Synthesis of [C2H3]methotrexate and [C2H3]7‐hydroxymethotrexate

Stable label analogues of methotrexate (MTX) and 7‐hydroxymethotrexate (7‐OH‐MTX) were required for use as internal standards for LC/MS quantitation. A minimum incorporation of stable isotopes to produce a mass increase of 3 in the non‐glutamate derived portion of the molecule was necessary for adequate MS detection. The commercial availability of des‐methyl MTX (aminopterin, 1 ) made methylation with C²H₃I an attractive option. Surprisingly, all attempted methylations of 1 and the dimethyl ester of 1 failed to provide a significant amount of the methylated aniline, apparently due to attenuated reactivity of the secondary amine towards alkylation. However, reductive amination of diacid 1 with C²H₂O and NaB²H₃CN gave [C²H₃]MTX in 52% yield. A previously reported method was utilized to convert [C²H₃]MTX to [C²H₃]7‐OH‐MTX. Preparative HPLC purification of [C²H₃]7‐OH‐MTX resulted in extremely low recovery from the column; this was resolved by switching to a column with few free silanols. Copyright © 2002 John Wiley & Sons, Ltd.     

Channel‐forming,self‐assembling,bishelical amphiphilic peptides: design,synthesis and crystal structure of Py(Aibn)2, n = 2, 3, 4

Abstract: The design, synthesis, characterization and self‐assembling properties of a new class of amphiphilic peptides, constructed from a bifunctional polar core attached to totally hydrophobic arms, are presented. The first series of this class, represented by the general structure Py(Aib_n)₂ (Py = 2,6‐pyridine dicarbonyl unit; Aib = α, α′‐dimethyl glycine; n = 1–4), is prepared in a single step by the condensation of commercially available 2,6‐pyridine dicarbonyl dichloride with the methyl ester of homo oligoAib peptide (Aib_n‐OMe) in the presence of triethyl amine. ¹H NMR VT and ROESY studies indicated the presence of a common structural feature of 2‐fold symmetry and an NH…N hydrogen bond for all the members. Whereas the Aib3 segment in Py(Aib3)₂ showed only the onset of a 3₁₀‐helical structure, the presence of a well‐formed 3₁₀‐helix in both Aib4 arms of Py(Aib4)₂ was evident in the ¹H NMR of the bispeptide. X‐ray crystallographic studies have shown that in the solid state, whereas Py(Aib2)₂ molecules organize into a sheet‐like structure and Py(Aib3)₂ molecules form a double‐stranded string assembly, the tetra Aib bispeptide, Py(Aib4)₂, is organized to form a tetrameric assembly which in turn extends into a continuous channel‐like structure. The channel is totally hydrophobic in the interior and can selectively encapsulate lipophilic ester (CH₃COOR, R = C₂H₅, C₅H₁₁) molecules, as shown by the crystal structures of the encapsulating channel. The crystal structure parameters are: 1b , Py(Aib2)₂, C₂₅H₃₇N₅O₈, sp. gr. P2₁2₁2₁, a = 9.170(1) Å, b = 16.215(2) Å, c = 20.091(3) Å, R = 4.80; 1c , Py(Aib3)₂, C₃₃H₅₁N₇O₁₀·H₂O, sp. gr. P, a = 11.040(1) Å, b = 12.367(1) Å, c = 16.959(1) Å, α = 102.41°, β = 97.29°, γ = 110.83°, R₁ = 6.94; 1 da, Py(Aib4)₂?et ac, C₄₁H₆₅N₉O₁₂?1.5H₂O·C₄H₈O₂, sp. gr. P, a = 16.064(4) Å, b = 16.156 Å, c = 21.655(5) Å, α = 90.14(1)°, β = 101.38(2)°, γ = 97.07(1)°, Z = 4, R₁ = 9.03; 1db, Py(Aib4)₂?amylac,C₄₁H₆₅N₉O₁₂?H₂O ·C₇H₁₄O₂, P2₁/c, a = 16.890(1) Å, b = 17.523(1) Å, c = 20.411(1) Å, β = 98.18 °, Z = 4, R = 11.1 (with disorder).     

