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New steroidal nitrosoureas
129
2846
NEW Claude
STEROIDAL
Chantal
Chavis+,
Jean-Louis
++
Borgna
NITROSOUREAS
and
de
Gourcy+,
Jean-Louis
+
Imbach
.
+
Laboratoire de Chimie Bio-Organique and Equipe de Recherche Associee no195 du CNRS, Universitg des Sciences et Techniques du Languedoc, Place E.Bataillon, 34090, Mvgtpellier, France. Unite d'Endocrinologie Cellulaire et Moleculaire, INSERM 34100 Montpellier, France. (U I4S), 60 rue de Navacelles, Received T-27-81 ABSTRACT 174 and 20 nitrosourea derivatives belonging to the dehydroepiandrosterone, estrone and pregnenolone series have been synthesized. The behavior of these compounds towards the calf uterine estradiol and pregnenolone receptors have been studied. INTRODUCTION During lating such
the
past
agents new
have
combination
between
(an
can
pointed
takes
can the
out
that
the
receptor
ted
function
steroid
xyl
group.
two
types
the
Thus, of
aim
steroidal
remain of
representative
function 20
3-OH
in position
(pregnenolone)
Volume
39,
Number
S
an
steroids.
The
target
drugs
and
a the
tissue
studies
on
have
to bind
a carbonyl has
with
(10,Il)
C-3 or
been
belonging
(ni-
association
Previous
such
fruitful moiety
an unblocked
work
estrone
178
could
2
this
as
nitrosoureas
transdehydroepiandrosterone, as
of
hor-
a lipophilic
nitrosocarbamates
least
for
against
estrogen)
such
alky-
search
a specific
receptor.
at
to
and or
By
effectivness
has
The
drugs
alkylating
androgen
to
steroidal
a possible
(7,8,9))
receptor.
and
of
cytotoxic
delivered
requires
which
(l-6).
advantage
active
be
of
anticancer
nitrosoureas
nitrosoureas
specific
synthesized
towards
agent
a number
potential
hormonally
deactivate
steroidal
years
a latent
or
selectivity
alkylating or
as
tumors
mustards
carrier high
been
compounds
mone-dependent
trogen
thirty
to
an
hydro-
synthesize
to each
pregnenolone
introduction
their
oxygena-
of
of
series the
(dehydroepiandrosterone,estrone) be
effected
T~ROXD=
from
the
the
urea or
corresponding
February,
1982
amino
or
tainable hoped
steroids
a-aminocyano from
the corresponding
part ot the molecule
might
be readily
functionality.
in an unusual
way
alcohol tives
prepared
and
compounds
:
Rl=R2=H
b
R+ =Thp;
c
R,z
prepared
which
R2xCN ; R2=CN
was
of the
17 amino
deriva-
data whereas
to a mixture
leads
are easily
re-
of (ZOR)
separated.
17 and 20 aminonitriles
lb, 2b, 3b, Ic -synthesis (16). This
amino-acid
in ammoniacal
cyanide, never
ting keto material
la, 2a, 3a, 4a were obtained oximino steroids with Na/
the literature
group
by a Strecker out
3N”2 R2
The stereochemistry
to be B from
carried
of potassium
H
steroids
of the 20 oximino
(20s) isomers
reaction,
a
of the corresponding
(12-15).
I
RR2 *R2
On the other hand, were
or
AND DISCUSSION
the following
17 and 20 amino
is known
duction
the
of the alkylating
CH
by reduction
It was
~1 to the nitrosourea
properties
RR2 R2
The
ob-
moiety.
RESULTS Ne have
group
influence
and pharmacological
carbamoytating
should
ketone
that the use of a cyano
chemical
which
appeared
always
methanol complete
detected
by
in the presence as some
t.1.c.
star-
of the reac-
S
131
TBICOSOI
NO 1
NH2
NH, C_N,CH2CH2Ci 6
y3
p H-~_N-CH2CH2Ct II
3a
tion mixture. well
However,
as a-cyanohydrins
carbonyl
compound
separation
been
that their
successful
_3b and _Ic were
2,
bamoylating
that a-aminonitriles equilibratewith
in such a way
has never
pounds 2,
it is known can readily
group
using
reacts
in a specific
to give excellent Thus compounds
car-
yields
in
p-nitrophenyl-N-
(18). This easily way on amino
prepared
functional
of the corresponding
groups
nitrosoureas.
9,
duced
compounds
yields
(Scheme
over
com-
nitrosocarbamoylated
the new reagent
Z-chloroethyl-N-nitrosocarbamate
2, -3a and -4a and the above reagent pro1, 6, 1, and 8 in very good to quantitative
1). These
previous
cultly-prepared
results
reports
on reaction
viz. &,
; therefore,
(17)
used as such for the next
may be directly
a one step procedure
ment
chromatographic
step.
The amino
reagent
as
the parent
with
where
are a significant
improve-
the same starting
material
N-nitrosocarbamoylazide,
and hazardous
reagent,
yielded
a diffi-
only 43% of 2.
However
-Ic does not react with p- nitrophenyl-N-2-chloroethyl-N-nitrosocarbamate probably owing to the weakened reactivity tain
of the amino
the desired
a-cyanonitrosourea namely
dure was employed, sation ning
group c1 to the nitrile.
of the urea.
a free
reaction
3-hydroxyl
(more than
3 days)
and incomplete
amounts
were
isocyanate
was used,
used,
group
nitrile
formation
pyrany;
was
titatively
chosen
takes
contai-
place
approximately a threefold from which
was
only
nitrosourea
deemed
in basic
The Strecker
stoiexcess
of
the
could be isolated.
be acid-labile
for this purpose
introduced.
by nitro-
the compound
of the steroid
sequence
should
18
an easier
at the 3-OH position
of the reaction
a protecting
resulted
compound
the aim of effecting
when
and when
a mixture
3R,l?B-diearbamoylated
gining
proce-
i.e. -Ic, showed that the isocyanate in DMF was very slow
chiometric
protection
two-step
followed
trials with
to ob-
function,
of N-2-chloroethyl
With
the usual
carbamoylation
Initial
In order
from
synthesis the be-
preferable.
since media
and tetrahydro-
as it is quickly procedure,
Such
the cc-amino-
when
and quanapplied
#!B WSlEOXDI
n t i v)
133
to the 3-0-tetrahydropyranylated aminonitriles next
lb, 3,
carbamoylation
and 46% yields
steroids
gave
the crude o-
-3b which were used directly for the step to produce 2, -10 and -II in 45, 40
respectively
purification.
Nitrosation
of the latter
three ureas,
after
column
by nitrosyl
chromatographic
chloride
in pyridine
ve yield
afforded
-12 and -14 in quantitatiRemoval of tetrahydropyranyl
was
Amberlite
and -13 in 72% yield. easily accomplished using
a dichloromethane-methanol titatively
STEREOCHEMISTRY
thought
to be c1 since
proceed
via an enamino
attacked
and gave -15 and -17 quan(Scheme 2).
OI' THE CYAN0 for the cyano
the Strecker
skeleton.
18-CH3'HNMR
chemical
from
group
which
at C-17
deduced
was partly
is
is known
(19) to
in turn will be
the less hindered
This hypothesis shifts
COMPOUNDS
reaction
intermediate
by CN- presumably
the steroid
in
mixture
and -16 in 85% yield
The stereochemistry
IR 120 (H+form)
~1 side of
confirmed
from
from Zurcher's
increments: Compound
0.73 0.77
5-Androsten-38-01 5-Androsten-3@,17B-diol 3H-(tetrahydropyran-2'yloxy)-17g-hydroxy-5androsten-17a-carbonitrile
3H-(tetrahydropyran-2'yloxy)-178-amino-5-androsten-17a-carbonitrile -lb The substituent
effect
Al7ol-CN= 0.93 Using
of the
of the
- 0.77=
the aminonitrile
is no deviation fect
reference
18-CH3 (dppm)
Al7B-NH22
If this Al7B-NH2
- 0.73
this work
is :
+ 0.16 ppm.
from the additivity
0.83
0.83
f7o-CN group
derivative
17fi-amino group
0.93
(20) (20) experimental value obtained by synthesis following (21)
should
be
- 0.16=
substituent
-lb and assuming there rule the substituent ef: - 0.06 ppm.
effect
is correct
chemical shift of 17fJ-amino+-androsten-36-01 CH3 equal to 0.73 - 0.06= 0.67 ppm. which is exactly
the
18-
should what was
be
S found
for
mental cyano
(see
(+ 0.13
value
deduced
found
these
experimental
literature group
sults
of
-la
in
study
offer
stereochemistry
series
only
one
for
is more
isomer
compound
when
at C20
conversion
shows
that
a downfield with
an
group
in
section) and (20o
results. (obtained 11, a
shift
upfield the
0.96
(ZOR)
reaction
20s) On
only
of
of
and
and the
from ppm.
2
mechanism
in
this
(208 the
reaction represent which
leading
to
pairs, or
ZOR)
18-CH3
in
of
the
preg-
there
is
the
18-CH3 for
is
compared
the
same
methyl
(experimental the
(ZOR)
derivatives
-8 fit
chemical
an upfield
there
concerning
is
to
data
(2OS)-NH2
viz,
product)
is
Literature
and
crude
stereochemical
shifts
study
value
steroids.
since
for
chemical
re-
that
is available
ppm.
the
suggest
for
of
ppm.
- 0.05
The
hand
stereochemistry
tool
17a
differentiation
spectra
isomer
nitrosourea
crude
NMR
to amide.
- 0.06
or -4a
other
These
one
0.02
found
and for
ZO-aminonitrile
the
a
theoretical
disubstituted
a conversion
0.05
were
(22)
experi-
of
available,
to ascertain
amine
(ZOR)-isomer.
which
the
no
effect
the
method
diagnostic
such
shift
was
whith
C,7
(from
of
for
(209)-amino or
of
Although
substituent
Apsimon
such
present
effect (23)
by
difficult
assignment the
the
agree
A possible
-3b).
section).
a convenient
stereochemistry
nane
135
experiments
calculated
effects
The
for
from
this
ppm.)
