Synthesis and antiviral activity of the nucleotide analogue (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine

The acyclic nucleotide analogue (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl] cytosine (2, HPMPC) was prepared on a multigram scale in 18% overall yield starting from (R)-2,3-O-isopropylideneglycerol. The key step in the nine-step synthetic route is coupling of cytosine with the side-chain derivative 8 which bears a protected phosphonylmethyl ether group. In vitro data showed that HPMPC has good activity against herpes simplex virus types 1 and 2, although it was 10-fold less potent than acyclovir [AVC, 9-[(2-hydroxyethoxy)methyl]guanine]. By comparison, HPMPC exhibited greater activity than ACV against a thymidine kinase deficient strain of HSV 1 and was more potent than ganciclovir [DHPG, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine] against human cytomegalovirus. In vivo, HPMPC showed exceptional potency against HSV 1 systemic infection in mice, having an ED50 of 0.1 mg/kg per day (ip) compared with 50 mg/kg per day for ACV. HPMPC was also more efficacious than ACV in the topical treatment of HSV 1 induced cutaneous lesions in guinea pigs.     

Synthesis and antiviral evaluation of carbocyclic analogues of 2-amino-6-substituted-purine 3'-deoxyribofuranosides

Carbocyclic analogues of 2-amino-6-substituted-purine 3'-deoxyribofuranosides were synthesized by beginning with (+/-)-(1 alpha,3 alpha,4 beta)-3-amino-4-hydroxycyclopentanemethanol and 2-amino-4,6-dichloropyrimidine. The route parallels the earlier syntheses of the corresponding ribofuranoside and 2'-deoxyribofuranoside analogues. The 2-amino-6-chloropurine, guanine, and 2,6-diaminopurine derivatives and the analogous 8-azapurines were prepared. The analogue (3'-CDG) of 3'-deoxyguanosine is active in vitro against a strain of type 1 herpes simplex virus (HSV-1) that induces thymidine kinase and is modestly active against a thymidine kinase inducing strain of type 2 HSV. 3'-CDG is not active against a strain of HSV-1 that lacks the thymidine kinase inducing capacity, whereas the carbocyclic analogue of 2-amino-6-chloropurine 3'-deoxyribofuranoside is active against that strain. The carbocyclic analogue of 2,6-diaminopurine 3'-deoxyribofuranoside displayed modest activity in vitro against influenza virus.     

Acyclic analogues of 2'-deoxynucleosides related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine as potential antiviral agents

A series of acyclic analogues of 2'-deoxynucleosides related in structure to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) have been synthesized and evaluated for antiviral activity against herpes simplex virus type 1 (F strain). Additionally, the ability of these analogues to function as substrates for the virus-specified thymidine kinase was examined. Phosphorylation by this kinase is essential for antiviral activity. Although the acyclic 4-oxopyrimidine nucleosides were substrates for the kinase, they were devoid of antiviral activity. In the purine series, most analogues similar in structure to DHPG did exhibit significantly lower antiviral activity, indicating that even small modifications in the purine substituents substantially reduce the antiviral potency. The most active agent, 2,6-diaminopurine 27, was only poorly phosphorylated by the viral kinase; therefore, its activity was most likely due to a prior enzymatic deamination to give DHPG. Evaluation of 27 in a mouse encephalitis model has shown it to be nearly as potent as DHPG (1).     

Phosphorylation of nucleoside analogs by equine herpesvirus type 1 pyrimidine deoxyribonucleoside kinase

Replication of equine herpesvirus type 1 (EHV-1) was sensitive to 9-(1,3-dihydroxy-2-propoxymethyl)guanine(DHPG) but relatively resistant to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). Likewise, plaque formation by EHV-1 was inhibited by DHPG, but not by BVDU. Plaque formation by a thymidine kinase-negative (tk-) mutant of EHV-1 was not inhibited by DHPG. In order to investigate biochemical mechanisms determining the differential sensitivity of EHV-1 to these drugs, the EHV-1-encoded thymidine kinase enzyme activity (TK)1 was partially purified from EHV-1-infected cells and analyzed. The EHV-1-induced enzyme utilized both ATP and CTP as phosphate donors and differed in relative electrophoretic mobility from the TKs of mock-infected and HSV-1-infected cells. Phosphorylation of 3H-dThd by the EHV-1 TK was inhibited by AraT, IdUrd, BVDU, and DHPG. The EHV-1 TK phosphorylated 125I-dCyd and 3H-ACV. The results indicate that EHV-1 encodes a pyrimidine deoxyribonucleoside kinase with broad nucleoside substrate specificity. These observations suggest that the failure of BVDU to inhibit EHV-1 replication is not attributable to an inability of the EHV-1 TK to phosphorylate BVDU, but may result from the incapacity of the viral TK to convert BVDU monophosphate to the triphosphate or from lack of inhibitory effect of BVDU triphosphate on viral DNA polymerase reactions.     

