Design,synthesis, and biological evaluation of deuterated phenylpropionic acid derivatives as potent and long-acting free fatty acid receptor 1 agonists

The free fatty acid receptor 1 (FFA1) is a potential target due to its function in enhancement of glucose-stimulated insulin secretion. Takeda’s compound 1 has robustly in vitro activity for FFA1, but it has been suffered from poor pharmacokinetic (PK) profiles because the phenylpropanoic acid is vulnerable to β-oxidation. To identify orally available agonists, we tried to interdict the metabolically labile group by incorporating two deuterium atoms at the α-position of phenylpropionic acid. Interestingly, the differences of physicochemical properties between hydrogen and deuterium are quite small, but there are many differences in the structure-activity relationship between phenylpropionic acid series and present deuterated series. Further optimizations of deuterated series led to the discovery of compound 18, which exhibited a superior balance in terms of in vitro activity, lipophilicity, and solubility. Better still, compound 18 revealed a lower clearance (CL = 0.44 L/h/kg), higher maximum concentration (C_max = 7584.27 μg/L), and longer half-life (T_1/2 = 4.16 h), resulting in a >23-fold exposure than compound 1. In subsequent in vivo pharmacodynamic studies, compound 18 showed a robustly glucose-lowering effect in rodent without the risk of hypoglycemia.     

4-epi-Isofagomine derivatives as pharmacological chaperones for the treatment of lysosomal diseases linked to β-galactosidase mutations: Improved synthesis and biological investigations

(5aR)-5a-C-pentyl-4-epi-isofagomine 1 is a powerful inhibitor of lysosomal β-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B. We report herein an improved synthesis of this compound and analogs (5a-C-methyl, pentyl, nonyl and phenylethyl derivatives), and a crystal structure of a synthetic intermediate that confirms its configuration resulting from the addition of a Grignard reagent. These compounds were evaluated as glycosidase inhibitors and their potential as chaperones for mutant lysosomal galactosidases determined. Based on these results and on docking studies, the 5-C-pentyl derivative 1 was selected as the optimal structure for further investigations: this compound induces the maturation of mutated β-galactosidase in fibroblasts of a GM1-gangliosidosis patient and promote the decrease of keratan sulfate and oligosaccharide load in patient cells. Compound 1 is clearly capable of restoring β-galactosidase activity and of promoting maturation of the protein, which should result in significant clinical benefit. These properties strongly support the development of compound 1 for the treatment of GM1-gangliosidosis and Morquio disease type B patients harboring β-galactosidase mutations sensitive to pharmacological chaperoning.     

3-deoxy-2,5-DI-O-p-nitrobenzoyl-α-D-threo-pentofuranosyl bromide,a starting material for the synthesis of nucleosides

Hydrogenation (Raney nickel) of methyl 2,3-anhydro-α- and β-D-lyxo-furanoside (3 and 12) for 12 h at $\text{60}\text{60}$ lb.in.-2 afforded methyl 3-deoxy-α- and β-D-threo pentofuranoside (4 and 13). These were p-nitrobenzoylated to give the 2,5-di-p-nitrobenzoates (5b and 14b) which, on treatment with hydrogen bromide in acetyl bromide—acetic acid—dichloromethane, afforded the title compound (6b). The structure of compound 4 was established by conversion into the previously prepared 2,5-dibenzoate 5a of known structure, and the anomeric configuration of 6b was established by comparison of its optical rotation and the signals of its anomeric proton with those of methyl 3-deoxy-D-threo-pentofuranosides.     

Design,synthesis, and biological evaluation of spiroindolines as novel inducers of oligodendrocyte progenitor cell differentiation—Use of a conformation-based hypothesis to facilitate compound design

Inducing oligodendrocyte progenitor cell (OPC) differentiation is a novel therapeutic strategy for the treatment of demyelinating diseases such as multiple sclerosis (MS). In the preceding article, we detailed the discovery of compound 1, a potent inducer of OPC differentiation possessing a characteristic spiroindoline structure. Also, we found that N-methylation and des-carbonyl compound 1 (4) led to a loss in potency. Herein, we describe our investigations of a conformation-based hypothesis for OPC differentiation activity based on the preferred conformation of the spiro core, and further structure–activity relationship (SAR) exploration led to the identification of 6-CF₃ derivative 8, which was more potent compared to compound 1.     

