Synthesis and neuromuscular blocking activity of 16β-piperidinosteroidal derivatives

Article abstract of DOI:10.1016/S0223-5234(00)01205-8

The synthesis and pharmacological profiles of some new steroidal mono- and bisquaternary ammonium derivatives have been described. The compounds featured have been conceptually derived structurally from two lead structures: pancuronium bromide 1 and chandonium iodide 2. In vitro and in vivo neuromuscular blocking studies have indicated the monoquaternary compound 15 to be less active than the bisquaternary compounds 10 and 11. The compound 11 has been found to be more active than d-tubocurarine.

Full text of DOI:10.1016/S0223-5234(00)01205-8

Eur. J. Med. Chem. 36 (2001) 195–202
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(00)01205-8/SCO
Short Communication
Synthesis and neuromuscular blocking activity of 16b-piperidinosteroidal
derivatives
Dharam Paul Jindala*, Poonam Piplania, Hassan Fajrakb, Chris Priorb, Ian G. Marshallb
aUniversity Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160 014, India
bDepartment of Physiology and Pharmacology, Royal College, University of Strathclyde, Glasgow, GI IXW,
Scotland, United Kingdom
Received 8 March 2000; revised 29 November 2000; accepted 29 November 2000
Abstract – The synthesis and pharmacological profiles of some new steroidal mono- and bisquaternary ammonium derivatives have
been described. The compounds featured have been conceptually derived structurally from two lead structures: pancuronium
bromide 1 and chandonium iodide 2. In vitro and in vivo neuromuscular blocking studies have indicated the monoquaternary
compound 15 to be less active than the bisquaternary compounds 10 and 11. The compound 11 has been found to be more active
´
than d-tubocurarine. © 2001 Editions scientifiques et me´dicales Elsevier SAS
monoquaternary / bisquaternary / piperidine / chick biventer / anaesthetized cat / d-tubocurarine / carbachol / nondepolarizing /
steroidal
1. Introduction
ation is that the compounds must have two tertiary
nitrogens
The search for new neuromuscular blocking agents
has been motivated by the long-time course of action
and pronounced side effects of the compounds avail-
able in clinical practice [1]. Since the discovery of
nondepolarizing neuromuscular blocking activity of
pancuronium bromide 1 [2–4], tremendous numbers
of steroidal [5–12] and nonsteroidal [1, 7, 13] neuro-
muscular blocking agents have been synthesized and
their muscle relaxation properties examined. Shortly
after the development of pancuronium 1, azasteroidal
compounds, in which the quaternary nitrogen atom is
situated in the ring D of the steroidal skeleton, were
synthesized and studied [5]. One of the compounds in
this series, chandonium iodide 2, is a powerful nonde-
polarizing neuromuscular blocking agent with short
duration and rapid onset being only slightly less ac-
tive than pancuronium 1 [14]. The general assumption
between these structures and skeletal muscle relax-
at appropriate distances (1.0–1.2 nm) of which one or
both the nitrogens should be quaternized. As the
* Correspondence and reprints.
interonium distance between two quaternary heads
falls below 1.0 nm, the skeletal muscle relaxation
E-mail addresses: dpjindal@USA.net (D.P. Jindal),
ppvohra@panjabuniv.chd.nic.in (P. Piplani).

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D.P. Jindal et al. / European Journal of Medicinal Chemistry 36 (2001) 195–202
property diminishes and the ganglion blocking activ-
ity becomes prominent [15]. Pipecuronium bromide 3
is an example that does not follow this structure
a boiling water bath provided the desired ester 9
(figure 1). Its NMR spectrum exhibited a three-pro-
ton singlet at l 2.13 (-OCOCH3), a single-proton dd
at l 3.03 (16a-H, J=9 Hz). IR spectrum showed a
band at 1725 cm–1 (CꢀO stretching).
The bisquaternary compounds 10 (DPJ-471) and 11
(DPJ-489) were prepared by treating 8 and 9 with
methyl iodide in dichloromethane at room tempera-
ture (figure 2). The N-methyl protons of the bisqua-
ternary compounds 10 and 11 appeared at l 3.0 and
1
3.1 in the H-NMR spectrum, respectively.