TBItOIDI
1.03 shift
can
be
understood
the
a-aminonitrile
-3a
the
above
shift
for
ppm.
and
and
argue
from from
-3b for for
the preg-
nenolone. EXPERIMENTAL Material
and
SECTION
methods
: Melting points are obtained in capillaries Chemical on a Buchi 510 melting range apparatus and are uncorrected. LJV spectra (imax in nm) were obtained on an Optica Model 10 spectrophotometer. Proton magnetic resonance spectra were recorded on Varian 90 or HA-100 instruments in CDCl all values are recorded in ppm. (6) with respect to TMS ai internal reference; band shape is indicated by s(singlet) ,d(doublet), t(triplet), m(multiplet). Thin layer chromatography was performed using Merck Kieselgel 60F 254 as 0.2 mm layers on aluminium foil. Merck silica (0.063 - 0.2 mm) was used for co-
lumn chromatography (solvent ratios are expressed as v/v). Optical rotations were taken on a Roussel-Jouan instrument with concentrations expressed in gram/100 ml of solution. IR spectra (v in cm-l) were recorded on a Perkin Elmer 450 instrumen??xMass spectra were obtained with a Jeol J.M.S. DlOO instrument. Elemental analysis were performed by the Service Central d'Analyse du C.N.R.S. Biochemical
:
Non radioactive R was kindly provided by Roussel-Uclaf 5P2?. (France). Stock so u ions (5 to 50 mM) of unlabelled compounds were made in ethanol or dimethylformamide and they were then diluted with ethanol ; to avoid degradation of the compounds, these solutions were kept at O'C and used during the following 24 h. Preparation of soluble extracts : uteri (stored at80°C) from immature calf and lamb were washed then homogenized in TE buffer (Tris-HCI IO mM pH : 7.4, EDTA 1.5 mM) The homogenates were centrifuged at with an ultraturrax. 105,000 g for 60 min. The supernatants defined as cytosols were adjusted to 2 mg protein/ml and used for binding studies. Determination of relative binding affinity for R (estrogen receptor) and R (progesterone receptor) : Eytoincubated for 4Por 24 h at 0-2°C with 3.5 to 7.5 and increasing concentrations of the teste
TE buffer) at 0°C. Tritiated steroid remaining in 2Q0 i.liof DCC treated cytosols were counted in 3 ml ethanol and 10 ml : 0.3 X) and POPOP of a toluene, PPO (2,5-diphenyloxazole -benzene : 0.01 X) mixture (1,4-bis Z-(5-phenyloxazolyl) using a SL30 intertechnique scintillation counter with a 20-25 % efficiency. Chemical synthesis : The N-2-chloroethyl-N-nitrosocarbamoylp-nitrophenylester was synthesized according to the were preliterature method (18). 178-or 20-aminosteroids pared following the known literature procedure by sodiumalcohol reduction of oximes. The separation of ,the (20 R)and (20 S)-amino-pregnane derivatives was done by chromatography on a silicagel column impregnated with ammonia. 17@-Amino-5-androsten-3H--01 (la) : mP 158-1590c (from ethyl acetate), lit. (12) : 160 V C ; TH NMR- (~~~l~~~,~H~;67 3H, 18-H), 1.03 (s, 3H, 19-H), 2.65 (t, (s,
S J = 8Hz),
3.47
137
'PBEOXDI
(m, IH, 3a-H),
5.33
(m, lH, 6-H).
(Za) : mp 17fi-Amino-l, 3, 5(10)-estratrien-3-01 222-224°C (from methanol) lit (13) : 222-226°C ; ‘H NMR (CDCI ) : 0.67 (s, 3H, 18-H), 2.60 (t, IH, 17a-H, J = 8Hz), 6.50 &H, 4-H), 6.56 (IH, 2-H), 7.07 (d, IH, l-H, J = 9nz). (2OS)-Amino-5-pregnen-3B-o1 (3a) : mp 172-174°C (from methanol), lit. (14) : 172-174°C ; IH NMR (CDC13) : 0.65 (s, 3H, 18-H), 0.98 (s, 3H, 19-H), 1.09 (d, 3H, 21-H), J= 6Hz), 2.77 (m, IH, 17a-H), 3.49 (m, lH, 3a-H), 5.33 (m, lH, 6-H). (2OR)-Amino-5-pregnen-3B-ol (4a) : mp 215-217'C (from methanol) lit. (15): 218-219°C ; 1H NMR (CDCI ) : 0.76 (s, 3H, 18-H), 1.00 (d, 3H, 21-H, J = 6Hz), ,.a3 (s, 3H, 19-H), 2.78 (m, IH, 17a-H), 3.51 (m, IN, 3a-H), 5.33 (m, lH, 6-H). Synthesis of 17@-and 20-N-aitrosoureas : In a typical exneriment N-2-chloroethyl-N-nltrosocarbamoyl-D-nitro-phenylester (0.828 mmole) was added to a solution of aminosteroid (0.715 mmole) in pyridine (5ml) at - IO'C. After vigorsus stirring was maintained for 3 h, the reaction mixture evaporated under vacuum then coevaporated three times with toluene. The oily residue was dissolved in ethanol (5ml) and allowed to crystaliize and then recrystallized.
17@-(N-2Tchloroethyl-N '-nitrosoureyl)-5-androsten-3R01. (5 ) : Following the above general procedure, the readtlon of la (0,2lg, 0.715 mmole) with the reagent (O,226g, 0.828 mmae) gave 5 after crystallization from ethanol
Mass spectrum : m/e/I, 316/27 (M-CICH2CH2N = NOH)?, 299/31 M-(H20 + ClCH2CH -N-NO+) 298/100 (M-Cl-Cfi -CH -N = NOH)t-H 0, 283184 (2$8-CH3)t Axial. talc. for 6 H gTp$4N~O$;7; C,g2.32 ; H,S.O8; N,9.91 ; found C,62.11 ; , ; ,a * 17@-(N-2-chloroethyl-N' -qit*osoureyl)-1,3,5 (lO)-estratrien-3-01 (6) : obtained from 2a in 92 % yield 88-9ooc (MeOH) ; Rf : 0.45 (CHC~~/E~~H, 9.5/0.5) + 12 (C 0.5, CHCl ) ; IR (CHCI ), 3590, 3420 294:&k! 1740 (C=O), 1610 &,, 1485 (N-30), 1340, ,296, 1160, lO;JO, 970, 900 ; UV (EtOH, 95) 220 nm (16,300), 277 nm (5,000), ,H NMR (CDC13) : 0.76 (6, 3H, IS-Y), 3.47 (t, 2H, CH -N-NO, J = 6,5 Hz), 3.96 (III,IH, 17a-H), 4.16 (t, 2H, CH2 -cb,
138
5.65
(m, OH), 6.53-7.10 (m, 3H, I-H, 2-H, 4-H). Anal. H28N303Cl ; C,62.13 ; H,6.95 ; N,10.35 ; found ; H,6.84 ; N,10.20.
talc.
20S-(N-2-chloroethyl-N '-nitrosoureyl)-5-pregnen-38-01 : The same above procedure gave compound 7 ( (7) mp 104-106*C(Et 0) ; Rf : 0.37 (CHCI /EtOH, 9,5/0.5) - 103 (C 0.5 CH& >. IR (CHC13), 3603, 3420, 1720, 1485, 1380,31350, 1160, 1090, 960, 900 (CDCI ) : 0.78 (s, 3H, 18-H), 1.03 (s, 3H, 19-H), 1.33 (d, J = 6.5~2), 3H, 21-H, J = 6Hz), 3.50 (t, 2~, CH~-N-NO, 4.18 (t, 2H, CH2-Cl), 3.40-4.20 (2m, 3a-H and l7o-H), 5.33 (m, 1H, 6-H), 6.77 (d, IH, NH, J = 9Hz), 6.90 (d, IH, J = : m/e/I, 345117 (M-C1CH2NNO), 344164 9Hz). Mass spectrum (M-CICH CH -N=NOH)+ = A? ; 329/25 (A-CH3)? ; 326/100 (M0 = B? ; 311/70 (B - CH )t Anal. talc. CICH -CA -~=NOH)+-H for z H2 NOCI; 6 ,63.76 ; H,8.47 ; N,9.23 ; found C,63.65 ,8.4024, 4:9?5& 20R-(N-2-chloroethyl-N '-nitrosoureyl)-5-pregnen-38-01 (8) in 93 % vield and crvstallized from methanol . , : Obtained * Fl40-142iC (cyclohexane) ; Rf : 0.37 (CH C1 /EtOH, 9.51 ";;;o;[;~~o-;;=~; 0;;&5C~;1a)0. IR (CHC13),23660, 3420, 2940 +amlde II), 1380, 1350, 1160, 1.00 1090, 960, 900 ; 'H NMR (CDCI ) : 0.77 (s, 3H, 18-H), 1.23 (d, 3H, 21-H, J = 6Hz), 3.52 (t, 2H, (s, 3H, 19-H), J = 6.5Hz), 4.20 (t, 2H, CH2C1), 3.30-4.30 (2m, CH2-N-NO, 3a-H and 17a-H), 5.35 (m, IH, 6-H), 6.73 (d, 1H, NH, J = 345/10 (M-C~CH cH -NNO)+, : m/e/I, + 9Hz). Mass spectrum 344/50+(M-CICH CH N = NOH)+, 326/100 (M-C1;H$&~H;. - H 0), 311/882(3$6-CH >+ Anal. talc. for C ; found C,63.99 ; , .4 ; N,9.44. C,6 5 .76 ; H,8.47 ; N,9?29 2
3B-(N-2-chloroethyl-carbamoyl)-l7~-(N-2-chloroethyl-N' -urevl)-5-androsten-17o-carbonitrile (18) : Potassium cyanichloride (log, 0.19 mole) de (log, _- 0.154 mole) and ammonium of 38-hydroxy-5-androsten-l7-one were added to a suspension in methanol (150 ml) saturated with ammo(1Og, 3.47 mmole) nia. The mixture was stirred for 24 h at room temperature whenadditional KCN (5g) and NH4Cl (5g) were added to the mixture which was then stirred an additional 4 days. The into cold water (11) containing reaction mixture was poured was washed with water acetic acid (20 ml) ; the precipitate and dried under vacuum over P 0 . To a solution of the abo(200 ml) ve solid material in anhydrous 2 aimethylformamide N-2-chloroethylisocyanate (II ml, at O'C was added dropwise After three days at room temperature the solu0.128 mole). tion was concentrated to half its initial volume and poured The precipitate was filtered, washed with into ice water. layer (500 ml) ; the organic water and dissolved in CHCI dried (d a2S0 ) and concentrated under was then separated, vacuum to give a oily residue (l$g) which was preabsorbed on silica and deposited on a silicagel column (6OOg, petro-
S
139
TDEOXDI
with a mixture of petroleum leum ether/ether l/l). Elution ether/ether 0.519.5 gave a solid product which was recrys; Rf : tallized from methanol (5.lg 28 X) ; mp 148-15O'C -55.8 (Cl, MeOH). IR(CHC13), 0.41 (Et O/MeOH, 0.8/0.2) i [p 3450, 32$0, 2950-2840, 2240 (Cl), 1700 (C=O), 1500-1550 (amide II), 1250-1200, 1140, 1080, 960. ]H NMR (CDCI ) : 0.77 (s, 3H, 18-H), 0.98 (s, 3H, 19-H), 3.30 and 3.53 (m, 4H, CH -CR Cl), 4.33 (m, 3a-H), 5.33 (m, IH, 6-H), 6.20 (t, IHf NH! J = 4.5 Hz), 6.65 (s, lH, NH), 7.25 (t, lH, NH,
; found
C,59.35;
: prepared according to Tetrahydropyranyl derivatives the literature procedure (24) in 95 % yield using p-toluene-sulfonic acid and dihydropyran in CH2C12. 3R-(tetrahydropyran-2'-yloxy)-5-androsten-l7-one 192-193°C (acetone), lit (25) 192-194'C.
