Synthesis and cytotoxic effect on cancer cell lines and macrophages of novel progesterone derivatives having an ester or a carbamate function at C-3 and C-17

Article abstract of DOI:10.1016/j.ejmech.2014.06.008

In this study we report the cytotoxic effect on human cancer cells of two series of novel progesterone derivatives; the first containing an aromatic ester (8a-e) or a carbamate functions both linked to C-3 (9a-e) on the pregn-4,16-diene-6,20-dione skeleto

Full text of DOI:10.1016/j.ejmech.2014.06.008

Accepted Manuscript
Synthesis and cytotoxic effect on cancer cell lines and macrophages of novel
progesterone derivatives having an ester or a carbamate function at C-3 and C-17
Alejandra Chávez-Riveros , Mariana Garrido , María Teresa Ramírez Apan , Armando
Zambrano , Mario Díaz , Eugene Bratoeff
PII:
S0223-5234(14)00527-3
10.1016/j.ejmech.2014.06.008
EJMECH 7051
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 February 2014
Revised Date: 4 June 2014
Accepted Date: 5 June 2014
Please cite this article as: A. Chávez-Riveros, M. Garrido, M.T. Ramírez Apan, A. Zambrano, M. Díaz,
E. Bratoeff, Synthesis and cytotoxic effect on cancer cell lines and macrophages of novel progesterone
derivatives having an ester or a carbamate function at C-3 and C-17, European Journal of Medicinal
Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.008.
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ACCEPTED MANUSCRIPT
Graphical abstract
Cytotoxic effect of novel compounds on cancer cell lines and macrophages
O
O
X
O
O
O
N
O
H
O
O
X
8a-8e
9a-9e
Series 1
O
O
O
O
MCF-7
O
O
X
N
H
X
O
O
13a-13e
14a-14e
Series 2
PC-3
HCT-15
X= a, H; b, F; c, Cl; d, Br; e, I

ACCEPTED MANUSCRIPT
Synthesis and cytotoxic effect on cancer cell lines and macrophages of
novel progesterone derivatives having an ester or a carbamate function at
C-3 and C-17
a
a
b
Alejandra Chávez-Riveros , Mariana Garrido , María Teresa Ramírez Apan ,
a
a
a
Armando Zambrano , Mario Díaz , Eugene Bratoeff .
a
Departamento de Farmacia, Facultad de Química, Universidad Nacional
Autónoma de México, D.F. 04510, México.
b
Instituto de Química, Universidad Nacional Autónoma de México, Circuito
Exterior, Ciudad Universitaria, DF 04510, D.F., México.
Address correspondence to:
Alejandra Chávez-Riveros
Departamento de Farmacia
Facultad de Química
Universidad Nacional Autónoma de México
Circuito Exterior, Ciudad Universitaria
México D.F. C.P. 04510, México
Phone: (011-52-55)56225290
FAX: (52-55)56225290
1

ACCEPTED MANUSCRIPT
Abstract
In this study we report the cytotoxic effect on human cancer cells of two series
of novel progesterone derivatives; the first containing an aromatic ester (8a−e)
or a carbamate functions both linked to C-3 (9a−e) on the pregn-4,16-diene-
6
,20-dione skeleton. In the second series, both functional groups (ester and
carbamate) are bound to C-17 on the pregn-4,6-diene-3,20-dione scaffold
13a−e and 14a−e). The panel cancer cell lines used in this study were the
(
following: PC-3 (human prostate cancer cell line), MCF-7 (human breast
cancer cell line), HCT-15 (human colon cancer cell line) and J774
(noncancerous murine macrophages) for comparison.
The results from this study showed that steroid 14a, having a carbamate
function at C-17, was the most potent against PC-3 cell line (96.6%) while 8c
and 8e showed much higher cytotoxic activity (100%) for MCF-7 cell line.
Finally, compounds 8c and 14a displayed selective properties towards tumor
cell lines than noncancerous murine macrophages.
2

ACCEPTED MANUSCRIPT
Key words: Cytotoxic effect; synthesis; progesterone derivatives; esters;
carbamates.
3

ACCEPTED MANUSCRIPT
1
. Introduction
The past decade has witnessed a quiet revolution in the field of cancer
hormone therapy. Although there have been no spectacular breakthroughs as
were seen in the 1940s with the discovery of penicillin and other antibiotics
for the treatment of infectious diseases, a wealth of basic knowledge,
improved investigation techniques, and a substantial number of new
antineoplastic agents have emerged.
Steroids are important physiological and pharmacological regulators of cell
growth and survival. There are several examples in the literature that describe
the use of natural [1] and synthetic [2] steroids for the treatment of sex
hormone-dependent cancer tumors, including breast cancer [3], endometrial
cancer [4], cervical cancer [5] and prostate cancer [6]. These hormone-
dependent tumor cells can control tumor growth through hormone receptors
antagonist whereas in sex hormone-independent cancer [3-6], some synthetic
steroids have been previously described as indicators of apoptosis [7].
The pharmacological studies using synthetic steroids offer an important
strategy for tumor control growth and tumor cell apoptosis. In this respect, 16-
dehydropregnenolone acetate (1, Fig. 1) offers possibilities for the synthesis of
a variety of compounds having anticancer activity [8]. The results from these
4

ACCEPTED MANUSCRIPT
investigations inspired us to synthesize a number of new progesterone (2, Fig.
1
) derivatives having an ester or a carbamate moiety at C-3 (series 1, Fig. 2)
and in the second series (Fig. 3), these groups are bound to C-17 of the
progesterone skeleton. In compounds 8b−e the ester moiety is a p-substituted
phenyl ring containing electronegative atoms (F, Cl, Br, I, and unsubstituted
phenyl ring 8a). Steroidal derivatives 8a−e and 9a−e have two
carbonyl function at C-6 and C-20. Compounds 13a−e and 14a−e have an
-conjugated carbonyl group at C-3.
α,β-unsaturated
α,β,γ,δ
The steroidal derivatives synthesized in this work were tested in vitro, against
a panel of human cancer cell lines such as PC-3 (prostate), MCF-7 (breast),
HCT-15 (colon); and J774 (noncancerous murine cells) for comparison.
In addition, ketoconazole (3, Fig. 1), an antimycotic drug that has previously
been described as an agent that inhibits cytochrome P450 enzymes and which
are required for the synthesis of androgens [9], was used as positive control.
This compound has also shown cytotoxic activity in prostate cancer cell lines
in the in vitro studies. Furthermore, it has been previously demonstrated that at
low dose, compound 3 can be used for the treatment of prostate cancer which
has failed androgen deprivation therapy [10-12].
5

