Synthesis of 11β-ether-17α-ethinyl-3,17β-estradiols with strong ER antagonist activities

Article abstract of DOI:10.1016/j.cclet.2014.01.015

We have previously found that several families of nonpolar short chain 11β-ethers and esters of estradiol are selective estrogen receptor modulators (SERMs). Surprisingly, the transformation from potent estrogen to anti-estrogen occurs when the 11β-side c

Full text of DOI:10.1016/j.cclet.2014.01.015

Chinese Chemical Letters 25 (2014) 567–570
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis of 11
b-ether-17
a
-ethinyl-3,17
b-estradiols with strong ER
antagonist activities
1
Jing-Xin Zhang 1, , David C. Labaree , Richard B. Hochberg
*
*
Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06520, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
We have previously found that several families of nonpolar short chain 11b-ethers and esters of estradiol
Received 3 August 2013
are selective estrogen receptor modulators (SERMs). Surprisingly, the transformation from potent
estrogen to anti-estrogen occurs when the 11 -side chain is increased slightly in length from four to five
non-hydrogen atoms. To generate strong antagonists for preclinical development, we have synthesized
other similar ER ligands with 11 -ethers and with an additional ethinyl group at the 17 -position in
order to slow metabolism of the steroidal moiety. Here we report the synthesis and biological activity of
Received in revised form 21 November 2013
Accepted 27 November 2013
Available online 14 January 2014
b
b
a
Keywords:
Estrogen receptor
SERM
two such compounds (11
antagonist activities.
b-i-PrO-propyl and 11b-t-BuO-propyl ethers) with extremely strong
ß 2014 Jing-Xin Zhang and Richard B. Hochberg. Published by Elsevier B.V. on behalf of Chinese Chemical
Antagonist
Estradiol
Society. All rights reserved.
1. Introduction
estra-1,3,5(10)-trien-3,17b-diol and 11b-(3-t-butoxypropyl)es-
tra-1,3,5(10)-trien-3,17 -diol (E11-3,i-Prether and E11-3,t-Buether
b
)
In recent years estrogen receptor antagonists have become very
important therapeutic agents for the treatment of estrogen
sensitive cancers, such as breast cancer [1]. Many anti-estrogenic
were especially potent anti-estrogens that were totally without
agonist activity. Consequently, in order to produce an ER
antagonist that would be longer lived and could be administered
compounds are typified by analogs of estradiol, substituted at C-7
a
orally, we introduced an ethinyl group at 17
to render the D-ring of the steroid resistant to oxidation [4].
These compounds are analogous to moxestrol, 11 -methoxy-
17 -ethinyl-estradiol, an extremely potent estrogen that is highly
resistant to metabolism [5]. The synthesis and biological
activity of these two compounds 11 -(3-isopropoxypropyl)estra-
17 -ethinyl-1,3,5(10)-trien-3,17 -diol and 11 -(3-t-butoxypro-
pyl)estra-17 -ethinyl-1,3,5(10)-trien-3,17 -diol (17 -ethinyl-
-ethinyl-E11-3,t-Buether, Fig. 2) are reported
a position of estradiol
(ICI 164,384) and C-11 (RU39411) or aromatic compounds, such
b
as Tamoxifen. They share some similar structural features, mainly
polar long chains containing tertiary amines or carboxylic groups
(Fig. 1).
b
a
We have identified a series of unusual estradiol analogs with
b
short and non-polar substituent groups at the 11
b
position [2,3].
a
b
b
One of the most remarkable examples is the simple and short chain
ester, E11-2,2 (Fig. 1). The highly unusual aspect of these results is
that a complete reversal of function occurs with a single methylene
group, lengthening the side-chain from 4 atoms of a methyl ester in
E11-2,1 to 5 atoms of an ethyl ester in E11-2,2 (Fig. 1). E11-2,1 is an
estrogen receptor agonist, while E11-2,2 an antagonist. This same
a
b
a
E11-3,i-Prether and 17
a
here.
2. Experimental
principle was found to apply to other substitutions at the 11
b
2.1. Chemistry
position, which include ketones, ethers (Fig. 1, E11-2,2ether) and
thiono esters [3].