Structure--toxicity relationships of 1-substituted-4-alkyl-2,6,7-trioxabicyclo[2.2.2.]octanes.

Many trioxabicyclooctanes [X(OCH₂)₃CY] at low doses administered ip produce tonic-clonic convulsions and death in mice. When X is P, OP, SP, HC, alkyl-C, and C₆H₅C, the optimal Y substituent is tC₄H₉ among alkyl groups examined. In ortho carboxylic acid esters, the optimal X substituent is either small (potency decreases in the order HC > CH₃C) or large (C₆H₅C > nC₄H₉ > nC₃H₇C) while intermediate-sized substituents (C₂H₅C and iC₃H₇C) reduce or negate the toxic properties. Limited literature data on the toxicity of silatranes [X(OCH₂CH₂)₃N] suggest that they may fit a similar pattern for the optimal X substituent (C₆H₅Si > HSi > alkylSi). These findings and particularly the discontinuous sizeactivity relationships of X indicate that the ortho carboxylic acid esters include compounds that may act at two different nerve receptors: one receptor accepts HC(OCH₂)₃CY and CH₃C(OCH₂)₃CY, which mimic P(OCH₂)₃CY, OP(OCH₂)₃CY, SP(OCH₂)₃CY, and possibly HSi(OCH₂CH₂)₃N; the other accepts nC₃H₇C(OCH₂)₃CY, nC₄H₉C(OCH₂)₃CY, and C₆H₅C(OCH₂)₃CY, which may mimic C₆H₅Si(OCH₂CH₂)₃N.     

Reaktionen ionisierter 4-Benzyl-2-methyl-1,2,3,4-tetrahydroisochinoline in der Gasphase

Reactions of Ionized 4-Benzyl-2-methyl-1,2,3,4-tetrahydroisoquinolines in the Gas Phase The M⁺* of 4-Benzyl-1,2,3,4-2-methyl-tetrahydroisoquinoline ( 1 ) decomposes by competing loss of H*- or H₃C*-radicals, of H₃C-NH₂, C₆H₆ and C₇H₈, respectively, or by RDA-fission. The fragmentation mechanisms are studied on the specifically deuterated analogues 2–8 . The elimination of C₆H₆ and C₇H₈ is preceded by extensive H/D-exchange, especially between the positions 1, 4, the benzylic methylene group, and the aromatic positions (pos. 5, 8, and phenyl).     

A comparative study on the accumulation of probenecid and analogues in rabbit kidney tubules in vitro

The characteristics of renal accumulation of probenecid (di-n-propylsulfamylbenzoic acid) and di-methyl [CH₃]₂R,di-ethyl [C₂H₅]₂R, and di-butyl [C₄H₉]₂R analogues by cortical slices suspended in an electrolyte medium have been compared. The compounds were accumulated both under aerobic and anaerobic conditions. Rate of initial active uptake increased in the order: [CH₃]₂R < [C₂H₅]₂R = [C₃H₇]₂R > [C₄H₉]₂-R. The compounds inhibited the aerobic uptake of PAH and phenol red with the following order of effectiveness: [CH₃]₂R < [C₂H₅]₂R < [C₃H₇]₂R < [C₄H₉]₂R. PAH affected the accumulation of probenecid and analogues in the reverse order. The accumulation of probenecid and analogues under anaerobic conditions could be accounted for by binding to various tissue constituents and exhibited close similarity to binding to human serum albumin and liposomes. Phenol red, in contrast to PAH, inhibited anaerobic binding of the compounds to various extents. Active accumulation of probenecid and analogues was markedly stimulated by acetate (10 mM), while fumarate and octanoate had a biphasic effect, except on the accumulation of dibutyl analogue which was little influenced by these metabolites. It is concluded that probenecid and analogues are actively transported by the organic anion system. The inhibitory potency is correlated with the hydrophobicity of these compounds as in the case of phenolsulphonphthalein dyes.     

