Discovery of WS-157 as a highly potent,selective and orally active EGFR inhibitor

EGFR tyrosine kinase inhibitor (EGFR-TKI) has been used successfully in clinic for the treatment of solid tumors. In the present study, we reported the discovery of WS-157 from our in-house diverse compound library, which was validated to be a potent and selective EGFR-TKI. WS-157 showed excellent inhibitory activities against EGFR (IC₅₀ = 0.81 nmol/L), EGFR^[d746−750] (IC₅₀ = 1.2 nmol/L) and EGFR^[L858R] (IC₅₀ = 1.1 nmol/L), but was less effective or even inactive against other nine kinases. WS-157 also displayed excellent antiproliferative activities against a panel of human cancer cell lines, and exhibited the ability to reduce colony formation and wound healing the same as gefitinib. We found that WS-157 upon oral administration showed better anti-tumor activity in A431 bearing xenograft mouse models compared to gefitinib. In addition, WS-157 showed better intestinal absorption than gefitinib and had favorable pharmacokinetic properties and microsomal metabolic stability in different species. These studies indicate that WS-157 has strong antitumor activity in vitro and in vivo, and could be used for the development of anti-lung cancer agent targeting EGFR.     

Discovery of novel 2,8-diazaspiro[4.5]decanes as orally active glycoprotein IIb-IIIa antagonists

In our efforts to develop orally active GPIIb-IIIa antagonists with improved pharmaceutical properties, we have utilized a novel 2,8-diazaspiro[4.5]decane scaffold as a template. We describe here our investigation of a variety of templates including spiropiperidinyl-gamma-lactams, spiropiperidinylimide, spiropiperidinylureas, and spiropiperidinylhydantoins. With the appropriate acidic and basic pharmacophores in place, each template yielded analogues with potent GPIIb-IIIa inhibitory activity. One of the compounds, 59 (CT50787), was also used to demonstrate for the first time the use of a pharmacological agent which is alphaIIbbeta3 specific to display biological activity in a lower species such as mouse and to extend bleeding times. Evaluation of the pharmacokinetic properties of selected compounds from each series in rat, dog, and cynomolgus monkey has led to the identification of 22 (CT51464), a double prodrug, with excellent pharmacokinetic properties. It exhibited good pharmacokinetic profile across species (F% = 33 (Cyno), 73 (dog), 22 (rat); t(1/2)(beta)() = 14.2 h (Cyno), 8.97 h (dog), 1.81 h (rat)). The biologically active form, 23 (CT50728), displayed inhibition of platelet aggregation in platelet rich plasma (PRP) with an IC(50) value of 53 nM in citrate buffer, 110 nM in PPACK anticoagulated PRP, and 4 nM in solid-phase GPIIb-IIIa competition binding assay (ELISA). Both 23 and 22 were stable in human liver microsomes, did not inhibit the P450 3A4 isozyme, and had low protein binding (18.22% for 23) and a desirable log P (0.45 +/- 0.06 for 22, and -0.91 +/- 0.32 for 23). It is predicted that the high oral bioavailability for these compounds in multiple species should translate into lower intra- and intersubject variability in man. The long plasma half-life of the lead is consistent with once or twice daily administration for chronic therapy. Analogue 22 (CT51464) thus appears to be a promising oral GPIIb-IIIa inhibitor with significantly improved pharmacokinetic properties over the previously described clinical candidates and may be found useful in the treatment of arterial occlusive disorders.     

Discovery of potent orally active thrombin receptor (protease activated receptor 1) antagonists as novel antithrombotic agents

Structurally novel thrombin receptor (protease activated receptor 1, PAR-1) antagonists based on the natural product himbacine are described. The prototypical PAR-1 antagonist 55 showed a Ki of 2.7 nM in the binding assay, making it the most potent PAR-1 antagonist reported. 55 was highly active in several functional assays, showed excellent oral bioavailability in rat and monkey models, and showed complete inhibition of agonist-induced ex vivo platelet aggregation in cynomolgus monkeys after oral administration.     

Synthesis and biological evaluation of novel (thio)urea derivatives as potential antitumor agents

A series of novel (thio)ureas containing the pyrimidinyl group was designed and synthesized. Their in-vitro antitumor activity against different human tumor cells was examined. Some of the compounds showed potential antitumor activity, which provided some hints for further studies on structure modification.     

