Abstract
The total synthesis and structural revision of antibiotic CJ-16,264 is described. Starting with citronellal, the quest for the target molecule featured anovel bis-transannular DielsAlder reaction that casted stereoselectively the decalin system and included the synthesis of six isomers before demystification of its true structure.

Isolated from fungus CL39457, antibiotic CJ-16,264 [1] (1, Figure 1A )w as reported to exhibit impressive activities against drug-resistant Gram-positive bacteria such as Staphylococcus aureus 01A1120 (MIC = 0.78 mgmL À1 )a nd Gramnegative bacteria such as Moraxella catarrhalis 87A1055 (MIC = 0.39 mgmL À1 )a nd Escherichia coli 51A1051 with altered permeability (MIC = 6.25 mgmL À1 ). Thea ssigned structure of this molecule is intriguing in that it is amongst the most complex of its relatives,C J-16,367 [2,F igure 1A, stereochemistry unassigned, isolated from the same fungus (CL39457)], [1] UCS1025A (3,F igure 1A) [2] and UCS1025B (4,F igure 1A), [2] 21-(S)-and 21-(R)-myceliothermophins C (5a) [3, 4] and D( 5b) [3, 4] ( Figure 1B ), and the most recently reported antibiotic pyrrolizilactone [5] (1a,F igure 1A). Antibiotic CJ-16,264 (1)p ossesses ac hallenging tetramethylated decalin system, whose relative and absolute configurations differ from those of its less complex siblings UCS compounds (3 and 4) , and the myceliothermophins C(5a)and D(5b)(see Figure 1 ). Antibiotic CJ-16,264 also appears dissimilar to its close relative antibiotic 1a.I nv iew of the biological properties of CJ-16,264 (1), the doubts over the correctness of its structure raised by the stereochemical differences from its siblings and the incomplete assignment of its absolute configuration, [1, 6] and the challenge presented by its unique polysubstituted decalin system, we embarked on its total synthesis in the hope of solving the puzzle presented by its molecular structure.H ere we describe our synthetic endeavors that led to the total synthesis and structural revision of this target molecule which set the stage for design, synthesis and biological evaluation of its analogs and structural elucidations within this emerging class of antibiotics.
Our retrosynthetic analysis of antibiotic CJ-16,264 (structure 1,F igure 1A)p ointed to (S)-citronellal, the more expensive [7] of the two enantiomeric forms of this convenient starting material. Mindful of the cost and given our doubts about the structure of our target molecule,weopted to use the less costly (R)-citronellal as the starting material for our initial studies,which were directed toward the total synthesis of the antipode of 1 (i.e. ent-1,S cheme 1A)a nd the confirmation of the relative stereochemical configuration of the molecule,i fn ot its absolute configuration. At that point we would have the option to apply our developed strategy using (S)-citronellal as as tarting material in order to obtain the correct enantiomer of CJ-16,264 should our findings dictated the endeavor. Thus,d isconnection of ent-1 (Scheme 1A)through the Reformatsky-type reaction employed by Lambert and Danishefsky in their synthesis of UCS1025A (3) [8, 9] led to TBS-protected iodide 6 (ideally racemic in order to obtain both diastereoisomers of the final product) and aldehyde 7,the latter being traced back to (R)-citronellal (12) through tricyclic lactone 8 (transannular Diels-Alder reaction; [10] see endo transition state TS-9,S cheme 1A)a nd hydroxy acid 11 a [macrolactonization, (Z)-a,b-unsaturated as required for the stereochemical relationship of the C1-and C2-substituents of the initially targeted ent-1].
