Laureates: awards and Honors, sCs FaLL Meeting 2012 CHIMIA 2013, 67, Nr. 4 227 doi:10.2533/chimia.2013.227 Chimia 67 (2013) 227–230 © Schweizerische Chemische Gesellschaft Total Synthesis of the Myxobacterial Macrolide Ripostatin B‡ Florian Glaus§ and Karl-Heinz Altmann* §SCS-Metrohm Foundation Award for best oral presentation Abstract: This article describes the total synthesis of ripostatin B, which is a 14-membered macrolide of myxobacterial origin that inhibits E. coli RNA polymerase by a different mechanism of action than the first-line anti-tuberculosis drug rifampicin. Structurally, ripostatin B features a labile and synthetically challenging doubly skipped triene motif embedded in the macrolactone ring. Key steps in the synthesis were a Paterson aldol reaction, a low-temperature Yamaguchi esterification and an alkene metathesis reaction to close the macrolide ring. The natural product was synthesized in a longest linear sequence of 21 steps and 3.6% overall yield. Keywords: Antibiotics · Ring-closing metathesis · Ripostatin · RNA polymerase · Total synthesis Ripostatins A and B (1) (Fig. 1) were pound binds to the hinge that mediates first isolated in 1995 by Reichenbach, opening and closing of the RNAP clamp, Höfle and co-workers from the myxobac- thus trapping the enzyme in a closed con- terium Sorangium cellulosum (strain So ce O O O formation and preventing access of the [1] [4,11] 377) found in a soil sample from Kenya. O OH dsDNA to the active-site cleft. This Both compounds are 14-membered macro- OH binding mode is fundamentally different lides that differ only in the oxidation state from that of the rifamycin-type inhibitors, at C(15) (ripostatin A exists as a hemiac- Ripostatin A(hemiacetal form) which prevent the extension of RNA be- etal/ketone mixture in a 4:3 ratio). The two yond a length of 2–3 nucleotides by bind- ripostatins exhibit a similar, albeit narrow ing to a site close to the active-site cleft. spectrum of antibiotic activity against Based on the location of RNAP mutations gram-positive bacteria, with minimum in- HO O O in myxopyronin- and ripostatin A-resistant hibitory concentrations (MICs) of less than E. coli, Ebright and co-workers have sug- 1 µg/mL only against S. aureus and the hy- O OH gested that ripostatin A (as well as a num- perpermeable E. coli strain DH21tolC.[1a] OH ber of other natural products) likewise Additionally, ripostatins A[1a] and B[2] both inhibit(s) bacterial RNAP by binding to inhibit the DNA-dependent RNA poly- Ripostatin B(1) the hinge region of the enzyme. merase (RNAP) from E. coli with sub-µM The attractive biological profile of activity, while being inactive against eu- Fig. 1. Ripostatins A and B. the ripostatins as well as their interesting karyotic RNA polymerase II.[1a] chemical structure (notably the C(2)–C(9) RNA polymerase is a suitable target for doubly skipped triene motif) prompted antibacterial therapy.[3] It is an essential en- mammalian RNA polymerases I, II and us to embark on the total synthesis of ri- zyme (permitting efficacy) and it is highly III (providing therapeutic selectivity).[4–6] postatin B[12] with the goal to provide a conserved across gram-positive and -nega- RNAP is also a clinically validated target, chemical basis for future SAR studies. No tive bacteria (permitting broad-spectrum since it is the target of the rifamycin-class of total synthesis of either ripostatin had been activity), while it differs significantly from antibiotics(themostprominentmemberbe- published prior to our initial communica- ing rifampicin).[7,8] The rifamycins are clin- tion. However, concurrent with our own ically used for the treatment of both gram- work total syntheses of ripostatin B were positive and gram-negative bacteria.[9] independently developed by Christmann They have proven particularly effective in and co-workers, and Prusov and Tang.[13a,b] the treatment of tuberculosis, where they In mid-2012, Prusov and Tang also pub- are used as first-line drugs.[6,7] However, lished a total synthesis of ripostatin A.[13c] the number of bacterial strains resis- Due to the anticipated tendency of tant to the rifamycins is increasing.[7,10] the skipped triene-motif (C(2)–C(9)) for *Correspondence: Prof. Dr. K.-H. Altmann Consequently, there is an urgent need for double-bond migration, we based our ret- Swiss Federal Institute of Technology (ETH) Zürich new RNAP-inhibitors that exhibit a differ- rosynthetic analysis on a ring-closing me- Institute of Pharmaceutical Sciences ent mode of action. tathesis (RCM), which, due to the absence HCI H 405, Wolfgang-Pauli-Strasse 10 of base in the ring-closing step, should CH-8093 Zürich Ebright and co-workers have recently Tel.: +41 44 633 73 90 reported an X-ray crystal structure of a minimize problems with the double-bond E-mail: [email protected] complex between T. thermophilus RNAP framework (Scheme 1).[14] After RCM, the ‡An original report on this work has been published and myxopyronin, another myxobacterial primary hydroxyl group would be depro- previously: F. Glaus, K.