The ethers were especially interesting as they were highly
active anti-estrogens and were more stable than other substituents
General: 1H NMR spectra were recorded with a Bruker Avance
400 spectrometer, and chemical shifts are reported relative to
residual CHCl3 (7.27 ppm). Purification by flash chromatography
was performed according to the procedure of Still [6], using
230–400 mesh silica gel (EM Science, Darmstadt Germany).
Unless otherwise noted, solvents (analytical or HPLC) and
reagents were used as supplied, and all reactions were carried
out under nitrogen. Thin-layer chromatography (TLC) was
performed using Merck silica gel plates (F254) (EM Science) and
visualized using phosphomolybdic acid or UV illumination. TLC
[3]. Among these ethers, two of them, 11b-(3-isopropoxypropyl)-
*
Corresponding authors.
E-mail addresses: zhang_jingxin@hotmail.com (J.-X. Zhang),
richard.hochberg@yale.edu (R.B. Hochberg).
1
These two authors contributed equally to this work.
1001-8417/$ – see front matter ß 2014 Jing-Xin Zhang and Richard B. Hochberg. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2014.01.015

568
J.-X. Zhang et al. / Chinese Chemical Letters 25 (2014) 567–570
HO
O
O
N
O
N
OH
S
OH
O
HO
N
Tamoxifen
RU39411
Raloxifene
O
O
O
O
O
OH
OH
OH
HO
HO
HO
E11-2,2ether
E11-2,2
E11-2,1
Fig. 1. The structures of the classical antiestrogens, raloxifene, tamoxifen, RU 39411; the estrogen E11-2,1 and the antiestrogens E11-2,2 and E11-2,2ether
.
system: T-1, hexanes/EtOAc (2:1); T-2, hexanes/EtOAc (1:1).
Analytical high performance liquid chromatography (HPLC) was
performed on a Beckman System Gold HPLC system (Beckman
Coulter, Inc., Fullerton, CA) consisting of a model 126 solvent
module and a model 168 diode array detector at 280 nm using the
following columns and systems: with an RP-18 column (LiChro-
To generate the 3,17-bis-protected E11-3,i-Prether (5a), iso-
propanol was first activated by reacting with a suspension of 35%
dispersion of KH in the presence of 18-crown-6. Reacting with this
anion of isopropanol in toluene at 80 8C converted tosylate (4) to
the protected ether (5a), which was then deprotected by
hydrogenolysis of the benzyl groups using 5% palladium on carbon
under an atmosphere of H2 to give 6a.
The 3,17-bis-protected E11-3,t-Buether (5b) was prepared from
the protected tosylate (4) using solid potassium t-butoxide
instead of KH and isopropanol. Deprotection and purification as
above gave 6b.
sorb RP-18, 5
CH3CN/H2O (50/50) at 3 mL/min, 280 nm; with a Diol column
(LiChrosphor Diol, 5
m
m, 4.6 mm  25 cm, EM Science), with H-1,
m
m, 4.6 mm  25 cm, EM Science) with, H-2,
CH2Cl2 at 1 mL/min, 280 nm.
Synthesis of 3-hydroxy, 17
b
-hydroxy, 17a-ethinyl, 11b-substi-
tuted estradiols is shown in Scheme 1.
The synthesis of 3,17 -dibenzyloxyestra-1,3,5(10)-trien-11-one
(1) was prepared as previously described in the literature [7].
Compound 1 was first converted to 11 -allyl-3,17 -dibenzylox-
yestra-1,3,5(10)-triene-11 -ol by addition of allylmagnesium bro-
To prepare 17a-ethinyl E11-3,i-Prether, another strategy of
b
protection/deprotection was employed. The 3-hydroxy group was
selectively protected by t-butyldiphenylsilyl chloride in CH2Cl2 in
the presence of 4-(dimethylamino)pyridine and triethylamine to
give the 3-protected ether (7a). The 17-hydroxy group was then
oxidized by pyridinium chlorochromate and sodium acetate in
CH2Cl2 to give the 3-protected-17-keton (8a). Deprotection of 3-
hyroxygroup by tetrabutylammonium fluroride in THF gave the 3-
hydroxy-17-keton (9a).
a
b
b
mide. Ally group was introduced from the less sterically hindered
alpha side. The hydroxyl group was then reduced by triethylsilane
and BF3ÁEt2O at 0 8C to yield 11
1,3,5(10)-triene (2), in which the configuration of 11-ally was
inversed from 11 to 11 . Hydroboration of the terminal olefin with
b-allyl-3,17b-dibenzyloxy estra-
a
b
Finally, to obtain 17a-ethinyl E11-3,i-Prether (10a), a solution of
LiBH4 and catecholborane followed by oxidation with H2O2 resulted
in propanol compound 3 [8]. Tosylation of the hydroxyl group by
p-toluenesulfonyl chloride in pyridine gave compound 4, which was
converted to the desired ethers in the next step.
