Changes of T Lymphocyte Subsets in Peripheral Blood of Patients with Intermediate and Advanced Cervical Cancer before and after Nimotuzumab Combined with Chemoradiotherapy.

INTRODUCTION: Cervical cancer (CC) is the main malignant tumor of gynecology with high mortality. This study aimed to explore the changes of T lymphocyte subsets in the peripheral blood of patients with intermediate and advanced CC (IACC) treated with nimotuzumab (Nimo) combined with chemoradiotherapy (CRT). METHODS: Peripheral blood was extracted before and after treatment, and patients were randomly divided into the CRT group (N = 68) or the Nimo + CRT group (N = 68). The levels of tumor markers squamous cell carcinoma antigen (SCCA), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA), tumor indexes leptin and insulin-like growth factor-2 (IGF2), and key secretory factors (interferon γ/tumor necrosis factor α/interleukin (IL)-4/IL-13) of Th1 and Th2 cells in the serum were detected. The levels of T lymphocyte subsets CD3+, CD4+, CD8+, and CD4+/CD8+ and the levels of Th1/Th2 and Th17/Treg in CD4+ cell subsets were determined by flow cytometry. The objective remission rate, progression-free survival (PFS), and overall survival (OS) of patients were analyzed, and Kaplan-Meier curves were drawn to show the prognosis of patients. RESULTS: After treatment, the levels of SCCA, CA125, CEA, leptin, and IGF2 in the serum of patients with IACC were decreased, the level of Th1/Th2 was increased, and the Th17/Treg level was decreased. The treatment effect of Nimo + CRT was more significant than that of CRT alone. Survival curve analysis showed that Nimo + CRT could prolong PFS and OS in patients with IACC. In addition, the follow-up results of patients showed that Nimo did not increase the incidence and severity of adverse reactions caused by CRT. CONCLUSION: Nimo combined with CRT can protect the immune function of patients, improve the effective rate, and prolong the survival rate of patients with IACC.

Circ_0022383 alleviates IL-1ß-induced apoptosis, inflammation and extracellular matrix degeneration in osteoarthritis cell model by miR-3619-5p/SIRT1 axis.

BACKGROUND: Circular RNAs (circRNAs) have been identified to play roles in cartilage homeostasis and chondrocyte development, and be associated with osteoarthritis (OA) pathophysiology. Here, we aimed to investigate the role and mechanism of circ_0022383 on OA progression. METHODS: Chondrocytes in functional groups were treated with interleukin (IL)-1ß. The levels of genes and proteins were assayed by qRT-PCR and western blotting. Cell proliferation and apoptosis were evaluated by cell counting kit-8 assay, 5-Ethynyl-2'-deoxyuridine (Edu) assay, and flow cytometry, respectively. The inflammation and extracellular matrix (ECM) degeneration were determined by assessing the activity of IL-6, tumor necrosis factor (TNF-α), Aggrecan (ACAN), collagen type II α 1 chain (COL2A1) and ADAMTS5 proteins. The binding between miR-3619-5p and circ_0022383 or silent information regulator 1 (SIRT1) was confirmed by dual-luciferase reporter, RIP and RNA pull-down assays. RESULTS: Circ_0022383 expression was lower in the cartilages of OA patients and IL-1ß-induced primary chondrocytes. Functionally, ectopic overexpression of circ_0022383 alleviated IL-1ß-induced proliferation arrest, apoptosis, the release of IL-6 and TNF-α, as well as the decrease of ACAN and COL2A1 and the increase of ADAMTS5 level in chondrocytes. Mechanistically, circ_0022383 acted as a sponge for miR-3619-5p, which was verified to target SIRT1. Rescue experiments showed that miR-3619-5p up-regulation reversed the protective effects of circ_0022383 on IL-1ß-stimulated chondrocytes. Additionally, miR-3619-5p inhibition abolished IL-1ß-induced apoptosis, inflammation and ECM degeneration in chondrocytes, which were counteracted by SIRT1 silencing. CONCLUSION: Circ_0022383 protected chondrocytes from IL-1ß-induced apoptosis, inflammation and ECM degeneration by miR-3619-5p/SIRT1 axis, inspiring future therapy development for OA prevention.

