In vivo evaluation of the uptake of [(123)I]FIAU, [(123)I]IVFRU and [(123)I]IVFAU by normal mouse brain: potential for noninvasive assessment of HSV-1 thymidine kinase gene expression in gliomas.

Radioiodinated 5-iodo-1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)uracil (F *IAU) is most commonly used for noninvasive assessment of herpes simplex virus type 1 thymidine kinase (HSV-1-tk) gene expression. However, it does not permeate the intact blood-brain barrier (BBB) because of its moderate lipophilicity. In this work, three iodo-nucleosides, FIAU, IVFRU, and IVFAU, were radiolabeled with iodine-123 and tested for permeation of the BBB in mice and for potential measurement of HSV-1-tk gene expression in gliomas. The results demonstrate that brain uptake and retention of these nucleosides is not directly related to their lipophilicity. The low brain uptake of IVFAU, in conjunction with its higher and constant brain/blood ratio, may reflect greater stability against hydrolysis of the N-glycosidic bond. In vivo PET evaluations of [(124)I]IVFRU and [(124)I]IVFAU in tumor-bearing mice are warranted.

Synthesis of unnatural 7-substituted-1-(2-deoxy-beta-D-ribofuranosyl)isocarbostyrils: "thymine replacement" analogs of deoxythymidine for evaluation as antiviral and anticancer agents.

A group of unnatural 1-(2-deoxy-beta-D-ribofuranosyl)isocarbostyrils having a variety of C-7 substituents [H, 4,7-(NO2)2, I, CF3, CN, (E)-CH=CH-I, -C triple bond CH, -C triple bond C-I, -C triple bond 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 triple bond 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.

Design and synthesis of 4,5-diphenyl-4-isoxazolines: novel inhibitors of cyclooxygenase-2 with analgesic and antiinflammatory activity.

4,5-Diphenyl-4-isoxazolines (13a-k) possessing a variety of substituents (H, F, MeS, MeSO2) at the para position of one of the phenyl rings were synthesized for evaluation as analgesic and selective cyclooxygenase-2 (COX-2) inhibitory antiinflammatory (AI) agents. Although the 4,5-phenyl-4-isoxazolines (13a-d,f), which do not have a C-3 Me substituent, exhibited potent analgesic and AI activities, those compounds evaluated (13a, 13b, 13h, and 13k) were not selective inhibitors of COX-2. In contrast, 2,3-dimethyl-5-(4-methylsulfonylphenyl)-4-phenyl-4-isoxazoline (13j) exhibited excellent analgesic and AI activities, and it was a potent and selective COX-2 inhibitor (COX-1, IC(50) = 258 microM; COX-2, IC(50) = 0.004 microM). A related compound 13k having a F substituent at the para position of the 4-phenyl ring was also a selective (SI = 3162) but less potent (IC(50) = 0.0316 microM) inhibitor of COX-2 than 13j. A molecular modeling (docking study) for 13j showed that the S atom of the MeSO2 substituent is positioned about 6.46 A inside the entrance to the COX-2 secondary pocket (Val(523)) and that a C-3 Me (13j, 13k) central isoxazoline ring substituent is crucial to selective inhibition of COX-2 for this class of compounds.

Design and synthesis of celecoxib and rofecoxib analogues as selective cyclooxygenase-2 (COX-2) inhibitors: replacement of sulfonamide and methylsulfonyl pharmacophores by an azido bioisostere.

Celecoxib (13) and rofecoxib (17) analogues, in which the respective SO2NH2 and SO2Me hydrogen-bonding pharmacophores were replaced by a dipolar azido bioisosteric substituent, were investigated. Molecular modeling (docking) studies showed that the azido substituent of these two analogues (13, 17) was inserted deep into the secondary pocket of the human COX-2 binding site where it undergoes electrostatic interaction with Arg(513). The azido analogue of rofecoxib (17), the most potent and selective inhibitor of COX-2 (COX-1 IC(50) = 159.7 microM; COX-2 IC(50) = 0.196 microM; COX-2 selectivity index = 812), exhibited good oral antiinflammatory and analgesic activities.

(E)-1-(2'-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-(2-iodovinyl)benzene.

This analysis of the title compound, C13H13F2IO3, establishes the orientation of (E)-5-(CH=CH-I) as antiperiplanar (ap) to the C-C bond (5-6 position) of the 2,4-difluorophenyl ring system, with the (E)-5-(CH=CH-I) H atom located in close proximity (2.17 A) to the F4 atom of the 2,4-difluorophenyl moiety.

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.

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.

