Simplified cyclic analogues of bastadin-5. Structure-activity relationships for modulation of the RyR1/FKBP12 Ca2+ channel complex

Article abstract of DOI:10.1021/jm050708u

Bastadin-5, a brominated macrodilactam from the marine sponge Ianthella basta, enhances release of Ca²⁺ from stores within the sarcoplasmic reticulum (SR) of muscle and nonmuscle cells by modulating RyR1/FKBP12 complex. Analogues of bastadin-5

Full text of DOI:10.1021/jm050708u

J. Med. Chem. 2006, 49, 4497-4511
4497
Simplified Cyclic Analogues of Bastadin-5. Structure-Activity Relationships for Modulation of
the RyR1/FKBP12 Ca2+ Channel Complex
Makoto N. Masuno,† Isaac N. Pessah,‡ Marilyn M. Olmstead,† and Tadeusz F. Molinski*,†,¶
Department of Chemistry and Biochemistry, and Skaggs School of Pharmacy and Pharmaceutical Sciences, UniVersity of California,
San Diego, 9500 Gilman DriVe, La Jolla, California 92093, and Department of Chemistry, Molecular Biosciences, Veterinary Medicine, and
Center for Children’s EnVironmental Health UniVersity of California, DaVis, California 95616
ReceiVed July 22, 2005
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 nonmuscle cells by modulating RyR1/
FKBP12 complex. Analogues 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 analogues
of bastadin-5 were synthesized from substituted benzaldehydes in a convergent manner, featuring an efficient
SNAr 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 analogue
(()-7, like bastadin-5, enhanced ryanodine binding to the RyR1/FKBP12 complex (EC50 11 µM), however,
unexpectedly, the corresponding achiral 18-membered ring analogue 14 potently inhibited binding (IC50
6
µM) under the same conditions. Structure-activity relationships of both families of cyclic analogues 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 analogues were those that conserved the dibromocatechol
ether moiety that corresponds to the ‘western edge’ of the bastadin-5 structure. These data suggest that
cyclic analogues 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. Regu-
lated 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 com-
pounds possessing newly identified properties: allosteric in-
teractions 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
)
Figure 1. Natural products that interact with FKBP12 or the RyR1/
FKBP12 complex.
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 macro-
dilactam. 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
* To whom correspondence should be addressed. Phone: 858-534-7115.
Fax: 858-822-0386. E-mail: tmolinski@ucsd.edu.
† Department of Chemistry, UC Davis.
‡ Molecular Biosciences, School of Veterinary Medicine.
¶ Department of Chemistry and Biochemistry, UCSD.
10.1021/jm050708u CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/30/2006

4498 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Masuno et al.
Scheme 1. Retrosynthetic Analysis for 14- and 18-Membered Ring Analogues of Bastadin-5
bonds to give a pseudo-tetrameric macrodilactam. Each of the
21 bastadins has an R-ketoxime that appears to derive from
R-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 tis-
sues.11,13 The RyR1/FKBP12 complex is a ∼2.2 MDa hetero-
tetrameric 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 composed of ∼5000 amino acid
residues folded into several transmembrane domains. Competi-
tion 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 dis-
sociation 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 prepara-
tions 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
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 analogues of
3 (e.g. 4-15), which embody the substituted catechol ethers of
the ‘western’ hemisphere of 3 within two families of macro-
cycles 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 analogue 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
analogue of 3, was a potent antagonist in the same binding assay
(IC50 ) 6 µM) which suggests brominated catechol ethers
exhibit a bimodal activity that varies with nonbonded interac-
tions. 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 analogues to the RyR1/FKBP12 receptor, which
suggests a bimodal model for Ca2+ gating, which may be tunable
by a selection of properly designed ‘second generation’ ana-
logues.
Synthesis
The retrosynthetic analysis shown in Scheme 1 reveals the
design for preparation of cyclic analogues. We envisioned the
14-membered cyclic analogues, a smaller, ring-constrained
macrocycle embodying a single amide bond, would derive from
common intermediates 16 and 17. The larger 18-membered
analogues would include an additional â-alanine as a spacer.
The units would be assembled by amide bond formation and
the pivotal macrocyclization would be accomplished by SNAr
coupling according to Zhu and co-workers.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.
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
nonbonded interactions (e.g. Br-steric interactions) in the
‘western’ and ‘eastern’ parts of the bastadins. Consequently,

