First Total Synthesis of ω-Phenyl Δ6 Fatty Acids and their Leishmanicidal and Anticancer Properties.

INTRODUCTION: The first total synthesis of ω-phenyl Δ6 fatty acids (FA) and their cytotoxicity (A549) and leishmanicidal (L. infantum) activities are described. The novel 16-phenyl-6-hexadecynoic acid (1) and the known 16-phenylhexadecanoic acid (2) were synthesized in 7-8 steps with overall yields of 46 % and 41 %, respectively. The syntheses of the unprecedented 10-phenyl-6-decynoic acid (3), 10-cyclohexyl-6-decynoic acid (4) and 10-(4-methoxyphenyl)-6-decynoic acid (5) was also performed in 3 steps with 73-76 % overall yields. The use of lithium acetylide coupling enabled the 4-step synthesis of 10-phenyl-6Z-decenoic acid (6) with a 100 % cis-stereochemistry. The cytotoxicity of these novel FA was determined against A549 cells and L. infantum promastigotes and amastigotes. Among the ω-phenylated FA, the best cytotoxicity towards A549 was displayed by 1, with an IC50 of 18 ± 1 µM. On the other hand, among the C10 acids, the ω-cyclohexyl acid 4 presented the best cytotoxicity (IC50 = 40 ± 2 µM) towards A549. RESULTS: Based on caspase-3/7 studies neither of the FA induced apoptosis in A549, thus implying other mechanisms of cell death. CONCLUSION: The antileishmanial studies were performed with the top Leishmania donovani topoisomerase IB (LdTopIB) inhibitors, namely 1 and 2 (EC50 between 14 and 36 µM, respectively), acids that did not stabilize the cleavage complexes between LdTopIB and DNA. Acids 1 and 2 displayed cytotoxicity towards L. infantum amastigotes (IC50 = 3-6 µM) and L. infantum promastigotes (IC50 = 60- 70 µM), but low toxicity towards murine splenocytes. Our results identified 1 as the optimum ω- phenylated acid of the series.

Biological and structural effects of the conjugation of an antimicrobial decapeptide with saturated, unsaturated, methoxylated and branched fatty acids.

The increasing bacterial resistance against conventional antibiotics has led to the search for new antimicrobial drugs with different modes of action. Cationic antimicrobial peptides (AMPs) and lipopeptides are promising candidates to treat infections because they act on bacterial membranes causing rapid destruction of sensitive bacteria. In this study, a decapeptide named A2 (IKQVKKLFKK) was conjugated at the N-terminus with saturated, unsaturated, methoxylated and methyl -branched fatty acids of different chain lengths (C8 - C20), the antimicrobial and structural properties of the lipopeptides being then investigated. The attachment of the fatty acid chain significantly improved the antimicrobial activity of A2 against bacteria, and so, endowed it with moderated antifungal activity against yeast strains belonging to genus Candida. Lipopeptides containing hydrocarbon chain lengths between C8 and C14 were the best antibacterial compounds (MIC = 0.7 to 5.8 µM), while the most active compounds against yeast were A2 conjugated with methoxylated and enoic fatty acids (11.1 to 83.3 µM). The improvement in antimicrobial activity was mainly related to the amphipathic secondary structure adopted by A2 lipopeptides in the presence of vesicles that mimic bacterial membranes. Peptide conjugation with long hydrocarbon chains (C12 or more), regardless of their structure, significantly increased toxicity towards eukaryotic cells, resulting in a loss of selectivity. These findings suggest that A2-derived lipopeptides are potential good candidates for the treatment of infectious diseases caused by bacteria and opportunistic pathogenic yeast belonging to genus Candida. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Voluntary running in young adult mice reduces anxiety-like behavior and increases the accumulation of bioactive lipids in the cerebral cortex.

