Agelastatin A Derivatives and Related Methods

Abstract

Agelastatin compounds, methods of making the agelastatin compounds, and methods of using the agelastatin compounds.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 63 / 187,297, filed May 11, 2021, the entire contents of which are expressly incorporated by reference herein.

[0002] Government License Rights Statement This invention was made with Government support under Grant Nos. R37 GM052964 and R35 GM134910 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] background Agelastatin A (AglA, Figure ​ (1),1) is one of the most prominent members of the pyrrole-2-aminoimidazole (P-2-AI) family of marine alkaloids, which has attracted great interest from both synthetic chemists and biologists due to its unique structure and wide range of biological activities. Isolated by Pietra in 1993, this tetracyclic marine alkaloid has demonstrated therapeutic potential as a drug indicator in the treatment of various cancers, including leukemia, breast cancer, lung cancer, and glioblastoma. In one study, AglA demonstrated the inhibition of osteopontin (OPN, encoded by SPP1), whose overexpression is believed to be associated with malignant transformation, cancer progression, and metastasis in various cancers. Furthermore, OPN is also significantly expressed by primary brain tumors, such as glioblastoma multiforme, astrocytoma, and primary central nervous system (CNS) lymphoma, making AglA particularly attractive for the treatment of brain tumors due to its excellent blood-brain barrier permeability. In addition, AglA also inhibits the expression of glycogen synthase kinase-3β.

[0004] Not surprisingly, since its discovery, extensive research has been carried out into the synthesis of AglA and the development of its structure-activity-relationship (SAR) profile towards cancer cell lines. Numerous total or formal syntheses have been reported, featuring many elegant synthetic strategies. These studies have led to an extensive understanding of the structural requirements for bioactivity and the development of several novel derivatives with improved potency. Recently, the cellular targets of AglA have been reported and a mechanism of action has been elucidated that explains its potent anticancer effects. In HeLa cells, AglA binds to the peptidyl transfer center (PTC) of the ribosome, resulting in protein synthesis inhibition and ultimately leading to apoptosis. Moreover, the X-ray structure of AglA in complex with the S80 subunit of the yeast ribosome has opened the possibility of designing novel AglA-based drug indicators. In detail, the X-ray structure revealed numerous important hydrogen bonds and n-n stacking interactions and rare halogen-n interactions. A biomimetic synthesis of AglA was also described, providing a concise entry into this class of alkaloids and supporting the proposed biosynthesis from acyclic precursors.

[0005] Despite advances in the synthesis of agelastatin derivatives, there remains a need for new synthetic methods for making agelastatin derivatives and for new agelastatin derivatives having improved therapeutic properties. The present disclosure seeks to meet these needs and further provides related advantages.

[0006] overview The present disclosure provides agelastatin compounds (eg, agelastatin A derivatives) and methods of making and using the compounds.

[0007] In one aspect, the present disclosure provides a compound of formula (I):

[0008] [ka]

[0009] (In the formula, R1 is selected from the group consisting of H, F, Cl, and Br; R2 is selected from the group consisting of H, F, Cl, and Br; R3 is selected from the group consisting of Br, CF3, SF5, SO2CF3, SO2CH3, CN, and NO2; R4 is selected from the group consisting of H, OH, F, Cl, Br, and CN; and R5 is H) or a stereoisomer, racemate, or pharma- ceutically acceptable salt thereof.

[0010] In another aspect, the disclosure provides a method of making a compound of formula (I). In certain embodiments, the method comprises converting a compound of formula (A) to a compound of formula (I) via a compound of formula (B).

[0011] [ka]

[0012] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a 7-hydroxyagelastatin compound described herein, or a stereoisomer, racemate, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0013] In another aspect of the present disclosure, a method for treating cancer is provided.In certain embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of 7-hydroxyagelastatin compound as described herein, or its stereoisomer, racemate, or pharmaceutically acceptable salt.

[0014] In a further aspect of the present disclosure, a method for inhibiting protein synthesis through interaction with the peptidyl transferase center of ribosome in a subject is provided.In certain embodiments, the method comprises administering to a subject an effective amount of 7-hydroxyagelastatin compound as described herein, or its stereoisomer, racemate, or pharmaceutically acceptable salt, in an amount effective to inhibit protein synthesis through interaction with the peptidyl transferase center of ribosome.

[0015] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 illustrates the structures of agelastatins (AglA) A-E and the unnatural derivative, 7-hydroxyAglA (7a), revealing the hidden pseudo-C2 symmetry of the bis-carbinolamine, bis-hydroxycyclopentane core. [Figure 2A] Illustrates docking of 7-hydroxy AglA (7a) with the X-ray structure of the AglA-yeast 80S ribosome complex using MOE. Panoramic view of the binding site showing a possible, but weak (3.52 Å) hydrogen bond between the C7-hydroxyl and U2875 (foreground, rRNA; background, protein). [Figure 2B] Separation of the pyrimidinedione of 7-hydroxy AglA (7a) and U2875. [Diagram 3] FIG. 10 Schematic illustrating the conformational exploration of AglA and 7-OH AglA by MOE and DFT calculations. [Figure 4] FIG. 1 is a schematic diagram of the retrosynthetic analysis of AglA(1) based on a cryptic C2 symmetric element that allows for late-stage pyrrole mutations upon addition of the C7-hydroxyl. [Diagram 5] FIG. 1 is a schematic representation of the stability study of pyrrole-derived carbinolamines 18a-18c. [Figure 6-1] FIG. 14. Schematic of the synthesis of dibromo 7-hydroxy AglA 24 (inset: X-ray structure of bis-carbinolamine 22). [Figure 6-2] FIG. 14. Schematic of the synthesis of dibromo 7-hydroxy AglA 24 (inset: X-ray structure of bis-carbinolamine 22). [Figure 7] FIG. 1 is a schematic representation of the attempt to reach the AglA core structure through an intramolecular Mitsunobu reaction. [Figure 8] FIG. 1 is a schematic diagram of the synthesis of AglA via base-promoted aza-Michael ring closure. [Figure 9] Schematic diagram of the synthesis of 13-nitroAglA (37). [Figure 10] 1 is a table summarizing the cytotoxicity (EC50, μM) of AglA derivatives against various cancer cell lines. [Figure 11] FIG. 1 illustrates the conformational exploration of 7-OH des-bromoAglA (24) by MOE and subsequent DFT calculations to determine the relative energies. [Figure 12] FIG. 1 is a schematic diagram of a retrosynthetic approach for introducing representative substituents R1 and R2 into agelastatin A compounds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description The present disclosure provides agelastatin compounds (eg, agelastatin A derivatives) and methods of making and using the compounds. In one aspect, the present disclosure provides a compound of formula (I):

[0018] [ka]

[0019] (In the formula, R1 is selected from the group consisting of H, F, Cl, and Br; R2 is selected from the group consisting of H, F, Cl, and Br; R3 is selected from the group consisting of Br, CF3, SF5, SO2CF3, SO2CH3, CN, and NO2; R4 is selected from the group consisting of H, OH, F, Cl, Br, and CN; and R5 is H) or a stereoisomer, racemate, or pharma- ceutically acceptable salt thereof.

[0020] In one embodiment, the disclosure provides a compound of formula (I), wherein R1, R2, R4, and R5 are H, and R3 is Br.

[0021] In another embodiment, the disclosure provides a compound of formula (I), wherein R1, R2, and R5 are H, and R3 and R4 are Br.

[0022] In a further embodiment, the disclosure provides a compound of formula (I), wherein R1-R5 are hydrogen.

[0023] In other embodiments, the disclosure provides compounds of formula (I), wherein R1 and R2 are independently selected from hydrogen and fluoro, hydrogen and chloro, or hydrogen and bromo, and R3-R5 are hydrogen. In certain of these embodiments, R1 is hydrogen and R2 is fluoro, and in other of these embodiments, R1 is fluoro and R2 is hydrogen (i.e., both are diastereomeric).

[0024] In another aspect, the disclosure provides methods of making compounds of formula (I). The methods described herein provide for the introduction of substituents R1-R5 into the agelastatin scaffold.

[0025] Substituents R1 and R2 can be incorporated into the product agelastatins by elaboration of furan (e.g., 15) or cyclopentanone (e.g., 12-14) intermediates. See Figures 4-6 and 12. A retrosynthetic analysis for the introduction of fluoro atoms at R1 and / or R2 is shown in Figure 12.

[0026] Introduction of mono- or dihalogens, including fluoro, chloro, or bromo, at R1 and / or R2 is accomplished with an electrophilic source of fluorine such as Selectfluor, or in the case of chloro or bromo, N-bromosuccinimide, N-chlorosuccinimide, chlorine (Cl2), bromine (Br2), or other electrophilic sources of Cl or Br, by halogenation of 4-cyclopentene-1,3-dione as shown in Figure 12, or to a greater extent, halogenation of cyclopentanone as shown in the synthesis described herein. For example, for fluorinated derivatives, the dione is treated with a base (e.g., sodium hydride, lithium diisopropylamide) to form an enolate, which is then reacted with Selectfluor to introduce one or two fluoro groups.

[0027] The present disclosure provides monohalo diastereomers:

[0028] [ka]

[0029] During the ceremony, (1) X1 is F and X2 is H; (2) X1 is H and X2 is F; (3) X1 is Cl and X2 is H; (4) X1 is H and X2 is Cl; (5) X1 is Br and X2 is H; and (6) X1 is H and X2 is Br; R3 to R5 are as described above.

[0030] Substituents R3, R4 and R5 can be incorporated into the product agelastatin by elaboration of a pyrrole (e.g., 11, 16) intermediate. See Figures 4-6, 8 and 12.

[0031] Details of the synthetic schemes are shown in Figures 4-6, 8, 9, and 12 and described in the experimental procedures below.

[0032] In certain embodiments, the method comprises converting a compound of formula (A) to a compound of formula (I) via a compound of formula (B).

[0033] [ka]

[0034] In certain embodiments of the method, the compound of formula (A) is

[0035] [ka]

[0036] to produce an amide (a):

[0037] [ka]

[0038] resulting in; The amide (a) is converted to a compound of formula (B) followed by ring closure to give the 7-hydroxy compound (b):

[0039] [ka]

[0040] resulting in; The 7-hydroxy compound (b) is converted to a compound of formula (I) by treatment with aqueous acid, During the ceremony, P is an alcohol protecting group; R is a methyl group; R1 is selected from the group consisting of H, F, Cl, and Br; R2 is selected from the group consisting of H, F, Cl, and Br; R3 is selected from the group consisting of Br, CF3, SF5, SO2CF3, SO2CH3, CN, and NO2; R4 is selected from the group consisting of H, OH, F, Cl, Br, and CN; and R5 is H.

