Synthesis of a carbocyclic sialic acid analogue for the inhibition of influenza virus neuraminidase.

The influenza virus neuraminidase (NA) is essential for viral infection and offers a potential target for antiviral drug development. We prepared a carbocyclic sialic acid analogue, potentially able to inhibit NA. Its structure is an analogue of the transition-state of the reaction catalysed by NA. As starting material, quinic acid was selected owing to its ready availability and its stereochemical feature suitable for the target structure. The quinic acid was first converted in the shikimic acid; then two of the three hydroxyl functions of this product were selectively functionalised to obtain the target molecule (3R,4S,5R)-4-acetamido-3-guanidino-5-hydroxycyclohex-1-ene-1-carboxylic acid.

Aromatic sialic acid analogues as potential inhibitors of influenza virus neuraminidase.

The influenza virus neuraminidase (NA) is an enzyme essential for viral infection and offers a potential target for antiviral drug development. We aimed our research at the synthesis of non-carbohydrate molecules able to inhibit NA as transition-state analogues. Aromatic sialic acid analogues (compound 5 and compound 10) were synthesised in good yields starting from commercially available benzoic acids using a suitable synthetic strategy.

Synthesis of 2-amino-5-sulfanyl-1,3,4-thiadiazole derivatives and evaluation of their antidepressant and anxiolytic activity.

Recently a series of 2-amino-5-sulfanyl-1,3,4-thiadiazole derivatives bearing different substituents were synthesized and screened pharmacologically in order to evaluate their central nervous system activity. The purpose of this study was to evaluate the effects of the title compounds on CNS activity by varying the substituents in the thiadiazole moiety. It was found that some of these compounds possess marked antidepressant and anxiolytic properties comparable in efficiency to the reference drugs Imipramine and Diazepam. The most potent compound 3k was further investigated to complete its pharmacological profile with respect to undesired side effects. Behavioral results showed that 3k is a very promising compound, characterized by a mixed antidepressant-anxiolytic activity accompanied by a therapeutic dose range that is essentially 2 orders of magnitude less than that at which side effects such as sedation and amnesia are evident.

Potentiation of cADPR-induced Ca(2+)-release by methylxanthine analogues.

Caffeine and other methylxanthines are known to induce Ca(2+)-release from intracellular stores via the ryanodine receptor. In the present work, a range of caffeine analogues, in which methyl groups at the 1 and 7 positions were replaced with alkyl chains containing different functional groups (oxo, hydroxyl, propargyl, ester, and acids), were synthesized. These compounds were then screened for their ability to potentiate Ca(2+)-release induced by cADPR (an endogenous modulator of ryanodine receptors) in sea urchin egg homogenates. Two of the synthesized methylxanthines, 1, 3-dimethyl-7-(7-hydroxyoctyl)xanthine (37) and 3-methyl-7-(7-oxooctyl)-1-propargylxanthine (66), were shown to be more potent than caffeine in potentiating cADPR-induced Ca(2+)-release, while 1,3-dimethyl-7-(5-ethylcarboxypentyl)xanthine (14) was shown to be more efficacious. The development of new methylxanthine analogues may lead to a better understanding of ryanodine receptor function and could possibly provide novel therapeutic agents.

"Back door" opening implied by the crystal structure of a carbamoylated acetylcholinesterase.

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline (MF268) is reported at 2.7 A resolution. In the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.

Synthesis, binding affinity and selectivity of new beta 1- and beta 2-adrenoceptor blockers.

The synthesis of a new series of sesamol derivatives with beta-adrenergic blocking activity is described. The affinity and selectivity of these compounds for beta 1- and beta 2-adrenoceptors were studied in comparison with those of ICI-118551 and propranolol. Some of the synthesized compounds show very good affinity for the beta 2-receptors of rat lung membranes and two of them provide interesting selectivity.

Inflammatory molecule release by beta-amyloid-treated T98G astrocytoma cells: role of prostaglandins and modulation by paracetamol.

Deposition of beta-amyloid in the brain triggers an inflammatory response which accompanies the neuropathologic events of Alzheimer's disease and contributes to the destruction of brain tissue. The present study shows that beta-amyloid can stimulate human astrocytoma cells (T98G) to secrete the proinflammatory factors interleukin-6 and prostaglandins. Furthermore, prostaglandins can stimulate T98G to secrete interleukin-6, which in turn triggers the formation of additional prostaglandins. Prostaglandins are, therefore, a key element in the induction and maintenance of a state of chronic inflammation in the brain which may exacerbate the fundamental pathology in Alzheimer patients. Paracetamol (0.01-1000 microM), an unusual analgesic/antipyretic drug which acts preferentially by reducing prostaglandin production within the central nervous system, and indomethacin (0.001-10 microM) caused a clear dose-dependent reduction of prostaglandin E2 production by stimulated T98G cells whereas interleukin-6 release was not affected. These data provide further evidence of the involvement of non-steroidal anti-inflammatory drugs in the inflammatory processes that can be generated by glial cells in intact brain.

