Expeditious and convenient synthesis of pregnanes and its glycosides as potential anti-dyslipidemic and anti-oxidant agents

Article abstract of DOI:10.1016/j.bmc.2007.04.022

A series of new pregnane derivatives and its glycosides were synthesized in order to find new 'leads' against some important targets. The 3β-hydroxy-16α-(2-hydroxy ethoxy) pregn-5-en-20-one (5) was synthesized from 3β-hydroxy-5,16-pregnadiene-20-one (2) by adopting general modified procedure using BF₃:Et₂O as a catalyst. Reduction of 5, with sodium borohydride yielded 3β,20β-dihydroxy-16α-(2-hydroxy ethoxy) pregn-5-en (7) as the major isolable product. O-alkylation of the C-20-oxime-pregnadiene (9) with 1,5-dibromopentane yielded 20-(O-5-bromopentyl)-oximino-3β-hydroxy-pregn-5,16-diene (11). Synthesis of C-16 substituted pregnane glycosides (20) and (21) were accomplished with the imidate method using BF₃:Et₂O. The synthesis of 4-chlorobenzoate (3) and 2-chlorobenzoate (4), derivatives of 2 were also accomplished. These compounds were evaluated for their anti-dyslipidemic and anti-oxidant activity and amongst them compounds 3 and 7 showed more lipid lowering and anti-oxidant activity.

Full text of DOI:10.1016/j.bmc.2007.04.022

Bioorganic & Medicinal Chemistry 15 (2007) 4520–4527
Expeditious and convenient synthesis of pregnanes and its
glycosides as potential anti-dyslipidemic and anti-oxidant agents
Arun Sethi,a,* Atul Maurya,a Vibha Tewari,a Sanjay Srivastava,c Shaheen Faridi,a
Gitika Bhatia,b M. M. Khan,b A. K. Khannab and J. K. Saxenab
aDepartment of Chemistry, University of Lucknow, Lucknow 226007, India
bDivision of Biochemistry, Central Drug Research Institute, Lucknow, India
cDepartment of Applied Sciences, Institute of Engineering and Technology, Lucknow, India
Received 23 January 2007; revised 11 April 2007; accepted 12 April 2007
Available online 19 April 2007
Abstract—A series of new pregnane derivatives and its glycosides were synthesized in order to find new ‘leads’ against some impor-
tant targets. The 3b-hydroxy-16a-(2-hydroxy ethoxy) pregn-5-en-20-one (5) was synthesized from 3b-hydroxy-5,16-pregnadiene-20-
one (2) by adopting general modified procedure using BF3:Et2O as a catalyst. Reduction of 5, with sodium borohydride yielded
3b,20b-dihydroxy-16a-(2-hydroxy ethoxy) pregn-5-en (7) as the major isolable product. O-alkylation of the C-20-oxime-pregnadiene
(9) with 1,5-dibromopentane yielded 20-(O-5-bromopentyl)-oximino-3b-hydroxy-pregn-5,16-diene (11). Synthesis of C-16
substituted pregnane glycosides (20) and (21) were accomplished with the imidate method using BF3:Et2O. The synthesis of 4-chlo-
robenzoate (3) and 2-chlorobenzoate (4), derivatives of 2 were also accomplished. These compounds were evaluated for their anti-
dyslipidemic and anti-oxidant activity and amongst them compounds 3 and 7 showed more lipid lowering and anti-oxidant activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
active compounds isolated chiefly from Asclepiada-
ceae13 family plants have been found to posses anti-can-
cer,14 anti-asthamatic,15 anti-tumor16 and anti-fungal
activities.17 The synthetic derivatives of this class of
compounds are larger, more hydrophilic, have increased
efficacy and reduced toxicity relative to the parent com-
pound (aglycone)18 and have primarily been used for the
treatment of ulcerative colitis,19 Crohn’s disease19 and
cardiac contractibility.20 Owing to the immense impor-
tance of these medicinally important pregnane deriva-
tives, considerable attention is being devoted to the
synthesis and biological evaluation of this important
class of compounds.21–27 In the search of newer deriva-
tives, the synthesis of novel pregnane derivatives and its
glycosides in excellent yields is herein reported.
Pregnanes, the C-21 steroid derivatives, are an impor-
tant class of bioactive compounds having diverse phar-
macological activity and which has provided
interesting ‘leads’ for the development of new drugs.
