Synthesis of ethyleneoxide modified 3-carboranyl thymidine analogues and evaluation of their biochemical, physicochemical, and structural properties

Eleven 3-carboranyl thymidine analogues (3CTAs) containing highly hydrophilic and flexible ethyleneoxide moieties were synthesized as potential agents for boron neutron capture therapy (BNCT) and their biochemical and physicochemical properties were evaluated. Based on specific structural features, this library of 3CTAs was divided into three subgroups. The first group contained 3CTAs with 1-4 ethyleneoxide units between the thymidine (Thd) scaffold and a carborane cluster. The second group of 3CTAs contained a pentylene spacer between Thd and the carborane and 2-4 ethyleneoxide units additionally attached to the carborane cluster. The third group contained three 3CTAs all with pentylene spacers and four ethylene units but with different carborane cages. The ethyleneoxide modified 3CTAs were good substrates of thymidine kinase 1 (TK1) and poor substrates of human mitochondrial thymidine kinase 2 (TK2) as determined in phosphoryl transfer assays. In the first group of 3CTAs, all the compounds were efficiently phosphorylated regardless of varying spacer lengths (37-42% of the activity of Thd). The second group of 3CTAs was less effectively phosphorylated (17-26% of the activity of Thd) probably due to a less favorable sterical orientation of Thd within the active site of TK1 and/or an increased lipophilicity compared with the first group. In the third group of structural isomers, no significant differences in phosphorylation rates were observed (17-25%). A structure-function hypothesis explaining these results is presented.     

Binding of ATP to TK1-like enzymes is associated with a conformational change in the quaternary structure

Human thymidine kinase 1 (hTK1) and structurally related TKs from other organisms catalyze the initial phosphorylation step in the thymidine salvage pathway. Though ATP is known to be the preferred phosphoryl donor for TK1-like enzymes, its exact binding mode and effect on the oligomeric state has not been analyzed. Here we report the structures of hTK1 and of the Thermotoga maritima thymidine kinase (TmTK) in complex with the bisubstrate inhibitor TP4A. The TmTK-TP4A structure reveals that the adenosine moiety of ATP binds at the subunit interface of the homotetrameric enzyme and that the majority of the ATP-enzyme interactions occur between the phosphate groups and the P-loop. In the hTK1 structure the adenosine group of TP4A exhibited no electron density. This difference between hTK1 and TmTK is rationalized by a difference in the conformation of their quaternary structure. A more open conformation, as seen in the TmTK-TP4A complex structure, is required to provide space for the adenosine moiety. Our analysis supports the formation of an analogous open conformation in hTK1 upon ATP binding.     

3′-(1,2,3-Triazol-1-yl)-3′-deoxythymidine analogs as substrates for human and Ureaplasma parvum thymidine kinase for structure–activity investigations

The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up thymidine kinase (UpTK) provides the necessary thymidine nucleotides. The anti-HIV compound 3?-azido-3′-deoxythymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3′-α-[1,2,3]triazol-3′-deoxythymidine analogs from AZT under Huisgen conditions are described. Thirteen 3′-analogues were tested with human cytosolic thymidine kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (K_m/V_max values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides.     

Biochemical and structural characterization of (South)-methanocarbathymidine that specifically inhibits growth of herpes simplex virus type 1 thymidine kinase-transduced osteosarcoma cells

Two analogs of the natural nucleoside dT featuring a pseudosugar with fixed conformation in place of the deoxyribosyl residue (carbathymidine analogs) were biochemically and structurally characterized for their acceptance by both human cytosolic thymidine kinase isoenzyme 1 (hTK1) and herpes simplex virus type 1 thymidine kinase (HSV1 TK) and subsequently tested in cell proliferation assays. 3'-exo-Methanocarbathymidine ((South)-methanocarbathymidine (S)-MCT), which is a substrate for HSV1 TK, specifically inhibited growth of HSV1 TK-transduced human osteosarcoma cells with an IC(50) value in the range of 15 microM without significant toxicity toward both hTK1-negative (TK(-)) and non-transduced cells. 2'-exo-Methanocarbathymidine ((North)-methanocarbathymidine (N)-MCT), which is a weak substrate for hTK1 and a substantial one for HSV1 TK, induced a specific growth inhibition in HSV1 TK-transfected cells comparable to that of (S)-MCT and ganciclovir. A growth inhibition activity was also observed with (N)-MCT and ganciclovir in non-transduced cells in a cell line-dependent manner, whereas TK(-) cells were not affected. The presented 1.95-A crystal structure of the complex (S)-MCT.HSV1 TK explains both the more favorable binding affinity and catalytic turnover of (S)-MCT for HSV1 TK over the North analog. Additionally the plasticity of the active site of the enzyme is addressed by comparing the binding of (North)- and (South)-carbathymidine analogs. The presented study of these two potent candidate prodrugs for HSV1 TK gene-directed enzyme prodrug therapy suggests that (S)-MCT may be even safer to use than its North counterpart (N)-MCT.     

