Pyrimidine Products Research Articles

Synthesis of pyrazolopyrano pyrimidines via one-pot solvent free greener approach utilizing lanthanum trifluoroacetate and trichloroacetate Lewis acids supported on mesoporous silica: An experimental and DFT studies

Herein, an economic, environment-benign and sustainable greener protocol was developed for the production of pyrazolopyrano pyrimidines utilizing water competent Lanthanum trifluoroacetate and trichloroacetate Lewis acids based over silica via one-pot reaction involving aromatic aldehydes, ethyl acetoacetate, hydrazine hydrate and barbituric acid. The capability to incorporate a broad spectrum of functional groups, fast reaction times, best product yield, gentle reaction medium, solvent-free synthesis and the easy recoverability and multiple-time reusability of catalysts are notable advantages of the present synthetic approach. Furthermore, DFT studies was employed to study the pyrazolopyrano pyrimidines (5a-l), highlighting role of computational methods in predicting and understanding reactivity. Consequently, the integration of DFT studies with synthesis has proven to be a potent strategy for creating the current synthesis method.

#2048 Exploring somatic mutation mechanisms in pre-cystic renal cells of autosomal dominant polycystic kidney disease patients

Abstract Background and Aims Genetic changes accumulate in our cells during a lifetime, at a rate that varies according to cell type and mutagen exposure [1]. Somatic mutations are the driving force of cancer and contribute to “second hit” disorders, such as Autosomal Dominant Polycystic Kidney Disease (ADPKD). ADPKD is caused by inherited mutations that disrupt one allele of PKD1 or PKD2 genes. Somatic loss of the second allele triggers tubule cell clonal expansion and the formation of renal cysts. Given the high number of cysts that form during a lifetime, somatic mutation rates may be abnormally high in the kidney of ADPKD patients. The identification of factors enhancing somatic mutation rates can lead to preventive strategies to limit cyst formation. Metabolic alterations, i.e. increased glutamine utilization and reduced urea cycle, have been shown to induce somatic mutations in cancer [2]. Loss of urea cycle increases pyrimidine synthesis, which unbalances the nucleotide pools and results in a distinct signature of single base substitutions (SBSs) [1]. Similar metabolic changes have been observed in ADPKD [3, 4]. Thus, we sought to test metabolism-driven somatic mutagenesis in pre-cystic kidneys and its contribution to second hit mutations in ADPKD. Method We clonally expanded normal kidney cells from human urine samples and performed a gene expression study by qPCR. We studied 31 clones from 4 ADPKD patients (age range: 25-45) with truncating mutations in PKD1 and normal kidney function, despite a clear cystic phenotype. ADPKD cells were compared to 55 clones from 5 healthy volunteers (age range: 24-53), and 32 clones from 6 patients with another cystic kidney disease, Von Hippel Lindau disease (VHL; age range: 29-56). A subset of clones (n = 19 controls, n = 12 ADPKD) was subjected to whole genome sequencing and somatic mutation analysis. Results Urines of ADPKD patients contained higher numbers of cells that expanded in vitro, compared to both control (p = 0.0008) and VHL individuals (p = 0.01). Gene expression analyses showed that irrespective of genetic background, all cultured clones originated from the kidney tubule epithelium (high PAX2 and PAX8), and some originated from damaged tubules (VCAM1, KIM1). We tested the hypothesized metabolic rewiring by analyzing expression levels of enzymes involved in glutamine utilization, urea cycle, and pyrimidine biosynthesis. Except for ASNS (Asparagine Synthetase), which was higher in ADPKD vs control clones (p = 0.0098), no gene-expression differences were observed. However, clones from ADPKD patients showed signs of the metabolic rewiring responsible for increased pyrimidine production, i.e. a positive correlation (r = 0.691; p < 0.0001) between urea cycle enzymes downregulation and upregulation of the pyrimidine synthesis enzyme CAD. Since excessive pyrimidines lead to mutation [2], we analyzed the number of somatic SBSs per genome, after filtering for germline variants. We did not find increased mutation rates in ADPKD compared to controls, but an analogous, linear increase of mutations with age and similar levels of the pyrimidine-rich mutational signature. Conclusion Urine-derived kidney tubule epithelial cells with heterozygous truncating mutations in PKD1 exhibit certain characteristics of metabolic reprogramming typical of kidneys from ADPKD patients with advanced pathology. Nevertheless, in the limited number of pre-cystic cells that we have analyzed, this metabolic reprogramming did not translate into an excess of somatic mutations.

