Synthesis and Preliminary Evaluation of 9-(4-[¹⁸F]-Fluoro-3-Hydroxymethylbutyl)Guanine ([¹⁸F]FHBG): A New Potential Imaging Agent for Viral Infection and Gene Therapy Using PET

Abstract

Synthesis and preliminary biological evaluation of 9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl)guanine ([¹⁸F]FHBG) is reported. 9-(4-Hydroxy-3-hydroxymethylbutyl)-guanine (penciclovir) 4 was converted to 9-[N², O-bis-(methoxytrityl)-3-(tosylmethybutyl)]guanine 7 by treatment with methoxytrityl chloride followed by tosylation. The tosylate 7 was reacted with either tetrabutylammonium fluoride or KF in the presence of kryptofix 2.2.2. to produce the 4-fluoro-N²-O-bis-(methoxytrityl) derivative 8. Removal of the methoxytrityl groups by acidic hydrolysis produced FHBG 5. Radiolabeled product [¹⁸F]FHBG was prepared by fluorination of the tosylate 7 with [¹⁸F]KF and kryptofix 2.2.2. The labeled product was isolated by HPLC purification on a reverse-phase C₁₈ column, and eluted at 12 min with 15% acetonitrile in water at a flow rate of 2.25 mL/min. Radiochemical yield was 8.0–22.3% with an average of 12% in 7 runs (corrected for decay). Synthesis time was 90 to 100 min including HPLC purification with radiochemical purity >99%, and average specific activity of 320 mCi/μmol. In vitro studies of the compound in HT-29 colon cancer cells revealed 18.2-fold higher uptake into transduced cells compared to control in 3 h. The agent may be useful for imaging viral infection or transfected cells in gene therapy.

Introduction

Many antiviral nucleoside analogues are known to localize selectively in herpes virus-infected cells due to a mechanism that calls for appreciable unique monophosphorylation by virus-encoded thymidine kinase (tk). Among these, acyclovir 1 and ganciclovir 2 were reported to be good antiviral agents with low-host toxicity 18, 20, ganciclovir being more active against herpes simplex virus type 1 and 2 21, 22, 23, 24(Fig. 1).

Acyclic nucleosides including acyclovir and ganciclovir are unusually selective as compared to other nucleoside antiviral agents 16, 18. The selectivity of these analogues is due, in part, to the fact that they are phosphorylated only in virus-infected cells, where a virus-specific thymidine kinase of low substrate specificity converts the nucleoside analogues to monophosphates 10, 13, 22. The monophosphates are converted to diphosphates, and then to the corresponding triphosphates by cellular enzymes 10, 18, 21. The triphosphates prevent viral replication by inhibition of the viral DNA polymerase. Additional selectivity is realized at this stage because the host polymerase is less sensitive than viral polymerase to the nucleoside triphosphate analogue.

These findings led to the synthesis of a number of analogues of ganciclovir (DHPG) in order to study further the effects of structural modifications on antiviral activity and toxicity [18]. Among these analogues, 9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine (FHPG) 3 was found to be biologically active, and is phosphorylated by the viral kinase at 67% the rate of thymidine. Synthesis of F-18-labeled FHPG has been reported 1, 2, 19, and it is currently under investigation both in our laboratory and in others [14]. Preliminary in vivo results in our facility demonstrate the absence of catabolism in primates and relative uptake into transduced cells 3, 4.

The carba-analogue of ganciclovir, 9-(4-hydroxy-3-hydroxymethylbutyl)guanine (penciclovir) 4, was reported to be active against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), and Epstein Barr virus (EBV) 12, 15, 25, and in some tests was more active than acyclovir, with no evidence of toxicity [7]. Like ganciclovir and acyclovir, penciclovir is also selectively phosphorylated by viral kinase to the monophosphate. The monophosphates are converted to di- and tri-phosphate by cellular enzymes. Penciclovir has higher phosphorylation rate and more effective viral DNA polymerase inhibition than does acyclovir [25].

In animal studies with HSV-1 or -2, penciclovir has been shown to be more effective than equivalent treatment with acyclovir, reflecting more resistant activity seen in cell culture, and a more stable triphosphate within the infected cell [8]. Although the efficacy of penciclovir is comparable with ganciclovir [25], the former is also useful in other viruses such as varicella-zoster virus and Epstein Barr virus. Comparison among acyclovir, ganciclovir, and penciclovir 22, 25reveals the following: (a) the phosphorylation rate by viral kinase followed by host kinase to form triphosphate of penciclovir is 12 times higher than ganciclovir, and 120 times higher than acyclovir in 4 h incubation; (b) the intracellular stability of the triphosphate of penciclovir is 7 times higher than acyclovir and 3 times higher than ganciclovir; and (c) inhibition of DNA polymerase by the penciclovir triphosphate is equal to or higher than acyclovir, and is 10 to 30 times higher than ganciclovir. This comparison suggests that the radiolabeled fluoro-analogue of penciclovir, FHBG, might be more suitable than the fluoro-analogue of ganciclovir, FHPG [11], or radiolabeled acyclovir 5, 26for imaging of viral infection and transfected cells by PET. In this article, we report a chemical and radiochemical synthesis of a new compound, 9-(4-[¹⁸F]fluoro-3-hydroxymethylbutyl)-guanine ([¹⁸F]FHBG), and its preliminary biological evaluation in HT-29 cells.