Simplified Bastadin-5 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4499
Scheme 3. Synthesis of Common Intermediate 17a
Scheme 2. Synthesis of Substituted Phenethylamine 16a
a Key: (a) RhCl(PPh3)3, H2 (3 atm), toluene, 8 h, 95%; (b) i. LiOH,
THF, H2O, MeOH (4:1:1); ii. HCl aq (0.5 M), 86%.
a Key: (a) i. Li2CO3, DMF, 45 °C, 1 h; ii. BnBr, 88% (b) i. malonic
acid, pyridine, piperidine (cat.), toluene, reflux, 12 h; ii. 1 N HCl, 86%; (c)
ethyl chloroformate, Hu¨nig’s base, acetone, 0 °C, 2 h; ii. NaN3, H2O, 0 °C;
iii. EtOH, toluene, reflux, 12 h, 65%; (d) i. TFA, Et3SiH, -10 °C, 0.5 h;
ii. NaHCO3 (aq), 94%; (e) hydrazine, KOH, 1,4-dioxane, reflux, 1 h, 92%
Scheme 4. Synthesis of 14-Membered Analogues 4, 5, and 6
via Macroetherificationa
Simple Dreiding molecular models predicted that the 14-
membered analogues would be locked into fixed conformations
as atropisomers due to severe torsional strain that arises from
rotation about the long axis of the p-O-aryl linkage, while the
18-membered ring analogues should retain relative conforma-
tional 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 phenethyl-
amine 16 was carried out using methodology we had optimized
earlier to satisfy these criteria.27
a 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. 1 N 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, 1 h, 58%; (f) i. AcOH/H2O, 9:1, NaNO2, 0 °C; ii. NaN3, 0 °C,
66%.
Scheme 5. Preparation of C-Aryl-Substituted 14-Membered
The method is amenable to radio-labeling of analogues 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 p-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 R,â-unsaturated
acid 22 in 86% yield which was converted to enamide 23 by a
Curtius rearrangement of the corresponding acyl azide (65%
over three 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%).
Ring Analoguesa
t
a Key: (a) i. CuBr2 (0.8 equiv), BuONO, CH3CN, 0 °C, 1 h; ii. 27 in
portions, 0 °C, 2 h then warmed to room temperature; iii. 1 N HCl, 66%
t
(b) i. CuBr2 (10 equiv), BuONO, CH3CN, 0 °C, 1 h; ii. 28 in portions, 0
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%) (Scheme 3).
°C, 2 h then warmed to room temperature; iii. 1 N HCl, 32%; (c) i. tBuONO,
THF, 0 °C, 1 h; ii. 28 in portions, 0 °C, 2 h then warmed to room
temperature; iii. 1 N HCl, 39%; (d) i. H2SO4, AcOH, NaNO2, 0 °C, 0.5 h;
ii. 28 in portions, 0 °C, 1 h then warmed to room temperature, 20 min; iii.
KI, H2O, rt, 15 min then warmed to 70 °C, 15 min, 16%; (e) BBr3, -78 °
C, 1 h, 91%; (f) TFA, rt, 24 h, 70%; (g) TFA, rt, 24 h, 75%.
Two possible routes to the cyclic intermediates were con-
sidered: SNAr 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 (2 mM, K2CO3, 4 Å sieves, DMSO, RT)
smoothly converted 18 to lactam 27 in high yield (85%).
Removal of the benzyl group provided the 14-membered ring-
constrained analogue 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 analogues 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 analogue 6 in
66% yield.33 The dibromo or tribromide compounds 29 and 30
were procured in acceptable yields by Sandmeyer-type reaction

