The effects of glutathione depletion on the biodistribution of Cu(PTSM) in rats

The tissue retention of radiocopper afforded by intravenous injection of the proposed blood flow imaging agent, ⁶²Cu-labeled copper(II) pyruvaldehyde bis(N⁴-methylthiosemicarbazone) [Cu(PTSM)], is thought to result from reductive decomposition of the copper(II) complex by intracellular sulfhydryls (e.g. glutathione, GSH). To determine if the tissue uptake and retention of this tracer adequately measures perfusion in tissues containing altered GSH concentrations, the biodistribution of copper-67 labeled Cu(PTSM) was determined in GSH-depleted rats. Despite treatment to induce relatively large reductions in tissue GSH levels, it was found that only very small changes in the biodistribution of copper-labeled Cu(PTSM) occurred in the treated rats compared to untreated controls.     

A novel side-bridged hybrid phosphonate/acetate pendant cyclam: Synthesis,characterization, and 64Cu small animal PET imaging

Copper-64 (t_1/2 = 12.7 h; β⁺: 0.653 MeV, 17.4%; β^?: 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik–Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with ⁶⁴Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the ⁶⁴Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in ⁶⁴Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.     

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [³H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-¹⁴C]palmitoyl-2-acetyl-sn-glycerol and [1-¹⁴C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[³H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Ternary copper(II) complexes involving 2-(aminomethyl)-benzimidazole and some bio-relevant ligands. Equilibrium studies and kinetics of hydrolysis for glycine methyl ester under complex formation

Formation equilibria of copper(II) complexes of 2-(aminomethyl)-benzimidazole (AMBI) and the ternary complexes Cu(AMBI)L (L = amino acid, amide, dicarboxylic acid or DNA constituents) have been investigated. Ternary complexes of amino acids or amides are formed by a simultaneous mechanism. Amino acids form the complex Cu(AMBI)L, whereas amides form two complex species Cu(AMBI)L and Cu(AMBI)(LH₋₁). The ternary complexes of copper(II) with AMBI and dicarboxylic acids or DNA units are formed by a stepwise mechanism, whereby binding of copper(II) to AMBI is followed by ligation of the dicarboxylic acids or DNA components. The values of Δ log K indicate that the ternary complexes containing aromatic amino acids are significantly more stable than the complexes containing alkyl- and hydroxyalkyl-substituted amino acids. This may be taken as an evidence for a stacking interaction between the aromatic moiety of AMBI and the aromatic side chains of the bio-active ligands. The solid complexes Cu(AMBI)L where L = 1,1-cyclobutanedicarboxylic acid (CBDCA) and malonic acid were separated and identified by elemental analysis and infrared spectroscopy and magnetic moment. The decomposition course and steps for the isolated complexes were analyzed and the kinetic parameters of the non-isothermal decomposition were calculated. The hydrolysis of glycine methyl ester (MeGly) is catalyzed by the Cu(AMBI)²⁺ complex. The kinetic data is fitted assuming that the hydrolysis reaction proceeds in two steps. The first step, involving coordination of the amino acid ester by the amino and carbonyl groups, is followed by rate-determining attack by OH⁻ ion. The second step involves the equilibrium formation of the hydroxo-complex Cu(AMBI)(MeGly)(OH) followed by intramolecular OH⁻ attack.     

Synthesis and characterization of 64Cu- and Cy5.5-labeled tetraiodothyroacetic acid derivatives for tumor angiogenesis imaging

It was previously reported that tetraiodothyroacetic acid (tetrac) inhibits angiogenesis by binding to the cell surface receptor for thyroid hormone on integrin α_Vβ₃. Therefore, we synthesized and evaluated two ⁶⁴Cu-labeled tetrac derivatives and a Cy5.5-labeled tetrac derivative for tumor angiogenesis imaging. Tetrac was structurally modified to conjugate with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) via its hydroxy or carboxylic acid end, and the resulting DOTA-conjugated tetrac derivatives were then labeled with ⁶⁴Cu. Tetrac was also conjugated with Cy5.5 via its carboxylic acid end. All three tetrac derivatives (1–3) exhibited greater inhibitory activity than tetrac against endothelial cell tube formation. The U87MG cell binding of [⁶⁴Cu]2 showed a time-dependent increase over 24 h and it was inhibited by 38% at 4 h in the presence of tetrac, indicating specificity of [⁶⁴Cu]2 to the thyroid hormone receptor site on integrin α_Vβ₃. Positron emission tomography (PET) images of U87MG tumor-bearing mice injected with [⁶⁴Cu]1 and [⁶⁴Cu]2 revealed that high radioactivity accumulated in the tumors, and that the tumor uptake and tumor-to-nontarget uptake ratio were higher in small tumors than in large tumors. In addition, the Cy5.5-labeled tetrac derivative (3) displayed a strong near-infrared (NIR) signal in the tumors. Taken together, these results suggest that these ligands hold promise as imaging agents for visualization of tumor angiogenesis.     