18% sodium acetylide in xylenes was first added to the above
deprotected ketone (9a) in DMSO. After reacting at r.t. for 3.5 h, the
reaction was poured into saturated aqueous NH4Cl and extracted
with EtOAc. Flash chromatography gave the 17a-ethinyl E11-3,
OH
OH
H
H
O
O
HO
HO
17α-ethinyl-E11-3,i-Prether
17α-ethinyl-E11-3,t-Buether
Fig. 2. The structures of 17a-ethinyl-E11-3,i-Prether and 17a-ethinyl-E11-3,t-Buether.

J.-X. Zhang et al. / Chinese Chemical Letters 25 (2014) 567–570
569
H2C
OBn
OBn
OBn
CH3
CH3
CH3
O
HOCH2CH2CH2
a
b
BnO
BnO
BnO
1
2
3
c
OH
OBn
OBn
CH3
CH3
CH3
R1OCH2CH2CH2
R1OCH2CH2CH2
TsOCH2CH2CH2
d
e
HO
BnO
BnO
6a,b
5a,b
4
f
OH
O
O
CH3
CH3
CH3
R1OCH2CH2CH2
R1OCH2CH2CH2
R1OCH2CH2CH2
g
h
TBDPSO
TBDPSO
HO
7a,b
8a,b
9a,b
i
OH
CH3
R1OCH2CH2CH2
CH
5a, 6a,7a, 8a, 9a, 10a, R1=i-Pr
1
5b 6b 7b 8b 9b 10b
,
,
,
,
,
, R =t-Bu
HO
10a,b
Scheme 1. Reagents and conditions: (a): (i) allylmagnesium bromide, THF, (ii) HSiEt3, BF3.Et2O, 0 8C (1–2). (b): (i) catecholborane, LiBH4, THF, (ii) NaOH, H2O2 (2–3). (c) TsCl,
Pyr, 0 8C (3–4). (d): (i) KH, 18-crown-6, ROH, toluene, r.t., (ii) 4, 80 8C (4–5a); (iii) t-BuOK (4–5b). (e) 5% Pd-C, H2, EtOAc-EtOH, r.t. (5–6). (f) t-BuDPSO, Et3N, 4-Me2N-pyridine,
CH2Cl2, r.t. (6–7). (g) pyridinium chlorochromate (PCC), NaOAc, CH2Cl2, r.t. (7–8). (h) tetrabutylammonium fluroride, THF, r.t. (8–9). (I) (i) 18% sodium acetylide, xylene, r.t., (ii)
NH4Cl (9–10).
i-Prether (10a). 17
prepared according to the above procedures.
Detailed experimental procedures and data for compounds 7a,
8a, 9a, 10a and 10b are listed in ref. [12].
a
-ethinyl E11-3,t-Buether (10b) was similarly
action of 10À9 mol/L E2 (EC50 = 0.2 nmol/L). Each compound was
analyzed in at least three separate experiments performed in
duplicate. The Ki were determined using the curve fitting program
Prism.
2.2. Biological studies
3. Results and discussion
The binding of the two 11
cytosol ER, human Estrogen Receptor
(hER -LBD) and human Estrogen Receptor
Domain (hER -LBD) [9] were determined by competition for the
b
-substituted ethers to rat uterine
ligand Binding Domain
ligand Binding
The biological properties of the two 11
b-ethers substituted
a
with a 17 -ethinyl group were analyzed in several different
a
a
b
assays: The affinity of the compounds for the estrogen receptor
(ER) was determined by their competition for the binding of [3H]E2:
b
binding of 1 nmol/L [3H] E2 as previously described [3]. Relative
binding affinity (RBA) was determined by analysis of the
displacement curves by the curve-fitting program Prism. The
results as RBAs compared to E2 represent the ratio of the EC50 of E2
to that of the steroid analogs  100.
