Total synthesis and evaluation of C26-hydroxyepothilone D derivatives for photoaffinity labeling of β-tubulin

Article abstract of DOI:10.1021/jo901752v

(Chemical Equation Presented) Three photaffinity labeled derivatives of epothilone D were prepared by total synthesis, using efficient novel asymmetric synthesis methods for the preparation of two important synthetic building blocks. The key step for the asymmetric synthesis of (S,E)-3-(tert- butyldimethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-yl)pent-4-enal involved a ketone reduction with (R)-Me-CBS-oxazaborolidine. For the synthesis of (5S)-5,7-di[(tert-butyldimethylsilyl)oxy]-4,4-dimethylheptan-3-one an asymmetric Noyori reduction of a β-ketoester was employed. The C26 hydroxyepothilone D derivative was constructed following a well-established total synthesis strategy and the photoaffinity labels were attached to the C26 hydroxyl group. The photoaffinity analogues were tested in a tubulin assembly assay and for cytotoxicity against MCF-7 and HCT-116 cancer cell lines. The 3- and 4-azidobenzoic acid analogues were found to be as active as epothilone B in a tubulin assembly assay, but demonstrated significantly reduced cellular cytotoxicity compared to epothilone B. The benzophenone analogue was inactive in both assays. Docking and scoring studies were conducted that suggested that the azide analogues can bind to the epothilone binding site, but that the benzophenone analogue undergoes a sterically driven ligand rearrangement that interrupts all hydrogen bonding and therefore protein binding. Photoaffinity labeling studies with the 3-azidobenzoic acid derivative did not identify any covalently labeled peptide fragments, suggesting that the phenylazido side chain was predominantly solvent-exposed in the bound conformation. 2009 American Chemical Society.

Full text of DOI:10.1021/jo901752v

pubs.acs.org/joc
Total Synthesis and Evaluation of C26-Hydroxyepothilone D Derivatives
for Photoaffinity Labeling of β-Tubulin
Emily A. Reiff,† Sajiv K. Nair,† John T. Henri,† Jack F. Greiner,† Bollu S. Reddy,†
Ramappa Chakrasali,† Sunil A. David,‡ Ting-Lan Chiu,§ Elizabeth A. Amin,§
Richard H. Himes,‡ David G. Vander Velde,† and Gunda I. Georg*,§
†Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, Kansas
66045, ‡Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence,
Kansas 66045, and §Department of Medicinal Chemistry, University of Minnesota, 717 Delaware Street SE,
Minneapolis, Minnesota 55414
georg239@umn.edu
Received August 24, 2009
Three photaffinity labeled derivatives of epothilone D were prepared by total synthesis, using
efficient novel asymmetric synthesis methods for the preparation of two important synthetic building
blocks. The key step for the asymmetric synthesis of (S,E)-3-(tert-butyldimethylsilyloxy)-4-methyl-
5-(2-methylthiazol-4-yl)pent-4-enal involved a ketone reduction with (R)-Me-CBS-oxazaborolidine.
For the synthesis of (5S)-5,7-di[(tert-butyldimethylsilyl)oxy]-4,4-dimethylheptan-3-one an asymme-
tric Noyori reduction of a β-ketoester was employed. The C26 hydroxyepothilone D derivative was
constructed following a well-established total synthesis strategy and the photoaffinity labels were
attached to the C26 hydroxyl group. The photoaffinity analogues were tested in a tubulin assembly
assay and for cytotoxicity against MCF-7 and HCT-116 cancer cell lines. The 3- and 4-azidobenzoic
acid analogues were found to be as active as epothilone B in a tubulin assembly assay, but demon-
strated significantly reduced cellular cytotoxicity compared to epothilone B. The benzophenone
analogue was inactive in both assays. Docking and scoring studies were conducted that suggested
that the azide analogues can bind to the epothilone binding site, but that the benzophenone analogue
undergoes a sterically driven ligand rearrangement that interrupts all hydrogen bonding and
therefore protein binding. Photoaffinity labeling studies with the 3-azidobenzoic acid derivative
did not identify any covalently labeled peptide fragments, suggesting that the phenylazido side chain
was predominantly solvent-exposed in the bound conformation.
Introduction
Since the isolation of the epothilones from Sorangium
for their potential as antitumor agents. The epothilones have
been the subject of numerous elegant total syntheses and
hundreds of analogues have been synthesized, some with
improved biological profiles.3-10 The epothilones have the
1
cellulosum by Hofle and collaborators at the GBF, and
€
the determination of their biological mode of action by
researchers at Merck,2 the epothilones have been explored
(3) Nicolaou, K. C.; Roschangar, F.; Vourloumis, D. Angew. Chem., Int.
Ed. 1998, 37, 2014–2045.
(1) Gerth, K.; Bedorf, N.; Hoefle, G.; Irschik, H.; Reichenbach, H.
J. Antibiot. 1996, 49, 560–563.
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Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Cancer Res. 1995, 55,
2325–2333.
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1535.
86 J. Org. Chem. 2010, 75, 86–94
Published on Web 12/02/2009
DOI: 10.1021/jo901752v
r
2009 American Chemical Society

Reiff et al.
JOCArticle
same mechanism of action, microtubule hyperstabilization,
as paclitaxel, which is a highly successful drug in the treat-
ment of a variety of cancers.2 The epothilones are cytotoxic
to cancer cell lines and exhibit excellent activity against
multidrug resistant cell lines, in which the activity of pacli-
taxel is diminished.2,11 Patupilone (epothilone B),12 sagopi-
lone (a synthetic analogue of epothilone B),13 21-amino
epothilone B,14 and 9,10-didehydroepothilone D15 are pre-
sently undergoing clinical trials and the aza analogue of
epothilone B, ixabepilone,16 has received FDA approval for
the treatment of metastatic breast cancer (Figure 1).16
SCHEME 1
FIGURE 1. The structures of epothilones A-D.
that are in close proximity to the epothilone molecule. With a
better understanding of the epothilone binding site, the struc-
ture of the epothilones can be modified to obtain the maximum
selectivity for a potential cancer chemotherapeutic agent. Here-
in, the synthetic approach to photoaffinity labels attached to
C26 hydroxyepothilone D is described (Scheme 1). This posi-
tion was selected because Nicolaou and collaborators had
shown that this site tolerates structural modifications without
loss of activity.24 The biological results obtained with C26
hydroxyepothilone D photoaffinity labels in tubulin assembly
and cytotoxicity assays are also presented.
The disconnections for the total synthesis of the C26
hydroxyepothilone D are detailed in Scheme 1 and follow a
well-precedented strategy,24,25 using three key precursors: the
C1-C6 ketone A, the C7-C12 Wittig ester B, and the
C13-C20 thiazole aldehyde C. Novel efficient asymmetric
synthesis methods for the preparation of two of the three
building blocks, required for the total synthesis, were devel-
oped. The key step for the asymmetric synthesis of (S,E)-
3-(tert-butyldimethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-
yl)pent-4-enal (C) involved a ketone reduction with (R)-Me-
CBS-oxazaborolidine.26 For the synthesis of (5S)-5,7-di[(tert-
butyldimethylsilyl)oxy]-4,4-dimethylheptan-3-one (A) anasym-
metric Noyori reduction27 of a β-ketoester was employed.
Information on the nature of the binding interactions
between the epothilones and β-tubulin comes from the
˚
2.9 A electron crystal structure of protofilament sheets of
bovine brain tubulin stabilized by epothilone A.17 Further
binding studies were carried out with NMR to analyze
epothilone bound to unpolymerized R,β-tubulin dimer in
solution.18 Photoaffinity labeling represents another app-
roach to identify sites of interaction between a small mole-
cule and a protein and has been used successfully in the study
of paclitaxel-tubulin interactions.19-23 This work focuses
on the design of photoaffinity labels of epothilone D that
could be used as tools to determine those amino acid residues
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Marsoni, S.; Corradino, I.; Gianni, L. Ann. Oncol. 2007, 18, 1548–1553.
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Cropp, G.; Zhong, Z.; Wells, S.; Hannah, A.; Burris, H. J. Clin. Oncol.
2007, 25, 2571.
Results and Discussion
The synthesis of thiazole aldehyde C28 began with the
reaction of aldehyde 229 with β-ketophosphonate 3 (Scheme
2).30 These precursors underwent the Paterson modification31
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46, 1864–1868.
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Chem. Commun. 1997, 2343–2344.
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J. Org. Chem. Vol. 75, No. 1, 2010 87

Reiff et al.
