Inhibition of antibiotic-resistant Staphylococcus aureus by the broad-spectrum dihydrofolate reductase inhibitor RAB1.

The bacterial burden on human health is quickly outweighing available therapeutics. Our long-term goal is the development of antimicrobials with the potential for broad-spectrum activity. We previously reported phthalazine-based inhibitors of dihydrofolate reductase (DHFR) with potent activity against Bacillus anthracis, a major component of Project BioShield. The most active molecule, named RAB1, performs well in vitro and, in a cocrystal structure, was found deep within the active site of B. anthracis DHFR. We have now examined the activity of RAB1 against a panel of bacteria relevant to human health and found broad-spectrum applicability, particularly with regard to gram-positive organisms. RAB1 was most effective against Staphylococcus aureus, including methicillin- and vancomycin-resistant (MRSA/VRSA) strains. We have determined the cocrystal structure of the wild-type and trimethoprim-resistant (Phe 98 Tyr) DHFR enzyme from S. aureus with RAB1, and we found that rotational freedom of the acryloyl linker region allows the phthalazine moiety to occupy two conformations. This freedom in placement also allows either enantiomer of RAB1 to bind to S. aureus, in contrast to the specificity of B. anthracis for the S-enantiomer. Additionally, one of the conformations of RAB1 defines a unique surface cavity that increases the strength of interaction with S. aureus. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development.

Effects of retinoids on cancerous phenotype and apoptosis in organotypic cultures of ovarian carcinoma.

BACKGROUND: Retinoic acid analogues, called retinoids, have shown promise in clinical trials in preventing breast and ovarian cancers. Classic retinoids bind to retinoic acid receptors, which regulate cell growth. Some novel retinoids, such as fenretinide, i.e., N-(4-hydroxyphenyl)retinamide (4-HPR), induce apoptosis through retinoic acid receptor-independent mechanisms; however, they appear to do so only at concentrations above those achieved in clinical chemoprevention trials. At lower concentrations (< or =1 microM), 4-HPR acts like classic retinoids, by inducing differentiation through a receptor-dependent mechanism. Our goal was to compare the effects of novel receptor-independent (apoptotic) retinoids with those of classic growth-inhibitory retinoids at clinically achievable doses on growth, differentiation, and apoptosis in ovarian tissue. METHODS: Four receptor-independent (apoptotic) and seven growth-inhibitory retinoids, including synthetic, low-toxicity compounds called heteroarotinoids, were administered at concentrations of 1 microM to organotypic cultures of ovarian primary and cancer cell lines: OVCAR-3, Caov-3, and SK-OV-3. After fixation, embedding, and sectioning, the growth fraction was quantified by measuring expression of the proliferation marker Ki-67/myb, differentiation was assessed by expression of mucin, and apoptosis was evaluated by the TUNEL assay. Spearman correlation analysis was performed on the data, and all P values were two-sided. RESULTS: All 11 retinoids reversed characteristics associated with the cancerous phenotype in all neoplastic cultures. Glandular structures were observed consistently in retinoid-treated, but not in untreated, OVCAR-3 and Caov-3 cultures. All retinoids decreased growth fractions, and some increased mucin expression. All receptor-independent retinoids and two receptor-dependent retinoids induced apoptosis, and the induction correlated significantly with increased expression of the mucin MUC1 (r =.83; P =.03). Retinoids with ester-linking groups did not induce apoptosis but decreased the growth fraction in correlation with MUC1 induction (r = -.93; P =.02). CONCLUSIONS: At clinically achievable concentrations, all retinoids tested decrease the growth fraction, induce differentiation and apoptosis. Induction of MUC1 expression is implicated in the mechanisms of action.

Water in normal muscle and muscle with a tumor.

The total water content, the amount of non-freezable water, and the Na-+ and K-+ contents in the gastrocnemius muscle of albino mice with and without a solid tumor were determined. The spin-lattice relaxation time (T1) for the water protons in the two kinds of muscle were measured at six resonance frequencies ranging from 4.5 to 60 MHz over the temperature range +37 to minus 65 degrees C. Quantitatively calculated T1 values are given. The difference in T1 for the two types of muscle at temperatures above minus 5 degrees C is attributed to the difference in the distribution ratio of water between hydration and free states, and bears no direct relation to the concentration of Na-+.

