Targeting the Hedgehog pathway with novel Gli1 hydrophobic tagging degraders.

The Hedgehog/Glioma-associated oncogene (Hh/Gli) signaling pathway plays an essential role in embryonic development and tissue homeostasis. Aberrant regulation of this pathway has been linked to various human malignancies. Gli1, the downstream transcription factor of the Hh pathway, is the ultimate effector of the canonical Hh pathway and has been identified as a common regulator of several tumorigenic pathways prevalent in Hh-independent cancers. Thus Gli1 represents a unique and promising drug target for a wide range of cancers. However, the identification and development of small molecules that directly target Gli1 protein have progressed slowly, due to an insufficient efficacy and selectivity. Herein, we developed novel small-molecule Gli1 degraders based on the hydrophobic tagging (HyT) strategy. The Gli1 HyT degrader 8e potently inhibited the proliferation of Gli1-overexpressed HT29 colorectal cancer cells, induced Gli1 degradation with a DC50 value of 5.4 µM in HT29 and achieved 70% degradation at 7.5 µM in MEFPTCH1-/- and MEFSUFU-/-cell lines, via proteasome pathway. Compared to the canonical Hh antagonist Vismodegib, 8e exhibited much stronger potency in suppressing the mRNA expression of Hh target genes in Hh-overactivated MEFPTCH1-/- and Vismodegib resistant MEFSUFU-/- cells. Our study provides small molecule Gli1 degraders effectively interfering with both canonical and noncanonical Hh signaling and overcoming current Smoothened (SMO) antagonists resistance, which might pave a new avenue for developing therapeutic modalities targeting Hh/Gli1 signaling pathway.

Discovery and synthesis of 6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-one-based novel chemotype CCR2 antagonists via scaffold hopping strategy.

The chemokine CC receptor subtype 2 (CCR2) has attracted intensive interest for drug development in diverse therapeutic areas, including chronic inflammatory diseases, diabetes, neuropathic pain, atherogenesis and cancer. By employing a cut-and-sew scaffold hopping strategy, we identified an active scaffold of 3,4-dihydro-2,6-naphthyridin-1(2H)-one as the central pharmacophore to derive novel CCR2 antagonists. Systematic structure-activity relationship study with respect to the ring size and the substitution on the naphthyridinone ring gave birth to 1-arylamino-6-alkylheterocycle-6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-ones as a brand new chemotype of CCR2 antagonists with nanomolar inhibitory activity. The best antagonism activity in this series was exemplified by compound 13a, which combined the optimal substitutions of 3,4-dichlorophenylamino at C-1 and 3-(4-(N-methylmethylsulfonamido)piperidin-1-yl)propyl at N-6 position, leading to an IC50 value of 61 nM and 10-fold selectivity for CCR2 over CCR5. Efficient and general synthesis was established to construct the innovative core structure and derive the compound collections. This is the first report on our designed 6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-one as novel CCR2 antagonist scaffold and its synthesis.

GPR40 agonists for the treatment of type 2 diabetes mellitus: The biological characteristics and the chemical space.

GPR40 belongs to the GPCR family and the activation of GPR40 has been shown to induce glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells as well as incretin secretion from intestinal endocrine cells. Therefore, GPR40 has emerged as a viable and promising therapeutic target for type 2 diabetes mellitus (T2DM) without the risk of hypoglycemia. However, the termination of TAK-875 in phase III clinical trials for the hepatotoxicity issue threw doubt over the long-term safety of targeting GPR40. Herein, we summarized the newly disclosed biological characteristics and the druglikeness-based structural evolution of GPR40 agonists to advance the development of GPR40-based anti-diabetic drugs.

Structure-based optimization and derivatization of 2-substituted quinolone-based non-nucleoside HCV NS5B inhibitors with submicromolar cellular replicon potency.

