Total synthesis of portimine A and its analogues as small molecule modulators of NMD3

Abstract

Disclosed herein are enantioselective synthetic methods for making cyclic imine (CI) toxins and their analogs, known as small molecule inhibitors of NMD3 with selective and potent anti-cancer activity.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 480,259, filed January 17, 2023, which is incorporated herein by reference in its entirety.

[0002] government support This invention was made with government support under GM118176 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Disclosed herein are enantioselective synthetic methods for making cyclic imine (CI) toxins and their analogs, known as small molecule inhibitors of NMD3 with selective and potent anti-cancer activity. [Background technology]

[0004] For decades, cyclic imine (CI) toxins have stimulated widespread interest from a broad scientific community based on their unique and potent biological activity combined with a trapping chemical structure. 1、2 pinnatoxin, 3 spirolides, 4 and gymnozymine 5 The therapeutic potential of larger members of this family, such as IL-1, is limited by their high neurotoxicity in vivo (LD 50 <100 μg / kg). More compact members of this family were isolated from the benthic dinoflagellate Vulcanodinium rugosum in 2013 and 2018, portimines A (1) and B (2), respectively (Figure 1; absolute composition confirmed in 2019). 6~8 In contrast to classical CI toxins, 1 exhibits lower toxicity (LD) coupled with potent biological activity. 50 = 1570 μg / kg), showing a promising therapeutic index. 6、9~12 In fact, 1 is a powerful anticancer agent, 6 Antifouling, 10 and anti-HIV-1 activity, 11This demonstrates the potential of these compounds as promising lead compounds. The Achilles' heel of such compounds is, of course, the means to scale up and obtain their complex structures. Since they are derived from dinoflagellates in low yield, 6 Chemical synthesis appears to be the only practical means of procuring such molecules.

[0005] Even if this could be achieved, semisynthetic analogs with deep structural modifications would be inaccessible. 1 and 2, featuring a five-membered cyclic imine spiro-fused within a highly oxidized, all-carbon tricyclic macrocyclic core, are extremely challenging synthetic targets. Oxidation adjacent to the imine carbon (C-5) and unusual peripheral oxidations, such as the adjacent unstable medium-sized cyclic ketal, add to this challenge.

[0006] Historically, CI toxins were synthesized from putative biogenes in which the acyclic structure with the greatest functionality was subjected to macrocyclization. 13~15 It has been constructed by patterning retrosynthetic analysis of 16~20 This pioneering approach was first achieved by Kishi et al. in the total synthesis of pinnatoxin A in 1998. 16 In the case of portimines, previous approaches have followed this dogma. 21~24 Thus, Brimble et al., 21、22 and Harran et al. 23 are fully functionalized polyketides, respectively. 3 and 4 We aimed to develop a biomimetic synthesis of 3 by mimicking polyketide synthases (PKSs), featuring a bold intramolecular cyclization of 3. The former approach demonstrated that ketalization of linear 3 was not facile, even with a well-functionalized scaffold. The latter Diels-Alder-based approach resulted in undesirable regioselectivity in the central cyclization of 4 to 6.

[0007] The difficulties encountered in these routes illustrate the challenges of forming the key linkages in such densely functionalized polycyclic alkaloids from acyclic precursors. From a broader perspective, this scenario is similar to that encountered in the synthesis of densely functionalized, highly oxidized terpene natural products. In these cases, it has been shown that a two-phase approach to synthesis can be beneficial by building a minimally oxidized carbon skeleton followed by strategic late-stage oxidation. 25~30 Therefore, there is a need in the art for improved methods for synthesizing cyclic imine toxins and their analogs as small molecule inhibitors of NMD3 with selective and potent anti-cancer activity. [Brief explanation of the drawings]

[0008] [Figure 1] Portimine A is non-toxic to primary human PBMCs. [Figure 2] Washout experiments reveal that PA has acute cytotoxicity. [Figure 3] Initial SAR studies: Evaluation of the activity of PAL-functionalized PTMA analogues. [Figure 4] Validation of NMD3 as a target of portimine A by chemical precipitation (ChP). [Figure 5] PA-related compounds induce cell apoptosis in Jurkat. [Figure 6] Portimine A inhibits S-phase entry in Jurkat cells. [Figure 7] NMD3 is downregulated by PA in Jurkat cells in a time- and dose-dependent manner. [Figure 8] Downregulation of NMD3 does not occur via proteosome-mediated degradation. [Figure 9] Portimine A exhibits potent antitumor activity in human and syngeneic mouse tumor models. Summary of the Invention

[0009] Disclosed herein is an enantioselective synthetic method for generating portimine A and its analogs as small molecule inhibitors of NMD3 with selective and potent anticancer activity. Remarkably, portimine A was found to be nontoxic to primary human PBMCs despite its high potency in multiple cancer cell lines (Figure 1).

[0010] The present application provides a process for preparing portimine A (1), [ka] The process is i) reduction of compound (2) to form portimine A (1): [ka] and, ii) Dess-Martin oxidation of compound (22) followed by hydrolysis to form compound (2): [ka] and, iii) Pd-catalyzed vinylation of compound (21) to form compound (22): [ka] and, iv) converting compound (20) to compound (21): [ka] and, v) forming compound (20) by silylation and oxidation of compound (19): [ka] and, vi) forming compound (19) by ring-chain tautomerization of compound (32): [ka] and, vii) reduction of compound (16) to compound (32): [ka] and, viii) ring formation and oxidation of compound (15) to form compound (16): [ka] and, ix) Mo-catalyzed ring-closing alkyne metathesis to form compound (15): [ka] and, x) Conversion of compound (7) into N-protected imine compound (31): [ka] and, xi) transmetallation of compound (13) and conjugate addition to compound (12) to form compound (7): [ka] and, xii) cyclization of compound (11) to form compound (12): [ka] and, xiii) Alcohol oxidation of compound (10) followed by Grignard addition and Dess-Martin oxidation to form compound (11): [ka] and, xiv) Diels-Alder cycloaddition of compound (8) to form compound (10): [ka] and, xv) conversion of compound (23) to compound (8): [ka] and,

[0011] The present application relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: [ka] (In the formula, R is (C6~C 10 ) aryl, (C2-C6) alkenyl or (C5-C 12 ) heteroaryl, each aryl optionally substituted with —NH(CH)C(—N═N—)(CH)(C≡CH); R' is H or Ac The present invention provides a process for preparing various analogs of Portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, including:

[0012] The present application provides a compound comprising: [ka] The present invention provides a compound prepared by the above process, wherein the compound is selected from the group consisting of:

[0013] The present application relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: [ka] (In the formula, R 1 is H, Me, Boc, or TES, R 2 H, halo, (C6~C 10 ) aryl, (C2-C6) alkenyl or (C5-C 12 ) heteroaryl, each aryl optionally substituted with —NH(CH)C(—N═N—)(CH)(C≡CH); R' is H or Ac The present invention provides a process for preparing various analogs of Portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, including:

[0014] The present application provides a compound comprising: [ka] The present invention provides a compound prepared by the above process, wherein the compound is selected from the group consisting of:

[0015] The present application relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: [ka] (In the formula, R 1 is H, Me, Boc, or TES, R 2 H, halo, (C6~C 10 ) aryl, (C2-C6) alkenyl or (C5-C 12 ) heteroaryl, each aryl optionally substituted with —NH(CH)C(—N═N—)(CH)(C≡CH); R' is H or Ac The present invention provides a process for preparing various analogs of Portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, including:

[0016] The present application provides a compound comprising: [ka] The present invention provides a compound prepared by the above process, wherein the compound is selected from the group consisting of:

[0017] The present application provides the process according to any one of the above embodiments, wherein the portimine A analog product is useful for forming an antibody-drug conjugate (ADC) for treating cancer.

[0018] The present application provides a process for forming an antibody-drug conjugate (ADC) for treating cancer, comprising the use of a compound according to any one of the above embodiments.

[0019] The present application provides a method of treating cancer comprising administering to a patient in need thereof a pharmaceutically effective amount of any one of the above embodiments capable of forming an antibody-drug conjugate (ADC). DETAILED DESCRIPTION OF THE INVENTION

[0020] Consistent with the logic of biphasic synthesis, we targeted a minimally oxidized macrocyclic intermediate to the portimine, under the assumption that the proper sequence of oxidation events would resolve both connectivity and stereochemistry issues. The only C-O bonds initially installed were those at C-4 (imine carbon) and C-10 (secondary alcohol). The triple bond at C-7 / 8 served as a surrogate for the final C-7 oxidation and, importantly, provided strategic cleavage to the macrocycle using ring-closing alkyne metathesis (RCAM). 31 Because four key oxidations (C-3, C-13, C-14, and C-15) occur after macrocyclization, such a strategy minimizes labile functional groups and redundant redox manipulations. To minimize protecting groups (PGs), inherent reactivity and conformational preferences are exploited through ring-chain reorganization / rearrangement steps. Unwinding the macrocycle returns the dialkyned precursor (7) to accessible building blocks (Figure 1B).

[0021] Finally, to maximize access to useful analogs, the unusually stable vinyl triflate was selected as the key functional group carried throughout the synthesis. The simplicity and scalability of this route will ultimately establish a method for elucidating the mechanism of action of 1 and 2 and fully evaluating their therapeutic potential.

[0022] Scheme 1: Total synthesis of portimines A and B [ka] Reagents and conditions: (1) Rawal's diene (1.2 equiv.), [Co] (1.0 mol%), MS4Å (50 wt%), DCM, 0°C, 2 h. (2) NaBH4 (1.0 equiv.), MeOH, 0°C, 1 h, then TBAF (1.03 equiv.), THF, RT, 30 min. (3) NaOCl (2.0 equiv.), KBr (0.1 equiv.), NaHCO3 buffer, TEMPO (1 mol%), DCM, 0°C to RT, 1 h, then 9, THF, -78°C, 2 h, then DMP (1.03 equiv.), DCM, RT, 1 h. (4) TFA / DCM (2:1 v / v), RT, then Na2CO3, NaOH overnight. (5) t-BuLi (2.08 equiv.) in EtO at −78°C for 2.5 hours, followed by [Cu] (1.17 equiv.), n-BuP (2.38 equiv.), THF at −78°C for 1 hour, followed by 12 (0.80 equiv.) at −78°C for 40 minutes, followed by Comins' reagent (1.20 equiv.) at −78°C to 0°C for 1 hour. (6) TrocCl (1.01 equiv.), pyridine (8.1 equiv.), toluene at 100°C for 15 minutes. (7) [Mo] (2 mol%), MS5Å (300 wt.%), toluene at 80°C for 2 hours, followed by PTSA (1.81 equiv.), DCM / MeOH / HO (20:2:1 v / v / v), overnight at 50°C. (8). (9) L-selectride (1.9 eq.), THF, -78°C, 30 min, then NaBH4 (5.5 eq.), MeOH, 0°C, 1 h. (10) NaOCl (1.8 eq.), KBr (3.8 eq.), NaHCO3 buffer, TEMPO (2.0 mol%), DCM, 0°C, then Zn (9.15 eq.), HOAc, 70-45°C, 5 hours. (11) TBSOTf (2.2 equiv.), TEA (5.0 equiv.), DCM, 40 °C, 1 h, then DMDO (excess), acetone, −78 to 0 °C, then AcO (6.2 equiv.), TEA (4.2 equiv.), DCM, reflux, 3 h.(12) LiOH·HO (0.1 M), THF, 0 °C, 45 min, then Pd(dppf)Cl (10 mol%), vinylBFK (2.0 equiv), TEA (3.5 equiv), n-PrOH, 100 °C, 1 h. (13) DMP (2.5 equiv), DCM, RT, 1 h, then methanolic NH, RT, 30 min (for the synthesis of 2); 2, NaBHCN (2.5 equiv), HOAc, RT, 3 h (for the synthesis of 1).

[0023] The synthesis is outlined in Scheme 1 and begins with a scalable asymmetric Diels-Alder cycloaddition developed by Rawal et al., which established C-3 chirality. Reduction of C-4 followed by removal of the carbamate auxiliary with TBAF afforded 10 in 88% yield and 94% ee on a 50-gram scale. Non-strategic reduction of the C-4 aldehyde was necessary due to its instability during the elimination step (see Supplementary Information; buffered TBAF, HF, HCl, CsF, etc. all resulted in decomposition). The required methyl-blocked alkyne side chain could be introduced via a sequence involving a Grignard addition and two oxidations, delivering 11 as the final product in 60% yield after a single purification. Treatment of 11 with TFA in DCM afforded the spirocyclic imine 12 in 72% yield on a gram scale.

[0024] The synthesis of fragment 13 was initiated from inexpensive (S)-solketal (approximately $1.1 / g, see Scheme 2 for details). To anchor this subunit to the established chiral spirocyclic core 12, a stereoselective copper-mediated conjugate addition was applied. The choice of copper(I) reagent was important, as switching to other common copper(I) salts, such as CuI, CuCN, and CuBr, showed little or no observable conversion. n-Bu3P was used as a ligand to promote nucleophilicity and increase the solubility of [Cu]. The stereochemical outcome in this step was controlled by the unique configuration of the spirocycle, with a side chain blocking the top surface. Direct treatment of the in situ generated enolate with Comins' reagent ensured the correct regiochemical olefinic outcome, smoothly delivering vinyl triflate 7 as a single product (6.5 g scale) bearing all the necessary backbone carbon atoms for 1 and 2.

[0025] Scheme 2: Synthesis of compound 13 [ka] Reagents and conditions: (1) TCCA (0.4 equiv.), 4-NHAc-TEMPO (1.0 mol%), NaOAc (1.2 equiv.), EtOAc, 0 °C for 1.5 h, followed by EtOCCHP(O)OEt (1.09 equiv.), KCO (3.0 equiv.), room temperature overnight. (2) LiMe·LiBr (1.5 equiv.), EtO, -78 °C for 2.5 h. (3) LAH (1.0 equiv.), THF, 0 °C to room temperature for 2 h, followed by PPh (1.2 equiv.), imidazole (1.3 equiv.), I (1.15 equiv.), DCM, room temperature, followed by TIPS acetylene (1.1 equiv.), n-BuLi (1.0 equiv.), DMPU (1.95 equiv.), THF, room temperature overnight. (4) HCl / THF (3 M, v / v = 2:1) at 75 °C for 12 hours, then NaH (2.5 equiv.) for 1 hour, then Ts-imidazole (1.2 equiv.) in THF for 45 minutes. (5) n-BuLi (3.0 equiv.) in propyne at -78 to 0 °C in THF for 30 minutes, then 29 in BF3·OEt2 (2.93 equiv.) in THF at -78 °C, then TBAF (1.65 equiv.) in THF at 45 °C overnight. Then TBSOTf (2.0 equiv.), TEA (1.8 equiv.), DCM at room temperature to reflux for 1.5 hours. (6) CpZrCl (1.51 equiv.), DIBAL-H (1.25 equiv.), THF, 0 °C, 1 h, then 30, overnight, then I (1.53 equiv.), 1 h.

[0026] We then focused on constructing a 14-membered macrocycle within the portimine framework via RCAM, a procedure that can be abruptly extended by imines, olefins, or vinyl triflates. Furstner's highly efficient canopy-shaped catalyst [Mo] was chosen at this point for its excellent functional compatibility and demonstrated robustness. 35 The rotary RCAM process could indeed be achieved by heating 7 with [Mo] in toluene in 53–65% yields. This is likely due to the aforementioned functional groups, especially the imine nitrogen. 36Relatively high catalyst loadings (12.5 mol%–15 mol%) and long reaction times were required. To solve this problem, the imine was masked with a Troc group, and the enamide was subsequently exposed to 2.0 mol% [Mo]. In this case, the formation of macrocycle 14 was complete in 1 h. Treatment of crude 14 with acidic wet methanol liberated the C-4 ketone and deprotected the TBS ether at C-10, 37, to afford 15 as a white powder (overall isolated yield of 68% over two steps, gram scale).

[0027] Reaching macrocycle 15 (the end of the "cyclase phase") was groundbreaking because all the core C-C bonds necessary to reach 1 and 2 were in place. All that remained was the installation of five oxygen atoms at C-5, C-7, C-13, C-14, and C-15. Because olefin oxidation is known to be compatible with alkynes, the "oxidase phase" began with the oxidation of C-14 / 15. Initial attempts involved the use of oxidizing agents (i.e., OsO, m-CPBA, etc.) that yielded a net two-electron result (epoxide or diol). Unfortunately, the stereochemical outcome at C-15 in all cases was undesirable. Therefore, a net six-electron oxidation catalyzed by ruthenium afforded the diketone, with a final strategic reduction to set the desired stereochemistry. 38 However, more powerful oxidants of this type do not tolerate the presence of alkynes. To this end, an internal protection strategy was designed. This goal was achieved upon backbone rearrangement by refluxing 15 and XPhosAuNTf2 (0.8 mol%) in DCM. The newly formed tricyclic ring system organized all potentially sensitive sites (C-1, C-4 ketone, C-7-C-8 alkyne, and nitrogen on the C-10 alcohol) into their inactive state with the newly rigidified backbone to control downstream stereochemical issues. Subsequent Ru-catalyzed six-electron oxidation afforded diketone 16 in 53% isolated yield (gram scale), thereby minimizing reliance on protecting groups.

[0028] With this newly constructed rigid polycyclic ring system in place, the correct oxidation state and stereochemistry at C-14 and C-15 were established. Site-specific reduction of C-14 was achieved using L-selectride, followed by treatment of the crude material with sodium borohydride (NaBH4) to afford the diol with the desired stereochemistry at C-15. C-14 was then selectively converted back to the ketone oxidation state with TEMPO / NaOCl. Other oxidizing agents tested for this step (e.g., DMP, IBX, activated DMSO, TPAP / NMO) showed poor selectivity. Remarkably, heating the crude product 17 with zinc powder in acetic acid decomposed the Troc group and spontaneously opened the polycyclic ring system (via 18) by ring-chain tautomerization, liberating the dideoxyporthimine triflate 19. The yield of this two-step sequence was 48%.

[0029] Only the oxygen atoms at C-13 and C-15 remained to be installed to complete the synthesis, and both oxidations could be achieved in a single step by treating the silyl enol ether of 19 with DMDO to give the nitrone intermediate. 39 Subsequent heating of the crude nitrone in the presence of Ac2O and TEA presumably forms N-oxyenamine 20, which spontaneously undergoes a Boekelheide-type rearrangement to deliver diacetate 21 as a single diastereomer in 76% yield on a 90 mg scale. 40~42 The preferential cis configuration of the C-4-C-5 double bond in 20 may explain the high stereochemical control. At this point, the vinyl triflate, which remained a silent observer throughout the synthesis, was now sought to append the final two carbon atoms of 1 and 2. Therefore, selective hydrolysis of the C-5 acetate using LiOH, followed by Suzuki coupling to introduce the exocyclic vinyl group, delivered diene 22 in 51% yield. 43To complete the synthesis, 22 could be oxidized with DMP followed by ammonia to give portimine B (2) in good yield (88%). During these studies, the originally assigned structure of 2 as the ring-open tautomer was suspected to be incorrect, and this was confirmed here to be the same ring-closed tautomer expressed in 1. To complete the synthesis of 1, crude 2 in solution could be chemoselectively reduced (NaBHCN) to the highly oxygenated natural product itself in high isolated yield (80%).

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Part 24. Nitrone Synthesis by Imine Oxidation Using Either a Peroxyacid or Dimethyldioxirane. J. Chem. Soc., Perkin Trans. 1 1990, 301. (40) Cummins, C.H.; Coates, R.M. α-Oxygenation of Aldehydes and Cyclic Ketones by Acylation-Rearrangement of Nitrones. J. Org. Chem. 1983, 48, 2070 - 2076. (41) Beshara, C.S.; Hall, A.; Jenkins, R.L.; Jones, K.L.; Jones, T.C.; Killeen, N.M.; Taylor, P.H.; Thomas, S.P.; Tomkinson, N.C. A General Method for the α-Acyloxylation of Carbonyl Compounds. Org. Lett. 2005, 7, 5729 - 5732. (42) Bartlett, S.; Keiter, K.; Zavesky, B.; Johnson, J. Formation of Complex α-Imino Esters via Multihetero-Cope Rearrangement of α-Keto Ester Derived Nitrones. Synthesis 2018, 51, 203 - 212. (43) Molander, G.A.; Rivero, M.R. Suzuki Cross-Coupling Reactions of Potassium Alkenyltrifluoroborates. Org. Lett. 2001, 4, 107 - 109. Detailed Description of the Drawings Figure 1. Portimine A is nontoxic to primary human PBMCs. Portimine A and its functional analogs exhibit stereoselective acute toxicity in cancer cells. (A) PA and Ph-PA have similar cytotoxicity in breast cancer cell lines (HCC1806) and leukemia cancer cell lines (Jurkat) after 36 hours of treatment, whereas PB and ePA show minimal effects. (B) Structures of portimine-related analogs: phenyl-portimine A (Ph-PA, 36) and epiportimine A (ePA, 38). Structures of fully functionalized portimine A-based epimeric probe pairs: portimine A-diazirine-alkyne (PA-DA, 35-2) and epiportimine A-diazirine-alkyne (ePA-DA, 35-1). (C) Cell viability analysis of portimine A and analogs. Jurkat cells were treated with increasing concentrations of portimine A and analogs for 24 hours. All data shown are the average of biologically replicated experiments (n = 3). (D) Immunoblot analysis of caspase 3 shows that PA, PA-DA, and Ph-PA, but not ePA, ePA-DA, or PB, induce cell apoptosis. Jurkat cells were treated with portimine A or analogs at 10 nM for 12 hours.