4500 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Masuno et al.
Scheme 6. Synthesis of 18-Membered Analogues by SNAr
Macroetherificationa
Figure 2. Representative NOEs of 29.
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 analogue 13 (69%), dibromo
analogue 14, and tribromide 15.
Structure and Biological Evaluation
For the purpose of discussion, the 14-membered analogues
are numbered according to Figure 2. The simple cyclic
analogues 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 analogues 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 analogues.
Varying the substitution on ring B may reveal electronic effects
upon the binding of bastadin analogues to the RyR1/FKBP12
complex.
a Key: (a) i. Boc2O, CH3CN, rt, 12 h; ii. Na2CO3, MeOH, H2O (4:1), rt,
76%; (b) TIPSCl, DMF, rt, 24 h, 95%; (c) i. TFA, CH2Cl2, (1:1), rt, 1h; ii.
NaHCO3 (aq), 95%; (d) i. N-Boc-â-alanine, EDCI, HOBt, CH2Cl2, rt, 12
h; ii. 0.5 N HCl, 92%; (e) i. TFA, CH2Cl2, (1:1), rt, 1 h; ii. NaHCO3 (aq),
97%; (f) i. 17, EDCI, HOBt, CH2Cl2, rt, 12 h; ii. 0.5 N HCl; 93%; (g) CsF,
DMSO (2 mM), 4 Å sieves, rt, 20 h, 84%; (h) BBr3, CH2Cl2, rt, 8 h, 82%;
(i) CrCl2, DMF, rt, 12h, 55%; (j) BBr3, CH2Cl2, rt, 8 h, 54%; (k) i. AcOH/
H2O, 9:1, NaNO2 0 °C; ii. NaN3, 0 °C, 69%; (l) i. CuBr2 (0.8 equiv),
tBuONO, CH3CN, 0 °C, 1 h; ii. 40 in portions, 0 °C, 2 h then warmed to
room temperature; iii. 1 N HCl, 25%; (m) BBr3, CH2Cl2, rt, 8 h, 94%; (l)
i. CuBr2 (3.0 equiv), tBuONO, CH3CN, 0 °C, 1 h; ii. 40 in portions, 0 °C,
2 h then warmed to room temperature; iii. 1 N HCl, 29%; (m) BBr3, CH2Cl2,
rt, 8 h, 72%.
Solution and solid-state conformation studies of the analogues
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 analogues 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).
1
The H NMR spectrum of 29 showed a diagnostic two-proton
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, presum-
ably 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
analogues 7, 8, and 9, respectively.
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 excep-
tionally high field H19 aryl proton signal (δ 5.06, 1H, J ) 2.0
Hz).
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 dia-
1
stereotopic methylene groups and, likewise, seen in the H
NMR spectra of 7 and other products derived from 28, except
9. The debromo analogue 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 analogue 29 was supported
by NOE experiments (Figure 2, Table 1). Representative NOEs
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-
Synthesis of the 18-membered ring analogues 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-â-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

