Isosteric conversion of protein carboxyl groups into carboxamide groups. I. Examination of alkyl and aryl esters as model compounds

With a view to substituting protein carboxyl groups by carboxamide groups the properties of a number of model compounds have been examined. The methyl ester of acetylglycine can be 97% converted into amide in 7 M ammonium acetate/ 7 M ammonia in 10 min. Amidation of the N-ethylsalicylamide ester of benzoyl-glycine, prepared by reaction with N-ethylbenzisoxazolium fluoroborate (EBI), is 100% complete in 2 M ammonium acetate/ammonia, pH 9.2, within 1 min; no rearrangement into imide occurred at all. Under the conditions of protein modification (0.1 M EBI, pH 4.2, 0°) acetyltyrosinamide is 13% converted into a compound, presumably containing the N-ethylsalicylimidate group, exhibiting an absorption maximum at 309 nm (e = 3300). This compound decomposes in ammonium acetate/ammonia, pH 9.1, within 1 min. Nα-acetylhistidinamide is not converted at all. Reaction with aminogroups can either be avoided by protection of these groups or be analyzed by spectroscopy of the amidine produced. Spectral data required for analysis have been measured for N-ethylsalicylamide (Λ^max295 nm, e = 3200 at pH 6, Λ_max324 nm, e = 5100 at pH 10, pK 8.15), for O-acetyl-N-ethylsalicylamide (e = 1200 at 250 nm, e = 180 at 280 nm) and for O-diethylsalicylamidine (Δ_max277nm, e = 2330 at pH 6, Λ_max302 nm, e = 3230 at pH 10, pK 7.90).     

Protected amino acid tetrafluorophenyl esters for peptide synthesis

New 2,3,5,6-tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second-order kinetics showed similarly large k_coupling/k_racemization ratios for both tetra- and pentafluorophenyl esters. In particular, the use of 2,3,5,6-tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N-tert.-butyloxycarbonyl-N^im-benzyl-histidine.     

Reactions of N-hydroxysulfosuccinimide active esters*

N-Hydroxysulfosuccinimide esters are reactive functional groups employed in a variety of protein modification reagents, especially cross-linking reagents. For these compounds, hydrolysis is the most important reaction competing for reaction of the esters with nucleophilic groups in proteins. We have employed model compounds to investigate the rates of hydrolysis of N-hydroxysulfosuccinimide esters and their reactions with the α-amino group and the side chains of naturally occurring amino acids, under conditions comparable to those used for protein modification studies. The rates of hydrolysis observed were found to be very low, as compared with their rates of reaction with nitrogen nucleophiles found in proteins. Further, within the ranges investigated, the rate of aminolysis was observed to increase more rapidly than the rate of hydrolysis with increasing pH or with increasing temperature. Four amino acid side chains and the α-amino group were found to react measurably with N-hydroxysulfosuccinimide esters. At pH 7.4 and room temperature, the order of reactivity was found to be N_α-Cbz-histidine < N_α-Cbz-lysine ≤ phenylalanine (α-amino group) ?N-acetylcysteine ?N-acetyltyrosine; however, the acylimidazole adduct formed with the side chain of histidine was found to be a transient product, subject to hydrolysis or reaction with another nucleophile.     

Kinetics and mechanisms of aqueous degradation of the anticancer agent,indicine N-oxide

The aqueous degradation of indicine N-oxide, an unsaturated pyrrolizidine alkaloid ester undergoing clinical testing as an anticancer agent, was studied as a function of pH, buffers and temperature using a colorimetric TLC assay. Isolation and identification of reaction products demonstrated that ester hydrolysis yielding retronecine N-oxide and trachelanthic acid represents the primary alkaline degradation pathway. No loss of indicine N-oxide could be detected in acidic conditions nor could any contribution from uncatalyzed water attack be observed. The pH-rate profiles in alkaline pH at several temperatures indicate that apparent first-order rate-constants may be defined as k_abs = k_OH-[OH^?]. The energy of activation (16.1 kcal/mol) is relatively high for the aqueous hydrolysis of an ester. The kinetics of hydrolysis are discussed relative to other esters and both steric hindrance and β-hydroxyl intramolecular catalysis are proposed. NMR and mass spectral details for indicine N-oxide, retronecine N-oxide and trachelanthic acid are reported.     

