Stereo-controlled synthesis of novel photoreactive γ-secretase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.117

The stereoselective synthesis of novel photoreactive γ-secretase inhibitors 2 and 3 has been achieved. Key steps of the strategy involve preparation of α-N-Boc-epoxide 8 and formation of lactone 14 in a practical and stereo-controlled fashion. Compounds 2

Full text of DOI:10.1016/j.bmcl.2008.11.117

Bioorganic & Medicinal Chemistry Letters 19 (2009) 922–925
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Stereo-controlled synthesis of novel photoreactive c-secretase inhibitors
Guangli Yang a, Ye Ingrid Yin b , Jiong Chun bà, Christopher C. Shelton b, Ouathek Ouerfelli a, Yue-Ming Li b,
*
a Organic Synthesis Core Facility, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
b Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The stereoselective synthesis of novel photoreactive
c
-secretase inhibitors 2 and 3 has been achieved.
Received 20 September 2008
Revised 25 November 2008
Accepted 26 November 2008
Available online 7 December 2008
Key steps of the strategy involve preparation of
tical and stereo-controlled fashion. Compounds 2 and 3 are potent
interact with presenilin-1, a catalytic subunit of -secretase.
a
-N-Boc-epoxide 8 and formation of lactone 14 in a prac-
c
-secretase inhibitors and directly
c
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Aspartyl proteases
Alzheimer disease
Photoaffinity labeling
Secretase
Dipeptide isostere
c
-Secretase cleaves the amyloid precursor protein (APP) to
generate b-amyloid (Ab) peptides, which are believed to play a
causative role in the pathogenesis of Alzheimer disease (AD).1
The synthesis of 2 and 3 started with the preparation of epoxide
8 using a modified Barrish–Polniaszek’s method11 (Scheme 1).
Methylation of Boc-p-Bz-Phe-OH (4) with TMSCHN2 in methanol12
provided methyl ester 5.
c
-
Secretase is an aspartyl protease composed of at least four proteins,
including presenilin, nicastrin, APH and Pen2.2 Genetic and bio-
chemical studies have indicated that presenilin is the catalytic core
However, an attempt that followed the same synthetic route for
preparation of Phe-BPA isostere10 to protect benzophenone 5 as a
dioxolane using ethylene glycol, p-TsOH and benzene at reflux
for 2 days failed to generate any product. Thus, we changed our
strategy by reducing ketone to an alcohol. We intended to find con-
ditions that allow for the stereo- and regioselective reduction the
ketone of benzophenone. Initially, we treated 5 with NaBH4 at
0 °C13 with favorable stereoselectivity (85:15 dr) and 70% yield,
but this condition also led to the formation of a small amount of
reduced methyl ester. However, when we performed the same
reaction at ꢀ60 °C, we obtained the stereoselective product
(85:15 dr) in 86% yield without reducing the methyl ester. Silyla-
tion of the resulting secondary alcohol produced 6, which led to
the generation of a chiral center at the benzylic carbon. The config-
of
been associated with early on-set of AD6 through alteration of the
specificity of -secretase. Furthermore, -secretase represents a
c
-secretase3–5 and as such, familial mutations of presenilin have
c
c
novel class of protease that hydrolyzes the scissile bond within
the transmembrane domain of substrate.7,8
L-685,458 (1) (Fig. 1), a potent
c
-secretase inhibitor9 that
contains a hydroxyethylene isostere, can be modified into a
photoreactive compound by replacing an unsubstituted phenyl
with a benzophenone (BP). These substitutions at P2, P10 and
P30 have been synthesized and utilized to study
c
-secretase.4,10
However, synthesis of a photoreactive dipeptide isostere at the
P1 position has not yet been achieved. In this study, we describe
the stereo-controlled synthesis of two new analogs of L-685,458
(2 and 3, Chart 1) with BPA (benzophenone alanine) at the P1
position and demonstrate that they directly interact with
P2
P1'
P3'
presenilin, the catalytic subunit of
c-secretase. Moreover, this
Ph
Ph
HO
O
novel BPA-Phe isostere could be useful as a functional unit to
synthesize active site directed inhibitors for profiling aspartyl
proteases.