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2
. Results and discussion
.1. Chemistry
The synthetic strategy for the preparation of the steroidal derivatives 8a−e,
a−e, 13a−e and 14a−e are outlined in Fig. 2 and Fig. 3, respectively.
2
9
Compounds 8a−e and 9a−e were prepared in the following manner:
commercially available 16-dehydropregnenolone acetate (1) was treated with
m-chloroperoxybenzoic acid to form the 5
oxidation of 4 with chromic anhydride in acetone-water afforded the 5
ketoalcohol 5. Elimination of the hydroxyl group at C-4 in 5 with thionyl
chloride yielded the -unsaturated carbonyl compound (6). The acetoxy
α,6α-epoxy compound 4. The
α
-
α,β
group in 6 was hydrolyzed with sodium hydroxide in methanol to afford the
alcohol 7. The final compounds 8a−e were obtained from 7 by using the
Steglich esterification method [13]. These esters were obtained with a high
yield (75-79%). The synthesis of 9a−e having a carbamate function at C-3 was
carried out with compound 7, the corresponding phenyl isocyanate and copper
bromide in tetrahydrofuran. The desired carbamates 9a−e were obtained with
a yield of 9-32%.
The second series of compounds 13a−e and 14a−e was prepared from the
commercially available 17α-acetoxyprogesterone (10). Treatment of 10 with
chloranil, acetic acid in toluene afforded the
α
,β,
γ,
δ
-unsaturated carbonyl
6

ACCEPTED MANUSCRIPT
derivative 11. Compound 11 upon treatment with sodium hydroxide in
methanol afforded the alcohol 12. Steroid 12 was reacted with the
corresponding acid using the Steglich procedure for ester formation 13a−e. On
the other hand when 12 was treated with the corresponding phenylisocyanate
(
this procedure is described above for the preparation of 9a−e) to form the
desired isocyanate, these compounds (14a−e) were obtained with a yield of
0-75%. These derivatives were isolated by column chromatography using as
5
a stationary phase florisil. On the other hand the carbamates 9a-e were
isolated by a silica gel column chromatography with a lower yield.
Apparently, the florisil stationary phase purifies much better the carbamate
derivatives as compared to silica gel. All intermediates and final compounds
1
13
are characterized by IR, H and C NMR and mass spectrometry.
7

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2
.2. Cytotoxic Effect
The differences in the cytotoxic activity of compounds 8c and 8e having an
ester moiety at C-3 for PC-3, MCF-7, and HCT-15 cancer cell lines and their
counterparts 13c and 13e (ester function at C-17) could be explained on the
ground of the higher electrophilic character of compounds 8c and 8e. It has
been reported that electrophilic centers in
readily with a variety of nucleophilic amino acids to form a Michael type
addition products [14]. Apparently, compounds 7 and 8a−e having two α,β-
α,β-unsaturated ketones react very
unsaturated ketone moieties could react very easily with the nucleophilic part
of the cell membrane and form an adduct, thus showing an important
cytotoxic effect. There are several nucleophilic amino acids having a free
nucleophilic group such as lysine (NH ), cysteine (SH) and serine (OH) which
2
is not involved in the peptide formation and thus it can react with the
electrophilic part of the steroid. As a result of this adduct formation, the
steroidal molecule shows a higher cytotoxic effect. This phenomenon explains
very well the higher cytotoxic effect of derivatives 8c and 8e for PC-3, MCF-
7
, and HCT-15 cell lines as compared to that of steroids 13c and 13e.
The low activity of compounds 9a−e (carbamate function at C-3) and 14a−e
carbamate function at C-17) could be explained on the basis of their low
(
solubility. On the other hand, compound 14a having an unsubstituted phenyl
8

ACCEPTED MANUSCRIPT
ring showed a potent cytotoxic activity against PC-3 cancer cell line (96.6%)
and also exhibited low inhibition of macrophages growth (48.2%). Apparently
this compound which lacks a halogen substituent on the phenyl ring (higher
solubility) shows certain selectivity for the inhibition of cancerous cells.
From this study, we can infer that compounds having an
α,β-unsaturated
function (electrophilic center) show high cytotoxic effect due to their ability to
react with the nucleophilic portion of the protein (lysine, cysteine and serine)
and thus can form a stable adduct. The compounds of series 1 which contain
two
α
,
β
-unsaturated carbonyl functions produce higher cytotoxicity as
-unsaturated carbonyl.
compared to those of series 2 having only one
α,β,γ,δ
9

ACCEPTED MANUSCRIPT
a
Table 1. Inhibitory activity on the growth of several cancer cell lines and murine
macrophages of series 8a−e, 9a−e, 13a−e and 14a−e.
Structure
Compound
3
PC-3
MCF-7
HCT-15
J774
Ketoconazole
88.48±3.9
95.16±4.8
90.59±1.2
53.95±0.6
7
70.6±3.3
2.9±0.9
63.5±3.4
32.7±1.2
20.8±4.5
100±2.5
86.2±13.7
100±4.3
67.7±5.9
48.7±15.1
22.8±7.7
88.2±6.9
55.9±31.4
67.3±4.8
98.9±0.2
89.3±0.8
50.0±3.6
38.78±1.5
60.87±1.2
61.15±3.6
8a (X= H)
8b (X= F)
8c (X= Cl)
8d (X= Br)
8e (X= I)
8.3±1.0
87.3±11.2
51.9±29.3
64.1±28
9a (X=H)
9b (X=F)
9c (X=Cl)
9d(X=Br)
9e (X=I)
4.5±2.0
34.7±6.2
34.6±1.7
13.7±5.3
14.0±2.5
33.1±3.9
41.4±8.8
60.1±6.8
65.3±16.1
30.0±0.8
43.2±6.4
73.8±0.2
78.3±2.2
92.4±2.1
80.5±0.6
70.7±1.0
33.0±5.4
63.4±10.9
26.9±2.4
42.8±1.5
1
1
1
1
1
1
2
39.6±2.7
42.49±4.7
30.92±3.5
46.09±6.4
84.71±8.1
30.8±3.0
27.8±1.9
26.80±2.3
29.16±9.7
39.76±3.2
44.44±3.7
22.6±4.7
25.0±4.9
50.82±4.3
35.59±1.7
39.32±0.6
53.87±4.2
27.5±3.5
58.5±0.5
100±3.8
3a (X= H)
3b (X= F)
3c (X= Cl)
3d (X= Br)
3e (X= I)
100±1.9
73.64±1.4
56.97±1.8
74.3±0.3
1
1
1
4a (X=H)
4b (X=F)
4c (X=Cl)
96.6±3.0
33.4±8.4
15.8±5.3
26.4±4.0
17.8± 2.7
51.3±9.5
20.0±0.1
11.2±3.4
13.1±2.0
45.3±3.8
NA
48.2±0.7
43.4±2.5
36.9±3.8
32.0±1.7
21.7±0.8
O
O
NA
O
X
N
H
14d (X=Br)
14e (X=I)
NA
b
1
4a-e
NA
NA
O
a
Results are expressed as a percentage of inhibition ± standard error.
These compounds were not active.
b
1
0

