WO1990001324A1 - PRODUCTION OF RADIOIODINATED 1-β-D-ARABINOFURANOSYL)-5(E)-(2-IODOVINYL)URACIL, AND USES THEREOF, AND RELATED ANALOGUES INCORPORATING ALTERNATIVE HALOGEN RADIONUCLIDES, THE GENERAL RADIOHALOGENATION PRECURSORS, 1-(2,3,5-TRI-O-ACETYL-β-D-ARABINOFURANOSYL)-5(Z and E)-(2-TRIMETHYLSILYLVINYL)URACIL, PROCESSES FOR RADIOHA - Google Patents

Abstract

Radioactive antiviral compounds, with 1-(β-D-arabinofuranosyl)-5(E)-(2-[*I]-iodovinyl)uracil, hereinafter referred to as '[*I]-IVaraU', as the prototype compound for the class of compounds designated as 1-β-D-arabinofuranosyl)-5(E)-(2-[*X]-halogenovinyl)uracil, hereinafter referred to as '[*X]-XVaraU', and novel precursors thereof, 1-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)-5(Z and E)-(2-trimethylsilylvinyl)uracil, hereinafter referred to as 'TMSVaraU', and processes for the preparation thereof, and uses thereof, wherein '*I' stands for a radionuclide of iodine, and '*X' stands for any other appropriate halogen radionuclide. Inclusion of the appropriate gamma or positron emitting iodine, or other appropriate halogen radionuclide, into the structure of [*I]-IVaraU, makes this agent useful as a diagnostic tool for detection of herpes virus infections in vitro and in vivo. Inclusion of the appropriate alpha- and/or beta- and/or gamma- emitting, and/or Auger electron decay-associated, (specifically-nuclear-toxic) isotopes of iodine, or other appropriate halogen radionuclides, into the structure of this agent makes the agent useful as a unique radiotherapeutic tool for herpes virus infections by precise targeting of the lethal effects of alpha and/or beta radiation and/or Auger electron decay effects to the site of viral infection.

Description

PRODUCTION OF RADTOTODINATED

1-(ß-D-ARAP,TNOFURANOSYL)-5(E)-(2-IODOVINYL) URACIL, AND

USES THEREOF, AND RELATED ANALOGUES INCORPORATING

ALTERNATIVE HALOGEN RADIONUCLIDES. THE GENERAL

RADTOHALOGENATION PRECURSORS, 1-( 2 ,3,5-TRT-O-ACETYL-ß- D-ARABINOFURANOSYL)-5(Z and E)-(2-

TRIMETHYLSILYLVTNYL)URACTL, PROCESSES FOR

RADIOHALOGENATION OF SUCH PRECURSORS. AND USES THEREOF

FIELD OF THE INVENTION

This invention pertains to novel radioactive antiviral compounds, with 1-(ß-D-arabinofuranosyl)-5(E)-(2-[*I]-iodovinyl) uracil, hereinafter referred to as w[*I]-IVaraU", as the prototype compound for the class of compounds designated as 1-(ß-D-arabinofuranosyl)-5(E)-(2-[*X]-halogenovinyl) uracil, hereinafter referred to as "[*X]-XVaraU", and novel precursors thereof, 1-(2,3,5-tri-O-acetyl-ß-D-arabinofuranosyl)-5( Z and E) - (2-trimethylsilylvinyl) uracil, hereinafter referred to as "TMSVaraU", and processes for the preparation thereof, and uses thereof, wherein "*I" stands for a

radionuclide of iodine, and *X stands for any other appropriate halogen radionuclide.

Inclusion of the appropriate gamma or

positron emitting iodine, or other appropriate halogen radionuclide, into the structure of [*I] -IVaraU, makes this agent useful as a diagnostic tool for detection of herpes virus infections in vitro and in vivo . Inclusion of the appropriate alpha- and/όr beta- and/or gamma-emitting, and/or Auger electron decay-associated,

(specifically nuclear-toxic) isotopes of iodine, or other appropriate halogen radionuclices, into the structure of this agent makes the agent useful as a unique radiotherapeutic tool for herpes virus

infections by precise targeting of the lethal effects of alpha and/or beta radiation and/or Auger electron decay effects to the site of viral infection.

BACKGROUND OF THE INVENTION Yamasa Shoyu Company Ltd. has developed a group of nonradioactive antiviral compounds known as 1- (ß-D-arabinofuranosyl)-5(E)-(2-halogenovinyl) uracils (XVaraϋ's) and methods of preparation of same. The prefix "halogeno", hereinafter designated as "X" in the abbreviated form, includes bromo, chloro, and iodo.

The bromo derivative, known as 1- (ß-D- arabinofuranosyl)-5(E)-(2-bromovinyl) uracil, or

"BVaraU", "BV-araU", or "BVAU", is easy to synthesize, owing to the high reactivity of the halogen, bromine. As an antiviral agent, BVaraU is effective against herpes virus infections of man and animals which are associated with viral-induced (deoxy) thymidine kinases.

A list of the known Yamasa patents protecting unlabeled BVaraU as an antiviral therapeutic agent are as follows:

United States Patent # 4,386,076

Canadian Patent # 115,024

Canadian Patent # 1,204,108

Japanese Patent # 1,157,897

European Patent # 3112

A list of the known publications describing BVaraU and/or other halogenovinyl-arauracils regarding antiviral activity in vitro and/or in vivo and/or their mechanisms of action are attached to this application as "Appendix A" [references 1-44]. None of these patents or publications disclose, consider, or reach the radiolabeling of XVaraU to [*X] -XVaraU, or related substances; nor do they disclose or provide a practical method of

synthesis which could be used to produce [*I] -IVaraU; nor do they disclose, imply, or teach the creation of a stable precursor to [*I] -IVaraU; nor do they discuss the use of XVaraU' s, [*X] -XVaraU' s, or related

substances or their radiolabeled analogues as potential diagnostic agents; nor do they mention the use of radiolabeled analogues of the XVaraU' s or related substances as targeted radiotherapeutic antiviral agents which derive their antiviral activity by precise targeting of the lethal effects of alpha and/or beta radiation and/or Auger electron decay effects to the site of viral infection.

