2′，4″-O-双（三甲基硅）红霉素A9-O-（1-异丙氧环己基）肟甲基化反应的副产物研究

目的改进克拉霉素合成工艺。方法鉴定甲基化反应的副产物并研究其影响因素。结果主要的副产物为E-2′-O-三甲基硅-6-O-甲基红霉素A9-O-（1-异丙氧基环己基）肟（1）。高温、反应时间的延长、三乙胺的加入、过量的去质子试剂均会导致副产物（1）的大量生成；而投料次序为先甲基化试剂后去质子试剂、甲基化试剂过量、THF／DMSO和toluene／DMSO混和溶剂不利于1的生成。结论通过优化甲基化工艺条件。甲基化反应收率最高可达100．2％，其中副产物1仅含0．76％。     

一锅煮合成2′,4″-O-双(三甲基硅) 红霉素A9-O-(1-异丙氧环己基)肟及其副产物

目的改进克拉霉素合成工艺。方法分离、鉴定红霉素A肟的醚化、硅烷化的一锅煮副产物。结果主产物为E-2′,4-″O-双(三甲基硅)红霉素A 9-O-(1-异丙氧基环己基)肟;4个副产物按含量从高到低依次为Z-2′,4-″O-双(三甲基硅)红霉素A 9-O-(1-异丙氧基环己基)肟、E-4-″O-三甲基硅-红霉素A 9-O-(1-异丙氧基环己基)肟、Z-4-″O-三甲基硅-红霉素A 9-O-(1-异丙氧基环己基)肟和E-2-′O-三甲基硅-红霉素A 9-O-(1-异丙氧基环己基)肟。结论六甲基二硅氨烷在酸性下生成三甲基硅正离子和NH3,由于3′-叔胺基在酸性下带正电,有排斥作用,硅烷化容易先发生在4-″OH上,该反应为SN1机制,相应的4-″OH比2-′OH表现出较高的活性。但由于NH3等碱性物质的干扰,提高了2-′OH的活性,硅烷化的区域选择性不高,却有助于得到2′-、4″-OH的双硅烷化产物。     

一锅煮合成2′，4＂-O-双（三甲基硅）红霉素A9-O-（1-异丙氧环己基）肟及其副产物

目的 改进克拉霉素合成工艺。方法 分离、鉴定红霉素A肟的醚化、硅烷化的一锅煮副产物。结果 主产物为E-2′，4＂-O-双（三甲基硅）红霉素A9-O-（1-异丙氧基环己基）肟；4个副产物按含量从高到低依次为Z-2′，4＂-O-双（三甲基硅）红霉素A9-O-（1-异丙氧基环己基）肟、E-4＂-O-三甲基硅-红霉素A9-O（1-异丙氧基环己基）肟、Z-4＂-O-三甲基硅-红霉素A9-O-（1-异丙氧基环己基）肟和B-2′-O-三甲基硅-红霉素A9-O-（1-异丙氧基环己基）肟。结论 六甲基二硅氨烷在酸性下生成三甲基硅正离子和NH3，由于3′-叔胺基在酸性下带正电，有排斥作用，硅烷化容易先发生在4＂-OH上，该反应为SN1机制，相应的4＂-OH比2′-OH表现出较高的活性。但由于NH，等碱性物质的干扰，提高了2′-OH的活性，硅烷化的区域选择性不高，却有助于得到2′、4＂-OH的双硅烷化产物。     

制备3-O-去红霉糖红霉素衍生物的新途径

目的 开发一种合成酮基内酯和酰基内酯常用中间体3-O-去红霉糖衍生物的新方法。方法 红霉素9(E)-（O-烷基)-肟衍生物与对甲基苯磺酸一水合物在DMF中于室温下反应选择性水解红霉素得到相应的3-O-去红霉糖衍生物。结果 高收率得到3-O-去红霉糖衍生物并利用MS、^13C-NMR、^1H—NMB和IR确定了其结构。结论 该方法是制备3-O-去红霉糖红霉素衍生物的简捷、有效的新途径。     