The therapeutic potential of targeting ABC transporters to combat multi-drug resistance

Introduction: Most disseminated cancers remain fatal despite the availability of a variety of conventional and novel treatments including surgery, chemotherapy, radiotherapy, immunotherapy, and biologically targeted therapy. A major factor responsible for the failure of chemotherapy in the treatment of cancer is the development of multidrug resistance (MDR). The overexpression of various ABC transporters in cancer cells can efficiently remove the anticancer drug from the cell, thus causing the drug to lose its effect.

Areas covered: In this review, we summarised the ongoing research related to the mechanism, function, and regulation of ABC transporters. We integrated our current knowledge at different levels from molecular biology to clinical trials. We also discussed potential therapeutic strategies of targeting ABC transporters to reverse MDR in cancer cells.

Expert opinion: Involvement of various ABC transporters to cancer MDR lays the foundation for developing tailored therapies that can overcome MDR. An ideal MDR reversal agent should have broad-spectrum ABC-transporter inhibitory activity, be potent, have good pharmacokinetics, have no trans-stimulation effects, and have low or no toxicity. Alternatively, nanotechnology-based drug delivery systems containing both the cytotoxic drug and reversing agent may represent a useful approach to reversing MDR with minimal off-target toxicity.     

Discovery of a novel orally active PDE-4 inhibitor effective in an ovalbumin-induced asthma murine model

Phosphodiesterase-4 (PDE-4) is responsible for metabolizing adenosine 3',5'-cyclic monophosphate that reduces the activation of a wide range of inflammatory cells including eosinophils. PDE-4 inhibitors are under development for the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease. Herein, we report a novel PDE-4 inhibitor, PDE-423 (3-[1-(3-cyclopropylmethoxy-4-difluoromethoxybenzyl)-1H-pyrazol-3-yl]-benzoic acid), which shows good in vitro and in vivo oral activities. PDE-423 exhibited in vitro IC(50)s of 140 nM and 550 nM in enzyme assay and cell-based assay, respectively. In vivo study using ovalbumin-induced asthmatic mice revealed that PDE-423 reduced methacholine-stimulated airway hyperreactivity in a dose-dependent manner by once daily oral administration (ED(50)=18.3 mg/kg), in parallel with decreased eosinophil peroxidase activity and improved lung histology. In addition, PDE-423 was effective in diminishing lipopolysaccharide-induced neutrophilia in vivo as well as in vitro. Oral administration of PDE-423 (100 mg/kg) had no effect on the duration of xylazine/ketamine-induced anesthesia and did not induce vomiting incidence in ferrets up to the dose of 1000 mg/kg. The present study indicates that a novel PDE-4 inhibitor, PDE-423, has good pharmacological profiles implicating this as a potential candidate for the development of a new anti-asthmatic drug.     

Discovery of phthalimide derivatives as novel inhibitors of a soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) inhibitors are effective in reducing blood pressure, inflammation, and pain in a number of mammalian disease models. As most classical urea-based sEH inhibitors suffer from poor solubility and pharmacokinetic properties, the development of novel sEH inhibitors with an improved pharmacokinetic specification has received a great deal of attention. In this study, a series of amide-based sEH inhibitors bearing a phthalimide ring as the novel secondary pharmacophore (P₂) was designed, synthesized, and evaluated. Docking results illustrated that the amide group as the primary pharmacophore (P₁) was placed at a suitable distance from the three key amino acids (Tyr383, Tyr466, and Asp335) for an effective hydrogen bonding. In agreement with these findings, most of the newly synthesized compounds demonstrated moderate to high sEH inhibitory activities, relative to 12-(3-adamantan-1-yl-ureido)dodecanoic acid as the reference standard. Compound 12e with a 4-methoxybenzoyl substituent exhibited the highest sEH inhibitory activity, with an IC₅₀ value of 1.06 nM. Moreover, the ADME properties of the compounds were evaluated in silico, and the results revealed appropriate predictions.     

Discovery of novel exceptionally potent and orally active c-MET PROTACs for the treatment of tumors with MET alterations

Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC₅₀ values and achieved picomolar DC₅₀ values and >99% of maximum degradation (D_max) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-MET^Y1230H and c-MET^D1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.     