Simplified Bastadin-5 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4501
Table 1. NMR Data for 29 (400 MHz, CDCl3)
no.
1H δ, m, J (Hz)
13C (ppm)
152.9
COSY
HMBC
NOE
1
3
4
5
6
7
8
9
154.6
136.4
117.9
125.8
142.8
30.5
6.88 dd (2.0, 1.2)
H19, H8
C5, C7, C19, C8
2.64 m
3.29 m
4.90 m
H9
H8; H10
H9
H6, H9, H10; H19
H8, H10; H18
H8, H9, H12, H19
39.7
10
11
171.1
12
13
2.29 m
3.00 m
41.0
31.7
H13
H12
C11, C13, C14
C11, C12, C14; C15, C18
H10, H13, H15, H18
H12, H15; H18
14
140.6
15
16
7.23 dd (8.4, 2.4)
7.05 d (8.4)
130.4
126.0
H16; H18
H15
C1, C13, C15; C17
C1, C18
H13, H16
H15, H19
17
118.7
18
19
20a
20b
21
7.51 d (2.4)
5.06 d (2.0)
5.34 d (10.8)
5.18 d (10.8)
134.6
113.1
75.2
75.2
H15
H6
H20b
H20a
C1, C13, C15; C17
C3, C7, C8, C6
C4, C21; C (22-26)
C4, C21, C (22-26)
H12, H13
H9, H10; H16
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)
(3), in particular, transport of Ca2+ across the channel and
alteration of channel gating (open state probability).13
Equilibrium binding of [3H]-ryanodine to skeletal JSR was
measured in the presence of analogue 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 analogues 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 2-fold loss of activity (EC50
) 21 µM), whereas the amino analogue 5 (EC50 ) 33 µM) and
the iodo analogue 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 dibromo-
catechol ether moiety at the western edge of the bastadin-5
structure. Despite almost identical conformations (X-ray),
analogues 4 and 9 showed the largest difference in ryanodine
binding among the 14-membered ring analogues (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 analogue 8 resulted in total loss of agonist activity
in this 14-membered ring analogue (EC50 < 100 µM) although,
curiously, this was not the case in the corresponding 18-
membered ring analogue (see below).
Figure 3. X-ray crystal structures of (()-4 and 9.
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 analogues 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 â-alanine linker.
The barrier to rotation in the 14-membered ring analogue 29
was briefly examined by temperature dependent H NMR (d6-
DMSO). At temperatures up to T ) 105 °C the H NMR line
shapes of the O-Bn signals in 29 remained sharp and revealed
no tendency toward coalescence or broadening which suggests
a barrier to rotation >17 kcal/mol.35
1
1
Biological Activity
Biological evaluation of the synthetic analogues 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
An unexpected trend was revealed by measurements of
ryanodine binding in the presence of the 18-membered ring
analogues: 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),

4502 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Masuno et al.
Table 2. Structure-Activity Relationships of Bastadin-5 (3) and Analoguesa
entry
compound
skeleton
X
Y
EC50 (µM)b
IC50 (µM)c
1
2
4
5
6
7
8
9
10
11
12
13
14
15
16
17
3
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A
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C
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C
C
D
D
-
-
H
H
H
H
Br
H
2.2 ( 0.1
20.9 (10.8
33.0 (12.2
-
-
NO2
NH2
N3
Br
-
-
63 (8.1
10.9 (2.6
>100
-
Br
H
I
NO2
NH2
N3
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Br
-
>100
-
28.6 (14.2
24 ( 3.9
21 (5.7
20.0 ( 2.3
-
-
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H
H
H
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-
12
13
14
-
-
6.2 (1.2
47 ( 15
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-
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6.5 (0.7
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49
52
53
Br
a Equilibrium binding of 1 nM [3H]-ryanodine to skeletal junctional sarcoplasmic reticulum (SR) was performed in assay buffer containing 250 mM KCl,
b
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
3
c
3
(enhanced [H]-ryanodine binding). Antagonist-like (reduced [H]-ryanodine binding)
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 (EC50s 24-58 µM).
These results suggest the interaction of simple bastadin-5
analogues with the RyR1/FKBP12 is more complex and
shows a broader range of action than expected. Each of
the active 14-membered ring analogues 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
analogue, 7.
Photoaffinity Analogues. With the ryanodine-binding prop-
erties of both 14-membered and 18-membered ring analogues
in hand, we asked the question, ‘can a simple photoaffinity
analogue of 3 be designed which retains high potency for the
Figure 4. Concentration-dependent enhancement/inhibition of the
RyR1/FKBP12 complex?’ Using the key design principles from
the 18-membered ring series, we replaced the â-alanine with a
â-lysine residue to prepare an 18-membered macrolactam that
contains a primary amino group that would could be acylated
at the ꢀ-NH2 group with a photoreactive azidobenzoic acid.
Photolysis of the derived azidobenzamide analogue of 3 would
produce a nitrene that may covalently bond to the RyR1/
FKPB12 complex. Tryptic digestion and MS analysis of the
photolabeled RyR1/FKPB12-derived peptides from the exposed
surface may reveal the consensus amino acid sequence of the
bastadin binding site.
binding of 1 nM [3H]-ryanodine to RyR1-FKBP12 in skeletal SR by
bastadin analogues (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.
Differentially N-protected â-lysine (41) was coupled (EDCI,
HOBt) to bromotyramine 34 (Scheme 7), followed by removal
of the â-N Fmoc protecting group (81%) and coupling to the