Novel pyrazolo, isoxazolo, and thiazolo steroidal systems and model analogs containing dimethyoxylaryl (or dihydroxylaryl) groups and derivatives. Synthesis, spectral properties, and biological activity.

The total syntheses of a series of vicinal-substituted dimethoxy and dihydroxy heterosteroids of the equilenin type and model analogs are described. A novel class of pyrazolo steroidal N-glucosides has also been synthesized. Compounds prepared were screened in vitro for growth inhibition of different microorganisms. Of these, 1-alpha-d-glucopyranosyl-4,5-dihydro-7-methyoxy-1H-benz[g]indazole tetraacetate (3) was quite active. For example, N-glucoside 13 inhibited the growth of Bacillus subtilis, Pseudomonas fluorescens, Staphylococcus aureus, and KB cells at moderate concentrations.

Synthesis and structure-activity relationships of heterocyclic compounds containing a trimethoxyarene function.

Pyrazole-, isoxazole-, and pyrazolone-containing systems were prepared from 3,4-dihydro-5,6,7-trimethoxy-1(2H)-naphthalenone, 3,4-dihydro-6,7,8-trimethoxy-1(2H)-naphthalenone, and 3,4-dihydro-6,7,8-trimethoxy-1(2H)-phenanthrone. Primarily, the pyrazoles displayed inhibition of growth in the microbial screens and in tissue culture. Correlation of the heteroatom distances between the oxygen atoms of two methoxy groups and a nitrogen atom in the pyrazole function with the percent plating efficiency on KB cell growth suggests increased inhibition as the (OA-N)/(OB-N) ratio deviates from one. No trend was observed in relating the OA-N-OB angle and activity for the examples studied.

Novel heteroarotinoids: synthesis and biological activity.

In this study, 13 heteroarotinoids were synthesized. The key step in each preparation was the condensation of the appropriate chroman-, thiochroman-, or benzothienyl-substituted phosphorus ylide, obtained from the independent synthesis of the corresponding phosphonium salts, with selected polyene-substituted aldehyde esters. Nine of these heterocycles contained a thiochroman group, two had a chroman group, and two others had a benzothienyl system. Screening of the compounds was with one of two assays. One assay measured the ability of a retinoid to inhibit the phorbol ester induced increase of mouse epidermal ornithine decarboxylase (ODC) activity. The other assay measured retinoid-induced differentiation of the human myoloid leukemia cell line HL-60. In the ODC assay, all thirteen compounds were screened. The most active heteroarotinoids were ester 10 [methyl (E)-4-[2-(2,2,4,4-tetramethylthiochroman-6-yl)-1- propenyl]benzoate] and acid 11 [(E)-4-[2-(2,2,4,4-tetramethyl-3,4- dihydro-2H-1- benzothiopyran-6-yl)-1-propenyl]benzoic acid]. Both of these retinoids had ID50 values (dose required for half-maximal inhibition of phorbol ester induced ODC activity) of about 0.3 nmol. In comparison, the ID50 value for trans-retinoic acid (1) was 0.12 nmol while the ID50 values for acids 7 and 9, namely (2Z,4E,6E)-3,7-dimethyl-7-(4,4-dimethyl-thiochroman -6-yl)-2,4,6-heptatrienoic acid and (2E,4E,6E)-3,7-dimethyl-7-(2,2,4,4-tetramethylthiochroman -6-yl)-2,4,6- heptatrienoic acid, respectively, were about 3.5 nmol. Heteroarotinoids 8 and 12-17 had ID50 values of 35 nmol or greater. With a thiochroman unit, the most active acids in decreasing order of activity in the ODC assay were 7 greater than 9 greater than 8. Thus, simple replacement of the terminal propenyl system [C(16,17,18)] in 7 with a cyclopropyl group produced acid 8 [(2E,4E,6E)-7-methyl-7-(4,4-dimethylthiochroman-6-yl)- 2,3-methylene-4,6-heptadienoic acid with markedly reduced activity. With a benzoic acid group as part of the structure attached to the thiochroman unit, the ODC activity was enhanced as shown in 10 and 11. The combination of the 2,2,4,4-tetramethylthiochroman group and the benzoic acid (or ester) terminal group seemed to enhance the biological action which resembles that found with (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)- 1-propenyl]benzoic acid (TTNPB, 6b), a well-known model system.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and antiarrhythmic properties of novel 3-selena-7-azabicyclo[3.3.1]nonanes and derivatives. Single-crystal X-ray diffraction analysis of 7-benzyl-3-selena-7-azabicyclo[3.3.1]nonan-9-one and 7-benzyl-3-selena-7-azabicyclo[3.3.1]nonane hydroperchlorate.