H
N
BocHN
NH2
N
H
O
O
* Corresponding author. Tel.: +1 646 888 2193; fax: +1 646 422 0640.
E-mail address: liy2@mskcc.org (Y.-M. Li).
P1
P2'
Figure 1. Structure of L-685,458 (1). The side chains corresponding to the P and P0
Present Address: Wellington Management, Boston, MA, USA.
à
Present Address: Symrise Inc., 300 North Street, Teterboro, NJ, USA.
sites are marked.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.117

G. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 922–925
923
Ph
O
Ph
R
OH
O
H
N
BocHN
N
H
O
Ph
(R)
O
(S)
2
3
R = OMe
(R)
R = biotinamidopentyl
Chart 1.
O
O
(R)
(S)
(S)
BocHN
(R)
Ph
O
O
O
BocHN
BocHN
BocHN
(S) Ph
OTBS
OMe
OMe
b,c
O
OH
a
12
Ph
Ph
Ph
Figure 2. X-ray crystallographic structure of 12: C37H49NSiO5, M 615.86, ortho-
rhombic P212121 (No. 19), a = 5.9239(12) Å, b = 12.923(3) Å, c = 46.419(9) Å,
OTBS
O
O
4
5
6 (85:15 dr)
V = 3553.0(12) Å3, Dc (Z = 4) = 1.151 g/cm3, T = 100 K,
l
= 0.106 cmꢀ1. The final R
I and 398 parameters.
OH
value is 0.2116 for 3442 independent reflections with I > 2
r
BocHN
Cl
f
BocHN
d,e
(The crystal structure of 12 has been deposited at the Cambridge Crystallographic
Data Centre with the deposition number: CCDC 710680.)
Ph
Ph
OTBS
OTBS
8
7 (9:1 dr)
on recovered starting material 6). Cyclization of chlorohydrin 7
produced epoxide 8 in 95% yield.14
Scheme 1. Synthesis of epoxide 8. Reagents and conditions: (a) TMSCHN2, MeOH,
0 °C to rt, 18 h, 90%; (b) NaBH4, MeOH, ꢀ60 °C, 86%; (c) TBSCl, CH2Cl2, imidazole, rt,
95%; (d) 4 equiv CH2ICl, 5 equiv LDA, THF, ꢀ78 °C; (e) 4 equiv NaBH4, MeOH,
ꢀ78–0 °C, 65%; (f) KOH, EtOH, 0 °C to rt, 95%.
Treatment of epoxide 8 with the sodium salt of diethyl malon-
ate directly provided lactone 9 as a mixture of stereoisomers
(Scheme 2).15 Hydrolysis of 9 with aqueous LiOH, followed by
decarboxylation gave lactone 10 in 60% yield. Aldol condensation
of 10 with benzaldehyde followed by dehydration with acetic
uration of 6 is assigned by X-ray crystallographic analysis of inter-
mediate 12 (Scheme 2) as described later in Figure 2. Treatment of
methyl ester 7 with excess LDA/CH2ICl provided an
tone, which was reduced with NaBH4 to give chlorohydrin 7 (9:1
dr) in favor of the desired stereoisomer as demonstrated by X-
ray analysis. The two diastereoisomers of 7 were separated by col-
umn chromatography (65% yield of the desired compound, based
anhydride-triethylamine at 120 °C gave the
a,b-unsaturated lac-
tone 11 in 80% yield.16 Hydrogenation of 11 with 10% Pd/C
(1 atm, 6 h) provided lactone 12 as the sole product. The assign-
ment of three chiral centers, as indicated in Scheme 2, was con-
firmed by the X-ray crystallographic analysis of 12 (Fig. 2).
