Steroid modulation of human serum albumin binding properties. A spin label study.

The binding isotherm and unique electron spin resonance spectral characteristics of a monoanionic spin label (1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) and a dianionic spin label (1-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) are used to prove the steroid modulation of serum albumin binding properties. Effects of a selected number of steroids (progesterone, testosterone, estradiol, aldosterone, estriol, corticosterone, deoxycorticosterone, hydrocortisone, and cortisone) on the binding isotherm of the monoanionic spin label binding to serum albumin have been determined. At the steroid/albumin ratio of 0.5 to 1, progesterone, testosterone, and estradiol enhance binding of the spin label at all concentrations studied. However, the remaining steroids exert an inhibitory effect at low spin label/albumin ratios and an enhancement effect at high spin label/albumin ratios. Progesterone and cortisone effects on the resonance spectra of the spin label bound to serum albumin confirm the enhancement and displacement properties of these ligands. Thus, like fatty acids, steroids may bind to either the primary or secondary bilirubin binding sites and also allosterically perturb the binding properties of serum albumin. The in vivo importance of the steroid-albumin interaction is discussed.     

Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl alpha-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59.10(5) M-1 at 25 degrees C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent with alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.     

Synthesis and conformational study of 3,4-carbocyclic bridged indole melatonin and serotonin analogues

Tetrahydrobenz[cd]indole, has been usually assumed to be a rigid scaffold of arylethylamines of pharmaceutical interest, such as melatonin and serotonin. A series of molecules containing this scaffold has been synthesized and their conformation in solution has been determined by 1H NMR. The values of the coupling constants show that the carbocycle fused with the indole ring is a mixture of the two conformers with substituent in equatorial or axial orientation. The molar fraction of the conformers appears to be sensibly affected by the bulkiness of the C-2 indole substituent. A pseudo-axial orientation of the C-3 alkylamido side chain is important for melatonin ligands to access the binding site and exhibit potent in vitro affinity, as illustrated for melatonin ligand 1 (pK(i)=9.32).     

Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.     

NAD(P)+ analogues: tools for the investigation of the active site of oestradiol 17beta-dehydrogenase from human placenta.

Oestradiol-17beta:NAD+ 17-oxidoreductase from human placenta can accept coenzyme analogues of NAD+ and NADP+ where the amide group is replaced by methyl ketone, nitrile or thioamide. The inhibition with analogues of NAD+ has been studied. The presence of a substituent at C-3 of the pyridinium ring is necessary for the binding. The inhibition by C-4 methylated analogues is very poor, and the effect of a methyl group at C-5 depends on the substituent at C-3. The 1,4,5,6-tetrahydronicotinamide adenine dinucleotide is a competitive inhibitor. Nicotinamide 8-bromoadenine dinucleotide and nicotinamide 8-thioadenine dinucleotide are efficient hydrogen acceptors.     

Bovine serum albumin. Study of the fatty acid and steroid binding sites using spin-labeled lipids.

Three spin-labeled derivatives of stearic acid and two derivatives of palmitic acid have been used to study the structure of the strong fatty acid binding site of bovine serum albumin. The steroid and indole binding sites have been studied using spin-labeled derivatives of androstol and indole, respectively. Paramagnetic resonance and fluorescence quenching data suggest that the fatty acid, steroid, and indole binding sites may be identical. The mobility of the nitroxyl group at C-8 of palmitic acid bound to albumin at a 1:1 molar ratio is unaffected when the carboxyl group is esterified. When the nitroxyl group is located at C-5 on this acid its motion is detectably increased by esterification of the carboxyl group but the magnitude of this change is small. This result suggests that the carboxyl group may play a minor role in the binding of fatty acids to the strongest fatty acid binding site of albumin. When stearic acid derivatives bearing the nitroxide at C-5, C-12, and C-16 are bound to albumin at a ligand to albumin ratio of 1, the order of mobility at 0-30 degrees is C-16 greater than C-12 congruent to C-5. Although motion at the methyl terminus is always greater than at the COOH terminus in the range 0-60 degrees, a simple monotonic increase in chain motion between the two termini is not observed. Arrhenius plots of the motion parameters for these bound fatty acids show two abrupt changes in slope. The temperature ranges for these changes are 15-23 degrees and 38-45 degrees. These results suggest that when one mole of spin-labeled fatty acid is bound to albumin, the protein undergoes a conformational change in each of these temperature ranges.     

