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Utilizing polymer-bound anthranilic acid derivatives, we were able to obtain 3,4-dihydro-1H-2,1-benzothiazine-4-one 2,2-dioxide derivatives through N-methanesulfonylation by use of sulfonyl chloride, sulfonic acid, or sodium sulfonate, N-alkylation under Mitsunobu condition, and the cyclative cleavage in 8–52% five-step overall isolated yields and 91–99% purities from Wang resin. The reactions on solid phase were monitored by on-bead ATR-FTIR spectroscopic method and checked by help of solution-phase model experiments.

Utilizing polymer-bound anthranilic acid derivatives 1, we were able to obtain the 1,4-benzodiazepine-2,5-dione derivatives 3 (R 3 = H, R 4 = H, MeO, Cl) through an unprecedented reaction sequence, reductive alkylation-N-protected amino acid coupling-deprotective cyclization, in 28-71% five-step overall isolated yields and 95-99% purities from Wang resin 4. Applying the novel protocol to the resin 2, the 7-benzamido-1,4-benzodiazepine-2,5-dione derivatives 3 (R 1 = Bn, R 4 = 7-BzNH) could be obtained in 19-42% seven- or eight-step overall isolated yields and 92-98% purities from AMEBA resin 7.

Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain.

Proopiomelanocortin (POMC)-expressing neurons in the hypothalamus integrate a variety of central and peripheral metabolic inputs, and regulate energy homeostasis by controlling energy expenditure and food intake. To accomplish this, a precise balance of production and degradation of α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide and product of the POMC gene, in the hypothalamus, is crucial. Prolyl carboxypeptidase (PRCP) is a key enzyme that degrades α-MSH to an inactive form unable to inhibit food intake. Because it represents a new therapeutic target for the treatment of metabolic disorders, such as obesity and diabetes, efforts have been made to generate potent, brain-penetrant PRCP inhibitors. Here, we discuss the role of PRCP on energy metabolism and the development of PRCP inhibitors.

Hypothalamic α-melanocyte-stimulating hormone (α-MSH) plays a central role in regulating energy uptake and expenditure. Prolyl carboxypeptidase (PRCP), a protease expressed in the hypothalamus, is responsible for the degradation of α-MSH. PRCP null animals (PRCP(gt/gt) mice) display elevated α-MSH in the hypothalamus, lower body weight, and are protected from diet induced obesity. Here, we report that PRCP(gt/gt) mice have a significant decrease in fat mass, although an increase in lean mass was also observed. In agreement with low fat accumulation, reduced leptin levels were found. Consistent with the effect of α-MSH on energy metabolism, PRCP(gt/gt) mice had increased energy expenditure with elevated circulating thyroid hormone levels and brown adipose tissue uncoupling protein 1 mRNA levels compared with control mice when exposed to regular diet. TRH mRNA levels in the PVN were significantly higher in fed PRCP(gt/gt) animals compared with fed wild-type controls. Fasting significantly decreased TRH mRNA levels in both PRCP(gt/gt) and wild-type (WT) mice. However, TRH mRNA levels in fasted PRCP(gt/gt) animals were significantly higher than those of fasted WT mice. Refeeding analysis after fasting showed a reduced food intake in PRCP(gt/gt) compared with WT mice. Finally, TRH mRNA levels in T(3)-treated hypothyroid PRCP(gt/gt) mice showed a non significant reduction compared with those of hypothyroid PRCP(gt/gt) mice, supporting the impairment of the hypothalamo-pituitary-thyroid axis in PRCP(gt/gt) mice. All together, these data confirm that PRCP plays a role in the regulation of energy metabolism.

Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prep(gt/gt)) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prep(gt/gt) and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prep(gt/gt) and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prep(gt/gt) mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus-PREP reversed the glucose-intolerant phenotype of the Prep(gt/gt) mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function.

Nat. Med. 17, 1121–1127 (2011); published online 28 August 2011; corrected after print 16 September 2011 In the version of this article initially published, the top electrophysiological trace of Figure 4a was inadvertently repeated as the bottom electrophysiological trace of Figure 4b. The scientific conclusions of the paper were not affected by the error.