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N-(Heteroarylmethyl)indol-3-ylglyoxylamides (1−26) were synthesized and evaluated as ligands of the benzodiazepine receptor (BzR) to probe the hydrogen bonding properties of the so-called S1site of the BzR by means of suitable heterocyclic side chains. SARs were developed in light of our hypothesis of binding modes A and B. Pyrrole and furan derivatives adopting mode A (2, 8, 10, 20, 22) turned out to be more potent (Kivalues < 35 nM) than their analogues lacking hydrogen bonding heterocyclic side chains. These data suggest that the most potent indoles interact with a hydrogen bond acceptor/donor (HBA/D) group located within the S1site of the BzR. Compounds 1, 2, 8, 19, 20, and 22, tested at recombinant rat 122, 222, and 532BzRs, elicited selectivity for the 122isoform. On the basis of published mutagenesis studies and the present SARs, we speculate that the S1HBA/D group might be identified as the hydroxyl of 1-Tyr209 or of other neighboring amino acids.

A series of 2-(quinazolin-4-ylamino)-[1,4] benzoquinone derivatives that function as potent covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2 (VEGFR-2) has been prepared by ceric ammonium nitrate oxidation of substituted (2,5-dimethoxyphenyl)(6,7-disubstituted-quinazolin-4-yl)amines and by displacement of the chlorine atom of substituted 2-chloro-5-(6,7-disubstituted-quinazolin-4-ylamino)-[1,4]benzoquinones with various amines, anilines, phenols, and alcohols. Enzyme studies were conducted in the absence and presence of glutathione and plasma. Several of the compounds inhibit VEGF-stimulated autophosphorylation in intact cells. Kinetic experiments were performed to study the reactivity of selected inhibitors toward glutathione. Reactivities correlated with LUMO energies calculated as averages of those of individual conformers weighted by the Boltzmann distribution. These results and molecular modeling were used to rationalize the biological observations. The compounds behave as non-ATP-competitive inhibitors. Unequivocal evidence, from mass spectral studies, indicates that these inhibitors form a covalent interaction with Cys-1045. One member of this series displays antitumor activity in an in vivo model.

Pyrrolobenzoxazepinones (PBOs) represent a new class of human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 5. Molecular modeling studies based on the X-ray structures of HIV-1 RT prompted the synthesis of novel analogues which were tested as anti-HIV agents. The PBO derivatives specifically designed to target the highly conserved amino acid residues within the β12-β13 hairpin, namely primer grip, proved to be very potent against the most common mutant enzymes, including the highly resistant K103N mutant strain. Structure−activity relationships (SARs) are discussed in terms of a possible interaction with the RT binding site, depending on the nature of the substituents at C-6. Among the pyrrolobenzoxazepines investigated, 15c appeared to be the most promising NNRTI of the series characterized by potent antiviral activity, broad spectrum, and low cytotoxicity. 15c showed synergistic antiviral activity with AZT.

Acetic acid derivatives of naphtho[1,2-d]isothiazole (NiT) were synthesized and tested as novel aldose reductase (ALR2) inhibitors. The parent compound 11 exhibited a fair inhibitory activity (IC50 = 10 μM), which was enhanced by 2 orders of magnitude by introducing a second carboxylic group at position 4 (13 and 14:  IC50 = 0.55 and 0.14 μM, respectively). Substitution of the acetic acid function with an apolar group gave inactive (29) or poorly active (25, 26, 30) compounds, thus demonstrating that the 2-acetic group is involved in the enzyme pharmacophoric recognition while the 4-carboxylic moiety has only an accessory role. The potent compounds 11, 13, 14, 26 all proved to be selective for ALR2, since none of them inhibited aldehyde reductase, sorbitol dehydrogenase, or glutathione reductase. The isopropyl ester 31, a prodrug of 14, was found to be effective in preventing cataract development in severely galactosemic rats, when administered as an eyedrop solution. The theoretical binding mode of 13 and 14, obtained by docking simulations into the ALR2 crystal structure, was fully consistent with the structure−activity relationships in the NiT series.

This study reports the synthesis of a number of 1- and 2-alkyl derivatives of the 4-aminopyrazolo[3,4-d]pyrimidine (APP) nucleus and their evaluation as inhibitors of ADA from bovine spleen. The 2-substituted aminopyrazolopyrimidines proved to be potent inhibitors, most of them exhibiting Ki values in the nanomolar/subnanomolar range. In this series the inhibitory activity is enhanced with the increase in length of the alkyl chain, reaching a maximum with the n-decyl substituent. Insertion of a 2‘-hydroxy group in the n-decyl chain gave 3k, whose (R)-isomer displayed the highest inhibitory potency of the series (Ki 0.053 nM), showing an activity 2 orders of magnitude higher than that of (+)-EHNA (Ki 1.14 nM), which was taken as the reference standard. Docking simulations of aminopyrazolopyrimidines into the ADA binding site were also performed, to rationalize the structure−activity relationships of this class of inhibitors.

