Synthesis of new symmetrical bis-steroidal pyrazine analogues from diosgenin

Article abstract of DOI:10.1016/j.steroids.2010.10.002

New symmetrical bis-steroidal pyrazine dimers that are cephalostatins/ ritterazines analogues have been prepared easily from a cheap, readily available natural steroid (diosgenin). These dimers were obtained by classical, condensation of α-amino ketones in order to construct the pyrazine rings. The three dimers differ in the functionalized diosgenin: (25R)-5α,6β- dihydroxy-5α-spirosta-3-one, (25R)-4,5α-epoxy-5β-spirosta-3,6- dione and (25R)-5α-hydroxy-5α-spirosta-3,6-dione respectively.

Full text of DOI:10.1016/j.steroids.2010.10.002

Steroids 76 (2011) 232–237
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of new symmetrical bis-steroidal pyrazine analogues from diosgenin
∗
Khaled Q. Shawakfeh , Naim H. Al-Said
Department of Applied Chemical Sciences, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan
a r t i c l e i n f o
a b s t r a c t
Article history:
New symmetrical bis-steroidal pyrazine dimers that are cephalostatins/ritterazines analogues have been
prepared easily from a cheap, readily available natural steroid (diosgenin). These dimers were obtained
by classical, condensation of ␣-amino ketones in order to construct the pyrazine rings. The three dimers
differ in the functionalized diosgenin: (25R)-5␣,6␤-dihydroxy-5␣-spirosta-3-one, (25R)-4,5␣-epoxy-
Received 23 August 2010
Received in revised form 4 October 2010
Accepted 6 October 2010
Available online 15 October 2010
5
␤-spirosta-3,6-dione and (25R)-5␣-hydroxy-5␣-spirosta-3,6-dione respectively.
©
2010 Elsevier Inc. All rights reserved.
Dedicated to Prof. John R. Williams for his
continuos support.
Keywords:
Steroidal
Pyrazine
Diosgenin
Dimers
Oxysterol
1
. Introduction
Marine species such as sponges, coelenterates, mollusks and
whereas the ritterazines have the more oxygenated left side. These
natural steroid dimers possess extremely potent inhibitory activ-
ity against a series of human cancer cell lines and the murine P388
lymphocytic leukemia cell line [7b,9]. Since the natural sources of
these compounds are extremely limited, and also the yield of these
compounds in nature is rather poor, the syntheses of a number
of various analogues of cephalostatines and ritterazines have been
reported in the literature [10–12].
There are many synthetic approaches for the central pyrazine
ring. Some of them describe the synthesis of unsymmetrical dimers
[13]. The symmetrical dimeric steroid-pyrazines can be obtained by
the classical condensation of ␣-amino ketones. This route is actu-
ally, the most efficient method of pyrazine rings construction. The
intermediate steroidal 2␣-amino-3-ketones are available by reduc-
tion of the corresponding 3-ketones with a nitrogen containing
substituent at C-2, such as azido [14] nitro [15], hydroxyimino [16]
and enamino [17] groups. The initially formed 2␣-amino-3-ketones
undergo spontaneous dimerization to a mixture of dihydropy-
razines, which are then oxidized by air to give pyrazine dimers
[3].
echinoderms are a rich source of a large variety of polyhydroxy
steroids [1]. The synthesis of polyhydroxy sterols has been a chal-
lenging subject for several years. Diosgenin is the steroidal saponin
that is isolated from the Mexican yam. Due to its structural simi-
larity to estrogen and progesterone precursors, diosgenin exhibts
estrogenic, progesterogenic and anti-inflammatory effects [2]. In
addition, diosgenin was used for the synthesis of nearly 50% of the
total steroid drugs in the world [2]. In 1988, a family of dimeric
steroid-pyrazine alkaloids and dimers was discovered by the Pettit
group [3]. Latter, as a continuation of their work on these dimers,
during the period from 1988 to 1998, the same group reported the
isolation and identification of compounds of this class, cephalo-
statines 2-19 [4–6]. For example Cephalostatin 1 (Fig. 1) was proved
to be one of the most powerful cancer cell growth inhibitors with
an ED50 value of 0.1–0.001 pM. This exceptional activity of cephalo-
statins has led to interest in the synthesis of compounds and their
analogues as potential anti-tumor agents [7].