one
: mp
3-(tetrahydropyran-2 '-yloxy)-1,3,5(10)-estratrien-17mp:147-149'C (ethylacetate), lit (26) 147-148°C.
3g-(tetrahydropyran-2 129-130°C (isopropylether),
'-yloxy)-5-pregnen-20-one lit (26) 129-13l'C.
: mp
3B-(tetrahydropyran-2'-yloxy)-l7~-(N-2-chloroethyl-N1ureyl)-5-androsten-17a-carbonitrile (9) : in a Typical experiment potassium cyanide (15.3 mmole, 0.995g), ammonium chloride (9.41 mmole, 0.498g) and saturated methanolic ammonia (10 ml) were added at O'C to a methanolic solution of 38-(tetrahydropyran-2 '-yloxy)-5-androsten-l7-one (1.27g, 3.41 mmole). The slurry was stirred at room temperature for then KCN (0.5g) and NH4C1 (0.252g) were added and 2 days, the mixture stirred for another 3 days. The KC1 was filtered off, washed with dry methanol and the filtrate concentrated under vacuum and dissolved in CHCl (50 ml) .I$-2-chloroethylisocyanate (0.307g, 3.5 mmole) was a a ded to the solution which was kept at room temperature for 17h. Then after removal of the solvent under vacuum, the oily residue was chromatographed (CH2~2/EtOH, 9.9/O.]) yn a silicagel column (60H) under low pressure (2.51b/in ) to give 9 (0.77g, 45% yield); 171-173'C (MeOH) ; Rf : 0.33 (CH Cl-/.EtOH, IR (CHCl ), 3386 $940, 2240 9.9/0.1) ; b]"SO -80 (Cl, CHC13). : 0.85 1500, 1135, 1025. 1H N& (CDCl;) (CN), 1690 (CgO), 1.00 (s, 3H, 19-H), 3.56 (m, 6H, ClCH-CH N-, (s, 3H, 18-H), 4.68 (m, IH, Thp), 5.33 (m, IH, 6-H), 0.60 ?m, Thp, 3a-H), : m/e/I, 383141 (M-RNHCONH)., 270/100 (M-RNCO-HCNAnal. talc. for C H N 0 Cl ; C, ;0,9.50 ; C1,7.0$8;4$02nJ C,66.50 ; H,8.33 ; N,8.34 ; 0,9.80 ; C1,7.10.
3-(tetrahydropyran-2 '-yloxy)-s73-(N-2-chIoxoethyl-N'ureylj-1,3,5(JO)-estratrien-J7a-carbonitrile (IO) : The above procedure was applied to 3-(tetrahydropyran-2'9yIoxy)r 1,3,5(10)-estratrien-17-one and JO -2as obtained as an oil
6.80 (dxd, 2-H), 7.45 (d, I-H, J=9Hz). Mass spectrum 364146 (M-RNHC0NH ) *,3/7/38 (N-RHco:~H~-HCN), 251/ 100 CH Cl? NCO-HCN--OH)'. Anal. ; N,8.64 ; found 66772'; H,7.46
: m/e/I (ccl-
3$-(tetrahydropyran-2'-yloxy)-20~-(~~-2-ch~o~oethy~-N~ureyl)-5-pregnen-20a-carbonitrile (!l) : Obtained from 3f3(tetrahydropyran-2'-yloxy)-S-pregnen-20-one in 4Oj: yield by
3B-(tetrahydropyran-2'-yloxy)-!7?-(~-2-chloroethyl-N_.'nitrosoureyl)-5-androsten-17a-carbonitrile (12) : Typically nltrosyl chloride was bubbled through a solution of 9 (ID~uo~&) maintained at O°C in anhydrous pyridine (15 nl) ; the reaction -.as complete within 15 mn. Cold ?ater (50 ml> was added until. precipitation occured ; the precipitate was filtared, washed with water and dissolved in dichloroaethane (150 ml.). The organic layer was separated,dried (Na2SOk), evaporated then coevaporated three times with toluene. ihe nitrosourea (12) was obtained as crystals (EtOH-Et20) in quEBtit2tiVe yield ; mp llg-120°C ; Rf : 0.55 (CHCX /EtOH, 9.810.2) ;[a]:" -47 (Cl, CHCl ). IR (CHCl > 29'-8, 2860, 2340 (CN), 3420 il35, 1020. H NXR fCDC13) 1740 (&==O), t290 (N-NO) 15;0-1456, 0.90 (s, 3H, 18-H), 1.06 (s, 38, 19-H), 3.50 (m, 2H, 3a-H,
found
C,62.99
3-(t~at~ahydr_o~yran-2'-~l~xy~-Ii~-(N-2-~h~~~~athy~~N1~ nittosourey~)-l,3,~(lO)-estratri~n-l7~-carhonit~~le (13) : compound 10 (2.3 mmole) gave I3 as 2211 0x1 1~ 72% weld after ; petroleum rapid colzn chromatog ether/ether 2930 ,
;
S
TICIICOID,I
141
uv (EtOH 95) 221 nm (11,250), 276 nm (2,200). IH NMR (CDCl ) 0.93 (8, 3H, 18-H), 3.50 (m, IH, Thp), 3.50 (t, 2H, CH -NNa ~=7Hs), 3.88 (m, IH, Thp), 4.16 (t, 2H, ClCH -, J=7Hz)'6.79 7.22 (s, (d, 4-H), 6.84 (dxd, Z-H), 7.16 (d, I-H, J=9&), Mass spectrum : m/e/I, 323/23 (M-C1CH2CH2N=NOHAnal talc. for C ; H;6*85 ; N,l0.88 ; found C,62.80 3~-(tetrahydropyran-2'-yloxy)-2O~-(N-2-chloroethyl-N1nitrosoureyl)-5-pregnen-2Oo-carbonitrile (14) : Compound
1135, 1025. (s, 3H, 21-H),
3.51
(m, 2H,,3a-H,
Thp),
3.90
-11
(m, IH,
. 17$-(N-2-chloroethyl-N' -nitrosoureyl)-3$-hydroxy-5androsten-17a-carbonitrile (15) : The tetrahydropyranylated derivative 12 (I mmole) was dissolved in a mixture+of di~hlorometha~-methanol (314) ; Amberlite IR 120 (H form) was added and after stirring 7h at room temperature, the resin was filtered and washed with dichloromethane. The filtrate was then stripped of solvent and the residue crystal-
1550-1540, 1090. H NMR (CDC13) : 0.90 (s, 3H, 3.52 (m, IH, 3a-H), 3.52 (t, 2H, CH2NNO, J=6.5Hz), 4.18 (t,2H, C1CH2-, J=6.5Hz), 5.36 (m, IH, 6-H), 7.15 (s,+lH, NH). Mass spectrum : m/efT, 341150 (Ma, 3231100 (M-cZCH,CH,N=NOH-OH).. Anal. talc. ; C,61.53 ; H,7,41 ; N,l2.48 ; found C,
17~-(N-2-chloroethyl-N '-nitrosoureyl)-3-hydroxy-1,3,5 (IO)-estratrien-17a-carbonitrile (16) : Obtained in 85% as an oil purrfled a florrsll column (2Og) eluted with petroleum ether/ether, 515 ; mp 135-137°C (ether) ; Rf : 0.40 (CH Cl /EtOH, 9.8/0.2) ;[aj2'+25.6 (Cl, CHCl >. IR (CHC13), 3606, 3420, 2930, 1740, 1618, 1490. UV GtOH 35) : 221 nm H NMR (CDCl ) : 0.93 (s, 3H (9,85(J), 277 nm (2,800) ; 18-H), 3.55 (t, 2H, CH2NN0, Jr7Hz), 4.20 (t, 2H, C1CH~I:=7Hz) 6.60 (d, 4-H), 6.65 (dxd, 2-H), 7.10 (d, I-H, J=9Hz), 7.65 (s, NH), 8.34 (s, OH). Mass spectrum : m/e/I, 323172 (M-ClCH CH2N=NOH) . Anal. talc. for C 6.51 ; N,l3,00 ; found C,61.28 ; ZOH-(N-2-chloroethyl-N '-nitrosoureyl)-3$-hydroxy-5pregnen-2Oa-carbonitrile (17) : Obtained In 95% after crys-
142
H NMR : 1.00 (s, ;IH, 1.93 (s, 3H, 21-H), (CDC13) 3.51 (m, IH, 3o-H), 3.51 (t, .J=7Hz), 4.16 (t, 2H, ClCH2-, J=7Hz), 5.33 (m, IH, 2H,CH2NN0, 6-H), 7.11 !s: IH, NH). Mass spectrum : rpW.3~;;;; ~~~CfCH~CH~N~N~~) 351/100 (M-ClCH CH2N=NOH-H20) ; H,7?553i 4 3 C,62.94 ; H,7.8$ ; N,ll.74 ; found C,62.88 N,11.55.
for
BIOLOGICAL
RESULTS
Compounds
-16
and
RE
compounds
while
efficient.
