Design and synthesis of a potential SH2 domain inhibitor bearing a stereodiversified 1,4-cis-enediol scaffold

Article abstract of DOI:10.1016/j.tet.2011.10.014

Synthesis of a potential Src family SH2 domain inhibitor incorporating a 1,4-cis-enediol scaffold is reported. The synthetic route offers straightforward and highly selective access to the enediol and its associated chiral centers. Key steps include stere

Full text of DOI:10.1016/j.tet.2011.10.014

Tetrahedron 67 (2011) 10216e10221
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design and synthesis of a potential SH2 domain inhibitor bearing
a stereodiversified 1,4-cis-enediol scaffold
y
*
Christine Marian, Rong Huang , Richard F. Borch
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a potential Src family SH2 domain inhibitor incorporating a 1,4-cis-enediol scaffold is re-
ported. The synthetic route offers straightforward and highly selective access to the enediol and its as-
sociated chiral centers. Key steps include stereocontrolled syn-aldol coupling, amide alkynylation, and
asymmetric ketone reduction.
Received 26 September 2011
Received in revised form 3 October 2011
Accepted 4 October 2011
Available online 12 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
cis-Enediol
SH2 domain
Stereodiversified
Alkynylation
1. Introduction
development of new and therapeutically useful SH2 domain in-
hibitors have met with limited success, and progress remains
Protein tyrosine kinase (PTK) signal transduction pathways
control a host of important cellular functions, including metabo-
lism, growth and proliferation, and apoptosis.1 As a result, aberrant
PTK signaling often results in disease, most notably cancer and
autoimmune diseases.2 The Src homology-2 (SH2) domain, com-
posed of w100 amino acids, is a non-catalytic phosphotyrosine
binding domain found in cytosolic PTKs which, by virtue of its
central role in mediating PTK signaling, has emerged as an impor-
tant target for therapeutic intervention.3,4 Small peptides con-
taining a phosphotyrosine (pTyr) can effectively inhibit SH2
domain binding of cognate proteins in the signal cascade, and
therefore provide a logical starting point for inhibitor design.5,6
Among PTKs of the Src family, pTyr-containing substrates gener-
ally adopt a similar binding mode, as exemplified by Lck kinase
slow.4,8,9 One major challenge has been the intracellular penetra-
tion of a free phosphate group. This challenge has been addressed
by various prodrug strategies, including a phosphoramidate-based
prodrug developed in our laboratory.10 Further issues to be
addressed include reducing peptide character, and expanding li-
gand diversity in order to improve potency and selectivity. An ad-
ditional consideration is the role of conformational restraint in
ligand binding.11 In summary, an ideal inhibitor would be non-
peptidic, allow for structural and stereochemical diversity, and pro-
vide a means to introduce conformational restraint.
An enediol-based scaffold meets all of these criteria, and
seemed to us an attractive choice for SH2 inhibitor design, espe-
cially given the report in the literature of a series of nanomolar
m
-opioid receptor agonists based on a 1,5-enediol system.12 We
binding to tetrapeptide Ac-pTyr-Glu-Glu-Ile, or pYEEI peptide.7
A
envisioned an inhibitor model that would incorporate a 1,4-cis-
enediol core, a phosphotyrosine mimic, and a hydrophobic sub-
stituent. As the linking segment, the cis-alkene would provide
a degree of conformational restraint and presumably serve to orient
the ligands into their respective pockets by wrapping around the
highly conserved pocket binds the pTyr, and a second pocket three
residues away (the ‘pYþ3’ pocket) binds a hydrophobic substituent.
The two pockets are connected by a narrow b-sheet region, which
protrudes into the solvent. This ‘plug-and-socket’ binding mode
can be used to derive a general model for inhibitor design (Fig. 1A).4
Despite the extensive structural data available, efforts toward the
central b-sheet region. The associated chiral centers would allow
for the systematic comparison of stereochemical preferences
within the SH2 domain. A benzyl group was chosen as the pYþ3
substituent based on previous reports in the literature highlighting
its suitability.13,14 The stereoisomer exhibiting the greatest binding
affinity will ultimately be synthesized as its corresponding pro-
drug10 for cell-based and in vivo studies (Fig. 2).
* Corresponding author. Fax: þ1 765 494 1414; e-mail address: borch@purdue.
edu (R.F. Borch).
y
Current address: Department of Medicinal Chemistry, Virginia Commonwealth
University, Richmond, VA 23284, USA.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.10.014

C. Marian et al. / Tetrahedron 67 (2011) 10216e10221
10217
2. Results and discussion
Computational docking of proposed inhibitors to the Lck SH2
domain (PDB 1Lkk) supported our reasoning and generated struc-
tures similar to pYEEI-bound Lck (Fig. 1B). Substituents at C-1
contributed little to binding, so they were not considered further.