Identification of Potent hDHODH Inhibitors for Lung Cancer via Virtual Screening of a Rationally Designed Small Combinatorial Library.

Cancer is characterized by altered cellular metabolism, and metabolic enzymes are considered as a promising target for anticancer therapy. Pyrimidine metabolism dysregulation is associated with various types of cancer, particularly lung cancer, which is one of the leading causes of cancer-related mortality worldwide. Recent studies have shown that small-cell lung cancer cells are particularly reliant on the pyrimidine biosynthesis pathway and are sensitive to its disruption. DHODH, the rate-limiting enzyme of the de novo pyrimidine production pathway, is essential in the production of RNA and DNA and is overexpressed in malignancies such as AML, skin cancer, breast cancer, and lung cancer, thereby highlighting DHODH as a viable target for developing drugs to combat lung cancer. Herein, rational drug design and computational techniques were used to discover novel DHODH inhibitors. A small combinatorial library was generated, and the top hits were synthesized and tested for anticancer activity against three lung cancer cell lines. Among the tested compounds, compound 5c possessed a stronger cytotoxicity (TC50 of 11 μM) compared to the standard FDA-approved drug (Regorafenib, TC50 of 13 μM) on the A549 cell line. Furthermore, compound 5c demonstrated potent inhibitory activity against hDHODH at a nanomolar level of 421 nM. DFT, molecular docking, molecular dynamic simulations, and free energy calculations were also carried out to understand the inhibitory mechanisms of the synthesized scaffolds. These in silico studies identified key mechanisms and structural features that will be crucial for future studies.

Design, synthesis, and insecticidal activity of pyrimidinamine derivatives containing 2‐pyridinyloxy moiety

AbstractAphis fabae is a widespread pest that has more than 200 hosts in the world, belongs to hemiptera, for stinging suction mouthparts, harm buds, young leaves, and flower buds. Pyrimidine derivatives have the advantages of insecticidal, bactericidal, herbicidal, regulating plant growth and other biological activities, and have a unique mechanism of action. Pyrimidinamine derivatives, one of pyrimidine derivatives, were reported as early as 1955, and all the world's major pesticide companies (Syngenta, Dow Yinon, Bayer, DuPont, and BASF) have carried out research studies, which had lasted for nearly 60 years. For example, commercial pyrimidine products such as flupyrimethanil (fungicide) and pyrimethanil (insecticidal and acaricidal agents) show excellent biological activity. So pyrimidinamine derivatives are considered promising agricultural compounds. In this study, a series of pyrimidinamine derivatives were designed and synthesized using pyrimidifen as a template according to bioisosterism. 5‐chloro‐N‐(1‐((3‐chloro‐5‐(trifluoromethyl)pyridin‐2‐yl)oxy)‐2‐methylpropan‐2‐yl)‐ 6‐ethylpyrimidin‐4‐amine, T23, had the best control effect on Aphis fabae with LC50 values of 1.46 mg/L, versus commercial insecticides spirotetramat and imidacloprid (2.45 and 0.53 mg/L). The synthesis, characterization, and activity data of the compounds are presented in the text, and the structure–activity relationships are discussed.

Front Cover Picture: Oxidative Annulation of Aldehydes, 5‐Aminopyrazoles, and Nitriles: Synthesis and Applications of Pyrazolo[3,4‐d]Pyrimidines (Adv. Synth. Catal. 8/2023)

The front cover illustrates an oxidative annulation reaction of aldehydes, 5-aminopyrazoles, and nitriles achieved under the oxidation of I2/O2. The theme picture of “Monkey king stealing elixirs” (from “Journey to the West”) was used to represent the one-pot generation of valuable pyrazolo[3,4-d]pyrimidine products that light up lipid droplets in living cells, similar to the piercing eyes gained after the Monkey king ingested the elixir. Details can be found in the Research Article by Hanhong Xu, Chuanle Zhu and co-workers (G. Yao, C. Zhu, T. Qin, M. Wang, Z. Sun, R.-Y. Tang, C. Zhao, H. Jiang, H. Xu, Adv. Synth. Catal. 2023, 365, XXXX–XXXX; DOI: 10.1002/adsc.202201212)

Clinical and genomic features of non-small cell lung cancer occurring in families.

