Discovery of Novel Non-Nucleoside Inhibitors Interacting with Dizinc Ions of CD73.

High extracellular concentrations of adenosine triphosphate (ATP) in the tumor microenvironment generate adenosine by sequential dephosphorylation of CD39 and CD73, resulting in potent immunosuppression to inhibit T cell and natural killer (NK) cell function. CD73, as the determining enzyme for adenosine production, has been shown to correlate with poor clinical tumor prognosis. Conventional inhibitors as analogues of adenosine 5'-monophosphate (AMP) may have a risk of further metabolism to adenosine analogues. Here, we report a new series of malonic acid non-nucleoside inhibitors coordinating with zinc ions of CD73. Compound 12f was found to be a superior CD73 inhibitor (IC50 = 60 nM) by structural optimization, and its pharmacokinetic properties were investigated. In mouse tumor models, compound 12f showed excellent efficacy and reversal of immunosuppression in combination with chemotherapeutic agents or checkpoint inhibitors, suggesting that it deserves further development as a novel CD73 inhibitor.

Tumor Microenvironment-Responsive Hydrogel for Direct Extracellular ATP Imaging-Guided Surgical Resection with Clear Boundaries.

Most solid tumors are clinically treated using surgical resection, and the presence of residual tumor tissues at the surgical margins often determines tumor survival and recurrence. Herein, a hydrogel (Apt-HEX/Cp-BHQ1 Gel, termed AHB Gel) is developed for fluorescence-guided surgical resection. AHB Gel is constructed by tethering a polyacrylamide hydrogel and ATP-responsive aptamers together. It exhibits strong fluorescence under high ATP concentrations corresponding to the TME (100-500 µm) but shows little fluorescence at low ATP concentrations (10-100 nm) such as those in normal tissues. AHB Gel can rapidly (within 3 min) emit fluorescence after exposure to ATP, and the fluorescence signal only occurs at sites exposed to high ATP, resulting in a clear boundary between the ATP-high and ATP-low regions. In vivo, AHB Gel exhibits specific tumor-targeting capacity with no fluorescence response in normal tissue, providing clear tumor boundaries. In addition, AHB Gel has good storage stability, which is conducive to its future clinical application. In summary, AHB Gel is a novel tumor microenvironment-targeted DNA-hybrid hydrogel for ATP-based fluorescence imaging. It can enable the precise imaging of tumor tissues, showing promising application in fluorescence-guided surgeries in the future.

Engineering near-infrared laser-activated gold nanorod vesicles with upper critical solution temperature for photothermal therapy and chemotherapy.

Multimodal synergistic therapy based on nanomedicine drug delivery systems can achieve accurate cancer treatment. The anisotropy of gold nanorods (AuNRs) allows the adjustment of the longitudinal localized surface plasmon resonance absorption to the near-infrared band, which shows potential application in the field of photothermal therapy of cancer. Here, we report a new type of thermal-sensitive gold nanorod drug-loaded vesicles (UGRV-DOX) via the self-assembly of AuNRs modified with the amphiphilic polymer (PEG45-b-PS150) and upper critical solution temperature (UCST) polymer (P(AAm-co-AN)). The hollow structure of the vesicle can increase the drug loading capacity, and the polymers on its surface are intertwined to reduce drug leakage. As-prepared UGRV-DOX vesicles exhibits excellent photothermal conversion efficiency and can achieve light-controlled drug release. In vivo anti-tumor experiments showed that UGRV-DOX could ablate HepG2 transplanted tumors significantly under 808 nm laser irradiation, and the inhibition rate was as high as 99.3 %. These tumor-specific nanovesicles prefigure great potentials for high-precision cancer treatment.

Correlation between fat infiltration of paraspinal muscle and L4 degenerative lumbar spondylolisthesis in asymptomatic adults.