Herpes simplex virus, herpes (varicella) zoster virus, cytomegalovirus, and Epstein Barr virus, hereinafter referred to as "HSV", "VZV", "CMV", and "EBV", respectively, are common human herpesviruses, which significantly contribute to a number of important ailments which afflict mankind. Most often, HSV and VZV are associated with skin or mucous membrane lesions which readily lend themselves to rapid and accurate viral diagnosis, generally using standard swab/culture techniques. By contrast, target organ and life-threatening infections with these viruses often evade diagnosis, because of the difficulty in achieving access to infected tissues on which to apply the culture techniques. The physician is often presented with the choice of either guessing what the patient has contracted based on symptoms and signs or obtaining a piece of deep tissue for analysis by surgical or endoscopic procedures (invasive testing). Recent advances in the chemotherapy of herpes virus infections have resulted in an expansion of the need to eliminate invasive testing, along with its inherent inaccuracies, delays, and morbidity. In certain clinical situations, invasive testing is not practical, e.g., with retinal or other deep ophthalmic infections, and for

neurological complications of herpes infections such as post-herpetic neuralgia or transverse myelitis. In an attempt to overcome the limitations of analyses by viral culture, a variety of nonivasive and/or rapid viral diagnostic techniques, including viral antigen or DNA detection, in cerebrospinal fluid (CSF),

quantitative comparisons of CSF to serum antibody ratios, and other methods have been established. Each technique, to date, has suffered from problems with sensitivity, specificity, and/or speed of detection. To get around these problems, several groups have suggested the possibility of diagnosis of HSV

encephalitis by localized uptake of radiolabeled antiviral nucleosides. Publications referring to such agents are listed in appendix "B" [references 45-50]. Such agents are phosphorylated by HSY-specified

deoxythymidine kinases, a group of herpesviral-specific enzymes, hereinafter designated as "dTK's". The first such hypothesis was published by Saito, et al [45,46], who studied HSV-specific localization with [¹⁴elabeled]-2'-fluoro-5-methyl-1-ß-D-arabinosyluracil ( [¹⁴C] -FMAU) . They demonstrated a high correlation between focal infection and increased [¹⁴C]-FMAU uptake in brain sections. [¹⁴C]-FMAU is phosphorylated by certain herpesviral dTK's, and was specifically

concentrated in areas of infection. These

investigators, however, found significant problems with background counts from [¹⁴C]-FMAU, especially when plasma nucleoside levels were high, and also found that uptake by the choroid plexus was disproportionate to the level of infection. Uptake by rapidly dividing cells was also a problem, since their compound was subject to significant nonspecific phosphorylation by cellular enzymes. Furthermore, the [¹⁴C] isotope, is an impractical radionuclide for clinical imaging.

Although the synthesis was not directly described, FMAU could be labeled with fluorine radionuclides, and the use of ¹⁸F for positron emission scanning was suggested. The authors also suggested the possibility of using 2'-fluoro-5-iodo-1-ß-D-arabinosylcytosine, FIAC. However, FIAC is known to be subject to nonspecific

decomposition, with resulting loss of iodine. In addition, this agent is excessively toxic and can be phosphorylated and incorporated to some extent by uninfected, rapidly dividing cells. This work is also described in and supported by US Patent #4,489,052.

The need for a positron or gamma-emitting radionuclide in order to make clinical diagnosis practical was discussed. US Patent #4,489,052 does not disclose, consider, or teach the use of [*X] -XVaraU or [*I]-IVaraU for this purpose, and was specifically limited to the use of 5-substituted-1-(2-deoxy-2-substituted-D-arabinofuranosyl) pyrimidine nucleosides. It also did not disclose, provide, or teach a method for

radiolabeling of their nucleoside, or any other

nucleoside, with a halogen radionuclide, but merely speculated on that possibility for their class of 5- substituted-1-(2-deoxy-2-substituted-D-arabinofuranosyl) pyrimidine nucleosides.

Acyclovir, 9-[(2-hydroxyethoxy) methyl] guanine, is a "nucleoside analogue" whose antiviral activity is dependent upon herpes virus-induced dTK's. Radiolabeled acyclovir was tested for its potential as a diagnostic agent, but those experiments were too insensitive to demonstrate acyclovir localization based on drug anabolism in infected tissues [47]. This may have resulted from passive uptake of acyclovir by

T-minfected cells, even though the phophorylation of acyclovir is likely enhanced in viral-infected cells. Acyclovir has also failed as a diagnostic agent because of difficulties in producing a stable or clinically practical radionuclide label, since this compound contains no halogen in its chemical structure.

A group from the University of Alberta has studied 5 (E) - (2- [¹³¹I] iodovinyl)-2'-deoxyuridine, herinafter designated as " [^13II] -IVdU", as a diagnostic agent [48]. [¹³¹I]-IVdU is structurally distinct from [*I]-IVaraU. The former is a derivative of 2' -deoxyuridine; the latter a derivative of 1-(ß-D- arabinofuranosyl) uracil. This major difference in the sugar moieties provides the basis for the predominance of metabolic resistance of [*I]-IVaraU to cleavage in vivo . Unlike the present [*I] -IVaraU, the former

[¹³¹I] -IVdU was rapidly cleaved biolocjically into a nonspecific metabolite, [¹³¹I] -iodovinyluracil,

hereinafter designated as " [¹³¹I]-IVU", or "[*I]-IVU" by pyrimidine nucleoside phosphorylases, a group of cellular enzymes, hereinafter designated as "PyNP's".

Attempts by the Alberta group to utilize [¹³¹I]-IVdU for noninvasive herpes diagnosis were thwarted by high background counts resulting from the formation of free [¹³¹1] -iodide (not readily explained) as well as [¹³¹I]- IVU, the cleavage product resulting from the activity of PyNP's [49]. Alternative radiolabeling procedures for this compound have also been described [50]. A second group studied [¹³¹I]-IVdU in HSV encephalitis in a rat model. They did show some correlation of radioactivity with sites of uptake in rat brain. However, their method was clinically impractical, as direct carotid injection, along with chemical

disruption of the blood-brain barrier was required

[51]. The University of Alberta group has also

described the potential use of [¹²⁵I]-, or [¹³¹I ]-5(E) - (2-iodovinyl)-1-(2-deoxy-2-fluoro-ß-D-ribofuranosyl) uracil and certain derivatives, as potential probes for non-invasive diagnosis of HSV encephalitis. That compound demonstrated resistance to glycosidic bond cleavage by PyNP's, although catabolism to an unidentified metabolite and free [¹³¹I] -iodide was observed [52].

Rand, et al [53] used ¹⁹F nuclear magnetic resonance in a murine hepatitis model of herpes simplex infection by probing with a dTK-dependent compound, trifluorothymidine. Trifluorothymidine has been associated with considerable systemic toxicity when, given in therapeutic doses. This method required the use of highly specialized equipment but did not require radiolabeling of the nucleoside probe.

