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Browsing Chemistry by Author "Bezuidenhout, B. C. B."
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Item Open Access Development of a viable protocol aimed at the synthesis of a selected natural product with possible application in the industry(University of the Free State, 2007) Jordaan, Lizette; Steenkamp, J. A.; Bezuidenhout, B. C. B.Since the introduction of synthetic analogues in both the health-related and cosmetic industry, a new generation has emerged in search of beneficial bioactivity compounds. This generation of “natural and green” focuses mainly on natural compounds and their health relating application. This research project focused on the natural polyphenolic compounds, Flavonoids. Flavonoids are known to be strong antioxidants, these are molecules that quenches reactive oxygen species (ROS). This generation of free radicals in the stratum corneum is the main factor in the development of skin damage and premature ageing. The two main sources of antioxidants are our body’s own in-house antioxidants or dietary antioxidants. Vitamins E and C were briefly discussed as antioxidants, but the main focus was the antioxidant activity of flavonoids. Through this study were unraveled the reaction pathways of natural antioxidants and their synthetic analogues, in chemical and biological systems. Emphasis was placed on their structure-activity relationship and correlated to their chemical and biological activities. Rooibos extract, known locally and overseas, was pursued not only for its bioactivity but rather its strong radical scavenging abilities. It is known that rooibos is not only unique to South Africa, but is hitherto the only natural source of the dihydrochalcone aspalathin (proven to be a very strong antioxidant). The uniqueness of this dihydrochalcone prompted the establishment of a viable synthetic route towards the construction of those crucial bonds in this target molecule, aspalathin. The first step would be the construction of the dihydrochalcone, 3,4,2’,4’,6’- pentahydroxy dihydrochalcone, which proved to be a challenging array of chemical reactivity. With acylations like Friedel-Craft and Fries, that is known to be very successful, it was decided to commence with the construction of the dihydrochalcone via an appropriate acylation step. Acylation of phenols can either occur via Cacylation (Friedel-Crafts reaction) or O-acylation (esterfication). This regioselectivity is governed by a set of principles incorporated in a theoretical premise, conveniently named as hard and soft acids and bases (HSAB). A new group of water tolerant Lewis acids, namely the lanthanide triflates have been introduced, and also the use of BF3·(C2H5)2O has proven success as catalyst in C-acylation. Simple phenolic substrates were used in the acylation process to assist the eventual establishment of a viable protocol. With these we were able to synthesize 1-hydroxy- 2-acetonaphthone and 3-(3,4-dihydroxy-phenyl)-1-(1-hydroxy-naphthalen-2-yl)- propan-1-one successfully, but in unsatisfactory yields (36 %). Despite many experiments under different conditions, starting with different model compounds, we were unable to improve the reaction yields. Within these reactions resorcinol produced the O-acylation product, 3’-O-hydroxy-phenyl 3-phenyl-propanoate and the C-acylation product, 2’,4’-dihydroxydihydrochalcone, whereas phloroglucinol only produced the O-acylated product, 3’,5’-dihydroxy-phenyl 3-phenyl-propanoate. From this analysis the conclusion can be made that, first occurring is the O-acylation followed by a Fries rearrangement in some cases. The neighboring hydroxy functionalities of phloroglucinol for example, posed a significant steric challenge for incoming electrophiles From the commencement of the project, replacement of the carboxylic acid group with the related, but with different chemical characteristics, nitrile groups was a necessary alternative. The Hoesch reaction was a good example of the HSAB principle, where in acid medium the nitrogen of the cyano group is protonated to afford the reactive electrophilic intermediate, the carbon of which is clearly a “softer” acidic site according to the HSAB theory. The C-acylated product, 2’,4’,6’- trihydroxy dihydrochalcone was produced in an impressive yield (73 %). During this reaction, an interesting result was also obtained, where the phenolic oxygen (“hard” base) as well as the aromatic ring (“soft” base) reacted with the nitrile to produce the product, 3’,5’-dihydroxy-4’-phenyl-propionic acid 1’-3-phenyl-propanoate. It is