Small Molecule Scaffolds Research Articles

Septin6 as a new approach for AD treatment

The number of Alzheimer’s disease (AD) patients is increasing and new therapeutic approaches need to be proposed urgently. In recent years, some researchers have focused on the relationship between calcium homeostasis and AD; however, selective regulation of abnormal calcium signaling pathways and related targets of action remain unclear, presenting a challenge. Gerard Griffioen’s team in Belgium has proposed a self-reinforcing amplification between cytoplasmic calcium concentration [Ca2+]cyto and AD pathology in previous studies, discovering a new kind of small-molecule scaffold protein. The protein, ReS19-T, can stabilize the structure of septin filaments and significantly improve the core pathology of AD by inhibiting the pathological activation of store-operated calcium entry (SOCE) and restoring calcium homeostasis, thereby suggesting a new avenue for therapeutic intervention. However, there is still a way to go before clinical application. There are some questions. SEP2/6/7 hexamer plays a role in maintaining immune function, so could ReS19-T affect this function and impact immune responses? Moreover, both Stim1 and Orai (affected by TRPC) contribute to SOCE. The TRPC-specific inhibitor SKF-96365 is highly selective, and its relationship with AD remains to be investigated. Future studies might use SKF-96365 to validate the therapeutic effect of Res19-T. In conclusion, Septin6 as a new approach to AD treatment expects more relevant research to emerge.

Corrigendum: Microwave Synthesized Hydrophilic 4‐Oxo‐2‐phenylquinazoline‐3(4H)–carboximidamide and –Carboxamide as Privileged Small Molecule Scaffolds with Anti‐HIV‐1 Activities

Corrigendum: Microwave Synthesized Hydrophilic 4‐Oxo‐2‐phenylquinazoline‐3(4H)–carboximidamide and –Carboxamide as Privileged Small Molecule Scaffolds with Anti‐HIV‐1 Activities

Microwave Synthesized Hydrophilic 4‐Oxo‐2‐phenylquinazoline‐3(4H)–carboximidamide and –Carboxamide as Privileged Small Molecule Scaffolds with Anti‐HIV‐1 Activities

AbstractOpportunistic infections like tuberculosis (TB) are prevalent in HIV‐infected individuals. In an approach to introducing an industrial‐scale microwave‐assisted production of agents having both antiviral and antibacterial properties, a set of hydrophilic 2,3‐substituted quinazolinones, 4‐oxo‐2‐phenylquinazoline‐3(4H)‐carboximidamide (Qg) and 4‐oxo‐2‐phenylquinazoline‐3(4H)‐carboxamide (Qu), were afforded in encouraging yields. They were obtained by treating 2‐benzamidobenzoyl chloride with guanidine hydrochloride or urea in the presence of potassium carbonate in DMF. DFT calculations were employed for geometrical optimization and vibrational frequency analysis of the compounds. Free energies of solvation (ΔGsol) of Qg, and Qu calculated using DFT lend evidence in support of their hydrophilicity The anti‐HIV‐1 activities of the small‐molecule quinazolinone derivatives Qg and Qu were investigated. The synthons under in vitro conditions inhibit the entry of HIV in receptor cells with low cytotoxicity (EC50=50.53 nM for Qu and EC50=97.92 nM for Qg). The study identifies the antiviral properties of the two benzo‐fused pyrimidine derivatives, which were earlier reported to have antitubercular activity. Our findings indicate the scope for further synthetic explorations of these quinazolinone scaffolds that can lead to developing promising alternatives in drug design, pharmaceutics, and clinical research.

Evaluating Known Zika Virus NS2B-NS3 Protease Inhibitor Scaffolds via In Silico Screening and Biochemical Assays.

