Synthetic Studies on Et-743. Assembly of the Pentacyclic Core and a
`Formal Total Synthesis
`
`Dan Fishlock† and Robert M. Williams*,†,‡
`Department of Chemistry, Colorado State UniVersity, Fort Collins, Colorado 80523, and UniVersity of
`Colorado Cancer Center, Aurora, Colorado 80045
`
`rmw@lamar.colostate.edu
`ReceiVed May 30, 2008
`
`A formal total synthesis of the potent anticancer agent Et-743 is described. The tetrahydroisoquinoline
`core is stereoselectively constructed using a novel radical cyclization of a glyoxalimine. Further elaboration
`of this core rapidly accessed the pentacyclic core of Et-743, but a mixture of regiosisomers was obtained
`in the key Pictet-Spengler ring closure. A known advanced intermediate in the synthesis of Et-743 was
`intercepted, constituting a formal synthesis of the molecule.
`
`Introduction
`
`Members of the tetrahydroisoquinoline family of alkaloids
`display a wide range of biological properties such as antitumor
`and antimicrobial activities.1 Of particular significance within
`this family is Ecteinascidin 743 (Et-743, 1, Figure 1,) which
`has been demonstrated to possess extremely potent cytotoxic
`activity with in vitro IC50 values in the 0.1-1 ng/mL range in
`several cell lines (as a measure of RNA, DNA, and protein
`synthesis inhibition).2 Et-743 is currently in phase II/III clinical
`trials for the treatment of ovarian, endometrial, and breast
`cancers and several sarcoma lines.3The scarcity of the natural
`product from marine sources renders Et-743 an important target
`for synthesis. Corey and co-workers reported the first total
`synthesis of Et-743 in 36 steps with an overall yield of 0.72%.4a
`
`† Colorado State University.
`‡ University of Colorado Cancer Center.
`(1) Scott, J. D.; Williams, R. M. Chem. ReV. 2002, 102, 1669–1730.
`(2) (a) Rinehart, K. L.; Holt, T. G.; Fregeau, N. L.; Keifer, P. A.; Wilson,
`G. R.; Perun, T. J.; Sakai, R.; Thompson, A. G.; Stroh, J. G.; Shield, L. S.;
`Seigler, D. S. J. Nat. Prod. 1990, 53, 771–792. (b) Rinehart, K. L.; Holt, T. G.;
`Fregeau, N. L.; Stroh, J. G.; Keifer, P. A.; Sun, F.; Li, L. H.; Martin, D. G. J.
`Org. Chem. 1990, 55, 4512–4515. (c) Wright, A. E.; Forleo, D. A.; Gunawardana,
`G. P.; Gunasekera, S. P.; Koehn, F. E.; McConnell, O. J. J. Org. Chem. 1990,
`55, 4508–4512. (d) Guan, Y.; Sakai, R.; Rinehart, K. L.; Wang, A. H.-J.
`J. Biomol. Struct. Dyn. 1993, 10, 793–818. (e) Aune, G. J.; Furuta, T.; Pommier,
`Y. Anti-Cancer Drugs 2002, 13, 545–555. (f) Rinehart, K. L. Med. Drug ReV.
`2000, 1–27.
`
`9594
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`J. Org. Chem. 2008, 73, 9594–9600
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`FIGURE 1. Ecteinascidin 743 (1).
`A second-generation synthesis improved the overall yield to
`2.04%, but still required 36 steps.4b Fukuyama and co-workers
`achieved a total synthesis of Et-743 in 50 steps and 0.56%
`overall yield.5 More recently, Zhu and co-workers reported a
`31 step synthesis in 1.7% overall yield.6 Most recently,
`Danishefsky and co-workers reported a formal total synthesis7via
`a pentacyclic compound that intercepted a late-stage intermediate
`of Fukuyama’s route.5 Despite the advancements in the state-
`of-the-art in total synthetic approaches to Et-743, the clinical
`supply of this complex drug is semisynthetically derived from
`natural cyanosafracin B, obtained by fermentation as reported
`by PharmaMar.8
`Our laboratory has been developing methodology for the
`assembly of tetrahydroisoquinoline natural products and has
`10.1021/jo801159k CCC: $40.75  2008 American Chemical Society
`Published on Web 08/08/2008
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`Synthetic Studies on Et-743
`
`SCHEME 1.
