Crystal Structure of the Human Cannabinoid Receptor CB1.

Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

Deacylative transformations of ketones via aromatization-promoted C-C bond activation.

Carbon-hydrogen (C-H) and carbon-carbon (C-C) bonds are the main constituents of organic matter. Recent advances in C-H functionalization technology have vastly expanded our toolbox for organic synthesis1. By contrast, C-C activation methods that enable editing of the molecular skeleton remain limited2-7. Several methods have been proposed for catalytic C-C activation, particularly with ketone substrates, that are typically promoted by using either ring-strain release as a thermodynamic driving force4,6 or directing groups5,7 to control the reaction outcome. Although effective, these strategies require substrates that contain highly strained ketones or a preinstalled directing group, or are limited to more specialist substrate classes5. Here we report a general C-C activation mode driven by aromatization of a pre-aromatic intermediate formed in situ. This reaction is suitable for various ketone substrates, is catalysed by an iridium/phosphine combination and is promoted by a hydrazine reagent and 1,3-dienes. Specifically, the acyl group is removed from the ketone and transformed to a pyrazole, and the resulting alkyl fragment undergoes various transformations. These include the deacetylation of methyl ketones, carbenoid-free formal homologation of aliphatic linear ketones and deconstructive pyrazole synthesis from cyclic ketones. Given that ketones are prevalent in feedstock chemicals, natural products and pharmaceuticals, these transformations could offer strategic bond disconnections in the synthesis of complex bioactive molecules.

Synthesis of Pyrazolo[3,4-b]pyridine Derivatives and Their In-Vitro and In-Silico Antidiabetic Activities.

In the current study, new pyrazolo[3,4-b]pyridine esters, hydrazides, and Schiff bases have been synthesized starting from 3-methyl-1-phenyl-1H-pyrazol-5-amine. The first step involved solvent-free synthesis of pyrazolo[3,4-b]pyridine-6-carboxylate derivatives (2a-d) with 55%-70% yield in the minimum time frame compared with the conventional refluxing method, which was followed by the synthesis of corresponding hydrazides (3a-d) and hydrazones (4a-e). The structures of the synthesized derivatives were confirmed using element analysis, FT-IR, 1H NMR, 13C NMR, and LC-MS techniques. Synthesized hydrazides (3a-d) and hydrazones (4a-e) were also tested for their in-vitro antidiabetic activity and found that all the compounds exhibited significant antidiabetic activity, while 3c (IC50 = 9.6 ± 0.5 µM) among the hydrazides and 4c (IC50 = 13.9 ± 0.7 µM) among the hydrazones were found to be more active in comparison to other synthesized derivatives. These in-vitro results were further validated via docking studies against the α-amylase enzyme using the reference drug acarbose (200.1 ± 10.0 µM). The results were greatly in agreement with their in-vitro studies and these derivatives can be encouraging candidates for further in-vivo studies in mice models.

RuBi-Ruxolitinib: A Photoreleasable Antitumor JAK Inhibitor.

We describe the synthesis and biological testing of ruthenium-bipyridine ruxolitinib (RuBiRuxo), a photoreleasable form of ruxolitinib, a JAK inhibitor used as an antitumoral agent in cutaneous T-cell lymphomas (CTCL). This novel caged compound is synthesized efficiently, is stable in aqueous solution at room temperature, and is photoreleased rapidly by visible light. Irradiation of RuBiRuxo reduces cell proliferation and induces apoptosis in a light- and time-dependent manner in a CTCL cell line. This effect is specific and is mediated by a decreased phosphorylation of STAT proteins. Our results demonstrate the potential of ruthenium-based photocompounds and light-based therapeutic approaches for the potential treatment of cutaneous lymphomas and other pathologies.

Mononuclear η6-arene ruthenium(II) complexes with pyrazolyl-pyridazine ligands: synthesis, CT-DNA binding, reactivity towards glutathione, and cytotoxicity.