Reduction of 5 with sodium borohydride and
acetylation of 12 with acetic anhydride in pyridine
yielded compounds 12 and 13, respectively (figure 3).
The NMR spectrum of 16b-piperidino-5-androstene-
3b, 17b-diol 12 exhibited signals at l 2.76 (dd; 1H,
J=9 Hz, 16a-H); 3.40 (d; 1H, J=9 Hz, 17a-H) and
5.33 (m; 1H, 6-CH). In the ester 13, the NMR signals
appeared at l 2.03 (s; 3H, 3b-OCOCH3); 2.10 (s; 3H,
17b-OCOCH3); 3.03 (dd; 1H, J=9 Hz, 16a-H) and
4.80 (d; 1H, J=9 Hz, 17a-H). Preparation of the
monoquaternary compound 14 was accomplished by
refluxing 5 in absolute ethanol with methyl iodide
whereas compounds 15 and 16 were prepared by
treating 12 and 13 with methyl iodide in
dichloromethane at room temperature (figure 4). The
16a-H of compound 14 appeared at l 4.89 in the
1H-NMR spectrum. The deshielding effect of quater-
nary nitrogen is the reason for downfield shift of
16a-H.
activity parameter [16]. In the present study, our basic
design concept was to combine structural features
taken from pancuronium 1 and chandonium 2 [5],
known to be essential for their nondepolarizing neu-
romuscular blocking activity, and to investigate the
role of interonium distances between two quaternary
heads at position 3 and 16 of the compounds. Fur-
thermore, it was desired to verify the structural re-
quirements in aminosteroids for skeletal muscle
relaxation properties. In this paper, we present the
synthesis and biological evaluation of the bisquater-
nary ammonium compounds 10 (DPJ-471) and 11
(DPJ-489) and monoquaternary compound 15 (DPJ-
464).
2. Chemistry
To prepare mono- and bisquaternary compounds,
16a-bromo-17-oxo-5-androsten-3b-ol 4 was the start-
ing material. It was prepared by a procedure reported
in the literature [17–19]. 16-Piperidino functionality
was introduced to obtain 5 by treating 4 with pipe-
ridine at refluxing temperature [18]. A one-proton
double doublet at l 3.12 in the NMR spectrum indi-
cated the 16a-H. The assignment of the configuration
at position 16 in compound 5 has been made on the
basis of earlier reports [18, 19].
Oppenauer oxidation of 5, using a cyclohexanone-
toluene system, afforded compound 6. In the NMR
spectrum, the vinylic proton appeared at l 5.70 (s;
4-CH). Compound 7 was prepared by refluxing the
4-en-3-one derivative 6 with pyrrolidine in methanol.
The NMR signals appeared at l 4.76 (s; 1H, 4-CH)
and 5.06 (m; 1H, 6-CH). The enamine 7 was subse-
quently reduced with sodium borohydride in
methanol to give the 3b-pyrrolidino derivative 8. The
esterification of 8 with acetic anhydride in pyridine in
3. Neuromuscular blocking activity
The pharmacological activity of compounds 10, 11
and 15 was examined on the isolated chick biventer
cervicis muscle preparation, a preparation selected for
its ability to elicit the potency of nicotinic antagonists
and its important indicative information on mecha-
nism of action. All three compounds reduced the
twitch response to nerve stimulation. There was no
evidence of any of the compounds producing contrac-
ture of the muscle, indicating a lack of depolarizing
activity. Each compound was added to the prepara-
tion in a concentration sufficient to produce a 75–
90% reduction of control twitch tension in ca. 30 min;
the concentrations required to produce these effects
are shown in table I, and compared to those for
tubocurarine as a standard. The effects of all three
test compounds were quickly reversed by washing

D.P. Jindal et al. / European Journal of Medicinal Chemistry 36 (2001) 195–202
197
Figure 1.