Synthesis and activity studies of N-[omega-N'-(adamant-1'-yl)aminoalkyl]- 2-(4'-dimethylaminophenyl)acetamides: in the search of selective inhibitors for the different molecular forms of acetylcholinesterase.

A series of N-[omega-N'-(adamant-1'-yl)aminoalkyl]-2-(4'- dimethylaminophenyl)acetamides were synthesized and tested as acetylcholinesterase inhibitors. A significant selectivity toward acetylcholinesterases from various natural sources, mainly differing in their quaternary structure and solubility, was pointed out. The interest of this kind of molecules as potential therapeutic agents for Alzheimer's disease is discussed.

Long chain analogs of physostigmine as potential drugs for Alzheimer's disease: new insights into the mechanism of action in the inhibition of acetylcholinesterase.

Heptylphysostigmine is in advanced clinical trial as a drug for Alzheimer's disease. 8-Morpholinooctylphysostigmine and 8-(cis-2,6-dimethylmorpholino)octylphysostigmine are currently undergoing pre-clinical evaluation. The mechanism of action of these compounds in the inhibition of acetylcholinesterase has been investigated. All the examined compounds display non competitive-like kinetics of inhibition. There are no reversible components in the observed inhibition: the whole inhibitory effect is due to the time-dependent pseudo-irreversible carbamylation of the active site. Yet the observed time course of the inhibition does not match a simple second order kinetics. An influence of the quaternary structure of the enzyme on the more complex kinetics of carbamylation is hypothesized. Reactivation experiments on the inhibited enzyme show long lasting inhibitory effects for these compounds. The higher duration of the anticholinesterase effect of the morpholino derivatives compared to heptylphysostigmine should provide the basis for their higher therapeutic potential.

Effects of MF-268, a new cholinesterase inhibitor, on acetylcholine and biogenic amines in rat cortex.

MF-268 bitartrate [(3a S, 8a R)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol- 5-ol[8-(cis2,-6-dimethyl-morpholin-4-yl)octyl]-carbamate L-bitartrate hydrate; Mediolanum Farmaceutici, Milan, Italy] is a pseudo-reversible carbamate-type cholinesterase inhibitor (ChEI) which interacts with the catalytic and regulatory anionic site of the enzyme. Its effects on extracellular levels of acetylcholine (ACh), norepinephrine (NE), dopamine (DA), and serotonin (5-HT, 5-hydroxytryptamine) were studied in rat cortex by using a microdialysis technique coupled with high-performance liquid chromatography-electrochemical detection (HPLC-ECD). Conscious, freely moving rats were systemically [per os (p.o.) and subcutaneously (s.c.)] administered MF-268 with no ChEI in the probe. Cholinesterase inhibition in brain was assayed in parallel experiments. Oral administration of MF-268 (0.5, 2.0, and 5.0 mg/kg) produced a significant increase of extracellular ACh in cortex; the maximal increase was 300% [not significant (n.s.)], 460% and 1,200%, respectively. Maximal cholinesterase (ChE) inhibition was 2.3% (n.s.) at 9 hr and 9.7% (P < .05) at 12 hr after the 2.0 and 5.0 mg/kg doses, respectively. Norepinephrine and DA levels were increased 180% and 100% after the 5.0 mg/kg dose, respectively; 100% and 60% after the 2.0 mg/kg dose, respectively; and 70% for both amines after the 0.5 mg/kg dose, respectively. The elevation lasted at least 5 hr with the 2.0 and 5.0 mg/kg doses. There were no major changes in 5-HT levels at these three doses. Subcutaneous administration (0.5 and 2.0 mg/kg) produced a maximal 360% (5.5 hr) and 2,500% (5 hr) increase in extracellular ACh, respectively. Maximal ChE inhibition was 13% (0.5 mg/kg) and 41% (2.0 mg/kg). Neither 0.5 nor 2.0 mg/kg produced a consistent modification of NE. Only a transient increase in DA was seen with the 0.5 mg/kg dose. There were no changes in 5-HT levels at these two doses. MF-268-treated animals showed slight cholinergic side effects (chewing, tremor) at both doses. MF-268 administered intracortically through the microdialysis probe at a concentration of 50 microM induced a 5,900% increase in ACh levels at 6 hr. This effect started 30 min after injection and continued throughout the period of administration. MF-268 produced a significant decrease in NE levels (-44%) starting at 30 min, and a slight but significant increase in DA levels of 45% at 2.5 hr. A significant increase of 5-HT (58%) was also observed starting at 4 hr. Slight symptoms of cholinergic toxicity were observed during intracortical administration.

Structure-activity relationships in open ansa-chain rifamycin S derivatives as inhibitors of HIV-1 reverse transcriptase.