[0031] PA and Ph-PA have an IC50 of approximately 2 nM against Jurkat cells after 12 hours. PA and Ph-PA are substantially less toxic to healthy PBMCs than the cancer T cell line Jurkat. The structurally related negative controls ePA and PB are non-toxic to all cell types at the current concentration range. PBMCs (25 kJ / well), Jurkat cells (10 kJ / well). Compound concentrations were increased from 0.23 pM to 1 uM, 4-fold per gradient, using Wesley-derived primary B cell medium (heat-inactivated) for PBMCs and regular RPMI medium for Jurkat cells, and incubated for 12 hours.

[0032] Figure 2. Washout experiments reveal that PA has acute cytotoxicity. Portimine A induces cell cycle arrest in Jurkat cells. Cell cycle analysis in Jurkat cells. Jurkat cells were treated with 10 nM of the indicated compounds for 24 h, and propidium iodide was used to identify the different cell phases by flow cytometry. Treatment conditions: Jurkat (50k / well); Portimine A concentration: 0.007nM to 480nM; washout time points: 0.5, 2, 4, 6, 12, 24 hours. Cells were washed once and suspended in PA-free RPMI medium. Cells were treated for a total of 36 hours. Cell viability was measured using the Cell Titerglo assay. An exposure-dependent decrease in IC50 was observed; compound removal after 30 minutes resulted in an approximately 5-fold decrease in IC50; IC50 after 30 minutes of exposure was approximately 5nM; at 4 hours it was approximately 2nM. Overall, this suggests a fast-acting cytotoxic mechanism.

[0033] Figure 3. Initial SAR studies: Evaluation of the activity of PAL-functionalized PTMA analogs. Portimine A and analogs do not induce apoptosis or affect cell viability in freshly isolated human PBMCs. (A) PA and Ph-PA demonstrate low toxicity to healthy PBMCs compared to Jurkat cells. Both ePA and PB were non-toxic to both cells in the pM-nM concentration range. (B) Immunoblot of caspase-3 in Jurkat and PBMCs with the indicated conditions. Both PA and PA-DA induced caspase-3 cleavage in a dose-dependent manner in Jurkat cells, but not in human PBMCs. PTMA-DA was found to have similar activity to PTMA → appropriate target ID probes; epi-PTMA-DA and epi-PTMA-Ac-DA had no activity in the concentration range, similar to epi-PTMA → useful inactive probes.

[0034] Figure 4. Validation of NMD3 as a target of portimine A by chemical precipitation (ChP). Portimine A mouse pharmacokinetic profile and fast-acting in vitro cell-based target engagement characteristics based on compound washout. (A) Mice intraperitoneally (ip) and orally (po) administered portimine (n=3). (B) Washout experiments performed in Jurkat and MC38 cells demonstrated that NMD3 was significantly increased in the IC 50 (C) Kaplan-Myer survival curves of WT MC38 tumor-bearing mice (n=6) after treatment with PA, as shown in (D). 3 Mice were euthanized when the concentration exceeded 0.05. Statistical analysis was performed using ANOVA followed by multiple comparisons. *p≤0.05. Treatment conditions: Jurkat (60 μM per condition); PA-DA / ePA-DA concentration: 0.5 μM, PA or ePA: 5 μM; 30-minute incubation with probe with or without competitor (PA, ePA); 20-minute UV irradiation (0.5 μM PA-DA was added to the no-UV condition); standard pull-down and biotin enrichment followed by Western blot analysis (elution conditions: 2x sample buffer + 500 μM biotin, boiling for 20 minutes); primary antibodies: NMD3 (ProteinTech, 1:150), actin (BioRad, 1:10,000). Conclusion: NMD3 can be selectively bound by portimine A; the interaction between NMD3 and portimine A is noncovalent; note: PA-DA binding to recombinantly overexpressed NMD3 in cells was not observed, but binding was observed only to endogenous NMD3. This may suggest that PA binds to NMD3 only when it is in the endogenous complex.

[0035] Figure 5. PA-related compounds induce cell apoptosis in Jurkat cells. Chemical proteomic analysis reveals that NMD3 is a target of portimine A. Chemical proteomic profiling using PA-DA in Jurkat cells (A, B, C, G) and HCC1806 cells (D, E, F, H) indicates that NMD3 is a major candidate target of portimine A. Volcano plots in panels A and D show protein competition in cells treated with the active photoaffinity probe PA-DA (500 nM) and DMSO or the active competitor portimine A (PA, 4 μM). The dotted line indicates a fold-change threshold of <4, p-value >0.05. Volcano plots in panels B and E show protein enrichment by the active probe PA-DA relative to the inactive probe ePA-DA (500 nM). The dotted line indicates a fold-change threshold of <2, p-value >0.05. Volcano plots in panels C and F show a comparison between the active competitor compound PA and the inactive control competitor ePA (4 μM, PA-DA + PA / PA-DA + ePA). The dotted line indicates a threshold for fold change of >4, p-value >0.05. (I, J) Confirmation of PA-DA engagement of NMD3 in Jurkat and HCC1806 cells. UV-dependent labeling of endogenous NMD3 by PA-DA was blocked by PA but not by ePA, and was virtually unlabeled by the inactive probe ePA-DA or PA-DA without UV irradiation. Treatment conditions: Jurkat (5 μM / well). Portimine A concentration: 2 nM–1000 nM; cells were treated for a total of 12 h. Conclusion: PA induces cell apoptosis in a dose-dependent manner; ePA does not induce significant caspase-3 activation within this concentration range.

[0036] Figure 6. Portimine A inhibits S-phase entry in Jurkat cells. Portimine A down-regulated NMD3 at the protein level but up-regulated it at the mRNA level, as well as several essential apoptotic proteins. (A) NMD3 and Bcl-xl were down-regulated after PA treatment, but not by proteasome-mediated degradation. Proteolysis of p53, Mcl1, Bid, Bim, and Noxa could be blocked by MG132 or epoxomicin. (B) NMD3 mRNA increased after PA treatment but not with the ePA control compound. Jurkat cells were treated with the indicated compounds for 24 hours, then fixed and stained with propidium iodide. Conclusion: S-phase entry was inhibited in Jurkat cells treated with 10 nM PA / PADA; PA / PADA does not affect G2M / sub-G1 (mainly apoptotic cells).

[0037] Figure 7. Portimine A targets NMD3 and prevents polysome formation. NMD3 is downregulated by PA in Jurkat cells in a time- and dose-dependent manner. (A) Immunoblot showing reduced NMD3 levels in shNMD3 Jurkat and HeLa cells. (B) PA has less toxic effects in cells in which NMD3 is knocked down (shNMD3) compared to shCtrl cells. Jurkat cells were treated with 12.5 nM PA for 12 or 24 hours. Jurkat and HeLa cells were treated with 5 nM or 150 nM PA for 24 hours. Data represent the mean ± SD of biologically replicated experiments (n = 3). (C) Polysome profiling reveals protein translation inhibition by portimine A. Jurkat cells were treated with DMSO or 50 nM portimine A for 6 hours. Cell lysates were fractionated on a sucrose gradient (5-50%), and each fraction was analyzed by Western blot. eIF6 levels increase in the 60S subunit fraction and decrease in the ribosome-free fraction after PA treatment. RPS6, a 40S and polysome subunit marker, decreases in the polysome fraction after PA treatment. Results are representative of three independent experiments. (D) Quantification of the 60S:80S and 80S:polysome ratios from the polysome profiling assay shown in Figure 7C. (E) Quantification of the relative protein distribution in the fractions shown in Figure 7C. Relative protein levels in each fraction were normalized to the peak fraction of the indicated protein from DMSO cells and plotted. Data represent the mean ± SD of biologically replicated experiments (n = 3). Statistical analysis was performed using a multiple independent Student's t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005; ****p ≤ 0.0001. Treatment conditions: Jurkat (5 M per condition); cells (1 M / mL) are incubated with inhibitors for 6 hours, 12 hours and 24 hours, respectively.Conclusions: NMD3 is downregulated by PA treatment at 10 nM at 12 hours; KPT-330 downregulates NMD3 at higher concentrations (above 50 nM) at 24 hours; XPO1 levels do not appear to be affected by PA; p53 can be downregulated by porcine A treatment; similar to KPT-330, PA does not affect Bcl2 levels at 6 / 12 hours.

[0038] Figure 8. Downregulation of NMD3 does not occur via proteasome-mediated degradation. Treatment conditions: Jurkat (10 M per condition); PA concentration: 10 nM; cells (1 M / mL) are preincubated with MG132 or epoxomicin for 2 hours. Cells are incubated with PA with or without MG132 or epoxomicin for an additional 12 hours. Conclusion: Downregulation of NMD3 is not due to proteasome-mediated degradation; it may be the result of transcriptional inhibition, similar to XPO1 inhibitors.

[0039] Figure 9. Portimine A exhibits potent antitumor activity in human and syngeneic mouse tumor models. (A) Systemic portimine A in an MC38 syngeneic tumor xenograft model; (B) Systemic portimine A in an HT-1080 tumor xenograft model.

[0040] Preliminary cell-based target engagement studies based on compound washout indicate that PA is rapidly acting with less than 1 hour of exposure and minimal loss of potency. Cmax-driven pharmacology, along with ease of synthesis and material availability, enabled evaluation of PA in both syngeneic (MC38 colon carcinoma) and human metastatic mesenchymal (HT-1080 fibrosarcoma) mouse tumor xenograft models. Potent antitumor activity was observed for PA at 0.3 or 1.0 mg / kg doses following tolerated systemic administration (ip delivery). Metabolic stability presents an opportunity for additional intellectual property positions.

[0041] The results indicate that portimine A may share similar anticancer effects with the XPO1 inhibitor KPT-330. [ka] i) NMD3 is a known adaptor for XPO1 and appears to be required for XPO1 localization. No NMD3 inhibitors have yet been reported; ii) KPT-330 is an FDA-approved first-in-class oral selective nuclear export inhibitor (SINE) compound by covalently targeting XPO1 protein, with IC50: 34~203nM; iii) XPO1 is frequently overexpressed and / or mutated in human cancers, functions as an oncogenic driver, and XPO1-mediated transport is increased in a variety of cancers; iv) KPT-330 induced rapid apoptosis at low nanomolar concentrations in a panel of 14 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines and, similar to PA, was largely nontoxic to normal mouse hematopoietic cells; v) KPT-330, like PA, inhibits proliferation and induced cell cycle arrest in cancer cells; vi) KPT-330 downregulates XPO1 in cells by reducing mRNA expression and protein synthesis; and vii) KPT-330 interfered with nucleolar rRNA processing and reduced the total levels of multiple mature rRNAs.

[0042] General experimental conclusions: i) NMD3 is the only target that emerged from the chemoproteomic target ID study in two cancer cell models; ii) endogenous NMD3 was confirmed as a target for portimine A and PA probes, but not for the inactive control; iii) NMD3 is thought to be a scaffolding protein for XPO1 / CRM1, but little is known about its exact role; iv) Portimine induces apoptosis and cell cycle arrest similar to XPO1 inhibitors; and v) Portimine A downregulates NMD3 protein levels, possibly not through degradation Embodiment Embodiment 1. Portimine A (1), [ka] 1. A process for preparing compound (10), comprising Diels-Alder cycloaddition of compound (8): [ka] .

[0043] Embodiment 2. The process of embodiment 1, further comprising alcohol oxidation of compound (10), followed by Grignard addition and Dess-Martin oxidation to form compound (11): [ka] .

[0044] Embodiment 3. The process of embodiment 2, further comprising cyclizing compound (11) to form compound (12): [ka] .

[0045] Embodiment 4. A process for preparing compound (13) from (S)-solketal, comprising the steps of: [ka] .

[0046] Embodiment 5. The process of embodiment 4, further comprising the steps of: [ka] .

[0047] Embodiment 6. The process of embodiment 5, further comprising the steps of: [ka] .

[0048] Embodiment 7. The process of embodiment 3, further comprising transmetallation of compound 13 and conjugate addition to compound 12 to form compound 7: [ka] .

[0049] Embodiment 8. The process of embodiment 7, further comprising converting compound (7) to N-protected imine compound (31): [ka] .

[0050] Embodiment 9. The process of embodiment 8, further comprising forming compound (15) by Mo-catalyzed ring-closing alkyne metathesis: [ka] .

[0051] Embodiment 10. The process of embodiment 9, further comprising cyclization and oxidation of compound (15) to form compound (16): [ka] .

[0052] Embodiment 11. The process of embodiment 10, further comprising reducing compound (16) to compound (32): [ka] .

[0053] Embodiment 12. The process of embodiment 11, further comprising forming compound (19) by ring-chain tautomerization of compound (32): [ka] .

[0054] Embodiment 13. The process of embodiment 12, further comprising silylation and oxidation of compound (19) to form compound (20): [ka] .

[0055] Embodiment 14. The process of embodiment 13, further comprising converting compound (20) to compound (21): [ka] .

[0056] Embodiment 15. The process of embodiment 14, further comprising Pd-catalyzed vinylation of compound (21) to form compound (22): [ka] .

[0057] Embodiment 16. The process of embodiment 15, further comprising Dess-Martin oxidation of compound 22 followed by hydrolysis to form compound 2: [ka] .

[0058] Embodiment 17. The process of embodiment 16, further comprising reducing compound (2) to form portimine A (1): [ka] .

[0059] Embodiment 18. Portimine A (1): [ka] 1. A process for preparing a compound of formula (I) comprising the reduction of compound (2) to form portimine A (1): [ka] .

[0060] Embodiment 19. Formula (2): [ka] or a scalemic or racemic mixture thereof.

[0061] Embodiment 20. A process for preparing a compound of embodiment 19, comprising Dess-Martin oxidation of compound (22) followed by hydrolysis to form compound (2): [ka] .

[0062] Embodiment 21. Formula (22): [ka] or a scalemic or racemic mixture thereof.

[0063] Embodiment 22. A process for preparing a compound according to embodiment 21, comprising Pd-catalyzed vinylation of compound (21) to form compound (22): [ka] .

[0064] Embodiment 23. Formula (21): [ka] or a scalemic or racemic mixture thereof.

[0065] Embodiment 24. A process for preparing a compound according to embodiment 23, comprising converting compound (20) to compound (21): [ka] .

[0066] Embodiment 25. Formula (20): [ka] or a scalemic or racemic mixture thereof.

[0067] Embodiment 26. A process for preparing a compound according to embodiment 25, comprising silylation and oxidation of compound 19 to form compound 20: [ka] .

[0068] Embodiment 27. Formula (19): [ka] or a scalemic or racemic mixture thereof.

[0069] Embodiment 28. A process for preparing a compound according to embodiment 27, comprising forming compound (19) by ring-chain tautomerism of compound (32): [ka] .

[0070] Embodiment 29. Formula (32): [ka] or a scalemic or racemic mixture thereof.

[0071] Embodiment 30. A process for preparing a compound according to embodiment 29, comprising reducing compound (16) to compound (32): [ka] .

[0072] Embodiment 31. Formula (16): [ka] or a scalemic or racemic mixture thereof.

[0073] Embodiment 32. A process for preparing a compound according to embodiment 31, comprising ring formation and oxidation of compound (15) to form compound (16): [ka] .

[0074] Embodiment 33. Formula (15) [ka] a compound having the structure or a scalemic or racemic mixture thereof.

[0075] Embodiment 34. A process for preparing a compound according to embodiment 33, comprising forming compound (15) by Mo-catalyzed ring-closing alkyne metathesis: [ka] .

[0076] Embodiment 35. Formula (31): [ka] or a scalemic or racemic mixture thereof.

[0077] Embodiment 36. A process for preparing a compound according to embodiment 35, comprising converting compound (7) to N-protected imine compound (31): [ka] .

[0078] Embodiment 37. Formula (7): [ka] or a scalemic or racemic mixture thereof.

[0079] Embodiment 38. A process for preparing a compound according to embodiment 37, comprising transmetallation of compound (13) and conjugate addition with compound (12) to form compound (7): [ka] .

[0080] Embodiment 39. Formula (28): [ka] or a scalemic or racemic mixture thereof.

[0081] Embodiment 40. The following steps: [ka] 40. A process for preparing a compound of embodiment 39 from (S)-solketal, comprising:

[0082] Embodiment 41. Formula (29): [ka] or a scalemic or racemic mixture thereof.

[0083] Embodiment 42. The following steps: [ka] 42. A process for preparing a compound of embodiment 41 from compound (28), comprising:

[0084] Embodiment 43. Formula (30): [ka] or a scalemic or racemic mixture thereof.

[0085] Embodiment 44. The following steps: [ka] 44. A process for preparing a compound of embodiment 43 from compound (28), comprising:

[0086] Embodiment 45. Formula (13): [ka] or a scalemic or racemic mixture thereof.

[0087] Embodiment 46. The following steps: [ka] 46. ​​A process for preparing the compound of embodiment 45 from (S)-solketal, comprising TIFF2026505889000075.tif70140.

[0088] Embodiment 47. Formula (12): [ka] or a scalemic or racemic mixture thereof.

[0089] Embodiment 48: A process for preparing the compound of embodiment 47, comprising cyclizing compound (11) to form compound (12): [ka] .

[0090] Embodiment 49. Formula (11): [ka] or a scalemic or racemic mixture thereof.

[0091] Embodiment 50. A process for preparing a compound according to embodiment 49, comprising the alcohol oxidation of compound (10), followed by Grignard addition and Dess-Martin oxidation to form compound (11): [ka] .

[0092] Embodiment 51. Formula (10): [ka] or a scalemic or racemic mixture thereof.

[0093] Embodiment 52. A process for preparing a compound according to embodiment 51, comprising Diels-Alder cycloaddition of compound (8) to form compound (10): [ka] .

[0094] Embodiment 53. Formula (8): [ka] or a scalemic or racemic mixture thereof.

[0095] Embodiment 54. A process for preparing a compound according to embodiment 53 having the formula of compound (8), comprising converting compound (23) to form compound (8): [ka] .

[0096] Embodiment 55. A process for preparing portimine A (1), comprising: [ka] The process is i) reduction of compound (2) to form portimine A (1): [ka] and, ii) Dess-Martin oxidation of compound (22) followed by hydrolysis to form compound (2): [ka] and, iii) Pd-catalyzed vinylation of compound (21) to form compound (22): [ka] and, iv) converting compound (20) to compound (21): [ka] and, v) forming compound (20) by silylation and oxidation of compound (19): [ka] and, vi) forming compound (19) by ring-chain tautomerization of compound (32): [ka] and, vii) reduction of compound (16) to compound (32): [ka] and, viii) ring formation and oxidation of compound (15) to form compound (16): [ka] and, ix) Mo-catalyzed ring-closing alkyne metathesis to form compound (15): [ka] and, x) Conversion of compound (7) into N-protected imine compound (31): [ka] and, xi) transmetallation of compound (13) and conjugate addition to compound (12) to form compound (7): [ka] and, xii) cyclization of compound (11) to form compound (12): [ka] and, xiii) Alcohol oxidation of compound (10) followed by Grignard addition and Dess-Martin oxidation to form compound (11): [ka] and, xiv) Diels-Alder cycloaddition of compound (8) to form compound (10): [ka] and, xv) conversion of compound (23) to compound (8): [ka] and, a process including.

[0097] Embodiment 56: Formula (33-1) [ka] A compound having the structure:

[0098] Embodiment 57. The following steps: [ka] 57. A process for preparing a compound of embodiment 56 from compound (33-0), comprising:

[0099] Embodiment 58. Formula (33-2) [ka] A compound having the structure:

[0100] Embodiment 59. The following steps: [ka] 59. A process for preparing a compound of embodiment 58 from compound (33-0), comprising:

[0101] Embodiment 60. Formula (34): [ka] or a scalemic or racemic mixture thereof.

[0102] Embodiment 61. The following steps: [ka] 61. A process for preparing a compound of embodiment 60 from compound (21), comprising:

[0103] Embodiment 62. Formula (35-1): [ka] or a scalemic or racemic mixture thereof.

[0104] Embodiment 63. The following steps: [ka] 63. A process for preparing a compound of embodiment 62 from compound (21), comprising:

[0105] Embodiment 64. Formula (35-2): [ka] or a scalemic or racemic mixture thereof.

[0106] Embodiment 65. The following steps: [ka] A process for preparing the compound of embodiment 64 from compound (35-2), comprising:

[0107] Embodiment 66. Formula (36): [ka] or a scalemic or racemic mixture thereof.