`
`Synthetic Plan
`
`reported syntheses of D,L-quinocarcinamide,9 (-)-tetrazomine,10
`(-)-renieramycin G,11 (-)-jorumycin,11 and cribrostatin 4
`(renieramycin H).12 As a part of this program, we have targeted
`Et-743 by a convergent route that envisioned coupling of a
`suitably functionalized tyrosine derivative13 with the complete
`tetrahydroisoquinoline core (Scheme 1.) We have successfully
`deployed this strategy, with the present objective of construction
`of pentacycle A, in the synthesis of (-)-renieramycin G and
`(-)-jorumycin.11,12
`We have previously reported a concise and highly diastereo-
`selective synthesis of the tetrahydroisoquinoline core of Et-743
`
`(3) (a) Zelek, L.; Yovine, A.; Etienne, B.; Jimeno, J.; Taamma, A.; Martı´n,
`C.; Spielmann, M.; Cvitkovic, E.; Misset, J. L. Ecteinacidin-743 in Taxane/
`Antracycline Pretreated AdVanced/Metastatic Breast Cancer Patients: Prelimi-
`nary Results with the 24 h Continuous Infusion Q3 Week Schedule; 36th Annual
`Meeting of the American Society of Clinical Oncology, New Orleans, May 20-
`23, 2000; Abstract number 592. (b) Delaloge, S.; Yovine, A.; Taamma, A.; Cottu,
`P.; Riofrio, M.; Raymond, E.; Brain, E.; Marty, M.; Jimeno, J.; Cvitkovic, E.;
`Misset, J. L. Preliminary EVidence of ActiVity Of Ecteinacidin-743 (ET-743) in
`HeaVily Pretreated Patients with Bone and Soft Tissue Sarcomas; 36th Annual
`Meeting of the American Society of Clinical Oncology, New Orleans, May 20-
`23, 2000; Abstract number 2181. (c) Le Cesne, A.; Judson, I.; Blay, J. Y.;
`Radford, J.; an Oosterom, A.; Lorigan, P.; Rodenhuis, E.; Donato Di Paoula,
`E.; Van Glabbeke, M.; Jimeno, J.; Verweij, J. Phase II of ET-743 in AdVance
`Soft Tissue Sarcoma in Adult:A STBSG-EORTC Trial; 36th Annual Meeting of
`the American Society of Clinical Oncology, New Orleans, May 20-23, 2000;
`Abstract number 2182. (d) Aune, G. J.; Furuta, T.; Pommier, Y. Anti-Cancer
`Drugs 2002, 13, 545–555.
`(4) (a) Corey, E. J.; Gin, D. Y.; Kania, R. S. J. Am. Chem. Soc. 1996, 118,
`9202–9203. (b) Martinez, E. J.; Corey, E. J. Org. Lett. 2000, 2, 993–996.
`(5) Endo, A.; Yanagisawa, A.; Abe, M.; Tohma, S.; Kan, T.; Fukuyama, T.
`J. Am. Chem. Soc. 2002, 124, 6552–6554.
`(6) Chen, J.; Chen, X.; Bois-Choussy, M.; Zhu, J. J. Am. Chem. Soc. 2006,
`128, 87–89.
`(7) Zheng, S.; Chan, C.; Furuuchi, T.; Wright, B. J. D.; Zhou, B.; Guo, J.;
`Danishefsky, S. J. Angew. Chem., Int. Ed. 2006, 45, 1754–1759.
`(8) Cuevas, C.; Pe´rez, M.; Martı´n, M. J.; Chicharro, J. L.; Ferna´ndez-Rivas,
`C.; Flores, M.; Francesch, A.; Gallego, P.; Zarzuelo, M.; de la Calle, F.; Garcı´a,
`J.; Polanco, C.; Rodrı´guez, I.; Manzanares, I. Org. Lett. 2000, 2, 2545–2548.
`(9) Flanagan, M. E.; Williams, R. M. J. Org. Chem. 1995, 60, 6791–6797.
`(10) (a) Scott, J. D.; Williams, R. M. Angew. Chem., Int. Ed. 2001, 40, 1463–
`1465. (b) Scott, J. D.; Williams, R. M. J. Am. Chem. Soc. 2002, 124, 2951–
`2956.
`(11) (a) Lane, J. W.; Chen, Y.; Williams, R. M. J. Am. Chem. Soc. 2005,
`127, 12684–12690. (b) Lane, J. W.; Estevez, A.; Mortara, K.; Callan, O.; Spencer,
`J. R.; Williams, R. M. Bioorg. Med. Chem. Lett. 2006, 16, 3180–3183. (c)
`Vincent, G.; Lane, J. W.; Williams, R. M. Tetrahedron Lett. 2007, 48, 3719–
`3722. (d) Jin, W.; Metobo, S.; Williams, R. M. Org. Lett. 2003, 5, 2095–2098.
`
`(E).14 This was achieved via an intramolecular 6-endo radical
`closure on a glyoxalimine, and the desired 1,3-cis-diastereomer
`was obtained exclusively. The synthesis of a tetrahydroiso-
`quinoline such as E can be problematic because of the acid
`sensitivity of the benzylic hydroxyl, particularly because it is
`ortho to the phenolic hydroxyl of the aromatic ring and thus
`has a high propensity for ortho-quinonemethide formation.
`Herein, we report a formal total synthesis of Et-743 as part of
`our ongoing efforts to devise a practical and scalable synthesis
`of this potent antitumor antibiotic that would be amenable to
`the construction of analogues with anticipated potent cytotoxic
`activity.
