New androst-4-en-17-spiro-1,3,2-oxathiaphospholanes. Synthesis, assignment of absolute configuration and in vitro cytotoxic and antimicrobial activities

Article abstract of DOI:10.1016/j.steroids.2012.01.021

The reactions of 17α-hydroxyprogesterone with Lawesson's reagent (LR) in toluene, CH₂Cl₂ and/or CCl₄ gave, depending on the duration of the reaction, two diastereoisomeric androst-4-en-17-spiro-1, 3,2-oxathiaphospholane-2-

Full text of DOI:10.1016/j.steroids.2012.01.021

Steroids 77 (2012) 558–565
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
New androst-4-en-17-spiro-1,3,2-oxathiaphospholanes. Synthesis, assignment
of absolute configuration and in vitro cytotoxic and antimicrobial activities
a,
a
b
c
d
⇑
´
´
´
´
Natalija M. Krstic , Mira S. Bjelakovic , Vladimir D. Pavlovic , Koen Robeyns , Zorica D. Juranic ,
d
a
b
´
´
´
Ivana Matic , Irena Novakovic , Dušan M. Sladic
a Center for Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentski trg 12-16, P.O. Box 473, 11001 Belgrade, Serbia
b Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, P.O. Box 158, 11001 Belgrade, Serbia
c Institute of Condensed Matter and Nanosciences (IMCN), Université Catholique de Louvain (UCL), Bâtiment Lavoisier, Place Louis Pasteur 1, bte L4.01.03,
1348 Louvain-la-Neuve, Belgium
d Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
The reactions of 17a-hydroxyprogesterone with Lawesson’s reagent (LR) in toluene, CH2Cl2 and/or CCl4
Received 20 December 2011
Received in revised form 20 January 2012
Accepted 30 January 2012
Available online 9 February 2012
gave, depending on the duration of the reaction, two diastereoisomeric androst-4-en-17-spiro-1,3,2-oxa-
thiaphospholane-2-sulfide pairs 2a,b and 3a,b in approximately 7:3 ratio, differing in configuration at the
phosphorus atom. A parallel analysis of heteronuclear 2D 1H–13C spectra (HSQC and HMBC) and homo-
nuclear 2D spectra (NOESY) enabled complete 1H and 13C assignments of each isomer. Also, analysis of
NOESY correlations provided evidence for the preferred conformation. X-ray analysis of 3a confirmed
the structure and absolute configuration on phosphorus. A pathway for the formation of 1,3,2-oxathia-
phospholane ring was proposed. Cytotoxic activity in vitro was tested against three tumor cell lines
(human cervix carcinoma HeLa cells and two human breast carcinoma MDA-MB-361 and MDA-MB-
453 cells). Compound 3a and mixture 3a,b showed a moderate activity against HeLa and MDA-MB-
453 cell lines while against MDA-MB-361 cell line all tested compounds exerted very weak cytotoxic
effect. Antimicrobial activity against Gram-positive, Gram-negative bacteria and fungal cells, toxicity
to brine shrimp Artemia salina, were evaluated. All tested compounds showed strong antifungal activity.
Ó 2012 Elsevier Inc. All rights reserved.
Keywords:
Phosphorus heterocycle
17a-Hydroxyprogesterone
Lawesson’s reagent
X-ray analysis
17-Spiro-androstene derivatives
Biological activity
1. Introduction
This has been proven by different ring modification studies of
steroidal molecules involving the A- and D-rings, whereby incorpo-
Modified steroids have attracted a great deal of attention
because of their ability to affect some of the most fundamental bio-
logical functions. Introduction of heteroatom and/or replacement
of one or more carbon atoms by a heteroatom, and addition of het-
erocyclic rings or new functional groups can extend the already
wide range of biological activities of those compounds [1]. Over
the past few decades development of new heterocyclic steroid
derivatives has been one of the major goals of the steroid chemis-
try. As expected, the addition of heterocycles to steroids has usu-
ally led to changes in their physiological properties and caused
new, interesting biological activities [2–4]. Hydrophobic steroid
units have an ability to interact with cell membranes and pass
through them. Therefore, they can improve transport and hybrid-
ization properties of such heterocyclic molecules.
ration of a heteroatom (N or O) has been reported to enhance the
biological activities of these molecules. Such systems have shown
a variety of anti-microbial, anti-inflammatory, hypotensive, hypo-
cholesterolemic and diuretic activities [5–10]. As a result, a num-
ber of different heterocyclic systems have been incorporated into
the core structure of steroids, particularly pyrazoles, pyrazolines,
isoxazolines, thiazoles, thiadiazoles, pyridines, pyrimidines and
imidazoles.
A great number of organophosphorus compounds in nature par-
ticipate in numerous biological activities; therefore, there is a
growing interest in these compounds. The monocyclic and con-
densed five- and six-membered P-heterocycles among them have
attracted considerable attention. 1,3,2-Dioxaphosphorinanes (six-
membered O-P-O-heterocycles) have become very interesting
since cyclic nucleotides, adenosine 30,50-cyclic monophosphate
and related compounds involved in the regulation of many biolog-
ical processes, contain such a framework [11,12]. On the other
hand the successful application of oxathiaphospholane and
dithiaphospholane derivatives (five-membered rings) as reactive
precursors of sulfur-modified oligonucleotides has raised interest
⇑
Corresponding author. Tel.: +381 11 3336 666; fax: +3811 11 2636 061.
E-mail addresses: nkrstic@chem.bg.ac.rs (N.M. Krstic´), mbjelak@chem.bg.ac.rs
(M.S. Bjelakovic´), pavlovic@chem.bg.ac.rs (V.D. Pavlovic´), koen.robeyns@
uclouvain.be (K. Robeyns), juranicz@ncrc.ac.rs (Z.D. Juranic´), ivanamatic@sbb.rs
(I. Matic´), irenan2003@yahoo.com (I. Novakovic´), dsladic@chem.bg.ac.rs (D.M.
Sladic´).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2012.01.021

´
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N.M. Krstic et al. / Steroids 77 (2012) 558–565
in their synthesis and molecular geometry, in particular in the con-
formation of the heterocyclic ring [13–15]. To the best of our
knowledge, amongst a number of potentially effective heterocyclic
steroid compounds only a few phosphorus-containing systems
have been synthesized so far [15–22].
Lawesson’s reagent (LR: 2,4-bis(p-methoxyphenyl)-1,3,2,
4-dithiadiphosphetane-2,4-disulfide) [23–25], commonly utilized
as a thionating agent, has been used to build five- and six-
membered phosphorus heterocycles, such as oxathiaphospholes,
oxathiaphospholidine-2-thiones, oxazaphosphorin-4-thione-2-
sulfides, and sulfur containing heterocycles thienothiazines and
benzothiazoles [24]. In our previous work on modified steroid
compounds we have reported the synthesis and antitumor activity
of some steroidal thiolactams using Lawesson’s reagent [26]. In
continuation of that research we have recently reported reactions
Varian Gemini-200 spectrometer (at 200 and 50 MHz, respectively)
and/or Bruker Avance 500 MHz spectrometer (1H at 500 MHz; 13C
at 125 MHz) in CDCl3 and/or C6D6 at room temperature using
TMS as internal standard. Chemical shifts are expressed in ppm
(d) values and coupling constants (J) in Hz. Mass spectra were
taken on Finnigan-MAT 8230. Thin-layer chromatography (TLC)
was performed on precoated Merck silica gel 60 F254 plates in tol-
uene/EtOAc (8/2), detection with 50% aq. H2SO4. Flash column
chromatography (FCC) was performed on silica gel Merck 0.040–
0.063 mm. Elemental analyses were performed on Vario EL III.