According
decreased bation ly
but
reached.
the
o-2Oc,
This
of
lateral
side
chain
results
were
(Fig.2)
all
bly of
do R
the
interact
cording
to
the
at
inhibition in Table
constant the
1.
ratio
affinity
KA= of
Only
with low
of
for
constant
kinetic
of
-6 incu-
with
uterus.
1
2 were
R
P The
not ineffi-
inhibition
constants after
that which
steroid
of 3 HR5020 proba-
binding
site
calculated 24 hrs
necessary
specific
the
-16 was
suggesting
the
the
affinity.
and the
by
at
Similar
For
a low
(27)
that,
site
competitors
rate,
occupancy
-6 occurs.
Again, time
KORENMAN
or[3H]R5020
the
binding
17,
concentrations
compounds
24 hrs
-6 (compound
affinity
affinity for 12LM-1 0.34 10 the
again
covalently
of [3H]E2
apparent
at
method
lamb
used.
apparent
0-2'C
the
derivatives
decreased
of -16 and
that
displayed
was
concentration
-8 were
suggesting
-16 and
with
tested
pregnane
relative
petition
high
obtained
to displace,
of
compounds
compound
binding
not
[I
alkylation of
also
while
P' The
no
compounds
efficient
after
-16 or 5 indicated site was reversible,
or
cient at 3 H R5020 [I was able
stabilized
3 H E2 II poorly
with
0-2“C, equilibrium was practical3 H E2 specific binding in the of
compounds
E 2 binding
tested)
at
increase
little
most
was
efficently
-17 or 5, 1 and the efficiency
time,
that,
DISCUSSION
-6 competed
-15 and to
markedly
suggesting
presence of
(Fig.1)
AND
binding
to
of
accom-
reach are
50%
listed
a relatively -16 and -6 displayed RE. On the basis of an affinity of E 2 for
constants
(neglecting
the
RE,
K+/Kpart
calculated (28),
of
the
from apparent
non-specifically
S
143
7FDEOSDI
;:-; \
2
Q a-v---V
A
l
0
l
0
X
0
X
500
50
5
Competitor
concentration
_ Figure.
Fig. I, : Relative binding of estradlol and compounds g,
1
5000
l
5oooo
( nH I
I_
affinity for the estrogen receptor -16 and -17. Calf uterine cytosol was incubated at 2; for 4 hrs (open symbols) H E2 and increasing conand 24 hrs (close symbols) with 3.5 nM : X ; 15 :AA; 16 :00; or 17 :VI centrations of unlabelled E 3H r binding was measul added in ethanol (final perzentage 72). red after 2 hrs charcoal adsorption at O'C. Ezch value, corrected for non specific binding, is expressed as a percentage of non(74000 cpm/ml of cytosol). inhibited 3H E2 specific binding
s
144
7.5
0
75 Competitor
Tacos=*
750
75000
7500
concentration
fnMl
.Figure.lI_
: Relative binding affinity for II of R5020 and compounds 5_, 2 and 7.
Fig. ceptor
the progesterone
re-
Calf uterine cytosol was incubated for 24 hrs with 7.5 nM 3H R5020 and increasing concentrations of unlabelled R :F added in ethanol (fina measured after 45 mn charcoal adsorption at binding is expressed as a percentage of non-inhibited bind?!g0(68000 cpm/ml of cytosol).
s bound
compound)
would
K*
(16)
= 0.58
109
LM-'
KA
(-2)
-
109
LM-1
and The be
apparent less
R5020 ted
affinity
than
for
RR
would
be
be
I .4
4 x
106
than
:
respectively
of the other -1 LM . Assuming
(29.), the less
145
T=EOSDI
apparent IO5 LM
-1
compounds a KA
affinity
tested
of
0.2
IO9 LM -'
of
the
ligands
tes-
I
Apparent relative binding for RE and RI, of calf
affinity uterus.
-RF
RE 100
3 T 8 13 16 17 -
of
.
TABLE
E2 ;5020
would
100 0.01
< 0.001
?I
0.4 < 0.001 < 0.001 < 0.001 0.17 < 0.001
0.06 < 0.01 < 0.01 < 0.01 N.D. < 0.01
For compounds 16 and 6, the apparent relative binding affinities were calculated-from concentration of unlabelled and competition necessary to reduce to 50 % specific binding after 24 hrsincuba~2J$$~~~dpH~~~o~o o the very inefficient competition of limit of their apparent re15, 17, 2: 7 and 8 only a upper lative affinity can be given. The high
estrone
affinity
absence large
of
derivatives
for
the
calf
and
178 hydroxyl
substituent
in
the
-16 and 5 lambuterine group
17o/$
and
evidence RE
kylation
concerning
in vitro of
any
RE
and
of
position.
could constitute a selective RE possessing an alkylating moiety ve
posses
a relatively
in
spite
the
presence
This
target
for
on
D-ring.
the
reaction
between
at
0-2'C.
However
RE may
occur.
Homologous
at
higher
of
of
suggests
such
the
that
derivatives We
such
do not
ha-
derivatives
temperature,
compounds
a
with
higher
al-
affinity group,
for RE (due to the presence
for instance)
could
In contrast,
homologous
and pregnane
series
constitute
derivatives
display
a very
of a 178 hydroxyl a more
specific
agent.
of -6 in the androstane low relative affinity
for RP, suggesting
that such derivatives
to act selectively
on RP.
would
not be useful
ACKNOWLEDGMENTS The authors wish to thank the "Ligue Nationale Fran$aise contre le Cancerit for appreciated financial support to one of us. We are also indebted to Professor RO~HEFORT for making possible the biological studies and to Dr. WINTERNITZ for helpful discussions. REFERENCES I. Niculescu-Duvaz, I., Elian, I., Ionescu, M. and Tarnauceanu, E., J. Prakt. Chemie 321, 522 (1979). 2. Niculescu-Duvaz, I., CambanirA. and Tarnauceanu, E., 3. Med. Chem. lo, 172 (1967). 3. Schneider, F., Hamsher, J. and Beyler, R.E., Steroids, 8, 553 (1966). 4. Jones, J.B., Adam, D.J. and Leman, J.D., J. Med. Chem. 2, 827 (1971). 5. Wall, M.E., Abernethy, G.S., Carroll, F-1. and Taylor, D.J., 3. Med. Chem. 12, 810 (1969). 6. Hamacher, H., Arzn. Forsh, Drug. Res. 2, 463 (1979). G.J., J. Med. 7. Lam, H.Y., Begleiter, R.A. and Goldenberg, Chem. 22, 200 (1979). 8. Bouveng, R., Ellman, M., Gunnarsson, P.O., Jensen, G., Liljekvist, J., and MGntzing, J., Eur. J. Cancer Is, 407 (1979). B., Shepherd, R. and Stamwik, A., "Nitro9. Hartley-Asp, soureas in Cancer Treatment" ; INSERT Symposium NO 19 ; Ed. by B, Serrou ; Elsevier Biomedical Press ; 1981, 105. 10. Carroll, F.I., Philip, A., Blackwell, J.T., Taylor, D-3. and Wall, M.E., J. Med. Chem. IS, 1158 (1972). G. and Stamvik, 11. K?inyves, I., P., HEgberg, B., Ensen, and Treatment of Human Tumors", A * f in "Characterization W. Davies and K.R. Harrap Editors, 303 (1978). 12. Shoppee. C.W. and.Sly, J.C.P., J. Chem. Sot. 345 (1959). 13. Wheeler, O.H. and Reyes-Zamora, C., Can. J. Chem., 5, 160 (1969). 14. Lucas, R.A., Dickel, D.F., Dziemian, M.J., Ceglowski, M.J., Henze, B.L. and Mac Phillamy, H.B., J. Amer. Chem. sot. 82, 5688 (1960). 15. Van de Woude, G. and Van-Hove, L., Bull. Sot. Chim. Beige, 76, 566 (1967). 16. Zelinsky, N., Stadnikoff, G., Ber. s, I722 (1906).
S 17.
18. 19. 20.
21. 22. 23. 24. 25. 26. 27. 28. 29.
ITDBLOIDI
147
Taillades, J. and Commeyras, A., Tetrahedron 30, 3407 (1974). Martinez, J., Oiry, .I., Imbach, J.L. and Winternitz, F., Eur. J. Med. Chem. 15, 211 (1980). A., Tetrahedron 30, 2493 Taillades, J. and Commeyras, (1974). Bhacca, N.S. and Williams, D.H. in "Applications of NMR Spectroscopy in Organic Chemistry ; illustration from ; HOLDEN-DAY San Francisco, 1964. the steroid field" P., J. Amer. Chem. Sot. 75, Ercoli, A. and De Ruggieri, 650 (1953). Apsimon, J.W., Beierbeck, H. and Todd, D.K., Can. J. Chem. 50, 2351 (1972). Robinson, C.H. and Hofer, P., Chem. and Ind. 377 (1966). Crabbe, P., Garcia, G.A. and Rius, C., J. Chem. Sot. Perkin I, 810 (1973). Ott, A.C., Murray, M,F. and Pederson, R.L., J, Amer. Chem. Sot. 2, 1239 (1952). R.V. and Gisvold, O., .J. Amer. Pharmacol. Petersen, Assoc. 45, 5727 (1956). Korenman, S.G., Endocrinol. 87, 1119 (1970). J.L. and Rochefort, H., Mol. Cell. Endocrinol. Borgna, 20, 7t (1980). Garcia, M. and Rochefort, H., Endocrinol. 104, 1797 (1979).