Organometallic η6-arene ruthenium(II) complexes with 3-chloro-6-(1H-pyrazol-1-yl)pyridazine (Ru1, Ru2, and Ru5) and 3-chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine (Ru3-4) N,N' heterocyclic and η6-arene (cymene (Ru1-4) or toluene (Ru 5)) have been synthesized. The ruthenium(II) complexes have common "three-legged piano-stool" pseudo-octahedral structures known for half-sandwich complexes. Evolution of their UV-Visible absorption spectra in PBS buffer or DMSO over 24 h confirmed their good solvolysis stability. Titrations of the complexes with the calf thymus DNA (CT-DNA) were monitored using UV-Visible absorption and fluorescence spectroscopies. The complexes interact moderately with CT-DNA and their binding constants are in the order of 104 M-1. Competitive binding of the complexes to a DNA-Hoechst 33,258 depicted competitive displacement of Hoechst from DNA's minor grooves. These complexes bind to glutathione forming GSH-adducts through S coordination by replacement of a halide, with the iodo-analogues having higher binding constants than the chloro-complexes. Cyclic voltammograms of the complexes exhibited one electron-transfer quasi-reversible process. Trends in the molecular docking data of Ru1-5/DNA were similar to those for DNA binding constants. Of the five, only Ru1, Ru3 and Ru5 showed some activity (moderate) against the MCF-7 breast cancer cells with IC50 values in the range of 59.2-39.9 for which Ru5 was the most active. However, the more difficult-to-treat cell line, MDA-MB 231 cell was recalcitrant to the treatment by these complexes.

A chemical platform for the efficient screening of arylazopyrazole-based photoswitchable CENP-E inhibitors using mild cyclization reactions.

A set of arylazopyrazole-based inhibitors targeting the mitotic motor protein CENP-E was discovered through the chemical platform using the quantitative cyclization of 1,3-diketone intermediate with various hydrazines under mild conditions. Through this efficient platform, the structure-activity relationship pertaining to the pyrazole photoswitch in photoswitchable CENP-E inhibitors not only in vitro but also in cells was successfully clarified.

Pyrazolines inhibiting the activity of the early growth response-1 DNA-binding domain.

To identify compounds inhibiting the activity of the Early Growth Response (EGR)-1 DNA-binding domain, thirty-seven pyrazolines were prepared and their EGR-1 DNA-binding activities were measured. Pharmacophores were derived based on quantitative structure-activity relationship calculations. As compound 2, 1-(5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-2-ol, showed the best inhibitory effects against the activity of the EGR-1 DNA-binding domain, the binding mode between compound 2 and EGR-1 was elucidated using in silico docking. The pharmacophores were matched to the binding modes. Electrophoretic mobility shift assays confirmed that compound 2 dose-dependently inhibited TNFα-induced EGR-1-DNA complex formation in HaCaT cells. Reverse transcription-polymerase chain reaction demonstrated that compound 2 effectively reduced the mRNA expression of EGR-1-regulated inflammatory genes, including thymic stromal lymphopoietin (TSLP), interleukin (IL)-1ß, IL-6, and IL-31, in TNFα-stimulated HaCaT cells. Therefore, compound 2 could be developed as an agent that inhibits the activity of the EGR-1 DNA-binding domain.

Design, synthesis and biological evaluation of thieno[3,2-c]pyrazol-urea derivatives as potent glycogen synthase kinase 3ß inhibitors based on the DFG-out conformation.

Glycogen synthase kinase 3ß (GSK-3ß) is a potential therapeutic target for the treatment of a variety of human diseases. Here, we report the design and synthesis of a series of thieno[3,2-c]pyrazol-urea derivatives and evaluation of their GSK-3ß inhibitory activity. Among these analogues, the compound without substitution on terminal phenyl ring (3a) was found to be the most potent GSK-3ß inhibitor with an IC50 of 74.4 nM, while substitution on the terminal phenyl (3b-3p) led to decreased potency, independent of the position, size, or electronic properties of the substituents. Kinase selectivity assay revealed that 3a showed good selectivity over a panel of kinases, but was less selective over CDK1, CDK2 and CDK5. Additionally, the pharmacological properties of the synthesized compounds were investigated computationally by the SwissADME and the results showed that most of the compounds have good ADME profiles.

Synthesis, fungicidal activity and molecular docking of novel pyrazole-carboxamides bearing a branched alkyl ether moiety.