In the two bisquaternary compounds in the series,
there was a 13-fold drop in potency with the change
from 17-acetoxy 11 to 17-hydroxy 10 showing the
crucial importance of the acetoxy moiety in combina-
tion with the 16-quaternary nitrogen for binding to
the nicotinic receptor. Removal of the A-ring quater-
nary group from 10 produced only a modest drop in
from the tissue bath. However, if left in the bath and
challanged with the anticholinesterase drug neostig-
mine (0.5 mM), only the effects of compound 11 were
reversed (to 65% control twitch tension) thus indicat-
ing competitive antagonism. This conclusion was
confirmed by the finding that although increasing
concentrations of compound 11 produced a parallel
rightwards shift of carbachol-induced dose–response
curves, yielding a slope value of close to unity in
Arunslakshana and Schild plots (table II). In con-
trast, the effect of compound 10 on twitches was not
reversed at all by neostigmine and that of compound
15 reversed to only about 20% of control twitch
tension. The lack of reversibility of the two weaker
compounds of the three tested suggests a mechanism
other than reversible competitive antagonism of nico-
tinic receptors. These observations for compounds 10
and 15 suggest a form of noncompetitive action,
perhaps at the level of endplate ion channel.
Figure 2.

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D.P. Jindal et al. / European Journal of Medicinal Chemistry 36 (2001) 195–202
potency (1.05–14-fold), again confirming the impor-
tance of the D-ring substituents.
Only compound 11 was considered to have the
appropriate mechanism and potency to warrant fur-
ther testing. This was confined to a single anaesthe-
sized cat experiment, using identical techniques to
those used to evaluate neuromuscular blockers such
as vecuronium and rocuronium whose values are
shown for comparison with compound 11 (table III).
Compound 11 produced a reduction of twitch tension
in the tibialis anterior and soleus muscles induced by
stimulation of the sciatic nerve. No pre-block twitch
augmentation was observed, again indicating a lack
of depolarizing activity. The onset of action was in
the rapid category, being the same as that of the
rapid-onset rocuronium and shorter than that of the
medium-onset vecuronium. Both the recovery and
duration times of the block were long. The compound
had no effect on the contractions of the pre-ganglion-
ically-stimulated nictitating membrane, indicating a
lack of ganglion blocking activity. There was no effect
on general arterial blood pressure, but the compound
produced a moderate reduction of the bradycardial
response of the heart rate to vagal stimulation, indi-
cating a probable block of cardiac muscarinic recep-
tors; such an effect is observed with pancuronium and
is associated with tachycardia in man. The effect of
compound 11 in the cat is shown in figure 5.
Figure 3.
Figure 4.
Table I. The neuromuscular blocking potency (75–90% twitch block in the chick biventer cervicis muscle, mm) of the quaternary
androstene compounds with tubocurarine as control drug.
Compound
Concentration for 75–95% inhibition (mM)
nb
Relative potencya
d-Tubocurarine
15 (DPJ-464)
10 (DPJ-471)
11 (DPJ-489)
1.090.13
2.4590.44
2.3290.16
0.1890.01
6
6
5
4
1
0.4
0.43
5.5
a
Indicates the relative potency to tubocurarine as 1×10–6 M=1
n, number of experiments.
b
Table II. Compared pA2 value of compound 11 (DPJ-489) in isolated chick biventer cervicis nerve muscle preparation with
tubocurarine as control.
Compound
pA2
Slope
KD
nb
(mean9S.E.M.)
(mean9S.E.M.)a
(mean9S.E.M.) mM
d-Tubocurarine
11 (DPJ-489)
6.090.13
7.690.25
1.190.03
1.0490.07
1.190.5
0.02590.02
8
4
a
S.E.M., standard error of mean.
n, number of experiments.
b

D.P. Jindal et al. / European Journal of Medicinal Chemistry 36 (2001) 195–202
199
Table III. Effective dose and course values for neuromuscular effects of the compound 11 (DPJ-489) investigated in an anaesthetized
cat. Values obtained in identical experiments are included for vecuronium and rocuronium [20].
