Synthesis of a phosphotyrosyl analogue having χ1, χ2 and φ angles constrained to values observed for an SH2 domain-bound phosphotyrosyl residue

Article abstract of DOI:10.1016/S0040-4020(03)00978-5

Src homology 2 (SH2) domains provide connectivity in protein-tyrosine kinase (PTK)-dependent signaling through their high affinity association with phosphotyrosyl (pTyr)-containing peptide sequences. Because recognition of pTyr residues is central to SH2

Full text of DOI:10.1016/S0040-4020(03)00978-5

Tetrahedron 59 (2003) 6087–6093
Synthesis of a phosphotyrosyl analogue having x1, x2 and f angles
constrained to values observed for an SH2 domain-bound
phosphotyrosyl residue
†
Xiang-Zhu Wang,a Zhu-Jun Yao,a Hongpeng Liu,b Manchao Zhang,b Dajun Yang,b
,
Clifford Georgec and Terrence R. Burke, Jr.a
,
*
aLaboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health, NCI-Frederick,
P.O. Box B, Bldg 376 Boyles St., Frederick, MD 21702-1201, USA
bDepartment of Hematology/Oncology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
cLaboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375, USA
Received 1 May 2003; revised 11 June 2003; accepted 19 June 2003
Abstract—Src homology 2 (SH2) domains provide connectivity in protein-tyrosine kinase (PTK)-dependent signaling through their high
affinity association with phosphotyrosyl (pTyr)-containing peptide sequences. Because recognition of pTyr residues is central to SH2
domain-binding affinity, design of pTyr-mimicking residues has been one component of SH2 domain signaling antagonist development.
Reported herein is the synthesis of (^)-(rel-1R,2R,5S)-3-acetyl-1,2,3,4,5,6-hexahydro-8-O-phosphoryl-1,5-methano-3-benzazocine-2-
carboxylic acid methyl ester (3c) as a monomeric pTyr-mimicking analogue that constrains three torsion angles (x1¼1688; x2¼2858;
f1¼21138) to values approximating those observed for a pTyr residue bound to the Grb2 SH2 domain (x1¼1828; x2¼2898; f1¼21328).
Compound 3c differs from our previously reported analogue, (^)-(rel-1R,2R,5S)-3-acetyl-1,2,3,4,5,6-hexahydro-1-methyl-1,5-methano-3-
benzazocin-8-ol, in lacking a methyl substituent at the bridgehead 1-position. Molecular modeling studies had indicated that this methyl
group could potentially hinder SH2 domain binding. Synthesis of the desmethyl derivative was achieved by formation of the
methanobenzazocine ring system using an intramolecular electrophilic cyclization that proceeds through an activated acyliminium
intermediate. Importantly, the correct relative (2R) stereochemistry at the ‘a-carboxyl’-bearing carbon is obtained through base-catalyzed
equilibration of a (2S/2R) diastereomeric mixture that results from intramolecular ring closure. Comparison of Grb2 SH2 domain-binding
affinity of 3c (IC50¼1167 mM) with conformationally flexible phosphorylated (^)-N-acetyl-tyrosine methyl ester (15; IC50¼1469 mM)
revealed no apparent enhancement in affinity. This apparent ineffectiveness of ‘local conformational constraint’ on SH2 domain-binding
affinity of the monomeric pTyr mimetic is consistent with previous reports obtained by conformationally constraining pTyr-mimicking
residues that were contained within peptide platforms. Although not providing high binding affinity in its current form, the novel 1,5-
methano-3-benzazocine ring system may afford a novel platform for further elaboration and development of small molecule SH2 domain
signaling antagonists.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction1
modules that serve downstream roles in PTK-dependent
signaling by binding phosphotyrosyl (pTyr)-containing
proteins in sequence-dependent fashions.8 Agents that
compete with cognate proteins for binding to select SH2
domains could disrupt PTK-dependent signaling and
thereby represent alternatives to kinase inhibitors as novel
therapeutics.4,9,10 The structural basis of SH2 domain
recognition of pTyr-containing ligands involves interactions
of pTyr residues in well-formed pockets that contain one or
two highly conserved arginine residues.11,12 The central role
of the pTyr residue (1) in the overall binding process12,13 has
made design of pTyr mimetics an important component of
SH2 domain-directed antagonist development.14–16 One
objective of these efforts has been reduction of binding
entropy penalties by introduction of conformational con-
straint into the pTyr-mimicking residue.17–20 Accordingly,
Aberrations in protein-tyrosine-kinase (PTK)-dependent
signaling are associated with proliferative disorders,
including certain cancers.2,3 Accordingly, recent studies
showing the promise of kinase inhibitors have begun to
validate PTK signaling blockade as an efficacious antic-
ancer therapeutic approach.4–7 Src homology 2 (SH2)
domains are a family of homologous non-catalytic protein
Keywords: phosphotyrosyl analogue; protein-tyrosine-kinase; SH2
domains.
