1-[2-Hydroxy-3-octadecan-1'-oate]propyl-2'',2'',5'',5''-tetramethyl Pyrolidine-N-oxyl-3''-carboxylate as a potential spin probe for membrane structure studies

Article abstract of DOI:10.1016/S0968-0896(99)00216-3

The synthesis of a new minimum steric perturbing proxyl nitroxide, which is a derivative of glycerol and contains a stearic acid moiety, has been carried out. Its localization in model membrane L-α-dipalmitoyl phosphatidyl choline (DPPC) was ascertained with the help of ESR, DSC, ¹H and ³¹P NMR techniques. The nitroxide was used for detecting the changes in the phase transition temperature of the model membranes in the presence and absence of drugs. The permeation of the vasodilating drug epinephrine has also been studied using this spin label. The results prove the potential applicability of the new spin probe in the spin labeling of biomembranes. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00216-3

Bioorganic & Medicinal Chemistry 7 (1999) 2753±2758
1-[2-Hydroxy-3-octadecan-10-oate]propyl-200,200,500,500-tetramethyl
Pyrolidine-N-oxyl-300-carboxylate as a Potential Spin Probe for
Membrane Structure Studies
Rita Katoch, a Girish K. Trivedi a,* and Ratna S. Phadke b
aDepartment of Chemistry, Indian Institute of Technology, Mumbai, 400076, India
bChemical Physics Group, Tata Institute of Fundamental Research, Mumbai, 400001, India
Received 27 April 1999; accepted 7 July 1999
AbstractÐThe synthesis of a new minimum steric perturbing proxyl nitroxide, which is a derivative of glycerol and contains a
stearic acid moiety, has been carried out. Its localization in model membrane l-a-dipalmitoyl phosphatidyl choline (DPPC) was
ascertained with the help of ESR, DSC, 1H and 31P NMR techniques. The nitroxide was used for detecting the changes in the phase
transition temperature of the model membranes in the presence and absence of drugs. The permeation of the vasodilating drug
epinephrine has also been studied using this spin label. The results prove the potential applicability of the new spin probe in the spin
labeling of biomembranes. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
of the membranes, is highly suitable for incorporation
into membranes. The nitroxide synthesized has been
employed as an ESR sensitive probe to monitor the
changes in ¯uidity and permeability of model mem-
branes made up of l-a-dipalmitoyl phosphatidyl cho-
line (DPPC) as such and in the presence of various
drugs.
Nitroxide labeled lipids and phospholipids have been
used extensively in ESR studies of the structure and
function of biological membranes.1±5 Despite the pro-
ven usefulness of the technique, key studies are some-
times precluded by the unavailability of site speci®c
labels having suitable spectral, physical or chemical
properties.6 Also, there is a constant concern that the
spin labeled biological system does not accurately re¯ect
the behavior of its naturally occurring analogue owing
to the steric bulk of the nitroxide moiety.7 Molecules
such as proteins, sugars, steroids and lipids are embed-
ded in membrane bilayers at random and can serve well
as parent molecules for nitroxide probes. These labels
can be embodied in the organized system without caus-
ing perturbations.
Results and Discussion
For the synthesis of the lipid nitroxide, mannitol (1) was
®rst protected and cleaved to be converted into 1,2-iso-
propylidene glycerol (3) which showed an M 15 peak
at m/z=117 in its mass spectrum and a hydroxy peak at
1
1
3450 cm in its IR spectrum. The H NMR spectrum
of this compound showed peaks which were comparable
with those reported in the literature.12 Isopropylidine
glycerol (3) hence obtained was condensed with stearic
acid. Product 4 obtained was connoted by the appear-
Our group has been involved in the synthesis of various
steroid based nitroxide spin labels.8±10 We, herein,
describe the synthesis and applications of a new non-
steroidal minimum steric perturbing proxyl nitroxide
lipid spin label. This nitroxide is a derivative of glycerol
and contains a stearyl moiety which, being a constituent
1
ance of an ester peak at 1738 cm in the IR spectrum
and con®rmed by the presence of multiplets at 4.31 (2-
1
H) and 4.05±4.20 ppm (1H2 and 3-H) in the H NMR
spectrum. The multiplets at 1.62 ppm were assigned to
the two 170 protons, those at 1.25 ppm to the 28 protons
(30-H2±160-H2) of the stearyl group and the triplet at
0.88 ppm (J=6.9 Hz) to the terminal methyl protons of
the same. The 13C NMR spectrum of compound 4 also
showed the presence of an ester carbonyl peak at 172 ppm
con®rming it to be isopropylidene glyceryl stearate.
