Novel fluorinated 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine analogues 7–12 were synthesized and tested for selective cellular uptake by folate receptors (FRs) α and β or the proton-coupled folate transporter (PCFT) and for antitumor efficacy. Compounds 8, 9, 11, and 12 showed increased in vitro antiproliferative activities (~11-fold) over the nonfluorinated analogues 2, 3, 5, and 6 toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds 8, 9, 11, and 12 also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, 9, 11, and 12 showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the de novo purine biosynthesis pathway. Molecular modeling studies validated in vitro cell-based results. NMR evidence supports the presence of an intramolecular fluorine−hydrogen bond. Potent in vivo efficacy of 11 was established with IGROV1 xenografts in severe compromised immunodeficient mice.

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There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.

Background: Tuberculosis (TB) and its evolving drug resistance have exerted severe threats on the global health, hence it is still essential to develop novel anti-TB antibiotics. Ilamycin-E1/E2 is a pair of cycloheptapeptide enantiomers obtained from a marine Streptomyces atratus SCSIO ZH16-ΔilaR mutant, and has presented significant anti-TB activities as promising drug lead compounds, but their clinical development has been hampered by low fermentation titers. Main methods and major results:By applying the statistical Plackett-Burman design (PBD) model, bacterial peptone was first screened out as the only significant but negative factor to affect the ilamycin-E1/E2 production. Subsequent single factor optimization in shaking flasks revealed that the replacement of bacterial peptone with malt extract could not only eliminate the accumulation of porphyrin-type competitive byproducts but also improve the titer of ilamycin-E1/E2 from original 13.6 ± 0.8 to 142.7 ± 5.7 mg L −1 , about 10.5-fold increase. Next, a pH coordinated feeding strategy was adopted in 30 L fermentor and obtained 169.8 ± 2.5 mg L −1 ilamycin-E1/E2, but further scaled-up production in 300 L fermentor only gave a titer of 131.5 ± 7.5 mg L −1 due to the unsynchronization of feeding response and pH change. Consequently, a continuous pulse feeding strategy was utilized in 300 L fermentor to solve the above problem and finally achieved 415.7 ± 29.2 mg L −1 ilamycin-E1/E2, representing a 30.5-fold improvement.Implication: Our work has provided a solid basis to acquire sufficient ilamycin-E1/E2 lead compounds and then support their potential anti-TB drug development.

Multitargeted agents with tumor selectivity result in reduced drug resistance and dose-limiting toxicities. We report 6substituted thieno [2,3-d]pyrimidine compounds (3−9) with pyridine (3, 4), fluorine-substituted pyridine (5), phenyl (6, 7), and thiophene side chains (8, 9), for comparison with unsubstituted phenyl (1, 2) and thiophene side chain (10, 11) containing thieno[2,3-d]pyrimidine compounds. Compounds 3−9 inhibited proliferation of Chinese hamster ovary cells (CHO) expressing folate receptors (FRs) α or β but not the reduced folate carrier (RFC); modest inhibition of CHO cells expressing the proton-coupled folate transporter (PCFT) by 4, 5, 6, and 9 was observed. Replacement of the side-chain 1′,4′-phenyl ring with 2′,5′-pyridyl, or 2′,5′-pyridyl with a fluorine insertion ortho to Lglutamate resulted in increased potency toward FR-expressing CHO cells. Toward KB tumor cells, 4−9 were highly active (IC 50 's from 2.11 to 7.19 nM). By metabolite rescue in KB cells and in vitro enzyme assays, de novo purine biosynthesis was identified as a targeted pathway (at 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase)). Compound 9 was 17-to 882-fold more potent than previously reported compounds 2, 10, and 11 against GARFTase. By targeted metabolomics and metabolite rescue, 1, 2, and 6 also inhibited mitochondrial serine hydroxymethyl transferase 2 (SHMT2); enzyme assays confirmed inhibition of SHMT2. X-ray crystallographic structures were obtained for 4, 5, 9, and 10 with human GARFTase. This series affords an exciting new structural platform for potent multitargeted antitumor agents with FR transport selectivity.

