Note: Descriptions are shown in the official language in which they were submitted.
1 HALOGEN LABELED COMPOUNDS INCLUDING
ESTRADIOL DERIVATIVES, ~YNl~r-l-IC INTERMEDIATES,
SYNTHESES THEREOF AND USES THEREOF
Background of the Invention
This invention relates to estradiol derivatives, their
syntheses, the preparation of their synthetic intermediates
or precursors and utilization thereof as tissue imaging
agents. In particular, this invention relates to the
preparation of certain substituted estradiols which have a
specific, preferred sterochemistry and their uses in
detecting tumor sites having high concentrations of
estradiol receptors.
The term "estradiol", as used herein, refers to
compounds having the following general structural formula:
18 011
1~
~16
~o 7
Estradiols similar to the compound I are, generically,
estra-1,3,5(10)-triene-3,17-diols. In the above drawing the
carbons in the steroid structure are numbered according to
the generally recognized nomenclature system for steroids,
~38l~
1 with the hydroxy substituents located at the 3- and 17-
positions. It will be understood that the substituent at
the 17 position may have either of two orientations,
referred to as alpha and beta. Generally, an alpha
substituent is one which projects beneath the plane of the
drawing shown above and a beta substituent is one which
projects above the plane of the steroidal ring drawing. The
same would be true for substituents located at C-16.
Substance II shown below is a form of estradiol
generally medically recognized to be of particular
importance and has a 17-hydroxy-substituent located beta.
A commonly used name for this substance is estra-1,3,5(10)-
triene-3,17-beta-diol (17-beta-estradiol or beta-
estradiol).
HO~J
II
Estradiols are naturally occuring substances whose
derivatives have been found to have medicinal use. For
example, the 3-methyl ether has been used for replacement
therapy in estrogen deficiency. Also, certain radioactively
labeled estradiols may be used in estrogen receptor assays.
In particular, the tritiated, iodine-125(I-125) and bromine-
77(Br-77) labeled substances have been tested.
It is known that estrogen receptors, i.e. binding
13 3
1 substrates for estradiols, may be found in certain Ani
tissues. It has also been found that the presence of
estrogen receptors may be connected with certain
abnormalities in the tissue. Estradiols, if properly
labeled, may be utilized to detect the presence of these
estrogen receptors in tissue. The medical profession
generally theorizes that these estrogen receptor analyses
may be conducted in vitro or in vivo. Further,
applicants foresee that appropriately labeled steroids may
be utilized to deliver a radioactive nucleus or a particular
reactive entity to a site of abnormality in tissue, in order
to promote a therapeutic effect or for diagnostic purposes.
For example, a reactive entity which would be selectively
carried to a particular type of cells and which wouid
destroy or modify such cells.
In particular, certain 17-beta-estradiols, which have
been substituted at the 16-position, are generally thought
to have an affinity for estrogen receptors which is both
significant and useful in performing estrogen receptor
analyses, including assays and imaging. It is foreseen that
numerous 16-substituted-17-beta-estradiols may be of
importance, particularly those in which the 16-substituent
is a halogen and most importantly when the halogen is
radioactive. It is foreseen that both 16-alpha- and 16-
beta-substituted-estradiols may be of use. However, for
any given substituent, the affinity of the 16-alpha-
substituted-17-beta-estradiol, for estrogen receptors,
is likely to differ from that for the analogous 16-beta-
substituted-17-betaestradiol.
Since both the 16-alpha-substituted and 16-beta-
- I 33~
1 substituted-17-beta-estradiols are foreseen to have
utility, it is prefered that methods of ~yntheses of each be
developed. It is preferable that each synthetic scheme
yield a desired isomer substantially stereospecifically, so
that problems of purification and problems from the
differences of affinity of the two isomers for binding
sites, are avoided. Thus, two general synthetic schemes are
needed, one which provides 16-alpha-substituted compound
with very little 16-beta-substituted compound being
present, and a second general reaction methodology which
yields 16-beta-substituted compound with very little 16-
alpha-substituted compound being present.
It is readily seen that it would be most desirable to
develop a single synthetic precursor or intermediate from
which either the 16-alpha-or the 16-beta-substituted
compound--~an-be--relatively very rapidly and easily formed.
That is, given a supply of the synthetic intermediate a
synthesis laboratory could easily prepare whichever 16-
substituted compound is desired. It is particularly
desirable to have alternate synthetic schemes which are
relatively easy to conduct and which are both very rapid-and- -
~very efficient and which produce a relatively high
percentage of the desired final product.
With present technology, two basic methods of detection
of labeled estradiols are most available. In one, a
radioactive substituent is introduced into the molecule and
standard methods of radioisotope detection are utilized to
determine the presence of the labeled estradiol in the
animal tissue, either in vitro or in vivo. In the
other, nuclear magnetic resonance (NMR) methods are utilized
1~3~
1 to detect certain nucleii, and generally non-radioactive
labels may be used. At the present time, only radioisotopic
techniques are widely available but it is foreseen that
other methods may become more available in the future.
If a radioactive isotope is used as the label, then the
stereospecificity of the reactions leading to the synthesis
of the 16-substituted-17-beta-estradiol may be critical.
.
Also, efficiency of the reaction, in terms of product yield
and the length of time it takes to introduce the radioactive
isotope into the molecule and then isolate the desired
product for diagnostic or therapeutic use, may be very
important.
The importance of the stereospecificity is easily
understood. Radioactive labels are very expensive and if
the reaction is not sufficiently stereospecific large
amounts of the label may be lost in undesired products.
Also, if the undesired products are to be discarded there
may be problems with dangerous residual waste-product
radioactivity. Finally, side products might not be easily
separable from the desired isomer, and they can-interfere
with the certainty of assay and imaging data collected when
the product is used in medicinal analyses.
If the product yields are not sufficiently high, much
of the radioactive isotope may not be incorporated into a
useful product, again wasting expensive isotope.
If the reactions involved in the introduction of the
radioisotope into the molecule, when coupled with any
further reactions or purifications necessary to isolate the
desired radioactive products, are not sufficiently rapid,
special problems may be encountered. It will be understood
133~1 81
1 that the radioactive label is constantly decaying: and, if
the isotope has a sufficiently short half-life, it must be
introduced into the molecule rapidly, and the compound must
be isolated for biological use relatively rapidly, or the
isotope will have passed through sufficient half-lives to
produce so little "hot" or radioactive substrate that
detection may be difficult.
In some instances the radioisotope used may be
contaminated with small amounts of other radioisotopes of
the same compound. For example, Iodine-123 (I-123) as it is
currently made, is often contaminated with some Iodine-124
(I-124). The half-life for I-123 is about 13.3 hours
whereas the half-life for I-124 is about 4.2 days. I-123 is
readily detectable and gives clear images whereas I-124
generally causes some scattering and images of low
resolution. Consider what happens if a mixture of 99 to 1,
I-123 to I-124, is utilized to label a substrate. If the
reaction takes too long, for example 26 hours, for
introduction of the isotope into the substrate molecule and
isolation of the desired product, then the I-123 will pass
through about two half-lives and only about 25% of it will
be left. The I-124, however, will have barely begun
decaying and nearly 100% of it will be left. The ratio of
I-123 to I-124, after the 26 hours, will have changed to
approximately 24 to 1. It is readily seen that this
enhancement of the amount of I-124 present, by ratio, may
cause difficulty since the I-124 might make resolution of
images difficult. Also, if much of the isotope mixture must
be given to accommodate imaging of I-123, residual
radioactivity from the I-124 component, with its long half-
, 3 ~
1 life, may be a problem. These types of problems are usuallypresent whenever an isotope of short half-life is used, if
the isotope is normally contaminated by a second isotope of
longer half-life.
In some instances, the decayed product may still be
active as far-as an estrogen receptor is concerned and the
labeled, but no longer hot, estradiol derivative may block
estrogen receptors from receiving the hot substrate, thus
interfering with the accuracy of any assay or imaging data
obtained. This problem cannot be overcome by using an
excess of the estradiol material since doing so may tend to
overload the estrogen receptors and send hot estradiol to
other locations, where it may be detected, generating
erroneous conclusions about the presence of estrogen
receptors.
When non-radioactive isotopes are used in chemical
syntheses, problems of low yield and low specificity are
often overcome by utilizing large amounts of starting
materials, and labels, and undergoing sufficient
purifications to allow for the isolation of significant
amounts of the desired products. It is clear that this
methodology is generally unacceptable when radioactive
labels are used. First, radioactive labels are usually too
expensive for an inefficient synthesis scheme to be
commercially utilizable. Secondly, radioactive isotopes can
be dangerous and large concentrations of them should be
avoided. Also, unreacted starting materials and
undesired side products may be radioactive, causing problems
with contamination during clean-up, isolation and waste
material disposal. Further, if the isolation of the desired
l 3 ~
1 product takes too long there may be problem~ with decay of
the isotope.
Radioactive isotopes of the halogens are generally
considered to be the most important types of labels for use
in labeling compounds for biological assays and imaging.
The isotopes generally considered to be of most importance
are fluorine-18 (F-18), bromine-77 (Br-77), iodine-123 (I-
123) and iodine-125 (I-125). Fluorine-18 is a positron
emitter and has a half-life of approximately 110 minutes
with a relatively high energy of decay. Detectors for
positron emission are not widely available at the present
time, so, while fluorine-18 may in the future become widely
used as an isotope label, its potential has yet to be
completely realized. It is foreseen, however, that
fluorine-18 compounds may be made-according to the present
invention, so as to provide the compound when the demand
therefor increases.
Bromine-77 decays with gamma emission and has a half-
life of approximately 56 hours.