Key words: 1-[2-Hydroxy-3-octadecan-10-oate]propyl-200,200,500,50-tet-
ramethyl pyrolidine-N-oxyl-300-carboxylate; proxyl stearate; spin label;
model membrane; phase transition.
* Corresponding author. Tel.: +91-22-576-7173; fax: +91-22-576-
7152; e-mail: chgktia@ether.chem.iitb.ernet.in
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00216-3

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R. Katoch et al. / Bioorg. Med. Chem. 7 (1999) 2753±2758
Scheme 1. Synthesis of 1-[2-hydroxy-3-octadecan-10-oate]propyl-200,200,500,500-tetramethyl pyrolidine-N-oxyl-300-carboxylate (6). Conditions: (i) ace-
tone, ZnCl2, K2CO3; (ii) NaIO4, NaBH4; (iii) stearic acid, DMAP, DCC; THF; (iv) Dowex 50WÂ8; (v) 3-carboxy proxyl, DCC, DMAP, THF, 16 h.
1
Product 4, hence formed, was then deprotected to yield
the corresponding diol 5 which showed a broad peak at
3320 cm in its IR spectrum. The elemental analysis
hydroxy peak at 3457 cm in its IR spectrum. The
mass spectrum of this compound showed M+ peak at
m/z 526 (C30H56NO6).
1
and M+ peak at m/z 358 established a molecular for-
mula of C21H42O4 for compound 5. The 1H NMR
spectrum of compound 5 was de®cient of the isopropyl
methyl singlets observed in the case of its parent com-
pound 4 at 1.44 and 1.37 ppm. The C-2 and C-3 signals
were shifted up®eld and appeared at 65.1 and 63.2 ppm,
respectively, in the 13C NMR spectrum of diol 5, further
establishing its formation.
1
The H NMR of compound 6 obtained after reduction
with phenylhydrazine showed four singlets at 1.35, 1.27,
1.21 and 1.06 ppm for the gemdimethyls of proxyl moi-
ety, con®rming the product to be 1-[2-hydroxy-3-octa-
decan-10-oate]propyl-200,200,500,500-tetramethyl pyrolidine-
N-oxyl-300-carboxylate. The ESR spectrum of the proxyl
compound
g0=2.0055 and A0=14.62 G.
6 showed a symmetrical triplet with
The primary hydroxy group in compound 5 was then
condensed selectively with 3-carboxy proxyl in presence
of 4-dimethyl amino pyridine (DMAP) and dicyclohexyl
carbodiimide (DCC) to obtain spin label 6. The forma-
tion of the nitroxide 6 was indicated by the presence of
The incorporation of the nitroxide 6 in liposomes was
ascertained by comparing the ESR spectra of the nitr-
oxide in rapidly tumbling solution state (CHCl3) with
that in DPPC liposomes (Fig. 1). The hyper®ne coup-
ling constant of SL in multilamellar vesicles was 14.50 G
1
two ester peaks at 1728 and 1650 cm along with a

R. Katoch et al. / Bioorg. Med. Chem. 7 (1999) 2753±2758
2755
Figure 1. ESR spectra of the nitroxide 6 in (a) solution state (CHCl3)
and (b) DPPC liposomes.
Figure 2. Plot of temperature (ꢀC) vs spectral parameter h‡1=ho of the
spin label 6 incorporated into pure DPPC liposomes (*) and in the
presence of diltiazem (~).
while that in rapidly tumbling solution phase was found
to be 14.62 G. The di€erences observed in the line
shapes of the highest and lowest ®eld lines in the ESR
spectrum of the SL in liposomes is due to the anisotropy
of the nuclear hyper®ne coupling tensor and g tensor
of the radical in the lipid.13 These results suggested that
the spin label 6 was completely incorporated into the
liposomes.
by other techniques. In the presence of diltiazem, the
sigmoidal curve indicated a transition temperature of
36ꢀC. This showed that the drug was bound to the
hydrophobic region in DPPC liposomes.17,18 Hence, it
can be concluded that the proxyl glyceryl stearate has
the ability to report the phase transition of pure lipids
and the lipids incorporated with external agents.
The DSC of DPPC incorporated with compound 6
showed a phase transition temperature of 40ꢀC, sug-
gesting that the incorporation did not cause any sig-
ni®cant perturbation to the system. The localization of
spin label 6 in DPPC liposomes was also evident from
The permeation of epinephrine in model DPPC mem-
branes was studied using ESR spin labeling technique.
