Tandem bispecific CD123/CLL-1 CAR-T cells exhibit specific cytolytic effector functions against human acute myeloid leukaemia.

OBJECTIVES: The treatment of refractory and recurrent acute myeloid leukaemia (AML) is still a challenge with poor response rates and short survival times. In an attempt to solve this problem, we constructed a tandem bispecific chimeric antigen receptor (CAR) targeting CD123 and C-type lectin-like molecule 1 (CLL-1), two different AML antigens, and verified its cytotoxic effects in vitro. METHODS: We established and cultured K562 cell lines expressing both CD123 and CLL1 antigens. Single-target CAR-T cells specific to CD123 and CLL1 were engineered, alongside tandem CD123/CLL1 bispecific CAR-T cells. Flow cytometry was used to determine cell phenotypes, transfection efficiencies, cytokine release, and CAR-T-cell proliferation, and an lactate dehydrogenase assay was used to detect the cytotoxicity of CD123/CLL-1 bispecific tandem CAR-T cells in vitro. RESULTS: Two types of tandem CAR-T cells exhibited significant killing effects on CLL-1 + CD123+ leukaemia cell lines and primary AML tumour cells. The killing efficiency of tandem CAR-T cells in the case of single antigen expression is comparable to that of single target CAR-T cells. When faced with dual target tumour cells, dual target CAR-T cells significantly surpass single target CAR-T cells. CD123/CLL-1 CAR-T cells in tandem targeted and killed CD123- and CLL-1-positive leukaemia cell lines and released a large number of cytokines. CONCLUSIONS: CD123/CLL-1 CAR-T cells in tandem can simultaneously target CD123 and CLL-1 on AML cells, demonstrating a significant ability to kill single antigens and multi-target tumour cells. This suggests that CD123/CLL-1 CAR-T cells exhibit significant advantages in the expression of multiple antigens in a wide range of target cells, which may help overcome the challenges posed by tumour heterogeneity and evasion mechanisms.

Identification of benzo[b]thiophene-1,1-dioxide derivatives as novel PHGDH covalent inhibitors.

The increased de novo serine biosynthesis confers many advantages for tumorigenesis and metastasis. Phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in serine biogenesis, exhibits hyperactivity across multiple tumors and emerges as a promising target for cancer treatment. Through screening our in-house compound library, we identified compound Stattic as a potent PHGDH inhibitor (IC50 = 1.98 ± 0.66 µM). Subsequent exploration in structural activity relationships led to the discovery of compound B12 that demonstrated the increased enzymatic inhibitory activity (IC50 = 0.29 ± 0.02 µM). Furthermore, B12 exhibited robust inhibitory effects on the proliferation of MDA-MB-468, NCI-H1975, HT1080 and PC9 cells that overexpress PHGDH. Additionally, using a [U-13C6]-glucose tracing assay, B12 was found to reduce the production of glucose-derived serine in MDA-MB-468 cells. Finally, mass spectrometry-based peptide profiling, mutagenesis experiment and molecular docking study collectively suggested that B12 formed a covalent bond with Cys421 of PHGDH.

Catalytic Asymmetric Hydroalkoxylation and Formal Hydration and Hydroaminoxylation of Conjugated Dienes.

The straightforward construction of stereogenic centers bearing unprotected functional groups, as in nature, has been a persistent pursuit in synthetic chemistry. Abundant applications of free enantioenriched allyl alcohol and allyl hydroxylamine motifs have made the asymmetric hydration and hydroaminoxylation of conjugated dienes from water and hydroxylamine, respectively, intriguing and efficient routes that have, however, been unachievable thus far. A fundamental challenge is the failure to realize transition-metal-catalyzed enantioselective C-O bond constructions via hydrofunctionalization of conjugated dienes. Here, we perform a comprehensive study toward the stereoselective formal hydration and hydroaminoxylation of conjugated dienes by synthesizing a set of new P,N-ligands and identifying an aryl-derived oxime as a surrogate for both water and hydroxylamine. Asymmetric hydroalkoxylation with new P,N-ligands is also elucidated. Furthermore, versatile derivatizations following hydration provide indirect but concise routes to formal hydrophenoxylation, hydrofluoroalkoxylation, and hydrocarboxylation of conjugated dienes that have been unreported thus far. Finally, a ligand-to-ligand hydrogen transfer process is proposed based on the results of preliminary mechanistic experiments.