Synthesis and immunological properties of bombesin analogs

Bombesin (Bn, pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂) is one of the most potent peptides, possessing a variety of physiological and pharmacological functions. We find from CD spectroscopy that the eight C-terminal residues of bombesin [Bn(7-14)NH₂] have an ordered structure, and replacement of His-12 with Pro of Bn(7-14)NH₂ changes the conformation from ordered to a more unordered form. Antibodies to Bn(7-14)NH₂ cross-react to Bn and gastrin releasing peptide (GRP) in a dose-dependent manner. Antibodies to the Pro-analog do not recognize Bn or GRP. Substitution of the C-terminal amide by isopropylamide [Bn(7-14)NHC₃H₇(i))] makes its antibodies more specific to Bn than to GRP. It appears that this region of the peptide is an important antigenic determinant, which makes these antibodies differentiate between BN and GRP.     

An innovative and efficient synthesis of stable isotope labelled 1‐methyl‐5‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)‐1H‐pyrazole via [13C42H3] N‐methylpyrazole

In support of a programme to develop a treatment for cancer, a stable isotope labelled version of the drug candidate was required. The key labelled intermediate was [¹³C₄²H₃] N‐methylpyrazole prepared by a novel bisacetal cyclisation. This was prepared from commercially available diethyl [¹³C₃] malonate and [¹³C²H₃] iodomethane. Copyright © 2012 John Wiley & Sons, Ltd.     

中国乌头的研究——Ⅹ.关白附子中的新生物碱

从关白附子(Aconitum koreanum R.Raymund)中共分得六种生物碱。其中一种是已知生物碱,卽次乌头碱,另五种为新生物碱,暂称为关附甲素C₂₄H₃₁O₆N、乙素C₂₂H₂₉O₅N、丙素C₂₂H₃₃O₂N、丁素C₂₄H₃₅O₃N及戊素C₂₉H_(43)O₇N。关附甲素是关附乙素的一乙酸酮。关附甲素、乙素、丙素的示性式分别定为:C₁₉H₂₀(OH)₂(CH₃COO)₂(CH₃)(∶N·),C₁₉H₂₀(OH)₃(CH₃COO)(CH₃)(∶N·),C₁₉H₂₃(OH)₂(CH₃)(N—C₂H₅)。后二种生物碱因量少尚待研究。     

Nuclear magnetic resonance analyses of side chain conformations of histidine and aromatic amino acid derivatives

Stereoselectively β-deuterated species were synthesized of Ac-His-NHMe, Ac-His-OEt, Ac-His-OH and H-His-NHMe, which are useful as models of histidine residues in peptides. From the spectral comparison of ¹H n.m.r., the β-proton resonances of the normal species were unambiguously assigned. In (C²H₃)₂SO, C₂²H₅O²H, C²H₃O²H, and C₅²H₅N solution and in aqueous solution, the lower-field and higher-field components of β-proton resonances of the four histidine derivatives are assigned to the pro-R and pro-S protons, respectively. The alternative assignments apply for Ac-His-NHMe, Ac-His-OEt and Ac-His-OH in non-polar solvents such as C²HCl₃. Vicinal coupling constants ³J_αβS and ³J_αβR were obtained for calculating the fractional populations of rotamers about the C_α–C_β bond. The rotamer populations depend little on the ionization states of the α-amino and carboxyl groups or the imidazole ring. The rotamer populations depend significantly on the solvent polarity, similar to those of Phe, Tyr and Trp derivatives. For the two β-proton resonances of His, Phe, Tyr, and Trp derivatives in a variety of solvents, linear relationships are found between the differences in chemical shifts and the differences in vicinal coupling constants.     

