Synthesis of novel heterocyclic systems as potential inhibitors of HIV-1 enzymes
- Authors: Sekgota, Khethobole Cassius
- Date: 2020
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , Quinoline , Amides , Nuclear magnetic resonance , Antiretroviral agents , AIDS vaccines , Nitrobenzaldehyde , Propylphosphonic acid anhydride
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/146502 , vital:38531
- Description: This study has focussed on the application of Baylis-Hillman methodology in the development of efficient synthetic pathways to libraries of novel 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones and indolizine-2-carboxamides and on an exploration of their medicinal potential. The approach to 3-[(N-cycloalkylbenzamido)methyl]-2(1H)-quinolones involved a six-step pathway comprising: Baylis-Hillman reaction of 2-nitrobenzaldehyde derivatives and methyl acrylate to afford nitro-Baylis-Hillman adducts; thermal cyclisation of the adducts to give a range of 3-(acetoxymethyl)-2(1H)-quinolones in good to excellent yields; hydrolysis of the acetates; conversion of the resulting alcohols to the 3-chloromethyl analogues; amination; and, finally, acylation to afford the target amides. Variable temperature NMR methods were used to facilitate analysis of the ¹H and ¹³C NMR spectra which were complicated by internal rotation and cycloalkyl ring-flipping effects. On the other hand, the indolizine-2-carboxamides were obtained in several steps commencing with the Baylis-Hillman reaction of pyridine-2-carboxaldehyde and methyl acrylate. Thermal cyclisation of the Baylis-Hillman adduct afforded indolizine esters, hydrolysis of which gave the corresponding acids which served as precursors to the target indolizine-2-carboxamides. The final amidation step, however, proved to be particularly challenging. Various coupling strategies were explored to access indolizine-2-carboxamides. These included the use of 2,2,2-trifluoroethyl borate which showed limited promise, but propylphosphonic acid anhydride (T3P) proved to be the most effective coupling agent, permitting the formation of 24 novel indolizine-2-carboxamides from hydrazines, aliphatic amines and a range of heterocyclic amines. A high-field NMR-based kinetic study of the mechanism of the Baylis-Hillman reaction of pyridine-4-carboxaldehyde and methyl acrylate in the presence of 3-hydroxyquinuclidine in deuterated chloroform was initiated, reaction progress being followed by the automated collection of ¹H and DEPT 135 NMR spectra over ca. 24 hours using a high-field (600 MHz) NMR instrument. The results have provided critical new insights into the mechanism. NMR analysis has also been used to elucidate the multiplicity of signals associated with rotameric equilibria observed at ambient probe temperature. Variable temperature 1D- and 2D-NMR spectra were used to facilitate the unambiguous characterisation of the 2-quinolone benzamides and some of the indolizine-2-carboxamides. The 3-[(N-cycloalkylbenzamido)methyl]-2(1H)-quinolones, together with selected precursors, and a number of the indolizine-2-carboxamides have been screened in vitro as potential HIV-1 enzyme inhibitors. A survey of the activity of the 2-quinolones against HIV-1 integrase, protease and reverse transcriptase revealed selective inhibition of HIV-1 integrase with the most active IN inhibitor, 3-[(cyclopentylamino)methyl-6-methoxy-2(1H)-quinolone 115e, producing residual enzyme activity of 40% at a concentration of 20 μM. Many of the 2-quinolones exhibited no significant cytotoxicity against HEK 293 cells at 20 μM concentrations. 3-[(N-Cyclohexylamino)methyl]-6-methoxy-2(1H)-quinolone 114e was the only compound to exhibit ant-plasmodial activity (55% pfLDH activity). The survey of indolizine-2-carboxamides also revealed encouraging inhibition against HIV-1 integrase. None of these compounds exhibited cytotoxicity at 20 μM against HEK 293 cells, while a number of them exhibited some activity against Plasmodium falciparum (3D7 strain) and Trypanosoma brucei. Selected indolizine-2-carboxamides exhibited significant anti-tubercular activity in the 7H9 CAS GLU Tx and 7H9 ADC GLU Tw media. In view of the inherent fluorescent character and biological potential of the synthesised indolizine-2-carboxamides, their photophysical properties were explored to establish their possible dual use as bio-imaging and therapeutic agents. The major absorption and corresponding emission bands, and the associated molar absorption coefficients (Ɛ) expressed in the form of log Ɛ were determined. Their high extinction coefficients, large Stokes shift and red-shifted emissions in the visible region indicate their potential for use as fluorophores.
