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The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

A highly convergent, stereocontrolled total synthesis of the potent antiproliferative agent (+)-phorboxazole A (1) has been achieved. Highlights of the synthesis include: modified Petasis−Ferrier rearrangements for assembly of both the C(11−15) and C(22−26) cis-tetrahydropyran rings; extension of the Julia olefination to the synthesis of enol ethers; the design, synthesis, and application of a novel bifunctional oxazole linchpin; and Stille coupling of a C(28) trimethyl stannane with a C(29) oxazole triflate. The longest linear sequence leading to (+)-phorboxazole A (1) was 27 steps, with an overall yield of 3%.

[formula: see text] In this, the second of two Letters, we describe the efficient assembly of (+)-4, a C(20-28) subtarget for the total synthesis of phorboxazoles A (1) and B (2). The synthesis was achieved in 12 linear steps (20% overall yield) via Petasis-Ferrier rearrangement of an E/Z mixture of trisubstituted enol ethers (15) to assemble the C(22-26) cis-tetrahydropyran. A mechanism for the observed diastereoconvergence of 15 is proposed. In addition, a new tactic for the synthesis of enol ethers (e.g., 15) based on the elegant work of Julia is described.

[formula: see text] In this, the first of two letters, we outline our overall strategy for the total synthesis of phorboxazoles A (1) and B (2), rare oxazole-containing macrolides possessing extraordinary antimitotic activity, and describe the assembly of a C(3-19) subtarget (-)-5 for the total synthesis of phorboxazole A. The synthesis of (-)-5 was achieved in 15 linear steps (12% overall yield), exploiting a modification of the Petasis-Ferrier rearrangement to construct the C(11-15) cis-tetrahydropyran. Dimethylaluminum chloride (Me2AlCl) proved to be the Lewis acid of choice for the Petasis-Ferrier rearrangement.

In this Letter, the first in a series of three, we outline our overall strategy and describe the assembly of a C(29–48) EF-ring advanced intermediate for the total synthesis of the spongistatins, rare and structurally unique polyether macrolides with unprecedented antitumor activity.

To improve receptor binding affinity and to investigate functional selectivity of 2,8-dimethyl-1-oxa-8-azaspiro[4.5]decan-3-one acetylhydrazone 2 at muscarinic receptor subtypes, a series of acylhydrazones A was synthesized. The SAR indicates that the binding affinity in the pirenzepine assay (M1) correlates well with lipophilicity. Intrinsic activity (30% of carbachol response) of agonists at M1 remains unchanged. Compounds with n = 0 and 6, where X = NHCO(CH 2 ) n Me, did not inhibit cAMP formation in rat heart membrane (M2). Most of the compounds are more efficacious at the M3 receptor than at M1. The results suggest that the M1 and M3 receptors can better tolerate bulky and long chained substituents than the M2 receptor.

6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943) is a novel PDE9A inhibitor identified using parallel synthetic chemistry and structure-based drug design (SBDD) and has advanced into clinical trials. Selectivity for PDE9A over other PDE family members was achieved by targeting key residue differences between the PDE9A and PDE1C catalytic site. The physicochemical properties of the series were optimized to provide excellent in vitro and in vivo pharmacokinetics properties in multiple species including humans. It has been reported to elevate central cGMP levels in the brain and CSF of rodents. In addition, it exhibits procognitive activity in several rodent models and synaptic stabilization in an amyloid precursor protein (APP) transgenic mouse model. Recent disclosures from clinical trials confirm that it is well tolerated in humans and elevates cGMP in cerebral spinal fluid of healthy volunteers, confirming that it is a quality pharmacological tool for testing clinical hypotheses in disease states associated with impairment of cGMP signaling or cognition.