The NS2B-NS3 protease (NS2B-NS3pro) is regarded as an interesting molecular target for drug design, discovery, and development because of its essential role in the Zika virus (ZIKV) cycle. Although no NS2B-NS3pro inhibitors have reached clinical trials, the employment of drug-like scaffolds can facilitate the screening process for new compounds. In this study, we performed a combination of ligand-based and structure-based in silico methods targeting two known non-peptide small-molecule scaffolds with micromolar inhibitory activity against ZIKV NS2B-NS3pro by a virtual screening (VS) of promising compounds. Based on these two scaffolds, we selected 13 compounds from an initial library of 509 compounds from ZINC15's similarity search. These compounds exhibited structural modifications that are distinct from previously known compounds yet keep pertinent features for binding. Despite promising outcomes from molecular docking and initial enzymatic assays against NS2B-NS3pro, confirmatory assays with a counter-screening enzyme revealed an artifactual inhibition of the assessed compounds. However, we report two compounds, 9 and 11, that exhibited antiviral properties at a concentration of 50 μM in cellular-based assays. Overall, this study provides valuable insights into the ongoing research on anti-ZIKV compounds to facilitate and improve the development of new inhibitors.

Machine learning and biological evaluation-based identification of a potential MMP-9 inhibitor, effective against ovarian cancer cells SKOV3

MMP-9, also known as gelatinase B, is a zinc-metalloproteinase family protein that plays a key role in the degradation of the extracellular matrix (ECM). The normal function of MMP-9 includes the breakdown of ECM, a process that aids in normal physiological processes such as embryonic development, angiogenesis, etc. Interruptions in these processes due to the over-expression or downregulation of MMP-9 are reported to cause some pathological conditions like neurodegenerative diseases and cancer. In the present study, an integrated approach for ML-based virtual screening of the Maybridge library was carried out and their biological activity was tested in an attempt to identify novel small molecule scaffolds that can inhibit the activity of MMP-9. The top hits were identified and selected for target-based activity against MMP-9 protein using the kit (Biovision K844). Further, MTT assay was performed in various cancer cell lines such as breast (MCF-7, MDA-MB-231), colorectal (HCT119, DL-D-1), cervical (HeLa), lung (A549) and ovarian cancer (SKOV3). Interestingly, one compound viz., RJF02215 exhibited anti-cancer activity selectively in SKOV3. Wound healing assay and colony formation assay performed on SKOV3 cell line in the presence of RJF02215 confirmed that the compound had a significant inhibitory effect on this cell line. Thus, we have identified a novel molecule that can inhibit MMP-9 activity in vitro and inhibits the proliferation of SKOV3 cells. Novel molecules based on the structure of RJF02215 may become a good value addition for the treatment of ovarian cancer by exhibiting selective MMP-9 activity. Communicated by Ramaswamy H. Sarma

Microwave-assisted Synthesis of 3-amino-2-phenylquinazolin-4(3H)-one (QH) and 4-oxo-2-phenylquinazoline-3(4H)-carbothioamide (QTh)

Background: Microwave synthesis has developed as a powerful tool for the cost-effective and greener synthesis of organic molecules, including quinazolines. Irradiation with microwave leads to the excitation of molecules and equitable distribution of thermal energy in a much shorter time than conventional synthesis. This results in shorter reaction time and, more often than not, higher efficiency Objective: The primary objective of the work presented in this article was to prepare hydrazine hydrate or thiourea derivative of quinazolines through microwave synthesis as small-molecule scaffolds for fur-ther need-based functionalisation, isolation, and characterisation. We, herein, report the synthesis of two quinazolinone derivatives of thiourea and hydrazine, 3-amino-2-phenylquinazolin-4(3H)-one (QH) and 4-oxo-2-phenylquinazoline-3(4H)-carbothioamide (QTh), respectively. Method: A multi-step synthetic strategy starting from anthranilic acid was employed to synthesise the small molecule quinazolinones 3-amino-2-phenylquinazolin-4(3H)-one (QH) and 4-oxo-2-phenylquinazoline-3(4H)-carbothioamide (QTh). The compounds were synthesised by reacting hydra-zine and thiourea with 2-benzamidobenzoyl chloride in DMF under microwave irradiation (800 W at 135 °C for 4 min) in the presence of potassium carbonate. The acid chloride was prepared by chlorination of 2-benzamidobenzoic acid, which in turn was synthesised from anthranilic acid by benzoylation. This method is an efficient alternative approach to synthesising quinazolinones from benzoxazin-4-ones. Results: We have successfully synthesised, isolated, and characterised the quinazolinone derivative QH (yield: 81%) and QTh (yield: 85%). The structures of the compounds were established through spectro-scopic techniques. Theoretical optimisation of the structures was also achieved using DFT. The HOMO-LUMO difference for QH and QTh was calculated to be 4.60 and 4.47 eV, respectively. Conclusion: The reported protocol is advantageous over conventional methods of quinazoline synthesis from benzoxazin-4-ones. The time required for the reaction is much less (4 min) as compared to the usual requirements of reflux (> 4 h); the higher energy gap of QH indicates greater stability than that of QTh.

