Synthesis and in vivo evaluation of [O-methyl-11C] N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide as an imaging probe for 5-HT6 receptors

The serotonin receptor 6 (5-HT(6)) is implicated in the pathophysiology of cognitive diseases, schizophrenia, anxiety and obesity and in vivo studies of this receptor would be of value for studying the pathophysiology of these disorders. Therefore, N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide (SB399885), a selective and high affinity (pK(i)=9.11) 5-HT(6) antagonist, has been radiolabeled with carbon-11 by O-methylation of the corresponding desmethyl analogue with [(11)C]MeOTf in order to determine the suitability of [(11)C]SB399885 to quantify 5-HT(6)R in living brain using PET. Desmethyl-SB399885 was prepared, starting from 1-(2-methoxyphenyl) piperazine hydrochloride, in excellent yield. The yield obtained for radiolabeling of [(11)C]SB399885 was 30±5% (EOS) and the total synthesis time was 30min at EOB. PET studies with [(11)C]SB399885 in baboon showed fast uptake followed by rapid clearance in the brain. Highest uptake of radioactivity of [(11)C]SB399885 in baboon brain were found in temporal cortex, parahippocampal gyrus, pareital cortex, amygdala, and hippocampus. Poor brain entry and inconsistent brain uptake of [(11)C]SB399885 compared to known 5-HT(6)R distribution limits its usefulness for the in vivo quantification of 5-HT(6)R with PET.     

Dual neurokinin NK(1)/NK(2) antagonists: N-[(R,R)-(E)-1-arylmethyl-3-(2-oxo-azepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamides and 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-arylmethyl-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamides.

Based on the structure of N-[(R,R)-(E)-1-(4-chlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (1), attempts to improve the NK(2) affinity have resulted in the discovery of N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (9, DNK333) exhibiting a 5-fold improved affinity to the NK(2) receptor in comparison to 1. Simplification of the structure via elimination of a chiral centre led to 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-(3,4-dichlorobenzyl)-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamide (22), a potent and fairly balanced NK(1)/NK(2) antagonist.     

Design,physico-chemical properties and biological evaluation of some new N-[(phenoxy)alkyl]- and N-{2-[2-(phenoxy)ethoxy]ethyl}aminoalkanols as anticonvulsant agents

A series of thirty N-(phenoxy)alkyl or N-{2-[2-(phenoxy)ethoxy]ethyl}aminoalkanols has been designed, synthesized and evaluated for anticonvulsant activity in MES, 6 Hz test, and pilocarpine-induced status epilepticus. Among the title compounds, the most promising seems R-(−)-2N-{2-[2-(2,6-dimethylphenoxy)ethoxy]ethyl}aminopropan-1-ol hydrochloride (22a) with proved absolute configuration with X-ray analysis and enantiomeric purity. The compound is effective in MES test with ED₅₀ = 12.92 mg/kg b.w. and its rotarod TD₅₀ = 33.26 mg/kg b.w. The activity dose is also effective in a neurogenic pain model—the formalin test. Within high throughput profile assay, among eighty one targets, the strongest affinity of the compound is observed towards σ receptors and 5-HT transporter and the compound does not bind to hERG. It also does not exhibit mutagenic properties in the Vibrio harveyi test. Moreover, murine liver microsomal assay and pharmacokinetics profile (mice, iv, p.o., ip) indicate that the liver is the primary site of biotransformation of the compound, suggesting that both 22a and its metabolite(s) are active, compensating probably low bioavailability of the parent molecule.     

11C]enzastaurin, the first design and radiosynthesis of a new potential PET agent for imaging of protein kinase C

Enzastaurin (LY317615) is a potent and selective protein kinase C (PKC) inhibitor with an IC₅₀ value of ∼6 nM. [¹¹C]Enzastaurin (3-(1-[¹¹C]methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-dione), a new potential PET agent for imaging of PKC, was first designed and synthesized in 20-25% decay corrected radiochemical yield and 370-555 GBq/μmol specific activity at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled maleic anhydride intermediate followed by the conversion to maleimide.     

