Cellular metabolism in lymphocytes of a novel thioether-phospholipid-AZT conjugate with anti-HIV-1 activity.

We previously synthesized a thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-hexadecylthio-2-methoxypropyl)-phosphothymidine, CP-102) with potent anti-HIV-1 activity and significant reduction in cell cytotoxicity compared to AZT alone. To study the cellular metabolism of the conjugate compound we synthesized a double-tritium-labeled thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-[9,10-3H]-S-octadecylthio-2-O-methoxypropyl)-phosphothymidine-[methyl-3H], [3H]CP-102). The intracellular radioactive metabolic products of [3H]CP-102 treated human lymphoblastoid CEM-SS cells were analyzed by HPLC and thin-layer chromatography. Results of this investigation provide evidence that a putative intracellular lipid cleavage enzyme metabolizes [3H]CP-102 to form a thioetherdiglyceride compound that migrates with an authentic 1-S-octadecyl-2-O-methyl-thioglycerol standard on TLC. The thioetherdiglyceride metabolite did not react with the ninhydrin reagent indicating it did not contain a primary amine such as that found on serine or ethanolamine containing phospholipids. Also, the product did not contain a phosphatidic acid group based on migration characteristics in the TLC plate. The other major hydrophilic metabolite was 3'-azido-3'-deoxythymidine-[methyl-3H]-monophosphate (AZT-MP) with lesser amounts of AZT, AZT-DP and AZT-TP. In summary, the best interpretation of these data is that the thioetherphospholipid-AZT conjugate, [3H]CP-102, is cleaved by a putative intracellular lipid cleavage enzyme to release a thioetherdiglyceride compound and AZT-MP. The resulting AZT-MP was either dephosphorylated to AZT or sequentially phosphorylated to AZT-DP and, ultimately, to AZT-TP, the known inhibitory metabolite against HIV-1 reverse transcriptase. Phospholipid-nucleoside conjugates may provide a unique approach for developing anti-HIV-1 prodrugs that do not have a strict requirement for a nucleoside kinase for initial activation of the prodrug to an antiviral form.

Structure-activity relationships for enhancement of paracellular permeability by 2-alkoxy-3-alkylamidopropylphosphocholines across Caco-2 cell monolayers.

The oral route is the preferred route of delivery for a large number of drug molecules. However, the intestinal epithelium presents a formidable barrier for delivery of drugs into systemic circulation. Phospholipids are among compounds that enhance the absorption of drugs across the intestinal epithelium. In this paper, we describe structure-activity relationships for phospholipid derivatives as enhancers of paracellular permeability across Caco-2 cell monolayers. In a series of 2-alkoxy-3-alkylamidopropylphosphocholine derivatives, compounds with a long chain at C-3 (R3) and short chain at C-2 (R2) were potent in causing a decrease in transepithelial electrical resistance (TEER) and an increase in mannitol transport, but also showed significant cytotoxicity. Compounds with 9-11 carbons at C-3 and 6-10 carbons at C-2 provided good separation (up to 2.7-fold) between activity and cytotoxicity. Notably, a good correlation (r2 = 0.93) was observed between EC(50) (TEER) [concentration that caused a drop in TEER to 50% of its control (untreated) value] and EC10x (mannitol) [concentration that caused 10-fold increase in mannitol transport over the control (untreated) value], confirming that a decrease in TEER is associated with enhanced permeability of the hydrophilic compounds across Caco-2 cell monolayers. Compounds with medium to long carbon chains at C-2 and C-3, and the total carbons in the alkyl chains > 20, showed poor activity and no cytotoxicity.

Three-dimensional quantitative structure-activity relationship study of nonsteroidal estrogen receptor ligands using the comparative molecular field analysis/cross-validated r2-guided region selection approach.

A newly developed comparative molecular field analysis (CoMFA) technique, the cross-validated r2-guided region selection (CoMFA/q2-GRS) method, has been used to build a quantitative structure-activity relationship (3D-QSAR) for nonsteroidal estrogen receptor (ER) ligands. Ligands included in this study belong to a series of diethylstilbestrol (DES) and indenestrol analogues whose affinities for the mouse ER (mER) have been determined in our laboratory. The final model utilized 30 compounds and yielded a q2GRS (cross-validated r2, guided region selection) of 0.796, as compared to a q2 of 0.720 for conventional CoMFA, with a standard error of prediction of 0.594 at 3 principal components. This model was used to visualize steric and electrostatic features of the ligands that correspond with ER binding affinity. Results obtained from the CoMFA steric and electrostatic plots of this model have also been compared to information from the ER binding affinities of substituted estradiol analogues. This is in an effort to determine structural features of compounds in the CoMFA analysis that may correspond to those of the estradiol analogues and to further clarify the mode of binding of nonsteroidal ER ligands.