Copper(I)-Catalyzed Asymmetric Hydrophosphination of 3,3-Disubstituted Cyclopropenes.

Herein, a copper(I)-catalyzed asymmetric hydrophosphination of 3,3-disubstituted cyclopropenes is reported. It provides a series of phosphine derivatives in high to excellent diastereo- and enantioselectivities. The methodology enjoys broad substrate scope on both 3,3-disubstituted cyclopropenes and diarylphosphines. The high stereoselectivity is attributed to both the high stability of the Cu(I)-(R,R)-QUINOXP* complex in the presence of stoichiometric HPPh2 and the produced phosphines, and the high-performance asymmetric induction of the Cu(I)-(R,R)-QUINOXP* complex. Finally, the method is used for the synthesis of new chiral phosphine-olefin compounds built on a cyclopropane skeleton, one of which serves as a wonderful ligand in Rh-catalyzed asymmetric conjugate addition of phenylboronic acid to various α,ß-unsaturated compounds.

Ligand-Dictated Regiodivergent Allylic Functionalizations via Palladium-Catalyzed Remote Substitution.

Different from classical allylic substitutions that require a vicinal leaving group, an olefin bearing a remote leaving group is scarcely viewed as a potential allylation substrate. Herein, we describe feasible protocols to achieve regiodivergent allylic C-H functionalizations via palladium-catalyzed remote substitution, which provides a novel strategy for the seldomly studied migratory Tsuji-Trost reaction. Dictated by a suitable ligand, a process that involved 4,3-hydrofunctionalization of the generated conjugated diene intermediate via metal walking is observed in generally >20 : 1 regioselectivity. Unexpectedly, a related 1,4-hydrofunctionalization pathway is found to be a major route with a newly synthesized electron-rich bisphosphine ligand, which challenges the conventional viewpoint on the potential regioselectivity of hydrofunctionalizations of linear internal conjugated dienes via η3 -substitution. A series of deuterium experiments and kinetic studies provide a preliminary insight into the potential catalytic cycle.

Hydroxylation with Unusual Stereoinversion Catalyzed by an FeII /2-OG Dependent Oxidase and 3,6-Diene-2,5-diketopiperazine Formation in the Biosynthesis of Brevianamide†K.

Natural products with the 3,6-diene-2,5-diketopiperazine core are widely distributed in nature; however, the biosynthetic mechanism of 3,6-diene-2,5-diketopiperazine in fungi remains to be further elucidated. Through heterologous expression and biochemical investigation of an FeII /2-oxoglutarate-dependent oxidase (AspE) and a heme-dependent P450 enzyme (AspF), we report that AspE, AspF and subsequent dehydration account for the formation of the 3,6-diene-2,5-diketopiperazine substructure of brevianamide K from Aspergillus sp. SK-28, a symbiotic fungus of mangrove plant Kandelia candel. More interestingly, in-depth investigation of the enzymatic mechanism showed that AspE promotes hydroxylation of brevianamide Q with unprecedented stereoinversion through hydrogen atom abstraction and water nucleophilic attack from the opposite face of the resultant iminium cation intermediate.

Asymmetric Substitution by Alkynyl Copper Driven Dearomatization and Rearomatization.

