Structure-function relationship of phenolic antioxidants in topical skin health products.

OBJECTIVES: The present work analysed the antioxidative activity of phenol-based antioxidants using an electron spin resonance method to predict the activity and stability of these antioxidants in cosmetic products. METHODS: The antioxidative power (AP) method was chosen to measure both the capacity and kinetics of an antioxidative reaction by detecting the DPPH (diphenylpicrylhydrazyl) radical. The antioxidative capacity (wc ) relates to the amount of free radicals that can be reduced, whereas the antioxidative reactivity (tr ) relates to the reaction speed and offers a fingerprinting of the redox state of the antioxidant molecules. Fifteen phenolic molecules have been analysed. They differed in the position of the hydroxyl groups and substituents on the aromatic ring. The AP of two distinct formulations containing hydroxytyrosol is presented as well as three phenol-based antioxidants within the same formulation vehicle. RESULTS: The rate at which phenol (ArOH) reacts with DPPH radicals, defined by the term reactivity (tr ) in this paper, was dependent upon the bond dissociation enthalpy (BDE) of the OH bond. Molecules having weak OH bonds and consequently low BDE values showed high antioxidant reactivity. On the other hand, the capacity factor (wc ), which is the concentration of phenol required to reduce a fixed concentration of DPPH radical, depends on the number and position of hydroxyl groups. The results showed that ortho and para positions of the two hydroxyl groups are important for higher capacity. If one of the two hydroxyl groups is blocked by methylation, both the antioxidative capacity and reactivity are reduced, mainly for ortho disubstituted compounds. The presence of a vinylic side chain improved reactivity and capacity tremendously. AP values may be useful in formulation design when identifying antioxidants that are likely to be physically and chemically stable. The importance of optimization of the formulation vehicle itself for a given antioxidant is also illustrated. CONCLUSION: Based on the presented findings, it is possible to predict the antioxidative performance of a phenol-based molecule and its stability and oxidation resistance within a cosmetic formulation. This is essential for antioxidant containing dermal formulations designed to combat skin ageing.

Structure-activity relationship for a series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles: potent subtype-selective inhibitors of N-methyl-D-aspartate (NMDA) receptors.

A series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles was synthesized as potential antagonists for the NR1A/2B subtype of N-methyl-D-aspartate (NMDA) receptors. Assayed by electrical recording under steady-state conditions, 7-hydroxy-2-(4-phenylbutyl)- 1,2,3,4-tetrahydropyrido-[3,4-b]indole (30) was the most potent compound in the series having an IC50 value of 50 nM at the NR1A/2B receptors.

Substituted 3beta-phenylethynyl derivatives of 3alpha-hydroxy-5alpha-pregnan-20-one: remarkably potent neuroactive steroid modulators of gamma-aminobutyric acidA receptors.

Neuroactive steroids are positive allosteric modulators of gamma-aminobutyric acidA (GABAA) receptor complexes. Synthetic modification generally does not increase neuroactive steroid potency beyond that of the naturally occurring progesterone metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-P). Recently, it has been shown that introduction of appropriately para-substituted phenylethynyl groups at the 3beta-position of 5beta steroids increases receptor potency. The present report presents the synthesis and pharmacological profile of an analogous series of 5alpha steroids. The most striking feature of this series is the further enhancement of in vitro and in vivo potency obtained. In particular, 3beta-(p-acetylphenylethynyl)-3alpha-hydroxy-5alpha-pr egnan-20-one (Co 152791) was 11-, 16- and 49-fold more potent than 3alpha, 5alpha-P in modulating the binding of [35S]TBPS, [3H]flunitrazepam and [3H]muscimol, respectively, in rat brain membranes (Co 152791 IC50 or EC50 = 2-7.5 nM). Similarly, Co 152791 was 3- to 20-fold more potent than 3alpha,5alpha-P as an inhibitor of [35S]TBPS binding in human recombinant receptor combinations containing alpha1, alpha2, alpha3 or alpha5 and beta2gamma2L subunits (Co 152791 IC50 1.4-5.7 nM). Co 152791 displayed low efficacy and 3alpha,5alpha-P had low potency at alpha4/6beta3gamma2L GABAA receptor complexes. Interestingly, Co 152791 demonstrated remarkable potency as a potentiator of GABA-evoked currents in Xenopus oocytes expressing alpha1beta2gamma2L receptors (EC50 0.87 nM), being 184-fold more potent than 3alpha,5alpha-P. High in vitro potency was also reflected in enhanced in vivo activity in that Co 152791 exhibited exceptional anticonvulsant potency, protecting mice from pentylenetetrazol-induced seizures at a approximately 5-fold lower dose than 3alpha,5alpha-P after i.p. administration (Co 152791 ED50 0.6 mg/kg). Moreover, Co 152791 was orally active (ED50 1.1 mg/kg) and exhibited a therapeutic index of 7 relative to rotorod impairment. The remarkable potency of Co 152791 as a positive allosteric modulator of GABAA receptors may be explained by its interaction with an auxiliary binding pocket in the neuroactive steroid binding site. In addition, modification at the 3beta-position probably hinders metabolism of the 3alpha-hydroxy group contributing to the exceptional anticonvulsant potency of this compound relative to other neuroactive steroids.