The cephalostatin 1 and the closely related ritterazines B share
many common structural features in which two highly oxygenated
C27 steroidal units are fused via a pyrazine ring at C-2 and C-3
and both chains of the steroidal units form spiroketals [8]. The
cephalostatins in general are more oxygenated on the right side,
A new methodology for the synthesis for four new cephalostatin
analogues was reported by Shawakfeh et al. [18]. The main com-
ponent of the steroid mixture was the known diosgenin, which
has a spiroketal ring in the side chain. As a continuation of our
studies and due to the structural similarities between cephalo-
statin/rittarazine and diosgenin, we report here the synthesis of
three new polyoxgenated symmetrical bis-steroidal pyrazine ana-
logues that are expected to show more biological effect due to the
increased presence of polar functional groups.
∗ Corresponding author. Tel.: +962 2 7201000; fax: +962 2 7095014.
E-mail address: shawakfa@just.edu.jo (K.Q. Shawakfeh).
0
039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.10.002

K.Q. Shawakfeh, N.H. Al-Said / Steroids 76 (2011) 232–237
233
3
.3 (t, J = 10.8 Hz, 1H, C26␤), 2.94 (d, J = 4.5 Hz, 1H, C6␤), 1.18 (s, 3H,
OH
13
O
O
C19). C NMR (CDCl3): ı 211 (C3), 109 (C22), 81 (C16), 67 (C26),
5 (C5), 59 (C6). Anal. Calcd. for C27H40O : C, 75.66; H, 9.41. Found:
HO
HO
6
4
C, 74.98; H, 9.38.
OH
H
H
H
N
N
H
2.2.3. (25R)-5˛, 6ˇ-dihydroxy-5˛-spirosta-3-one (4)
OH
H
To a solution of 3 (1.0 g, 2.33 mmol) in acetone (60 mL) and water
H
O
H
(20 mL), was added perchloric acid (70%, 0.4 mL) with vigorous stir-
ring for 2 days. The reaction mixture was extracted with chloroform
(3 × 25 mL) and the combined organic layer was dried over Na2SO4,
concentrated to dryness and purified by column chromatography
O
O
Cephalostatin 1
O
O
HO
H
(30% ethyl acetate/hexane) to give a white solid of 4. Yield: 0.55 g
OH
◦
−1
(
50%), mp 142–144 C. IR [KBr]: 3327, 2957, 1712, 1681, 1058 cm
.
H
1
H NMR (CDCl ): ı 4.37 (dt, J = 6.6, 8.1 Hz, 1H, C16␣), 3.5 (t, J = 2.7 Hz,
3
N
N
H
1
7
H, C6). 13C NMR (CDCl ): ı 212 (C3), 109 (C22), 81 (C16), 67 (C26),
3
H
H
H
9 (C6), 75 (C5). Anal. Calcd. for C27H42O5: C, 72.61; H, 9.48.Found:
H
H
C, 72.55; H, 9.43.
O
O
OH
2.2.4. (25R)-2˛-Bromo-5˛,6ˇ-dihydroxy-5˛-spirosta-3-one (5)
To a solution of dihydroxy ketone 4 (0.2 g, 0.45 mmol) in THF
OH
Ritterazine B
HO
(10 mL) at room temperature, phenyl trimethylammonium perbro-
mide (PTAB, 0.22 g, 0.59 mmol, 1.3 equiv) in THF (7 mL) was added
rapidly. The resulting orange solution deposited a copious pre-
cipitate which faded to a beige color within 10 min. The solution
was quenched with brine solution (10 mL), extracted with CHCl3
Fig. 1. Structure of a cephalostatin 1 and ritterazine B.
2
. Experimental
(
2 × 20 mL), dried over Na SO and concentrated. The residue was
2
4
2.1. General
purified by column chromatography (30% ethyl acetate/hexane) to
give a pale yellow solid of bromodihydroxy ketone 5. Yield: 0.16 g
◦
−1
Melting points (mp) were determined on an electrothermal dig-
(69%), mp 147–150 C. IR (KBr, cm ): 3319, 2951, 1687 and 1053.
1
ital melting point apparatus and are uncorrected. All the starting
materials and reagents were obtained from commercial sources
and were used without further purification. FT-IR spectra were
H NMR (200 MHz, CDCl ): ı 4.76 (dd, J = 7.2, 5.2 Hz, 1H, C2␤, 4.37
3
13
(dt, J = 6.8, 7.7 Hz, 1H, C16␣), 3.57 (t, J = 2.7 Hz, 1H, C6␣). C NMR
(CDCl ): ı 211 (C3), 109 (C22), 81 (C16), 79 (C6), 76 (C5), 67 (C26).