Acylated oligosaccharides from Klebsiella K63 capsular polysaccharide: Depolymerization by partial hydrolysis and by bacteriophage-borne enzymes

The extracellular polysaccharide from Klebsiella K63 is unique in having acetic and formic ester groups attached to the d-galactopyranosyluronic residues in the trisaccharide repeating-sequence. These O-acyl substituents are shown to be some what resistant to mild hydrolysis by both acid and alkali. Bacteriophage-induced depolymerization of the polysaccharide generated a series of acylated oligosaccharides comprising one, or more, repeating unit(s). By mild hydrolysis with acid, the same series of oligomers was released from the polysaccharide, together with the corresponding non-acylated compounds and the expected acylated and non-acylated aldobiouronic acids. A study of these oligosaccharides, as well as of a number of their related compounds, is described, with particular emphasis on the methods used to locate the formic and acetic ester groups. The location of the O-acyl substituents on the galactosyluronic residues was further supported by the results obtained from the high-resolution, 400-MHz, p.m.r. spectra and ¹³C-n.m.r. spectra of a number of the oligosaccharides.     

The palladium(II) promoted hydrolysis of the methyl esters of glycyl-L-leucine,glycyl-L-alanine and L-alanylglycine

The palladium(II)-promoted hydrolysis of the methyl esters of glycyl-L-leucine, glycyl-L-alanine and L-alanylglycine have been studied at 25 °C and I=0.1 M in the pH range 4–5. At a 1:1 metal to ligand ratio the peptide esters act as tridentate ligands, donation occurring via the terminal amino group, the deprotonated amide nitrogen, and the carbonyl group of the ester. Due to the high Lewis acidity of Pd(II) rapid hydrolysis of the ester function by water and hydroxide ion occurs. Rate constants k_OH and k_H2O have been obtained for base hydrolysis and water hydrolysis of the coordinated peptide esters at 25 °C. The rate constants for base hydrolysis are 3.4 X 10⁶ M⁻¹ s⁻¹ (L-alaglyOMe), 6.4 X 10⁶ M⁻¹ s⁻¹ (gly-L-alaOMe) and 2.3 X 10⁷ M⁻¹ s⁻¹ (gly-L-leuOMe). Base hydrolysis of the coordinated peptide esters is at least 10⁶ times that of the free unprotonated ligand. Activation parameters have been obtained for both water and base hydrolysis of the Pd(II) complex of methyl L-alanylglycinate and possible mechanisms for the hydrolyses are considered.     

Structure and binding properties of hen lysozyme modified at tryptophan 62

The structure of a derivative of hen egg-white lysozyme (EC 3.2.1.17) modified by N-bromosuccinimide at Trp62 has been studied by both ¹H nuclear magnetic resonance spectroscopy and X-ray crystallography. It was shown that this modification, changing the tryptophan residue to an oxindolealanine² residue, only causes minor structural changes at the site of the modification, and that the overall structure of the native enzyme is maintained in the derivative. Both diastereomers of the oxindolealanine-62 lysozyme were observed by the two methods employed, in accordance with previous observations (Norton & Allerhand, 1976). The pK values of the catalytically important carboxyl groups of Glu35 and Asp52 were identical in the native enzyme and its derivative. However, the modified enzyme is virtually inactive in the hydrolysis of the cell-wall mucopolysaccharide of Micrococcus lysodeikticus. The binding of N-acetylglucosamine oligosaccharides to both native lysozyme and Ox-62 lysozyme was studied by nuclear magnetic resonance spectroscopy, observing the perturbations on the lysozyme ¹H n.m.r. resonances, and differences in the perturbations of the two systems demonstrated that binding of (GlcNAc)₃ in particular was not identical in the two systems. The structure of Ox-62 lysozyme-(GlcNAc)₃ was studied by X-ray crystallography and it was shown that only two GlcNAc residues make contact with the enzyme, binding the reducing end residue in a similar mode as the α-anomeric form of GlcNAc binds to the native enzyme (Blake et al., 1967a). On the basis of the results obtained by X-ray crystallography and ¹H n.m.r. spectroscopy, the lack of enzymatic activity of the Ox-62 lysozyme arises from the obstruction by the oxindolealanine residue of sub-site B of the active site, preventing productive binding of the substrate.     

