AU715811B2 - Composition use of nucleoside analogues and gene transfection for tissue imaging and therapy - Google Patents

Description

WO 96/12508 PCT/CA95/00593 COMPOSITION USE OF NUCLEOSIDE ANALOGUES AND GENE TRANSFECTION FOR TISSUE IMAGING AND THERAPY TECHNICAL FIELD This invention relates to diagnostic, radiotherapy and chemotherapy methods for use in conjunction with gene therapy techniques and to the use of certain compounds in performing these methods.

BACKGROUND ART The utilization of gene therapy techniques to express foreign proteins within tissues and cell populations is providing insights into their function and plasticity. These techniques have been successfully used to investigate and treat a broad range of physiological processes. Progress in manipulating transgenic products in vivo and achieving cell-specific delivery of genetic material provides encouragement for enhancing the value of these techniques and their therapeutic potential for treating human and animal disorders.

One aspect of gene therapy involves the transfer of DNA to introduce a sensitivity gene into a target tissue. This can be achieved by direct injection of the DNA into the target tissue, delivery of DNA via liposomes, or via a viral vector that transfers the gene to the target tissue. In the latter case, the viral vector is genetically modified to include the new sensitivity gene in its genome. Such vectors are capable of "transducing" mammalian cells, resulting in expression of a protein which is encoded by the new gene. This expressed protein sensitizes the target tissue to a drug which is a substrate for the protein expressed. The enzymatic process induced by the drug leads to death of target tissue cells expressing the protein. -Since proteins that are present in non-transduced cells have a very low affinity for the drug, systemic toxicity related to this mechanism is not observed.

Gene transfer can be retrovirus-mediated, which provides gene integration only into target cells that are actively synthesizing DNA, with the result that adjacent non-proliferating normal target tissue or cells should not acquire the WO 96/12508 PCT/CA95/00593 -2gene and should remain insensitive to the drug. All of the transduced target cells and viral vector producing cells will be killed by the drug treatment and/or host immune response eliminating potential concern regarding insertional mutagenesis that could give rise to malignant cells.

Gene therapy has been used to treat malignant tumors by in vivo genetic manipulation of the tumor's genome. For example, the efficacy of the herpes simplex virus type 1 thymidine kinase (HSV-1 TK) gene as a "suicide vector" in gene therapy of cancer has been demonstrated P. Short et al., J. Neurosci. Res., 27, 427 (1990); Y. Takamiya et al., J. Neurosurg. 79, 104 (1993)]. One of the most promising approaches involves HSV-1 TK gene transduction in brain tumors followed by intravenous ganciclovir {9-[1,3-dihydroxy-2-propoxy)methyl]guanine, GCV} treatment which is selectively toxic to transduced cells due to selective phosphorylation by HSV-1 TK W. Culver et al., Science, 256, 1550 (1992)]. GCVmonophosphate is subsequently converted by endogenous mammalian kinases to GCV-triphosphate which is a potent inhibitor of viral DNA polymerase. In this therapy, delivery of the Moloney murine retrovirus vector has been achieved through stereotactic implantation of mouse fibroblast producer cells into the brain tumor mass.

Transduction efficiency and subsequent expression of HSV-1 TK in neoplastic tissue is variable and optimal treatment time is currently unknown. However, the dramatic tumor regressions (or cures) observed in animal models, and the lack of systemic toxicity, has prompted the initiation of clinical trials in humans H. Oldfield et al., Hum. Gene Ther., 4, 60 (1993); S. M. Freeman et al., Hum. Gene Ther., 3, 342 (1992)].

Other studies have also indicated the value of gene therapy techniques in the treatment of cancers L. Moolten et al., J. Natl. Cancer Inst., 82,297(1990); S. Freeman et al., Cancer Res., 53, 5274 (1993); D. Barba et al., Proc. Natl. Acad.

Sci. 91 4348 (1994)], including types of cancer such as human lung cancers Hasegawa et al., Am. J. Resp. Cell and Mol. Biol.. 8. 655 (1993)] and breast I cdacers Manome et al., Cancer Res., 54, 5408 (1994)]. Related studies using ,.e

S,

WO 96/12508 PCT/CA95/00593 -3adenovirus HSV-TK gene transfer into cancer cells has also provided encouraging results for thoracic Roy Symthe et al., Cancer Res., 54, 2055 (1994)], brain Chen et al., Proc. Natl. Acad. Sci. 91, 3054 (1994)] and head and neck W.

O'Malley et al., Cancer Res., 55, 1080 (1995)] cancers. It has also been suggested that an adenovirus vector encoding for HSV-TK in porcine arteries produces cells that are sensitive to GCV treatment and may have value to limit smooth muscle cell proliferation in response to arterial injury Ohno et al., Science, 265, 781 (1995)].

However, it has also been shown that 5-(thien-2-yl)- and 5-(furan-2-yl)-2'-deoxyuridine are at least 100-fold more cytostatic to HSV-TK gene-transfected FM3A cells than wild-type FM3A/O cells, and that viral TK expressed in the HSV-1 TK genetransfected tumor cells merely acts as an activating enzyme, whereas thymidylate synthase serves as the target enzyme for the cytostatic action of these compounds Bohman et al., J. Biol. Chem., 269, 8036 (1994)]. Other viral genes may be employed similarly for gene therapy, in conjunction with an anti-viral nucleoside in a prodrug form that is biotransformed to a cytotoxic form by the protein encoded by the selected transfected viral gene.

One significant limitation associated with any gene therapy technique is that one cannot be certain that gene transfer has been restricted to the tumor or other target tissue, and that it has not also occurred in other sensitive dividing cells such as those of bone marrow or intestinal lining. A second major limitation is that even in the target tissue, gene transfer does not necessarily mean that the gene is actually expressed to give the active protein throughout the target tissue. Currently, an invasive technique, which requires obtaining a biopsy sample of the transduced target tissue, is used to determine the extent of gene transfer by employing an in vitro technique such as beta-galactosidase staining Ram et al., Cancer Res., 53, 83 (1993); R. G. Vile et al., Cancer Res., 53, 962 (1993)]. Other techniques such as Doppler color-flow and ultrasound imaging only provide images of the tumor vasculature and tumor volume but no information regarding gene transfer efficacy [Z.

Ram et al., J. Neurosurg., 81, 256 (1994)]. Morphological imaging techniques such WO 96/12508 PCT/CA95/00593 -4as computed tomography (CT) and magnetic resonance imaging (MRI) also do not provide information regarding gene transfer efficacy.

Accordingly, there is a need for a diagnostic method which may be used in conjunction with gene therapy techniques to monitor the transfer of a foreign gene throughout a population of cells including target tissue, which method when performed in vivo is non-invasive. There is also a need to identify compounds possessing specific properties which are suitable for use in performing this diagnostic method. Such a diagnostic method and the use of such compounds in performing this diagnostic method may also be adapted to satisfy a need for radiotherapy and chemotherapy methods which may be used in like manner in conjunction with gene therapy techniques.

DISCLOSURE OF INVENTION The present invention provides for a diagnostic method, a radiotherapy method, and a chemotherapy method, which methods may be used in conjunction with gene therapy techniques. The invention also provides for the use of labelled and unlabelled compounds in performing these methods, which labelled and unlabelled compounds have specific physical and chemical properties.

The invention is applicable to populations of cells into which a foreign gene has been transferred, which foreign gene expresses a protein which preferably is not naturally occurring within the cells. A compound is chosen which will interact selectively with the protein expressed by the foreign gene to produce a product which is trapped within the cells, is cytotoxic or cytostatic to the cells, or both, depending upon whether the compound is being used for diagnostic purposes or for radiotherapy or chemotherapy purposes. In the case of diagnostic applications, trapping of the product, which is labelled, permits the product to accumulate in those of the cells in which the protein has been expressed by the foreign gene, thus facilitating detection of the labelled product in those cells. In the case of radiotherapy applications, WO 96/12508 PCT/CA95/00593 trapping of the product, which is radioactive as a result of the compound being radiolabelled, permits the product to accumulate in those of the cells in which the protein has been expressed by the foreign gene, thus facilitating radiotherapeutic effects directed specifically at those cells. In the case of chemotherapy applications, interaction of the protein with the compound has a cytotoxic or cytostatic effect on the cells, which is enhanced if the product is trapped within those of the cells in which the protein has been expressed.

In a first embodiment relating to diagnostic applications of the invention, the invention comprises a method for monitoring the transfer of a foreign gene throughout a population of cells, comprising the steps of administering to the cells an effective dose of a labelled compound so that the labelled compound interacts selectively with a protein expressed by the foreign gene to produce a labelled product, and then detecting the labelled product, wherein the labelled compound is selected to interact selectively with the protein expressed by the foreign gene such that the labelled product becomes trapped within those of the cells in which the protein has been expressed by the foreign gene. In this first embodiment, the invention also comprises the use of labelled compounds in performing this diagnostic method.

The foreign gene may be a gene selected from any eucaryotic or procaryotic cells, or from a virus, including a virus from the group of viruses consisting of herpes simplex virus, human cytomegalovirus, varicella zoster virus and Epstein-Barr virus. A preferred foreign gene is a gene which expresses herpes simplex virus thymidine kinase.

The labelled compound is preferably radiolabelled, but any other form of labelling which facilitates detection of the labelled product may also be suitable.