The adrenergic neurotransmitter epinephrine, known to
exhibit cardiac and respiratory stimulations by com-
plexing with appropriate receptors that are usually
located in the plasma membranes, is also capable of
quenching paramagnetism of a spin label.19,20 This
made it possible to study the dynamics of the permea-
tion process in DPPC model membrane by monitoring
the ESR signal heights of the spin label 6 incorporated
in the system. The spin label dissolved entirely into the
lipid phase of the water/lipid system and when incorpo-
rated into unilamellar vesicles the label was dispersed
both in the outer and inner monolayers of the lipid
bilayer. When epinephrine was introduced into the
DPPC system, it di€used into the bilayer and quenched
the paramagnetism of the spin label molecules present
in the outer monolayer at a faster rate as compared to
those residing in the inner region. As a result, the ESR
signal height decreased with time and the plot of signal
height with time showed an exponential decay (Fig. 3).
The half-life times for the reduction of the spin label in
1
the H and 31P NMR spectral studies. It was observed
1
that, on incorporation of the spin label, the H NMR
resonances of di€erent regions underwent line broad-
ening to di€erent extent.
The NMe3 peak showed more broadening due to the
possible proximity of the proxyl group as well as the
hydrogen bonding of the hydroxy group of the SL with
the polar head group of the lipid, while the broadening
of the terminal Me signal was markedly less (Table 1).
The increase in the 31P line widths also indicated the
same.14±16
In order to explore the potential of nitroxide 6 as a
`reporter group', phase transition temperature of DPPC
dispersions incorporated with compound 6 as an ESR
sensitive probe was determined. Alterations in the phase
transition temperature induced by a vasodilating drug,
diltiazem, were also studied (Fig. 2). The transition
temperature obtained for pure DPPC incorporated with
spin label 6 was in agreement with the values reported
Table 1. Comparative line widths of the 1H and 31P resonances of the DPPC liposomes as such and in presence of spin label 6
Half line widths (Hz)
N+Me3
Terminal Me
31P
WL
WLS
Pure DPPC
6+DPPC
3.36505
4.12795
WLS/WL
1.2715
1.3206
1.3846
WLS/WL
1.0484
1.9362
2.4542
WLS/WL
1.2675

2756
R. Katoch et al. / Bioorg. Med. Chem. 7 (1999) 2753±2758
MS Engine 5989-A spectrometer was used to record the
mass spectra. 1H NMR spectra were recorded on a
Varian VXR 300S spectrometer using about 5 mg of the
sample dissolved in 0.6 mL of the solvent. The 1H NMR
spectra of nitroxides were recorded after in situ reduc-
tion of their CDCl3 solutions with 1.5 equivalents of
1
freshly distilled phenyl hydrazine (PhNHNH2). The H
NMR and 31P NMR for determination of the localiza-
tion of the spin label in DPPC (2:10 mmol) was taken in
0.6 mL of CDCl3. The DSC experiments were carried
out on a France Setaram instrument and the volume of
the sample taken was 0.85 mL. ESR spectra were recor-
ded at ambient temperature or at 50ꢀC on Varian E-112
spectrometer operating in the X-band with tetra-
cyanoethylene as internal standard (go=2.00277).
Deoxygenated solvents were used for ESR measure-
ments. The concentrations of the nitroxides were main-
tained ca. 10 5 M. Multilamellar dispersions of DPPC
were prepared using Hill's method11 in the following
way.
Figure 3. Plot of time (s) vs the signal height S(t) of the ESR spectrum
of the spin label 7 incorporated into DPPC liposomes in the presence
of epinephrine.
Chloroform solutions of the required spin label and the
lipid were taken in a small tube. The solvent was eva-
porated slowly with a stream of nitrogen gas to get a
thin ®lm on the walls of the tube. Traces of solvent were
removed by drying under vacuum for three hours. The
dried ®lm was then hydrated with the required amount
of 10 mM phosphate bu€er (pH=7.2) for 20 min and
then vortexed at 50ꢀC for 10 min to get multilamellar
vesicles. The concentration of the lipid in the bu€er
solution was 80 mM and that of the spin label was
0.8 mM. The samples were taken in 50 mL glass capil-
laries sealed at both ends and mounted in the variable
temperature accessory of the spectrometer.