Cyclic‐RGD penta‐peptides cRGDyK derivatized with cyclopentadienyl complexes of technetium and rhenium as radiopharmaceutical probes

The present study reports the syntheses of half‐sandwich complexes of the type [M(η⁵‐C₅H₄CONH‐R)(CO)₃] (M═Re,^99mTc;R═cyclic RGD peptide (cRGDyK) for potential imaging of α_vβ₃ integrin expression. The ^99mTc complex was prepared directly from the reaction of [^99mTc(OH₂)₃(CO)₃]⁺ with cRGDyK, doubly conjugated to Thiele's acid [(C₅H₅COOH)₂] in water. This approach extends the viability of metal‐mediated retro Diels‐Alder reactions for the preparation of small molecules such as linear tripeptides to a more complex cyclic peptide carrying a [(η⁵‐C₅H₄)^99mTc(CO)₃] tag. The Diels‐Alder product [(C₅H₅CONH‐cRGDyK)₂] was prepared from Thiele's acid via double peptide coupling. The Re‐complex [Re(η⁵‐C₅H₄CONH‐cRGDyK)(CO)₃] was obtained by attaching [Re(η⁵‐C₅H₄COOH)(CO)₃] directly to the N‐terminus of cRGDyK. The identity of the ^99mTc‐complex is confirmed by chromatographic comparison with the corresponding rhenium complex, fully characterized by spectroscopic techniques.     

国药中黄碱类之研究 Ⅴ. 黄芩化学成分的研究(第一报)黄芩素新提取法及其新甲基化物

黄芩是属于唇形科(Labiatae),植物学名为Scutellaria baicalensis Georg。早已收载于神农本草经,在中国已于2000年以前作为藥用。一般主要藥效为退热治头痛,于1953年的調查,本藥是常用中藥之一。于1950年在苏联由夏斯氏發表謂黄芩酊有治疗高血压的作用,并能消除高血压的主观症状如头痛失眠等,这是与我国本草上的記載相符合的。据报告,使用黄芩酊剂来治疗高血压症病人,均發現有良好的降压作用,不但与苏联的临床报告相符合,而且对本草中記載的用黃芩酒浸治太陽少陽头痛的事实相符。黄芩的煎剂由本所藥理組做降压的急性試驗,結果降压作用相当显     

In silico molecular docking of niloticin with acetylcholinesterase 1 (AChE1) of <Emphasis Type="Italic">Aedes aegypti</Emphasis> L. (Diptera: Culicidae): a promising molecular target

Synthetic insecticides used in mosquito control program are harmful to environment and also affect other associated organisms. As a choice, plant-based natural compounds proved to be a good alternative to synthetic insecticides. In a study, we had reported niloticin (C₃₀H₄₈O₃) from the plant Limonia acidissima L. was effective and disturbed the larval growth of A. aegypti. The main molecular target for many commercially available synthetic mosquitocides is acetylcholinesterase (AChE), which plays a major role in larval knockdown/resistant mechanisms. AChE1 is a serine protease, which fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the present study, we performed molecular docking studies with acetylcholinesterase 1 (AChE1) of A. aegypti with niloticin (C₃₀H₄₈O₃) and compared with commercially available chemical larvicidal compound temephos (C₁₆H₂₀O₆P₂S₃). The docking results revealed that the binding affinities and energy values of niloticin (?8.4 kcal/mol) were found to be significantly higher than temephos (?4.75 kcal/mol). Both niloticin (C₃₀H₄₈O₃) and temephos (C₁₆H₂₀O₆P₂S₃) showed the same binding residues (THR’58 and HIS’62) on AChE1. Further, niloticin produced least binding energy (?8.4 kcal/mol), good inhibition constant value (695.18 μM) and high ligand efficiency (0.25) than temephos, suggesting that niloticin (C₃₀H₄₈O₃) could be used against the larvae of A. aegypti as an effective AChE1 inhibitor.     

