Novel Small Molecule Inhibitors Targeting the IL-6/STAT3 Pathway or IL-1ß.

Development of small molecules that inhibit inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases such as rheumatoid arthritis (RA). Following up a previous study, we synthesized 10 novel compounds with a 2,5-diaminobenzoxazole moiety and evaluated their biological activities. Among them, compound 3e showed potent inhibitory activity on Interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling inhibition (71.5%), and 3a showed excellent inhibitory activity on Interleukin 1 (IL-1ß) (92.1%). To test in vivo anti-inflammatory activity, compounds 3a and 3e were administered by intraperitoneal (IP) injection after subcutaneous (SC) injection of zymosan A into the right footpad of mice. Inflammation on the footpad was reduced after administration of compounds 3a and 3e. Especially, compound 3a showed a significant ameliorative effect on zymosan-induced inflammation. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective on the RA animal model through inhibition of the IL-6/STAT3 signaling pathway. Since drugs developed with small molecule inhibitors have several advantages over biological drugs, further study on these compounds is needed for the development of potent SMI drugs on RA.

Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1ß mRNA Expression.

LPS induces inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, and causes an inflammatory response. The development of small molecules that have suppressive effect on those inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases. We synthesized 12 novel compounds with 4-amino-N-(4-(benzo[d]oxazol-2-ylamino)phenyl)butanamide moiety and evaluated their biological activities. Among them, 4 compounds (compound 5d, 5c, 5f, 5m and synthetic intermediate 4d) showed potent inhibition activities on IL-1ß and IL-6 mRNA expression in vitro. Further, in vivo activity was evaluated with two compounds (5f and 4d) and mRNA levels of IL-1ß, IL-6, and TNF-α were significantly decreased without hepatotoxicity. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective for suppression of representative inflammatory cytokines.

Pharmacokinetic Alteration of Paclitaxel by Ferulic Acid Derivative.

P-glycoprotein (P-gp) is known to be involved in multidrug resistance (MDR) and modulation of pharmacokinetic (PK) profiles of substrate drugs. Here, we studied the effects of synthesized ferulic acid (FA) derivatives on P-gp function in vitro and examined PK alteration of paclitaxel (PTX), a well-known P-gp substrate drug by the derivative. Compound 5c, the FA derivative chosen as a significant P-gp inhibitor among eight FA candidates by in vitro results, increased PTX AUCinf as much as twofold versus the control by reducing PTX elimination in rats. These results suggest that FA derivative can increase PTX bioavailability by inhibiting P-gp existing in eliminating organs.

Effects of Piperazine Derivative on Paclitaxel Pharmacokinetics.

Paclitaxel (PTX) is an anticancer agent that is used to treat many cancers but it has a very low oral bioavailability due, at least in part, to the drug efflux transporter, P-glycoprotein (P-gp). Therefore, this study was performed to enhance oral bioavailability of PTX. In this study, we investigated the effects of several piperazine derivatives on P-gp function in vitro. Compound 4 was selected as the most potent P-gp inhibitor from the in vitro results for examining the pharmacokinetic (PK) changes of PTX in rats. Compound 4 increased the AUCinf of PTX without alterations in the Cmax value. The elimination half-life was extended and the oral clearance decreased. Additionally, the Tmax was delayed or widened in the treatment groups. Therefore, the bioavailability (BA) of PTX was improved 2.1-fold following the co-administration of 5 mg/kg of the derivative. A piperazine derivative, compound 4, which was confirmed as a substantial P-gp inhibitor in vitro increased the BA of PTX up to 2-fold by a lingering absorption, in part due to inhibition of intestinal P-gp and a low oral clearance of PTX. These results suggest that co-administering compound 4 may change the PK profile of PTX by inhibiting P-gp activity in the body.

NF-κB signaling regulates cell-autonomous regulation of CXCL10 in breast cancer 4T1 cells.