A series of N-(arylalkyl)indol-3-ylglyoxylylamides (4−8) was synthesized as ligands of the benzodiazepine receptor (BzR) and tested for their ability to displace [³H]flumazenil from bovine brain membranes. The new compounds, bearing a branched (4) or a geometrically constrained benzyl/phenylethyl amide side chain (5−8), represent the continuation of our research on N-benzylindol-3-ylglyoxylylamides 1 (Da Settimo et al., 1996), N‘-phenylindol-3-ylglyoxylohydrazides 2 (Da Settimo et al., 1998), and N-(indol-3-ylglyoxylyl)alanine derivatives 3 (Primofiore et al., 1989). A few indoles belonging to the previously investigated benzylamides 1 and phenylhydrazides 2 were synthesized and tested to enrich the SARs in these two series. The affinities and the GABA ratios of selected compounds for clonal mammalian α1β2γ2, α3β2γ2, and α5β3γ2 BzR subtypes were also determined. It was hypothesized that the reduced flexibility of indoles 4−8 would both facilitate the mapping of the BzR binding cleft and increase the chances of conferring selectivity for the considered receptor subtypes. In the series of indoles 4, the introduction of a methyl group on the benzylic carbon with the R configuration improved affinity of the 5-substituted (5-Cl and 5-NO2) derivatives, whereas it was detrimental for their 5-unsubtituted (5-H) counterparts. All S enantiomers were less potent than the R ones. Replacement of the methyl with hydrophilic substituents on the benzylic carbon lowered affinity. The isoindolinylamide side chain was tolerated if the 5-position was unsubstituted (Ki of 5a = 123 nM), otherwise affinity was abolished (5b, c). All the 2-indanylamides 6 and (S)-1-indanylamides 8 were devoid of any appreciable affinity. The 5-Cl and 5-NO2 (R)-1-indanylamides 7b (Ki 80 nM) and 7c (Ki 28 nM) were the most potent among the indoles 5−8 geometrically constrained about the side chain. The 5-H (R)-1-indanylamide 7a displayed a lower affinity (Ki 675 nM). The SARs developed from the new compounds, together with those collected from our previous studies, confirmed the hypothesis of different binding modes for 5-substituted and 5-unsubstituted indoles, suggesting that the shape of the lipophilic pocket L1 (notation in accordance with Cook's BzR topological model) is asymmetric and highlighted the stereoelectronic and conformational properties of the amide side chain required for high potency. Several of the new indoles showed selectivity for the α1β2γ2 subtype compared with the α3β2γ2 and α5β3γ2 subtypes (e.g.:  4t and 7c bind to these three BzR isoforms with Ki values of 14 nM, 283 nM, 239 nM, and 9 nM, 1960 nM, 95 nM, respectively). The GABA ratios close to unity exhibited by all the tested compounds on each BzR subtype were predictive of an efficacy profile typical of antagonists.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure−activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Radioligand binding assays using bovine cortical membrane preparations and biochemical in vitro studies revealed that various 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (ATBI) derivatives, previously reported by us as ligands of the central benzodiazepine receptor (BzR) (Primofiore, G.; et al. J. Med. Chem. 2000, 43, 96−102), behaved as antagonists at the A1 adenosine receptor (A1AR). Alkylation of the nitrogen at position 10 of the triazinobenzimidazole nucleus conferred selectivity for the A1AR vs the BzR. The most potent ligand of the ATBI series (10-methyl-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 12) displayed a Ki value of 63 nM at the A1AR without binding appreciably to the adenosine A2A and A3 nor to the benzodiazepine receptor. Pharmacophore-based modeling studies in which 12 was compared against a set of well-established A1AR antagonists suggested that three hydrogen bonding sites (HB1 acceptor, HB2 and HB3 donors) and three lipophilic pockets (L1, L2, and L3) might be available to antagonists within the A1AR binding cleft. According to the proposed pharmacophore scheme, the lead compound 12 engages interactions with the HB2 site (via the N2 nitrogen) as well as with the L2 and L3 sites (through the pendant and the fused benzene rings). The results of these studies prompted the replacement of the methyl with more lipophilic groups at the 10-position (to fill the putative L1 lipophilic pocket) as a strategy to improve A1AR affinity. Among the new compounds synthesized and tested, the 3,10-diphenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (23) was characterized by a Ki value of 18 nM which represents a 3.5-fold gain of A1AR affinity compared with the lead 12. A rhodopsin-based model of the bovine adenosine A1AR was built to highlight the binding mode of 23 and two well-known A1AR antagonists (III and VII) and to guide future lead optimization projects. In our docking simulations, 23 receives a hydrogen bond (via the N1 nitrogen) from the side chain of Asn247 (corresponding to the HB1 and HB2 sites) and fills the L1, L2, and L3 lipophilic pockets with the 10-phenyl, 3-phenyl, and fused benzene rings, respectively.