A preliminary synthesis of compound 2 was carried out, using
a published procedure to access the cis-enediol core via Grubbs
ring-closing metathesis.15 We found the synthesis to be laborious
and inefficient, but testing of 2 in a competitive fluorescence
binding assay gave an IC50 comparable to that of pYEEI peptide
(Huang and Borch, unpublished). These results suggested that the
cis-enediol scaffold could be used to generate high-affinity SH2
domain ligands, and encouraged us to develop an improved syn-
thetic pathway to the proposed compounds.
Our new synthetic approach is focused on efficient conversions
and installation of the three chiral centers with high selectivity. In
addition, the synthesis allows for further modification of the linker
region, which may be of use in future studies. Computational
modeling studies showed the terminal amide of 2 directed outward
into the solvent rather than interacting with the protein, so it was
replaced with a hydroxyl group. The pathway to all eight stereo-
isomers is outlined in Scheme 1. 1,4-cis-Enediol compounds 1 can
be made by coupling a protected chiral alkyne and Weinreb amide.
The alkyne can in turn be synthesized by Evans syn16 or Masamune
anti17 stereocontrolled aldol coupling of TMS-propynal with the
appropriate homobenzyl substituted chiral auxiliary. Herein we
describe the synthesis of the (R,R,R)-stereoisomer.
Synthesis of ynone 7 is shown in Scheme 2. Introduction of the
chiral centers at carbons 5 and 6 was achieved by Evans asymmetric
syn-aldol addition of trimethylsilylpropynal to chiral auxiliary
(S,R)-3.18 Subsequent silyl cleavage with TBAF yielded aldol product
4, which was isolated with a diastereomeric ratio of 96:4 after
column chromatography. Reductive cleavage of the chiral auxiliary
with sodium borohydride and acetonide protection of the resulting
diol afforded alkyne 5. The lithium alkynylide was then added to
Weinreb amide 6 to give ynone 7 in 68% yield. An alkyne to amide
ratio of 2:1 was necessary to obtain reasonable yields for this step,
but unreacted alkyne was easily recovered. Zinc-mediated addition
of the alkyne to Boc-glycinal was also tried under numerous con-
ditions, but did not yield any product.
Fig. 1. A. General ligand binding mode for Src family SH2 domains. Figure adapted
from Machida and Mayer.4 B. Ribbon diagram of cis-enediol inhibitor docked to Lck
SH2 domain (PDB 1Lkk).
Fig. 2. cis-Enediol phosphoramidate prodrug.
Scheme 1. Retrosynthetic analysis of modified 1,4-cis-enediol compounds (PG¼protecting group).

10218
C. Marian et al. / Tetrahedron 67 (2011) 10216e10221
Scheme 4. Completion of (R,R,R)-1.
Scheme 2. Preparation of ynone 7.
3. Conclusion
In conclusion, we have synthesized a novel Src family SH2 do-
main inhibitor based on a 1,4-cis-enediol core. Our synthesis offers
straightforward and highly selective access to a privileged molec-
ular scaffold, and will allow the study of stereochemical prefer-
ences within the SH2 domain. Synthesis of the remaining seven
diastereomers is underway, and binding data for the entire series
will be reported shortly.
The next step required asymmetric reduction of ynone 7 to give
the desired propargyl alcohol (Scheme 3). Reduction with methyl-
CBS-oxazaborolidine gave poor selectivity, but asymmetric trans-
fer hydrogenation with Noyori’s chiral ruthenium catalyst19 in-
stalled the third and final chiral center at carbon 2 with high yield
and selectivity (dr 9:1). Stereochemical assignment was determined
by Mosher analysis of the O-MTPA esters.20 Propargyl alcohol 9 was
then reduced to cis-alkene 10 via hydrogenation with Lindlar cata-
lyst. At this point a protecting group exchange became necessary to
ensure selective removal of the Boc group in the next step, so the 1,3-
acetonide was cleaved and the resulting triol was protected by in-
troduction of TBS groups to give protected aminotriol 11.