Nimesulide, a COX-2 inhibitor, sensitizes pancreatic cancer cells to TRAIL-induced apoptosis by promoting DR5 clustering †

ABSTRACT Nimesulide is a nonsteroidal anti-inflammatory drug and a COX-2 inhibitor with antitumor and antiproliferative activities that induces apoptosis in oral, esophagus, breast, and pancreatic cancer cells. Despite being removed from the market due to hepatotoxicity, nimesulide is still an important research tool being used to develop new anticancer drugs. Multiple studies have been done to modify the nimesulide skeleton to develop more potent anticancer agents and related compounds are promising scaffolds for future development. As such, establishing a mechanism of action for nimesulide remains an important part of realizing its potential. Here, we show that nimesulide enhances TRAIL-induced apoptosis in resistant pancreatic cancer cells by promoting clustering of DR5 in the plasma membrane. In this way, nimesulide acts like a related compound, DuP-697, which sensitizes TRAIL-resistant colon cancer cells in a similar manner. Our approach applies a time-resolved FRET-based biosensor that monitors DR5 clustering and conformational states in the plasma membrane. We show that this tool can be used for future high-throughput screens to identify novel, nontoxic small molecule scaffolds to overcome TRAIL resistance in cancer cells.

A Machine Learning Language to Build a QSAR Model of Pyrazoline Derivative Inhibitors Targeting Mycobacterium tuberculosis Strain H37Rv

Background:Machine learning has become an essential tool for drug research to generate pertinent structural information to design drugs with higher biological activities. Quantitative structureactivity relationship (QSAR) is considered one technique. QSAR study involves two main steps: first is the generation of descriptors, and the second is building and validating the models.Aim:By using a Python program language for building the QSAR model of pyrazoline derivatives, the data were collected from diverse literature for the inhibition of Mycobacterium tuberculosis. Pyrazoline, a small molecule scaffold, could block the biosynthesis of mycolic acids, resulting in mycobacteria death and leading to anti-tubercular drug discovery.Methods:We have developed a new Python script that effectively uses CDK descriptor as the independent variable and anti-tubercular bioactivity as the dependent variable in building and validating the best QSAR model. The built QSAR model was further cross-validated by using the external test set compounds. Then, the three algorithms, viz. multiple linear regression, support vector machine, and partial least square classifiers, were used to differentiate and compare their r2 values.Results:Our generated QSAR model via an open-source python program predicted well with external test set compounds. The generated statistical model afforded the ordinary least squares (OLS) regression as R2 value of 0.514, F value of 5.083, the adjusted R2 value of 0.413, and std. error of 0.092. Moreover, the multiple linear regression showed the R2 value of 0.5143, reg.coef_ of, -0.07795 (PC1), 0.01619 (PC2), 0.03763 (PC3), 0.07849 (PC4), -0.09726 (PC5), and reg.intercept_ of 4.8324. The performance of the model was determined by the support vector machine classifier of sklearn, module and it provided a model score of 0.5901. Further, the model performance was supported by a partial least square regression, and it showed the R2 value of 0.5901. The model performance was validated, and the model predicted similar values when compared to that of the train set, and the plotted linear curve between the predicted and actual pMIC50 value showed all data to fall over the middle linear line.Conclusion:We have found that the model score obtained using this script via three diverse algorithms correlated well, and there was not much difference between them; the model may be useful in the design of a similar group of pyrazoline analogs as anti-tubercular agents.

Coumaryl-sulfonamide moiety: Unraveling their synthetic strategy and specificity towardhCA IX/XII, facilitating anticancer drug development.

