Discovery,Synthesis, and Optimization of Diarylisoxazole‐3‐carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

The mitochondrial permeability transition pore (mtPTP) is a Ca²⁺‐requiring mega‐channel which, under pathological conditions, leads to the deregulated release of Ca²⁺ and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1 , 5‐(3‐hydroxyphenyl)‐N‐(3,4,5‐trimethoxyphenyl)isoxazole‐3‐carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC₅₀<0.39 μM ) and showed no interference on the inner mitochondrial membrane potential (rhodamine 123 uptake EC₅₀>100 μM ). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N‐(3‐chloro‐2‐methylphenyl)‐5‐(4‐fluoro‐3‐hydroxyphenyl)isoxazole‐3‐carboxamide ( 60 ), was validated in a biologically relevant zebrafish model of collagen VI congenital muscular dystrophies.     

N‐Phenylbenzamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

Persistent opening of the mitochondrial permeability transition pore (PTP), an inner membrane channel, leads to mitochondrial dysfunction and renders the PTP a therapeutic target for a host of life‐threatening diseases. Herein, we report our effort toward identifying small‐molecule inhibitors of this target through structure–activity relationship optimization studies, which led to the identification of several potent analogues around the N‐phenylbenzamide compound series identified by high‐throughput screening. In particular, compound 4 (3‐(benzyloxy)‐5‐chloro‐N‐(4‐(piperidin‐1‐ylmethyl)phenyl)benzamide) displayed noteworthy inhibitory activity in the mitochondrial swelling assay (EC₅₀=280 nm ), poor‐to‐very‐good physicochemical as well as in vitro pharmacokinetic properties, and conferred very high calcium retention capacity to mitochondria. From the data, we believe compound 4 in this series represents a promising lead for the development of PTP inhibitors of pharmacological relevance.     

New Insight into the Structure–Activity Relationships of the Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors UCPH‐101 and UCPH‐102

In the present study, we made further investigations on the structure–activity requirements of the selective excitatory amino acid transporter 1 (EAAT1) inhibitor, 2‐amino‐4‐(4‐methoxyphenyl)‐7‐(naphthalen‐1‐yl)‐5‐oxo‐5,6,7,8‐tetrahydro‐4H‐chromene‐3‐carbonitrile (UCPH‐101), by exploring 15 different substituents (R¹) at the 7‐position in combination with eight different substituents (R²) at the 4‐position. Among the 63 new analogues synthesized, we identified a number of compounds that unexpectedly displayed inhibitory activities at EAAT1 in light of understanding the structure–activity relationship (SAR) of this inhibitor class extracted from previous studies. Moreover, the nature of the R¹ and R² substituents were observed to contribute to the functional properties of the various analogues in additive and non‐additive ways. Finally, separation of the four stereoisomers of analogue 14 g (2‐amino‐4‐([1,1′‐biphenyl]‐4‐yl)‐3‐cyano‐7‐isopropyl‐5‐oxo‐5,6,7,8‐tetrahydro‐4H‐chromene) was carried out, and in agreement with a study of a related scaffold, the R configuration at C4 was found to be mandatory for inhibitory activity, while both the C7 diastereomers were found to be active as EAAT1 inhibitors. A study of the stereochemical stability of the four pure stereoisomers 14 g ‐ A – D showed that epimerization takes places at C7 via a ring‐opening, C?C bond rotation, ring‐closing mechanism.     

Structural Investigation of the Binding of 5‐Substituted Swainsonine Analogues to Golgi α‐Mannosidase II

Golgi α‐mannosidase II (GMII) is a key enzyme in the N‐glycosylation pathway and is a potential target for cancer chemotherapy. The natural product swainsonine is a potent inhibitor of GMII. In this paper we characterize the binding of 5α‐substituted swainsonine analogues to the soluble catalytic domain of Drosophila GMII by X‐ray crystallography. These inhibitors enjoy an advantage over previously reported GMII inhibitors in that they did not significantly decrease the inhibitory potential of the swainsonine head‐group. The phenyl groups of these analogues occupy a portion of the binding site not previously seen to be populated with either substrate analogues or other inhibitors and they form novel hydrophobic interactions. They displace a well‐organized water cluster, but the presence of a C(10) carbonyl allows the reestablishment of important hydrogen bonds. Already approximately tenfold more active against the Golgi enzyme than the lysosomal enzyme, these inhibitors offer the potential of being extended into the N‐acetylglucosamine binding site of GMII for the creation of even more potent and selective GMII inhibitors.     