3
1
recorded on a Nicolet-Impact 410 spectrophotometer. Both H and
Anal. Calcd. for C27H41O Br: C, 61.71; H, 7.86. Found: C, 61.57; H,
5
13
C NMR spectra were recorded on Bruker DPX-400 instruments.
The chemical shifts (ı) are reported in ppm relative to TMS used as
an internal standard.
7.68.
ꢀ
ꢀ
2.2.5. Di (25R-6˛-4,6ˇ-dihydroxy-5˛-spirostano[2,3-b:2 ,3 -e])
pyrazine (6)
2
2
.2. Chemical synthesis
The bromodihydroxy ketone 5 (0.2 g, 0.38 mmol) was dissolved
in DMF (30 mL) and a few mg of KI were added followed by addi-
tion of NaN3 (0.24 g, 3.8 mmol, 10 equiv.). The mixture was stirred
.2.1. (25R)-5-En-spirosta-3-one (2)
Pyridinum chlorochromate (1.7 g, 4.0 mmol) was added to
◦
for 3 h at 50 C. The solvent was evaporated under reduced pres-
a mixture of powdered CaCO3 (4.0 g, 4.0 mmol) and 1 (2.0 g,
.8 mmol) in CH Cl (50 mL) at room temperature. The reaction
sure. The residue was dissolved in CHCl3 (50 mL) and the resulting
solution was washed with brine solution (2 × 20 mL), dried over
Na SO and evaporated to give the azido ketone intermediate. To
4
2
2
mixture was stirred for 30 min. The reaction mixture was diluted
with diethyl ether (50 mL) and filtered through a short column of
florisil. The solvent was concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (10%
ethyl acetate in hexane) to give a ketone 2 as a white solid. Yield:
2
4
the intimate mixture of azido ketone in THF (5 ml) was added triph-
enylphosphine (0.33 g, 1.24 mmol, 3 equiv.) under N . The solution
2
was stirred for 5 h under N2 until the evolving gas from the solu-
tion ceased. Then THF (5 mL) and water (0.2 mL, 11 mmol) were
added and the reaction mixture was stirred overnight. After con-
centration, the yellow residue was azeotroped with toluene and
absolute ethanol (10 mL) and TsOH (catalytic amount) were added.
The orange mixture was stirred vigorously at room temperature
under atmospheric pressure for further 2 days. The fine solid was
filtered over a bed of celite and washed with CHCl3 followed by
evaporation to give a dimeric pyrazine 6. The product was purified
by crystallization from methanol and give pure dimeric pyrazine 6.
◦ ◦
.6 g (80%), mp 194–195 C, lit. mp 194–196 C [14]. IR (KBr): 2961,
1
1
−1 1
726 and 1681 cm
. H NMR (CDCl ): ı 5.3 (s, 1H, C6), 4.4 (dt,
3
J = 6.8, 8.0 Hz, 1H, C16␣), 3.48 (m, 1H, C26␣), 3.38 (t, J = 10.7 Hz, 1H,
1
3
C26␤). C NMR (CDCl ): ı 211 (C3), 140.6 (C5), 122 (C6), 80.1 (C17),
3
6
6.7 (C26), 109 (C22), 62.5 (C17).
2.2.2. (25R)-5,6-˛-Epoxy-5˛-spirosta-3-one (3)
A solution of 2 (1.0 g, 2.4 mmol) in dichloromethane (25 mL) was
◦
◦
−1
cooled to 0 C and a solution of m-chloroperoxybenzoic acid (1.8 g,
Yield: 0.11 g, 64%), m.p. 260 C(dec). IR (KBr, cm ): 3383, 2950, and
1
1
0.4 mmol) in dichloromethane (20 mL) was added dropwise. The
1417. H NMR (300 MHz, CDCl ): ı 4.40 (dt, J = 6.8, 8.1 Hz, 1H, C16␣),
3.46 (t, J = 2.7 Hz, 1H, C6␣), 1.09 (s, 3H, C19). C NMR (CDCl ): ı
3
13
reaction mixture was stirred at room temperature for 22 hours,
and successively washed with 5% solution of Na SO , saturated
3
143, 141 (pyrazine carbons), 109 (C22), 81 (C16), 79 (C6), 76 (C5),
67 (C26). Anal. Calcd. for C54H80N O : C, 73.27; H, 9.11; N, 3.16.
