Synthesis of (Z)-2-((1H-indazol-3-yl)methylene)-6-[11C]methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one as a new potential PET probe for imaging of the enzyme PIM1

(Z)-2-((1H-Indazol-3-yl)methylene)-6-methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one is a potent and selective proviral integration site in moloney murine leukemia virus kinase 1 (PIM1) inhibitor with an IC₅₀ value of 3 nM. (Z)-2-((1H-Indazol-3-yl)methylene)-6-[¹¹C]methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one, a new potential PET probe for imaging of the enzyme PIM1, was first designed and synthesized in 20–30% decay corrected radiochemical yield and 370–740 GBq/μmol specific activity at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled Boc-protected intermediate followed by a quick acidic de-protection.     

3-(Cyclopropylmethyl)-7-((4-(4-[11C]methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine: Synthesis and preliminary evaluation for PET imaging of metabotropic glutamate receptor subtype 2

Selective metabotropic glutamate receptor 2 (mGluR2) inhibitors have been demonstrated to show therapeutic effects by improving alleviating symptoms of schizophrenic patients in clinical studies. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography (PET) tracer originating from a mGluR2 inhibitor, 3-(cyclopropylmethyl)-7-((4-(4-methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (CMTP, 1a). [¹¹C]CMTP ([¹¹C]1a) was synthesized by O-[¹¹C]methylation of desmethyl precursor 1b with [¹¹C]methyl iodide in 19.7 ± 8.9% (n = 10) radiochemical yield (based on [¹¹C]CO₂) with >98% radiochemical purity and >74 GBq/μmol molar activity. Autoradiography study showed that [¹¹C]1a possessed moderate in vitro specific binding to mGluR2 in the rat brain, with a heterogeneous distribution of radioactive accumulation in the mGluR2-rich brain tissue sections, such as the cerebral cortex and striatum. PET study indicated that [¹¹C]1a was able to cross the blood–brain barrier and enter the brain, but had very low specific binding in the rat brain. Further optimization for the chemical structure of 1a is necessary to increase binding affinity to mGluR2 and then improve in vivo specific binding in brain.     

Synthesis and preliminary evaluation of 4-hydroxy-6-(3-[11C]methoxyphenethyl)pyridazin-3(2H)-one,a 11C-labeled d-amino acid oxidase (DAAO) inhibitor for PET imaging

Selective DAAO inhibitors have demonstrated promising therapeutic effects in clinical studies, including clinically alleviating symptoms of schizophrenic patients and ameliorating cognitive function in Alzheimer’s patients with early phase. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography ligand based on a DAAO inhibitor, DAO-1903 (8). ¹¹C-Isotopologue of 8 was prepared in high radiochemical yield with high radiochemical purity (>99%) and high molar activity (>37 GBq/µmol). In vitro autoradiography studies indicated that the ligand possessed high in vitro specific binding to DAAO, while in vivo dynamic PET studies demonstrated that [¹¹C]8 failed to cross the blood–brain barrier possibly due to moderate brain efflux mechanism. Further chemical scaffold optimization is necessary to overcome limited brain permeability and improve specific binding.     

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.     

4-[6-(2-Aminoethyl)naphthalen-2-yl]benzonitriles are potent histamine H3 receptor antagonists with high CNS penetration

4-[6-(2-Tertiaryaminoethyl)naphthalen-2-yl]benzonitriles are conformationally constrained histamine H3 receptor antagonists with high potency and selectivity. The analogs were designed around a naphthalene core, with the goal of enhancing lipophilicity and CNS penetration, as compared to a previously reported benzofuran series. The SAR of the tertiary amine moiety is similar to that reported for the benzofuran series, with analogs bearing a 2-methylpyrrolidine substituent possessing the greatest rat and human H3 receptor binding affinities.     

1-((3S,4S)-4-amino-1-(4-substituted-1,3,5-triazin-2-yl) pyrrolidin-3-yl)-5,5-difluoropiperidin-2-one inhibitors of DPP-4 for the treatment of type 2 diabetes

A 3-amino-4-substituted pyrrolidine series of dipeptidyl peptidase IV (DPP-4) inhibitors was rapidly developed into a candidate series by identification of a polar valerolactam replacement for the lipophilic 2,4,5-trifluorophenyl pharmacophore. The addition of a gem-difluoro substituent to the lactam improved overall DPP-4 inhibition and an efficient asymmetric route to 3,4-diaminopyrrolidines was developed. Advanced profiling of a subset of analogs identified 5o with an acceptable human DPP-4 inhibition profile based on a rat PK/PD model and a projected human dose that was suitable for clinical development.     