SUMMARY OF THE INVENTION

Processes for preparing radiohalogen compounds of the formula:

wherein X is a radioisotope of iodine, selected from the group consisting of radioactive ^{1 23}I, ¹²⁵I, ¹²⁷I, ¹³¹I, or, alternatively, a radiohalogen selected from the group consisting of radioactive ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ³⁴Cl, or other appropriate radionuclides which comprises: (a) converting uridine to its arabino analogue; (b) protecting the arabino sugar moiety against substitution or degradation with

suitable protecting substances; (c) halogenating the protected analogue at the C5 position; (d) coupling the halogenated analogue with an appropriate compound to form a vinyl compound at the C5 position; (e) radiohalogenating the coupled compound at the terminal carbon atom of the C5-vinyl group; and (f) removing the protecting substance from the arabino sugar moiety. Processes for preparing radiohalogen

compounds of the formula:

wherein X is a radiohalogen isotope selected from the group consisting of radioactive ¹²³I, ¹²⁵I, ¹²⁷I, ¹³¹I, or, alternatively, from the group consisting of ⁷⁵Br, ^7βBr, ⁷⁷Br, ⁸²Br, ³⁴C1, or other appropriate radionuclides which comprises: (a) converting uridine to its arabino analogue; (b) protecting the arabino analogue by acetylation; (c) halogenating the

protected analogue at the C5 position; (d) coupling the halogenated protected analogue with

trimethylsilylacetylene; (e) reducing the coupled compound to a vinyl silane; (f) radiohalogenating the reduced compound; (g) removing the acetyl groups from the arabino ring.

A radiohalogen compound of the formula :

wherein X is a radioisotope of iodine selected from the group consisting of radioactive ¹²³I, ¹²⁵I,- ¹²⁷I, ¹³¹I, or alternatively, a radiohalogen selected from the group consisting of radioactive ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ³⁴Cl, or other appropriate

radionuclides.

DRAWINGS and FIGURES

Figure 1 illustrates in graphical format the process of the invention for synthesizing a precursor compound, 1- (2, 3, 5-tri-O-acetyl-ß-D-arabinofuranosyl)-5(Z) -(2-trimethylsilylvinyl) uracil, followed by preparation of 1-(ß-D-arabinofuranosyl)-5(E) -(2-[*I]-iodovinyl) uracil, as the prototype compound for the class of compounds designated as 1-(ß-D-arabinofuranosyl)-5(E)-(2-[*X]-halogenovinyl) uracil, wherein any halogen radionuclide is used in the process.

Figure 2. Trapping of [¹²⁵I] -IVaraU in HSV-1-infected PRK cells. A variety of HSV-1 mutants are compared demonstrating the dependence of trapping upon

expression of viral dTK's.

Figure 3. Reversibility of trapping [¹²⁵I ] -IVaraU in HSV-1-infected PRK cells. Reversibility of trapping was noted, although incomplete at 24 hours post drugwithdrawal.

Figure 4. Trapping of [¹²⁵I] -IVaraU into HSV-1-infected PRK cells : Effects of varying inocula and times of virus exposure. These results demonstrate that at an inoculum of 1 PFU per cell, [¹²⁵I-] IVaraU trapping will detect growth of HSV-1 as early as 4.0 hours

Figure 5. Localization of [¹²³I] -IVaraU in HSV-1-infected areas of the New Zealand White rabbit infected onto the cribriform plate. A. A photograph of a positive scan from an animal scanned 7 days post-infection which is acutely infected in the nasopharynx and olfactory bulb. B. Scan 7 weeks post-infection of a control animal latently, but not actively infected. Olfactory bulb necrotic but no virus present. Similar appearance to an uninfected control animal.

Figure 6. Radiographic display of a brain scan of HSV-1 (KOS) -infected New Zealand White rabbit infected onto the cribriform plate and scanned 9 days post-infection. Four serial scans, each with three views, are shown, designated A-D in chronological order. Scan 6D was obtained 14.5 hours post-injection with [¹²³I] -IVaraU. An area of intense localization (2.2 X = target :back-ground ratio) is demonstrated in the region of the olfactory bulb.

DETAILED DESCRIPTION OF SPECTFTC EMBODIMENTS OF THE INVENTION

We have demonstrated that cells infected with HSV (and by extrapolation this applies to VZV, EBV, or any dTK-producing herpes virus of human clinical and/or veterinary importance) produce enzyme activity that phosphorylates [*I] -IVaraU, or alternatively, any of the antiviral 5 (E)-(2-halogenovinyl)-1(-ß-D-arabinofuranosyl) uracils, designated herein as "[*X]-XVaraU's". It may also apply to viruses which induce uptake through cellular enzymes without producing a recognizable viral dTK, e.g., CMV. This substrate selectivity for antiviral nucleosides by viral dTK's has been observed for many antiviral compounds. We have observed, in this case, however, that normal, uninfected cells do not exhibit significant uptake of [*I] -IVaraU. In contrast, we have found that cells that are infected with virus rapidly effect uptake of [*I] -IVaraU. Once passage into the infected cell and conversion to its phosphorylated product has occurred, nonspecific breakdown to [*I] -IVUracil was not

observed. Specifically, we have found that [*I] -IVaraU is taken up by infected cells at approximately one to two thousand times the concentrations observed inside normal cells treated with this agent. This highly specific targeting to infected cells is dependent upon the action of herpes virus-induced dTK's which cause the phosphorylation of the nucleoside, and in so doing, trap the resulting phosphate ester inside the cell. At our level of detection, [*I] -IVaraU is not

phosphorylated and/or trapped inside of normal,

uninfected cells.

The tentatively proposed mechanism of action of this agent class is summarized in the graphic below, where. [*I] -IVaraU is used as the prototypic example. l

As shown above, [*I] -IVaraUMP is an

abbreviation for [*I] -IVaraU 5' -monophosphate, the

first nucleotide product made by the herpes virus- specific enzymes known as the dTK's. [*I] -IVaraUMP is

again phosphorylated by the same herpes protein (dTK)

which (for selected herpes viruses) also displays

herpes virus-specific thymidylate kinase activity that

catalyzes the formation of [*I] -IVaraU 5' -diphosphate,

abbreviated [*I] -IVaraUDP. The cell then, may further

process the nucleotide. The PyNP's are widely

distributed human enzymes which have severely thwarted

development of nucleoside antiviral agents. PyNP's

quickly cleave most nucleosides and nucleotides of this class. We have discovered that PyNP's are relatively

inactive against [*I] -IVaraU, owing to the unnatural

sugar moiety, arabinose. PyNP's actions result in

"phosphorolysis" which cleaves the nucleoside into the

sugar and base components. If this activity were

functional with [*I] -IVaraU, it would be converted into

[*I] -IVU, an inactive metabolite. Using our detection

methods, no [*I]-IVU was observed after treatment with

[*I] -IVaraU. Thus, if any is formed, it is in

insignificant quantities. BVaraU is also resistant to

PyNP's cleavage, suggesting that all [*X] -XVaraU's will have similar targeting characteristics. Thus, the physician can tailor the medicine according to its use by selecting the radionuclide for optimal imaging and safety characteristics for gamma scans (for example, by selecting ¹²³I, ¹³¹I, or ⁸²Br), vs. optimal imaging and safety characteristics for positron emission scans (for example, by selecting ¹²³I, ¹²⁷I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ³⁴Cl), vs. prolonged half-life and safety for ease of use in vitro (for example, by selecting ¹²⁵I), vs.

emission of lethal effects of alpha and/or beta

radiation to the site of viral infection resulting in radiotherapeutic antiviral effects mediated by viral dTK's for the targeting of lethal radiation (for example by selecting ¹³¹I, Br), vs. emission of lethal effects of Auger electron decay to the intranuclear site of viral DNA replication resulting in

radiotherapeutic antiviral effects mediated by viral dTK's for the targeting of lethal radiation (for example, by selecting ¹²⁵I, ⁷⁷Br).