Stability and Efficiency of Mixed Aryl Phosphonate Prodrugs

A set of phosphonate prodrugs of a butyrophilin ligand was synthesized and evaluated for plasma stability and cellular activity. The mixed aryl acyloxy esters were prepared either via a standard sequence through the phosphonic acid chloride, or through the more recently reported, and more facile, triflate activation. In the best of cases, this class of prodrugs shows cellular potency similar to that of bis-acyloxyalkyl phosphonate prodrugs and plasma stability similar to that of aryl phosphonamidates. For example, {[((3E)-5-hydroxy-4-methylpent-3-en-1-yl) (naphthalen-2-yloxy)phosphoryl]oxy}methyl 2,2-dimethylpropanoate can activate BTN3A1 in K562 cells after just 15 minutes of exposure (at an EC₅₀ value of 31 nm ) and is only partially metabolized (60 % remaining) after 20 hours in human plasma. Other related novel analogues showed similar potency/stability profiles. Therefore, mixed aryl acyloxyalkyl phosphonate prodrugs are an exciting new strategy for the delivery of phosphonate-containing drugs.     

O‐(Triazolyl)methyl Carbamates as a Novel and Potent Class of Fatty Acid Amide Hydrolase (FAAH) Inhibitors

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O‐aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O‐(1,2,3‐triazol‐4‐yl)methyl carbamate derivatives were designed and synthesized exploiting a copper‐ catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure–activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC₅₀ values in the single‐digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug‐like properties.     

Improvement of SNAr Reaction Rate by an Electron‐Withdrawing Group in the Crosslinking of DNA Cytosine‐5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor

DNA cytosine 5‐methyltransferase (DNMT) catalyzes methylation at the C5 position of the cytosine residues in the CpG sequence. Aberrant DNA methylation patterns are found in cancer cells. Therefore, inhibition of human DNMT is an effective strategy for treating various cancers. The inhibitors of DNMT have an electron‐deficient nucleobase because this group facilitates attack by the catalytic Cys residue in DNMTs. Recently, we reported the synthesis and properties of mechanism‐based modified nucleosides, 2‐amino‐4‐halopyridine‐C‐nucleosides (d^XP), as inhibitors of DNMT. To develop a more efficient inhibitor of DNMT for oligonucleotide therapeutics, oligodeoxyribonucleotides (ODNs) containing other nucleoside analogues, which react more quickly with DNMT, are needed. Herein, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐3‐cyano‐4‐halopyridine‐C‐nucleosides (d^XP^CN) and ODNs containing d^XP^CN, as more reactive inhibitors of DNMTs. Nucleophilic aromatic substitution (S_NAr) of the designed nucleosides, d^XP^CN, was faster than that of d^XP, and the ODN containing d^XP^CN effectively formed a complex with DNMTs. This study suggests that the incorporation of an electron‐withdrawing group would be an effective method to increase reactivity toward the nucleophile of the DNMTs, while maintaining high specificity.     

Preparation of fatty 3,5-disubstituted isoxazole compounds from FA esters

Long-chain fatty compounds containing an isoxazole heterocyclic ring system substituted at the 3- and 5-ring positions were prepared in moderate to good yields (40–64%) in one pot by condensing FA esters such as methyl palmitate, stearate, oleate, or linoleate with the lithiated anion of N-(isopropylidene)isopropylamine followed by dehydrative cyclization. This approach allows readily available FA esters to be utilized and incorporated into the construction of the isoxazole ring system. These novel products were isolated then characterized by NMR, IR spectroscopy, GC-MS, and m.p. Mass spectra of the fatty isoxazole compounds, derived utilizing EI ionization, showed distinctive cleavage patterns that occurred uniformly along the fatty alkyl chain allowing the position of double bonds to be readily located. Two prominent ions at m/z 97 and 110 were common to all the fatty isoxazole compounds examined and were presumably from a McLafferty rearrangement and a cyclization displacement reaction, respectively.     

Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca2+ into Neurons

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, −4, and −5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, −4, and −5 hetero- or homomers at the plasma membrane and to the influx of Ca²⁺ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and −4 or TRPC1 and −5. NCAM-stimulated Ca²⁺ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca²⁺ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, −4, and −5 contributes to the NCAM-stimulated and PSA-dependent Ca²⁺ entry into neurons thereby influencing essential neural functions.     

Terpendole E and its Derivative Inhibit STLC‐ and GSK‐1‐Resistant Eg5

Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5–microtubule interaction and in several Eg5 mutants. 11‐Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule‐stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S‐trityl‐L ‐cysteine (STLC) and GSK‐1, both terpendole E and 11‐ketopaspaline only partially inhibited Eg5–microtubule interaction. Furthermore, terpendole E and 11‐ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5^D130A, Eg5^L214A) or GSK‐1 (Eg5^I299F, Eg5^A356T), but with the same extent of inhibition against wild‐type Eg5. Because Eg5^D130A and Eg5^L214A show cross‐resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop‐binding type Eg5 inhibitors.     

Highly Selective and Potent Human β-Secretase 2 (BACE2) Inhibitors against Type 2 Diabetes: Design,Synthesis, X-ray Structure and Structure–Activity Relationship Studies

Herein we present the design, synthesis, and biological evaluation of potent and highly selective β-secretase 2 (memapsin 1, beta-site amyloid precursor protein cleaving enzyme 2, or BACE 2) inhibitors. BACE2 has been recognized as an exciting new target for type 2 diabetes. The X-ray structure of BACE1 bound to inhibitor 2 a {N³-[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)propyl]amino]propyl]-5-[methyl(methylsulfonyl)amino]-N¹-[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide} containing a hydroxyethylamine isostere was determined. Based on this structure, a computational docking study was performed which led to inhibitor 2 a -bound BACE2 models. These were used to optimize the potency and selectivity of inhibitors. A systematic structure–activity relationship study led to the identification of determinants of the inhibitors’ potency and selectivity toward the BACE2 enzyme. Inhibitors 2 d [N³-[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)pentyl]amino]propyl]-N¹-methyl-N¹-[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide; K_i=0.031 nm , selectivity over BACE1: ≈174 000-fold] and 3 l [N¹-((2S,3R)-3-hydroxy-1-phenyl-4-((3-(trifluoromethyl)benzyl)amino)butan-2-yl)-N³,5-dimethyl-N³-((R)-1-phenylethyl)isophthalamide; K_i=1.6 nm , selectivity over BACE1: >500-fold] displayed outstanding potency and selectivity. Inhibitor 3 l is nonpeptide in nature and may pave the way to the development of a new class of potent and selective BACE2 inhibitors with clinical potential.     

Diarylheterocycle core ring features effect in selective COX-1 inhibition

The COX-1 isoenzyme plays a significant role in a variety of diseases, as it catalyzes the bioprocesses behind many health problems. Among the diarylheterocycle class of COX inhibitors, the isoxazole ring has been widely used as a central heterocycle for the preparation of potent and selective COX-1 inhibitors such as P6 [3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole]. The role of the isoxazole nucleus in COX-1 inhibitor selectivity has been clarified by preparing a set of new diarylheterocycles with various heterocycle cores. Replacement of isoxazole with isothiazole or pyrazole gave a drastic decrease in COX-1 inhibitory activity, whereas the introduction of an electron-donating group (EDG) on the N-aryl pyrazole allowed recovery of COX-1 inhibitory activity and selectivity. The EDG-equipped 5-(furan-2-yl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (17) selectively inhibits COX-1 activity (IC(50) =3.4 μM; 28% COX-2 inhibition at 50 μM), in contrast to its inactive analogue, 3-(furan-2-yl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazole, which does not bear the methoxy EDG. Molecular docking studies of compound 17 into the binding site of COX-1 shed light on its binding mode.     