4. Experimental
4.1. General procedures
All NMR spectra were recorded on a Bruker Avance DPX-300
spectrometer equipped with a multinuclear (31P, 13C, 1H, and 19F)
probe. Chemical shifts are given in parts per million with reference
to tetramethylsilane. All 31P NMR spectra were acquired using
broadband gated decoupling. 31P chemical shifts are reported in
parts per million using 1% triphenylphosphine oxide in benzene-d6
as the coaxial reference set at 0. Mass spectral data were obtained
from the Purdue University Mass Spectrometry Service, West
Lafayette, IN. IR spectra were recorded on a Perkin Elmer Spectrum I
FTIR spectrometer using a NaCl plate. Optical rotations were mea-
sured on a Perkin Elmer 341 polarimeter with 0.5 dm path length
cell. Analytical HPLC analysis was done using a Beckman System
Gold equipped with a 168 detector set at 215 nm and a Zorbax
Eclipse 5-
m
analytical C18 column (4.6ꢀ250 mm) from Agilent
Technologies Inc. Lck GST-SH2 protein was a kind gift from Dr.
Robert Geahlen. All moisture-sensitive reactions were carried out
under an atmosphere of argon, and all organic solvents were dis-
tilled prior to use unless otherwise specified. Flash chromatography
using silica gel grade 60 (230e400 mesh) was carried out for all
chromatographic separations. Thin layer chromatography was
performed using Analtech glass plates precoated with silica gel
(250 nm), and visualized using UV or phosphomolybdic acid stain.
All reagents were purchased from Aldrich, Alfa-Aesar, or TCI
America, unless stated otherwise.
Scheme 3. Synthesis of protected aminotriol 11.
Completion of (R,R,R)-1 is detailed in Scheme 4. tert-Butyl
phosphoryl acid 13 was synthesized in two steps from commer-
cially available methyl 4-hydroxyphenylacetate.21 Boc cleavage of
11 with TBS-triflate followed by coupling to acid 13 with EDC/HOBt
afforded acylation product 14 in good yield (Scheme 4). Global
deprotection under acidic conditions completed the synthesis and
gave (R,R,R)-1 in essentially quantitative yield.
4.2. Chemistry
4.2.1. (4S,5R)-3-((2S,3S)-2-Benzyl-3-hydroxypent-4-ynoyl)-4-
methyl-5-phenyloxazolidin-2-one (4). (S,R)-3 (6.56 g, 21.2 mmol)
was dissolved in 100 mL dry CH2Cl2 and cooled to 0 ꢁC. Dibu-
tylboron triflate (23 mL, 1.0 M in CH2Cl2, 23.3 mmol) was added

C. Marian et al. / Tetrahedron 67 (2011) 10216e10221
10219
dropwise, and the light orange reaction mixture allowed to stir for
10 min at 0 ꢁC. Diisopropylethylamine (4.42 mL, 25.4 mmol) was
then added dropwise, and the resulting clear solution allowed to
stir for 30 min at 0 ꢁC. The mixture was then cooled to ꢂ78 ꢁC, and
a solution of trimethylsilylpropynal (3.1 mL, 21.0 mmol) in 30 mL
CH2Cl2 at ꢂ78 ꢁC was added via cannula. After 20 min stirring at
ꢂ78 ꢁC, the reaction mixture was warmed to 0 ꢁC and allowed to
stir for an additional 2 h at that temperature, then quenched by
dropwise addition of the following, in the order listed: 25 mL
phosphate buffer (pH 7.4), 65 mL methanol, and 65 mL of a 2:1
solution of methanol and 30% aqueous H2O2, cooled to 0 ꢁC. The
mixture was allowed to stir at 0 ꢁC for 1 h, then extracted 3ꢀ with
CH2Cl2. The combined organic layers were washed with NaHCO3
and brine, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure. The crude product was dissolved in
135 mL THF and 9 mL MeOH and cooled to ꢂ20 ꢁC. Tetrabuty-
lammonium fluoride (21 mL, 1.0 M in THF, 21.2 mmol) was added
dropwise, and the mixture allowed to stir for 2 h at 0 ꢁC. The re-
action was quenched with satd NH4Cl solution and extracted with
CH2Cl2. The organic layer was washed with brine and dried over
anhydrous Na2SO4, followed by removal of solvent under reduced
pressure. The crude product was purified by column chromatog-
raphy (20% ethyl acetate/hexane) to yield 5.8 g (60% over two steps)
of major diastereomer (S,R)-4 as an off-white foam. TLC 50% ethyl
acetate/hexane Rf¼0.7 visualized with PMA or UV. Diastereomeric
ratio after flash column chromatography was 96:4, as determined
described by Morwick et al.22 1H NMR (CDCl3, 300 MHz)
d
5.29 (br,
1H), 4.01 (d, J¼4.0 Hz, 2H), 3.65 (s, 3H), 3.14 (s, 3H), 1.38 (s, 9H). 13C
NMR (CDCl3, 75 MHz)
d 170.1, 155.8, 79.5, 61.3, 41.6, 32.2, 28.2. CI
(þ) LRMS (m/z): 219 [MþH]þ.