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SCHEME 2a
SCHEME 3a
aReagents and conditions: (a) Ba(OH)2 8H2O, THF, then 2 in THF:
3
H2O (40:1) 0 °C; (b) (R)-2-Me-CBS-oxazaborolidine (0.5 equiv),
BH3 Me2S (1.5 equiv), CH2Cl2, 0 °C, then 4, 0 °C, 2 h, then ethanol-
3
amine; (c) TBSOTf, 2,6-lutidine, CH2Cl2, -78 °C; (d) HF (40% aq),
glass splinters, MeCN:Et2O (1:1), 0 °C; (e) Dess-Martin periodinane,
CH2Cl2.
aReagents and conditions: (a) lithium isopropylcyclohexylamine, THF,
-78 °C, 3-(benzyloxy)propanoyl chloride; (b) RuBr2(S)-binap, H2,
MeOH, 60 psi, 60 °C, 96 h; (c) H2, 10% Pd/C, THF; (d) TBSOTf, 2,6-
lutidine, CH2Cl2; (e) PhSO2Et, n-BuLi, -78 °C; (f) Na(Hg), Na2HPO4,
MeOH; (g) LDA, -78 °C, ketone 11, -78 to -40 °C, aldehyde 7, 2 min.
of the Horner-Emmons-Wadsworth reaction with barium
hydroxide and provided the desired enone 4 with >20:1 E:
Z selectivity. The stereochemistry at the C15 position was
then established by using Corey’s (R)-Me-CBS-oxazaboro-
lidine26 to reduce the enone 4. On a small scale the reduc-
tion was carried out in good yield and with high enantio-
meric excess (95% ee), using (R)-Me-CBS-oxazaborolidine
and borane dimethylsulfide. Upon scale-up, however, a
decrease in ee to 89% was observed. The reaction condi-
tions were studied and the catalyst loading and the addition
rate were determined to be the key parameters for obtaining
high enantiomeric excess. (R)-Me-CBS-oxazaborolidine
(0.5 equiv) and borane dimethylsulfide (1.5 equiv) were
combined and enone 4 was transferred dropwise via syringe
pump over 15 h. Following workup and chromatography
the required S-alcohol 5 was obtained from the optimized
conditions in 98% yield and 92-95% enantiomeric excess.
The enantiomeric excess was determined on a chiral column
with racemic 5 as the standard. The resulting secondary
alcohol 5 was protected as its TBS ether, followed by
selective deprotection of the primary TBS group using HF
in the presence of glass splinters.32,33 The primary alcohol
was then oxidized with the Dess-Martin periodinane
and provided known aldehyde 6.34 We then followed the
Nicolaou group protocol for the synthesis of intermedi-
ate 7.35,36
The key step for the synthesis of fragment A was the
asymmetric Noyori reduction27 of β-ketoester 9 as shown
in Scheme 3. The anion of methyl isobutyrate was acylated
with 3-(benzyloxy)propanoyl chloride to furnish the β-keto
ester 9. The best results for the asymmetric Noyori reduc-
tion37,38 were obtained when the catalyst was generated in
situ from bis(2-methylallyl)cycloocta-1,5-diene ruthenium
and (S)-BINAP in deoxygenated acetone in the presence of
HBr. High enantiomeric excess (>95% ee) was consistently
obtained through hydrogenation with low catalyst loading
(1.4 mol %) in deoxygenated methanol; however, due to the
hindered nature of the β-ketoester the conversion was in-
complete. The optimized hydrogenation conditions for the
hindered β-ketoester required heating at 60 °C at 60 psi
for 110 h to maximize the yield. Following chromatography,
(35) Nicolaou, K. C.; Hepworth, D.; King, N. P.; Finlay, M. R. V.;
Scarpelli, R.; Pereira, M. M. A.; Bollbuck, B.; Bigot, A.; Werschkun, B.;
Winssinger, N. Chem.;Eur. J. 2000, 6, 2783–2800.
(32) Pilcher, A. S.; DeShong, P. J. Org. Chem. 1993, 58, 5130–5134.
(33) Schinzer, D.; Bauer, A.; Bohm, O. M.; Limberg, A.; Cordes, M.
Chem.;Eur. J. 1999, 5, 2483–2491.
(34) Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.;
Vallberg, H.; Finlay, M. R. V.; Yang, Z. J. Am. Chem. Soc. 1997, 119, 7974–
7991.
(36) Nicolaou, K. C.; Hepworth, D.; Finlay, M. R. V.; Paul King, N.;
Werschkun, B.; Bigot, A. Chem. Commun. 1999, 519–520.
(37) Genet, J. P.; Ratovelomanana-Vidal, V.; Cano de Andrade, M. C.;
Pfister, X.; Guerreiro, P.; Lenoir, J. Y. Tetrahedron Lett. 1995, 36, 4801–
4804.
(38) Taber, D. F.; Silverberg, L. J. Tetrahedron Lett. 1991, 32, 4227–4230.
88 J. Org. Chem. Vol. 75, No. 1, 2010

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SCHEME 4
TABLE 1. Cytotoxicity and Tubulin Assembly ED50 Valuesa
ED50, nM ED50, μM
ED50, nM (MCF-7) (HCT-116) (tubulin assembly)
compd
14
15
16
>200
>200
>1000
400
500
>1000
2.0
2.5
>20
2.0
epothilone B
1.0
1.8
aAssays were carried out as described in ref 41.
β-hydroxyester 10 was obtained in 92% yield and 95%
enantiomeric excess as determined by chiral HPLC. β-Hydro-
xyester 10 was subsequently debenzylated via catalytic hydro-
genation with 10% Pd/C and the resulting diol was protected
as the bis-TBS ether. Conversion of the ester to the correspond-
ing ethylphenyl sulfone39 followed by reductive cleavage of the
sulfone provided the C1-C6 ketone 11.34,40 This synthesis
constitutes one of the shortest ways to access the C1-C6
fragment. Another advantage of this method is low catalyst
loading and the fact that the catalyst can be recycled.
The subsequent chemistry shown in Scheme 3 for the
synthesis of 26-hydroxyepothilone D follows the work by
Nicolaou and collaborators.24,25 The primary alcohol in 13
was acylated with 3-azidobenzoic acid, using the peptide
coupling reagent O-(benzotriazol-1-yl)-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate (HBTU) in the pre-
sence of diisopropylethylamine (DIPEA) (Scheme 4). The
TBS groups were then removed with TFA to provide the
desired 3-azido photoaffinity analogue 14 of C26 hydroxy-
epothilone D. This process was repeated to generate the
4-azido photoaffinity analogue 15, and the benzophenone
analogue 16 (Scheme 4).
The 3- and 4-azido analogues and the benzophenone
analogues of 14-16 were tested for tubulin assembly pro-
moting activity and cytotoxicity against the MCF-7 breast
cancer and the HCT-116 colon cancer cell lines (Table 1).
The azido compounds were as active as epothilone B in the
tubulin assembly assay, and demonstrated cytotoxicity
against both cell lines but were significantly less active than
epothilone B. This may be due to differences in transport
across the cell membrane. The benzophenone analogue did
not promote tubulin assembly, nor did it show cytotoxicity in
either cancer cell line. Photolabeling studies were carried out
with the 3-azido analogue with bovine brain tubulin polym-
erized in the presence of the analogue. After UV irradiation
and subsequent examination of tryptic digests of the protein
for adducts by LC-MS and LC-MS/MS studies no cova-
lently labeled peptide fragments could be identified, suggest-
ing that the phenylazido side chain was predominantly
solvent-exposed in the bound conformation.
FIGURE 2. Docked configurations (Surflex-Dock, Tripos, Inc.)
of epothilone B (a), compound 14 (b), compound 15 (c), and
compound 16 (d) shown with MOLCAD (Tripos, Inc.) electron
density surfaces of the tubulin active site (1TVK1), onto which
hydrogen bond donor and acceptor regions have been mapped.
Red areas represent hydrogen bond donors; blue areas represent
hydrogen bond acceptors; and gray indicates regions in which no
hydrogen bonding takes place. Key ligand-receptor interactions
are shown in these pictures. In the docked conformation of
epothilone B (a), hydrogen bonding occurs between the thiazole
nitrogen and the imidazole NH of His227, between the C1
carbonyl and two amino groups in Arg276, between the C3
carbonyl and backbone NH of Thr274, and between C5 OH
and one amino group in Arg282. In the docked conformation of
14 (b), hydrogen bonding occurs between the thiazole nitrogen
and the imidazole NH of His227, between the C1 carbonyl and
two amino groups in Arg276, between the C7 OH and backbone
carbonyl of Pro272, and between C7 OH and backbone NH of
Thr274. In the docked configuration of 15 (c), hydrogen bonding
occurs between the thiazole nitrogen and one of the amino groups
of Arg276, between the C3 OH and the backbone carbonyl of
Thr274, between the C5 carbonyl and backbone NH of Thr274,
between the C7 OH and the two amino groups in Arg282, and
between the C7 OH and backbone carbonyl of Pro272. Overall,
the hydrogen bonding networks between receptor and ligand are
preserved in panels b and c. However, all of these interactions
disappear in the docked conformation of 16 (d) due to sterically
driven ligand rearrangement. As shown, the molecule rearranges
to avoid steric clashes between the receptor and the bulky group
on C12.
Docking and scoring studies were conducted to probe
tubulin binding modes and ligand-receptor interactions
(39) Paquette, L. A.; Galemmo, R. A. Jr.; Caille, J. C.; Valpey, R. S.