OBJECTIVE: To explore the relationship between different indicators of the degree of fat infiltration and L4 Degenerative lumbar spondylolisthesis (DLS). METHODS: 128 patients received annual health check-up underwent lumbar lateral Digital Radiography (DR) and abdominal Computed tomography (CT) imaging were enrolled. The DLS group included 60 patients diagnosed with DLS, and the control group included 68 patients without DLS. The data collected included vertebral density of L4-L5, fat infiltration ratio (FIR) of paravertebral muscle (PM) and psoas major muscle (PMM), skeletal muscle density of PM and PMM, low attenuation muscle ratio (LTR) of PM and PMM, paraspinal muscle density (PMD), psoas major muscle density (PMMD), low attenuation muscle density (LMD) of PM and PMM, facet joint angle (FJA), facet joint degeneration (FJD), etc. RESULTS: PM FIR and PM LTR were weakly positively correlated with the degree of L4 DLS, and there was a weak negative correlation between PMD and the degree of L4 DLS in asymptomatic adults (P < 0.05). Logistic regression analysis showed that PM FIR was an independent related factor of L4 DLS (Q3 vs. Q1, OR = 3.746, 95% CI: 1.076-13.048, p = 0.038). ROC curve analysis showed that the PM FIR has a high predictive value for L4 DLS in asymptomatic adults. CONCLUSION: The indicator of PM FIR was an independent related factor of L4 DLS in asymptomatic adults. It has a high predictive value for L4 DLS and can be applied as a potential target for clinical treatment of L4 DLS in asymptomatic adults.

Discovery and optimization of 4-anilinoquinazoline derivatives spanning ATP binding site and allosteric site as effective EGFR-C797S inhibitors.

Epidermal growth factor receptor (EGFR) is an effective drug target for the treatment of non-small cell lung cancer (NSCLC). However, a tertiary point mutation (C797S) at the ATP binding pocket of the EGFR induces resistance to the third-generation EGFR inhibitors, due to the loss of covalent interaction with Cys797. Here, we designed a series of 4-anilinoquinazoline derivatives that simultaneously occupied the ATP binding pocket and the allosteric site. The newly-synthesized compounds displayed high potency against EGFR-C797S resistance mutation. Among them, compound 14d presented high anti-proliferative effect against BaF3-EGFRL858R/T790M/C797S (IC50 = 0.75 µM) and BaF3-EGFR19del/T790M/C797S (IC50 = 0.09 µM) cells. Moreover, 14d resulted in obvious inhibition activities against EGFR and its downstream signaling pathways in a dose-dependent manner in BaF3-EGFR19del/T790M/C797S cells. Finally, 14d significantly inhibited tumor growth in BaF3-EGFR19del/T790M/C797S xenograft model (30 mg/kg, TGI = 67.95%). These results demonstrated that 14d is a novel and effective EGFR-C797S inhibitor which spanning the ATP binding pocket and the allosteric site and effective both in vitro and in vivo.

Dynamic Compartmentalization of Peptide-Oligonucleotide Conjugates with Reversible Nanovesicle-Microdroplet Phase Transition Behaviors.

Developing artificial microsystems based on liquid-liquid phase separation (LLPS) to mimic cellular dynamic compartmentalization has gained increasing attention. However, limitations including complicated components and laborious fabrication techniques have hindered their development. Herein, we describe a new single-component dynamic compartmentalization system using peptide-oligonucleotide conjugates (POCs) produced from short elastin-like polypeptides (sELPs) and oligonucleotides (ONs), which can perform thermoreversible phase transition between a nanovesicle and a microdroplet. The phase transition of sELP-ONs is thoroughly investigated, of which the transition temperature can be controlled by concentration, length of sELPs and ONs, base sequences, and salt. Moreover, the sELP-ON microcompartment can enrich a variety of functional molecules including small molecules, polysaccharides, proteins, and nucleic acids. Two sELP-ON compartments are used as nano- and microreactors for enzymatic reactions, separately, in which chemical activities are successfully regulated under different-scaled confinement effects, demonstrating their broad potential application in matter exchange and artificial cells.

An Endoplasmic Reticulum (ER)-Targeting DNA Nanodevice for Autophagy-Dependent Degradation of Proteins in Membrane-Bound Organelles.

Targeted protein degradation via proteasomal and lysosomal pathways is a promising therapeutic approach, and proteins in cytoplasm or on the cell membrane can be easily contacted and have become the major targets. However, degradation of disease-related proteins that exist in membrane-bound organelles (MBO) such as the endoplasmic reticulum (ER) remains unsolved due to the membrane limits. Here we describe a DNA nanodevice that shows ER targeting capacity and undergoes new intracellular degradation via the autophagy-dependent pathway. Then the DNA nanostructure functionalized with specific ligands is used to selectively catch ER-localized proteins and then transport them to the lysosome for degradation. Through this technique, the degradation of both exogenous ER-resident protein (ER-eGFP) and endogenous overexpressed molecular chaperone (glucose-regulated protein 78) in cancer cells has been successfully executed with high efficiency.