Combinatorial therapies using voluntary exercise and diet supplementation with polyunsaturated fatty acids have synergistic effects benefiting brain function and behavior. Here, we assessed the effects of voluntary exercise on anxiety-like behavior and on total FA accumulation within three brain regions: cortex, hippocampus, and cerebellum of running versus sedentary young adult male C57/BL6J mice. The running group was subjected to one month of voluntary exercise in their home cages, while the sedentary group was kept in their home cages without access to a running wheel. Elevated plus maze (EPM), several behavioral postures and two risk assessment behaviors (RABs) were then measured in both animal groups followed immediately by blood samplings for assessment of corticosterone levels. Brains were then dissected for non-targeted lipidomic analysis of selected brain regions using gas chromatography coupled to mass spectrometry (GC/MS). Results showed that mice in the running group, when examined in the EPM, displayed significantly lower anxiety-like behavior, higher exploratory and risky behaviors, compared to sedentary mice. Notably, we found no differences in blood corticosterone levels between the two groups, suggesting that the different EPM and RAB behaviors were not related to reduced physiological stress in the running mice. Lipidomics analysis revealed a region-specific cortical decrease of the saturated FA: palmitate (C16:0) and a concomitant increase of polyunsaturated FA, arachidonic acid (AA, omega 6-C20: 4) and docosahexaenoic acid (DHA, omega 3-C22: 6), in running mice compared to sedentary controls. Finally, we found that running mice, as opposed to sedentary animals, showed significantly enhanced cortical expression of phospholipase A2 (PLA2) protein, a signaling molecule required in the production of both AA and DHA. In summary, our data support the anxiolytic effects of exercise and provide insights into the molecular processes modulated by exercise that may lead to its beneficial effects on mood.

Synthesis of marine α-methoxylated fatty acid analogs that effectively inhibit the topoisomerase IB from Leishmania donovani with a mechanism different from that of camptothecin.

Sponges biosynthesize α-methoxylated fatty acids with unusual biophysical and biological properties and in some cases they display enhanced anticancer activities. However, the antiprotozoal properties of the α-methoxylated fatty acids have been less studied. In this work, we describe the total synthesis of (5Z,9Z)-(±)-2-methoxy-5, 9-eicosadienoic acid (1) and its acetylenic analog (±)-2-methoxy-5,9-eicosadiynoic acid (2), and report that they inhibit (EC50 values between 31 and 22 µM) the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB). The inhibition of LdTopIB (EC50 = 53 µM) by the acid (±)-2-methoxy-6-icosynoic acid (12) was studied as well. The potency of LdTopIB inhibition followed the trend 2 > 1 > 12, indicating that the effectiveness of inhibition depends on the degree of unsaturation. All of the studied α-methoxylated fatty acids failed to inhibit the human topoisomerase IB enzyme (hTopIB) at 100 µM. However, the α-methoxylated fatty acids were capable of inhibiting an active but truncated LdTopIB with which camptothecin (CPT) cannot interact suggesting that the methoxylated fatty acids inhibit LdTopIB with a mechanism different from that of CPT. The diunsaturated fatty acids displayed low cytotoxicity towards Leishmania infantum promastigotes (EC50 values between 260 and 240 µM), but 12 displayed a better cytotoxicity towards Leishmania donovani promastigotes (EC50 = 100 µM) and a better therapeutic index.

Recent developments in the antiprotozoal and anticancer activities of the 2-alkynoic fatty acids.