J. Org. Chem. 1986, 51, 686–695.
(40) Mulzer, J.; Mantoulidis, A.; Oehler, E. J. Org. Chem. 2000, 65, 7456–
7467.
(41) Liu, Y.; Ali, S. M.; Boge, T. C.; Georg, G. I.; Victory, S.; Zygmunt, J.;
Marquez, R. T.; Himes, R. H. Comb. Chem. High Throughput Screening
2002, 5, 39–48.
J. Org. Chem. Vol. 75, No. 1, 2010 89

Reiff et al.
JOCArticle
for epothilone B as well as for compounds 14, 15, and 16.
Docking validation was carried out by comparing the
docked configuration of epothilone A to the experimental
(E)-5-(tert-Butyldimethylsilyloxy)-2-methyl-1-(2-methylthiazol-
4-yl)pent-1-en-3-one (4). Barium hydroxide octahydrate (12.7 g,
40.2 mmol, 0.800 equiv) was added to β-ketophosphonate 3
(17.7 g, 50.2 mmol, 1.00 equiv) in THF (143 mL) and the
solution was stirred at room temperature until it dissolved.
The mixture was then cooled to 0 °C and aldehyde 229 (6.40 g,
50.2 mmol, 1.00 equiv) dissolved in THF (143 mL) and water
(3.27 mL) was added dropwise. The reaction mixture was stirred
at 0 °C for 2 h. The mixture was quenched with saturated
aqueous sodium bicarbonate and passed through Celite. After
the addition of brine, the aqueous layer was extracted five times
with ethyl acetate and the combined organics were dried over
sodium sulfate and concentrated. Flash column chromatogra-
phy of the crude material on silica gel with a gradient of 20-30%
˚
configuration (rmsd=1.575 A). The initial conformations of
epothilone B and compounds 14, 15, and 16 were prepared
by modifying the experimental (X-ray) bound configuration
of epothilone A,17 and optimizing the resulting geometries by
using the MMFF94s force field in MOE (Chemical Compu-
ting Group, Inc.). Figure 2a shows the docked configuration
of epothilone B in the tubulin binding site; panels b and c in
Figure 2 show the docked configurations of compounds 14
and 15, respectively. More details on the docking and scoring
protocols are provided in the Experimental Section. Com-
paring all three predicted bound configurations illustrates
that epothilone B, 14, and 15 all exhibit similar key hydro-
gen-bonding interactions with the tubulin active site. The
relatively minor rearrangements of azido compounds 14 and
15 in the active site with respect to epothilone B do not
disrupt these interactions to any significant extent, hence
their similar activities in the tubulin assembly assay.
Although hydrogen bonding between the thiazole ring and
His227 is not predicted to occur in compound 15, the dock-
ing suggests that the loss of this hydrogen bond is not
detrimental to activity as long as the other key interactions
are preserved. However, in the case of compound 16
(Figure 2d), van der Waals clashes between the receptor
and the benzophenone moiety lead to sterically driven ligand
rearrangement that disrupts the entire hydrogen-bonding
network and consequently destroys tubulin assembly pro-
moting activity for this compound.
1
ether in hexanes gave 11.15 g of the enone 4 (68%): H NMR
(400 MHz, CDCl3) δ 7.56 (s, 1 H), 7.36 (s, 1 H), 4.00 (t, J=6.7
Hz, 2 H), 3.05 (t, J=6.7 Hz, 2 H), 2.78 (s, 3 H), 2.25 (s, 3 H), 0.88
(s, 9 H), 0.07 (s, 6 H); 13C NMR (100 MHz, CDCl3) δ 201.5,
165.8, 152.3, 138.0, 131.9, 121.7, 60.1, 41.1, 26.3, 19.7, 18.7, 13.7;
IR (neat) 3100, 2960, 2940, 2860, 1665, 1625, 1468 cm-1; MS
(þFAB) 326 (M þ Hþ); HRMS m/e calcd for C16H28NO2SSi (M
þ Hþ) 326.1610, found 326.1625.
(S,E)-5-(tert-Butyldimethylsilyloxy)-2-methyl-1-(2-methylthia-
zol-4-yl)pent-1-en-3-ol (5)40. (R)-Me-CBS-oxazaborolidine (16.6
mL, 16.6 mmol, 1.0 M solution in toluene, 0.500 equiv) was
placed in a 500 mL flask and the toluene was removed in vacuo.
The residual solid was dissolved in CH2Cl2 (76 mL) and cooled to
0 °C. Borane dimethylsulfide complex (50.0 mL, 49.8 mmol, 1.0
M in CH2Cl2, 1.50 equiv) was added dropwise and this mixture
was stirred for 1 h. Enone 4 (11.0 g, 33.2 mmol, 1.00 equiv)
dissolved in CH2Cl2 (65 mL) was added dropwise via syringe
pump over a period of 15 h, followed by a CH2Cl2 rinse (20 mL)
over 4 h. The reaction was allowed to warm to room temperature
over 4 h, and methanol (20 mL) was carefully added followed by
ethanolamine (20 mL). Twelve hours later additional ethanol-
amine (20 mL) was added. The reaction mixture was stirred for
another 24 h at room temperature and then quenched with
saturated aqueous ammonium chloride. The organic and aqu-
eous layers were separated and the aqueous layer was extracted
three times with CH2Cl2. The organic layer was subsequently
washedwithbrine. The combined aqueous layer was washedonce
with CH2Cl2. The combined organic layer was then dried over
sodium sulfate and concentrated. The crude material was then
purified by column chromatography on silica gel, using a
20-30% ethyl acetate in hexanes gradient as eluent. The desired
alcohol was obtained in 98% yield (10.8 g) and in 93% enantio-
meric excess. The enantiomeric excess was determined by chiral
HPLC on a Chiracel OD-H column at 254 nm, using 90:10
hexanes/isopropanol at 0.8 mL/min as the mobile phase. The
retention times (major: 5.5 min; minor: 7.5 min) were compared
to the racemic alcohol for verification: 1H NMR (400 MHz,
CDCl3) δ 6.95 (s, 1 H), 6.63 (s, 1 H), 4.42-4.38 (m, 1 H),
3.95-3.86 (m, 2 H), 3.55 (d, J=2.7 Hz, 1 H), 2.73 (s, 3 H), 2.07
(s, 3 H), 1.85(q, J=5.7Hz, 2 H), 0.93(s, 9 H), 0.11 (s, 3 H), 0.10 (s,
3 H); 13C NMR (100 MHz, CDCl3) δ 164.8, 153.6, 142.1, 118.6,
115.8, 62.7, 37.4, 26.2, 19.7, 18.6, 15.3, -5.1; IR(neat) 3400-3350
(b), 2950, 2940, 2850, 1510, 1470, 1256 cm-1; MS (þFAB) 328 (M
þ Hþ); HRMS m/e calcd for C16H30NO2SSi (M þ Hþ) 328.1767,
Experimental Section
Diethyl 5-(tert-Butyldimethylsilyloxy)-3-oxopentan-2-ylphos-
phonate (3). Diethyl ethanephosphonate (38.6 g, 232 mmol,
˚
3.20 equiv) was placed in THF (150 mL) along with 4 A
molecular sieves. The reaction temperature was lowered to
-78 °C and n-BuLi (160 mL, 225 mmol, 3.10 equiv) was added
dropwise over 2 h via a dropping funnel. The reaction mixture
was stirred at -78 °C for an additional hour and then methyl
3-(tert-butyldimethylsilyloxy)propanoate (prepared by silyla-
tion42 of commercially available methyl 3-hydroxypropanoate)
was added dropwise via cannula. The reaction was stirred at
-78 °C for 2 h and then quenched with methanol (10 mL).
Saturated aqueous NH4Cl (80 mL) was added and the mixture
was allowed to warm to room temperature. The organic layer
was decanted and concentrated. The aqueous layer was filtered
to remove the molecular sieves. Brine was added and the
aqueous layer was washed four times with EtOAc. The organic
layer was dried over Na2SO4 and concentrated. Three columns
on silica gel with a gradient of 10-25% ethyl acetate in hexanes
provided 17.9 g of the β-ketophosphonate (70% yield). This was
necessary as the product ran together with the starting phos-
phonate and the mixed fractions were subjected to further
column chromatography. Distillation was attempted on a small
scale, but resulted in decomposition: 1H NMR (400 MHz,
CDCl3) δ 4.17-4.12 (m, 4 H), 3.93-3.90 (t, J=6.3 Hz, 2 H),
3.40-3.25 (dq, J = 7.1, 7.1 Hz, 1 H), 2.94-2.84 (m, 2 H),
1.38-1.33 (m, 6 H), 0.94 (s, 3 H), 0.89 (s, 9 H), 0.06 (s, 6 H);
13C NMR (100 MHz, CDCl3) δ 205.2, 62.8, 59.2, 48.1, 46.9,
46.3, 26.1, 18.4, 16.6, -5.2; IR (neat) 2960, 2925, 2850, 1711,
1480, 1396 cm-1; MS (CI) 353 (M þ Hþ); HRMS m/e calcd for
C15H34O5PSi (M þ Hþ) 353.1905, found 353.1911.
found 328.1789; [R]20 -7.2 (c 0.76, CHCl3).