Scheme 1B summarizes the construction of the required hydroxy acid 11 a starting from (R)-citronellal (12) . Reaction of 12 with the ylide generated from Ph 3 Pa nd CBr 4 gave the expected 1,1-dibromoolefin (98 %y ield) whose exposure to nBuLi led to the corresponding terminal acetylene 14 (95 % yield). [11] Alternatively,a cetylene 14 could be prepared from 12 by reaction with the Bestmann-Ohira reagent [12] (85 % yield) as shown in Scheme 1B.The latter compound was then converted to vinyl iodide 15 through reductive ozonolysis (O 3 ; then NaBH 4 ,8 5% yield) followed by as equence involving carboalumination/iodination [13] (Cp 2 ZrCl 2 -AlMe 3 ;t hen I 2 , overall 84 %y ield). Subsequent oxidation of 15 (SO 3 ·py, [14] 95 %yield) was then followed by selective (Z)-olefination of the resulting aldehyde with bis-trifluoroethoxy phosphonate 16 [15] in the presence of KHMDS and 18-crown-6 to furnish (Z)-a,b-unsaturated ethyl ester 17 (92 %y ield, ! 20:1 Z:E d.r.). Thev inyl iodide 17 was then coupled with hydroxy vinylstannane 18 using CuTC [16] to afford hydroxy ester 19 (88 %yield) which, upon hydrolysis (aq. LiOH), provided the desired (Z)-hydroxy acid 11 a (99 %yield). We note here that attempted use of palladium-catalyzed [17] coupling reactions to unite vinyl iodide 17 with stannane 18 led to significant amounts of homo-coupling side-products.
With (Z)-hydroxy acid 11 a readily available,i ts macrolactonization was then attempted under various conditions, [18] including Yamaguchi, [19] Keck [20] and Shiina [21] protocols,all of which failed to produce any of the desired 13-membered macrolide 10 (see Scheme 2) . Instead, and under high dilution Shiina conditions (MNBA, Et 3 N, DMAP cat.), we observed the formation of the 26-membered macrolide 22 (37 %y ield plus 8% yield of the corresponding triolide). That the (2Z)-olefinic bond of 11 a had isomerized to the (2E)-olefinic bond within the diolide 22 was evident from the 1 HNMR spectrum of the latter (J a,b = 15.7 Hz). AM ichael addition/bond rotation/elimination of DMAP [22] or Et 3 N [23] under the reaction conditions may account for this observed isomerization of the (2Z)-olefinic bond of 11 a to the (2E)-olefinic bond within the diolide 22.The same macrolide (22) was prepared from (2E)-a,b-unsaturated hydroxy acid 11 b (synthesized from hydroxy vinyl iodide 15 in three steps and 79 %overall yield as shown in Scheme 1C)b yt he same procedure as that used for the macrodimerization of 11 a in 40 %yield (plus 8% yield of the corresponding triolide).
Our inability to produce the targeted Diels-Alder precursor 10,c oupled with the failure of hydroxy acids 11 a and 11 b to undergo intramolecular [4+ +2] cycloaddition, [24] [25] [26] [27] led us to attempt the double intramolecular Diels-Alder reaction of the now readily available macrolide 22.T he anticipated incorrect configuration of the product at C1/C1' (see structure . . Angewandte Zuschriften 9336 www.angewandte.de 25,S cheme 2) was expected to be invertable through epimerization. Pleasantly,upon heating 22 in m-xylene at 220 8 8Cfor 12 h, 10-membered ring macrodiolide 25 was obtained exclusively and in 48 %y ield, presumably through the exo transition state exo-TS-24 (Scheme 2). Theracemic form of 25 [that is,( AE)-25 a,o btained from racemic citronellal via the 6,6' '-anti diolide 22 a (not shown)] crystallized nicely from hexane (colorless crystals,m.p.119-123 8 8C), yielding to X-ray crystallographic analysis [28] (see ORTEP representation, Scheme 2), which confirmed the relative configurations of the decalin moiety as depicted in 25. [29] Exposure of diolide 25 to NaOMe in THF:wet MeOH at 65 8 8Cl ed to the formation of g-lactone 8 (epimerized at C1, colorless crystals,m.p.103-105 8 8C, diethyl ether, see ORTEP representation, Scheme 2) [28] as the major product and hydroxy acid 26 (not epimerized at C1) as the minor product (85 %yield, 8:26 ca 3:2, chromatographically separated) (see Scheme 2). Thed esired diastereoisomer (8)f or the synthesis of ent-1 was then converted to TBS ether decalin system 27 through af our-step sequence involving 1) LAH reduction (diol, 98 %y ield);2 )selective reaction of the less hindered hydroxy group with the bulky 2,4,6-triisopropylbenzenesulfonyl chloride (TPBSCl) in the presence of Ag 2 O
[30] and catalytic amounts of KI [to afford the corresponding sulfonate at the less hindered site (appendage at C2), 63 %yield, 79 % yield based on 80 %c onversion];3 )silylation (TBSCl, imidazole,9 0% yield) of the remaining hydroxy moiety (on C1 appendage);a nd 4) reductive removal of the sulfonate group (LiEt 3 BH, [31] 82 %yield). Thelatter compound (i.e. 27) was then desilylated (TBAF,9 1% yield) and the resulting alcohol was oxidized with DMP [32] to the targeted aldehyde 7 in 81 %yield as shown in Scheme 2.