*
Corresponding author. Tel.: þ1-301-846-5906; fax: þ1-301-846-6033;
e-mail: tburke@helix.nih.gov
Present address: Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, People’s Republic of China.
†
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00978-5

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X.-Z. Wang et al. / Tetrahedron 59 (2003) 6087–6093
Figure 1. Structure of pTyr (1) showing locations of torsion angles as well as conformationally constrained pTyr mimetics. Structure 2 highlights the pTyr sub-
structure within the constrained analogues.
we had previously proposed the tricyclic methanobenzazo-
cino—2 as containing within its framework a tyrosyl
substructure having x1, x2 and f angles constrained to
values observed for an SH2 domain-bound pTyr residue
(Fig. 1).21 For reasons of synthetic simplicity, the
structurally abbreviated compound 3a, lacking an a-
carboxyl equivalent and having an undesired 1-methyl
substituent, was initially prepared using a Mannich
condensation approach.21 Subsequently, the more highly
functionalized 3b, containing an a-carboxyl equivalent was
prepared in its unphosphorylated form. Unfortunately, as
with the earlier synthesis, this analogue also retained the
unwanted 1-methyl substituent.22 In both syntheses, incor-
poration of the 1-methyl group was required to block
unwanted alkylation at this reactive position. However,
molecular modeling studies had indicated that this methyl
group could potentially hinder SH2 domain binding, and it
was highly desirable to derive synthetic approaches that
could circumvent its use. For this purpose we have devised
an alternate synthetic route that obviates the unwanted 1-
methyl group. Reported herein is the culmination of this
work, wherein the fully elaborated, carboxyl-containing 3c
is prepared by a novel approach that eliminates retention of
the unwanted 1-methyl group. Included are the synthesis of
3c in its phosphorylated form, verification of structure by X-
ray crystallography and evaluation of Grb2 SH2 domain-
binding potency.
2. Results and discussion
Mannich-type reactions have been extensively utilized for
the synthesis of nitrogen-containing heterocycles, natural
products and drug-like biomimetics.23–27 The confor-
mationally constrained tricyclic mimetic 2 can be viewed
as a pTyr residue in which the amino acid a-nitrogen is
methano-bridged to a tetrahydronaphthalenol ring system.
Following introduction of a methyl group at the 1-position
of 6-methoxy-2-tetolone (4), previous approaches to this
tricyclic ring system have utilized either Mannich reac-
tions,21 or intramolecular 1,4-Michael-type reaction of a
carboxamido group onto an exocyclic double bond.22 This
ultimately has resulted in retention of an unwanted 1-methyl
substituent in the final products (3a and 3b, respectively). In
order to eliminate this methyl substituent, tetralone 4 was
subjected to an initial aminomethylation of silyl enol ethers
5a and 5b,28 which had been obtained from 4 as an
inseparable mixture (Scheme 1). The ratio of 5a to 5b was
1
determined by integration of vinylic H NMR signals at
5.66 ppm and 5.05 ppm, respectively. Depending on reac-
tion conditions, the thermodynamically favored 5a could be
obtained as a single regioisomer if passed through basic
aluminum oxide. However passage through silica gel
resulted in significant generation of 5b (5a:5b<1:1).
Subjecting a mixture of 5a and 5b to aminomethylation
provided the desired primary amine 6 as a single
Scheme 1. Reagents and Conditions: (i) NEt3, TIPSiOTf, benzene, rt, 1 h (.90% yield). (If purified through basic Al2O3, only 5a is observed to 1H NMR,
however if purified through silica gel, 5a:5b¼52:48.); (ii) Me3SiCH2N3, AlCl3, CH2Cl2.

X.-Z. Wang et al. / Tetrahedron 59 (2003) 6087–6093
6089
Scheme 2. Reagents and Conditions: (i) OHCCO2Me, MgSO4; (ii) AcCl, toluene (68% yield over 3 steps); (iii) HSCH2CH2SH, BF3·Et2O, CH2Cl2 (80%
yield); (iv) Raney nickel, EtOH (90% yield); (v) NH3, MeOH.
regioisomer. The reaction occurred in high yield in spite of
the fact that formation of 6 proceeded from 5b, which was a
minor component in the starting reaction mixture. This
implied that during aminomethylation a reversible equili-
brium between 5a and 5b was maintained with depletion of
5b occurring through transformation to product 6.
its structure and provided a measure of x1, x2 and f torsion
angles (Fig. 2C). Comparison of these values (x1¼1688;
x2¼2858; f¼21138) with the corresponding torsion angles
of a pTyr residue bound to the Grb2 SH2 domain as part of a
larger peptide ligand30 (Fig. 2A: x1¼1828; x2¼2898;
f¼21328) showed remarkable congruence that can be
appreciated visually through overlay of the two structures
(Fig. 2B).