noteworthy to mention the fact that phloroglucinol was by far the most potent Cand O-nucleophile in a ‘normal’ series of model phenolic entries (phenol, resorcinol, catechol etc.) and resulted in the formation of the biphenyl, 3,5-dihydroxy-phenyl- 2’,4’,6’-trihydroxy-phenylether. Since the formation of a biphenyl ether is a rare occurrence, extensive methylation was employed to confirm the structure. Another part of this study includes the investigation and comparison of similar reactions under the influence of microwaves. Microwave reactions are known for their very short reaction times, higher product yields, less solvent utilized and more cost-effective energy consumption, but it was proved that selectivity was not increased. BF3·(C2H5)2O was the catalyst of choice for the selective C-acylation of phloroglucinol, rather than the water soluble Hf(OTf)4 Lewis acid. Different carboxylic acids were reacted with resorcinol and phloroglucinol with both Lewis acids as catalyst. In the one reaction between resorcinol and 3-phenylpropanoic acid with Hf(OTf)4 as catalyst, a reaction mixture was produced. The reaction mixture was acetylated to give both the O- and C-acetylated products, and from this result it was indicated that Hf(OTf)4 can act as both a Brønsted and Lewis acid in a catalytic cycle. The use of protecting groups was not only to optimize the yields obtained but also to understand BF3·(C2H5)2O and Hf(OTf)4 as catalysts. The low yields for the synthesis of the unprotected dihydrochalcones can be ascribed to: the formation of 3,5- dihydroxy-phenyl-2’,4’,6’-trihydroxy-phenylether, and the formation of 6,7- dimethoxy-indan-1-one and 5,6-dihydroxy-indan-1-one (intramolecular cyclization). At last the C-glycosylated flavonoid, aspalathin was synthesized. The best reaction result of phloroglucinol and 3,4-dihydroxyhydrocinnamic acid was catalyzed by BF3·(C2H5)2O to produce 3,4,2’,4’,6’-pentahydroxy dihydrochalcone, which resulted in a 20 % yield. A reliable method for the direct C-glycosylation of 3,4,2’,4’,6’- pentahydroxy dihydrochalcone with an unprotected sugar, D-glucose in aqueous media was used and yielded synthetic aspalathin (10.7%). Not only was this reported as the first 2 step synthesis of aspalathin, but was distinguished as the first complete free phenolic synthesis of a C-glycosylated flavonoid being reported. Combining this unique synthesis with a global industry such as cosmetics was possible. A study was conducted by Miao-Juei Huang and according to their results it was confirmed that aspalathin would be ideal for the use in topically applied cosmetic products, due to the accumulation of aspalathin in the stratum corneum. This causes a barrier on the skin with strong antioxidant properties, which protects the skin from harmful UV rays, reduce reactive oxygen species and slow down the aging process. Finally the potential of the desired compound to act as an active ingredient in commercial products was confirmed.Item Open Access Development of new "green" methodology for the synthesis of substituted phenylacetic acid derivatives as precursors to isoflavonoids and related compounds(University of the Free State, 2013-01) Pieterse, Tanya; Bezuidenhout, B. C. B.; Marais, C.English: Flavonoids and isoflavonoids are known to exhibit many important physiological properties and are especially promising candidates for cancer chemoprevention. Similar to most natural products, studies directed at the synthesis of flavonoids have, therefore, emerged from the search for new compounds with beneficial biological properties. Metabolic studies related to flavonoids are, however, frequently hampered by the inaccessibility of a variety of optically active compounds. While a single method for the synthesis of enantiomerically enriched isoflavonoids has been published, this process utilizes phenylacetic acid derivatives which are not always readily available in all naturally occurring substitution patterns. Even though the synthesis of phenylacetic acids are possible via a number of routes, these are based on ancient low yielding chemical processes utilizing harsh reaction conditions, stoichiometric quantities of reagents and in many cases, poisonous heavy metals like lead and thallium. In order to address the availability of phenylacetic acid derivatives of variable substitution patterns, the current study was aimed at the development of methodology for the synthesis of phenylacetic acid derivatives that would be high yielding, environmentally benign, have a limited number of process steps, and are applicable to all naturally occurring flavonoid