Separation of indenestrol A and B isomers and enantiomers by high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) methods have been developed for the separation of substituted indenestrol A and B isomers on different columns. The isomers were separated by normal-phase liquid chromatography with a silica gel column. Enantiomers of these compounds were separated by chiral HPLC and the most successful separations were achieved with a Chiralcel OJ column.

In vitro evaluation and characterization of newly designed alkylamidophospholipid analogues as anti-human immunodeficiency virus type 1 agents.

Our laboratories first reported two novel classes of complex synthetic lipids, including alkylamidophosphocholines (PC lipid; CP-51) and alkylamidophosphate ester-linked lipid-AZT conjugates (lipid-AZT conjugates; CP-92), with selective and potent activity against human immunodeficiency virus type 1 (HIV-1). To extend these observations, we synthesized additional PC lipids and lipid-AZT conjugates (INK and INK-AZT conjugate) to evaluate their structure-activity relationships by testing for selectivity against infectious wild-type (wt) and drug-resistant HIV-1 replication, virus fusogenic activity and toxicity for mouse bone marrow cells. PC lipid compounds with medium chain lengths at positions 1 and 2 gave an improved selective index (SI). INK-3, with 12 and 8 carbons and INK-15, with 10 and 12 carbons were among the most selective when evaluated in CEM-SS cells. INK-14, a lipid-AZT conjugate where AZT replaced the choline in PC lipid INK-3, gave the highest SI of > 1250 against both infectious wt HIV-1 replication in CEM-SS cells and a clinical isolate in peripheral blood leukocytes. Notably, the PC lipid compounds INK-3 and INK-15, but not the lipid-AZT conjugate INK-14, were potent inhibitors of matched pairs of AZT-sensitive and AZT-resistant HIV-1 clinical isolates. INK-3 also inhibited replication of HIV-2 and TIBO-resistant HIV-1, and inhibited HIV-1-mediated fusogenic activity by 78, 41 and 9% in a dose-dependent manner. The TC50 for mouse bone marrow cells was > 100 micrograms/ml for INK-3 compared to 9.15-14.17 micrograms/ml for CP-51 and 0.142-0.259 microgram/ml for AZT. These data suggest that optimum PC lipid compounds are significantly less toxic than AZT and have high potential as novel therapeutic agents for AIDS.

Synthesis and evaluation of novel thymidine analogs as antitumor and antiviral agents.

Two series of thymidine analogs with a hydroxyalkylammonium(amine) moiety have been synthesized and evaluated for antitumor and antiviral activities. The hydroxyalkylammonium-(amine) group was introduced at the 5' position of the 2'-deoxyribose residue of thymidine or at a corresponding position in acyclic thymidine analogs. In order to increase the lipophilicity of these compounds and potentially enable them to cross the cell membrane, the free hydroxy group also was esterified with a long hydrocarbon chain. The hexadecanoyl analogs (compounds 1c, 1d, 7c, and 7d) showed moderate antitumor cytotoxicity against SV-28 and KB cell lines (IC50 approximately 20 microM). Compound 1d showed moderate anti-HIV activity (EC50 = 6.8 microM), while compound 5 showed weak anti-HIV activity (EC50 = 55 microM). None of the compounds showed antiherpes simplex virus activity.

Membrane-interactive phospholipids inhibit HIV type 1-induced cell fusion and surface gp160/gp120 binding to monoclonal antibody.

Membrane-interactive phospholipids (PLs), previously evaluated for activity against HIV-1 in vitro, are known to affect late steps in viral replication. Studies were done to determine the effects of PL analogs on post-translational processing of HIV-1 proteins, binding of viral surface gp160/gp120 to CD4 receptor, and HIV-1-induced cell fusion. Results of this investigation indicated that PL alone (1-octadecanamido-2-ethoxypropyl-rac-3-phosphocholine, CP-51) and PL-AZT conjugate (1-octadecanamido-2-ethoxypropyl-rac-3-phospho-3'- azido-3'-deoxythymidine, CP-92) have no effect on HIV-1-induced syntheses or processing of gp160/gp120, pr51, p24, or p17 (including myristoylation) in infected cells. Progeny HIV-1 particles made in CP-92-treated H9IIIB cells contained gp120, pr51, and p24; however, these virus particles had reduced capacity to bind to CD4+ cells. Both CP-51 and CP-92 inhibited syncytium (cell fusion) formation between treated HIV-1-infected cells and uninfected CD4+ cells, and, they reduced HIV-1 gp160/gp120 binding to CD4+ cells and monoclonal antibody. These results suggest that anti-HIV-1 activity of PL compounds involves alteration of cell surface membranes and viral envelopes. Phospholipid compounds are a novel class of membrane interactive compounds with potential use in blocking the spread of HIV-1 infection and pathogenesis in AIDS.

