SYNTHESIS OF UNNATURAL 7-SUBSTITUTED-1-(2-DEOXY-β-D-RIBOFURANOSYL)ISOCARBOSTYRILS†: “THYMINE REPLACEMENT” ANALOGS OF DEOXYTHYMIDINE FOR EVALUATION AS ANTIVIRAL AND ANTICANCER AGENTS

A group of unnatural 1-(2-deoxy-β-D-ribofuranosyl)isocarbostyrils having a variety of C-7 substituents [H, 4,7-(NO₂)₂, I, CF₃, CN, (E)-CH=CH-I, -C═CH, -C═C-I, -C═C-Br, -C═C-Me], designed as nucleoside mimics, were synthesized for evaluation as anticancer and antiviral agents. This class of compounds exhibited weak cytotoxicity in a MTT assay (CC₅₀=10^?3 to 10^?5 M range) with the 4,7-dinitro derivative being the most cytotoxic, relative to thymidine (CC₅₀=10^?3 to 10^?5 M range), against a variety of cancer cell lines. The 4,7-dinitro, 7-I and 7-C═CH compounds exhibited similar cytotoxicity against non-transfected (KBALB, 143B), and HSV-1 TK⁺ gene transfected (KBALB-STK, 143B-LTK) cancer cell lines possessing the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (TK⁺). This observation indicates that these compounds are not substrates for HSV type-1 TK, and are therefore unlikely to be useful in gene therapy based on the HSV gene therapy paradigm.

     

Synthesis of 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes: "thymine replacement" analogs of thymidine for evaluation as anticancer and antiviral agents

A group of unnatural 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluorobenzenes having a variety of C-5 two-carbon substituents [-C...C-X, X = I, Br; -C...CH; (E)-CH=CH-X, X = I, Br; -CH=CH2; -CH2CH3; -CH(N3) CH2Br], designed as nucleoside mimics, were synthesized for evaluation as anticancer and antiviral agents. The 5-substituted (E)-CH=CH-I and -CH2CH3 compounds exhibited negligible cytotoxicity in a MTT assay (CC50 = 10(-3) to 10(-4)M range), relative to thymidine (CC50 = 10(-3) to 10(-5)M range), against a variety of cancer cell lines. In contrast, the C-5 substituted -C...C-I and -CH(N3)CH2Br compounds were more cytotoxic (CC50 = 10(-5) to 10(-6)M range). The -C...C-I and -CH2CH3 compounds exhibited similar cytotoxicity against non-transfected (KBALB, 143B) and HSV-1 TK+ gene transfected (KBALB-STK, 143B-LTK) cancer cell lines expressing the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (TK+). This observation indicates that expression of the viral TK enzyme did not provide a gene therapeutic effect. The parent group of 5-substituted compounds, that were evaluated using a wide variety of antiviral assay systems [HSV-1, HSV-2, varicella-zoster virus (VZV), vaccinia virus, vesicular stomatitis, cytomegalovirus (CMV), and human immunodeficiency (HIV-1, HIV-2) viruses], showed that this class of unnatural C-aryl nucleoside mimics are inactive and/or weakly active antiviral agents.     

Virus Type-Specific Thymidine Kinase in Cells Biochemically Transformed by Herpes Simplex Virus Types 1 and 2

Transformation of mouse cells (Ltk(-)) and human cells (HeLa Bu) from a thymidine kinase (TK)-minus to a TK(+) phenotype (herpes simplex virus [HSV]-transformed cells) has been induced by infection with ultraviolet-irradiated HSV type 2 (HSV-2), as well as by HSV type 1 (HSV-1). Medium containing methotrexate, thymidine, adenine, guanosine, and glycine was used to select for cells able to utilize exogenous thymidine. We have determined the kinetics of thermal inactivation of TK from cells lytically infected with HSV-1 or HSV-2 and from HSV-1- and HSV-2-transformed cells. Three hours of incubation at 41 C produces a 20-fold decrease in the TK activity of cell extracts from HSV-2-transformed cells and Ltk(-) cells lytically infected with HSV-2. The same conditions produce only a twofold decrease in the TK activities from HSV-1-transformed cells and cells lytically infected with HSV-1. This finding supports the hypothesis that an HSV structural gene coding for TK has been incorporated in the HSV-transformed cells.     