Compound
Dose to produce 80–95% Onset time injection to
Duration: injection to 90% Recovery: 25–75%
twitch block (soleus)
max. block (min)
recovery (min)
twitch-height recovery
(min)
(mg kg–1)
Vecuronium
Rocuronium 311
11 (DPJ-489) 150
38
5.0
2.9
2.9
17.4
13.7
26
5.2
5.2
9.9
Figure 5. Chloralose-anesthetized cat. Effect of DPJ-489 (150 mg kg–1) on blood pressure (B.P.), heart rate (H.R.), response of the
nictitating membrane (Nic. Mem) to preganglionic stimulation and responses of the soleus and tibialis anterior muscles to indirect
stimulation at 0.1 Hz.

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4. Experimental protocols
4.1. Chemistry
15 min and the solution was concentrated to induce
crystallization. The crystalline material was filtered,
washed with methanol and dried to obtain 7. Yield: 1.50
g (87.21%); m.p.: 165–170 °C; UVmax (MeOH): 274 nm
(logꢀ4.36); IR (KBr): 2920, 2825, 1725, 1580, 1390,
Melting points reported are uncorrected. 1H-NMR
spectra were recorded on AC-300F, 300 MHz, Varian
EM-390, 90 MHz and EM-360, 60 MHz NMR instru-
ments using tetramethylsilane (TMS) as the internal
standard (chemical shifts in l, ppm). IR and UV spectra
were recorded on Perkin-Elmer 882 and Lamda 15
spectrophotometer models, respectively. The purity of
the compounds was established by thin layer chro-
matography (TLC) and by elemental analysis (C, H, N).
Elemental analyses were carried out on a Perkin-Elmer-
2400. Mass spectra were recorded on a V6-11-250 J 70S.
Anhydrous sodium sulphate was used as a drying agent.
UV spectra were recorded in methanol (umax in nm). IR
spectra were obtained with potassium bromide pellets
(Vmax in cm–1).
1
1340, 1165, 1025 and 818 cm–1; H-NMR (CDCl3): l
0.93 (s; 3H, 18-CH3); 1.03 (s; 3H, 19-CH3); 2.96 (dd; 1H,
16a-CH); 3.10 (m; 4H, N-methylenes of pyrrolidino
function); 4.76 (s; 1H, 4-CH) and 5.06 (m; 1H, 6-CH).
Anal. for C28H42N2O: C, 79.57; H, 10.02; N, 6.63.
Found: C, 79.41; H, 10.01; N, 6.56.
4.1.3. 16i-Piperidino-3i-pyrrolidino-5-androsten-17i-ol
8
Sodium borohydride (1.0 g, 26.43 mmol) was added
to a stirred suspension of 16b-piperidino-3-pyrrolidino-
3,5-androstadien-17-one
7 (1.0 g, 2.37 mmol) in
methanol (150 mL) in small quantities at room tempera-
ture. Stirring was continued for 2 h, the reaction mixture
was poured into ice-cold water (800 mL) and the
aqueous suspension was extracted with chloroform (4×
100 mL). The combined chloroform extract was washed
with water, dried and solvent removed under reduced
pressure to give a solid residue which was crystallized
from acetone to afford 8. Yield: 0.60 g (59.46%); m.p.:
168–170 °C; IR (KBr): 2930, 2870, 1440, 1340, 1125,
4.1.1. 16i-Piperidino-4-androstene-3,17-dione 6
17-Oxo-16b-piperidino-5-androsten-3b-ol
5 (2.0 g,
5.38 mmol) was dissolved in a mixture of cyclohexanone
(20 mL, 92.97 mmol) and dry toluene (200 mL) and the
solution was subjected to azeotropic distillation to re-
move traces of moisture. The distillation was continued
at a slow rate, during dropwise addition of a solution of
aluminium isopropoxide (2.0 g, 9.79 mmol) in dry tolu-
ene (20 mL). The reaction mixture was refluxed for 4 h
and allowed to stand at room temperature for 12 h. The
slurry was filtered and filtrate was steam-distilled until
the complete removal of organic solvents was effected.
