Complete resolution of the microscopic protonation equilibria of N-methyl-D-aspartic acid and related compounds

Protonation equilibria of N-methyl-D-aspartate (NMDA, a specific glutamate receptor agonist) and its derivatives are characterized at the macroscopic and microscopic levels. (1)H NMR-pH and pH-potentiometric titrations were carried out to determine the macroconstants. Microconstants were obtained by appropriate combination of acidity and NMR parameters of the parent compound and its three synthetic derivatives. These derivatives were close models of the NMDA minor microspecies, allowing the calculation of all the 12 microconstants, the 8 microspecies concentrations and 3 site interactivity parameters. Reliability of the microconstants was assessed by three independent test methods. It was found that protonation of the secondary amino site decreases the beta- and alpha-carboxylate basicities almost exactly by one and two orders of magnitude, respectively, whereas protonation of one of the carboxylates lessens the basicity of the other one by a factor of 3. NMR-pH profiles, macro- and microscopic protonation schemes and species-specific distribution diagrams are presented.     

Microspeciation of amphoteric molecules of unusual acid-base properties

The phisico-chemical properties of bio- and drug molecules greatly influence their interactions in the body and strongly effect the mechanism of drug action. Among these properties, macroscopic and site-specific protonation constants are of crucial importance. Latter one is the tool to calculate the relative concentration of the various microspecies in the compartments of the body at different pH values, and also, it is the versatile parameter to improve the pharmacokinetic properties of a new molecule in a particular family of drugs. In the present thesis work, the microspeciation of three molecules of great pharmaceutical importance and unusual acid-base properties, were carried out. The microconstants of tenoxicam, the non-steroidal anti-inflammatory drug, were described, introducing a novel deductive method using Hammett constants. For this purpose, a total of 8 tenoxicam and piroxicam derivatives were synthesised. To the best of our knowledge, the log k(N)O microconstant of tenoxicam obtained thus is the lowest enolate basicity value, which, however, can be well explained by the effects of the intramolecular environment. The developed evaluation procedure is suitable for microconstant determination of compounds in other molecule families. Besides, prodrug-type compounds and analogues similar to the structures of selective COX-2 isoenzyme inhibitors were synthesised. The other two molecules studied, the 6-aminopenicillanic acid and 7-cephalosporanic acid, the core molecules of the two most important beta-lactam antibiotic-types were derivatised and investigated by 1D and 2D NMR techniques. The NMR-pH titration on the parent compounds and their ester derivatives, combined with in situ pH-measurements allowed the microspeciation of these easily decomposing molecules. One of the protonation constant of 7-ACA (log kN(O) = 4.12), to the best of our knowledge, is the least non-aromatic basic amino-site among the natural compounds.     

Electrodeless, accurate pH determination in highly basic media using a new set of (1)H NMR pH indicators

A set of indicator molecules was selected and applied to elaborate an NMR-based pH determination method, free of glass electrode errors in highly basic media. Accurate measurement of pH values and protonation constants was achieved by a successive build-up of overlapping, increasingly high pH solutions, using a collection of 8 compounds of appropriately incremented basicities. In order to verify the method, acid-base properties were quantified for two compounds with very high basicities in conflicting reports: two pharmaceutically important biguanidine drugs, metformin and phenformin.     

The complete microspeciation of arginine and citrulline

(1)H NMR-pH titrations of arginine, the most basic natural amino acid and citrulline, its neutral counterpart were carried out. Two other closely related auxiliary compounds were also studied. The 8 macroscopic protonation constants were determined. Combining the four datasets in a deductive method, all the 12 microconstants of arginine, the 4 microconstants of citrulline and arginine amide were calculated. An error-propagation analysis and the pH-dependent distribution of the 8 arginine microspecies are provided.     