[Effectiveness of limited middle and posterior column osteotomy via transvertebral space approach for old thoracolumbar compression fracture].

OBJECTIVE: To investigate the effectiveness of limited middle and posterior column osteotomy via transvertebral space approach in treatment of old thoracolumbar compression fracture. METHODS: A clinical data of 47 patients with old thoracolumbar compression fractures, who met the selection criteria between January 2010 and March 2018, was retrospectively analyzed. Twenty-five patients (group A) underwent the limited middle and posterior column osteotomy via transvertebral space approach, and 22 patients (group B) underwent the pedicle subtraction osteotomy (PSO). There was no significant difference in gender, age, cause of injury, time from injury to operation, fracture segment, and preoperative Cobb angle, sagittal vertical axis (SVA), visual analogue scale (VAS) score, Japanese Orthopaedic Association (JOA) score, and Oswestry disability index (ODI) between the two groups ( P>0.05). The operation time, intraoperative blood loss, and postoperative complications, as well as postoperative Cobb angle, SVA, VAS score, JOA score, ODI and the differences of all indexes between pre- and post-operation were recorded and compared between the two groups. The neurological function was evaluated by Frankel scale. RESULTS: The operations of both groups were successfully completed. The operation time and intraoperative blood loss in group A were significant lower than those in group B ( P<0.05). All incisions healed by first intetion. All patients were followed up 23-27 months (mean, 24.2 months) in group A and 24-28 months (mean, 24.8 months) in group B. At last follow-up, the VAS score, JOA score, ODI, Cobb angle, and SVA of the two groups were compared with those before operation, and the differences were significant ( P<0.05). There was no significant difference between the two groups ( P>0.05) in the indexes at last follow-up and the difference between pre- and post-operation. The lower extremity neurological symptoms (Frankel grade D) in 3 patients of group A before operation relieved (Frankel grade E) at last follow-up. The other patients were Frankel grade E. At last follow-up, CT showed bony fusion in the grafted area without any complications such as failure of internal fixation or pseudarthrosis. CONCLUSION: For patients with old thoracolumbar compression fractures, the limited middle and posterior column osteotomy via transvertebral space approach has a satisfactory effectiveness. Compared with PSO, it can reduce surgical trauma on the basis of achieving the same degree of deformity correction.

Effect of Acellular Amnion With Increased TGF-ß and bFGF Levels on the Biological Behavior of Tenocytes.

The human amniotic membrane has been a subject for clinical and basic research for nearly 100 years, but weak rejection has been reported. The purpose of this research is to remove the cellular components of the amnion for eliminating its immune-inducing activity to the utmost extent. The amniotic membrane treated by acid removed the epithelial cell, fibroblast, and sponge layers and retained only the basal and dense layers. In vitro, biological effects of the new material on tenocytes were evaluated. The levels of transforming growth factor (TGF-ß1), fibroblast growth factor (bFGF) proteins were measured. In vivo, the tendon injury model of chickens was constructed to observe effects on tendon adhesion and healing. The acellular amniotic membrane effectively removed the cell components of the amnion while retaining the fibrous reticular structure. Abundant collagen fibers enhanced the tensile strength of amnion, and a 3D porous structure provided enough 3D space structure for tenocyte growth. In vitro, acellular amnion resulted in the fast proliferation trend for tenocytes with relatively static properties by releasing TGF-ß1 and bFGF. In vivo, the experiment revealed the mechanism of acellular amnion in promoting endogenous healing and barrier exogenous healing by evaluating tendon adhesion, biomechanical testing, and labeling fibroblasts/tendon cells and monocytes/macrophages with vimentin and CD68. The acellular amnion promotes endogenous healing and barrier exogenous healing by releasing the growth factors such as TGF-ß1 and bFGF, thereby providing a new direction for the prevention and treatment of tendon adhesion.

Ginsenoside CK induces apoptosis and suppresses proliferation and invasion of human osteosarcoma cells through the PI3K/mTOR/p70S6K1 pathway.

