Structure-activity relationship studies of 3-aroylindoles as potent antimitotic agents

The concise synthesis and structure-activity relationship (SAR) studies of 3-aroylindoles were carried out in an effort to improve the potency and solubility of anticancer drug candidate BPR0L075 (8) by exploring structure modifications through three regimens: substitution of the B ring, at the N1 position, and of the 3-carbonyl linker. The SAR information revealed that the methoxy group of the B ring could be replaced with an electron-donating group such as methyl (in compound 9) or N,N-dimethylamino (in compound 13) while retaining both strong cytotoxic and antitubulin activities. The introduction of amide (compounds 30-33) and carbamate (compounds 34-37) functionalities at the N1 position of 8 gave analogues with potent antiproliferative activities. The cytotoxic potency of 8 was improved by replacing the carbonyl group with sulfide (compound 41) or oxygen (compound 43), indicating that the carbonyl moiety is important but not essential. The N,N-dimethylamino derivative 13 not only displayed potent cytotoxicity and antitubulin activity, but also showed a markedly improved physicochemical profile relative to the parent compound.     

Structure-activity studies on the corticotropin releasing factor antagonist astressin, leading to a minimal sequence necessary for antagonistic activity

Corticotropin Releasing Factor (CRF) antagonists are considered promising for treatment of stress-related illnesses such as major depression and anxiety-related disorders. We report here the design, synthesis and biological evaluation of 91 truncated astressin analogues in order to deduce the pharmacophoric amino acid residues. Such truncated peptides may serve as valuable lead structures for the development of new small, non-peptide-based CRF antagonists. N-Terminal truncation of astressin led to active CRF antagonists that are substantially reduced in size and are selectively active at the human CRF receptor type 1 in vitro and in vivo. Subsequently, an alanine scan in combination with further truncated derivatives led to the proposal of a new pharmacophoric model of peptide-based CRF antagonists. It was found that the astressin(27-41)C sequence is the shortest active CRF antagonist. The first eight N-terminal amino acid residues were found to be an important structural determinant and were replaceable by alanine residues, thus enhancing the alpha-helical propensity. A covalent structural constraint is of utmost importance for the preorganization of the C-terminal amino acid residues. The C-terminal heptapeptide sequence, however, was found to be crucial for the antagonistic activity, since substitution or deletion of any residue led to inactive compounds.     

Improved 2,4‐Diarylthiazole‐Based Antiprion Agents: Switching the Sense of the Amide Group at C5 Leads to an Increase in Potency

Amide derivatives of 2,4‐diarylthiazole‐5‐carboxylic acids were synthesised and tested for efficacy in a cell line model of prion disease. A number of compounds demonstrating antiprion activity were thereby identified from the screening libraries, showing improved potency and reproducibility of results relative to amide derivatives of the related 2,4‐diphenyl‐5‐aminothiazole, which have been documented previously. Thus, 'switching' the sense of the amide bond at thiazole C5 revealed a more promising lead series of potential prion disease therapeutics. Furthermore, 3,5‐diaryl‐1,2,4‐thiadiazoles isolated as by‐products during library synthesis provided a handful of additional examples possessing an antiprion effect, thereby augmenting the set of newly identified active compounds. Evaluation of binding to cellular prion protein (PrP^C) showed only weak affinities at best, suggesting that the newly identified antiprion agents do not mediate their biological effect through direct interaction with PrP^C.     

Synthesis, enantiomeric resolution, and structure-activity relationship study of a series of 10,11-dihydro-5H-dibenzo[a,d]cycloheptene MT2 receptor antagonists

Racemic N-(8-methoxy-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-10-ylmethyl)acetamide (compound 5) was previously identified as a novel selective MT(2) antagonist fulfilling the requirements of pharmacophore and 3D QSAR models. In this study the enantiomers of 5 were separated by medium-pressure liquid chromatography and behaved as the racemate. Compound 5 was modified at the acylaminomethyl side chain and at position C8. The resulting analogues generally behaved as melatonin receptor antagonists (GTPgammaS test) with a modest degree of selectivity (up to 10-fold) for the MT(2) receptor. Changes at the amide side chain led to a decrease in binding affinity, whereas 8-acetyl and 8-methyl derivatives 12 and 11, respectively, were as potent as the 8-methoxy parent compound 5. Docking experiments with an MT(2) receptor model suggested binding modes consistent with the observed SARs and with the lack of selectivity of the enantiomers of 5.     