`
`Results and Discussion
`The synthesis began with Borchardt’s catechol 315 that was
`regioselectively brominated to generate 4 (92% yield) (Scheme
`2.) Conversion of catechol 4 to the methylenedioxy aldehyde 5
`was accomplished using bromochloromethane in a sealed vessel
`(69% yield). Baeyer-Villiger oxidation using m-CPBA provided
`bromophenol 6 as an off-white solid following hydrolysis of
`the resulting formate intermediate (73% yield). Stereoselective
`aldol condensation of the titanium phenolate of 6 with (R)-
`Garner’s aldehyde (7)16 using a modification of Casiraghi’s
`method17 provided the anti-product 8 followed by allyl protec-
`tion of the phenolic oxygen delivering 9 (65% yield, two steps).
`Subsequent hydrolysis of the oxazolidine and formation of the
`trans-acetonide (84% yield, two steps) provided 10 as an oil
`that cleanly underwent N-Boc deprotection using Ohfune’s
`protocol18 (76% yield) to afford free amine 11 as a stable
`crystalline solid. From 11, the glyoxalimine intermediate 13 (see
`Scheme 3) was readily obtained by condensation with ethyl
`glyoxalate. Following isolation by filtration through Celite and
`concentration, the radical ring closure commenced with slow
`addition of Bu3SnH and AIBN via syringe pump to a refluxing
`dilute solution of the glyoxalimine (13). Concentration and KF/
`silica chromatography19 of the crude reaction mixture provided
`solid 12 as a single diastereomer (58% yield, two steps). The
`relative stereochemistry of 12 was secured 1H NMR data and
`corroborated by X-ray crystallography. Examination of the crude
`1H NMR revealed the formation of a single diastereomer in the
`radical closure and exclusive 6-endo regioselectivity. In addition
`to 12 and tin impurities visible in the 1H NMR spectrum, an
`aromatic proton arising from hydride quenching of the aryl
`radical revealed a ∼6.6:1 ratio of 12 to reduced substrate. Slower
`addition rates (over 18 or 36 h) did not improve the isolated
`yield of 12.
`
`(12) (a) Vincent, G.; Williams, R. M. Angew. Chem., Int. Ed. 2007, 46, 1517–
`1520. (b) Vincent, G.; Chen, Y.; Lane, J. W.; Williams, R. M. Heterocycles
`2007, 72, 385–398.
`(13) Jin, W.; Williams, R. M. Tetrahedron Lett. 2003, 44, 4635–4639.
`(14) Fishlock, D.; Williams, R. M. Org. Lett. 2006, 8, 3299–3301.
`(15) Prep a red from 2,3-dimethoxytoluene according to: Shinhababu, A. K.;
`Ghosh, A. K.; Borchardt, R. T. J. Med. Chem. 1985, 28, 1273–1279.
`(16) (R)-Garner‘s aldehyde was synthesized from D-serine according to:
`Garner, P.; Park, J. M. Org. Synth. 1992, 70, 18–28.
`(17) Casiraghi, G.; Cornia, M.; Rassu, G. J. Org. Chem. 1988, 53, 4919–
`4922. In our case, sonication was not required as described in the original paper.
`(18) Sakaitani, M.; Ohfune, Y. J. Org. Chem. 1990, 55, 870–876.
`(19) Effective removal of tin impurities from Bu3SnH-mediated reactions:
`Harrowven, D. C.; Guy, I. L. Chem. Commun. 2004, 1968–1969.
`(20) (a) De Paolis, M.; Chiaroni, A.; Zhu, J. Chem. Commun. 2003, 2896–
`2897. (b) Chen, X.; Chen, J.; De Paolis, M.; Zhu, J. J. Org. Chem. 2005, 70,
`4397–4408.
`(21) (a) Herberich, B.; Kinugawa, M.; Vazquez, A.; Williams, R. M.
`Tetrahedron Lett. 2001, 42, 543–546. (b) Jin, W; Metobo, S.; Williams, R. M.
`Org. Lett. 2003, 5, 2095–2098.
`
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`SCHEME 2. Tetrahydroisoquinoline Core of Et-743
`
`Fishlock and Williams
`
`SCHEME 3. Pentacycle Construction
`
`The diastereoselectivity of this reaction stands apart from
`numerous Pictet-Spengler cyclizations on related substrates that
`provide tetrahydroisoquinolines exclusively as the 1,3-trans-
`diastereomers.11,20,21 We qualitatively rationalize the cis-dias-
`tereoselectivity of this radical process using the Beckwith-Houk
`chairlike transition state model for intramolecular radical ring
`closures (Figure 2).22 The lowest-energy chair conformation (A)
`
`adopted by the trans-acetonide of the substrate (13) results in
`both the glyoxalimine and aryl substituent being in an equatorial
`disposition. In this conformation, 1,3-diaxial steric effects and
`allylic strain interactions are minimized in the ring-forming
`transition state. To further examine the stereocontrol imparted
`the cis-acetonide substrate 14 was
`by the acetonide ring,
`prepared (using Casiraghi’s method from the magnesium
`phenolate of 6).17 Substrate 14 resulted in a 1:1 mixture of 1,3-
`
`(22) (a) Beckwith, A. L. J.; Schiesser, C. H. Tetrahedron 1985, 41, 3925–
`3941. (b) Spellmeyer, D. C.; Houk, K. N. J. Org. Chem. 1987, 52, 959–974.