Osteosarcoma is one of the most malignant bone tumors, and its major threats are aggressive invasion and early tumor metastasis, which result in a poor prognosis and high mortality. Accumulating evidence indicates that ginsenoside compound K (CK) has a significant antitumor effect, particularly on the inhibition of proliferation and invasion of numerous human tumors. In the present study, it was revealed that CK inhibited the viability and proliferation of osteosarcoma cells. Moreover, it was demonstrated that CK induced apoptosis and inhibited the migration and invasion of osteosarcoma cells via apoptotic staining, Annexin V/PI staining, and Transwell invasion assays. Furthermore, at the molecular level, the present results confirmed that apoptosis and invasion­related proteins were regulated by CK, which was possibly related to the blockade of the PI3K/mTOR/p70S6K1 signaling pathway. In summary, the present findings indicated that CK inhibited viability and proliferation, induced apoptosis, and inhibited the migration and invasion of osteosarcoma cells through the PI3K/mTOR/p70S6K1 signaling pathway.

Mechanism of miR-143-3p inhibiting proliferation, migration and invasion of osteosarcoma cells by targeting MAPK7.

Objective: To investigate the effects of miR-143-3p and MAPK7 on the proliferation, migration, and invasion of U2OS human osteosarcoma (OS) cells. Methods: The expression of miR-143-3p and MAPK7 in U2OS cells were detected by qRT-PCR, and the protein level of MAPK7 was measured by western blot assay. The targeting relationship between miR-143-3p and MAPK7 was predicted by TargetScan and verified by dual luciferase reporter assay. MTT and Transwell assays were used to detect cell viability, migrated cells and invaded cells of U2OS cells. Results: Compared with hFOB1.19 cells, the expression of miR-143-3p was down-regulated and MAPK7 was up-regulated in U2OS cells. Cell viability, migration and invasion ability significantly decreased induced by miR-143-3p overexpression or MAPK7 knockdown in U2OS cells. The results of dual luciferase reporter assay indicated that miR-143-3p interacted with MAPK7. Furthermore, overexpression of MAPK7 could reverse the inhibitory effects on cell proliferation, migration and invasion in U2OS cells induced by miR-143-3p mimics. Conclusion: miR-143-3p could inhibit proliferation, migration and invasion of U2OS cells by targeting MAPK7.

Anterior decompression and fusion versus laminoplasty for cervical myelopathy due to ossification of posterior longitudinal ligament: A meta-analysis.

BACKGROUND: Both anterior decompression and fusion (ADF) and laminoplasty (LAMP) are frequently used for the treatment of cervical myelopathy due to ossification of the posterior longitudinal ligament (OPLL). However, some controversies still remained in surgical options. We investigated whether ADF had better neurological outcome than LAMP in the treatment of cervical myelopathy due to OPLL. Secondary outcomes included operation time, blood loss, rate of complication and reoperation. METHODS: PubMed, EMBASE and the Cochrane Register of Controlled Trials database were searched to identify potential clinical studies compared ADF with LAMP for treatment of cervical myelopathy owing to OPLL. We also manually searched the reference lists of articles and reviews for possible relevant studies. Quality assessment was performed according to Cochrane Handbook and meta-analysis was conducted using Stata 12.0 software. RESULTS: Nine studies involving 712 patients were finally included in this analysis. Compared with LAMP, ADF was associated with an increase of the Japanese Orthopaedic Association (JOA) score (WMD = 1.86, 95% CI 0.43 to 3.29, P = .011) and recovery JOA score at final follow-up (WMD = 30.94, 95% CI 20.56 to 41.33, P = .000). And, ADF was associated with a decrease of the late neurologic deterioration than LAMP group (RR = 0.34, 95% CI 0.12 to 0.92, P = .003). However, ADF was associated with an increase of the postoperative cervical lordosis (WMD = 4.47, 95% CI 1.58 to 7.36, P = .002) than LAMP. There was no significant difference between the complication, reoperation rate (P > .05). What's more, ADF was associated with an increase of the operation time than LAMP (P < .05). CONCLUSIONS: ADF yields better neurological improvement, but higher cervical lordosis and longer operation time compared with LAMP for cervical myelopathy caused by OPLL. No significant difference was found in the complication and re-operation rate.