Nonenzymatic hydrolysis reactions of adenosine 5′-triphosphate and related compounds: II. Kinetic studies of the hydrolysis of ATP and related compounds with various polyamines

Various structural features of polyamines which are responsible for the acceleration of the hydrolysis of ATP to ADP and P_i at pH 3–4 were surveyed by means of kinetic studies, leading to the following conclusions: 1) The ethyleneimine chain of the polyamines should be as long as possible; 2) the number of methylene carbon atoms between the two adjacent nitrogen atoms of the polyamine has to be two; 3) the terminal groups of the ethyleneimine chain should be primary amino groups.The rate of ATP hydrolysis in the presence of pentaethylenehexamine (pentaen), which possesses the above properties, was found to be 15 times as high as that of hydrolysis at pH 3.5 in the absence of amines. The kinetic data support the previous assumption that there is formation of an ATP-pentaen complex in the hydrolysis reaction. The formation constant of the complex has been calculated to be K = 1.9 × 10⁴M⁻¹ at pH 3.5 and 50°C from the kinetic data. From the temperature dependence of the rates for pentaen or tetraethylenepentamine, the thermodynamic data of these reactions have been obtained.On the other hand, it has been found that pentaen enhances the hydrolysis of GTP and UTP as well as ATP. No phosphate ester bonds of AMP, p-nitrophenylphosphate and α-d-glucose-1-phosphate were hydrolyzed. Therefore, it may be concluded that hydrolysis of the phosphate ester bond with the polyamine is characteristic of ATP, GTP and UTP.     

Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves

The properties of acyl hydrolase enzymes purified from the leaves of Phaseolus multiflorus have been studied. Hydrolase I which deacylates phosphatidylcholine and oleoylglycerol had a pH optimum towards phosphatidylcholine of 5.3. Hydrolase II which deacylates glycosylglycerides and oleoylglycerol showed pH optima of 7.3 (monogalactosyldiglyceride, MGDG) and 4.3 (sulphoquinovosyldiglyceride, SQDG). Both enzymes showed activity peaks towards oleoylglycerol at pH 6.8 and 8.8. Unesterified fatty acids and Triton X-100 inhibited the rate of SQDG hydrolysis while bovine serum albumin increased activity. An apparent K_m for SQDG of 0.15 mM was found. Hydrolase II catalysed transmethylation of liberated fatty acids during the hydrolysis of oleoylglycerol when methanol was included in the assay system. A number of salts inhibited SQDG hydrolysis but their effect on oleoylglycerol was less consistent. The position of ester cleavage of oleoylglycerol was determined by the use of H₂¹⁸O. Cell-free extracts from P. multiflorus leaves degraded SQDG as far as sulphoquinovose.     

Incorporation of methanol into pectic substance

Others have shown that l-methionine is utilized in the biosynthesis of methyl ester groups in pectic substance. Methanol, like l-methionine, is used for methyl ester biosynthesis by detached parsley leaves (Petroselinum crispum). When a combination of methanol-³H and methanol-¹⁴C is given to parsley leaves, methanol recovered from pectic substance by alkaline hydrolysis has a ³H/¹⁴C ratio about one-fourth that of the mixture administered. Unlike l-methionine, methanol is oxidized prior to its utilization as a carbon source for methyl ester biosynthesis.     

