CA1338187C - Halogen labeled compounds including estradiol derivatives, synthetic intermediates, syntheses thereof and uses thereof - Google Patents

Halogen labeled compounds including estradiol derivatives, synthetic intermediates, syntheses thereof and uses thereof

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CA1338187C
CA1338187C CA 493461 CA493461A CA1338187C CA 1338187 C CA1338187 C CA 1338187C CA 493461 CA493461 CA 493461 CA 493461 A CA493461 A CA 493461A CA 1338187 C CA1338187 C CA 1338187C
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beta
estradiol
process according
group
ions
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French (fr)
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William P. Duncan
Gary A. Rotert
Jay Spicer
Richard J. Baranczuk
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Bio Medical Research Laboratories Inc
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Bio Medical Research Laboratories Inc
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Abstract

A method of preparation of estradiol derivatives labeled by iodine, especially 123I, substitution at C-16 are synthesized according to the present invention. A
triflate intermediate is prepared from which either 16-alpha-123I-17-beta-estradiol or 16-beta-123I-17-beta-estradiol are prepared by described methods. Both 16-alpha-123I- and 16-beta-123I-17-beta-estradiol made according to the methods described herein have a high relative specific activity. The methods are sufficiently rapid so that the relatively short half-life of 123I is readily accommodated without substantial radioactive decay of the label. A process for detecting a presence of steroid receptors in tissue especially tissue having estrogen receptors, includes the step of treating the tissue by injection, surface application or the like, with a labeled steroid. The labeled steroid may include a moiety which is readily imageable on a gamma radiation detector, on x-ray film, on a fluoroscope, through NMR devices or the like. An especially suitable labeled steroid has been found to be 16-123I-17-beta-estradiol having a specific activity of greater than 2,000 curies per millimole and preferably at least 5,000 curies per millimole.

Description

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:

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 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 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 ~

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 ' ~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 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

- 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 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 . .
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 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

-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

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:

-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 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

0~

. .
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 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 (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 -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, 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:

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 -.
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.
-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~

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 ~

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 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 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 -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 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 ., .

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 ..

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 -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:

.

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 ~

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 ~, 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~

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).

B. Oxidation of the Enolate (XI) to Form 16-keto-estradiol, 3,17-beta-diacetate (XII).

Ac~ Nllll[D3 AC,,J`~
XI XII

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

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

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 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 ~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 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 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 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.

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 , 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.

Claims (14)

1. 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:

wherein OR" 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 bromine, chlorine, fluorine and iodine ions and mixtures thereof, until substantial substitution of said -OR" by said halide ion occurs.
2. The process according to claim 1 wherein: (a) said -OR" is a triflate moiety; and (b) said halide ion is an iodine ion.
3. The process according to claim 2 wherein: (a) said halide ion is an 123I ion.
4. The process according to claim 3 wherein: (a) said R is selected from a group consisting essentially of hydrogen wherein OR" 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 reacting until substantial substitution of said halide ions for said -OR" moiety occurs, with inversion of stereochemistry.

15. The process according to claim 14 wherein: (a) said leaving group -OR" is a triflate group, -OSO2CF3.

16. The process according to claim 15 wherein: (a) said halide ion is an ion of iodine.

17. The process according to claim 16 wherein: (a) said ion of iodine is an ion of a radioisotope of iodine having an atomic mass number of 123.

18. The process according to claim 17 wherein: (a) said source of halide ions is an ammonium salt of said iodine-123 ions, having a general formula NH4123I.