Acid-base properties of thymopoietin-type tri- and tetrapeptides and their derivatives

The submolecular basicities of 21 immuno-modulating, thymopoietin-type di-, tri-, and tetrapeptides were studied and characterized in terms of group constants and partial microconstants. All compounds were derivatives of the H-Arg-Lys-Asp-OH tripeptide. Modifications within four covalent bonds of the basic site (esterification, acylation, curtailment or addition at C-terminal end, exchange of amino acids) cause significant changes in the scheme of protonation and in the individual basicity of proton binding sites. Configurational changes of the component amino acids, however, do not cause significantly different basicities in the diastereomers.     

Nitrosative guanine deamination: ab initio study of deglycation of N-protonated 5-cyanoimino-4-oxomethylene-4,5-dihydroimidazoles

5-Cyanoimino-4-oxomethylene-4,5-dihydroimidazoles (1) (R at N1) have been discussed as possible intermediates in nitrosative guanine deamination, which are formed by dediazoniation and deprotonation of guaninediazonium ion. The parent system 1 (R = H) and its N1 derivatives 2 (R = Me) and 3 (R = MOM) are considered here. Protonation of 1-3, respectively, may occur either at the cyano-N to form cations 4 (R = H), 6 (R = Me), and 8 (R = MOM) or at the imino-N to form cations 5 (R = H), 7 (R = Me), and 9 (R = MOM), respectively. This protonation is the first step in the acid-catalyzed water addition to form 5-cyanoimino-imidazole-4-carboxylic acid, which then leads to oxanosine. There also exists the option of a substitution reaction by water at the R group of 6-9, and this dealkylation forms N-[4-(oxomethylene)-imidazol-5-yl]carbodiimide (10) and N-[4-(oxomethylene)-imidazol-5-yl]cyanamide (11). In the case of DNA, the R group is a deoxyribose sugar, and attack by water leads to deglycation. To explore this reaction option, the S(N)1 and S(N)2 reactions of 6-9 with water were studied at the MP2/6-31G*//RHF/6-31G* and CCSD/6-31G*//RHF/6-31G* levels, with the inclusion of implicit solvation at the IPCM(MP2/6-31G*)//RHF/6-31G* level, and the electron density distributions of tautomers 1, 10, and 11 were analyzed. The low barriers determined for the MOM transfer show that the deglycation could occur at room temperature but that the process cannot compete with water addition.     

Cyclodextrin/imatinib complexation: binding mode and charge dependent stabilities.

Host-guest interactions in various protonation forms of the anticancer drug imatinib with beta-cyclodextrin (CD) and randomly methylated beta-CD (RAMEB) have been investigated using techniques of proton magnetic resonance spectroscopy ((1)H NMR), phase solubility, pH-potentiometry and electrospray ionization mass spectrometry (ESI-MS). Phase-solubility analysis showed A(L)-type diagram with beta-CD, which suggested the formation of 1:1 inclusion complexes. The 1:1 stoichiometry was confirmed by potentiometry in aqueous solution and by ESI-MS in the gas phase. Charge-specific stability constants of the neutral, mono-, di-, and tricationic forms of imatinib were determined for both the beta-CD and RAMEB. Stability of the beta-CD complexes shows an unexpected minimum at the monoprotonated form, while a stepwise decrease with increasing guest charge was observed for RAMEB. The 1:1 complex stoichiometry and stability constants of selected imatinib protonation species were verified by (1)H NMR titrations. Two-dimensional rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments were carried out to identify the interacting host-guest moieties. The observed ROESY cross-peaks indicated spatial proximities between several aromatic hydrogens of imatinib and beta-CD protons, revealing that the inclusion occurs by accommodation of the benzamide ring of imatinib.     