Catalytic conformation of carboxypeptidase A. The structure of a true reaction intermediate stabilized at subzero temperatures

The structure of the mixed anhydride, acyl-enzyme intermediate of the esterolytic reaction of carboxypeptidase A is characterized by application of cryoenzymologic, magnetic resonance, and molecular graphics methods with use of the Co²⁺-substituted enzyme and the specific spin-label ester substrate O-3-(2,2,5,5-tetra-methylpyrrolinyl-1-oxyl)-propen-2-oyl-l-β-phenyllactate. A radial separation of 7·7 Å between the active site Co²⁺ and the nitroxide group in the low temperature-stabilized acyl-enzyme intermediate is determined on the basis of their spin-spin (dipole-dipole) interactions. Application of molecular graphics techniques shows that the only configuration of the substrate that is sterically accommodated by the active site yields a calculated metal ion-to-nitroxide distance of 7·8 Å. Steric accommodation of the spin-label in the active site requires severe torsional distortion around the aliphatic double bond of the propenoyl side-chain. Examination of the structure of the enzyme: spin-label intermediate reveals that the distortion arises from steric interactions of the pyrrolinyl group with the protein at a position that corresponds to the site occupied by the penultimate amide residue of an oligopeptide substrate from the site of cleavage. Together with kinetic data showing that hydrolysis of the spin-label is governed by rate-limiting deacylation, the results indicate that geometric distortion of substrates by secondary interactions with the enzyme, in general, is an obligatory part of the catalytic action of carboxypeptidase A. When viewed with respect to requirements for stereoelectronic control of bond cleavage in tetrahedral adducts of esters and amides (Deslongchamps, 1975) the results suggest that torsional distortion during catalysis results in rotation around the scissile bond of the substrate, and that this rotation is required to form the mixed anhydride reaction intermediate. These findings further support the interpretation that the hydrolysis of esters and amides catalyzed by carboxypeptidase A proceeds according to similar mechanisms except that formation of the mixed anhydride is rate-determining in peptide hydrolysis while deacylation of the mixed anhydride is rate-limiting in ester hydrolysis.Additionally, in this study application of the extension of the theory of the Solomon-Bloembergen-Morgan equations derived by Lindner (1965) for paramagnetic metal ions with S ≥ 1 demonstrates that the zero-field splitting of the high-spin Co²⁺ in the metal-substituted enzyme has no significant influence in determination of the relaxation enhancement of solvent protons by the active site metal ion.     

A rate-limiting step of enteropeptidase hydrolysis

It has been shown for the first time that deacylation is the rate-limiting step in the enteropeptidase-catalyzed hydrolysis of highly effective oligopeptide substrates containing four Asp residues in positions P2–P5. On the other hand, the rate-limiting step in the hydrolysis of low-efficiency peptide substrates containing less than four Asp or Glu residues in positions P2–P5 is acylation, as it has previously been suggested for all amide and peptide substrates of serine proteases on the basis of classical works of Bender et al. The method of introduction of an additional nucleophile or another effector that selectively affects the deacylation step was used to determine the rate-limiting step in the enteropeptidase hydrolysis of N ^α-benzyloxycarbonyl-L-lysine thiobenzyl ester, the highly efficient amide substrate GlyAsp₄-Lys β-naphthyl amide, and the low-efficiency peptide substrate VLSAADK-GNVKAAWG (where a hyphen denotes the hydrolysis site).     

Sulfhydrolase Activity in Sediments of Wintergreen Lake, Kalamazoo County, Michigan

The hydrolysis of p-nitrophenyl sulfate, p-nitrocatechol sulfate, and [³⁵S]sodium dodecyl sulfate was examined in anoxic sediments of Wintergreen Lake, Michigan. Significant levels of sulfhydrolase activity were observed in littoral, transition, and profundal sediment samples. Rates of sulfate formation suggest that the sulfhydrolase system would represent a major source of sulfate within these sediments. Sulfate formed by ester sulfate hydrolysis can support dissimilatory sulfate reduction as shown by the incorporation of ³⁵S from labeled sodium dodecyl sulfate into H₂³⁵S. Sulfhydrolase activity varied with sediment depth, was greatest in the littoral zone, and was sensitive to the presence of oxygen. Estimations of ester sulfate concentrations in sediments revealed large quantities of ester sulfate (~30% of total sulfur). Both total sulfur and ester sulfate concentrations varied with the sediment type and were two to three orders of magnitude greater than the inorganic sulfur concentration.     

