Synthesis, structure-affinity relationships, and biological activities of ligands binding to retinoic acid receptor subtypes.

The retinoic acid receptors (RARs) transduce retinoid dependant gene regulation, and many biological effects of retinoids are mediated through binding and activation of three closely related receptor subtypes (RAR alpha, RAR beta, and RAR gamma). In order to investigate the role of receptor subtypes, we have carried out a chemical synthesis program to seek selective retinoids for these receptors. We measured receptor binding affinity using recombinant RAR alpha, -beta, and -gamma proteins and assessed cellular differentiating activity in F9 murine teratocarcinoma cells (F9 cells). This research has identified the 4-substituted-3-(1-adamantyl)phenyl moiety as a new pharmacophore which can replace the beta-cyclogeranylidene ring of the naturally occurring all-trans-retinoic acid. Two chemical series derived from the general structures 6-(3-tertioalkylphenyl)-2-naphthoic acid (series I) and 4-[(E)-2-(3-tertioalkylphenyl)propenyl]benzoic acid (series II) were developed. In particular, we have obtained the RAR gamma selective derivatives 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthoic acid (7) [Ki(RAR alpha) = 6500 nM, Ki(RAR beta) = 2480 nM, Ki(RAR gamma) = 77 nM] and 4-[(E)-2-[3-(1-adamantyl)-4-hydroxyphenyl]propenyl]benzoic acid (19) [Ki(RAR alpha) = 1,144 nM, Ki(RAR beta) = 1245 nM, Ki(RAR gamma) = 53 nM]. In series I, the presence of a phenol group, irrespective of the nature of tertioalkyl group, imparted at least partial RAR gamma selectivity, whereas in series II, the presence of both adamantyl and phenol groups is needed to confer RAR gamma selectivity. The RAR gamma selective ligands induce differentiation in F9 cells (7, AC50 = 33 nM; 19, AC50 = 66 nM). From series I, a mixed RAR beta-gamma agonist with potent cellular differentiating activity was selected for development as a topical antiacne agent, 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid (5, CD 271) [Ki(RAR alpha) = 1100 nM, Ki-(RAR beta) = 34 nM, Ki(RAR gamma) = 130 nM, AC50(F9) = 37 nM]. Finally, from series II, we have obtained a weak antagonist in the F9 cellular differentiation assay, 4-[(E)-2-(3-tert-butyl-4-hydroxyphenyl)propenyl]benzoic acid (15, IC50 = 700 nM).

A new, ring closing metathesis-based synthesis of (-)-fumagillol.

[reaction: see text]. A new strategy to access the fumagillin/fumagillol skeleton is proposed. An Evans aldolization and a RCM involving an enone are used for the preparation of a key cyclohexanone intermediate, which was readily converted to fumagillol. The synthesis also features an efficient preparation of isogeraniol and isogeranic acid.

Typhoid perforation of the intestine.

We reviewed 91 cases of intestinal perforation complicating typhoid fever treated at a rural hospital in Haiti over a ten-year period. Surgical management involved simple primary closure of the perforation (80 patients), small-bowel resection with anastomosis (two patients), simple drainage of the peritoneal cavity (two patients), and serosal patching of the perforation (one patient). Six patients died before surgery. The mortality was 30.8% for all 91 cases but 21.2% for those treated with primary closure of the perforation. We also reviewed the literature pertaining to the management of intestinal perforation complicating typhoid fever.

On the interaction between anthralin and DNA: a revision.

The interaction between anthralin and DNA in vitro was examined. According to our data, there is no evidence for a specific interaction between these substances. However, we found that the addition of small quantities of DNA or albumin significantly enhanced the stability of aqueous solutions of anthralin and markedly affected the rate of anthralin decomposition.

New acyclic analogues of lipid A: synthesis of 4-phosphonoxybutyl and 3-phosphonoxypropyl glycosides of 2-amino-2-deoxy-D-glucose.

Several analogues of lipid A have been synthesized, in which the reducing monosaccharide moiety of the parent molecule has been replaced by an acyclic spacer. The new compounds show high endotoxic activity and are able to protect neutropenic mice against pseudomonas infection, two properties characteristic of LPS-like molecules.

Use of the chick embryo for pharmacological screening of retinoids.

Retinoic acid and some selected analogs were tested to evaluate their effect on skin morphogenesis and toxicity in the chick embryo. Retinoids dissolved in dimethyl sulfoxide were injected at doses varying from 10 pmol to 10 mumol into the amniotic cavity of 10-day-old chick embryos (n = 20). At 16 days of incubation, the eggs were opened to record the number of dead embryos and the number of embryos presenting club-shaped feathers. A lethal embryotoxic dose (LED50d16) which, at 16 days of incubation, provokes the death of 50% of the embryos and an effective dose (ED50) which induces production of club-shaped feathers in 50% of surviving embryos were then calculated using log-probit analysis. Retinoids could be classified according to their ED50. For example, arotinoid Ro 13-7410 appears approximately 1,000 times more active than all transretinoic acid. However, the analogs which are more active are also more toxic. The assay described in the present study appears to be a simple and useful model for the screening of retinoids.