`
`(23) Chen, X.; Zhu, J. Angew. Chem., Int. Ed 2007, 46, 3962–3965.
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`Synthetic Studies on Et-743
`
`FIGURE 2. Transition state models to rationalize the observed 1,3 relative stereochemistry in the tetrahydroisoquinoline radical ring closure.
`
`trans- and 1,3-cis-tetrahydroisoquinolines (15 and 16, both are
`known componds),20 which suggests the energy difference
`between transition state conformations B and C (axial aryl group
`versus axial glyoxalimine) is negligible.
`As shown in Scheme 3, reduction of the tetrahydroisoquino-
`line ester (12)14 with LAH, followed by immediate protection
`as the benzyl ether (17), proceeded cleanly in 77% yield over
`two steps. The substituted tyrosine amino acid component (18)
`has been previously reported by us, utilizing the oxazinone
`template technology developed in our laboratory that was
`benzylated with the advanced aromatic side chain.13 Thus,
`acylation of the tetrahydroisoquinoline (17) was achieved via
`the N-Fmoc-protected amino acid chloride (18) to give amide
`19a without epimerization. The use of the N-Boc free acid with
`a variety of coupling agents (DCC, HOBt, HATU) all resulted
`in very sluggish reactions with poor isolated yields, as did the
`attempted use of the N-Boc acid fluoride.
`Treatment of 19a with diethylamine provided the free amine,
`which was not isolated in favor of immediate evaporation of
`excess base and solvent and subsequent Boc protection of the
`crude material. Isolation following chromatography provided
`compound 19b in 90% yield. Removal of the acetonide from
`19b was accomplished using the extremely mild, albeit slow,
`method of stirring with Dowex 50W-X8 cationic resin in
`methanol. Complete deprotection took 8-12 h, but the yield
`was quantitative following simple filtration and concentration.
`Instead of providing the usual diol product,
`this substrate
`incorporated methanol at the benzylic position thus providing
`the methyl ether as a ∼1:1 mixture of diastereomers. Not
`unexpectedly, the benzylic stereogenic center loses stereochem-
`ical integrity since the methanol is incorporated via the incipient
`ortho-quinonemethide species arising from the acidic depro-
`tection conditions.
`Alternatively, we found that the use of water/dichloromethane
`with cationic resin on 19b could provide the corresponding free
`
`diol, but oxidation of the primary alcohol (in the presence of
`the free benzylic alcohol) could not, in our hands, be cleanly
`accomplished. The methyl ether was thus a fortuitous selective
`protection of the benzylic alcohol, ultimately simplifying the
`subsequent manipulations.
`Facile deprotection of the O-TBS-protected phenol using
`TBAF was followed by oxidation of the primary alcohol using
`Swern conditions in high yield. This oxidation product (20)
`existed as an equilibrium mixture of the aldehyde and the
`corresponding hemiaminal species (illustrated) as observed by
`1H NMR, which was otherwise additionally complicated by
`amide and carbamate rotamers. The attempted oxidation using
`either Dess-Martin periodinane or TPAP/NMO both failed,
`leading to extensive decomposition. Following filtration of crude
`20 through a plug of silica gel, this substance was immediately
`subjected to the Pictet-Spengler conditions.
`The objective at this stage was to achieve the Pictet-Spengler
`reaction via N-Boc deprotection, iminium ion formation, and
`electrophilic aromatic substitution to provide the desired pen-
`tacyclic core of Et-743. This meant that the aromatic substitution
`must occur ortho to the free phenol, and the benzylic methyl
`ether must survive these conditions. Unfortunately, it had already
`been demonstrated above that the electron-rich aromatic ring
`of the tetrahydroisoquinoline component was highly sensitive
`to protic conditions, leading to ortho-quinonemethide formation.
`Indeed, when substrate 20 was treated with trifluoroacetic
`acid in methylene chloride, it cleanly underwent the expected
`pentacycle formation furnishing 21 + 22 as a ∼0.72:1 ortho:
`para mixture of regioisomers in 72% combined yield. As
`anticipated, the benzylic methoxy group was eliminated presum-
`ably via the incipient ortho-quinonemethide species that forms
`under these conditions. In a fruitless effort to circumvent the
`vexing olefin formation, pentacycle formation with TFA in dry
`methanol resulted in extensive decomposition of the substrate.