Exposure to environmental carcinogens, such as through smoking, is a major factor in the carcinogenesis of non-small cell lung cancer (NSCLC). However, genetic factors may also contribute. To identify candidate tumor suppressor genes for NSCLC, we included 23 patients (10 related pairs and 3 individuals) with NSCLC who had other NSCLC-affected first-degree relatives in a local hospital. Exome analyses for both germline and somatic (NSCLC specimens) DNA were performed for 17 cases. Germline exome data of these 17 cases revealed that most of the short variants were identical to the variants in 14KJPN (a Japanese reference genome panel of more than 14 000 individuals) and only a nonsynonymous variant in the DHODH gene, p.A347T, was shared between a pair of NSCLC patients in the same family. This variant is a known pathogenic variant of the gene for Miller syndrome. Somatic genetic alterations in the exome data of our samples showed frequent mutations in the EGFR and TP53 genes. Principal component analysis of the patterns of 96 types of single nucleotide variants (SNVs) suggested the existence of unique mechanisms inducing somatic SNVs in each family. Delineation of mutational signatures of the somatic SNVs with deconstructSigs for the pair of germline pathogenic DHODH variant-positive cases showed that the mutational signatures of these cases included SBS3 (homologous recombination repair defect), SBS6, 15 (DNA mismatch repair), and SBS7 (ultraviolet exposure), suggesting that disordered pyrimidine production causes increased errors in DNA repair systems in these cases. Our results suggest the importance of the detailed collection of data on environmental exposure along with genetic information on NSCLC patients to identify the unique combinations that cause lung tumorigenesis in a particular family.

P864: CTPS1 IS A NOVEL THERAPEUTIC TARGET IN MYELOMA - SELECTIVE SMALL MOLECULE INHIBITION DELIVERS SINGLE AGENT ACTIVITY AND SYNERGISES WITH ATR INHIBITION

Background: Recent years have seen major improvements in the treatment of myeloma; however, relapse occurs in the majority of patients. Novel treatment approaches are moving away from cytotoxic chemotherapy towards targeted therapies with defined mechanisms of action. Malignant cells have an increased demand for key cellular components and a reliance on de novo synthesis pathways. CTP synthetase 1 (CTPS1) plays a pivotal role in pyrimidine production, by catalysing the rate limiting step in the de novo synthesis of CTP, which is required for DNA, RNA and phospholipids. Human genetic studies have identified an essential and non-redundant role for CTPS1 in lymphoid cell proliferation which is complemented by the homologous CTPS2 isoform outside the haemopoietic system. Aims: To evaluate the role of CTPS1 as a novel target in myeloma, elucidate the molecular consequences of CTPS1 inhibition and test rationally designed combination therapy. Methods: STP938 is a potent small molecule CTPS1 inhibitor with >1,300-fold selectivity for CTPS1 over CTPS2. The effects of STP938 on cell proliferation (metabolic activity, tetrazolium indicator), apoptosis (annexin V) and cell cycle (propidium iodide) were assessed in vitro using 12 myeloma cell lines. The role of CTPS1 was further assessed in CRISPR knock-out (KO) experiments. Activation of proteins in the replication stress and DNA damage response pathways was analysed by western blotting. Additive/synergistic effects of combining STP938 with two different ATR inhibitors were assessed on cell line proliferation and enumerated by Bliss score (Synergyfinder 2.0). Results: STP938 showed single agent activity against 6 of 12 cell lines tested, with IC50 values for sensitive lines ranging from 19 to 128 nM (Figure A). STP938 showed cytotoxic activity against sensitive lines, evidenced by induction of apoptosis. KO experiments confirmed that MM cell lines depend on CTPS1 for proliferation. Exposure to STP938 was associated with accumulation of cells in S phase, induction of a replication stress response as evidenced by activation (phosphorylation) of CHEK1, and induction of the DNA damage response pathway as evidenced by activation of CHEK2 (albeit to a lesser extent than activation of CHEK1). Importantly, S phase accumulation, induction of replication stress and induction of DNA damage response was observed in cell lines where STP938 did not produce significant anti-proliferative activity (Figure B). Given the ability of STP938 to activate CHEK1, STP938 was tested in combination with an ATR inhibitor, as ATR is the main upstream activator of CHEK1. The ATR inhibitor ceralasertib demonstrated additive or synergistic activity when combined with STP938 (Figure C). Notably, synergy between STP938 and ATR inhibition, along with induction of apoptosis, was observed in cell lines resistant to single agent STP938. Results were confirmed with a second ATR inhibitor (VE-821, Figure C). Image:Summary/Conclusion: Inhibition of CTPS1 by single agent STP938 showed anti-proliferative activity against 6 of 12 myeloma cell lines, and induced replication stress in all cell lines. Combined STP938 and ATR inhibition showed anti-proliferative activity in all cell lines, including those resistant to single agent STP938. These data suggest a model whereby myeloma cells with high background replication stress are sensitive to killing by single agent STP938, whereas cells with lower background replication stress are sensitised by STP938 to cell death induced by ATR inhibition (Figure D). STP938 will shortly enter clinical development for patients with late stage lymphoid neoplasms.