- 20 Iodine-123 is a gamma emitter having a half-life of
approximately 13.3 hours. Its energy of gamma decay i~
relatively high, approximately 159 kilo-electron-volts
(KeV). The toxicity of iodine is generally well understood,
and I-123 is generally considered to be an almost ideal
radioisotope for use in biological studies. In particular,
its relatively short half-life makes radioactivity
contamination a relatively minor problem, while at the same
time, its relatively high energy of decay makes detection
relatively easy, even in vivo.
Iodine-125 has a half-life of approximately 60 days and
1338187
1 is a gamma emitter having a gamma decay energy of
approximately 35.48 KeV. Its relatively long half-life
makes it undesirable for many uses since there will be
residual radioactivity for a considerable period of time.
Its relatively low gamma emission energy makes it hard to
detect, with most instruments, especially when used in
vivo, although even in vitro detection can be a problem.
While there are many other radioisotopes of the
halogens, the above discussed radioisotopes are the ones
most generally considered for use in diagnostic medicine.
Of them, I-123 is presently considered to be the most
generally desirable and practical isotope for use in
labeling estradiols.
In the past, 17-beta-estradiols labeled with I-123 at
the 16-position have been unavailable in amounts and
purities generally considered to be useful in assays, and
other diagnostic work, either in vitro or in vivo, due
to problems in their syntheses. Generally, these problems
result from the length of time formerly required to
introduce an I-123 label into the 16-position,
stereospecifically, and the length of time required to
complete the synthesis and isolate and purify the desired
product. In addition, those synthetic methodologies which
were considered in the past were often of low yield and
often resulted in the waste of large amounts of I-123
label.
The advantages of the present invention, in preparing
labeled compounds, will be most apparent if an examination
is first made of previously known methods of introducing
halo-substituents into the 16-position of 17-beta-
13~8187
1 estradiol. Such an examination, of the major known methods,follows:
A highly publicized method of preparing 16-alpha-
halo-substituted-17-beta-estradiol is that published by R.
B. Hochberg and will be generally referred to as Hochberg's
method or synthesis. The final step of Hochberg's synthesis
is shown below and comprises Iodo-substitution on the 16-
beta-bromo-compound, i.e. a Finkelstein reaction:
0~ Of I
H~ Br Ho~```I
In this spcification and the claims: a wedge indicates
a substituent projecting above the plane of the drawing; a
dotted line indicates projection below; and, a curve
indicates a mixture of both. These are conventional
methods of indicating stereochemistry. Also, Ac is used to
indicate an acetyl group, -C(O)CH3.
The substitution reaction is generally run in 2-
butanone for anywhere from 12-to 24 hours. Although the
reaction has been utilized to introduce the radioisotope I-
125 into the 16-alpha position of 17-beta-estradiol, it
is generally considered to be of too low a yield and too
long a length of time to allow efficient production of a
compound by the substitution of a radioisotope, such as I-
123, which has a relatively short half-life.
Even if conditions are found which allow for an
increase in the rate of the substitution reaction, it
appear~ unlikely that the synthesis and purification method
proposed by Hochberg can be readily and economically
1 ~
1338187
1 utilized to prepare commercially useful 16-alpha-
substituted radioactive estradiol derivatives when the
radioactive isotope has a very short half-life. For
example, the Hochberg synthesis may require time-consuming
product isolations and purifications. Further, the reaction
does not appear to be adaptable to substantially
stereospecific preparation of 16-beta-substituted
compound.
The following scheme shows the overall Hochberg method
10 of synthesis: . /1
HO
O
.- , .
O~t OH
Ho~ f~o~_~r
se~*On
9 '
01
~Br
q 2 ~ ~tltanonC
OH
~`"I
1 1 '
1~38187
1 There are certain problems with the above reaction
scheme. For example, the bromination step yields two
compounds, mostly the beta form, and the reduction step
gives a mixture of all four possible bromohydrins, from
which the desired isomer has to be isolated. No single
intermediate is formed from which the 16-alpha-substituted
compound can be rapidly formed and from which the 16-beta-
substituted compound can also be rapidly formed. Also, the
starting material for the final Finkelstein, 16-beta-Br-
17-beta-estradiol, and the product of the final
substitution, 16-alpha-I-17-beta-estradiol, have similar
characteristics for chromatographic purposes, so their clean
separation from one another, in the event that the final
substitution does not go to completion, can be difficult.
Further, during the Finkelstein reaction and work-up there
may be epimerization of starting material or product, thus
decreasing efficiency. Also, one epimerization product, 16-
alpha-Br-17-beta-estradiol, has very similar
chromatographic properties to the desired product, making
purification somewhat difficult.
Accordingly, researchers' initial attempts to form 16-
alpha-125I-17-beta-estradiol via the Hochberg method
resulted in products having specific activities which were
very low, approximately 95 to 140 Curies per millimole
(Ci/mmole), as opposed to the theoretical specific activity
of approximately 2,000 Ci/mmole. It is reported in the
literature that meticulous purification of the 16-beta-Br-
17-beta-estradiol used in the Finkelstein reaction has
resulted in some increased specific activity; however,
purity of products still appears to be a problem and seems
1338187
1 to keep specific activity down.
Another, practical, problem is asso~iated with
Hochberg's method. Most radioactive halogen anions are
commercially available in the form of an ammonium salt or a
sodium salt. In the case of radioactive iodides both
ammonium salts and sodium salts are usually available
whereas, in the case of the bromides and fluorides, usually
only the sodium salt is available. These radioisotopes are
normally shipped in water, which does not readily evaporate,
and significant amounts of base, either sodium hydroxide or
ammonium hydroxide, will be present. The Finkelstein
reaction, and its starting material, may be sensitive to the
presence of either base or water. Therefore, the
commercially available isotope, in its basic storage
mixture, usually must be scrupulously neutralized and dried
before it can be utilized. It is evident that it would be
desirable ~o develop a reaction methadology which is at
least relatively insensitive to the presence of water and
preferably can tolerate base.
An alternative method of labeling 17-beta-estradiol
with halogens at the 16-position has been developed and
utilized by Katzenellenbogen et al. Their general reaction
methodology is shown below:
~c 0 2q- X ~R~Ac O
d j ~ à ~ x
R 2 2d . x - cl; R-f~ RO~V--4Q X--Br3 R
4 4~ x c c~; R=H
~ - J /
~ 3b . x r Br j R = J~; R '= Ot~ ,~,H
3c~.X=B~ja~RcY,~ J X 1 J~Br
,~3c.X=Cl,R~ R'=tt ~ `~
Ho~ 3d. X ' Cl ;R ~, H~J
13
- 1~38187
1 Katzenellenbogen et al. J. Med. Chem., 23, p. 994-
1002(1980) describe bromination of the enol acetate (1)
followed by hydrolysis to give 16-alpha-Br-3-hydroxy-
1,3,5(10)-triene-17-one(16-alpha-Br-estrone; (2b) as
proceding with relatively high yield and relatively high
specificity for the alpha-product (2b). Formation of the
16-beta-Br-compound, 4a, was accomplished by epimerization
of the 16-alpha-compound (2b) in acid. The epimeric ratio
was 1/1.8 (2b/4a). In commercial use, such a mixture would
have to be separated by chromatography and clean separation
can be expected to be difficult and time-consuming.
16-alpha-Br-17-beta-estradiol (3a) was formed from
reduction of the ketone (2a) with lithium aluminum hydride.
(LiAlH4). The reduction yields both the 17-alpha-and
17-beta-alcohols (3a and 3b) in a ratio of 2/1
(17-beta/17-alpha; 3a/3b). Such a mixture can, in
theory, be separated by chromatography; however, again,
since the products are so similar, chromatographic
separation may be expected to be difficult and time-
consuming~
Katzenellenbogen prepares Hochberg'~ precursor,compound 5 above, by reduction of a mixture of 16-alpha-
and 16-beta-Br-estrones with zinc borohydride (ZnBH4).
All four bromohydrins are formed from such a reduction,
since the reduction is not stereospecific. Again, although
chromatographic means may in theory be utilzed to separate
the four bromohydrins, they are similar enough in properties
so that the separation can be expected to be very
difficult.
16-alpha-Cl-estrone (2c) was formed by
14
- 1338187
1 Katenellenbogen et al., by treating the enol acetate (1)
with tert-butyl hypochlorite. A mixture of products is
generally formed from such a reaction so some purification
is necessary for isolation of the chloride.
16-alpha-Cl-17-beta-estradiol (3c) and 16-alpha-
Cl-17-alpha-estradiol (3d) are formed from treatment of
the estrone (2c) with LiAlH4. Isolation of either product
requires purification generally by chromatography, which can
be difficult with such similar reaction products.
It may be appreciated from the above that the variation
of the Hochberg method utilized by Katzenellenbogen et al.
is generally inefficient for 16-substituted compounds,
especially when radioisotopes are being used. First, in
nearly all instances, product mixtures are formed, which
wastes expensive label and which can be hard to separate.
Also, too much time may be needed to easily handle
radioisotopes of relatively low half-lives.
Longcope et al. have developed a method of synthesis
for 16-beta-I-17-beta-estradiol which is significantly
different from Hochberg's synthesis and the Katzenellenbogen
variations. Their synthesis begins with 16-alpha-17-
beta-estriol and is shown below:
triphenylphosphite methiodide
~ '`o~ C~ tetrahydrofuran
H
mixture including- OH
Ho ~ H
133818~
1 The Longcope method yields a product mixture which
includes the desired beta-product together with an
elimination product, 80, again, it is inefficient.
Secondly, it cannot be readily adapted for formation of 16-
alpha products. Also, radioactive triphenylphosphite
methiodide is expected to be difficult to prepare. In
addition, it does not appear to be easily adaptable to the
utilization of other halogens and their radioactive
isotopes.