rat uterine cytosol (native ER, predominantly ER
a [11]); ligand
binding domain (LBD) of human ER and the LBD of human ERb.
a
Anti-estrogenic potency was measured by inhibition of the
stimulation of alkaline phosphatase in Ishikawa cells by 1 nmol/L
E2. The results summarized in Table 1 are compared to the
previously reported results [3] of the parent compounds (unsub-
The anti-estrogenic potency of the two 11b-substituted ethers
were determined by the inhibition of the effect of 1 nmol/L E2 on
the estrogen sensitive marker, alkaline Phosphatase, in Ishikawa
cells [10] using the procedure previously described [3]. For
antagonists, the effect (Ki) of each compound tested at a range
from 10À6 to 10À12 mol/L was measured for the inhibition of the
stituted at 17
ethinyl-E11-3,t-Buether and 17
strongly to both ER and ER
approximately equal to that of E2. In each case, the 17
substituted compounds bound equally, or slightly weaker, than the
a
). As shown in Table 1, these two compounds, 17
-ethinyl-E11-3,i-Prether, bind very
, with relative binding affinities
-ethinyl
a-
a
b
a
a

570
J.-X. Zhang et al. / Chinese Chemical Letters 25 (2014) 567–570
Table 1
Biological activities.
Compound
Rat uterine ER RBAa
hERa
-LBD RBAa
hERb
-LBD RBAa
Inhibition Ki (nmol/L)b
c
E11-3,i-Prether
114 Æ 25
110 Æ 14
81 Æ 18
56 Æ 24
220 Æ 62
97 Æ 64
93 Æ 18
116
0.2 Æ 0.2
0.09 Æ 0.07
0.09 Æ 0.06
0.06 Æ 0.03
17
a-ethinyl-E11-3,i-Prether
c
E11-3,t-Buether
128 Æ 54
117 Æ 46
82 Æ 16
124
17a-ethinyl-E11-3,t-Buether
a
RBA: relative binding activity where E2 = 100.
b
Inhibition: effect on the estrogenic stimulation of 1 nmol/L E2 on alkaline phosphatase activity in Ishikawa cells. Experiments were performed in duplicate in at least 3
separate experiments (with the exception of hERb-LBD which was done only once). The values were determined using the curve fitting using program, GraphPad Prism.
Values are mean Æ S.D.
c
See ref. [3].
chromatography on a 2 cm  17 cm column of silica gel using 2:1 hexanes/EtOAc
as eluent gave 14.4 mg (54%) of 7a. Data for 7a: TLC, T-1, Rf 0.35.3-t-Butyldi-
phenylsiloxy-11b-(3-isopropoxypropyl)estra-1,3,5(10)-trien-17-one (8a). A so-
lution of 7a, NaOAc (1 mg) in CH2Cl2 (2 mL) was stirred at r.t. as PCC (8 mg,
0.035 mmol) was added. The reaction was stirred at r.t. for 2 h, poured into H2O
(30 mL) and extracted with EtOAc (3Â 20 mL). Combined organic extracts were
dried over Na2SO4 and concentrated in vacuo giving a brown film. Purification by
flash chromatography on a 2Â 17 cm column of silica gel using 4:1 hexanes/EtOAc
as eluent gave 10.3 mg (71%) of 8a. Data for 8a: TLC, T-1, Rf 0.55.3-Hydroxy-11b-
parents. More importantly, both compounds are extremely potent
anti-estrogens with potencies equal to, or greater than, that of the
unsubstituted parent compounds; most likely reflecting their
resistance to metabolism in the cell. Thus, it is highly likely that
both compounds are promising for antiestrogen drug development.
4. Conclusion
(3-isopropoxypropyl)estra-1,3,5(10)-trien-17-one (9a).