Comparison of the effectiveness and safety of topical versus intravenous tranexamic acid in primary total knee arthroplasty: a meta-analysis of randomized controlled trials.

BACKGROUND: This study aims to compare the effectiveness and safety of topical versus intravenous tranexamic acid (TXA) in reducing blood loss in primary total knee arthroplasty (TKA). METHODS: PubMed, Embase, the Cochrane Library, Web of Science, Chinese Biomedicine Literature (CBM), Wanfang Database and China National Knowledge Infrastructure (CNKI), and Google Scholar were searched for randomized controlled studies (RCTs) that compared topical versus intravenous TXA in terms of reducing blood loss during TKA from their inception to September 2015. This systematic review and meta-analysis was performed according to PRISMA criteria. RESULTS: Twelve studies reporting 12 RCTs comprising 1130 patients were included. Compared with the intravenous administration of TXA, the topical administration of TXA showed no significant differences in total blood loss (MD 2.08, 95% CI -68.43 to 72.60, P = 0.95), blood loss in drainage (MD 18.49, 95% CI -40.01 to 76.98, P = 0.54), hidden blood loss (MD 4.75, 95% CI -337.94 to 347.44, P = 0.99), need for transfusion (RR = 0.92, 95% CI 0.67~1.25, P = 0.58), hemoglobin (Hb) decline (MD -0.42, 95% CI -0.89 to 0.05, P = 0.08), and DVT occurrence (RR = 1.17, 95% CI 0.55~2.50, P = 0.68). CONCLUSIONS: Compared with intravenous administration TXA, topical administration TXA exhibits comparable effectiveness and safety in terms of reducing blood loss during TKA. Due to the poor quality of the included studies, more high-quality RCTs are needed to identify the optimal method and dose of TXA after TKA.

Metabolism of 5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737): identification of an unusual N-acetylglucosamine conjugate in the cynomolgus monkey.

5-Isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737) is a potent and selective vascular endothelial growth factor receptor-2 antagonist. In this study, liquid chromatography/tandem mass spectrometry and NMR were used to investigate the biotransformation of BMS-645737 in vitro and in the cynomolgus monkey, dog, mouse, and rat. Metabolic pathways for BMS-645737 included multistep processes involving both oxidation and conjugation reactions. For example, the 2-methyl-1H-pyrrolo moiety underwent cytochrome P450-catalyzed hydroxylation followed by oxidation to a carboxylic acid and then conjugation with taurine. Alternatively, the 5-methyl-1,3,4-oxadiazol-2-yl moiety was metabolized by hydroxylation and then conjugation with sulfate. The pyridin-5-yl group underwent direct glucuronidation in hepatocytes (dog, monkey, human) and conjugation with N-acetylglucosamine in the monkey. Conjugation with glutathione and processing along the mercapturic acid pathway was a minor metabolic pathway in vivo, although BMS-645737 did not form conjugates in the presence of glutathione-supplemented liver microsomes. Other minor biotransformation pathways included oxidative dehydrogenation, dihydroxylation, and hydrolytic opening of the oxadiazole ring followed by either deacetylation or hydrolysis of the resulting diacyl hydrazide. Whereas previous studies have shown the formation of N-acetylglucosamine conjugates of alcohols, arylamines, and other small molecules, this report describes the biotransformation of a heterocyclic aromatic amine via direct conjugation with N-acetylglucosamine.

Discovery and preclinical studies of 5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737), an in vivo active potent VEGFR-2 inhibitor.

We report herein a series of substituted N-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amines as inhibitors of vascular endothelial growth factor receptor-2 tyrosine kinase. Through structure-activity relationship studies, biochemical potency, pharmacokinetics, and kinase selectivity were optimized to afford BMS-645737 (13), a compound with good preclinical in vivo activity against human tumor xenograft models.

Discovery of brivanib alaninate ((S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate), a novel prodrug of dual vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 kinase inhibitor (BMS-540215).

