Concise synthesis of (8Z,11Z,14Z)-8,11,14-heptadecatrienal, (7Z,10Z,13Z)-7,10,13-hexadecatrienal, and (8Z,11Z)-8,11-heptadecadienal, components of the essential oil of marine green alga Ulva pertusa

The long-chain aldehydes, (8Z,11Z,14Z)-8,11,14-heptadecatrienal, (7Z,10Z,13Z)-7,10,13-hexadecatrienal, and (8Z,11Z)-8,11-heptadecadienal, were concisely synthesized by using Grignard coupling, catalytic hydrogenation with the Lindlar catalyst, and oxidation with Dess-Martin periodinane as the key steps. Particularly, (8Z,11Z,14Z)-8,11,14-heptadecatrienal and (7Z,10Z,13Z)-7,10,13-hexadecatrienal both possessed a seaweed-like odor.     

Synthesis of (6Z,9Z,11E)-octadecatrienoic and (8Z,11Z,13E)-eicosatrienoic acids and their [1-(14)C]-radiolabeled analogs

In order to study the metabolic pathway and the physiological effects of 9c,11t-18:2 (major isomer of conjugated linoleic acid) and its C(18:3) and C(20:3) metabolites, 6c,9c,11t-18:3 and 8c,11c,13t-20:3 and their [1-(14)C]-radiolabeled analogs were prepared stereoselectively by total synthesis. The 8c,11c,13t-20:3 was obtained in 11 steps. The synthesis involves a highly stereoselective Wittig reaction between 3-(t-butyldiphenylsilyloxy)propanal and the ylide of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt which gave (3Z)-1-(t-butyldiphenylsilyloxy)-10-(2-tetrahydropyranyloxy)dec-3-ene in a first step. Then the t-butyldiphenylsilyl derivative was deprotected selectively and the resulting alcohol function was converted via a bromide into a phosphonium salt. The second stereoselective Wittig condensation between the phosphonium salt and commercial (2E)-non-2-enal under cis-olefinic conditions using Lithium hexamethyldisilazide as base afforded the (7Z,10Z,12E)-1-(2-tetrahydropyranyloxy)nonadeca-7,10,12-triene in a very good isomeric purity. The intermediate product was brominated and transformed by reaction with magnesium into Grignard reagent, which was one-carbon elongated by unlabeled or labeled carbon dioxide to obtain the 8c,11c,13t-20:3 in good isomeric purity (95%) and high radiochemical purity for its [1-(14)C]-radiolabeled analog (99%). 6c,9c,11t-18:3 was synthesized in a similar way by using 5-(2-tetrahydropyranyloxy)pentanylphosphonium salt in place of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt in a first step. Other reactions were unchanged and products were obtained in similar yields. Similar to 8c,11c,13t-20:3, the 6c,9c,11t-18:3 was obtained in a very good isomeric purity (95%) and its [1-(14)C]-radiolabeled analog in a high radiochemical purity (95%).     

Synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-3H8]-analogues through a common synthetic route

Total synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-³H₈]-analogues via the corresponding p-toluenesulphonates is reported. This synthetic approach allows the preparation of radioactively labelled arachidonic acid derivatives following a common synthetic route. Activity assays indicated that 15-lipoxygenases may tolerate the azido group in the substrate binding pocket and thus, radioactively labelled azido compounds may be used as photo-affinity probes to investigate mechanistic features of eicosanoid biosynthesis.     

First total syntheses of (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid

The first total syntheses for the (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid were accomplished in seven steps and in 31-32% overall yields. The (trimethylsilyl)acetylene was the key reagent in both syntheses. It is proposed that the best synthetic strategy towards monounsaturated iso methyl-branched fatty acids with double bonds close to the omega end of the acyl chain is first acetylide coupling of (trimethylsilyl)acetylene to a long-chain bifunctional bromoalkane followed by a second acetylide coupling to a short-chain iso bromoalkane, since higher yields are thus obtained. Spectral data is also presented for the first time for these two unusual fatty acids with potential as biomarkers and as topoisomerase I inhibitors.     