- Full Text:
- Date Issued: 2020
- Authors: Sekgota, Khethobole Cassius
- Date: 2020
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , Quinoline , Amides , Nuclear magnetic resonance , Antiretroviral agents , AIDS vaccines , Nitrobenzaldehyde , Propylphosphonic acid anhydride
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/146502 , vital:38531
- Description: This study has focussed on the application of Baylis-Hillman methodology in the development of efficient synthetic pathways to libraries of novel 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones and indolizine-2-carboxamides and on an exploration of their medicinal potential. The approach to 3-[(N-cycloalkylbenzamido)methyl]-2(1H)-quinolones involved a six-step pathway comprising: Baylis-Hillman reaction of 2-nitrobenzaldehyde derivatives and methyl acrylate to afford nitro-Baylis-Hillman adducts; thermal cyclisation of the adducts to give a range of 3-(acetoxymethyl)-2(1H)-quinolones in good to excellent yields; hydrolysis of the acetates; conversion of the resulting alcohols to the 3-chloromethyl analogues; amination; and, finally, acylation to afford the target amides. Variable temperature NMR methods were used to facilitate analysis of the ¹H and ¹³C NMR spectra which were complicated by internal rotation and cycloalkyl ring-flipping effects. On the other hand, the indolizine-2-carboxamides were obtained in several steps commencing with the Baylis-Hillman reaction of pyridine-2-carboxaldehyde and methyl acrylate. Thermal cyclisation of the Baylis-Hillman adduct afforded indolizine esters, hydrolysis of which gave the corresponding acids which served as precursors to the target indolizine-2-carboxamides. The final amidation step, however, proved to be particularly challenging. Various coupling strategies were explored to access indolizine-2-carboxamides. These included the use of 2,2,2-trifluoroethyl borate which showed limited promise, but propylphosphonic acid anhydride (T3P) proved to be the most effective coupling agent, permitting the formation of 24 novel indolizine-2-carboxamides from hydrazines, aliphatic amines and a range of heterocyclic amines. A high-field NMR-based kinetic study of the mechanism of the Baylis-Hillman reaction of pyridine-4-carboxaldehyde and methyl acrylate in the presence of 3-hydroxyquinuclidine in deuterated chloroform was initiated, reaction progress being followed by the automated collection of ¹H and DEPT 135 NMR spectra over ca. 24 hours using a high-field (600 MHz) NMR instrument. The results have provided critical new insights into the mechanism. NMR analysis has also been used to elucidate the multiplicity of signals associated with rotameric equilibria observed at ambient probe temperature. Variable temperature 1D- and 2D-NMR spectra were used to facilitate the unambiguous characterisation of the 2-quinolone benzamides and some of the indolizine-2-carboxamides. The 3-[(N-cycloalkylbenzamido)methyl]-2(1H)-quinolones, together with selected precursors, and a number of the indolizine-2-carboxamides have been screened in vitro as potential HIV-1 enzyme inhibitors. A survey of the activity of the 2-quinolones against HIV-1 integrase, protease and reverse transcriptase revealed selective inhibition of HIV-1 integrase with the most active IN inhibitor, 3-[(cyclopentylamino)methyl-6-methoxy-2(1H)-quinolone 115e, producing residual enzyme activity of 40% at a concentration of 20 μM. Many of the 2-quinolones exhibited no significant cytotoxicity against HEK 293 cells at 20 μM concentrations. 3-[(N-Cyclohexylamino)methyl]-6-methoxy-2(1H)-quinolone 114e was the only compound to exhibit ant-plasmodial activity (55% pfLDH activity). The survey of indolizine-2-carboxamides also revealed encouraging inhibition against HIV-1 integrase. None of these compounds exhibited cytotoxicity at 20 μM against HEK 293 cells, while a number of them exhibited some activity against Plasmodium falciparum (3D7 strain) and Trypanosoma brucei. Selected indolizine-2-carboxamides exhibited significant anti-tubercular activity in the 7H9 CAS GLU Tx and 7H9 ADC GLU Tw media. In view of the inherent fluorescent character and biological potential of the synthesised indolizine-2-carboxamides, their photophysical properties were explored to establish their possible dual use as bio-imaging and therapeutic agents. The major absorption and corresponding emission bands, and the associated molar absorption coefficients (Ɛ) expressed in the form of log Ɛ were determined. Their high extinction coefficients, large Stokes shift and red-shifted emissions in the visible region indicate their potential for use as fluorophores.
