R.J. Griffin

Structural studies on bio-active compounds. Part XV. Structure-activity relationships for pyrimethamine and a series of diaminopyrimidine analogues versus bacterial dihydrofolate reductase

The phenylpyrimidine derivative pyrimethamine and its congeners inhibit the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and are of interest as antiproliferative agents. In this study the equilibrium conformations of some pyrimethamine derivatives, and their interactions with Escherichia coli dihydrofolate reductase, were investigated using molecular modelling techniques. In each case the phenyl ring avoided coplanarity with the pyrimidine ring and attained a position approximately perpendicular to it, in agreement with crystal structures. A meta substituent could be placed either side of the pyrimidine plane, forming two non-equivalent, slowly interconverting solution conformations. Except for meta-azidopyrimethamine, both conformations of all the inhibitors were able to bind to the active site cleft of the enzyme with the diaminopyrimidine moiety, making the normal pattern of enzyme/inhibitor hydrogen bonds. One such conformation of the meta-azido compound failed to bind because of unacceptable steric clashes, whilst the other showed enhanced binding energy attributable to the occupation of a hydrophobic pocket by the azido group. The enhanced binding of 2,4-diamino-6-ethyl-5-phenylpyrimidine over its 6-methyl analogue was also related to attractive hydrophobic interactions.

Direct 19F NMR observation of the conformational selection of optically active rotamers of the antifolate compound fluoronitropyrimethamine bound to the enzyme dihydrofolate reductase.

The molecular basis of the binding of the lipophilic antifolate compound fluoronitropyrimethamine [2,4‐diamino‐5‐(4‐fluoro‐3‐nitrophenyl)‐6‐ethylpyrimidine] to its target enzyme dihydrofolate reductase has been investigated using a combination of 19F NMR spectroscopy and molecular mechanical calculations. 19F NMR reveals the presence of two different conformational states for the fluoronitropyrimethamine‐Lactobacillus casei enzyme complex. MM2 molecular mechanical calculations predict restricted rotation about the C5‐C1′ bond of the ligand and this gives rise to two slowly interconverting rotamers which are an enantiomeric pair. The results of 19F NMR spectroscopy reveal that both these isomers bind to the enzyme, with different affinities. There is no detectable interconversion of the bound rotamers themselves on the NMR timescale. The effect of the addition of co‐enzyme to the sample is to reverse the preference the enzyme has for each rotamer.

The percutaneous absorption of m-azidopyrimethamine: A soft antifolate for topical use

Abstract The in vitro transdermal transport of the soft dihydrofolate reductase inhibitor m-azidopyrimethamine (MZP), a potential antipsoriatic drug, has been studied from a range of aqueous propylene glycol vehicles. The determination of solubilities in the various vehicles used and the measurement of skin-vehicle and lipid-vehicle partition coefficients were also undertaken. Although permeation rates were slow, measurable fluxes across hairless mouse skin were observed which were dependent upon the composition of the vehicle. In nonbuffered aqueous propylene glycol the flux decreased with increasing propylene glycol while in buffered systems it increased as the pH was lowered. Here, the basic diaminopyrimidine is more fully protonated and suggests the involvement of the ionic form in transport. Oleic acid was the only member of a series of enhancers of percutaneous transport to significantly increase the flux of MZP. This effect is probably mediated by both ion-pair facilitation and by direct modification of epidermal barrier properties.

The reduction of aryl azides by dithiothreitol: a model for bioreduction of aromatic azido-substituted drugs☆

Abstract The relative rates of reduction of a series of simple phenyl azides by dithiothreitol (DTT) were studied and the results modelled using the Hammett equation. The rate of reduction was found to be dependent on electronic effects and the steric environment of the azido group, with electron-deficient aryl azides being reduced at a faster rate. These findings were extended to study the reduction rates of model azido-2,4-diaminopyrimidine drugs by DTT, and showed similar dependence on electronic and steric considerations. The findings have implications for the design of aromatic azido pro-drugs and “soft drugs”, from the point of view of bioreductive susceptibility.

Percutaneous absorption of azidoprofen, a model for a soft anti-inflammatory drug for topical application

The penetration of azidoprofen through excised hairless mouse skin was investigated. Formulation factors influencing skin permeation, such as pH, solute and cosolvent concentration were studied and were related to physico-chemical parameters including pKa, solubility and partition. In addition, the effect of a range of penetration enhancers on the transport of azidoprofen was also assessed. The flux of azidoprofen from solution formulations was dependent on vehicle pH and permeant concentration and was directly related to the degree of ionisation of the solute. Increased permeant concentration in the vehicle resulted in a greater level of saturation and, hence, a higher rate of penetration. In contrast, over the range used (0–30%), propylene glycol concentration in the vehicle had little effect. Flux from suspensions was independent of pH, since the level of unionised drug, the predominant diffusing species, was maintained at the intrinsic saturated solubility at all pH values. Pretreatment of the skin with a range of enhancers only moderately promoted permeation. Pretreatment with Azone in propylene glycol resulted in an increased flux with increasing pH and thus appeared to facilitate penetration of the ionised species.