Synthesis and biological investigations of 5-substituted pyrimidine nucleosides coupled to a dihydropyridine/pyridinium salt redox chemical delivery system.

The syntheses, antiviral activities, and partition coefficients (P) of 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-coupled nucleosides are described. These novel compounds were designed in an effort to enhance the lipophilicity, and thereby the delivery to the CNS, without compromising the anti-HSV-1 activity of the parental nucleosides. We have previously reported the synthesis of 3'-O-(1-methyl-1,4-dihydropyridyl-3- carbonyl) analogs of 5-iodo-(5), 5-vinyl-(6), and (E)-5-(2-iodovinyl)-2'-deoxyuridines (7). We now report the synthesis of 5-iodo-3'-O-(1-methyl-1,4-dihydropyridyl-3- carbonyl)-5'-O-acetyl-2'-deoxyuridine (15) and 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-deoxyuridine (17). Quarternization of the 3'-O-(3-pyridylcarbonyl) compounds (10,12) using iodomethane afforded the corresponding 1-methyl pyridinium salts (13,14) which were reduced with sodium dithionite to yield the corresponding 3'-O-1-methyl-1,4-dihydropyridyl-3-carbonyl compounds (15,16). The deprotection of 3'-O-(1-methyl-1,4-dihydropyridyl- 3-carbonyl)-5'-O-t-butyldimethylsilyl-2'-deoxyuridine (16) with Bu4N+F- afforded 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-deoxyuridine (17). Compounds 5-7 and 15 were evaluated for their antiviral activity in vitro against HSV-1, HSV-2, HCMV, and VZV, and were found to retain anti-HSV-1, HSV-2 and VZV activity as compared to their parental nucleosides (1-3). In addition, the cellular toxicity of 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-coupled compounds (5-7 and 15) was found to be lower than the parent nucleosides. The lipophilicity of compounds (5-7,15,17) are enhanced substantially, compared to the parent nucleosides, as indicated by an increase in corresponding P values (1-octanol-water) upon replacement of the C-3' hydroxyl by 1-methyl-1,4-dihydropyridyl-3-carbonyl moiety.

Enzyme-targeted, nucleoside-based radiopharmaceuticals for scintigraphic monitoring of gene transfer and expression.

Enzymes, the expression products of transferred or native genes, offer unique windows of opportunity for clinical diagnosis and therapy. Although some expression products can be monitored in plasma, nuclear medicine imaging (SPECT and PET) offers the unique ability to selectively measure the intensity and regional/spatial distribution of gene expression both in vivo, in situ. Importantly, the superior sensitivity and moderate spatial resolution of the nuclear techniques also enable in vivo kinetic characterization of enzyme-substrate interaction. Indeed, the non-invasive, whole-body assessment of gene expression can only be achieved through imaging techniques. Given today's technology, nuclear imaging techniques uniquely provide the necessary sensitivity required to evaluate the success of the gene delivery and expression (transcription and translation), and to detect unwanted expression by non-target tissues. Enzymes are a special class of proteinacious gene expression products that selectively bind specific substrates for the purpose of molecular biotransformation rather than for signal transduction. In general, enzymes have received much less attention for imaging than receptors and antibodies, despite the enzymes' high substrate specificity and the potential for kinetic evaluation. Enzymes are attractive targets for diagnostic imaging and radioisotope radiotherapy because they convert multiple molecular copies of the substrate (radiotracer) per molecule of enzyme, thereby greatly increasing the ultimate sensitivity relative to the sensitivity offered by receptors that bind with 1:1 stoichiometry. Not surprisingly, enzymes have been the preferred molecular targets to date for scintigraphic imaging of gene therapy. This overview describes opportunities and advances in the utilization of radiolabelled nucleosides and nucleoside bases for imaging in gene therapy, with emphasis on the exploitation of enzyme systems for scintigraphic imaging of gene expression in gene therapy of cancer. Herpes simplex virus type-1 thymidine kinase and bacterial/fungal cytosine deaminase are discussed within the context of gene therapy issues such as gene vectors for targeting and delivery, the bystander effect, and radionucleoside delivery. The utilization of nucleosides as markers of tissue proliferation is discussed with respect to selected enzyme targets.

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).

Evaluation of methylthio-, methylsulfinyl-, and methylsulfonyl-analogs of alkanes and alkanoic acids as cardiac inotropic and antifungal agents.