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3
. Experimental
3
.1. General
Reagents and solvents were purchased from commercial sources and were
used without further purification. Melting points were determined on a Fisher
1
13
Johns melting point apparatus and are uncorrected. H and C NMR were
taken on a Varian Gemini 200 and a VRX-300, respectively. Chemical shifts
are given in ppm relative to that of Me Si (δ=0) in CDCl . High Resolution
4
3
Mass Spectra (HRMS) were obtained with a Thermo DFS spectrometer by
+
direct infusion and using FAB ionization mode. IR spectra were recorded on
a Perkin-Elmer 200 spectrometer.
3
3
.2. Synthesis of esters and carbamates 8a−e and 9a−e
.2.1. 3β-Acetoxy-5α,6α-epoxypregn-16-ene-20-one (4)
A solution of steroid 1 (1 g, 2.82 mmol) and m-chloroperbenzoic acid (1.62 g,
.4 mmol) in chloroform (50 mL) was stirred at room temperature for 30 min.
9
After this time, a saturated aqueous solution of sodium bicarbonate (84 mL)
containing sodium bisulfite (1 g) was added. The reaction mixture was
extracted three times with chloroform; the organic phase was washed with
1
1

ACCEPTED MANUSCRIPT
water, dried over sodium sulfate and the solvent was removed in vacuum. The
crude product was recrystallized from methanol to give 4. Yield 97% (1.01g),
−1
1
mp 170-172 °C. IR (KBr) cm : 2943, 1734, 1665 and 1588. H NMR
CDCl3) : 0.8 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.0 (s, 3H, H-21), 2.2 (s, 3H,
acetoxy), 2.9 (d, 1H, J = 2 Hz, H-6), 4.7 (d, 1H, J = 2 Hz, H-3). C NMR
CDCl3) : 15.8 (C-18), 16.9 (C-19), 21.3 (C-21), 27.2 (acetoxy), 63.2 (C-6),
5.3 (C-5), 144.1 (C-16), 155.2 (C-17), 170.5 (acetoxy carbonyl), 196.7 (C-
0). HRMS cal for C H O 372.2301, found 372.2311.
(
δ
1
3
(
δ
6
2
23
32
4
3
.2.2. 3β-Acetoxy-5α-hydroxypregn-16-ene-6,20-dione (5)
To a solution of steroid 4 (1 g, 2.7 mmol) in acetone (50 mL) was added
dropwise a solution of chromium trioxide (1.05 g, 10.5 mmol) in water (5 mL)
at 0 °C during 10 min. The resulting mixture was allowed to warm up to room
temperature and after 30 minutes the same amount of chromium trioxide was
added in the same manner. The mixture was diluted with cold water (150 mL)
and the precipitated product was filtered and dried. The product was
recrystallized from methanol. Yield 81% (0.848 g), mp 244-245 °C. IR (KBr)
−1
1
cm : 3409, 2940, 1730, 1700, 1685 and 1600. H NMR (CDCl )
δ
: 0.8 (s, 3H,
3
H-18), 1.1 (s, 3H, H-19), 2.0 (s, 3H, H-21), 2.2 (s, 3H, acetoxy), 4.8(m, 1H,
13
H-3), 6.6 (q, 1H, J = 2 Hz, H-16). C NMR (CDCl ) δ: 15.8 (C-18), 17.0 (C-
3
1
2

ACCEPTED MANUSCRIPT
1
9), 22.0 (C-21), 28.0 (acetoxy), 80.0 (C-5), 143.8 (C-16), 155.0 (C-17), 173.0
(
acetoxy carbonyl), 197.2 (C-20), 212.8 (C-6). HRMS cal for C H O
2
3
32
5
3
88.2550, found 388.2542.
3
.2.3. 3β-Acetoxypregn-4,16-diene-6,20-dione (6)
To a cold solution of steroid 5 (1 g, 2.57 mmol) in a mixture of pyridine (10
mL) and chloroform (5 mL) was added dropwise thionyl chloride (1 mL)
under a nitrogen atmosphere. The resulting solution was stirred at room
temperature for 45 min. Iced water (100 mL) was added and it was extracted
three times with ethyl acetate. The organic phase was washed with 10%
aqueous hydrochloric acid, 5% aqueous sodium bicarbonate and water. It was
dried over sodium sulfate and the solvent was evaporated in vacuum. The
crude product was recrystallized from ethyl acetate-hexane. Yield 66%
−
1
1
(
0.64g), mp 193–195 °C. IR (KBr) cm : 2942, 1735, 1691 and 1635. H
NMR (CDCl3) : 0.9 (s, 3H, H-18), 1.0 (s, 3H, H-19), 2.1 (s, 3H, H-21), 2.3 (s,
H, acetoxy), 5.1 (m, 1H, H-3), 6.0 (q, 1H, J = 2 Hz, H-4), 6.7 (q, 1H, J = 2
δ
3
1
3
Hz, H-16). C NMR (CDCl )
δ: 15.7 (C-18), 19.6 (C-19), 22.0 (C-21), 29.5
3
(acetoxy), 70.1 (C-3), 129.0 (C-4), 143.7 (C-16), 147.2 (C-5), 154.6 (C-17),
1
3
70.6 (acetoxy carbonyl), 195.0 (C-20), 201.7 (C-6). HRMS cal for C H O
2
3
30
4
70.2144, found 370.2551.
1
3

