Design, synthesis and preliminary evaluation of a series of histone deacetylase inhibitors carrying a benzodiazepine ring.

A series of new histone deacetylase inhibitors were designed and synthesized based on hybridization between SAHA or oxamflatin and 5-phenyl-1,4-benzodiazepines. The compounds were tested for their enzyme inhibitory activity on HeLa nuclear extracts, and on human recombinant HDAC1 and HDAC6. Antiproliferative activity was tested on different cancer cells types, while proapoptotic activity was primarily tested on NB4 cells. The compounds showed IC50 values similar to those of SAHA. Compound (S)-8 displayed interesting activity against hematological and solid malignancies.

Influence of ring size on the cognition-enhancing activity of DM235 and MN19, two potent nootropic drugs.

A series of analogs of DM235 and MN19, characterized by rings with different size, have been prepared and evaluated for their nootropic activity in the mouse passive-avoidance test. It was found that the optimal ring size for the analogs of DM235, showing endocyclic both amidic groups, is 6 or 7 atoms. For the compounds structurally related to MN19, carrying an exocyclic amide group, the piperidine ring is the moiety which gives the most interesting compounds.

N,N-bis(cyclohexanol)amine aryl esters: the discovery of a new class of highly potent inhibitors of transporter-dependent multidrug resistance (MDR).

Multidrug resistance (MDR) is a kind of acquired resistance of microorganisms and cancer cells to chemotherapeutic drugs that are characterized by different chemical structure and different mechanism of action. Classic MDR is due to a lower intracellular concentration of cytotoxic drugs that is associated with accelerated efflux of the chemotherapeutic drugs and is the consequence of the over expression of transporter proteins that act as extrusion pumps. P-glycoprotein (P-gp/ABCB1) is the most important and studied member of such proteins belonging to the ATP Binding Cassette (ABC) superfamily of transporters that use ATP as energy source. Inhibition of the functions of P-gp and other ABC proteins could represent a way to circumvent appearance of MDR in cancer cells and the most classical pharmacological strategy is the administration of agents able to modulate the P-gp function. On the basis of the known characteristics of the recognition site of P-gp, we have designed a new class of P-gp-mediated MDR reverters. These compounds are flexible molecules carrying a basic nitrogen atom flanked, at properly modulated distance, by two aromatic moieties; most of them possess MDR inhibitory activity on anthracycline-resistant erytroleukemia K562 cells. By applying the frozen analog approach to that series of very flexible MDR reverters, we identified a new series of N,N-bis(cyclohexanol)amine aryl esters that show very interesting MDR-reversing properties. Among them, compound 15d, that consistently shows low nanomolar potency and high efficacy in all the tests used, appears as a new pharmacological tool for P-gp studies and a promising lead for the development of potent, efficient and safe MDR reverters.

Design, synthesis and preliminary biological evaluation of new hydroxamate histone deacetylase inhibitors as potential antileukemic agents.

This study concerns the synthesis of new histone deacetylase inhibitors (HDACi) characterized by a 1,4-benzodiazepine ring used as the cap, joined through an amide function or a triple bond as connection units, to a linear alkyl chain bearing the hydroxamate function as Zn2+-chelating group. Biological tests performed in human acute promyelocytic leukemia NB4 cells showed that new hybrids can induce histone H3/H4 acetylation, growth arrest, and also apoptosis. Notably, chiral compounds exhibit stereoselective activity.

The functions and structure of ABC transporters: implications for the design of new inhibitors of Pgp and MRP1 to control multidrug resistance (MDR).

