Simplified Cyclic Analogs of Bastadin-5. Structure Activity Relationships for Modulation of the RyR1/FKBP12 Ca2+ Channel Complex

Makoto N Masuno; Isaac N Pessah; Marilyn M Olmstead; Tadeusz F Molinski

doi:10.1021/jm050708u

. Author manuscript; available in PMC: 2014 Apr 15.

Published in final edited form as: J Med Chem. 2006 Jul 27;49(15):4497–4511. doi: 10.1021/jm050708u

Simplified Cyclic Analogs of Bastadin-5. Structure Activity Relationships for Modulation of the RyR1/FKBP12 Ca²⁺ Channel Complex

Makoto N Masuno ^†, Isaac N Pessah ^‡, Marilyn M Olmstead ^†, Tadeusz F Molinski ^*,^†,^£,^#

PMCID: PMC3987770 NIHMSID: NIHMS61207 PMID: 16854055

Abstract

Bastadin-5, a brominated macro-dilactam from the marine sponge Ianthella basta, enhances release of Ca²⁺ from stores within the sarcoplasmic reticulum (SR) of muscle and non-muscle cells by modulating RyR1/FKBP12 complex. Analogs of bastadin-5 present desirable targets for SAR studies to shed light on the gating mechanism and locus of bastadin-5 binding on these heteromeric channels that mediate essential steps in early coupling of membrane excitation to Ca²⁺ signaling cascades. Simple, ring-constrained analogs of bastadin-5 were synthesized from substituted benzaldehydes in a convergent manner, featuring an efficient S_NAr macroetherification, and evaluated in an assay that measures [³H]-ryanodine that is known to correlate with the functional open state of the Ca²⁺ channel. The simplified 14-membered ring, atropisomeric analog (±)-7, like bastadin-5, enhanced ryanodine binding to the RyR1/FKBP12 complex (EC₅₀ 11 μM), however, unexpectedly, the corresponding achiral 18-membered ring analog 14 potently inhibited binding (IC₅₀ 6 μM) under the same conditions. Structure-activity relationships of both families of cyclic analogs showed activity in a ryanodine binding assay that varied with substitutions of the Br atom on the trisubstituted aryl ring by various functional groups. The most active analogs were those that conserved the dibromocatechol ether moiety that corresponds to the ‘western edge’ of the bastadin-5 structure. These data suggest that cyclic analogs of bastadin-5 interact with the channel complex in a complex manner that can either enhance or inhibit channel activity.

Introduction

Natural products have played a major role in our current understanding of how FKBP12, an immunophilin, regulates both cellular signal processing and Ca²⁺ efflux within the junctional sarcoplasmic reticulum (JSR) of skeletal muscle tissue. Regulated Ca²⁺ release from the SR is critical for normal striated muscle contractility and dysfunctional Ca²⁺ channel conductance carries important consequences in disease states of skeletal and cardiac muscle. The immunophillins rapamycin (1) and FK506 (2) are two macrolide immunosuppressant compounds that bind to FKPB12 with high affinity and have been used to explore the RyR1/FKBP12 complex.^1-8 Bastadins are known compounds possessing newly identified properties – allosteric interactions with the RyR1/FKBP12 complex.^9-13

Bastadin-5 (3) is one member of a family of over 20 bromotyrosine-derived macrolactams that have been isolated from the Verongid marine sponges Ianthella basta (Pallas), I. flabeliformis, I. quadrangulata, I. sp. and Psammaplysilla purpurea.^12,14-24 Our previous studies show that several, but not all, bastadins stimulate Ca²⁺ release from stores in the JSR by binding to the RyR1/FKBP12 channel in skeletal muscle. The most active member of the family is bastadin-5 (3, EC₅₀= 2.2 μM), while a constitutional isomer of 3, bastadin-19 (3a), does not mobilize Ca²⁺ from the channel (EC₅₀ >100 μM), but competes for the binding site of bastadin-5.¹³ Bastadin-5 also facilitates FK506-induced release of FKBP12 from RyR1, indicating the former may influence stability of the RyR1/FKBP12 complex.¹³ Although these effects are concentration-dependent and follow saturable sigmoidal binding isotherms, the locus of binding of bastadin-5 to the RyR1/FKBP12 complex is not yet known.

The structure of 3 is comprised of four brominated, modified tyrosines and tyramines arrayed within a 28-membered macrodilactam. Biosynthetically, each half of the 28-membered ring macrocycle that is common to most bastadins appears to derive from oxidative coupling of one unit each of bromotyramine and bromotyrosine. The two halves are united through two amide bonds to give a pseudo-tetrameric macrodilactam. Each of the 21 bastadins has an α-ketoxime that appears to derive from α-oxidation of the bromotyrosine. Oxidative modification may also occur at C5/C6 to introduce a vinyl bond (e.g. bastadin-4) or a C6 hydroxyl group (e.g. bastadin-7). The most active member of this family, bastadin-5 (3), stimulates release of Ca²⁺ from stores in the SR by binding to an as-yet unidentified site of the intact RyR1/FKBP12 complex in skeletal muscle tissues.^11,13 The RyR1/FKBP12 complex is a ~2.2 MDalton heterotetrameric protein anchored within the junctional sarcoplasmic reticulum (JSR) and spans the 100 nm gap between the JSR and transverse tubule membranes. The RyR1 complex is therefore essential for orchestrating physiological Ca²⁺ release during excitation contraction coupling of skeletal muscle. Each monomeric polypeptide is comprised of ~5000 amino acid residues folded into several transmembrane domains. Competition studies have revealed that the binding site of 3 is distinct from sites on the channel surface that are recognized as other SR Ca²⁺ channel effectors, such as Ca²⁺, Mg²⁺, ATP, caffeine or the plant alkaloid ryanodine.¹³ Bastadins mediate their effects upon the Ca²⁺ channel without compromising the RyR1/FKBP12 association, unlike FK506 which promotes the dissociation of FKBP12 from RyR1. While the macrolide FK506 was useful in revealing the nature of interaction of the large tetrameric channel complex with FKBP12, the bastadins have provided information on the intact RyR1/FKBP12 Ca²⁺ channel complex. For example, addition of 3 to Ca²⁺ channel preparations revealed the function of the RyR1/FKBP12 complexes as a regulator in the filling capacity of the Ca²⁺ stores by influencing the “leak state” conformation of RyR1.¹⁰

Members of the bastadin family show a range of potency (EC₅₀ 2.3 to >100 μM) for binding to the RyR1/FKBP12 complex. Since the functional groups present in each bastadin are essentially the same, we hypothesize that this structural activity relationship (SAR) is attributable to different preferred solution conformers of bastadins as a result of differences in non-bonded interactions (e.g. Br-steric interactions) in the ‘western’ and ‘eastern’ parts of the bastadins. Consequently, the altered torsional angles and even the overall shape of the molecule, may affect the receptor binding energy. We proposed the synthesis of simple rationally designed cyclic analogs of 3 (e.g. 4-15), which embody the substituted catechol ethers of the ‘western’ hemisphere of 3 within two families of macrocycles bearing 14- and 18-membered ring sizes. Ring-constrained macrocycles with constrained aryl C-O-C-C torsional angles may reveal preferential ryanodine-binding agonism, which in turn may shed light on the minimum pharmacophore of 3. We report here the results of a study which shows the minimal analog 7 (EC₅₀= 11 μM), which embodies the bromocatechol ether unit found in 3, has comparable activity to the natural product 3 in the ryanodine binding assay. Unexpectedly, compound 14, a synthetic 18-membered ring analog of 3, was a potent antagonist in the same binding assay (IC₅₀= 6 μM) which suggests brominated catechol ethers exhibit a bi-modal activity that varies with non-bonded interactions. Interestingly, the ketoximino group found in the natural products appears not to be required for activity. Our studies suggest a complex interplay of stereoelectronic factors in binding of bastadin-5 analogs to the RyR1/FKBP12 receptor, which suggests a bi-modal model for Ca²⁺ gating, which may be tunable by a selection of properly designed ‘second generation’ analogs.

Synthesis

The retro-synthetic analysis shown in Scheme 1 reveals the design for preparation of cyclic analogs. We envisioned the 14-membered cyclic analogs, a smaller, ring-constrained macrocycle embodying a single amide bond, would derive from common intermediates 16 and 17. The larger 18-membered analogs would include an additional β-alanine as a spacer. The units would be assembled by amide bond formation and the pivotal macrocyclization would be accomplished by S_NAr coupling according to Zhu and coworkers.^25,26 The nitro group in compounds 18 and 19 would activate the leaving group and serve as a handle to introduce variable functionality at this position through diazonium salt displacements.

Simple Dreiding molecular models predicted that the 14-membered analogs would be locked into fixed conformations as atropisomers due to severe torsional strain that arises from rotation about the long axis of the para-O-aryl linkage, while the 18-membered ring analogs should retain relative conformational mobility.

The synthesis of common intermediate 16 is illustrated in Scheme 2. Key considerations in the synthesis included management of the reductively-sensitive aryl Br groups and O-benzyl protecting groups. Synthesis of substituted phenethylamine 16 was carried out using methodology we had optimized earlier to satisfy these criteria.²⁷

The method is amenable to radio-labeling of analogs by insertion of tritium.²⁸ Selective benzylation of the more acidic hydroxyl of 20 was performed through a modification of a published procedure.²⁹ Pretreatment of catechol 20 with base (DMF, Li₂CO₃, 45 °C, 1 h) prior to the addition of benzyl bromide achieved selective benzylation of the para-OH group to provide 21 in good yield (88%). The position of benzylation was confirmed by nOe experiments. A Doebner-modified Knoevenagel reaction of 21 led to the requisite α,β-unsaturated acid 22 in 86% yield which was converted to enamide 23 by a Curtius rearrangement of the corresponding acyl azide (65% over 3 steps).³⁰ Cationic hydrogenation of enamide 23 gave carbamate 24 (94%).²⁷ Standard conditions for hydrolysis of methyl/ethyl carbamates (e.g. (KOH, H₂O, EtOH, reflux) were ineffective when applied to 24 – either starting material or the corresponding oxazolidone, formed by cyclization/elimination of MeOH/EtOH, were returned. More forcing conditions (NH₂NH₂, KOH, 1,4-dioxane, 80 °C) gave phenethylamine 16 in good yield (92%).

Hydrogenation of methyl 4-fluoro-3-nitrocinnamate (25) in the presence of Wilkinson's catalyst³¹ with careful control of H₂ pressure and temperature gave methyl ester 26 in excellent yield (95%)³² without reduction of the nitro functionality. Saponification of ester 26 completed the synthesis of the dihydrocinnamic acid 17 (86%).

Two possible routes to the cyclic intermediates were considered; S_NAr coupling of a suitable aryl fluoride with a phenol followed by macrolactamization, or the reverse sequence of reactions. In the event, the latter method proved to be the most efficient (Scheme 4) whereas the former resulted in uniformly poor yields of cyclic product (<10%). Amide coupling of intermediates 16 and 17 afforded the macroetherification precursor 18 in 72% yield. Macroetherification of 18 under dilute conditions (2mM, K₂CO₃, 4Å sieves, DMSO, RT) smoothly converted 18 to lactam 27 in high yield (85%). Removal of the benzyl group provided the 14-membered ring-constrained analog 4. Reduction of lactam 27 to the aniline derivative 28 under conditions that preserved the reductively sensitive aryl Br groups (CrCl₂, DMF, room temperature, 73%) and set the stage for the synthesis of several analogs via diazonium salts (Scheme 5). Removal of the benzyl group from 28 gave aniline 5. The diazonium salt prepared from 5 was quenched with sodium azide to give the azido analog 6 in 66% yield.³³ The dibromo or tribromide compounds 29 and 30 were procured in acceptable yields by Sandmeyer-type reaction of the corresponding diazonium salts prepared from 28 (tert-BuNO₂, CH₃CN) and use of carefully controlled amounts of CuBr₂ (0.8 or 10 equiv; respectively). The use of sub-stoichiometric CuBr₂ (0.8 equiv) resulted in formation of 29, however, excess CuBr₂ (10 equiv) gave the brominated product 30.³⁴ Compound 28 suffered unexpected reductive deamination when the corresponding diazonium salt was prepared with tert-BuNO₂ in the presence of THF to give compound 31, presumably by hydride abstraction from the solvent.^34b Quenching the latter diazonium salt potassium iodide resulted in aryl iodide 10. Removal of the benzyl protecting group in 29, 30, and 31, as before, yielded the corresponding phenolic 14-membered analogs 7, 8, and 9, respectively.

Scheme 4 — Synthesis of 14-membered analogs **4, 5** and 6 via macroetherification.

Key: (a) i. EDCI, HOBT, CH₂Cl₂, RT, 12 h; ii. 0.5 N HCl, 72%; (b) i. K₂CO₃, DMSO (2 mM), 4 Å sieves, 2 h; ii. 1N HCl, 85%; (c) i. CrCl₂, DMF, RT, 12 h; ii. 1 N HCl, 73%; (d) BBr₃, –78 °C, 1 h, 91%; (e) BBr₃ –78 °C, 1h, 58%; (f) i. AcOH/H₂O, 9:1, NaNO₂, 0 °C; ii. NaN₃, 0 °C, 66%

Scheme 5 — Preparation of C-aryl-substituted 14-membered ring analogs.

Key: (a) i. CuBr₂ (0.8 equiv), ^tBuONO, CH₃CN, 0 °C, 1h; ii. 27 in portions, 0 °C, 2h then warmed to rt; *iii*. 1N HCl, 66% (b) i. CuBr₂ (10 eq), ^tBuONO, CH₃CN, 0 °C, 1h; ii. 28 in portions, 0 °C, 2h then warmed to rt; *iii*. 1N HCl, 32%; (c) i. ^tBuONO, THF, 0 °C, 1h; ii. 28 in portions, 0 °C, 2h then warmed to rt; *iii*. 1N HCl, 39%; (d) i. H₂SO₄, AcOH, NaNO₂, 0 °C, 0.5 h; ii. 28 in portions, 0 °C, 1 h then warmed to rt, 20 min; *iii*. KI, H₂O, rt, 15 min then warmed to 70 °C, 15 min, 16%; (e) BBr₃, –78 ° C, 1h, 91%; (f) TFA, rt, 24h, 70%; (g) TFA, rt, 24h, 75%.

Synthesis of the 18-membered ring analogs is illustrated in Scheme 6. Phenol 32 was protected as a triisopropylsilyl ether (95%) followed by removal of the N-Boc under standard conditions to provide amine 34 (97%), which was coupled, in turn, with N-Boc-β-alanine to afford amide 35 in 92% yield. Removal of the Boc group from 35 as before (97%) followed by coupling of the resultant free-amine 36 to acid 17 (EDCI, HOBt, CH₂Cl₂) provided the precursor for cyclization 19 in 92% yield. One-pot, tandem removal of the TIPS protecting group and macroetherification of 19 with CsF smoothly produced the 18-membered cyclic product 37 in excellent yield (84%). The nitro group in lactam 37 was reduced as before to provide the aniline 38 (58%) which was deprotected to provide phenolic amine 12 (54%). Diazotization, substitution and deprotection reactions were carried out using similar sequences to those described above to provide azido analog 13 (69%), dibromo analog 14 and tribromide 15.

Structure and Biological Evaluation

For the purpose of discussion, the 14-membered analogs are numbered according to Figure 2. The simple cyclic analogs were designed to embody the ‘western’ portion of 3 but with different ring sizes and substitutions on ring B (C17). It was hypothesized that the ‘western’ portion of 3 composed the minimum pharmacophore, where variation of the torsional angle (C1-O2-C3-C4) would modulate activity. The highly constrained 14-membered analogs would contain fewer degrees of freedom and simultaneously constrain both the biphenyl ether angle (C1-O2-C3) and the torsional angle (C1-O2-C3-C4) compared to the larger 18-membered analogs. Varying the substitution on ring B may reveal electronic effects upon the binding of bastadin analogs to the RyR1/FKBP12 complex.