Structure-activity relationships of progesterone derivatives that bind to the digitalis receptor: modifications in A and B rings

A number of progesterone derivatives, having a 17 alpha-acetoxy group and various functions at C-3 and C-6, interact at the cardiac glycoside (CG) binding site, using [3H]ouabain in a radioligand binding assay (RBA) with membranes from dog myocardium. We now report on results of structure-activity studies concerned with modification of the A and B rings as they influence potency in the RBA. Some progesterone derivatives with 5 alpha- or 5 beta-stereochemistry show weak receptor competing activity. Among the congeners highest potency is associated with the presence of C-4 or C-4,6 unsaturation and a C-6 substituent (CH3, Cl, Br) whose importance appears to reside in its steric rather than electronic character. The C-3 function may be carbonyl, 3 beta-hydroxy or 3 beta-acetoxy when associated with C-4 or C-4,6 unsaturation. In compounds with other substituents that promote activity, C-6 alpha substitution with -CH3, -Cl, or -Br strongly enhances activity; -F, -OCH3, carbonyl, or the unsubstituted compound promotes weak binding; and -OC2H5, -OAc, -OCOOCH3, or -OH eliminates binding activity. Receptor interaction with the double bond at C-4, but not C-5, appears to be particularly important for binding. The most potent analog identified thus far is chlormadinone acetate (17 alpha-acetoxy-6-chloropregna-4,6-diene-3,20-dione), which has 1/20 the potency of ouabain in the RBA. Studies to determine optimal structural requirements for CG-receptor binding by these hormonal steroid congeners, in conjunction with appropriate biological assays, may provide insight into the nature of a putative endogenous counterpart, lead to a better understanding of the mode of action of the CG and yield CG-like compounds with superior therapeutic properties.     

Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.     

Structural requirements for the action of steroids as quenchers of albumin fluorescence.

1. Androgens, corticoids, gestagens, estrogens and related steroids are effective quenchers of the intrinsic fluorescence of bovine serum albumin. The quenching effect involves the formation of a steroid albumin complex which formation constant (Kf) and free energy of formation (delta G 0) can be determined by fluorescence titration. The fluorimetrically determined delta G 0 values range from -6.5 to -7.5 kcal/mol. 2. 5 alpha-Androstane and 5 alpha-pregnane are effective quenchers of albumin fluorescence, in accord with the essentially hydrophobic nature of the steroid-albumin interaction. Introduction of hydroxy or oxo groups in 5 alpha-androstane decreases the fluorescence quenching action, but the effect of each group declines when other polar groups are present in the steroid molecule. Similar effects occur with 5 alpha-pregnane except that 20-hydroxy (or oxo) duo-polar derivatives are more effective than the parent hydrocarbon. 3. Comparison of delta G 0 values for steroids differing in a single grouping shows that the steroid-albumin interaction is increased by (a) the benzenoid A-ring; (b) sulfate or carboxylate ions in the vicinity of C-3; (c) the 3-oxo group in place of the 3 alpha-hydroxyl (with 5 beta-pregnane derivatives; not with 5 alpha-androstane derivatives); (d) 17 beta-acetyl or 17 beta-hydroxyethyl residues; (e) acetylated or propionated 17 beta-hydroxy groups; (f) acetylated or methylated hydroxy groups at the C-3 of estrogens; (g) delta 5 and delta 6 double bonds; and (h) the 19 beta-methyl group. The maximal variation of delta G 0 determined by affinity-enhancing groups is -0.8 kcal/mol. Conversely, the steroid-albumin interaction is decreased by introduction of (i) oxygen atoms at C-3, C-6, C-11, C-16, and C-17; (j) 17 alpha-ethynyl and 17 alpha-acetoxyl residues; (k) benzoylated or hexahydro-benzoylated beta-hydroxy groups at C-17; (l) acetylated and benzoylated hydroxy groups at C-3; and delta 1 (conjugated) double bond. Oxo groups at C-3, C-6, C-16 and the 16 alpha, 17 alpha-epoxy group are more effective than the corresponding alpha-hydroxyl in decreasing affinity, while at C-11 and C-17, the alpha-hydroxyl is more effective than the beta-hydroxyl and the oxo group. The effect of substituents is influenced by the whole molecular structure, particularly, by the stereostructure at the A/B juncture, and the presence of an oxo group at C-17. 4. The stereospecific effect of substituents at different positions in the steroid molecule suggests that with non-aromatic, A/B trans (planar) steroids, binding to albumin primarily involves the (alpha) rear surface of the B-, C- and D-ring, and possibly, the 17 beta-side chain. With estrogens and A/B cis (dihedral) steroids, the benzenoid A-ring and electron attracting groups at C-3, respectively, may participate in binding.     