Pharmacokinetics and pharmacodynamics of a novel orally active angiotensin converting enzyme inhibitor (HOE 498) in healthy subjects

Summary The pharmacokinetics and pharmacodynamics of the angiotensin converting enzyme inhibitor HOE 498 were investigated in 10 healthy normotensive male subjects. Serum levels of the active metabolite M 1 (dicarboxylic acid) of HOE 498 were measured by HPLC up to 14 days after a single oral dose of 10 m g HOE 498. Peak serum concentration of M 1 between 5–50 ng/ml was observed 1.5–3.0 h after administration. The serum concentration-time curve of M 1 was polyphasic and exhibited a prolonged terminal phase with a half-life of approximately 110 h. Despite the long terminal half-life M 1 could not be detected in urine later than 72 h after administration. The activity of the angiotensin converting enzyme in plasma was completely suppressed for up to 12 h, and 72 h after dosing 50% inhibition of the enzyme was still observed.     

Pharmacokinetics and safety of JTP-4819, a novel specific orally active prolyl endopeptidase inhibitor, in healthy male volunteers

Aims To investigate the pharmacokinetics and safety profile of JTP-4819, (-)-(2S)-1-benzylaminocarbonyl-[ (2S)-2-glycoloylpyrrolidinyl]-2-pyrrolidinecarboxamide, a novel specific orally active prolyl endopeptidase (PEP) inhibitor. Methods JTP-4819 was given orally to 28 healthy male volunteers at single doses of 30 mg (n=6), 60 mg (n=6), 120 mg (n=6) and placebo (n=3) and multiple doses of 60 mg three times daily (n=5) and placebo (n=2) for 7 days to investigate its safety and pharmacokinetics following a preliminary safety evaluation of 3, 10 and 30 mg doses in six healthy volunteers. With the single dose of 60 mg, a cross-over study was conducted to examine the effect of food on the bioavailability of the drug. The concentrations of JTP-4819 in plasma and urine were determined by electrospray ionization-liquid chromatography/mass spectrometry (ESI-LC/MS) method. Results In the multiple–dose study, the cholinesterase activity was gradually increased and reached above the normal range on days 4 to 8 in all five subjects given JTP-4819 and gradually returned to normal range after completion of dosing. The elevation of plasma cholinesterase activity was considered to be an action of JTP-4819, but this remains to be verified. There were no other abnormal findings in objective symptoms and laboratory findings including blood pressure, heart rate, electrocardiogram, body temperature, haematology, blood chemistry and urinalysis. The C_max of JTP-4819 at 30, 60 and 120 mg in fasting state were 474, 887 and 1,649 ng ml⁻¹, respectively, at 1 h after administration, and the t_1/2 was about 2 h. AUC increased in proportion to the given doses. The cumulative urinary recoveries within 24 h were approximately 66%. C_max, AUC, t_1/2 and urinary recovery were not affected by food intake. In the multiple-dose study, there was no drug accumulation trend in plasma. Conclusions These results indicate that JTP-4819 has acceptable pharmacodynamic and pharmacokinetics profiles for clinical use without any serious adverse events as we verified in healthy young male volunteers.     

Construction and characterization of intestinal oligopeptide transporter PepT1-targeted polymeric micelles for enhanced intestinal absorption of poorly water-soluble agents

To overcome the main barrier of intestinal epithelium for the oral absorption of poorly water-soluble drugs and further improve their oral absorption, Gly-Sar, the substrate of the oligopeptide transporter PepT1 widely distributed in the small intestine,conjugated poly(ethylene glycol)-block-poly(D,L-lactide) (Gly-Sar-PEG-b-PLA) was designed and synthesized, and PepT1-targetedpolymeric micelles were prepared and characterized. The structure of the synthesized Gly-Sar-PEG-b-PLA was confirmed by use of TLC and ¹H-NMR. The average molecular weight measured by GPC was 5954 g/mol with PDI of 1.34. The DiI-loaded polymeric micelles from Gly-Sar-PEG-b-PLA with drug loading content of 0.076% were characterized to exhibit 40.36 nm in diameter with PDI of 0.294, and well-defined spherical shape observed by TEM. Furthermore, the PepT1-targeted polymeric micelles profoundly enhanced intestinal absorption of poorly water-soluble drug. Therefore, the designed PepT1-targeted polymeric micelles might have a promising potential for oral delivery of water-insoluble drugs.     

Discovery of novel 1,2,4-thiadiazole derivatives as potent, orally active agonists of sphingosine 1-phosphate receptor subtype 1 (S1P(1))

A novel series of 1,2,4-thiadiazole compounds was discovered as selective S1P(1) agonists. The extensive structure-activity relationship studies for these analogues were reported. Among them, 17g was identified to show high in vitro potency with reasonable free unbound fraction in plasma (F(u) > 0.5%), good brain penetration (BBR > 0.5), and desirable pharmacokinetic properties in mouse and rat. Oral administration of 1 mg/kg 17g resulted in significant peripheral lymphocytes reduction at 4 h after dose and rapid lymphocytes recovery at 24 h. 17g showed a transient lymphopenia profile in the repeated dose study in mouse. In addition, 17g also demonstrated efficacy comparable to that of FTY720 (1) in the mouse EAE model of MS.     