Currently, cancer is the most grieving threat to society. The cancer-related death rate has had an ascending trend, despite the implementation of numerous treatment strategies or the discovery of an array of potent molecules against several pathways of cancer growth. The need of the hour is to prevent the multidrug resistance toll, and the current efforts have been bestowed upon a versatile small molecule scaffold, coumarin (benz[α]pyrone), a natural compound possessing interesting affinity towardthe cancer target human carbonic anhydrase (hCA), focusing on hCA I, II, IX, and XII. Along with coumarin, the age-old known antibacterial drug sulfonamide, when conjugated at positions 3, 7, and 8 of coumarin either with a linker group or as a single entity, has been reported to enhance the affinity of coumarin towardthe overexpressed enzymes in tumor cell lines. The sulfonamides have been listed as obsolete drugs due to the severe side effects caused by them; however, their affinity towardthe hCA-zinc-binding core has attracted the attention of researchers. Hence, in the process of drug development, coumarin and sulfonamides have remained the choice of last resort. To unveil the synthetic strategy of coumarin-sulfonamide conjugation, their rationale for inhibiting cancer cells/enzymes, and their affinity towardvarious types of carcinoma have been the sole goal of the researchers. This review specifically focuses on the mechanism of action and the structure-activity relationship through synthetic strategies and the binding affinity of coumaryl-sulfonamide conjugates with the anticancer targets possessing the highest enzyme affinity, since 2008.

Recent Advances in Gold(I)-Catalyzed Approaches to Three-Type Small-Molecule Scaffolds via Arylalkyne Activation.

Gold catalysts possess the advantages of water and oxygen resistance, with the possibility of catalyzing many novel chemical transformations, especially in the syntheses of small-molecule skeletons, in addition to achieving the rapid construction of multiple chemical bonds and ring systems in one step. In this feature paper, we summarize recent advances in the construction of small-molecule scaffolds, such as benzene, cyclopentene, furan, and pyran, based on gold-catalyzed cyclization of arylalkyne derivatives within the last decade. We hope that this review will serve as a useful reference for chemists to apply gold-catalyzed strategies to the syntheses of related natural products and active molecules, hopefully providing useful guidance for the exploration of additional novel gold-catalyzed approaches.

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution changes

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed "fixation-driven chemical cross-linking of exogenous ligands (FixEL)," which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOIs such as the scaffold of PET tracer derivatives or drug-like small molecules.

Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen.

Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator.

Gold-Mediated Multiple Cysteine Arylation for the Construction of Highly Constrained Bicycle Peptides.

Bicycles are constrained bicyclic peptides formed through reaction of three cysteine residues within a linear sequence with a trivalent, symmetrical small molecule scaffold. Bicycles with high binding affinities to therapeutically important targets can be discovered using screening technologies such as phage display. Increasing the chemical diversity of Bicycles should improve the probability of finding hits to new targets and can be achieved by expanding the toolbox of Bicycle forming chemistries. Gold(III) S-arylation has recently been described as a method for the efficient bioconjugation of cysteine residues under conditions compatible with phage display. Herein, we explore the scope and generality of this methodology for Bicycle construction through the synthesis and evaluation of four novel tris-Gold complexes. These new scaffolds were systematically reacted with a variety of peptide sequences, varying in amino acid loop lengths. All four scaffolds proved to be capable and selective reactive partners for each peptide sequence and afforded the desired Bicycle products in 13-48% isolated yield. This work exemplifies Gold-mediated arylation as a general approach for construction of novel, highly constrained Bicycles.

Pharmacophore-Based Discovery of Viral RNA Conformational Modulators.

New RNA-binding small-molecule scaffolds are needed to unleash the pharmacological potential of RNA targets. Here we have applied a pharmacophore-based virtual screening approach, seldom used in the RNA recognition field, to identify novel conformational inhibitors of the hepatitis C virus internal ribosome entry site. The conformational effect of the screening hits was assessed with a fluorescence resonance energy transfer assay, and the affinity, specificity, and binding site of the ligands were determined using a combination of fluorescence intensity and NMR spectroscopy experiments. The results indicate that this strategy can be successfully applied to discover RNA conformational inhibitors bearing substantially less positive charge than the reference ligands. This methodology can potentially be accommodated to other RNA motifs of pharmacological interest, facilitating the discovery of novel RNA-targeted molecules.