The above three general synthetic methodologies
illustrate many of the problems associated with syntheses of
16-substituted, either alpha- or beta-, 17-beta-
- estradiols. Generally, reaction mixtures including numerous
products-are formed. Also, no synthesis is readily
adaptable to yield, stereospecifically, either the 16-
alpha or the-16-beta-isomer as--desired.- Also, the
lengths of time required for the introduction of halogen
label and isolation of the products tend to make the
utilization of the above schemes for radioactive isotopes,
especially isotopes with relatively short half-lives, very
difficult, if not impossible.
Medical science has often found the use of non-invasive
techniques to study various tissues within a body to be very
helpful in diagnosis of various diseases or injuries. ~or
this purpose, various detectable materials have been
developed over the years which are designed to be applied to
living tissue to be studied, so as to provide an image of
the tissue outside of the body. Preferably, such materials
bind with the particular tissue to be studied. It has also
been found that such imaging materials often help in
l~
v~1~381~7
1 studying tissues outside of a body, as for example in assays
in which an amount of binding site present is measured.
Thus, the detectable materials are often usable for both in
vivo and in vitro studies.
Various detectable materials are imageable or
detectable in different ways. For example, some materials
that have been suggested for imaging or assay purposes
release radiation which can be detected. Other materials
are suitable for detection by nuclear magnetic resonance
(NMR) devices or the like.
One of the major limitations on materials used for
detection within living animals is that the material should
be relatively benign to the animal host. Therefore,
although high concentrations of strongly radioactive
materials may often provide a good imaging source, they are
not generally compatible with use in living tissue. In
addition, where a carrier mechanism is utilized to
preferentially carry the detectable material to a particular
tissue of interest, certain labels may render the carrier
- 20 generally ineffective in preferentially binding to that
particular tissue or the receptors therein to be studied.
Steroids have been heavily studied for use as carriers
for detectable materials. One particularly effective group
of steroids has been estrogen and its derivatives, which
seek out organs in the body having estrogen receptors,
including such organs as the ovaries, cervix, uterus,
breast, and brain. Suitable estrogen receptors are also
sometimes present in some types of cancerous lung tissue, as
well as skin, bones, and testes.
Various halogens have often been suggested as effective
_ 17
1338187
. .
1 detectable agents, since the halogens often are readily
combinable with estrogen. While certain combination~ of
estrogens and halides may affect the binding ability of an
estrogen carrier,-certain other combinations do not
substantially modify the binding ability of the estrogen
with estrogen receptors. One particular type of halogen-
that has been proposed as a label is radioactive iodine.
For example, Hochberg has developed a process to make
estrogens labled with iodine 125.
Problems have arisen, however, with the use of iodine,
even though the affect of iodine and iodinated steroids in
animals has been heavily studied. One of the major problems
associated with the use of certain common radioactive
isotopes of iodine has been that they often do not have
sufficient energy associated with their radioactive decay to
substantially penetrate the animal body, when used in
vivo, and consequently do not provide clear images on a
detector. In addition, the readily available radioactive
isotope 125I has a fairly long half-life and it is
generally not desirable to place long half-life
radioisotopes within an animal body, since damage from
radioactivity is more likely to occur with the longer lived
isotopes. Also, a greater amount of the radioactive iodine
may need to be injected when the half-life is long, in order
to provide enough decay to be readily detectable.
Iodine isotope I-123 substantially overcomes the
problem with the long half-life, since it has a half-life of
only approximately 13.3 hours; and, further has a fairly
high energy gamma radiation decay, generally making it
readily detectable. In addition, it is found that images
.. ~ hl 3~8 1 8~
1 produced by 123I decay are generally sharp or clear.
Unfortunately, the short half-life of 123I has effectively
prevented previous use of 123I-iodinated estradiols, since
conventional methods of producing them have typically taken
between 20 to 40 hours, conventional methods of synthesis
generally produce mixtures, and conventional syntheses have
generally led to relatively low yields. Thus, by the end of
the production of the 123I-iodonated estradiol, a
substantial number of half-lives may have already run and,
especially if the yield is relatively low, the iodinated
product may not be generally readily utilizable for imaging
or assays.
With general availability of the estradiol effectively
iodonated with the I-123 isotope, there has been a desire to
produce imaging methods for uti-lization of the I-123
Objects of the Invention
The principal objects of the present invention are: to
provide a method of rapidly introducing a radioactive
halogen into an organic molecule, especially steroids and
steroid derivatives; to provide a general method of
preparation of 16-halo-substituted-17-beta-estradiols; to
provide such a method by which radioactive substituents, of
relatively short half-lives, can be quickly and efficiently
introduced into the 16-position of 17-beta-estradiols; to
provide such a method by which substitution of the
substituent into the 16-position of 17-beta-estradiols can
be made either alpha or beta as desired, either
substitution being made with nearly complete
-- 1338187
1 stereospecificity; to provide a synthetic intermediate for
use in such a method of synthesis from which either 16-
alpha-substituted-,or, 16-beta-substituted-17-beta-
estradiols can be relatively quickly, stereospecifically and
efficiently derived: to provide a method by which 16-
alpha-123I-17-beta-estradiol, having a relatively high
specific activity, suitable for use in estrogen receptor
assays and imaging studies can be quickly, efficiently, and
easily manufactured; to provide a method of synthesis by
which 16-beta-123I-17-beta-estradiol, having a
relatively high specific activity and being suitable for use
in estrogen receptor assays and imaging studies can be
relatively quickly, stereospecifically and efficiently
manufactured; to provide the products of such syntheses; to
provide the specific product 16-alpha-123I-17-beta-
estradiol having a specific activity of at least 5,000
Ci/mmole and being collected in sufficient amounts for
utilization in estrogen receptor assays and imaging studies;
to provide the specific product 16-beta-123I-17-beta-
estradiol to provide such methods of syntheses which are
relatively easy to utilize, are economical and which are
particularly well suited for their proposed usages; to
provide a method of imaging or treating tissue, especially
imaging selected animal body tissue as compared to other
tissue within the animal; to provide such a method utilizing
labeled steroids which are selectively targeted for body
tissue having receptors for such steroids; to provide such a
method wherein the steroid is an estrogen carrier suitably
preferential for binding with various estrogen receptors in
selective body tissue; to provide such a method wherein the
~ h 1 33`81~87
1 label is a halogen, in particular, radioactive iodine-123:
to provide such a method which allows assay or imaging of
tissue both in vitro and _ vivo; to provide such a
method utilizing an 123I estradiol composition, which has
a relatively high specific activity and which i8
sufficiently concentrated to permit use of sufficient
radioactive iodine to provide suitable imaging, and yet not
have a substantial amount of non-radioactive estradiol
present; and to provide such a method which is easy to use
and particularly well adapted for the proposed usage
thereof.
According to the present invention there is provided a
process for the production of a 16-halo-17-beta-estradiol
comprising the step of: (a) reacting a compound having the
following general formula:
~OR~
R'o~J
wherein 0RII is an ester leaving group moiety of
relatively low nucleophilicity;
R is selected from a group consisting essentially
of hydrogen and hydroxy protecting moieties and
R' is selected from a group consisting essentially
of hydrogen and hydroxy protecting moieties,
with a source of halide ion such that said halide ion
is selected from the group consisting essentially of
~I
1~8187
1 bromine, chlorine, fluorine and iodine ions and
mixture~ thereof, until substantial substitution of
said -OR 11 by said halide ion occurs.
Also according to the present invention there is
provided a composition comprising 16-beta-123I-17-beta
estradiol.
Further according to the present invention there i8
provided a composition comprising 16-alpha-123I-17-
beta-estradiol having a specific activity at time of
production of greater than 2,000 Ci/mmole.
Further according to the present invention there is
provided a pharmaceutical composition comprising at least
0.5 millicuries of 16-123I-17-beta-estradiol having a
specific activity of greater than 2,000 Ci/mmole at time of
production.
Further according to the present invention there is
provided a process for preparing of iodo-substituted organic
compounds comprising a step of reacting an organic compound,
RX, with a source of iodide ions, I-, to form a compound RI;
wherein, (a) X is a triflate leaving group having a formula:
-OSO2CF3 (b) R is an organic substrate having ability to
undergo nucleophilic substitution at a carbon substituted by
X; and, R is otherwise generally insensitive to iodide
ions.
Further according to the present invention there is
provided a process for preparing 16-alpha-halo-
substituted-17-beta-estradiol compounds having a general
structural formula:
'~ h 1 ~ 3 8 1'~7
,~"Y
R'O
wherein:
Y is a halide substituent selected from a group
consisting essentially of fluorine, chlorine,
bromine and iodine;
R is a non-beta directing substituent selected
from a group consisting essentially of hydrogen(H)
and non-beta directing hydroxy protecting
moities; and
Rl is a substituent selected from a group
consisting essentially of hydrogen ion (H) and
hydroxy protecting moities;
said process comprising the steps of: (a) reacting a
compound having a general structural formula:
0~ ,
~_OR.
R~OJ~J
wherein ORll is an ester leaving group
moiety of low nucleophilicity,
with a source of halide ions, said halide ions
being selected from a group consisting essentially
of anions of fluorine, chlorine, bromine, iodine
and mixtures thereof, and (b) continuing said
~ 3
''h~338~87
1 reacting until substantial substituion of said halide
ions for said -ORll moiety occurs, with inversion of
stereochemistry.