Solution and solid-state conformation studies of the analogs 4-15 were ascertained from NMR nOe and chemical shift analysis (Figure 2, Table 1), and X-Ray crystal structure analysis (Figure 3). With the exception of compound 9, the 14-membered cyclic analogs are chiral and exhibit atropisomerism due to restricted rotation. Consequently the latter compounds were synthesized as racemates. Evidence for atropisomerism is seen in the NMR data as illustrated by spectral interpretation of lactam 29 (the precursor to 7) as follows (Figure 2, Table 1). The ¹H NMR spectrum of 29 showed a diagnostic two-proton signal with an ‘AB quartet’ J coupling pattern (δ 5.18, d, 1H, J=11 Hz; δ 5.34, d, 1H, J=11 Hz) corresponding to diastereotopic O-benzyloxy protons. Restricted rotation about the catechol ether linkage (C1-O2-C3) was also evident from diamagnetism due to ring current effects leading to an exceptionally high field H19 aryl proton signal (δ 5.06, 1H, J=2.0 Hz).

Table 1.

NMR data for 29 (400 MHz, CDCl₃)

#	¹H δ, m, J (Hz)	¹³C (ppm)	COSY	HMBC	nOe
1		152.9
3		154.6
4		136.4
5		117.9
6	6.88 dd (2.0, 1.2)	125.8	H19, H8	C5, C7, C19, C8
7		142.8
8	2.64 m	30.5	H9		H6, H9, H10; H19
9	3.29 m	39.7	H8; H10		H8, H10; H18
10	4.90 m		H9		H8, H9, H12, H19
11		171.1
12	2.29 m	41.0	H13	C11, C13, C14	H10, H13, H15, H18
13	3.00 m	31.7	H12	C11, C12, C14; C15, C18	H12, H15; H18
14		140.6
15	7.23 dd (8.4, 2.4)	130.4	H16; H18	C1, C13, C15; C17	H13, H16
16	7.05 d (8.4)	126.0	H15	C1, C18	H15, H19
17		118.7
18	7.51 d (2.4)	134.6	H15	C1, C13, C15; C17	H12, H13
19	5.06 d (2.0)	113.1	H6	C3, C7, C8, C6	H9, H10; H16
20a	5.34 d (10.8)	75.2	H20b	C4, C21; C (22- 26)
20b	5.18 d (10.8)	75.2	H20a	C4, C21, C (22- 26)
21		137.0
22-26	7.3- 7.4 m; 7.61- 7.64 m	128.8, 128.4, 128.2	H22-26	C20, C22-C26)

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The constrained conformation of 29 places H19 in ring A within the shielding region that is close to a perpendicular extended from center of ring B. Complexity in ¹H NMR signals of other CH₂ signals in 29 was also consistent with diastereotopic methylene groups and, likewise, seen in the ¹H NMR spectra of 7 and other products derived from 28, except 9. The debromo analog 9, where free rotation about the 1,4-axis of the disubstitued phenyl ring is allowed and the barrier to macrocyclic torsional inversion is relaxed, shows no atropisomerism.

A rigid, compact conformation of analog 29 was supported by nOe experiments (Figure 2, Table 1). Representative nOe's of 29 are consistent with a conformation where ring B lies close to parallel with a plane that bisects ring A (Figure 2). The solid-state conformations of 4 and 9 were determined by X-ray crystallography (Figure 3). The catechol ether torsional angles in 4 are 83.0° (C3-O2-C1-C17) and 152.4° (C1-O2-C3-C4). The bond angle (C1-O2-C3) of 110.7° deviated slightly from that expected from sp³ hybridization. Slight bending of the aromatic ring B from planarity (C15-C14-C18-C17, 9.5°) was also evident, compensating in part for the ring strain. Few differences between the solution state conformation and the solid conformation were ascertained; this is not unexpected due to the rigidity imposed by torsional strain within these compounds. Comparison of the X-ray crystal structures of analogs 4 and 9 revealed identical bond angles and distances of their respective carbon skeletons. As expected, the 18-membered counterparts 11-15 did not show atropisomerism due to the additional degrees of freedom conferred by the β-alanine linker.

The barrier to rotation in the 14-membered ring analog 29 was briefly examined by temperature dependent ¹H NMR (d₆-DMSO). At temperatures up to T=105 °C the ¹H NMR lineshapes of the O-Bn signals in 29 remained sharp and revealed no tendency towards coalescence or broadening which suggests a barrier to rotation >17 kcal/mol.³⁵

Biological Activity

Biological evaluation of the synthetic analogs 4-15, together with bastadin-5 (3) was carried out using the [³H]-ryanodine binding assay (Table 2). The assay provides a ‘readout’ of the open state of the Ca²⁺ channel by detection of [³H]-ryanodine bound to the protein which occurs only in the open state.¹³ In earlier studies, we showed that [³H]-ryanodine binding correlates with the functional properties of bastadin-5 (3), in particular, transport of Ca²⁺ across the channel and alteration of channel gating (open state probability).¹³

Table 2.

Structure-activity relationships of bastadin-5 (3) and analogues.^a


Entry	Compound	Skeleton	X	Y	EC₅₀ (μM)^b	IC₅₀ (μM)^c
1	3	A	–	–	2.2 ± 0.1	–
2	(±)-4	B	NO₂	H	20.9 ±10.8	–
4	(±)-5	B	NH₂	H	33.0 ±12.2	–
5	(±)-6	B	N₃	H	–	63 ±8.1
6	(±)-7	B	Br	H	10.9 ±2.6	–
7	(±)-8	B	Br	Br	>100	–
8	(±)-9	B	H	H	>100	–
9	(±)-10	B	I		28.6 ±14.2	–
10	11	C	NO₂	H	24 ± 3.9	–
11	12	C	NH₂	H	21 ±5.7	–
12	13	C	N₃	H	20.0 ± 2.3	–
13	14	C	Br	H	–	6.2 ±1.2
14	(±)-15	C	Br	Br	–	47 ± 15
15	49	D	Br	H	>100
16	52	D	Br	Br	6.5 ±0.7
17	53				>100

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Equilibrium binding of 1 nM [³H]-ryanodine to skeletal junctional sarcoplasmic reticulum (SR) was performed in assay buffer containing 250 mM KCl, 15 mM NaCl, 20 mM HEPES, 20 μM Ca²⁺, pH 7.4. Nonspecific binding was determined in the presence of 1 μM cold ryanodine.

agonist-like activity (enhanced ³[H]-ryanodine binding)

antagonist-like (reduced ³[H]- ryanodine binding)

Equilibrium binding of [³H]-ryanodine to skeletal JSR was measured in the presence of analog and solvent DMSO, or solvent alone (DMSO, 0.5% v/v final concentration), together with assay buffer containing 250 mM KCl, 15 mM NaCl, 20 mM HEPES, 20 μM Ca²⁺, pH 7.4. Nonspecific binding was determined in the presence of 1 μM cold ryanodine.

Most of the 14-membered ring analogs retained at least some of the potency of 3 on the ryanodine binding to the RyR1/FKBP12 complex. The most potent agonist was 7 (EC₅₀= 11 μM) with a structure corresponding to the substitution pattern of bastadin-5. Substitution of the bromine at C17 in ring B by different functional groups (NO₂, NH₂, H, I, N₃) diminished ryanodine binding. Replacement of the bromine with a nitro group in compound 4 resulted in a twofold loss of activity (EC₅₀= 21 μM), whereas the amino analog 5 (EC₅₀= 33 μM) and the iodo analog 10 (EC₅₀= 28 μM) retained only a third of the activity of 7. The debromo analog 9 (EC₅₀< 100 μM) was nearly inactive underscoring the importance of the dibromocatechol ether moiety at the western edge of the bastadin-5 structure. Despite almost identical conformations (X-ray), analogs 4 and 9 showed the largest difference in ryanodine binding among the 14-membered ring analogs (EC₅₀= 21 and >100 μM, respectively). Since the nitro group is expected to occupy space equivalent to the van der Waals radius of a Br atom, it is tempting to speculate that stereoelectronic effects may be responsible for differences in biological activity. Unfortunately, we cannot be more definitive without additional data on the locus of binding of bastadin-5 at the receptor surface and knowledge of the putative amino acid residue contacts that mediate the binding contacts. The addition of a third Br atom on ring B in analog 8 resulted in total loss of agonist activity in this 14-membered ring analog (EC₅₀< 100 μM) although, curiously, this was not the case in the corresponding 18-membered ring analog (see below).

An unexpected trend was revealed by measurements of ryanodine binding in the presence of the 18-membered ring analogs – two of the five compounds were inhibitors of ryanodine binding, which suggests inhibition of Ca²⁺ channel opening. While the amino- and azido-substituted 18-membered ring compounds (12 and 13, respectively) showed weak activity in promoting channel activation (EC₅₀ 21 μM and 24 μM), compound 14 with the bastadin-5-like substitution pattern, was a more potent inhibitor (IC₅₀= 6 μM, Figure 4), in diametric opposition to that of the 14-membered ring 7 with the same aryl substitution pattern which is agonist-like (EC₅₀ 11 μM). The remaining compounds in the 18-membered ring series showed weaker agonist-like activity (EC₅₀'s 24-58 μM).

Concentration-dependent enhancement/inhibition of the binding of 1 nM [³H]-ryanodine to RyR1-FKBP12 in skeletal SR by bastadin analogs (a) 14 and (b) 52. Equilibrium binding of 1 nM [³H]-ryanodine to skeletal junctional sarcoplasmic reticulum (SR) was performed in assay buffer containing 250 mM KCl, 15 mM NaCl, 20 mM HEPES, 20 μM Ca²⁺, pH 7.4. Control (1% DMSO aq) = 0.175 pmol [³H]-ryanodine/mg SR. Nonspecific binding was determined in the presence of 1 μM cold ryanodine. Measurements were carried out in triplicate; error bars represent ±SD.

These results suggest the interaction of simple bastadin-5 analogs with the RyR1/FKBP12 is more complex and shows a broader range of action than expected. Each of the active 14-membered ring analogs were synthesized as racemic mixtures, but we predict that specific protein-drug contacts with the binding site should favor only one of the two enantiomers of each compound. Consequently, it would be of interest to investigate the ryanodine binding and Ca²⁺ transport activity of an enantiopure preparation of the most active analog, 7.

Photoaffinity Analogues

With the ryanodine-binding properties of both 14-membered and 18-membered ring analogs in hand, we asked the question, ‘can a simple photoaffinity analog of 3 be designed which retains high potency for the RyR1/FKBP12 complex?’ Using the key design principles from the 18-membered ring series, we replaced the β-alanine with a β-lysine residue to prepare an 18-membered macrolactam that contains a primary amino group that would could be acylated at the ε-NH₂ group with a photoreactive azidobenzoic acid. Photolysis of the derived azidobenzamide analog of 3 would produce a nitrene that may covalently bond to the RyR1/FKPB12 complex. Tryptic digestion and MS analysis of the photolabelled RyR1/FKPB12-derived peptides from the exposed surface may reveal the consensus amino acid sequence of the bastadin binding site.

Differentially N-protected β-lysine (41) was coupled (EDCI, HOBt) to bromotyramine 34 (Scheme 7), followed by removal of the β-N Fmoc protecting group (81%) and coupling to the substituted dihydrocinnamic acid 17 to give the N,N’-diacyl β-lysine 44 (86% over 2 steps). Exposure of 44 to standard S_NAr macroetherification conditions gave cyclic analog 45 in very good yield (75%). Transformation of the NO₂ group in 44 to the corresponding analogs (replacement of NO₂ by Br (47) and the overbrominated product 50) was achieved, as before, by reduction-diazonium salt displacements. Finally, simultaneous deprotection of the aryl ethers and ε-N-Boc protecting group in each of the latter compounds, followed by coupling to 2-azido-5-iodobenzoic acid³⁷ (EDCI, HOBt) gave the corresponding photoaffinity analogs 49 and 52, respectively, in acceptable yields (two steps, 33% and 48%, respectively).

When tested in the ryanodine binding assay, each of the photoaffinity analogs gave very different results. Compound 49 was essentially inactive (EC₅₀ >100 μM), however, dibromo compound 52 showed very potent agonist-like activity (EC₅₀ = 6 μM, Figure 4). It should be noted that the methyl ester 53 prepared from 2-azido-5-iodobenzoic acid (CH₂N₂, Et₂O, 0°C), was inactive in this assay. Compound 52 shows binding affinity for the receptor similar to that of bastadin-5 (3), and its synthesis is amenable to introduction of radiolabel for preparation of [¹²⁵I]-52. Consequently, 52 should provide a suitable probe for photoaffinity labeling of the RyR-FKBP12 complex.

Conclusion

The synthesis of simple, ring-constrained cyclic analogs of bastadin-5, with structures that embody the ‘western’ edge of 3, was accomplished by an efficient, macrocyclization based on intramolecular S_NAr substitution. Assay of two classes of analogs that differ in ring size and aryl substitution patterns in the [³H]-ryanodine binding assay – which detect the open state of the RyR1/FKBP12 Ca²⁺ channel – revealed an interesting bi-modal range of activity represented by compounds with agonistic and antagonistic properties. In each of the two families, the compound structures that embodied the same ‘western edge’ substitution pattern found in native bastadin-5 (dibromocatechol ether) showed the highest activities. Members of the smaller 14-membered ring family, constrained by a conformationally rigid macrocycle, exhibited atropisomerism. The synthesis of an active photoaffinity probe 52, based on the structure of bastadin-5, has been achieved. The synthetic design of 52 should facilitate preparation of [¹²⁵I]-52 for use in defining the bastadin-5 binding locus on the surface of the heterotetrameric RyR1/ FKBP12 complex that constitutes the membrane bound Ca²⁺ channel of the SR. The unexpected antagonistic activity of the 18-membered ring analogs suggests more complex interactions occur between the receptor site and this family of ligands. A bi-modal gating model that accommodates binding-activation-inactivation of the RyR1/ FKBP12 complex by bastadin-5 analogs suggests the possibility that Ca²⁺ release from the SR may be modulated by ‘fine tuning’ of stereoelectronic factors. Tuned release of Ca²⁺ from stores by custom-designed ligand-gate interactions between small molecule analogs of 3 and the RyR1/FKBP12 complex is an attractive idea that may have practical benefits in treatment of disease conditions that arise from complications of defective SR Ca²⁺ channel activity, including arrythmias, heart failure, and malignant hypothermia.³⁸

Supplementary Material

Supporting Info_rev

NIHMS61207-supplement-Supporting_Info_rev.pdf^{(20.8MB, pdf)}

Natural products that interact with FKBP12 or the RyR1/FKBP12 complex

Scheme 3 — Synthesis of Common Intermediate 17

Key: (a) RhCl(PPh₃)₃, H₂ (3 atm), toluene, 8 h, 95%; (b) i. LiOH, THF, H₂O, MeOH (4:1:1); *ii.* HCl aq. (0.5M), 86%.

Acknowledgements

We thank S. Goth and T-A. Ta for assistance with some of the binding assays. Funding for this study was provided by the U.S. National Institutes of Health (GM57560 to TFM, and ES11269 to INP) and the U.S. Environmental Protection Agency (R829388 to INP).

Experimental

General

TLC was carried out on aluminum plates coated with silica (0.2 mm) containing a fluorescent indicator. Spots were visualized was under a UV lamp then sprayed with a solution of vanillin in ethanolic–H₂SO₄, or ninhydrin in ethanol followed by heating. Purity of each compound was established as >95% by ¹H NMR and HPLC. Pyridine, triethylamine, dimethylsulfoxide (DMSO), and dimethylformamide (DMF) were distilled from glass over CaH₂. Dichloromethane, acetonitrile, toluene, tetrahydrofuran, and 1,4-dioxane were dried through commercial alumina cartridge. Optical rotations were recorded on a Jasco DIP-370 instrument. IR spectra were recorded on a Mattson Galaxy FTIR. . ¹H and ¹³C NMR were recorded at 400 MHz and 100 MHz, respectively, in the stated solvent (≥ 99.5% atom D) and referenced to residual protonated solvent signal (δ_Η CDCl₃ 7.24 ppm; CD₃OD, 3.30 ppm; δ_CCDCl₃ 77.00 ppm, CD₃OD, 49.00 ppm).