Synthesis and initial PET imaging of new potential dopamine D3 receptor radioligands (E)-4,3,2-[11C]methoxy-N-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides

D3 receptor radioligands (E)-4,3,2-[11C]methoxy-N-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides (4-[11C]MMC, [11C]1a; 3-[11C]MMC, [11C]1b; and 2-[11C]MMC, [11C]1c) were synthesized for evaluation as novel potential positron emission tomography (PET) imaging agents for brain D3 receptors. The new tracers 4,3,2-[11C]MMCs were prepared by O-[11C]methylation of corresponding precursors (E)-4,3,2-hydroxy-N-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides (4,3,2-HMCs) using [11C]methyl triflate and isolated by the solid-phase extraction (SPE) purification procedure with 40-65% radiochemical yields, decay corrected to end of bombardment (EOB), and a synthesis time of 15-20 min. The PET dynamic studies of the tracers [11C]1a-c in rats were performed using an animal PET scanner, IndyPET-II, developed in our laboratory. The results show that the brain uptake sequence was 4-[11C]MMC > 3-[11C]MMC > 2-[11C]MMC, which is consistent with their in vitro biological properties. The initial PET blocking studies of the tracers 4,3,2-[11C]MMCs with corresponding pretreatment drugs (E)-4,3,2-methoxy-N-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides (4,3,2-MMCs, 1a-c) had no effect on 4,3,2-[11C]MMCs-PET rat brain imaging. These results suggest that the localization of 4,3,2-[11C]MMCs in rat brain is mediated by nonspecific processes, and the visualization of 4,3,2-[11C]MMCs-PET in rat brain is related to nonspecific binding.     

Diaryldimethylpiperazine ligands with mu- and delta-opioid receptor affinity: Synthesis of (+)-4-[(alphaR)-alpha-(4-allyl-(2S,5S)-dimethylpiperazin-1-yl)-(3-hydroxyphenyl)methyl]-N-ethyl-N-phenylbenzamide and (-)-4-[(alphaR)-alpha-(2S,5S)-dimethylpiperazin-1-yl)-(3-hydroxyphenyl)methyl]-N-ethyl-N-phenylbenzamide

We have explored the synthesis of compounds that have good affinity for both mu- and delta-opioid receptors from the (alphaR,2S,5S) class of diaryldimethylpiperazines. These non-selective compounds were related to opioids that have been found to interact selectively with mu- or delta-opioid receptors as agonists or antagonists. In our initial survey, we found two compounds, (+)-4-[(alphaR)-alpha-(4-allyl-(2S,5S)-dimethylpiperazin-1-yl)-(3-hydroxyphenyl)methyl]-N-ethyl-N-phenylbenzamide (14) and its N-H relative, (-)-4-[(alphaR)-alpha-(2S,5S)-dimethylpiperazin-1-yl)-(3-hydroxyphenyl)methyl]-N-ethyl-N-phenylbenzamide (15), that interacted with delta-receptors with good affinity, and, as we hoped, with much higher affinity at mu-receptors than SNC80. The relative configuration of the benzylic position in (+)-4-[(alphaR)-alpha-(4-allyl-(2S,5S)-dimethyl-1-piperazinyl)-(3-methoxyphenyl)methyl]-benzyl alcohol (10) was determined by X-ray crystallographic analysis of a crystal that was an unresolved twin. The absolute stereochemistry of that benzylic stereogenic center was unequivocally derived by the X-ray crystallographic analysis from the two other centers of asymmetry in the molecule that were known. Those were established from the synthesis via a dipeptide cyclo-L-Ala-L-Ala in which the absolute stereochemistry was established.     

Closing in on the AMPA receptor: synthesis and evaluation of 2-acetyl-1-(4'-chlorophenyl)-6-methoxy-7-[11C]methoxy-1,2,3,4-tetrahydroisoquinoline as a potential PET tracer

2-Acetyl-1-(4'-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, one of the most potent non-competitive AMPA antagonists described to date, has been labelled with carbon-11 and tritium and evaluated as a potential ligand for in vivo imaging of AMPA receptors using PET. The carbon-11 labelled compound showed good initial brain uptake in rats, but with rapid clearance and relatively homogenous distribution. In saturation binding studies, the tritiated racemic ligand was found to be highly potent with a Kd of 14.8+/-1.8 nM. We conclude that the low receptor density labelled with this compound, its rapid clearance from the CNS and low specific binding makes it unsuitable as an in vivo PET imaging agent for AMPA receptors.     

Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ～330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure–activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.     

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.     

Anticancer potential of 3-(arylideneamino)-2-phenylquinazoline-4(3H)-one derivatives

Different quinazoline derivatives have showed wide spectrum of pharmacological activities. Some 3-(arylideneamino)-phenylquinazoline-4(3H)-ones have been reported to possess antimicrobial activity. The present study has been undertaken to evaluate the anticancer effect of these quinazolinone derivatives. The quinazolinone derivatives were synthesized as reported earlier. Compounds containing NO(2), OH, OCH(3), or OH and OCH(3) as substituent(s) on the arylideneamino group were named as P(3a), P(3b), P(3c), and P(3d) respectively. Out of these, P(3a) and P(3d) showed better cytotoxic activity than P(3b) and P(3c) on a panel of six cancer cell lines of different origin, namely, B16F10, MiaPaCa-2, HCT116, HeLa, MCF7, and HepG2, though the effect was higher in B16F10, HCT116, and MCF7 cells. P(3a) and P(3d) induced death of B16F10 and HCT116 cells was associated with characteristic apoptotic changes like cell shrinkage, nuclear condensation, DNA fragmentation, and annexin V binding. Also, cell cycle arrest at G1 phase, alteration of caspase-3, caspase-9, Bcl-2 and PARP levels, loss of mitochondrial membrane potential, and enhanced level of cytosolic cytochrome c were observed in treated B16F10 cells. Treatment with multiple doses of P(3a) significantly increased the survival rate of B16F10 tumor bearing BALB/c mice by suppressing the volume of tumor while decreasing microvascular density and mitotic index of the tumor cells.     

Synthesis and biological evaluation of the metabolites of 2-(1-{3-[(6-chloronaphthalen-2-yl)sulfonyl]propanoyl}piperidin-4-yl)-5-methyl-1,2-dihydro-3H-imidazo[1,5-c]imidazol-3-one

We have recently discovered imidazo[1,5-c]imidazol-3-one derivative 1 as a potent, selective, and orally bioavailable factor Xa (FXa) inhibitor. In this study, we have synthesized metabolites of 1 and evaluated their biological activities. As a result, we identified the active metabolites S-5 and 6 with a potent FXa inhibitory activity comparable to 1 and a favorable pharmacokinetic profile in monkeys.     

Radiosynthesis and evaluation of [11C]EMPA as a potential PET tracer for orexin 2 receptors

EMPA is a selective antagonist of orexin 2 (OX₂) receptors. Previous literature with [³H]-EMPA suggest that it may be used as an imaging agent for OX₂ receptors; however, brain penetration is known to be modest. To evaluate the potential of EMPA as a PET radiotracer in non-human primate (as a step to imaging in man), we radiolabeled EMPA with carbon-11. Radiosynthesis of [¹¹C]N-ethyl-2-(N-(6-methoxypyridin-3-yl)-2-methylphenylsulfonamido)-N-(pyridin-3-ylmethyl)acetamide ([¹¹C]EMPA), and evaluation as a potential PET tracer for OX₂ receptors is described. Synthesis of an appropriate non-radioactive O-desmethyl precursor was achieved from EMPA with sodium iodide and chlorotrimethylsilane. Selective O-methylation using [¹¹C]CH₃I in the presence of cesium carbonate in DMSO at room temp afforded [¹¹C]EMPA in 1.5–2.5% yield (non-decay corrected relative to trapped [¹¹C]CH₃I at EOS) with ?95% chemical and radiochemical purities. The total synthesis time was 34–36 min from EOB. Studies in rodent suggested that uptake in tissue was dominated by nonspecific binding. However, [¹¹C]EMPA also showed poor uptake in both rats and baboon as measured with PET imaging.     