17a-Hydroxyprogesterone was purchased from Galenika AD (man-
ufactured by Diosynth, Oss) and was recrystallized from MeOH.
2.1.1. General procedure for thionation with LR
LR (808 mg, 2 mmol) was added to a solution of 17a-hydroxy-
of
a,b-unsaturated steroidal ketones (several cholestane, andro-
progesterone (660 mg, 2 mM) in dichloromethane, toluene and/or
carbon tetrachloride (30 mL). The reaction mixture was heated to
reflux (reaction times are given in Scheme 2) with stirring and pro-
gress of the reaction was monitored by TLC (toluene/EtOAc (8/2)).
After the completion of the reaction excess of the reagent was re-
moved by filtration and solvent evaporated under reduced pres-
sure. The residue was purified by FCC using toluene/EtOAc (99/1
and 95/5) as eluent. Compounds 2a and 2b as well as 3a and 3b
were obtained as a mixture of two diastereoisomers, but 3a was
also isolated as a pure isomer after several FCC.
stane and pregnane carbonyl derivatives were chosen) with LR.
We synthesized several sulfur-containing compounds, including
dithiones Ic–d,
a,b-unsaturated 3-thiones IIa–e, dimer-sulfides
IIIa–e, 1,2,4-trithiolanes IVa–e and some sulfur and phosphorus
containing compounds i.e. phosphonotrithioates Vb–e (products
vary with changes of solvent and reaction times) [27] (Scheme 1).
As a follow-up our goal was to extend our research on the syn-
thesis and characterization of new modified steroid compounds.
This paper describes the simple syntheses and configurational
study of the new androst-4-en-17-spiro-1,3,2-oxathiaphospho-
The mixture 2a,b: Rf = 0.82. Mp > 137 °C (decomp.). IR (C6D6):
3442, 2942, 1592, 1258, 1108, 916, 703. Anal. calcd for
lane-2-sulfides via the reaction of 17a-hydroxyprogesterone with
LR used here both as thionating and phosphorylating agent. The
reactions were carried out in toluene, dichloromethane and carbon
tetrachloride with varying reaction times. As far as we know Kvas-
nica et al. [28] have described reactions of hydroxyketones with LR
in which they obtained dithiaphospholane and dioxaphospholane,
but 1,3,2-oxathiaphospholane ring formation in the reactions with
LR has not yet been reported. The screening for antimicrobial and
cytotoxic activity was also performed.
C
28H35O2PS3: C 63.36; H 6.65; S 18.12. Found: C 63.06; H 6.83, S
18.11. ESI-TOF-MS: m/z calcd for
C28H35O2PS3: 531.16096
[M + H]+, found 531.16171. NMR (1H at 200 MHz; 13C at 50 MHz)
spectra were taken with a mixture of 2a and 2b:
2.1.1.1. (20S)-20-(Methoxyphenyl)-40-methylene-3-thioxo-spiro[and-
rost-4-en-17,50-[1,3,2]-oxathiaphospholane]-20-sulfide
NMR: 0.65 (s, 3H, H3C(18)), 0.79 (s, 3H, H3C(19)), 2.68 (dtd,
(2a). 1H
J = 18.6, 13.6, 4.6, 1H, Hb–C(2)), 3.13 (dt, J = 17.4, 3.4, 1H, Ha–
C(2)), 3.23 (s, 3H, OCH3), 5.04 and 5.13 (2br.s, 2H, H2C@C(40)),
4
2. Experimental
6.70 (s, 1H, H–C(4)), 7.12 (dd, JPH = 3.5, JHH = 9.0, 2H, H–C(300)
3
and H–C(500)), 8.08 (dd, JPH = 15.2, JHH = 9.0, 2H, H–C(200) and H–
4
2.1. Chemistry
C(600)). 13C NMR: 237.3 (s, C(3)), 163.1 (sd, JPC = 3.6, C(400)), 161.0
2
(s, C(5)), 144.7 (sd, JPC = 2.8, C(40)), 135.5 (d, C(4)), 132.9 (dd,
Removal of solvents was carried out under reduced pressure. IR
spectra were recorded on a Perkin–Elmer spectrophotometer FT-IR
3JPC = 14.4, C(300) and C(500)), 131.7 (sd, JPC = 117.4, C(100)), 113.7
2
3
(dd, JPC = 16.5, C(200) and C(600), 113.4 (td, JPC = 9.1, CH2@C(40)),
2
1725X:
m
in cmꢀ1
.
1H and 13C NMR spectra were recorded on a
107.6 (sd, JPC = 10.0, C(17)), 54.9 (q, OCH3), 52.8 (d, C(9)), 50.3
Me
S
Me
Me
Me
R1
R2
Me
R1
R2
S
H
Me
R
R
S
S
S
S
2
Ic
Me
Id
IIa-e
Me
IIIa-e
Me
Me
R1
R2
R1
R2
R1
R2
R1
R2
R
R
R
R
S S
S
S
S
S
P
OCH3
IVa-e
Vb-e
a) R=CH3, R1=C8H17, R2=H
b) R=CH3, R1R2=O
c) R=H, R1R2=O
d
) R=CH3, R1=COCH3, R2=H,
e) R=CH3, R1=COCH3, 16α,17α-epoxy
Scheme 1. The products obtained in the reactions of some a,b-unsaturated steroidal ketones with LR.

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N.M. Krstic et al. / Steroids 77 (2012) 558–565
21
1M8 e
20
CH3
O
OH
S
S P
P S
S
19
17
Me
OMe
H
+
MeO
H
H
1
3
O
4
LR
a) Toluene, 45 min
b) Toluene, 5 h
c) CH2Cl2, 45 min
d) CH2Cl2, 7 h
e) CCl4, 45 min
f) CCl4, 8h
18
CH2
CH2
Me
12 Me
19
S3'
4'
6'' 5''
2'' 3''
5'
Me11
S
O P
S
13
14
17
Me
9 H
OCH3
+
1
16 O 2'P
4'' OCH3 +
SM
1
1''
2
3
10
H 8 7 H
15 1'
S
5
S
O
6
4
2a,b
3a,b
a) 18%
a) 8%
b) 16%
c) 15%
d) 46%
e) 27%
f) 13%
a) 48%
b)
c) 58%
d)
e) 50%
f)
b)
-
-
c) 6%
d)
e) 8%
f)
-
-
-
-
Scheme 2. The reaction of 17a-hydroxyprogesterone with an equimolar amount of LR.
(d, C(14)), 48.8 (s, C(13)), 43.5 (t, C(2)), 38.3 (s, C(10)), 36.3 (d,
C(8)), 35.8 (t, C(16)), 35.7 (t, C(6)), 32.7 (t, C(12)), 32.6 (t, C(7)),
31.7 (t, C(1)), 23.6 (t, C(15)), 21.4 (t, C(11)), 16.7 (q, C(19)), 16.1
(q, C(18)).