Succinate dehydrogenase inhibitors are essential fungicides used in agriculture. To explore new pyrazole-carboxamides with high fungicidal activity, a series of N-substitutedphenyl-3-di/trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamides bearing a branched alkyl ether moiety were designed and synthesized. The in vitro bioassay indicated that some target compounds displayed appreciable fungicidal activity. For example, compounds 5d and 5e showed high efficacy against S. sclerotiorum with EC50 values of 3.26 and 1.52 µg/mL respectively, and also exhibited excellent efficacy against R. solani with EC50 values of 0.27 and 0.06 µg/mL respectively, which were comparable or superior to penflufen. The further in vivo bioassay on cucumber leaves demonstrated that 5e provided strong protective activity of 94.3 % against S. sclerotiorum at 100 µg/mL, comparable to penflufen (99.1 %). Cytotoxicity assessment against human renal cell lines (239A cell) revealed that 5e had low cytotoxicity within the median effective concentrations. Docking study of 5e with succinate dehydrogenase illustrated that R-5e formed one hydrogen bond and two π-π stacking interactions with amino acid residues of target enzyme, while S-5e formed only one π-π stacking interaction with amino acid residue. This study provides a valuable reference for the design of new succinate dehydrogenase inhibitor.

Discovery of a potent orally available pyrazolopyridone derivative as a novel selective bromodomain and extra-terminal domain (BET)-first bromodomain (BD1) inhibitor.

Clinical studies have shown that inhibitors of bromodomain and extra-terminal domain (BET) proteins, particularly BRD4, have antitumor activity and efficacy. The BET protein has two domains, BD1 and BD2, and we previously focused on BD1 and reported orally bioavailable BD1-selective inhibitors. In this study, we obtained a BD1 inhibitor, a more potent and highly selective pyrazolopyridone derivative 13a, and confirmed its in vivo efficacy.

Design and discovery of carboxamide-based pyrazole conjugates with multifaceted potential against Triple-Negative Breast cancer MDA-MB-231 cells.

We report the design, synthesis, and validation of carboxamide-based pyrazole and isoxazole conjugates with a multifaceted activity against Breast Cancer Cell Line MDA-MB-231. The study established that amongst the series, N-(3,5-bis(trifluoromethyl)benzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (5g) exhibits the highest potency in inhibiting Breast Cancer Cell Line MDA-MB-231 with an IC50 value of 15.08 ± 0.04 µM. The MDA-MB-231 cells, upon treatment with compound 5g, exhibited characteristic apoptotic specific activities such as nuclear fragmentation, phosphatidylserine translocation to the outer plasma membrane, release of lactate dehydrogenase (LDH), and upregulation of caspase 3 and caspase 9 activities. Also, the modulation of pro and antiapoptotic proteins in 5g treated MDA-MB-231 cells was revealed by membrane array analysis. More importantly, the combination of paclitaxel and compound 5g has exhibited improved activity by several folds via their synergistic effects.

In-cell chemical construction of a photoswitchable CENP-E using a photochromic covalent inhibitor.

An arylazopyrazole-based covalent inhibitor targeting the mitotic motor protein of centromere-associated protein E (CENP-E) was developed. Using this photoswitchable inhibitor, a photoswitchable CENP-E was chemically constructed in cells, which enabled to local control of mitotic cell division with light illumination.

Structure optimization of Cmpd-15 as negative allosteric modulators for the ß2-adrenergic receptor.

19 derivatives of 1-benzyl-3-arylpyrazole-5-carboxamides (H1-H19) and 5 derivatives of 1-benzyl-5-arylpyrazole-3-carboxamides (J1-J5) have been designed and synthesized as potential negative allosteric modulators (NAMs) for the ß2-adrenergic receptor (ß2AR). The new pyrazole derivatives were screened on the classic G-protein dependent signaling pathway at ß2AR. The majority of 1-benzyl-3-aryl-pyrazole-5-carboxamide derivatives show more potent allosteric antagonistic activity against ß2AR than Cmpd-15, the first reported ß2AR NAM. However, the 1-benzyl-5-arylpyrazole-3-carboxamide derivatives exhibit very poor or even no allosteric antagonistic activity for ß2AR. Furthermore, the active pyrazole derivatives have relative better drug-like profiles than Cmpd-15. Taken together, we discovered a series of derivatives of 1-benzyl-3-arylpyrazole-5-carboxamides as a novel scaffold of ß2AR NAM.

Discovery, synthesis and SAR of 2-acyl-1-biarylmethyl pyrazolidines, dual orexin receptor antagonists designed as fast and short-acting sleeping drugs.