The residual aqueous suspension was allowed to stand
at room temperature and extracted with chloroform
(4×50 mL). The combined chloroform extract was
washed with water, dried and the solvent removed to
obtain a semisolid residue 6 which could not be crystal-
lized and was used as such for further reaction;
UVmax(MeOH): 239.2 nm; IR (KBr): 2910, 1725, 1660,
1
1070 and 885 cm–1; H-NMR (CDCl3): l 0.70 (s; 3H,
18-CH3); 1.03 (s; 3H, 19-CH3); 2.30–2.60 (m; 8H, N-
methylenes of piperidino and pyrrolidino functions);
2.83 (dd; 1H, J=9 Hz, 16a-CH); 3.26 (d; 1H, J=9 Hz,
17a-CH) and 5.23 (m; 1H, 6-CH); [a]2D2: 22.39° (C 0.046
g 100 mL–1, CHCl3); MS: m/z: 427 [M+]. Anal. for
C28H46N2O: C, 78.82; H, 10.87; N, 6.57. Found: C,
78.60; H, 10.83; N, 6.39.
4.1.4. 16i-Piperidino-3i-pyrrolidino-5-androsten-17i-yl
acetate 9
16b-Piperidino-3b-pyrrolidino-5-androsten-17b-ol
8
(0.5 g, 1.17 mmol) was heated in a mixture of pyridine
(0.25 mL, 3.09 mmol) and acetic anhydride (0.5 mL,
5.30 mmol) for 1 h on steam bath. The reaction mixture
was poured into ice-cold water basified with ammonia
and the precipitated product was filtered, washed with
water and dried. The product obtained was crystallized
from acetone to give 9. Yield: 0.30 g (54.64%); m.p.:
215 °C; IR (KBr): 2925, 2745, 1725, 1365, 1232, 1135
1600, 1430, 1220 and 855 cm–1; H-NMR (CDCl3): l
0.90 (s; 3H, 18-CH3); 1.26 (s; 3H, 19-CH3); 2.30–2.90
(m; 4H, N-methylenes of piperidino function); 3.16 (dd;
1H, 16a-CH) and 5.70 (s; 1H, 4-CH).
1
4.1.2.
16i-Piperidino-3-pyrrolidino-3,5-androstadien-17-one 7
Freshly distilled pyrrolidine (9 mL, 107.82 mmol) was
added dropwise to a refluxing solution of 16b-pipe-
ridino-4-androstene-3,17-dione 6 (1.5 g, 4.06 mmol) in
methanol (15 mL). Refluxing was further continued for
1
and 1025 cm–1; H-NMR (CDCl3): l 0.90 (s; 3H, 18-
CH3); 1.03 (s; 3H, 19-CH3); 2.13 (s; 3H, -OCOCH3);
2.16–2.76 (dd; 8H, N-methylenes of piperidino and
pyrrolidino functions); 3.03 (q; 1H, J=9 Hz, 16a-CH);

D.P. Jindal et al. / European Journal of Medicinal Chemistry 36 (2001) 195–202
201
4.83 (d; 1H, J=9 Hz, 17a-CH) and 5.30 (m; 1H,
6-CH). Anal. for C30H48N2O2: C, 76.87; H, 10.32; N,
5.98. Found: C, 76.51, H, 9.92; N, 6.17.
to afford 14. Yield: 0.5 g (72.4%); m.p.: 248–250 °C; IR
(KBr): 3400, 1730, 1460 and 1045 cm–1; 1H-NMR
(CDCl3): l 3.18 (s; 3H, N-CH3 of piperidino moiety);
4.89 (dd; 1H, J=9 Hz, 16a-CH). Anal. for
C25H40NO2I: C, 58.47; H, 7.85, N, 2.73. Found: C,
58.39; H, 7.95, N, 2.35.
4.1.5. 16i-Piperidino-3i-pyrrolidino-5-androsten-17i-ol
dimethiodide 10 (DPJ-471)
Methyl iodide (4 mL, 64.25 mmol) was added to a
solution of 16b-piperidino-3b-pyrrolidino-5-androsten-
17b-ol 8 (0.30 g, 0.703 mmol) in dichloromethane (20
mL) and allowed to stand for 5 days at room tempera-
ture with occasional stirring. The reaction mixture was
concentrated to dryness and crystallized from acetone.
