Synthesis and evaluation of novel 4-amino-4,6-androstadiene-3,17-dione: An analog of formestane

Article abstract of DOI:10.1016/j.bmcl.2008.09.018

Synthesis of 4-amino-4,6-androstadiene-3,17-dione 7, an analog of formestane used in breast cancer therapy as an aromatase inhibitor, from 4-acetoxy-4-androstene-3,17-dione 2 is described. This is the first report of a 4-amino diene (4,6) system in this series of molecules. The new (7) and reported molecules were screened by the National Cancer Institute (NCI, Bethesda, USA) for in vitro antitumor activity against 60 human cancer cell lines. Molecule 7 showed best activity against breast cancer cell line (MCF-7).

Full text of DOI:10.1016/j.bmcl.2008.09.018

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5563–5566
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel 4-amino-4,6-androstadiene-3,17-dione:
An analog of formestane
Sanjay K. Sharma a,b, , Weizhong Zheng , Alummoottil V. Joshua a,b, Douglas N. Abrams a,b
a
,
*
a
Alexander J. B. McEwan
a
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2
Edmonton Radiopharmaceutical Centre, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2008
Revised 26 August 2008
Accepted 3 September 2008
Available online 22 September 2008
Synthesis of 4-amino-4,6-androstadiene-3,17-dione 7, an analog of formestane used in breast cancer
therapy as an aromatase inhibitor, from 4-acetoxy-4-androstene-3,17-dione 2 is described. This is the
first report of a 4-amino diene (4,6) system in this series of molecules. The new (7) and reported
molecules were screened by the National Cancer Institute (NCI, Bethesda, USA) for in vitro antitumor
activity against 60 human cancer cell lines. Molecule 7 showed best activity against breast cancer cell line
(MCF-7).
Keywords:
Aromatase
Formestane
Ó 2008 Elsevier Ltd. All rights reserved.
4
-OHA
Breast cancer
Aromatization
Mammary tumors represent the most widespread type of
malignant neoplasms and are the second major cause of mortality
in women. About half of these malignancies require a source of
estrogens for their growth and development. Estrogens are biosyn-
thesized from androgens by the microsomal cytochrome P-450 en-
zyme system termed aromatase. Aromatase represents an enzyme
complex made up of two major components: a flavoprotein
18
O
12
1
17
16
1
9
11 13
1
C
D
9
B
14
2
1
5
1
4
0
8
7
A
5
O
3
6
(
NADPH-cytochrome P-450 reductase) and a specific form of cyto-
R
chrome P-450 commonly known as aromatase cytochrome P-450.
This protein is involved in the binding of C-19 atom of the steroid
substrate and catalyzes the multistep reaction resulting in the aro-
1. R = OH, Formestane (4-OHA)
2. R = OAc, Formestane acetate (4-OAcA)
2
matization of the ring A of the steroid. Current endocrine therapy
Figure 1.
relies upon the use of either antiestrogens, which act on tumor
cells directly or an alternative strategy consisting of the application
of aromatase inhibitors (AI) which suppress production of estro-
gens in the organ. A potent aromatase inhibitor would be a possible
alternative to endocrine ablation in the treatment of advanced
estrogen dependent mammary carcinoma. Since aromatase cataly-
ses the final step in the biosynthesis of estrogens, the potent aro-
matase inhibitor formestane 1 (4-hydroxy-4-androstene-3,17-
dione, 4-OHA) has been proven very effective in the treatment of
advanced estrogen dependent breast cancer (Fig. 1).3
The mechanism of the inhibitory effect of 4-OHA on aromatase
has not been completely elucidated. An inactivation mechanism,
which involves the formation of a covalent bond between the en-
zyme and position 4 of the steroid substrate has been proposed
4
by Covey et al. Protonation of the hydroxy group at position 4 of
the substrate and its elimination as a water molecule occurs in
the enzyme bound intermediate. Inactivation of the enzyme fol-
lows aromatization of the ring A of the steroid. Although 4-OHA
is a potent aromatase inhibitor, it suffers from a low bioavailability
and short term action and manifests in vivo activity only on subcu-
taneous administration mainly due to its high rate of glucuronida-
*
Corresponding author. Address: Department of Oncology (ERC), 11560 Univer-
sity Avenue, Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2. Tel.: +1
5
tion. These limitations of 4-OHA has lead to the quest for the
7
0
80 4328868; fax: +1 780 4328637.