A rapid and simple test system for the evaluation of the inhibitory activity of retinoids on induced ornithine decarboxylase activity in the hairless rat epidermis.

In mouse skin, antiproliferative agents including retinoids inhibit induction of ornithine decarboxylase activity by a variety of hyperproliferative stimuli. In the hairless rat skin, ornithine decarboxylase activity was induced by ten successive strippings with cellotape and by topical application of 12-O-tetradecanoyl-phorbol-13-acetate. Topical application of all trans-retinoic acid (25 nmol/cm2) immediately after the tape stripping of the skin significantly inhibited the induction of ornithine decarboxylase activity at all time points measured. The inhibition by all trans-retinoic acid of ornithine decarboxylase induced by cellotape stripping was dose dependent as was found to be the case for arotinoid, retinol, Ro-10-1670, motretinid, 13-cis-retinoic acid, etretinate, and vitamin A. Oral administration of all trans-retinoic acid also inhibited the ornithine decarboxylase activity induced by cellotape stripping. We propose the assay of ornithine decarboxylase activity in the hairless rat epidermis after tape stripping for a rapid evaluation of new retinoids.

Bioassays for retinoic acid-like substances using cultured human keratinocytes.

Using cultured human keratinocytes, three bioassays for retinoic acid-like substances have been developed. They are based on the ability of these substances (1) to inhibit cross-linked envelope formation, (2) to inhibit the synthesis of the K1 keratin, and (3) to stimulate the synthesis of the K19 keratin. The data, expressed as 50% inhibitory or activating concentrations were used to rank compounds according to their activity.

An improved assay procedure and a new chemically stable ligand for cytosolic retinoic acid binding protein.

A high through-put method to assay for binding of retinoids to their cytosolic binding protein is described. The protein-bound retinoid is quantified following its complete separation from the free ligand by a single gel filtration chromatography step on Sephadex G-25 columns. The method allows a single person to process up to 100 samples per day. A new, substituted benzo[b]thiophene carboxylic acid derivative (tritiated) is proposed as an alternative ligand for cytosolic retinoic acid binding protein. This new analog (CD270), which contains no olefinic double bonds, is characterized by its chemical stability to light and atmospheric oxidation. This molecule shows binding characteristics similar to those of retinoic acid, both in terms of binding affinity and binding specificity (Kd of about 2 nM), and offers the additional advantage that the unbound molecule is absorbed on Sephadex G-25, while the protein-bound ligand is unaffected by the carbohydrate gel matrix. When the affinities of retinoic acid and several analogs were determined by competition binding experiments, similar results were obtained when either tritiated retinoic acid or tritiated CD270 were used as the labeled ligand. The tritiated, heterocyclic retinoic acid analog, CD270, is thus proposed as an alternative ligand for cytosolic retinoic acid binding protein.

Conformationally constrained NO synthase inhibitors: rigid analogs of L-N-iminoethylornithine.

The synthesis of eight rigid analogs of L-N-iminoethylornithine (L-NIO) is described. The compounds have been evaluated for their inhibition of inducible nitric oxide synthase. Preliminary structure-activity relationships are discussed.

New synthetic analogs of lipid A as lipopolysaccharide agonists or antagonists of B lymphocyte activation.

We have studied the ability of synthetic analogs of lipid A to mimic lipopolysaccharide (LPS) for activation of 70Z/3 pre-B cells (expression of surface Igs) or to antagonize this effect. The results indicate that the presence of glucosamine (mono- or disaccharide) as a 'backbone' for the attachment of fatty acids is not necessary for activation of cells of the B lineage. Phosphate groups are not necessary either. Other structural features such as the configuration of particular asymmetric carbons, and the distance between an anionic group and an N-acyl chain, seem to be much more critical parameters for activation of B cells. Among the synthetic lipids which were unable to activate 70Z/3 cells, one compound, consisting of N,N-acylated and bisphosphorylated 2,3-dideoxy-2,3-diamino-D-glucose, behaved as a specific LPS antagonist and blocked also the activation triggered by the other synthetic inducers. The influence of the synthetic lipids on the entry of mature mouse B lymphocytes into the G1A phase of the cell cycle (cell enlargement) was also investigated. A high correlation was observed between the potency to activate pre-B cells and the ability to induce blast formation in mature B cells.