A group of alkane and alkanoic acid compounds of general formula MeS(O)m(CH2)nR [m = 0-2; n = 1, 5, 13; R = Me, CO2H(Na)] were synthesized for evaluation as cardiac inotropic and antifungal agents. Inotropic activity was determined as the ability of the test compound to modulate in vitro guinea pig atrium contractility. The oxidation state of the S-atom was an important determinant of inotropic modulation since the thio (m = 0) analogs exhibited a positive inotropic effect. In contrast, the sulfinyl (m = 1) and sulfonyl (m = 2) analogs exhibited a negative inotropic effect. A pentyl spacer (n = 5) provided the largest positive or negative inotropic effect. The relative positive, and negative, inotropic potency orders with respect to the R-substituent were Me > or = CO2H, and CO2Na > or = Me, respectively. The most potent positive inotrope MeS(CH2)5Me (EC50 = 4.49 x 10(-6) M) could serve as a useful lead-compound for the design of a new class of positive inotropic agents. In a broad spectrum antifungal screen, the minimal inhibitory concentration (MIC) range for the five most active compounds was MeSO2(CH2)5Me (0.46-1.83 mM), MeS(CH2)13Me (0.31-1.23 mM), MeSO(CH2)13Me (< 0.009-1.87 mM), MeSO2(CH2)13Me (0.27-1.09 mM), and MeS(CH2)13CO2H (0.27-1.09 mM), relative to the reference drug Ampotericin B (< 0.0002-0.002 mM). The most active antifungal agent MeSO(CH2)13Me was selective against C. guillermondi, C. neoformans, S. cerevisiae, and A. fumigatus (strain TIMM 1776).

Novel approaches for designing 5'-O-ester prodrugs of 3'-azido-2', 3'-dideoxythymidine (AZT).

3'-Azido-2',3'-dideoxythymidine (AZT, 1, zidovudine, RetrovirTM) is used to treat patients with human immunodeficiency virus (HIV) infection. AZT, after conversion to AZT-5'-triphosphate (AZT-TP) by cellular enzymes, inhibits HIV-reverse transcriptase (HIV-RT). The major clinical limitations of AZT are due to clinical toxicities that include bone marrow suppression, hepatic abnormalities and myopathy, absolute dependence on host cell kinase-mediated activation which leads to low activity, limited brain uptake, a short half-life of about one hour in plasma that dictates frequent administration to maintain therapeutic drug levels, low potential for metabolic activation and/or high susceptibility to catabolism, and the rapid development of resistance by HIV-1. These limitations have prompted the development of strategies for designing prodrugs of AZT. A variety of 5'-O-substituted prodrugs of AZT constitute the subject of this review. The drug-design rationale on which these approaches are based is that the ester conjugate will be converted by hydrolysis and/or enzymatic cleavage to AZT or its 5′-monophosphate (AZT-MP). Most prodrug derivatives of AZT have been prepared by derivatization of AZT at its 5'-O position to provide two prominent classes of compounds that encompass: A) 5'-O-carboxylic esters derived from 1) cyclic 5'-O-carboxylic acids such as steroidal 17b-carboxylic acids, 1-adamantanecarboxylic acid, bicyclam carboxylic acid derivatives, O-acetylsalicylic acid, and carbohydrate derivatives, 2) amino acids, 3) 1, 4-dihydro-1-methyl-3-pyridinylcarboxylic acid, 4) aliphatic fatty acid analogs such as myristic acid containing a heteroatom, or without a heteroatom such as stearic acid, and 5) long chain polyunsaturated fatty acid analogs such as retinoic acid, and B) masked phosphates such as 1) phosphodiesters that include monoalkyl or monoaryl phosphate, carbohydrate, ether lipid, ester lipid, and foscarnet derivatives, 2) a variety of phosphotriesters that include dialkylphosphotriesters, diarylphosphotriesters, glycolate and lactate phosphotriesters, phosphotriester approaches using simultaneous enzymatic and chemical hydrolysis of bis(4-acyloxybenzyl) esters, bis(S-acyl-2-thioethyl) (SATE) esters, cyclosaligenyl prodrugs, glycosyl phosphotriesters, and steroidal phosphotriesters, 3) phosphoramidate derivatives, 4) dinucleoside phosphate derivatives that possess a second anti-HIV moiety such as AZT-P-ddA, AZT-P-ddI, AZTP2AZT, AZTP2ACV), and 5) 5'-hydrogen phosphonate and 5'-methylene phosphonate derivatives of AZT. In these prodrugs, the conjugating moiety is linked to AZT via a 5'-O-ester or 5'-O-phosphate group. 5'-O-Substituted AZT prodrugs have been designed with the objectives of improving anti-HIV activity, enhancing blood-brain barrier penetration, modifying pharmacokinetic properties to increase plasma half-life and improving drug delivery with respect to site-specific targeting or drug localization. Bypassing the first phosphorylation step, regulating transport and conferring sustained release of AZT prolong its duration of action, decrease toxicity and improve patient acceptability. The properties of these prodrugs and their anti-HIV activities are now reviewed.