E-mail address: sanjaysh@cancerboard.ab.ca (S.K. Sharma).
synthesis and analysis of its various analogues with improved
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.018

5
564
S. K. Sharma et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5563–5566
aromatase inhibitory activity with fewer limitations. The chemical
synthesis of new AI’s is a big challenge employing original ap-
proaches based on a combination of several methods. Various
R1
6
structural modifications of 4-OHA have consisted of the introduc-
R3
tion of substituents into ring A or at adjacent positions with simul-
taneous preservation, in the majority of cases, of polar functional
groups at C(3), and C(17). The sites for substitution have been C
R1
(
1), C (4), C (6), C (7) and C (19).
Highly active 4-amino androstenediones present special inter-
R2
est. They appeared to be efficient aromatase inhibitors. Studies of
structure activity relationship revealed the enhanced rate and
selectivity of aromatase inhibition upon the introduction of 1,2-
and 6,7-double bonds into the molecules of 4-amino androstenedi-
ones. Further conjugation of the 3-keto-4-ene system caused more
rapid inactivation of aromatase in rat ovarian microsomes than the
parent compound 4-OHA. Conjugated systems such as 4-amino
1
2
3
8
9
1
1
. R = S, R = OAc, R = CH3
. R = O, R = H, R = CH F
0. R = O, R = OC H , R = CH
1. R = O, R = OCH CH , R = CH
1
2
3
2
1
2
3
6
5
3
1
2
3
2
3
3
Figure 2.
1
,4,6-triene-3,17-dione have been synthesized by a patented and
7
lengthy chemical procedure and evaluated in detail, but no at-
tempts have been made in the past to synthesize 4-amino-4,6-
from the presence of 6,7 olefinic protons (5.15 and 5.65 ppm) in
the H NMR. The yield of 4 after column chromatography was
1
3
,17-dione molecule (7). Considering the significance of extended
74%. The final step of deprotection of the acetyl group was done
by reacting 4 with 2.5 M KOH in methanol: dichloromethane
(20:1) at room temperature for 2 h to give 5 as a colorless solid
conjugation in 4-OHA in breast cancer therapy, herein we report
a new methodology for the synthesis of 7 which will facilitate
the design of novel aromatase inhibitors for the treatment of breast
cancer.
1
after silica gel column chromatography. The H NMR spectrum
showed the olefinic protons at C6 and C7 in 5 at 6.92 and
1
3
Attempts to synthesize reported 4-hydroxy-4,6-androstadiene-
6.03 ppm, the C NMR showed the C6 and C7 carbons at 115.13
3
,17-dione 5 from 4-acetoxy-4-androstenene-3,17-dione 2 using
and 135.69 ppm, respectively. Conversion of 5 to the methanesul-
fonyl derivative 6 followed by treatment with NH OH (28% NH )
4 3
8
triethyl orthoformate and p-toulenesulfonic acid as well as direct
oxidation using the patented procedure with chloranil and acetic
9
yielded the title compound 7 as an oil, which was precipitated
out as salt by passing HCl gas to the DCM solution of the oil.
We also synthesized some compounds 8-11 (Fig. 2) for compar-
ing the in vitro activity with our amino compound 7. All the syn-
thesized analogs have been evaluated by the National Cancer
Institute (NCI, Bethesda, USA), on all the 60 human cancer cell lines
organized into subpanel derived from nine different human cancer
acid in toulene were unsuccessful. These procedures work well
when the 4-hydroxy group is protected with an electron donating
group but not with parent 4-OHA (1) or its acetyl derivative (2).