Syntheses of 1-[(2-hydroxyethoxy)methyl]- and 1-[(1,3-dihydroxy-2-propoxy)methyl]- derivatives of 5-substituted-2,4-difluorobenzene: unnatural acyclo thymidine mimics for evaluation as anticancer and antiviral agents

A group of 1-[(2-hydroxyethoxy)methyl]- (12) and 1-[(1,3-dihydroxy-2-propoxy)methyl]- (13) derivatives of 2,4-difluorobenzene possessing a variety of C-5 substituents (R = Me, H, I, NO2) were designed with the expectation that they may serve as acyclic 5-substituted-2'-deoxyuridine (thymidine) mimics. Compounds 12 and 13 (R = Me, H, I) were inactive as anticancer agents (CC50 = 10(-3) to 10(-4) M range), whereas the 5-nitro compounds (12d, 13d) exhibited weak-to-moderate cytotoxicity (CC50 = 10(-5) to 10(-6) M range) against a variety of cancer cell lines. All compounds prepared (12a-d, 13a-d) were inactive as antiviral agents in a broad-spectrum antiviral screen that also included the human immunodeficiency virus (HIV-1 and HIV-2) and herpes simplex virus (HSV-1 and HSV-2).     

Herpes Simplex Virus Thymidine Kinase Gene-Transfected Tumor Cells; Sensitivity to Antiherpetic Drugs

Abstract

A series of antiherpetic 5-substituted 2′-deoxyuridine derivatives (i. e. BVDU) and guanine derivatives (i. e. ganciclovir) have been evaluated for their cytostatic activity against murine mammary carcinoma FM3A cell lines that are deficient in cytosol thymidine kinase, but transfected by the herpes simplex virus type 1 (HSV-1)- or type 2 (HSV-2)-specified thymidine kinase gene. Most compounds were endowed with a markedly higher cytostatic activity against the HSV TK gene-transfected tumor cells than against wild-type tumor cells. The principal target for cytostatic activity of the BVDU derivatives proved thymidylate synthase, whereas the guanine derivatives inhibited HSV TK gene-transfected tumor cell proliferation by competing with cellular DNA polymerase(s) and subsequent incorporation into the cellular genome.

     

A cytotoxic effect associated with 9-(1,3-dihydroxy-2-propoxymethyl)-guanine is observed during the selection for drug resistant human cells containing a single herpesvirus thymidine kinase gene

A cytotoxic effect associated with 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (DHPG) was discovered while searching for spontaneous mutations in a single copy, integrated HSV-1 thymidine kinase (TK) gene in the human 143 TK- cell line. It was found that spontaneous DHPGR mutations could not be selected while other anti-TK drugs resulted in selectable mutation frequencies of 10(-4) to 10(-3). When 143 TK- cells were mixed with these HSV-1 TK+ cells and subjected to DHPG, a 90% to 100% decrease in recoverable TK- colonies was observed. In addition, the media from the HSV-1 TK+ cells metabolizing DHPG was shown to inhibit the growth of the TK- cells.     

Synthesis and characterization of a C6 nucleoside analogue for the in vivo imaging of the gene expression of herpes simplex virus type-1 thymidine kinase (HSV1 TK)

The synthesis and biological evaluation of '6-(1,3-dihydroxyisobutyl)thymine' (DHBT; 1), which corresponds to 6-[3-hydroxy-2-(hydroxymethyl)propyl]-5-methylpyrimidine-2,4(1H,3H)-dione, is reported. DHBT (1) was designed as a new substrate for herpes simplex virus type-1 thymidine kinase (HSV1 TK). The compound was found to be exclusively phosphorylated by HSV1 TK, and to exhibit good binding affinity (Ki = 35.3+/-1.3 microM). Cell-proliferation assays with HSV1-TK-transduced human osteosarcoma cells (143B-TK+-HSV1-WT) and with both human-thymidine-kinase-1-negative (143B-TK-) and non-transduced parental (MG-63) cells indicate that 1 is less cytotoxic than the standard drug Ganciclovir. Thus, DHBT (1) represents a promising precursor of a nontoxic reporter probe for the monitoring of HSV1 TK gene expression by means of positron-emission tomography (PET).     

