AviTrap: A novel solution to achieve complete biotinylation.

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.

Base-promoted triple cleavage of CCl2Br: a direct one-pot synthesis of unsymmetrical oxalamide derivatives.

We present a novel, eco-friendly and one-pot approach for synthesizing unsymmetrical oxalamides with the aid of dichloroacetamide and amine/amides in the presence of CBr4 in a basic medium. The use of water as a potent supplement for the oxygen atom source and the detailed mechanism have been disclosed. Moreover, the protocol involves triple cleavage of CCl2Br and the formation of new C-O/C-N bonds, with the advantage of achieving selective bromination using CBr4 with good to excellent yield under mild conditions. The method also demonstrates promise for industrial use, as proven by its effective implementation in gram-scale synthesis conducted in a batch process, along with its utilization in a continuous-flow system.

Novel Selective Quantification of Zinpentraxin Alfa Biotherapeutic in the Presence of Endogenous Isomer in Plasma Samples of Idiopathic Pulmonary Fibrosis Patients Using Immunoaffinity LC-MS.

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal interstitial lung disease that affects three million patients worldwide and currently without an effective cure. Zinpentraxin alfa, a recombinant human pentraxin-2 (rhPTX-2) protein, has been evaluated as a potential drug candidate for the treatment of IPF. Clinical pharmacokinetic analysis of zinpentraxin alfa has been challenging historically due to interference from serum amyloid P component (SAP), an endogenous human pentraxin-2 protein. These molecules share an identical primary amino acid sequence and glycan composition; however, zinpentraxin alfa possesses α2,3-linked terminal sialic acid residues while SAP is an α2,6-linked isomer. By taking advantage of this only structural difference, we developed a novel assay strategy where α2,3-sialidase was used to selectively hydrolyze α2,3-linked sialic acid residues, resulting in desialylated zinpentraxin alfa versus unchanged sialylated SAP, following an immunoaffinity capture step. Subsequent tryptic digestion produced a unique surrogate asialo-glycopeptide from zinpentraxin alfa and allowed specific quantification of the biotherapeutic in human plasma. In addition, a common peptide shared by both molecules was selected as a surrogate to determine total hPTX-2 concentrations, i.e., sum of zinpentraxin alfa and SAP. The quantification methods for both zinpentraxin alfa and total hPTX-2 were validated and used in pharmacokinetic assessment in IPF patients. The preliminary results suggest that endogenous SAP levels remained largely constant in IPF patients throughout the treatment with zinpentraxin alfa. Our novel approach provides a general bioanalytical strategy to selectively quantify α2,3-sialylated glycoproteins in the presence of their corresponding α2,6-linked isomers.

An Active-Matrix Organic Light-Emitting Diode Pixel Circuit Featuring Mobility Compensation for Portable Applications.

A 6T1C pixel circuit based on low-temperature polycrystalline oxide (LTPO) technology for portable active-matrix organic light-emitting diode (AMOLED) display applications is proposed in this paper. For superior high-end portable applications including 4K high resolution and high PPI (pixels per inch), the proposed pixel circuit employs a single storage capacitor and signal sharing switch-control design and provides low-voltage driving and immunity to the IR-drop issue and OLED degradation. Furthermore, the threshold voltage and mobility-compensating capabilities are improved by both compensation mechanisms, which are based on a negative feedback system, and mobility-related compensation parameters. Simulation results reveal that threshold voltage variations of ±0.33 V in the driving thin-film transistors can be well sensed and compensated while the maximum OLED current shift is 4.25%. The maximum variation in OLED currents within all gray levels is only 1.05% with mobility variations of ±30%. As a result, the proposed 6T1C pixel circuit is a good candidate for portable AMOLED display usage.

Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

FeCl3-Catalyzed Decyanative [4 + 2] Annulation of α-Aminonitriles with Alkynes: Access to 2,4-Diaryl Quinolines in Batch and Continuous-Flow Processes.

FeCl3-catalyzed decyanation of α-aminonitriles followed by a [4 + 2] annulation with terminal alkynes has been developed to synthesize 2,4-diaryl quinolines. A broad range of aniline, aldehyde, and arylacetylene derivatives were well tolerated to access 2,4-diaryl quinolines in moderate to good yields. The control experiment studies suggested that the reaction proceeds through a nonradical pathway involving Povarov-type [4 + 2] annulation from the in situ generated iminium species. The synthetic application of this strategy (i) includes gram-scale synthesis and (ii) a continuous-flow process for a few representative compounds in a shorter reaction time (22 min) and (iii) worked well with styrene as a proof of concept.