Design,synthesis, and biological evaluation of deuterated apalutamide with improved pharmacokinetic profiles

A series of deuterated apalutamide were designed and prepared. Compared to its prototype compound 18, deuterated analogues 19 and 21 showed obviously higher plasma concentrations and better PK parameters after oral administration in mice. In rats, N-trideuteromethyl compound 19 displayed 1.8-fold peak concentration (C_max), and nearly doubled its drug exposure in plasma (AUC_0–∞) compared to compound 18. Unsurprisingly, compounds 18 and 19 had similar affinity for AR in vitro. In summary, the deuteration strategy could obviously improve PK parameters of apalutamide.     

Potent,orally bioavailable,liver-selective stearoyl-CoA desaturase (SCD) inhibitors

Two structurally distinct series of SCD (Δ9 desaturase) inhibitors (1 and 2) have been previously reported by our group. In the present work, we merged the structural features of the two series. This led to the discovery of compound 5b (CVT-12,012) which is highly potent in a human cell-based (HEPG2) SCD assay (IC₅₀ = 6 nM). This compound has 78% oral bioavailability in rats and is preferentially distributed into liver (76 times vs plasma) with relatively low brain penetration. In a five-day study (sucrose fed rats) compound 5b significantly reduced SCD activity in a dose-dependent manner as determined by GC analysis of fatty acid composition in plasma and liver, and significantly reduced liver triglycerides versus the control group (～50%).     

Identification and hit-to-lead optimization of a novel class of CB1 antagonists

The discovery, synthesis and preliminary structure–activity relationships (SARs) of a novel class of CB1 antagonists is described. Initial optimization of benzimidazole-based screening hit 4 led to the identification of ‘inverted’ indole-based lead compound 18c with improved properties versus compound 4 including reduced A log P, improved microsomal stability and improved aqueous solubility. Compound 18c demonstrates in vivo CB1 antagonist efficacy (CB1 agonist induced hypothermia model) and is orally bioavailable in rat.     

Design,synthesis and bio-evaluation of C-1 alkylated tetrahydro-β-carboline derivatives as novel antifungal lead compounds

The field of antifungal agent has become static and development of resistance by the pathogen as well as limited clinical efficacy of marketed drugs demand the constant development of new antifungals. The presence of hydrocarbon chain of specific length linked with various different heterocycles was found to be an important structural feature in various antifungal lead compounds. Based on the prominent antimicrobial activity of β-carboline derivatives, a set of C1 alkylated tetrahydro-β-carboline derivatives were proposed to be active against fungi. To validate and confirm the role of suitable alkyl chains linked to a β-carboline scaffold, few related analogues having C1 aryl substituents were also synthesized in one step via classic Pictet-Spengler reaction. The synthesized library was evaluated for its antifungal activity against C. albicans, C. krusei, C. parapsilosis, C. kefyr, C. glabrata, C. tropicalis and C. neoformans. One of the library members (compound 12c), with n-alkyl chain of eight carbons exhibited potent antifungal activity against C. glabrata and C. kefyr. The lead compound, being selectively toxic also demonstrated prominent synergy enhancing the potency of antifungal drugs up to 10-fold. The time kill kinetic studies confirmed the efficacy of compound 12c, where the results obtained were comparable to that of Amp B. FE-SEM analysis revealed the increased asymmetry, disintegration and roughness of cell surface which could be because of the possible interaction of compound 12c at membrane level or interference in cell wall structure. Apoptosis/necrosis detection assay confirmed the significant apoptotic activity in C. glabrata cells after 12c treatment which was responsible for the rapid killing of C. glabrata cells.     

Synthesis,biological evaluation and molecular modeling of 2-amino-2-phenylethanol derivatives as novel β2-adrenoceptor agonists

A novel series of 2-amino-2-phenylethanol derivatives were developed as β₂-adrenoceptor agonists. Among them, 2-amino-3-fluoro-5-(2-hydroxy-1-(isopropylamino)ethyl)benzonitrile (compound 2f) exhibited the highest activity (EC₅₀ = 0.25 nM) in stimulating β₂-adrenoceptor-mediated cellular cAMP production with a 763.6-fold selectivity over the β₁-adrenoceptor. The (S)-isomer of 2f was subsequently found to be 8.5-fold more active than the (R)-isomer. Molecular docking was performed to determine the putative binding modes of this new class of β₂-adrenoceptor agonists. Taken together, these data show that compound 2f is a promising lead compound worthy of further study for the development of β₂-adrenoceptor agonists.     