Removal of the silyl group in lactone 12 with n-Bu4NF (TBAF) led
a-chloroke-
to epimerization at the a-lactone position, perhaps due to the ba-
sicity of the TBAF reagent. However, we were able to find that
treatment of 12 with pyridine/HF overnight successfully removed
the silyl protecting group to give 13 without any epimerization.17
Oxidation of the benzylic alcohol with MnO218 gave benzophenone
14 in 76% yield.19 Hydrolysis of lactone 14 with LiOH and silylation
of the resulting hydroxy acid produced 15 in 79% yield.
O
O
O
O
d,e
b,c
BocHN
a
CO2Et
BocHN
8
Ph
Esterification of Leu-Phe-OH with TMSCl in MeOH,20 followed
by coupling of the resulting amine with acid 15, and deprotection
of the resulting silyl ether with TBAF, produced the desired com-
Ph
OTBS
O
9
10
OTBS
O
O
O
O
O
h
BocHN
f
BocHN
BocHN
Ph
Ph
Ph
Ph
H
N
Ph
O
Ph
RO
O
a,b
BocHN
OMe
d,e
Leu-Phe-OH
N
H
Ph
OTBS
Ph
OR
O
14
O
11
12 R = TBS
13 R = H
Ph
g
16
R = TBS
c
O
O
2
R = H
Ph
NH
TBSO
HN
Ph
Ph
i
BocHN
OH
HO
O
H
N
H
N
H
N
BocHN
S
O
O
N
H
O
O
O
O
Ph
3
Ph
15
Scheme 2. Synthesis of acid 15. Reagents and conditions: (a) CH2(CO2Et)2, NaOEt,
EtOH, rt, 70%; (b) LiOH/DME–H2O, 50 °C, 6 h; (c) toluene, reflux, 8 h, 60%; (d) LDA,
PhCHO, THF, ꢀ78 °C; (e) Ac2O, Et3N, 120 °C, 80% for 2 steps; (f) H2, 10% Pd/C, EtOAc,
rt, 6 h, 90%; (g) HFꢁPy, THF, 18 h, 83%; (h) MnO2, CH2Cl2, 18 h, 76%; (i) ia—LiOH/
DME–H2O, rt; ib—TBSCl, imidazole, DMF, rt; ic—MeOH, 79% for 3 steps.
Scheme 3. Synthesis of compounds 2 and 3. Reagents and conditions: (a) TMSCl,
MeOH, 0 °C to rt, 18 h, 80%; (b) 15, EDC, HOBt, i-Pr2NEt, DMF, 57%; (c) n-Bu4NF, THF,
rt, 85%; (d) LiOH, THF/H2O, rt, 90%; (e) 5-(biotinamido)pentylamine, EDC, HOBt,
DMF, rt, 50%.

924
G. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 922–925
Acknowledgments
This work was supported by NIH Grant AG026660 (Y.M.L.), the
100
80
60
40
20
0
kDa
37
3
2
Alzheimer’s Association (Zenith Fellows Award to Y.M.L.), Mr. Wil-
liam H. Goodwin and Mrs. Alice Goodwin and the Commonwealth
Foundation for Cancer Research, the Experimental Therapeutics
Center of MSKCC, and the William Randolph Hearst Fund in Exper-
imental Therapeutics. C.C.S. is supported by an NIH NRSA predoc-
toral fellowship 5F31NS053218-02. We thank Dr. George
Sukenick, Ms. Sylvi Rusli (NMR Core Facility, Sloan-Kettering Insti-
tute) for mass spectral analyses and Dr. Louis Todaro (Hunter Col-
lege, New York) for X-ray structure analyses.