We used an alternative approach (Scheme 1) by synthesizing a
stable 4-acetoxy-6-bromo-4-androstene-3,17-dione 3 by reacting
4
-OHA with N-bromosuccinamide in refluxing carbon tetrachloride
1
10
for 20 min. in 94% yield. The H NMR spectrum showed a multiplet
at 5.48 ppm for one proton (C6), while this carbon appears at
types : leukemia, melanoma, lung, colon, renal, ovarian, breast,
-5
prostate and CNS, at one dose (10 M concentration) primary
anti-cancer assay (Table 1).
2.12 ppm in the 1 C NMR. The HRMS showed peak at M +Na at
45.09 which confirms the structure of 3. Compound 3 yielded
-acetoxy-4,6-androstadiene-3,17-dione 4 on treatment with so-
3
+
4
4
4
A compound is considered active when it reduces the mean
growth percentage of any cell lines to 32% or less (negative num-
bers indicate cell kill). Compound 7 have shown significant activity
dium iodide in acetone. The double bond formation in 4 is evident
O
O
O
a
b
O
O
O
OAc
OAc
OAc Br
3
2
4
O
O
O
c
d
e
O
O
O
OH
OMs
NH2.HCl
5
6
7
o
Scheme 1. Reagents and conditions: (a) NBS/CCl
4 2 2
, reflux, 20 min., 94% (b) NaI/acetone, refluxed, 4 h, 74% (c) 2.5 M KOH, MeOH: CH Cl (20:1), rt, 2 h, 26% (d) MsCl, Py, 0 C,
1
h, 76% (e) NH OH, 1,4-dioxane, 96 h.
4

S. K. Sharma et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5563–5566
5565
Table 1
(6.15), SW-620 (15.36), KM12 (39.52), Breast cancer MCF-7
À15.97), Ovarian cancer OVCAR-8 (44.03), Leukemia CCRF-CEM
In vitro one dose primary anticancer assay for compounds
(
Panel/Cell line
7
8
9
10
11
(6.62), K-562 (À26.94), SR (À18.11) and also Prostate cancer PC-3
(
46.51) as compared to the other compounds.
NSC lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
103.34
103.40
65.58
102.14
83.39
113.39
100.35
92.32
99.49
89.15
99.26
94.85
100.25
104.06
106.47
87.06
96.55
97.57
106.72
77.42
87.31
97.79
In conclusion we have synthesized a new 4-amino-4,6-andro-
1
1
97.99
111.88
61.94
96.30
74.07
88.38
83.77
76.59
111.14
103.52
92.93
98.58
101.11
95.97
83.36
91.00
stadiene-3,17-dione (7)
compound, that exhibits potential
123.96
113.59
111.29
106.48
101.55
108.88
87.35
in vitro cytotoxicity in number of cancer cell lines. The detailed
anticancer activity and mechanism of action of 7 will be published
in due course.
Acknowledgments
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
This work was supported by the grant to S.K.S. (Project #
3051) from Alberta Cancer Foundation (ACF). We thank National
Cancer Institute (NCI) for screening our compounds in human can-
cer cell lines.
121.26
96.01
6.15
72.99
51.17
39.52
15.36
110.35
126.51
86.59
105.90
111.91
109.64
107.43
134.97
139.86
110.19
106.17
116.40
117.01
125.89
115.44
128.78
99.87
132.65
125.75
103.20
110.45
98.16
2
94.74
100.27
115.72
120.45
123.89
120.57
References and notes
Breast cancer
BT-549
HS578T
1
2
.
.
Young, J. L. Breast Cancer. In Liss, A. R., Ed.; Acadmic Press: New York, 1989; p 1.
(a) Thompson, E. A.; Siiteri, P. K. J. Biol. Chem. 1974, 249, 5373; (b) Bellino, P. L. J.
Steroid Biochem. 1982, 17, 261; (c) DiSalle, E.; Giudici, D.; Briatico, G.; Ornati, G.
Ann. N.Y. Acad. Sci. 1990, 595, 357.
(a) Hofken, K.; Jonat, W.; Possinger, K.; Kolbel, M.; Kunz, T. H.; Wagner, H.;
Becher, R.; Callies, R.; Friederich, P.; Willmanns, W.; Maass, H.; Schmidt, C. G. J.
Clin. Oncol. 1990, 90, 875; (b) Pickles, T.; Perry, L.; Murray, P.; Plowman, P. Br. J.