- Full Text:
- Date Issued: 2020
Characterization of amide bond hydrolysis in novel hydantoinase-producing bacteria
- Authors: Skepu, Zoleka G
- Date: 2000
- Subjects: Amides , Hydrolysis , Hydantoin , Imides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3911 , http://hdl.handle.net/10962/d1003970 , Amides , Hydrolysis , Hydantoin , Imides
- Description: This thesis describes a series of investigations into the amide bond-hydrolyzing activity of bacterial strains RU-KM1, RU-KM3L, RU-KM3S, and RU-OR, which were previously isolated for their ability to hydrolyze hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids and related compounds. Several compounds may be used as substrates by biocatalysts for the production of amino acids, such as hydantoins, amino nitriles, and amides. These compounds are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as 2- arylpropionic acids, which are non-steroidal anti-inflammatory drugs. Thus, the ability of the above-mentioned strains to hydrolyze these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. The compounds used as substrates in the investigation are all essentially amides. Thus, the ability of the strains to hydrolyze imides, hydantoins, and amides, was investigated. In particular, imides have a structure which is very similar to that of hydantoins, and thus it was an objective of the study to determine whether these strains could hydrolyze imides. Imidehydrolyzing activity has only recently been discovered in microorganisms. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an Ncarbamylamino acid intermediate, and subsequently, an "-amino acid. The hydantoinhydrolyzing enzymes of a Pseudomonas putida strain, RU-KM3S, were characterized in a crude extract preparation and reaction conditions for its biocatalytic application were optimized. The optimum conditions for conversion of 5-methylhydantoin were found to be 3 hours at 40°C, with conversion yields greater than 50% achieved. The enzymes of RU-KM3S demonstrated considerable stability, retaining 80% of their activity after incubation at 40°C for 3 hours. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamylase of RUKM3S suggested that these enzymes required metal ions for activity, with metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ resulting in activation of the enzymes. However, Cu²⁺ and Fe²⁺ caused inactivation of these enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was non-selective, whereas the N-carbamylase was L-selective. The hydantoin substrate selectivity of RU-KM3S was compared to that of three other hydantoinase-producing bacteria, RU-KM1, RU-KM3L, and RU-OR. The four strains were able to hydrolyze all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing higher activity than whole cells, except in the case of RU-KM1. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolyzed p-hydroxyphenylhydantoin. RU-KM3L whole cells achieved a higher conversion yield with isobutylhydantoin, whereas the crude extract achieved a higher yield with 5-t-butylhydantoin. RU-KM3S whole cells and crude extract preferentially hydrolyzed 5-n-butylhydantoin, although the yield was greater with the crude extract. The highest conversion yields were observed with RU-KM3S crude extract, with conversion yields of 71.6% and 100% for n-butylhydantoin and phydroxyphenylhydantoin, respectively.The ability of RU-KM1, RU-KM3L, and RU-KM3S to hydrolyze nitriles, initially to amides and subsequently to carboxylic acids, was investigated. These strains were demonstrated to be unable to utilize acrylonitrile, propionitrile and benzonitrile as nitrogen sources, but were able to hydrolyze acrylonitrile, propionitrile and acetonitrile, in resting cell reactions. Nitrile hydrolysis was demonstrated to be inducible in all three strains, and the enzyme system responsible for nitrile hydrolysis was proposed to be a nitrile hydratase-amidase system. Amidase activity in the four bacterial strains was investigated. The ability of RU-KM1, RUKM3L, RU-KM3S, and RU-OR to utilize amides as a nitrogen source was investigated, and the results showed that propionamide was a good nitrogen source for all four of the strains. Amide-hydrolyzing activity, by resting cells, was shown to be inducible by propionamide in all four strains. RU-KM3S demonstrated superior amide-hydrolyzing ability in that it hydrolyzed propionamide, acetamide, and acrylamide to a greater extent than the other strains. Resting cells of RU-KM1 and RU-OR were demonstrated to have the ability to hydrolyze the imide substrate, succinimide, and this imidase activity was found to be inducible. These strains were also able to utilize this imide as the sole source of nitrogen for growth, which is a novel finding, as to date, bacteria have only be reported to utilize imides as a carbon source. The identity of the enzyme system responsible for succinimide hydrolysis is not yet clear. In conclusion, the hydantoin-hydrolyzing enzymes of RU-KM3S have been shown to be possibly membrane associated, which is a novel finding that has also been proposed in three other hydantoinase-producing strains in our laboratory. This study has shown that the Ncarbamylase of RU-KM3S is L-stereoselective, which, to our knowledge, is the first report of an L-stereospecific N-carbamylase in a Pseudomonas putida. Publication of these findings is already in progress. This is the first report on the study of imide hydrolysis in either an Agrobacterium tumefaciens or a Pseudomonas sp., and publications reporting these results are in preparation.