Dual orexin receptor antagonists (DORAs) are approved for the treatment of sleep onset and/or sleep maintenance insomnia. In the present disclosure, we report the discovery of a new class of DORAs designed to treat sleep disorders requiring a fast onset and a short duration of action (<4 h). We used early human pharmacokinetic-pharmacodynamic (PK-PD) predictions and in vivo experiments to identify DORAs eliciting this specific hypnotic profile. A high-throughput screening campaign revealed hits based on a rarely precedented tricyclic pyrazolidine scaffold. After unsuccessful structure-activity-relationship (SAR) studies on this hit series, a scaffold hopping exercise, aimed at reducing the molecular complexity of the tricyclic scaffold, resulted in the discovery of the 2-acyl-1-biarylmethylpyrazolidine series. SAR studies on this achiral series gave rise to the lead compound DORA 42. In vitro and in vivo parameters of DORA 42, and its PK-PD simulation for human use are detailed.

Kinetic studies, molecular docking, and antioxidant activity of novel 1,3-diphenyl pyrazole-thiosemicarbazone with anti-tyrosinase and anti-melanogenesis properties.

This study reports the Design Hypothesis of a novel series of 1,3-diphenyl pyrazole-thiosemicarbazone as novel tyrosinase inhibitors (TYRI). The designed compounds were prepared and their TYRI activity and mechanisms were studied. The results showed that the selected compounds exhibited potent tyrosinase inhibitory activities greater than that of kojic acid (KA). Lead candidates, denoted as 6g and 6n, with a para-hydroxyphenyl group attached to the 3-position of the pyrazole ring demonstrated IC50 values of 2.09 and 3.18 µM, respectively. The potency of these compounds was approximately 5-8 times higher than that of KA. The in vitro melanin content of 6g or 6n-treated melanoma cells resulted in significant efficacy in melanin reduction. The DPPH assay result revealed that the tyrosinase inhibition mechanism for these derivatives was independent of a redox effect and corresponded to the interaction with tyrosinase. According to the Lineweaver-Burk plot, the most potent compounds, 6g and 6n, exhibit a mixed type of inhibition, primarily noncompetitive inhibition. In silico molecular docking studies were employed to determine the binding mode and explore the Design Hypothesis in detail. The results suggested that these compounds could be considered promising leads for the further development of novel inhibitors to treat disorders related to tyrosinase.

Design, synthesis, antineoplastic activity of new pyrazolo[3,4-d]pyrimidine derivatives as dual CDK2/GSK3ß kinase inhibitors; molecular docking study, and ADME prediction.

In the current study, novel pyrazolo[3,4-d]pyrimidine derivatives 5a-h were designed and synthesized as targeted anti-cancer agents through dual CDK2/GSK-3ß inhibition. The designed compounds demonstrated moderate to potent activity on the evaluated cancer cell lines (MCF-7 and T-47D). Compounds 5c and 5 g showed the most promising cytotoxic activity against the tested cell lines surpassing that of the used reference standard; staurosporine. On the other hand, both compounds showed good safety and tolerability on normal fibroblast cell line (MCR5). The final compounds 5c and 5 g showed a promising dual CDK2/GSK-3ß inhibitory activity with IC50 of 0.244 and 0.128 µM, respectively, against CDK2, and IC50 of 0.317 and 0.160 µM, respectively, against GSK-3ß. Investigating the effect of compounds 5c and 5 g on CDK2 and GSK-3ß downstream cascades showed that they reduced the relative cellular content of phosphorylated RB1 and ß-catenin compared to that in the untreated MCF-7 cells. Moreover, compounds 5c and 5 g showed a reasonable selective inhibition against the target kinases CDK2/GSK-3ß in comparison to a set of seven off-target kinases. Furthermore, the most potent compound 5 g caused cell cycle arrest at the S phase in MCF-7 cells preventing the cells' progression to G2/M phase inducing cell apoptosis. Molecular docking studies showed that the final pyrazolo[3,4-d]pyrimidine derivatives have analogous binding modes in the target kinases interacting with the hinge region key amino acids. Molecular dynamics simulations confirmed the predicted binding mode by molecular docking. Moreover, in silico predictions indicated their favorable physicochemical and pharmacokinetic properties in addition to their promising cytotoxic activity.