Several members of the heterocyclic family 3-selena-7-azabicyclo[3.3.1]nonane have been synthesized and characterized via IR, 1H, 13C, 15N, and 77Se NMR spectroscopy and, in some cases, by X-ray diffraction analysis. Select members, namely the hydroperchlorates of the amines, were examined for antiarrhythmic properties in anesthetized dogs in which myocardial infarctions were induced by techniques previously described. In the predrug, or control state, sustained ventricular tachycardia were induced by ventricular paced beats at rates above 300/min. When 7-benzyl-3-selena-7-azabicyclo[3.3.1]nonane hydroperchlorate was administered at 3 and 6 mg/kg, the sustained ventricular tachycardia could no longer be induced. Similar doses of lidocaine, a commonly used antiarrhythmic, caused slowing of the sustained ventricular tachycardia below 300/min but did not abolish their inducibility. In addition, select members of the hydroperchlorates caused a moderate 10-20% increase in mean blood pressure whereas lidocaine caused either no change in or slightly reduced mean blood pressure. Some general conclusions are delineated concerning the structural requirements that appear to be necessary for activity in this family of heterocycles and that have not been reported previously.

Synthesis and characterization of selected heteroarotinoids. Pharmacological activity as assessed in vitamin A deficient hamster tracheal organ cultures. Single-crystal X-ray diffraction analysis of 4,4-dimethylthiochroman-6-yl methyl ketone 1,1-dioxide and ethyl (E)-p-[2-(4,4-dimethylthiochroman-6-yl)propenyl]benzoate.

There is reported the first four members of heteroarotinoids, the names of which are ethyl (E)-p-[2-(4,4-dimethylthiochroman-6-yl)propenyl]benzoate (1b), ethyl (E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoate (1c), ethyl (E)-p-[2-(4,4-dimethyl-1-oxothiochroman-6-yl)propenyl]benzoate (1d), and (E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid (1e). IR, 1H NMR and 13C NMR data have been recorded for each compound and support the structural assignments. To provide a firm basis for comparison purposes of future analogues, an X-ray analysis was performed on a single crystal of ethyl (E)-p-[2-(4,4-dimethylthiochroman-6-yl)propenyl]benzoate (1b) and a precursor 4,4-dimethylthiochroman-6-yl methyl ketone 1,1-dioxide (18). These data for the heteroarotinoid 1b revealed that the two aryl ring systems were nearly perpendicular in each of the two molecules present in the unit cell (86.37 degrees and 84.17 degrees, respectively). The space group for both molecules was P1 in triclinic systems. Unit cell dimensions (at 15 degrees C) are as follows: for 1b, a = 20.568 (6) A, b = 14.760 (3) A, c = 7.679 (2) A, alpha = 113.33 (2) degrees, beta = 79.45 (2) degrees, gamma = 79.98 (2) degrees, Z = 4; for 18, a = 9.292 (5) A, b = 9.291 (5) A, c = 7.951 (3) A, alpha = 102.16 (3) degrees, beta = 77.49 (3) degrees, gamma = 79.60 (4) degrees, Z = 2. The sulfur-containing ring is in a distorted half-chair in 1b and the methyl carbon C(12) is shown to be trans to H(13) at the C(11)-C(13) bond. The biological activity of these arotinoids was determined in the tracheal organ culture assay and compared with trans-retinoic acid for ability to reverse keratinization in vitamin A deficient hamsters. The ester 1b displayed activity about one-half log unit less than that of the reference while 1c and 1e had activity nearly one log until less than trans-retinoic acid. The sulfoxide was the least active of the heteroretinoids.