Synthesis and coordination behaviors of 14-membered tetraaza macrocyclic copper(II) complexes bearing two N-propionic acid or N-propionic methyl ester groups

A di-N-functionalized 14-membered tetraaza macrocycle, [H₄L³](ClO₄)₂ (L³ = 1,8-bis(2-carboxyethyl)-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane), has been synthesized by acid hydrolysis of 1,8-bis(2-cyanoethyl)-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane (L²). The copper(II) complexes [CuL²](ClO₄)₂ and [Cu(H₂L³)](ClO₄)₂ were prepared and characterized. The complex [Cu(H₂L³)]²⁺ readily reacts with methanol to yield [CuL⁴]²⁺ (L⁴ = 1,8-bis(2-carbomethoxyethyl)-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane). The N-CH₂CH₂COOH groups of [Cu(H₂L³)](ClO₄)₂ are not coordinated to the metal ion in the solid state but are involved in coordination in various non-aqueous solvents or in aqueous solutions of pH ? 1.0. Interestingly, [CuL⁴](ClO₄)₂ exists as two stable structural isomers, 1 (the pendant ester groups are not involved in coordination) and 2 (one of the two ester groups is coordinated to the metal ion), in the solid state; the two isomers can be prepared selectively by controlling ionic strength of a methanol solution of the complex. Crystal structures and coordination behaviors of the two isomers are described. The di-N-cyanoethylated macrocyclic complex [CuL²](ClO₄)₂ is rapidly decomposed in 0.1 M NaOH solution even at room temperature. On the other hand, [Cu(H₂L³)](ClO₄)₂ and [CuL⁴](ClO₄)₂ are quite inert against decomposition under similar basic conditions. In acidic or basic aqueous solutions, [CuL⁴]²⁺ is hydrolyzed to [Cu(H₂L³)]²⁺ or [CuL³].     

The uncatalysed and the copper(II) promoted hydrolysis of 4-nitrophenyl L-leucinate

The uncatalysed hydrolysis of 4-nitrophenyl L-leucinate has been studied in detail over a range of pH and temperature at I=0.1 M (KNO₃). Base hydrolysis of the ester is strongly promoted by copper(II) ions. Rate constants have been obtained for the following reactions (where EH⁺ is the N- protonated ester and E is the free base form) EH⁺ + OH⁻ → products E + OH⁻ → products E + H₂O → products CuE²⁺ + OH⁻ → products Base hydrolysis of the copper(II) complex CuE²⁺ is 3.8 × 10⁵ times faster than that of E and 75 times faster than that of EH⁺ at 25 °C and I=0.1 M. Activation parameters for these reactions have been determined and possible mechanisms are considered.     

Strategies for enhancement of radioimmunotherapy

Two new strategies for increasing tumor uptake have been investigated. First the effect of interleukin-2 (IL-2) on tumor uptake of ¹²⁵I-Lym-l antibody in nude mice was investigated. Secondly, the use of ⁶⁷Cu-labeled Lym-1 was evaluated in patients. In nude mice implanted with Raji human lymphoma, a greater than 2-fold enhancement of tumor uptake of ¹²⁵I-Lym-1 was observed after administration of PEG-interleukin-2 (PEG-IL-2). The macrocycle 1,4,8,11-tetraazacylcotetradecane-N,N′,N′,N'''-tetraacetic acid (TETA), synthesized specifically for copper chelation, has been conjugated to Lym-1 for ⁶⁷Cu labeling of the monoclonal antibody (MoAb). There was no evidence for bone or normal marrow uptake and the residence time in the tumor was prolonged. Surprisingly, a dose of 4.4 mCi that was intended for imaging induced substantial tumor regression in a patient.     