胸苷激酶基因在人二倍体成纤维细胞衰老过程中的表达调控

为研究人胸苷激酶 (humanthymidinekinase ,hTK)基因在复制衰老细胞及早衰细胞中表达下调的分子机制 ,构建了含hTK启动子的荧光素酶报告基因载体 .转染结果显示 ,复制衰老细胞与早衰细胞中hTK启动子的转录活性比年轻细胞中下降了近 3倍 ,表明转录水平的调控是hTK在衰老细胞中表达下降的主要调控机制 .定点突变的结果显示 ,转录因子Sp1、NF Y结合位点的突变可使hTK启动子活性降低近 5 0 % ,而E2F结合位点的突变可使其活性升高 2倍多 ,提示Sp1和NF Y是hTK基因的转录活化因子 ,而E2F为转录抑制因子 .电泳迁移率变更实验发现 ,与年轻细胞相比 ,Sp1、NF Y与hTK启动子的DNA结合活性在复制衰老细胞和早衰细胞中无明显改变 ,提示转录活化因子Sp1、NF Y并非hTK在衰老细胞中下调的主要因素 .染色质免疫共沉淀结果显示 ,在细胞内Rb结合在hTK启动子上 ,且同年轻细胞相比 ,复制衰老细胞及早衰细胞中的hTK启动子结合着更多的Rb ,这提示细胞衰老过程中Rb的去磷酸化可能与hTK基因在衰老过程中的下调有关 .     

Antiviral activity of the 3''-amino derivative of (E)-5-(2-bromovinyl)-2''-deoxyuridine.

3'-NH2-BV-dUrd, the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3'-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3'-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5'-triphosphate of 3'-NH2-BV-dUrd (3'-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3'-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3'-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5'-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3'-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.     

Pyrrolidin-3-yl-N-methylbenzamides as potent histamine 3 receptor antagonists

On the basis of the previously reported benzimidazole 1,3'-bipyrrolidine benzamides (1), a series of related pyrrolidin-3-yl-N-methylbenzamides were synthesized and evaluated as H(3) receptor antagonists. In particular, compound 32 exhibits potent H(3) receptor binding affinity, improved pharmaceutical properties and a favorable in vivo profile.     

Kinetic analysis and ligand-induced conformational changes in dimeric and tetrameric forms of human thymidine kinase 2

Recombinant human thymidine kinase 2 (hTK2) expressed in Escherichia coli has been found to bind tightly a substoichiometric amount of deoxyribonucleoside triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Incubation of hTK2 with the substrate dThd was able to release the dNTPs from the active site during purification from E. coli and thus allowed the kinetic characterization of the noninhibited enzyme, with the tetrameric hTK2 showing slightly higher activity than the most abundant dimeric form. The unliganded hTK2 revealed a lower structural stability than the inhibitor-bound enzyme forms, being more prone to aggregation, thermal denaturation, and limited proteolysis. Moreover, intrinsic tryptophan fluorescence (ITF), far-UV circular dichroism (CD), and limited proteolysis have revealed that hTK2 undergoes distinct conformational changes upon binding different substrates and inhibitors, which are known to occur in the nucleoside monophosphate kinase family. The CD-monitored thermal denaturation of hTK2 dimer/tetramer revealed an irreversible process that can be satisfactorily described by the two-state irreversible denaturation model. On the basis of this model, the parameters of the Arrhenius equation were calculated, providing evidence for a significant structural stabilization of the enzyme upon ligand binding (dCyd < MgdCTP < dThd < dCTP < dTTP < MgdTTP), whereas MgATP further destabilizes the enzyme. Finally, surface plasmon resonance (SPR) was used to study in real time the reversible binding of substrates and inhibitors to the immobilized enzyme. The binding affinities for the inhibitors were found to be 1-2 orders of magnitude higher than for the corresponding substrates, both by SPR and ITF analysis.     