An orally active reversible inhibitor of cathepsin S inhibits human trans vivo delayed-type hypersensitivity

Adefovir is transported by the organic anion transporter (OAT1) and the multidrug resistant protein (MRP2, 4 and 5). We studied adefovir clearance in rat after inhibition of transporters by probenecid and in mutant transport-deficient (TR−) rats, in which MRP2 is lacking. After treatment by probenecid or placebo, pharmacokinetics of adefovir 10 mg/kg was studied via population nonlinear mixed effect modeling. The fraction of drug excreted in the urine was low. Renal clearance of adefovir was significantly lower (P < 0.05) in probenecid TR− rats (0.03 ± 0.02 l/h) than in normal control (0.09 ± 0.05 l/h), in normal probenecid (0.10 ± 0.07 l/h) and in TR− control rats (0.13 ± 0.07 l/h). In vivo in rats MRP2 mutation alone did not affect adefovir clearance suggesting that MRP2 does not play a critical role in the secretion of adefovir. Additional pharmacological inhibition of transporters decreased renal clearance, which may reflect inhibition of compensating transport mechanisms activated when MRP2 is lacking.     

Discovery of N-benzyl hydroxypyridone carboxamides as a novel and potent antiviral chemotype against human cytomegalovirus (HCMV)

Current drugs for treating human cytomegalovirus (HCMV) infections are limited by resistance and treatment-associated toxicities. In developing mechanistically novel HCMV antivirals, we discovered an N-benzyl hydroxypyridone carboxamide antiviral hit (8a) inhibiting HCMV in submicromolar range. We describe herein the structure–activity relationship (SAR) for 8a, and the characterization of potent analogs for cytotoxicity/cytostatic property, the preliminary mechanism of action, and the absorption, distribution, metabolism and excretion (ADME) properties. The SAR revealed a few pharmacophore features conferring optimal antiviral profile, including the 5-OH, the N-1 benzyl, at least one –CH₂− in the linker, and a di-halogen substituted phenyl ring in the amide moiety. In the end, we identified numerous analogs with sub-micromolar antiviral potency and good selectivity index. The preliminary mechanism of action characterization used a pUL89-C biochemical endonuclease assay, a virus entry assay, a time-of-addition assay, and a compound withdrawal assay. ADME profiling measuring aqueous solubility, plasma and liver microsomal stability, and parallel artificial membrane permeability assay (PAMPA) permeability demonstrated largely favorable drug-like properties. Together, these studies validate the N-benzyl hydroxypyridone carboxamide as a viable chemotype for potent and mechanistically distinct antivirals against HCMV.KEY WORDS: Human cytomegalovirus, N-Benzyl hydroxypyridone carboxamides, Structure–activity relationship, Mechanism of action     

D‐420720, A novel orally active sulfonamide compound dipeptidyl peptidase IV inhibitor: structure and activity relationship of arylsulfonamide to dipeptidyl peptidase IV inhibition

Diabetes mellitus is a chronic disease characteristic of poor glucose homeostasis that requires constant monitoring and adjustment of blood glucose levels by exogenous intervention. Glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic peptide (GIP) are two incretin peptide hormones secreted from the intestine to synergize insulin's function at lowering blood glucose. The effects of GLP‐1 or GIP administration are short‐lived because they are rapidly inactivated by circulating dipeptidyl peptidase IV (DPP‐IV). Therefore, DPP‐IV inhibitors have been suggested to be a new class of molecule for treating hyperglycemic conditions in diabetic patients. The recent approval of Merck's Sitagliptin (a DPP‐IV‐specific inhibitor) indicates that DPP‐IV inhibition is a good target for new therapeutic agent development. The present study was conducted to evaluate the efficacies of a series of dipeptidyl derivatives with a sulfonamide moiety as DPP‐IV inhibitors. Among these compounds, D‐420720 was a potent inhibitor (K_i=39 nM), with a selectivity of 9160‐fold over the DPP‐II isozyme and elicits a hypoglycemic effect on oral glucose tolerance test with normal male ICR mice. Drug Dev Res 69: 514–525, 2008. © 2008 Wiley‐Liss, Inc.     

