Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

Biochemical aromatization of 2-methyleneandrostenedione: stereochemistry of hydrogen removal at the C-1 position

To explore a stereochemistry of hydrogen removal at C-1 of the powerful aromatase inhibitor 2-methyleneandrostenedione (1), of which the A-ring conformation is markedly different from that of the natural substrate androstenedione (AD), in the course of the aromatase-catalyzed A-ring aromatization producing 2-methylestrone (2), we synthesized [1-²H]labeled steroid 1 and its [1β-²H]stereoisomer, and the metabolic fate of the C-1 deuterium in aromatization was analyzed by gas chromatography–mass spectrometry (GC–MS) in each. Parallel experiments with the natural substrates [1-²H] and [1β-²H]ADs were also carried out. The GC–MS analysis indicated that 2-methyl estrogen 2 produced from [1-²H]labeled substrate 1 retained completely the 1-deuterium (1β-H elimination), while product 2 obtained from [1β-²H]isomer 1 lost completely the 1β-deuterium. Stereospecific 1β-hydrogen elimination was also observed in the parallel experiments with the labeled ADs as established previously. The results indicate that biochemical aromatization of the 2-methylene steroid 1 proceeds through the 1β-hydrogen removal concomitant with cleavage of the C₁₀–C₁₉ bond, yielding 1(10),4-dienone 9, in a similar manner to that involved in AD aromatization. This would give additional evidence for the stereomechanisms for the last step of aromatization of AD, requiring the stereospecific 1β-hydrogen abstraction and cleavage of the C₁₀–C₁₉ bond, and for the enolization of a carbonyl group at C-3 in the A-ring aromatization.     

Synthesis, NMR study, and X-ray structure of 2-(N-methyl-N′-cyano-N″-ethylguanidino) thiotriophenylphosphinegold(I)

The reaction between cimetidine in a methanolic solution of KOH and a dichloromethane solution of PPh₃AuCl affords a new compound with formula [L-Au-PPh₃] (I) (L = 2-(N-methyl-N′-cyano-N″-ethylguanidino)thiolate), the thiolato ligand resulting from cleavage of one of the thioether bonds of cimetidine. (I) has been characterized by elemental analysis, infrared, and ¹H and ¹³C NMR spectroscopy. Single crystal x-ray structure determination shows that the gold atom is linearly coordinated by a phosphine ligand (Au-P 2.258(1) Å) and by an S atom (Au-S 2.282(1) Å) of the thiolato ligand. Crystal data: triclinic, space group P with a = 8.848(1), b = 11.343(3), c = 12.107(3)Å, = 87.63(1), β = 85.24(1), γ = 79.89(1)°, R = 0.024 for 3673 reflections with I > 3 δ (I).     

Feeding of xenobiotic ω-phenylalkanoic acids remarkably changes the chemistry and toxicity of the defensive secretion of Oxytelus sculpturatus Grav. (Coleoptera: Staphylinidae: Oxytelinae)

Feeding experiments with 12-phenyl[2,2-²H₂]dodecanoic acid and the correponding methylester resulted in the formation of 2-phenylethanol (probably produced via β-oxidation) and high amounts of 2-phenylethylesters of free fatty acids from the defensive secretion of Oxytelus sculpturatus rove beetles. The extraordinarily high amount of the metabolites occurring in the glands after administration of methyl-12-phenyl[2,2-²H₂]dodecanoate had a significant effect on the toxicity of the toluquinone-saturated defensive secretion mixture. Analogous experiments with 11-phenyl-[2-²H]undecanoic acid revealed a less efficient incorporation of this precursor leading to esters of 1-phenylethanol and 4-phenylbutan-2-ol and traces of 10-phenyl-1-decene probably formed via oxidative decarboxylation.     

Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7-³H₂,4-¹⁴C]estradiol, 7-ethoxycoumarin, and [N-methyl-³H₃]cocaine. 6-Hydroxy[7-³H,4-¹⁴C]estradiol was isolated as the metabolite of estradiol and the ³H-water release method based on the 6-³H label was established. The initial rate kinetics of the 6-hydroxylation gave K_m of 4.3 μM, V_max of 4.02 nmol min⁻¹mg⁻¹, and turnover rate of 0.27 min⁻¹. Testosterone competed dose-dependently with the 6-hydroxylation and showed the K_i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K_m of 200 μM, V_max of 12.5 nmol min⁻¹mg⁻¹ and turnover rate of 1.06 min⁻¹. The N-demethylation of cocaine was analysed by the ³H-release method, giving K_m of 670 μM, V_max of 4.76 nmol min⁻¹mg⁻¹, and turnover rate of 0.49 min⁻¹. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min⁻¹mg⁻¹ (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min⁻¹.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Selective addressing of heteroditopic ligands by iron(II) and platinum(II)

The heteroditopic ligand 4′-(4,7,10-trioxadec-1-yn-10-yl)-2,2′:6′,2″-terpyridine, 2, contains an N,N′,N″-donor metal-binding domain that recognizes iron(II), and a terminal alkyne site that selectively couples to platinum(II). This selectivity has been used to investigate routes to the formation of heterometallic systems. The single crystal structures of ligand 2 and the complex [Fe(2)₂][PF₆]₂ are reported.     

Pullulans produced by strains of Cryphonectria parasitica—II. Nuclear magnetic resonance evidence

The structure of pullullan-like polysaccharides produced as exocellular material by different strains of Cryphonectria parasitica, the fungus responsible for chestnut tree cankers, was investigated with nuclear magnetic resonance (NMR) techniques. ¹³C, mono- and bidimensional ¹H, and ¹H–¹³C heteronuclear correlated NMR spectra (HSQC and HMBC) were recorded. Advanced analysis of the NMR spectra allowed the main resonance of the atoms in the maltotriose and in the maltotetraose repeat units of pullulan-like polysaccharides from C. parasitica to be recognised with confidence. In all cases investigated, the presence of large amounts of -(1→6) maltotetraose subunits was evidenced, in addition to the -(1→6) maltotriose subunits, corresponding to the repeating unit of pullulan produced by Aureobasidium pullulans and other fungi. The results were in agreement with other data from this laboratory, obtained with independent techniques. The belief that in ‘pullulans’ the maximum amount of -(1→6) maltotetraose subunits is about 7% can thus be considered as definitely outdated.     

Metabolism of some possible ecdysone precursors in Calliphora stygia

A study has been made, using Calliphora stygia at the time of puparium formation, of the incorporation of a number of labelled sterols into β-ecdysone. [1-³H]-Cholesterol and [4-¹⁴C]-cholesterol are incorporated to a similar extent (0·01-0·02%). [1-³H]-7-Dehydrocholesterol is better incorporated (0·025%) than cholesterol while [1-³H]-cholesterol sulphate, (22R)-22-hydroxy-[22-³H]-cholesterol, and 25-hydroxy-[26-¹⁴C]-cholesterol are not incorporated to a significant extent.     

Biochemical properties of fat body DNA of the silkworm, Bombyx mori

Some properties of DNA, especially of pupal fat body of the silkworm, Bombyx mori, were studied. Pupal fat body DNA was separated into at least three components called -DNA, β₁-DNA, and β₂-DNA on methylated albumin kieselguhr (MAK) column chromatography. All of these classes of DNA were demonstrated to be pure DNA, neither contaminated nor hybridized with RNA, by their being positive to the diphenylamine reaction, sensitive to DNase, resistant to RNase, and incorporating thymidine-6-³H but not uridine-5-³H. The GC contents calculated from T_m values were around 38 per cent for all of these three components, almost coinciding with that of bulk DNA. But the molecular weight of -DNA, roughly calculated from the sedimentation coefficient on a sucrose density gradient centrifugation was several-fold larger than that of β₁-DNA.