Catalytic asymmetric transformations by dearomatization have developed into a widely applicable synthetic strategy, but heavily relied on the use of arenes bearing a heteroatom. In this case, the dearomatization is facilitated by the involvement of a p-orbital electron of the heteroatom. Different from the conventional substrate-dependent model, here we demonstrate that the activation by a d-orbital electron of the transition-metal center can serve as a driving force for dearomatization, and is applied to the development of a novel asymmetric alkynyl copper facilitated remote substitution reaction. A newly modified PyBox chiral ligand enables the construction of valuable diarylmethyl and triarylmethyl skeletons in high enantioselectivities. An unexpected tandem process involving sequential remote substitution/cyclization/1,5-H shift leads to the formation of the enantioenriched C-N axis. A gram-scale reaction and various downstream transformations highlight the robustness of this method and the potential transformations of the products. Preliminary mechanistic studies reveal a mononuclear Cu-catalyzed remote substitution process.

Analysis of the Amine Submetabolome Using Novel Isotope-Coded Pyrylium Salt Derivatization and LC-MS: Herbs and Cancer Tissues as Cases.

The amine submetabolome, including amino acids (AAs) and biogenic amines (BAs), is a class of small molecular compounds exhibiting important physiological activities. Here, a new pyrylium salt named 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate ([d0]-DMMIC) with stable isotope-labeled reagents ([d3]-/[d6]-DMMIC) was designed and synthesized for amino compounds. [d0]-/[d3]-/[d6]-DMMIC-derivatized had a charged tag and formed a set of molecular ions with an increase of 3.02 m/z and the characteristic fragment ions of m/z 204.1:207.1:210.1. When DMMIC coupled with liquid chromatography-mass spectrometry (LC-MS), a systematic methodology evaluation for quantitation proved to have good linearity (R2 between 0.9904 and 0.9998), precision (interday: 2.2-21.9%; intraday: 1.0-19.7%), and accuracy (recovery: 71.8-108.8%) through the test AAs. Finally, the methods based on DMMIC and LC-MS demonstrated the advantaged application by the nontargeted screening of BAs in a common medicinal herb Senecio scandens and an analysis of metabolic differences among the amine submetabolomes between the carcinoma and paracarcinoma tissues of esophageal squamous cell carcinoma (ESCC). A total of 20 BA candidates were discovered in S. scandens as well as the finding of 13 amine metabolites might be the highest-potential differential metabolites in ESCC. The results showed the ability of DMMIC coupled with LC-MS to analyze the amine submetabolome in herbs and clinical tissues.

AgNTf2-Catalyzed Regioselective C-H Alkenylation of N,N-Dialkylanilines with Ynamides.

Regioselective C-H alkenylation of N,N-dialkylanilines with ynamides was developed using AgNTf2 as a catalyst. This approach represents a facile hydroarylation of ynamides, allowing for the introduction of an alkenyl group exclusively at the para position of aniline derivatives. As a result, a series of 4-alkenyl N,N-dialkylanilines were synthesized with excellent regioselectivities.

Synthesis of tetrasubstituted allenes via a 1,4-palladium migration/carbene insertion/ß-H elimination sequence.

A palladium-catalyzed synthesis of tetrasubstituted allenes from aryl bromides and aryl diazoacetates is developed. This transformation proceeded via an aryl to alkenyl 1,4-palladium migration/carbene insertion/ß-H elimination sequence under mild reaction conditions.

Base-controlled dearomative [3 + 2] cycloadditions between 3-nitro-indoles and fumaric acid amide esters.

The base-controlled dearomative [3 + 2] cycloaddition reaction between 3-nitroindoles and fumaric acid amide esters has been disclosed by using the dearomatization and aromatization strategy. Three kinds of diverse functionalized pyrrolo[2,3-b]indole derivatives were obtained respectively with excellent chemoselectivities and good diastereoselectivities using different bases.

Discovery of PHGDH inhibitors by virtual screening and preliminary structure-activity relationship study.