ACCEPTED MANUSCRIPT
3
.2.4. 3
β
-Hydroxypregn-4,16-diene-6,20-dione (7)
To a solution of steroid 6 (1 g, 2.7 mmol) in methanol (150 mL) was added an
aqueous sodium hydroxide solution (2%, 10 mL) and stirred at room
temperature for 20 min. Upon termination of the hydrolysis, the methanol was
evaporated and water (200 mL) was added. The precipitated product was
filtered and recrystallized from methanol. Yield 60% (0.53g), mp 168–170 °C.
−1
1
IR (KBr) cm : 3430, 2941, 1688, 1663 and 1630. H NMR (CDCl )
δ: 0.9 (s,
3
3
H, H-18), 1.0 (s, 3H, H-19), 2.2 (s, 3H, H-21), 4.3 (t, 1H, J = 2 Hz, H-3), 6.2
1
3
(
q, 1H, J = 2, H-4), 6.7 (q, 1H, J=2, H-16). C NMR (CDCl )
δ
: 15.7 (C-18),
9.8 (C-19), 27.1(C-21), 67.1 (C-3), 133.2 (C-4), 143.9 (C-16), 146.5 (C-5),
54.8 (C-17), 196.6 (C-20), 202.4 (C-6). HRMS cal for C H O 328.2038,
3
1
1
21
28
3
found 328.2032.
3
.2.5. General procedure for the synthesis of esters 8a−e
To solution of steroid 7 (1 mmol), DCC (3 mmol) and DMAP (2 mmol) in
chloroform (6 mL) was added the corresponding acid (5 mmol). The resulting
solution was stirred at room temperature for 2 h. Ethyl acetate (5 mL) was
added and the precipitated dicyclohexyl urea was filtered. The organic phase
1
4

ACCEPTED MANUSCRIPT
was washed three times with 10% aqueous hydrochloric acid, 5% aqueous
sodium bicarbonate, water and was dried over anhydrous sodium sulfate. The
crude ester was recrystallized from methanol.
3
.2.6. 3β-Benzoyloxypregn-4,16-diene-6,20-dione (8a)
−1
Yield 77% (0.335g), mp 168-170 °C. IR (KBr) cm : 3072, 2944, 1718, 1686
1
and 1662. H NMR (CDCl )
δ: 0.9 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.3 (s, 3H,
3
H-21), 5.5 (t, 1H, J = 2 Hz, H-3), 6.2 (q, 1H, J = 2 Hz, H-4), 6.7 (m, 1H, H-
1
3
1
6), 7.4 (m, 1H, Ar-H), 7.5 (m, 1H, Ar-H) and 8.0 (m, 2H, Ar-H). C NMR
CDCl3) : 15.7 (C-18), 19.6 (C-19), 27.1 (C-21), 70.0 (C-3), 128.3 (Ar-C),
29.1 (C-4), 129.7 (Ar-C), 130.0 (Ar-C), 133.0 (Ar-C), 143.7 (C-16), 147.0
(
δ
1
(C-5), 154.8 (C-17), 166.1 (ester carbonyl), 196.5 (C-20), 201.5 (C-6). HRMS
cal for C H O 432.2301, found 432.2298.
28
32
4
3
.2.7. 3β-p-Fluorobenzoyloxpregn-4,16-diene-6,20-dione (8b)
−1
Yield 75% (0.34 g), mp 129-131 °C. IR (KBr) cm : 3086, 2944, 1720 and
1
1
2
691. H NMR (CDCl )
δ
: 0.9 (s, 3H, H-18), 1.0 (s, 3H, H-19), 2.2 (s, 3H, H-
3
1), 5.5 (t, 1H, J = 2 Hz, H-3), 6.2 (q, 1H, J = 2 Hz, H-4), 6.7 (t, 1H, J= 2 Hz,
1
3
H-16), 7.1 (t, 1H, J=8.6 Hz, Ar-H), 8.1 (m, 1H, Ar-H). C NMR (CDCl )
δ:
3
1
5.8 (C-18), 19.6 (C-19), 27.1 (C-21), 70.0 (C-3), 115.4 (Ar-C), 126.3 (Ar-C),
1
5

ACCEPTED MANUSCRIPT
1
28.9 (C-4), 132.2 (Ar-C), 143.7 (C-16), 148.1 (C-5), 154.9 (C-17), 165.2
(ester carbonyl), 167.1 (Ar-C), 196.5 (C-20), 201.8 (C-6). HRMS cal for
C H FO 450.2206, found 450.2209.
2
8
31
4
3
.2.8. 3
β
-p-Chlorobenzoyloxypregn-4,16-diene-6,20-dione (8c)
−1
Yield 76% (0.354 g), mp 238–240 °C. IR (KBr) cm : 3077, 2940, 1719, 1692
1
and 1660. H NMR (CDCl )
δ: 0.9 (s, 3H, H-18), 1.8 (s, 3H, H-19), 2.2 (s, 3H,
3
H-21), 5.5 (m, 1H, H-3), 6.2 (s, 1H, H-4), 6.7 (m, 1H, H-16), 7.4 (d, 2H, J=
1
3
8
1
.6 Hz, Ar-H), 7.9 (m, 2H, Ar-H). C NMR (CDCl )
δ
: 15.8 (C-18), 19.6 (C-
3
9), 27.1 (C-21), 70.5 (C-3), 128.5 (Ar-C), 128.7 (Ar-C), 131.1 (Ar-C), 139.5
(Ar-C), 128.9 (C-4), 143.7 (C-16), 148.0 (C-5), 154.9 (C-17), 165.3 (ester
carbonyl), 196.5 (C-20), 201.8 (C-6). HRMS cal for C H ClO 466.1911,
2
8
31
4
found 466.1903.
3
.2.9. 3β-p-Bromobenzoyloxypregn-4,16-diene-6,20-dione (8d)
−1
Yield 79% (0.4 g), mp 235–237 °C. IR (KBr) cm : 3074, 2940, 1719, 1691
1
and 1660. H NMR (CDCl )
δ: 0.9 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.2 (s, 3H,
3
H-21), 5.6 (m, 1H, H-3), 6.2 (s, 1H, H-4), 6.7 (m, 1H, H-16), 7.6 (d, 2H, J=
1
3
7
1
.8 Hz, Ar-H), 7.9 (m, 2H, Ar-H). C NMR (CDCl )
δ
: 15.8 (C-18), 19.6 (C-
3
9), 27.1 (C-21), 70.1 (C-3), 128.2 (Ar-C), 128.9 (Ar-C) 128.9 (C-4), 131.2
1
6