In vitro evaluation of phosphocholine and quaternary ammonium containing lipids as novel anti-HIV agents.

A series of synthetic lipids containing a two- or three-carbon backbone substituted with a thio, oxy, or amidoalkyl functionality and either a phosphocholine or quaternary ammonium moiety was evaluated as potential anti-HIV-1 agents. Several analogues were identified as possessing activity with the most promising compound being rac-3-octadecanamido-2-ethoxypropylphosphocholine (8). Compound 8 exhibited an IC50 for the inhibition of plaque formation of 0.16 microM which was 84-fold lower than the IC50 value determined for CEM-SS cell growth inhibition. Initial mechanistic studies have indicated that these compounds, unlike AZT, are not reverse transcriptase (RT) inhibitors, but instead appear to inhibit a late step in HIV replication involving virus assembly and infectious virus production. Since these lipids are acting via a different mechanism, they represent an alternative approach to the chemotherapeutic treatment of AIDS as well as candidates for combination therapy with AZT.

Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV-1 activity.

Combinations of an amidoalkylphosphocholine, 8, and AZT have been found to cause an apparent synergistic action in suppressing infectious HIV-1 replication. In addition, amidoalkyl, oxyalkyl, and thioalkyl ether lipids have been chemically linked to anti-HIV-1 nucleosides (AZT and DDI) through phosphate and phosphonate linkages. These conjugates have shown promising in vitro anti-HIV-1 activity. Also, the conjugates have a 5-10-fold reduction in cell cytotoxicity compared to AZT alone. The most active compound, an amidoalkyl ether lipid-AZT conjugates, 4A, was found to have a differential selectivity of 1793 in a syncytial plaque assay. In comparison, AZT alone has a value of 1281.

Synthesis and biological activity of novel quaternary ammonium derivatives of alkylglycerols as potent inhibitors of protein kinase C.

Alkylglycerols such as rac-1-O-octadecyl-2-O-methylglycerophosphochocholine (Et-18-OMe) have shown an inhibitory effect on the metastasis and growth of various cancer cell lines. Alkyl phospholipids have been shown to accumulate at the surface in several cell lines, the selectivity of which is still not clearly understood. A consequence of this action may lead to the inhibition of cell membrane related protein kinase C (PKC). The goal of this research was to develop ether lipid inhibitors of PKC to augment antineoplastic activity. This led to the synthesis and in vitro testing of a series of novel quaternary ammonium derivatives of alkylglycerols. The biological testing of these analogues on PKC stimulated with rac-1-O-oleoyl-2-O-acetylglycerol showed several analogues with inhibition comparable to that of Et-18-OMe.

Synthesis and biological evaluation of ether-linked derivatives of phosphatidylinositol.

The synthesis of two novel glycero-3-phosphoinositol ether lipid analogues, rac-1-O-octadecyl-2-O-methylglycero-3-phospho-myo-inositol 6 (an ether lipid analogue of rac-1-O-octadecyl-2-O-methylglycero-3-phosphocholine; ET-18-OMe) and rac-1-O-octadecyl-2-O-acetylglycero-3-phospho-myo-inositol 11 (an ether lipid analogue of platelet-activating factor), is described. The two target compounds and the synthetic intermediates were evaluated for inhibition of HL60, BG1, and BG3 human malignant cells in vitro and inhibition of protein kinase C. Tumor inhibitory activity was found for compounds 6 and 11 in all systems but not for their synthetic intermediates. However, compounds 6 and 11 as well as the synthetic intermediates 5 and 13, but not 9, exhibited protein kinase C inhibitory activity.

Novel lipid analogs with cytostatic and cytocidal activity.

1-0-Alkyl diol and glyceryl ether lipids with a quaternary ammonium polar head group were synthesized and the cytotoxicity (IC50) tested against KB cells, with low 1-0-alkyl-cleavage activity, and rat hepatoma 77 cells with relatively high 1-0-alkyl-cleavage activity. The original premise was that the compounds would be inactivated by the cleavage enzyme and thus be selectively toxic to cells with less of the enzyme. Results with two other cell lines with equivalent cleavage enzyme, HL-60 and K562-4, however, are not consistent with this premise.