Simplified Bastadin-5 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4503
Scheme 7. Synthesis of Photoaffinity Analogues by SNAr Macroetherificationa
a a. i. EDCI, HOBt, CH2Cl2, 41;36 ii. H+, 81%; b. N,N,N,N-tetramethylethylenediamine, CH2Cl2; c. EDCI, HOBt, CH2Cl2, 17 86% two steps; d. K2CO3,
4 Å mol. sieves, DMSO, 75%; e. CrCl2, DMF, 85%; f. t-BuONO, CuBr2, CH3CN, 38% of 47, 42% of 50; g. BBr3, CH2Cl2; h. 2-azido-5-iodobenzoic acid,37
EDCI, HOBt, DMF, 48, 33% two steps; i. BBr3, CH2Cl2 j. 2-azido-5-iodobenzoic acid,37 EDCI, HOBt, DMF, 51, 48% two steps.
substituted dihydrocinnamic acid 17 to give the N,N′-diacyl
â-lysine 44 (86% over two steps). Exposure of 44 to standard
SNAr macroetherification conditions gave cyclic analogue 45
in very good yield (75%). Transformation of the NO2 group in
44 to the corresponding analogues (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 ꢀ-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 analogues 49 and 52, respec-
tively, in acceptable yields (two steps, 33% and 48%, respec-
tively).
When tested in the ryanodine binding assay, each of the
photoaffinity analogues 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.
of 3, was accomplished by an efficient, macrocyclization based
on intramolecular SNAr substitution. Assay of two classes of
analogues that differ in ring size and aryl substitution patterns
in the [3H]-ryanodine binding assay, which detects the open state
of the RyR1/FKBP12 Ca2+ channel, revealed an interesting
bimodal 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 analogues suggests more complex
interactions occur between the receptor site and this family of
ligands. A bimodal gating model that accommodates binding-
activation-inactivation of the RyR1/FKBP12 complex by
bastadin-5 analogues suggests the possibility that Ca2+ release
from the SR may be modulated by ‘fine-tuning’ of stereoelec-
tronic factors. Tuned release of Ca2+ from stores by custom-
designed ligand-gate interactions between small molecule
analogues of 3 and the RyR1/FKBP12 complex is an attractive
idea that may have practical benefits in treatment of disease
Conclusion
The synthesis of simple, ring-constrained cyclic analogues
of bastadin-5, with structures that embody the ‘western’ edge

4504 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Masuno et al.
conditions that arise from complications of defective SR Ca2+
channel activity, including arrythmias, heart failure, and ma-
lignant hypothermia.38
extracted with EtOAc (3 × 5 mL). The organic layers were
combined, washed with brine, and 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
1
solid: IR (ZnSe, neat) ν 3371, 2927, 1635, 1502, 1434 cm-1; H
Experimental Section
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.
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, dimethyl sulfoxide
(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.
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). NaNO2
(0.2 mg, 3 µmol) was added in one portion into a chilled solution
(0 °C, ice bath) of arylamine 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, and dried (Na2SO4), and the volatiles were removed to
give crude 6. HPLC purification (C18 5 µm Microsorb 10 × 250
mm, MeOH/H2O, 3:2, 4 mL/min, rt, 6.4 min) provided 6 (0.7 mg,
1
IR spectra were recorded on a Mattson Galaxy FTIR. H and 13C
NMR were recorded at 400 and 100 MHz, respectively, in the stated
solvent (g99.5% atom D) and referenced to residual protonated
solvent signal (δΗ CDCl3 7.24 ppm; CD3OD, 3.30 ppm; δCCDCl3
77.00 ppm, CD3OD, 49.00 ppm).
[3H]-Ryanodine Binding Asssay. Specific binding of [3H]-
ryanodine to high affinity sites on rabbit skeletal membrane
vesicles13,39 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), and 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 concentration ∼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), and 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 non-
specific binding was determined in the presence of 100-fold ‘cold’
ryanodine.
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[12.2.2.10,0]-
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 (1 h, -78 °C), quenched with a NaHCO3 (aq, satd), and
extracted with EtOAc (3 × 5 mL). The organic layers were
combined, washed with brine, and 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
1
solid: IR (ZnSe, neat) ν 3291, 2933, 1637, 1504, 1224 cm-1; H
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-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). 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 (1 h, -78 °C), quenched with NaHCO3 (aq, satd), and
extracted with EtOAc (3 × 10 mL). The organic layers were
combined, washed with brine, and 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,
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). Trifluoro-
acetic 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 traces of trifluoro-
acetic 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.
1
2933, 1642, 1531, 1346 cm-1; H 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[12.2.2.10,0]-
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 (1 h, -78 °C), quenched with a NaHCO3 (aq, satd), and