19. The process according to claim 18 including the steps of: (a) forming a solution of said compound and said halide source with t-BuOH present as a solvent during said reacting; and (b) adding a crown ether to said solution during said reacting.

and non-beta directing moieties.
5. The process according to claim 3 wherein: (a) said R is a beta directing moiety; and (b) thereafter hydrolyzing with an acid.
6. A composition comprising 16-beta-123I-17-beta-estradiol, together with a suitable diluent or carrier.
7. A composition comprising 16-alpha-123I-17-beta-estradiol having a specific activity at time of production of greater than 2,000 Ci/mmole, together with a suitable diluent or carrier.
8. The composition according to claim 7 wherein said specific activity is at least 5,000 Ci/mmole.
9. 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 together with a suitable diluent or carrier.
10. The composition according to claim 9 wherein: (a) said estradiol is 16-alpha-123I-beta-estradiol; and (b) said specific activity is at least 5,000 Ci/mmole.
11. The composition according to claim 9 wherein: (a) said estradiol is 16-beta-123I-17-beta-estradiol.
12. 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.
13. A process according to claim 12 wherein: (a) the source of iodide ions is selected from a group consisting essentially of NH4I, NaI and mixtures thereof; and (b) a substantial portion of the iodide ions utilized are ions formed from a radioactive isotope of iodine having an atomic mass number of 123.
14. A process for preparing 16-alpha-halo-substituted-17-beta-estradiol compounds having a general structural formula:

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 R' is a substituent selected from a group consist-ing essentially of hydrogen ion (H) and hydroxy protecting moieties;
said process comprising the steps of: (a) reacting a compound having a general structural formula:

20. The process according to claim 19 wherein: (a) said crown ether is 18-Crown-6.

21. The process according to claim 17 wherein: (a) said -OR is a hydroxy group, -OH; and (b) said -OR' is a hydroxy group, -OH.

22. A process for preparing 16-beta-halo-substituted-17-beta-estradiol compounds having a general formula:

wherein Y is a halide substituent selected from a group consisting essentially of fluorine, chlorine, bromine and iodine:
R' is a substituent selected from a group consist-ing essentially of hydrogen ion (H), and hydroxy protecting groups; and R''' is a beta directing hydroxy protecting group;
said process comprising the steps of: (a) reacting a compound having a general structural formula:

wherein OR is an ester leaving group moiety of low nucleo-philicity 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 ORIV moiety occurs to produce a product with retention of stereochemistry.

23. The process according to claim 22 including an additional step of: (a) after said reacting, hydrolyzing said product with acid, so as to form 16-beta-halo-17-beta-estradiol.

24. The process according to claim 23 wherein: (a) said leaving group -OR is a triflate group -OSO2CF3.

25. The process according to claim 24 wherein: (a) said halide ion is an ion of iodine.

26. The process according to claim 25 wherein: (a) said ion of iodine is an ion of a radioisotope of iodine having an atomic mass number of 123.

27. The process according to claim 26 wherein: (a) said source of halide ions is an ammonium salt of said iodine-123 ions having a general formula NH4123I.

28. The process according to claim 27 including the steps of: (a) forming a solution of said compound and said halide source with t-BuOH present as a solvent during said reacting; and (b) adding a crown ether to said solution during said reacting.

29. The process according to claim 28 wherein: (a) said crown ether is 18-Crown-6.

30. The process according to claim 29 wherein: (a) said -OR''' is an acetoxy group, -OC(O)CH3.

31. The process according to claim 30 wherein: (a) said -OR' is an acetoxy group, -OC(O)CH3.

32. A process for 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:

wherein RV is a hydroxy protecting group, and, RVI is a hydroxy protecting group, until said hydrolyzing converts said protected compound to the following diol:

(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 -OSO2CF3 moiety occurs, with inversion of stereochemistry.

33. The process according to claim 32 wherein: (a) said halide ion is an ion of iodine.

34. The process according to claim 33 wherein: (a) said ion of iodine is an anion of a radioisotope of iodine having an atomic mass number of 123.

35. The process according to claim 34 wherein: (a) said source of halide ions is an ammonium salt, of said iodine-123 ions, having a general formula NH4123I.

36. The process according to claim 35 including the steps of: (a) forming a solution of said diol and said source of halide ions with t-BuOH present as a solvent during said reacting; and (b) adding a crown ether to said solution during said reacting.