The crystalline material was filtered, washed and dried
to give 10. Yield: 0.33 g (66.13%); m.p.: 300 °C; IR
4.1.9. 16i-Piperidino-5-androstene-3i, 17i-diol
methiodide 15 (DPJ-464)
Quaternization
of
16b-piperidino-5-androstene-
3b,17b-diol 12 (0.5 g, 1.34 mmol) was done in
dichloromethane to afford 15. Yield: 0.45 g (65.03%);
m.p.: 218–220 °C; IR (KBr): 3300, 2910, 1445, 1270,
1
1140 and 1045 cm–1; H-NMR (CDCl3): l 3.09 (s; 3H,
1
(KBr): 3215, 1445, 1145, 1025 and 920 cm–1; H-NMR
N-CH3 of piperidino moiety) and 4.60 (dd; 1H, J=9
Hz, 16a-CH). Anal. for C25H42NO2I: C, 58.24; H, 8.21;
N, 2.72. Found: C, 58.41; H, 8.04; N, 2.37.
(CDCl3-DMSO-d6): l 2.99 (s; 3H, N-CH3 of pyrrolidino
moiety) and 3.11 (s; 3H, N-CH3 of piperidino moiety).
Anal. for C30H52N2OI2: C, 50.71; H, 7.38; N, 3.94.
Found: C, 50.52; H, 7.32; N, 3.82.
4.2. 16i-Piperidino-5-androstene-3i,17i-diol diacetate
methiodide 16 (DPJ-465)
4.1.6. 16i-Piperidino-3i-pyrrolidino-5-androsten-17i-yl
acetate dimethiodide 11 (DPJ-489)
Quaternization
of
16b-piperidino-5-androstene-
3b,17b-diol diacetate 13 (0.8 g, 1.75 mmol) was done in
dichloromethane to afford 16. Yield: 0.63 g (60.1%);
m.p.: 290–292 °C; IR (KBr): 2900, 2840, 1722, 1430,
Quaternization of 16b-piperidino-3b-pyrrolidino-5-an-
drosten-17b-yl acetate
9 (0.30 g, 0.64 mmol) in
dichloromethane was done as above to afford 11. Yield:
0.20 g (41.49%). IR (KBr): 1735, 1440 and 1210 cm–1;
1H-NMR (CDCl3-DMSO-d6): l 2.21 (s; 3H, 17b-
OCOCH3); 3.00 (s; 3H, N-CH3 of pyrrolidino moiety)
and 3.19 (s; 3H, N-CH3 of piperidino moiety). Anal. for
C32H54N2O2I2: C, 51.07; H, 7.23, N, 3.72. Found: C,
51.12; H, 7.23; N, 3.61.
1352, 1230 and 1010 cm–1; H-NMR (CDCl3): l 2.02 (s;
1
3H, 3b-OCOCH3); 2.22 (s; 3H, 17b-OCOCH3); 3.41 (s;
3H, N-CH3 of piperidino moiety) and 4.73 (dd; 1H,
J=9 Hz, 16a-CH). Anal. for C29H46NO4I: C, 58.09; H,
7.33; N, 2.34. Found: C, 58.13; H, 7.83; N, 2.31.
5. Pharmacological methods
4.1.7. 16i-Piperidino-5-androstene-3i,17i-diol
diacetate 13
5.1. Chick biventer cervicis muscle preparation
16b-Piperidino-5-androstene-3b, 17b-diol 12 (1.0 g,
2.68 mmol) was acetylated as in compound 9 to afford
13. Yield: 0.8 g (65.04%); m.p.: 208–210 °C; IR (KBr):
Biventer cervicis nerve-muscle preparations [21] were
dissected from young chickens (3–10 days) and
mounted in a 10 mL tissue bath maintained at 32 °C
and containing Krebs solution of the following composi-
tion (mmol L–1): NaCl, 118; KCl, 5; CaCl2, 2.5;
NaHCO3, 30; KH2PO4, 1; MgSO4, 1; glucose, 11 and
pH 7.4 when aspirated with 5% carbon dioxide in
oxygen. The motor nerves were stimulated at a fre-
quency of 0.1 Hz with rectangular pulses of 0.2 ms
duration and a voltage greater than that required to
produce maximal twitches. Tension responses were
recorded by Grass FT03C force displacement
transducers.
