Chronic cocaine exposure during pregnancy increases postpartum neuroendocrine stress responses.

The cycle of chronic cocaine (CC) use and withdrawal results in increased anxiety, depression and disrupted stress-responsiveness. Oxytocin and corticosterone (CORT) interact to mediate hormonal stress responses and can be altered by cocaine use. These neuroendocrine signals play important regulatory roles in a variety of social behaviours, specifically during the postpartum period, and are sensitive to disruption by CC exposure in both clinical settings and preclinical models. To determine whether CC exposure during pregnancy affected behavioural and hormonal stress response in the early postpartum period in a rodent model, Sprague-Dawley rats were administered cocaine daily (30 mg/kg) throughout gestation (days 1-20). Open field test (OFT) and forced swim test (FST) behaviours were measured on postpartum day 5. Plasma CORT concentrations were measured before and after testing throughout the test day, whereas plasma and brain oxytocin concentrations were measured post-testing only. The results obtained indicated increased CORT response after the OFT in CC-treated dams (P ≤ 0.05). CC-treated dams also exhibited altered FST behaviour (P ≤ 0.05), suggesting abnormal stress responsiveness. Peripheral, but not central, oxytocin levels were increased by cocaine treatment (P ≤ 0.05). Peripheral oxytocin and CORT increased after the FST, regardless of treatment condition (P ≤ 0.05). Changes in stress-responsiveness, both behaviourally and hormonally, may underlie some deficits in maternal behaviour; thus, a clearer understanding of the effect of CC on the stress response system may potentially lead to treatment interventions that could be relevant to clinical populations. Additionally, these results indicate that CC treatment can have long-lasting effects on peripheral oxytocin regulation in rats, similar to changes observed in persistent social behaviour and stress-response deficits in clinical populations.

Synthesis and unusual chemical reactivity of certain novel 4,5-disubstituted 7-benzylpyrrolo[2,3-d][1,2,3]triazines.

The fact that only two pyrrolo[2,3-d][1,2,3]triazines heterocycles had been reported in the literature prompted us to initiate studies designed to provide additional members of this ring system. Initial attempts to prepare additional derivatives of the 7-unsubstituted pyrrolo[2,3-d][1,2,3]triazin-4-ones were limited by their low chemical reactivity. Subsequently, 7-benzyl-5-carboxamidopyrrolo[2,3-d][1,2,3]triazin-4-one (16) was prepared from diethyl 2-nitropyrrole-3,4,-dicarboxylate via an alkylation, ammonolysis, reduction and intramolecular diazocoupling sequence. Conversion of 16 into 7-benzyl-4-(1,2,4-triazol-1-yl)pyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile (17) was accomplished, and nucleophilic displacements of the 4-triazol-1-yl group were studied. Treatment of 17 with NH3/CH3CN gave a mixture of 4-amino-7-benzylpyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile (19) and 2-amino-1-benzylpyrrole-3,4-dicarbonitrile (21). A mechanism to account for the formation of this mixture is described along with studies on the effect that ammonia concentration and a TFA catalyst have on the product ratio. Compound 19 was converted into the 5-carboxamide and 5-thioamide derivatives of 19.

Dimetalation of pyrazines. a one-pot synthesis of multisubstituted pyrazine C-nucleosides.

As a part of our efforts to pursue direct, convergent, and concise methodologies for the synthesis of pyrazine C-nucleosides, we have successfully established a sequential dilithiation-addition method, which allows one to introduce two different functional groups to a pyrazine ring in a one-pot fashion. 2,6-Dichloropyrazine was dilithiated at -100 degrees C and then allowed to react with an electrophile, such as bromine, iodine, or disulfides, followed by a reaction with a protected ribonolactone to afford C-nucleosides. After reduction and deprotection, tetrasubstituted pyrazine C-nucleosides, including 2,6-dichloro-3-iodo-5-(beta-D-ribofuranosyl)pyrazine and 2-bromo-3,5-dichloro-6-(beta-D-ribofuranosyl)pyrazine, were obtained. A tandem reaction sequence occurred when disulfides were used, resulting in the formation of 5,6-bis-methylthio-2-chloro-3-(beta-D-ribofuranosyl)pyrazine and 6-(beta-D-ribofuranosyl)-2,3,5-tris-phenylthiopyrazine.