Targeting the Plasmodium falciparum's Thymidylate Monophosphate Kinase for the Identification of Novel Antimalarial Natural Compounds.

Recent reports of resistance to artemisinin-based combination drugs necessitate the need to discover novel antimalarial compounds. The present study was aimed at identifying novel antimalarial compounds from natural product libraries using computational methods. Plasmodium falciparum is highly dependent on the pyrimidine biosynthetic pathway, a de novo pathway responsible for the production of pyrimidines, and the parasite lacks the pyrimidine salvage enzymes. The P. falciparum thymidylate monophosphate kinase (PfTMPK) is an important protein necessary for rapid DNA replication; however, due to its broad substrate specificity, the protein is distinguished from its homologs, making it a suitable drug target. Compounds from AfroDB, a database of natural products originating from Africa, were screened virtually against PfTMPK after filtering the compounds for absorption, distribution, metabolism, excretion, and toxicity (ADMET)-acceptable compounds with FAF-Drugs4. Thirteen hits with lower binding energies than thymidine monophosphate were selected after docking. Among the thirteen compounds, ZINC13374323 and ZINC13365918 with binding energies of −9.4 and −8.9 kcal/mol, respectively, were selected as plausible lead compounds because they exhibited structural properties that ensure proper binding at the active site and inhibitory effect against PfTMPK. ZINC13374323 (also called aurantiamide acetate) is known to exhibit anti-inflammatory and antiviral activities, and ZINC13365918 exhibits antileishmanial activity. Furthermore, aurantiamide acetate, which is commercially available, is a constituent of Artemisia annua, the herb from which artemisinin was derived. The compound also shares interactions with several residues with a potent thymidine analog inhibitor of PfTMPK. The anti-plasmodial activity of aurantiamide acetate was evaluated in vitro, and the mean half-maximal inhibitory concentration (IC50) was 69.33 μM when synchronized P. falciparum 3D7 culture was used as compared to IC50 > 100 μM with asynchronized culture. The significance of our findings within the context of malaria treatment strategies and challenges is discussed.

Fullerene‐C60/NH2: A recyclable heterogeneous catalyst for the green synthesis of chromene and pyrimidine derivatives and antibacterial evaluation

AbstractAn effective procedure for the synthesis of amino‐functionalized fullerene with diethylenetriamine (C60‐NH2) is described. The obtained material was characterized by Fourier‐transform infrared spectroscopy, X‐ray diffraction, scanning electron microscopy, energy dispersive X‐ray spectroscopy, elemental analysis, thermogravimetric measurements, and X‐ray photoelectron spectroscopy. The catalytic activity of C60‐NH2 was studied in multicomponent reactions for the synthesis of chromene and pyrimidine derivatives. The results revealed that C60‐NH2 was very effective and the desired chromene and pyrimidine products were isolated in good to excellent yields 78%–97% and 81%–98%, respectively. Moreover, the C60‐NH2 catalyst could be recycled and reused for at least eight runs without substantial loss in its activity, which makes our procedure environmentally benign. Antibacterial behavior of some of the synthesized products was studied against Gram‐positive and Gram‐negative bacteria using disk and well diffusion methods. The chromene 4g showed a good antibacterial activity against both bacterial strains Staphylococcus aureus and Serratia marcescens. A good antibacterial activity was also detected for chromene 4o and pyrimidine 7j against S. aureus. The chromene 4m, 4n and pyrimidine 7i compounds displayed a good activity against Micrococcus luteus as a Gram‐positive bacterium.