[0041] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a 7-hydroxyagelastatin compound described herein, or a stereoisomer, racemate, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0042] In another aspect of the present disclosure, a method for treating cancer is provided.In certain embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of 7-hydroxyagelastatin compound as described herein, or its stereoisomer, racemate, or pharmaceutically acceptable salt.In certain embodiments, the cancer is breast cancer (triple negative breast cancer or estrogen receptor positive breast cancer) or glioblastoma.

[0043] In a further aspect of the present disclosure, a method for inhibiting protein synthesis through interaction with the peptidyl transferase center of ribosome in a subject is provided.In certain embodiments, the method comprises administering to a subject an effective amount of 7-hydroxyagelastatin compound as described herein, or its stereoisomer, racemate, or pharmaceutically acceptable salt, in an amount effective to inhibit protein synthesis through interaction with the peptidyl transferase center of ribosome.

[0044] As described above, the present disclosure provides agelastatin A (AglA) derivatives with improved solubility and a synthetic strategy based on the strategies described herein that renders the pyrrole moiety more amenable to further probing the nn interactions leading to the hidden C2-symmetry, leading to the conception of 7-hydroxy AglA (7a) and related derivatives as synthetic targets.

[0045] Structurally, AglA has four nitrogen atoms in a central C ring bound in a syn-anti-syn relationship. It was recognized that the addition of the C7-hydroxyl group confers pseudosymmetry to AglA, resulting in a second carbinolamine, which is derived from the addition of the pyrrole nitrogen to the pendant ketone, leading to a hydroxyl-substituted dihydropyrazinones (Fig. 1, 7a). The addition of the additional hydroxyl group is also speculated to confer greater aqueous solubility to AglA (clogP: 7-OH AglA(7a), -2.13; AglA(1), -1.23), while potentially providing additional hydrogen bonds at the binding site. We docked 7-OH AglA into the X-ray structure of the ribosome-AglA complex (McClary, B.; Zinshteyn, B.; Meyer, M.; Jouanneau, M.; Pellegrino, S.; Yusupova, G.; Schuller, A.; Reyes, JC; Lu, J.; Guo, Z.; Romo, D.; Yusupov, M., Green, R.; Liu, JCell Chem. Bio. 2017, vol. 24, 605-613) and, using Molecular Operating Environment (MOE) software, found a possible weak (extended, 3.52 Å) hydrogen bond between the pyrimidinedione of uracil 2875 (U2875) and the C7-OH that could be formed in a low-energy pose similar to that of AglA.

[0046] We also wanted to confirm that the introduction of the C7-hydroxyl does not significantly affect the overall topology of AglA and that a low-energy conformation corresponding to AglA is also achievable with 7-OH AglA. Molecular dynamics and minimization in MOE as well as density functional theory (DFT) calculations also showed two similar low-energy conformers (A / B and A' / B') for both AglA and 7-OH AglA, differing only by the envelope conformation adopted by the cyclopentane ring (C-ring) of these otherwise extremely rigid molecules (see FIG. 3). In the case of AglA, the two lowest-energy conformers A / B differed by about 4-5 kcal / mol. The ΔG of approximately 0.5 kcal / mol (MOE) at 25 °C for the two conformers A' / B' of 7-OH AglA suggests that they are both readily accessible at physiological temperatures and that one envelope conformation corresponds to the lower energy conformation of AglA.

[0047] These preliminary computational studies supported the synthetic strategy we described, including a late-stage pyrrole cyclization strategy that makes this region of the molecule more easily variable and extends the known SAR of agelastatin. This provides further impetus, based on the described hidden C2-symmetry element found in this targeted derivative, to explore the synthesis of 7-hydroxy AglA, forming the basis of our retrosynthetic strategy (Figure 4). In addition, this strategy could be followed by reduction of C7-carbinolamine 7a / 7b, again leading to non-C7-hydroxylated AglA derivatives with mutations in the pyrrole ring. The C7-carbinolamine is derived from intramolecular cyclization of the pyrrole NH of the ketone precursor 9. The bicyclic urea 9 can then be synthesized from three sub-moieties: pyrrole 11, azide 12 and N-methyl isocyanate (10) through acylation with an isocyanate followed by cyclization on the ketone. Azide 12 can be introduced by ring cleavage of aziridine 13, which in turn is derived from the known cyclopentenone iodide 14, readily accessible from furfural by reported procedures ((a) Saitman, A.; Theodorakis, E. A. Org. Lett. 2013, 15, 2410-2413. (b) Yang P.; Yao M.; Li J.; Li Y.; Li A. Angew. Chem. Int. Ed. 2016, 55, 6964-6968. (c) Truax, N. J.; Ayinde, S.; Van, K.; Liu, J. O.; Romo, D. Org. Lett. 2019, 21, 7394-7399).

[0048] Carbinolamines can theoretically be formed reversibly and exist in equilibrium with the aldehyde and amine components. Pyrrolocarbinolamines are formed by the nucleophilic addition of pyrrole to aldehydes or ketones, are reasonably stable, can be purified and isolated, and serve as aldehyde protecting groups, but can revert back to their carbonylpyrrole precursors upon treatment with base. The C5-carbinolamine of AglA is known to exist primarily in a closed ring form, leading to hydroxyimidazolidinones. However, we were not confident of the same equilibrium for the proposed hydroxydihydropyrazinones 7 derived from the cyclization of the pyrrole nitrogen addition to the pendant ketone. To address this question, we first performed model studies to probe the stability of such ketopyrrole-derived carbinolamines leading to dihydropyrazinones.

[0049] We synthesized pyrrole carboxylic acid amides 17a-c and studied the equilibria between the corresponding carbinolamines and pyrroloketones (Figure 5). Reduction of 2-azidocyclopentanone and condensation with pyrrole-1H-carboxylic acid gave amide 17b. Upon treatment of ketone 17b with Et3N, cyclization afforded the corresponding carbinolamine 18b in 73% yield (>19:1 dr, 600 MHz). 1 H NMR). Carbinolamine 18b was very stable and could be purified by silica gel chromatography and was stable in CD3OD for several days. Retreatment with NEt3 / CH2Cl2 at ambient temperature (22 °C) for 16 h produced only small amounts of the ring-opened ketopyrrole 17b, resulting in an equilibrium ratio of 11:1 (18b / 17b), 1As judged by H NMR, the cyclized form predominated. Similarly, the 4-bromopyrrole-derived carbinolamine 18c was stable under neutral conditions (e.g., in CD3OD), but in NEt3 / CH2Cl2 at 22 °C afforded a mixture of 18c and 17c in an 8:1 ratio, again with the cyclized form predominance. In contrast, the 5-bromopyrrole analog 17a most closely mimicked the targeted 7-hydroxyAglA, with the closed form 18a not predominance. Not only did the precursor ketopyrrole 17a not cyclize to 18a under similar conditions to 17b and 17c, but even if the cyclic carbinolamine 18a was targeted through bromination of the carbinolamine 18b, the brominated adduct 18a rapidly opened in CD3OD to form a mixture of 17a / 18a in an 8.7:1 ratio, with the ketopyrrole 17a predominance. Overall, these results suggest that the presence of a substituent on C5 of the pyrrole ring is unfavorable for cyclic carbinolamines, and therefore Applicants first explored the non-brominated C7-OH AglA 7b.

[0050] The synthesis of racemic 7-hydroxy-13-des-bromoAglA (24) begins with the aziridination of the known iodide 14 and can be obtained in three steps from furfuryl alcohol (Figure 6) ((a) Saitman, A.; Theodorakis, E. A. Org. Lett. 2013, 15, 2410-2413. (b) Yang P.; Yao M.; Li J.; Li Y.; Li A. Angew. Chem. Int. Ed. 2016, 55, 6964-6968. (c) Truax, N. J.; Ayinde, S.; Van, K.; Liu, J. O.; Romo, D. Org. Lett. 2019, 21, 7394-7399). Aziridination with p-toluenesulfonamide under basic conditions incorporated aziridine 13 by the method of Maycock (Silva, S.; Rodrigues, P.; Bento, I.; Maycock, CDJ Org. Chem. 2015, Vol. 80, 3067-3074). Next, aziridine cleavage with azide anion was investigated, but instability of both the aziridine and the derived azide towards both acidic and basic conditions was observed. After extensive experimentation, trimethylsilyl azide was found to be optimal for aziridine ring opening. This afforded azidocyclopentanone 12 as a mixture of diastereomers (43%, dr 1.2:1) and was likely due to nonselective a-protonation of the intermediate silyl enol ether. The desired non-diastereomer 12b was isolated by column chromatography, while the undesired diastereomer 12a was re-equilibrated over 4 Å molecular sieves in acetonitrile for 6 days, resulting in an approximately 1:1 ratio, leading to the isolation of an additional amount of the desired non-diastereomer. Hydrogenation of the azide 12a in the presence of N-methylisocyanate (10) afforded the bicyclic intermediate 20 through a known cyclization in a single step to give the cyclic urea. To introduce the pyrrole moiety, the N-tosyl group was cleaved using SmI2 / H2O / NEt3 (Ankner T.; Hilmersson, G. Org. Lett. 2009, 11, 503-506) to provide the primary amine 21.Subsequent amide coupling of pyrrole 2-carboxylic acid with lithium carboxylate was initially problematic and could not proceed to completion due to the insolubility of primary amine 21, which was found to be only partially soluble in typical organic solvents for amide coupling (e.g., CH2Cl2, DMF). Eventually, a suspension of amine 21 in DMF was found to completely dissolve upon heating to approximately 100 °C to give a homogenous solution. After cooling to ambient temperature, the coupling proceeded without incident to afford the desired amide 22 in 82% yield.

[0051] Deprotection of the TBS ether was achieved with HCl in MeOH, which also served to shield the hemiaminal as methoxy aminal 22. Swern oxidation afforded an intermediate ketone, which cyclized in the presence of excess triethylamine to directly afford carbinolamine 23 in 46% yield. This is consistent with our modeling studies, which suggested that the equilibrium favored the cyclic form of the C7-carbinolamine (see Figure 5, 18b), which was further confirmed by X-ray crystallography of carbinolamine 23 (inset, Figure 6). Interestingly, the solid-state conformation corresponds to the lower energy conformation found through conformational exploration (see above). Hydrolysis of methoxy aminal 15 was achieved under mild conditions to afford the targeted 7-hydroxy-13-des-bromoAglA (24), which, when stored in CD3OD or DMSO-d6 at ambient temperature (22 °C) for 7 days, 1 It was found to be very stable as determined by 1 H NMR.

[0052] In an effort to convert mono-carbinolamine 23 to the core structure of AglA, we explored several straightforward methods; however, direct reduction was not successful under a variety of Lewis acid / hydride addition conditions. Alternatively, we considered direct intramolecular stereoinversion displacement of alcohols 25a and 25b derived from amine 21 (see 25a, b) to form the B ring via a Mitsunobu reaction. However, although cyclization did occur, it was the amide oxygen atom that acted as the nucleophile, resulting in oxazolines 26a and 26b, as confirmed by X-ray crystallography in the case of oxazoline 26b (Figure 7).