Population, acid-base, and redox properties of N-acetylcysteine conformers

Rotamers of N-acetyl-L-cysteine (NAC, the most popular mucolytic drug) are characterized in terms of populations, site- and conformer-specific acid-base properties, reducing strength, and molecular pharmacology. A new, general relationship between the bulk- and rotamer-specific basicities is introduced. NAC at high pH predominantly exists in a trans thiolate-carboxylate rotameric form, whereas protonation promotes the occurrence of intramolecular hydrogen bond-forming isomers. Distribution curves of the rotamers are depicted as a function of pH. Rotamer-dependent thiolate basicities differ by up to 0.5 log k units. Carboxylate basicities show slight conformation-dependence only. The membrane-penetrating capabilities from various compartments of the body are assessed on the basis of the pH-dependent charge of the molecule. The thiol-disulfide half-cell potential is calculated, using the correlation between the thiolate basicity and oxidizability. The oxidation-reduction properties of NAC are compared to those of other biological thiols in their definite microscopic forms. The pharmacokinetic behavior is interpreted in terms of the physicochemical parameters, providing molecular/submolecular explanation for several therapeutic properties of NAC.     

Theoretical study of the protonation and tautomerization of adenosine, formycin, and their 2-NH2 and 2-F derivatives: functional implications in the mechanism of reaction of adenosine deaminase

A quantum chemical study of adenosine, formycin, and their 2-NH2 and 2-F derivatives is performed. The tautomerism of neutral and protonated species as well as the protonation of adenosine, formycin, and their derivatives are theoretically studied using semiempirical MNDO and AM1, as well as ab initio STO-3G methods. Calculations have been performed on a reduced model, in which the ribose moiety has been substituted by a hydroxy-methyl group. Results indicate that adenosine is mainly protonated at the N1 atom, whereas formycin can be protonated on N1 or N3, depending on the tautomeric form (N8-H or N7-H). The quantum chemical study of the N1-protonated molecules shows that a second protonation of adenosine is mainly on the N3 atom, whereas formycin can be protonated on N8 or N3, depending on the tautomeric form. On the other hand, results indicate that the protonation of formycin and its derivatives at the N1 atom leads to a change in their tautomeric preference from N7-H to N8-H. The importance of both tautomerism and protonation reactions in the mechanism of action of adenosine deaminase is studied by means of a quantitative structure activity relationships strategy. Significant correlations were found between several electronic parameters and the logarithm of the maximum rate of deamination (log Vm) of the studied compounds. For formycin and its derivatives, it was necessary to consider their N8-H tautomeric forms. The electronic parameters giving good correlations were as follows: energy of the minimum of the ab initio molecular electrostatic potential on N1, net charge over purine (pyrazolo-pyrimidine) and pyrimidine rings, and the N1 protonation energy. It must be noted that all these parameters are informative in relation to a proton attack. Adenosine and purine ribosides have been studied largely because of their high biological relevance. They are constituents of nucleic acids, intermediates in secondary metabolism, neuromodulators, and neurohormones. Their analogues have been extensively used because of their wide range of pharmacological effects (1). Formycin A (Fig. 1) is one of the most studied analogues of adenosine. It is a natural product extracted from Nocardia interforma (2) with proven antiviral (3-5), antibiotic (2), immunodepressant (6), antitumor (6), and antimetabolic (5) activities.     

Protonation equilibria of quinolone antibacterials

The acid-base properties of seven antibacterial 7-piperazinyl fluoroquinolone derivatives were studied by potentiometry and UV and NMR spectroscopy. These molecules contain two proton-binding sites of similar basicity, namely, the piperazine amino and the carboxylate groups, as proven by 1H NMR spectroscopy. The basicities are quantitated at the molecular level in terms of macroconstants, and also at the submolecular level in terms of microconstants. The microconstants are then used to calculate the concentration of the positive, zwitterionic, neutral, and negatively charged species (microspeciation). The zwitterionic forms always predominate over their neutral protonation isomers, but the zwitterionic:neutral concentration ratio is considerably different for the examined fluoroquinolone derivatives.     