These potent pregnane derivatives have been found to
posses cytotoxic,1 antifeedant,2 anti-inflammatory,3,4
anti-asthamatic,5 lipid lowering6 and anti-viral7 activi-
ties. They also find use as neurosteroids8 and as inhibi-
tors of testosterone 5a reductase9 which helps in the
treatment of androgen sensitive prostate cancer in
men.10 Besides this, a number of pregnanes are not only
similar to cardiac glycosides with respect to molecular
and cellular site of action, but they have enhanced
cardiac contractibility.11
Pregnane glycosides because of their remarkable phar-
macological activities are one of the major components
of traditional Chinese medicine.12 These biologically
2. Chemistry
During the course of our studies towards the synthesis
of C-16-substituted pregnane derivatives we employed
an earlier reported method28 and carried out a number
of modifications in order to increase the total yield of
the desired product. The modified procedure involved
the stirring of the compound 3b-hydroxy pregn-5,16-
diene-20-one 2 with nucleophilic reagent ethylene glycol
Keywords: Pregnanes; Glycosides; Oximo ether; Anti-dyslipidemic;
Anti-oxidant.
*
Corresponding author. Tel.: +91 522 2454108; e-mail: alkaarunsethi@
rediffmail.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.022

A. Sethi et al. / Bioorg. Med. Chem. 15 (2007) 4520–4527
4521
in the presence of BF3:Et2O as a catalyst at different
temperatures. The best results were obtained when the
reaction mixture was stirred at 30 ꢁC and that too for
lesser period (55–60 h). Employing this modified proce-
dure compound 5 was synthesized. The yield of the de-
sired derivative was not only excellent (80%) but also
It is also well recognized that L-rhamnose containing
serogroups of mycobacterium are closely related to
opportunistic pulmonary infection40 and AIDS dissemi-
nate infections.39b A number of naturally occurring
D-mannose related glycosides have also shown marked
biological activities.41
1
free from any other position being substituted. The H
NMR spectrum not only conclusively proved the struc-
ture of compound 5 but it also helped in ascertaining the
orientation of side chain at C-16. A well-defined doublet
at d 2.58 was attributed to the methine proton at C-17.
The magnitude of the coupling constant of this doublet,
J = 6.4 Hz due to J16bH–17aH confirms the orientation of
this side chain at C-16 to be a.29 Reduction of 5 with so-
dium borohydride afforded 7 in good yield. The stereo-
chemistry of the orientation of hydroxyl group at C-20,
which was postulated to be b,30 was confirmed by the
presence of small NOE31 between C-13 and H-20.
Acetylation of 5and 7yielded their respective acetyl deriv-
atives 6 and 8. Guggulsterone and related compound 80–
574 have been found to act as antagonists of the bile acid
receptor32 (BAR). But the compound 80–574 is very
poorly absorbed through the intestines. In order to impart
betterabsorption,32b,2c the synthesis oftwo newer benzoyl
derivatives 3 and 4 was accomplished by treating com-
pound 2 with 4-chlorobenzoyl chloride and 2-chloro-
benzoyl chloride, respectively (Scheme 1).
In our effort to prepare these biologically important
L-rhamnose/D-mannose-related pregnane glycosides, tri-
chloroimidate method42 was adopted in which 2,3,4-tri-
O-acetyl-a-L-rhamnopyranosyl trichloroacetimidate 15/
2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl trichloro-
acetimidate 19, derivatives of L-rhamnose and D-man-
nose were treated with pregnane genin 5 in presence of
Lewis acid catalyst BF3:Et2O affording glycosides 20
1
and 21, respectively43 (Scheme 2). The H NMR spec-
trum not only confirmed the structure but also helped
in ascertaining the configuration of the glycosidic link-
age in 20. The anomeric proton was observed as a singlet
at d 4.76. The negligible diequatorial coupling confirmed
the glycosidic linkage to be a. Besides this, the downfield
shifting of the methylene protons of the side chain at C-
16 by ca. 0.3 ppm suggested that the sugar is glycosidi-
cally linked to the hydroxyl group of the side chain at
C-16 and not to C-3 hydroxyl group. In the FAB MS
of 20, fragments at m/z 544 (Retro Diels Alder rear-
rangement at C-2 and C-3 of sugar), m/z 510 (Retro
Diels Alder rearrangement at C-5 and C-6 of aglycone)
and at m/z 429 (due to C-2 C-3 bond cleavage of sugar
followed by loss of C-3 OH of aglycone as water mole-
cule)44 further confirm that the sugar is glycosidically
linked to the hydroxyl group of the side chain and not
Steroidal oximino ether derivatives which posses contra-
gestational activity,33a anti-androgenic activity,33b neu-
romuscular blocking activity33c have also been used
for the treatment of breast cancer in women34–36 by
functioning as inhibitors of enzyme aromatase cyto-
chrome P450. A novel steroidal oximino ether derivative
11 was synthesized by treating 9 with 1,5-dibromopen-
tane in dry DMF in presence of base NaH (Scheme 1).
1
to the C-3 hydroxyl group. Similarly in the H NMR
spectrum of 21 the anomeric proton was also observed
as a singlet at d 5.02, suggesting the linkage to be a.