Further according to the present invention there is
provided a process for preparing 16-beta-halo-substituted-
17-beta-estradiol compounds having a general formula:
0~ ~
,~Y
~'
wherein:
Y is a halide substituent selected from a group
consisting essentially of fluorine, chlorine,
bromine and iodine;
Rl is a substituent selected from a group
consisting essentially of hydrogen ion (H), and
hydroxy protecting groups; and
Rlll is a beta directing hydroxy protecting
group;
said process comprising the steps of: (a) reacting a
compound having a general structural formula: -
~Of~v
R'O
-
1338187
wherein:
0RIV is an ester leaving group moiety of low
nucleophilicity and beta-substitution
potential,
with a source of halide ions, said halide ions
being selected from a group consisting essentially
of anions of fluorine, chlorine, bromine, iodine
and mixtures thereof; and (b) continuing said
reacting until substantial substitution of said halide ions
for said 0RIV moiety occurs to produce a product with
retention of stereochemistry.
Further according to the present invention there i~
provided a process for the preparing 16-alpha-halo-
substituted 17-beta-estradiols, said process comprising
the steps of: (a) hydrolyzing, with acid, a protected
compound having the following general formula:
~OSO2C~3
~'
wherein:
RV is a hydroxy protecting group, and, RVI is
a hydroxy protecting group,
until said hydrolyzing converts said protected
~S
1338187
1 compound to the following diol:
~~t '
OzCF3
~0
(b) reacting said diol with a source of halide ions, (i)
said halide ions being selected from a group consisting
essentially of anions of fluorine, chlorine, bromine, iodine
and mixtures thereof; and, (c) continuing said reacting
until substantial substitution of said halide ions for said
-OS02CF3 moiety occurs, with inversion of
stereochemistry.
Further according to the present invention is provided
a radioactive composition for use in diagnostic and
therapeutic medicine, said composition including: (a) a
radioactive estradiol component comprising 16-alpha-
123I-17-beta-estradiol; (i) said estradiol component
having a specific activity of substantially greater than
2,000 curies per millimole.
Further according to the present invention is provided
a radioactive composition for use in diagnostic medicine,
said composition including: (a) a radioactive estradiol
component comprising 16-beta-123I-17-beta-estradiol.
Further according to the present invention is provided
a process for preparing an intermediate for use in preparing
16-halo-substituted-17-beta-estradiol derivatives, said
intermediate having the following general formula:
26
-
1338187
c~YIl
~ OR~X
RVI
wherein:
-RVII comprises a beta-directing, hydroxy-
protecting substituent;
-RVIII comprises a hydroxy-protecting
substituent and
-0RIX comprises an ester leaving group moiety of
low neucleophilicity;
said process comprising the steps of: (a) reducing a
ketone having the following general structural formula, with
a hydride source,
C~R~IJ
''~0
.. - ,~r
.
Rvllb
to form the following alcohol, after aqueous work-
up:
~7
1338187
O~?~J
(b) thereafter, reacting said alcohol with an acid anhydride
of the general formula (RXo)2o~ in solution including a
base of low nucleophilicity, wherein RXO- is an ester
leaving-group of low nucleophilicity, to form an ester of
the following general formula:
- OR
~'
O~X
01 J
0~
- 1338187
. .
1 ~ Further according to the present invention is provided
a process for preparing an intermediate for use in making
16-halo-substituted-17-beta-estradiol derivatives, said
intermediate having the following general formula:
ORVII
~ORX
RVJU
- wherein:
-RVll comprises a beta-directing, hydroxy-
protecting substituent
-RVlll comprises a hydroxy-protecting
substituent and
_ORX comprises an ester leaving group moiety of
low nucloephilicity:
said process comprising the steps of: (a) oxidizing,
by reaction with an oxidizing reagent, the following
enolate:
ORxl
ie.X~o~
wherein:
~q
1338187
1 RXl is an enolate hydroxy protecting group of
expoxide rearranging proclivity: and RXll i8 a
hydroxy-protecting group,
so as to form the following ketone:
OR
R Xll o~S
(b) converting said ketone to:
OR
'RVII
wherein:
-RVII comprises a beta-directing,
hydroxy-protecting substituent; and
-RVII compries a hydroxy-protecting
substituent;
(c) reducing said ketone with a hydride source, to form the
following alcohol, after aqueous work-up:
ORVII
R VJI o~
~-o
1338187
(d) reacting said alcohol with an acid anhydride of the
general formula (RXo)2o~ in a solution including a base
of low nucleophilicity, to form the desired product:
1 0 . ORVII
,^~
- ~~rRX
R V/JJo~
wherein:
_ORX is a leaving group of high leaving
group potential and relatively low
nucleophilicity.
Further according to the present invention is provided
a pharmaceutical composition for imaging comprising between
0.5 and 10 millicurries of time of use of a 16-123I-17-
beta-estradiol component and said component having a
specific activity of at least 5,000 curies per millimole, in
combination with a physiologically tolerable pharmaceutical
diluent.
Further according to the present invention is provided
a process for preparing 16-alpha-123I-17-beta-
~1
-
- 1338187
1 estradiol, for use in diagnostic and therapeutic medicine,
said process comprising the steps of: (a) oxidizing, by
reaction with meta-chloroperbenzoic acid in a solution
system including NaHC03, the followng enolate:
~Ac
Ac~ - ~ .
_,
to form the following ketone:
. c~ _
AC~
(b) thereafter, reducing said ketone with NaBH4 to form
the following alcohol, after aqueous work-up:
O~c
f~cO~ OH
(c) thereafter reacting said alcohol with triflic anhydride,
32
1338187
1 (CF3S02)0, in a solution including pyridine, to form the
following triflate:
O~c
~OSO2CF3
Aco
(d) thereaftér, hydrolyzing said triflate with acid to form
- the following diol:
~ OSOz CFa
(e) thereafter reacting said diol with a souce of I-123 ions
to form 16-alpha-123I-17-beta-estradiol by
substitution of I-123 ions for triflate ions, with inversion
of stereochemistry.
Further according to the present invention is provided
a process for preparing 16-beta-123I-17-beta-
estradiol, for use in diagnostic and therapeutic medicine,
said process comprising the steps of: (a) oxidizing, by
reaction with meta-chloroperbenzoic acid in a solution
system including NaHC03, the following enolate:
1~38187
,~,3 C
Aco~
to form the following ketone:
Q~c
~o
Aco
(b) thereafter, reducing said ketone with NaBH4 to form
the following alcohol, after aqueous work-up:
~~
Acc~J
(c) thereafter, reacting said alcohol with triflic
anhydride, (CF3S02)20, in a solution including
pyridine, to form the following triflate-diacetate:
- 1338187
Q~c
~OSo2 cF3
Aco~ '
(d) thereafter, reacting said triflate-diacetate with a
source of I-123 ions to form the following iodide by overall
substitution of I-123 ions for triflate ions, with retention
of stereochemistry:
a~c
o~
Ac
and (e) thereafter hydrolyzing said iodide with acid to form
16-beta-123I-17-beta-estradiol.
Further according to the present invention is provided
a method of imaging a tissue in vivo, said tissue having
steroid receptors present therein; said method comprising
the steps of: (a) applying a labeled steroid material to
said tissue said material including a steroid labeled by a
radioactive component having a minimum specific activity of
5,000 curies per millimole and said component upon decay
releasing radiation of sufficient energy to penetrate said
body (b) allowing a portion of said labeled steroid
material to substantially join with said receptor~ in said
tissue and (c) thereafter obtaining an image of said
labeled steroid material in said tissue by utilization of
-
1338187
.
1 detection means appropriately positioned to detect said
radiation from said radioactive component.
Further according to the present invention i8 provided
a method of imaging a tissue within a human body wherein
said tissue includes estrogen receptors said method
comprising the steps of: (a) applying 123I labeled
estradiol to said tissue; said 123I labeled estradiol
having a specific activity at a time of application to said
body of at least 5,000 curies per millimole (b) allowing a
substantial portion of said 123I labeled estradiol to join
with said receptors; (c) utilizing gamma radiation detection
means outside said body to detect a location and relative
strength of radiation radiating from said tissue so as to
allow imaging thereof.
Other objects and advantages of the present invention
will become apparent from the following descriptions,
wherein are set forth by way of illustration and example
certain embodiments of the present invention. As required,
detailed embodiments and examples of the present invention
are disclosed herein. However, it is to be understood that
the disclosed embodiments and examples are merely exemplary
of the invention, which may be embodied in various forms.
Therefore, specific details disclosed herein are not to be
interpreted as limiting but rather merely as a basis of the
claims and a representative basis for teaching those skilled
in the art to variously employ the present invention.
Summary of the Invention
It will be understood that certain superscripts used
~,hl 3381 87
1 herein are also used in the Claims. The super~cripts are
generally used consistently between the specification and
Claims. Therefor, the superscripts may not appear in
numerical order in the Claims or the specification.
The present invention is directed to a new synthetic
method for preparing halogen-substituted organic compounds.
More particularly, the method is particularly suitable for
preparing 16-substituted-steroids, for example 16-
substituted-17-beta-estradiols. Specifically, a synthetic
scheme is presented which permits the introduction of
Iodine-123 (I-123) into either the 16-alpha- or 16-beta-
~ position of 17-beta-estradiol, essentially
stereospecifically, as desired. The nature of the synthesis
is such that the desired product can be isolated, in
relatively large and useful amounts, before the I-123 label,
which has a relatively short half-life of approximately 13.3
hours, has decayed to such a point that the product mixture
is no longer hot enough to be desirable for use in
biological assays. Generally, I-123 can be introduced into
17-beta-estradiol in approximately one to two hours so its
half-life of 13.3 hours is readily accommodated by the
present synthetic scheme.
Also, it is foreseen that the general synthetic scheme
developed is readily adaptable to the syntheses of other 16-
substituted-17-beta-estradiol products. For example, it
is anticipated tha 16-substituted fluorine, chlorine and
bromine derivatives can be formed. Accordingly, it is
foreseen that radioisotopes of the various halogens can be
rapidly introduced into the 16-position of 17-beta-
estradiols.