2
3
NaHCO , and water. Extraction with CHCl (2 × 50 mL) and then the
3
3
2
8
organic layer was dried over Na SO , evaporated to dryness under
Found: C, 73.07; H, 8.98; N, 3.01.
2
4
reduced pressure. The crude product was purified by crystalliza-
tion from acetone and gave a white solid of 3. Yield: 0.5 g (95%),
2.2.6. (25R)-4-en-Spirosta-3,6-dione (7)
Freshly prepared Jones reagent was added dropwise to a solu-
tion of 1 (2.0 g, 4.82 mmol) in acetone (150 mL) at 10 C. The reaction
◦
−1
1
mp 171–173 C. IR [KBr]: 2943, 1715, 1681, 1059 cm
.
H NMR
◦
(
CDCl ): ı 4.39 (dt, J = 6.8, 8.1 Hz, 1H, C16␣), 3.41 (m, 1H, C26␣),
3

2
34
K.Q. Shawakfeh, N.H. Al-Said / Steroids 76 (2011) 232–237
◦
mixture was stirred below 20 C for 30 min. The reaction mixture
2.2.12. (25R)-2˛-Bromo-5˛-hydroxy-spirosta-3,6-dione (13)
Procedure as described for 5 gave a white solid of bromo dike-
was washed with brine solution, extracted with CHCl3 (3 × 25 mL),
◦
−1
dried over Na SO4 and concentrated. The residue was purified
tone 13. Yield: 0.26 g (74%), mp 195–197 C. IR (KBr, cm ): 3340,
2
by column chromatography (20% ethyl acetate/hexane) to give a
2951, 1711, 1705, and 840. 1H NMR (300 MHz, CDCl ): ı 4.70 (1H,
3
◦
13
white solid of pure 7. Yield: 1.95 g (95%), mp. 190–193 C, lit. mp
dd, H2␤), 4.45 (1H, dt, 16␣), 3.48 (2H, m, H 26). C NMR (300 MHz,
◦
−1
1
90–192 C [19]. IR (KBr, cm ): 2943, 1681, 1610, and 1450. 1
H
CDCl ): ı 209 (C3), 208 (C6), 109 (C22), 82 (C5), 54 (C2). Anal. Calcd.
3
NMR (400 MHz, CDCl ): ı 6.3 (s, 1H, C4), 4.36 (dt, J = 6.8, 8.1 Hz, 1H,
C16-␣H), 3.45 (m, 2H, C26), 3.37 (t, 2H, C26). C NMR (400 MHz,
for C27H39O5Br: C, 61.95; H, 7.51. Found: C, 61.77; H, 7.45.
3
13
CDCl ): ı 202 (C3), 199 (C6), 163 (C5), 127 (C4), 109 (C22), 80.6
3
2
.2.13. Di (25R-3,6
(
C16), 67 (C26), 62.7 (C17), 56 (C14).
dioxo-5˛-hydroxy-5˛-spirostano[2,3-b:2’,3’-e]) pyrazine (14)
Procedure as described for 6 gave pure dimeric pyrazine 14.
2.2.7. (25R)-4,5˛-Epoxy-5ˇ-spirosta-3,6-dione (8)
◦
−1
Yield: 0.16 g (62%), m.p. 290 C(d). IR (KBr, cm ): 3353, 2944, 1706,
To a solution of diketone 7 (0.2 g, 0.47 mmol) in MeOH (20 mL)
and 1404. 1H NMR (300 MHz, CDCl ): ı 4.4 (dt, 1H, C16␣), 3.45
3
◦
at 15 C, hydrogen peroxide (30% H O , 5.0 mL) was added rapidly
13
2
2
(m, 2H, C26), 1.14 (s, 3H, C19). C NMR (300 MHz, CDCl ): ı 203
3
followed by dropwise addition of sodium hydroxide solution
(C6), 139, 138 (pyrazine carbons), 108 (C22), 79 (C5), 67 (C26). Anal.
(
NaOH, 4 M, 3.0 mL). The reaction mixture was stirred keeping the
Calcd. for C54H76N O : C, 73.60; H, 8.69; N, 3.18. Found: C, 73.52;
H, 8.60; N, 3.06.