Equilibrium binding of spin-labeled fatty acids to bovine serum albumin: suitability as surrogate ligands for natural fatty acids

Electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies were used to characterize the binding of spin-labeled fatty acid (SLFA) to bovine serum albumin (BSA). Association constants of three stearic acid derivatives labeled with a nitroxyl radical at C-5, C-12, or C-16 were estimated by EPR spectroscopy as the ratio of SLFA to BSA was increased from about 0 to 9. The values were compared to those for unmodified stearate. With all three SLFA, it was apparent that the nitroxyl residue modified the binding pattern. For SLFA:BSA ratios up to 1, which probably involves the site(s) on BSA most specific for long-chain FA, the C-16 derivative bound with an affinity similar to that of the natural FA. At higher ratios, the association constants for this SLFA were lower than those for stearate. The C-12 and C-5 derivatives showed only low-affinity binding relative to stearate. The spectral parameter, W, was constant for SLFA:BSA ratios between 0 and 1 in the case of C-16 compound, indicating physical homogeneity of the high-affinity binding site. At higher ratios, the spectra changed progressively, indicating inhomogeneity of the lower affinity binding sites although parallel changes in association constants were not observed. Changes in W due to Heisenberg spin exchange were ruled out. By examining the mobility profile of the bound SLFA by both EPR and ST-EPR techniques, it was shown that the nitroxyl group was maximally immobilized when attached near the center of the carbon chain of the bound SLFA.     

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.     

Conformational preferences in glycosylamines. implications for the exo-anomeric effect

The conformational preferences about the C-N bond in N-(4-methoxyphenyl)-2,3,4,6-tetra-O-acetyl-alpha (1) and beta-D-glucopyranosylamine (2), in the solid state and in solution, have been investigated. The crystal structure of the axially substituted alpha anomer (1) indicates a conformational preference about the C-1-N bond in which nN-->sigma*C-O exo-anomeric interactions may be expressed, although this conformational preference is not displayed in solution. The solution conformation relieves steric interactions that result from expression of the exo-anomeric effect in the solid-state conformation. The conformational preference in the equatorially substituted beta anomer (2) both in solution and in the solid state is similar and permits expression of nN-->sigma*C-O exo-anomeric interactions. The structural data for 1 and 2 indicate significant differences in O-5-C-1-N-1 bond angles but insignificant differences in each of the O-5-C-1 or C-1-N-1 bond lengths. The J(C-1-H-1 coupling constants in 1 and 2 indicate a greater coupling constant for the alpha anomer that is consistent with a dominant nO-->sigma*C-H orbital interaction in the beta anomer that weakens the C-1-H-1 bond.     

Determination of the relative and absolute stereochemistry of a potent and alpha1A-selective adrenoceptor antagonist

The binding affinities and selectivities of antagonists 1-4 for the alpha1A-adrenoceptor are dependent on the stereochemical orientation of the groups at the C-4 and C-5 positions of the oxazolidinone ring. The unambiguous assignment of the relative and absolute configurations of the diastereomers of SNAP 7915 (1) is reported.     