Lipophilic and electrostatic forces encoded in IAM-HPLC indexes of basic drugs: their role in membrane partition and their relationships with BBB passage data

The membrane phospholipid affinity data, log k(w)(IAM), for 14 basic drugs spanning a wide lipophilicity range were measured by HPLC on two different phospholipid stationary phases, i.e. IAM.PC.MG and IAM.PC.DD2. These data related weakly with log P(N) values, the n-octanol/water partition coefficients of the neutral forms; poorer relationships were found with log D(7.0) values, the n-octanol/water partition coefficients of the mixtures of neutral and ionized forms at pH 7.0. The lack of collinearity confirms that, differently from partition in n-octanol/water, partition in phospholipids encodes not only lipophilic/hydrophobic intermolecular recognition forces but also ionic bonds, due to electrostatic interactions between electrically charged species and phospholipids, according to the "pH-piston hypothesis". This component of interaction was parameterized by Δ log k(w)(IAM) values; they are the differences between the log k(w)(IAM) values experimentally measured and the values expected for neutral isolipophilic compounds. Δ log k(w)(IAM) values of the various analytes changed almost linearly from positive to negative values at increasing lipophilicity. This behavior is consistent with an interaction mechanism with membrane phospholipids including two intermolecular interaction forces: (i) lipophilic/hydrophobic interactions, which decrease on ionization proportionally to the lipophilicity of the neutral forms, and (ii) electrostatic interactions, which increase on ionization and are quite constant for all the analytes at a given ionization degree. Since BBB passage of the considered compounds is supposed to be based on passive mechanisms, we investigated the possible relationships between log BB values, i.e. the logarithms of the ratio between brain and blood concentrations, and three physico-chemical parameters, i.e. (i) log P(N) (lipophilic interaction of the neutral form), (ii) log k(w)(IAM) (global interaction with phospholipids), and (iii) Δ log k(w)(IAM) (electrostatic component of interaction with phospholipids). The results suggest that the electrostatic interactions encoded in log k(w)(IAM) values might act as trapping forces in a phospholipid barrier. Actually, we observed an inverse linear dependence of log BB on Δ log k(w)(IAM) values, but only for the compounds showing positive Δ log k(w)(IAM) values. We conclude that the driving force for BBB passage is the lipophilicity of the neutral forms, log P(N), and not the lipophilicity actually displayed at the experimental pH, log D(7.0). Indeed, the latter does not adequately take into account the role played by protonation in the analyte/membrane interactions because protonation, although hindering membrane passage, can either reduce or enhance partition in phospholipids, depending on analyte lipophilicity.     

Proton Speciation and Microspeciation of Vinpocetine and Related Compounds in Aqueous and Biomimetic Media

Purpose. The determination of protonation macroconslants of twelve compounds in the vincamine drug family and the determination of protonation microconstants of cis- and trans-apovincaminic acid in media of various solvent composition to characterise their site-specific basicity and to estimate the concentration of the membrane-penetrating and receptor-binding forms. Methods. UV-pH titrations have been used to determine the protonation macroconstants in 10–43 wt% methanol/water mixtures. Yasuda-Shedlovsky extrapolation was applied to obtain aqueous log values for compounds sparingly soluble in water. Protonation microconstants were also determined by deductive methods for compounds of free carboxylic group. Results. In the case of the two water-soluble compounds the extrapolated and the directly measured aqueous log K values were in good agreement, verifying all other extrapolated data. Compounds of cis-D/E ring anellation are 0.4-0.8 log K units more basic than their epimeric,trans counterparts. The pH-dependent distribution of apovincaminic acid microspecies in aqueous and membrane-like media is depicted in microspeciation diagrams. Conclusions. The N(4) nitrogen is more shielded by the adjacent ethyl group in trans-D/E ring anellation eburnanes than in cis ones, as reflected by the protonation constants. Solvent-dependent basicity data predict superiority of trans isomers in lipophilicity and membrane-penetrating ability.     

