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Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1 complex-mediated phosphorylation of a protein substrate with IC(50)s in the low micromolar range. On the basis of preliminary structure-activity relationships (SAR), a model was proposed in which these inhibitors occupy the ATP-binding site of the enzyme, forming critical hydrogen bonds to the same residue (Val96) to which the amino group in ATP is presumed to bind. X-ray diffraction studies on a later derivative bound to CDK2 support this binding mode. Iterative cycles of synthesis and screening lead to a novel series of potent, CDK2-selective 6-(arylmethyl)pyrazolopyrimidinones. Placement of a hydrogen-bond donor in the meta-position on the 6-arylmethyl group resulted in approximately 100-fold increases in CDK4 affinity, giving ligands that were equipotent inhibitors of CDK4 and CDK2. These compounds exhibit antiproliferative effects in the NCI HCT116 and other cell lines. The potency of these antiproliferative effects is enhanced in anilide derivatives and translates into tumor growth inhibition in a mouse xenograft model.

[reaction: see text] New methods for the palladium-catalyzed cyanation of aryl and heteroaryl chlorides have been developed, featuring sterically demanding, electron-rich phosphines. Highly challenging electron-rich aryl chlorides, in addition to electron-neutral and electron-deficient substrates, as well as nitrogen- and sulfur-containing heteroaryl chlorides can all undergo efficient cyanation under relatively mild conditions using readily available materials. In terms of substrate scope and temperature, these methods compare very favorably with the state-of-the-art cyanations of aryl chlorides.

Lysophosphatidic acid (LPA) functions through activation of LPA receptors (LPARs). LPA-LPAR signaling has been implicated in development of fibrosis. However, the role of LPA-LPAR signaling in development of diabetic nephropathy (DN) has not been studied. We examined whether BMS002, a novel dual LPAR1 and LPAR3 antagonist, affects development of DN in endothelial nitric oxide synthase-knockout mice. Treatment of these mice with BMS002 from 8 to 20 weeks of age led to a significant reduction in albuminuria, similar to that observed with renin-angiotensin system inhibition (losartan plus enalapril). LPAR inhibition also prevented the decline in GFR observed in vehicle-treated mice, such that GFR at week 20 differed significantly between vehicle and LPAR inhibitor groups (<0.05). LPAR inhibition also reduced histologic glomerular injury; decreased the expression of profibrotic and fibrotic components, including fibronectin, -smooth muscle actin, connective tissue growth factor, collagen I, and TGF-; and reduced renal macrophage infiltration and oxidative stress. Notably, LPAR inhibition slowed podocyte loss (podocytes per glomerulus ±SEM at 8 weeks: 667±40, =4; at 20 weeks: 364±18 with vehicle,=7, and 536±12 with LPAR inhibition, =7;<0.001 versus vehicle). Finally, LPAR inhibition minimized the production of 4-hydroxynonenal (4-HNE), a marker of oxidative stress, in podocytes and increased the phosphorylation of AKT2, an indicator of AKT2 activity, in kidneys. Thus, the LPAR antagonist BMS002 protects against GFR decline and attenuates development of DN through multiple mechanisms. LPAR antagonism might provide complementary beneficial effects to renin-angiotensin system inhibition to slow progression of DN.

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One important factor influencing the affinity of a flexible ligand for a receptor is the internal strain energy required to attain the bound conformation. Calculation of fully equilibrated ensembles of bound and free ligand and receptor conformations are computationally not possible for most systems of biological interest; therefore, the qualitative evaluation of a novel structure as a potential high-affinity ligand for a given receptor can benefit from taking into account both the bound and unbound (usually aqueous) low-energy geometries of the ligand and the difference in their internal energies. Although many techniques for computationally generating and evaluating the conformational preferences of small molecules are available, there are a limited number of studies of complex organics that compare calculated and experimentally observed conformations. To assess our ability to predict a priori favored conformations of cyclic HIV protease (HIV-1 PR) inhibitors, conformational minima for nine 4,7-bis(phenylmethyl)-2H-1,3-diazepin-2-ones I (cyclic ureas) were calculated using a high temperature quenched dynamics (QD) protocol. Single crystal X-ray and aqueous NMR structures of free cyclic ureas were obtained, and the calculated low-energy conformations compared with the experimentally observed structures. In each case the ring conformation observed experimentally is also found in the lowest energy structure of the QD analysis, although significantly different ring conformations are observed at only slightly higher energy. The 4- and 7-benzyl groups retain similar orientations in calculated and experimental structures, but torsion angles of substituents on the urea nitrogens differ in several cases. The data on experimental and calculated cyclic urea conformations and their binding affinities to HIV-1 PR are proposed as a useful dataset for assessing affinity prediction methods.

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