We have invented a novel radiolabeled

nucleoside, 1-ß-D-arabinofuranosyl-5(E)-(2-[*I]- iodovinyl) uracil ( [*I] -IVaraU) as well as novel

synthetic methods and general precursors, 1-(2,3,5-tri- O-acetyl-ß-D-arabinofuranosyl)-5(Z and E) - (2- trimethylsilylvinyl) uracil, for preparing this agent, wherein *I represents an iodine radionuclide of choice according to the specific use for the product, and where *I may be further replaced by *X, representing a halogen radionuclide of choice according to the

specific use for the product. The precursor compounds named above can be reacted in the penultimate synthetic step with radiolabeled iodine, using the generally available compounds, Na¹²⁵I, Na¹²³I,and Na¹³¹I or, in the alternative, a less-widely used iodine radionuclide, Na¹²⁷I, or, in the alternative, other halogen

radionuclides, e.g., Na⁷⁵Br, Na⁷⁶Br, Na⁷⁷Br, Na⁸²Br, Na³⁴Cl, or alternative salts of these radiohalogens. Since the mechanism of localization of the nucleoside to infected sites is not dependent upon the nature of the radionuclide, per se, it is quite certain that other suitable halogen radionuclides will become available for use with this invention as future

developments occur in the science and art of clinically applicable radionuclides. It should be noted that the methods we have formulated to produce these novel radiolabeled. agents are very mild, i.e., chemical reactions are conducted in common solvents at or below room temperature. The vinyl-halogen bond is covalent and stable to

manipulation conditions. Other approaches that have been used to introduce radiolabeled halogens into compounds have often involved harsh chemical and/or reactor conditions [49,50]. As ¹²³I will probably be the radionuclide of choice for use as an in vivo diagnostic tool, because of its high degree of

specificity as a gamma emitter and its very short half-life, it was necessary to devise a synthetic method which allowed a short time between acquisition of the radionuclide (as a sodium or ammonium salt) and

completion of the synthesis of the diagnostic agent in a state of pharmacological purity.

By our process, the radiopharmaceutical agents of this invention can be generated quickly in a small laboratory in a hospital's nuclear medicine department. The conditions are mild, standard, and easy to reproduce, and employ inexpensive and

disposable equipment. The success of our processes has been achieved via general precursor agents that react rapidly to give exchange of halogen lor trimethylsilyl. Initial experiments were performed with iodine radionuclides, since ¹²³I, ¹²⁵I, and ¹³¹I provide an ideal combination of clinically useful, safe with long half-life, and lethal radionuclides, respectively.

Our novel method was required to give IVaraU in high yields, with the flexibility required

clinically to give practical on-site synthesis of

IVaraU from the stable but highly reactive precursor, as described above. This general method is not

specific for a particular halogen, and is therefore readily applied to other halogens. As outlined in Figure 1, iodine monochloride reacts with the

vinylsilane at C5 to substitute iodine in place of trimethylsilyl. Iodine monochloride is synthesized in situ as an intermediate from Na*I which is purchased in radioactive form. The overall reaction is rapid. In addition, we have made novel use of phenyliodine (III) dichloride to effect formation of iodine monochloride as a stoichiometric source of iodine radionuclides for such reactions. Alternatively, xenon difluoride has been used to generate the mixed halogens IF, BrF, and ClF in situ, for general radiohalogenation in such reactions.

Example 1

Novel Method of Preparation of IVaraU and its

vinylsilane precursor

(a) Method, of The Vinylsilane Precursor

The reaction sequence of the process of the invention is graphically depicted in Figure 1 of the drawings.

As displayed in Figure 1, commercially available uridine is converted into its arabino

analogue by published methods in high yield [54]. This compound is protected by acetylation, and then

iodinated at C5, and coupled with trimethylsilyl-acetylene, according to our published procedure [55-57]. Careful reduction with a Lindlar catalyst gives good conversions to TMSVaraU.

(C) Unlaheled Tordination of Tri-O-aoetyl-TMSV-araU:

(i) With phenyliodine (III) dichloride

[*I] -IVaraU was synthesized from the precursor, TMSVaraU, with iodine monochloride (ICl) produced in situ from sodium iodide and

phenyliodine (III) dichloride. Phenyliodine (III) dichloride serves as oxidant and chlorine donor, resulting in the formation of iodine monochloride, which subsequently effects replacement of the TMS group by iodine on the vinyl side chain (at C2) with the trans (E) configuration. Sodium iodide (12 mg, 0.08 mMole, 1.3 eq) was added to a 3 ml reacti-vial equipped with a stir bar and wrapped in aluminum foil to protect it from light. 150 μi of water was added followed by 1 ml of analytical grade benzene. The vial was placed in an ice bath and 20 mg (0.074 mMole, 1.2 eq) of

phenyliodine (III) dichloride was added and the reaction was stirred for 20-30 minutes at -10ºC. The remaining ice was removed from the bath and 28 mg (0.06 mMole) of TMSVaraU was added. The reaction mixture was allowed to warm from 10ºC to 22°C (ambient) over the course of 1/2 hour and stirring was continued for an additional 2 hours. This reaction time can be reduced. Based on HPLC studies, the reaction was rapid, but was extended to this time because of the dilute solution. At 55 minutes there was 70% Iodo and 30% unreacted TMSVaraU. The reaction was quenched by the addition of 1 ml of an aqueous 5% NaHSO₃ (sodium bisulfite) solution and allowed to stir for 20-30 minutes, to destroy unreacted ICl. Shorter stirring times resulted in a colored product eluting from the silica gel column. The benzene layer was removed and applied to a dry column of silica gel prepared by filling a 10" Pasteur pipet 1/2 full of silica gel. The aqueous bisulfite layer was washed with an additional 1 ml of benzene and this organic layer was also applied to the column . The column was then washed with 2 column volumes (~2 ml) of dichloromethane to remove the phenyliodine (III) dichloride by-product followed by elution with 3 column volumes of dichloromethane: ethyl acetate [CH₂CI₂ :EtOAc (2:1)]. The product was found in the first two 1 ml fractions containing ethyl acetate. Solvent was removed by passing an inert gas over the reacti-vial containing the desired fractions.