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

A series of new substrate analogue inhibitors of the WNV NS2B–NS3 protease containing decarboxylated arginine mimetics at the P1 position was developed. Among the various analogues, trans‐(4‐guanidino)cyclohexylmethylamide (GCMA) was identified as the most suitable P1 residue. In combination with dichloro‐substituted phenylacetyl groups at the P4 position, three inhibitors with inhibition constants of <0.2 μM were obtained. These GCMA inhibitors have a better selectivity profile than the previously described agmatine analogues, and possess negligible affinity for the trypsin‐like serine proteases thrombin, factor Xa, and matriptase. A crystal structure in complex with the WNV protease was determined for one of the most potent inhibitors, 3,4‐dichlorophenylacetyl‐Lys‐Lys‐GCMA (K_i=0.13 μM ). The inhibitor adopts a horseshoe‐like conformation, most likely due to a hydrophobic contact between the P4 phenyl ring and the P1 cyclohexyl group, which is further stabilized by an intramolecular hydrogen bond between the P1 guanidino group and the P4 carbonyl oxygen atom. These inhibitors are stable, readily accessible, and have a noncovalent binding mode. Therefore, they may serve as suitable lead structures for further development.     

Direct synthesis and characterization of crosslinked polysiloxanes via anionic ring‐opening copolymerization with octaisobutyl‐polyhedral oligomeric silsesquioxane and octamethylcyclotetrasiloxane

The crosslinked polysiloxanes were directly synthesized by anionic ring‐opening copolymerization of octaisobutyl‐polyhedral oligomeric silsesquioxane (POSS) as a multifunctional monomer with octamethylcyclotetrasiloxane (D₄) under base catalysts such as potassium hydroxide (KOH) or tetramethylammonium hydroxide (Me₄ NOH) siloxanolate. The mechanism of anionic ring‐opening copolymerization of octaisobutyl‐POSS and D₄ was discussed and the influences of the polar additive N,N‐dimethylacetamide on gelation time at different temperatures were investigated. The results of gel content and swelling ratio, GPC, solid‐state ²⁹Si and ¹³C NMR, FTIR, XRD show that octaisobutyl‐POSS is reacted and most of the product is crosslinked. The DSC and TG results indicate that the crosslinked polysiloxanes exhibit distinct glass transition temperatures (T_g) and excellent thermal stability. Compared to that under KOH siloxanolate, the crosslinked polysiloxane synthesized with Me₄NOH siloxanolate has better preferable thermal stability. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 3848–3856, 2006     

Structure–Activity Relationships and Molecular Docking of Novel Dihydropyrimidine‐Based Mitotic Eg5 Inhibitors

Dihydropyrimidine‐based compounds belong to the first discovered inhibitors of the human mitotic kinesin Eg5. Although they are used by many research groups as model compounds for chemical genetics, considerably less emphasis has been placed on the improvement of this type of inhibitor, with the exception of two recent studies. Dihydropyrimidines can be divided into class I (analogues that bind in the S configuration) and class II type inhibitors, which bind in the R configuration. Herein we report the synthesis and optimization of novel class II type dihydropyrimidines using a combination of in vitro and docking techniques.     

Structure–Activity Relationship Studies on (R)‐PFI‐2 Analogues as Inhibitors of Histone Lysine Methyltransferase SETD7

SETD7 is a histone H3K4 lysine methyltransferase involved in human gene regulation. Aberrant expression of SETD7 has been associated with various diseases, including cancer. Therefore, SETD7 is considered a good target for the development of new epigenetic drugs. To date, few selective small‐molecule inhibitors have been reported that target SETD7, the most potent being (R)‐PFI‐2. Herein we report structure–activity relationship studies on (R)‐PFI‐2 and its analogues. A library of 29 structural analogues of (R)‐PFI‐2 was synthesized and evaluated for inhibition of recombinantly expressed human SETD7. The key interactions were found to be a salt bridge and a hydrogen bond formed between (R)‐PFI‐2′s NH₂⁺ group and SETD7′s Asp256 and His252 residue, respectively.     