The 2-alkynoic fatty acids are an interesting group of synthetic compounds that display antimycobacterial, antifungal, anticancer, and pesticidal activities but their antiprotozoal activity has received little attention until recently. In this review we have summarized our present knowledge of the biomedical potential of the 2-hexadecynoic acid (2-HDA) and 2-octadecynoic acid (2-ODA) together with several mechanistic pieces of work attesting to the fact that these compounds, and their metabolites, are good fatty acid biosynthesis inhibitors. The antiprotozoal activity of 2-HDA and 2-ODA against Leishmania donovani and Plasmodium falciparum, parasites responsible for visceral leishmaniasis and malaria, respectively, is also reviewed. The evidence obtained so far supports the fact that these fatty acids are good inhibitors of the L. donovani DNA topoisomerase IB enzyme (LdTopIB) and the potency of LdTopIB inhibition is chain length dependent. We also demonstrate the generality of the antiprotozoal activity of 2-HDA and 2-ODA against P. falciparum, and review our present knowledge of their inhibition of key P. falciparum enzymes such as PfFabZ, PfFabG, and PfFabI together with some possible modes of inhibition. Recent research by our group has also demonstrated that 2-ODA displays antineoplastic activity, specifically against the neuroblastoma SH-SY5Y cell line via lactate dehydrogenase (LDH) release, which is a cell death mechanism principally associated to necrosis. This is the first comprehensive review of the medicinal chemistry of this interesting group of acetylenic fatty acids.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to ß-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.

First total synthesis of the (±)-2-methoxy-6-heptadecynoic acid and related 2-methoxylated analogs as effective inhibitors of the leishmania topoisomerase IB enzyme.

The fatty acids (±)-2-methoxy-6Z-heptadecenoic acid (1), (±)-2-methoxy-6-heptadecynoic acid (2) and (±)-2-methoxyheptadecanoic acid (3) were synthesized and their inhibitory activity against the Leishmania DNA topoisomerase IB enzyme (LdTopIB) determined. Acids 1 and 2 were synthesized from 4-bromo-1-pentanol, the former in ten steps and in 7% overall yield, while the latter in seven steps and in 14% overall yield. Acid 3 was prepared in six steps and in 42% yield from 1-hexadecanol. Acids 1-3 inhibited the LdTopIB enzyme following the order 2 > 1 ⪢ 3, with 2 displaying an EC(50) = 16.6 ± 1.1 µM and 3 not inhibiting the enzyme. Acid 1 preferentially inhibited the LdTopIB enzyme over the human TopIB enzyme. Unsaturation seems to be a prerequisite for effective inhibition, rationalized in terms of weak intermolecular interactions between the active site of LdTopIB and either the double or triple bonds of the fatty acids. Toxicity towards Leishmania donovani promastigotes was also investigated resulting in the same order 2 > 1 > 3, with 2 displaying an EC(50) = 74.0 ± 17.1 µM. Our results indicate that α-methoxylation decreases the toxicity of C(17:1) fatty acids towards L. donovani promastigotes, but improves their selectivity index.

2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani.

2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC(50)=5.3±0.7µM. The potency of LdTopIB inhibition follows the trend 2-ODA>2-HDA>2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC(50)=11.0µM), but it was less effective against other protozoa, Trypanosoma cruzi (IC(50)=48.1µM) and Trypanosoma brucei rhodesiense (IC(50)=64.5µM). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA>2-HDA>2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound.

Synthesis and antifungal properties of alpha-methoxy and alpha-hydroxyl substituted 4-thiatetradecanoic acids.

4-thiatetradecanoic acid exhibited weak antifungal activities against Candida albicans (ATCC 60193), Cryptococcus neoformans (ATCC 66031), and Aspergillus niger (ATCC 16404) (MIC=4.8-12.7 mM). It has been demonstrated that alpha-methoxylation efficiently blocks beta-oxidation and significantly improve the antifungal activities of fatty acids. We examined whether antifungal activity of 4-thiatetradecanoic acid can be improved by alpha-substitution. The unprecedented (+/-)-2-hydroxy-4-thiatetradecanoic acid was synthesized in four steps (20% overall yield), while the (+/-)-2-methoxy-4-thiatetradecanoic acid was synthesized in five steps (14% overall yield) starting from 1-decanethiol. The key step in the synthesis was the hydrolysis of a trimethylsilyloxynitrile. In general, the novel (+/-)-2-methoxy-4-thiatetradecanoic acid displayed significantly higher antifungal activities against C. albicans (ATCC 60193), C. neoformans (ATCC 66031), and A. niger (ATCC 16404) (MIC=0.8-1.2 mM), when compared with 4-thiatetradecanoic acid. In the case of C. neoformans the (+/-)-2-hydroxy-4-thiatetradecanoic acid was more fungitoxic (MIC=0.17 mM) than the alpha-methoxylated analog, but not as effective against A. niger (MIC=5.5 mM). The enhanced fungitoxicity of the (+/-)-2-methoxy-4-thiatetradecanoic acid, as compared to decylthiopropionic acid, might be the result of a longer half-life in the cells due to a blocked beta-oxidation pathway which results in more time to exert its toxic effects. Thus, these novel fatty acids may have applications as probes to study fatty acid metabolic routes in human cells.

