Novel 5- and 6-subtituted benzothiazoles with improved physicochemical properties: potent S1P1 agonists with in vivo lymphocyte-depleting activity.

An SAR campaign designed to increase polarity in the 'tail' region of benzothiazole 1 resulted in two series of structurally novel 5-and 6-substituted S1P(1) agonists. Structural optimization for potency ultimately delivered carboxamide (+)-11f, which in addition to possessing improved physicochemical properties relative to starting benzothiazole 1, also displayed good S1P(3) selectivity and acceptable in vivo lymphocyte-depleting activity.

Benzodiazepine binding site occupancy by the novel GABAA receptor subtype-selective drug 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) in rats, primates, and humans.

The GABA(A) receptor alpha2/alpha3 subtype-selective compound 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023; also known as MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors and has partial agonist efficacy at the alpha2 and alpha3 but not the alpha1 or alpha5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo [(3)H]flumazenil binding or [(11)C]flumazenil small-animal positron emission tomography (microPET) in rats, [(123)I]iomazenil gamma-scintigraphy in rhesus monkeys, and [(11)C]flumazenil PET in baboons and humans. For each study, plasma-occupancy curves were derived, and the plasma concentration of TPA023 required to produce 50% occupancy (EC(50)) was calculated. The EC(50) values for rats, rhesus monkeys, and baboons were all similar and ranged from 19 to 30 ng/ml, although in humans, the EC(50) was slightly lower at 9 ng/ml. In humans, a single 2-mg dose of TPA023 produced in the region of 50 to 60% occupancy in the absence of overt sedative-like effects. Considering that nonselective full agonists are associated with sedation at occupancies of less than 30%, these data emphasize the relatively nonsedating nature of TPA023.

Phase I and Biomarker Study of the Wnt Pathway Modulator DKN-01 in Combination with Gemcitabine/Cisplatin in Advanced Biliary Tract Cancer.

PURPOSE: Dickkopf-1 (DKK1) modulates Wnt signaling, promoting tumor growth, metastasis, and immunosuppression. High DKK1 expression has been detected in various tumor types-including biliary tract cancer (BTC)-and is associated with poor prognosis. DKN-01-a humanized mAb targeting DKK1-was evaluated in a phase I multicenter study in combination with gemcitabine and cisplatin in patients with unresectable or metastatic BTC with no prior systemic therapy for advanced disease. PATIENTS AND METHODS: This study included a dose-escalation phase assessing DKN-01 at two dose levels (150 mg and 300 mg) combined with gemcitabine (1,000 mg/m2) and cisplatin (25 mg/m2) followed by dose expansion. Primary endpoints evaluated safety and tolerability; secondary endpoints evaluated efficacy, pharmacokinetics, and circulating biomarkers. RESULTS: Fifty-one patients with intrahepatic cholangiocarcinoma (63%), extrahepatic cholangiocarcinoma (8%), and gallbladder cancer (29%) were enrolled. No dose-limiting toxicities were seen, and the expansion phase proceeded with DKN-01 300 mg (N = 47). The most frequent grade 3/4 treatment-emergent adverse events included neutropenia (60%), thrombocytopenia (34%), and anemia (23%). The objective response rate was 21.3% and median progression-free survival was 8.7 months (95% confidence interval, 5.4-10.3 months). Better outcomes were associated with biomarkers of angiogenesis inhibition (increased sVEGFR1 and lower VEGF-C) and reduced inflammation (lower IL6 and decreased TNFα). CONCLUSIONS: DKN-01 300 mg was well tolerated in this combination but did not appear to have additional activity beyond historically reported efficacy with gemcitabine/cisplatin alone. Exploratory pharmacokinetic and biomarker data indicate potential antiangiogenic and immunomodulatory activity of DKN-01/chemotherapy and the need for increased dose/intensity. A study with DKN-01 600 mg in combination with a PD-1 inhibitor in BTC is ongoing.

Evaluation of a series of naphthamides as potent, orally active vascular endothelial growth factor receptor-2 tyrosine kinase inhibitors.