Comparative molecular field analysis and comparative molecular similarity indices analysis of boron-containing human thymidine kinase 1 substrates

Three-dimensional quantitative structure-activity relationship (3D-QSAR) using CoMFA and CoMSIA techniques was applied to evaluate 56 pyrimidine nucleosides as substrates of human thymidine kinase 1 (hTK1), 27 of them containing a carborane substituent either at the 3-, 5-, or 3'-position of the 2'-deoxyuridine scaffold. This is the first report describing 3D-QSAR studies of compounds containing boron atoms. Both CoMFA and CoMSIA models were derived from a training set of 47 molecules and the predictive capacity of the CoMSIA model was successfully validated by accurately calculating known phosphorylation rates of both boronated and non-boron hTK1 substrates that were not included in the training set. The optimal CoMSIA model provided the following values: q(2) 0.622, r(2) 0.983, s 0.165, and F 187.5. Contour maps obtained from the CoMSIA model were in agreement with the experimentally determined biological data.     

High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His(6)-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (approximately 35 mg) and truncated hTK1 (approximately 23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP-MgCl(2) incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (T(m)) of 71.7 and 73.4 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K(M) of 0.51 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM and k(cat) of 1.65 s(-1), thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.     

G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover.

The 545-residue Cln2 protein, like the other G1 cyclins of Saccharomyces cerevisiae, is a very unstable protein. This instability is thought to play a critical role in regulating cell cycle progression. The carboxyl-terminal domains of Cln2 and the other G1 cyclins contain sequences rich in Pro, Glu (and Asp), Ser, and Thr (so-called PEST motifs) that have been postulated to make up the signals that are responsible for the rapid degradation of these and other unstable proteins. To test this hypothesis, the carboxyl-terminal 178 residues of Cln2 were fused to the C terminus of a reporter enzyme, a truncated form of human thymidine kinase (hTK delta 40). The resulting chimeric protein (hTK delta 40-Cln2) retained thymidine kinase activity but was markedly less stable than hTK, hTK delta 40, or an hTK-beta-galactosidase fusion protein, as judged by enzyme assay, immunoblotting with anti-hTK antibodies, pulse-chase analysis of the radiolabeled polypeptides, and ability to support the growth of a thymidylate auxotroph (cdc21 mutant) on thymidine-containing medium. Thus, the presence of the Cln2 PEST domain was sufficient to destabilize a heterologous protein. Furthermore, the half-life of hTK delta 40-Cln2 was similar to that of authentic Cln2, and the rate of degradation of neither protein was detectably enhanced by treatments known to cause G1 arrest, including exposure of MATa haploids to alpha-factor mating pheromone and shifting cdc28ts and cdc34ts mutants to the restrictive temperature. These results suggest that the major signals responsible for Cln2 instability are confined to its C-terminal third. Because hTK delta 40-Cln2 and Cln2 were expressed from heterologous promoters yet their half-lives both in asynchronous cultures and when arrested at various cell cycle stages were always similar, the Cln2 PEST domain contains a signal for rapid protein turnover that is constitutively active and operative throughout the cell cycle. Removal of the 37 codons that encode the most prominent PEST-like segment from either hTK delta 40-Cln2 or Cln2 decreased the turnover rate of the resulting proteins, as expected; however, an hTK delta 40 chimera containing only this 37-residue segment was not detectably destabilized, suggesting that this PEST sequence, when removed from its normal context, is not a self-contained determinant of protein instability.     

Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli. Purification and partial characterization of its dimeric and tetrameric forms

Human thymidine kinase 2 (hTK2) phosphorylates pyrimidine deoxyribonucleosides to the corresponding nucleoside monophosphates, using a nucleotide triphosphate as a phosphate donor. In this study, hTK2 was cloned and expressed at high levels in Escherichia coli as a fusion protein with maltose-binding protein. Induction of a heat-shock response by ethanol and coexpression of plasmid-encoded GroEL/ES chaperonins at 28 degrees C minimized the nonspecific aggregation of the hybrid protein and improved the recovery of three homooligomeric forms of the properly folded enzyme, i.e., dimer > tetramer > hexamer. The dimer and the tetramer were isolated in stable and highly purified forms after proteolytic removal of the fusion partner. Both oligomers contained a substoichiometric amount of deoxyribonucleotide triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Steady-state kinetic studies were consistent with the presence of endogenous inhibitors, and both oligomeric forms revealed a lag phase of at least approximately 5 min, which was abolished on preincubation with substrate (dThd or dCyd). The rather similar kinetic properties of the two oligomeric forms indicate that the basic functional unit is a dimer. Molecular docking experiments with a modeled hTK2 three-dimensional structure accurately predicted the binding positions at the active site of the natural substrates (dThd, dCyd, and ATP) and inhibitors (dTTP and dCTP), with highly conserved orientations obtained for all ligands. The calculated relative nonbonded interaction energies are in agreement with the biochemical data and show that the inhibitor complexes have lower stabilization energies (higher affinity) than the substrates.     

