Final Report Summary - DAPHNISYN (Total Synthesis of Daphlongeranine B) PEOPLEMARIE CURIE ACTIONSIntra-European Fellowships (IEF)Call: FP7-PEOPLE-2012-IEFTotal Synthesis of Daphlongeranine B“DAPHNISYN” TOTAL SYNTHESIS OF DAPHLONGERANINE BDr. Christopher Rose and Prof. Dr. Darren J. Dixon(University of Oxford)1. WORK PROGRESS AND ACHIEVEMENTS DURING THE PERIODOur proposal is aimed at the first total synthesis of Daphniphyllum alkaloid Daphlongeranine B (Figure 1). To date, more than 200 members of this family of natural products have been isolated and structurally characterised; some of them have been shown to possess interesting biological activity, such as cytotoxic- or antioxidative properties. However, not least due to their limited availability from natural sources, the biological functions and pharmacological properties of Daphniphyllum alkaloids have been only poorly studied until now. All the more surprising, the overall number of completed total syntheses until now is relatively little. Our target molecule, Daphlongeranine B (1) (Figure 1) was isolated from the fruits of Daphniphyllum longeracemosum in 2007 and was shown to exhibit in vitro platelet aggregation. Its unprecedented hexacyclic core with seven stereogenic centres, three of which are fully substituted, presents a formidable and as yet unmet synthetic challenge. In our endeavours to elaborate a synthetic route towards the architecturally complex skeleton of our target, we identified spirocyclic pyrrolidone 2 (Scheme 1) as a key building block with two stereogenic centers of Daphlongeranine B, including one fully substituted carbon atom already in place. 1.1 Attempts towards a Michael-addition reductive cyclisation strategy for the synthesis of XXInitial studies as proposed in our fellowship application were centered on a catalytic Michael-addition – cyclisation strategy for elaborating nitrone 3, an important intermediate en route to spirocycle 2 (Scheme 1). To this end, -ketoester 5 and nitropropene 6 were prepared according to literature procedures and subjected to a range of Lewis-acidic and Lewis-basic catalysts. Disappointingly, in my hands, none of the evaluated conditions proved successful, as formation of the desired -nitro ketone 4 could not be achieved under any of the applied conditions. Instead, the formation of oligomers, as well as 1,2-silyl migration in the ketoester coupling partner 5 proved to be the major problems associated with this strategy. It might be worth mentioning, that in existing related reports, a successful catalytic Michael-addition between a -oxygenated -ketoester and nitropropene (or similar aliphatic nitroolefins) has not yet been reported to the best of our knowledge.1.2 Development and optimization of a synthetic route towards highly functionalized pyrrolidine 8Given the failure of our originally proposed strategy, we quickly turned our attention to identifying an alternative route to key intermediate 2. Our retrosynthetic analysis is shown in Scheme 3: the spirocyclic enone 2 should be accessible in four steps from 3, including a [3+2] cycloaddition, ketone synthesis, N-O bond cleavage and an olefination reaction (see also Scheme 6). Synthesis of the nitrone 3 by oxidation of the corresponding pyrrolidine is expected to be feasible, given the results on a very similar model system. For the key carbon-carbon bond forming event, we envisaged an catalytic isocyanoacetate aldol reaction between enantiomerically pure aldehyde 11 and methyl isocyanoacetate (10) using Ag2O in combination with bifunctional organocatalyst 7, which would be followed by hydrolysis of the resulting oxazoline and subsequent cyclisation. The starting materials for our planned approach are either commercially available (in case of 10), or conveniently accessible in few steps from commercially available compounds (in case of 11). Therefore, in the ideal case, we hypothesized densely functionalized pyrrolidine 8 should be accessible in only four steps from simple and