Synthesis and characterization of heteroarotinoids demonstrate structure specificity relationships.

Heteroarotinoids are synthetic retinoids derived from trans-retinoic acid and the arotinoid structures and include a heteroatom in a five- or six-membered cyclic ring. This is the first systematic study of influences of the heteroatom, ring size, number of aryl groups, and terminal side chain on retinoid receptor specificity. Two new heteroarotinoids were synthesized and characterized. Although all heteroarotinoids activated RAR receptors, two dominant associations between structure and specificity were identified across all compounds. The six-membered ring conferred increased RARbeta specificity over the five-membered ring. The sulfur atom conferred greater specificity for RARgamma than the oxygen atom. RARalpha specificity was attenuated by a combination of influences from the heteroatom and aryl groups. In summary, the heteroatom and cyclic ring size exerted dominant effects, while the number of aryl rings and terminal side chain had attenuating effects on retinoid receptor specificity of heteroarotinoids.

Synthesis and antimicrobial activity of azasteroid-type compounds and related systems. Effect of hydrophilic and lipophilic groups on activity.

Pyrazole-, pyrazolone- and isoxazole-containing systems were prepared from 3,4-dihydro-6-(hexyloxy)-1(2H)-naphthalenone, 3,4-dihydro-6-(hexadecyloxy)-1(2H)-naphthalenone,3,4-dihydro-6(2-dimethylaminoethyloxy)-1-(2H)-naphthalenone, 3,4-dihydro-7-hexyloxy-1(2H)-phenanthrone, and 3,4-dihydro-7-(2-dimethylaminoethyloxy)-1(2H)-phenanthrone. A number of compounds derived from 7, 8-dihydro-5(6H)-quinolinone were also synthesized and characterized. Both hydrophilic and lipophilic groups were incorporated into certain systems as well as cidal groups. The compounds were screened for their in vitro inhibitory activity against Bacillus subtilis and Pseudomonas fluorescens. Structure-acitivity relationships among the molecular systems are discussed.

Synthesis and biological activity of some derivatives of thiochroman-4-one and tetrahydrothiapyran-4-one.

A small series of pyrazoles and isoxazoles derived from thiochroman-4-one has been synthesized and characterized. The compounds were examined for their in vitro inhibitory activity against Bacillus subtilis and Pseudomonas fluorescens. Among the tested compounds the pyrazole derivative from thiochroman-4-one was found to be the most effective inhibitor of growth of B. subtilis. Extensive H NMR analysis was recorded for all compounds.

Heteroarotinoids. Synthesis, characterization, and biological activity in terms of an assessment of these systems to inhibit the induction of ornithine decarboxylase activity and to induce terminal differentiation of HL-60 cells.

