was introduced by two different ways: via reductive amination (Scheme 2) and via N-alkylation (Scheme 3).
Homoallylic alcohol 2 was obtained by Barbier reaction of 2,2-dimethoxyacetaldehyde with allyl bromide and indium powder in water (95% yield). 7 Attempts at direct deprotection of the dimethyl acetal under acidic conditions led to polymerization of 2; 8 therefore, the alcohol had to be protected. Acetylation of the alcohol to give compound 3, 7 followed by dimethyl acetal deprotection using LiBF 4 in MeCN-H 2 O, 9 gave the aldehyde 4 in excellent yield.
Reductive amination of 4 with aniline 5 10 required special conditions due to the poor nucleophilicity of the aniline (which is deactivated by the methyl ester group in the ortho position): thus, reaction of 4, 5, phenylsilane, and dibutyltin dichloride under microwave irradiation for short reaction times gave the aminoalkene 6a in 67% yield. 11,12 Attempts at protecting the aniline NH in 6a as a t Bu carbamate failed due to its poor reactivity; therefore, 6a was treated with K 2 CO 3 in MeOH to give the deacetylated derivative 6b. However, all attempts at oxidizing 6b to its ketone derivative resulted in decomposition of the starting material. 13 Thus, the aniline had to be protected, but this was not possible in the presence of the unprotected alcohol. Exploiting the lack of reactivity of the aromatic amine toward Boc protection, and using standard conditions, 6b was converted into its t Bu carbonate derivative 6c in 43% yield. 14 The aniline group of 6c was then orthogonally protected using (CF 3 CO) 2 O in pyridine to afford the trifluoroacetamide derivative 6d in quantitative yield. Treatment of 6d with 10% TFA in CH 2 Cl 2 to remove the carbonate gave the free alcohol 6e in quantitative yield. Compound 6e was then oxidized with Dess-Martin periodinate (DMP) 15 to yield the ketone 7 in 93% yield. Slow addition of 7 to N-methylmorpholine oxide (NMO) and a catalytic amount of OsO 4 in acetone-H 2 O to generate the corresponding diol 8 while preventing double-bond isomerization gave 8 in good yield. Diol 8 was further protected by conversion into its 2, 2-dimethyl-1,3-dioxolane derivative 9 using 2,2-dimethoxypropane plus pyridinium p-toluenesulfonate (PPTS) as catalyst (quantitative yield). Deprotection of the amine in 9 via mild basic hydrolysis gave the free amine 10 in 97% yield.
A faster and better yielding synthesis of 10 (Scheme 3) was done in parallel to the route described above. The first step was dihydroxylation of isobutyl but-3-enoate. The introduction of the bromomethyl residue was planned for a later step. The oxidation conditions described above afforded isobutyl 3,4-dihydroxybutanoate (11), which was then further protected as the 2,2-dimethyl-1,3-dioxolane derivative 12, in excellent overall yield for both steps. The key step, transformation of 12 into the bromoketone 13 using bromomethyllithium, gave 13 in 49% yield. N-Alkylation of 5 with 13 under microwave irradiation gave 10.
Wittig chemistry was employed to introduce the ethylidene chain. Reaction of 10 with the Wittig ylide derived from ethyltriphenylphosphonium bromide yielded 14 (43%) as a mixture of Z/E diastereomers. 16 Z/E-14 was transformed into 1 in three successive reactions: hydrolysis of the methyl ester, acetonide deprotection under acidic conditions, and macrocyclization. The acid Z-15 was obtained by purification of the Z/E mixture of acids by semipreparative HPLC. 17 Racemic Z-1 was obtained in 35% yield by acetal deprotection followed by macrocyclization using EDC 3 HCl and   (11) Kangasmets€ a, J. J.; Johnson, T. Org. Lett. 2005, 7, 5653-5655. (12) Other reduction conditions proved unsuccessful. These included NaBH-(OAc) 3 in THF at room temperature for 16 h, NaBH(OAc) 3 in CH 2 Cl 2 /AcOH at room temperature for 5 h, and NaBH(OAc) 3 in toluene at 110°C for 2 h.
(13) The aniline 5 was isolated from the oxidation degradation mixture. Its formation could be rationalized through hydrolysis of the enamine resulting from enolization of the keto compound.