4.2.4. tert-Butyl-4-((4S,5R)-5-benzyl-2,2-dimethyl-1,3-dioxan-4-yl)-
2-oxobut-3-ynylcarbamate (7). Alkyne 5 (3.27 g, 14.2 mmol) was
dissolved in 68 mL dry THF and cooled to ꢂ78 ꢁC. n-BuLi (8.37 mL,
1.78 M in hexane, 14.9 mmol) was added dropwise and the result-
ing mixture stirred at ꢂ78 ꢁC for 1 h. The mixture was then added
via cannula to a solution of Weinreb amide 2 (1.55 g, 7.10 mmol) in
35 mmol THF cooled to ꢂ78 ꢁC. The mixture was then stirred at 0 ꢁC
for 3 h, cooled to ꢂ78 ꢁC, and quenched by slow addition of satd
NH4Cl solution. The resulting mixture was extracted 3ꢀ with Et2O,
and the combined organic layers washed with NaHCO3 and brine
and dried over anhydrous Na2SO4. Purification of the crude oil by
column chromatography (10%/20% ethyl acetate/hexane) yielded
1.87 g ynone 7 as a yellow oil (68%). Starting alkyne (1.33 g (82%))
was also recovered. TLC 30% ethyl acetate/hexane Rf¼0.5 visualized
25
with PMA. [
d
a
]
D
þ34.0 (c 1.00, CHCl3). 1H NMR (CDCl3, 300 MHz)
7.16e7.32 (m, 5H), 5.20 (br, 1H), 5.02 (d, J¼3.4 Hz, 1H), 4.15 (d,
J¼5.3 Hz, 2H), 3.82 (dd, J¼3.0, 12.1 Hz, 1H), 3.64 (dd, J¼4.0, 12.1 Hz,
1H), 2.91e2.98 (m, 2H), 1.96e2.00 (m, 1H), 1.53 (s, 3H), 1.44 (s, 12H).
13C NMR (CDCl3, 75 MHz)
d 182.5, 155.4, 139.0, 129.2, 128.5, 126.3,
99.7, 91.9, 83.4, 80.2, 64.3, 60.9, 52.1, 39.1, 32.2, 28.2, 27.7, 21.5. ESI
(þ) HRMS (m/z): [MþNa]þ calcd for C22H29NO5 410.1943; found,
410.1946.
by reverse phase HPLC. HPLC, Zorbax XDB-C18 (250ꢀ4.6 mm, 5
m);
flow¼1.0 mL/min;
jor isomer tR¼7.15 min. [
300 MHz)
l
¼215 nm; isocratic (80:20 MeCN/ddH2O); Ma-
a
]
25 ꢂ34.0 (c 1.00, CHCl3). 1H NMR (CDCl3,
4.2.5. tert-Butyl-((R)-4-((4S,5R)-5-benzyl-2,2-dimethyl-1,3-dioxan-
4-yl)-2-hydroxybut-3-yn-1-yl)carbamate (9). To a stirred solution
of ynone 7 (480 mg, 1.24 mmol) in 9.5 mL anhydrous dime-
thylformamide under argon atmosphere was added (S,S)-Noyori
catalyst 8 (16 mg, 0.025 mmol) at room temperature. In a separate
flask, diisopropylethylamine (208 mg, 1.61 mmol) was cooled to
ꢂ10 ꢁC, and 98% formic acid (188 mg, 4.09 mmol) was added
dropwise, keeping the temperature below 0 ꢁC. This solution was
added dropwise to the ynone, and the reaction mixture allowed to
stir overnight at room temperature. The mixture was then cooled to
0 ꢁC and quenched by addition of satd NaHCO3, extracted 3ꢀ with
ethyl acetate, washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The crude residue was pu-
rified by column chromatography (10%/50% ethyl acetate/hexane)
to give 420 mg of propargyl alcohol 9 as a light yellow oil (87%, dr
9:1). Stereochemical assignment of the alcohol configuration was
determined by Mosher analysis of the (R) and (S)-MTPA esters.16
Diastereomeric ratio was determined by 19F NMR of the MTPA es-
D
d
7.16e7.39 (m, 10H), 5.60 (d, J¼7.4 Hz, 1H), 4.66e4.80
(m, 2H), 4.64 (s,1H), 3.18 (d, J¼6.4 Hz, 2H), 2.71 (br,1H), 2.62 (d,1H),
0.60 (d, J¼6.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz)
d 172.6, 152.8,
137.6, 133.0, 129.3, 128.7, 128.6, 128.3, 126.6, 125.6, 81.8, 78.7, 77.2,
75.0, 63.1, 54.7, 49.8, 34.5, 14.1. ESI (þ) HRMS (m/z): [MþNa]þ calcd
for C22H21NO4 386.1368; found, 386.1370.