PS-1
25
20
3
+
+
L-685,458 -
+
0.01
0.1
1
10
100
Concentration (nM)
References and notes
1. Hardy, J.; Allsop, D. Trends Pharmacol. Sci. 1991, 12, 383.
2. De Strooper, B. Neuron 2003, 38, 9.
3. Wolfe, M. S.; Xia, W.; Ostaszewski, B. L.; Diehl, T. S.; Kimberly, W. T.; Selkoe, D.
J. Nature 1999, 398, 513.
Binding
+
4. Li, Y. M.; Xu, M.; Lai, M. T.; Huang, Q.; Castro, J. L.; DiMuzio-Mower, J.; Harrison,
T.; Lellis, C.; Nadin, A.; Neduvelil, J. G.; Register, R. B.; Sardana, M. K.; Shearman,
M. S.; Smith, A. L.; Shi, X. P.; Yin, K. C.; Shafer, J. A.; Gardell, S. J. Nature 2000,
405, 689.
5. Esler, W. P.; Kimberly, W. T.; Ostaszewski, B. L.; Diehl, T. S.; Moore, C. L.; Tsai, J.
Y.; Rahmati, T.; Xia, W.; Selkoe, D. J.; Wolfe, M. S. Nat. Cell Biol. 2000, 2, 428.
6. Sherrington, R.; Rogaev, E. I.; Liang, Y.; Rogaeva, E. A.; Levesque, G.; Ikeda, M.;
Chi, H.; Lin, C.; Li, G.; Holman, K., et al Nature 1995, 375, 754.
7. Wolfe, M. S.; Kopan, R. Science 2004, 305, 1119.
3
Target Protein
Photolysis
8. Brown, M. S.; Ye, J.; Rawson, R. B.; Goldstein, J. L. Cell 2000, 100, 391.
9. Shearman, M. S.; Beher, D.; Clarke, E. E.; Lewis, H. D.; Harrison, T.; Hunt,
P.; Nadin, A.; Smith, A. L.; Stevenson, G.; Castro, J. L. Biochemistry 2000,
39, 8698.
10. Chun, J.; Yin, Y. I.; Yang, G.; Tarassishin, L.; Li, Y. M. J. Org. Chem. 2004, 69, 7344.
11. Chen, P.; Cheng, P. T. W.; Spergel, S. H.; Zahler, R.; Wang, X. B.; Thottathil, J.;
Barrish, J. C.; Polniaszek, R. P. Tetrahedron Lett. 1997, 38, 3175.
12. Chai, W.; Murray, W. V. Tetrahedron Lett. 1999, 40, 7185.
13. Orito, K.; Sato, S.; Suginome, H. J. Chem. Soc., Perkin Trans. 1 1995, 1, 63.
Western
Blotting
Streptavidin
Column
Figure 3. Both 2 and 3 are potent
presenilin-1. (A) Inhibitory potencies of compounds 2 and 3 against
Scheme of photoaffinity labeling procedure. After photo-crosslinking, the biotinyl-
ated proteins were captured, eluted and analyzed by Western analysis. (C) Analysis
of photolabeled proteins. The photo-crosslinked proteins were resolved by SDS–
PAGE and probed with PS-1-NTF (N-terminal fragment) antibody.
c
-secretase inhibitors that directly bind to
-secretase. (B)
14. Preparion
of
8:
tert-Butyl
(S)-2-(4-(S)-tert-butyldimethylsilyloxy
phenylmethylphenyl)-1-((S)-oxiran-2-yl)ethylcarbamate (8). To an ice-cold
solution of 7 (1.25 g, 2.41 mmol) in EtOH (30 mL) was added KOH (163 mg,
2.9 mmol), and the reaction mixture was stirred at rt for 2 h. The reaction
mixture was concentrated under reduced pressure, and the residue was
partitioned between EtOAc (200 mL) and H2O (50 mL). The organic layer was
washed with saturated NH4Cl solution, H2O, and brine, dried with Na2SO4, and
concentrated under reduced pressure. Purification of the residue by column
chromatography (20% EtOAc in hexane) gave 8 (1.11 g, 95%) as a yellow syrup:
c
pound 2 in reasonable yield (Scheme 3).21 In order to facilitate the
purification of the labeled proteins or fragments thereof, biotinyl-
ated compound 3 was prepared (Scheme 3). Mild saponification
of the methyl ester in 2 led to the corresponding carboxylic acid,
which was coupled with 5-(biotinamido)pentylamine in the pres-
ence of EDC and HOBt and resulted in compound 3.
