Discovery of a Potent, Selective, and Orally Available MTHFD2 Inhibitor (DS18561882) with in Vivo Antitumor Activity.

We report the discovery of a potent and isozyme-selective MTHFD2 inhibitor, DS18561882 (2). Through investigation of the substituents on our tricyclic coumarin scaffold (1,2,3,4-tetrahydrochromeno[3,4-c]pyridin-5-one), MTHFD2 inhibitory activity was shown to be elevated by incorporating an amine moiety at the 8-position and a methyl group at the 7-position of the initial lead 1. X-ray structure analysis revealed that a key interaction for enhanced potency was salt bridge formation between the amine moiety and the diphosphate linker of an NAD+ cofactor. Furthermore, ortho-substituted sulfonamide in place of benzoic acid of 1 significantly improved cell permeability and cell-based growth inhibition against a human breast cancer cell line. The thus-optimized DS18561882 showed the strongest cell-based activity (GI50 = 140 nM) in the class, a good oral pharmacokinetic profile, and thereby tumor growth inhibition in a mouse xenograft model upon oral administration.

Structure-Based Design and Synthesis of an Isozyme-Selective MTHFD2 Inhibitor with a Tricyclic Coumarin Scaffold.

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays a key role in one-carbon (1C) metabolism in human mitochondria, and its high expression correlates with poor survival of patients with various types of cancer. An isozyme-selective MTHFD2 inhibitor is highly attractive for potential use in cancer treatment. Herein, we disclose a novel isozyme-selective MTHFD2 inhibitor DS44960156, with a tricyclic coumarin scaffold, which was initially discovered via high-throughput screening (HTS) and improved using structure-based drug design (SBDD). DS44960156 would offer a good starting point for further optimization based on the following features: (1) unprecedented selectivity (>18-fold) for MTHFD2 over MTHFD1, (2) a molecular weight of less than 400, and (3) good ligand efficiency (LE).

Discovery of Novel Pyrido-pyridazinone Derivatives as FER Tyrosine Kinase Inhibitors with Antitumor Activity.

To obtain a new anticancer drug, we focused on FER tyrosine kinase. Starting with high-throughput screening with our in-house chemical library, compound 1, which has a pyridine moiety, was found. Referring to their X-ray crystal structure with FES proto-oncogene tyrosine kinase, as a surrogate of FER followed by chemical modification including scaffold hopping of the pyridine template, we discovered pyrido-pyridazinone derivatives with potent FER kinase inhibitory activity. Here, we disclose the structure-activity relationship on the scaffold and representative compound 21 (DS21360717), which showed in vivo antitumor efficacy in a subcutaneous tumor model.

Preclinical therapeutic efficacy of a novel blood-brain barrier-penetrant dual PI3K/mTOR inhibitor with preferential response in PI3K/PTEN mutant glioma.

Glioblastoma (GBM) is an ideal candidate disease for signal transduction targeted therapy because the majority of these tumors harbor genetic alterations that result in aberrant activation of growth factor signaling pathways. Loss of heterozygosity of chromosome 10, mutations in the tumor suppressor gene PTEN, and PI3K mutations are molecular hallmarks of GBM and indicate poor prognostic outcomes in many cancers. Consequently, inhibiting the PI3K pathway may provide therapeutic benefit in these cancers. PI3K inhibitors generally block proliferation rather than induce apoptosis. To restore the sensitivity of GBM to apoptosis induction, targeted agents have been combined with conventional therapy. However, the molecular heterogeneity and infiltrative nature of GBM make it resistant to traditional single agent therapy. Our objectives were to test a dual PI3K/mTOR inhibitor that may cross the blood-brain barrier (BBB) and provide the rationale for using this inhibitor in combination regimens to chemotherapy-induced synergism in GBM. Here we report the preclinical potential of a novel, orally bioavailable PI3K/mTOR dual inhibitor, DS7423 (hereafter DS), in in-vitro and in-vivo studies. DS was tested in mice, and DS plasma and brain concentrations were determined. DS crossed the BBB and led to potent suppression of PI3K pathway biomarkers in the brain. The physiologically relevant concentration of DS was tested in 9 glioma cell lines and 22 glioma-initiating cell (GIC) lines. DS inhibited the growth of glioma tumor cell lines and GICs at mean 50% inhibitory concentration values of less than 250 nmol/L. We found that PI3K mutations and PTEN alterations were associated with cellular response to DS treatment; with preferential inhibition of cell growth in PI3KCA-mutant and PTEN altered cell lines. DS showed efficacy and survival benefit in the U87 and GSC11 orthotopic models of GBM. Furthermore, administration of DS enhanced the antitumor efficacy of temozolomide against GBM in U87 glioma models, which shows that PI3K/mTOR inhibitors may enhance alkylating agent-mediated cytotoxicity, providing a novel regimen for the treatment of GBM. Our present findings establish that DS can specifically be used in patients who have PI3K pathway activation and/or loss of PTEN function. Further studies are warranted to determine the potential of DS for glioma treatment.