4.2.2. (4S,5R)-5-Benzyl-4-ethynyl-2,2-dimethyl-1,3-dioxane
(5). Aldol product 4 (415 mg, 1.14 mmol) was dissolved in 30 mL
THF and cooled to 0 ꢁC. NaBH4 (173 mg, 4.56 mmol) was dissolved
in 7 mL H2O and added dropwise to the THF solution. The mixture
was allowed to warm to room temperature and stirred for 1 h, after
which it was cooled to 0 ꢁC and quenched with NH4Cl. The mixture
was again brought to room temperature and stirred for 1 h. The
layers were separated, and the aqueous layer extracted 3ꢀ with
EtOAc. The combined organic layer was washed with NaHCO3 and
brine, dried over anhydrous Na2SO4, and concentrated under re-
duced pressure. The chiral auxiliary was recovered by re-
crystallization of the crude residue with ethyl acetate/hexanes
(w1:1). The remaining oil was then dissolved in 23 mL 2,2-
dimethoxypropane. p-Toluenesulfonic acid monohydrate (22 mg,
0.114 mmol) was added in one portion, and the mixture stirred for
1 h at room temperature. The mixture was then diluted with CH2Cl2
and quenched with NaHCO3 at 0 ꢁC. The layers were separated and
the aqueous layer extracted 3ꢀ with CH2Cl2. The combined organic
layers were dried over anhydrous Na2SO4 and concentrated under
reduced pressure to yield 236 mg pure alkyne 5 as a light yellow oil
ters. TLC 30% ethyl acetate/hexane Rf¼0.4 visualized with PMA.
23
[a
]
þ22.0 (c 1.00, CHCl3). 1H NMR (CDCl3, 300 MHz)
d 7.15e7.29
D
(m, 5H), 5.21 (t, J¼5.5 Hz, 1H), 4.91 (s, 1H), 4.54 (br, 1H), 3.78 (dd,
J¼2.6, 11.8 Hz, 1H), 3.59 (dd, J¼3.1, 12.0 Hz, 1H), 3.30e3.46 (m, 3H),
2.90e3.04 (m, 2H), 1.80 (m, 1H),1.50 (s, 3H),1.42 (s, 3H), 1.41 (s, 9H).
13C NMR (CDCl3, 75 MHz)
d 156.6, 139.9, 129.3, 128.3, 126.0, 99.4,
85.7, 83.0, 79.8, 77.3, 64.3, 62.0, 60.9, 46.5, 39.6, 31.8, 28.4, 20.7. ESI
(þ) HRMS (m/z): [MþNa]þ calcd for C22H31NO5, 412.2100; found,
412.2093.
(90% over two steps). TLC 30% ethyl acetate/hexane Rf¼0.9 visual-
4.2.6. tert-Butyl-((R,Z)-4-((4R,5R)-5-benzyl-2,2-dimethyl-1,3-
dioxan-4-yl)-2-hydroxybut-3-en-1-yl)carbamate (10). Propargyl al-
cohol 9 (271 mg, 0.693 mmol) was dissolved in 8.5 mL ethyl acetate
and flushed with argon. Lindlar catalyst (5% palladium on calcium
carbonate, poisoned with lead; 52 mg, 0.485 mmol) and quinoline
þ22.6 (c 1.00, CHCl3). 1H NMR (CDCl3,
25
ized with PMA. [
a
]
D
300 MHz)
d
7.21e7.34 (m, 5H), 4.92 (t, 1H), 3.80 (dd, J¼2.8, 12.1 Hz,
1H), 3.65 (dd, J¼3.3, 12.0 Hz, 1H), 2.99e3.11 (m, 2H), 2.62 (d,
J¼2.4 Hz, 1H), 1.82e1.87 (m, 1H), 1.57 (s, 3H), 1.47 (s, 3H). 13C NMR
(CDCl3, 75 MHz)
d
140.0, 129.3, 128.4, 126.1, 99.4, 81.4, 75.2, 64.2,
(6 mL, 0.049 mmol) were added. The flask was flushed with argon
60.8, 39.6, 31.6, 28.4, 20.8. CI (þ) LRMS (m/z): 231 [MþH]þ.