Optimization of 2-(1H-imidazo-2-yl)piperazines series of Trypanosoma brucei growth inhibitors as potential treatment for the second stage of HAT

A previous publication from our laboratory reported the identification of a new class of 2-(1H-imidazo-2-yl)piperazines as potent T. brucei growth inhibitors as potential treatment for Human African Trypanosomiasis (HAT). This work describes the structure–activity relationship (SAR) around the hit compound 1, which led to the identification of the optimized compound 18, a single digit nanomolar inhibitor (EC₅₀ 7 nM), not cytotoxic and with optimal in vivo profile that made it a suitable candidate for efficacy studies in a mouse model mimicking the second stage of disease.     

The tyrosinase inhibitory effects of isoxazolone derivatives with a (Z)-β-phenyl-α, β-unsaturated carbonyl scaffold

Thirteen (Z)-4-(substituted benzylidene)-3-phenylisoxazol-5(4H)-ones were designed to confirm the geometric effect of the double bond of the β-phenyl-α, β-unsaturated carbonyl scaffold on tyrosinase inhibitory activity. Compounds 1a–1m, which all possessed the (Z)-β-phenyl-α, β-unsaturated carbonyl scaffold, were synthesized using a tandem reaction consisting of an isoxazolone ring formation and a Knoevenagel condensation, and three starting materials, ethyl benzoylacetate, hydroxylamine and benzaldehydes. Some of the compounds showed inhibitory activity against mushroom tyrosinase as potent as compounds containing the “(E)”-β-phenyl-α, β-unsaturated carbonyl scaffold. Compounds 1c and 1m showed greater inhibitory activity than kojic acid: IC₅₀?=?32.08?±?2.25?μM for 1c; IC₅₀?=?14.62?±?1.38?μM for 1m; and IC₅₀?=?37.86?±?2.21?μM for kojic acid. A kinetic study indicated that 1m inhibited tyrosinase in a competitive manner and that it probably binds to the enzyme’s active site. In silico docking simulation supported binding of 1m (?7.6?kcal/mol) to the active site of tyrosinase with stronger affinity than kojic acid (?5.7?kcal/mol). Similar results were obtained using cell-based assays, and in B16F10 cells, compound 1m dose-dependently inhibited tyrosinase activity and melanogenesis. These results indicate the anti-melanogenic effect of compound 1m is due to the inhibition of tyrosinase and (Z)-isomer of the β-phenyl-α, β-unsaturated carbonyl scaffold can, like its congener the (E)-isomer, act as an excellent scaffold for tyrosinase inhibition.     

Orally bioavailable,liver-selective stearoyl-CoA desaturase (SCD) inhibitors

We discovered a structurally novel SCD (Δ9 desaturase) inhibitor 4a (CVT-11,563) that has 119 nM potency in a human cell-based (HEPG2) SCD assay and selectivity against Δ5 and Δ6 desaturases. This compound has 90% oral bioavailability (rat) and excellent plasma exposure (dAUC 935 ng h/mL). Additionally, 4a shows moderately selective liver distribution (three times vs plasma and adipose tissue) and relatively low brain penetration. In a five-day study (high sucrose diet, rat) compound 4a significantly reduced SCD activity as determined by GC analysis of fatty acid composition in plasma and liver. We describe the discovery of 4a from HTS hit 1 followed by scaffold replacement and SAR studies focused on DMPK properties.     

Formosusin A,a novel specific inhibitor of mammalian DNA polymerase β from the fungus Paecilomyces formosus

Variotin (1) and three novel compounds, formosusin A (2), B (3), and C (4), were isolated from the cultures of the fungus Paecilomyces formosus, and their structures were determined by spectroscopic analyses. Compound 2 is (6Z,8E,10E)-variotin, a new cis-olefin analog of compound 1. Compound 2 selectively inhibited the activity of mammalian DNA polymerase β (pol β) in vitro, with an IC₅₀ of 35.6 μM. By contrast, compounds 1, 3, and 4 did not influence the activity of pol β. These four compounds showed no effect on the activities of other 10 mammalian pols (i.e., pols α, γ, δ, ε, η, ι, κ, λ, and μ, and terminal deoxynucleotidyl transferase). These compounds also did not inhibit the activities of fish, insect, plant, and prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 2 could be a selective inhibitor of mammalian pol β. The compound 2-induced inhibition of rat pol β activity was competitive and non-competitive with respect to the DNA template–primer substrate and the dNTP substrate, respectively. On the basis of these results, the relationship between the three-dimensional structure and pol β inhibitory mechanism of compound 2 is discussed.     