HCV NS5B polymerase is an attractive and validated target for anti-HCV therapy. Starting from our previously identified 2-aryl quinolones as novel non-nucleoside NS5B polymerase inhibitors, structure-based optimization furnished 2-alkyl-N-benzyl quinolones with improved antiviral potency by employing privileged fragment hybridization strategy. The N-(4-chlorobenzyl)-2-(methoxymethyl)quinolone derivative 5f proved to be the best compound of this series, exhibiting a selective sub-micromolar antiviral effect (EC50=0.4µM, SI=10.8) in Huh7.5.1 cells carrying a HCV genotype 2a. Considering the undesirable pharmacokinetic property of the highly substituted quinolones, a novel chemotype of 1,6-naphthyridine-4,5-diones were evolved via scaffold hopping, affording brand new structure HCV inhibitors with compound 6h (EC50 (gt2a)=2.5µM, SI=7.2) as a promising hit. Molecular modeling studies suggest that both of 2-alkyl quinolones and 1,6-naphthyridine-4,5-diones function as HCV NS5B thumb pocket II inhibitors.

A novel proteolysis-resistant cyclic helix B peptide ameliorates kidney ischemia reperfusion injury.

Helix B surface peptide (HBSP), derived from erythropoietin, displays powerful tissue protection during kidney ischemia reperfusion (IR) injury without erythropoietic side effects. We employed cyclization strategy for the first time, and synthesized thioether-cyclized helix B peptide (CHBP) to improve metabolic stability and renoprotective effect. LC-MS/MS analysis was adopted to examine the stability of CHBP in vitro and in vivo. The renoprotective effect of CHBP in terms of renal function, apoptosis, inflammation, extracellular matrix deposition, and histological injury was also detected in vivo and in vitro. Antibody array and western blot were performed to analyze the signal pathway of involvement by CHBP in the IR model and renal tubular epithelial cells. In this study, thioether-cyclized peptide was significantly stable in vivo and in vitro. One dose of 8nmol/kg CHBP administered intraperitoneally at the onset of reperfusion improved renal protection compared with three doses of 8nmol/kg linear HBSP in a 48h murine IR model. In a one-week model, the one dose CHBP-treated group exhibited remarkably improved renal function over the IR group, and attenuated kidney injury, including reduced inflammation and apoptosis. Interestingly, we found that the phosphorylation of autophagy protein mTORC1 was dramatically reduced upon CHBP treatment. We also demonstrated that CHBP induced autophagy via inhibition of mTORC1 and activation of mTORC2, leading to renoprotective effects on IR. Our results indicate that the novel metabolically stable CHBP is a promising therapeutic medicine for kidney IR injury treatment.

Discovery of potent 1H-imidazo[4,5-b]pyridine-based c-Met kinase inhibitors via mechanism-directed structural optimization.

Starting from our previously identified novel c-Met kinase inhibitors bearing 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one scaffold, a global structural exploration was conducted to furnish an optimal binding motif for further development, directed by the enzyme inhibitory mechanism. First round SAR study picked two imidazonaphthyridinone frameworks with 1,8- and 3,5-disubstitution pattern as class I and class II c-Met kinase inhibitors, respectively. Further structural optimization on type II inhibitors by truncation of the imidazonaphthyridinone core and incorporation of an N-phenyl cyclopropane-1,1-dicarboxamide pharmacophore led to the discovery of novel imidazopyridine-based c-Met kinase inhibitors, displaying nanomolar enzyme inhibitory activity and improved Met kinase selectivity. More significantly, the new chemotype c-Met kinase inhibitors effectively inhibited Met phosphorylation and its downstream signaling as well as the proliferation of Met-dependent EBC-1 human lung cancer cells at submicromolar concentrations.

Metal-free TEMPO-promoted C(sp³)-H amination to afford multisubstituted benzimidazoles.

An efficient TEMPO-air/cat. TEMPO-O2 oxidative protocol was developed to synthesize multisubstituted or fused tetracyclic benzimidazoles via a metal-free oxidative C-N coupling between the sp(3) C-H and free N-H of readily available N(1)-benzyl/alkyl-1,2-phenylenediamines.