A series of 3-substituted [1,2,4]triazino[4,3-c]benzimidazoles V were prepared and tested at the central benzodiazepine receptor (BzR). These compounds were designed as rigid analogues of the previously described N-benzylindolylglyoxylylamide derivatives IV. The title compounds V showed an affinity which depended directly on the presence of the N(10)-H group and an aromatic ring at position 3. Some of them elicited a 2- or 3-fold higher affinity with respect to that of the indolylglyoxylylamide derivatives IV (R = H). The GABA ratio and [³⁵S]-tert-butylcyclophosphorothionate binding data revealed an efficacy profile of partial inverse agonists/antagonists for compounds 1c,e,f,j,k, and of a partial agonist for 2c. This last compound proved to be effective in antagonizing pentylenetetrazole-induced seizures in mice. Attempts were made to interpret the structure−affinity relationships of compounds V in the light of possible tautomeric equilibria involving the ligands.

Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose prototype is (±)-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-7(6H)-one (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis and biological evaluation of novel derivatives and analogues of 6 featuring a meta-substituted phenyl or a 2-thienyl ring at C-6 and a pyridine system in place of the fused-benzene ring to yield pyrrolopyridooxazepinones (PPOs). Compared with the lead 6 and nevirapine, several of the synthesized compounds (PBOs 13a−d and PPOs 13i−k) displayed higher inhibitory activity against wild-type RT and clinically relevant mutant RTs containing the single amino acid substitutions L100I, K103N, V106A, Y181I, and Y188L. The most potent inhibitors were further evaluated for in vitro antiviral activity on lymphocytes and monocyte-macrophages, for cytotoxicity on a panel of cell lines, and for potential synergistic antiviral activity with AZT. Pharmacokinetic studies performed on 13b, 13c, and 13i showed that these compounds achieve high concentrations in the brain. The results of the biological and pharmacokinetic experiments suggest a potential clinical utility of analogues such as 13b−d, 13i, and 13j, in combination with nucleoside RT inhibitors, against strains of HIV-1 bearing those mutations that confer resistance to known NNRTI.

A series of N‘-phenylindol-3-ylglyoxylohydrazides, isosters of the N-benzylindol-3-ylglyoxylamide derivatives previously described by us, were synthesized and tested for their ability to displace [³H]Ro 15-1788 from bovine brain membranes. These compounds were designed with the aim of obtaining products which could exert an in vivo activity, thanks to a higher hydrosolubility and consequently a better bioavailability. Affinity was restricted to the derivatives unsubstituted in the 5 position of the indole nucleus (1, 6, 9, 12, 15, 18, 23, and 26), with Ki values ranging from 510 to 11 nM. The most active compounds (6, 9, 23, and 29) proved to be effective in antagonizing pentylenetetrazole-induced seizures. Molecular modeling studies were performed to rationalize the lack of affinity of hydrazides with a chloro or a nitro group in the 5 position of the indole nucleus. It was hypothesized that the conformational preference of the hydrazide side chain, characterized by a gauche disposition of lone pairs and substituents about the N−N bond, prevents all hydrazides from binding to the receptor similarly to other classes of indole analogues previously investigated. The potency of 5-H hydrazides was attributed to a binding mode which is not feasible for 5-Cl and 5-NO2 counterparts. This theoretical model of ligand−receptor interaction permitted a more stringent interpretation of structure−affinity relationships of hydrazides and of recently described benzylamide derivatives (Da Settimo et al. J. Med. Chem. 1996, 39, 5083−5091).

A number of N-(indol-3-ylglyoxylyl)benzylamine derivatives were synthesized and tested for [³H]flunitrazepam displacing activity in bovine brain membranes. Some of these derivatives (9, 12, 14, 15, 17, 27, 34, 35, 38, 41, and 45) exhibited high affinity for the benzodiazepine receptor (BzR) with Ki values ranging from 67 to 11 nM. The GABA ratio and [³⁵S]-tert-butylbicyclophosphorothionate binding data, determined for the most active compounds, showed that they elicit an efficacy profile at the BzR which depends on the kind of substituent present on the phenyl ring of the benzylamine moiety. Moreover, lengthening (propylamine derivatives 1−3) and shortening (aniline derivatives 46−54) of the distance between the phenyl ring and the amide group of the side chain gave compounds with a drastically lower binding potency. The biological results are discussed in the light of a recently proposed pharmacophore model and compared, by molecular modeling studies, with those obtained from effective BzR ligands.