(ii) With xenon difluoride

Experiments with xenon difluoride as the oxidant and fluorine donor work equally well with NaI, and additionally, give ge eral access to other

radiohalogens for synthesis of the radiobromo- and radiochloro-vinyl compounds

(iii) Other oxidants such as, but not limited to, N-halosuccinimides, chloramine-T, or polymer-bound derivatives can also be used with a radioiodide salt and TMSVaraU. (d) Removal of the Acetyl Blocking

Groups to Yield IV-ara-U: The above tri-O-acetyl compound (ca. 23 mg) was dissolved in HPLC grade methanol and a small piece of sodium metal (somewhat larger than a pin head) was added. The solution was stirred for 40 minutes at room temperature while the reaction proceeded. This

reaction was monitored by TLC (chloroform: methanol,

8:2). The deprotected compound was found to have an R_f of ~0.75. The methanol solution was neutralized by the careful addition of a solution of two drops of

concentrated HCl in 2.5 ml of 100% ethanol and checked with pH paper, range: 6.0-8.0. The solvent was removed by evaporation. The solid remaining had a yellow colour associated with it, which could be removed to a great extent by stirring with ethyl acetate or dry ether. This treatment results in -15 mg of an off-white product containing some sodium chloride. HPLC on the deprotected compound showed a retention time of 2.2 minutes under the following conditions: Cis (4 mm × 25 cm); 47% B in A [A: 0.1% trifluoroacetic acid (TFA) in H₂O; B: TFA in 60% acetonitrile (MeCN) in H₂O]. This compound had identical spectroscopic and chromatographic mobility properties when compared directly with an authentic sample of IVaraU. It melted with decomposition from approximately 175ºC. It had

ultraviolet absorption maxima at 256 and 300 nm and gave a mass spectral molecular ion at m/z 396. The elemental analytical data for this compound agreed to ±0.4% of theoretical values. The compound had ¹³C nuclear magnetic resonance chemical shifts in dimethyl sulfoxide solution at δ 149.17 (C2), 161.81 (C4),

110.02 (C5), 140.96 (C6), 137.06 and 84.70 (ethenyl Cl and C2), 85.29 (Cl'), 75.23 (C2'), 74.93 (C3'), 77.54 (C4'), 60.44 (C5').

Example 2

(a) Prepa rat ion of radio labeled ¹²⁵IVaraU from the vinylsilane precursor. TMSVaraU.

The previous reaction (Example 1) yielded large

quantities of IVaraU in the presence of sodium iodide. In order to synthesize radiolabeled material, however, the reaction required further dilution, to accomodate the small quantities of Na[*I]-I as pure radionuclide. One "carrier-added" product was made at low specific activity, in order to avoid altering the total

concentrations of the reactants. This was accomplished by mixing a commercial sample of Na [¹²⁵I] -I/NaOH (2 mCi in 20 μl : specific activity 17 Ci/mg) with unlabeled Nal in approximately 1:5000 ratio prior to the

reaction. This solution was neutralized with an equal volume of phosphate buffered physiological saline (pH 7.4), by adding the saline solution to the V-vial containing the radionuclide. Subsequently, 25 μl of spectral quality benzene was added, a.long with 0.5 mg of NaI and 1.0 mg of phenyliodine (III) dichloride. The reaction mixture was shaken vigorously and placed in the plastic safety container (which is supplied with the ¹²²I-sodium iodide), to shield it from light. The reaction was allowed to proceed for 15 minutes at room temperature, with frequent shaking. Two layers were visible; a dark red upper organic layer, and a slightly yellow-colored aqueous layer. 1.4 mg of TMSVaraU was dissolved in 25 μl of benzene, and added to the V-vial using a glass capillary pipette. The vial was returned to the safety container and shaken every 15 minutes for 1.5 hours. At the end of this reaction, 0.5 ml of 5% aqueous bisulfite was added to the reaction vial.

After 15', additional benzene was added to facilitate the extraction process. The contents were removed with a pasteur pipette, and the layers allowed to separate. The benzene layer was added to a column of dry silica gel in the terminal portion of a glass Pasteur pipette. The aqueous bisulfite layer was extracted with benzene, and the benzene was added to the column. The column was washed with one column volume of dichloromethane. The product was then eluted with dichloromethane:ethyl acetate (2:1) mixture, and collected in one vial.

Solvent was removed by passing a stream of N₂ gas over the vial. In order to remove the acetyl protecting groups, a small piece of elemental sodium was added to 1 ml of HPLC grade methanol to form a sodium methoxide solution. 0.5 ml of this solution was added to the dried compound. After 15 minutes, the progress of the deprotection reaction was checked using silica coated TLC plates against a standard. The running solvent was chloroform: methanol, (8:2). The deprotected compound has an R_f of ~0.75. The solution was not neutralized, and the methanol was allowed to evaporate overnight. The dried residue was dissolved in 0.5 ml PBS, pH 7.4, for biological studies.

Example 3

Product analysis of first, radiolabeled synthesis

In order to determine the concentration of product and its specific activity, a standard curve was established for IVaraU at OD₂₉₀. The following were the results of that analysis:

This yields a regression formula as follows:

[Concentration] = 0.07x - 0.009. From this it was determined that 305 μg of material had been

synthesized. A regression curve was also created for the gamma activity of commercially available 5-[¹²⁵I]- iododeoxycytidine (2200 Ci/mmol,375 μCi/ml), where μCi/ml = 5.451e-7x - 3.746e-4. Therefore, it was determined that the specific activity of the product, [¹²⁵I] -IVaraU was 463 μCi/μmol. This material has been used to perform all of the experiments reported herein.

Example 4

Uptake of ¹²⁵IVaraU into HSV-1-infected Prima ry Rabb it

Kidney (PRK) Cells

Using the "carrier-added" [¹²⁵I] -IVaraU, an experiment was performed to confirm our findings regarding the specificity of uptake of unlabeled IVaraU into infected vs. uninfected cells, and to confirm that this uptake specificity could be detected by gamma counting. Isolate #615 is quite resistant in vitro to acyclovir. Three resistant plaque-purified substrains of 615 have been characterized for another purpose. Two are pure DNA polymerase mutants (615.5, 615.8) which express normal amounts of dTK and yet show in vitro and in vivo resistance to acyclovir. The third isolate (615.3) is a dTK deficient strain which does not phosphorylate acyclovir and phosphorylates

deoxythymidine very poorly. The pretreatment "wildtype" isolate (sensitive to drugs as for any other HSV-1 isolate) was also plaque-purified (294.1) and used as a sensitive control. A laboratory-induced acyclovirresistant mutant of strain KOS, known as ACG^r4 was also used [an HSV-1 reference strain which was artificially induced to lack the dTK enzyme (viral dTK's are

completely absent from this strain) through laboratory exposure to the antiviral agent, acyclovir. It lacks the dTK polypeptide, and was supplied to our lab by Dr. Don Coen, Department of Pharmacology, Harvard