2'''',4″-O-二(三甲基硅)红霉素A肟的合成研究

目的深入研究2′,4″-O-二(三甲基硅)红霉素A肟(EMAO)的合成反应.方法通过对反应过程的监测和产品分析,考察溶剂、硅烷化试剂、催化剂对反应的影响,研究Z、E异构体的反应活性.结果反应可在室温下进行,CH3CN作溶剂最好,用量是EMAO的5～6倍(体积与质量比);硅烷化试剂HMDS为EMAO的3倍摩尔质量较好;催化剂Py*HCl和NH4Cl分别为EMAO的0.3～0.5倍和1.2～1.5倍摩尔质量较好.产品收率达95%,纯度达90%.结论 EMAO的2′、4″位的两个羟基硅烷化反应是分步进行的,反应活性E-EMAO大于Z-EMAO.优化反应后产品收率和纯度较文献均有显著提高.     

吗啡拮抗剂——纳洛酮的合成

纳洛酮为吗啡专一性拮抗剂,临床用为吗啡中毒解救药。本文报道从蒂巴因开始经八步反应合成盐酸纳洛酮的方法。在去氧甲基化反应中采用以三溴化硼为试剂的新工艺;在去氮甲基化反应中,发现了文献上未报道的副产物,通过改变加料方式和试剂配比,避免了副产物的生成。二步改革可使收率显著提高。     

三苄糖苷的合成工艺优化

对三苄糖苷(1)的合成工艺进行了改进。1,2-O-异亚丙基-α-D-呋喃葡萄糖苷(2)在氢氧化钠/DMSO条件下,与氯化苄反应得3,5,6-三苄基-1,2-O-异亚丙基-α-D-呋喃葡萄糖苷(3)。3不经纯化,在乙酸/硫酸条件下水解后,经2次重结晶得到3,5,6-三苄基-D-呋喃葡萄糖苷(4),4在三氟乙酸/原甲酸三乙酯条件下与乙醇成苷制得1,纯度99.57%,总收率66%(以2计)。改进后的工艺反应条件温和,操作简便,已经过中试验证。     

1,2:5,6-二-O-异亚丙基-α-D-木型-己呋喃-3-酮糖的简便合成法

半乳糖可在极性非质子传递溶剂及 CuSO_4的存在下生成1,2:5,6-二-O-异亚丙基-α-D-半乳呋喃糖(9),后者可用氯铬酸吡啶鎓/氧化铝(PCC/Al_2O_3)载体氧化剂氧化制得1,2:5,6-二-O-异亚丙基-α-D-木型-己呋喃-3-酮糖(1),氧化收率85%。本方法步骤简短,原料易得,后处理操作简便,适用于大量制备。     

2,3,5-三-O-苄基-1-O-对硝基苯甲酰基-D-阿拉伯呋喃糖的合成

D-阿拉伯糖经甲基化、苄基化得1-O-甲基-2，3，5-三-O-苄基-D-阿拉伯呋喃糖，再经水解、由对硝基苯甲酰氯酰化制得核苷类药物中间体2，3，5-三-D-苄基-1-D-对硝基苯甲酰基-D-阿拉伯呋喃糖，总收率约45％。     

10-O-(1-β-D-葡萄糖基)喜树碱的制备

五乙酰葡萄糖经溴化后与10-羟基喜树碱反应，再经脱乙酰保护基后生成水溶性提高、内酯结构不变的新化合物10-O-（1-β-D-葡萄糖基）喜树碱，总收率32％。     

A galpha(s) carboxyl-terminal peptide prevents G(s) activation by the A(2A) adenosine receptor