½ ꢂ
a 2D5 9.1 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): 7.38 (d, J = 7.4 Hz, 2H), 7.33
(m, 4H), 7.22 (t, J = 7.1 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H), 5.75 (s, 1H), 4.47 (br s,
1H), 3.70 (br s, 1H), 3.93 (m, 2H), 2.80 (m, 3H), 1.38 (s, 9H), 0.94 (s, 9H), 0.01 (s,
3H), 0.00 (s, 3H); 13C NMR: 155.2, 145.1, 143.7, 135.3, 129.3, 128.1, 126.9,
126.5, 126.2, 79.5, 76.4, 53.2, 46.8, 37.2, 28.2, 25.8, 18.3, ꢀ4.8; EIMS: 506.3
[M+Na+], HRMS (ESI) Calcd for C28H41NSiO4Na: 506.2703, found 506.2698.
15. Evans, B. E.; Rittle, K. E.; Homnick, C. F.; Springer, J. P.; Hirshfield, J.; Veber, D. F.
J. Org. Chem. 1985, 50, 4615.
We next examined the biological activities of 2 and 3. First, we
16. Nadin, A.; Lopez, J. M. S.; Neduvelil, J. G.; Thomas, S. R. Tetrahedron 2001, 57, 1861.
17. Preparation of 13: tert-Butyl (S)-1-((2R,4R)-4-benzyl-5-oxotetrahydrofuran-2-
yl)-2-(4-(S)-hydroxyphenyl methylphenyl) ethyl carbamate (13). A solution of
determined their inhibitory potency against c-secretase using an
in vitro assay.22 The IC50 values of 2 and 3 are 0.7 and 0.6 nM,
respectively (Fig. 3A), which is similar to the parent compound,
L-685,458 (1). These findings have demonstrated that incorporat-
ing BPA into the P1 position and attaching a biotin tag at the C-ter-
silyl ether 12 (390 mg, 0.63 mmol) in THF (10 mL) was transferred to
a
polyethylene vial and PyꢁHF (70% HF, 1.5 mL) was dropwise added at 0 °C. The
reaction mixture was stirred at rt overnight. The reaction mixture was
quenched with saturated NaHCO3 solution, and extracted with EtOAc (3ꢃ
50 mL). The combined organic layer was washed with brine, dried with
Na2SO4. The residue was purified by column chromatography (50% EtOAc in
minus do not affect their potency for inhibition of
Second, we tested whether 3 was capable of photo-crosslinking
to -secretase. HeLa cell membranes were incubated with 3 at a fi-
nal concentration of 10 nM in the absence and the presence of
M of L-685,458 for 2.5 h. Then samples were irradiated with
c-secretase.
hexane) to give 13 (264 mg, 83%) as a white solid: mp 59–60 °C; ½a D25
ꢂ
ꢀ58.8 (c
0.7, CHCl3); 1H NMR (400 MHz, CDCl3): 7.35–7.21 (m, 10 H), 7.15 (d, J = 7.2 Hz,
2 H), 7.10 (d, J = 7.9 Hz, 2H), 5.77 (d, J = 2.7 Hz, 1H), 4.43 (br, 1H), 4.09 (br, 1H),
3.85 (br, 1H), 3.24 (dd, J = 4.0, 13.8 Hz, 1H), 2.87–2.68 (m, 4H), 2.63 (d,
J = 3.3 Hz, 1H, OH), 2.18 (m, 1H), 1.75 (m, 1H), 1.31 (s, 9H); 13C NMR: 177.6,
155.3, 143.9, 142.5, 138.4, 135.9, 129.6, 128.8, 128.7, 128.5, 128.4, 127.5, 127.3,
127.2, 126.8, 126.7, 126.5, 79.8, 78.8, 75.9, 54.3, 43.0, 36.1, 31.6, 28.2; EIMS:
524.1 [M+Na+], HRMS (ESI) Calcd for C31H35NO5Na: 524.2413, found 524.2402.