Aminomethylation product 6 was reacted with methyl
glyoxalate in the presence of magnesium sulfate to form
imine 7 (Scheme 2). This was acetylated without purifi-
cation to form an activated acyliminium intermediate (8)
that underwent intramolecular electrophilic cyclization to
yield the key tricyclic ketone 9 in 68% overall yield from
6.29 Of note, the 2-carbomethoxy group in 9 was obtained as
an endo/exo-diastereomeric mixture. Reductive deoxygena-
tion of the keto group of 9 could be achieved in a two-step
manner by initial thioketalization to spiro bisdithiane 10,
followed by desulfurization to 11 using Raney Nickel in
refluxing EtOH. Although undesired endo (2S)-2-carbo-
methoxy-containing material could be removed chromato-
graphically at this point, it was found that treatment of 11
with mild base (2 M methanolic ammonia) resulted in
epimerization at the 2-position to yield the desired exo
isomer (2R)-11a as a single product. Remarkably, formation
of carboxamide side product was not detected under these
conditions.
The Grb2 SH2 domain-binding affinity of 3c was examined
using an ELISA-based assay as previously described.31 For
comparison, unconstrained phosphorylated N-acetyl-tyro-
sine methyl ester was also examined in parallel in both
racemic and (L)-forms (15 and 16, respectively). Consistent
with earlier findings,21 affinities for monomeric pTyr
analogues were markedly reduced as compared with values
obtained when pTyr residues are contained within peptide or
peptide mimetic display platforms (Table 1).13 Of primary
interest however, were potential relative increases in affinity
resulting from reduction of binding entropy penalties
incurred through conformational constraint. Here it was
observed by comparison of IC50 values obtained for 3c
relative to 15 and 16, that all three analogues were
approximately equipotent within statistical error. Although
this contrasts with marked binding enhancement achieved
through ‘global’ conformational constraint of Grb2 SH2
domain-binding molecules,32–34 the apparent ineffective-
ness of ‘local conformational constraint’ of the pTyr residue
in enhancing SH2 domain-binding affinity has also been
observed with conformationally constrained pTyr-mimick-
ing residues contained within peptide platforms.18,20,35 In
one study the ineffectiveness of local conformational
constraint in enhancing overall peptide binding was in
spite of successful realization of an intended decrease in
Selective demethylation of 11a to 12 without de-esterifica-
tion could be achieved in good yield using BBr3. Finally,
phosphorylation to final product 3c was accomplished in
two steps by initial conversion to protected phosphoester 13
followed by acidic cleavage of tert-butyl-groups (Scheme 3).
Subjecting 3c to X-ray crystallographic analysis confirmed
Scheme 3. Reagents and Conditions: (i) BBr3, CH2Cl2 (85% yield); (ii) (ButO)2P(NPri)2, tetrazole, ButOOH (47% yield); (iii) CF3COOH, THF (92% yield).

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X.-Z. Wang et al. / Tetrahedron 59 (2003) 6087–6093
Figure 2. (A) Grb2 SH2 domain-bound pTyr residue derived from data presented in Ref. 30 showing relevant torsion angles; (B) Overlay of A and C; (C) X-ray
crystal structure of constrained pTyr mimetic 3c showing relevant torsion angles.
entropy penalties.35 This failure of entropic advantage to be
reflected in total binding free energy was attributed to an
offsetting reduction in binding enthalpy, which is an
example of ‘entropy–enthalpy compensation’36,37 that has
been observed in other peptide–SH2 domain binding
interactions.38 Although a detailed calorimetric binding
analysis is beyond the scope of the current work, it can
be speculated that some component of entropy–
enthalpy compensation may underlie the absence of
conformationally induced binding enhancement of 3c
relative to 15 and 16.
straint, the tricyclic methano-3-benzazocine structure may
potentially afford an anchoring platform from which to
append additional recognition functionality that may lead to
more potent small molecule, non-peptidic SH2 domain
signaling antagonists.
3. Experimental
3.1. Single-crystal X-ray diffraction analysis of
compound 3c
In conclusion, reported herein is a novel and direct synthesis
of a new amino acid analogue that is remarkable in
simultaneously constraining three torsion angles to values
observed for a Grb2 SH2 domain-bound pTyr residue. An
important aspect of the current synthetic approach is its
ability to prepare this new constrained pTyr mimetic in the
absence of a potentially undesirable methyl substituent at
the bridgehead 1-position. Although only modest binding
affinity was achieved through such conformational con-
C16H20NO7P, Mw¼369.30, monoclinic space group P21/c,
˚
a¼7.614(1), b¼23.416(1), c¼9.785(1) A, b¼98.122(1)8,
3
˚
V¼1727.16(15) A ,
Z¼4,
rcalc¼1.420 mg mm23
,
l(Cu Ka)¼1.54184 A, m¼1.786 mm21, F(000)¼776,
T¼228C. A 0.18£0.15£0.02 mm3 crystal was used for
data collection on an automated Bruker 6K CCD equipped
with a Gobel mirrors monochromator. Lattice parameters
were determined from 2856 centered reflections within
7.55#2u#113.278. The data collection range of hkl
was: 27#h# 8, 225#k#24, 210#l# 10, with
[(sin u/l]max¼0.55. A set of 7593 reflections was collected
using v scans. There were 2322 unique reflections, and 1440
were observed with I.2s(I). The structure was solved and
refined with a full-matrix least squares39,40 on F 2. The
refinement varied 223 parameters: atom coordinates and
anisotropic thermal parameters for all non-H atoms. H
atoms were included using a riding model [coordinate shifts
of C applied to attached H atoms, C–H distances set to
˚
Table 1. Extracellular ELISA-derived Grb2 SH2 domain binding affinities
of synthetic residues
No.