63.71; H 7.19; S 11.73. Found: C 63.97; H 7.02, S 12.59. ESI-TOF-
MS: m/z calcd for
515.17894.
C
28H35O3PS2: 515.18380 [M + H]+, found
2.1.1.4. (20R)-20-(Methoxyphenyl)-40-methylene-3-oxo-spiro[androst-
4-en-17,50-[1,3,2]-oxathiaphospholane]-20-sulfide (3b) (from the 3a,b
mixture). Rf = 0.63, (toluene/EtOAc, 8:2, double development).
Mp > 123 °C (decomp.). IR (CHCl3): 3438, 2945, 1666, 1592, 1107,
919, 705. 1H NMR (500 MHz, CDCl3): 0.91 (s, 3H, H3C(18)), 1.19
(s, 3H, H3C(19)), 3.88 (s, 3H, OCH3), 5.57 and 5.76 (br.s and t,
J = 1.5 Hz, 2H, H2C@C(40)), 5.70 (s, 1H, H–C(4)), 7.00 (dd,
4JPH = 3.5, JHH = 9.0, 2H, H–C(300) and H–C(500)), 8.10 (dd,
3JPH = 15.2, JHH = 8.8, 2H, H–C(200) and H–C(600)). 13C NMR
2.1.1.2. (20R)-20-(Methoxyphenyl)-40-methylene-3-thioxo-spiro[and-
rost-4-en-17,50-[1,3,2]-oxathiaphospholane]-20-sulfide
(2b). 1H
NMR: 0.68 (s, 3H, H3C(18)), 0.75 (s, 3H, H3C(19)), 2.68 (dtd,
J = 18.6, 13.6, 4.6, 1H, Hb–C(2)), 3.13 (dt, J = 17.4, 3.4, 1H, Ha–
C(2)), 3.20 (s, 3H, OCH3), 5.26 and 5.53 (2br.s 2H, H2C@C(40)),
4
6.63 (s, 1H, H–C(4)), 7.20 (dd, JPH = 3.5, JHH = 9.0, 2H, H–C(300)
3
and H-C(500)), 8.36 (dd, JPH = 15.2, JHH = 9.0, 2H, H–C(200) and H–
4
C(600)). 13C NMR: 237.1 (s, C(3)), 163.9 (sd, JPC = 3.1, C(400)), 161.4
4
(s, C(5)), 142.7 (sd, JPC = 2.4, C(40)), 135.2 (d, C(4)), 132.9 (dd,
3JPC = 14.4, C(300) and C(500)), 131.7 (sd, JPC = 117.4, C(100)), 116.4
(125 MHz): 199.3 (s, C(3)), 170.8 (s, C(5)), 163.7 (sd, JPC = 3.1,
2
3
C(400)), 141.9 (sd, JPC = 1.6, C(40)), 134.9 (dd, JPC = 15.1, C(300) and
C(500)), 122.9 (sd, JPC = 121.1, C(100)), 123.9 (d, C(4)), 116.5 (td,
3
2
(td, JPC = 8.0, CH2@C(40)), 113.9 (dd, JPC = 16.5, C(200) and C(600)),
2
2
104.0 (sd, JPC = 7.4, C(17)), 55.0.4 (q, OCH3), 52.8 (d, C(9)), 51.0
3JPC = 9.4, CH2@C(40)), 113.9 (dd, JPC = 16.5, C(200) and C(600)),
104.1 (sd, JPC = 7.1, C(17)), 55.5 (q, OCH3), 52.8 (d, C(9)), 50.7
2
(d, C(14)), 48.9 (s, C(13)), 43.5 (t, C(2)), 39.3 (t, C(16)), 38.3 (s
C(10)), 36.2 (t, C(6)), 36.1 (d, C(8)), 32.5 (t, C(12)), 32.6 (t, C(7)),
31.7 (t, C(1)), 23.6 (t, C(15)), 21.0 (t, C(11)), 16.9 (q, C(19)), 15.8
(q, C(18)).
C(14)), 48.9 (s, C(13)), 39.0 (t, C(16)), 38.6 (s, C(10)), 36.3 (d,
C(8)), 35.6 (t, C(1)), 33.8 (t, C(2)), 32.3 (t, C(6)), 31.7 (t, C(7)), 31.1
(t, C(12)), 23.6 (t, C(15)), 20.6 (t, C(11)), 17.3 (q, C(19)), 15.8 (q,
C(18)). Anal. calcd for C28H35O3PS2ꢁ0.5 CH3OH: C 64.50; H 7.03; S
12.08. Found: C 64.86; H 7.10, S 12.06. ESI-TOF-MS: m/z calcd for
2.1.1.3. (20S)-20-(Methoxyphenyl)-40-methylene-3-oxo-spiro[androst-
C
28H35O3PS2: 515.18380 [M + H]+, found 515.17880
4-en-17,50-[1,3,2]-oxathiaphospholane]-20-sulfide
(3a). Rf = 0.66,
(toluene/EtOAc, 8:2, double development). Mp > 123 °C (decomp.).
IR (CHCl3): 3438, 2945, 1660, 1106, 918, 704. 1H NMR (500 MHz,
CDCl3): 0.84 (s, 3H, H3C(18)), 1.20 (s, 3H, H3C(19)), 3.84 (s, 3H,
OCH3), 5.43 and 5.49 (br.s and t, J = 1.5, 2H, H2C@C(40)), 5.73 (s,
2.2. X-ray crystallography
The crystals were obtained by slow evaporation of methanol.
Colorless translucent plates like crystals were obtained, of which
one was selected and flash frozen at 120 K prior to the measure-
ment. The Mo (Ka) radiation was generated by a Rigaku UltraX
18 generator (with Zr filter) and the reflections were collected on
a MAR345 image plate. Data were integrated by the Crysalis soft-
ware (Oxford Diffraction) and the data were corrected by the mul-
ti-scan absorption correction implemented in SCALE3 ABSPACK
scaling algorithm. The internal R (Rint) value after integration is
4.45% for 18,700 reflections (96.7% data completeness). The struc-
ture was solved by directs methods (SHELXS). Full matrix least-
squares refinement based on |F2| was performed (SHELXL) [29].
4
1H, H–C(4)), 6.92 (dd, JPH = 3.5, JHH = 9.0, 2H, H–C(300) and
3
H–C(500)), 7.85 (dd, JPH = 15.0, JHH = 9.0, 2H, H–C(200) and H–
C(600)). 13C NMR (125 MHz): 199.5 (s, C(3)), 170.9 (s, C(5)), 162.7
4
2
(sd, JPC = 3.2, C(400)), 144.0 (sd, JPC = 2.4, C(40)), 132.4 (dd,
3JPC = 14.4, C(300) and C(500)), 130.7 (sd, JPC = 120.3, C(100)), 124.0 (d,
3
2
C(4)), 113.8 (td, JPC = 11.6, CH2@C(40)), 113.7 (dd, JPC = 16.5,
2
C(200) and C(600)), 107.5 (sd, JPC = 10.0, C(17)), 55.4 (q, OCH3), 52.7
(d, C(9)), 50.1 (d, C(14)), 48.6 (s, C(13)), 38.5 (s C(10)), 36.4 (d,
C(8)), 35.7 (t, C(16)), 35.5 (t, C(1)), 33.9 (t, C(2)), 32.3 (t, C(12)),
32.7 (t, C(6)), 31.7 (t, C(7)), 23.6 (t, C(15)), 20.9 (t, C(11)), 17.4 (q,
C(19)), 16.1 (q, C(18)). Anal. calcd for C28H35O3PS2ꢁCH3OH: C

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N.M. Krstic et al. / Steroids 77 (2012) 558–565
Hydrogen atoms were placed at calculated positions with temper-
ature factors 1.2 times higher than those of the parent atoms (1.5
for methyl groups) and were refined in riding mode. The absolute
configuration was assigned relative to the unchanged androstane
moiety as the flack parameter was inconclusive, friedel pairs were
merged for the final refinement cycle.