Discovery of pyrrolo[1,2-a]quinoxalin-4(5H)-one derivatives as novel non-covalent Bruton's tyrosine kinase (BTK) inhibitors.

Bruton's tyrosine kinase (BTK) is a promising target in the treatment of B cell malignancies and autoimmune disorders. Developing selective non-covalent BTK inhibitors is an important strategy to overcome the side effects and drug resistance induced by covalent BTK inhibitors. In this article, we designed and synthesized pyrrolo[1,2-a]quinoxalin-4(5H)-one and imidazo[1,2-a]quinoxalin-4(5H)-one based selective noncovalent BTK inhibitors via scaffold hopping from BMS-986142 and investigated their biological activities. Among the synthesized compounds, pyrrolo[1,2-a]quinoxalin-4(5H)-one derivatives 2 and 4 showed great BTK inhibition potency with IC50 value at 7.41 nM and 11.4 nM, respectively. Besides, they showed equivalent or even better potency in U937 and Ramos cells than BMS-986142. The kinase selectivity profiling study illustrated the excellent selectivity of compound 2 against a panel of 468 kinases. In U937 xenograft models, compound 2 could significantly inhibit tumor growth with TGI = 65.61%. In all, we provided a new scaffold as non-covalent selective BTK inhibitors and the representative compounds exhibited potency both in vitro and in vivo.

Pentraxin 3 depletion (PTX3 KD) inhibited myocardial fibrosis in heart failure after myocardial infarction.

BACKGROUND: HF is a common complication of MI. The underlying mechanisms of myocardial fibrosis in HF after MI are incompletely defined. Here, this study aims to investigate the role of PTX3 KD in HF after MI. METHODS: Bioinformatics analysis based on GSE86569 dataset was performed to explore the potential role of PTX3 in HF. Male C57/BL6J mice were administered with lentiviral vector encoding PTX3 KD or empty vector, and then underwent either coronary ligation or sham surgery. Echocardiography, Masson staining, and immunofluorescence counterstaining were conducted to evaluate the cardiac function and fibrosis. Cardiac fibroblasts were isolated and transfected with lentiviral vector encoding PTX3 KD in vitro to verify the in vivo findings. RESULTS: Bioinformatics analysis based on GSE86569 revealed the aberrant expression of PTX3 in HF patients. Echocardiography showed that PTX3 KD reversed the HF-induced cardiac dysfunction with better cardiac function parameters. Masson staining demonstrated that the obvious infarct and high fibrosis ratio in HF mice were remarkably improved after PTX3 KD. Immunofluorescence staining indicated that the HF-induced increase expression of α-SMA was significantly suppressed by PTX3 KD. Additionally, both in vivo and in vitro results confirmed that PTX3 KD decreased the fibrosis-related up-regulation of collagen I, collagen III, and p-STAT3. However, the result was opposite after IL-6 treatment. CONCLUSIONS: PTX3 KD protects the cardiac function and counteracts the myocardial fibrosis by down-regulating IL-6/STAT3 pathway in HF.

Engineering naphthalimide-cyanine integrated near-infrared dye into ROS-responsive nanohybrids for tumor PDT/PTT/chemotherapy.

Photodynamic (PDT) and photothermal therapies (PTT) are emerging treatments for tumour ablation. Organic dyes such as porphyrin, chlorin, phthalocyanine, boron-dipyrromethene and cyanine are the clinically or preclinically used photosensitizer or photothermal agents. Development of structurally diverse near-infrared dyes with long absorption wavelength is of great significance for PDT and PTT. Herein, we report a novel near-infrared dye ML880 with naphthalimide modified cyanine skeleton. The introduction of naphthalimide moiety results in stronger electron delocalization and larger redshift in emission compared with IR820. Furthermore, ML880 is co-loaded with chemotherapeutic drug into ROS-responsive mesoporous organosilica (RMON) to construct nanomedicine NBD&ML@RMON, which exhibits remarkable tumor inhibition effects through PDT/PTT/chemotherapy in vivo.

NIR Activated Upper Critical Solution Temperature Polymeric Micelles for Trimodal Combinational Cancer Therapy.