Coupling of aldehyde 7 with racemic pyrrolizidinone iodide 6 under the influence of BEt 3 (via the corresponding boron enolate of 6) [8] proceeded stereoselectively as shown in Scheme 3A,furnishing amixture of alcohols 30 and 31 (87 % yield, 30:31 ca 3.5:1 d.r.,chromatographically separated). The absolute stereochemical configuration of compound 30 (colorless crystals from ethyl acetate/hexane,m .p.1 75-178 8 8C) was confirmed by X-ray crystallographic analysis (see ORTEP representation, Scheme 3A). [28] Removal of the TBS group (TASF,8 1% yield) from the targeted diastereoisomer 30 (major) followed by DMP oxidation of the secondary alcohol of the resulting diol (32)t hen furnished corrected-ent-1 (epimerized at C7',S cheme 3A,8 3% yield). While the mass spectrum of synthetic correctedent-1 matched that reported for natural CJ-16,264, its 1 H-and 13 CNMR data differed significantly from those reported for the natural product, suggesting that the originally assigned structure was in error.The observed epimerization at C7' in corrected-ent-1 upon oxidation at C8' of its precursor (i.e. 32)c onfirmed by NMR spectroscopy (J 7',7'a = 0Hzf or corrected-ent-1; J 7',7'a = 4.1 Hz for 32)i sn oteworthy.M anual molecular modeling of corrected-ent-1 reveals ad ihedral angle closer to 908 8 in its most sterically favorable conformation (anti relationship of H7' and H7'a; [2b] for model study and Ref. [5] for structural assignment of pyrrolizilactone 1a), whereas similar modeling of 32 shows its most sterically favorable conformation exhibiting ad ihedral angle of closer to 308 8 (syn relationship of H7' and H7'a; see Ref. [2] for model study). Thel atter conformation is supported by the X-ray analysis of 30 (see ORTEP representation, Scheme 3A,X-ray dihedral angle for H7'-C7'-C7'a-H7'a = 38.68 8). It is presumed that thermodynamics spontaneously drive this epimerization to the most stable diastereoisomer (i.e.c orrected-ent-1).
Faced with this predicament, and in order to decipher the true structure of antibiotic CJ-16,264, we then opted to synthesize the diastereoisomeric structure of corrected-ent-1, isomer 34 (Scheme 3A)d erived from enantiomerically pure 7,a nd the antipode of the shown enantiomer of 6 via intermediate 31 as shown in Scheme 3A.T hus,c hromatographically separated diastereoisomer 31 was converted to 34 through the same two-step sequence used for the conversion of 30 to corrected-ent-1,a nd in similar yields,a sd epicted in Scheme 3A.H owever,t he spectroscopic data of 34 again failed to match those reported for natural CJ-16,264, prompting us to target the remaining two diastereoisomers of this series of compounds,n amely those reachable from hydroxy acid 26 (Scheme 2).
To this end, reaction conditions were first optimized to obtain the desired hydroxy acid 26 as the major product of the hydrolysis of 25 (Scheme 2). Thus,treatment of 25 with LiOH in THF:MeOH:H 2 Of urnished am ixture of 26 and 8 (93 % yield) in which the desired hydroxy acid 26 predominated (26:8 ca 2:1, chromatographically separated). Ther equired aldehyde 29 (anti C1/C2) was then synthesized from hydroxy acid 26 through asequence involving selective methylation of the carboxylic acid moiety (K 2 CO 3 ,M eI, 85 %y ield), mesylation of the hydroxy group (MsCl, Et 3 N, 90 %y ield), and reduction of the resulting mesylate methyl ester with Zn dust [33] in the presence of NaI to afford tetramethylated decalin system 28 (83 %yield). Themethyl ester of the latter was then reduced (DIBAL, 93 %y ield) and the resulting alcohol was oxidized (DMP,87% yield) to afford the desired aldehyde 29 as shown in Scheme 2.