[0053] We next considered an alternative strategy to form the B-ring that we had successfully utilized in our previous biomimetic strategy for AglA (Reyes, JCP; Romo, D. Angew. Chem., Int. Ed. 2012, vol. 51, 6870-6873), namely, the 5-exo, aza-Michael ring closure. Attempted dehydration of alcohol 25a to directly incorporate the C6-C7 alkene was in very low yield due to multiple side reactions (not shown). We therefore returned to the initial intermediate, silyl ether 20, and after generation of methoxycarbinolamine and desilylation, we introduced the required C6-C7 alkene via Grieco elimination, affording cyclopentene 28 in 42% yield in two steps (Figure 8). Cleavage of the tosyl group with SmI2 and acylation with pyrrolic acid chloride afforded the amide 30, which was hydrolyzed to the carbinolamine 31, yielding the same intermediate as in our previous biomimetic synthesis of AglA. Mild heating of the carbinolamine with silica gel initiated the aza-Michael addition, which again succeeded in affording AglA. However, as it was not easily scalable, we explored alternative reaction conditions for this transformation. We anticipated that condensation of the opened C-ring enone of the carbinolamine 31, if achieved under mildly basic conditions, would facilitate the desired aza-Michael reaction. After several experiments with various bases, we found that a substoichiometric amount of base, i.e., 0.25 equivalents of K2CO3 in MeOH, afforded AglA (33) in 43% yield (two steps) along with the known 3,4-bis-epi-AglA (34). The spectra of both products were consistent with those previously reported.

[0054] Previous SAR studies of AglA have shown that electron-withdrawing groups on the pyrrole moiety are beneficial to the potency of derivatives (e.g., Cl, CF3) (Stout, EP; Choi, MY; Castro, JE; Molinski, TFJ Med. Chem. 2014, 57, 5085-5093; Jouanneau, M.; McClary, B.; Reyes, JCP; Chen, R.; Chen, Y.; Plunkett, W.; Cheng, X.; Milinichik, AZ; Albone, EF; Liu, Jun O. Bioorg. & Med. Chem. Lett. 2016, 26, 2092-2097). This observation is consistent with the nn-stacking observed with nucleotide bases from ribosomal RNA in the X-ray structure of AglA bound to the A-site of the ribosomal peptidyl transfer center. We therefore targeted the electron-withdrawing nitro group to demonstrate the utility of this late-stage pyrrole cyclization strategy, despite the potential hindrance of the nitro group from a medicinal chemistry perspective. The substrate for the key aza-Michael cyclization was easily prepared from amine 29, in a similar manner to that described above for AglA, but to avoid bis-epimerization at C1, C5 of the bicyclic imidazolidinone 29 (see 34), which was found to occur readily even under mildly acidic conditions, we used the N-hydroxysuccinimide (NHS) ester of the nitro-substituted pyrrole 35 (Figure 9). In a similar manner, the aza-Michael cyclization proceeded unhindered to give 13-nitro AglA (37) in 41% yield over two steps. Interestingly, this AglA derivative 27 precipitated from the reaction mixture as a pale yellow solid, and simple filtration and washing with methanol afforded the nitro AglA derivative 37 in pure form.

[0055] The cytotoxicity of the synthesized novel AglA derivatives was determined against four cancer cell lines in comparison to both (-)-AglA and (±)-AglA (Figure 10). The bis-carbinolamine 24 lacks the C13-bromo substituent and showed no activity against MCF7, Caco2, and MDA-MB-231 cell lines up to 250 μM. This is not surprising based on the apparent halogen-p interactions observed in the X-ray structure and the previously demonstrated approximately 500-fold reduction in potency of 13-de-bromoAglA HeLa cells compared to AglA. Unfortunately, as suggested by our modeling studies (see above, Figures 5, 17a), 7-OH AglA (24) had a C5-bromo substituent and was found to exist primarily as the ring-open ketopyrrole tautomer, which could not be assayed due to instability. However, bis-carbinolamine 24 did not show any measurable EC 50 A value of (171.0±8.0 μM) could be obtained. Oxazoline 26a showed no activity against any of the cell lines studied.

[0056] Prompted by the reduced bioactivity of the de-bromo mutant of 7-OH AglA (24), we performed a conformational analysis of this derivative (Fig. S11). Of particular interest was the comparison of the conformational preferences of the C-ring cyclopentane compared to AglA. Indeed, the envelope ring conformer A'' was determined to be lower in energy (i1E = 3.4 kcal / mol) than the lowest energy cyclopentyl conformer A of AglA (see Fig. 3), and therefore, the lower concentration of the AglA-like envelope conformer may contribute to the reduced cytotoxicity.

[0057] This disclosure provides a novel synthetic strategy based on hidden symmetry elements that results in bis-carbinolamine derivatives of AglA (e.g., C7-hydroxydibromoAglA (24)) with key features that allow late-stage mutation of the pyrrole moiety. The design was guided by X-ray structure and molecular modeling of the AglA-ribosome complex. The targeted 7-hydroxyAglA was found to exist predominantly in the ring-opened ketopyrrole form and to be unstable, whereas 7-hydroxyde-bromoAglA could be synthesized and isolated. In addition, a new set of conditions for the final 6-exo-trig, aza-Michael ring closure was developed and utilized to arrive at 13-nitroAglA (37). The biological activity of the new AglA derivatives was measured against four cancer cell lines, and the novel C5-nitroAglA showed activity (1.16-35.4 μM) against all cell lines studied except the colon cancer line, Caco-2. However, 7-OH des-bromoAglA (24) was cytotoxic to the glioblastoma cell lines tested (U87, EC 50 =171.0±8.0μM). The following examples are offered for illustrative purposes and not by way of limitation. EXAMPLES

[0058] Working Example General information Unless otherwise stated, all non-aqueous reactions were carried out in flame-dried glassware under a N2 atmosphere. All solvents used were dried by using a solvent purification system based on activated molecular sieves. Triethylamine was distilled over CaH2 prior to use. All other commercially available reagents were used as received. 1H NMR spectra were measured at 600 MHz, 500 MHz or 400 MHz and chemical shifts are reported as 8 values ​​in ppm relative to CDCl3 (7.26 ppm) or CD3OD (3.31 ppm). Coupling constants (J) are reported in Hertz (Hz) and multiplicities follow conventional practice. The abbreviations brs (broad singlet), s, d, t, q, p, hept and m (or any combination thereof) represent the multiplicity of the resonance, broad singlet, singlet, doublet, triplet, quartet, quintet, sextet and multiplet. The designation app represents an appearance of splitting that was observed but not necessarily expected, broad represents a broadening of the peak that cannot be distinguished, possibly due to additional smaller couplings. Deuterated solvents (CDCl3, 77.16 ppm, CD3OD, 49.00 ppm, or DMSO, 39.52 ppm) were added. 13 100 nm was used as an internal standard for C NMR spectra. Flash column chromatography was performed using 60 Å silica gel (Silicycle, 230-400 mesh) as the stationary phase, using a gradient solvent system or an automated flash chromatography system. High-resolution mass spectra were acquired at the Center for Mass Spectrometry (Baylor University). Thin-layer chromatography (TLC) was performed using TLC plates with pre-coated glass backs, Silica Gel F 254 (Silicycle, 250 μm thick). Fourier transform infrared spectroscopy (FTIR) spectra were recorded as thin films on NaCl plates. X-ray structures were obtained in the X-ray diffraction lab at Baylor University. List of Abbreviations (COCl)2Oxalyl chloride DEAD Diethyl azodicarboxylate DMAP 4-Dimethylaminopyridine DMF N,N-Dimethylformaldehyde DMP Dess-Martin Periodinane DMSO Dimethyl sulfoxide HBTU Hexafluorophosphate Benzotriazole Tetramethyluronium NBS N-Bromosuccinimide NEt3 Triethylamine 2-NO2PhSeCN 2-Nitrophenyl selenocyanate PBu3 Tributylphosphine PPh3 Triphenylphosphine SmI2 Samarium diiodide TBAF Tetrabutylammonium fluoride TBSCl tert-Butyldimethylsilyl chloride THF Tetrahydrofuran TMSN3 Trimethylsilyl azide TsNH2p-Toluenesulfonamide

[0059] Experimental procedure Provided below is a description of the synthesis of the compounds described herein, the schematics of which are illustrated in Figures 4-9.

[0060] [ka]

[0061] To azide 15 (Dede, D. Regioselective synthesis of functionalized salicylates, isotetronic acids, and alkylidene-isobenzofurans based on one-pot cyclization of enol ethers; Rostock, Germany, 2008) (0.30 g, 2.4 mmol, 1.0 equiv.) in methanol (2.4 mL) was added 10% palladium on carbon (133 mg, 0.28 mmol, 0.12 equiv.). A H2 balloon attached to the flask via a 3-way adapter was connected and the flask was sequentially placed under vacuum and filled with H2 (3 times). 1 M hydrogen chloride in diethyl ether (4.08 mL, 4.08 mmol, 1.7 equiv.) was added and the reaction was stirred for 21 h. A second portion of palladium on carbon (66.5 mg, 0.14 mmol, 0.06 equiv) and methanol (0.6 mL) were added and the reaction was stirred for an additional 17 h with a fresh hydrogen balloon. TLC indicated the disappearance of starting material, and the mixture was filtered through a cotton plug, washed with methanol, and concentrated in vacuo to give the amine hydrochloride salt 15a as a black solid (0.3 g), which was used directly in the next step.

[0062] A 10 mL round bottom flask was charged with pyrrole-1H-2-carboxylic acid (214.2 mg, 1.93 mmol, 1.5 equiv) and THF (6.0 mL). To this clear solution was added hexafluorophosphate benzotriazole tetramethyluronium (HBTU, 731.2 mg, 1.93 mmol, 1.5 equiv) and triethylamine (0.54 mL, 3.87 mmol, 2.0 equiv) at ambient temperature (22 °C), followed by DMF (1.5 mL) to form a clear, colorless solution. The mixture was stirred for 1 h and then cooled to 0 °C. A solution of 15a (174.3 mg, 1.29 mmol, 1.0 equiv) prepared as above in N,N-dimethylformaldehyde (3.3 mL) was added and the reaction was allowed to warm to ambient temperature (22 °C) by removing the ice-water bath. The reaction was stirred for an additional 6 h and concentrated in vacuo to remove the solvent. The organic residue was purified by MPLC on silica (ethyl acetate / hexanes, 0-60%) to give amide 17b (89.0 mg, 36%) as a white solid.