Analogues of substance P. Peptides containing D-amino acid residues in various positions of substance P and displaying agonist or receptor selective antagonist effects

Agonist and antagonist analogues of substance P were synthesized by replacing at least two of the amino acid residues with D-Trp, D-Phe, D-Val, or D-Pro residues. The syntheses of these compounds were achieved by solid-phase methodology using the hydroxymethyl resin. The analogues were tested for agonist and antagonist activity on guinea pig ileum and rat spinal cord preparations. Two types of antagonists were obtained. The first type of compounds, e.g., [N alpha-Z-Arg1,N epsilon-Z-Lys3,D-Trp7,8,D-Met11]-SP-OMe (1), antagonized SP and SP(6-11)-hexapeptide on the ileum but only SP(6-11)-hexapeptide on the spinal cord. The second type of antagonists, e.g., [N alpha-Z-Arg1,N epsilon-Z-Lys3,D-Pro9,10]-SP-OMe (17), were inactive on the ileum but were potent antagonists of the hexapeptide on the spinal cord. Two of the antagonists, [N alpha-Z-Arg1,N epsilon-Z-Lys3,D-Trp7,8,D-Met11]-SP (3) and [D-Trp7,8,9]-SP (43), were also tested in vivo. Both of these depressed hypotensive responses to SP and SP(6-11)-hexapeptide in rabbits.     

In vitro blood distribution and plasma protein binding of the tyrosine kinase inhibitor imatinib and its active metabolite, CGP74588, in rat, mouse, dog, monkey, healthy humans and patients with acute lymphatic leukaemia

AIMS: To determine blood binding parameters of imatinib and its metabolite CGP74588 in humans and non-human species. METHODS: The blood distribution and protein binding of imatinib and CGP74588 were determined in vitro using (14)C labelled compounds. RESULTS: The mean fraction of imatinib in plasma (f(p)) was 45% in dog, 50% in mouse, 65% in rat, 70% in healthy humans and up to 92% in acute lymphatic leukaemia (AML) patients. Similarly, f(p) for CGP74588 was low in dog and monkey (30%), higher in rat, mouse and humans (70%) and highest in some AML patients (90%). The unbound fraction of imatinib and CGP74588 in plasma was lower in rat, mouse, healthy humans and AML patients (2.3-6.5% at concentrations < or = 5000 ng ml(-1)) compared to monkey and dog (7.6-19%). Both compounds displayed high binding to human alpha(1)-acid glycoprotein. AML patients had a reduced haematocrit and showed greatest variability in their blood binding parameters. CONCLUSION: Imatinib and CGP74588 displayed very similar blood binding parameters within all species/groups investigated. The five species clustered into two distinct groups with rat, mouse and humans being clearly different from dog and monkey. For both compounds, higher protein binding was associated with a decreased partitioning into blood cells.     

Synthesis and investigation of protonation properties and lipophilicity of some new quinazolone-3-carboxylic acid derivatives]

Several new quinazolone-carboxylic acid derivatives as potential NMDA and AMPA receptor antagonists have been synthesized, and the protonation properties and lipophilicity of some representative molecules have also been studied. The protonation macroconstants (logK) of 4(3H)-quinazolone (Q0) and two 2-methyl-4-oxo-3H-quinazoline-3-carboxylic acids (Q1, Q2) were determined by pH-potentiometry. The acid-base chemistry of Q1 and Q2, where protein-bindings take place in an overlapping fashion, was described in terms of protonation microconstants (logk) as well. Microspeciation was carried out by UV-pH titration and deductive method. Microspeciation revealed remarkable differences between the two homologue compounds (Q1 and Q2), namely insertion of a second methylene moiety into the aliphatic acid side-chain reversed the predominantly zwitterion-involved protonation pathway into neutral form-involved one. Lipophilicity of our molecules was described by the octanol-water partition coefficients. The apparent partition coefficients of Q1 and Q2 were determined by shake-flask method and converted into true logP values using the protonation microconstants. The unexpected differences between their true logP values were explained, similarly to the different protonation pathways with conformational preferences and formation of intramolecular interactions. Out of the other 15 monoprotic quinazolone compounds the lipophilicity of 10 molecules (Q8-Q17, experimental set) was determined by RP-TLC method with the help of a calibration set consisting of 12 standard molecules, five quinazolones (Q3-Q7, determined by shake-flask method) and seven pyrido[1,2a]pyrimidines (PP1-PP7). The obtained logP values proved mostly the expected structure-property relationships. These physico-chemical investigations are pieces of predictive information for the pharmacokinetics of our compounds. These are also discussed in the paper.     

