Protonation-deprotonation equilibria in tetrapyrroles Part 4: Mono- and diprotonations of deutero-, hemato-, meso-, and protoporphyrin IX dimethyl esters in methanolic hydrochloric acid

The N-protonations in the deutero, hemato, meso and protoporphyrin IX dimethyl esters (DME) were investigated by spectrophotometric titrations using HCl as the acid and methanol as the solvent. Two spectroscopically different protonated species were observed for each porphyrin DME in addition to the neutral form. These were assigned to the N-protonated monocation and dication. There were no difficulties encountered in observing the monocation formation in the HCl – MeOH system. Very sharp isosbestic points were characteristic of each protonation stage. The p K3 values for the porphyrins in the above order were 3.23, 4.70, 2.93 and 3.37; the p K4 values were 2.48, 2.62, 2.41 and 2.64, respectively. For all porphyrins studied, no further spectral changes were observed after the dication was completely formed. This was interpreted as indicating that the formation of more highly protonated species is not possible in fullydelocalized porphyrins possessing the 18 π-electron [18]diazaannulene delocalization pathway. When the titration was performed on the free dicarboxylic acid porphyrins, aggregation obscured the first protonation step and no clear monocation spectrum could be distinguished. However, also in that case the titration ended up to a UVvis spectrum typical of the dication and the effect of aggregation on the p K4 values was negligible. The UVvis spectrometric parameters are given for the neutral forms and for the protonated species of the porphyrin DMEs. The results are discussed in terms of the NH tautomerization connected to the π-electron delocalization pathway (aromaticity), which tends to hinder outofplane distortions in the porphyrin plane, and in terms of solvation and counterion stabilization of the protonated forms.

Protonation-deprotonation equilibria in tetrapyrroles Part 3: Mono- and diprotonations of the trimethyl esters of chlorin e6 and the 71-acetal of rhodin g7 in methanolic hydrochloric acid

Spectrophotometric protonation titrations were performed for the trimethyl esters (TME) of chlorin e6 (31,32-didehydrorhodochlorin-15-acetic acid) and the 71-acetal of rhodin g7 (31,32-didehydrorhodochlorin-71-oxo-15-acetic acid) using HCl as the acid and methanol as the solvent. For rhodin g7 TME, the 71-acetal formation could be clearly detected as the first step in the titration. Only two spectroscopically different protonated species were observed for each chlorin derivative in addition to the neutral forms. The two protonated species were assigned to the monocation and dication of each chlorin derivative. The following p K a values were obtained: p K3 = 4.63 and p K4 = 0.62 for chlorin e6 TME and p K3 = 4.40 and p K4 = 0.60 for the acetal of rhodin g7 TME. The protonation titration for chlorin e6 TME with HCl in acetic acid afforded UV-vis spectra similar to those obtained with HCl in methanol. The UV-vis spectrometric parameters are given for the neutral forms of chlorin e6 TME, rhodin g7 TME and its 71-acetal, as well as for the mono- and diprotonated species of chlorin e6 TME and rhodin g7 TME acetal. The protonation titration results of the chlorin e6 derivatives are compared with those previously obtained for phytyl/methyl pyropheophorbide a.

Protonation-deprotonation equilibria in tetrapyrroles Part 2: Mono- and diprotonation of methyl pyropheophorbide a in methanolic hydrochloric acid as verified by the 1H, 13C HSQC and 1H, 15N HMBC NMR experiments

The two-dimensional 1H, 13C and 1H, 15N heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR techniques were applied to investigate the formation of N-protonated cationic species of methyl pyropheophorbide a in methanolic hydrochloric acid (CD3OH-HCl). The 1H, 13C HSQC and 1H, 15N HMBC NMR spectra, recorded at the temperature of 278 K, verified that the CD3OH-HCl solution with [H]+ = 0.021 M, contained the N22-protonated monocations of methyl pyropheophorbide a, whereas the CD3OH-HCl solution with [H]+ = 5.0 M contained the N22, N24-protonated dications of the phorbin. No further protonations to form the trication or tetracation were observed. Consequently, the two middle electronic spectra, which were previously tentatively interpreted as representing the dication and trication, were now attributed to conformational alterations originating from steric hindrance between the central hydrogens and changes in counter-ion and solvation interactions and/or conformational alterations intimately connected to NH-tautomerism. These possibilities to explain the previously observed middle electronic spectra are briefly discussed.

