Design and synthesis of potential new apoptosis agents: hybrid compounds containing perillyl alcohol and new constrained retinoids

Novel retinoids 1-3 containing perillyl alcohol were synthesized through the addition of perillyl alcohol to the activated carboxylic acids (retinoids) promoted by DCC (N,N′-dicyclohexyl carbodiimide). A set of structurally and functionally diverse perillyl alcohol derivatives of retinoids were obtained in good yields (78-82%). Biological evaluation of these novel hybrid compounds (containing retinoids and perillyl alcohol) is currently underway in our laboratory.     

Qualitative and quantitative liquid chromatographic determination of natural retinoids in biological samples.

Liquid chromatography continues to be the preferred method for determining retinoids in biological samples. The highly unstable nature of retinoids and the real possibility of artifacts or erroneous results have led to the development of rapid and highly automated protocols for retinoid extraction, separation and detection. Due to strong light absorbance in the ultraviolet region, UV detectors still predominate although mass spectrometric detection is gaining increased popularity. This paper reviews recent advances and provides major guidelines for using liquid chromatography to identify and quantify retinoids in biological samples.     

Design and synthesis of guanidine-containing novel retinoids

Guanidine-containing new retinoids 1–4 were synthesized through the addition of guanidine to the activated carboxylic acids (retinoids) promoted by carbonyldiimidazole (CDI). A set of structurally and functionally diverse guanidine derivatives of retinoids were obtained in high yields (78–82%). We are in the process of studying the biological effect of these molecules on retinoic acid signaling pathways.     

Design and synthesis of α-aminonitrile-functionalized novel retinoids

We have developed an expedient one-pot method for the synthesis of α-aminonitrile-functionalized new retinoids via a three-component condensation of β-cyclocitral, amine, and TMSCN (trimethyl silylcyanide) in the presence of a catalytic amount of indium(III) chloride in water. The reactions proceeded smoothly at room temperature in water to generate the corresponding retinoids in moderate to excellent yields (85–92%). In addition, the utility of this reaction was demonstrated to synthesize boron-containing retinoids.     

Linear dichroism study of retinoids

Linear dichroism spectra of several retinoids and related polyenes incorporated in stretched polyethylene films were determined. It is suggested that the retinoids are oriented with the plane of the ring parallel to the stretching direction of the film, the long polyene chain being displaced from that direction.     

Delivery of 9-Cis retinal to photoreceptors from bovine serum albumin

Retinoids are labile compounds with less than micromolar solubility in aqueous solutions. They are normally associated with a carrier protein, interphotoreceptor retinoid binding protein, in the interstitial spaces of the retina. In the past, experimental retinoids have been delivered to isolated retinas and photoreceptors using either unprotected ethanolic suspensions or phospholipid vesicles that protect but require burdensome preparation. The objective of these experiments was to characterize the protection of retinoids and their delivery to isolated photoreceptor cells at high concentrations using fatty-acid-free bovine serum albumin (BSA) as a convenient vehicle and protectant. Our results confirm that BSA is a useful protectant and vehicle for delivery of retinoids to isolated retinas and photoreceptors.     

Structural basis for engineering of retinoic acid receptor isotype-selective agonists and antagonists.

BACKGROUND: Many synthetic retinoids have been generated that exhibit a distinct pattern of agonist/antagonist activities with the three retinoic acid receptors (RARalpha, RARbeta and RARgamma). Because these retinoids are selective tools with which to dissect the pleiotropic functions of the natural pan-agonist, retinoic acid, and might constitute new therapeutic drugs, we have determined the structural basis of their receptor specificity and compared their activities in animal and yeast cells. RESULTS: There are only three divergent amino acid residues in the ligand binding pockets (LBPs) of RARalpha, RARbeta and RARgamma. We demonstrate here that the ability of monospecific (class I) retinoid agonists and antagonists to bind to and induce or inhibit transactivation by a given isotype is directly linked to the nature of these residues. The agonist/antagonist potential of class II retinoids, which bind to all three RARs but depending on the RAR isotype have the potential to act as agonists or antagonists, was also largely determined by the three divergent LBP residues. These mutational studies were complemented by modelling, on the basis of the three-dimensional structures of the RAR ligand-binding domains, and a comparison of the retinoid agonist/antagonist activities in animal and yeast cells. CONCLUSIONS: Our results reveal the rational basis of RAR isotype selectivity, explain the existence of class I and II retinoids, and provide a structural concept of ligand-mediated antagonism. Interestingly, the agonist/antagonist characteristics of retinoids are not conserved in yeast cells, suggesting that yeast co-regulators interact with RARs in a different way than the animal cell homologues do.     