2
8
◦
temperature below 15 C for 1 h, then concentrated followed by
extraction with CHCl3 (2 × 20 mL). The extract was washed with
water (2 × 15 mL), dried over Na SO , concentrated to get a white
2
4
3
. Results and discussion
solid. The residue was purified by column chromatography (20%
ethyl acetate/hexane) to give a white solid of pure 8 (0.13 g, 63%).
◦
−1
1
In this paper, the synthesis of dimer 6, which has two hydroxyl
M.p. 167–169 C. IR (KBr): 2949, 1726, 1705, and 1054 cm
. H
groups at C5 and C6, was carried out, as shown in Scheme 1. Oxi-
dation of diosgenin 1 with PCC in the presence of CaCO3 gave the
known ketone 2 [18]. Next, the reaction of m-chloroperoxybenzoic
acid (mCPBA) with the ketone 2 gives the 5␣, 6␣-epoxide 3 with
the epoxidation taking place predominantly from the ␣-face of the
molecule. The selective epoxidation from the ␣-face was attributed
to the equatorial attack of mCPBA to the double bond of diosgenin is
destabilized by the steric interaction between the reagent and the
NMR (CDCl ): ı 4.37 (dt, J = 6.6, 8.1 Hz, 1H, C16␣), 2.98 (s, 1H, C4␤),
3
13
3
.48 (m, 2H, C26), 3.35 (t, 2H, C26), 1.26 (s, 3H, C19). C NMR
(
CDCl ): ı 203 (C3), 201 (C6), 109 (C22), 81 (C16), 70 (C5), 67 (C26),
3
5
8 (C4). Anal. Calcd. for C27H38O5: C, 73.27; H, 8.65. Found: C, 72.97;
H, 8.58.
2.2.8. (25R)-2˛-Bromo-4,5˛-epoxy-5ˇ-spirosta-3,6-dione (9)
Procedure as described for 5 gave a pale yellow solid of bromodi-
1
◦
−1
axial CH3 group at C-19. The H NMR spectrum for product 3 shows
one 9. Yield: 0.17 g (72%), mp 170–172 C. IR (KBr, cm ): 2951,
−
. H NMR (300 MHz, CDCl ): ı 4.68
3
1
1
doublet of triplet peak at 4.39 ppm (J = 6.6, 8.1) for one ␣ proton at
C16 and a doublet peak at 2.94 ppm (J = 4.5 Hz) for one ␤ proton at
C6. The 13C NMR spectrum shows new peaks at 59 and 65 ppm for
C6 and C5, respectively. Moreover, the signal representing C3 was
observed at 211 ppm. The protonation of the epoxide 3 will lead
to the formation of oxonium intermediate, which is susceptible to
nucleophilic attack by water will lead to the formation of the diol 4.
1
720, 1704, 1054, and 838 cm
(
(
dd, J = 6.9, 5.2 Hz, 1H, C2␤), 4.36 (dt, J = 6.6, 8.1 Hz, 1H, C16␣), 3.88
s, 1H, C4␤), 3.48 (m, 2H, C26), 3.35 (t, 2H, C26), 1.26 (s, 3H, C19). 13
C
NMR (CDCl ): ı 209 (C3), 206 (C6), 109 (C22), 81 (C16), 70 (C5), 69
3
(
C4), 54 (C2). Anal. Calcd. for C27H37O5Br: C, 62.19; H, 7.15. Found:
C, 61.97; H, 7.07.
1
The H NMR spectrum for product 4 shows doublet of triplet peak
ꢀ
ꢀ
2
.2.9. Di (25R-6 oxo-4,5˛-epoxy-5ˇ-spirostano[2,3-b:2 ,3 -e])
pyrazine (10)
Procedure as described for 6 gave a white solid of pure dimeric
at 4.37 ppm (J = 6.6, 8.1) for one ␣ proton at C16 and a triplet peak at
3
.5 ppm (J = 2.7 Hz) for one ␣ proton at C6. The 13C NMR spectrum
shows new peaks at 79 and 76 ppm for C6 and C5, respectively, in
addition to the peak at 212 ppm for C3.