Phosphoglycerate dehydrogenase (PHGDH) is abnormally expressed in numerous malignant tumor cells and catalyzes the first step of serine biosynthesis, thus becoming a key drug target for antitumor treatment. In this study, compound B2 bearing a benzene-1,3-diamine scaffold was identified by structure-based virtual screening as a novel PHGDH inhibitor with moderate enzymatic activity. The structure-activity relationship study led to the discovery of compound C25 possessing improved enzymatic inhibitory activity and potent inhibitory activity on the proliferation of cells overexpressing PHGDH. The enzyme kinetic assay confirmed that C25 inhibited PHGDH in a nicotinamide adenine dinucleotide (NAD+) competitive manner. Molecular docking and mutagenesis experiment on PHGDH collectively revealed the binding site and key interaction residues of C25 in the PHGDH catalytic site. Taken together, this study provides information on the structural diversity for a further development of potent PHGDH inhibitors.

Stereodivergent Synthesis of Tertiary Fluoride-Tethered Allenes via Copper and Palladium Dual Catalysis.

Herein we describe a protocol for the unprecedented stereodivergent synthesis of tertiary fluoride-tethered allenes bearing a stereogenic center and stereogenic axis via Cu/Pd synergistic catalysis. A broad scope of conjugated enynes are coupled with various α-fluoroesters in high yields with high diastereoselectivities and generally >99% ee. In addition, the four stereoisomers of the allene products ensure precise access to the corresponding four stereoisomers of the fluorinated hydrofurans via a novel stereodivergent axial-to-central chirality transfer process.

Triptolide: reflections on two decades of research and prospects for the future.

Covering: 2000 to 2020 Triptolide is a bioactive diterpene triepoxide isolated from Tripterygium wilfordii Hook F, a traditional Chinese medicinal plant whose extracts have been used as anti-inflammatory and immunosuppressive remedies for centuries. Although triptolide and its analogs exhibit potent bioactivities against various cancers, and inflammatory and autoimmune diseases, none of them has been approved to be used in the clinic. This review highlights advances in material sourcing, molecular mechanisms, clinical progress and new drug design strategies for triptolide over the past two decades, along with some prospects for the future course of development of triptolide.

Multiplexed Analysis of Endogenous Guanidino Compounds via Isotope-Coded Doubly Charged Labeling: Application to Lung Cancer Tissues as a Case.

Endogenous guanidino compounds (GCs), nitrogen-containing metabolites, have very important physiological activities and participate in biochemical processes. Therefore, accurately characterizing the distribution of endogenous GCs and monitoring their concentration variations are of great significance. In this work, a new derivatization reagent, 4,4'-bis[3-(dimethylamino)propyl]benzyl (BDMAPB), with isotope-coded reagents was designed and synthesized for doubly charged labeling of GCs. BDMAPB-derivatized GCs not only promote the MS signal but also form multicharged quasimolecular ions and abundant fragment ions. With this reagent, an isotope-coded doubly charged labeling (ICDCL) strategy was developed for endogenous GCs with high-resolution liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF MS). The core of this methodology is a 4-fold multiplexed set of [d0]-/[d4]-/[d8]-/[d12]-BDMAPB that yields isotope-coded derivatized GCs. Following a methodological assessment, good linear responses in the range of 25 nM to 1 µM with correlation coefficients over 0.99 were achieved. The limit of detection and the limit of quantitation were below 5 and 25 nM, respectively. The intra- and interday precisions were less than 18%, and the accuracy was in the range of 77.3-122.0%. The percentage recovery in tissues was in the range of 85.1-113.7%. The results indicate that the developed method facilitates long-term testing and ensures accuracy and reliability. Finally, the method was applied for the simultaneous analysis of endogenous GCs in four types of lung tissues (solid adenocarcinoma, solid squamous-cell carcinoma, ground-glass carcinoma, and paracancerous tissues) for absolute quantification, nontargeted screening, and metabolic difference analysis. It is strongly believed that ICDCL combined with isotope-coded BDMAPB will benefit the analysis and study of endogenous GCs.

Nickel-Catalyzed Regioselective Hydroamination of Ynamides with Secondary Amines.