[0063] 17b: White solid. 1 H NMR (400 MHz, CD3OD) δ 6.91 (dd, J = 2.6, 1.4 Hz, 1H), 6.79 (dd, J = 3.8, 1.5 Hz, 1H), 6.16 (dd, J = 3.7, 2.5 Hz, 1H), 4.37 - 4.13 (m, 1H), 2.45 - 2.33 (m, 2H), 2.32 - 2.22 (m, 1H), 2.14 - 2.02 (m, 1H), 2.00 - 1.80 (m, 2H), two NH protons were not observed; 13 C NMR (100 MHz,CD3OD) δ 217.6,163.5,126.4,123.1,112.0,110.2,58.1,36.7,30.32,19.41; IR (thin film,cm -1 ) 1737,1626,1557,1519,1129; HRMS (ESI) C 10 H 13 N2O2 + [M+H] + Calculated value: 193.0972, measured value: 193.0979.

[0064] [ka]

[0065] A 10 mL round bottom flask was charged with 5-bromopyrrole-1H-2-carboxylic acid (118.8 mg, 0.625 mmol, 1.5 equiv) and hexafluorophosphate benzotriazole tetramethyluronium (HBTU, 237.0 mg, 0.625 mmol, 1.5 equiv). THF (2.0 mL) was added followed by triethylamine (0.23 mL, 1.67 mmol, 4.0 equiv) at ambient temperature (22 °C). DMF (0.5 mL) was added and the suspension became homogenous. The mixture was stirred for 1 h and cooled to 0 °C. A solution of 15a (56.5 mg, 0.42 mmol, 1.0 equiv) in N,N-dimethylformaldehyde (1.1 mL), prepared as described above for 17b, was added and the reaction was allowed to warm to ambient temperature (22 °C) by removing the ice-water bath. The reaction was stirred for an additional 1.5 h and concentrated in vacuo to remove the solvent. The organic residue was purified by MPLC on silica (ethyl acetate / hexanes, 0-60%) to give the amide 17a (53.0 mg, 47%) as a white solid. 17a: White solid. 1 H NMR (500 MHz, CD3OD) δ 6.73 (d, J = 3.8 Hz, 1H), 6.13 (d, J = 3.8 Hz, 1H), 4.25 (dd, J = 11.3, 8.7 Hz, 1H), 2.42 - 2.31 (m, 2H), 2.31 - 2.20 (m, 1H), 2.13 - 2.02 (m, 1H), 1.98 - 1.81 (m, 2H), two NH protons were not observed; 13 C NMR (125 MHz,CD3OD) δ 217.4,162.3,128.3,113.5,112.5,104.6,58.1,36.6,30.3,19.4; IR (thin film,cm -1 ) 3183,1744,1623,1555,1455,1394; HRMS (ESI) C 10 H 11 79 BrN2NaO2+ [M+Na] + The calculated value was 292.9896, the actual value was 292.9901.

[0066] [ka]

[0067] To a 10 mL round bottom flask was added 4-bromopyrrole-1H-2-carboxylic acid (130.3 mg, 0.69 mmol, 1.5 equiv) and hexafluorophosphate benzotriazole tetramethyluronium (HBTU, 260.1 mg, 0.69 mmol, 1.5 equiv) followed by THF (2.0 mL) and triethylamine (0.26 mL, 1.83 mmol, 4.0 equiv) at 22° C. DMF (0.5 mL) was added and the suspension became a clear solution. The mixture was stirred for 1 h and cooled to 0° C. In a separate 1.5 dram vial, 15a (62 mg, 0.46 mmol, 1.0 equiv) was dissolved in DMF (1.1 mL) and the solution was transferred to the reaction vessel via cannula, then the reaction was allowed to warm to ambient temperature (22° C.) by removing the ice-water bath. The reaction was stirred for an additional 1.5 h and concentrated in vacuo to remove the solvent. The organic residue was purified by MPLC on silica (ethyl acetate / hexanes, 0-60%) to give amide 17c (17.0 mg, 14%) as an off-white solid. 17c: Off-white solid. 1 H NMR (600 MHz, CD3OD) δ 6.92 (br t, J = 1.3 Hz, 1H), 6.78 (d, J = 1.5 Hz, 1H), 4.25 (dd, J = 11.4, 8.2 Hz, 1H), 2.41 - 2.34 (m, 2H), 2.27 (ddd, J = 19.7, 11.0, 9.3 Hz, 1H), 2.10 (m, 1H), 2.00 - 1.84 (m, 2H), one NH proton was not observed; 13 C NMR (150 MHz,CD3OD) δ 217.3,162.3,127.1,123.0,113.5,97.5,58.1,36.6,30.2,19.4; IR (thin film,cm-1 ) 3219,1746,1633,1563,1523; HRMS (ESI) C 10 H 79 BrN2O2[MH] - Calculated value: 268.9931, measured value: 268.9935.

[0068] [ka]

[0069] To a stirred solution of amide 17b (13.0 mg, 0.068 mmol, 1.0 equiv) in dichloromethane (1.3 mL) was added triethylamine (28 ul, 0.20 mmol, 3.0 equiv) at ambient temperature (22 °C). The mixture was stirred for 22 h and the reaction was concentrated. The residue was purified by column chromatography (silica, methanol / dichloromethane 0-15%) to give cyclic carbinolamine 18b (9.5 mg, 73%) as a white solid.

[0070] 18b: White solid. 1 H NMR (600 MHz, CD3OD) δ 7.21 (dd,J = 2.7,1.6 Hz,1H), 6.88 (dd,J = 3.8,1.6 Hz,1H), 6.29 (dd,J = 3.8,2.7 Hz,1H), 3.88 (dd,J = 7.2,5.9 Hz,1H), 2.32 (ddd,J = 13.9,9.6,5.7 Hz,1H), 2.29 - 2.22 (m,1H), 2.19 (ddd,J = 13.8,9.5,6.5 Hz,1H), 1.87 (m,1H), 1.78 (m,1H), 1.67 (m,1H); no OH and NH protons observed; 13 C NMR (150 MHz,CD3OD) δ 161.8,123.4,122.3,115.3,111.5,90.5,62.7,38.3,32.1,20.5; IR (thin film,cm -1 ) 3272,1633,1558,1458,1330; HRMS (ESI) C 10 H 12 N2NaO2+ [M+Na] + Calculated value: 215.0791, measured value: 215.0801.

[0071] [ka]

[0072] To a stirred solution of amide 17c (6.0 mg, 0.022 mmol, 1.0 equiv) in dichloromethane (0.2 mL) was added triethylamine (9 mL, 0.066 mmol, 3.0 equiv) at ambient temperature (22 °C). The mixture was stirred for 28 h and the reaction was concentrated. The residue was purified by column chromatography (silica, methanol / dichloromethane, 0-15%) and the cyclized product 18c (4.0 mg, 67%) was isolated as a white solid.

[0073] 18c: White solid. 1 H NMR (600 MHz,CD3OD) δ 7.23 (d,J = 1.8 Hz,1H),6.82 (d,J = 1.8 Hz,1H),3.88 (dd,J = 7.2,6.0 Hz,1H),2.31 (ddd,J = 13.7,9.5,5.6 Hz,1H),2.28 - 2.22 (m,1H),2.19 (ddd,J = 13.9,9.4,6.5 Hz,1H),1.92 - 1.84 (m,1H),1.84 - 1.73 (m,1H),1.68 (m,1H); 13 C NMR (150 MHz,CD3OD) δ 160.4,124.5,121.9,116.4,99.9,90.9,62.6,38.2,32.1,20.5; IR (thin film,cm -1 ) 3295,1637,1553,1470; HRMS (ESI) C 10 H 10 79 BrN2O2[MH] - Calculated value: 268.9931, measured value: 268.9935.

[0074] [ka]

[0075] To a stirred solution of carbinolamine 18b (3.0 mg, 0.016 mmol, 1.0 equiv) in dichloromethane (0.32 mL) was added triethylamine (6.6 μl, 0.047 mmol, 3.0 equiv). 1 The reaction was stirred at ambient temperature (22 °C) for 16 h as monitored by H NMR, which indicated that a mixture of ketone 18b and cyclic carbinolamine 17b had formed in a 12:1 ratio, respectively, which remained unchanged over this period.

[0076] [ka]

[0077] To a stirred solution of carbinolamine 18c (1.2 mg, 0.0044 mmol, 1.0 equiv) was added a solution of triethylamine in dichloromethane (0.1 mL, the solution was made by mixing 19 μl of triethylamine with 1.0 mL of dichloromethane). 1 The reaction was stirred for 16 h as monitored by H NMR, which showed that a mixture of ketone 18c and cyclic carbinolamine 17c had formed in a ratio of 8:1, respectively, which remained unchanged over this period.

[0078] [ka]

[0079] A 1.0 dram vial was charged with 17a (2.7 mg, 0.01 mmol, 1.0 equiv) and a solution of triethylamine in dichloromethane (0.23 mL, 0.03 equiv, 3.0 equiv, the solution was made by mixing 19 μl of triethylamine with 1.0 mL of dichloromethane). The reaction was stirred for 2 days. 11 H NMR showed that a mixture of ketone 17a and cyclic carbinolamine 18a in a ratio of 1:0.11, respectively, was formed, and this ratio remained unchanged over this period.

[0080] [ka]

[0081] In a 1.5 dram vial, carbinolamine 18b (2.6 mg, 0.014 mmol, 1.0 equiv) was dissolved in THF (0.14 mL) and MeOH (0.14 mL) and the mixture was cooled to 0 °C. N-Bromosuccinamide (2.5 mg, 0.0142 mmol, 1.05 equiv) was added and the reaction was allowed to warm to ambient temperature (22 °C). After stirring for 4 h, TLC showed no more starting material, and the reaction mixture was concentrated and purified by column chromatography on silica (MeOH / CHCl, 0-20%) to give a mixture of 18a / 18c / 17a (2.4 mg, 65%) in a ratio of 1:0.6:0.44. This mixture was monitored by NMR to probe the stability of the 5-bromo derivative in CDOD. After 6 hours the mixture gave a ratio of 18a / 18c / 17a=0.15:0.6:1.30, which remained unchanged over 16 hours.

[0082] [ka]

[0083] Iodide 14 was synthesized using the procedure described below ((a) Yang, P.; Yao, M.; Li, J.; Li, Y.; Li, A. Total Synthesis of Rubriflordilactone B. Angew. Chem. Int. Ed. 2016, Volume 55, 6964~6968. (b) Saitman, A.; Theodorakis, EASynthesis of a Highly Functionalized Core of Verrillin.Org.Lett.2013, Volume 15, 2410~2413 (c)Truax,NJ;Ayinde,S.;Van,K.;Liu,JO;Romo,D.Pharmacophore-Directed Retrosynthesis Applied to Rameswaralide:Synthesis and Bioactivity of Sinularia Natural Product Tricyclic. Cores. Org. Lett. 2019, Vol. 21, 7394-7399).