Crystallographic data (excluding structure factors) for the struc-
tures in this paper have been deposited with the Cambridge Crys-
tallographic Data Centre as supplementary publication CCDC-ID:
832161. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK,
(fax: +44 (0)1223 336033 or e-mail: deposit@ccdc.cam.ac.Uk).
Streptosporangium longisporum (ATCC 25212), Micrococcus flavus
(ATCC 10240), Sarcina lutea (ATCC 9341), Klebsiella pneumoniae
(ATCC 10031) and Staphylococcus aureus (ATCC 6538). The Gram-
negative bacteria used were: Escherichia coli (ATCC 25922), Pseudo-
monas aeruginosa (ATCC 9027), Salmonella enteritidis (ATCC 13076)
and Proteus vulgaris (ATCC 13315). The fungi tested were: Candida
albicans (ATCC 10231) and Saccharomyces cerevisiae (ATCC 9763).
Sterile 96-well polystyrene microtitre plates with well capacities
of 300 lL were used and 100 lL of fresh Mueller Hinton broth were
added to each well of the plate. One hundred microlitres of the
compound stock solution were added to each well of the first col-
umn. Then, 100
umn and mixed thoroughly with the broth in the corresponding
wells of the second column. Subsequently, a 100 L aliquot was re-
lL of the solution were removed from the first col-
2.3. Cytotoxic activity
l
moved from each well in this column and mixed with contents of
the corresponding well of the next column. This doubling dilution
was performed in all rows across the plate. Two rows in each plate
were used as controls. One row was used as a positive control and
contained a broad-spectrum antibiotic amikacin to determine the
sensitivity of Gram-positive and Gram-negative bacterial species
and an antimycotic nystatin to determine the sensitivity of fungal
species. The other row contained the solvent (DMSO) as a negative
2.3.1. Preparation of solutions of tested compounds
Stock solutions of the compounds were prepared in dimethyl
sulfoxide (DMSO) at a concentration of 10 mM and afterwards
diluted with a nutrient medium (RPMI 1640), supplemented with
L-glutamine (3 mM), streptomycin (100 lg/mL) and penicillin
(100 IU/mL), 10% heat inactivated (56 °C) fetal bovine serum
(FBS) and 25 mM Hepes, adjusted to pH 7.2 by bicarbonate solu-
tion, with the addition of 2 mg/mL glucose and applied to target
cells to various final concentrations. MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide) was dissolved (5 mg/
mL) in phosphate buffer saline. RPMI 1640 cell culture medium,
FBS and MTT were Sigma Chemicals products.
control. In each well of the plate 10
l
L of bacterial cultures (106
L of fungi cultures
cells per mL) for antibacterial activity and 10
l
(105 spores per mL) were inoculated. The microtitre plate was
incubated at 37 °C for 24 h for bacteria or at 28 °C for 48 h for fungi.
After that, bacterial and fungi growth were measured. The MIC was
determinated as the lowest concentration that resulted in inhibi-
tion of bacterial and fungal growth.
2.3.2. Cell culture
Human cervix adenocarcinoma HeLa, human breast carcinoma
MDA-MB-453 and human breast adenocarcinoma MDA-MB-361
cells were cultured as monolayers in the nutrient medium. The
cells were grown at 37 °C in an atmosphere of 5% CO2 and humid-
ified air.
2.5. The brine shrimp test
The brine shrimp test was performed against freshly hatched
nauplii of Artemia salina [33]. Briefly, the compounds were dis-
solved in DMSO and diluted by artificial seawater so that an appro-
priate range of concentrations (0.05–0.30 mg/mL) was obtained.
The final concentration of DMSO was 1%, and the number of nauplii
was 10. Surviving nauplii were counted after 24 h, and LC50 (con-
centration lethal to 50% of the nauplii) were determined after sta-
tistical analysis. All the tests were performed in triplicate.
2.3.3. Treatment of cancer cell lines
HeLa (2000 cells per well), MDA-MB-453 (3000 cells per well)
and MDA-MB-361 cells (10,000 cells per well), were seeded into
96-well microtiter plates. Twenty hours later, after the cell adher-
ence, five different concentrations of investigated compounds were
added to the wells (range 12.5–200 lM), except for the control
cells to which a nutrient medium only was added.
3. Results and discussion
2.3.4. Determination of target cell survival
Survival of target cells was determined by MTT test, according
to the method of Mosmann, which is modified by Ohno and Abe
3.1. Chemistry
[30,31]. Briefly, 72 h after continuous agents action, 10
lL of MTT
The reaction of 17a-hydroxyprogesterone (1) with an equimo-
solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium
bromide) was added to each well. Samples were incubated for a
further 4 h and then 100 lL of 10% SDS was added to the wells.
Absorbance (A) at 570 nm was measured the next day.
To get cell survival (S%), A of a sample with cells grown in the
presence of various concentrations of the investigated compounds
was divided with control optical density (the A of control cells
grown only in the nutrient medium), and multiplied by 100. It
was implied that A of the blank was always subtracted from A of
the corresponding sample with target cells. IC50 concentration
was defined as the concentration of an agent inhibiting cell sur-
vival by 50%, compared to a control.
lar amount of LR resulted in a complex mixture of products
(Scheme 2). Two major products, 3-thioxo-androst-4-en-17-
spiro-oxathiaphospholane 2, and 3-oxo-androst-4-en-17-spiro-
oxathiaphospholane 3 were isolated. The reaction conditions were
optimized by varying solvent/temperature (reflux in toluene,
dichloromethane and/or carbon tetrachloride), and duration of
the reaction. Compound 2 was obtained in each solvent but only
when the reactions were quenched after 45 min (while the solu-
tion was still purple indicating the presence of 3-thioketones),
and before complete consumption of 1. Oxathiaphospholane 3
was formed in all cases and the highest yield was obtained when
reaction was carried out in dichloromethane for 7 h (46%). With
a prolonged reaction time (reflux 24 h) more by-products were
formed making the mixture more complex and it was not possible
to isolate either of the products 2 or 3.