[0108] Embodiment 67. The following steps: [ka] 67. A process for preparing a compound of embodiment 66 from compound (39), comprising:

[0109] Embodiment 68. Formula (37): [ka] or a scalemic or racemic mixture thereof.

[0110] Embodiment 69. The following steps: [ka] 69. A process for preparing a compound of embodiment 68 from compound (22), comprising:

[0111] Embodiment 70. Formula (38) [ka] A compound having the structure:

[0112] Embodiment 71. The following steps: [ka] 71. A process for preparing a compound of embodiment 70 from compound (21), comprising:

[0113] Embodiment 72. Formula (39) [ka] A compound having the structure:

[0114] Embodiment 73. The following steps: [ka] 73. A process for preparing a compound of embodiment 72 from compound (21), comprising:

[0115] Embodiment 74. Formula (40): [ka] or a scalemic or racemic mixture thereof.

[0116] Embodiment 75. The method of claim 7, further comprising the steps of: [ka] 75. A process for preparing a compound of embodiment 74 from compound (39), comprising:

[0117] Embodiment 76. A process for preparing various analogs of Portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, comprising the steps of: [ka] (Wherein, R is (C6 to C 10 ) aryl, (C2-C6) alkenyl or (C5-C 12 ) heteroaryl, wherein each aryl is optionally substituted with —NH(CH 2 ) 2 C(—N═N—)(CH 2 ) 2 (C≡CH), and R′ is H or Ac.

[0118] Embodiment 77. The compound is [ka] 77. The compound prepared by the process of claim 76, selected from the group consisting of:

[0119] Embodiment 78. A process for preparing various analogs of Portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, comprising the steps of: [ka] (In the formula, R 1is H, Me, Boc, or TES, R 2 H, halo, (C6~C 10 ) aryl, (C2-C6) alkenyl or (C5-C 12 ) heteroaryl, each aryl optionally substituted with —NH(CH)C(—N═N—)(CH)(C≡CH); R' is H or Ac.

[0120] Embodiment 79. The compound is [ka] 79. The compound prepared by the process of claim 78, selected from the group consisting of:

[0121] Embodiment 80. A process for preparing various analogs of portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, comprising the steps of: [ka] (In the formula, R 1 is H, Me, Boc, or TES, R 2 H, halo, (C6~C 10 ) aryl, (C2-C6) alkenyl or (C5-C 12 ) heteroaryl, each aryl optionally substituted with —NH(CH)C(—N═N—)(CH)(C≡CH); R' is H or Ac.

[0122] Embodiment 81. The compound is [ka] 81. The compound prepared by the process of claim 80, selected from the group consisting of:

[0123] Embodiment 82. The process of any one of claims 76, 78, or 80, wherein the portimine A analog product is useful for forming an antibody-drug conjugate (ADC) for treating cancer.

[0124] Embodiment 83. A method of forming an antibody-drug conjugate (ADC) for treating cancer, comprising the use of a compound according to any one of claims 76 to 81.

[0125] Embodiment 84. A method of treating cancer, comprising administering to a patient in need thereof a pharmaceutically effective amount of any one of claims 76 to 81 capable of forming an antibody-drug conjugate (ADC).

[0126] Embodiment 85. Any process, compound, method, or mixture described herein.

[0127] definition As used herein, the phrase "a" or "an" entity refers to one or more of that entity. For example, a compound refers to one or more compounds or at least one compound. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0128] The phrase "as defined herein above" refers to the broadest definition of each group, as set forth in the Summary of the Invention, the Detailed Description of the Invention, the Experimental, or the broadest claim. In all other embodiments provided below, substituents that may be present in each embodiment and that are not explicitly defined retain the broadest definition provided in the Summary of the Invention.

[0129] As used herein, whether in transitional phrases or in the body of a claim, the terms "comprising" and "including" should be construed as having an open-ended meaning. That is, these terms should be interpreted synonymously with the phrases "having at least" or "including at least." When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term "comprising" means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

[0130] As used herein, unless specifically stated otherwise, the word "or" is used in the inclusive sense of "and / or" and not in the exclusive sense of "either / or."

[0131] The term "independently" is used herein to indicate that a variable applies at any instance regardless of the presence or absence of variables with the same or different definitions within the same compound. Thus, in a compound where "R" appears twice and is defined as "independently selected from," each instance of that R group is separately identified as one member of the set that follows in the definition of that R group. For example, "each R 1 and R 2 are independently selected from carbon and nitrogen" means that R 1 and R 2 Both can be carbon or R 1 and R 2 Both of R and R may be nitrogen. 1 or R 2 This means that one can be carbon and the other nitrogen, or vice versa.

[0132] When any variable occurs more than one time in any moiety or formula depicting and describing the compounds used or claimed in this invention, its definition at each occurrence is independent of its definition at every other occurrence, and combinations of substituents and / or variables are permissible only if such compounds result in stable compounds.

[0133] The symbol "*" at the end of a bond, or a line drawn through a bond, or "~~~~" drawn through a bond, respectively, indicates the point at which a functional group or other chemical moiety is attached to the rest of the molecule of which it is a part.

[0134] A bond drawn into a ring system (as opposed to being attached at a separate vertex) indicates that the bond may be attached to any of the suitable ring atoms.

[0135] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance can occur, but need not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally substituted" means that the "optionally substituted" moiety may incorporate a hydrogen or a substituent.

[0136] The phrase "any bond" means that the bond may or may not be present, and that the description includes single, double, or triple bonds. When a substituent is designated as a "bond" or "absent," the atoms linked to the substituents are directly connected.

[0137] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 20%.

[0138] Certain compounds disclosed herein may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertible species. The prototypical tautomer results from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate individual tautomers usually produce mixtures whose chemical and physical properties match the mixture of compounds. The position of the equilibrium depends on the chemical features within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. Common protic tautomers include: TIFF2026505889000126.tif24153. The latter two are particularly common in heteroaryl and heterocyclic rings, and the present invention encompasses all tautomeric forms of the compounds.

[0139] Unless otherwise defined, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Reference is made herein to various methods and materials known to those skilled in the art. A standard reference describing the general principles of pharmacology is Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10 th Ed., McGraw Hill Companies Inc., New York (2001). Any suitable materials and / or methods known to those skilled in the art can be utilized in practicing the present invention. However, preferred materials and methods are described. Materials, reagents, and the like referred to in the following description and examples may be obtained from commercial sources unless otherwise noted.

[0140] The definitions set forth herein may be added to form chemically relevant combinations, such as "heteroalkylaryl," "haloalkylheteroaryl," "arylalkylheterocyclyl," "alkylcarbonyl," "alkoxyalkyl," etc. When the term "alkyl" is used as a suffix following another term, such as "phenylalkyl" or "hydroxyalkyl," it is intended to refer to an alkyl group as defined above substituted with one to two substituents selected from other specifically named groups. Thus, for example, "phenylalkyl" refers to an alkyl group having one to two phenyl substituents and thus includes benzyl, phenylethyl, and biphenyl. An "alkylaminoalkyl" is an alkyl group having one to two alkylamino substituents. "Hydroxyalkyl" includes 2-hydroxyethyl, 2-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 2,3-dihydroxybutyl, 2-(hydroxymethyl), 3-hydroxypropyl, and the like. Thus, as used herein, the term "hydroxyalkyl" is used to define a subset of the heteroalkyl groups defined below. The term -(ar)alkyl refers to either an unsubstituted alkyl or an aralkyl group. The term (hetero)aryl or (het)aryl refers to either an aryl or a heteroaryl group.

[0141] As used herein, the term "acyl" refers to a group of the formula -C(=O)R, where R is hydrogen or lower alkyl as defined herein. As used herein, the term "alkylcarbonyl" refers to a group of the formula C(=O)R, where R is alkyl as defined herein. 1~6 The term acyl refers to a group -C(=O)R containing 6 carbon atoms. As used herein, the term "arylcarbonyl" refers to a group of the formula C(=O)R where R is an aryl group, and as used herein, the term "benzoyl" is an "arylcarbonyl" group where R is phenyl.

[0142] As used herein, the term "alkyl" refers to an unbranched or branched-chain saturated monovalent hydrocarbon residue containing 1 to 12 carbon atoms. As used herein, the term "lower alkyl" or "C1-C6 alkyl" refers to a straight-chain or branched-chain hydrocarbon residue containing 1 to 6 carbon atoms. As used herein, "C 1~12 "Alkyl" refers to an alkyl composed of 1 to 12 carbons. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t-butyl or lower alkyl groups including pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

[0143] When the term "alkyl" is used as a suffix following another term, such as "phenylalkyl" or "hydroxyalkyl," it is intended to refer to an alkyl group as defined above, substituted with one to two substituents selected from the other specifically named group. Thus, for example, "phenylalkyl" refers to the radical R'R"-, where R' is a phenyl radical and R" is an alkylene radical as defined herein with the understanding that the point of attachment of the phenylalkyl moiety is on the alkylene radical. Examples of arylalkyl groups include, but are not limited to, benzyl, phenylethyl, and 3-phenylpropyl. The terms "arylalkyl" or "aralkyl" are interpreted similarly except that R' is an aryl radical. The terms "(het)arylalkyl" or "(het)aralkyl" are interpreted similarly except that R' may be an aryl or heteroaryl radical.

[0144] When a range of values ​​is listed, it is intended to encompass each value and subrange within the range. For example, "C 1~6 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C 1~6 , C 1~5 , C 1~4 , C 1~3 , C 1~2 , C 2~6 , C2~5 , C 2~4 , C 2~3 , C 3~6 , C 3~5 , C 3~4 , C 4~6 , C 4~5 , and C 5~6 Alkyl is intended to be included.

[0145] "Alkyl" means a radical of a linear or branched saturated hydrocarbon group having 1 to 20 carbon atoms ("C 1~20 In some embodiments, an alkyl group has 1 to 15 carbon atoms ("C 1~15 In some embodiments, the alkyl group has 1 to 14 carbon atoms ("C 1~14 In some embodiments, the alkyl group has 1 to 13 carbon atoms ("C 1~13 In some embodiments, the alkyl group has 1 to 12 carbon atoms ("C 1~12 In some embodiments, the alkyl group has 1 to 11 carbon atoms ("C 1~11 In some embodiments, an alkyl group has 1 to 10 carbon atoms ("C 1~10 In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C 1~9 In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C 1~8 In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C 1~7 In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C 1~6 In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C 1~5 In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C 1~4 In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C 1~3In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C 1~2 In some embodiments, the alkyl group has 1 carbon atom ("C alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C 2~6 alkyl). C 1~6 Examples of alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Further examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like.

[0146] "Alkenyl" or "olefin" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds ("C 2~10 In some embodiments, an alkenyl group has 2 to 9 carbon atoms ("C 2~9 In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("C 2~8 In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("C 2~7 In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("C 2~6 In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("C 2~5 In some embodiments, the alkenyl group has 2 to 4 carbon atoms ("C 2~4 In some embodiments, the alkenyl group has 2 to 3 carbon atoms ("C 2~3In some embodiments, the alkenyl group has two carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (such as 2-butenyl) or terminal (such as 1-butenyl). C 2~4 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. 2~6 Examples of alkenyl groups include the above-mentioned C 2~4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Further examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), etc.

[0147] "Alkynyl" refers to the radical of a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) ("C 2~10 In some embodiments, an alkynyl group has 2 to 9 carbon atoms ("C 2~9 In some embodiments, an alkynyl group has 2 to 8 carbon atoms ("C 2~8 In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C 2~7 In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C 2~6 In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C 2~5 In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C 2~4 In some embodiments, the alkynyl group has 2 to 3 carbon atoms ("C 2~3 In some embodiments, the alkynyl group has two carbon atoms ("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). C 2~4Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. 2~6 Examples of alkenyl groups include the above-mentioned C 2~4 Alkynyl groups include pentynyl (C5), hexynyl (C6), etc. Further examples of alkynyl include heptynyl (C7), octynyl (C8), etc.

[0148] The terms "haloalkyl" or "halo-lower alkyl" or "lower haloalkyl" refer to a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms, in which one or more carbon atoms are replaced by one or more halogen atoms.

[0149] As used herein, unless otherwise specified, the term "alkylene" or "alkylenyl" refers to a divalent saturated straight-chain hydrocarbon radical of 1 to 10 carbon atoms (e.g., (CH) n ) or a branched, saturated divalent hydrocarbon radical of 2 to 10 carbon atoms (e.g., -CHMe- or -CHCH(i-Pr)CH-). Except in the case of methylene, the open valences of an alkylene group are not attached to the same atom. Examples of alkylene radicals include, but are not limited to, methylene, ethylene, propylene, 2-methyl-propylene, 1,1-dimethyl-ethylene, butylene, and 2-ethylbutylene.

[0150] As used herein, the term "alkoxy" refers to an -O-alkyl group, where alkyl is as defined above, including isomers thereof, such as methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy, pentyloxy, hexyloxy, and the like. As used herein, "lower alkoxy" refers to an alkoxy group having a "lower alkyl" group as defined above. As used herein, "C 1~10 "Alkoxy" is a group in which the alkyl is C 1~10 It refers to -O-alkyl, where

[0151] As used herein, the term "hydroxyalkyl" refers to an alkyl radical, as defined herein, in which one to three hydrogen atoms on different carbon atoms are replaced by hydroxyl groups.

[0152] As used herein, the terms "alkylsulfonyl" and "arylsulfonyl" refer to a group of formula -S(=O)R, where R is each alkyl or aryl, where alkyl and aryl are as defined herein. As used herein, the term "heteroalkylsulfonyl" refers to a group of formula -S(=O)R, where R is "heteroalkyl" as defined herein.

[0153] As used herein, the terms "alkylsulfonylamino" and "arylsulfonylamino" refer to groups of formula -NR'S(=O)R, where R is independently alkyl or aryl, and R' is hydrogen or C 1~3 alkyl, where alkyl and aryl are as defined herein.

[0154] As used herein, the term "cycloalkyl" refers to a saturated carbocyclic ring containing from 3 to 8 carbon atoms, i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl. 3~7 "Cycloalkyl" refers to a cycloalkyl composed of 3 to 7 carbons in the carbocyclic ring.

[0155] As used herein, the term carboxy-alkyl refers to an alkyl moiety in which one hydrogen atom is replaced with carboxyl, and it is understood that the point of attachment of the heteroalkyl radical is through a carbon atom. The terms "carboxy" or "carboxyl" refer to the moiety -COH.

[0156] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a monocyclic or bicyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing 4 to 8 atoms per ring, incorporating one or more N, O, or S heteroatoms, the remaining ring atoms being carbon, with the point of attachment of the heteroaryl radical being understood to be on the aromatic ring. As is well known to those skilled in the art, heteroaryl rings are less aromatic than their all-carbon counterparts. Thus, for purposes of this invention, a heteroaryl group need only possess some degree of aromatic character. Examples of heteroaryl moieties include monocyclic aromatic heterocycles having 5 to 6 ring atoms and 1 to 3 heteroatoms, including, but not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, imidazolyl, oxazole, isoxazole, thiazole, isothiazole, triazoline, thiadiazole, and oxadiaxoline, which may be optionally substituted with one or more, preferably one or two, substituents selected from hydroxy, cyano, alkyl, alkoxy, thio, lower haloalkoxy, alkylthio, halo, lower haloalkyl, alkylsulfinyl, alkylsulfonyl, halogen, amino, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, and dialkylaminoalkyl, nitro, alkoxycarbonyl, and carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, alkylcarbonylamino, and arylcarbonylamino. Examples of bicyclic moieties include, but are not limited to, quinolinyl, isoquinolinyl, benzofuryl, benzothiophenyl, benzoxazole, benzisoxazole, benzothiazole, and benzisothiazole. Bicyclic moieties may be optionally substituted on either ring, but the point of attachment is on the ring containing the heteroatom.

[0157] As used herein, the terms "heterocyclyl," "heterocycloalkyl," or "heterocycle" refer to a heterocyclic group consisting of one or more rings, preferably 1 to 2 rings, including spiro ring systems, of 3 to 8 atoms per ring, containing one or more ring heteroatoms (N, O, or S(O)). 0~2and n is 0 or 1; and n is 1 or 2; and n is 2 or 3; and n is 3 or 4; and n is 4 or 5; and n is 5 or 6; and n is 6 or 7; and n is 7 or 8; and n is 8 or 9; and n is 1 or 2 ... Examples of heterocyclic groups include, but are not limited to, azetidinyl, pyrrolidinyl, hexahydroazepinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, oxazolidinyl, thiazolidinyl, isoxazolidinyl, morpholinyl, piperazinyl, piperidinyl, tetrahydropyranyl, thiomorpholinyl, quinuclidinyl, and imidazolinyl.

[0158] "Heterocyclyl" or "heterocyclic" refers to a group or radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur (a "3- to 14-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon atom or a nitrogen atom, where valence permits. Heterocyclyl groups may be either monocyclic (a "monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged, or spiro ring systems, such as bicyclic (a "bicyclic heterocyclyl") or tricyclic (a "tricyclic heterocyclyl") ring systems), saturated or containing one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring as defined above is fused to one or more carbocyclyl groups, and the point of attachment is on either the carbocyclyl or heterocyclyl ring, or in which a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, and the point of attachment is on the heterocyclyl ring, in which case the number of ring members continues to specify the number of ring members in the heterocyclyl ring system.

[0159] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur (a "5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heterocyclyl"). In some embodiments, a 5- to 6-membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, a 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0160] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl.Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydro-benzo-thienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, Examples thereof include 1,4,5,7-tetrahydro-pyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo-[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-furo[3,2-c]pyridinyl, 4,5,6,7-tetrahydro-thieno[3,2-b]pyridinyl, and 1,2,3,4-tetrahydro-1,6-naphthyridinyl.

[0161] "Aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared within the cyclic array) having 6 to 14 ring carbon atoms and 0 heteroatoms provided to the aromatic ring system ("C 6~14 In some embodiments, an aryl group has 6 ring carbon atoms ("C aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 Aryl"; for example, naphthyl, e.g., 1-naphthyl (α-naphthyl) and 2-naphthyl (β-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14"Aryl"; e.g., anthracyl). "Aryl" also includes ring systems in which an aryl ring, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups, and the radical or point of attachment is on the aryl ring; in such cases, the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system.

[0162] "Heteroaryl" refers to a radical of a 5- to 14-membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared within the cyclic array) having ring carbon atoms and containing 1 to 4 ring heteroatoms provided to the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 14-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be at a carbon atom or a nitrogen atom, valence permitting. Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring; in such cases, the number of ring members continues to designate the number of ring members in the heteroaryl ring system. "Heteroaryl" also refers to a heteroaryl ring, as defined above, fused to one or more aryl groups, and the point of attachment can be on either the aryl or heteroaryl ring; in such cases, the number of ring members refers to the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., either the ring containing a heteroatom (e.g., 2-indolyl) or the ring without a heteroatom (e.g., 5-indolyl).

[0163] In some embodiments, heteroaryl groups are 5-10 membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In some embodiments, heteroaryl groups are 5-8 membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some embodiments, heteroaryl groups are 5-6 membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl"). In some embodiments, 5-6 membered heteroaryls have 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, 5-6 membered heteroaryls have 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0164] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0165] "Saturated" refers to a ring moiety that does not contain any double or triple bonds, ie, the ring contains all single bonds.

[0166] Alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups may be substituted. Optionally substituted refers to a group that may be substituted or unsubstituted. Generally, the term "substituted" means that at least one hydrogen atom present on the group is replaced with a non-hydrogen substituent, resulting in a stable compound upon substitution, e.g., a compound that does not spontaneously undergo transformation, such as rearrangement, cyclization, elimination, or other reaction. Heteroatoms such as nitrogen, oxygen, and sulfur may have hydrogen and / or non-hydrogen substituents that satisfy the valence of the heteroatom and result in the formation of a stable compound.