D
(()-(E)-5-(tert-Butyldimethylsilyloxy)-2-methyl-1-(2-methyl-
thiazol-4-yl)pent-1-en-3-ol ((()-5). The enone 4 (10 mg, 0.031
mmol, 1.0 equiv) was dissolved in CH2Cl2 (0.5 mL) and the
temperature was lowered to -78 °C. DIBAL-H (0.062 mL,
0.062 mmol, 1.0 M, 2.0 equiv) was added dropwise and the
reaction was stirred for 1.25 h. The reaction was quenched with
methanol (0.5 mL) and additional CH2Cl2 (5.0 mL) and satu-
rated potassium sodium tartrate solution (5.0 mL) were
added. The mixture was stirred for 2 h and then extracted four
(42) Li, L.-S.; Wu, Y.-L. Tetrahedron Lett. 2002, 43, 2427–2430.
90 J. Org. Chem. Vol. 75, No. 1, 2010

Reiff et al.
JOCArticle
times with CH2Cl2. The combined organic layer was dried
over Na2SO4 and concentrated. Purification by preparative
chromatography on silica gel with 70:30 ethyl acetate/hexanes
gave 7 mg (70% yield) of the racemic material, which was used as
an HPLC standard to monitor the enantiomeric excess of the
CBS reduction: 1H NMR (500 MHz, CDCl3) δ 6.96 (s, 1 H), 6.64
(s, 1 H), 4.42-4.39 (m, 1 H), 3.93-3.86 (m, 2 H), 3.77 (t, J=6.6
Hz, 1 H), 2.74 (s, 3 H), 2.08 (s, 3 H), 1.89-1.87 (m, 2 H), 0.93 (s, 9
H), 0.11 (s, 6 H); 13C NMR (125 MHz, CDCl3) δ 164.8, 153.6,
142.1, 118.6, 115.8, 62.7, 37.4, 26.2, 19.7, 18.6, 15.3, -5.1; IR
(neat) 3400-3350 (b), 2924, 2854, 1463, 1259, 1094 cm-1; MS
(þFAB) 328 (M þ Hþ); HRMS m/e calcd for C16H30NO2SSi
(M þ Hþ) 328.1767, found 328.1776.
concentrated and subjected to column chromatography with
30% ether in hexanes. This provided 6.5 g (98% yield) of the
desired aldehyde 6: 1H NMR (400 MHz, CDCl3) δ 9.81 (s, 1 H),
6.97 (s, 1 H), 6.59 (s, 1 H), 4.71 (dd, J=8.3, 4.0 Hz, 1 H), 2.75 (dd,
J=7.1, 3.0 Hz, 1 H), 2.74 (s, 3 H), 2.54 (dd, J=4.0, 2.0 Hz, 1 H),
2.07 (s, 3 H), 0.90 (s, 9 H), 0.11 (s, 3H), 0.06 (s, 3H); IR (neat)
2960, 2920, 2855, 2715, 1728, 1500, 1468, 1466 cm-1; MS (þFAB)
326 (M þ Hþ) HRMS m/e calcd for C16H28NO2SSi (M þ Hþ)
326.1610, found 326.1606; [R]20D -17 (c 3.0, CHCl3).
Methyl 5-(Benzyloxy)-2,2-dimethyl-3-oxopentanoate (9). To a
solution of isopropylcyclohexylamine (7.3 mL, 45 mmol 1.5
equiv) in THF (40 mL) at -30 °C was added dropwise n-
butyllithium (16.1 mL, 38.6 mmol, 1.3 equiv) and the mixture
was stirred for 30 min. The temperature was raised to 0 °C for 15
min and then cooled to -78 °C for 15 min. Methyl isobutyrate
(3.75 mL, 32.7 mmol, 1.10 equiv) dissolved in THF (5 mL) was
added dropwise to the reaction mixture. After the mixure was
stirred for 30 min, 3-(benzyloxy)propanoyl chloride (5.0 g, 29.7
mmol, 1 equiv) in THF (5 mL) was added dropwise. The
reaction mixture was stirred for an additional hour until the
disappearance of the starting material (TLC: 80:20 hexanes/
EtOAc). The reaction was quenched with HCl (20 mL, 20%)
and warmed to room temperature. The reaction mixture was
extracted with ether (3 times) and the combined organic phase
was washed twice with sodium bicarbonate and once with brine.
The combined aqueous layer was cross-extracted twice with
ether. The two combined organic layers were dried (Na2SO4)
and concentrated under reduced pressure. Flash column chro-
matography on silica gel (gradient hexanes/EtOAc) provided
5.1 g (65%) of the target compound. 1H NMR (400 MHz,
CDCl3) δ 7.38-7.32 (m, 5 H), 4.52 (s, 2 H), 3.74 (t, J=6 Hz, 2 H),
3.69 (s, 3 H), 2.76 (t, J=6 Hz, 2 H), 1.40 (s, 6 H); 13C NMR (100
MHz, CDCl3) δ 206.5, 174.4, 138.6, 128.8, 128.0, 127.2, 73.7,
65.8, 52.9, 38.8, 22.2; IR (neat) 2940, 2850, 1740, 1710, 1450,
1360, 1145, 1100 cm-1; MS (CI) 282 (M þ NH4þ); HRMS m/e
calcd for C15H21O4 (M þ Hþ) 265.1423, found 265.1155.
Methyl (3S)-5-(Benzyloxy)-2,2-dimethyl-3-hydroxypentano-
ate (10). Acetone and MeOH were degassed five times with
the freeze-thaw method and placed under argon. Bis(2-
methylallyl)cycloocta-1,5-diene ruthenium(II) complex (91
mg, 0.28 mmol, 1.0 equiv) and (S)-BINAP (177 mg, 0.284 mmol,
1.00 equiv) were combined in a Schlenk flask with acetone (24
mL) and HBr solution [2.0 mL, (0.25 mL 48% HBr, 5.1 mL
acetone)]. The mixture was stirred for 1.5 h and the acetone was
removed under reduced pressure. A solution of β-ketoester 9
(5.36 g, 22.7 mmol, 71.0 equiv) in MeOH (22.8 mL) was
degassed four times and transferred to a Parr hydrogenation
flask. The catalyst was then added to the Parr flask with MeOH.
The reaction mixture was hydrogenated for 110 h at 60 psi and
60 °C. The resulting suspension was concentrated under reduced
pressure and then taken up in ether. The reaction mixture was
filtered twice to remove the catalyst and concentrated. Final
purification by column chromatography (silica gel, 5-15%
EtOAc in hexane, 1% MeOH, 1% TEA) provided 4.57 g
(85% yield) of β-hydroxy ester 10 in 95% enantiomeric excess.
The enantiomeric excess was determined by chiral HPLC on a
Chiracel OD-H column at 254 nm, using 99:1 hexanes/isopro-
panol at 0.8 mL/min; retention times (major: 19.5 min; minor:
22.3 min) were compared to the racemic alcohol for verification.
1H NMR (400 MHz, CDCl3) δ 7.29-7.21 (m, 5 H), 4.56 (d, J=
11.9 Hz, 1 H), 4.53 (d, J=11.8 Hz, 1 H), 3.85 (m, 1 H), 3.68-3.58
(m, 2 H), 3.62 (s, 3 H), 3.11 (d, J=4.0 Hz, 1 H), 1.65-1.61 (m, 2
H), 1.13 (s, 3 H), 1.11 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ
178.2, 138.4, 128.8, 128.1, 76.0, 73.8, 69.7, 52.3, 47.4, 31.8, 22.1,
20.8; IR (neat) 3500, 2950, 2860, 1721, 1468, 1450, 1431, 1385,
1365, 1265, 1190, 1135, 1090 cm-1; MS (CI) 267 (M þ Hþ);
HRMS m/e calcd for C15H23O4 (M þ Hþ) 267.1596, found
267.1617; [R]20D -2.8 (c 2.0, CHCl3).
(S,E)-4-(3,5-Bis(tert-butyldimethylsilyloxy)-2-methylpent-
1-enyl)-2-methylthiazole (5a).33 Alcohol 5 (10.8 g, 33.0 mmol,
1.00 equiv) dissolved in CH2Cl2 (200 mL) was cooled to 0 °C and
2,6-lutidine (8.20 mL, 70.3 mmol, 2.13 equiv) was added fol-
lowed by TBSOTf (11.8 mL, 51.2 mmol, 1.55 equiv). The
temperature was gradually warmed to room temperature and
after stirring for 1.5 h the reaction mixture was quenched with
saturated aqueous ammonium chloride, followed by washing
with saturated NaHCO3 and brine. The aqueous layer was
extracted once with dichloromethane. The combined organics
were dried over sodium sulfate and concentrated. Flash column
chromatography of the crude with silica gel and 95:5 hexanes/
EtOAc as eluent provided 13.70 g (94% yield) of the bis-silyl
ether of 5: 1H NMR (400 MHz, CDCl3) δ 6.92 (s, 1 H), 6.49 (s, 1
H), 4.35-4.32 (dd, J=8.0, 4.6 Hz, 1 H), 3.69-3.64 (m, 2 H), 2.71
(s, 3 H), 2.00 (s, 3 H), 1.78-1.73 (m, 2 H), 0.91 (s, 18 H), 0.08 (s, 3
H), 0.07 (s, 3 H), 0.05 (s, 3 H), 0.03 (s, 3 H); 13C NMR (100 MHz,
CDCl3) δ 164.8, 153.6, 143.0, 119.0, 115.4, 75.5, 60.1, 40.3, 26.3,
26.3, 26.1, 19.6, 18.6, 14.2, -4.2, -4.7, -4.9, -4.9; IR (neat)
2952, 2940, 2855, 1470, 1380, 1360, 1256 cm-1; MS (þFAB) 441
(M þ Hþ); HRMS m/e calcd for C22H43NO2SSi2 (M þ Hþ)
441.2553, found 441.2525; [R]20 -4.9 (c 0.88, CHCl3).