Anxiolytic and anticonvulsant activity of a synthetic neuroactive steroid Co 3-0593.

Endogeneously occurring neuroactive steroids, metabolites of progesterone and deoxycorticosterone, have been shown previously to interact with the GABAA receptor with great specificity in vitro and to have anticonvulsant, anxiolytic and sedative activity in vivo. However, these endogenously occurring steroids are not useful as therapeutic agents due to their potential metabolism to hormonally active steroids and their poor oral bioavailability. In an attempt to develop therapeutic agents which would maintain the pharmacological profiles of endogeneous neuroactive steroids but with increased oral bioavailability and reduced metabolic liability, we explored simple substitutions at the 3 beta-position of the endogenous neuroactive steroid, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P). This report describes part of the in vitro and in vivo pharmacological profile of a 3 beta-substituted analog, 3 beta-ethenyl-3 alpha-hydroxy-5 alpha-pregnan-20-one (Co 3-0593). The compound exhibited anticonvulsant activity against pentylenetrazol-induced seizures in mice and rats (ED50 = 5.6 and 11.5 mg/kg, i.p., respectively). Co 3-0593 showed robust anxiolytic effects, comparable to benzodiazepines in the Geller-Seifter test after both SC and oral administration. Furthermore, the anxiolytic activity was maintained after chronic administration suggesting an absence of tolerance. The compound did not affect the acquisition of a learned response at both anticonvulsant and anxiolytic doses. However, at higher doses the compound showed rotorod deficit which was further enhanced by ethanol. In summary, 3 beta-ethenyl-substituted 3 alpha, 5 alpha-P appeared to maintain the pharmacological activities of the endogenous neuroactive steroid with apparent oral activity.

Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.

Two naturally occurring metabolites of progesterone, 3 alpha-hydroxy-5 alpha- and 5 beta-pregnan-20-one (1 and 2), are potent allosteric modulators of the GABAA receptor. Their therapeutic potential as anxiolytics, anticonvulsants, and sedative/hypnotics is limited by rapid metabolism. To avoid these shortcomings, a series of 3 beta-substituted derivatives of 1 and 2 was prepared. Small lipophilic groups generally maintain potency in both the 5 alpha- and 5 beta-series as determined by inhibition of [35S]TBPS binding. In the 5 alpha-series, 3 beta-ethyl, -propyl, -trifluoromethyl and -(benzyloxy)methyl, as well as substituents of the form 3 beta-XCH2, where X is Cl, Br, or I or contains unsaturation, show limited efficacy in inhibiting [35S]TBPS binding. In the 5 beta-series, the unsubstituted parent 2 is a two-component inhibitor, whereas all of the 3 beta-substituted derivatives of 2 inhibit TBPS via a single class of binding sites. In addition, all of the 3-substituted 5 beta-sterols tested are full inhibitors of [35S]TBPS binding. Electrophysiological measurements using alpha 1 beta 2 gamma 2L receptors expressed in oocytes show that 3 beta-methyl- and 3 beta-(azidomethyl)-3 alpha-hydroxy-5 alpha-pregnan-20-one (6 and 22, respectively) are potent full efficacy modulators and that 3 alpha-hydroxy-3 beta-(trifluoromethyl)-5 alpha-pregnan -20-one (24) is a low-efficacy modulator, confirming the results obtained from [35S]TBPS binding. These results indicate that modification of the 3 beta-position in 1 and 2 maintains activity at the neuroactive steroid site on the GABAA receptor. In animal studies, compound 6 (CCD 1042) is an orally active anticonvulsant, while the naturally occurring progesterone metabolites 1 and 2 are inactive when administered orally, suggesting that 3 beta-substitution slows metabolism of the 3-hydroxyl, resulting in orally bioavailable steroid modulators of the GABAA receptor.