Synthesis of chalcone-based pyrimidine derivatives and their antimicrobial activity

A series of substituted chalcones and pyrimidine derivatives on their base was synthesized. Obtained compounds were tested for antimicrobial activity and ability to increase activity of known antimicrobial substances. Pyrimidine products shown good activity against Staphylococcus aureus and Candida tropicalis. These compounds also could increase activity of modified polyguanidines against Staphylococcus aureus.

ASS1 and ASL suppress growth in clear cell renal cell carcinoma via altered nitrogen metabolism

BackgroundKidney cancer is a common adult malignancy in the USA. Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, is characterized by widespread metabolic changes. Urea metabolism is one such altered pathway in ccRCC. The aim of this study was to elucidate the contributions of urea cycle enzymes, argininosuccinate synthase 1 (ASS1), and argininosuccinate lyase (ASL) towards ccRCC progression.MethodsWe employed a combination of computational, genetic, and metabolomic tools along with in vivo animal models to establish a tumor-suppressive role for ASS1 and ASL in ccRCC.ResultsWe show that the mRNA and protein expression of urea cycle enzymes ASS1 and ASL are reduced in ccRCC tumors when compared to the normal kidney. Furthermore, the loss of ASL in HK-2 cells (immortalized renal epithelial cells) promotes growth in 2D and 3D growth assays, while combined re-expression of ASS1 and ASL in ccRCC cell lines suppresses growth in 2D, 3D, and in vivo xenograft models. We establish that this suppression is dependent on their enzymatic activity. Finally, we demonstrate that conservation of cellular aspartate, regulation of nitric oxide synthesis, and pyrimidine production play pivotal roles in ASS1+ASL-mediated growth suppression in ccRCC.ConclusionsccRCC tumors downregulate the components of the urea cycle including the enzymes argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL). These cytosolic enzymes lie at a critical metabolic hub in the cell and are involved in aspartate catabolism and arginine and nitric oxide biosynthesis. Loss of ASS1 and ASL helps cells redirect aspartate towards pyrimidine synthesis and support enhanced proliferation. Additionally, reduced levels of ASS1 and ASL might help regulate nitric oxide (NO) generation and mitigate its cytotoxic effects. Overall, our work adds to the understanding of urea cycle enzymes in a context-independent of ureagenesis, their role in ccRCC progression, and uncovers novel potential metabolic vulnerabilities in ccRCC.

Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine.

Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y.lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine-5'-phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y.lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed-batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y.lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y.lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.

DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes

AbstractDihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.

Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells.

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.

3093 – DHODH INHIBITION SYNERGIZES WITH DNA-DEMETHYLATING AGENTS IN THE TREATMENT OF MYELODYSPLASTIC SYNDROMES

<h3>Background</h3> Myelodysplastic syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. Although DNA methyltransferase inhibitors have been used as chemotherapeutic agents for high-risk MDS, the disease become uncontrolled in many cases. Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We herein investigated the efficacy of PTC299, a novel DHODH inhibitor, for MDS. <h3>Methods</h3> Anti-MDS efficacy of PTC299 was studied using human MDS cell lines and primary MDS cells in vitro. In addition, we assessed the efficacy of PTC299 in xenograft mouse models of MDS by AkaBLI system, which is composed of AkaLumine-HCl and Akaluc. AkaBLI system has improved performance by a factor of 100 to 1,000 over the conventional bioluminescence imaging system composed of D-luciferin and firefly luciferase. Mechanistic studies were conducted via LC-MS/MS, cell cycle analysis, RNA-seq and RT-qPCR. <h3>Results</h3> In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents to inhibit the growth of MDS cells. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. <h3>Conclusions</h3> Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine.