In the FAB MS, fragments at m/z 483 (Retro Diels
Alder rearrangement at C-5 and C-6 of aglycone) and
m/z 357 (Retro Diels Alder rearrangement at C-2
and C-3 of sugar) further supported the glycosidic
nature of compound 21.
L-Rhamnose containing oligosaccharides are widely dis-
tributed in nature as triterpenoid glycosides,37 K-anti-
gens38 and phenolic glycolipids from mycobacteria.39
Scheme 1. Reagents: (a) HOCH2CH2OH, BF3Et2O; (b) NH2OHÆHCl–pyridine; (c) Br(CH2)5Br, NaH, DMF; (d) NaBH4; (e) 4/2 chloro benzoyl
chloride.

4522
A. Sethi et al. / Bioorg. Med. Chem. 15 (2007) 4520–4527
Scheme 2. Reagents: (a) Ac2O–pyridine; (b) N,N-DMF–NH2NHAc; (c) Cl3C–CN/K2CO3; (d) BF3:Et2O.
3. Biological activity
2500g) at 4 ꢁC for 10 min and plasma was separated.
Plasma was diluted with normal saline (ratio of 1:3)
and used for analysis of total cholesterol (Tc), triglycer-
ides (Tg) and phospholipids (Pl) by standard enzymatic
methods46–48 using Beckmann auto-analyzer and stan-
dard kits purchased from Beckmann Coulter Interna-
tional, USA.
The present study has been undertaken to evaluate
the antidyslipidemic activity of pregnane derivatives
in triton model. The anti-oxidant activity of these
derivatives was also evaluated by generating free rad-
icals in vitro in the absence and presence of pregnane
derivatives.
Anti-oxidant activity (generation of free radicals). Super-
oxide anions (Oꢀ2) were generated enzymatically49 by
xanthine (160 mM), xanthine oxidase (0.04 U) and
nitroblue tetrazolium (320 lM) in absence or presence
of compounds 3, 4, 5, 6, 7, 8, 11, 20, and 21 (400 lg/ml)
in 100 mM phosphate buffer (pH 8.2). Fractions were
sonicated well in phosphate buffer before use. The reac-
tion mixtures were incubated at 37 ꢁC and after 30 min
the reaction was stopped by adding 0.5 mL glacial
acetic acid. The amount of formazone formed was mea-
sured at 560 nm on a spectrophotometer. Percentage
inhibition was calculated taking absorption coefficient
of formazone as 7.2 · 103 Mꢀ1 cmꢀ1. In another set of
experiment, effects of compounds 3, 4, 5, 6, 7, 8, 11,
20, and 21 on generation of hydroxyl radicals (OHÆ)
was also studied by non-enzymic reactants.50 Briefly
Animals used. Rats (Charles Foster strain, male, adult,
body weight 200–225 g) were kept in a room with con-
trolled temperature (25–26 ꢁC), humidity (60–80%) and
12/12 h light/dark cycle (light on from 8.00 A.M. to
8.00 P.M.) under hygienic conditions. Animals, which
were acclimatized for one week before starting the
experiment, had free access to the normal diet and
water.
Lipid lowering activity. Rats were divided into ten
groups—control, triton induced, triton plus 3, 4, 5, 6,
7, 8, 11 and Gemfibrozil (100 mg/kg) treated groups
containing six rats in each group. In this experiment of
18 h, hyperlipidemia was developed by administration
of triton WR-1339 (Sigma chemical company, St. Louis,
MO, USA) at a dose of 400 mg/kg. b.w. intraperitone-
ally to animals of all the groups except the control. Se-
ven pregnane derivatives were macerated with gum
acacia (0.2% w/v), suspended in water and fed simulta-
neously with triton with a dose of 250 mg/kg po to the
animals of treated group and the diet being withdrawn.
Animals of control and triton group without treatment
with pregnane compounds were given same amount of
gum acacia suspension (vehicle). After 18 h of treatment
the animals were anaesthetized with thiopentone solu-
tion (50 mg/kg b.w.) prepared in normal saline and then
1 mL blood was withdrawn from retro-orbital sinus
using glass capillary in EDTA coated Eppendorf tube
(3.0 mg/ml blood). The blood was centrifuged (at
OHÆ were generated in
a
non-enzymic system
comprised of deoxy ribose (2.8 mM), FeSO4.7H2O
(2 mM), sodium ascorbate (2.0 mM) and H2O2
(2.8 mM) in 50 mM KH2PO4 buffer, pH 7.4 to a final
volume of 2.5 mL. The above reaction mixtures in the
absence or presence of compound 3, 4, 5, 6, 7, 8, 11,
20, and 21 (400 lg/ml) were incubated at 37 ꢁC for
90 min. Reference tubes and reagent blanks were also
run simultaneously.