A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

Efferocytosis is a phagocytic process by which apoptotic cells are cleared by professional and nonprofessional phagocytic cells. In tumors, efferocytosis of apoptotic cancer cells by tumor-associated macrophages prevents Ag presentation and suppresses the host immune response against the tumor. Therefore, reactivating the immune response by blockade of tumor-associated macrophage-mediated efferocytosis is an attractive strategy for cancer immunotherapy. Even though several methods have been developed to monitor efferocytosis, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study, we describe a real-time efferocytosis assay with an imaging system for live-cell analysis. Using this assay, we successfully discovered potent anti-MerTK Abs that block tumor-associated macrophage-mediated efferocytosis in mice. Furthermore, we used primary human and cynomolgus monkey macrophages to identify and characterize anti-MerTK Abs for potential clinical development. By studying the phagocytic activities of different types of macrophages, we demonstrated that our efferocytosis assay is robust for screening and characterization of drug candidates that inhibit unwanted efferocytosis. Moreover, our assay is also applicable to investigating the kinetics and molecular mechanisms of efferocytosis/phagocytosis.

Iodine-Promoted Oxidative Cyclization of Acylated and Alkylated Derivatives from Epoxides toward the Synthesis of Aza Heterocycles.

A new method for directly synthesizing acylated and alkylated quinazoline derivatives by the epoxide ring-opening reaction in the presence of I2/DMSO with 2-aminobenzamide is described herein. The developed mild protocol is efficient and displays a wide variety of functional group tolerance and substrate-controlled high selectivity, and the application of a continuous flow technique allows for faster reaction time and higher yields. Moreover, the robustness of the method is applicable in gram-scale synthesis.

The Development and Characterization of a Highly Sensitive Mature TGFß3 Assay to Evaluate Anti-TGFß3 Target Engagement.

MTBT 1466A is a monoclonal antibody designed to bind to mature human TGFß3 in human tissue and systemic circulation. To evaluate binding of this therapeutic, a mature TGFß3 assay was needed to be able to monitor pharmacodynamic responses in non-human primate (NHP) studies. However, mature TGFß3 levels in systemic circulation are very low and require development of a highly sensitive assay for detection. This study describes the development of a highly sensitive, drug-tolerant pharmacodynamic biomarker assay for demonstrating target engagement in a pre-clinical study using MTBT1466A. Since mature TGFß3 is a dimer, a single MAb was used as both the capture and detection antibodies. This assay was developed on the SMCxPRO platform and qualified based on current accepted criteria for biomarker assays. The assay demonstrated specificity to mature TGFß3, with a lower limit of quantification of 31.3pg/mL. Although baseline levels of mature TGFß3 were below the assay detection limit in 40% of animals within our study, 2- to 16-fold increases were observed in many of the animals following multiple-dosing regimen.

Extract of Ficus septica modulates apoptosis and migration in human oral squamous cell carcinoma cells.

According to the alarming statistical analysis of global cancer, there are over 19 million new diagnoses and more than 10 million deaths each year. One such cancer is the oral squamous cell carcinoma (OSCC), which requires new therapeutic strategies. Ficus septica extract has been used in traditional medicine to treat infectious diseases. In this study, we examined the anti-proliferative effects of an extract of F. septica bark (FSB) in OSCC cells. Our results showed that FSB caused a concentration-dependent reduction in the viability of SCC2095 OSCC cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and was less sensitive to fibroblasts. In addition, FSB induced apoptosis by activating caspases, accompanied by the modulation of Akt/mTOR/NF-κB and mitogen-activated protein kinase signaling. Moreover, FSB increased reactive oxygen species generation in a concentration-dependent manner in SCC2095 cells. Furthermore, FSB inhibited cell migration and modulated the levels of the cell adhesion molecules including E-cadherin, N-cadherin, and Snail in SCC2095 cells. Pinoresinol, a lignan isolated from FSB, showed antitumor effects in SCC2095 cells, implying that this compound might play an important role in FSB-induced OSCC cell death. Taken together, FSB is a potential anti-tumor agent against OSCC cells.