- Full Text:
- Date Issued: 2000
- Authors: Skepu, Zoleka G
- Date: 2000
- Subjects: Amides , Hydrolysis , Hydantoin , Imides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3911 , http://hdl.handle.net/10962/d1003970 , Amides , Hydrolysis , Hydantoin , Imides
- Description: This thesis describes a series of investigations into the amide bond-hydrolyzing activity of bacterial strains RU-KM1, RU-KM3L, RU-KM3S, and RU-OR, which were previously isolated for their ability to hydrolyze hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids and related compounds. Several compounds may be used as substrates by biocatalysts for the production of amino acids, such as hydantoins, amino nitriles, and amides. These compounds are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as 2- arylpropionic acids, which are non-steroidal anti-inflammatory drugs. Thus, the ability of the above-mentioned strains to hydrolyze these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. The compounds used as substrates in the investigation are all essentially amides. Thus, the ability of the strains to hydrolyze imides, hydantoins, and amides, was investigated. In particular, imides have a structure which is very similar to that of hydantoins, and thus it was an objective of the study to determine whether these strains could hydrolyze imides. Imidehydrolyzing activity has only recently been discovered in microorganisms. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an Ncarbamylamino acid intermediate, and subsequently, an "-amino acid. The hydantoinhydrolyzing enzymes of a Pseudomonas putida strain, RU-KM3S, were characterized in a crude extract preparation and reaction conditions for its biocatalytic application were optimized. The optimum conditions for conversion of 5-methylhydantoin were found to be 3 hours at 40°C, with conversion yields greater than 50% achieved. The enzymes of RU-KM3S demonstrated considerable stability, retaining 80% of their activity after incubation at 40°C for 3 hours. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamylase of RUKM3S suggested that these enzymes required metal ions for activity, with metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ resulting in activation of the enzymes. However, Cu²⁺ and Fe²⁺ caused inactivation of these enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was non-selective, whereas the N-carbamylase was L-selective. The hydantoin substrate selectivity of RU-KM3S was compared to that of three other hydantoinase-producing bacteria, RU-KM1, RU-KM3L, and RU-OR. The four strains were able to hydrolyze all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing higher activity than whole cells, except in the case of RU-KM1. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolyzed p-hydroxyphenylhydantoin. RU-KM3L whole cells achieved a higher conversion yield with isobutylhydantoin, whereas the crude extract achieved a higher yield with 5-t-butylhydantoin. RU-KM3S whole cells and crude extract preferentially hydrolyzed 5-n-butylhydantoin, although the yield was greater with the crude extract. The highest conversion yields were observed with RU-KM3S crude extract, with conversion yields of 71.6% and 100% for n-butylhydantoin and phydroxyphenylhydantoin, respectively.The ability of RU-KM1, RU-KM3L, and RU-KM3S to hydrolyze nitriles, initially to amides and subsequently to carboxylic acids, was investigated. These strains were demonstrated to be unable to utilize acrylonitrile, propionitrile and benzonitrile as nitrogen sources, but were able to hydrolyze acrylonitrile, propionitrile and acetonitrile, in resting cell reactions. Nitrile hydrolysis was demonstrated to be inducible in all three strains, and the enzyme system responsible for nitrile hydrolysis was proposed to be a nitrile hydratase-amidase system. Amidase activity in the four bacterial strains was investigated. The ability of RU-KM1, RUKM3L, RU-KM3S, and RU-OR to utilize amides as a nitrogen source was investigated, and the results showed that propionamide was a good nitrogen source for all four of the strains. Amide-hydrolyzing activity, by resting cells, was shown to be inducible by propionamide in all four strains. RU-KM3S demonstrated superior amide-hydrolyzing ability in that it hydrolyzed propionamide, acetamide, and acrylamide to a greater extent than the other strains. Resting cells of RU-KM1 and RU-OR were demonstrated to have the ability to hydrolyze the imide substrate, succinimide, and this imidase activity was found to be inducible. These strains were also able to utilize this imide as the sole source of nitrogen for growth, which is a novel finding, as to date, bacteria have only be reported to utilize imides as a carbon source. The identity of the enzyme system responsible for succinimide hydrolysis is not yet clear. In conclusion, the hydantoin-hydrolyzing enzymes of RU-KM3S have been shown to be possibly membrane associated, which is a novel finding that has also been proposed in three other hydantoinase-producing strains in our laboratory. This study has shown that the Ncarbamylase of RU-KM3S is L-stereoselective, which, to our knowledge, is the first report of an L-stereospecific N-carbamylase in a Pseudomonas putida. Publication of these findings is already in progress. This is the first report on the study of imide hydrolysis in either an Agrobacterium tumefaciens or a Pseudomonas sp., and publications reporting these results are in preparation.
- Full Text:
- Date Issued: 2000
- «
- ‹
- 1
- ›
- »