D
(S,E)-3-(tert-Butyldimethylsilyloxy)-4-methyl-5-(2-methylthi-
azol-4-yl)pent-4-en-1-ol (5b).33 (S,E)-4-(3,5-bis(tert-butyldime-
thylsilyloxy)-2-methylpent-1-enyl)-2-methylthiazole (13.16 g,
29.79 mmol) was placed in a 500 mL plastic bottle with aceto-
nitrile (120 mL) and ether (120 mL) and the temperature was
lowered to 0 °C. Glass splinters (133 mg, catalytic) were added
followed by 40% aqueous HF (20 mL). The reaction was stirred
at 0 °C for 2 h and 40% aqueous HF (20 mL) was again added.
After the mixture was stirred for 1 h, solid sodium bicarbonate
(84.0 g) was added over 15 min. The mixture was stirred for
30 min and water was then added to dissolve the solids (800 mL).
Brine was also added (100 mL). The mixture was extracted four
times with CH2Cl2, and the organic layer was again washed with
brine. The organic layer was dried over sodium sulfate and
concentrated. Column chromatography on silica gel with a
gradient of 20-50% ether in hexanes provided 8.0 g (82% yield)
of the primary alcohol (S,E)-3-(tert-butyldimethylsilyloxy)-4-
methyl-5-(2-methylthiazol-4-yl)pent-4-en-1-ol: 1H NMR (400
MHz, CDCl3) δ 6.93 (s, 1 H), 6.52 (s, 1 H), 4.42-4.39 (dd, J=
7.3, 4.6 Hz, 1 H), 3.80-3.72 (m, 2H), 2.72 (s, 3 H), 2.43 (m, 1 H),
2.04 (s, 3 H), 1.91-1.80 (m, 2 H), 0.93 (s, 9 H), 0.12 (s, 3 H), 0.05
(s, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.9, 165.0, 153.4,
142.0, 119.2, 115.8, 109.5, 60.8, 39.0, 26.2 (3 C), 19.6, 18.5, 14.8,
-4.2, -4.8; IR (neat) 3400-3350 (b), 2960, 2940, 2860, 1500,
1475, 1256, 1082 cm-1; MS (þFAB) 328 (M þ Hþ); HRMS m/e
calcd for C16H30NO2SSi (M þ Hþ) 328.1767, found 328.1738;
[R]20 -33 (c 2.5, CHCl3).
D
(S,E)-3-(tert-Butyldimethylsilyloxy)-4-methyl-5-(2-methylthi-
azol-4-yl)pent-4-enal (6)34. (S,E)-3-(tert-Butyldimethylsilyloxy)-
4-methyl-5-(2-methylthiazol-4-yl)pent-4-en-1-ol (6.70 g, 20.5
mmol, 1.00 equiv) was dissolved in CH2Cl2 and the Dess-Martin
periodinane (11.3 g, 26.6 mmol, 1.30 equiv) was added. After
being stirred at roomtemperaturefor 6 h the reactionmixturewas
J. Org. Chem. Vol. 75, No. 1, 2010 91

Reiff et al.
JOCArticle
Methyl (3S)-3,5-Di[(tert-butyldimethylsilyl)oxy]-2,2-dimethyl-
pentanoate (10a)40. β-Hydroxy ester 10 (6.90 g, 1.00 equiv) was
dissolved in THF (75 mL) and transferred to a Parr hydrogena-
tion vessel under argon. Then 10% Pd/C (1.73 g, 0.250 equiv)
was added and the flask was purged for an additional 10 min
with argon. The hydrogenation reaction was carried out at
58-52 psi for 22 h. The resulting mixture was filtered and the
residue washed with EtOAc (300 mL). The filtrate was concen-
trated under reduced pressure to provide 4.50 g (99% yield) of
the crude diol, which was carried on to the next step without
further purification. The diol (4.50 g, 25.6 mmol, 1.00 equiv) was
dissolved in CH2Cl2 (85 mL) and the temperature was lowered
to 0 °C. 2,6-Lutidine (14.9 mL, 128 mmol, 5.00 equiv) was added
followed by TBSOTf (17.6 mL, 76.7 mmol, 3.00 equiv). The
reaction mixture was gradually warmed to room temperature
and stirred overnight. The reaction was quenched with ammo-
nium chloride (25 mL). The reaction mixture was extracted four
times with CH2Cl2 and the combined organic layer was washed
with brine, dried over Na2SO4, and concentrated. Purification
via column chromatography on silica gel with a gradient of
0-5% EtOAc in hexanes provided 9.27 g (90% yield) of 10a: 1H
NMR (400 MHz, CDCl3) δ 4.07 (dd, J = 2.3, 2.2 Hz, 1 H),
3.72-3.59 (m, 2 H), 3.67 (s, 3 H), 1.66-1.55 (m, 2 H), 1.18 (s,
3 H), 1.11 (s, 3 H), 0.90 (s, 9 H), 0.89 (s, 9 H), 0.10 (s, 3 H), 0.06 (s,
6 H), 0.05 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 178.0, 73.8,
60.7, 52.1, 48.7, 37.3, 26.4, 26.3, 22.1, 20.7, 18.7, -3.5, -3.9,
-4.9; IR (neat) 2940, 2914, 2870, 2840, 1721, 1458, 1422, 1379,
1351, 1242, 1180, 1122, 1087 cm-1; MS (CI) 405 (M þ Hþ);
HRMS m/e calcd for C20H45O4Si2 (M þ Hþ) 405.2856, found
405.2850; [R]20D -5.6 (c 3.0, CHCl3).
(5S)-5,7-Di[(tert-butyldimethylsilyl)oxy]-4,4-dimethylheptan-
3-one (11)40. Ethylphenyl sulfone (3.4 g, 20 mmol, 5.5 equiv) was
dissolved in THF (50 mL) and the temperature was lowered to
-78 °C. n-BuLi (13 mL, 18 mmol, 5.0 equiv) was added
dropwise and the pale yellow solution that formed was stirred
at -78 °C for 1.5 h. The ester 10a in THF (15 mL) was added
dropwise at -78 °C. The bath was removed and the reaction was
stirred at room temperature for 20 h. The reaction was quenched
with 1:1 saturated NH4Cl and water (30 mL) and the aqueous
layer was extracted five times with Et2O. The organic layer was
dried over Na2SO4 and concentrated. The crude was then
dissolved in MeOH (50 mL) and then temperature was lowered
to 0 °C. NaH2PO4 (2.00 g, 14.4 mmol, 4.00 equiv) was added
followed by Na(Hg) (4.39 g). The pink colored suspension,
which developed, was stirred at 0 °C for 1 h and 15 min, and
1:1 saturated NH4Cl and water was added followed by dilution
with Et2O. The mercury metal was filtered with a funnel and a
plug of glass wool. The filtrate was extracted five times with
Et2O. The combined organic layer was washed with H2O and
brine, dried over Na2SO4, and concentrated. Column chroma-
tography on silica gel with 5% Et2O in hexanes provided 1.3 g
(90%) of the ketone: 1H NMR (400 MHz, CDCl3) δ 4.08 (dd, J=
3.0, 3.0 Hz, 1 H), 3.66-3.63 (m, 2 H), 2.60-2.50 (m, 2 H),
1.56-1.49 (m, 2 H), 1.13 (s, 3 H), 1.07 (s, 3 H), 1.01 (t, J=7.1 Hz,
3 H), 0.91 (s, 9 H), 0.90 (s, 9 H), 0.11 (s, 3 H), 0.07 (s, 3 H), 0.06 (s,
3 H), 0.05 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 216.0, 73.8,
60.5, 53.4, 37.7, 32.0, 26.5, 26.3, 22.6, 20.4, 18.7, 18.6, 8.1, -3.6,
-3.7, -4.9; IR (neat) 2939, 2910, 2840, 1698, 1459, 1380, 1355,
1248, 1087 cm-1; MS (CI) 403 (M þ Hþ); HRMS m/e calcd for
C21H47O3Si2 (M þ Hþ) 403.3064, found 403.3051; [R]20D -7.0 (c
1.8, CHCl3).