The chemokine CXCL10 and its receptor CXCR3 play a role in breast cancer metastasis to bone and osteoclast activation. However, the mechanism of CXCL10/CXCR3-induced intracellular signaling has not been fully investigated. To evaluate CXCL10-induced cellular events in the mouse breast cancer cell line 4T1, we developed a new synthetic CXCR3 antagonist JN-2. In this study, we observed that secretion of CXCL10 in the supernatant of 4T1 cells was gradually increased during cell growth. JN-2 inhibited basal and CXCL10-induced CXCL10 expression and cell motility in 4T1 cells. Treatment of 4T1 cells with CXCL10 increased the expression of P65, a subunit of the NF-κB pathway, via activation of the NF-κB transcriptional activity. Ectopic overexpression of P65 increased CXCL10 secretion and blunted JN-2-induced suppression of CXCL10 secretion, whereas overexpression of IκBα suppressed CXCL10 secretion. These results indicate that the CXCL10/CXCR3 axis creates a positive feedback loop through the canonical NF-κB signaling pathway in 4T1 cells. In addition, treatment of osteoblasts with conditioned medium from JN-2-treated 4T1 cells inhibited the expression of RANKL, a crucial cytokine for osteoclast differentiation, which resulted in an inhibitory effect on osteoclast differentiation in the co-culture system of bone marrow-derived macrophages and osteoblasts. Direct intrafemoral injection of 4T1 cells induced severe bone destruction; however, this effect was suppressed by the CXCR3 antagonist via downregulation of P65 expression in an animal model. Collectively, these results suggest that the CXCL10/CXCR3-mediated NF-κB signaling pathway plays a role in the control of autonomous regulation of CXCL10 and malignant tumor properties in breast cancer 4T1 cells.

5-Lipoxygenase inhibitors suppress RANKL-induced osteoclast formation via NFATc1 expression.

5-Lipoxygenase synthesizes leukotrienes from arachidonic acid. We developed three novel 5-LO inhibitors having a benzoxazole scaffold as a potential anti-osteoclastogenics. They significantly suppressed RANKL-induced osteoclast formation in mouse bone marrow-derived macrophages. Furthermore, one compound, K7, inhibited the bone resorptive activity of osteoclasts. The anti-osteoclastogenic effect of K7 was mainly attributable to reduction in the expression of NFATc1, an essential transcription factor for osteoclast differentiation. K7 inhibited osteoclast formation via ERK and p38 MAPK, as well as NF-κB signaling pathways. K7 reduced lipopolysaccharide (LPS)-induced osteoclast formation in vivo, corroborating the in vitro data. Thus, K7 exerted an inhibitory effect on osteoclast formation in vitro and in vivo, properties that make it a potential candidate for the treatment of bone diseases associated with excessive bone resorption.

3-Benzamides and 3,4,5-trimethoxyphenyl amines as calcium channel blockers.

T- and N-type calcium channels have known for relating to therapy of neuropathic pain which is chronic, debilitating pain state. Neuropathic pain is caused by damage of the somatosensory system. It may be associated with abnormal sensations and pain produced by normally non-painful stimuli (allodynia). Neuropathic pain is very difficult to treat, and only some 40-60% of patients achieve partial relief. For a neuropathic pain therapy, anticonvulsant like Lamotrigine, Carbamazepine and a topical anesthetic such as Lidocaine are used. We synthesized 15 novel amine derivatives and evaluated their activities against T-type and N-type calcium channels by whole-cell patch clamp recording on HEK293 cells. Among the tested compounds, compound 10 showed good inhibitory activity for both T-type and N-type calcium channels with the IC50 value of 1.9 µM and 4.3 µM, respectively. Compound 10 also showed good analgesic activity on rat spinal cord injury model.

Synthesis and biological evaluation of benzoisothiazole derivatives possessing N,N-dimethylformimidamide group as 5-HT6 receptor antagonists.

A series of novel N,N-dimethyl-N'-(5-(Ar-sulfonamido) benzo[d]isothiazol-3-yl)formimidamides was designed and synthesized as 5-HT(6) ligands. Here N,N-dimethyl formimidamides was used as a replacement for an aminoethyl moiety. In vitro functional assays demonstrated compounds 9b and 9i significantly inhibited the 5-HT-induced Ca(2+) increases (9b; IC(50)=0.36 µM and 9i; IC(50)=0.44 µM), indicating that 9b and 9i were potent 5-HT(6) receptor antagonists. Compounds 9i also showed good selectivity on the 5-HT(6) over 5-HT(4) and 5-HT(7) receptors.