Simultaneous Determination of Ascorbic Acid and Acetylsalicylic Acid in Pharmaceutical Formulations

A direct, simple, and practical first-derivative spectrophotometric method is described for simultaneous determination of ascorbic acid and acetylsalicylic acid. The effects of the solvent, excipients, and spectral variables on the analytical signal were investigated. The drugs were determined simultaneously with a 0.01M methanolic hydrochloric acid solution as the solvent, and the signals were evaluated directly by using the zero-crossing method at 245.0 and 256.0 nm for acetylsalicylic acid and ascorbic acid, respectively. The method allows the simultaneous determinations of acetylsalicylic acid and ascorbic acid in the ranges of 6.6 × 10−6 to 1.5 × 10−4M and 3.4 × 10−6 to 2.0 × 10−4M, respectively, with standard deviation of <2.0%. The proposed method was applied to determinations of these drugs in tablets.

Aza analogues of pteridine. VIII. 3-Amino- from 3-Alkoxy-pyrimido[5,4-e]-as-triazin- 5-ones prepared by cyclization of 3-Ethoxy-1,2,4-triazines or other means

Syntheses of hitherto unknown 3-aminopyrimido[5,4-e]-as-triazin-5-ones are described. One route involves selective hydrolysis of 3,5-di- or 3,5,7-tri-alkoxypyrimidotriazines at the 5-position followed by aminolysis. Another is exemplified thus: 3-ethoxy-5-methoxypyrimidotriazine (2a) is converted by methanolic hydrochloric acid into methyl 6-amino-3-ethoxy-1,2,4-triazine-5-carboxylate (3a) which then undergoes successive ammonolysis to the corresponding amide (3b), cyclization by triethyl orthoformate-acetic anhydride to 3-ethoxypyrimidotriazin-5-one (1d) and final ammonolysis to the corresponding amine (1a). The second route may be modified to furnish 3-aminopyrimidotriazine- 5,7-dione (5c) and its 6-methyl derivative (5d). Dehydration of the above amide (3b) with phosphoryl chloride gives both the corresponding nitrile (3h) and an unusual hydrolytic product, 6-amino-3- ethoxytriazin-5-one (6). The ionization constants and the u.v., N.M.R. and mass spectra are discussed.

An assessment of methanolysis and other factors used in the analysis of carbohydrate-containing materials

The stability of monosaccharides in methanolic hydrochloric acid of different strengths and at different temperatures was determined. They are generally stable for 24h in methanolic 1m- and 2m-hydrochloric acid at both 85°C and 100°C, but undergo considerable destruction in methanolic 4m- and 6m-hydrochloric acid at 100°C. Analysis of glycopeptides and oligosaccharides of known composition showed that release of carbohydrate was complete within 3h in methanolic 1m-hydrochloric acid at 85°C. Removal of methanolic hydrochloric acid by rotary evaporation resulted in considerable losses of monosaccharides, which could be prevented by prior neutralization. Methanolysis caused extensive de-N-acetylation of acetamidohexoses, so that a re-N-acetylation step is necessary in the analytical procedure. The addition of acetic anhydride for this purpose also prevented loss of internal standard by adsorption on the insoluble silver salts used in neutralization. Several trimethylsilylating agents were studied and suitable conditions are recommended. The effects on the analytical system of water and some common organic and inorganic contaminants are assessed.

Reactions of primulagenin A. II. Acid-catalysed transformation

Treatment of primulagenin A with methanolic hydrochloric acid gave a complex mixture of products from which the known aegiceradiol (oleana- 12,15-diene-3β,28-dial) and aegiceradienol (28-noroleana-12,17(18)- dien-3β-ol) were obtained. In addition, three new products, a double- bond isomer of aegiceradiol, the structure of which has been established by synthesis, a C30 aromatic alcohol, and a hydroxy ketone containing a cyclopropane ring, have been isolated.