Acid-catalyzed dehydrative cyclization of 4-(D-galacto -pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole. synthesis and anomeric configuration of D-lyxo-C-nucleoside analogs.

Dehydration of 4-(D-galacto-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole with 20% methanolic sulfuric acid afforded the anomeric pairs of nucleosides, 4-(alpha-D-lyxopyranosyl)-2-phenyl-2H-1,2,3-triazole (major component) and its beta-anomer, as well as 4-(alpha-D-lyxofuranosyl)-2H-1,2,3-triazole and its beta-anomer. The four anomeric C-nucleosides were separated by chromatography, and their structure and anomeric configuration were determined by periodate oxidation, acylation, and NMR spectroscopy as well as mass spectrometry. The anomeric assignment from optical rotation was not in agreement with final structure assignment and represented a violation of the Hudson isorotation rules. NOE studies and X-ray diffraction measurements confirmed the anomeric configuration.

Design, synthesis, and biological evaluation of tricyclic nucleosides (dimensional probes) as analogues of certain antiviral polyhalogenated benzimidazole ribonucleosides.

The polyhalogenated benzimidazole nucleosides 2,5, 6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and the 2-bromo analogue (BDCRB) were synthesized in our laboratory and established as potent and selective inhibitors of human cytomegalovirus (HCMV) with a novel mode of action. In an effort to study the behavior of the key substructure in a dimensionally extended manner and probe the spatial limitation of the target enzyme(s), a series of 2-substituted 6, 7-dichloro-1-(beta-D-ribofuranosyl)naphtho¿2,3-dimidazoles and the N1- and N3-ribonucleosides of 2-substituted 6,7-dichloroimidazo¿4, 5-bquinolines were prepared. The nucleosides 6, 7-dichloro-1-(beta-D-ribofuranosyl)imidazo¿4,5-bquinolin-2-one and 6,7-dichloro-3-(beta-D-ribofuranosyl)imidazo¿4,5-bquinolin-2-one were selected and used as the key synthetic intermediates in the imidazo¿4,5-bquinoline series. Evaluation of the compounds for activity against HCMV and herpes simplex virus type 1 revealed that the trichloro analogues of TCRB (2a, 3a) were nearly as active against HCMV as TCRB but were more cytotoxic. The results suggest that extending the heterocycle of TCRB affected the affinity for the HCMV target only slightly but increased the affinity for cellular enzymes.

Deoxy sugar analogues of triciribine: correlation of antiviral and antiproliferative activity with intracellular phosphorylation.

Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic activity at low micromolar or submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore requirements for the number of hydroxyl groups on the ribosyl moiety for biological activity. 2'-Deoxytriciribine (2'-dTCN), 3'-deoxytriciribine (3'-dTCN), 2', 3'-epoxytriciribine (2',3'-epoxyTCN), 2',3'-dideoxy-2', 3'-didehydrotriciribine (2',3'-d4TCN), and 2',3'-dideoxytriciribine (2',3'-ddTCN) were synthesized and evaluated for activity against human immunodeficiency virus (HIV-1), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV). Antiproliferative activity of the compounds also was tested in murine L1210 cells and three human tumor cell lines. All compounds were either less active than TCN and TCN-P or inactive at the highest concentration tested (100 microM) in both antiviral and antiproliferative assays. Reverse-phase HPLC of extracts from uninfected cells treated with the deoxytriciribine analogues only detected the conversion of 3'-dTCN and 2',3'-ddTCN to their respective monophosphates. Therefore, either the deoxytriciribine analogues were not transported across the cell membrane or, more likely, they were not substrates for a nucleoside kinase or phosphotransferase. We have concluded that the hydroxyl groups on the ribosyl ring system of TCN and TCN-P must be intact in order to obtain significant antiviral and antineoplastic activity.

Synthesis and antiviral evaluation of trisubstituted indole N-nucleosides as analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB).