substitution patterns. In this regard it was envisaged that ozonolysis of substituted allylbenzenes would comply with all of the stated criteria and was therefore investigated as methodology for the synthesis of phenylacetic acid derivatives that could serve as building blocks during isoflavonoid preparations. Since substituted allylbenzenes of all oxygenation patterns are not available commercially, the allylic moiety was introduced into the required phenols by means of a allyl phenyl ether intermediate, through utilization of Williamson ether synthesis (allyl bromide; K2CO3, refluxing CH3CN) followed by Claisen rearrangement of the neat allyl phenyl ethers, allyl 3- methoxyphenyl ether and allyl 3,5-dimethoxyphenyl ether, under microwave irradiation (at 200 ºC in 15 min. intervals and 0-200 W variable power) to obtain the desired allylphenols, 1-allyl-2-hydroxy-4-methoxybenzene and 1-allyl-2-hydroxy-4,6-dimethoxy-benzene, in 44 and 88 % yield, respectively. Apart from the desired allylphenol, Claisen rearrangement of allyl 3-methoxyphenyl ether, however, also led to the formation of 1-allyl-2-hydroxy-6- methoxybenzene in 45% yield, indicating a lack of selectivity towards the formation of the sterically less hindered product under the prevailing reaction conditions. Since free phenolic substituents on the aromatic rings of the envisaged substrates might have a negative effect during ozonolysis reactions, the commercially available allylphenols as well as the two substrates prepared by allylation and Claisen rearrangement (vide supra) were subjected to methylation (MeI; K2CO3; refluxing acetone or acetonitrile) and the respective fully methylated analogues, 1-allyl-3,4-dimethoxybenzene, 1-allyl-2,4-dimethoxybenzene, 1-allyl- 2,4,6-trimethoxy-benzene, and 1-allyl-3,4,5-trimethoxybenzene, obtained in 79, 96, 80, and 77% yield, respectively. Ozonolysis [O3 (6-8 min.), DCM, 0 °C] with reductive work-up [N-methylmorpholine-Noxide (NMMO)] of 1-allyl-2-methoxybenzene, 1-allyl-4-methoxybenzene, and 1-allyl-3,4- dimethoxybenzene afforded the corresponding phenylacetaldehydes in 58, 88 and 15% yield, respectively. Ozonolysis of the highly oxygenated substrates, 1-allyl-2-hydroxy-4- methoxybenzene, 1-allyl-2,4,6-trimethoxybenzene, and 1-allyl-3,4,5-trimethoxybenzene, however, only led to cleavage of the aromatic ring and formation of unidentifiable product mixtures. Cleavage of the aromatic rings of these substrates was confirmed by 1H NMR analysis of the reaction mixture [O3 (6-8 min.), CDCl3, -78 °C] where the formation of the 1,2,4-trioxolane intermediate could be detected for the substrates that gave the desired phenylacetaldehydes but not for the highly oxygenated analogues. In order to reduce electron density on the aromatic ring of the highly oxygenated substrates and prevent ring ozonolysis in this way, the free hydroxy function on each substrate was changed into a trifluoromethanesulfonyl ester and the subtrates, 1-allyl-2- trifluoromethanesulfonyloxy-4-methoxybenzene, 1-allyl-4-trifluoromethanesulfonyloxy-3- methoxybenzene, 1-allyl-4-trifluoromethanesulfonyloxy-3,5-dimethoxybenzene, and 1-allyl- 2-trifluoromethanesulfonyloxy-4,6-dimethoxybenzene submitted to ozonation with NMMO work-up again. While the phloroglucinol based sulfonyl ester gave the desired phenylacetaldehyde in 71% yield, the other three substrates furnished NMMO induced double bond migration with the subsequent formation of the benzaldehyde equivalent products. When the ozonation reaction was repeated on the resorcinol, catechol and pyrogallol trifluoromethanesulphonyl esters, with replacement of the NMMO with dimethyl sulphide (DMS) as reductant, the desired phenylacetaldehydes or trioxolanes were, however, obtained in 63, 32 and 31% yield, respectively. Ozonolysis with oxidative work-up [(i) O3/MeOH; (ii) Ac2O-Et3N] applied to the monomethoxy substrates, 1-allyl-2-methoxybenzene and 1-allyl-4-methoxybenzene, afforded the desired methyl phenylacetates in 91 and 32% yield, respectively. Similar to what was found for the reductive work-up procedure, the higher oxygenated substrates had to be converted to their respective triflates before ozonolysis of the allylic double bond could be effected successfully and the phenylacetic acid esters, methyl 4-trifluoromethanesulfonyloxy- 3-methoxyphenyl acetate, methyl 4-trifluoromethanesulfonyloxy-3,5- dimethoxyphenyl acetate, and methyl 2-trifluoromethanesulfonyloxy-4,6-dimethoxyphenyl acetate obtained in 9, 17 and 65% yield, respectively. Since the published process for the stereoselective synthesis of isoflavonoids would