Stereoselective synthesis of (6Z,10E,12Z)-octadeca-6,10,12-trienoic acid, (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid,and their [1-14C]-radiolabeled analogs

To study the metabolic fate of conjugated linoleic acid isomers, we synthesized, in seven steps, from 1-heptyne, (6Z,10E,12Z)-octadeca-6,10,12-trienoic acid, (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid, and their [1-(14)C]-analogs. In the case of (6Z,10E,12Z)-octadecatrienoic acid, a series of palladium-catalyzed cross-coupling reactions between 1-heptyne and (E)-1,2-dichloro-ethene, a coupling reaction with a Grignard reagent and cleavage of the dioxolane gave (E)-dodec-4-en-6-ynal 3. Stereoselective Wittig reaction between aldehyde 3 and triphenyl-[5-(tetrahydro-pyran-2-yloxy)-pentyl]-phosphonium provided a dienyne. Stereocontrolled reduction of the triple bond and replacement of the tetrahydropyranyl group by a bromine gave (5Z,9E,11Z)-1-bromo-heptadeca-5,9,11-triene 10. Formation of the alkenyl lithium derivative and carbonation with CO(2) furnished (6Z,10E,12Z)-octadecatrienoic acid. (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid was obtained by the same route but using triphenyl-[5-(tetrahydro-pyran-2-yloxy)-heptyl]-phosphonium iodide for the Wittig reaction. [1-(14)C]-analogs were obtained from the bromides by carbonation with (14)CO2. In all cases, chemical or radiochemical purities were found to be better than 95% after purification by flash chromatography on silica gel (>99% after additional purification by RP-HPLC). Metabolism studies in animals are in progress.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Synthesis of (8Z,11Z,14Z)-eicosatrien-5-ynoic (5,6-dehydroarachidonic) acid and its transformation into [5,6-3H]arachidonic acid and [5,6-3H]prostaglandins E2 and F2 alpha

8Z,11Z,14Z-Eicosatriene-5-ynoic acid and its tritium-labelled analogue, [5,6-3H]arachidonic acid, have been synthesized on the basis of acetylenic compounds. [5,6-3H]Arachidonic acid has been used as substrate for the enzymatic synthesis of [5,6-3H]PGE2 and [5,6-3H]PGF2 alpha.     

Synthesis of methyl (5S,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate,an analogue of 15R-lipoxin A4

We describe a method for the synthesis of methyl (5S,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate, a compound that has been described as a metabolically stable analogue of 15R-lipoxin A(4).     

Spatial requirements for 15-(R)-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2. Why acetylated COX-1 does not synthesize 15-(R)-hete

The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (15-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11- and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-1 equivalents resulted in abrogation of 11- and 15-HETE production after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the omega-end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site-directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel. Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.     

Synthesis of (3alpha,7beta,17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-triol, a metabolite of ORG OD14, and its 7-epimer

The syntheses of the 7beta-hydroxy metabolite of ORG OD14 (Livial((R))), (3alpha,7beta,17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-triol (35), and its 7-epimer, (3alpha,7alpha,17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-triol (11), are described.     

Stereoselective Synthesis of a Promising Flower-Inducing KODA Analog, (9R,12S,13R,15Z)-9-Hydroxy-12,13-methylene-10-oxooctadec-15-enoic Acid

Stereoselective synthesis of a promising flower-inducing 9,10-ketol octadecadienoic acid (KODA) analog, (9R,12S,13R,15Z)-9-hydroxy-12,13-methylene-10-oxooctadec-15-enoic acid, was designed to obtain the desired stereoisomer via coupling between chiral sulfone and aldehyde segments. A known chiral cyclopropane derivative was converted to the sulfone segment via carbon-chain elongation and sulfonylation. Dec-9-en-1-ol was converted to the aldehyde segment, whose C-9 configuration was introduced by Sharpless asymmetric dihydroxylation. Coupling of the both segments and subsequent assembly gave the desired (9R,12S,13R,15Z)-analog. The (9S,12S,13R,15Z)-analog was also synthesized by using the enatiomeric aldehyde segment. This strategy made it possible to synthesize the remaining stereoisomeric analogs.     