Bis(benzoyloxybenzyl)‐DiPPro Nucleoside Diphosphates of Anti‐HIV Active Nucleoside Analogues

Nucleoside analogues are extensively used as antiviral and anticancer agents. Their efficiency is dependent on their metabolism into the ultimately active nucleoside triphosphates. Often one step or even more in the metabolism of the nucleoside to the triphosphate is inefficient. To overcome this hurdle, prodrugs of the nucleotides are needed. Bis(acyloxybenzyl)nucleoside diphosphates have been reported by us as a first example of an efficient nucleoside diphosphate prodrug (DiPPro nucleotides). Here, the synthesis and the properties of bis(benzoyloxybenzyl)nucleoside diphosphates of the nucleoside analogues d4T and AZT are disclosed. The synthesis was achieved by using a phosphoramidite/oxidation route. In chemical hydrolysis studies, most of the compounds formed a nucleoside diphosphate. This was confirmed in CEM cell extracts, although the prodrug stability in extracts was lower than in phosphate buffer. Furthermore, the stability and the amount of nucleoside diphosphate formed were dependent on the substituent in the benzoyl moiety. Some of the compounds were more active against HIV in thymidine kinase‐deficient CEM/TK^? cells than were d4T or AZT.     

Action of Dipeptidyl Peptidase-4 Inhibitors on SARS-CoV-2 Main Protease

In a recent publication, Eleftheriou et al. proposed that inhibitors of dipeptidyl peptidase-4 (DPP-4) are functional inhibitors of the main protease (M^pro) of SARS-CoV-2. Their predictions prompted the authors to suggest linagliptin, a DPP-4 inhibitor and approved anti-diabetes drug, as a repurposed drug candidate against the ongoing COVID-19 pandemic. We used an enzymatic assay measuring the inhibition of M^pro catalytic activity in the presence of four different commercially available gliptins (linagliptin, sitagliptin, alogliptin and saxagliptin) and several structural analogues of linagliptin to study the binding of DPP-4 inhibitors to M^pro and their functional activity. We show here that DPP-4 inhibitors like linagliptin, other gliptins and structural analogues are inactive against M^pro.     

Oxidative Cycloaddition of Aldoximes with Maleimides using Catalytic Hydroxy(aryl)iodonium Species

A mild catalytic procedure for the efficient oxidative cyclization of aldoximes with maleimides mediated by hypervalent iodine(III) active species has been developed. This catalytic cyclization affords the corresponding pyrrolo‐isoxazole products in generally good yields. The catalytic cycle involves active hydroxy(aryl)iodonium species generated in situ from 2‐iodobenzoic acid as precatalyst and m‐chloroperoxybenzoic acid (m‐CPBA) as terminal oxidant in the presence of trifluoromethanesulfonic acid. The presence of active hydroxy(aryl)iodonium species in this reaction has been confirmed by ESI‐mass spectrometry and ¹H NMR spectroscopy.

     

Mapping the Melatonin Receptor. 8. Selective MT2 Agonists Derived from 5,6-Dihydroindolo[2,1-a]isoquinolines and Related Systems

A series of substituted indolo[2,1-a]isoquinolines and indolo[1,2-a]benzoxazines have been prepared, as melatonin analogues, to investigate the nature of the binding site of the melatonin receptor. Agonist and antagonist potency of all the analogues was measured using the [35S]GTPγS binding assay protocol. The binding affinity of the analogues were measured by competition binding studies against the human MT1 (hMT1) and MT2 (hMT2) receptors stably transfected in Chinese Hamster Ovarian (CHO) cells, using 2-[¹²⁵I]-iodomelatonin, as a ligand. N-Acetyl 2-(10-methoxy-5,6-dihydroindolo[2,1-a]isoquinolin-12-yl)propyl-1-amine (12 a) binds strongly to both the hMT1 and hMT2 receptors, and shows a preference for the hMT2, as does its propanamido counterpart 12 b . The introduction of two methyl groups into their side chain, analogues 15 a and 1 5 b , leads to antagonism, in the case of the former, and drastically diminishes its hMT1 binding; an analogous profile is seen for 15 b , which, however, is a partial agonist. Introduction of chlorine or methoxy groups into ring 4 gives compounds, that are weakly binding, with a preference for MT2. Substitution of oxygen for carbon at position 5 gives the indolo[1,2-c]benzoxazines 33 , 36 a and b , that bind strongly to the human receptors, 33 , 36 b being potent agonists at the melatonin receptors, but do not discriminate between hMT1 and hMT2.