Synthesis, 18F-radiolabelling and biological evaluations of C-6 alkylated pyrimidine nucleoside analogues

Synthesis of pyrimidine derivatives with a side-chain attached to the C-6 of pyrimidine ring (6-14) is reported. Target compounds 8 and 12 were subjected to in vitro phosphorylation tests, determination of their binding affinities to herpes simplex virus (HSV-1) thymidine kinase (TK) and catalytic turnover constants. Fluorinated pyrimidine derivative 12 (40 microM) exhibited better binding affinity for HSV-1 TK than acyclovir (ACV, 170 microM) and ganciclovir (GCV, 48 microM). Catalytic turnover constant (k(cat)) of 12 (0.08 s(-1)) was close to the k(cat) values of ACV (0.10 s(-1)) and GCV (0.10 s(-1)). Furthermore, compounds 8 and 12 showed no cytotoxic effects in HSV-1 TK-transduced and non-transduced cell lines. Besides, compounds 8 and 12 did not exhibit antiviral or cytostatic activities against several viruses and malignant tumor cell lines that were evaluated. The new fluorinated pyrimidine derivative 16 that is phosphorylated by HSV-1 TK could be developed as non-toxic PET-tracer molecule. Thus, 18F labelling of the precursor 14 was performed by nucleophilic substitution using [18F] tetrabutylammonium fluoride as the fluorinating reagent.     

Transfer of Thymidine Kinase to Thymidine Kinaseless L Cells by Infection with Ultraviolet-Irradiated Herpes Simplex Virus

L cells lacking thymidine kinase (TK) activity (Ltk(-) cells) have been stably transformed to a TK-positive phenotype by infection with ultraviolet-irradiated herpes simplex virus (HSV-UV). The highest frequency of the Ltk(-) to Ltk(+) transformation observed in these experiments was approximately 10(-3), whereas no measurable transformation was observed (less than 10(-8)) in the absence of HSV-UV infection. Cell lines of HSV-transformed Ltk(+) cell lines contain 7 to 24 times as much TK activity as do the parental Ltk(-) cells, and they have been maintained in culture for a period exceeding 8 months. The kinetics of thermal inactivation of the TK activity derived from an Ltk(+) HSV-transformed cell line and the TK activity from Ltk(-) cells lytically infected with infectious HSV are similar. Both of these TK activities are much more thermolabile than the TK activity present in wild-type L cells. A mutant strain of HSV which does not induce TK activity during lytic infection does not cause the Ltk(-) to Ltk(+) transformation. These data suggest that either an HSV TK gene has been transferred to Ltk(-) cells or that an HSV gene product has caused the expression of a previously repressed cellular enzyme.     

Fluorosubstitution and 7-alkylation as prospective modifications of biologically active 6-aryl derivatives of tricyclic acyclovir and ganciclovir analogues

A series of fluorine containing tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2) were synthesized and evaluated for their activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and cytostatic activity against HSV-1 thymidine kinase (TK) gene-transduced human osteosarcoma tumour cells. It was found that fluorine substitution reduced the antiviral activity, but most of the new compounds were pronounced cytostatic agents with potency and selectivity similar to those of parental ACV and GCV. Compounds 12, 13 and 16 seem to be promising as labeled substrates for (19)F NMR studies of the HSV TK-ligand interaction and/or monitoring of their metabolites in cells expressing HSV TK.     

Synthesis of 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzene thymidine mimics, some related alpha-anomers, and their evaluation as antiviral and anticancer agents

A group of unnatural 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluorobenzenes having a variety of C-5 substituents (H, Me, F, Cl, Br, I, CF3, CN, NO2, NH2), designed as thymidine mimics, were synthesized for evaluation as anticancer and antiviral agents. The coupling reaction of 3,5-bis-O-(p-chlorobenzoyl)-2-deoxy-alpha-D-ribofuranosyl chloride with an organocadmium reagent [(2,4-difluorophenyl)2Cd] afforded a mixture of the alpha- and beta-anomeric products (alpha:beta = 3:1 to 10:1 ratio). Treatment of the alpha-anomer with BF3.Et2O in nitroethane at 110-120 degrees C for 30 min was developed as an efficient method for epimerization of the major alpha-anomer to the desired beta-anomer. The 5-substituted (H, Me, Cl, I, NH2) beta-anomers exhibited negligible cytotoxicity in a MTT assay (CC50 = 10(-3)-10(-4) M range), relative to thymidine (CC50 = 10(-3)-10(-5) M range), against a variety of cancer cell lines. In contrast, the 5-NO2 derivative was more cytotoxic (CC50 = 10(-5)-10(-6) M range). A number of 5-substituted beta-anomers, and some related alpha-anomers, that were evaluated using a wide variety of antiviral assay systems [HSV-1, HSV-2, varicella-zoster virus (VZV), vaccinia virus, vesicular stomatitis, cytomegalovirus (CMV) and human immunodeficiency (HIV-1, HIV-2) viruses], showed that this class of unnatural C-aryl nucleoside mimics are inactive antiviral agents.     