Malondialdehyde (MDA) content in both experimental
and reference tubes were estimated spectrophotometri-
cally by thiobarbituric acid method as mentioned
above.51

A. Sethi et al. / Bioorg. Med. Chem. 15 (2007) 4520–4527
4523
Statistical evaluation. Data were analyzed using Stu-
dent’s t-test. The hyperlipidemic groups were compared
with control and 3, 4, 5, 6, 7, 8, and 11 treated groups.
Similarly the generation of oxygen free radicals with dif-
ferent fractions of compounds 3, 4, 5, 6, 7, 8, 11, 20, and
21 were compared with that of their formation without
fractions. P < 0.05 was considered to be significant.
tive damage to lipoproteins, which is responsible for
initiation and progression of atherosclerosis in hyper-
lipidemic subjects.52 Hyperlipidemia may also induce
other abnormalities like oxidation of fatty acids, leading
to the formation of ketone bodies as well as masking li-
ver and muscle resistance to insulin which initiates the
progress of diabetes in patients.53 Furthermore, due to
hyperglycemia, increase in non-enzymic glycosylation
occurs, accompanied with glucose oxidation and these
reactions being cataꢁlyzed by Cu2+ and Fe2+, resulting
in formation of O2 and OHÆ radicals which further
accelerates the risk of cardiac diseases in dyslipidemic
patients.54
4. Results and discussion
Effect of pregnane derivatives on hyperlipidemia:
Administration of triton WR-1339 in rats induced
marked hyperlipidemia as evidenced by increase in the
plasma level of Tc (3.67-fold) Tg (3.39-fold) and Pl
(3.97-fold) as compared to control (Table 1). Treatment
of hyperlipidemic rats with pregnane derivatives at the
dose of 400 mg/kg po reversed the plasma levels of lipid
with varying extents. The effect of 3 causing decrease in
plasma levels of Tc, Tg, and Pl by 27, 25, 27 and effect of
7 causing decrease in plasma levels of Tc, Tg, and Pl by
23, 26, and 25 and the effect of compound 4 showed Tc,
Tg, Pl by 21, 14, 17%, respectively, while compound 6
and 8 showed mild lipid lowering activity as compared
to triton which was more significant than other frac-
tions. These data compared with standard drug Gemfi-
brozil at the dose of 100 mg/kg showed decrease in
plasma levels of Tc, Tg, Pl by 37,32,36%, respectively.
The order of lipid lowering activity by these fractions
in above model was 3 > 7(Table 1).
Table 2. Anti-oxidant activity of pregnane derivatives in-vitro
Treatment Concentration Formation of
superoxide
anionsa
Formation of
hydroxyl
radicalsb
3
4
None
400
15.01 1.06
11.10 0.96*
17.91 1.44
14.33 1.22
14.27 2.08
7.83 1.60**
23.20 2.44
21.50 1.77
14.03 1.43
9.33 1.10*
13.60 1.12
12.63 1.24
16.13 1.76
41.62 1.24
27.16 1.54**
40.40 3.66
35.01 2.82
41.52 1.73
36.12 2.92*
44.70 4.77
41.50 3.72
40.19 2.32
31.09 3.12**
35.12 3.62
33.90 3.00
41.52 2.12
None
400
5
None
400
6
None
400
7
None
400
8
None
400
Effect of 3, 4, 5, 6, 7, 8, 11, 20, and 21 on oxygen free
radical generation in vitro: the scavenging potential of
pregnane derivatives at 400 lg/ml against formation of
11
20
21
None
400
15.33 3.00 NS 38.52 3.12 NS
14.25 0.49 42.13 1.39
12.8 1.02 NS 39.02 3.01 NS
15.32 1.32 39.00 2.12
13.22 2.30 NS 35.05 1.12 NS
None
400
O2 and OHÆ in non-enzymic systems were also studied
ꢀ
(Table 2). The significant decrease in superoxide anions
by 3 and 7 and hydroxyl radicals (17%, 40%, 39%, 18%,
8%, and 16%) by fraction 3 and 7 were found at concen-
tration of 400 lg/ml. The 3 and 7 derivatives showed
more anti-oxidant activity in above test system as com-
pared to that of 4, 5, 6, 8, 11, 20, and 21.
None
400
Each value is means SD of four separate observations.
NS, non significant as compared to the systems without drug
treatment.
a nmol formazone formed/minute.
b nmole MDA/h.
* P < 0.05.