2,6-hexadecadiynoic acid and 2,6-nonadecadiynoic acid: novel synthesized acetylenic fatty acids as potent antifungal agents.

The hitherto unknown 2,6-hexadecadiynoic acid, 2,6-nonadecadiynoic acid, and 2,9-hexadecadiynoic acid were synthesized in two steps and in 11-18% overall yields starting from either 1,5-hexadiyne or 1,8-nonadiyne. Among all the compounds 2,6-hexadecadiynoic acid displayed the best overall antifungal activity against both the fluconazole-resistant Candida albicans strains ATCC 14053 and ATCC 60193, with a minimum inhibitory concentration (MIC of 11 microM), and against Cryptococcus neoformans ATCC 66031 (MIC < 5.7 microM). 2,9-Hexadecadiynoic acid did not display any significant cytotoxicity against the fluconazole-resistant C. albicans strains, but it showed fungitoxicity against C. neoformans ATCC 66031 with a MIC value of < 5.8 microM. Other FA, such as 2-hexadecynoic acid, 5-hexadecynoic acid, 9-hexadecynoic acid, and 6-nonadecynoic acid were also synthesized and their antifungal activities compared with those of the novel acetylenic FA. 2-Hexadecynoic acid, a known antifungal FA, exhibited the best antifungal activity (MIC = 9.4 microM) against the fluconazole-resistant C. albicans ATCC 14053 strain, but it showed a MIC value of only 100 microM against C. albicans ATCC 60193. 2,6-Hexadecadiynoic acid and 2-hexadecynoic acid also displayed a MIC of 140-145 microM toward Mycobacterium tuberculosis H37Rv in Middlebrook 7H12 medium. In conclusion, 2,6-hexadecadiynoic acid exhibited the best fungitoxicity profile compared with other analogues. This diynoic FA has the potential to be further evaluated for use in topical antifungal formulations.

Total synthesis and further scrutiny of the in vitro antifungal activity of 6-nonadecynoic acid.

The antifungal, naturally occurring acetylenic fatty acid 6-nonadecynoic acid was synthesized in three steps (18 % overall yield), for the first time starting with commercially available 1-tetradecyne. The synthesis developed herein will facilitate the further study of the antifungal properties of this naturally occurring acetylenic fatty acid. The 6-nonadecynoic acid exhibited the best antifungal activity (< 4.3 microM) against Cryptococcus neoformans ATCC 66031 in Sabouraud Dextrose Broth (SDB) media. In our hands, it was not active against Candida albicans ATCC 14053 and Candida albicans ATCC 60193.

2-methoxylated fatty acids in marine sponges: defense mechanism against mycobacteria?

A series of saturated 2-methoxylated FA having even-numbered chains with 8-14 carbons were synthesized, and their spectroscopic data are presented for the first time. The 2-methoxylated C10-C14 acids were prepared from the corresponding 2-hydroxylated FA, whereas the 2-methoxyoctanoic acid was synthesized starting with heptaldehyde. All of the methoxylated FA displayed some degree of inhibition (between 2 and 99%) of Mycobacterium tuberculosis H(37)Rv at 6.25 microg/mL. The most inhibitory FA was 2-methoxydecanoic acid, with a minimum inhibitory concentration of 200-239 microM against M. tuberculosis H(37)Rv as determined by both the microplate Alamar Blue assay and the green fluorescent protein microplate assay. These results are discussed in terms of the possible role of the 2-methoxylated FA as antimicrobial lipids produced either by marine sponges, or the associated marine symbiotic bacteria, as a defense mechanism in a highly competitive environment.