[³H]-Ryanodine Binding Asssay

Specific binding of [³H]-ryanodine to high affinity sites on rabbit skeletal membrane vesicles^13,39 was determined by incubating SR protein (25 μg), containing the RyR1-FKBP12 complex, with [³H]-ryanodine (1 nM) for 3.5 h at 37° C in binding assay buffer containing KCl (250 mM), NaCl (15mM), HEPES (20 mM), CaCl₂ (20 μM) and at pH 7.4 (500 μL, final volume). The binding reaction was initiated by addition of a solution of the drug in DMSO (final DMSO conc. ~1%) to the complete assay medium and the incubation was terminated by filtration through Whatman GF/B glass fiber filters using a Brandel cell harvester (Gaithersburg, MD). Separation of bound and free [³H]-ryanodine was performed by washing the filters with ice-cold buffer (3 × 500 μL) containing Tris-HCl (20 mM), KCl (250 mM), NaCl (15 mM) at pH 7.4. Filters were placed in scintillation vials containing scintillant (5 mL). Treatments and controls were measured in triplicate and bound radioactivity (dpm) was measured by scintillation counting and corrected for background. Ryanodine affinity curves were plotted and fitted to sigmoidal functions (Origin, Microcal Software, Inc., Northampton, MA). Error bars represented in Figure 4 are ±1 standard deviation. Positive controls were bastadin-5 (EC₅₀ 2.0 μM) and PCB95 (2,2',3,5',6-pentachlorobiphenyl)^40,41 and nonspecific binding was determined in the presence of 100-fold ‘cold’ ryanodine.

5-Bromo-4-hydroxy-17-nitro-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (4)

BBr₃ (20 μL, 0.21 mmol) was added to a solution of lactam 27 (20 mg, 0.04 mmol) in CH₂Cl₂ (300 μL) at –78 C. The orange solution was stirred (1h, –78 °C), quenched with a NaHCO₃ (aq., satd.) and extracted with EtOAc (3 × 10 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 4. Flash chromatography (SiO₂, CH₂Cl₂/MeOH 20:1) gave 4 (15.0 mg, 91%) as an amorphous solid: mp 259-260 °C (CH₂Cl₂/MeOH); IR (ZnSe, neat) ν 3282, 2933, 1642, 1531, 1346 cm^-1; ¹H NMR (300 MHz, CD₃COCD₃) δ 2.40 (t, J= 6.3 Hz, 2H), 2.60-2.64 (m, 2H), 3.00-3.20 (m, 4H), 5.28 (d, J= 2.0 Hz, 1H), 6.89 (d, J= 2.0 Hz, 1H), 6.93 (brs, 1H) 7.29 (d, J= 8.4 Hz, 1H), 7.63 (dd, J= 8.4, 2.4 Hz, 1H), 8.00 (d, J= 2.4 Hz, 1H), 8.90 (brs, 1H); ¹³C NMR (75 MHz, CD₃COCD₃) δ 30.8 (CH₂), 32.0 (CH₂), 40.2 (CH₂), 40.6 (CH₂), 110.3 (C), 114.3 (CH), 126.7 (CH), 127.4 (CH), 127.9 (CH), 134.0 (C), 137.4 (C), 141.9 (C), 142.3 (C), 144.9 (C), 150.3 (C), 151.1 (CH), 170.9 (C); HRMS (DEI) found m/z 406.0179 [M]⁺, C₁₇H₁₅N₂O₅Br requires 406.0164.

17-Amino-5-bromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (5)

BBr₃ (2 μL, 0.02 mmol) was added to a solution of lactam 28 (3 mg, 0.006 mmol) in CH₂Cl₂ (300 μL) at –78 C. The orange solution was stirred (1h, –78 °C), quenched with a NaHCO₃ (aq., satd.) and extracted with EtOAc (3 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 5. Flash chromatography (SiO₂, CH₂Cl₂/MeOH 20:1) gave 5 (1.4 mg, 58%) as an amorphous solid: IR (ZnSe, neat) ν 3371, 2927, 1635, 1502, 1434 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.15-2.23 (m, 1H), 2.32-2.38 (m, 1H), 2.57-2.71 (m, 2H), 2.86-2.94 (m, 2H), 3.25-3.57 (m, 2H), 4.89 (brs, 1H), 5.36 (d, J= 1.6 Hz, 1H), 5.79 (brs, 1H), 6.62-6.67 (m, 2H), 6.82-6.84 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 30.2 (CH₂), 32.2 (CH₂), 39.7 (CH₂), 41.1 (CH₂), 109.2, 112.7, 120.2, 124.7, 125.06, 131.3, 132.4, 140.3, 141.0, 142.2, 171.8; HRMS (DEI) found m/z 376.0434 [M]⁺, C₁₇H₁₇N₂O₃Br requires 376.0423.

17-Azido-5-bromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (6)

NaNO₂ (0.2 mg, 3 μmol) was added in one portion into a chilled solution (0 °C, ice bath) of aryl amine 5 (1.0 mg, 0.003 mmol) in AcOH/H₂O (20 μL, 9:1). The solution was allowed to stir 15 min followed by the addition of NaN₃ (1.0 mg, 0.015 mmol) in one portion. After 0.5 h, the reaction was quenched with H₂O and was extracted with CH₂Cl₂ (3 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 6. HPLC purification (C₁₈ 5μm Microsorb 10 × 250 mm, MeOH/H₂O, 3:2, 4 mL/min, rt, 6.4 min) provided 6 (0.7 mg, 66%) as an oil: IR (neat) ν 3241, 2923, 2115, 1639, 1500 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.33-2.38 (m, 2H), 2.63-2.67 (m, 2H), 2.94-2.99 (m, 2H), 3.13-3.17 (m, 2H), 5.18 (d, J= 2.0 Hz, 1H), 6.87 (d, J= 2.0 Hz, 1H), 7.01-7.11 (m, 3H), 7.80 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 30.7 (CH₂), 32.6 (CH₂), 40.5 (CH₂), 41.4 (CH₂), 114.0 (CH), 123.3 (CH), 126.7 (CH), 127.1 (CH), 128.6 (CH), 134.0 (C), 136.2 (C), 141.9 (C), 174.4 (C); HRMS (DEI) found m/z 402.0336 [M]⁺, C₁₇H₁₅N₄O₃Br requires 402.0327.

5,17-Dibromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (7)

BBr₃ (2 μL, 0.02 mmol) was added to a solution of lactam 29 (4.0 mg, 7 μmol) in CH₂Cl₂ (200 μL) at –78 C. The orange solution was stirred (1h, –78 °C), quenched with a NaHCO₃ (aq., satd.) and extracted with EtOAc (3 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 7. Flash chromatography (SiO₂, CH₂Cl₂/MeOH 20:1) gave 7 (3.0 mg, 91%) as an amorphous solid: IR (ZnSe, neat) ν 3291, 2933, 1637, 1504, 1224 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.18-2.25 (m, 1H), 2.32-2.38 (m, 1H), 2.61-2.64 (m, 2H), 2.98-3.02 (m, 2H), 3.20-3.36 (m, 2H), 4.85 (brs, 1H), 5.07 (d, J= 2.0 Hz, 1H), 5.85 (brs, 1H), 6.85 (d, J= 2.0 Hz, 1H), 7.11 (d, J= 8.4 Hz, 1H), 7.24 (dd, J= 8.4, 2.0 Hz, 1H), 7.49 (d, J= 2.0 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 30.3 (CH₂), 31.6 (CH₂), 39.5 (CH₂), 41.0 (CH), 112.7 (CH), 118.5 (C), 125.6 (CH), 126.0 (CH), 130.3 (CH), 132.0 (C), 134.3 (CH), 140.9 (C), 148.5 (C); HRMS (DCI/NH₃) found m/z 439.9493 [M]⁺, C₁₇H₁₆O₃NBr₂ requires 439.9497.

5,15,17-Tribromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (8)

Trifluoroacetic acid (0.5 mL) was added to lactam 30 (5 mg, 8 μmol) and was allowed to stir at room temperature for 24 h. The trifluoroacetic acid was removed under reduced pressure and trace trifluoroacetic acid were removed by reevaporation from toluene to give crude 8. Reversed phase HPLC purification (C₁₈, 5 μm Microsorb, 10 × 250 mm, MeOH/H₂O, 60:40, 3 mL/min) provided 8 (3.2 mg, 75%) as a colorless amorphous solid: IR (ZnSe, neat) ν 3284, 2937, 1644, 1503, 1463, 1232, 1058 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.28-2.36 (m, 1H), 2.47-2.70 (m, 3H), 3.00-3.09 (m, 1H), 3.20-3.26 (m, 2H), 3.38-3.47 (m, 1H), 4.90 (d, J= 6.8 Hz, 1H), 5.14 (d, J= 1.6 Hz, 1H), 5.78 (brs, 1H), 6.87 (d, J= 1.6 Hz, 1H), 7.35 (s, 1H), 7.53 (s, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 30.5 (CH₂), 32.0 (CH₂), 37.1 (CH₂), 39.9 (CH₂), 109.6 (C), 112.5 (CH), 118.1 (C), 122.5 (C), 125.9 (CH), 129.4 (CH), 132.6 (C), 136.8 (CH), 139.5 (C), 140.6 (C), 148.1 (C), 153.2 (C), 170.8 (C); HRMS (DCI/NH₃) found m/z 517.8594 [M]⁺, C₁₇H₁₅O₃NBr₃ requires 517.8602.

5-Bromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (9)

Trifluoroacetic acid (0.5 mL) was added to lactam 31 (4 mg, 9 μmol) and was allowed to stir at room temperature for 24 h. The trifluoroacetic acid was removed under reduced pressure and trace trifluoroacetic acid were removed by reevaporation from toluene. Reversed phase HPLC purification (C₁₈, 5μm Microsorb, 10 × 250 mm, MeOH/H₂O, 60:40, 3 mL/min) provided 9 (2.2 mg, 70%) as a colorless solid: mp 250-251 °C (CH₂Cl₂/MeOH); IR (ZnSe, neat) ν 2929, 1627, 1501, 1429 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.34-2.37 (m, 2H), 2.58-2.63 (m, 2H), 2.96-3.00 (m, 2H), 3.10-3.13 (m, 2H), 4.60 (brs, 1H), 5.03 (d, J= 2.0 Hz, 1H), 6.79 (dd, J= 2.0, 0.8 Hz, 1H), 7.02, (d, J= 8.4 Hz, 2H), 7.30 (d, J= 8.4 Hz, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 30.7 (CH₂), 32.7 (CH₂), 40.7 (CH₂), 41.4 (CH₂), 110.8 (C), 115.4 (CH), 125.4 (CH), 125.8 (CH), 132.2 (CH), 133.8 (C), 140.0 (C), 153.3 (C), 158.3 (C), 174.7 (C); HRMS (DCI/NH₃) found m/z 362.0399 [M+H]⁺, C₁₇H₁₇O₃NBr requires 362.0392.

5-Bromo-4-hydroxy-17-iodo-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (10)

Sodium nitrite (1.6 mg, 0.023 mmol) was added in one portion to concentrated sulfuric acid (50 μL) chilled with an ice bath. AcOH (60 μL) was added dropwise to this solution at 0 °C. The solution was stirred for 30 min and then treated with lactam 28 (9.4 mg, 0.021 mmol) in portions over 1 h. This mixture was stirred for 1 h at 0 °C, then room temperature for 20 min. KI (5.6 mg, 0.034 mmol) in 2M HCl (0.2 mL) was added to this mixture and the mixture was stirred at room temperature for 15 min, then 70 °C for 10 min. The mixture was chilled by ice bath and treated with Na₂SO₃ (aq., satd.) followed by extraction with EtOAc (4 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 10. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) followed by reversed phase HPLC (C₁₈, 5μm Microsorb, 10 × 250 mm, MeOH/H₂O, 65:35, 3 mL/min) gave 10 (1.6 mg, 16%) as a pale yellow amorphous solid: IR (ZnSe, neat) ν cm^-1 3289, 2928, 1643, 1502, 1433, 1217; ¹H NMR (400 MHz, CD₃OD) δ 2.50-2.54 (m, 2H), 2.78- 2.84 (m, 2H), 3.10-3.16 (m, 2H), 3.30-3.36 (m, 2H), 5.20 (d, J= 2.0 Hz, 1H), 7.02, (d, J= 2.0 Hz, 1H), 7.27 (d, J= 8.4 Hz, 1H), 7.49 (dd, J= 2.0, 8.4 Hz, 1H), 7.92, (d, J= 2.4 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 24.7 (CH₂), 26.1 (CH₂), 34.6 (CH₂), 35.4 (CH₂), 108.4 (CH), 119.9 (CH), 120.5 (CH), 126 (C), 135.8 (CH), 135.9 (CH), 151.9 (C); HRMS (DEI) found m/z 486.9296 [M]⁺, C₁₇H₁₅NO₃Br requires 486.9280.

5-Bromo-4-hydroxy-20-nitro-2-oxa-10,14-diaza-tricyclo[16.2.2.1^0,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (11)

BBr₃ (4 μL, 0.042 mmol) was added to a solution of lactam 37 (8 mg, 0.014 mmol) in CH₂Cl₂ (100 μL) at –78 ° C. The orange solution was stirred (1h, –78 °C), quenched with NaHCO₃ (aq., satd.) and extracted with EtOAc (3 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 11. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 20:1) gave 11 (6 mg, 82%) as an oil: IR (ZnSe, neat) ν 3405, 3303, 1648, 1533 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.16-2.13 (m, 2H), 2.40-2.34 (m, 2H), 2.65-2.60 (m, 2H), 3.10-3.00 (m, 2H), 3.31-3.27 (m, 2H), 3.51-3.46 (m, 4H), 5.29-5.25 (m, 1H), 5.95 (d, J= 2.0 Hz, 1H), 6.20 (bs, 1H), 6.23 (brt, J= 2.0 Hz, 1H), 6.99 (d, J= 2.0 Hz, 1H), 7.17 (d, J= 8.4 Hz, 1 H), 7.47 (dd, J= 8.4, 2.4 Hz, 1H), 7.81 (d, J= 2.4 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 31.3 (CH₂), 33.4 (CH₂), 34.0 (CH₂), 35.4 (CH₂), 38.9 (CH₂), 41.0 (CH₂), 110.3 (C), 114.2 (CH), 124.7 (CH), 126.1 (CH), 126.9 (CH), 131.8 (C), 135.1 (CH), 139.8 (C), 141.8 (C), 142.1(C), 145.7 (C), 145.9 (C), 170.6 (C), 171.9 (C); HRMS (DCI) found m/z 478.0629 [M+H]⁺, C₂₀H₂₁O₆N₃Br requires 478.0614.