The molecular mechanisms of interaction between G(s) and the A(2A) adenosine receptor were investigated using synthetic peptides corresponding to various segments of the Galpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [(3)H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxam idoade nosine ([(3)H]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The Galpha(s) peptides stimulated specific binding both in the presence and absence of 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS). Three peptides, Galpha(s)(378-394)C(379)A, Galpha(s)(376-394)C(379)A, and Galpha(s)(374-394)C(379)A, were the most effective. In the presence of GTPgammaS, peptide Galpha(s)(374-394)C(379)A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A(2A) adenosine receptor for [(3)H]CGS21680. Binding assays with a radiolabeled selective antagonist, [(3)H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH58261), showed that the addition of the Galpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPgammaS, the displacement curve was right-shifted, whereas the addition of Galpha(s)(374-394)C(379)A caused a partial left-shift. Both curves were fitted by one-site models. This same Galpha(s) peptide was also able to disrupt G(s)-coupled signal transduction as indicated by inhibition of the A(2A) receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from Galpha(s) and Galpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Galpha(s)(374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of Galpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.     

A(1)-, A(2A)- and A(3)-subtype adenosine receptors modulate intraocular pressure in the mouse

Despite the potential importance of the mouse in studying the pharmacology of aqueous dynamics, measurement of intraocular pressure (IOP) in its very small eye has been problematic. Utilizing a novel servo-null electrophysiologic approach recently applied to the mouse, we have identified a diversity of adenosine-receptor mechanisms in modulating IOP in this species. We report the first evidence that A(3) receptors increase IOP in any species, and verify in the mouse reports with larger mammals that A(1) receptors lower and A(2A) receptors increase IOP.     

Local hemostatics (A review)

Information on the hemostatic agents of local action, which are most widely used in leading countries of the world, is summarized. The mechanisms of action of various local hemostatics are considered and possible variants of their combinations in particular ready-to-use drugs are presented. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 7, pp. 9–15, July, 2006.     

2-Chloro-N(6)-cyclopentyladenosine,adenosine A(1) receptor agonist,antagonizes the adenosine A(3) receptor

The potent adenosine A(1) receptor agonists, N(6)-cyclopentyladenosine (CPA) and 2-chloro-N(6)-cyclopentyladenosine (CCPA), were studied in Chinese hamster ovary (CHO) cells expressing the human adenosine A(3) receptor. CPA, but not CCPA, induced phosphoinositide turnover. CPA inhibited forskolin-stimulated cyclic AMP production (EC(50) value of 242+/-47 nM). CCPA competitively antagonized the effects of agonist Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine) with K(B) value of 5.0 nM. CPA competition curves versus the A(3) antagonist radioligand [3H]PSB-11 (8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one) were right-shifted four-fold by 100 microM GTP, which had no effect on binding of CCPA or the antagonist MRS 1220 (N-[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-yl]benzene-acetamide). Thus, CCPA is a moderately potent antagonist (K(i)=38 nM) of the human A(3) adenosine receptor.     

A single amino acid residue on the alpha(5) subunit (Ile215) is essential for ligand selectivity at alpha(5)beta(3)gamma(2) gamma-aminobutyric acid(A) receptors

Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABA(A) receptors containing an alpha(5) subunit. A single amino acid residue (alpha(5)Ile215) has been identified that plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of alpha(5)Ile215 with the cognate amino acid contained in the alpha(1) subunit (Val211) reduced the selectivity of RY-80 for alpha(5)beta(3)gamma(2) receptors from approximately 135- to approximately 8-fold compared with alpha(1)beta(3)gamma(2) receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for alpha(5)beta(3)gamma(2) receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the alpha(1) subunit with the cognate amino acid contained in the alpha(5) subunit (i.e., alpha(1)V211I) increased the affinities of alpha(5)-selective ligands by a approximately 20-fold and reduced by 3-fold the affinity of an alpha(1)-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of alpha(5)215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for alpha(5)-containing GABA(A) receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater than that produced by the alpha(5)I215V mutation. These data indicate that residue alpha(5)215 may not participate in formation of the lipophilic L(2) pocket that has been proposed to contribute to the unique pharmacological properties of alpha(5)-containing GABA(A) receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type alpha(5)beta(3)gamma(2) and mutant alpha(5)I215Kbeta(3)gamma(2) receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant alpha(5)I215Vbeta(3)gamma(2) and alpha(5)I215Tbeta(3)gamma(2) receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue alpha(5)215 is a determinant of both ligand affinity and efficacy at GABA(A) receptors containing an alpha(5) subunit.     