◦
−1
pyrazine 10. Yield: 0.12 g (65%), m.p. 300 C(d). IR (KBr, cm ):
922, 1719, and 1417. H NMR (300 MHz, CDCl ): ı 4.40 (dt, J = 6.8,
.1 Hz, 1H, C16␣), 3.84 (s, 1H, C4␤), 1.07 (s, 3H, C19). C NMR
1
2
8
3
Selective bromination at C2 by phenyl trimethylammonium
13
perbromide (PTAB) gave the ␣-bromo ketone 5 as the thermody-
(
CDCl ): ı 203 (C6), 142, 141 (pyrazine carbons), 109 (C22), 81
3
namic product. The 1H NMR spectrum shows doublet of doublets
(
C16), 70 (C5), 67 (C4). Anal. Calcd. for C54H72N O : C, 73.94; H,
2
8
at 4.76 ppm (J = 7.2, 5.2 Hz) for ␤ proton at C2 and doublet of triplet
peak at 4.37 ppm for one ␣ proton at C16. The 13C NMR spectrum
shows a new peak at 54 ppm for C2, in addition to the peaks at 211,
8
.27; N, 3.19. Found: C, 73.69; H, 8.18; N, 3.04.
2.2.10. (25R)-5,6˛-Epoxy-5˛-spirosta-3ˇ-ol (11)
7
9 and 76 ppm for C3, C6 and C5 respectively.
Procedure as described for 3 gave a white solid of the epoxy
Next, completion of the total synthesis required direct conver-
◦
1
(
1. Yield: 0.95 g (92%), mp 200–202 C, lit. m.p 202–204 [20]. IR
sion of ␣-bromodione 5 into the targeted bis-steroidal pyrazine
dimer 6. Therefore substitution of bromide with an azido group
using sodium azide in the presence of catalytic amount of sodium
iodide furnished the corresponding ␣-azido ketone. This ␣-azido
ketone was not stable at room temperature and it was reduced by
aqueous triphenyl phosphine (Staudinger reaction) in THF for 24 h.
Addition of a catalytic amount of p-toluenesulfonic acid in ethanol
and oxidation in air afforded the bis steroidal pyrazine dimer 6 in
KBr, cm ): 3510, 3400, 2950, 1645, and 1053. 1H NMR (300 MHz,
−1
CDCl ): ı 4.36 (m, 1H, C16), 3.90 (m, 1H, C3,), 3.48 (m, 2H, C26), 2.9
3
13
(
d, J = 14 Hz, 1H, C6), 1.08 (s, 3H, C19), 0.76 (s, 3H, C18). C NMR
(
300 MHz, CDCl : ı 109 (C22), 81 (C16), 70 (C3), 67 (C26), 66 (C5),
3
5
9 (C6).
2.2.11. (25R)-5˛-Hydroxy-5˛-spirosta-3,6-dione (12)
Procedure as described for 7 but with a solution of 11 (2.0 g,
.65 mmol) gave a white solid of pure 12. Yield: 1.8 g (87%), mp
91–192 C. IR (KBr, cm ): 3346, 2949, 1713, and 1705. H NMR
4% yield. The 1H NMR spectrum shows a triplet peak at 3.46 ppm
6
4
1
(
4
J = 2.7 Hz) for one ␣ proton at C6 and a doublet of triplet peak at
.4 ppm for one ␣ proton at C16. The C NMR spectrum shows two
◦
−1
1
13
(
(
300 MHz, CDCl ): ı 4.48 (m, 1H, C16), 3.48 (t, 2H, C26), 2.80–2.90
3
peaks at 141 and 143 ppm characteristics for the pyrazine carbons,
in addition to the C5 and C6 peaks at 76 and 79 ppm respectively.
Oxysterols represent a class of potent regulatory molecules
with remarkably diverse, important biological activities [19]. They
13
s, 2H, C4), 2.26 (m, 2H, C2). C NMR (300 MHz, CDCl ): ı 210 (C6),
3
2
09 (C3), 109 (C22), 82 (C5), 67 (C26), 63 (C17), 56 (C14). Anal.
Calcd. for C27H40O5: C, 72.94; H, 9.07. Found: C, 72.77; H, 8.95.