Spectral properties of topical retinoids prevent DNA damage and apoptosis after acute UV-B exposure in hairless mice

We showed in a recent study that topical retinyl palmitate prevented UV-B-induced DNA damage and erythema in humans. Given that retinyl palmitate is a precursor of retinoic acid, the biological form of vitamin A that acts through nuclear receptors, we wondered whether these protective effects toward UV-B exposure were either receptor dependent or linked to other properties of the retinoid molecule such as its spectral properties. We determined the epidermal retinoid profile induced by topical retinoic acid in hairless mice and analyzed its effect on markers of DNA photodamage (thymine dimers) and apoptosis following acute UV-B exposure; we compared these effects to those induced by other natural topical retinoids (retinaldehyde, retinol and retinyl palmitate) which do not directly activate the retinoid receptors. We then analyzed the direct action of these retinoids on UV-B-induced DNA damage and apoptosis in cultured A431 keratinocytes. Topical retinoic acid significantly decreased (approximately 50%) the number of apoptotic cells, as well as the formation of thymine dimers in the epidermis of mice exposed to acute UV-B. Interestingly, the other topical retinoids decreased apoptosis and DNA damage in a similar way. On the other hand, neither retinoic acid nor the other retinoids interfered with the apoptotic process in A431 keratinocytes exposed to UV-B, whereas DNA photodamage was slightly decreased. We conclude that the decrease of apoptotic cells in hairless mouse epidermis following topical retinoids and UV-B irradiation reflects a protection of the primary targets of UV-B (DNA) by a mechanism independent of the activation of retinoid nuclear receptors, rather than a direct inhibition of apoptosis.     

Detection of some retinoid radicals using high-performance liquid chromatography with electron spin resonance spectroscopy or electrochemical detection

Radical species were detected in the incubation mixtures of some retinoids (retinoic acid, retinal, retinol and retinyl acetate) by using the spin-trapping technique. The spin-adducts were resolved by high-performance liquid chromatography on a reversed-phase column with isocratic elution and detected by electron spin resonance spectroscopy and electrochemical detection. The spin-adducts were eluted in the order retinoic acid, retinol, retinyl acetate, in a similar manner to the retinoids themselves. These results suggest that the spin-adducts are products of nitrosobenzene with retinoid radicals in which the retinoid radicals retain their original chemical structures.     

A Comparison of the Retention Behaviour of Vitamin A and Some Metabolites Eluted from Four Different Reversed Phase High Performance Liquid Chromatographic Stationary Phases

Five retinoids, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, all-trans-retinol and 13-cis-retinal were isocratically separated from four different reversed phase high performance liquid chromatographic stationary phases. By taking advantage of the different retention mechanisms, present between the stationary phases and the analytes, the retinoids were separated with different elution orders using the same mobile phase composition. Two of the stationary phases appeared to have more possibilities to interact with the analytes than the usual hydrophobic interactions. The stationary phase with embedded polar groups showed hydrogen bonding properties and the calix[4]arene based stationary phase showed possibilities to form inclusion complexes with the analytes. These additional interactions appeared to benefit the separations of the analytes. This publication shows the benefits by isocratically separate retinoids employing other stationary phases than the conventional C₁₈ stationary phase.     

Topical delivery of retinoids counteracts the UVB-induced epidermal vitamin A depletion in hairless mouse

UVB irradiation depletes all-trans-retinol (ROL) and all-trans-retinyl esters (RE) from the hairless mouse epidermis. Prevention of this may be of relevance in counter-acting the long-term side effects of UVB exposure. We studied the effects of a topical treatment with natural retinoids before and after UVB exposure on three parameters involved in vitamin A metabolism: the amount of epidermal ROL and RE, the level of functional cellular retinol-binding protein I (CRBP-I), which is likely to protect ROL from UVB, as well as the cytosolic and microsomal enzyme activities which generate ROL and RE, i.e. all-trans-retinaldehyde (RAL) reductase, acylCoA:retinol acyltransferase (ARAT) and retinyl-ester hydrolase (REH). Topical pretreatment with retinoids promoted a dramatic increase of epidermal ROL, RE and CRBP-I levels, a transient increase of RAL reductase and ARAT activities as well as a decreased activity of REH, indicating a direction of epidermal vitamin A metabolism toward storage. In untreated mice UVB irradiation induced a depletion of epidermal ROL and RE in 10 min and a 50% decrease of CRBP-I after 24 h. In mice treated with topical retinoids, and then exposed to UVB, epidermal RE levels were higher than in vehicle-treated, nonirradiated mice. In contrast, ROL was as much depleted after UVB in pretreated as in untreated animals in spite of an induction of CRBP-I, indicating that CRBP-I does not actually protect ROL from UVB-induced depletion in this model. However, the reconstitution of both epidermal ROL and RE, after their depletion induced by UVB, was accelerated by previous topical treatment with RAL. Our results indicate that topical delivery of retinoids partly counteracts UVB-induced vitamin A depletion and promotes recovery.     