2.4. Antimicrobial activity
A micro-broth dilution assay [32] was used to evaluate the anti-
microbial efficacy against Gram-positive, Gram-negative bacteria
and fungal cells. The Gram-positive bacteria used were: Bacillus
subtilis (ATCC 6633), Clostridium sporogenes (ATCC 19404),
The 1H and 13C NMR analysis for both compounds, 2 and 3,
revealed the presence of two pairs of signals in approx. 7:3 ratio
(deduced by comparing the peak areas of the H-4 and exocyclic
methylene H2C@C(40) in the corresponding 1H NMR spectra)

´
N.M. Krstic et al. / Steroids 77 (2012) 558–565
562
indicating the existence of two diastereoisomers differing in the
configuration at the phosphorus atom. In the case of the compound
2 all our efforts to separate the isomers by crystallization or by
chromatography on silica failed (no eluent tested caused separa-
tion of the diastereoisomers). For that reason all NMR spectra taken
were of the mixtures of 2a and 2b.
13C NMR spectrum of 3a the new signals were the triplet at
3
113.8 (CH2@C(40), td, JPC = 11.6 Hz), singlet at 144.0 (C-40, sd,
2
2JPC = 2.4 Hz) and the singlet at 107.5 (C-17), sd, JPC = 10.0 Hz).
The signals attributed to the same C-atoms in 3b were found at
3
2
116.5 (td, JPC = 9.4 Hz), 141.9 sd, JPC = 1.6 Hz) and 104.1 (sd,
2JPC = 7.1 Hz) ppm, respectively. The presence of 4-methoxyphenyl
group was confirmed on the basis of NMR spectral data for both
pure 3a and the mixture 3a,b and these signals had corresponding
JHH, JPH and JPC coupling constants (Table 1). Furthermore, the ele-
mental analysis for 3a was in agreement with the proposed
structures.
In the IR spectrum of 2a,b the absorption bands for carbonyls at
C-3 and C-20, as well as for the 17-OH group, of 17a-hydroxypro-
gesterone (1) were absent. The 1H NMR spectrum of the mixture 2a
and 2b showed two broad singlet pairs for the exocyclic methylene
H2C@C(40) group at 5.04, 5.13 ppm and 5.26, 5.53 ppm, respec-
tively. Two singlets for H-4 at 6.70 for 2a and 6.63 ppm for 2b were
situated downfield from the resonance for the corresponding pro-
ton in the starting compound 1, which was attributed to the strong
The absolute configuration on the phosphorus atom (R or S) was
determined by the detailed analysis of the NOESY correlation. The
most intensive NOESY interaction, such as the correlation between
exocyclic methylene H2C@C(40) and Hb-16 and 18-Me group, were
found not to be significantly different. The most important cross-
peaks for the configurational investigation were those involving
aromatic protons from 4-methoxyphenyl group H-200 and H-600. In
the 3a (major isomer) these protons at 7.85 ppm showed NOESY
C@S anisotropy. In addition, both protons at C-2 (a-position to the
C@S group) resonate well separated downfield from the other ring
protons (Hax, td, 3.13 ppm; Heq, dtd, 2.68 ppm), both with corre-
sponding JHH coupling constants.
The characteristic signals in the 13C NMR spectrum were two
singlets at 237.3 and 237.1 ppm for C(3)@S, triplets at 113.4 and
cross-peak with the Ha-16 at 2.16 ppm and no correlation was ob-
3
116.4 ppm (CH2@C(40) with JPC = 9.1 and 8.0 Hz) and the singlets
served with the H -12 proton. According to Cahn–Ingold–Prelog
a
2
at 144.7 and 142.7 ppm (C-40 with JPC = 2.8 and 2.4 Hz) for 2a
[34] rules the absolute configuration at phosphorus atom is S. On
and 2b, respectively. Also, there were signals at 107.6 and
104.0 ppm (C-17) for 2a and 2b, respectively, and these singlets
the other hand, in the minor isomer 3b H-200 and H-600 protons at
8.10 ppm showed NOESY cross-peak correlation only with Ha-12
at 1.62 ppm, indicating R configuration at phosphorus atom (Table
2
resembled doublets with JPC = 10 and 7.4 Hz, respectively. The
presence of 4-methoxyphenyl group with corresponding JHH, JPH
and JPC coupling constants was evident in both 1H and 13C NMR
spectra.
1 and Fig. 1).
The structure of the major isomer 3a and its absolute configura-
tion on phosphorus were confirmed by the X-ray analysis (Fig. 2
and Table 2). In this isomer the 4-methoxyphenyl group occupies
its prefered axial position with respect to the five-membered het-
erocyclic ring, while the conformation of the ring was found to be
half-chair. This explains the higher proportion of the conformer 3a.
It is assumed that its predominance is due to the steric repulsion
between 4-methoxyphenyl group and sterane skeleton in the 3b
isomer where 4-methoxyphenyl group adopts pseudo-equatorial
position with respect to the five-membered heterocyclic ring. Fur-
thermore, the values of bond lenghts and bond angles (Table 3)
show that endocyclic angles at S and P have considerably lower
values than other angles in the hetreocyclic ring, and these findings
are in accordance with the available literature data concerning
compounds with 1,3,2-oxathiaphospholane ring [15,35–37]. This
reflects the strain in the five-membered ring formed and its insta-
bility which can explain the low conversion of 1 to 2a,b and 3a,b
and moderate yield [18]. As the androstane moiety, which is iden-
In the case of the isomers of the compound 3 we were more for-
tunate and after several consecutive column chromatographies
diastereomerically pure major isomer 3a was obtained. Unfortu-
nately, all our attempts to get the minor isomer 3b in pure form
have failed, and its structure was determined on the basis of the
spectral data (IR, 1H NMR, 13C NMR) for the mixture of 3a and 3b.
All NMR spectra for the mixture of 3a,b, as well as the spectra
for pure 3a were taken on the Bruker Avance 500 MHz spectrome-
ter. Combined use of HSQC, HMBC and NOESY spectra in CDCl3
solution enabled complete 1H and 13C assignments and determina-
tion of absolute configurations on the phosphorus atom of each
isomer.
In the IR spectrum the absorption bands for C(20)@O at
1702 cmꢀ1 and for the C(17)–OH group at 3490 cmꢀ1 of 17
a-
hydroxyprogesterone (1) disappeared, but the C(3)@O absorption
band at 1660 cmꢀ1 was unchanged. The 1H NMR spectrum of 3a
had a broad singlet at 5.43 and a triplet at 5.49 ppm for the exo-
cyclic H2C@C(40) group and a singlet at 5.73 for H-4. The signals
for the corresponding protons in 3b (from the spectrum of the
mixture) were at 5.57, 5.76 and 5.70 ppm, respectively. In the
tical to the parent compound – 17a-hydroxyprogesterone (1), was
not altered by the synthesis, the chiral carbon atoms were used to
undoubtedly assign the configuration of the remaining chiral
centers.
Table 1
Characteristic 1H NMR and 13C NMR spectral data of 3a and 3b in CDCl3.
1H NMR (d in ppm, J in Hz)
13C NMR (d in ppm, J in Hz)
No.