Allosteric interaction of zinc with recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) GABA(A) receptors

In a recent study we have provided evidence that inhibition of native GABA(A) receptors by zinc depends primarily on the allosteric modulation of receptor gating. Both the kinetics and the sensitivity of the GABA(A) receptor to zinc depend on subunit composition, especially on the presence of the gamma(2) subunit. To analyze the mechanism of action of zinc its effects have been tested on recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors expressed in HEK 293 cells. The currents produced by ultrafast application of GABA have been measured to assess the impact of zinc ions on GABA(A) receptor gating with resolution corresponding to the time scale of synaptic currents. While, as expected, zinc markedly reduced the peak amplitude of alpha(1)beta(2)-mediated currents, its effect on kinetics was significantly different from that observed for alpha(1)beta(2)gamma(2). In particular, unlike alpha(1)beta(2)gamma(2), zinc did not affect the onset of alpha(1)beta(2)-mediated responses. Moreover, zinc increased the extent of desensitisation of alpha(1)beta(2)gamma(2) receptors and reduced desensitisation of alpha(1)beta(2) ones. Quantitative analysis suggests that zinc exerts an allosteric modulation on both alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors. Zinc effects on alpha(1)beta(2)gamma(2) were qualitatively similar to those reported for native receptors.     

Binding of the prototypical adenosine A(2A) receptor agonist CGS 21680 to the cerebral cortex of adenosine A(1) and A(2A) receptor knockout mice

1. 2-p-(2-carboxyethylphenethylamino-5'-ethylcarboxamidoadenosine) (CGS 21680) is considered the reference compound to study adenosine A(2A) receptors. However, CGS 21680 binding in the cerebral cortex, where adenosine A(1) receptors are predominant, displays a mixed A(2A)/A(1) receptor pharmacology. We now use adenosine A(1) and A(2A) receptor knockout mice to investigate the characteristics of cortical [(3)H]CGS 21680 binding. 2. [(3)H]CGS 21680 binding to the cerebral cortex was strongly reduced in adenosine A(1) receptor knockout mice, but only slightly reduced in A(2A) receptor knockout mice compared with the corresponding wild-type littermates. 3. Another selective A(2A) receptor ligand, [(3)H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH 58261), displayed a saturable binding to mouse cortical membranes, albeit with a binding density 20 times lower than that of striatal membranes, and this [(3)H]SCH58261 binding was abolished in both striatal and cortical membranes of A(2A) receptor knockout mice and unchanged in A(1) receptor knockout mice. 4. The presence of A(2A) receptors in cortical neurons was further confirmed by Western blot in mouse cortical nerve terminal membranes. 5. It is concluded that, although A(2A) receptors are present in the cerebral cortex, the purportedly selective A(2A) receptor agonist [(3)H]CGS 21680 binds in the cerebral cortex to an entity that requires the presence of adenosine A(1) receptors. Thus, CGS 21680 should be used with care in all preparations where adenosine A(1) receptors out-number A(2A) receptors.     

Acute paw oedema induced by local injection of adenosine A(1), A(2) and A(3) receptor agonists