2,5,6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) are nucleosides that exhibit strong and selective activity against human cytomegalovirus (HCMV). Selected polyhalogenated indole nucleosides have now been synthesized as 3-deaza analogues of the benzimidazole nucleosides using the sodium salt glycosylation method. 2-Benzylthio-1-¿2-deoxy-3, 5-bis-O-(4-methylbenzoyl)-beta-D-erythro-pentofuranosyl-5, 6-dichloroindole (8) was prepared stereoselectively via the coupling of a 2-deoxyribofuranosyl alpha-chloride derivative with the sodium salt of 2-benzylthio-5,6-dichloroindole (5). Compound 8 was then elaborated into the targeted 2-benzylthio-1-(beta-D-ribofuranosyl)-5, 6-dichloroindole (18) in five steps. 2,5, 6-Trichloro-(1-beta-D-ribofuranosyl)indole (19) was prepared using the same synthetic route with 2,5,6-trichloroindole (6) as the starting material. We were subsequently able to prepare 19 in three steps using a modification of the sodium salt glycosylation method. 2-Bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)indole (25) was also prepared using the same procedures. Target compounds were tested for activity against HCMV, herpes simplex virus type 1 (HSV-1), and human herpes virus six (HHV-6) and for cytotoxicity. All of the compounds were less active against HCMV than TCRB and weakly active or inactive against HSV-1 and HHV-6.

6-N-Acyltriciribine analogues: structure-activity relationship between acyl carbon chain length and activity against HIV-1.

Triciribine (TCN) and triciribine-5'-monophosphate (TCN-P) are active against HIV-1 at submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore the tolerance of TCN to structural modifications at the 6-position. A number of 6-N-acyltriciribine analogues were synthesized and evaluated for antiviral activity and cytotoxicity. The cytotoxicity of these compounds was minimal in three human cell lines (KB, CEM-SS cells, and human foreskin fibroblasts (HFF)). The compounds were marginally active or inactive against herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). In contrast, most of the compounds exhibited moderate to high activity against human immunodeficiency virus type 1 (HIV-1), IC(50)'s = 0.03 to 1 microM. This structure-activity relationship study identified the N-heptanoyl group as having the optimal carbon chain length. This compound was as active against HIV-1 as TCN and TCN-P. Reverse phase HPLC of extracts from uninfected cells treated with 6-N-acyltriciribines detected sufficient TCN-P to account for anti-HIV activity thereby suggesting a prodrug effect. Studies in an adenosine kinase deficient cell line showed that the 6-N-acyl derivative was not phosphorylated directly but first was metabolized to triciribine which then was converted to TCN-P.

Synthesis and antiviral evaluation of halogenated beta-D- and -L-erythrofuranosylbenzimidazoles.

A series of 2-substituted benzimidazole D- and L-erythrofuranosyl nucleosides were synthesized and tested for activity against herpesviruses and for cytotoxicity. The D-nucleosides 2,5, 6-trichloro-1-(beta-D-erythrofuranosyl)benzimidazole (8a) and 2-bromo-5,6-dichloro-1-(beta-D-erythrofuranosyl)benzimidazole (8b) were prepared by coupling 1,2,3-tri-O-acetyl-beta-D-erythrofuranose (D-6) with the appropriate benzimidazole, followed by removal of the acetyl protecting groups. The 2-isopropylamino (9), 2-cyclopropylamino (10), and 2-mercaptobenzyl (11) derivatives were synthesized by nucleophilic displacements of the C-2 chlorine in the benzimidazole moiety of 8a. The D-nucleoside 4-bromo-5, 6-dichloro-2-isopropylamino-1-(beta-D-erythrofuranosyl)benzimid azo le (17) was prepared by coupling D-6 with the appropriate benzimidazole. The L-erythrofuranosyl derivatives, 5, 6-dichloro-2-isopropylamino-1-(beta-L-erythrofuranosyl)benzimid azo le (21a), its 2-cyclopropylamino analogue (21b), and the 2-isopropylamino analogue (25), were prepared by coupling L-6 with the appropriate benzimidazole. Several of these new derivatives had very good activity against HCMV in plaque and yield reduction assays (IC(50) = 0.05-19 microM against the Towne strain of HCMV) and DNA hybridization assays. Very little activity was observed against other herpesviruses. This pattern is similar to the antiviral activity profile observed for the corresponding ribofuranosides 2,5, 6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (4a), its 2-bromo analogue (4b), and the 2-cyclopropylamino analogue (4c). In comparison, 8a was 15-fold more active against HCMV than 4a, and 8b was 4-fold more active against HCMV than 4b. The 5, 6-dichloro-2-isopropylamino-1-(beta-L-erythrofuranosyl)benzimid azo le (21a) was less active than 4c, which is now in clinical trials for HCMV infection. Both 8a,b had comparable HCMV activity to 4c. Mode of action studies with the D-erythrose analogues established that 8b acted by inhibition of viral DNA processing whereas 9 and 10 may act via a different mechanism. The lack of a 5'-hydroxymethyl group in all members of this series established that antiviral activity occurred without 5'-phosphorylation, a feature required for the activity of most nucleoside analogues.