K.Q. Shawakfeh, N.H. Al-Said / Steroids 76 (2011) 232–237
235
Me
Me
Me
Me
Me
Me
Me
O
O
Me
Me
O
O
O
O
Me
H
Me
H
Me
H
PCC, CaCO3
MCBPA,
CH Cl , 2 h
H
H
H
H
H
H
2
2
CH Cl , 1.5 h
HO
2
2
O
O
O
Diosgenin (1)
3
2
HClO , acetone
4
2
days
Me
Me
Me
Me
Me
Me
O
O
O
O
Me
H
PTAB, THF
R.T, 10 min
Me
H
Br
H
H
H
H
O
O
HO
HO
OH
5
OH
4
o
1
2
3
. NaN , DMF, KI, 50 C, 4h
3
. PPh , THF, H O, 24 h
. Ethanol, TsOH, 24h
3
2
Me
Me
Me
O
O
OH
H
OH
Me
H
N
N
H
H
H
H
Me
HO
O
O
OH
6
Me
Me
Me
Scheme 1. Synthesis of dimer 6.
exhibit a number of biological activities, including inhibition of
cellular proliferation and cytotoxicity associated with induction
of apoptosis [20]. For this reason, we decided to synthesize new
polyoxgenated pyrazine dimers taking advantages of epoxidation
methodology presented in Scheme 1.
same protocol for compound 4 by selective bromination at C2 of
compound 8 by phenyl trimethylammonium perbromide (PTAB)
gave the ␣-bromodione epoxide 9 as the thermodynamic prod-
uct. The 1H NMR spectrum shows doublet of doublets at 4.68 ppm
(J = 6.9, 5.2 Hz) for ␤ proton at C2 and doublet of triplet peak at
4.36 ppm for one ␣ proton at C16. The 13C NMR spectrum shows a
new peak at 54 ppm for C2, in addition to the peaks at 209, 206, 69
and 70 ppm for C3, C6, C4 and C5 respectively. The dimerization of
the bromoketone 9 involved two steps to achieve the formation of
the pyrazine dimer 10. The first step is the substitution of the bromo
atom with an azido group using sodium azide in the presence of
catalytic amount of sodium iodide. The next step is reduction to
␣-amino ketone by triphenylphosphine in dry THF followed by
addition of water to hydrolyze the aza-Wittig intermediate. The
resulting amino ketone intermediate was stirred in ethanol and
toluene containing a catalytic amount of p-toluenesulfonic acid,
and open to the atmosphere to facilitate aromatization to yield the
pyrazine dimer 10 in good overall yield. The 1H NMR spectrum
shows a doublet of triplet peak at 4.4 ppm for one ␣ proton at C16
and a singlet peak at 3.84 ppm for one proton at C4. The 13C NMR
spectrum shows two peaks at 141 and 142 ppm characteristics for
the pyrazine carbons. It also shows the epoxide carbon peaks at 67
and 70 ppm and the carbonyl carbon peak at 203 ppm.
Here, we present a chemically simple, rapid, economical and
4
diverse one-pot synthesis for generation of ꢀ -3, 6-dione function-
alized diosgenin as shown in Scheme 2. Utilization of the modified
Jones oxidation methodology afforded 4-ene-3, 6-dione compound
7
in 95% yields. The 1H NMR spectrum for compound 7 showed a
13
singlet resonance signal at 6.3 ppm for the C4 proton. The C NMR
spectrum of 7 showed two new peaks at 202 and 199 ppm for car-
bonyl groups at C3 and C6, respectively. The nature of the double
bond values changed significantly for the C4 and C5 value to 127
and 163 ppm respectively. Both the H and C NMR melting points
were consistent with the literature reported values [21,22]. Having
successfully prepared compound 7, we next turned to introduce an
epoxide ring at C4/C5, thus compound 7 was epoxdized with 30%
H O and NaOH solution in methanol to give the 4␣, 5␣-epoxide
1
13
2
2
8
, with the epoxidation taking place predominantly from the ␣-
1
face of the molecule. The H NMR spectrum for product 8 showed
doublet of triplet peak at 4.37 ppm (J = 6.6, 8.1) for one ␣ proton at
C16 and a singlet peak at 2.98 ppm for one ␤ proton at C4, in addi-
tion to the significantly shifted singlet peak at 1.26 ppm for three
protons at C19. The 1 C NMR spectrum shows two peaks at 203
and 201 ppm for carbonyl groups at C3 and C6, respectively. The
epoxide carbons at C4 and C5 appear at 67 and 70 ppm, respec-
tively. These data are consistent with reported values for systems
with 4␣,5-epoxy-5␣ and 4␤,5-epoxy-5␤-sterol [23]. Following the
For the last dimer, we decided to have free hydroxyl group at
C5 keeping the ketone functionality at C6 as outlined in Scheme 2.