Multidrug resistance (MDR) is a kind of acquired resistance of microorganisms and cancer cells to chemotherapic drugs that are characterized by different chemical structure and different mechanism of action. Classic MDR is the consequence of the over-expression of a variety of proteins that extrude the chemotherapic from the cell, lowering its concentration below the effective one. The ABC (ATP Binding Cassette) is a ubiquitous and important family of such transporter proteins. Members of this super family are present in mammals as well as in prokaryotic organisms and use ATP as the energy source to activate the extrusion process. P-glycoprotein (Pgp) and Multidrug Resistance Proteins (MRP1 and sister proteins) are the most important and widely studied members of ABC super family. Our knowledge about the structures and functions of transporter proteins has definitely improved in recent years, following the resolution of the structure of bacterial pumps which opened the way to the building of homology models for the more complex Pgp and MRP. It can be anticipated that these results will have a strong impact on the design of more potent and safer MDR reverters. A huge number of small molecules, many of natural origin, are able to reverse multidrug resistance by inhibiting the functions of Pgp, MRP1 and sister proteins and their action has been considered a possible way to reverse MDR. However, while a few compounds have reached clinical trials, none of them has, so far, been cleared for therapeutic use. Two main reasons are at the base of this difficulty: i) MDR is a complex phenomenon that may arise from several different biochemical mechanisms, with the consequence that inhibition of transporter proteins may be insufficient to reverse it; ii) the physiological role of Pgp and sister proteins requires more potent modulators with proper selectivity and pharmacokinetic in order to avoid unwanted side effects. This paper first reviews the most recent discoveries on the structures and functions of the ABC super family, in particular Pgp and MRP. Then, the medicinal chemistry of MDR reverters, in light of these findings, is discussed and the molecules that are presently in development are reviewed.

The medicinal chemistry of multidrug resistance (MDR) reversing drugs.

Multidrug resistance (MDR) is a kind of resistance of cancer cells to multiple classes of chemotherapic drugs that can be structurally and mechanistically unrelated. Classical MDR regards altered membrane transport that results in lower cell concentrations of cytotoxic drug and is related to the over expression of a variety of proteins that act as ATP-dependent extrusion pumps. P-glycoprotein (Pgp) and multidrug resistance protein (MRP1) are the most important and widely studied members of the family that belongs to the ABC superfamily of transporters. It is apparent that, besides their role in cancer cell resistance, these proteins have multiple physiological functions as well, since they are expressed also in many important non-tumoural tissues and are largely present in prokaryotic organisms. A number of drugs have been identified which are able to reverse the effects of Pgp, MRPI and sister proteins, on multidrug resistance. The first MDR modulators discovered and studied in clinical trials were endowed with definite pharmacological actions so that the doses required to overcome MDR were associated with unacceptably high side effects. As a consequence, much attention has been focused on developing more potent and selective modulators with proper potency, selectivity and pharmacokinetics that can be used at lower doses. Several novel MDR reversing agents (also known as chemosensitisers) are currently undergoing clinical evaluation for the treatment of resistant tumours. This review is concerned with the medicinal chemistry of MDR reversers, with particular attention to the drugs that are presently in development.

Structure-affinity relationships of a unique nicotinic ligand: N(1)-dimethyl-N(4)-phenylpiperazinium iodide (DMPP).

DMPP is a well-known nicotinic agonist that does not fit any proposed pharmacophore for nicotinic binding and represents a unique ligand among the hundreds of nicotinic agonists studied in the past decades. A systematic modulation of the chemical structure of DMPP, aimed to establish its structure-affinity relationships, is reported. The research has allowed to identify molecules such as 11c, 13c, 14c, and 28c, with affinities for alpha(4)beta(2) receptors in the low nanomolar range, some 2 orders of magnitude lower than the lead compound. The agonistic properties of the most interesting compounds have been assessed by measuring their analgesic activity on mice (hot-plate test). Another result of the research was the identification of DMPP analogues, such as 3a (K(i) = 90 nM) and 14b (K(i) = 180 nM), that maintain affinity for the central nicotinic receptor when the ammonium function is changed into an aminic one and are therefore possible leads for drug development in neurodegenerative diseases.

Structure-activity relationships and optimisation of the selective MDR modulator 2-(3,4-dimethoxyphenyl)-5-(9-fluorenylamino)-2-(methylethyl) pentanenitrile and its N-methyl derivative.