Total synthesis and in vitro-antifungal activity of (+/-)-2-methoxytetradecanoic Acid.

The marine fatty acid (+/-)-2-methoxytetradecanoic acid was synthesized in two steps (71% overall yield) starting from commercially available methyl-2-hydroxy-tetradecanoate. The title compound was antifungal against Candida albicans (ATCC 14053) in RPMI medium and Aspergillus niger (ATCC 16404) and Cryptococcus neoformans (ATCC 66031) in SDB medium at the minimum inhibitory concentration (MIC) of 100 mM, which compares to the fungitoxicity of a 2-iodotetradecanoic acid against the same fungi. The title compound was also five to ten times more cytotoxic than capric acid to C. albicans and A. niger in the tested medium but comparable in cytotoxicity to either capric acid and its 2-methoxylated analog to C. neoformans. The antifungal activity of (+/-)-2-methoxytetradecanoic acid is explained in terms of inhibition of N-myristoyltransferase.

The first total synthesis of the marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid: a cytotoxic fatty acid to leukemia cells.

The recently discovered marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid was synthesized for the first time in six steps (26% overall yield) starting from commercially available methyl 12-methyltridecanoate. The synthetic approach provided enough material to corroborate the structure of the acid, which was recently identified in the sponge Amphimedon complanata from Aguadilla, Puerto Rico, and to test its cytotoxicity to three leukemia cell lines. The key step in the synthesis was the addition of trimethylsilyl cyanide to 12-methyltridecanal under triethylamine catalysis. Nuclear magnetic resonance data are provided for the first time for this methoxylated fatty acid and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other methyl-branched 2-methoxylated fatty acids. We also report that the acid (+/-)-2-methoxy-13-methyltetradecanoic acid is cytotoxic to human chronic myelogenous leukemia K-562 (EC50=238 microM), histiocytic lymphoma U-937 (EC50=250 microM), and promielocytic leukemia HL-60 (EC50=476 microM) in RPMI 1640 medium.

The first total synthesis of the marine fatty acid (+/-)-9-methoxypentadecanoic acid: a synthetic route towards mid-chain methoxylated fatty acids.

The marine fatty acid (+/-)-9-methoxypentadecanoic acid was synthesized for the first time in seven steps (7.8% overall yield) starting from commercially available 9-decen-1-ol. The key step in the synthesis was the coupling of pentylmagnesium bromide with 1-benzyloxy-9,10-epoxydecane under 1,5-cyclooctadiene copper (I) chloride catalysis. Nuclear magnetic resonance data are provided for the first time for this type of methoxylated fatty acids and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other mid-chain methoxylated fatty acids. This synthetic methodology should afford sufficient quantities of these fatty acids for biological evaluation. The spectral data obtained for the title compound will also be helpful in subsequent characterizations of other mid-chain methoxylated fatty acids using nuclear magnetic resonance spectroscopy.

Phospholipid fatty acid composition of Gorgonia mariae and Gorgonia ventalina.

The phospholipid fatty acid composition of the Caribbean gorgonians Gorgonia mariae (Bayer) and Gorgonia ventalina (Linnaeus) is described for the first time. The main phospholipids identified were phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. The main fatty acids were 14:0, 16:0, 18:3(n-6), 18:4(n-3), 18:2(n-6), 20:4(n-6), 22:6(n-3), and 24:5(n-6). In both G. mariae and G. ventalina n-6 polyunsaturated fatty acids predominated over the n-3 family. In addition, the 7-methyl-6(E)-hexadecenoic acid was identified in both gorgonians. The occurrence of tetracosapolyenoic fatty acids in the genus Gorgonia is also reported for the first time.