require the phenyl acetates prepared through ozonolysis to be transformed into the corresponding amides, the possibility of direct formation of the nitrogen derivatives, like anilides, during the ozonolysis reaction was subsequently investigated. While first attempts at having the aniline present during the ozonolysis reaction only led to nitrogen oxidation, the process was amended to addition of the nitrogen nucleophile after formation of the 1,2,4-trioxolane, which resulted in the desired 2-methoxyphenylacetanilide being formed in 37% yield. The scope of this novel reaction was subsequently extended to the reaction of 1-allyl-4- methoxybenzene with acetamide leading to the product being obtained in 39% yield. While this reaction gave indications that deactivated nitrogen nucleophiles could also be used in this process, the reaction with 2-imidazolidinone, a secondary amide, did not succeed indicating that the new reaction still needs to be optimized to be useful in the enantioselective synthesis of isoflavonoids. Finally, it was shown during the current study that the phenylacetic acid derivatives prepared via ozonolysis could be transformed into deoxybenzoins, another isoflavonoid precursor, through formation of the acid chloride followed by reaction with a phenyl Grignard reagent. Thus phenylacetyl chloride could be reacted successfully with phenylmagnesiumbromide at - 78 oC in diethyl ether to give the deoxybenzoin in almost 60% yield.Item Open Access Structure and synthesis of bioactive polyphenols from Cyclopia subternata (Honeybush) and Aspalathus linearis (Rooibos) tea: conformational analysis of selected chiral derivatives(University of the Free State, 2009-10-23) Brand, Dirk Jacobus; Brandt, E. V.; Bezuidenhout, B. C. B.; Steenkamp, J. A.English: Endorsed by results from our initial investigations of the unique South African tea, brewed from Cyclopia intermedia (Honeybush), demonstrating the presence of polyphenolic flavonoids and glycosides, the tea is gaining popularity as a health beverage. The presence of these compounds which are claimed to have beneficial pharmacological properties, supported by the fact that the tea contains no caffeine and has low tannin content, as well as its usage as a medicinal plant by the indigenous people of the Western and Eastern Cape, prompted continued investigations also into the metabolic content of Cyclopia subternata. Selected extracts of the unfermented shoots and stems of C. subternata were subjected to chromatographic separations that afforded a novel flavan, flavonols, flavanones, flavones, isoflavone and C6.C2- and C6.C1-type compounds. The content of the acetone extract has previously been reported as well as the exploration of various synthetic routes in an attempt to confirm the structure of the novel flavan O- glucoside isolated from the tea. A continuation of the investigation of fractions from the methanol extract of the plant yielded a few compounds that were not found in the acetone extract. This work has been summarized in Chapter one and published as the phenolic profile of Cyclopia subternata. The unambiguous confirmation of the structure of the flavan O-glucoside and essentially the novel aglycone isolated from the tea, necessitated the use of synthetic, Nuclear Magnetic Resonance spectrometric (1H NMR), mass spectroscopic and circular dichroism methods. Mass spectrometry (‘soft’ ionization) was utilized to produce signature fragment ions of the synthesized flavan and the aglycone of the natural flavan for direct comparison. Hydroxylation patterns of the synthetic flavan and the natural flavan aglycone, as well as the relative configuration and point of attachment of the sugar moiety was deduced from NMR experiments. The absolute configuration of the aglycone was determined by circular dichroism. The total synthesis of Aspalathin, a potent antioxidant found in the South African plant, Aspalathus linearis (Rooibos Tea), as the free phenol, was demonstrated. The aglycone of aspalathin, phloretin, also a good antioxidant, was synthesized in aqueous medium without the need for protective groups. This one-step synthesis of phloretin, utilizing a recyclable and environmentally friendly catalyst, has potential commercial applications. For the past 30 years, Mosher esters have extensively been utilized as chiral derivatizing agents (CDA’s) for secondary alcohols and amides with great success. The empirical model employed to deduce the absolute stereochemistry of the resultant ester or amide requires a certain degree of conformational rigidity for the CDA, the origin of which has