Three types of open ansa-chain rifamycin S derivatives have been prepared: derivatives with the ansa-chain open at C(29) and the original dihydrofuranone ring; derivatives with the ansa-chain open at C(29) and a furane ring; derivatives with the ansa-chain at open NH-C(15). Only derivatives of the first type are weak inhibitors of HIV-1 reverse transcriptase (IC50 ca.300 microM) while derivatives of the two other types are inactive. It has been hypothesized that the active derivatives inhibit the viral enzyme interacting through the groups C(14)H3, C(13)H3, and C(1)O at the same site as the well-known inhibitors TIBO and Nevirapine. In particular C(13)H3 must be unhindered and in an appropriate position out of the plane containing the chromophore-rings. The open ansa-chain seems to play the role of a lipophylic substituent.

Synthesis of 2'-heptylcarbamoyloxy-2-methyl-6,7-benzomorphan: a new analogue of heptylphysostigmine (MF 201).

The synthesis of 2'-heptylcarbamoyloxy-2-methyl-6,7-benzomorphan is described. The compound is structurally related to the cholinesterase inhibitor heptylphysostigmine (MF 201) because the angular methyl group of the esoroline nucleus has been changed into a bridging carbon and the anilinic nitrogen has been replaced by a methylene group. This compound proved to be a potent cholinesterase in vitro inhibitor.

Rifamycins as inhibitors of retroviral reverse transcriptase from M-MuLV, RAV-2, and HIV-1.

29 Rifamycins were tested for inhibition of Reverse Transcriptase (RT) as potential anti HIV drugs. Two purified commercial enzymes from M-MuLV and RAV-2 were used. Anti-RT activity was also measured on a crude lysate of HIV-1. The results show that some derivatives have interesting levels of activity on isolated M-MuLV and RAV-2 RTs, while they are less active on the RT in the crude HIV-1 lysate. The active derivatives include oximes and hydrazones, alkylaminoderivatives, open ansa-chain derivatives and derivatives carrying a modified nucleoside.

Acetazolamide-like carbonic anhydrase inhibitors with topical ocular hypotensive activity.

New carbonic anhydrase (EC 4.2.1.1) inhibitors were synthesized as potential drugs for the topical treatment of glaucoma. They were obtained by substituting the acetyl group of acetazolamide and methazolamide with bicarboxylic acids of different chain length (C4-C6). The terminal carboxyl was either kept free or esterified with alcohols of different size (C1-C12). A gamma-aminovaleric derivative was also prepared. All compounds proved active as carbonic anhydrase inhibitors in vitro, with an average IC50 of about 0.5 microM. Some proved also to be topically active in vivo in lowering the artificially elevated intraocular pressure in rabbits. The most active compound, carrying a succinic acid side chain, is the most soluble in aqueous buffers. Its duration of action is about 8 h and it is under evaluation as a topical antiglaucoma drug. It is hypothesized that the duration of action could be longer in compounds having both the same high water solubility and partition coefficient.

New 1,2,4-oxadiazole derivatives: synthesis and adrenergic receptors binding studies.

In order to obtain derivatives with simultaneous alpha- and beta-adrenergic blocking activity, compounds having the phenoxypropanolaminic structure of beta-adrenergic blockers have been synthesised, as well as 1,2,4-oxadiazole moiety, which could imitate the imidazolinic nucleus characteristic of drugs acting on alpha-adrenergic receptors. The synthesised compounds have been submitted to alpha and beta receptor binding assays. Some derivatives showed an alpha-adrenoceptor binding activity higher than labetalol and similar to prazosin, but with a poor beta-adrenoceptor binding activity.

Synthesis of phenothiazine derivatives as potential inhibitors of phospholipase C.

In order to study the structure-activity relationships of phenothiazine derivatives inhibiting phosphatidylinositol-specific phospholipase C (PI-PLC), the synthesis of some phenothiazine amide, amine and ester derivatives was performed mainly by reacting 10H-phenothiazine-10-propanoyl chloride with some amines and alcohols; the resulting amides were reduced with borane to yield the corresponding amines. Starting from 2-chloro and 2-trifluoromethyl-10H-phenothiazine-10-propanoyl chloride two amides were synthesized. The inhibiting activity on PI-PLC from human platelets is reported.

Synthesis of some carbamates of myo-inositol.

The syntheses are described of the 1-O-carbamoyl (11), 1-O-carbamoyl-2-O-stearoyl (10), 1-O-(acetylcarbamoyl)-2-O-stearoyl (12), 1-O-(heptylcarbamoyl) (13), 2-O-(heptylcarbamoyl) (14) 1,2-di-O-(heptylcarbamoyl) (15), and 1-O-(octadecylcarbamoyl) (16) derivatives of myo-inositol. None of these compounds had significant activity against phospholipase C.

Synthesis of 2-hydroxyacetyl-7-acetyl-xanthone, a new xanthone derivative endowed with antianaphylactic, analgesic, and antiinflammatory activities.

2-Hydroxyacetyl-7-acetylxanthone (V), a new xanthone derivative, was synthesized in four steps starting from xanthene, by two synthetic approaches. The new compound displayed antianaphylactic activity in the PCA test and in the anaphylaxis shock test in rats. It also displayed analgesic activity in the writhing test in mice, and antiinflammatory activity in the carrageenin edema test in rats. The activity of the new compound has been tentatively interpreted on the basis of its chemical and structural analogy with known drugs.