Where the foreign gene is a gene which expresses herpes simplex virus thymidine kinase, the preferred labelled compound is a compound of the following formula: WO 96/12508 PCrCA9S/00593 formula (1) or a pharmaceutically acceptable salt thereof, wherein X is a radioactive halogeno substituent, wherein R, is a hydrogen, hydroxyl or fluoro substituent, wherein R 2 is a hydrogen or fluoro substituent, wherein R 3 is a substituent selected from the group consisting of hydrogen, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, 1methyl-1, 4-dihydropyridyl-3-carbonyl, 3-7C cycloalkylcarbonyl, and alkylcarbonyls with a straight or branched chain having from 1 to 8 carbon atoms, and wherein R 4 is a substituent selected from the group consisting of hydrogen, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, 1-methyl-1, 4-dihydropyridyl-3-carbonyl, 3-7C cycloalkylcarbonyl, and alkylcarbonyls with a straight or branched chain having from 1 to 8 carbon atoms. Preferably, at least one of R, and R 4 are hydrogen, and most preferably, R 4 is hydrogen. Preferably X is selected from the group consisting of 1231, 1241, '31, 75 Br, 76 Br, and and most preferably X is 1231. The substituents for some specific preferred labelled compounds for use with this foreign gene are as follows: 1 WO 96/12s50 s PCT/CA95M00S93 -7- 1. X 1231, 1241 or 1311, most preferably 1231 R, hydrogen

R

2 hydrogen

R

3 hydrogen or 1-methyl-1, 4-dihydropyridyl-3-carbonyl R4 hydrogen 2. X 1231, 1241 or 131l, most preferably 1231 R, hydrogen R2= fluorine R3 hydrogen or 1-methyl-1, 4-dihydropyridyl-3-carbonyl

R

4 hydrogen 3. X 123 1, 1241 or 1311, most preferably 1231 R, fluorine

R

2 hydrogen

R

3 hydrogen or 1-methyl-1, 4-dihydropyridyl-3-carbonyl

R

4 hydrogen 4. X 3 1, 1241 or 1311, most preferably 1231 R, hydroxyl R2= hydrogen R, hydrogen or 1-methyl-1, 4-dihydropyridyl-3-carbonyl R4 hydrogen In a second embodiment relating to radiotherapy applications of the invention, the invention comprises a method of radiotherapy for use with a population of cells into which a foreign gene has been transferred, comprising the step of administering to the cells an effective radiotherapeutic dose of a radiolabelled compound so that the radiolabelled compound interacts selectively with a protein expressed by the foreign gene to produce a radiolabelled product, wherein the radiolabelled compound is selected to interact selectively with the protein expressed by the foreign gene such that the radiolabelled product becomes trapped within those of the cells in which the protein has been expressed by the foreign gene. In this second embodiment, the invention also comprises the use of radiolabelled compounds in performing this radiotherapy method.

r,:rL WO 96/12508 PCT/CA95/00593 -8- The parameters for choosing a foreign gene for use in the radiotherapy method of the invention are the same as for the diagnostic method of the invention, and a preferred foreign gene is a gene which expresses herpes simplex virus thymidine kinase.

Where the foreign gene is a gene which expresses herpes simplex virus thymidine kinase, the preferred radiolabelled compound is of the same general formula as for the diagnostic method of the invention, except that X is a radioactive halogeno substituent, preferably selected from the group consisting of 1231, 1251 and 1311. Most preferably X is 1311. Preferably, at least one of R 3 and R 4 are hydrogen and most preferably R 4 is hydrogen. The substituents for the specific preferred radiolabelled compounds for use with this foreign gene are the same as for the diagnostic method of the invention, except that X in these specific radiolabelled compounds is selected from the group consisting of 1231, 1251 and 1311.

In a third embodiment relating to chemotherapy applications of the invention, the invention comprises a method of chemotherapy for use with a population of cells into which a foreign gene has been transferred, comprising the step of administering to the cells an effective chemotherapeutic dose of a compound so that the compound interacts selectively with a protein expressed by the foreign gene to produce a product, wherein the compound is selected to interact selectively with the protein expressed by the foreign gene such that the product is cytotoxic or cytostatic to those of the cells in which the protein has been expressed by the foreign gene. In this third embodiment, the invention also comprises the use of compounds in performing this chemotherapy method.

The parameters for choosing a foreign gene for use in the chemotherapy method of the invention are the same as for the diagnostic method of the invention, and a preferred foreign gene is a gene which expresses herpes simplex virus thymidine kinase.

WO 96/12508 WO 96/12508 PCT/CA95/00593 -9- Where the foreign gene is a gene which expresses herpes simplex virus thymidine kinase, the preferred compound is of the same general formula as for the diagnostic method of the invention, except that X is a halogeno substituent, preferably selected from the group consisting of bromo, chloro, fluoro and iodo. Most preferably X is either a bromo, chloro, or iodo substituent. Preferably, at least one of

R

3 and R 4 are hydrogen, and most preferably R 4 is hydrogen. The substituents for the specific preferred compounds for use with this foreign gene are the same as for the diagnostic method of the invention, except that in these specific preferred compounds, X may be described generally as an iodo substituent.

BRIEF DESCRIPTION OF DRAWINGS Embodiments of the invention will now be described with reference to the accompanying drawings, in which: Figure 1 is a graph showing the in vitro cellular uptake of 25 1]-IVDU in KBALB-LNL and KBALB-STK cells for example 12; Figure 2 is a graph showing the in vitro cellular uptake of 125 1]-IVFRU in KBALB, KBALB-LNL, and KBALB-STK cells for example 12; Figure 3 is a graph showing the in vivo biodistribution of 131 1]-IVFRU in mice bearing KBALB-STK tumors for example 13; Figure 4 is a graph showing the in vivo biodistribution of 131 1]-IVFRU in mice bearing KBALB tumors for example 13; Figure 5 is a graph showing the in vivo tumor to blood ratios in mice bearing KBALB or KBALB-STK tumors for example 13; WO 96/12508 PCT/CA95/00593 Figure 6 is a scintigram image of KBALB-STK tumor expressing HSV-1 TK at 8 hours after injection of 1 31 1]-IVFRU, but before Ganciclovir treatment, for example 14; and Figure 7 is a scintigram image of KBALB-STK tumor expressing HSV-1 TK at 8 hours after injection of 1 31 1]-IVFRU following 4 days of Ganciclovir treatment for example 14.

BEST MODE FOR CARRYING OUT THE INVENTION In a first embodiment of the invention, the invention is comprised of a diagnostic use of a labelled compound and a diagnostic method using that compound. The use and the method are for monitoring the transfer of a foreign gene throughout a population of cells. The method is comprised of the steps of first administering an effective dose of a labelled compound to the cells in order to produce a labelled product by the selective interaction described below and then detecting the labelled product.

The foreign gene is preferably deliberately transferred, transduced or transfected into the population of cells or a portion of the population of cells, as desired, by gene therapy techniques. The foreign gene is encoded to express a protein. When an effective dose of the labelled compound is administered to the cells including the transferred foreign gene, the labelled compound interacts selectively with the expressed protein to produce the labelled product. Transfer of the foreign gene into the cells does not necessarily mean that every transferred foreign gene is actively expressing the protein into the cell in which that specific foreign gene is located. Some foreign genes may be actively expressing the protein, while others may be dormant. As a result, the labelled product is produced only within those cells in which the protein has actually been expressed by the foreign gene. The labelled product may then be detected in order to monitor the transfer of the foreign gene in the cells.

t WO 96/12508 PCT/CA95/00593 11 The labelled compound is selected to interact selectively with, or be acted on by, the specific protein expressed by the foreign gene in order to produce a labelled product which is trapped and thus localized within, and which does not readily escape from, the cells in which the protein has been expressed. Thus a preferential accumulation or localization or a selective metabolic trapping of the labelled product occurs in the protein expressing cells, as compared to cells which either do not include the foreign gene or which include a dormant foreign gene. This selective trapping permits the specific detection of those cells which both include the foreign gene and in which the specific protein has been expressed. Modification of the labelled compound, such as by phosphorylation, occurs in the presence of the protein, which results in the formation of the labelled product inside the cell. The resulting labelled product does not readily leave the cell and therefore accumulates or is localized within that cell. The labelled product that is trapped within the cell may be any product resulting from the interaction which satisfies the above noted requirements, and which includes the label, as well as the label itself in isolation when the label alone is capable of being selectively trapped in the cell.

The diagnostic use and method may be used both in vitro and in vivo to monitor the transfer of the foreign gene throughout the population of cells. More specifically, the method may be useful to determine the location or site, the extent and the kinetics of the transfer of the foreign gene throughout the cells, to determine the optimal time for initiation of chemotherapy in mammalian subjects using a prodrug to destroy gene transduced tumors in cancer treatment, to effect radiotherapy of a specific population of cells using radiopharmaceuticals, or to study gene modulation processes. Further, the diagnostic method may be used in clinical studies to assess treatment efficacy and whether repeat treatment is required in the event that regrowth occurs in the cellular population.

Accordingly, the population of cells of the within invention may be any in vitro or in vivo population of cells or a smaller specific portion of a larger cellular population. In the preferred embodiment, the population of cells is either a specific WO 96/12508 PCr/CA9500593 12tissue of a mammalian subject, such as a human (eg: brain, liver, breast, etc.) or a portion of a specific tissue, such as a cancerous tumor within the tissue. Given the manner of the foreign gene transference or integration into the cells, the diagnostic method is particularly useful when the cells or tissue in which the transference of the foreign gene is to be monitored are actively synthesizing DNA, as is found in human conditions resulting in cell proliferation. Such cell proliferative conditions include cancer and the repair of tissues after injuries, such as smooth muscle cell proliferation in response to arterial injury.

The transferred gene may be any gene selected from any eucaryotic or procaryotic cell or any virus, which is preferably readily uptaken by the population of cells in which the transference of the foreign gene is to be monitored. Further, the gene must express a protein which, preferably, is not naturally occurring within that population of cells. For this reason, the transferred gene is preferably a foreign gene.