the outer and inner re¯ect the rates of permeation of the
drug for the outer and inner monolayer. These half
times were calculated to be 28.88 s and 69.33 s, respec-
tively, according to the equation14
kt
kt
Sꢀt† ˆ S0ꢀ0†e0 ‡ Siꢀ0†ei
where sꢁt† is the ESR signal height due to the total spin
label present at time t and S0ꢁ0† and Siꢁ0† are signal
heights due to initial spin label concentration in the
outer and inner monolayers, respectively. These results
showed that the di€usion process enables the drug to
move through the membrane structure by lateral di€u-
sion at a moderate rate to reach its receptor sites.21,22
To prepare unilamellar vesicles, a lipid dispersion of
DPPC in 10 mM phosphate bu€er with spin label 6 was
prepared according to the above mentioned method.
After a vortex of 10 min at 50ꢀC, the dispersion was
subjected to soni®cation using soni®er, 1210 BRAN-
SON (Model 1210E-DTH, working frequency 47
KHz‹6%, HF-output power nom. 35 W) at 40ꢀC.
Soni®cation for 30 min produced clear homogeneous
solution, which was used for the ESR experiments.
Conclusively, 1-[2-hydroxy-3-octadecan-1(-oate]propyl-
200,200,500,500-tetramethyl
pyrolidine-N-oxyl-300-carbox-
ylate (6) was synthesized by selective esteri®cation of the
primary hydroxy group of glyceryl stearate. To ascer-
tain its utility as a spin probe, this new proxyl spin label
was incorporated in DPPC liposomes. The ESR studies
indicated that this spin label can be successfully used for
observing phase transition in pure DPPC and in the
presence of drugs, as well as in determining the permea-
tion of drugs through membranes, and, hence, shows a
strong potential for probing biomembrane properties.
For phase transition experiments using diltiazem, the
multilamellar dispersion was hydrated as usual using an
appropriate amount of 10 mM phosphate bu€er (pH
7.2) containing the drug. The system was allowed to
equilibrate for 20 min before vortexing. The molar
ratios of the drug:lipid:spin label were maintained at
40:100:1.
Experimental
For permeation studies with epinephrine, at time t=0,
to 50 mL of the sonicated preparation, the required
amount of drug solution in bu€er was added. Subse-
quently, the decay of the ESR signal with time was
monitored. The samples were taken in glass capillaries
sealed at both ends and mounted in the variable tem-
perature accessory of the spectrometer.
Melting points are reported uncorrected. All solvents
were predried according to standard procedures. Petro-
leum ether refers to the fraction having bp 60±80ꢀC. 3-
Carboxy proxyl, DPPC and epinephrine were purchased
from Sigma-Aldrich Chemical Company. Diltiazem was
received as a gift from Iswtituto di Science Fisiche,
Unversity di Ancona, Italy. Elemental analysis was car-
ried out on CEST 1106, IR spectra on a Nicolet Impact
400 FT IR spectrophotometer, while a Hewlett±Packard
In all the permeation experiments, the concentration of
the lipid in the bu€er solution was 100 mM and that of

R. Katoch et al. / Bioorg. Med. Chem. 7 (1999) 2753±2758
2757
the spin label was 1 mM. The molar ratios of the drug:
lipid:spin label were maintained at 20:100:1. The
quencher solutions were prepared in 10 mM phosphate
bu€er (pH 7.2). The temperature was kept at 50ꢀC to
ensure that the lipid remained in the liquid crystalline
phase. The microwave power of the instrument was set
at a low value of 0.5 mW with a microwave frequency of
9.1 GHz. The modulation amplitude was set at
2.0 mWÂ1G and the time constant of the detector unit
at 0.128 s. The scan time for each spectrum was 4 min.
The receiver gain at the start of the experiment was kept
at a fairly large value and was not altered throughout
the experiment to give a strong signal whose decay
could be monitored with time. Only the ®rst line in the
ESR spectrum and its decay with time were recorded.
1H, 1-H), 3.68 (dd, J=4.0, 11.6, 1H, 3-H), 3.58 (dd,
J=3.6, 11.6, 1H, 3-H), 1.44 (s, 3H, >C(CH3)), 1.36 (s,
3H, >C(CH3)).
2,3-Isopropylidene glyceryl stearate (4). Stearic acid
(0.75 g, 2.65 mmol) was dissolved in dry THF (20 mL)
to which DCC (0.54 g, 2.65 mmol), DMAP (0.32 g,
2.65 mmol) and isopropylidine glycerol (3; 0.42 g,
3.19 mmol) were added. The reaction mixture was stir-
red for 24 h, the insoluble urea ®ltered o€ and the sol-
vent removed in vacuo. The resulting oil on puri®cation
over silica gel column yielded product 4 (1.6 g) as a
white solid having low melting point in 70% yield.