Coupling of the now readily available aldehyde 29 with racemic pyrrolizidinone iodide 6 under BEt 3 -mediated Reformatsky-type coupling, [8] as shown in Scheme 3B,a gain proceeded stereoselectively,providing amixture of crystalline alcohols 35 (colorless crystals from ethyl acetate/hexane,m.p. 142-145 8 8C) and 36 (colorless crystals from ethyl acetate/ hexane,m .p.1 30-134 8 8C) (83 %y ield, 35:36 ca 2.5:1 d.r., chromatographically separated). Both 35 and 36 yielded to Xray crystallographic analysis [28] (see ORTEP representations, Scheme 3B)t hat proved their configurational arrangements as shown in Scheme 3B.The syn arrangement of H7' and H7'a in 35 and 36 was consistent with their 1 HNMR data (J 7',7'a = 3.9 Hz) as it was for their diastereoisomeric counterpart 30 (J 7',7'a = 4.0 Hz). Subsequent removal of the TBS group from intermediates 35 and 36 provided diols 37 and 39,respectively (TASF,7 8% yield for 37,9 1% yield for 39). Thet argeted diastereoisomeric structures of CJ-16,264 were then obtained from 37 and 39 through DMP oxidation to afford (À)-38 and 40 in 83 %a nd 82 %y ield, respectively.A gain, we observed epimerization at C7' in both final products (À)-38 and 40 as evidenced by the J 7',7'a = 0Hzcoupling constants between H7' and H7'af or both compounds.T he same argument and supporting evidence provided above for corrected-ent-1 and 32 apply here for (À)-38 and 40 and their precursors 37 and [1] pointing to the enantiomer of (À)-38 as the true structure of antibiotic CJ-16,264. With this information in our possession, we then sought to synthesize the antipode of (À)-38,c ompound (+ +)-38 (Scheme 4), in order to fully confirm the absolute configuration and obtain as ynthetic sample of the naturally occurring antibiotic CJ-16,264.
Thefinal drive toward the targeted molecule [(+ +)-38](via intermediates ent-29 plus 6, ent-35 and ent-37)p roceeded from (S)-citronellal (41)a long the same lines and in similar yields as the synthesis of (À)-38 (Scheme 3B)assummarized in Scheme 4. The 1 H-and 13 CNMR spectroscopic and spectrometric data for synthetic CJ-16,264 matched those reported [1] and provided by Dr.Y .S ugie for the natural product and so did the sign of optical rotation ([a] D =+7.78 8 in MeOH). Attempts to recrystallize (+ +)-38 from av ariety of solvents thus far led to partial formation of the C7' À C8' enol (enol proton, d = 12.72 ppm, keto:enol ca. 4:1) form and semicrystallization. Thee nolization of (+ +)-38 could be completely reversed by passage of the mixture through as ilica gel column (EtOAc:h exanes,h exanes!50 % EtOAc in hexanes). Thed euterated form (deuteration at C7')o f( + +)-38 was also detected through a 1 HNMR experiment ( [D 6 ]benzene , D 2 Oe xchange) in which the C7' proton signal decreased significantly.T he corresponding diastereoisomer of (+ +)-38,compound ent-40,was also synthesized from intermediate ent-36,f ormed in the coupling of ent-29 and racemic 6,via ent-39 (see Scheme 4) . Interestingly,the proven structure of antibiotic CJ-16,264 [(+ +)-38]r esembles closely that proposed for antibiotic 1a (pyrrolizilactone,F igure 1A), although the latter was not precisely defined with regard to the relative stereochemistry between the decalin and pyrrolizidinone domains and the absolute stereochemistry of the molecule (structure 1arepresented four stereostructures:two enantioisomers and two diastereoisomers). [5] Ther eported chemistry led to an enantioselective total synthesis and structural revision of antibiotic CJ-16,264 as structure (+ +)-38 and sets the foundation for the full structural elucidation of other members of this growing class of antitumor antibiotics.T he devised synthetic strategy features anovel macrolactonization/dimerization and astereoselective intramolecular bis-[4+ +2] cycloaddition to forge the highly substituted decalin system of the molecule in its proper stereochemical configuration. At otal of six isomeric structures of the antibiotic have been rendered readily available for biological investigations,u nderscoring the importance of total synthesis to both biology and structural elucidation. Most significantly,t he reported total synthesis of antibiotic CJ-16,264 paves the way for analog design, synthesis,a nd biological evaluation within this class of natural products.
Keywords: antibiotic ·n atural products ·structural revision · total synthesis ·transannular Diels-Alder reaction

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