3a
3b
3a
3b
3
4
12
–
–
199.5, s
124.0, d
32.3, t
199.3,s
123.9, d
31.1, t
5.73, s
5.70, s
H
a
, 1.80, m
Hb, 2.24, m
, 2.16, m
H
a
, 1.62, m
Hb, 2.23, m
, 2.16, m
16
H
a
H
a
35.7, t
39.0, t
Hb, 2.45, m
–
–
Hb, 2.42, m
–
–
2
2
17
40
107.5, sd, JPC = 10.0
104.1, sd, JPC = 7.1
2
2
144.0, sd, JPC = 2.4
141.9, sd, JPC = 1.6
3
3
CH2@C(40)
100
5.43, br.s, 5.49, t, J = 1.5
5.57, br.s, 5.76, t, J = 1.5
113.8, td, JPC = 11.6
116.5, td, JPC = 9.4
1
1
–
–
130.7, sd, JPC = 120.3
122.9, sd, JPC = 121.1
3
3
2
2
200 and 600
300 and 500
400
7.85, dd, JPH = 15.0, JHH = 9.0
6.92, dd, JPH = 3.5, JHH = 9.0
8.10, dd, JPH = 15.2, JHH = 8.8
7.00, dd, JPH = 3.5, JHH = 9.0
113.7, dd, JPC = 16.5
113.9, dd, JPC = 16.5
4
4
3
3
132.4, dd, JPC = 14.4
134.9, dd, JPC = 15.1
4
4
–
–
162,7, sd, JPC = 3.2
163,7, sd, JPC = 3.1

´
563
N.M. Krstic et al. / Steroids 77 (2012) 558–565
Fig. 1. 3D representation of the proposed structures of 3a and 3b on the basis of NOESY connectivities.
Fig. 2. ORTEP representation of 3a, with thermal displacement ellipsoids drawn at the 50% probability level.
The proposed mechanism of the formation of these products is
as follows. In the first step simultaneous attacks of a LR sulfur on C-
20 and the hydroxy oxygen on LR phosphorus create 1,3,2-oxathia-
phospholane ring. After the proton transfer and elimination of a
water molecule oxathiaphospholanes 2a,b and 3a,b were formed
(Scheme 3).
MDA-MB-453 cells could be explained only by their specific ac-
tion on signal pathways which regulate cell division. Also, it is
obvious that the replacement of 3-carbonyl group with 3-thioxo
group resulted in decrease in cytotoxicity which is in accordance
with our previous finding concerning antitumor activity of azaho-
mosteroids [26]. The antiproliferative actitivity of investigated
compounds is presented in Table 4 along with the activity of cis-
platin [38].
3.2. Biological evaluation
The antimicrobial activity of new spirosteroids was evaluated
against seven Gram-positive, four Gram-negative bacteria and
two fungi using a micro-broth dilution assay. A weak inhibitory
activity was exhibited by 3a and 3a,b against Gram-positive bacte-
ria B. subtilis and M. flavus; and by 2a,b only against M. flavus. Sim-
ilarly, all compounds had a weak activity against Gram-negative
bacteria P. aeruginosa while compound 3a showed activity against
P. aeruginosa and S. enteritidis. Unfortunately, in all cases the MIC
values were much higher than those for amikacin, showing that
these compounds are not promising antibacterial compounds for
further research (Table 5).
Cytotoxic activity in vitro was tested against three tumor cell
lines: human cervix adenocarcinoma HeLa, and two human breast
cancer cell lines: originally estrogen receptor-positive and proges-
terone receptor-positive MDA-MB-361 cells and estrogen recep-
tor-negative and progesterone receptor-negative MDA-MB-453
cells. The weak activity against HeLa cells was exhibited by both
mixtures of isomers 2a,b and 3a,b while compound 3a showed a
moderate activity with IC50 of 84.8 lM. Against ER and PR-nega-
tive MDA-MB-453 cells compound 3a and mixture of isomers
3a,b exhibited more pronounced cytotoxic activity with IC50 val-
ues of 55.0 and 74.3
l
M, respectively, while mixture of isomers
However, all new spirosteroids were found to have activity
comparable with nystatin against both fungi tested, C. albicans
andS. cerevisiae. The MIC values ranged from 2.36 to 4.86 mM, sim-
ilar to 0.89–2.70 mM for nystatin. As there was no significant dif-
ference in antifungal activity between 3-oxo and 3-thioxo
2a,b was less active. Against ER and PR-positive MDA-MB-361
cell line all tested compounds exerted very weak cytotoxic effect.
These differences in the extent of the cytotoxic action of exam-
ined compounds on MDA-MB361 and on more proliferative

´
564
Table 2
N.M. Krstic et al. / Steroids 77 (2012) 558–565
Table 5
Crystal data and structure refinement for 3a.
Antibacterial activity of compounds 2a,b, 3a,b and 3a.
Empirical formula
Formula weight (g/mol)
Temperature (K)
Wavelength (Å)
Crystal system
Space group
C28 H35 O3PS2
514.65
120 (2)
0.71073
Orthorhombic
C 2 2 21
a = 7.66888 (15)
b = 12.5544 (2)
Bacteria
MIC (mM)
2a,b
3a,b
3a
Amikacin
C. sporogenes
B. subtilis
S. longisporum
K. pneumoniae
S. aureus
S. lutea
M. flavus
P. aeruginosa
E. coli
S. enteritidis
P. vulgaris
–
–
–
–
–
–
2.36
4.72
–
–
4.86
–
–
–
–
2.43
–
–
–
0.0256
0.0717
0.1110
0.0034
0.0188
0.0034
0.0034
0.0854
0.0085
0.0137
0.0120
Unit cell dimensions (Å)
–
–
c = 53.1980 (9)
5121.81 (16)
Volume (Å3)
2.43
4.86
–
–
–
2.43
2.43
–
2.43
–
Z
8
Calculated density (gcmꢀ3
)
1.335 Mg/m3
Absorption coefficient (mmꢀ1)
F(000)
0.299
2192
–
–
Crystal size (mm)
2hmax (deg)
0.61 ꢂ 0.47 ꢂ 0.22
50.92
Limiting indices
Measured reflections
Unique reflections
ꢀ8 6 h 6 8, 15 6 k 6 15,64 6 l 6 64
Table 6
Antifungal activity of compounds 2a,b, 3a,b and 3a.
18700
2594
Observed reflections (Io > 2
Parameters refined
R1
a
(Io))
2474
310
Fungi
MIC (mM)
2a,b
3a,b
3a
Nystatin
0.0571
0.1170
0.0603
0.1182
1.205
x
R2
R1 (all data)
R2 (all data)
GOOF
Largest diff. peak and hole (e–Å–3
S. cerevisiae
C. albicans
2.36
2.36
2.43
4.86
2.43
2.43
1.89
2.70
x
)
0.620/ꢀ0.383
Table 7
Brine shrimp test results of 2a,b, 3a,b and 3a.
Table 3
Bond lengths (Å) and bond angles (°) in 1,3,2-oxathiaphospholane ring in 3a.
Compound
IC50 (lM)
Atoms
Bond lengths
Atoms
Bond angles
2a,b
3a,b
3a
301.8 1.9
252.8 4.6
136.1 3.8
PꢀO
1.594(2)
2.0989(12)
1.9223(12)
1.795(3)
1.790(3)
1.514(5)
1.472(4)
OꢀPꢀC100
OꢀP@S
106.27(12)
115.07(9)
97.47(9)
106.39(11)
115.50(9)
89.41(12)
128.1(3)
120.30(3)
111.5(2)
107.3(2)
PꢀS
P@S
OꢀPꢀS
PꢀC100
SꢀC40
C50ꢀC40
C50ꢀO
C100ꢀPꢀS
S@PꢀS
compounds we assume that the 1,3,2-oxathiaphospholane ring at
C-17 position is responsible for this activity (Table 6).