3 7
-
1338187
1 The synthetic scheme developed i~ readily adaptable
for the highly stereospecific synthesis of either 16-
alpha-halo-substituted-17-beta-estradiols or 16-beta-
halo-substituted-17-beta-estradiols. In particular, a
product solution including substantially only 16-alpha-
halo-substituted-17-beta-estradiol can be obtained. Also,
a product solution including substantially only 16-beta-
halo-substituted-17-beta-estradiol can be obtained via a
minor modification in the reaction scheme. The term
"stereospecific", as used herein, means that the product
mixture includes substantially only the desired
stereoisomer, be it 16-alpha or 16-beta.
The following reaction scheme outlines a general
synthesis of either 16-alpha-halo-substituted or 16-
beta-halo-substituted-17-beta-estradiols according to
the present invention:
3~
1338187
Co~H
HOJ~ ~
OAC
~, ~ C
AC~} ~4 ~ 5Z)2
XII XIII
- . . OAc
~52 C f:~
XIV H~
AY~ ~ o~
,~ r H~0502 CF3
XV ~X
Ht . ~-- ~ r
XVI XXI
WHEREIN: Ac is C(O)CH3
AY is a salt of a halide with:
. Y being halide ion; and A being cation
_ 3 ~
1338187
For ease of reference the intermediates and products in
the reaction scheme will be referred to, as indicated above,
beginning with Roman Numeral X.
An important step in the synthesis involves the
utilization of a synthetic intermediate XIV, a 16-beta-
triflate-estradiol in which both the 3-and i7-beta-hydroxy
groups have been protected with an acetate group. This
intermediate is relatively stable and can be easily purified
~ by high pressure liquid chromotography (HPLC). Also, it is
relatively easily characterized and is relatively stable to
the presence of water.
As shown in the reaction scheme, according to one
series of reactions, the triflate diacetate XIV is
hydrolized in acid to yield a 16-beta-triflate of 17-
beta-estradiol, XX. Following this, the diol XX is
treated with a nucleophile source, AY, to yield a product
XXI in which the halogen substituent, Y, is located alpha
on the 16-position. This latter substitution reaction
occurs relatively rapidly, as will be seen from the examples
below, and in generally high enough yield to make the
synthesis commercially utilizable. Also, the substitution,
which is believed to proceed via an SN2-type mechanism, is
generally observed to be relatively stereospecific.
Also as seen in the above reaction scheme, a 16-beta-
halo-substituted-17-beta-estradiol derivative XVI can also
be readily prepared from the fiame triflate intermediate XIV.
According to the method of the invention, if the triflate
~0
1338187
1 diacetate XIV i8 reacted with a halide anion source AY,
before hydrolysis to an estradiol, a 16-beta-halo-
substituted-17-beta-estradiol, 3,17-beta-diacetate (XV)
is formed. While the mechanism for this substitution is not
fully understood, the reaction appears to be highly
stereospecific and yields almost exclusively the 16-
beta-halo-substituted product (XV). Presumably the 17-
beta protecting group interacts with the triflate group at
the 16-beta position to cause the substitution to occur in
the manner observed.
If the diacetate product, XV, is then hydrolized, 16-
beta-halo-substituted-17-beta-estradiol, XVI is readily
isolated.
Numerous features of the above mentioned substitutions
and hydrolyses make the synthetic scheme of the present
invention highly advantageous. First, the 16-substituted
halo-compounds are readily separated from the 16-beta-
triflates, regardless of whether dihydroxy or diacetal, so
the products from the substitutions can be easily separated
from the starting material. This is due to the large
difference in chromatographic properties between triflates
and halogen compounds.
Secondly, the substitution reactions are relatively
rapid so anions of isotopes which have relatively short
half-lives can be utilized as the nucieophiles displacing-
the triflate group. In part, this is probably due to the
nature of the triflate anion as a very good leaving group
for substitution reactions.
Also, as mentioned above, the triflate group and the
halo-substitution reactions thereon have been found to be
~1
38187
1 relatively stable to the presence of water in the reaction
mixture. It was also noted above that radioisotopes are
normally only commercially available in aqueous suspension.
Thus, the insensitivity of the triflate to water means that
scrupulous drying of the isotope reagent is not necessary.
However, some drying is preferred and normally is
conducted.
It has been found that the substitutions for the
triflate group can be made to proceed more rapidly when an
appropriate crown ether is utilized with the nucleophile.
Crown ethers are macrocyclic polyethers which complex with
- certain cations. The rings may be of various size and
complex with different cations accordingly. In the case of
the substitution reactions, it is believed that the crown
ether complexes with the cation associated with the halogen
anion, thus freeing the halogen anion for the substitution
reactions.
The general source of radioactive halide ions is the
sodium halide or the amonium halide salt. It has been found
that a useful crown ether for complexing with the sodium and
amonium cations is 18-Crown-6. In the syntheses of the
radio-labeled estradiols discussed herein, the crown ether
is normally utilized in excess, since a very small amount of
the halide is present in the reaction mixture. The excess
of crown ether has not been found to cause any complications
in the syntheses. Applicants foresee that the presence of
crown ethers may facilitate many halo-substitutions at the
16-position of estradiols or in steroids generally.
Another advantage to the present reaction scheme i8
that for the halo-substitution of the triflate anion, the
- 13~8187
1 conditions are such that epimerizations of the products are
kept to a minimum. Generally, the triflate anion is a very
poor nucleophile and the conditions are such that
epimerization is unlikely. By "poor nucleophile", it is
meant that relative to the halogens, the poor nucleophile is
unlikely to attack substrate and cause multiple
substitutions. A result of this is that in addition to the
fact that the mechanisms of the substitution reactions are
highly stereospecific, to yield virtually only the desired
stereoisomer, the reaction conditions and product mixtures
are such that epimerizations, which lead to undesired
products, are generally avoided. In Finkelstein reactions,
by contrast, epimerizations of starting materials and
products can be a problem.
It is apparent that the 16-beta-triflate of
estradiol,3,17-beta-diacetate (XIV) is a very useful
intermediate for the introduction of halogens,
stereospecifically, into the 16-position of estradiol. It
is readily seen that the disclosed synthesis of this
triflate as an intermediate or precursor for the syntheses
of the desired products is very useful and important. It
will be under~tood that when radioactive substituents of
relatively short half-lives are used, the triflate
intermediate may be prepared and sent to a location near a
facility where the use of the radioactively labeled
estradiol is to take place. The halogen substitution
reaction discussed above would then be performed at this
location, the product purified, and the labeled estradiol
would then be ready for use, before the radioisotope has had
a chance to decay significantly.
~ 3
1338187
-
1 It is foreseen that certain pharmaceutical compositions
including a 16-123I-17-beta-estradiol may be used for
imaging estrogen receptor locations in mammals. It i8
foreseen that the estradiol component will generally be
introduced into the mammal by injection into the blood
stream in an amount sufficient to introduce between 0.5 and
10.0 millicurries of radioactivity. Generally, compounds
having a specific activity of at least 5,000 curries per
millimole are foreseen to be preferred. It is further
foreseen that the pharmaceutical composition will generally
comprise the radioactive component dissolved in a
physiologically tolerable pharmaceutical diluent. For
example, a mixture of ethanol and aqueous saline solution
may be used. It is foreseen that a preferred concentration
of the radioactive estradiol component in the dilutent is
between 1.0 and 10.0 millicurries per milliliter, so that
all of the radioactive compound may be injected in a
relatively small volume.
A general synthesis of a triflate, XIV, was outlined in
the reaction scheme illustrated above. The starting
material, estrone (X) is readily available and upon
treatment with isopropenyl acetate in acid readily forms
an enol acetate XI.
The next step in the synthesis, that is, the oxidation
with a peroxy acid to yield ketone XII, is both critical and
somewhat unexpected. Upon treatment with
meta-chloroperbenzoic acid, under base conditions, i.e. with
sodium bicarbonate, enolate XI was observed to form ketone
XII. This is in contrast to the chemical literature which
suggests that oxidation of enolate XI with peroxy acids will
1~38187
1 form relatively stable epoxides. It will be understood that
the isolation of ketone XII from the reaction with the
peroxy acid facilitates a simple synthesis of the desired
triflate XIV, since the oxidation yields only the 17-beta-
acetate shown.
The next step in the synthesis, the reduction of the
ketone XII to form alcohol XIII was performed with sodium
borohydride (NaBH4) and was observed to be rapid, of
relatively high yield and highly stereospecific, giving
reduction only from the alpha face to yield the 16-beta-
hydroxy compound XIII. When lithium aluminum hydride
(LiAlH4) is used as the reducing agent, a mixture of
alcohols is formed. It is believed that the reduction is
controlled by steric factors and as long as a reagent which
is primarily sensitive to steric approach to reduction is
utilized, the amount of alpha attack can be maximized and
essentially only the 16-beta-hydroxy compound isolated.
The next step in the synthesis of the triflate XIV is
its formation from the hydroxy compound XIII. This is
readily achieved with triflic anhydride, (CF3SO2)2 in
the presence of pyridine. As reported above, the triflate
XIV is relatively stable and can be easily purified by HPLC.
Thus, if any undesired side products are present from the
previous reaction steps, they can be easily separated from
the desired triflate product.
It is foreseen that the above synthesis is a
generally useful, synthetic scheme for the generally
stereospecific production of certain 16-halo-substituted-
estradiols.
Beginning with estrone (XXX), the first part of the
1338187
1 overall synthesis involves conversion to an enolate and
protection of the 3-hydroxy group. In general:
O ' c~x
HO ~ K
XXX XXXI
In the specific example discussed earlier RXI and
RXII were both acetyl groups. However, it is foreseen
that other groups may be utilized and RXI and RXII need
not be identical. For example, the 3-hydroxy group of
estrone XXX might be protected by conversion to an ether or
ester other than acetate before the 17-keto group is
converted to an enolate. What is required, generally, is
that RXII be a hydroxy-protecting group which can be
readily hydrolyzed with acid for later removal and which is
stable to the conditions of oxidation of the 17-enolate
group. It is expected that certain silyl ethers, and esters
other than acetates, may be used.