commercially available compounds as a single diastereomer in high chemical yield. Moreover, the implementation in a complex molecule synthesis would clearly highlight the synthetic potential of a highly stereoselective catalytic isocyanoacetate aldol reaction, which has been developed in the Dixon group (see ref. 7).Our synthesis starts from commercially available (R)-(-)-Roche Ester (12, Scheme 4). Tosylation under standard conditions, , followed by reduction gave rise to alcohol 13 in 89% over 2 steps. a) TsCl (1.2 equiv), NEt3 (1.3 equiv), DMAP (0.15 equiv), DCM, 0° C to r.t. 15h, 90%; b) DIBALH (2.5 equiv), THF, -78° C to r.t. 3h, 99%; c) SO3-pyridine complex (2.9 equiv), DIPEA (2.9 equiv), DMSO, DCM, 0° C, 80min; d) methyl isocyanoacetate (10, 1.3 equiv), 7 (10 mol%), Ag2O, EtOAc, 4A molecular sieves, -40° C to -20° C, 22h; e) HCl in MeOH (dry), 3h; f) sat. aq. K2CO3 solution, CHCl3/iso-propanol (3:1), r.t. 15 min then extraction ; g) TBSOTf (2.5 equiv), 2,6-lutidine (5 equiv), DCM, 0° C to r.t. 14h, 32% over 5 steps, single diastereomer.For the synthesis of the aldehyde 11, we were taking advantage of a Parikh-Doering oxidation using SO3-pyridine complex. This procedure was efficiently performed on up to 1 g scale. After performing an aqueous workup, crude 11 was sufficiently pure and because of its instability, it was directly subjected to the subsequent isocyanoacetate aldol reaction. Other methods for the synthesis of aldehyde 11 involve direct reduction from the tosylated ester, IBX oxidation, as well as TEMPO oxidation of 13. These methods have been evaluated and were found to be inferior in terms of reproducibility and scaleability, as well as regarding the grade of purity of the crude aldehyde. Gratifyingly, reacting 11 with methyl isocyanoacetate (10) in ethyl acetate under silver/bifunctional organocatalysis resulted in a clean formation of oxazoline 9. Cinchonidine-derived catalyst 7 in combination with catalytic amounts of Ag2O provided the desired product in good diastereomeric ratio (10:1 by 1H-NMR). Remarkably, there is no need for further purification as the crude oxazoline 9 is obtained sufficiently pure after filtration over a short pad of silica gel. Treatment of this compound with a freshly prepared solution of HCl in dry MeOH leaded to the expected opening of the heterocycle, which upon subjection to mildly basic conditions (i.e. a saturated aqueous K2CO3 solution) afforded the desired pyrrolidine 14. , Exposing crude 14 to an excess of TBSOTf and 2,6-lutidine in dry DCM ultimately leaded to the desired protected pyrrolidine 8 as a single diastereomer in a good chemical yield of 32% over five steps. Pleasingly, the whole sequence only requires one final purification by column chromatography. The oxidation of the pyrrolidine 8 to the corresponding nitrone proved non-trivial. Our current best conditions take advantage of Davis oxaziridine 15 as oxidant and give rise to the nitrone 3 in acceptable chemical yields of 57% - 67% (Scheme 5). Attempts to use other oxidants have so far proven to be less efficient and/or providing mixtures of regioisomeric nitrones. Nevertheless, endeavours to further optimize this transformation are currently ongoing in the Dixon group.In conclusion, an efficient synthetic procedure for the synthesis of densely functionalized pyrrolidine 8 could be established during this fellowship. Our approach gives rise to the desired product as a single diastereomer in good chemical yield. Notably, the five-step sequence from alcohol 13 to the final product 8 only requires one single purification by column chromatography. Since the developed sequence is scaleable, it will provide the required quantities of material for consecutive studies. These are currently ongoing in the Dixon group and are expected to culminate in the first total synthesis of our target molecule, Daphlongeranine B in due course.1.2 Synthesis of chiral side chains bearing different electron withdrawing groupsAs depicted in Scheme 6, our