The synthesis of certain heteroarotinoids has been achieved, namely the systems (2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimethyl-6 -thiochromanyl)-2,4,6-heptatrienoic acid (1a), ethyl (2E,4E,6E)-3,7-dimethyl-7- (1,2,3,4-teterahydro-4,4-dimethyl-6-thiochromanyl)-2,4,6- heptatrienoate (1b), (2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimethyl-6 -chromanyl)-2,4,6-heptatrienoic acid (1c), 2-phthalimidoethyl 3,7-dimethyl-7-(1,2,3,4-tetrahydro-4 4-dimethyl-6-thiochromanyl)-2,4,6-heptatrienoate (1d), methyl (E)-p-[2-(4,4- dimethyl-6-chromanyl)-1-propenyl]benzoate (2a), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzyl alcohol (2b), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzonitrile (2c), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzaldehyde (2d), methyl 4-[2-(2,3-dihydro-3,3-dimethyl-5-benzofuranyl)-1-propenyl] benzoate (3a), and (E)-p-[2-(2,3-dihydro-3,3-dimethyl-5-benzofuranyl)-1- propenyl]benzoic acid (3b). Characterization via elemental, IR, 1H NMR, and 13C NMR analyses was completed for these heterocycles. The biological activity of these heteroarotinoids was assayed by either the suppression of the 12-O-tetradecanoylphorbol 13-acetate (TPA) induced synthesis of ornithine decarboxylase (ODC) in mouse skin or the induction of differentiation of human (HL-60) promyelocytic cells. In the ODC assay, systems 1a-c exhibited strong activity (within 10% of or less than the control) whereas alcohols 2b and 3a showed good activity (within 50% of the control) as compared to either 13-cis-retinoic acid or trans-retinoic acid. Moderate activity was observed with 2a and 2b while 1d and 2c were essentially inactive. With the HL-60 assay, 1a and 1c were approximately 2- and 5-fold less active, respectively, than trans-retinoic acid. In contrast, 2a, 3a, and 3b induced differentiation of only a very small percentage of the cells. Acids 1a and 1c were the most active heteroarotinoids in the two biological assays. Consequently, the presence of the heteroatom does not eradicate the activity of the heteroarotinoids and thus they may have potential as chemotherapeutic agents.

Synthesis, structure-activity relationships, and RARgamma-ligand interactions of nitrogen heteroarotinoids.

Three heteroarotinoids containing a nitrogen atom in the first ring and a C-O linking group between the two aryl rings were synthesized and evaluated for RAR and RXR retinoid receptor transactivation, tumor cell growth inhibition, and transglutaminase (TGase) induction. Ethyl 4-(N,4,4-trimethyl-1,2,3,4-tetrahydroquinolinyl)benzoate (1) contained an N-CH(3) group and activated all retinoid receptors except for RARgamma. Inceasing the hydrophobicity around the rings with analogues ethyl 4-(N,4,4,7-tetramethyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (2) [7-methyl group added] and ethyl 4-(4,4-dimethyl-N-isopropyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (3) [NCH(CH(3))(2) group at C-4] increased the potency and specificity for RARalpha, RARbeta, and RXRalpha, compared to 1, but had little effect on RXRbeta and RXRgamma activation. Although 1 and 3 were unable to activate RARgamma, 2 did activate this receptor with efficacy and high potency equal to that of 9-cis-retinoic acid (9-c-RA). All three heteroarotinoids exhibited 5-8-fold greater specificities for RARbeta over RARalpha. In addition, esters 1-3 inhibited the growth of two cell lines each derived from cervix, vulvar, ovarian, and head/neck tumors with similar efficiencies to that of 9-c-RA through a mechanism independent of apoptosis. The vulvar cell lines were the most sensitive, and the ovarian lines were the least sensitive. Ester 2 was similar to 1 and 3 except that 2 was a much more potent growth inhibitor of the two vulvar cell lines, which is consistent with strong RARgamma activation by 2 (but not by 1 and 3) and the high levels of RARgamma expression in skin. All three heteroarotinoids induced production of TGase, a marker of retinoid activity in human erythroleukemic cells. Esters 2 and 3 were the more potent TGase activators than 1, in agreement with the stronger activation of the RAR receptors by 2 and 3. The biological activities of these agents, and the RARgamma potency of 2 in particular, demonstrate the promise of these compounds as pharmaceutics for cancer and skin disorders.

Novel 3,7-diheterabicyclo[3.3.1]nonanes that possess predominant class III antiarrhythmic activity in 1-4 day post infarction dog models: X-ray diffraction analysis of 3-[4-(1H-imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo[3.3.1]nona ne dihydroperchlorate.