[0084] [ka]

[0085] To a stirred solution of iodide 14 (1.0 g, 2.96 mmol, 1 equiv.) in dichloromethane (anhydrous, 25 mL) in a 50 mL round bottom flask, 1,10-phenanthroline (598 mg, 3.32 mmol, 1.12 equiv.) and p-toluenesulfonamide (1.01 g, 5.91 mmol, 2 equiv.) were added. The mixture was then cooled in an ice bath and cesium carbonate (982 mg, 3.02 mmol, 1.02 equiv.) was added in 5 batches over 20 min to form a pale yellow suspension. After stirring for 10 min, the ice bath was removed and the reaction was stirred at ambient temperature (22 °C) for 5 h. The reaction was quenched by adding water (30 mL), brine (20 mL), and extracted with dichloromethane (30 mL x 3). The organic phases were combined, washed with brine (30 mL), dried over Na2SO4, and concentrated in vacuo. The residue was purified by automated column chromatography on silica (wet-loaded, acetone / hexanes, 0-40%) to give aziridine 13 (0.68 g, 60%, 70% based on recovered starting material) as an off-white solid and recovered iodide (0.14 g).

[0086] When the reaction was scaled up to 1,10-phenanthroline (5.24 g), p-toluenesulfonamide (8.89 g), and cesium carbonate (8.63 g) with 8.8 g of iodoenone 14 in dichloromethane (200 mL), this gave aziridine 13 (5.05 g, 51%, BRSM 60%) along with recovered iodoenone 14 (1.32 g).

[0087] 13: Off-white solid. 1 H NMR (CDCl3,400 MHz) δ 7.80 (d,J = 8.3 Hz,2H),7.36 (d,J = 8.3 Hz,2H),4.55 (d,J = 5.5 Hz,1H),3.71 (d,J = 4.5 Hz,1H),3.30 (d,J = 4.4 Hz,1H),2.50 (dd,J = 18.3,5.6 Hz,1H),2.45 (s,3H),1.93 (d,J = 18.3 Hz,1H),0.87 (s,9H),0.11 (s,3H),0.07 (s,3H);13 IR (thin film, cm -1 ) 3069,2930,2857,1757,1337,1159,1086; HRMS (ESI) C 18 H 28 N7O4SSi + [M+Na] + Calculated value: 404.1322, measured value: 404.1327.

[0088] [ka]

[0089] To a stirred solution of aziridine 13 (5.05 g, 13.2 mmol, 1 equiv.) in a 350 mL pressure vessel (sealed flask) was added acetonitrile (anhydrous, 104 mL), and trimethylsilyl azide (2.08 mL, 15.8 mmol, 1.2 equiv.). The flask was sealed and heated at 39 °C for 24 h. The brown mixture was then concentrated and purified by MPLC on silica using acetone / hexanes (wet-packed, 0-50%) to give the desired diastereomer 12b (1.5 g, 27%, BRSM 47%) as a yellow solid, along with recovered starting material 13 (2.25 g, 46%), and the undesired diastereomer 12a (0.87 g, 16%, BRSM 27%) as a yellow solid. 12a: Yellow solid. 11H NMR (600 MHz, CDCl3) δ 7.75 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 4.95 (d, J = 3.0 Hz, 1H), 4.65 (apparent dt, J = 5.5, 1.7 Hz, 1H), 4.23 (dd, J = 6.4, 1.4 Hz, 1H), 3.57 - 3.40 (m, 1H), 2.69 (dd, J = 19.1, 5.4 Hz, 1H), 2.44 (s, 3H), 2.42 (d, J = 19.2 Hz, 1H), 2.22 (d, J = 19.2 Hz, 1H), 0.84 (s, 9H), 0.09 (s, 3H), 0.07 (s, 3H); 13 13C NMR (150 MHz, CDCl3) δ 210.1, 144.4, 135.7, 130.1, 127.4, 70.1, 62.8, 58.9, 42.7, 25.7, 21.7, 17.9, -4.8, -5.0; IR (film, cm-1): 2113, 1699, 1162, 833; HRMS (ESI) C 18 H 28 N4NaO4SSi + [M+Na] + calculated value 447.1493, found 447.1496.

[0090] 12b: Yellow solid. 1 1H NMR (600 MHz, CDCl3) δ 7.80 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 5.53 (d, J = 6.7 Hz, 1H), 4.25 (q, J = 7.1 Hz, 1H), 3.76 (apparent d, J = 8.9 Hz, 1H), 3.43 (dt, J = 9.1, 6.8 Hz, 1H), 2.77 (ddd, J = 18.8, 7.2, 1.6 Hz, 1H), 2.43 (s, 3H), 2.26 (dd, J = 18.8, 7.3 Hz, 1H), 0.82 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H); 13C NMR (150 MHz,CDCl3) δ 206.4,144.2,137.3,130.0 (2),127.3 (2),70.5,67.7,63.7,45.3,25.7 (3),21.7,18.0,-4.72,-4.73; IR (thin film, cm-1): 3238,2110,1759,1328,1151; HRMS (ESI) C 18 H 28 N4NaO4SSi + [M+Na] + Calculated value: 447.1493, measured value: 447.1496.

[0091] [ka]

[0092] A 100 mL round bottom flask was charged with azide 12a (3.45 g, 8.12 mmol) and acetonitrile (anhydrous, 68 mL), followed by 4A molecular sieve powder (10.35 g) at ambient temperature (22 °C), and the mixture was stirred for 6 days. NMR indicates that a mixture of two diastereomers was formed in approximately 1:1 ratio. The mixture was filtered and washed with acetonitrile (50 m). The solvent was concentrated in vacuo and purified by flash column chromatography using acetone / hexanes (wet pack, 0-50%) to give the desired diastereomer 12b (1.35 g, 39% yield) and 12a (1.15 g, 33% recovery).

[0093] [ka]

[0094] A 250 mL round bottom flask was charged with 12b (2.19 g, 5.16 mmol, 1.0 equiv) and THF (103 mL) followed by 10% palladium on carbon (1.19 g, 2.58 mmol, 0.5 equiv) at ambient temperature (22 °C). A H2 bag (equipped with a valve) was connected and the flask was placed under vacuum and filled with H2 three times. Methyl isocyanate (0.35 mL, 5.67 mmol, 1.1 equiv) was added and another H2 balloon was connected. The reaction was stirred at ambient temperature for 48 h and TLC showed no more starting material was present. The mixture was filtered through a cotton plug, washed with THF (10 mL) and concentrated in vacuo. The residue was purified by MPLC on silica (dry loading, MeOH / CHCl 0-5%) to give 20 (1.7 g, 72% yield) as a pale yellow solid.

[0095] 20: Pale yellow solid. 1 H NMR (600 MHz,CD3OD) δ 7.79 (d,J = 8.1 Hz,2H),7.39 (d,J = 8.2 Hz,2H),4.23 - 4.01 (m,1H),3.59 (dd,J = 2.9,1.0 Hz,1H),3.13 (ddd,J = 4.0,2.8,1.0 Hz,1H),2.71 (s,3H),2.43 (s,3H),2.16 (dd,J = 13.8,5.2 Hz,1H),1.99 (ddd,J = 13.8,4.5,1.2 Hz,1H),0.77 (s,9H),-0.05 (s,3H),-0.10 (s,3H); 13 IR (thin film, cm -1 ) 3093,1690,1327,1160,1083; HRMS (ESI) C 20 H 33 N3NaO5SSi + [M+Na] + The calculated value was 478.1802, the measured value was 478.1807.

[0096] [ka]

[0097] A 500 mL round bottom flask was charged with 20 (0.83 g, 1.82 mmol, 1.0 equiv.) and the flask was placed under vacuum and backfilled with argon three times. Degassed DI water (freeze-pump-thaw, 0.98 mL, 54.7 mmol, 30 equiv.) and samarium iodide solution (0.078 M in THF, 234 mL, 18.2 mmol, 10 equiv.) were added sequentially at ambient temperature (22 °C). Triethylamine (freshly distilled over CaH2, 5.1 mL, 36.4 mmol, 20 equiv.) was added dropwise over 10 min. TLC showed that all the starting material was consumed. The reaction mixture was filtered and the liquid phase was collected while the solids were transferred to six centrifuge tubes (10 mL / tube) and extracted with 10 mL of CH3OH-CH2Cl2 (10%, v / v) in each tube. The suspension was centrifuged and the clear supernatant was collected while the solid was extracted again. This process was repeated 6-10 times until no more product was seen by TLC. The organic solutions were combined and concentrated and the residue was purified by MPLC on silica (dry-packed method with ELSD detector) using CH3OH / CH2Cl2 (0-10%) to give amine 21 (0.5 g, 73%) as a white powder. 21: White solid; 1 H NMR (600 MHz, CD3OD) δ 4.02 (apparent q, J = 6.5 Hz, 1H), 3.33 (d, J = 5.5 Hz, 1H), 2.84 (apparent t, J = 5.9 Hz, 1H), 2.75 (s, 3H), 2.27 (dd, J = 13.4, 6.0Hz, 1H), 1.91 (dd, J = 13.3, 7.2 Hz, 1H), 0.90 (s, 9H), 0.11 (dd, J = 11.0, 1.4 Hz, 6H); 13 C NMR (150,MHz,CD3OD) δ 161.0,94.7,77.2,67.91,67.85,44.0,26.3 (3),24.5,18.8,-4.6,-4.7; IR (thin film,cm -1) 2920,1660,1467; HRMS (ESI) C 13 H 28 N3O3Si + [M+H] + Calculated value: 302.1894, measured value: 302.1898.

[0098] [ka]

[0099] A 100 mL round bottom flask was charged with pyrrole-2-carboxylic acid (191.6 mg, 1.73 mmol, 2.0 equiv), DI water (17 mL), followed by lithium hydroxide monohydrate (74.5 mg, 1.78 mmol, 2.06 equiv). The mixture was stirred for 15 minutes to dissolve the solids. The solution was then dried under high vacuum to give lithium pyrrole carboxylate as a white solid. To this solid was added DMF (20 mL) and hexafluorophosphate benzotriazole tetramethyluronium (HBTU, 654.0 mg, 1.73 mmol, 2.0 equiv) and the mixture was stirred at ambient temperature (22 °C) for 1 hour. To a separate 100 mL flask was added sequentially the amine (260 mg, 0.86 mmol, 1.0 equiv) and DMF (anhydrous, 60 mL) and the mixture was heated at approximately 100 °C to give a clear solution. The mixture was cooled back to ambient temperature (22 °C) and added via cannula to a flask containing the acid, pyrrole-2-carboxylic acid, and HBTU. The mixture was stirred for 18 h until TLC analysis indicated complete consumption of amine 21. The reaction was placed under high vacuum to remove the solvent and the organic residue was purified by MPLC (dry loading) on ​​silica using ethyl acetate / hexanes (0-100%) followed by methanol / dichloromethane (0-10%) to give amide 21a (278.8 mg, 82% yield) as a white solid that was carried on directly to the next step.