It is foreseen that under certain circumstances it may
be desirable to leave the 3-hydroxy group unprotected, in
which case RXII = H. It is foreseen that the presence of
an -OH group at the 3-position will not significantly
interfere with the later reactions, although it may be
converted to an ester, for example a triflate, when the acid
anhydride/pyridine or analogous reaction is run. This,
-
- 1~38187
1 however, is not foreseen to be a problem since whatever
ester group is placed on the 3-position is likely to be
easily removable during the final acid hydrolysis. Also,
during the hydride reduction step, if the 3-hydroxy group is
not protected, it may react with the hydride reagent,
however, it is foreseen that this should not be a
substantial problem if an excess of hydride reagent is used.
Protecting group RXI~ on the other hand, is an
enolate protecting group of epoxide rearranging proclivity.
Its general characteristics are that it can be used to trap
an enol, as an enolate, and it can be removed by treatment
with acid, once the enolate has been converted to a
protected hydroxy group as in the next step. Also for the
present purpose, group RXI~ should be able to function in
the following oxidation and rearrangement:
oRX
f~, 0~ ~ ~=
~ XXXI XXXII
The mechanism of the oxidation/rearrangement reaction
is not fully understood, however it appears to yield,
substantially stereospecifically, the 17-beta-16-keto
compound XXXII. It is foreseen that enolate protecting
., .
- 1338187
1 groups other than -C(O)CH3 may be utilized as RXI.
The oxidation, ~O], described above was conducted with
meta-chloro perbenzoic acid (m-CPBA) as the oxidizing
reagent. It is foreseen that other epoxide forming
oxidizing reagentfi may be used, especially other peroxy
acids. The conditions of the m-CPBA oxidations were
generally basic with NaHCO3, to protect the groups -ORXI
and -ORXII from hydrolysis. Generally, neutral or basic
conditions for the oxidation are preferred.
The next major step in the syntheses of 16-halo-
substituted estradiols, according to the present invention,
is the reduction, [R], of the 16-keto-3,17-beta-
diprotected substrate (XXXIII), to the 16-beta-hydroxy-
compound (XXXIV):
o~v ~ORV
lo~=O ~R~ ~_OH
XXXIII XXXIV
In most instances, the groups RV and RVI will be
the ~ame as RXI and RXII respectively. However, it is
not necessary that they be so. It is foreseen that compound
XXXII could be converted to a compound XXXIII, having a
different pair of protecting groups, through deprotection of
- - 1338187
..
1 the 3,17-beta-hydroxy groups and re-protection with
protecting groups RVI and RV. Such a conversion may be
desirable to maximize certain aspects of either the
preceding or following reactions, or to aid in the syntheses
of derivatives with functional groups, not discussed herein,
located at other positions in the substrate. It is foreseen
that RVI may be almost any protecting group which can be
readily removed by conventional acid hydrolysis techniques;
and, RV is preferably a protecting group which, in
addition, will not interfere with the direction of hydride
attack during the reduction. Also, -ORV is preferably a
relatively poor leaving group so it will not be displaced
during reduction.
As mentioned above, the reduction [R] is accomplished
with a hydride source or hydride reducing agent.
Preferably, a hydride reagent is used which gives exclusive
or generally exclusive alpha-attack to yield substantially
only the 16-beta-hydroxy compound XXXIV upon aqueous work-
up. NaBH4 has been found to work well, while LiAlH4
gives a mixture. It is believed that reducing agents which
are principally affected by steric factors will tend to
maximize alpha-hydride attack.
The next general step in the reaction sequence is
conversion of the 16-beta-hydroxy group into an
appropriate leaving group for displacement by the halide
ions:
~'O
XXXV XXXVI
4q
-
- 1338187
It will be understood that R and RI, in XXXV, need
not be identical to RV and RVI, respectively, in XXXIV,
although generally they will be. RI is preferably a
protecting group which is readily hydrolyzed with acid. R
is preferably a readily hydrolyzable protecting group which
does not interfere with the formation of -ORII. Generally
this requires -OR to be relatively non-bulky. Also, -OR, if
compound XXVI is to be used for the halogen-displacement of
-ORII at C-16, is preferably a poor enough leaving group
so that substitution at C-17 will not compete with
substitution at C-16 in the next step. It is believed that
if -OR is a carboxy group, this latter requirement will
generally be fulfilled.
On the other hand, -ORII is preferably an excellent
leaving group in substitution reactions. The currently
preferred group is a triflate group, -OS02CF3, formed
from reaction of the 16-alcohol, XXXV, with triflic
anhydride and a base, preferably pyridine. Generally, a
weak base of low nucleophilicity is preferred so that
completing substitution and elimination reactions are
minimized. In general, -ORII will usually be an ester
leaving group, generally an ester of a sulfonic acid. It is
preferred that the group -ORII not be too readily
hydrolyzable or deprotection may occur before de~ired.
If the 16-alpha-halo compound is desired, the next
step in the synthesis is as follows:
.
- 1338187
OR ~ O~ ~ -
a,o~--OR
XXX~l XXXVII
OR ' / - O~J \
R10~ or
X~X~lII XXXIX
If R is a protecting group which is non-beta
directing, that is, it will allow nucleophilic attack at C-
16 to proceed with inversion, then the substitution reaction
with AY can be conducted on the protected compound XXVI. It
will be recalled, however, that when -OR is -OC(O)CH3,
attack on XXVI, at least when -ORII is a triflate,
proceeds with retention at C-16. Under these conditions,
compound XXXVI should be hydrolyzed to deprotect at C-17 and
give XXXVII. It is expected that if a protecting group was
left on the 3-hydroxy substituent, the substitution reaction
will proceed with inversion also. However, in most
circumstances, hydrolysis at C-17 will be sufficient to
deprotect at C-3 as well.
It will be understood that differing combinations of
-OR and -ORII will yield attack with inversion. If -OR is
-OAc and -ORII is triflate, then attack with AY, where AY
S ~
1338187
1 is NH4I or NaI, proceeds with inversion at C-16.
If the 16-beta-halo-substituted compound is desired,
the reaction will be as follows:
OR ~
R R
XL XLI
In XL, -ORIII is a protected hydroxy with beta-
directing capabilities, when associated with -ORII. Such
a combination, as stated above, is when -ORIII is -OAc and
-ORII is -OS02CF3. It is foreseen that other
combinations with similar chemical features may also be
used. The group - ORIII may be a group -ORII from the
previously discussed series.
It will be understood that hydrolyses of the protected
C-3 and C-17 groups may be conducted, whenever appropriate,
for the desired diols to be formed. These will be generally
acid hydrolyses so that the halo-substituent at C-16 will be
undisturbed.
The following examples illustrate the high efficiency
and ver~atility of the present invention in application to
52
~, h ~ 338 t 87
. . ~
1 form certain 16-substituted estradiols. In addition, the
following examples are for purposes of illustration of the
invention and should not be interpreted as limiting the
scope of applicants' invention.
Experiment 1, Synthesis of the 16-beta-triflate of
Estradiol, 3,17-beta-diacetate (XIV).
A. Formation of the Enol Acetate (XI).-
O 'p~c
2504 Aco~
X - XI
A solution of 10.0 grams (g.) (37.0 millimoles) of
estrone (x) was dissolved in 70 milliliters (ml.) of
isopropenyl acetate and 10 ml. of catalyst solution. The
catalyst solution comprised 0. 2 ml. of concentrated
H2S04 dissolved in 10 ml. of isopropenyl acetate. The
reaction mixture was heated to boiling and approximately 10
ml. of distillate was taken off over a period of 0.5 hours.
An additional 30 ml. of isopropenyl acetate was added along
5~
1338187
1 with approximately 1 ml. of catalyst solution and boiling
was continued until approximately 50 ml. of distillate was
taken off. At this time another 30 ml. of isopropenyl
acetate and 1 ml. of catalyst solution was added and boiling
continued until a second 30 ml. of distillate was taken
off.
The reaction mixture was cooled to room temperature and
dilluted with 200 ml. of ether. The ether solution was
washed with cold concentrated sodium bicarbonate solution
The solution was then washed with water, dried and the
solvent was evaporated. The residue was taken up in 50 ml.
of acetone and passed through a silica gel column (500 grams
in a 5 cm. by 30 cm. column) developed with hexane acetone
4/1. Upon removal from the column, the solid was
recrystallized from ethanol (120 ml.). The product was a
yellow solid (9.88 grams, 75% yield, melting point 145 to
148 degrees centigrade). An NMR spectrum was run of the
resulting product and it was consistent with the structure
for the desired product (XI).
20-
B. Oxidation of the Enolate (XI) to Form 16-keto-
estradiol, 3,17-beta-diacetate (XII).
CO3H
Ac~ Nllll[D3 AC,,J`~
XI XII
1338187
A 0.5 millimolar aqueous sodium bicarbonate solution
was prepared and 100 ml. of the solution was added to 150
ml. of chloroform in a 500 ml. round bottom flask. The
diacetate (XII) (9.88 grams, 27.9 millimoles) was added to
the chloroform solution in one portion. After the steroid
had dissolved, 6.90 grams (40.0 millimoles) of meta-
chloroperbenzoic acid was added in one portion and thereaction mixture was stirred for lS hours at room
- temperature. The aqueous phase was then removed in a
separatory funnel and the organic phase was washed with two
50 ml. portions of 10~ sodium bisulfate solution and then
two 50 ml. portions of saturated aquèous sodium bicarbonate
solution. The organic solution was dried over sodium
sulfate, filtered, and evaporated to dryness to give a
yellow semisolid which was recrystallized from enthanol to
give a white solid (5.50 grams, 53% yield, melting point 146
to 149 degrees centigrade.) This solid was recrystallized
again from ethanol to give a white solid (3.54 grams, 34%
yield, melting point 151 to 153 degrees centigrade,
[alpha]D = 54.5 (EtOH), one spot by TLC (hexane:
acetone, 4/1, silica gel) (NMR analysis confirmed that only
isomer XII was formed.)