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`SCHEME 4. Pictet-Spengler Regioselectivity
`
`Fishlock and Williams
`
`As part of these synthetic investigations, the intermediate 23
`was prepared (in parallel with the O-benzyl-protected synthesis)
`bearing an O-allyl-protected hydroxymethyl at C1 of the THIQ
`core. This substrate was used to examine the regioselectivity
`of the pentacycle-forming ring closure and was utilized to
`acquire detailed 1H NMR data, while the O-benzyl material 21
`was carried forward in the synthesis. One interesting observation
`was the behavior of compound 25 containing the O-Boc
`carbonate-protected phenolic oxygen. Treatment of 25 under
`the same reaction conditions provided the pentacycles 26 + 27
`in a 2:3 ratio of ortho:para regioisomers. The O-Boc carbonate
`would presumably be deprotected quickly under these conditions
`to reveal
`the free phenol-containing reactive species,
`thus
`resulting in a comparable regioselectivity as observed with
`substrate 20 (beginning with a free phenol on the aryl nucleo-
`phile moiety). Notably, however, when substrate 25 was treated
`the O-Boc carbonate was selectively
`with K2CO3/MeOH,
`removed (28) with apparent olefin formation prior to the
`Pictet-Spengler reaction and pentacycle formation. Treatment
`of 28 with TFA in dichloromethane produced the pentacycles
`26 + 27 in a 1:3 ratio of ortho:para regioisomers, supporting
`the hypothesis that some regioselectivity in the closure might
`arise from an intramolecular H bond with a heteroatom at the
`benzylic position.11c
`In their synthesis of renieramycin H, the Zhu group has
`interestingly reported control of Pictet-Spengler regioselectivity
`in a related system by variation of acid concentration (Scheme
`4).23 It was found in that case that lowering the concentration
`of methanesulfonic acid to 0.01% in CH2Cl2 could invert the
`ortho:para selectivity from 3.4:1 to 2:3. Furthermore, the use
`of acetonitrile as the solvent instead of dichloromethane favored
`the undesired isomer, giving ortho:para selectivity of 1:10. Our
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`attempt to reproduce the Zhu conditions on substrate 24 using
`the
`0.01% methanesulfonic acid in CH2Cl2 did not affect
`regioselectivity of this reaction. The substrate was consumed
`to provide some material that appeared to still contain the N-Boc
`protecting group, but the 1H NMR of the crude product was
`prohibitively complex. Subsequent treatment of this reaction
`crude with a TFA/anisole/CH2Cl2 mixture provided the penta-
`cycles 26 + 27 with ∼1:1 regioselectivity. The same ratio is
`obtained if the TFA/anisole conditions are used directly on
`substrate 24.
`In order to redeem the synthetic utility of the olefinic products
`(21 or 26), our attention was captured by the recent formal
`synthesis of Et-743 reported by the Danishefsky group7 in which
`the olefin (29, Scheme 5) underwent facile oxidation using
`DMDO and immediate hydride reduction delivering the benzylic
`alcohol 30. With the availability of this methodology in the
`literature, our efforts were briefly redirected to convert our
`synthetic pentacycle 21 into compound 29 which would
`constitute a formal total synthesis of Et-743 by relay through
`the Danishefsky7 and then Fukuyama5 syntheses, respectively.
`In the event, the desired pentacycle 21 (Scheme 3) was
`N-protected as the trichloroethyl carbamate (Troc), and the
`phenolic residue was protected as the corresponding O-benzyl
`ether in 85% yield for the two steps (Scheme 5). Removal of
`the O-allyl group under standard conditions followed by
`reprotection as the corresponding MOM ether provided com-
`pound 29 (56% yield for the two steps). Compound 29 perfectly
`matched Danishefsky’s substrate by 1H, 13C NMR, and optical
`rotation, confirming the structure of compound 29.
`Since Danishefsky has previously converted7 compound 29
`into a late-stage intermediate in Fukuyama’s total synthesis5
`(namely, compound 30, Scheme 5), this two-stage relay of our
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`Synthetic Studies on Et-743
`
`SCHEME 5. Formal Synthesis of Et-743 via 21 to 29 and Danishefsky to Fukuyama Relay
`
`synthetic 21 thus constitutes a formal total synthesis of Et-743
`and provides firm structural corroboration of our synthetic
`material and methods.
`While the present formal synthesis reveals that our glyoxal-
`imine radical cyclization technology14 holds considerable
`potential for the efficient total synthesis of Et-743 and congeners,
`we are currently endeavoring to improve the regioselectivity
`of the key pentacycle formation (20 to 21) as well as refining
`the overall synthetic efficiency of our approach. These objectives
`are currently under study in our laboratory and will be reported
`in due course.
`
`Experimental Section
`
`For general methods and considerations, see Supporting Informa-
`tion.
`Compound 19. The Fmoc-amino acid (410 mg, 0.727 mmol,
`1.2 equiv) was dissolved in dry toluene and concentrated (×2),
`and then dried under high vacuum. This oil was dissolved in dry
`CH2Cl2 (4 mL) to which was added oxalyl chloride (1 mL) at room
`temperature, followed by dry DMF (20 µL). After stirring for 20
`min, the solution was concentrated and reconcentrated from dry
`toluene (×2) and then dried under high vacuum. This acid chloride
`18 was dissolved in CH2Cl2 (4 mL) and cooled to 0 °C. THIQ(OBn)
`17 (275 mg, 0.61 mmol, 1 equiv) was dissolved in dry CH2Cl2 (2
`mL) and 2,6-lutidine (77 µL 0.67 mmol, 1.1 equiv). This solution
`was transferred into the acid chloride solution slowly dropwise,
`and the resulting mixture was warmed to rt and stirred 7 h (TLC
`showed consumption of the THIQ(OBn) starting material). The
`reaction was quenched with saturated NH4Cl (aq) and then extracted
`to EtOAc (×3). The combined organic fractions were dried
`(Na2SO4), filtered, and concentrated to provide a crude orange oil.