Biallelic TET2 mutations confer sensitivity to 5'-azacitidine in acute myeloid leukemia.

Precision medicine can significantly improve outcomes for patients with cancer, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here, we describe somatic biallelic TET2 mutations in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine but acutely sensitive to 5'-azacitidine (5'-Aza) hypomethylating monotherapy, resulting in long-term morphological remission. Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5'-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5'-Aza compared with cells with monoallelic mutations. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first-line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for patients with cancer.

The association between eosinophil count, serum lipids and metabolic syndrome in Taiwanese.

BACKGROUND: Eosinophil count, dyslipidemia, and metabolic syndrome (MetS) are associated with systemic inflammation. We conducted this large population-based study to investigate the association between elevated eosinophil count, serum lipids, and MetS in the Taiwanese population. METHODS: A cross-sectional study of 10,357 adults who underwent health checkups at Shin Kong Wu Ho-Su Memorial Hospital in Taiwan between January 2006 and December 2016 was conducted. MetS was defined according to criteria modified by the International Diabetes Federation specifically for the Chinese population. The measurement of serum lipids included high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A-I (ApoA-I), and apolipoprotein B (ApoB). Pearson's correlation and linear regression were used to determine the association of eosinophil count with waist circumference, blood pressure, fasting glucose, and serum lipids. Multivariate logistic regression analysis was used to determine the odds ratio of MetS and abnormal serum lipid levels in each eosinophil count quartile. RESULTS: Eosinophil count was positively associated with triglycerides levels and negatively associated with HDL-C levels; however, it was not significantly associated with ApoA-I and ApoB. The odds ratio of MetS increased significantly across eosinophil count quartiles. The adjusted odds ratios of MetS for the second, third, and fourth quartiles were 1.28, 1.38, and 1.42, respectively, with reference to the first quartile. CONCLUSIONS: High eosinophil count is an independent risk factor for MetS. Hypertriglyceridemia and hypo-HDL-cholesterolemia might partly contribute to this result.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Bladder Management Strategies for Urological Complications in Patients with Chronic Spinal Cord Injury.

Neurogenic lower urinary tract dysfunction, common in patients with chronic spinal cord injury, inevitably results in urological complications. To address neurogenic lower urinary tract dysfunction after spinal cord injury, proper and adequate bladder management is important in spinal cord injury rehabilitation, with the goal and priorities of the protection of upper urinary tract function, maintaining continence, preserving lower urinary tract function, improvement of SCI patients' quality of life, achieving compatibility with patients' lifestyles, and decreasing urological complications. This concise review aims to help urologists address neurogenic lower urinary tract dysfunction by focusing on the risks of long-term urological complications and the effects of different bladder management strategies on these complications based on scientifically supported knowledge.

Kefir peptides ameliorate osteoporosis in AKR1A1 knockout mice with vitamin C deficiency by promoting osteoblastogenesis and inhibiting osteoclastogenesis.

The AKR1A1 protein is a member of the aldo-keto reductase superfamily that catalyzes the transformation of D-glucuronate to L-gulonate in the synthesis of L-ascorbic acid (vitamin C, Vit C). We previously demonstrated that AKR1A1 knockout mice (AKR1A1eGFP/eGFP) with Vit C deficiency exhibited aberrant bone formation and osteoporosis. In this study, we aimed to evaluate the osteoprotective effects of kefir peptides (KPs) in AKR1A1eGFP/eGFP mice and uncover the underlying mechanism of KPs in the modulation of bone remodeling. Six male CD-1 mice and 24 male AKR1A1eGFP/eGFP mice were used in this study, in which the AKR1A1eGFP/eGFP mice were randomly divided into four groups (n = 6). KPs treatment for 12 weeks exerted several effects in AKR1A1eGFP/eGFP mice including the reduction of serum proinflammatory cytokines (IL-1ß, IL-6, TNF-α), bone resorption markers (CTX-1, RANKL), and the increase of serum bone formation markers (P1NP, OPG, OC). µ-CT analysis indicated that KPs prevented the bone loss in the femurs of AKR1A1eGFP/eGFP mice by significantly increasing the trabecular parameters of bone mineral density, bone volume and bone number. Nanoindentation analysis demonstrated that KPs enhanced the elasticity and hardness of femoral cortical bones in AKR1A1eGFP/eGFP mice. KPs promoted bone marrow mesenchymal stem cells (BMMSCs)-derived osteoblast differentiation and mineralization by upregulating positive regulators of osteoblastogenesis (Runx2, ß-catenin, BMP-2, NFATc1). Conversely, KPs inhibited bone marrow macrophages (BMMs)-derived osteoclast differentiation and bone resorption, which was demonstrated by the facts that KPs suppressed RANKL-induced p38, NF-κB, Akt, PLCγ2 and CREB-1 phosphorylation, decreased the nuclear translocation of NFATc1 and c-Fos. Our findings demonstrate the efficacy of KPs in the prevention of osteoporosis in AKR1A1eGFP/eGFP mice and also unveil the dual effects of KPs in osteogenic promotion and osteoclastic inhibition. This study supports the use of KPs as nutritional supplements for the prevention of osteoporosis.