Radiochemical investigations of (99m)Tc-N(3)S-X-BBN[7-14]NH(2): an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop (99m)Tc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N(3)S-BBN (N(3)S = dimethylglycyl-l-seryl-l-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = 0 (no spacer), omega-NH(2)(CH(2))(2)COOH, omega-NH(2)(CH(2))(4)COOH, omega-NH(2)(CH(2))(7)COOH, or omega-NH(2)-(CH(2))(10)COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N(3)S-X-BBN[7-14]NH(2) (X = 0 carbons, beta-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) with gentle heating. Re-N(3)S-5-Ava-BBN[7-14]NH(2) was also prepared by the reaction of [Re(V)dimethylglycyl-l-seryl-l-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH(2). ES-MS was used to determine the molecular constitution of the new Re(V) conjugates. The (99m)Tc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[(99m)TcO(4)] with excess SnCl(2), sodium gluconate, and corresponding ligand. The (99m)Tc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.     

Optimization of Large Scale Preparation of 13-(S)-Hydroperoxy-9Z, 11E-Octadecadienoic Acid Using Soybean Lipoxygenase. Application to the Chemoenzymatic Synthesis of (+)-Coriolic Acid

The optimization of 13-S-hydroperoxy-9Z, 11E-octadecadienoic acid synthesis is described using lipoxygenase-1 from soybeans at high substrate concentration. The optimal values of the tested parameters are as follows: oxygen pressure 2.5 bar, temperature 5°C, pH 11, enzyme concentration 4 mg/ml and substrate concentration 0.1 M. All these values were used in a single reaction, allowing chemoenzymatic synthesis of gram amounts of (+)-coriolic acid (99%, e.e. 97%) with a 54% yield starting from linoleic acid.     

A New Synthesis of (E,E)-8, 10-Dodecadien-1-ol,Sex Pheromone of Codling Moth

A methanol-utilizing bacterium, which produced red to pink pigments and assimilated methanol via icl^- serine pathway, was isolated from soil and tentatively designated as Pseudomonas MS 31. This bacterium produced L-serine when glycine was added to the growth medium at the late exponential phase of growth. The cells showed high L-serine degradation activity. Chelating agents and some metal ions, which inhibited L-serine degradation, stimulated the L-serine accumulation. In the presence of 0.1 ? 1 mM EDTA, o-phenanthroline, 8-hydroxyquinoline, α,α'-dipyridyl, cobalt sulfate or nickel sulfate, this bacterium produced 0.7?2.1mg L-serine from 4mg glycine per ml culture.     

13C NMR spectra of 1, 3, 6 -trihydroxy-7-methoxy-8-(3, 7 -dimethyl-2,6-octadienyl) xanthone and its dimethyl derivative

The ¹³C NMR spectra of 1, 3, 6-trihydroxy-7-methoxy-8-(3, 7-dimethyl-2, 6-octadienyl)xanthone and its dimethyl derivative are discussed. The data obtained confirmed the assigned structures. The geometrical configuration of the C₁₀ dienyl side-chain has been deduced as trans.     

Interaction of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with relaxed circular pBR322 DNA

The interaction of (+/-)-BPDE (1) with DNA at neutral pH was investigated by the application of relaxed circular pBR322 DNA. (+/-)-BPDE causes a rapid positive supercoiling of this DNA followed by a slower spontaneous relaxation. The results indicate that there are two clearly discernible types of chemical interactions between 1 and DNA, a rapid intercalative covalent binding and a slower strand breakage. The implications of these findings are discussed.     