Formulation of Vaccine Adjuvant Muramyldipeptides (MDP). 2. The Thermal Reactivity and pH of Maximum Stability of MDP Compounds in Aqueous Solution

The degradation of muramyldipeptides (MDPs) in aqueous solution obeys the rate law k _obs = k _H+_H+ + k _o + k _HO- a _HO- and the Arrhenius equation. For example, the rate constants for degradation of N-acetylmuramyl-L-threonyl-D-isoglutamine, 3, at 25°C are k _H+ = 2.3 × 10^–6 M ^–1 sec^–1, k _o = 8.2× 10^–10 sec^–1, and k _HO– = 0.19 M ^–l sec^–1. The degradation rates are dependent on the side-chain substituents; it is predicted that sterically hindered MDP compounds will show an extended shelf life in aqueous solution. Product studies in the weakly acid pH region (where the pH of maximum stability occurs) show that MDP compounds degrade largely by hydrolysis of the dipeptide side chain. These data show that MDP 3 exhibits a shelf life (t ₉₀) of greater than 2 years in aqueous solutions of pH 4–4.5, the pH of maximum stability.     

Synthesis of β and γ-fluorenylmethyl esters of respectively Nα-Boc-L-aspartic acid and Nα-Boc-L-glutamic acid

The orthogonal synthesis of N^x-Boc-L-aspartic acid-γ-fluorenylmethyl ester and N^α-Boc-L-glutamic acid-δ-fluorenylmethyl ester is reported. This is a four-step synthesis that relies on the selective esterification of the side-chain carboxyl groups on N^x-CBZ-l -aspartic acid and N^α-CBZ-l -glutamic acid. Such selectivity is accomplished by initially protecting the a-carboxyl group through the formation of the corresponding 5-oxo-4-oxazolidinone ring. Following side-chain esterification, the α-carboxyl and α-amino groups are deprotected with acidolysis. Finally, the α-amino group is reprotected with the t-butyl-oxycarbonyl (Boc) group. Thus aspartic acid and glutamic acid have their side-chain carboxyl groups protected with the base-labile fluorenylmethyl ester (OFm) and their α-amino groups protected with the acid-labile Boc group. These residues, when used in conjunction with N^x-Boc-N^ε-Fmoc-l -lysine, are important in the formation of side-chain to side-chain cyclizations, via an amide bridge, during solid-phase peptide synthesis.     

The Relationship of Diastereomer Hydrolysis Kinetics to Shelf-Life Predictions for Cefuroxime Axetil

Cefuroxime axetil, an ester prodrug of cefuroxime, is comprised of a 50:50 mixture of diastereomers A and B. The first-order hydrolysis kinetics of cefuroxime axetil were investigated as a function of pH, temperature, buffers, and ionic strength. Chromatographically identified hydrolysis products were cefuroxime, ²-cefuroxime axetil, and ,-sulfoxides. Buffer catalysis was observed in acetate and phosphate buffers. No significant kinetic effect was observed for ionic strength in the range µ = 0.1-1.0. The pH–rate profiles for hydrolysis of cefuroxime axetil isomeric mixture were obtained at 45, 35, and 25°C. The equation defining the cefuroxime axetil hydrolysis rate constant as a function of pH was k _obs = k _H(a _H) + k _s + k _OH(K _w/a _H), exhibiting maximal stability in the pH range 3.5 to 5.5. The predicted profile at 5°C was in excellent agreement with experimental data in the pH range 3.6 to 5.5. In the pH range 1 to 9, the maximum difference observed for individual hydrolysis constants of isomers was 27%. Shelf-life estimates based on the hydrolysis rate constants for cefuroxime axetil as an isomeric mixture were shown to be equivalent to those based on individual hydrolysis rate constants for isomers A and B.     

Preparation of partially protected des-alanineB30 -insulin-B-chain-disulphide and its use in semisynthesis*

The preparation of Nα¹N29-bis-methylsulphonylethyloxycarbonyl-des-alanine^B30-B-chain-disulphide-OΛ¹³OΛ²¹-dimethyl ester is described. Starting with the di-S-sulphonate-B-chain it was prepared by i) removal of the C-terminal amino acid by digestion with carboxypeptidase A, ii) reduction and oxidation to form the cyclic disulphide, iii) esterification of the three carboxyl groups (αCOOH = Lys^B29, ΛCOOH = Glu^B13 and Glu^B21) followed by selective tryptic hydrolysis of the α-carboxyl ester, and iv) the protection of both amino groups (αNH₂= Phe^B1, NH₂= Lys^B29). This B-chain derivative was activated selectively at the C-terminal carboxyl group by formation of a mixed anhydride, and condensed with the dipeptide Thr-Arg. All protecting groups were removed by treatment with alkali. The human-B-chain C-terminal elongated with Arg was obtained in a yield of 30% based on the protected des-Ala^B30-B-chain derivative.     