again, and then flushed with H2 gas 3ꢀ. A hydrogen balloon was
then added, and the mixture allowed to stir overnight at room
temperature. The following day, the reaction mixture was filtered
through CeliteÒ, rinsing with ethyl acetate. The organic filtrate was
4.2.3. tert-Butyl 2-(methoxy(methyl)amino)-2-oxoethylcarbamate
(6). Weinreb amide
6 was synthesized from Boc-glycine as

10220
C. Marian et al. / Tetrahedron 67 (2011) 10216e10221
washed with 1 M HCl, satd NaHCO3, and brine, then dried over
anhydrous Na2SO4 and concentrated to yield 273 mg alkene 10 as
a light brown foam (quantitative). TLC 30% ethyl acetate/hexane
LiOH (10.6 mL, 3.18 mmol) was added dropwise, and the mixture
allowed to stir at 0 ꢁC for 30 min. The reaction mixture was acidi-
fied with 1 M aqueous HCl to a pH of 3.0, after which it was
extracted 3ꢀ with ethyl acetate, dried over anhydrous sodium
sulfate, and concentrated to yield 365 mg carboxylic acid 13 as
Rf¼0.4 visualized with PMA. 1H NMR (CDCl3, 300 MHz)
d 7.14e7.28
(m, 5H), 5.59e5.62 (m, 2H), 5.15 (br, 1H), 4.97 (br, 1H), 4.54 (q,
J¼5.3 Hz, 1H), 3.85 (d, J¼11.6 Hz, 1H), 3.53 (d, J¼11.9 Hz, 1H),
3.20e3.32 (m, 3H), 2.77e3.02 (m, 2H), 1.66 (d, J¼11.6 Hz, 1H), 1.49
a white solid (quantitative). 1H NMR (CDCl3, 300 MHz)
d
7.13e7.26
(m, 4H), 5.68 (br, 1H), 3.58 (s, 2H), 1.50 (s, 18H). 13C NMR (CDCl3,
(s, 6H), 1.43 (s, 9H). 13C NMR (CDCl3, 75 MHz)
d
156.6, 140.5, 132.2,
75 MHz)
(CDCl3)
367.1286; found, 367.1277.
d
175.5, 150.4, 130.3, 129.6, 119.9, 84.0, 40.2, 29.8. 31P NMR
131.3, 129.3, 128.3, 125.9, 99.0, 79.5, 70.0, 67.9, 61.6, 46.4, 39.9, 30.2,
29.7, 28.3, 19.1. ESI (þ) HRMS (m/z): [MþNa]þ calcd for C22H33NO5,
414.2256; found, 414.2273.
d
ꢂ41.1. ESI (þ) HRMS (m/z): [MþNa]þ calcd for C16H25O6P,
4.2.10. 4-(2-(((2R,5R,6R,Z)-6-Benzyl-2,5,7-tris((tert-butyldime-
thylsilyl)oxy)hept-3-en-1-yl)amino)-2-oxoethyl)phenyl di-tert-butyl
phosphate (14). Protected aminotriol 11 (19 mg, 0.027 mmol) was
4.2.7. tert-Butyl-((2R,5R,6R,Z)-6-benzyl-2,5,7-tris((tert-butyldime-
thylsilyl)oxy)hept-3-en-1-yl)carbamate (11). Alkene 10 (216 mg,
0.552 mmol) was dissolved in 3.5 mL THF and cooled to 0 ꢁC.
dissolved in 500
m
L anhydrous CH2Cl2 and cooled to ꢂ78 ꢁC. TBS-
Phosphoric acid of 85% (566
mL, 8.28 mmol) was added dropwise,
trifluoromethanesulfonate (8 mL, 0.030 mmol) was added drop-
and the resulting solution stirred at room temperature for 3 h. The
mixturewas then cooled back down to 0 ꢁC and diluted with H2O. Aq
NaOH of 50 wt % was added dropwise to bring the solution to pH 8,
and the solutionwas poured into brine and extracted 3ꢀ with EtOAc,
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The crude oil was then dissolved in 9 mL anhydrous CH2Cl2
wise, and the reaction mixture stirred at 0 ꢁC for 2 h. Diisopropy-
lethylamine (0.030 mmol, 5 L) was then added dropwise, and the
m
solution poured into H2O and extracted 3ꢀ with CH2Cl2. The or-
ganic layer was dried over anhydrous Na2SO4, concentrated in
vacuo, and used immediately in the next step.