4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC50 values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2-10 fold selectivity over a panel of PTP's. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.

Design and discovery of flavonoid-based HIV-1 integrase inhibitors targeting both the active site and the interaction with LEDGF/p75.

HIV integrase (IN) is an essential enzyme for the viral replication. Currently, three IN inhibitors have been approved for treating HIV-1 infection. All three drugs selectively inhibit the strand transfer reaction by chelating a divalent metal ion in the enzyme active site. Flavonoids are a well-known class of natural products endowed with versatile biological activities. Their ß-ketoenol or catechol structures can serve as a metal chelation motif and be exploited for the design of novel IN inhibitors. Using the metal chelation as a common pharmacophore, we introduced appropriate hydrophobic moieties into the flavonol core to design natural product-based novel IN inhibitors. We developed selective and efficient syntheses to generate a series of mono 3/5/7/3'/4'-substituted flavonoid derivatives. Most of these new compounds showed excellent HIV-1 IN inhibitory activity in enzyme-based assays and protected against HIV-1 infection in cell-based assays. The 7-morpholino substituted 7c showed effective antiviral activity (EC50=0.826 µg/mL) and high therapeutic index (TI>242). More significantly, these hydroxyflavones block the IN-LEDGF/p75 interaction with low- to sub-micromolar IC50 values and represent a novel scaffold to design new generation of drugs simultaneously targeting the catalytic site as well as protein-protein interaction domains.

Investigation on the 1,6-naphthyridine motif: discovery and SAR study of 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one-based c-Met kinase inhibitors.

The 1,6-naphthyridine motif is a multivalent scaffold in medicinal chemistry presenting various bioactivities when properly substituted. By incorporating a cyclic urea pharmacophore into the 1,6-naphthyridine framework through conformationally constraining the 7,8-positions, the resulting 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one was identified as a new class of c-Met kinase inhibitor. A comprehensive SAR study indicated that an N-1 alkyl substituent bearing a terminal free amino group, a hydrophobic substituted benzyl group at the N-3 position and the tricyclic core were essential for retaining effective Met inhibition of the 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one chemotype. Further introduction of a 4'-carboxamide phenoxy group at the C-5 position significantly improved the potency. The best c-Met kinase inhibitory activity was exemplified by 2t with an IC(50) = 2.6 µM, which also displayed effective inhibition against TPR-Met phosphorylation and the proliferation of the BaF3-TPR-Met cells at low micromolar concentrations.

Discovery of a novel 5-carbonyl-1H-imidazole-4-carboxamide class of inhibitors of the HIV-1 integrase-LEDGF/p75 interaction.

Though much progress has been made in the inhibition of HIV-1 integrase catalysis, clinical resistance mutations have limited the promise of long-term drug prescription. Consequently, allosteric inhibition of integrase activity has emerged as a promising approach to antiretroviral discovery and development. Specifically, inhibitors of the interaction between HIV-1 integrase and cellular cofactor LEDGF/p75 have been validated to diminish proviral integration in cells and deliver a potent reduction in viral replicative capacity. Here, we have contributed to the development of novel allosteric integrase inhibitors with a high-throughput AlphaScreen-based random screening approach, with which we have identified novel 5-carbonyl-1H-imidazole-4-carboxamides capable of inhibiting the HIV-1 integrase-LEDGF/p75 interaction in vitro. Following a structure-activity relationship analysis of the initial 1H-imidazole-4,5-dicarbonyl core, we optimized the compound's structure through an industrial database search, and we went further to synthesize a selective and non-cytotoxic panel of inhibitors with enhanced potency.

Discovery of orally effective and safe GPR40 agonists by incorporating a chiral, rigid and polar sulfoxide into ß-position to the carboxylic acid.