AMOLED Pixel Circuit Using LTPO Technology Supporting Variable Frame Rate from 1 to 120 Hz for Portable Displays.

This paper proposes a new 6T1C pixel circuit based on low-temperature polycrystalline oxide (LTPO) technology for portable active-matrix organic light-emitting diode (AMOLED) displays with variable refresh rates ranging from 1 to 120 Hz. The proposed circuit has a simple structure and is based on the design of sharing lines of switch-controlling signals. It also provides low-voltage driving and immunity to OLED degeneration issues. The calculation and analysis of programming time are discussed, and the optimal storage capacitor for the proposed circuit's high-speed driving is selected. The results of the simulation reveal that threshold voltage variations in driving thin-film transistors of ±0.33 V can be well sensed and compensated with a 1.8% average shift of OLED currents in high-frame-rate operation (120 Hz), while the maximum variation in OLED currents within all gray levels is only 3.56 nA in low-frame-rate operation (1 Hz). As a result, the proposed 6T1C pixel circuit is a good candidate for use in portable AMOLED displays.

Surgical outcome predictor analysis following hand-assisted or pure laparoscopic transperitoneal nephroureterectomy using the Taiwan upper urinary tract urothelial carcinoma database.

Purpose: Taiwan has a high incidence of upper tract urothelial carcinoma (UTUC). This study aimed to compare the surgical outcomes following transperitoneal hand-assisted laparoscopic nephroureterectomy (TP-HALNU) and transperitoneal pure laparoscopic nephroureterectomy (TP-LNU) from the Taiwan nationwide UTUC collaboration database using different parameters, including surgical volumes. Materials and methods: The nationwide UTUC collaboration database includes 14 hospitals in Taiwan from the Taiwan Cancer Registry. We retrospectively reviewed the records of 622 patients who underwent laparoscopic nephroureterectomy between July 1988 and September 2020. In total, 322 patients who received TP-LNU or TP-HALNU were included in the final analysis. Clinical and pathological data and oncological outcomes were compared. Results: Of the 322 patients, 181 and 141 received TP-LNU and TP-HALNU, respectively. There were no differences in clinical and histopathological data between the two groups. No differences were observed in perioperative and postoperative complications. There were no significant differences in oncological outcomes between the two surgical approaches. In the multivariate analysis, the cohort showed that age ≥70 years, positive pathological lymph node metastasis, tumors located in the upper ureter, and male sex were predictive factors associated with an increased risk of adverse oncological outcomes. A surgical volume of ≥20 cases showed a trend toward favorable outcomes on cancer-specific survival [hazard ratio (HR) 0.154, p = 0.052] and marginal benefit for overall survival (HR 0.326, p = 0.019) in the multivariate analysis. Conclusion: Although different approaches to transperitoneal laparoscopic nephroureterectomy showed no significant differences in surgical outcomes, age, sex, lymph node metastasis, and tumor in the upper ureter in the following period were predictive factors for oncological outcomes. Higher surgical volume did not impact disease-free survival and bladder recurrence-free survival but was associated with improved overall survival and cancer-specific survival. Exploration of unknown influencing factors is warranted.

Mixed lineage kinase 3 requires a functional CRIB domain for regulation of blood pressure, cardiac hypertrophy, and left ventricular function.