retrosynthetic analysis of our target Daphlongeranine B (1) involves a chiral side chain of general structure 17 being coupled to spirocyclic enone 2. Also, our synthetic plan involves a Michael addition of an enolate (generated from the amide in 16) onto the spirocyclic Michael acceptor. Taking this into account, we considered it as beneficial to have a set of different acceptor substituted acid derivatives in place. In the course of this fellowship, syntheses of derivatives 26 and 28, bearing two different electron-withdrawing groups alpha to the carboxylic acid function, were developed. Both syntheses start from enantiomerically pure alcohol 22. A Johnson-Claisen rearrangement using triethyl orthoacetate gives rise to ester 23 in 60% yield, which is then reduced to alcohol 24. Transformation into a good leaving group using TsCl affords tosylate 25 in high yield, which can then be transformed into malonic acid derivatives 26 and 28, respectively. Under SN2-conditions, dimethyl malonate derivative 26 is accessible in 87% chemical yield, whereas the corresponding methyl malonomononitrile 28 is obtained in 39%. A selective monohydrolysis of diester 26 gives rise to -keto acid 27 in 66%. Hydrolysis of the mononitrile 28 was shown to be possible, however it was not optimized. a) MeC(OEt)3, 2-nitrophenol (5 mol %), 120° C, 14h, 60%; b) LiAlH4 (2.5 equiv), Et2O, 0° C to r.t. 2h, 89%; c) TsCl (1.4 equiv), NEt3 (9.4 equiv), DMAP (0.1 equiv), DCM, r.t. 16h, 88%; d) Dimethyl malonate (3.0 equiv), KOtBu (3.0 equiv), KI (1.0 equiv), DMF/THF (1:1), r.t. 4d, 87%; e) Methyl 2-cyanoacetate (3.0 equiv), KOtBu (3.0 equiv), KI (1.0 equiv), DMF/THF (1:1), r.t. 4d, 39%; f) Aq. KOH (1M) (0.9 equiv), MeOH, r.t. 3h, 39%. g) LiOH (5.0 equiv), MeOH/H2O (1:1), r.t. 6h, 44%, unoptimised2. SUMMARY OF THE PROGRESS OF THE RESEARCH TRAINING2.1 Attendance of seminars and lecturesDuring my fellowship, I have had the opportunity to attend the first “Chemistry Careers Conference”, held by the department of Organic Chemistry. In this one day seminar, I could learn about the different career pathways for researchers, both in academia and industry. Also, I had the opportunity to participate in a workshop entitled “managing your mind”. Furthermore, I attended numerous lectures held by leading academic researchers from all over the world, providing me with the opportunity to broaden my chemistry-related knowledge significantly. One highlight of the academic year was the Newton Abraham Symposium, including a lecture by Nobel Prize awardee Prof. Richard Schrock entitled “Olefin Metathesis: A Long-Lived Reaction”.2.2 Research skills and techniquesThis Marie Curie fellowship provided me with the opportunity to develop new skill and abilities, which complement those, I already possessed. In particular, I developed new skills related to the field of total synthesis, analytical methodology (NMR, HPLC, MS, IR), organic synthesis methods and techniques (especially reaction scale-up, asymmetric synthesis). The fellowship project in combination with the outstanding research infrastructure at Oxford University was an ideal training environment for me to develop as a research scientist.2.3 Communication and professional skillsApart from the scientific part of the project, I had the opportunity to develop my abilities as a communicator and professional research scientist. Research results, progress and the work plan for the near future was discussed on a weekly basis. The presentation of research updates in front of the whole group including my supervisor Prof. Dixon was done on a weekly basis. These group meetings provided the opportunity for in-depth discussions of my research involving all post-doctoral and post-graduate co-workers of the Dixon group. In addition, I had the opportunity to discuss strategy and direction of my project in one-to-one meetings with Prof. Dixon on a weekly basis, ensuring progress and smooth-running of the fellowship. 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