The reduction of azidoprofen : a model for a soft anti-inflammatory drug for topical application

Abstract Azidoprofen ( RS -2-(4-azidophenyl)propionic acid) and a series of esters, together with the corresponding amino analogues, have been synthesised as a model anti-inflammatory agents susceptible to reductive bioinactivation. As monitored by 1 H-NMR and HPLC, azidoprofen undergoes a rapid, base-catalysed reduction by dithiothreitol (DTT) in vitro in aqueous solution at physiological temperature. This suggest that a thiol-mediated reduction, possibly induced by endogenous thiols acting as in vivo reducing agents, is a potential intracellular deactivation mechanism which may be utilised in the design of soft drugs. The conditions of the in vitro model with respect to DTT concentration and pH may be modified to facilitate kinetic modelling, and the base-dependence of the reaction supports the role of the nucleophilic thiolate anion (RS − ) as the predominant reducing species. In acqueous acetonitrile, reactions are significantly slower, oxidation of DTT becomes significant and anaerobic conditions are required for reliable rate estimates.

The hydrolysis of azidoprofen esters: A model for a soft anti-inflammatory drug for topical application

Abstract A series of ester prodrugs of azidoprofen was investigated for susceptibility towards in vitro enzymatic and chemical hydrolysis. In each case, the parent acid was regenerated with the rate and extent of the process being dependent on the nature of the ester moiety. All esters, with the exception of the glycolamide derivative, exhibited much greater lability towards enzyme-mediated hydrolysis compared to chemical decomposition. In particular, the tetrahydropyranylmethyl ester showed the largest difference in sensitivity towards these two processes. This specificity is an important requirement in prodrug design and indicates the capacity to undergo rapid metabolic activation while exhibiting stability under conditions encountered during formulation and storage. The ability of skin to activate these prodrugs was demonstrated by hydrolysis of the methyl ester of azidoprofen on incubation with a hairless mouse skin homogenate. In addition, when enzymatic hydrolysis was monitored in aqueous alcoholic systems competitive hydrolysis and enzyme-mediated transesterification were observed. The rate of both processes increased with a longer chain ester and transesterification was the predominant reaction. Eventually, the new ester also underwent enzyme-mediated hydrolysis to yield azidoprofen.

Uptake, cytotoxicity and metabolism of m-azidopyrimethamine and related lipophilic antifolates in SV-K14 human keratinocytes in vivo

The growth-inhibitory properties of a series of lipophilic diaminopyrimidine antifolates were evaluated in comparison with methotrexate (MTX) against SV-K14 human keratinocytes in vitro under folate-dependent and folate-independent conditions. Under folate-dependent conditions metoprine (DDMP) proved more cytotoxic than MTX, despite the greater inhibitory activity of the latter compound against mammalian dihydrofolate reductase (DHFR), possibly reflecting differences in cellular accumulation. The significantly lower activity of both compounds under folate-independent conditions indicated DHFR as the primary target. Pyrimethamine (PYM), m-azidopyrimethamine (MZP) and m-aminopyrimethamine (MAP), a metabolite of MZP, were approximately equiactive but less cytotoxic than MTX or DDMP. The unexpected activity of MAP, an inferior DHFR inhibitor, suggests differences in the mechanism of action or cellular transport of the drug, although the reduction of cytotoxicity observed under folate-independent conditions indicate folate metabolism as the cytotoxic locus. In contrast, the cytotoxicity of PYM or MZP was not reduced under folate-independent conditions implying an alternative mechanism of action. The uptake of 2-[14C]pyrimethamine by SV-K14 keratinocytes was rapid with steady-state intracellular concentrations being observed after approximately 100 min, partition of drug into the plasma membrane preceding redistribution and extensive accumulation within the particulate cell components. The previously reported NADPH-dependent metabolism of MZP to MAP by murine liver microsome preparations was not observed with SV-K14 keratinocytes nor with murine skin homogenates in the present study.

Regioselective deacylation of 2,4-diacylaminopyrimidine derivatives by Lewis acids and crystal structures of two products

Deacylation of 2,4-diacylamino derivatives of pyrimethamine and related diaminopyrimidines with tin(II) chloride or zinc chloride, in ethanol or propan-2-ol, affords 2-acyl-4-aminopyrimidines exclusively. Regioselective 4-deacylation was observed by 1H NMR spectroscopy and established by crystallographic analysis of the 2,4-dipropionylpyrimidine 11 and the corresponding 4-amino-2-propionylpyrimidine deacylation product 17. The latter exists in the solid state as an unusual base-pair dimer linked by two pairs of equivalent hydrogen bonds.