Integration site(s) of herpes simplex virus type 1 thymidine kinase gene and regional assignment of the gene for aminoacylase-1 in human chromosomes

To investigate the chromosomal sites of integration of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in HSV-1-transformed human HeLa(BU25)/KOS 8-1 cells, the biochemically transformed cells were fused with TK-negative mouse LM(TK-) cells, and human-mouse somatic cell hybrid lines (LH81) were isolated using a HATG-ouabain selection system. The presence of HSV-1 TK activity in the hybrid lines was verified by disc polyacrylamide gel electrophoresis (PAGE) and by enzyme neutralization with type-specific rabbit anti-HSV-1 TK immunoglobulin. Karyotype analyses of several somatic cell hybrid clones using G-banding, Hoechst 33258 staining, and combined G-banding and Hoechst staining demonstrated that they retained only a few human chromosomes. A marker chromosome, M7, consisting of a chromosome 17 translocated to the short arm of 3, occurred in 25 of the 28 metaphases examined. Also chromosomes 8 and X were found in a minority of metaphases. Isozyme analyses showed that all 19 hybrid clones analyzed expressed human aminoacylase-1 (ACY1) and esterase D (ESD), markers for 3 and 13, respectively. Back-selection of somatic cell hybrid clones with 5-bromodeoxyuridine resulted in the isolation of several subclones lacking HSV-1 TK activity, human ACY1, human ESD, and the human chromosomes. These experiments suggest that the HSV-1 TK gene is associated with either M7 or a segment of 13, or both, in biochemically transformed HeLa(BU25)/KOS 8-1 cells. These experiments also permit localization of the ACY1 structural gene to the pter leads to p12 region of 3.     

Expression of the Viral Thymidine Kinase Gene in Herpes Simplex Virus-Transformed L Cells

In these studies, the expression of thymidine kinase (TK) in normal and herpes simplex virus (HSV)-transformed L cells has been compared. In asynchronously dividing cultures of L cells, the TK activity rose and declined rapidly and coordinately with DNA synthesis. When net cell increase stopped, TK activity was at a minimum. In contrast, TK activity of HSV-transformed cells remained at a minimum during rapid DNA synthesis and gradually increased as the rate of DNA synthesis decreased. When net cell increase stopped, TK activity was at a maximum. In synchronous cultures of L cells, TK activity rose and fell coordinately with the rate of DNA synthesis. In synchronous cultures of HSV-transformed cells, no increase in TK activity was observed during the period of rapid DNA synthesis, i.e., the S phase. These findings indicated that the viral TK gene in HSV-transformed cells was not placed under the control of the cellular mechanisms which normally modulate the host cell TK gene. Lytic infection of HSV-transformed cells with a TK(-) mutant of HSV-1 induced a four-to fivefold increase in viral TK. The TK of HSV-1 was induced in the HSV-1-transformed cells and HSV-2 in the HSV-2-transformed cells by this TK(-) mutant. The same infection of normal L cells decreased the cellular TK activity by 80%. This stimulation, rather than inhibition, suggest that the viral gene in HSV-transformed cells retain some of its original viral characteristics.     

Identification of an Epstein-Barr virus-coded thymidine kinase.

We have demonstrated the presence of an Epstein-Barr virus (EBV)-coded thymidine kinase (TK) by producing biochemically transformed, TK-positive mammalian cell lines using either microinjection of whole EBV virions or calcium phosphate-mediated transfection of the SalI-B restriction endonuclease fragment of EBV DNA. Analysis of these cell lines showed that: (i) EBV DNA was present in the cell lines, (ii) sequences from the SalI-B restriction endonuclease fragment of EBV were expressed, (iii) a TK activity was present and (iv) a protein with antigenic cross-reactivity with the herpes simplex virus (HSV) TK was produced. The identity of the EBV TK gene was determined by demonstrating that a recombinant plasmid, which expressed the protein product of the BXLF1 open reading frame as a fusion protein, could complement TK- strains of E. coli. A comparison of the predicted amino acid sequences of the TK proteins of EBV and HSV-1 revealed significant regions of homology.     