In the pupal stage, fat body DNA was mainly composed of β₁- and β₂-DNA with a minor amount of -DNA, while in larval stage, it consisted only of -DNA. Larval fat body type DNA was observed in the larval silk gland, and in pupal and/or pharate adult tissues like the integument, muscles, and gonads. On the other hand, pupal fat body type DNA was detected in the tissues destined to degenerate or in the process of degeneration, such as pupal silk gland and midgut. These facts indicate that β₁- and β₂-DNA may be the degradation products of -DNA.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

Metabolic switching patterns provide definitive evidence of substrate-specificity and delineate subtle differences in positioning at the aromatase active site [Poster 06]

We found that extensive metabolic switching can be triggered in aromatization. Up to 20–35% of (19-³H₃)-labeled substrate is diverted to non-estrogenic product, namely 1β- and 2β-hydroxy androgens, negating usefulness for [³H]water assays. The strikingly substrate-specific metabolite patterns observed reflect positioning at the active site, and the large kinetic isotope effects involved. This switching is unusual in that it involves: 1) tritium labeling, 2) a multistep enzymic process, and 3) the potential unraveling of an enzymic mechanism.     

Incorporation of 5-bromodeoxyuridine into DNA in newborn rat tissues

The incorporation of bromodeoxyuridine (BUdR) into newborn rat tissue DNA has been determined after i.p. injection of 5-bromo-2′-deoxy[6-³H]uridine. Incorporation of the unchanged nucleoside was shown by hydrolysis and ion exchange chromatography of extracted DNA. In all tissues examined, more than 90% of the radioactivity incorporated was in the form of bromodeoxyuridine.     

Investigations into the biosynthetic pathways for classical and ring B-unsaturated oestrogens in equine placental preparations and allantochorionic tissues

In on-going studies of ‘classical’ and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-²H₅]-testosterone and [16,16,17-²H₃]-5,7-androstadiene-3β,17β-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [²H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography–mass spectrometry (GC–MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell™ cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [²H₅]-Testosterone was converted into [²H₄]-oestradiol-17β, [²H₄]-oestrone and [²H₃]-6-dehydro-oestradiol-17 in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse.

On the basis of GC–MS characteristics (M⁺ m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M⁺). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [²H₄]-17β-dihydroequilin was also shown, and a biosynthetic pathway is proposed.

In static incubations of placental microsomal fractions, the 17β-dihydro forms of both equilin and equilenin were shown to be major metabolites of [²H₃]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17β-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17β-isomer of dihydroequilenin. Confirmation of the identity of 17β-dihydroequilin and 17β-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17β-dihydroequilin) and 412 (17β-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3β,17β-diol are proposed.     

Hinokiresinol is not a precursor of agatharesinol in the norlignan biosynthetic pathway in Japanese cedar

The biosynthetic relationship between the two norlignans agatharesinol and trans-hinokiresinol was investigated. Fresh sapwood sticks of Cryptomeria japonica were fed with stable isotope-labeled compounds, namely p-coumaryl alcohol-[9,9-²H], p-coumaryl alcohol-[9-¹⁸O] and trans-hinokiresinol-[1-²H], and then incubated under high-humidity for approximately 20 days, during which the two norlignans were produced simultaneously. While trans-hinokiresinol was strongly deuterium-labeled after feeding with p-coumaryl alcohol-[9,9-²H], agatharesinol was only lightly labeled after feeding with either p-coumaryl alcohol-[9,9-²H] or -[9-¹⁸O]. These results suggest that p-coumaryl alcohol, which is a precursor of hinokiresinol, is not involved in the biosynthesis of agatharesinol. Therefore, the norlignan carbon skeleton of agatharesinol must be framed from different types of phenylpropanoid monomers compared to those utilized by the trans-hinokiresinol pathway. The biosynthesis of these two norlignans seems to branch at an early stage, i.e., before the framing of the norlignan carbon skeleton. Furthermore, agatharesinol was not labeled with deuterium after feeding with ²H-labeled trans-hinokiresinol, which has the simplest norlignan structure. This result strongly supports the suggestion that the conversion of trans-hinokiresinol to agatharesinol is not part of the biosynthesis of norlignans and that early branching occurs instead.     