and the temperature was lowered to -20 °C. Formic acid (1.0 mL)
was added dropwise over 15 min. The reaction was stirred at
-10 °C for 10 min and then at -5 to 0 °C for 6 h. The reaction
was quenched with water (2 mL) and solid sodium bicarbonate
was added until the pH was neutral. The mixture was extracted
four times with ether. The organic layer was dried over MgSO4
and concentrated. Column chromatography on silica gel with
a gradient of 20-50% ether in hexanes gave 22 mg (60% yield)
of the monodeprotected product 13 that was fully characterized
and found to match the results obtained by the Nicolaou
group25 for this derivative: 1H NMR (500 MHz, CDCl3) δ
6.99 (s, 1 H), 6.60 (s, 1 H), 5.51 (dd, J=9.4, 6.8 Hz, 1 H), 5.05
(d, J=8.4 Hz, 1 H), 4.18-4.13 (m, 1 H), 4.06 (d, J=9.0 Hz, 1 H),
4.02 (d, J=13.0 Hz, 1 H), 3.92 (d, J=13.6 Hz, 1 H), 3.08-3.01
(m, 1 H), 2.80-2.78 (m, 1 H), 2.78-2.76 (m, 1 H), 2.74 (s, 3 H),
2.71 (dd, J=16.3, 10.1 Hz, 1 H), 2.47-2.41 (m 1 H), 2.23-2.18
(m, 1 H), 2.13 (s, 3 H), 2.03-1.97 (m, 1 H), 1.85-1.63 (m, 2 H),
1.62-1.50 (m, 3 H), 1.21 (s, 3 H), 1.16 (s, 3 H), 1.12 (d, J=6.8 Hz,
3 H), 0.99 (d, J=6.9 Hz, 3 H), 0.96 (s, 9 H), 0.86 (s, 9 H), 0.13 (s, 3
H), 0.12 (s, 3 H), 0.09 (s, 3 H), -0.01 (s, 3 H); 13C NMR (125
MHz, CDCl3) δ 215.1, 171.0, 164.6, 152.3, 143.8, 138.3, 128.5,
127.7, 120.3, 119.6, 116.0, 79.4, 76.0, 66.4, 64.3, 53.3, 48.1, 39.3,
37.3, 32.0, 31.3, 28.1, 27.3, 26.3 (3 C), 26.0 (3 C), 24.3, 19.1, 18.5
(2 C), 17.7, 15.1, -3.4, -3.7, -3.8, -5.7; IR (neat) 2929, 2856,
1742, 1695, 1463, 1381, 1255 cm-1; MS (þFAB) 736.4 (M þ
Hþ); HRMS m/e calcd for C39H70NO6SSi2 (M þ Hþ) 736.4462,
found 736.4463; [R]20D -22 (c 0.40, CHCl3).
((2S,9S,10S,11R,14S,E)-10,14-Dihydroxy-9,11,13,13-tetra-
methyl-2-((E)-1-(2-methylthiazol-4-yl)prop-1-en-2-yl)-12,16-di-
oxooxacyclohexadec-4-en-5-yl)methyl 3-Azidobenzoate (14).
Alcohol 13 (11.0 mg, 0.0150 mmol, 1.00 equiv) was placed in a
flask with m-azidobenzoic acid (10.0 mg, 0.0600 mmol, 4.00
equiv), HBTU (18.0 mg, 0.0450 mmol, 3.00 equiv), and CH3CN
(0.40 mL). The temperature was lowered to 0 °C and DIEA
(0.020 mL, 0.090 mmol, 6.0 equiv) was added. The reaction was
gradually warmed to room temperature and stirred overnight.
The reaction was quenched with saturated NaHCO3, extracted
three times with ether, dried over MgSO4, and concentrated.
Column chromatography on silica gel with a gradient of 5-20%
ether in hexanes gave 9 mg (69% yield) of the acylated product:
1H NMR (500 MHz, CDCl3) δ 7.84 (d, J=7.2 Hz, 1 H), 7.73 (s, 1
H), 7.49 (t, J=7.8 Hz, 1 H), 7.23 (d, J=8.0 Hz, 1 H), 7.00 (s, 1 H),
6.61 (s, 1 H), 5.64 (dd, J=12.1, 9.0 Hz, 1 H), 5.12 (d, J=9.5 Hz, 1
H), 4.87 (d, J=12.5 Hz, 1 H), 4.70 (d, J=12.5 Hz, 1 H), 4.10 (d,
J = 8.9 Hz, 1 H), 3.92 (d, J = 8.9 Hz, 1 H), 3.06 (m, 1 H),
2.84-2.79 (m, 2 H), 2.72-2.69 (m, 1 H), 2.74 (s, 3 H), 2.49-2.42
(m, 1 H), 2.31-2.22 (m, 1 H), 2.16 (s, 3 H), 2.10-2.08 (m, 1 H),
1.80-1.73 (m, 1 H), 1.70-1.54 (m, 1 H), 1.35-1.28 (m, 3 H),
1.22 (s, 3 H), 1.16 (s, 3 H), 1.13 (d, J=6.7 Hz, 3 H), 0.99 (d, J=6.8
Hz, 3 H), 0.97 (s, 9 H), 0.87 (s, 9 H), 0.13 (s, 3 H), 0.11 (s, 3 H),
0.07 (s, 3 H), -0.09 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ
215.1, 170.9, 165.0, 151.7, 140.5, 138.1, 132.0, 129.7 (2 C), 125.9
(2 C), 123.3, 120.0 (2 C), 119.8, 116.2, 78.1, 75.9, 68.2, 67.9, 53.3,
40.5, 38.6, 37.9, 32.0, 31.8, 30.2, 28.6, 26.3 (3 C), 26.0 (3 C), 24.4,
22.6, 19.1, 18.5 (2 C), 17.7, 15.1, 14.0, -3.6 (2 C), -3.8 (2 C); IR
(neat) 2958, 2925, 2854, 2111, 1728, 1462, 1259, 1096, 1020, 801
cm-1; MS (þFAB) 881.5 (M þ Hþ); HRMS m/e calcd for
C46H73N4O7SSi2 (M þ Hþ) 881.4739, found 881.4759; [R]20
D
-76 (c 0.075, CHCl3). The acylated product (4.0 mg, 0.0045
mmol, 1.0 equiv) was placed in a flask and the temperature was
lowered to -20 °C. A TFA solution (0.100 mL, 20% v/v TFA in
CH2Cl2) was added and the reaction was stirred at -20 °C for 6
h. The reaction was then quenched into saturated sodium
bicarbonate. The mixture was extracted three times with ethyl
acetate, dried over Na2SO4, and concentrated. Column chro-
matography on silica gel with a gradient of 0-1% CH3OH in
(4S,7R,8S,9S,16S,E)-4,8-Bis(tert-butyldimethylsilyloxy)-13-
(hydroxymethyl)-5,5,7,9-tetramethyl-16-((E)-1-(2-methylthi-
azol-4-yl)prop-1-en-2-yl)oxacyclohexadec-13-ene-2,6-dione (13)25.
(4S,7R,8S,9S,16S,E)-4,8-Bis(tert-butyldimethylsilyloxy)-5,5,7,
9-tetramethyl-16-((E)-1-(2-methylthiazol-4-yl)prop-1-en-2-yl)-
13-(trityloxymethyl)oxacyclohexadec-13-ene-2,6-dione25 (50.0
mg, 0.0512 mmol, 1.00 equiv) was placed in ether (1.0 mL)
1
CH2Cl2 gave 2.0 mg (68% yield) of the final product 14: H
NMR (500 MHz, CDCl3) δ 7.83-7.81 (m, 1 H), 7.70 (t, J=1.9
92 J. Org. Chem. Vol. 75, No. 1, 2010

Reiff et al.
JOCArticle
Hz, 1 H), 7.45 (t, J=7.9 Hz, 1 H), 7.22 (dd, J=2.3, 2.3 Hz, 1 H),
6.94 (s, 1 H), 6.61 (s, 1 H), 5.60 (dd, J=9.4, 5.6 Hz, 1 H), 5.31 (d,
J=9.1 Hz, 1 H), 4.84 (d, J=12.6 Hz, 1 H), 4.71 (d, J=12.7 Hz,
1 H), 4.34 (d, J=9.8 Hz, 1 H), 3.72-3.71 (m, 1 H), 3.63 (d, J=
5.5 Hz, 1 H), 3.19 (dq, J=6.9, 2.2 Hz, 1 H), 3.00 (br s, 1 H),
2.74-2.68 (m, 1 H), 2.71 (s, 3 H), 2.52-2.47 (m, 1 H), 2.46-2.40
(m, 1 H), 2.38-2.31 (m, 1 H), 2.29 (dd, J=14.5, 2.6 Hz, 1 H),
2.20-2.16 (m, 1 H), 2.09 (s, 3 H), 1.78-1.76 (m, 2 H), 1.42-1.38
(m, 3 H), 1.38 (s, 3 H), 1.20 (d, J=6.8 Hz, 3 H), 1.09 (s, 3 H), 1.03
(d, J=7.0 Hz, 1 H); 13C NMR (125 MHz, CDCl3) δ 220.5, 170.2,
165.3, 165.0, 151.7, 140.5, 138.5, 136.7, 131.9, 129.8, 125.9,
125.2, 119.9, 119.3, 115.6, 77.9, 73.9, 72.0, 68.3, 53.6, 41.5,
39.6, 37.9, 32.0, 31.5, 30.0, 28.4, 25.2, 22.8, 19.0, 17.6, 15.9,
15.7, 13.2; IR (neat) 2160, 1705, 1695, 1325 cm-1; MS (þFAB)
653.4 (M þ Hþ); HRMS m/e calcd for C34H45N4O7S (M þ Hþ)
653.3009, found 653.2984; [R]20D -42 (c 0.15, CHCl3).