University]. For this study, Primary Rabbit Kidney Cells (PRK) were grown to confluence overnight in tissue culture grade roller tubes, kept rolling at 1 rpm at 37°C. Tubes contained approximately 1.4 × 10⁶ cells/tube. Each tube was infected with an moi of 10 in 1.0 ml of media and incubated for 6 hours at 37°C. Approximately 4.8 × 10⁶ counts per minute, hereinafter referred to as "cpm", of [¹²⁵I] -IVaraU (100 μl) were then added to each tube and incubation continued for 0.75 hours at 37°C. The medium was then removed from the tubes and replaced with 2.0 ml of 0.25% trypsin in EDTA, and incubated at 37°C until cells were separated from the plastic, transferred into 12 × 75 mm plastic tubes, and centrifuged to a pellet at 800 × 9 and washed 3 times in PBS, pH 7.4, 4°C, and counted in a gamma counter (Beckman).

The results of this study are displayed in Figure 2.

As shown in Figure 2, background only was detected in the cells infected with the dTK-negativestrain, ACG^r4. By contrast, large quantities of uptake were detected in cells infected with the wild-type and the DNA polymerase-altered (dTK-normal) mutants. Low, but positive uptake was seen in the 615.3 (dTK-altered) strain as predicted on the basis of its acyclovir and deoxythymidine phosphorylating ability. Example 5

Reversibility of Uptake of ¹²⁵IVaraU into HSV-1-infected Primary Rabbit Kidney Cells.

The "carrier-added" material was also utilized in order to determine to what extent reversal of uptake of this agent occurs, (dephosphorylation to nucleoside and loss from the infected cell). To this end, roller tubes were again seeded with PRK cells as described in example 4, and infected with 10 moi of the wild-type isolate, 294.1 and ACG^r4 (dTK negative) in parallel. After 6 hours of incubation at 37°C, 4 × 10⁴ cpm of [¹²⁵I] -IVaraU were added and the incubation continued for a further 0.75 hours. The media were then removed, the monolayers washed with PBS, pH 7.4, 37°C, and the media were replaced with no nucleoside added. Incubation was restarted in the roller

apparatus at 37°C. At 0,1,2,3,4,8,12,16,20,24 hours the process was halted. Cells were pelleted, washed, and counted in the gamma counter. The results are displayed in Figure 3. Reversibility of uptake was noted. It continued in an exponential fashion

throughout the 24 hour period. At 12 hours, however, the uptake retention ratio between 294.1 and ACG^r4 of approximately two-fold persisted. Indeed, the

differential was still apparent at 24 hours. Exampl e 6

Uptake of [^I25I] -IVaraU into HSV-1-infected Primary Rabbit Kidney Cells as a Function of The Mnltiplicity of Infection and The Time Post-Infection.

In order to assess the trapping of [¹²⁵I]-IVaraU into HSV-infected cells as a function of time and inoculum size, PRK cells were grown to confluence at the bottom of tissue culture grade plastic roller tubes. For this experiment, the number of cells were 1.29 × 10⁶. Inocula per cell, of 1, 0.1, 0.01, 0.001, and 0.0001 plaque-forming units, hereinafter referred to as "PFU", of HSV-1 strain 294.1 (Wild type) were used. The media were removed and replaced with Hank's "199", containing 2% inactivated fetc.1 calf serum and the various viral inocula. After addition of virus, the tubes were spun at room temperature for 1800 revolutions per minute for 30 minutes, and incubated at 37°C. for 2,3,4,5,6,7,8,10,12,20,22,24 hours. Forty-five minutes prior to completion of the incubation period, 4.0 × 10⁶ cpm of [¹²⁵I] -IVaraU were added to the viral growth media in 100 μl of PBS. Cells were then trypsinized with 2.0 ml of 0.25% trypsin and

transferred to 12 × 75 mm plastic tubes where they were centrifuged at 800 × 9 for 10 minutes and washed with cold PBS × 3, pH 7.4. The final pellet was resuspended in 1.0 ml of distilled water and counted in the gamma counter. The results of this experiment are shown in figure 4.

These results demonstrate that at an inoculum of 1 PFU per cell, [¹²⁵I] -IVaraU trapping will detect growth of HSV-1 as early as 4.0 hours. This is at least 4 hours in advance of any visibly detectable alteration in the monolayer. dTK activity is detected at 20 hours by this assay using an moi of 0.0001 PFU per cell (approximately 130 infective particles per tube). Although not apparent visually in Figure 4, uptake at 20 hours at moi = 0.0001 is 3-fold above background. It is, therefore, likely that future experiments will allow for even earlier detection of viral infections of low inocula.

Example 7

Measurement of the Targeted Antiviral Radiotherapeutic Effects of [¹²⁵I] -IVaraU for HSV infections In vi t ro .

In order to measure the specific effects of the radiotherapeutic component of [¹²⁵I] -IVaraU to induce viral-specific lethality, the antiviral effects of identical molar (mass) quantities of [¹²⁵I] -IVaraU were compared with those of unlabeled IVaraU in vitro .

The antiviral assay methods are previously described [41]. Briefly, VERO cells were obtained from the American Type Culture Collection. The cells were grown to confluence and infected with HSV-1, strain F; or HSV-2, strain G, used as reference; strains. These strains were obtained from Dr. Bernard Roizman,

University of Chicago (Chicago IL) . We define 1 "DUo" as the amount of virus required to give > 95%

cytopathic effects, hereinafter referred to as "CPE" in 48 hours where no treatment is used to inhibit

infection. Ninety-six well plates were seeded with 2 × 10⁴ cells per well. Approximately 18 h afterward, virus stocks were diluted (one at a time) to a final concentrations of 1 DUo and added to each of the wells (except for the cell control which contains drug dilutions without virus infection) in 50 μl volumes. The plates were then incubated at 37°C, in an atmosphere of 5% CO₂ for 1 hour. Antiviral drug dilutions were made during this period, beginning at the highest concentration of IVaraU 0.065 μg/ml which was equal to a radiation dose in wells treated with [¹²⁵I] -IVaraU, of 0.50 μCi/ml and then serially 2-fold diluted 16 times, and added to the wells at the end of the 1 h adsorption period in a volume of 50 μl. All experiments were performed in quadruplicate, and results are expressed as the average of those four wells at each drug dilution. The unlabeled IVaraU and the [¹²⁵I] -IVaraU were compared at equal concentrations and dilutions in parallel. Two types of controls are run on each 96 well plate. One column receives mock virus inoculation [50 μl of Medium 199 (with

supplements) alone]; one other column receives mock drug treatment [50 μl of Medium 199 (with supplements) alone]. The plates are returned to the incubator and kept at 37°C, in an atmosphere of 5% CO₂ for 2 days, followed by the dye uptake and analysis. Fifty μl of 15% neutral red in PBS, pH 6.0 was then added to each well, and then incubated for 45 min at 37°C, in an atmosphere of 5% CO₂. The dye was then aspirated and the monolayer washed X 2 with PBS, pH 6.0. The medium was removed and the plate blotted to dryness. Plates were then frozen until ready for analysis. At that time, 100 μl of lysis buffer was added and the

absorbance of each well at 570nm/410nm determined on a Dynatech® plate reader.