2846
NEW Claude
STEROIDAL
Chantal
Chavis+,
Jean-Louis
++
Borgna
NITROSOUREAS
and
de
Gourcy+,
Jean-Louis
+
Imbach
.
+
Laboratoire de Chimie Bio-Organique and Equipe de Recherche Associee no195 du CNRS, Universitg des Sciences et Techniques du Languedoc, Place E.Bataillon, 34090, Mvgtpellier, France. Unite d'Endocrinologie Cellulaire et Moleculaire, INSERM 34100 Montpellier, France. (U I4S), 60 rue de Navacelles, Received T-27-81 ABSTRACT 174 and 20 nitrosourea derivatives belonging to the dehydroepiandrosterone, estrone and pregnenolone series have been synthesized. The behavior of these compounds towards the calf uterine estradiol and pregnenolone receptors have been studied. INTRODUCTION During lating such
the
past
agents new
have
combination
between
(an
can
pointed
takes
can the
out
that
the
receptor
ted
function
steroid
xyl
group.
two
types
the
Thus, of
aim
steroidal
remain of
representative
function 20
3-OH
in position
(pregnenolone)
Volume
39,
Number
S
an
steroids.
The
target
drugs
and
a the
tissue
studies
on
have
to bind
a carbonyl has
with
(10,Il)
C-3 or
been
belonging
(ni-
association
Previous
such
fruitful moiety
an unblocked
work
estrone
178
could
2
this
as
nitrosoureas
transdehydroepiandrosterone, as
of
hor-
a lipophilic
nitrosocarbamates
least
for
against
estrogen)
such
alky-
search
a specific
receptor.
at
to
and or
By
effectivness
has
The
drugs
alkylating
androgen
to
steroidal
a possible
(7,8,9))
receptor.
and
of
cytotoxic
delivered
requires
which
(l-6).
advantage
active
be
of
anticancer
nitrosoureas
nitrosoureas
specific
synthesized
towards
agent
a number
potential
hormonally
deactivate
steroidal
years
a latent
or
selectivity
alkylating or
as
tumors
mustards
carrier high
been
compounds
mone-dependent
trogen
thirty
to
an
hydro-
synthesize
to each
pregnenolone
introduction
their
oxygena-
of
of
series the
(dehydroepiandrosterone,estrone) be
effected
T~ROXD=
from
the
the
urea or
corresponding
February,
1982
amino
or
tainable hoped
steroids
a-aminocyano from
the corresponding
part ot the molecule
might
be readily
functionality.
in an unusual
way
alcohol tives
prepared
and
compounds
:
Rl=R2=H
b
R+ =Thp;
c
R,z
prepared
which
R2xCN ; R2=CN
was
of the
17 amino
deriva-
data whereas
to a mixture
leads
are easily
re-
of (ZOR)
separated.
17 and 20 aminonitriles
lb, 2b, 3b, Ic -synthesis (16). This
amino-acid
in ammoniacal
cyanide, never
ting keto material
la, 2a, 3a, 4a were obtained oximino steroids with Na/
the literature
group
by a Strecker out
3N”2 R2
The stereochemistry
to be B from
carried
of potassium
H
steroids
of the 20 oximino
(20s) isomers
reaction,
a
of the corresponding
(12-15).
I
RR2 *R2
On the other hand, were
or
AND DISCUSSION
the following
17 and 20 amino
is known
duction
the
of the alkylating
CH
by reduction
It was
~1 to the nitrosourea
properties
RR2 R2
The
ob-
moiety.
RESULTS Ne have
group
influence
and pharmacological
carbamoytating
should
ketone
that the use of a cyano
chemical
which
appeared
always
methanol complete
detected
by
in the presence as some
t.1.c.
star-
of the reac-
S
131
TBICOSOI
NO 1
NH2
NH, C_N,CH2CH2Ci 6
y3
p H-~_N-CH2CH2Ct II
3a
tion mixture. well
However,
as a-cyanohydrins
carbonyl
compound
separation
been
that their
successful
_3b and _Ic were
2,
bamoylating
that a-aminonitriles equilibratewith
in such a way
has never
pounds 2,
it is known can readily
group
using
reacts
in a specific
to give excellent Thus compounds
car-
yields
in
p-nitrophenyl-N-
(18). This easily way on amino
prepared
functional
of the corresponding
groups
nitrosoureas.
9,
duced
compounds
yields
(Scheme
over
com-
nitrosocarbamoylated
the new reagent
Z-chloroethyl-N-nitrosocarbamate
2, -3a and -4a and the above reagent pro1, 6, 1, and 8 in very good to quantitative
1). These
previous
cultly-prepared
results
reports
on reaction
viz. &,
; therefore,
(17)
used as such for the next
may be directly
a one step procedure
ment
chromatographic
step.
The amino
reagent
as
the parent
with
where
are a significant
improve-
the same starting
material
N-nitrosocarbamoylazide,
and hazardous
reagent,
yielded
a diffi-
only 43% of 2.
However
-Ic does not react with p- nitrophenyl-N-2-chloroethyl-N-nitrosocarbamate probably owing to the weakened reactivity tain
of the amino
the desired
a-cyanonitrosourea namely
dure was employed, sation ning
group c1 to the nitrile.
of the urea.
a free
reaction
3-hydroxyl
(more than
3 days)
and incomplete
amounts
were
isocyanate
was used,
used,
group
nitrile
formation
pyrany;
was
titatively
chosen
takes
contai-
place
approximately a threefold from which
was
only
nitrosourea
deemed
in basic
The Strecker
stoiexcess
of
the
could be isolated.
be acid-labile
for this purpose
introduced.
by nitro-
the compound
of the steroid
sequence
should
18
an easier
at the 3-OH position
of the reaction
a protecting
resulted
compound
the aim of effecting
when
and when
a mixture
3R,l?B-diearbamoylated
gining
proce-
i.e. -Ic, showed that the isocyanate in DMF was very slow
chiometric
protection
two-step
followed
trials with
to ob-
function,
of N-2-chloroethyl
With
the usual
carbamoylation
Initial
In order
from
synthesis the be-
preferable.
since media
and tetrahydro-
as it is quickly procedure,
Such
the cc-amino-
when
and quanapplied
#!B WSlEOXDI
n t i v)
133
to the 3-0-tetrahydropyranylated aminonitriles next
lb, 3,
carbamoylation
and 46% yields
steroids
gave
the crude o-
-3b which were used directly for the step to produce 2, -10 and -II in 45, 40
respectively
purification.
Nitrosation
of the latter
three ureas,
after
column
by nitrosyl
chromatographic
chloride
in pyridine
ve yield
afforded
-12 and -14 in quantitatiRemoval of tetrahydropyranyl
was
Amberlite
and -13 in 72% yield. easily accomplished using
a dichloromethane-methanol titatively
STEREOCHEMISTRY
thought
to be c1 since
proceed
via an enamino
attacked
and gave -15 and -17 quan(Scheme 2).
OI' THE CYAN0 for the cyano
the Strecker
skeleton.
18-CH3'HNMR
chemical
from
group
which
at C-17
deduced
was partly
is
is known
(19) to
in turn will be
the less hindered
This hypothesis shifts
COMPOUNDS
reaction
intermediate
by CN- presumably
the steroid
in
mixture
and -16 in 85% yield
The stereochemistry
IR 120 (H+form)
~1 side of
confirmed
from
from Zurcher's
increments: Compound
0.73 0.77
5-Androsten-38-01 5-Androsten-3@,17B-diol 3H-(tetrahydropyran-2'yloxy)-17g-hydroxy-5androsten-17a-carbonitrile
3H-(tetrahydropyran-2'yloxy)-178-amino-5-androsten-17a-carbonitrile -lb The substituent
effect
Al7ol-CN= 0.93 Using
of the
of the
- 0.77=
the aminonitrile
is no deviation fect
reference
18-CH3 (dppm)
Al7B-NH22
If this Al7B-NH2
- 0.73
this work
is :
+ 0.16 ppm.
from the additivity
0.83
0.83
f7o-CN group
derivative
17fi-amino group
0.93
(20) (20) experimental value obtained by synthesis following (21)
should
be
- 0.16=
substituent
-lb and assuming there rule the substituent ef: - 0.06 ppm.
effect
is correct
chemical shift of 17fJ-amino+-androsten-36-01 CH3 equal to 0.73 - 0.06= 0.67 ppm. which is exactly
the
18-
should what was
be
S found
for
mental cyano
(see
(+ 0.13
value
deduced
found
these
experimental
literature group
sults
of
-la
in
study
offer
stereochemistry
series
only
one
for
is more
isomer
compound
when
at C20
conversion
shows
that
a downfield with
an
group
in
section) and (20o
results. (obtained 11, a
shift
upfield the
0.96
(ZOR)
reaction
20s) On
only
of
of
and
and the
from ppm.
2
mechanism
in
this
(208 the
reaction represent which
leading
to
pairs, or
ZOR)
18-CH3
in
of
the
preg-
there
is
the
18-CH3 for
is
compared
the
same
methyl
(experimental the
(ZOR)
derivatives
-8 fit
chemical
an upfield
there
concerning
is
to
data
(2OS)-NH2
viz,
product)
is
Literature
and
crude
stereochemical
shifts
study
value
steroids.
since
for
chemical
re-
that
is available
ppm.
the
suggest
for
of
ppm.
- 0.05
The
hand
stereochemistry
tool
17a
differentiation
spectra
isomer
nitrosourea
crude
NMR
to amide.
- 0.06
or -4a
other
These
one
0.02
found
and for
ZO-aminonitrile
the
a
theoretical
disubstituted
a conversion
0.05
were
(22)
experi-
of
available,
to ascertain
amine
(ZOR)-isomer.
which
the
no
effect
the
method
diagnostic
such
shift
was
whith
C,7
(from
of
for
(209)-amino or
of
Although
substituent
Apsimon
such
present
effect (23)
by
difficult
assignment the
the
agree
A possible
-3b).
section).
a convenient
stereochemistry
nane
135
experiments
calculated
effects
The
for
from
this
ppm.)