GPR40 is primarily expressed in pancreatic islet ß-cells, and its activation by endogenous ligands of medium to long-chain free fatty acids or synthetic agonists is clinically proved to improve glycemic control by stimulating glucose-dependent insulin secretion. However, most of the reported agonists are highly lipophilic, which might cause lipotoxicity and the off-target effects in CNS. Particularly, the withdrawal of TAK-875 from clinical trials phase III due to liver toxicity concern threw doubt over the long-term safety of targeting GPR40. Improving the efficacy and the selectivity, thus enlarging the therapeutic window would provide an alternative to develop safe GPR40-targeted therapeutics. Herein, by employing an innovative "three-in-one" pharmacophore drug design strategy, the optimal structural features for GPR40 agonist was integrated into one functional group of sulfoxide, which was incorporated into the ß-position of the propanoic acid core pharmacophore. As a result, the conformational constraint, polarity as well as chirality endowed by the sulfoxide significantly enhanced the efficacy, selectivity and ADMET properties of the novel (S)- 2-(phenylsulfinyl)acetic acid-based GPR40 agonists. The lead compounds (S)-4a and (S)-4s exhibited robust plasma glucose-lowering effects and insulinotropic action during an oral glucose tolerance test in C57/BL6 mice, excellent pharmacokinetic profile and little hepatobiliary transporter inhibition, marginal cell toxicities against human primary hepatocyte at 100 µM.

Synthesis of the pyridinyl analogues of dibenzylideneacetone (pyr-dba) via an improved Claisen-Schmidt condensation, displaying diverse biological activities as curcumin analogues.

An efficient and easy procedure to synthesize the pyridinyl analogues of dibenzylideneacetone (pyr-dba) was developed by the condensation of substituted nicotinaldehyde and acetone in the presence of K(2)CO(3) in toluene-EtOH-H(2)O solvent system. Structurally diverse pyr-dba, including quinolinyl dba, can be prepared conveniently in moderate to excellent yields under mild conditions with this method. The resulting pyr-dba functioned as the enone analogs of curcumin and efficiently inhibited the activation of NF-κB and the growth of colorectal carcinoma HCT116 p53+/+ cells as well as the HIV-1 IN-LEDGF/p75 interaction.

Recent advances in the discovery of protein tyrosine phosphatase SHP2 inhibitors.

Src homology 2 domain-containing protein tyrosine phosphatase (SHP2) is a non-receptor protein tyrosine phosphatase encoded by the Ptpn11 gene, which regulates cell growth, differentiation and apoptosis via modulating various signaling pathways, such as the RAS/ERK signaling pathway, and participates in the PD-1/PD-L1 pathway governing immune surveillance. It has been recognized as a breakthrough antitumor therapeutic target. Besides, numerous studies have shown that SHP2 plays an important role in the regulation of inflammatory diseases. However, inhibitors targeting the active site of SHP2 lack drug-likeness due to their low selectivity and poor bioavailability, thus none has advanced to clinical development. Recently, allosteric inhibitors that stabilize the inactive conformation of SHP2 have achieved breakthrough progress, providing the clinical proof for the druggability of SHP2 as an antitumor drug target. This paper reviews the recently reported design and discovery of SHP2 small molecule inhibitors, focused on the structure-activity relationship (SAR) analysis of several representative SHP2 inhibitors, outlining the evolution and therapeutic potential of the small molecule inhibitors targeting SHP2.

On-Demand Activation of a Bioorthogonal Prodrug of SN-38 with Fast Reaction Kinetics and High Releasing Efficiency In Vivo.