The dose required to achieve a 50% reduction of CPE compared with controls (ID₅₀) was then calculated using a log-dose response curve. No antiviral effects were observed with unlabeled IVaraU against HSV-1 or HSV-2 at the highest dose tested (0.065 μg/ml). This is consistent with previous data from our laboratory showing the antiviral ID50 for HSV-1 to be ≥ 1.0 μg/ml with unlabeled IVaraU, with no observed antiviral activity up to 20 μg/ml against HSV-2, using this type of assay in VERO cells. In the present experiment, no antiviral effect of [¹²⁵I] -IVaraU was observed at 0.065 μg/ml against HSV-2. In contrast, however, the ID₅₀ for [¹²⁵I] -IVaraU against HSV-1, strain F was 0.017 μg/ml (0.13 μCi/ml).

We conclude that [¹²⁵I] -IVaraU displays significant radiotherapeutic antiviral effects at concentrations far below those accounted for on the basis of the antiviral activity of the unlabeled compound. This results from specific targeting of the radiation effects of this compound. Further work to define the optimal isotope for such targeted

radiotherapy is underway.

Example 8

Nuclear Medicine Brain Scan of herpes simplex virus encephalitis in New Zealand White Rabbits using ^[123I, ¹²⁵I]-IVaraU.

In order to assess the potential for the use of [ l ] -IVaraU as a diagnostic scanning agent in vivo, an animal model of herpes simplex virus encephalitis was established in New Zealand White rabbits. Five days post-inoculation with 100 μl of HSV-1, strain KOS (3.7 × 10⁶ pfu/ml) onto the right cribriform plate.

[¹²⁵I] -IVaraU (136 μCi; 37 Ci/mmol) were given to 3 infected rabbits 1 hour prior to in situ perfusion and sacrifice. 5-7 μm sections were stained with HSV-1 monoclonal antibodies, labeled by immunoperoxidase, counted by 0.0058 mm² grids, and assessed for peroxidase positive hits per total number of grids. Maximal infection was seen in the right olfactory bulb with 32.9% peroxidase positive. [¹²⁵I] -IVaraU uptake was also maximal in this region, with a mean of 13,872 cpm per gram of tissue. In contrast, minimal uptake was seen (2,938 cpm per gram) in the dorsal/left brain, where immunoperoxidase staining was not found. A rabbit sacrificed 7 weeks post infection and infused with 5.4 μCi of tracer, was found to have localized right temporal lobe necrosis (28.5% peroxidase

positive) with uptake of 8,226 cpm per gram compared with 268 cpm per gram in the left temporal lobe.

[¹²³I] -IVaraU (21.5-87.5 Ci/mmol) was then

administered to 2 five day post infection rabbits and an uninfected control. Images obtained on a small head gamma camera at 3.5 hours showed an anterior region of enhanced uptake in the infected animals . This region of interest was drawn by computer and ratios of counts per pixel compared between an identical region in the posterior brain of one infected animal

(target/background = 1.6:1) and the anterior region of its uninfected control (target/background = 2.7:1). In a separate experiment, the same rabbit model was infected with a more virulent strain of HSV-1 (Whitley 1) from a patient with HSV encephalitis, and scans performed 7 days post infection. Lateral and anterior views from this scan are shown in figure 5A. In these scans, a clear nuclear image was demonstrated in the olfactory bulb region and in the nasopharynx where acute HSV-1 infection was occurring. Additional uptake was seen in the central portion of the nasopharynx, and the thyroid. Uninfected animals and (as demonstrated in figure 5B, latently infected animals) demonstrated uptake only in this central region and thyroid.

Enhancement of uptake in the infected olfactory bulb in this animal post-sacrifice confirmed the potential for this agent in nuclear scanning studies of CNS infection in man. Figure 6 displays in radiographic print form, a series of nuclear scans over time from an animal with HSV-1 (KOS) encephalitis infected via the cribriform plate 9 days prior to the scans. After infusion with [¹²³I] -IVaraU three scans in different head views were obtained. A localized area of enhanced uptake was seen in the region of the left olfactory bulb which appears early in the post-infusion period anc (6A-D with increasing time post-infusion) becomes relatively more dense with time to 14 hours (6D) as the background uptake clears from the animal, providing a target to background ratio of 2.2 in scan 6D . SUMMARY OF USES FOR THE INVENTION

We would expect that radiolabeled [*I] -IVaraU will be made with any or all feasible isotopes of iodine, and that [*X] -XVaraU will be made with any or all feasible isotopes of halogens. Inclusion of the appropriate gamma or positron emitting iodine, or other appropriate halogen radionuclide, into the structure of [^*I] -IVaraU or [*X] -XVaraU, makes this agent useful as a diagnostic tool for detection of herpes virus

infections in vitro and in vivo . Inclusion of the appropriate alpha- and/or beta- and/or gamma-emitting, and/or Auger electron decay-associated, (specifically nuclear-toxic) isotopes of iodine, or other appropriate halogen radionuclides, into the structure of this agent makes the agent useful as a unique radiotherapeutic tool for herpes virus infections by precise targeting of the lethal effects of alpha and/or beta radiation and/or Auger electron decay effects to the site of viral infection. The uses for the agent include, but are not restricted to, the following:

1. In Vitro Diagnosis

Because the infected: uninfected cell ratio of trapping of the agent is so high, minute quantities of virus growth are detectable with this agent.

Accordingly, the compound is useful as a marker of viral growth in vitro . This allows for the following: (a) Automation of viral cultures;

(b) Enhancement of the speed the process of viral culture to the point where it can be completed in several hours, which, in turn, may be applied to a process for screening for herpes in pregnancy, whereby a culture test can be done during labour on all women to prevent the transmission of herpes infections to the neonate. Furthermore, rapid testing will aid in the therapeutics of mucocutaneous infections where speed of diagnosis is critical in terms of outcome.

(c) Rapid detection of viral

susceptibilities to antiviral compounds which depend upon dTK activity for their action

(d) A method for measuring HSV growth in any circumstance where applicable, including laboratory research studies, antiviral susceptibility testing, or any situation where measurements of viral growth are required.