Amounts and compositional analysis of glycosaminoglycans in the tissue of fish.

We isolated GAGs from the inedible parts; head, skin, internal organs, fins, scales and spine, of atlantic mackerel (Scomber scombrus), japanese jack mackerel (Trachurus japonicus), pacific bluefin tuna (Thunnus orientalis), yellowfin sole (Limanda aspera), broadbanded thornyhead (Sebastolobus macrochir), golden threadfin bream (Nemipterus virgatus), and nile tilapia (Oreochromis niloticus). We also investigated deep-sea fish, eelpouts (Bothrocara hollandi, Lycodes toyamensis, and Lycodes nakamurae), rough snailfish (Careproctus trachysoma), and squids (Watasenia scintillans, Enoploteuthis chunii, and Berryteuthis magister). Enzymatic digestion of the GAGs enabled a compositional analysis of CS, DS, and HA including the sulfation patterns of CS and DS, as well as the amount of each GAG. The molecular weights and distributions of these GAGs were also examined. The amounts of GAGs contained in the tissues and CS/DS ratios differed remarkably among the fish. The dorsal fin of the yellowfin sole contained more than 1300mg of CS-DS per 100g of defatted-dry tissue. Although the fish generally contained A-type rich CS-DS, bottom fish and deep-sea fish often possessed C-type CS-DS in larger ratios. Squid characteristically had E-type CS-DS which was normally less common in fish except in cartilaginous fish. These analytical results had no relation to the biological classification.

Spongipyran Synthetic Studies. Total Synthesis of (+)-Spongistatin 2.

Evolution of a convergent synthetic strategy to access (+)-spongistatin 2 (2), a potent cytotoxic marine macrolide, is described. Highlights of the synthesis include: development of a multicomponent dithiane-mediated linchpin union tactic, devised and implemented specifically for construction of the spongistatin AB and CD spiro ring systems; application of a Ca(II) ion controlled acid promoted equilibration to set the thermodynamically less stable axial-equitorial stereogenicity in the CD spiroketal; use of sulfone addition/Julia methylenation sequences to unite the AB and CD fragments and introduce the C(44)-C(51) side chain; and fragment union and final elaboration to (+)-spongistatin 2 (2) exploiting Wittig olefination to unite the advanced ABCD and EF fragments, followed by regioselective Yamaguchi macrolactonization and global deprotection. Correction of the CD spiro ring stereogenicity was subsequently achieved via acid equilibration in the presence of Ca(II) ion to furnish (+)-spongistatin 2 (2). The synthesis proceeded with a longest linear sequence of 41 steps.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate.

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, substituted at the 2-position with piperidines bearing quaternary ammonium salt side chains, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Attachment of the charged entity using an N-ethylcarbamoyloxy linker led to the discovery of the highly soluble compound 22 (D13-9001), which maintained good potency in vitro and displayed excellent activity in vivo in a rat pneumonia model of P. aeruginosa.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 6: exploration of aromatic substituents.