Synthesis of fluorosugar analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole as antivirals with potentially increased glycosidic bond stability.

The metabolic instability in vivo of the glycosidic bond of 2,5, 6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) prompted us to design and synthesize the hitherto unreported fluorinated benzimidazole nucleosides 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole , 2,5, 6-trichloro-1-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)benzimidazole, and 2-bromo-5, 6-dichloro-1-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)benzimidazole. TCRB was converted into the 2',5'-ditrityl and 3',5'-ditrityl derivatives, which were fluorinated with DAST and deprotected to yield 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole and 2,5, 6-trichloro-1-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)benzimidazole. The resulting low overall yield (5%) of 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole encouraged us to develop an alternative route. The heterocycle 2,5, 6-trichlorobenzimidazole was condensed with 1-bromo-3, 5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-D-arabinofuranose to give, after deprotection, 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole in a 50% overall yield. The 2'-deoxy-2'-fluoro-beta-D-ribofuranosyl compounds were prepared using 2'-deoxy-2'-fluorouridine, N-deoxyribofuranosyl transferase, and 5,6-dichlorobenzimidazole. Functionalization of the C2 position then gave the desired derivatives. Antiviral and cytotoxicity testing revealed that the deoxy fluoro arabinofuranosyl, xylofuranosyl, and ribofuranosyl derivatives were less active against human cytomegalovirus and more cytotoxic than TCRB.

Synthesis of 4-substituted imidazo[4,5-d][1,2,3]triazine (2-azapurine)nucleosides.

Several methods for functionalization of the 4-position of imidazo[4,5-d][1,2,3]triazin-4-one were investigated. These investigations were successful and led to the preparation of 4-amino, 4-triazol-1-yl, 4-methoxy, 4-methylthio, 4-methylamino, 4-thio, 4-nitrobenzyl, and 4-unsubstituted 9-(beta-D-ribofuranosyl)-imidazo-[4,5-d][1,2,3]triazine (2-azapurine ribosides). The 4-unsubstituted compound (19) was slightly active against HCMV in plaque and yield reduction experiments and was not cytotoxic at 100 microM. The methylamino (15), hydrazino (16), and p-nitrobenzylthio (20) were inactive against HCMV but slightly cytotoxic. The thiomethyl-substituted analog (21) was the most active with activity comparable to ganciclovir but with greater cytotoxicity. We conclude that even though none of the tested compounds had antiviral activity superior to ganciclovir, the new synthetic methods will provide a route to more interesting compounds.

Synthesis and evaluation of a series of 2'-deoxy analogues of the antiviral agent 5,6-dichloro-2-isopropylamino-1-(beta-L-ribofuranosyl)-1H-benzimidazole (1263W94).

A series of 2'-deoxy analogues of the antiviral agent 5,6-dichloro-2-isopropylamino-1-(beta-L-ribofuranosyl)-1H-benzimidazole (1263W94) were synthesized and evaluated for activity against human cytomegalovirus (HCMV) and for cytotoxicity. The 2-substituents in the benzimidazole moiety correspond to those that were used in the 1263W94 series. In general, as was found in the 1263W94 series, cyclic and branched alkylamino groups were needed for potent activity against HCMV. Three analogues 3a, 3b and 3d were as potent as 1263W94. Further evaluation of two analogues, 3a and 3b, suggested that these 2'-deoxy analogues may act via a novel mechanism of action similar to that of 1263W94. These 2'-deoxy analogues generally lacked cytotoxicity in vitro. Pharmacokinetic parameters in mice and protein binding properties of 3a were quite similar to 1263W94. However, the oral bioavailability of 3a was only half of that observed for 1263W94.