18. Fauq, A. H.; Cherif-Ziani, C.; Richelson, E. Tetrahedron: Asymmetry 1998, 9, 2333.
19. Preparation of 14: tert-Butyl (S)-2-(4-benzoylphenyl)-1-((2R,4R)-4-benzyl-5-
oxotetrahydrofuran-2-yl) ethyl carbamate (14). To an ice-cold solution of 13
(240 mg, 0.478 mmol) in CH2Cl2 (10 mL) was added MnO2 (415 mg,
4.78 mmol). The suspension was stirred at rt overnight. The reaction mixture
was filtered through Celite, washed with EtOAc. The combined organic layer
was concentrated, the residue was purified by column chromatography (40%
c
2
l
UV light (>350 nm) and the labeled proteins were solubilized and
isolated with streptavidin beads.4
The biotinylated proteins were eluted and analyzed by Western
blotting with antibodies against presenilin-1 (PS-1). Inhibitor 3 di-
rectly photolabels PS-1 (Fig. 3C). Moreover, an excess of L-685,458
is able to block photoinsertion of this probe into presenilin-1.
Taken together, these results have demonstrated that compounds
2 and 3 are potent
c-secretase inhibitors that can specifically label
EtOAc in hexane) to give 14 (185 mg, 76%) as a white solid: mp 59–60 °C; ½a D25
ꢂ
the catalytic core of
c-secretase. Therefore, compounds 2 and 3
ꢀ68.7 (c 0.7, CHCl3); 1H NMR (400 MHz, CDCl3): 7.74 (dd, J = 7.2, 11.9 Hz, 4H),
7.57 (t, J = 7.5 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.28 (m, 4 H), 7.17 (m, 3H), 4.68
(d, J = 9.4 Hz, 1H), 4.32 (br, 1H), 3.93 (br, 1H), 3.26 (dd, J = 4.1, 13.9 Hz, 1H),
should be valuable probes for mapping the active site of
secretase.
c-

G. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 922–925
925
3.03–2.73 (4H), 2.30 (m, 1H), 1.86 (m, 1H), 1.34 (s, 9H); 13C NMR: 196.4, 177.6,
155.3, 142.3, 138.4, 137.7, 136.1, 132.5, 130.4, 130.0, 129.5, 128.9, 128.8, 128.4,
126.9, 80.0, 79.2, 54.6, 42.5, 36.5, 36.2, 31.4, 28.3; EIMS: 522.2 [M+Na+], HRMS
(ESI) Calcd for C31H33NO5Na: 522.2256, found 522.2250.
3H), 0.06 (s, 3H); 13C NMR: 196.6, 174.9, 171.9, 171.4, 155.7, 144.2, 139.5, 137.9,
136.1, 135.9, 132.4, 130.5, 130.2, 129.5, 129.4, 129.2, 128.8, 128.8, 128.5, 127.3,
126.7, 79.9, 72.5, 54.9, 53.5, 52.5, 51.9, 45.5, 40.8, 38.9, 38.1, 36.0, 33.9, 28.6, 26.1,
25.0, 22.7, 22.6, 18.2, ꢀ4.2, ꢀ4.7; EIMS: 928.2 [M+Na+], Calcd for C53H71N3O8Si:
905.50.To an ice-cold solution of 16 (20 mg, 0.022 mmol) in THF (2 mL) was
added a solution of TBAF in THF (1.0 M, 0.2 mL). The reaction mixture was stirred
at rt overnight. The reaction mixture was diluted with EtOAc and washed with
citric acid and brine, dried with Na2SO4, and concentrated under reduced
pressure. Purification of the residue by column chromatography (5% MeOH in
20. Kokotos, G.; Padrón, J. M.; Martín, T.; Gibbons, W. A.; Martín, V. S. J. Org. Chem.
1998, 63, 3741.