`Purification by flash chromatography (hexanes:EtOAc 5:1, silica
`gel) gave the peptide 19a as a pale yellow oil (426 mg, 70%); Rf
`) 0.34 (3:1 hexanes:EtOAc, UV, CAM); [R]25
`D -22.8 (c 1.14,
`CH2Cl2); IR (thin film) 3289, 2929, 2858, 1717, 1634 cm-1; 1H
`and 13C NMR spectra are extremely complex due to amide and
`carbamate rotamers. See the rt (CDCl3) and 373 K (DMSO-d6) 1H
`spectra and rt (CDCl3) 13C spectra in the Supporting Information;
`HRMS(ESI/APCI+) m/z calcd for C58H68N2O11NaSi (M + Na)+
`1019.4485, found 1019.4499.
`Compound 19b. Fmoc (OBn) peptide 19a (146 mg, 0.146 mmol)
`was dissolved in a 20% v/v solution of Et2NH in CH2Cl2 [CH2Cl2
`(2.5 mL) and diethylamine (0.6 mL]. After stirring for 6 h, the
`solution was concentrated and then reconcentrated from toluene
`and dried under high vacuum. The crude material was dissolved in
`EtOH:CH2Cl2 (2:0.5 mL) to which was added Boc2O (370 mg, 10
`
`equiv). After stirring for 12 h, the reaction was concentrated and
`immediately purified by flash chromatography (9:1 to 5:1 hexanes:
`EtOAc, silica gel) to provide 19b as a clear colorless oil (115 mg,
`90% over 2 steps): Rf ) 0.43 (3:1 hexanes:EtOAc, UV, CAM);
`D -26.6 (c 1.0, CH2Cl2); IR (thin film) 3319, 2930, 2858, 1711,
`[R]25
`1646 cm-1; 1H and 13C NMR spectra are extremely complex due
`to amide and carbamate rotamers; see the 1H spectra (CDCl3, rt)
`and (DMSO-d6, 373 K) and 13C spectrum (CDCl3, rt) in the
`Supporting Information; HRMS(ESI/APCI+) m/z calcd for
`C48H66N2O11NaSi (M + Na)+ 897.4328, found 897.4310.
`Compounds 21 and 22. Boc (OBn) peptide 19b (115 mg, 0.132
`mmol) was dissolved in dry MeOH (5 mL), and Dowex 50W-X8
`cationic resin (100 mg) was added (the resin was first rinsed with
`dry methanol and dried under a stream of argon). After 65 h, the
`reaction was complete by TLC and a single streak was observed
`(during the course of the reaction, two streaks initially arise due to
`a mixture of diol and methyl ether/alcohol products). The reaction
`was filtered through a plug of Celite, eluting with dry MeOH, and
`the filtrate was combined to provide the methyl ether as clear,
`colorless oil (100 mg, 90% yield): Rf ) 0 to 0.35 streak (3:1
`hexanes:EtOAc, UV, CAM); HRMS(FAB+) m/z calcd for
`C46H65N2O11Si (M + H)+ 849.4358, found 849.4354. The methyl
`ether (100 mg) was dissolved in THF (3 mL), and TBAF (1 M in
`THF, 125 µL, 1.06 equiv) was added in one portion. After 20 min,
`the reaction was concentrated by rotary evaporation and passed
`through a silica plug (eluting with 3:1 to 1:1 hexanes:EtOAc) to
`provide the free phenol as a clear, colorless oil (82 mg, 95% yield):
`Rf ) 0 to 0.43 streak (3:1 hexanes:EtOAc, UV, CAM); HRMS-
`(FAB+) m/z calcd for C40H51N2O11 (M + H)+ 735.3493, found
`735.3490. Oxalyl chloride (15 µL 1.5 equiv) was added carefully
`to a solution of DMSO (25 µL, 3.2 equiv) in CH2Cl2 (1 mL)
`previously cooled to -78 °C. A solution of the above alcohol (82
`mg, 0.11 mmol) in CH2Cl2 (2 mL) was added dropwise, and the
`resulting mixture was stirred at -78 °C for 40 min. The reaction
`was quenched with Et3N (125 µL, 8 equiv) and then allowed to
`warm to rt. The reaction was diluted with CH2Cl2 and washed with
`brine, and then the combined organic fractions were dried (Na2SO4),
`filtered, and concentrated. The crude material was passed through
`a silica gel plug (eluting with hexanes:EtOAc 1:1) to provide a
`yellow oil/foam (82 mg, quant.) of hemiaminal 20 which was used
`without further purification: Rf ) 0.5 (hexanes:EtOAc 1:1, UV,
`CAM). Hemiaminal 20 (232 mg, 0.32 mmol) was dissolved in
`CH2Cl2 (3 mL) to which were added TFA (3 mL) and anisole (0.350
`mL) at rt. The reaction was stirred for 14 h and then concentrated
`to remove TFA, then redissolved in CH2Cl2 and washed with
`saturated aq NaHCO3. The organic fraction was dried (Na2SO4),
`filtered, and concentrated. Crude 1H NMR shows 0.72:1 ortho (21)
`to para (22) regioisomers. Purification by PTLC (2% MeOH in
`
`J. Org. Chem. Vol. 73, No. 24, 2008 9599
`
` P. 6
`
`UT Ex. 2028
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`

`
`Fishlock and Williams
`
`69.8, 60.5, 54.4/53.7, 50.9/50.1, 47.3/47.2, 32.8/32.4, 16.0, 9.5;
`HRMS(ESI/APCI+) m/z calcd for C44H42N2O9Cl3 (M + H)+
`847.1950, found 847.1949.