[0167] As used herein, a moiety "optionally substituted" means that the moiety can be substituted with any additional moiety selected from the group consisting of, but not limited to, exemplary non-hydrogen substituents: halogen, -CN, -NO, -N, -SOH, -SOH, -OH, -OR. aa , -N(R bb )2, -N(OR cc )R bb , -SH, -SR aa , -C(=O)R aa , -CO2H, -CHO, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb ) OR aa , -OC(=NR bb )R aa , -OC(=NR bb ) ORaa , -C(=NR bb )N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -S(=O)R aa , -OS(=O)R aa , -B(OR cc )2, C 1~10 Alkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, C 3~14 Carbocyclyl, 3-14 membered heterocyclyl, C 6~14 Aryl and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of 0, 1, 2, 3, 4, or 5 R dd groups, or two geminal hydrogens on the carbon atom are replaced with groups =O); aa Each instance of 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, C 3~14 Carbocyclyl, 3-14 membered heterocyclyl, C 6~14 aryl and 5- to 14-membered heteroaryl, or two R aa groups are linked to form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring (each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl may have 0, 1, 2, 3, 4, or 5 R dd independently substituted with R groups; bb Each instance of is independently hydrogen, -OH, -OR aa , -N(R cc)2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -SO2N(R cc )2, -SOR aa , C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, C 3~14 Carbocyclyl, 3-14 membered heterocyclyl, C 6~14 aryl and 5- to 14-membered heteroaryl, or two R bb groups joined to form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring (each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl may have 0, 1, 2, 3, 4, or 5 R dd independently substituted with R groups; cc Each instance of is independently hydrogen, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, C 3~14 Carbocyclyl, 3-14 membered heterocyclyl, C 6~14 aryl and 5- to 14-membered heteroaryl, or two R cc The groups combine to form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring (each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl may have 0, 1, 2, 3, 4, or 5 R dd independently substituted with R groups; dd Each example is independently a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1~6 Alkyl, -ON(C 1~6 alkyl)2, -N(C 1~6 alkyl)2, -N(OC 1~6 Alkyl)(C 1~6 alkyl), -N(OH)(C 1~6alkyl), -NH(OH), -SH, -SC 1~6 Alkyl, -C(=O)(C 1~6 alkyl), -CO2H, -CO2(C 1~6 alkyl), -OC(=O)(C 1~6 alkyl), -OCO2(C 1~6 alkyl), -C(=O)NH2, -C(=O)N(C 1~6 alkyl)2, -OC(=O)NH(C 1~6 alkyl), -NHC(=O)(C 1~6 alkyl), -N(C 1~6 alkyl)C(=O)(C 1~6 alkyl), -NHCO2(C 1~6 alkyl), -NHC(=O)N(C 1~6 alkyl)2, -NHC(=O)NH(C 1~6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1~6 alkyl), -OC(=NH)(C 1~6 alkyl), -OC(=NH)OC 1~6 Alkyl, -C(=NH)N(C 1~6 alkyl)2, -C(=NH)NH(C 1~6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1~6 alkyl)2, -OC(NH)NH(C 1~6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1~6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1~6 alkyl), -SO2N(C 1~6 alkyl)2, -SO2NH(C 1~6 alkyl), -SO2NH2, -SO2C 1~6 Alkyl, -B(OH)2, -B(OC 1~6 Alkyl)2, C 1~6 Alkyl, C 1-6 Perhaloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~10 Carbocyclyl, C 6~10 Aryl, 3- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl; or two geminal R on a carbon atom ddThe substituents may be linked to form =O.

[0168] "Halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br) or iodine (iodo, -I).

[0169] As used herein, the term "composition" is intended to encompass a product that includes the specified ingredients, as well as any product that results directly or indirectly from the combination of the specified ingredients.

[0170] As used herein, the term "adjuvant" refers to a compound or composition that enhances a medical treatment, such as an additional pharmacological agent added to a drug to increase or supplement its effect, such as an immunological agent that increases the antigenic response or otherwise contributes to or enhances an existing medical regimen (e.g., Freund's adjuvant).

[0171] The term "salt" includes any and all salts. A "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and that is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts include salts derived from inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N-methyl-N ... + (C 1~4Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0172] As used herein, the term "amino terminal modification group" refers to any molecule that can be attached to a terminal amine group. By way of example, such a terminal amine group may be at the end of a polymer molecule, including, but not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of example only, terminal modification groups include polyethylene glycol or serum albumin. Terminal modification groups may be used to modify the therapeutic properties of the polymer molecule, including, but not limited to, increasing the serum half-life of a peptide.

[0173] As used herein, the term "antibody fragment" refers to any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components present within a full-length antibody, as well as engineered antibodies. Antibody fragments include, but are not limited to, Fv, Fe, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, as well as alternative scaffold non-antibody molecules, bispecific antibodies, etc. (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is the single-chain Fv (scFv), which consists of the variable regions of immunoglobulin heavy and light chains covalently linked by a peptide linker (Sz Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules. Unless otherwise specified, descriptions and claims using the term "antibody" or "antibody" specifically include "antibody fragment" and "antibody fragments."

[0174] As used herein, "antibody-drug conjugate," or "ADC," as used herein, refers to an antibody molecule or fragment thereof covalently linked to one or more biologically active molecule(s). The biologically active molecule may be conjugated to the antibody via a linker, polymer, or other covalent bond.

[0175] Unless otherwise specified, the compounds described herein may contain one or more asymmetric centers and, therefore, may exist in various stereoisomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of an individual enantiomer, diastereomer, or geometric isomer, or in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC). The compounds described herein may be in the form of an individual isomer substantially free of other isomers, or may be as a mixture of various isomers.

[0176] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms, for example, the replacement of hydrogen by deuterium or tritium. 19 F 18 Substitution by F, 13 C or 14 Substitution of carbon with C-rich carbon, and / or 18 Compounds having the present structure except for the replacement of the oxygen atom by O are within the scope of this disclosure. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, 36 Cl and 123 Compounds having such isotopically enriched atoms are useful, for example, as analytical tools or probes in biological assays.

[0177] Certain isotopically labeled compounds (e.g., 3 H and 14 C) are useful in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e., 14C) Isotopes are particularly preferred due to their ease of preparation and detectability. Certain isotopically labeled compounds of formula (I) may be useful for medical imaging purposes, for example: 11 C or 18 Those labeled with positron-emitting isotopes such as F may be useful for applications in positron emission tomography (PET), 123 Those labeled with gamma-ray emitting isotopes such as I may be useful for applications in single photon emission computed tomography (SPECT). Additionally, deuterium (i.e., 2 Substitution with heavier isotopes, such as H), may offer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and therefore may be preferable in some situations. Additionally, substitution with deuterium (i.e., 2 Substitution with heavier isotopes, such as H, may confer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may therefore be preferred in some circumstances. Furthermore, isotopic substitution at sites where epimerization occurs may slow or reduce the epimerization process, thereby retaining a more active or efficacious form of the compound for a longer period of time. Isotopically labeled compounds of formula (I), particularly those with longer half-lives (t 1 / 2 Isotopically labeled compounds containing isotopes with a valence of >1 day can generally be prepared by procedures analogous to those disclosed in the schemes and / or examples herein below, by substituting the appropriate isotopically labeled reagent for the non-isotopically labeled reagent.

[0178] In the event of a discrepancy between a drawn structure and a name given to that structure, the drawn structure controls. Furthermore, if the stereochemistry of a structure or portion of a structure is not indicated, for example, by bold or dashed lines, the structure or portion of a structure should be interpreted as encompassing all stereoisomers thereof. However, in some cases, when two or more chiral centers are present, the structure and name may be represented as a single enantiomer to help explain the relative stereochemistry. Those skilled in the art of organic synthesis will know whether compounds are prepared as single enantiomers from the methods used to prepare them.

[0179] [Example] Abbreviation Commonly used abbreviations include: acetyl (Ac), azo-bis-isobutyrylnitrile (AIBN), atmosphere (Atm), 9-borabicyclo[3.3.1]nonane (9-BBN or BBN), tert-butoxycarbonyl (Boc), di-tert-butylpyrocarbonate or boc anhydride (BOC2O), benzyl (Bn), butyl (Bu), Chemical Abstracts Registration Number (CAL). Carbonyl Carbonyl Fluoride (CBZ or Z), Carbonyldiimidazole (CDI), 1,4-Diazabicyclo[2.2.2]octane (DABCO), Diethylaminosulfur trifluoride (DAST), Dibenzylideneacetone (dba), 1,5-Diazabicyclo[4.3.0]non-5-ene (DBN), 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), N,N'-Dicyclohexylcarbodiimide (DCC), 1,2-Dichloroethane (DCE), Dichloromethane (DCM), Diethylazodicarboxylate (DEAD), Di-isopropylazodicarboxylate (DIAD), Di-isobutylaluminum hydride (DIBAL or DIBAL-H), 1,3-Diisobutylazodicarboxylate (DIBAL), Di ... Isopropylcarbodiimide (DIC), di-isopropylethylamine (DIPEA), N,N-dimethylacetamide (DMA), 4-N,N-dimethylaminopyridine (DMAP), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,1'-bis-(diphenylphosphino)ethane (dppe), 1,1'-bis-(diphenylphosphino)ferrocene (dppf), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), ethyl acetate (Et), ethyl acetate (EtOAc), ethanol (EtOH), 2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (EEDQ), diethyl ether (EtO), O-(7-azabenzotriazol-1-yl)-N,N,N'N'-Tetramethyluronium hexafluorophosphate acetate (HATU), acetic acid (HOAc), 1-N-hydroxybenzotriazole (HOBt), high pressure liquid chromatography (HPLC), isopropanol (IPA), lithium hexamethyldisilazane (LiHMDS), methanol (MeOH), melting point (mp), MeSO2- (mesyl or Ms), methyl (Me), acetonitrile (MeCN), m-chloroperbenzoic acid (MCPBA), mass spectrum (ms), methyl t-butyl ether (MTBE), N-bromosuccinimide (NBS), N-carboxyanhydride (NCA), N-chlorosuccinimide (NCS), N-methylmorpholine (NMM), N-methylpyrrolidone (NMP), pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), phenyl (Ph), propyl (Pr), iso-propyl (i- Pr), pounds per square inch (psi), pyridine (pyr), room temperature (rt or RT), tert-butyldimethylsilyl or t-BuMe2Si (TBDMS), triethylamine (TEA or Et3N), 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), triflate or CF3SO2- (Tf), trifluoroacetic acid (TFA), 1,1'-bis-2,2,6,6-tetramethylheptane-2,6-di N-(2-methyl- ...

[0180] compound 23 [ka] To a solution of 4-aminobutyraldehyde diethyl acetal (32.2 g, 200 mmol) in CHCl (200 mL) was added BocO (43.8 g, 201 mmol) dropwise at room temperature. The reaction was monitored by TLC (stain: ninhydrin in ethanol). Upon completion, the solvent was removed under reduced pressure, and the residue was dissolved in THF (200 mL) under argon at 0 °C. To this solution, NaHMDS (0.6 M in toluene, 400 ml, 240 mmol) was added dropwise with vigorous stirring. The resulting solution was kept stirring at the same temperature, followed by the slow addition of BocO (43.8 g, 201 mmol). Upon completion, saturated aqueous NHCl was added and stirred overnight. The organic layer was separated, and the aqueous layer was extracted with EtO. The combined organic layers were dried over MgSO and then concentrated to give the crude Boc-carbamate as the product.

[0181] The crude carbamate obtained above was dissolved in acetone / HO (4:1, 500 ml). PTSA monohydrate (14.0 g, 73.6 mmol) was added and stirred at room temperature for 16 hours. The mixture was neutralized with saturated aqueous NaHCO and extracted with EtO. The combined organic layers were dried over MgSO, filtered, and concentrated. The crude product was purified by chromatography (5% EtOAc in hexane to 15% EtOAc in hexane) to give compound 23 (51.7 g, 90%) as a colorless oil.

[0182] Physical state: colorless oil TLC:R f =0.41 (25% ethyl acetate in hexane, stains brown when stained with KMnO4) 1 H-NMR(500 MHz,CDCl3)δ 9.74(t,J=2.2 Hz,1H),3.58(t,J=7.2 Hz,2H),2.43(t,J=7.2 Hz,2 H),1.86(quin,J=7.2 Hz,2H),1.47(s,18H) 13C-NMR(125 MHz,CDCl3)δ 201.6,152.7,82.6,45.5,41.1,28.1,21.6 All spectral data are consistent with those reported. 1 Large-scale synthesis of Rawal's diene. [ka] To a solution of BnNH2 (30.6 g, 286 mmol) in methanol (200 mL) at 0 °C was added 4-(trimethylsilyl)-3-butyn-2-one (21.6 mL, 143 mmol) dropwise. After stirring at room temperature for 1.0 h, the solvent was removed in vacuo. The crude product was suspended in EtOAc and rinsed with aqueous NaH2PO4 to remove benzylamine. The organic layer was separated and the aqueous phase was extracted three times with EtOAc. The combined organic layers were dried over MgSO4 and concentrated under reduced pressure to give the crude enamine. 2 was obtained and used directly in the next step.

[0183] The crude enamine obtained above was dissolved in THF (10 mL / g) and n-BuLi (2.5 M, 60 mL, 150 mmol) was added over 30 min at 0 °C under argon. After 10 min, ClCOMe (11.6 mL, 14.2 g, 150 mmol) was added slowly and the solution was stirred for 2 h. Upon completion, the mixture was poured into HO (500 mL). The mixture was extracted three times with EtOAc and dried over MgSO. Compound 24 (27.9 g, 120 mmol, 84%) could be obtained by chromatography (0–40% EtOAc in hexanes).

[0184] Physical state: pale yellow oil TLC:R f = 0.28 (30% EtOAc in hexane, UV active, stains brown when stained with KMnO4) 1H-NMR(400 MHz,CDCl3)δ 8.17(d,J=14.4 Hz,1H),7.36-7.03(m,5H),5.50(d,J=14.4 Hz,1H),4.76(s,2H),3.84(s,3H),2.13(s,3H) 13 C-NMR(100 MHz,CDCl3)δ 197.2,154.5,141.9,135.3,128.8,127.6,126.4,109.9,54.4,48.2 HRMS(ESI-TOF):C 13 H 15 NO3[M+H] + Calculated value: 234.1125, Measured value: 234.1126 Pure compound 24 (52 g, 223 mmol) was dissolved in EtO (650 mL) under argon. To this solution, TEA (45.5 g, 450 mmol) was added in one portion at −78°C. TBSOTf (59.5 g, 225 mmol) was slowly added dropwise to the solution. A cloudy mixture formed during the addition. After stirring at −78°C for 30 minutes, the flask was gradually warmed to 0°C for an additional 30 minutes. The reaction mixture was quenched with hexane and saturated aqueous NaHCO, followed by extraction with hexane. The organic phase was dried over MgSO and concentrated to give Lawal's diene (purity as indicated by NMR) which was used directly in the next step.

[0185] Physical state: pale yellow solid TLC:R f =0.43 (hexane + 1% TEA, UV active, stains brown when stained with KMnO4) 1 H-NMR(400 MHz,CDCl3)δ 7.72-7.42(m,1H),7.32(t,J=7.3 Hz,2H),7.27-7.13(m,3H),5.36(d,J=14.1 Hz,2H),4.80(s,2H),4.13(s,1H),4.07(s,1H),3.83(s,3H),1.00(s,9H),0.18(s,6H) 13C-NMR(100 MHz,CDCl3)δ 155.1,154.5,136.9,128.8,127.3,126.7,126.3,108.4,93.0,53.8,48.2,26.0,18.4,-4.5 HRMS(ESI-TOF):C 19 H 29 NO3Si[M+H] + Calculated value: 348.1990, Measured value: 348.1986 compound 25 [ka] To a mixture of aqueous formaldehyde (13 mL, 37% aqueous formaldehyde) and compound 23 (46.8 g, 162.9 mmol) in i-PrOH (95 mL) was added propionic acid (1.20 g, 16.2 mmol, 0.1 equiv.) and pyrrolidine (1.15 g, 16.2 mmol, 0.1 equiv.). The reaction mixture was stirred at 55 °C for 1.0 h. Upon completion, saturated aqueous NaHCO3 and brine were added, and the mixture was extracted with Et2O. The combined organic layers were dried over MgSO4 and concentrated in vacuo to remove all volatiles. The resulting crude acrolein (compound 8) was used directly in the Diels-Alder cycloaddition.

[0186] Compound 8 was dissolved in DCM (200 mL), followed by the addition of MS4Å (25 g, oven-dried) and Lawal's diene (70.5 g, 10.7 mmol, 1.2 equiv.) under argon. After stirring the mixture at 0 °C for 15 min, [Co(salen)]SbF6 (984 mg, 1.63 mmol, 0.01 equiv.) was added in one portion, and the mixture was stirred overnight in an ice bath. Upon completion, the reaction was diluted with DCM and silica gel was added. After concentration in vacuo, the crude product was purified by chromatography (dry loading, 0–20% EtOAc in hexanes) to give compound 25 (92.7 g, 143.3 mmol, 88%).

[0187] Physical state: White powder TLC:R f= 0.35 (20% ethyl acetate in hexane, UV active, stains brown when stained with KMnO4) [α] 25 D :-55.4(c=1.0, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 9.89-9.59(m,1H),7.28-7.24(m,2H),7.21-7.14(m,1H),7.14-6.98(m,2H),5.12-4.75(m,1H),4.59(d,J=5.7 Hz,1H),4.52(d,J=16.4 Hz,1H),4.37(d,J=16.4 Hz,1H),3.79-3.47(m,4H),3.40-3.30(m,1H),2.23-2.02(m,3H),2.01-1.83(m ,2H),1.63-1.56(m,1H),1.51(s,18H),0.83(s,9H),-0.04(s,3H),-0.13(s,3H) 13 C-NMR(150 MHz,CDCl3)δ 204.8,158.2,155.7,152.1,139.5,128.6,126.7,126.2,99.4,82.8,57. 4,54.2,53.0,49.8,48.1,42.2,30.9,28.3,25.8,25.6,20.3,18.0,-4.7 HRMS(ESI-TOF):C 34 H 54 N2O8Si[M+H] + Calculated value: 647.3728, Measured value: 647.3736 compound 10 [ka] reduction To a solution of compound 25 (81.4 g, 126 mmol) in a mixture of MeOH (400 ml) and DCM (100 ml) was added NaBH (4.7 g, 124 mmol) portionwise at 0 °C. The reaction was kept stirring for 1 h and quenched with NH Cl (sat) and HO. The mixture was extracted with DCM and dried over Na SO . The organic phase was concentrated under reduced pressure to give the crude product, which was used directly in the next step.

[0188] E1cb The crude product was dissolved in 500 ml of THF and TBAF (130 ml, 1 M in THF) was added. The reaction mixture was stirred for 30 minutes and quenched with NH4Cl (saturated) and HO. The mixture was extracted with DCM and dried over Na2SO4. The organic phase was concentrated under reduced pressure. The crude product was purified by chromatography (30% ethyl acetate in hexane to 80% ethyl acetate in hexane) to give compound 10 (46 g, 126 mmol, quantitative yield).

[0189] *Note: Compound 10 is bench stable.

[0190] Physical state: pale yellow oil TLC:R f =0.25 (30% ethyl acetate in hexane, UV active, stains brown when stained with KMnO4) [α] 25 D :+37.8(c=1.0, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 6.82(d,J=10.2 Hz,1H),6.01(d,J=10.2 Hz,1H),3.71-3.61(m,3H),3.56(d,J=11.6 Hz,1H),2.93(t,J=6.4 Hz,1H),2.55-2.38(m,2H),1.95-1.76(m,4H),1.51(s,18H) 13 C-NMR(150 MHz,CDCl3)δ 199.3,154.3,152.8,129.5,83.2,66.8,42.0,40.3,33.9,33.1,28.4,28.2 HRMS(ESI-TOF):C 19 H 31 NO6[M-Boc+2H] + Calculated value: 270.1705, Measured value: 270.1704 compound 11 [ka] Alcohol oxidation To a solution of compound 10 (16.1 g, 43.6 mmol) in DCM (60 ml) was added NaHCO (15 g) and water (50 ml). To the resulting mixture, KBr (539 mg, 4.5 mmol) and TEMPO (68 mg, 0.44 mmol) were added at room temperature (open system). The reaction was cooled to 0 °C, and NaOCl solution (6 wt%, 105 ml) was added slowly at the same temperature. The reaction mixture immediately turned red and was gradually warmed to ambient temperature by removing the ice bath.

[0191] After 1 hour, the reaction mixture was quenched with saturated aqueous NaSO and extracted with DCM. The organic phase was dried over MgSO and concentrated under reduced pressure. The product (approximately 16 g) was used directly in the next step without purification.

[0192] Grignard reagent preparation 3 To a suspension of magnesium (3.1 g, 129 mmol) and LiCl (6.0 g, 141 mmol) in THF (29 ml) was subsequently added DIBAL-H (1.3 ml, 1.0 M in toluene) dropwise at 0° C. under argon.

[0193] Meanwhile, a solution of 3-pentynyl bromide (19.0 g, 129 mmol) in 72.8 mL of THF was prepared at room temperature. A small amount (8.7 mL) of this stock solution was added to the above suspension at 0°C to initiate the Grignard reaction. After 15 minutes, the remaining stock solution was slowly added. Residual bromide was rinsed three times with THF (34 mL total). The reaction mixture was kept under vigorous stirring at 0°C for 30 minutes and then allowed to warm to ambient temperature for an additional 30 minutes. The Grignard reagent was obtained as 0.54 M (theoretical 0.95 M) as indicated by titration with iodine.

[0194] Grignard addition To a solution of the aldehyde (16 g, 43.6 mmol, in 320 mL of THF) at −78° C., freshly prepared Grignard reagent (110 mL, 59.4 mmol) was added dropwise over 1 h (under argon). The reaction mixture was continued to stir at the same temperature for another 1 h, and AcOH (10 mL) was added to quench the reaction. The resulting solution was gradually warmed to room temperature, and brine and saturated aqueous NaHCO3 were added. The mixture was extracted with ethyl acetate and dried over MgSO4. The resulting organic phase was concentrated under reduced pressure to give the crude product, which was used in the next step without purification.