37. The process according to claim 36 wherein: (a) said crown ether is 18-Crown-6; and including the step of (b) adding said crown ether in an excess molar amount when compared to said diol having the following general formula:

38. 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, in admixture with a suitable diluent or carrier.

39. A radioactive composition according to claim 38 wherein: (a) said specific activity is greater than 5,000 Ci/mmole; and (b) said estradiol component includes 16-beta-123I-17-beta-estradiol and a ratio of said 16-alpha-123I
component to said 16-beta-123I component is at least 25 to 1 by weight, in admixture with a suitable diluent or carrier.

40. A radioactive composition according to claim 39 wherein: (a) said estradiol component is present in a total amount at production to provide at least .5 millicuries.

41. A radioactive composition for use in diagnostic medicine, said composition including: (a) a radioactive estradiol component comprising 16-beta-123I-17-beta-estradiol.

42. A radioactive composition according to claim 41 wherein: (a) said estradiol component has a specific activity of substantially greater than 2,000 curies per millimole; and (b) said estradiol component is present in a total amount to provide at least 0.5 millicuries immediately following production.

43. The product made by the process according to claim 31.

44. A process for preparing 16-halo-substituted-17-beta-estradiol derivatives as defined in claim 1 which process includes the step of preparing an intermediate having the following general formula:

wherein -RVII comprises a beta-directing, hydroxy-protecting substituent;
-RVIII comprises a hydroxy-protecting substituent;
and -ORIX comprises an ester leaving group moiety of low neucleophilicity;
by: (a) reducing a ketone having the following general structural formula, with a hydride source, to form the following alcohol, after aqueous work-up:

(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:

45. The process according to claim 44 wherein: (a) said hydride source is NaBH4.

46. The process according to claim 45 wherein: (a) (RXO)2O is triflic anhydride, (F3CSO2)2O: and (b) said base is pyridine.

47. The process according to claim 46 wherein: (a) -ORVII is an acetoxy group, -OC(O)CH3.

48. The process according to claim 47 wherein: (a) -ORVIII is an acetoxy group, -OC(O)CH3.

49. A process for preparing 16-halo-substituted-17-beta-estradiol derivatives as defined in claim 1 which process includes the step of preparing an intermediate having the following general formula:

wherein -RVII comprises a beta-directing, hydroxy-protecting substituent;
-RVIII comprises a hydroxy-protecting substituent;
and -ORX comprises an ester leaving group moiety of low nucleophilicity;
by: (a) oxidizing, by reaction with an oxidizing reagent, the following enolate:

wherein RXI is an enolate hydroxy-protecting group of expoxide rearranging proclivity; and RXII is a hydroxy-protecting group, so as to form the following ketone:

(b) converting said ketone to:

wherein -RVII comprises a beta-directing hydroxy-protecting substituent; and -RVIII comprises a hydroxy-protecting substituent;
(c) reducing said ketone with a hydride source, to form the following alcohol, after aqueous work-up:

(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:

wherein -ORX is a leaving group of high leaving group potential and relatively low nucleophilicity.

50. The process according to claim 49 wherein: (a) -ORXI is an acetoxy group, -OC(O)CH3.

51. The process according to claim 50 wherein: (a) -ORVII is an acetoxy-group, -OC(O)CH3.

52. The process according to claim 51 wherein: (a) said hydride source is NaBH4.

53. The process according to claim 52 wherein: (a) said acid anhydride is triflic anhydride, (CF3SO2)2O; and (b) said base is pyridine.

54. The process according to claim 53 wherein: (a) said oxidizing agent is meta-chloroperbenzoic acid; and including the steps of (b) conducting said oxidation in the presence of sodium bicarbonate.

55. The process according to claim 54 wherein: (a) -ORXII is an acetoxy group, -OC(O)CH3, and (b) -ORVIII is an acetoxy group, -OC(O)CH3.

56. The process according to claim 55 including: (a) forming said enolate, having the following formula:

by treating 17-beta-estradiol with isopropenyl acetate in the presence of acid.