Simplified Bastadin-5 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4505
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). 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 traces of trifluoroacetic acid
were removed by reevaporation from toluene. Reversed phase
HPLC purification (C18, 5 µm 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,
solution was combined, washed with brine, and dried (Na2SO4),
and the volatiles were removed to give crude 12. Flash chroma-
tography (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.
1
1627, 1501, 1429 cm-1; H 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.
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-di-
one (13). NaNO2 (0.8 mg, 12 µmol) was added in one portion to
a chilled solution (0 °C, ice bath) of arylamine 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, and 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, tR 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[16.2.2.10,0]-
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
(1 h, -78 °C), quenched with a NaHCO3 (aq, satd), and extracted
with EtOAc (3 × 10 mL). The organic layers were combined,
washed with brine, and 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-di-
one (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 (1 h, -78 °C), quenched with NaHCO3 (aq, satd), and
extracted with EtOAc (3 × 10 mL). The organic layers were
combined, washed with brine, and 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-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). 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 and then room temperature for 20 min. KI (5.6 mg, 0.034
mmol) in 2 M 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, and dried
(Na2SO4), and the volatiles were removed to give crude 10. Flash
chromatography (SiO2, CH2Cl2/MeOH, 9:1) followed by reversed
phase HPLC (C18, 5 µm 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,
1
1433, 1217; H 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-
[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-di-
one (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 (1 h, -78 °C), quenched with NaHCO3
(aq, satd), and extracted with EtOAc (3 × 20 mL). The organic
layers were combined, washed with brine, and 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-
[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-di-
one (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 was stirred (1 h, -78 °C), quenched with NaHCO3
(aq, satd), and extracted with EtOAc (3 × 10 mL). The organic
5-(2-Aminoethyl)-2-benzyloxy-3-bromophenol (16). KOH (70
mg, 1.3 mmol) 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 (1 N) until
the mixture was neutral by pH paper and was extracted with EtOAc