Mitotic degradation of human thymidine kinase 1 is dependent on the anaphase-promoting complex/cyclosome-CDH1-mediated pathway

The expression of human thymidine kinase 1 (hTK1) is highly dependent on the growth states and cell cycle stages in mammalian cells. The amount of hTK1 is significantly increased in the cells during progression to the S and M phases, and becomes barely detectable in the early G(1) phase by a proteolytic control during mitotic exit. This tight regulation is important for providing the correct pool of dTTP for DNA synthesis at the right time in the cell cycle. Here, we investigated the mechanism responsible for mitotic degradation of hTK1. We show that hTK1 is degraded via a ubiquitin-proteasome pathway in mammalian cells and that anaphase-promoting complex/cyclosome (APC/C) activator Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. Furthermore, a KEN box sequence located in the C-terminal region of hTK1 is required for its mitotic degradation and interaction capability with Cdh1. By in vitro ubiquitinylation assays, we demonstrated that hTK1 is targeted for degradation by the APC/C-Cdh1 ubiquitin ligase dependent on this KEN box motif. Taken together, we concluded that activation of the APC/C-Cdh1 complex during mitotic exit controls timing of hTK1 destruction, thus effectively minimizing dTTP formation from the salvage pathway in the early G(1) phase of the cell cycle in mammalian cells.     

Synthesis and pharmacological evaluation of carbamic acid 1-phenyl-3-(4-phenyl-piperazine-1-yl)-propyl ester derivatives as new analgesic agents

A series of carbamic acid 1-phenyl-3-(4-phenyl-piperazine-1-yl)-propyl ester derivatives were synthesized through discovery strategies for balancing target-based in vitro screening and phenotypic in vivo screening. All the newly synthesized compounds were screened for their analgesic activities and compared with standard drug morphine. Among them, compound 44r, a potent analgesic agent that has favorable pharmacokinetic properties in rats and most importantly, has a wide safety margin. We demonstrated with in vitro and in vivo functional assays that its analgesic activity might be through 5-HT(2A) antagonism to some extent. Hence, it is concluded that there is ample scope for further study in developing compound 44r as a good lead candidate for an analgesic agent.     

Synthesis,anti-HIV and cytostatic evaluation of 3′-deoxy-3′-fluorothymidine (FLT) pro-nucleotides

A series of pro-nucleotide phosphoramidates and phosphorodiamidates of the antiviral lead compound 3′-deoxy-3′-fluorothymidine (FLT) have been designed and synthesized. In vitro antiretroviral and cytostatic studies revealed potent (sub-micromolar) inhibition of HIV-1 and HIV-2 replication, with retention of activity in thymidine kinase-negative cell models, as predicted by the ProTide concept.     

Stereochemical considerations in the enzymatic phosphorylation and antiviral activity of acyclonucleosides. I. Phosphorylation of 2'-nor-2'-deoxyguanosine

The antiviral compound 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (2'-nor-2'-deoxyguanosine, 2'-NDG) is phosphorylated by the HSV-1-induced thymidine kinase to the monophosphate (2'-NDG-MP) and this is further phosphorylated by cellular kinases to the triphosphate (2'-NDG-TP) which is a potent inhibitor of DNA polymerases. Since phosphorylation of 2'-NDG creates a chiral center in the molecule, it was of interest to examine whether both monophosphate enantiomers were produced by the viral thymidine kinase, whether they both could be further phosphorylated by cellular kinases and, if so, whether the respective triphosphates were equally inhibitory to the DNA polymerases. The time course of the phosphorylation by GMP kinase of a chemically synthesized, racemic 2'-NDG-MP was compared to that of a 2'-NDG-MP preparation obtained by enzymatic phosphorylation of 2'-NDG with HSV-1 thymidine kinase. The results indicated that the two enantiomeric monophosphates were phosphorylated by GMP kinase with different rates and that phosphorylation of 2'-NDG by HSV-1 thymidine kinase gave only one of the isomers, whose structure was determined to be S. Both enantiomeric diphosphates were further phosphorylated to the respective triphosphates and it was shown that, in contrast to the triphosphate obtained from the 2'-NDG-MP prepared by viral thymidine kinase which was a potent inhibitor of HSV-1 DNA polymerase, the triphosphate obtained from the slow-reacting R isomer had little or no inhibitory activity against this enzyme.     