21. Preparation of 2: {1S-Benzoyl-phenyl-4R-[1-(1S-methyl-oxycarbonyl-2-phenyl-
ethylcarbamoyl)-3-(1S)-methyl-butylcarbamoyl]-2R-hydroxy-5-
phenyl-pentyl}-carbamic acid tert-butyl ester (2). A solution of 15 (20 mg,
0.0316 mmol), Leu-Phe-OMe (16 mg, 0.054 mmol), 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (11 mg, 0.057 mmol) and 1-
hydroxylbenzotriazole (8 mg, 0.057 mmol) in DMF (2 mL) was stirred at rt. The
reaction mixture was diluted with EtOAc, and the organic layer was washed with
aqueous citricacid, saturated NaHCO3 solution, and brine, driedwith Na2SO4, and
concentrated under reduced pressure. Purification of the residue by column
chromatography (50% EtOAc in hexane) gave 16 (20 mg, 70%) as a white foam: 1H
NMR (400 MHz, CDCl3): 7.76 (d, J = 7.1 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H), 7.59 (t,
J = 7.4 Hz, 1H), 7.48 (d, J = 7.7 Hz, 2H), 7.27 (m, 4H), 7.20 (m, 4H), 7.11 (d,
J = 7.0 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 6.49 (t, J = 8.05 Hz, 2H), 4.78 (m, 1H), 4.55
(d, J = 8.0 Hz, 1H), 4.43 (m, 1H), 3.84 (m, 1H), 3.69 (s, 3H), 3.65 (m, 1H), 3.12–3.00
(m, 2H), 2.65 (dd, J = 5.7, 13.6 Hz, 1H), 2.59 (m, 1H), 2.45 (m, 1H), 1.87 (m, 1H),
1.79–1.58 (m, 3H), 1.37 (s, 9H), 0.91 (s, 9H), 0.86 (dd, J = 6.7, 11.1 Hz, 6H), 0.07 (s,
CH2Cl2) gave 2 (17 mg, 85%) as a white solid: mp 147–148 °C; ½a D25
ꢀ12.1 (c 0.7,
ꢂ
CHCl3); 1H NMR (400 MHz, CDCl3, a few drop of d4-MeOH): 7.77 (d, J = 7.3 Hz,
2H), 7.72 (d, J = 8.1 Hz, 2H), 7.58 (t, J = 7.4 Hz, 1H), 7.47 (t, J = 7.7 Hz, 2H), 7.33–
7.17 (m, 6H), 7.13 (t, J = 7.5 Hz, 2H), 6.69 (d, J = 7.1 Hz, 1H), 6.17 (d, J = 7.2 Hz, 1H),
4.78 (q, J = 7.3 Hz, 1H), 4.71 (d, J = 8.5 Hz, 1H), 4.26 (q, J = 7.3 Hz, 1H), 3.70 (m+s,
6H), 3.09 (m, 2H), 2.93 (dd, J = 7.2, 13.5 Hz, 1H), 2.86–2.62 (m, 4H), 1.83 (m, 1H),
1.74 (m, 1H), 1.56 (m, 1H), 1.46 (m, 2H), 1.39 (s, 9H), 0.84 (dd, J = 6.3, 8.5 Hz, 6H);
13C NMR: 196.6, 175.8, 172.2, 171.8, 156.2, 143.6, 139.0, 137.9, 136.0, 135.9,
132.5, 130.5, 130.1, 129.5, 129.16, 129.0, 128.8, 128.8, 128.7, 128.4, 127.3, 126.8,
79.9, 73.3, 56.6, 53.5, 52.5, 47.0, 40.8, 39.3, 38.1, 35.7, 35.5, 29.9, 28.4, 26.0, 24.8,
23.0, 22.1; HRMS (ESI) Calcd for C47H58N3O8: 792.4224, found 792.4229.
22. Li, Y. M.; Lai, M. T.; Xu, M.; Huang, Q.; DiMuzio-Mower, J.; Sardana, M. K.; Shi, X.
P.; Yin, K. C.; Shafer, J. A.; Gardell, S. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6138.