Design and synthesis of new 2,5-disubstituted-1,3,4-oxadiazole analogues as anticancer agents

In continuance of our search for new anticancer agents, we report herein the design, synthesis, and anticancer evaluation of oxadiazole analogues. Two series (4a-h and 4i-q) of new oxadiazole analogues were designed based on heterocyclic (1,3,4-oxadiazole)-linked aryl core of IMC-038525 (tubulin polymerization inhibitor), NSC 776715, and NSC 776715 and synthesized. All the compounds were fully characterized by infrared, nuclear magnetic resonance spectroscopy, and mass spectral data and the purity of compounds was checked by elemental analysis (C, H, and N analysis). Further seven compounds were evaluated for anticancer activity on nine different panels of 60 cell lines (60 NCI cancer cell lines) according to the National Cancer Institute screening protocol and percent growth and percent growth inhibition was calculated at 10?µM drug concentration. Ten compounds were evaluated for anticancer activity on two cancer cell lines (HeLa and MDA-MB-435) as per the standard protocol reported at four different drug concentrations (10^?7, 10^?6, 10^?5, and 10^?4?µM) and GI₅₀, LC₅₀, and TGI dose-related parameters were calculated. The compound 4j showed maximum anticancer activity at 10?µM, and was found to have higher sensitivity against MOLT-4, IGROV1, HCT-116, and K-562 with percent growth inhibitions of 50.38, 48.45, 46.26, and 46.26 respectively. The compound 4j showed superior anticancer activity than imatinib on 41 human cancer cell lines. The compound 4p showed anticancer activity with GI₅₀ of 36.7 and 46.5?µM against HeLa and MDA-MB-435 cell lines, respectively.     

In the search for new anticancer drugs. 19. A predictive design of N,N:N',N':N',N'-tri-1,2-ethanediylphosphorothioic triamide (TEPA) analogues

The nitroxyl-labeled analogues of N,N:N',N':N",N"-tri-1,2-ethanediylphosphoric triamide (TEPA), N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,6,6-tetramethyl-1-oxypiperidi n-4- yl)amino]carbonyl]phosphoric triamide (5a) and N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,5,5-tetramethyl-1-oxypyrrolid in-3- yl)amino]carbonyl]phosphoric triamide (11a), possess therapeutic indexes that are 8-12 times higher than those of thio-TEPA (1) and TEPA (2). The introduction of methyl groups into the aziridine ring, or the replacement of the nitroxyl moiety with hydroxylamine or amine derivatives, or with an adamantane moiety, results in compounds of lesser activity. An attempt is made to rationalize these results using a lipophilicity scale. A predictive design pattern is established.     

Similar pyridinone compounds with different activities of anti-HIV-1 reverse transcriptase

Among the structurally diverse NNRTIs, pyridinone scaffolds demonstrate high potency against HIV-1 wild type and drug-resistant strains. During the optimization of our pyridinone compound 1 (LAM-trans),we found that the introduction of the N atoms in the C-4 position could dramatically improve the water solubility (7b), whereas protonation of the piperidine N atom resulted in a decrease in its hydrophobic interaction with the binding pocket. In particular, protonation altered the orientation of the alicyclic rings in the hydrophobic pocket, thus impeding the formation of key halogen bond and eventually leading to a huge change in anti-HIV-1 RT activity. These results provided theoretical and experimental basis for the subsequent structural modification of pyridinone compounds.