Molecular Docking Study and Hirshfeld Surface Analysis of Formamide Derivative

Formamide (HCONH2), a formic acid amide, has its versatile properties as an intermediate in the agrochemical and pharmaceutical industries and for the production of vitamins and pyrimidines. N-(4-Bromo-Phenyl)-Formamide is a novel derivative of formamide with bromo-phenyl moiety substituted at nitrogen. The title molecule is a moderate antimicrobial agent. The synthesized compound, C7H6BrNO has been confirmed by single crystal X-ray diffraction studies. This molecule crystallizes in centric orthorhombic crystal system and space-group Pbca with crystal parameters a=10.8560(19) Ǻ, b=9.8166(17) Ǻ, c=13.912(40) Ǻ and Z=8. The number of weak but significant C–H…O, N–H…O and π–π interactions take part in the stability of crystal packing. Qualitative and quantitative contributions of intermolecular interactions in molecular structure are investigated by Hirshfeld surface analysis and 2D fingerprint plots. In order to study the structure-function relationship of the ligand with different receptors and to determine the lowest free energy structures for the receptor ligand complex, docking study has been carried out. The different receptors are docked with title compound and the energy value calculated.

Phosphoramidates as Steering Elements for Highly Selective Access to Complementary Imidazo[1,2-a]pyrimidine Isomers.

We report that selective N-phosphorylation of aminoimidazoles results in a key steering element that controls isomeric selectivity in the condensation of β-ethoxy acrylamides and aminoimidazoles to furnish imidazo[1,2-a]pyrimidines. We identified conditions that provide highly selective (99:1) phosphorylation at the endo- or exocyclic nitrogen. Either the 2-amino or 4-amino isomer of the (benzo)imidazo[1,2-a]pyrimidine products could be isolated in 64-95% yield. Mass spectrometric analysis and computational studies give insight into the mechanism of this exceptionally selective transformation.

Macrophages throw tumour cells a lifeline.

Pancreatic ductal adenocarcinoma cells induce macrophage production of pyrimidines that mediate tumour cell resistance to chemotherapy.

Preliminary Results of a Phase 2a Dose Optimization Study of ASLAN003 (DHODH inhibitor) in Acute Myeloid Leukemia (AML) Patients Who Are Ineligible for Standard Therapy; Early Signs of Activity