[0195] Alcohol oxidation The crude product obtained from the Grignard addition was dissolved in DCM (100 ml, open system) and cooled to 0 °C. NaHCO (10 g) was added to the solution, followed by Dess-Martin periodinane (19.0 g, 44.8 mmol). The reaction mixture was allowed to warm to ambient temperature. After 1 h, the reaction mixture was diluted with DCM (approximately 50 ml) and quenched with saturated NaSO solution and saturated aqueous NaHCO solution. The resulting mixture was stirred for 45 min. The mixture was extracted with DCM and dried over MgSO. The organic phase was concentrated under reduced pressure. The crude product was purified by chromatography (5% EtOAc in hexane to 20% EtOAc in hexane) to give compound 11 (11.3 g, 26.1 mmol, 60%).

[0196] *Note: Compound 11 is bench stable.

[0197] Physical state: light yellow oil TLC:R f = 0.60 (30% ethyl acetate in hexane, UV active, stains brown when stained with KMnO4) [α] 25 D :-32.2(c=1.0, CHCl3) 1H-NMR(600 MHz,CDCl3)δ 7.01(d,J=10.3 Hz,1H),6.09(d,J=10.3 Hz,1H),3.60-3.45(m,2H),2.81-2.68(m,2H),2.54-2.35(m,5H),2.06-1.93(m,3H),1.72,(s,3H),1.50(s,18H) 13 C-NMR(150 MHz,CDCl3)δ 207.5,198.3,152.4,150.1,130.6,83.0,77.6,76.6,52.4,42.1,38.8,36.5,34.8,29.8,28.3,13.6,3.6 HRMS(ESI-TOF):C 24 H 35 NO6[M+Na] + Calculated value: 456.2362, Measured value: 456.2350 compound 12 [ka] To a solution of compound 11 (8.8 g, 20.3 mmol) in DCM (62.8 ml) was added TFA (31.4 ml) at room temperature. The resulting red solution was stirred at the same temperature for 1.5 h. The reaction mixture was carefully added (using a dropping funnel) to a mixture of Na2CO3 (64 g in 120 ml of HO) and 4 M NaOH (24 ml), followed by vigorously stirring for 24 h. The mixture was extracted with Et2O (first portion) and DCM (remaining portions). The organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by chromatography (DCM → 30% EtOAc + 1% TEA in DCM) to give compound 12 (3.14 g, 14.6 mmol, 72%).

[0198] *Note: Compound 12 is unstable on long-term storage (gradual decomposition occurred even after 1 week at -20°C) and should be used as soon as possible.

[0199] Physical state: viscous orange oil TLC:R f = 0.37 (pure EtOAc, high UV activity, stains brown when stained with KMnO4) [α] 25 D -155.8 (c=0.5, CHCl3) 1 H-NMR(600 MHz, CDCl3)δ 6.62(dd,J=10.1,1.6 Hz,1H),6.05(d,J=10.1 Hz,1H),3.96(dddt,J=16.0,8.6,3.8,1.9 Hz,1H),3.81(dtt,J=15.6,7.6,2.4 Hz,1H),2.58-2.48(m,4H),2.48-2.34(m,2H),2.20-2.09(m,2H),1.96(dddd,J=13.0,8.7,7.7,1.0 Hz,1H),1.86(dtd,J=13.5,4.9,1.7 Hz,1H),1.75(t,J=2.5 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 198.3,178.0,152.0,129.7,78.4,76.2,58.3,55.7,35.2,30.1,29.3,16.0,3.7 HRMS (ESI-TOF):C 14 H 17 NO[M+H] + Calculated value: 216.1388, measured value: 216.1382 Compound 26 (Ref. 5)

change

[0200] Physical state: colorless oil TLC:R f = 0.40 (10% EtOAc in hexane, UV active, stains brown when stained with KMnO4) [α] 25 D :-36.4(c=1.0, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 6.88(dd,J=15.6,5.6 Hz,1H),6.10(dd,J=15.6,1.4 Hz,1H),4.66(tdd,J=7.0,5.6,1.4 Hz,1H),4.26-4.14(m,3H),3.68(dd,J=8.3,7.0 Hz,1H),1.48-1.43(m,3H),1.41(d,J=0.8 Hz,3H),1.29(t,J=7.1 Hz,3H) 13 C-NMR(150 MHz,CDCl3)δ 166.2,110.4,75.1,69.0,60.8,26.6,25.9,14.4 HRMS(ESI-TOF):C 10 H 16 O4[M+Na] +Calculated value: 223.0941, Measured value: 223.0942 Compound 27 (reference 6) [ka] To a solution of compound 26 (21.0 g, 104 mmol) in EtO (550 mL) was added MeLi·LiBr (1.6 M in EtO, 71.5 mL, 157 mmol) dropwise over 2.5 h via syringe pump at −78 °C. Upon completion, the solution was quenched with MeOH (10 mL) and saturated aqueous NaHCO at −78 °C and then slowly warmed to room temperature. The resulting mixture was extracted with EtO and dried over MgSO. The solvent was removed under reduced pressure, and the crude product was purified by chromatography (0% to 20% EtOAc in hexanes) to give compound 27 (18.5 g, 85.5 mmol, 82%).

[0201] Physical state: colorless oil TLC:R f =0.47 (20% EtOAc in hexane) [α] 25 D :-9.60(c=1.0, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 4.14(q,J=7.2 Hz,2H),4.05-3.96(m,2H),3.66-3.61(m,1H),2.40(dd,J=14.9,5.0 Hz,1H),2.24-2.17(m,1H),2.13(dd,J=14.9,8.8 Hz,1H),1.25(s,3H),1.22(s,3H),1.26(t,J=7.2 Hz,3H),1.00(d,J=6.8,3H) 13 C-NMR(150 MHz,CDCl3)δ 172.8,109.1,79.0,67.0,60.6,37.8,33.2,26.6,25.4,15.6,14.4 HRMS(ESI-TOF):C 11 H 20 O4[M+Na] + Calculated value: 239.1259, Measured value: 239.1254 compound 28 [ka] To a solution of compound 27 (10.4 g, 48.1 mmol) in THF (90 ml) was added LAH (1.83 mg, 48.2 mmol) in small portions at 0° C. The ice bath was removed, and the reaction mixture was then stirred at ambient temperature for 2 hours. Upon completion, EtOAc (100 ml) was slowly added, followed by water (2 ml) to quench the remaining LAH. After stirring for 30 minutes, saturated aqueous Rochelle's salt solution was added to the mixture and stirred overnight. After separating the organic layer, the aqueous phase was extracted with EtOAc. The combined organic layers were dried over MgSO4 and concentrated under reduced pressure to give the crude alcohol.

[0202] The alcohol obtained above was dissolved in DCM (50 mL) under argon, and imidazole (4.25 g, 62.5 mmol) and PPh3 (15.1 g, 57.7 mmol) were added. To this mixture, I2 (14.0 g, 55.3 mmol) was slowly added. The red color faded as all I2 was consumed. Upon completion (as indicated by TLC), methanol was added to quench the excess iodine, followed by bubbling air through the solution to quench the excess PPh3 (typically taking 30 min). The volatiles were then removed under reduced pressure, and pentane (150 mL) was added and stirred vigorously (sonication if necessary). After 1 h, the solids were removed using a Buchner funnel, and the residue was washed twice with pentane. The filtrate was then collected, and the pentane was removed under reduced pressure to yield the crude iodide (containing approximately 6% TPPO).

[0203] In a separate flame-dried round-bottom flask, TIPS acetylene (9.65 g, 52.9 mmol) was dissolved in THF (86 ml) and n-BuLi (2.5 M in hexane, 19.2 ml, 48.1 mmol) was added dropwise under argon. The mixture was then stirred at 0 °C for 30 min and cooled to -78 °C. DMPU (12 ml) was added to this solution in one portion. Subsequently, a solution of the above iodide (in 20 ml of THF) was added dropwise to the freshly prepared lithium acetylide. The mixture was stirred overnight at ambient temperature and quenched with water. The aqueous phase was extracted with EtO and combined. The combined organic phase was rinsed three times with water to remove residual DMPU and dried over MgSO. After solvent removal, the residue was purified by chromatography (hexane to 5% EtOAc in hexane) to give compound 28 (12.2 g, 36.1 mmol, 75%).

[0204] Physical state: colorless oil TLC:R f = 0.21 (30% DCM in hexane, stains brown when stained with KMnO4) [α] 25 D :-16.5(c=1.0, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 4.02(dd,J=7.6,6.3 Hz,1H),3.92(q,J=6.3 Hz,1H),3.64(t,J=7.6 Hz,1H),2.35(ddd,J=17.0,7.5,5.7 Hz,1H),2.27(ddd,J=17.0,8.2,7.3 Hz,1H),1.87-1.75(m,1H),1.61-1.54(m,1H),1.39(s,3H),1.35(s,3H),1.36-1.29(m,1H),1.10-1.00(m,21H),0.99(d,J=6.6 Hz,3H) 13 C-NMR(150 MHz,CDCl3)δ 108.9,108.6,80.8,79.9,67.9,35.6,32.2,26.7,25.7,18.8,17.8,15.0,11.5 HRMS(ESI-TOF):C 20 H 38 O2Si[M+H]+ Calculated value: 339.2719, Measured value: 339.2711 compound 29 [ka] A solution of compound 28 (18.5 g, 54.5 mmol) in a mixture of HCl (3 M, 50 mL) and THF (100 mL) was heated at 75 °C for 12 h. The mixture was then cooled to room temperature, and saturated aqueous NaHCO was slowly added to neutralize the HCl. The mixture was extracted with ether, and the organic phase was dried over MgSO. The crude product was obtained by concentration under reduced pressure and used directly.

[0205] NaH (5.4 g, 60 wt% in mineral oil, 135 mmol) was suspended in THF (100 ml) under an argon atmosphere. The mixture was then cooled to 0 °C. To this solution, the crude product obtained above (dissolved in 20 ml of THF) was added dropwise. Upon completion, the ice bath was removed and the mixture was allowed to warm. The mixture was then stirred at ambient temperature for 1.5 h, and tosylimidazole (14.4 g, 65 mmol) was added portionwise. The resulting mixture was vigorously stirred at the same temperature for 45 min. Upon completion, water was added, and the mixture was stirred for 15 min. The organic layer was separated, and the aqueous phase was extracted three times with EtO. The combined organic phases were dried over MgSO and concentrated. The crude product was purified by chromatography (hexane to 30% DCM in hexane) to give epoxide 29 (13.3 g, 47.4 mmol, 87%).

[0206] Physical state: colorless oil TLC:R f = 0.33 (40% DCM in hexane, stains brown when stained with KMnO4) [α] 25 D :+13.3(c=1.0, CHCl3) 1H-NMR(600 MHz,CDCl3)δ 2.77(dd,J=5.0,3.2 Hz,1H),2.70(dt,J=6.8,3.2 Hz,1H),2.59(dd,J=5.0,3.2 Hz,1H),2.39-2.26(m,2H),1.64(tt,J=11.3,4.7 Hz,1H),1.56-1.47(m,2H),1.13-0.96(m,24H) 13 C-NMR(150 MHz,CDCl3)δ 108.4,80.9,56.7,47.1,35.3,32.8,18.8,17.9,16.9,11.4 HRMS(ESI-TOF):C 17 H 32 OSi[M+H] + Calculated value: 281.2301, Measured value: 281.2296 compound 30 [ka] A solution of n-BuLi (73.0 ml in hexane, 182 mmol) in THF (300 ml) was cooled to −78°C under argon. To this solution, a balloon of propyne was added via needle, and the solution began to absorb the gas. Upon completion (solution became cloudy), the balloon was removed. The resulting mixture was stirred at −78°C for 30 minutes.

[0207] To this freshly prepared lithium acetylide, a solution of compound 29 (17.0 g, 60.6 mmol) in THF (30 ml) was slowly added at the same temperature. BF etherate (22.0 ml, 178 mmol) was then added, and the reaction mixture was stirred at −78° C. for 1 h. The reaction was quenched with saturated aqueous NaHCO, diluted with EtO, and then stirred for 1 h. The mixture was extracted with EtOAc, and the organic layer was dried over MgSO. The solvent was removed in vacuo, and the crude product was used directly in the next transformation.

[0208] To the crude product obtained above, TBAF (100 ml, 1 M in THF, 100 mmol) was added under argon. The resulting mixture was stirred at 45 °C overnight. Upon completion, saturated aqueous NH4Cl and EtOAc were added. The mixture was extracted with EtOAc and dried over MgSO4. After removing the solvent under reduced pressure, the crude product was dissolved in DCM (120 ml), and then TEA (18.2 g, 25.1 ml, 180 mmol) was added. To this solution, TBSOTf (31.7 g, 27.6 ml, 120 mmol) was slowly added at room temperature. The mixture was stirred at ambient temperature for 30 minutes and heated to reflux for 1 hour. Upon completion, saturated aqueous NaHCO3 was added, and the mixture was extracted with DCM. The combined organic layers were dried over MgSO4 and concentrated. The crude product was purified by chromatography (0–5% EtOAc in hexanes) to give compound 30 (15.5 g, 55.7 mmol, 92%).

[0209] Physical state: colorless oil TLC:R f = 0.50 (5% EtOAc in hexane, stains brown when stained with KMnO4) [α] 25 D :-10.5(c=1.0, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 3.71(td,J=6.7,2.8 Hz,1H),2.30-2.22(m,2H),2.18(dtd,J=16.9,7.8,2.6 Hz,1H),1.93(t,J=2.6 Hz,1H),1.92-1.85(m,1H),1.77(t,J=2.6 Hz,3H),1.67(dtd,J=13.3,7.9,5.3 Hz,1H),1.38(dtd,J=13.3,8.4,6.0 Hz,1H),0.88(s,9H),0.85(d,J=6.8 Hz,3H),0.08(s,3H),0.06(s,3H) 13 C-NMR(150 MHz,CDCl3)δ 84.8,77.2,76.8,74.5,68.3,36.6,32.3,26.0,25.0,18.3,16.7,13.0,3.70,-4.1,-4.6 HRMS(ESI-TOF):C 17 H 30 OSi[M+H] + Calculated value: 279.2139, Measured value: 279.2142 compound 13 [ka] To a solution of CpZrCl (13.6 g, 46.5 mmol) in THF (100 ml) was added DIBAL-H (38.6 ml, 38.6 mmol, 1.0 M in hexane) dropwise at 0 °C. A white precipitate formed. The mixture was stirred for 1 h, and a solution of the compound (8.58 g, 30.8 mmol) in THF (10 ml) was slowly added. The reaction mixture (cloudy) was slowly warmed to room temperature and continued to stir overnight. A solution of I (12.0 g, 47.2 mmol) in THF (20 ml) was added at 0 °C, and the resulting dark solution was stirred at the same temperature for 1 h. The reaction was quenched with saturated aqueous NaSO and filtered to remove undissolved impurities (if necessary). The resulting mixture was extracted with EtO, and the organic phase was dried over MgSO and concentrated under reduced pressure to give the crude compound. Pure compound 13 (12.4 g, 30.4 mmol, 99%) was obtained by chromatography (2% EtOAc in hexane).

[0210] *Note: Compound 13 is very stable and can be stored neat (5 °C) for several months.

[0211] Physical state: pale yellow oil TLC:R f = 0.44 (3% EtOAc in hexane, UV active, stains brown when stained with KMnO4) [α] 25 D :-13.6(c=1.0, CHCl3) 1H-NMR(600 MHz,CDCl3)δ 6.51(dt,J=14.3,7.2 Hz,1H),5.99(dd,J=14.3,1.5 Hz,1H),3.68(td,J=6.8,2.8 Hz,1H),2.29-2.18(m,2H),2.17-1.99(m,2H),1.78(t,J=2.6 Hz,3H),1.77-1.70(m,1H),1.55-1.46(m,1H),1.28-1.18(m,1H),0.88(s,9H),0.83(d,J=6.8 Hz,3H),0.07(s,3H),0.04(s,3H) 13 C-NMR(150 MHz,CDCl3)δ 146.8,77.3,76.7,74.6,74.4,36.4,34.1,32.0,26.0,25.0,18.3,13.3,3.8,-4.1,-4.5 HRMS(ESI-TOF):C 17 H 31 IOSi [M+H] + Calculated value: 407.1267, Measured value: 407.1260 compound 7 [ka] Lithium-halogen exchange To a solution of compound 13 (7.42 g, 18.3 mmol, in 46 mL of EtO) at −78°C, t-BuLi (23.8 mL, 1.6 M in pentane, 38.1 mmol) was added dropwise. A bright yellow solution immediately formed, followed by a precipitate. The reaction was allowed to stir at −78°C for at least 2.5 hours to give a solution of vinyllithium.

[0212] Preparation of copper(I)-phosphine complexes Meanwhile, [Cu] (2.86 g, 21.5 mmol) was suspended in THF (16 mL) under argon. To this suspension, n-Bu3P (10.9 mL, 8.82 g, 43.6 mmol) was added dropwise. Dissolution of the yellow precipitate (participate) occurred and was complete within 15–30 min.

[0213] Transmetalation and conjugate addition The prepared copper-phosphine complex was added dropwise to the resulting vinyllithium solution over 5 minutes at −78° C. After the addition, the orange mixture was stirred at −78° C. for 1 hour.

[0214] A solution of compound 12 (3.14 g, 14.6 mmol, in 10 ml of THF) was added dropwise to the above solution at −78° C. The resulting mixture was stirred at the same temperature for 40 minutes. Then, a solution of Comins' reagent (8.59 g, 21.9 mmol, in 20 ml of THF) was added, and the resulting solution was warmed to 0° C. The reaction mixture was stirred at 0° C. for 1 hour, quenched with 5% aqueous NH3, Na2CO3, and bubbled with air. The resulting mixture was vigorously stirred for 45 minutes and extracted three times with ether. The organic layer was dried over MgSO4. The solvent was removed under reduced pressure, and the resulting residue was purified by gradient chromatography (20% Et2O in hexane to 40% Et2O in hexane + 1% TEA) to give compound 7 (6.41 g, 10.2 mmol, 70%).

[0215] *Note: Compound 7 slowly decomposes in the crude product, so it is better to purify the crude product as soon as possible (on the same day). If there are impurities in this step, the RCAM step will not work.

[0216] Physical state: pale yellow oil TLC:R f =0.58 (50% Et-2O in hexane, stains brown when stained with KMnO4) [α] 25 D :-53.1(c=1.0, CHCl3) 1H-NMR(600 MHz, CDCl3)δ 5.68(d,J=2.0 Hz,1H),5.46(dt,J=14.3,6.8 Hz,1H),5.21(ddt,J=14.3,7.44,1.49 Hz,1H),3.85-3.77(m,1H),3.67(td,J=6.96,2.80 Hz,1H),3.52(tdd,J=10.3,8.3,4.1 Hz,1H),3.17-3.06(m,1H),2.56-2.51(m,2H),2.50-2.34(m,4H),2.28-2.18(m,2H),2.10-1.93(m,3H),1.93-1.86(m,1H), 1.71-1.65(m,1H),1.81-1.73(m,6H),1.65-1.53(m,2H),1.46-1.39(m,1H),1.21-1.12(m,1H),0.88(s,9H),0.81(d,J=6.8 Hz,3H),0.07(s,3H),0.04(s,3H) 13 C-NMR(150 MHz, CDCl3)δ 177.8,148.9,134.4,126.3,120.6,118.7(q,J C-F =316 Hz),79.0,77.1,76.9,76.0,74.7,58.8,56.7,43.1,37.0,33.2,31.5,30.9,30.1,29.0,26.0,25.5,25.1,18.3,15.9,13.2,3.7,-4.0,-4.5 1 H-COSY, 1 H- 13 C-HSQC 1 H- 13 C-HMBC, NOESYスペクトルが can be used HRMS (ESI-TOF):C 32 H 48 F3NO4SSi[M+H] + Calculated value: 628.3104, measured value: 628.3093 Compound 31

change

[0217] The crude product was dissolved in a mixture of DCM (2.4 ml), MeOH (24 ml), and HO (1.2 ml). PTSA (1 g, 5.3 mmol) was added to the solution and heated to 50 °C overnight. The reaction mixture was quenched with saturated aqueous NaHCO, brine was added, and extracted with DCM. The resulting organic phase was dried over MgSO and concentrated under reduced pressure. The crude product was purified by chromatography (DCM → 20% EtO in DCM) to give compound 15 (1.38 g, 2.11 mmol, 72%).

[0218] *Note: Compound 15 is bench stable.