Structure
IC50 (mM)
1167^465
3c
˚
0.96 A, H angles idealized, Uiso(H) were set to 1.2 Ueq(C)
hydroxyl hydrogen coordinates were refined. Final residuals
were R1¼0.054 for I.2s(I) and wR2¼0.159 for all data
with final difference Fourier excursions of 0.27 and
–3
˚
20.25 eA
15
16
1469^326
1419^28
3.2. General synthetic
Reactions were carried out under argon in oven-dried
glassware using standard gas-tight syringes, cannulas and
septa. Anhydrous solvents were purchased from Aldrich
Chemical Corporation and used without further drying.
Melting points were measured using a MEL-TEMP II
apparatus and are uncorrected. Elemental analyses were
Assays were conducted as described in Ref. 35.

X.-Z. Wang et al. / Tetrahedron 59 (2003) 6087–6093
6091
1
obtained from Atlantic Microlab, Inc., Norcross, Ga. H
NMR data were recorded on a Varian 400 MHz spec-
trometer and are reported in ppm relative to TMS and
referenced to the solvent in which they were run. Fast atom
bombardment mass spectra (FABMS) were acquired with a
VG analytical 7070E mass spectrometer under the control of
a VG 2035 data system. Where indicated, Preparative HPLC
purification was conducted with a Waters Prep LC4000
system using photodiode array detection and binary solvent
systems as indicated, where A¼0.1% aqueous TFA and
B¼0.1% TFA in acetonitrile and an Advantage C18 (5m)
column (preparative size, 20 mm dia.£250 mm long with a
flow rate of 10 mL/min).
(18H); IR (neat): 3455, 2945, 2866, 1636, 1498, 1173,
882 cm21; FABMS (m/z) 362 (MþH)þ.
3.2.3. (6)-(rel-1R,2R/S,5S)-3-Acetyl-1,2,3,4,5,6-hexahy-
dro-8-methoxy-11-oxo-1,5-methano-3-benzazocine-2-
carboxylic acid methyl ester (9). A mixture of crude 6
(3.39 g, 9.37 mmol) and methyl glyoxalate (924 mg,
10.5 mmol) in anydrous CH2Cl2 (25 mL) was stirred with
MgSO4 at room temperature (1.5 h). Solid was removed by
filtration, the filter pad was washed with CH2Cl2 and the
combined filtrate was concentrated under vacuum to afford
crude 7 as an oil (4.13 g). This was dissolved in anhydrous
toluene (20 mL), cooled to 2788C and acetyl choride
(12.5 mmol) was added under argon. The mixture was
stirred at 708C (10 h), then solvent was removed under
vacuum and residue was purified by silica gel flash
chromatography to provide tricyclic 9 (2.07 g, 68% yield
3.2.1. [(3,4-Dihydro-6-methoxy-2-naphthalenyl)oxy]-
tris(1-methylethyl)-silane (5a) and [(1,4-dihydro-6-
methoxy-2-naphthalenyl)oxy]tris(1-methylethyl)-silane
(5b). A solution of 6-methoxy-2-teralone (4) (1.86 g,
10.0 mmol) in anhydrous benzene (30 mL) under argon at
room temperature was treated with triethylamine (1.52 g,
15.0 mmol) and triisopropylsilyl trifluomethanesulfonate
(3.37 g, 11.0 mmol) and the mixture was stirred at room
temperature (1 h). The reaction was quenched by addition of
saturated aqueous NaHCO3 and the organic layer was
collected and the aqueous phase was extracted with EtOAc
(twice) and the combined organic layers were washed with
brine and dried (MgSO4). Solvent was removed under
vacuum and residue was purified through basic aluminum
oxide to provide 5a as an oil (3.24 g, 98% yield). [Note:
Purification through silica gel provided a mixture of 5a and
1
over 3 steps). H NMR (CDCl3) d 6.97 (m, 1H), 6.74 (m,
1H), 6.63 (m, 1H), 5.18 (m, 1H), 4.20 (m, 1H), 3.97 (dd, 1H,
J¼1.7 and 7.8 Hz), 3.82 (m, 1H), 3.78 (s, 3H), 3.63 (s, 3H),
3.27 (m, 1H), 3.11 (d, 2H, J¼15.2 Hz), 2.17 (s, 3H); IR
(neat): 742, 1646, 1219 cm21; FABMS (m/z) 318 (MþH)þ.
Anal. Calcd (C17H19NO5·H2O): C, 60.89; H, 5.71; N, 4.17.
Found: C, 61.11; H, 5.72; N, 3.99.