C. Reduction of Ketone XII to Form 16-beta-
hydroxy-17-beta-estradiol, 3,17-beta-
diacetate XIII.
~S
1338187
A't~ NaBH4 ~, ~oH
~ XII XIII
10The 16-oxo-estradiaol-3,17-diacetate (XII) (1.04
grams, 2.81 millimoles) was dissolved in 80 ml. of
- isopropanol and 35 milligrams (0.92 millimoles) of sodium
borohydride was added in one portion at room temeprature.
After 0.5 hours the reaction mixture was treated with 3
drops of concentrated hydrochloric acid and evaporated to
dryness. The residue was purified by preparative TLC
(hexane:acetone, 3:1, silica gel, eight 1,000 microplates)
with the highest Rf component eluted and isolated as a
white solid (144 milligrams, 14% yield, melting point 75 to
80 degrees centigrade; NMR in CDC13, consistent with the
above stated structure XIII).
D. Formation of Triflate XIV from Diacetate XIII.
Ac~ S o~ (Cf~s~2)~o ~050ZcF3
XIII . XIV
~G
1338187 -
Fifty-four milligrams (0.145 millimoles) of the 16-
beta-17-beta-estriol-3,17-diacetate (XIII) was dissolved
in 0.5 ml. of deutero-chloroform in a septum top vial and
21 microliters (21 milligrams, 0.260 millimoles) of pyridine
was added. The vial was cooled in an ice bath and 40
microliters (65 milligrams, 0.237 millimoles) of triflic
anhydride was added via syringe. After one hour the
reaction mixture was filtered and the filtrate purified by
~ preparative TLC (hexane:acetone, 3:1 silica gel, one 1,000
microplate) to give a colorless solid (59 milligrams, 81%
yield, melting point 50 to 58 degrees; NMR in CDC13,
consistent with the above stated structùre XIV).
The deprotected diol is readily isolated from the
diacetate by hydrolysis with concentrated HCl in tert-butanol
(t-BuOH) analogously to the hydrolysis conducted in
experiment 3 below.
Experiment 2
Preparation of 16-alpha-123I-17-beta-estradiol
The radioisotope I-123 is generally available as either
NH4123I or Nal23I. In the experiments described
below, NH4123I was utilized as the source of the iodine
isotope, however analogous experiments have been run with
Nal23I, which was found to function satisfactorily for the
present purpose. The experimental procedure for either is
1338187
1 substantially identical.
The NH4123I was received from Atomic Energy of
Canada, Ltd.(AECL) in a solution of one percent amonium
hydroxide. The volume of the solution was between 0.5 and
1.0 milliliter (ml). The container was a sealed 10 ml. vial
with a rubber septum.
The vial was placed in a lead container and equipped
with a gas and vacuum line via a 19 guage needle. A 25-
guage needle was inserted through the septum so that an air
flow through the vial could be maintained. The apparatus
was heated, at 80 degrees centigrade, until dryness was
-- obtained in the vial.
An acidic solution of t-butanol was prepared by adding
1 ml. of concentrated H2SO4 to 99 ml. of t-butanol.
A sufficient amount of the one percent H2SO4 solution
was added to the NH4123I vial to neutralize the sodium
hydroxide. This generally required the same volume of
acid solution (0.5 to 1.0 ml.) as the amount of base
solution that was originally present in the vial. Testing
paper was utilized to test the pH and the acid solution was
added until the pH paper showed the pH range to be between
6.5 and 7.5 or about neutral.
After the above, the amount of radioactivity contained
in the solution was generally between 8 and 10 millicurries
(mCi). Approximately one-half of the solution (4 to 5 mCi)
was transferred to a 0.30 ml. micro vial fitted with a cap.
200 micrograms of 16-beta-triflate-17-beta-
estradiol was dissolved in 200 microliters of t-Butanol and
transferred to the reaction vial already containing the
NH4123I. One crystal of 18-crown-6 ether (500 to 1,000
58
~hl 3 3 8 ~ 87
1 micrograms) was added to the reaction solution.
By evaporation with an air stream passing through the
vial, the reaction mixture was concentrated to a volume of
approximately 100 microliters. At this point, the vial was
sealed and heated to boiling for 90 minutes. After heating,
the vial was cooled and the contents transferred to a micro-
injection vial and injected into an HPLC. The HPLC
conditions utilized are described below.
After the product was collected from the HPLC the
solvent was evaporated by an air stream and the residue
dissolved in a 5 percent absolute ethanol/saline solution.
The solution was filtered through a 0.22 micron filter for
sterilization.
The HPLC conditions were as follows:
1. A Waters model 720 instrument was used in
association with a WISP 710B automatic injector.
2. The detector was a model 440 UV/Vis absorbence
detector with the W detector wave length being at
254 or 280 nanometers, as selected.
3. The column was a C18 RP 10 micron, 8 millimeter
internal diameter, radial packed column, available
from Waters.
4. The solvent system utilized was a 50/50
acetonitrile/water mixture with the flow rate set
at 1.0 ml. per minute.
Under the above conditions, the reaction products came
off the column after approximately 19 minutes.
When the substitution was conducted, as described
above, for between 1 and 1.5 hours, the yield of substituted
product was about 40-50% (based on measurements with non-
3381~7
1 radioactive NH4I). When the heating for substitution wasallowed to continue for an extra 2-3 hours, yields were
increased to about 70~ (again based on experiments with non-
radioactive materials).
Although limits of detection require an estimate of
specific activity of 30,000 Ci/mmole, it is expected that
the product, immediately off the column, had a specific
activity of near maximum, 237,000 Ci/mmole.
Experiment 3
- Preparation of 16-beta-123I-17-beta-estradiol
Commercially available I-123 in the form of
NH4123I, in one percent sodium hydroxide solution, was
neutralized with acid and prepared for reaction with a
substrate, in a manner as described above for preparation of
the 16-alpha isomer. The neutralized NH4123I solution
was diluted with 500 microliters of t-butanol and
approximately one-half of the solution-(4 to 5 milliCuries)
was transferred to a micro reaction vial.
Two hundred micrograms of estradiol, 16-beta-
triflate,3,~17-beta-diacetate was dissolved in 200
microliters of t-butanol and transferred to the micro
reaction vial. One crystal of 18-crown-6 ether (500 to
1,000 micrograms) was added to the reaction solution. The
reaction mixture was concentrated, by drying with an air
stream, to a volume of approximately 100 microliters, and
was heated to boiling for aproximately 90 minutes.
After substitution, 100 microliters of concentrated HCl
~ 60
1 3~8 1 87
1 was added to the solution and heating was continued for
about 60 minutes. After the hydrolysis, the reaction vial
was cooled to room temperature and the contents of the vial
were injected to the HPLC system described above and the
desired product collected from the column.
Yields of the 16-beta-isomer appeared comparable to
the 16-alpha-isomer described in Experiment 2 above. The
specific activity of the product was also comparable.
A method is provided for binding detectable labels to
receptors so as to allow imaging of their locations in
various living and non-living tissue, as compared to
- surrounding tissue. The imaging is especially effective on
living tissue and generally results in little substantial
damage to the tissue. Also, the method can be used to
determine certain features about certain tissues within a
living body without invasive surgery. The method includes
application of a detectable labeled material to the tissue
to be studied. The application may be by intravenous or
subcutaneous injection, by oral administration, or by
application to a surface, such as by vaginal douche or the
like. For detection of receptors in non-living tissue,
application may be directly to the surface thereof or on
homogenization therewith.
Preferably, the method includes the application of a
labeled steroid to the tissue such that the labeled fiteroid
becomes joined or bound to certain binding cites or
receptors in a specific tissue, in preference to other
locations in the surrounding tissue or in other parts of the
body. After binding, suitable detection or imaging
techniques can be used to analyze the presence of the
_ 61
- 1338187
1 receptors at the binding sites.
For example, estrogen-receptor-active estrogens will
generally selectively bind to that tissue which has
relatively greater quantities of such estrogen receptors.
It is noted that in certain early stages of cancer, at least
for certain types of tissue, there has been found to be a
stage in which there are more estrogen receptors present
than are present in normal, healthy tissue. The relatively
high level of estrogen receptors in these cancerous tissues
or tumors and metasteses continues until the cancer reaches
a certain stage in its development, after which the relative
- quantity of estrogen receptors therein are generally
observed to become substantially reduced. The early rapid
increase in the number of estrogen receptors, in cancerous
tissue, may allow labeled estrogen to be used to image such
cancerous tissue, or the tumors therein, to distinguish the
cancerous portion from surrounding healthy tissue. This
generally allows simple and rapid determination of the
location of the original tumor and any metasteses thereof as
well as a potentially easy determination of whether
chemotherapy-type treatment or the like is likely to be
effective in reducing or retarding additional tumor growth.
Also, the decrease or complete absence of estrogen receptors
in a known tumor may appear as a non-image, which may form
medical personnel that the tumor has undergone modification
and that a change of treatment is probably warranted.
Further, if detection is by count, rather than imaging, then
regardless of whether the detection is conducted in vivo
or in vitro, the assay performed may lead to a relatively
rapid determination of the relative amount of receptor
~ 62
~ ,i 1 3~ J ~
1 present, 80 as to aid in certain diagnoses.