Antitumor effects of a novel benzonaphthofurandione derivative (8e) on the human colon cancer cells in vitro and in vivo through cell cycle arrest accompanied with the modulation of EGFR and mTOR signaling.

Benzonaphthofurandione has been considered as an important class of naturally occurring and synthetic compounds having a variety of biological functions. In this study, we evaluated the antitumor effects of 3-[2-(dimethylamino)isopropoxy]-1-hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (8e), a novel benzonaphthofurandione derivative, on the growth of colorectal cancer HCT 116 cells both in vitro culture and an in vivo animal model. Compound 8e exhibited the potential growth inhibition of the colon cancer cells in a concentration-dependent manner. The anti-proliferative activity of 8e was also associated with the induction of cell cycle arrest in the G(0)/G(1) phase. The 8e-induced cell cycle arrest was well correlated with the suppression of cyclin-dependent kinase 2 (CDK2), CDK4, cyclin D1, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb). The tumor growth in xenograft nude mice bearing HCT 116 cells by compound 8e (10mg/kg) also significantly inhibited without any overt toxicity. In addition, the down-regulation of epidermal growth factor receptor (EGFR), Akt, and mTOR signalings were associated with the anti-proliferative activity of compound 8e in colon cancer cells. Taken together, these findings suggested that cell cycle arrest and modulation of cell signal transduction pathways might be the plausible mechanisms of actions for the anti-proliferative activity of 8e, and thus 8e might be used as an effective chemotherapeutic agent in human colon cancer.

Synthesis, biological evaluation, and molecular docking study of 3-(3'-heteroatom substituted-2'-hydroxy-1'-propyloxy) xanthone analogues as novel topoisomerase IIα catalytic inhibitor.

Epoxide ring-opened xanthone derivatives were synthesized and tested for their topoisomerase inhibitory activity and cytotoxicity. Most of the compounds showed topo IIα specific inhibitory activity. To clarify the mechanism of action of these compounds, the most potent compound (compound 14) of the synthesized analogues was further studied by testing its ATPase inhibitory activity and through molecular docking experiments. The results showed that the topo IIα inhibitory activity of compound 14 was inversely proportional to ATP concentration. In the ATPase inhibitory test, ATP hydrolysis was reduced less efficiently by compound 14 (28.5±4.6%) than novobiocin (60.4±8.1%). Molecular docking study revealed compound 14 to have a stable binding pattern to the ATP-binding domain of human topo II.

Synthesis and biological evaluation of (phenylpiperazinyl-propyl)arylsulfonamides as selective 5-HT(2A) receptor antagonists.

A novel series of 5-HT(2A) ligands that contain a (phenylpiperazinyl-propyl)arylsulfonamides skeleton was synthesized. Thirty-seven N-(cycloalkylmethyl)-4-methoxy-N-(3-(4-arylpiperazin-1-yl)propyl)-arylsulfonamide and N-(4-(4-arylpiperazin-1-yl)butan-2-yl)-arylsulfonamide compounds were obtained. The binding of these compounds to the 5-HT(2A), 5-HT(2C), and 5-HT(7) receptors was evaluated. Most of the compounds showed IC(50) values of less than 100 nM and exhibited high selectivity for the 5-HT(2A) receptor. Among the synthesized compounds, 16a and 16d showed good affinity at 5-HT(2A) (IC(50)=0.7 nM and 0.5 nM) and good selectivity over 5-HT(2C) (50-100 times) and 5-HT(7) (1500-3000 times).

Preparation of piperazine derivatives as 5-HT7 receptor antagonists.