Several 3,7-diheterabicyclo[3.3.1]nonanes (DHBCNs) were prepared and screened in the Harris dog model for their ability to abolish pace-induced and sustained ventricular tachycardia (SVT) or prevent induction of ventricular tachycardia. In addition, an electrophysiological examination was made in the infarcted hearts of each animal to determine if more than one class activity was present. The examples exhibited predominately class III antiarrhythmic activity via a prolongation of the ventricular effective refractory period (VERP) in the models, although there may well be an underlying class Ib action present as exemplified by the ability of several of the agents to slow conduction in the myocardial infarcted dog hearts. 3-[4-(1H-Imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo[3.3.1]nonan e dihydroperchlorate displayed powerful class III activity in the model systems while several other DHBCNs exhibited various degrees of class III action. An X-ray diffraction analysis revealed that this compound has a 3,7-diazabicyclo[3.3.1]nonane bicyclic unit in a chair-chair conformation.

Synthesis, conformational analysis, and antiarrhythmic properties of 7-benzyl-3-thia-7-azabicyclo[3.3.1]nonan-9-one, 7-benzyl-3-thia-7-azabicyclo[3.3.1]nonane hydroperchlorate, and 7-benzyl-9-phenyl-3-thia-7-azabicyclo[3.3.1]nonan-9-ol hydroperchlorate and derivatives: single-crystal X-ray diffraction analysis and evidence for chair-chair and chair-boat conformers in the solid state.

The synthesis of the title ketone has been completed via a type of Mannich reaction starting from 4- thianone . An X-ray diffraction analysis has revealed that the solid system is a chair-boat conformer with the sulfur atom in the boat portion of the bicyclic ring compound. Wolff- Kishner reduction of the ketone group gave 7-benzyl-3-thia-7-azabicyclo [3.3.1]nonane, which was isolated as the hydroperchlorate . However, X-ray diffraction analysis of the salt showed this solid to be a chair-chair conformer. Addition of phenylmagnesium bromide to the ketone gave a tertiary alcohol with the C-C6H5 bond being equatorial with respect to the thiane ring and axial with respect to the piperidine ring. The reaction of the Grignard reagent with the ketone to give this alcohol seems to be very stereospecific. An X-ray analysis of the hydroperchlorate of the alcohol confirmed the system to be a chair-chair form in the solid. The title compounds were screened for antiarrhythmic activity in anesthetized mongrel dogs in which myocardial infarctions had been created when the left anterior descending coronary artery was ligated. Vagal-induced slowing of the sinus mode firing rate was used to determine the underlying ventricular automaticity in the dogs, which averaged 164 +/- 27 beats/min. Ventricular pacing was initiated to rates between 240 and 390/min. This technique resulted in the induction of rapid and sustained ventricular tachycardia. At doses of 3 and 6 mg/kg of body weight, 7-benzyl-3-thia-7-azabicyclo [3.3.1]nonane hydroperchlorate in alcohol (the solution was administered intravenously) was able to suppress markedly the induced ventricular tachycardia in five of six dogs. The compound also caused a 10-15% increase in blood pressure within a few minutes. The antiarrhythmic properties of this compound and others of related structure are discussed, and some comparison is made with the action of lidocaine in similar dog preparations.

Heteroarotinoids inhibit head and neck cancer cell lines in vitro and in vivo through both RAR and RXR retinoic acid receptors.