A series of amino acid ester prodrugs of the dual VEGFR-2/FGFR-1 kinase inhibitor 1 (BMS-540215) was prepared in an effort to improve the aqueous solubility and oral bioavailability of the parent compound. These prodrugs were evaluated for their ability to liberate parent drug 1 in in vitro and in vivo systems. The l-alanine prodrug 8 (also known as brivanib alaninate/BMS-582664) was selected as a development candidate and is presently in phase II clinical trials.

Synthesis, SAR, and Evaluation of 4-[2,4-Difluoro-5-(cyclopropylcarbamoyl)phenylamino]pyrrolo[2,1-f][1,2,4]triazine-based VEGFR-2 kinase inhibitors.

Introduction of the 2,4-difluoro-5-(cyclopropylcarbamoyl)phenylamino group at the C-4 position of the pyrrolo[2,1-f][1,2,4] triazine scaffold led to the discovery of a novel sub-series of inhibitors of VEGFR-2 kinase activity. Subsequent SAR studies on the 1,3,5-oxadiazole ring appended to the C-6 position of this new sub-family of pyrrolotriazines resulted in the identification of low nanomolar inhibitors of VEGFR-2. Antitumor efficacy was observed with compound 37 against L2987 human lung carcinoma xenografts in athymic mice.

Discovery and evaluation of N-cyclopropyl- 2,4-difluoro-5-((2-(pyridin-2-ylamino)thiazol-5- ylmethyl)amino)benzamide (BMS-605541), a selective and orally efficacious inhibitor of vascular endothelial growth factor receptor-2.

Substituted 3-((2-(pyridin-2-ylamino)thiazol-5-ylmethyl)amino)benzamides were identified as potent and selective inhibitors of vascular endothelial growth factor receptor-2 (VEGFR-2) kinase activity. The enzyme kinetics associated with the VEGFR-2 inhibition of 14 (Ki=49+/-9 nM) confirmed that the aminothiazole-based analogues are competitive with ATP. Analogue 14 demonstrated excellent kinase selectivity, favorable pharmacokinetic properties in multiple species, and robust in vivo efficacy in human lung and colon carcinoma xenograft models.

Discovery and preclinical studies of (R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5- methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan- 2-ol (BMS-540215), an in vivo active potent VEGFR-2 inhibitor.

A series of substituted 4-(4-fluoro-1H-indol-5-yloxy)pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of vascular endothelial growth factor receptor-2 kinase is reported. Structure-activity relationship studies revealed that a methyl group at the 5-position and a substituted alkoxy group at the 6-position of the pyrrolo[2,1-f][1,2,4]triazine core gave potent compounds. Biochemical potency, kinase selectivity, and pharmacokinetics of the series were optimized and in vitro safety liabilities were minimized to afford BMS-540215 (12), which demonstrated robust preclinical in vivo activity in human tumor xenograft models. The l-alanine prodrug of 12, BMS-582664 (21), is currently under evaluation in clinical trials for the treatment of solid tumors.

Design, synthesis, and evaluation of orally active 4-(2,4-difluoro-5-(methoxycarbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]triazines as dual vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 inhibitors.

A series of substituted 4-(2,4-difluoro-5-(methoxycarbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]triazines was identified as potent and selective inhibitors of the tyrosine kinase activity of the growth factor receptors VEGFR-2 (Flk-1, KDR) and FGFR-1. The enzyme kinetics associated with the VEGFR-2 inhibition of compound 50 (K(i) = 52 +/- 3 nM) confirmed that the pyrrolo[2,1-f][1,2,4]triazine analogues are competitive with ATP. Several analogues demonstrated low-nanomolar inhibition of VEGF- and FGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation. Replacement of the C6-ester substituent of the pyrrolo[2,1-f][1,2,4]triazine core with heterocyclic bioisosteres, such as substituted 1,3,5-oxadiazoles, afforded compounds with excellent oral bioavailability in mice (i.e., 50 F(po) = 79%). Significant antitumor efficacy was observed with compounds 44, 49, and 50 against established L2987 human lung carcinoma xenografts implanted in athymic mice. A full account of the synthesis, structure-activity relationships, pharmacology, and pharmacokinetic properties of analogues within the series is presented.