[0219] Physical state: white solid TLC:R f =0.45 (the color is brown when dyed with 20% Et-2O and KMnO4 in the dyeing method) [α] 25 D -41.4 (c=0.5, CHCl3) 1 H-NMR(600 MHz, CDCl3)δ 5.65(ddd,J=15.3,9.7,4.7 Hz,1H),5.60(d,J=2.3 Hz,1H),5.27(dd,J=15.3,8.6 Hz,1H),5.17(t,J=5.8 Hz,1H),4.74(d,J=12.0 Hz,1H),4.67(d,J=12.0 Hz,1H),3.56-3.50(m,1H),3.45(s,1H),3.34-3.27(m,1H),3.26-3.20(m,1H),2.80(ddd,J=18.8,11.2,3.0 Hz,1H),2.64-2.46(m,4H),2.46-2.28(m,3H),2.20-2.01(m,4H),1.95(dd,J=14.0,5.8 Hz,1H),1.85(dq,J=9.4,6.6 Hz,1H),1.64(ddd,J=14.0,9.4,5.0 Hz,1H),1.50-1.39(m,2H),1.23-1.15(m,1H),0.93(d,J=6.6 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 209.9,154.7,147.6,133.9,126.5,120.5,118.6(q,J C-F =320 Hz),95.7,81.5,75.8,74.7,73.0,52.0,46.2,37.7,35.7,33.0,32.3,28.3,28.0,27.2,25.6,24.9,13.6,13.1 HRMS (ESI-TOF):C 25 H 31 Cl3F3NO7S[M+H] + Calculated value: 652.0917, measured value: 652.0919 Compound 16

change

[0220] The resulting polycyclic compound was dissolved in MeCN / PhMe / HO (5:5:1, 112 ml), and TBAI (808 mg, 2.19 mmol) was added in one portion to obtain a clear solution. Ru(PPh3)3Cl2 (191 mg, 0.2 mmol) was then added to this solution, producing a gray-green mixture. TBHP (5-6 M in decane, 10.9 ml) was then added dropwise. The yellow-green solution turned dark (approximately 15 min; shielded with aluminum foil because the product is light-sensitive). The reaction was stirred at ambient temperature for 1.5 h (open system, water bath to maintain constant temperature). Upon completion, the reaction was diluted with Et2O and quenched with a mixture of water, solid NaHSO3, and NaCl. The resulting cloudy mixture was stirred for 1 h, resulting in good phase separation. After collecting the organic phase, the aqueous phase was extracted four times with Et2O. The combined organic phases were dried over MgSO4, concentrated under reduced pressure and the crude product was purified by chromatography (DCM to 5% Et2O in DCM) to give compound 16 (1.67 g, 2.44 mmol, 52%).

[0221] *Note: Compound 16 is photosensitive and highly unstable in solvents (CHCl3, benzene, etc.). The solvent must be removed for storage.

[0222] Physical state: yellow foam TLC:R f =0.45 (20% Et-2O in hexane, yellow in light, brown when stained with KMnO4) [α] 25 D :+40.4(c=1.0, MeOH) 1H-NMR(600 MHz,CD3CN)δ 5.57(m,1H),4.82(d,J=12.3 Hz,1H),4.70(d,J=12.3 Hz,1H),4.48(dt,J=4.9,2.5 Hz,1H),3.78(ddd,J=10.1,6.1,3.8 Hz,1H),3.68-3.53(m,1H),3.47(td,J=11.1,7.1 Hz,1H),3.15-3.06(m,1H),2.77(dt,J=12.6,10.0 Hz,1H),2.68(dddd,J=13.6,11.5,9.7,1.8 Hz,1H),2.63-2.54(m,1H),2.38(ddt,J=18.3,5.3,2.3 Hz,1H),2.32-2.20(m,3H),2.02(ddd,J=13.6,8.6,3.0 Hz,1H),1.96-1.93(m,1H),1.88(dd,J=13.2,7.0 Hz,1H),1.81-1.73(m,1H),1.72-1.64(m,3H),1.63-1.53(m,2H),0.83(d,J=7.2 Hz,3H) 13 C-NMR(150 MHz,CD3CN)δ 199.8,199.0,152.7,150.6,119.7(q,J C-F =320 Hz),119.0,117.9,105.5,96.9,84.3,75.0,52.4,45.2,40.36,37.6,37.4,37.0,32.9,29.1,27.4,25.4,25.4,24.1,23.8,18.9 HRMS(ESI-TOF):C 25 H 29 Cl3F3NO9S[M+H] + Calculated value: 682.0659, Measured value: 682.0668 compound 32 [ka] To a solution of compound 16 (2.43 g, 3.53 mmol) in THF (135 ml) was added L-selectride (1.0 M in THF, 6.7 ml) under argon at −78° C. The yellow solution turned colorless upon completion (usually taking 30 minutes). The reaction was quenched according to the following procedure: i. MeOH (3.8 ml) was added to the mixture at -78°C and stirred at this temperature for 5 min.

[0223] The cooling bath was removed, and a mixture of H2O (50 ml), EtOAc (10 ml), and Na2CO3 (3.0 g) was added successively. The resulting mixture was stirred for 5 minutes.

[0224] iii. H2O2 solution (50 wt% in H2O, 2 ml) was added dropwise and the mixture was stirred at room temperature for 60 min.

[0225] After workup, the mixture was extracted three times with EtOAc, and the combined organic layers were dried over MgSO. After concentration under reduced pressure, the residue was dissolved in MeOH (100 ml) and cooled to 0 °C. NaBH (excess, ca. 500 mg) was added portionwise at this temperature. The reaction was stirred for 1 h. After complete conversion, aqueous NHCl was added, and the mixture was extracted three times with DCM. The combined organic phases were dried over MgSO and concentrated, and the crude product was purified by chromatography (5% EtOAc in DCM to 15% EtOAc in DCM) to give compound 32 (2.03 g, 83%).

[0226] Physical state: White foam TLC:R f = 0.23 (30% EtOAc in hexane, stains brown when stained with KMnO4) [α] 25 D :+17.9(c=1.0, CHCl3) 1 H-NMR(500 MHz,CDCl3)δ 5.86(s,1H),4.81(d,J=12.0 Hz,1H),4.64(d,J=12.0 Hz,1H),4.06(td,J=8.9,8.3,4.0 Hz,1H),3.92-3.82(m,1H),3,60(t,J=10.4 Hz,1H),3.56-3.10(m,3H),3.03-2.81(m,1H),2.57-2.22(m,5H),2.11-1.74(m,10H),1.73-1.52(m,4H),0.86(d,J=7.2 Hz,3H) 13C-NMR(125 MHz,CDCl3)δ 152.3,149.3,124.0,118.7(q,J C-F =320 Hz),116.2,106.5,96.0,86.2,81.4,75.0,74.6,66.0,49.7,44.8,38.0,36.4,35.8,34.5,30.0,30.0,28.7,26.6,25.1,24.1,18.1 HRMS(ESI-TOF):C 25 H 33 Cl3F3NO9S [M-OH] + Calculated value: 668.0866, Measured value: 668.0869 compound 19 [ka] To a solution of compound 32 (1.95 g, 2.84 mmol) in DCM (37 ml) were added TEMPO (55.5 mg, 0.059 mmol), KBr (1.3 g, 10.9 mmol), and saturated aqueous NaHCO (9.3 ml), followed by cooling to 0 °C. The mixture was then stirred for 5 min, and NaOCl solution (6%, ca. 0.8 M, 5.1 ml) was added dropwise. The resulting red biphasic solution was stirred at the same temperature for 3.5 h and then quenched by adding saturated aqueous NaSO. The yellow mixture became colorless within a few seconds and was subsequently extracted several times with DCM. The combined organic layers were dried over MgSO and concentrated under reduced pressure to give a white foam (C-14 oxidation product).

[0227] The crude compound obtained above was mixed with zinc powder (1.7 g, 26 mmol), then dissolved in a mixture of AcOH (70 mL) and HO (23 mL) and heated to 70 °C under argon. The mixture was stirred at the same temperature for 3 h and then cooled to 45 °C. After stirring at 45 °C for 2 h, the mixture was cooled to ambient temperature, diluted with EtOAc, and added dropwise to a solution of KCO (70 g in 100 mL of HO). After quenching the acetic acid, the biphasic mixture was extracted three times with DCM and dried over MgSO. After removing the solvent, the crude product was purified by chromatography (EtOAc to 10% MeOH + 1% TEA in EtOAc) to give compound 19 (1.03 g, 2.10 mmol, 74%).

[0228] Physical state: pale yellow powder TLC:R f =0.23 (3% MeOH in EtOAc, stains brown when stained with KMnO4) [α] 25 D :-35.6(c=0.5, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 5.09(q,J=1.7 Hz,1H),4.29(dt,J=10.3,6.2 Hz,1H),4.04(d,J=12.0 Hz,1H),4.00-3.94(m,1H),3.89-3.81(m,1H),3.65-3.58(dtt,J=17.2,6.9,3.6 Hz,1H),2.74-2.56(m,3H),2.55-2.43(m,4H),2.38-2.28(m,1H),2.19- 2.07(m,2H),2.00-1.80(m,4H),1.79-1.68(m,3H),1.49(dt,J=12.6,6.3 Hz,1H),1.43(dd,J=14.6,8.1 Hz,1H),0.83(d,J=7.1 Hz,3H) 13 C-NMR(150 MHz,CDCl3)δ 206.4,182.0,148.4,119.1,118.6(q,J C-F=320 Hz),110.7,84.6,75.9,56.0,53.6,48.7,40.4,37.2,36.5,34.1,28.6,28.3,27.0,26.3,24.8,24.4,17.7 HSQC available HRMS(ESI-TOF):C 22 H 28 F3NO6S:[M+H] + Calculated value: 492.1668, Measured value: 492.1663 compound 20 [ka] Silylation To a solution of compound 19 (505 mg, 1.03 mmol) in refluxing DCM (5.0 mL) was added TEA (1.07 mL, 777 mg, 7.68 mmol) and TBSOTf (0.75 mL, 862 mg, 3.26 mmol). The reaction mixture was then stirred for 45 minutes. Upon completion, the mixture was rinsed with saturated aqueous NaHCO3, followed by extraction with DCM. The organic phase was dried over MgSO4, filtered, and concentrated to give the crude silyl enol ether as the product.

[0229] DMDO oxidation The crude product from above was dissolved in DCM (3 ml) and a DMDO solution (freshly prepared, approximately 0.08 M in acetone, 50 ml) was added dropwise at 0°C. After stirring at 0°C for 1 h, the solvent was removed in vacuo. The residue was redissolved in acetone / HO (v / v = 1:1, 20 ml) and solid NaHCO was added. Oxone® (550 mg) was added to the mixture and the reaction was stirred at ambient temperature for 1 h. Upon completion, a mixture of HO and DCM was added. The aqueous phase was extracted four times with DCM, and the organic layer was dried and concentrated. The crude product was purified by chromatography (EtOAc to 10% MeOH in EtOAc) to give compound 20 (387.1 mg, 0.74 mmol, 73%).

[0230] Physical state: White powder TLC:R f=0.30 (10% MeOH in EtOAc, UV activity, KMnO4 staining results in brown color) [α] 25 D +49.0 (c=0.1, CHCl3) 1 H-NMR(600 MHz, CDCl3)δ 5.23(s,1H),4.78(br s,1H),4.59(d,J=12.0 Hz,1H),4.25(dt,J=11.2,6.0 Hz,1H),4.09-4.02(m,1H),4.00(dd,J=11.3,2.8 Hz,1H),3.95-3.86(dt,J=13.4,6.4 Hz,2H),3.45(td,J=14.3,7.3 Hz,1H),2.60-2.43(m,3H),2.30(dt,J=14.1,11.0 Hz,1H),2.26-2.18(m,2H),2.14-2.04(m,2H),2.04-1.92(m,2H),1.92-1.84(m,2H),1.82(dd,J=15.2,7.2 Hz,1H),1.73(dt,J=12.7,7.0 Hz,1H),1.59(dt,J=12.7,6.3 Hz,1H),1.54(dd,J=14.1,2.8 Hz,1H),0.88(d,J=6.9 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 204.2,153.4,147.8,118.6(q,J C-F =320 Hz),118.0,110.7,84.9,78.7,70.9,59.5,49.3,40.2,37.7,34.3,33.9,33.6,29.0,25.6,24.4,22.7,18.4,17.6 HRMS(ESI-TOF):calc'd for:C 22 H 28 F3NO8S:[M+H] + Calculated value: 524.1561, measured value: 524.1561 Compound 21

change

[0231] Physical state: white solid TLC:R f = 0.42 (5% MeOH in DCM, stains brown when stained with KMnO4) [α] 25 D :+5.30(c=1.0, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 5.78(dd,J=10.9,8.0 Hz,1H),5.19(dd,J=11.1,2.8 Hz,1H),5.05(q,J=1.7 Hz,1H),4.36(d,J=12.0 Hz,1H),4.25(dt,J=10.0,6.0 Hz,1H),4.18-4.08(m,1H),3.92(dd,J=16.2,9.1 Hz,1H),3.77(ddd,16.2,10.2,6.9 Hz,1H),2.64-2.41(m,3H),2.41-2.26(m,3H),2.23-2.13(m,1H),2.12-1.9 5(m,2H),2.08(s,3H),2.05(s,3H),1.93-1.73(m,4H),1.60(td,J=12.5,7.6 Hz,1H),1.52(dd,J=13.2,2.8 Hz,1H),0.88(d,J=6.5 Hz,3H) 13 C-NMR(150 MHz,CDCl3)δ 199.8,177.2,169.3,169.0,148.8,118.6(q,J C-F=320 Hz),117.8,108.1,84.0,79.9,73.0,72.1,56.4,53.7,40.4,39.1,36.7,34.0,31.6,29.4,28.7,26.5,24.5,21.3,20.9,17.4 HRMS(ESI-TOF):C 26 H 32 F3NO 10 S: [M+H] + Calculated value: 608.1777, Measured value: 608.1776 The crude product 39 obtained above was dissolved in THF (3.1 ml) and HO (0.31 ml), and a solution of LiOH (1.4 M in HO, 0.23 ml) was added at 0 °C. The resulting solution was then stirred at 0 °C and carefully monitored by TLC (5% MeOH in DCM). The reaction was quenched with a mixture of saturated aqueous NaHCO and DCM, followed by extraction three times with DCM. The organic layers were combined and dried over MgSO. After removal of the solvent, the crude product was purified by chromatography (pure DCM to 5% MeOH in DCM) to give compound 21 (88.1 mg, 0.16 mmol, 64%).

[0232] Physical state: White powder TLC:R f =0.25 (5% MeOH in DCM, stains brown when stained with KMnO4) [α] 25 D :+14.0(c=0.1, CHCl3) 1H-NMR(600 MHz, CDCl3)δ 5.19(dd,J=11.4,2.7 Hz,1H),5.06(q,J=1.7 Hz,1H),4.75(dd,J=11.1,7.6 Hz,1H),4.34(d,J=12.2 Hz,1H),4.29-4.20(m,2H),3.81(dd,J=15.7,8.9 Hz,1H),3.61(ddt,J=16.2,10.5,5.8 Hz,1H),2.72(dd,J=14.9,11.2 Hz,1H),2.57-2.25(m,5H),2.19(dd,J=15.0,7.6 Hz,1H),2.04(s,3H),2.03-1.98(m,1H),1.98-1.93(m,1H),1.91-1.81(m,2H),1.77(dt,J=12.1,7.1 Hz,1H),1.70-1.67(m,1H),1.58(dd,J=12.6,7.7 Hz,1H),1.52(dd,J=13.6,2.8 Hz,1H),0.88(d,J=6.9 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 199.6,183.2,169.1,148.6,118.7(q,J C-F =319 Hz),118.0,108.4,84.0,80.0,72.9,70.3,55.4,53.5,42.7,38.9,37.0,34.0,31.5,29.5,29.1,26.5,24.4,21.0,17.5 HRMS (ESI-TOF):C 24 H 30 F3NO9S[M+H] + Calculated value: 566.1666, measured value: 566.1658 Compound 22

change

[0233] Physical state: white solid TLC:R f =0.43 (40% acetone in hexane) [α] 25 D :+18.0(c=0.1, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 6.18(dd,J=17.5,10.7 Hz,1H),5.24-5.19(m,1H),5.10(d,J=17.5 Hz,1H),4.91-4.99(m,2H),4.75(dd,J=11.2,7.4 Hz,1H),4.31(d,J=12.1 Hz,1H),4.26(dt,J=10.7,5.6 Hz,1H),4.16(d,J=12.1 Hz,1H),3.76(dd,J=16.2,8.8 Hz,1H),3.61(ddd,J=16.2,10.4,6.8 Hz,1H),2.81(dd,J=14.8,11.3 Hz,1H),2.42-2.22(m,5H),2.19-2.14(m,1H),2.07(s,3H),2.03(dd,J=12.3,7.0 Hz,1H),1.91-1.71(m,4H),1.71-1.63(m,1H),1.57(td,J=12.5,7.6 Hz,1H),1.53-1.46(m,1H),0.87(d,J=7.0 Hz,3H) 13C-NMR(150 MHz,CDCl3)δ 200.1,184.0,168.9,139.0,135.4,127.7,111.6,108.2,83.7,80.1,73.7,70. 8,55.4,54.2,42.5,39.8,36.9,34.0,31.6,29.6,29.2,24.3,22.7,21.1,17.4 HRMS(ESI-TOF):C 25 H 33 NO6[M+H] + Calculated value: 444.2381, Measured value: 444.2377 Compound 2 (Portimine B) [ka] To a solution of compound 22 (16.7 mg, 0.038 mol) in DCM (0.3 mL) was added DMP (55.1 mg, 0.13 mmol) and NaHCO powder (25 mg). The resulting mixture was stirred at ambient temperature for 1 h. Upon completion, saturated aqueous KCO (0.2 mL) and saturated aqueous NaSO (0.1 mL) were added, followed by dilution with DCM. The biphasic mixture was vigorously stirred for 30 min, and the organic layer was separated. The aqueous phase was extracted with DCM, and the combined DCM layers were dried over MgSO and filtered. After concentration, the residue was dissolved in MeOH (0.2 mL), and NHOH (37% in H2O, 0.1 mL) was added. The progress of the hydrolysis can be monitored by TLC (5% MeOH in DCM). Upon completion, saturated aqueous NaCl was added to the mixture to aid in phase separation. The solution was extracted with DCM (three times), and the combined DCM layers were dried and concentrated. Portimine B(2) (13.3 mg, mmol, 88%) was obtained by chromatography (pure DCM to 5% MeOH in DCM).

[0234] Physical state: white solid TLC:R f = 0.22 (3% MeOH in DCM, UV active, stains brown with KMnO4) [α] 25 D :+42.9(c=0.08, CHCl3) 1H-NMR(600 MHz, CDCl3)δ 6.21(dd,J=17.4,10.8 Hz,1H),5.11(d,J=17.4 Hz,1H),5.07(d,J=2.0 Hz,1H),4.97(d,J=10.8 Hz,1H),4.62(d,J=12.2 Hz,1H),4.281(dt,J=10.1,6.0 Hz,1H),4.15(d,J=11.3 Hz,1H),4.08(dd,J=16.2,9.1 Hz,1H),3.83(ddd,J=16.6,10.4,6.7 Hz,1H),3.78(d,J=12.8 Hz,1H),3.43(d,J=17.5 Hz,1H),2.79(d,J=17.5 Hz,1H),2.50(s,1H),2.42-2.24(m,4H),2.12(dd,J=12.5,7.1 Hz,1H),2.03-1.90(m,2H),1.87(dd,J=12.5,6.6 Hz,1H),1.78(dt,J=12.4,6.4 Hz,2H),1.73-1.65(m,1H),1.63-1.53(m,1H),0.91(d,J=7.1 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 204.4,200.9,182.5,138.6,136.0,126.7,112.4,107.1,84.7,78.9,72. 2,58.1,54.7,53.0,40.9,38.2,34.3,34.1,28.7,28.6,24.4,22.6,17.7 HRMS (ESI-TOF):C 23 H 29 NO5[M+H] + Calculated value: 400.2119, measured value: 400.2117 Table 1 Compound 1 (Hydron A)

change

[0235] Physical state: white solid TLC:R f = 0.54 (10% MeOH in DCM, UV active, stains brown when stained with KMnO4) [α] 25 D :+5.8(c=1.0, CHCl3) 1 H-NMR(600 MHz,CDCl3)δ 6.21(dd,J=17.6,10.8 Hz,1H),5.17-5.01(m,2H),4.93(d,J=10.8 Hz,1H),4.71(br s,1H),4.52(s,1H),4.47(d,J=12.0 Hz,1H),4.18(dt,J=11.2,5.6 Hz,1H),4.08(dd,J=11.2,2.7 Hz,1H),3.86(dd,J=15.2,8.8 Hz,1H),2.81(dd,J=15.9,5.1 Hz,1H),2.43-2.19(m,4H),2.09(d,J=15.8 Hz,1H),2.01-1.92(m,2H),1.93-1.79(m,2H),1.79-1.61(m,5H),1.50(dd,J=13.7,2.6 Hz,1H),0.88(d,J=6.9 Hz,3H) 13 C-NMR(150 MHz,CDCl3)δ 204.6,185.0,138.8,135.2,127.8,111.7,109.1,83.2,79.3,71.6,65. 6,54.9,52.7,43.1,40.6,37.6,34.3,33.8,29.6,29.0,24.6,22.6,17.6 HRMS(ESI-TOF):C23 H 31 NO5[M+H] + Calculated value: 402.2275, Measured value: 402.2265 [Table 2] Compound 33-1 [ka] To a solution of the amine (16 mg, 0.12 mmol) in DMF (0.2 ml) was added HATU (44 mg, 0.12 mmol) and acid (38 mg, 0.15 mmol). The mixture was stirred at room temperature, followed by the addition of DIPEA (10 drops). The resulting yellow solution was stirred under argon for 16 h and diluted with EtO. Saturated aqueous NaHCO was added to the mixture to quench excess benzoic acid. The resulting biphasic mixture was extracted with EtO, dried over MgSO, and concentrated under reduced pressure. PTLC (12% EtO in DCM) afforded pure compound 33-1 (32 mg, 0.087 mmol, 73%).