Nanostructure formation enhances the activity of LPS-neutralizing peptides

Peptides that interact with lipopolysaccharide (LPS) can provide the basis for the development of new antisepsis agents. In this work, several LPS-neutralizing acyl peptides derived from LALF, BPI, and SAP were prepared, structurally characterized, and biologically evaluated. In all cases, peptides with long acyl chains showed greater LPS-neutralizing activities than the original acetylated peptides. Structural analysis of these peptides revealed that N-acylation with long acyl chains promotes the formation of micellar or fibril-like nanostructures, thus proving a correlation between anti-LPS activity and nanostructure formation. The results of this study provide useful structural insight for the future design of new acyl peptides that strongly bind LPS and therefore act as antisepsis drugs. Furthermore, this nanostructure-biological activity correlation can be translated into other therapeutic areas.     

Probing novel 1-aza-9-oxafluorenes as selective GSK-3beta inhibitors

Within the histopathology of Alzheimer's disease (AD) certain hallmarks are beeing observed. The occurance of protein deposits belong to such characteristic features. Such deposits can be found extracellular as beta-amyloid (Abeta) plaques and intracellular as neurofibrillary tangles (NFTs). In the search for novel AD therapeutics it became of great interest to investigate the formation of NFTs and their contribution to the AD symptomatic. NFTs consist of hyperphosphorylated tau protein. Within the phosphorylation process of tau protein two kinases are of great importance: cyclin dependent kinase 5 (cdk5) and its truncated regulatory subunit p25 and glycogen synthase kinase 3beta (GSK-3beta). The role of both kinases within the NFT formation process is still under debate. To better understand the pathophysiological process highly selective inhibitors of both kinases are of value. Known inhibitors lack the necessary selectivity. We developed novel 1-aza-9-oxafluo-renes as selective GSK-3beta inhibitors. Structure-activity relationships of a series of 4-phenyl substituted derivatives are discussed. Variation of the 3-side chain led to selective carbonyl amide derivatives with selectivity factors of more than 100 at the tested ATP competitor concentrations. Such selectivities permit specific investigation of the role of GSK-3beta within the NFT formation processes.     

Aspartic proteases involved in Alzheimer's disease

Alzheimer's disease afflicts every tenth human aged over 65. Despite the dramatic progress that has been made in understanding the disease, the exact cause of Alzheimer's disease is still unknown. Most gene mutations associated with Alzheimer's disease point at the same culprits: amyloid precursor protein and ultimately amyloid beta. The enigmatic proteases alpha-,beta-, and gamma-secretase are the three executioners of amyloid precursor protein processing, and disruption of their delicate balance is suspected to result in Alzheimer's disease. Significant progress has been made in the selective control of these proteases, regardless of the availability of structural information. Not even the absence of a robust cell-free assay for gamma-secretase could hamper the identification of nonpeptidic inhibitors of this enzyme for long. Within five years, four distinctly different structural moieties were developed and the first drug candidates are in clinical trials. Unfortunately, selective inhibition of amyloid beta formation remains a crucial issue because fundamental fragments of the gamma-secretase complex are important for other signaling events. This problem makes beta-secretase inhibition and alpha-secretase induction even more appealing.     

Design, synthesis, and biological activity of urea derivatives as anaplastic lymphoma kinase inhibitors

In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells.     

Discovery and structure-activity relationship studies of a unique class of HIV-1 integrase inhibitors

HIV-1 integrase (IN) is an essential enzyme for viral replication and a validated target for the development of drugs against AIDS. Currently there are no approved drugs that target IN. However, new IN inhibitors are under clinical investigation. As more IN inhibitors enter human drug trials, there is a growing need for the design of novel lead compounds with diverse structural scaffolds and promising pharmacokinetic properties to counteract the difficulties observed with first-generation IN inhibitors. We have identified a novel class of IN inhibitors through the systematic exploration of structure-activity relationships in a series of linomide analogues. The predicted bound conformation of the most active analogues inside the IN active site also supports the observed structure-activity correlation in this new compound class.     

Structure-activity relationships of phenylalkylamines as agonist ligands for 5-HT(2A) receptors

Agonist activation of central 5-HT(2A) receptors results in diverse effects, such as hallucinations and changes of consciousness. Recent findings indicate that activation of the 5-HT(2A) receptor also leads to interesting physiological responses, possibly holding therapeutic value. Selective agonists are needed to study the full therapeutic potential of this receptor. 5-HT(2A) ligands with agonist profiles are primarily derived from phenylalkylamines, indolealkylamines, and certain piperazines. Of these, phenylalkylamines, most notably substituted phenylisopropylamines, are considered the most selective agonists for 5-HT(2) receptors. This review summarizes the structure-activity relationships (SAR) of phenylalkylamines as agonist ligands for 5-HT(2A) receptors. Selectivity is a central theme, as is selectivity for the 5-HT(2A) receptor and for its specific signaling pathways. SAR data from receptor affinity studies, functional assays, behavioral drug discrimination as well as human studies are discussed.     