Several ring-substituted derivatives of previously studied MDR inhibitors 2-(3,4-dimethoxyphenyl)-5-(9-fluorenylamino)-2-(methylethyl)pentanenitrile and 2-(3,4-dimethoxyphenyl)-5-[(9-fluorenyl)-N-methylamino]-2-(methylethyl)pentanenitrile have been synthesised and studied with the aim of optimising activity and selectivity. The results show that MDR inhibition is scarcely sensitive to modulation of the electronic properties of the fluorene ring. Even if dramatic improvement was not obtained, one of the compounds (2) showed improved potency and selectivity with respect to the leads and appears to be a better candidate for drug development.

Structure-activity relationships in 2,2-diphenyl-2-ethylthioacetic acid esters: unexpected agonistic activity in a series of muscarinic antagonists.

As a continuation of previous research on anticholinergic drugs derived from 2,2-diphenyl-2-ethylthioacetic acid, several 5,5-diphenyl-5-ethylthio-2-pentynamines (2-11) were synthetised and their antimuscarinic activity on M(1-4) receptor subtypes was evaluated by functional tests and binding experiments. One of the compounds obtained showed unexpected agonistic activity in functional experiments on M(2) receptors. Since the compound carried a phenylpiperazine moiety, other similar compounds (12-17) were prepared and found to be endowed with similar behaviour. These ligands, although possessing the bulky structure characterising muscarinic antagonists, display agonistic activity at M(2) subtypes while, as expected, behaving as antagonists on M(3) and M(4) subtypes. On M(1) subtypes, they show agonistic activity which, however, is not blocked by atropine. The peculiar pharmacological profile of these compounds is of interest for studying muscarinic receptor subtypes.

Design, synthesis, and preliminary pharmacological evaluation of 1, 4-diazabicyclo[4.3.0]nonan-9-ones as a new class of highly potent nootropic agents.

Several 4-substituted 1,4-diazabicyclo[4.3.0]nonan-9-ones have been synthesized and tested in vivo on mouse passive avoidance test, to evaluate their nootropic activity. The results show that they represent a new class of nootropic drugs with a pharmacological profile very similar to that of piracetam, showing much higher potency with respect to the reference. Among the compounds studied, 7 (DM 232) shows outstanding potency, being active at the dose of 0. 001 mg kg(-1) sc.

Antinociceptive effect of R-(+)-hyoscyamine on the conjunctival reflex test in rabbits.

R-(+)-Hyoscyamine (1-10 microg/kg, s.c.) dose-dependently increased the local anesthetic effect of procaine (50 microg/ml) and lidocaine (50 microg/ml) in the conjunctival reflex test in the rabbit. This potentiating effect is completely prevented by the M1 antagonist dicyclomine (10 mg/kg, s.c.). The intensity of R-(+)-hyoscyamine antinociception was comparable to that induced by morphine (2 mg/kg, s.c.) and minaprine (15 mg/kg, s.c.), used as analgesic reference drugs. In the same experimental conditions, the S-(-)-enantiomer of atropine (0.1-10 microg/kg, s.c.), was completely ineffective. The present results confirm the ability of R-(+)-hyoscyamine to produce a paradoxical antinociceptive effect mediated by a cholinergic mechanism not only in rodents but also in the rabbit.

Synthesis and binding properties of photoactivable biotin-conjugated verapamil derivatives for the study of P-170 glycoprotein.

The design and synthesis of two photoactivable biotin-labeled analogues of verapamil (6 and 7) is reported. Preliminary evaluation of the biological profile of 6 (EDP 137) and 7 (EDP 141) shows that they have comparable affinities to that of verapamil for P-170, the protein responsible for multidrug resistance (MDR). Since both appear to bind irreversibly to the protein and the presence of biotin in their structure makes them easily detectable by avidin, they promise to be of great help in studying the protein and its mechanism of action.