[0236] Physical state: colorless oil TLC:R f = 0.55 (30% EtOAc in hexane, UV active, stains brown when stained with KMnO4) 1 H-NMR(600 MHz,CDCl3)δ 7.87(d,J=8.2 Hz,2H),7.77(d,J=8.2 Hz,2H),6.35(t,J=5.8 Hz,1H),3.31(q,J=6.6 Hz,2H),2.03(td,J=7.2,2.6 Hz,2H),1.98(t,J=2.6 Hz,1H),1.82(t,J=6.6 Hz,2H),1.68(t,J=7.2 Hz,2H),1.35(s,12H) 13 C-NMR(150 MHz,CDCl3)δ 167.7,136.7,135.2,126.2,84.3,82.9,69.6,35.1,32.7,32.3,29.9,27.1,25.0,13.4 HRMS(ESI-TOF):C 20 H26 BN3O3[M+H] + Calculated value: 368.2140, Measured value: 368.2136 Compound 33-2 [ka] To a solution of the amine (10.0 mg, 0.12 mmol) in DMF (0.2 ml) was added HATU (36 mg, 0.12 mmol) and acid (16 mg, 0.15 mmol). The mixture was stirred at room temperature, followed by the addition of DIPEA (0.05 ml). The resulting yellow solution was stirred under argon for 16 h, followed by the addition of HO. The resulting biphasic mixture was extracted with EtOAc, dried over MgSO, and concentrated under reduced pressure. Pure compound 33-2 (17.2 mg, 0.087 mmol, 73%) was obtained by PTLC (60% EtOAc in hexane).

[0237] Physical state: white solid TLC:R f = 0.65 (60% EtOAc in hexane, UV active, stains brown when stained with KMnO4) 1 H-NMR(500 MHz,CD3OD)δ 7.79(d,J=7.6 Hz,2H),7.70(d,J=7.8 Hz,2H),2.27(t,J=2.7 Hz,1H),2.05(td,J=7.4,2.7 Hz,2H),1.75(t,J=7.1 Hz,2H),1.68(t,J=7.4 Hz,2H) 13 C-NMR(126 MHz,CD3OD)δ 169.0,133.5,125.8,82.2,69.0,37.5,34.6,32.1,31.8,26.6,12.5 HRMS(ESI-TOF):C14H16BN3O3[M+H] + Calculated value: Measured value: 286.1354 compound 34 [ka] To a solution of compound 21 (5.0 mg, 0.0088 mmol) and compound 33-2 (5.1 mg, 0.018 mmol) in 1,4-dioxane (0.35 ml), Pd(PPh3)4 (0.7 mg, 0.00091 mmol) and K3PO4 (3 mg) were added. The resulting yellow solution was heated to 85 °C. Upon completion (typically 2 h), HO was added to the mixture and extracted with DCM. Pure compound 34 (4.5 mg, 0.0069 mmol, 78%) was obtained by PTLC (45% acetone in hexanes).

[0238] Physical state: white solid TLC:R f =0.25 (10% MeOH in DCM, UV active, stains brown when stained with KMnO4) 1 H-NMR(600 MHz,CDCl3)δ 7.70(d,J=8.5 Hz,2H),7.36(d,J=8.5 Hz,2H),6.29(t,J=5.9 Hz,1H),5.41(s,1H),5.25-5.20(m,1H),4.80(dd,J=11.1,7.5 Hz,1H),4.39(d,J=12.1 Hz,1H),4.29-4.20(m,2H),3.82(dd,J=15.6,8.6 Hz,1H),3.68(ddd,J=16.0,10.1,7.0 Hz,1H),3.31(q,J=6.4 Hz,2H),2.84(dd,J=14.8,11.2 Hz,1H),2.64-2.52(m,2H),2.47-2.32(m,3H),2.21(dd,J=14.9,7.5 Hz,1H),2.04(s,3H),2.04-2.02(m,2H),1.98(t,J=2.6 Hz,2H),1.98-1.84(m,3H),1.82(t,J=6.6 Hz,2H),1.81-1.74(m,3H),1.68(t,J=7.3 Hz,2H),1.59(td,J=12.6,7.7 Hz,1H),1.53(dd,J=13.1,2.9 Hz,1H),0.89(d,J=6.2 Hz,3H) 13C-NMR(150 MHz,CDCl3)δ 200.3,184.5,169.1,167.3,144.1,135.6,133.1,127.2,125.2,124.9,108.4,83.9,82. 9,80.3,73.8,70.8,69.7,55.4,53.8,42.7,40.1,37.0,35.1,34.2,32.7,32.3(d,J=5.55 Hz),31.7,30.1,29.9,29.3,27.1,26.0,24.5,21.3,17.6 HRMS(ESI-TOF):C 37 H 44 N4O7[M+H] + Calculated value: 657.3283, Measured value: 657.3291 Compound 35-1 [ka] To a solution of compound 21 (5.1 mg, 0.0091 mmol) and compound 33 (6.6 mg, 0.018 mmol) in DME (0.2 mL) was added Pd(PPh3)-4 (1.0 mg, 0.00091 mmol). The resulting yellow solution was added with saturated aqueous NaHCO3 (10 drops) with stirring, followed by heating to 100 °C. Upon completion (typically 2 h), H2O was added to the mixture and extracted with DCM. The combined organic layers were dried over MgSO4 and concentrated. The residue was dissolved in MeOH (0.1 mL) and treated with K2CO3 (10 mg). After vigorously stirring at room temperature for 30 min, the mixture was extracted with DCM and purified by PTLC (8% MeOH in DCM) to give compound 35 (4.4 mg, 0.0072 mmol, 79%) as product.

[0239] Physical state: white solid TLC:R f = 0.21 (10% MeOH in DCM, UV active, stains brown when stained with KMnO4) 1H-NMR(600 MHz, CDCl3)δ 7.78(d,J=8.0 Hz,2H),7.46(d,J=8.0 Hz,2H),7.12(br s,1H),5.63(s,1H),4.74(t,J=9.3 Hz,1H),4.62(d,J=11.9 Hz,1H),4.31(s,1H),4.14-4.03(m,1H),3.92(d,J=12.0 Hz,1H),3.59(q,J=6.5 Hz,1H),3.54-3.45(m,1H),3.42-3.30(m,1H),2.96(q,J=9.1 Hz,1H),2.85(t,J=12.9 Hz,1H),2.43-2.29(m,2H),2.29-2.20(m,2H),2.18-2.09(m,1H),2.05-1.87(m,6H),1.86-1.70(m,4H),1.68(td,J=7.2,4.5 Hz,2H),1.65-1.55(m,3H),1.54-1.44(m,1H),0.95(d,J=6.5 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 207.8,185.1,167.7,142.3,133.8,132.9,127.4,124.7,124.4,108.1,84.3,82.9,79.6,71.5,70.8,69.6,54 .7,54.0,53.8,42.4,40.3,37.1,34.9,34.6,33.0,32.7,32.3,29.8,29.4,28.9,27.2,25.5,25.1,24.5,17.8 HRMS (ESI-TOF):C 35 H 42 N4O6[M+H] + Calculated value: 615.3177, measured value: 615.3170 Compound 35-2

change

[0240] Physical state: white solid TLC:R f = 0.23 (10% MeOH in DCM, UV active, stains brown when stained with KMnO4) 1H-NMR(600 MHz, CDCl3)δ 7.69(d,J=8.1 Hz,2H),7.40(d,J=8.2 Hz,2H),6.31(s,1H),5.54(d,J=2.2 Hz,1H),4.56(d,J=12.0 Hz,1H),4.43(s,1H),4.21(dt,J=11.2,5.9 Hz,1H),4.10(dd,J=11.3,2.7 Hz,1H),3.90-3.83(m,1H),3.72-3.62(m,2H),3.30(q,J=6.4 Hz,2H),2.80(dd,J=15.9,5.1 Hz,1H),2.54(d,J=7.8 Hz,2H),2.39-2.32(m,1H),2.31-2.25(m,1H),2.13(d,J=15.9 Hz,1H),2.07-1.96(m,5H),1.92(dd,J=12.6,6.7 Hz,1H),1.89-1.83(m,1H),1.81(t,J=6.6 Hz,2H),1.75(td,J=12.3,5.1 Hz,2H),1.72-1.63(m,3H),1.61-1.51(m,2H),0.90(d,J=6.9 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 205.5,185.2,167.3,143.7,135.0,133.0,127.1,125.4,109.2,83.5,82.9,79.4,71.6,69.7,65.7, 55.0,52.1,43.2,40.8,37.8,35.1,34.5,33.8,32.6,32.3,29.9,29.5,29.3,27.2,25.7,24.8,17.8 HRMS (ESI-TOF):C 35 H 42 N4O6[M+H] + Calculated value: 615.3177, measured value: 615.3193 Compound 36 (Ref. 7)

change

[0241] Physical state: White powder TLC:R f = 0.46 (10% MeOH in DCM, stains brown when stained with KMnO4) 1 H-NMR(600 MHz,CDCl3)δ 7.39(d,J=7.4 Hz,2H),7.29(t,J=7.4 Hz,2H),7.24(t,J=7.4 Hz,1H),5.47(s,1H),4.53(d,J=12.1 Hz,1H),4.37(s,1H2),4.21(dt,J=11.2,6.0 Hz,1H),4.09(dd,J=11.2,2.6 Hz,1H),3.84(dd,J=15.3,9.0 Hz,1H),3.71-3.61(m,1H),3.55(d,J=12.1 Hz,1H),2.78(dd,J=15.9,5.1 Hz,1H),2.58-2.41(m,2H),2.39-2.24(m,2H),2.11(d,J=15.9 Hz,1H),2.01-1.93(m,2H),1.93-1.81(m,2H),1.76-1.70(m,1H),1.71-1.63(m,3H),1.55(dd,J=14.1,2.7 Hz,1H),0.91(d,J=6.9 Hz,3H) 13C-NMR(150 MHz,CDCl3)δ 206.1,140.4,135.4,128.5,127.6,125.2,123.4,109.2,83.4,79.6,71.7,65. 7,54.8,52.1,43.1,40.5,37.7,34.5,33.5,29.9,29.4,29.1,25.6,24.8,17.8 ( 13 One carbon (C=N) was missing in the C NMR HRMS(ESI-TOF):C 27 H 33 NO5[M+H] + Calculated value: 452.2432, Measured value: 452.2435 compound 37 [ka] Compound 37 (6.3 mg, 0.014 mmol) was prepared from compound 22 (9.0 mg, 0.020 mmol) by oxidation with DMP and reduction with NaBHCN. Purification was performed by PTLC (5% i-PrOH in DCM).

[0242] Physical state: White powder TLC:R f = 0.58 (10% MeOH in DCM, UV active, stains brown when stained with KMnO4) 1H-NMR(600 MHz, CDCl3)δ 6.16(dd,J=17.5,10.8 Hz,1H),5.21-5.16(m,1H),5.10(d,J=17.5 Hz,1H),4.96(d,J=10.8 Hz,1H),4.90(s,1H),4.52(t,J=4.2 Hz,1H),4.29(d,J=11.9 Hz,1H),4.20(dt,J=10.8,5.4 Hz,1H),3.88(dd,J=15.3,8.9 Hz,1H),3.75(d,J=12.4 Hz,1H),3.70(ddd,J=14.7,7.1,3.7 Hz,1H),2.81(dd,J=16.0,5.1 Hz,1H),2.45-2.26(m,4H),2.11(dd,J=16.1,1.9 Hz,1H),2.06(s,3H),1.99(dd,J=12.2,7.1 Hz,2H),1.93(dd,J=12.5,6.9 Hz,1H),1.88-1.63(m,5H),1.49(dd,J=12.9,2.8 Hz,1H),0.87(d,J=6.6 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 200.5,185.8,169.0,138.8,135.6,127.3,112.2,109.4,83.3,80.2,73.6,65.7,54.8(d,J=2.8 Hz),52.8,43.3,40.7,37.8,34.1,31.6,29.5,29.0,24.7,22.7,21.3,17.6 HRMS (ESI-TOF):C 25 H 33 NO6[M+H] + Calculated value: 444.2381, measured value: 444.2379 Compound 38

change

[0243] Physical state: white solid TLC:R f = 0.44 (10% MeOH in DCM, UV active, stains brown when stained with KMnO4) [α] 25 D :-8.0(c=0.1, CHCl3) 1H-NMR(600 MHz, CDCl3)δ 6.21(dd,J=17.5,10.8 Hz,1H),5.08(d,J=17.5 Hz,1H),5.04(s,1H),4.93(d,J=10.8 Hz,1H),4.78(dd,J=11.1,7.5 Hz,1H),4.47(d,J=12.0 Hz,1H),4.23(dt,J=10.4,6.0 Hz,1H),4.14(d,J=12.0 Hz,1H),4.08(dd,J=11.2,2.7 Hz,1H),3.75(dd,J=16.0,8.8 Hz,1H),3.63(ddd,J=16.0,10.3,7.0 Hz,1H),2.80(dd,J=14.6,11.1 Hz,1H),2.40-2.21(m,5H),2.15(dd,J=14.0,6.7 Hz,1H),2.01(dd,J=12.1,7.1 Hz,1H),1.92-1.82(m,2H),1.79(dd,J=12.5,6.7 Hz,1H),1.76-1.66(m,2H),1.58-1.49(m,2H),0.88(d,J=6.0 Hz,3H) 13 C-NMR(150 MHz, CDCl3)δ 204.7,184.8,139.1,135.3,128.2,111.6,108.2,83.9,79.3,71.9,70. 7,55.3,54.5,42.5,40.1,37.0,34.6,33.9,29.8,29.2,24.5,22.8,17.7 HRMS (ESI-TOF):C 23 H 31 NO5[M+H] + Calculated value: 402.2275, measured value: 402.2266 Compounds 39 and 40

change

[0244] Physical state: white solid TLC:R f =0.53 (10% MeOH in DCM, stains brown when stained with KMnO4) [α] 25 D :(c=0.1, CHCl3) 1H-NMR(600 MHz,CDCl3)δ 5.18(dd,J=11.3,2.8 Hz,1H),5.04(s,1H),4.66(br s,1H),4.48(s,1H),4.34(d,J=12.0 Hz,1H),4.21(dt,J=11.1,5.9 Hz,1H),3.94(dd,J=15.5,9.0 Hz,1H),3.86(d,J=12.3 Hz,1H),3.79-3.70(m,1H),2.74(dd,J=16.2,5.1 Hz,1H),2.60-2.46(m,2H),2.38-2.25(m,2H),2.18-2.05(m,3H),2.04(s,3H),2.01(dt,J=12.4,6.3 Hz,1H),1.88-1.78(m,3H),1.78-1.66(m,2H),1.52(dd,J=13.6,2.9 Hz,1H),0.88(d,J=6.7 Hz,3H) 13 C-NMR(150 MHz,CDCl3)δ 200.0,169.1,148.5,117.9,109.5,83.6,80.0,72.8,65.6,55.0,52.0,43.4,39.6,37.8,34.0,31.5,29.6,28.8,26.4,24.7,21.0,17.6 (Two carbons (C=N and CF3) 13 C NMR was missing but is shown in compound 39) HRMS(ESI-TOF):C 24 H 30 F3NO9S[M+H] + Calculated value: 566.1666, Measured value: 566.1641 [ka] Compound 40 (11.1 mg, mmol) was obtained by treating 39 (15.5 mg, mmol) with MeOH (1.0 ml) and K2CO3 (58 mg).

[0245] Physical state: white solid TLC:R f =0.50 (10% MeOH in DCM, stains brown when stained with KMnO4) [α]25 D :-15.0 (c = 0.1, CHCl3) 1 1H-NMR (600 MHz, CDCl3) δ 5.16 (s, 1H), 4.52 (d, J = 12.0 Hz, 1H), 4.48 (s, 1H), 4.19 (dt, J = 11.1, 5.8 Hz, 1H), 4.08 - 4.00 (m, 1H), 3.92 (dd, J = 15.5, 9.0 Hz, 1H), 3.81 (d, J = 12.0 Hz, 1H), 3.78 - 3.68 (m, 1H), 2.71 (dd, J = 16.1, 5.1 Hz, 1H), 2.63 - 2.52 (m, 1H), 2.50 - 2.42 (m, 1H), 2.32 - 2.20 (m, 2H), 2.15 - 2.04 (m, 2H), 2.04 - 1.93 (m, 2H), 1.84 (dq, J = 13.1, 8.9, 6.2 Hz, 3H), 1.73 (dt, J = 12.2, 6.9 Hz, 1H), 1.66 (td, J = 12.7, 7.6 Hz, 1H), 1.52 (dd, J = 13.2, 2.7 Hz, 1H), 0.89 (d, J = 6.5 Hz, 3H) 13 13C-NMR (150 MHz, CDCl3) δ 204.8, 148.3, 118.7, 118.6 (q, J C-F = 320 Hz), 109.3, 83.6, 79.0, 70.6, 65.6, 54.8, 52.0, 43.2, 39.7, 37.7, 34.2, 33.4, 29.9, 29.4, 28.7, 26.2, 24.7, 17.7 HRMS (ESI-TOF): C 22 H 28 F3NO8S [M + H] + Calculated value: 524.1561, Measured value: 524.1547 References 1. Synthesis of Medium-Sized Cyclic Amines by Selective Ring Cleavage of Sulfonylated Bicyclic Amines. Iradier, F.; Gomez Arrayas, R.; Carretero, J. C. Org. Lett. 2001, 3, 2957 - 2960. 2.Microwave-Assisted Palladium-Catalyzed Allylation of β-Enaminones.Erray,I.;Rezgui,F.;Oble,J.;Poli,G.Synlett 2014,25,2196-2200. 3.Activation of Mg Metal for Safe Formation of Grignard Reagents on Plant Scale.Organic Process Research&Development 2002,6,906-910. Biological assays 1) Portimine A and its functional analogs exhibit stereoselective acute toxicity in cancer cells (Figure 1). (A) PA and Ph-PA have similar cytotoxicity in breast cancer cell lines (HCC1806) and leukemia cancer cell lines (Jurkat) after 36 h of treatment, whereas PB and ePA show minimal effects. (B) Structures of portimine-related analogs: phenyl-portimine A (Ph-PA, 36) and epiportimine A (ePA, 38). Structures of fully functionalized portimine A-based epimeric probe pairs: portimine A-diazirine-alkyne (PA-DA, 35-2) and epiportimine A-diazirine-alkyne (ePA-DA, 35-1). (C) Cell viability analysis of portimine A and analogs. Jurkat cells were treated with increasing concentrations of portimine A and analogs for 24 h. All data presented are the average of biologically replicated experiments (n=3). (D) Immunoblots for caspase 3 show that PA, PA-DA, and Ph-PA induce cell apoptosis, but ePA, ePA-DA, and PB do not. Jurkat cells were treated with portimine A or analogs at 10 nM for 12 hours. PA and Ph-PA have an IC50 of approximately 2 nM for Jurkat cells after 12 hours. PA and Ph-PA are substantially less toxic to healthy PBMCs compared with the cancer T cell line Jurkat. The structurally related negative controls ePA and PB are nontoxic to all cell types at the current concentration range. PBMCs (25 kJ / well), Jurkat cells (10 kJ / well). Compound concentrations were increased from 0.23 pM to 1 uM, 4-fold per gradient, and incubated for 12 hours in Wesley-derived primary B cell medium (heat inactivated) for PBMCs and regular RPMI medium for Jurkats.

[0246] 2) Portimine A induces cell cycle arrest in Jurkat cells. Cell cycle analysis in Jurkat cells (Figure 2). Jurkat cells were treated with 10 nM of the indicated compound for 24 hours and then analyzed by flow cytometry using propidium iodide to identify different cell phases. Treatment conditions: Jurkat (50 kJ / well); Portimine A concentrations: 0.007 nM to 480 nM; washout time points: 0.5, 2, 4, 6, 12, and 24 hours. Cells were washed once and suspended in PA-free RPMI medium. Cells were treated for a total of 36 hours. Cell viability was measured using the Cell Titerglo assay. An exposure-dependent decrease in IC50 was observed; compound removal after 30 minutes resulted in an approximately 5-fold decrease in IC50; IC50 after 30 minutes of exposure was approximately 5 nM; at 4 hours it was approximately 2 nM. Overall, this suggests a rapid-acting cytotoxic mechanism.