We have previously shown N-arylnaphthamides can be potent inhibitors of vascular endothelial growth factor receptors (VEGFRs). N-Alkyl and N-unsubstituted naphthamides were prepared and found to yield nanomolar inhibitors of VEGFR-2 (KDR) with an improved selectivity profile against a panel of tyrosine and serine/threonine kinases. The inhibitory activity of this series was retained at the cellular level. Naphthamides 3, 20, and 22 exhibited good pharmacokinetics following oral dosing and showed potent inhibition of VEGF-induced angiogenesis in the rat corneal model. Once-daily oral administration of 22 for 14 days led to 85% inhibition of established HT29 colon cancer and Calu-6 lung cancer xenografts at doses of 10 and 20 mg/kg, respectively.

Naphthamides as novel and potent vascular endothelial growth factor receptor tyrosine kinase inhibitors: design, synthesis, and evaluation.

A series of naphthyl-based compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptors. Investigations of structure-activity relationships led to the identification of a series of naphthamides that are potent inhibitors of the VEGF receptor tyrosine kinase family. Numerous analogues demonstrated low nanomolar inhibition of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, and of these several compounds possessed favorable pharmacokinetic (PK) profiles. In particular, compound 48 demonstrated significant antitumor efficacy against established HT29 human colon adenocarcinoma xenografts implanted in athymic mice. A full account of the preparation, structure-activity relationships, pharmacokinetic properties, and pharmacology of analogues within this series is presented.

Structural characterization of novel adenine dinucleotide phosphate conjugates of imatinib in incubations with rat and human liver microsomes.

Imatinib, a potent and selective protein tyrosine kinase inhibitor, has been approved for the treatment of chronic myelogenous leukemia and metastatic and unresectable malignant gastrointestinal stromal tumors. In vitro metabolism of imatinib was investigated in rat and human liver microsomes. Besides several oxidative metabolites and an N-desmethyl metabolite, as previous reported, a novel metabolite with a mass addition of 621 atomic mass units to the parent was detected as the major metabolite in the incubations with rat liver microsomes, using NADPH as a cofactor. The analysis of MS(2) and MS(n) data revealed that this metabolite corresponded to adenine dinucleotide phosphate (ADP+) conjugate of imatinib. The ADP+ adduct was scaled up from rat liver microsomal incubations and isolated for NMR analysis. NMR data confirmed and conclusively showed the conjugation had occurred between the pyridine nitrogen of imatinib to the ribose ring of ADP+ moiety. The formation of this adduct was enzymatic and required NADP+ as a reactant. In addition, ADP+ adducts of imatinib N-oxide and desmethyl imatinib were also detected as minor metabolites in the incubations with rat liver microsomes. In contrast, only trace levels of ADP+ adducts of imatinib and desmethyl imatinib were detected in the incubations with human liver microsomes. Imatinib-ADP+ adducts have been observed only in in vitro studies to date. The physiological role of these adducts is not clear, nor is their in vivo relevance.

Discovery of amido-benzisoxazoles as potent c-Kit inhibitors.

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

Discovery of a potent and selective c-Kit inhibitor for the treatment of inflammatory diseases.

A potent and selective c-Kit inhibitor 20 was identified through a structure-activity relationship study. In an in vivo mouse model of mast cell activation, 20 blocked the SCF-induced histamine release with an EC(50) of 26 nM.

Benzofuran Derivatives as Potent, Orally Active S1P1 Receptor Agonists: A Preclinical Lead Molecule for MS.

We have discovered novel benzofuran-based S1P1 agonists with excellent in vitro potency and selectivity. 1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl) azetidine-3-carboxylic acid (18) is a potent S1P1 agonist with >1000× selectivity over S1P3. It demonstrated a good in vitro ADME profile and excellent oral bioavailability across species. Dosed orally at 0.3 mg/kg, 18 significantly reduced blood lymphocyte counts 24 h postdose and demonstrated efficacy in a mouse EAE model of relapsing MS.

Discovery and optimization of a series of benzothiazole phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitors.

