Fig 1.
Chemical structures of 1, 3-dibenzylurea, benzyl glucosinolate and isothiocyante.
Fig 2.
NMR spectroscopic data (1H 800 MHz, 13C 201 MHz) for compound 2 (DMSO-d6) and compound 3 (CDCl3).
Table 1.
Concentration of 1, 3-dibenzylurea (compound 1) in plant samples.
Fig 3.
Key HMBC and 1H-1H COSY correlations of compound 2 and 3.
Fig 4.
sEH inhibition potency of ureas found in Brassicales and related compounds.
Fig 5.
Topical treatment of compound 3 effectively reduces carrageenan-induced inflammatory pain in rat.
(A) Carrageenan (CAR) induces a stable hyperalgesic response (enhanced pain perception) throughout the duration of the experiment. Treatment with compound 3 (○, 6 mg/paw) or synthetic sEH inhibitor (TCC) (▼, 6 mg/paw) significantly reduced pain levels (Kruskal-Wallis One Way ANOVA on Ranks, p ≤ 0.001, Tukey’s post hoc test (compound 3 vs vehicle, TCC vs vehicle, compound 3 vs TCC, p < 0.05). Mean ± SE (n = 6) of mechanical withdrawal threshold (% of naïve baseline) are shown. (B) Plasma concentration of compound 3 in the pain assay after dermal treatment of compound 3 (6 mg/paw). Concentration of demethoxy metabolite compounds 6 & 7 were below 200 nM. Blood samples were processed by the method described for pharmacokinetic analysis. Mean ± SE (n = 6) are shown.
Fig 6.
Compound 3 is orally bioavailable, and rapidly metabolized into demethoxy-metabolite compound 6.
Male Swiss Webster mice were orally treated (by gavage) with 10 mg/kg of compound 3 and 1-(adamantan-1-yl)-3-(5-(2-(2-ethoxyethoxy) ethoxy) pentyl) urea (AEPU), and 0.3 mg/kg of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) formulated in PEG300 by cassette dosing (all three compounds given in one cassette). Compound 3, a demethoxy-form metabolite of compound 3 (○, compound 6), AEPU and TPPU were measured by LC-MS/MS. Bis-demethoxy compound (compound 7) was not detected (LOQ < 9.8 nM in blood). Mean ± SE (n = 4) of blood concentrations are shown. Recently developed synthetic sEH inhibitor TPPU is much more bioavailable than many other compounds tested and is more metabolically stable. The data show compound 3 to be of sufficient oral availability and stability to be of possible pharmacological interest.
Fig 7.
Proposed biosynthetic pathway of 1, 3-dibenzylurea derivatives.
Table 2.
Effect of incubation conditions and trapping experiment on concentration of compounds 1 and 4 from papaya seed.
Fig 8.
Screening results of plant library in the order Brassicales against human sEH inhibitory potency.
IC50 was measured using cyano (2-methoxynaphthalen-6-yl) methyl trans-(3-phenyl-oxyran-2-yl) methyl carbonate (CMNPC) as a substrate. The fluorescent-based assay as performed here has a standard error between 10% and 20%, suggesting that differences of two-fold or greater are significant. Inhibitory potency was expressed as -log (IC50 (μg plant extract/mL)/100). Dotted line represents IC50 = 12.6 μg/mL. Extracts with IC50 above this value (below the dotted line on the graph) are considered not active. IC50 values and plant names are given in S3 Table.
Table 3.
Summary of the screening of Brassicales library for the occurrence of urea derivatives.
Fig 9.
sEH inhibitory potency of HPLC fractions of water cress extract.
Crude extract of water cress sprout (approximately mg) was injected into a reverse phase HPLC column (Waters SunFire Prep C18, 5 μm, 10x100 mm) and eluted with 10% acetonitrile in water with a flow rate of 2 mL/min for 10 min, followed by a linear gradient elution of acetonitrile 10% to 100% at a flow rate of 2 mL/min for 25 min, and eluted with 100% acetonitrile for 15 min at a flow rate of 2 mL/min. The relative intensity of the UV absorption at the wavelength of 210 and 280 nm are shown (top and middle). The retention time of the synthetic urea (compound 8) is indicated by an arrow in the figure. Fractions were collected for every 4 mL of eluent. After the solvent was evaporated the residue was reconstituted in 50 μL DMSO. The inhibition percentage by each fractions (100 times dilution of reconstituted solution) was measured using the CMNPC assay with recombinant human sEH (bottom). The black circles (●) represent the inhibition percentage by each of the fractions. The crude extract (2 mg extract/mL) showed complete inhibition of sEH activity.
Table 4.
Concentration of compound 8 in various parts of water cress.