Novel and Potential Small Molecule Scaffolds as DYRK1A Inhibitors by Integrated Molecular Docking-Based Virtual Screening and Dynamics Simulation Study.

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a novel, promising and emerging biological target for therapeutic intervention in neurodegenerative diseases, especially in Alzheimer’s disease (AD). The molMall database, comprising rare, diverse and unique compounds, was explored for molecular docking-based virtual screening against the DYRK1A protein, in order to find out potential inhibitors. Ligands exhibiting hydrogen bond interactions with key amino acid residues such as Ile165, Lys188 (catalytic), Glu239 (gk+1), Leu241 (gk+3), Ser242, Asn244, and Asp307, of the target protein, were considered potential ligands. Hydrogen bond interactions with Leu241 (gk+3) were considered key determinants for the selection. High scoring structures were also docked by Glide XP docking in the active sites of twelve DYRK1A related protein kinases, viz. DYRK1B, DYRK2, CDK5/p25, CK1, CLK1, CLK3, GSK3β, MAPK2, MAPK10, PIM1, PKA, and PKCα, in order to find selective DYRK1A inhibitors. MM/GBSA binding free energies of selected ligand–protein complexes were also calculated in order to remove false positive hits. Physicochemical and pharmacokinetic properties of the selected six hit ligands were also computed and related with the proposed limits for orally active CNS drugs. The computational toxicity webserver ProTox-II was used to predict the toxicity profile of selected six hits (molmall IDs 9539, 11352, 15938, 19037, 21830 and 21878). The selected six docked ligand–protein systems were exposed to 100 ns molecular dynamics (MD) simulations to validate their mechanism of interactions and stability in the ATP pocket of human DYRK1A kinase. All six ligands were found to be stable in the ATP binding pocket of DYRK1A kinase.

Identification and Optimization of a Novel HIV-1 Integrase Inhibitor.

Human immunodeficiency virus-1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV-1, like all retroviruses, stably integrates its vDNA copy into host chromatin, a process allowing for permanent infection. This essential step for HIV-1 replication is catalyzed by viral integrase (IN) and aided by cellular protein LEDGF/p75. In addition, IN is also crucial for proper virion maturation as it interacts with the viral RNA genome to ensure encapsulation of ribonucleoprotein complexes within the protective capsid core. These key functions make IN an attractive target for the development of inhibitors with various mechanisms of action. We conducted a high-throughput screen (HTS) of ∼370,000 compounds using a homogeneous time-resolved fluorescence-based assay capable of capturing diverse inhibitors targeting multifunctional IN. Our approach revealed chemical scaffolds containing diketo acid moieties similar to IN strand transfer inhibitors (INSTIs) as well as novel compounds distinct from all current IN inhibitors including INSTIs and allosteric integrase inhibitors (ALLINIs). Specifically, our HTS resulted in the discovery of compound 12, with a novel IN inhibitor scaffold amenable for chemical modification. Its more potent derivative 14e similarly inhibited catalytic activities of WT and mutant INs containing archetypical INSTI- and ALLINI-derived resistant substitutions. Further SAR-based optimization resulted in compound 22 with an antiviral EC50 of ∼58 μM and a selectivity index of >8500. Thus, our studies identified a novel small-molecule scaffold for inhibiting HIV-1 IN, which provides a promising platform for future development of potent antiviral agents to complement current HIV-1 therapies.

A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure-activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action.