It is foreseen that such imaging may be particularly
useful to image areas for determination of where surgical
removal of tissue will be desired. For exampl-e, if the
patient is diagnosed to have breast cancer, the imaging may
indicate what portion should be removed during surgery.
It is foreseen that the labeled steroid may be any of a
variety of steroids and that the label may be any of a
variety of imageable materials which can be easily detected.
For example, concentrations of estrogen receptor active
estrogens labeled with fluorine may be imaged by use of
nuclear magnetic resonance (NMR) devices which can detect
the presence of certain fluorine nucleii. Certain
radioactive halogens may also be utilized for imaging
depending on whether the imaging is to be on the surface of
the tissue or internal. In the latter case, the radiation
energy of the radioactive decay should be sufficient to
substantially penetrate the surrounding body and give clear
images on a detector.
As used with respect to the method of labelizing
described herein, the term estrogen refers to any of the
estrogen-receptor-active estrogen derivatives, and in
particular to e~tradiol. The binding affinity of the
labeled estradiol, for estrogen receptors, may vary
considerably from that for estradiol itself and still be
substantial enough for reliable detection. The 16-123I-
17-beta-estradiols have been found to be a class of
particularly effective labeled steroids. 16-alpha-
iodinated-17-beta estradiol is likely to be slightly more
effective in binding than the 16-beta-iodinated-17-beta-
_ 63
- 1338187
1 estradiol, since it has a somewhat greater affinity for
estrogen receptors, although both are fairly close in
efficiency in binding with estrogen receptor sites and it i8
foreseen that either may be effective.
Preferably, I-123-labeled estradiol used is relatively
free from by-products and precursors, immediately following
its manufacture. By having a relatively pure labeled
estrogen material, fewer non-labeled estrogens, or undesired
labeled estrogen derivatives, are present with the desired
labeled estrogens, to compete therewith for estrogen
receptor sites. If too many unlabeled estrogens are present
within the material, too few desirably labeled estrogens may
be able to bind to the estrogen receptor sites, at the
location where imaging is desired, to provide a satisfactory
image on detection equipment. Further, side products or
cont~m;n~nts may have an affinity for binding sites other
than estrogen receptors and could lead to unreliable assay
results, particularly if they cause detectable label to
appear at non-estrogen receptor sites. Further, even the
preferred 16-123I-17-beta-estradiols may have some
affinity for sites that are not desired to be imaged, and
could, in time, bind to or be captured by such sites if they
lose in-competition-for---estrogen receptor binding sites_with
the side products or contaminants.
Small doses of the labeled estrogen are also preferably
used, so that not all of the receptor sites in the body will
be occupied. A reason for this is that, if excess labeled
estrogen is present, quantities of a labeled estrogens may
circulate in the blood and effectively show as a single
image throughout the entire body, or they may partially blur
6y
1338187
1 or blind the actual tissue which is desired to be imaged.
Preferably, where a radioactive component is used, the
specific activity is relatively high 80 that a small
quantity can provide sufficient radiation for detectable
images. When I-123 estradiol is utilized a specific
activity substantially greater than 2,000 curies per
millimole and preferably at least 5,000 curies per millimole
is desired. Generally, higher specific activities are
feasible if the iodinated estradiol is utilized relatively
soon after manufacture thereof. As used herein, specific
activity refers to the radioactive decay of a quantity of
the labeled material only. Thus, when labeled material is
placed in a carrier solution for injection, the specific
activity value remains unchanged.
For imaging, the dosage for use in mammals is foreseen
to be typically in a range from one-half to ten millicuries.
It is foreseen that higher doses are feasible when the
labeled material is to be used for therapeutic rather than
simple imaging purposes. Generally, for imaging, doses on
the order of one-half to two millicuries are preferable for
most purposes. Normally, the labeled estrogen will be
diluted, with a solvent, to approximately between one and
ten millicuries per milliliter, so that a relatively high
amount of radioactively decaying material may be contained
in relatively small volume, in order to make injection
simpler and to avoid dispersion of the label over too great
a volume during injection. When injection is the method of
utilization of the imaging material, a suitable carrier
fluid, or diluent, for the labeled material, is a mixture,
by volume, having about 95% of a normal saline solution and
l338187
1 about 5% of 95% ethanol. Certain adjustment may be made if
the particular steroid used has a low solubility in water.
When imaging, normally the time frame during which
binding or joining occurs, in order to reach a detectable or
an optimal imaging level, varies depending upon what is to
be imaged and the labeled material. Very soon after
injection of some steroids, the gallbladder and gut of the
subject animal will typically quickly collect a large amount
of the material. Such collection is normally not associated
with the steroid binding to a receptor.
Detectable images due to binding at receptors are often
present very quickly and normally within one-half to two
hours after injection of labeled ~aterial into a body.
However, longer periods of time may sometimes be necessary
to let areas like the gut become relatively clear of the
imaging material, so that other organs aligned with the gut
become more visible. Sometimes the bowel must be cleaned of
degradation products of labeled materials from the gut in
order to remove partly degradatéd but still labeled,
material that had originally entered the gut and generally
enable ready detection of images on either side thereof.
It is foreseen that under certain conditions it may be
desirable to flood the body with non-radioactive label prior
to injection of radioactively labeled steroid material. For
example, when radioactive iodine is incorporated into the
steroid, it may be desirable to first increase the level of
free iodine within the body being treated, so that the
thyroid is somewhat saturated with non-radioactive iodine,
and therefore less likely to pick up radioactive iodine
resulting from degradation of the labeled steroid material.
1338187
1 As was previously mentioned, it is foreseen that it i8
feasible to use the concepts of the present invention,
especially the I-123 labeled estradiol material, for imaging
of non-living tissue or the like. One usage of such
material is in autoradiography. Conventionally for this
procedure a slice of tissue is placed upon a slide and
allowed to mix with a steroid carrier having tritium
attached thereto, washed and retained tritium bound to
receptors is allowed to expose a photographic emulsion.
However, such a process is very slow often taking many
months.
In accordance with the present invention a receptive
carrier labeled with iodine 123 may be utilized for this
purpose. For example, a tissue having estrogen receptors is
coated with an I-123-estradiol after which excess estradiol
is washed away. A photographic emulsion is then placed on
the surface of the tissue and allowed to develop.
Steroid material having a therapeutic label may be
utilized for treatment of a tissue in a body. For example,
a radioactive label may be utilized to provide radioactive
treatment of a cancerous tumor at the site of the tumor.
In accordance with the present invention, methods are
provided for imaging a selected tissue, through detection
means remote from such tissue, and for therapeutically
treating such tissue. The imaging may be accomplished by
detecting radioactive decay or by other detection methods
such as NMR. In the present particular embodiment, an
iodinated estradiol having a high percentage of iodine-123,
especially as 16-alpha-I-123-17-beta-estradiol, is
applied to a tissue to be imaged. For example, the imaging
~7
~ vhl 3 ~ 7
1 material is dissolved in one-half milliliter of a duluent
comprising, by volume, 95~ of normal saline solution and 5%
of 95% ethanol, and is injected into a blood stream of an
animal host to the particular tissue which is desired to be
imaged. The imageable material is preferably selected to
preferentially seek out certain receptors in the tissue and
to bind thereto, for later detection.
Preferably, a small quantity of a radioactive material
having a relatively high specific activity is used. Where
I-123 labeled estradiol is utilized, it is preferred that
the specific activity of the labeled steroid be at least
5,000 curies per millimole. Generally, the higher the
specific activity the better, since small quantities must be
used to produce the minimum radiation necessary to be
detectable.
A theoretical upper limit on activity for the I-123
estradiol is about 237,000 curies per millimole. It is
foreseen that the number of millicuries injected will vary
with the body weight of the animal or person being
administered the steroid. Also, the amount of radioactive
substrate injected is foreseen to be variable depending upon
the location and type of receptor involved, the intensity of
image needed, and if therapy is intended, the therapeutic
effect desired. It is expected that a normal usage in
humans for imaging would be in the range of one-half to two
millicuries, but this range may, for numerous reasons, vary
substantially, as discussed above.
Following injection, detection will often be delayed to
allow the imaging material to reach the desired tissue
location where detection or therapy is to occur. After
68
, h l 338 ~ B7
1 allowing the labeled material to bind to receptors, the
animal or human subject is placed under an appropriate
detector for the type of label used. For example, where
gamma radiation is released, as by iodine 123, a suitable
gamma radiation detector is positioned close to the body and
over the tissue to be imaged. The detector effectively
determines the patern of radiation emission to image the
tissue being studied. Where I-123 labeled estradiol is
utilized, the I-123 fairly rapidly degrades due to the short
half-life thereof. The estradiol is also eventually
degraded by the body possibly even before the iodine totally
decays.
The following example is for the purpose of
illustration of the invention and should not be interpreted
as limiting the scope of applicants' invention.
Example 1
A plurality of laboratory rabbits are injected with
1.92 millicuries of I-123 estradiol. The 123I-estradiol
is contained within and is administered within, an aliquot
of solution. The diluent comprises a mixture, by volume,
comprising 95% of normal saline solution and 5% of 90%
ethanol. The I-123-labeled estradiol had an apparent
specific activity in excess of 30,000 curies per millimole.
A gamma radiation detector was placed immediately ex~erior
to each of the rabbits, and over a region directly adjacent
the ovaries and uterus. Within a few minutes the detector
was able to begin to show an outline of the uterus. After
about ~ne-half hour images of the uterus and ovaries were
~q
u~1~3~187
1 readily dete~table. Slight improvement~ in the images were
seen over the next few hours. The ovaries and uterus of
each of the rabbits was clearly visible against the other
body organs of the associated rabbit.
It is to be understood that while certain forms of the
present invention have been illustrated and described
herein, it is not be limited to the specific processes,
forms or compositions described.