The involvement of hydroxyl free radicals (OHÆ) has
been found to be a major causative factor for peroxida-
** P < 0.001.
Table 1. Effect of pregnane derivatives on biochemical parameters in hyperlipidemic rats
Treatment
Total cholesterol (Tc)
Triglyceride (Tg)
Phospholipids (Pl)
Control
Triton treated
86.44 7.21
82.66 6.77
71.49 5.62
318.82 26.71 (+3.6F)***
232.17 15.66 (ꢀ27)***
251.77 18.66 (ꢀ21)**
275.11 23.44 (ꢀ13)*
276.41 24.92 (ꢀ13)*
244.00 16.44 (ꢀ23)**
260.10 16.88 (ꢀ18)*
305.55 27.7 (ꢀ4) NS
200.00 13.22 (ꢀ37)***
280.41 22.22 (+3.39F)***
210.00 14.73 (ꢀ25)***
240.12 21.11 (ꢀ14)*
250.55 18.73 (ꢀ11) NS
235.52 20.22 (ꢀ16)*
208.33 18.23 (ꢀ26)***
244.22 19.92 (ꢀ13)*
266.22 22.00 (ꢀ5) NS
190.01 17.00 (ꢀ32)***
283.87 24.00 (+3.97F)***
206.28 12.93 (ꢀ27)***
236.12 16.66 (ꢀ17)*
260.44 20.82 (ꢀ8) NS
248.12 16.66 (ꢀ13)*
212.24 15.55 (ꢀ25)***
244.44 17.81 (ꢀ14)*
270.35 20.66 (ꢀ5) NS
180.11 12.44 (ꢀ36)***
Triton + 3
Triton + 4
Triton + 5
Triton + 6
Triton + 7
Triton + 8
Triton + 11
Triton + gemfibrozil (100 mg/Kg) standard drug
Unit: mg/dl. Each value is means SD of six rats.
NS, non-significant.
Hyperlipidemic group was compared with control, hyperlipidemic + pregnane derivatives treated with hyperlipidemic.
* P < 0.05.
** P < 0.01.
*** P < 0.001.

4524
A. Sethi et al. / Bioorg. Med. Chem. 15 (2007) 4520–4527
In order to overcome these ailments, a drug having mul-
tifold properties such as anti-oxidant, anti-diabetic and
lipid lowering activities is in great demand. In our pres-
ent study, we have investigated these properties in differ-
ent analogues of pregnanes. Both 3 and 7 caused
significant decrease in the plasma level of lipid in triton
models of hyperlipidemia. Triton WR-1339 acts as sur-
factant, suppresses the action of lipase and blocks the
uptake of lipoproteins from the circulation by extrahe-
patic tissues resulting an increase in the levels of circulat-
ing lipid.55 These test samples inhibited cholesterol
biosynthesis and potentiated the activity of lipolytic en-
zymes to early clearance of lipids from circulation in tri-
ton-induced hyperlipidemia. We have successfully used
this model for evaluation of lipid lowering activity of
pregnane derivatives.
with aqueous Na2CO3, water, dried over anhydrous so-
dium sulphate, filtered and concentrated. The crude
product was purified by column chromatography on
silica gel (hexane/ethyl acetate 99:1) to afford the pure
compounds.
5.2.1. Pregn-5,16-diene-20-one 3b-yl-4-chlorobenzoate
25
D
(3). White solid, yield: 90%; mp 180–182 ꢁC; ½aꢂ ꢀ7.0
(c 0.50, MeOH); IR (KBr) mmax 2921, 1722, 1592,
1
1278, 1096, 770 cmꢀ1; H NMR (CDCl3, 300 MHz) d
(ppm) 8.05–8.02 (m, 2H, Ar–H), 7.39–7.37 (m, 2H,
Ar–H), 6.72 (m, 1H, H-16), 5.43 (m, 1H, H-6), 4.87
(m, 1H, H-3), 2.27 (s, 3H, CH3-21), 1.107 (s, 3H, CH3-
19), 0.93 (s, 3H, CH3-18). MS (m/z) 452 (M+,Cl35), 454
(M+,Cl37). Anal. Calcd for C28H33ClO3: C, 74.24; H,
7.34; Cl, 7.83. Found: C, 74.12; H, 7.28; Cl, 7.78.
To access the anti-oxidant activity of pregnane deriva-
tives, we have used in-vitro model of non-enzymic and
enzymic superoxide and hydroxyl radicals generation.
The properties of 3 and 7 as anti-oxidant and scavenger
of oxygen free radicals appears to be mediated through
activity like metal ion chaelators and xanthine oxidase
inhibitors.
5.2.2. Pregn-5,16-diene-20-one 3b-yl-2-chlorobenzoate
25
D
(4). White solid, yield: 90%; mp 192–195 ꢁC; ½aꢂ
ꢀ12.0 (c 0.25, MeOH); IR (KBr) mmax 2919, 1716,
1536, 1230, 1096, 593 cmꢀ1
;
1H NMR (CDCl3,
300 MHz) d (ppm) 8.04–8.01 (m, 2H, Ar–H,) 7.46–7.37
(m, 2H, Ar–H), 6.71 (m, 1H, H-16), 5.5 (m, 1H, H-6),
4.82 (m, 1H, H-3), 2.27 (s, 3H, CH3-21), 1.10 (s, 3H,
CH3-19), 0.88 (s, 3H, CH3-18). MS (m/z) 452(M+). Anal.