As expected, the results of the brine shrimp test are in good cor-
relation with cytotoxicity against tumor cell lines. The most active
compound 3a showed a moderate activity, while the mixtures of
isomers were less active (Table 7).
C40ꢀSꢀP
H2C@C40ꢀC50
H2C@C40ꢀS
C50ꢀC40ꢀS
OꢀC50ꢀC40
C50ꢀOꢀP
119.96(19)
4. Conclusion
CH3
O
CH3
O
CH3
CH3
S
Ar
Ar
S
S
In conclusion, it has been shown that the reactions of 17a-
P
P
O
H
O
H
hydroxyprogesterone (1) with LR in toluene, CH2Cl2 and/or CCl4
gave, depending on the duration of the reaction, mainly two com-
pounds, actually two diastereoisomeric androst-4-en-17-spiro-
1,3,2-oxathiaphospholane pairs 2a,b and 3a,b in approx. 7:3 ratio,
differing in configuration at the phosphorus atom. The best results
for 2a,b were obtained when the reaction was performed in boiling
toluene for 45 min (18%) and for 3a,b when the reaction was car-
ried out in CH2Cl2 for 7 h (46% yield). The low conversion of 1 to
2a,b and 3a,b and moderate yield can be explained by the ring
strain in the five-membered ring formed.
S
HO CH3
S
CH2
CH3
CH3
S
proton
transfer
Ar
O
P
-H2O
O
P
S
Ar
S
2a,b; 3a,b
Ar =
OCH3
Scheme 3. Proposed mechanism for the formation of oxathiaphospholanes 2a,b
and 3a,b.
Careful analysis of IR, 13C- and 1H-NMR spectroscopic data for
2a,b and 3a,b, revealed spectral features which correlated with
the 17-spiro-1,3,2-oxathiaphospholane structure. A parallel analy-
sis of heteronuclear 2D 1H–13C spectra (HSQC and HMBC) and
homonuclear 2D spectra (NOESY) enabled complete 1H and 13C
assignments of each isomer. Also, analysis of NOESY correlations
provided evidence for the preferred conformations. The ratio of
the isomers was deduced from the 1H NMR spectra by the peak
areas of H-4 and H2C@C(40). In addition, compound 3a was fully
characterized by the X-ray analysis which confirmed the structure
and absolute configuration on phosphorus. A pathway for the for-
mation of 1,3,2-oxathiaphospholane ring was proposed.
Table 4
The in vitro cytotoxic activity of compounds 2a,b, 3a,b and 3a.
Cell line
IC50 SD [lM]
2a,b
3a,b
3a
cisplatin
HeLa
MDA-MB-361
MDA-MB-453
161.8 37.4
>200
194.5 4.5
151.2 25.2
>200
74.3 19.4
84.8 14.3
>200
55.0 8.3
2.1 0.2
17.1 1.2
3.6 0.5

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N.M. Krstic et al. / Steroids 77 (2012) 558–565
diastereomers of thymidine phenyl cyclic 30,50-monophosphate. J Am Chem
Soc 1987;109:4058–64.
The weak cytotoxic activity against HeLa cells was exhibited by
both mixtures of isomers 2a,b and 3a,b while compound 3a
showed a moderate activity. Against MDA-MB-453 cells compound
3a and mixture of isomers 3a,b exhibited more pronounced cyto-
toxic activity, while mixture of isomers 2a,b was less active.
Against MDA-MB-361 cell line all tested compounds exerted very
weak cytotoxic effect. Similarly, mixtures 2a,b and 3a,b and pure
compound 3a showed week inhibitory activity against Gram-posi-
tive bacteria B. subtilis and M. flavus; and against Gram-negative
bacteria P. aeruginosa and S. enteritidis, indicating that these com-
pounds are not promising antibacterial compounds for further re-
search. Unlike cytotoxic and antibacterial, the antifungal activity of
the mixtures of isomers 2a,b and 3a,b as well as of compound 3a
against C. albicans and S. cerevisiae, was found to be very good, sim-
ilar to that of nystatin, in some cases even stronger. However low
cytotoxic and antibacterial activities indicate a selective activity of
these new 17-spiro-1,3,2-oxathiaphospholanes. Thus, further anal-
yses of structure–activity relationships of these compounds would
make it possible to design novel synthetic antifungal agents.
[13] Błaszczyk J, Wieczorek MW, Okruszek A. Conformation of oxathiaphospholane
and dithiaphospholane rings in the solid state. J Mol Struct 1996;374:85–96.
[14] Nawrot B, Re˛bowska B, Michalak O, Bulkowski M, Błaziak D, Guga P, et al.
1,3,2-Oxathiaphospholane approach to the synthesis of P-chiral stereodefined
analogs of oligonucleotides and biologically relevant nucleoside polyphos-
phates. Pure Appl Chem 2008;80:1859–71.
´
[15] Błaszczyk J, Wieczorek MW, Okruszek A, Sierzchała A, Kobylanska A, Stec WJ.
The synthesis and X-ray structural studies of 2-cholesteryl-2-thio-1,3,2-
oxathiaphospholane and 2-cholesteryl-2-thio-1,3,2-dithiaphospholane.
Chem Crystallogr 1996;26:33–42.
J
[16] Foster EL, Blickenstaff RT. Synthesis of steroidal cyclophosphamides. Steroids
1976;27:353–60.
[17] Frank É, Kazi B, Ludányi K, Keglevich G. Synthesis of some novel D-ring-fused
dioxa- and oxazaphosphorinanes in the estrone series. Tetrahedron Lett
2006;47:1105–8.
[18] Frank É, Schäfer B, Mucsi Z, Keglevich G. Synthesis and conformational study of
P-heterocyclic androst-5-ene derivatives. Heteroat Chem 2008;19:7–14.
[19] Frank É, Körtvélyesi T, Czugler M, Mucsi Z, Keglevich G. New steroid-fused P-
heterocycles
Part
I.
Synthesis
and
conformational
study
of
dioxaphosphorino[16,17-d]estrone derivatives. Steroids 2007;72:437–45.
[20] Frank É, Sipos L, Wölfling J, Schneider G. Synthesis and conformational
preferences of novel steroidal 16-spiro-1,3,2-dioxaphosphorinanes. Lett Org
Chem 2009;6:340–4.
[21] Mohamed NR, Elmegeed GA, Younis M. Studies on organophosphorus
compounds VII: transformation of steroidal ketones with Lawesson’s reagent
into thioxo and heterofused steroids. Results of antimicrobial and antifungal
activity. Phosphorus Sulfur Silicon Relat Elem 2003;178:2003–17.
[22] Mohamed NR, Elmegeed GA, Abd-ElMalek HA, Younis M. Synthesis of
biologically active steroid derivatives by the utility of Lawesson’s reagent.
Steroids 2005;70:131–6.
Acknowledgement
This work was supported by the Ministry of Education and Sci-
ence of the Republic of Serbia (Grant 172055).
