STEROID DERIVATIVES AND THEIR USE IN RADIOIMMUNOASSAYS

DERIVES DE STEROIDES UTILISES DANS LES DOSAGES PAR LA METHODE RADIO-IMMUNOLOGIQUE

English Abstract

ABSTRACT
Radiolabeled steroid derivatives having the
formula
<IMG>
wherein St is a des-hydroxy steroid moiety of (i) a
hydroxy steroid intended for radioimmunoassay or (ii)
a hydroxy containing derivative of a steroid intended
for radioimmunoassay, said derivative having a strong
affinity for the antibodies of the steroid intended for
radioimmunoassay: R is hydrogen or alkyl of 1 to 3 car-
bon atoms; n is 0, 1, 2, 3, or 4 and the asterisk (*)
indicates tagging with a radioisotope, are useful as
tracers in radioimmunoassays.

Claims

The embodiments of the invention in which an exclusive
property or privilege is claimed are defined as follows:-
1. A compound selected from the group consisting of
a) a steroid compound having the formula
<IMG>
wherein R is hydrogen or an alkyl group of 1 to 3 carbon
atoms; n is 0, 1, 2, 3, or 4; and St is a des-hydroxy steroid
moiety of (i) a hydroxy steroid intended for radioimmunoassay
or (ii) a hydroxy containing derivative of a steroid intended
for radioimmunoassay, said derivative having a strong affinity
for the antibodies of the steroid intended for radioimmuno-
assay;
b) a steroid compound having the formula
<IMG>
wherein R is hydrogen or alkyl having 1 to 3 carbon atoms;
n is 0, 1, 2, 3 or 4; the asterisk (*) indicates labeling
with a radioisotope; and St is a des-hydroxy steroid moiety
of (i) a hydroxy steroid intended for radioimmunoassay or
28

(ii) a hydroxy containing derivative of a steroid intended for
radioimmunoassay, said derivative having a strong affinity for
the antibodies of the steroid intended for radioimmunoassay;
c) compound having the formula
<IMG>
wherein R is hydrogen or an alkyl group having 1 to 3 carbon
atoms; R1 is alkanoyl having 2 to 6 carbon atoms and n is 0,
1, 2, 3 or 4;
d) a compound having the formula
<IMG>
wherein R is hydrogen or an alkyl group having 1 to 3 carbon
atoms and n is 1, 2, 3 or 4; and
e) a compound having the formula
<IMG>
29

wherein R is hydrogen or alkyl having 1 to 3 carbon atoms and
n is 1, 2, 3 or 4.
2. A steroid compound having the formula
<IMG>
wherein R is hydrogen or an alkyl group of 1 to 3 carbon atoms;
n is 0, 1, 2, 3, or 4; and St is a des-hydroxy steroid moiety
of (i) a hydroxy steroid intended for radioimmunoassay or (ii)
a hydroxy containing derivative of a steroid intended for
radioimmunoassay, said derivative having a strong affinity
for the antibodies of the steroid intended for radioimmuno-
assay.
3. A steroid compound in accordance with claim 2 where-
in n is 0.
4. A steroid compound in accordance with claim 2 where-
in n is 1.
5. A steroid compound in accordance with claim 2 where-
in n is 2.
6. A steroid compound in accordance with claim 2 where-
in n is 3.
7. A steroid compound in accordance with claim 2 where-
in n is 4.

8. A steroid compound in accordance with claim 2 where-
in R is hydrogen.
9. A steroid compound in accordance with claim 2 where-
in R is an alkyl group of 1 to 3 carbon atoms.
10. A steroid compound in accordance with claim 2 where-
in St is a des-hydroxy derivative of a steroid selected from
the group consisting of cholesterol, cortisol, cortisone,
corticosterone, prednisolone, methylprednisolone, triamcino-
lone, betamethasone, dexamethasone, triamcinolone acetonide,
betamethasone valerate, halcinonide, aldosterone, estrone,
estradiol, estriol, testosterone, 19-nortesterone, methyl-
testosterone, pregnenolone, digoxigenin, digitoxigenin and
11.alpha.-hydroxyprogesterone.
11. The steroid compound in accordance with claim 2,
which is (3.beta.,5.beta.,12.beta.)-3-[4-carboxy-3-(4-hydroxyphenyl)-1-
oxobutoxy]-12,14-dihydroxycard-20(22)-enolide.
12. The steroid compound in accordance with claim 2,
which is (3.beta.,5.beta.,12.beta.)-3-[4-carboxy-3-[(4-hydroxyphenyl)methyl]-
1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide.
13. The steroid compound in accordance with claim 2,
which is (3.beta.,5.beta.,12.beta.)-3-[[3-(carboxymethyl)-5-(4-hydroxy-
phenyl)-1-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide.
14. The steroid compound in accordance with claim 2,
which is (3.beta.,5.beta.,12.beta.)-3[[3-(carboxymethyl)-6-(4-hydroxy-
phenyl)-1-oxohexyl]oxy]-12,14-dihydroxycard-20(22)-enolide.
31

15. The steroid compound in accordance with claim 2,
which is (3.beta.,5.beta.,12.beta.)-3-[[3-(carboxymethyl)-7-(4-hydroxy-
phenyl)-1-oxoheptyl]-12,14-dihydroxycard-20(22)-enolide.
16. The steroid compound in accordance with claim 2,
which is 21-[4-carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-
11.beta.,18-epoxy-18-hydroxypregn-4-ene-3,20-dione.
17. The steroid compound in accordance with claim 2,
which is 21-[4-carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-
11.beta.,17-dihydroxypregn-4-ene-3,20-dione.
18. A compound having the formula
<IMG>
wherein R is hydrogen or an alkyl group having 1 to 3 carbon
atoms; R1 is alkanoyl having 2 to 6 carbon atoms and n is 0,
1, 2, 3 or 4.
19. A compound in accordance with claim 18 wherein R1
is acetyl and R is hydrogen.
20. A compound in accordance with claim 18 wherein n
is 0.
21. A compound in accordance with claim 18 wherein n
is 1.
32

22. A compound in accordance with claim 18 wherein n
is 2.
23. A compound in accordance with claim 18 wherein n
is 3.
24. A compound in accordance with claim 18 wherein n
is 4.
25. A compound having the formula
<IMG>
wherein R is hydrogen or an alkyl group having 1 to 3 carbon
atoms and n is 1, 2, 3 or 4.
26. A compound having the formula
<IMG>
wherein R is hydrogen or alkyl having 1 to 3 carbon atoms
and n is 1, 2, 3 or 4.
27. A steroid compound having the formula
33