[a]d= 22.222 (0.045% in CHCl3). MS: m/z 398 (M+,
22%), 297 (54), 265 (12). IR (KBr): n 2924, 2850, 1738,
1374, 1385 cm 1. Elemental analysis: calculated for
C24H46O4: C, 72.31; H, 11.63%. Found: C, 72.23; H,
11.39%.
1,2:5,6-Di-O-isopropylidene-D-mannitol (2). Acetone
(500 mL) was saturated with anhydrous zinc chloride
(150 g, 1.1 mol) and the milky solution so formed was
allowed to settle down. The saturated solution was
transferred in two portions under N2 pressure to a
round bottom ¯ask containing d-mannitol (1, 72 g,
395 mmol). The ¯ask was ®tted with a mechanical stirrer
with a calcium chloride drying tube and its contents
stirred at room temperature till the solution became
clear (2 h). The mixture was poured in saturated K2CO3
solution (400 mL), stirred for half an hour and ®ltered.
The residue and ®ltrate were extracted with CHCl3
(300 mLÂ3), dried over anhydrous Na2SO4, ®ltered and
evaporated under vacuum to a crude solid (93.2 g). The
solid thus obtained was again dissolved in hot hexane:
dichloromethane (9:1) and kept overnight for crystal-
lization. The crystals formed were ®ltered o€, washed
with cold hexane and dried to a constant weight in
vacuo over P2O5. Yield: 88 g (85%). Mp: 120ꢀC (lit.
120±121ꢀC). MS: m/z 247 (M+ 15). IR (KBr): n 3450±
1H NMR (CDCl3, 300 MHz): d 4.31 (m, 1H, 2-H), 4.05±
4.2 (m, 3H, 1-H2, 3-H), 3.74 (dd, J=6.3, 8.4, 1H, 3-H),
2.34 (t, J=7.5, 2H, 20-H2), 1.62 (m, 2H, 170-H2), 1.44,
1.37 (3, gemdimethyl), 1.25 (m, 28H, 30-H2-160-H2), 0.88
(t, J=6.9, 3H, 180-H3). 13C NMR (CDCl3, 50 MHz): d
172 (C-10), 108 (C-20), 72 (C-1), 64.7 (C-2), 62 (C-3), 12
(C-180).
Glyceryl stearate (5). Compound 4 (0.1 g, 0.25 mmol)
was dissolved in methanol (60 mL) and Dowex 50WÂ8
(1±2% w/w) was added to it. The reaction mixture was
stirred for 4 h, ®ltered and the solvent was evaporated
under reduced pressure. The crude product on puri®ca-
tion over silica gel column yielded the required diol 5
(0.05 g) in 56% yield. Mp: 70±72ꢀC. [a]d= 36.363
(0.055% in CHCl3). MS: m/z 358 (M+, 6%), 340 (M 18,
15%), 322 (12%), 265 IR (KBr): n 3320, 2924, 2850,
1738, 1374, 1385 cm 1. Elemental analysis: calculated
for C21H42O4: C, 70.35; H, 11.81%. Found: C, 70.29; H,
11.77%.
1
3300, 1395, 1385 cm 1. H NMR (CDCl3, 300 MHz): d
4.23±4.17 (m, 2H, 2-H, 5-H), 4.01 (dd, J=8.2, 6.6 Hz,
2H, 3-H, 6-H), 3.76 (dd, J=8.2, 6.6 Hz, 2H, 1-H, 6-H),
3.67 (dd, J=11.7, 3.9 Hz, 1-H, 3H/4H), 3.56 (dd,
J=11.5, 5.3 Hz, 1H, 3H/4H), 2.5 (bs, 2H, 3-OH and 4-
OH), 1.41 (s, 6H, >C(CH3)2), 1.35 (s, 6H, >C(CH3)2).
1H NMR (CDCl3, 300 MHz): d 4.19 (m, 2H, 1-H2,),
3.94 (m, 1H, 2-H), 3.65 (m, 2H, 3-H2), 2.35 (t, J=7.5,
2H, 20-H2), 1.63 (m, 2H, 170-H2), 1.25 (m, 28H, 30-H2±
160-H2), 0.88 (t, J=6.7, 3H, 180-H3). 13C NMR (CDCl3,
75 MHz): d 172 (C-10), 70.2 (C-1), 65.1 (C-2), 63.2 (C-3),
13.9 (C-180).