Cancer 1990, 62, 309.
82.63
112.26
À15.97
100.87
112.52
68.71
94.20
117.57
97.31
107.68
116.00
104.07
102.16
97.23
127.96
130.43
117.71
100.91
107.98
39.44
110.10
103.50
103.22
109.86
113.61
90.34
93.87
118.13
108.56
106.73
104.79
97.45
MCF7
MDA-MB- 231/ATCC
MDAMB-435
NCI/ADR-RES
T-47D
3
.
73.66
116.70
117.94
4
5
.
.
Covey, D. F.; Hood, W. F. Cancer Res. (Suppl. 8) 1982, 42, 3327.
(a) Foster, A. B.; Jarman, M.; Mann, J.; Parr, I. B. J. Steroid Biochem. 1986, 24, 607;
Ovarian cancer
IGROV1
49.46
64.57
61.98
107.69
44.03
120.27
36.69
105.69
102.43
108.39
89.90
62.42
119.07
98.85
118.37
94.26
55.73
121.39
100.72
97.47
93.48
117.40
85.75
128.63
96.98
101.66
98.96
(
b) Brodie, A. M. H.; Wing, L. Y. Steroids 1987, 50, 245; (c) Brodie, A. M. H.;
Garrett, W. M.; Hendrickson, J. P.; Tsai-Morris, C. H. Cancer Res. (Suppl. 8) 1982,
2, 3360.
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
4
6
7
.
.
Levina, I. S. Russian Chem. Rev. 1998, 6711, 975.
(a) DiSalle, E.; Briatico, G.; Giudici, D.; Ornati, G.; Zaccheo, T. J. Steroid. Biochem.
Mol. Biol. 1992, 43, 137. Br. P. 2,284605, Chem Abstract 123, 228, 634, 1995; (b)
Giudici, D.; Ornati, G.; Briatico, G.; Buzzetti, F.; Lombardi, P.; Disalle, E. J. Steroid.
Biochem. 1988, 30, 391; (c) DiSalle, E.; Giudici, D.; Ornati, G.; Briatico, G.;
D’Alessio, R.; Villa, V.; Lombardi, P. J. Steroid. Biochem. Mol. Biol. 1990, 37, 369;
118.54
120.50
111.30
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
6.62
52.53
92.56
97.15
68.18
114.9
57.95
88.29
88.21
112.8
93.48
106.1
—
90.45
105.95
86.08
105.63
87.07
91.63
112.2
94.09
98.59
—
(
d) Zaccheo, T.; DiSalla, E. Cancer Chemother. Pharmacol. 1993, 31, 308.
À26.94
8
9
.
.
Whitesides, G. M.; Sadowski, J. S.; Lilliburn, J. J. Am. Chem. Soc. 1974, 96, 2829.
Specht, H.; Jahn, H.; Stachowiak, A. East German Patent, 41938, Chem. Abs. 1966,
57.20
À18.11
6
4, 14245.
118.7
66.91
104.9
10. (a) Grever, M. R.; Schepartz, S. A.; Chabner, B. A. The National Cancer
Institute: Cancer Drug Discovery and Development Programme. Seminars in
Oncology 1992, 196, 622; b The human tumor cell lines of the cancer
screening panel are grown in RPMI 1640 medium containing 5% fetal bovine
Renal cancer
7
86-0
89.28
99.19
88.16
84.08
95.21
88.53
85.59
64.59
97.68
110.9
110.1
112.9
104.6
180.9
97.59
79.41
105.6
111.1
119.9
111.0
67.71
113.5
114.8
87.76
100.78
103.00
105.72
112.48
107.93
90.07
103.4
105.6
105.1
115.2
117.9
110.6
100.6
85.82
A498
serum and 2 mM
plates in 100 L at plating densities ranging from 5000 to 40,000 cells/well.