Calcd for C28H33ClO3: C, 74.24; H, 7.34; Cl, 7.83.
Found: C, 74.14; H, 7.30; Cl, 7.76.
5. Experimental
IR spectra were recorded on Beckmann Aculab-10, Per-
kin-Elmer 881 and FTIR 8210 PC, Schimadzu spectro-
photometers using KBr discs. Nuclear magnetic
resonance (NMR) spectra were recorded on either Bru-
ker advance DRX-300 MHz or Bruker DPX 200 FT
spectrometers using TMS as an internal reference.
FAB mass spectra were recorded on JEOL SX 102/DA
6000 whereas ESI was recorded on MICROMASS
QUATTRO II triple quadrupole mass spectrometer.
Optical rotations were recorded on ORIBA, SEPA-300
digital polarimeter. Chemical analysis was carried out
on Carlo-Erba-1108 instrument. The melting points
are recorded on an electrically heated melting point
apparatus and are uncorrected.
5.3. General procedure for the synthesis of compounds 5
550 mg (1.75 mmol) of compound 2 was dissolved in
4.2 mL (67 mmol) of freshly distilled ethylene glycol
and then 0.5 mL of boron trifluoride etherate was added
to it. The reaction mixture was stirred at 30 ꢁC for 55–
60 h (reaction was monitored by t.l.c during this period).
Ice-cold solution of sodium bicarbonate was added to
light brown solution of reaction mixture and the aque-
ous phase was extracted with dichloromethane. The or-
ganic layer was washed with water and then dried over
anhydrous sodium sulphate. The crude product was
purified by column chromatography on silica gel (hex-
ane/ethyl acetate 80:20) to afford the pure compound.
5.1. General procedure for the synthesis of compounds
5.3.1. 3b-Hydorxy-16a-(2-hydroxy ethoxy)pregn-5-en-20-
25
D
one (5). White solid, yield: 80%; mp 170–172 ꢁC; ½aꢂ
5.1.1. 3b-Acetoxy-5,16-pregnadiene-20-one (1). Dios-
genin was converted into compound 1 by reported meth-
od.45 It was identified56 by its mp 166–170 ꢁC, 1H NMR
and MS.
ꢀ26.6 (c 0.75, MeOH); IR (KBr) mmax 3432, 1658,
1
1057 cmꢀ1; H NMR (CDCl3, 300 MHz) d (ppm) 5.34
(1H, m, H-6), 4.53 (1H, m, H-16), 3.68–3.64 (2H, m,
OCH2CH2OH), 3.64–3.38 (3H, m, OCH2CH2OH and
H-3), 2.58 (1H, d, H-17, J = 6.4 Hz), 2.18 (3H, s, CH3-
21), 0.85 (3H, s, CH3-19), 0.63 (3H, s, CH3-18); 13C
NMR (75 MHz, CDCl3) d 208.6 (C-20), 140.9 (C-5),
121.4 (C-6), 80.0 (C-16), 71.84 (C-3), 71.77 (OCH2CH2),
70.77 (OCH2CH2OH), 67.47 (C-17), 54.68 (C-14), 49.98
(C-9), 44.6 (C-13), 42.4 (C-4), 38.9 (C-12), 37.3 (C-1),
36.7 (C-10), 34.15 (C-15), 32.42 (C-8), 31.87 (C-7),
31.72 (C-2), 31.64 (C-21), 20.9 (C-11), 19.6 (C-19),
14.6 (C-18). MS (m/z) 377 (M++1). Anal. Calcd for
C23H36O4: C, 73.37; H, 9.64. Found: C, 73.22; H, 9.56.
5.1.2. 3b-Hydroxy-5,16-pregnadiene-20-one (2). Deacety-
lation of compound 1 by zemplen method57 yielded
compound 2, identified by its mp 212–214 ꢁC (lit mp
216 ꢁC),58 1H NMR and MS.
5.2. General procedure for the synthesis of compounds 3
and 4
To a solution of 2 (0.5 g, 1.59 mmol) in dry pyridine
(2.5 mL) at 0 ꢁC was added 0.5 mL of 4/2-chloro ben-
zoyl chlorides. The reaction mixture was stirred at room
temperature for 2 h. Ice water (10 mL) was added and
the mixture extracted with dichloromethane, washed
5.3.2. 3b-Acetoxy-16a-(2-acetoxy ethoxy)pregn-5-en-20-
one (6). Conventional acetylation of compound 5 with
acetic anhydride and pyridine yielded compound

A. Sethi et al. / Bioorg. Med. Chem. 15 (2007) 4520–4527
4525
25
6.White solid, yield: 90%; mp 154–156 ꢁC; ½aꢂ ꢀ31.66
reported method.59 It was identified by its mp 225–
228 ꢁC, H NMR and MS.