Background:Dihydroorotate dehydrogenase (DHODH), catalyzing the ubiquinone-mediated oxidation of dihydroorotate to orotate, is the rate-limiting enzyme in the de novo synthesis of pyrimidines, and as such may control the rate of cell division. The enzyme is localised on the outer side of the inner mitochondrial membrane, and links both the electron transport chain to pyrimidine production and thus to the maintenance of cell viability. DHODH inhibitors were identified in a myeloid differentiation screen using an ER-HoxA9 GMP cell line, and ASLAN003, a novel small molecule DHODH oral inhibitor, has been found to be a potent inducer of myeloid differentiation in AML cell lines. ASLAN003 also demonstrated the ability to reduce leukemic burden and extend survival in AML xenograft models. ASLAN003 has previously been shown to exhibit a safe and tolerable profile in prior phase I studies in healthy volunteers.Methods:A multicenter, single arm phase IIA study was initiated to evaluate ASLAN003 monotherapy administered as a 28-day cycle in patients with AML who are ineligible for standard therapy. The primary objective is to determine the optimum dose of ASLAN003 in this AML cohort based on efficacy (Overall Complete Remission Rate, % of complete remission [CR] + CR with incomplete hematologic recovery [CRi]), tolerability and safety. The secondary objective is to assess the pharmacokinetics (PK) of ASLAN003 and its metabolites and to further assess the efficacy based on relapse-free survival and clinical benefit rate (CBR, % of partial remission + CR + CRi). Exploratory objectives are to examine the myeloid differentiation effects of ASLAN003 ex vivo and explore possible relationships between the clinical response and molecular profile of leukemic cells. The study contains 3 cohorts for the optimum dose determination (100 mg, 200 mg, and 300 mg once daily [QD], with planned enrollment for 6 patients for each cohort), and an additional expansion cohort with the selected optimum dose (20 patients).Results:Enrollment started in December 2017. As of 12 July 2018, 10 patients were enrolled and treated (6 in the 100 mg QD cohort and 4 in the 200 mg QD cohort). Although the data are immature, two patients to date have exhibited some evidence of clinical activity (one per cohort). A patient in the 100 mg QD cohort, with a baseline peripheral blood blast of 27%, experienced a reduction to 6% on Cycle 3 Day 10 (C3D10), coupled with an upward trend in the percentage of neutrophils (16% at baseline, 64% on C3D10). The patient had underlying chronic lung infection, but also showed suspected symptoms of differentiation syndrome (dyspnea, leukocytosis). After 126 days of ASLAN003 initiation, the patient expired due to AML related clinical deterioration. The second patient in the 200 mg QD cohort experienced a decrease in bone marrow blasts from 26% at baseline, to 12% on C2D1. Concomitantly, there was a slight decrease in peripheral blood blasts with an increase in neutrophil percentages from 7% to 51%. The patient remains on study with further efficacy data awaited. To date, the safety profile is consistent with the previous healthy volunteer studies. Treatment-related adverse events (AEs) are listed in Table 1. The most frequent treatment-related AEs include nausea (n=2, 20%) and leukocytosis (n=2, 20%). Two treatment-related Grade ≥ 3 AEs were observed (Grade 3 anaemia and Grade 3 leukocytosis). The study continues and is currently enrolling to the 200 mg cohort.Conclusion:At the date of submission, this is the first report of clinical activity of a DHODH inhibitor in AML patients. Monotherapy with ASLAN003 was well tolerated and has showed encouraging signs of clinical activity in AML patients. This study is on-going and the optimal dose for AML has not yet been determined. Safety, PK and efficacy data will be updated at the time of presentation. Clinical trial information: NCT03451084 [Display omitted] DisclosuresChng:ASLAN Pharmaceuticals: Research Funding. McHale:ASLAN Pharmaceuticals: Employment, Equity Ownership. Hsieh:ASLAN Pharmaceuticals: Employment, Equity Ownership. Shih:ASLAN Pharmaceuticals: Employment; AstraZeneca Taiwan: Employment. McIntyre:ASLAN Pharmaceuticals: Consultancy. Kwek:ASLAN Pharmaceuticals: Employment. Chang:ASLAN Pharmaceuticals: Employment. Lindmark:ASLAN Pharmaceuticals: Employment, Equity Ownership.

Novel synthesis, ring transformation and anticancer activity of 1,3-thiazine, pyrimidine and triazolo[1,5-a]pyrimidine derivatives

Synthesis, heterocyclization and anticancer activity of a new series of heterocyclic compounds are described. Aminothiazine 1 was obtained from the base induced condensation of thiourea, benzaldehyde and ethyl cyanoacetate. The synthesis of N-phenyl amino pyrimidine derivative 2 was obtained as a result of reaction of aniline with compound 1. Compound 2 underwent ring opening and recyclization upon reaction with HCl or H2O2/ NaOH to afford the acid derivative 3 or oxazine 4, respectively. Thiazine 1 undergoes ring transformation upon the effect of NH2OH.HCl to produce pyrimidine derivative 5. Heterocyclization of compound 1 with thiosemicarbazide followed by oxidation with I2/AcOH afforded triazolopyrimidine 6 and 7, respectively. Alkylation of compound 1 was promoted by reaction of 1 with ethyl iodide to give alkylated thiazine 8 which in turn undergo ring transformation when subjected to reaction with hydrazine hydrate to give pyrazole derivative 9. Refluxing of amino-1,3-thiazine derivative 1 with ethyl bromoacetate in the presence of Et3N produce the alkylated pyrimidine product 10. Hydrazonolysis of 1,3-thiazine 1 with hydrazine or phenyhydrazine gave pyrimidine derivatives 11a,b, respectively. Compound 11b was cyclized with carbon disulfide or formaldehyde to produce triazolopyrimidines 12 and 13, respectively. Some of the new compounds were screened for anticancer activity and significant results were found for some compounds. KEY WORDS: 1,3-Thiazine, Pyrimidine, Triazole, Pyrazole, Anticancer activity Bull. Chem. Soc. Ethiop. 2018, 32(3), 513-522.DOI: https://dx.doi.org/10.4314/bcse.v32i3.10