Kinetics of peptide synthesis studied by fluorescence of fluorophenyl esters

The kinetics of the reaction of Boc-alanine-trifluorophenyl, Boc-alanine-tetrafluorophenyl, Boc-alanine-pentafluoropbenyl, and Boc-alanine-p-chlorotetrafluorophenyl esters (BocAlaOTrf, BocAlaOTfp, BocAlaOPfp, and BocAlaTfc, respectively) with leucine amide and with valine methyl ester have been measured using changes in fluorophenyl chromophore emission at 375 nm. The kinetic data cannot be well fit with a simple second-order reaction scheme. Measurements of the reaction kinetics at different concentrations of the reagents showed that the expression for the reaction rate is in which k is the reaction rate constant, C_N is the concentration of either LeuNH₂ or ValOCH₃, and C_AE is the concentration of the fluorophenyl ester. This reaction equation indicates a complex, probably chain-like, reaction mechanism. The order of reactivity for these active esters with ValOCH₃ is BocAlaOTfc > BocAlaOPfp > BocAlaOTfp > BocAlaTrf. The apparent rate constant, k, for the reaction with LeuNH₂ is higher than that for the reaction with ValOCH₃.     

Acyloxymethyl as a Drug Protecting Group. Part 3. Tertiary O-Amidomethyl Esters of Penicillin G: Chemical Hydrolysis and Anti-Bacterial Activity

Purpose. O-(N-alkylamido)methyl esters of penicillin G were studied as a new class of prodrugs. Methods. Their hydrolysis in aqueous buffers containing 20 % (v/v) of acetonitrile was investigated by HPLC. Results. A U-shaped pH-rate profile was seen with a pH-independent process extending from pH ca. 2 to pH ca. 10. This pathway is characterised by kinetic data that are consistent with a uni-molecular mechanism involving rate-limiting iminium ion formation and penicillinoate expulsion. Penicillin G and the corresponding amide are the ultimate products detected and isolated, indicating that β-lactam ring opening is much slower than ester hydrolysis. The O-(N-alkylamido)methyl esters of penicillin G displayed similar in vitro antibacterial activity to penicillin G itself. Conclusions. Compared to the penicillin G derivatives, the much higher stability of the O-(N-methylbenzamido)methyl benzoate, acetate and valproate esters (which gave rise to a Bronsted β_lg value of ca. -1) suggests that tertiary N-acyloxymethylamides may be useful prodrugs for carboxylic acid drugs with pK_a >4.     

Resolution of proline acylation problem for thiol capture strategy by use of a chloro-dibenzofuran template

The acyl transfer rate for proline, in the prior thiol capture strategy, was enhanced by changing the electronic character of the dibenzofuran template. The rate of amide bond formation between proline and cysteine by the 1-chloro-4-hydroxy-6-mercaptodibenzofuran was measured to be 0.012 min^-1, which translates to a half-life of 53 min. Further enhancement of the reaction rate was accomplished by the use of a 1,3-dichloro-dibenzofuran template. The k₁ for the reaction was measured to be 0.093 min^-1, and the half-life was calculated to be 7 min. To test the applicability of the activated template, 1-chloro-4-hydroxy-6-mercaptodibenzofuran, in peptide synthesis, the 34 amino acid long peptide, H-RPDFCLEPPYTGPCRKARNNFKSADECMRTCGGA-OH, was synthesized. This peptide represents the condensation of the N-terminal 13-mer and the C-terminal 21-mer of the basic pancreatic trypsin inhibitor.     

Design of novel prodrugs for the enhancement of the transdermal penetration of indomethacin

A series of esters of indomethacin containing tertiary amine functional groups and designed for transdermal delivery were synthesized. The rates of chemical hydrolysis of all the esters in pH 7.4 phosphate buffer were determined. For N,N-diethylaminopropyl N-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indole acetate (ester 4), the rates of chemical hydrolysis in pH 5.5–11.25 buffers were investigated in detail. Ester 4 was chemically stable and had a higher n-octanol/water partition coefficient and a greater solubility in water at pH 7.4 than indomethacin. Furthermore, the rate of transdermal penetration of ester 4 through cadaver skin in Franz cells was found to be more than 10-times faster than that of indomethacin itself. Ester 4 was found to possess surface-active properties (CMC = 0.5 mg/ml). Assuming that micelles cannot penetrate biologic membranes, a corrected permeability coefficient was calculated for ester 4 using only the monomer concentration. This value, 3.6 × 10⁻² cm/h, was 100-times greater than that of indomethacin. These results suggest that prodrugs with structures similar to that of ester 4 may be useful for enhancing transdermal penetration of other carboxylic acid-containing anti-inflammatory agents.     

