Synthesis and Biological Activity Evaluation of Novel α‑Amino Phosphonate Derivatives Containing a Pyrimidinyl Moiety as Potential Herbicidal Agents

To find novel high-activity and low-toxicity herbicide lead compounds with novel herbicidal mode of action, series of novel α-amino phosphonate derivatives containing a pyrimidinyl moiety, <b>I</b>, <b>II</b>, <b>III</b>, and <b>IV</b>, were designed and synthesized by Lewis acid (magnesium perchlorate) catalyzed Mannich-type reaction of aldehydes, amines, and phosphites. Their structures were clearly identified by spectroscopy data (IR, ¹H NMR, ³¹P NMR, EI-MS) and elemental analyses. The bioassay [in vitro, in vivo (GH1 and GH2)] showed that most compounds <b>I</b> exhibited good herbicidal activities; for example, the activities of compounds <b>Ib</b>, <b>Ic</b>, <b>Ig</b>, <b>Ii</b>, <b>Ik</b>, and <b>Im</b> were as good as the positive control herbicides (acetochlor, atrazine, mesotrione, and glyphosate). However, their structural isomers <b>II</b> and <b>III</b> and analogues <b>IV</b> did not display any herbicidal activities in vivo, although some of them possessed selective inhibitory activity against Arabidopsis thaliana in vitro. Interestingly, it was found that compounds <b>IVs</b>, <b>IVt</b>, and <b>IVl</b> showed selective insecticidal activities against Aphis species or Plutella xylostella, respectively. Their preliminary herbicidal mode of action and structure–activity relationships were also studied

Efficacy of AMG 837 in Zucker fatty (<i>fa/fa</i>) rats following a single dose.

<p>8-week old Zucker fatty rats were administered a single bolus of AMG 837 (at 0.3, 1 and 3 mg/kg, n = 6/group) by oral gavage 30-minutes prior to an intraperitoneal glucose challenge at t = 0 minutes. (A) Blood glucose during the IPGTT (black circle = vehicle, blue triangle = 0.3 mg/kg AMG 837, green diamond = 1 mg/kg AMG 837 and purple square = 3 mg/kg AMG 837) (B) Glucose AUC (from −30 to 120 minutes) during the IPGTT. (C) Plasma insulin levels during the IPGTT (black circle = vehicle, blue triangle = 0.3 mg/kg AMG 837, green diamond = 1 mg/kg AMG 837 and purple square = 3 mg/kg AMG 837). Statistical significance compared to vehicle treated animals is denoted by * (p<0.5), ** (p<0.01), *** (p<0.001) and **** (p<0.001) as determined by one-way or two-way ANOVA and colors match the corresponding groups in the figure legend.</p

AMG 837 Potentiates Insulin Secretion from Islets.

<p>Islets were isolated from mice and the activity of AMG 837 on insulin secretion was determined. (A) The dose response relationship of AMG 837 and insulin secretion on mouse islets at 16.7 mM glucose was evaluated. (B) In order to determine whether the activity of AMG 837 was GPR40/FFA1 dependent, islets were isolated from GPR40 null mice (<i>gpr40^−/−</i>). AMG 837 potentiated glucose stimulated insulin secretion from wild type islets (black bar), but not <i>gpr40^−/−</i> islets (blue bar). (C) Glucose dependence of AMG 837 on glucose stimulated insulin secretion was determined by incubating islets in buffer containing either 0.1% DMSO (black bar) or 1 µM AMG 837 in 0.1% DMSO (blue bar) in the presence of increasing concentrations of glucose. Statistical significance is denoted by * (p<0.5), ** (p<0.01) and *** (p<0.001) as determined by one-way or two-way ANOVA.</p

Efficacy of AMG 837 in Zucker fatty (<i>fa/fa</i>) rats following daily dosing for 21-days.