not yet been determined. The stabilizing atomic orbital hyperconjugative interactions and the location of the prevalent molecular orbitals in such esters were identified with the aid of ab initio calculations, 1H, 19F NMR and infrared spectrometry. Although flavonoid crystal structures are very rare, a novel (-)-epicatechin-Mosher ester crystal was obtained and analyzed by x-ray crystallography with interesting conformational observations. The theoretical model used to investigate the hyperconjugative effects in the Mosher ester, was published and utilized on a recently developed novel CDA that shows a greater degree of rigidity. This CDA, α-cyano-α-fluoro-p-tolyl-acetic acid (CFTA) has greater reactivity and was utilized in cases where the Mosher acid proved inadequate. The investigation regarding the employment of CFTA was carried out with Japanese collaboration and commercial production of the reagent is envisaged. The hyperconjugative molecular model used in the modeling of the Mosher reagent was refined and extended to CFTA. The origin of the very large fluorine chemical shift difference between the epicatechin-CFTA diastereomers was investigated and the results published.Item Open Access The synthesis of an internal standard for bicalutamide(University of the Free State, 2008) Jordaan, Maryam Amra; Van der Westhuizen, J. H.; Bezuidenhout, B. C. B.(R,S)-Bicalutamide [N-(4-cyano-3-trifluoromethylphenyl)-α-methyl-α-hydroxy-β-(4- fluorophenylsulfonyl)propanamide], sold as Casodex®, is the leading antiandrogen currently used to treat prostate cancer. It binds to androgen receptors and blocks cancer growth. This work aims to develop internal standards for the bio-analytical component of clinical trials that are required to detect bicalutamide and derivatives. An internal standard is added to the body fluid sample (mostly blood) at the beginning of the sample work up at about the same concentration of the analyte to be quantified. An ideal internal standard has a similar extraction recovery and a similar retention time in HPLC. For quantification with mass spectrometry it should have a difference of at least 3 mass units from the analyte and a similar ionization response. The internal standard is used to calibrate the total ion current of the metabolite. We did not have access to deuterium labeled starting materials and investigated structural analogues as an alternative strategy to obtain internal standards. De novo synthesis of structural analogues failed because we could not deprotonate the methyl sulfone in the presence of aromatic amides. We ascribed this to incomplete disclosure in the patented methods. Treatment of bicalutamide with palladium on activated charcoal under the right conditions did not give the usually produced amide but gave smooth reduction of the C≡N group to a CH3 group. This unusual reduction gave ready access to a good internal standard in good yield. Elimination of the tertiary aliphatic hydroxy group of bicalutamide would give an alkene with similar polarity that could serve as an internal standard. Acid catalysis using 1M HCl or p-toluene sulfonic acid failed, but treatment of bicalutamide with H2SO4 in benzene gave hydrolysis of the nitrile to an amide. This provides a second internal standard in good yield. Bicalutamide did not react with weak base. Strong base such as LDA led to fission of the aliphatic moiety and isolation of aromatic sulfone and amide fragments. Derivitization of the tertiary aliphatic hydroxy group of bicalutamide with 3- nitrobenzoyl chloride gave a benzoyl ester that allows facile thermal elimination of nitrobenzoic acid at 40 ºC to form an alkene. This represents a third potential internal standard. The NOESY experiment proves that the alkene has a Z-configuration. This indicates that the pro-R aliphatic hydrogen of bicalutamide was eliminated stereoselectively via a syn-periplanar cyclic transition state. Efforts to eliminate the hydroxy group of bicalutamide photolytically at 300 nm in ethanol yielded an unexpected replacement of the aromatic CF3 group with an ethoxy group. We could not find a similar transformation in the literature and believe it to be a novel reaction. We used 19F NMR to prove the absence or presence of fluorine and CF3 moieties in the products. We also used the magnitude of 13C-19F coupling constants in proton decoupled 13C spectra for accurate resonance assignment and structure elucidation. We will test these novel analogues of bicalutamide in cancer bioassays and use them as internal standards for the quantification of bicalutamide and analogues in body fluids.