<IMG>
wherein R is hydrogen or alkyl having 1 to 3 carbon atoms; n
is 0, 1, 2, 3 or 4; the asterisk (*) indicates labeling with
a radioisotope; and St is a des-hydroxy steroid moiety of
(i) a hydroxy steroid intended for radioimmunoassay or (ii)
a hydroxy containing derivative of a steroid intended for
radioimmunoassay, said derivative having a strong affinity
for the antibodies of the steroid intended for radioimmuno-
assay.
28. A steroid compound in accordance with claim 27
wherein the radioisotope label is iodine-125 or iodine-131.
29. A steroid compound in accordance with claim 27
wherein the radioisotope label is iodine-125.
30. A process for preparing a steroid compound which
can be readily labeled with a radioisotope and used as a
tracer is a radioimmunoassay which comprises:
(a) reacting a hydroxy steroid intended for radio-
immunoassay or a hydroxy containing derivative of a steroid
intended for radioimmunoassay, said derivative having a
strong affinity for the antibodies of the steroid intended
for radioimmunoassay, with a compound having the formula
34

<IMG>
wherein R is hydrogen or an alkyl group having 1 to 3 carbon
atoms; R1 is an alkanoyl group having 2 to 6 carbon atoms
and n is 0, 1, 2, 3, or 4; and (b) saponifying the resultant
steroid.
31. A process in accordance with claim 30 wherein n
is 0.

Description

3409
RB50a
~STEl~OID DERIV~TIVES AND THEIR USE
IN RADIOIMMUNOASSAYS
s
The measurement of various substances by the use
of radioimmunoassay techniaues has achieved widespread
acceptance in recent years. Yalow and Berson, In Vitro
Procedures With Radioisotopes in Medicine, International
Atomic Energy Agency, Vienna (1970) pgs. 455 et seq.,
express the principle of radioimmunoassay in the follow-
ing terms:
"Unlabelled antigen in unknown samples competes
against labelled antigen ("tracer") for binding
to antibody and thereby diminishes the binding
of labelled antigen. The degree of competitive
inhibition observed in unknown samples is com-
pared with that obtained in known standard
solutions for determination of concentration
of antigen in unknowns."
Radioimmunoassay tests require a specific anti-
body, a radioisotope-labeled (hereinafter referred to
as "radio-labeled") antigen, a pure sample of the anti-
gen to be measured to serve as a reference standard, and
means for the separation of free antigen from antibody-
bound antigen. Radioimmunoassays follow the basic prin-
ciple of saturation analysis, i.e., competition between
labeled and unlabeled antigen for a fixed number of
antibody binding sites.

3409
RB5Oa
When radiolabeled antigen, unlabeled antigen, and
anti-body are brought together, the amount of radio-
labeled antigen bound to antibody and the amount of
radiolabeled antigen ramaining unbound (free) has a
direct relationship to the amount of unlabeled antigen
present when a given amount of antibody is present.
Thus, by using a constant amount of antibody and
radiolabeled antigen, and using known concentrations
of unlabeled antigen, a standard (calibration) curve
can be plotted showing antigen concentration versus the
amount of radiolabeled antigen bound or versus radlo-
labeled antigen unbound, or versus a ratio of the two
measurements. The concentration of antigen in an un-
known sample can be read from the standard curve by
determining the amount of bound or free radiolabeled
antigen (or ratio of the two measurements) resulting when
the unknown sample is mixed with the amount of radio-
labeled antigen and antibody used to prepare the curve.
In all radioimmunoassay procedures, it is necessary to
provide means for separating the bound from the free
labeled tracer material. Many widely varied procedures
have been developed and used; exemplary procedures
are electrophoresis; chromatography; ion exchange;
adsorption to dextran coated charcoal, talc, or aelu-
lose; and a number of solid-phase antibody techniques.
The term "antigen", as used in the field of radio-
immunoassays, may cover substances of limited immuno-
genicity (ability to generate antibodies). In those cases
where the ~ubstance to be measured is of limited immu-
nogencity, the substance can be coupled with an immu-
nogenic carrier, usually a protein, to increase its

34~9
RB50a
immunogenicity. A substance that is nonimmunogenic,
but requires immunogenicity when linked with a carrier
is referred to as a "hapten".
Radioimmunoassay techniques have been used to de-
termine the concentration in body fluids of variousendogenous and exogenous steroids. In the development
of radioimmunoassays for the various steroids, the
preparation of a radiolabeled antigen is of primary
concern. Possible radioisotope lables are tritium, car-
bon-14, iodine-125, iodine-131, and others. However,
because tritium and carbon-14 must be counted by liquid
scintillation ta time-consuming and expensive process),
iodine-125 and iodine-131 are more desirable. For
reasons well-recognized in the art (e.g.,half-life,
radiation hazard, counting efficiency and others) iodine-
125 has become the radioisotope of choice for use in
steroid radioimmunoassays.
The chemical structure of steroids is such that it
is generally not possible to radioiodinate them directly.
It is necessary, therefore, to utilize as a precursor
of the radiolabeled antigen a derivative of the steroid
to be assayed which is readily iodinated. In choosing
or developing such a derivative, the primary concern
is the affinity of the derivative for the antibodies
of the steroid to be assayed; the affinity of the deri-
vative for the anti-bodies should, of course, be as
close to the affinity of the steroid for the antibodies
as possible.
Compounds having the formula

RBSOa
O
Il 11
St-O-C-CH2- ~ H-CH2-C-O~
~C~2)n
S
_ ~ J
R
OH
are readily tagged with a radioisotope and can be used
(when radiolabeled) as a tracer in radioimmunoassay
procedures fox the determination of steroid levels in a
body fluid. In formula I, and throughout the specifi-
cation, R is hydrogen or an alkyl group of 1 to 3 carbon
atoms; n is 0, 1, 2, 3, or 4 and St is a des-hydroxy
steroid moiety of (i) a hydroxy steroid intended for
radioimmunoassay or (ii) a hydroxy containting deriva-
tive of a steroid intended for radioimmunoassay, said
derivative having a strong affinity for the antibodies
of the steroid intended for radioimmunoassay.
Exemplary of hydroxy steroids intended for radio-
immunoassay which may be modified structurally as shown
in formula I are: cholesterol, cortisol, cortisone,
corticosterone, prednisolone, methylprednisolone, tri-
amcinolone, betamethasone, dexamethasone, triamcinoloneacetonide, betamethasone valerate, halcinonide, al-
dosterone, estrone, estradiol, estriol, testosterone,
l9-nortestosterone, methyltestosterone, and pregnenono-
lone. Exemplary of hydroxy containing derivatives of
a steroid intended for radioimmunoassay (which deriva-
tives have a strong affinity for the antibodies of the
steroid intended for radioimmunoassay) are digoxigenin,
.