A solution of 1 mol/L
tetra-n-butylammonium fluoride in THF (1 mL) was added to 8a (10.3 mg,
0.0169 mmol) and stirred at r.t. for 1.5 h. The reaction was poured into H2O
(70 mL) and extracted with EtOAc (3Â 20 mL). Combined organic extracts were
dried over Na2SO4 and concentrated in vacuo giving a clear colorless oil. Purifi-
cation by flash chromatography on a 2 cm  17 cm column of silica gel using 2:1
We have generated two estradiols with 11
b-t-BuO-propyl ethers substitutions and demonstrated their
extremely strong antagonist activities.
b-i-PrO-propyl and
11
hexanes/EtOAc as eluent gave 5 mg (79%) of 9a. Data for 9a: TLC, T-1, Rf 3.75; 1
H
References
NMR (400 MHz, CDCl3): d 1.05 (s, 3H, H-18), 1.11 (d, 3H, J = 6.1 Hz, –CH3), 1.12 (d,
3H, J = 6.1 Hz, –CH3), 2.18 (dd, 1H, J = 13.9, 1.6 Hz, H-12b), 2.50–2.55 (m, 1H, H-
11), 2.54 (dd, 1H, J = 18.9, 8.6 Hz, H-16b), 2.60 (dd, 1H, J = 10.9, 4.6 Hz, H-9), 2.72–
2.87 (m, 2H, H-6), 3.28–3.37 (m, 2H, CH2O), 3.51 (septet, 1H, J = 6.1 Hz, CH(CH3)2),
4.70 (br s, 1H, OH), 6.56 (d, 1H, J = 2.7 Hz, H-4), 6.65 (dd, 1H, J = 8.5, 2.7 Hz, H-2),
7.04 (d, 1H, J = 8.5 Hz, H-1).11b-(3-Isopropoxypropyl)estra-17a-ethinyl-
1,3,5(10)-trien-3,17b-diol (10a). A solution of 9a (5 mg) in DMSO (2 mL) was
stirred at r.t. as an 18% solution of sodium acetylide in xylene/mineral oil (1 mL)
was added over 5 min. The reaction was stirred at r.t. for 3.5 h, poured into
saturated aqueous NH4Cl (30 mL) and extracted with EtOAc (3Â 30 mL). Com-
bined organic extracts were dried over Na2SO4 and concentrated in vacuo giving a
yellow oil (DMSO was azeotroped off with toluene). Purification by flash chro-
matography on a 1 cm  17 cm column of silica gel using 1:1 hexanes/EtOAc as
eluent gave product contaminated with a nonpolar impurity. Further purification
in 6 portions by semiprep HPLC (RP-18) eluting at 3 mL/min with 50/50 CH3CN/
H2O (tR, 15 min) followed by crystallization from acetone-petroleum ether gave
3.4 mg (63%) of 10a. Data for 10a: TLC, T-1, 0.35; 1H NMR (400 MHz, CDCl3): d 1.04
(s, 3H, H-18), 1.12 (d, 3H, J = 6.1 Hz, –CH3), 1.13 (d, 3H, J = 6.1 Hz, –CH3), 2.51–2.56
(m, 1H, H-11), 2.60 (dd, 1H, J = 10.5, 4.3 Hz, H-9), 2.64 (s, 1H, ethinyl-H), 2.67–2.83
(m, 2H, H-6), 3.32 (t, 2H, J = 6.9 Hz, CH2O), 3.52 (septet, 1H, J = 6.1 Hz, –CH(CH3)2),
6.54 (d, 1H, J = 2.7 Hz, H-4), 6.64 (dd, 1H, J = 8.5, 2.7 Hz, H-2), 7.05 (d, J = 8.5 Hz, H-
1); HPLC system: H-2, tR = 11.16 min; system H-1, tR 15 min, >99% pure.11b-(3-
t-Butoxypropyl)estra-17a-ethinyl-1,3,5(10)-trien-3,17b-diol (10b). A solution of
9b (5.3 mg, 0.01378 mmol) in DMSO (2 mL) was stirred at r.t. as an 18% solution of
sodium acetylide in xylene/mineral oil (2 mL) was added dropwise over 5 min.