Although a myriad of bioorthogonal prodrugs have been developed, very few of them present both fast reaction kinetics and complete cleavage. Herein, we report a new bioorthogonal prodrug strategy with both fast reaction kinetics (k2: ∼103 M-1 s-1) and complete cleavage (>90% within minutes) using the bioorthogonal reaction pair of N-oxide and boron reagent. Distinctively, an innovative 1,6-elimination-based self-immolative linker is masked by N-oxide, which can be bioorthogonally demasked by a boron reagent for the release of both amino and hydroxy-containing payload in live cells. Such a strategy was applied to prepare a bioorthogonal prodrug for a camptothecin derivative, SN-38, resulting in 10-fold weakened cytotoxicity against A549 cells, 300-fold enhanced water solubility, and "on-demand" activation upon a click reaction both in vitro and in vivo. This novel bioorthogonal prodrug strategy presents significant advances over the existing ones and may find wide applications in drug delivery in the future.

Metal-free hypervalent iodine-promoted tandem carbonyl migration and unactivated C(Ph)-C(Alkyl) bond cleavage for quinolone scaffold synthesis.

An unexpected iodine(III)-mediated C(sp3)-C(sp2) bond cleavage of 3-(methylamino)-2-(2-substitutedbenzoyl)acrylates for efficient synthesis of privileged scaffold 4-quinolones was described. Notably, a wide range of alkyl groups (e.g. methyl, tert-butyl or alkyl chain) can be conveniently cleaved in this system. The detailed mechanism studies revealed that the transformation proceeded through cascade ipso-cyclization and 1,2-carbonyl migration, the smaller bond energy determined ortho C-C bond cleavage rather than C-H bond cleavage, via an enamine radical intermediate.

Convergent synthesis of tetrahydropyranyl side chain of verucopeptin, an antitumor antibiotic active against multidrug-resistant cancers.

A concise synthesis of the tetrahydropyranyl side chain of verucopeptin, an antitumor antibiotic cyclodepsipeptide efficacious against MDR cancers in vivo, is achieved using 12 steps in the longest linear sequence and 21 total steps, in which Julia-Kocienski olefination for the segments coupling, asymmetric hydroxylation as well as stereoselective synthesis of stable tetrahydropyran ring from a D-isoascorbic acid derivative are key steps. This convergent synthetic strategy enables the structural modification and mechanism study of verucopeptin for its clinical applications.

Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups.

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 µM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 µM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.

Discovery of novel (S)-alpha-phenyl-gamma-amino butanamide containing CCR5 antagonists via functionality inversion approach.

By using functionality inversion approach, we identified a new scaffold containing (S)-alpha-phenyl-gamma-amino butanamide as CCR5 antagonists derived from the 1,3-propanediamine carboxamide pharmacophore protocol. The (2S)-2-phenyl-4-(8-aza-bicyclo[3.2.1]octan-8-yl)-butanamide derivatives display significantly high potency to antagonize CCR5 receptor with nanomolar IC(50) values.

Targeting inactive enzyme conformation: aryl diketoacid derivatives as a new class of PTP1B inhibitors.

There has been considerable interest in protein tyrosine phosphatase 1B (PTP1B) as a therapeutic target for diabetes, obesity, as well as cancer. Identifying inhibitory compounds with good bioavailability is a major challenge of drug discovery programs targeted toward PTPs. Most current PTP active site-directed pharmacophores are negatively charged pTyr mimetics which cannot readily enter the cell. This lack of cell permeability limits the utility of such compounds in signaling studies and further therapeutic development. We identify aryl diketoacids as novel pTyr surrogates and show that neutral amide-linked aryl diketoacid dimers also exhibit excellent PTP inhibitory activity. Kinetic studies establish that these aryl diketoacid derivatives act as noncompetitive inhibitors of PTP1B. Crystal structures of ligand-bound PTP1B reveal that both the aryl diketoacid and its dimeric derivative bind PTP1B at the active site, albeit with distinct modes of interaction, in the catalytically inactive, WPD loop open conformation. Furthermore, dimeric aryl diketoacids are cell permeable and enhance insulin signaling in hepatoma cells, suggesting that targeting the inactive conformation may provide a unique opportunity for creating active site-directed PTP1B inhibitors with improved pharmacological properties.