lllS409
RB5Oa
digitoxigenin and ll~-hydroxyprogesterone.
The steroids of formula I are prepared from the
precursor hydroxy steroid having the formula St-OH
and a glutaric anhydride derivative having the formula
II O
Rl 0 ~3 (C~2)n C
In formula II, and throughout the specification, Rl is
an alkanoyl group having 2 to 6 carbon atoms, acetyl
being the preferred group.
The anhydrides of Formula II are novel compounds,
and as such constitute an integral part of this inven-
tion. They can be prepared by first reacting a 4-
methoxyphenylaliphatic aldehyde having the formula
III
R
CH30 ~ (CH2)n CH
with at least 2 molar equivalents of cyanoacetic acid
in the presence of a base (e.g., sodium hydroxide)
to yield, on acid hydrolysis, a compound having the
formula

8409
RB50a
IV R C
C~l30 _ ~3 CH2- -OH
CH2-1CI--OH
0
An alternative preparation for the compound of formula
IV wherein n is O and R is hydrogen, i.e., 3-(4-methoxy-
phenyl)glutaric acid, is disclosed by Smith et al.,
J.A.C.S., 72, 1877 (1950). In that procedure, anisalde-
hyde is condensed with ethyl acetoacetate in the pre-
sence of piperidine to give ethyl anisal-bis-acetoace-
tate. Cleavage of this product to give the desired 3-
(4-methoxyphenyl)glutaric acid can be accomplished
with boiling alcoholic sodium hydroxide solution.
Demethylation of the glutaric acid derivatives of
formula IV results in glutaric acid derivatives having
the formula
V O
R
HO ~ (CH2)n C~
CH2-1CI-OH
and can be accomplished by following one of the several
procedures known in the art for the demethylation of
aryl methyl etherq. One such procedure, described by
Feutrill et al., Aust. J. Chem., 25, 1719 (1972), in-
volves the treatment of the aryl methyl ether with
thioethoxide ion (readily prepared in situ from ethane-
thiol and sodium hydride) in a polar aprotic solvent,

3409
RB5Oa
preferably dimethylformamide.
'I'he phenolic hydroxy group of a compound of formula
V can be protected with an alkanoyl group using art-
recognized procedures. One such procedure comprises
reacting the glutaric acid derivative with the appro-
priate acid anhydride (acetic anhydride is preferred).
The preferred method of preparing a glutaric anhydride
derivative of formula II from the glutaric acid deriva-
tive of formula V is to combine the conversion of the acid
to anhydride and the protection of the phenolic hydroxy
group into a single step. When the Rl protecting group
is acetyl, this would involve heating a glutaric acid
derivative of formula V in acetic anhydride.
The reaction of a steroid precursor having the
lS formula St-OH and a glutaric anhydride derivative of
formula II to yield a steroid having the formula
VI R 1
St-O-C-CH -CH-CH -C-OH
(CH2)n
R
O-Rl
can be run in the presence of an organic base. Exem-
plary organic bases are nitrogen containing hetero-
cyclics, e.g., pyridine, and tertiary amines, e.g.,
triethylamine. The reaction will preferably be run at
an elevated temperature.
In those instances wherein the steroid precursor
has more than one hydroxy substitutent, it will be possi-
ble to monoacylate the steroid with a glutanic anhy-
dride derivative of formula II because of the varying

~118409
RB5Oa
reactivites of the steroid's hydroxyl substituents. If
it is desirable to acylate a hydroxy substituent other
than the most reactive one, conventional blocking
techniques should be used to protect the more reactive
substituents.
~r~al of the phenolic hydroxyl protecting group
in a compound of formula VI yields the corresponding pro-
duct of formula I.
The compounds of formula I can be labeled ("tagged")
10 with a radioisotope, preferably iodine-125 or iodine-131,
and most preferably iodine-125, using procedures well
known in the artl to yield a radiolabeled hapten having
the formula O O
VII St O C CH2 CH C 2 C
(CH2)n
~,
R /
OH
The asterisk (*) in formula VII indicates tagging with
a radioisotope. Exemplary of the methods known in the
art is the method of Hunter and Greenwood; see Nature,
194:495 (1962). The radiolabeled compounds of formula
VII form an integral part of this invention.
The radiolabeled compounds of formula VII can be
used as tracers in radioimmunoassay procedures following
the general principles set forth hereinabove. Exemplary
detailed procedures are described in Jaffe
et al., "Methods of Hormone Radioimmunoassay", Academic
Press, New York (1974) and Berson et al., "Methods in
Investigative and Diagnostic Endocrinology", Vol. 3
on "Steroid Hormones", North Holland, Amsterdam (1975).

3409
RB50a
The radiolabeled compounds of this invention are parti-
cularly useful as reagents in the automated radioimmu-
noassay system of Brooker et al. disclosed in United
States patent 4,022,577 issued May 10, 1977.
The following examples are specific embodiments
of this invention.
Example 1
(3~,5~,12~)-3-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]
-12,14-dihydroxycard-20(22)-enolide
A) 3-(4-Methoxyphenyl)glutaric acid
A mixture of ~-anisaldehyde (27.2g), ethyl aceto-
acetate (52.1g) and piperidine (4 ml) in 95% ethanol
(10 ml) is stirred at room temperature for 5.0 hours
while a solid forms. The solid is isolated by filtration,
washed with 25% ethanol and crystallized from 95% etha-
nol to afford ethyl 2,2'-(4-methoxybenzal)-bls-aceto-
acetate (31.4g), melting point 138-141C. The filtrate
on dilution with an equal amount of water gives a solid
which is crystallized from 95% ethanol to afford another
crop of material (8.5g), melting point 137-142C.
A mixture of ethyl 2,2'-(4-methoxybenzal(-bis-
acetoacetate (30g), ethanol (450 ml) and 50~ sodium
hydroxide (450g) is refluxed vigorously for 1.0 hour.
Water (150 ml) is added and most of the ethanol is re-
moved by distillation in vacuo. The concentrate is
acidified with concentrated hydrochloric acid and is
extracted with ethyl acetate. The ethyl acetate solution
is wa~hed with brine, dried, evaporated, and the residue
is crystallized from benzene-methanol to afford 3.3g of
3-(4-methoxyphenyl)glutaric acid, melting point 147-150C.