Synthesis, design, and antiproliferative evaluation of 6-(N-Substituted-methyl)pyrazolo[3,4-d]pyrimidines as the potent anti-leukemia agents.

Pyrazolopyrimidine derivatives, including pyrazolopyrimidines, 6-aminopyrazolopyrimidines, 6-[(formyloxy)methyl]pyrazolopyrimidines, 6-(hydroxymethyl)pyrazolopyrimidine, and 6-(aminomethyl)pyrazolopyrimidines have been successfully prepared and tested against NCI-H226, NPC-TW01, and Jurkat cancer cell lines. Among the tested pyrazolopyrimidine compounds, we found 6-aminopyrazolopyrimidines and 6-(aminomethyl)pyrazolopyrimidines with essential o-ClPh or p-ClPh substituted moieties on N-1 pyrazole ring exhibited the best IC50 inhibition activity for Jurkat cells. Furthermore, optimization of the SAR study on the C-6 position of pyrazolopyrimidine ring demonstrated that 6-(N-substituted-methyl)pyrazolopyrimidines 17b, 17d, and 19d possessed the significant IC50 inhibitory activity for the different leukemia cell lines, especially for Jurkat, K-562, and HL-60. On the other hand, further SAR inhibition and docking model studies revealed that compound 19d, which has a 3-(1H-imidazol-1-yl)propan-1-amino side-chain on the C-6 position, was able to form four hydrogen bonds with residues Ala226, Leu152, and Glu194 and specifically extended into the P1 pocket subsite with Aurora A, resulting in improved inhibitory activity almost similar to SNS-314. To explore the anti-cancer mechanism, compound 19d was measured by Western blot analysis in Jurkat T-cells, however, it showed non-responsibility to Aurora B. For the further structural modifications on the lateral chain of compound 19d, compounds 24 with longer lateral chain were designed and synthesized for testing leukemia cell lines. However, compounds 24 was significantly decrease inhibition potency against leukemia cell lines. Based on the in-vitro results, compounds 17b and 19d could be considered to be the best potential lead drug in our study for the development of new and effective therapies for leukemia treatment. On the other hand, the DHFR inhibition results indicated compound 19d possessed good inhibitory activity and better than the reported naphthalene derivative. Through further comparisons of the model superposition of three-dimensional (3D) conformations in DHFR, compound 19d presented a similar structural alignment to Methotrexate and the reported naphthalene derivative and led to similar drug-like functional relationships. As a results, compound 19d would be a potential DHFR inhibitor for anti-leukemia drug candidate.

Molecular docking approach for the design and synthesis of new pyrazolopyrimidine analogs of roscovitine as potential CDK2 inhibitors endowed with pronounced anticancer activity.

Cyclin-dependent kinase 2 (CDK2) is a vital protein for controlling cell cycle progression that is critically associated with various malignancies and its inhibition could offer a convenient therapeutic approach in designing anticancer remedies. Consequently, this study aimed to design and synthesize new CDK2 inhibitors featuring roscovitine as a template model. The purine ring of roscovitine was bioisosterically replaced with the pyrazolo[3,4-d]pyrimidine scaffold, in addition to some modifications in the side chains. A preliminary molecular docking study for the target chemotypes in the CDK2 binding domain revealed their ability to accomplish similar binding patterns and interactions to that of the lead compound roscovitine. Afterwards, synthesis of the new derivatives was accomplished. Then, the initial anticancer screening at a single dose by the NCI revealed that compounds 7a, 9c, 11c, 17a and 17b achieved the highest GI% values reaching up to 150 % indicating their remarkable activity. These derivatives were subsequently selected to undertake five-dose testing, where compounds 7a, 9c, 11c and 17a unveiled the most pronounced activity against almost the full panel with GI50 ranges; 1.41-28.2, 0.116-2.39, 0.578-60.6 and 1.75-42.4 µM, respectively and full panel GI50 (MG-MID); 8.24, 0.6, 2.46 and 6.84 µM, respectively. CDK2 inhibition assay presented compounds 7a and 9c as the most potent inhibitors with IC50 values of 0.262 and 0.281 µM, respectively which are nearly 2.4 folds higher than the reference ligand roscovitine (IC50 = 0.641 µM). Besides, flow cytometric analysis on the most susceptible and safe cell lines depicted that 7a caused cell cycle arrest at G1/S phase in renal cancer cell line (RXF393) while 9c led to cell growth arrest at S phase in breast cancer cell line (T-47D) along with pronounced apoptotic induction in the mentioned cell lines. These findings afforded new anticancer pyrazolo[3,4-d]pyrimidine, roscovitine analogs, acting via CDK2 inhibition.