Synthesis and biological characterization of novel hybrid 7-[[2-(4-phenyl-piperazin-1-yl)-ethyl]-propyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol and their heterocyclic bioisosteric analogues for dopamine D2 and D3 receptors

In a recent preliminary communication we described the development of a series of hybrid molecules for the dopamine D2 and D3 receptor subtypes. The design of these compounds was based on combining pharmacophoric elements of aminotetralin and piperazine molecular fragments derived from known dopamine receptor agonist and antagonist molecules. Molecules developed from this approach exhibited high affinity and selectivity for the D3 receptor as judged from preliminary [(3)H]spiperone binding data. In this report, we have expanded our previous finding by developing additional novel molecules and additionally evaluated functional activities of these novel molecules in the [(3)H]thymidine incorporation mitogenesis assay. The binding results indicated highest selectivity in the bioisosteric benzothiazole derivative N6-[2-(4-phenyl-piperazin-1-yl)-ethyl]-N6-propyl-4,5,6,7-tetrahydro-benzothiazole-2,6-diamine (14) for the D3 receptor whereas the racemic compound 7-([2-[4-(2,3-dichloro-phenyl)-piperazin-1-yl]-ethyl]-propyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol (10c) showed the strongest potency. Mitogenesis studies to evaluate functional activity demonstrated potent agonist properties in these novel derivatives for both D2 and D3 receptors. In this regard, compound 7-[[4-(4-phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol (7b) exhibited the most potent agonist activity at the D3 receptor, 10 times more potent than quinpirole and was also the most selective compound for the D3 receptor in this series. Racemic compound 10a was resolved; however, little separation of activity was found between the two enantiomers of 10a. The marginally more active enantiomer (-)-10a was examined in vivo using the 6-OH-DA induced unilaterally lesioned rat model to evaluate its activity in producing contralateral rotations. The results demonstrated that in comparison to the reference compound apomorphine, (-)-10a was quite potent in inducing contralateral rotations and exhibited longer duration of action.     

中华蜜蜂化学感受蛋白AcerCSP3的配基结合功能分析

为研究中华蜜蜂Apis cerana cerana化学感受蛋白AcerCSP3在化学感受系统中的生理功能, 本实验通过对AcerCSP3进行原核表达、分离纯化后, 利用荧光法研究了体外重组AcerCSP3与1-NPN以及候选化学配基的结合特征。Scatchard方程显示AcerCSP3与1-NPN的解离常数K_D为8.29 μmol/L, 结合位点数约等于1。在候选配基竞争1-NPN与AcerCSP3结合的实验中, 5种配基均能在200 μmol/L浓度下使1-NPN的相对荧光强度下降至50%以下, 其中β-紫罗兰酮甚至能使1-NPN的相对荧光强度下降至10%左右, 表明候选配基均与AcerCSP3有较强的结合能力, 而3, 4-二甲基苯甲醛与中蜂AcerCSP3的结合能力最强, K_D达到18.77 μmol/L。本研究所用化学配基均为植物花与叶片的挥发性的次生代谢产物, 表明AcerCSP3可能作为中蜂化学感受系统的一部分, 在其搜寻某些植物花粉蜜源时作为气味分子运载体发挥一定的作用。     

Synthesis of long-chain amide analogs of the cannabinoid CB1 receptor antagonist N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716) with unique binding selectivities and pharmacological activities

An extended series of alkyl carboxamide analogs of N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxamide (SR141716; 5) was synthesized. Each compound was tested for its ability to displace the prototypical cannabinoid ligands ([3H]CP-55,940, [3H]2; [3H]SR141716, [3H]5; and [3H]WIN55212-2, [3H]3), and selected compounds were further characterized by determining their ability to affect guanosine 5'-triphosphate (GTP)-gamma-[35S] binding and their effects in the mouse vas deferens assay. This systematic evaluation has resulted in the discovery of novel compounds with unique binding properties at the central cannabinoid receptor (CB1) and distinctive pharmacological activities in CB1 receptor tissue preparations. Specifically, compounds with nanomolar affinity which are able to fully displace [3H]5 and [3H]2, but unable to displace [3H]3 at similar concentrations, have been synthesized. This selectivity in ligand displacement is unprecedented, in that previously, compounds in every structural class of cannabinoid ligands had always been shown to displace each of these radioligands in a competitive fashion. Furthermore, the selectivity of these compounds appears to impart unique pharmacological properties when tested in a mouse vas deferens assay for CB1 receptor antagonism.