Alkyl-linked diglycerides inhibit protein kinase C activation by diacylglycerols.

Alkylacylglycerols are synthesized when choline-phospholipids are degraded by a phospholipase C. This class of compounds has been shown to have biological activities; however, the mechanism of action is unknown. A series of alkyl-linked diglycerides were synthesized and tested for activity in an in vitro assay for protein kinase C. When protein kinase C activity was stimulated with the synthetic diacylglyceride analog 1-oleoyl-2-acetyl-sn-glycerol, the addition of alkyl glycerides caused a concentration-dependent inhibition of protein kinase C activity. Comparison of the protein kinase C inhibition by this series of 1-O-alkyl-2-acyl analogs revealed that both saturated and unsaturated long-chain groups in position 1 were effective and that dietherglycerols with short-chain moieties in position 2 were also effective. It is concluded from these studies that the biological activity of alkyl-linked glycerides may be expressed through protein kinase C inhibition.

Compounds that inhibit chymotrypsin and cell replication.

Several compounds have been tested for their ability to inhibit bovine pancreatic alpha-chymotrypsin (Ki) and their ability to inhibit cell replication (IC50). There is good agreement over three orders of magnitude between the Ki and the IC50 values of these compounds. The data support the hypothesis that a cellular, chymotrypsin-like activity is necessary for cell replication.

Synthesis of active site-directed organometallic irreversible protease inhibitors.

p-Antimonybenzenesulfonyl fluoride and p-mercurybenzenesulfonyl fluoride irreversibly inhibit chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), and chromosomal protease, and these inhibitors appear to be as active as phenylmethanesulfonyl fluoride. The pretreatment of the proteases interferes with the phosphorylation of the active-site serine by diisopropylfluorophosphate suggesting that the organometallic inhibitors may also interact with the active site serine. The organometallic inhibitors may be used for localization of proteases in different parts of the cell by electron microscopy and p-mercurybenzenesulfonyl fluoride could also be used for isolation of proteases by sulfhydryl affinity chromatography.

The inhibition of rat liver chromatin protease by congeners of the phenylboronic acids.

A group of arylalkylboronic acids were synthesized in order to investigate the inhibitory potential of these compounds for rat liver chromatin protease (EC 3.4--). The effect of side chain length, side chain substitution and aromatic substitution on proteolytic activity in chromatin dissociated in salt and urea was assayed. It was determined that a side chain length two carbons long provided the greatest inhibitory effect with complete inhibition attainable at 20 mM concentration of phenylethylboronic acid. Aryl substitution in the ortho position proved to be the most potent structural change with complete inhibition attained by 1 mM concentration of 0-methylphenylethylboronic acid. The binding of these two inhibitors proved to be reversible.

Hypocholesterolemic activity of 1,3-bis(substituted phenoxy)-2-propanones.

A series of 1,3-bis(substituted phenoxy)-2-propanones was found to be active hypocholesterolemic agents at 10 mg/kg/day. The p-chloro- and p-methyl-substituted phenoxy compounds possess the highest activity. These compounds did not possess the estrogenic and antifertility activities of the related previously reported derivatives of the bis(beta-phenylethyl) ketone series. The 1,3-bis(p-methylphenoxy)-2-propanone (7) also lowered serum triglycerides and glycerol which appeared to be due to increased levels of serum lipase and reduced activity of liver lipase. There was reduced incorporation of free fatty acids into complex lipids by the liver. Cholesterol was excreted faster in the treated animals.

Incorporation and subcellular distribution of an unnatural phospholipid base-analog, N-isopropyl-ethanolamine, in rat liver.

An unnatural phospholipid, phosphatidyl-N-isopropylethanolamine, was isolated from rat liver after intraperitoneal injections of N-isopropylethanol-amine; it was identified on the basis of enzymic, chemical, and chromatographic analyses. Although this phospholipid was formed at the expense of phosphatidylcholine and phosphatidylethanolamine, its fatty acid composition did not resemble either of these lipids. Microsomes, mitochondria, and plasma membranes contained significant amounts (up to 9%) of this unusual phospholipid. Radioisotope incorporation experiments suggest that the N-isopropylethanol-amine containing phospholipid is rapidly equilibrated between microsomes and mitochondria and more slowly with surface membranes.