A foreign gene is any gene which is not present in that exact or specific form in the population of cells in which the transference of the foreign gene is to be monitored.

In other words, a foreign gene is either not present at all in those cells or is present in the cells in a differing form such that the protein expressed by the foreign gene is not naturally expressed in those cells. The protein expressed by the foreign gene may be an enzyme.

As stated, the foreign gene is preferably readily uptaken by the cells into which the foreign gene is to be transferred. For this reason, the foreign gene is preferably selected from a virus. Further, the foreign gene is preferably selected from the group of viruses consisting of herpes simplex virus, human cytomegalovirus, varicella zoster virus and Epstein-Barr virus. Of these viruses, the herpes simplex virus is presently commonly used for the selection of a gene for use in gene therapy.

Therefore, in the preferred embodiment of the within diagnostic method and use, the foreign gene is selected from the herpes simplex virus, and more specifically, the 0 herpes simplex virus type 1. The specific foreign gene of the preferred embodiment ,expresses herpes simplex virus thymidine kinase (HSV-TK). However, as indicated WO 96/12508 PCT/CA95/00593 13previously, any foreign gene may be used as long as the protein expressed by the foreign gene is matched with an appropriate labelled compound to selectively interact with to produce the required labelled product.

The compound may be labelled by any suitable conventional means as long as the interaction with the expressed protein results in a labelled product which is capable of detection in the cells, preferably in a non-invasive manner. In the preferred embodiment, the labelled compound is radiolabelled and upon the selective interaction of the radiolabelled compound with the expressed protein, a radiolabelled product is produced. The radiolabelled product is detected in the preferred embodiment using known nuclear medicine imaging technology. The specific radioactive label used to radiolabel the compound is selected with reference to the specific type of imaging technology to be used for detection of the resulting radiolabelled product, which selection process is known to those skilled in the art.

For example, for single photon emission computed tomography (SPECT), the radioactive isotopes 1231 and 1311 are both suitable as labels, although, for clinical use, 1231 is preferred. For positron emission tomography (PET), suitable labels include the radioactive isotopes 124, 75 Br, 76 Br and 18

F.

For many foreign genes, any suitably labelled nucleoside or nucleobase may be chosen as the compound. For other foreign genes, a compound other than a nucleoside or nucleobase may be appropriate. As discussed above, to be considered to be suitable, the protein expressed by the transferred foreign gene and the labelled nucleoside or nucleobase must selectively interact to produce a labelled product which is trapped within those of the cells in which the protein has been expressed.

For example, the nucleoside 5-fluoro-2'-deoxyuridine, suitably labelled, may be employed for detecting cells including a gene expressing thymidylate synthase, or fluorocytosine may be employed for cells including a gene expressing cytosine deaminase.

WO 96/12508 14- PCT/CA95/00593 As well, different compounds may possess different biodistribution properties and may therefore have different efficacies or other characteristics which make a particular compound more suitable for detecting and imaging a specific population of cells brain, lung, breast, ovary, colon, pancreas, etc.). Therefore, when selecting the compound, consideration should also be given to the properties of that particular compound as they relate to the specific cellular population in which the transfer of the foreign gene is to be monitored.

In the preferred embodiment, in which the transferred foreign gene expresses HSV-TK, a compound of the following formula or a pharmaceutically acceptable salt thereof, is preferably used:

NH

NIO

OR

3

R

2 formula (1) Referring to formula X is the label of the compound, which is preferably a radiolabel as previously described. The radiolabel is preferably a radioactive halogeno substituent. The radioactive halogeno substituent is selected, as stated above, with reference to the type of nuclear medicine imaging technology to be used for detection of the radiolabelled product. Suitable radioactive halogeno WO96/12508 PCTICA95/00593 substituents include 31, 124, 1251, 131, 75 Br, 76 Br and 18 F. Of this group, 1251 is least preferred for diagnostic purposes, 1241 and 131 are more preferred, while 1231 is most preferred for clinical use. Therefore in the preferred embodiment, X in the compound of formula is 'al. The radiolabels are incorporated into the compound by conventional techniques.

When X is a radioactive halogeno substituent, the compound of formula is preferably a high-specific-activity no-carrier-added compound. In other words, in the preferred embodiment, the concentration of the radioactive 1231 is as high as possible relative to any source iodine or iodine salt contained in the compound in order to increase the specific activity.

R, of formula is a hydrogen, hydroxyl or fluoro substituent and R 2 is a hydrogen or fluoro substituent. It has been found that compounds of formula (1) that possess a R, or R 2 substituent other than hydrogen at the C-2' position of the sugar moiety are more stable to pyrimidine phosphorylase which cleaves the glycosidic C-N bond of most C-2' unsubstituted pyrimidine nucleosides. For example, it is known that a 2'-ribo fluoro R 2 -substituent R. Mercer et al., J. Med.

Chem., 30, 670 (1987)], a 2'-arabino fluoro R,-substituent Lopez et al., Antimicrob. Agents Chemother., 17, 803 (1980); J. A. Codere et. al., J. Med. Chem., 26, 1149 (1983)] or a 2'-arabino hydroxyl R,-substituent J. Robins et al., J. Med.

Chem., 34, 2275 (1991); N. K. Ayusi et al., Mol. Pharmacol., 31, 422 (1987); T. C.

Chou et al., Antimicrob. Agents Chemother., 31, 1355 (1987)] confers resistance to glycosidic bond cleavage, which causes deactivation.

Further, in formula R 3 is a substituent selected from the group consisting of hydrogen, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, 1methyl-1, 4 dihydropyridyl-3-carbonyl, 3-7C cycloalkylcarbonyl, and alkylcarbonyls with a straight or branched chain having from 1 to 8 carbon atoms. Similarly, R 4 is a substituent selected from the group consisting of hydrogen, arylcarbonyl, S-i\ ./heteroarylcarbonyl, heterocyclocarbonyl, 1-methyl-i, 4-dihydropyridyl-3-carbonyl, 3-7C WO 96/12508 WO 96/12508 PCT/CA95/00593 -16cycloalkylcarbonyl, and alkylcarbonyls with a straight or branched chain having from 1 to 8 carbon atoms. When R 3 and R 4 are both hydrogen, a parent compound is formed. When R 3

R

4 or both are other than hydrogen, a derivative compound is formed. It is preferred that at least one of R 3 and R 4 is hydrogen, preferably R 4 as found in the preferred embodiments of formula discussed below.

Where R 3 of formula is 1-methyl-I, 4 dihydropyridyl-3-carbonyl and

R

4 is hydrogen, a more lipophilic derivative compound is formed in which R 3 is referred to as a chemical delivery system (CDS) or a CDS moiety. CDS derivatives may have an enhanced ability to cross the blood-brain-barrier (BBB) and localize in transduced or transfected brain cells or tissue. Due to their polar nature, pyrimidine nucleosides or acyclic nucleosides such as GCV do not readily cross the BBB.

Increasing the lipophilic character of the nucleosides of formula while still retaining their antiviral properties, is a viable method to enhance brain localization.

One of the more successful approaches currently used to design lipophilic drugs is the dihydropyridine*pyridinium salt chemical delivery system (CDS) Bodor et al., Science, 214, 1370 (1981)]. This brain-targeted concept involves the coupling of a lipophilic 1-methyl-i, 4-dihydropyridyl promoiety to a drug molecule through an ester or amide linkage that is readily hydrolyzed. The compounds of formula wherein the R 3 substituent is a 3'-[O-(1-methyl-1, 4-dihydropyridyl-3-carbonyl)] chemical delivery system (CDS) and R 4 is hydrogen cross the BBB more readily, and then undergo oxidation in a manner analogous to the NAD-NADH redox system in brain tissue. The resulting pyridinium salt is highly polar which results in its cerebral trapping leading to an elevated and sustained concentration in brain tissue.

Clearance from blood is facilitated, since any oxidation product in the periphery is rapidly cleared. Hydrolysis of the ester linkage of the trapped pyridinium salt releases the parent compound of formula wherein the R 3 and the R 4 substituents are hydrogen and R, and R 2 are as defined above, and the oxidized promoiety, trigonelline. Compounds of formula wherein R 4 is hydrogen possess a free hydroxyl group which is desirable for selective phosphorylation to the monophosphate derivative by the viral encoded enzyme produced in transfected WO 96/12508 CA 93 PCT/CA95/00593 -17tissue or cells which results in its selected metabolic trapping thereby preventing its egress.

Thus, nucleosides containing the CDS moiety as the R 3 substituent may have increased lipophilicity and an increased ability to penetrate the population of cells. As well, nucleosides containing the CDS moiety as the R 3 substituent may have the advantage of the additional polar trapping effect described above, in addition to the selective trapping occurring as a result of the selective interaction of the expressed protein with the compound. Various other moieties may also be added to the nucleosides to increase lipophilicity and tissue penetration. For example, esterification may be used, for example, with alkoxycarbonyl chains having up to 7 or 8 carbon atoms.

Of the various compounds included within formula as described above, the following compounds have been found to be the most preferred when the foreign gene expresses HSV-TK: IVDU, IVDU-CDS, IVFRU, IVFRU-CDS, IVFAU, IVFAU-CDS, IVAU and IVAU-CDS. All of these preferred compounds are of formula and include the substituents described below. In addition, in all of these preferred compounds, when used for diagnosis and imaging, X is an iodine, and is preferably 1231, as discussed above. The other substituents of each preferred compound are as follows: IVDU R, is hydrogen, R 2 is hydrogen, R 3 is hydrogen and R, is hydrogen.

IVDU-CDS R, is hydrogen, R 2 is hydrogen, R 3 is 1-methyl-1, 4dihydropyridyl-3-carbonyl and R 4 is hydrogen.