Discovery of a highly selective VEGFR2 kinase inhibitor CHMFL-VEGFR2-002 as a novel anti-angiogenesis agent

Angiogenesis is an essential process in tumor growth, invasion and metastasis. VEGF receptor 2 (VEGFR2) inhibitors targeting tumor angiogenic pathway have been widely used in the clinical cancer treatment. However, most of currently used VEGFR2 kinase inhibitors are multi-target inhibitors which might result in target-associated side effects and therefore limited clinical toleration. Highly selective VEGFR inhibitors are still highly demanded from both basic research and clinical application point of view. Here we report the discovery and characterization of a novel VEGFR2 inhibitor (CHMFL-VEGFR2-002), which exhibited high selectivity among structurally closed kinases including PDGFRs, FGFRs, CSF1R, etc. CHMFL-VEGFR2-002 displayed potent inhibitory activity against VEGFR2 kinase in the biochemical assay (IC₅₀ = 66 nmol/L) and VEGFR2 autophosphorylation in cells (EC₅₀s ∼100 nmol/L) as well as potent anti-proliferation effect against VEGFR2 transformed BaF3 cells (GI₅₀ = 150 nmol/L). In addition, CHMFL-VEGFR2-002 also displayed good anti-angiogenesis efficacy in vitro and exhibited good in vivo PK (pharmacokinetics) profile with bioavailability over 49% and anti-angiogenesis efficacy in both zebrafish and mouse models without apparent toxicity. These results suggest that CHMFL-VEGFR2-002 might be a useful research tool for dissecting new functions of VEGFR2 kinase as well as a potential anti-angiogenetic agent for the cancer therapy.     

Rational design and synthesis of an orally active indolopyridone as a novel conformationally constrained cannabinoid ligand possessing antiinflammatory properties

A series of unique indazoles and pyridoindolones have been rationally designed and synthesized as novel classes of cannabinoid ligands based on a proposed bioactive amide conformation. This has led to the discovery of the novel indolopyridone 3a as a conformationally constrained cannabinoid ligand that displays high affinity for the CB2 receptor (K(i)(CB2) = 1.0 nM) and possesses antiinflammatory properties when administered orally in an in vivo murine inflammation model.     

Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel

Aim:

Ca²⁺-release-activated Ca²⁺ (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors.

Methods:

HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca²⁺ influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP.

Results:

A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl)urea inhibited Ca²⁺ influx with IC₅₀ of 3.25±0.17 μmol/L. SAR study on its derivatives showed that the alkyl substituent on the α-position of the left-side benzylic amine (R1) was essential for Ca²⁺ influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N–H of urea was not necessary to maintain the high potency of Ca²⁺ influx inhibition. The N,N′-disubstituted or N′-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity.

Conclusion:

1-Phenyl-3-(1-phenylethyl)urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a new chemical scaffold for design and development of CRAC channel inhibitors with improved Ca²⁺ influx inhibition, immune inhibition and low cytotoxicity.     

Evaluation of WO2015042088 A1 - a novel urea-based scaffold for TrkA inhibition

Tropomyosin receptor kinases (TrkA/B/C) are involved in the development and maintenance of the nervous system. TrkA is a target for chronic pain treatment due to the central position of the nerve growth factor (NGF)/TrkA pathway in nociception. Clinical evidence points toward mutated oncogenic Trk fusion proteins retaining intact kinase domains as relevant targets for cancer treatment. Merck pursues Trk inhibitors for inflammatory and neuropathic pain treatment and has previously reported type I and II selective pan-Trk inhibitors. This is the fifth filing by Merck disclosing urea-based Trk inhibitor series. This application claims nonsymmetric 1-(9H-fluoren-9-yl)urea and 1-(9H-xanthen-9-yl)urea derivatives containing a wide range of 5- and 6-membered bi- or tri-heterocyclic fragments as TrkA inhibitors for the treatment of Trk-related conditions. The exemplified compounds display IC₅₀ values ranging from 27 to 4800 nM against TrkA. The TrkA inhibitors claimed confirm the emergence of nonsymmetric ureas lacking the hinge-binding motif as a favored Trk inhibitor structure. The compounds exemplified will likely be structurally optimized in the future. Despite the lack of selectivity profiling, the progression of the Trk inhibitor scaffold exploration by Merck also suggests that the compounds disclosed in this patent likely constitute non–adenosine triphosphate (ATP) competitive type III pan-Trk inhibitors.