1118409
1~1350a
B) 3-(4-Hydroxyphenyl)glutaric acid
To a stirred suspension of 57% sodium hydride-
paraffin (6.45g), in dry dimethylformamide (70 ml) is
slowly added ethanethiol (11.89 ml) in dry dimethyl-
- S formamide (20 ml). After stirring the resultant slurry
for 15 minutes, a salution of 3-(4-methoxyphenyl)glu-
taric acid (3.0g) in dry dimethylformamide (20 ml) is
added. The slurry is heated in a bath at 165C for 5.0
hours and most of the solvent is removed by distillation
ln vacuo. The residue is diluted with water, acidified
with concentrated hydrochloric acid and extracted twice
with ether (the extracts are discarded). The solution is
saturated with sodium chloride and extracted with ethyl
acetate. The ethyl acetate solution is washed once with
brine, dried and the residue crystallized from chloro-
form-hexane to afford 2.3g of 3-(4-hydroxyphenyl)-
glutaric acid, melting point 168-170C.
C) 3-(4-Acetyloxyphenyl)glutaric anhydride
A solution of 3-(4-hydroxyphenyl)glutaric acid
(800 mg) in acetic anhydride (15 ml) is heated at 100C
for 2.5 hours and evaporated to dryness in vacuo. The
residual solid is crystallized from chloroform-hexane
to afford 600 mg of 3-(4-acetyloxyphenyl)glutaric an-
hydride, melting point 140-143C.
D) 12~-(Acetyloxy)-3~,5~)-3-[4carboxy-3-[4-(acetyloxy)-
phenyl]-l-oxobutoxy]-14-hydroxycard-20(22)-enolide
A solution of digoxigenin-12-acetate (130 mg) and
3-(4-acetyloxyphenyl)glutaric anhydride (286 mg) in dry
pyridine (4.0 ml) i8 heated under nitrogen in a bath at
120C for 12 hours. Water (0.5 ml) is added and after 5
minutes, the mixture is evaporated in vacuo. The residue
is dissolved in chloroform, washed with 10% hydrochloric

11184(~9
RB50a
acid and brine, dried and evaporated. This residue is
subjected to preparative thin-layer chromatography (tlcl
on a silica gel plate (2.0 X 200 X 200 mm) using chloro-
formmethanol (9:1) for development to afford 110 mg of
the title compound as an amorphous solid, melting point
118-135C.
E) (3~,5~,12~)-3-[4-Carboxy-3-(4-hydroxyphenyl)-1-
oxobutoxy]-12,14-dlhydroxycard-2o(22)-enolide
To a solution of 12~-(acetyloxy)-(3~,5~)-3-[4-
carboxy-3-[4-(acetyloxy)phenyl]-1-oxobutoxy]-14-hydroxy-
card-20 (22)-enolide (68 mg) in methanol (3.0 ml) is
added a solution of potassium carbonate (83 mg) in water
(1.0 ml) and the mixture is stirred at room temp rature
for 2.5 hours. The solution is then acidified with 5%
hydrochloric acid and the methanol is evaporated ln vacuo.
The residue is diluted with water (10 ml) and extracted
with ethyl acetate. The ethyl acetate solution is wash-
ed with brine, dried over anhydrous magnesium sulfate,
evaporated and the residue subjected to preparative tlc
on silica gel plates (1 X 200 X 200 mm) using chloro-
form-methanol (3:1) for development to afford 22 mg of
the title compound as an amorphous solid, melting point
110-118C (melting completely to liquid at 144C).
Example 2
(3~,5~,12~)-3-[4-Carboxy-3-[(4-hydroxyphenyl)methyl]-1-
oxobutoxy]-12,14-dihydroxycard-20(22)-enolide
A) 3-1(4-Methoxyphenyl)methyl]glutaric acid
To a solution of cyanoacetic acid (3.57g), in water

~118409
RB50a
20 ml is added a solution of sodium hydroxide (2.08g) in
water (20ml). The solution is diluted with glyme
(70 ml), 4-methoxyphenyl acetaldehyde (3.0g) is added
and the mixture is left at room temperature for 6.0
hours. The glyme is evaporated in vacuo, and the resi-
due is mixed with I0% hydrochloric acid (200 ml) and re-
fluxed for 6.0 hours. After cooling, the mixture is
extracted with ethyl acetate. The ethyl acetate ex-
tract is washed once with brine, dried, evaporated and
the residue subjected to chromatography on a column
of silica gel using chloroform-ethyl acetate mixtures
for elution to afford 2.2g of 3-[(4-methoxyphenyl)
methyl]glutaric acid. Crystallization from a benzene-
ethyl acetate-hexane mixture gives a specimen having
a melting point 109-110C.
B) 3-[(4-HydroxYphenyl)methyl]glutaric acid
To a stirred suspension of 57% sodium hydride-
paraffin (1.4g) in dry dimethylformamide (30 ml) is
added dropwise a solution of ethanethiol (4.0 ml~ in
dry dimethylformamide (10 ml). After 15 minutes, a
solution of 3-[(4-methoxyphenyl)methyl]glutaric acid
(800 mg) in dry dimethylformamide (20 ml) is added.
The resulting slurry is heated in a bath at 165C for
20 hours and evaporated in vacuo. The residue is acidi-
fied with 20~ hydrochloric acid, extracted with etherand the extracts are discarded. The aqueous solution
is saturated with salt and extracted with ethyl acetate.
The extracts are combined, washed once with brine,
dried, evaporated and the residue crystallized from a
mixture of ethyl acetate-chloroform-hexane to give 600
mg of 3-[(4-hydroxyphenyl)methyl]glutaric acid, melting
point 117-119C.
.
.