Design, synthesis, and in vitro activity of catamphiphilic reverters of multidrug resistance: discovery of a selective, highly efficacious chemosensitizer with potency in the nanomolar range.

On the basis of the results obtained in previous research, three series of compounds (A-C), derived from verapamil, were designed and synthesized to obtain drugs able to revert multidrug resistance (MDR), an acquired resistance that frequently impairs cancer chemotherapy. The ability of the obtained compounds to revert MDR was evaluated on anthracycline-resistant erythroleukemia K 562 cells, measuring the uptake of THP-adriamycin (pirarubicin) by continuous spectrofluorometric monitoring of the decrease of the fluorescence signal of the anthracycline at 590 nm (lambdaex = 480 nm), after incubation with cells. Cardiovascular activity, which is responsible for unwanted side effects, was also evaluated. The results obtained show that many of the compounds studied are potent reverters of MDR and are endowed with reduced cardiovascular activity. One of the compounds (7, MM36) presents a pharmacological profile (unprecedented nanomolar potency, high reversal of MDR, low cardiovascular activity) that makes it a promising drug candidate to treat MDR and a useful tool for studying P-glycoprotein.

Hybridized and isosteric analogues of N1-acetyl-N4-dimethyl-piperazinium iodide (ADMP) and N1-phenyl-N4-dimethyl-piperazinium iodide (DMPP) with central nicotinic action.

A series of piperazine derivatives, obtained by hybridization of N1-acetyl-N4-dimethyl-piperazinium iodide (1, ADMP) and N1-phenyl-N4-dimethyl-piperazinium iodide (3, DMPP) or of the corresponding tertiary bases (2, 4) with arecoline (5) and arecolone (6) or by isosteric substitution of the phenyl ring of DMPP, has been synthesized. Hybridization afforded compounds that, both as tertiary bases and as iodomethylates, have no affinity for the nicotinic receptor. On the contrary, isosteric substitution gave compounds that maintain affinity for the receptor; among them, two tertiary bases (37, 38), show affinity in the nanomolar range for the nicotinic receptor. The pharmacological profile of these isomeric compounds is quite interesting as they present differences in their peripheral and central effects, suggesting that they interact with different subtypes of the nicotinic receptor.

Further structure-activity relationships in the series of tropanyl esters endowed with potent antinociceptive activity.

Several analogs of the alpha-tropanyl esters of 2-(4-chlorophenoxy)butyric acid (SM21) and 2-phenylthiobutyric acid (SM32), endowed with potent antinociceptive and cognition enhancing activity, were synthesized, aimed at obtaining more potent and safe drug candidates. Variation of the acyl moiety (4-11), as well as the conformational restriction of atropine to give the alpha-tropanyl ester of 2,3-dihydrobenzofurane-3-carboxylic acid (18), practically abolished activity. In the case of 18, the antimuscarinic activity was also severely affected by the conformation restrain. On the contrary, conformational restriction of phenoxybutyric and phenylthiobutyric acid derivatives to give the alpha-tropanyl ester of 2,3-dihydro-benzofurane-2-carboxylic acid and 2,3-dihydro-benzothiophene-2-carboxylic acid (12-17), afforded potent analgesic drugs that unfortunately were too toxic to be reliable drug candidates. A series of related esters of benzofurane-3-carboxylic acid (20-27) and benzothiophene-3-carboxylic acid (28) were also studied and found to be potent but toxic analgesics.

SAR studies on the potent and selective muscarinic antagonist 2-ethylthio-2,2-diphenylacetic acid N,N-diethylaminoethyl ester.

Molecular modification of the potent and selective muscarinic antagonist 2-ethylthio-2,2-diphenylacetic acid N,N-diethylaminoethyl ester was performed in order to identify M2 selective antagonists able to cross the blood brain barrier and potentially useful in the treatment of Alzheimer's disease. Modifications included substitution or hydrogenation of one of the phenyl rings as well as their incorporation in a tricyclic system. In general the changes introduced were detrimental for both affinity and selectivity. Only a modest M2 selectivity is present in some compounds that, on the other hand, carry a quaternary ammonium group which precludes their penetration into the brain.