21-Amino-4-hydroxy-5-bromo-2-oxa-10,14-diaza-tricyclo[16.2.2.1^0,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (12)

BBr₃ (4 μL, 0.042 mmol) was added to a solution of lactam 38 (7 mg, 0.013 mmol) in CH₂Cl₂ (100 μL) at –78 ° C. The orange solution stirred (1h, –78 °C), quenched with NaHCO₃ (aq., satd.) and extracted with EtOAc (3 × 10 mL). The organic solution was combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 12. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 20:1) gave 12 (3 mg, 51%) as an oil: IR (ZnSe, neat) ν 3328, 2929, 1648, 1509, 1423, 1278 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.07-2.10 (m, 2H), 2.35-2.38 (m, 2H), 2.52-2.55 (m, 2H), 2.81-2.84 (m, 2H), 3.19-3.21 (m, 2H), 3.28-3.20 (m, 2H), 3.37-3.40 (m, 2H), 6.10 (d, J= 2.0 Hz, 1H), 6.54 (dd, J= 2.0, 8.4 Hz, 1H), 6.70 (d, J= 2.0 Hz, 1H), 6.81 (d, J= 8.4, 1H), 6.96 (d, J= 2.0 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 32.8, (CH₂), 34.5 (CH₂), 35.0 (CH₂), 36.3 (CH₂), 39.5 (CH₂), 42.4 (CH₂), 111.4 (C), 115.7 (CH), 118.9 (CH), 120.3 (CH), 123.3 (CH), 126.8 (CH), 133.4 (C), 139.9 (C), 140.8 (C), 141.8 (C), 143.8 (C), 148.2 (C), 173.8 (C), 174.9 (C); LRMS (ESI) found m/z 448.0 [M+H]⁺, C₂₀H₂₃N₃BrO₃ requires 448.0.

21-Azido-4-hydroxy-5-bromo-2-oxa-10,14-diaza-tricyclo[16.2.2.1^0,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (13)

NaNO₂ (0.8 mg, 12 μmol) was added in one portion to a chilled solution (0 °C, ice bath) of aryl amine 12 (4.8 mg, 11 μmol) in AcOH/ H₂O (60 μL, 9:1). The solution was stirred for 15 min and treated with NaN₃ (4.0 mg, 62 μmol) in one portion. After 0.5 h, the reaction was quenched with H₂O and was extracted with CH₂Cl₂ (3 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 13. Purification by HPLC (C₁₈ 5 μm Microsorb 10 × 250 mm, MeOH/H₂O, 3:2, 4 mL/min, rt. 6.0 min) provided 13 (3.5 mg, 68%) as an oil: IR (neat) ν 3303, 2925, 2117, 1648, 1500 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.05-2.09 (m, 2H), 2.40-2.45 (m, 2H), 2.58-2.62 (m, 2H), 2.91-2.96 (m, 2H), 3.21-3.27 (m, 2H), 3.37-3.42 (m, 2H), 6.05 (d, J= 2.0 Hz, 1H), 6.95-7.10 (m, 4H), 7.67-7.74 (m, 2H); ¹³C NMR (100 MHz, CDCl₃/CD₃OD, 1:1) δ 31.9 (CH₂), 33.8 (CH₂), 34.1 (CH₂), 35.4 (CH₂), 39.1 (CH₂), 41.3 (CH₂), 111.0 (CH), 115.3 (CH), 121.6 (CH), 123.7 (CH), 127.0 (CH), 127.1 (CH), 132.3 (C), 133.0 (C), 139.7 (C), 143.0 (C), 145.7 (C), 147.5 (C), 173.1 (C), 173.4 (C); HRMS (DEI) found m/z 473.0694 [M]⁺, C₂₀H₂₀N₅O₄Br requires 473.0699.

5,21-Dibromo-4-hydroxy-2-oxa-10,14-diaza-tricyclo[16.2.2.1^0,0]tricosa- 1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (14)

BBr₃ (1 μL, 5 μmol) was added to a solution of lactam 39 (1 mg, 2 μmol) in CH₂Cl₂ (20 μL) at –78 °C. The orange solution was stirred (1h, –78 °C), quenched with a NaHCO₃ (aq., satd.) and extracted with EtOAc (3 × 10 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 14. Flash chromatography (SiO₂, CH₂Cl₂/MeOH 20:1) gave 14 (0.8 mg, 94%) as an amorphous solid: ¹H NMR (400 MHz, CD₃OD) δ 2.08-2.11 (m, 2H), 2.41-2.44 (m, 2H), 2.58-2.61 (m, 2H), 2.93-2.96 (m, 2H), 3.23-3.24 (m, 2H), 3.38-3.41 (m, 2H), 5.98 (d, J= 1.6 Hz, 1H), 7.05 (d, J= 2.0 Hz, 1H), 7.07 (d, J= 8.4 Hz, 1H), 7.21 (dd, J= 1.6, 8.4 Hz, 1H), 7.53 (d, J= 2.0 Hz, 1H); HRMS (DCI) found m/z 510.9853 [M+Na]⁺, C₂₀H₂₁O₄N₂Br₂ requires 510.9868.

5,20,22-Tribromo-4-hydroxy-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (15)

BBr₃ (1M 50 μL) was added to a solution of lactam 40 (4 mg, 6 μmol) in CH₂Cl₂ (50 μL) at –78 °C. The orange solution was stirred (1h, –78 °C), was quenched with NaHCO₃ (aq., satd.) and extracted with EtOAc (3 × 10 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 15. Flash chromatography (SiO₂, CH₂Cl₂/MeOH 20:1) gave 15 (2.5 mg, 72%) as an amorphous solid: ¹H NMR (400 MHz, CD₃OD) δ 1.92-2.00 (m, 1H), 2.26-2.32, (m, 1H), 2.40-2.50 (m, 2H), 2.54-2.71 (m, 2H), 2.86-2.92 (m, 1H), 3.03-3.23 (m, 3H), 3.55-3.72 (m, 2H), 5.98 (d, J= 2.0 Hz, 1H), 7.07 (d, J= 2.0 Hz, 1H), 7.50 (s, 1H), 7.56 (s, 1H); LRMS (DCI) found m/z 610.9 [M+Na]⁺, C₂₀H₁₉O₄N₂Br₃Na requires 610.9.

5-(2-Aminoethyl)-2-benzyloxy-3-bromophenol (16)

KOH (70 mg, 1.3 mmoles) and hydrazine (cat.) were added to a solution of carbamate 24 (22 mg, 0.05 mmol) dissolved in dry 1,4-dioxane (1 mL). This heterogeneous mixture was rapidly stirred and heated to 80 °C. After 0.5 h, the mixture was treated with HCl (1N) until the mixture was neutral by pH paper and was extracted with EtOAc (3 × 25 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to yield 16 as an oil (14 mg 92% crude). The product was carried forward without purification. ¹H NMR (400 MHz, CDCl₃) δ 2.58 (bs, 2H), 2.85 (bs, 2H), 4.20 (bs, 2H), 4.97 (s, 2H), 6.65 (bs, 1H), 6.89 (bs, 1H), 7.2-7.6 (m, 5H); HRMS (DCI/NH₃) found m/z 322.0445 [M+H]⁺, C₁₅H₁₇O₂NBr requires 322.0443.

3-(4-Benzyloxy-3-bromo-5-hydroxyphenyl)-N-[2-(4-fluoro-3-nitro-phenyl)-ethyl]propionamide (18)

Acid 17 (83 mg, 0.39 mmol), HOBt (55 mg, 0.41 mmol), and EDCI (78 mg, 0.41 mmol) were added sequentially to a solution of amine 16 (120 mg, 0.37 mmol) in CH₂Cl₂ (5 mL) at room temperature. After 1 h, the reaction was quenched with HCl (1N, 20 mL) and extracted with CH₂Cl₂ (4 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to provide crude 18 which was purified by flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) yielding 18 (140 mg, 72%) as a amorphous solid: IR (KBr, pellet) ν 3392, 3075, 1639, 1529, 1425, 1349 cm^-1; ¹H NMR (300 MHz, CD₃COCD₃) δ 2.52 (t, J= 7.2 Hz, 2H), 2.65 (t, J= 7.2 Hz, 2H), 3.01 (t, J= 7.2 Hz), 3.38 (dt, J= 7.2, 6.0 Hz, 2H), 5.01 (s, 2H), 6.78 (d, J= 1.8 Hz, 1H), 6.88 (d, J= 1.8 Hz, 1H), 7.25 (brs, 1H), 7.30-7.45 (m, 4H), 7.50-7.60 (m, 2H), 7.64-7.69 (m, 1H), 7.99 (dd, J= 6.9, 2.1 Hz, 1H), 8.68 (bs, 1H); ¹³C NMR (75 MHz, CD₃COCD₃) δ 31.0 (CH₂), 35.0 (CH₂), 37.6 (CH₂), 41.2 (CH₂), 74.8 (CH₂), 117.3 (CH), 117.6 (C), 118.7 (CH, J= 21.0 Hz), 124.3 (CH), 126.2 (CH, J= 3.0 Hz), 128.5 (CH), 128.8 (2 CH), 136.7 (CH, J= 8.0 Hz), 137.7 (C), 138.1 (C, J= 7.0 Hz), 139.6 (C, J= 4.3 Hz), 142.8 (C), 151.9 (C), 154.2 (C, J= 258.0 Hz), 171.9 (C); HRMS (DEI) found m/z 516.0686 [M]⁺, C₂₄H₂₂N₂O₅BrF requires 516.0696.

N-(2-{2-[4-Benzyloxy-3-bromo-5-triisopropyl-silanyloxy)-phenyl]-ethylcarbamoyl}-ethyl-3-(4-fluoro-3-nitro-phenyl)-propionamide (19)

EDCI (76 mg, 0.40 mmol) was added to a solution of amine 36 (110 mg, 0.20 mmol), HOBt (54 mg, 0.40 mmol), and acid 17 (47 mg, 0.22 mmol) in CH₂Cl₂ (5.0 mL). The solution stirred overnight at room temperature, quenched with HCl (1N) and extracted with CH₂Cl₂ (3 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 19. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 20:1) gave 19 (139 mg, 93%) as an oil: IR (NaCl, neat) ν 3293, 2944, 2867, 1644, 1538 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.07 (d, 7.2 Hz, 18H), 1.26 (sept, J= 7.2 Hz, 3H), 2.28 (t, J= 6.0 Hz, 2H), 2.45 (t, J= 7.2 Hz, 2H), 2.67 (t, J= 7.2 Hz, 2H), 2.99 (t, J= 7.2 Hz, 2H), 3.50-3.38 (m, 4H), 4.98 (s, 2H), 5.54 (brs, 1H), 6.39 (brs, 1H), 6.63 (d, J= 2.0 Hz, 1H), 6.94 (d, J= 2.0 Hz, 1H), 7.15 (dd, J= 10.8, 8.4 Hz, 1H), 7.38-7.29 (m, 3H), 7.48-7.42 (m, 1H), 7.52-7.48 (m, 2H), 7.86 (dd, J= 7.2, 2.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 12.9 (CH), 17.9 (CH₃), 30.2 (CH₂), 34.8 (CH₂), 35.2 (CH₂), 35.4 (CH₂), 37.2 (CH₂), 40.5 (CH₂), 74.4 (CH₂), 118.0 (C), 118.3 (d, J= 6.2 Hz, CH), 119.7 (CH), 125.1 (CH), 125.3 (d, J= 2.9 Hz, CH), 127.8 (CH), 128.0 (CH), 128.1 (CH), 135.5 (C), 135.6 (d, J= 5.1 Hz, CH), 136.0 (d, C), 136.9 (C), 137.8 (d, J= 4.3 Hz, C), 145.4 (C), 150.3 (C), 153.8 (d, J= 260.9 Hz, C), 171.0 (C), 171.4 (C); HRMS (FAB) found m/z 744.2478 [M+H]⁺, C₃₆H₄₈O₆N₃FSiBr requires 744.2478.

4-(Benzyloxy)-3-bromo-5-hydroxybenzaldehyde (21)

Li₂CO₃ (85 mg, 1.2 mmol) was added to a solution of 3-bromo-4,5-dihydroxybenzaldehyde 20 (100 mg, 0.46 mmol) in DMF (2 mL). This solution was vigorously stirred and heated (45 °C, 1h) followed by dropwise addition of benzylbromide (0.14 mL, 1.2 mmol). After 45 min, the reaction was quenched with HCl (aq., 1N) resulting in precipitation of the crude product 21. The precipitate was filtered, washed with water, dried under high vacuum and was purified by flash chromatography (SiO₂, CH₂Cl₂/hexane, 9:1) to yield 21 (125 mg, 88%) as a pale yellow solid: mp 92-93 °C; IR (NaCl, neat) ν 3235, 1683 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 5.14 (s, 2H), 5.85 (s, 1H), 7.34 (d, J= 2.0 Hz, 1H), 7.38-7.46 (m, 5H), 7.63 (d, J= 2.0 Hz, 1H), 9.79 (s, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 76.1 (CH₂), 115.5 (CH), 117.0 (C), 126.7 (CH), 128.6 (CH), 129.0 (CH), 129.2 (CH), 133.9 (C), 148.3 (C), 150.9 (C), 190.0 (CH); HRMS (DCI/NH₃) found m/z 306.9963 [M+H]⁺, C₁₄H₁₂O₃Br requires 306.9969.

4-Benzyloxy-3-bromo-5-hydroxycinnamic acid (22)

Pyridine (0.55 mL, 6.8 mmol) and piperidine (0.16 mL, 1.62 mmol) were added to a solution of 4-benzyloxy-3-bromo-5-hydroxybenzaldehyde 21 (2.0 g, 6.5 mmol) and malonic acid (0.7 g, 6.8 mmol) in toluene (100 mL) within a round-bottom flask equipped with a Dean Stark trap and heated under reflux for 5h. The reaction was quenched with HCl (1N, 300 mL) resulting in precipitation of 22. This compound was collected and washed with water and dried under high vacuum to provide 22 (2.0 g, 88%) as a colorless solid: mp 173-174 °C; IR (NaCl, neat) ν 3249, 1650, 1633, 1616 cm^-1; ¹H NMR (300 MHz, CDCl₃) δ 5.09 (s, 2H), 6.3 (d, J= 15.9 Hz, 1H), 7.03 (d, J= 1.8 Hz, 1 H), 7.29 (d, J= 1.8 Hz, 1H), 7.38-7.44 (m, 5H), 7.57 (d, J= 15.9 Hz, 1 H); ¹³C NMR (75 MHz, CDCl₃/ drop of DMSO) δ 74.9 (CH₂), 114.9 (CH), 117.5 (C), 118.7 (CH), 123.9 (CH), 128.3 (CH), 128.4 (CH), 128.5 (CH), 131.9 (C), 136.5(C), 143.3 (CH), 145.2 (C), 151.1 (C), 169.0 (C); HRMS (DCI/NH₃) found m/z 349.0087 [M]⁺, C₁₆H₁₄O₄Br requires 349.0075.

Ethyl(4-benzyloxy)-3-bromo-5-(ethoxycarbonyloxy)styrylcarbamate (23)

Diisopropylethylamine (1.4 mL) was added dropwise to a chilled solution (–10 °C) of 4-benzyloxy-5-bromo-3-hydroxy-cinnamic acid 22 (1.00 g) dissolved in acetone (40 mL), followed by the dropwise addition of ethylchloroformate (0.6 mL). After stirring for 2hr (–10 °C), a chilled aqueous solution of sodium azide (560 mg, 10 mL H₂O) was added dropwise to the reaction. After stirring for 5 h at 0 °C, the solution was extracted with CH₂Cl₂ (3 × 100 mL). Extracts were combined, dried over anhydrous MgSO₄, filtered, and volatiles were removed to give a colorless solid. The resulting solid was azeotroped dried with toluene (3 × 20 mL). Ethanol (5 mL) and toluene (50 mL) were added and this solution was heated to 80 °C for 12 h. The volatiles were removed to give crude 23 that was purified by flash chromatography (SiO₂, CH₂Cl₂/EtOAc, 98:2) to yield 23 (0.89 g, 66%) as an amber viscous oil: IR (NaCl, neat) ν 3324, 2981, 1766, 1729, 1660 1525, 1475, 1257, 1224 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.29 (t, J= 7.2 Hz, 6H), 4.21 (q, J= 7.2 Hz, 4H), 4.98 (s, 2H), 5.80 (d, J= 14.4 Hz, 1H,), 6.53 (d, J= 10.4 Hz, 1H,), 7.02 (d, J= 2.0 Hz, 1H), 7.14 (dd, J= 14.4, 10.4 Hz, 1H,), 7.5-7.3 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 14.5 (CH₃), 61.8 (CH₂), 65.2 (CH₂), 75.6 (CH₂), 107.7 (CH), 118.3 (C), 118.7 (CH), 125.6 (CH), 127.5 (CH), 128.2 (CH), 128.3 (CH), 128.4 (CH), 134.5 (C), 136.4 (C), 145.3 (C), 146.3 (C), 152.9 (C), 153.5 (C); HRMS (DCI/NH₃) found m/z 463.0614 [M]⁺, C₂₁H₂₂O₆NBr requires 463.0630.