The present study used plethysmometry to examine oedema following local injection of selective adenosine A(1), A(2) and A(3) receptor agonists and inhibitors of adenosine metabolism into the hindpaw of the rat. N(6)-Cyclopentyladenosine and L-N(6)-phenylisopropyladenosine (A(1)), 2-[p(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680) (A(2A)) and N(6)-benzyl-5'-N-ethylcarboxamido adenosine (N(6)-B-NECA) (A(3)) all produced an increase in paw volume (N(6)N(6)-cyclopentyladenosine, L-N(6)CGS21680). At the highest dose, each agent also produced a systemically mediated suppression of oedema. Oedema by N(6)-cyclopentyladenosine was blocked by caffeine, 8-cyclopentyl-1,3-dimethylxanthine and enprofylline. Oedema by CGS21680 was blocked by caffeine and 8-cyclopentyl-1, 3-dimethylxanthine. Oedema by N(6)-B-NECA was blocked by enprofylline, but not by caffeine or 8-cyclopentyl-1, 3-dimethylxanthine, or by systemic administration of MRS 1191. Oedema by both N(6)-cyclopentyladenosine and N(6)-B-NECA was blocked by mepyramine, ketanserin and phentolamine, but that by CGS21680 was not. The adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine and the adenosine deaminase inhibitor 2'-deoxycoformycin produced only a limited increase in paw volume, and this was blocked by caffeine. This study demonstrates an acute paw oedema response following local administration of adenosine A(1), A(2) and A(3) receptor agonists, which likely results from different mechanisms of action in each case.     

Adenosine negatively regulates duodenal motility in mice: role of A(1) and A(2A) receptors

BACKGROUND AND PURPOSE

Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved.

EXPERIMENTAL APPROACH

RT-PCR was used to investigate the expression of mRNA encoding for A₁, A_2A, A_2B and A₃ receptors. Contractile activity was examined in vitro as changes in isometric tension.

KEY RESULTS

In mouse duodenum, all four classes of adenosine receptors were expressed, with the A_2B receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A₁ receptor antagonist and mimicked by N⁶-cyclopentyladenosine (CPA), selective A₁ agonist. The relaxation induced by A₁ receptor activation was insensitive to tetrodotoxin (TTX) or N^ω-nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A₁ receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A_2A receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A_2A receptor antagonist or l-NAME.

CONCLUSIONS AND IMPLICATIONS

Adenosine can negatively regulate mouse duodenal motility either by activating A₁ inhibitory receptors located post-junctionally or controlling neurotransmitter release via A₁ or A_2A receptors. Both receptors are available for pharmacological recruitment, even if only A_2A receptors appear to be preferentially stimulated by endogenous adenosine.

LINKED ARTICLE

This article is commented on by Antonioli et al., pp. 1577–1579 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01529.x     

Mechanisms of anabolic androgenic steroid modulation of alpha(1)beta(3)gamma(2L) GABA(A) receptors

Modulation of GABA(A) receptors induced by both anabolic androgenic steroids (AAS) and the benzodiazepine (BZ) site agonist, zolpidem, show equivalent dependence upon gamma subunit composition suggesting that both compounds may be acting at a shared allosteric site. Here we have characterized modulation induced by the AAS, 17alpha-methyltestosterone (17alpha-MeT), for responses elicited from alpha(1)beta(3)gamma(2L) GABA(A) receptors and compared it to modulation induced by the BZ site agonists, zolpidem and diazepam. For responses elicited by brief pulses of 20 microM GABA, both the AAS and the BZ site compounds significantly increased the peak current amplitudes and total charge transfer, although 17alpha-MeT was an appreciably weaker agonist than either diazepam or zolpidem at alpha(1)beta(3)gamma(2L) receptors. Neither class of modulator enhanced peak current amplitudes for responses elicited by mM concentrations of GABA. BZ site compounds altered time constants of deactivation, desensitization, and recovery from desensitization, however 17alpha-MeT had no overall effect on these parameters. Experiments in which 17alpha-MeT and BZ site ligands were applied concomitantly indicated that potentiation elicited by 17alpha-MeT and zolpidem were additive and that potentiation by 17alpha-MeT could be elicited in the presence of concentrations of flumazenil that blocked BZ potentiation. Finally, kinetic modeling suggests that while effects of 17alpha-MeT can be simulated by altering receptor affinity, the data for these alpha(1)beta(3)gamma(2L) receptors were best fitted by simulations in which 17alpha-MeT increases transitions into the singly liganded open state. Taken together, our results suggest that 17alpha-MeT does not act at the high-affinity BZ site, but may elicit some of its effects at the low affinity BZ site or at a novel site.