Phosphoryl acid 13 (11 mg, 0.032 mmol) was dissolved in 200 mL
and cooled to ꢂ78 ꢁC. 2,6-Lutidine (322
m
L, 2.76 mmol) was added
CH2Cl2 and stirred at 0 ꢁC. EDC (6 mg, 0.032 mmol) was added,
followed by HOBt hydrate (5 mg, 0.032 mmol) and DMAP (4 mg,
0.032 mmol). The resulting mixture was stirred for 20 min. The
dropwise, followed by TBS-trifluoromethanesulfonate (507
mL,
2.21 mmol). The reaction mixture was stirred overnight at room
temperature, after which it was quenched with MeOH at 0 ꢁC. The
mixture was then poured into brine and extracted 3ꢀ with CH2Cl2,
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The crude oil was purified by column chromatography (3%
ether/pentane) toyield 190 mg pure tris-TBS aminotriol 11 as a clear,
colorless oil (50% over two steps). TLC 10% ethyl acetate/hexane
crude amine was dissolved in 200 mL CH2Cl2 and added to the acid
solution dropwise. The reaction mixture was warmed to room
temperature and allowed to stir for two days. The solvent was then
removed in vacuo and the crude residue redissolved in ethyl acetate
and washed with the following: water, 5% aqueous citric acid so-
lution, satd aqueous NaHCO3, and brine. The organic layer was then
dried over anhydrous Na2SO4, concentrated, and purified by col-
umn chromatography (20%/30% ethyl acetate/hexanes) to yield
12 mg 14 as a light yellow oil (50% over two steps). TLC 20% ethyl
acetate/hexane Rf¼0.2 visualized with PMA. 1H NMR (CDCl3,
Rf¼0.7 visualized with PMA. 1H NMR (CDCl3, 300 MHz)
d 7.12e7.26
(m, 5 Hz), 5.50e5.57 (m, 1H), 5.19e5.26 (m, 1H), 4.80e4.83 (d,
J¼8.4 Hz, 1H), 4.66 (br, 1H), 4.46e4.52 (m, 1H), 3.09e3.51 (m, 4H),
2.80 (dd, J¼4.6, 14.1 Hz, 1H), 2.43 (dd, J¼9.8, 14.0 Hz, 1H), 1.92 (br,
1H),1.44 (s, 9H), 0.85e0.92 (m, 27H), ꢂ0.06e0.09 (m,18H). 13C NMR
300 MHz)
d 7.09e7.26 (m, 9H), 5.68 (br, 1H), 5.45e5.52 (m, 1H),
(CDCl3, 75 MHz)
d
155.6, 141.1, 134.9, 129.6, 128.9, 128.1, 125.6, 78.8,
5.09e5.15 (m, 1H), 4.77 (d, J¼9 Hz, 1H), 4.46 (br, 1H), 3.34e3.58 (m,
5H), 3.02e3.11 (m, 1H), 2.74e2.81 (m, 1H), 2.37e2.45 (m, 1H), 1.88
(br, 1H), 1.50 (s, 27H), 0.90 (s, 9H), 0.83 (s, 9H), 0.77 (s, 9H),
68.1, 66.7, 61.7, 50.5, 46.4, 31.1, 28.3, 25.9,18.1, ꢂ4.0, ꢂ4.1, ꢂ4.6, ꢂ4.9,
ꢂ5.6. ESI (þ) LRMS (m/z): 381 [MþNa]þ.
ꢂ0.08e0.10 (m, 18H). 13C NMR (CDCl3, 75 MHz)
d 170.3, 150.7, 141.1,
134.8, 130.5, 129.2, 128.9, 128.1, 125.6, 125.2, 120.3, 83.7, 67.9, 66.6,
4.2.8. Methyl-2-(4-((di-tert-butoxyphosphoryl)-oxy)phenyl)acetate
(12). To a stirred solution of imidazole (188 mg, 2.76 mmol) in
61.6, 50.5, 45.3, 43.1, 31.1, 30.3, 29.8, 25.9, 25.6, 18.1, 17.7, ꢂ3.7, ꢂ4.0,
750
mL anhydrous THF at room temperature under argon atmo-
ꢂ4.7, ꢂ5.0, ꢂ5.6. 31P NMR (CDCl3)
d
ꢂ41.2. ESI (þ) HRMS (m/z):
sphere was added trifluoroacetic acid (210
m
L, 2.76 mmol) drop-
[MþNa]þ calcd for C48H86NO8PSi3, 942.5277; found, 942.5297.