Synthesis and preclinical characterization of 1-(6′-deoxy-6′-[18F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-6′-[18F]FAZAL) as a positron emission tomography radiotracer to assess tumor hypoxia

Positron emission tomography (PET) using fluorine-18 (¹⁸F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6′-deoxy-6′-[¹⁸F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-[¹⁸F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [¹⁸F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (β-6) in a final radiochemical yield of 12 ± 8% (n = 10, based on [¹⁸F]fluoride starting activity) in a total synthesis time of 60 min with a specific activity at end of synthesis of 218 ± 58 GBq/μmol (n = 10). Both radiolabeling precursor β-6 and unlabeled reference compound β-1 were prepared in multistep syntheses starting from 1,2:5,6-di-O-isopropylidene-α-d-allofuranose. In vitro experiments demonstrated an interaction of β-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as hypoxia specific retention of β-[¹⁸F]1 in tumor cell lines. In biodistribution studies in healthy mice β-[¹⁸F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13 ± 0.22 (n = 4) at 2 h after administration of β-[¹⁸F]1. In ex vivo autoradiography experiments β-[¹⁸F]1 distribution closely matched staining with the hypoxia marker pimonidazole. In conclusion, β-[¹⁸F]1 shows potential as PET hypoxia radiotracer which merits further investigation.     

Design,synthesis, and evaluation of novel inhibitors for wild-type human serine racemase

Most of the endogenous free d-serine (about 90%) in the brain is produced by serine racemase (SR). d-Serine in the brain is involved in neurodegenerative disorders and epileptic states as an endogenous co-agonist of the NMDA-type glutamate receptor. Thus, SR inhibitors are expected to be novel therapeutic candidates for the treatment of these disorders. In this study, we solved the crystal structure of wild-type SR, and tried to identify a new inhibitor of SR by in silico screening using the structural information. As a result, we identified two hit compounds by their in vitro evaluations using wild-type SR.Based on the structure of the more potent hit compound 1, we synthesized 15 derivatives and evaluated their inhibitory activities against wild-type SR. Among them, the compound 9C showed relatively high inhibitory potency for wild-type SR. Compound 9C was a more potent inhibitor than compound 24, which was synthesized by our group based upon the structural information of the mutant-type SR.     

Genome Wide Association Study of Fetal Hemoglobin in Sickle Cell Anemia in Tanzania

Background

Fetal hemoglobin (HbF) is an important modulator of sickle cell disease (SCD). HbF has previously been shown to be affected by variants at three loci on chromosomes 2, 6 and 11, but it is likely that additional loci remain to be discovered.

Methods and Findings

We conducted a genome-wide association study (GWAS) in 1,213 SCA (HbSS/HbSβ⁰) patients in Tanzania. Genotyping was done with Illumina Omni2.5 array and imputation using 1000 Genomes Phase I release data. Association with HbF was analysed using a linear mixed model to control for complex population structure within our study. We successfully replicated known associations for HbF near BCL11A and the HBS1L-MYB intergenic polymorphisms (HMIP), including multiple independent effects near BCL11A, consistent with previous reports. We observed eight additional associations with P<10⁻⁶. These associations could not be replicated in a SCA population in the UK.

Conclusions

This is the largest GWAS study in SCA in Africa. We have confirmed known associations and identified new genetic associations with HbF that require further replication in SCA populations in Africa.     

Molecular Determination of Vascular Endothelial Growth Factor,miRNA-423 Gene Abnormalities by Utilizing ARMS-PCR and Their Association with Fetal Hemoglobin Expression in the Patients with Sickle Cell Disease