滇产植物的皂素成分研究 Ⅰ.滇吉祥草的甾体成分(1)

从滇吉祥草(Reineckea yunnanensis W.W.Smith)中分得两个甾体皂甙元,其一为薯蓣皂甙元(diosgenin),另一皂甙元暂名为滇吉祥草皂甙元(yunnanogenin),熔点275—277℃,[α]_D^17°—64.1(CHCl₃,c=0.156),制成双乙酰化物,熔点181—182℃,双苯甲酰化物,熔点236—239℃,23-溴代双乙酰化物,熔点196—199℃。由上述数据及红外吸收光谱数据,推测为一正系饱和的双羟基皂甙元。     

Synthesis and Antifungal Activity of 1‐Aryl‐3‐phenethylamino‐1‐propanone Hydrochlorides and 3‐Aroyl‐4‐aryl‐1‐phenethyl‐4‐piperidinols

Mono‐Mannich bases, 1‐aryl‐3‐phenethylamino‐1‐propanone hydrochlorides, 1a, 2a , 3a , 4a , 5a , 6a , 7a , 8a , 9a , and semi‐cyclic mono‐Mannich bases, 3‐aroyl‐4‐aryl‐1‐phenethyl‐4‐piperidinols, 1b , 2b , 3b , 4b , 5b , 6b , 7b , 8b , 9b , were synthesized by a non‐classical Mannich reaction. The aryl part was: C₆H₅ for 1a , 1b ; 4‐CH₃C₆H₄ for 2a , 2b ; 4‐CH₃OC₆H₄ for 3a , 3b ; 4‐ClC₆H₄ for 4a , 4b ; 4‐FC₆H₄ for 5a , 5b ; 4‐BrC₆H₄ for 6a , 6b ; 2,4‐(Cl)₂C₆H₃ for 7a , 7b ; 4‐NO₂C₆H₄ for 8a , 8b ; and C₄H₃S(2‐yl) i. e., 2‐thienyl for 9a , 9b . Piperidinol compounds 2b , 3b , 4b , 5b , 7b , 8b , and 9b are reported here for the first time. The synthesized compounds were tested against seven types of plant pathogenic fungi and three types of human pathogenic fungi using the agar dilution assay. Itraconazole was tested against Candida parapsilosis as the reference compound, while Nystatin was tested as the reference compound against the other fungi. Compounds 1a , 1b , 2a , 4a , 4b , 5a , 5b , 6a , 7a , 8a , 9a , and 9b can be selected as model compounds to develop new antifungal agents against the human pathogen Microsporum canis. Compounds 8a and 8b , which had a similar antifungal activity compared with the reference compound Nystatin against the plant pathogen Aspergillus flavus, can serve as model compounds to develop new antifungal agents to solve agricultural problems.     

Comparison of early injury to liver endoplasmic reticulum by halomethanes,hexachloroethane, benzene,toluene, bromobenzene,ethionine, thioacetamide and dimethylnitrosamine

This study compares the effects in vivo of CH₂Cl₂, CH₂Br₂, CHCl₃, CH₂I₂, CHBr₃, CCl₄, CHI₃, CBr₄, CI₄, C₂Cl₆, C₂HCl₅, C₆H₆, C₇H₈, C₆H₅Br, ethionine, thioaeetamide and dimethylnitrosamine on the functions and composition of liver endoplasmic reticulum 2 hr after poisoning. Within the halomethane series, the effects on lipid diene conjugate content, oxidative demethylase, ¹⁴C-glycine incorporation into protein and glucose 6-phosphatase of liver microsomes and cell sap RNA content, increase with decreasing effective negative charge on the halogen atoms (?), an indicator of increasing halomethane free radical reactivity (cf.B. P. Dailey, J. chem. Phys.33, 1641,1960). Peak toxic effect is reached following CCl₄ and CHI₃. Glycine incorporation into protein is also decreased 2 hr after thioacetamide and dimethylnitrosamine. After dimethylnitrosamine, suppression of protein synthesis is concomitant with increases in lipid diene conjugate content and cell sap RNA. C₆H₅Br and ethionine, both known hepatotoxins, do not effect microsomal composition and function at this time, nor do C₆H₆ or C₇H₈ both organic solvents with solubility properties similar to CCl₄. The findings support the hypothesis that free radical halomethane metabolites injure the endoplasmic reticulum by reacting with and chemically altering its constituents. While dimethylnitrosamine may act similarly, thioaeetamide, bromobenzene and ethionine apparently do not.     