Efficacy of 2'-nor-cyclicGMP in treatment of experimental herpes virus infections

9-[(2-Hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide (2'-nor-cGMP), the cyclic phosphate of 2'-nor-deoxyguanosine (2'-NDG) was synthesized by phosphorylation of 2'-NDG and evaluated for antiherpetic activity in cell cultures and in animal protection studies. 2'-nor-cGMP was effective in cell culture against both thymidine kinase deficient and wild-type herpes simplex virus type 1 strains and also against herpes simplex virus type 2. The anti-herpes activity of 2'-nor-cGMP against thymidine kinase deficient HSV-1 was confirmed by animal protection studies. Also, in comparative cell culture protection studies, the ED50 (microM) of 2'-nor-cGMP was approximately 10-fold lower than that of 2'-NDG against three strains of varicella zoster virus. In addition, 2'-nor-cGMP was effective orally in preventing HSV-1 orofacial infection and HSV-2 genital infection of mice. Topical therapeutic applications of 2'-nor-cGMP prevented orofacial HSV-1 lesion development in mice and development of HSV-2 genital lesions in guinea pigs. Subcutaneous application of 2'-nor-cGMP to intracerebral HSV-1 challenged weanling mice significantly prolonged survival. These studies indicate that 2'-nor-cGMP is not dependent on viral thymidine kinase for its antiviral activity and is highly effective in preventing experimental HSV infections.     

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses

The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6-99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.     

Synthesis and biological activity of dansyl thymidines

The synthesis of thymidines selectively dansylated in the 3'- and 5'-positions is reported. The biological investigation showed that these fluorescent nucleosides behave as competitive inhibitors of thymidine kinase (TK) from herpes simplex virus (HSV) and are endowed with a certain degree of antiviral activity against HSV-2 and HSV-1.     

Anti-herpesvirus activity of carbocyclic oxetanocin G in vitro

A series of new compounds, carbocyclic oxetanocins, have been synthesized and their anti-herpesvirus activity determined. Carbocyclic oxetanocin G (OXT-G) was most active against herpes simplex virus (HSV) and human cytomegalovirus (HCMV) among carbocyclic oxetanocins tested; the median effective concentrations (EC50) for HSV-1, -2, and HCMV were 0.23, 0.04 and 0.40 micrograms/ml, respectively. The EC50 value of carbocyclic OXT-G against HSV-2 was significantly lower than those of acyclovir, ganciclovir (DHPG) and OXT-G, while the value for HCMV was comparable to those of DHPG and OXT-G. Carbocyclic OXT-G showed much higher activity against TK+ HSV-2 than against a TK- mutant, suggesting that this compound is a good substrate for HSV-2-induced TK. The antiviral activity of the compound was only partially reversed even by the addition of 100-fold excess deoxyguanosine. The results suggest that the mode of action of carbocyclic OXT-G is different from that of OXT-G.     

Broad-spectrum antiviral activity of the acyclic guanosine phosphonate (R,S)-HPMPG