[23] Pedersen BS, Scheibye S, Nilsson NH, Lawesson SO. Studies on organophos-
phorus compounds. XX. Syntheses of thioketones. Bull Soc Chim Belg 1978;
87:223–8.
Appendix A. Supplementary data
[24] Pedersen BS, Lawesson SO. Studies on organophosphorus compounds—XXVIII:
Syntheses of 3H–1,2-dithiole-3-thiones and 4H–1,3,2,-oxazaphosphorine
derivatives from the dimer of p-methoxyphenyl-thionophosphine sulfide
and derivatives of 3-oxo carboxylic acids. Tetrahedron 1979;35:2433–7.
[25] Cava MP, Levinson MI. Thionation reaction of Lawesson’s reagent. Tetrahedron
1985;41:5061–87.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2012.01.021.
References
ˇ
ˇ
ˇ
´
´
´
[26] Krstic´ NM, Bjelakovic´ MS, Zizak Z, Pavlovic MD, Juranic ZD, Pavlovic VD.
Synthesis of some steroidal oximes, lactams, thiolactams and their antitumor
activities. Steroids 2007;72:406–14.
[1] Suginome H, Yamada S, Wang JB. Photoinduced molecular transformations.
107. A versatile substitution of a carbonyl group of steroidal ketones by a
heteroatom. The synthesis of Aza-, Oxa-, Thia-, Selena-, and Tellurasteroids. J
Org Chem 1990;55:2170–6.
[2] Tietze LF, Schneider G, Wölfling J, Nöbel T, Wulff C, Schubert I, et al. Efficient
synthesis of a novel estrone–talaromycin hybrid natural product. Angew Chem
Int Ed 1998;37:2469–70.
[27] Krstic a, b-
´ NM, Bjelakovic´ MS, Dabovic´ MM, Pavlovic´ VD. Thionation of some
unsaturated steroidal ketones. Molecules 2010;15:3462–77.
[28] Kvasnica M, Rudovska I, Cisarova I, Sarek J. Reaction of lupane and oleanane
triterpenoids with Lawesson’s reagent. Tetrahedron 2008;64:3736–43.
[29] Sheldrick GM.
Crystallogr 2008;A64:112–22.
[30] Mosmann T. Rapid colorimetric assay for cellular growth and survival:
A short history of SHELX. Acta Crystallogr Sect A Found
[3] Tietze LF, Schneider G, Wölfling J, Fecher A, Nöbel T, Petersen S, et al. A novel
approach in drug discovery: synthesis of estrone
product hybrids. Chem Eur J 2000;6:3755–60.
–
talaromycin natural
application to proliferation and cytotoxicity assays.
1983;65:55–63.
J Immunol Methods
[4] Wölfling J, Hackler L, Mernyák E, Schneider G, Tóth I, Szécsi M, et al.
Neighboring group participation Part 15. Stereoselective synthesis of some
[31] Ohno M, Abe T. Rapid colorimetric assay for the quantification of leukemia
inhibitory factor (LIF) and interleukin-6 (IL-6). J Immunol Methods 1991;145:
199–203.
steroidal tetrahydrooxazin-2-ones, as novel presumed inhibitors of human 5
a-
reductase. Steroids 2004;69:451–60.
[32] NCCLS (National Committee for Clinical Laboratory Standards): Approval
standard document M7–A5, Vilanova, Pa, USA, 2000.
[33] Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL.
Brine shrimp; a convenient general bioassay for active plant constituents.
Planta Med 1982;45:31–4.
[5] Banday AH, Mir BP, Lone IH, Suri KA, Kumar HMS. Studies on novel D-ring
substituted steroidal pyrazolines as potential anticancer agents. Steroids
2010;75:805–9.
[6] Hirschmann R, Buchschacher P, Steinberg NG, Fried JH, Ellis R, Kent GJ, et al.
Synthesis and structure of steroidal Pregn-4-eno- and
c]pyrazoles. A novel class of potent anti-inflammatory steroids. J Am Chem
Soc 1964;86:1520–7.
5
a
-Pregnano[3,2-
[34] Cahn RS, Ingold C, Prelog V. Spezifikation der molekularen Chiralität. Angew
Chem 1966;78:413–47.
[35] Uznanski B, Grajkowski A, Krzyzanowska B, Kazmierkowska A, Stec WJ,
Wieczorek MW, et al. Stereochemistry of base-catalyzed ring opening of 1,3,2-
[7] Gupta R, Pathak D, Jindal DP. Synthesis and biological activity of azasteroidal
[3,2-c]- and [17,16-c]pyrazoles. Eur J Med Chem 1996;31:241–7.
[8] Manson AJ, Stonner FW, Neumann HC, Christiansen RG, Clarke RL, Ackerman
JH, et al. Steroidal heterocycles. VII. Androstano[2,3-d]isoxazoles and related
compounds. J Med Chem 1963;6:1–9.
oxathiaphospholanes. Absolute configuration of 2-{N-[(Rc)-1-(
ethyl]amino}-2-thiono-1,3,2-oxathiaphospholanes and 0, S-Dimethyl N-[(Rc)-
1-( -Naphthyl)ethyl]phosphoramidothioates. Am Chem Soc 1992;114:
10197–202.
a-Naphthyl)
a
J
[9] Tremblay MR, Luu-The V, Leblanc G, Noël P, Breton E, Labrie F, et al.
[36] Błaszczyk J, Wieczorek MW, Okruszek A, Olesiak M, Karwowski B. Geometry
and conformation of crystalline forms of b-naphthoxy-substituted 2-thio-
1,3,2-oxathiaphospholanes and 2-thio-1,3,2-dithiaphospholanes. Heteroat
Chem 1994;5:519–27.
Spironolactone-related
inhibitors
of
Type
II
17b-hydroxysteroid
dehydrogenase: chemical synthesis, receptor binding affinities, and
proliferative/antiproliferative activities. Bioorg Med Chem 1999;7:1013–23.
[10] Koizumi N, Takegawa S, Mieda M, Shibata K. Antiandrogen. IV. C-17 spiro 2-
oxasteroids. Chem Pharm Bull 1996;44:2162–4.
[11] Gerlt JA, Gutterson NI, Datta P, Belleau B, Penney CL. Thermochemical
identification of the structural factors responsible for the thermodynamic
instability of 30,50-cyclic nucleotides. J Am Chem Soc 1980;102:1655–60.
[12] Nelson KA, Bentrude WG, Setzer WN, Hutchinson JP. The question of chair–
twist equilibria for the phosphate rings of nucleoside cyclic 30,50-
monophosphates. 1H NMR and X-ray crystallographic study of the
_
[37] Krzyzanowska B, Stec WJ, Wieczorek MW, Błaszczyk J. Synthesis and
assignment of absolute configuration of enantiomeric dicyclohexylammo-
nium 2-oxo-2-thioxo-1,3,2-oxathiaphospholanes. Heteroat Chem 1994;5:
533–9.
[38] Stanojkovic TP, Kovala-Demertzi D, Primikyri A, Garcia-Santos I, Castineiras A,
Juranic Z, et al. Zinc(II) complexes of 2-acetyl pyridine 1-(4-fluorophenyl)-
piperazinyl thiosemicarbazone: synthesis, spectroscopic study and crystal
structures – Potential anticancer drugs. J Inorg Biochem 2010;104:467–76.