3 alpha-Hydroxy-3 beta-(phenylethynyl)-5 beta-pregnan-20-ones: synthesis and pharmacological activity of neuroactive steroids with high affinity for GABAA receptors.

Neuroactive steroids that allosterically modulate GABAA receptors have potential uses as anticonvulsants, anxiolytics, and sedative-hypnotic agents. Recently, a series of pregnanes substituted with simple alkyl groups at the 3 beta-position were synthesized and found to be active in vitro. The present report describes the synthesis of a series of substituted 3 alpha-hydroxy-3 beta-(phenylethynyl)pregnan-20-ones and their in vitro structure-activity relationship determined by their potency for inhibition of [35S]TBPS binding in rat brain membranes. Appropriate substitution of the phenyl group results in ligands with particularly high affinity for the neuroactive steroid site on GABAA receptors (e.g., 4-acetyl 28, IC50 10 nM). The potency of selected steroids was confirmed electrophysiologically in oocytes expressing cloned human GABAA alpha 1 beta 2 gamma 2L receptors (e.g., compound 28, EC50 6.6 nM). Consistent with their in vitro activity, some of the 3 beta-(phenylethynyl)-substituted steroids displayed anticonvulsant activity in the pentylenetetrazol (PTZ) and maximal electroshock (MES) tests following ip administration in mice. Notably, the 3 beta-[(4-acetylphenyl)ethynyl]-19-nor derivative 36 demonstrated an attractive anticonvulsant profile (PTZ and MES ED50 values of 2.8 and 9.2 mg/kg, respectively). A new pharmacophore for the neuroactive steroid site of GABAA receptors is proposed based upon the high affinity of certain substituted 3 beta-(phenylethynyl) steroids.

In vitro and in vivo activity of 16,17-dehydro-epipregnanolones: 17,20-bond torsional energy analysis and D-ring conformation.

PURPOSE: Certain neuroactive pregnane steroids (also known as "epalons") are allosteric modulators of the GABA, receptor and have been shown to be potent anticonvulsants, anxiolytics, sedative/hypnotics, and anesthetic agents. The purpose of this study was to calculate the structural consequences of introduction of a double bond in the 16,17-position and to determine if this modification would selectively reduce sedative activity, but maintain the potent anticonvulsant activity of neuroactive steroids. METHODS: We have studied the biochemical and behavioral effects of introducing a 16,17 double bond into the naturally occurring neuroactive steroids, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) and three synthetic neuroactive steroid derivatives, 3 alpha-hydroxy-3 beta-methyl-5 alpha-pregnan-20-one (3 alpha,3 beta Me,5 alpha-P), 3 alpha-hydroxy-5 alpha-androstane (3 alpha, 5 alpha-A), and alphaxalone (3 alpha,5 alpha-11-one-P). RESULTS: The 16-ene analogs of most of these neuroactive steroids were found to be 7- and 16-fold less potent in inhibiting [35S]TBPS binding to GABAA receptors and in a similar fashion, had reduced anticonvulsant and sedative potency in proportional amounts. The exception was the androstane (3 alpha,5 alpha-A) without a 17-acetyl group, that had virtually identical IC50 and ED50 values for the saturated and unsaturated derivatives. Calculation of the torsional energy profile for each of the 17-acetyl side chain conformations showed that the conformational energy minima found in the alpha,beta-unsaturated keto systems, produce an orientation of the 20-keto group that is rotated by 165 degrees when compared to the non-conjugated acetyl group (determined by X-ray crystallography and its minimum energy conformation). CONCLUSIONS: The modified orientation of the 20-keto group of neuroactive steroids containing a 16-ene, provides an explanation for their decreased biological activity overall, but did not lead to an enhanced protective index.