A class of less toxic retinoids, called heteroarotinoids, was evaluated for their molecular mechanism of growth inhibition of two head and neck squamous cell carcinoma (HNSCC) cell lines SCC-2 and SCC-38. A series of 14 heteroarotinoids were screened for growth inhibition activity in vitro. The two most active compounds, one that contained an oxygen heteroatom (6) and the other a sulfur heteroatom (16), were evaluated in a xenograph model of tumor establishment in nude mice. Five days after subcutaneous injection of 10(7) SCC-38 cells, groups of 5 nu/nu mice were gavaged daily (5 days/week for 4 weeks) with 20 mg/kg/day of all-trans-retinoic acid (t-RA, 1), 10 mg/kg/day of 6, 10 mg/kg/day of 16, or sesame oil. After a few days, the dose of t-RA (1) was decreased to 10 mg/kg/day to alleviate the side effects of eczema and bone fracture. No significant toxic effects were observed in the heteroarotinoid groups. All three retinoids caused a statistically significant reduction in tumor size as determined by the Student t-test (P < 0. 05). Complete tumor regression was noted in 3 of 5 mice treated with t-RA (1), 4 of 5 mice treated with 16, 1 of 5 mice treated with 6, and 1 of 5 mice treated with sesame oil. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine that the expression levels of RARalpha, RXRalpha, and RXRbeta were similar in the two cell lines, while RARbeta expression was higher in SCC-2 over SCC-38, and RARgamma expression was higher in SCC-38 over SCC-2. Receptor cotransfection assays in CV-1 cells demonstrated that 16 was a potent activator of both RAR and RXR receptors, while 6 was selective for the RXR receptors. Transient cotransfection assays in CV-1 cells using an AP-1 responsive reporter plasmid demonstrated that t-RA (1), 6, and 16 each inhibited AP-1-driven transcription in this cell line. In conclusion, the growth inhibition activity of the RXR-selective 6 and the more potent growth inhibition activity of the RAR/RXR pan-agonist 16 implicate both RARs and RXRs in the molecular mechanism of retinoid growth inhibition. Moreover, the chemoprevention activity and the lack of toxicity of heteroarotinoids demonstrate their clinical potential in head and neck cancer chemoprevention.

Biologically active heteroarotinoids exhibiting anticancer activity and decreased toxicity.

A series of retinoids, containing heteroatoms in a cyclic ring and called heteroarotinoids, were synthesized, and their biological activity was evaluated using tissue culture lines that have measurable responses to trans-retinoic acid (t-RA). Transglutaminase (TGase) was assessed in the human erythroleukemia cell line (GMO6141A) as an indicator of differentiation and apoptosis. Proliferation was evaluated in a human cervical cell line, CC-1, which exhibits dose-dependent alterations in growth rate in response to treatment with trans-retinoic acid. Activation of nuclear retinoic acid receptors was determined in a reporter cell line established from CC-1. The reporter line, called CC-B, contains a reporter gene controlled by a retinoic acid responsive element (RARE) and a thymidine kinase (tk) promoter. Treatment of the CC-B line with the heteroarotinoids resulted in a dose-responsive and retinoid-dependent regulation of reporter gene expression. The heteroarotinoids exhibited activity in all assays and correlated in a statistically significant manner between assays. RARE transactivation activity in CC-B cells correlated with induction of TGase in GMO6141A (R = 0.96) and with a decrease in the growth rate of CC-1 cells (R = -0.90). The ability of the selected heteroarotinoids to induce differentiation, inhibit proliferation, and activate nuclear receptors demonstrates the chemotherapeutic potential of these agents. In view of the biological activity cited, an in vivo toxicity study was conducted on male B6D2F1 mice with three heteroarotinoids, namely 8 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylthiochroman-6-yl)-2,4,6-heptatrienoic acid], 10 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylchroman-6-yl)-2, 4,6-heptatrienoic acid], and 13 [(E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid]. The mice were used with gavage of heteroarotinoids in corn oil [0.1, 0.2, 0.4, or 0.8 mg/kg] and with 0.01 or 0.05 mg/kg of TTNPB (5) [(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl]benzoic acid] as reference controls. The target organs affected in the mice by the three heteroarotinoids were those typically associated with t-RA (1) toxicity. The maximum tolerated dose (MTD) of 13 was 9.4 mg/kg/day, which was equal in toxicity to that of t-RA (1) and 1000-fold less toxic than TTNPB (5). The MTDs of 8 and 10 were 34 and 32 mg/kg/day, respectively, which is 3-fold less toxic than t-RA (1) and 3000-fold less toxic than TTNPB (5). The 3000-fold reduced toxicity, compared with only a 27% reduction biological activity of 8 and 10 with respect to that of TTNPB, observed in our assays indicates a good therapeutic ratio of these heteroarotinoids over the parent compound. The biological activity and reduced toxicity of these heteroartinoids demonstrate the potential efficacy as anticancer agents.