3.2.4. (6)-(rel-10R,20R/S,50S)-30-Acetyl-10,20,30,40,50,60-
hexahydro-80-methoxy-spiro[1,3-dithiolane-2,110-
[1,5]methano[3]benzazocine]-20-carboxylic acid methyl
ester (10). To a solution of 9 (1.41 g, 4.41 mmol) and
ethane-1,2-dithiol (4.85 mmol) in anhydrous CH2Cl2
(10 mL) was slowly added BF3·Et2O (6.60 mmol) under
argon, then the mixture was stirred at room temperature
(18 h). The reaction was quenched by addition of dilute
aqueous NaOH and the mixture was washed with H2O and
brine and dried (MgSO4). Purification by silica gel flash
chromatography afforded 10 as a foam (1.27 g, 73% yield).
1H NMR (CDCl3) d 7.0 (d, 1H, J¼8.4 Hz), 6.68 (d, 1H,
J¼8.4 Hz), 6.60 (s, 1H), 4.98 (m, 1H), 3.97 (m, 1H), 3.77 (s,
3H), 3.61 (m, 1H), 3.50 (m, 1H), 3.48 (s, 3H), 3.37 (m, 3H),
3.19 (m, 2H), 2.80 (m, 1H), 2.66 (m, 1H), 2.08 (m, 3H); IR
(neat): 1742, 1652 cm21; FABMS (m/z) 394 (MþH)þ.
Anal. Calcd (C19H23NO4S2): C, 57.99; H, 5.89; N, 3.56.
Found: C, 57.78; H, 6.12; N, 3.42.
1
5b in an approximate 1:1 ratio.]. H NMR (CDCl3) 5a: d
6.68 (d, 1H, J¼7.8 Hz), 6.66 (m, 2H), 5.66 (s, 1H), 3.78 (s,
3H), 2.88 (t, 2H, J¼8.2 Hz), 2.38 (t, 2H, J¼8.2 Hz), 1.24
(m, 3H), 1.12 (d, 18H, J¼7.0 Hz). 5b: d 7.03 (d, 1H,
J¼8.3 Hz), 6.73 (dd, 1H, J¼2.7 and 8.5 Hz), 6.64 (dd, 1H,
J¼2.7 and 8.5 Hz), 5.02 (m, 1H), 3.79 (s, 3H), 3.44 (m, 2H),
3.36 (m, 2H), 1.24 (m, 3H), 1.12 (d, 18H, J¼7.0 Hz); IR
(neat) 2938, 2865, 1642, 1498, 1257, 1175, 1119, 882 cm21
;
FABMS (m/z) 333 (MþH)þ. Anal. Calcd (C20H32O2Si): C,
72.23; H, 9.70. Found: C, 72.47; H, 9.82.
3.2.2. 1,2-Dihydro-7-methoxy-3-[[tris(1-methylethyl)si-
lyl]oxy]-2-naphthalenemethanamine (6). To a suspension
of aluminum chloride (1.46 g, 11.4 mmol) in anhydrous
CH2Cl2 (25 mL) at 2788C under argon was added
TMSCH2N3 (1.32 g, 12.4 mmol) under argon. The mixture
was stirred at 2788C (30 min), then at 08C (1 h) and then the
mixture was warmed to room temperature and stirred (1 h)
to provide a clear solution. The solution was re-cooled to
2788C and a mixture of silyl enol ethers 5a/5b (3.04 g,
9.14 mmol) in CH2Cl2 (10 mL) was added dropwise, then
the mixture was warmed to room temperature and stirred
overnight. The reaction was quenched by cautious addition
of 2N NaOH (2£30 mL) at 08C. The organic layer was
collected and the aqueous layer was extracted with CH2Cl2
and the combined organic layer was washed with brine and
dried (MgSO4). Solvent was removed under reduced
pressure to provide crude 6 as a light brown solid (3.39 g).
This was used directly without purification due to
3.2.5. (6)-(rel-1R,2R/S,5S)-3-Acetyl-1,2,3,4,5,6-hexahy-
dro-8-methoxy-1,5-methano-3-benzazocine-2-carboxylic
acid methyl ester (11). A solution of 10 (1.37 g,
3.48 mmol) in absolute EtOH (20 mL) was refluxed under
argon with two spoonfuls of activated Raney nickel (24 h).