Reporter gene imaging: effects of ganciclovir treatment on nucleoside uptake, hypoxia and perfusion in a murine gene therapy tumour model that expresses herpes simplex type-1 thymidine kinase.

Perfusion, hypoxia and nucleoside uptake during ganciclovir therapy were determined in a murine HSV-1 TK-expressing tumour model (KBALB-STK). HSV-1 TK mRNA transcription in this cell line was confirmed by RT-PCR. BALB/c mice bearing KBALB-STK tumours accumulated (E)-5-(2-[125I]iodovinyl)-2'-fluoro-2'-deoxyuridine ([125I]IVFRU) (2.54% injected dose.g-1) and could be readily detected with planar imaging following administration of [131I]IVFRU. However, a single dose of ganciclovir (100 mg.kg-1 intraperitoneally) decreased tumour uptake of [125I]IVFRU to 0.33% injected dose.g-1. Subsequent single daily doses of ganciclovir over 3 consecutive days had a negligible effect on [125I]IVFRU uptake, which remained low. Tumour perfusion during 3 days of ganciclovir treatment was monitored with intravenous [99Tcm]HMPAO. Tumour perfusion increased from day 0 (no ganciclovir treatment) with 1.83% injected dose.g-1 tumour, to a maximum at day 2 (3.77% injected dose.g-1). In the same animals, accumulation of [3H]misonidazole decreased from 0.70% injected dose.g-1 at day 0 to a minimum at day 3 (0.24% injected dose.g-1), indicating that tumour tissue had become less hypoxic over the ganciclovir regimen. The uptake of [125I]IVFRU into the acid insoluble fraction of KBALB-STK cells in vitro in the presence of ganciclovir (2.0 microM) was completely inhibited, leading to a 57% decrease in total cellular accumulation of radioactivity. However, cytosolic entrapment of [125I]IVFRU was not affected by the presence of ganciclovir. These results indicate that the mechanisms leading to IVFRU exclusion during ganciclovir treatment of HSV-1 TK-expressing tumours can be attributed, at least partially, to inhibition of [125I]IVFRU-nucleotide incorporation into DNA.

Synthesis and calcium channel antagonist activity of dialkyl 1,4-dihydro-2,6-dimethyl-4-[4-(1-methoxycarbonyl-1,4-dihydropyridyl)]-3 ,5- pyridinedicarboxylates.

A novel class of dialkyl 1,4-dihydro-2,6-dimethyl-4-[4-(1- methoxycarbonyl-1,4-dihydropyridyl)]-3,5-pyridinedicarboxylates (8-14) were synthesized and evaluated as calcium channel antagonists. The differences in activity among members of this new class of compounds was less than one log unit (IC50 range of 1.12 x 10(-6) to 8.57 x 10(-6) M), relative to the reference drug nifedipine (IC50 = 1.43 x 10(-8) M). The small differences in potency, irrespective of the size of the dialkyl (Me, Et, i-Pr, i-Bu) ester substituents, is attributed to the fact that the N-CO2Me substituent is too far removed from the C-3 and C-5 ester substituents to undergo non-bonded steric interactions. The 4-[4-(1-methoxycarbonyl-1,4-dihydropyridyl) moiety in this new class of compounds is bioisosteric with a C-4 4-nitrophenyl, or a 4-pyridyl, substituent in classical 1,4-dihydropyridines.

Synthesis, calcium channel antagonist activity, and anticonvulsant activity of 3-ethyl 5-methyl 1,4-dihydro-2-[(2-hydroxyethoxy) methyl]-6-methyl-4-(2,3-dichlorophenyl)-3,5-pyridinedicarboxylate coupled to a 1-methyl-1,4-dihydropyridyl-3-carbonyl chemical delivery system.