ACCEPTED MANUSCRIPT
(Ar-C), 131.7 (Ar-C), 143.7 (C-16), 148.2 (C-5), 154.8 (C-17), 165.6 (ester
carbonyl), 196.5 (C-20), 201.5 (C-6). HRMS cal for C H BrO 510.1406,
2
8
31
4
found 510.1399.
3
.2.10. 3β-p-Iodobenzoyloxypregn-4,16-diene-6,20-dione (8e)
−1
Yield 76% (0.424g), mp 229-231 °C. IR (KBr) cm : 3097, 2946, 1709, 1687
1
and 1657. H NMR (CDCl )
δ: 0.9 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.2 (s, 3H,
3
H-21), 5.6 (m, 1H, H-3), 6.2 (s, 1H, H-4), 6.7 (m, 1H, H-16), 7.7 (s, 2H, Ar-
1
3
H), 7.7 (m, 2H, Ar-H). C NMR (CDCl )
δ
: 15.7 (C-18), 19.6 (C-19), 27.1
3
(C-21), 70.0 (C-3), 100.9 (Ar-C), 128.7 (Ar-C), 129.5 (C-4), 131.1 (Ar-C),
1
1
5
37.6 (Ar-C), 143.7 (C-16), 147.9 (C-5), 154.8 (C-17), 165.6 (ester carbonyl),
96.5 (C-20), 201.5 (C-6). HRMS cal for C H IO 558.1267, found
2
8
31
4
58.1258.
3
.2.11. General procedure for the synthesis of carbamates 9a−e
To a solution of compound 7 (1 mmol) and CuI (0.1 mmol) in dry THF (4
mL) under nitrogen atmosphere was added the corresponding isocyanate (3
mmol). The reaction mixture was stirred at room temperature for 24 h.
Chloroform was added and the catalyst CuI was filtered. The organic layer
was washed with 10% aqueous hydrochloric acid, 5% of aqueous sodium
bicarbonate, water and was dried over anhydrous sodium sulfate. Purification
1
7

ACCEPTED MANUSCRIPT
by silica gel column chromatography using a mixture of hexane and ethyl
acetate (9:1) afforded the 9a−e carbamate derivatives.
3
.2.12. 3β-Phenylcarbamoiloxypregn-4,16-diene-6,20-dione (9a)
−1
Yield 32% (0.143g), mp 252-254 °C. IR (KBr) cm : 3331, 1721, 1687 and
1
1
2
662. H NMR (CDCl )
δ
: 0.9 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.2 (s, 3H, H-
3
1), 5.3 (m, 1H, H-3), 6.1 (s, 1H, H-4), 6.7 (s, 1H, H-16), 6.9 (s, 1H, N-H), 7.0
13
(
m, 1H, Ar-H), 7.3 (m, 2H, Ar-H), 7.3(m, 2H, Ar-H). C NMR (CDCl ) δ:
3
1
1
5.7 (C-18), 19.5 (C-19), 27.1 (C-21), 70.0 (C-3), 118.7 (Ar-C), 129.0 (Ar-C),
29.1 (C-4), 129.3 (Ar-C), 137.6 (Ar-C), 143.8 (C-16), 147.9 (C-5), 152.9
(carbamate carbonyl), 154.8 (C-17), 196.6 (C-20) and 201.9 (C-6). HRMS cal
for C H NO 447.2410, found 447.2415.
28
33
4
3
.2.13. 3
β
-p-Fluorophenylcarbamoiloxypregn-4,16-diene-6,20-dione (9b)
−1
Yield 29% (0.135g), mp 224-227 °C. IR (KBr) cm : 3335, 1724, 1685 and
1
1
2
7
2
662. H NMR (CDCl )
δ
: 0.9 (s, 3H, H-18), 1.0 (s, 3H, H-19), 2.1 (s, 3H, H-
3
1), 5.2 (m, 1H, H-3), 6.0 (s, 1H, H-4), 6.9 (s, 1H, H-16), 7.1 (m, 2H, Ar-H),
13
.4 (m, 2H, Ar-H). C NMR (CDCl )
δ
: 15.6 (C-18), 19.6 (C-19), 27.1 (C-
1), 74.3 (C-3), 120.3 (Ar-C), 120.5 (Ar-C), 128.5 (C-4), 133.6 (Ar-C), 141.1
C-16), 150.3 (C-5), 152.8 (carbamate carbonyl), 156.7 (C-17), 163.7 (Ar-C),
3
(
1
8

ACCEPTED MANUSCRIPT
2
4
01.3 (C-20) and 206.0 (C-6). HRMS cal for C H FNO 465.2315, found
2
8
32
4
65.2306.
3
.2.14. 3β-p-Chlorophenylcarbamoiloxypregn-4,16-diene-6,20-dione (9c)
−1
Yield 23% (0.11 g), mp 221-223 °C. IR (KBr) cm : 3365, 1726, 1683 and
1
1
2
662. H NMR (CDCl )
δ
: 0.9 (s, 3H, H-18), 1.0 (s, 3H, H-19), 2.2 (s, 3H, H-
3
1), 5.3 (m, 1H, H-3), 6.2 (s, 1H, H-4), 6.7 (s, 1H, H-16), 6.8 (s, 1H, N-H), 7.2
1
3
(
m, 2H, Ar-H), 7.3(m, 2H, Ar-H). C NMR (CDCl )
δ
: 15.6 (C-18), 19.6 (C-
9), 27.1 (C-21), 70.2 (C-3), 119.9 (Ar-C), 128.9 (C-4), 129.0 (Ar-C), 135.0
Ar-C), 136.4 (Ar-C), 143.8 (C-16), 148.0 (C-5), 152.9 (carbamate carbonyl),
3
1
(
1
4
54.9 (C-17), 196.6 (C-20) and 201.8 (C-6). HRMS cal for C H ClNO
2
8
32
4
81.2020, found 481.2030.
3
.2.15. 3β-p-Bromophenylcarbamoiloxypregn-4,16-diene-6,20-dione (9d)
−1
Yield 9% (0.047 g), mp 201-203 °C. IR (KBr) cm : 3294, 1727, 1692 and
1
1
2
7
7
663. H NMR (CDCl )
δ
: 0.9 (s, 3H, H-18), 1.2 (s, 3H, H-19), 2.1 (s, 3H, H-
3
1), 5.3 (m, 1H, H-3), 5.9 (s, 1H, H-4), 6.0 (s, 1H, H-16), 7.4 (m, 2H, Ar-H),
13
.5(m, 2H, Ar-H). C NMR (CDCl )
δ
: 15.6 (C-18), 19.6 (C-19), 27.1 (C-21),
5.31 (C-3), 120.0 (Ar-C), 121.0 (Ar-C), 127.0 (C-4), 133.6 (Ar-C), 139.2
Ar-C), 144.1 (C-16), 152.7 (C-5), 156.7 (carbamate carbonyl), 153.2 (C-17),
3
(
1
9