[0100] 21a: White solid. 1H NMR (600 MHz,CD3OD) δ 6.92 (dd,J = 2.5,1.4 Hz,1H),6.83 (dd,J = 3.7,1.4 Hz,1H),6.17 (dd,J = 3.7,2.5 Hz,1H),4.70 (s,1H),4.37 (dt,J = 7.3,6.5 Hz,1H,1H),4.00 (dd,J = 7.1,5.9 Hz,1H),3.64 (d,J = 5.9 Hz,1H),2.78 (s,3H),2.31 (dd,J = 13.4,6.3 Hz,1H),1.99 (dd,J = 13.4,7.9 Hz, 1H), 0.85 (s, 9H), 0.08 (s,3H),0.04 (s,3H); 13 IR (thin film, cm -1 ): 3318,1671,1637,1408,1070; HRMS (ESI) C 18 H 30 N4NaO4Si + [M+Na] + Calculated value: 417.1929, measured value: 417.1932.

[0101] [ka]

[0102] To a stirred solution of crude 21a (398 mg, 0.755 mmol, 1.0 equiv) was added methanol (18.0 mL), followed by 1 N aqueous hydrochloric acid (0.75 mL, 0.75 mmol, 1.0 equiv). A cloudy suspension resulted which became clear as the reaction proceeded. After stirring for 6 h, TLC indicated complete consumption of starting material and the reaction was neutralized with pH 7 buffer. The reaction was then concentrated and the residue was purified by MPLC on silica (dry loading, methanol / dichloromethane (0-10%)) to give alcohol 22 (193.4 mg, 87%) as a white solid.

[0103] 22: White solid. 1 H NMR (500 MHz, CD3OD) δ 6.92 (dd,J = 2.6,1.4 Hz,1H),6.86 (dd,J = 3.8,1.4 Hz,1H),6.17 (dd,J = 3.7,2.5 Hz,1H),4.22 (ddd,J = 9.2,8.2,6.8 Hz,1H),3.91 (dd,J = 8.3,6.5 Hz,1H),3.74 (d,J = 6.4 Hz,1H),3.07 (s,3H),2.73 (s,3H),2.36 (ddd,J = 13.3,6.8,0.9 Hz,1H),2.00 (dd,J = 13.4,9.2 Hz,1H); 13 C NMR (125 MHz, CD3OD) δ 164.3, 161.7, 126.6, 123.1, 112.1, 110.2, 97.8, 72.9, 66.7, 61.5, 49.6, 43.5, 24.6; IR (thin film, cm -1 ): 3290,1679,1625,1405,1064; HRMS (ESI) C 13 H 18 N4NaO4 + [M+Na] + The calculated value is 317.1220, and the measured value is 317.1224.

[0104]

change

[0105] In a 2.0 dram vial, alcohol 22 (31.0 mg, 0.11 mmol, 1.0 equiv) was dissolved in THF (1.2 mL) and dimethylsulfoxide (anhydrous, 0.12 mL) and the mixture was cooled to -78°C. In a separate 1.0 dram vial, oxalyl chloride (21 uL, 0.24 mmol, 2.0 equiv) was dissolved in dichloromethane (0.6 mL), the mixture was cooled to -78°C, and DMSO (21 uL, 0.03 mmol, 2.8 equiv) was added. After stirring for 30 min, this solution was transferred via cannula to the 2.0 dram vial containing 22 at -78°C. The reaction was stirred for 1 h, freshly distilled triethylamine (0.15 mL, 1.07 mmol, 10 equiv) was added, and the reaction was warmed to -30°C within 1 h. A second batch of triethylamine (0.15 mL, 1.07 mmol, 10 equiv) was added and the reaction was allowed to warm to ambient temperature (22 °C) and stirred for 18 h. The reaction was filtered through a cotton plug, washed with THF (1 mL) and the combined organic phases were concentrated under high vacuum to remove DMSO. The residue was purified by column chromatography on silica (dry loaded) using methanol / dichloromethane (0-10%) to give carbinolamine 23 (14.2 mg, 46%) as an off-white solid.

[0106] 23: Off-white solid. 1 H NMR (500 MHz,CD3OD) δ 7.22 (dd,J = 2.8,1.6 Hz,1H),6.93 (dd,J = 3.8,1.6 Hz,1H),6.30 (dd,J = 3.8,2.7 Hz,1H),3.80 (d,J = 4.4 Hz,1H),3.74 (d,J = 4.5 Hz,1H),2.98 (s,3H),2.76 (s,3H),2.75 (d,J = 13.5 Hz,3H),2.63 (d,J = 14.4 Hz,1H); 13 IR (cm -1, thin film): 3251,1687,1646,1557,1459,1315,1072; HRMS (ESI): C 13 H 16 N4NaO4 + [M+Na] + Calculated value: 315.1064, measured value: 315.1066.

[0107] [ka]

[0108] A 1.5 dram vial was charged with aminal 23 (6.8 mg, 0.023 mmol, 1.0 equiv), THF (1.5 mL), and 0.5 N aqueous hydrochloric acid (23 uL, 0.012 mmol, 0.5 equiv). The reaction was stirred at ambient temperature (22 °C) for 3 h. The reaction was then adjusted to pH 7 with pH 7 buffer and concentrated in vacuo. The residue was purified by column chromatography on silica (dry loaded) using methanol / dichloromethane (0-10%) to give hemiaminal 24 (3.3 mg, 51%, 65% based on recovered starting material) as a white solid, and 23 (1.5 mg, 22%) recovered.

[0109] 24: White solid. 1 H NMR (600 MHz,CD3OD) δ 7.22 (dd,J = 2.7,1.6 Hz,1H),6.93 (dd,J = 3.8,1.6 Hz,1H),6.31 (dd,J = 3.8,2.7 Hz,1H),3.77 (apparently d,J = 4.4 Hz,1H),3.59 (d,J = 4.4 Hz,1H),2.80 (s,3H),2.73 (d,J = 14.3 Hz,1H),2.60 (d,J = 14.4 Hz,1H); 13 C NMR (150 MHz,CD3OD) δ 161.4,161.2,123.23,123.19,116.1,111.7,93.7,88.0,69.2,69.0,47.4,24.5; -1): 3305,1679,1643,1557,1462; HRMS (ESI): C 12 H 14 N4NaO4 + [M+Na] + The calculated value was 301.0907, and the measured value was 301.0910.

[0110] [ka]

[0111] Following a procedure similar to that described for the synthesis of amide 22, 5-bromopyrrole-2-carboxylic acid (109.9 mg, 0.58 mmol, 3.8 equiv), DI water (3 mL), lithium hydroxide monohydrate (25.0 mg, 0.59 mmol, 3.92 equiv), hexafluorophosphate benzotriazole tetramethyluronium (HBTU, 219.4 mg, 0.58 mmol, 3.8 equiv), and DMF (16.6 mL) were used and the reaction was stirred for 36 h. Column chromatography on silica using methanol / dichloromethane (0–10%) afforded 22a (57.7 mg, 80%) as a white solid.

[0112] 22a: White solid. 1 H NMR (600 MHz,CD3OD) δ 6.77 (apparently d,J = 3.8 Hz,1H),6.14 (apparently d,J = 3.9 Hz,1H),4.29 (q,J = 6.8 Hz,1H),3.97 (t,J = 6.2 Hz,1H),3.80 (d,J = 5.6 Hz,1H),3.08 (s,3H),2.73 (s,3H),2.29 (dd,J = 13.6,6.2 Hz,1H),2.03 (dd,J = 13.6,7.3 Hz,1H),0.85 (d,J = 1.4 Hz,9H),0.07 (s,3H),0.05 (s,3H); 13IR (thin film, cm -1 ) 3271,1687,1632,1396,1102,1082; HRMS (ESI): C 19 H 30 79 BrNOSi-[MH] - Calculated value: 485.1225, measured value: 485.1246.

[0113] [ka]

[0114] To a stirred solution of 22a (12.5 mg, 0.026 mmol, 1.0 equiv) in methanol (0.25 mL) was added hydrochloric acid (1N aqueous solution, 31 ul, 0.031 mmol, 1.2 equiv) at ambient temperature. The reaction was stirred for 3.5 h and no more starting material was present as shown by TLC. The reaction was neutralized to pH 7 with pH 7 buffer and the solution was concentrated under high vacuum. The residue was purified by MPLC on silica (dry loading, MeOH / CHCl 0-15%) to give alcohol 25a (7.3 mg, 76% yield) as a white solid.

[0115] 25a: White solid. 1 H NMR (500 MHz,CD3OD) δ 6.81 (d,J = 3.9 Hz,1H),6.14 (d,J = 3.9 Hz,1H),4.20 (td,J = 8.9,7.0 Hz,1H),3.89 (dd,J = 8.3,6.5 Hz,1H),3.72 (d,J = IR (thin film, cm -1) 3311,1670,1638,1529,1066; HRMS (ESI) C 13 H 17 79 BrN4NaO4 + [M+Na] + Calculated value: 395.0325, measured value: 395.0327.

[0116] [ka]

[0117] A 1.5 dram vial was charged with 25a (7.0 mg, 0.019 mmol, 1.0 equiv), triphenylphosphine (20.0 mg, 0.076 mmol, 4.0 equiv) and THF (0.5 mL). The mixture was stirred for 5 min and cooled to 0 °C. A solution of diethyl azodicarboxylate (9 uL, 0.057 mmol, 3.0 equiv) in THF (0.03 mL) was added. After stirring for 1 h, the starting material 25a was completely consumed as shown by TLC. The reaction mixture was concentrated in vacuo and the organic residue was purified by column chromatography on silica (dry loading, methanol / dichloromethane, 0-10%) to give oxazoline 26a (4.0 mg, 60%) as a white solid.

[0118] 26a: White solid. 1 H NMR (600 MHz,CD3OD) δ 6.74 (d,J = 3.8 Hz,1H),6.18 (d,J = 3.8 Hz,1H),5.13 (td,J = 7.8,5.4 Hz,1H),4.39 (d,J = 7.9 Hz,1H),3.99 (d,J = 1.7 Hz,1H),3.12 (s,3H),2.86 - 2.49 (m,4H),2.19 (dd,J = 15.1,5.5 Hz,1H); 13C NMR (150 MHz,CD3OD) δ 161.9,159.4,121.8,116.2,113.1,105.1,102.1,83.1,79.9,63.4,51.1,42.4,24.7; IR (thin film, cm-1) 1683,1648,1434,1393,1075,1019; HRMS (ESI) C 13 H 16 79 BrNO3 + [M+H] + Calculated value 355.0400, measured value 355.0410.