Synthesis of 11‐cis‐Retinoids by Hydrosilylation–Protodesilylation of an 11,12‐Didehydro Precursor: Easy Access to 11‐ and 12‐Mono‐ and 11,12‐Dideuteroretinoids

An expeditious, highly efficient approach to 11‐cis‐retinoids was achieved by semihydrogenation of a readily available 11‐yne precursor through a hydrosilylation–protodesilylation protocol. The complete chemo‐, regio‐, and syn‐stereoselectivity of the method also allowed direct access to 11‐ and 12‐monodeutero‐, and 11,12‐dideutero‐11‐cis‐retinoids. The analogous trans series was not accessible by this route, and was synthesized by means of Hiyama coupling.     

Cross‐Coupling Reactions of Organosilicon Compounds in the Stereocontrolled Synthesis of Retinoids

This paper presents a full account of the use of Hiyama cross‐coupling reactions in a highly convergent approach to retinoids in which the key step is construction of the central C10? C11 bond. Representatives of two families of oxygen‐activated dienyl silanes (ethoxysilanes and silanols) and of all reported families of “safety‐catch” silanols (siletanes, silyl hydrides, allyl‐, benzyl‐, aryl‐, 2‐pyridyl‐ and 2‐thienylsilanes) were regio‐ and stereoselectively prepared and stereospecifically coupled to an appropriate electrophile by treatment with a palladium catalyst and a nucleophilic activator. Both all‐trans and 11‐cis‐retinoids, and their chain‐demethylated analogues, were obtained in good yields regardless of the geometry (E/Z) and of the steric congestion in each fragment. This comprehensive study conclusively establishes the Hiyama cross‐coupling reaction, with its mild reaction conditions and stable, easily prepared, ecologically advantageous silicon‐based coupling partners, as the most effective route to retinoids reported to date.     

Synthetic Retinoids: Structure–Activity Relationships

Retinoid signalling pathways are involved in numerous processes in cells, particularly those mediating differentiation and apoptosis. The endogenous ligands that bind to the retinoid receptors, namely all‐trans‐retinoic acid (ATRA) and 9‐cis‐retinoic acid, are prone to double‐bond isomerisation and to oxidation by metabolic enzymes, which can have significant and deleterious effects on their activities and selectivities. Many of these problems can be overcome through the use of synthetic retinoids, which are often much more stable, as well as being more active. Modification of their molecular structures can result in retinoids that act as antagonists, rather than agonists, or exhibit a large degree of selectivity for particular retinoid‐receptor isotypes. Several such selective retinoids are likely to be of value as pharmaceutical agents with reduced toxicities, particularly in cancer therapy, as reagents for controlling cell differentiation, and as tools for elucidating the precise roles that specific retinoid signalling pathways play within cells.     

A versatile synthesis of retinoids via condensation of the side-chain to cyclic ketones

A simple synthesis of ethyl 3,7-dimethyl-2,4,6-nonatriene-8-ynoates and their corresponding alcohols is presented; codensation with various cyclic ketones can lead to retinoids having modified ring structures.     