3-Ethyl 5-methyl 1,4-dihydro-2-[(2-hydroxyethoxy) methyl]-6-methyl-4-(2,3-dichlorophenyl)-3,5-pyridinedicarboxylate (13), a bioisostere of amlodipine, was prepared by the reaction of ethyl 4-(2-hydroxyethoxy)acetoacetate (11) with methyl 2-(2,3-dichlorobenzylidene)acetoacetate (12) and NH4OAc. Compound 13 was elaborated to the target product 3-ethyl 5-methyl 1,4-dihydro-2-[2- [(1-methyl-1,4-dihydropyridyl-3-carbonyloxy)ethoxy]methyl]- 6-methyl-4-(2,3-dichlorophenyl)-3,5-pyridinedicarboxylate (16). The C-2 CH2OCH2CH2OH compound (13, IC50 = 6.56 x 10(-9) M) was about 44-fold more active as a calcium channel antagonist than the reference drug nimodipine (IC50 = 1.49 x 10(-8) M), but 4-fold less potent than felodipine (IC50 = 1.45 x 10(-9) M). Compound 16, possessing the 1-methyl-3-pyridylcarbonyloxy chemical delivery system moiety is a slightly less potent calcium channel antagonist (IC50 = 2.99 x 10(-8) M) than the parent compound 13. Compounds 13, 16, felodipine and nimodipine are highly lipophilic (Kp = 227, 344, 442 and 187, respectively). The C-2 CH2OCH2CH2OH compound (13) exhibited equipotent anticonvulsant activity to nimodipine in the maximal electroshock (MES) anticonvulsant screen. Unlike nimodipine, 13 provided modest protection in the subcutaneous metrazol (scMet) anticonvulsant screen. In contrast, compound 16 was inactive in both the MES and scMet screens.

Synthesis and biological evaluation of butanoate, retinoate, and bis(2,2,2-trichloroethyl)phosphate derivatives of 5-fluoro-2'-deoxyuridine and 2',5-difluoro-2'-deoxyuridine as potential dual action anticancer prodrugs.

A group of 3'-O-butanoyl, 5'-O-butanoyl, and 3',5'-di-O-butanoyl esters of 5-fluoro-2'-deoxyuridine (FDU), and 2',5-difluoro-2'-deoxyuridine (DFDU), 3'-O-retinoyl, and 3',5'-di-O-retinoyl esters of FDU, and 5'-O-bis(2,2,2-trichloroethyl)phosphoryl-FDU and its 3'-O-butanoyl ester, was synthesized. These compounds were designed to act as double prodrugs that would serve as a depot to release two active drugs that act through different mechanisms. Thus, a nucleotide derivative of FDU or DFDU could act as a competitive inhibitor for thymidylate synthase, whereas retinoic acid and butyric acid would be expected to induce cell differentiation. The in vitro anticancer activities for these prodrugs were determined against a panel of nine tumor types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, breast) that encompassed about 60 human tumor cell lines. Structure-activity relationships indicate that O-butanoyl esters of FDU are approximately equipotent to FDU, the O-butanoyl esters of DFDU are less active than FDU, and the retinoyl and bis(2,2,2-trichloroethyl)phosphate derivatives of FDU exhibit comparable activity to FDU. In addition to their cytotoxic effect, 3'-O-retinoyl-FDU (12) and 3'-O-butanoyl-5'-O-bis(2,2,2-trichloroethyl)phosphoryl-FD U (16) also induced in vitro cell differentiation of promyelocytic leukemia HL60 cells. These combined cytotoxic and cell differentiation effects exhibited by 12 and 16 produced greater morphological drug-induced granulation and neutrophil vacuolation, and more cell apoptosis, than observed upon exposure to either retinoic acid or sodium butanoate. Dose-escalation studies in mice showed that 12 or 16 did not induce any acute or chronic toxicity, change in plasma clinical chemistry parameters, or gross histapathological changes at 60 days following an initial dosage regimen of 0.013 mmol/kg i.p. for 7-consecutive days. The in vivo growth delay response of murine mammary EMT6 solid tumors suggests that 3'-O-retinoyl-FDU (12) delays tumor growth relative to the other prodrugs investigated, sodium butyrate, retinoic acid, FDU, or a combination of retinoic acid and FDU. These preliminary results suggest that 3'-O-retinoyl-FDU (12) warrants further in vivo investigation to determine its tissue biodistribution and pharmacokinetic parameters that would be of value in assessing its potential usefulness as an anticancer prodrug.

The design of (-)-(S)-2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate: a cardioselective positive inotropic derivative of Bay K 8644.

The title compound, (-)-(S)-9, is a novel cardioselective calcium channel modulator that exhibits a calcium channel agonist effect on heart, a weak calcium channel antagonist effect on smooth muscle, and releases nitric oxide in vitro. (-)-(S)-9 is a useful lead-compound for the design of positive inotropic agents to treat congestive heart failure, and to study the structure-function relationship of calcium channel modulation.