-H. Altmann, Angew. Chem. [4] Int. Ed. 2012, 51, 3405. RNA polymerase inhibitor. The com- tected selectively and oxidized, thereby 228 CHIMIA 2013, 67, Nr. 4 Laureates: awards and Honors, sCs FaLL Meeting 2012 installing the vinylogous malonate-type C(1)-C(3)-C(28) framework. The RCM- metathesis precursor 2 could be accessible by Stille- coupling between an allylstannane and 11 vinyl iodide 3. The Stille reaction was spe- HO O 5 O TBSO O stille cifically chosen because it is usually per- O 1 3 28 OH O OTBS formed at neutral pH, which would mini- 15 OH OTBS mize double-bond migration in the dienoic ester. Vinyl iodide 3 should be accessible Ripostatin B(1) 2 by esterification between alcohol 4 and io- doacrylic acid 5.[15] In principle, perform- ing the Stille coupling with acid 5 (or a anti 1,3- suitably protected derivative thereof) prior reduction TBSO O I TBSO to esterification should lead to a more con- aldol vergent approach toward the natural prod- O OTBS OH O I uct. This approach, however, had failed in OTBS O HO OTBS the hands of Kirschning and co-workers, esterification 5 due to the pronounced tendency for dou- 3 4 ble-bond migration in the dienoic acid un- der a variety of basic esterification condi- O tions (under neutral or acidic conditions, O [16] + yields were generally unsatisfactory). TBSO PMBO We intended to set the C(13)- and C(15)- 6 7 8 stereocenters by an aldol reaction between O ketone 6 and aldehyde 7 (stereocenter at Scheme 1. Retrosynthetic analysis of ripostatin B. PMB = para-methoxybenzyl,TBS = tert-butyldi- C(13)), followed by an anti 1,3-reduc- methylsilyl. tion (stereocenter at C(15)). Compound 7 would in turn be accessible from epoxide 8[17] by epoxide opening and subsequent MeMgBr, 1) Mg,PhCH2CHO O O elaboration of the diene moiety. Br CH3NHOCH3·HCl The synthesis of ketone 6 started 2) CH CH COOH, OEt 3 2 78% with the addition of the Grignard reagent CH C(OEt) , 3 3 9 6 derived from 2-bromopropene to phen- 49% ylacetaldehyde (Scheme 2). The result- ing secondary alcohol was submitted to Scheme 2. Synthesis of ketone 6. a Johnson-Claisen rearrangement with triethyl orthoacetate in the presence of a Scheme 3. Synthesis 1) TBSCl, OH ® catalytic amount of propionic acid at 145 Red-Al®, of acid 5. Red-Al = °C, giving γ,δ-unsaturated ester 9[18] in imidazole,92% sodium bis(2- OH methoxyethoxy)alu- 49% yield for the two-step sequence. By thenI using Williams’ procedure, which involves 2) nBuLi, OTBS 2 minum dihydride. 74% treatment of the ester with an excess of (CH2O)n,93% 10 the Grignard-reagent in presence of N,O- dimethylhydroxylamine hydrochloride, 9 I 1) MnO2 O I could be transformed into methyl ketone HO OTBS 2) Pinnickox. HO OTBS 6 in one pot.[19] The known carboxylic acid 5[15] was 11 57%(twosteps) 5 synthesized from 3-butyn-1-ol, which was TBS-protected and homologated with paraformaldehyde in 86% yield (two steps, by treatment of the diol with base (NaH) sulting primary alcohol 14 was converted Scheme 3). The triple bond was then re- and PMBBr (Scheme 4). PMB-protected into the corresponding allylic bromide us- ® ing Appel’s conditions (CBr , PPh ) in the duced stereoselectively with Red-Al and epoxy alcohol 8 was obtained in 61% 4 3 the intermediate aluminate was quenched yield for the three-step sequence. Epoxide presence of 2,6-lutidine;[22] this bromide with iodine to give vinyl iodide 11 in 74% opening with the anion derived from ethyl was then converted into 1,4-diene 15 by Stille reaction with Bu SnCH=CH using yield. A two-step oxidation involving propiolate followed by TBS-protection of 3 2 MnO -mediated conversion of the allylic the resulting secondary alcohol then gave Pd (dba) (12 mol%) as palladium source 2 2 3 and 0.5 equiv. AsPh .[23] PMB-removal alcohol into the aldehyde, followed by a TBS-ether 12 in 83% yield (two steps). 3 Pinnick reaction afforded then iodoacrylic The trisubstituted E-configured double from 15 then turned out to be rather acid 5 in 57% yield from 11. bond was installed next by treatment of 12 challenging. Deprotection could not be The synthesis of aldehyde 7 departed with thiophenol and a catalytic amount of achieved under standard DDQ conditions, from d-(–)-aspartic acid; this was convert- sodium methoxide,[21] to provide thioether which might be attributed to the presence ed into epoxide 8 via a known three-step 13 (85%), followed by displacement of the of the 1,4-diene moiety.[24] After the inves- sequence,[17,20] which involved displace- phenylsulfenyl moiety by a methyl group tigation of a range of methods, we finally ment of the amino group by bromide un- with full retention of configuration by found that TMSI[25] cleaves the PMB-ether der retention of configuration, reduction treatment with dimethylcuprate at −78 °C very efficiently, leading, after cleavage of the ensuing TMS-ether with K CO in of both carboxylic acid groups to the al- and warming to −30 °C over 30–40 min- 2 3 cohol stage, and finally epoxide formation utes.[21] After DIBAL-H reduction, the re- methanol, to the desired primary alcohol Laureates: awards and Honors, sCs FaLL Meeting 2012 CHIMIA 2013, 67, Nr.