D
1
(c 0.12, CHCl3); IR (KBr) mmax 2946, 1730, 1662, 1248,
1
1037 cmꢀ1; H NMR (CDCl3, 200 MHz) d (ppm) 5.36
(m, 1H, H-6), 5.005 (m, 1H, H-16), 4.64–4.44 (m, 3H,
OCH2CH2OAc and H-3), 3.64–3.57 (m, 2H, OCH2-
CH2OAc), 2.26 (s, 3H, CH3-21), 2.03 (s, 3H, oAc),
1.97 (s, 3H, OAc), 1.05 (s, 3H, CH3-19), 0.88 (s, 3H,
CH3-18). MS (m/z) 460 (M+). Anal. Calcd for
C27H40O6: C, 70.41; H, 8.75. Found: C, 70.34; H, 8.80.
5.5.2. 3b-Hydroxy-5,16-pregnadiene-20-one oxime (10).
Deacetylation of compound 9 by zemplen method57
yielded compound 10.59b White solid, mp 210–214 ꢁC;
1
IR (KBr) mmax 3422, 2927, 1654, 1560, 1111 cmꢀ1; H
NMR (CDCl3, 300 MHz) d (ppm) 6.05 (m, 1H, H-16),
5.36 (m, 1H, H-6), 3.53 (m, 1H, H-3), 1.99 (s, 3H,
CH3-21), 1.04 (s, 3H, CH3-19), 0.94 (s, 3H, CH3-18).
MS (m/z) 330(M++1). Anal. Calcd for C21H31NO2: C,
76.55; H, 9.48; N, 4.25. Found: C, 76.41; H, 9.60; N,
4.09.
5.4. General procedure for the synthesis of compound 7
Compound 5 (435 mg, 1.15 mmol) in dry tetrahydrofu-
ran (7.1 mL) and dry methanol (2.1 mL) was treated
with sodium borohydride (84 mg, 2.21 mmol) portion-
wise during 10 min at 5 ꢁC; reaction mixture was stirred
at the same temperature for 1 h, when t.l.c showed
completition of reaction. Acetic acid was added drop-
wise until the reaction mixture became neutral. Water
was added and the resulting mixture concentrated under
reduced pressure. The aqueous phase was extracted with
dichloromethane. The organic layer was washed with
water and then dried over anhydrous sodium sulphate.
The crude product was purified by column chromatog-
raphy on silica gel (hexane/ethyl acetate 75:25) to afford
the pure compound.
5.6. General procedure for the synthesis of compound 1160
Compound
9 (500 mg, 1.40 mmol), NaH (96 mg,
4.01 mmol) in dry DMF (25 mL) was stirred at
0 ꢁC for 30 min. To the reaction mixture 1,5-dibrom-
opentane (0.5 mL, 2.2 mmol) was added and the reac-
tion mixture further stirred at room temperature for
3 h. DMF was evaporated under reduced pressure
and the solid residue obtained was extracted with
chloroform, washed with water, dried over anhydrous
sodium sulphate and concentrated in vaccuo. Column
chromatography of the resultant residue (hexane/ethyl
acetate, 99:1)afforded compound 11.
5.4.1. 3b,20b-Dihydorxy-16a-(2-hydroxy ethoxy)pregn-5-
25
D
en (7). White solid, yield: 85%; mp 199–201 ꢁC; ½aꢂ
5.6.1. 20-(O-5-Bromopentyl)-oximino-3b-hydorxy-pregn-
5,16-diene (11). Light brownish solid, yield: 60%; mp
ꢀ38.0 (c 0.50, MeOH); IR (KBr) mmax 3308, 2926,
25
1654, 1055 cmꢀ1
;
1H NMR (CDCl3, 300 MHz) d
148–152 ꢁC; ½aꢂ ꢀ4.0 (c 0.5, MeOH); IR (KBr) mmax
D
1
(ppm) 5.36 (m, 1H, H-6), 4.23 (m, 1H, H-16), 3.92 (q,
1H, H-20, J = 6.0 Hz), 3.70–3.65 (m, 2H, OCH2-
CH2OH), 3.61–3.43 (m, 3H,OCH2CH2OH and H-3),
1.29 (d, 3H, CH3-21, J = 6.0 Hz) 1.01 (s, 3H,CH3-19),
0.79 (3H, s, CH3-18); 13C NMR (75 MHz, MeOD) d
142.42 (C-5), 122.34 (C-6), 84.80 (C-16), 72.47 (C-3),
71.81 (OCH2CH2), 69.94 (OCH2CH2OH), 66.29
(C-20), 62.48 (C-17), 54.86(C-14), 51.90 (C-9), 44.10
(C-13), 43.07 (C-4), 40.95 (C-12), 38.54 (C-1), 37.80
(C-10), 33.42 (C-15), 33.09 (C-8), 32.49 (C-7), 30.78
(C-2),23.88 (C-21), 21.80 (C-11), 19.93 (C-19), 14.16
(C-18). MS (m/z) 379 (M++1). Anal. Calcd for
C23H38O4: C, 72.98; H, 10.12. Found: C, 70.92; H,
10.06.