Carboxyester hydrolysis promoted by Cu(II) complexes of pyridyl-amine carboxylate-pendant ligands

Multidentate ligands containing tripodal pyridyl-amine moieties tethered to a carboxylate group by alkyl linkers of varying lengths were synthesized to obtain a series of water-soluble ligands to elucidate the effects of the differing coordination environments on the properties of the resulting metal complexes. These new, water-soluble ligands, [bis-(2-pyridin-2-yl-ethyl)-amino]-acetic acid (L¹), 3-[bis-(2-pyridin-2-yl-ethyl)-amino]-propionic acid (L²), 4-[bis-(2-pyridin-2-yl-ethyl)-amino]-butyric acid (L³), and 6-[bis-(2-pyridin-2-yl-ethyl)-amino]-hexanoic acid (L⁴), were treated with copper(II) perchlorate hexahydrate to yield the corresponding Cu(II) complexes, which have all been characterized by X-ray crystallography. L¹ binds Cu(II) to form the tetrameric complex {[Cu(μ-¹)][ClO₄] · 4H₂O}₄ (1) in the solid state, whereas the Cu(II) complexes of ligands L²-L⁴ form long-chain one-dimensional polymeric complexes {[Cu(μ-L²)][ClO₄] · H₂O}_n (2), {[Cu(μ-L³)][ClO₄] · H₂O}_n (3), and {[Cu(μ-L²)][ClO₄]  · H₂O}_n (4), respectively, in the solid state. Complexes 1-4 dissolved in 10% (v/v) CH₃CN aqueous solution were tested for their ability to promote the hydrolysis of the activated ester compound 4-nitrophenylacetate (NA), with 3 being the most active complex and 1 being the least active, possibly due to differences in the ability of the carboxylate moiety to act as either a general base or a nucleophile in the hydrolysis of NA as dictated by the tether length. The pK_a values of the copper-bound aquo ligands in solution were measured by spectrophotometric titration.     

Chlorzoxazone esters of some non-steroidal anti-inflammatory (NSAI) carboxylic acids as mutual prodrugs: Design,synthesis, pharmacological investigations and docking studies

The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. New chlorzoxazone ester prodrugs (6–8) of some acidic NSAIDs (1–3) were designed, synthesized and evaluated as mutual prodrugs with the aim of improving the therapeutic potency and retard the adverse effects of gastrointestinal origin. The structure of the synthesized mutual ester prodrugs (6–8) were confirmed by IR, ¹H NMR, mass spectroscopy (MS) and their purity was ascertained by TLC and elemental analyses. In vitro chemical stability revealed that the synthesized ester prodrugs (6–8) are chemically stable in hydrochloric acid buffer pH 1.2 as a non-enzymatic simulated gastric fluid (SGF) and in phosphate buffer pH 7.4 as non-enzymatic simulated intestinal fluid (SIF). In 80% human plasma, the mutual prodrugs were found to be susceptible to enzymatic hydrolysis at relatively faster rate (t_1/2 ≈ 37 and 34 min for prodrugs 6 and 7, respectively). Mutual ester prodrugs (6–8) were evaluated for their anti-inflammatory and muscle relaxation activities. Scanning electromicrographs of the stomach showed that the ester prodrugs induced very little irritancy in the gastric mucosa of rats after oral administration for 4 days. In addition, docking of the mutual ester prodrugs (6–8) into COX-2 active site was conducted in order to predict the affinity and orientation of these prodrugs at the enzyme active site.     

Novel Cu(II)Cd(II) macrocyclic complex that hydrolyzes an activated phosphate diester

The dinucleating macrocyclic ligand 3,6,9,17,20,23-hexaaza-29,30-dihydroxy-13,27-dimethyl-tricyclo[23,3,1,1^11,15] triaconta-1(28),11,13,15(30),25,26,-hexaene(BDBPH) was synthesized from [2+2] condensation between a diethylenetriamine lead(II) mononuclear complex and diformal-p-cresol. By stepwise synthesis, the heterodinuclear Cu(II)Cd(II) complex was obtained. The single crystal was triclinic, space group P1, with cell constants a=13.2675(10) Å, b=16.4655(13) Å, c=17.9502(14) Å, α=87.78(10), β=68.69(10), γ=74.81(10), V=3517.6(3) Å and Z=4. Potentiometric titration reveals that Cu(II)Cd(II)BDBPHOH species dominate in basic solution from pH 7 to 12. In the hydrolysis of BNP, this dinuclear complex can provide both Lewis acid and base sites to an active phosphate diester in which nucleophilic OH⁻ attacks the substrate to fulfill the hydrolysis cycle. In this system the synergic two functional groups in one catalyst molecule exerted remarkable catalytic activity towards hydrolysis of BNP.