The balance between drug efficiency and its side effects on normal tissues is still a challenging problem to be solved in current cancer therapies. Among different strategies, cancer therapeutic methods based on nanomedicine delivery systems have received extensive attention due to their unique advantages such as improved circulation and reduced toxicity of drugs in the body. Herein, we constructed dual-responsive polymeric micelles DOX&ALS@MFM based on an upper critical solution temperature (UCST) polymer to simultaneously combine chemotherapy, photothermal therapy (PTT), and photodynamic therapy (PDT). Amphiphilic block copolymer P(AAm-co-AN)-b-PEI-ss-PEG-FA with a critical point of 42 °C was able to self-assemble into polymeric micelles under physiological conditions, which further encapsulated anticancer drug doxorubicin (DOX) and photosensitizer ALS to obtain drug-loaded micelles DOX&ALS@MFM. Micelles aggregated at tumor sites due to folate targeting and an enhanced permeability retention (EPR) effect. After that, the high intracellular concentration of glutathione (GSH) and near-infrared (NIR) light prompted disassembly of the polymer to release DOX and ALS. ALS not only plays a role in PTT but also produces singlet oxygen, therefore killing tumor cells by PDT. Both in vitro and in vivo studies demonstrated the photothermal conversion and reactive oxygen species generation ability of DOX&ALS@MFM micelles, at the same time as the excellent inhibitory effect on tumor growth with NIR light irradiation. Thus, our research substantiated a new strategy for the biomedical application of UCST polymers in the cited triple modal tumor therapy.

Discovery of Pteridine-7(8H)-one Derivatives as Potent and Selective Inhibitors of Bruton's Tyrosine Kinase (BTK).

Bruton's tyrosine kinase (BTK) is an attractive therapeutic target in the treatment of cancer, inflammation, and autoimmune diseases. Covalent and noncovalent BTK inhibitors have been developed, among which covalent BTK inhibitors have shown great clinical efficacy. However, some of them could produce adverse effects, such as diarrhea, rash, and platelet dysfunction, which are associated with the off-target inhibition of ITK and EGFR. In this study, we disclosed a series of pteridine-7(8H)-one derivatives as potent and selective covalent BTK inhibitors, which were optimized from 3z, an EGFR inhibitor previously reported by our group. Among them, compound 24a exhibited great BTK inhibition activity (IC50 = 4.0 nM) and high selectivity in both enzymatic (ITK >250-fold, EGFR >2500-fold) and cellular levels (ITK >227-fold, EGFR 27-fold). In U-937 xenograft models, 24a significantly inhibited tumor growth (TGI = 57.85%) at a 50 mg/kg dosage. Accordingly, 24a is a new BTK inhibitor worthy of further development.

Discovery of pyrido[3,4-b]indol-1-one derivatives as novel non-covalent Bruton's tyrosine kinase (BTK) inhibitors.

Bruton's tyrosine kinase (BTK) is an attractive target for the treatment of malignancy and inflammatory/autoimmune diseases. Most of the covalent BTK inhibitors would induce off-target side effects and drug resistance. To improve the drug safety of BTK inhibitors, non-covalent inhibitors have attracted more and more attention. We designed a series of novel pyrido[3,4-b]indol-1-one derivatives (N-A and N-B) via scaffold hopping from CGI-1746. The structure-activity relationship (SAR) of the newly-synthesized compounds was explored. The results showed that compounds 12 and 18 exhibited potent enzymatic potency against BTK with IC50 values of 0.22 µM and 0.19 µM, respectively. In lymphoma cell lines U-937 cells and Ramos cells, compounds 12 and 18 displayed comparative antiproliferative activity with Ibrutinib. Moreover, compound 12 induced G1-phase cell cycle arrest and apoptosis in U-937 cells. And it could effectively inhibit tumor growth in U-937 xenograft mouse model (TGI = 41.90% at 50 mg/kg). In all, the new pyrido[3,4-b]indol-1-one derivatives have the antitumor potency by BTK inhibition and were worthy of further exploration.

Discovery, Optimization, and Structure-Activity Relationship Study of Novel and Potent RSK4 Inhibitors as Promising Agents for the Treatment of Esophageal Squamous Cell Carcinoma.