Prodrugs of Peptides. 11. Chemical and Enzymatic Hydrolysis Kinetics of N-Acyloxymethyl Derivatives of a Peptide-like Bond

Various carboxylic acid esters of the N-hydroxymethyl derivative of N-benzyloxycarbonylglycine benzylamide, used as a peptide-like model, were prepared and their decomposition kinetics studied in aqueous solution and in human plasma solutions. These N-acyloxymethylamide derivatives were found to undergo a facile decomposition by a pH-independent process in the pH range 4–8.5, the half-lives being 1–11hr at 37°C. The cause of this limited stability was suggested to be due to the occurrence of an elimination-addition mechanism involving a reactive N-acyliminium ion intermediate. In alkaline solutions (pH > 10) the derivatives showed a normal ester stability. The ester group in the N-acyloxymethyl derivatives was readily hydrolyzed by plasma enzymes to yield the N-hydroxymethyl amide, which subsequently decomposed to the parent amide. The results obtained suggest that N-acyloxymethylation of a peptide bond may be a useful prodrug approach to obtain derivatives with varying lipophilicities and a ready ability to undergo conversion to the parent peptide in vivo. However, the stability of the derivatives in aqueous solutions is limited.     

9-Fluorenylmethyl esters

N ^α-Protected amino acid 9-fluorenylmethyl esters (Fm esters) were prepared by imidazole-catalyzed transesterification of active esters with 9-fluorenylmethanol (9-hydroxymethylfluorene). The new carboxyl protection is unaffected by acids, but is efficiently removed by β-elimination under the influence of secondary and tertiary amines. Primary amines and ammonia can cause slight amide formation. Deblocking was achieved also by catalytic hydrogenation.     

High-Performance Liquid Chromatographic (HPLC) and HPLC-Mass Spectrometric (MS) Analysis of the Degradation of the Luteinizing Hormone-Releasing Hormone (LH-RH) Antagonist RS-26306 in Aqueous Solution

The kinetics of the degradation of an LH-RH antagonist, RS-26306,1, in aqueous solution from pH 1 to pH 11 were studied by reverse-phase HPLC. The pH–rate profiles at 50, 60, and 80°C were U-shaped with the rate law of k _obs = k _H a _H + k _w + k _OH a _OH. The predicted 25°C shelf life at the pH of maximum stability, pH 5, is greater than 10 years. The products from the degradation were analyzed by HPLC-MS using thermospray ionization. Below pH 3, the primary product, 2, forms from the acid-catalyzed deamidation of the C-terminal amide. Above pH 7, epimerization of the individual amino acids is the principal reaction. Between pH 4 and pH 6, intramolecular serine-catalyzed peptide hydrolysis becomes important, yielding a tripeptide, 3, and a heptapeptide, 4. At the pH of maximum stability all three pathways for degradation are observed.     

Water-Soluble,Solution-Stable,and Biolabile N-Substituted (Aminomethyl)benzoate Ester Prodrugs of Acyclovir

Various N-substituted 3- or 4-(aminomethyl)benzoate esters of acyclovir were synthesized and evaluated as water-soluble prodrug forms with the aim of improving the delivery characteristics of acyclovir, in particular its parenteral administration. The esters showed a high solubility in weakly acidic solutions and, as demonstrated with the 3-(N,N-dipropylaminomethyl)benzoate ester, a high stability in such solutions, allowing storage for several years. The esters combine these properties with a high susceptibility to undergo enzymatic hydrolysis in plasma. The half-lives of hydrolysis in 80% human plasma ranged from 0.8 to 57 min, the rate being highly dependent on the position (3 or 4) of the aminomethyl group relative to the ester moiety. All esters were more lipophilic than acyclovir in terms of octanol–pH 7.4 buffer partition coefficients. These properties make N-substituted (aminomethyl)-benzoate esters a promising new prodrug type for acyclovir to enhance its delivery characteristics.     