Synthesis and biological evaluation of novel 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of novel S-DABO analogues of 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were compounds 6c1,6c6, and 6b1 (EC(50)=0.24 ± 0.05, 0.38 ± 0.13, 0.39 ± 0.05 μM, respectively), which possess improved or similar HIV-1 inhibitory activity compared with nevirapine (NVP) (EC(50)=0.21 μM) and delavirdine (DLV) (EC(50)=0.32 μM). None of these compounds were active against HIV-2 replication. Furthermore, enzyme inhibitory assays were performed with selected derivatives against HIV-1 wtRT, confirming that the main target of these compounds is the HIV-1 RT and these new S-DABOs are acting as NNRTIs. The preliminary structure-activity relationship (SAR) of these new congeners is discussed briefly and rationalized by docking studies.     

Discovery of (R)-5-(benzo[d][1,3]dioxol-5-yl)-7-((1-(vinylsulfonyl)pyrrolidin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (B6) as a potent Bmx inhibitor for the treatment of NSCLC

Described as a Btk inhibitor, ibrutinib also potently inhibits Bmx and EGFR, two good targets for lung cancer. Owing to its high CLogP (4.07) and low aqueous solubility (<0.01 mg/ml), resulting in unfavorable bioavailability, ibrutinib requires high dosages to achieve good clinical response in the treatment of non-small cell lung cancer (NSCLC). In our effort to improve the CLogP of ibrutinib by structural optimization led to the discovery of a potent anti-cancer agent B6, with beneficial physicochemical parameters (CLogP = 2.56, solubility in water ≈ 0.1 mg/ml) meeting the principles of oral drugs. B6 exhibited anti-proliferation activities against EGFR-expressing cells, especially the mutant ones, such as H1975 (L858R/T790M, IC₅₀ = 0.92 ± 0.19 μM) and HCC827 (Del119 IC₅₀ = 0.014 ± 0.01 μM). Moreover, B6 significantly slowed down H1975 tumor growth with anti-tumor rate of 73.9% (p < 0.01). Enzyme potencies assay demonstrated B6 moderately selectively inhibited Bmx (IC₅₀ = 35.7 ± 0.1 nM) over other kinases. So, as a potent Bmx inhibitor, B6 has the potential to be an efficacious treatment for NSCLC with acquired drug resistance.     

Design,synthesis and evaluation of 6-(4-((substituted-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)phenanthridine analogues as antimycobacterial agents

Focus in this Letter is made to design and synthesize a series of nineteen new 6-(4-((substituted-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)phenanthridine analogues employing click chemistry and evaluated for their anti-tubercular activity against Mycobacterium tuberculosis H₃₇Rv. Among the tested compounds, 7f and 7j exhibited good activity (MIC = 3.125 μg/mL), while 8a displayed excellent activity (MIC = 1.56 μg/mL) against the growth of M. tuberculosis H₃₇Rv. In addition, 7f, 7j and 8a compounds were subjected to cytotoxic studies against mouse macrophage (RAW264.7) cell lines and the selectivity index values are >15 indicating suitability of compounds for further drug development.     

Discovery of ((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone, PF-4254196, a CCR2 antagonist with an improved cardiovascular profile

We describe the systematic optimization, focused on the improvement of CV-TI, of a series of CCR2 antagonists. This work resulted in the identification of 10 (((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone) which possessed a low projected human dose 35-45 mg BID and a CV-TI = 3800-fold.     

Discovery of (1R,6S)-5-[4-(1-cyclobutyl-piperidin-4-yloxy)-phenyl]-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one (R,S-4a): Histamine H3 receptor inverse agonist demonstrating potent cognitive enhancing and wake promoting activity

A series of fused cyclopropyl-4,5-dihydropyridazin-3-one (3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one) phenoxypiperidine analogs was designed and synthesized, leading to the identification of (1R,6S)-5-[4-(1-cyclobutyl-piperidin-4-yloxy)-phenyl]-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one (R,S-4a) as a second-generation pyridazin-3-one H₃R antagonist. Compound R,S-4a was a potent H₃R functional antagonist in vivo in the rat dipsogenia model, demonstrated potent wake activity in the rat EEG/EMG model, and enhanced short-term memory in the rat social recognition memory model at doses as low as 0.03–0.3 mg/kg po.     

Optimization of the aminopyridopyrazinones class of PDE5 inhibitors: Discovery of 3-[(trans-4-hydroxycyclohexyl)amino]-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one

We describe efforts to improve the pharmacokinetic profile of the aminopyridopyrazinone class of PDE5 inhibitors. These efforts led to the discovery of 3-[(trans-4-hydroxycyclohexyl)amino]-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one, a potent and selective inhibitor of PDE5 with an excellent PK profile.