Phosphoinositide 3-kinase α (PI3Kα) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3Kα has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.

Discovery of a Potent, S1P3-Sparing Benzothiazole Agonist of Sphingosine-1-Phosphate Receptor 1 (S1P1).

Optimization of a benzofuranyl S1P1 agonist lead compound (3) led to the discovery of 1-(3-fluoro-4-(5-(2-fluorobenzyl)benzo[d]thiazol-2-yl)benzyl)azetidine-3-carboxylic acid (14), a potent S1P1 agonist with minimal activity at S1P3. Dosed orally at 0.3 mg/kg, 14 significantly reduced blood lymphocyte counts 24 h postdose and attenuated a delayed type hypersensitivity (DTH) response to antigen challenge.

Discovery of AMG 369, a Thiazolo[5,4-b]pyridine Agonist of S1P1 and S1P5.

The optimization of a series of thiazolopyridine S1P1 agonists with limited activity at the S1P3 receptor is reported. These efforts resulted in the discovery of 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5,4-b]pyridin-2-yl)benzyl)azetidine-3-carboxylic acid (5d, AMG 369), a potent dual S1P1/S1P5 agonist with limited activity at S1P3 and no activity at S1P2/S1P4. Dosed orally at 0.1 mg/kg, 5d is shown to reduce blood lymphocyte counts 24 h postdose and delay the onset and reduce the severity of experimental autoimmune encephalomyelitis in rat.

Catalytic turnover of pyrene by CYP3A4: evidence that cytochrome b5 directly induces positive cooperativity.

The metabolism of pyrene to hydroxypyrene by CYP3A4 was investigated to determine the effect of cytochrome b5 (b5) on turnover kinetics. In the absence of b5, formation of hydroxypyrene in in vitro incubations showed a biphasic substrate-velocity curve where K(m1) and V(max1) were 1.3 microM and 0.5 pmol/min/pmol P450, respectively. The addition of testosterone to the incubation mixture completely abolished the second phase to yield a typical, hyperbolic curve, presumably through the disruption in the formation of a pi-pi stacked pyrene complex within the CYP3A4 active site. Finally, the addition of b5 yielded an increase hydroxypyrene formation that resulted in a sigmoidal substrate velocity curve. The V(max) was 15.7 pmol/min/pmol P450, the K(m) was 7.5 microM, and the Hill coefficient was greater than two. This demonstrated that b5 could directly induce positive cooperativity on CYP3A4 and that this biological factor needs to be carefully considered when included in in vitro P450 reactions.

Solvent effect on cDNA-expressed human sulfotransferase (SULT) activities in vitro.

Sulfation is an important reaction in the biotransformation of steroid hormones, neurotransmitters, drugs, and other xenobiotics, yet little is known about the effects of organic solvents on sulfotransferase (SULT) activities in vitro. Initial experiments found that surprisingly low levels of solvent had dramatic effects on sulfotransferase activity. Consequently, we evaluated the effects of five commonly used solvents (methanol, ethanol, acetonitrile, dimethyl sulfoxide, and dimethyl formamide) on activities of cDNA-expressed sulfotransferase isozymes 1A1 (4-nitrophenol sulfation), 1A3 (dopamine sulfation), 1E1 (ethynylestradiol sulfation), and 2A1 (dehydroepiandrosterone sulfation). In addition, 1-hydroxypyrene was used as a general fluorescent probe for all four sulfotransferase isoforms examined. When substrates were present at their respective isoform-specific Km values, methanol and ethanol (0.4%, v/v) generally had less effect than acetonitrile, dimethyl sulfoxide, and dimethyl formamide on sulfotransferase activities. Acetonitrile, a commonly used solvent in cytochrome P450 studies, inhibited SULT1A1 activities (approximately 40%) at 0.4% (v/v), but activated SULT1E1-mediated 1-hydroxypyrene sulfation approximately 2.6-fold. Assuming a two-site kinetic model, studies revealed that solvent affected Vmax1, Vmax2, and the Ki value of 1-hydroxypyrene sulfation mediated by SULT1E1. In contrast, the Km value was not affected, suggesting that solvent may potentially alter binding interactions of the second substrate molecule, but not the first. Additional experiments with expressed SULT1A1, supplemented with control protein, revealed that the inhibitory effect of solvent (0.4%, v/v) was reduced to <15% for all solvents examined. Thus, it is recommended that ethanol is used as the preferred solvent vehicle and that incubations with expressed enzyme contain >12 microg/ml total protein.