`The allyl-protected pentacycle obtained above (20 mg, 0.024
`mmol) was dissolved in CH2Cl2 (400 µL), and pyrrolidine (6 µL,
`3 eq) was added, followed by Pd(PPh3)4 (2 mg, 0.002 mmol) under
`Ar. After 16 h, the reaction was still not complete, so additional
`portions of pyrrolidine and palladium catalyst were added. After
`stirring an additional 4 h (20 h total), the dark green reaction was
`applied directly to flash chromatography (silica gel, hexanes:EtOAc
`3:1). The pure fractions were combined to provide the phenol as
`yellow oil (11 mg 56%), used without characterization: Rf ) 0.26
`(hexanes:EtOAc 3:1, UV, CAM). Phenol (11 mg, 0.014 mmol) was
`dissolved in CH2Cl2 (200 µL) to which were added iPr2NEt (12
`µL, 0.07 mmol, 5 equiv) and MOMBr (3.3 µL, 0.042 mmol, 3
`equiv). The mixture was stirred for 30 min at rt and then quenched
`with water and extracted with CH2Cl2 (×3). The combined organic
`fractions were dried (Na2SO4), filtered, and concentrated. Flash
`chromatography (hexanes:EtOAc 3:1) provided the protected pen-
`tacycle 29 (11.5 mg, quant.): Rf ) 0.41 (hexanes:EtOAc 3:1, UV,
`CAM); [R]25
`D +45.4 (c 0.8, CHCl3) [lit. +50 (c 1.0, CHCl3)]; IR
`(thin film) 2932, 1723, 1681, 1654, 1432, 1371 cm-1. 1H and 13C
`NMR spectra perfectly match the data provided by the Danishefsky
`group for this intermediate in their formal synthesis (copies of their
`spectra included in the Supporting Information):7 1H NMR (400
`MHz, CDCl3, mixture of carbamate rotamers) δ 7.56-7.31 (m, 5H),
`7.13-7.23 (m, 3H), 6.96 (app br d, J ) 6.9 Hz, 2H), 6.46 (d, J )
`9.4 Hz, 1H), 6.01-6.15 (m, 3H), 5.86 (app d, J ) 3.0 Hz, 2H),
`5.82 (br s, 1H), 4.97-5.19 (m, 4H), 4.86 (d, J ) 11.9 Hz, 1H),
`4.79 (A of AB quart, J ) 12.0 Hz, 1H), 4.68 (B of AB quart, J )
`11.9 Hz, 1H), 4.49-4.60 (m, 2H), 4.43 (app d, J ) 6.1 Hz, 1H),
`4.01 (d of A of AB quart, J ) 11.8, 4.4 Hz, 1H), 3.85 (B of AB
`quart, J ) 12.1 Hz, 1H), 3.71 (app d, J ) 10.6 Hz, 3H), 3.38
`(rotomeric s, 3H), 3.03-3.29 (m, 5H), 2.11 (s, 6H); 13C NMR (100
`MHz, CDCl3, mixture of carbamate rotamers) δ 166.0/165.9, 151.6/
`151.4, 149.9, 148.7/148.2, 147.3, 146.2/146.1, 139.8, 138.4/138.4,
`137.8/137.7, 132.6/132.5, 131.1/131.1, 128.8, 128.2, 127.9, 127.2,
`126.8, 126.6/126.5, 125.2/125.0, 117.0/116.8, 113.7/113.6, 108.5/
`108.4, 103.3/102.7, 101.6, 100.4/100.4, 95.3/95.2, 75.4/75.3, 74.4/
`74.0, 72.6/72.6, 69.9/69.9, 60.4, 57.6/57.5, 54.4/53.7, 50.8/50.1,
`47.4/47.3, 32.7/32.3, 16.0, 9.9; HRMS(ESI/APPI+) m/z calcd for
`C43H42N2O10Cl3 (M + H)+ 851.1900, found 851.1897.
`Acknowledgment. This paper is fondly dedicated to the
`memory of the late Professor A.I. Meyers. We gratefully
`acknowledge financial support from the National Institutes of
`Health (Grant CA85419). We are grateful to Prof. Samuel J.
`Danishefsky of the Memorial Sloan-Kettering Cancer Research
`Institute for kindly providing characterization data for compound
`29.
`
`Note Added after ASAP Publication. Reference 6 contained
`an incorrect publication date and the description of the condi-
`tions used by Zhu et al. (below Scheme 4) was erroneous in
`the version published ASAP August 8, 2008; the corrected
`version was published ASAP September 17, 2008.