3417, 1594, 1065 cmꢀ1; H NMR(CDCl3, 200 MHz) d
(ppm) 6.0 (m, 1H, H-16), 5.36 (m,1H, H-6); 4.08 (t,
2H, NAOACH2CH2, J = 6.2 Hz), 3.54–3.49 (m,1H,
H-3), 3.41 (t, 2H, CH2CH2Br), 1.98 (s, 3H, CH3-21),
1.69–1.39 (m, 6H, CH2CH2CH2CH2Br), 1.05 (s, 3H,
CH3-19), 0.96 (s, 3H, CH3-18): 13C (75 MHz, CDCl3):
166.1 (C-20), 152.6 (C-17), 145.6 (C-16), 140.8 (C-5),
121.4 (C-6), 72.8 (@NAOCH2), 71.8 (C-3), 56.63 (C-
14), 50.46 (C-9), 44.7 (C-13), 42.29 (C-4), 38.6 (C-12),
37.11 (C-1), 36.8 (C-10), 35.7 (@NAOCH2CH2
CH2CH2), 33.8 (@NAOCH2CH2CH2CH2Br), 32.58
(C-15), 31.8 (C-8), 31.61 (C-7), 30.28 (C-2), 28.7
(@NAOCH2CH2), 24.7 (@NAOCH2CH2CH2), 20.93
(C-11), 19.3 (C-19), 15.88 (C-21), 14.3 (C-18). MS
(m/z) 478 (M++1, Br79), 480 (M++1, Br81). Anal.
Calcd for C26H40BrNO2: C, 65.26; H, 8.43; Br,
16.70; N, 2.93. Found: C, 65.06; H, 8.46; Br, 16.56;
N, 2.91.
5.4.2. 3b,20b-Diacetoxy-16a-(2-acetoxy ethoxy)pregn-5-
en (8). Conventional acetylation of compound 7 with
acetic anhydride and pyridine yielded compound 8.
25
White solid, yield: 90%; mp 144–146 ꢁC; ½aꢂ ꢀ66 (c
D
0.1,CHCl3); IR (KBr) mmax 2932, 1729, 1250, 1044 cmꢀ1
;
5.7. General procedure for the synthesis of compound 2061
and 2162
1H NMR (CDCl3, 200 MHz) d (ppm) 5.37 (m, 1H, H-6),
5.005 (m, 1H, H-20), 4.60 (m, 1H, H-16), 4.18–4.10 (m,
3H, OCH2CH2OAc and H-3), 3.65–3.54 (m, 2H, OCH2-
CH2OAc), 2.33 (s, 3H, OAc); 2.03 (s, 6H, OAc), 1.25 (d,
3H, CH3-21, J = 6.4 Hz), 1.00 (s, 3H, CH3-19), 0.65
(3H, s, CH3-18). MS (m/z) 505 (M++1). Anal. Calcd for
C29H44O7: C, 69.02; H, 08.79. Found: C, 68.95; H, 8.74.
Conventional acetylation of L-rhamnose (2 g,
12.2 mmol) with acetic anhydride (7.2 mL, 70.6 mmol)
and pyridine (25 mL) yielded compound 13 (1,2,3,4
tetra-O-acetyl-L-rhamnose) (2.8 g). Compound 13
(2 g, 6.02 mmol) in N,N-DMF (20 mL) was stirred
with hydrazine acetate (830 mg, 1.5 meq) at 50 ꢁC
for 2 h (reaction mointored by tlc) and then concen-
trated. Ethyl acetate (20 mL) was added and the or-
ganic layer was washed with water, dried and finally
concentrated. Column chromatography of the residue
5.5. General procedure for the synthesis of compound 9
5.5.1. 3b-Acetoxy-5,16-pregnadiene-20-one oxime (9).
Compound 1 was converted into compound 9 by

4526
A. Sethi et al. / Bioorg. Med. Chem. 15 (2007) 4520–4527
gave 14 (1 g, 57.4%) (2,3,4-tri-O-acetyl-L-rhamnose) as
a syrup. To a solution 14 (700 mg, 2.4 mmol) in dry
dichloromethane (25 mL) was added anhydrous potas-
sium carbonate (350 mg) and trichloroacetonitrile
(2.4 mL, 24 mmol). The suspension was stirred for
48 h at room temperature under nitrogen atmosphere.
The mixture was filtered over Celite, washed with
dichloromethane (20 mL) and the filtrate concentrated
under reduced pressure. The residue was purified to
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25
D
chloroacetimidate) as a yellow syrup. ½aꢂ ꢀ50.0 (c
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25
D
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