Cytochrome P450 2C8 (CYP2C8)-mediated hydroxylation of an endothelin ETA receptor antagonist in human liver microsomes.

In vitro studies were performed to identify the human cytochrome P450 enzyme(s) involved in the hydroxylation (isopropyl moiety) of a previously reported endothelin ET(A) receptor antagonist, compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-(4-methoxy-2-[(2R)-3-methoxy-2-methylpropyl])-5-(3,4-methylenedioxyphenyl)cyclopenteno(1,2-b) pyridine 6-carboxylic acid]. Several lines of evidence indicated that the reaction was mainly catalyzed by CYP2C8. Of the 10 recombinant cytochrome P450 isoforms tested, only CYP2C8 exhibited hydroxylase activity. In agreement, inhibitory antibodies selective for CYP2C8 attenuated (>95%) the hydroxylase activity in human liver microsomes, whereas antibodies and chemical inhibitors selective for other cytochrome P450 isoforms had a minor or no effect on the reaction. In addition, the formation of the hydroxy metabolite correlated well with CYP2C8-selective paclitaxel 6alpha-hydroxylation (r(2) approximately 0.92; p < 0.0001) and amodiaquine N-de-ethylation (r(2) approximately 0.91; p < 0.0001) in a bank of human liver microsomes (n = 15 organ donors). Finally, compound A hydroxylase activity conformed to Michaelis-Menten kinetics, and the K(m) (Michaelis constant) in human liver microsomes was similar to that of CYP2C8 ( approximately 10 microM). It is concluded that the hydroxylation of compound A is mainly catalyzed by CYP2C8, and thus the reaction can possibly serve as an alternative marker assay for CYP2C8 in human liver microsomes.

Heterotropic modulation of sulfotransferase 2A1 activity by celecoxib: product ratio switching of ethynylestradiol sulfation.

The major sulfated product of 17alpha-ethynylestradiol (EE) after incubations with 3'-phosphoadenosine-5'-phosphosulfate and recombinant human sulfotransferase 2A1 (SULT2A1), or liver cytosol, is the 3-O-sulfate of EE. However, when celecoxib is also present in the incubation, sulfation is switched (in a concentration-dependent manner) from the 3-O-position to the 17beta-O-position of ethynylestradiol. In incubations with recombinant SULT2A1, increasing concentrations of celecoxib decreased the Vmax of 3-O-sulfate product formation by 3- to 4-fold, with no major change in the Km value. For 17beta-O-sulfate formation, increasing concentrations of celecoxib resulted in an 8-fold decrease in the Km and a 7-fold increase in Vmax. Celecoxib not only modulated the regioselectivity of the enzyme, but also activated the enzyme such that total sulfated product exceeded product formation by the native enzyme, 3- to 4-fold (at 250 microM celecoxib). Finally, IC50 values obtained by varying celecoxib concentrations (0-250 microM) at fixed concentrations of EE showed that 3-O-sulfation was inhibited by celecoxib to the same extent, independent of the concentration of EE. In addition, the apparent kinetic constant for celecoxib (as measured by EE 17beta-O-sulfation) decreased 2-fold in the presence of high concentrations of EE, consistent with the potential for celecoxib to bind to either the enzyme-EE complex or to free enzyme. Taken as a whole, these data suggest that celecoxib is acting as a heterotropic modulator of SULT2A1 activity, most likely involving a separate noncompetitive binding site.

Pyrene.pyrene complexes at the active site of cytochrome P450 3A4: evidence for a multiple substrate binding site.

Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates.