The reaction was stirred at r.t. for 3.5 h, poured into saturated aqueous NH4Cl
(60 mL) and extracted with EtOAc (2Â 50 mL). Combined organic extracts were
dried over Na2SO4 and concentrated in vacuo (DMSO was azeotroped off with
toluene). Purification by flash chromatography on a 1Â 17 cm column of silica gel
using 2:1 hexanes/EtOAc as eluent gave 3.7 mg of product which was further
purified by semiprep HPLC using an RP-18 column eluting with 50/50 CH3CN/H2O
giving 2.7 mg 10b. Crystallization from acetone-petroleum ether gave 1.9 mg
(33%) of 10b as a white solid. Data for 10b: TLC, T-2, Rf 0.67; 1H NMR (400 MHz,
CDCl3): d 1.04 (s, 3H, CH3), 1.15 (s, 9H, tBu), 2.52 (m, 1H, H-11), 2.59 (dd, 1H,
J = 10.2, 4.9 Hz, H-9), 2.63 (s, 1H, ethinyl-H), 2.67–2.83 (m, 2H, H-6), 3.20–3.29 (m,
2H, CH2O), 6.54 (d, 1H, J = 2.7 Hz, H-4), 6.64 (dd, 1H, J = 8.6, 2.7 Hz, H-2), 7.05 (d,
1H, J = 8.6 Hz, H-1); HPLC system: H-2, tR = 10.6 min, >92% pure; system: H-1, tR
17.2 min, >99% pure.
[1] S.X. Lin, J. Chen, M. Mazumdar, et al., Molecular therapy of breast cancer: progress
and future directions, Nat. Rev. Endocrinol. 6 (2010) 485–493.
[2] J.X. Zhang, D.C. Labaree, G. Mor, et al., Estrogen to antiestrogen with a single
methylene group resulting in an unusual steroidal selective estrogen receptor
modulator, J. Clin. Endocrinol. Metab. 89 (2004) 3527–3535.
[3] J.X. Zhang, D.C. Labaree, R.B. Hochberg, Nonpolar and short side chain groups at
C-11b of estradiol result in antiestrogens, J. Med. Chem. 48 (2005) 1428–1447.
[4] S.M. Hyder, C. Chiappetta, G.M. Stancel, Synthetic estrogen 17a-ethinyl estradiol
induces pattern of uterine gene expression similar to endogenous estrogen,
J. Pharmacol. Exp. Ther. 290 (1990) 740–747.
[5] J. Salmon, D. Coussediere, C. Cousty, J.P. Raynaud, Pharmaco kinetics and metab-
olism of moxestrol in animals (rat, dog, monkey), J. Ster. Biochem. 19 (1983)
1223–1234.
[6] W.C. Still, M. Kahn, A. Mitra, Rapid chromatographic technique for preparative
separations with moderate resolution, J. Org. Chem. 43 (1978) 2923–2925.
[7] D.C. Labaree, J.X. Zhang, H.A. Harris, et al., Synthesis and evaluation of B-, C-, and
D-ring-substituted estradiol carboxylic acid esters as locally active estrogens,
J. Med. Chem. 46 (2003) 1886–1904.
[8] R. Tedesco, R. Fiaschi, E. Napolitano, Novel stereoselective synthesis of 11
b-carbon-substituted estradiol derivatives, J. Org. Chem. 60 (1995) 5316–5318.
[9] H.A. Harris, A.R. Bapat, D.S. Gonder, D.E. Frail, The ligand binding profiles of estrogen
receptors alpha and beta are species dependent, Steroids 67 (2002) 379–384.
[10] B.A.Littlefield, E. Gurpide,L.Markiewicz, etal.,Asimpleandsensitivemicrotiterplate
estrogen bioassay based on stimulation of alkaline phosphatase in Ishikawa cells:
estrogenic action of delta 5 adrenal steroids, Endocrinology 127 (1990) 2757–2762.
[11] G.G. Kuiper, B. Carlsson, K. Grandien, et al., Comparison of the ligand binding
specificity and transcript tissue distribution of estrogen receptors alpha and beta,
Endocrinology 138 (1997) 863–870.
[12] Detailed experimental procedures and data for compounds 7a, 8a, 9a, 10a and
10b:3-t-Butyldiphenylsiloxy-11b-(3-isopropoxypropyl)estra-1,3,5(10)-trien-
17b-ol (7a). A solution of 16 mg (0.0432 mmol) of E11-3,i-Prether (6a), t-butyldi-
phenylsilyl chloride (124 mL, 0.475 mmol), dimethylaminopyridine (10 mg,
0.08 mmol) and triethylamine (200 mL, 1.434 mmol) in CH2Cl2 (1 mL) was
allowed to stir at r.t. overnight. The reaction was poured into H2O (50 mL) and
extracted with EtOAc (3Â 50 mL). Combined organic extracts were dried over
Na2SO4 and concentrated in vacuo giving a clear colorless oil. Purification by flash