<p>8-week old Zucker fatty rats were administered a single bolus of AMG 837 (at 0.03, 0.1 and 0.3 mg/kg, n = 6/group) by oral gavage 30-minutes prior to an intraperitoneal glucose challenge at t = 0 minutes. (A) Blood glucose during the IPGTT (black circle = vehicle, blue triangle = 0.03 mg/kg AMG 837, green diamond = 0.1 mg/kg AMG 837 and purple square = 0.3 mg/kg AMG 837). (B) Glucose AUC (from −30 to 120 minutes) during the IPGTT. (E) Plasma insulin levels during the IPGTT (black bar = vehicle, blue bar = 0.03 mg/kg AMG 837, green bar = 0.1 mg/kg AMG 837 and purple bar = 0.3 mg/kg AMG 837). Once daily dosing was continued for 21-days. On day 21, an IPGTT was performed in an identical manner to that on day 1. (C) Blood glucose (D) glucose AUC (from −30 to 120 minutes) and (F) plasma insulin levels were measured from the day 21 IPGTT. Figure legends are identical to those of the day 1 figures. (G) Body weights of the animals were followed through the course of the 21-day study; no difference in BW was observed between the groups. (H) Total plasma concentration of AMG 837 30-minutes following the final dose on day 21. Statistical significance compared to vehicle treated animals is denoted by * (p<0.5), ** (p<0.01) and *** (p<0.001) as determined by one-way or two-way ANOVA.</p

Improvement in glucose tolerance and potentiation of insulin secretion in Sprague-Dawley rats treated with AMG 837.

<p>8-week old Sprague-Dawley rats were treated with a single bolus of AMG 837 (at 0.03, 0.1 and 0.3 mg/kg, n = 6/group) by oral gavage 30-minutes prior to an intraperitoneal glucose challenge at t = 0 minutes. (A) Blood glucose measurements were taken during prior to and following glucose challenge. Black circle = vehicle, blue triangle = 0.03 mg/kg AMG 837, green diamond = 0.1 mg/kg AMG 837 and purple square = 0.3 mg/kg AMG 837 (B) The glucose AUC (from −30 to 120 minutes) during the course of the experiments were calculated. (C) Plasma insulin levels were measured using ELISA. Black circle = vehicle, blue triangle = 0.03 mg/kg AMG 837, green diamond = 0.1 mg/kg AMG 837 and purple square = 0.3 mg/kg AMG 837 (D–D) Two successive glucose challenges were conducted in Sprague-Dawley rats following a single oral dose of vehicle (n = 4, black circle) or AMG 837 at 0.3 mg/kg (n = 4, purple diamond). AMG 837 was dosed at −30 minutes, and glucose was administered by <i>ip</i> injection at 0 and 180 minutes. Blood glucose (D), calculated glucose AUC (from 0–60 minutes following glucose challenge (E, black bars = vehicle, purple bars = 0.3 mg/kg AMG 837) and plasma insulin (F) were determined. Statistical significance is denoted by * (p<0.5), ** (p<0.01) and *** (p<0.001) as determined by one-way or two-way ANOVA.</p

<i>In vitro</i> characterization of AMG 837.

<p>(A) The chemical structure of AMG 837 is shown. (B–D) The activity of AMG 837 in various GPCR assays was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027270#s4" target="_blank">Materials and Methods</a>. Dose response relationships of AMG 837 in GTPγS binding (B), inositol phosphate accumulation (C) and aequorin Ca²⁺ flux assays (D) in cell lines overexpressing GPR40/FFA1 were determined. (D–G) In order to compare the activity of AMG 837 to fatty acids, plasmid titration experiments where either 5000 ng (D), 500 ng (E), 50 ng (F) or 5 ng (G) of GPR40 expression plasmid was co-transfected with aequorin expression plasmids into CHO cells. Activity of AMG 837 (blue diamond) was compared to the naturally occurring GPR40/FFA1 ligand docosahexaenoic acid (DHA, green square) in aequorin Ca²⁺ flux. (H) The activity of AMG 837 in the aequorin Ca²⁺ flux assays in the presence of 0.01% (v/v) purified human serum albumin (HSA, blue diamond), 0.625% (w/v) HSA (green square) or human serum (100% v/v, black circle) was determined.</p

Aequorin Ca²⁺ Flux Activity (EC₅₀, nM) of AMG 837 on Various Receptors.

<p>CHO cells were co-transfected with expression plasmids of a given receptor along with the Ca²⁺ sensitive bioluminescent reporter aequorin, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027270#s4" target="_blank">Materials and Methods</a>. Response to AMG 837 was measured with a luminometer and the EC₅₀ (nM) ± SEM was determined.</p