(R,S)-9-(3-hydroxy-2-phosphonomethoxypropyl)guanine [(R,S)-HPMPG] exhibits broad spectrum antiviral activity with an ED50 of less than 1 microM against herpes simplex virus (HSV) types 1 and 2, varicella zoster virus, human cytomegalovirus (HCMV) and vaccinia in plaque reduction assays. Wild type HSV-2 and its thymidine kinase deficient variant are equally sensitive to (R,S)-HPMPG. (R,S)-HPMPG is 100-fold more potent than acyclovir (ED50 = 0.45 microM vs. 44 microM, respectively) against HCMV in cell culture, and 10-fold more active than acyclovir in extending survival time in mice intraperitoneally infected with 70 LD50 HSV-1. However, (R,S)-HPMPG is toxic when administered repeatedly at 44 mg/kg/day in uninfected adult mice. The diphosphoryl derivative of HPMPG was enzymatically synthesized and is a competitive inhibitor of HSV-1 DNA polymerase relative to dGTP (K1 = 0.03 microM). HPMPG-PP is 70-fold less active at inhibiting HeLa DNA polymerase alpha than HSV-1 DNA polymerase. At concentrations between 0.3 and 1.5 microM (R,S)-HPMPG inhibited HSV-1 DNA replication greater than or equal to 50% in infected cells as measured by nucleic acid hybridization. Consistent with inhibition of viral DNA synthesis, 6 to 30 microM (R,S)-HPMPG reduces late viral polypeptide synthesis in HSV-1 infected cells. These data indicate that (R,S)-HPMPG is a thymidine kinase independent broad spectrum antiviral drug which is capable of inhibiting viral DNA polymerase.     

Synthesis and biological evaluation of 5-substituted derivatives of the potent antiherpes agent (north)-methanocarbathymine

The conformationally locked nucleoside, (north)-methanocarbathymine (1a), is a potent and selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2) viruses. Hereby, we report on the synthesis and biological evaluation of a small set of 5-substituted pyrimidine nucleosides belonging to the same class of bicyclo[3.1.0]hexane nucleosides. Both the 5-bromovinyl (4) and the 5-bromo analogue (3) appeared to be exclusive substrates of HSV1 thymidine kinase (TK), contrasting with the 5-iodo analogue (2), which was significantly phosphorylated by the human cytosolic TK. The binding affinity constant and catalytic turnover for HSV1 TK were measured to assess the influence of the substitution on these parameters. In the plaque reduction and cytotoxicity assays, the 5-bromo analogue (3) showed good activity against HSV1 and HSV2 with less general toxicity than 1a. Against varicella-zoster virus (VZV), the north-locked 5-bromovinyl analogue (4) proved to be as potent as its conformationally unlocked 2'-deoxyriboside equivalent BVDU. The three compounds were also tested in vitro as prodrugs used in a gene therapy context on three osteosarcoma cell lines, either deficient in TK (TK(-)), nontransduced, or stably transduced with HSV1 TK. The 5-iodo compound (2, CC(50) 25 +/- 7 microM) was more efficient than ganciclovir (GCV, CC(50) 75 +/- 35 microM) in inhibiting growth of HSV1-TK transfected cells and less inhibitory than GCV toward TK(-) cells, whereas compound 3 inhibited transfected and nontransfected cell lines in a relatively similar dose-dependent manner.     

Incorporation of the carbocyclic analog of 2'-deoxyguanosine into the DNA of herpes simplex virus and of HEp-2 cells infected with herpes simplex virus.

The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus. In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of [3H]CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7). Incubation of HSV-1-infected HEp-2 cells with [3H]CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA. These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases. Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs. The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with [3H]thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA. CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM). CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma. Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase. Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension. These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions. In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation.     

Comparison of antimicrobial activity of nuclear-substituted aromatic esters of 5-dimethylamino-1-phenyl-3-pentanol and 3-dimethylamino-1-phenyl-1-propanol with related cyclic analogs.

A series of six aromatic esters of both 5-dimethyl-amino-1-phenyl-3-pentanol and 3-dimethylamino-1-(2-phenylcyclohexyl)-1-propanol was prepared. Antimicrobial evaluation showed that the cyclic analogs had approximately twice the activity of the open chain series; in particular, the o-chlorophenyl ester showed pronounced activity against three pathogenic fungi at approximately 10 ppm. Aromatic esters of 3-dimethylamino-1-phenyl-1-propanol were prepared and demonstrated lower activity than two esters of 2-dimethylamino-1-phenylcyclohexanol. The screening results showed that the best activity was found when a dimethylene chain was present between the phenyl ring and the carbon atom bearing the acyloxy function and that the cyclic derivatives were more active than their more flexible counterparts.     