Synthesis and Biological Evaluation of Ω-(N,N,N-Trialkylammonium)alkyl Esters and Thioesters of Carboxylic Acid Nonsteroidal Antiinflammatory Agents

A series of novel -(N,N,N-trialkylammonium)alkyl ester and thioester derivatives [RCOM(CH₂) _n NR ₃ ⁺ X ^–, M = O or S, n = 2–6, X = I or Cl] of 11 nonsteroidal antiinflammatory carboxylic acid agents (naproxen, ketorolac, indomethacin, ibuprofen, sulindac, ketoprofen, flufenamic acid, mefenamic acid, zomepirac, etodolac, and tifurac) was prepared and evaluated for their antiinflammatory, analgesic, and gastrointestinal erosive properties. In general, each prodrug retained the antiinflammatory activity characteristic of the corresponding parent drug but exhibited moderately to greatly reduced gastrointestinal erosive properties and significantly reduced analgetic potencies. This profile is likely due to a combination of factors including the rate of hydrolysis of the esters in the stomach, gut, and plasma, changes in the locus of absorption of the prodrug or nonsteroidal antiinflammatory drug (NSAID), and altered metabolic disposition patterns resulting from these changes. The results obtained from the compounds of this series indicate that esters of this general class may offer a means to modulate both the aqueous/lipid solubility and the hydrolytic/enzymatic cleavage indices of NSAID prodrugs which potentially possess a more favorable therapeutic ratio of antiinflammatory to gastrointestinal erosive activities.     

Structural effects of the selective reduction of amide carbonyl groups in motilin 1–12 as determined by nuclear magnetic resonance

Motilin is a 22-residue peptide stimulating stomach and intestinal motility. The motilin 1–12 fragment displays biological effects similar to the native peptide. Selective reduction of the amide carbonyl groups to form CH₂NH analogs leads to a significant reduction in activity for the first two N-terminal positions and to a complete loss of activity for all other positions. The structures of motilin 1–12 and ten reduced analogs were investigated using the temperature dependence of the amide NH chemical shifts. In all the analogs, the structure of the N-terminal region (residues 1–5) was different from the structure of motilin 1–12, which is characterized by hydrogen bonding between Phe¹ and Ile⁴. The structure of the C-terminal region of analogs was similar to the structure of moth 1–12 for the first two reduction positions only (1–2 and 2–3), indicating that the C-terminal portion of motilin 1–12 is more critical for biological activity. Complete structural characterizations of motilin 1–12, [CH₂NH]^1–2, and [CH₂NH]^4–5-motilin 1–12 were performed by two-dimensional NMR spectroscopy and molecular modeling. The structural features observed confirm the differences based on the temperature dependence of the amide NH chemical shifts. These results demonstrate that conservation of the amide bond rigidity is essential for the activity of non-hydrolyzable analogs. © Munksgaard 1995.     

[d-Met2, Pro5] enkephalin [N1.5-β-d-glucopyranosyl] amide: a glycosylpeptide with high antinociceptive activity

Different synthetic strategies have been attempted for the synthesis of a glycosylpeptide resulting from the covalent bonding of a sugar residue to the C-terminal carboxyl group of an enkephalin related pentapeptide. The final structure is: Tyr-d -Met-Gly-Phe-Pro [N¹⁵-β-d -glucopyranosyl] amide. The in vitro potency on the GPI test of this analogue was IC₅₀= 64.0 nm . However, its antinociceptive activity by tail immersion tests, after intraperitoneal administration, was 2000 and 200 times higher than morphine in rats and mice, respectively.     

Stabilizing effect of citrate buffer on the photolysis of riboflavin in aqueous solution

In the present investigation the photolysis of riboflavin (RF) in the presence of citrate species at pH 4.0–7.0 has been studied. A specific multicomponent spectrophotometric method has been used to assay RF in the presence of photoproducts during the reactions. The overall first-order rate constants (k_obs) for the photolysis of RF range from 0.42 to 1.08×10^–2 min⁻¹ in the region. The values of k_obs have been found to decrease with an increase in citrate concentration indicating an inhibitory effect of these species on the rate of reaction. The second-order rate constants for the interaction of RF with total citrate species causing inhibition range from 1.79 to 5.65×10^–3 M⁻¹ min⁻¹ at pH 4.0–7.0. The log k–pH profiles for the reactions at 0.2–1.0 M citrate concentration show a gradual decrease in k_obs and the value at 1.0 M is more than half compared to that of k₀, i.e., in the absence of buffer, at pH 5.0. Divalent citrate ions cause a decrease in RF fluorescence due to the quenching of the excited singlet state resulting in a decrease in the rate of reaction and consequently leading to the stabilization of RF solutions. The greater quenching of fluorescence at pH 4.0 compared to that of 7.0 is in accordance with the greater concentration of divalent citrate ions (99.6%) at that pH. The trivalent citrate ions exert a greater inhibitory effect on the rate of RF photolysis compared to that of the divalent citrate ions probably as a result of excited triplet state quenching. The values of second-order rate constants for the interaction of divalent and trivalent citrate ions are 0.44×10^–2 and 1.06×10^–3 M^–1 min^–1, respectively, indicating that the trivalent ions exert a greater stabilizing effect, compared to the divalent ions, on RF solutions.