ACCEPTED MANUSCRIPT
2
5
05.9 (C-20) and 212.6 (C-6). HRMS cal for C H BrNO 525.1515, found
2
8
32
4
25.1507.
3
.2.16. 3β-p-Iodophenylcarbamoiloxypregn-4,16-diene-6,20-dione (9e)
−1
Yield 9% (0.051 g), mp 197-199 °C. IR (KBr) cm : 3327, 1726, 1695 and
1
1
2
7
2
663. H NMR (CDCl )
δ
: 0.9 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.2 (s, 3H, H-
3
1), 5.3 (m, 1H, H-3), 5.7 (s, 1H, H-4), 6.1 (s, 1H, H-16), 7.3 (m, 2H, Ar-H),
13
.5 (m, 2H, Ar-H). C NMR (CDCl )
δ
: 15.7 (C-18), 19.6 (C-19), 27.1 (C-
1), 72.1 (C-3), 119.5 (Ar-C), 128.1 (C-4), 129.1 (Ar-C), 134.5 (Ar-C), 139.3
Ar-C), 144.2(C-16), 148.6 (C-5), 152.1 (carbamate carbonyl), 154.0 (C-17),
3
(
1
5
96.2 (C-20) and 202.0 (C-6). HRMS cal for C H INO 573.1376, found
2
8
32
4
73.1384.
3
3
.3. Synthesis of esters and carbamates 13a−e and 14a−e
.3.1. 17α-acetoxypregn-4,5-dien-3,20-dione (11)
Compound 10 (1 g, 2.68mmol) and chloranil (0.7 g, 2.8 mmol) were dissolved
in a mixture of toluene (2 mL) and glacial acetic acid (8 mL) and was allowed
to reflux for 4 h. To the reaction was added cold water (10 mL) and the
precipitated tetrachlorohydroquinone was filtered. The compound was
extracted with ethyl acetate and the organic layer was washed with an aqueous
2
0

ACCEPTED MANUSCRIPT
solution of sodium hydroxide (11%) and distilled water. The organic phase
was dried over sodium sulfate and evaporated under reduced pressure. The
compound was recrystallized from ethyl acetate. Yield 70% (0.696 g), mp
−
1
1
2
24-226 °C. IR (KBr) cm : 2978, 1733, 1718, 1652 and 1620. H NMR
CDCl3) : 0.7 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.0 (s, 3H, acetoxy), 2.1 (s,
H, H-21), 5.7 (s, 1H, H-4), 6.1 (dd, 1H, J= 10 and 1.2 Hz, H-6) and 6.1 (dd,
(
δ
3
1
2
1
3
H, J= 9.6 and 2 Hz, H-7). C NMR (CDCl )
δ
: 14.3 (C-18), 16.3 (C-19),
3
6.4 (C-21), 123.9 (C-4), 128.3 (C-6), 140.0 (C-7), 163.1 (C-5), 170.6
(
acetoxy carbonyl), 199.3 (C-3) and 203.8 (C-20). HRMS cal for C H O
2
3
30
4
3
70.2144, found 370.2138.
3
.3.2. 17α-Hidroxypregn-4,5-dien-3,20-dione (12)
Compound 11 (1g, 2.703 mmol) was dissolved in methanol (50 mL) and an
aqueous solution of sodium hydroxide (2%, 50 mL) was added. The mixture
was heated under reflux for 2 h, the excess of methanol was evaporated and
the compound was precipitated with water and filtered. The desired product
was recrystallized from methanol. Yield 62% (0.55 g), mp 254-256 °C. IR
−1
1
(
KBr) cm : 3012, 3026, 2945, 1704 and 1644. H NMR (CDCl )
δ
: 0.8 (s,
3
3
2
H, H-18), 1.1 (s, 3H, H-19), 4.9 (s, 1H, H-17), 5.6 (s, 1H, H-4) and 6.1 (s,
13
H, H-6 y H-7). C NMR (CDCl ) δ: 15.2 (C-18), 16.2 (C-19), 27.8 (C-21),
3
2
1

ACCEPTED MANUSCRIPT
8
9.5 (C-17), 123.7 (C-4), 128.1 (C-6), 140.5 (C-7), 163.4 (C-5), 199.4 (C-3)
and 211.2 (C-20). HRMS cal for C H O 328.2038, found 328.2047.
2
1
28
3
3
.3.3. General procedure for the synthesis of esters 13a−e
To a solution of the corresponding acid (2.1 mmol) and PTSA (0.33 mmol) in
chloroform was added trifluoroacetic anhydride (8.15 mmol) and the mixture
was stirred for 3 h. After this time, the compound 12 (1 mmol) was added and
stirred until the reaction was completed. Chloroform was added (3 mL) and
subsequently was washed with a saturated aqueous solution of sodium
bicarbonate, the organic phase was separated and dried over sodium sulfate.
The solvent was evaporated under reduced pressure. Purification by basic
aluminum column chromatography using a mixture of hexane and ethyl
acetate (8:2) gave the series 13a−e.
3
.3.4. 17α-Benzoiloxypregn-4,6-dien-3,20-dione (13a)
−1
Yield 34% (0.146 g), mp 230-231 °C. IR (KBr) cm : 3054, 3025, 2946, 1713
1
and 1658. H NMR (CDCl )
δ: 0.8 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.1 (s, 3H,
3
H-21), 5.7 (s, 1H, H-4), 6.1 (s, 2H, H-6 y H-7), 7.4 (t, 2H, J = 7.8 and 1.6 Hz,
Ar-H),7.6 (t, 1H, J = 7.4 and 1.44 Hz, Ar-H) and 8.0 (d, 2H, J= 8.4 and 1.2
1
3
Hz, Ar-H). C NMR (CDCl )
δ: 14.4 (C-18), 16.4 (C-19), 26.4 (C-21), 124.0
3
(C-4), 128.4 (C-6), 128.6 (Ar-C), 129.7 (Ar-C), 129.8 (Ar-C), 133.6 (Ar-C),
2
2