Arabinose furanosyl thymidine: uptake, phosphorylation and incorporation into DNA of mammalian cells

The naturally occurring nucleoside analogue arabinosyl thymidine is known as an anti-herpes and anti-cancer agent. The biologically active form is arabinosyl thymidine triphosphate (Ara-TTP), which inhibits cellular and viral DNA-polymerases and thus interferes with DNA replication. Using two murine erythroleukemia cell lines, Friend cell clone F4-6 and F4-12N, the latter being thymidine kinase deficient (TK-) cells transformed to a TK+ phenotype with the HSV TK gene, we have determined 1) the role of cellular and herpes simplex virus thymidine kinase (HSV TK) in the uptake of Ara-T into the cells; 2) the subsequent phosphorylation of intracellular Ara-T to Ara-TMP, Ara-TDP and Ara-TTP; 3) the incorporation of Ara-TTP into the DNA. Incorporation into DNA was studied under different conditions, including selective inhibition of the different cellular DNA polymerases by aphidicolin (that inhibits polymerases alpha and delta) and dideoxythymidine (that preferentially inhibits polymerases beta and gamma). The uptake of Ara-T into the methanol soluble pool of the cells depends upon its phosphorylation to Ara-TMP, which is more efficiently performed by the HSV TK than by the cellular TK, thus explaining the sensitivity of HSV infected cells to Ara-T. However, using increasing concentrations of Ara-T, we have shown that phosphorylation also occurs in normal control cells due to the cellular thymidine kinase. More than 90% of Ara-T is phosphorylated in the cell, and more than 60% of total Ara-T(MP, DP, TP) exists in the triphosphate form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of some 5′-amino-2′,5′-dideoxy-5-iodouridine derivatives and their antiviral properties against herpes simples virus

In an attempt to improve the antiviral efficacy of 5′-amino-2′,5′-dideoxy-5-iodouridine (AIdU) the N-acetyl and N,3′-O-diacetyl derivatives were prepared. N-Acetylation of AIdU increased its ability to inhibit the phosphorylation of thymidine by the deoxypyrimidine kinase of herpes simplex virus type 1 (HSV1) while diacetylation had the converse effect. The affinity of the corresponding compounds containing uracil or thymine for virus deoxypyrimidine kinase was also determined. A range of N-acyl-, N-sulphonyl- and N,3′-O-diacyl- derivatives of AIdU were synthesised; enhanced inhibition of deoxypyrimidine kinase by a number of these compounds was observed. The previous observation that 5′-azido-2′,5′-dideoxy-5-iodouridine has antiherpetic activity in vivo led us to investigate its 3′-O-acetyl derivative as well as the corresponding compound containing uracil. None of the derivatives described showed antiviral activity in cell culture against HSV1; acylation failed to enhance the potency of AIdU against HSV1 in vivo.     

Synthesis and evaluation of cis-1-[4-(hydroxymethyl)-2-cyclopenten-1-yl]-5-[(124)I]iodouracil: a new potential PET imaging agent for HSV1-tk expression

In our pursuit to find an appropriate reporter probe for herpes simplex virus type-1 thymidine kinase (HSV1-tk), a carbocyclic nucleoside analogue, cis-1-[4-(hydroxymethyl)-2-cyclopenten-1-yl]-5-[124I]iodouracil, has been efficiently synthesized. A Pd(0)-catalyzed coupling reaction together with organotin and exchange reactions for radiolabeling gave more than 80% radiochemical yield with greater than 95% radiochemical purity and 1.15 GBq/mumol specific activity. Biological data reveal that the analogue is stable in vitro, less toxic than ganciclovir (GCV), and selective to HSV1-tk-expressed cells based on micro positron emission tomography (microPET) image analyses. Thus, this new carbocyclic nucleoside, referred to in this paper as carbocyclic 2',3'-didehydro-2',3'-dideoxy-5-iodouridine (carbocyclic d4IU) is a potential imaging probe for HSV1-tk.     