4506 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Masuno et al.
(3 × 25 mL). The organic layers were combined, washed with brine,
and dried (Na2SO4), and the volatiles were removed to yield 16 as
an oil (14 mg 92% crude). The product was carried forward without
(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-hydroxybenzalde-
hyde 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 (1 N, 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:
1
purification. H 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 (1 N, 20 mL) and extracted with
CH2Cl2 (4 × 20 mL). The organic layers were combined, washed
with brine and 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 an
amorphous solid: IR (KBr, pellet) ν 3392, 3075, 1639, 1529, 1425,
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)styryl-
carbamate (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 2 h (-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,
1
1349 cm-1; H 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)-phen-
yl]-ethylcarbamoyl}-ethyl-3-(4-fluoro-3-nitro-phenyl)-propion-
amide (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 (1 N)
and extracted with CH2Cl2 (3 × 20 mL). The organic layers were
combined, washed with brine, and 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,
1
1257, 1224 cm-1; H 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.
1
neat) ν 3293, 2944, 2867, 1644, 1538 cm-1; H 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.
Carbonic acid 2-benzyloxy-3-bromo-5-(2-methoxycarbonyl-
amino-vinyl)-phenyl ester methyl ester: IR (KBr, neat) ν 3336,
1
2956, 1770, 1734, 1660, 1477, 1261, 943 cm-1; H 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-methoxycarbonyl-
amino-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 cannula 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,
and dried (Na2SO4), and the volatiles were removed to provide 24
as a viscous oil (47 mg, 94%): IR (NaCl, neat) ν 3343, 2956, 1770,
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, 1 h)
followed by dropwise addition of benzyl bromide (0.14 mL, 1.2
mmol). After 45 min, the reaction was quenched with HCl (aq, 1
N) resulting in precipitation of the crude product 21. The precipitate
was filtered, washed with water, dried under high vacuum, and
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
1
(NaCl, neat) ν 3235, 1683 cm-1; H NMR (400 MHz, CDCl3) δ
1
1722, 1481, 1257 cm-1; H NMR (400 MHz, CDCl3) δ 2.75 (t, J
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
) 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

Simplified Bastadin-5 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4507
(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]+, C21H25O6-
NBr requires 466.0865.
5 mL). The organic layers were combined, washed with brine, and
dried (Na2SO4), and the volatiles were removed to give crude 29.
Flash chromatography (SiO2, CH2Cl2/MeOH, 9:1) followed by
reversed phase HPLC (C18, 5 µm 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
3-(4-Fluoro-3-nitro-phenyl)-propionic Acid Methyl Ester (26).
In a glovebox, 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 glovebox 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,
1
cm-1; H 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[12.2.2.10,0]-
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 dropwise over 20 min at 0
°C. After stirring for 2 h, the mixture was allowed to warm to room
temperature and quenched with HCl (1 N, 4 mL) and extracted
with EtOAc (4 × 5 mL). The organic layers were combined, washed
with brine, and dried (Na2SO4), and the volatiles were removed to
give crude 30. Flash chromatography (SiO2, CH2Cl2/MeOH, 9:1)
followed by reversed phase HPLC (C18, 5 µm 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]+, C24H20-
NO3Br3 requires 606.8993.
1
1537, 1349 cm-1; H 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[12.2.2.10,0]-
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,
and 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.
4-Benzyloxy-5-bromo-2-oxa-10-aza-tricyclo[12.2.2.10,0]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 1 h, 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 (1 N, 4 mL), and extracted with
EtOAc (4 × 5 mL). The organic layers were combined, washed
with brine, and dried (Na2SO4), and the volatiles were removed to
give crude 31. Flash chromatography (SiO2, CH2Cl2/MeOH, 9:1)
followed by reversed phase HPLC (C18, 5 µm 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,
17-Amino-4-benzyloxy-5-bromo-2-oxa-10-aza-tricyclo[12.2.2.10,0]-
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 and 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.
1
1501, 1202 cm-1; H 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.
5,17-Dibromo-4-benzyloxy-2-oxa-10-aza-tricyclo[12.2.2.10,0]-
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 1 h, 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,
quenched with HCl (1 N, 4 mL), and extracted with CH2Cl2 (4 ×
[2-(4-Benzyloxy-3-bromo-5-hydroxy-phenyl)-ethyl]-carbam-
ic 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-butyl dicarbonate (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