ACCEPTED MANUSCRIPT
1
40.1 (C-7), 163.1 (C-5), 165.1 (C-17), 199.4 (C-3) and 203.7 (C-20). HRMS
cal for C H O 432.2301, found 432.2307.
28
32
4
3
.3.5. 17α-p-Fluorobenzoiloxypregn-4,6-dien-3,20-dione (13b)
−1
Yield 33% (0.148 g), mp 248-251 °C. IR (KBr) cm : 3080, 2940, 1718 and
1
1
2
660. H NMR (CDCl )
δ
: 0.7 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.0 (s, 3H, H-
3
1), 5.7 (s, 1H, H-4), 6.1 (d, 1H, 3J = 8.8 Hz, H-6), 6.1 (t, 1H, J = 8.8 and 2.4
1
3
Hz, H-7) , 7.1 (m, 2H, Ar-H), 8.0 (m, 2H, Ar-H). C NMR (CDCl )
δ
: 14.9
C-18), 16.8 (C-19), 26.9 (C-21), 116.1 (d, JC-F = 21.9 Hz, C-F, Ar-C), 124.2
C-4), 128.5 (C-6), 132.4 (d, J = 9.1 Hz, C-F, Ar-C), 140.2 (C-7), 163.2 (C-
3
(
(
C-F
5
), 165.1 (C-17), 162.2 (d, J = 253.3 Hz, C-F, Ar-C), 199.4 (C-3) and 203.7
C-F
(
C-20). HRMS cal for C H FO 450.2206, found 450.2219.
2
8
31
4
3
.3.6. 17α-p-Chlorobenzoiloxypregn-4,6-dien-3,20-dione (13c)
−1
Yield 28% (0.130 g), mp 216-218 °C. IR (KBr) cm : 3078, 3004, 2970, 1717
1
and 1677. H NMR (CDCl )
δ: 0.8 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.1 (s, 3H,
3
H-21), 5.7 (s, 1H, H-4), 6.1 (m, 2H, H-6 y H-7), 7.4 (d, 2H, J = 8.8 Hz, Ar-H),
1
3
7
.9 (d, 2H, J = 8.4, Ar-H). C NMR (CDCl )
δ: 14.5 (C-18), 16.4 (C-19), 26.5
3
(C-21), 124.2 (C-4), 128.5 (C-6), 129.1 (Ar-C), 131.0 (Ar-C), 139.7 (C-7),
2
3

ACCEPTED MANUSCRIPT
1
40.2 (C-5), 165.1 (C-17), 199.0 (C-3) and 201.9 (C-20). HRMS cal for
C H ClO 466.1911, found 466.1904.
2
8
31
4
3
.3.7. 17
α
-p-Bromobenzoiloxypregn-4,6-dien-3,20-dione (13d)
−1
Yield 22.5% (0.112 g), mp 196-199 °C. IR (KBr) cm : 3061, 2947, 1780 and
1
1
2
663. H NMR (CDCl )
δ
: 0.8 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.1 (s, 3H, H-
3
1), 5.7 (s, 1H, H-4), 6.1 (m, 2H, H-6 y H-7), 7.6 (d, 2H, J = 8.2 and 2.2 Hz,
1
3
Ar-H), 7.8 (d, 2H, J = 8.8 and 2.1 Hz, Ar-H). C NMR (CDCl )
δ
: 14.5 (C-
8), 16.4 (C-19), 26.5 (C-21), 124.2 (C-4), 128.8 (C-6), 129.1 (Ar-C), 131.1
Ar-C), 132.1 (Ar-C), 139.7 (C-7), 139.8 (Ar-C) 157.6 (C-5), 165.9 (C-17),
3
1
(
1
5
97.2 (C-3) and 213.7 (C-20). HRMS cal for C H BrO 510.1406, found
2
8
31
4
10.1416.
3
.3.8. 17α-p-Iodobenzoiloxy-4,6-pregnadien-3,20-dione (13e)
−1
Yield 20% (0.11 g), mp 267-269 °C. IR (KBr) cm : 3060, 2940, 1716 and
1
1
2
665. H NMR (CDCl )
δ
: 0.8 (s, 3H, H-18), 1.2 (s, 3H, H-19), 2.1 (s, 3H, H-
3
1), 5.8 (s, 1H, H-4), 6.1 (m, 2H, H-6 y H-7), 7.5 (m, 2H, Ar-H), 8.1 (m, 2H,
13
Ar-H). C NMR (CDCl )
δ: 14.9 (C-18), 17.4 (C-19), 26.3 (C-21), 97.0 (C-
3
1
7), 97.9 (Ar-C), 123.7 (C-4), 128.1 (C-6), 128.6 (Ar-C), 129.6 (Ar-C), 135.5
2
4

ACCEPTED MANUSCRIPT
(Ar-C), 140.5 (C-7), 157.5 (C-5), 199.3 (C-3) and 208.8 (C-20). HRMS cal for
C H IO 558.1267, found 558.1258.
2
8
31
4
3
.3.9. General procedure for the synthesis of carbamates 14a−e
Compound 12 (1 mmol), copper bromide-dimethyl sulfide complex (3 mmol)
and the corresponding isocyanate (6 mmol) were dissolved in anhydrous THF
(3 mL). The mixture was stirred under nitrogen atmosphere for 48 h at room
temperature. Upon termination of the reaction, chloroform (5 mL) was added
and the complex was filtered. The organic layer was washed with water and
dried over sodium sulfate. The solvent was evaporated under reduced
pressure. Purification by florisil column chromatography using a mixture of
hexane and ethyl acetate gave the desired carbamates 14a−e.
3
.3.10. 17α-Phenylcarbamoiloxypregn-4,6-dien-3,20-dione (14a)
−1
Yield 65% (0.290 g), mp 224-226 °C. IR (KBr) cm : 3264, 2944, 1728 and
1
1
2
7
1
660. H NMR (CDCl )
δ
: 0.7 (s, 3H, H-18), 1.2 (s, 3H, H-19), 2.2 (s, 3H, H-
3
1), 5.8 (s, 1H, H-4), 6.0 (dd, 1H, J=13.2 and 1.2 Hz, H-6), 6.2 (m, 1H, H-7),
.1 (dd, 1H, J= 12.8 and 3.2 Hz, Ar-H), 7.3 (d, 1H, J = 17.2 Hz, Ar-H), 7.4 (d,
H, J = 10 Hz, Ar-H), 7.5 (d, 1H, J = 10 Hz, Ar-H), 7.6 (d, 1H, J= 9.2 Hz, Ar-
13
H), 10.6 (s, 1H, N-H). C NMR (CDCl ) δ: 13.6 (C-18), 15.9 (C-19), 26.1 (C-
3
2
5