Recent studies have indicated that microRNA and VEGF are considered to be genetic modifiers and are associated with elevated levels of fetal haemoglobin HbF, and thus they reduce the clinical impact of sickle haemoglobin (HbS) patients. This cross-sectional study was performed on clinical confirmed subjects of SCD cases. miR-423-rs6505162 C>T and VEGF-2578 C>A genotyping was conducted by ARMS-PCR in SCD and healthy controls. A strong clinical significance was reported while comparing the association of miR-423 C>T genotypes between SCD patients and controls (p = 0.031). The microRNA-423 AA genotype was associated with an increased severity of SCD in codominant model with odd ratio (OR = 2.36, 95% CI, (1.15–4.84), p = 0.018) and similarly a significant association was observed in recessive inheritance model for microRNA-423 AA vs (CC+CA) genotypes (OR = 2.19, 95% CI, (1.32–3.62), p < 0.002). The A allele was associated with SCD severity (OR = 1.57, 95% CI, (1.13–2.19), p < 0.007). The distribution of VEGF-2578 C>A genotypes between SCD patients and healthy controls was significant (p < 0.013). Our results indicated that in the codominant model, the VEGF-2578-CA genotype was strongly associated with increased SCD severity with OR = 2.56, 95% CI, (1.36–4.82), p < 0.003. The higher expression of HbA1 (65.9%), HbA2 (4.40%), was reported in SCD patients carrying miR-423-AA genotype than miR-423 CA genotype in SCD patients carrying miR-423 CA genotype HbA1 (59.98%), HbA2 (3.74%) whereas SCD patients carrying miR-423 CA genotype has higher expression of HbF (0.98%) and HbS (38.1%) than in the patients carrying AA genotype HbF (0.60%), HbS (36.1%). ARMS-PCR has been proven to be rapid, inexpensive and is highly applicable to gene mutation screening in laboratories and clinical practices. This research highlights the significance of elucidating genetic determinants that play roles in the amelioration of the HbF levels that is used as an indicator of severity of clinical complications of the monogenic disease. Further well-designed studies with larger sample sizes are necessary to confirm our findings.     

Observations on the possible application of glycosyl disulphides,sulphenic esters,and sulphones in the synthesis of glycosides

Partial desulphuration of tetra-O-acetyl-β-d-glucopyranosyl phenyl disulphide with a phosphine derivative gave 40% of phenyl 2,3,4,6-tetra-O-acetyl-1-thio-α-d- glucopyranoside and a similar proportion of β-d-glucopyranosyl 1-thio-α-d-glucopyranoside octa-acetate, showing that this procedure is of limited value in α-d-thio-glucoside synthesis. Similar treatment of allyl tetra-O-acetyl-,β-d-glucopyranosyl sulphoxide caused abstraction of oxygen, rather than of sulphur, from the derived allyl glucosylsulphenate. The phenylsulphonyl group was not readily displaced from β-d-glucopyranosyl phenyl sulphone, except intramolecularly, nor could it be displaced from the tetrabenzyl ether. Elimination of benzyl alcohol from this compound afforded a new 1-(phenylsulphonyl)glycal derivative.     

Photo-induced synthesis of Axinastatin 3 analogs,the secondary structures and their in vitro antitumor activities

Cyclic peptides combine several favorable properties such as good binding affinity, target selectivity and low toxicity that make them an attractive modality for drug development. In an effort to identify what conformation could be accounting for the bioactive disparity of natural and synthetic cyclic peptides, some structurally-constrained analogs of cyclopeptide Axinastatin 3 were prepared by photo-induced single electron transfer (SET) reaction. Detailed stereochemistry study was performed by experimental electronic circular dichroism combined with theoretical calculations. Our study suggested that the cyclopeptide 1 with βI-turn presented stronger antitumor activity comparing with those without such secondary structures. Moreover, a rare ‘π helix unit’ (compound 3) was realized because of the constrained cyclic structure, which could be considered an important research object for future study of unique helix secondary structures.     

Novel naphtho[2,1-d]oxazole-4,5-diones as NQO1 substrates with improved aqueous solubility: Design,synthesis, and in vivo antitumor evaluation

A new series of ortho-naphthoquinone analogs of β-lapachone were designed, synthesized and evaluated. The biological results indicated that most of our compounds were efficient substrates for NQO1. The new scaffold with water-soluble side chain resulted in greater solubility under acidic condition compared to β-lapachone. Thus avoiding the use of hydroxylpropyl β-cyclodextrin which would finally cause the rapid drug clearance from the blood and dose-limiting toxicity in the form of hemolytic anemia. The most soluble and promising compound in this series was 2-((4-benzylpiperazin-1-yl)methyl)naphtho[2,1-d]oxazole-4,5-dione (3k), which inhibited cancer cell (NQO1-rich A549 cell line) growth at IC₅₀ values of 4.6 ± 1.0 μmol·L⁻¹. Furthermore, compound 3k had in vivo antitumor activity in an A549 tumor xenografts mouse model comparable to the activity obtained with β-lapachone. The results indicated that these ortho-naphthoquinones could serve as promising leads for further optimization as novel substrates for NQO1.