Development of 2-substituted-N-(naphth-1-ylmethyl) and N-benzhydrylpyrimidin-4-amines as dual cholinesterase and Aβ-aggregation inhibitors: Synthesis and biological evaluation

A group of 2-substituted N-(naphth-1-ylmethyl)pyrimidin-4-amines (6a-k) and N-benzhydrylpyrimidin-4-amines (7a-k) in conjunction with varying steric and electronic properties at the C-2 position were designed, synthesized and evaluated as dual cholinesterase and amyloid-β (Aβ)-aggregation inhibitors. The naphth-1-ylmethyl compound 6f (2-(4-cyclohexylpiperazin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine) exhibited optimum dual ChE (AChE IC(50)=8.0 μM, BuChE IC(50)=3.9 μM) and hAChE-promoted Aβ-aggregation inhibition (30.8% at 100 μM), whereas in the N-benzhydryl series, compound 7f (N-benzhydryl-2-(4-cyclohexylpiperazin-1-yl)pyrimidin-4-amine) exhibited optimum combination of dual ChE (AChE IC(50)=10.0 μM, BuChE IC(50)=7.6μM) and hAChE-promoted Aβ-aggregation inhibition (32% at 100 μM). These results demonstrate that a 2,4-disubstituted pyrimidine ring serves as a suitable template to target multiple pathological routes in AD, with a C-2 cyclohexylpiperazine substituent providing dual ChE inhibition and potency whereas a C-4 diphenylmethane substituent provides Aβ-aggregation inhibition.     

Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols

The relationship between the structure and activity of meta- and para-hydroxylated monophenols was studied during their tyrosinase-catalysed hydroxylation and the rate-limiting steps of the reaction mechanism were identified. The para-hydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxylated substrates) or for electrophilic attack (meta-hydroxylated substrates). The electron-attracting effect of the -CH2OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V(M)Max) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K(M)m), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(M)cat/K(M)m is much greater for thepara-hydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the meta-hydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K(M)m of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the para-hydroxylated substrates, the binding constant decreases, leading to an increase in K(M)m. The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V(M)max. The small difference between the absolute values of V(M)max suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.     

Enantioselectivity of Candida Rugosa Lipase Toward Carboxylic Acids: A Predictive Rule from Substrate Mapping and X-Ray Crystallography

We used substrate mapping to develop a rule that predicts which enantiomer of chiral carboxylic acid esters reacts faster in hydrolyses catalyzed by lipase from Candida rugosa (CRL, triacylglycerol hydrolase, E. C. 3.1.1.3). This rule, based on the size of the substituents at the stereocenter, is not reliable for crude CRL. It predicts the favoured enantiomer for only 23 out of 34 examples, 68% reliability. However, this rule is completely reliable for purified CRL; it predicts the favoured enantiomer for all 16 examples correctly. The examples include arylpropanoicacids, aryloxypropanoic acids, α-halophenylacetic acids, mandelic acid and O-methylmandelic acid. Further, purified CRL did not catalyse the hydrolysis of N-CBZ-phenylalanine methyl ester and N-CBZ-norleucine methyl ester. These two substrates were exceptions to the rule with crude CRL as the catalyst. Besides eliminating several exceptions, purification also raised the enantioselectivity of CRL toward carboxylic acid esters. To provide a structural basis for this proposed rule we examined the x-ray crystal structure of CRL containing transition state analogs of ester hydrolysis. We suggest that the large substituent of chiral carboxylic acids binds in a tunnel that normally binds the alkyl chain of a fatty acid. The phenyl rings of Phe 345 and Phe 415 lie close to the stereocenter, thereby fixing the orientation of the medium substituent. The three-dimensional orientation of these proposed binding sites is consistent with the rule derived from substrate mapping.     

P.M.R studies on fully methylated aldohexopyranosides and their 6-deoxy analogues using lanthanide shift reagents

The complexes of lanthanide shift reagents (LSR) with permethylated aldo-hexopyranosides and their 6-deoxy analogues having the gluco, galacto, and manno configurations have been studied. On the basis of shift data from Eu(fod)₃ and Pr(fod)₃, and broadening data from Gd(fod)₃, it was found that the LSR bind preferentially to two neighbouring MeO-oxygens having the axial-equatorial relationship. Because of its steric requirements, the C-5 substituent hinders the binding increasingly in the following order: O-2(ax)-O-3(eq)<O-1(ax)-O-2(eq)<O-4(ax)-O-3(eq). Equatorial groups bind the LSR only weakly. Strong binding to O-6 was found when MeO-6 is predominantly “axially” oriented; when this group has the “equatorial” position, O-6 is not favoured over any other equatorial oxygen. In view of the preference of the LSR to bind to an O(ax)-O(eq) site, it is proposed that O-5 is involved in the binding to the axial O-6. Eu(fod)₃ seems to have less tendency to bind to the O-6(ax)-O-5 site than the other two LSR.     