ACCEPTED MANUSCRIPT
2
1), 89.5 (C-17), 123.6 (Ar-C), 123.9 (C-4), 127.8 (Ar-C), 128.1 (C-6), 128.7
(Ar-C), 131.4 (C-7), 139.3 (Ar-C), 154.3 (carbamate carbonyl), 162.7 (C-5),
1
4
99.1 (C-3) and 202.9 (C-20). HRMS cal for C H NO 447.2410, found
2
8
33
4
47.2405.
3
.3.11. 17α-p-Fluorophenylcarbamoiloxypregn-4,6-dien-3,20-dione (14b)
−1
Yield 55% (0.256 g), mp 220-221 °C. IR (KBr) cm : 3264, 2944, 1728 and
1
1
2
661. H NMR (CDCl )
δ
: 0.7 (s, 3H, H-18), 1.1 (s, 3H, H-19), 2.3 (s, 3H, H-
3
1), 5.9 (s, 1H, H-4), 6.0 (dd, 1H, J=13.2 and 1.2 Hz, H-6), 6.2 (dd, 1H, J=9.6
and 3.0 Hz, H-7), 7.3 (d, 1H, J = 17.2 Hz, Ar-H), 7.4 (d, 1H, J = 10 Hz, Ar-
H), 7.5 (d, 1H, J = 9.2 Hz, Ar-H), 7.6 (d, 1H, J= 9.2 Hz, Ar-H), 10.6 (s, 1H,
1
3
N-H). C NMR (CDCl )
δ
: 13.6 (C-18), 15.9 (C-19), 26.1 (C-21), 89.5 (C-
7), 124.0 (C-4), 128.9 (C-6), 129.3 (Ar-C), 129.7 (Ar-C), 130.4 (Ar-C),
34.5 (C-7), 135.9 (Ar-C), 154.5 (carbamate carbonyl), 162.4 (C-5), 199.0
3
1
1
(
C-3) and 202.5 (C-20) HRMS cal for C H FNO 466.2394, found 466.2402.
28
32
4
3
.3.12. 17α-p-Chlorophenylcarbamoiloxypregn-4,6-dien-3,20-dione (14c)
−1
Yield 75% (0.361 g), mp 170-172 °C. IR (KBr) cm : 3264, 2944, 1728 and
1
1
2
661. H NMR (CDCl )
δ
: 0.6 (s, 3H, H-18), 1.0 (s, 3H, H-19), 2.3 (s, 3H, H-
3
1), 5.7 (s, 1H, H-4), 5.9 (dd, 1H, J=13.2 and 1.2 Hz, H-6), 6.2 (dd, 1H, J=10
2
6

ACCEPTED MANUSCRIPT
and 3.2 Hz, H-7), 7.1 (d, 1H, J= 9.8 Hz, Ar-H), 7.2 (d, 1H, J= 8.8 Hz, Ar-H),
7
.3 (d, 1H, J= 8.8 Hz, Ar-H), 7.4 (d, 1H, J= 8.8 Hz, Ar-H), 10.5 (s, 1H, N-H).
1
3
C NMR (CDCl3) δ: 13.9 (C-18), 16.3 (C-19), 26.1 (C-21), 99.1 (C-17),
1
7
2
24.2 (C-4), 128.6 (C-6), 129.1 (Ar-C), 129.5 (Ar-C), 130.1 (Ar-C), 134.5 (C-
), 135.8 (Ar-C), 154.5 (carbamate carbonyl), 162.7 (C-5), 199.4 (C-3) and
02.9 (C-20). HRMS cal for C H ClNO 482.2098, found 482.2104.
28
32
4
3
.3.13. 17α-p-Bromophenylcarbamoiloxypregn-4,6-dien-3,20-dione (14d)
−1
Yield 58% (0.305 g), mp 206-208 °C. IR (KBr) cm : 3287, 2949, 1721 and
1
1
2
659. H NMR (CDCl )
δ
: 0.6 (s, 3H, H-18), 1.0 (s, 3H, H-19), 2.1 (s, 3H, H-
3
1), 5.7 (s, 1H, H-4), 5.9 (dd, 1H, J=13.2 and 1.2 Hz, H-6), 6.2 (dd, 1H, J=9.6
and 3.1 Hz, H-7), 7.1 (d, 1H, J= 8.8 Hz, Ar-H), 7.3 (d, 1H, J= 10 Hz, Ar-H),
.4 (d, 1H, J= 10.4 Hz, Ar-H), 7.6 (d, 1H, J= 9.2 Hz, Ar-H), 10.5 (s, 1H, N-
7
1
3
H). C NMR (CDCl )
δ
: 14.0 (C-18), 16.5 (C-19), 26.5 (C-21), 90.3 (C-17),
3
1
7
2
24.0 (C-4), 128.7 (C-6), 129.0 (Ar-C), 130.1 (Ar-C), 130.7 (Ar-C), 135.3 (C-
), 136.1 (Ar-C), 154.7 (carbamate carbonyl), 162.9 (C-5), 199.5 (C-3) and
02.9 (C-20). HRMS cal for C H BrNO 526.1593, found 526.1584.
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4
2
7

ACCEPTED MANUSCRIPT
3
.3.14. 17
α
-p-Iodophenylcarbamoiloxypregn-4,6-dien-3,20-dione (14e)
−1
Yield 50% (0.286 g), mp 217-219 °C. IR (KBr) cm : 3287, 2949, 1726 and
1
1
2
659. H NMR (CDCl )
δ
: 0.6 (s, 3H, H-18), 1.0 (s, 3H, H-19), 2.1 (s, 3H, H-
3
1), 5.7 (s, 1H, H-4), 5.9 (dd, 1H, J=10 and 1.2 Hz, H-6), 6.2 (dd, 1H, J=2.4
and 9.6 Hz, H-7), 6.9 (d, 1H, J= 8.4 Hz, Ar-H), 7.3 (d, 1H, J= 9.2 Hz, Ar-H),
.3 (d, 1H, J= 9.2 Hz, Ar-H), 7.6 (d, 1H, J= 8.8 Hz, Ar-H), 10.5 (s, 1H, N-H).
7
1
3
C NMR (CDCl3) δ: 13.8 (C-18), 16.0 (C-19), 26.2 (C-21), 90.0 (C-17),
1
7
2
24.1 (C-4), 128.5 (C-6), 129.3 (Ar-C), 129.8 (Ar-C), 130.0 (Ar-C), 135.2 (C-
), 135.9 (Ar-C), 154.6 (carbamate carbonyl), 162.9 (C-5), 199.5 (C-3) and
02.9 (C-20). HRMS cal for C H INO 573.1376, found 573.1371.
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32
4
3
3
.4 Cytotoxicity Assay
.4.1. Biological material
The panel cells used in this study were the following: PC-3 (human prostate
cancer cell line), MCF-7 (human breast cancer cell line), HCT-15 (human
colon cancer cell line) and J774 (murine macrophages) and were supplied by
the National Cancer Institute (NCI, USA). The ketoconazol used as a
reference in this experiment was purchased from Sigma Life Science.
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8