[0119] [ka]

[0120] The procedure for making 22 was followed using 4-bromopyrrole-2-carboxylic acid (16.6 mg, 0.087 mmol, 3.6 equiv), DI water (0.4 mL), lithium hydroxide monohydrate (3.7 mg, 0.087 mmol, 3.6 equiv), hexafluorophosphate benzotriazole tetramethyluronium (HBTU, 34 mg, 0.087 mmol, 3.6 equiv), amine 21 (7.3 mg, 0.024 mmol, 1.0 equiv) and DMF (0.7 mL) and the reaction was stirred for 18 h. Column chromatography on silica using CHCl / MeOH (0-10%) afforded 22b (5.0 mg, 42% yield) as a white solid.

[0121] 22b: White solid. 1 H NMR (400 MHz,CD3OD) δ 6.92 (d,J = 1.6 Hz,1H),6.83 (d,J = 1.6 Hz,1H),4.59 (s,1H),4.36 (q,J = 7.2 Hz,1H),4.02 - 3.93 (m,1H),3.63 (d,J = 5.9 Hz,1H),2.77 (s,3H),2.30 (dd,J = 13.3,6.3 Hz,1H),1.98 (dd,J = 13.4,8.0 Hz,1H),0.85 (s,9H),0.06 (d,J = 11.2 Hz,6H).

[0122] [ka]

[0123] To a stirred solution of 22b (28 mg, 0.057 mmol) in THF (1.0 mL) was added TBAF (1 M solution in THF, 0.11 mL, 0.11 mmol, 2.0 equiv) at ambient temperature (22 °C). The mixture was stirred for 12 h and all starting material was consumed as shown by TLC. The mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica using MeOH / CH2Cl2 (0-10%) to give 25b (15.0 mg, 70% yield) as a colorless oil.

[0124] 25b: Colorless oil. 1 H NMR (600 MHz,CD3OD) δ 6.93 (d,J = 1.6 Hz,1H),6.87 (d,J = 1.6 Hz,1H),4.20 (td,J = 9.0,6.9 Hz,1H),3.89 (dd,J = 8.4,6.6 Hz,1H),3.73 (d,J = 6.5 Hz,1H),3.07 (s,3H),2.73 (s,3H),2.36 (dd,J = 13.4,6.8 Hz,1H),1.99 (dd,J = 13.4,9.3 Hz,1H).

[0125] [ka]

[0126] A 1.0 dram vial was charged with alcohol 25b (2.8 mg, 0.0075 mmol, 1.0 equiv), triphenylphosphine (5.9 mg, 0.0225 mmol, 3.0 equiv), and THF (0.2 mL). The mixture was cooled to 0° C. and a stock solution of diethyl azodicarboxylate (DEAD, 10 ul, 0.0225 mmol, 3.0 equiv) was added (a stock solution of DEAD was made by dissolving 35 ul of DEAD in 0.1 mL of THF). Two more batches of DEAD solution (10 ul) were added every hour thereafter, and 25b was completely consumed as indicated by TLC. The reaction was concentrated and the residue was purified by column chromatography on silica (dry loaded, methanol / dichloromethane 0-10%). The product was further purified by semi-preparative HPLC to give oxazoline 26b (1.1 mg, 41%) as a colorless oil for characterization.

[0127] 26b: Colorless oil. 1 H NMR (600 MHz,CD3OD) δ 6.96 (d,J = 1.7 Hz,1H),6.76 (d,J = 1.7 Hz,1H),5.15 (td,J = 7.8,5.4 Hz,1H),4.41 (dd,J = 8.3,1.8 Hz,1H),3.99 (d,J = 1.8 Hz,1H),3.12 (s,3H),2.75 (dd,J = 15.1,7.5 Hz,1H),2.70 (s,3H),2.19 (dd,J = 15.1,5.4 Hz,1H); 13 C NMR (150 MHz,CD3OD) δ 161.9,159.3,123.7,121.1,116.1,102.1,98.0,83.3,79.9,63.4,51.1,42.4,24.7.

[0128] [ka]

[0129] A 100 mL round bottom flask was charged with silyl ether 20 (1.40 g, 3.07 mmol, 1.0 equiv) and methanol (61 mL). The mixture was cooled to 0° C. and a 1 M solution of hydrochloric acid in diethyl ether (0.31 mL, 0.31 mmol, 0.1 equiv) was added dropwise. The solution was warmed to ambient temperature (22° C.) for 1 h and a white solid formed. THF (10 mL) was added to dissolve the solid. After stirring for 0.5 h, all of the starting material had reacted as indicated by TLC and the reaction was concentrated in vacuo to remove all solvent. The resulting solid was redissolved in THF (61 mL) and the solution was cooled to −20° C. and a tetrabutylammonium fluoride (TBAF) solution (1 M in THF, 3.38 mL, 3.38 mmol, 1.1 equiv) was added and the reaction was warmed to ambient temperature (22° C.) and stirred for 21 h. The reaction was complete as indicated by TLC and was concentrated in vacuo, and the residue was purified by MPLC on silica (wet loading, methanol / dichloromethane 0-10%) to give alcohol 27 (1.0 g, 92%) as a white solid.

[0130] 27: White solid. 1 H NMR (600 MHz,CD3OD) δ 7.81 (d,J = 8.0 Hz,2H),7.38 (d,J = 8.0 Hz,2H),3.98 (q,J = 6.9 Hz,1H),3.70 (d,J = 5.0 Hz,1H),3.14 - 2.92 (m,4H),2.66 (s,3H),2.43 (s,3H),2.22 (dd,J = 13.6,6.3 Hz,1H),1.89 (dd,J = 13.6,7.8 Hz,1H); 13 IR (thin film, cm -1 ) 3393,3147,1703,1159; HRMS (ESI) C 15 H 21 N3NaO5S + [M+Na] +Calculated value: 378.1094, measured value: 378.1099.

[0131] [ka]

[0132] In a 25 mL round bottom flask, alcohol 27 (210 mg, 0.59 mmol, 1.0 equiv) was dissolved in anhydrous N,N-dimethylformaldehyde (5.9 mL). The reaction flask was kept at ambient temperature in a water bath and 2-nitrophenyl selenocyanate (536.7 mg, 2.36 mmol, 4.0 equiv) was added, the mixture becoming a clear red solution. To this solution was added tributylphosphine (0.59 mL, 2.36 mmol, 4.0 equiv) dropwise and the mixture was held for 1 h, at which point all of 27 was consumed as indicated by TLC. The mixture was then cooled to 0° C. and methanol (11.8 mL) was added. After stirring at ambient temperature (22 °C) for 10 min, the mixture was concentrated under high vacuum to remove the solvent and the residue was purified by MPLC on silica (dry loading, CH3OH / CH2Cl2, 0-5%) to give a mixture of the selenide intermediate and tributylphosphine oxide (0.88 g). This intermediate was then dissolved in THF (4.62 mL) at 0 °C and hydrogen peroxide (35%, 0.12 mL, 1.58 mmol) was added dropwise. After warming to ambient temperature (22 °C) over 1 h, the mixture was stirred for an additional 6 h. Sodium sulfite (1.0 g) was added solid and the reaction was stirred for 5 min and concentrated in vacuo. The residue was purified by MPLC on silica (dry loading, MeOH / CH2Cl2, 0-10%) to give a mixture of alkene 28 and POBu3 (284 mg) (containing ca. 83.8 mg of desired product, 42% over two steps). The product was repurified by MPLC (dry loading, acetone / dichloromethane, 0-50%) to give pure 28 as a pale yellow oil for characterization purposes.

[0133] 28: Pale yellow oil. 11H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 6.04 (dd, J = 5.9, 2.2 Hz, 1H), 5.66 (dd, J = 5.9, 2.2 Hz, 1H), 5.14 (s, 1H), 5.03 (d, J = 8.6 Hz, 1H), 4.03 (dq, J = 8.5, 2.3 Hz, 1H), 3.88 (q, J = 2.0 Hz, 1H), 3.15 (s, 3H), 2.72 (s, 3H), 2.44 (s, 3H); 13 13C NMR (100 MHz, CDCl3) δ 158.6, 144.4, 136.9, 133.3, 133.0, 130.2 (2), 127.3 (2), 102.8, 66.7, 63.9, 50.1, 25.2, 21.7; IR (thin film, cm-1) 3374, 1694, 1447, 1331, 1160; HRMS (ESI) C 15 H 19 N3NaO4S + [M+Na] + Calculated value for [M+Na] 360.0988, found 360.0991.

[0134]

Chem.

[0135] A 100 mL round bottom flask was charged with 28 (0.8 g mixture containing 0.24 g POBu, 0.56 g, 1.66 mmol, 1.0 equiv.) and the flask was placed under vacuum and backfilled with argon three times. Degassed water (freeze-pump-thaw, 0.6 mL, 33.2 mmol, 20 equiv.) and samarium iodide solution (0.1 M in THF, 166 mL, 16.6 mmol, 10 equiv.) were added sequentially at ambient temperature (22 °C). Triethylamine (freshly distilled over CaH2, 6.9 mL, 49.8 mmol, 30 equiv.) was added dropwise over 10 min. TLC showed all starting material had been consumed. The reaction was filtered and the mother liquor was collected while the solid was collected and transferred to six 10 mL centrifuge tubes and extracted with 10 mL of CH3OH-CH2Cl2 (20%, v / v, containing 1% NEt3) in each tube. This process was repeated 6-10 times until no more product was seen by TLC. The liquid was collected and concentrated and the residue was purified by MPLC on silica (equipped with ELSD detector, dry packed, 0-10% CH3OH / CH2Cl2 containing 1% NEt3) to give amine 29 (214.4 mg, 71%) as a colorless oil.

[0136] 29: Colorless oil. 1 H NMR (400 MHz,CD3OD) δ 6.04 (dd,J = 6.4,1.4 Hz,1H),6.00 (dd,J = 6.0,2.0 Hz,1H),3.73 (d,J = 2.1 Hz,1H),3.68 (d,J = 2.2 Hz,1H),3.18 (s,3H),2.72 (s,3H); 13 C NMR (100 MHz,CD3OD) δ 161.4,138.9,131.3,105.2,66.3,64.9,50.2,25.3; IR (thin film,cm -1 ) 3280,2360,2341,1698; HRMS (ESI) C8H 13 N3NaO2 + [M+Na]+ calculated value 206.0900, found value 206.0900.

[0137] [ka]

[0138] In a 1.0 dram vial, amine 29 (24.2 mg, 0.13 mmol, 1.0 equiv) was dissolved in dichloromethane (0.6 mL), triethylamine (55 ul, 0.4 mmol, 3.0 equiv) was added, and the mixture was cooled to -20°C. In another 1.0 dram vial, 2-bromo-1H-pyrrole (37.6 mg, 0.2 mmol, 1.5 equiv) was dissolved in dichloromethane (0.7 mL) at 15°C, N,N-dimethylformaldehyde (0.5 drops) was added, followed by oxalyl chloride (25 μl, 0.30 mmol, 2.25 equiv). The mixture was stirred for 30 min and transferred by cannula to another vial containing substrate 29 and triethylamine at -20°C. The mixture became a cloudy pale yellow solution. After stirring for 15 min, the reaction was allowed to warm to ambient temperature. Methanol (1.0 mL) was added and stirred for 20 min. The reaction was concentrated in vacuo and the residue was purified by MPLC (dry loading) on ​​silica using 0-5% methanol / dichloromethane to give the amide 30 as a white solid (27 mg, 58%).