After cell inoculation, the microtiter plates are incubated at 37 °C, 5% CO
5% air and 100% relative humidity for 24 h prior to addition of drugs. The
L-glutamine. Cells are inoculated into 96-well microtiter
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
l
2
,
9
drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final
concentration and stored frozen prior use. At the time of drug addition, an
aliquot of frozen concentrate is thawed and diluted to twice the desired
122.99
76.98
final concentration with complete medium containing 50
Additional four, 10-folds or half log serial dilutions are made to provide a
total five drug concentrations plus control. Aliquots of 100 L of these
different drugs dilutions (single dose of 10 M) are added to the appropriate
microtiter wells already containing 100 of medium, resulting in the
required final drug concentration. Following drug addition, the plates are
incubated for an additional 48 h at 37 °C C, 5% CO , 95% air and 100%
relative humidity. Cells are fixed in situ by the gentle addition of 50 L of
lg/mL gentamicin.
Melanoma
LOX IMVI
M14
MALME-3M
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
60.57
79.51
70.81
74.41
103.4
69.71
86.69
81.02
95.59
101.6
75.28
88.46
119.4
100.9
114.4
105.6
96.65
102.8
94.17
97.19
116.5
108.7
97.04
114.6
101.44
102.62
120.52
82.37
119.81
94.23
100.3
103.8
87.47
94.48
135.2
99.39
117.1
90.78
l
-5
lL
2
l
108.45
87.46
cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min
at 4 °C. The supernatant is discarded, and plates are washed five times with
tap water and air dried. Sulforhodamine B (SRB) solution (100
lL) at 0.4%
Prostate cancer
DU-145
PC-3
(
w/v) in 1% acetic acid is added to each well, and plates are incubated for
62.36
46.51
115.4
89.28
116.2
—
107.32
89.25
123.2
96.50
10 min at room temperature. After staining unbound dye is removed by
washing (five times with 1% acetic acid) and plates air dried. Bound stain is
subsequently solubilized with 10 mM trizma base, and the absorbance is
read on an automated plate reader at a wavelength of 515 nm.
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
89.03
105.1
82.75
102.8
94.19
54.19
111.7
124.3
105.8
110.1
98.00
102.0
109.8
106.3
105.8
109.3
90.07
111.4
113.44
124.31
106.75
114.92
91.11
128.3
114.0
111.3
112.3
79.71
1
1. 4-Acetoxy-6-bromo-4-androstene-3,17-dione (3): To a solution of 2 (120 mg,
0
1
.35 mmol) in carbon tetrachloride (20 ml) was added NBS (240 mg,
.35 mmol) and benzoyl peroxide (13 mg, 0.05 mmol). The reaction mixture
was stirred and refluxed for 20 min. After being cooled to room temperature,
the mixture was filtered and the filtrate was washed with a solution of aqueous
sodium bicarbonate and water. The organic phase was dried with sodium
sulfate and concentrated under reduced pressure. The residue was purified by
91.35
2
column chromatography on SiO using hexane-ethyl acetate (2:1) as eluent.
Evaporation of the appropriate fraction yielded 3 as colorless solid (140 mg,
by reducing the growth of cells in wide range of cell lines (nine
panels). The most sensitive cell lines are colon cancer HCT-116
4%).%). 1H NMR (CDCI
1.61(3 H, s, 19-CH ), 0.98(3 H, s, 18-CH
9
3
, 500 MHz) d 5.48(1 H, t, 6-H), 2.30(3 H, s, COCH
3
),
13
3
3
).
C NMR (CDCI
3
, 125 MHz)
d

5
566
S. K. Sharma et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5563–5566
2
9
1
19.78(C-17), 190.60(C-3), 167.97(COCH
), 50.27(C-14), 42.12(C-6), 29.85(C-8), 22.45(COCH
8). ES-MS (in positive ionization) m/z calcd for C21 27BrO
3
), 150.04(C-4), 141.01(C-5), 52.64(C-
), 20.40(C-19), 13.79(C-
Na 445.11 (M + Na),
(3:1) as eluent. Evaporation of the appropriate fraction yielded 5 as colorless
solid of (8 mg, 26%). 1H NMR (CDCI
, 500 MHz) d 6.92(1 H, dd, J7,6 = 8.5 Hz,
J7,8 = 3.5 Hz, 7-H), 6.15(1 H, br, s, OH), 6.03(1 H, dd, J6,7 = 8.5 Hz, J6,8 = 3.5 Hz, 6-
3
3
H
4
13
measured 445.1.