Anti-herpes simplex virus activity of alkaloids isolated from Stephania cepharantha

By screening water and MeOH extracts of 30 Chinese medicinal plants for their anti-herpes simplex virus (HSV)-1 activity, a MeOH extract of the root tubers of Stephania cepharantha HAYATA showed the most potent activity on the plaque reduction assay with an IC50 value of 18.0 microg/ml. Of 49 alkaloids isolated from the MeOH extract, 17 alkaloids were found to be active against HSV-1, including 13 bisbenzylisoquinoline, 1 protoberberine, 2 morphinane and 1 proaporphine alkaloids, while benzylisoquinoline and hasubanane alkaloids were inactive. Although N-methylcrotsparine was active against HSV-1, as well as HSV-1 thymidine kinase deficient (acyclovir resistant type, HSV-1 TK-) and HSV-2 (IC50 values of 8.3, 7.7 and 6.7 microg/ml, respectively), it was cytotoxic. FK-3000 was found to be the most active against HSV-1, HSV-1 TK- and HSV-2 (IC50 values of 7.8, 9.9 and 8.7 microg/ml) with in vitro therapeutic indices of 90, 71 and 81, respectively. FK-3000 was found to be a promising candidate as an anti-HSV agent against HSV-1, acyclovir (ACV) resistant-type HSV-1 and HSV-2.     

Synthesis and in vitro evaluation of some modified 4-thiopyrimidine nucleosides for prevention or reversal of AIDS-associated neurological disorders

Oxygen-sulfur exchange at the C-4 carbonyl of several modified pyrimidine nucleosides, including 3'-azido-3'-deoxythymidine (AZT), is described in an effort to enhance the lipophilicity and, thereby, the delivery to the central nervous system of the sulfur analogues without compromising the anti-HIV activities of the parental structures. Preparation of 3'-azido-3'-deoxy-4-thiothymidine (3) proceeded from 4-thiothymidine (1) and utilized the same methodology developed for the initial synthesis of AZT. Thiation of 2',3'-didehydro-3'-deoxythymidine (4a) and 2',3'-didehydro-2',3'-dideoxyuridine (4c) was carried out with Lawesson's reagent on the corresponding 5'-O-benzoate esters, 4b and 4d, to give 5a and 5c, respectively. The latter, on alkaline hydrolysis, gave 2',3'-didehydro-3'-deoxy-4-thiothymidine (5b) and 2',3'-didehydro-2',3'-dideoxyuridine (5d), respectively. The same series of reactions were applied to the 5'-O-benzoate esters of 2',3'-dideoxyuridine (6a) and 3'-deoxythymidine (6b) to give 2',3'-dideoxy-4-thiouridine (7d) and 3'-deoxy-4-thiothymidine (7b), respectively. Characterization of the saturated and unsaturated thionucleosides included mass spectrometric studies. Under electron impact conditions, the thiated analogues gave more intense parent ions than the corresponding oxygen precursors. The lipophilicity of thymidine and the 3'-deoxythymidine derivatives are enhanced significantly, as indicated, by increases in corresponding P values (1-octanol-0.1 M sodium phosphate) upon replacement of the 4-carbonyl oxygens by sulfur. Compounds 5b, 5d, 7b, and 7d were evaluated for their effects on HIV-induced cytopathogenicity of MT-2 and CEM cells. Only 5b and 7b were moderately active in protecting both cell lines against the cytolytic effect of HIV. The inhibitory effects of analogues 5b, 5d, 7b, and 7d on thymidine phosphorylation by rabbit thymus thymidine kinase were evaluated. Only 3 showed moderate affinity (Ki = 54 microM) for the enzyme. The generally weak anti-HIV activities of the remaining thio analogues are consistent with correspondingly low susceptibilities to thymidine kinase phosphorylation as estimated from the respective Ki values of the synthetic nucleosides. However, the phosphorylation of the 5'-monophosphate derivatives to their respective 5'-triphosphates must also be considered in connection with the weak in vitro anti-HIV effects of these thiated compounds.     

Synthesis and antineoplastic activity of some cyano-, carboxy-, carbomethoxy-, and carbamoylborane adducts of heterocyclic amines.