409
RBSOa
C) 3-[[(4-Acetyloxy)phenyl]methyl]glutaric anhydride
3-[(4-Hydroxyphenyl)methyl]glutaric acid (500 mg)
in acetic anhydride (15 ml) is heated in a bath at 120C
for 2 hours. The solution is then evaporated to dry-
ness in vacuo and the residue triturate~ with chloro-
form~exane to afford 490 mg of the title compound,
melting point 88-89C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[4-carboxy-3-[[4-(acetyl-
oxy)-phenyl]methyl]-l-oxobutoxy]-14-hydroxycard-
20(22)-enolide
A solution of digoxigenin-12-acetate (216 mg) and
3-[[(4-acetyloxy)phenyl]methyl]glutaric anhydride (500
mg) in dry pyridine (6.0 ml) is refluxed for 20 hours.
Most of the pyridin~ is removed by distillation in vacuo
and the residue is diluted with water (20 ml) and aci-
dified with concentrated hydrochloric acid. The mix-
ture is extracted with ethyl acetate, the extract washed
twice with small amounts of brine, dried, evaporated and
the residue subjected to preparative thin-layer chromato-
graphy (as in Example ID) to isolate 323 mg of 12B-
(acetyloxy)-(3~,5~)-3-[4-carboxy-3-[[4-(acetyloxy)
pheyny]methyl]-l-oxobutoxy]-14-hydroxycard-20(22)-
enolide, melting point 70-7gC.
E) (3B,5B,12~)-[4-Carboxy-3-[(4-hydroxyphenyl)methyl]
1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide
A mixture of 12B-(acetyloxy)-(3~,5B)-3-[4-carboxy-
3-[[4-(acetyloxy)phenyl]methyl]-1-oxobutoxy]-14-hydroxy-
card-20(22)-enolide (160 mg), methanol (20 ml), water
(7.0 ml) and potassium carbonate (207 mg) is stirred
at 15C for 2.5 hours. The mixture is acidified with
concentrated hydrochloric acid and most of the methanol
is evaporated ln vacuo at room temperature. The concen-
trate is extracted with ethyl acetate, the extract washed

~184~g
RB50a
with small amounts of brine, dried (MgSO4anhydrous)
evaporated and the residue subjected to a preparative
thin-layer chromatography on silica gel plates (as
in Example lE) to afford 55 mg of (3~,5~,12~)-[4-carboxy-
3-[(4-hydroxyphenyl)methyl]-1-oxobutoxy]-12,14-dihydroxy-
card-20(22)-enolide, melting point 155-165C.
Example 3
(3~,5~,12~)-3-[[3-tCarboxymethyl)-5-(4-hydroxyphenyl)-1-
oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide
A) 3-[2-(4-Methoxyphenyl)ethyl]glutaric acid
To a solution of cyanoacetic acid (850 mg) in
water (5.0 ml) is added a solution of sodium hydroxide
(440 mg) in water (10 ml). The solution is diluted with
glyme (15 ml) and 3-(4-methoxyphenyl)propionaldehyde
(820 mg) is added. The solution is kept at room tempera-
ture for 10 hours and then evaporated to remove the glyme.
To the residue is added 10% hydrochloric acid (50 ml)
and the mixtureis thereafter refluxed for 6 hours with
stirring. It is then cooled and extracted with ethyl
acetate. The extract is washed with brine, dried over
anhydrous magnesium sulfate and evaporated. The resi-
due is subjected to chromatography over silica gel (15 g)to isolate in chloroform-ethyl acetate (80/20) fraction,
600 mg of the title compound. Crystallization from ethyl
acetate-benzene gives a specimen having a melting point
of 98-100C.
B) 3-[2-(4-Hydroxyphenyl)ethyl]glutaric acid
To a suspension of 57% sodium hydride-paraffin
(2.2g) in dry dimethylformamide (70 ml) in an atmosphere

34()9
- RB50a
of nitrogen is slowly added ethanethiol (4.0 ml). The
mixture is stirred at room temperature until a clear
solution results. To this solution is added a solution
of 3-[2-(4-methoxyphenyl)ethyl]-glutaric acid (1.2 g)
in dry dimethylformamide (10 ml) and the mixture is
heated with stirring in a bath at 165C for 6.0 hours.
The mixture is then evaporated to dryness ln vacuo
and the resulting residue is dissolved in water (50 ml),
extracted with ether (two 50 ml portions) and the ether
extract is discarded. The aqueous solution is acidified
with concentrated hydrochloric acid and extracted with
ethyl acetate. The ethyl acetate solution is washed with
brine, dried with anhydrous magnesium sulfate, and evapor-
ated to a residue. Crystallization of this from chloro-
form-hexane affords l.Og of the title compound, melting
point 142-144C.
C) 3-[2-[4-Acetyloxy)phenyl]ethyl]glutaric anhydride
A solution of 3-~2-(4-hydroxyphenyl)ethyl]glutaric
acid (600 mg) in acetic anhydride (15 ml) is heated
in a bath at 120C for 2.0 hours. It is evaporated in
vacuo and the residue is crystallized from dichlorometh-
ane-hexane to afford 495 mg of 3-[2-[4-(acetyloxy)phenyl]-
ethyl]glutaric anhydride, melting point 97-99C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[[5-[4-(acetyloxy)phenyl]
-3-(carboxymethyl)-1-oxopentyl]oxycard-20(22)-
enolide
A solution of digoxigenin-12-acetate (200 mg)
and 3-[2-[4-(acetyloxy)phenyl]ethyl]glutaric anhydride
(470 mg) in dry pyridine (8.0 ml) is refluxed for 20
hours. The pyridine is evaporated ln vacuo, the residue
diluted with water (25 ml), acidified with concentrated
,,~

34()g
RBSOa
16
hydrochloric acid and extracted with chloroform. The
chloroform solution is washed with small amounts of brine,
dried, evaporated and purified by preparative thin-
layer chromatography (as in Example ID) to isolate 253
mg of 12~-(acetyloxy)-(3~,5~)-3-1[5-[4-(acetyloxy(phen-
yl]-3-(carboxymethyl)-1-oxopentyl]-oxy]card-20(22)-
enolide.
E) (3~,5~,12~)-3-[[3-(Carboxymethyl)-5-(4-hydroxyphen-
yl)-l-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-
enolide.
A solution of 12~-(acetyloxy)-(3~,5~)-3-[[5-[4-
(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxopentyl]oxy]
card-20(22)-enolide (230 mg) in a mixture of methanol
(10 ml) and water (2.5 ml) containing potassium carbonate
(210 mg) is stirred at 10 to 15C for 2.5 hours. It is
then acidified with concentrated hydrochloric acid.
Isolation and purification of the product as described
in Example lE yields 44 mg of (3~,5~,12~)-3-[[3-(carbo-
xymethyl)-5-(4-hydroxy-phenyl)-1-oxopentyl]oxy]-12,14-
dihydroxycard-20(22)-enolide, melting point 126-134C.
Example 4
(3~,5~,12~)-3-1[3-(carboxymethyl)-6-(4-hydroxyphenyl)-1-
oxohexyl]oxy]-12,14-dihydroxycard-20(22)-enolide
A) 3-[3-(4-Methoxyphenyl)propyl]glutaric acid
4-(4-Methoxyphenyl)butyraldehyde (5.34 g) is re-
acted with cyanoacetic acid (5.15 g) and sodium hydroxide
(2.74 g) in a mixture of glyme (50 ml) and water (60 ml).
The mixture is treated with hydrochloric acid and pro-
cessed as described in Example 2A to afford the title
co~xNnd (2.lg) which on crystallization from ethyl ace-