Twenty-four compounds of 4-methoxy-N-[3-(4-substituted phenyl-piperazine-1-yl)propyl] benzene sulfonamides and N-[3-(4-substituted phenyl-piperazine-1-yl)propyl] naphthyl sulfonamides were prepared and evaluated as 5-HT(7) receptor antagonists. Most of the compounds showed the IC(50) values of 12-580nM. Four methyl branched analogues were also obtained, but the activity for methyl branched analogues was almost same as its straight chain congeners. Among the synthesized compounds, 3c showed a good activity on 5-HT(7) receptors and a good selectivity on 5-HT(1a), 5-HT(2a), 5-HT(2c), and 5-HT(6) receptors.

Synthesis of 1-/2-substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones and evaluation of their cytotoxicity and topoisomerase II inhibition.

Studies on antitumor heterocyclic quinones containing nitrogens revealed that the number and position of nitrogens on the heterocyclic ring have significance on cytotoxicity of quinones. In our continuous effort to find more cytotoxic quinone compounds, we designed triazolophthalazine analogues in order to introduce more nitrogens on the heterocyclic quinones. 1-/2-Substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones were synthesized by 1,3-dipolar addition of phthalazine-5,8-dione and 4-methoxybenzyl azide by modification of previously reported method. The cytotoxicity of the synthesized compounds was evaluated by a SRB (sulforhodamine B) assay against nine types of human cancer cell lines and inhibition against topoisomerase II (Topo II) of them was assessed by a decatenation assay. Most of the synthesized compounds showed considerably higher cytotoxicity than that of doxorubicin. Also, topoisomerase II inhibitory activity of the tested compounds was higher than that of etoposide and IC(50) values of the compounds were 19.4-64.5 microM.

Synthesis and cytotoxicity evaluation of substituted pyridazino[4,5-b]phenazine-5,12-diones and tri/tetra-azabenzofluorene-5,6-diones.

The substituted pyridazino[4,5-b]phenazine-5,12-diones and tri/tetra-azabenzo[a]fluorene-5,6-diones were synthesized from 6,7-dichlorophthalazine-5,8-dione and 6,7-dichloroquinoline-5,8-dione, respectively. The cytotoxic activities of the prepared compounds were evaluated by an SRB (Sulforhodamine B) assay against the following human cancer cell lines: A549 (lung), SK-OV-3 (ovarian), SK-MEL-2 (melanoma), XF 498 (CNS), and HCT 15 (colon). Almost all synthesized pyridazino[4,5-b]phenazine-5,12-diones (7a-j) presented higher cytotoxicity than that of doxorubicin (IC(50)=0.097-0.225 microM) against the cancer cell lines. In particular, the cytotoxicity of compounds 7f (R(1)=Et) and 7h (R(1), R(2)=Me) against all human cancer cell lines examined was about 10 times higher than that of doxorubicin. However, the cytotoxicities of several synthesized azabenzo[a]fluorene-5,6-diones (12a, 12c, 12d, 12e, and 12g) against the cancer cell lines in vitro were comparable to those of doxorubicin.

The development of 3D-QSAR study and recursive partitioning of heterocyclic quinone derivatives with antifungal activity.

It was reported that some 1,4-quinone derivatives such as 6-(N-arylamino)-7-chloro/6,7-bis[S-(aryl)thio]-5,8-quinolinedione and 6-arylthio-/5,6-arylamino-4,7-dioxobenzothiazoles have antifungal effects. To understand the structural basis for antifungal activity and guide in the design of more potent agents, we performed three-dimensional quantitative structure-activity relationship studies for a series of compounds using comparative molecular field analysis (CoMFA). The MIC values of 1,4-quinone derivatives on Aspergillus niger exhibited a strong correlation with steric and electrostatic factors of the 3D structure of molecules. The statistical results of the training set, cross-validated q(2) (0.683) and conventional r(2) (0.877) values, gave reliability to the prediction of inhibitory activity of a series of compounds. We also performed recursive partitioning (RP) analysis, used for the classification of molecules with activity using CART methods. Physicochemical, structural, and topological connectivity indices and E-state key descriptors were used for obtaining the decision tree models. The decision tree could classify the inhibitory activity of 1,4-quinone derivatives and its essential descriptors were S_aaN, Hbond donor, and Kappa-3.