Design, synthesis, and structure-activity relationships of tetrahydroquinoline-based farnesyltransferase inhibitors.

Tetrahydroquinoline-based small molecule inhibitors of farnesyltransferase (FT) have been identified. Lead compounds were shown to have nanomolar to sub-nanomolar activity in biochemical assays with excellent potency in a Ras-mutated cellular reversion assay. BMS-316810 (9e), a 0.7 nM FT inhibitor, was orally-active in a nude mouse tumor allograft efficacy study.

Synthesis and SAR of 4-(3-hydroxyphenylamino)pyrrolo[2,1-f][1,2,4]triazine based VEGFR-2 kinase inhibitors.

A versatile synthesis of the suitably functionalized pyrrolo[2,1-f][1,2,4]triazine nucleus is described. SAR at the C-5 and C-6 positions of the 4-(3-hydroxy-4-methylphenylamino)pyrrolo[2,1-f][1,2,4]triazine template led to compounds with good in vitro potency against VEGFR-2 kinase. Glucuronidation of the phenol group is mitigated by incorporation of a basic amino group on the C-6 side chain of the pyrrolotriazine nucleus.

N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent.

N-Acyl-2-aminothiazoles with nonaromatic acyl side chains containing a basic amine were found to be potent, selective inhibitors of CDK2/cycE which exhibit antitumor activity in mice. In particular, compound 21 [N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide, BMS-387032], has been identified as an ATP-competitive and CDK2-selective inhibitor which has been selected to enter Phase 1 human clinical trials as an antitumor agent. In a cell-free enzyme assay, 21 showed a CDK2/cycE IC(50) = 48 nM and was 10- and 20-fold selective over CDK1/cycB and CDK4/cycD, respectively. It was also highly selective over a panel of 12 unrelated kinases. Antiproliferative activity was established in an A2780 cellular cytotoxicity assay in which 21 showed an IC(50) = 95 nM. Metabolism and pharmacokinetic studies showed that 21 exhibited a plasma half-life of 5-7 h in three species and moderately low protein binding in both mouse (69%) and human (63%) serum. Dosed orally to mouse, rat, and dog, 21 showed 100%, 31%, and 28% bioavailability, respectively. As an antitumor agent in mice, 21 administered at its maximum-tolerated dose exhibited a clearly superior efficacy profile when compared to flavopiridol in both an ip/ip P388 murine tumor model and in a s.c./i.p. A2780 human ovarian carcinoma xenograft model.

Discovery of aminothiazole inhibitors of cyclin-dependent kinase 2: synthesis, X-ray crystallographic analysis, and biological activities.

High throughput screening identified 2-acetamido-thiazolylthio acetic ester 1 as an inhibitor of cyclin-dependent kinase 2 (CDK2). Because this compound is inactive in cells and unstable in plasma, we have stabilized it to metabolic hydrolysis by replacing the ester moiety with a 5-ethyl-substituted oxazole as in compound 14. Combinatorial and parallel synthesis provided a rapid analysis of the structure-activity relationship (SAR) for these inhibitors of CDK2, and over 100 analogues with IC(50) values in the 1-10 nM range were rapidly prepared. The X-ray crystallographic data of the inhibitors bound to the active site of CDK2 protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues displayed potent and broad spectrum antiproliferative activity across a panel of tumor cell lines in vitro. In addition, A2780 ovarian carcinoma cells undergo rapid apoptosis following exposure to CDK2 inhibitors of this class. Mechanism of action studies have confirmed that the phosphorylation of CDK2 substrates such as RB, histone H1, and DNA polymerase alpha (p70 subunit) is reduced in the presence of compound 14. Further optimization led to compounds such as water soluble 45, which possesses a favorable pharmacokinetic profile in mice and demonstrates significant antitumor activity in vivo in several murine and human models, including an engineered murine mammary tumor that overexpresses cyclin E, the coactivator of CDK2.