The reaction mixture was cooled to room temperature and
carefully filtered through celite. The filter pad was washed
well with EtOH and the combined filtrate was taken to
dryness under vacuum and the residue was purified by silica
gel flash chromatography to afford 11 as a foam (997 mg,
95% yield). 1H NMR (CDCl3) (2S)-11: d 6.98 (d, H,
J¼8.4 Hz), 6.64 (m, 2H), 4.87 (d, 1H, J¼7.2 Hz), 3.91 (m,
1H), 3.75 (s, 3H), 3.58 (s, 3H), 3.55 (m, 1H), 3.22 (m, 1H),
2.91 (m, 1H), 2.60 (m 2H), 2.05 (s, 2.4H), 1.78 (m, 2H), 1.63
(s, 0.6H). (2R)-11: d 7.0 (m, 1H), 6.62 (m, 1H), 6.57 (m,
1H), 5.08 (s, 0.6H), 4.57 (d, 0.4H, J¼13.7 Hz), 4.20 (s,
0.4H), 3.90 (m, 7.4H), 3.43 (m, 1H), 3.07 (m, 0.7H), 2.98
(m, 1.H), 2.70 (m, 1H), 2.17 (m, 1H), 1.98 (s, 1.8H), 1.90
(m, 0.5H), 1.78 (m, 1.5H), 1.46 (s, 1.2H); IR(neat): 2933,
1
instability. H NMR (CDCl3) d 6.76 (d, 1H, J¼8.2 Hz),
6.62 (m, 2H), 5.61 (s, 1H), 3.78 (s, 3H), 3.02 (dd, 1H, J¼7.2
and 16.0 Hz), 2.83 (m, 2H), 2.72 (dd, 1H, J¼7.0 and
13.1 Hz), 2.35 (m, 1H), 1.60 (br, 2H), 1.21 (m, 3H), 1.07

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X.-Z. Wang et al. / Tetrahedron 59 (2003) 6087–6093
1736, 1650, 1423, 1233 cm21; FABMS (m/z) 304 (MþH)þ.
Anal. Calcd (C17H21NO4·0.5H2O): C, 65.37; H, 6.78; N,
4.48. Found: C, 65.38; H, 7.05; N, 4.30.
3.2.9. (6)-(rel-1R,2R,5S)-3-Acetyl-1,2,3,4,5,6-hexahy-
dro-1,5-methano-8-phosphoryloxy-3-benzazocine-2-car-
boxylic acid methyl ester (3c). To a solution of 13 (97 mg,
0.2 mmol) in anhydrous CH2Cl2 (4 mL) at room tempera-
ture under argon was added a solution of CF3CO2H
(1.0 mL) in H2O (0.2 mL) and the mixture was stirred
(1 h). Solvent was removed under vacuum and residue was
purified by preparative HPLC (Advantage J-sphere ODS-
H80 column; flow¼10 mL/min; gradient elution 10–25% B
over 20 minutes; retention time¼24.1 min) to provide 3c as
3.2.6. (6)-(rel-1R,2R,5S)-3-Acetyl-1,2,3,4,5,6-hexahy-
dro-8-methoxy-1,5-methano-3-benzazocine-2-carboxylic
acid methyl ester (11a). To a solution of 11 (1.35 g,
4.45 mmol) in MeOH (5 mL) was added 2 M NH3 in MeOH
(5 mL) and the mixture was stirred at room temperature
(24 h). Removal of solvent under vacuum provided 11a
in quantitative yield. Confirmation of the relative stereo-
chemistries was achieved by single crystal X-ray crystal-
lography as described previously in Section 3 and as shown
in Fig. 2C.
1
a white solid (75 mg, 92% yield); m.p 2328C (dec.). H
NMR (CDCl3) d 7.16 (m, 1H), 6.93 (m, 2H), 5.02 (s, 0.6H),
4.51 (s, 0.4H), 4.50 (d, 0.4H, J¼9.8 Hz), 3.86 (0.6H), 3.80
(s, 1H), 3.77 (s, 2H), 3.58 (m, 2H), 3.28 (m, 2H), 3.07 (m,
1H), 2.76 (m, 1H), 2.20 (m, 1H), 1.98 (s, 2H), 1.86 (m, 2H),
3.2.7. (6)-(rel-1R,2R,5S)-3-Acetyl-1,2,3,4,5,6-hexahy-
dro-8-hydroxy-1,5-methano-3-benzazocine-2-carboxylic
acid methyl ester (12). A solution of 11a (715 mg,
2.36 mmol) in anhydrous CH2Cl2 (6 mL) at 2788C was
treated with BBr3, 1.0 M in ether (4.6 mmol) (1 h) and then
warmed to 08C and stirred (3 h). Excess BBr3 was destroyed
by addition of MeOH, then solvent was removed under
vacuum and the residue was purified by silica gel flash
chromatography to provide 12 as a solid (584 mg, 86%
1.67 (s, 1H); IR (neat): 2947, 1737, 1490, 962 cm21
;
FABMS (m/z) 368 (M2H)2. Anal. Calcd (C16H20NO7-
P·0.2CF3CO2H): C, 50.23; H, 5.19; N, 3.57. Found: C,
50.35; H, 5.17; N, 3.60.