Design and stereoselective synthesis of retinoids with ferrocene or N-butylcarbazole pharmacophores that induce post-differentiation apoptosis in acute promyelocytic leukemia cells

New ferrocene and N‐alkylcarbazole retinoids were designed and synthesized stereoselectively in good yields. A number of these synthesized ligands, in particular 2 , 3 , and 11 , were found to exhibit a high RARα activation potential and to effectively induce post‐differentiation apoptosis in NB4 acute promyelocytic leukemia (APL) cells. Increasing the length of the side chain attached to the heterocycle of the carbazole arotinoids creates new opportunities for altered compound catabolism and for fine‐tuning of the apoptosis‐inducing potential of the ligand. In the carbazole series of new retinoids, maximal activity was established for N‐butylcarbazole analogue 11 in all assays (i.e., RARα activation, differentiation induction, and apoptosis induction). Study of the mechanism of apoptosis revealed an activation of initiator caspases‐8 and ‐9, followed by efficient cleavage of effector caspase‐3 on day 6 of treatment. Subsequent induction of a caspase cascade in NB4 cells triggered ultimate leukemic cell death. The selected ligands 2 , 3 , and 11 may provide alternate options for the treatment of APL in cases of life‐threatening ATRA syndrome, resistance, and high toxicity to conventionally used retinoids.     

Structure-activity studies on suramin analogues as inhibitors of NAD+-dependent histone deacetylases (sirtuins)

Suramin is a symmetric polyanionic naphthylurea originally used for the treatment of trypanosomiasis and onchocerciasis. Suramin and diverse analogues exhibit a broad range of biological actions in vitro and in vivo, including, among others, antiproliferative and antiviral activity. Suramin derivatives usually target purinergic binding sites. Class III histone deacetylases (sirtuins) are amidohydrolases that require nicotinamide adenine dinucleotide (NAD(+)) as a cofactor for their catalytic mechanism(.) Deacetylation of the target proteins leads to a change in conformation and alters the activity of the proteins in question. Suramin was reported to inhibit human sirtuin 1 (SIRT1). We tested a diverse set of suramin analogues to elucidate the inhibition of the NAD(+)-dependent histone deacetylases SIRT1 and SIRT2 and discovered selective inhibitors of human sirtuins with potency in the two-digit nanomolar range. In addition, the structural requirements for the binding of suramin derivatives to sirtuins were investigated by molecular docking. The recently published X-ray crystal structure of human SIRT5 in complex with suramin and the human SIRT2 structure were used to analyze the interaction mode of the novel suramin derivatives.     

Discovery of a Class of Potent and Selective Non-competitive Sentrin-Specific Protease 1 Inhibitors

Sentrin-specific proteases (SENPs) are responsible for the maturation of small ubiquitin-like modifiers (SUMOs) and the deconjugation of SUMOs from their substrate proteins. Studies on prostate cancer revealed an overexpression of SENP1, which promotes prostate cancer progression as well as metastasis. Therefore, SENP1 has been identified as a novel drug target against prostate cancer. Herein, we report the discovery and biological evaluation of potent and selective SENP1 inhibitors. A structure-activity relationship (SAR) of the newly identified pyridone scaffold revealed allosteric inhibitors with very attractive in vitro ADMET properties regarding plasma binding and plasma stability for this challenging target. This study also emphasizes the importance of biochemical mode of inhibition studies for de novo designed inhibitors.     

Structure-activity relationship studies on the immune stimulatory effects of base-modified CpG toll-like receptor 9 agonists

Synthetic oligodeoxynucleotides containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are able to stimulate potent immune responses through a signaling pathway involving Toll-like receptor 9 (TLR9). We have investigated the structure-activity relationship (SAR) of base-modified CpG oligonucleotides with TLR9 by measuring TLR9 activation by 20-mer oligonucleotides having just a single human recognition motif (5'-GTCGTT-3') in functional cell-based TLR9 assays. Substitution of guanine by hypoxanthine and 6-thioguanine resulted in activity similar to the unmodified parent molecule, whereas purine, 2-aminopurine, 2,6-diaminopurine, and 8-oxo-7,8-dihydroguanine substitution resulted in approximately 40-60 % reduction in activity, and 7-deazaguanine substitution led to the strongest (80 %) reduction in TLR9 stimulation. Furthermore, none of the investigated modifications at C5 and N4 of cytosine were well tolerated with respect to human TLR9 stimulation. Our results are compatible with a SAR model in which guanine is recognized by the Hoogsteen site, and C5 is most critical for recognition of cytosine. In addition, we found significant species-specific differences between human and murine TLR9 recognition, which demonstrates the importance of choosing appropriate assay systems for SAR studies.     

Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor

The Eph-ephrin system, including the EphA2 receptor and the ephrinA1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist that is able to inhibit EphA2 receptor activation; it is therefore potentially useful as a novel EphA2 receptor-targeting agent. Herein we explore the structure-activity relationships of a focused set of lithocholic acid derivatives based on molecular modeling investigations and displacement binding assays. Our exploration shows that while the 3-α-hydroxy group of lithocholic acid has a negligible role in recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrinA1 to EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits the EphA2-ephrinA1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand binding domain of EphA2, with a steady-state dissociation constant (K(D) ) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 as well as cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrinA1 ligand. These findings suggest that cholanic acid can be used as a template structure for the design of effective EphA2 antagonists, and may have potential impact in the elucidation of the role played by this receptor in pathological conditions.     

Synthesis and structure-activity relationship studies of HIV-1 virion infectivity factor (Vif) inhibitors that block viral replication

The human immunodeficiency virus 1 (HIV-1) virion infectivity factor (Vif) protein, essential for in vivo viral replication, protects the virus from innate antiviral cellular factor apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G; A3G) and is an attractive target for the development of novel antiviral therapeutics. We have evaluated the structure-activity relationships of N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (RN-18), a small molecule recently identified as an inhibitor of Vif function that blocks viral replication only in nonpermissive cells expressing A3G, by inhibiting Vif-A3G interactions. Microwave-assisted cross-coupling reactions were developed to prepare a series of RN18 analogues with diverse linkages and substitutions on the phenyl rings. A dual cell-based assay system was used to assess antiviral activity against wild-type HIV-1 in both nonpermissive (H9) and permissive (MT4) cells that also allowed evaluation of specificity. In general, variations of phenyl substitutions were detrimental to antiviral potency and specificity, but isosteric replacements of amide and ether linkages were relatively well tolerated. These structure-activity relationship data define structural requirements for Vif-specific activity, identify new compounds with improved antiviral potency and specificity, and provide leads for further exploration to develop new antiviral therapeutics.     

From high-throughput cell culture screening to mouse model: identification of new inhibitor classes against prion disease

Transmissible spongiform encephalopathies (TSE) or prion diseases belong to a category of fatal and so far untreatable neurodegenerative conditions. All prion diseases are characterized by both degeneration in the central nervous system (CNS) in humans and animals and the deposition and accumulation of Proteinase K-resistant prion protein (PrP(res)). Until now, no pharmaceutical product has been available to cure these diseases or to alleviate their associated symptoms. Here, a cell-culture screening system is described that allows for the large-scale analysis of the PrP(res) inhibitory potential of a library of compounds and the identification of structural motifs leading potent compounds able to cause PrP(res) clearance at the cellular level. Based on different scrapie-infected cell lines, 10,000 substances were tested, out of which 530 potential inhibitors were identified. After re-screening and validation using a series of dilutions, 14 compounds were identified as the most effective. These 14 compounds were then used for therapeutic studies in a mouse bioassay to test and verify their in vivo potency. Two compounds exhibited therapeutic potential in the mouse model by significantly extending the survival time of intracerebrally infected mice, when treated 90 days after infection with scrapie.     

Virtual screening leads to the discovery of an effective antagonist of lymphocyte function-associated antigen-1

The binding of lymphocyte function-associated antigen-1 (LFA-1) to its ligand on endothelial cells, intercellular adhesion molecule-1 (ICAM-1), is a crucial step in the migration of leukocytes during the early stages of inflammation and is also involved in T-cell activation. In this paper, we report the identification of a series of novel antagonists of the LFA-1/ICAM-1 interaction using ligand-based virtual screening (VS), analogue design, and structure-activity relationship (SAR) analysis. Candidate compounds were evaluated in protein binding and cell adhesion assays. Experimental evaluation of only 25 candidates selected from a pool of approximately 2.5 million database compounds identified an initial hit that could be expanded and converted into a lead that effectively blocked the interaction between LFA-1 and ICAM-1.