(17) The NOESY correlations between 4 CH 2 (2.27 and 2.41 ppm) and the vinyl proton (5.58 ppm) confirmed the stereochemistry of (Z)-15 (see NOESY interactions in the Supporting Information). solid-supported DMAP in a 5 mM CH 2 Cl 2 solution. The 1 H NMR spectrum of Z-1 showed two doublets for the CH 3 linked to the double bond (1.42 ppm and 1.45 ppm) and two quadruplets for the vinylic proton (5.51 and 5.53 ppm). 18 These data could be explained by the presence of two highly populated conformations of Z-1 at room temperature. Therefore, we studied peak coalescence by 1 H NMR run at different temperatures. Spectra from the initial experiments, run up to 55°C in CDCl 3 as solvent, exhibited this trend, but coalescence was not reached at this temperature limit. Finally, coalescence was almost reached in DMSO-d 6 as solvent at 145°C (see Table 4 in the Supporting Information). Moreover, comparison of spectroscopic data for barmumycin with those for Z-1 revealed dramatic differences in the chemical shifts (see Tables 2 and 3 in the Supporting Information). This discrepancy, despite the conflicting stereochemistry of the two compounds (Z-1 and E-barmumycin), led us to pursue a new structural assignment.
Re-evaluation of all possible alternative structures led us to systematic elucidation of E-16 as a novel structure for barmumycin ( Figure 3). Interestingly, the very close structural resemblance of 16 to the pretomaymycin and oxotomaymycin isolated from the extract (Figure 1) suggests that all three molecules could derive from the same biogenetic pathway.
In order to confirm that the structure of barmumycin is actually that of E-16, we synthesized the latter and subsequently compared it to an authentic sample of the former. This began with selective silyl protection of the primary alcohol in the commercially available N-Boc-trans-4-hydroxy-L-prolinol followed by oxidation of the secondary alcohol in the derivative 17, which afforded ketone 18 in 62% yield over two steps (Scheme 4). Wittig chemistry was again employed to introduce the ethylidene chain: reaction of 18 with the Wittig ylide derived from ethyltriphenylphosphonium bromide yielded 19 as a 9:1 mixture of Z/ E-diastereomers. Z/E-19 was used directly without separation, as a single purification was planned for the final step of the synthesis. The TMS ether and the tert-butyl carbamate of Z/E-19 were deprotected with 10% TFA in CH 2 Cl 2 to give the pyrrolidine derivative Z/E-20. Condensation of Z/E-20 to vanillic acid using (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIEA) gave a 9:1 mixture of Z/E-16 diastereomers. The configuration of the double bond was established by the NOESY correlations in Z/E-16 between 5 CH 2 (4.00-4.20 ppm) and the CH 3 (1.60 ppm) (see NOESY correlations in the Supporting Information).
None of the Horner-Wadsworth-Emmons reactions tested (using the appropriate phosphonate and different bases 19 ) gave SCHEME 1. Retrosynthetic Analysis of Compound 1 SCHEME 2. Route A for the Synthesis of 10 (18) The NOESY correlations between 2 CH 2 (4.35 and 4.82 ppm) and CH 3 (1.42 and 1.45 ppm), and between 4 CH 2 (2.09-2.20 ppm) and the vinyl proton (5.51 and 5.53 ppm), confirmed the stereochemistry of (Z)-1 (see NOESY interactions in the Supporting Information).
(19) The reaction was performed with (EtO) 2 P(O)CH 2 CH 3 and either LDA, K t BuO, or NaHMDS as base.
the desired E-19. However, the Kocienski variant of the Julia-Lythgoe olefination 20 afforded a 2:1 mixture of E/Z-19. This process entails nucleophilic addition of 5-(ethylsulfonyl)-1-phenyl-1H-tetrazole anion to the ketone followed by transposition and elimination to give the double bond. Again, deprotection of the hydroxyl group and the amine group was obtained using 10% TFA in CH 2 Cl 2 , and condensation of E/Z-20 to vanillic acid using PyBOP and DIEA gave a 2:1 mixture of E/Z-16. This mixture was purified by semipreparative HPLC to obtain E-16 as a single diastereomer. The configuration of the double bond was established by the NOESY correlations in E-16 between 5 CH 2 (4.00-4.22 ppm) and the vinyl proton (5.30-5.38 ppm) (see the NOESY correlations in the Supporting Information).
Comparison of spectroscopic data obtained for E-16 and barmumycin confirmed that the revised structure is indeed the structure of the natural product.
In summary, the previously unreported marine compound barmumycin was isolated, and its chemical formula was determined via mass spectrometry. On the basis of preliminary NMR data, barmumycin was initially assigned the structure of compound 1. To confirm this assignment, compound 1 was synthesized following two different strategies starting from an o-aminobenzoic ester: one based on reductive amination, and one based on N-alkylation, which was shorter and higher yielding. However, comparison of the NMR spectra for 1 with those for isolated barmumycin showed dramatic differences. The structure of barmumycin was reassessed, and most probable option conceived was compound E-16, which was subsequently prepared (in five steps and 18% overall yield) for comparison with the natural compound. The spectroscopic data for E-16 fully coincided with that for barmumycin, thereby confirming that the two structures are equivalent. This work is a new example of the importance of total synthesis for structural characterization and confirmation of natural products. 21