1,2-Isopropylidine glycerol (3). To an ice-cold solution
of NaIO4 (15 g, 70 mmol) in 250 mL distilled water,
1,2:5,6-O-isopropylidene-d-mannitol (2; 15 g, 57 mmol)
was added in four portions with constant stirring till the
solution became clear. Ethanol (500 mL) was added and
stirred for half an hour at 0ꢀC. The reaction mixture
thus obtained was ®ltered and the residue was washed
with precooled ethanol. To the cold ®ltrate, NaBH4 (5 g,
131.6 mmol) was added at 0ꢀC in portions over a period
of half an hour and left stirring for 2 h at room tem-
perature. The reaction mixture was then neutralized
with NH4Cl to pH 7 and concentrated under reduced
pressure to a ®nal volume of 250 mL. The turbid solu-
tion was saturated with NaCl and evaporated to dryness
in vacuo at room temperature. It was distilled to 6.6 g
(88%) of a colorless oil at 70ꢀC/8±9 mm of Hg. MS:
m/z: 117 (M 15, 20%), 114 (M 18, 24), 101 (20). IR
1-[2-Hydroxy-3-octadecan-10-oate]propyl-
200,200,500,500- tetramethyl pyrolidine-N-oxyl-300-carboxy-
late (6). The diol 5 (0.1 g, 0.28 mmol) obtained was dis-
solved in dry THF (10 mL) and to this solution was
added 3-carboxy proxyl (0.05 g, 0.28 mmol), DCC
(0.06 g, 0.28 mmol) and DMAP (0.034 g, 0.28 mmol).
The reaction mixture was stirred for 30 h followed by
®ltration of the insoluble urea formed and the removal
of the solvent under reduced pressure. The crude residue
was column chromatographed to get the spin label 6
(0.75 g) in 53.5% yield. Mp=204ꢀC. [a]d= 28.282
(0.055% in CHCl3). MS: m/z 526 (M+, 4%), 496
(M 30, 12), 391 (25). IR (KBr): n 2929, 2855, 1728,
1650, 1456, 1373 cm 1. Elemental analysis: calculated
for C30H56O6N: C, 68.40; H, 10.72; N, 2.66%. Found:
C, 68.29; H, 10.57; N, 2.56%.
1
(neat): n 3450, 1390, 1380, 1050, 840 cm 1. H NMR
(CDCl3, 300 MHz): d 4.27±4.19 (m, 1H, 2-H), 4.03 (ddd,
J=1.0, 4.1, 7.8, 1H, 1-H), 3.78 (ddd, J=1.0, 3.8, 7.8,

2758
R. Katoch et al. / Bioorg. Med. Chem. 7 (1999) 2753±2758
1H NMR (CDCl3 with 1.5 equivalents of PhNHNH2): d
4.16 (m, 2H,1-H2,), 3.74 (m, 1H, 2-H), 3.51 (m, 2H, 3-
H2), 2.35 (m, 2H, 20-H2), 1.26 (bs, 28H, 30-H2±160-H2),
0.88 (t, J=6.9, 3H, 180-H3), 1.35 (s, 3H, gemdimethyl),
1.27 (s, 3H, gemdimethyl), 1.21 (s, 3H, gemdimethyl),
1.06 (s, 3H, gemdimethyl). ESR spectrum (10 M in
CHCl3): symmetrical triplet with g0=2.0055 and
A0=14.62 G.
Feig, E., Ed.; Ablex Publishing Company; Norwood, New
Jersey, 1989; p 49.
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8. Katoch, R.; Korde, S. S.; Deodhar, K. D.; Trivedi, G. K.
Tetrahedron 1999, 55, 1741.
5
9. Banerjee, S.; Desai, U. R.; Trivedi, G. K. Tetrahedron 1992,
48, 133.
10. Banerjee, S.; Trivedi, G. K.; Srivastava, S.; Phadke, R. S.
Steroids 1994, 59, 377.
Acknowledgements
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We thank C.S.I.R., New Delhi and B.R.N.S., Trombay
for the ®nancial assistance. The spectral facilities pro-
vided by R.S.I.C., I.I.T., Mumbai and National Facility
for High Field NMR, T.I.F.R., Mumbai are gratefully
acknowledged. We also wish to thank Prof. Nand
Kishore, I.I.T., Mumbai for providing the DSC facility.
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