3-(Cyclopropylmethyl)-7-((4-(4-[11C]methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine: Synthesis and preliminary evaluation for PET imaging of metabotropic glutamate receptor subtype 2

Selective metabotropic glutamate receptor 2 (mGluR2) inhibitors have been demonstrated to show therapeutic effects by improving alleviating symptoms of schizophrenic patients in clinical studies. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography (PET) tracer originating from a mGluR2 inhibitor, 3-(cyclopropylmethyl)-7-((4-(4-methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (CMTP, 1a). [¹¹C]CMTP ([¹¹C]1a) was synthesized by O-[¹¹C]methylation of desmethyl precursor 1b with [¹¹C]methyl iodide in 19.7 ± 8.9% (n = 10) radiochemical yield (based on [¹¹C]CO₂) with >98% radiochemical purity and >74 GBq/μmol molar activity. Autoradiography study showed that [¹¹C]1a possessed moderate in vitro specific binding to mGluR2 in the rat brain, with a heterogeneous distribution of radioactive accumulation in the mGluR2-rich brain tissue sections, such as the cerebral cortex and striatum. PET study indicated that [¹¹C]1a was able to cross the blood–brain barrier and enter the brain, but had very low specific binding in the rat brain. Further optimization for the chemical structure of 1a is necessary to increase binding affinity to mGluR2 and then improve in vivo specific binding in brain.     

Synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-H8]-analogues through a common synthetic route

Total synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-³H₈]-analogues via the corresponding p-toluenesulphonates is reported. This synthetic approach allows the preparation of radioactively labelled arachidonic acid derivatives following a common synthetic route. Activity assays indicated that 15-lipoxygenases may tolerate the azido group in the substrate binding pocket and thus, radioactively labelled azido compounds may be used as photo-affinity probes to investigate mechanistic features of eicosanoid biosynthesis.     

Synthesis and biological properties of 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl- and 16alpha-[125I]iodo-estradiols

The synthesis, receptor binding affinity, estrogenic potency and tissue distribution of the 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl-(CIVE) and 16alpha-[125I]iodo-estradiols (CIE) are reported. The iodovinyl derivatives were prepared via the (17alpha,20E/Z)-tri-n-butylstannyl intermediates, derived from the addition of tri-n-butyl tin hydride to the 17alpha-ethynyl group of the 7alpha-cyano-17alpha-ethynylestradiol, using triethylborane as a catalyst. The no-carrier-added [125I]-CIVE isomers were prepared via the same stereospecific reaction. [125I]-CIE was prepared from 7alpha-cyano-16beta-bromoestradiol via halogen exchange with Na125I. Addition of the 7alpha-cyano group to 16alpha-iodoestradiol did not affect estrogen receptor binding affinity (RBA of CIE is 115). However the estrogenic potential of CIE, as measured by the capacity to stimulate the expression of the pS2 gene, was reduced to 1% as compared to that of estradiol. Addition of a 7alpha-cyano group to the (17alpha,20E/Z)-IVE isomers reduced the RBA to 21 and 36, respectively, while the estrogenic potential was reduced to 2-3% of that of estradiol. Uterus uptake in immature rats of the 125I-labeled CIVE 20E-isomer and the 16alpha-iodo CIE peaked at 0.5h post injection while the (17alpha,20Z)-CIVE isomer showed a maximum only past 5h post injection. Uptake of all three 125I-labeled 7alpha-cyanoestrogens was suppressed by the co-injection of non-radioactive estradiol confirming the role of estrogen receptors in the localization process. Uterus retention pattern differ substantially from those of the analogues 7alpha-methylestrogens, which were previously shown to give high maximum 125I-uptake values at 2h post injection. Overall our data indicate that addition of a 7alpha-cyano group to 123I-labeled estrogens does not improve their potential to serve as SPECT agents for the imaging of estrogen receptor densities in breast cancer.