IVFRU R, is hydrogen, R 2 is fluorine, R 3 is hydrogen and R, is hydrogen.

IVFRU-CDS R, is hydrogen, R 2 is fluorine, R 3 is 1-methyl-1, 4dihydropyridyl-3-carbonyl and R 4 is hydrogen.

IVFAU R, is fluorine, R 2 is hydrogen, R 3 is hydrogen and R 4 is hydrogen.

WO 96/12508 PCT/CA95/00593 -18- IVFAU-CDS R, is fluorine, R, is hydrogen, R 3 is 1-methyl-i, 4dihydropyridyl-3-carbonyl and R 4 is hydrogen.

IVAU R, is hydroxyl, R 2 is hydrogen, R 3 is hydrogen and R, is hydrogen.

IVAU-CDS R, is hydroxyl, R 2 is hydrogen, R 3 is 1-methyl-1, 4dihydropyridyl-3-carbonyl and R, is hydrogen.

Of these compounds, it is preferable to use those compounds which show greater stability. For example, it has been found that of the parent compounds, IVDU is less stable than IVFRU, IVFAU and IVAU. However, it has been further found that IVDU becomes more stable as a IVDU-CDS derivative.

All of the parent and derivative compounds of formula for which R, is hydrogen, except for those derivative compounds in which R 3 is CDS, can be prepared by reacting in an inert solvent, a 5-iodouracil nucleoside of the formula formula (2) wherein R, and R 3 are as defined above, with (E)-1-(tri-n-butylstannyl)-2- (trimethylsilyl)ethene of formula i WO 96/12508 PCT/CA95/00593 19- H /SiMea

C=C/

(n-Bu)3-Sn

\H

formula (3) in the presence of a suitable cross-coupling catalyst, preferably of formula [(Ph) 3

P]

2 Pd(lI)Cl2 formula (4) allowing the reaction to occur (normally at 50°C) in an inert solvent such as tetrahydrofuran to convert to a (E)-5-(2-trimethylsilylvinyl)-2'-deoxyuridine or -arabinouridine compound of formula formula WO 96/12508 DfTIF A nI/Ann 20 UU3 wherein R 2 and R, are as defined above. Electrophilic halogenation of the compound of formula wherein R 2 and R 3 are as defined above using a halogenation agent of formula l3

X-Y

formula (6) wherein X is as defined above and Y is selected from a group consisting of iodo, bromo, chloro, fluoro, acetoxy and trifluoromethoxy, in an inert solvent such as dry acetonitrile, allowing the reaction to occur (normally at 25 0 C) to convert to a product of formula x 0

NH

N O

RI

HO

R

3 2 formula (7) wherein X, R 2 and R 3 are as defined above.

The derivative compounds of formula in which R 3 is CDS and R 4 is hydrogen can be prepared by quaternization of a compound of formula WO 96/12508 PCT/r A O/nn0 -21 MesSi 0

NH

HO 0

R

(N

formula (8) wherein R, and R 2 are as defined above, with iodomethane or bromomethane in an inert solvent such as acetone (preferably at reflux temperature), to convert to a compound of formula

NO

Me3Si N

O

HO O Ri c- R2 N I- (or Br- Me formula (9) WO 96/12508 -22wherein R, and R 2 are as defined above. Electrophilic halogenation of the compound of formula wherein R, and R 2 are as defined above using a halogenation agent of formula

X-Y

formula wherein X is as defined as above and Y is selected from a group consisting of iodo, bromo, chloro or fluoro, in an inert solvent such as acetonitrile, allowing the reaction to occur (normally at 250C) to convert to a compound of formula (11): X

O

NO

HO 0 200 _8 r

N

I I Me formula (11) wherein X, R, and R 2 are as defined above. Reduction of the compound of formula (11) using a suitable reducing agent such as preferably sodium dithionite in the presence of a suitable base as sodium bicarbonate in a suitable inert two-phase solvent system (preferably water-ethyl acetate, 1:1, v/v) preferably at 25°C to convert to a product of formula (12): WO 96/12508 -23 rL/Ay/UUS3 X 0

NH

HO 0 Me formula (12) wherein X, R, and R 2 are as defined above.

The starting materials for the preparation of compounds of formula viz the 5-iodouracil nucleosides of formula (E)-1-(tri-n-butylstannyl)-2trimethylsilyl)ethene of formula bis(triphenylphosphine)palladium(ll) chloride of formula and electrophilic halogenation reagents of formula are either known or are conveniently prepared from starting materials by methods known per se.

All of the compounds of formula can be administered either parentally, preferably by injection, or orally. As a liquid carrier, a carrier such as water, ethyl alcohol or polyethyleneglycol, liposomes, or other physiologically acceptable solvents or dispersing liquids can be used. For oral administration, either solid or liquid carriers may be used. One commonly used solid carrier is gum acacia, but others are also suitable.

Those skilled in medical diagnostic imaging will be able to calculate an effective dose of the labelled compound for the particular use, including human use, WO 96/12508 PCT/CA95/00593 -24based on their experience with other compounds carrying similar labels. However, in general, when dealing with diagnostic uses, any radiolabelled compound should be kept to a small dosage in order to avoid any toxicity to the subject.

In a second embodiment of the invention, the invention is comprised of a radiotherapeutic use of a radiolabelled compound and a method of radiotherapy using that radiolabelled compound. The method and use involve a population of cells into which a foreign gene, which expresses a protein, has been transferred. The method is comprised of the step of administering to the cells an effective radiotherapeutic dose of the radiolabelled compound so that the radiolabelled compound interacts selectively with the protein to produce a radiolabelled product.

The radiolabelled product achieves or performs the desired therapeutic function or effect. The radiolabelled compound is selected to interact selectively with the protein such that the radiolabelled product becomes trapped within those of the cells in which the protein has been expressed by the foreign gene. The radiotherapy method may be used in conjunction with the diagnostic and chemotherapy methods, or may be used separately.

All of the considerations, parameters, properties, methods of preparation and administration and other characteristics provided above for the population of cells, the foreign gene, the compound and the product, and the preferred embodiments of each, with respect to the diagnostic method and use are applicable to the radiotherapeutic method and use except as hereafter specified.

The population of cells of the radiotherapeutic method and use may be of the same type as the population of cells indicated for the diagnostic method and use. Specifically, at least a portion of the population of cells is preferably actively synthesizing DNA. Therefore, the most likely cells for the application of the radiotherapeutic method and use are cancerous cells or tissue or other cell proliferative conditions.

WO 96/12508 PCT/CA95/00593 In the radiotherapeutic method and use, the compound and the product are radiolabelled as in the preferred embodiment of the diagnostic method. Thus, the X substituent of formula being the radiolabel, may similarly be any suitable radioactive halogeno substituents. Suitable radioactive halogeno substituents include 1231, 1251, and 1311. Of this group, for radiotherapeutic purposes, 1311 is the most preferred. It has been found that 1241, 75 Br, 7 Br and "F are less appropriate or suitable for radiotherapeutic purposes. Therefore in the preferred embodiment of the radiotherapeutic method and use, X in the compound of formula is 1311. Further, as in the diagnostic method and use, the compound of formula is preferably a high-specific-activity no-carrier-added compound.

Those skilled in medical radiotherapeutic methods and uses will be able to calculate a suitable effective dose of the radiolabelled compound for human or other uses based on their experience with other compounds carrying similar radiolabels. However, as indicated previously, when the radiolabelled compound is used for diagnostic purposes, as small a dosage as possible should be used in order to minimize any toxicity to the population of cells or surrounding tissue. When using the compound for radiotherapeutic purposes, an effective radiotherapeutic dose of the radiolabelled compound must be used. Typically, the dosage of the radiolabelled compound for therapeutic purposes will be greater than that used for diagnostic purposes in order to achieve the desired radiotherapeutic effect. When used on cancerous cells, the desired radiotherapeutic effect will be destruction of the cells in which the protein has been expressed by the foreign gene.

As discussed previously, given the manner of the foreign gene transference into the population of cells, the radiotherapeutic method and use are particularly useful when at least a portion of the population of cells is actively synthesizing DNA. Non-proliferating cells or surrounding tissue should not acquire the foreign gene and will thus remain insensitive to the radiolabelled compound. The result is that systemic toxicity should not be observed upon use of the radiolabelled compound.

WO 96/12508 PCT/CA95/00593 -26- In a third embodiment of the invention, the invention is comprised of a chemotherapeutic use of a compound and a method of chemotherapy using that compound. The chemotherapeutic use and method also involve a population of cells into which a foreign gene, which expresses a protein, has been transferred. The chemotherapeutic method is comprised of the step of administering to the cells an effective chemotherapeutic dose of a compound so that the compound interacts selectively with the protein to produce a product. The compound is selected to interact selectively with the protein such that the product which is produced is cytotoxic or cytostatic to those of the cells in which the protein has been expressed by the foreign gene. The chemotherapy method may be used in conjunction with the diagnostic and radiotherapy methods, or may be used separately.

All of the considerations, parameters, properties, methods of preparation and administration and other characteristics described above for the population of cells, the foreign gene, the compound and the product, and the preferred embodiment of each, with respect to the diagnostic method and use are applicable to the chemotherapeutic method and use except as hereafter specified.

As in the diagnostic method and in the radiotherapeutic method, at least a portion of the population of cells is preferably tissue actively synthesizing DNA.

Therefore, again, the most likely cells for the application of the chemotherapeutic method and use are cancerous cells or tissue or other cell proliferative conditions.

As indicated, the product is cytotoxic or cytostatic to the dividing cells.

In the chemotherapeutic method and use, it is not necessary that either the compound or the product be labelled. Thus, in the chemotherapeutic method and use, the X substituent of formula is any suitable halogeno substituent. Suitable halogeno substituents include iodo, bromo, chloro, and fluoro. Of this group, for chemotherapeutic purposes, fluoro is the least preferred halogeno substituent.