The Photochemistry of the Retinoids as Studied by Steady-State and Pulsed Methods

The retina and retinal pigment epithelium contain a number of retinoids in a metabolic pathway that eventually forms the visual pigments. This study investigates the photochemistry of those retinoids that may contribute to light-induced damage to the retina. These include retinal (RAL), retinol (ROL), retinylpalmitate (ROLpal) and the protonated Schiff-base of retinal (RAL.,). Their photochemistry was followed by both EPR spin-trapping techniques and the direct detection of singlet oxygen via its luminescence at 1270 nm. Irradiation (>300 nm) of RAL, ROL in methanol (MeOH) or RALpal in dimeth-ylformamide, produces free radicals from both solvents. Illumination of RAL., in MeOH containing NADH with light above 400 nm (and even above 455 nm) generates the superoxide radical. We also determined that the quantum yields for singlet oxygen sensitization by RAL, ROL or RALpal in MeOH are 0.05, 0.03 and 4.01, respectively. These values are at least 75% less than those previously found using chemical methods. These observations indicate that a major photochemical process for these retinoids may be an electron (or hydrogen) process that will lead to radical products, and that the singlet oxygen mechanism is of relatively minor importance in protic solvents. These results may explain the action spectra obtained from light-induced damage to the retina.     

Comparative studies on radical cation formation from carotenoids and retinoids

A comparative study of radical cation formation from selected polyenes, namely carotenoids (C₄₀) and retinoids (C₂₀), has been carried out by treatment with the Lewis acids BF₃ as its etherate or SbCl₃. The reaction in chloroform was monitored by vis/NIR and EPR spectroscopy at variable temperature. β,β-Carotene, β,β-caroten-4-ol, retinol (vitamin A), retinyl acetate and anhydroretinol were used as substrates. It is concluded that whereas BF₃-diethyl etherate or SbCl₃ is capable of effecting one-electron transfers to produce radical cations from the longer polyenes (carotenoids), no radical cations were obtained from the retinoids. The results of SbCl₃ treatment of the retinoids have a bearing on the current studies on the mechanism of the Carr-Price blue colour reaction previously used for quantitative analysis of vitamin A.     

Single-run analysis of isomers of retinoyl-beta-D-glucuronide and retinoic acid by reversed-phase high-performance liquid chromatography

Reversed-phase HPLC methods capable of separating several retinoic acid isomers are generally not designed for simultaneous analysis of isomers of other classes of retinoids. A reversed-phase HPLC method is presented which allows the separation of at least four retinoic acid (RA) isomers (13-cis-RA, 9,13-dicis-RA, 9-cis-RA, all-trans-RA)and of all isomers of retinoyl-beta-D-glucuronide (RAG), which have been observed in vivo (13-cis-RAG, 9-cis-RAG,all-trans-RAG). The recovery of retinoids was generally between 80 and 90%. Intra-day reproducibility (expressed as relative standard deviation) was less than or equal to 7%. As little as 0.25 ng of RA isomers and of all-trans-RAG could be detected. This method allowed the study of the metabolism of 9-cis-retinoids, where isomerization reactions play a predominant role.     

Quantification of retinoid concentrations in human serum and brain tumor tissues

Retinoic acid signaling is essential for central nervous system (CNS) differentiation and appears to be impaired in tumors. Thus far, there are no established methods to quantify relevant retinoids (all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis retinoic acid, and retinol) in human brain tumors. We developed a single step extraction and quantification procedure for polar and apolar retinoids in normal tissue, lipid-rich brain tumor tissues, and serum. This quantification procedure is based on high performance liquid chromatography (HPLC) with diode-array detection (DAD) using all-trans-acitretin as an internal standard and extraction by liquid–liquid partition with ethyl acetate and borate buffer at pH 9. Recovery with this extraction procedure was higher than earlier (two-step) liquid–liquid extraction procedures based on hexane, NaOH, and HCl. The overall quantification procedure was validated according to Food and Drug Administration (FDA) guidelines and fulfilled all criteria of accuracy, precision, selectivity, recovery, and stability. The overall method accuracy varied between −5.6% and +5.4% for serum and −3.8% and +6.2% for tissues, and overall precision ranged from 3.1% to 6.9% for serum and 2.1% to 8.3% for tissues (%CV batch-to-batch). The lower limit of quantification for all compounds in tumor tissue (and serum) was 3.9 ng g⁻¹ (ng mL⁻¹). Using this assay, photodegradation of the retinoids was evaluated and endogenous polar and apolar retinoids were quantified in sera and brain tumor tissues of patients and compared with serum and tonsil tissue concentrations of controls. It may thus serve as a suitable method for the characterization of retinoid uptake and metabolism in the respective compartments.     

Solution-phase synthesis of diaryl selenides using polymer-supported borohydride

[reaction: see text] A new series of selenium-containing diaryl retinoids have been prepared by a new direct nickel(II)-catalyzed coupling of a diselenide with an iodoaryl in the presence of polymer-supported borohydride.