Design, synthesis, and antiviral evaluation of 2-deoxy-D-ribosides of substituted benzimidazoles as potential agents for human cytomegalovirus infections.

Stereoselective glycosylation of 2,5,6-trichlorobenzimidazole (1b), 2-bromo-5,6-dichlorobenzimidazole (1c), 5,6-dichlorobenzimidazole (1d), 5,6-dichlorobenzimidazole-2-thione (1e), 5,6-dichloro-2-(methylthio)benzimidazole (1f), 2-(benzylthio)-5,6-dichlorobenzimidazole (1g), and 2-chloro-5,6-dimethylbenzimidazole (1h) with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride was achieved to give the desired beta nucleosides 2b-h. Subsequent deprotection afforded the corresponding free beta-D-2-deoxyribosides 3b-h. The 2-methoxy derivative 3i was synthesized by the treatment of 2b with methanolic sodium methoxide. Displacement of the 2-chloro group of 2b with lithium azide followed by a removal of the protective groups gave the 2-azido-5,6-dichlorobenzimidazole derivative (5). The 2-amino derivative (6) was obtained by hydrogenolysis of 5 over Raney nickel. 5,6-Dichloro-2-isopropylamino-1-(2-deoxy-beta-D-erythro- pentofuranosyl)benzimidazole (10) was prepared using 2'-deoxyuridine (7), N-deoxyribofuranosyl transferase and 1d followed by functionalization of the C2 position. Antiviral evaluation of target compounds established that compounds 3b and 3c were active against human cytomegalovirus (HCMV) at non-cytotoxic concentrations. The activity of these 2-deoxy ribosides, however, was less than the activity of the parent riboside, 2,5,6-trichloro-1-beta-D-ribofuranosylbenzimidazole (TCRB). Compared to TCRB, 3b and 3c were somewhat more cytotoxic and active against herpes simplex virus type 1. Compounds 3d-i with other substituents in the 2-position were inactive against both viruses and non-cytotoxic. In contrast, compounds with amine substituents in the 2-position (5, 6, 10) were active against HCMV albeit less so than TCRB. These results establish that 2-deoxy-D-ribosyl benzimidazoles are less active against the DNA virus HCMV than are the corresponding D-ribosides.

Differences in DNA packaging genes and sensitivity to benzimidazole ribonucleosides between human cytomegalovirus strains AD169 and Towne.

The AD169 strain of human cytomegalovirus was approximately twofold more sensitive to polyhalogenated benzimidazole ribonucleosides than Towne strain. Sequence differences between the two strains have been identified in genes UL51, UL52, UL56, UL77, UL89 and UL104. Because these genes are involved in cleavage and packaging of viral DNA and the benzimidazole ribonucleosides inhibit this process, these sequence differences may be involved in the difference in drug sensitivity.

Nonnucleoside pyrrolopyrimidines with a unique mechanism of action against human cytomegalovirus.

Based upon a prior study which evaluated a series of nonnucleoside pyrrolo[2,3-d]pyrimidines as inhibitors of human cytomegalovirus (HCMV), we have selected three active analogs for detailed study. In an HCMV plaque-reduction assay, compounds 828, 951, and 1028 had 50% inhibitory concentrations (IC(50)s) of 0.4 to 1.0 microM. Similar results were obtained when 828 and 951 were examined by HCMV enzyme-linked immunosorbent assay (IC(50)s = 1.9 and 0.4 microM, respectively) and when 828 was tested in a viral DNA-DNA hybridization assay (IC(50) = 1.3 microM). In yield-reduction assays with a low multiplicity of infection (MOI), all three compounds caused multiple log(10) reductions in virus titer, and the activities of these compounds were comparable to the activity of ganciclovir (GCV; IC(90) = 0.2 microM). In contrast to the reduction of viral titers by GCV, the reduction of viral titers by 828, 951, and 1028 decreased with increasing MOI. Cytotoxicity in human foreskin fibroblasts and KB cells ranged from 32 to >100 microM. In addition, 828 (the only compound tested) was less toxic against human bone marrow progenitor cells than GCV. Time-of-addition and time-of-removal studies established that the three pyrrolopyrimidines inhibited HCMV replication before GCV had an effect on viral DNA synthesis but after viral adsorption. Compound 828 was equally effective against GCV-sensitive and GCV-resistant HCMV clinical isolates. Combination studies with 828 and GCV showed that the effects of the two compounds on HCMV were additive but not synergistic. Taken together, the data indicate that these pyrrolopyrimidines target a viral protein that is required in an MOI-dependent manner and that is expressed early in the HCMV replication cycle.