Study of P-glycoprotein functionality in living resistant K562 cells after photolabeling with a verapamil analogue.

To our knowledge, this is the first study to investigate the modification of P-glycoprotein functionality in living resistant cells after photolabeling. For this purpose, four new photoactive verapamil analogues were synthesized. These compounds have the same efficacy as verapamil to increase pirarubicin (pira) incorporation into living multidrug resistant (MDR) K562 cells and to sensitize them to the cytotoxic effect of this anthracycline derivative, indicating that they act as typical MDR modifiers in MDR cells. These compounds were used to photolabel P-glycoprotein (P-gp) in living resistant cells. Irradiation did not result in photodamage to cells, and P-gp functionality was verified by the ability of living cells to incorporate pira. The irradiation of resistant cells, 10(6)/mL, in the presence of a verapamil analogue at concentrations equal to or higher than 3 microM yielded 70% inhibition of P-gp functionality. Our data provide the first evidence that the binding of a verapamil analogue to P-gp is not sufficient to completely inhibit the efflux of this anthracycline. The cells were, subsequently, cultured for several days. Resistance was progressively recovered with time, with the treated cells being just as resistant as before photolabeling after 6 days.

Reduced flexibility analogs of analgesic and cognition enhancing alpha-tropanyl esters.

A series of semirigid analogs of compounds 1 and 2, two potent analgesics and cognition enhancers, have been synthesized and tested for antinociceptive activity (hot plate test) and for muscarinic affinity (binding in rat cerebral cortex). They were found to be in general less potent than the reference compounds; only one of them (22) shows a good affinity for the muscarinic receptor and an antinociceptive efficacy comparable with those of the reference compounds. At a dose of 30 mg/kg 22 is also able to reverse the amnesic effect of dicyclomine. Since the analgesic effect of these compounds is affected by the 5-HT4 antagonist SDZ 205557, the possible role of this receptor is discussed.

Synthesis and enantioselectivity of the enantiomers of PG9 and SM21, new potent analgesic and cognition-enhancing drugs.

The enantiomers of two alpha-tropanyl esters, SM21 (1) and PG9 (2), derived from (+)-R-hyoscyamine, that act by increasing the central cholinergic tone, were obtained by esterification after resolution of the corresponding racemic acids [(-)-S-1, (-)-R-2 and (+)-S-2] and by stereospecific synthesis [(+)-R-1]. Their analgesic and cognition-enhancing activities were tested in mice and their ACh-releasing properties determined on rat parietal cortex. These compounds show enantioselectivity in analgesic and cognition-enhancing tests on mice, the eutomers being the isomers which possess the same spatial arrangement of the groups on the chiral atom as (+)-R hyoscyamine [(+)-R-SM21, (+)-S-PG9]. The ACh-releasing effect of the enantiomers of SM21 in rats is in agreement with the results in mice, while PG9 enantiomers do not show any appreciable enantioselectivity in this test. On the basis of the different effects of the 5-HT4 antagonist SDZ 205557 on analgesia induced by the enantiomers of 1 and 2 and by (+)-R-hyoscyamine and the alpha-tropanyl ester of 2-phenylpropionic acid 3, a mechanism of action is proposed for this class of compounds.

Chiral synthesis and pharmacological evaluation of the enantiomers of SM32, a new analgesic and cognition-enhancing agent.

The enantiomers of 3-alpha-tropyl 2-(phenylthio)butyrate (SM32, 1) were prepared by chiral synthesis and tested for analgesic, cognition-enhancing, and ACh-releasing properties. They show enantioselectivity in some of the tests, the eutomer being related in configuration to R-(+)-hyoscyamine.