[0247] 3) Portimine A and analogs do not induce apoptosis or affect cell viability in freshly isolated human PBMCs (Figure 3). (A) PA and Ph-PA demonstrate low toxicity to healthy PBMCs compared with Jurkat cells. Both ePA and PB were non-toxic to both cells in the pM-nM concentration range. (B) Immunoblot analysis of caspase-3 in Jurkat and PBMCs under the indicated conditions. Both PA and PA-DA induced caspase-3 cleavage in a dose-dependent manner in Jurkat cells but not in human PBMCs. PTMA-DA was found to have similar activity to the PTMA → appropriate target ID probe; epi-PTMA-DA and epi-PTMA-Ac-DA had no activity in the concentration range, similar to the epi-PTMA → useful inactive probe.

[0248] 4) Mouse pharmacokinetic and fast-acting in vitro cell-based target engagement properties based on washout of portimine compounds (Figure 4). (A) Mice were administered portimine intraperitoneally (ip) and orally (po) (n=3). (B) Washout experiments performed in Jurkat and MC38 cells showed IC 50(C) Kaplan-Myer survival curves of WT MC38 tumor-bearing mice (n=6) after treatment with PA, as shown in (D). 3 Mice were euthanized when the concentration exceeded 0.05. Statistical analysis was performed using ANOVA followed by multiple comparisons. *p≤0.05. Treatment conditions: Jurkat (60 μM per condition); PA-DA / ePA-DA concentration: 0.5 μM, PA or ePA: 5 μM; 30-minute incubation with probe with or without competitor (PA, ePA); 20-minute UV irradiation (0.5 μM PA-DA was added to the no-UV condition); standard pull-down and biotin enrichment followed by Western blot analysis (elution conditions: 2x sample buffer + 500 μM biotin, boiling for 20 minutes); primary antibodies: NMD3 (ProteinTech, 1:150), actin (BioRad, 1:10,000). Conclusion: NMD3 can be selectively bound by portimine A; the interaction between NMD3 and portimine A is noncovalent; note: PA-DA binding to recombinantly overexpressed NMD3 in cells was not observed, but binding was observed only to endogenous NMD3. This may suggest that PA binds to NMD3 only when it is in the endogenous complex.

[0249] 5) Chemical proteomic analysis reveals that NMD3 is a target of portimine A (Figure 5). Chemical proteomic profiling using PA-DA in Jurkat cells (A, B, C, G) and HCC1806 cells (D, E, F, H) indicates that NMD3 is a major candidate target of portimine A. Volcano plots in panels A and D show protein competition in cells treated with the active photoaffinity probe PA-DA (500 nM) and DMSO or the active competitor portimine A (PA, 4 μM). The dotted line indicates a fold-change threshold of <4, p-value >0.05. Volcano plots in panels B and E show protein enrichment by the active probe PA-DA relative to the inactive probe ePA-DA (500 nM). The dotted line indicates a fold-change threshold of <2, p-value >0.05. Volcano plots in panels C and F show a comparison between the active competitor compound PA and the inactive control competitor ePA (4 μM, PA-DA + PA / PA-DA + ePA). The dotted lines indicate the threshold for a fold change of >4 and a p-value >0.05. (I, J) Confirmation of PA-DA engagement of NMD3 in Jurkat and HCC1806 cells. UV-dependent labeling of endogenous NMD3 by PA-DA was blocked by PA but not by ePA, and was virtually unlabeled by the inactive probe ePA-DA or PA-DA without UV irradiation. Treatment conditions: Jurkat (5 μM / well). Portimine A concentration: 2 nM–1000 nM; cells were treated for a total of 12 h. Conclusion: PA induces cell apoptosis in a dose-dependent manner; ePA does not induce significant caspase-3 activation within this concentration range.

[0250] 6) Portimine A down-regulated NMD3 at the protein level but up-regulated its mRNA level in cells, as well as several essential apoptotic proteins (Figure 6). (A) NMD3 and Bcl-xl were down-regulated after PA treatment, but not by proteasome-mediated degradation. Proteolysis of p53, Mcl1, Bid, Bim, and Noxa could be blocked by MG132 or epoxomicin. (B) NMD3 mRNA was increased after PA treatment but not by ePA control compounds. Jurkat cells were treated with the indicated compounds for 24 hours, then fixed and stained with propidium iodide. Conclusion: S-phase entry was inhibited in Jurkat cells treated with 10 nM PA / PADA; PA / PADA does not affect G2M / sub-G1 (mainly apoptotic cells).

[0251] 7) NMD3 is downregulated by PA in Jurkat cells in a time- and dose-dependent manner (Figure 7). (A) Immunoblot showing reduced NMD3 levels in shNMD3 Jurkat and HeLa cells. (B) PA has less toxic effects in cells in which NMD3 is knocked down (shNMD3) compared to shCtrl cells. Jurkat cells were treated with 12.5 nM PA for 12 or 24 hours. Jurkat and HeLa cells were treated with 5 nM or 150 nM PA for 24 hours. Data represent the mean ± SD of biologically replicated experiments (n = 3). (C) Polysome profiling reveals protein translation inhibition by portimine A. Jurkat cells were treated with DMSO or 50 nM portimine A for 6 hours. Cell lysates were fractionated on a sucrose gradient (5-50%), and each fraction was analyzed by Western blot. eIF6 levels increase in the 60S subunit fraction and decrease in the ribosome-free fraction after PA treatment. RPS6, a 40S and polysome subunit marker, decreases in the polysome fraction after PA treatment. Results are representative of three independent experiments. (D) Quantification of the 60S:80S and 80S:polysome ratios from the polysome profiling assay shown in Figure 7C. (E) Quantification of the relative protein distribution in the fractions shown in Figure 7C. Relative protein levels in each fraction were normalized to the peak fraction of the indicated protein from DMSO cells and plotted. Data represent the mean ± SD of biologically replicated experiments (n = 3). Statistical analysis was performed using a multiple independent Student's t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005; ****p ≤ 0.0001. Treatment conditions: Jurkat (5 μM per condition); cells (1 μM / mL) were incubated with the inhibitors for 6, 12, and 24 hours, respectively. Conclusions: NMD3 is downregulated by PA treatment at 10 nM at 12 hours; KPT-330 downregulates NMD3 at higher concentrations (above 50 nM) at 24 hours; XPO1 levels do not appear to be affected by PA; p53 can be downregulated by porcine A treatment; similar to KPT-330, PA does not affect Bcl-2 levels at 6 / 12 hours.

[0252] 8) Downregulation of NMD3 is not due to proteasome-mediated degradation (Figure 8). Treatment conditions: Jurkat (10 M per condition); PA concentration: 10 nM; cells (1 M / mL) are preincubated with MG132 or epoxomicin for 2 hours. Cells are further incubated with PA with or without MG132 or epoxomicin for 12 hours. Conclusion: Downregulation of NMD3 is not due to proteasome-mediated degradation; it may be the result of transcriptional inhibition, similar to XPO1 inhibitors.

[0253] 9) Portimine A exhibits potent antitumor activity in humans and syngeneic mouse tumor models (Figure 9). (A) Systemic Portimine A in an MC38 syngeneic tumor xenograft model; (B) Systemic Portimine A in an HT-1080 tumor xenograft model. Preliminary cell-based target engagement studies based on compound washout demonstrate that PA acts rapidly with less than 1 hour of exposure and minimal loss of efficacy. Cmax-driven pharmacology, along with ease of synthesis and availability of materials, enabled evaluation of PA in both syngeneic (MC38 colon carcinoma) and human metastatic mesenchymal (HT-1080 fibrosarcoma) mouse tumor xenograft models. Potent antitumor activity was observed for PA at doses of 0.3 or 1.0 mg / kg after systemic administration (ip delivery) of tolerated doses. Metabolic stability presents an opportunity for additional intellectual property positions.

[0254] Wide-area cell line profiling data.

[0255] Portimine A exhibits cytotoxic activity across a wide range of cancer cell types at pM to single-digit nM concentrations A panel of human and mouse cancer cell types used to assess the breadth of PA activity, including multiple treatment-resistant metastatic mesenchymal tumor cell lines and primary glioblastoma patient-derived metastatic stem / tumor-initiating cell populations. [Table 3] The foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding. It will be apparent to those skilled in the art that changes and modifications may be practiced within the scope of the appended claims. It is therefore to be understood that the foregoing description is intended to be illustrative, and not limiting. The scope of the present disclosure should, therefore, be determined not with reference to the above description, but instead with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled.

[0256] This application refers to various issued patents, published patent applications, journal articles, and other publications, each of which is incorporated herein by reference.

Claims

1. Portimine A (1), 【Chemistry 1】 1. A process for preparing compound (10), comprising Diels-Alder cycloaddition of compound (8): 【Chemistry 2】 。

2. 10. The process of claim 1, further comprising: forming compound (11) by alcohol oxidation of compound (10), followed by Grignard addition, and Dess-Martin oxidation: 【Transformation 3】 。

3. 3. The process of claim 2, further comprising cyclizing compound (11) to form compound (12): 【Chemistry 4】 。

4. A process for preparing compound (13) from (S)-solketal, comprising the steps of: 【Transformation 5】 。

5. 5. The process of claim 4 further comprising the steps of: 【Transformation 6】 。

6. 6. The process of claim 5 further comprising the steps of: 【Transformation 7】 。

7. 4. The process of claim 3, further comprising transmetallation of compound (13) and conjugate addition to compound (12) to form compound (7): 【Transformation 8】 。

8. 8. The process of claim 7, further comprising converting compound (7) to an N-protected imine compound (31): 【Chemistry 9】 。

9. 9. The process of claim 8, further comprising forming compound (15) by Mo-catalyzed ring-closing alkyne metathesis: 【Chemistry 10】 。

10. 10. The process of claim 9, further comprising ring formation and oxidation of compound (15) to form compound (16): 【Chemistry 11】 。

11. 11. The process of claim 10, further comprising reducing compound (16) to compound (32): 【Chemistry 12】 。

12. 12. The process of claim 11, further comprising forming compound (19) by ring-chain tautomerization of compound (32): 【Chemistry 13】 。

13. 13. The process of claim 12, further comprising silylation and oxidation of compound (19) to form compound (20): 【Chemistry 14】 。

14. 14. The process of claim 13, further comprising converting compound (20) to compound (21): 【Chemistry 15】 。

15. 15. The process of claim 14, further comprising Pd-catalyzed vinylation of compound (21) to form compound (22): 【Chemistry 16】 。

16. 16. The process of claim 15, further comprising Dess-Martin oxidation of compound (22) followed by hydrolysis to form compound (2): 【Chemistry 17】 。

17. 17. The process of claim 16, further comprising reducing compound (2) to form portimine A (1): [Chemistry 18] 。

18. Portimine A (1): 【Chemistry 19】 1. A process for preparing a compound of formula (I) comprising the reduction of compound (2) to form portimine A (1): 【Chemistry 20】 。

19. Formula (2): 【Chemistry 21】 or a scalemic or racemic mixture thereof.

20. 20. A process for preparing the compound of claim 19, comprising Dess-Martin oxidation of compound (22) followed by hydrolysis to form compound (2): 【Chemistry 22】 。

21. Formula (22): 【Chemistry 23】 or a scalemic or racemic mixture thereof.

22. 22. A process for preparing the compound of claim 21, comprising Pd-catalyzed vinylation of compound (21) to form compound (22): 【Chemistry 24】 。

23. Formula (21): 【Chemistry 25】 or a scalemic or racemic mixture thereof.

24. 24. A process for preparing the compound of claim 23, comprising converting compound (20) to compound (21): 【Chemistry 26】 。

25. Formula (20): 【Chemistry 27】 or a scalemic or racemic mixture thereof.

26. 26. A process for preparing the compound of claim 25, comprising silylation and oxidation of compound (19) to form compound (20): 【Chemistry 28】 。

27. Formula (19): 【Chemistry 29】 or a scalemic or racemic mixture thereof.

28. 28. A process for preparing the compound of claim 27, comprising ring-chain tautomerization of compound (32) to form compound (19): 【Transformation 30】 。

29. Formula (32): 【Chemistry 31】 or a scalemic or racemic mixture thereof.

30. 30. A process for preparing the compound of claim 29, comprising reducing compound (16) to compound (32): 【Chemistry 32】 。

31. Formula (16): 【Transformation 33】 or a scalemic or racemic mixture thereof.

32. 32. A process for preparing the compound of claim 31, comprising ring formation and oxidation of compound (15) to form compound (16): 【Transformation 34】 。

33. Formula (15): 【Chemistry 35】 or a scalemic or racemic mixture thereof.

34. 34. A process for preparing the compound of claim 33, comprising forming compound (15) by Mo-catalyzed ring-closing alkyne metathesis: 【Transformation 36】 。

35. Formula (31): 【Chemistry 37】 or a scalemic or racemic mixture thereof.

36. 36. A process for preparing the compound of claim 35, comprising converting compound (7) to N-protected imine compound (31): 【Transformation 38】 。

37. Formula (7): 【Chemistry 39】 or a scalemic or racemic mixture thereof.

38. 38. A process for preparing the compound of claim 37, comprising transmetallation of compound (13) and conjugate addition with compound (12) to form compound (7): 【Chemistry 40】 。

39. Formula (28): 【Chemistry 41】 or a scalemic or racemic mixture thereof.

40. The following steps: 【Chemistry 42】 40. A process for preparing the compound of claim 39 from (S)-solketal, comprising:

41. Formula (29): 【Chemistry 43】 or a scalemic or racemic mixture thereof.

42. The following steps: 【Chemistry 44】 42. A process for preparing the compound of claim 41 from compound (28), comprising:

43. Formula (30): 【Chemistry 45】 or a scalemic or racemic mixture thereof.

44. The following steps: 【Chemistry 46】 44. A process for preparing the compound of claim 43 from compound (28), comprising:

45. Formula (13): 【Chemistry 47】 or a scalemic or racemic mixture thereof.

46. The following steps: 【Chemistry 48】 46. ​​A process for preparing the compound of claim 45 from (S)-solketal, comprising:

47. Formula (12): 【Chemistry 49】 or a scalemic or racemic mixture thereof.

48. 48. A process for preparing the compound of claim 47, comprising cyclizing compound (11) to form compound (12): [Transformation 50] 。

49. Formula (11): 【Chemistry 51】 or a scalemic or racemic mixture thereof.

50. 50. A process for preparing the compound of claim 49, comprising alcohol oxidation of compound (10), followed by Grignard addition and Dess-Martin oxidation to form compound (11): 【Chemistry 52】 。

51. Formula (10) 【Chemistry 53】 or a scalemic or racemic mixture thereof.

52. 52. A process for preparing the compound of claim 51, comprising Diels-Alder cycloaddition of compound (8) to form compound (10): 【Chemistry 54】 。

53. Formula (8) 【Transformation 55】 or a scalemic or racemic mixture thereof.

54. 54. A process for preparing a compound of claim 53 having the formula of compound (8), comprising converting compound (23) to form compound (8): 【Transformation 56】 。

55. 1. A process for preparing portimine A (1), comprising: 【Chemistry 57】 The process comprises: i) Reduction of compound (2) to form portimine A (1): 【Chemistry 58】 and, ii) Dess-Martin oxidation of compound (22) followed by hydrolysis to form compound (2): 【Chemistry 59】 and, iii) Pd-catalyzed vinylation of compound (21) to form compound (22): 【Transformation 60】 and, iv) Step of converting compound (20) into compound (21): 【Chemistry 61】 and, v) silylation and oxidation of compound (19) to form compound (20): 【Transformation 62】 and, vi) forming compound (19) by ring-chain tautomerization of compound (32): 【Transformation 63】 and, vii) Reduction step of compound (16) to compound (32): 【Chemistry 64】 and, viii) ring formation and oxidation of compound (15) to form compound (16): 【Transformation 65】 and, ix) Mo-catalyzed ring-closing alkyne metathesis to form compound (15): 【Chemical Formula 66】 and, x) Conversion of compound (7) into N-protected imine compound (31): 【Transformation 67】 and, xi) transmetallation of compound (13) and conjugate addition to compound (12) to form compound (7): 【Transformation 68】 and, xii) cyclization of compound (11) to form compound (12): 【Transformation 69】 and, xiii) Alcohol oxidation of compound (10) followed by Grignard addition and Dess-Martin oxidation to form compound (11): 【Transformation 70】 and, xiv) Diels-Alder cycloaddition of compound (8) to form compound (10): 【Chemistry 71】 and, xv) Step of converting compound (23) into compound (8): 【Chemistry 72】 and, a process including.

56. Formula (33-1) 【Transformation 73】 A compound having the structure:

57. The following steps: 【Chemistry 74】 A process for preparing the compound according to claim 55 from compound (33-0), comprising:

58. Formula (33-2) 【Chemistry 75】 A compound having the structure:

59. The following steps: 【Transformation 76】 A process for preparing the compound of claim 57 from compound (33-0), comprising:

60. Formula (34): 【Chemical Formula 77】 or a scalemic or racemic mixture thereof.

61. The following steps: 【Transformation 78】 61. A process for preparing the compound of claim 60 from compound (21), comprising:

62. Formula (35-1): 【Transformation 79】 or a scalemic or racemic mixture thereof.

63. The following steps: 【Chemistry 80】 63. A process for preparing the compound of claim 62 from compound (21), comprising:

64. Formula (35-2): 【Chemistry 81】 or a scalemic or racemic mixture thereof.

65. The following steps: 【Chemistry 82】 A process for preparing the compound according to claim 64 from compound (35-2), comprising:

66. Formula (36): 【Chemistry 83】 or a scalemic or racemic mixture thereof.

67. The following steps: 【Chemical 84】 Compound (39) 67. A process for preparing the compound of claim 66.

68. Formula (37): 【Chemical 85】 or a scalemic or racemic mixture thereof.

69. The following steps: 【Chemical 86】 69. A process for preparing the compound of claim 68 from compound (22), comprising:

70. Formula (38) 【Transformation 87】 A compound having the structure:

71. The following steps: 【Chemical 88】 71. A process for preparing the compound of claim 70 from compound (21), comprising:

72. Formula (39) 【Chemistry 89】 A compound having the structure:

73. The following steps: [Chemical 90] 73. A process for preparing the compound of claim 72 from compound (21), comprising:

74. Formula (40): 【Chemistry 91】 or a scalemic or racemic mixture thereof.

75. The following steps: 【Chemistry 92】 75. A process for preparing the compound of claim 74 from compound (39), comprising:

76. 1. A process for preparing various analogs of Portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, comprising the steps of: 【Chemistry 93】 (In the formula, R is (C ６ ~C １０ ) aryl, (C ２ ~C ６ ) alkenyl or (C ５ ~C １２ ) heteroaryl, each aryl being —NH(CH ２ ) ２ C(-N=N-)(CH ２ ) ２ optionally substituted with (C≡CH); R' is H or Ac. The process includes:

77. The compound is 【Chemical 94】 77. A compound prepared by the process of claim 76 selected from the group consisting of:

78. 1. A process for preparing various analogs of Portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, comprising the steps of: 【Chemical 95】 (In the formula, R １ is H, Me, Boc, or TES; R ２ is H, halo, (C ６ ~C １０ ) aryl, (C ２ ~C ６ ) alkenyl or (C ５ ~C １２ ) heteroaryl, each aryl being —NH(CH ２ ) ２ C(-N=N-)(CH ２ ) ２ optionally substituted with (C≡CH); R' is H or Ac. The process includes:

79. The compound is 【Chemistry 96】 79. A compound prepared by the process of claim 78 selected from the group consisting of:

80. 1. A process for preparing various analogs of Portimine A, including enantiomers, scalemic and racemic mixtures, and pharmaceutically acceptable salts thereof, comprising the steps of: 【Chemistry 97】 (In the formula, R １ is H, Me, Boc, or TES; R ２ is H, halo, (C ６ ~C １０ ) aryl, (C ２ ~C ６ ) alkenyl or (C ５ ~C １２ ) heteroaryl, each aryl being —NH(CH ２ ) ２ C(-N=N-)(CH ２ ) ２ optionally substituted with (C≡CH); R' is H or Ac. The process includes:

81. The compound is 【Chem.98】 81. The compound prepared by the process of claim 80, selected from the group consisting of:

82. 81. The process of any one of claims 76, 78, or 80, wherein the portimine A analogue product is useful for forming antibody-drug conjugates (ADCs) for treating cancer.

83. 82. A method of forming an antibody-drug conjugate (ADC) for treating cancer, comprising the use of a compound according to any one of claims 76 to 81.

84. 82. A method for treating cancer, comprising administering to a patient in need thereof a pharmaceutically effective amount of any one of claims 76 to 81 capable of forming an antibody-drug conjugate (ADC).

85. Any process, compound, method, or mixture described herein.

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