2. In Vivo Diagnosis

Deep seated infections will be detectable with this agent. Currently, unless on the skin or mucous membrane, where a sore is visible and accessible to the swab, viral diagnosis is often missed. Often, the physician may not consider the diagnosis, or considers it, but is afraid of the risk of the test. Moreover, in some settings, for example, deep eye infections, biopsy is clinically impossible.

It is expected that the doses required for specific in vivo diagnosis of herpes virus-related clinical syndromes will be determined by physicians in accordance with the clinical situation at hand.

Specifically, however, it is expected that in most cases, in vivo diagnosis will be performed via

intravenous injections of isotopes in the range of 1 to 20 mCi, total body dose. Because this requires only a very small amount of the nucleoside, the dose may be administered at one time by intravenous bolus

injection. The amount of the agent in molar quantities required will be determined by the specific activity of the synthesized material. Regardless, the actual quantity of the agent in its nucleoside form

administered will be less than 1 mg per day. In certain clinical situations, it may be preferable to apply the diagnostic agent either topically or

ophthalmologically, Thus, ophthalmic drops or

ointments or dermatological salves or ointments may be preferred with such doses as would otherwise be

administered intravenously (1 to 20 mCi), applied to directly to the area of infection, followed by an assay for retained (trapped) nucleoside, sometime following natural body clearance or physical removal of the agent.

Herpes virus-related clinical syndromes which present diagnostic problems and which might benefit from the enhanced diagnostic capabilities described herein, include, but are not limited to, the following:

(a) HSV and VZV encephalitis.

(b) HSV and VZV visceral infections.

(c) HSV esophagitis.

(d) HSV and VZV pneumonia. (e) HSV and VZV infection of the neonate.

(f) HSV and VZV infections of the eye, including: retinitis, keratitis, iritis, uveitis, retinal necrosis and zoster ophthalmicus. (g) HSV meningitis often associated with genital herpes infections.

(h) HSV and VZV myelitis or radiculomyelopathy.

(i) Herpes simplex virus-induced temporal lobe epilepsy. (j) Bell's Palsy/Ramsay Hunt syndrome and other forms of Herpes zoster oticus.

(k) Infectious Mononucleosis and other EBV-related infections associated with productive

replication of the virus.

(1) HSV and VZV sensory nerve, root and ganglionic infections. (m) Other clinical situations which are considered as possibly related to HSV or VZV or CMV infection are also included, since the agent could enhance understanding of HSV infection enough to clarify their relationship to HSV, if any. A partial list of possibly-related syndromes includes multiple sclerosis, schizophrenia, migraine and other severe headache, Alzheimer's disease, and others. It is expected that new knowledge to be gained through the use of this agent will expand the knowledge of

associated illness.

(n) There are also a variety of herpes virus-induced diseases in the veterinary setting, each specific to a certain type of animal. Many of these viruses express the herpes virus enzyme, dTK, which specifically phosphorylates [*I] -IVaraU, and/or [*X]-XVaraU. In such situations, the veterinarian may elect to diagnose a herpes viral disease after intravenous, or intraperitoneal administration to the animal of from 0.01 to 0.50 μCi per kg of total body weight.

3. in Vivo Therapy

Through utilization of halogen radionuclides, including ¹³¹I, ⁸²Br, and others with suitable cytotoxic alpha and/or beta emission characteristics, and/or ¹²⁵I, or ⁷⁷Br, or other halogen radionuclides which display suitable Auger electron decay phenomena, localized, and thereby targeted destruction of cells actively or latently infected with HSV, VZV, or EBV will be

achieved. Such therapy may be used in conjunction with any antiherpesviral antiviral agent, since the

mechanism of antiviral action of [*I]-IVaraU, and/or [*X] -XVaraU is unique and will, therefore act

synergistically with other available agents in

achieving safe, but lethal radiotherapeutic antiviral effects.

The precise form of the agent and mode of

administration and dosage of this agent as an antiviral drug, will be determined by the physician in ccordance with the specific clinical condition. Based on the potent in vitro antiviral effects observed, it is possible, however, to predict a dosage range of from 5 to 150 mCi total dose as the isotope, administered by intravenous injection, or 5 to 150 mCi total dose as the isotope, administered orally. Either mode of administration may be possible, depending on whether long-term administration of the agent is required. The amount of the agent in molar quantities required will be determined by the specific activity of the

synthesized material. Regardless, the actual quantity of the agent in its nucleoside form administered will be less than 1 mg per day. In certain veterinary situations where in vivo therapy is required, either intravenous or oral or intraperitoneal therapy may be used with a total dosage range of 0.05 to 3 mCi/kg.

As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

Claims

A radiohalogen compound of the formula:

wherein X is a radioisotope of iodine, selected from the group consisting of radioactive ¹²³I, ¹²⁵I, ¹²⁷I, ¹³¹I, or, alternatively, a radiohalogen selected from the group consisting of radioactive ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ³⁴Cl, or other appropriate radionuclides and the processes of radioiodination of TMSVaraU using a salt of radioactive iodide isotopes (e.g., Na[*I]-I) with an oxidant and chlorine donor such as

phenyliodine (III) dichloride ( syn : iodobenzene

dichloride) or radiohalogenation of TMSVaraU using the salt of radioactive halogen isotopes (e.g., Na[*X]-X) with alternative oxidant and halogen donors such as xenon difluoride, including acylated derivatives and other prodrug forms of the radiohalogen compound of the above formula which might provide alternative

biodistribution qualities.

2. A vinylsilane precursor compound (for the

radiohalogen formula defined in claim 1) known as 1- (2, 3, 5-tri-O-acetyl-ß-D-arabinofuranosyl)-5( Z and E) - (2-trimethylsilylvinyl) uracil, also referred to in this text as "TMSVaraU", of the formula:

wherein "TMS" is used to refer to the trimethylsilyl component of the molecule and "Ac" is used to refer to the protecting "O-acetyl" groups and processes for preparing the precursor compounds

which comprises:

(a) converting uridine to its arabino analogue;

(b) protecting the arabino sugar moiety against substitution or degradation;

(c) halogenating the protected analogue at the C5 position;

(d) coupling the halogenated protected analogue with trimethylsilylacetylene and;

(e) selectively reducing the coupled

compound to 1-(2,3,5-tri-O-acetyl-ß-D- arabinofuranosyl)-5( Z and E) - (2- trimethylsilylvinyl) uracil.

3. A method of diagnosing herpesviral infections in vivo, using radiation scanning techniques, or in vitro. using radiation detection, which is based on the viral-specific enhanced uptake and consequent localization of radiation from the radiohalogen compounds described in claims 1 and/or 2.

4. A method of treating herpesviral infections in vi vo, which is based on the viral-specific enhanced uptake and consequent localization of radiation from the radiohalogen compounds described in claim 1 and/or 2.