MACHINE LEARNING ALGORITHM USED TO BUILD A QSAR MODEL FOR PYRAZOLINE SCAFFOLD AS ANTI-TUBERCULAR AGENT

Machine learning has become an essential tool for drug research to generate pertinent structural information to design drugs with higher biological activities. In this paper, we used python program language on pyrazoline scaffold, which is collected from diverse literature for the inhibition of Mycobacterium tuberculosis. Pyrazoline, a small molecule scaffold could block the biosynthesis of mycolic acids, resulting in mycobacteria death and leading to anti-tubercular drug discovery. The generated QSAR model afforded the ordinary least squares (OLS) regression as R2 = 0.380, F=4.909, and Q2 =0.303, reg. coef_ developed were of 0.00651593 (molecular weight), -0.0069445 (hydrogen bond acceptor), - 0.07576775 (hydrogen bond donor), -0.239021 (LogP) and reg. intercept of 3.10331589018553 developed through statsmodels.formula module. The support vector machine of the sklearn module generated the model score of 0.6294242262068762, the developed model was cross-validated by using the test set compounds and plotting the linear curve between the predicted and actual pMIC50 value. We have found that the values obtained using this script correlated well and may be useful in the design of a similar group of pyrazoline analogs as anti-tubercular agents.

Discovery and biological evaluation of phthalazines as novel non-kinase TGFβ pathway inhibitors.

TGFβ is crucial for the homeostasis of epithelial and neural tissues, wound repair, and regulating immune responses. Its dysregulation is associated with a vast number of diseases, of which modifying the tumor microenvironment is one of vital clinical interest. Despite various attempts, there is still no FDA-approved therapy to inhibit the TGFβ pathway. Major mainstream approaches involve impairment of the TGFβ pathway via inhibition of the TGFβRI kinase. With the purpose to identify non-receptor kinase-based inhibitors to impair TGFβ signaling, an in-house chemical library was enriched, through a computational study, to eliminate TGFβRI kinase activity. Selected compounds were screened against a cell line engineered with a firefly luciferase gene under TGFβ-Smad-dependent transcriptional control. Results indicated moderate potency for a molecule with phthalazine core against TGFβ-Smad signaling. A series of phthalazine compounds were synthesized and evaluated for potency. The most promising compound (10p) exhibited an IC50 of 0.11±0.02μM and was confirmed to be non-cytotoxic up to 12μM, with a selectivity index of approximately 112-fold. Simultaneously, 10p was confirmed to reduce the Smad phosphorylation using Western blot without exhibiting inhibition on the TGFβRI enzyme. This study identified a novel small-molecule scaffold that targets the TGFβ pathway via a non-receptor-kinase mechanism.

Stable analogs of 13‑hydroxy-9,10-trans-epoxy-(11E)-octadecenoate (13,9-HEL), an oxidized derivative of linoleic acid implicated in the epidermal skin barrier

Hydroxy-epoxy- and trihydroxy derivatives of linoleic acid are proposed to play an essential function in formation of the mammalian skin permeability barrier, which could account for the essential nature of its precursor, linoleic acid. Recent literature suggests that a specific oxidized enone derivative of LA esterified in ceramides facilitates binding to proteins, potentially serving a structural role in formation of the epidermal skin barrier. However, it is still to be established if other linoleic acid derivatives are also required for skin barrier formation, and whether the essential role is performed exclusively by an esterified, structural lipid or as an unesterified, labile signaling lipid, or by some combination of these derivatives. Progress in this domain is limited by lack of availability of hydroxy‑epoxy-and trihydroxy- and octadecenoate derivatives of linoleic acid and related compounds, and challenges in maintaining them in the unesterified lipid pool. Here we describe methods for the total synthesis of hydroxy‑epoxy-octadecenoate derivatives of linoleic acid (HEL11HEL=hydroxy epoxide linoleic acid derivativeTHL=trihydroxy linoleic acid derivativeSA=sebaleic acid), and stable analogs that are designed to be resistant to inactivation by: (a) acylation/esterification (thus trapping these lipids in the free acid pool), (b) dehydrogenation, and (c) analogs combining both modifications. We further provide a total synthesis of corresponding hydroxy‑epoxy- derivatives of sebaleic acid (a regioisomer of linoleic acid present in skin), and of small molecule scaffolds containing the allylic and non-allylic epoxide 7-carbon substructures shared by both families of hydroxy‑epoxy-and trihydroxy- octadecenoates. Finally, we demonstrate that 2,2-dimethyl analogs of hydroxy‑epoxy-and trihydroxy- octadecenoates are resistant to esterification with an in vitro assay and thus provide a novel template for stabilizing labile, bioactive lipids as free acids by preventing acylation/esterification.