[0139] 30: White solid. 1 H NMR (500 MHz,CD3SO2CD3) δ 12.23 (s,1H),8.34 (d,J = 7.4 Hz,1H),7.19 (d,J = 2.0 Hz,1H),6.84 (dd,J = 3.8,2.2 Hz,1H),6.22 - 6.08 (m,2H),5.95 (dd,J = 5.9,2.1 Hz,1H),4.61 (tt,J = 4.6,2.3 Hz,1H),3.78 (t,J = 2.3 Hz,1H),3.06 (s,3H),2.62 (s,3H); 13 IR (thin film, cm -1) 3325,2258,1691,1628,1440,1056; HRMS (ESI) C13H15BrN4NaO3+ [M+Na]+ calcd 377.0220, found 377.0222.

[0140] [ka]

[0141] To a stirred solution of 30 (14.7 mg, 0.041 mmol, 1.0 equiv) in THF (4.1 mL) in a 10 mL round bottom flask at 0° C. was added aqueous hydrochloride solution (0.1 M, 0.2 mL, 0.02 mmol, 0.5 equiv). The reaction was stirred for 10 min and warmed to ambient temperature (22° C.) for 30 min. After stirring for 2 h, the reaction was cooled back to 0° C., the pH was adjusted to 7 by addition of saturated NaHCO3 solution, and the solvent was removed under high vacuum to give crude intermediate 31 as a pale yellow solid.

[0142] Crude 31 was dissolved in anhydrous methanol (1.3 mL) and the mixture was cooled to 0 °C. Saturated K2CO3 / MeOH solution (45 uL, 0.01 mmol, 0.25 equiv) was added and the reaction was warmed to ambient temperature (22 °C) for 1 h. The solid was then filtered and washed with methanol (0.5 mL x 2). The mother liquors were combined and concentrated and the residue was purified by column chromatography on silica (dry loading, 0-10% methanol / dichloromethane) to give agelastatin A as an off-white solid (1.7 mg). The solid cake from the reaction was dried, redissolved in DMSO (1.0 mL) and filtered through a cotton plug. The DMSO was removed under high vacuum to give agelastatin A (4.3 mg) in pure form as an off-white solid (combined yield: 43%).

[0143] 33: Off-white solid. 1H NMR (600 MHz,CD3OD) δ 6.91 (d,J = 4.1 Hz,1H),6.33 (d,J = 4.1 Hz,1H),4.60 (apparently dt,J = 12.1,6.1 Hz,1H),4.09 (d,J = 5.5 Hz,1H),3.88 (s,1H),2.81 (s,3H),2.65 (dd,J = 13.1,6.5 Hz,1H),2.10 (t,J = 12.6 Hz,1H); 13 C NMR (150 MHz, CD3OD) δ 161.4, 161.1, 124.1, 116.0, 113.8, 107.2, 95.7, 67.4, 62.2, 57.5, 54.4, 40.0, 24.2. Data were consistent with those previously reported.

[0144] [ka]

[0145] A 10 mL round bottom flask was charged with amine 29 (53.0 mg, 0.29 mmol, 1 equiv) and DMF (anhydrous, 2.9 mL). The solution was cooled to 0° C. and triethylamine (anhydrous, 0.20 mL, 1.43 mmol, 5.0 equiv) was added. Solid 35 (87.9 mg, 0.35 mmol, 1.2 equiv) was added in one batch and the mixture was allowed to warm to ambient temperature (22° C.) for 1 h. After stirring for an additional 5 h, all of the starting material was consumed as indicated by TLC. To the reaction mixture was added CH3OH (1.0 mL) and a solid precipitated. The solid was collected by filtration and washed with CH3OH (0.2 mL) to give the product 36 as a pale yellow solid (62.0 mg, 67%). The product is pure enough for the next step.

[0146] 36: Pale yellow solid. 1H NMR (500 MHz,CD3SOCD3) δ 12.77 (s,1H),8.76 (d,J = 7.5 Hz,1H),7.92 (d,J = 1.7 Hz,1H),7.55 (d,J = 1.7 Hz,1H),7.23 (d,J = 1.9 Hz,1H),6.18 (dd,J = 5.9,2.0 Hz,1H),5.96 (dd,J = 5.9,2.1 Hz,1H),4.63 (dq,J = 7.1,2.3 Hz,1H),3.82 (t,J = 2.4 Hz,1H),3.07 (s,3H),2.62 (s,3H); 13 C NMR (125 MHz,CD3SOCD3) δ 158.9,158.4,136.3,134.8,131.6,126.5,122.9,105.8,102.4,62.7,61.5,49.1,24.7; -1 ) 1677,1638,1331,1045; HRMS (ESI) C 13 H 15 N5NaO5 + [M+Na] + Calculated value: 344.0965, measured value: 344.0966.

[0147] [ka]

[0148] To a stirred suspension of 36 (10.6 mg, 0.033 mmol, 1.0 equiv) in THF (1.5 mL) and 1,3-dioxane (1.8 mL) at 0° C. was added aqueous HCl (0.1 M, 0.17 mL, 0.017 mmol, 0.5 equiv). The reaction was stirred for 10 min and warmed to ambient temperature (22° C.). After stirring for an additional hour, the reaction was complete and the pH was adjusted to 8 by addition of saturated NaHCO3 solution and the solvent was removed under high vacuum to give a solid as the crude intermediate.

[0149] The crude intermediate was dissolved in methanol (1.7 mL) and N,N-dimethylformaldehyde (0.2 mL) and the mixture was cooled to 0° C. Saturated K2CO3 / MeOH solution (freshly prepared by stirring K2CO3 in MeOH overnight, 37 uL, 0.0083 mmol, 0.25 equiv). After 30 min, another batch of K2CO3 / MeOH solution (144 uL, ca. 0.033 mmol, 1.0 equiv) was added and the reaction was warmed to ambient temperature (22° C.) and stirred for 20 h, at which time a pale yellow solid precipitated. The solid was filtered and washed with methanol (1.0 mL×3). The solid cake was dried, redissolved in DMSO (0.5 mL) and filtered through a cotton plug. The liquid was collected and dried under high vacuum to give the desired 37 in high purity as a pale yellow solid (4.4 mg, 41% over two steps).

[0150] 37: Pale yellow solid. 1 H NMR (500 MHz,CD3SOCD3) δ 8.43 (s,1H),8.22 (d,J = 1.9 Hz,1H),7.15 (d,J = 1.9 Hz,1H),7.09 (d,J = 2.5 Hz,1H),4.69 (dt,J = 11.3,5.8 Hz,1H),3.93 (d,J = 5.2 Hz,1H),3.70 (s,1H),2.64 (s,3H),2.58 (dd,J = 13.0,6.5 Hz,1H),2.15 (dd,J = 13.0,10.8 Hz,1H); 13 C NMR (125 MHz,CD3SOCD3) δ 158.4,157.1,136.1,124.2,123.2,107.1,93.4,65.6,60.4,54.2,39.9,23.7; -1 ) 3184,1654,1494,1373,1307; HRMS (ESI) C 12 H 12 N5O5 - [MH] - Calculated value: 306.0844, measured value: 308.0846.

[0151] Cancer cell line cytotoxicity assay Cancer cell lines (MDA-MB-231, MCF7, Caco2, or U87) were plated at 2,000 cells per well in 96-well plates and incubated at 37° C. for 24 hours in 5% CO2. AglA and derivatives were dissolved in DMSO and diluted to appropriate concentrations, and compound solutions or equivalent concentrations of solvent (vehicle) were added to the plates and incubated at 37° C. for 72 hours in 5% CO2. 20 μL of CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega) or CellTiter-Blue® Cell Viability Assay (resazurin, Promega) were added according to the manufacturer's protocol. Plates were developed for 1-4 hours at 37°C in 5% CO2 and evaluated on an AccuSkan Go (Fisher Scientific) at 490 nm or a VarioSkan Lux Multimode Microplate Reader (Fisher Scientific) at 560 nm excitation and 590 nm emission.

[0152] While exemplary embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. Formula (I): [Formula 1] (In the formula, R １ is selected from the group consisting of H, F, Cl, and Br; R ２ is selected from the group consisting of H, F, Cl, and Br; R ３ Br, CF ３ , S.F. ５ , S.O. ２ CF ３ , S.O. ２ CH ３ , C.N., and N.O. ２ selected from the group consisting of: R ４ is selected from the group consisting of H, OH, F, Cl, Br, and CN; and R ５ is H) or a stereoisomer, racemate, or pharma- ceutically acceptable salt thereof.

2. R １ , R ２ , R ４ , and R ５ is H, and R ３ The compound of claim 1 , wherein is Br.

3. R １ , R ２ , and R ５ is H, and R ３ and R ４ The compound of claim 1 , wherein is Br.

4. R １ ~R ５ The compound of claim 1 , wherein is hydrogen.

5. R １ and R ２ is independently selected from hydrogen and halo (e.g., fluoro, chloro, bromo); R ３ ~R ５ The compound of claim 1 , wherein is hydrogen.

6. A method for making a compound of formula (I), comprising converting a compound of formula (A) to a compound of formula (I) via a compound of formula (B): [chemical 2] The compound of formula (A) [C3] to produce an amide (a): [C4] resulting in; The amide (a) is converted to a compound of formula (B) above, followed by ring closure to give the 7-hydroxy compound (b): [C5] resulting in; The 7-hydroxy compound (b) is converted to the compound of formula (I) by treatment with aqueous acid. (In the formula, P is an alcohol protecting group; R is a methyl group; R １ is selected from the group consisting of H, F, Cl, and Br; R ２ is selected from the group consisting of H, F, Cl, and Br; R ３ Br, CF ３ , S.F. ５ , S.O. ２ CF ３ , S.O. ２ CH ３ , C.N., and N.O. ２ selected from the group consisting of: R ４ is selected from the group consisting of H, OH, F, Cl, Br, and CN; and R ５ is H) method.

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5, or a stereoisomer, racemate, or pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

8. 13. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 5, or a stereoisomer, racemate, or pharma- ceutically acceptable salt thereof.

9. 9. The method of claim 8, wherein the cancer is breast cancer (triple-negative breast cancer or estrogen receptor positive breast cancer) or glioblastoma.

10. A method for inhibiting protein synthesis through interaction with the peptidyl transferase center of the ribosome in a subject, comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 5, or a stereoisomer, racemate, or pharma- ceutically acceptable salt thereof, in an amount effective to inhibit protein synthesis through interaction with the peptidyl transferase center of the ribosome.

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