Carbonic acid 2-benzyloxy-3-bromo-5-(2-methoxycarbonylamino-vinyl)-phenyl ester methyl ester

IR (KBr, neat) ν 3336, 2956, 1770, 1734, 1660, 1477, 1261, 943 cm^-1; ¹H NMR (300 MHz, CDCl₃) δ 3.73 (s, 3H), 3.80 (s, 3H), 4.98 (s, 2H), 5.75 (d, J= 14.0 Hz, 1H), 6.87 (bd, J= 11.0 Hz, 1H), 7.01 (d, J= 2.0 Hz, 1H), 7.11 (bd, J= 11.0 Hz, 1H), 7.3-7.5 (m, 4H); ¹³C NMR (75 MHz, CDCl₃) δ 52.7 (CH₃), 55.7 (CH₃), 75.6 (CH₂), 107.8 (CH), 118.2 (C), 118.5 (CH), 125.5 (CH), 127.4 (CH), 128.1 (CH), 128.2 (2CH), 134.3 (C), 136.3 (C), 145.1 (C), 146.0 (C), 153.4 (C), 153.8 (C); HRMS (DCI/NH₃) found m/z 349.0087 [M]^+., C₁₆H₁₄O₄Br requires 349.0075.

Carbonic acid 2-benzyloxy-3-bromo-5-(2-methoxycarbonylamino-ethyl)-phenyl ester ethyl ester (24)

Triethylsilane (265 uL, 1.67 mmol) was added to 23 (50 mg, 0.17 mmol) and the resulting heterogeneous mixture was rapidly stirred (–10 °C). Chilled (–10 °C) neat trifluoroacetic acid (1 mL) was rapidly transferred via canula to the reaction mixture. The heterogeneous mixture was allowed to rapidly stir for 20 min. The reaction was quenched with a NaHCO₃ (aq., satd.) and was extracted with CH₂Cl₂ (4 × 25 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to provide 24 as a viscous oil (47 mg, 94%): IR (NaCl, neat) ν 3343, 2956, 1770, 1722, 1481, 1257 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 2.75 (t, J= 7.0 Hz, 2H), 3.40 (dt, J= 7.0, 7.0 Hz, 2H), 3.65 (s, 3H), 3.80 (s, 3H), 4.74 (brs, 1H), 4.98 (s, 2H), 6.96 (d, J= 2.0 Hz, 1H), 7.29 (d, J= 2.0 Hz, 1H), 7.32-7.5 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 35.2 (CH₂), 41.8 (CH₂), 52.1 (CH₃), 55.7 (CH₃), 75.5 (CH₂), 118.0 (C), 122.4 (CH), 128.1 (CH), 128.2 (2CH), 131.0 (CH), 136.2 (C), 136.4 (C), 144.9 (C), 146.6 (C), 153.3 (C), 156.7 (C); HRMS (DCI/NH₃) found m/z 466.0864 [M+H]⁺, C₂₁H₂₅O₆NBr requires 466.0865.

3-(4-Fluoro-3-nitro-phenyl)-propionic acid methyl ester (26)

In a glove box, olefin 25 (300 mg, 1.3 mmol) and Wilkinson's catalyst (44 mg, 0.05 mmol) were dissolved in toluene (25 mL) in a pressure vessel. The vessel was removed from the glove box and purged with H₂ and pressurized with H₂ (3 atm). The mixture was rapidly stirred and heated (60 °C). After 8 h, the toluene was removed resulting in a brown residue that was passed through a flash column (SiO₂, EtOAc/CH₂Cl₂, 1:50) to provide 26 as an amorphous solid (290 mg, 95%): IR (NaCl, neat) ν 2954, 1735, 1537, 1349 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 7.86 (dd, J= 7.2, 2.4 Hz, 1H), 7.45 (m, 1H), 7.17 (dd, J= 10.0, 8.4 Hz, 1H), 3.63 (s, 3H), 2.97 (t, J= 7.6 Hz, 2H), 2.63 (t, J= 7.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 172.2 (s, C), 153.9 (d, J= 348 Hz, C), 137.4 (d, J= 5.7 Hz, C) 136.8 (brs, C), 135.5 (d, J= 11 Hz, CH), 125.4 (d, J= 3.8 Hz, CH), 118.2 (d, J= 28 Hz, CH), 51.8 (s, CH₃), 34.9 (s, CH₂), 29.6 (s, CH₂); HRMS (EI) found m/z 227.0601 [M]⁺, C₁₀H₁₀FNO₄ requires 227.0593.

4-Benzyloxy-5-bromo-16-nitro-2-oxa-11-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-10-one (27)

K₂CO₃ (500 mg, 3.6 mmol) was added to a solution of phenol 18 (100 mg, 0.19 mmol) in DMSO (100 mL, 2 mM) containing 4 Å sieves at room temperature. After 3 h of vigorous stirring, the reaction mixture was diluted with water (100 mL) and extracted with EtOAc (5 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to provide crude 27 that was purified by flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) yielding 27 (86 mg, 85%) as a viscous oil: IR (NaCl, neat) ν 3272, 2933, 1639, 1531, 1346 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 2.2-2.5 (m, 3H), 3.0-3.4 (m, 5H), 5.05 (d, J= 1.6 Hz, 1H), 5.15 (d, J= 10.4 Hz, 1H), 5.30 (d, J= 10.4 Hz, 1H), 5.34 (brs, 1H), 6.86 (d, J= 1.6 Hz, 1H), 7.09 (d, J= 8.4 Hz, 1H), 7.3-7.4 (m, 3H), (dd, J= 8.4, 2.0 Hz, 1H), 7.6-7.62 (m, 2H), 7.92 (d, J = 2.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 30.3 (CH₂), 31.5 (CH₂), 39.6 (CH₂), 39.9 (CH₂), 75.2 (CH₂), 113.2 (CH), 117.8 (C), 126.3 (CH), 126.4 (CH), 127.4 (CH), 128.2 (CH), 128.3 (CH), 128.6 (CH), 136.8 (C), 136.8 (CH), 137.3 (C), 140.5 (C), 142.6 (C), 143.9 (C), 149.4 (C), 155.2 (C), 171.0 (C); HRMS (DEI) found m/z 496.0624 [M]⁺, C₂₄H₂₁N₂O₅Br requires 496.0634.

17-Amino-4-benzyloxy-5-bromo-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (28)

CrCl₂ (110 mg, 0.89 mmol) was added to a solution of lactam 27 (34 mg, 0.07 mmol) in DMF (1 mL) and the mixture stirred at room temperature. After 12 h, the volatiles were removed to give a residue that was dissolved in EtOAc (10 mL). The organic solution was washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 28 that was purified by flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) yielding 28 (23 mg, 73%) as a viscous gum: IR (NaCl, neat) ν 3291, 2927, 1639, 1504, 1270, 1187 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 2.2-2.3 (m, 2H), 2.6-2.7 (m, 2H), 2.8-2.9 (m, 2H), 3.2-3.3 (m, 2H), 4.85 (brs, 1H), 5.17 (d, J= 10.4 Hz, 1H), 5.27 (d, J= 10.4 Hz, 1H), 5.41 (s, 1H), 6.63 (brd, J= 7.2 Hz, 1H), 6.79 (d, J= 7.2 Hz, 1H), 6.88 (s, 1H), 7.3-7.4 (m, 3H), 7.5-7.6 (m, 2H), 7.99 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 30.4 (CH₂), 32.2 (CH₂), 39.4 (CH₂), 41.1 (CH₂), 75.3 (CH₂), 113.8 (CH), 117.6 (CH), 120.0 (CH), 124.5 (CH), 125.7 (CH), 128.3 (CH), 128.4 (CH), 128.8 (CH), 136.4 (C), 136.9 (C), 140.0 (C), 141.0 (C), 142.4 (C), 142.7 (C), 154.6 (C), 171.9 (C); HRMS (DEI) found m/z 466.0908 [M]⁺, C₂₄H₂₃N₂O₃Br requires 466.0892.

5,17-Dibromo-4-benzyloxy-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19) ,4,6,14(18),15-hexaen-11-one (29)

tert-Butyl nitrite (20 μL, 17 μmol) was added to a solution of CuBr₂ (3 mg, 14 μmol) in CH₃CN (0.2 mL) at 0 °C. After stirring for 1h, a heterogeneous mixture of lactam 28 (8 mg, 17 μmol) in CH₃CN (0.8 mL) was added dropwise over 20 min at 0 °C. After stirring for 2 h, the solution was allowed to warm to room temperature and quenched with HCl (1N, 4 mL) and extracted with CH₂Cl₂ (4 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 29. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) followed by reversed phase HPLC (C₁₈, 5μm Microsorb 10 × 250 mm, MeOH/H₂O, 65: 35, 3 mL/min) gave 29 (6 mg, 66%) as a colorless amorphous solid: IR (NaCl, neat) ν 3286, 2928, 1640, 1480, 1214 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 2.2-2.4 (m, 2H), 2.6-2.7 (m, 2H), 2.9-3.1 (m, 2H), 3.2-3.4 (m, 2H), 4.90 (brs, 1H), 5.06 (d, J= 2.0 Hz, 1H), 5.18 (d, J= 10.4 Hz, 1H), 5.34 (d, J= 10.4 Hz, 1H), 6.88 (dd, J= 2.0, 1.2 Hz, 1H), 7.05 (d, 8.4 Hz, 1H), 7.23 (dd, J= 8.4, 2.4 Hz, 1 H), 7.3-7.4 (m, 3H), 7.51 (d, J= 2.4 Hz, 1H), 7.6-7.5 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 30.5 (CH₂), 31.7 (CH₂), 39.7 (CH₂), 41.0 (CH₂), 75.2 (CH₂), 113.1 (CH), 117.9 (C), 118.7 (CH), 125.8 (CH), 125.9 (CH), 128.2 (CH), 128.4 (CH), 128.8 (CH), 130.4 (CH), 134.6 (C), 136.4 (C), 137.0 (C), 140.6 (C), 142.8 (C), 152.9 (C), 154.6 (C), 171.1 (C); HRMS (DEI) found m/z 532.9852 [M]⁺, C₂₄H₂₁NO₃Br₂ requires 532.9847.

4-Benzyloxy-5,15,17-tribromo-2-oxa-10-azatricyclo[12.2.2.1^0,0]nonadeca-1 (17),3(19),4,6,14,(18),15-hexaene-11-one (30)

tert-Butyl nitrite (12 μL, 0.09 mmol) was added to a solution of CuBr₂ (56 mg, 0.25 mmol) in CH₃CN (0.5 mL) was added at 0 °C. After stirring for 1 h, a heterogeneous mixture of lactam 28 (12 mg, 0.03 mmol) in CH₃CN (0.8 mL) was added drop-wise over 20 min at 0 °C. After stirring for 2 h, the mixture was allowed to warm to room temperature and quenched with HCl (1N, 4 mL) and extracted with EtOAc (4 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 30. Flash chromatography (SiO₂, CH₂Cl₂/ MeOH, 9:1) followed by reversed phase HPLC (C₁₈, 5μm Microsorb, 10 × 250 mm, MeOH/ H₂O, 75:25, 3 mL/ min) gave 30 (5 mg, 32%) as a colorless amorphous solid: IR (NaCl, neat) ν 3273, 2928, 1638, 1463, 1218 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 2.2- 2.8 (m, 4H), 3.0-3.5 (m, 4H), 4.93 (bd, J= 7.2 Hz, 1H), 5.16 (d, J= 10.4 Hz, 1H), 5.31 (d, J= 10.4 Hz, 1), 6.92 (d, J= 2.0 Hz, 1H), 7.25 (s, 1H), 7.3- 7.4 (m, 3H), 7.55 (s, 1H), 7.59-7.63 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 30.5 (CH₂), 32.1 (CH₂), 37.1 (CH₂), 39.7 (CH₂), 75.3 (CH₂), 112.9 (CH), 118.1 (C), 118.7 (C), 122.7 (C), 126.2 (CH), 128.3 (CH), 128.4 (CH), 128.8 (CH), 129.4 (CH), 136.5 (CH), 136.7 (C), 136.8 (C), 139.3 (C), 142.7 (C), 153.2 (C), 154.1 (C), 170.9 (C); HRMS (DEI) found m/z 606.9021 [M]⁺, C₂₄H₂₀NO₃Br₃ requires 606.8993.

4-Benzyloxy-5-bromo-2-oxa-10-aza-tricyclo[12.2.2.1^0,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (31)

tert-Butyl nitrite (40 μL, 0.34 mmol) was added to THF (0.3 mL) at 0 °C. After stirring for 1h, a heterogeneous mixture of lactam 28 (10 mg, 0.02 mmol) in THF (0.4 mL) was added dropwise over 20 min at 0 °C. After stirring for 2 h, the solution was allowed to warm to room temperature, quenched with HCl (1N, 4 mL) and extracted with EtOAc (4 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 31. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) followed by reversed phase HPLC (C₁₈, 5μm Microsorb, 10 × 250 mm, MeOH/H₂O, 65:35, 3 mL/min) gave 31 (3.8 mg, 39%) as a colorless powder: IR (NaCl, neat) ν 3286, 2925, 1640, 1567, 1501, 1202 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 2.26-2.29 (m, 2H), 2.58-2.62 (m, 2H), 3.00-3.04 (m, 2H), 3.24-3.28 (m, 2H), 4.77 (brs, 1H), 5.03 (d, J= 2.4 Hz, 1H), 5.22 (s, 2H), 6.83 (d, J= 2.4 Hz, 1H), 6.97 (d, J= 8.4 Hz, 2H), 7.27 (d, J= 8.4 Hz, 2H), 7.30-7.42 (m, 3H), 7.59-7.61 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 30.5 (CH₂), 32.0 (CH₂), 39.6 (CH₂), 41.2 (CH₂), 75.2 (CH₂), 114.6 (CH), 117.6 (C), 124.4 (CH), 125.0 (CH), 128.1 (CH), 128.3 (CH), 128.7 (CH), 130.8 (CH), 135.9 (C), 136.8 (C), 138.7 (C), 142.2 (C), 156.3 (C), 156.6 (C), 171.4 (C); HRMS (DEI) found m/z 451.0795 [M]⁺, C₂₄H₂₂NO₃Br requires 451.0783.

[2-(4-Benzyloxy-3-bromo-5-hydroxy-phenyl)-ethyl]-carbamic acid tert-butyl ester (32)

Phenethylamine 16, (28 mg, 86 μmol) was dissolved in CH₃CN (0.75 mL) and was treated with a solution of di-tert-butyldicarbonate (46 mg, 210 μmol) in CH₃CN (0.25 mL). After 3 h, the volatiles were removed and the resulting residue was redissolved in MeOH (0.5 mL), water (0.2 mL) and treated with sodium carbonate (50 mg). After stirring 12 h, the solution was extracted with CH₂Cl₂ (4 × 25 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to yield 32 as an oil (28 mg, 76%): IR (NaCl, neat) ν 3359, 2977, 1685, 1500, 1367, 1166 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.42 (s, 9H), 2.65 (bt, J= 4.8 Hz, 2H), 3.31 (bd, 2H), 4.59 (bs, 1H), 5.01 (s, 2H), 5.94 (bs, 1H), 6.69 (bs, 1H), 6.90 (bs, 1H), 7.3-7.5 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 28.3 (CH₃), 35.4 (CH₂), 41.4 (CH₂), 79.4 (C), 115.5 (CH), 116.3 (C), 124.6 (CH), 128.5 (CH), 128.7 (CH), 128.7 (CH), 136.4 (C), 137.3 (C), 141.8 (C), 150.2 (C), 155.9 (C); HRMS (DCI/NH₃) found m/z 439.1221 [M+NH₄]⁺, C₂₀H₂₈O₄N₂Br requires 439.1232.