Mixed lineage kinase 3 (MLK3) modulates blood pressure and left ventricular function, but the mechanisms governing these effects remain unclear. In the current study, we therefore investigated the role of the MLK3 Cdc42/Rac interactive binding (CRIB) domain in cardiovascular physiology. We examined baseline and left ventricular pressure overload responses in a MLK3 CRIB mutant (MLK3C/C) mouse, which harbors point mutations in the CRIB domain to disrupt MLK3 activation by Cdc42. Male and female MLK3C/C mice displayed increased invasively measured blood pressure compared with wild-type (MLK3+/+) littermate controls. MLK3C/C mice of both sexes also developed left and right ventricular hypertrophy but normal baseline LV function by echocardiography and invasive hemodynamics. In LV tissue from MLK3C/C mice, map3k11 mRNA, which encodes MLK3, and MLK3 protein were reduced by 74 ± 6% and 73 ± 7%, respectively. After 1-wk LV pressure overload with 25-gauge transaortic constriction (TAC), male MLK3C/C mice developed no differences in LV hypertrophy but displayed reduction in the LV systolic indices ejection fraction and dP/dt normalized to instantaneous pressure. JNK activation was also reduced in LV tissue of MLK3C/C TAC mice. TAC induced MLK3 translocation from cytosolic fraction to membrane fraction in LV tissue from MLK3+/+ but not MLK3C/C mice. These findings identify a role of the MLK3 CRIB domain in MLK3 regulation of basal blood pressure and cardiac morphology, and in promoting the compensatory LV response to pressure overload.NEW & NOTEWORTHY Here, we identified that the presence of two discrete point mutations within the Cdc42/Rac interaction and binding domain of the protein MLK3 recapitulates the effects of whole body MLK3 deletion on blood pressure, cardiac hypertrophy, and left ventricular compensation after pressure overload. These findings implicate the CRIB domain, and thus MLK3 activation by this domain, as critical for maintenance of cardiovascular homeostasis.

Survival Impact of Nephroureterectomy for De Novo Stage IV Nonmetastatic and Metastatic Upper Tract Urothelial Carcinoma.

Background: Whether nephroureterectomy (NU) provides survival benefits in patients with stage IV upper tract urothelial carcinoma (UTUC) remains unclear. We compared the effect of chemotherapy (CT) alone with that of CT combined with NU (CT + NU) on the overall survival (OS) of patients with stage IV nonmetastatic UTUC (nmUTUC) and metastatic UTUC (mUTUC). Patients and Methods: This multicenter retrospective cohort study included the data of patients with UTUC undergoing CT alone or CT + NU from the Chang Gung Cancer Database (2002-2015) and followed them until August 2017. OS and hazard ratios (HRs) were assessed using the Kaplan-Meier method and Cox proportional hazards model, respectively. Results: This study included 308 patients with stage IV UTUC, comprising 139 with nmUTUC and 169 with mUTUC. Moreover, 91 (74.6%) patients with nmUTUC and 31 (25.4%) patients with mUTUC received NU. The CT + NU group had a higher 3-year OS rate (41.0.% vs 16.7%, p < 0.001), longer median OS duration (20.7 vs 9.0 months, p < 0.001), and lower risk of death (HR, 0.48; 95% confidence interval, 0.36-0.66; p < 0.001) than did the CT-alone group. Similarly, patients with mUTUC who underwent CT + NU had a longer median OS duration (25.0 vs 7.8 months, p < 0.001) and lower risk of death (HR, 0.37; 95% confidence interval, 0.23-0.59; p < 0.001) than did those who received CT alone. Conclusion: Compared with CT alone, NU + CT can provide survival benefits to patients with nonmetastatic and metastatic stage IV UTUC.

Metal-Free N-H/C-H Carbonylation by Phenyl Isocyanate: Divergent Synthesis of Six-Membered N-Heterocycles.

We disclose a method using phenyl isocyanate to synthesize carbonyl-containing N-heterocycles. The metal-free novel approach for both N-H and C-H carbonylation processes was successfully refined, delivering a range of synthetically valuable derivatives of quinazoline-2,4(1H,3H)-dione, 2H-benzo[e] [1,2,4] thiadiazin-3(4H)-one 1,1-dioxide, and pyrrolo[1,2-a] quinoxalin-4(5H)-one. The protocol features broad substrates with diverse reactions suitable for excellent yields, mild conditions, and good functional group compatibility. Moreover, the applicability of the reaction was characterized by gram-scale synthesis and synthetic transformations for drug molecules.