3 3 3
H), 1.15(3 H, s, 19-CH ), 0.90(3 H, s, 18-CH ). C NMR (CDCI , 125 MHz) d
4
1
-Acetoxy-4,6-androstadiene-3,17-dione (4): To
.49 mmol) in acetone (150 ml) was added sodium iodide (7.0 g, 46.70 mmol).
a
solution of
3
(630 mg,
220.21(C-17), 184.01(C-3), 146.26(C-4), 140.64(C-5), 135.69(C-7), 115.13(C-6),
51.43(C-9), 49.26(C-14), 30.74(C-8), 21.66(C-19), 13.55(C-18). ES-MS (in
The reaction mixture was stirred and refluxed for 4 h. The solvent was removed
under reduced pressure. The residue was partitioned between dichloromethane
24 3
positive ionization) m/z calcd for C19H O Na 323.17 (M+Na), measured 323.2.
4-Methanesulfonyl-4,6-androstadiene-3,17-dione (6): To a cool solution of 4
(125 mg, 0.42 mmol) in dichloromethane-triethylamine (30:1) (15 ml) was
added methanesulfonyl chloride (0.035 mL, 0.45 mmol). After the reaction
mixture was stirred at 0 °C for 1 h, ice-water (20 mL) was added. The organic
solvent was separated, washed with water, dried with sodium sulfate and
concentrated under reduced pressure. The residue was purified by column
(
150 mL) and an aqueous sodium thiosulfate solution. Dichloromethane was
washed with a solution of aqueous sodium thiosulfate (2 Â 50 mL), water, dried
with sodium sulfate and concentrated under reduced pressure. The residue was
purified by column chromatography on SiO
2
using hexane-ethyl acetate (2:1) as
eluent. Evaporation of the appropriate fraction yielded 4 as colorless solid
1
(
377 mg, 74%). H NMR (CDCI
3
, 500 MHz) d 5.15 and 5.65 (2 H, m, 6-H and 7-H),
chromatography on SiO
2
using hexane-ethyl acetate (2:1) as eluents.
1
3
2
1
5
2
C
.38(3 H, s, COCH
25 MHz) d 219.74(C-17), 191.43(C-3), 168.31(COCH
), 124.74(C-7), 123.88(C-6), 53.13(C-9), 50.96(C-14), 29.42(C-8), 22.42(COCH
0.25(C-19), 13.93(C-18). ES-MS (in positive ionization) m/z calcd for
Na 365.18 (M + Na), measured 365.2.
-Hydroxy-4,6-androstadiene-3,17-dione (5): To a solution of 4 (35 mg, 0.1 mmol)
3
), 1.26(3 H, s, 19-CH
3
), 0.92(3 H, s, 18-CH
), 148.02(C-4), 140.67(C-
),
3 3
). C NMR (CDCI ,
Evaporation of the appropriate fraction yielded a yellowy solid (121 mg, 76%).
+
3
EI-MS (in positive ionization) m/z calcd for C20
H
26
O
5
S 378.15(M ), measured
3
378.1498.
4-Amino-4,6-androstadiene-3,17-dione (7): NH
4
OH [NH (28%), 16 mL] was
3
21
H
26
O
4
added to a stirred solution of 5 (96 mg, 0.25 mmol) in dry 1, 4-dioxane
(8 mL), and the reaction was allowed to stir at room temperature for 96 h.
The solvents were removed in vacuo, the oil was redissolved in dry
4
in methanol-dichloromethane (20:1) (20 ml) was added a solution of 2.5 M KOH
in water (1.5 mL). The reaction mixture was stirred for 2 h at ambient
temperature. The solvent was removed under reduced pressure. The residue
dichloromethane and to it HCL gas was passed, the salt of 7 precipitated out.
+
EI-MS (in positive ionization) m/z calcd C19
H25NO
2
299.1(M ), measured
was purified by column chromatography on SiO
2
using hexane-ethyl acetate
300.16 (M+1).