1
2910, 1722, 1435, 1352, 1235 and 1030 cm–1; H-NMR
(CDCl3): l 0.82 (s; 3H, 18-CH3); 1.03 (s; 3H, 19-CH3);
2.03 (s; 3H, 3b-OCOCH3); 2.10 (s; 3H, 17b-OCOCH3);
2.38–2.54 (m; 4H, N-methylenes of piperidino func-
tion); 3.03 (dd; 1H, J=9 Hz, 16a-CH); 4.60 (m; 1H,
3a-CH); 4.80 (d; 1H, J=9 Hz, 17a-CH) and 5.30 (d;
1H, 6-CH). Anal. for C28H43NO4: C, 73.48; H, 9.47; N,
3.06. Found: C, 73.21; H, 9.21; N, 3.13.
4.1.8. 17-Oxo-16i-piperidino-5-androsten-3i-ol
methiodide 14 (DPJ-458)
Quaternization of 17-oxo-16b-piperidino-5-androsten-
3b-ol 5 (0.5 g, 1.34 mmol) was done in absolute ethanol
In some experiments dose–response curves were con-
structed by adding increasing concentrations of the ago-

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D.P. Jindal et al. / European Journal of Medicinal Chemistry 36 (2001) 195–202
nist carbachol until maximal responses were obtained.
Carbacol dose–response curves were constructed in the
absence and the presence of three concentrations of the
test compounds. Affinity constants (KD values) were
calculated from plots of log (dose ratio-1) against molar
concentration of antagonist [22]. Slope values from the
plots were used to assess competitive antagonism or
otherwise.
Research, New Delhi, and Panjab University for provid-
ing the research facilities. The authors express apprecia-
tion to Panacea Biotech Limited, New Delhi for
financial support for this investigation, and Cipla, Bom-
bay for the supply of DHA acetate.
References
[1] P. Taylor, in: J.G. Hardman, L.E. Limbird, P.B. Molinoff,
R.W. Ruddon, A.G. Gilman (Eds.), Goodman and Gilman’s
The Pharmacological Basis of Therapeutics, 9th ed, McGraw-
Hill, New York, 1996 pp. 177–197.
5.2. Anaesthetized cat
A cat was anaesthetized with a mixture of a-chlo-
ralose 80 mg/kg–1 and pentobarbitone 5 mg/kg–1 in-
jected i.p. Lungs were ventilated with air at a rate of 26
b.p.m. at a tidal volume of approximately 13 mL kg–1,
adjusted to maintain arterial pH at 7.37–7.47.
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The right hind limb was immobilized and the contrac-
tile responses of the tibialis anterior and soleus muscles
to single shock stimulation of the sciatic nerve were
recorded. The sciatic nerve is stimulated at a rate of 0.1
Hz using rectangular pulses of 0.2 ms duration and of
length greater than that required to produce maximal
twitch. This stimulation frequency was chosen to allow
comparison of the results with test compounds with
those for compounds reported previously in the litera-
ture. Contractions of the nictitating membrane were
evoked in response to preganglionic stimulation of the
cervical sympathetic nerve with trains (frequency 5 Hz,
duration 10 s) of strength sufficient to produce maximal
concentrations of nictitating membrane.
Arterial pressure was recorded from the carotid artery
using a Statham PC45 pressure transducer. The arterial
pressure pulse triggered a cardiachograph to display a
heart rate. Both vagus nerves were ligated and, at 100 s
intervals, the right vagus nerve was stimulated at a
frequency of 2–5 Hz and with pulses of 0.5 ms duration
and strength greater than that required to produce a
maximal reduction in heart rate. Contractile responses
of muscle were recorded using Grass FTO3Cand FT10
displacement transducers. All responses were displayed
on a Grass model 5 ink oscillograph.
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Steroid Biochemistry and Pharmacology, Academic Press, Lon-
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Acknowledgements
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We are pleased to acknowledge the financial support
of the project by the Council of Scientific and Industrial
[22] Arunlakshana O., Schild H.O., Br. J. Pharmacol. Chemother.
14 (1959) 48–58.
.