Evaluation of 5-[1-(2-halo(or nitro)ethoxy-2-iodoethyl)]-2'-deoxyuridines as inhibitors of herpes simplex virus

A series of 5-[1-(2-haloethyl(or nitro)ethoxy-2-iodoethyl)]-2'-deoxyuridines (3-7) and related uracil analogs (9-10) were prepared using 5-vinyl-2-deoxyuridine (2) and 5-vinyl uracil (8) as starting materials. The regiospecific reaction of 2 and 8 with iodine monochloride and an alcohol provided the target compounds 3-10. These analogs were evaluated in vitro for inhibitory activity against thymidine-kinase (TK) positive and negative strains of herpes simplex virus type-1. The compounds 3-10 were either weak or non-inhibitory to HSV-1 replication. All compounds investigated exhibited low host cell cytotoxicity.     

Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Murine mammary carcinoma (FM3A TK-/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ng/ml, respectively; i.e., a concentration 5000 to 20 000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action of BVDU and related compounds.     

Characterization of pyrimidine deoxyribonucleoside kinase (thymidine kinase) and thymidylate kinase as a multifunctional enzyme in cells transformed by herpes simplex virus type 1 and in cells infected with mutant strains of herpes simplex virus.

Pyrimidine deoxyribonucleoside kinase (thymidine kinase [TK]) was purified from two herpes simplex virus type 1 (HVS-1)-transformed TK-deficient mouse (LMTK-) cell lines and from LMTK- cells infected with HSV-1 mutant viruses coding for variant TK enzymes. These preparations exhibited normal or variant virus-induced thymidylate kinase activities correlating with their relative TK activities. Neither virus-induced activity was detected in LMTK- cells infected with an HSV-1 TK-deficient mutant. These results suggest that HSV-1 thymidylate kinase activity and TK activity are mediated by the same protein.     

Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2.

It is well-known that viral thymidine kinase (TK) expression is important for the maximum demonstration of virulence of herpes simplex virus (HSV). In this study, we have investigated interactions of a TK- mutant of virulent HSV type 2 (HSV-2) (syn+) and a nonneuroinvasive HSV-1 (syn) in mice. When the mice were inoculated with each virus alone in their rear footpads, no mice were killed even after infection with high doses of viruses (greater than 10(6) PFU per mouse), whereas 100% of the mice died when inoculated with 10(5) PFU of a 1:1 mixture of HSV-2 TK- mutant and nonneuroinvasive HSV-1. The 1:1 mixture exhibited even more virulence than the parental HSV-2; the mean survival time of coinfected mice was significantly shorter than that of mice inoculated with 10(5) PFU of the virulent HSV-2. We have also examined the genotypes and phenotypes of viruses isolated from the central nervous system of coinfected mice. Of 50 isolates, 7 were judged to be recombinants from their restriction endonuclease cleavage patterns, but all were nonneuroinvasive. In addition, all syn+ viruses (23 clones) tested were found to have TK- phenotypes, indicating that the majority of viruses present in the central nervous system were TK- viruses, since about 90% of viruses detected in spinal cords and brains exhibited syn+ phenotypes. These results strongly suggest that the lethal invasion of the central nervous system by HSV-2 TK- and nonneuroinvasive HSV-1 was the consequence of in vivo complementation between the two viruses.     

Evaluation of 5-[1-(2-Halo(or nitro)ethoxy-2-iodoethyl)]-2′-deoxyuridines as Inhibitors of Herpes Simplex Virus

A series of 5-[1-(2-haloethyl(or nitro)ethoxy-2-iodoethyl)]-2′-deoxyuridines (3 - 7) and related uracil analogs (9 - 10) were prepared using 5-vinyl-2′-deoxyuridine (2) and 5-vinyl uracil (8) as starting materials. The regiospecific reaction of 2 and 8 with iodine monochloride and an alcohol provided the target compounds 3 - 10. These analogs were evaluated in vitro for inhibitory activity against thymidine-kinase (TK) positive and negative strains of herpes simplex virus type-1. The compounds 3 - 10 were either weak or non-inhibitory to HSV-1 replication. All compounds investigated exhibited low host cell cytotoxicity.     

An optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases

The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.