WO 96/12508 PCT/CA95/00593 -27- Although the preferred embodiment of the chemotherapeutic method and use uses the herpes simplex virus gene and specifically the gene expressing HSV-TK, other genes may be employed for the chemotherapeutic method and use as long as the protein expressed by the gene is matched with an appropriate compound to produce the required product which is cytotoxic or cytostatic to the cells. For example, the human cytomegalovirus (HCMV) UL97 gene which encodes a protein that phosphorylates GCV offers potential for treating HCMV infections Littler et al., Nature, 358, 160 (1992); V. Sullivan et al., Nature, 358, 162 (1992)]. In addition, transfer of genes that encode for thymidylate synthase polymerase or cytosine deaminase from eucaryotic or procaryotic organisms could be employed.

Those skilled in medical chemotherapeutic methods and uses will be able to calculate a suitable effective dose of the compound for human and other uses based on their experience with other similar compounds. As indicated previously, the transference of the foreign gene is preferably into those cells actively synthesizing DNA, so that non-proliferating cells or surrounding tissue do not acquire the foreign gene and thus remain insensitive to the chemotherapeutic compound. Thus, systemic toxicity should not be observed in use of the chemotherapeutic compound.

In addition, it is known that only about 10 percent of cancerous cells need to be transduced with the HSV-TK gene to obtain effective tumor destruction using an antiviral drug such as GCV. This important observation is due to a by-stander effect where non-transduced cells are also killed. Although the mechanism of this effect is not fully understood, explanations offered to explain this phenomenon include: (i) continued viral infection; (ii) transfer of an integrated HSV-TK gene during mytosis; (iii) transfer of the toxic GCV-triphosphate via gap junctions or apoptotic vesicles; or (iv) immune related effects Freeman et al., Cancer Res., 53, 5274 (1993); W. Roy Symthe et al., Cancer Res., 54, 2055 (1994)].

The following non-limitative examples illustrate some selective methods for producing the compounds according to the present invention, as well as comparative data illustrating the in vitro physicochemical and biological properties WO 96/12508 PCT/CA95/00593 -28and in vitro biodistribution and imaging data of representative compounds according to the present invention.

Preparation EXAMPLE 1 (E)-5-(2-iodovinyl)-2'-fluoro-2'-deoxyuridine

(IVFRU)

(See schematic representation of reaction) Bis(triphenylphosphine)palladium(ll) chloride (37 mg, 0.053 mmol) and (E)-l-(tri-n-butylstannyl)-2-(trimethylsilyl)ethene (412 mg, 1.06 mmol) were added to a solution of 5-iodo-2'-fluoro-2'-deoxyuridine (197 mg, 0.53 mmol) in dry THF (5 ml) with stirring at 50°C. The reaction mixture was stirred for 16 h at 50°C under an argon atmosphere at which time TLC analysis indicated that the reaction was complete. The solvent was removed in vacuo and the residue obtained was purified by silica gel column chromatography using MeOH-CH 2 Cl 2 (1:25, v/v) as eluent to yield 2 -trimethylsilylvinyl)-2'-fluoro-2'-deoxyuridine as a white solid (146 mg, yield) after recrystallization from ethyl acetate; mp 164-165oC, 1 H NMR (DMSO-d 6 ):6 0.08 9H, SiMe 3 3.64 1H, Jgem 9.9 Hz, 3.86 1H, 3.89 1H, J3,4. 8.8 Hz, 4.21 1H, 24.2 Hz, 5.04 (dd, 1H, J 2 53.3 Hz, 3.8 Hz, 5.43 1H, JOH, 3 6.6 Hz, C-3' OH, exchanges with deuterium oxide), 5.92 1H J,,F 17.0 Hz, 6.52 and 6.59 (two d, 1H each, 19.8 Hz, CH=CHSiMe 3 8.33 1H, uracil 11.51 1H, NH, exchanges with deuterium oxide); Exact Mass Calcd for C, 4

H

2 1 FN20OSi:344.1204. Found (HRMS): 344.1208 Anal. Calcd for C 14

H

21

FN

2 O0Si:C, 48.82; H, 6.15; N, 813.

Found: C, 48.68; H, 6.16; N, 7.87.

(E)-5-(2-trimethylsilylvinyl)-2'-fluoro-2'-deoxyuridine (40 mg, 0.116 mmol) was dissolved in acetonitrile (2 MI) and then iodine monochloride (19 mg, 0.116 mmol) was added with stirring at 25°C. The reaction mixture was stirred for 30 min at 250C at which time TLC analysis indicated that the reaction was completed. The solvent was removed in vacuo and the residue obtained was purified by silica gel WO 96/12508 PCTICA95IOOS93 29 column chromatography using MeOH-0H 2 01 2 (7:93, v/v) as eluent to afford iodovinyl)-2'-fluor-2'-deoxyuridine (IVFRU, 36 mg, 78% yield) as a white solid after recrystallization from methanol; mp 108-110OOC; 1 H NMR (DMSO-d 6 3.65 1 H, igem 12Hz, 3.82-3.96 (in, 2H, 4.20 (dd, 1H, J 3 23 Hz, J 2 3 6 Hz, 5.06 (dd, I H, J 2 'F 54 Hz, J 2 3 6 Hz, 5.47 (br s, 1 H, C-5' OH, exchanges with deuterium oxide), 5.72 (br s, 1 H, C-3' OH, exchanges with deuterium oxide), 5.92 1H, J1lF 18 Hz, 7.09 1H, J,.n 16 Hz, CH=CHI), 7.20 (d, 1 H, Jtmn 16 Hz, CH=CHI), 8.23 1IH, uracil 11.6 (br s, 1IH, NH, exchanges with deuterium oxide); Exact Mass Calcd for C, 1

H

12 F1N 2 0 5 397.9775. Found (HRMS): 397.9773 Anal. Calcd. for C, 11 H1 12 F1N 2 0 5 C, 33.19; H, 3.04; N, 7.04.

Found: C, 33.83; H, 3.32; N, 7.00.

Schematic for ExamDle 1 0 117 NH~ H\CCSIMe3 [(Ph) 3

P]

2 Pd(I)CI 2 HO-k 0 (4) (n-Bu) 3 Sn H

THF

(3) OH F (2) Me 3 Si 0 0 I NH ICI

NH

HO 0 Nk e HO 0 OH F OH F

IVFRU

WO 96/12508 PCTIC AQ~Iflh1~O2 EXAMPLE 2 The related (E)-5-(2-iod ovinyl)-2'-fl uoro-2-deoxya rab inou rid ine (IVFAU), (E)-5-(2-iodovinyl)arabinouridine (IVAU), and (E)-5-(2-iodovinyl)-2'-deoxyu rid ine (IVDU) -compounds have been prepared, using a procedure similar to that used in Example 1, as illustrated in the schematic for Example 2 shown below using an equivalent quantity of the (E)-5-iodouracil nucleoside of formula in place of (E)-5-iodo-2'-fluoro-2'-deoxyuridine in Example 1, to afford IVFAU, IVAU and IVDU which had melting points of 176-178 IC, 171-175 *C and 166-170 respectively.

Schematic for Example 2 H SiMe 3 (n-BU) 3 Sn H [(Ph) 3

P]

2 Pd(l)CI 2 (4)

THF

ICI

(6) MeCN IVFAU (RI F, R 2

R

3

=H)

IVAU (RI OH, R2 H, R 3

=H)

IVDU (Ri H, R 2

R

3

=H)

WO 96/12508 PCT/CAos/f53OI -31 EXAMPLE 3 Carrier Added Synthesis of 31 1]-(E)-5-(2-iodovinyl)-2'-fluoro-2'deoxyuridine 31 1]-IVFRU} (See schematic presentation following example) A solution of 13 1]-Nal (74 MBq) in 0.1 N NaOH (5 pL) was placed in a Wheaton vial and then a solution of ICI (124 pg, 0.765 pmol) in acetic acid-acetonitrile v/v; 10 pL) was added. A solution of (E)-5-(2-trimethylsilylvinyl)- 2'-fluoro-2'-deoxyuridine (500 pg, 1.53 pmol) in acetic acid-acetonitrile v/v, pL) was then added to the contents in the Wheaton vial and the reaction was allowed to proceed for 15 min at 25 The product was purified by preparative reverse phase HPLC using a Whatman Partisil M9 10/25 C8 column by isocratic elution with acetonitrile-water (70:30, v/v) at a flow rate of 1.5 MI/min. The product [1311]-IVFRU had a retention time of 11.76 min under these conditions whereas unreacted (E)-5-(2-trimethylsilylvinyl)-2'-fluoro-2'-deoxyuridine had a retention time of 24.19 min.

131 ]-(E)-5-(2-iodovinyl)-2'-fluoro-2'-deoxyuridine (63 Mbq, 85 radiochemical yield, 98 radiochemical purity, specific activity 252 GBq/mmol) prepared using this procedure displayed identical chromatographic retention times to that observed with an authentic sample of unlabelled IVFRU under a variety of chromatographic conditions.