Efficient biocatalytic synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol by a newly isolated Trichoderma asperellum ZJPH0810 using dual cosubstrate: ethanol and glycerol

(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol is a crucial intermediate for the synthesis of Aprepitant. An efficient biocatalytic process for (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol was developed via the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone, catalyzed by whole cells of newly isolated Trichoderma asperellum ZJPH0810 using ethanol and glycerol as dual cosubstrate for cofactor recycling. A fungal strain ZJPH0810, showing asymmetric biocatalytic activity of 3,5-bis(trifluoromethyl) acetophenone to its corresponding (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol, was isolated from a soil sample. Based on its morphological and physiological characteristics and internal transcribed spacer sequence, this isolate was identified as T. asperellum ZJPH0810, which afforded an NADH-dependent (R)-stereospecific carbonyl reductase and was a promising biocatalyst for the synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. Some key reaction parameters involved in the bioreduction catalyzed by T. asperellum ZJPH0810 were subsequently optimized. The effectiveness of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol production was significantly enhanced by employing a novel dual cosubstrate-coupled system for cofactor recycling. The established efficient bioreduction system contained 50 mM of 3,5-bis(trifluoromethyl) acetophenone and 60 g l^?1 of resting cells, employing ethanol (6.0 %, v/v) and glycerol (0.5 %, v/v) as dual cosubstrate. The bioreduction was performed in distilled water medium, at 30 °C and 200 rpm. Under the above conditions, a best yield of 93.4 % was obtained, which is nearly a 3.5-fold increase in contrast to no addition of cosubstrate. The ee value of the product reached above 98 %. This biocatalytic process shows great potential in the production of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol, a valuable chiral building block in the pharmaceutical industry.     

N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea and N'-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]-thiourea as potent inhibitors of multidrug-resistant human immunodeficiency virus-1.

We have replaced the pyridyl ring of trovirdine with an alicyclic cyclohexenyl, adamantyl or cis-myrtanyl ring. Only the cyclohexenyl-containing thiourea compound N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]- thiourea (HI-346) (as well as its chlorine-substituted derivative N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]- thiourea/HI-445) showed RT inhibitory activity. HI-346 and HI-445 effectively inhibited recombinant RT with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cell-free RT inhibition assays was: HI-346 (IC50 = 0.4 microM) > HI-445 (IC50 = 0.5 microM) > trovirdine (IC50 = 0.8 microM) > MKC-442 (IC5 = 0.8 microM) = delavirdine (IC50 = 1.5 microM) > nevirapine (IC50 = 23 microM). In accord with this data, both compounds inhibited the replication of the drug-sensitive HIV-1 strain HTLV(IIIB) with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cellular HIV-1 inhibition assays was: HI-445 = HI-346 (IC50 = 3 nM) > MKC-442 (IC50 = 4 nM) = AZT (IC50 = 4 nM) > trovirdine (IC50 = 7 nM) > delavirdine (IC50 = 9 nM) > nevirapine (IC50 = 34 nM). Surprisingly, the lead compounds HI-346 and HI-445 were 3-times more effective against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V,41L, and 215Y) than they were against HTLV(IIIB) with wild-type RT. HI-346 and HI-445 were 20-times more potent than trovirdine, 200-times more potent than AZT, 300-times more potent than MKC-442, 400-times more potent than delavirdine, and 5000-times more potent than nevirapine against the multidrug resistant HIV-1 strain RT-MDR. HI-445 was also tested against the RT Y181C mutant A17 strain of HIV-1 and found to be >7-fold more effective than trovirdine and >1,400-fold more effective than nevirapine or delavirdine. Similarly, both HI-346 and HI-445 were more effective than trovirdine, nevirapine, and delavirdine against the problematic NNI-resistant HIV-1 strain A17-variant with both Y181C and K103N mutations in RT, although their activity was markedly reduced against this strain. Neither compound exhibited significant cytotoxicity at effective concentrations (CC50 >100 microM). These findings establish the lead compounds HI-346 and HI-445 as potent inhibitors of drug-sensitive as well as multidrug-resistant stains of HIV-1.     

Rapid C-carboxylation of nitro[(11)C]methane for the synthesis of ethyl nitro[2-(11)C]acetate

The labelling synthesis of ethyl nitro[2-(11)C]acetate, a synthetic intermediate feasible for (11)C-labelled PET tracers, by C-carboxylation of [(11)C]MeNO(2) with 1-ethoxycarbonylbenzotriazole, and its simple application are presented.     