{2-[4-Benzyloxy-3-bromo-5-(triisopropyl-silanyloxy)-phenyl]-ethyl}-carbamic acid tert- butyl ester (33)

TIPSCl (75 μL, 0.35 mmol) was added to a solution of amide 32 (122 mg, 0.299 mmol), and imidazole (50 mg, 0.72 mmol) in DMF (0.5 mL). The yellow solution was stirred overnight at room temperature, quenched with water and extracted with EtOAc (3 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 33. Flash chromatography (SiO₂, CH₂Cl₂) gave 33 (164 mg, 95%) as an oil: IR (NaCl, neat) ν 3434, 3359, 2944, 2867, 1718, 1477, 1170 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.08 (d, J= 7.6 Hz, 18H), 1.27 (sept, J=7.6, 3H), 1.42 (s, 9H), 2.65 (t, J= 6.4, 2H), 3.36-3.29 (m, 2H), 4.49 (brs, 1H), 4.98 (s, 2H), 6.64 (d, J= 2.0 Hz, 1H), 6.95 (d, J= 2.0 Hz, 1H), 7.40-7.30 (m, 3H), 7.53-7.50 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 13.0 (CH), 18.0 (CH₃), 28.4 (CH₃), 74.4 (CH₂), 79.2 (C), 35.5 (CH₂), 41.6 (CH₂), 118.4 (C), 119.9 (CH), 125.3 (CH), 127.8 (CH), 128.0 (CH), 128.1 (CH), 136.0 (C), 137.0 (C), 145.4 (C), 150.2 (C), 155.6 (C); HRMS (FAB) found m/z 600.2131 [M+Na]⁺, C₂₉H₄₄O₄NNaSiBr requires 600.2121.

2-[4-Benzyloxy-3-bromo-5-(triisopropyl-silanyloxy)-phenyl]-ethyl amine (34)

TFA (1.0 mL) was added to a solution of carbamate 33 (142 mg, 0.246 mmol) in CH₂Cl₂ (1.0 mL). The solution was stirred for 0.5 h at 0 °C, quenched with NaHCO₃ (aq., satd.) and extracted with CH₂Cl₂ (3 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to yield 34 (112 mg, 95%) as an oil: IR (NaCl, neat) ν 2944, 2867, 1556, 1475 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.08 (d, J= 7.2 Hz, 18H), 1.27 (sept, J= 7.2 Hz, 3H), 2.62 (t, J= 6.8 Hz, 2H), 2.92 (bt, J= 6.8 Hz, 2H), 4.98 (s, 2H), 6.66 (d, J= 2.0 Hz, 1H), 6.96 (d, J= 2.0 Hz, 1H), 7.40-7.25 (m, 3H), 7.55-7.49 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 13.0 (CH), 18.0 (CH₃), 39.1 (CH₂), 43.3 (CH₂), 74.4 (CH₂), 118.3 (C), 119.9 (CH), 125.3 (CH), 127.7 (CH), 128.0 (CH), 128.1 (CH), 136.6 (C), 137.1 (C), 145.2 (C), 150.2 (C); HRMS (DCI/NH₃) found m/z 478.1759 [M+H]⁺, C₂₄H₃₇O₂NSiBr requires 478.1777.

(2-{2-[4-Benzyloxy-3-bromo-5-(triisopropyl-silanyloxy)-phenyl]-ethylcarbamoyl}-ethyl)-carbamic acid tert- butyl ester (35)

EDCI (88 mg, 0.460 mmol) was added to a solution of amine 34 (110 mg, 0.23 mmol), HOBt (62 mg, 0.46 mmol), and N-Boc-β-alanine (65 mg, 0.35 mmol) in CH₂Cl₂ (1.0 mL). The solution was stirred overnight at room temperature, quenched with HCl (1N) and extracted with CH₂Cl₂ (3 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 34. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 20:1) gave 34 (138 mg, 92%) as an oil: IR (NaCl, neat) ν 3315, 2945, 2867, 1691, 1558, 1477 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.07 (d, J= 7.2 Hz, 18H), 1.26 (sept, J= 7.2 Hz, 3H), 1.41 (s, 9H), 2.34 (t, J= 6.0 Hz, 2H), 2.67 (t, J= 7.2 Hz, 2H), 3.37 (dt, J= 6.0, 6.0 Hz, 2H), 3.44 (dt, J= 7.2, 6.0 Hz, 2H), 4.99 (s, 2H), 5.16 (bs, 1H), 5.73 (bs, 1H), 6.64 (d, J= 2.0 Hz, 1H), 6.95 (d, J= 2.0 Hz, 1H), 7.40-7.29 (m, 3H), 7.52-7.48 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 12.8 (CH), 17.8 (CH₃), 28.3 (CH₃), 34.8 (CH₂), 36.1 (CH₂), 36.5 (CH₂), 40.4 (CH₂), 74.3 (CH₂), 79.2 (C), 118.5 (C), 119.7 (CH), 125.2 (CH), 127.9 (CH), 128.1 (CH), 128.2 (CH), 135.8 (C), 137.0 (C), 145.6 (C), 150.4 (C), 156.1 (C), 171.3 (C); HRMS (FAB) found m/z 671.2512 [M+Na]⁺, C₃₂H₄₉O₅N₂NaSiBr requires 671.2492.

3-Amino-N-{2-[4-Benzyloxy-3-bromo-5-(triisopropyl-silanyloxy)-phenyl]-ethyl-propionamide (36)

TFA (1.0 mL) was added to a solution of carbamate 35 (136 mg, 0.209 mmol) in CH₂Cl₂ (1.0 mL) at room temperature. The solution was stirred for 0.5 h at 0 °C, quenched with NaHCO₃ (aq., satd.) and extracted with CH₂Cl₂ (3 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to yield 36 (112 mg, 97%) as a oil: IR (NaCl, neat) ν 3315, 2945, 2867, 1691, 1646, 1558, 1477 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.08 (d, J= 7.2 Hz, 18H), 1.27 (sept, J= 7.2 Hz, 3H), 2.29 (bs, 2H), 2.68 (t, J= 6.8 Hz, 2H), 2.97 (bs, 2H), 3.44 (dt, J= 6.8, 6.4 Hz, 2H), 4.99 (s, 2H), 6.65 (d, J= 2.0 Hz, 1H), 6.96 (d, J= 2.0 Hz, 1H), 7.08 (bs, 1H), 7.40-7.28 (m, 3H), 7.52-7.48 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 12.8 (CH), 17.9 (CH₃), 28.3 (CH₂), 34.9 (CH₂), 37.9 (CH₂), 40.2 (CH₂), 74.4 (CH₂), 118.3 (C), 119.8 (CH), 125.4 (CH), 127.9 (CH), 128.1 (CH), 128.2 (CH), 136.2 (C), 137.1 (C), 145.5 (C), 150.4 (C), 172.4 (C); HRMS (FAB) found m/z 549.2142 [M+H]⁺, C₂₇H₄₂O₃N₂NaSiBr requires 549.2148.

4-Benzyloxy-5-bromo-21-nitro-2oxa-10,14-diaza-tricyclo[16.2.2.1^0,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (37)

CsF (43 mg, 0.29 mmol) was added to a solution of amide 19 (102 mg, 0.140 mmol) and 4 Å sieves in DMSO (75 mL, 2 mM). The solution was rapidly stirred overnight at room temperature, quenched with water and extracted with CH₂Cl₂ (5 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 37. Flash chromatography (SiO₂, CH₂Cl₂/ MeOH, 20:1) gave 37 (65 mg, 84%) as an oil. IR (NaCl, neat) ν 3299, 2931, 1647, 1533 cm^-1; ¹H NMR (300 MHz, CDCl₃) δ 2.12-2.16 (m, 2H), 2.33-2.40 (m, 2H), 2.63-2.69 (m, 2H), 3.05-3.08 (m, 2H), 3.28-3.37 (m, 2H), 3.46-3.52 (m, 2H), 5.14 (s, 2H), 5.36 (brs, 1H), 5.96 (d, J= 1.8 Hz, 1H), 6.22 (brs, 1H), 7.04 (d, J= 1.8 Hz, 1H), 7.07 (d, J= 8.4 Hz, 1H), 7.30-7.38 (m, 3H), 7.45 (dd, J= 8.4 Hz, 2.1 Hz), 7.54-7.58 (m, 2H), 7.82 (d, J= 2.1 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 31.2 (CH₂), 33.6 (CH₂), 33.8 (CH₂), 35.3 (CH₂), 38.9 (CH₂), 40.8 (CH₂), 75.3 (CH₂), 115.0 (CH), 118.9 (C), 124.6 (CH), 125.8 (CH), 127.1 (CH), 128.2 (CH), 128.4 (CH), 128.6 (CH), 135.2 (CH), 136.6 (C), 136.7 (C), 139.6 (C), 142.6 (C), 143.8 (C), 146.0 (C), 152.0 (C), 170.8 (C), 172.0 (C); HRMS (FAB) found m/z 568.1061 [M+H]⁺, C₂₇H₂₇N₃O₆Br requires 568.1083.

21-Amino-4-benzyloxy-5-bromo-2-oxa-10,14-diaza-tricyclo[16.2.2.1^0,0]-tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (38)

CrCl₂ (80 mg, 0.65 mmol) was added to a solution of lactam 37 (40 mg, 0.07 mmol) in DMF (1 mL) at room temperature. After 12 h of stirring, the DMF was removed under reduced pressure followed by dissolving the residue in EtOAc (10 mL). The organic solution was washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 38. Flash chromatography (SiO₂, CH₂Cl₂/ MeOH, 9:1) gave 38 (21 mg, 55%) as a yellow viscous oil: IR (NaCl, neat) ν 3320, 2928, 1644, 1479, 1196 cm^-1; ¹H NMR (400 MHz, CD₃OD) δ 2.10-2.14 (m, 2H), 2.38-2.63 (m, 2H), 2.60-2.63 (m, 2H), 2.84-2.87 (m, 2H), 3.25-3.27 (m, 2H), 3.40-3.43 (m, 2H), 5.16 (s, 2H), 6.21 (d, J= 2.0 Hz, 1H), 6.60 (dd, J= 8.0, 2.0 Hz, 1H), 6.75 (d, J= 8.0, 2.0 Hz, 1H), 6.75 (d, J= 2.0 Hz, 1H), 6.83 (d, J= 8.0 Hz, 1H), 7.11 (d, J= 2.0 Hz, 1H), 7.30-7.39 (m, 3H), 7.55-7.58 (m, 2H), 7.62 (bt, J= 6.0 Hz, 1H), 7.74 (bt, J= 6.0 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 32.8 (CH₂), 34.5 (CH₂), 35.2 (CH₂), 36.3 (CH₂), 39.5 (CH₂), 42.1 (CH₂), 76.4 (CH₂), 117.2 (CH), 119.0 (CH), 119.1 (C), 120.8 (CH), 123.0 (CH), 127.2 (CH), 129.3 (CH), 129.4 (CH), 129.8 (CH), 138.4 (C), 139.1 (C), 140.1 (C), 140.2 (C), 141.8 (C), 144.7 (C), 153.4 (C), 173.8 (C), 174.9 (C); HRMS (DCI/NH₃) found m/z 538.1330 [M+H]⁺, C₂₇H₂₉O₄N₃Br requires 466.0865.

4-Benzyloxy-5,21-dibromo-2-oxa-10,14-diaza-tricyclo[16.2.2.1^0,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (39)

tert-Butyl nitrite (10 μL, 9 μmol) was added to a solution of CuBr₂ (2.5 mg, 10 μmol) in CH₃CN (0.5 mL) at 0 °C and allowed to stir for 1h. A mixture of lactam 38 (10 mg, 0.02 mmol) in CH₃CN (0.8 mL) was then added drop-wise into the above solution over 20 min at 0 °C. After stirring for 2 h, the solution was allowed to warm to room temperature, quenched with HCl (1 N, 4 mL) and extracted with CH₂Cl₂ (4 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 39. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) followed by reversed phase HPLC (C₁₈, 5μm Microsorb, 10 × 250 mm, MeOH/H₂O, 65:35, 3 mL/min) gave 39 (2.7 mg, 21%) as a colorless amorphous solid: ¹H NMR (400 MHz, CD₃OD) δ 2.15-2.13 (m, 2H), 2.45-2.41 (m, 2H), 2.66-2.63 (m, 2H), 2.98-2.95 (m, 2H), 3.27-3.23 (m, 2H), 3.44-3.36 (m, 2H), 5.18 (s, 2H), 6.05 (d, J= 1.6 Hz, 1H), 7.08 (d, J= 8.4 Hz, 1H), 7.15 (d, J= 1.6 Hz, 1H), 7.24 (dd, J= 8.4, 1.6 Hz, 1H), 7.32-7.40 (m, 3H), 7.54-7.57 (m, 3H); HRMS (FAB) found m/z 601.0312 [M+H]⁺, C₂₇H₂₇O₄N₂Br₂ requires 601.0338.

4-Benzyloxy-5,20,22-tribromo-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (40)

tert-Butyl nitrite (3.6 μL, 0.04 mmol) was added to a solution of CuBr₂ (14 mg, 0.06 mmol) in CH₃CN (0.5 mL) at 0 °C and allowed to stir for 1h. A mixture of lactam 38 (11 mg, 0.02 mmol) in CH₃CN (0.5 mL) was added drop-wise into the above solution over 20 min at 0 °C. After stirring for 2 h, the solution was warmed to room temperature, quenched with HCl (1 N, 4 mL) and extracted with CH₂Cl₂ (4 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 40. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) followed by reversed phase HPLC (C₁₈, 5μm Microsorb, 10 × 250 mm, MeOH/H₂O, 70:30, 3 mL/min) gave 40 (4 mg, 29%) as a colorless amorphous solid: IR (neat) ν 3307, 2925, 1648 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.94-2.02 (m, 1H), 2.09-2.16, (m, 1H), 2.31-2.36 (m, 1H), 2.55-2.63 (m, 2H), 2.73-2.81 (m, 2H), 3.08-3.19 (m, 2H), 3.81-3.86 (m, 1H), 3.96-4.02 (m, 3H), 5.23 (d, J= 10.8 Hz, 1H), 5.24 (bs, 1H), 5.28 (d, J= 10.8 Hz, 1H), 5.85 (d, J= 2.0 Hz, 1H), 6.18 (bd, J= 6.8 hz, 1H), 7.03 (d, J= 2.0 Hz, 1H), 7.32-7.40 (m, 3H), 7.34 (s, 1H), 7.56-7.59 (m, 2H), 7.58 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 31.4 (CH₂), 33.7 (CH₂), 33.9 (CH₂), 35.6 (CH₂), 36.9 (CH₂), 41.5 (CH₂), 75.1 (CH₂), 114.6 (CH), 115.1 (C), 119.0 (C), 123.2 (C), 126.4 (CH), 128.3 (CH), 128.4 (CH), 128.6 (2 × CH), 135.6 (CH), 136.5 (C), 136.8 (C), 139.6 (C), 143.4 (C), 149.9 (C), 151.7 (C), 171.1 (C), 171.9 (C); LRMS (ESI) found m/z 701.1 [M+Na]⁺, C₂₇H₂₅N₂O₄Br₃ requires 700.9.