Schematic for Example 3 MeSI 131 0 N O1311]-Nal

N

o -ICI

N

HO 0 HO 0 OH F OH F [1311]-IVFRU WO 96/12508 32 PCT/CA95/00593 EXAMPLE 4 No Carrier Added Synthesis of ['131 l]-(E)-5-(2-iodovinyl)-2'-fluoro-2'deoxyuridine {[131 l]-IVFRU} (See schematic presentation following example) A solution of 5 2 -trimethylsilylvinyl)-2'-fluoro-2'-deoxyuridine (100 Pg, 0.306 pmol) in acetic acid-acetonitrile v/v, 10 pL) was added to a solution of [1 131 1]-Nal (11.3 Mbq) in 0.1 N NaOH (5 pL) in a Wheaton vial. A solution of N-chlorosuccinimide (100 pg, 0.749 pmol) in acetic acid-acetonitrile v/v, 10 pL) was then added, the reaction was allowed to proceed for 30 min at 25 OC, and the reaction was terminated by the addition of sodium thiosulfate (100 pg, 0.632 /umol) in water (10 pL). The reaction mixture was separated by HPLC using the procedure described in Example 3 to afford 131 I]-(E)-5-(2-iodovinyl)-2'-fluoro-2'-deoxyuridine Mbq, 71 radiochemical yield, 98 radiochemical purity, specific activity 5.29 TBq/mmol) as a no carrier added product.

Schematic for Example 4 Me 3 Si 0 1311 0 NH

NH

HO3 N O [1311]-Nal N "kO 0 N-chlorosuccinimide HO 0 OH F OH F [1311].IVFRU WO 96/12508 1Dr'rr/ WO 96/12508 33 EXAMPLE The related [1241]-, [1251]_. and 131 ]-labelled IVFAU, IVAU and IVDU compounds can be prepared, using a procedure similar to that used in Example 3 using an equivalent quantity of the (E)-5-(2-trimethylsilylvinyl)uracil nucleoside of formula in place of (E)-5-(2-trimethylsilylvinyl)-2'-fl u oro-2'-deoxyu rid ine in Example 3, to afford [12311-, [12411-, [1251]-, and 131 -labelled IVFAU, IVAU and IVDU.

For example, [1 2 1 ]-IVDU was prepared using this procedure (59% radiochemical yield, 98% radiochemical purity, specific activity of 12.7 Gbq/mmoi).

Schematic for Examrle Me 3 Si 00 NH [11]IINH HO aUO HO 0O

R

1

R

OR

3

R

2 OR 3

R

2 VFAU (R F, R 2

R

3

H)

IVAU (RI OH, R 2

R

3

H)

1~~VDU (RI H, R2= H, R3= H) 1231, 1241, 1251, 1311 WO 96/12508 PCTICA9/00593 -34- EXAMPLE 6 (E)-5-(2-iodovinyl)-3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-fluoro- 2'-deoxyuridine (IVFRU-CDS) (See schematic presentation following example) lodomethane (165 mg, 1.16 mmol) was added to a solution of (E)-5-(2-trimethylsilylvinyl)-3'-O-(3-pyridylcarbonyl)-2'-fluoro-2'-deoxyuridine (26 mg, 0.058 mmol) in acetone (3 MI) and the reaction mixture was heated at reflux for 16 h.

Removal of the solvent in vacuo and trituration of the residue obtained with ether (3 x MI) gave (E)-5-(2-trimethylsilylvinyl)-3'-O-(1-methylpyridinium-3-carbonyl) fluoro-2'-deoxyuridine iodide (31 mg, 90 as yellow crystals, mp 172-174 1H NMR (DMSO-d 6 6 0.1 9H, SiMe 3 3.7-3.9 2H, 4.44 (br s, 4H, NCH 3 5.44 1H, JOH.5 3 Hz, C-5' OH, exchanges with deuterium oxide), 5.55-5.58 1H, 5.58-5.80 1H, 6.12 (dd, 1H, Jr., 17.0, J1, 2 1.8 Hz, 6.60 2H, CH=CHTMS), 8.22 1H, uracil 8.29 (dd, 1H, J 4 7.6, J.

6 Hz, pyridinium 9.06 1H, J4. 7.6 Hz, pyridinium 9.23 1H, Js. Hz, pyridinium 9.66 1H, pyridinium 11.62 (s,1H, NH, exchanges with deuterium oxide). Anal. Calcd for C 2 1

H

27 FIN30 6 Si.1/2 H 2 0: C, 42.00; H, 4.71; N, 6.99. Found: C, 42.37; H, 4.57; N, 6.57.

Iodine monochloride (1.7 mg, 0.01 mmol) was added to a solution of (E)-5-(2-trimethylsilylvinyl)-3'-O-(1-methylpyridinium-3-carbonyl)-2'-fluoro-2'deoxyuridine iodide (6 mg, 0.0101 mmol) in acetonitrile (1 MI), immediately after its preparation in the previous reaction, and the reaction mixture was stirred at 25 OC for min. Removal of the solvent in vacuo gave (E)-5-(2-iodovinyl)-3'-O-(1methylpyridinium-3-carbonyl)-2'-fluoro-2'-deoxyuridine iodide as a yellow solid which was used immediately without further purification in the subsequent reaction. This yellow solid was dissolved in a two phase solvent system comprised of water-ethyl acetate (1 MI each), sodium dithionite (10 mg, 0.057 mmol) and sodium bicarbonate (4 mg, 0.048 mmol) were added and the reaction was allowed to proceed at 25 °C :O with stirring for 15 min. The ethyl acetate fraction was washed with water (1 MI) and the ethyl acetate solution was dried (Na 2

SO

4 The solvent was removed in vacuo WO 96/12508 PCTCA95IOOS93 35 and the product was purified using a short neutral aluminum oxide column with chloroform-methanol (90:10, v/v) as eluent to afford 2 -iodovinyl)3'o(l1methyll, 4 -d ihyd ropyridyl3ca rbonyl2'fluoro2'-deoxyu rid ine (IVFRU-CDS, 3 mg, 59%) as a yellow solid after recrystallization from methanol, mp 131-133 00; IH NMVR (CDCl 3 6 3.02 3H, NCH.), 3.11 (br s, 2H, dihydropyridyl 3.86 and 4. 10 (two d, 1IH each, Jgem 12.5 Hz, 4.27 1IH, J 3 4 7.5 Hz, 4.87 (dt, 1IH, J 56 3.8 Hz, dihydropyridyl 5.18 (in, 1H, J 2 51 Hz, 5.32 (in, 1IH, 5.68 1IH, J 5 ,6 8.0 Hz, dihydropyridyl H-6) 6.05 1H, J1',F 16 Hz, H-i1), 7.06 (d, 1H, Jtmn 15 Hz, CH=CHI), 7.11 1H, dihydropyridyl H-2) 7.40 1H, Jtrans Hz, CH=CHI), 8.12 1H, uracil Anal. Calcd. for C 18

H

19 F1N306. H20: C, 40.24; H, 3.94; N, 7.82. Found: C, 40.52; H, 4.09; N, 7.61.

WO 96/12508 PCTCA95/00593 36 Schematic for Examole 6 Mel Acetone ICI, Acetonitrile Na 2

S

2

O

4 NaHCO 3

H

2 0-EtOAc IVF RU-C DS EXAMPLE 7 The related (E)-5-(2-iodovinyl)-3'-O-( 1-methyl-I ,4-dihyd ropyridyl-3carbonyl)-2'-fl uoro-2'-deoxya rabinou rid ine (IVFAU-CDS) and (E)-5-(2-iodovinyl)-3'-O- (1 -methyl-i 1,4-d ihyd ropyrid yl-3-ca rbonyl)-2'-d eoxyu rid ine (IVDU-CDS) compounds WO 96/12508 IPCTICA95/00593 37 have been prepared by a procedure similar to that used in Example 6 using an equivalent quantity of a (E)-5-(2-trimethylsilylvinyl)uraci nucleoside of formula in place of (E)-5-(2-trimethylsilylvinyl)-3'-o-(3-pyridylcarbonyl)-2'fluoro-2'-deoxyuridine in Example 6, to afford IVFAU-CDS and IVDU-CDS with melting points of 148-1 52 00 and 165-168 00, respectively.

Schematic for Example 7 Mel Acetone ICI, Acetonitrile Na 2

S

2 0 4 NaHCO 3 IVFAU-CDS (RI F, R 2

H)

IVDU-CDS (Rj R 2

H)

WO 96/12508 PCT/CA95/00593 -38- EXAMPLE 8 Carrier Added Synthesis of 131 1]-(E)-5-(2-iodovinyl)-3'-O-(1-methyl-1,4dihydropyridyl-3-carbonyl)-2'-fluoro-2'-deoxyuridine 31 1]-IVFRU-CDS} (See schematic presentation following example) A solution of (E)-5-(2-trimethylsilylvinyl)-3'-O-(1-methylpyridinium-3carbonyl)-2'-fluoro-2'-deoxyuridine bromide (1 mg, 0.00184 mmol) was dissolved in acetonitrile (100 pL) which was immediately added to a stirred solution of Icl (30 pg, 0.185 pmol) and [1 3 11]-Nal (26.8 Mbq) in acetonitrile (20 pL). The reaction was allowed to proceed for 15 min at 25 the solvent was evaporated over a stream of nitrogen gas and the residue obtained was then dissolved in degassed water (200 and ethyl acetate (200 pL). Sodium dithionite (4 mg, 0.0229 mmol) and sodium bicarbonate (2 mg, 0.0238 mmol) were added and the reaction was allowed to proceed with stirring for 20 min at 25 OC. The ethyl acetate layer was then separated and the solvent was evaporated over a stream of nitrogen. The residue obtained was dissolved in methanol and purified by preparative reverse phase HPLC using a Whatman Partisil M9 10/25 C8 column. Isocratic elution with acetonitrile:water (60:40, v/v) at a flow rate of 2.0 MI/min gave pure 13 1 1]-(E)-5-(2-iodovinyl)-3'-O(1methyl-1, 4 -dihydropyridyl-3-carbonyl)-2'-fluroro-2'deoxyuridine {[131 I]-IVFRU-CDS}, retention time of 19.54 min, in 14% isolated radiochemical yield (3.8 Mbq), having a specific activity of 4.3 Gbq/mmol and 98% radiochemical purity after HPLC purification.