A highly convergent, stereocontrolled total synthesis of the potent antiproliferative agent (+)phorboxazole A (1) has been achieved. Highlights of the synthesis include: modified Petasis-Ferrier rearrangements for assembly of both the C(11-15) and C(22-26) cis-tetrahydropyran rings; extension of the Julia olefination to the synthesis of enol ethers; the design, synthesis, and application of a novel bifunctional oxazole linchpin; and Stille coupling of a C(28) trimethyl stannane with a C(29) oxazole triflate. The longest linear sequence leading to (+)-phorboxazole A (1) was 27 steps, with an overall yield of 3%. Marine sponges comprise a rich source of architecturally complex, biomedically important natural products; examples include the spongistatins, discodermolide, and the tedanolides.1 Despite the structural complexity, the scarcity of these molecules in conjunction with their medicinal importance continues to prompt intense synthetic campaigns. During a recent search for novel marine antifungals, Searle and Molinski2 identified a methanolic extract from the sponge Phorbas sp. which displayed significant activity against Candida albicans. Bioassay-guided extraction, flash chromatography, and subsequent reverse-phase HPLC afforded two isomeric macrolides termed (+)-phorboxazoles A (1) and B (2). The structures of the phorboxazoles, including relative and absolute stereochemistry, were determined via a combination of NMR analyses, degradation studies, and synthetic correlations.3 The bioactivity profile of the phorboxazoles proved exceptional. In addition to the antifungal activity, the phorboxazoles displayed antibiotic activity against saccharomyces carlsberensis. However, it was the antiproliferative activity that elevated the phorboxazoles to the level of premier medicinal targets. Bioassays against the National Cancer Institute panel of 60 human solid tumor cell lines revealed extraordinary activity against the entire panel;2 the mean GI50 value was 1.58 × 10-9 M for both 1 and 2.3a Some cell lines were completely inhibited at the lowest level tested.2 Particularly noteworthy, phorboxazole A (1) inhibited the human colon tumor cell line HCT-116 and the breast cancer cell line MCF7 with GI50 values of 4.36 × 10-10 M and 5.62 × 10-10 M, respectively. These data place the phorboxazoles in the company of the spongistatins,1a collectively the most potent cytostatic agents discovered to date. Although the precise biochemical mode of action remains undefined, (+)-phorboxazole A (1) is known to arrest the cell cycle in S phase but does not inhibit tubulin polymerization or interfere with the integrity of microtubules. Unfortunately, further biological analysis is not possible, because access to the producing sponge is currently restricted.4 Thus, the phorboxazoles will be only available via total synthesis. Not surprisingly, the novel architecture combined with the impressive bioactivity has attracted wide attention in the synthetic community,5 including our own interest.6 In 1998, Forsyth and co-workers4 published the first total synthesis of (+)-phorboxazole A (1); shortly thereafter, Evans and Fitch reported completion of (+)(1) (a) Spongistatin: Pettit, G. R.; Cichacz, Z. A.; Gao, F.; Herald, C. L.; Boyd, M. R.; Schmidt, J. M.; Hooper, J. N. A. J. Org. Chem. 1993, 58, 1302. (b) Discodermolide: Gunasekera, S. P.; Gunasekera, M.; Longley, R. E.; Schulte, G. K. J. Org. Chem. 1990, 55, 4912. Correction: Gunasekera, S. P.; Gunasekera, M.; Longley, R. E.; Schulte, G. K. J. Org. Chem. 1991, 56, 1346. (c) Tedanolide: Schmitz, F. J.; Gunasekera, S. P.; Yalamanchili, G.; Hossain, M. B.; van der Helm, D. J. Am. Chem. Soc. 1984, 106, 7251. (2) Searle, P. A.; Molinski, T. F. J. Am. Chem. Soc. 1995, 117, 8126. (3) (a) Searle, P. A.; Molinski, T. F.; Brzezinski, L. J.; Leahy, J. W. J. Am. Chem. Soc. 1996, 118, 9422. (b) Molinski, T. F. Tetrahedron Lett. 1996, 37, 7879. (4) Forsyth, C. J.; Ahmed, F.; Cink, R. D.; Lee, C. S. J. Am. Chem. Soc. 1998, 120, 5597. (5) (a) Lee, C. S.; Forsyth, C. J. Tetrahedron Lett. 1996, 37, 6449. (b) Cink, R. D.; Forsyth, C. J. J. Org. Chem. 1997, 62, 5672. (c) Ahmed, F.; Forsyth, C. J. Tetrahedron Lett. 1998, 39, 183. (d) Ye, T.; Pattenden, G. Tetrahedron Lett. 1998, 39, 319. (e) Pattenden, G.; Plowright, A. T.; Tornos, J. A.; Ye, T. Tetrahedron Lett. 1998, 39, 6099. (f) Paterson, I.; Arnott, E. A. Tetrahedron Lett. 1998, 39, 7185. (g) Wolbers, P.; Hoffmann, H. M. R. Tetrahedron 1999, 55, 1905. (h) Misske, A. M.; Hoffmann, H. M. R. Tetrahedron 1999, 55, 4315. (i) Williams, D. R.; Clark, M. P.; Berliner, M. A. Tetrahedron Lett. 1999, 40, 