δ 221.1, 170.7, 165.8, 165.5, 152.2, 141.0, 139.0, 137.1, 132.3,
130.3, 126.4, 125.7, 120.4, 119.7, 116.1, 78.4, 74.4, 72.5, 68.8,
54.1, 42.0, 40.0, 38.4, 32.4, 32.0, 30.1, 28.8, 25.7, 23.3, 19.4, 18.0,
16.5, 16.1, 13.6; IR (neat) 2950, 2140, 1730, 1305, 1265 cm-1; MS
(þFAB) 653.4 (M þ Hþ); HRMS m/e calcd for C34H45N4O7S (M
þ Hþ) 653.3009, found 653.3017; [R]20D -60 (c 0.10, CHCl3).
((2S,9S,10S,11R,14S,E)-10,14-Dihydroxy-9,11,13,13-tetra-
methyl-2-((E)-1-(2-methylthiazol-4-yl)prop-1-en-2-yl)-12,16-dio-
xooxacyclohexadec-4-en-5-yl)methyl 4-Benzoylbenzoate (16).
Alcohol 13 (12.0 mg, 0.0163 mmol, 1.00 equiv) was placed in a
flask with benzoylbenzoic acid (15.0 mg, 0.0653 mmol, 4.00
equiv), HBTU (18 mg, 0.049 mmol, 3.0 equiv), and CH3CN
(0.20 mL). The temperature was lowered to 0 °C and DIEA
(0.020 mL, 0.098 mmol, 6.0 equiv) was added. The reaction was
gradually warmed to room temperature and stirred overnight.
The reaction was quenched with saturated NaHCO3, extracted
three times with ethyl acetate, dried over Na2SO4, and concen-
trated. Column chromatography on silica gel with a gradient of
0-50% ether in hexanes gave 8 mg (53% yield) of the acylated
product: 1H NMR (500 MHz, CDCl3) δ 8.18 (d, J=8.3 Hz, 2 H),
7.84 (dd, J=16.1, 8.3 Hz, 4 H), 7.64 (t, J=7.4 Hz, 1 H), 7.53 (t,
J=7.4 Hz, 2 H), 7.00 (s, 1 H), 6.61 (s, 1 H), 5.67 (dd, J=9.4, 6.6
Hz, 1 H), 5.12 (d, J=9.3 Hz, 1 H), 4.90 (d, J=12.5 Hz, 1 H), 4.74
(d, J=12.6 Hz, 1 H), 4.09 (d, J=8.5 Hz, 1 H), 3.93 (d, J=8.9 Hz,
1 H), 3.07 (m, 1 H), 2.80-2.74 (2 H), 2.74 (s, 3 H), 2.70-2.68 (m,
1 H), 2.56-2.49 (m, 1 H), 2.20-2.12 (m, 1 H), 2.20 (s, 3 H),
2.10-2.03 (m, 1 H), 1.82-1.76 (m, 2 H), 1.38-1.18 (m, 3 H),
1.22 (s, 3 H), 1.17 (s, 3 H), 1.13 (d, J=6.8 Hz, 3 H), 1.10 (d, J=6.9
Hz, 3 H), 0.97 (s, 9 H), 0.87 (s, 9 H), 0.14 (s, 3 H), 0.13 (s, 3 H),
0.10 (s, 3 H), -0.08 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ
215.1, 195.9, 170.9, 165.5, 164.6, 152.3, 141.2, 138.5, 136.9,
136.7, 133.3, 132.9, 130.0 (2 C), 129.7 (2 C), 129.5 (2 C), 128.4
(2 C), 119.8 (2 C), 116.2, 79.1, 75.8, 68.3, 67.9, 53.3, 39.5, 37.9,
32.0, 31.8, 30.9, 30.5, 27.5, 26.9, 26.3 (3 C), 26.0 (3 C), 24.4, 22.6,
19.1, 18.6, 18.5, 15.1, 14.0, -3.3, -3.6, -3.8, -5.6; IR (neat)
((2S,9S,10S,11R,14S,E)-10,14-Dihydroxy-9,11,13,13-tetra-
methyl-2-((E)-1-(2-methylthiazol-4-yl)prop-1-en-2-yl)-12,16-
dioxooxacyclohexadec-4-en-5-yl)methyl 4-Azidobenzoate (15).
Alcohol 13 (12.0 mg, 0.0160 mmol, 1.00 equiv) was placed in a
flask with p-azidobenzoic acid (11.0 mg, 0.0650 mmol, 4.00
equiv), HBTU (18.0 mg, 0.0490 mmol, 3.00 equiv), and CH3CN
(0.30 mL). The temperature was lowered to 0 °C and DIEA
(0.019 mL, 0.098 mmol, 6.0 equiv) was added. The reaction was
gradually warmed to room temperature and stirred overnight.
The reaction was quenched with saturated NaHCO3, extracted
three times with ethyl acetate, dried over Na2SO4, and concen-
trated. Column chromatography on silica gel with a gradient of
10-50% ether in hexanes gave 8.5 mg (65% yield) of the
1
acylated product: H NMR (500 MHz, CDCl3) δ 7.84 (d, J=
7.5 Hz, 1 H), 7.72 (s, 1 H), 7.45 (t, J=7.8 Hz, 1 H), 7.23 (d, J=8.6
Hz, 1 H), 7.00 (s, 1 H), 6.60 (s, 1 H), 5.63 (dd, J=12.1, 9.0 Hz,
1 H), 5.10 (d, J=9.3 Hz, 1 H), 4.86 (d, J=12.8 Hz, 1 H), 4.69 (d,
J=12.3 Hz, 1 H), 4.08 (d, J=9.1 Hz, 1 H), 3.91 (d, J=9.0 Hz,
1 H), 3.04 (m, 1 H), 2.85-2.76 (m, 2 H), 2.74 (s, 3 H), 2.72-2.69
(m, 1 H), 2.50-2.45 (m, 1 H), 2.28-2.25 (m, 1 H), 2.16 (s, 3 H),
2.10-2.03 (m, 1 H), 1.80-1.73 (m, 1 H), 1.70-1.51 (m, 1 H),
1.34-1.18 (m, 3 H), 1.22 (s, 3 H), 1.16 (s, 3 H), 1.13 (d, J=6.8 Hz,
3 H), 0.99 (d, J=7.0 Hz, 3 H), 0.96 (s, 9 H), 0.86 (s, 9 H), 0.13 (s, 3
H), 0.12 (s, 3 H), 0.10 (s, 3 H), -0.09 (s, 3 H); 13C NMR (125
MHz, CDCl3) δ 215.5, 170.2, 165.3, 151.2, 140.5, 132.0, 129.8 (2
C), 125.9 (2 C), 123.7, 123.3, 120.0 (2 C), 119.8, 116.2, 79.1, 75.9,
68.2, 67.9, 53.3, 39.4, 38.6, 37.6, 32.0, 31.8, 30.1, 28.6, 26.3 (3 C),
26.0 (3 C), 24.7, 22.6, 19.1, 18.6, 18.5, 17.1, 15.1, 14.0, -3.4,
-3.6, -3.8, -5.6; IR (neat) 2954, 2925, 2112, 1732, 1463, 1259,
1094, 798 cm-1; MS (þFAB) 881.4 (M þ Hþ); HRMS m/e calcd
for C46H73N4O7SSi2 (M þ Hþ) 881.4739, found 881.4746;
3019, 2926, 2854, 1720, 1662, 1463, 1259, 1215, 1102, 769 cm-1
;
MS (þFAB) 944.4 (M þ Hþ); HRMS m/e calcd for
C53H78NO8SSi2 (M þ Hþ) 944.4987, found 944.5009; [R]20
D
-5.3 (c 0.15, CHCl3). The acylated product (8.0 mg, 0.0085
mmol, 1.0 equiv) was placed in a flask and the temperature was
lowered to -20 °C. TFA solution (0.100 mL, 20% v/v TFA in
CH2Cl2) was added and the reaction was stirred at -20 °C for 5
h and stored in the freezer at -4 °C overnight. The reaction was
then quenched into saturated sodium bicarbonate. The mixture
was extracted three times with ethyl acetate, dried over Na2SO4,
and concentrated. Column chromatography on silica gel with a
gradient of 0-1% CH3OH in CH2Cl2 gave 4.0 mg (67% yield)
of the final product 16: 1H NMR (500 MHz, CDCl3) δ 8.16 (d,
J=6.7 Hz, 2 H), 7.85 (t, J=7.6 Hz, 4 H), 7.66 (t, J=7.4 Hz, 1 H),
7.53 (t, J=7.1 Hz, 2 H), 6.95 (s, 1 H), 6.61 (s, 1 H), 5.63 (dd, J=
9.5, 5.3 Hz, 1 H), 5.31 (d, J=9.0 Hz, 1 H), 4.88 (d, J=12.7 Hz,
1 H), 4.74 (d, J=12.8 Hz, 1 H), 4.34 (d, J=9.8 Hz, 1 H), 3.74 (br
s, 1 H), 3.62 (br s, 1 H), 3.19 (dq, J=14.0, 2.2 Hz, 1 H), 3.01 (br s,
1 H), 2.72-2.69 (m, 1 H), 2.70 (s, 3 H), 2.52-2.47 (m, 1 H),
2.46-2.40 (m, 1 H), 2.38-2.33 (m, 1 H), 2.30 (dd, J=14.5, 2.6
Hz, 1 H), 2.23-2.19 (m, 1 H), 2.10 (s, 3 H), 1.81-1.78 (m, 2 H),
1.44-1.39 (m, 3 H), 1.38 (s, 3 H), 1.21 (d, J=6.8 Hz, 3 H), 1.10 (s,
3 H), 1.04 (d, J=7.0 Hz, 1 H); 13C NMR (125 MHz, CDCl3)
δ 220.5, 195.9, 170.2, 165.4, 165.1, 151.7, 141.3, 138.5, 136.9,
136.7, 133.2, 132.9, 130.0 (2 C), 129.7 (2 C), 129.4 (2 C), 128.4
(2 C), 125.0, 119.4, 115.7, 78.0, 73.9, 72.1, 68.2, 53.5, 41.6, 39.5,
37.9, 32.0, 31.6, 28.3, 25.2, 22.8, 19.0, 17.8, 15.9, 15.7, 13.2; IR
(neat) 2995, 2955, 1730, 1665, 1280 cm-1; MS (þFAB) 716.4
(M þ Hþ); HRMSm/e calcd for C41H50NO8S(M þ Hþ) 716.3257,
found 716.3254; [R]20D -60 (c 0.20, CHCl3).