Ribosomal S6 protein kinase 4 (RSK4) was identified to be a promising target for the treatment of esophageal squamous cell carcinoma (ESCC) in our previous research, whose current treatments are primarily chemotherapy and radiotherapy due to the lack of targeted therapy. However, few potent and specific RSK4 inhibitors are reported. In this study, a series of 1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-ones derivatives were designed and synthesized as novel and potent RSK4 inhibitors. Compound 14f was identified with potent RSK4 inhibitory activity both in vitro and in vivo. 14f significantly inhibited the proliferation and invasion of ESCC cells in vitro with IC50 values of 0.57 and 0.98 µM, respectively. It dose dependently inhibited the phosphorylation of RSK4 downstream substrates while exerting little effect on the substrates of RSK1-3 in ESCC cells. The markedly suppressed tumor growth and no observed toxicity to main organs in the ESCC xenograft mouse model suggested 14f to be a promising RSK4-targeting agent for ESCC treatment.

Enzyme/GSH dual-responsive biodegradable nanohybrid for spatiotemporally specific photodynamic and hypoxia-augmented therapy against tumors.

Photodynamic therapy (PDT) efficacy has been severely limited by the hypoxia in tumor microenvironment. A multitherapy modality was developed, integrating the advantages of each therapy and a nanocarrier: PDT and PDT-induced hypoxia-activated chemotherapy. Following PDT-induced hypoxia augmented in the periphery of the tumors, chemotherapy was locally activated. To this end, new indocyanine green (IR820) and a hypoxia-activated prodrug tirapazamine (TPZ) were loaded in glutathione (GSH) decomposable mesoporous organic silica nanoparticles (GMONs), tethered by hyaluronic acid (HA). This nanohybrid showed a tendency to accumulate and be retained in tumors, due to passive and active targeting. The IR820 produced singlet oxygen (1O2) under near-infrared (NIR) laser irradiation and concomitantly tumorous abnormality exacerbated hypoxia. TPZ-mediated hypoxia-activated chemotherapy acted to kill more tumor cells. In vivo results indicated that the tumor inhibition rate of dual-loaded nanohybrids was up to 76% under NIR laser irradiation. The immunofluorescence staining of tumor slices demonstrated that the superficial part of tumors experienced exacerbated hypoxia with laser irradiation, resulting in TPZ exerting powerful chemotherapy effects. This nanohybrid is expected to be valuable as spatiotemporally specific therapy for cancer.

Dual-responsive nanohybrid based on degradable silica-coated gold nanorods for triple-combination therapy for breast cancer.

Multi-modal combination therapy has attracted great attention, owing to the unsatisfactory therapeutic efficacy of conventional chemotherapy. Mesoporous silica-coated gold nanorods possess great potential in photothermal therapy and drug delivery. In this work, we fabricate a dual-responsive nanohybrid for combination treatment of the malignant tumor. In this system, gold nanorods are coated with the degradable mesoporous silica, and the chemotherapy drug doxorubicin (DOX) and photosensitizer (IR820) are co-loaded inside the pores of the silica. The encapsulation of hyaluronic acid (HA) endow the nanohybrids with mammary carcinoma targeting ability and better biocompatibility, owning to CD44+ receptor overexpressed in some cancer cells. As-prepared nanohybrids exhibit high responsiveness to a high glutathione (GSH) level and degrade rapidly in the presence of hyaluronidase (HAase) and GSH after endocytosis by 4T1 cells, allowing the efficient release of loaded DOX and IR 820 in tumor sites. Interestingly, near-infrared (NIR) laser not only triggers the generation of reactive oxygen species, but also remarkable photothermal efficacy originating from GNRs. Therefore, upon the irradiation of 808 nm NIR light, the combinatorial photodynamic, photothermal and chemotherapy is achieved, accordingly leading to a highly efficient antitumor outcome in vitro and in vivo. This strategy provides an ideal approach to constructing multimodal cancer therapy system. STATEMENT OF SIGNIFICANCE: • Dual-responsive nanohybrids for combinatorial therapy of breast cancer. • The nanohybrids exhibit both HAase and GSH stimuli-responsive behavior. • The nanohybrids exhibit light-activated PDT/PTT/chemotherapy. • The nanohybrids show good biosafety for potential clinical application.

Short intrinsically disordered polypeptide-oligonucleotide conjugates for programmed self-assembly of nanospheres with temperature-dependent size controllability.