Investigating Monoclonal Antibody Aggregation Using a Combination of H/DX-MS and Other Biophysical Measurements

To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the C_H2 domain and the hinge region between C_H1 and C_H2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.     

中国乌头的研究 Ⅷ.黄草乌根中的生物碱

从云南黄草乌根中分得三种生物碱,其中两种暂称为黄草乌碱甲及乙.甲碱C₃₃H₄₇O₉N,熔点为182-184℃;乙碱C₂₁H₃₃O₄N,熔点为184-185℃;和另一微量生物碱,熔点151-152℃.其中甲碱为主要成分,经官能团测定后,定其示性式为C₁₉H₂₁(OH)₂(OCH₃)₄-(CH₃OC₆H₄COO)(N-C₂H₅).     

Synthesis,toxicity, and percutaneous activity of silicon glycerolates and related hydrogels

Silicon glycerolates of the general formula Si(C₃H₇O₃)₄ ⋅ xC₃H₈O₃(3 ≤ x ≤ 10) and related hydrogels of the general formula Si(C₃H₇O₃)₄ ⋅ xC₃H₈O₃ ≤ yH₂O (3 ≤ x ≤ 10, 20 ≤ y ≤ 40) were synthesized and some of their pharmacological properties were studied. The laws of gel formation were investigated and optimum conditions for the process were determined. High percutaneous (transdermal) activity of the synthesized compounds was revealed by measuring the diffusion of drugs through intact skin membrane in vitro. The acute and chronic toxicity was studied. It was established that all substances are nontoxic. The experimental results show that silicon glycerolates and related hydrogels can be recommended for further testing and investigation as effective biologically active percutaneous vehicles of medicinal preparations. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 11, pp. 5–9, November, 2008.     

Protein A does not induce allosteric structural changes in an IgG1 antibody during binding

Affinity chromatography is widely used for antibody purification in biopharmaceutical production. Although there is evidence suggesting that affinity chromatography might induce structural changes in antibodies, allosteric changes in structure have not been well-explored. Here, we used hydrogen exchange-mass spectrometry (HX-MS) to reveal conformational changes in the NIST mAb upon binding with a protein A (ProA) matrix. HX-MS measurements of NIST mAb bound to in-solution and resin forms of ProA revealed regions of the C_H2 and C_H3 domains with increased protection from HX upon ProA binding, consistent with the known ProA binding region. In-solution ProA experiments revealed regions in the F_ab with increased HX uptake when the ProA:mAb molar ratio was increased to 2:1, suggesting an allosterically induced increase in backbone flexibility. Such effects were not observed with lower ProA concentration (1:1 molar ratio) or when ProA resin was used, suggesting some kind of change in binding mode. Since all pharmaceutical processes use ProA bound to resin, our results rule out reversible allosteric effects on the NIST mAb during interaction with resin ProA. However, irreversible effects cannot be ruled out since the NIST mAb was previously exposed to ProA during its original purification.     

Massenspektrometrie einiger substituierter α-Ethoxy-Pyridine und -Pyrimidine

Mass Spectrometry of Substituted α-Ethoxypyridines and -Pyrimidines The molecular ions of the α-ethoxy heterocycles 1–8 lose * CH₃ and C₂H₄ or * C₂H₅ from the α-ethoxy group. Also, fragmentation of the esters 2b, 3b, 4–8 occurs by degradation of the ethoxycarbonyl group.