wise. The resulting slurry was stirred at room temperature for
10 min, after which di-tert-butyl N,N-diisopropylphosphoramidite
4.2.11. 4-(2-(((2R,5R,6R,Z)-6-Benzyl-2,5,7-trihydroxyhept-3-en-1-yl)
amino)-2-oxoethyl)phenyl dihydrogen phosphate (1). Acylation
product 14 (23 mg, 0.025 mmol) was dissolved in 1 mL of a 1:1
mixture of anhydrous methanol and THF. The solution was cooled
(285
ditional 10 min, a solution of methyl 4-hydroxyphenylacetate
(100 mg, 0.60 mmol) in 300 L anhydrous THF was added drop-
mL, 0.90 mmol) was added dropwise. After stirring for an ad-
m
wise over 15 min. The solution was allowed to stir at room tem-
perature for 30 min, then cooled to ꢂ40 ꢁC. Aqueous tert-butyl
hydroperoxide solution of 14% (1 mL) was added dropwise, and the
mixture stored at 4 ꢁC overnight. The reaction was quenched with
NaHCO3 at 0 ꢁC, poured into water, extracted 3ꢀ with EtOAc, dried
over anhydrous Na2SO4, and concentrated in vacuo. The crude
product was purified by column chromatography (5%/10% ethyl
acetate/CH2Cl2) to yield 200 mg pure phosphoryl methyl ester 12 as
a clear, colorless oil (93%). TLC 20% ethyl acetate/CH2Cl2 Rf¼0.7 vi-
to 0 ꢁC, and 230
mL of 3 M HCl in MeOH was added. The reaction
mixture was warmed to room temperature and allowed to stir
overnight. Solvents were then removed in vacuo, and the product
triturated with hexane and ether, and co-evaporated 2ꢀ with tol-
23
uene to yield 12 mg cis-enediol phosphate 1 (quantitative). [a]
D
ꢂ20.6 (c 1.00, CH3OH). 1H NMR (CD3OD, 300 MHz)
d 7.14e7.30 (m,
9H), 5.48e5.68 (m, 2H), 4.96 (br, phosphate), 4.52e4.56 (m, 2H),
3.20e3.66 (m, 10H), 2.59e3.21 (m, 2H), 1.79 (br, 1H). 13C NMR
(CD3OD, 75 MHz)
d 150.3, 140.7, 133.9, 131.8, 131.3, 129.9, 128.8,
sualized with PMA. 1H NMR (CDCl3, 300 MHz)
d
7.08e7.17 (m, 4H),
127.9, 125.4, 119.9, 67.4, 65.7, 59.5, 48.7, 45.0, 41.4, 31.8. 31P NMR
3.61 (s, 3H), 3.52 (s, 2H), 1.44 (s, 18H). 13C NMR (CDCl3, 75 MHz)
(CDCl3)
d
ꢂ30.1. ESI (þ) HRMS (m/z): [MþH]þ calcd for C22H28NO8P
d
171.7, 150.5, 130.1, 129.7, 119.9, 83.5, 51.8, 40.2, 29.7. 31P NMR
466.1631; found, 466.1609.
(CDCl3)
d
ꢂ40.8. ESI (þ) LRMS (m/z): 716 [MþNa]þ.
Acknowledgements
4.2.9. 2-(4-((Di-tert-butoxyphosphoryl)oxy)
phenyl)acetic
acid
(13). Methyl ester 12 (380 mg, 1.06 mmol) was dissolved in 5 mL
Financial support from the National Cancer Institute (Grant R01
CA34619) and from the Purdue Research Foundation is gratefully
H2O and 13 mL THF, and cooled to 0 ꢁC. A 0.3 M aqueous solution of

C. Marian et al. / Tetrahedron 67 (2011) 10216e10221
10221
11. DeLorbe, J. E.; Clements, J. H.; Teresk, M. G.; Benfield, A. P.; Plake, H. R.; Mill-
spaugh, L. E.; Martin, S. F. J. Am. Chem. Soc. 2009, 131, 16758e16770.
12. Harrison, B. A.; Gierasch, T. M.; Neilan, C.; Pasternak, G. W.; Verdine, G. L. J. Am.
Chem. Soc. 2002, 124, 13352e13353.
acknowledged. Support from the Purdue Cancer Center Support
Grant P30 CA231168 for services provided by the NMR and Mass
Spectrometry Shared Resources is appreciated.
13. Llinas-Brunet, M.; Beaulieu, P. L.; Cameron, D. R.; Ferland, J.-M.; Gauthier, J.;
Ghiro, E.; Gillard, J.; Gorys, V.; Poirier, M.; Rancourt, J.; Wernic, D. J. Med. Chem.
1999, 42, 722e729.
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