Studies designed to increase the stability and antiviral activity (HCMV) of the active benzimidazole nucleoside, TCRB.

The potent activity of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) against Human Cytomegalovirus with the concomitant low cellular toxicity at concentrations that inhibit viral growth prompted considerable interest in this research area. This interest was moderated by the pharmacokinetic studies of TCRB in rats and monkeys that revealed the instability of TCRB in vivo. These studies suggested that the instability was due to a cleavage of the glycosidic bond in vivo which released the heterocycle (2,5,6-trichlorobenzimidazole) into the bloodstream. This prompted us to initiate synthetic studies designed to increase the stability of the glycosidic bond of TCRB and BDCRB. Several synthetic approaches to address this and other problems are presented.

Total synthesis of the naturally occurring antibiotic toyocamycin using new and improved synthetic procedures.

Starting with commercially available tetracyanoethylene, we describe a more efficient and higher yielding synthesis of toyocamycin with regards to convenience, overall yield, and total reaction time than those syntheses previously reported.

Acyclic sugar analogs of triciribine: lack of antiviral and antiproliferative activity correlate with low intracellular phosphorylation.

Triciribine and triciribine monophosphate have antiviral and antiproliferative activity at low or submicromolar concentrations. In an effort to improve and better understand this activity, we have synthesized a series of acyclic analogs and evaluated them for activity against select viruses and cancer cell lines. We conclude that the rigid ribosyl ring system of triciribine must be intact in order to be phosphorylated and to obtain significant antiviral and antiproliferative activity.

An unprecedented nitrogen elimination reaction: mechanistic studies using 15N-labeled 4-amino-7-benzylpyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile.

[formula: see text] In the course of our work on novel pyrrolo[2,3-d][1,2,3]triazines we have discovered that 1 undergoes an elimination of nitrogen at 250 degrees C to give 2. We have conducted 15N labeling studies that establish that the loss of N-2 and N-3 from 1 had occurred rather than N-1 and N-2, presumably via a retro Diels-Alder reaction of the imino tautomer 7. This study provides an alternative to the commonly accepted mechanism which involves the loss of N-1 and N-2 via the transient formation of a diazonium compound generated from 4-amino- or 4-oxo-substituted 1,2,3-triazines condensed with carbocycles or heterocycles.

Phosphorylation of triciribine is necessary for activity against HIV type 1.

Triciribine (TCN) is a tricyclic nucleoside with known antineoplastic and antiviral activity. It is a potent and selective inhibitor of HIV-1 and HIV-2, including strains known to be resistant to AZT or TIBO. TCN is phosphorylated to its 5'-monophosphate (TCN-P) by intracellular adenosine kinase (AK), but is not converted to di- or triphosphates. We now report that 5'-phosphorylation is requisite for the activity of TCN against HIV-1. CEM cells incubated with TCN at concentrations ranging from 0.1 to 330 microM gave intracellular TCN-P concentrations from 27 to 775 microM, respectively. There was no difference in the amount of intracellular TCN-P detected in uninfected compared with HIV-1-infected CEM cells. The antiviral effect of TCN against HIV-1 was strongly antagonized by the AK inhibitor 5-iodotubercidin (ITu). In contrast, TCN and ITu only exhibited additive cytotoxicity. The 5'-deoxy analog of TCN, which cannot be phosphorylated, had no antiviral effect against HIV-1 at a concentration more than 100 times higher than the IC50 of TCN. Similarly, TCN was not active against HIV-1 in an AK-deficient cell line (AA-2) at concentrations shown to inhibit the virus by >95% in CEM cells. Consistent with its AK-deficient phenotype, this cell line phosphorylated TCN to only 3% of the extent observed in CEM cells. We conclude that TCN must be phosphorylated to TCN-P for activity against HIV-1.