Acknowledgements
1
Appreciation is expressed to Dr James Kelley of the LMC
for mass spectral analysis and to the Komen Foundation for
Breast Cancer for financial support (to D. Y.). Work was
supported in part (for C. G.) by the Office of Naval
Research.
yield): mp 117.5–119.08C. H NMR (CDCl3) [Note: N-
Acetyl rotomers, observed in an approximate 4:6 ratio,
resulted in partial proton counts for certain peaks.] d 6.92 (t,
1H, J¼8.1 Hz), 6.53 (dd, 0.4H, J¼2.4 and 8.3 Hz), 6.45 (d,
0.6H, J¼2.4 Hz), 6.40 (dd, 0.6H, J¼2.4 and 8.3 Hz), 6.34
(d, 0.4H, J¼2.4 Hz), 5.18 (d, 0.6H, J¼2.4 Hz), 4.68 (dd,
0.4H, J¼1.5 and 13.7 Hz), 4.20 (s, 0.4H), 3.76 (d, 0.6H,
J¼13.7 Hz), 3.77 (s, 1H), 3.73 (s, 2H), 3.67 (m, 1H), 3.44
(m, 1H), 3.0 (m, 1H), 2.68 (m, 1H), 2.14 (m, 1H), 1.96 (s,
2H), 1.90 (m, 0.6H), 1.81 (m, 1.4H), 1.47 (s, 1H), 1.20 (s,
References
1H); IR (neat): 3212, 1738, 1614, 1434, 1207 cm21
;
1. A preliminary account of this work has been presented:Yao,
Z. J.; Barchi, J. J.; Burke, T. R., Jr. Peptides for the Millenium:
Proceedings of the 16th American Peptide Symposium; Fields,
G. B., Tam, J. P., Barany, G., Eds.; Kluwer: Dordrecht, 2000;
pp 575–578.
FABMS (m/z) 290 (MþH)þ. Anal. Calcd (C16H19NO4):
C, 66.42; H, 6.62; N, 4.84. Found: C, 66.29; H, 6.77; N, 4.80.
3.2.8. (6)-(rel-1R,2R,5S)-3-Acetyl-1,2,3,4,5,6-hexahy-
dro-8-(di-(tert-butyl)phosphoryloxy)-1,5-methano-3-
benzazocine-2-carboxylic acid methyl ester (13). To a
solution of 12 (130 mg, 0.45 mmol) in anhydrous THF
(3 mL) was added tetrazole (97 mg, 1.35 mmol) and di-
(tert-butyl) diisopropylphosphoramidite at room tempera-
ture under argon and the mixture was stirred (overnight).
The mixture was cooled to 08C and a solution of 14% tert-
butyl hydroperoxide was added slowly and the mixture was
stirred (30 min), then the reaction was quenched by addition
of saturated aqueous Na2SO3. The mixture was extracted
(CH2Cl2) and the combined organic extracts were washed
with H2O and brine and dried (MgSO4). Concentration
under vacuum provided a residue, which was purified by
silica gel flash chromatography to afford 13 as an oil
(103 mg, 47% yield). 1H NMR (CDCl3) d 7.11 (m, 1H), 6.97
(m, 2H), 5.11 (d, 0.6H, J¼2.2 Hz), 4.64 (d, 0.4H,
J¼13.7 Hz), 4.36 (s, 0.4H), 3.82 (s, 1H), 3.80 (d, 0.6H,
J¼13.8 Hz), 3.79 (s, 3H), 3.73 (m, 0.6H), 3.72 (s, 1H), 3.54
(d, 1H, J¼15.1 Hz), 3.14 23.02 (m, 1H), 2.77 (t, 1H,
21.2 Hz), 2.23 (m, 1H), 2.01 (s, 2H), 1.94 (m, 1H), 1.85 (m,
1H), 1.57 (s, 1H), 1.55 (s, 18H); IR (neat): 2979, 2928, 1740,
1652, 991 cm21; FABMS (m/z) 482 (MþH)þ. Anal. Calcd
(C24H36NO7P): C, 59.86; H, 7.54; N, 2.91. Found: C, 59.88;
H, 7.82; N, 2.85.
2. Tsatsanis, C.; Spandidos, D. A. Int. J. Mol. Med. 2000, 5,
583–590.
3. Renhowe, P. A. Annual Reports on Medicinal Chemistry;
Doherty, A. M., Ed.; Academic: London, UK, 2001; Vol. 36,
pp 109–118.
4. Morin, M. J. Oncogene 2000, 19, 6574–6583.
5. Levitzki, A. Top. Curr. Chem. 2000, 211, 1–15.
6. Sawyers, C. L. Curr. Opin. Genet. Dev. 2002, 12, 111–115.
7. Traxler, P.; Bold, G.; Buchdunger, E.; Caravatti, G.; Furet, P.;
Manley, P.; O’Reilly, T.; Wood, J.; Zimmermann, J. Med. Res.
Rev. 2001, 21, 499–512.
8. Pawson, T.; Gish, G. D.; Nash, P. Trends Cell Biol. 2001, 11,
504–511.
9. Garcia-Echeverria, C. Curr. Med. Chem. 2001, 8, 1589–1604.
10. Sawyer, T. K.; Bohacek, R. S.; Dalgarno, D. C.; Eyermann,
C. J.; Kawahata, N.; Metcalf, C. A., III; Shakespeare, W. C.;
Sundaramoorthi, R.; Wang, Y.; Yang, M. G. Mini-Rev. Med.