57. A pharmaceutical composition for imaging comprising between 0.5 and 10 millicuries 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.

58. A pharmaceutical composition according to claim 57 wherein said pharmaceutical diluent is a solution comprising ethanol and saline solution and said estradiol component is present in a concentration of between 1 and 10 millicuries per milliliter at the time of usage thereof.

59. A pharmaceutical composition according to claim 58 wherein said estradiol component comprises at least 96 percent by weight 16-beta-123I-17-beta-estradiol.

60. A pharmaceutical composition according to claim 58 wherein said estradiol component comprises at least 96 percent by weight 16-alpha-123I-17-beta-estradiol.

61. A process for preparing 16-alpha-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 NaHCH3, the following enolate:

to form the following ketone:

(b) thereafter, reducing said ketone with NaBH4 to form the following alcohol, after aqueous work-up:

(c) thereafter reacting said alcohol with triflic anhydride, (CF3SO2)O, in a solution including pyridine, to form the following triflate:

(d) thereafter, hydrolyzing said triflate with acid to form the following diol:

(e) thereafter reacting said diol with a source of I-123 ions to form 16-alpha-123I-17-beta-estradiol by substitution of I-123 ions for triflate ions, with inversion of stereo-chemistry.

62. The process according to claim 61 wherein: (a) said source of I-123 ions is NH4123I; and including the step of (b) said reacting of said diol from 61(d), with said NH4123I, is conducted in solution with excess 18-Crown-6 ether present.

63. The process according to claim 61 wherein: (a) said source of I-123 ions is Na123I; and (b) reacting said diol with said Na123I in a solution having excess 18-Crown-6 ether present.

64. 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 NaHCO3, the following enolate:

to form the following ketone:

(b) thereafter, reducing said ketone with NaBH4 to form the following alcohol, after aqueous work-up:

(c) thereafter, reacting said alcohol with triflic anhydride, (CF3SO2)2O, in a solution including pyridine, to form the following triflate-diacetate:

(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:

and (e) thereafter hydrolyzing said iodide with acid to form 16-beta-123I-17-beta-estradiol.

65. The process according to claim 64 wherein: (a) said source of I-123 ions is NH4123I; and including the step of (b) conducting said reacting of said triflate-diacetate with said NH4123I in a solution with excess 18-Crown-6 ether present.

66. The process according to claim 64 wherein: (a) said source of I-123 ions is Na123I; and including the step of (b) conducting said reacting of said triflate-diacetate with said Na123I in a solution with excess 18-Crown-6 ether present.

67. 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 receptors in said tissue; and (c) thereafter obtaining an image of said labeled steroid material in said tissue by utilization of detection means appropriately positioned to detect said radiation from said radioactive component.

68. The process according to claim 67 wherein: (a) said steroid is an estrogen; and (b) said radioactive component is a radioactive halogen.

69. The process according to claim 68 wherein: (a) said labeled steroid is substantially 16-123I-17-beta-estradiol; and including the step of: (b) injecting said labeled material into a circulatory system of an animal body.

70. The process according to claim 69 wherein: (a) said body is a human body and including the step of: (b) injecting an amount in the range from about 0.5 to about 10.0 millicuries of said labeled steroid material into said body.

71. The method according to claim 70 wherein: (a) said estradiol is 16-beta-123I-17-beta-estradiol and said injected amount is between 0.5 and 2 millicuries.

72. The method according to claim 70 wherein: (a) said labeled steroid material is allowed to join with said receptors for a time period of between about 0.5 to about 24 hours.

73. The method according to claim 70 wherein: (a) said specific activity is greater than 30,000 curies per millimole at the time said material is applied to said tissue.

74. 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.

75. The method according to claim 74 including the step of: (a) injecting between about 0.5 and about 10.0 millicuries of said 123I labeled estradiol into said body.
CA 493461 1984-10-22 1985-10-21 Halogen labeled compounds including estradiol derivatives, synthetic intermediates, syntheses thereof and uses thereof Expired - Fee Related CA1338187C (en)

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