WO 96/12508 PCTICA95/rj0593 39 Schematic for Example 8 Me 3 IdJ, [1 3 11]-NaI, Acetonitrile Na 2

S

2 0 4 NaHCO 3

H

2 0-EtOAc ''Br- Me (9) [131QJ-IVFRU-CDS EXMPLE 9 The related 1 12411-, 1251]-. and 13 l]-labelled IVFAU-CDS, IVAU-CDS and IVDU-CDS compounds can be prepared, using a procedure similar to that used in Example 8 using an equivalent quantity of the (E)-5-(2-trimethylsilylvinyl)uracil nucleoside of formula in place of (E)-5-(2-trimethylsilylvinyl)-3'-O-( 1methylpyrid inium-3-carbonyl)-2'-fluoro-2'-deoxyu rid ine bromide in Example 8, to afford 1231]-, [1241].. [125 11-, and 1 31 1]-labelled IVFAU-CDS, IVAU-CDS and IVDU-CDS. For example, ['1]-IVDU-CDS was prepared using this procedure (59% radiochemical yield, 98% radiochemical purity, specific activity of 12.7 Gbq/mmol).

WO 96/12508 PrT/r A o/iAnnln 40 Schematic for Example 9 Me 3 0

NH

N" O ICI, l]-Nal, Acetonitrile

O

H s Na2S 2 04, NaHCO 3 H20-EtOAc H 0 Ri c-o Rz R2 I Br- Me

I

Me rQ-IVFAUCDS (Ri F, R2= H) r-IVAU-CDS (Ri O, R2= H) Q-VDU-CDS (R 1 R2=H) 1231, 1241, 1251, 1311 BIOLOGICAL EVALUATIONS PERTINENT TO THE INVENTION EXAMPLE Partition Coefficients and Pseudo-first Order Oxidation Rate Constants Partition coefficients (log P) were measured by determining the distribution of the test compound between a presaturated mixture of n-octanol and water The analytical method consisted of a modified shake-flask technique and ultraviolet (UV) spectrometry quantitation after centrifugal separation of the two phases. The concentration of the test compound in the octanol phase prior to distribution was 0.5 mM. The dihydropyridine chromophore (Amax 360 nm) was used for quantitative UV analysis of IVDU-CDS and IVFRU-CDS, for which the results are shown in Table 1. The log P values for IVDU-CDS and IVFRU-CDS were significantly higher than those for IVDU (log P 1.10) and IVFRU (log P 1.21).

WO 96/12508 PCT/CA95/00593 -41 Pseudo-first order rate constants and half-lives for the oxidation of IVDU-CDS and IVFRU-CDS were determined in 50% mouse blood, 20% mouse brain homogenate and 20% mouse liver homogenate. A solution of the test compound in DMSO (10 Mm) was added to each matrix and incubated at 37 At various times after test compound addition, an aliquot was removed and added to acetonitrile (100 The samples were immediately centrifuged and the supernatant was analyzed by quantitative reverse phase HPLC. The rate of disappearance of the 1-methyl-1,4dihydropyridine-CDS was determined by UV detection at 360 nm. The results, which are illustrated in Table 1, demonstrate that the 1-methyl-1,4-dihydropyridine promoiety undergoes facile oxidation to a pyridinium salt in selected biological tissues and fluids.

TABLE 1 Oxidation Rates in Mouse Tissues and Partition Coefficients k x 10 3 min-1 tl/2 (min) Compound blood brain liver blood brain liver log P IVDU-CDS 5.22 9.61 10.81 133 72 3 1.77 IVFRU-CDS 2.70 3.10 5.31 256 223 130 1.83 EXAMPLE 11 In Vitro Antiviral Activity Against Herpes Viruses.

The in vitro antiviral activities for some selected test compounds against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type-2 (HSV-2) have been determined using a cytopathic effect (CPE) inhibition assay using cultured human foreskin fibroblasts (HFF). Antiviral activity against varicella zoster virus (VZV) has been determined using a plaque reduction assay. A cell proliferation WO 96/12508 PCT/CA95/00593 -42assay using uninfected HFF cells was employed for the cytotoxicity assay. The results indicated that the test compounds are potent antiviral compounds against a battery of herpes viruses, and that the test compounds exhibited minimal host cell toxicity.

KBALB Cell Models for Gene Therapy Studies The KBALB sarcoma models used in these studies are derived by exposing wild-type KBALB cells to Moloney murine leukemia virus (MMLV) producer cell supernatants containing replication incompetent ecotropic retroviruses. The KBALB-STK cell line was transduced with a vector possessing the HSV-1 TK and neomycin resistance genes, whereas the KBALB-LNL cells were transduced with a vector possessing only the neomycin resistance gene. These transduced cell lines are cultured in media containing the antibiotic G-418 to select for those cells expressing the neomycin resistance gene. These rapidly growing sarcomas have been characterized extensively and do not produce replication competent retrovirus particles Freeman et al., Cancer Res., 53, 5274 (1993)].

EXAMPLE 12 In Vitro Cellular Uptake of Radiopharmaceuticals in Transduced or Non- Transduced Sarcoma Cell Lines KBALB-STK, KBALB-LNL, or wild type KBALB cells were grown to confluency in 24 well culture plates. 125 1]-IVDU (sp. act. 12 Gbq/mmol), [1251]- IVFRU (sp. act. 11 Gbq/mmol), 13 '1]-IVDU-CDS (sp. act. 2.8 Gbq/mmol) or [1311]- IVFRU-CDS (sp. act. 4.3 Gbq/mmol) were added to each well in 100 pL saline solution and incubated at 37 OC. At varying times after exposure to the test compound, the supernatant was removed, the cells rinsed with saline, and the adherant cells were then trypsinized prior to their removal. Cellular uptake was determined by gamma counting using a Beckmann 8000 gamma counter. The results, which are presented in Figures 1 and 2, indicate that selective uptake occurred for 125 1]-IVDU and 125 1]-IVFRU in KBALB-STK cells that express HSV-1 TK relative to KBALB or KBALB-LNL cells.

WO 96/12508 PCT/CA95/00593 -43- EXAMPLE 13 Biodistribution of 131 1]-IVFRU in Mice Bearing KBALB or KBALB-STK Tumors KBALB or KBALB-STK cells (1 x 10 5 cells) were injected subcutaneously into the flank of male Balb-c mice. Palpable tumors appeared 14 days after injection.

[13 1 1]-IVFRU (370 KBq, sp. act. 59 Gbq/mmol) was injected via the tail vein into each tumor bearing animal. Three animals were sacrificed and dissected at each time interval. The radioactivity uptake by selected tissues of interest were determined using a Beckman 8000 gamma counter. Tissue uptake is expressed as a percentage of the administered dose per gram of tissue versus time in Figures 3 and 4. The tumor to blood ratio is presented for both tumor models in Figure 5. Preferential tumor uptake is evident in tumors expressing HSV-1 TK resulting in a peak tumor/blood ratio of approximately 3 for animals bearing KBALB-STK tumors at 8 hours post injection. In contrast, wild type KBALB tumor bearing animals showed significantly less tumor radioactivity and much lower tumor/blood ratios were observed at all time points relative to those for KBALB-STK tumors.

EXAMPLE 14 Scintigraphic Imaging of KBALB-STK Tumors BALB-STK cells (1 x 10' cells) were injected subcutaneously into the flank of 12 Balb-c mice. All inoculated animals developed palpable tumors suitable for imaging after 14 days at which time the tumors were approximately 10 mm in diameter. Static images were obtained following injection of 3.7 Mbq of [1 31 1]-IVFRU (sp. act. 252 Gbq/mmol) via the tail vein. Animals were placed in the prone position under a pinhole collimator using a Searle gamma camera (Scintiview) with data manipulated on an ADAC computer (DPS 3300). Images were acquired over minutes using a 256 x 256 matrix. After initial imaging, a group of animals (n=6) were administered an intraparietoneal (ip) injection of ganciclovir (100 mg/kg) once daily for seven consecutive days. The control group received daily saline injections. After four days, the animals were administered 13 1 1]-IVFRU as described previously and imaging was performed using the same acquisition protocol described above. It was noted that the treatment group tumor size had shrunk to an average WO 96/12508 PCT/CA95/00593 -44size of less than 5 mm. After 7 days of ganciclovir treatment, the KBALB-STK tumors had completely regressed in all treated animals. The tumors in the saline administered control animals continued to grow until termination of the experiment.

The scintigram, presented as Figure 6, for an animal prior to ganciclovir treatment, which was imaged 8 hours after 3 1 1]-IVFRU administration, illustrates selective uptake of 13 'I]-IVFRU into KBALB-STK tumors expressing HSV-1 TK. The scintigram, presented as Figure 7, is for the same animal 8 hours after 13

I]-IVFRU

administration following 4 days of ganciclovir treatment. The majority of the radioactivity present in the scintigram image shown in Figure 7 was present in the urinary bladder, since the mouse had not voided, while the region of the shrinking tumor remnant was relatively devoid of activity.

Claims (47)

1. The use of a labelled compound to monitor the extent and location of a foreign gene throughout a population of cells following transfection, by administering to the cells an effective dose of the labelled compound so that the labelled compound interacts selectively with a protein expressed by the foreign gene to produce a labelled product and then detecting the labelled product, wherein the labelled compound is selected to interact selectively with the protein expressed by the foreign gene such that the labelled product becomes trapped within those of the cells in which the protein has been expressed by the foreign gene.