S-(–)-(6-{2-[4-Benzyloxy-3-bromo-5-(triisopropyl-silanyloxy)-phenyl]-ethylcarbamoyl}-5-tert butoxycarbonylamino-hexyl)-carbamic acid 9H-fluoren-9-ylmethyl ester (42)

A stirred solution of amine 34 (102 mg, 0.213 mmol) and acid 41³⁶ (108 mg, 0.224 mmol) in CH₂Cl₂ (2 mL) was treated with HOBt (58 mg, 0.426 mmol) and EDCI (81 mg, 0.426 mmol) at room temperature. After 3 h, the reaction was quenched with HCl (0.5 N), extracted with EtOAc (3 × 10 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 42. Flash chromatography (SiO₂, CH₂Cl₂/MeOH 5%) gave 42 (163 mg, 81%) as an oil: [α]²⁵_D –3.1° (c 0.75, CHCl₃); IR (neat) ν 3313, 2944, 2867, 1687, 1641, 1538cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.08 (d, J= 7.2 Hz, 18H), 1.20-1.34 (m, 4H), 1.41(s, 9H), 2.31-2.43 (m, 2H), 2.66 (dd, J= 6.8 Hz, 2H), 2.85-3.15 (m, 2H), 3.29-3.49 (m, 2H), 3.80-3.90 (m, 1H), 4.19 (dd, J= 7.2 Hz, 1H), 4.35 (d, J=8.4 Hz, 2H), 4.65 (bs, 1H), 4.99 (s, 2H), 5.87 (bd, J= 8.0 Hz, 1H), 6.18 (bs, 1H), 6.65 (d, J=1.6 Hz, 1H), 6.94 (d, J= 1.6 Hz, 1H), 7.28-7.37 (m, 8H), 7.51 (d, J= 7.2 Hz, 4H), 7.58 (d, J= 7.2 Hz, 4H), 7.73 (d, J= 7.2 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 12.8 (CH), 17.8 (CH₃), 23.1 (CH₂), 28.3 (CH₂), 29.6 (CH₂), 33.7(C), 34.8 (CH₂), 39.9 (CH₂), 40.4 (CH₂), 40.5 (CH₂), 47.1 (CH), 48.6 (CH), 66.5 (CH₂), 74.3 (CH₂), 79.0 (C) , 118.4 (C), 119.7 (CH), 119.9 (CH), 125.0 (CH), 125.3 (CH), 126.9 (CH), 127.6 (CH), 127.8 (CH), 128.1 (CH), 135.8 (C), 137.1 (C), 141.2 (C), 143.9 (C), 143.9 (C), 145.6 (C), 150.4 (C), 156.1 (C), 156.1 (C), 170.9 (C); HRMS (MALDI) found m/z 964.3905 [M+Na]⁺, C₅₁H₆₈N₃O₇BrSiNa requires 964.3902.

S-(–)-{6-{2-[4-benzyloxy-3-bromo-5-(triisopropyl-silanyloxy)-phenyl]-ethylcarbamoyl}-5-{3-(4-fluoro-3-nitro-phenyl)-propionylamino]-hexyl}-carbamic acid tert-butyl ester (44)

A stirred solution of amine 42 (72 mg, 0.076 mmol) in CH₂Cl₂ (1 mL) was treated with tris-(2-amino-ethyl)-amine (TAEA) (0.57 mL, 3.81 mmol) at room temperature. After 5 minutes, the reaction was quenched with NaCl (sat.) and extracted with CH₂Cl₂ (10 mL). The organic layers were combined, washed with brine (3 × 20 mL), phosphate buffer (pH= 5.5) (3 × 20mL), dried (Na₂SO₄), and the volatiles were removed to give crude amine 43. The crude amine 43 was carried forward without further purification. A stirred solution of amine 43 (55 mg, 0.076 mmol), and acid 17 (96 mg, 0.45 mmol) in CH₂Cl₂ (3 mL) was treated with HOBt (120 mg, 0.92 mmol) and EDCI (180 mg, 0.92 mmol) at room temperature. After 6 h, the reaction was quenched with HCl (0.5 N) and extracted with CH₂Cl₂ (3 × 10 mL). The organic layers were combined, washed with KOH (0.5 N) (3 × 30 mL) and brine, dried (Na₂SO₄), and the volatiles were removed to give crude amide 44. Flash chromatography (SiO₂, CH₂Cl₂/EtOAc 20% then CH₂Cl₂/MeOH 5%) gave 44 (60 mg, 86%) as an amorphous solid: [α]²⁵_D –6.1° (c 0.58, CHCl₃); IR (neat) ν 3295, 2942, 2867, 1697, 1646, 1538 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.06 (d, J= 7.6 Hz, 18H), 1.21-1.30 (m, 3H), 1.40 (s, 9H), 2.21-2.35 (m, 2H), 2.48 (t, J= 7.2 Hz, 2H), 2.67 (t, J= 7.2 Hz, 2H), 2.98 (t, J= 7.2 Hz, 2H), 3.00-3.07 (m, 2H), 3.41 (q, J= 6.0 Hz, 2H), 4.00-4.10 (m, 1H), 4.58-4.62 (m, 1H), 4.97 (s, 2H), 6.25 (bs, 1H), 6.64 (d, J= 2.0 Hz, 1H), 6.95 (d, J= 2.0 Hz, 1H), 6.98 (bd, J= 7.6 Hz, 1H), 7.15 (dd, J= 8.4, 10.4 Hz, 1H), 7.37-7.28 (m, 3H), 7.44-7.51 (m, 3H), 7.87 (dd, J= 7.2, 2.0 Hz, 1Hz); ¹³C NMR (100 MHz, CDCl₃) δ 12.9 (CH), 17.9 (CH₃), 23.1 (CH₂), 28.4 (CH₃), 29.6 (CH₂), 30.2 (CH₂), 33.3 (CH₂), 34.8 (CH₂), 37.4 (CH₂), 39.4 (CH₂), 39.8 (CH₂), 40.4 (CH₂), 46.6 (CH), 74.4 (CH), 79.2 (C), 118.2 (d, J= 20.5, CH) ; 118.5 (C), 119.8 (CH), 125.3 (CH), 125.5 (d, J= 2.3 Hz, CH), 127.9 (CH), 128.2 (CH), 128.2 (CH), 135.6 (d, J= 8.4 Hz, CH), 135.7 (CH), 137.1 (d, J= 8.0 Hz, C), 137.1 (C), 138.0 (d, J= 4.5 Hz, C), 145.7 (C), 150.5 (C), 154.0 (d, J= 261 Hz, C), 156.3 (C), 170.6 (C), 171.3 (C); HRMS (MALDI) found m/z 937.3572 [M+Na]⁺, C₄₅H₆₄N₄O₈BrSiNa requires 937.3553.

S-(–)-[4-(4-Benzyloxy-5-bromo-21-nitro-11,15-dioxo-2-oxa-10,14-diaza-tricyclo[16.2.2.1^0,0tricosa-1(21), 3(23), 4, 6, 18(22), 19-hexaen-13-yl)-butyl]-carbamic acid tert-butyl ester (45)

A stirred solution of amide 44 (52 mg, 0.057 mmol) in DMSO (30 mL, 2 mM) containing 4 Å sieves was treated with CsF (86 mg, 0.57 mmol) at room temperature. After 3 h of rapid stirring, the reaction mixture was diluted with water (250 mL) and extracted with CH₂Cl₂ (5 × 20 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude amide 45. Flash chromatography (SiO₂, 9.4:0.6 CH₂Cl₂/MeOH) gave 45 (31 mg, 75%) as an viscous oil: [α]²⁵_D –65.0° (c 0.600, CHCl₃); IR (KBr, neat) ν 3301, 2929, 1683, 1644, 1531, 1284, 1236 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.14-1.38 (m, 6H), 1.40 (s, 9H), 1.50-1.56 (m, 1H), 1.94-2.18 (m, 3H), 2.56-2.67 (m, 2H), 2.87-3.05 (m, 5H), 3.15-3.23 (m, 1H), 3.74-3.79 (m, 1H), 4.02-4.16 (m, 1H), 4.50-4.58 (m, 1H), 5.09-5.15 (m, 2H), 5.54-5.62 (m, 1H), 6.21 (s, 1H), 6.83 (bd, J= 7.2 Hz, 1H), 7.00 (d, J= 8.4 Hz, 1H), 7.05 (d, J= 2.0 Hz, 1H), 7.29-7.36 (m, 3H), 7.52-7.54 (m, 2H), 7.80 (d, J= 2.0 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 23.5 (CH₂), 28.4 (CH₂), 29.6 (CH₂), 31.6 (CH₂), 32.7 (CH₂), 32.8 (CH₂), 38.1 (CH₂), 39.3 (CH₂), 39.4 (CH₂), 40.1 (CH₂), 45.3 (CH), 75.4 (CH₂), 79.1 (C), 115.5 (CH), 118.6 (C), 124.1 (C), 125.7 (CH), 127.6 (CH), 128.2 (CH), 128.3 (CH), 128.6 (CH), 135.1 (CH), 136.3 (C), 136.7 (C), 138.9 (C), 142.4 (C), 144.3 (C), 146.6 (C), 151.8 (C), 156.1 (C), 170.5 (C), 171.5 (C); HRMS (MALDI) found m/z 761.2150 [M+Na]⁺, C₃₆H₄₃N₄O₈BrNa requires 761.2157.

S-[4-(20-Amino-4-benzyloxy-5-bromo-11,15-dioxo-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaen-13-yl)-butyl]-carbamic acid tert-butyl ester (46)

CrCl₂ (140 mg, 1.139 mmol) was added to a solution of lactam 45 (50 mg, 0.066 mmol) in DMF (1 mL) at room temperature. After 12 h of stirring, the DMF was removed under reduced pressure and the residue dissolved in EtOAc (10 mL). The organic solution was washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 46. Flash chromatography (SiO₂, CH₂Cl₂/ MeOH, 9:1) gave 46 (40 mg, 85%) as a yellow viscous oil: IR (NaCl, neat) ν 3289, 2929, 1643, 1509, 1278 cm^-1; ¹H NMR (400 MHz, CD₃OD/CDCl₃, 1:1) δ 1.25-1.5 (m, 6H), 1.38 (s, 9H), 1.90-2.0 (m, 2H), 2.1-2.3 (m, 1H), 2.50-2.63 (m, 3H), 2.70-3.13 (m, 8H), 3.38-3.49 (m, 1H), 3.88-4.03 (m, 1H), 5.10 (s, 2H), 6.32 (s, 1H), 6.56 (d, J= 7.2 Hz, 1H), 6.65 (s, 1H), 6.81 (d, J= 7.2 Hz, 1H), 7.03 (s, 1H), 7.25-7.35 (m, 3H), 7.40-7.55 (m, 3H); ¹³C NMR (100 MHz, CD₃OD/CDCl₃, 1:1) δ 23.7 (CH₂), 28.2 (CH₃), 29.5 (CH₂), 32.0 (CH₂), 32.9 (CH₂), 33.0 (CH₂), 38.5 (CH₂), 38.9 (CH₂), 39.6 (CH₂), 40.2 (CH₂), 45.9 (CH), 75.7 (CH₂), 79.1 (C), 116.5 (CH), 118,1 (CH), 120.4 (C), 122.1 (CH), 126.8 (CH), 128.5 (CH), 128.9 (CH), 136.8 (C), 137.3 (C), 137.8 (C), 138.3 (C), 141.6 (C), 144.0 (C), 152.0 (C), 157.2 (C), 172.3 (C), 172.9 (C); HRMS (MALDI) found m/z 731.2386 [M+Na]⁺, C₃₆H₄₅O₆N₄BrNa requires 731.2415.

S-(+)-[4-(4-Benzyloxy-5,20-dibromo-11,15-dioxo-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaen-13-yl)-butyl]-carbamic acid tert-butyl ester (47)

^tBuONO (2.5 μL, 0.025 mmol) was added to a stirred solution of CuBr₂ (5.6 mg, 0.01 mmol) in CH₃CN (0.1 mL) at 0 °C. After 1 h, a mixture of lactam 46 (12 mg, 0.02 mmol) in CH₃CN (0.8 mL) was added drop-wise into the above solution over 10 min at 0 °C. After stirring for 2 h, the mixture was allowed to warm to room temperature, quenched with HCl (1 N, 1 mL) and extracted with CH₂Cl₂ (4 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude lactam 47 and 50. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 9:1) followed by silca HPLC (SiO₂, 5μm Microsorb, 10 × 250 mm, CH₂Cl₂/MeOH, 99:1.5, 3 mL/min) gave 47 (4.8 mg, 38%) as a colorless amorphous solid and 50 (5.4 mg, 42%) as a colorless amorphous solid. 47: [α]_D²⁵ +69.4° (c 0.320, CHCl₃); IR (KBr) ν 3303, 2927, 1681, 1644, 1527, 1488, 1280 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.41 (s, 9H), 1.18-1.68 (m, 5H), 2.00-2.18 (m, 3H), 2.53-3.20 (m, 9H), 3.68-3.79 (m, 1H), 4.12-4.24 (m, 1H), 4.46-4.58 (m, 1H), 5.16 (d, J= 10.8 Hz, 1H), 5.25 (d, J= 10.8 Hz, 1H), 5.34 (bs, 1H), 5.99 (bs, 1H), 6.85 (bd, J=10 Hz, 1H), 7.01 (d, J= 8.4 Hz, 1H), 7.01 (d, J=2.0 Hz, 1H), 7.17 (dd, J= 8.4, 2.0 Hz, 1H), 7.29-7.38 (m, 3H); 7.48 (d, J= 2.0 Hz,1H), 7.58-7.59 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 23.6 (CH₂), 28.4 (CH₃), 29.6 (CH₂), 31.7 (CH₂), 32.7 (CH₂), 32.9 (CH₂), 38.2 (CH₂), 39.7 (CH₂), 40.2 (CH₂), 40.5 (CH₂), 45.2 (CH), 75.2 (CH₂), 79.0 (C), 115.1 (CH), 116.2 (C), 118.6 (C), 123.7 (CH), 126.2 (CH), 128.2 (CH), 128.4 (CH), 128.7 (CH), 129.6 (CH), 134.2 (CH), 136.1 (C), 136.9 (C), 140.1 (C), 143.6 (C), 150.0 (C), 152.0 (C), 156.1 (C), 171.7 (C); HRMS (MALDI) found m/z 794.1425 [M+Na]⁺, C₃₆H₄₃N₃O₆Br₂Na requires 794.1441.

S-(+)-[4-(4-Benzyloxy-5,20,22-tribromo-11,15-dioxo-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaen-13-yl)-butyl]-carbamic acid tert-butyl ester (50)

[α]_D²⁵ +38.3° (c 0.360, CHCl₃); IR (KBr) ν 3334, 2925, 1697, 1652, 1513, 1247 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 1.41 (s, 9H), 1.18-1.68 (m, 7H), 2.00-2.22 (m, 3H), 2.50-2.66 (m, 2H), 2.78-2.88 (m, 1H), 2.96-3.24 (m, 4H), 3.78-3.86 (m, 1H), 4.18-4.28 (m, 1H), 4.48-4.60 (m, 1H), 5.11 (d, J= 10.4 Hz, 1H), 5.28 (d, J= 10.4 Hz, 1H), 5.28 (bs, 1H), 5.95 (d, J= 2.0 Hz, 1H), 6.87 (bd, J= 9.6 Hz, 1H), 7.03 (d, J= 2.0 Hz, 1H), 7.32-7.39 (m, 3H); 7.36 (s,1H), 7.52 (s, 1H), 7.56-7.59 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 23.7 (CH₂), 28.4 (CH₃), 29.7 (CH₂), 31.9 (CH₂), 32.7 (CH₂), 32.9 (CH₂), 37.3 (CH₂), 38.3 (CH₂), 40.8 (CH₂), 45.2 (CH), 75.2 (CH₂), 79.0 (C), 114.5 (CH), 114.9 (C), 118.8 (C), 123.0 (C), 126.5 (CH), 128.2 (CH), 128.4 (CH), 128.7 (CH), 128.7 (CH), 135.4 (CH), 136.1 (C), 136.9 (C), 139.4 (C), 143.4 (C), 150.0 (C), 151.9 (C), 156.0 (C), 171.8 (C); HRMS (MALDI) found m/z 872.0502 [M+Na]⁺, C₃₆H₄₂N₃O₆Br₃Na requires 872.0516.