`
`Supporting Information Available: Complete experimental
`procedures and spectroscopic data. This material is available
`free of charge via the Internet at http://pubs.acs.org.
`
`JO801159K
`
`EtOAc) provided the ortho (63 mg) and para products (69 mg) for
`a combined yield of 72%. Data for 21: Rf ) 0.61 (EtOAc:MeOH
`95:5, UV, CAM); [R]25
`D -18.0 (c 1.0, CH2Cl2); IR (thin film) 3295,
`2932, 1672, 1632, 1455, 1428 cm-1; 1H NMR (400 MHz, CDCl3)
`δ 7.14-7.30 (m, 3H), 6.98 (s, 1H), 6.97 (s, 1H), 6.24 (s, 1H), 6.19
`(s, 1H), 6.12 (dddd, J ) 16.0, 11.0, 5.4, 5.4 Hz, 1H), 6.05 (dd, J
`) 7.2, 5.0 Hz, 1H), 5.85 (br s, 1H), 5.82 (br s, 1H), 5.78 (v br s,
`1H), 5.45 (app dd, J ) 17.1, 1.1 Hz, 1H), 5.29 (app dd, J ) 10.3,
`0.8 Hz, 1H), 4.9 (s, 1H), 4.30 (app d of AB quartet, J ) 12.3, 5.4
`Hz, 2H), 4.03 (d, J ) 6.1 Hz, 1H), 3.87 (AB quartet, J ) 12.1 Hz,
`2H), 3.63 (s, 3H), 2.95-3.2 (m, 5H), 2.11 (s, 3H), 2.09 (s, 3H);
`13C NMR (100 MHz, CDCl3) δ 168.8, 147.6, 145.6 (×2), 143.4,
`139.7, 138.7, 134.5, 133.9, 129.4, 128.8, 128.0 (×2), 127.1, 126.8
`(×2), 122.5, 119.3, 117.7, 117.5, 113.0, 108.7, 101.5, 100.2, 75.3,
`72.6, 70.0, 60.8, 54.4, 50.0, 46.9, 33.4, 15.9, 9.4. HRMS(ESI/
`APCI+) m/z calcd for C34H35N2O7 (M + H)+ 583.2439, found
`583.2441. Data for 22: Rf ) 0.5 (EtOAc:MeOH 95:5, UV, CAM);
`[R]25
`D +47.8 (c 1.45, CH2Cl2); IR (thin film) 3298, 2931, 1671,
`1631, 1430, 1409 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.11-7.22
`(m, 3H), 6.94 (s, 1H), 6.92 (s, 1H), 6.41 (s, 1H), 6.11 (dddd, J )
`16.1, 10.6, 5.5, 5.5 Hz, 1H), 6.08 (s, 1H), 6.03 (dd, J ) 6.6, 5.1
`Hz, 1H), 5.86 (br s, 1H), 5.83 (br s, 1H), 5.45 (app dd, J ) 17.1,
`1.1 Hz, 1H), 5.30 (app dd, J ) 10.4, 0.8 Hz, 1H), 4.65 (s, 1H),
`4.36 (app d of A of AB quartet, J ) 12.5, 5.5 Hz, 1H), 4.24 (app
`d of B of AB quartet, J ) 12.5, 5.5 Hz, 2H), 4.01 (d, J ) 6.0 Hz,
`1H), 3.91 (AB quartet, J ) 12.2 Hz, 1H), 3.56 (s, 3H), 2.95-3.24
`(m, 5H), 2.27 (s, 3H), 2.12 (s, 3H); 13C NMR (100 MHz, CDCl3)
`δ 168.7, 148.0, 147.6, 145.9, 144.3, 139.7, 138.4, 134.6, 133.7,
`129.6, 128.7, 128.1 (×2), 127.2, 126.9 (×2), 124.9, 117.8, 117.1,
`113.8, 113.0, 108.8, 101.5, 100.5, 75.4, 72.8, 70.1, 61.0, 54.3, 52.6,
`46.8, 35.4, 12.0, 9.4; HRMS (ESI/APCI+) m/z calcd for
`C34H35N2O7 (M + H)+ 583.2439, found 583.2429.
`Preparation of Compound 29. The desired ortho-regioisomer
`21 (55 mg, 0.095 mmol) was dissolved in CH2Cl2 (2 mL) and
`pyridine (11 µL, 0.14 mmol, 1.5 equiv) at 0 °C. TrocCl (13.5 µL,
`0.1 mmol, 1.0 equiv) was added and the reaction maintained at 0
`°C for 2 h, and then diluted with CH2Cl2 and washed with saturated
`aq NH4Cl. The organic layer was dried (Na2SO4), filtered, and then
`concentrated. The crude oil was passed through a plug of silica gel
`eluting with EtOAc, and then concentrated and dried under vacuum.
`The resulting oil was dissolved in CH2Cl2 (600 µL), and MeOH
`(200 µL) and K2CO3 (52 mg, 0.38 mmol, 4 equiv) were added
`followed by benzyl bromide (22 µL, 0.19 mmol, 2 equiv) and a
`catalytic amount of tetrabutylammonium iodi