Boron analogues of piperidine, piperazine, morpholine, and imidazole proved to be cytotoxic against the growth of murine and human tissue culture cells. Significant activity was demonstrated for single-cell suspensions of L1210 lymphoid leukemia, Tmolt3 lymphoblastic leukemia, and HeLa-S3 cervical carcinoma. Trimethylamine-imidazole carbonyldihydroborane 17 demonstrated activity against solid tumor growth of human colorectal adenocarcinoma, KB nasopharynx, and osteosarcoma. In addition, 4-methylpiperidine-carbomethoxyborane 12, 2-methylimidazole-3-cyanoborane 16, and 1-methylimidazole-3-(N-ethylcarbamoyl)borane 19 were active against the KB nasopharynx growth. Piperidine-cyanoborane 2, piperidine-carboxyborane 4, and 1-methylimidazole-3-(N-ethylcarbamoyl)borane 19 were effective in reducing the growth of osteosarcoma cells. The imidazole derivatives 13-19, as well as 4-methylpiperidine-carboxyborane 11 and carbomethoxyborane 12, demonstrated good activity against lung bronchogenic and glioma growth. In the in vivo studies, N-methylmorpholine-carboxyborane 7,4-phenylpiperidine-carboxyborane 9, 4-phenylpiperidine-carbomethoxyborane 10, 4-methylpiperidine-carboxyborane 11, imidazole cyanoborane 14, and 1-methylimidazole-3-carbomethoxyborane 18 demonstrated the best activity against Lewis Lung growth and P388 lymphocytic leukemia growth in mice. Mode of action studies in L1210 leukemia cells demonstrated that piperidine-carboxyborane 4 and N-methylmorpholine-carboxyborane 7 inhibited DNA synthesis, purine synthesis at PRPP amido transferase and IMP dehydrogenase sites, and thymidine kinase and thymidine diphosphate kinase activities, while lowering d(NTP) pool levels. Also, DNA strand scission was evident after incubation with these drugs.     

Prolactin stimulation of ornithine decarboxylase and mitogenesis in Nb2 node lymphoma cells: the role of protein kinase C and calcium mobilization

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination with calcium ionophores has been shown to bypass the requisite antigen- or lectin-induced signal for lymphocyte mitogenesis. This suggests that protein kinase C activation and calcium mobilization may be early events required for lymphocyte proliferation. Therefore, the relationship(s) of protein kinase C activation and calcium mobilization to ornithine decarboxylase induction and cellular proliferation were examined in a rat node lymphoma cell line (Nb2) which is dependent upon prolactin (PRL) for mitogenesis. TPA enhanced PRL-stimulated Nb2 node lymphoma cell ornithine decarboxylase induction and [3H]thymidine incorporation. Addition of a calcium ionophore (A23187) to cultures containing TPA plus PRL increased ornithine decarboxylase above PRL alone or PRL plus TPA but inhibited proliferation compared to the PRL plus TPA regimen. Exposure of cells to TPA or TPA plus A23187 increased [3H]thymidine incorporation in a similar manner to that demonstrated for low-dose PRL. However, optimal concentrations were only 20-25% as effective as mitogens as was optimal PRL. Protein kinase C and calmodulin antagonists inhibited PRL-stimulated ornithine decarboxylase induction and proliferation. Ca2+ chelation or cation channel antagonism inhibited both PRL-stimulated responses. The cyclic AMP analogue, 8Br-cAMP, inhibited PRL-stimulated ornithine decarboxylase activity as well as cellular proliferation processes assessed by [3H]thymidine incorporation. Finally, tumor-promoting phorbol esters inhibited 125I-rPRL binding. These data strongly suggest that protein kinase C activation and calcium mobilization are requisite events for PRL-stimulated ornithine decarboxylase induction and cellular proliferation in Nb2 node lymphoma cells. An additional component that is linked to alterations in K+ channeling is also implicated. These data support a role for protein kinase C in PRL-coupled mitogenesis. However, other critical Ca2+ and/or ion-induced events are also required.     

Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus

The in vitro antiviral activity of antimicrobial cationic peptides: cecropin A, melittin, magainin I and II and indolicidin against the arenavirus Junin virus (JV), and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) was evaluated. Cecropin A effectively inhibited JV multiplication and failed to affect HSV replication whereas melittin impeded the multiplication of JV and HSV, but was highly toxic for the host cell. Magainins I and II exhibited inhibitory action toward HSV-1 and HSV-2 but were inactive against JV. Only indolicidin showed a direct inactivation effect on cell-free virus stocks. Besides its inhibitory effect on JV replication cecropin A also was active against the arenaviruses Tacaribe and Pichinde, mainly affecting late events of arenavirus multiplication cycle by preventing viral morphogenesis and egress from infected cells.