2. A method for monitoring the extent and location of a foreign gene throughout a population of cells following transfection, comprising the following steps: administering to the cells an effective dose of a labelled compound so that the labelled compound interacts selectively with a protein expressed by the foreign gene to produce a labelled product; and detecting the labelled product; wherein the labelled compound is selected to interact selectively with the protein expressed by the foreign gene such that the labelled product becomes trapped within those of the cells in which the protein has been expressed by the foreign gene.

3. A method as claimed in claim 2, wherein the protein expressed by the foreign gene is not naturally occurring within the cells.

4. A method as claimed in claim 2 or claim 3, wherein the labelled compound is a radiolabelled compound which interacts with the protein expressed by the foreign gene to produce a radiolabelled product which can be detected using nuclear medicine imaging techniques. A method as claimed in any one of claims 2 to 4, wherein the foreign gene is a gene selected from eucaryotic or procaryotic cells.

6. A method as claimed in any one of claims 2 to 4, wherein the foreign gene is selected from a virus.

7. A method as claimed in claim 6, wherein the foreign gene is selected from the group of viruses consisting of herpes simplex virus, human cytomegalovirus, varicella zoster virus and Epstein-Barr virus.

8. A method as claimed in claim 7, wherein the foreign gene is a gene which expresses herpes simplex virus thymidine kinase.

9. A method as claimed in any one of claims 4 to 8, wherein the radiolabelled compound is a compound of the formula: X 0 NH N O R 4 0 0 R, OR R 2 or a pharmaceutically acceptable salt thereof, wherein X is a radioactive halogeno substituent, wherein R 1 is a hydrogen, hydroxyl or fluoro substituent, wherein Rz is a hydrogen or fluoro substituent, wherein R 3 is a substituent selected from the group consisting of hydrogen, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, 1-methyl-1,4-dihydropyridyl-3- carbonyl, 3-7C cycloalkylcarbonyl, and alkylcarbonyls with a straight or branched chain having from 1 to 8 carbon atoms, and wherein R 4 is a substituent selected from the group consisting of hydrogen, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, 1-methyl-1,4-dihydropyridyl-3- carbonyl, 3-7C cycloalkylcarbonyl, and alkylcarbonyls with a straight or branched chain having from 1 to 8 carbon atoms. 47 A method as claimed in claim 9, wherein X is a radioactive halogeno substituent selected from the group consisting of 123I, 124j, 131, 75 Br, 78Br and 18F.

11. A method as claimed in claim 10, wherein X is 1231.

12. A method as claimed in claim 9, wherein X is a radioactive halogeno substituent selected from the group consisting of 123, 124I and 131.

13. A method as claimed in claim 12, wherein R, is hydrogen, wherein R 2 is hydrogen, wherein R 3 is hydrogen and wherein R 4 is hydrogen.

14. A method as claimed in claim 13, wherein X is 123I. A method as claimed in claim 12, wherein R, is hydrogen, wherein R 2 is hydrogen, wherein R 3 is 1-methyl-1,4-dihydropyridyl-3-carbonyl, and wherein R 4 is hydrogen.

16. A method as claimed in claim 15, wherein X is 123I.

17. A method as claimed in claim 12, wherein Ri is hydrogen, wherein R 2 is fluorine, wherein R 3 is hydrogen and wherein R 4 is hydrogen.

18. A method as claimed in claim 17, wherein X is 123I.

19. A method as claimed in claim 12, wherein R, is hydrogen, wherein R 2 is fluorine, wherein R 3 is 1-methyl-1,4-dihydropyridyl-3-carbonyl, and wherein R 4 is hydrogen. A method as claimed in claim 19, wherein X is 1 23 I.

21. A method as claimed in claim 12, wherein R, is fluorine, wherein R 2 is hydrogen, wherein R 3 is hydrogen and wherein R 4 is hydrogen.

22. A method as claimed in claim 21, wherein X is 123I.

23. A method as claimed in claim 12, wherein R 1 is fluorine, wherein R 2 is hydrogen, wherein R 3 is 1-methyl-1,4-dihydropyridyl-3-carbonyl, and wherein R 4 is hydrogen.

24. A method as claimed in claim 23, wherein X is 123I. A method as claimed in claim 12, wherein R 1 is hydroxyl, wherein R 2 is hydrogen, wherein R 3 is hydrogen and wherein R 4 is hydrogen.

26. A method as claimed in claim 25, wherein X is 123I.

27. A method as claimed in claim 12, wherein R 1 is hydroxyl, wherein R 2 is hydrogen, wherein R 3 is 1-methyl-1,4-dihydropyridyl-3-carbonyl, and wherein R 4 is hydrogen.

28. A method as claimed in claim 27, wherein X is 123I.

29. A method as claimed in claim 9, wherein at least one of R 3 and R 4 is hydrogen. A method as claimed in claim 9, wherein R 4 is hydrogen.

31. The use of a radiolabelled compound in radiotherapy involving a population of cells into which a foreign gene has been transferred, by administering to the cells an effective radiotherapeutic dose of the radiolabelled compound so that the radiolabelled compound interacts selectively with a protein expressed by the foreign gene to produce a radiolabelled product, wherein the radiolabelled compound is selected to interact selectively with the protein expressed by the foreign gene such that the radiolabelled product becomes trapped within those of the cells in which the protein has been expressed by the foreign gene.

32. A method of radiotherapy for use with a population of cells into which a foreign gene has been transferred, comprising the step of administering to the cells an effective radiotherapeutic dose of the radiolabelled compound so that the radiolabelled compound interacts selectively with a protein expressed by the foreign gene to produce a radiolabelled product, wherein the radiolabelled compound is selected to interact selectively with the protein expressed by the foreign gene such that the radiolabelled product becomes trapped within those of the cells in which the protein has been expressed by the foreign gene.

33. A method as claimed in claim 32, wherein the protein expressed by the foreign gene is not naturally occurring within the cells.

34. A method as claimed in claim 32 or claim 33, wherein the foreign gene is a gene selected from eucaryotic or procaryotic cells. A method as claimed in claim 33, wherein the foreign gene is selected from a virus.

36. A method as claimed in claim 35, wherein the foreign gene is selected from the group of viruses consisting of herpes simplex virus, human cytomegalovirus, varicella zoster virus and Epstein-Barr virus.

37. A method as claimed in claim 36, wherein the foreign gene is a gene which expresses herpes simplex virus thymidine kinase.

38. A method as claimed in any one of claims 32 to 37, wherein the radiolabelled compound is a compound of the formula: NH NX O R 4 0 OR 3 R z or a pharmaceutically acceptable salt thereof, wherein X is a radioactive halogeno substituent, wherein R 1 is a hydrogen, hydroxyl or fluoro substituent, wherein R 2 is a hydrogen or fluoro substituent, wherein R 3 is a substituent selected from the group consisting of hydrogen, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, 1-methyl-1,4-dihydropyridyl-3- carbonyl, 3-7C cycloalkylcarbonyl, and alkylcarbonyls with a straight or branched chain having from 1 to 8 carbon atoms, and wherein R 4 is a substituent selected from the group consisting of hydrogen, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, 1-methyl-1,4-dihydropyridyl-3- carbonyl, 3-7C cycloalkylcarbonyl, and alkylcarbonyls with a straight or branched chain having from 1 to 8 carbon atoms.

39. A method as claimed in claim 38, wherein X is a radioactive halogeno substituent selected from the group consisting of 123, 125I and 13 1 I. A method as claimed in claim 39, wherein X is 1311.

41. A method as claimed in claim 39, wherein R, is hydrogen, wherein R 2 is hydrogen, wherein R, is hydrogen, and wherein R 4 is hydrogen.

42. A method as claimed in claim 41, wherein X is 1311.

43. A method as claimed in claim 39, wherein R, is hydrogen, wherein R 2 is hydrogen, wherein R 3 is 1-methyl-1,4-dihydropyridyl-3-carbonyl, and wherein R 4 is hydrogen.

44. A method as claimed in claim 43, wherein X is 311. A method as claimed in claim 39, wherein Ri is hydrogen, wherein R 2 is fluorine, wherein R, is hydrogen and wherein R 4 is hydrogen.

46. A method as claimed in claim 45, wherein X is 131 I.

47. A method as claimed in claim 39, wherein R, is hydrogen, wherein R 2 is fluorine, wherein R 3 is 1-methyl-1,4-dihydropyridyl-3-carbonyl, and wherein R 4 is hydrogen.

48. A method as claimed in claim 47, wherein X is 131I. 51

49. A method as claimed in claim 39, wherein R 1 is fluorine, wherein R 2 is hydrogen, wherein R 3 is hydrogen and wherein R 4 is hydrogen. A method as claimed in claim 49, wherein X is 1311.

51. A method as claimed in claim 39, wherein R, is fluorine, wherein R 2 is hydrogen, wherein R 3 is 1-methyl-1,4-dihydropyridyl-3-carbonyl, and wherein R 4 is hydrogen.

52. A method as claimed in claim 51, wherein X is 131I.

53. A method as claimed in claim 39, wherein R, is hydroxyl, wherein R 2 is hydrogen, wherein R 3 is hydrogen and wherein R 4 is hydrogen.

54. A method as claimed in claim 53, wherein X is 131I. A method as claimed in claim 39, wherein R, is hydroxyl, wherein R 2 is hydrogen, wherein R 3 is 1-methyl-1,4-dihydropyridyl-3-carbonyl, and wherein R 4 is hydrogen.

56. A method as claimed in claim 55, wherein X is 131i.

57. A method as claimed in claim 38, wherein at least one of R 3 and R 4 is hydrogen.

58. A method as claimed in claim 38, wherein R 4 is hydrogen. Dated this sixth day of December 1999 THE GOVERNORS OF THE UNIVERSITY OF ALBERTA Patent Attorneys for the Applicant: F B RICE CO