Chem. 2002, 2, 475–489.
11. Eck, M. J.; Shoelson, S. E.; Harrison, S. C. Nature 1993, 362,
87–91.
12. Bradshaw, J. M.; Grucza, R. A.; Ladbury, J. E.; Waksman, G.
Biochemistry 1998, 37, 9083–9090.
13. Bradshaw, J. M.; Mitaxov, V.; Waksman, G. J. Mol. Biol.
1999, 293, 971–985.

X.-Z. Wang et al. / Tetrahedron 59 (2003) 6087–6093
6093
14. Burke, T. R., Jr.; Yao, Z.-J.; Smyth, M. S.; Ye, B. Curr.
Pharm. Des. 1997, 3, 291–304.
29. Takahashi, M.; Tanino, K.; Kuwajima, I. Chem. Lett. 1993,
1655–1658.
15. Burke, T. R., Jr.; Yao, Z.-J.; Liu, D.-G.; Voigt, J.; Gao, Y.
Biopolymers 2001, 60, 32–44.
30. Rahuel, J.; Gay, B.; Erdmann, D.; Strauss, A.; Garcia-
Echeverria, C.; Furet, P.; Caravatti, G.; Fretz, H.; Schoepfer,
J.; Grutter, M. G. Nature Struct. Biol. 1996, 3, 586–589.
31. Wei, C-Q.; Li, B.; Guo, R.; Yang, D.; Burke, T. R., Jr. Bioorg.
Med. Chem. Lett. 2002, 12, 2781–2784.
16. Burke, T. R., Jr.; Lee, K. Acc. Chem. Res. 2003, 36, 426–433.
17. Liu, W. Q.; Carreaux, F.; Meudal, H.; Roques, B. P.; Garbay-
Jaureguiberry, C. Tetrahedron 1996, 52, 4411–4422.
18. Davidson, J. P.; Martin, S. F. Tetrahedron Lett. 2000, 41,
9459–9464.
32. Gao, Y.; Voigt, J.; Wu, J. X.; Yang, D.; Burke, T. R., Jr.
Bioorg. Med. Chem. Lett. 2001, 11, 1889–1892.
33. Wei, C-Q.; Gao, Y.; Lee, K.; Guo, R.; Li, B.; Zhang, M.; Yang,
D.; Burke, T. R., Jr. J. Med. Chem. 2003, 46, 244–254.
34. Ettmayer, P.; France, D.; Gounarides, J.; Jarosinski, M.;
Martin, M-S.; Rondeau, J. M.; Sabio, M.; Topiol, S.;
Weidmann, B.; Zurini, M.; Bair, K. W. J. Med. Chem. 1999,
42, 971–980.
19. Plake, H. R.; Sundberg, T. B.; Woodward, A. R.; Martin, S. F.
Tetrahedron Lett. 2003, 44, 1571–1574.
20. Liu, D-G.; Wang, X-Z.; Gao, Y.; Li, B.; Yang, D.; Burke, T. R.,
Jr. Tetrahedron 2002, 58, 10423–10428.
21. Burke, T. R., Jr.; Barchi, J. J., Jr.; George, C.; Wolf, G.;
Shoelson, S. E.; Yan, X. J. Med. Chem. 1995, 38, 1386–1396.
22. Ye, B.; Yao, Z.-J.; Burke, T. R., Jr. J. Org. Chem. 1997, 62,
5428–5431.
35. Davidson, J. P.; Lubman, O.; Rose, T.; Waksman, G.; Martin,
S. F. J. Am. Chem. Soc. 2002, 124, 205–215.
23. Arend, M.; Westermann, B.; Risch, N. Angew. Chem. Int. Ed.
1998, 37, 1044–1070.
36. Liu, L.; Guo, Q-X. Chem. Rev. 2001, 101, 673–695.
37. Sharp, K. Protein Sci. 2001, 10, 661–667.
24. Muller, R.; Rottele, H.; Henke, H.; Waldmann, H. Chem. Eur.
J. 2000, 6, 2032–2043.
38. Dekker, F. J.; de Mol, N. J.; Bultinck, P.; Kemmink, J.;
Hilbers, H. W.; Liskamp, R. M. J. Bioorg. Med. Chem. 2003,
11, 941–949.
25. Martin, S. F. Acc. Chem. Res. 2002, 35, 895–904.
26. List, B. Tetrahedron 2002, 58, 5573–5590.
27. Padwa, A.; Heidelbaugh, T. M.; Kuethe, J. T.; McClure, M. S.;
Wang, Q. J. Org. Chem. 2002, 67, 5928–5937.
28. Tanino, K.; Takahashi, M.; Murayama, K.; Kuwajima, I.
J. Org. Chem. 1992, 57, 7009–7010.
39. SMART and SAINT data collection and reduction software
for the SMART system. Bruker-AXS, Madison, Wisconsin,
USA, 1995.
40. Sheldrick, G. M. SHELXTL Version 5.1. Bruker Analytical
X-ray Instruments: Madison, 1997.