Interactions of anilinoacridines with nucleic acids: effects of substituent modifications on DNA-binding properties

Spectroscopic methods are used to probe the interactions of several anilinoacridine analogues with calf thymus DNA over a wide range of temperatures and sodium chloride concentrations. The structurally similar compounds m-AMSA, AMSA (both active as antitumor agents), and o-AMSA (inactive as an antitumor agent) have been widely studied in their abilities to bind DNA in an intercalative manner. Recent studies from this laboratory reveal distinct differences in the thermodynamic binding mechanisms between m-AMSA and o-AMSA (Wadkins & Graves, 1989), with the m-AMSA-DNA interaction being an enthalpy-driven process while the binding of o-AMSA to DNA is characterized by more positive entropy values. To further examine the physical chemical properties associated with these compounds and their correlation with antitumor activities, an in-depth investigation into the thermodynamic parameters of these compounds and structurally related anilinoacridine analogues was performed. These studies demonstrate that substituent type and position on the aniline ring of the anilinoacridines greatly influences both the affinities of these drugs in binding to DNA and dictates whether the DNA binding is an enthalpy- or entropy-driven process. The differences in thermodynamic mechanisms of binding between the two isomers along with molecular modeling studies reveal the electronic and/or steric factors resulting from the positioning of the methoxy substituent group on the anilino ring directs the DNA-binding properties through orientation of the methanesulfonamido group at the 1' position of the aniline ring. The orientation of this substituent group may result in favorable contacts through hydrogen bonding with neighboring base pairs and ultimately influence the biological effectiveness as an antitumor agent.     

Substituted thiophene-anthranilamides as potent inhibitors of human factor Xa

A series of thiophene-containing non-amidine factor Xa inhibitors is described. Simple methyl-substituted thiophene analogs were relatively weak inhibitors. However, introduction of hydrophilic substituents at C-4 or C-5 of the thiophene afforded inhibitors with low nanomolar potency. Optimization of the thiophene substituent at C-4 afforded subnanomolar inhibitors with improved in vitro anticoagulant activity. Incorporating basic amine substituents on the thiophene increased hydrophilicity and improved anticoagulant activity. The pharmacokinetic profile of one inhibitor was evaluated in dogs, and the X-ray crystal structure of this compound bound to factor Xa provides insight into the observed SAR for binding to factor Xa.     

Comparison of ivermectin, doramectin, selamectin, and eleven intermediates in a nematode larval development assay.

Chemical substitutions at pharmacologically relevant sites such as C-5, C-13, C-22,23, and C-25 were examined in ivermectin, doramectin, selamectin, and a series of 11 other intermediates using a larval development assay with Haemonchus contortus. A range of activities spanning 5 orders of magnitude were manifest with small changes in the substituents to the 14 avermectins. Within this compound series, there was no major potency advantage or disadvantage to a disaccharide over a monosaccharide substituent at C-13. Ivermectin and doramectin were each fully effective at a concentration of 0.001 microg/ml, and both were similar to their respective monosaccharide homologs. Specific patterns emerged among the analogs with substituents at C-5. Analogs possessing hydroxyl groups at C-5 were superior in activity by several orders of magnitude over those with oxo substituents. Replacement of the oxo with an oxime (NOH) restored activity to some degree but did not restore it to the level of those possessing the hydroxyl substituent. Consequently, ivermectin and doramectin that possess hydroxyl moieties at C-5 were superior against H. contortus to those like selamectin that have oxime substituents. There was no advantage for analogs with a single or double bond at C-22,23 within the cyclohexyl series, and these analogs had equivalent activity as those with a single bond at C-22,23 in the sec-butyl/isopropyl series. However, there was superior activity for the analog series that possessed the combination of a double-bond at C-22,23 and a sec-butyl/isopropyl substituent at C-25. As a result, the most potent compound in this test was not any of the 3 commercialized avermectins but was a monosaccharide with a double bond at C-22,23, an hydroxyl at C-5, and a sec-butyl/isopropyl moiety at C-25.