4~)9
RB5Oa
17
tate-chloroform-hexane had a melting poin~ of 71-73C.
B) 3-[3-(4-Hydroxyphenyl)propyl]glutaric acid
3-[3-(4-Methoxyphenyl)propyl]glutaric acid (2.0g)
is demethylated by the procedure described in Example
5 lB to afford, after cyrstallization of the product from
ethyl acetate-chloroform-hexane, 1.2 g of 3-[3-(4-hydro-
xyphenyl)-propyl3glutaric acid, melting point 107-108C.
C) 3-[3-14-(Acetyloxy)phenyl]propyl]glutaric anhydride
3-[3-(4-Hydroxyphenyl)propyllglutaric acid (533
10 mg) is reacted with acetic anhydride as described in
Example lC to afford 3-[3-~4-(acetyloxy)phenyl]propyl]
glutaric anhydride, melting point 55-57C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[[6-[4-~acetyloxy)phenyl]
3-(carboxymethyl)-1-oxohexyl]oxy]card-20(22)-enolide
A mixture of digoxigenin-12-acetate (216 mg) and
3-[13-(4-acetyloxy)phenyl]propyl]glutaric anhydride
(610 mg) in dry pyridine (9.0 ml) is reacted for 18 hours
and the product is isolated and purified as described in
Example lD to afford 245 mg of 12B~acetyloxy)-(3B,5B)-3-
20 [[6-[4-(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxohexyl]
- oxy]card-20(22)-~nolide, melting point 98-113C.
E) (3B,5B,12~)-3-[[3-~Carboxymethyl)-6-(4-hydroxy-
phenyl)-l-oxohexyl]oxy]-12,14-dihydroxycard-20(22)
-enolide
A mixture of 12~-(acetyloxy) (3B,5~)-3-[[6-~4-
(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxohexyl]oxy]
card-20(22)-enolide (150 mg) and potassium carbonate
(172 mg) is reacted in a mixture of methanol (5.0 ml)
and water (2 ml). The mixture is processed and purified
30 as described in Example lE to afford 58 mg of (3B,5B,
12B)-3-[[3-(carboxymethyl)-6-(4-hydroxyphenyl)-1-oxo-
hexyl]oxy]-12,14-dihydroxycard-20(22)-enolide, melting
point 115-128C.

409 RB50a
18
Example 5
(3~',5~,12p,)-3-[[3-(Carboxymethyl)-7-(4-hydroxyphenyl)
-1-oxoheptyl]oxy-12,14-dihydroxycard-20~22)-enolide
Following the procedure of Example 4, but sub-
stituting 5-(4-methoxyphenyl)valeraldehyde for 4-(4-
methoxyphenyl)-butyraldehyde yields the title compound.
Detailed Procedure for Radioiodination
of Digoxigenin Derivatives
Sodium radioiodide (I125) aqueous solution (10 ml;
approximately 6mCi) is added to a reaction vial contain-
15 ing a methanolic solution of (3~,5~,12~)-3-~4-carboxy-3-
(4-hydroxyphenyl)-1-oxobutoxy]-12,14-dihydroxyca~d-20
(22)-enolide (10 ml 1 mg/ml). The vial is stoppered
and a chloramine T solution (25 ml; 2 mg/ml in 0.5M
phosphate buffer, pH 7.5) is injected through the stoppër
20 The vial is mixed well by shaking and allowed to stand for
five minutes. Sodium metabisulfite solution (20 ml;
3 mg/ml in 0.5M phosphate buffer, pH 7.5) is injected
through the stopper to quench the reaction.
The reaction mixture is applied to a 40 ml Sephadex
25 G-10 column and eluted with tris acetate buffer (0.05M,
pH 6.5). Forty-four drops per tube are collected with the
aid of a fraction collector. Free iodide comes off around
tube #38 and the iodinated product elutes off in two
fractions, tubes 110 through 130 (fraction I) and tubes
30 131 through 150 (fraction II?. Fraction II is found to
be superior and is used in radioimmunoassays.
* Trade Mark
B~

~ 4~9 RB50a
19
Example 6
21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-11~,18-
epoxy-18-hydroxypregn-4-ene-3,20-dione
~) 21-L3-t4-Acetyloxyphenyl)-4-carboxy-1-oxobutoxy]
-11~ 18-e~oxv-18-hvdroxv~regn-4-ene-3,20-dione
Aldosterone (100 mg) is refluxed in dry pyridine
10 (6.0 ml) with 3-(4-acetyloxyphenyl)glutaric anhydride
(300 mg) for 2.0 hours. The solution is then cooled
to room temperature, water is added and after standing for
a few minutes evaporated in vacuo. The residue is dis-
solved in ethyl acetate (20 ml), washed with 15% hydro-
15 chloric acid (6.0 ml) and brine, dried and evaporated toafford 408 mg of a gum. A thin-layer chromatography
examination of this material shows the presence of three
steroidal products. It is then subjected to preparative
thin-layer chromatography on two 2.0 mm silica gel plates
20 (with two developments of the plates with chloroform-
methanol (9:1) to isolate 135 mg of the title compound,
melting point 145-160C with consistent spectral data.
B) 21-~4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]~
18-epoxy-18-hydroxypregn-4-ene-3,20-dione
A solution of 21-[3-(4-acetyloxyphenyl)-4-carboxy-
l-oxobutoxy]~ ,18-epoxy-18-hydroxypregn-4-ene-3,20-
dione (130 mg) in anhydrous methanol (15 ml) containing
triethylamine (0.2 ml) is allowed to stand at room temp-
erature for 24 hours. The methanol is then evaporated
30 in vacuo, the residue is diluted with water (5.0 ml),
acidified with 10~ hydrochloric acid and extracted with
ethyl acetate (three 5.0 ml portions). The ethyl acetate