The cardiac electrophysiological effects of sparteine and its analogue BRB-I-28 in the rat.

This study compares the cardiovascular and antiarrhythmic effects of sparteine and a 3,7-diheterobicyclo[3.3.1]nonane analogue of sparteine, BRB-I-28, in pentobarbitone-anaesthetized rats subjected to left-ventricle electrical stimulation and occlusion of the left anterior descending coronary artery. Sparteine and BRB-I-28 produced a dose-dependent reduction in heart rate and blood pressure over the dose range 1-64 mumol/kg/min. As well, the P-R and Q-aT intervals of the electrocardiogram (ECG) were prolonged. The thresholds for induction of premature beats and ventricular fibrillation were dose-dependently increased and both drugs increased refractoriness. While sparteine and BRB-I-28 (at 16 and 64 mumol/kg/min, respectively) did not change the incidence of premature beats or ventricular tachycardia with coronary occlusion, both drugs equally reduced the incidence of ventricular fibrillation. We characterized the actions of sparteine and BRB-I-28 on cardiac Na+, transient outward and sustained outward plateau K+ currents of rat myocytes using the whole-cell patch-clamp. Sparteine and BRB-I-28 produced a concentration-dependent reduction in Na+ current with EC50 values of 110 and 230 microM, respectively. Both drugs produced hyperpolarizing shifts of 8 and 11 mV, respectively, for Na+ channel inactivation while neither produced a change in channel activation. Both drugs produced a concentration-dependent block of the sustained plateau K+ current and increased the rate of decay of the transient outward K+ current. Thus, sparteine and BRB-I-28 possess Na+ and K+ channel blocking properties which may account for their antiarrhythmic actions against electrical and ischaemic arrhythmias.

Effect of GLG-V-13, a class III antiarrhythmic agent, on potassium currents in rabbit ventricular myocytes.

The effects of a new Class III antiarrhythmic drug, GLG-V-13, on the 4-aminopyridine sensitive transient outward current, on the inward rectifier potassium current, on the ATP sensitive potassium current and on the rapid and slow components of the delayed rectifier potassium current were studied in single rabbit ventricular myocytes using the whole-cell voltage-clamp technique. GLG-V-13 blocked the rapid component of the delayed rectifier potassium current in a dose-dependent manner, with an estimated EC50 value of 0.36 microM. At high concentration, the slow component of the delayed rectifier potassium current was also depressed by the drug (40% effect at 10 microM concentration). The transient outward current, the inward rectifier potassium current and the ATP sensitive potassium current were not influenced by GLG-V-13, even at 10 microM concentration. Thus, GLG-V-13 blocks predominantly the rapid component of the delayed rectifier potassium current which may play a significant role in the prolongation of repolarization by the drug in ventricular tissue.

Antibacterial activity of 15-azasteroids alone and in combination with antibiotics.

A new class of 15-azasteroid analogues has been synthesized and tested for antimicrobial activity. The compounds 1, 10, 11, 11a-tetrahydro-7-methoxy-11a-methyl-2H-naphth (1,2-g) indol (methoxyimine) and 1,10,11,11a-tetrahydro-11a-methyl-2H-naphth (1,2-g) indol-7-ol (hydroxyimine) inhibit the growth of Bacillus subtillis and Escherichia coli at concentrations as low as 10-5 M. Addition of either compound to the growth medium casued a rapid inhibition in the transport of radioactive glucose, uracil and several amino acids. The inhibition of growth and substrate transport was reversed following removl of the steroid from the medium. The evidence is consistent with a site of steroid action at the cell periphery. Combining the methoxyimine with polyor circulin at subinhibitory concentrations produced greatly enhanced antimicrobial activity against Pseudomonas fluorescens. Similar action was observed against B. subtilis when the azasteroid was combined with vancomycin or chloramphenicol. The inhibitory action of other antibiotics such as penicillin or erythromycin was not affected by addition of the test compound. The results suggest formation of a molecular complex between the azasteroid and antibiotic which is responsible for the enhanced biological activity.