The first Ni(OTf)2-catalyzed hydroamination of ynamides 2 was developed by reacting with secondary amines (1 and 4). This protocol features excellent regioselectivity, a broad substrate scope of secondary aryl amines, and good functional group tolerance for ynamides. Using this method, a variety of substituted ethene-1,1-diamine compounds were prepared in moderate to excellent yields with high regioselectivities.

Synthesis of Functionalized Bicyclo[2.2.2]octan-2-ones Skeleton via Tandem Process of Amino Acid Involved Formal [4 + 2] and Decarboxylative-Mannich Sequence.

An efficient approach to a functionalized bicyclo[2.2.2]octan-2-one scaffold has been developed through a one-pot cascade process including amino acid involved successive Michael addition and decarboxylative-Mannich sequence. Starting from α,ß-unsaturated ketones and amino acids, a series of desired products 7a-7m and 8a-8o were obtained with moderate yields. In addition, the tandem process was reasonably explained by the results of DFT calculations.

A One-Pot Approach to 2-Substituted-2-(Dimethoxyphosphoryl)-Pyrrolidines from Substituted tert-Butyl 4-Oxobutylcarbamates and Trimethyl Phosphite.

A novel approach to 2-substituted-2-(dimethoxyphosphoryl)-pyrrolidines 7a-7o and 9a-9r has been developed, which features a TMSOTf-mediated one-pot intramolecular cyclization and phosphonylation of substituted tert-butyl 4-oxobutylcarbamates. The major advantages of this method include simple operation under mild reaction conditions, the use of cheap Lewis acid, and good to excellent yields with high diastereoselectivities (dr up to 99:1).

Non-targeted screening of pyranosides in Rhodiola crenulata using an all ion fragmentation-exact neutral loss strategy combined with liquid chromatography-quadrupole time-of-flight mass spectrometry.

INTRODUCTION: Pyranosides as one kind of natural glycosides contain a pyran ring linked to an aglycone in the structure. They occur widely in plants and possess diverse biological activities. The discovery of new pyranosides not only contributes to research on natural products but also may promote pharmaceutical development. OBJECTIVES: A non-targeted liquid chromatography-quadrupole time-of-flight mass spectrometry method coupled with an all ion fragmentation-exact neutral loss (AIF-ENL) strategy was developed for the screening of pyranosides in plants. METHODS: Pyranosides in various types were collected as a model. The AIF-ENL strategy comprised three steps: AIF spectrum acquisition and generation, ENL-based searching and identification, and confirmation of structural type using target second-stage mass spectrometry (MS/MS). The strategy was systematically evaluated based on the matrix effects, fragmentation stability, scan rate and screening efficiency and finally applied to Rhodiola crenulata (Hook. f. et Thoms) H. Ohba. RESULTS: The method was proved to be an efficient tool for the screening of pyranosides. When it was applied to R. crenulata, a total of 24 pyranoside candidates were detected. Among them, six were tentatively identified on the basis of the agreement of their elemental composition with the reported. The other 18 were detected in R. crenulata for the first time. CONCLUSION: The method offers a new platform for discovering pyranosides. In addition, the developed non-targeted strategy can also be used for other natural products, such as flavonoids and coumarins, as long as there is a common fragmentation behaviour in their MS/MS to generate characteristic neutral losses or fragments.

Synthesis of Amide Enol Carbamates and Carbonates through Cu(OTf)2-Catalyzed Reactions of Ynamides with t-Butyl Carbamates/Carbonates.

A highly regioselective approach to access amide enol carbamates and carbonates 5a-5c', 7a-7h, and 9 was developed through Cu(OTf)2-catalyzed reactions of ynamides 4 with t-butyl carbamates 2 and 8 and t-butyl carbonates 6. Moreover, this strategy was successfully applied to generate amide enol carbamates 11a-11s and 14a-14f from imides 10 and 13 with ynamides through an N-Boc cleavage-addition ring-opening process. A range of substituents was amenable to this transformation, and the desired amide enol carbamates and carbonates were obtained in moderate to good yields.