~8409
RB50a
solution is washed with brine, dried over anhydrous
magnesium sulfate, evaporated and the residue is subject-
ed to preparative thin-layer chromatography on silica
gel plates (using chloroform-methanol, 9:1 for develop-
5 ment) to isolate 83 mg of the title compound, meltingpoint 145-152Cr with consistent spectral data.
Detailed Procedure for Radioiodination
of Aldosterone Derivative
. . .
To a solution of 21-[4-Carboxy-3-(4-hydroxyphenyl)-
30 1-oxobutoxy]-11~,18-epoxy-18-hydroxypregn-4-ene-3,20-
dione (5.0,ug) in dioxane-0.5M borate buffer (1:9, 50 ul)
at pH 8.5 are added successively 20 ~1 of aqueous solutions
of sodium radioiodide (I125, 4mCi) and freshly prepared

111~3409
RB5Oa
21
chloramine-T (80 ~g). After 90 seconds, the reaction is
stopped by the addition of sodium bisulfite (80,ug) in
water (20 ~1). The solution is applied on a 5 X 20
cm silica gel plate which is developed with chloroform-
5 methanol (9:1). The band of radioiodinated product islocated using a scanner and is isolated by extraction
with ethanol. The concentrated ethanol solution is
stored at 15C.
Example 7
21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-11~,17-
dihydroxypregn-4-ene-3,20-dione
A) 21-[3-(4-Acetyloxyphenyl)-4-carboxy-1-oxobutoxy-
11~,17-dihydroxypregn-4-ene-3,20-dione
A solution of cortisol (346 mg) and 3-(4-acetyloxy-
phenyl)glutaric anhydride (496 mg) in dry pyridine (5.0
ml) is refluxed for 40 minutes. The pyridine is evapor-
ated in vacuo, the residue is diluted with ethylacetate
20 (30 ml), washed with 15% hydrochloric acid (10 ml) and
brine, dried over anhydrous magnesium sulfate and evapor-
ated to yield 840 mg of a powder. Examination of the
NMR spectrum and TLC behavior of this material shows
that it is a 1:1 mixture of the title compound and 3-
25 (4-acetyloxyphenyl)glutaric acid.
B) 21-[4-Carboxy-3-(4-hydroxyphenyl)l-oxobutoxy]-11~,
17- dihyroxypregn-4-ene-3,20-dione
The mixture obtained above (700 mg) is dissolved
in anhydrous methanol (30 ml) containing triethyamine
30 (1.0 ml) and the solution is refluxed for 5.0 hours.
The solution is cooled, acidified with 10~ hydrochloric
acid and most of the methanol is evaporated in vacuo.
The resulting slurry is extracted with ethyl acetate,

409
RBSOa
22
the ethyl acetate solution is washed with brine,
dried, and evaporated. The residue is subjected to pre-
parative thin-layer chromatography on two 2.0 mm silica
gel plates u3ing chloroform-methanol (9:1) for develop-
5 ment and the major band is isolated with chloroform-
methanol (8:2) to afford 300 mg of the title compound,
melting point 98-110C.
Description for Radioiodination
of Cortisol Derivative
21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-
llr~,17-dihydroxypregn-4-ene-3,20-dione is radiolabeled
with I125 using the procedure described above for the
15 radioiodination of aldosterone derivatives.
Example 8
(3r"5r~,12f~)-3-[4-Carboxy-3-(4-hydroxy-3- methylphenyl)
20 -1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide
A) 3-(4-Methoxy-3-methylphenyl)glutaric acid
A mixture of 4-methoxy-3-methylbenzaldehyde
(100 mmole), cyanoacetic acid (210 mmole) and potassium
25 hydroxide (250 mmole( in water SOO ml) is stirred at
room temperature for 20 hours. The resulting solution
is acidified with concentrated hydrochloric acid (100 ml)
and the mixture is refluxed for 60 hours. After Gooling,
it is saturated with sodium chloride and extracted with
30 ethyl acetate to afford the title compound.
.~.

8409
RBSOa
23
;
B) 3-(4-Elydroxy-3-methylphenyl)glutaric acid
Followin~ the procedure described in Example lB,
but substituting 3-(4-methoxy-3-methylphenyl)glutaric
acid for 3-(4-methoxyphenyl)glutaric acid, yields the
5 title compound.
C) 3-[4-(Acetyloxy)-3-methylphenyl)]glutaric anhydride
Following the procedure described in Example lC,
but substituting 3-(4-hydroxy-3-methylphenyl)glutaric
acid for 3-(4-hydroxyphenyl)glutaric acid, yields the
10 title compound.
D) 12~-(Acetyloxy)-(3~,5~)-3-[4-carboxy-3-[4-(acetyl-
oxy)-3-methylphenyl]-1-oxobutoxy]-14-hydroxycard
-20(22)-enolide
Following the procedure described in Example lD,
but substituting 3-~4-(acetyloxy)-3-methylphenyl)]glu-
taric anhydride for 3-(4-acetyloxyphenyl)glutaric anhy-
dride, yields the title compound.
; E) (3~,5~,12~)-3-t4-Carboxy-3-](4-hydroxy-3-methyl-
phenyl)-l-oxobutoxy]-12,14-dihydroxycard-20(22)-
enolide
Following the procedure described in Example lE,
but substituting 12~-(acetyloxy)-(3~,5~)-3-t4-carboxy
-3-[4-(acetyloxy)-3-methylphenyl]-1-oxobutoxy]-14-hy-
droxycard-20(22)-enolide for 12~-(acetyloxy)-(3~,5~)-3-
5 [4-carboxy-3-[4-(acetyloxy)phenyl]-1-oxobutoxy]-14-hy-
droxycard-20(22)-enolide, yields the title compound.
Examples 9-26
Following the procedure described in Example 6,
but ~ubstituting the steroid listed in Column I for al-

111~3409
RB50a
24
do~terone and the anhydride reagent listed in ColumnII for 3-(4-acetyloxyphenyl)glutaric anhydride, yields
the product listed in Column III.