Discovery of new 2-(3-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)thiazole derivatives with potential analgesic and anti-inflammatory activities: In vitro, in vivo and in silico investigations.

Joining the global demand for the discovery of potent NSAIDs with minimized ulcerogenic effect, new pyrazole clubbed thiazole derivatives 5a-o were designed and synthesized. The new derivatives were initially evaluated for their analgesic activity. Eight compounds 5a, 5c, 5d, 5e, 5f, 5h, 5m, and 5o showed higher activity than Indomethacin (potency = 105-130 % vs. 100 %). Subsequently, they were picked for further evaluation of their anti-inflammatory activity, ulcerogenic liability as well as toxicological studies. Derivatives 5h and 5m showed a potential % edema inhibition after 3 h (79.39 % and 72.12 %, respectively), with a promising safety profile and low ulcer indices (3.80 and 3.20, respectively). The two compounds 5h and 5m were subjected to in vitro COX-1 and COX-2 inhibition assay. The candidate 5h showed nearly equipotent COX-1 inhibition (IC50 = 38.76 nM) compared to the non-selective reference drug Indomethacin (IC50 = 35.72 nM). Compound 5m expressed significant inhibitory activities and a higher COX-2 selectivity index (IC50 = 87.74 nM, SI = 2.05) in comparison with Indomethacin (SI = 0.52), with less selectivity than Celecoxib (SI = 8.31). Simulation docking studies were carried out to gain insights into the binding interaction of compounds 5h and 5m in the vicinity of COX-1 and COX-2 enzymes that illustrated the importance of pyrazole clubbed thiazole core in hydrogen bonding interactions. The thiazole motif of compounds 5h and 5m exhibited a well orientation toward COX-1 Arg120 key residue by hydrogen bonding interactions. Compound 5h revealed an additional arene-cation interaction with Arg120 that could rationalize its superior COX-1 inhibitory activity. Compounds 5h and 5m overlaid the co-crystallized ligand Celecoxib I differently in the active site of COX-2. Compound 5m showed an enhanced accommodation with binding energy of - 6.13 vs. - 1.70 kcal/mol of compounds 5h. The naphthalene ring of compound 5m adopted the Celecoxib I benzene sulfonamide region that is stabilized by hydrogen-arene interactions with the hydrophobic sidechains of the key residues Ser339 and Phe504. Further, the core structure of compound 5m, pyrazole clubbed thiazole, revealed deeper hydrophobic interactions with Ala513, Leu517 and Val509 residues. Finally, a sensitive and accurate UPLC-MS/MS method was developed for the simultaneous estimation of some selected promising pyrazole derivatives in rat plasma. Accordingly, compounds 5h and 5m were suggested to be promising potent analgesic and anti-inflammatory agents with improved safety profiles and a novel COX isozyme modulation activity.

Discovery of novel pyrazolo[1,5-a]pyrimidine derivatives as selective ROCK2 inhibitors with anti-breast cancer migration and invasion activities.

Rho-associated coiled-coil kinase (ROCK) is involved in multiple cellular activities regulating the actin cytoskeleton, such as cell morphology, adhesion, and migration. The inhibition of ROCK is a feasible strategy to suppress breast cancer metastasis. Herein, based on Belumosudil, a series of pyrazolo[1,5-a]pyrimidine derivatives as selective ROCK2 inhibitors were designed and synthesized. Through systematic investigation of SARs, the piperazine analog 7u was identified with optimum ROCK2 inhibitory activity (IC50 = 36.8 nM) and excellent selectivity over the isoform protein ROCK1 (>250-fold). Intriguingly, upon treatment with 7u, the arrangement of the MDA-MB-231 cytoskeleton was affected accompanied by the alteration of morphology. Furthermore, cell scratch and transwell assays indicated that 7u inhibited MDA-MB-231 cell migration and invasion in a dose-dependent manner. Ultimately, the binding model of 7u with ROCK2 well accounted for the superior activities of 7u as a promising ROCK2 inhibitor with the potential application in breast cancer metastasis treatment.