S-2-Azido-N-[4-(5,20-dibromo-4-hydroxy-11,15-dioxo-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaen-13-yl)-butyl]-5-iodo-benzamide (49)

BBr₃ (1 M, 10 μL, 10 μmol) was added to a solution of lactam 47 (3 mg, 3.8 μmol) in CH₂Cl₂ (50 μL) and the orange solution was allowed to stir overnight at room temperature. The solution was directly transferred onto a flash column (SiO₂, CH₂Cl₂/MeOH/NH₂OH, 10:1:0.1) to give the crude amine 48 (2.2 mg) as an amorphous solid that was immediately carried forward. EDCI (2 mg, 10 μmol) was added to a stirred solution of the crude amine (2.2 mg), HOBt (1.5 mg, 10 μmol), and 2-azido-5-iodo-benzoic acid³⁷ (3 mg, 10 μmol) in DMF (5.0 mL) at room temperature. After 5 h, the reaction was quenched with HCl (1N) and extracted with CH₂Cl₂ (3 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 49. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 20:1) followed by reversed phase HPLC (C₁₈, MeOH/H₂O, 3:1, 4 mL/min) gave pure 49 (0.56 mg, 33% over two steps) as a colorless amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 1.20-1.60 (m, 7H), 1.94-2.18 (m, 3H), 2.48-2.64 (m, 2H), 2.76-3.00 (m, 3H), 3.04-3.14 (m, 1H), 3.22-3.40 (m, 2H), 3.61-3.74 (m, 1H), 4.06-4.19 (m, 1H), 5.41 (bs, 1H), 5.98 (s, 1H), 6.06 (d, J= 2.0 Hz, 1H), 6.92 (d, J= 8.4 Hz, 1H), 6.96 (d, J= 2.0 Hz, 1H), 7.09 (d, J= 8.0 Hz, 1H), 7.19 (dd, J= 8.0, 2.0 Hz, 1H), 7.33 (bs, 1H), 7.75 (dd, J= 8.4, 2.0 Hz, 1H), 8.37 (d, J= 2.0 Hz, 1H); HRMS (MALDI) found m/z 874.9709 [M+Na]⁺, C₃₁H₃₁N₆O₅Br₂NaI requires 874.9660.

S-2-Azido-5-iodo-N-[4-(5,20,22-tribromo-4-hydroxy-11,15-dioxo-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaen-13-yl)-butyl]-benzamide (52)

BBr₃ (1 M, 10 μL, 10 μmol) was added to a stirred solution of lactam 50 (2 mg, 2.4 μmol) in CH₂Cl₂ (50 μL) at room temperature. The solution was directly transferred onto a flash column (SiO₂, CH₂Cl₂/MeOH/NH₂OH, 10:1:0.1) to give crude amine 51 (1.8 mg) as an amorphous solid that was immediately carried forward. EDCI (2 mg, 10 μmol) was added to a stirred solution of crude amine 51 (2.2 mg), HOBt (1.5 mg, 10 μmol), and 2-azido-5-iodo-benzoic acid³⁷ (3 mg, 10 μmol) in DMF (5.0 mL) at room temperature. After 5 h, the reaction was quenched with HCl (1N) and extracted with CH₂Cl₂ (3 × 5 mL). The organic layers were combined, washed with brine, dried (Na₂SO₄), and the volatiles were removed to give crude 52. Flash chromatography (SiO₂, CH₂Cl₂/MeOH, 20:1) followed by reversed phase HPLC (C₁₈, MeOH/H₂O, 3:1, 4 mL/min) gave 52 (0.80 mg, 48% over two steps) as a colorless amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 1.20-1.60 (m, 4H), 1.91-2.24 (m, 5H), 2.48-2.62 (m, 2H), 2.91-3.22 (m, 4H), 3.28-3.48 (m, 2H), 3.74-3.84 (m, 1H), 4.12-4.24 (m, 1H), 5.24 (bt, J= 5.6 Hz, 1H), 5.93 (s, 1H), 6.02 (d, J= 2.0 Hz, 1H), 6.92 (d, J= 8.4 Hz, 1H), 6.96 (dd, J= 8.4, 2.0 Hz, 1H), 6.99 (d, J= 2.0 Hz, 1H), 7.40 (bt, J= 5.6 Hz, 1H), 7.46 (s, 1H), 7.48 (s, 1H), 7.75 (dd, J= 8.4, 2.0 Hz, 1H), 8.37 (d, J= 2.0 Hz, 1H); HRMS (MALDI) found m/z 952.8767 [M+Na]⁺, C₃₁H₃₀N₆O₅Br₃NaI requires 952.8765.

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25.Bigot A, Zhu J. Synthesis of cycloisodityrosine revisited: A selective ring forming process. Tetrahedron Lett. 1998;39:551–554. [Google Scholar]
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28.Masuno MN, Molinski TF. Cationic Reduction of Bastadin-4 to Bastadin-5. Preparation of 5-[2H]-Bastadin-5 by Site-Specific Isotopic Labeling. J. Nat. Prod. 2003;66:112–114. doi: 10.1021/np020382h. [DOI] [PubMed] [Google Scholar]
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30.Jessup PJ, Petty CB, Roos J, Overman LE. 1-N-Acylamino-1,3-dienes from 2,4-pentadienoic acids by the Curtius rearrangement: benzyl trans-1,3-butadiene-1-carbamate. Org. Synth. 1980;59:1–9. [Google Scholar]
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32.Harmon RE, Parsons JL, Cooke DW, Gupta SK, Schoolenberg J. Homogeneous catalytic hydrogenation of unsaturated organic compounds. J. Org. Chem. 1969;34:3684–3685. [Google Scholar]
33.Hosoya T, Aoyama H, Ikemoto T, Hiramatsu T, Kihara Y, et al. [125I]-N-[(3-Azido-5-iodo)benzyl]dantrolene and [125I]-N-{[3-Iodo-5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl}dantrolene: photoaffinity probes specific for the physiological Ca2+ release from sarcoplasmic reticulum of skeletal muscle. Bioorg. Med. Chem. Lett. 2002:3263–3265. doi: 10.1016/s0960-894x(02)00701-1. [DOI] [PubMed] [Google Scholar]
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36.Guichard G, Abele S, Seebach D. Preparation of N-Fmoc-protected β2- and β3-amino acids and their use as building blocks for the solid-phase synthesis of β-peptides. Helv. Chim. Acta. 1998;81:187–206. [Google Scholar]
37.Latli B, Tomizawa M, Casida JE. Synthesis of a Novel [125I]Neonicotinoid Photoaffinity Probe for the Drosophila Nicotinic Acetylcholine Receptor. Bioconjugate Chem. 1997;8:7–14. doi: 10.1021/bc960066c. [DOI] [PubMed] [Google Scholar]
38.Berchtold MW, Brinkmeier H, Muntener M. Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. Physiol. Rev. 2000;80:1215–1265. doi: 10.1152/physrev.2000.80.3.1215. [DOI] [PubMed] [Google Scholar]
39.Saito A, Seiler S, Chu A, Fleischer S. Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J. Cell Biol. 1984;99:875–885. doi: 10.1083/jcb.99.3.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Wong PW, Pessah IN. Ortho-substituted polychlorinated biphenyls alter calcium regulation by a ryanodine receptor-mediated mechanism: structural specificity toward skeletal- and cardiac-type microsomal calcium release channels. Mol. Pharmacol. 1996;49:740–751. [PubMed] [Google Scholar]
41.Wong PW, Pessah IN. Noncoplanar PCB 95 alters microsomal calcium transport by an immunophilin FKBP 12-dependent mechanism. Mol. Pharmacol. 1997;51:693–702. doi: 10.1124/mol.51.5.693. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Info_rev

NIHMS61207-supplement-Supporting_Info_rev.pdf^{(20.8MB, pdf)}

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[R19] 19.Carney JR, Scheuer PJ, Kelly-Borges M. A new bastadin from the sponge Psammaplysilla purpurea. J. Nat. Prod. 1993;56:153–157. doi: 10.1021/np50091a025. [DOI] [PubMed] [Google Scholar]

[R20] 20.Pordesimo EO, Schmitz FJ. New bastadins from the sponge Ianthella basta. J. Org. Chem. 1990;55:4704–4709. [Google Scholar]

[R21] 21.Miao S, Andersen RJ, Allen TM. Cytotoxic metabolites from the sponge Ianthella basta collected in Papua New Guinea. J. Nat. Prod. 1990;53:1441–1446. doi: 10.1021/np50072a007. [DOI] [PubMed] [Google Scholar]

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[R24] 24.Kazlauskas R, Lidgard RO, Murphy PT, Wells RJ, Blount JF. Brominated tyrosine-derived metabolites from the sponge Ianthella basta. Aust. J. Chem. 1981;34:765–786. [Google Scholar]

[R25] 25.Bigot A, Zhu J. Synthesis of cycloisodityrosine revisited: A selective ring forming process. Tetrahedron Lett. 1998;39:551–554. [Google Scholar]

[R26] 26.Bigot A, Dau METH, Zhu J. Total Synthesis of an Antitumor Agent RA-VII via an Efficient Preparation of Cycloisodityrosine. J. Org. Chem. 1999;64:6283–6296. [Google Scholar]

[R27] 27.Masuno MN, Molinski TF. Regioselective cationic reduction of 2-aryl-1-N-(ethoxycarbonyl)enamines to 2-arylethylamine carbamates. Tetrahedron Lett. 2001;42:8263–8266. [Google Scholar]

[R28] 28.Masuno MN, Molinski TF. Cationic Reduction of Bastadin-4 to Bastadin-5. Preparation of 5-[2H]-Bastadin-5 by Site-Specific Isotopic Labeling. J. Nat. Prod. 2003;66:112–114. doi: 10.1021/np020382h. [DOI] [PubMed] [Google Scholar]

[R29] 29.Wymann WE, Davis R, Patterson JW, Jr., Pfister JR. Selective alkylations of certain phenolic and enolic functions with lithium carbonate-alkyl halide. Synth. Commun. 1988;18:1379–1384. [Google Scholar]

[R30] 30.Jessup PJ, Petty CB, Roos J, Overman LE. 1-N-Acylamino-1,3-dienes from 2,4-pentadienoic acids by the Curtius rearrangement: benzyl trans-1,3-butadiene-1-carbamate. Org. Synth. 1980;59:1–9. [Google Scholar]

[R31] 31.Park H, Cao B, Joullie MM. Regioselective asymmetric aminohydroxylation approach to a β-hydroxyphenylalanine derivative for the synthesis of ustiloxin D. J. Org. Chem. 2001;66:7223–7226. doi: 10.1021/jo010482c. [DOI] [PubMed] [Google Scholar]

[R32] 32.Harmon RE, Parsons JL, Cooke DW, Gupta SK, Schoolenberg J. Homogeneous catalytic hydrogenation of unsaturated organic compounds. J. Org. Chem. 1969;34:3684–3685. [Google Scholar]

[R33] 33.Hosoya T, Aoyama H, Ikemoto T, Hiramatsu T, Kihara Y, et al. [125I]-N-[(3-Azido-5-iodo)benzyl]dantrolene and [125I]-N-{[3-Iodo-5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl}dantrolene: photoaffinity probes specific for the physiological Ca2+ release from sarcoplasmic reticulum of skeletal muscle. Bioorg. Med. Chem. Lett. 2002:3263–3265. doi: 10.1016/s0960-894x(02)00701-1. [DOI] [PubMed] [Google Scholar]

[R34] 34.a Doyle MP, Siegfried B, Dellaria JF., Jr Alkyl nitrite-metal halide deamination reactions. 2. Substitutive deamination of arylamines by alkyl nitrites and copper(II) halides. A direct and remarkably efficient conversion of arylamines to aryl halides. J. Org. Chem. 1977;42:2426–2431. [Google Scholar]; b Doyle MP, Dellaria JF, Jr., Siegfried B, Bishop SW. Reductive deamination of arylamines by alkyl nitrites in N,N-dimethylformamide. A direct conversion of arylamines to aromatic hydrocarbons. J. Org. Chem. 1977;42:3494–3498. [Google Scholar]

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[R36] 36.Guichard G, Abele S, Seebach D. Preparation of N-Fmoc-protected β2- and β3-amino acids and their use as building blocks for the solid-phase synthesis of β-peptides. Helv. Chim. Acta. 1998;81:187–206. [Google Scholar]

[R37] 37.Latli B, Tomizawa M, Casida JE. Synthesis of a Novel [125I]Neonicotinoid Photoaffinity Probe for the Drosophila Nicotinic Acetylcholine Receptor. Bioconjugate Chem. 1997;8:7–14. doi: 10.1021/bc960066c. [DOI] [PubMed] [Google Scholar]

[R38] 38.Berchtold MW, Brinkmeier H, Muntener M. Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. Physiol. Rev. 2000;80:1215–1265. doi: 10.1152/physrev.2000.80.3.1215. [DOI] [PubMed] [Google Scholar]

[R39] 39.Saito A, Seiler S, Chu A, Fleischer S. Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J. Cell Biol. 1984;99:875–885. doi: 10.1083/jcb.99.3.875. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] 40.Wong PW, Pessah IN. Ortho-substituted polychlorinated biphenyls alter calcium regulation by a ryanodine receptor-mediated mechanism: structural specificity toward skeletal- and cardiac-type microsomal calcium release channels. Mol. Pharmacol. 1996;49:740–751. [PubMed] [Google Scholar]

[R41] 41.Wong PW, Pessah IN. Noncoplanar PCB 95 alters microsomal calcium transport by an immunophilin FKBP 12-dependent mechanism. Mol. Pharmacol. 1997;51:693–702. doi: 10.1124/mol.51.5.693. [DOI] [PubMed] [Google Scholar]

PERMALINK

Simplified Cyclic Analogs of Bastadin-5. Structure Activity Relationships for Modulation of the RyR1/FKBP12 Ca2+ Channel Complex

Makoto N Masuno

Isaac N Pessah

Marilyn M Olmstead

Tadeusz F Molinski

Abstract

Introduction

Synthesis

Scheme 1.

Scheme 2.

Scheme 4.

Scheme 5.

Scheme 6.

Structure and Biological Evaluation

Figure 2.

Table 1.

Figure 3.

Biological Activity

Table 2.

Figure 4.

Photoaffinity Analogues

Scheme 7.

Conclusion

Supplementary Material

Figure 1.

Scheme 3.

Acknowledgements

Experimental

General

[3H]-Ryanodine Binding Asssay

5-Bromo-4-hydroxy-17-nitro-2-oxa-10-aza-tricyclo[12.2.2.10,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (4)

17-Amino-5-bromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.10,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (5)

17-Azido-5-bromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.10,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (6)

5,17-Dibromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.10,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (7)

5,15,17-Tribromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.10,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (8)

5-Bromo-4-hydroxy-2-oxa-10-aza-tricyclo[12.2.2.10,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (9)

5-Bromo-4-hydroxy-17-iodo-2-oxa-10-aza-tricyclo[12.2.2.10,0]nonadeca-1(17),3(19),4,6,14(18),15-hexaen-11-one (10)

5-Bromo-4-hydroxy-20-nitro-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (11)

21-Amino-4-hydroxy-5-bromo-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (12)

21-Azido-4-hydroxy-5-bromo-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (13)

5,21-Dibromo-4-hydroxy-2-oxa-10,14-diaza-tricyclo[16.2.2.10,0]tricosa- 1(21),3(23),4,6,18(22),19-hexaene-11,15-dione (14)