Structure-activity relationships of adenosines with heterocyclic N6-substituents

Two series of N⁶-substituted adenosines with monocyclic and bicyclic N⁶ substituents containing a heteroatom were synthesized in good yields. These derivatives were assessed for their affinity ([³H]CPX), potency, and intrinsic activity (cAMP accumulation) at the A₁ adenosine receptor in DDT₁ MF-2 cells. In the monocyclic series, the N⁶-tetrahydrofuran-3-yl and thiolan-3-yl adenosines (1 and 26, respectively) were found to possess similar activities, whereas the corresponding selenium analogue 27 was found to be more potent. A series of nitrogen containing analogues showed varying properties, N⁶-((3R)-1-benzyloxycarbonylpyrrolidin-3-yl)adenosine (30) was the most potent at the A₁AR; IC₅₀ = 3.2 nM. In the bicyclic series, the effect of a 7-azabicyclo[2.2.1]heptan-2-yl substituent in the N⁶-position was explored. N⁶-(7-Azabicyclo[2.2.1]heptan-2-yl)adenosine (38) proved to be a reasonably potent A₁ agonist (K_i = 51 nM, IC₅₀ = 35 nM) while further substitution on the 7″-nitrogen with tert-butoxycarbonyl (31, IC₅₀ = 2.5 nM) and 2-bromobenzyloxycarbonyl (34, IC₅₀ = 9.0 nM) gave highly potent A₁AR agonists.     

Synthesis and solution behavior of the trinuclear palladium(II) unsaturated carboxylate complexes triangle-Pd3[μ-O2CC(R′) = CHMe]6 (R′ = Me, H): X-ray structure of palladium(II) tiglate (R′ = Me)

The first examples of binary palladium(II) derivatives of unsaturated carboxylic acids are reported. It was found that the interaction of Pd₃(μ-OAc)₆ with the ,β-unsaturated 1-methylcrotonic (tiglic) and crotonic acids leads to the corresponding carboxylates of composition Pd₃[μ-O₂CC(R′) = CHMe]₆, where R′ = Me (1) or H (2). The new compounds have been characterized by elemental analysis, solid and solution IR, ¹H and ¹³C NMR, and ESI mass spectrometry. The crystal structure of 1 has been determined. This molecule displays a central Pd₃ cyclic core with Pd–Pd distances of 3.093–3.171 Å. Each Pd–Pd bond is bridged by a pair of carboxylate ligands, one above and the other below the Pd₃ plane, providing a square planar coordination for each Pd atom in an approximate D_3h overall symmetry arrangement. Solution spectroscopic data show that the bridging η¹:η¹:μ₂ interaction of the carboxylates of 1 and 2 is readily displaced, with a change of the ligand to the terminal (η¹) coordination mode.     

4,5-Bis(diphenylthiophosphinoyl)-1,2,3-triazole, L: a new varidentate ligand containing diphenylthiophosphinoyl moieties

The novel ligand 4,5-bis(diphenylthiophosphinoyl)-1,2,3-triazole, L^T-S2H, has been synthesized, converted to the triethylamine salt, and to the palladium complexes Pd[L^T-S2]₂ and Pd[L^T-S2][η³-methallyl]. Structures of L^T-S2H, of its 2-acetyl derivative, of Pd[L^T-S2]₂ and Pd[L^T-S2][η³-methallyl] were determined by X-ray crystallography. In the last two complexes the L^T-S2 ligand was N,S-bonded.     

Synthesis, crystal structures and magnetic studies of dicyanamide bridged two new 1D copper(II) complexes

Two new dicyanamide bridged 1D polynuclear copper(II) complexes [Cu(L¹){μ_1,5-N(CN)₂}]_n (1) [L¹H = C₆H₅C(O)NHNC(CH₃)C₅H₄N] and [Cu(L²){μ_1,5-N(CN)₂}]_n (2) [L²H=C₆H₅C(O)CHC(CH₃)NCH₂CH₂N(CH₃)₂] have been synthesised and structures of both the complexes and their crystal packing arrangements have been established by X-ray crystallography. For complex 1, a tridentate hydrazone ligand (L¹H) obtained by the condensation of benzhydrazide and 2-acetylpyridine is used, whereas a tridentate Schiff base (L²H) derived from benzoylacetone and 2-dimethylaminoethylamine is employed for the preparation of complex 2. Variable temperature magnetic susceptibility measurement studies indicate there are weak antiferromagnetic interactions with J values −0.10 and −1.41 cm⁻¹ for 1 and 2, respectively.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.