Diosgenin 1 was treated with MCPBA and gave epoxysterol 11 in
92% yields. The 1H and C NMR as well as melting point were
consistent with literature reported values [2]. The next step was
to increase the oxygen content by further oxidation in order to
3
13

2
36
K.Q. Shawakfeh, N.H. Al-Said / Steroids 76 (2011) 232–237
Me
Me
O
Me
Me
Me
O
O
Me
O
H
O
O
Me
H
Me
H
N
N
Br
o
H
H
H
1. NaN , DMF, KI, 50 C, 4h
2. PPh , THF, H O, 24 h
3. Ethanol, TsOH, 24h
H
H
3
O
3
2
H
Me
O
O
O
O
O
O
1
0
9
Me
Me
Me
PTAB, THF
R.T, 10 min
Me
Me
Me
Me
Me
Me
O
O
O
O
Me
H
H
Me
H
H O NaOH
MeOH, 15 C, 1hr
2
2,
H
H
H
o
O
O
O
7
O
8
O
Jones Reagent,
o
1
5 C, 30 min
Diosgenin (1)
MCBPA,
Me
Me
Me
Me
Me
Me
CH Cl , 2 h
O
O
O
O
2
2
Me
H
Me
H
Jones Reagent,
H
H
H
H
o
15 C, 2 h
HO
O
O
HO
O
1
1
12
PTAB, THF
R.T, 10 min
Me
Me
Me
O
O
Me
Me
Me
O
O
H
OH
Me
H
N
N
O
Me
H
H
H
H
Br
o
1
. NaN , DMF, KI, 50 C, 4h
3
H
O
H
H
Me
HO
2. PPh3, THF, H2O, 24 h
. Ethanol, TsOH, 24h
O
O
O
3
HO
Me
O
Me
14
Me
13
Scheme 2. Synthesis of dimers 10 and 14.
generate new oxysterols. Therefore, Jones oxidation of epoxysterol
steroidal pyrazine dimer 14. In our case, substitution by sodium
azide in the presence of catalytic amount of sodium iodide, fol-
lowed by addition of a catalytic amount of p-toluenesulfonic acid in
ethanol and further oxidation in air afforded bis steroidal pyrazine
dimer 14 in 62% yield. The 1H NMR spectrum shows a doublet of
triplet peak at 4.4 ppm for one ␣ proton at C16 and a singlet peak
at 1.24 ppm for three protons at C19. The 13C NMR spectrum shows
two peaks at 139 and 138 ppm characteristics for the pyrazine car-
1
1
1 furnished in excellent yield the diketosterol 12. The H NMR
spectrum shows two peaks at 2.9 and 2.26 ppm for the C4 and C2
methylene protons. The 1 C NMR spectrum shows three new peaks
at 210, 219 and 82 ppm for ca rbonyl carbons at C3 and C6 and
3
◦
for the 3 -alcohol carbon at C5, respectively. Selective bromina-
tion at C2 by phenyl trimethylammonium perbromide (PTAB) gave
the ␣-bromodione 13 as the thermodynamic product. Interestingly
bromination did not occur at C7 ␣ to the C6 ketone. The 1H NMR
spectrum shows doublet of doublets at 4.70 ppm for ␤ proton at C2
and doublet of triplet peak at 4.45 ppm for one ␣ proton at C16. The
◦
bons. It also shows the 3 -alcoholic carbon peak at 79 ppm and the
carbonyl carbon peak at 203 ppm.
13
C NMR spectrum shows a new peak at 54 ppm for C2, in addition
4. Conclusion
to the peaks at 209, 208 and 82 ppm for C3, C6 and C5 respectively.
As mentioned earlier, the dimerization steps required direct
conversion of ␣-bromodione 13 into our target which is the bis-
A convenient synthesis for three new symmetrical bis-steroidal
pyrazine dimers from a cheap, available and natural steroid (dios-

K.Q. Shawakfeh, N.H. Al-Said / Steroids 76 (2011) 232–237
237
genin) is reported. These polyoxygenated symmetrical bis steroidal
pyrazine analogues were obtained by classical condensation of ␣-
amino ketones that is the most efficient method of pyrazine rings
construction.
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Acknowledgements
We would like to thank Jordan University of Science and Tech-
nology for the opportunity to do this research during a sabbatical
leave at University of Dammam-Saudia Arabia. Also many thanks
to Dr. Abedlatif Ibdah at King Fahed for Petroleum and Minerals for
doing some spectroscopic analysis.
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