lllB~9
RB50a
~5
,_/
o ~o I I
o o~ ~ ~ o c o ~ o~ o ~ o
x x~ ~ X ~ X ~x :~ X ~ X
o o o ~ I o ~1 o ~o X o c~ o ~
r ~1 ~ ~1 ~~ ~ ~I X ~ X
I ~ I ~ ~rl ~0 ~0
C C I E~ C C (d S~ h C h C ~1
H XX ~r X ~>1 X ~ X IX ~ X ~ O X ~O O
C ~ ~ C h O h X~ ~o X~O ~ O I ~ I
t~ ~ O ~ O~ ~ C ~ ~
O ~ `O ~ `O
~ I ~ X ~ ~ ~ ~ ~1 C~ I O ~ I I ~ I
I u)I 0 1 ~1 1 ~ I ~ oI o ~ I O ~ I O
I ~ I ~ I I I ~ I r~ ~ I o ~ I o ~ I o a~
X ~ X ~ X ~ X ~ X ~OX ~ ~ X--I ~ X
0 ~10 1 0 X O ~Q O ~ ~O ~ :: O q~ I O ~ I
.q O ~ 0 .4 ~1 ~ R I a) R I ~ 5~
l ~ l ~ l o ' ~ l x~ ~ l x~ l x~ ~ l x c
~ o ~ o ~ o c --~ o o~ o ~ o ~l ~ o ~ --~ o ~
c ~ c ~ c a) C a~
H~ hQ~ h~ r~ h
H ~ X ~ XO ~ X ~1 X >X ~1
0~ 0~ ~1~ 0~ 0~ O.C 0~ 0~
C~1 C ~ ~ -I C ~1 C~1 C ~ ~ ~1 C
~ ~ ~ ~ C ~Id ~ ~-d ~ ~td
oa) u~u u~ ~ u a) u~ u ~ o a) u
U~r~U~ U~-lUrlU~rlU~rlU~
O ~ ~rl C C C
~0 ~ O
H ~ ~Ol Q)
C ~ O o ta ~ ~1
~ a) ,~Q ,U~ ac)
~1 ~ ~ .{ O
o o ~ 1 ri ~ X
V U U U ~ E~ ~, ,R
a~ o ~

3409
R~350a
26
o
~ ~1~ 0 ~ ~ O I X
S L ~ X ~ ~ O ~ I O I
o s I I I X ~ ~ ~ x
I~ o ~ ~ I
H S ~ ~rl o I ~ S O I ~ ~ Rl I ~ ) I S
~ ~ ~ s ~1 ~ s~ o
H ~ ` I P~ X 1 ~1 0 N~ ~rl X .t: Q)~ JJ ~1 ~
X ~ o :~ o Irl X ~ O ~I Q. r~X 0~ X ~J
O ~1 ~ X Ll ~ O ~1 ~) ~ h-- ~1 51 O O O I
~: ~ ~ ` O ~ tJ~ 1 1 X I ~ 1~-) X ~ h h h ~r
3 ~ h ~ ~ O-- ~ ` O 1
~1 :~ O I ~ S h ~ 1 0 S ~1 h--
O S h ~ S I a)~ ~1 1 ` ~ o ~ 1~ S
I O ~ ~ C~ ~ I O ~
~ ~I rl ~r ~I X ~ O I I ' ~ ~ ~ X ~ h
-- I O I ~I d'~1~ ~ I h 'r ` I O I ~
O ~ ~ S ` -- ` ~ ~ --~ ~ ~ ~ 1::
I I ` I 0~ ~ I I ~~ I ~O ~1 ~R >1SI
X ~1 ~ ) X ~ I I X O X O
X ~ I X ~1 ~ O ~ S~ O ~ (d O X O
O X t~ O ~ P~ ~ I tJ~ OX ~ A X X h A O A I
R O ~: A I O ~ h ~ ~ 0 0 h 0 0 ~ h I h
5) O h a~ X ~ 10 ~ h OA O 1~ ~ .q u~
0 ~ 1 1 0 0 O X ~ Oh I
t) R h rl C) ~-- I ,1 I O ~ ~ I R
O Q~ I ~1 ~ ~ ~ X It) ::~ ~ O O ~
~ X ~ o ~r X ~ I ~ ~ O 1~I X ~ X I X ~- ~ ~ X
--~ O X ~ ~ O I oI R ~1 ~1 ~ O I O er O ~ I ~ I O
o a) I IJ ~` ~. Ctl O ~ ` -' ~ ~ I ~ ~ O
a 1~ ~ R I ~ _I o s ~1~ R --I I ~ A 'a~1 ~ O --I R
~ 111 ~ -- h Q) ~
H D. S~rl S ~rl S r1 ~ ) S ~ S S ~--I
H ~o Q~ ~ h l~h X ~ Q~h ~ ~1
X~ ~ ~ ~ O
1:: O h X :~ X ~ X :~ ~ ~1 X :>~X--' X ::~
f~ ~/ ~ OS 0~ 0~: ~ OS 0~1 OS
o ~ ,, ~ ~ ~ ~ o ~ ~ ~ ~ a) a) ~
u ~ ~ u ~ u al u ~ s ~ ~ u Q~ s~a ~ u
U~r~ U~rl C)~r~ I ~'~1 U'~1U ~'~1 U'~
0 ~ h 'r ~ h (d ~1 ~I h 1~ h
--~ ~ S -- ~ 1 ~ ~ S ~ ~ S -- :~
O O ~ ~ O
0 ~)
H O a~ 1 u
~ o o a~
C~ ~ l ~ ~ O ~ 'rl ~ ~ ~
U ~ ~ O 0 h O
3 ~ o e ~ O ~ O O ~
~1 ~ U h h h ~ ~: O
O I h
U h U O 0 d La u~
,~
1-- 00 ~ O ~ ~ ~ ~r

:~ii8409
RB50a
27
I I~1 ~ I
~ ~o a~
~ ~U7 ~ o
S ~ S ~
~
X O
o s~I ~ o
H ~ X
H S ~i~1 0
H
.C.C ~1 e~' I
I ~
:~ ~ E~ E~ C (~ C:
I I~a) I ~
O ~ X ~ ~ .
~) Xl- O O X
O--I~ X O
.4 1~ O R I
X-- I U X
l Ol ^ l O
I ~~ O I ~
~L O ~ I aJ I ~
r~ X ~ CQ S O --I X
~1 0 o ~ ~ _I O
,~
,
c ~ a~
a) Y~ ~s
~ ~1Q. 10 _-,1
H
H :>1~X O X ~
X -10~1 0 ~1
~ O,S~ S
O ~U U~ U U'
S~
O ~ ~
H ~ ~ o 0
O O
~1 .C o ~ I t~
O ~ ~ 0 25 o
C)
~ ~ ~ .