The differences between Chinese and non-Chinese cultures in multinational enterprises in China are introduced, and how communication in different cultural contexts can be efficient and effective, avoiding unnecessary communicative conflicts and thus leading to increased efficiency. The theory of speech act is used to explore the characteristics of multicultural communication in multinational enterprises. By analyzing the speech act characteristics of employees in multinational companies, we can raise people's awareness of different cultural backgrounds, different thinking habits and different values, and help them to control the appropriateness and accuracy of their speech acts in cross-cultural communication, so as to promote cost-effective communication and efficient communication in the company.

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One-carbon (C1) metabolism supports a number of physiological and pathophysiological processes ranging from stem cell renewal to cancer progression. Clinically used antifolates are transported into both tumor and normal cells by the ubiquitously expressed reduced folate carrier (RFC). Uptake of targeted agents via tumor-specific folate receptors (FRs) over RFC would permit tumor-selectivity, while limiting dose-limiting toxicities associated with standard chemotherapy. Serine catabolism in mitochondria is the major source of glycine and C1 units for cytosolic biosynthesis, preserves redox balance and minimizes reactive oxygen species, and is an important source of ATP. Among the mitochondrial C1 enzymes, serine hydroxymethyltransferase 2 (SHMT2) and 5,10-methylene tetrahydrofolate (me-THF) dehydrogenase 2 (MTHFD2) are highly expressed in tumors versus normal tissues. SHMT2 has been suggested to be an important oncodriver. However, there are no clinically relevant inhibitors of these enzymes. To generate potential inhibitors of these enzymes, we synthesized 5-substituted pyrrolo[3,2-d]pyrimidine analogs as structural hybrids of cytotoxic 5-substituted pyrrolo[2,3-d]pyrimidines and me-THF. The 5-substituted pyrrolo[3,2-d] pyrimidine with a four carbon bridged phenyl side chain AGF300 afforded selective uptake via FRα over RFC, with inhibition of mitochondrial C1 metabolism and de novo purine biosynthesis, resulting in inhibition of KB human tumor cell proliferation. Inhibition of KB cells by AGF300 was reversed by glycine and adenosine. As previous studies of related 5-substituted pyrrolo[2,3-d]pyrimidines established that the nature and length of the bridge plays an important role in determining tumor cell potency and transport selectivity, we replaced the carbon adjacent to the phenyl ring in AGF300 with heteroatoms, including O (AGF323), S (AGF346) or NH (AGF350). These compounds were tested as growth inhibitors against engineered Chinese hamster ovary (CHO) cells singly expressing human FRα (RT16) or RFC (PC43-10). Incorporation of the O, S and NH in the pyrrolo[3,2-d]pyrimidine analogs preserved excellent inhibition of FRα-containing CHO cells (IC50s of 57 nM, 77 nM and 50 nM, respectively); there was no inhibition of cells with RFC uptake up to 1000 nM. AGF323, AGF346 and AGF350 inhibited proliferation of KB cells which was reversed by excess glycine and adenosine. This establishes that for AGF323, AGF346 and AGF350, both mitochondrial and cytosolic C1 metabolism were inhibited. The development of novel compounds targeting mitochondrial and cytosolic C1 pathways with tumor-selective uptake is highly significant in that this would overcome the drawbacks of currently used cytotoxic agents for cancer. Citation Format: Nian Tong, Khushbu Shah, Aleem Gangjee, Carrie O’Connor, Adrianne W. Porvirk, Aamod Dekhne, Zhanjun Hou, Larry H. Matherly. Multi-targeted novel 5-substituted pyrrolo[3,2-d]pyrimidines with tumor-selective targeting and inhibition of cytosolic de novo purine biosynthesis and mitochondrial one-carbon metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 789.