[R]20 -11 (c 0.15, CHCl3). The acylated compound (8.5 mg,
D
0.0097 mmol, 1.0 equiv) was placed in a flask and the tempera-
ture was lowered to -20 °C. TFA solution (0.200 mL, 20% v/v
TFA in CH2Cl2) was added and the reaction was stirred at
-20 °C for 5 h. The reaction was then placed in the freezer
overnight at -4 °C. The reaction was quenched into saturated
sodium bicarbonate. The mixture was extracted three times with
ethyl acetate, dried over Na2SO4, and concentrated. Column
chromatography on silica gel with a gradient of 0-1% CH3OH
in CH2Cl2 gave 4.5 mg (71% yield) of the final product 15: 1H
NMR (500 MHz, CDCl3) δ 7.82 (d, J=6.7 Hz, 1 H), 7.70 (t, J=
1.7 Hz, 1 H), 7.44 (t, J=7.9 Hz, 1 H), 7.23 (dd, J=2.2, 2.3 Hz,
1 H), 6.95 (s, 1 H), 6.60 (s, 1 H), 5.60 (dd, J=11.7, 5.6 Hz, 1 H),
5.29 (d, J=7.2 Hz, 1 H), 4.84 (d, J=12.6 Hz, 1 H), 4.71 (d, J=
12.7 Hz, 1 H), 4.34 (d, J = 10.9 Hz, 1 H), 3.71 (br s, 1 H),
3.69-3.67 (m, 1 H), 3.18 (dq, J=13.6, 2.1 Hz, 1 H), 3.02 (br s,
1 H), 2.72-2.69 (m, 1 H), 2.71 (s, 3 H), 2.52-2.46 (m, 1 H),
2.45-2.38 (m, 1 H), 2.37-2.32 (m, 1 H), 2.28 (dd, J=14.5, 2.6
Hz, 1 H), 2.21-2.17 (m, 1 H), 2.09 (s, 3 H), 1.78-1.70 (m, 2 H),
1.42-1.38 (m, 3 H), 1.38 (s, 3 H), 1.20 (d, J=6.8 Hz, 3 H), 1.09 (s,
3 H), 0.94 (d, J=7.0 Hz, 3 H); 13C NMR (125 MHz, CDCl3)
(43) Algaier, J.; Himes, R. H. Biochem. Biophys. Res. Commun. 1988, 954,
235–243.
J. Org. Chem. Vol. 75, No. 1, 2010 93

Reiff et al.
JOCArticle
Photoaffinity Labeling of Tubulin. Tubulin was purified
from bovine brains by a procedure described previously.43
Photoaffinity labeling of tubulin was performed as described.44
To a solution of 0.1 mmol of tubulin (10 mg) in 100 mM Pipes,
pH 7 containing 1 mM MgSO4, 1 mM EGTA, and 1 mM
dithiothreitol at 0 °C was added 0.5 mmol of a concentrated
stock solution in DMSO of 14, 15, or 16 such that the DMSO
content did not exceed 0.1%. The mixture was then spread on
plastic weighing boats (to maximize surface area) and irradiated
by exposure to UV light of 253.7 nm (Rayonet photochemical
reactor 253.7 nm lamp) for 30 min at a distance of 6 cm,
achieving 600-700 mW/cm2.
Preparation of Tryptic Peptides. The pH of the tubu-
lin-epothilone derivative mixture was raised to pH 8.5 with
the addition of small aliquots of a 1 M NH4HCO3 solution and
tryptic digestion was performed directly with TPCK-treated
proteomics grade trypsin at 37 °C for 24 h at an enzyme/
substrate ratio of 1:100. The tryptic digest was taken to dryness,
dissolved in a small volume of 50 mM glycine-HCL buffer, pH
10, containing 1.0 mM MgC12, and analyzed by reverse-phase
HPLC (4.6 ꢀ 150 mm C18 column; 5-95% H2O/0.1%CF3CO-
OH-CH3CN/CF3COOH gradient; 220 nm detection). Frac-
tions corresponding to chromatographic peaks were pooled,
lyophilized, and examined by mass spectrometry with a Q-Tof-2
instrument. The masses of all MHþ and MH2þ ions were parsed
and compared against a library of tryptic fragments of all
isoforms of tubulin present in bovine tubulin compiled pre-
viously by us (data obtained from two-dimensional LC-ion trap
MS/MS experiments performed on a LTQ Linear ion-trap MS
instrument). All masses that were flagged as a mismatch
(deviation of greater than 50 ppm from expected mass) were
subjected to MS/MS experiments on the Q-Tof2 instrument,
and neutral loss, b and y ion assignments were attempted. No
MS or MS/MS ions indicative of true adduction of the epothi-
lone derivatives were found.
Docking and Scoring. Docking and scoring calculations were
carried out with Surflex-Dock45-48 and CScore49 in the SYBYL
8.0 discovery software suite (Tripos, Inc.). In Surflex-Dock, the
1TVK.pdb cocrystallized ligand (epothilone A)17 was used to
guide the protomol generation process. Default parameters of
0.5 and 0 were used for docking threshold and bloat, respec-
tively. The maximum number of conformations per compound
fragment and the maximum number of poses per ligand were
both set to their default values of 20, and the maximum number
of rotatable bonds per molecule was set to 100. Molecule
fragmentation was disabled. Postdock minimizations were done
on each molecule to enhance the quality of results, and all four
CScore consensus scoring functions were implemented.
Acknowledgment. This work was supported by National
Institute of Health grants CA79641 and CA105305. E.A.R.
and J.F.G. were supported by NIH Training Grant GM
07775. E.A.R. was also supported by the Army Breast
Cancer Initiative Predoctoral Fellowship Program DAMD
17-001-0303 and the American Association for Pharmaceu-
tical Education Predoctoral Fellowship Program.
1
Supporting Information Available: H and 13C NMR spectra
are provided for compounds 3, 4, 9, 10, 14, 15, and16. This material
is available free of charge via the Internet at http://pubs.acs.org.
(45) Jain, A. N. J. Comput.-Aided Mol. Des. 2007, 21, 281–306.
(46) Pham, T. A.; Jain, A. N. J. Med. Chem. 2006, 49, 5856–5868.
(47) Jain, A. N. Curr. Opin. Drug Discovery Dev. 2004, 7, 396–403.
(48) Jain, A. N. J. Med. Chem. 2003, 46, 499–511.
(44) Nath, J. P.; Eagle, G. R.; Himes, R. H. Biochemistry 1985, 24, 1555–
1560.
(49) Meng, E. C.; Shoichet, B. K.; Kuntz, I. D. J. Comput. Chem. 1992, 13,
505–524.
94 J. Org. Chem. Vol. 75, No. 1, 2010