A series of short intrinsically disordered polypeptide conjugated oligonucleotides (IDPOCs) were rationally developed and assembled into well-defined nanospheres. The nanospheres exhibited excellent reversible thermoresponsive regulation of their contraction and expansion. Furthermore, the nanospheres showed biocompatibility, drug encapsulation and effective cellular uptake.

Self-accelerating H2O2-responsive Plasmonic Nanovesicles for Synergistic Chemo/starving therapy of Tumors.

Rationale: Nanoscale vehicles responsive to abnormal variation in tumor environment are promising for use in targeted delivery of therapeutic drugs specifically to tumor sites. Herein, we report the design and fabrication of self-accelerating H2O2-responsive plasmonic gold nanovesicles (GVs) encapsulated with tirapazamine (TPZ) and glucose oxidase (GOx) for synergistic chemo/starving therapy of cancers. Methods: Gold nanoparticles were modified with H2O2-responsive amphiphilic block copolymer PEG45-b-PABE330 by ligand exchange. The TPZ and GOx loaded GVs (TG-GVs) were prepared through the self-assembly of PEG45-b-PABE330 -grafted nanoparticles together with TPZ and GOx by solvent displacement method. Results: In response to H2O2 in tumor, the TG-GVs dissociate to release the payloads that are, otherwise, retained inside the vesicles for days without noticeable leakage. The released GOx enzymes catalyze the oxidation of glucose by oxygen in the tumor tissue to enhance the degree of hypoxia that subsequently triggers the reduction of hypoxia-activated pro-drug TPZ into highly toxic free radicals. The H2O2 generated in the GOx-catalyzed reaction also accelerate the dissociation of vesicles and hence the release rate of the cargoes in tumors. The drug-loaded GVs exhibit superior tumor inhibition efficacy in 4T1 tumor-bearing mice owing to the synergistic effect of chemo/starvation therapy, in addition to their use as contrast agents for computed tomography imaging of tumors. Conclusion: This nanoplatform may find application in managing tumors deeply trapped in viscera or other important tissues that are not compatible with external stimulus (e.g. light).

pH-Activatable tumor-targeting gold nanoprobe for near-infrared fluorescence/CT dual-modal imaging in vivo.

One hallmark of solid tumors, regardless of its type or stage, is the existence of an unsual acidic microenvironment, which has been considered a specific and ideal target for cancer imaging. Therefore, we developed a pH-activatable nanoprobe GNPs-CKL-FA for near-infrared fluorescence (NIR) and computed tomography (CT) imaging of tumors. This nanoprobe consists of a near-infrared fluorophore (Cy5.5), a pH-sensitive ketal linker, and gold nanoparticles (GNPs) decorated with folates that could bind to tumor cells' surface receptors to promote cellular internalization. This ability of folate to mediate tumor targeting and accelerate internalization has been confirmed by in vitro experiments with HeLa cells. The fluorescence of the nanoprobes successfully activated by low intracellular pH, especially in more acidic organelles. Furthermore, fluorescence signals increased to a greater extent when the pH in tumors was lowered by injection of acetate buffer and isoproterenol. The CT contrast of GNPs-CKL-FA was obtained after administering intravenously to HeLa subcutaneous tumor-bearing mice. These results suggest that GNPs-CKL-FA has the potential to be a pH-activatable fluorescent nanoprobe combined with CT contrast agent for tumor targeted imaging.

Design, synthesis and structure-activity relationship study of aminopyridine derivatives as novel inhibitors of Janus kinase 2.

Janus Kinase 2 (JAK2) is a kind of intracellular non-receptor protein tyrosine kinase and has been certified as an important target for the treatment of myeloproliferative neoplasms and rheumatoid arthritis. However, the low selectivity and potential safety issues restrict the clinical applications of JAK2 inhibitors. Here we found that crizotinib showed good inhibitory activity against JAK2 by enzymatic assays (IC50 = 27 nM). Then we carried out structure-based drug design and synthesized a series of compounds with an aminopyridine scaffold. Finally, compound 12k and 12l were identified as the promising inhibitors of JAK2, which exhibited high inhibitory activity (IC50 = 6 nM and 3 nM, respectively) and selectivity for JAK2 over JAK1 and JAK3, and showed potent antiproliferative activities toward HEL human erythroleukemia cells. Moreover, 12k suppressed symptoms of the collagen-induced arthritis (CIA) model in rats.