Synthesis and in vitro antitubercular activity of some 1-[(4-sub)phenyl]-3-(4-{1-[(pyridine-4-carbonyl)hydrazono]ethyl}phenyl)thiourea

Various isonicotinyl hydrazones were prepared by reacting isonicotinyl hydrazide [INH] with 1-(4-acetylphenyl)-3-[(4-sub)phenyl]thiourea and were tested for their antimycobacterial activity in vitro against Mycobacterium tuberculosis H37Rv and INH-resistant M. tuberculosis using the BACTEC 460 radiometric system. Among the synthesized compounds, 1-(4-fluorophenyl)-3-(4-{1-[(pyridine-4-carbonyl)-hydrazono]ethyl}phenyl)thiourea (4d) was found to be the most potent compound with a minimum inhibitory concentration of 0.49 microM against M. tuberculosis H37Rv and INH-resistant M. tuberculosis. When compared to INH, 4d was found to be 3 and 185 times more active against M. tuberculosis H37Rv and INH-resistant M. tuberculosis, respectively, with a selectivity index of >300.     

3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-substituted-3,6-diazabicyclo[3.1.1]heptanes as novel potent dopamine uptake inhibitors

A series of analogues 2a-i related to 3-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-8-(1H-indol-2-ylmethyl)-3,8-diazabicyclo[3.2.1]octane (1) in which the 3,8-diazabicyclo[3.2.1]octane core was replaced by 3,6-diazabicyclo[3.1.1]heptane ring system has been synthesized and evaluated for their ability to inhibit DA reuptake into striatal nerve endings (synaptosomes). Biological data showed that compound 2a, the closest analogue of lead 1, possessed an increased reuptake inhibition activity over 1 (2a, K(i)=5.5 nM). Replacement of the indole ring with bioisosteric aromatic rings--benzothiophene (2b), benzofurane (2c), or indene (2d)--resulted, with the exception of 2d, in a double digit nanomolar activity. Changing the indenyl moiety of 2d with simplified aryl groups led to compounds 2e-h which displayed a similar or slightly decreased activity with respect to the ground term. Naphthalene derivative (2i) demonstrated a weaker activity than aromatic analogues.     

Identification and SAR around N-{2-[4-(2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl)-[1,4]diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide, a selective alpha2C adrenergic receptor antagonist

The discovery of the CNS-penetrant and selective alpha(2C) adrenergic receptor antagonist N-{2-[4-(2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl)-[1,4]diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide, 13 is described. Structure-activity studies demonstrate the structural requirements for binding affinity, functional activity, and selectivity over other alpha(2)-AR subtypes.     

2-[N1-2-pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate: a potent antitumor agent

2-[N'-2-Pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate has been prepared by reaction of chlorambucil with sulfadiazine derivative. Schiffs base has been used as the protective group of the aromatic amine in the synthesis. It can be completely removed by the irradiation of 365 nm UV light at room temperature. The title compound exhibits a high antitumor activity with a therapeutic index (TI) of 47.55 which is twice that of chlorambucil's (TI: 22.84).     

Identification of 1-{2-[4-chloro-1′-(2,2-dimethylpropyl)-7-hydroxy-1,2-dihydrospiro[indole-3,4′-piperidine]-1-yl]phenyl}-3-{5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl}urea,a potent,efficacious and orally bioavailable P2Y1 antagonist as an antiplatelet agent

Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y₁ receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y₁ antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y₁₂ antagonist clopidogrel in rat efficacy/bleeding models.     

Asymmetric biocatalytic reduction of 3,5-bis(trifluoromethyl) acetophenone to (<Emphasis Type="Italic">1R</Emphasis>)-[3,5-bis(trifluoromethyl)phenyl] ethanol using whole cells of newly isolated <Emphasis Type="Italic">Leifsonia xyli</Emphasis> HS0904

A novel bacterial strain HS0904 was isolated from a soil sample using 3,5-bis(trifluoromethyl) acetophenone as the sole carbon source. This bacterial isolate can asymmetrically reduce 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol with high enantiometric excess (ee) value. Based on its morphological, physiological characteristics, Biolog, 16S rDNA sequence and phylogenetic analysis, strain HS0904 was identified as Leifsonia xyli HS0904. To our knowledge, this is the first reported case on the species L. xyli exhibited R-stereospecific carbonyl reductase and used for the preparation of chiral (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. The optimization of parameters for microbial transformation of 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol catalyzed by whole cells of L. xyli HS0904 was carried out by examining some key factors including buffer pH, reaction temperature, shaking speed, substrate concentration, and reaction time. The obtained optimized conditions for the bioreduction are as follows: buffer pH 8.0, 70 mM of 3,5-bis(trifluoromethyl) acetophenone, 100 g l⁻¹ of glucose as co-substrate, 200 g l⁻¹ of resting cells as biocatalyst, reaction for 30 h at 30 °C and 200 rpm. Under above conditions, 99.4% of product ee and best yield of 62% were obtained, respectively. The results indicated that isolate L. xyli HS0904 is a novel potential biocatalyst for the production of (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol.     

Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides

Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.     

Discovery of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide as a selective T-type calcium channel (Cav3.2) inhibitor

The T-type calcium channel inhibitor Mibefradil was reported to protect the heart from atrial remodeling, a key process involved in the development of atrial fibrillation and arrhythmias. Mibefradil is not a selective T-type calcium channel inhibitor and also affects the function of different ion channels. Our aim was to develop a selective T-type calcium channel inhibitor to validate the importance of T-type-related pharmacology in atrial fibrillation. Structural optimisation of a previously disclosed hit series focussed on minimising exposure to the central nervous system and improving pharmacokinetic properties, while maintain adequate potency and selectivity. This resulted in the design of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide, a novel, selective, peripherally restricted chemical probe to verify the role of T-type calcium channel inhibition on atrial fibrillation protection.     

Synthesis, in vitro and in vivo evaluation of [O-methyl-11C] 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]-triazine-3,5-dione: a novel agonist 5-HT1A receptor PET ligand

Synthesis and in vivo evaluation of 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (5 or MMT), a high affinity and selective serotonin 5-HT1AR agonist PET tracer, are described. GTPgammaS assay shows that MMT is an agonist with an EC50 comparable to 5-HT. Radiolabeling of 5 was achieved in 30% yield (EOS) from desmethyl-MMT (4) with >99% chemical and radiochemical purities and a specific activity >1000 Ci/mmol. PET studies in baboon show that [11C]5 penetrates the blood-brain barrier but, because of low specific binding and fast clearance of radioactivity it is not a suitable PET tracer for the in vivo quantification of 5-HT1AR.     

Rationally designed PKA inhibitors for positron emission tomography: Synthesis and cerebral biodistribution of N-(2-(4-bromocinnamylamino)ethyl)-N-[11C]methyl-isoquinoline-5-sulfonamide

Protein kinase A (PKA) is an important signal transduction target for drug development because it influences critical cellular processes implicated in neuropsychiatric illnesses such as major depressive disorder. The goal of the present study was to develop the first imaging agent for measuring the levels of PKA with positron emission tomography (PET). By rational derivatization of 5-isoquinoline sulfonamides, it was found that the introduction of a methyl group to the sulphonamidic nitrogen on the known PKA inhibitors N-(2-aminoethyl)isoquinoline-5-sulfonamide (H-9, 1) and N-(2-(4-bromocinnamylamino)ethyl)isoquinoline-5-sulfonamide (H-89, 2), (yielding N-(2-aminoethyl)-N-methyl-isoquinoline-5-sulfonamide (4) and N-(2-(4-bromocinnamylamino)ethyl)-N-methyl-isoquinoline-5-sulfonamide (5), respectively) does not appreciably reduce in vitro potency toward PKA. We have facilitated the synthesis of 4 by reacting isoquinoline-5-sulfonyl chloride with N-methylethylenediamine (20% yield). Several techniques were used to thoroughly characterize 4 including multi (¹H, ¹³C and ¹⁵N) NMR spectroscopy and X-ray crystallography. Compound 4 and 1-(4-bromophenyl)-1-propen-3-yl bromide were reacted to produce 5 in 16% yield. Compound 2 was reacted with [¹¹C]CH₃I to prepare N-(2-(4-bromocinnamylamino) ethyl)-N-[¹¹C]methyl-isoquinoline-5-sulfonamide ([¹¹C]5), with a decay-corrected radiochemical yield of 32%, based on [¹¹C]CO₂. [¹¹C]5 was produced with >98% radiochemical purity and 1130 mCi/μmol specific activity after 40 min (end of synthesis). Conscious rats were administered [¹¹C] 5 and sacrificed at 5, 15, 30 and 60 min after injection. Radioactivity from all excised brain regions was <0.2%ID/g at all time points. The modest brain penetration of [¹¹C]5 may limit its use for studying PKA in the central nervous system.     

The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR

A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3, 5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.