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with aromatic substituents, were synthesized by the Suzuki cross-coupling method and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. By incorporating hydrophilic substituents onto the aryl nucleus, we found a morpholine analogue that possessed improved solubility, retained activity in vitro, and displayed potentiation activity in vivo in a rat model of P. aeruginosa pneumonia.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 5: Carbon-substituted analogues at the C-2 position.

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with carbon-linked substituents, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Palladium-catalyzed cross-coupling methods were applied for the incorporation of aliphatic and aromatic substituents.

Insonation facilitates plasmid DNA transfection into the central nervous system and microbubbles enhance the effect.

Many of the diseases which affect the central nervous system are intractable to conventional therapies and therefore require alternative treatments such as gene therapy. Therapy requires safety, since the central nervous system is a critical organ. Choice of nonviral vectors such as naked plasmid DNA may have merit. However, transfection efficiencies of these vectors are low. We have investigated the use of 210.4 kHz ultrasound and found that 5.0 W/cm(2) of insonation for 5 s most effectively transfected a plasmid DNA into culture slices of mouse brain (147.68-fold increase compared with 0 W/cm(2) of insonation for 5 s). The effect was reinforced by combination with echo contrast agent, Levovist. One hundred fifty mg/mL of Levovist significantly increased gene transfection by ultrasound (5.23-fold when insonated at 5.0 W/cm(2) for 5 s). When DNA was intracranially injected, Levovist also enhanced gene transfection in newborn mice (4.49-fold increase when insonated at 5.0 W/cm(2) for 5 s). Since ultrasound successfully transfected naked plasmid DNA into the neural tissue and Levovist enhanced the effect, this approach may have a significant role in gene transfer to the central nervous system.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 4: Addressing the problem of poor stability due to photoisomerization of an acrylic acid moiety.

Exchange of the ethylene tether in a series of pyridopyrimidine-based MexAB-OprM specific efflux pump inhibitors to an amide bond stabilized the olefin of the acrylic acid moiety, preventing facile photoisomerization to the Z-isomer. Furthermore, the activity was drastically improved in the amide tether variants, providing extremely potent acrylic acid and vinyl tetrazole analogues.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 3: Optimization of potency in the pyridopyrimidine series through the application of a pharmacophore model.

The addition of substituents to the pyridopyrimidine scaffold of MexAB-OprM specific efflux pump inhibitors was explored. As predicted by a pharmacophore model, the incorporation substituents at the 2-position improved potency. Piperidines were found to be optimal, and further introduction of polar groups without compromising the activity was shown to be feasible. Careful positioning of the essential acidic moiety of the pharmacophore relative to the scaffold led to the discovery of vinyl tetrazoles with still greater potency.

MexAB-OprM-specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 1: discovery and early strategies for lead optimization.

The identification of a series of compounds that specifically inhibit efflux by the MexAB-OprM pump system in Pseudomonas aeruginosa is described. Synthesis and in vitro structure-activity relationships (SARs) are outlined. Early leads lacked activity in animal models, and efforts to improve solubility and reduce serum protein binding by the introduction of polar groups are discussed.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 2: achieving activity in vivo through the use of alternative scaffolds.

Problems of low solubility, high serum protein binding, and lack of efficacy in vivo in first generation MexAB-OprM specific efflux pump inhibitors were addressed. Through the use of pharmacophore modelling, the key structural elements for pump inhibition were defined. Use of alternative scaffolds upon which the key elements were arrayed gave second generation leads with greatly improved physical properties and activity in the potentiation of antibacterial quinolones (levofloxacin and sitafloxacin) versus Pseudomonas aeruginosa in vivo.

Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa.

Conformational restriction of the ornithine residue of the efflux pump inhibitor D-ornithine-D-homophenylalanine-3-aminoquinoline (MC-02,595, 2) furnished bioisosteric proline derivatives that were less toxic in vivo and as active as the lead in potentiating the activity of the fluoroquinolone levofloxacin via the inhibition of efflux pumps in Pseudomonas aeruginosa.