TBItOIDI
1.03 shift
can
be
understood
the
a-aminonitrile
-3a
the
above
shift
for
ppm.
and
and
argue
from from
-3b for for
the preg-
nenolone. EXPERIMENTAL Material
and
SECTION
methods
: Melting points are obtained in capillaries Chemical on a Buchi 510 melting range apparatus and are uncorrected. LJV spectra (imax in nm) were obtained on an Optica Model 10 spectrophotometer. Proton magnetic resonance spectra were recorded on Varian 90 or HA-100 instruments in CDCl all values are recorded in ppm. (6) with respect to TMS ai internal reference; band shape is indicated by s(singlet) ,d(doublet), t(triplet), m(multiplet). Thin layer chromatography was performed using Merck Kieselgel 60F 254 as 0.2 mm layers on aluminium foil. Merck silica (0.063 - 0.2 mm) was used for co-
lumn chromatography (solvent ratios are expressed as v/v). Optical rotations were taken on a Roussel-Jouan instrument with concentrations expressed in gram/100 ml of solution. IR spectra (v in cm-l) were recorded on a Perkin Elmer 450 instrumen??xMass spectra were obtained with a Jeol J.M.S. DlOO instrument. Elemental analysis were performed by the Service Central d'Analyse du C.N.R.S. Biochemical
:
Non radioactive R was kindly provided by Roussel-Uclaf 5P2?. (France). Stock so u ions (5 to 50 mM) of unlabelled compounds were made in ethanol or dimethylformamide and they were then diluted with ethanol ; to avoid degradation of the compounds, these solutions were kept at O'C and used during the following 24 h. Preparation of soluble extracts : uteri (stored at80°C) from immature calf and lamb were washed then homogenized in TE buffer (Tris-HCI IO mM pH : 7.4, EDTA 1.5 mM) The homogenates were centrifuged at with an ultraturrax. 105,000 g for 60 min. The supernatants defined as cytosols were adjusted to 2 mg protein/ml and used for binding studies. Determination of relative binding affinity for R (estrogen receptor) and R (progesterone receptor) : Eytoincubated for 4Por 24 h at 0-2°C with 3.5 to 7.5 and increasing concentrations of the teste
TE buffer) at 0°C. Tritiated steroid remaining in 2Q0 i.liof DCC treated cytosols were counted in 3 ml ethanol and 10 ml : 0.3 X) and POPOP of a toluene, PPO (2,5-diphenyloxazole -benzene : 0.01 X) mixture (1,4-bis Z-(5-phenyloxazolyl) using a SL30 intertechnique scintillation counter with a 20-25 % efficiency. Chemical synthesis : The N-2-chloroethyl-N-nitrosocarbamoylp-nitrophenylester was synthesized according to the were preliterature method (18). 178-or 20-aminosteroids pared following the known literature procedure by sodiumalcohol reduction of oximes. The separation of ,the (20 R)and (20 S)-amino-pregnane derivatives was done by chromatography on a silicagel column impregnated with ammonia. 17@-Amino-5-androsten-3H--01 (la) : mP 158-1590c (from ethyl acetate), lit. (12) : 160 V C ; TH NMR- (~~~l~~~,~H~;67 3H, 18-H), 1.03 (s, 3H, 19-H), 2.65 (t, (s,
S J = 8Hz),
3.47
137
'PBEOXDI
(m, IH, 3a-H),
5.33
(m, lH, 6-H).
(Za) : mp 17fi-Amino-l, 3, 5(10)-estratrien-3-01 222-224°C (from methanol) lit (13) : 222-226°C ; ‘H NMR (CDCI ) : 0.67 (s, 3H, 18-H), 2.60 (t, IH, 17a-H, J = 8Hz), 6.50 &H, 4-H), 6.56 (IH, 2-H), 7.07 (d, IH, l-H, J = 9nz). (2OS)-Amino-5-pregnen-3B-o1 (3a) : mp 172-174°C (from methanol), lit. (14) : 172-174°C ; IH NMR (CDC13) : 0.65 (s, 3H, 18-H), 0.98 (s, 3H, 19-H), 1.09 (d, 3H, 21-H), J= 6Hz), 2.77 (m, IH, 17a-H), 3.49 (m, lH, 3a-H), 5.33 (m, lH, 6-H). (2OR)-Amino-5-pregnen-3B-ol (4a) : mp 215-217'C (from methanol) lit. (15): 218-219°C ; 1H NMR (CDCI ) : 0.76 (s, 3H, 18-H), 1.00 (d, 3H, 21-H, J = 6Hz), ,.a3 (s, 3H, 19-H), 2.78 (m, IH, 17a-H), 3.51 (m, IN, 3a-H), 5.33 (m, lH, 6-H). Synthesis of 17@-and 20-N-aitrosoureas : In a typical exneriment N-2-chloroethyl-N-nltrosocarbamoyl-D-nitro-phenylester (0.828 mmole) was added to a solution of aminosteroid (0.715 mmole) in pyridine (5ml) at - IO'C. After vigorsus stirring was maintained for 3 h, the reaction mixture evaporated under vacuum then coevaporated three times with toluene. The oily residue was dissolved in ethanol (5ml) and allowed to crystaliize and then recrystallized.
17@-(N-2Tchloroethyl-N '-nitrosoureyl)-5-androsten-3R01. (5 ) : Following the above general procedure, the readtlon of la (0,2lg, 0.715 mmole) with the reagent (O,226g, 0.828 mmae) gave 5 after crystallization from ethanol
Mass spectrum : m/e/I, 316/27 (M-CICH2CH2N = NOH)?, 299/31 M-(H20 + ClCH2CH -N-NO+) 298/100 (M-Cl-Cfi -CH -N = NOH)t-H 0, 283184 (2$8-CH3)t Axial. talc. for 6 H gTp$4N~O$;7; C,g2.32 ; H,S.O8; N,9.91 ; found C,62.11 ; , ; ,a * 17@-(N-2-chloroethyl-N' -qit*osoureyl)-1,3,5 (lO)-estratrien-3-01 (6) : obtained from 2a in 92 % yield 88-9ooc (MeOH) ; Rf : 0.45 (CHC~~/E~~H, 9.5/0.5) + 12 (C 0.5, CHCl ) ; IR (CHCI ), 3590, 3420 294:&k! 1740 (C=O), 1610 &,, 1485 (N-30), 1340, ,296, 1160, lO;JO, 970, 900 ; UV (EtOH, 95) 220 nm (16,300), 277 nm (5,000), ,H NMR (CDC13) : 0.76 (6, 3H, IS-Y), 3.47 (t, 2H, CH -N-NO, J = 6,5 Hz), 3.96 (III,IH, 17a-H), 4.16 (t, 2H, CH2 -cb,
138
5.65
(m, OH), 6.53-7.10 (m, 3H, I-H, 2-H, 4-H). Anal. H28N303Cl ; C,62.13 ; H,6.95 ; N,10.35 ; found ; H,6.84 ; N,10.20.
talc.
20S-(N-2-chloroethyl-N '-nitrosoureyl)-5-pregnen-38-01 : The same above procedure gave compound 7 ( (7) mp 104-106*C(Et 0) ; Rf : 0.37 (CHCI /EtOH, 9,5/0.5) - 103 (C 0.5 CH& >. IR (CHC13), 3603, 3420, 1720, 1485, 1380,31350, 1160, 1090, 960, 900 (CDCI ) : 0.78 (s, 3H, 18-H), 1.03 (s, 3H, 19-H), 1.33 (d, J = 6.5~2), 3H, 21-H, J = 6Hz), 3.50 (t, 2~, CH~-N-NO, 4.18 (t, 2H, CH2-Cl), 3.40-4.20 (2m, 3a-H and l7o-H), 5.33 (m, 1H, 6-H), 6.77 (d, IH, NH, J = 9Hz), 6.90 (d, IH, J = : m/e/I, 345117 (M-C1CH2NNO), 344164 9Hz). Mass spectrum (M-CICH CH -N=NOH)+ = A? ; 329/25 (A-CH3)? ; 326/100 (M0 = B? ; 311/70 (B - CH )t Anal. talc. CICH -CA -~=NOH)+-H for z H2 NOCI; 6 ,63.76 ; H,8.47 ; N,9.23 ; found C,63.65 ,8.4024, 4:9?5& 20R-(N-2-chloroethyl-N '-nitrosoureyl)-5-pregnen-38-01 (8) in 93 % vield and crvstallized from methanol . , : Obtained * Fl40-142iC (cyclohexane) ; Rf : 0.37 (CH C1 /EtOH, 9.51 ";;;o;[;~~o-;;=~; 0;;&5C~;1a)0. IR (CHC13),23660, 3420, 2940 +amlde II), 1380, 1350, 1160, 1.00 1090, 960, 900 ; 'H NMR (CDCI ) : 0.77 (s, 3H, 18-H), 1.23 (d, 3H, 21-H, J = 6Hz), 3.52 (t, 2H, (s, 3H, 19-H), J = 6.5Hz), 4.20 (t, 2H, CH2C1), 3.30-4.30 (2m, CH2-N-NO, 3a-H and 17a-H), 5.35 (m, IH, 6-H), 6.73 (d, 1H, NH, J = 345/10 (M-C~CH cH -NNO)+, : m/e/I, + 9Hz). Mass spectrum 344/50+(M-CICH CH N = NOH)+, 326/100 (M-C1;H$&~H;. - H 0), 311/882(3$6-CH >+ Anal. talc. for C ; found C,63.99 ; , .4 ; N,9.44. C,6 5 .76 ; H,8.47 ; N,9?29 2
3B-(N-2-chloroethyl-carbamoyl)-l7~-(N-2-chloroethyl-N' -urevl)-5-androsten-17o-carbonitrile (18) : Potassium cyanichloride (log, 0.19 mole) de (log, _- 0.154 mole) and ammonium of 38-hydroxy-5-androsten-l7-one were added to a suspension in methanol (150 ml) saturated with ammo(1Og, 3.47 mmole) nia. The mixture was stirred for 24 h at room temperature whenadditional KCN (5g) and NH4Cl (5g) were added to the mixture which was then stirred an additional 4 days. The into cold water (11) containing reaction mixture was poured was washed with water acetic acid (20 ml) ; the precipitate and dried under vacuum over P 0 . To a solution of the abo(200 ml) ve solid material in anhydrous 2 aimethylformamide N-2-chloroethylisocyanate (II ml, at O'C was added dropwise After three days at room temperature the solu0.128 mole). tion was concentrated to half its initial volume and poured The precipitate was filtered, washed with into ice water. layer (500 ml) ; the organic water and dissolved in CHCI dried (d a2S0 ) and concentrated under was then separated, vacuum to give a oily residue (l$g) which was preabsorbed on silica and deposited on a silicagel column (6OOg, petro-
S
139
TDEOXDI
with a mixture of petroleum leum ether/ether l/l). Elution ether/ether 0.519.5 gave a solid product which was recrys; Rf : tallized from methanol (5.lg 28 X) ; mp 148-15O'C -55.8 (Cl, MeOH). IR(CHC13), 0.41 (Et O/MeOH, 0.8/0.2) i [p 3450, 32$0, 2950-2840, 2240 (Cl), 1700 (C=O), 1500-1550 (amide II), 1250-1200, 1140, 1080, 960. ]H NMR (CDCI ) : 0.77 (s, 3H, 18-H), 0.98 (s, 3H, 19-H), 3.30 and 3.53 (m, 4H, CH -CR Cl), 4.33 (m, 3a-H), 5.33 (m, IH, 6-H), 6.20 (t, IHf NH! J = 4.5 Hz), 6.65 (s, lH, NH), 7.25 (t, lH, NH,
; found
C,59.35;
: prepared according to Tetrahydropyranyl derivatives the literature procedure (24) in 95 % yield using p-toluene-sulfonic acid and dihydropyran in CH2C12. 3R-(tetrahydropyran-2'-yloxy)-5-androsten-l7-one 192-193°C (acetone), lit (25) 192-194'C.