2287. (j) Williams, D. R.; Clark, M. P. Tetrahedron Lett. 1999, 40, 2291. (k) Wolbers, P.; Hoffmann, H. M. R. Synthesis 1999, 797. (l) Evans, D. A.; Cee, V. J.; Smith, T. E.; Santiago, K. J. Org. Lett. 1999, 1, 87. (m) Wolbers, P.; Misske, A. M.; Hoffmann, H. M. R. Tetrahedron Lett. 1999, 40, 4527. (n) Wolbers, P.; Hoffmann, H. M. R.; Sasse, F. Synlett 1999, 11, 1808. (o) Pattenden, G.; Plowright, A. T. Tetrahedron Lett. 2000, 41, 983. (p) Schaus, J. V.; Panek, J. S. Org. Lett. 2000, 2, 469. (q) Rychnovsky, S. D.; Thomas, C. R. Org. Lett. 2000, 2, 1217. (r) Williams, D. R.; Clark, M. P.; Emde, U.; Berliner, M. A. Org. Lett. 2000, 2, 3023. (s) Greer, P. B.; Donaldson, W. A. Tetrahedron Lett. 2000, 41, 3801. (t) Evans, D. A.; Cee, V. J.; Smith, T. E.; Fitch, D. M.; Cho, P. S. Angew. Chem., Int. Ed. 2000, 39, 2533. (u) Huang, H.; Panek, J. S. Org. Lett. 2001, 3, 1693. 10942 J. Am. Chem. Soc. 2001, 123, 10942-10953 10.1021/ja011604l CCC: $20.00 © 2001 American Chemical Society Published on Web 10/13/2001 phorboxazole B (2).7 Herein, we disclose a full account of the total synthesis of (+)-phorboxazole A (1) recently completed in our laboratory.6c A central feature of this synthetic venture was the exploitation of the Petasis-Ferrier rearrangement for the construction of the two 2,6-cis-tetrahydropyran rings resident in the phorboxazole macrolide ring. Petasis-Ferrier Rearrangement. In 1996, Petasis reported that the acid-promoted rearrangement of enol acetals to tetrahydropyranones (e.g., 3f4, Scheme 1) proceeds via fragmentation, followed by endo cyclization onto an oxocarbenium (ii),8 a reaction closely related to the earlier Ferrier Type-II9 enol ether rearrangement (e.g., 5f6) induced by mercuric ion. Inspection of the Petasis-Ferrier rearrangement in the context of complex molecule synthesis revealed two important attributes. First, construction of the enol acetal rearrangement substrates comprises an ideal linchpin tactic for complex fragment assembly; second, the latent element of symmetry inherent in the target cis-tetrahydropyranones permits rearrangement of either enol acetal 8 or 9 (Scheme 2). Both attributes provide considerable latitude for fragment union and thereby cistetrahydropyranone construction. Synthetic Analysis. In addition to the two 2,6-cis-fused tetrahydropyrans (vide supra), the phorboxazoles present a wide array of architectural features, including a 21-membered macrolactone, a trans-fused tetrahydropyran, two oxazoles, and six olefinic units: one Z and two E alkenes, an exomethylene, a diene, and an E-vinyl bromide. From the retrosynthetic perspective (Scheme 3), we envisioned disconnection of the phorboxazoles at C(2-3), C(19-20), and C(28-29) to reveal three subtargets (10, 11, and 12) of comparable structural complexity. In the synthetic sense, fragments 11 and 12 would be united via a Wittig reaction. Continuing with this analysis, disconnection of side chain 10 at C(40-41) and C(32-33) would furnish subtargets 13, 14, and 15. In the forward sense, vinyl stannane 14 and vinyl iodide 13 could be coupled via a Stille reaction. For union of the side chain to the macrocycle, we planned to exploit oxazole triflate 15a,b as a novel bidirectional linchpin (vide infra). Finally, the cornerstone for construction of the central C(20-28) tetrahydropyran, 11, and bistetrahydropyran 12 would be the Petasis-Ferrier rearrangements, respectively, of vinyl acetals 16 and 17. Importantly, the overall synthetic strategy held the promise of considerable flexibility for fragment assembly, their union, endgame operations (vide infra), and the construction of analogues. Bistetrahydropyran 12: The C(3-19) Subtarget. To implement the first Petasis-Ferrier rearrangement, we sought enol acetal 17. Our point of departure entailed preparation of trans-tetrahydropyran 24 from known aldehyde 18 (Scheme 4).10 (6) (a) Smith, A. B., III; Verhoest, P. R.; Minbiole, K. P.; Lim, J. J. Org. Lett. 1999, 1, 909. (b) Smith, A. B., III; Minbiole, K. P.; Verhoest, P. R.; Beauchamp, T. J. Org. Lett. 1999, 1, 913. (c) Smith, A. B., III; Verhoest, P. R.; Minbiole, K. P.; Schelhaas, M. J. Am. Chem. Soc. 2001, 123, 4834. (7) (a) Evans, D. A.; Fitch, D. M. Angew. Chem., Int. Ed. 2000, 39, 2536. (b) Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. J. Am. Chem. Soc. 2000, 122, 10033. (8) Petasis, N. A.; Lu, S.-P. Tetrahedron Lett. 1996, 37, 141. (9) Ferrier, R. J.; Middleton, S. Chem. ReV. 1993, 93, 2779. (10) Evans, D. A.; Kaldor, S. W.; Jones, T. K.; Clardy, J.; Stout, T. J. J. Am. Chem. Soc. 1990, 112, 7001. Scheme 1 Scheme 2 Scheme 3 Total Synthesis of (+)-Phorboxazole A J. Am. Chem. Soc., Vol. 123, No. 44, 2001 10943