4508 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Masuno et al.
was extracted with CH2Cl2 (4 × 25 mL). The organic layers were
combined, washed with brine, and 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, and 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]-
ethylamine (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, and 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.
(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, and 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-
[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-di-
one (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 temper-
ature, quenched with water, and extracted with CH2Cl2 (5 × 20
mL). The organic layers were combined, washed with brine, and
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
1
cm-1; H 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-
[16.2.2.10,0]tricosa-1(21),3(23),4,6,18(22),19-hexaene-11,15-di-
one (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 and 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
;
(2-{2-[4-Benzyloxy-3-bromo-5-(triisopropyl-silanyloxy)-phen-
yl]-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-â-
alanine (65 mg, 0.35 mmol) in CH2Cl2 (1.0 mL). The solution was
stirred overnight at room temperature, quenched with HCl (1 N),
and extracted with CH2Cl2 (3 × 20 mL). The organic layers were
combined, washed with brine, and 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,
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.
1
neat) ν 3315, 2945, 2867, 1691, 1558, 1477 cm-1; H 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.
4-Benzyloxy-5,21-dibromo-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 (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 1 h. A mixture of lactam 38 (10 mg, 0.02 mmol) in CH3CN
(0.8 mL) was then added dropwise 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, and dried (Na2SO4), and the volatiles
were removed to give crude 39. Flash chromatography (SiO2,
3-Amino-N-{2-[4-Benzyloxy-3-bromo-5-(triisopropyl-silanyl-
oxy)-phenyl]-ethyl-propionamide (36). TFA (1.0 mL) was added
to a solution of carbamate 35 (136 mg, 0.209 mmol) in CH2Cl2

Simplified Bastadin-5 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4509
CH2Cl2/MeOH, 9:1) followed by reversed phase HPLC (C18, 5 µm
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.
The organic layers were combined, washed with brine (3 × 20
mL), phosphate buffer (pH ) 5.5) (3 × 20 mL), and 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, and dried
(Na2SO4), and the volatiles were removed to give crude amide 44.
Flash chromatography (SiO2, CH2Cl2/EtOAc 20% then CH2Cl2/
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-di-
one (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 1 h. A mixture of lactam 38 (11 mg, 0.02
mmol) in CH3CN (0.5 mL) was added dropwise 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, and dried (Na2SO4), and the volatiles
were removed to give crude 40. Flash chromatography (SiO2,
CH2Cl2/MeOH, 9:1) followed by reversed phase HPLC (C18, 5 µm
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,
MeOH 5%) gave 44 (60 mg, 86%) as an amorphous solid: [R]25
D
-6.1° (c 0.58, CHCl3); IR (neat) ν 3295, 2942, 2867, 1697, 1646,
1
1538 cm-1; H 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, 1 Hz); 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[16.2.2.10,0tricosa-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, and dried (Na2SO4), and the volatiles were
removed to give crude amide 45. Flash chromatography (SiO2, 9.4:
1
2925, 1648 cm-1; H 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) and extracted with EtOAc (3 × 10 mL). The organic layers
were combined, washed with brine, and dried (Na2SO4), and the
volatiles were removed to give crude 42. Flash chromatography
0.6 CH2Cl2/MeOH) gave 45 (31 mg, 75%) as an viscous oil: [R]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.
(SiO2, CH2Cl2/MeOH 5%) gave 42 (163 mg, 81%) as an oil: [R]25
D
-3.1° (c 0.75, CHCl3); IR (neat) ν 3313, 2944, 2867, 1687, 1641,
1
1538 cm-1; H 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)-pro-
pionylamino]-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 min, the reaction was
quenched with NaCl (sat.) and extracted with CH2Cl2 (10 mL).
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 and 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-

4510 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Masuno et al.
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.
(5.0 mL) at room temperature. After 5 h, the reaction was quenched
with HCl (1 N) and extracted with CH2Cl2 (3 × 5 mL). The organic
layers were combined, washed with brine, and 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-(+)-[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
dropwise 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, and
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, 5 µm 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
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 (1 N) and extracted
with CH2Cl2 (3 × 5 mL). The organic layers were combined,
washed with brine, and 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
25
amorphous solid. 47: [R]D +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.
1
colorless amorphous solid. H 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]+, C31H30N6O5-
Br3NaI requires 952.8765.
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): [R]D
Acknowledgment. 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 T.F.M., and ES11269 to I.N.P.) and the U.S.
Environmental Protection Agency (R829388 to I.N.P.).
25
+38.3° (c 0.360, CHCl3); IR (KBr) ν 3334, 2925, 1697, 1652,
1
1513, 1247 cm-1; H 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
Supporting Information Available: Spectral and X-ray crys-
tallographic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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