one
: mp
3-(tetrahydropyran-2 '-yloxy)-1,3,5(10)-estratrien-17mp:147-149'C (ethylacetate), lit (26) 147-148°C.
3g-(tetrahydropyran-2 129-130°C (isopropylether),
'-yloxy)-5-pregnen-20-one lit (26) 129-13l'C.
: mp
3B-(tetrahydropyran-2'-yloxy)-l7~-(N-2-chloroethyl-N1ureyl)-5-androsten-17a-carbonitrile (9) : in a Typical experiment potassium cyanide (15.3 mmole, 0.995g), ammonium chloride (9.41 mmole, 0.498g) and saturated methanolic ammonia (10 ml) were added at O'C to a methanolic solution of 38-(tetrahydropyran-2 '-yloxy)-5-androsten-l7-one (1.27g, 3.41 mmole). The slurry was stirred at room temperature for then KCN (0.5g) and NH4C1 (0.252g) were added and 2 days, the mixture stirred for another 3 days. The KC1 was filtered off, washed with dry methanol and the filtrate concentrated under vacuum and dissolved in CHCl (50 ml) .I$-2-chloroethylisocyanate (0.307g, 3.5 mmole) was a a ded to the solution which was kept at room temperature for 17h. Then after removal of the solvent under vacuum, the oily residue was chromatographed (CH2~2/EtOH, 9.9/O.]) yn a silicagel column (60H) under low pressure (2.51b/in ) to give 9 (0.77g, 45% yield); 171-173'C (MeOH) ; Rf : 0.33 (CH Cl-/.EtOH, IR (CHCl ), 3386 $940, 2240 9.9/0.1) ; b]"SO -80 (Cl, CHC13). : 0.85 1500, 1135, 1025. 1H N& (CDCl;) (CN), 1690 (CgO), 1.00 (s, 3H, 19-H), 3.56 (m, 6H, ClCH-CH N-, (s, 3H, 18-H), 4.68 (m, IH, Thp), 5.33 (m, IH, 6-H), 0.60 ?m, Thp, 3a-H), : m/e/I, 383141 (M-RNHCONH)., 270/100 (M-RNCO-HCNAnal. talc. for C H N 0 Cl ; C, ;0,9.50 ; C1,7.0$8;4$02nJ C,66.50 ; H,8.33 ; N,8.34 ; 0,9.80 ; C1,7.10.
3-(tetrahydropyran-2 '-yloxy)-s73-(N-2-chIoxoethyl-N'ureylj-1,3,5(JO)-estratrien-J7a-carbonitrile (IO) : The above procedure was applied to 3-(tetrahydropyran-2'9yIoxy)r 1,3,5(10)-estratrien-17-one and JO -2as obtained as an oil
6.80 (dxd, 2-H), 7.45 (d, I-H, J=9Hz). Mass spectrum 364146 (M-RNHC0NH ) *,3/7/38 (N-RHco:~H~-HCN), 251/ 100 CH Cl? NCO-HCN--OH)'. Anal. ; N,8.64 ; found 66772'; H,7.46
: m/e/I (ccl-
3$-(tetrahydropyran-2'-yloxy)-20~-(~~-2-ch~o~oethy~-N~ureyl)-5-pregnen-20a-carbonitrile (!l) : Obtained from 3f3(tetrahydropyran-2'-yloxy)-S-pregnen-20-one in 4Oj: yield by
3B-(tetrahydropyran-2'-yloxy)-!7?-(~-2-chloroethyl-N_.'nitrosoureyl)-5-androsten-17a-carbonitrile (12) : Typically nltrosyl chloride was bubbled through a solution of 9 (ID~uo~&) maintained at O°C in anhydrous pyridine (15 nl) ; the reaction -.as complete within 15 mn. Cold ?ater (50 ml> was added until. precipitation occured ; the precipitate was filtared, washed with water and dissolved in dichloroaethane (150 ml.). The organic layer was separated,dried (Na2SOk), evaporated then coevaporated three times with toluene. ihe nitrosourea (12) was obtained as crystals (EtOH-Et20) in quEBtit2tiVe yield ; mp llg-120°C ; Rf : 0.55 (CHCX /EtOH, 9.810.2) ;[a]:" -47 (Cl, CHCl ). IR (CHCl > 29'-8, 2860, 2340 (CN), 3420 il35, 1020. H NXR fCDC13) 1740 (&==O), t290 (N-NO) 15;0-1456, 0.90 (s, 3H, 18-H), 1.06 (s, 38, 19-H), 3.50 (m, 2H, 3a-H,
found
C,62.99
3-(t~at~ahydr_o~yran-2'-~l~xy~-Ii~-(N-2-~h~~~~athy~~N1~ nittosourey~)-l,3,~(lO)-estratri~n-l7~-carhonit~~le (13) : compound 10 (2.3 mmole) gave I3 as 2211 0x1 1~ 72% weld after ; petroleum rapid colzn chromatog ether/ether 2930 ,
;
S
TICIICOID,I
141
uv (EtOH 95) 221 nm (11,250), 276 nm (2,200). IH NMR (CDCl ) 0.93 (8, 3H, 18-H), 3.50 (m, IH, Thp), 3.50 (t, 2H, CH -NNa ~=7Hs), 3.88 (m, IH, Thp), 4.16 (t, 2H, ClCH -, J=7Hz)'6.79 7.22 (s, (d, 4-H), 6.84 (dxd, Z-H), 7.16 (d, I-H, J=9&), Mass spectrum : m/e/I, 323/23 (M-C1CH2CH2N=NOHAnal talc. for C ; H;6*85 ; N,l0.88 ; found C,62.80 3~-(tetrahydropyran-2'-yloxy)-2O~-(N-2-chloroethyl-N1nitrosoureyl)-5-pregnen-2Oo-carbonitrile (14) : Compound
1135, 1025. (s, 3H, 21-H),
3.51
(m, 2H,,3a-H,
Thp),
3.90
-11
(m, IH,
. 17$-(N-2-chloroethyl-N' -nitrosoureyl)-3$-hydroxy-5androsten-17a-carbonitrile (15) : The tetrahydropyranylated derivative 12 (I mmole) was dissolved in a mixture+of di~hlorometha~-methanol (314) ; Amberlite IR 120 (H form) was added and after stirring 7h at room temperature, the resin was filtered and washed with dichloromethane. The filtrate was then stripped of solvent and the residue crystal-
1550-1540, 1090. H NMR (CDC13) : 0.90 (s, 3H, 3.52 (m, IH, 3a-H), 3.52 (t, 2H, CH2NNO, J=6.5Hz), 4.18 (t,2H, C1CH2-, J=6.5Hz), 5.36 (m, IH, 6-H), 7.15 (s,+lH, NH). Mass spectrum : m/efT, 341150 (Ma, 3231100 (M-cZCH,CH,N=NOH-OH).. Anal. talc. ; C,61.53 ; H,7,41 ; N,l2.48 ; found C,
17~-(N-2-chloroethyl-N '-nitrosoureyl)-3-hydroxy-1,3,5 (IO)-estratrien-17a-carbonitrile (16) : Obtained in 85% as an oil purrfled a florrsll column (2Og) eluted with petroleum ether/ether, 515 ; mp 135-137°C (ether) ; Rf : 0.40 (CH Cl /EtOH, 9.8/0.2) ;[aj2'+25.6 (Cl, CHCl >. IR (CHC13), 3606, 3420, 2930, 1740, 1618, 1490. UV GtOH 35) : 221 nm H NMR (CDCl ) : 0.93 (s, 3H (9,85(J), 277 nm (2,800) ; 18-H), 3.55 (t, 2H, CH2NN0, Jr7Hz), 4.20 (t, 2H, C1CH~I:=7Hz) 6.60 (d, 4-H), 6.65 (dxd, 2-H), 7.10 (d, I-H, J=9Hz), 7.65 (s, NH), 8.34 (s, OH). Mass spectrum : m/e/I, 323172 (M-ClCH CH2N=NOH) . Anal. talc. for C 6.51 ; N,l3,00 ; found C,61.28 ; ZOH-(N-2-chloroethyl-N '-nitrosoureyl)-3$-hydroxy-5pregnen-2Oa-carbonitrile (17) : Obtained In 95% after crys-
142
H NMR : 1.00 (s, ;IH, 1.93 (s, 3H, 21-H), (CDC13) 3.51 (m, IH, 3o-H), 3.51 (t, .J=7Hz), 4.16 (t, 2H, ClCH2-, J=7Hz), 5.33 (m, IH, 2H,CH2NN0, 6-H), 7.11 !s: IH, NH). Mass spectrum : rpW.3~;;;; ~~~CfCH~CH~N~N~~) 351/100 (M-ClCH CH2N=NOH-H20) ; H,7?553i 4 3 C,62.94 ; H,7.8$ ; N,ll.74 ; found C,62.88 N,11.55.
for
BIOLOGICAL
RESULTS
Compounds
-16
and
RE
compounds
while
efficient.