Sulfotransferase 1E1 is a low km isoform mediating the 3-O-sulfation of ethinyl estradiol.

Sulfation of ethinyl estradiol (EE) is a major pathway of first pass metabolism in both the intestine and liver. Consequently, we sought to identify the human sulfotransferases (SULTs) involved in the 3-O-sulfation of EE (EE-SULT). Based on the results described herein, cDNA-expressed human cytosolic SULT1A3 and SULT1E1 were identified as low Km isoforms (18.9 and 6.7 nM, respectively) mediating the sulfation of EE. In contrast, the EE-SULT catalyzed by other recombinant SULTs (SULT1A1 and 2A1) was a relatively high Km process (Km > or = 230 nM). The kinetics of EE-SULT in human intestine (Km1 = 24 nM; Km2 = 1206 nM) and liver (Km1 = 8 nM; Km2 = 2407 nM) cytosol was biphasic and conformed to a two-Km model with both low and high Km components. At a low EE concentration (3 nM), inhibition of EE-SULT activity (intestinal) was characterized with 2,6-dichloro p-nitrophenol (DCNP) (IC50 = 15.6 microM) and quercetin (IC50 = 0.4 microM). When these IC50 values were compared with those derived from expressed enzyme, inhibition of EE-SULT was consistent with the SULT1E1 (DCNP, IC50 = 20 microM; quercetin, IC50 = 0.6 microM), but not SULT1A3 (DCNP, IC50 = 12.4; quercetin, IC50 = 7 microM). Moreover, when estrone (which selectively inhibits expressed SULT1E1 and SULT1A3) was included in intestinal incubations, the high-affinity component of the Eadie-Hofstee plot for EE sulfation was inhibited, converting the plot from biphasic to monophasic. Collectively, these data are consistent with SULT1E1 as the primary sulfotransferase involved in EE sulfation at clinically relevant concentrations (<10 nM).

Mechanism-based inhibition of human liver microsomal cytochrome P450 1A2 by zileuton, a 5-lipoxygenase inhibitor.

Zileuton, a 5-lipoxygenase inhibitor, was evaluated as an inhibitor of cytochrome P450 activity in human liver microsomes. In the absence of preincubation, the racemate was found to be a weak inhibitor (IC50 > 100 microM) of phenacetin O-deethylation (POD) (CYP1A2), paclitaxel 6alpha-hydroxylation (CYP2C8), diclofenac 4'-hydroxylation (CYP2C9), (S)-mephenytoin 4'-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), testosterone 6beta-hydroxylation (CYP3A4), chlorzoxazone 6-hydroxylation (CYP2E1), and bupropion hydroxylation (CYP2B6). When preincubated with NADPH-fortified human liver microsomes in the absence of substrate, zileuton (racemate) was shown to inhibit POD. The effect was NADPH-, time-, and concentration-dependent, and was characterized by a kinact (maximal rate of enzyme inactivation) and apparent KI(inhibitor concentration that supports half the maximal rate of inactivation) of 0.035 min(-1) and 117 microM, respectively (kinact/KIratio of 0.0003 min-1 microM(-1)). Preincubation-dependent inhibition of POD activity was also observed with the individual (S)-(-)- and (R)-(+)-enantiomers of zileuton [(S)-(-)-zileuton; kinact, 0.037 min(-1), KI, 98.2 microM, kinact/KIratio, 0.0004 min(-1) microM(-1); (R)-(+)-zileuton; kinact, 0.012 min(-1), KI, 66.6 microM, kinact/KIratio, 0.0002 min(-1) microM(-1)]. In addition, the inhibition of CYP1A2 was not reversed in the presence of reduced glutathione, catalase, and superoxide dismutase and was refractory to dialysis. Therefore, zileuton was characterized as a mechanism-based inhibitor of human liver microsomal CYP1A2. Mechanism-based inhibition of CYP1A2 may explain why zileuton decreases the oral clearance of antipyrine, propranolol, (R)-warfarin, and theophylline, at doses that have a minimal effect on the pharmacokinetics of (S)-warfarin, phenytoin, and terfenadine.