According
decreased bation ly
but
reached.
the
o-2Oc,
This
of
lateral
side
chain
results
were
(Fig.2)
all
bly of
do R
the
interact
cording
to
the
at
inhibition in Table
constant the
1.
ratio
affinity
KA= of
Only
with low
of
for
constant
kinetic
of
-6 incu-
with
uterus.
1
2 were
R
P The
not ineffi-
inhibition
constants after
that which
steroid
of 3 HR5020 proba-
binding
site
calculated 24 hrs
necessary
specific
the
-16 was
suggesting
the
the
affinity.
and the
by
at
Similar
For
a low
(27)
that,
site
competitors
rate,
occupancy
-6 occurs.
Again, time
KORENMAN
or[3H]R5020
the
binding
17,
concentrations
compounds
24 hrs
-6 (compound
affinity
affinity for 12LM-1 0.34 10 the
again
covalently
of [3H]E2
apparent
at
method
lamb
used.
apparent
0-2'C
the
derivatives
decreased
of -16 and
that
displayed
was
concentration
-8 were
suggesting
-16 and
with
tested
pregnane
relative
petition
high
obtained
to displace,
of
compounds
compound
binding
not
[I
alkylation of
also
while
P' The
no
compounds
efficient
after
-16 or 5 indicated site was reversible,
or
cient at 3 H R5020 [I was able
stabilized
3 H E2 II poorly
with
0-2“C, equilibrium was practical3 H E2 specific binding in the of
compounds
E 2 binding
tested)
at
increase
little
most
was
efficently
-17 or 5, 1 and the efficiency
time,
that,
DISCUSSION
-6 competed
-15 and to
markedly
suggesting
presence of
(Fig.1)
AND
binding
to
of
accom-
reach are
50%
listed
a relatively -16 and -6 displayed RE. On the basis of an affinity of E 2 for
constants
(neglecting
the
RE,
K+/Kpart
calculated (28),
of
the
from apparent
non-specifically
S
143
7FDEOSDI
;:-; \
2
Q a-v---V
A
l
0
l
0
X
0
X
500
50
5
Competitor
concentration
_ Figure.
Fig. I, : Relative binding of estradlol and compounds g,
1
5000
l
5oooo
( nH I
I_
affinity for the estrogen receptor -16 and -17. Calf uterine cytosol was incubated at 2; for 4 hrs (open symbols) H E2 and increasing conand 24 hrs (close symbols) with 3.5 nM : X ; 15 :AA; 16 :00; or 17 :VI centrations of unlabelled E 3H r binding was measul added in ethanol (final perzentage 72). red after 2 hrs charcoal adsorption at O'C. Ezch value, corrected for non specific binding, is expressed as a percentage of non(74000 cpm/ml of cytosol). inhibited 3H E2 specific binding
s
144
7.5
0
75 Competitor
Tacos=*
750
75000
7500
concentration
fnMl
.Figure.lI_
: Relative binding affinity for II of R5020 and compounds 5_, 2 and 7.
Fig. ceptor
the progesterone
re-
Calf uterine cytosol was incubated for 24 hrs with 7.5 nM 3H R5020 and increasing concentrations of unlabelled R :F added in ethanol (fina measured after 45 mn charcoal adsorption at binding is expressed as a percentage of non-inhibited bind?!g0(68000 cpm/ml of cytosol).
s bound
compound)
would
K*
(16)
= 0.58
109
LM-'
KA
(-2)
-
109
LM-1
and The be
apparent less
R5020 ted
affinity
than
for
RR
would
be
be
I .4
4 x
106
than
:
respectively
of the other -1 LM . Assuming
(29.), the less
145
T=EOSDI
apparent IO5 LM
-1
compounds a KA
affinity
tested
of
0.2
IO9 LM -'
of
the
ligands
tes-
I
Apparent relative binding for RE and RI, of calf
affinity uterus.
-RF
RE 100
3 T 8 13 16 17 -
of
.
TABLE
E2 ;5020
would
100 0.01
< 0.001
?I
0.4 < 0.001 < 0.001 < 0.001 0.17 < 0.001
0.06 < 0.01 < 0.01 < 0.01 N.D. < 0.01
For compounds 16 and 6, the apparent relative binding affinities were calculated-from concentration of unlabelled and competition necessary to reduce to 50 % specific binding after 24 hrsincuba~2J$$~~~dpH~~~o~o o the very inefficient competition of limit of their apparent re15, 17, 2: 7 and 8 only a upper lative affinity can be given. The high
estrone
affinity
absence large
of
derivatives
for
the
calf
and
178 hydroxyl
substituent
in
the
-16 and 5 lambuterine group
17o/$
and
evidence RE
kylation
concerning
in vitro of
any
RE
and
of
position.
could constitute a selective RE possessing an alkylating moiety ve
posses
a relatively
in
spite
the
presence
This
target
for
on
D-ring.
the
reaction
between
at
0-2'C.
However
RE may
occur.
Homologous
at
higher
of
of
suggests
such
the
that
derivatives We
such
do not
ha-
derivatives
temperature,
compounds
a
with
higher
al-
affinity group,
for RE (due to the presence
for instance)
could
In contrast,
homologous
and pregnane
series
constitute
derivatives
display
a very
of a 178 hydroxyl a more
specific
agent.
of -6 in the androstane low relative affinity
for RP, suggesting
that such derivatives
to act selectively
on RP.
would
not be useful
ACKNOWLEDGMENTS The authors wish to thank the "Ligue Nationale Fran$aise contre le Cancerit for appreciated financial support to one of us. We are also indebted to Professor RO~HEFORT for making possible the biological studies and to Dr. WINTERNITZ for helpful discussions. REFERENCES I. Niculescu-Duvaz, I., Elian, I., Ionescu, M. and Tarnauceanu, E., J. Prakt. Chemie 321, 522 (1979). 2. Niculescu-Duvaz, I., CambanirA. and Tarnauceanu, E., 3. Med. Chem. lo, 172 (1967). 3. Schneider, F., Hamsher, J. and Beyler, R.E., Steroids, 8, 553 (1966). 4. Jones, J.B., Adam, D.J. and Leman, J.D., J. Med. Chem. 2, 827 (1971). 5. Wall, M.E., Abernethy, G.S., Carroll, F-1. and Taylor, D.J., 3. Med. Chem. 12, 810 (1969). 6. Hamacher, H., Arzn. Forsh, Drug. Res. 2, 463 (1979). G.J., J. Med. 7. Lam, H.Y., Begleiter, R.A. and Goldenberg, Chem. 22, 200 (1979). 8. Bouveng, R., Ellman, M., Gunnarsson, P.O., Jensen, G., Liljekvist, J., and MGntzing, J., Eur. J. Cancer Is, 407 (1979). B., Shepherd, R. and Stamwik, A., "Nitro9. Hartley-Asp, soureas in Cancer Treatment" ; INSERT Symposium NO 19 ; Ed. by B, Serrou ; Elsevier Biomedical Press ; 1981, 105. 10. Carroll, F.I., Philip, A., Blackwell, J.T., Taylor, D-3. and Wall, M.E., J. Med. Chem. IS, 1158 (1972). G. and Stamvik, 11. K?inyves, I., P., HEgberg, B., Ensen, and Treatment of Human Tumors", A * f in "Characterization W. Davies and K.R. Harrap Editors, 303 (1978). 12. Shoppee. C.W. and.Sly, J.C.P., J. Chem. Sot. 345 (1959). 13. Wheeler, O.H. and Reyes-Zamora, C., Can. J. Chem., 5, 160 (1969). 14. Lucas, R.A., Dickel, D.F., Dziemian, M.J., Ceglowski, M.J., Henze, B.L. and Mac Phillamy, H.B., J. Amer. Chem. sot. 82, 5688 (1960). 15. Van de Woude, G. and Van-Hove, L., Bull. Sot. Chim. Beige, 76, 566 (1967). 16. Zelinsky, N., Stadnikoff, G., Ber. s, I722 (1906).
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Taillades, J. and Commeyras, A., Tetrahedron 30, 3407 (1974). Martinez, J., Oiry, .I., Imbach, J.L. and Winternitz, F., Eur. J. Med. Chem. 15, 211 (1980). A., Tetrahedron 30, 2493 Taillades, J. and Commeyras, (1974). Bhacca, N.S. and Williams, D.H. in "Applications of NMR Spectroscopy in Organic Chemistry ; illustration from ; HOLDEN-DAY San Francisco, 1964. the steroid field" P., J. Amer. Chem. Sot. 75, Ercoli, A. and De Ruggieri, 650 (1953). Apsimon, J.W., Beierbeck, H. and Todd, D.K., Can. J. Chem. 50, 2351 (1972). Robinson, C.H. and Hofer, P., Chem. and Ind. 377 (1966). Crabbe, P., Garcia, G.A. and Rius, C., J. Chem. Sot. Perkin I, 810 (1973). Ott, A.C., Murray, M,F. and Pederson, R.L., J, Amer. Chem. Sot. 2, 1239 (1952). R.V. and Gisvold, O., .J. Amer. Pharmacol. Petersen, Assoc. 45, 5727 (1956). Korenman, S.G., Endocrinol. 87, 1119 (1970). J.L. and Rochefort, H., Mol. Cell. Endocrinol. Borgna, 20, 7t (1980). Garcia, M. and Rochefort, H., Endocrinol. 104, 1797 (1979).